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Beauchamp 2015 Biosci Rep + (Intrauterine growth restriction is associa … Intrauterine growth restriction is associated with an increased risk of developing obesity, insulin resistance, and cardiovascular disease. However its effect on energetics in heart remains unknown. In this study, we examined respiration in cardiac muscle and liver from adult mice that were undernourished ''in utero''. We report that ''in utero'' undernutrition is associated with impaired cardiac muscle energetics, including decreased fatty acid oxidative capacity, decreased maximum oxidative phosphorylation rate, and decreased proton leak respiration. No differences in oxidative characteristics were detected in liver. We also measured plasma acylcarnitine levels and found that short-chain acylcarnitines are increased with ''in utero'' undernutrition. Results reveal the negative impact of suboptimal maternal nutrition on adult offspring cardiac energy metabolism, which may have lifelong implications for cardiovascular function and disease risk. cardiovascular function and disease risk.)
McKee 2010 Abstract IOC60 + (Introduction: It is well established that … Introduction: It is well established that an appropriately balanced deoxynucleotide (dNTPs) pools are required for normal mitochondrial DNA replication. Disorders in dNTP metabolism that affect these pools lead to mitochondria DNA depletion diseases which have devastating consequences. However, little is known concerning the mechanisms by which these mitochondrial pools are regulated. In earlier work we demonstrated that isolated intact mitochondria were capable of synthesizing all four deoxynucleotide triphosphates (dNTPs) from radiolabeled deoxynucleoside precursors. The aim of this study was to determine the extent that dNTP pools in isolated mitochondria depend on the concentration of deoxynucleosides in the medium and the extent of transport of the mitochondrially synthesized dNTP pools from the matrix to the medium. We are also interested in investigating the extent that anti-viral nucleoside analogs may perturb these systems. In this regard we have already demonstrated that AZT is a competitive inhibitor of thymidine phosphorylation in mitochondria isolated from heart, liver, and brain [1, 2] and that AZT significantly decreases the size of the mitochondrial TTP pool. Methods: Tightly coupled mitochondria isolated from adult rat heart were incubated at 300C in an appropriate medium with varying concentrations of deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC), and thymidine (dT). At various time-points, the levels of all four dNTPs in the medium and in the mitochondria were measured using a template driven assay as described [3] that limits the interference with rNTPs. Results: The results demonstrated that the initial level of the dNTP pools and their response to medium concentration of deoxynucleosides varied considerably with dGTP being the predominant dNTP. TTP was the only dNTP shown to be absolutely dependent on the concentration of its precursor, thymidine in the medium and was also the only mitochondrially synthesized nucleotide to freely equilibrate with the medium. The other mitochondrially synthesized dNTPs appear to be primarily sequestered within the matrix. Conclusions: These results suggest that the mitochondrial TTP pool is likely to be much more sensitive to perturbation in precursor thymidine levels than the other mitochondrial dNTPs.[1] McKee EE, Bentley AT, Hatch M, Gingerich J, Susan-Resiga D (2004) Phosphorylation of thymidine and AZT in heart mitochondria: elucidation of a novel mechanism of AZT cardiotoxicity. Cardiovasc. Toxicol. 4: 155-167.[2] Lynx MD, McKee EE (2006) 3'-Azido-3'-deoxythymidine (AZT) is a competitive inhibitor of thymidine phosphorylation in isolated rat heart and liver mitochondria. Biochem. Pharmacol. 72: 239-243.[3] Ferraro P, Franzolin E, Pontarin G, Reichard P, Bianchi V (2010) Quantitation of cellular deoxynucleoside triphosphates. Nucleic Acids Res. 38: e85.triphosphates. Nucleic Acids Res. 38: e85.)
Iakovou 2022 Front Aging Neurosci + (Introduction: Aging is a normal, inevitabl … Introduction: Aging is a normal, inevitable, irreversible, and progressive process which is driven by internal and external factors. Oxidative stress, that is the imbalance between prooxidant and antioxidant molecules favoring the first, plays a key role in the pathophysiology of aging and comprises one of the molecular mechanisms underlying age-related diseases. However, the oxidative stress theory of aging has not been successfully proven in all animal models studying lifespan, meaning that altering oxidative stress/antioxidant defense systems did not always lead to a prolonged lifespan, as expected. On the other hand, animal models of age-related pathological phenotypes showed a well-correlated relationship with the levels of prooxidant molecules. Therefore, it seems that oxidative stress plays a more complicated role than the one once believed and this role might be affected by the environment of each organism. Environmental factors such as UV radiation, air pollution, and an unbalanced diet, have also been implicated in the pathophysiology of aging and seem to initiate this process more rapidly and even at younger ages.Aim: The purpose of this review is to elucidate the role of oxidative stress in the physiology of aging and the effect of certain environmental factors in initiating and sustaining this process. Understanding the pathophysiology of aging will contribute to the development of strategies to postpone this phenomenon. In addition, recent studies investigating ways to alter the antioxidant defense mechanisms in order to prevent aging will be presented.Conclusions: Careful exposure to harmful environmental factors and the use of antioxidant supplements could potentially affect the biological processes driving aging and slow down the development of age-related diseases. Maybe a prolonged lifespan could not be achieved by this strategy alone, but a longer healthspan could also be a favorable target.althspan could also be a favorable target.)
Misra 2019 J Postgrad Med + (Introduction: Body adiposity measured by p … Introduction: Body adiposity measured by percentage of body fat (BF%) is found to be better predictor of cardiovascular morbidity and mortality than body mass index (BMI). Limited information exists showing relationship between BMI and BF% in North Indian population.Objectives: To study the relationship between BMI and BF% among North Indian adult females across various age strata and level of BMI.Materials and Methods: This was a cross-sectional study conducted at Ballabgarh Health and Demographic Surveillance Site (HDSS) among randomly selected females. BMI using standard techniques and BF% using bioelectrical impedance analysis was estimated. Linear regression was performed using general linear model with BF% as dependent variable and BMI as main independent variable.Results: Mean (±2 SD) age of participants was 41.3 ± 15.7 years. Mean BMI (±SD) was 23.3 (±4.6) kg/m2, whereas mean fat mass (±2 SD) and BF% (±95 % CI) was 19.2 (±7.9) kg and 33.6 (±6.9) %. BMI and BF% were highly correlated among obese (''r'' = 0.77), whereas least correlated (''r'' = 0.32) in underweight females. Across age strata, correlation between BMI and BF% was maximum in 18-35 years age group (''r'' = 0.95), whereas least in females ≥56 years (''r'' = 0.67). Age and BMI together predicted 73 % of variability in BF% in hierarchical linear regression model.Conclusions: In this population, we have found strong correlation between BMI and BF% particularly at higher level of BMI and in younger females. There is need to conduct more robust prospective longitudinal studies to assess BF%, which is a better predictor of cardiovascular morbidity and mortality.of cardiovascular morbidity and mortality.)
Turton 2021 Expert Opinion Orphan Drugs + (Introduction: Coenzyme Q10 (CoQ10) is a ub … Introduction: Coenzyme Q10 (CoQ10) is a ubiquitous organic molecule with a significant role in the mitochondrial electron transport chain (ETC). As a result of its role in such an important biological process, CoQ10 deficiency has been implicated in the pathogenesis of numerous diseases such as Parkinson’s disease (PD) and multiple sclerosis (MS). This has led to multiple attempts to use CoQ10 supplementation as a treatment or pre-treatment with varying degrees of success.Areas covered: The present review will identify evidence of mitochondrial dysfunction in MS, PD and mitochondrial ETC disorders. In addition, the inability of Co10 supplementation to elicit significant clinical outcome in these disorders and possible flaws in these studies will be discussed. The databases utilized for this review were the Web of science and PubMed, with inclusive dates (1957–2021).Expert opinion: A lack of improved neurological outcome in these disorders post treatment with CoQ10 may be attributable to the limited ability of CoQ10 to cross the blood–brain barrier. Thus, CoQ10 alternatives should also be considered. Other factors including time of disease diagnosis, dosage, administration, and duration of CoQ10 therapy may have a significant influence on the efficacy of this treatment.fluence on the efficacy of this treatment.)
Roden 2023 MiP2023 + (Introduction: Common metabolic diseases su … Introduction: Common metabolic diseases such obesity and type 2 diabetes associate with insulin resistance (1), but also with impaired mitochondrial function in several tissues such as skeletal muscle and adipose tissue (2,3). Assessment of hepatic mitochondrial functionality in the liver is more challenging because of invasive and complex methodology, which limits large-scale studies in human. Results and Discussion: Previously, employing noninvasive multitracer magnetic resonance spectroscopy we found reduction in hepatic ATP concentration and synthesis in humans with type 2 diabetes, but not in those with obesity (4). Subsequent high-resolution respirometry studies allowed us to detect an adaptation of hepatic oxidative capacity to excessive lipid availability (mitochondrial plasticity) in humans with obesity, which is lost with progressive non-alcoholic fatty liver disease, i. e. steatohepatitis (NASH) (5). The loss of mitochondrial plasticity goes along impaired antioxidant defense leading to increased H202 production and lipid peroxidation. These alterations are even more pronounced in people with type 2 diabetes or with increasing liver fibrosis (6). The alterations of mitochondrial function are preceded by changes in mitochondrial quality control and help to explain ultrastructural features of NASH such megamitochondria. Adipose tissue dysfunction (1), specifically impairment of insulin sensitivity and mitochondrial oxidative capacity in visceral adipose tissue (7), could be the initial driving force of altered hepatic energy metabolic and common metabolic diseases. # Roden M, Shulman GI (2019) The integrative biology of type 2 diabetes. doi: 10.1038/s41586-019-1797-8. Epub 2019 Dec 4.# Fromenty B, Roden M. Mitochondrial alterations in fatty liver diseases. J Hepatol. 2023 Feb;78(2):415-429. https://doi.org/10.1016/j.jhep.2022.09.020. Epub 2022 Oct 7.# Targher G, Corey KE, Byrne CD, Roden M (2021) The complex link between NAFLD and type 2 diabetes mellitus - mechanisms and treatments. https://doi.org/10.1038/s41575-021-00448-y. Epub 2021 May 10.# Szendroedi J, Chmelik M, Schmid AI, Nowotny P, Brehm A, Krssak M, Moser E, Roden M (2009) Abnormal hepatic energy homeostasis in type 2 diabetes. https://doi.org/10.1002/hep.23093.# Koliaki C, Szendroedi J, Kaul K, Jelenik T, Nowotny P, Jankowiak F, Herder C, Carstensen M, Krausch M, Knoefel WT, Schlensak M, Roden M (2015). Adaptation of hepatic mitochondrial function in humans with non-alcoholic fatty liver is lost in steatohepatitis. https://doi.org/10.1016/j.cmet.2015.04.004.# Pafili K, et al. (2022) Mitochondrial respiration is decreased in visceral but not subcutaneous adipose tissue in obese individuals with fatty liver disease. https://doi.org/10.1016/j.jhep.2022.08.010. Epub 2022 Aug 19.# Gancheva S, et al. (2022) Impaired Hepatic mitochondrial capacity in nonalcoholic steatohepatitis associated with type 2 diabetes. https://doi.org/10.2337/dc21-1758.type 2 diabetes. https://doi.org/10.2337/dc21-1758. )
Khan 2020 Cureus + (Introduction: Depression is one of the mos … Introduction: Depression is one of the most incapacitating psychiatric diseases that disturb life of millions of people round the globe. Its major causes include stressful life events, bereavement, social abuses or certain biological and genetic factors with complex causal mechanisms. Higher salivary cortisol levels for a long period lead to dyslipidemias which increase body mass index (BMI), elevate adiposity and waist-to-hip ratio (WHR). Such individuals with high quartiles of BMI have considerably higher risk of major depressive disorder. The aim of this study was to establish a correlation between major depression, BMI and salivary cortisol. Methods: This cross-sectional analysis was accomplished in the Physiology Department, Sheikh Zayed Federal Postgraduate Medical Institute, Lahore as well as in Punjab Institute of Mental Health, Lahore, Pakistan, over a period of six months. A total of 60 participants aged between 18 and 60 years were included in this study; they were divided equally into two groups as normal healthy individuals with no physical or mental illness and severely depressed groups. The patients were categorized as cases of severe depression on outdoor clinical assessment and further confirmed by ICD-10. Patient's BMI was estimated by measuring height in meters (m) and weight in kilograms (kg), and then dividing weight with square height. Early morning saliva samples were collected. Estimation of cortisol levels in saliva was done through ELISA. SPSS version 20.0 (IBM Corp., Armonk, NY) was used to analyze the data and ''p'' ≤ 0.05 was considered statistically significant. Results: The mean BMI in normal healthy group was 22.02 ± 4.21, while the mean BMI in severely depressive group was 24.64 ± 3.58. The difference was statistically significant (''p'' = 0.012). The mean salivary cortisol level was significantly raised in patients with major depression (2.23 ± 1.69 nmol/L) in contrast to healthy normal individuals (1.46 ± 0.91 nmol/L), with ''p''-value = 0.031. Conclusion: BMI and depression has a very noteworthy correlation and there is a remarkable link between raised salivary cortisol, greater BMI and development of major depression.r BMI and development of major depression.)
Garcia-Souza 2013 Abstract IOC80 + (Introduction: Evidence has indicated that … Introduction: Evidence has indicated that pro-coagulant factors modulate platelet energy and redox metabolism pathways. However, the involvement of mitochondria during platelet activation remain poorly understood and was investigated in the present work. Materials and methods: Human platelets were collected from healthy volunteers, isolated in M199 medium, and subsequently challenged with different thrombin concentrations. Several parameters were analyzed in activated platelets such as P-selectin externalization, respiration, lactate and nitric oxide (NO) production. The assessment of oxygen flow and metabolic states in platelets was carried out in the presence of several mitochondrial modulators by titrating each one of them in the following order: Oligomycin, FCCP, rotenone and Antimycin A in the presence or absence of thrombin. Results and discussion: During platelet activation, CD62p expression increased and was followed by an increase in lactate secretion and NO production with high correlation between each other. Analyzing mitochondrial states we could detect changes in several states, mainly ROUTINE, OXPHOS, proton leak and reserve capacity. However, normalizing our signal by the highest uncoupled oxygen consumption (ET-pathway), we detected differences in residual oxygen consumption (ROX) alongside other states. Conclusions: Our data indicate that mitochondria from human platelets are affected by their ativation. This raises the concern of using platelets as models of indirect studies such as mitochondrial diseases and Alzheimer. The mitochondria play a very important role in platelet physiology and is not an inert within the cell, is affected by a number of substances that are present at the time of activation, such as calcium and nitric oxide. Therefore, it is necessary to evaluate the changes in mitochondrial human platelets against different agonists and inhibitors of coagulation.nt agonists and inhibitors of coagulation.)
Liufu 2023 Front Physiol + (Introduction: Mitochondrial disease is a s … Introduction: Mitochondrial disease is a spectrum of debilitating disorders caused by mutations in the mitochondrial DNA (mtDNA) or nuclear DNA that compromises the respiratory chain. Mitochondrial 3243A>G (m.3243 A>G) is the most common mutation showing great heterogeneity in phenotype. Previous studies have indicated that NADH: ubiquinone oxidoreductase (complex I) deficiency accompanied by a decreased nicotinamide adenine dinucleotide (NAD+)/reduced NAD+ (NADH) ratio may play a pivotal role in the pathogenesis of m.3243A>G mutation. Methods: To evaluate the potential effects of strategies targeting the imbalanced NAD+/NADH ratio in m.3243A>G mutation, we treated fibroblasts derived from patients with the m.3243 A>G mutation using nicotinamide riboside (NR) or mitochondria-targeted H2O-forming NADH oxidase (mitoLbNOX). Results: M.3243 A>G fibroblasts showed a significant reduction in complex I core subunit 6, complex I enzymatic activity, complex I-dependent oxygen consumption rate (OCR), and adenosine triphosphate (ATP) production compared to the controls. The NAD+/NADH ratio was also significantly reduced in m.3243 A>G fibroblasts, and, using fluorescence lifetime imaging microscopy, we also found that the NADH level was elevated in m.3243 A>G fibroblasts. After NR treatment, the NAD+/NADH ratio, complex I-dependent OCR, and ATP levels increased, whereas NADH levels remained unchanged. More excitingly, after treatment with mitoLbNOX, the NAD+/NADH ratio, complex I-independent OCR, and ATP levels increased more pronouncedly compared with the NR treatment group, accompanied by significantly reduced NADH levels. Discussion: The present study suggests that compared with repletion of NAD+ alone, the combination of this therapeutic modality with alleviation of NADH overload may amplify the treatment effect of restoring NAD+/NADH balance in m.3243A>G fibroblasts.nt effect of restoring NAD+/NADH balance in m.3243A>G fibroblasts.)
Weiss 2022 Shock + (Introduction: Peripheral blood mononuclear … Introduction: Peripheral blood mononuclear cells (PBMCs) are commonly used to compare mitochondrial function in patients with versus without sepsis, but how these measurements in this mixed cell population vary by composition of immune cell subtypes is not known, especially in children. We determined the effect of changing immune cell composition on PBMC mitochondrial respiration and content in children with and without sepsis.Methods: PBMC mitochondrial respiration and citrate synthase (CS) activity, a marker of mitochondrial content, were measured in 167 children with sepsis at three timepoints (day 1-2, 3-5, and 8-14) and once in 19 nonseptic controls. The proportion of lymphocytes and monocytes and T, B, and NK cells was measured using flow cytometry. More specific CD4+ and CD8+ T cell subsets were measured from 13 sepsis patients and 6 controls. Spearman's correlation and simple and mixed effects linear regression were used to determine the association of PBMC mitochondrial measures with proportion of immune cell subtypes.Results: PBMC mitochondrial respiration and CS activity were correlated with proportion of monocytes, lymphocytes, T B, and NK cells in controls, but not in sepsis patients. PBMC mitochondrial respiration was correlated with CD4+ and CD8+ T cell subsets in both groups. After controlling for differences in immune cell composition between groups using linear regression models, PBMC respiration and CS activity remained lower in sepsis patients than controls.Conclusions: Mitochondrial measurements from PBMCs varied with changes in immune cell composition in children with and without sepsis. However, differences in PBMC mitochondrial measurements between sepsis patients and controls were at least partially attributable to the effects of sepsis rather than solely an epiphenomena of variable immune cell composition.omena of variable immune cell composition.)
Karabatsiakis 2022 Abstract Bioblast + (Introduction: Post-traumatic stress disord … Introduction: Post-traumatic stress disorder (PTSD) is a mental disorder associated with the exposure to chronic and/or traumatic psychological stress. PTSD causes severe individual suffering, impairments in everyday-life functioning and psychosomatic complaints. Using cross-sectional study designs, we [1] and others [2,3] provided evidence for impaired mitochondrial function in different biological samples including intact peripheral blood mononuclear cells (PBMC) collected from patients with major depressive disorder (MDD), one highly prominent psychiatric comorbidity of PTSD. However, data on the possible reversibility effects following psychiatric treatments are sparse but represent an aspect of highest interest for translational biomarker research in the fields of clinical psychology and psychiatry. Here, we present initial data from a pilot study reporting treatment effects of eye-movement desensitization and reprocessing (EMDR) therapy, a clinically approved and specialized treatment option for PTSD, on mental functioning in combination with mitochondrial bioenergetics and biogenesis.Methods: Three female patients diagnosed with PTSD and comorbid MDD were recruited in the Psychiatry Unit of University Hospital in Ulm (Germany), which also provided blood samples for an initial cross-sectional investigation of mitochondrial function in PBMC. One patient agreed to be also followed longitudinally and blood samples were collected at two additional time points across treatment with EMDR. All participants provided written informed consent before participation in the pilot study. Psychiatric impairments related to depression were measured with the Beck Depression Inventory (BDI) [4] as previously reported [1]. To characterize mitochondrial function, PBMC were isolated from non-fasting EDTA-buffered whole blood using density gradient centrifugation procedures. Isolated intact PBMC were cryopreserved using standardized procedures and thawed samples were used for high-resolution respirometry using the O2k (Oroboros Instruments, Austria). Following the measurement of oxygen consumption rates, mitochondrial density was measured in shock-frozen samples using spectrophotometrical assessment of citrate synthase activity (CSA). Respiration data and CSA results were compared to a group of previously characterized individuals free of any history of mental disorders (control group, ''n''=38).Results: While respiration levels and mitochondrial mass were highly stable over a five-weeks interval in the control group, the samples from the three patients with PTSD showed a significant reduction in these mitochondrial parameters before EMDR treatment. A significant improvement in the clinical severity of depressive symptoms was found with EMDR treatment. In addition, mitochondrial oxygen consumption and mitochondrial mass normalized to the level of the mentally-stable control subjects.Conclusions: Effects of EMDR treatment on patients with PTSD and comorbid MDD seem to include not only an improvement in the level of mental functioning but can also be associated with a normalization of mitochondrial respiration and mitochondrial density in PBMC. The underlying biomolecular processes that lead to these normalization processes associated with EMDR treatment need further investigation. Here, changes in inflammation and bioenergetic metabolism are of special interest for future studies. Towards a clinically-applicable biomarker in the field of clinical psychology and psychiatry, the robustness of our first observation requires another full study cohort with a longitudinal design to demonstrate and confirm mitochondrial bioenergetics and biogenesis as two interrelated biomarker candidates to be used in psychotherapy and psychopharmacological research.# Karabatsiakis A, Böck C, Salinas-Manrique J, Kolassa S, Calzia E, Dietrich DE, Kolassa IT (2014) Mitochondrial respiration in peripheral blood mononuclear cells correlates with depressive subsymptoms and severity of major depression. https://doi.org/10.1038/tp.2014.44# Kuffner K, Triebelhorn J, Meindl K, Benner C, Manook A, Sudria-Lopez D, Siebert R, Nothdurfter C, Baghai TC, Drexler K, Berneburg M, Rupprecht R, Milenkovic VM, Wetzel CH (2020) Major depressive disorder is associated with impaired mitochondrial function in skin fibroblasts. https://doi.org/10.3390/cells9040884# Hroudová J, Fišar Z, Kitzlerová E, Zvěřová M, Raboch J (2013) Mitochondrial respiration in blood platelets of depressive patients. https://doi.org/10.1016/j.mito.2013.05.005# Hautzinger M, Keller F, Kühner C (2006) Beck depressions inventar revision—Manual. Frankfurt, Germany: Harcourt Test Services.kfurt, Germany: Harcourt Test Services. )
Koizumi 2023 Front Cardiovasc Med + (Introduction: Recent studies have demonstr … Introduction: Recent studies have demonstrated that sodium-glucose co-transporter-2 inhibitors (SGLT2-i) reduce the risk of atrial fibrillation (AF) in patients with diabetes mellitus (DM), in which oxidative stress due to increased reactive oxygen species (ROS) contributes to the pathogenesis of AF. We aimed to further investigate this, and examine whether the SGLT2-i empagliflozin suppresses mitochondrial-ROS generation and mitigates fibrosis.Methods: A high-fat diet and low-dose streptozotocin treatment were used to induce type-2 DM (T2DM) in Sprague-Dawley rats. The rats were randomly divided into three groups: control, DM, and DM treated with empagliflozin (30 mg/kg/day) for 8 weeks. The mitochondrial respiratory capacity and ROS generation in the atrial myocardium were measured using a high-resolution respirometer. Oxidative stress markers and protein expression related to mitochondrial biogenesis and dynamics as well as the mitochondrial morphology were examined in the atrial tissue. Additionally, mitochondrial function was examined in H9c2 cardiomyoblasts. Atrial tachyarrhythmia (ATA) inducibility, interatrial conduction time (IACT), and fibrosis were also measured.Results: Inducibility of ATA, fibrosis, and IACT were increased in rats with DM when compared to controls, all of which were restored by empagliflozin treatment. In addition, the rats with DM had increased mitochondrial-ROS with an impaired complex I-linked oxidative phosphorylation capacity. Importantly, empagliflozin seemed to ameliorate these impairments in mitochondrial function. Furthermore, empagliflozin reversed the decrease in phosphorylated AMPK expression and altered protein levels related to mitochondrial biogenesis and dynamics, and increased mitochondrial content. Empagliflozin also improved mitochondrial function in H9c2 cells cultured with high glucose medium.Discussion: These data suggest that empagliflozin has a cardioprotective effect, at least in part, by reducing mitochondrial ROS generation through AMPK signaling pathways in the atrium of diabetic rats. This suggests that empagliflozin might suppress the development of AF in T2DM.ht suppress the development of AF in T2DM.)
Shim 2022 Cancer Cell Int + (Introduction: The importance of fatty acid … Introduction: The importance of fatty acid oxidation (FAO) in the bioenergetics of glioblastoma (GBM) is being realized. Etomoxir (ETO), a carnitine palmitoyltransferase 1 (CPT1) inhibitor exerts cytotoxic effects in GBM, which involve interrupting the FAO pathway. We hypothesized that FAO inhibition could affect the outcomes of current standard temozolomide (TMZ) chemotherapy against GBM.Methods: The FAO-related gene expression was compared between GBM and the tumor-free cortex. Using four different GBM tumorspheres (TSs), the effects of ETO and/or TMZ was analyzed on cell viability, tricarboxylate (TCA) cycle intermediates and adenosine triphosphate (ATP) production to assess metabolic changes. Alterations in tumor stemness, invasiveness, and associated transcriptional changes were also measured. Mouse orthotopic xenograft model was used to elucidate the combinatory effect of TMZ and ETO.Results: GBM tissues exhibited overexpression of FAO-related genes, especially CPT1A, compared to the tumor-free cortex. The combined use of ETO and TMZ further inhibited TCA cycle and ATP production than single uses. This combination treatment showed superior suppression effects compared to treatment with individual agents on the viability, stemness, and invasiveness of GBM TSs, as well as better downregulation of FAO-related gene expression. The results of in vivo study showed prolonged survival outcomes in the combination treatment group.Conclusion: ETO, an FAO inhibitor, causes a lethal energy reduction in the GBM TSs. When used in combination with TMZ, ETO effectively reduces GBM cell stemness and invasiveness and further improves survival. These results suggest a potential novel treatment option for GBM. potential novel treatment option for GBM.)
Phillips 2017 Cancer Research + (Introduction: We have reported aberrant ov … Introduction: We have reported aberrant overexpression of CBS in human colorectal cancers, where its activity (H2S production) promotes tumor progression and metastasis. New data show that CBS expression increases in adenomatous polyps. The aims of this study were to identify possible mechanisms for CBS upregulation and determine the biological impact of its upregulation using a premalignant colonic epithelial cell line NCM356.Methods: Induction of CBS in response to LPS, TLR2&4 agonists, and hypoxia was assessed by immunoblotting. CBS overexpression was achieved using lentiviral expression vector (pReceiver-Lv103, GeneCopoeia™) in NCM356 cells. Global metabolic profiling (Metabolon, Durham) was performed on cellular extracts from NCM365 overexpressing cells (CBS-hi) and vector-transduced controls. High-resolution respirometry (Orobors Oxygraph-2k) assessed bioenergetics. H2S levels were measured using the fluorogenic probe 7-Azido-4-methylcoumarin. A Coulter counter was used to assess cell proliferation rates. Transwell assays were used to assess cell migration. CBS activity was inhibited with aminooxyacetic acid (AOAA).Results: CBS protein expression was increased by ~3-fold in pNCM after 2 and 24 hr of hypoxia (1% O2). Treatment with LPS but not the TLR agonists increased CBS expression 2-fold. Metabolic profiling of CBS-hi cells and controls identified 85 metabolites that were differentially expressed (65 increased and 20 decreased; p≤0.05 Welch’s 2-Sample t-Test). The metabolite lanthionine, which is produced by the CBS-dependent condensation of cysteine to produce H2S, showed the largest increase (12.3-fold increase, p=1.5E-06). A 4-fold increase in basal H2S production was measured in CBS-hi cells compared to control cultures. High-resolution respirometry showed that CBS-hi cells exhibited a significant increase in maximum respiration rate, reserve respiratory capacity and increased citrate synthase activity, suggesting increased cellular mitochondria mass. Treatment of CBS-hi cells either with pentose phosphate pathway inhibitor (oxythiamine) or lactate dehydrogenase inhibitor (XF-11) reducing growth by 90% and ~25% at day 5, respectively; the inhibitors had no effect on the vector control cells. Exposure of NCM356 cells to hypoxic conditions resulted in increased migration to conditioned media (p<0.001, NCM356 normoxia vs. hypoxia); inhibition of CBS using AOAA attenuated the hypoxia-induced migration.Conclusions: Hypoxia and LPS can induce CBS expression in a premalignant colonic epithelial cell line. Upregulation of CBS has a board impact on cellular metabolism including enhanced flux through the transsulfuration, glycolytic and pentose phosphate pathways, resulting in enhanced cellular bioenergetics, growth and migration. These data suggest that enhance CBS activity may be involved the adenoma to carcinoma sequence.nd migration. These data suggest that enhance CBS activity may be involved the adenoma to carcinoma sequence.)
Pernas MiP2010 + (Invasion with the intracellular parasite T … Invasion with the intracellular parasite Toxoplasma gondii results in the formation of a parasitophorous vacuole (PV), which constitutes a safe niche for parasite survival. The PV membrane (PVM) fails to fuse with endocytic organelles yet exhibits a remarkable and specific association with host mitochondria. Previous work has modelled rhoptry protein 2 (ROP2) as the physical link that tethers host mitochondria to the parasitophorous vacuole membrane (PVM). A recent analysis of the ROP2 family protein structures however, suggests that the ROP2 transmembrane domain postulated to mediate mitochondrial recruitment to the PVM is actually buried in the core of the protein’s conserved serine/threonine kinase fold. To determine if ROP2 is necessary for PVM-mitochondrial association, we created a parasite line deficient in ROP2 and ROP8 expression and analyzed the PVM-mitochondrial association of this knockout mutant. We show that mutants deficient in ROP2 and ROP8 expression retain the ability to recruit host mitochondria in a manner that is indistinguishable from the parental strain re-opening two important questions: what mediates mitochondrial recruitment to the Toxoplasma PVM and what effect, if any, is this having on host cells?Fig. 1. Vacuoles containing Toxoplasma RHDrop2/8 tachyzoites show no defect in mitochondrial association by light microscopy. Human foreskin fibroblasts (HFFs) were labelled for 30 min at 37 oC with 50 nM MitoTracker and infected with (A) RH wild type; (B) Neospora caninum (NC-1); (C) RHΔrop2/8, or co-infected with (D) RHgfpluc and RHΔrop2/8. Four hours p.i. monolayers were fixed in 3.7% formaldehyde for 15 min at 37 oC. Scale bar, 5 µm. Colour figure: p. 122.. Scale bar, 5 µm. Colour figure: p. 122.)
Blatzer 2015 Antioxid Redox Signal + (Invasive fungal infections have significan … Invasive fungal infections have significantly increased over the past decades in immune-compromised individuals and high-risk patients. Amphotericin B (AmB) exerts a powerful and broad activity against a vast array of fungi and has a remarkably low rate of microbial resistance. However, most isolates of ''A. terreus'' developed an intrinsic resistance against AmB and during this study we characterized the mode of action of this polyene antifungal drug in more detail in resistant (ATR) and rare susceptible (ATS) clinical isolates of A. terreus.We illustrate that AmB treatment changes cellular redox status and promotes the generation of high levels of reactive oxygen species (ROS) in ATS. In contrast, ATR were able to cope better with AmB-induced oxidative stress.Most importantly, we demonstrate in this study that co-application of anti- and pro-oxidants significantly affects AmB efficacy in an antithetic manner - antioxidants and ROS-scavenging agents increase AmB tolerance in susceptible strains, while pro-oxidants render formerly resistant isolates considerably susceptible to the antifungal drug also ''in vivo'' in a Galleria animal model.Thereby, our study provides novel therapeutic options to treat formerly resistant fungal strains by a combination of AmB and pro-oxidant compounds. of AmB and pro-oxidant compounds. )
Senyilmaz 2015 Fatty Acid Oxidation O2k-Network Discussion Forum + (Inverted Drosophila larvae which are permeabilized by digitonin. Their body walls are more or less made of muscles.)
Hughey 2011 J Vis Exp + (Investigation of mitochondrial function re … Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, oxidative stress, apoptosis, aging, mitochondrial encephalomyopathies and drug toxicity. Given this, technologies to measure cardiac mitochondrial function are in demand. One technique that employs an integrative approach to measure mitochondrial function is respirometric oxidative phosphorylation (OXPHOS) analysis.The principle of respirometric OXPHOS assessment is centered around measuring oxygen concentration utilizing a Clark electrode. As the permeabilized fiber bundle consumes oxygen, oxygen concentration in the closed chamber declines. Using selected substrate-inhibitor-uncoupler titration protocols, electrons are provided to specific sites of the electron transport chain, allowing evaluation of mitochondrial function. Prior to respirometric analysis of mitochondrial function, mechanical and chemical preparatory techniques are utilized to permeabilize the sarcolemma of muscle fibers. Chemical permeabilization employs saponin to selectively perforate the cell membrane while maintaining cellular architecture.This paper thoroughly describes the steps involved in preparing saponin-skinned cardiac fibers for oxygen consumption measurements to evaluate mitochondrial OXPHOS. Additionally, troubleshooting advice as well as specific substrates, inhibitors and uncouplers that may be used to determine mitochondria function at specific sites of the electron transport chain are provided. Importantly, the described protocol may be easily applied to cardiac and skeletal tissue of various animal models and human samples.f various animal models and human samples.)
Hartmann 2017 Crit Care Med + (Investigation of the effects of hyperoxia … Investigation of the effects of hyperoxia during resuscitation from hemorrhagic shock in swine with preexisting coronary artery disease.Prospective, controlled, randomized trial.University animal research laboratory.Nineteen hypercholesterolemic pigs with preexisting coronary artery disease.Anesthetized, mechanically ventilated, and surgically instrumented pigs underwent 3 hours of hemorrhagic shock (removal of 30% of the calculated blood volume and subsequent titration of mean arterial blood pressure ≈40 mm Hg). Postshock resuscitation (48 hr) comprised retransfusion of shed blood, crystalloids (balanced electrolyte solution), and norepinephrine support. Pigs were randomly assigned to "control" (FIO2 0.3, adjusted for arterial oxygen saturation ≥ 90%) and "hyperoxia" (FIO2 1.0 for 24 hr) groups.Before, at the end of shock and every 12 hours of resuscitation, datasets comprising hemodynamics, calorimetry, blood gases, cytokines, and cardiac and renal function were recorded. ''Postmortem'', organs were sampled for immunohistochemistry, western blotting, and mitochondrial high-resolution respirometry. Survival rates were 50% and 89% in the control and hyperoxia groups, respectively (p = 0.077). Apart from higher relaxation constant τ at 24 hours, hyperoxia did not affect cardiac function. However, troponin values were lower (2.2 [0.9-6.2] vs 6.9 [4.8-9.8] ng/mL; p < 0.05) at the end of the experiment. Furthermore, hyperoxia decreased cardiac 3-nitrotyrosine formation and increased inducible nitric oxide synthase expression. Plasma creatinine values were lower in the hyperoxia group during resuscitation coinciding with significantly improved renal mitochondrial respiratory capacity and lower 3-nitrotyrosine formation.Hyperoxia during resuscitation from hemorrhagic shock in swine with preexisting coronary artery disease reduced renal dysfunction and cardiac injury, potentially resulting in improved survival, most likely due to increased mitochondrial respiratory capacity and decreased oxidative and nitrosative stress. Compared with our previous study, the present results suggest a higher benefit of hyperoxia in comorbid swine due to an increased susceptibility to hemorrhagic shock.ncreased susceptibility to hemorrhagic shock.)
Burtscher 2011Abstract Mitochondrial Medicine + (Investigations of oxidative phosphorylatio … Investigations of oxidative phosphorylation (OXPHOS) play an increasingly important role in relating mitochondrial health to life style (exercise and nutrition), mitochondrial haplogroups and heteroplasmy, and individual training programs of competitive athletes.training programs of competitive athletes.)
Vrbacky 2007 Biochim Biophys Acta + (Involvement of mammalian [[Glycerophosphate dehydrogenase complex |mitochondrial glycerophosphate dehydrogenase]] … Involvement of mammalian [[Glycerophosphate dehydrogenase complex |mitochondrial glycerophosphate dehydrogenase]] (mGPDH, EC 1.1.99.5) in reactive oxygen species (ROS) generation was studied in brown adipose tissue mitochondria by different spectroscopic techniques. Spectrofluorometry using ROS-sensitive probes CM-H2 DCFDA and Amplex Red was used to determine the glycerophosphate- or succinate-dependent ROS production in mitochondria supplemented with respiratory chain inhibitors antimycin A and myxothiazol. In case of glycerophosphate oxidation, most of the ROS originated directly from mGPDH and coenzyme Q while complex III was a typical site of ROS production in succinate oxidation. Glycerophosphate-dependent ROS production monitored by KCN-insensitive oxygen consumption was highly activated by one-electron acceptor ferricyanide, whereas succinate-dependent ROS production was unaffected. In addition, superoxide anion radical was detected as a mGPDH-related primary ROS species by fluorescent probe dihydroethidium, as well as by electron paramagnetic resonance (EPR) spectroscopy with DMPO spin trap. Altogether, the data obtained demonstrate pronounced differences in the mechanism of ROS production originating from oxidation of glycerophosphate and succinate indicating that electron transfer from mGPDH to coenzyme Q is highly prone to electron leak and superoxide generation.ghly prone to electron leak and superoxide generation.)
Evinova 2024 J Bioenerg Biomembr + (Inward rectifying potassium channels sensi … Inward rectifying potassium channels sensitive to ATP levels (KATP) have been the subject of investigation for several decades. Modulators of KATP channels are well-established treatments for metabolic as well as cardiovascular diseases. Experimental studies have also shown the potential of KATP modulation in neurodegenerative disorders. However, to date, data regarding the effects of KATP antagonists/agonists in experiments related to neurodegeneration remain inconsistent. The main source of confusion in evaluating available data seems to be the choice of experimental models. The present study aims to provide a comprehensive understanding of the effects of both opening and blocking KATP channels in two forms of SH-SY5Y cells. Our results offer valuable insights into the significance of metabolic differences between differentiated and non-differentiated SH-SY5Y cells, particularly in the context of glibenclamide and diazoxide effects under normal conditions and during the initiation of pathological events simulating Parkinson's disease ''in vitro''. We emphasize the analysis of mitochondrial functions and changes in mitochondrial network morphology. The heightened protein expression of KATP channels identified in non-differentiated SH-SY5Y cells seems to be a platform for a more significant impact of KATP modulators in this cell type. The efficiency of rotenone treatment in inducing morphological changes in the mitochondrial network depends on the differentiation status of SH-SY5Y cells.e differentiation status of SH-SY5Y cells.)
Peruzzo 2016 Abstract Mito Xmas Meeting Innsbruck + (Ion channels are emerging as new oncologic … Ion channels are emerging as new oncological targets. Indeed, several ion channels show a different expression pattern in normal and cancer cells. The potassium channel Kv1.3 has a multiple sub-cellular localization, including both in the plasma membrane and in the inner mitochondrial membrane. Pharmacological inhibition of the mitochondrial channel (mtKv1.3), but not of the plasma membrane channel, by membrane permeant blockers, Psora-4, PAP-1 and clofazimine, triggered apoptosis in different cancer cells. Cell death occured even in the absence of Bax and Bak, by inducing mitochondrial membrane depolarization, production of mitochondrial ROS and release of cytochrome c. Downregulation by siRNA of Kv1.3 prevented all these effects, indicating specificity. Since membrane permeant Kv1.3 inhibitors are characterized by poor water solubility, in order to increase their bioavailability as well as their solubility, we have recently synthesized a few more soluble PAP-1 derivatives. The new derivatives have been found to selectively kill cancer cells ''in vitro'' and even ''in vivo'' in a mouse melanoma preclinical model, without inducing any side effect.l model, without inducing any side effect.)
Rieger 2014 Nat Commun + (Ion-driven ATP synthesis by rotary F0F1 AT … Ion-driven ATP synthesis by rotary F0F1 ATP-synthase powers aerobic life. Since Mitchell's seminal hypothesis, this synthesis has been discussed in terms of the proton-motive force between two bulk phases, each in equilibrium. In active mitochondria, a steady proton flow cycles between pumps and the distant ATP synthase. Here we determine the lateral pH profile along the p-side of cristae in situ by attaching a ratiometric fluorescent pH-sensitive GFP variant to OXPHOS complex IV, a proton pump, and the dimeric F0F1 ATP-synthase, a proton consumer. In respiring HeLa cells, we observe that the local pH at F0F1 dimers is 0.3 units less acidic than that at complex IV. This finding is consistent with the calculated pH profile for steady proton diffusion from CIV to F0F1. The observed lateral variation in the proton-motive force necessitates a modification to Peter Mitchell's chemiosmotic proposal. The experimental technique can be extended to other pH-dependent reactions in membrane microcompartments.t reactions in membrane microcompartments.)
Bunkin 2016 J Phys Chem B + (Ion-stabilized gas nanobubbles (the so-ter … Ion-stabilized gas nanobubbles (the so-termed "bubstons") and their clusters are investigated in bulk aqueous solutions of NaCl at different ion concentrations by four independent laser diagnostic methods. It turned out that in the range of NaCl concentration 10(-6) < C < 1 M the radius of bubston remains virtually unchanged at a value of 100 nm. Bubstons and their clusters are a thermodynamically nonequilibrium phase, which has been demonstrated in experiments with magnetic stirrer at different stirring rates. Different regimes of the bubston generation, resulting from various techniques of processing the liquid samples, were explored.of processing the liquid samples, were explored.)
Brun 2022 Plant Direct + (Iron (Fe) is an essential metal ion that p … Iron (Fe) is an essential metal ion that plays a major role as a cofactor in many biological processes. The balance between the Fe2+ and Fe3+ forms is central for cellular Fe homeostasis because it regulates its transport, utilization, and storage. Contrary to Fe3+ reduction that is crucial for Fe uptake by roots in deficiency conditions, ferroxidation has been much less studied. In this work, we have focused on the molecular characterization of two members of the MultiCopper Oxidase family (MCO1 and MCO3) that share high identity with the ''Saccharomyces cerevisiae'' ferroxidase Fet3. The heterologous expression of MCO1 and MCO3 restored the growth of the yeast fet3fet4 mutant, impaired in high and low affinity Fe uptake and otherwise unable to grow in Fe deficient media, suggesting that MCO1 and MCO3 were functional ferroxidases. The ferroxidase enzymatic activity of MCO3 was further confirmed by the measurement of Fe2+-dependent oxygen consumption, because ferroxidases use oxygen as electron acceptor to generate water molecules. In planta, the expression of MCO1 and MCO3 was induced by increasing Fe concentrations in the medium. Promoter-GUS reporter lines showed that MCO1 and MCO3 were mostly expressed in shoots and histochemical analyses further showed that both promoters were highly active in mesophyll cells. Transient expression of MCO1-RFP and MCO3-RFP in tobacco leaves revealed that both proteins were localized in the apoplast. Moreover, cell plasmolysis experiments showed that MCO1 remained closely associated to the plasma membrane whereas MCO3 filled the entire apoplast compartment. Although the four knock out mutant lines isolated (mco1-1, mco1-2, mco3-1, and mco3-2) did not display any macroscopic phenotype, histochemical staining of Fe with the Perls/DAB procedure revealed that mesophyll cells of all four mutants overaccumulated Fe inside the cells in Fe-rich structures in the chloroplasts, compared with wild-type. These results suggested that the regulation of Fe transport in mesophyll cells had been disturbed in the mutants, in both standard condition and Fe excess. Taken together, our findings strongly suggest that MCO1 and MCO3 participate in the control of Fe transport in the mesophyll cells, most likely by displacing the Fe2+/Fe3+ balance toward Fe3+ in the apoplast and therefore limiting the accumulation of Fe2+, which is more mobile and prone to be transported across the plasma membrane.ore limiting the accumulation of Fe2+, which is more mobile and prone to be transported across the plasma membrane.)
Volani 2016 Abstract MitoFit Science Camp 2016 + (Iron is a fundamental co-factor for severa … Iron is a fundamental co-factor for several cell processes, including oxidative phosphorylation, and mitochondria are the major sites of iron-utilization [1]. Besides being central part of mitochondrial complex I-IV enzymes of the electron transport system, iron also regulates citric acid cycle activity by modulating mitochondrial aconitase expression [2,3]. However, so far, little information is available on how mitochondrial function is affected by alterations of iron homeostasis, and how to best measure tissue mitochondrial activity and its interaction with iron metabolism ''in vivo''; therefore we aimed at investigating the impact of tissue iron loading on mitochondria as well as the presence of mitochondrial blood markers, that might represent good surrogates for tissue iron imbalances.To access the impact of iron on mitochondrial respiration we studied mitochondrial function in liver samples of 10-week old FVB mice and C57BL/6N mice, receiving either normal- or high iron (25 g/kg)-diet two weeks before being sacrificed. The liver was collected and stored in Custadiol prior to homogenization in MiR05. Mitochondrial LEAK respiration, Complex I and II maximal oxidative phosphorylation together with ET-pathway of the liver homogenates (2 mg) were assessed by means of high resolution respirometry (Oroboros Instruments, Austria).Our ongoing experiments indicate that mitochondrial function testing can be successfully performed in mouse tissues as well as in isolated PBMCs from rats. Analysis of liver samples from mice indicate that dietary iron supplementation triggers changes in oxidative phosphorylation, having an impact on the activity of the electron transfer-pathway complexes. Analysis of mitochondrial PBMCs activity in rat samples is currently under investigation and might provide useful information on mitochondrial tissue activity.ormation on mitochondrial tissue activity.)
Volani 2017 Metallomics + (Iron is an essential co-factor for several … Iron is an essential co-factor for several metabolic processes, including mitochondrial respiration, and mitochondria are the major sites of iron-utilization. Cellular iron homeostasis must be tightly regulated, as intracellular iron deficiency can lead to insufficient energy production, whereas iron overload triggers ROS (reactive oxygen species) formation via the Fenton reaction. So far little is known on how iron imbalances affect mitochondrial function ''in vivo'' and the impact of the genotype on that, we studied the effects of dietary iron loading on mitochondrial respiratory capacity in liver by comparing two genetically divergent mouse strains, namely C57BL/6N and FVB mice. Both mouse strains differed in their basal iron levels and their metabolic responses to iron loading as determined by expression of iron trafficking proteins (ferritin was increased in livers of animals receiving high iron diet) as well as tissue iron content (2-fold increase, FVB p = 0.0013; C57BL/6N p = 0.0022). Dietary iron exposure caused a significant impairment of mitochondrial oxidative phosphorylation, especially regarding OXPHOS capacity (FVB p = 0.0006; C57BL/6N p = 0.0087) and S-ET capacity (FVB p = 0.0281; C57BL/6N p = 0.0159). These effects were more pronounced in C57BL/6N than in FVB mice and were paralleled by an iron mediated induction of oxidative stress in mitochondria. The increased susceptibility of C57BL6/N mice to iron loading may be due to reduced expression of anti-oxidant defense mechanisms and altered iron trafficking upon dietary challenge pointing to a role of genetic modifiers for cellular and mitochondrial iron trafficking. Finally, iron-mediated induction of mitochondrial oxidative stress and reduction of oxidative phosphorylation may underlie fatigue in subjects with iron loading diseases.ue in subjects with iron loading diseases.)
Fischer 2022 Metabolites + (Iron is an essential component for metabol … Iron is an essential component for metabolic processes including oxygen transport within hemoglobin, tricarboxylic acid (TCA) cycle activity and mitochondrial energy transformation. Iron deficiency can thus lead to metabolic dysfunction and eventually result in iron deficiency anemia (IDA) which affects approximately 1.5 billion people worldwide. Using a rat model of IDA induced by phlebotomy, we studied the effects of IDA on mitochondrial respiration in peripheral blood mononuclear cells (PBMCs) and liver. Furthermore, we evaluated whether mitochondrial function evaluated by high-resolution respirometry in PBMCs reflects corresponding alterations in the liver. Surprisingly, mitochondrial respiratory capacity was increased in PBMCs from rats with IDA compared to controls. In contrast, mitochondrial respiration remained unaffected in livers from IDA rats. Of note, citrate synthase activity indicated an increased mitochondrial density in PBMCs, whereas it remained unchanged in the liver, partly explaining the different responses of mitochondrial respiration in PBMCs and liver. Taken together, these results indicate that mitochondrial function determined in PBMCs cannot serve as a valid surrogate for respiration in the liver. Metabolic adaptions to iron deficiency resulted in different metabolic reprogramming in the blood cells and liver tissue. the blood cells and liver tissue. )
Oexle 1999 Biochim Biophys Acta + (Iron modulates the expression of the criti … Iron modulates the expression of the critical citric acid cycle enzyme aconitase via a translational mechanism involving iron regulatory proteins. Thus, the present study was undertaken to investigate the consequences of iron perturbation on citric acid cycle activity, oxidative phosphorylation and mitochondrial respiration in the human cell line K-562. In agreement with previous data iron increases the activity of mitochondrial aconitase while it is reduced upon addition of the iron chelator desferrioxamine (DFO). Interestingly, iron also positively affects three other citric acid cycle enzymes, namely citrate synthase, isocitric dehydrogenase, and succinate dehydrogenase, while DFO decreases the activity of these enzymes.Consequently, iron supplementation results in increased formation of reducing equivalents (NADH) by the citric acid cycle, and thus in increased mitochondrial oxygen consumption and ATP formation via oxidative phosphorylation as shown herein. This in turn leads to downregulation of glucose utilization. In contrast, all these metabolic pathways are reducedupon iron depletion, and thus glycolysis and lactate formation are significantly increased in order to compensate for the decrease in ATP production via oxidative phosphorylation in the presence of DFO. Our results point to a complex interaction between iron homeostasis, oxygen supply and cellular energy metabolism in human cells.cellular energy metabolism in human cells.)
Cejvanovic 2018 Free Radic Biol Med + (Iron promotes formation of hydroxyl radica … Iron promotes formation of hydroxyl radicals by the Fenton reaction, subsequently leading to potential oxidatively generated damage of nucleic acids. Oxidatively generated damage to RNA, measured as 8-oxo-7,8-dihydroguanosine (8-oxoGuo) in urine, is increased in patients with genetic iron overload, which have led us to test the hypothesis that high iron status, assessed by iron biomarkers and genetic disposition, increases urinary excretion of 8-oxoGuo. In a general Danish population study we used a Mendelian randomization design with HFE genotypes as a proxy for iron status and supplemented with ex vivo experiments in mice muscle tissue exposed to iron(II) sulfate to attempt to clarify this hypothesis. The biomarkers ferritin, transferrin, and transferrin saturation (TS) were associated with 8-oxoGuo (in linear univariable and multivariable regression analyses: P < 0.001). Mendelian randomization indicated a causal pathway between genetically elevated iron biomarkers (assessed by ferritin and TS) and high levels of 8-oxoGuo. The ''ex vivo'' experiments showed a monotonically increase in 8-oxoGuo with increased iron concentration (ANOVA: P = 0.0008) that was prevented with iron chelation (P = 0.01). Our results indicate a causal relationship between iron biomarkers and 8-oxoGuo. Furthermore, the ''ex vivo'' experiment shows a mechanistic link between iron and 8-oxoGuo formation. Both iron overload and the biomarker 8-oxoGuo have been linked to e.g. diabetes, which merits future studies to investigate if iron induced 8-oxoGuo is involved in disease development. 8-oxoGuo is involved in disease development.)
Melenovsky 2016 Eur J Heart Fail + (Iron replacement improves clinical status … Iron replacement improves clinical status in iron-deficient patients with heart failure (HF), but the pathophysiology is poorly understood. Iron is essential not only for erythropoiesis, but also for cellular bioenergetics. The impact of myocardial iron deficiency (MID) on mitochondrial function, measured directly in the failing human heart, is unknown.Left ventricular samples were obtained from 91 consecutive HF patients undergoing transplantation and 38 HF-free organ donors (controls). Total myocardial iron content, mitochondrial respiration, citric acid cycle and respiratory chain enzyme activities, respiratory chain components (complex I-V), and protein content of reactive oxygen species (ROS)-protective enzymes were measured in tissue homogenates to quantify mitochondrial function. Myocardial iron content was lower in HF compared with controls (156 ± 41 vs. 200 ± 38 µg·g-1 dry weight, P < 0.001), independently of anaemia. MID (the lowest iron tercile in HF) was associated with more extensive coronary disease and less beta-blocker usage compared with non-MID HF patients. Compared with controls, HF patients displayed reduced myocardial oxygen2 respiration and reduced activity of all examined mitochondrial enzymes (all P < 0.001). MID in HF was associated with preserved activity of respiratory chain enzymes but reduced activity of aconitase and citrate synthase (by -26% and -15%, P < 0.05) and reduced expression of catalase, glutathione peroxidase, and superoxide dismutase 2.Myocardial iron content is decreased and mitochondrial functions are impaired in advanced HF. MID in HF is associated with diminished citric acid cycle enzyme activities and decreased ROS-protecting enzymes. MID may contribute to altered myocardial substrate use and to worsening of mitochondrial dysfunction that exists in HF.© 2016 The Authors. European Journal of Heart Failure © 2016 European Society of Cardiology.Journal of Heart Failure © 2016 European Society of Cardiology.)
Read 2021 Redox Biol + (Iron-sulfur (Fe-S) clusters are essential … Iron-sulfur (Fe-S) clusters are essential cofactors most commonly known for their role mediating electron transfer within the mitochondrial respiratory chain. The Fe-S cluster pathways that function within the respiratory complexes are highly conserved between bacteria and the mitochondria of eukaryotic cells. Within the electron transport chain, Fe-S clusters play a critical role in transporting electrons through Complexes I, II and III to cytochrome c, before subsequent transfer to molecular oxygen. Fe-S clusters are also among the binding sites of classical mitochondrial inhibitors, such as rotenone, and play an important role in the production of mitochondrial reactive oxygen species (ROS). Mitochondrial Fe-S clusters also play a critical role in the pathogenesis of disease. High levels of ROS produced at these sites can cause cell injury or death, however, when produced at low levels can serve as signaling molecules. For example, Ndufs2, a Complex I subunit containing an Fe-S center, N2, has recently been identified as a redox-sensitive oxygen sensor, mediating homeostatic oxygen-sensing in the pulmonary vasculature and carotid body. Fe-S clusters are emerging as transcriptionally-regulated mediators in disease and play a crucial role in normal physiology, offering potential new therapeutic targets for diseases including malaria, diabetes, and cancer.s including malaria, diabetes, and cancer.)
Shepherd 2023 J Am Chem Soc + (Iron-sulfur cluster (ISC) assembly occurs … Iron-sulfur cluster (ISC) assembly occurs in both mitochondria and cytosol. Mitochondria are thought to export a low-molecular-mass (LMM) iron and/or sulfur species which is used as a substrate for cytosolic ISC assembly. This species, called X-S or (Fe-S)int, has not been directly detected. Here, an assay was developed in which mitochondria were isolated from 57Fe-enriched cells and incubated in various buffers. Thereafter, mitochondria were separated from the supernatant, and both fractions were investigated by ICP-MS-detected size exclusion liquid chromatography. Aqueous 54FeII in the buffer declined upon exposure to intact 57Fe-enriched mitochondria. Some 54Fe was probably surface-absorbed but some was incorporated into mitochondrial iron-containing proteins when mitochondria were activated for ISC biosynthesis. When activated, mitochondria exported/released two LMM nonproteinaceous iron complexes. One species, which comigrated with an Fe-ATP complex, developed faster than the other Fe species, which also comigrated with phosphorus. Both were enriched in 54Fe and 57Fe, suggesting that the added 54Fe entered a pre-existing pool of 57Fe, which was also the source of the exported species. When 54Fe-loaded 57Fe-enriched mitochondria were mixed with isolated cytosol and activated, multiple cytosolic proteins became enriched with Fe. No incorporation was observed when 54Fe was added directly to the cytosol in the absence of mitochondria. This suggests that a different Fe source in mitochondria, the one enriched mainly with 57Fe, was used to export a species that was ultimately incorporated into cytosolic proteins. Iron from buffer was imported into mitochondria fastest, followed by mitochondrial ISC assembly, LMM iron export, and cytosolic ISC assembly. a species that was ultimately incorporated into cytosolic proteins. Iron from buffer was imported into mitochondria fastest, followed by mitochondrial ISC assembly, LMM iron export, and cytosolic ISC assembly.)
Schettino 2017 Substantia + (Isaac Newton dedicated a good part of his … Isaac Newton dedicated a good part of his activity to alchemical experiments. This article tries to discuss the motivations that drove Newton to spend so much of his time in the laboratory: the search for a unitary vision of the forces acting in the macrocosm and in the microcosm, the belief on a hidden ''prisca sapientia'' in the occult philosophy to be rediscovered with a scientific approach and the dispute with materialistic philosophy.the dispute with materialistic philosophy.)
Ren 2023 Cell Discov + (IscU2 is a scaffold protein that is critic … IscU2 is a scaffold protein that is critical for the assembly of iron-sulfur (Fe-S) clusters and the functions of Fe-S-containing mitochondrial proteins. However, the role of IscU2 in tumor development remains unclear. Here, we demonstrated that IscU2 expression is much higher in human pancreatic ductal adenocarcinoma (PDAC) tissues than in adjacent normal pancreatic tissues. In PDAC cells, activated KRAS enhances the c-Myc-mediated IscU2 transcription. The upregulated IscU2 stabilizes Fe-S cluster and regulates the activity of tricarboxylic acid (TCA) cycle enzymes α-ketoglutarate (α-KG) dehydrogenase and aconitase 2, which promote α-KG catabolism through oxidative and reductive TCA cycling, respectively. In addition to promoting mitochondrial functions, activated KRAS-induced and IscU2-dependent acceleration of α-KG catabolism results in reduced α-KG levels in the cytosol and nucleus, leading to an increase in DNA 5mC due to Tet methylcytosine dioxygenase 3 (TET3) inhibition and subsequent expression of genes including DNA polymerase alpha 1 catalytic subunit for PDAC cell proliferation and tumor growth in mice. These findings underscore a critical role of IscU2 in KRAS-promoted α-KG catabolism, 5mC-dependent gene expression, and PDAC growth and highlight the instrumental and integrated regulation of mitochondrial functions and gene expression by IscU2 in PDAC cells.nd gene expression by IscU2 in PDAC cells.)
Sorby-Adams 2024 Redox Biol + (Ischaemia-reperfusion (IR) injury is the p … Ischaemia-reperfusion (IR) injury is the paradoxical consequence of the rapid restoration of blood flow to an ischaemic organ. Although reperfusion is essential for tissue survival in conditions such as myocardial infarction and stroke, the excessive production of mitochondrial reactive oxygen species (ROS) upon reperfusion initiates the oxidative damage that underlies IR injury, by causing cell death and inflammation. This ROS production is caused by an accumulation of the mitochondrial metabolite succinate during ischaemia, followed by its rapid oxidation upon reperfusion by succinate dehydrogenase (SDH), driving superoxide production at complex I by reverse electron transport. Inhibitors of SDH, such as malonate, show therapeutic potential by decreasing succinate oxidation and superoxide production upon reperfusion. To better understand the mechanism of mitochondrial ROS production upon reperfusion and to assess potential therapies, we set up an ''in vitro'' model of IR injury. For this, isolated mitochondria were incubated anoxically with succinate to mimic ischaemia and then rapidly reoxygenated to replicate reperfusion, driving a burst of ROS formation. Using this system, we assess the factors that contribute to the magnitude of mitochondrial ROS production in heart, brain, and kidney mitochondria, as well as screening for inhibitors of succinate oxidation with therapeutic potential.nate oxidation with therapeutic potential.)
Chouchani 2014 Nature + (Ischaemia-reperfusion injury occurs when t … Ischaemia-reperfusion injury occurs when the blood supply to an organ is disrupted and then restored, and underlies many disorders, notably heart attack and stroke. While reperfusion of ischaemic tissue is essential for survival, it also initiates oxidative damage, cell death and aberrant immune responses through the generation of mitochondrial reactive oxygen species (ROS). Although mitochondrial ROS production in ischaemia reperfusion is established, it has generally been considered a nonspecific response to reperfusion. Here we develop a comparative in vivo metabolomic analysis, and unexpectedly identify widely conserved metabolic pathways responsible for mitochondrial ROS production during ischaemia reperfusion. We show that selective accumulation of the citric acid cycle intermediate succinate is a universal metabolic signature of ischaemia in a range of tissues and is responsible for mitochondrial ROS production during reperfusion. Ischaemic succinate accumulation arises from reversal of succinate dehydrogenase, which in turn is driven by fumarate overflow from purine nucleotide breakdown and partial reversal of the malate/aspartate shuttle. After reperfusion, the accumulated succinate is rapidly re-oxidized by succinate dehydrogenase, driving extensive ROS generation by reverse electron transport at mitochondrial complex I. Decreasing ischaemic succinate accumulation by pharmacological inhibition is sufficient to ameliorate in vivo ischaemia-reperfusion injury in murine models of heart attack and stroke. Thus, we have identified a conserved metabolic response of tissues to ischaemia and reperfusion that unifies many hitherto unconnected aspects of ischaemia-reperfusion injury. Furthermore, these findings reveal a new pathway for metabolic control of ROS production in vivo, while demonstrating that inhibition of ischaemic succinate accumulation and its oxidation after subsequent reperfusion is a potential therapeutic target to decrease ischaemia-reperfusion injury in a range of pathologies.erfusion injury in a range of pathologies.)
Wu 2018 Br J Pharmacol + (Ischaemic stroke is a leading cause of dea … Ischaemic stroke is a leading cause of death and long-term disability. Promising neuroprotective compounds are urgently needed to overcome clinical therapeutic limitations. Neuroprotective agents are limited to single-target agents, which further limit their clinical effectiveness. Due to the brain's particular energy requirements, the energy micro-environment, centred in mitochondria, is a new research hotspot in the complex pathology of ischaemic stroke. Here, we studied the effects of neferine (Nef), a bis-benzylisoquinoline alkaloid extracted from the seed embryo of ''Nelumbo nucifera'' Gaertn, on ischaemic stroke and its underlying mitochondrial protective mechanisms.Rats with permanent middle cerebral artery occlusion (pMCAO)-induced focal cerebral ischaemia and tert-butyl hydroperoxide (t-BHP)-injured PC12 cells were used to investigate the neuroprotective effects of Nef, particularly with regard to energy micro-environment regulation by mitochondria and its mechanism ''in vivo'' and ''in vitro''.Nef protected t-BHP-injured PC12 cells ''in vitro'' and ameliorated neurological score, infarct volume, regional cerebral blood flow, cerebral microstructure and oxidant-related enzyme deficits in pMCAO rats ''in vivo''. Nef also prevented mitochondrial dysfunction both ''in vivo'' and ''in vitro''. The underlying mechanism of the mitochondrial protective effect of Nef might be attributed to the increased translocation of Nrf2 to the nucleus. Furthermore, the translocation of Nrf2 to nucleus was also decreased by sequestosome 1 (p62) knockdown.Our results demonstrated that Nef might have therapeutic potential for ischaemic stroke and may exert its protective role through mitochondrial protection. This protection might be attributed to the modulation of Nrf2 signalling.© 2018 The British Pharmacological Society.018 The British Pharmacological Society.)
Jespersen 2020 Sci Rep + (Ischemia reperfusion (IR) injury may be at … Ischemia reperfusion (IR) injury may be attenuated through succinate dehydrogenase (SDH) inhibition by dimethyl malonate (DiMAL). Whether SDH inhibition yields protection in diabetic individuals and translates into human cardiac tissue remain unknown. In isolated perfused hearts from 24 weeks old male Zucker diabetic fatty (ZDF) and age matched non-diabetic control rats and atrial trabeculae from patients with and without diabetes, we compared infarct size, contractile force recovery and mitochondrial function. The cardioprotective effect of a 10 minutes DiMAL administration prior to global ischemia and ischemic preconditioning (IPC) was evaluated. In non-diabetic hearts exposed to IR, DiMAL 0.1 mM reduced infarct size compared to IR (55 ± 7% vs. 69 ± 6%, p < 0.05). Mitochondrial respiration was reduced by DiMAL 0.6 mM compared to sham and DiMAL 0.1 mM (p < 0.05). In diabetic hearts an increased concentration of DiMAL (0.6 mM) was required for protection compared to IR (64 ± 13% vs. 79 ± 8%, p < 0.05). Mitochondrial function remained unchanged. In trabeculae from humans without diabetes, IPC and DiMAL improved contractile force recovery compared to IR (43 ± 12% and 43 ± 13% vs. 23 ± 13%, p < 0.05) but in patients with diabetes only IPC provided protection compared to IR (51 ± 15% vs. 21 ± 8%, p < 0.05). Neither IPC nor DiMAL modulated mitochondrial respiration in patients. Cardioprotection by SDH inhibition is possible in human tissue, but depends on diabetes status. The narrow therapeutic range and discrepancy in respiration between experimental and human studies may limit clinical translation.imental and human studies may limit clinical translation.)
Wall 2006 Am J Physiol Heart Circ Physiol + (Ischemia-reperfusion (I/R) has critical co … Ischemia-reperfusion (I/R) has critical consequences in the heart. Recent studies on the functions of I/R-activated kinases, such as p38 mitogen-activated protein kinase (MAPK), showed that I/R injury is reduced in the hearts of transgenic mice that overexpress the p38 MAPK activator MAPK kinase 6 (MKK6). This protection may be fostered by changes in the levels of many proteins not currently known to be regulated by p38. To examine this possibility, we employed the multidimensional protein identification technology MudPIT to characterize changes in levels of proteins in MKK6 transgenic mouse hearts, focusing on proteins in mitochondria, which play key roles in mediating I/R injury in the heart. Of the 386 mitochondrial proteins identified, the levels of 58 were decreased, while only 2 were increased in the MKK6 transgenic mouse hearts. Among those that were decreased were 21 mitochondrial oxidative phosphorylation complex proteins, which was unexpected because p38 is not known to mediate such decreases. Immunoblotting verified that proteins in each of the five oxidative phosphorylation complexes were reduced in MKK6 mouse hearts. On assessing functional consequences of these reductions, we found that MKK6 mouse heart mitochondria exhibited 50% lower oxidative respiration and I/R-mediated reactive oxygen species (ROS) generation, both of which are predicted consequences of decreased oxidative phosphorylation complex proteins. Thus the cardioprotection observed in MKK6 transgenic mouse hearts may be partly due to decreased electron transport, which is potentially beneficial, because damaging ROS are known to be generated by mitochondrial complexes I and III during reoxygenation. complexes I and III during reoxygenation.)
Moriyama 2016 J Surg Res + (Ischemia-reperfusion (I/R) injury is one o … Ischemia-reperfusion (I/R) injury is one of the most important pathologic processes causing acute kidney injury. Human atrial natriuretic peptide (hANP) has various effects, including renal protection. The purpose of the present work was to study the effects of intrarenal angiotensin II (Ang II) and investigate the potential of hANP to prevent kidney injury.Male Sprague-Dawley rats were divided into three groups as follows: (1) sham; (2) I/R (30 min of bilateral renal ischemia followed by 6 h reperfusion); and (3) I/R + hANP (I/R injury + continuous intravenous infusion of hANP at 0.025 μg/kg/min). After 6 h of reperfusion, both renal and plasma Ang II concentrations were measured. Urinary angiotensinogen and neutrophil gelatinase-associated lipocalin were measured before ischemia and 2, 4, and 6 h after reperfusion. To evaluate the renal-protective effects of hANP, serum creatinine was determined 6 and 24 h after reperfusion. In addition, mitochondrial oxygen consumption in kidney cortex was measured in the presence of Ang II and hANP.Renal Ang II concentrations were 24.5 ± 3.9 and 14.2 ± 3.4 pg/mg renal weight in the I/R and I/R + hANP groups, respectively. Urinary angiotensinogen and neutrophil gelatinase-associated lipocalin excretions were elevated after I/R injury. Treatment with hANP significantly attenuated this effect after 4 and 6 h. Oxygen consumption in renal mitochondria increased with the addition of Ang II, which was also attenuated by hANP.Production of intrarenal Ang II was attenuated by hANP, indicating a potential to diminish renal I/R injury.Copyright © 2016 Elsevier Inc. All rights reserved. 2016 Elsevier Inc. All rights reserved.)
Lemoine 2015 Transplantation + (Ischemia-reperfusion (IR) injury leads to … Ischemia-reperfusion (IR) injury leads to mitochondrial permeability transition pore opening, which contributes to cell death. The aim of this study is to determine whether ischemic or pharmacological postconditioning with cyclosporine A (CsA) might protect the kidney from lethal reperfusion injury.Male mice underwent a unilateral (right) nephrectomy followed by 30 minutes of contralateral (left) clamping of the renal artery. We studied 4 groups at 20 minutes and 24 hours of reperfusion: a sham group (n = 4), an ischemic group (n = 6), CsA-postconditioned group (postcond-CsA, injection of 3 mg/kg of CsA 5 minutes before the end of ischemia, (n = 6), and an ischemic postconditioning (IPC) group (n = 6), consisting of 3 cycles of 30 seconds of renal ischemia with 30 seconds intervening reperfusion. After 24 hours of reperfusion, we measured plasma creatinine, urea, and histological kidney injury. The kidney mitochondria were isolated to assess the mitochondria calcium retention capacity and oxidative phosphorylation.At 24 hours after reperfusion, serum creatinine decreased in postcond-CsA and IPC compared to ischemic group. The histological score was also significantly improved with postcond-CsA and IPC. At 20 minutes and 24 hours of reperfusion, calcium retention capacity was decreased significantly in the ischemic group. The mitochondrial respiration stay decreased in the ischemic group at 24 hours of reperfusion, whereas the respiration was improved significantly in the postcond-CsA and IPC group. Bax and cleaved caspase 3 decreased in PostCsA and IPC group.Our results suggest that IPC and CsA, administered immediately before reperfusion, protect the kidney from lethal injury.on, protect the kidney from lethal injury.)
Doerrier 2016b Abstract Mito Xmas Meeting Innsbruck + (Ischemia-reperfusion damage during the tra … Ischemia-reperfusion damage during the transplant process occurs mainly in three steps: (i) warm ischemia (WI), (ii) cold ischemia, (iii) reperfusion. WI is defined as the time interval between a tissue remaining at body temperature after blood supply has been reduced or interrupted. Oxidative stress is considered to be one of the main causes of injury during ischemia-reperfusion.In the present work we used high-resolution respirometry (O2k-Fluorometer; Oroboros , Innsbruck, Austria) [1] to investigate simultaneously respiration and hydrogen peroxide production (H2O2) of mitochondria isolated from the hearts of C57BL/6 mice. By using inhibitors of the main mitochondrial H2O2 scavengers (DNCB for glutathione and AF for thioredoxin) we evaluated the total H2O2 production compared to net H2O2 production in the absence of these inhibitors [2]. [[OXPHOS]] and [[Electron transfer pathway]] respiratory capacities of isolated mitochondria (normalized per mg mt-protein) were decreased after 1-h WI of the excised heart. A significant injury of the outer mt-membrane is consistent with ischemia-induced mt-permeability transition [3], which can explain a general respiratory defect. In addition, application of a newly developed substrate-uncoupler-inhibitor titration (SUIT) protocol [4] revealed a specific defect of fatty acid β-oxidation (FAO) [5] H2O2 flux based on the Amplex red assay more than doubled after application of AF and DNCB inhibitors to the controls. The glutathione and thioredoxin antioxidant system did not protect mitochondria after WI from this increased H2O2 production. Taken together, standardized respiratory SUIT protocols combined with SOPs in the fluorometric assay of H2O2 production offer a sensitive diagnostic tool for comprehensive OXPHOS analysis.ized respiratory SUIT protocols combined with SOPs in the fluorometric assay of H2O2 production offer a sensitive diagnostic tool for comprehensive OXPHOS analysis.)
Timon-Gomez 2023 EUROMIT2023 Bologna + (Ischemia-reperfusion damage occurs when th … Ischemia-reperfusion damage occurs when there is a disruption and restoration of blood supply to an organ and it underlies many pathologies, mostly heart attack and stroke. Warm ischemia is defined as the time interval after the interruption of blood flow while the tissue remains at body temperature. The evaluation of mitochondrial damage caused by ischemia-reperfusion is essential to assess treatments for protecting or rescuing the tissue from an injury. We studied brain homogenate and isolated cardiac mitochondria from C57BL/6N mice, and HEK 293T cells by high-resolution respirometry. Warm ischemia (WI) was induced by incubating brains and hearts in preservation buffer BIOPS for 1 h at 37 °C, followed by homogenization or mitochondria isolation. HEK 293T cells were exposed to anoxia in the O2k chamber for 1 h. For OXPHOS analysis, we used two substrate-uncoupler-inhibitor titration (SUIT) reference protocols, RP1 and RP2, interrogating 20 respiratory states. These include fatty acid oxidation F-, NADH N-, succinate S-, glycerol phosphate Gp- electron transfer (ET) pathways. Fluxes were normalized by tissue or protein mass, or by cell count. Respirometric OXPHOS analysis does not focus on single enzymes but provides a broad diagnostic spectrum on multiple pathways and coupling control. In all models, OXPHOS and ET capacities were decreased in all pathways (F, N, S) and their combinations (NS, FNS, FNSGp, SGp) after 1 h warm ischemia, compared to controls. In cardiac mitochondria, flux control ratios showed a specific defect in OXPHOS and ET capacities of fatty acid oxidation, and a significant cytochrome c effect revealed an injury of the outer mitochondrial membrane. In contrast, in brain tissue and HEK 293T cells, there was no cytochrome c effect and flux control ratios indicated a major damage in N-linked respiration after warm ischemia. Our results show that warm ischemia produces tissue-specific mitochondrial damages. In carbohydrate-dependent tissues, such as brain, the damage is highest in the N-pathway, similarly as in HEK 293T cells. Whereas the F-pathway is mainly affected in tissues that are more dependent on fatty acid oxidation, such as heart. SUIT protocols provide a broad scope to detect specific OXPHOS defects, offering a more sensitive diagnostic approach compared to respiratory protocols with non-permeabilized living cells.ocols with non-permeabilized living cells.)
Suchadolskiene 2014 BMC Neurosci + (Ischemic brain injury due to stroke and/or … Ischemic brain injury due to stroke and/or cardiac arrest is a major health issue in modern society requiring urgent development of new effective therapies. The aim of this study was to evaluate mitochondrial, microcirculatory, and histological changes in a swine model of global cerebral ischemia.In our model, significant microcirculatory changes, but only negligible histological cell alterations, were observed 3 h after bilateral carotid occlusion, and were more pronounced if the vascular occlusion was combined with systemic hypotension. Analysis of mitochondrial function showed that LEAK respiration (measured in the presence of pyruvate + malate but without ADP) was not affected in any model of global cerebral ischemia in pigs. The OXPHOS capacity with pyruvate + malate as substrates decreased compared with the control levels after bilateral carotid artery occlusion, and bilateral carotid artery occlusion + hypotension by 20% and 79%, respectively, resulting in decreases in the respiratory control index of 14% and 73%, respectively. OXPHOS capacity with succinate as a substrate remained constant after unilateral carotid artery occlusion or bilateral carotid artery occlusion, but decreased by 53% after bilateral carotid artery occlusion and hypotension compared with controls (''p'' < 0.05, ''n'' = 3-6). Addition of exogenous cytochrome c to mitochondria isolated from ischemia brains had no effect on respiration in all models used in this study.We found a decrease in microcirculation and mitochondrial oxidative phosphorylation activity, but insignificant neuronal death, after 3 h ischemia in all our pig models of global cerebral ischemia. Dysfunction of the mitochondrial oxidative phosphorylation system, particularly damage to complex I of the respiratory chain, may be the primary target of the ischemic insult, and occurs before signs of neuronal death can be detected.fore signs of neuronal death can be detected.)
Kahl 2018 Stroke + (Ischemic brain injury is characterized by … Ischemic brain injury is characterized by 2 temporally distinct but interrelated phases: ischemia (primary energy failure) and reperfusion (secondary energy failure). Loss of cerebral blood flow leads to decreased oxygen levels and energy crisis in the ischemic area, initiating a sequence of pathophysiological events that after reoxygenation lead to ischemia/reperfusion (I/R) brain damage. Mitochondrial impairment and oxidative stress are known to be early events in I/R injury. However, the biochemical mechanisms of mitochondria damage in I/R are not completely understood.We used a mouse model of transient focal cerebral ischemia to investigate acute I/R-induced changes of mitochondrial function, focusing on mechanisms of primary and secondary energy failure.Ischemia induced a reversible loss of flavin mononucleotide from mitochondrial complex I leading to a transient decrease in its enzymatic activity, which is rapidly reversed on reoxygenation. Reestablishing blood flow led to a reversible oxidative modification of mitochondrial complex I thiol residues and inhibition of the enzyme. Administration of glutathione-ethyl ester at the onset of reperfusion prevented the decline of complex I activity and was associated with smaller infarct size and improved neurological outcome, suggesting that decreased oxidation of complex I thiols during I/R-induced oxidative stress may contribute to the neuroprotective effect of glutathione ester.Our results unveil a key role of mitochondrial complex I in the development of I/R brain injury and provide the mechanistic basis for the well-established mitochondrial dysfunction caused by I/R. Targeting the functional integrity of complex I in the early phase of reperfusion may provide a novel therapeutic strategy to prevent tissue injury after stroke.© 2018 The Authors.troke. © 2018 The Authors.)
Kancirova 2016 Nove Strategicke Pristupy v experimentalnej Kardioprotekcii + (Ischemic heart disease is one of the most … Ischemic heart disease is one of the most frequent causes of severe myocardial injury occuring in the developed countries. Therefore, the emphasis is focused on the new possibilities of the cardioprotection usage in order to avoid the lethal heart injury and to improve its functional parameters. Different types of preconditioning preparing heart on the increased load induce protective functional and structural changes participating in processes of endogenous protection. The aim of our study was to elucidate the role of heart mitochondria as the final effector of noninvasive form of remote ischemicpreconditioning applied at the distance location from the heart. Remote ischemic preconditioning consisted of three cycles of 5-minutes ischemiaand 5-minutes reperfusion (200 mmHg) of the right hind limb using a pressure cuff. Mitochondria were isolated by differential centrifugation usingthe protease (P 6141) from the excited hearts subjected to ischemia-reperfusion Langendorff test (15 minutes of stabilization, 30 minutes of global ischemia, 40 minutes of reperfusion). Following parameters were determined in theisolated heart mitochondria: the activity of mitochondrial respiration using respirometer Oxygraph-2k (Oroboros Instruments, Austria), the mitochondrial ATP synthase activity determined as the amount of inorganic phosphate released byATP splitting per unit of time, the mitochondrial membrane fluidity by spectrofluorometry using the fluorescent probe 1,6-diphenyl-1,3,5-hexatrienand the content of oxidised isoforms coenzyme Q(CoQ9ox) by HPLC method. Isolated heart exposed to ischemic-reperfusion injury resulted in a reductionof the mitochondrial ATP synthase activity as well as decrease in the ADP-stimulated respiration without affecting the basal mitochondrial respiration. Increase the content of coenzyme CoQ9ox oxidised isoforms reflecting respiratory chain damage during heart global ischemia and reperfusion accompanied by the mitochondrial membrane fluidity decrease.Remote ischemic preconditioning partly prevented the decrease of mitochondrial ATP synthase activity as well as decrease of ADP-stimulated respirationdue to ischemia-reperfusion injury whereas the content of coenzyme CoQ9ox oxidized isoforms was increased after 15 minutes stabilization of ischemiareperfusion test. Mitochondrial membrane fluidity was increased in all phases of ischemia-reperfusion test. Despite of increased generation of free oxygen radicals during the stabilization phase, rigidization of the mitochondrial membrane was not observed. This findings suggest a signallig role of free oxygen radicals. Our results confirm damaging consequence of ischemia-reperfusion test on the functional and structural properties of the heart mitochondria. Remote ischemic preconditioning trought the generation of free oxygen radicals by the mitochondrial respiratory chain probably indicates the mechanisms of heart endogenous protection that lead to the alleviation of the lethal damaged induced by ischemic-reperfusion injury.ed induced by ischemic-reperfusion injury.)
Pchelin 2018 IOC130 + (Ischemic lesions remain to be one of the m … Ischemic lesions remain to be one of the main causes of physical disability and mortality worldwide. Furthermore, stroke is known to be followed by mitochondrial dysfunction and impaired cell respiration. Mounting evidence demonstrates that the cytokine hormone erythropoietin (EPO) is capable of activating signaling pathways that increase the brain’s resistance to ischemia/reperfusion stress. After the discovery of EPO’s heteroreceptor that promotes tissue protection [1], a number of attempts were made to develop non-hematopoietic EPO’s derivatives, including CdEPO. However, the precise mechanisms implicated into protective CdEPO effect, notably on brain mitochondria, are still to be elucidated.The purpose of current research is to elucidate the effect of non-hematopoietic derivative of erythropoietin (CdEPO) on brain mitochondria respiration rate on 4, 10 and 20 day after local acute ischemia/reperfusion in mice.Male C57BL/6 mice (2 months old, weighing 18-23 g) were used in the study. Local acute ischemia in mice was induced with transient middle cerebral artery occlusion (tMCAO). Following 6 hours after ischemic exposure a fivefold intravenous СdEPO administration was carried out. In a control group the administration of sodium chloride was performed in the same conditions. On 4, 10 and 20 day after reperfusion forebrains of animals were dissected to obtain isolated mitochondria. Bioenergetic studies were carried out using high-resolution respirometry (Oroboros Oxygraph-2k). Significant difference (at least p<0.05) was tested by one-way ANOVA and Holm-Sidak post hoc.It was revealed that ischemia/reperfusion with tMCAO did not lead to significant alterations in LEAK (glutamate and malate) respiration (Fig. 1 A). Intravenous CdEPO administration following 6 hours after ischemia/reperfusion did not exert any effect on LEAK respiration rate compared to control level. However, on day 20 after reperfusion brain mitochondrial OXPHOS respiration showed a significant decrease by 41% (p=0.01) in the control group compared to intact level (Fig. 1 B). Along with that on day 20 OXPHOS respiration rate was increased by 35% (p=0.027) in the CdEPO group in comparison with control level.The observed effect of CdEPO on forebrain mitochondrial bioenergetics might be implicated in the realization of protective mechanisms, which was induced by EPO’s heteroreceptor activation, and resulted in postponed improvement of mitochondrial respiration after ischemia/reperfusion. Other effects of CdEPO on different parameters of mitochondrial bioenergetics require further investigation. bioenergetics require further investigation.)
Kolb 2018 J Am Soc Nephrol + (Ischemic preconditioning confers organ-wid … Ischemic preconditioning confers organ-wide protection against subsequent ischemic stress. A substantial body of evidence underscores the importance of mitochondria adaptation as a critical component of cell protection from ischemia. To identify changes in mitochondria protein expression in response to ischemic preconditioning, we isolated mitochondria from ischemic preconditioned kidneys and sham-treated kidneys as a basis for comparison. The proteomic screen identified highly upregulated proteins, including NADP+-dependent isocitrate dehydrogenase 2 (IDH2), and we confirmed the ability of this protein to confer cellular protection from injury in murine S3 proximal tubule cells subjected to hypoxia. To further evaluate the role of IDH2 in cell protection, we performed detailed analysis of the effects of ''Idh2'' gene delivery on kidney susceptibility to ischemia-reperfusion injury. Gene delivery of IDH2 before injury attenuated the injury-induced rise in serum creatinine (P<0.05) observed in controls and increased the mitochondria membrane potential (P<0.05), maximal respiratory capacity (P<0.05), and intracellular ATP levels (P<0.05) above those in controls. This communication shows that gene delivery of ''Idh2'' can confer organ-wide protection against subsequent ischemia-reperfusion injury and mimics ischemic preconditioning.eperfusion injury and mimics ischemic preconditioning.)
Wang 2023 Clin Exp Pharmacol Physiol + (Ischemic reperfusion injury, caused by oxi … Ischemic reperfusion injury, caused by oxidative stress during reperfusion, is an inevitable outcome of organ transplantation, especially when the organ preservation time is prolonged. Prolonged ischaemic preservation is a valuable technique for improving the success of organ transplantation, but numerous challenges remain. 3-nitro-N-methyl salicylamide (3-NNMS), an inhibitor of mitochondrial electron transport chain complex III, can be used to reduce reactive oxygen species production during blood reperfusion by slowing the electron flow rate of the respiratory chain. Based on this property, a novel preservation solution was developed for the preservation of isolated rat heart and its cardioprotective effect was investigated during an 8-h cold ischaemia preservation time for the first time. For comparison, 3-NNMS was also included in the histidine-tryptophan-ketoglutarate (HTK) solution. Compared to HTK, HTK supplemented with 3-NNMS significantly improved the heart rate of isolated rat hearts after 8 h of cold storage. Both 3-NNMS solution and HTK supplemented with 3-NNMS solution decreased cardiac troponin T and lactate dehydrogenase levels in perfusion fluid and reduced reactive oxygen species and malondialdehyde levels in the myocardium. The 3-NNMS also maintained the membrane potential of myocardial mitochondria and significantly increased superoxide dismutase levels. These results showed that the new 3-NNMS solution can protect mitochondrial and cardiomyocyte function by increasing antioxidant capacity and reducing oxidative stress in cryopreserved rat hearts during a prolonged preservation time, resulting in less myocardial injury and better heart rate.s myocardial injury and better heart rate.)
Arandarcikaite 2019 ESCI2019 + (Ischemic stroke causes disability or morta … Ischemic stroke causes disability or mortality with limited therapeutic options. Ischemia leads to inhibition of mitochondrial respiration rate and opening of mitochondrial permeability transition pore (MPTP) and cell death processes initiation. Previously we reported that ischemia inhibits mitochondrial respiration, but ischemia- induced MPTP does not cause the release of cytochrome c. The inhibition of MPTP formation just partly protected against necrosis [1]. The brain are constituted from two types of cells neurons and glial. And different parts of brain are different composition of neurons and glial cells, for example in cerebellum there are common neurons, while in cortex there are just 20 % of neurons. The risk for stroke increases with aging. Mostly for the brain ischemic experiments are performed by using young adult animals. What for, we performed a comparative analysis of respiratory functions of mitochondria isolated from cortex and cerebellum of rats at various ages: neonatal (7 days), young adults (2-3 months), mature adults (7-10 months), and aged (24 months). We found that ischemia caused persistent inhibition of respiration of mitochondria isolated from cortex at all ages of animals. Ischemia induced inhibition of cerebellar mitochondrial respiration at the age of 7 days, 2-3 and 24 months, except group 7-10 months there were no statistically significant effect of ischemia.Summarizing the data suggest that ischemia induced injuries to mitochondria respiration are related with age of the animal and region of the brain.age of the animal and region of the brain.)
Makhnyeva 2015 Abstract MiPschool Greenville 2015 + (Ischemic stroke is one of the leading caus … Ischemic stroke is one of the leading causes of human death worldwide. It occurs due to the high susceptibility of neurons to anoxia and reoxygenation. Unlike mammals, the fruit fly Drosophila melanogaster withstands low oxygen levels without pathology [1]. It was previously shown that the fruit flies carrying the Rover allele of the foraging gene (forR), express a cGMP-dependent protein kinase (PKG) with high enzymatic activity, whereas fruit flies with the Sitter allele of for (fors) express a low-enzymatic activity PKG; it was also demonstrated that upon return to normal oxygen levels after anoxia, the forR flies survive better than fors flies [2]. From mammalian research, it is also known that the activated NO/PKG signaling cascade induces mitochondrial biogenesis but the exact mechanism is not known [3].The present study attempts to elucidate whether different levels of enzymatic activity of PKG have an effect on mitochondrial content in ''Drosophila'' brains and whole bodies before and after exposure to anoxic stress. Here, the mitochondrial content was assessed by measuring citrate synthase enzymatic activity and by determining protein expression levels of cytochrome c oxidase subunit IV (COX IV) and cytochrome c (Cyt c) by Western blotting. From preliminary results, forR fruit flies had higher citrate synthase enzymatic activity rates in brains when subjected to normal oxygen levels; in addition, these fruit flies showed higher basal levels of COX IV and cytochrome c in whole body homogenates compared to the fors flies. Interestingly, mitochondrial protein levels increased upon reoxygenation in both types of ''Drosophila''.Results of this study may lead to a better understanding of fruit fly’s innate anoxia-tolerance strategies and the protective role of increased PKG signaling. Subsequently, this knowledge may be used to identify potential therapeutic targets to prevent detrimental neurological effects of an ischemic stroke in humans.l effects of an ischemic stroke in humans.)
Arandarcikaite 2022 Abstract Bioblast + (Ischemic stroke is one of the leading caus … Ischemic stroke is one of the leading causes of disability and mortality worldwide, but therapeutic approach are limited. Ischemia causes inhibition of mitochondrial respiration, mitochondrial permeability transition pore (MPTP) opening and subsequent cell death processes. The risk for ischemic stroke is increasing with aging, but there is very little information about aging-related changes in mitochondrial functions and proteomics.In this study, we investigated ischemic lesions to 7 days, 2-3, 7-10 and 24-26 months-old rats brain mitochondria respiration and MPTP sensitivity to Ca2+ with particular focus on mitochondrial Complex I. Results have shown that hypoxia inhibited cortical mitochondrial respiration rate of animals from all age groups and reduced mitochondrial calcium retention capacity (CRC) in 2-3, 7-10 and 24-26 months animals groups. Hypoxia induced inhibition of cerebellar mitochondrial respiration in 7 days, 2-3 and 24 month-old groups, but in the 7-10 month-old group there were no statistically significant effect compared to control. CRC after hypoxia were reduced in 10 and 24-26 months - old rats cerebellum.Further injury investigation revealed that hypoxia inhibits the activity of Complex I in 2-3, 7-10 and 24-26 month-animals. Mitochondrial protein expression study showed an age-related decrease of Complex I protein NDUFS2 levels and subsequent increase in mitochondrial respiration in aged animals. Altogether, we demonstrated that hypoxia induces MPTP opening and inhibits mitochondrial Complex I activity in adult and aged animals groups.l Complex I activity in adult and aged animals groups.)
Stepanova 2017 J Cereb Blood Flow Metab + (Ischemic stroke is one of the most prevale … Ischemic stroke is one of the most prevalent sources of disability in the world. The major brain tissue damage takes place upon the reperfusion of ischemic tissue. Energy failure due to alterations in mitochondrial metabolism and elevated production of reactive oxygen species (ROS) is one of the main causes of brain ischemia-reperfusion (IR) damage. Ischemia resulted in the accumulation of succinate in tissues, which favors the process of reverse electron transfer (RET) when a fraction of electrons derived from succinate is directed to mitochondrial complex I for the reduction of matrix NAD+. We demonstrate that in intact brain mitochondria oxidizing succinate, complex I became damaged and was not able to contribute to the physiological respiration. This process is associated with a decline in ROS release and a dissociation of the enzyme's flavin. This previously undescribed phenomenon represents the major molecular mechanism of injury in stroke and induction of oxidative stress after reperfusion. We also demonstrate that the origin of ROS during RET is flavin of mitochondrial complex I. Our study highlights a novel target for neuroprotection against IR brain injury and provides a sensitive biochemical marker for this process.vides a sensitive biochemical marker for this process.)
Hals 2013 J Diabetes Res + (Islet transplantation in diabetes is hampe … Islet transplantation in diabetes is hampered by the need of life-long immunosuppression. Encapsulation provides partial immunoprotection but could possibly limit oxygen supply, a factor that may enhance hypoxia-induced beta cell death in the early post-transplantation period. Here we tested susceptibility of alginate microencapsulated human islets to experimental hypoxia (0.1-0.3 % O2 for 8 h, followed by re-oxygenation) on viability and functional parameters. Hypoxia reduced viability as measured by MTT by 33.8 ± 3.5 % in encapsulated and 42.9 ± 5.2 % in non-encapsulated islets (''p'' < 0.2). Non-encapsulated islets released 37.7% (median) more HMGB1 compared to encapsulated islets after hypoxic culture conditions (''p'' < 0.001). Glucose-induced insulin release was marginally affected by hypoxia. Basal oxygen consumption was equally reduced in encapsulated and non-encapsulated islets, by 22.0 ± 6.1 % vs. 24.8 ± 5.7 %. Among 27 tested cytokines/chemokines, hypoxia increased the secretion of IL-6 and IL-8/CXCL8 in both groups of islets whereas an increase of MCP-1/CCL2 was seen only with non-encapsulated islets. Conclusion: alginate microencapsulation of human islets does not increase susceptibility to acute hypoxia. This is a positive finding in relation to potential use of encapsulation for islet transplantation. use of encapsulation for islet transplantation.)
Lim 2015 Am J Cancer Res + (Isoflavonoids have been shown to inhibit t … Isoflavonoids have been shown to inhibit tumor proliferation and metastasis by activating cell death pathways.As such, they have been widely studied as potential therapies for cancer prevention. The second generationsynthetic isoflavan analogues ME-143 and ME-344 also exhibit anti-cancer effects, however their specific moleculartargets have not been completely defined. To identify these targets, we examined the effects of ME-143 and ME-344 on cellular metabolism and found that they are potent inhibitors of mitochondrial oxidative phosphorylation(OXPHOS) complex I (NADH: ubiquinone oxidoreductase) activity. In isolated HEK293T mitochondria, ME-143 andME-344 reduced complex I activity to 14.3% and 28.6% of control values respectively. In addition to the inhibitionof complex I, ME-344 also significantly inhibited mitochondrial complex III (ubiquinol: ferricytochrome-c oxidoreductase)activity by 10.8%. This inhibition of complex I activity (and to a lesser extent complex III activity) wasassociated with a reduction in mitochondrial oxygen consumption. In permeabilized HEK293T cells, ME-143 andME-344 significantly reduced the maximum ADP-stimulated respiration rate to 62.3% and 70.0% of control levelsrespectively in the presence of complex I-linked substrates. Conversely, complex II-linked respiration was unaffectedby either drug. We also observed that the inhibition of complex I-linked respiration caused the dissipation of themitochondrial membrane potential (ΔΨm). Blue native (BN-PAGE) analysis revealed that prolonged loss of ΔΨm resultsin the destabilization of the native OXPHOS complexes. In particular, treatment of 143B osteosarcoma, HeLaand HEK293T human embryonic kidney cells with ME-344 for 4 h resulted in reduced steady-state levels of maturecomplex I. Degradation of the complex I subunit NDUFA9, as well as the complex IV (ferrocytochrome c: oxygen oxidoreductase)subunit COXIV, was also evident. The identification of OXPHOS complex I as a target of ME-143 andME-344 advances our understanding of how these drugs induce cell death by disrupting mitochondrial metabolism,and will direct future work to maximize the anti-cancer capacity of these and other isoflavone-based compounds.hese and other isoflavone-based compounds.)
Alston 2018 Am J Hum Genet + (Isolated complex I deficiency is a common … Isolated complex I deficiency is a common biochemical phenotype observed in pediatric mitochondrial disease and often arises as a consequence of pathogenic variants affecting one of the ∼65 genes encoding the complex I structural subunits or assembly factors. Such genetic heterogeneity means that application of next-generation sequencing technologies to undiagnosed cohorts has been a catalyst for genetic diagnosis and gene-disease associations. We describe the clinical and molecular genetic investigations of four unrelated children who presented with neuroradiological findings and/or elevated lactate levels, highly suggestive of an underlying mitochondrial diagnosis. Next-generation sequencing identified bi-allelic variants in NDUFA6, encoding a 15 kDa LYR-motif-containing complex I subunit that forms part of the Q-module. Functional investigations using subjects' fibroblast cell lines demonstrated complex I assembly defects, which were characterized in detail by mass-spectrometry-based complexome profiling. This confirmed a marked reduction in incorporated NDUFA6 and a concomitant reduction in other Q-module subunits, including NDUFAB1, NDUFA7, and NDUFA12. Lentiviral transduction of subjects' fibroblasts showed normalization of complex I. These data also support supercomplex formation, whereby the ∼830 kDa complex I intermediate (consisting of the P- and Q-modules) is in complex with assembled complex III and IV holoenzymes despite lacking the N-module. Interestingly, RNA-sequencing data provided evidence that the consensus RefSeq accession number does not correspond to the predominant transcript in clinically relevant tissues, prompting revision of the NDUFA6 RefSeq transcript and highlighting not only the importance of thorough variant interpretation but also the assessment of appropriate transcripts for analysis.t of appropriate transcripts for analysis.)
Jung 1989 Anal Biochem + (Isolated heart mitochondria hydrolyze the … Isolated heart mitochondria hydrolyze the acetoxymethyl esters of the Ca2+-sensitive fluorescent probe fura-2 and the pH-sensitive 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). The resulting charged forms of the probes are retained in the mitochondrial matrix and appear well-suited for the estimation of pCa and pH in this compartment. The mitochondria esterase activity is stimulated by Ca2+, inhibited by butacaine and quinine, and shows an alkaline pH optimum. The esterase has a similar affinity for the two probes (Km about 1.5 microM) and a somewhat higher Vmax for BCECF. Intramitochondrial pH can be determined by recording the ratio of the fluorescence of matrix BCECF at its excitation maximum of 509 nm to that at 450 nm, an excitation wavelength that is unresponsive to pH. A calibration plot relating the fluorescence ratio to pH is constructed using detergent-lysed mitochondria and the excitation maximum of 500 nm for BCECF in aqueous solution. Estimates of matrix pH by BCECF fluorescence in its useful range (pH 6 to 8) agree well with values obtained using the distribution of 5,5-dimethyl-2,4-oxazolidenedione. In protocols in which the fluorescence with excitation at 450 nm does not vary, a direct recording of BCECF fluorescence with excitation at 509 nm can be used to follow the kinetics of matrix pH changes. follow the kinetics of matrix pH changes.)
Iglesias-Gonzalez 2013 J Neurosci Methods + (Isolated mitochondria are widely used in m … Isolated mitochondria are widely used in metabolic and oxidative stress studies for neurodegenerative diseases. In the present work, the influence of EGTA and EDTA has been tested on a sucrose-based differential centrifugation protocol in order to establish the optimal concentrations to be used in this process. Our results showed alterations in both active and resting respiration, which were dependent on both the addition of EDTA or EGTA to the isolation buffer and the chelator concentration used. However, the addition of chelator to the isolation medium does not modify the mitochondria structure as assessed by both distribution of biological markers and electron micrography in the final pellet. Our results endorse this protocol as the method of choice for metabolic and oxidative stress experiments with fresh isolated rat brain mitochondria.ith fresh isolated rat brain mitochondria.)
Fisar 2017 Indian J Biochem Biophys + (Isolated mitochondria serve as a biologica … Isolated mitochondria serve as a biological model for analysis of oxidative phosphorylation and understanding of mitochondrial respiration control, effects of various biologically active substances, and pathophysiology of mitochondrial diseases. The aim of our study was to develop protocols of high-resolution respirometry optimized for pig brain mitochondria. Optimal and/or minimal concentrations of components controlling oxidative phosphorylation were determined by titration of isolated mitochondria with digitonin, adenosine diphosphate, respiratory substrates (malate, pyruvate, succinate, TMPD), inhibitors (antimycin, azide, cyanide, malonate, oligomycin, rotenone), and uncoupler. Multiple substrate-uncoupler-inhibitor titration (SUIT) protocols were proposed for Complex I-, Complex II-, Complex I AND II-, and Complex IV linked respiration. Crude mitochondrial fraction and mitochondria purified on sucrose gradient were compared. We confirmed that pig brain mitochondria can serve as a biological model for research of mitochondrial respiration using SUIT protocols analogous to those, which were previously published for rodent mitochondria and permeabilized cells. We suppose that difference between a substance effect on respiration of crude and purified mitochondria is due to higher content of mitochondrial enzymes and lower content of the non-mitochondrial contaminants in purified mitochondria. High-resolution respirometry of pig brain mitochondria can be recommended as a suitable assay in research of neuroprotective effects and/or mitochondrial toxicity of novel medical drugs.chondrial toxicity of novel medical drugs.)
Pless-Petig 2018 Cell Transplant + (Isolated primary hepatocytes, which are wi … Isolated primary hepatocytes, which are widely used for pharmacological and clinical purposes, usually undergo certain periods of cold storage in suspension during processing. While adherent hepatocytes were shown previously to suffer iron-dependent cell death during cold (4 °C) storage and early rewarming, we previously found little iron-dependent hepatocyte death in suspension but severely decreased attachment ability unless iron chelators were added. Here, we focus on the role of mitochondrial impairment in this nonattachment of hepatocyte suspensions. Rat hepatocyte suspensions were stored in a chloride-poor, glycine-containing cold storage solution with and without iron chelators at 4 °C. After 1 wk of cold storage in the basic cold storage solution, cell viability in suspension was unchanged, while cell attachment was decreased by >80%. In the stored cells, a loss of mitochondrial membrane potential (MMP), a decrease in adenosine triphosphate (ATP) content (2 ± 2 nmol/106 cells after cold storage, 5 ± 3 nmol/106 cells after rewarming vs. control 29 ± 6 nmol/106 cells), and a decrease in oxygen consumption (101 ± 59 pmol sec-1 per 106 cells after rewarming vs. control 232 ± 83 pmol sec-1 per 106 cells) were observed. Addition of iron chelators to the cold storage solution increased cell attachment to 53% ± 20% and protected against loss of MMP, and cells were able to partially regenerate ATP during rewarming (15 ± 10 nmol/106 cells). Increased attachment could also be achieved by addition of the inhibitor combination of mitochondrial permeability transition, trifluoperazine + fructose. Attached hepatocytes displayed normal MMP and mitochondrial morphology. Additional experiments with freshly isolated hepatocytes confirmed that impaired energy production-as elicited by an inhibitor of the respiratory chain, antimycin A-can decrease cell attachment without decreasing viability. Taken together, these results suggest that mitochondrial impairment with subsequent energy deficiency is a key factor for the lack of attachment of cold-stored hepatocyte suspensions. mitochondrial impairment with subsequent energy deficiency is a key factor for the lack of attachment of cold-stored hepatocyte suspensions.)
Zhang 2006 J Physiol + (Isolated whole skeletal muscles fatigue mo … Isolated whole skeletal muscles fatigue more rapidly than isolated single muscle fibres. We have now employed this difference to study mechanisms of skeletal muscle fatigue. Isolated whole soleus and extensor digitorum longus (EDL) muscles were fatigued by repeated tetanic stimulation while measuring force production. Neither application of 10 mm lactic acid nor increasing the [K+] of the bath solution from 5 to 10 mm had any significant effect on the rate of force decline during fatigue induced by repeated brief tetani. Soleus muscles fatigued slightly faster during continuous tetanic stimulation in 10 mM K+. Inhibition of mitochondrial respiration with cyanide resulted in a faster fatigue development in both soleus and EDL muscles. Single soleus muscle fibres were fatigued by repeated tetani while measuring force and myoplasmic free [Ca2+] ([Ca2+]i). Under control conditions, the single fibres were substantially more fatigue resistant than the whole soleus muscles; tetanic force at the end of a series of 100 tetani was reduced by about 10 % and 50 %, respectively. However, in the presence of cyanide, fatigue developed at a similar rate in whole muscles and single fibres, and tetanic force at the end of fatiguing stimulation was reduced by approximately 80 %. The force decrease in the presence of cyanide was associated with a approximately 50 % decrease in tetanic [Ca2+]i, compared with an increase of approximately 20 % without cyanide. In conclusion, lactic acid or [K+] has little impact on fatigue induced by repeated tetani, whereas hypoxia speeds up fatigue development and this is mainly due to an impaired Ca2+ release from the sarcoplasmic reticulum.ereas hypoxia speeds up fatigue development and this is mainly due to an impaired Ca2+ release from the sarcoplasmic reticulum.)
Gross 2011 Anal Biochem + (Isolation of functional and intact mitocho … Isolation of functional and intact mitochondria from solid tissue is crucial for studies that focus on the elucidation of normal mitochondrial physiology and/or mitochondrial dysfunction in conditions such as aging, diabetes and cancer. There is growing recognition of the importance of mitochondria as both targets for drug development and as off-target mediators of drug side effects. Unfortunately, mitochondrial isolation from tissue is generally carried out using homogenizer-based methods that require extensive operator experience to obtain reproducible, high-quality preparations. These methods limit dissemination, impede scale-up, and contribute to difficulties in reproducing experimental results over time and across laboratories. Here we describe semi-automated methods to disrupt tissue, using kidney and muscle mitochondria preparations as exemplars. These methods utilize either a Barocycler, or The PCT Shredder, or both. The PCT-Shredder is a mechanical grinder that quickly breaks up tissue without significant risk of over-homogenization. Mitochondria isolated using The PCT-Shredder are shown to be comparable to controls. The Barocycler generates controlled pressure pulses that can be adjusted to lyse cells and release organelles. The mitochondria subjected to pressure cycling-mediated tissue disruption are shown to retain functionality, enabling combinations of The PCT-Shredder and Barocycler to be used to purify mitochondrial preparations.used to purify mitochondrial preparations.)
Kabiri 2021b Methods Mol Biol + (Isolation of mitochondria is a crucial met … Isolation of mitochondria is a crucial method for examining molecular details of this organelle's manifold functions. Historically, mitochondrial isolations required large amounts of sample material which impeded their isolation from cultured cells. We have therefore developed a method allowing for controlled and reproducible isolation of intact and functional mitochondria from diverse cell types in culture. Here we provide a methodological update of this approach together with a protocol for the subsequent analysis of such isolated mitochondria by electron microscopy. Combining the isolation procedure with this powerful imaging method can reveal ultrastructural mitochondrial peculiarities in disease settings that might not be evident in intact cells and allows for assessment of mitochondrial membrane integrity and sample purity.rial membrane integrity and sample purity.)
Teh 2015 Oncogene + (Isoprenylcysteine carboxylmethyltransferas … Isoprenylcysteine carboxylmethyltransferase (Icmt) catalyzes the last of the three-step posttranslational protein prenylation process for the so-called CaaX proteins, which includes many signaling proteins, such as most small GTPases. Despite extensive studies on Icmt and its regulation of cell functions, the mechanisms of much of the impact of Icmt on cellular functions remain unclear. Our recent studies demonstrated that suppression of Icmt results in induction of autophagy, inhibition of cell growth and inhibition of proliferation in various cancer cell types, prompting this investigation of potential metabolic regulation by Icmt. We report here the findings that Icmt inhibition reduces the function of mitochondrial oxidative phosphorylation in multiple cancer cell lines. In-depth oximetry analysis demonstrated that functions of mitochondrial complex I, II and III are subject to Icmt regulation. Consistently, Icmt inhibition decreased cellular ATP and depleted critical tricarboxylic acid cycle metabolites, leading to suppression of cell anabolism and growth, and marked autophagy. Several different approaches demonstrated that the impact of Icmt inhibition on cell proliferation and viability was largely mediated by its effect on mitochondrial respiration. This previously unappreciated function of Icmt, which can be therapeutically exploited, likely has a significant role in the impact of Icmt on tumorigenic processes.e impact of Icmt on tumorigenic processes.)
Hochachka 1976 Eur J Appl Physiol Occup Physiol + (It has been demonstrated in several diving … It has been demonstrated in several diving vertebrates that succinate, a component of the Krebs cycle, accumulates in blood during breath-hold dives. The production of succinate is thought to result from amino acid catabolism. Our purpose was to determine whether succinate accumulation occurs in man during muscular activity requiring anaerobic energy contribution. Experiments using an endurance athlete included apneic work on an underwater ergometer and treadmill running to exhaustion. During 1 min breath-hold "dives" in cold water while exercising at a work rate equivalent to 62% of VO2max, venous succinate increased from 42 mumoles/l (M X 10(-6)) at rest to 125 M X 10(-6). The treadmill run elicited VO2max and increased succinate from a similar resting value to 93 M X 10(-6). Increases in alanine, lactate, and pyruvate were observed for both types of exercise. The findings confirm that succinate accumulation also occurs in man. It was suggested that amino acid catabolism may provide a source of anaerobic energy production in addition to glycolysis. However, the importance of the proposed energy pathway remains to be quantified.d energy pathway remains to be quantified.)
Khmil 2016 International Symposium Mitochondrial Motility + (It has been determined that 80% of populat … It has been determined that 80% of population in Russia lack selenium [1,2]. At the same time, the problem of diseases caused by deficit of selenium, and as a result the development of oxidative stress in patients, has been left unsettled. The data shows that various organoselenium compounds are able to demonstrate antitoxic, antibacterial, antioxidant, anantiviral, antiarrhythmic and fungicidal activity. During the past few years, a number of laboratories have synthesized the organic forms of selenium with the purpose of preventing deficit of selenium and other diseases. In the recent decade, several selenium-containing compounds have been synthesized at the Taras Shevchenko Luhansk National University [3–5]. It has been shown that some of them can be of strong antioxidant effect [6,7]. It is known that the positive effect of antioxidants is achieved through their ability to remove excessive peroxide: up to 85–90% of peroxide are formed in the electron transport chain of mitochondrion. Thereby, the relationship study between the structure and biological activity of these compounds and their influence on energy and oxidative metabolism in the mitochondria undoubtedly is of interest. Thus, the aim of this work was to study and compare the influence of six different selenium-containing newly synthesized compounds on respiratory and phosphorylation parameters, as well as on the concentration of peroxide lipids in liver mitochondria of rats.xide lipids in liver mitochondria of rats.)
Kovalcikova 2018 Thesis + (It has been estimated that the mammalian m … It has been estimated that the mammalian mitochondrial proteome consists of ~1500 distinct proteins and approximately one quarter of them is still not fully characterized. One of these proteins is TMEM70, protein involved in the biogenesis of the eukaryotic F1Fo-ATP synthase. TMEM70 mutations cause isolated deficiency of ATP synthase often resulting in a fatal neonatal mitochondrial encephalocardiomyopathies in patients. To understand the molecular mechanism of TMEM70 action, we generated constitutive ''Tmem70'' knockout mice, which led to embryonic lethal phenotype with disturbed ATP synthase biogenesis. Subsequently generated inducible ''Tmem70'' mouse knockout was lethal by the week 8 post induction. It exhibited primarily impaired liver function, which contrasts with the predominantly cardiologic phenotype at disease onset in humans. Liver mitochondria revealed formation of labile ATP synthase subcomplexes lacking subunit c. Thus, in case of TMEM70 deficiency c-oligomer was not incorporated into ATP synthase, which led to critical impairment of mitochondrial energy provision, analogous to TMEM70 dysfunction in humans. In TMEM70 deficient models, the ATP synthase deficiency reached the ‘threshold’ for its pathologic presentation, which we quantified at 30 %. We observed compensatory increases in the content of most OXPHOS complexes but unexpectedly also of ANT and PiC, components of ATP synthasome, which should associate with ATP synthase. We also studied ATP synthase subunit DAPIT (coded by ''Usmg5'' gene). We generated DAPIT deficient rats, which were fully viable but had lower body weight, pronounced decrease of fat tissue and right ventricular hypertrophy. We observed normal levels of assembled ATP synthase, however, it was predominantly present in the monomeric form, pointing at the role of DAPIT in formation of ATP synthase dimers. ATP synthase function was reduced by ~10 % in both liver and heart. Its higher sensitivity to inhibitor oligomycin than to aurovertin indicated that DAPIT shields oligomycin binding site at Fo moiety. In conclusion, we generated unique models of mitochondrial proteins deficiency and characterised TMEM70 and DAPIT function.mitochondrial proteins deficiency and characterised TMEM70 and DAPIT function.)
Murphy 1997 Exp Parasitol + (It has been hypothesized that ''Plasmodium … It has been hypothesized that ''Plasmodium'' parasites utilize a branched chain respiratory pathway, consisting of a classical cyanide-sensitive branch and an alternative cyanide-resistant branch. To further explore this hypothesis, the effect of cyanide on ''Plasmodium falciparum'' was determined using a polarographic assay. The rate of oxygen consumption by saponin-freed parasites was approximately 5% that of control human white blood cells or of ''Toxoplasma gondii'',consistent with an anabolic role for ''P. falciparum'' respiration. However, while all of the oxygen consumption of the control white blood cells and of ''T. gondii'' could be inhibited by cyanide, 25% of the oxygen consumption of the ''P. falciparum'' parasites was found to be insensitive to high concentrations of cyanide. The cyanide-resistant portion of the parasite oxygen consumption was completely inhibited by two inhibitors of alternative oxidase activities in other systems, propyl gallate and salicyclhydroxamic acid. These studies provide the first direct evidence for a branched chain respiratory pathway in ''P. falciparum''.Furthermore, salicyclhydroxamic acid, propyl gallate, and related inhibitors of alternative oxidase activities were shown to inhibit the growth of ''P. falciparum'' ''in vitro''.These results support the need for further investigation of alternative oxidase activity as an antimalarial chemotherapeutic target.s an antimalarial chemotherapeutic target.)
Welker 2017 Thesis + (It has been observed that highly-prolifera … It has been observed that highly-proliferating cells, such as cancer cells, rely mainly on glycolysis for ATP production, regardless of presence of oxygen. This effect, however, can be reversed by changing the main energy substrate in the medium from glucose to galactose. The oxidation of galactose in glycolysis yields less net ATP, presumably forcing the cell into OXPHOS. This has been established in many cell lines, including HeLA, HepG2, and skeletal muscle cells. As of yet, this has not been reproduced in neuronal cells. Using Neuro2a, a murine neuroblastoma cell line, this study exposes neuronal cells to galactose medium, and measures effect on neurite outgrowth, cell proliferation, and other indicators of metabolic function. An increase in neurite length and overall growth was observed in galactose-grown cells, as was an increase in doubling time (n = 3, p < .05). Oxygen consumption shows an increase of 20% in galactose grown cells (n=5-10, p < .05). Mitochondrial protein shows an increase in galactose-grown cells (n=3, p < .05).ncrease in galactose-grown cells (n=3, p < .05).)
Lagendijk 1996 J Lipid Res + (It has been postulated that lipid peroxida … It has been postulated that lipid peroxidation plays a crucial role in the pathogenesis of atherosclerosis. As CoQ10H2 (reduced form of coenzyme Q10) is easily oxidized to CoQ10 (oxidized form of coenzyme Q10), it has been proposed that the CoQ10H2/CoQ10 ratio may be used as a possible marker of in vivo oxidative stress. However, sample preparation has an important effect on the redox status of coenzyme Q10 due to the extreme sensitivity of CoQ10H2 towards oxidation. We now report a rapid, simple isocratic HPLC procedure for the determination of CoQ10H2 and CoQ10 in plasma isopropanol extracts, and we used this method to investigate conditions by which the CoQ10H2/CoQ10 ratio can be reliably measured. Our results indicate that CoQ10H2 is unstable in whole blood, plasma, and isopropanol extracts; subsequently the CoQ10H2/CoQ10 ratio changes considerably soon after a blood sample has been obtained. The time period since blood sampling and HPLC analysis, as well as the sample pretreatment procedure, are two factors that have a profound effect on the pre-analytical variation in the determination of the CoQ10H2/CoQ10 ratio. If these two factors are properly controlled, the CoQ10H2/CoQ10 ratio may be a sensitive and practical way to measure in vivo oxidative stress. Furthermore, this indicator is independent from plasma total cholesterol concentrations, implying that groups who differ with respect to cholesterol levels may be compared directly.olesterol levels may be compared directly.)
Pasdois 2006 J Bioenerg Biomembr + (It has been proposed that activation of th … It has been proposed that activation of the mitochondrial ATP-sensitive potassium channel (mitoKATP) is part of signaling pathways triggering the cardioprotection afforded by ischemic preconditioning of the heart. This work was to analyze the mitochondrial function profile of Langendorff-perfused rat hearts during the different phases of various ischemia-reperfusion protocols. Specifically, skinned fibers of ischemic preconditioned hearts exhibit a decline in the succinate-supported respiration and complex II activity during ischemia, followed by a recovery during reperfusion. Meanwhile, the apparent affinity of respiration for ADP (which reflects the matrix volume expansion) is increased during preconditioning stimulus and, to a larger extent, during prolonged ischemia. This evolution pattern is mimicked by diazoxide and abolished by 5-hydroxydecanoate. It is concluded that opening the mitoKATP channel mediates the preservation of mitochondrial structure-function via a mitochondrial matrix shrinkage and a reversible inactivation of complex II during prolonged ischemic insult.mplex II during prolonged ischemic insult.)
Lund 2018 Acta Physiol (Oxf) + (It has been proposed, but not yet demonstr … It has been proposed, but not yet demonstrated by convincing evidence in published articles, that insulin resistance and mitochondrial respiratory function are causally related physiological phenomena. Here, we tested the prediction that weight loss-induced increase in insulin sensitivity will correlate with a corresponding change in mitochondrial respiratory capacity over the same time period.Insulin sensitivity was evaluated using the hyperinsulinaemic-euglycaemic clamp technique, and skeletal muscle mitochondrial respiratory capacity was evaluated by high-resolution respirometry in 26 patients with obesity. Each experiment was performed ~2 months and 1-2 weeks before, and ~4 and ~19 months after Roux-en-Y gastric bypass (RYGB) surgery.A substantial weight loss was observed in all patients, and insulin sensitivity increased in all patients over the 21-months time period of the study. In contrast, skeletal muscle mitochondrial respiratory capacity, intrinsic mitochondrial respiratory capacity and mitochondrial content remained unchanged over the same time period.Among obese patients with and without type 2 diabetes undergoing RYGB surgery, intrinsic mitochondrial respiratory capacity in skeletal muscle is not correlated with insulin sensitivity before or after the surgical intervention. Mitochondrial respiratory function may not be germane to the pathophysiology and/or aetiology of obesity and/or type 2 diabetes.© 2018 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.ciety. Published by John Wiley & Sons Ltd.)
Bezuidenhout 2016a Abstract MitoFit Science Camp 2016 + (It has been reported that the oxygen (O< … It has been reported that the oxygen (O2) dependence of mitochondrial respiration in permeabilized fibers (Pfi) is ~100-fold higher compared to isolated mitochondria [1]. Pfi are sensitive to low oxygen supply due to diffusion restrictions that limit the supply of oxygen to the core of the fiber bundle. Therefore, hyperoxic conditions (oxygen range 400-250 μM) have been employed to counteract this limitation. Further studies have shown that the addition of a myosin II-specific inhibitor, blebbistatin (BLEB) in the respiration medium reduces this sensitivity and allows the study of ADP kinetics in Pfi at normoxic oxygen levels (250-200 μM). Moreover, it has been described that the use of BLEB prevents fiber contraction and yields high Km values for ADP [2].In the present study, high-resolution respirometry (HRR) was used to test ADP respiratory kinetics in skeletal muscle Pfi from wild-type C57BL/6 mice under three different oxygen regimens: hypoxia (100-80 μM), normoxia (250-200 μM) and hyperoxia (400 μM) comparing two respiration media: Buffer Z with catalase (Ctl), creatine (Cr) and BLEB (BufferZCtlCr+BLEB) and MiR06+Cr (MiR06Cr) at a physiological temperature of 37 °C. Data were normalized to wet weight (''W''w) (mg of tissue), complex IV (COX) and citrate synthase (CS) activities.In our experiments, we found no differences in O2 flux when comparing rates determined in Buffer ZCtlCr + BLEB and MiR06Cr. However, our results do show that different O2 regimens such as hypoxic and normoxic conditions in the O2k-chamber may lead to a relevant underestimation of OXPHOS capacity due to the oxygen limitation in both media (BufferZCtlCr+BLEB and MiR06Cr). It would therefore appear that, in our hands, the use of BufferZCtlCr with the addition of BLEB does not alleviate O2 dependency in permeabilized skeletal muscle fibers.ot alleviate O2 dependency in permeabilized skeletal muscle fibers.)
Larsen 2014 Biochim Biophys Acta + (It has been suggested that human mitochond … It has been suggested that human mitochondrial variants influence maximal oxygen uptake (VO2max). Whether mitochondrial respiratory capacity per mitochondrion (intrinsic activity) in human skeletal muscle is affected by differences in mitochondrial variants is not known. We recruited 54 males and determined their mitochondrial haplogroup, mitochondrial oxidative phosphorylation capacity (OXPHOS), mitochondrial content (citrate synthase (CS)) and VO2max. Intrinsic mitochondrial function is calculated as mitochondrial OXPHOS capacity divided by mitochondrial content (CS). Haplogroup H showed a 30% higher intrinsic mitochondrial function compared with the other haplo group U. There was no relationship between haplogroups and VO2max. In skeletal muscle from men with mitochondrial haplogroup H, an increased intrinsic mitochondrial function is present.trinsic mitochondrial function is present.)
Yin 2014 J Clin Endocrinol Metab + (It has been suggested that mitochondrial d … It has been suggested that mitochondrial dysfunctional in adipocytes contribute to obesity-related metabolic complications. However, obesity results in adipocyte hypertrophy, large and small adipocytes from the same depot have different characteristics, raising the possibility that obesity-related mitochondrial defects are an inherent function of large adipocytes.Objective:to examine whether obesity, independent of fat cell size and fat depot, is associated with mitochondria dysfunction.Design:cross-sectional comparison.Setting:Academic medical center.Patients or Other Participants:omental (OM) and/or abdominal subcutaneous (SQ) adipose samples were collected from 20, age-matched obese and non-obese non-diabetic men and women undergoing either elective abdominal surgery or research needle biopsy.Intervention:None.Main Outcome Measures:mitochondrial DNA abundance, oxygen consumption rates (OCR) and citrate synthase (CS) activity from populations of large and small adipocytes (separated with differential floatation).Results:For both omental and subcutaneous adipocytes, at the cell and organelle level, OCR and CS activity were significantly reduced in cells from obese compared with non-obese volunteers, even when matched for cell size by comparing large adipocytes from non-obese and small adipocytes from obese. Adipocyte mitochondrial content was not significantly different between obese and non-obese volunteers. Mitochondrial function and content parameters were not different between small and large cells, omental and subcutaneous adipocytes from the same person.Conclusion:Adipocyte mitochondrial oxidative capacity is reduced in obese compared to non-obese adults and this difference is not due to cell size differences. Adipocyte mitochondrial dysfunction in obesity is therefore related to overall adiposity rather than adipocyte hypertrophy.iposity rather than adipocyte hypertrophy.)
Yao 2018 PLoS Biol + (It has been suggested that some cancer cel … It has been suggested that some cancer cells rely upon fatty acid oxidation (FAO) for energy. Here we show that when FAO was reduced approximately 90% by pharmacological inhibition of carnitine palmitoyltransferase I (CPT1) with low concentrations of etomoxir, the proliferation rate of various cancer cells was unaffected. Efforts to pharmacologically inhibit FAO more than 90% revealed that high concentrations of etomoxir (200 μM) have an off-target effect of inhibiting complex I of the electron transport chain. Surprisingly, however, when FAO was reduced further by genetic knockdown of CPT1, the proliferation rate of these same cells decreased nearly 2-fold and could not be restored by acetate or octanoic acid supplementation. Moreover, CPT1 knockdowns had altered mitochondrial morphology and impaired mitochondrial coupling, whereas cells in which CPT1 had been approximately 90% inhibited by etomoxir did not. Lipidomic profiling of mitochondria isolated from CPT1 knockdowns showed depleted concentrations of complex structural and signaling lipids. Additionally, expression of a catalytically dead CPT1 in CPT1 knockdowns did not restore mitochondrial coupling. Taken together, these results suggest that transport of at least some long-chain fatty acids into the mitochondria by CPT1 may be required for anabolic processes that support healthy mitochondrial function and cancer cell proliferation independent of FAO.cer cell proliferation independent of FAO.)
Kluckova 2022 Hemasphere + (It has been unclear what role metabolism i … It has been unclear what role metabolism is playing in the pathophysiology of chronic lymphocytic leukemia (CLL). One reason is that the study of CLL metabolism is challenging due to the resting nature of circulating CLL cells. Also, it is not clear if any of the genomic aberrations observed in this disease have any impact on metabolism. Here, we demonstrate that CLL cells in proliferation centers exhibit upregulation of several molecules involved in glycolysis and mitochondrial metabolism. Comparison of CXCR4/CD5 intraclonal cell subpopulations showed that these changes are paralleled by increases in the metabolic activity of the CXCR4lowCD5high fraction that have recently egressed from the lymph nodes. Notably, anti-IgM stimulation of CLL cells recapitulates many of these metabolic alterations, including increased glucose uptake, increased lactate production, induction of glycolytic enzymes, and increased respiratory reserve. Treatment of CLL cells with inhibitors of B-cell receptor (BCR) signaling blocked these anti-IgM-induced changes ''in vitro'', which was mirrored by decreases in hexokinase 2 expression in CLL cells from ibrutinib-treated patients ''in vivo''. Interestingly, several samples from patients with 17p-deletion manifested increased spontaneous aerobic glycolysis in the unstimulated state suggestive of a BCR-independent metabolic phenotype. We conclude that the proliferative fraction of CLL cells found in lymphoid tissues or the peripheral blood of CLL patients exhibit increased metabolic activity when compared with the bulk CLL-cell population. Although this is due to microenvironmental stimulatory signals such as BCR-engagement in most cases, increases in resting metabolic activity can be observed in cases with 17p-deletion.ing metabolic activity can be observed in cases with 17p-deletion.)
Rocha 2018 Antioxidants (Basel) + (It has long been accepted that mitochondri … It has long been accepted that mitochondrial function and morphology is affected in Parkinson's disease, and that mitochondrial function can be directly related to its morphology. So far, mitochondrial morphological alterations studies, in the context of this neurodegenerative disease, have been performed through microscopic methodologies. The goal of the present work is to address if the modifications in the mitochondrial-shaping proteins occurring in this disorder have implications in other cellular pathways, which might constitute important pathways for the disease progression. To do so, we conducted a novel approach through a thorough exploration of the available proteomics-based studies in the context of Parkinson's disease. The analysis provided insight into the altered biological pathways affected by changes in the expression of mitochondrial-shaping proteins via different bioinformatic tools. Unexpectedly, we observed that the mitochondrial-shaping proteins altered in the context of Parkinson's disease are, in the vast majority, related to the organization of the mitochondrial cristae. Conversely, in the studies that have resorted to microscopy-based techniques, the most widely reported alteration in the context of this disorder is mitochondria fragmentation. Cristae membrane organization is pivotal for mitochondrial ATP production, and changes in their morphology have a direct impact on the organization and function of the oxidative phosphorylation (OXPHOS) complexes. To understand which biological processes are affected by the alteration of these proteins we analyzed the binding partners of the mitochondrial-shaping proteins that were found altered in Parkinson's disease. We showed that the binding partners fall into seven different cellular components, which include mitochondria, proteasome, and endoplasmic reticulum (ER), amongst others. It is noteworthy that, by evaluating the biological process in which these modified proteins are involved, we showed that they are related to the production and metabolism of ATP, immune response, cytoskeleton alteration, and oxidative stress, amongst others. In summary, with our bioinformatics approach using the data on the modified proteins in Parkinson's disease patients, we were able to relate the alteration of mitochondrial-shaping proteins to modifications of crucial cellular pathways affected in this disease.ellular pathways affected in this disease.)
Zeng 2014 Br J Nutr + (It has not been established which specific … It has not been established which specific measures of obesity might be most appropriate for predicting CVD risk in Asians. The objectives of the present study were to determine the associations of BMI, waist circumference (WC) and waist:height ratio (WHtR) with CVD risk factors and to evaluate the optimal cut-off values to define overweight or obesity in Chinese adults. Data collected from seven nationwide health examination centres during 2008 and 2009 were analysed. The BMI, WC and WHtR of 244 266 Chinese adults aged ≥ 20 years included in the study were measured. Logistic regression models were fit to evaluate the OR of each CVD risk factor according to various anthropometric indices. Receiver operating characteristic (ROC) analyses were conducted to assess the optimal cut-off values to predict the risk of diabetes, hypertension, dyslipidaemia and the metabolic syndrome. WHtR had the largest areas under the ROC curve for all CVD risk factors in both sexes, followed by WC and BMI. The optimal cut-off values were approximately 24·0 and 23·0 kg/m2 for BMI, 85·0 and 75·0 cm for WC, and 0·50 and 0·48 for WHtR for men and women, respectively. According to well-established cut-off values, BMI was found to be a more sensitive indicator of hypertension in both men and women, while WC and WHtR were found to be better indicators of diabetes and dyslipidaemia. A combination of BMI and central obesity measures was found to be associated with greater OR of CVD risk factors than either of them alone in both sexes. The present study demonstrated that WHtR and WC may be better indicators of CVD risk factors for Chinese people than BMI. risk factors for Chinese people than BMI.)
Larsen S 2013 Abstract MiP2013 + (It has previously been reported that mitoc … It has previously been reported that mitochondrial ADP sensitivity decreases (higher Km’) with endurance training in human skeletal muscle [1]. This decrease was accompanied by an increased maximal ADP stimulated mt-respiration (Jmax) and an increased maximal oxygen uptake (VO2max). It is not known whether high intensity training (HIT) has the same effect on mitochondria and if these changes also occur in mitochondria from human adipose tissue. The aim of this project was to investigate mitochondrial ADP sensitivity and maximal ADP stimulated respiration in human adipose and skeletal muscle tissue after six weeks of HIT. Twelve healthy overweight subjects (7 F/5 M) (age: 40±2 yrs, BMI: 32±2, VO2max: 2612±166 ml/min) were included in the study. The subjects underwent six weeks (3 times a week) of HIT. VO2max was measured pre and post training. Mitochondrial ADP sensitivity (Km’) and Jmax was measured pre and post HIT in adipose tissue and permeabilized muscle fibers by high-resolution respirometry (Oxygraph-2k, Oroboros, Insbruck, Austria). The adipose tissue was permeabilized with digitonin and the skeletal muscle fibers with saponin. The protocol used for respirometry was as follows: Malate (2 mM), Glutamate (10 mM) and ADP titrated in the following steps (0.05 – 0.10 – 0.25 – 0.50 – 1.00 – 2.50 – 5.00 mM). SigmaPlot was used to determine Km’ for ADP and Jmax.VO2max was significantly improved after HIT. Mitochondrial ADP sensitivity was significantly (P<0.05) decreased after HIT in permeabilized skeletal muscle (0.14±0.02 mM vs. 0.29±0.03 mM), and the same trend was seen in adipose tissue although not significant (P=0.056; 0.11±0.02 mM vs. 0.16±0.04 mM). Jmax was similar after HIT in permeabilized skeletal muscle (21±1 pmol∙s-1∙mg-1 vs. 22±1 pmol∙s-1∙mg-1) as well as in adipose tissue (0.36±0.03 pmol∙s-1∙mg-1 vs. 0.36±0.03 pmol∙s1∙mg-1), before vs. after, respectively.ADP sensitivity decreased in permeabilized human skeletal muscle and the same trend was seen in adipose tissue. This was accompanied by a similar maximal ADP stimulated respiration pre and post training in both skeletal muscle and adipose tissue. This is the first time that ADP sensitivity has been investigated in adipose tissue. Interestingly the HIT training adaptation in mitochondria from adipose tissue is similar to that observed in skeletal muscle although not with the same magnitude. muscle although not with the same magnitude.)
Loew 1961 Biochem Biophys Res Commun + (It has recently been reported from several … It has recently been reported from several laboratories that mitochondrial DPN is reduced by succinate and that this reduction requires the addition of ATP. All of these studies involved intact mitochondria and bound DPN.In the present communication some experiments will be described which deal with the reduction of added DPN by a submitochondrial particle from beef heart muscle, ETPH. This particle enjoys several advantages for the study of DPN reduction; it contains little or no bound DPN, and does not have a functional Krebs cycle (which excludes any reduction of DPN by substrates other than succinate).f DPN by substrates other than succinate).)
Boushel 2014 Acta Physiol (Oxf) + (It is an ongoing discussion the extent to … It is an ongoing discussion the extent to which oxygen delivery and oxygen extraction contribute to an elevated muscle oxygen uptake during dynamic exercise. It has been proposed that local muscle factors including the capillary bed and mitochondrial oxidative capacity play a large role in prolonged low intensity training of a small muscle group when the cardiac output capacity is not directly limiting. The purpose of this study was to investigate the relative roles of circulatory and muscle metabolic mechanisms by which prolonged low-intensity exercise training alters regional muscle ''V''O2.In 9 healthy volunteers (7 male, 2 female), hemodynamic and metabolic responses to incremental arm cycling were measured by the Fick method and biopsy of the deltoid and triceps muscles before and after 42 days of skiing for 6 h.day-1 at 60 % max heart rate.Peak pulmonary ''V''O2 during arm crank was unchanged after training (2.38±0.19 vs. 2.18±0.2 L.min-1 pre-training) yet arm ''V''O2 (1.04±0.08 vs. 0.83±0.1 L.min-1, ''P''<0.05) and power output (137±9 vs. 114±10 W) were increased along with a higher arm blood flow (7.9±0.5 vs. 6.8±0.6 L.min-1, ''P''<0.05) and expanded muscle capillary volume (76±7 vs. 62±4 mL, ''P''<0.05). Muscle O2 diffusion capacity (16.2±1 vs. 12.5 ±0.9 mL.min-1.mmHg-1, ''P''<0.05) and O2 extraction (68±1 vs. 62±1 %, ''P''<0.05) were enhanced at a similar mean capillary transit time (569±43 vs. 564±31 ms) and ''p''50 (35.8±0.7 vs. 35±0.8), whereas mitochondrial O2 flux capacity was unchanged (147±6 mL.min-1.kg-1 vs. 146±8 mL.min-1.kg-1).The mechanisms underlying the increase in peak arm ''V''O2 with prolonged low intensity training in previously untrained subjects are an elevated convective O2 delivery specifically to the muscles of the arm combined with a larger capillary-muscle surface area that enhance diffusional O2 conductance, with no apparent role of mitochondrial respiratory capacity. This article is protected by copyright. All rights reserved. an elevated convective O2 delivery specifically to the muscles of the arm combined with a larger capillary-muscle surface area that enhance diffusional O2 conductance, with no apparent role of mitochondrial respiratory capacity. This article is protected by copyright. All rights reserved. )
Kidane 1997 J Thermal Anal + (It is claimed, though not without dispute, … It is claimed, though not without dispute, that genetically engineered mammalian cells grow more slowly than their progenitor cells because the recombinant gene system causes a metabolic burden. This was found to be the case for CHO cells transfected with expression vectors forcytochrome b5. The slower growth was associated with lower metabolic activity measured by heat flux and mitochondrial activity (rhodamine 123 fluorescence). The calorimetric-respirometric ratio was similar for all cell types, implying that the greater fluxes of glucose and glutamine in the recombinant cells was channelled to biosynthesis. This demand probably restricted the supply of pyruvate to the mitochondria in these cells.ruvate to the mitochondria in these cells.)
Dungel 2011 J Biochem Mol Toxicol + (It is commonly accepted that the major eff … It is commonly accepted that the major effect of nitroglycerin (NG) is realized through the release of nitric oxide (NO) catalyzed by aldehyde dehydrogenase-2 (ALDH2). In addition, it has been shown that NG inhibits mitochondrial respiration. The aim of this study was to clarify whether NG-mediated inhibition of mitochondrial respiration is mediated by NO. In rat liver mitochondria, NG inhibited complex-I-dependent respiration and induced reactive oxygen species (ROS) production, preferentially at complex I. Both effects were insensitive to chloral hydrate, an ALDH2 inhibitor. Nitrite, an NG intermediate, had no influence on either mitochondrial respiration or the production of ROS. NO inhibited preferentially complex I but did not elevate ROS production. Hemoglobin, an NO scavenger, and blue light had contrary effects on mitochondria inhibited by NO or NG. In summary, our data suggest that although NG induces vasodilatation via NO release, it causes mitochondrial dysfunction via an NO-independent pathway.dysfunction via an NO-independent pathway.)
Jang 2018 J Med Toxicol + (It is conservatively estimated that 5000 d … It is conservatively estimated that 5000 deaths per year and 20,000 injuries in the USA are due to poisonings caused by chemical exposures (e.g., carbon monoxide, cyanide, hydrogen sulfide, phosphides) that are cellular inhibitors. These chemical agents result in mitochondrial inhibition resulting in cardiac arrest and/or shock. These cellular inhibitors have multi-organ effects, but cardiovascular collapse is the primary cause of death marked by hypotension, lactic acidosis, and cardiac arrest. The mitochondria play a central role in cellular metabolism where oxygen consumption through the electron transport system is tightly coupled to ATP production and regulated by metabolic demands. There has been increasing use of human blood cells such as peripheral blood mononuclear cells and platelets, as surrogate markers of mitochondrial function in organs due to acute care illnesses. We demonstrate the clinical applicability of measuring mitochondrial bioenergetic and dynamic function in blood cells obtained from patients with acute poisoning using carbon monoxide poisoning as an illustration of our technique. Our methods have potential application to guide therapy and gauge severity of disease in poisoning related to cellular inhibitors of public health concern.lular inhibitors of public health concern.)
Kozlov 2010 Abstract IOC60 + (It is difficult to clarify mechanisms unde … It is difficult to clarify mechanisms underlying cellular dysfunction induced by shock in in vivo models, because of their multiple physiological/ pathological regulations. Shock comprises two major pathologic stimuli, namely low levels of oxygen and high levels of inflammatory mediators. In our experiments we used hepatocyte cell line (HEP), liver slices (LS), and liver tissue (LT) to simulate either ischemia or inflammatory response. Confocal and electron microscopy examinations revealed that mitochondria in LS and LT are fragmented while in HEP they built up a network. Hypoxia induced the loss of mitochondrial respiratory activity (in LT & LS), total decrease in ATP levels (LT & LS), the release of ALT/AST, cytoplasmic enzymes, (in LS & HEP), strong degradation of intracellular RNA (in LS & HEP) and an increase in numbers of necrotic, but not apoptotic cells (HC). In LT and LS these changes occurred after 2 h of hypoxia, while HEP required 24 h of ischemia to induce damage to a similar extend. Mitochondrial dysfunction mediated by hypoxia was fully recovered by exogenous cytochrome c (LT). Exposure to naturally generated inflammatory mediators under normoxic conditions resulted in a moderate decrease in ATP levels (LS, LT) and Δψ (HEP), fragmentation of mitochondrial network, and increased levels of mitochondrial ROS (LT & HEP). Experiments with radical scavengers revealed that the major target for mitochondrial ROS are not mitochondria but the expression of specific genes regulating inflammatory and unfolded protein responses. Our data suggest that the lost of cytochrome c and disrupted ATP synthesis are the predominant mitochondrial response to hypoxia but not to inflammatory mediators. The predominant mitochondrial response to inflammatory mediators is increased levels of mitochondrial ROS. Fragmentation of mitochondria is likely of marginal pathological significance in these models. likely of marginal pathological significance in these models.)
Mootha 1997 Am J Physiol + (It is difficult to estimate the maximum in … It is difficult to estimate the maximum in vivo aerobic ATP production rate of the intact heart independent of limitations imposed by blood flow, oxygen delivery, and maximum mechanical power. This value is critical for establishing the kinetic parameters that control oxidative phosphorylation, as well as for providing insights into the limits of myocardial performance. In this study, the maximum ADP-P(i)-driven heart mitochondrial respiratory rate (MV(O2 mito)) was determined with saturating levels of oxygen, substrates, and cofactors at 37 degrees C. These rates were normalized to cytochrome alpha1 alpha3 (cytochrome oxidase; Cyt a) content. To extrapolate this rate to the intact heart, the Cyt a content of the myocardium (nmol Cyt a/g wet wt myocardium) was determined in the same hearts. The maximum ADP-P(i)-driven mitochondrial respiratory rates were 676 +/- 31 and 665 +/- 65 nmol O2 x min(-1) x nmol Cyt a(-1) in the dog and pig, respectively. The Cyt a content in the two species was 43.6 +/- 2.4 and 36.6 +/- 3.1 nmol Cyt a/g wet wt, respectively. With these values, the MV(O2 mito) was calculated to be 29.5 (dog) and 24.3 (pig) micromol O2 x min(-1) x g wet wt myocardium(-1). Comparison with in vivo studies shows that the exercising heart can utilize 80-90 % of its maximum oxidative capacity, implying there is little aerobic ATP production reserve in the mammalian heart.production reserve in the mammalian heart.)
Sullivan 2015 Abstract MiPschool Greenville 2015 + (It is hypothesized that cardiolipin molecu … It is hypothesized that cardiolipin molecules in the inner mitochondrial membrane form spatially distinct lipid microdomains that facilitate the formation of supercomplexes and allow for efficient electron transport and ATP production. Cardiolipin-enriched microdomains from mitochondria can potentially be isolated biochemically in the presence of 1% Triton-X 100 [1]. Previous studies show that the amount and type of detergent are critical variables in the isolation of lipid microdomains in other organelles. Therefore, we first investigated the influence of different detergents at different concentrations on the isolation of biochemical cardiolipin- enriched microdomains [2]. Next, we determined if supercomplexes were associated with the detergent resistant cardiolipin-enriched microdomains. Biochemical isolation of mitochondrial microdomains was adapted from a previous protocol [1]. Briefly, isolated mitochondria were subjected to a range of detergents (Triton X-100, NP-40 or digitonin) at varying concentrations (0.01%, 0.05% or 0.50%). Next, mitochondria were centrifuged at 20,000 x g, which separated the detergent resistant membranes (DRM) microdomains from the non-microdomain fraction, known as detergent soluble membranes (DSM). Both DSM and DRM fractions were subjected to lipid extraction; the lipids were separated via thin layer chromatography (TLC), visualized by phosphoric charring and quantified via densitometry. Blue native PAGE (BN-PAGE) determined whether the DSM or DRM fraction contained supercomplexes. BN-PAGE protocol was adapted from the NativePAGE system from Life Technologies. Briefly, DSM and DRM fractions underwent solubilization with digitonin. Samples were centrifuged at 16,875 x g and the supernatants containing supercomplexes were collected. Protein was loaded onto the native gel as directed and ran until completion. Gels were fixed, and then destained overnight until the desired background was reached.Analysis of TLC plates strikingly revealed that DRM fractions were not just enriched in cardiolipin but also contained phosphatidylcholine and phosphatidylethanolamine. Increasing the concentration of each detergent resulted in a decreased amount of phosphatidylethanolamine, cardiolipin and phosphatidylcholine in the DRMs. This decrease in phospholipids in the DRMs was paralleled by an increase in lipid content of the DSMs with one exception. No decrease in lipids was detected in the DRMs from 0.01% digitonin to 0.05% digitonin. Preliminary data with BN-PAGE results from each detergent at the 0.05% concentration showed supercomplexes to only be present in the DRM fraction.Taken together, our results reveal no differences in the type of detergent used to isolate DRMs. These results also suggest that lipid microdomains formed in the mitochondria are not simply enriched in cardiolipin and likely contain several other lipid species. Although more precise analytical tools may be required for more in-depth mitochondrial membrane analysis, the supercomplexes housed in the inner mitochondrial membrane were associated with DRMs implying supercomplexes may rely on distinct lipid microdomains for optimal function.t lipid microdomains for optimal function.)
Gorbacheva 2016 International Symposium Mitochondrial Motility + (It is known that cardiovascular diseases a … It is known that cardiovascular diseases are the main problem of present medicine. Recently, for the treatment of these diseases have been widely used various modulators of calcium and potassium channels, as well as inhibitors of the renin-angiotensin system [1].With the collaboration of laboratories O.O. Moibenko and L.M. Yagupolskii developed technological scheme of synthesis of a new cardioprotective fluorine-containing pinacidil derivative preparation floсalin [2]. It was shown that this preparation at experimental acute ischemia/reperfusion has pronounced cardioprotective properties in animals and reduces myocardial infarct size relative to control without treatment by 42% [3]. It is assumed that one of the mechanisms cardiopro-tection in this case may be to increase the proportion cNOS/iNOS activity. In addition, it is shown that flocalin promotes normalization of bioenergetic metabolism in the ischemic brain, restoring the content of adenine nucleotides and creatine phosphate against decrease metabolic acidosis and growth of glucose [4].Since flocalin is a fluorine-containing analog of pinacidil - known opener of ATP-sensitive potassium channel both cytoplasmic (sarcKATP) and mitochondrial (mitoKATP) membranes [5], the aim of work was to investigate the effect of this preparation on the functioning of mitochondria, and in particular, on work of mitoKATP channel.n particular, on work of mitoKATP channel.)
Shigaeva 2013 Abstract MiP2013 + (It is known that different people and anim … It is known that different people and animals within the same species have different resistance to the stress damage. It is believed that these differences are largely genetically determined or acquired during the life of the differences in the activity of the stress system implementing the organism's response to stress-factor. Our objective was to determine the role of mitochondrial ATP-dependent potassium channel (mtKATP), which plays a key role in protecting the myocardium from ischemia [1], in protecting of the organism against some types of stress and adaptation to it.The work was carried out on heart and liver mitochondria of rats of different genetic lines. To perform the first part of the study we selected purebred white rats having high and low resistance to acute hypoxic stress. Part of the low resistant animals were then artificially adapted to acute hypobaric hypoxia. The second part of the work was carried out on rats Wistar and August lines, having different resistance to the stress damage of the circulatory system [2] and to hypoxia [3] (August rats are resistant to stress influences, Wistar rats are less resistant).When using outbred rats we have shown that the rate of ATP-dependent potassium transport was higher (by 15% in liver mitochondria and 30% in heart mitochondria) in mitochondria of high resistant to hypoxia rats compared with a low resistance. In adapted to oxygen deficiency rats rate of ATP-dependent potassium transport in mitochondria of both tissues is increased by 45-50%. It is accompanied by a decrease in the amount of K+ in the mitochondria of both tissues by an average of 30%, which may indicate the activation also K+/H+-antiporter in mitochondria. Thus, the potassium cycle is activated in mitochondria. The same effect was observed in the study of potassium transport in mitochondria of rats Wistar and August lines. In more resistant to stress August line rats potassium uptake into the mitochondria via mtKATP and the potassium amount were significantly higher compared to Wistar line rats.A high rate of mitochondrial potassium transport led to a marked reduction (by 25-30%) of the rate of generation of H2O2 in the mitochondria. We presume that the effect of reducing the formation of reactive oxygen species in the mitochondria as a result of the activation mtKATP may mediate the positive effect of hypoxic training as well as genetic resistance to oxidative stress.may mediate the positive effect of hypoxic training as well as genetic resistance to oxidative stress.)
Brierley 1994 J Bioenerg Biomembr + (It is now well established that mitochondr … It is now well established that mitochondria contain three antiporters that transport monovalent cations. A latent, allosterically regulated K+/H+ antiport appears to serve as a cation-extruding device that helps maintain mitochondrial volume homeostasis. An apparently unregulated Na+/H+ antiport keeps matrix [Na+] low and the Na(+)-gradient equal to the H(+)-gradient. A Na+/Ca2+ antiport provides a Ca(2+)-extruding mechanism that permits the mitochondrion to regulate matrix [Ca2+] by balancing Ca2+ efflux against influx on the Ca(2+)-uniport. All three antiports have well-defined physiological roles and their molecular properties and regulatory features are now being determined. Mitochondria also contain monovalent cation uniports, such as the recently described ATP- and glibenclamide-sensitive K+ channel and ruthenium red-sensitive uniports for Na+ and K+. A physiological role of such uniports has not been established and their properties are just beginning to be defined.operties are just beginning to be defined.)
Pham 2016 Sensors (Basel) + (It is now well established that, even with … It is now well established that, even within a single cell, multiple copies of the mitochondrial genome may be present (genetic heteroplasmy). It would be interesting to develop techniques to determine if and to what extent this genetic variation results in functional variation from one mitochondrion to the next (functional heteroplasmy). Measuring mitochondrial respiration can reveal the organelles' functional capacity for Adenosine triphosphate (ATP) production and determine mitochondrial damage that may arise from genetic or age related defects. However, available technologies require significant quantities of mitochondria. Here, we develop a technology to assay the respiration of a single mitochondrion. Our "micro-respirometer" consists of micron sized chambers etched out of borofloat glass substrates and coated with an oxygen sensitive phosphorescent dye Pt(II) meso-tetra(pentafluorophenyl)porphine (PtTFPP) mixed with polystyrene. The chambers are sealed with a polydimethylsiloxane layer coated with oxygen impermeable Viton rubber to prevent diffusion of oxygen from the environment. As the mitochondria consume oxygen in the chamber, the phosphorescence signal increases, allowing direct determination of the respiration rate. Experiments with coupled vs. uncoupled mitochondria showed a substantial difference in respiration, confirming the validity of the microchambers as single mitochondrial respirometers. This demonstration could enable future high-throughput assays of mitochondrial respiration and benefit the study of mitochondrial functional heterogeneity, and its role in health and disease.neity, and its role in health and disease.)
Salin 2015 Proc Biol Sci + (It is often assumed that an animal's metab … It is often assumed that an animal's metabolic rate can be estimated through measuring the whole-organism oxygen consumption rate. However, oxygen consumption alone is unlikely to be a sufficient marker of energy metabolism in many situations. This is due to the inherent variability in the link between oxidation and phosphorylation; that is, the amount of adenosine triphosphate (ATP) generated per molecule of oxygen consumed by mitochondria (P/O ratio). In this article, we describe how the P/O ratio can vary within and among individuals, and in response to a number of environmental parameters, including diet and temperature. As the P/O ratio affects the efficiency of cellular energy production, its variability may have significant consequences for animal performance, such as growth rate and reproductive output. We explore the adaptive significance of such variability and hypothesize that while a reduction in the P/O ratio is energetically costly, it may be associated with advantages in terms of somatic maintenance through reduced production of reactive oxygen species. Finally, we discuss how considering variation in mitochondrial efficiency, together with whole-organism oxygen consumption, can permit a better understanding of the relationship between energy metabolism and life history for studies in evolutionary ecology.story for studies in evolutionary ecology.)
Brown 2012 Mitochondrion + (It is often assumed that mitochondria are … It is often assumed that mitochondria are the main source of reactive oxygen species (ROS) in mammalian cells, but there is no convincing experimental evidence for this in the literature. What evidence there is suggests mitochondria are a significant source for ROS, which may have physiological and pathological effects. But quantitatively, endoplasmic reticulum and peroxisomes have a greater capacity to produce ROS than mitochondria, at least in liver. In most cells and physiological or pathological conditions there is a lack of evidence for or against mitochondria being the main source of cellular ROS. Mitochondria can rapidly degrade ROS and thus are potential sinks for ROS, but whether mitochondria act as net sources or sinks within cells in particular conditions is unknown.cells in particular conditions is unknown.)
Sender 2016 Cell + (It is often presented as common knowledge that, in the human body, bacteria outnumber human cells by a ratio of at least 10:1. Revisiting the question, we find that the ratio is much closer to 1:1.)
Schönfeld 2013 J Cereb Blood Flow Metab + (It is puzzling that hydrogen-rich fatty ac … It is puzzling that hydrogen-rich fatty acids are used only poorly as fuel in the brain. The long-standing belief that a slow passage of fatty acids across the blood-brain barrier might be the reason. However, this has been corrected by experimental results. Otherwise, accumulated nonesterified fatty acids or their activated derivatives could exert detrimental activities on mitochondria, which might trigger the mitochondrial route of apoptosis. Here, we draw attention to three particular problems: (1) ATP generation linked to β-oxidation of fatty acids demands more oxygen than glucose, thereby enhancing the risk for neurons to become hypoxic; (2) β-oxidation of fatty acids generates superoxide, which, taken together with the poor anti-oxidative defense in neurons, causes severe oxidative stress; (3) the rate of ATP generation based on adipose tissue-derived fatty acids is slower than that using blood glucose as fuel. Thus, in periods of extended continuous and rapid neuronal firing, fatty acid oxidation cannot guarantee rapid ATP generation in neurons. We conjecture that the disadvantages connected with using fatty acids as fuel have created evolutionary pressure on lowering the expression of the β-oxidation enzyme equipment in brain mitochondria to avoid extensive fatty acid oxidation and to favor glucose oxidation in brain.n and to favor glucose oxidation in brain.)
Rich 1984 Biochim Biophys Acta + (It is the aim of this article to discuss t … It is the aim of this article to discuss the details of electron and proton transfers through quinones and cytochrome ''bc'' complexes. Emphasis will be placed on the molecular organisation and mobility of components, on the chemistry of the individual redox steps, and on the relation of these factors to overall protonmotive ability.e factors to overall protonmotive ability.)
Noone 2023 J Physiol + (It is unclear how skeletal muscle metaboli … It is unclear how skeletal muscle metabolism and mitochondrial function adapt to long duration bed rest and whether changes can be prevented by nutritional intervention. The present study aimed (1) to assess the effect of prolonged bed rest on skeletal muscle mitochondrial function and dynamics and (2) to determine whether micronutrient supplementation would mitigate the adverse metabolic effect of bed rest. Participants were maintained in energy balance throughout 60 days of bed rest with micronutrient supplementation (INT) (body mass index: 23.747 ± 1.877 kg m-2 ; 34.80 ± 7.451 years; n = 10) or without (control) (body mass index: 24.087 ± 2.088 kg m-2 ; 33.50 ± 8.541 years; n = 10). Indirect calorimetry and dual-energy x-ray absorptiometry were used for measures of energy expenditure, exercise capacity and body composition. Mitochondrial respiration was determined by high-resolution respirometry in permeabilized muscle fibre bundles from ''vastus lateralis'' biopsies. Protein and mRNA analysis further examined the metabolic changes relating to regulators of mitochondrial dynamics induced by bed rest. INT was not sufficient in preserving whole body metabolic changes conducive of a decrease in body mass, fat-free mass and exercise capacity within both groups. Mitochondrial respiration, OPA1 and Drp1 protein expression decreased with bed rest, with an increase pDrp1s616. This reduction in mitochondrial respiration was explained through an observed decrease in mitochondrial content (mtDNA:nDNA). Changes in regulators of mitochondrial dynamics indicate an increase in mitochondrial fission driven by a decrease in inner mitochondrial membrane fusion (OPA1) and increased pDrp1s616.inner mitochondrial membrane fusion (OPA1) and increased pDrp1s616.)
Galli 2021 Front Physiol + (It is well established that adult vertebra … It is well established that adult vertebrates acclimatizing to hypoxic environments undergo mitochondrial remodeling to enhance oxygen delivery, maintain ATP, and limit oxidative stress. However, many vertebrates also encounter oxygen deprivation during embryonic development. The effects of developmental hypoxia on mitochondrial function are likely to be more profound, because environmental stress during early life can permanently alter cellular physiology and morphology. To this end, we investigated the long-term effects of developmental hypoxia on mitochondrial function in a species that regularly encounters hypoxia during development-the common snapping turtle (''Chelydra serpentina''). Turtle eggs were incubated in 21 % or 10 % oxygen from 20 % of embryonic development until hatching, and both cohorts were subsequently reared in 21 % oxygen for 8 months. Ventricular mitochondria were isolated, and mitochondrial respiration and reactive oxygen species (ROS) production were measured with a microrespirometer. Compared to normoxic controls, juvenile turtles from hypoxic incubations had lower LEAK respiration, higher P:O ratios, and reduced rates of ROS production. Interestingly, these same attributes occur in adult vertebrates that acclimatize to hypoxia. We speculate that these adjustments might improve mitochondrial hypoxia tolerance, which would be beneficial for turtles during breath-hold diving and overwintering in anoxic environments. and overwintering in anoxic environments.)
Groennebaek 2018 Front Physiol + (It is well established that high-load resi … It is well established that high-load resistance exercise (HLRE) can stimulate myofibrillar accretion. Additionally, recent studies suggest that HLRE can also stimulate mitochondrial biogenesis and respiratory function. However, in several clinical situations, the use of resistance exercise with high loading may not constitute a viable approach. Low-load blood flow restricted resistance exercise (BFRRE) has emerged as a time-effective low-load alternative to stimulate myofibrillar accretion. It is unknown if BFRRE can also stimulate mitochondrial biogenesis and respiratory function. If so, BFRRE could provide a feasible strategy to stimulate muscle metabolic health. To study this, 34 healthy previously untrained individuals (24 ± 3 years) participated in BFRRE, HLRE, or non-exercise control intervention (CON) 3 times per week for 6 weeks. Skeletal muscle biopsies were collected; (1) before and after the 6-week intervention period to assess mitochondrial biogenesis and respiratory function and; (2) during recovery from single-bout exercise to assess myocellular signaling events involved in transcriptional regulation of mitochondrial biogenesis. During the 6-week intervention period, deuterium oxide (D2O) was continuously administered to the participants to label newly synthesized skeletal muscle mitochondrial proteins. Mitochondrial respiratory function was assessed in permeabilized muscle fibers with high-resolution respirometry. Mitochondrial content was assessed with a citrate synthase activity assay. Myocellular signaling was assessed with immunoblotting. Mitochondrial protein synthesis rate was higher with BFRRE (1.19%/day) and HLRE (1.15%/day) compared to CON (0.92%/day) (P < 0.05) but similar between exercise groups. Mitochondrial respiratory function increased to similar degree with both exercise regimens and did not change with CON. For instance, coupled respiration supported by convergent electron flow from complex I and II increased 38% with BFRRE and 24% with HLRE (P < 0.01). Training did not alter citrate synthase activity compared to CON. BFRRE and HLRE elicited similar myocellular signaling responses.These results support recent findings that resistance exercise can stimulate mitochondrial biogenesis and respiratory function to support healthy skeletal muscle and whole-body metabolism. Intriquingly, BFRRE produces similar mitochondrial adaptations at a markedly lower load, which entail great clinical perspective for populations in whom exercise with high loading is untenable.populations in whom exercise with high loading is untenable.)
Chen 2016 Nat Chem Biol + (It is well established that lactate secret … It is well established that lactate secreted by fermenting cells can be oxidized or used as a gluconeogenic substrate by other cells and tissues. It is generally assumed, however, that within the fermenting cell itself, lactate is produced to replenish NAD+ and then is secreted. Here we explore the possibility that cytosolic lactate is metabolized by the mitochondria of fermenting mammalian cells. We found that fermenting HeLa and H460 cells utilize exogenous lactate carbon to synthesize a large percentage of their lipids. Using high-resolution mass spectrometry, we found that both 13C and 2-2H labels from enriched lactate enter the mitochondria. The lactate dehydrogenase (LDH) inhibitor oxamate decreased respiration of isolated mitochondria incubated in lactate, but not of isolated mitochondria incubated in pyruvate. Additionally, transmission electron microscopy (TEM) showed that LDHB localizes to the mitochondria. Taken together, our results demonstrate a link between lactate metabolism and the mitochondria of fermenting mammalian cells.between lactate metabolism and the mitochondria of fermenting mammalian cells.)
Rodriguez 2013 Int J Mol Sci + (It is well established that melatonin exer … It is well established that melatonin exerts antitumoral effects in many cancer types, mostly decreasing cell proliferation at low concentrations. On the other hand, induction of apoptosis by melatonin has been described in the last few years in some particular cancer types. The cytotoxic effect occurs after its administration at high concentrations, and the molecular pathways involved have been only partially determined. Moreover, a synergistic effect has been found in several cancer types when it is administered in combination with chemotherapeutic agents. In the present review, we will summarize published work on the pro-apoptotic effect of melatonin in cancer cells and the reported mechanisms involved in such action. We will also construct a hypothesis on how different cell signaling pathways may relate each other on account for such effect.ate each other on account for such effect.)
Ferraretti 2023 bioRxiv + (It is well established that several ''Homo … It is well established that several ''Homo sapiens'' populations experienced admixture with extinct human species during their evolutionary history. Sometimes, such a gene flow could have played a role in modulating their capability to cope with a variety of selective pressures, thus resulting in archaic adaptive introgression events. A paradigmatic example of this evolutionary mechanism is offered by the EPAS1 gene, whose most frequent haplotype in Himalayan highlanders was proved to reduce their susceptibility to chronic mountain sickness and to be introduced in the gene pool of their ancestors by admixture with Denisovans. In this study, we aimed at further expanding the investigation of the impact of archaic introgression on more complex adaptive responses to hypobaric hypoxia evolved by populations of Tibetan and Sherpa ancestry, which have been plausibly mediated by soft selective sweeps and/or polygenic adaptations rather than by hard selective sweeps. For this purpose, we used a combination of composite-likelihood and gene network-based methods to detect adaptive loci in introgressed chromosomal segments from Tibetan whole genome sequence data and to shortlist those enriched for Denisovan-like derived alleles that participate to the same functional pathways. According to this approach, we identified multiple genes putatively involved in archaic introgression events and that, especially as regards EP300 and NOS2, have plausibly contributed to shape the adaptive modulation of angiogenesis and nitric oxide induction in high-altitude Himalayan peoples. These findings provided unprecedented evidence about the complexity of the adaptive phenotype evolved by these human groups to cope with challenges imposed by hypobaric hypoxia, offering new insights into the tangled interplay of genetic determinants that mediates the physiological adjustments crucial for human adaptation to the high-altitude environment.aptation to the high-altitude environment.)
Joergensen 2015 J Cereb Blood Flow Metab + (It is well known that few weeks of high fa … It is well known that few weeks of high fat (HF) diet may induce metabolic disturbances and mitochondrial dysfunction in skeletal muscle. However, little is known about the effects of long-term HF exposure and effects on brain mitochondria are unknown. Wistar rats were fed either chow (13E% fat) or HF diet (60E% fat) for 1 year. The HF animals developed obesity, dyslipidemia, insulin resistance, and dysfunction of isolated skeletal muscle mitochondria: state 3 and state 4 were 30% to 50% increased (''p''<0.058) with palmitoyl carnitine (PC), while there was no effect with pyruvate as substrate. Adding also succinate in state 3 resulted in a higher substrate control ratio (SCR) with PC, but a lower SCR with pyruvate (''p''<0.05). The P/O2 ratio was lower with PC (''p''<0.004). However, similar tests on isolated brain mitochondria from the same animal showed no changes with the substrates relevant for brain (pyruvate and 3-hydroxybutyrate). Thus, long-term HF diet was associated with obesity, dyslipidemia, insulin resistance, and significantly altered mitochondrial function in skeletal muscle. Yet, brain mitochondria were unaffected. We suggest that the relative isolation of the brain due to the blood-brain barrier may play a role in this strikingly different phenotype of mitochondria from the two tissues of the same animal.notype of mitochondria from the two tissues of the same animal.)
Dos Santos Escaliante 2021 Int J Biol Macromol + (It is well known that the chemical structu … It is well known that the chemical structure of polysaccharides is important to their final biological effect. In this study we investigated the cytotoxic effect of xyloglucan from ''Copaifera langsdorffii'' seeds (XGC) and its complex with oxovanadium (XGC:VO) on hepatocellular carcinoma cells (HepG2). After 72 h of incubation, XGC and XGC:VO (200 μg/mL) reduced cell viability in ~20% and ~40%, respectively. At same conditions, only XGC:VO increased in ~20% the LDH enzyme release. In permeabilized cells, incubated with XGC and XGC:VO (200 μg/mL) for 72 h, NADH oxidase activity was reduced by ~45% with XGC and XGC:VO. The succinate oxidase activity was reduced by ~35% with XGC and ~65% with XGC:VO, evidencing that polysaccharide complexation with vanadium could intensify its effects on the respiratory chain. According to this result, the mitochondrial membrane potential was also reduced by ~9% for XGC and ~30% for XGC:VO, when compared to the control group. Interestingly, ATP levels were more elevated for XGC:VO in respect to XGC, probably due the enhance in glycolytic flux evidenced by increased levels of lactate. These results show that the xyloglucan complexation with oxovanadium (IV/V) potentiates the cytotoxic effect of the native polysaccharide, possibly by impairment of oxidative phosphorylation.y impairment of oxidative phosphorylation.)
Duicu 2013 Can J Physiol Pharmacol + (It is widely recognized that mitochondrial … It is widely recognized that mitochondrial dysfunction is a key component of the multifactorial process of ageing. The effects of age on individual components of mitochondrial function vary across species and strains. In the present study we investigated and compared oxygen consumption, membrane potential (Δψ), the sensitivity of the mitochondrial permeability transition pore (mPTP) to calcium overload and production of reactive oxygen species (ROS) in heart mitochondria isolated from old vs. adult healthy Sprague-Dawley (SD) rats.Respirometry studies and Δψ measurements were performed with the Oxygraph-2k (Oroboros, Austria) equipped with a tetraphenylphosphonium electrode. ROS production and calcium retention capacity (CRC) were measured spectrofluorimetrically.Our results showed an important decline for all bioenergetic parameters for both Complex I and Complex II supported-respiration, a decreased Δ''ψ'' in mitochondria energized with Complex I substrates and an increased mitochondrial ROS production in old vs. the adult group. Mitochondrial sensitivity to Ca2+-induced mtPTP opening was also increased in the old vs. adult animals. Moreover, the protective effect of cyclosporine A on mtPTP opening was significantly reduced in the old group.Healthy ageing is associated with heart mitochondria dysfunction in Sprague Dawley rats.ondria dysfunction in Sprague Dawley rats.)
Baker 2016b Nature + (It may not be sexy, but quality assurance is becoming a crucial part of lab life. © 2016 Macmillan Publishers Limited. All rights reserved )
Chance 1962 Science + (It now appears to be possible to continuou … It now appears to be possible to continuously record changes in intracellular oxidation-reduction levels in terms of the fluorescence of reduced pyridine nucleotide in mitochondria of various tissues and organs in situ. Studies of kidney and brain cortex in the rat show that changes in fluorescence are not measurably affected by the presence of oxyhemoglobin. Nitrogen, sulfide, cyanide, and carbon monoxide cause increases in fluorescence to very nearly the same levels, and the increases are attributed to larger reduction of mitochondrial diphosphopyridine nucleotide. Amytal at a low blood concentration causes increased reduction in the kidney cortex, and at a high blood concentration, in the brain cortex. The qualitative response of the pyridine nucleotide to low oxygen concentrations shows the brain to be more sensitive than the kidney. The first measurable increase in pyridine nucleotide reduction observed on the brain occurs at a concentration of inspired oxygen of 8 percent. Breathing stops when the percentage increase of pyridine nucleotide reduction on the brain reaches about 90; at this point the percentage increase for the kidney is only about 30. This difference corresponds roughly to a tenfold difference in oxygen tension. Half-maximal increase in pyridine nucleotide reduction on the brain occurs at a concentration of inspired oxygen of about 4 percent and corresponds to an intracellular oxygen tension of about 0.2 mm.racellular oxygen tension of about 0.2 mm.)
Robach 2012 Br J Sports Med + (It remains unclear by which mechanism 'liv … It remains unclear by which mechanism 'live high-train low' (LHTL) altitude training increases exercise performance. Haematological and skeletal muscle adaptations have both been proposed. To test the hypotheses that (i) LHTL improves maximal oxygen uptake (VO2max ) and (ii) this improvement is related to hypoxia-induced increases in total haemoglobin mass (Hb(mass)) and not to improved maximal oxidative capacity of skeletal muscle, we determined VO2max before LHTL and after LHTL, before and after the altitude-induced increases in Hb(mass) (measured by carbon-monoxide rebreathing) had been abolished by isovolumic haemodilution. We obtained skeletal muscle biopsies to quantify mitochondrial oxidative capacity and efficiency. Sixteen endurance-trained athletes were assigned (double-blinded, placebo controlled) to ≥16;h/day over 4;weeks to normoxia (placebo, n=6) or normobaric hypoxia equivalent to 3000;m altitude (LHTL, n=10). Four-week LHTL did not increase VO2max , irrespective of treatment (LHTL: 1.5%; placebo: 2.0%). Hb(mass) was slightly increased (4.6%) in 5 (of 10) LHTL subjects but this was not accompanied by a concurrent increase in VO2max . In the subjects demonstrating an increase in Hb(mass), isovolumic haemodilution elicited a 5.8% decrease in VO2max . Cycling efficiency was altered neither with time nor by LHTL. Neither maximal capacity of oxidative phosphorylation nor mitochondrial efficiency was modified by time or LHTL. The present results suggest that LHTL has no positive effect on VO2max in endurance-trained athletes because (i) muscle maximal oxidative capacity is not improved following LHTL and (ii) erythrocyte volume expansion after LHTL, if any, is too small to alter O2 transport.ing LHTL and (ii) erythrocyte volume expansion after LHTL, if any, is too small to alter O2 transport.)
Trewin 2018 Am J Physiol Regul Integr Comp Physiol + (It remains unclear whether high-intensity … It remains unclear whether high-intensity interval exercise (HIIE) elicits distinct molecular responses to traditional endurance exercise relative to the total work performed. We aimed to investigate the influence of exercise intensity on acute perturbations to skeletal muscle mitochondrial function (respiration and reactive oxygen species), metabolic and redox signaling responses. In a randomized, repeated measures crossover design, eight recreationally active individuals (24 ± 5 years; VO2peak 48 ± 11 mL.kg-1.min-1) undertook continuous moderate-intensity (CMIE: 30 min, 50% peak power output [PPO]), high-intensity interval (HIIE: 5x4 min, 75% PPO, work-matched to CMIE), and low-volume sprint interval (SIE: 4x30 s) exercise, ≥7 days apart. Each session included muscle biopsies at baseline, immediately and 3 h post-exercise for high-resolution mitochondrial respirometry (JO2) and H2O2 emission (JH2O2), gene and protein expression analysis. Immediately post-exercise and irrespective of protocol, JO2 increased during complex I+II leak/state-4 respiration but JH2O2 decreased (p<0.05). AMP-activated protein kinase (AMPK) and acetyl co-A carboxylase (ACC) phosphorylation increased ~1.5 and 2.5-fold respectively, while thioredoxin-reductase-1 protein abundance was ~35% lower after CMIE vs. SIE (p<0.05). At 3 hours post-exercise, regardless of protocol, JO2 was lower during both ADP-stimulated state-3 OXPHOS and uncoupled respiration (p<0.05) but JH2O2 trended higher (p<0.08); PPARGC1A mRNA increased ~13-fold, and peroxiredoxin-1 protein decreased ~35%. In conclusion, intermittent exercise performed at high intensities has similar dynamic effects on muscle mitochondrial function compared with endurance exercise, irrespective of whether total workload is matched. This suggests exercise prescription can accommodate individual preferences while generating comparable molecular signals known to promote beneficial metabolic adaptations.irrespective of whether total workload is matched. This suggests exercise prescription can accommodate individual preferences while generating comparable molecular signals known to promote beneficial metabolic adaptations.)
Holloway 2018 Cell Rep + (It remains unknown if mitochondrial bioene … It remains unknown if mitochondrial bioenergetics are altered with aging in humans. We established an ''in vitro'' method to simultaneously determine mitochondrial respiration and H2O2 emission in skeletal muscle tissue across a range of biologically relevant ADP concentrations. Using this approach, we provide evidence that, although the capacity for mitochondrial H2O2 emission is not increased with aging, mitochondrial ADP sensitivity is impaired. This resulted in an increase in mitochondrial H2O2 and the fraction of electron leak to H2O2, in the presence of virtually all ADP concentrations examined. Moreover, although prolonged resistance training in older individuals increased muscle mass, strength, and maximal mitochondrial respiration, exercise training did not alter H2O2 emission rates in the presence of ADP, the fraction of electron leak to H2O2, or the redox state of the muscle. These data establish that a reduction in mitochondrial ADP sensitivity increases mitochondrial H2O2 emission and contributes to age-associated redox stress.muscle. These data establish that a reduction in mitochondrial ADP sensitivity increases mitochondrial H2O2 emission and contributes to age-associated redox stress.)
Cook 2013 J Exp Biol + (It was hypothesised that chronic hypoxia a … It was hypothesised that chronic hypoxia acclimation (preconditioning) would alter the behavioural low-O2 avoidance strategy of fish as a result of both aerobic and anaerobic physiological adaptations. Avoidance and physiological responses of juvenile snapper (''Pagrus auratus'') were therefore investigated following a 6 week period of moderate hypoxia exposure (10.2–12.1 kPa PO2, 21±1°C) and compared with those of normoxic controls (PO2=20–21 kPa, 21±1°C). The critical oxygen pressure (Pcrit) limit of both groups was unchanged at ~7 kPa, as were standard, routine and maximum metabolic rates. However, hypoxia-acclimated fish showed increased tolerances to hypoxia in behavioural choice chambers by avoiding lower PO2 levels (3.3±0.7 vs 5.3±1.1 kPa) without displaying greater perturbations of lactate or glucose. This behavioural change was associated with unexpected physiological adjustments. For example, a decrease in blood O2 carrying capacity was observed after hypoxia acclimation. Also unexpected was an increase in whole-blood P50 following acclimation to low-O2, perhaps facilitating Hb–O2 off-loading to tissues. In addition, cardiac mitochondria measured ''in situ'' using permeabilised fibres showed improved O2 uptake efficiencies. The proportion of the anaerobic enzyme lactate dehydrogenase, at least relative to the aerobic marker enzyme citrate synthase, also increased in heart and skeletal red muscle, indicating enhanced anaerobic potential, or ''in situ'' lactate metabolism, in these tissues. Overall, these data suggest that a prioritization of O2 delivery and O2 utilisation over O2 uptake during long-term hypoxia may convey a significant survival benefit to snapper in terms of behavioural low-O2 tolerance.;2 delivery and O2 utilisation over O2 uptake during long-term hypoxia may convey a significant survival benefit to snapper in terms of behavioural low-O2 tolerance.)
Galina 2013 Abstract MiP2013 + (It was proposed that the alkylating agent … It was proposed that the alkylating agent 3-bromopyruvate (3-BrPA) could act as an antitumor agent in different cell lines of hepatocellular carcinoma mainly by targeting the mitochondrial hexokinase type 2 that is overexpressed in many tumor cells. Several revisions of drug therapy for cancer treatment have taken this as the main mechanism of action. Despite the potent negative effects of 3-BrPA on cell viability of the tumors, the analogue of pyruvate/lactate alkyl is oxidizing, as expected, other enzymes in energy transducing metabolism in tumor cells. However, little attention has been given to these side effects of 3-BrPA on tumor mitochondria, glycolysis and calcium pump SERCA. A dataset of high-resolution respirometry, analysis of flux, recovery of enzyme activities and metabolomics evaluated by our group in human hepatoma HepG2, isolated liver mitochondria and activities measured of calcium transport in sarcoplasmic reticulum vesicles mediated by SERCA, point to different metabolic targets with significant implications for the mechanism of cell death in tumor. Among the enzymes as targets we list the main ones: monocarboxylate transporter (MCT), glyceraldehyde dehydrogenase (GA3PDH); 3-phosphoglycerate kinase (3PGK); succinate dehydrogenase (SDH); pyruvate dehydrogenase (PDH); glutamate dehydrogenase (GDH); malate dehydrogenase (MDH) and SERCA 2a. Interestingly, mt-HK 1 and 2 are not significantly inhibited by 3-BrPA, but on the contrary contribute to depletion of the cytosolic ATP pool of the ATP-consuming path of glycolysis. Importantly, the mt-HK acts by modulating the rate of oxidative phosphorylation putting succinate dehydrogenase in a state of greater reactivity and inhibition by 3-BrPA. Given these observations we postulate that the mitochondrial hexokinase is not the primary molecular target of tumor cells but a potent depletory agent of cellular ATP and modulator of succinate dehydrogenase inhibition in mitochondrial supported respiration and inducer of permeability transition pore formation involved in cell death in tumor cells.ion involved in cell death in tumor cells.)
Chinopoulos 2011 J Neurosci Res + (It was recently shown that, in progressive … It was recently shown that, in progressively depolarizing mitochondria, the F(0) -F(1) ATP synthase and the adenine nucleotide translocase (ANT) may change directionality independently from each other (Chinopoulos et al. [2010] FASEB J. 24:2405). When the membrane potentials at which these two molecular entities reverse directionality, termed reversal potential (Erev), are plotted as a function of matrix ATP/ADP ratio, an area of the plot is bracketed by the Erev_ATPase and the Erev_ANT, which we call "B space". Both reversal potentials are dynamic, in that they depend on the fluctuating values of the participating reactants; however, Erev_ATPase is almost always more negative than Erev_ANT. Here we review the conditions that define the boundaries of the "B space". Emphasis is placed on the role of matrix substrate-level phosphorylation, because during metabolic compromise this mechanism could maintain mitochondrial membrane potential and prevent the influx of cytosolic ATP destined for hydrolysis by the reversed F(0) -F(1) ATP synthase.s by the reversed F(0) -F(1) ATP synthase.)
West 1996 J Appl Physiol + (It would be valuable to have model atmosph … It would be valuable to have model atmospheres that allow barometric pressures (PB) to be predicted at high altitudes. Attempts to do this in the past using the International Civil Aviation Organizations or United States Standard Atmosphere model have brought such models into disrepute because the predicted pressures at high altitudes are usually much too low. However, other model atmospheres have been developed by geophysicists. The critical variable is the change of air temperature with altitude, and, therefore, model atmospheres have been constructed for different latitudes and seasons of the year. These different models give a large range of pressures at a given altitude. For example, the maximum difference of pressure at an altitude of 9 km is from 206 to 248 Torr, i.e., approximately 20%. However, the mean of the model atmospheres for latitude of 15 degrees (in all seasons) and 30 degrees (in the summer) predicts PB at many locations of interest at high altitude very well, with predictions within 1%. The equation is PB (Torr) = exp (6.63268 - 0.1112 h - 0.00149 h2), were h is the altitude in kilometers. The predictions are good because many high mountain sites are within 30 degrees of the equator and also many studies are made during the summer. Other models should be used for latitudes of 45 degrees and above. Model atmospheres have considerable value in predicting PB at high altitude if proper account is take of latitude and season of the year.er account is take of latitude and season of the year.)
Nemeth 2016 FASEB J + (Itaconate is a nonamino organic acid exhib … Itaconate is a nonamino organic acid exhibiting antimicrobial effects. It has been recently identified in cells of macrophage lineage as a product of an enzyme encoded by immunoresponsive gene 1 (Irg1), acting on the citric acid cycle intermediate cis-aconitate. In mitochondria, itaconate can be converted by succinate-coenzyme A (CoA) ligase to itaconyl-CoA at the expense of ATP (or GTP), and is also a weak competitive inhibitor of complex II. Here, we investigated specific bioenergetic effects of increased itaconate production mediated by LPS-induced stimulation of Irg1 in murine bone marrow-derived macrophages (BMDM) and RAW-264.7 cells. In rotenone-treated macrophage cells, stimulation by LPS led to impairment in substrate-level phosphorylation (SLP) of ''in situ'' mitochondria, deduced by a reversal in the directionality of the adenine nucleotide translocase operation. In RAW-264.7 cells, the LPS-induced impairment in SLP was reversed by short-interfering RNA(siRNA)-but not scrambled siRNA-treatment directed against Irg1. LPS dose-dependently inhibited oxygen consumption rates (61-91%) and elevated glycolysis rates (>21%) in BMDM but not RAW-264.7 cells, studied under various metabolic conditions. In isolated mouse liver mitochondria treated with rotenone, itaconate dose-dependently (0.5-2 mM) reversed the operation of adenine nucleotide translocase, implying impairment in SLP, an effect that was partially mimicked by malonate. However, malonate yielded greater ADP-induced depolarizations (3-19%) than itaconate. We postulate that itaconate abolishes SLP due to 1) a "CoA trap" in the form of itaconyl-CoA that negatively affects the upstream supply of succinyl-CoA from the α-ketoglutarate dehydrogenase complex; 2) depletion of ATP (or GTP), which are required for the thioesterification by succinate-CoA ligase; and 3) inhibition of complex II leading to a buildup of succinate which shifts succinate-CoA ligase equilibrium toward ATP (or GTP) utilization. Our results support the notion that Irg1-expressing cells of macrophage lineage lose the capacity of mitochondrial SLP for producing itaconate during mounting of an immune defense.aconate during mounting of an immune defense.)
Belosludtsev 2020 Biochimie + (Itaconic acid (methylene-succinic acid, It … Itaconic acid (methylene-succinic acid, ItA) is an unsaturated dicarboxylic acid that is secreted by mammalian macrophages in response to a pro-inflammatory stimulus and shows an anti-inflammatory/antibacterial effect. Being a mitochondrial metabolite, it exhibits an inhibitory activity on succinate dehydrogenase and subsequently induces mitochondrial dysfunction. The present study has shown that ItA dose-dependently inhibited ADP- and DNP-stimulated (uncoupled) respiration of rat liver mitochondria energized with succinate. This effect of ItA could be related to the suppression of the activity of complex II and the combined activity of complexes II + III of the respiratory chain. At the same time, ItA had no effect on the activity of the dicarboxylate carrier, which catalyzes the transport of succinate across the inner mitochondrial membrane. It was found that 4 mM ItA diminished the rates of ADP- and DNP-stimulated mitochondrial respiration supported by the substrates of complex I glutamate and malate. A study of the effect of ItA on the activity of complexes of the respiratory chain showed that it significantly decreases the activity of complex IV. It was observed that 4 mM ItA inhibited the rate of H2O2 production by mitochondria. At the same time, ItA promoted the opening of the cyclosporin A-sensitive Ca2+-dependent permeability transition pore. The latter was revealed as the decrease in the calcium retention capacity of mitochondria and the stimulation of release of cytochrome c from the organelles. ItA by itself promotes the cytochrome c release from mitochondria. Possible mechanisms of the effect of ItA on mitochondrial function are discussed.sible mechanisms of the effect of ItA on mitochondrial function are discussed.)
Nemeth 2017 Thesis + (Itaconic acid, matrix substrate-level phos … Itaconic acid, matrix substrate-level phosphorylation (SLP) and macrophages represent the main focus of this thesis. From a more general point of view, it is about immune cell specific metabolism.Usually, metabolism is viewed as the function of cells generating a store of energy by catabolism, and to synthesizing macromolecules for cell maintenance and growth through anabolic pathways. However, today we know that there are some disorders, such as diabetes, atherosclerosis, cancer, inflammatory conditions, in which there are obvious dysfunctions in metabolism....re obvious dysfunctions in metabolism. ...)
Reynolds 2016 Am J Physiol Endocrinol Metab + (Iβ-cell insulin secretion is dependent on … Iβ-cell insulin secretion is dependent on proper mitochondrial function. Various studies have clearly shown that the Nr4a family of orphan nuclear receptors is essential for fuel utilization and mitochondrial function in liver, muscle and adipose. We have previously demonstrated that overexpression of Nr4a1 or Nr4a3 is sufficient to induce proliferation of pancreatic β-cells. In this study we examined whether Nr4a expression impacts pancreatic β-cell mitochondrial function. Here we show that β-cell mitochondrial respiration is dependent on the nuclear receptors Nr4a1 and Nr4a3. Mitochondrial respiration in permeabilized cells was significantly decreased in β-cells lacking Nr4a1 or Nr4a3. Furthermore, respiration rates of intact cells deficient for Nr4a1 or Nr4a3 in the presence of 16mM glucose resulted in decreased glucose mediated oxygen consumption. Consistent with this reduction in respiration, a significant decrease in glucose stimulated insulin secretion rates is observed with deletion of Nr4a1 or Nr4a3. Interestingly, the changes in respiration and insulin secretion occur without a reduction in mitochondrial content, suggesting decreased mitochondrial function. We establish that knockdown of Nr4a1 and Nr4a3 results in decreased expression of the mitochondrial dehydrogenase subunits Idh3g and Sdhb. We demonstrate that loss of Nr4a1 and Nr4a3 impedes production of ATP, and ultimately inhibits glucose stimulated insulin secretion. These data demonstrate for the first time that the orphan nuclear receptors Nr4a1 and Nr4a3 are critical for β-cell mitochondrial function and insulin secretion.Copyright © 2016, American Journal of Physiology - Endocrinology and Metabolism.Physiology - Endocrinology and Metabolism.)
JASIS 2017 Chiba JP + (JASIS - Japan Analytical & Scientific Instruments Show, Chiba, Japan)
Weir 2005 N Engl J Med + (JOSEPH PRIESTLEY, ONE OF THE THREE SCIENTI … JOSEPH PRIESTLEY, ONE OF THE THREE SCIENTISTS CREDITED WITH THEdiscovery of oxygen, described the death of mice that were deprived of oxygen. However, hewas also well aware of the toxicity of too much oxygen, stating, “For as a candle burns muchfaster in dephlogisticated [oxygen-enriched] than in common air, so we might live out too fast,and the animal powers be too soon exhausted in this pure kind of air. A moralist, at least, maysay, that the air which nature has provided for us is as good as we deserve.”1In this review we examine the remarkable mechanisms by which different organs detect andrespond to acute changes in oxygen tension. Specialized tissues that sense the local oxygentension include glomus cells of the carotid body, neuroepithelial bodies in the lungs, chromaffincells of the fetal adrenal medulla, and smooth-muscle cells of the resistance pulmonary arteries,fetoplacental arteries, systemic arteries, and the ductus arteriosus. Together, they constitute aspecialized homeostatic oxygen-sensing system. Although all tissues are sensitive to severehypoxia, these specialized tissues respond rapidly to moderate changes in oxygen tensionwithin the physiologic range (roughly 40 to 100 mm Hg in an adult and 20 to 40 mm Hg in afetus) (Fig. 1).t and 20 to 40 mm Hg in a fetus) (Fig. 1).)
Indian Academy of Pediatrics Growth Charts Committee 2015 Indian Pediatr + (JUSTIFICATION: The need to revise Indian A … JUSTIFICATION: The need to revise Indian Academy of Pediatrics (IAP) growth charts for 5- to 18-year-old Indian children and adolescents was felt as India is in nutrition transition and previous IAP charts are based on data which are over two decades old.PROCESS: The Growth Chart Committee was formed by IAP in January 2014 to design revised growth charts. Consultative meeting was held in November 2014 in Mumbai. Studies performed on Indian children's growth, nutritional assessment and anthropometry from upper and middle socioeconomic classes in last decade were identified. Committee contacted 13 study groups; total number of children in the age group of 5 to 18 years were 87022 (54086 boys). Data from fourteen cities (Agartala, Ahmadabad, Chandigarh, Chennai, Delhi, Hyderabad, Kochi, Kolkata, Madurai, Mumbai, Mysore, Pune, Raipur and Surat) in India were collated. Data of children with weight for height Z scores >2 SD were removed from analyses. Data on 33148 children (18170 males, 14978 females) were used to construct growth charts using Cole's LMS method.OBJECTIVE: To construct revised IAP growth charts for 5-18 year old Indian children based on collated national data from published studies performed on apparently healthy children and adolescents in the last 10 years.RECOMMENDATIONS: The IAP growth chart committee recommends these revised growth charts for height, weight and body mass index (BMI) for assessment of growth of 5-18 year old Indian children to replace the previous IAP charts; rest of the recommendations for monitoring height and weight remain as per the IAP guidelines published in 2007. To define overweight and obesity in children from 5-18 years of age, adult equivalent of 23 and 27 cut-offs presented in BMI charts may be used. IAP recommends use of WHO standards for growth assessment of children below 5 years of age. assessment of children below 5 years of age.)
Chow 2016 Photosynth Res + (Joan Mary Anderson, known to most people a … Joan Mary Anderson, known to most people as Jan, was born on May 12, 1932 in Dunedin, New Zealand. She died on August 28, 2015 in Canberra, Australia. To celebrate her life, we present here a brief biography, some comments on her discoveries in photosynthesis during a career spanning more than half a century, and reminiscences from family and friends. We remember this wonderful person who had an unflagging curiosity, creative ability to think laterally, enthusiasm, passion, generosity and love of color and culture. generosity and love of color and culture.)

ASMRM & J-mit 2019 Fukuoka JP + (Joint ASMRM and J-mit Conference, Fukuoka, Japan, 2019)

Yamada 2016 J Physiol + (KEY POINTS: Mitochondrial respiration is r … KEY POINTS:Mitochondrial respiration is regulated by multiple elaborate mechanisms. It has been shown that muscle specific O2 binding protein, Myoglobin (Mb), is localized in mitochondria and interacts with respiratory chain complex IV, suggesting that Mb could be a factor that regulates mitochondrial respiration. Here, we demonstrate that muscle mitochondrial respiration is improved by Mb overexpression via up-regulation of complex IV activity in cultured myoblasts; in contrast, suppression of Mb expression induces a decrease in complex IV activity and mitochondrial respiration compared with the overexpression model. The present data are the first to show the biological significance of mitochondrial Mb as a potential modulator of mitochondrial respiratory capacity.ABSTRACT:Mitochondria are important organelles for metabolism, and their respiratory capacity is a primary factor in the regulation of energy expenditure. Deficiencies of cytochrome c oxidase complex IV, which reduces O2 in mitochondria, are linked to several diseases, such as mitochondrial myopathy. Moreover, mitochondrial respiration in skeletal muscle tissue tends to be susceptible to complex IV activity. Recently, we showed that the muscle-specific protein myoglobin (Mb) interacts with complex IV. The precise roles of mitochondrial Mb remain unclear. Here, we demonstrate that Mb facilitates mitochondrial respiratory capacity in skeletal muscles. Although mitochondrial DNA copy numbers were not altered in Mb-overexpressing myotubes, O2 consumption was greater in these myotubes than that in mock cells (Mock vs. Mb-Flag::GFP: state 4, 1.00 ± 0.09 vs. 1.77 ± 0.34; state 3, 1.00 ± 0.29; Mock: 1.60 ± 0.53; complex 2-3-4: 1.00 ± 0.30 vs. 1.50 ± 0.44; complex IV: 1.00 ± 0.14 vs. 1.87 ± 0.27). This improvement in respiratory capacity could be because of the activation of enzymatic activity of respiratory complexes. Moreover, mitochondrial respiration was up-regulated in myoblasts transiently overexpressing Mb; complex IV activity was solely activated in Mb-overexpressing myoblasts, and complex IV activity was decreased in the myoblasts in which Mb expression was suppressed by Mb-siRNA transfection (Mb vector transfected vs. Mb vector, control siRNA transfected vs. Mb vector, Mb siRNA transfected: 0.15 vs. 0.15 vs. 0.06). Therefore, Mb enhances the enzymatic activity of complex IV to ameliorate mitochondrial respiratory capacity, and could play a pivotal role in skeletal muscle metabolism.iratory capacity, and could play a pivotal role in skeletal muscle metabolism.)
Montero 2015 J Physiol + (KEY POINTS: This study assessed the respec … KEY POINTS:This study assessed the respective contributions of haematological and skeletal muscle adaptations to any observed improvement in peak oxygen uptake (VO2 peak ) induced by endurance training (ET). VO2 peak , peak cardiac output (Q̇ peak ), blood volumes and skeletal muscle biopsies were assessed prior (pre) to and after (post) 6 weeks of ET. Following the post-ET assessment, red blood cell volume (RBCV) reverted to the pre-ET level following phlebotomy and VO2 peak and Q̇ peak were determined again. We speculated that the contribution of skeletal muscle adaptations to an ET-induced increase in VO2 peak could be identified when offsetting the ET-induced increase in RBCV. VO2 peak , Q̇ peak , blood volumes, skeletal muscle mitochondrial volume density and capillarization were increased after ET. Following RBCV normalization, VO2 peak and Q̇ peak reverted to pre-ET levels. These results demonstrate the predominant contribution of haematological adaptations to any increase in VO2 peak induced by ET.ABSTRACT:It remains unclear whether improvements in peak oxygen uptake (V̇O2 peak ) following endurance training (ET) are primarily determined by central and/or peripheral adaptations. Herein, we tested the hypothesis that the improvement in V̇O2 peak following 6 weeks of ET is mainly determined by haematological rather than skeletal muscle adaptations. Sixteen untrained healthy male volunteers (age = 25 ± 4 years, V̇O2 peak = 3.5 ± 0.5 l min-1 ) underwent supervised ET (6 weeks, 3-4 sessions per week). V̇O2 peak , peak cardiac output (Q̇ peak ), haemoglobin mass (Hbmass ) and blood volumes were assessed prior to and following ET. Skeletal muscle biopsies were analysed for mitochondrial volume density (MitoVD ), capillarity, fibre types and respiratory capacity (OXPHOS). After the post-ET assessment, red blood cell volume (RBCV) was re-established at the pre-ET level by phlebotomy and V̇O2 peak and Q̇ peak were measured again. We speculated that the contribution of skeletal muscle adaptations to the ET-induced increase in V̇O2 peak would be revealed when controlling for haematological adaptations. V̇O2 peak and Q̇ peak were increased (P < 0.05) following ET (9 ± 8 and 7 ± 6%, respectively) and decreased (P < 0.05) after phlebotomy (-7 ± 7 and -10 ± 7%). RBCV, plasma volume and Hbmass all increased (P < 0.05) after ET (8 ± 4, 4 ± 6 and 6 ± 5%). As for skeletal muscle adaptations, capillary-to-fibre ratio and total MitoVD increased (P < 0.05) following ET (18 ± 16 and 43 ± 30%), but OXPHOS remained unaltered. Through stepwise multiple regression analysis, Q̇ peak , RBCV and Hbmass were found to be independent predictors of V̇O2 peak . In conclusion, the improvement in V̇O2 peak following 6 weeks of ET is primarily attributed to increases in Q̇ peak and oxygen-carrying capacity of blood in untrained healthy young subjects.g 6 weeks of ET is primarily attributed to increases in Q̇ peak and oxygen-carrying capacity of blood in untrained healthy young subjects.)
Airik 2023 Antioxidants (Basel) + (Karyomegalic interstitial nephritis (KIN) … Karyomegalic interstitial nephritis (KIN) is a genetic adult-onset chronic kidney disease (CKD) characterized by genomic instability and mitotic abnormalities in the tubular epithelial cells. KIN is caused by recessive mutations in the FAN1 DNA repair enzyme. However, the endogenous source of DNA damage in FAN1/KIN kidneys has not been identified. Here we show, using FAN1-deficient human renal tubular epithelial cells (hRTECs) and FAN1-null mice as a model of KIN, that FAN1 kidney pathophysiology is triggered by hypersensitivity to endogenous reactive oxygen species (ROS), which cause chronic oxidative and double-strand DNA damage in the kidney tubular epithelial cells, accompanied by an intrinsic failure to repair DNA damage. Furthermore, persistent oxidative stress in FAN1-deficient RTECs and FAN1 kidneys caused mitochondrial deficiencies in oxidative phosphorylation and fatty acid oxidation. The administration of subclinical, low-dose cisplatin increased oxidative stress and aggravated mitochondrial dysfunction in FAN1-deficient kidneys, thereby exacerbating KIN pathophysiology. In contrast, treatment of FAN1 mice with a mitochondria-targeted ROS scavenger, JP4-039, attenuated oxidative stress and accumulation of DNA damage, mitigated tubular injury, and preserved kidney function in cisplatin-treated FAN1-null mice, demonstrating that endogenous oxygen stress is an important source of DNA damage in FAN1-deficient kidneys and a driver of KIN pathogenesis. Our findings indicate that therapeutic modulation of kidney oxidative stress may be a promising avenue to mitigate FAN1/KIN kidney pathophysiology and disease progression in patients.ology and disease progression in patients.)
Frey 2016 Biochim Biophys Acta + (Ketogenic Diet used to treat refractory ep … Ketogenic Diet used to treat refractory epilepsy for almost a century may represent a treatment option for mitochondrial disorders for which effective treatments are still lacking. Mitochondrial complex I deficiencies are involved in a broad spectrum of inherited diseases including Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes syndrome leading to recurrent cerebral insults resembling strokes and associated with a severe complex I deficiency caused by mitochondrial DNA (mtDNA) mutations. The analysis of MELAS neuronal cybrid cells carrying the almost homoplasmic m.3243A>G mutation revealed a metabolic switch towards glycolysis with the production of lactic acid, severe defects in respiratory chain activity and complex I disassembly with an accumulation of assembly intermediates. Metabolites, NADH/NAD+ ratio, mitochondrial enzyme activities, oxygen consumption and BN-PAGE analysis were evaluated in mutant compared to control cells. A severe complex I enzymatic deficiency was identified associated with a major complex I disassembly with an accumulation of assembly intermediates of 400kDa. We showed that Ketone Bodies (KB) exposure for 4weeks associated with glucose deprivation significantly restored complex I stability and activity, increased ATP synthesis and reduced the NADH/NAD+ ratio, a key component of mitochondrial metabolism. In addition, without changing the mutant load, mtDNA copy number was significantly increased with KB, indicating that the absolute amount of wild type mtDNA copy number was higher in treated mutant cells. Therefore KB may constitute an alternative and promising therapy for MELAS syndrome, and could be beneficial for other mitochondrial diseases caused by complex I deficiency.Copyright © 2016 Elsevier B.V. All rights reserved.ex I deficiency. Copyright © 2016 Elsevier B.V. All rights reserved.)
Geffroy 2018 Biochim Biophys Acta + (Ketogenic diet (KD) which combined carbohy … Ketogenic diet (KD) which combined carbohydrate restriction and the addition of ketone bodies has emerged as an alternative metabolic intervention used as an anticonvulsant therapy or to treat different types of neurological or mitochondrial disorders including MELAS syndrome. MELAS syndrome is a severe mitochondrial disease mainly due to the m.3243A > G mitochondrial DNA mutation. The broad success of KD is due to multiple beneficial mechanisms with distinct effects of very low carbohydrates and ketones. To evaluate the metabolic part of carbohydrate restriction, transmitochondrial neuronal-like cybrid cells carrying the m.3243A > G mutation, shown to be associated with a severe complex I deficiency was exposed during 3 weeks to glucose restriction. Mitochondrial enzyme defects were combined with an accumulation of complex I (CI) matrix intermediates in the untreated mutant cells, leading to a drastic reduction in CI driven respiration. The severe reduction of CI was also paralleled in post-mortem brain tissue of a MELAS patient carrying high mutant load. Importantly, lowering significantly glucose concentration in cell culture improved CI assembly with a significant reduction of matrix assembly intermediates and respiration capacities were restored in a sequential manner. In addition, OXPHOS protein expression and mitochondrial DNA copy number were significantly increased in mutant cells exposed to glucose restriction. The accumulation of CI matrix intermediates appeared as a hallmark of MELAS pathophysiology highlighting a critical pathophysiological mechanism involving CI disassembly, which can be alleviated by lowering glucose fuelling and the induction of mitochondrial biogenesis, emphasizing the usefulness of metabolic interventions in MELAS syndrome.ss of metabolic interventions in MELAS syndrome.)
Dilliraj 2022 Nutrients + (Ketone bodies are small compounds derived … Ketone bodies are small compounds derived from fatty acids that behave as an alternative mitochondrial energy source when insulin levels are low, such as during fasting or strenuous exercise. In addition to the metabolic function of ketone bodies, they also have several signaling functions separate from energy production. In this perspective, we review the main current data referring to ketone bodies in correlation with nutrition and metabolic pathways as well as to the signaling functions and the potential impact on clinical conditions. Data were selected following eligibility criteria accordingly to the reviewed topic. We used a set of electronic databases (Medline/PubMed, Scopus, Web of Sciences (WOS), Cochrane Library) for a systematic search until July 2022 using MeSH keywords/terms (i.e., ketone bodies, BHB, acetoacetate, inflammation, antioxidant, etc.). The literature data reported in this review need confirmation with consistent clinical trials that might validate the results obtained in in vitro and in vivo in animal models. However, the data on exogenous ketone consumption and the effect on the ketone bodies' brain uptake and metabolism might spur the research to define the acute and chronic effects of ketone bodies in humans and pursue the possible implication in the prevention and treatment of human diseases. Therefore, additional studies are required to examine the potential systemic and metabolic consequences of ketone bodies.d metabolic consequences of ketone bodies.)
Wojtala 2014 Abstract MiP2014 + (Key mitochondrial energy-providing reactio … Key mitochondrial energy-providing reactions are carried out by the oxidative phosphorylation system (OXPHOS), involving the electron transfer and phosphorylation systems including F1Fo-ATP synthase. The most common OXPHOS disorder in humans is associated with Complex I deficiency, leading to fatal encephalomyopathies of early childhood- Leigh-like syndrome [1]. The growth factor adaptor protein p66Shc has a substantial impact on mitochondrial metabolism through regulation of cellular responses to oxidative stress. A low level of p66Shc protein or its complete ablation protects against numerous age-related disorders and may partially prevent pathologies caused by reactive oxygen species (ROS). On the other hand, a high level of p66Shc phosphorylation is correlated with increased intracellular ROS production [2,3].Organs from NDUFS4-/- mice with Complex I deficiency were used as a model of self-propelling intracellular oxidative stress. The status of the antioxidant defense system, oxidative stress markers and the p66Shc-Ser36 phosphorylation pathway were measured in these tissues. Mass spectrometry analysis was also performed for selected NDUFS4-/- mouse tissues. In our study, mice with defective Complex I were characterized by attenuated intracellular oxidative stress, connected with increased p66Shc phosphorylation. Mass spectrometry revealed aberrations in the level of Complex I proteins and oxidative stress- related proteins, as well as other proteins involved in metabolic processes.ative stress- related proteins, as well as other proteins involved in metabolic processes.)
Ortiz-Jimenez 2019 MethodsX + (Key mitochondrial processes are known to b … Key mitochondrial processes are known to be widely conserved throughout the eukaryotic domain. However, the scarce availability of working materials may restrict the assessment of such mitochondrial activities in several working models. Pollen tube mitochondrial studies represent one example of this, where tests have been often restricted due the physical impossibility of performing experiments with isolated mitochondria in enough quantities. Here we detail a method to measure ''in situ'' mitochondrial respiratory chain activity and calcium transport in tobacco pollen tubes. Digitonin-mediated plasmalemma permeabilization allows efficient assessment of mitochondrial respiration and calcium uptake. This method allows quick, reliable and portable measurements from low to high cellular densities, versus methods requiring intracellular calcium reporters.requiring intracellular calcium reporters.)
Keystone Symposia 2014 + (Keystone Symposia - Mitochondrial Dynamics and Physiology (Q5), Santa Fee, New Mexico, USA [http://www.keystonesymposia.org/index.cfm?e=web.meeting.program&meetingid=1266 Keystone Symposia 2014])
Keystone Symposia 2015 + (Keystone Symposia - Obesity and the Metabolic Syndrome: Mitochondria and Energy Expenditure (X7) Whistler, CA [http://www.keystonesymposia.org/15X7 Keystone Symposia 2015])
Keystone symposium: “Hypoxia: from basic mechanisms to therapeutics” + (Keystone Symposium: “Hypoxia: from basic mechanisms to therapeutics” Dublin, Ireland [http://www.keystonesymposia.org/index.cfm?e=web.Meeting.Program&meetingid=1323 Keystone Symposium: “Hypoxia: from basic mechanisms to therapeutics”])
Kezic 2016 Oxid Med Cell Longev + (Kidney ischemia/reperfusion injury emerges … Kidney ischemia/reperfusion injury emerges in various clinical settings as a great problem complicating the course and outcome. Ischemia/reperfusion injury is still an unsolved puzzle with a great diversity of investigational approaches, putting the focus on oxidative stress and mitochondria. Mitochondria are both sources and targets of ROS. They participate in initiation and progression of kidney ischemia/reperfusion injury linking oxidative stress, inflammation, and cell death. The dependence of kidney proximal tubule cells on oxidative mitochondrial metabolism makes them particularly prone to harmful effects of mitochondrial damage. The administration of antioxidants has been used as a way to prevent and treat kidney ischemia/reperfusion injury for a long time. Recently a new method based on mitochondria-targeted antioxidants has become the focus of interest. Here we review the current status of results achieved in numerous studies investigating these novel compounds in ischemia/reperfusion injury which specifically target mitochondria such as MitoQ, Szeto-Schiller (SS) peptides (Bendavia), SkQ1 and SkQR1, and superoxide dismutase mimics. Based on the favorable results obtained in the studies that have examined myocardial ischemia/reperfusion injury, ongoing clinical trials investigate the efficacy of some novel therapeutics in preventing myocardial infarct. This also implies future strategies in preventing kidney ischemia/reperfusion injury.enting kidney ischemia/reperfusion injury.)
Bugarski 2018 Am J Physiol Renal Physiol + (Kidney proximal tubules (PTs) are densely … Kidney proximal tubules (PTs) are densely packed with mitochondria, and defects in mitochondrial function are implicated in many kidney diseases. However, little is known about intrinsic mitochondrial function within PT cells. Here, using intravital multiphoton microscopy and live slices of mouse kidney cortex, we show that autofluorescence signals provide important functional readouts of redox state and substrate metabolism and that there are striking axial differences in signals along the PT. Mitochondrial NAD(P)H intensity was similar in both PT segment (S)1 and S2 and was sensitive to changes in respiratory chain (RC) redox state, whereas cytosolic NAD(P)H intensity was significantly higher in S2. Mitochondrial NAD(P)H increased in response to lactate and butyrate but decreased in response to glutamine and glutamate. Cytosolic NAD(P)H was sensitive to lactate and pyruvate and decreased dramatically in S2 in response to inhibition of glucose metabolism. Mitochondrial flavoprotein (FP) intensity was markedly higher in S2 than in S1 but was insensitive to changes in RC redox state. Mitochondrial FP signal increased in response to palmitate but decreased in response to glutamine and glutamate. Fluorescence lifetime decays were similar in both S1 and S2, suggesting that intensity differences are explained by differences in abundance of the same molecular species. Expression levels of known fluorescent mitochondrial FPs were higher in S2 than S1. In summary, substantial metabolic information can be obtained in kidney tissue using a label-free live imaging approach, and our findings suggest that metabolism is tailored to the specialized functions of S1 and S2 PT segments.alized functions of S1 and S2 PT segments.)
Shrum 2019 Biomolecules + (Kidneys from deceased donors used for tran … Kidneys from deceased donors used for transplantation are placed in cold storage (CS) solution during the search for a matched recipient. However, CS causes mitochondrial injury, which may exacerbate renal graft dysfunction. Here, we explored whether adding NS11021, an activator of the mitochondrial big-conductance calcium-activated K+ (mitoBK) channel, to CS solution can mitigate CS-induced mitochondrial injury. We used normal rat kidney proximal tubular epithelial (NRK) cells as an ''in vitro'' model of renal cold storage (18 h) and rewarming (2 h) (CS + RW). Western blots detected the pore-forming α subunit of the BK channel in mitochondrial fractions from NRK cells. The fluorescent K+-binding probe, PBFI-AM, revealed that isolated mitochondria from NRK cells exhibited mitoBK-mediated K+ uptake, which was impaired ~70% in NRK cells subjected to CS + RW compared to control NRK cells maintained at 37 °C. Importantly, the addition of 1 M NS11021 to CS solution prevented CS + RW-induced impairment of mitoBK-mediated K+ uptake. The NS11021-treated NRK cells also exhibited less cell death and mitochondrial injury after CS + RW, including mitigated mitochondrial respiratory dysfunction, depolarization, and superoxide production. In summary, these new data show for the first time that mitoBK channels may represent a therapeutic target to prevent renal CS-induced injury.hat mitoBK channels may represent a therapeutic target to prevent renal CS-induced injury.)
Stefan 2022 Abstract Bioblast + (Kinases function as molecular switches for … Kinases function as molecular switches for coordinating spatiotemporal signal transmission. Genomic alterations affect kinase abundance and/or their activities which contribute to the etiology and progression of diseases such as distinct cancers and Parkinson’s disease (PD). Thus, major drug discovery efforts aim to identify lead molecules targeting the clinically relevant kinase entity. The concept of personalized medicine aims to apply the therapeutic agent with the highest efficacy towards a patient-specific target protein mutation. We have recently implemented a cell-based reporter system which fosters the decision-making process for identifying and selecting efficient lead molecules. We have engineered a modular kinase conformation (KinCon) biosensor platform for live-cell analyses of kinase activity states. This biosensor facilitates the recording of kinase activity conformations of the wild-type and the respective mutated kinase upon lead-molecule or approved-drug exposure. First, in proof-of-principle studies we have demonstrated that this technology is suitable for the systematic determination of melanoma drug efficacies using the full-length KinCon reporters for BRAF and MEK which harbor distinct cancer patient mutations (Röck et al., Science Advances 2019, Mayrhofer et al., PNAS 2020, Fleischmann et al., Biomolecules 2021). Second, we have extended KinCon reporters to quantify the activity-relevant formation of multimeric kinase complexes, involving members of the RIPK [inflammation] or CDK [cancer] kinase families. Third, recently we have engineered mitochondria associated biosensors to analyze and categorize PD kinase mutations. Thus, with new KinCon reporters we are setting out to characterize PD causing kinase gain-of-function mutations and to reactivate a PD kinase displaying a collection of loss-of-function mutations, directly in an intact cell setting. Finally, we would like to underline that this precision-medicine-oriented KinCon biosensor concept is not restricted to recordings of kinase drug efficacies/specificities. We have first evidence that such conformation reporter can extended to other (pseudo)enzyme categories.tended to other (pseudo)enzyme categories.)
Klinische Mitochondrienmedizin und Umweltmedizin 2017 Heidelberg DE + (Klinische Mitochondrienmedizin und Umweltmedizin 2017, Heidelberg, Germany.)
Ernster 1981 J Cell Biol + (Known for over a century, mitochondria hav … Known for over a century, mitochondria have become during the last three decades an important subject of research within several disciplines of experimental biology. For the cytologist, they represented the ideal test objects for applying electron microscopy to the exploration of cellular ultrastructure and for the elaboration of tissue-fractionation techniques with the aim of isolating cytoplasmic organelles. For the biochemist, the identification of mitochondria as the site of cell respiration and respiration-linked phosphorylation implied a decisive step towards the resolution and reconstitution of these processes at a molecular level and the elucidation of their relationship to cellular membranes. For the physiologist, mitochondria afforded the first opportunity for an experimental approach to structure-function relationships, in particular those involved in active transport, vectorial metabolism, and metabolic control mechanisms on a subcellular level. And for the molecular biologist, the discovery of mitochondrial DNA and protein synthesis and the study of mitochondrial biogenesis opened up a new chapter of eukaryotic gene expression. The purpose of this review is to give a brief account of these developments by selecting some of the highlights of the long and eventful history of mitochondrial research.ventful history of mitochondrial research.)
MiPNet20.06 IsolationMouseHeart-mt + (Komlodi T, Cardoso LHD, Gnaiger E (2021) … Komlodi T, Cardoso LHD, Gnaiger E (2021) Laboratory protocol: Isolation of mouse heart mitochondria. Mitochondr Physiol Network 20.06(02):1-4. <div style="padding:0px;border: 1px solid #aaaaaa;margin-bottom:0px;margin-right:10px"><div style="font-size:100%;font-weight:bold;padding:0.2em;padding-right: 0.4em;padding-left: 0.4em;background-color:#eeeeee;border-bottom:1px solid #aaaaaa;text-align:left;">[[Image:O2k-support system.jpg|right|150px|link=http://wiki.oroboros.at/index.php/O2k-technical_support_and_open_innovation|O2k-technical support and open innovation]]: <big>Open the '''pdf document''' above.</big></div><div style="background-color:#ffffff;padding-top:0.2em;padding-right: 0.4em;padding-bottom: 0.2em;padding-left: 0.4em;">::::» Current O2k-series: '''[https://www.oroboros.at/index.php/product-category/products/o2k-packages/ NextGen-O2k Series XB and O2k Series J]'''::::» Current software versions DatLab 8.0: [[MitoPedia: DatLab]]::::* ''Further details:'' '''» [[MitoPedia: O2k-Open Support]]'''</div></div>:» Product: [[Oroboros O2k]], [[O2k-Catalogue]]k-Catalogue]])
MiPNet24.10 H2O2 flux analysis + (Komlodi T, Cardoso LHD, Gnaiger E (2021) H … Komlodi T, Cardoso LHD, Gnaiger E (2021) Hydrogen peroxide flux analysis using Amplex UltraRed assay in MiR05-Kit with DatLab 7.4. Mitochondr Physiol Network 24.10(03):1-5. <div style="padding:0px;border: 1px solid #aaaaaa;margin-bottom:0px;margin-right:10px"><div style="font-size:100%;font-weight:bold;padding:0.2em;padding-right: 0.4em;padding-left: 0.4em;background-color:#eeeeee;border-bottom:1px solid #aaaaaa;text-align:left;">[[Image:O2k-support system.jpg|right|150px|link=http://wiki.oroboros.at/index.php/O2k-technical_support_and_open_innovation|O2k-technical support and open innovation]]: <big>Open the '''pdf document''' above.</big></div><div style="background-color:#ffffff;padding-top:0.2em;padding-right: 0.4em;padding-bottom: 0.2em;padding-left: 0.4em;">::::» Current O2k-series: '''[https://www.oroboros.at/index.php/product-category/products/o2k-packages/ NextGen-O2k Series XB and O2k Series J]'''::::» Current software versions DatLab 8.0: [[MitoPedia: DatLab]]::::* ''Further details:'' '''» [[MitoPedia: O2k-Open Support]]'''</div></div>itoPedia: O2k-Open Support]]''' </div> </div>)
MiPNet24.09 Data analysis of mt-membrane potential + (Komlodi T, Cardoso LHD, Gnaiger E (2021) D … Komlodi T, Cardoso LHD, Gnaiger E (2021) Data analysis of mitochondrial membrane potential estimation using various fluorescence dyes. Mitochondr Physiol Network 24.09(03):1-6. <div style="padding:0px;border: 1px solid #aaaaaa;margin-bottom:0px;margin-right:10px"><div style="font-size:100%;font-weight:bold;padding:0.2em;padding-right: 0.4em;padding-left: 0.4em;background-color:#eeeeee;border-bottom:1px solid #aaaaaa;text-align:left;">[[Image:O2k-support system.jpg|right|150px|link=http://wiki.oroboros.at/index.php/O2k-technical_support_and_open_innovation|O2k-technical support and open innovation]]: <big>Open the '''pdf document''' above.</big></div><div style="background-color:#ffffff;padding-top:0.2em;padding-right: 0.4em;padding-bottom: 0.2em;padding-left: 0.4em;">::::» Current O2k-series: '''[https://www.oroboros.at/index.php/product-category/products/o2k-packages/ NextGen-O2k Series XB and O2k Series J]'''::::» Current software versions DatLab 8.0: [[MitoPedia: DatLab]]::::* ''Further details:'' '''» [[MitoPedia: O2k-Open Support]]'''</div></div>:» Product: [[DatLab]], [[Oroboros O2k]], [[Oroboros O2k-Catalogue |O2k-Catalogue]]oboros O2k-Catalogue |O2k-Catalogue]])
MiPNet24.12 NextGen-O2k: Q-Module + (Komlodi T, Cardoso LHD, Gollner M, Merth A, Niedenzu W, Haider M, Doerrier C, Tindle-Solomon L, Schwaninger H, Walter-Vracevic M, Gradl P, Moore AL, Rich P, Gnaiger E (2021) Mitochondr Physiol Network 24.12(04):1-20.)
MiPNet24.08 Safranin Analysis Template + (Komlodi T, Gnaiger E (2020) Excel template … Komlodi T, Gnaiger E (2020) Excel template for safranin data analysis. Mitochondr Physiol Network 24.08(02):1-8. <div style="padding:0px;border: 1px solid #aaaaaa;margin-bottom:0px;margin-right:10px"><div style="font-size:100%;font-weight:bold;padding:0.2em;padding-right: 0.4em;padding-left: 0.4em;background-color:#eeeeee;border-bottom:1px solid #aaaaaa;text-align:left;">[[Image:O2k-support system.jpg|right|150px|link=http://wiki.oroboros.at/index.php/O2k-technical_support_and_open_innovation|O2k-technical support and open innovation]]: <big>Open the '''pdf document''' above.</big></div><div style="background-color:#ffffff;padding-top:0.2em;padding-right: 0.4em;padding-bottom: 0.2em;padding-left: 0.4em;">::::» Current O2k-series: '''[https://www.oroboros.at/index.php/product-category/products/o2k-packages/ NextGen-O2k Series XB and O2k Series J]'''::::» Current software versions DatLab 8.0: [[MitoPedia: DatLab]]::::* ''Further details:'' '''» [[MitoPedia: O2k-Open Support]]'''</div></div>:» Product: [[DatLab]], [[Oroboros O2k]], [[Oroboros O2k-Catalogue |O2k-Catalogue]]oboros O2k-Catalogue |O2k-Catalogue]])
MiPNet24.16 DatLab8.0: CV-Module + (Komlodi T, Niedenzu W, Haider M, Cardoso L … Komlodi T, Niedenzu W, Haider M, Cardoso LHD, Tindle-Solomon L, Gnaiger E (2021) DatLab8.0:Cyclic voltammetry manual. Mitochondr Physiol Network 24.16(03):1-4. <div style="padding:0px;border: 1px solid #aaaaaa;margin-bottom:0px;margin-right:10px"><div style="font-size:100%;font-weight:bold;padding:0.2em;padding-right: 0.4em;padding-left: 0.4em;background-color:#eeeeee;border-bottom:1px solid #aaaaaa;text-align:left;">[[Image:O2k-support system.jpg|right|150px|link=http://wiki.oroboros.at/index.php/O2k-technical_support_and_open_innovation|O2k-technical support and open innovation]]: <big>Open the '''pdf document''' above.</big></div><div style="background-color:#ffffff;padding-top:0.2em;padding-right: 0.4em;padding-bottom: 0.2em;padding-left: 0.4em;">::::» For O2k-series: '''[https://wiki.oroboros.at/index.php/NextGen-O2k NextGen-O2k]'''::::» For software version: [[MitoPedia: DatLab |'''DatLab 8''']]::::* ''Further details:'' '''» [[MitoPedia: O2k-Open Support]]'''</div></div>:>> Product: [[Oroboros O2k]], [[NextGen-O2k]]extGen-O2k]])
MiPNet15.03 O2k-MultiSensor-ISE + (Komlodi T, Schmitt S, Gnaiger E (2021) O2k … Komlodi T, Schmitt S, Gnaiger E (2021) O2k-MultiSensor system with ion selective electrodes (ISE). Mitochondr Physiol Network 15.03(09): 1-21. <div style="padding:0px;border: 1px solid #aaaaaa;margin-bottom:0px;margin-right:10px"><div style="font-size:100%;font-weight:bold;padding:0.2em;padding-right: 0.4em;padding-left: 0.4em;background-color:#eeeeee;border-bottom:1px solid #aaaaaa;text-align:left;">[[Image:O2k-support system.jpg|right|150px|link=http://wiki.oroboros.at/index.php/O2k-technical_support_and_open_innovation|O2k-technical support and open innovation]]: <big>Open the '''pdf document''' above.</big></div><div style="background-color:#ffffff;padding-top:0.2em;padding-right: 0.4em;padding-bottom: 0.2em;padding-left: 0.4em;">::::» Current O2k-series: '''[https://www.oroboros.at/index.php/product-category/products/o2k-packages/ NextGen-O2k Series XB and O2k Series J]'''::::» Current software versions DatLab 8.0: [[MitoPedia: DatLab]]::::* ''Further details:'' '''» [[MitoPedia: O2k-Open Support]]'''</div></div>:» Product: [[O2k-TPP+ ISE-Module]], [[Oroboros O2k-Catalogue]]oboros O2k-Catalogue]])
Murphy 2018 Cell + (Krebs cycle intermediates traditionally li … Krebs cycle intermediates traditionally link to oxidative phosphorylation whilst also making key cell components. It is now clear that some of these metabolites also act as signals. Succinate plays an important role in inflammatory, hypoxic, and metabolic signaling, while itaconate (from another Krebs cycle intermediate, cis-aconitate) has an anti-inflammatory role.-aconitate) has an anti-inflammatory role.)
MiPNet20.13 Safranin mt-membranepotential + (Krumschnabel G, Fasching M, Gnaiger E (201 … Krumschnabel G, Fasching M, Gnaiger E (2019) O2k-FluoRespirometry: HRR and simultaneous determination of mt-membrane potential with [[safranin]] or [[TMRM]]. Mitochondr Physiol Network 20.13(03):1-5. <div style="padding:0px;border: 1px solid #aaaaaa;margin-bottom:0px;margin-right:10px"><div style="font-size:100%;font-weight:bold;padding:0.2em;padding-right: 0.4em;padding-left: 0.4em;background-color:#eeeeee;border-bottom:1px solid #aaaaaa;text-align:left;">[[Image:O2k-support system.jpg|right|150px|link=http://wiki.oroboros.at/index.php/O2k-technical_support_and_open_innovation|O2k-technical support and open innovation]]: <big>Open the '''pdf document''' above.</big></div><div style="background-color:#ffffff;padding-top:0.2em;padding-right: 0.4em;padding-bottom: 0.2em;padding-left: 0.4em;">::::» Current O2k-series: '''[https://www.oroboros.at/index.php/product-category/products/o2k-packages/ NextGen-O2k Series XB and O2k Series J]'''::::» Current software versions DatLab 8.0: [[MitoPedia: DatLab]]::::* ''Further details:'' '''» [[MitoPedia: O2k-Open Support]]'''</div></div>itoPedia: O2k-Open Support]]''' </div> </div>)
MiPNet20.14 AmplexRed H2O2-production + (Krumschnabel G, Fontana-Ayoub M, Fasching … Krumschnabel G, Fontana-Ayoub M, Fasching M, Gnaiger E (2019) O2k-FluoRespirometry: HRR and simultaneous determination of H2O2 production with Amplex UltraRed. Mitochondr Physiol Network 20.14(04):1-6. <div style="padding:0px;border: 1px solid #aaaaaa;margin-bottom:0px;margin-right:10px"><div style="font-size:100%;font-weight:bold;padding:0.2em;padding-right: 0.4em;padding-left: 0.4em;background-color:#eeeeee;border-bottom:1px solid #aaaaaa;text-align:left;">[[Image:O2k-support system.jpg|right|150px|link=http://wiki.oroboros.at/index.php/O2k-technical_support_and_open_innovation|O2k-technical support and open innovation]]: <big>Open the '''pdf document''' above.</big></div><div style="background-color:#ffffff;padding-top:0.2em;padding-right: 0.4em;padding-bottom: 0.2em;padding-left: 0.4em;">::::» Current O2k-series: '''[https://www.oroboros.at/index.php/product-category/products/o2k-packages/ NextGen-O2k Series XB and O2k Series J]'''::::» Current software versions DatLab 8.0: [[MitoPedia: DatLab]]::::* ''Further details:'' '''» [[MitoPedia: O2k-Open Support]]'''</div></div>itoPedia: O2k-Open Support]]''' </div> </div>)
MiPNet21.12 Comparison mt respiration media + (Krumschnabel G, Hiller E, Gnaiger E (2016) … Krumschnabel G, Hiller E, Gnaiger E (2016) O2k-MultiSensor: Mitochondrial respiration media for HRR and simultaneous O2k-Fluorometry. Mitochondr Physiol Network 21.12(01):1-11. <div style="padding:0px;border: 1px solid #aaaaaa;margin-bottom:0px;margin-right:10px"><div style="font-size:100%;font-weight:bold;padding:0.2em;padding-right: 0.4em;padding-left: 0.4em;background-color:#eeeeee;border-bottom:1px solid #aaaaaa;text-align:left;">[[Image:O2k-support system.jpg|right|150px|link=http://wiki.oroboros.at/index.php/O2k-technical_support_and_open_innovation|O2k-technical support and open innovation]]: <big>Open the '''pdf document''' above.</big></div><div style="background-color:#ffffff;padding-top:0.2em;padding-right: 0.4em;padding-bottom: 0.2em;padding-left: 0.4em;">::::» Current O2k-series: '''[https://www.oroboros.at/index.php/product-category/products/o2k-packages/ NextGen-O2k Series XB and O2k Series J]'''::::» Current software versions DatLab 8.0: [[MitoPedia: DatLab]]::::* ''Further details:'' '''» [[MitoPedia: O2k-Open Support]]'''</div></div>itoPedia: O2k-Open Support]]''' </div> </div>)
MiPNet21.14 Reference sample HRR + (Krumschnabel G, Lamberti G, Hiller E, Hans … Krumschnabel G, Lamberti G, Hiller E, Hansl M, Gnaiger E (2016) Development of a reference sample for HRR. Mitochondr Physiol Network 21.14(02):1-10. <div style="padding:0px;border: 1px solid #aaaaaa;margin-bottom:0px;margin-right:10px"><div style="font-size:100%;font-weight:bold;padding:0.2em;padding-right: 0.4em;padding-left: 0.4em;background-color:#eeeeee;border-bottom:1px solid #aaaaaa;text-align:left;">[[Image:O2k-support system.jpg|right|150px|link=http://wiki.oroboros.at/index.php/O2k-technical_support_and_open_innovation|O2k-technical support and open innovation]]: <big>Open the '''pdf document''' above.</big></div><div style="background-color:#ffffff;padding-top:0.2em;padding-right: 0.4em;padding-bottom: 0.2em;padding-left: 0.4em;">::::» Current O2k-series: '''[https://www.oroboros.at/index.php/product-category/products/o2k-packages/ NextGen-O2k Series XB and O2k Series J]'''::::» Current software versions DatLab 8.0: [[MitoPedia: DatLab]]::::* ''Further details:'' '''» [[MitoPedia: O2k-Open Support]]'''</div></div>itoPedia: O2k-Open Support]]''' </div> </div>)
MiPNet06.06 Chemical O2 background + (Kuznetsov AV, Gnaiger E (2010) Oxygraph assay of cytochrome ''c'' oxidase activity: chemical background correction. Mitochondr Physiol Network 06.06(07):1-4. :» Product: O2k-Catalogue: [[O2k-MultiSensor]], [[O2k-Core]], [[O2k-Catalogue]])
Boyle 2015 Abstract MiP2015 + (Kv1.3 is a member of the delayed rectifier … Kv1.3 is a member of the delayed rectifier family of voltage-activated potassium channels and has become a major therapeutic target because of its role in autoimmune diseases, in leukaemia, atherosclerosis and obesity and type 2 diabetes. Kv1.3 is not only expressed on the plasma membrane but also on the inner mitochondrial membrane [1] suggesting that some of its actions might be via modulation of mitochondrial function.This was investigated in HEK293/Kv1.3 cells and human saphenous vein smooth muscle cells (HSVSMCs), using proliferation assays, immunocytochemistry and high resolution respirometry.HEK293/Kv1.3 cells had significantly increased rates of proliferation compared to WT HEK293 cells. PAP-1, a selective, cell permeant Kv1.3 inhibitor, reduced proliferation in both HEK293/Kv1.3 and HSVSMCs. Channel expression in both the plasma membrane and mitochondria was confirmed using mitotracker in conjunction with immunocytochemical detection of Kv1.3. Mitochondrial expression of the channel was confirmed in both cell types. In addition, the functional expression of the Kv1.3 channel in the plasma membrane was confirmed using patch clamp electrophysiology. High resolution respirometry demonstrated that HEK293/Kv1.3 cells were significantly more metabolically active than WT HEK cells with both increased OXPHOS and glycolytic activity.Thus mitochondrial Kv1.3 may contribute to increased mitochondrial respiration. This will be further investigated using additional permeant and impermeant inhibitors of the Kv1.3 channel.mpermeant inhibitors of the Kv1.3 channel.)
Traufeller 2004 Biochim Biophys Acta + (L-Aminocarnitine (L-AC) has been shown to … L-Aminocarnitine (L-AC) has been shown to inhibit carnitine palmitoyltransferases (CPT) in rat muscle and in rat liver. However, there are no reports on interactions of L-AC with CPT II and CPT I of human muscle. Therefore, the aim of the present work was to characterize the inhibition of human muscle CPT I and CPT II by L-AC in muscle mitochondria, skinned fibers and muscle homogenates in comparison to the established action of malonyl-CoA. Both isoenzymes were inhibited by L-AC, but sensitivity was different (CPT I, Kd=3.8 mM L-AC; CPT II, Kd=21.3 μM L-AC). A mixed inhibition type in respect to carnitine was detected (Ki=3.5 μM L-AC). At 0.5 mM L-AC, CPT II was completely inhibited without affection of CPT I. In contrast, CPT I was completely inhibited by 0.4 mM malonyl-CoA (Kd=0.5 μM), whereas CPT II was nearly not affected by this inhibitor. Using these inhibitors in muscle homogenates, activities of CPT II and CPT I were detected to be 38±10% and 63±10% of total, respectively (n=21). In intact mitochondria and different fractions of muscle homogenates after selective solubilization of CPT II by Tween 20, the extent of specific CPT inhibition changed in relation to the accessible isoenzyme pattern. Palmitoyl-carnitine-dependent respiration in skinned fibers was inhibited by high concentrations of L-AC, indicating that the inhibitor can be transported via the acyl-carnitine transporter, too. The combined use of both inhibitors (L-AC and malonyl-CoA) allows the kinetic characterization of CPT I and CPT II in human muscle homogenates. In addition, it has been shown that L-AC can be used for the study of metabolic consequences of CPT II deficiency on function of intact mitochondria.ciency on function of intact mitochondria.)
Pecina 2015 Abstract MiP2015 + (L-asparaginase (ASNase), a key component i … L-asparaginase (ASNase), a key component in the treatment of childhood acute lymphoblastic leukemia (ALL), hydrolyzes plasma asparagine and glutamine and thereby disturbs metabolic homeostasis of leukemic cells. The efﬁcacy of such therapeutic strategy will depend on the capacity of cancer cells to adapt to the metabolic challenge, which could relate to the activation of compensatory metabolic routes. Therefore, we studied the impact of ASNase on the main metabolic pathways in leukemic cells. Treating leukemic cells with ASNase increased fatty-acid oxidation (FAO) and cell respiration and inhibited glycolysis. FAO, together with the decrease in protein translation and pyrimidine synthesis, was positively regulated through inhibition of the RagB-mTORC1 pathway, whereas the effect on glycolysis was RagB-mTORC1 independent. As FAO has been suggested to have a pro-survival function in leukemic cells, we tested its contribution to cell survival following ASNase treatment. Pharmacological inhibition of FAO signiﬁcantly increased the sensitivity of ALL cells to ASNase. Moreover, constitutive activation of the mammalian target of rapamycin pathway increased apoptosis in leukemic cells treated with ASNase but did not increase FAO. Our study uncovers a novel therapeutic option based on the combination of ASNase and FAO inhibitors. combination of ASNase and FAO inhibitors.)
Volska 2013 Abstract MiP2013 + (L-carnitine takes part in the regulation o … L-carnitine takes part in the regulation of cellular energy metabolism. Recently it has been shown that mildronate, an inhibitor of L-carnitine biosynthesis, improves the neurological outcome after ischemic damage of brain tissue [1]. The aim of the present study was to investigate the effects of mildronate treatment on brain mitochondrial function using an in vitro model of anoxia-reoxygenation.Wistar rats were treated daily with mildronate (per os; 100 mg/kg) for 14 days. Control animals received water. The mitochondrial respiration measurements were performed in isolated brain mitochondria with a Clark-type oxygen sensor. OXPHOS capacity was measured using ADP and various substrates to evaluate respiration of all respiratory complexes. In order to investigate anoxia-reoxygenation damage, brain mitochondria were subjected to 5 min anoxia, followed by 5 min reoxygenation. In parallel, isolated mitochondria were treated under the same conditions but without 5 min anoxia to obtain control (normoxic) measurements. Respiratory parameters were determined: LEAK respiration in the absence of ADP (LN); OXPHOS capacity (P); LEAK respiration after phosphorylation of ADP to ATP (LT); respiratory control ratio (P/LT, RCR).Under normoxic conditions, mildronate treatment did not affect LN and P. However, LT was increased by 30%, resulting in a 28% decreased RCR. Anoxia-reoxygenation induced a significant 2.8-fold decrease in P and a 1.6-fold increase LT. These effects of anoxia-reoxygenation resulted in 4-fold reduction of the RCR. The mildronate treatment significantly diminished the anoxia-reoxygenation-induced decrease in P and increase in LT by 20% and 36%, respectively. After anoxia-reoxygenation the RCR was almost 2 times higher in the mildronate treated group compared to controls.These results demonstrate that mildronate treatment induces uncoupling preconditioning-like effect and improves tolerance against anoxia-reoxygenation.es tolerance against anoxia-reoxygenation.)
De Bari 2015 Abstract MiPschool London 2015 + (L-lactate (L-LAC) and D-lactate (D-LAC) ha … L-lactate (L-LAC) and D-lactate (D-LAC) have been often considered waste products of anaerobic glycolysis and methylglyoxal pathway, respectively. Contrarily, mammalian mitochondria have been shown to metabolize both lactate isomers, due to the existence of isomer specific mitochondrial carriers and enzymes [1,2]. Nothing is known on the possible role of lactate isomers in the overall metabolism of cancer cells, in which both lactate isomers are endogenously formed and considered to be exclusively released to the extracellular phase. Then we examined whether and how the mitochondrial metabolism of L-LAC and D-LAC occurs in human cancer cells, as compared to normal cells.At this aim we used human cultured androgen-insensitive, aggressive prostate cancer (PC-3) cells and normal immortalized prostate (PNT1A) cells, used as a control, grown and handled as described in [3]. Isolation of mitochondria from cultured cells and preparation of cell homogenates, mitochondrial membrane-enriched and cell soluble fractions were carried out as in [3,4 and refs therein]. Polarographic measurements of oxygen consumption, fluorimetric detection of mitochondrial membrane potential (ΔΨ) generation and photometric measurements of mitochondrial ATP production by OXPHOS were carried out as in [3,4 and refs therein]. Both L- and D-lactate dehydrogenase activity and protein level were monitored by kinetic and immunological analysis, respectively, as described in [3,4 and refs therein].We found that mitochondria from both prostate cancer and normal cells metabolize both L-LAC and D-LAC [3,4]. Both the isomers can enter mitochondria in a carrier-mediated manner, probably in symport with protons. Moreover, externally added D-LAC causes the efflux of malate from prostate mitochondria, at a higher rate in PC-3 than in PNT1A cells, probably due to the existence of the D-LAC/malate antiporter, already shown in rat liver mitochondria [2,4]. L-LAC and D-LAC are oxidized inside mitochondria by the mitochondrial L-lactate dehydrogenase (mL-LDH) located in the matrix [1,3] and the flavoprotein D-lactate dehydrogenase (D-LDH), associated to the inner face of the inner mitochondrial membrane [2,4], respectively. The mitochondrial metabolism of both lactate isomers was found to be more active in cancer than in normal cells, likely due to the higher mL-LDH and D-LDH expression and activity found in the former. We propose that the mitochondrial metabolism of lactate isomers in prostate cancer cells could account for anaplerosis of Krebs cycle intermediates in the case of L-LAC, and for energy production and export of malate, this ultimately supporting cytosolic NADPH-dependent processes crucial for cell viability and proliferation, in the case of D-LAC.y and proliferation, in the case of D-LAC.)
Garcia-Corzo 2014 Thesis + (La Coenzima (ubiquinona o CoQ) fue aislada por primera vez en 1955 a partir de fraccion insaponificable de mucosa intestinal de caballo y cerdo (Federstein et a., 1955). Dos anos mas tarde, Crane y colbs....)
Geyer 1951 J Biol Chem + (Labeled carbon dioxide and acetoacetate ar … Labeled carbon dioxide and acetoacetate are two of the chief products formed during the incubation of radioactive fatty acids with rat tissue slices (1, 2). The quantitative aspects of the distribution of radiocarbon in these products can be affected by (a) the kind of tissue, (b) the length of the carbon chain of the substrate, and (c) the odd or even character of the substrate carbon chain (1). In addition, it has been shown that the amount of C1402 produced can be altered by a number of inhibitors (3). In the case of malonic acid, it was found that the decrease in total C1402 which resulted was accompanied by an increase in the Cl4 content of the carboxyl group of the acetoacetic acid formed. This finding suggested that the breakdown of fatty acids to the 2-carbon fragment stage did not depend on the operation of the intact tricarboxylic acid cycle, and, furthermore, supported the concept that the 2-carbon fragments formed were capable of following several different metabolic pathways. The nature of the data available, however, made it impossible to deduce whether one or both species of acetic acid radicals (CHSCO- and -CH&O-) were subject to this choice of pathways. Since the degree of asymmetry in the labeling of the acetoacetic acid formed involves the relative numbers of each of these species present (4, 5), information concerning the flexibility of the pathway of each type of fragment can be gained by the simultaneous study of the respired carbon dioxide and acetoacetic acid formed in the presence and absence of various inhibitors. Such studies are reported in the present paper. The experiments involved the incubation of rat liver with various labeled fatty acids in the presence of various inhibitors. Of the latter compounds, malonic acid was studied the most intensively.malonic acid was studied the most intensively.)
Liang 2016 J Biol Chem + (Lactate dehydrogenase (LDH) catalyzes the … Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate, which are critical fuel metabolites of skeletal muscle particularly during exercise. However, the physiological relevance of LDH remains poorly understood. Here we show that Ldhb expression is induced by exercise in human muscle and negatively correlated with changes in intramuscular pH levels, a marker of lactate production, during isometric exercise. We found that the expression of Ldhb is regulated by exercise-induced peroxisome proliferator-activated receptor-g coactivator 1α (PGC-1α). Ldhb gene promoter reporter studies demonstrated that PGC-1α activates Ldhb gene expression through multiple conserved estrogen-related receptor (ERR) and myocyte enhancer factor 2 (MEF2) binding sites. Transgenic mice overexpressing Ldhb in muscle (muscle creatine kinase [MCK]-Ldhb) exhibited increased exercise performance and enhanced oxygen consumption during exercise. MCK-Ldhb muscle was shown to have enhanced mitochondrial enzyme activity and increased mitochondrial gene expression, suggesting an adaptive oxidative muscle transformation. In addition, mitochondrial respiration capacity was increased and lactate production decreased in MCK-Ldhb skeletal myotubes in culture. Together, these results identified a previously unrecognized Ldhb-driven alteration in muscle mitochondrial function and suggested a mechanism for the adaptive metabolic response induced by exercise training.Copyright © 2016, The American Society for Biochemistry and Molecular Biology.ty for Biochemistry and Molecular Biology.)
George 2015 BA Thesis + (Lactate has been considered a “dead-end” w … Lactate has been considered a “dead-end” waste product of anaerobic metabolism. However, research continually demonstrates that lactate serves as an important metabolic fuel for many tissues, including skeletal and cardiac muscles, liver and brain. The intracellular lactate shuttle hypothesis posits that lactate generated in the cytosol is oxidized by mitochondrial lactate dehydrogenase (LDH) of the same cell. The details of the shuttle have not been made entirely clear. It has been proposed that such a shuttle operates similarly to the malate-aspartate shuttle; it has also been proposed that the two shuttles are necessarily interconnected in a lactate-malate-aspartate shuttle. We hypothesized that experimental support for robust mitochondrial lactate oxidation requires detailed attention to methodological detail. More specifically, an optimal malate concentration was expected to elicit maximal lactate oxidation in skeletal muscle mitochondria ''in vitro''. To test this hypothesis, rates of mitochondrial respiratory oxygen flux (JO2) were continuously monitored during titration of increasing concentrations of lactate (5-30 mM) atop 5 mM ADP, 1 mM NAD+ and either 0.5 mM or 4 mM malate in saponin-permeabilized red gastrocnemius muscle fibres from Sprague-Dawley (N = 3) and Wistar rats (N = 3). Net lactate-supported JO2 was significantly greater with 0.5 mM malate (two-way ANOVA with repeated measures; main effect for malate concentration, P = .0156). The results of this study suggest that mitochondrial lactate oxidation may indeed depend on multi-dehydrogenase activity outside of the mitochondrial matrix. These results also highlight malate concentration as an important variable to consider when interpreting ''in vitro'' phenomena in the context of muscle physiology ''in vivo''.vitro'' phenomena in the context of muscle physiology ''in vivo''.)
Jacobs 2013 Am J Physiol Endocrinol Metab + (Lactate is an important intermediate metab … Lactate is an important intermediate metabolite in human bioenergetics and is oxidized in many different tissues including the heart, brain, kidney, adipose tissue, liver and skeletal muscle. The mechanism(s) explaining the metabolism of lactate in these tissues, however, remains unclear. Here, we analyze the ability of skeletal muscle to respire lactate using an in situ mitochondrial preparation that leaves the native tubular reticulum and subcellular interactions of the organelle unaltered. Skeletal muscle biopsies were obtained from the m. vastus lateralis in 16 human subjects. Samples were chemically permeabilized with saponin, which selectively perforates the sarcolemma and facilitates the loss of cytosolic content without altering mitochondrial membranes, structure, and subcellular interactions. High-resolution respirometry was performed on permeabilized muscle biopsy preparations. Using four separate and specific substrate titration protocols the respirometric analysis revealed that mitochondria were capable of oxidizing lactate in the absence of exogenous LDH. The titration of lactate and NAD(+) into the respiration medium stimulated respiration (p ≤ 0.003). The addition of exogenous LDH failed to increase lactate-stimulated respiration (p = 1.0). The results further demonstrate that human skeletal muscle mitochondria cannot directly oxidize lactate within the mitochondrial matrix. Alternately, these data support previous claims that lactate is converted to pyruvate within the mitochondrial intermembrane space with the pyruvate subsequently taken into the mitochondrial matrix where it enters the TCA cycle and is ultimately oxidized. the TCA cycle and is ultimately oxidized.)
Kane 2014 Abstract MiP2014 + (Lactate serves as an important metabolic i … Lactate serves as an important metabolic intermediate for many tissues including skeletal and cardiac muscles, liver and brain. It is thought that a primary purpose of cytosolic lactate production from pyruvate by lactate dehydrogenase (LDH) is to regenerate NAD+ for continued glycolytic ATP production [1]. Considered in isolation, the NAD+ recycling afforded by cytosolic lactate production does not reconcile with the traditional view of aerobic glycolysis in which pyruvate, generated by glycolysis, enters the mitochondria directly for subsequent oxidation in the TCA cycle. The intracellular lactate shuttle hypothesis posits that lactate, generated in the cytosol, is oxidized by mitochondrial LDH of the same cell [2]. The details of the shuttle, however, are not entirely clear. Evidence is presented which supports that in skeletal muscle, extra-matrix LDH is strategically positioned within the cell to functionally interact with mitochondria [3]. A model incorporating mitochondrial lactate oxidation makes sense of aerobic glycolysis by permitting, among other things, cytosolic NAD+ regeneration, locally. However, experimental support requires attention to methodological detail. Important experimental conditions for assessing mitochondrial lactate oxidation in permeabilized fibers are discussed. Proper malate concentration [4] is necessary for robust NAD+-dependent lactate oxidation, suggesting that a functional malate-aspartate shuttle is essential to the assay. The cytochrome ''c'' test, a convenient means of confirming the integrity of the mitochondrial outer membrane [5], may not accurately reflect the integrity of mitochondrial preparations when assessing lactate oxidation. Indeed, parallel experiments in high-resolution respirometry reveal that in permeabilized rat skeletal muscle fibers, exogenous cytochrome ''c'' stimulates respiration with lactate but not with pyruvate as substrate. These findings highlight the importance of optimizing seemingly trivial experimental variables. findings highlight the importance of optimizing seemingly trivial experimental variables.)
Chang 2021 J Fungi (Basel) + (Lactoferricin (Lfcin) is an amphipathic, c … Lactoferricin (Lfcin) is an amphipathic, cationic peptide derived from proteolytic cleavage of the N-lobe of lactoferrin (Lf). Lfcin and its derivatives possess broad-spectrum antibacterial and antifungal activities. However, unlike their antibacterial functions, the modes of action of Lfcin and its derivatives against pathogenic fungi are less well understood. In this study, the mechanisms of LfcinB15, a derivative of bovine Lfcin, against ''Candida albicans'' were, therefore, extensively investigated. LfcinB15 exhibited inhibitory activity against planktonic cells, biofilm cells, and clinical isolates of ''C. albicans'' and non-''albicans Candida'' species. We further demonstrated that LfcinB15 is localized on the cell surface and vacuoles of ''C. albicans'' cells. Moreover, LfcinB15 uses several different methods to kill ''C. albicans'', including disturbing the cell membrane, inducing reactive oxygen species (ROS) generation, and causing mitochondrial dysfunction. Finally, the Hog1 and Mkc1 mitogen-activated protein kinases were both activated in ''C. albicans'' cells in response to LfcinB15. These findings help us to obtain more insight into the complex mechanisms used by LfcinB15 and other Lfcin-derived peptides to fight fungal pathogens.erived peptides to fight fungal pathogens.)
Rubio-Villena 2018 Hum Mol Genet + (Lafora disease (LD) is a fatal form of pro … Lafora disease (LD) is a fatal form of progressive myoclonus epilepsy characterized by the accumulation of insoluble poorly branched glycogen-like inclusions named Lafora bodies (LBs) in the brain and peripheral tissues. In the brain, since its first discovery in 1911, it was assumed that these glycogen inclusions were only present in affected neurons. Mouse models of LD have been obtained recently, and we and others have been able to report the accumulation of glycogen inclusions in the brain of LD animals, what recapitulates the hallmark of the disease. In this work we present evidence indicating that, although in mouse models of LD glycogen inclusions co-localize with neurons, as originally established, most of them co-localize with astrocytic markers such as glial fibrillary acidic protein (GFAP) and glutamine synthase. In addition, we have observed that primary cultures of astrocytes from LD mouse models accumulate higher levels of glycogen than controls. These results suggest that astrocytes may play a crucial role in the pathophysiology of Lafora disease, as the accumulation of glycogen inclusions in these cells may affect their regular functionality leading then to a possible neuronal dysfunction.g then to a possible neuronal dysfunction.)
Ballot 2010 Apoptosis + (Lamellarin D (Lam D), a marine alkaloid, e … Lamellarin D (Lam D), a marine alkaloid, exhibits a potent cytotoxicity against many different tumors. The pro-apoptotic function of Lam D has been attributed to its direct induction of mitochondrial permeability transition (MPT). This study was undertaken to explore the mechanisms through which Lam D promotes changes in mitochondrial function and as a result apoptosis. The use of eight Lam derivatives provides useful structure-apoptosis relationships. We demonstrate that Lam D and structural analogues induce apoptosis of cancer cells by acting directly on mitochondria inducing reduction of mitochondrial membrane potential, swelling and cytochrome c release. Cyclosporin A, a well-known inhibitor of MPT, completely prevents mitochondrial signs of apoptosis. The drug decreases calcium uptake by mitochondria but not by microsomes indicating that Lam D-dependent permeability is specific to mitochondrial membranes. In addition, upon Lam D exposure, a rapid decline of mitochondrial respiration and ATP synthesis occurs in isolated mitochondria as well as in intact cells. Evaluation of the site of action of Lam D on the electron-transport chain revealed that the activity of respiratory chain complex III is reduced by a half. To determine whether Lam D could induce MPT-dependent apoptosis by inhibiting mitochondrial respiration, we generated respiration-deficient cells (rho0) derived from human melanoma cells. In comparison to parental cells, rho0 cells are totally resistant to the induction of MPT-dependent apoptosis by Lam D. Our results indicate that functional mitochondria are required for Lam D-induced apoptosis. Inhibition of mitochondrial respiration is responsible for MPT-dependent apoptosis of cancer cells induced by Lam-D.poptosis of cancer cells induced by Lam-D.)
Serteyn 2014 Bioenergetics + (Laminitis is a common and debilitating dis … Laminitis is a common and debilitating disease affecting horses and ponies. It often leads to the demise of theanimal. Energy deficiency is suspected to entrain the disruption of the hemidesmosomes leading to the failure of thedermal-epidermal interface. The aim of this study was to measure the muscle mitochondrial function by highresolution respirometry. Muscle micro-biopsies were obtained from 11 horses affected by acute metabolic laminitis,6 horses affected by acute laminitis resulting from a systemic inflammation response syndrome and 28 healthyhorses distributed in 2 control groups: 17 horses with a body condition score [BSC, ranging from 0 (emaciated) to 5(obese)] of 2 to 3 and 11 horses with a BSC of 4 to 5. During the acute phase of laminitis, a significant reduction ofthe muscle mitochondrial respiration was observed. The muscle mitochondrial dysfunction occurred independently ofthe etiology (metabolic disorder or systemic inflammation) leading to laminitis. The reduction of the oxidativephosphorylation and of the maximal respiratory capacity (after uncoupling) may induce depletion of the cell’s ATPcontent. If the same mitochondrial alteration occurs in the foot lamina, mitochondria targeting should be consideredfor the future, not only to better understand the physiopathology of the disease but also to maintain and to supportthe mitochondrial function before reaching the « mitochondrial dysfunction threshold » that may lead to the failure ofthe dermal-epidermal interface.failure of the dermal-epidermal interface.)
MiPNet19.14 SOP Hamilton microsyringes + (Laner V, Timón-Gómez A, Baglivo E, Gnaiger … Laner V, Timón-Gómez A, Baglivo E, Gnaiger E (2023) SOP for manual O2k-titrations with Hamilton microsyringes. Mitochondr Physiol Network 19.14(07):1-4. <div style="padding:0px;border: 1px solid #aaaaaa;margin-bottom:0px;margin-right:10px"><div style="font-size:100%;font-weight:bold;padding:0.2em;padding-right: 0.4em;padding-left: 0.4em;background-color:#eeeeee;border-bottom:1px solid #aaaaaa;text-align:left;">[[Image:O2k-support system.jpg|right|150px|link=http://wiki.oroboros.at/index.php/O2k-technical_support_and_open_innovation|O2k-technical support and open innovation]]: <big>Open the '''pdf document''' above.</big></div><div style="background-color:#ffffff;padding-top:0.2em;padding-right: 0.4em;padding-bottom: 0.2em;padding-left: 0.4em;">::::» Current O2k-series: '''[https://www.oroboros.at/index.php/product-category/products/o2k-packages/ NextGen-O2k Series XB and O2k Series J]'''::::» Current software versions DatLab 8.0: [[MitoPedia: DatLab]]::::* ''Further details:'' '''» [[MitoPedia: O2k-Open Support]]'''</div></div>:» Product: [[O2k-Titration Set]], [[OROBOROS O2k-Catalogue | O2k-Catalogue]]OROS O2k-Catalogue | O2k-Catalogue]])
Welker 2013 Comp Biochem Physiol A Mol Integr Physiol + (Large changes in oxygen availability in aq … Large changes in oxygen availability in aquatic environments, ranging from anoxia through to hyperoxia, can lead to corresponding wide variation in the production of reactive oxygen species (ROS) by animals with aquatic respiration. Therefore, animals living in marine, estuarine and freshwater environments have developed efficient antioxidant defenses to minimize oxidative stress and to regulate the cellular actions of ROS. Changes in oxygen levels may lead to bursts of ROS generation that can be particularly harmful. This situation is commonly experienced by aquatic animals during abrupt transitions from periods of hypoxia/anoxia back to oxygenated conditions (e.g. intertidal cycles). The strategies developed differ significantly among aquatic species and are (i) improvement of their endogenous antioxidant system under hyperoxia (that leads to increased ROS formation) or other similar ROS-related stresses, (ii) increase in antioxidant levels when displaying higher metabolic rates, (iii) presence of constitutively high levels of antioxidants, that attenuates oxidative stress derived from fluctuations in oxygen availability, or (iv) increase in the activity of antioxidant enzymes (and/or the levels of their mRNAs) during hypometabolic states associated with anoxia/hypoxia. This enhancement of the antioxidant system - coined over a decade ago as "preparation for oxidative stress" - controls the possible harmful effects of increased ROS formation during hypoxia/reoxygenation. The present article proposes a novel explanation for the biochemical and molecular mechanisms involved in this phenomenon that could be triggered by hypoxia-induced ROS formation. We also discuss the connections among oxygen sensing, oxidative damage and regulation of the endogenous antioxidant defense apparatus in animals adapted to many natural or man-made challenges of the aquatic environment.ade challenges of the aquatic environment.)
Remor 2011 Biochim Biophys Acta + (Large scale clinical trials have demonstra … Large scale clinical trials have demonstrated that an intensive antihyperglycemic treatment in diabetes mellitus (DM) in individuals reduces the incidence of micro- and macrovascular complications, e.g. nephropathy, retinopathy, DM-accelerated atherosclerosis, myocardial infarction, or limb amputations. Here, we investigated the effect of short- and long-term insulin administration on mitochondrial function in peripheral tissues of streptozotocin (STZ)-induced hyperglycemic rats. In addition, the ''in vitro'' effect of methylglyoxal (MG), advanced glycation end products (AGEs) and human diabetic plasma on mitochondrial activity was investigated in skeletal muscle and liver mitochondria and in rat skin primary fibroblasts. Hyperglycemic STZ rats showed tissue-specific patterns of energy deficiency, evidenced by reduced activities of complexes I, II and/or IV after 30 days of hyperglycemia in heart, skeletal muscle and liver; moreover, cardiac tissue was found to be the most sensitive to the diabetic condition, since energy metabolism was impaired after 10 days of the hyperglycemia. Insulin-induced tight glycemic control was effective in protecting against the hyperglycemia-induced inhibition of mitochondrial enzyme activities. Furthermore, the long-term hormone replacement (30 days) also increased these activities in kidney from STZ-treated animals, where the hyperglycemic state did not modify the electron transport activity. Results from ''in vitro'' experiments indicate that mitochondrial impairment could result from oxidative stress-induced accumulation of MG and/or AGEs. Further investigations demonstrated that human plasma AGE accumulation elicits reduced mitochondrial function in skin fibroblast. These data suggest that persistent hyperglycemia results in tissue-specific patterns of energy deficiency and that early and continuous insulin therapy is necessary to maintain proper mitochondrial metabolism. maintain proper mitochondrial metabolism.)
Herledan 2017 Thesis + (Le muscle squelettique est un tissu plasti … Le muscle squelettique est un tissu plastique ayant la capacité d’adapter sa taille suivant les demandes fonctionnelles extérieures. Il peut s’atrophier (perte de volume musculaire), ou s’hypertrophier (gain de volume musculaire) par une augmentation de la synthèse protéique ou par l’intervention des cellules satellites (SC). Les mécanismes moléculaires de réarrangement de la taille des fibres musculaires demeurent encore peu connus. Les cellules souches du muscle adulte, appelées les cellules satellites, sont quiescentes et sont localisées le long des fibres musculaires. Elles sont amenées à s’activer lors d’une lésion, d’une surcharge de travail ou d’une pathologie. Les SC activées (myoblastes), prolifèrent puis se différencient et fusionnent entre elles ou avec les myofibres préexistantes. Ce processus est appelé la myogenèse adulte. D’une part, nous avons étudié l’implication du facteur de transcription SRF dans l’atrophie du muscle squelettique par arrêt d’activité mécanique induite par dénervation. Se basant sur un modèle murin d’inactivation conditionnelle et inductible de SRF dans les fibres musculaires, nous montrons le rôle de SRF dans la régulation de voies cataboliques comme l’autophagie. De plus, nos données suggèrent l’intervention de SRF dans la régulation énergétique mitochondriale via l’activité des complexes de la chaîne respiratoire. D’autre part, un modèle conditionnel et inductible d’inactivation de SRF dans les SC, nous a permis d’évaluer le rôle de SRF dans les SC. Des travaux réalisés dans l’équipe ont montré que la perte de SRF perturbe fortement les processus de régénération musculaire et d’hypertrophie par surcharge de travail, deux situations au cours desquelles les SC sont mobilisées. Au niveau cellulaire nous avons montré que la perte de SRF dans les SC n’altère ni la prolifération, ni l’entrée en différenciation des myoblastes ; cependant elle entraîne un défaut de motilité et de fusion. Se basant sur une analyse transcriptionnelle, l’équipe a identifié plusieurs gènes cibles de SRF susceptibles d’être responsables du défaut de motilité et/ou de fusion, dont l’alpha-actine. Par une approche de diminution de l’expression de gènes par transfection de siRNA dans les myoblastes, nous avons identifié quelques gènes dont la diminution d’expression phénocopie le défaut de motilité et/ou de fusion des SC. Dans cette étude, nous avons retenu les gènes codant pour FHL2 et HIC5. Une approche « gain de fonction », par surexpression d’actine dans les SC dépourvues de SRF, nous permet d’améliorer le phénotype de fusion hétérotypique/asymétrique, suggérant que la restauration seule du cytosquelette d’actine est suffisante pour rétablir ce type de fusion et l’hypertrophie du muscle. Cependant la fusion homotypique/symétrique et la régénération ne sont pas restaurées ; ainsi nous émettons l’hypothèse de l’intervention d’autres acteurs, tels que FHL2 ou HIC5.n d’autres acteurs, tels que FHL2 ou HIC5.)
Yu-Wai-Man 2011 Prog Retin Eye Res + (Leber hereditary optic neuropathy (LHON) a … Leber hereditary optic neuropathy (LHON) and autosomal-dominant optic atrophy (DOA) are the two most common inherited optic neuropathies in the general population. Both disorders share striking pathological similarities, marked by the selective loss of retinal ganglion cells (RGCs) and the early involvement of the papillomacular bundle. Three mitochondrial DNA (mtDNA) point mutations; m.3460G>A, m.11778G>A, and m.14484T>C account for over 90% of LHON cases, and in DOA, the majority of affected families harbour mutations in the OPA1 gene, which codes for a mitochondrial inner membrane protein. Optic nerve degeneration in LHON and DOA is therefore due to disturbed mitochondrial function and a predominantly complex I respiratory chain defect has been identified using both in vitro and in vivo biochemical assays. However, the trigger for RGC loss is much more complex than a simple bioenergetic crisis and other important disease mechanisms have emerged relating to mitochondrial network dynamics, mtDNA maintenance, axonal transport, and the involvement of the cytoskeleton in maintaining a differential mitochondrial gradient at sites such as the lamina cribosa. The downstream consequences of these mitochondrial disturbances are likely to be influenced by the local cellular milieu. The vulnerability of RGCs in LHON and DOA could derive not only from tissue-specific, genetically-determined biological factors, but also from an increased susceptibility to exogenous influences such as light exposure, smoking, and pharmacological agents with putative mitochondrial toxic effects. Our concept of inherited mitochondrial optic neuropathies has evolved over the past decade, with the observation that patients with LHON and DOA can manifest a much broader phenotypic spectrum than pure optic nerve involvement. Interestingly, these phenotypes are sometimes clinically indistinguishable from other neurodegenerative disorders such as Charcot-Marie-Tooth disease, hereditary spastic paraplegia, and multiple sclerosis, where mitochondrial dysfunction is also thought to be an important pathophysiological player. A number of vertebrate and invertebrate disease models has recently been established to circumvent the lack of human tissues, and these have already provided considerable insight by allowing direct RGC experimentation. The ultimate goal is to translate these research advances into clinical practice and new treatment strategies are currently being investigated to improve the visual prognosis for patients with mitochondrial optic neuropathies.for patients with mitochondrial optic neuropathies.)
Wallace 1988 Science + (Leber's hereditary optic neuropathy is a m … Leber's hereditary optic neuropathy is a maternally inherited disease resulting in optic nerve degeneration and cardiac dysrhythmia. A mitochondrial DNA replacement mutation was identified that correlated with this disease in multiple families. This mutation converted a highly conserved arginine to a histidine at codon 340 in the NADH dehydrogenase subunit 4 gene and eliminated an Sfa NI site, thus providing a simple diagnostic test. This finding demonstrated that a nucleotide change in a mitochondrial DNA energy production gene can result in a neurological disease.gene can result in a neurological disease.)
Gomez-Duran 2012 Biochim Biophys Acta + (Leber's hereditary optic neuropathy is a m … Leber's hereditary optic neuropathy is a maternally inherited optic atrophy caused by mitochondrial DNA point mutations. Previous epidemiological studies have shown that individuals from mitochondrial genetic backgrounds (haplogroups) J/Uk and H have a higher and a lower risk, respectively, of suffering this disorder. To analyze the bases of these associations at cellular and molecular levels, functional studies with cybrids provide high quality evidence. Cybrids from haplogroup J contain less mitochondrial deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and synthesize a smaller amount of mitochondrial DNA-encoded polypeptides than those from haplogroup H. Haplogroup J cybrids also display lower oxygen consumption, mitochondrial inner membrane potential and total adenosine-5'-triphosphate (ATP) levels. Moreover, mitochondrial DNA levels correlate with many parameters of the oxidative phosphorylation system. These results suggest that the mitochondrial DNA amount determines oxidative phosphorylation capacity and, along with other recently published observations, support the possibility that mitochondrial DNA levels may be responsible for the bias of the disorder toward males, for the incomplete penetrance of mutations causing Leber's hereditary optic neuropathy and for the association of the disease with particular mitochondrial DNA haplogroups. particular mitochondrial DNA haplogroups.)
Sheremet 2016 Biochemistry (Moscow) + (Leber’s hereditary optic neuropathy (LHON) … Leber’s hereditary optic neuropathy (LHON) refers to a group of mitochondrial diseases and is characterized by defects of the mitochondrial electron transport chain and decreased level of oxidative phosphorylation. The list of LHON primary mtDNA mutations is regularly updated. In this study we describe the homoplasmic nucleotide substitution m.3472T>C in the MT-ND1 gene and specific changes in cell metabolism in a patient with LHON and his asymptomatic sister. To confirm the presence of mutation-related mitochondrial dysfunction, respiration of skin fibroblasts and platelets from the patient and his sister was studied, as well as the mitochondrial potential and production of reactive oxygen species in the skin fibroblasts. In addition, based on characteristics of the toxic effect of paraquat, a new approach was developed for detecting the functional activity of complex I of the mitochondrial respiratory chain.lex I of the mitochondrial respiratory chain.)
Lane 2015 Phil Trans R Soc B + (Leeuwenhoek's 1677 paper, the famous 'lett … Leeuwenhoek's 1677 paper, the famous 'letter on the protozoa', gives the first detailed description of protists and bacteria living in a range of environments. The colloquial, diaristic style conceals the workings of a startlingly original experimental mind. Later scientists could not match the resolution and clarity of Leeuwenhoek's microscopes, so his discoveries were doubted or even dismissed over the following centuries, limiting their direct influence on the history of biology; but work in the twentieth century confirmed Leeuwenhoek's discovery of bacterial cells, with a resolution of less than 1 µm. Leeuwenhoek delighted most in the forms, interactions and behaviour of his little 'animalcules', which inhabited a previously unimagined microcosmos. In these reflections on the scientific reach of Leeuwenhoek's ideas and observations, I equate his questions with the preoccupations of our genomic era: what is the nature of Leeuwenhoek's animalcules, where do they come from, how do they relate to each other? Even with the powerful tools of modern biology, the answers are far from resolved-these questions still challenge our understanding of microbial evolution. This commentary was written to celebrate the 350th anniversary of the journal Philosophical Transactions of the Royal Society.ophical Transactions of the Royal Society.)
Korre 2016 Am J Cardiol + (Left ventricular (LV) mass is a strong pre … Left ventricular (LV) mass is a strong predictor of cardiovascular disease (CVD) events; increased LV mass is common among US firefighters and plays a major role in firefighter sudden cardiac death. We aim to identify significant predictors of LV mass among firefighters. Cross-sectional study of 400 career male firefighters selected by an enriched randomization strategy. Weighted analyses were performed based on the total number of risk factors per subject with inverse probability weighting. LV mass was assessed by echocardiography (ECHO) and cardiac magnetic resonance, and normalized (indexed) for height. CVD risk parameters included vital signs at rest, body mass index (BMI)-defined obesity, obstructive sleep apnea risk, low cardiorespiratory fitness, and physical activity. Linear regression models were performed. In multivariate analyses, BMI was the only consistent significant independent predictor of LV mass indexes (all, ''p'' <0.001). A 1-unit decrease in BMI was associated with 1-unit (g/m1.7) reduction of LV mass/height1.7 after adjustment for age, obstructive sleep apnea risk, and cardiorespiratory fitness. In conclusion, after height-indexing ECHO-measured and cardiac magnetic resonance-measured LV mass, BMI was found to be a major driver of LV mass among firefighters. Our findings taken together with previous research suggest that reducing obesity will improve CVD risk profiles and decrease on-duty CVD and sudden cardiac death events in the fire service. Our results may also support targeted noninvasive screening for LV hypertrophy with ECHO among obese firefighters.sive screening for LV hypertrophy with ECHO among obese firefighters.)
Thapa 2022 Physiol Rep + (Left ventricular diastolic dysfunction is … Left ventricular diastolic dysfunction is a structural and functional condition that precedes the development of heart failure with preserved ejection fraction (HFpEF). The etiology of diastolic dysfunction includes alterations in fuel substrate metabolism that negatively impact cardiac bioenergetics, and may precipitate the eventual transition to heart failure. To date, the molecular mechanisms that regulate early changes in fuel metabolism leading to diastolic dysfunction remain unclear. In this report, we use a diet-induced obesity model in aged mice to show that inhibitory lysine acetylation of the pyruvate dehydrogenase (PDH) complex promotes energetic deficits that may contribute to the development of diastolic dysfunction in mouse hearts. Cardiomyocyte-specific deletion of the mitochondrial lysine acetylation regulatory protein GCN5L1 prevented hyperacetylation of the PDH complex subunit PDHA1, allowing aged obese mice to continue using pyruvate as a bioenergetic substrate in the heart. Our findings suggest that changes in mitochondrial protein lysine acetylation represent a key metabolic component of diastolic dysfunction that precedes the development of heart failure.precedes the development of heart failure.)
Iyer 2022 Abstract Bioblast + (Leigh Syndrome (LS), is a severe neuro-met … Leigh Syndrome (LS), is a severe neuro-metabolic disorder and has no current cure or adequate cellular models to understand the rapid fatality associated with the disease [1,2]. Other symptoms are widespread tissue malfunction in brain stem and muscle in LS patients. We hypothesize that altered bioenergetic function caused by mitochondrial genome mutations in the electron transfer system (ETS) may lead to rapid fatality in LS. The extent to which pathogenic mtDNA variants regulate disease severity in LS is currently not well understood. To better understand this relationship, we computed the mitochondrial bioenergetics health index (mtBHI) and glycolytic bioenergetics health index (glycoBHI) for measuring overall mitochondrial dysfunction in LS patient fibroblast cells harboring varying percentages of pathogenic mutant mtDNA (T8993G, T9185C) exhibiting deficiency in ATP synthase or Complex I (T10158C, T12706C). The mtBHI was based on four key aspects of mitochondrial respiration: ET capacity minus ROUTINE respiration (''E''-''R''), net ROUTINE respiration (''R''-''L''), residual oxygen consumption ''Rox'' after inhibition by rotenone and antimycin A (ROX state), and LEAK respiration ''L''. The glycoBHI was based on four key aspects of cellular proton efflux rate linked to glycolysis [3]. Our results indicated that (''1'') high heteroplasmy was detected in disease lines affecting ATP synthase and low heteroplasmy was detected in disease lines affecting NADH dehydrogenase; (''2'') levels of defective enzyme activities of the ETS correlated with the percentage of pathogenic mtDNA; (''3'') mitochondrial respiration was disrupted in diseased lines with variable ''E''-''R''; (''4'') mitochondrial ATP synthesis rate was decreased while glycolytic ATP synthesis rate was elevated in diseased cell lines.Based on the overall analysis of the five diseased patient-specific fibroblasts, the glycoBHI emerged as a sensitive indicator of mitochondrial defects because the cells had switched ‘on’ the glycolytic pathway. GlycoBHI was significantly increased in all cell lines compared to control BJ-FB and was indeed sensitive to mitochondrial dysfunction. We also computed the ‘composite BHI ratio’ (OXPHOS/Glycolysis) by dividing mtBHI/glycoBHI values because the cell lines were utilizing both OXPHOS (although highly defective) and glycolysis pathways to maintain the energy requirements in the individual cell line.Overall, these results suggest that as long as the precise mechanism of LS has not been elucidated, a multi-pronged approach that takes into consideration the specific pathogenic mtDNA variant, along with a composite BHI ratio, can aid in better diagnosis and understanding the factors influencing disease severity and rapid fatality in LS. Future experiments will determine whether mitochondrial morphology depend on mtDNA mutation load and whether they influence bioenergetics within a cell. Our ongoing studies are focused on evaluating mutation burden in human induced pluripotent stem cells (hiPSCs) reprogrammed from these patient fibroblast cells, followed by bioenergetic analyses in differentiated neurons and muscle cells derived from hiPSCs. Results from these studies will address the knowledge gaps that exist in the understanding of relationships among mtDNA mutations, morphology, function, and cell fate that may ultimately contribute to devastating mitochondrial disorders.# Bakare AB, Lesnefsky EJ, Iyer S (2021) Leigh Syndrome: a tale of two genomes. https://doi.org/10.3389/fphys.2021.693734# Grace HE, Galdun P, Lesnefsky EJ, West FD, Iyer S (2019) mRNA reprogramming of T8993G Leigh's Syndrome fibroblast cells to create induced pluripotent stem cell models for mitochondrial disorders. https://doi.org/10.1089/scd.2019.0045# Bakare AB, Dean J, Chen Q, Thorat V, Huang Y, LaFramboise T, Lesnefsky EJ, Iyer S (2021) Evaluating the bioenergetics health index ratio in Leigh Syndrome fibroblasts to understand disease severity. https://doi.org/10.3390/ijms221910344. https://doi.org/10.3390/ijms221910344 )
Inak 2021 Nat Commun + (Leigh syndrome (LS) is a severe manifestat … Leigh syndrome (LS) is a severe manifestation of mitochondrial disease in children and is currently incurable. The lack of effective models hampers our understanding of the mechanisms underlying the neuronal pathology of LS. Using patient-derived induced pluripotent stem cells and CRISPR/Cas9 engineering, we developed a human model of LS caused by mutations in the complex IV assembly gene SURF1. Single-cell RNA-sequencing and multi-omics analysis revealed compromised neuronal morphogenesis in mutant neural cultures and brain organoids. The defects emerged at the level of neural progenitor cells (NPCs), which retained a glycolytic proliferative state that failed to instruct neuronal morphogenesis. LS NPCs carrying mutations in the complex I gene NDUFS4 recapitulated morphogenesis defects. SURF1 gene augmentation and PGC1A induction via bezafibrate treatment supported the metabolic programming of LS NPCs, leading to restored neuronal morphogenesis. Our findings provide mechanistic insights and suggest potential interventional strategies for a rare mitochondrial disease.rategies for a rare mitochondrial disease.)
Zhou 2022 J Hum Genet + (Leigh syndrome (LS)/Leigh-like syndrome (L … Leigh syndrome (LS)/Leigh-like syndrome (LLS) is one of the most common mitochondrial disease subtypes, caused by mutations in either the nuclear or mitochondrial genomes. Here, we identified a novel intronic mutation (c.82-2 A > G) and a novel exonic insertion mutation (c.290dupT) in TMEM126B from a Chinese patient with clinical manifestations of LLS. ''In silico'' predictions, minigene splicing assays and patients' RNA analyses determined that the c.82-2 A > G mutation resulted in complete exon 2 skipping, and the c.290dupT mutation provoked partial and complete exon 3 skipping, leading to translational frameshifts and premature termination. Functional analysis revealed the impaired mitochondrial function in patient-derived lymphocytes due to severe complex I content and assembly defect. Altogether, this is the first report of LLS in a patient carrying mutations in TMEM126B. Our data uncovers the functional effect and the molecular mechanism of the pathogenic variants c.82-2 A > G and c.290dupT, which expands the gene mutation spectrum of LLS and clinical spectrum caused by TMEM126B mutations, and thus help to clinical diagnosis of TMEM126B mutation-related mitochondrial diseases.f TMEM126B mutation-related mitochondrial diseases.)
Barca 2018 Hum Mol Genet + (Leigh syndrome is a frequent, heterogeneou … Leigh syndrome is a frequent, heterogeneous pediatric presentation of mitochondrial oxidative phosphorylation (OXPHOS) disease, manifesting with psychomotor retardation and necrotizing lesions in brain deep gray matter. OXPHOS occurs at the inner mitochondrial membrane through the integrated activity of 5 protein complexes, of which complex V (CV) functions in a dimeric form to directly generate adenosine triphosphate (ATP). Mutations in several different structural CV subunits cause Leigh syndrome; however, dimerization defects have not been associated with human disease. We report four Leigh syndrome subjects from three unrelated Ashkenazi-Jewish families harboring a homozygous splice-site mutation (c.87 + 1G>C) in a novel CV subunit disease gene, ''USMG5''. The Ashkenazi population allele frequency is 0.57%. This mutation produces two ''USMG5'' transcripts, wild-type and lacking exon 3. Fibroblasts from two Leigh syndrome probands had reduced wild-type ''USMG5'' mRNA expression and undetectable protein. The mutation did not alter monomeric CV expression, but reduced both CV dimer expression and ATP synthesis rate. Rescue with wild-type ''USMG5'' cDNA in proband fibroblasts restored USMG5 protein, increased CV dimerization and enhanced ATP production rate. These data demonstrate that a recurrent ''USMG5'' splice-site founder mutation in the Ashkenazi Jewish population causes autosomal recessive Leigh syndrome by reduction of CV dimerization and ATP synthesis.duction of CV dimerization and ATP synthesis.)
Morales-Garcia 2016 Abstract MitoFit Science Camp 2016 + (Leigh syndrome is a hereditary disease cha … Leigh syndrome is a hereditary disease characterized by progressive neurological degeneration. The underlying cause of the disease is defective oxidative phosphorylation. Leigh syndrome may result from defects in different respiratory complexes. One such case is the disfunction of chaperonin SURF1, which catalyzes incorporation of subunit I into Complex IV. This defect results in deficient cytochrome oxidase activity and most likely leads to the clinical manifestations. In yeast, the orthologue of SURF1 is SHY1 [1,2]. SHY1 possesses two transmembrane domains and remains in the inner mitochondrial membrane, even after it has fulfilled its assembly role. Thus, it would be of interest to determine whether SHY1 has other functions besides being a chaperonin.We examined mitochondrial function in a ∆Shy1 ''Saccharomyces cerevisiae'' mutant strain grown in YPGal for 24 h at 30ºC. Cells were disrupted using a Bead-beater (Biospec Product Inc., USA) and 0.5 mm diameter glass beads.[3] From the homogenate, mitochondria were isolated by differential centrifugation. Protein concentration was determined by biuret.The rate of oxygen consumption was measured in an oxymeter, the resting state and in the phosphorylating state; transmembrane potential was determined using safranine-O, following the absorbance changes at 511-533nm in a DW2000 Aminco/Olis spectrophotometer in dual mode[4] and mitochondrial swelling was measured at 540 nm in the same spectrophotometer in split mode. The concentrations of Pi were 0.1, 0.4,1, 2 and 4 mM. As expected, the respiratory chain was highly inhibited as a result of a defective complex IV activity. No other functions, such as the permeability transition seemed to be affected. It is still necessary to keep studying SHY1 to determine whether it has additional functions.rmine whether it has additional functions.)
Alahmad 2020 EMBO Mol Med + (Leigh syndrome is a progressive neurodegen … Leigh syndrome is a progressive neurodegenerative disorder, most commonly observed in paediatric mitochondrial disease, and is often associated with pathogenic variants in complex I structural subunits or assembly factors resulting in isolated respiratory chain complex I deficiency. Clinical heterogeneity has been reported, but key diagnostic findings are developmental regression, elevated lactate and characteristic neuroimaging abnormalities. Here, we describe three affected children from two unrelated families who presented with Leigh syndrome due to homozygous variants (c.346_*7del and c.173A>T p.His58Leu) in NDUFC2, encoding a complex I subunit. Biochemical and functional investigation of subjects' fibroblasts confirmed a severe defect in complex I activity, subunit expression and assembly. Lentiviral transduction of subjects' fibroblasts with wild-type NDUFC2 cDNA increased complex I assembly supporting the association of the identified NDUFC2 variants with mitochondrial pathology. Complexome profiling confirmed a loss of NDUFC2 and defective complex I assembly, revealing aberrant assembly intermediates suggestive of stalled biogenesis of the complex I holoenzyme and indicating a crucial role for NDUFC2 in the assembly of the membrane arm of complex I, particularly the ND2 module.rm of complex I, particularly the ND2 module.)
Meldau 2024 Mol Genet Metab Rep + (Leigh syndrome is a severe progressive mit … Leigh syndrome is a severe progressive mitochondrial disorder mainly affecting children under the age of 5 years. It is caused by pathogenic variants in any one of more than 75 known genes in the nuclear or mitochondrial genomes. A 19-week-old male infant presented with lactic acidosis and encephalopathy following a 2-week history of irritability, neuroregression and poor weight gain. He was hypotonic with pathological reflexes, impaired vision, and nystagmus. Brain MRI showed extensive bilateral symmetrical T2 hyperintense lesions in basal ganglia, thalami, and brainstem. Metabolic workup showed elevated serum alanine, and heavy lactic aciduria with increased ketones, fumarate, malate, and alpha-ketoglutarate as well as reduced succinate on urine organic acid analysis. Lactic acidemia persisted, with only a marginally elevated lactate:pyruvate ratio (16.46, ref. 0-10). He demised at age 7 months due to respiratory failure. Exome sequencing followed by virtual gene panel analysis for pyruvate metabolism and mitochondrial defects could not identify any nuclear cause for Leigh syndrome. Mitochondrial DNA (mtDNA) genome sequencing revealed 88% heteroplasmy for a novel variant, NC_012920.1(MT-ND6):m.14430A>C p.(Trp82Gly), in blood DNA. This variant was absent from the unaffected mother's blood, fibroblast, and urine DNA, and detected at a level of 5% in her muscle DNA. Mitochondrial respiratory chain analysis revealed markedly reduced mitochondrial complex I activity in patient fibroblasts (34% of parent and control cells), and reduced NADH-linked respirometry (less than half of parental and control cells), while complex II driven respirometry remained intact. The combined clinical, genetic, and biochemical findings suggest that the novel MT-ND6 variant is the likely cause of Leigh syndrome in this patient. The mitochondrial ND6 protein is a subunit of complex I. An interesting finding was the absence of a significantly elevated lactate:pyruvate ratio in the presence of severe lactatemia, which directed initial diagnostic efforts towards excluding a pyruvate metabolism defect. This case highlights the value of a multidisciplinary approach and complete genetic workup to diagnosing mitochondrial disorders in South African patients.hondrial disorders in South African patients.)
Nuskova 2011 Abstract IOC61 + (Leigh syndrome is most frequently caused b … Leigh syndrome is most frequently caused by mutations in SURF1 gene which encodes cytochrome c oxidase (COX) specific assembly factor. Our previous studies focused on fibroblasts from patients harbouring SURF1 mutations (1, 2). To further characterize the mitochondrial energetics affected by SURF1 mutation, we used immortalised fibroblasts originated from SURF1 knockout (KO) mice (3). The COX content was decreased to 58 % of the control, which was in accordance with 38% decline of COX activity measured spectrophotometrically. However, there was no change in the rate of endogenous respiration or in the rate of ascorbate+TMPD-dependent respiration of permeabilised cells. In contrast, mitochondrial membrane potential generated by COX achieved 92 % of maximal membrane potential in the control cells, but only 73 % in the KO cells. Furthermore using ascorbate and TMPD, the decrease of membrane potential at state 3 (ADP) compared to state 4 (oligomycin) was more profound in the KO fibroblasts. Therefore, the proton-pumping activity of COX was partially impaired unlike the electron-transporting activity, suggesting a decrease in the H+/e− ratio. Since the p50 value of the KO cells was approximately 2-fold increased in all metabolic states measured, the oxygen affinity of COX was also decreased. Taken together, the KO cells from mice showed similar but milder functional manifestations of COX impairment than the cells of Surf1-deficient patients, which indicates that the Surf1 protein is not as essential for mouse as for human.(1) Pecina P, Capkova M, Chowdhury SKR, Drahota Z, Dubot A, Vojtiskova A, Hansikova H, Houstkova H, Zeman J, Godinot C, Houstek J (2003) Functional alteration of cytochrome c oxidase by SURF1 mutations in Leigh syndrome. BBA 1639: 53-63.(2) Pecina P, Gnaiger E, Zeman J, Pronicka E, Houstek J (2004) Decreased affinity for oxygen of cytochrome-c oxidase in Leigh syndrome caused by SURF1 mutations. Am. J. Physiol. Cell. Physiol. 287: C1384-C1388.(3) Dell’Agnello C, Leo S, Agostino A, Szabadkai G, Tiveron C, Zulian A, Prelle A, Roubertoux P, Rizzuto R, Zeviani M (2007) Increased longevity and refractoriness to Ca2+-dependent neurodegeneration in Surf1 knockout mice. Hum. Mol. Gen. 16: 431-444.knockout mice. Hum. Mol. Gen. 16: 431-444.)
Alston 2020 Am J Hum Genet + (Leigh syndrome is one of the most common n … Leigh syndrome is one of the most common neurological phenotypes observed in pediatric mitochondrial disease presentations. It is characterized by symmetrical lesions found on neuroimaging in the basal ganglia, thalamus, and brainstem and by a loss of motor skills and delayed developmental milestones. Genetic diagnosis of Leigh syndrome is complicated on account of the vast genetic heterogeneity with >75 candidate disease-associated genes having been reported to date. Candidate genes are still emerging, being identified when "omics" tools (genomics, proteomics, and transcriptomics) are applied to manipulated cell lines and cohorts of clinically characterized individuals who lack a genetic diagnosis. NDUFAF8 is one such protein; it has been found to interact with the well-characterized complex I (CI) assembly factor NDUFAF5 in a large-scale protein-protein interaction screen. Diagnostic next-generation sequencing has identified three unrelated pediatric subjects, each with a clinical diagnosis of Leigh syndrome, who harbor bi-allelic pathogenic variants in NDUFAF8. These variants include a recurrent splicing variant that was initially overlooked due to its deep-intronic location. Subject fibroblasts were found to express a complex I deficiency, and lentiviral transduction with wild-type NDUFAF8-cDNA ameliorated both the assembly defect and the biochemical deficiency. Complexome profiling of subject fibroblasts demonstrated a complex I assembly defect, and the stalled assembly intermediates corroborate the role of NDUFAF8 in early complex I assembly. This report serves to expand the genetic heterogeneity associated with Leigh syndrome and to validate the clinical utility of orphan protein characterization. We also highlight the importance of evaluating intronic sequence when a single, definitively pathogenic variant is identified during diagnostic testing.Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.ished by Elsevier Inc. All rights reserved.)
Mesquita 2016 Thesis + (Leishmania protozoan parasites need iron t … Leishmania protozoan parasites need iron to generate infective forms, colonize host cells and develop skin lesions in mice. Iron is also essential for the production of energy through the electron transport chain and the oxidative phosphorylation. This thesis aimed to analyze the effect of iron depletion on the cell growth, ultrastructure, protein expression and interaction of ''L. (V.) braziliensis'' with synthetic collagen matrices. Our results showed that iron depletion stops cell proliferation and impairs mitochondrial ultraestructure. This impairment is characterized by decreased activity of the electron transport chain complex IV which leads to both the reduction of oxygen consumption and the depolarization of mitochondrial membrane potential. However, while some parasites die due to mitochondrial dysfunction, others can resist nutritional stress and survive. It was observed that promastigotes increased mRNA expression of iron (LIT1) and heme (LHR1) transporters in iron depleted medium. The parasites also showed increased LIT1 and iron ferric reductase (LFR1) protein levels. Probably, the increased expression of transporters can enhance or maintain the intracellular concentrations of the metal, contributing to the parasites\2019 survival The increased expression of iron superoxide dismutase (FeSOD) mRNA should also play an important role on the parasites\2019 resistance to oxidative stress caused by iron depletion. During the interaction with collagen matrices it was observed that ''L. (V.) braziliensis'' promastigotes make contact with the fibers by both the flagellum and the cellular body, but their remodeling due to the action of parasites may start after longer periods of incubation. Also, the peptidases secreted in the conditioned medium and during the interaction with 3D collagen matrices exhibited identical profiles showing that collagen does not modulate the proteolytic profile of ''L. (V.) braziliensis''. However, metalopeptidases probably contribute to matrix degradation because one was identified directly from the enzymatic halo suggesting that it is an active enzyme responsible for the degradation of the substrate.ible for the degradation of the substrate.)
MiPNet12.13 Q-JunctionSCR + (Lemieux H, Gnaiger E (2007) MitoPathways c … Lemieux H, Gnaiger E (2007) MitoPathways compilation: Additive effect of succinate with substrates for Complexes I+II/Complex I. Mitochondr Physiol Network 12.13:1-5. <div style="padding:0px;border: 1px solid #aaaaaa;margin-bottom:0px;margin-right:10px"><div style="font-size:100%;font-weight:bold;padding:0.2em;padding-right: 0.4em;padding-left: 0.4em;background-color:#eeeeee;border-bottom:1px solid #aaaaaa;text-align:left;">[[Image:O2k-support system.jpg|right|150px|link=http://wiki.oroboros.at/index.php/O2k-technical_support_and_open_innovation|O2k-technical support and open innovation]]: <big>Open the '''pdf document''' above.</big></div><div style="background-color:#ffffff;padding-top:0.2em;padding-right: 0.4em;padding-bottom: 0.2em;padding-left: 0.4em;">::::» Current O2k-series: '''[https://www.oroboros.at/index.php/product-category/products/o2k-packages/ NextGen-O2k Series XB and O2k Series J]'''::::» Current software versions DatLab 8.0: [[MitoPedia: DatLab]]::::* ''Further details:'' '''» [[MitoPedia: O2k-Open Support]]'''</div></div>itoPedia: O2k-Open Support]]''' </div> </div>)
MiPNet12.23 FiberRespiration + (Lemieux H, Votion DM, Doerrier C, Gnaiger … Lemieux H, Votion DM, Doerrier C, Gnaiger E (2019) Mitochondrial respiration in permeabilized fibers: Needle biopsies from horse skeletal muscle. Mitochondr Physiol Network 12.23(08):1-4.:» O2k-Protocols:[[SUIT-011| SUIT-011]]:» Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]]Oroboros O2k-Catalogue | O2k-Catalogue]])
Holmstroem 2013 Metabolism + (Leptin stimulates peripheral lipid oxidati … Leptin stimulates peripheral lipid oxidation, but the influence on mitochondrial function is partly unknown. We investigated tissue-specific mitochondrial function in leptin-deficient obese C57BL/6J-''ob/ob'' mice compared to lean littermates following leptin treatment.Lean and obese ''ob/ob'' mice were treated with saline or leptin for 5days. At day six, liver, extensor digitorum longus (EDL) and soleus muscle were dissected and mitochondrial respiration analyzed in freshly dissected tissues. Expression of key proteins in the regulation of mitochondrial function was determined.In liver, mitochondrial respiration was reduced in ''ob/ob'' mice compared to lean mice. Expression of mitochondrial transcription factor A (TFAM) was decreased in ''ob/ob'' mice, but increased with leptin treatment. In glycolytic EDL muscle, mitochondrial respiration was increased in ''ob/ob'' mice. Protein markers of complex II, IV and ATP synthase were increased in EDL muscle from both saline- and leptin-treated ''ob/ob'' mice. TFAM protein abundance was decreased, while dynamin-1-like protein was increased in EDL muscle from saline-treated ''ob/ob'' mice and restored by leptin treatment. In oxidative soleus muscle, mitochondrial respiration and electron transport system protein abundance were unchanged, while TFAM was reduced in ''ob/ob'' mice.In conclusion, leptin-deficient ''ob/ob'' mice display tissue-specific mitochondrial adaptations under basal conditions and in response to leptin treatment. Mitochondrial respiration was decreased in liver, increased in glycolytic muscle and unaltered in oxidative muscle from ob/ob mice. Insight into the tissue-specific regulation of mitochondrial function in response to energy supply and demand may provide new opportunities for the treatment of insulin resistance.s for the treatment of insulin resistance.)
Viana 2019 Sci Rep + (Leucine can stimulate protein synthesis in … Leucine can stimulate protein synthesis in skeletal muscle, and recent studies have shown an increase in leucine-related mitochondrial biogenesis and oxidative phosphorylation capacity in muscle cells. However, leucine-related effects in tumour tissues are still poorly understood. Thus, we described the effects of leucine in both ''in vivo'' and ''in vitro'' models of a Walker-256 tumour. Tumour-bearing Wistar rats were randomly distributed into a control group (W; normoprotein diet) and leucine group (LW; leucine-rich diet [normoprotein + 3% leucine]). After 20 days of tumour evolution, the animals underwent 18-fludeoxyglucose positron emission computed tomography (18F-FDG PET-CT) imaging, and after euthanasia, fresh tumour biopsy samples were taken for oxygen consumption rate measurements (Oroboros Oxygraph), electron microscopy analysis and RNA and protein extraction. Our main results from the LW group showed no tumour size change, lower tumour glucose (18F-FDG) uptake, and reduced metastatic sites. Furthermore, leucine stimulated a shift in tumour metabolism from glycolytic towards oxidative phosphorylation, higher mRNA and protein expression of oxidative phosphorylation components, and enhanced mitochondrial density/area even though the leucine-treated tumour had a higher number of apoptotic nuclei with increased oxidative stress. In summary, a leucine-rich diet directed Walker-256 tumour metabolism to a less glycolytic phenotype profile in which these metabolic alterations were associated with a decrease in tumour aggressiveness and reduction in the number of metastatic sites in rats fed a diet supplemented with this branched-chain amino acid.tic sites in rats fed a diet supplemented with this branched-chain amino acid.)
Brunetta 2019 J Funct Foods + (Leucine is an essential amino acid that ha … Leucine is an essential amino acid that has been investigated by its participation in the regulation of whole-body metabolism and mitochondrial function. Here, we evaluated acute leucine effects on mitochondrial respiration of skeletal muscle from male Swiss mice ''in vitro''. Additionally, we further investigated the effects of 4-wk leucine ingestion (2.5% on drinking water) on skeletal muscle mitochondrial respiration and morphology of diet-induced obesity (DIO) mice. ''In vitro'', acute leucine increased mitochondrial respiration, and these effects were abolished in the presence of rapamycin. In DIO mice, ingestion of leucine for 4-wk improved glucose tolerance and insulin responsivity. Leucine supplementation also prevented the reduction in mitochondrial respiration, size, and complexity in the soleus skeletal muscle. We conclude that the positive effects of leucine on whole-body metabolism in DIO mice are associated with improvements in skeletal muscle mitochondrial function and morphology. Furthermore, leucine acute effects on mitochondrial respiration are mTORC1 dependent.hondrial respiration are mTORC1 dependent.)
Hashimi 2013 Abstract MiP2013 + (Leucine zipper EF-hand containing transmem … Leucine zipper EF-hand containing transmembrane protein 1 (Letm1) is evolutionarily conserved in diverse eukaryotic lineages having energized mitochondria, from opisthokonts, comprising metazoa and fungi, to plastid containing plants and apicomplexans. Its deletion from the human genome has been implicated in causing the seizure symptoms of Wolf-Hirschhorn syndrome. Research in predominantly opsithokont model systems has resulted in disparate roles of Letm1 such as a cation/proton antiporter, translocating either potassium or calcium across the inner membrane (mtIM), and anchoring of mitochondrial ribosomes to facilitate mtIM incorporation of hydrophobic respiratory system subunits. We have undertaken functional analysis of Letm1 in the highly evolutionarily diverged Trypanosoma brucei, member of the excavate order Kinetoplastida, taking advantage of the existence of in vitro cultured forms bearing mitochondria with different physiological states, including a petite mutant-like organelle. RNAi-interference in all of these cell types results in swelling that can be mitigated by the ionophore nigericin, suggesting that it plays a role in the efflux of the cation from the matrix. An observed effect on mitochondrial translation is a downstream effect of potassium accumulation in the organelle. By comparison of our results with those from other systems, we conclude that the basal function of Letm1 is to regulate potassium efflux from the matrix to maintain mitochondrial volume. The protein shows remarkable conservation of function not only across wide evolutionary distances, but also in different mitochondrial states. T. brucei is an appealing model for study of mitochondrial biology due to its amenability to reverse genetics, existence of in vitro cultures bearing different mitochondria and its diverged position in the eukaryotic tree of life, bringing another perspective for comparative biochemical approaches.ve for comparative biochemical approaches.)
Manfredi 2002 Methods + (Levels of phosphorylated adenosine nucleot … Levels of phosphorylated adenosine nucleotides, including the universal energy carrier adenosine 5(')-triphosphate (ATP) and its metabolites adenosine 5(')-diphosphate (ADP) and adenosine 5(')-monophosphate (AMP), define the energy state in living cells and are dependent mainly on mitochondrial function. In this article, we describe a method based on the luciferase-luciferin system used to measure mitochondrial ATP synthesis continuously in permeabilized mammalian cells and mitochondria isolated from animal tissues. We also describe a technique that uses the expression of recombinant targeted luciferase to report ATP content in different cell compartments. Finally, we describe an HPLC-based method for accurate measurement of ATP, ADP, and AMP in cultured cells and animal tissues. AMP in cultured cells and animal tissues.)
Bristot 2014 Abstract MiP2014 + (Li-Fraumeni Syndrome (LFS) and Li-Fraumeni … Li-Fraumeni Syndrome (LFS) and Li-Fraumeni-like Syndrome (LFL) are inherited disorders, associated to TP53 germline mutations and characterized by increased predisposition to multiple early-onset cancers [1]. Studies in families from Southern and Southeastern Brazil with LFS/LFL phenotype have identified a germline founder mutation in the TP53 gene, the p.R337H mutation (c.1010G>A), in a high population prevalence (~0.3%) [2]. Unlike the majority of the mutations in TP53, which are missense mutations located in the DNA-binding domain (DBD) of the protein (exons 5-8), the TP53 p.R337H (c.1010G>A) is located in exon 10, corresponding to the oligomerization domain (OD). The p53 nuclear phosphoprotein is known for its functions in the DNA damage response and apoptosis. Recently, this protein has been shown to regulate many aspects of energy metabolism as well as enzymes that are involved in cell responses to oxidative stress, manly through TIGAR activation [3].In a previous work, we analyzed the levels of several markers of oxidative stress responses in blood samples of p.R337H mutation carries and non-carries. We observed oxidative damage in lipids and proteins. Moreover, there was increased erythrocyte GPx activity, as well as increased total antioxidant status in the p.R337H mutation carries [4]. Therefore, our study was able to establish the relationship of oxidative stress with the loss of function of p53. The aim of this work was to evaluate the association between TP53 germline mutations with deregulation of cell bioenergetics. For this purpose, we performed high-resolution respirometry (HHR) of intact human fibroblast cells, derived from patients. Preliminary results showed a distinct pattern of HHR in different TP53 germline mutations genotypes. Fibroblasts from carriers of DBD mutations and wt/p.R337H showed higher ROUTINE, total and extramitochodrial respiration, as well as LEAK respiration, compared to p.R377H/p.R337H mutants and WT/WT cells. In agreement with HHR results, cells with DBD mutation showed increased ROS (reactive oxygen species) by DCF assay. On the other hand an unexpectedly high production was found of ROS by p.R377H/p.R337H. These data were correlated with antimetabolic drug sensitivity, mitochondrial membrane potential and cellular doubling time to better evaluate the potential role of these findings for the increased predisposition to multiple early-onset cancers presented by Li-Fraumeni patients.onset cancers presented by Li-Fraumeni patients.)
Life Science PhD Meeting 2019 Innsbruck AT + (Life Science PhD Meeting, Innsbruck, Austr … Life Science PhD Meeting, Innsbruck, Austria, 2019__TOC__== General information== :::: This meeting is a joint event of the Medical University of Innsbruck (MUI) and the Leopold Franzens University Innsbruck and is organized by the MUI PhD representatives, the Center for Molecular Biosciences Innsbruck (CMBI), the three MUI excellence PhD programs, i.e. Molecular Cell Biology and Oncology (MCBO), Signal Processing in Neurons (SPIN) and Host Response in Opportunistic Infections (HOROS) and the SFB F-44.:::: The program will cover a broad selection of topics ranging from Analytical and Diagnostic Methods and Models, over Biochemistry and Cell Biology, Developmental Biology and Aging, Genetics and Genomics, Immunity, Infectious Diseases and Clinical Medicine to Pharmacology and Neuroscience. :::: Please note that not only PhD students, but everyone in the Life Science community in Innsbruck (including undergraduate students) is most welcome to join and also to present, so spread the word to anyone interested!== Venue == :::: Center for Chemistry and Biomedicine (CCB):::: Innrain 80, 6020 Innsbruck:::: [http://biocenter.i-med.ac.at/ Location]== Organizers ==:::: Medical University of Innsbruck and Leopold Franzens University Innsbruck== Registration ==:::: For participation, please register at: [http://www.meduni-ibk.at/page.cfm?vpath=anmeldung&veranstaltung_fk=165 here]== Lecturers ==:::: Andrea Pauli (IMP, Vienna, Austria):::: Ralf Amann (University of Tübingen, Germany):::: Robert Messing (University of Texas at Austin, USA) :::: Patrick Eyers (University of Liverpool, UK)==Oroboros presentation ==[[File:2019-04-25 Life Science PhD Meeting.png|right|400px|Poster presentation Bastos Ana]]:::: '''[[Sant'Anna-Silva ACB]]''': [[Bastos Sant'Anna Silva 2019 Life Sciences Meeting 2019 Innsbruck AT|Cellular succinate transport and mitochondrial respiratory function in prostate cancer]][Bastos Sant'Anna Silva 2019 Life Sciences Meeting 2019 Innsbruck AT|Cellular succinate transport and mitochondrial respiratory function in prostate cancer]])
Life Sciences Partnering 2023 Innsbruck AT + (Life Sciences Partnering 2023, Innsbruck, Austria, 2023)
Salin 2013 Abstract MiP2013 + (Life histories describe how organisms grow … Life histories describe how organisms grow, mature, reproduce and senesce, and evidence is accumulating that most of the variation in the pace of life of animals can be arranged along a slow-fast continuum. Natural variation in life history traits has largely been studied from an evolutionary viewpoint, highlighting their links to ecological factors and their fitness consequences [1]. It is only recently that researchers have focused their interest on the physiological mechanisms underlying variation in the pace of life [2]. In this context, we proposed that mitochondrial function, the main generator of both energy (adenosine triphosphate, ATP) and reactive oxygen species (ROS), may act as the keystone between the optimization of energy availability for life-history traits and the resulting oxidative cost.Using both descriptive and experimental approaches, we investigated the relationship between energy flow, oxidative stress and growth in an amphibian anuran model, the common frog Rana temporaria. We hypothesized that mitochondrial uncoupling (resulting in less ATP but also reduced ROS production) induces a slow pace of life (slow growth and slow ageing). Chronic exposure to an uncoupler (2,4-dinitrophenol) during tadpole development induced a lower growth rate associated with a decreased mitochondrial efficiency of energy transduction and an improved oxidative status (lower ROS production and low oxidative damage despite low antioxidant defenses). In addition, a comparison of physiological parameters of wild frogs exhibiting dissimilar body size showed a higher energy efficiency transduction in frogs exhibiting high growth rate.Our results suggest that (i) the mitochondrial efficiency of energy transduction could drive the amount of energy available for animal growth, and (ii) studying both facets of mitochondrial function (ATP and ROS production) allows a better understanding of the proximate mechanisms underlying life history trade-offs. Mitochondrial function under the regulation of numerous metabolites and endocrine factors, might represent the physiological link between environmental condition (temperature, resource, stressor events…) and energy allocated to animal performance. This approach is opening new avenues within an evolutionary ecological framework that aims at explaining the diversification of paces-of-life in response to environmental conditions.e in response to environmental conditions.)
Chung 2018 Integr Comp Biol + (Life history strategies, physiological tra … Life history strategies, physiological traits and behavior are thought to covary along a "pace of life" axis, with organisms at the fast end of this continuum having higher fecundity, shorter lifespan, and more rapid development, growth, and metabolic rates. Countergradient variation represents a special case of pace of life variation, in which high-latitude organisms occupy the fast end of the continuum relative to low-latitude conspecifics when compared at a common temperature. Here, we use Atlantic killifish (''Fundulus heteroclitus'') to explore the role of mitochondrial properties as a mechanism underlying countergradient variation, and thus variation in the pace of life. This species is found along the Atlantic coast of North America, through a steep latitudinal thermal gradient. The northern subspecies has faster development, more rapid growth, higher routine metabolic rate, and higher activity than the southern subspecies when compared at a common temperature. The northern subspecies also has greater mitochondrial respiratory capacity in the liver, although these differences are not evident in other tissues. The increased respiratory capacity of liver mitochondria in northern fish is associated with increases in the activity of multiple electron transport complexes, which largely reflects an increase in the amount of inner mitochondrial membrane per mitochondrion in the northern fish. There are also differences in the lipid composition of liver mitochondrial membranes, including differences in cardiolipin species, which could also influence respiratory capacity. These data suggest that variation in mitochondrial properties could, at least in part, underlie variation in the pace of life in Atlantic killifish.in the pace of life in Atlantic killifish.)
Perin 2023 Proc Natl Acad Sci U S A + (Life on earth depends on photosynthetic pr … Life on earth depends on photosynthetic primary producers that exploit sunlight to fix CO2 into biomass. Approximately half of global primary production is associated with microalgae living in aquatic environments. Microalgae also represent a promising source of biomass to complement crop cultivation, and they could contribute to the development of a more sustainable bioeconomy. Photosynthetic organisms evolved multiple mechanisms involved in the regulation of photosynthesis to respond to highly variable environmental conditions. While essential to avoid photodamage, regulation of photosynthesis results in dissipation of absorbed light energy, generating a complex trade-off between protection from stress and light-use efficiency. This work investigates the impact of the xanthophyll cycle, the light-induced reversible conversion of violaxanthin into zeaxanthin, on the protection from excess light and on biomass productivity in the marine microalgae of the genus Nannochloropsis. Zeaxanthin is shown to have an essential role in protection from excess light, contributing to the induction of nonphotochemical quenching and scavenging of reactive oxygen species. On the contrary, the overexpression of zeaxanthin epoxidase enables a faster reconversion of zeaxanthin to violaxanthin that is shown to be advantageous for biomass productivity in dense cultures in photobioreactors. These results demonstrate that zeaxanthin accumulation is critical to respond to strong illumination, but it may lead to unnecessary energy losses in light-limiting conditions and accelerating its reconversion to violaxanthin provides an advantage for biomass productivity in microalgae.ge for biomass productivity in microalgae.)
Sarovar 2010 Nature Phys + (Light-harvesting components of photosynthe … Light-harvesting components of photosynthetic organisms are complex, coupled, many-body quantum systems, in which electronic coherence has recently been shown to survive for relatively long timescales, despite the decohering effects of their environments. Here, we analyse entanglement in multichromophoric light-harvesting complexes, and establish methods for quantification of entanglement by describing necessary and sufficient conditions for entanglement and by deriving a measure of global entanglement. These methods are then applied to the Fenna–Matthews–Olson protein to extract the initial state and temperature dependencies of entanglement. We show that, although the Fenna–Matthews–Olson protein in natural conditions largely contains bipartite entanglement between dimerized chromophores, a small amount of long-range and multipartite entanglement should exist even at physiological temperatures. This constitutes the first rigorous quantification of entanglement in a biological system. Finally, we discuss the practical use of entanglement in densely packed molecular aggregates such as light-harvesting complexes.egates such as light-harvesting complexes.)
Ghysels 2013 PLoS One + (Like a majority of photosynthetic microorg … Like a majority of photosynthetic microorganisms, the green unicellular alga Chlamydomonas reinhardtii may encounter O2 deprived conditions on a regular basis. In response to anaerobiosis or in a respiration defective context, the photosynthetic electron transport chain of Chlamydomonas is remodeled by a state transition process to a conformation that favours the photoproduction of ATP at the expense of reductant synthesis. In some unicellular green algae including Chlamydomonas, anoxia also triggers the induction of a chloroplast-located, oxygen sensitive hydrogenase, which accepts electrons from reduced ferredoxin to convert protons into molecular hydrogen. Although microalgal hydrogen evolution has received much interest for its biotechnological potential, its physiological role remains unclear. By using specific Chlamydomonas mutants, we demonstrate that the state transition ability and the hydrogenase function are both critical for induction of photosynthesis in anoxia. These two processes are thus important for survival of the cells when they are transiently placed in an anaerobic environment.iently placed in an anaerobic environment.)
Ayari 2020 Commun Biol + (Like all obligate intracellular pathogens, … Like all obligate intracellular pathogens, influenza A virus (IAV) reprograms host cell's glucose and lipid metabolism to promote its own replication. However, the impact of influenza infection on white adipose tissue (WAT), a key tissue in the control of systemic energy homeostasis, has not been yet characterized. Here, we show that influenza infection induces alterations in whole-body glucose metabolism that persist long after the virus has been cleared. We report depot-specific changes in the WAT of IAV-infected mice, notably characterized by the appearance of thermogenic brown-like adipocytes within the subcutaneous fat depot. Importantly, viral RNA- and viral antigen-harboring cells are detected in the WAT of infected mice. Using in vitro approaches, we find that IAV infection enhances the expression of brown-adipogenesis-related genes in preadipocytes. Overall, our findings shed light on the role that the white adipose tissue, which lies at the crossroads of nutrition, metabolism and immunity, may play in influenza infection.immunity, may play in influenza infection.)
Huetter 2004 Biochem J + (Limitation of lifespan in replicative sene … Limitation of lifespan in replicative senescence is related to oxidative stress, which is probably both the cause and consequence of impaired mitochondrial respiratory function. The respiration of senescent human diploid fibroblasts was analysed by [[high-resolution respirometry]]. To rule out cell-cycle effects, proliferating and growth-arrested young fibroblasts were used as controls. [[Noncoupled respiration]], as normalized to citrate synthase activity, remained unchanged, reflecting a constant mitochondrial [[Electron transfer pathway]] (ET-pathway) capacity. Oligomycin-inhibited [[LEAK respiration]], however, was significantly increased in mitochondria of senescent cells, indicating a lower coupling of electron transfer to phosphorylation of ADP to ATP. In contrast, growth-arrested young fibroblasts exhibited a higher coupling control compared with proliferating controls. In living cells, partial uncoupling ([[dyscoupling]]) may lead to either decreased oxidative ATP production or a compensatory increase in [[ROUTINE respiration]]. To distinguish between these alternatives, we subtracted oligomycin-inhibited respiration from ROUTINE respiration, which allowed us to determine the part of respiratory activity coupled with ATP production. Despite substantial differences in the [[respiratory acceptor control ratio]], ranging from 4 to 11 in the different experimental groups, a fixed proportion of ET-capacity was maintained for coupled oxidative phosphorylation in all the experimental groups. This finding indicates that the senescent cells fully compensate for increased proton leakage by enhanced electron-transfer activity in the ROUTINE state. These results provide a new insight into age-associated defects in mitochondrial function and compensatory mechanisms in living cells.d compensatory mechanisms in living cells.)
Sorochkina 2012 PLoS One + (Limited uncoupling of oxidative phosphoryl … Limited uncoupling of oxidative phosphorylation could be beneficial for cells by preventing excessive generation of reactive oxygen species. Typical uncouplers are weak organic acids capable of permeating across membranes with a narrow gap between efficacy and toxicity. Aimed at designing a nontoxic uncoupler, the protonatable amino acid residue Glu was substituted for Val at the N-terminus of the pentadecapeptide gramicidin A (gA). The modified peptide [Glu1]gA exhibited high uncoupling activity in isolated mitochondria, in particular, abolishing membrane potential at the inner mitochondrial membrane with the same or even larger efficacy as gA. With mitochondria in cell culture, the depolarizing activity of [Glu1]gA was observed at concentrations by an order of magnitude lower than those of gA. On the contrary, [Glu1]gA was much less potent in forming proton channels in planar lipid bilayers than gA. Remarkably, at uncoupling concentrations, [Glu1]gA did not alter cell morphology and was nontoxic in MTT test, in contrast to gA showing high toxicity. The difference in the behavior of [Glu1]gA and gA in natural and artificial membranes could be ascribed to increased capability of [Glu1]gA to permeate through membranes and/or redistribute between different membranes. Based on the protective role of mild uncoupling, [Glu1]gA and some other proton-conducting gA analogues may be considered as prototypes of prospective therapeutic agents.totypes of prospective therapeutic agents.)
Pichaud 2012 Evolution + (Linking the mitochondrial genotype and the … Linking the mitochondrial genotype and the organismal phenotype is of paramount importance in evolution of mitochondria. In this study, we determined the differences in catalytic properties of mitochondria dictated by divergences in the siII and siIII haplogroups of Drosophila simulans using introgressions of siII mtDNA type into the siIII nuclear background. We used a novel in situ method (permeabilized fibers) that allowed us to accurately measure the consumption of oxygen by mitochondria in constructed siII-introgressed flies and in siIII-control flies. Our results showed that the catalytic capacity of the electron transport system is not impaired by introgressions, suggesting that the functional properties of mitochondria are tightly related to the mtDNA haplogroup and not to the nuclear DNA or to the mito-nuclear interactions. This is the first study, to our knowledge, that demonstrates a naturally occurring haplogroup can confer specific functional differences in aspects of mitochondrial metabolism. This study illustrates the importance of mtDNA changes on organelle evolution and highlights the potential bioenergetic and metabolic impacts that divergent mitochondrial haplogroups may have upon a wide variety of species including humans. wide variety of species including humans.)
Maekawa 2019 Cell Commun Signal + (Linoleic acid is the major fatty acid moie … Linoleic acid is the major fatty acid moiety of cardiolipin, which is central to the assembly of components involved in mitochondrial oxidative phosphorylation (OXPHOS). Although linoleic acid is an essential nutrient, its excess intake is harmful to health. On the other hand, linoleic acid has been shown to prevent the reduction in cardiolipin content and to improve mitochondrial function in aged rats with spontaneous hypertensive heart failure (HF). In this study, we found that lower dietary intake of linoleic acid in HF patients statistically correlates with greater severity of HF, and we investigated the mechanisms therein involved.HF patients, who were classified as New York Heart Association (NYHA) functional class I (n = 45), II (n = 93), and III (n = 15), were analyzed regarding their dietary intakes of different fatty acids during the one month prior to the study. Then, using a mouse model of HF, we confirmed reduced cardiolipin levels in their cardiac myocytes, and then analyzed the mechanisms by which dietary supplementation of linoleic acid improves cardiac malfunction of mitochondria.The dietary intake of linoleic acid was significantly lower in NYHA III patients, as compared to NYHA II patients. In HF model mice, both CI-based and CII-based OXPHOS activities were affected together with reduced cardiolipin levels. Silencing of CRLS1, which encodes cardiolipin synthetase, in cultured cardiomyocytes phenocopied these events. Feeding HF mice with linoleic acid improved both CI-based and CII-based respiration as well as left ventricular function, together with an increase in cardiolipin levels. However, although assembly of the respirasome (i.e., CI/CIII2/CIV complex), as well as assembly of CII subunits and the CIII2/CIV complex statistically correlated with cardiolipin levels in cultured cardiomyocytes, respirasome assembly was not notably restored by dietary linoleic acid in HF mice. Therefore, although linoleic acid may significantly improve both CI-based and CII-based respiration of cardiomyocytes, respirasomes impaired by HF were not easily repaired by the dietary intake of linoleic acid.Dietary supplement of linoleic acid is beneficial for improving cardiac malfunction in HF, but is unable to completely cure HF.ng cardiac malfunction in HF, but is unable to completely cure HF.)
Watts 2017 Genetics + (Lipid and carbohydrate metabolism are high … Lipid and carbohydrate metabolism are highly conserved processes that affect nearly all aspects of organismal biology. Caenorhabditis elegans eat bacteria, which consist of lipids, carbohydrates, and proteins that are broken down during digestion into fatty acids, simple sugars, and amino acid precursors. With these nutrients, C. elegans synthesizes a wide range of metabolites that are required for development and behavior. In this review, we outline lipid and carbohydrate structures as well as biosynthesis and breakdown pathways that have been characterized in C. elegans. We bring attention to functional studies using mutant strains that reveal physiological roles for specific lipids and carbohydrates during development, aging, and adaptation to changing environmental conditions.tion to changing environmental conditions.)
Yang 2023 Redox Biol + (Lipid peroxidation and redox imbalance are … Lipid peroxidation and redox imbalance are hallmarks of ferroptosis, an iron-dependent form of cell death. Growing evidence suggests that dysregulation in glycolipid metabolism and iron homeostasis substantially contribute to the development of hepatocellular carcinoma (HCC). However, there is still a lack of comprehensive understanding regarding the specific transcription factors that are capable of coordinating glycolipid and redox homeostasis to initiate the onset of ferroptosis. We discovered that overexpression of SOX8 leads to impaired mitochondria integrate, increased oxidative stress, and enhanced lipid peroxidation. These effects can be attributed to the inhibitory impact of SOX8 on ''de novo'' lipogenesis, glycolysis, the tricarboxylic acid cycle (TCA), and the pentose phosphate pathway (PPP). Additionally, upregulation of SOX8 results in reduced synthesis of NADPH, disturbance of redox homeostasis, disruption of mitochondrial structure, and impairment of the electron transport chain. Furthermore, the overexpression of SOX8 enhances the process of ferroptosis by upregulating the expression of genes associated with ferroptosis and elevating intracellular levels of ferrous ion. Importantly, the overexpressing of SOX8 has been observed to inhibit the proliferation of HCC in immunodeficient animal models. In conclusion, the findings suggest that SOX8 has the ability to alter glycolipid and iron metabolism of HCC cells, hence triggering the process of ferroptosis. The results of our study present a novel strategy for targeting ferroptosis in the therapy of HCC.rgeting ferroptosis in the therapy of HCC.)
Katunga 2015 Mol Metab + (Lipid peroxides and their reactive aldehyd … Lipid peroxides and their reactive aldehyde derivatives (LPPs) have been linked to obesity-related pathologies, but whether they have a causal role has remained unclear. Glutathione peroxidase 4 (GPx4) is a selenoenzyme that selectively neutralizes lipid hydroperoxides, and human gpx4 gene variants have been associated with obesity and cardiovascular disease in epidemiological studies. This study tested the hypothesis that LPPs underlie cardio-metabolic derangements in obesity using a high fat, high sucrose (HFHS) diet in gpx4 haploinsufficient mice (GPx4(+/-)) and in samples of human myocardium.Wild-type (WT) and GPx4(+/-) mice were fed either a standard chow (CNTL) or HFHS diet for 24 weeks, with metabolic and cardiovascular parameters measured throughout. Biochemical and immuno-histological analysis was performed in heart and liver at termination of study, and mitochondrial function was analyzed in heart. Biochemical analysis was also performed on samples of human atrial myocardium from a cohort of 103 patients undergoing elective heart surgery.Following HFHS diet, WT mice displayed moderate increases in 4-hydroxynonenal (HNE)-adducts and carbonyl stress, and a 1.5-fold increase in GPx4 enzyme in both liver and heart, while gpx4 haploinsufficient (GPx4(+/-)) mice had marked carbonyl stress in these organs accompanied by exacerbated glucose intolerance, dyslipidemia, and liver steatosis. Although normotensive, cardiac hypertrophy was evident with obesity, and cardiac fibrosis more pronounced in obese GPx4(+/-) mice. Mitochondrial dysfunction manifesting as decreased fat oxidation capacity and increased reactive oxygen species was also present in obese GPx4(+/-) but not WT hearts, along with up-regulation of pro-inflammatory and pro-fibrotic genes. Patients with diabetes and hyperglycemia exhibited significantly less GPx4 enzyme and greater HNE-adducts in their hearts, compared with age-matched non-diabetic patients.These findings suggest LPPs are key factors underlying cardio-metabolic derangements that occur with obesity and that GPx4 serves a critical role as an adaptive countermeasure.itical role as an adaptive countermeasure.)
Kiebish 2012 J Biol Chem + (Lipidomic regulation of mitochondrial card … Lipidomic regulation of mitochondrial cardiolipin content and molecular species composition is a prominent regulator of bioenergetic efficiency. However, the mechanisms controlling cardiolipin metabolism during health or disease progression have remained elusive. Herein, we demonstrate that cardiac myocyte-specific transgenic expression of cardiolipin synthase results in accelerated cardiolipin lipidomic flux that impacts multiple aspects of mitochondrial bioenergetics and signaling. During the postnatal period, cardiolipin synthase transgene expression results in marked changes in the temporal course of maturation of cardiolipin molecular species during development. In adult myocardium, cardiolipin synthase transgene expression leads to a marked increase in symmetric tetra18:2 molecular species without a change in total cardiolipin content. Mechanistically, these alterations result from increased cardiolipin remodeling by sequential phospholipase and transacylase/acyltransferase activities in conjunction with a decrease in phosphatidylglycerol content. Moreover, cardiolipin synthase transgene expression results in alterations in signaling metabolites including a marked increase in the cardioprotective eicosanoid 14,15 EET. Examination of mitochondrial bioenergetic function by high resolution respirometry demonstrated that cardiolipin synthase transgene expression resulted in improved mitochondrial bioenergetic efficiency demonstrated by enhanced electron transport chain coupling using multiple substrates as well as by salutary changes in Complex III and IV activities. Furthermore, transgenic expression of cardiolipin synthase attenuated maladaptive cardiolipin remodeling and bioenergetic inefficiency in myocardium rendered diabetic by streptozotocin treatment. Collectively, these results demonstrate the unanticipated role of cardiolipin synthase in maintaining physiologic membrane structure and function even under metabolic stress thereby identifying cardiolipin synthase as a novel therapeutic target to attenuate mitochondrial dysfunction in diabetic cardiomyopathy.al dysfunction in diabetic cardiomyopathy.)
Chambers 2021 JCI Insight + (Lipin 1 is a bifunctional protein that is … Lipin 1 is a bifunctional protein that is a transcriptional regulator and has phosphatidic acid (PA) phosphohydrolase activity, which dephosphorylates PA to generate diacylglycerol. Human lipin 1 mutations lead to episodic rhabdomyolysis, and some affected patients exhibit cardiac abnormalities, including exercise-induced cardiac dysfunction and cardiac triglyceride accumulation. Furthermore, lipin 1 expression is deactivated in failing heart, but the effects of lipin 1 deactivation in myocardium are incompletely understood. We generated mice with cardiac-specific lipin 1 KO (cs-Lpin1-/-) to examine the intrinsic effects of lipin 1 in the myocardium. Cs-Lpin1-/- mice had normal systolic cardiac function but mild cardiac hypertrophy. Compared with littermate control mice, PA content was higher in cs-Lpin1-/- hearts, which also had an unexpected increase in diacylglycerol and triglyceride content. Cs-Lpin1-/- mice exhibited diminished cardiac cardiolipin content and impaired mitochondrial respiration rates when provided with pyruvate or succinate as metabolic substrates. After transverse aortic constriction-induced pressure overload, loss of lipin 1 did not exacerbate cardiac hypertrophy or dysfunction. However, loss of lipin 1 dampened the cardiac ionotropic response to dobutamine and exercise endurance in association with reduced protein kinase A signaling. These data suggest that loss of lipin 1 impairs cardiac functional reserve, likely due to effects on glycerolipid homeostasis, mitochondrial function, and protein kinase A signaling.fects on glycerolipid homeostasis, mitochondrial function, and protein kinase A signaling.)
Schweitzer 2019 FASEB J + (Lipin 1 regulates glycerolipid homeostasis … Lipin 1 regulates glycerolipid homeostasis by acting as a phosphatidic acid phosphohydrolase (PAP) enzyme in the triglyceride-synthesis pathway and by regulating transcription factor activity. Mutations in human lipin 1 are a common cause of recurrent rhabdomyolysis in children. Mice with constitutive whole-body lipin 1 deficiency have been used to examine mechanisms connecting lipin 1 deficiency to myocyte injury. However, that mouse model is confounded by lipodystrophy not phenocopied in people. Herein, 2 muscle-specific mouse models were studied: 1) Lpin1 exon 3 and 4 deletion, resulting in a hypomorphic protein without PAP activity, but which preserved transcriptional coregulatory function; and 2) Lpin1 exon 7 deletion, resulting in total protein loss. In both models, skeletal muscles exhibited a chronic myopathy with ongoing muscle fiber necrosis and regeneration and accumulation of phosphatidic acid and, paradoxically, diacylglycerol. Additionally, lipin 1-deficient mice had abundant, but abnormal, mitochondria likely because of impaired autophagy. Finally, these mice exhibited increased plasma creatine kinase following exhaustive exercise when unfed. These data suggest that mice lacking lipin 1-mediated PAP activity in skeletal muscle may serve as a model for determining the mechanisms by which lipin 1 deficiency leads to myocyte injury and for testing potential therapeutic approaches. testing potential therapeutic approaches.)
Song 2018 Proc Natl Acad Sci U S A + (Lipocalin-2 (Lcn2), a critical component o … Lipocalin-2 (Lcn2), a critical component of the innate immune response which binds siderophores and limits bacterial iron acquisition, can elicit spillover adverse proinflammatory effects. Here we show that holo-Lcn2 (Lcn2-siderophore-iron, 1:3:1) increases mitochondrial reactive oxygen species (ROS) generation and attenuates mitochondrial oxidative phosphorylation in adult rat primary cardiomyocytes in a manner blocked by N-acetyl-cysteine or the mitochondria-specific antioxidant SkQ1. We further demonstrate using siderophores 2,3-DHBA (2,3-dihydroxybenzoic acid) and 2,5-DHBA that increased ROS and reduction in oxidative phosphorylation are direct effects of the siderophore component of holo-Lcn2 and not due to apo-Lcn2 alone. Extracellular apo-Lcn2 enhanced the potency of 2,3-DHBA and 2,5-DHBA to increase ROS production and decrease mitochondrial respiratory capacity, whereas intracellular apo-Lcn2 attenuated these effects. These actions of holo-Lcn2 required an intact plasma membrane and were decreased by inhibition of endocytosis. The hearts, but not serum, of Lcn2 knockout (LKO) mice contained lower levels of 2,5-DHBA compared with wild-type hearts. Furthermore, LKO mice were protected from ischemia/reperfusion-induced cardiac mitochondrial dysfunction. Our study identifies the siderophore moiety of holo-Lcn2 as a regulator of cardiomyocyte mitochondrial bioenergetics.cardiomyocyte mitochondrial bioenergetics.)
Saillan-Barreau 2008 J Acquir Immune Defic Syndr + (Lipodystrophic syndrome is a major side ef … Lipodystrophic syndrome is a major side effect of antiviral therapy leading to profound disturbances in adipose tissue. Human preadipocyte primary culture represents a model to understand mechanisms by which antiretroviral drugs alter adipocyte biology. The aim of this study was to evaluate the effects of various protease and nucleoside reverse transcriptase inhibitors in this model. We tested the effect of drugs on triglyceride accumulation and expression of specific genes by real-time polymerase chain reaction. To determine differential mechanisms by which the efficient drugs operate, we studied mitochondrial effects by evaluating oxygen consumption rates and nuclear lamina alteration by immunocytology. Only stavudine and nelfinavir, both at 10 microM, altered human adipose cell differentiation, as shown by reduced triglyceride accumulation. Our studies revealed that stavudine increased expression of genes such as PGC1 and LPL and affected mitochondrial respiration. Cells treated with nelfinavir had a lower expression of PPARgamma, LPL, and ap2 and presented disorganization of lamin A/C. Our data suggest for the first time in a model of human adipocytes differentiated in vitro that stavudine and nelfinavir interfere with the process of differentiation by 2 distinct mechanisms. This may be particularly relevant in understanding the physiopathologic mechanisms underlying the lipodystrophic syndrome.ms underlying the lipodystrophic syndrome.)
Hansen 2015 Shock + (Lipopolysaccharides (LPS) are prevalent pa … Lipopolysaccharides (LPS) are prevalent pathogenic molecules that are found within tissues and blood. Elevated circulating LPS is a feature of obesity and sepsis, both of which are associated with mitochondrial abnormalities that are key pathological features of LPS excess. However, the mechanism of LPS-induced mitochondrial alterations remains poorly understood. Herein we demonstrate the necessity of sphingolipid accrual in mediating altered mitochondrial physiology in skeletal muscle following LPS exposure. In particular, we found LPS elicited disparate effects on the sphingolipids dihydroceramides (DhCer) and ceramides (Cer) in both cultured myotubes and in muscle of LPS-injected mice. Although LPS-treated myotubes had reduced DhCer and increased Cer as well as increased mitochondrial respiration, muscle from LPS-injected mice manifested a reverse trend, namely elevated DhCer, but reduced Cer as well as reduced mitochondrial respiration. In addition, we found that LPS treatment caused mitochondrial fission, likely via dynamin-related protein 1, and increased oxidative stress. However, inhibition of ''de novo'' sphingolipid biosynthesis via myriocin protected normal mitochondrial function in spite of LPS, but inhibition of DhCer desaturase 1, which increases DhCer, but not Cer, exacerbated mitochondrial respiration with LPS. In an attempt to reconcile the incongruent effects of LPS in isolated muscle cells and whole muscle tissue, we incubated myotubes with conditioned medium from treated macrophages. In contrast to direct myotube LPS treatment, conditioned medium from LPS-treated macrophages reduced myotube respiration, but this was again mitigated with sphingolipid inhibition. Thus, macrophage sphingolipid production appears to be necessary for LPS-induced mitochondrial alterations in skeletal muscle tissue.ial alterations in skeletal muscle tissue.)
Kolleritsch 2019 Cardiovasc Res + (Lipotoxic cardiomyopathy in diabetic and o … Lipotoxic cardiomyopathy in diabetic and obese patients typically encompasses increased cardiac fatty acid (FA) uptake eventually surpassing the mitochondrial oxidative capacity. Lowering FA utilization via inhibition of lipolysis represents a strategy to counteract the development of lipotoxic heart dysfunction. However, defective cardiac triacylglycerol (TAG) catabolism and FA oxidation in humans (and mice) carrying mutated ATGL alleles provokes lipotoxic heart dysfunction questioning a therapeutic approach to decrease cardiac lipolysis. Interestingly, decreased lipolysis via cardiac overexpression of Perilipin 5 (Plin5), a binding partner of ATGL, is compatible with normal heart function and lifespan despite massive cardiac lipid accumulation. Herein, we decipher mechanisms that protect Plin5 transgenic mice from the development of heart dysfunction.We generated mice with cardiac-specific overexpression of Plin5 encoding a serine-155 to alanine exchange (Plin5-S155A) of the protein kinase A phosphorylation site, which has been suggested as a prerequisite to stimulate lipolysis and may play a crucial role in the preservation of heart function. Plin5-S155A mice showed a substantial increase in cardiac TAG and ceramide levels, which was comparable to mice overexpressing non-mutated Plin5. Lipid accumulation was compatible with normal heart function even under mild stress. Plin5-S155A mice showed reduced cardiac FA oxidation but normal ATP production and changes in the Plin5-S155A phosphoproteome compared to Plin5 transgenic mice. Interestingly, mitochondrial recruitment of dynamin-related protein 1 (Drp1) was markedly reduced in cardiac muscle of Plin5-S155A and Plin5 transgenic mice accompanied by decreased phosphorylation of mitochondrial fission factor, a mitochondrial receptor of Drp1.This study suggests that low cardiac lipolysis is associated with reduced mitochondrial fission and may represent a strategy to combat the development of lipotoxic heart dysfunction.© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.f of the European Society of Cardiology.)
Bernardinelli 2017 PLoS One + (Lipoyl(Octanoyl) Transferase 2 (LIPT2) is … Lipoyl(Octanoyl) Transferase 2 (LIPT2) is a protein involved in the post-translational modification of key energy metabolism enzymes in humans. Defects of lipoic acid synthesis and transfer start to emerge as causes of fatal or severe early-onset disease. We show that the first 31 amino acids of the N-terminus of LIPT2 represent a mitochondrial targeting sequence and inhibition of the transit of LIPT2 to the mitochondrion results in apoptotic cell death associated with activation of the apoptotic volume decrease (AVD) current in normotonic conditions, as well as over-activation of the swelling-activated chloride current (IClswell), mitochondrial membrane potential collapse, caspase-3 cleavage and nuclear DNA fragmentation. The findings presented here may help elucidate the molecular mechanisms underlying derangements of lipoic acid biosynthesis. derangements of lipoic acid biosynthesis.)
Alegre 2019 Am J Transplant + (Literature Watch 969 BY MARIA-LUISA ALEGRE … Literature Watch 969BY MARIA-LUISA ALEGRE, MD, PHDRegulatory T cells that express the transcription factor FoxP3 (Tregs) are essential for the maintenance of immune homeostasis. A lack of Tregs at birth, Treg deletion in adulthood or the selective ablation of the T cell receptor (TCR) in Tregs or of key membrane-associated or signaling molecules, such as CTLA-4, CD28 or PTEN, have been shown in mice to lead to dramatic lymphoproliferative disease, tissue infiltration by activated conventional T cells (Tconvs) and, in many cases, animal death. This underscores the importance of continuous and proper activation of Tregs throughout life. Tregs have a unique metabolic profile, including greater mitochondrial metabolism than Tconvs. Weinberg and colleagues investigate whether mitochondrial respiration (Figure 1) is necessary for the ability of Tregs to maintain homeostasis, and find that tampering with Treg–mitochondrial complex III triggers fatal autoimmunity.l complex III triggers fatal autoimmunity.)
D'Amico 2019 Mol Cell + (Little information is available about how … Little information is available about how post-transcriptional mechanisms regulate the aging process. Here, we show that the RNA-binding protein Pumilio2 (PUM2), which is a translation repressor, is induced upon aging and acts as a negative regulator of lifespan and mitochondrial homeostasis. Multi-omics and cross-species analyses of PUM2 function show that it inhibits the translation of the mRNA encoding for the mitochondrial fission factor (''Mff''), thereby impairing mitochondrial fission and mitophagy. This mechanism is conserved in ''C. elegans'' by the PUM2 ortholog PUF-8. ''puf-8'' knock-down in old nematodes and Pum2 CRISPR/Cas9-mediated knockout in the muscles of elderly mice enhances mitochondrial fission and mitophagy in both models, hence improving mitochondrial quality control and tissue homeostasis. Our data reveal how a PUM2-mediated layer of post-transcriptional regulation links altered ''Mff'' translation to mitochondrial dynamics and mitophagy, thereby mediating age-related mitochondrial dysfunctions.Copyright © 2018 Elsevier Inc. All rights reserved. 2018 Elsevier Inc. All rights reserved.)
Triska 2019 Cancer Res + (Little is known about the spectrum of mito … Little is known about the spectrum of mitochondrial DNA (mtDNA) mutations across pediatric malignancies. In this study, we analyzed matched tumor and normal whole genome sequencing data from 616 pediatric patients with hematopoietic malignancies, solid tumors, and brain tumors. We identified 391 mtDNA mutations in 284 tumors including 45 loss-of-function mutations, which clustered at four statistically significant hotspots in MT-COX3, MT-ND4, and MT-ND5, and at a mutation hotspot in MT-tRNA-MET. A skewed ratio (4.83) of nonsynonymous versus synonymous (dN/dS) mtDNA mutations with high statistical significance was identified on the basis of Monte Carlo simulations in the tumors. In comparison, opposite ratios of 0.44 and 0.93 were observed in 616 matched normal tissues and in 249 blood samples from children without cancer, respectively. mtDNA mutations varied by cancer type and mtDNA haplogroup. Collectively, these results suggest that deleterious mtDNA mutations play a role in the development and progression of pediatric cancers. SIGNIFICANCE: This pan-cancer mtDNA study establishes the landscape of germline and tumor mtDNA mutations and identifies hotspots of tumor mtDNA mutations to pinpoint key mitochondrial functions in pediatric malignancies.drial functions in pediatric malignancies.)
Laehteenmaeki 2022 Physiol Rep + (Little is known how acute exercise-induced … Little is known how acute exercise-induced inflammation and metabolic stress affect immune cell bioenergetics and the portion of its components. Therefore, we investigated acute effects of eccentric-only (E), concentric-only (C) and combined eccentric-concentric resistance exercise (E + C) bouts on cellular respiration of peripheral blood mononuclear cells (PBMCs). Twelve strength-trained young men performed bench press resistance exercises in randomized order. Venous blood samples were drawn at pre-, 5 min post- and 24 h post-exercise. Several PBMC respiration states were measured using high-resolution respirometry. Levels of leukocytes, interleukin 6 (IL-6), C-reactive protein (CRP), creatine kinase (CK), blood lactate and maximum voluntary isometric force were measured from the same time points. Effects of blood lactate and pH change on bioenergetics of PBMCs were investigated ''ex vivo''. PBMC routine respiration (p = 0.017), free routine capacity (p = 0.025) and ET-capacity (p = 0.038) decreased immediately after E + C. E responded in opposite manner 5 min post-exercise compared to E + C (p = 0.013) and C (p = 0.032) in routine respiration, and to E + C in free routine activity (p = 0.013). E + C > C > E was observed for increased lactate levels and decreased isometric force that correlated with routine respiration (R = -0.369, p = 0.035; R = 0.352, p = 0.048). Lactate and pH change did not affect bioenergetics of PBMCs. Acute resistance exercise affected cellular respiration of PBMCs, with training volume and the amount of metabolic stress appear influential. Results suggest that acute inflammation response does not contribute to changes seen in cellular respiration, but the level of peripheral muscle fatigue and metabolic stress could be explaining factors.nd metabolic stress could be explaining factors.)
Bellance 2009 Int J Biochem Cell Biol + (Little is known on the metabolic profile o … Little is known on the metabolic profile of lung tumors and the reminiscence of embryonic features. Herein, we determined the bioenergetic profiles of human fibroblasts taken from lung epidermoid carcinoma (HLF-a) and fetal lung (MRC5). We also analysed human lung tumors and their surrounding healthy tissue from four patients with adenocarcinoma. On these different models, we measured functional parameters (cell growth rates in oxidative and glycolytic media, respiration, ATP synthesis and PDH activity) as well as compositional features (expression level of various energy proteins and upstream transcription factors). The results demonstrate that both the lung fetal and cancer cell lines produced their ATP predominantly by glycolysis, while oxidative phosphorylation was only capable of poor ATP delivery. This was explained by a decreased mitochondrial biogenesis caused by a lowered expression of [[PGC1α]] (as shown by RT-PCR and Western blot) and mtTFA. Consequently, the relative expression of glycolytic versus OXPHOS markers was high in these cells. Moreover, the re-activation of mitochondrial biogenesis with resveratrol induced cell death specifically in cancer cells. A consistent reduction of mitochondrial biogenesis and the subsequent alteration of respiratory capacity was also observed in lung tumors, associated with a lower expression level of bcl2. Our data give a better characterization of lung cancer cells' metabolic alterations which are essential for growth and survival. They designate mitochondrial biogenesis as a possible target for anti-cancer therapy.a possible target for anti-cancer therapy.)
Robach 2018 Scand J Med Sci Sports + (Live high - train low (LHTL) using hypobar … Live high - train low (LHTL) using hypobaric hypoxia was previously found to improve sea-level endurance performance in well-trained individuals, however confirmatory controlled data in athletes are lacking. Here we test the hypothesis that natural-altitude LHTL improves aerobic performance in cross-country skiers, in conjunction with expansion of total hemoglobin mass (Hbmass, carbon-monoxide rebreathing technique) promoted by accelerated erythropoiesis. Following duplicate baseline measurements at sea level over the course of two weeks, nineteen Norwegian cross-country skiers (three women, sixteen men, age 20±2 yr, maximal oxygen uptake (VO2 max) 69±5 ml.min-1.kg-1) were assigned to 26 consecutive nights spent either at low (1035m, Control, n=8) or moderate altitude (2207m, daily exposure 16.7±0.5 hours, LHTL, n=11). All athletes trained together daily at a common location ranging from 550-1500m (21.2% of training time at 550m, 44.2% at 550-800m, 16.6% at 800-1100m, 18.0% at 1100-1500m). Three test sessions at sea level were performed over the first three weeks after intervention. Despite the demonstration of nocturnal hypoxemia at moderate altitude (pulse oximetry), LHTL had no specific effect on serum erythropoietin, reticulocytes, Hbmass, VO2 max or 3000-m running performance. Also LHTL had no specific effect on i) running economy (VO2 assessed during steady-state submaximal exercise), ii) respiratory capacities or efficiency of the skeletal muscle (biopsy), and iii) diffusing capacity of the lung. The present study, showing similar physiological responses and performance improvements in the two groups following intervention, suggests that in young cross-country skiers, improvements in sea-level aerobic performance associated with LHTL may not be due to moderate altitude acclimatization. This article is protected by copyright. All rights reserved.ed with LHTL may not be due to moderate altitude acclimatization. This article is protected by copyright. All rights reserved.)
Siow 2022 J Cell Sci + (Liver cancers, including hepatocellular ca … Liver cancers, including hepatocellular carcinoma (HCC), are the second leading cause of cancer death worldwide, and novel therapeutic strategies are still highly needed. Recently, the endolysosomal cation channel TRPML1 (also known as MCOLN1) has gained focus in cancer research because it represents an interesting novel target. We utilized the recently developed isoform-selective TRPML1 activator ML1-SA1 and the CRISPR/Cas9 system to generate tools for overactivation and loss-of-function studies on TRPML1 in HCC. After verification of our tools, we investigated the role of TRPML1 in HCC by studying proliferation, apoptosis and proteomic alterations. Furthermore, we analyzed mitochondrial function in detail by performing confocal and transmission electron microscopy combined with SeahorseTM and Oroboros® functional analysis. We report that TRPML1 overactivation mediated by a novel, isoform-selective small-molecule activator induces apoptosis by impairing mitochondrial function in a Ca2+-dependent manner. Additionally, TRPML1 loss-of-function deregulates mitochondrial renewal, which leads to proliferation impairment. Thus, our study reveals a novel role for TRPML1 as regulator of mitochondrial function and its modulators as promising molecules for novel therapeutic options in HCC therapy. promising molecules for novel therapeutic options in HCC therapy.)
Hoene 2021 Mol Metab + (Liver mitochondria adapt to high calorie i … Liver mitochondria adapt to high calorie intake. We investigated how exercise alters the early compensatory response of mitochondria and thus prevents fatty liver disease as a long-term consequence of overnutrition.We compared the effects of a steatogenic high-energy diet (HED, for 6 weeks) on mitochondrial metabolism of sedentary and treadmill-trained C57BL/6N mice. We applied multi-OMICs analyses to study the alterations in the proteome, transcriptome and lipids in isolated mitochondria of liver and skeletal muscle as well as in whole tissue and examined the functional consequences by high resolution respirometry.HED increased the respiratory capacity of isolated liver mitochondria, both in sedentary and in trained mice. However, proteomics analysis of the mitochondria and transcriptomics indicated that training modified the adaptation of the hepatic metabolism to HED on the level of respiratory complex I, glucose oxidation, pyruvate and acetyl-CoA metabolism and lipogenesis. Training also counteracted the HED-induced increase in fasting insulin, glucose tolerance, and liver fat. This was accompanied by lower diacylglycerol species and JNK phosphorylation in the livers of trained HED-fed mice, two mechanisms that can reverse hepatic insulin resistance. In skeletal muscle, the combination of HED and training improved the oxidative capacity to a greater extent than training alone by increasing respiration of isolated mitochondria and total mitochondrial protein content.We provide a comprehensive insight into the early adaptations of mitochondria in liver and skeletal muscle to HED and endurance training. Our results suggest that exercise disconnects the HED-induced increase in mitochondrial substrate oxidation from pyruvate and acetyl-CoA-driven lipid synthesis. This could contribute to the prevention of deleterious long-term effects of high fat and sugar intake on hepatic mitochondrial function and insulin sensitivity.hondrial function and insulin sensitivity.)
Ma 2017 J Membr Biol + (Liver mitochondria are involved in several … Liver mitochondria are involved in several important life processes; mitochondrial dysfunction and disorders are implicated in several human diseases. Alcohol permeates all tissues of the body and exerts some intrinsic hepatotoxicity. In this work, our results demonstrated that ethanol caused a series of mitochondria permeability transition pore (MPTP) opening factors such as mitochondrial swelling, increased permeability of H+ and K+, collapsed membrane potential, and increased membrane fluidity. Furthermore, mitochondrial ultrastructure alternation observed clearly by transmission electron microscopy and the release of Cytochrome c could explain the MPTP opening from another aspect. Moreover, ethanol damaged the mitochondrial respiration system and induced disturbance of mitochondrial energy metabolism which was monitored by polarographic and microcalorimetric methods, respectively. Considered together, these damages may promote both apoptotic and necrotic cell death and contribute to the onset or progression alcohol-induced liver diseases.rogression alcohol-induced liver diseases.)
Sobotka 2017 Thesis + (Liver mitochondria play a crucial role in … Liver mitochondria play a crucial role in intermediary metabolism and main metabolic pathways. We evaluated the pharmacological effect on liver mitochondria ''in vitro'' using two novel anticancer drugs: 3-bromopyruvate and α-tocopheryl succinate. Metabolic influence on liver mitochondria was performed ''in vivo'' by high fat and high cholesterol diet. Toxicity of both drugs was evaluated in cell cultures of hepatocytes isolated from rat and mouse liver. The effect of anticancer drugs on liver mitochondrial functions ''in vitro'' was studied on suspensions of isolated liver mitochondria, tissue homogenate and permeabilized hepatocytes. Mitochondrial respiration was measured using high-resolution respirometry. 3-bromopyruvate caused morphological and functional damage of primary rat and mouse hepatocytes in cell cultures; this toxic effect was accompanied by an increase of reactive oxygen species production and mitochondrial dysfunction. 3-bromopyruvate decreased the oxygen consumption of mitochondria energized by substrates for complex I and complex II. α-Tocopheryl succinate caused a decrease of succinate-dependent respiration in all experimental models both in coupled and in uncoupled states. The most pronounced effect of α-tocopheryl succinate was apparent in isolated mitochondria and the least pronounced effect was observed in permeabilized hepatocytes. High fat and high cholesterol diet caused changes of liver mitochondrial functions which were dependent on duration of the treatment. The maximal capacity of oxidative phosphorylation was increased after three weeks of the experiment, but followed by a decline after 6 weeks and later in comparison to normal diet controls. The oxidation of Krebs cycle substrates was significantly inhibited after 12 and more weeks of high fat diet feeding. On the other hand β- oxidation of fatty acids was increased already after one week of high fat diet feeding. The capacity of fatty acids oxidation returned to control level after 24 weeks of experimental diet.level after 24 weeks of experimental diet.)
Mukherjee 2021 JCI Insight + (Liver regeneration is critical to survival … Liver regeneration is critical to survival after traumatic injuries, exposure to hepatotoxins, or surgical interventions, yet the underlying signaling and metabolic pathways remain unclear. In this study, we show that hepatocyte-specific loss of the mitochondrial deacetylase SIRT3 drastically impairs regeneration and worsens mitochondrial function after partial hepatectomy. Sirtuins, including SIRT3, require NAD as a cosubstrate. We previously showed that the NAD precursor nicotinamide riboside (NR) promotes liver regeneration, but whether this involves sirtuins has not been tested. Here, we show that despite their NAD dependence and critical roles in regeneration, neither SIRT3 nor its nuclear counterpart SIRT1 is required for NR to enhance liver regeneration. NR improves mitochondrial respiration in regenerating WT or mutant livers and rapidly increases oxygen consumption and glucose output in cultured hepatocytes. Our data support a direct enhancement of mitochondrial redox metabolism as the mechanism mediating improved liver regeneration after NAD supplementation and exclude signaling via SIRT1 and SIRT3. Therefore, we provide the first evidence to our knowledge for an essential role for a mitochondrial sirtuin during liver regeneration and insight into the beneficial effects of NR.insight into the beneficial effects of NR.)
Liu 2021 Front Physiol + (Living at high altitudes is extremely chal … Living at high altitudes is extremely challenging as it entails exposure to hypoxia, low temperatures, and high levels of UV radiation. However, the Tibetan population has adapted to such conditions on both a physiological and genetic level over 30 000-40 000 years. It has long been speculated that fetal growth restriction is caused by abnormal placental development. We previously demonstrated that placentas from high-altitude Tibetans were protected from oxidative stress induced by labor compared to those of European descent. However, little is known about how placental mitochondria change during high-altitude adaptation. In this study, we aimed to uncover the mechanism of such adaptation by studying the respiratory function of the placental mitochondria of high-altitude Tibetans, lower-altitude Tibetans, and lower-altitude Chinese Han. We discovered that mitochondrial respiration was greater in high-altitude than in lower-altitude Tibetans in terms of OXPHOS via Complexes I and I+II, ETSmax capacity, and non-phosphorylating respiration, whereas non-ETS respiration, LEAK/ETS, and OXPHOS via Complex IV did not differ. Respiration in lower-altitude Tibetans and Han was similar for all tested respiratory states. Placentas from high-altitude Tibetan women were protected from acute ischemic/hypoxic insult induced by labor, and increased mitochondrial respiration may represent an acute response that induces mitochondrial adaptations.acute response that induces mitochondrial adaptations.)
Joshi 2022 Abstract Bioblast + (Living eukaryotic cells typically contain … Living eukaryotic cells typically contain large quantities of highly dynamic mitochondria, which sustain the cells’ energy and redox homeostasis. Growing evidence suggests that mitochondria can functionally differ among but also within cells. The extent and biological significance of mitochondrial diversity is still largely unexplored, due to technical limitations that hamper profiling of individual organelles. Previous measurements of the cell’s interior have shown that membrane-bound compartments respond to metabolic manipulation by changes in their surface stiffness, suggesting that mechano-physical properties are a valuable readout of mitochondrial function. We here present the establishment of a robust multi-step analysis pipeline that allows one to profile mechano-physical properties of single mitochondria at the nanoscale using Atomic Force Microscopy (AFM) [1]. Firstly, we developed a rapid cell-type specific isolation protocol (mRACE), which selectively functionalizes mitochondria with biotin, facilitating isolation by streptavidin decorated microbeads. We established the technique for human and rat cell cultures, the invertebrate Caenorhabditis elegans, and the model plant Arabidopsis thaliana. Based on this versatile tool, we detected diversity of mitochondrially associated proteins among different tissues, reflecting the trophic condition of the source material. Secondly, a rapid filtration-based mitochondria isolation protocol was established, which was combined with mRACE. Lastly, we established an AFM analysis platform, which generates 3D maps of the nano-topography and mechano-physical properties of individual mitochondria. The comparison of mitochondria with each other revealed an unprecedented diversity in their mechano-physical properties and suggests that shape is not the sole determining parameter for mitochondrial outer membrane stiffness. We expect our results to not only introduce a new dimension for basic mitochondrial research, but in addition to open the door for the exploitation of individual mitochondria for diagnostic characterization [1].# Saurabh J, Hater F, Eirich J, Palovaara J, Ellinghaus H, Heinkow P, Callenius H, Peter A, Schweser O, Kubitschke M, Madduri MK, Mathew AJ, Ciacchi LC, Kirstein J, Maedler K, Masseck OA, Finkemeier I, Radmacher M, Groß-Hardt R (2021) A versatile mitochondria isolation- and analysis-pipeline generates 3D nano-topographies and mechano-physical surface maps of single organelles. https://doi.org/10.1101/2021.10.31.466655 - The present abstract is a copy from the preprint.t abstract is a copy from the preprint. )
Heinonen 2016 Hum Exp Toxicol + (Local anesthetic toxicity is thought to be … Local anesthetic toxicity is thought to be mediated partly by inhibition of cardiac mitochondrial function. Intravenous (i.v.) lipid emulsion may overcome this energy depletion, but doses larger than currently recommended may be needed for rescue effect. In this randomized study with anesthetized pigs, we compared the effect of a large dose, 4 mL/kg, of i.v. 20% Intralipid® (n = 7) with Ringer's acetate (n = 6) on cardiovascular recovery after a cardiotoxic dose of bupivacaine. We also examined mitochondrial respiratory function in myocardial cell homogenates analyzed promptly after needle biopsies from the animals. Bupivacaine plasma concentrations were quantified from plasma samples. Arterial blood pressure recovered faster and systemic vascular resistance rose more rapidly after Intralipid than Ringer's acetate administration (p < 0.0001), but Intralipid did not increase cardiac index or left ventricular ejection fraction. The lipid-based mitochondrial respiration was stimulated by approximately 30% after Intralipid (p < 0.05) but unaffected by Ringer's acetate. The mean (standard deviation) area under the concentration-time curve (AUC) of total bupivacaine was greater after Intralipid (105.2 (13.6) mg·min/L) than after Ringer's acetate (88.1 (7.1) mg·min/L) (p = 0.019). After Intralipid, the AUC of the lipid-un-entrapped bupivacaine portion (97.0 (14.5) mg·min/L) was 8% lower than that of total bupivacaine (p < 0.0001). To conclude, 4 mL/kg of Intralipid expedited cardiovascular recovery from bupivacaine cardiotoxicity mainly by increasing systemic vascular resistance. The increased myocardial mitochondrial respiration and bupivacaine entrapment after Intralipid did not improve cardiac function.© The Author(s) 2016.ot improve cardiac function. © The Author(s) 2016.)
Crawford 2006 Blood + (Local vasodilation in response to hypoxia … Local vasodilation in response to hypoxia is a fundamental physiologic responseensuring oxygen delivery to tissues under metabolic stress. Recent studies identify a role for the red blood cell (RBC), with hemoglobin the hypoxic sensor. Herein, we investigate the mechanisms regulating this process and explore the relative roles of adenosine triphosphate, S-nitrosohemoglobin, and nitrite as effectors. We provide evidence that hypoxic RBCs mediate vasodilation by reducing nitrite to nitric oxide (NO) and ATP release.NO dependence for nitrite-mediated vasodilation was evidenced by NO gas formation, stimulation of cGMP production, and inhibition of mitochondrial respiration in a process sensitive to the NO scavenger C-PTIO. The nitrite reductase activity of hemoglobin is modulated by heme deoxygenation and heme redox potential, with maximal activity observed at 50% hemoglobin oxygenation (''p''50). Concomitantly, vasodilation is initiated at the ''p''50, suggesting that oxygen sensing by hemoglobin is mechanistically linked to nitrite reduction and stimulation ofvasodilation. Mutation of the conserved β93cys residue decreases the heme redox potential (ie, decreases E1/2), an effect that increases nitrite reductase activity and vasodilation at any given hemoglobin saturation. These data support a function for RBC hemoglobin as an allosterically and redox-regulated nitrite reductase whose “enzyme activity” couples hypoxia to increased NO-dependent blood flow.me activity” couples hypoxia to increased NO-dependent blood flow.)
Long Night of Research 2022 Innsbruck AT + (Long Night of Research 2022: The diagnostic bioenergetic report – a milestone on the way to mitochondrial fitness and physical well-being.)
Long Night of Research 2024 Innsbruck AT + (Long Night of Research 2024)
Cecatto 2016 Thesis + (Long-chain 3-hydroxy-acyl-CoA dehydrogenas … Long-chain 3-hydroxy-acyl-CoA dehydrogenase (LCHAD) and mitochondrialtrifunctional protein (MTP) deficiencies are inborn errors of fatty acid oxidation. Affected patients present accumulation of long-chain hydroxylated fatty acids (LCHFA), particularly 3-hydroxytetradecanoic (3HTA) and 3-hydroxypalmitic (3HPA) acids, in blood and other tissues. The symptomatology is varied, including severe cardiomyopathy and muscular symptoms such as weakness, muscle pain and recurrent episodes of rhabdomyolysis, as well as hepatopathy, retinopathy, hypotonia, peripheral neuropathy, speech and development delay, leading to premature death. Considering that the pathogenesis of cardiac and skeletal muscle damage presented by the patients are still not established, the aim of the present work was to investigate the ''in vitro'' effects of 3HTA and 3HPA on important parameters of mitochondrial bioenergetics, namely the respiratory parameters state 3, state 4, respiratory control ratio (RCR) and uncoupled respiration, as well as mitochondrial membrane potential (ΔΨm), swelling, Ca2+ retention capacity and NAD(P)H redox state in cardiac and skeletal muscle mitochondria isolated from young rats. Initially, we observed that 3HTA and 3HPA at lower concentrations (10-30 UM) increased state 4 and decreased RCR in skeletal muscle mitochondria, indicating an uncoupling effect. At higher concentrations (50-100 UM), these fatty acids decreased state 4, state 3 and uncoupled respiration, suggesting metabolic inhibition. Furthermore, we observed that 3HPA was capable to provoke similar effects on mitochondrial respiration in permeabilized skeletal muscle fibers, validating the results obtained in isolated mitochondria. We also demonstrated that 3HPA and 3HTA (30 UM) strongly decreased the ΔΨm, NAD(P)H content, Ca2+ retention capacity and ATP production, besides inducing swelling, in mitochondria obtained from both tissues and supplemented with Ca2+. These effects were prevented by cyclosporin A and ADP, as well as by ruthenium red, indicating the involvement of mitochondrial permeability transition and Ca2+, respectively. The fact that 3HPA strongly increased the mitochondrial membrane fluidity in skeletal muscle indicates that this mechanism may be involved in the mitochondrial bioenergetics impairment caused by these compounds. Finally, we verified that the 3HTA dicarboxylic analogue, 3-hydroxytetradecanodioic acid, which also accumulates in the affected patients, did not alter the tested parameters, indicating a selective action of the monocarboxylic acids. Taken together, we demonstrated that the major LCHFA accumulated in LCHAD and MTP deficiencies impair mitochondrial homeostasis in cardiac and skeletal muscle. We presume that these mechanisms may explain, at least in part, the severe cardiomyopathy, symptomatology and muscle alterations characteristics of the patients affected by these disorders.tology and muscle alterations characteristics of the patients affected by these disorders.)
Grevengoed 2015 J Lipid Res + (Long-chain acyl-CoA synthetase 1 (ACSL1) c … Long-chain acyl-CoA synthetase 1 (ACSL1) contributes more than 90% of total cardiac ACSL activity, but its role in phospholipid synthesis has not been determined. Mice with an inducible knockout of ACSL1 (''Acsl1T-/-'') have impaired cardiac fatty acid oxidation and rely on glucose for ATP production. Because ACSL1 exhibited a strong substrate preference for linoleate, we investigated the composition of heart phospholipids. ''Acsl1T-/-'' hearts contained 83% less tetralinoleoyl-cardiolipin (CL), the major form present in control hearts. A stable knockdown of ACSL1 in H9c2 rat cardiomyocytes resulted in low incorporation of linoleate into CL and in diminished incorporation of palmitate and oleate into other phospholipids. Overexpression of ACSL1 in H9c2 and HEK-293 cells increased incorporation of linoleate into CL and other phospholipids. To determine whether increasing the content of linoleate in CL would improve mitochondrial respiratory function in ''Acsl1T-/-'' hearts, control and ''Acsl1T-/-'' mice were fed a high-linoleate diet; this diet normalized the amount of tetralinoleoyl-CL but did not improve respiratory function. Thus, ACSL1 is required for the normal composition of several phospholipid species in heart. Although ACSL1 determines the acyl-chain composition of heart CL, a high tetralinoleoyl-CL content may not be required for normal function.ion of heart CL, a high tetralinoleoyl-CL content may not be required for normal function.)
Krause 2023 J Transl Med + (Long-chain acyl-carnitines (ACs) are poten … Long-chain acyl-carnitines (ACs) are potential arrhythmogenic metabolites. Their role in atrial fibrillation (AF) remains incompletely understood. Using a systems medicine approach, we assessed the contribution of C18:1AC to AF by analysing its ''in vitro'' effects on cardiac electrophysiology and metabolism, and translated our findings into the human setting.Human iPSC-derived engineered heart tissue was exposed to C18:1AC. A biphasic effect on contractile force was observed: short exposure enhanced contractile force, but elicited spontaneous contractions and impaired Ca2+ handling. Continuous exposure provoked an impairment of contractile force. In human atrial mitochondria from AF individuals, C18:1AC inhibited respiration. In a population-based cohort as well as a cohort of patients, high C18:1AC serum concentrations were associated with the incidence and prevalence of AF.Our data provide evidence for an arrhythmogenic potential of the metabolite C18:1AC. The metabolite interferes with mitochondrial metabolism, thereby contributing to contractile dysfunction and shows predictive potential as novel circulating biomarker for risk of AF.tential as novel circulating biomarker for risk of AF.)
Zvejniece 2023 Biomed Pharmacother + (Long-chain acylcarnitines (LCACs) are inte … Long-chain acylcarnitines (LCACs) are intermediates of fatty acid oxidation and are known to exert detrimental effects on mitochondria. This study aimed to test whether lowering LCAC levels with the anti-ischemia compound 4-[ethyl(dimethyl)ammonio]butanoate (methyl-GBB) protects brain mitochondrial function and improves neurological outcomes after transient middle cerebral artery occlusion (MCAO). The effects of 14 days of pretreatment with methyl-GBB (5 mg/kg, p.o.) on brain acylcarnitine (short-, long- and medium-chain) concentrations and brain mitochondrial function were evaluated in Wistar rats. Additionally, the mitochondrial respiration and reactive oxygen species (ROS) production rates were determined using ''ex vivo'' high-resolution fluorespirometry under normal conditions, in models of ischemia-reperfusion injury (reverse electron transfer and anoxia-reoxygenation) and 24 h after MCAO. MCAO model rats underwent vibrissae-evoked forelimb-placing and limb-placing tests to assess neurological function. The infarct volume was measured on day 7 after MCAO using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Treatment with methyl-GBB significantly reduced the LCAC content in brain tissue, which decreased the ROS production rate without affecting the respiration rate, indicating an increase in mitochondrial coupling. Furthermore, methyl-GBB treatment protected brain mitochondria against anoxia-reoxygenation injury. In addition, treatment with methyl-GBB significantly reduced the infarct size and improved neurological outcomes after MCAO. Increased mitochondrial coupling efficiency may be the basis for the neuroprotective effects of methyl-GBB. This study provides evidence that maintaining brain energy metabolism by lowering the levels of LCACs protects against ischemia-induced brain damage in experimental stroke models.rain damage in experimental stroke models.)
Divakaruni 2018 Cell Metab + (Long-chain fatty acid (LCFA) oxidation has … Long-chain fatty acid (LCFA) oxidation has been shown to play an important role in interleukin-4 (IL-4)-mediated macrophage polarization (M(IL-4)). However, many of these conclusions are based on the inhibition of carnitine palmitoyltransferase-1 with high concentrations of etomoxir that far exceed what is required to inhibit enzyme activity (EC90 < 3 μM). We employ genetic and pharmacologic models to demonstrate that LCFA oxidation is largely dispensable for IL-4-driven polarization. Unexpectedly, high concentrations of etomoxir retained the ability to disrupt M(IL-4) polarization in the absence of Cpt1a or Cpt2 expression. Although excess etomoxir inhibits the adenine nucleotide translocase, oxidative phosphorylation is surprisingly dispensable for M(IL-4). Instead, the block in polarization was traced to depletion of intracellular free coenzyme A (CoA), likely resulting from conversion of the pro-drug etomoxir into active etomoxiryl CoA. These studies help explain the effect(s) of excess etomoxir on immune cells and reveal an unappreciated role for CoA metabolism in macrophage polarization.or CoA metabolism in macrophage polarization.)
Pereyra 2021 J Lipid Res + (Long-chain fatty acid oxidation is frequen … Long-chain fatty acid oxidation is frequently impaired in primary and systemic metabolic diseases affecting the heart, thus therapeutically increasing reliance on normally minor energetic substrates, such as ketones and medium chain fatty acids, could benefit cardiac health. However, the molecular fundamentals of this therapy are not fully known. Here, we explored the ability of octanoate, an eight-carbon medium-chain fatty acid known as an unregulated mitochondrial energetic substrate, to ameliorate cardiac hypertrophy in long-chain fatty acid oxidation deficient hearts due to carnitine palmitoyltransferase 2 deletion (Cpt2M-/-). CPT2 converts acylcarnitines to acyl-CoAs in the mitochondrial matrix for oxidative bioenergetic metabolism. In Cpt2M-/- mice, high octanoate-ketogenic diet failed to alleviate myocardial hypertrophy, dysfunction, and acylcarnitine accumulation suggesting that this alternative substrate is not sufficiently compensatory for energy provision. Aligning this outcome, we identified a major metabolic distinction between muscles and liver, wherein heart and skeletal muscle mitochondria were unable to oxidize free octanoate but liver was able to oxidize free octanoate. Liver mitochondria, but not heart or muscle, highly expressed medium-chain acyl-CoA synthetases, potentially enabling octanoate activation for oxidation and circumventing acylcarnitine-shuttling. Conversely, octanoylcarnitine was oxidized by liver, skeletal muscle, and heart, with rates in heart 4-fold greater than liver and, in muscles, was not dependent upon CPT2. Together, these data suggest that dietary octanoate cannot rescue CPT2-deficient cardiac disease. These data also suggest the existence of tissue-specific mechanisms for octanoate oxidative metabolism, with liver being independent of free carnitine availability while cardiac and skeletal muscles depend on carnitine but not on CPT2. cardiac and skeletal muscles depend on carnitine but not on CPT2.)
Oliveira 2015 Biotechniques + (Long-chain free fatty acids (FFAs) are im … Long-chain free fatty acids (FFAs) are important metabolic substrates for energy production and lipid synthesis that are also involved in signaling processes (1,2). Two of the most common fatty acids in humans are the long-chain saturated FFA palmitate (C16:0) and the monounsat-urated oleate (C18:1). Western diets rich in fatty acids are associated with increased levels of plasma cholesterol, hepatic steatosis, and a greater risk of cardiovascular disease (3,4). High levels of circulating FFAs, in particular of saturated FFAs, are implicated in insulin resistance and pancreatic b-cell dysfunction, and are predictive of diabetes development (5–8). In vitro exposure to high levels of FFAs leads to lipotoxicity, causing cellular dysfunction and death (5,9)....ellular dysfunction and death (5,9). ...)
Ma 2019 Biochem Biophys Res Commun + (Long-term high salt intake leads to cardia … Long-term high salt intake leads to cardiac hypertrophy, but the mechanism remains elusive. Transient receptor potential channel, canonical 3(TRPC3), located in mitochondria, regulates mitochondrial calcium and reactive oxygen species(ROS) production. Herein, we investigated whether TRPC3 participates in high salt-induced cardiac hypertrophy by impairing cardiac mitochondrial function. High salt treatment increased the expression of mitochondrial TRPC3 in cardiomyocytes, accompanied by enhanced mitochondrial calcium uptake and elevated ROS production. Inhibition of TRPC3 significantly reduced high salt-induced ROS generation, promoted ATP production by stimulating oxidative phosphorylation, and increased enzyme activity in mitochondria in cardiomyocytes. Additionally, TRPC3 deficiency inhibited high salt-induced cardiac hypertrophy ''in vivo''. A long-term high salt diet increased cardiac mitochondrial TRPC3 expression, elevated expression of cardiac hypertrophic markers atrial natriuretic peptide (ANP),brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) and decreased ATP production and mitochondrial complex I and II enzyme activity in a TRPC3-dependent manner. TRPC3 deficiency antagonises high salt diet-mediated cardiac hypertrophy by ameliorating TRPC3-mediated cardiac mitochondrial dysfunction. TRPC3 may therefore represent a novel target for preventing high salt-induced cardiac damage.Copyright © 2019 Elsevier Inc. All rights reserved. 2019 Elsevier Inc. All rights reserved.)
Kuznetsov 2003 Anal Biochem + (Long-term preservation of muscle mitochond … Long-term preservation of muscle mitochondria for consequent functional analysis is an important and still unresolved challenge in the clinical study of metabolic diseases and in the basic research of mitochondrial physiology. We here present a method for cryopreservation of mitochondria in various muscle types including human biopsies. Mitochondrial function was analyzed after freeze–thawing permeabilized muscle fibers using glycerol and dimethyl sulfoxide as cryoprotectant. Using optimal freeze–thawing conditions, high rates of adenosine 5′-diphosphate-stimulated respiration and high respiratory control were observed, showing intactness of mitochondrial respiratory function after cryopreservation. Measurement of adenosine 5′-triphosphate (ATP) formation showed normal rates of ATP synthesis and ATP/O ratios. Intactness of the outer mitochondrial membrane and functional coupling between mitochondrial creatine kinase and oxidative phosphorylation were verified by respiratory cytochrome c and creatine tests. Simultaneous confocal imaging of mitochondrial flavoproteins and nicotinamide adenine dinucleotide revealed normal intracellular arrangement and metabolic responses of mitochondria after freeze–thawing. The method therefore permits, after freezing and long-term storage of muscle samples, mitochondrial function to be estimated and energy metabolism to be monitored in situ. This will significantly expand the scope for screening and exchange of human biopsy samples between research centers, thus providing a new basis for functional analysis of mitochondrial defects in various diseases.mitochondrial defects in various diseases.)
Shrum 2016 J Kidney + (Long-term renal function is compromised in … Long-term renal function is compromised in patients receiving deceased donor kidneys which require cold storage exposure prior to transplantation. It is well established that extended cold storage induces renal damage and several labs, including our own, have demonstrated renal mitochondrial damage after cold storage alone. However, to our knowledge, few studies have assessed renal and mitochondrial function after transplantation of rat kidneys exposed to short-term (4 hr) cold storage compared to transplant without cold storage (autotransplantation). Our data reveal that cold storage plus transplantation exacerbated renal and mitochondrial dysfunction when compared to autotransplantation alone.hen compared to autotransplantation alone.)
Damiano 2014 PLoS One + (Loss of Parkin, encoded by PARK2 gene, is … Loss of Parkin, encoded by PARK2 gene, is a major cause of autosomal recessive Parkinson's disease. In Drosophila and mammalian cell models Parkin has been shown in to play a role in various processes essential to maintenance of mitochondrial quality, including mitochondrial dynamics, biogenesis and degradation. However, the relevance of altered mitochondrial quality control mechanisms to neuronal survival ''in vivo'' is still under debate. We addressed this issue in the brain of PARK2-/- mice using an integrated mitochondrial evaluation, including analysis of respiration by polarography or by fluorescence, respiratory complexes activity by spectrophotometric assays, mitochondrial membrane potential by rhodamine 123 fluorescence, mitochondrial DNA content by real time PCR, and oxidative stress by total glutathione measurement, proteasome activity, SOD2 expression and proteins oxidative damage. Respiration rates were lowered in PARK2-/- brain with high resolution but not standard respirometry. This defect was specific to the striatum, where it was prominent in neurons but less severe in astrocytes. It was present in primary embryonic cells and did not worsen ''in vivo'' from 9 to 24 months of age. It was not associated with any respiratory complex defect, including complex I. Mitochondrial inner membrane potential in PARK2-/- mice was similar to that of wild-type mice but showed increased sensitivity to uncoupling with ageing in striatum. The presence of oxidative stress was suggested in the striatum by increased mitochondrial glutathione content and oxidative adducts but normal proteasome activity showed efficient compensation. SOD2 expression was increased only in the striatum of PARK2-/- mice at 24 months of age. Altogether our results show a tissue-specific mitochondrial defect, present early in life of PARK2-/- mice, mildly affecting respiration, without prominent impact on mitochondrial membrane potential, whose underlying mechanisms remain to be elucidated, as complex I defect and prominent oxidative damage were ruled out.prominent oxidative damage were ruled out.)