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• Kotiadis 2012 J Cell Sci  + (ADF/cofilin family proteins are essential … ADF/cofilin family proteins are essential regulators of actin cytoskeletal dynamics. Recent evidence also implicates cofilin in the regulation of mitochondrial function. Here, we identify new functional surfaces of cofilin that are linked with mitochondrial function and stress responses in the budding yeast ''S. cerevisiae''. Our data links surfaces of cofilin that are involved in separable activities of actin filament disassembly or stabilisation, to the regulation of mitochondrial morphology and the activation status of Ras respectively. Importantly, charge alterations to conserved surfaces of cofilin that do not interfere with its actin regulatory activity leads to a dramatic increase in respiratory function that triggers a retrograde signal to up-regulate a battery of ABC transporters and concurrent metabolic changes that support multi-drug resistance. We hypothesise that cofilin functions within a novel bio-sensing system that connects the cytoskeleton and mitochondrial function to environmental challenge.drial function to environmental challenge.)
• Scheibye-Knudsen 2009 Eur J Appl Physiol  + (ADP is generally accepted as a key regulat … ADP is generally accepted as a key regulator of oxygen consumption both in isolated mitochondria and in permeabilized fibers from skeletal muscle. The present study explored inorganic phosphate in a similar regulatory role. Saponin permeabilized fibers and isolated mitochondria from type-I and type-II muscle from male Wistar rats were prepared. Respiration was measured while the medium Pi concentration was gradually increased. The apparent Km values for Pi were 607 ± 17 µM and 405 ± 15 μM (P < 0.0001) for type-I and type-II fibers, respectively. For isolated mitochondria the values were significantly lower than type-1 permeabilized fibers, 338 ± 130 μM and 235 ± 30 μM (P < 0.05), but not different with respect to fiber type. The reason for this difference in Km values in the permeabilized muscle is unknown, but a similar pattern has been observed for K m of ADP. Our data indicate that phosphate may play a role in regulation of oxygen consumption ''in vitro'' and ''in vivo''.oxygen consumption ''in vitro'' and ''in vivo''.)
• Ara 2011 Int J Obes  + (AIM/HYPOTHESIS: The aim of this study was … AIM/HYPOTHESIS: The aim of this study was to investigate mitochondrial function, fibre-type distribution and substrate oxidation during exercise in arm and leg muscles in male postobese (PO), obese (O) and age- and body mass index (BMI)-matched control (C) subjects. The hypothesis of the study was that fat oxidation during exercise might be differentially preserved in leg and arm muscles after weight loss.</br></br>METHODS: Indirect calorimetry was used to calculate fat and carbohydrate oxidation during both progressive arm-cranking and leg-cycling exercises. Muscle biopsy samples were obtained from musculus deltoideus (m. deltoideus) and m. vastus lateralis muscles. Fibre-type composition, enzyme activity and O2 flux capacity of saponin-permeabilized muscle fibres were measured, the latter by high-resolution respirometry.</br></br>RESULTS: During the graded exercise tests, peak fat oxidation during leg cycling and the relative workload at which it occurred (FatMax) were higher in PO and O than in C. During arm cranking, peak fat oxidation was higher in O than in C, and FatMax was higher in O than in PO and C. Similar fibre-type composition was found between groups. Plasma adiponectin was higher in PO than in C and O, and plasma leptin was higher in O than in PO and C.</br></br>CONCLUSIONS: In O subjects, maximal fat oxidation during exercise and the eliciting relative exercise intensity are increased. This is associated with higher intramuscular triglyceride levels and higher resting non esterified fatty acid (NEFA) concentrations, but not with differences in fibre-type composition, mitochondrial function or muscle enzyme levels compared with Cs. In PO subjects, the changes in fat oxidation are preserved during leg, but not during arm, exercise. during leg, but not during arm, exercise.)
• Nakhostin-Roohi 2008 J Sports Med Phys Fitness  + (AIM: Low levels of physical activity and c … AIM:</br>Low levels of physical activity and cardio respiratory fitness are both associated with higher risk of all-cause and disease-specific mortality. The purpose of this study is to examine obesity and fitness of the female staff of Ardebil Azad University in the northwest of Iran.</br></br>METHODS:</br>Thirty seven staff (medium age: 32.97+/-5.81 year, height: 158.21+/-5.88 cm, Body Mass Index [BMI]: 26.59+/-4.02 kg/m(2)) of Ardebil Azad University participated in this study voluntarily. Primary measurements of interest in the present study were height, BMI, subcutaneous skin folds, and cardio respiratory fitness determined by 1 609 meter (one mile) walk test.</br></br>RESULTS:</br>The subjects of the present study are more obese than some other population (fat percentage: 28.68+/-5.33) and cardio respiratory fitness of them is rather low (VO(2max): 33.43+/-6.90 mL/kg/min).</br></br>CONCLUSION:</br>Social/lifestyle factors such as the level of education, marital status, exercise, dietary and smoking habits may be related to overweight/obesity and cardio respiratory fitness in female staff of Ardebil Azad University.n female staff of Ardebil Azad University.)
• Larsen 2012 Acta Physiol (Oxf)  + (AIM: Mitochondrial function has previously … AIM: Mitochondrial function has previously been studied in ageing, but never in humans matched for maximal oxygen uptake (V·O2max). Furthermore, the influence of ageing on mitochondrial substrate sensitivity is not known.</br></br>METHODS: Skeletal muscle mitochondrial respiratory capacity and mitochondrial substrate sensitivity was measured by respirometry in young (23±3 years) and middle-aged (53±3 years) male subjects with similar V·O2max. Protocols for respirometry included titration of substrates for complexI (glutamate), complexII (succinate) and both (octanoyl-carnitine) for calculation of substrate sensitivity (C(50) ). Myosin Heavy Chain (MHC) isoforms, citrate synthase (CS) and β-hydroxy-acyl-CoA-dehydrogenase (HAD) activity, mitochondrial DNA (mtDNA) content, protein levels of complexes I-V and antioxidant defense system (manganese superoxide dismutase (MnSOD)) was measured.</br></br>RESULTS: No differences were found in maximal mitochondrial respiration or C(50) with glutamate (2.0±0.3 and 1.8±0.3 mmol/l), succinate (3.7±0.2 and 3.8±0.4 mmol/l) or octanoyl-carnitine (47±8 and 56±7 μmol/l) in young and middle-aged subjects, respectively. Normalising mitochondrial respiration to mtDNA young subjects had a higher (P<0.05) respiratory capacity per mitochondrion compared to middle-aged subjects. HAD activity and mtDNA per mg tissue were higher in middle-aged compared to young subjects. Middle-aged had a higher MHC I isoform and a lower MHC IIX isoform content compared to young subjects.</br></br>CONCLUSION: Mitochondrial substrate sensitivity is not affected by ageing. When young and middle-aged men are carefully matched for V·O2max, mitochondrial respiratory capacity is also similar. However, per mitochondrion respiratory capacity was lower in middle-aged compared to young subjects. Thus, when matched for V·O2max middle-aged seems to require a higher mitochondrial content than young subjects.er mitochondrial content than young subjects.)
• Raboel 2009 Diabetes Obes Metab  + (AIM: Several mechanisms have been targeted … AIM: Several mechanisms have been targeted as culprits of weight gain during antihyperglycaemic treatment in type 2 diabetes (T2DM). These include reductions in glucosuria, increased food intake from fear of hypoglycaemia, the anabolic effect of insulin, decreased metabolic rate and increased efficiency in fuel usage. The purpose of the study was to test the hypothesis that mitochondrial efficiency increases as a result of insulin treatment in patients with type 2 diabetes.</br></br>METHODS: We included ten patients with T2DM (eight males) on oral antidiabetic treatment, median age: 51.5 years (range: 39-67) and body mass index (BMI): 30.1 +/- 1.2 kg/m2 (mean +/- s.e.). Muscle biopsies from m. vastus lateralis and m. deltoideus were obtained before and after seven weeks of intensive insulin treatment, and mitochondrial respiration was measured using high-resolution respirometry. State 3 respiration was measured with the substrates malate, pyruvate, glutamate, succinate and ADP. State 4o was measured with addition of oligomycine. An age, sex and BMI-matched control group was also included.</br></br>RESULTS: HbA1c improved significantly and the patients gained on average 3.4 +/- 0.9 kg. Before treatment, respiratory control ratios (RCRs) of the T2DM were lower than the obese controls [2.6 vs. 3.2 (p < 0.05)], but RCR returned to the levels of the control subjects during treatment. Average state 4o of arm and leg declined by 14% (p < 0.05) during insulin treatment.</br></br>CONCLUSIONS: Tight glycaemic control leads to reductions in inner mitochondrial membrane leak and increased efficiency of mitochondria. This change in mitochondrial physiology could contribute to the weight gain seen with antihyperglycaemic treatment.ght gain seen with antihyperglycaemic treatment.)
• Melzer 2010 Ann Nutr Metab  + (AIM: The resting metabolic rate (RMR) vari … AIM: The resting metabolic rate (RMR) varies among pregnant women. The factors responsible for this variability are unknown. This study aimed to assess the influence of the prepregnancy body mass index (BMI) on the RMR during late pregnancy.</br></br>METHODS: RMR, height, weight, and total (TEE) and activity (AEE) energy expenditures were measured in 46 healthy women aged 31 ± 5 years (mean ± SD) with low (<19.8), normal (19.8-26.0), and high (>26.0) prepregnancy BMI at 38.2 ± 1.5 weeks of gestation (t(gest)) and 40 ± 7 weeks postpartum (t(post)) (''n'' = 27).</br></br>RESULTS: The mean t(gest) RMR for the low-, normal-, and high-BMI groups was 1,373, 1,807, and 2,191 kcal/day, respectively (''p'' = 0.001). The overall mean t(gest) RMR was 316 ± 183 kcal/day (21%), higher than the overall mean t(post) value and this difference was correlated with gestational weight gain (''r'' = 0.78, ''p'' < 0.001). The scaled metabolic rate by allometry (RMR/kilograms⁰·⁷³) was similar in the low-, normal-, and high-BMI groups, respectively (''p'' = 0.45). Changes in t(gest) TEE closely paralleled changes in t(gest) RMR (''r'' = 0.84, 'p'' < 0.001). AEE was similar among the BMI groups.</br></br>CONCLUSION: The RMR is significantly increased in the third trimester of pregnancy. The absolute gestational RMR is higher in women with high prepregnancy BMI due to increased body weight. The scaled metabolic rate (RMR/kilograms⁰·⁷³) is similar among the BMI groups of pregnant women.⁷³) is similar among the BMI groups of pregnant women.)
• WHO 2006 Acta Paediatr  + (AIM: To describe the methods used to const … AIM: To describe the methods used to construct the WHO Child Growth Standards based on length/height, weight and age, and to present resulting growth charts.</br></br>METHODS: The WHO Child Growth Standards were derived from an international sample of healthy breastfed infants and young children raised in environments that do not constrain growth. Rigorous methods of data collection and standardized procedures across study sites yielded very high-quality data. The generation of the standards followed methodical, state-of-the-art statistical methodologies. The Box-Cox power exponential (BCPE) method, with curve smoothing by cubic splines, was used to construct the curves. The BCPE accommodates various kinds of distributions, from normal to skewed or kurtotic, as necessary. A set of diagnostic tools was used to detect possible biases in estimated percentiles or z-score curves.</br></br>RESULTS: There was wide variability in the degrees of freedom required for the cubic splines to achieve the best model. Except for length/height-for-age, which followed a normal distribution, all other standards needed to model skewness but not kurtosis. Length-for-age and height-for-age standards were constructed by fitting a unique model that reflected the 0.7-cm average difference between these two measurements. The concordance between smoothed percentile curves and empirical percentiles was excellent and free of bias. Percentiles and z-score curves for boys and girls aged 0-60 mo were generated for weight-for-age, length/height-for-age, weight-for-length/height (45 to 110 cm and 65 to 120 cm, respectively) and body mass index-for-age.</br></br>CONCLUSION: The WHO Child Growth Standards depict normal growth under optimal environmental conditions and can be used to assess children everywhere, regardless of ethnicity, socio-economic status and type of feeding.socio-economic status and type of feeding.)
• Larsen 2011 Acta Physiol (Oxf)  + (AIM: To study whether the phenotypical cha … AIM: To study whether the phenotypical characteristics (exercise intolerance; reduced spontaneous activity) of the AMPKα2 kinase-dead (KD) mice can be explained by a reduced mitochondrial respiratory flux rates (JO(2) ) in skeletal muscle. Secondly, the effect of the maturation process on JO(2) was studied.</br></br>METHODS: In tibialis anterior (almost exclusively type 2 fibres) muscle from young (12-17 weeks, n = 7) and mature (25-27 weeks, n = 12) KD and wild-type (WT) (12-17 weeks, n = 9; 25-27 weeks, n = 11) littermates, JO(2) was quantified in permeabilized fibres ex vivo by respirometry, using a substrate-uncoupler-inhibitor-titration (SUIT) protocol: malate, octanoyl carnitine, ADP and glutamate (GMO(3) ), + succinate (GMOS(3) ), + uncoupler (U) and inhibitor (rotenone) of complex I respiration. Citrate synthase (CS) activity was measured as an index of mitochondrial content.</br></br>RESULTS: Citrate synthase activity was highest in young WT animals and lower in KD animals compared with age-matched WT. JO(2) per mg tissue was lower (P < 0.05) in KD animals (state GMOS(3) ). No uncoupling effect was seen in any of the animals. Normalized oxygen flux (JO(2) /CS) revealed a uniform pattern across the SUIT protocol with no effect of KD. However, JO(2) /CS was higher [GMO(3) , GMOS(3) , U and rotenone (only WT)] in the mature compared with the young mice - irrespective of the genotype (P < 0.05).</br></br>CONCLUSION: Exercise intolerance and reduced activity level seen in KD mice may be explained by reduced JO(2) in the maximally coupled respiratory state. Furthermore, an enhancement of oxidative phosphorylation capacity per mitochondrion is seen with the maturation process.tochondrion is seen with the maturation process.)
• Paglialunga 2012 Diabetologia  + (AIMS/HYPOTHESIS: High-fat, high-sucrose d … AIMS/HYPOTHESIS: </br>High-fat, high-sucrose diet (HF)-induced reactive oxygen species (ROS) levels are implicated in skeletal muscle insulin resistance and mitochondrial dysfunction. Here we investigated whether mitochondrial ROS sequestering can circumvent HF-induced oxidative stress; we also determined the impact of any reduced oxidative stress on muscle insulin sensitivity and mitochondrial function.</br>METHODS: </br>The Skulachev ion (plastoquinonyl decyltriphenylphosphonium) (SkQ), a mitochondria-specific antioxidant, was used to target ROS production in C2C12 muscle cells as well as in HF-fed (16 weeks old) male C57Bl/6 mice, compared with mice on low-fat chow diet (LF) or HF alone. Oxidative stress was measured as protein carbonylation levels. Glucose tolerance tests, glucose uptake assays and insulin-stimulated signalling were determined to assess muscle insulin sensitivity. Mitochondrial function was determined by high-resolution respirometry.</br>RESULTS: </br>SkQ treatment reduced oxidative stress in muscle cells (-23% p < 0.05), but did not improve insulin sensitivity and glucose uptake under insulin-resistant conditions. In HF mice, oxidative stress was elevated (56% vs LF p < 0.05), an effect completely blunted by SkQ. However, HF and HF+SkQ mice displayed impaired glucose tolerance (AUC HF up 33%, p < 0.001; HF+SkQ up 22%; p < 0.01 vs LF) and disrupted skeletal muscle insulin signalling. ROS sequestering did not improve mitochondrial function.</br>CONCLUSIONS/INTERPRETATION:</br>SkQ treatment reduced muscle mitochondrial ROS production and prevented HF-induced oxidative stress. Nonetheless, whole-body glucose tolerance, insulin-stimulated glucose uptake, muscle insulin signalling and mitochondrial function were not improved. These results suggest that HF-induced oxidative stress is not a prerequisite for the development of muscle insulin resistance.site for the development of muscle insulin resistance.)
• Friederich-Persson 2012 Diabetologia  + (AIMS/HYPOTHESIS: Increased oxygen consump … AIMS/HYPOTHESIS: </br>Increased oxygen consumption results in kidney tissue hypoxia, which is proposed to contribute to the development of diabetic nephropathy. Oxidative stress causes increased oxygen consumption in type 1 diabetic kidneys, partly mediated by uncoupling protein-2 (UCP-2)-induced mitochondrial uncoupling. The present study investigates the role of UCP-2 and oxidative stress in mitochondrial oxygen consumption and kidney function in db/db mice as a model of type 2 diabetes.</br></br>METHODS: </br>Mitochondrial oxygen consumption, glomerular filtration rate and proteinuria were investigated in db/db mice and corresponding controls with and without coenzyme Q10 (CoQ10) treatment.</br></br>RESULTS: </br>Untreated db/db mice displayed mitochondrial uncoupling, manifested as glutamate-stimulated oxygen consumption (2.7 ± 0.1 vs 0.2 ± 0.1 pmol O(2) s(-1) [mg protein](-1)), glomerular hyperfiltration (502 ± 26 vs 385 ± 3 μl/min), increased proteinuria (21 ± 2 vs 14 ± 1, μg/24 h), mitochondrial fragmentation (fragmentation score 2.4 ± 0.3 vs 0.7 ± 0.1) and size (1.6 ± 0.1 vs 1 ± 0.0 μm) compared with untreated controls. All alterations were prevented or reduced by CoQ10 treatment. Mitochondrial uncoupling was partly inhibited by the UCP inhibitor GDP (-1.1 ± 0.1 pmol O(2) s(-1) [mg protein](-1)). UCP-2 protein levels were similar in untreated control and db/db mice (67 ± 9 vs 67 ± 4 optical density; OD) but were reduced in CoQ10 treated groups (43 ± 2 and 38 ± 7 OD).</br></br>CONCLUSIONS/INTERPRETATION: </br>db/db mice displayed oxidative stress-mediated activation of UCP-2, which resulted in mitochondrial uncoupling and increased oxygen consumption. CoQ10 prevented altered mitochondrial function and morphology, glomerular hyperfiltration and proteinuria in db/db mice, highlighting the role of mitochondria in the pathogenesis of diabetic nephropathy and the benefits of preventing increased oxidative stress. of preventing increased oxidative stress.)
• Flachs 2011 Diabetologia  + (AIMS/HYPOTHESIS: Calorie restriction is an … AIMS/HYPOTHESIS: Calorie restriction is an essential component in the treatment of obesity and associated diseases. Long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) act as natural hypolipidaemics, reduce the risk of cardiovascular disease and could prevent the development of obesity and insulin resistance. We aimed to characterise the effectiveness and underlying mechanisms of the combination treatment with LC n-3 PUFA and 10% calorie restriction in the prevention of obesity and associated disorders in mice.</br></br>METHODS: Male mice (C57BL/6J) were habituated to a corn-oil-based high-fat diet (cHF) for 2 weeks and then randomly assigned to various dietary treatments for 5 weeks or 15 weeks: (1) cHF, ad libitum; (2) cHF with LC n-3 PUFA concentrate replacing 15% (wt/wt) of dietary lipids (cHF + F), ad libitum; (3) cHF with calorie restriction (CR; cHF + CR); and (4) cHF + F + CR. Mice fed a chow diet were also studied. </br></br>RESULTS: We show that white adipose tissue plays an active role in the amelioration of obesity and the improvement of glucose homeostasis by combining LC n-3 PUFA intake and calorie restriction in cHF-fed mice. Specifically in the epididymal fat in the abdomen, but not in other fat depots, synergistic induction of mitochondrial oxidative capacity and lipid catabolism was observed, resulting in increased oxidation of metabolic fuels in the absence of mitochondrial uncoupling, while low-grade inflammation was suppressed, reflecting changes in tissue levels of anti-inflammatory lipid mediators, namely 15-deoxy-Δ(12,15)-prostaglandin J(2) and protectin D1.</br></br>CONCLUSIONS/INTERPRETATION: White adipose tissue metabolism linked to its inflammatory status in obesity could be modulated by combination treatment using calorie restriction and dietary LC n-3 PUFA to improve therapeutic strategies for metabolic syndrome.apeutic strategies for metabolic syndrome.)
• Rosca 2008 Cardiovasc Res  + (AIMS: Mitochondrial dysfunction is a maj … AIMS: </br></br>Mitochondrial dysfunction is a major factor in heart failure (HF). A pronounced variability of mitochondrial electron transport chain (ETC) defects is reported to occur in severe acquired cardiomyopathies without a consistent trend for depressed activity or expression. The aim of this study was to define the defect in the integrative function of cardiac mitochondria in coronary microembolization-induced HF.</br></br>METHODS AND RESULTS:</br></br>Studies were performed in the canine coronary microembolization-induced HF model of moderate severity. Oxidative phosphorylation was assessed as the integrative function of mitochondria, using a comprehensive variety of substrates in order to investigate mitochondrial membrane transport, dehydrogenase activity and electron-transport coupled to ATP synthesis. The supramolecular organization of the mitochondrial ETC also was investigated by native gel electrophoresis. We found a dramatic decrease in ADP-stimulated respiration that was not relieved by an uncoupler. Moreover, the ADP/O ratio was normal, indicating no defect in the phosphorylation apparatus. The data point to a defect in oxidative phosphorylation within the ETC. However, the individual activities of ETC complexes were normal. The amount of the supercomplex consisting of complex I/complex III dimer/complex IV, the major form of respirasome considered essential for oxidative phosphorylation, was decreased.</br></br>CONCLUSIONS:</br></br>We propose that the mitochondrial defect lies in the supermolecular assembly rather than in the individual components of the ETC.n in the individual components of the ETC.)
• Wen 2017 Antioxid Redox Signal  + (AIMS: We investigated the effects of mit … AIMS: </br></br>We investigated the effects of mitochondrial reactive oxygen species (mtROS) on nuclear factor (erythroid 2)-like 2 (NFE2L2) transcription factor activity during Trypanosoma cruzi (Tc) infection and determined whether enhancing the mtROS scavenging capacity preserved the heart function in Chagas disease.</br></br>RESULTS: </br></br>C57BL/6 wild type (WT, female) mice infected with Tc exhibited myocardial loss of mitochondrial membrane potential, complex II (CII)-driven coupled respiration, and ninefold increase in mtROS production. In vitro and in vivo studies showed that Tc infection resulted in an ROS-dependent decline in the expression, nuclear translocation, antioxidant response element (ARE) binding, and activity of NFE2L2, and 35-99% decline in antioxidants' (gamma-glutamyl cysteine synthase [γGCS], heme oxygenase-1 [HO1], glutamate-cysteine ligase modifier subunit [GCLM], thioredoxin (Trx), glutathione S transferase [GST], and NAD(P)H dehydrogenase, quinone 1 [NQO1]) expression. An increase in myocardial and mitochondrial oxidative adducts, myocardial interventricular septum thickness, and left ventricle (LV) mass, a decline in LV posterior wall thickness, and disproportionate synthesis of collagens (COLI/COLIII), αSMA, and SM22α were noted in WT.Tc mice. Overexpression of manganese superoxide dismutase (MnSOD) in cultured cells (HeLa or cardiomyocytes) and MnSODtg mice preserved the NFE2L2 transcriptional activity and antioxidant/oxidant balance, and cardiac oxidative and fibrotic pathology were significantly decreased in MnSODtg.Tc mice. Importantly, echocardiography finding of a decline in LV systolic (stroke volume, cardiac output, ejection fraction) and diastolic (early/late peak filling ratio, myocardial performance index) function in WT. Tc mice was abolished in MnSODtg. Tc mice. Innovation and Conclusion: The mtROS inhibition of NFE2L2/ARE pathway constitutes a key mechanism in signaling the fibrotic gene expression and evolution of chronic cardiomyopathy. Preserving the NFE2L2 activity arrested the mitochondrial and cardiac oxidative stress, cardiac fibrosis, and heart failure in Chagas disease.osis, and heart failure in Chagas disease.)
• Meinild Lundby 2018 Acta Physiol (Oxf)  + (AIMS: 1) determine whether exercise induce … AIMS: 1) determine whether exercise induced increases in muscle mitochondrial volume density (MitoVD ) is related to enlargement of existing mitochondria or de novo biogenesis, 2) establish if measures of mitochondrial-specific enzymatic activities are valid biomarkers for exercise induced increases in MitoVD .</br></br>METHOD: Skeletal muscle samples were collected from twenty-one healthy males prior to and following 6 weeks of endurance training. Transmission electron microscopy was used for estimation of mitochondrial densities and profiles. Biochemical assays, western blotting and high resolution respirometry were applied to detect changes in specific mitochondrial functions.</br></br>RESULT: MitoVD increased with 55 ± 9% (''P'' < 0.001), whereas the number of mitochondrial profiles per area of skeletal muscle remained unchanged following training. Citrate synthase activity (CS) increased (44 ± 12%, ''P'' < 0.001) however, there were no functional changes in oxidative phosphorylation capacity (OXPHOS, CI+IIP ) or cytochrome c oxidase (COX) activity. Correlations were found between MitoVD and CS (''P''=0.01; ''r''=0.58), OXPHOS, CI+CIIP (''P''=0.01; ''r''=0.58) and COX (''P''=0.02; ''r''=0.52) before training, after training a correlation was found between MitoVD and CS activity only (''P''=0.04; ''r''=0.49). Intrinsic respiratory capacities decreased (''P'' < 0.05) with training when respiration was normalized to MitoVD. This was not the case when normalized to CS activity although the percentage change was comparable. </br></br>CONCLUSIONS: MitoVD was increased by inducing mitochondrial enlargement rather than de novo biogenesis. CS activity may be appropriate to track training induced changes in MitoVD.priate to track training induced changes in MitoVD.)
• Hafstad 2011 J Appl Physiol  + (AIMS: Although exercise training induces h … AIMS: Although exercise training induces hypertrophy with improved contractile function, the effect of exercise on myocardial substrate metabolism and cardiac efficiency is less clear. High intensity training has been shown to produce more profound effects on cardiovascular function and aerobic capacity than isocaloric low and moderate intensity training. The aim of the present study was to explore metabolic and mechanoenergetic changes in the heart following endurance exercise training of both high and moderate intensity.</br></br>METHODS AND RESULTS: C57BL/6J mice were subjected to 10 wk treadmill running, either high intensity interval training (HIT) or distance-matched moderate intensity training (MIT), where HIT led to a pronounced increase in maximal oxygen uptake. Although both modes of exercise were associated with a 10% increase in heart weight-to-body weight ratio, only HIT altered cardiac substrate utilization, as revealed by a 36% increase in glucose oxidation and a concomitant reduction in fatty acid oxidation. HIT also improved cardiac efficiency by decreasing work-independent myocardial oxygen consumption. In addition, it increased cardiac maximal mitochondrial respiratory capacity.</br></br>CONCLUSION: This study shows that high intensity training is required for induction of changes in cardiac substrate utilization and energetics, which may contribute to the superior effects of high compared with moderate intensity training in terms of increasing aerobic capacity.g in terms of increasing aerobic capacity.)
• Bouitbir 2016 Antioxid Redox Signal  + (AIMS: Although statins are the most widely … AIMS: Although statins are the most widely used cholesterol-lowering agents, they are associated with a variety of muscle complaints. The goal of this study was to characterize the effects of statins on the mitochondrial apoptosis pathway induced by mitochondrial oxidative stress in skeletal muscle using human muscle biopsies as well as ''in vivo'' and ''in vitro'' models.</br></br>RESULTS:</br>Statins increased mitochondrial H₂O₂ production, the Bax/Bcl-2 ratio and TUNEL staining in deltoid biopsies of patients with statin-associated myopathy. Furthermore, atorvastatin treatment for two weeks at 10 mg/kg/day in rats increased H₂O₂ accumulation, and mRNA levels and immunostaining of the Bax/Bcl-2 ratio, as well as TUNEL staining and caspase 3 cleavage in glycolytic (plantaris) skeletal muscle but not in oxidative (soleus) skeletal muscle, which has a high antioxidative capacity. Atorvastatin also decreased the GSH/GSSG ratio, but only in glycolytic skeletal muscle. Co-treatment with the antioxidant quercetin at 25 mg/kg/d abolished these effects in plantaris. An ''in vitro'' study with L6 myoblasts directly demonstrated the link between mitochondrial oxidative stress following atorvastatin exposure and activation of the mitochondrial apoptosis signaling pathway.</br></br>INNOVATION:</br>Treatment with atorvastatin is associated with mitochondrial oxidative stress, which activates apoptosis and contributes to myopathy. Glycolytic muscles are more sensitive to atorvastatin than oxidative muscles, which may be due to the higher antioxidative capacity in oxidative muscles.</br></br>CONCLUSION:</br>There is a link between statin-induced mitochondrial oxidative stress and activation of the mitochondrial apoptosis signaling pathway in glycolytic skeletal muscle, which may be associated with statin-associated myopathy.ay in glycolytic skeletal muscle, which may be associated with statin-associated myopathy.)
• Lancel 2012 PLoS One  + (AIMS: Metabolic syndrome induces cardiac d … AIMS: Metabolic syndrome induces cardiac dysfunction associated with mitochondria abnormalities. As low levels of carbon monoxide (CO) may improve myocardial and mitochondrial activities, we tested whether a CO-releasing molecule (CORM-3) reverses metabolic syndrome-induced cardiac alteration through changes in mitochondrial biogenesis, dynamics and autophagy.</br> </br>METHODS AND RESULTS: Mice were fed with normal diet (ND) or high-fat diet (HFD) for twelve weeks. Then, mice received two intraperitoneal injections of CORM-3 (10 mg x kg(-1)), with the second one given 16 hours after the first. Contractile function in isolated hearts and mitochondrial parameters were evaluated 24 hours after the last injection. Mitochondrial population was explored by electron microscopy. Changes in mitochondrial dynamics, biogenesis and autophagy were assessed by western-blot and RT-qPCR. Left ventricular developed pressure was reduced in HFD hearts. Mitochondria from HFD hearts presented reduced membrane potential and diminished ADP-coupled respiration. CORM-3 restored both cardiac and mitochondrial functions. Size and number of mitochondria increased in the HFD hearts but not in the CORM-3-treated HFD group. CORM-3 modulated HFD-activated mitochondrial fusion and biogenesis signalling. While autophagy was not activated in the HFD group, CORM-3 increased the autophagy marker LC3-II. Finally, ''ex vivo'' experiments demonstrated that autophagy inhibition by 3-methyladenine abolished the cardioprotective effects of CORM-3.</br> </br>CONCLUSION: CORM-3 may modulate pathways controlling mitochondrial quality, thus leading to improvements of mitochondrial efficiency and HFD-induced cardiac dysfunction.iency and HFD-induced cardiac dysfunction.)
• Haram 2009 Cardiovasc Res  + (AIMS: The recent development of a rat mode … AIMS: The recent development of a rat model that closely resembles the metabolic syndrome allows to study the mechanisms of amelioration of the syndrome by exercise training. Here, we compared the effectiveness for reducing cardiovascular risk factors by exercise training programmes of different exercise intensities.</br></br>METHODS AND RESULTS: Metabolic syndrome rats were subjected to either continuous moderate-intensity exercise (CME) or high-intensity aerobic interval training (AIT). AIT was more effective than CME at reducing cardiovascular disease risk factors linked to the metabolic syndrome. Thus, AIT produced a larger stimulus than CME for increasing maximal oxygen uptake (VO_2max; 45 vs. 10%, ''P'' < 0.01), reducing hypertension (20 vs. 6 mmHg, ''P'' < 0.01), HDL cholesterol (25 vs. 0%, ''P'' < 0.05), and beneficially altering metabolism in fat, liver, and skeletal muscle tissues. Moreover, AIT had a greater beneficial effect than CME on sensitivity of aorta ring segments to acetylcholine (2.7- vs. 2.0-fold, ''P'' < 0.01), partly because of intensity-dependent effects on expression levels of nitric oxide synthase and the density of caveolae, and a greater effect than CME on the skeletal muscle Ca²⁺ handling (50 vs. 0%, ''P'' < 0.05). The two exercise training programmes, however, were equally effective at reducing body weight and fat content.</br></br>CONCLUSION: High-intensity exercise training was more beneficial than moderate-intensity exercise training for reducing cardiovascular risk in rats with the metabolic syndrome. This was linked to more superior effects on VO_2max, endothelial function, blood pressure, and metabolic parameters in several tissues. These results demonstrate that exercise training reduces the impact of the metabolic syndrome and that the magnitude of the effect depends on exercise intensity.of the metabolic syndrome and that the magnitude of the effect depends on exercise intensity.)
• Garcia-Roves 2008 J Biol Chem  + (AMP-activated protein kinase (AMPK) is a h … AMP-activated protein kinase (AMPK) is a heterotrimeric complex, composed of a catalytic subunit (α) and two regulatory subunits (β and γ), that works as a cellular energy sensor. The existence of multiple heterotrimeric complexes provides a molecular basis for the multiple roles of this highly conserved signaling system. The AMPK_γ3 subunit is predominantly expressed in skeletal muscle, mostly in type II glycolytic fiber types. We determined whether the AMPK_γ3 subunit has a role in signaling pathways that mediate mitochondrial biogenesis in skeletal muscle. We provide evidence that overexpression or ablation of the AMPK_γ3 subunit does not appear to play a critical role in defining mitochondrial content in resting skeletal muscle. However, overexpression of a mutant form (R225Q) of the AMPK_γ3 subunit (Tg-AMPK_γ3^225Q) increases mitochondrial biogenesis in glycolytic skeletal muscle. These adaptations are associated with an increase in expression of the co-activator [[PGC-1α]] and several transcription factors that regulate mitochondrial biogenesis, including NRF-1, NRF-2, and TFAM. Succinate dehydrogenase staining, a marker of the oxidative profile of individual fibers, was also increased in transversal skeletal muscle sections of white gastrocnemius muscle from Tg-AMPK_γ3^225Q mice, independent of changes in fiber type composition. In conclusion, a single nucleotide mutation (R225Q) in the AMPK gamma3 subunit is associated with mitochondrial biogenesis in glycolytic skeletal muscle, concomitant with increased expression of the co-activator [[PGC-1α]] and several transcription factors that regulate mitochondrial proteins, without altering fiber type composition. and several transcription factors that regulate mitochondrial proteins, without altering fiber type composition.)
• Mungai 2011 Mol Cell Biol  + (AMP-activated protein kinase (AMPK) is an … AMP-activated protein kinase (AMPK) is an energy sensor activated by increases in [AMP] or by oxidant stress (reactive oxygen species [ROS]). Hypoxia increases cellular ROS signaling, but the pathways underlying subsequent AMPK activation are not known. We tested the hypothesis that hypoxia activates AMPK by ROS-mediated opening of calcium release-activated calcium (CRAC) channels. Hypoxia (1.5% O2) augments cellular ROS as detected by the redox-sensitive green fluorescent protein (roGFP) but does not increase the [AMP]/[ATP] ratio. Increases in intracellular calcium during hypoxia were detected with Fura2 and the calcium-calmodulin fluorescence resonance energy transfer (FRET) sensor YC2.3. Antioxidant treatment or removal of extracellular calcium abrogates hypoxia-induced calcium signaling and subsequent AMPK phosphorylation during hypoxia. Oxidant stress triggers relocation of stromal interaction molecule 1 (STIM1), the endoplasmic reticulum (ER) Ca2+ sensor, to the plasma membrane. Knockdown of STIM1 by short interfering RNA (siRNA) attenuates the calcium responses to hypoxia and subsequent AMPK phosphorylation, while inhibition of L-type calcium channels has no effect. Knockdown of the AMPK upstream kinase LKB1 by siRNA does not prevent AMPK activation during hypoxia, but knockdown of CaMKKβ abolishes the AMPK response. These findings reveal that hypoxia can trigger AMPK activation in the apparent absence of increased [AMP] through ROS-dependent CRAC channel activation, leading to increases in cytosolic calcium that activate the AMPK upstream kinase CaMKKβ. activate the AMPK upstream kinase CaMKKβ.)
• Zachariah 2014 Diabetes  + (AMP-activated protein kinase (AMPK) is a h … AMP-activated protein kinase (AMPK) is a heterotrimeric complex, composed of a catalytic subunit (α) and two regulatory subunits (β and γ), which act as a metabolic sensor to regulate glucose and lipid metabolism. A mutation in the γ3 subunit (AMPKγ3(R225Q)) increases basal AMPK phosphorylation, while concomitantly reducing sensitivity to AMP. AMPKγ3(R225Q) (γ3(R225Q)) transgenic mice are protected against dietary-induced triglyceride accumulation and insulin resistance. We determined whether skeletal muscle-specific expression of AMPKγ3(R225Q) prevents metabolic abnormalities in leptin-deficient ob/ob (ob/ob-γ3(R225Q)) mice. Glycogen content was increased, triglyceride content was decreased, and diacylglycerol and ceramide content were unaltered in gastrocnemius muscle from ob/ob-γ3(R225Q) mice, whereas glucose tolerance was unaltered. Insulin-stimulated glucose uptake in extensor digitorum longus muscle during the euglycemic-hyperinsulinemic clamp was increased in lean γ3(R225Q) mice, but not in ob/ob-γ3(R225Q) mice. Acetyl-CoA carboxylase phosphorylation was increased in gastrocnemius muscle from γ3(R225Q) mutant mice independent of adiposity. Glycogen and triglyceride content were decreased after leptin treatment (5 days) in ob/ob mice, but not in ob/ob-γ3(R225Q) mice. In conclusion, metabolic improvements arising from muscle-specific expression of AMPKγ3(R225Q) are insufficient to ameliorate insulin resistance and obesity in leptin-deficient mice. Central defects due to leptin deficiency may override any metabolic benefit conferred by peripheral overexpression of the AMPKγ3(R225Q) mutation.rexpression of the AMPKγ3(R225Q) mutation.)
• Dorigatti 2021 Geroscience  + (AMP-activated protein kinase (AMPK) is a c … AMP-activated protein kinase (AMPK) is a central regulator of both lifespan and health across multiple model organisms. β-Guanidinopropionic acid (GPA) is an endogenous AMPK activator previously shown to improve metabolic function in young and obese mice. In this study, we tested whether age of administration significantly affects the physiological outcomes of GPA administration in mice. We report that intervention starting at 7-8 months (young) results in activation of AMPK signaling and a phenotype consisting of lower body mass, improved glucose control, enhanced exercise tolerance, and altered mitochondrial electron transport chain flux similar to previous reports. When GPA treatment is started at 18-19 months (old), the effect of GPA on AMPK signaling is blunted compared to younger mice despite similar accumulation of GPA in skeletal muscle. Even so, GPA administration in older animals delayed age-related declines in lean mass, improved measures of gait performance and circadian rhythm, and increased fat metabolism as measured by respiratory exchange ratio. These results are likely partially driven by the relative difference in basal function and metabolic plasticity between young and old mice. Our results suggest that age-related declines in AMPK sensitivity may limit potential strategies targeting AMPK signaling in older subjects and suggest that further research and development is required for AMPK activators to realize their full potential.ctivators to realize their full potential.)
• Coelho 2015 Abstract Thyroid Cancer  + (AMP-activated protein kinase (AMPK) is a s … AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that acts directly on cell proliferation and on transition from anaerobic to aerobic metabolic state. Our group showed that the activated form of AMPK (pAMPK) is overexpressed in papillary thyroid carcinoma (PTC) cases by immunohistochemistry. AMPK presents anti proliferative effects, so the biological meaning of AMPK activation in thyroid tumor cells and the consequences of its further activation on PTC metabolism is not known. </br></br>We used the PTC-derived BCPAP cell line to analyze cellular responses to a further stimulation of AMPK with the pharmacological activator 5-aminoimidazole-4 carboxamide ribonucleoside (1 mM AICAR). Cell viability was measured by MTT, and apoptosis was analyzed by the expression of Annexin V by Muse cell analyser. ROS was measured by DCFHD probe by FACS. Oxygen consumption was measured using high-resolution respirometry (Oroboros). Hexokinase (HK) tertiary structure was evaluated on a spectrofluorometer (Jasco).</br></br>BCPAP cells constitutively express high pAMPK levels, which correlates with the high glucose consumption rate (2 fold) and lactate production (2 fold), but lower oxygen consumption (30%) in these cells when compared to the nontumoral NTHYori cell line. AICAR exposure further stimulated these metabolic parameters, but decreased BCPAP cell viability (4 fold), with increased (1.5 fold) production of reactive oxygen species (ROS). HK is an enzyme of the glycolysis pathway that, upon binding to mitochondria, protects against ROS. We observed an increase in HK activity (1.4 fold) produced by AICAR, but not in its association with HK-mitochondrial activity. Purified HK experiments confirmed that ROS (100 μM Z₂O₂) alters the tertiary structure of HK decreasing its mitochondrial binding. The presence of NAC prevented cell death induced by AICAR treatment. </br></br>Overall, these results suggest that, despite the upregulation of glucose metabolism by AMPK, chronic activation of the enzyme with AICAR increases ROS levels promoting a negative regulation of HK-mitochondrial binding. The further increase in ROS production induced by AICAR might play a role in the induction of tumor cell apoptosis. AICAR might play a role in the induction of tumor cell apoptosis.)
• Canto 2009 Nature  + (AMP-activated protein kinase (AMPK) is a m … AMP-activated protein kinase (AMPK) is a metabolic fuel gauge conserved along the evolutionary scale in eukaryotes that senses changes in the intracellular AMP/ATP ratio. Recent evidence indicated an important role for AMPK in the therapeutic benefits of metformin, thiazolidinediones and exercise, which form the cornerstones of the clinical management of type 2 diabetes and associated metabolic disorders. In general, activation of AMPK acts to maintain cellular energy stores, switching on catabolic pathways that produce ATP, mostly by enhancing oxidative metabolism and mitochondrial biogenesis, while switching off anabolic pathways that consume ATP. This regulation can take place acutely, through the regulation of fast post-translational events, but also by transcriptionally reprogramming the cell to meet energetic needs. Here we demonstrate that AMPK controls the expression of genes involved in energy metabolism in mouse skeletal muscle by acting in coordination with another metabolic sensor, the NAD+-dependent type III deacetylase SIRT1. AMPK enhances SIRT1 activity by increasing cellular NAD+ levels, resulting in the deacetylation and modulation of the activity of downstream SIRT1 targets that include the [[PGC-1alpha|peroxisome proliferator-activated receptor-gamma coactivator 1alpha]] and the forkhead box O1 (FOXO1) and O3 (FOXO3a) transcription factors. The AMPK-induced SIRT1-mediated deacetylation of these targets explains many of the convergent biological effects of AMPK and SIRT1 on energy metabolism.ts of AMPK and SIRT1 on energy metabolism.)

• ASMRM 2021 Singapore SG  + (ASMRM 2020, Singapore, SG, 2021)

• Li 2014 Mol Cell Biol  + (ATAD3 is a vital ATPase of the inner mitoc … ATAD3 is a vital ATPase of the inner mitochondrial membrane of pluri-cellular eucaryotes with largely unknown functions. Invalidation of ATAD3 blocks organism development at early stages requiring mitochondrial mass increase. Since ATAD3 knock-down (KD) in C. elegans inhibits first of all the development of adipocyte-like intestinal tissue, we used mouse adipocyte model 3T3-L1 cells to analyze ATAD3 functions during adipogenesis. By stable and transient modulation of ATAD3 expression in adipogenesis-induced 3T3-L1 cells, we show that (i) an increase in ATAD3 is preceding mitochondrial biogenesis and remodelling; (ii) down-regulation of ATAD3 inhibits adipogenesis, lipogenesis, and impedes overexpression of many mitochondrial proteins; (iii) ATAD3 re-expression rescues the phenotype of ATAD3 KD, and (iv) differentiation and lipogenesis are accelerated by ATAD3 overexpression, but inhibited by expression of a dominant-negative mutant. We further show that the ATAD3 KD phenotype is not due to altered insulin signal, but involves a limitation of mitochondrial biogenesis and remodelling linked to Drp1. These results demonstrate that ATAD3 is limiting for ''in vitro'' adipogenesis and lipogenesis.''in vitro'' adipogenesis and lipogenesis.)
• Koopman 2015 Abstract MiP2015  + (ATP can be produced in the cytosol by glyc … ATP can be produced in the cytosol by glycolytic conversion of glucose (GLC) into pyruvate (PYR). The latter can be metabolized into lactate (LAC), which is released by the cell, or taken up by mitochondria to fuel ATP production by the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) system. Altering the balance between glycolytic and mitochondrial ATP generation is crucial for cell survival during mitoenergetic dysfunction, which is observed in a large variety of human disorders including cancer [1].</br></br>To gain insight into the kinetic properties of this adaptive mechanism we here determined how acute (30 min) inhibition of OXPHOS affected cytosolic GLC homeostasis. GLC dynamics were analyzed in single living C2C12 myoblasts expressing the fluorescent biosensor FLII12Pglu-700µδ6 (FLII, [2]). Following ''in situ'' FLII calibration, the kinetic properties of GLC uptake (V1) and GLC consumption (V2) were determined independently and used to construct a minimal mathematical model of cytosolic GLC dynamics [3].</br></br>After validating the model, it was applied to quantitatively predict V1 and V2 at steady-state (i.e. when V1=V2=Vsteady-state) in the absence and presence of OXPHOS inhibitors. Integrating model predictions with experimental data on LAC production, cell volume and oxygen consumption revealed that glycolysis and mitochondria equally contribute to cellular ATP production in control myoblasts. Inhibition of OXPHOS induced a 2-fold increase in Vsteady-state and glycolytic ATP production flux. Both in the absence and presence of OXPHOS inhibitors, GLC was consumed at near maximal rates, meaning that GLC consumption is rate-limiting under steady-state conditions.</br></br>Taken together, we here demonstrate that OXPHOS inhibition increases steady-state GLC uptake and consumption in C2C12 myoblasts [3]. The latter activation fully compensates for the reduction in mitochondrial ATP production, thereby maintaining the balance between cellular ATP supply and demand. The underlying mechanistic aspects and further consequences of this phenomenon [e.g. 4,5] are currently investigated.non [e.g. 4,5] are currently investigated.)
• Liemburg-Apers 2015 Biophys J  + (ATP can be produced in the cytosol by glyc … ATP can be produced in the cytosol by glycolytic conversion of glucose (GLC) into pyruvate. The latter can be metabolized into lactate, which is released by the cell, or taken up by mitochondria to fuel ATP production by the tricarboxylic acid cycle and oxidative phosphorylation (OXPHOS) system. Altering the balance between glycolytic and mitochondrial ATP generation is crucial for cell survival during mitoenergetic dysfunction, which is observed in a large variety of human disorders including cancer. To gain insight into the kinetic properties of this adaptive mechanism we determined here how acute (30 min) inhibition of OXPHOS affected cytosolic GLC homeostasis. GLC dynamics were analyzed in single living C2C12 myoblasts expressing the fluorescent biosensor FLII(12)Pglu-700μδ6 (FLII). Following ''in situ'' FLII calibration, the kinetic properties of GLC uptake (V1) and GLC consumption (V2) were determined independently and used to construct a minimal mathematical model of cytosolic GLC dynamics. After validating the model, it was applied to quantitatively predict V1 and V2 at steady-state (i.e., when V1 = V2 = Vsteady-state) in the absence and presence of OXPHOS inhibitors. Integrating model predictions with experimental data on lactate production, cell volume, and O2 consumption revealed that glycolysis and mitochondria equally contribute to cellular ATP production in control myoblasts. Inhibition of OXPHOS induced a twofold increase in Vsteady-state and glycolytic ATP production flux. Both in the absence and presence of OXPHOS inhibitors, GLC was consumed at near maximal rates, meaning that GLC consumption is rate-limiting under steady-state conditions. Taken together, we demonstrate here that OXPHOS inhibition increases steady-state GLC uptake and consumption in C2C12 myoblasts. This activation fully compensates for the reduction in mitochondrial ATP production, thereby maintaining the balance between cellular ATP supply and demand.</br></br>Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.shed by Elsevier Inc. All rights reserved.)
• Pesout MiP2010  + (ATP decreases rapidly during ischemia. It … ATP decreases rapidly during ischemia. It is then degraded to adenosine, which moves to the extracellular space and activates adenosine receptors (ADOR). ADOR play a unique role in cardioprotection against ischemia reperfusion injury, because their activation is responsible for cardioprotection by ischemic preconditioning and ischemic postconditioning.onditioning and ischemic postconditioning.)
• Bundgaard 2019 Sci Rep  + (ATP depletion and succinate accumulation d … ATP depletion and succinate accumulation during ischemia lead to oxidative damage to mammalian organs upon reperfusion. In contrast, freshwater turtles survive weeks of anoxia at low temperatures without suffering from oxidative damage upon reoxygenation, but the mechanisms are unclear. To determine how turtles survive prolonged anoxia, we measured ~80 metabolites in hearts from cold-acclimated (5 °C) turtles exposed to 9 days anoxia and compared the results with those for normoxic turtles (25 °C) and mouse hearts exposed to 30 min of ischemia. In turtles, ATP and ADP decreased to new steady-state levels during fasting and cold-acclimation and further with anoxia, but disappeared within 30 min of ischemia in mouse hearts. High NADH/NAD⁺ ratios were associated with succinate accumulation in both anoxic turtles and ischemic mouse hearts. However, succinate concentrations and succinate/fumarate ratios were lower in turtle than in mouse heart, limiting the driving force for production of reactive oxygen species (ROS) upon reoxygenation in turtles. Furthermore, we show production of ROS from succinate is prevented by re-synthesis of ATP from ADP. Thus, maintenance of an ATP/ADP pool and low succinate accumulation likely protects turtle hearts from anoxia/reoxygenation injury and suggests metabolic interventions as a therapeutic approach to limit ischemia/reperfusion injury in mammals.roach to limit ischemia/reperfusion injury in mammals.)
• Ley-Ngardigal 2022 FEBS J  + (ATP is the most universal and essential en … ATP is the most universal and essential energy molecule in cells. This is due to its ability to store cellular energy in form of high-energy phosphate bonds, which are extremely stable and readily usable by the cell. This energy is key for a variety of biological functions such as cell growth and division, metabolism, and signaling, and for the turnover of biomolecules. Understanding how ATP is produced and hydrolyzed with a spatiotemporal resolution is necessary to understand its functions both in physiological and in pathological contexts. In this review, first we will describe the organization of the electron transport chain and ATP synthase, the main molecular motor for ATP production in mitochondria. Second, we will review the biochemical assays currently available to estimate ATP quantities in cells, and we will compare their readouts, strengths, and weaknesses. Finally, we will explore the palette of genetically encoded biosensors designed for microscopy-based approaches, and show how their spatiotemporal resolution opened up the possibility to follow ATP levels in living cells.lity to follow ATP levels in living cells.)
• Nuskova 2015 Abstract MiPschool London 2015  + (ATP produced by the mitochondrial FoF1-ATP … ATP produced by the mitochondrial FoF1-ATP synthase represents a major source of energy for aerobic organisms. Unsurprisingly, ATP synthase deficiencies are associated with severe pathologic phenotypes. To shed light on the functional consequences of ATP synthase deficiencies, we utilised a model of HEK293 cell line and explored the effect of RNAi mediated knockdown of the three subunits (γ, δ and ε) forming the central stalk of the enzyme connecting Fo and F1 domains.</br></br>For functional evaluations of ATP synthase deficiencies, 10 stable knockdown clones with down-regulated subunits γ (ATP5C1 gene), δ (ATP5D gene), or ε (ATP5E gene) have been selected. The protein content of ATP synthase subunit α among the knockdown clones covers the range of 40–100 % as compared to controls. Further characterization of these clones revealed 2–78 % oligomycin-sensitive ATPase hydrolytic activity that parallels a decrease in the content of fully assembled ATP synthase complex.</br></br>Two aspects of cellular energetics have been examined in detail, specifically respiration and glycolysis, using the Seahorse XFe24 analyser. Our results indicate that the clones with less than 30 % of residual ATPase activity switched their metabolism to enhanced glycolysis. There is a decrease in their basal respiration rate relatively to their respiratory capacity (47 vs 61 % in controls) and in parallel, their basal glycolytic rates utilise by up to 20 % more of their glycolytic capacity. These findings clearly demonstrate metabolic adaptations of these cells. On the other hand, the clones with more than 30 % residual ATPase activity displayed a change neither in the respiration nor in their basal glycolytic rate. </br></br>In the case of ATP synthase deficiency, the mitochondrial membrane potential is expected to rise, which would then stimulate the production of reactive oxygen species (ROS). Indeed, the γ knockdown clones with a very low residual ATPase activity exhibit elevated ROS production. With respect to the role of the oxidative stress in ATP synthase deficiencies, we aim to examine oxidative damage of cell structures and the content of antioxidant enzymes.</br></br>In conclusion, using these model clones, we are planning on investigating the effect of ATP synthase deficiency on the mitochondrial energetics, oxidative stress, energy state, and cell viability and define the threshold residual activity of ATP synthase for the presentation of pathological phenotype. At this moment, our data suggest that the threshold for metabolic remodelling equals to approximately 30 % of ATPase activity. to approximately 30 % of ATPase activity.)
• Cortassa 2022 J Mol Cell Cardiol  + (ATP synthase (F1Fo) is a rotary molecular … ATP synthase (F1Fo) is a rotary molecular engine that harnesses energy from electrochemical-gradients across the inner mitochondrial membrane for ATP synthesis. Despite the accepted tenet that F1Fo transports exclusively H+, our laboratory has demonstrated that, in addition to H+, F1Fo ATP synthase transports a significant fraction of ΔΨm-driven charge as K+ to synthesize ATP. Herein, we utilize a computational modeling approach as a proof of principle of the feasibility of the core mechanism underlying the enhanced ATP synthesis, and to explore its bioenergetic consequences. A minimal model comprising the 'core' mechanism constituted by ATP synthase, driven by both proton (PMF) and potassium motive force (KMF), respiratory chain, adenine nucleotide translocator, Pi carrier, and K+/H+ exchanger (KHEmito) was able to simulate enhanced ATP synthesis and respiratory fluxes determined experimentally with isolated heart mitochondria. This capacity of F1Fo ATP synthase confers mitochondria with a significant energetic advantage compared to K+ transport through a channel not linked to oxidative phosphorylation (OxPhos). The K+-cycling mechanism requires a KHEmito that exchanges matrix K+ for intermembrane space H+, leaving PMF as the overall driving energy of OxPhos, in full agreement with the standard chemiosmotic mechanism. Experimental data of state 4➔3 energetic transitions, mimicking low to high energy demand, could be reproduced by an integrated computational model of mitochondrial function that incorporates the 'core' mechanism. Model simulations display similar behavior compared to the experimentally observed changes in ΔΨm, mitochondrial K+ uptake, matrix volume, respiration, and ATP synthesis during the energetic transitions at physiological pH and K+ concentration. The model also explores the role played by KHEmito in modulating the energetic performance of mitochondria. The results obtained support the available experimental evidence on ATP synthesis driven by K+ and H+ transport through the F1Fo ATP synthase.+ transport through the F1Fo ATP synthase.)
• Juhaszova 2021 Function (Oxf)  + (ATP synthase (F₁F&l … ATP synthase (F₁F_o) synthesizes daily our body's weight in ATP, whose production-rate can be transiently increased several-fold to meet changes in energy utilization. Using purified mammalian F₁F_o-reconstituted proteoliposomes and isolated mitochondria, we show F₁F_o can utilize both Δ''Ψ''_mt-driven H⁺- and K⁺-transport to synthesize ATP under physiological pH = 7.2 and K⁺ = 140 mEq/L conditions. Purely K⁺-driven ATP synthesis from single F₁F_o molecules measured by bioluminescence photon detection could be directly demonstrated along with simultaneous measurements of unitary K⁺ currents by voltage clamp, both blocked by specific F_o inhibitors. In the presence of K⁺, compared to osmotically-matched conditions in which this cation is absent, isolated mitochondria display 3.5-fold higher rates of ATP synthesis, at the expense of 2.6-fold higher rates of oxygen consumption, these fluxes being driven by a 2.7:1 K⁺: H⁺ stoichiometry. The excellent agreement between the functional data obtained from purified F₁F_o single molecule experiments and ATP synthase studied in the intact mitochondrion under unaltered OxPhos coupling by K⁺ presence, is entirely consistent with K⁺ transport through the ATP synthase driving the observed increase in ATP synthesis. Thus, both K⁺ (harnessing Δ''Ψ''_mt) and H⁺ (harnessing its chemical potential energy, Δ''μ''_H) drive ATP generation during normal physiology.istent with K⁺ transport through the ATP synthase driving the observed increase in ATP synthesis. Thus, both K⁺ (harnessing Δ''Ψ''_mt) and H⁺ (harnessing its chemical potential energy, Δ''μ''_H) drive ATP generation during normal physiology.)
• Juhaszova 2019 bioRxiv  + (ATP synthase (F₁F&l … ATP synthase (F₁F_o) synthesizes daily our body’s weight in ATP, whose production-rate can be transiently increased several-fold. Using purified mammalian F₁F_o-reconstituted proteoliposomes and isolated mitochondria, we show that F₁F_o utilizes both H⁺- and K⁺-transport (because of >10⁶-fold K⁺ excess vs H⁺) to drive ATP synthesis with the H⁺:K⁺ permeability of ~10⁶:1. F₁F_o can be upregulated by endogenous survival-related proteins (Bcl-xL, Mcl-1) and synthetic molecules (diazoxide, pinacidil) to increase its chemo-mechanical efficiency via IF₁. Increasing K⁺- and H⁺-driven ATP synthesis enables F₁F_o to operate as a primary mitochondrial K⁺-uniporter regulating energy supply-demand matching, and as the recruitable mitochondrial K_ATP-channel that can limit ischemia-reperfusion injury. Isolated mitochondria in the presence of K⁺ can sustain ~3.5-fold higher ATP-synthesis-flux (vs K⁺ absence) driven by a 2.7:1 K⁺:H⁺ stoichiometry with unaltered OxPhos coupling. Excellent agreement between F₁F_o single-molecule and intact-mitochondria experiments is consistent with K⁺-transport through ATP synthase driving a major fraction of ATP synthesis. (vs K⁺ absence) driven by a 2.7:1 K⁺:H⁺ stoichiometry with unaltered OxPhos coupling. Excellent agreement between F₁F_o single-molecule and intact-mitochondria experiments is consistent with K⁺-transport through ATP synthase driving a major fraction of ATP synthesis.)
• Walker 1994 Curr Opin Struct Biol  + (ATP synthase is regulated so as to prevent … ATP synthase is regulated so as to prevent futile hydrolysis of ATP when the transmembrane proton electrochemical gradient, delta mu H+, falls. Mitochondria and chloroplasts have different mechanisms for inhibition of ATP synthase: by binding an inhibitor protein, and by stabilization of the ADP-inhibited state by making an intramolecular disulphide bond, respectively. The recently determined structure of bovine F1-ATPase is locked in a conformation that probably represents the ADP-inhibited state of the enzyme.nts the ADP-inhibited state of the enzyme.)
• Klusch 2017 Elife  + (ATP synthases produce ATP by rotary cataly … ATP synthases produce ATP by rotary catalysis, powered by the electrochemical proton gradient across the membrane. Understanding this fundamental process requires an atomic model of the proton pathway. We determined the structure of an intact mitochondrial ATP synthase dimer by electron cryo-microscopy at near-atomic resolution. Charged and polar residues of the a-subunit stator define two aqueous channels, each spanning one half of the membrane. Passing through a conserved membrane-intrinsic helix hairpin, the lumenal channel protonates an acidic glutamate in the c-ring rotor. Upon ring rotation, the protonated glutamate encounters the matrix channel and deprotonates. An arginine between the two channels prevents proton leakage. The steep potential gradient over the sub-nm inter-channel distance exerts a force on the deprotonated glutamate, resulting in net directional rotation.te, resulting in net directional rotation.)
• Morciano 2017 Nat Protoc  + (ATP, the energy exchange factor that conne … ATP, the energy exchange factor that connects anabolism and catabolism, is required for major reactions and processes that occur in living cells, such as muscle contraction, phosphorylation and active transport. ATP is also the key molecule in extracellular purinergic signaling mechanisms, with an established crucial role in inflammation and several additional disease conditions. Here, we describe detailed protocols to measure the ATP concentration in isolated living cells and animals using luminescence techniques based on targeted luciferase probes. In the presence of magnesium, oxygen and ATP, the protein luciferase catalyzes oxidation of the substrate luciferin, which is associated with light emission. Recombinantly expressed wild-type luciferase is exclusively cytosolic; however, adding specific targeting sequences can modify its cellular localization. Using this strategy, we have constructed luciferase chimeras targeted to the mitochondrial matrix and the outer surface of the plasma membrane. Here, we describe optimized protocols for monitoring ATP concentrations in the cytosol, mitochondrial matrix and pericellular space in living cells via an overall procedure that requires an average of 3 d. In addition, we present a detailed protocol for the in vivo detection of extracellular ATP in mice using luciferase-transfected reporter cells. This latter procedure may require up to 25 d to complete.cedure may require up to 25 d to complete.)
• Bayot 2014 Biochimie  + (ATP-dependent proteases are currently emer … ATP-dependent proteases are currently emerging as key regulators of mitochondrial functions. Among these proteolytic systems, Lon protease is involved in the control of selective protein turnover in the mitochondrial matrix. In the absence of Lon, yeast cells have been shown to accumulate electron-dense inclusion bodies in the matrix space, to loose integrity of mitochondrial genome and to be respiratory deficient. In order to address the role of Lon in mitochondrial functionality in human cells, we have set up a HeLa cell line stably transfected with a vector expressing a shRNA under the control of a promoter which is inducible with doxycycline. We have demonstrated that reduction of Lon protease results in a mild phenotype in this cell line in contrast with what have been observed in other cell types such as WI-38 fibroblasts. Nevertheless, deficiency in Lon protease led to an increase in ROS production and to an accumulation of carbonylated protein in the mitochondria. Our study suggests that Lon protease has a wide variety of targets and is likely to play different roles depending of the cell type.ifferent roles depending of the cell type.)
• Kahancova 2018 FEBS Lett  + (ATPase Inhibitory factor 1 (IF1) is an end … ATPase Inhibitory factor 1 (IF1) is an endogenous regulator of mitochondrial ATP synthase, which is involved in cellular metabolism. Although great progress has been made, biological roles of IF1 and molecular mechanisms of its action are still to be elucidated. Here, we show that IF1 is present in pancreatic β-cells, bound to the ATP synthase also under normal physiological conditions. IF1 silencing in model pancreatic β-cells (INS-1E) increases insulin secretion over a range of glucose concentrations. The left-shifted dose-response curve reveals excessive insulin secretion even under low glucose, corresponding to fasting conditions. A parallel increase in cellular respiration and ATP levels is observed. To conclude, our results indicate that IF1 is a negative regulator of insulin secretion involved in pancreatic β-cell glucose sensing.lved in pancreatic β-cell glucose sensing.)
• ATSPB 2023 Hall AT  + (ATSPB 2023, Hall in Tirol, Austria, 2023)
• Gnaiger 2000 Transplant Proc  + (AUTOOXIDATION reactions of highly reduced … AUTOOXIDATION reactions of highly reduced organic compounds are a source of reactive oxygen species that contribute to ischemia/reperfusion injury. Antioxidants such as reduced glutathione (GSH; g-glutamylcysteinylglycine) are added to organ preservation solutions to reduce oxidative stress.^1,2 GSH is unstable, however, in University of Wisconsin (UW) solution in the presence of oxygen.³ To our knowledge, no reports are available on the stability of GSH in other organ preservation solutions, such as histidine-tryptophan-ketoglutarate (HTK) or Celsior solution. The present study reports for the first time a quantitative comparison of autooxidation of GSH in a variety of established preservation solutions, demonstrating in particular the high stability of GSH in HTK solution.nstrating in particular the high stability of GSH in HTK solution.)
• ARVO 2018 Honolulu HI US  + (AVRO - Association for Research in Vision and Ophthalmology, Honolulu, Hawaii, USA, 2018)
• Abcam Mitochondria Meeting 2014  + (Abcam Mitochondria Meeting 2014, London, UK; [http://www.abcam.com/index.html?pageconfig=resource&rid=16185&viapagetrap=mitochondriafeb Abcam Mitochondria Meeting 2014])
• Bricambert 2018 Nat Commun  + (Aberrant histone methylation profile is re … Aberrant histone methylation profile is reported to correlate with the development and progression of NAFLD during obesity. However, the identification of specific epigenetic modifiers involved in this process remains poorly understood. Here, we identify the histone demethylase Plant Homeodomain Finger 2 (Phf2) as a new transcriptional co-activator of the transcription factor Carbohydrate Responsive Element Binding Protein (ChREBP). By specifically erasing H3K9me2 methyl-marks on the promoter of ChREBP-regulated genes, Phf2 facilitates incorporation of metabolic precursors into mono-unsaturated fatty acids, leading to hepatosteatosis development in the absence of inflammation and insulin resistance. Moreover, the Phf2-mediated activation of the transcription factor NF-E2-related factor 2 (Nrf2) further reroutes glucose fluxes toward the pentose phosphate pathway and glutathione biosynthesis, protecting the liver from oxidative stress and fibrogenesis in response to diet-induced obesity. Overall, our findings establish a downstream epigenetic checkpoint, whereby Phf2, through facilitating H3K9me2 demethylation at specific gene promoters, protects liver from the pathogenesis progression of NAFLD.rom the pathogenesis progression of NAFLD.)
• Wu 2018 Adv Sci  + (Aberrant mitochondrial energy transfer und … Aberrant mitochondrial energy transfer underlies prevalent chronic health conditions, including cancer, cardiovascular, and neurodegenerative diseases. Mitochondrial transplantation represents an innovative strategy aimed at restoring favorable metabolic phenotypes in cells with dysfunctional energy metabolism. While promising, significant barriers to ''in vivo'' translation of this approach abound, including limited cellular uptake and recognition of mitochondria as foreign. The objective is to functionalize isolated mitochondria with a biocompatible polymer to enhance cellular transplantation and eventual ''in vivo'' applications. Herein, it is demonstrated that grafting of a polymer conjugate composed of dextran with triphenylphosphonium onto isolated mitochondria protects the organelles and facilitates cellular internalization compared with uncoated mitochondria. Importantly, mitochondrial transplantation into cancer and cardiovascular cells has profound effects on respiration, mediating a shift toward improved oxidative phosphorylation, and reduced glycolysis. These findings represent the first demonstration of polymer functionalization of isolated mitochondria, highlighting a viable strategy for enabling clinical applications of mitochondrial transplantation.ications of mitochondrial transplantation.)
• Scrima 2020 bioRxiv  + (Abnormal hemoglobins can have major conseq … Abnormal hemoglobins can have major consequences for tissue delivery of oxygen. Correct diagnosis of hemoglobinopathies with altered oxygen affinity requires a determination of hemoglobin oxygen dissociation curve (ODC), which relates the hemoglobin oxygen saturation to the partial pressure of oxygen in the blood. Determination of the ODC of human hemoglobin is typically carried out under conditions in which hemoglobin is in equilibrium with O₂ at each partial pressure. However, in the human body due to the fast transit of RBCs through tissues hemoglobin oxygen exchanges occur under non-equilibrium conditions. We describe the determination of non-equilibrium ODC, and show that under these conditions Hb cooperativity has two apparent components in the Adair, Perutz, and MWC models of Hb. The first component, which we call sequential cooperativity, accounts for ∼70% of Hb cooperativity, and emerges from the constraint of sequential binding that is shared by the three models. The second component, which we call conformational cooperativity, accounts for ∼30% of Hb cooperativity, and is due either to a conformational equilibrium between low affinity and high affinity tetramers (as in the MWC model), or to a conformational change from low to high affinity once two of the tetramer sites are occupied (Perutz model).two of the tetramer sites are occupied (Perutz model).)
• Szabo 2020 Int J Mol Sci  + (Abnormal tau protein aggregation in the br … Abnormal tau protein aggregation in the brain is a hallmark of tauopathies, such as frontotemporal lobar degeneration and Alzheimer's disease. Substantial evidence has been linking tau to neurodegeneration, but the underlying mechanisms have yet to be clearly identified. Mitochondria are paramount organelles in neurons, as they provide the main source of energy (adenosine triphosphate) to these highly energetic cells. Mitochondrial dysfunction was identified as an early event of neurodegenerative diseases occurring even before the cognitive deficits. Tau protein was shown to interact with mitochondrial proteins and to impair mitochondrial bioenergetics and dynamics, leading to neurotoxicity. In this review, we discuss in detail the different impacts of disease-associated tau protein on mitochondrial functions, including mitochondrial transport, network dynamics, mitophagy and bioenergetics. We also give new insights about the effects of abnormal tau protein on mitochondrial neurosteroidogenesis, as well as on the endoplasmic reticulum-mitochondria coupling. A better understanding of the pathomechanisms of abnormal tau-induced mitochondrial failure may help to identify new targets for therapeutic interventions.new targets for therapeutic interventions.)
• Valis 2017 Oncotarget  + (Abnormalities in cancer metabolism represe … Abnormalities in cancer metabolism represent potential targets for cancer therapy. We have recently identified a natural compound Quambalarine B (QB), which inhibits proliferation of several leukemic cell lines followed by cell death. We have predicted ubiquinone binding sites of mitochondrial respiratory complexes as potential molecular targets of QB in leukemia cells. Hence, we tracked the effect of QB on leukemia metabolism by applying several omics and biochemical techniques. We have confirmed the inhibition of respiratory complexes by QB and found an increase in the intracellular AMP levels together with respiratory substrates. Inhibition of mitochondrial respiration by QB triggered reprogramming of leukemic cell metabolism involving disproportions in glycolytic flux, inhibition of proteins O-glycosylation, stimulation of glycine synthesis pathway, and pyruvate kinase activity, followed by an increase in pyruvate and a decrease in lactate levels. Inhibition of mitochondrial complex I by QB suppressed folate metabolism as determined by a decrease in formate production. We have also observed an increase in cellular levels of several amino acids except for aspartate, indicating the dependence of Jurkat (T-ALL) cells on aspartate synthesis. These results indicate blockade of mitochondrial complex I and II activity by QB and reduction in aspartate and folate metabolism as therapeutic targets in T-ALL cells. Anti-cancer activity of QB was also confirmed during ''in vivo'' studies, suggesting the therapeutic potential of this natural compound.peutic potential of this natural compound.)
• Roy Chowdhury 2018 Mol Cell Neurosci  + (Abnormalities in mitochondrial function un … Abnormalities in mitochondrial function under diabetic conditions can lead to deficits in function of cortical neurons and their support cells exhibiting a pivotal role in the pathogenesis of several neurodegenerative disorders, including Alzheimer's disease. We aimed to assess mitochondrial respiration rates and membrane potential or H₂O₂ generation simultaneously and expression of proteins involved in mitochondrial dynamics, ROS scavenging and AMPK/SIRT/PGC-1α pathway activity in cortex under diabetic conditions.</br></br>Cortical mitochondria from streptozotocin (STZ)-induced type 1 diabetic rats or mice, and aged-matched controls were used for simultaneous measurements of mitochondrial respiration rates and mitochondrial membrane potential (mtMP) or H₂O₂ using Oroboros oxygraph. Measurements of enzymatic activities of respiratory complexes were performed using spectophotometry. Protein levels in cortical mitochondria and homogenates were determined by Western blotting.</br></br>Mitochondrial coupled respiration rates and FCCP-induced uncoupled respiration rates were significantly decreased in mitochondria of cortex of STZ-diabetic rats compared to controls. The mtMP in the presence of ADP was significantly depolarized and succinate-dependent respiration rates and H₂O₂ were significantly diminished in cortical mitochondria of diabetic animals compared to controls, accompanied with reduced expression of CuZn- and Mn-superoxide dismutase. The enzymatic activities of Complex I, II, and IV and protein levels of certain components of Complex I and II, mitofusin 2 (Mfn2), dynamin-related protein 1 (DRP1), P-AMPK, SIRT2 and PGC-1α were significantly diminished in diabetic cortex.</br></br>Deficits in mitochondrial function, dynamics, and antioxidant capabilities putatively mediated through sub-optimal AMPK/SIRT/PGC-1α signaling, are involved in the development of early sub-clinical neurodegeneration in the cortex under diabetic conditions.</br></br><small>Copyright © 2018. Published by Elsevier Inc.</small>degeneration in the cortex under diabetic conditions. <small>Copyright © 2018. Published by Elsevier Inc.</small>)