Biochemical thresholds for pathological presentation of ATP synthase deficiencies.

Mitochondrial ATP synthase is responsible for production of the majority of cellular ATP. Disorders of ATP synthase in humans can be caused by numerous mutations in both structural subunits and specific assembly factors. They are associated with variable pathogenicity and clinical phenotypes ranging from mild to the most severe mitochondrial diseases. To shed light on primary/pivotal functional consequences of ATP synthase deficiency, we explored human HEK 293 cells with a varying content of fully assembled ATP synthase, selectively downregulated to 15-80% of controls by the knockdown of F1 subunits γ, δ and ε. Examination of cellular respiration and glycolytic flux revealed that enhanced glycolysis compensates for insufficient mitochondrial ATP production while reduced dissipation of mitochondrial membrane potential leads to elevated ROS production. Both insufficient energy provision and increased oxidative stress contribute to the resulting pathological phenotype. The threshold for manifestation of the ATP synthase defect and subsequent metabolic remodelling equals to 10-30% of residual ATP synthase activity. The metabolic adaptations are not able to sustain proliferation in a galactose medium, although sufficient under glucose-rich conditions. As metabolic alterations occur when the content of ATP synthase drops below 30%, some milder ATP synthase defects may not necessarily manifest with a mitochondrial disease phenotype, as long as the threshold level is not exceeded.

Metabolic profile of leukemia cells influences treatment efficacy of L-asparaginase.

BACKGROUND: Effectiveness of L-asparaginase administration in acute lymphoblastic leukemia treatment is mirrored in the overall outcome of patients. Generally, leukemia patients differ in their sensitivity to L-asparaginase; however, the mechanism underlying their inter-individual differences is still not fully understood. We have previously shown that L-asparaginase rewires the biosynthetic and bioenergetic pathways of leukemia cells to activate both anti-leukemic and pro-survival processes. Herein, we investigated the relationship between the metabolic profile of leukemia cells and their sensitivity to currently used cytostatic drugs. METHODS: Altogether, 19 leukemia cell lines, primary leukemia cells from 26 patients and 2 healthy controls were used. Glycolytic function and mitochondrial respiration were measured using Seahorse Bioanalyzer. Sensitivity to cytostatics was measured using MTS assay and/or absolute count and flow cytometry. Mitochondrial membrane potential was determined as TMRE fluorescence. RESULTS: Using cell lines and primary patient samples we characterized the basal metabolic state of cells derived from different leukemia subtypes and assessed their sensitivity to cytostatic drugs. We found that leukemia cells cluster into distinct groups according to their metabolic profile. Lymphoid leukemia cell lines and patients sensitive to L-asparaginase clustered into the low glycolytic cluster. While lymphoid leukemia cells with lower sensitivity to L-asparaginase together with resistant normal mononuclear blood cells gathered into the high glycolytic cluster. Furthermore, we observed a correlation of specific metabolic parameters with the sensitivity to L-asparaginase. Greater ATP-linked respiration and lower basal mitochondrial membrane potential in cells significantly correlated with higher sensitivity to L-asparaginase. No such correlation was found in the other cytostatic drugs tested by us. CONCLUSIONS: These data support that cell metabolism plays a prominent role in the treatment effect of L-asparaginase. Based on these findings, leukemia patients with lower sensitivity to L-asparaginase with no specific genetic characterization could be identified by their metabolic profile.

ROS production in brown adipose tissue mitochondria: the question of UCP1-dependence.

Whether active UCP1 can reduce ROS production in brown-fat mitochondria is presently not settled. The issue is of principal significance, as it can be seen as a proof- or disproof-of-principle concerning the ability of any protein to diminish ROS production through membrane depolarization. We therefore undertook a comprehensive investigation of the significance of UCP1 for ROS production, by comparing the ROS production in brown-fat mitochondria isolated from wildtype mice (that display membrane depolarization) or from UCP1(-/-) mice (with a high membrane potential). We tested the significance of UCP1 for glycerol-3-phosphate-supported ROS production by three methods (fluorescent dihydroethidium and the ESR probe PHH for superoxide, and fluorescent Amplex Red for hydrogen peroxide), and followed ROS production also with succinate, acyl-CoA or pyruvate as substrate. We studied the effects of the reverse electron flow inhibitor rotenone, the UCP1 activity inhibitor GDP, and the uncoupler FCCP. We also examined the effect of a physiologically induced increase in UCP1 amount. We noted GDP effects that were not UCP1-related. We conclude that only ROS production supported by exogenously added succinate was affected by the presence of active UCP1; ROS production supported by any other tested substrate (including endogenously generated succinate) was unaffected. This conclusion indicates that UCP1 is not involved in control of ROS production in brown-fat mitochondria. Extrapolation of these data to other tissues would imply that membrane depolarization may not necessarily decrease physiologically relevant ROS production. This article is a part of a Special Issue entitled: 18th European Bioenergetics Conference (Biochim. Biophys. Acta, Volume 1837, Issue 7, July 2014).

Cytochrome c oxidase subunit 4 isoform 2-knockout mice show reduced enzyme activity, airway hyporeactivity, and lung pathology.

Cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial electron transport chain. The purpose of this study was to analyze the function of lung-specific cytochrome c oxidase subunit 4 isoform 2 (COX4i2) in vitro and in COX4i2-knockout mice in vivo. COX was isolated from cow lung and liver as control and functionally analyzed. COX4i2-knockout mice were generated and the effect of the gene knockout was determined, including COX activity, tissue energy levels, noninvasive and invasive lung function, and lung pathology. These studies were complemented by a comprehensive functional screen performed at the German Mouse Clinic (Neuherberg, Germany). We show that isolated cow lung COX containing COX4i2 is about twice as active (88 and 102% increased activity in the presence of allosteric activator ADP and inhibitor ATP, respectively) as liver COX, which lacks COX4i2. In COX4i2-knockout mice, lung COX activity and cellular ATP levels were significantly reduced (-50 and -29%, respectively). Knockout mice showed decreased airway responsiveness (60% reduced P(enh) and 58% reduced airway resistance upon challenge with 25 and 100 mg methacholine, respectively), and they developed a lung pathology deteriorating with age that included the appearance of Charcot-Leyden crystals. In addition, there was an interesting sex-specific phenotype, in which the knockout females showed reduced lean mass (-12%), reduced total oxygen consumption rate (-8%), improved glucose tolerance, and reduced grip force (-14%) compared to wild-type females. Our data suggest that high activity lung COX is a central determinant of airway function and is required for maximal airway responsiveness and healthy lung function. Since airway constriction requires energy, we propose a model in which reduced tissue ATP levels explain protection from airway hyperresponsiveness, i.e., absence of COX4i2 leads to reduced lung COX activity and ATP levels, which results in impaired airway constriction and thus reduced airway responsiveness; long-term lung pathology develops in the knockout mice due to impairment of energy-costly lung maintenance processes; and therefore, we propose mitochondrial oxidative phosphorylation as a novel target for the treatment of respiratory diseases, such as asthma.

Hypolipidemic Effects of Beetroot Juice in SHR-CRP and HHTg Rat Models of Metabolic Syndrome: Analysis of Hepatic Proteome.

Recently, red beetroot has attracted attention as a health-promoting functional food. Studies have shown that beetroot administration can reduce blood pressure and ameliorate parameters of glucose and lipid metabolism; however, mechanisms underlying these beneficial effects of beetroot are not yet fully understood. In the current study, we analysed the effects of beetroot on parameters of glucose and lipid metabolism in two models of metabolic syndrome: (i) transgenic spontaneously hypertensive rats expressing human C-reactive protein (SHR-CRP rats), and (ii) hereditary hypertriglyceridemic (HHTg) rats. Treatment with beetroot juice for 4 weeks was, in both models, associated with amelioration of oxidative stress, reduced circulating lipids, smaller visceral fat depots, and lower ectopic fat accumulation in the liver compared to the respective untreated controls. On the other hand, beetroot treatment had no significant effects on the sensitivity of the muscle and adipose tissue to insulin action in either model. Analyses of hepatic proteome revealed significantly deregulated proteins involved in glycerophospholipid metabolism, mTOR signalling, inflammation, and cytoskeleton rearrangement.

Omega-3 PUFAs prevent bone impairment and bone marrow adiposity in mouse model of obesity.

Obesity adversely affects bone and fat metabolism in mice and humans. Omega-3 polyunsaturated fatty acids (omega-3 PUFAs) have been shown to improve glucose metabolism and bone homeostasis in obesity. However, the impact of omega-3 PUFAs on bone marrow adipose tissue (BMAT) and bone marrow stromal cell (BMSC) metabolism has not been intensively studied yet. In the present study we demonstrated that omega-3 PUFA supplementation in high fat diet (HFD + F) improved bone parameters, mechanical properties along with decreased BMAT in obese mice when compared to the HFD group. Primary BMSCs isolated from HFD + F mice showed decreased adipocyte and higher osteoblast differentiation with lower senescent phenotype along with decreased osteoclast formation suggesting improved bone marrow microenvironment promoting bone formation in mice. Thus, our study highlights the beneficial effects of omega-3 PUFA-enriched diet on bone and cellular metabolism and its potential use in the treatment of metabolic bone diseases.

Mitochondrial ATP synthase deficiency due to a mutation in the ATP5E gene for the F1 epsilon subunit.

F1Fo-ATP synthase is a key enzyme of mitochondrial energy provision producing most of cellular ATP. So far, mitochondrial diseases caused by isolated disorders of the ATP synthase have been shown to result from mutations in mtDNA genes for the subunits ATP6 and ATP8 or in nuclear genes encoding the biogenesis factors TMEM70 and ATPAF2. Here, we describe a patient with a homozygous p.Tyr12Cys mutation in the epsilon subunit encoded by the nuclear gene ATP5E. The 22-year-old woman presented with neonatal onset, lactic acidosis, 3-methylglutaconic aciduria, mild mental retardation and developed peripheral neuropathy. Patient fibroblasts showed 60-70% decrease in both oligomycin-sensitive ATPase activity and mitochondrial ATP synthesis. The mitochondrial content of the ATP synthase complex was equally reduced, but its size was normal and it contained the mutated epsilon subunit. A similar reduction was found in all investigated F1 and Fo subunits with the exception of Fo subunit c, which was found to accumulate in a detergent-insoluble form. This is the first case of a mitochondrial disease due to a mutation in a nuclear encoded structural subunit of the ATP synthase. Our results indicate an essential role of the epsilon subunit in the biosynthesis and assembly of the F1 part of the ATP synthase. Furthermore, the epsilon subunit seems to be involved in the incorporation of subunit c to the rotor structure of the mammalian enzyme.

Genetic Complementation of ATP Synthase Deficiency Due to Dysfunction of TMEM70 Assembly Factor in Rat.

Mutations of the TMEM70 gene disrupt the biogenesis of the ATP synthase and represent the most frequent cause of autosomal recessive encephalo-cardio-myopathy with neonatal onset. Patient tissues show isolated defects in the ATP synthase, leading to the impaired mitochondrial synthesis of ATP and insufficient energy provision. In the current study, we tested the efficiency of gene complementation by using a transgenic rescue approach in spontaneously hypertensive rats with the targeted Tmem70 gene (SHR-Tmem70ko/ko), which leads to embryonic lethality. We generated SHR-Tmem70ko/ko knockout rats expressing the Tmem70 wild-type transgene (SHR-Tmem70ko/ko,tg/tg) under the control of the EF-1α universal promoter. Transgenic rescue resulted in viable animals that showed the variable expression of the Tmem70 transgene across the range of tissues and only minor differences in terms of the growth parameters. The TMEM70 protein was restored to 16-49% of the controls in the liver and heart, which was sufficient for the full biochemical complementation of ATP synthase biogenesis as well as for mitochondrial energetic function in the liver. In the heart, we observed partial biochemical complementation, especially in SHR-Tmem70ko/ko,tg/0 hemizygotes. As a result, this led to a minor impairment in left ventricle function. Overall, the transgenic rescue of Tmem70 in SHR-Tmem70ko/ko knockout rats resulted in the efficient complementation of ATP synthase deficiency and thus in the successful genetic treatment of an otherwise fatal mitochondrial disorder.

Novel thiazolidinedione analog reduces a negative impact on bone and mesenchymal stem cell properties in obese mice compared to classical thiazolidinediones.

OBJECTIVE: The use of thiazolidinediones (TZDs) as insulin sensitizers has been shown to have side effects including increased accumulation of bone marrow adipocytes (BMAds) associated with a higher fracture risk and bone loss. A novel TZD analog MSDC-0602K with low affinity to PPARγ has been developed to reduce adverse effects of TZD therapy. However, the effect of MSDC-0602K on bone phenotype and bone marrow mesenchymal stem cells (BM-MSCs) in relation to obesity has not been intensively studied yet. METHODS: Here, we investigated whether 8-week treatment with MSDC-0602K has a less detrimental effect on bone loss and BM-MSC properties in obese mice in comparison to first generation of TZDs, pioglitazone. Bone parameters (bone microstructure, bone marrow adiposity, bone strength) were examined by µCT and 3-point bending test. Primary BM-MSCs were isolated and measured for osteoblast and adipocyte differentiation. Cellular senescence, bioenergetic profiling, nutrient consumption and insulin signaling were also determined. RESULTS: The findings demonstrate that MSDC-0602K improved bone parameters along with increased proportion of smaller BMAds in tibia of obese mice when compared to pioglitazone. Further, primary BM-MSCs isolated from treated mice and human BM-MSCs revealed decreased adipocyte and higher osteoblast differentiation accompanied with less inflammatory and senescent phenotype induced by MSDC-0602K vs. pioglitazone. These changes were further reflected by increased glycolytic activity differently affecting glutamine and glucose cellular metabolism in MSDC-0602K-treated cells compared to pioglitazone, associated with higher osteogenesis. CONCLUSION: Our study provides novel insights into the action of MSDC-0602K in obese mice, characterized by the absence of detrimental effects on bone quality and BM-MSC metabolism when compared to classical TZDs and thus suggesting a potential therapeutical use of MSDC-0602K in both metabolic and bone diseases.

Mitochondrial respiration supports autophagy to provide stress resistance during quiescence.

Mitochondrial oxidative phosphorylation (OXPHOS) generates ATP, but OXPHOS also supports biosynthesis during proliferation. In contrast, the role of OXPHOS during quiescence, beyond ATP production, is not well understood. Using mouse models of inducible OXPHOS deficiency in all cell types or specifically in the vascular endothelium that negligibly relies on OXPHOS-derived ATP, we show that selectively during quiescence OXPHOS provides oxidative stress resistance by supporting macroautophagy/autophagy. Mechanistically, OXPHOS constitutively generates low levels of endogenous ROS that induce autophagy via attenuation of ATG4B activity, which provides protection from ROS insult. Physiologically, the OXPHOS-autophagy system (i) protects healthy tissue from toxicity of ROS-based anticancer therapy, and (ii) provides ROS resistance in the endothelium, ameliorating systemic LPS-induced inflammation as well as inflammatory bowel disease. Hence, cells acquired mitochondria during evolution to profit from oxidative metabolism, but also built in an autophagy-based ROS-induced protective mechanism to guard against oxidative stress associated with OXPHOS function during quiescence.Abbreviations: AMPK: AMP-activated protein kinase; AOX: alternative oxidase; Baf A: bafilomycin A1; CI, respiratory complexes I; DCF-DA: 2',7'-dichlordihydrofluorescein diacetate; DHE: dihydroethidium; DSS: dextran sodium sulfate; ΔΨmi: mitochondrial inner membrane potential; EdU: 5-ethynyl-2'-deoxyuridine; ETC: electron transport chain; FA: formaldehyde; HUVEC; human umbilical cord endothelial cells; IBD: inflammatory bowel disease; LC3B: microtubule associated protein 1 light chain 3 beta; LPS: lipopolysaccharide; MEFs: mouse embryonic fibroblasts; MTORC1: mechanistic target of rapamycin kinase complex 1; mtDNA: mitochondrial DNA; NAC: N-acetyl cysteine; OXPHOS: oxidative phosphorylation; PCs: proliferating cells; PE: phosphatidylethanolamine; PEITC: phenethyl isothiocyanate; QCs: quiescent cells; ROS: reactive oxygen species; PLA2: phospholipase A2, WB: western blot.

A suggested role for mitochondria in Noonan syndrome.

Noonan syndrome (NS) is an autosomal dominant disorder, and a main feature is congenital heart malformation. About 50% of cases are caused by gain-of-function mutations in the tyrosine phosphatase SHP2/PTPN11, a downstream regulator of ERK/MAPK. Recently it was reported that SHP2 also localizes to the mitochondrial intercristae/intermembrane space (IMS), but the role of SHP2 in mitochondria is unclear. The mitochondrial oxidative phosphorylation (OxPhos) system provides the vast majority of cellular energy and produces reactive oxygen species (ROS). Changes in ROS may interfere with organ development such as that observed in NS patients. Several phosphorylation sites have been found in OxPhos components including cytochrome c oxidase (CcO) and cytochrome c (Cytc), and we hypothesized that OxPhos complexes may be direct or indirect targets of SHP2. We analyzed mitochondrial function using mouse fibroblasts from wild-types, SHP2 knockdowns, and D61G SHP2 mutants leading to constitutively active SHP2, as found in NS patients. Levels of OxPhos complexes were similar except for CcO and Cytc, which were 37% and 28% reduced in the D61G cells. However, CcO activity was significantly increased, as we also found for two lymphoblast cell lines from NS patients with two independent mutations in PTPN11. D61G cells showed lower mitochondrial membrane potential and 30% lower ATP content compared to controls. ROS were significantly increased; aconitase activity, a marker for ROS-induced damage, was decreased; and catalase activity was increased in D61G cells. We propose that decreased energy levels and/or increased ROS may explain, at least in part, some of the clinical features in NS that overlap with children with mitochondrial disorders.

POLRMT mutations impair mitochondrial transcription causing neurological disease.

While >300 disease-causing variants have been identified in the mitochondrial DNA (mtDNA) polymerase γ, no mitochondrial phenotypes have been associated with POLRMT, the RNA polymerase responsible for transcription of the mitochondrial genome. Here, we characterise the clinical and molecular nature of POLRMT variants in eight individuals from seven unrelated families. Patients present with global developmental delay, hypotonia, short stature, and speech/intellectual disability in childhood; one subject displayed an indolent progressive external ophthalmoplegia phenotype. Massive parallel sequencing of all subjects identifies recessive and dominant variants in the POLRMT gene. Patient fibroblasts have a defect in mitochondrial mRNA synthesis, but no mtDNA deletions or copy number abnormalities. The in vitro characterisation of the recombinant POLRMT mutants reveals variable, but deleterious effects on mitochondrial transcription. Together, our in vivo and in vitro functional studies of POLRMT variants establish defective mitochondrial transcription as an important disease mechanism.

Phosphomimetic substitution of cytochrome C tyrosine 48 decreases respiration and binding to cardiolipin and abolishes ability to trigger downstream caspase activation.

Mammalian cytochrome c (Cytc) transfers electrons from the bc(1) complex to cytochrome c oxidase (CcO) as part of the mitochondrial electron transport chain, and it also participates in type II apoptosis. Our recent discovery of two tyrosine phosphorylation sites in Cytc, Tyr97 in bovine heart and Tyr48 in bovine liver, indicates that Cytc functions are regulated through cell signaling. To characterize the role of Cytc tyrosine phosphorylation in detail using an independent approach, we here overexpressed and purified a Tyr48Glu mutant Cytc, mimicking the in vivo Tyr48 phosphorylation found in cow liver, along with wild-type and Tyr48Phe variants as controls. The midpoint redox potential of the phosphomimetic mutant was decreased by 45 mV compared to control (192 vs 237 mV). Similar to Tyr48 in vivo phosphorylated Cytc, direct kinetic analysis of the Cytc reaction with isolated CcO revealed decreased V(max) for the Tyr48Glu mutant by 30% compared to wild type or the Tyr48Phe variants. Moreover, the phosphomimetic substitution resulted in major changes of Cytc functions related to apoptosis. The binding affinity of Tyr48Glu Cytc to cardiolipin was decreased by about 30% compared to wild type or the Tyr48Phe variants, and Cytc peroxidase activity of the Tyr48Glu mutant was cardiolipin-inducible only at high cardiolipin concentration, unlike controls. Importantly, the Tyr48Glu Cytc failed to induce any detectable downstream activation of caspase-3. Our data suggest that in vivo Tyr48 phosphorylation might serve as an antiapoptotic switch and highlight the strategic position and role of the conserved Cytc residue Tyr48 in regulating multiple functions of Cytc.

Role of Mitochondrial Glycerol-3-Phosphate Dehydrogenase in Metabolic Adaptations of Prostate Cancer.

Prostate cancer is one of the most prominent cancers diagnosed in males. Contrasting with other cancer types, glucose utilization is not increased in prostate carcinoma cells as they employ different metabolic adaptations involving mitochondria as a source of energy and intermediates required for rapid cell growth. In this regard, prostate cancer cells were associated with higher activity of mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH), the key rate limiting component of the glycerophosphate shuttle, which connects mitochondrial and cytosolic processes and plays significant role in cellular bioenergetics. Our research focused on the role of mGPDH biogenesis and regulation in prostate cancer compared to healthy cells. We show that the 42 amino acid presequence is cleaved from N-terminus during mGPDH biogenesis. Only the processed form is part of the mGPDH dimer that is the prominent functional enzyme entity. We demonstrate that mGPDH overexpression enhances the wound healing ability in prostate cancer cells. As mGPDH is at the crossroad of glycolysis, lipogenesis and oxidative metabolism, regulation of its activity by intramitochondrial processing might represent rapid means of cellular metabolic adaptations.

Cytochrome c Oxidase Subunit 4 Isoform Exchange Results in Modulation of Oxygen Affinity.

Cytochrome c oxidase (COX) is regulated through tissue-, development- or environment-controlled expression of subunit isoforms. The COX4 subunit is thought to optimize respiratory chain function according to oxygen-controlled expression of its isoforms COX4i1 and COX4i2. However, biochemical mechanisms of regulation by the two variants are only partly understood. We created an HEK293-based knock-out cellular model devoid of both isoforms (COX4i1/2 KO). Subsequent knock-in of COX4i1 or COX4i2 generated cells with exclusive expression of respective isoform. Both isoforms complemented the respiratory defect of COX4i1/2 KO. The content, composition, and incorporation of COX into supercomplexes were comparable in COX4i1- and COX4i2-expressing cells. Also, COX activity, cytochrome c affinity, and respiratory rates were undistinguishable in cells expressing either isoform. Analysis of energy metabolism and the redox state in intact cells uncovered modestly increased preference for mitochondrial ATP production, consistent with the increased NADH pool oxidation and lower ROS in COX4i2-expressing cells in normoxia. Most remarkable changes were uncovered in COX oxygen kinetics. The p50 (partial pressure of oxygen at half-maximal respiration) was increased twofold in COX4i2 versus COX4i1 cells, indicating decreased oxygen affinity of the COX4i2-containing enzyme. Our finding supports the key role of the COX4i2-containing enzyme in hypoxia-sensing pathways of energy metabolism.

Mitochondrial dysfunction in a neural cell model of spinal muscular atrophy.

Mutations of the survival motor neuron (SMN) gene in spinal muscular atrophy (SMA) lead to anterior horn cell death. The cause is unknown, but motor neurons depend substantially on mitochondrial oxidative phosphorylation (OxPhos) for normal function. Therefore, mitochondrial parameters were analyzed in an SMA cell culture model using small interfering RNA (siRNA) transfection that decreased Smn expression in NSC-34 cells to disease levels. Smn siRNA knock-down resulted in 35% and 66% reduced Smn protein levels 48 and 72 hr posttransfection, respectively. ATP levels were reduced by 14% and 26% at 48 and 72 hr posttransfection, respectively, suggesting decreased ATP production or increased energy demand in neural cells. Smn knock-down resulted in increased mitochondrial membrane potential and increased free radical production. Changes in activity of cytochrome c oxidase (CcO), a key OxPhos component, were observed at 72 hr with a 26% increase in oxygen consumption. This suggests a compensatory activation of the aerobic pathway, resulting in increased mitochondrial membrane potentials, a condition known to lead to the observed increase in free radical production. Further testing suggested that changes in ATP at 24 hr precede observable indices of cell injury at 48 hr. We propose that energy paucity and increased mitochondrial free radical production lead to accumulated cell damage and eventual cell death in Smn-depleted neural cells. Mitochondrial dysfunction may therefore be important in SMA pathology and may represent a new therapeutic target.

High efficiency of ROS production by glycerophosphate dehydrogenase in mammalian mitochondria.

We investigated hydrogen peroxide production in mitochondria with low (liver, heart, brain) and high (brown adipose tissue, BAT) content of glycerophosphate dehydrogenase (mGPDH). ROS production at state 4 due to electron backflow from mGPDH was low, but after inhibition of electron transport with antimycin A high rates of mGPDH-dependent ROS production were observed in liver, heart and brain mitochondria. When this ROS production was related to activity of mGPDH, many-fold higher ROS production was found in contrast to succinate- (39-, 28-, 3-fold) or pyruvate plus malate-dependent ROS production (32-, 96-, 5-fold). This specific rate of mGPDH-dependent ROS production was also exceedingly higher (28-, 66-, 22-fold) compared to that in BAT. mGPDH-dependent ROS production was localized to the dehydrogenase+CoQ and complex III, the latter being the highest in all mitochondria but BAT. Our results demonstrate high efficiency of mGPDH-dependent ROS production in mammalian mitochondria with a low content of mGPDH and suggest its endogenous inhibition in BAT.

Mitochondrial targets of metformin-Are they physiologically relevant?

Metformin is the most widely prescribed treatment of hyperglycemia and type II diabetes since 1970s. During the last 15 years, its popularity increased due to epidemiological evidence, that metformin administration reduces incidence of cancer. However, despite the ongoing effort of many researchers, the molecular mechanisms underlying antihyperglycemic or antineoplastic action of metformin remain elusive. Most frequently, metformin is associated with modulation of mitochondrial metabolism leading to lowering of blood glucose or activation of antitumorigenic pathways. Here we review the reported effects of metformin on mitochondrial metabolism and their potential relevance as effective molecular targets with beneficial therapeutic outcome.

Pleiotropic Effects of Biguanides on Mitochondrial Reactive Oxygen Species Production.

Metformin is widely prescribed as a first-choice antihyperglycemic drug for treatment of type 2 diabetes mellitus, and recent epidemiological studies showed its utility also in cancer therapy. Although it is in use since the 1970s, its molecular target, either for antihyperglycemic or antineoplastic action, remains elusive. However, the body of the research on metformin effect oscillates around mitochondrial metabolism, including the function of oxidative phosphorylation (OXPHOS) apparatus. In this study, we focused on direct inhibitory mechanism of biguanides (metformin and phenformin) on OXPHOS complexes and its functional impact, using the model of isolated brown adipose tissue mitochondria. We demonstrate that biguanides nonspecifically target the activities of all respiratory chain dehydrogenases (mitochondrial NADH, succinate, and glycerophosphate dehydrogenases), but only at very high concentrations (10-2-10-1 M) that highly exceed cellular concentrations observed during the treatment. In addition, these concentrations of biguanides also trigger burst of reactive oxygen species production which, in combination with pleiotropic OXPHOS inhibition, can be toxic for the organism. We conclude that the beneficial effect of biguanides should probably be associated with subtler mechanism, different from the generalized inhibition of the respiratory chain.