Spontaneous nonsense mutation in the tuftelin 1 gene is associated with abnormal hair appearance and amelioration of glucose and lipid metabolism in the rat.

Recently, we have identified a recessive mutation, an abnormal coat appearance in the BXH6 strain, a member of the HXB/BXH set of recombinant inbred (RI) strains. The RI strains were derived from the spontaneously hypertensive rat (SHR) and Brown Norway rat (BN-Lx) progenitors. Whole genome sequencing of the mutant rats identified the 195875980 G/A mutation in the tuftelin 1 (Tuft1) gene on chromosome 2, which resulted in a premature stop codon. Compared with wild-type BXH6 rats, BXH6-Tuft1 mutant rats exhibited lower body weight due to reduced visceral fat and ectopic fat accumulation in the liver and heart. Reduced adiposity was associated with decreased serum glucose and insulin and increased insulin-stimulated glycogenesis in skeletal muscle. In addition, mutant rats had lower serum monocyte chemoattractant protein-1 and leptin levels, indicative of reduced inflammation. Analysis of the liver proteome identified differentially expressed proteins from fatty acid metabolism and ß-oxidation, peroxisomes, carbohydrate metabolism, inflammation, and proteasome pathways. These results provide evidence for the important role of the Tuft1 gene in the regulation of lipid and glucose metabolism and suggest underlying molecular mechanisms.NEW & NOTEWORTHY A new spontaneous mutation, abnormal hair appearance in the rat, has been identified as a nonfunctional tuftelin 1 (Tuft1) gene. The pleiotropic effects of this mutation regulate glucose and lipid metabolism. Analysis of the liver proteome revealed possible molecular mechanisms for the metabolic effects of the Tuft1 gene.

Mitochondrial HER2 stimulates respiration and promotes tumorigenicity.

BACKGROUND: Amplification of HER2, a receptor tyrosine kinase and a breast cancer-linked oncogene, is associated with aggressive disease. HER2 protein is localised mostly at the cell membrane, but a fraction translocates to mitochondria. Whether and how mitochondrial HER2 contributes to tumorigenicity is currently unknown. METHODS: We enriched the mitochondrial (mt-)HER2 fraction in breast cancer cells using an N-terminal mitochondrial targeting sequence and analysed how this manipulation impacts bioenergetics and tumorigenic properties. The role of the tyrosine kinase activity of mt-HER2 was assessed in wild type, kinase-dead (K753M) and kinase-enhanced (V659E) mtHER2 constructs. RESULTS: We document that mt-HER2 associates with the oxidative phosphorylation system, stimulates bioenergetics and promotes larger respiratory supercomplexes. mt-HER2 enhances proliferation and invasiveness in vitro and tumour growth and metastatic potential in vivo, in a kinase activity-dependent manner. On the other hand, constitutively active mt-HER2 provokes excessive mitochondria ROS generation, sensitises to cell death, and restricts growth of primary tumours, suggesting that regulation of HER2 activity in mitochondria is required for the maximal pro-tumorigenic effect. CONCLUSIONS: mt-HER2 promotes tumorigenicity by supporting bioenergetics and optimal redox balance.

Adipose-Derived Stem Cells in Reinforced Collagen Gel: A Comparison between Two Approaches to Differentiation towards Smooth Muscle Cells.

Scaffolds made of degradable polymers, such as collagen, polyesters or polysaccharides, are promising matrices for fabrication of bioartificial vascular grafts or patches. In this study, collagen isolated from porcine skin was processed into a gel, reinforced with collagen particles and with incorporated adipose tissue-derived stem cells (ASCs). The cell-material constructs were then incubated in a DMEM medium with 2% of FS (DMEM_part), with added polyvinylalcohol nanofibers (PVA_part sample), and for ASCs differentiation towards smooth muscle cells (SMCs), the medium was supplemented either with human platelet lysate released from PVA nanofibers (PVA_PL_part) or with TGF-ß1 + BMP-4 (TGF + BMP_part). The constructs were further endothelialised with human umbilical vein endothelial cells (ECs). The immunofluorescence staining of alpha-actin and calponin, and von Willebrand factor, was performed. The proteins involved in cell differentiation, the extracellular matrix (ECM) proteins, and ECM remodelling proteins were evaluated by mass spectrometry on day 12 of culture. Mechanical properties of the gels with ASCs were measured via an unconfined compression test on day 5. Gels evinced limited planar shrinkage, but it was higher in endothelialised TGF + BMP_part gel. Both PVA_PL_part samples and TGF + BMP_part samples supported ASC growth and differentiation towards SMCs, but only PVA_PL_part supported homogeneous endothelialisation. Young modulus of elasticity increased in all samples compared to day 0, and PVA_PL_part gel evinced a slightly higher ratio of elastic energy. The results suggest that PVA_PL_part collagen construct has the highest potential to remodel into a functional vascular wall.

TMEM70 facilitates biogenesis of mammalian ATP synthase by promoting subunit c incorporation into the rotor structure of the enzyme.

Biogenesis of F1Fo ATP synthase, the key enzyme of mitochondrial energy provision, depends on transmembrane protein 70 (TMEM70), localized in the inner mitochondrial membrane of higher eukaryotes. TMEM70 absence causes severe ATP-synthase deficiency and leads to a neonatal mitochondrial encephalocardiomyopathy in humans. However, the exact biochemical function of TMEM70 remains unknown. Using TMEM70 conditional knockout in mice, we show that absence of TMEM70 impairs the early stage of enzyme biogenesis by preventing incorporation of hydrophobic subunit c into rotor structure of the enzyme. This results in the formation of an incomplete, pathologic enzyme complex consisting of F1 domain and peripheral stalk but lacking Fo proton channel composed of subunits c and a. We demonstrated direct interaction between TMEM70 and subunit c and showed that overexpression of subunit c in TMEM70-/- cells partially rescued TMEM70 defect. Accordingly, TMEM70 knockdown prevented subunit c accumulation otherwise observed in F1-deficient cells. Altogether, we identified TMEM70 as specific ancillary factor for subunit c. The biologic role of TMEM70 is to increase the low efficacy of spontaneous assembly of subunit c oligomer, the key and rate-limiting step of ATP-synthase biogenesis, and thus to reach an adequately high physiologic level of ATP synthase in mammalian tissues.-Kovalcíková, J., Vrbacký, M., Pecina, P., Tauchmannová, K., Nusková, H., Kaplanová, V., Brázdová, A., Alán, L., Eliás, J., Cunátová, K., Korínek, V., Sedlacek, R., Mrácek, T., Houstek, J. TMEM70 facilitates biogenesis of mammalian ATP synthase by promoting subunit c incorporation into the rotor structure of the enzyme.

Knockout of Tmem70 alters biogenesis of ATP synthase and leads to embryonal lethality in mice.

TMEM70, a 21-kDa protein localized in the inner mitochondrial membrane, has been shown to facilitate the biogenesis of mammalian F1Fo ATP synthase. Mutations of the TMEM70 gene represent the most frequent cause of isolated ATP synthase deficiency resulting in a severe mitochondrial disease presenting as neonatal encephalo-cardiomyopathy (OMIM 604273). To better understand the biological role of this factor, we generated Tmem70-deficient mice and found that the homozygous Tmem70-/- knockouts exhibited profound growth retardation and embryonic lethality at ∼9.5 days post coitum. Blue-Native electrophoresis demonstrated an isolated deficiency in fully assembled ATP synthase in the Tmem70-/- embryos (80% decrease) and a marked accumulation of F1 complexes indicative of impairment in ATP synthase biogenesis that was stalled at the early stage, following the formation of F1 oligomer. Consequently, a decrease in ADP-stimulated State 3 respiration, respiratory control ratio and ATP/ADP ratios, indicated compromised mitochondrial ATP production. Tmem70-/- embryos exhibited delayed development of the cardiovascular system and a disturbed heart mitochondrial ultrastructure, with concentric or irregular cristae structures. Tmem70+/- heterozygous mice were fully viable and displayed normal postnatal growth and development of the mitochondrial oxidative phosphorylation system. Nevertheless, they presented with mild deterioration of heart function. Our results demonstrated that Tmem70 knockout in the mouse results in embryonic lethality due to the lack of ATP synthase and impairment of mitochondrial energy provision. This is analogous to TMEM70 dysfunction in humans and verifies the crucial role of this factor in the biosynthesis and assembly of mammalian ATP synthase.

Acadian variant of Fanconi syndrome is caused by mitochondrial respiratory chain complex I deficiency due to a non-coding mutation in complex I assembly factor NDUFAF6.

The Acadian variant of Fanconi Syndrome refers to a specific condition characterized by generalized proximal tubular dysfunction from birth, slowly progressive chronic kidney disease and pulmonary interstitial fibrosis. This condition occurs only in Acadians, a founder population in Nova Scotia, Canada. The genetic and molecular basis of this disease is unknown. We carried out whole exome and genome sequencing and found that nine affected individuals were homozygous for the ultra-rare non-coding variant chr8:96046914 T > C; rs575462405, whereas 13 healthy siblings were either heterozygotes or lacked the mutant allele. This variant is located in intron 2 of NDUFAF6 (NM_152416.3; c.298-768 T > C), 37 base pairs upstream from an alternative splicing variant in NDUFAF6 chr8:96046951 A > G; rs74395342 (c.298-731 A > G). NDUFAF6 encodes NADH:ubiquinone oxidoreductase complex assembly factor 6, also known as C8ORF38. We found that rs575462405-either alone or in combination with rs74395342-affects splicing and synthesis of NDUFAF6 isoforms. Affected kidney and lung showed specific loss of the mitochondria-located NDUFAF6 isoform and ultrastructural characteristics of mitochondrial dysfunction. Accordingly, affected tissues had defects in mitochondrial respiration and complex I biogenesis that were corrected with NDUFAF6 cDNA transfection. Our results demonstrate that the Acadian variant of Fanconi Syndrome results from mitochondrial respiratory chain complex I deficiency. This information may be used in the diagnosis and prevention of this disease in individuals and families of Acadian descent and broadens the spectrum of the clinical presentation of mitochondrial diseases, respiratory chain defects and defects of complex I specifically.

Tissue- and species-specific differences in cytochrome c oxidase assembly induced by SURF1 defects.

Mitochondrial protein SURF1 is a specific assembly factor of cytochrome c oxidase (COX), but its function is poorly understood. SURF1 gene mutations cause a severe COX deficiency manifesting as the Leigh syndrome in humans, whereas in mice SURF1(-/-) knockout leads only to a mild COX defect. We used SURF1(-/-) mouse model for detailed analysis of disturbed COX assembly and COX ability to incorporate into respiratory supercomplexes (SCs) in different tissues and fibroblasts. Furthermore, we compared fibroblasts from SURF1(-/-) mouse and SURF1 patients to reveal interspecies differences in kinetics of COX biogenesis using 2D electrophoresis, immunodetection, arrest of mitochondrial proteosynthesis and pulse-chase metabolic labeling. The crucial differences observed are an accumulation of abundant COX1 assembly intermediates, low content of COX monomer and preferential recruitment of COX into I-III2-IVn SCs in SURF1 patient fibroblasts, whereas SURF1(-/-) mouse fibroblasts were characterized by low content of COX1 assembly intermediates and milder decrease in COX monomer, which appeared more stable. This pattern was even less pronounced in SURF1(-/-) mouse liver and brain. Both the control and SURF1(-/-) mice revealed only negligible formation of the I-III2-IVn SCs and marked tissue differences in the contents of COX dimer and III2-IV SCs, also less noticeable in liver and brain than in heart and muscle. Our studies support the view that COX assembly is much more dependent on SURF1 in humans than in mice. We also demonstrate markedly lower ability of mouse COX to form I-III2-IVn supercomplexes, pointing to tissue-specific and species-specific differences in COX biogenesis.

ROS generation and multiple forms of mammalian mitochondrial glycerol-3-phosphate dehydrogenase.

Overproduction of reactive oxygen species (ROS) has been implicated in a range of pathologies. Mitochondrial flavin dehydrogenases glycerol-3-phosphate dehydrogenase (mGPDH) and succinate dehydrogenase (SDH) represent important ROS source, but the mechanism of electron leak is still poorly understood. To investigate the ROS production by the isolated dehydrogenases, we used brown adipose tissue mitochondria solubilized by digitonin as a model. Enzyme activity measurements and hydrogen peroxide production studies by Amplex Red fluorescence, and luminol luminescence in combination with oxygraphy revealed flavin as the most likely source of electron leak in SDH under in vivo conditions, while we propose coenzyme Q as the site of ROS production in the case of mGPDH. Distinct mechanism of ROS production by the two dehydrogenases is also apparent from induction of ROS generation by ferricyanide which is unique for mGPDH. Furthermore, using native electrophoretic systems, we demonstrated that mGPDH associates into homooligomers as well as high molecular weight supercomplexes, which represent native forms of mGPDH in the membrane. By this approach, we also directly demonstrated that isolated mGPDH itself as well as its supramolecular assemblies are all capable of ROS production.

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).

Mitochondrial ATP synthasome: Expression and structural interaction of its components.

Mitochondrial ATP synthase, ADP/ATP translocase (ANT), and inorganic phosphate carrier (PiC) are supposed to form a supercomplex called ATP synthasome. Our protein and transcript analysis of rat tissues indicates that the expression of ANT and PiC is transcriptionally controlled in accordance with the biogenesis of ATP synthase. In contrast, the content of ANT and PiC is increased in ATP synthase deficient patients' fibroblasts, likely due to a post-transcriptional adaptive mechanism. A structural analysis of rat heart mitochondria by immunoprecipitation, blue native/SDS electrophoresis, immunodetection and MS analysis revealed the presence of ATP synthasome. However, the majority of PiC and especially ANT did not associate with ATP synthase, suggesting that most of PiC, ANT and ATP synthase exist as separate entities.

Effects of mtDNA in SHR-mtF344 versus SHR conplastic strains on reduced OXPHOS enzyme levels, insulin resistance, cardiac hypertrophy, and systolic dysfunction.

Common inbred strains of the laboratory rat can be divided into four major mitochondrial DNA (mtDNA) haplotype groups represented by the BN, F344, LEW, and SHR strains. In the current study, we investigated the metabolic and hemodynamic effects of the SHR vs. F344 mtDNA by comparing the SHR vs. SHR-mt(F344) conplastic strains that are genetically identical except for their mitochondrial genomes. Altogether 13 amino acid substitutions in protein coding genes, seven single nucleotide polymorphisms in tRNA genes, and 12 single nucleotide changes in rRNA genes were detected in F344 mtDNA compared with SHR mtDNA. Analysis of oxidative phosphorylation system (OXPHOS) in heart left ventricles (LV), muscle, and liver revealed reduced activity and content of several respiratory chain complexes in SHR-mt(F344) conplastic rats compared with the SHR strain. Lower function of OXPHOS in LV of conplastic rats was associated with significantly increased relative ventricular mass and reduced fractional shortening that was independent of blood pressure. In addition, conplastic rats exhibited reduced sensitivity of skeletal muscles to insulin action and impaired glucose tolerance. These results provide evidence that inherited alterations in mitochondrial genome, in the absence of variation in the nuclear genome and other confounding factors, predispose to insulin resistance, cardiac hypertrophy and systolic dysfunction.

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.

Impairment of adrenergically-regulated thermogenesis in brown fat of obesity-resistant mice is compensated by non-shivering thermogenesis in skeletal muscle.

OBJECTIVE: Non-shivering thermogenesis (NST) mediated by uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) can be activated via the adrenergic system in response to cold or diet, contributing to both thermal and energy homeostasis. Other mechanisms, including metabolism of skeletal muscle, may also be involved in NST. However, relative contribution of these energy dissipating pathways and their adaptability remain a matter of long-standing controversy. METHODS: We used warm-acclimated (30 °C) mice to characterize the effect of an up to 7-day cold acclimation (6 °C; CA) on thermoregulatory thermogenesis, comparing inbred mice with a genetic background conferring resistance (A/J) or susceptibility (C57BL/6 J) to obesity. RESULTS: Both warm-acclimated C57BL/6 J and A/J mice exhibited similar cold endurance, assessed as a capability to maintain core body temperature during acute exposure to cold, which improved in response to CA, resulting in comparable cold endurance and similar induction of UCP1 protein in BAT of mice of both genotypes. Despite this, adrenergic NST in BAT was induced only in C57BL/6 J, not in A/J mice subjected to CA. Cold tolerance phenotype of A/J mice subjected to CA was not based on increased shivering, improved insulation, or changes in physical activity. On the contrary, lipidomic, proteomic and gene expression analyses along with palmitoyl carnitine oxidation and cytochrome c oxidase activity revealed induction of lipid oxidation exclusively in skeletal muscle of A/J mice subjected to CA. These changes appear to be related to skeletal muscle NST, mediated by sarcolipin-induced uncoupling of sarco(endo)plasmic reticulum calcium ATPase pump activity and accentuated by changes in mitochondrial respiratory chain supercomplexes assembly. CONCLUSIONS: Our results suggest that NST in skeletal muscle could be adaptively augmented in the face of insufficient adrenergic NST in BAT, depending on the genetic background of the mice. It may provide both protection from cold and resistance to obesity, more effectively than BAT.

Nonsynonymous variants in mt-Nd2, mt-Nd4, and mt-Nd5 are linked to effects on oxidative phosphorylation and insulin sensitivity in rat conplastic strains.

Common inbred strains of the laboratory rat can be divided into four different mitochondrial DNA haplotype groups represented by the SHR, BN, LEW, and F344 strains. In the current study, we investigated the metabolic and hemodynamic effects of the SHR vs. LEW mitochondrial genomes by comparing the SHR to a new SHR conplastic strain, SHR-mt(LEW); these strains are genetically identical except for their mitochondrial genomes. Complete mitochondrial DNA (mtDNA) sequence analysis comparing the SHR and LEW strains revealed gene variants encoding amino acid substitutions limited to a single mitochondrial enzyme complex, NADH dehydrogenase (complex I), affecting subunits 2, 4, and 5. Two of the variants in the mt-Nd4 subunit gene are located close to variants known to be associated with exercise intolerance and diabetes mellitus in humans. No variants were found in tRNA or rRNA genes. These variants in mt-Nd2, mt-Nd4, and mt-Nd5 in the SHR-mt(LEW) conplastic strain were linked to reductions in oxidative and nonoxidative glucose metabolism in skeletal muscle. In addition, SHR-mt(LEW) conplastic rats showed increased serum nonesterified fatty acid levels and resistance to insulin stimulated incorporation of glucose into adipose tissue lipids. These results provide evidence that inherited variation in mitochondrial genes encoding respiratory chain complex I subunits, in the absence of variation in the nuclear genome and other confounding factors, can influence glucose and lipid metabolism when expressed on the nuclear genetic background of the SHR strain.

Expression and processing of the TMEM70 protein.

TMEM70 protein represents a novel ancillary factor of mammalian ATP synthase. We have investigated import and processing of this factor in human cells using GFP- and FLAG-tagged forms of TMEM70 and specific antibodies. TMEM70 is synthesized as a 29kDa precursor protein that is processed to a 21kDa mature form. Immunocytochemical detection of TMEM70 showed mitochondrial colocalization with MitoTracker Red and ATP synthase. Western blot of subcellular fractions revealed the highest signal of TMEM70 in isolated mitochondria and mitochondrial location was confirmed by mass spectrometry analysis. Based on analysis of submitochondrial fractions, TMEM70 appears to be located in the inner mitochondrial membrane, in accordance with predicated transmembrane regions in the central part of the TMEM70 sequence. Two-dimensional electrophoretic analysis did not show direct interaction of TMEM70 with assembled ATP synthase but indicated the presence of dimeric form of TMEM70. No TMEM70 protein could be found in cells and isolated mitochondria from patients with ATP synthase deficiency due to TMEM70 c.317-2A>G mutation thus confirming that TMEM70 biosynthesis is prevented in these patients.

Generation and characterization of human U-2 OS cell lines with the CRISPR/Cas9-edited protoporphyrinogen oxidase IX gene.

In humans, disruptions in the heme biosynthetic pathway are associated with various types of porphyrias, including variegate porphyria that results from the decreased activity of protoporphyrinogen oxidase IX (PPO; E.C.1.3.3.4), the enzyme catalyzing the penultimate step of the heme biosynthesis. Here we report the generation and characterization of human cell lines, in which PPO was inactivated using the CRISPR/Cas9 system. The PPO knock-out (PPO-KO) cell lines are viable with the normal proliferation rate and show massive accumulation of protoporphyrinogen IX, the PPO substrate. Observed low heme levels trigger a decrease in the amount of functional heme containing respiratory complexes III and IV and overall reduced oxygen consumption rates. Untargeted proteomics further revealed dysregulation of 22 cellular proteins, including strong upregulation of 5-aminolevulinic acid synthase, the major regulatory protein of the heme biosynthesis, as well as additional ten targets with unknown association to heme metabolism. Importantly, knock-in of PPO into PPO-KO cells rescued their wild-type phenotype, confirming the specificity of our model. Overall, our model system exploiting a non-erythroid human U-2 OS cell line reveals physiological consequences of the PPO ablation at the cellular level and can serve as a tool to study various aspects of dysregulated heme metabolism associated with variegate porphyria.

Uncoupling protein-1 is not leaky.

The activity of uncoupling protein-1 (UCP1) is rate-limiting for nonshivering thermogenesis and diet-induced thermogenesis. Characteristically, this activity is inhibited by GDP experimentally and presumably mainly by cytosolic ATP within brown-fat cells. The issue as to whether UCP1 has a residual proton conductance even when fully saturated with GDP/ATP (as has recently been suggested) has not only scientific but also applied interest, since a residual proton conductance would make overexpressed UCP1 weight-reducing even without physiological/pharmacological activation. To examine this question, we have here established optimal conditions for studying the bioenergetics of wild-type and UCP1-/- brown-fat mitochondria, analysing UCP1-mediated differences in parallel preparations of brown-fat mitochondria from both genotypes. Comparing different substrates, we find that pyruvate (or palmitoyl-L-carnitine) shows the largest relative coupling by GDP. Comparing albumin concentrations, we find the range 0.1-0.6% optimal; higher concentrations are inhibitory. Comparing basic medium composition, we find 125 mM sucrose optimal; an ionic medium (50-100 mM KCl) functions for wild-type but is detrimental for UCP1-/- mitochondria. Using optimal conditions, we find no evidence for a residual proton conductance (not a higher post-GDP respiration, a lower membrane potential or an altered proton leak at highest common potential) with either pyruvate or glycerol-3-phosphate as substrates, nor by a 3-4-fold alteration of the amount of UCP1. We could demonstrate that certain experimental conditions, due to respiratoty inhibition, could lead to the suggestion that UCP1 possesses a residual proton conductance but find that under optimal conditions our experiments concur with implications from physiological observations that in the presence of inhibitory nucleotides, UCP1 is not leaky.

Effect of metformin therapy on cardiac function and survival in a volume-overload model of heart failure in rats.

Advanced HF (heart failure) is associated with altered substrate metabolism. Whether modification of substrate use improves the course of HF remains unknown. The antihyperglycaemic drug MET (metformin) affects substrate metabolism, and its use might be associated with improved outcome in diabetic HF. The aim of the present study was to examine whether MET would improve cardiac function and survival also in non-diabetic HF. Volume-overload HF was induced in male Wistar rats by creating ACF (aortocaval fistula). Animals were randomized to placebo/MET (300 mg·kg(-1) of body weight·day(-1), 0.5% in food) groups and underwent assessment of metabolism, cardiovascular and mitochondrial functions (n=6-12/group) in advanced HF stage (week 21). A separate cohort served for survival analysis (n=10-90/group). The ACF group had marked cardiac hypertrophy, increased LVEDP (left ventricular end-diastolic pressure) and lung weight confirming decompensated HF, increased circulating NEFAs (non-esterified 'free' fatty acids), intra-abdominal fat depletion, lower glycogen synthesis in the skeletal muscle (diaphragm), lower myocardial triacylglycerol (triglyceride) content and attenuated myocardial (14)C-glucose and (14)C-palmitate oxidation, but preserved mitochondrial respiratory function, glucose tolerance and insulin sensitivity. MET therapy normalized serum NEFAs, decreased myocardial glucose oxidation, increased myocardial palmitate oxidation, but it had no effect on myocardial gene expression, AMPK (AMP-activated protein kinase) signalling, ATP level, mitochondrial respiration, cardiac morphology, function and long-term survival, despite reaching therapeutic serum levels (2.2±0.7 µg/ml). In conclusion, MET-induced enhancement of myocardial fatty acid oxidation had a neutral effect on cardiac function and survival. Recently reported cardioprotective effects of MET may not be universal to all forms of HF and may require AMPK activation or ATP depletion. No increase in mortality on MET supports its safe use in diabetic HF.

Loss of COX4I1 Leads to Combined Respiratory Chain Deficiency and Impaired Mitochondrial Protein Synthesis.

The oxidative phosphorylation (OXPHOS) system localized in the inner mitochondrial membrane secures production of the majority of ATP in mammalian organisms. Individual OXPHOS complexes form supramolecular assemblies termed supercomplexes. The complexes are linked not only by their function but also by interdependency of individual complex biogenesis or maintenance. For instance, cytochrome c oxidase (cIV) or cytochrome bc1 complex (cIII) deficiencies affect the level of fully assembled NADH dehydrogenase (cI) in monomeric as well as supercomplex forms. It was hypothesized that cI is affected at the level of enzyme assembly as well as at the level of cI stability and maintenance. However, the true nature of interdependency between cI and cIV is not fully understood yet. We used a HEK293 cellular model where the COX4 subunit was completely knocked out, serving as an ideal system to study interdependency of cI and cIV, as early phases of cIV assembly process were disrupted. Total absence of cIV was accompanied by profound deficiency of cI, documented by decrease in the levels of cI subunits and significantly reduced amount of assembled cI. Supercomplexes assembled from cI, cIII, and cIV were missing in COX4I1 knock-out (KO) due to loss of cIV and decrease in cI amount. Pulse-chase metabolic labeling of mitochondrial DNA (mtDNA)-encoded proteins uncovered a decrease in the translation of cIV and cI subunits. Moreover, partial impairment of mitochondrial protein synthesis correlated with decreased content of mitochondrial ribosomal proteins. In addition, complexome profiling revealed accumulation of cI assembly intermediates, indicating that cI biogenesis, rather than stability, was affected. We propose that attenuation of mitochondrial protein synthesis caused by cIV deficiency represents one of the mechanisms, which may impair biogenesis of cI.

Succinimidyl oleate, established inhibitor of CD36/FAT translocase inhibits complex III of mitochondrial respiratory chain.

The functional role of CD36 protein detected in mitochondrial fractions in long chain fatty acid (LCFA) oxidation is unclear due to conflicting results obtained in Cd36 knockout mice and experiments using sulfo-N-succinimidyl oleate (SSO) for inhibition of CD36 mediated LCFA transport. We investigated effect of SSO on mitochondrial respiration and found that SSO substantially inhibits not only LCFA oxidation, but also oxidation of flavoprotein- and NADH-dependent substrates and generation of mitochondrial membrane potential. Experiments in rat liver, heart and kidney mitochondria demonstrated a direct effect on mitochondrial respiratory chain with the most pronounced inhibition of the complex III (IC(50) 4microM SSO). The results presented here show that SSO is a potent and irreversible inhibitor of mitochondrial respiratory chain.