Cystine/glutamate antiporter xCT controls skeletal muscle glutathione redox, bioenergetics and differentiation.

Cysteine, the rate-controlling amino acid in cellular glutathione synthesis is imported as cystine, by the cystine/glutamate antiporter, xCT, and subsequently reduced to cysteine. As glutathione redox is important in muscle regeneration in aging, we hypothesized that xCT exerts upstream control over skeletal muscle glutathione redox, metabolism and regeneration. Bioinformatic analyses of publicly available datasets revealed that expression levels of xCT and GSH-related genes are inversely correlated with myogenic differentiation genes. Muscle satellite cells (MuSCs) isolated from Slc7a11sut/sut mice, which harbour a mutation in the Slc7a11 gene encoding xCT, required media supplementation with 2-mercaptoethanol to support cell proliferation but not myotube differentiation, despite persistently lower GSH. Slc7a11sut/sut primary myotubes were larger compared to WT myotubes, and also exhibited higher glucose uptake and cellular oxidative capacities. Immunostaining of myogenic markers (Pax7, MyoD, and myogenin) in cardiotoxin-damaged tibialis anterior muscle fibres revealed greater MuSC activation and commitment to differentiation in Slc7a11sut/sut muscle compared to WT mice, culminating in larger myofiber cross-sectional areas at 21 days post-injury. Slc7a11sut/sut mice subjected to a 5-week exercise training protocol demonstrated enhanced insulin tolerance compared to WT mice, but blunted muscle mitochondrial biogenesis and respiration in response to exercise training. Our results demonstrate that the absence of xCT inhibits cell proliferation but promotes myotube differentiation by regulating cellular metabolism and glutathione redox. Altogether, these results support the notion that myogenesis is a redox-regulated process and may help inform novel therapeutic approaches for muscle wasting and dysfunction in aging and disease.

Accessory subunit NDUFB4 participates in mitochondrial complex I supercomplex formation.

Mitochondrial electron transport chain complexes organize into supramolecular structures called respiratory supercomplexes (SCs). The role of respiratory SCs remains largely unconfirmed despite evidence supporting their necessity for mitochondrial respiratory function. The mechanisms underlying the formation of the I1III2IV1 "respirasome" SC are also not fully understood, further limiting insights into these processes in physiology and diseases, including neurodegeneration and metabolic syndromes. NDUFB4 is a complex I accessory subunit that contains residues that interact with the subunit UQCRC1 from complex III, suggesting that NDUFB4 is integral for I1III2IV1 respirasome integrity. Here, we introduced specific point mutations to Asn24 (N24) and Arg30 (R30) residues on NDUFB4 to decipher the role of I1III2-containing respiratory SCs in cellular metabolism while minimizing the functional consequences to complex I assembly. Our results demonstrate that NDUFB4 point mutations N24A and R30A impair I1III2IV1 respirasome assembly and reduce mitochondrial respiratory flux. Steady-state metabolomics also revealed a global decrease in citric acid cycle metabolites, affecting NADH-generating substrates. Taken together, our findings highlight an integral role of NDUFB4 in respirasome assembly and demonstrate the functional significance of SCs in regulating mammalian cell bioenergetics.

The SARS-CoV-2 spike glycoprotein interacts with MAO-B and impairs mitochondrial energetics.

SARS-CoV-2 infection is associated with both acute and post-acute neurological symptoms. Emerging evidence suggests that SARS-CoV-2 can alter mitochondrial metabolism, suggesting that changes in brain metabolism may contribute to the development of acute and post-acute neurological complications. Monoamine oxidase B (MAO-B) is a flavoenzyme located on the outer mitochondrial membrane that catalyzes the oxidative deamination of monoamine neurotransmitters. Computational analyses have revealed high similarity between the SARS-CoV-2 spike glycoprotein receptor binding domain on the ACE2 receptor and MAO-B, leading to the hypothesis that SARS-CoV-2 spike glycoprotein may alter neurotransmitter metabolism by interacting with MAO-B. Our results empirically establish that the SARS-CoV-2 spike glycoprotein interacts with MAO-B, leading to increased MAO-B activity in SH-SY5Y neuron-like cells. Common to neurodegenerative disease pathophysiological mechanisms, we also demonstrate that the spike glycoprotein impairs mitochondrial bioenergetics, induces oxidative stress, and perturbs the degradation of depolarized aberrant mitochondria through mitophagy. Our findings also demonstrate that SH-SY5Y neuron-like cells expressing the SARS-CoV-2 spike protein were more susceptible to MPTP-induced necrosis, likely necroptosis. Together, these results reveal novel mechanisms that may contribute to SARS-CoV-2-induced neurodegeneration.

GDF15 promotes weight loss by enhancing energy expenditure in muscle.

Caloric restriction that promotes weight loss is an effective strategy for treating non-alcoholic fatty liver disease and improving insulin sensitivity in people with type 2 diabetes1. Despite its effectiveness, in most individuals, weight loss is usually not maintained partly due to physiological adaptations that suppress energy expenditure, a process known as adaptive thermogenesis, the mechanistic underpinnings of which are unclear2,3. Treatment of rodents fed a high-fat diet with recombinant growth differentiating factor 15 (GDF15) reduces obesity and improves glycaemic control through glial-cell-derived neurotrophic factor family receptor α-like (GFRAL)-dependent suppression of food intake4-7. Here we find that, in addition to suppressing appetite, GDF15 counteracts compensatory reductions in energy expenditure, eliciting greater weight loss and reductions in non-alcoholic fatty liver disease (NAFLD) compared to caloric restriction alone. This effect of GDF15 to maintain energy expenditure during calorie restriction requires a GFRAL-ß-adrenergic-dependent signalling axis that increases fatty acid oxidation and calcium futile cycling in the skeletal muscle of mice. These data indicate that therapeutic targeting of the GDF15-GFRAL pathway may be useful for maintaining energy expenditure in skeletal muscle during caloric restriction.

16p11.2 haploinsufficiency reduces mitochondrial biogenesis in brain endothelial cells and alters brain metabolism in adult mice.

Neurovascular abnormalities in mouse models of 16p11.2 deletion autism syndrome are reminiscent of alterations reported in murine models of glucose transporter deficiency, including reduced brain angiogenesis and behavioral alterations. Yet, whether cerebrovascular alterations in 16p11.2df/+ mice affect brain metabolism is unknown. Here, we report that anesthetized 16p11.2df/+ mice display elevated brain glucose uptake, a phenomenon recapitulated in mice with endothelial-specific 16p11.2 haplodeficiency. Awake 16p11.2df/+ mice display attenuated relative fluctuations of extracellular brain glucose following systemic glucose administration. Targeted metabolomics on cerebral cortex extracts reveals enhanced metabolic responses to systemic glucose in 16p11.2df/+ mice that also display reduced mitochondria number in brain endothelial cells. This is not associated with changes in mitochondria fusion or fission proteins, but 16p11.2df/+ brain endothelial cells lack the splice variant NT-PGC-1α, suggesting defective mitochondrial biogenesis. We propose that altered brain metabolism in 16p11.2df/+ mice is compensatory to endothelial dysfunction, shedding light on previously unknown adaptative responses.

Novel Homozygous Variant in COQ7 in Siblings With Hereditary Motor Neuropathy.

Background and Objectives: Coenzyme Q10 (CoQ10) is an important electron carrier and antioxidant. The COQ7 enzyme catalyzes the hydroxylation of 5-demethoxyubiquinone-10 (DMQ10), the second-to-last step in the CoQ10 biosynthesis pathway. We report a consanguineous family presenting with a hereditary motor neuropathy associated with a homozygous c.1A > G p.? variant of COQ7 with abnormal CoQ10 biosynthesis. Methods: Affected family members underwent clinical assessments that included nerve conduction testing, histologic analysis, and MRI. Pathogenicity of the COQ7 variant was assessed in cultured fibroblasts and skeletal muscle using a combination of immunoblots, respirometry, and quinone analysis. Results: Three affected siblings, ranging from 12 to 24 years of age, presented with a severe length-dependent motor neuropathy with marked symmetric distal weakness and atrophy with normal sensation. Muscle biopsy of the quadriceps revealed chronic denervation pattern. An MRI examination identified moderate to severe fat infiltration in distal muscles. Exome sequencing demonstrated the homozygous COQ7 c.1A > G p.? variant that is expected to bypass the first 38 amino acid residues at the n-terminus, initiating instead with methionine at position 39. This is predicted to cause the loss of the cleavable mitochondrial targeting sequence and 2 additional amino acids, thereby preventing the incorporation and subsequent folding of COQ7 into the inner mitochondrial membrane. Pathogenicity of the COQ7 variant was demonstrated by diminished COQ7 and CoQ10 levels in muscle and fibroblast samples of affected siblings but not in the father, unaffected sibling, or unrelated controls. In addition, fibroblasts from affected siblings had substantial accumulation of DMQ10, and maximal mitochondrial respiration was impaired in both fibroblasts and muscle. Discussion: This report describes a new neurologic phenotype of COQ7-related primary CoQ10 deficiency. Novel aspects of the phenotype presented by this family include pure distal motor neuropathy involvement, as well as the lack of upper motor neuron features, cognitive delay, or sensory involvement in comparison with cases of COQ7-related CoQ10 deficiency previously reported in the literature.

Parkin coregulates glutathione metabolism in adult mammalian brain.

We recently discovered that the expression of PRKN, a young-onset Parkinson disease-linked gene, confers redox homeostasis. To further examine the protective effects of parkin in an oxidative stress model, we first combined the loss of prkn with Sod2 haploinsufficiency in mice. Although adult prkn-/-//Sod2± animals did not develop dopamine cell loss in the S. nigra, they had more reactive oxidative species and a higher concentration of carbonylated proteins in the brain; bi-genic mice also showed a trend for more nitrotyrosinated proteins. Because these redox changes were seen in the cytosol rather than mitochondria, we next explored the thiol network in the context of PRKN expression. We detected a parkin deficiency-associated increase in the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) in murine brain, PRKN-linked human cortex and several cell models. This shift resulted from enhanced recycling of GSSG back to GSH via upregulated glutathione reductase activity; it also correlated with altered activities of redox-sensitive enzymes in mitochondria isolated from mouse brain (e.g., aconitase-2; creatine kinase). Intriguingly, human parkin itself showed glutathione-recycling activity in vitro and in cells: For each GSSG dipeptide encountered, parkin regenerated one GSH molecule and was S-glutathionylated by the other (GSSG + P-SH [Formula: see text] GSH + P-S-SG), including at cysteines 59, 95 and 377. Moreover, parkin's S-glutathionylation was reversible by glutaredoxin activity. In summary, we found that PRKN gene expression contributes to the network of available thiols in the cell, including by parkin's participation in glutathione recycling, which involves a reversible, posttranslational modification at select cysteines. Further, parkin's impact on redox homeostasis in the cytosol can affect enzyme activities elsewhere, such as in mitochondria. We posit that antioxidant functions of parkin may explain many of its previously described, protective effects in vertebrates and invertebrates that are unrelated to E3 ligase activity.

Minimal adaptation of the molecular regulators of mitochondrial dynamics in response to unilateral limb immobilisation and retraining in middle-aged men.

PURPOSE: Mitochondrial dynamics are regulated by the differing molecular pathways variously governing biogenesis, fission, fusion, and mitophagy. Adaptations in mitochondrial morphology are central in driving the improvements in mitochondrial bioenergetics following exercise training. However, there is a limited understanding of mitochondrial dynamics in response to inactivity. METHODS: Skeletal muscle biopsies were obtained from middle-aged males (n = 24, 49.4 ± 3.2 years) who underwent sequential 14-day interventions of unilateral leg immobilisation, ambulatory recovery, and resistance training. We quantified vastus lateralis gene and protein expression of key proteins involved in mitochondrial biogenesis, fusion, fission, and turnover in at baseline and following each intervention. RESULTS: PGC1α mRNA decreased 40% following the immobilisation period, and was accompanied by a 56% reduction in MTFP1 mRNA, a factor involved in mitochondrial fission. Subtle mRNA decreases were also observed in TFAM (17%), DRP1 (15%), with contrasting increases in BNIP3L and PRKN following immobilisation. These changes in gene expression were not accompanied by changes in respective protein expression. Instead, we observed subtle decreases in NRF1 and MFN1 protein expression. Ambulatory recovery restored mRNA and protein expression to pre-intervention levels of all altered components, except for BNIP3L. Resistance training restored BNIP3L mRNA to pre-intervention levels, and further increased mRNA expression of OPA-1, MFN2, MTFP1, and PINK1 past baseline levels. CONCLUSION: In healthy middle-aged males, 2 weeks of immobilisation did not induce dramatic differences in markers of mitochondria fission and autophagy. Restoration of ambulatory physical activity following the immobilisation period restored altered gene expression patterns to pre-intervention levels, with little evidence of further adaptation to resistance exercise training.

Exercise training enhances muscle mitochondrial metabolism in diet-resistant obesity.

BACKGROUND: Current paradigms for predicting weight loss in response to energy restriction have general validity but a subset of individuals fail to respond adequately despite documented diet adherence. Patients in the bottom 20% for rate of weight loss following a hypocaloric diet (diet-resistant) have been found to have less type I muscle fibres and lower skeletal muscle mitochondrial function, leading to the hypothesis that physical exercise may be an effective treatment when diet alone is inadequate. In this study, we aimed to assess the efficacy of exercise training on mitochondrial function in women with obesity with a documented history of minimal diet-induced weight loss. METHODS: From over 5000 patient records, 228 files were reviewed to identify baseline characteristics of weight loss response from women with obesity who were previously classified in the top or bottom 20% quintiles based on rate of weight loss in the first 6 weeks during which a 900 kcal/day meal replacement was consumed. A subset of 20 women with obesity were identified based on diet-resistance (n=10) and diet sensitivity (n=10) to undergo a 6-week supervised, progressive, combined aerobic and resistance exercise intervention. FINDINGS: Diet-sensitive women had lower baseline adiposity, higher fasting insulin and triglycerides, and a greater number of ATP-III criteria for metabolic syndrome. Conversely in diet-resistant women, the exercise intervention improved body composition, skeletal muscle mitochondrial content and metabolism, with minimal effects in diet-sensitive women. In-depth analyses of muscle metabolomes revealed distinct group- and intervention- differences, including lower serine-associated sphingolipid synthesis in diet-resistant women following exercise training. INTERPRETATION: Exercise preferentially enhances skeletal muscle metabolism and improves body composition in women with a history of minimal diet-induced weight loss. These clinical and metabolic mechanism insights move the field towards better personalised approaches for the treatment of distinct obesity phenotypes. FUNDING: Canadian Institutes of Health Research (CIHR-INMD and FDN-143278; CAN-163902; CIHR PJT-148634).

Targeting skeletal muscle mitochondrial health in obesity.

Metabolic demands of skeletal muscle are substantial and are characterized normally as highly flexible and with a large dynamic range. Skeletal muscle composition (e.g., fiber type and mitochondrial content) and metabolism (e.g., capacity to switch between fatty acid and glucose substrates) are altered in obesity, with some changes proceeding and some following the development of the disease. Nonetheless, there are marked interindividual differences in skeletal muscle composition and metabolism in obesity, some of which have been associated with obesity risk and weight loss capacity. In this review, we discuss related molecular mechanisms and how current and novel treatment strategies may enhance weight loss capacity, particularly in diet-resistant obesity.

A recurrent de novo ATP5F1A substitution associated with neonatal complex V deficiency.

Mitochondrial disorders are a heterogeneous group of rare, degenerative multisystem disorders affecting the cell's core bioenergetic and signalling functions. Spontaneous improvement is rare. We describe a novel neonatal-onset mitochondriopathy in three infants with failure to thrive, hyperlactatemia, hyperammonemia, and apparent clinical resolution before 18 months. Exome sequencing showed all three probands to be identically heterozygous for a recurrent de novo substitution, c.620G>A [p.(Arg207His)] in ATP5F1A, encoding the α-subunit of complex V. Patient-derived fibroblasts exhibited multiple deficits in complex V function and expression in vitro. Structural modelling predicts the observed substitution to create an abnormal region of negative charge on ATP5F1A's ß-subunit-interacting surface, adjacent to the nearby ß subunit's active site. This disorder, which presents with life-threatening neonatal manifestations, appears to follow a remitting course; the long-term prognosis remains unknown.

Cardiomyocyte-specific Srsf3 deletion reveals a mitochondrial regulatory role.

Serine-rich splicing factor 3 (SRSF3) was recently reported as being necessary to preserve RNA stability via an mTOR mechanism in a cardiac mouse model in adulthood. Here, we demonstrate the link between Srsf3 and mitochondrial integrity in an embryonic cardiomyocyte-specific Srsf3 conditional knockout (cKO) mouse model. Fifteen-day-old Srsf3 cKO mice showed dramatically reduced (below 50%) survival and reduced the left ventricular systolic performance, and histological analysis of these hearts revealed a significant increase in cardiomyocyte size, confirming the severe remodeling induced by Srsf3 deletion. RNA-seq analysis of the hearts of 5-day-old Srsf3 cKO mice revealed early changes in expression levels and alternative splicing of several transcripts related to mitochondrial integrity and oxidative phosphorylation. Likewise, the levels of several protein complexes of the electron transport chain decreased, and mitochondrial complex I-driven respiration of permeabilized cardiac muscle fibers from the left ventricle was impaired. Furthermore, transmission electron microscopy analysis showed disordered mitochondrial length and cristae structure. Together with its indispensable role in the physiological maintenance of mouse hearts, these results highlight the previously unrecognized function of Srsf3 in regulating the mitochondrial integrity.

Grx2 Regulates Skeletal Muscle Mitochondrial Structure and Autophagy.

Glutathione is an important antioxidant that regulates cellular redox status and is disordered in many disease states. Glutaredoxin 2 (Grx2) is a glutathione-dependent oxidoreductase that plays a pivotal role in redox control by catalyzing reversible protein deglutathionylation. As oxidized glutathione (GSSG) can stimulate mitochondrial fusion, we hypothesized that Grx2 may contribute to the maintenance of mitochondrial dynamics and ultrastructure. Here, we demonstrate that Grx2 deletion results in decreased GSH:GSSG, with a marked increase of GSSG in primary muscle cells isolated from C57BL/6 Grx2-/- mice. The altered glutathione redox was accompanied by increased mitochondrial length, consistent with a more fused mitochondrial reticulum. Electron microscopy of Grx2-/- skeletal muscle fibers revealed decreased mitochondrial surface area, profoundly disordered ultrastructure, and the appearance of multi-lamellar structures. Immunoblot analysis revealed that autophagic flux was augmented in Grx2-/- muscle as demonstrated by an increase in the ratio of LC3II/I expression. These molecular changes resulted in impaired complex I respiration and complex IV activity, a smaller diameter of tibialis anterior muscle, and decreased body weight in Grx2 deficient mice. Together, these are the first results to show that Grx2 regulates skeletal muscle mitochondrial structure, and autophagy.

SMN Depleted Mice Offer a Robust and Rapid Onset Model of Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is considered a health epidemic with potential devastating effects on the patients and the healthcare systems. Current preclinical models of NAFLD are invariably imperfect and generally take a long time to develop. A mouse model of survival motor neuron (SMN) depletion (Smn2B/- mice) was recently shown to develop significant hepatic steatosis in less than 2 weeks from birth. The rapid onset of fatty liver in Smn2B/- mice provides an opportunity to identify molecular markers of NAFLD. Here, we investigated whether Smn2B/- mice display typical features of NAFLD/nonalcoholic steatohepatitis (NASH). METHODS: Biochemical, histologic, electron microscopy, proteomic, and high-resolution respirometry were used. RESULTS: The Smn2B/- mice develop microvesicular steatohepatitis within 2 weeks, a feature prevented by AAV9-SMN gene therapy. Although fibrosis is not overtly apparent in histologic sections of the liver, there is molecular evidence of fibrogenesis and presence of stellate cell activation. The consequent liver damage arises from mitochondrial reactive oxygen species production and results in hepatic dysfunction in protein output, complement, coagulation, iron homeostasis, and insulin-like growth factor-1 metabolism. The NAFLD phenotype is likely due to non-esterified fatty acid overload from peripheral lipolysis subsequent to hyperglucagonemia compounded by reduced muscle use and insulin resistance. Despite the low hepatic mitochondrial content, isolated mitochondria show enhanced ß-oxidation, likely as a compensatory response, resulting in the production of reactive oxygen species. In contrast to typical NAFLD/NASH, the Smn2B/- mice lose weight because of their associated neurological condition (spinal muscular atrophy) and develop hypoglycemia. CONCLUSIONS: The Smn2B/- mice represent a good model of microvesicular steatohepatitis. Like other models, it is not representative of the complete NAFLD/NASH spectrum. Nevertheless, it offers a reliable, low-cost, early-onset model that is not dependent on diet to identify molecular players in NAFLD pathogenesis and can serve as one of the very few models of microvesicular steatohepatitis for both adult and pediatric populations.

Daily protein supplementation attenuates immobilization-induced blunting of postabsorptive muscle mTORC1 activation in middle-aged men.

Disuse-induced muscle atrophy is accompanied by a blunted postprandial response of the mammalian target of rapamycin complex 1 (mTORC1) pathway. Conflicting observations exist as to whether postabsorptive mTORC1 pathway activation is also blunted by disuse and plays a role in atrophy. It is unknown whether changes in habitual protein intake alter mTORC1 regulatory proteins and how they may contribute to the development of anabolic resistance. The primary objective of this study was to characterize the downstream responsiveness of skeletal muscle mTORC1 activation and its upstream regulatory factors, following 14 days of lower limb disuse in middle-aged men (45-60 yr). The participants were further randomized to receive daily supplementation of 20 g/d of protein (n = 12; milk protein concentrate) or isocaloric carbohydrate placebo (n = 13). Immobilization reduced postabsorptive skeletal muscle phosphorylation of the mTORC1 downstream targets, 4E-BP1, P70S6K, and ribosomal protein S6 (RPS6), with phosphorylation of the latter two decreasing to a greater extent in the placebo, compared with the protein supplementation groups (37% ± 13% vs. 14% ± 11% and 38% ± 20% vs. 25% ± 8%, respectively). Sestrin2 protein was also downregulated following immobilization irrespective of supplement group, despite a corresponding increase in its mRNA content. This decrease in Sestrin2 protein was negatively correlated with the immobilization-induced change in the in silico-predicted regulator miR-23b-3p. No other measured upstream proteins were altered by immobilization or supplementation. Immobilization downregulated postabsorptive mTORC1 pathway activation, and 20 g/day of protein supplementation attenuated the decrease in phosphorylation of targets regulating muscle protein synthesis.

The lifecycle of skeletal muscle mitochondria in obesity.

Skeletal muscle possesses dramatic metabolic plasticity that allows for the rapid adaptation in cellular energy transduction to meet the demands of the organism. Obesity elicits changes in skeletal muscle structure and function, resulting in the accumulation of intramuscular lipids. The accumulation of intramuscular lipids in obesity is associated with impaired skeletal muscle mitochondrial content and function. Mitochondria exist as a dynamic network that is regulated by the processes of biogenesis, fusion, fission, and mitophagy. In this review, we outline adaptations in molecular pathways that regulate mitochondrial structure and function in obesity. We highlight the emerging role of dysregulated skeletal muscle macroautophagy and mitochondrial turnover in obesity. Future research should further elucidate the role of mitophagy in observed reductions in mitochondrial content and function during obesity.

Glutaredoxin-2 and Sirtuin-3 deficiencies impair cardiac mitochondrial energetics but their effects are not additive.

Altered redox biology and oxidative stress have been implicated in the progression of heart failure. Glutaredoxin-2 (GRX2) is a glutathione-dependent oxidoreductase and catalyzes the reversible deglutathionylation of mitochondrial proteins. Sirtuin-3 (SIRT3) is a class III histone deacetylase and regulates lysine acetylation in mitochondria. Both GRX2 and SIRT3 are considered as key in the protection against oxidative damage in the myocardium. Knockout of either contributes to adverse heart pathologies including hypertrophy, hypertension, and cardiac dysfunction. Here, we created and characterized a GRX2 and SIRT3 double-knockout mouse model, hypothesizing that their deletions would have an additive effect on oxidative stress, and exacerbate mitochondrial function and myocardial structural remodeling. Wildtype, single-gene knockout (Sirt3-/-, Grx2-/-), and double-knockout mice (Grx2-/-/Sirt3-/-) were compared in heart weight, histology, mitochondrial respiration and H2O2 production. Overall, the hearts from Grx2-/-/Sirt3-/- mice displayed increased fibrosis and hypertrophy versus wildtype. In the Grx2-/- and the Sirt3-/- we observed changes in mitochondrial oxidative capacity, however this was associated with elevated H2O2 emission only in the Sirt3-/-. Similar changes were observed but not worsened in hearts from Grx2-/-/Sirt3-/- mice, suggesting that these changes were not additive. In human myocardium, using genetic and histopathological data from the human Genotype-Tissue Expression consortium, we confirmed that SIRT3 expression correlates inversely with heart pathology. Altogether, GRX2 and SIRT3 are important in the control of cardiac mitochondrial redox and oxidative processes, but their combined absence does not exacerbate effects, consistent with the overall conclusion that they function together in the complex redox and antioxidant systems in the heart.

SGCG rs679482 Associates With Weight Loss Success in Response to an Intensively Supervised Outpatient Program.

Weight loss in response to energy restriction is highly variable, and identification of genetic contributors can provide insights into underlying biology. Leveraging 1000 Genomes imputed genotypes, we carried out genome-wide association study (GWAS) analysis in 551 unrelated obese subjects of European ancestry who participated in an intensively supervised weight loss program with replication of promising signals in an independent sample of 1,331 obese subjects who completed the program at a later date. By single nucleotide polymorphism-based and sib-pair analysis, we show that that weight loss is a heritable trait, with estimated heritability (h 2 = 0.49) within the range reported for obesity. We find rs679482, intronic to SGCG (sarcoglycan γ), highly expressed in skeletal muscle, to concordantly associate with weight loss in discovery and replication samples reaching GWAS significance in the combined meta-analysis (ß = -0.35, P = 1.7 × 10-12). Located in a region of open chromatin, rs679482 is predicted to bind DMRT2, and allele-specific transcription factor binding analysis indicates preferential binding of DMRT2 to rs679482-A. Concordantly, rs679482-A impairs native repressor activity and increases basal and DMRT2-mediated enhancer activity. These findings confirm that weight loss is a heritable trait and provide evidence by which a novel variant in SGCG, rs679482, leads to impaired diet response.

Comparison of the Acute Postprandial Circulating B-Vitamin and Vitamer Responses to Single Breakfast Meals in Young and Older Individuals: Preliminary Secondary Outcomes of a Randomized Controlled Trial.

B-vitamin deficiency is common in ageing populations either due to altered dietary habits or altered digestive and metabolic functions. There is limited data on the acute circulating concentrations of B-vitamins and their various forms (vitamers), following ingestion of realistic meals. This study compared the acute circulating B-vitamin and vitamer responses to either an energy-dense (ED) or a nutrient-dense (ND) breakfast meal, consumed in a randomized cross-over sequence, in older and younger adults (n = 15 and 15, aged 67.3 ± 1.5 and 22.7 ± 0.5 years (mean ± SEM), respectively). Eleven differing B-vitamins and vitamers were determined in plasma samples by ultra-high-performance liquid chromatography-tandem mass spectrometry, in the fasting and postprandial state (hourly for 5 h). While postprandial thiamine concentration increased following both meals, riboflavin increased only following a ND meal in both age groups. Many vitamins including nicotinic acid, pantothenic acid, pyridoxal, pyridoxamine, pyridoxal-5'phosphate, and 4-pyridoxic acid remained unaltered, and flavin mononucleotide (FMN), nicotinamide and nicotinuric acid concentrations reduced following both meals. Biological age and food composition had minimal impact on postprandial B-vitamin concentrations, yet the differences between the ED and ND meals for riboflavin highlight the importance of riboflavin intake to achieve adequacy.

SIRT3 controls brown fat thermogenesis by deacetylation regulation of pathways upstream of UCP1.

OBJECTIVE: Brown adipose tissue (BAT) is important for thermoregulation in many mammals. Uncoupling protein 1 (UCP1) is the critical regulator of thermogenesis in BAT. Here we aimed to investigate the deacetylation control of BAT and to investigate a possible functional connection between UCP1 and sirtuin 3 (SIRT3), the master mitochondrial lysine deacetylase. METHODS: We carried out physiological, molecular, and proteomic analyses of BAT from wild-type and Sirt3KO mice when BAT is activated. Mice were either cold exposed for 2 days or were injected with the ß3-adrenergic agonist, CL316,243 (1 mg/kg; i.p.). Mutagenesis studies were conducted in a cellular model to assess the impact of acetylation lysine sites on UCP1 function. Cardiac punctures were collected for proteomic analysis of blood acylcarnitines. Isolated mitochondria were used for functional analysis of OXPHOS proteins. RESULTS: Our findings showed that SIRT3 absence in mice resulted in impaired BAT lipid use, whole body thermoregulation, and respiration in BAT mitochondria, without affecting UCP1 expression. Acetylome profiling of BAT mitochondria revealed that SIRT3 regulates acetylation status of many BAT mitochondrial proteins including UCP1 and crucial upstream proteins. Mutagenesis work in cells suggested that UCP1 activity was independent of direct SIRT3-regulated lysine acetylation. However, SIRT3 impacted BAT mitochondrial proteins activities of acylcarnitine metabolism and specific electron transport chain complexes, CI and CII. CONCLUSIONS: Our data highlight that SIRT3 likely controls BAT thermogenesis indirectly by targeting pathways upstream of UCP1.