The beneficial metabolic effects of insulin sensitizers are not attenuated by mitochondrial pyruvate carrier 2 hypomorphism.

NEW FINDINGS: What is the central question of this study? The antidiabetic effects of thiazolidinedione (TZD) drugs may be mediated in part by a molecular interaction with the constituent proteins of the mitochondrial pyruvate carrier complex (MPC1 and MPC2). We examined the ability of a mutant mouse strain expressing an N-terminal truncation of MPC2 (Mpc2Δ16 mice) to respond to TZD treatment. What is the main finding and its importance? The response of Mpc2Δ16 mice to TZD treatment was not significantly different from that of wild-type C57BL6/J control animals, suggesting that the 16 N-terminal amino acids of MPC2 are dispensable for the effects of TZD treatment. Rosiglitazone and pioglitazone are thiazolidinedione (TZD) compounds that have been used clinically as insulin-sensitizing drugs and are generally believed to mediate their effects via activation of the peroxisome proliferator-activated receptor Î³ (PPARγ). Recent work has shown that it is possible to synthesize TZD compounds with potent insulin-sensitizing effects and markedly diminished affinity for PPARγ. Both clinically used TZDs and investigational PPARγ-sparing TZDs, such as MSDC-0602, interact with the mitochondrial pyruvate carrier (MPC) and inhibit its activity. The MPC complex is composed of two proteins, MPC1 and MPC2. Herein, we used mice expressing a hypomorphic MPC2 protein missing 16 amino acids in the N-terminus (Mpc2Δ16 mice) to determine the effects of these residues in mediating the insulin-sensitizing effects of TZDs in diet-induced obese mice. We found that both pioglitazone and MSDC-0602 elicited their beneficial metabolic effects, including improvement in glucose tolerance, attenuation of hepatic steatosis, reduction of adipose tissue inflammation and stimulation of adipocyte browning, in both wild-type and Mpc2Δ16 mice after high-fat diet feeding. In addition, truncation of MPC2 failed to attenuate the interaction between TZDs and the MPC in a bioluminescence resonance energy transfer-based assay or to affect the suppression of pyruvate-stimulated respiration in cells. Collectively, these data suggest that the interaction between TZDs and MPC2 is not affected by loss of the N-terminal 16 amino acids nor are these residues required for the insulin-sensitizing effects of these compounds.

Quaternary structural parameters of the congenital cataract causing mutants of αA-crystallin.

Pediatric cataract of the congenital type is the most common form of childhood blindness and it is clinically and genetically heterogeneous. Mutations in 22 different genes have been identified to be associated with congenital cataracts, and among them, eight mutants belong to αA-crystallin. To explain how mutations in αA-crystallin lead to the development of cataract, quaternary structural parameters, and chaperone function have been investigated in αA-wt and in the following mutants: R12C, R21L, R21W, R49C, R54C, R116C, and R116H. Average molar mass, mass at the RI peak, mass across the peak, hydrodynamic radius (R(h)), and polydispersity index (PDI) were determined by dynamic light-scattering measurements. The average molar mass and mass across the peak showed major increase in R116C and R116H, moderate increase in R12C, R21W, and R54C, and no increase in R21L and R49C as compared to αA-wt. PDI and R(h) values were significantly increased only in R116C and R116H. Significant secondary structural changes, as determined by CD measurements, were seen in R21W, R21L, R116C, and R116H, and tertiary structural changes were evident in R21W, R54C, R116C, and R116H. Non-reducing SDS-PAGE has shown the presence of dimers presumably formed by inter-polypeptide disulfide bonds. Chaperone activity, as measured with ADH as the target protein, appeared normal in R49C and R54C, while R12C, R21L, and R21W showed moderate loss and R116C and R116H showed significant loss. Although a specific change in the αA-crystallin behavior that is common to all the mutants was not evident, each mutant showed one or more perturbation as the end effect that leads to cataract.

Male-specific association of the 2p25 region with suicide attempt in bipolar disorder.

We previously conducted a genome-wide association study (GWAS) of attempted suicide within bipolar disorder, which implicated common variation in the 2p25 region primarily in males. The top association signal from our GWAS occurred in an intergenic region of 2p25 (p = 5.07 × 10-8) and was supported by two independent studies. In the current study, to better characterize the association of the 2p25 region with attempted suicide, we sequenced the entire 350kb 2p25 region in 476 bipolar suicide attempters and 473 bipolar non-attempters using targeted next-generation sequencing. This fine-mapping project identified 4,681 variants in the 2p25 region. We performed both gene-level and individual-variant tests on our sequencing results and identified 375 variants which were nominally significant (p < 0.05) and three common variants that were significantly associated with attempted suicide in males (corrected p = 0.035, odds ratio (OR) = 2.13). These three variants are in strong linkage disequilibrium with the top variant from our GWAS. Our top five variants are also predicted expression quantitative trait loci (eQTL) for three genes in the 2p25 region based on publicly available brain expression databases. Our sequencing and eQTL data implicate these three genes - SH3YL1, ACP1, and FAM150B - and three additional pathways - androgen receptor, Wnt signaling, and glutamatergic/GABAergic signaling - in the association of the 2p25 region with suicide. The current study provides additional support for an association of the 2p25 region with attempted suicide in males and identifies several candidate genes and pathways that warrant further investigation to understand their role in suicidal behavior.

Two human patient mitochondrial pyruvate carrier mutations reveal distinct molecular mechanisms of dysfunction.

The Mitochondrial Pyruvate Carrier (MPC) occupies a central metabolic node by transporting cytosolic pyruvate into the mitochondrial matrix and linking glycolysis with mitochondrial metabolism. Two reported human MPC1 mutations cause developmental abnormalities, neurological problems, metabolic deficits, and for one patient, early death. We aimed to understand biochemical mechanisms by which the human patient C289T and T236A MPC1 alleles disrupt MPC function. MPC1 C289T encodes two protein variants, a mis-spliced, truncation mutant (A58G) and a full length point mutant (R97W). MPC1 T236A encodes a full length point mutant (L79H). Using human patient fibroblasts and complementation of CRISPR-deleted, MPC1 null mouse C2C12 cells, we investigated how MPC1 mutations cause MPC deficiency. Truncated MPC1 A58G protein was intrinsically unstable and failed to form MPC complexes. The MPC1 R97W protein was less stable but when overexpressed formed complexes with MPC2 that retained pyruvate transport activity. Conversely, MPC1 L79H protein formed stable complexes with MPC2, but these complexes failed to transport pyruvate. These findings inform MPC structure-function relationships and delineate three distinct biochemical pathologies resulting from two human patient MPC1 mutations. They also illustrate an efficient gene pass-through system for mechanistically investigating human inborn errors in pyruvate metabolism.

Disrupting Mitochondrial Pyruvate Uptake Directs Glutamine into the TCA Cycle away from Glutathione Synthesis and Impairs Hepatocellular Tumorigenesis.

Hepatocellular carcinoma (HCC) is a devastating cancer increasingly caused by non-alcoholic fatty liver disease (NAFLD). Disrupting the liver Mitochondrial Pyruvate Carrier (MPC) in mice attenuates NAFLD. Thus, we considered whether liver MPC disruption also prevents HCC. Here, we use the N-nitrosodiethylamine plus carbon tetrachloride model of HCC development to test how liver-specific MPC knock out affects hepatocellular tumorigenesis. Our data show that liver MPC ablation markedly decreases tumorigenesis and that MPC-deficient tumors transcriptomically downregulate glutathione metabolism. We observe that MPC disruption and glutathione depletion in cultured hepatomas are synthetically lethal. Stable isotope tracing shows that hepatocyte MPC disruption reroutes glutamine from glutathione synthesis into the tricarboxylic acid (TCA) cycle. These results support a model where inducing metabolic competition for glutamine by MPC disruption impairs hepatocellular tumorigenesis by limiting glutathione synthesis. These findings raise the possibility that combining MPC disruption and glutathione stress may be therapeutically useful in HCC and additional cancers.

Impaired skeletal muscle mitochondrial pyruvate uptake rewires glucose metabolism to drive whole-body leanness.

Metabolic cycles are a fundamental element of cellular and organismal function. Among the most critical in higher organisms is the Cori Cycle, the systemic cycling between lactate and glucose. Here, skeletal muscle-specific Mitochondrial Pyruvate Carrier (MPC) deletion in mice diverted pyruvate into circulating lactate. This switch disinhibited muscle fatty acid oxidation and drove Cori Cycling that contributed to increased energy expenditure. Loss of muscle MPC activity led to strikingly decreased adiposity with complete muscle mass and strength retention. Notably, despite decreasing muscle glucose oxidation, muscle MPC disruption increased muscle glucose uptake and whole-body insulin sensitivity. Furthermore, chronic and acute muscle MPC deletion accelerated fat mass loss on a normal diet after high fat diet-induced obesity. Our results illuminate the role of the skeletal muscle MPC as a whole-body carbon flux control point. They highlight the potential utility of modulating muscle pyruvate utilization to ameliorate obesity and type 2 diabetes.

Hepatic Mitochondrial Pyruvate Carrier 1 Is Required for Efficient Regulation of Gluconeogenesis and Whole-Body Glucose Homeostasis.

Gluconeogenesis is critical for maintenance of euglycemia during fasting. Elevated gluconeogenesis during type 2 diabetes (T2D) contributes to chronic hyperglycemia. Pyruvate is a major gluconeogenic substrate and requires import into the mitochondrial matrix for channeling into gluconeogenesis. Here, we demonstrate that the mitochondrial pyruvate carrier (MPC) comprising the Mpc1 and Mpc2 proteins is required for efficient regulation of hepatic gluconeogenesis. Liver-specific deletion of Mpc1 abolished hepatic MPC activity and markedly decreased pyruvate-driven gluconeogenesis and TCA cycle flux. Loss of MPC activity induced adaptive utilization of glutamine and increased urea cycle activity. Diet-induced obesity increased hepatic MPC expression and activity. Constitutive Mpc1 deletion attenuated the development of hyperglycemia induced by a high-fat diet. Acute, virally mediated Mpc1 deletion after diet-induced obesity decreased hyperglycemia and improved glucose tolerance. We conclude that the MPC is required for efficient regulation of gluconeogenesis and that the MPC contributes to the elevated gluconeogenesis and hyperglycemia in T2D.

Mutations in human αA-crystallin/sHSP affect subunit exchange interaction with αB-crystallin.

BACKGROUND: Mutation in αA-crystallin contributes to the development of congenital cataract in humans. Heterooligomerization of αA-crystallin and αB-crystallin is essential for maintaining transparency in the eye lens. The effect of congenital cataract causing mutants of αA-crystallin on subunit exchange and interaction with αB-crystallin is unknown. In the present study, interaction of the mutants of αA-crystallin with αB-crystallin was studied both in vitro and in situ by the fluorescence resonance energy transfer (FRET) technique. METHODOLOGY/PRINCIPAL FINDINGS: In vitro FRET technique was used to demonstrate the rates of subunit exchange of αB-wt with the following αA-crystallin mutants: R12C, R21L, R21W, R49C, R54C, and R116C. The subunit exchange rates (k values) of R21W and R116C with αB-wt decreased drastically as compared to αA-wt interacting with αB-wt. Moderately decreased k values were seen with R12C, R49C and R54C while R21L showed nearly normal k value. The interaction of αA- mutants with αB-wt was also assessed by in situ FRET. YFP-tagged αA mutants were co-expressed with CFP-tagged αB-wt in HeLa cells and the spectral signals were captured with a confocal microscope before and after acceptor laser photobleaching. The interaction of R21W and R116C with αB-wt was decreased nearly 50% as compared to αA-wt while the rest of the mutants showed slightly decreased interaction. Thus, there is good agreement between the in vitro and in situ FRET data. CONCLUSIONS/SIGNIFICANCE: Structural changes occurring in these mutants, as reported earlier, could be the underlying cause for the decreased interaction with αB may contribute to development of congenital cataract.