Summary 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.
SUMMARYHepatocellular 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.
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.
Pyruvate is a central metabolic intermediate and plays a prominent role in nervous system function. Neurons are highly reliant on pyruvate oxidation for maintenance of cellular energetics. Disorders in pyruvate metabolism may result in severe neurological defects. Pyruvate is imported into the mitochondrial matrix by the mitochondrial pyruvate carrier (MPC) for oxidation by the TCA cycle to support oxidative phosphorylation. Understanding the function of the MPC requires specialized methods for investigating mitochondrial pyruvate metabolism. Multiple instruments are available for measuring respiration of animal tissues, intact cells, permeabilized cells and tissue, and isolated mitochondria. Compared to other platforms, the comparative advantage of the Seahorse extracellular flux analyzer is the ability to sequentially administer treatment compounds and observe the effects on cellular respiration in a multiplexed 96-well format. Here we describe the methods and procedures for: (1) assessing mitochondrial pyruvate oxidation by intact cultured neuronal cells; (2) by mitochondria isolated from HEK293T cells, and (3) by mitochondria isolated from mouse liver, which are useful as a general model of mitochondrial function.
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 (Rh), 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 Rh 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 SDSPAGE 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.
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 γ (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. Two proteins, MPC1 and MPC2, comprise the MPC complex. 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 improving glucose tolerance, attenuating hepatic steatosis, reducing adipose tissue inflammation, and stimulating adipocyte browning, in both WT 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 BRET-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.
BackgroundMutation 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/SignificanceStructural 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.
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