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Pyruvate is a critical intermediary metabolite in gluconeogenesis, lipogenesis, as well as NADH production. As a result, there is growing interest in targeting the mitochondrial pyruvate carrier (MPC) complex in liver and metabolic diseases. However, recent in vitro data indicate that MPC inhibition diverts glutamine/glutamate away from glutathione synthesis and toward glutaminolysis to compensate for loss of pyruvate oxidation, possibly sensitizing cells to oxidative insult. Here, we explored this using the clinically relevant acetaminophen (APAP) overdose model of acute liver injury, which is driven by oxidative stress. We report that MPC inhibition does indeed sensitize the liver to APAP-induced injury in vivo, but only with concomitant loss of alanine aminotransferase 2 (ALT2). Pharmacologic and genetic manipulation of neither MPC2 nor ALT2 alone affected APAP toxicity, but liver-specific double knockout (DKO) of these proteins significantly worsened the liver damage. Further investigation confirmed that DKO impaired glutathione synthesis and increased urea cycle flux, consistent with increased glutaminolysis. Furthermore, APAP toxicity was exacerbated by inhibition of both the MPC and ALT in vitro. Thus, increased glutaminolysis and susceptibility to oxidative stress requires loss of both the MPC and ALT2 in vivo and exacerbates them in vitro. Finally, induction of ALT2 reduced APAP-induced injury.

ObjectiveThe mitochondrial pyruvate carrier (MPC) has emerged as a therapeutic target for treating insulin resistance, type 2 diabetes, and nonalcoholic steatohepatitis (NASH). We evaluated whether MPC inhibitors (MPCi) might correct impairments in branched chain amino acid (BCAA) catabolism, which are predictive of developing diabetes and NASH.MethodsCirculating BCAA concentrations were measured in people with NASH and type 2 diabetes, who participated in a recent randomized, placebo-controlled Phase IIB clinical trial to test the efficacy and safety of the MPCi MSDC-0602K (EMMINENCE;NCT02784444). In this 52-week trial, patients were randomly assigned to placebo (n = 94) or 250 mg MSDC-0602K (n = 101). Human hepatoma cell lines were used to test the direct effects of various MPCi on BCAA catabolism in vitro. Lastly, we investigated how hepatocyte-specific deletion of MPC2 affects BCAA catabolism in the liver of obese mice.ResultsIn patients with NASH, MSDC-0602K treatment, which led to marked improvements in insulin sensitivity and diabetes, decreased plasma concentrations of BCAAs compared to baseline while placebo had no effect. The rate-limiting enzyme in BCAA catabolism is the mitochondrial branched chain ketoacid dehydrogenase (BCKDH), which is deactivated by phosphorylation. In multiple human hepatoma cell lines, MPCi markedly reduced BCKDH phosphorylation and stimulated branched chain keto acid catabolism; an effect that required the BCKDH phosphatase PPM1K. Mechanistically, the effects of MPCi were linked to activation of the energy sensing AMP-dependent protein kinase (AMPK) and mechanistic target of rapamycin (mTOR) kinase signaling cascades. Finally, BCKDH phosphorylation was reduced in liver of obese, hepatocyte-specific MPC2 knockout (LS-Mpc2-/-) mice compared to wild-type controls concomitant with activation of mTOR signaling in vivo.ConclusionsThese data demonstrate novel cross talk between mitochondrial pyruvate and BCAA metabolism and suggest that MPC inhibition leads to lower plasma BCAA concentrations and BCKDH phosphorylation by activating the AMPK/mTOR axis.

Hepatic gluconeogenesis from amino acids contributes significantly to diabetic hyperglycemia, but the molecular mechanisms involved are incompletely understood. Alanine transaminases (ALT1 and ALT2) catalyze the interconversion of alanine and pyruvate, which is required for gluconeogenesis from alanine. We find that ALT2 is overexpressed in the liver of diet-induced obese and db/db mice and that the expression of the gene encoding ALT2 (GPT2) is downregulated following bariatric surgery in people with obesity. The increased hepatic expression of Gpt2 in db/db liver is mediated by activating transcription factor 4, an endoplasmic reticulum stress-activated transcription factor. Hepatocyte-specific knockout of Gpt2 attenuates incorporation of

SUMMARYHepatic gluconeogenesis from amino acids contributes significantly to diabetic hyperglycemia, but the molecular mechanisms involved are incompletely understood. Alanine transaminases (ALT1 and ALT2) catalyze the interconversion of alanine and pyruvate, which is required for gluconeogenesis from alanine. We found that ALT2 was overexpressed in liver of diet-induced obese and db/db mice and that the expression of the gene encoding ALT2 (GPT2) was downregulated following bariatric surgery in people with obesity. The increased hepatic expression of Gpt2 in db/db liver was mediated by activating transcription factor 4; an endoplasmic reticulum stress-activated transcription factor. Hepatocyte-specific knockout of Gpt2 attenuated incorporation of 13C-alanine into newly synthesized glucose by hepatocytes. In vivo Gpt2 knockdown or knockout in liver had no effect on glucose concentrations in lean mice, but Gpt2 suppression alleviated hyperglycemia in db/db mice. These data suggest that ALT2 plays a significant role in hepatic gluconeogenesis from amino acids in diabetes.

Enhancer of zeste homolog 2 (EZH2), an epigenetic regulator that plays a key role in cell differentiation and oncogenesis, was reported to promote adipogenic differentiation in vitro by catalyzing trimethylation of histone 3 lysine 27. However, inhibition of EZH2 induced lipid accumulation in certain cancer and hepatocyte cell lines. To address this discrepancy, we investigated the role of EZH2 in adipogenic differentiation and lipid metabolism using primary human and mouse preadipocytes and adipose-specific EZH2 knockout (KO) mice. We found that the EZH2-selective inhibitor GSK126 induced lipid accumulation in human adipocytes, without altering adipocyte differentiation marker gene expression. Moreover, adipocyte-specific EZH2 KO mice, generated by crossing EZH2 floxed mice with adiponectin-Cre mice, displayed significantly increased body weight, adipose tissue mass, and adipocyte cell size and reduced very low-density lipoprotein (VLDL) levels, as compared with littermate controls. These phenotypic alterations could not be explained by differences in feeding behavior, locomotor activity, metabolic energy expenditure, or adipose lipolysis. In addition, human adipocytes treated with either GSK126 or vehicle exhibited comparable rates of glucose-stimulated triglyceride accumulation and fatty acid uptake. Mechanistically, lipid accumulation induced by GSK126 in adipocytes was lipoprotein-dependent, and EZH2 inhibition or gene deletion promoted lipoprotein-dependent lipid uptake in vitro concomitant with up-regulated apolipoprotein E (ApoE) gene expression. Deletion of ApoE blocked the effects of GSK126 to promote lipoprotein-dependent lipid uptake in murine adipocytes. Collectively, these results indicate that EZH2 inhibition promotes lipoprotein-dependent lipid accumulation via inducing ApoE expression in adipocytes, suggesting a novel mechanism of lipid regulation by EZH2.

The mitochondrial pyruvate carrier (MPC) has emerged as a therapeutic target for treating insulin resistance, type 2 diabetes, and nonalcoholic steatohepatitis (NASH). However, the molecular mechanisms by which MPC inhibition leads to metabolic improvements are incompletely understood. We evaluated whether MPC inhibitors might correct impairments in branched chain amino acid (BCAA) catabolism, which are predictive of developing diabetes and NASH. BCAA concentrations were assessed in plasma collected in EMMINENCE (NCT02784444), a randomized, placebo-controlled trial of the MPC inhibitor MSDC-0602K in people with NASH. MSDC-0602K treatment, which led to marked improvements in insulin sensitivity and diabetes endpoints, decreased plasma concentrations of BCAAs compared to baseline while placebo had no effect. The rate-limiting enzyme in BCAA catabolism is the mitochondrial branched chain ketoacid dehydrogenase (BCKDH). BCKDH activity is suppressed by phosphorylation mediated by a kinase (BDK) and activated by the phosphatase (PPM1K). In Huh7 or HepG2 cells, MPC inhibitors markedly reduced BCKDH phosphorylation, suggesting increased BCKDH activity. Chemical inhibition or siRNA-mediated silencing of BDK had no effect on suppression of BCKDH phosphorylation by MPC inhibitors. In contrast, PPM1K knockdown prevented the effects of MPC inhibition on phospho-BCKDH. Lastly, we quantified BCKDH phosphorylation in liver of wild-type and hepatocyte-specific MPC2 knockout (LS-Mpc2-/-) mice after 20 weeks of high fat diet. LS-Mpc2-/- mice exhibited reduced phosphorylation of BCKDH compared to wild-type mice. Lean LS-Mpc2-/- mice also exhibited reduced plasma BCAA concentrations after an overnight fast. These data demonstrate novel cross talk between mitochondrial pyruvate and BCAA metabolism and suggest that MPC inhibition leads to lower plasma BCAA concentrations and BCKDH phosphorylation via the phosphatase PPM1K. Disclosure D. Ferguson: None. N. K. H. Yiew: None. K. S. Mccommis: None. J. R. Colca: Board Member; Self; Metabolic Solutions Development Company, LLC, Employee; Self; Cirius Therapeutics. B. N. Finck: Advisory Panel; Self; Cirius Therapeutics, Stock/Shareholder; Self; Cirius Therapeutics. Funding National Institutes of Health (R01DK104735, T32DK007120)

Objective Inflammation in adipose tissues in obesity promotes insulin resistance and metabolic disease. The Duffy antigen receptor for chemokines (DARC) is a promiscuous non-signaling receptor expressed on erythrocytes and other cell types that modulates tissue inflammation by binding chemokines such as monocyte chemoattractant protein-1 (MCP-1) and by acting as a chemokine reservoir. DARC allelic variants are common in humans, but the role of DARC in modulating obesity-related metabolic disease is unknown. Methods We examined body weight gain, tissue adiposity, metabolic parameters and inflammatory marker expression in wild-type and DARC knockout mice fed a chow diet (CD) and high fat diet (HFD). Results Compared to wild-type mice, HFD-fed DARC knockout mice developed glucose intolerance and insulin resistance independent of increases in body weight or adiposity. Interestingly, insulin sensitivity was also diminished in lean male DARC knockout mice fed a chow diet. Insulin production was not reduced by DARC gene deletion, and plasma leptin levels were similar in HFD fed wild-type and DARC knockout mice. MCP-1 levels in plasma rose significantly in the HFD fed wild-type mice, but not in the DARC knockout mice. Conversely, adipose tissue MCP-1 levels were higher, and more macrophage crown-like structures were detected, in the HFD fed DARC knockout mice as compared with the wild-type mice, consistent with augmented adipose tissue inflammation that is not accurately reflected by plasma levels of DARC-bound MCP-1 in these mice. Conclusions These findings suggest that DARC regulates metabolic function and adipose tissue inflammation, which may impact obesity-related disease in ethnic populations with high frequencies of DARC allelic variants.

Visceral adipose tissue (VAT) inflammation and metabolic dysregulation are key components of obesity-induced metabolic disease. Upregulated arginase, a ureahydrolase enzyme with two isoforms (A1-cytosolic and A2-mitochondrial), is implicated in pathologies associated with obesity and diabetes. This study examined A2 involvement in obesity-associated metabolic and vascular disorders. WT and globally deleted A2(&minus, /&minus, ) or A1(+/&minus, ) mice were fed either a high fat/high sucrose (HFHS) diet or normal diet (ND) for 16 weeks. Increases in body and VAT weight of HFHS-fed WT mice were abrogated in A2&minus, but not A1+/&minus, mice. Additionally, A2&minus, HFHS-fed mice exhibited higher energy expenditure, lower blood glucose, and insulin levels compared to WT HFHS mice. VAT and adipocytes from WT HFHS fed mice showed greater A2 expression and adipocyte size and reduced expression of PGC-1&alpha, PPAR-&gamma, and adiponectin. A2 deletion blunted these effects, increased levels of active AMPK-&alpha, and upregulated genes involved in fatty acid metabolism. A2 deletion prevented HFHS-induced VAT collagen deposition and inflammation, which are involved in adipocyte metabolic dysfunction. Endothelium-dependent vasorelaxation, impaired by HFHS diet, was significantly preserved in A2&minus, mice, but more prominently maintained in A1+/&minus, mice. In summary, A2 is critically involved in HFHS-induced VAT inflammation and metabolic dysfunction.

Impaired adipogenic differentiation during diet-induced obesity (DIO) promotes adipocyte hypertrophy and inflammation, thereby contributing to metabolic disease. Adenomatosis polyposis coli down-regulated 1 (APCDD1) has recently been identified as an inhibitor of Wnt signaling, a key regulator of adipogenic differentiation. Here we report a novel role for APCDD1 in adipogenic differentiation via repression of Wnt signaling and an epigenetic linkage between miR-130 and APCDD1 in DIO. APCDD1 expression was significantly up-regulated in mature adipocytes compared with undifferentiated preadipocytes in both human and mouse subcutaneous adipose tissues. siRNA-based silencing of APCDD1 in 3T3-L1 preadipocytes markedly increased the expression of Wnt signaling proteins (Wnt3a, Wnt5a, Wnt10b, LRP5, and β-catenin) and inhibited the expression of adipocyte differentiation markers (CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ)) and lipid droplet accumulation, whereas adenovirus-mediated overexpression of APCDD1 enhanced adipogenic differentiation. Notably, DIO mice exhibited reduced APCDD1 expression and increased Wnt expression in both subcutaneous and visceral adipose tissues and impaired adipogenic differentiation in vitro. Mechanistically, we found that miR-130, whose expression is up-regulated in adipose tissues of DIO mice, could directly target the 3′-untranslated region of the APCDD1 gene. Furthermore, transfection of an miR-130 inhibitor in preadipocytes enhanced, whereas an miR-130 mimic blunted, adipogenic differentiation, suggesting that miR-130 contributes to impaired adipogenic differentiation during DIO by repressing APCDD1 expression. Finally, human subcutaneous adipose tissues isolated from obese individuals exhibited reduced expression of APCDD1, C/EBPα, and PPARγ compared with those from non-obese subjects. Taken together, these novel findings suggest that APCDD1 positively regulates adipogenic differentiation and that its down-regulation by miR-130 during DIO may contribute to impaired adipogenic differentiation and obesity-related metabolic disease.