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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.

The peptide hormone adropin regulates energy metabolism in skeletal muscle and plays important roles in the regulation of metabolic homeostasis. Besides muscle, the liver has an essential role in regulating glucose homeostasis. Previous studies have reported that treatment of diet-induced obese (DIO) male mice with adropin(34–76) (the putative secreted domain) reduces fasting blood glucose independently of body weight changes, suggesting that adropin suppresses glucose production in the liver. Here, we explored the molecular mechanisms underlying adropin's effects on hepatic glucose metabolism in DIO mice. Male DIO B6 mice maintained on a high-fat diet received five intraperitoneal injections of adropin(34–76) (450 nmol/kg/injection) over a 48-h period. We found that adropin(34–76) enhances major intracellular signaling activities in the liver that are involved in insulin-mediated regulation of glucose homeostasis. Moreover, treatment with adropin(34–76) alleviated endoplasmic reticulum stress responses and reduced activity of c-Jun N-terminal kinase in the liver, explaining the enhanced activities of hepatic insulin signaling pathways observed with adropin(34–76) treatment. Furthermore, adropin(34–76) suppressed cAMP activated protein kinase A (PKA) activities, resulting in reduced phosphorylation of inositol trisphosphate receptor, which mediates endoplasmic reticulum calcium efflux, and of cAMP-responsive element–binding protein, a key transcription factor in hepatic regulation of glucose metabolism. Adropin(34–76) directly affected liver metabolism, decreasing glucose production and reducing PKA-mediated phosphorylation in primary mouse hepatocytes in vitro. Our findings indicate that major hepatic signaling pathways contribute to the improved glycemic control achieved with adropin(34–76) treatment in situations of obesity.

During prolonged fasting, the liver plays a central role in maintaining systemic energy homeostasis by producing glucose and ketones in processes fueled by oxidation of fatty acids liberated from adipose tissue. In mice, this is accompanied by transient hepatic accumulation of glycerolipids. We found that the hepatic expression of monoacylglycerol acyltransferase 1 (Mogat1), an enzyme with monoacylglycerol acyltransferase (MGAT) activity that produces diacyl­glycerol from monoacylglycerol, was significantly increased in the liver of fasted mice compared with mice given ad libitum access to food. Basal and fasting-induced expression of Mogat1 was markedly diminished in the liver of mice lacking the transcription factor PPARα. Suppressing Mogat1 expression in liver and adipose tissue with antisense oligonucleotides (ASOs) reduced hepatic MGAT activity and triglyceride content compared with fasted controls. Surprisingly, the expression of many other PPARα target genes and PPARα activity was also decreased in mice given Mogat1 ASOs. When mice treated with control or Mogat1 ASOs were gavaged with the PPARα ligand, WY-14643, and then fasted for 18 h, WY-14643 administration reversed the effects of Mogat1 ASOs on PPARα target gene expression and liver triglyceride content. In conclusion, Mogat1 is a fasting-induced PPARα target gene that may feed forward to regulate liver PPARα activity during food deprivation.

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)

Lipin 1 is a bifunctional protein that is a transcriptional regulator and has phosphatidic acid (PA) phosphohydrolase activity, which dephosphorylates PA to generate diacylglycerol. Human lipin 1 mutations lead to episodic rhabdomyolysis, and some affected patients exhibit cardiac abnormalities, including exercise-induced cardiac dysfunction and cardiac triglyceride accumulation. Furthermore, lipin 1 expression is deactivated in failing heart, but the effects of lipin 1 deactivation in myocardium are incompletely understood. We generated mice with cardiac-specific lipin 1 KO (cs-Lpin1-/-) to examine the intrinsic effects of lipin 1 in the myocardium. Cs-Lpin1-/- mice had normal systolic cardiac function but mild cardiac hypertrophy. Compared with littermate control mice, PA content was higher in cs-Lpin1-/- hearts, which also had an unexpected increase in diacylglycerol and triglyceride content. Cs-Lpin1-/- mice exhibited diminished cardiac cardiolipin content and impaired mitochondrial respiration rates when provided with pyruvate or succinate as metabolic substrates. After transverse aortic constriction-induced pressure overload, loss of lipin 1 did not exacerbate cardiac hypertrophy or dysfunction. However, loss of lipin 1 dampened the cardiac ionotropic response to dobutamine and exercise endurance in association with reduced protein kinase A signaling. These data suggest that loss of lipin 1 impairs cardiac functional reserve, likely due to effects on glycerolipid homeostasis, mitochondrial function, and protein kinase A signaling.

Objective Monoacylglycerol acyltransferase (MGAT) enzymes catalyze the synthesis of diacylglycerol from monoacylglycerol. Previous work has suggested the importance of MGAT activity in the development of obesity-related hepatic insulin resistance. Indeed, antisense oligonucleotide (ASO)-mediated knockdown of Mogat1 mRNA, which encodes MGAT1, reduced hepatic MGAT activity and improved glucose tolerance and insulin resistance in high-fat diet (HFD)-fed mice. However, recent work has suggested that some ASOs may have off-target effects on body weight and metabolic parameters via activation of the interferon alpha/beta receptor 1 (IFNAR-1) pathway. Methods Mice with whole-body Mogat1 knockout or a floxed allele for Mogat1 to allow for liver-specific Mogat1-knockout (by either a liver-specific transgenic or adeno-associated virus-driven Cre recombinase) were generated. These mice were placed on an HFD, and glucose metabolism and insulin sensitivity were assessed after 16 weeks on diet. In some experiments, mice were treated with control scramble or Mogat1 ASOs in the presence or absence of IFNAR-1 neutralizing antibody. Results Genetic deletion of hepatic Mogat1, either acutely or chronically, did not improve hepatic steatosis, glucose tolerance, or insulin sensitivity in HFD-fed mice. Furthermore, constitutive Mogat1 knockout in all tissues actually exacerbated HFD-induced obesity, insulin sensitivity, and glucose intolerance on an HFD. Despite markedly reduced Mogat1 expression, liver MGAT activity was unaffected in all knockout mouse models. Mogat1 overexpression in hepatocytes increased liver MGAT activity and TAG content in low-fat-fed mice but did not cause insulin resistance. Multiple Mogat1 ASO sequences improved glucose tolerance in both wild-type and Mogat1 null mice, suggesting an off-target effect. Hepatic IFNAR-1 signaling was activated by multiple Mogat1 ASOs, but its blockade did not prevent the effects of either Mogat1 ASO on glucose homeostasis. Conclusion These results indicate that genetic loss of Mogat1 does not affect hepatic MGAT activity or metabolic homeostasis on HFD and show that multiple Mogat1 ASOs improve glucose metabolism through effects independent of targeting Mogat1 or activation of IFNAR-1 signaling., Graphical abstract Image 1, Highlights • Mogat1 liver-specific KO or KD does not improve metabolism in HFD-fed mice. • Whole-body Mogat1 deletion impairs insulin tolerance in HFD-fed mice. • Mogat1 ASOs improve whole-body metabolism independently of gene knockdown. • Blockade of the INFAR1 response does not prevent off-target effects of Mogat1 ASOs.

Nicotinamide adenine dinucleotide (NAD+) is an essential coenzyme that regulates cellular energy metabolism in many cell types. The major purpose of the present study was to test the hypothesis that NAD+ in white adipose tissue (WAT) is a regulator of whole-body metabolic flexibility in response to changes in insulin sensitivity and with respect to substrate availability and use during feeding and fasting conditions. To this end, we first evaluated the relationship between WAT NAD+ concentration and metabolic flexibility in mice and humans. We found that WAT NAD+ concentration was increased in mice after calorie restriction and exercise, 2 enhancers of metabolic flexibility. Bariatric surgery-induced 20% weight loss increased plasma adiponectin concentration, skeletal muscle insulin sensitivity, and WAT NAD+ concentration in people with obesity. We next analyzed adipocyte-specific nicotinamide phosphoribosyltransferase (Nampt) knockout (ANKO) mice, which have markedly decreased NAD+ concentrations in WAT. ANKO mice oxidized more glucose during the light period and after fasting than control mice. In contrast, the normal postprandial stimulation of glucose oxidation and suppression of fat oxidation were impaired in ANKO mice. Data obtained from RNA-sequencing of WAT suggest that loss of NAMPT increases inflammation, and impairs insulin sensitivity, glucose oxidation, lipolysis, branched-chain amino acid catabolism, and mitochondrial function in WAT, which are features of metabolic inflexibility. These results demonstrate a novel function of WAT NAMPT-mediated NAD+ biosynthesis in regulating whole-body metabolic flexibility, and provide new insights into the role of adipose tissue NAD+ biology in metabolic health.

The myocardium is metabolically flexible; however, impaired flexibility is associated with cardiac dysfunction in conditions including diabetes and heart failure. The mitochondrial pyruvate carrier (MPC) complex, composed of MPC1 and MPC2, is required for pyruvate import into the mitochondria. Here we show that MPC1 and MPC2 expression is downregulated in failing human and mouse hearts. Mice with cardiac-specific deletion of Mpc2 (CS-MPC2−/−) exhibited normal cardiac size and function at 6 weeks old, but progressively developed cardiac dilation and contractile dysfunction, which was completely reversed by a high-fat, low-carbohydrate ketogenic diet. Diets with higher fat content, but enough carbohydrate to limit ketosis, also improved heart failure, while direct ketone body provisioning provided only minor improvements in cardiac remodelling in CS-MPC2−/− mice. An acute fast also improved cardiac remodelling. Together, our results reveal a critical role for mitochondrial pyruvate use in cardiac function, and highlight the potential of dietary interventions to enhance cardiac fat metabolism to prevent or reverse cardiac dysfunction and remodelling in the setting of MPC deficiency. Impaired myocardial metabolic flexibility is associated with cardiac dysfunction in diabetes and heart failure. McCommis et al. reveal a critical role for mitochondrial pyruvate use in cardiac function, and use dietary interventions to enhance cardiac fat metabolism in dilated cardiomyopathy.

ObjectiveMonoacylglycerol acyltransferase (MGAT) enzymes catalyze the synthesis of diacylglycerol from monoacylglycerol. Previous work has suggested the importance of MGAT activity in the development of obesity-related hepatic insulin resistance. Indeed, antisense oligonucleotide (ASO)-mediated knockdown of the gene encoding MGAT1, Mogat1, reduced hepatic MGAT activity and improved glucose tolerance and insulin resistance in high fat diet (HFD) fed mice. However, recent work has suggested that some ASOs may have off-target effects on body weight and metabolic parameters via activation of the interferon alpha/beta receptor 1 (IFNAR-1) pathway.MethodsMice with whole-body Mogat1 knockout or a floxed allele for Mogat1 to allow for liver-specific Mogat1-knockout (by either a liver-specific transgenic or adeno-associated virus-driven Cre recombinase) were generated. These mice were placed on a high fat diet and glucose metabolism and insulin sensitivity was assessed after 16 weeks on diet. In some experiments, mice were treated with control or Mogat1 or control ASOs in the presence or absence of IFNAR-1 neutralizing antibody.ResultsGenetic deletion of hepatic Mogat1, either acutely or chronically, did not improve hepatic steatosis, glucose tolerance, or insulin sensitivity in HFD-fed mice. Furthermore, constitutive Mogat1 knockout in all tissues actually exacerbated HFD-induced weight gain, insulin resistance, and glucose intolerance on a HFD. Despite markedly reduced Mogat1 expression, liver MGAT activity was unaffected in all knockout mouse models. Mogat1 overexpression hepatocytes increased liver MGAT activity and TAG content in low-fat fed mice, but did not cause insulin resistance. Interestingly, Mogat1 ASO treatment improved glucose tolerance in both wild-type and Mogat1 null mice, suggesting an off target effect. Inhibition of IFNAR-1 did not block the effect of Mogat1 ASO on glucose homeostasis.ConclusionThese results indicate that genetic loss of Mogat1 does not affect hepatic MGAT activity or metabolic homeostasis on HFD and show that Mogat1 ASOs improve glucose metabolism through effects independent of targeting Mogat1 or activation of IFNAR-1 signaling.Abstract FigureHighlightsMogat1 liver-specific KO or KD does not improve metabolism in HFD fed mice.Whole-body Mogat1-deletion impairs insulin tolerance in HFD fed mice.Mogat1 ASOs improves whole body metabolism independently of gene knockdown.Blockade of the INFR response does not prevent off-target effects of Mogat1 ASOs.

Emerging evidence suggests that the capacity of adipocytes to appropriately store lipids and prevent their ectopic accumulation in liver and skeletal muscle may be an important regulator of systemic insulin sensitivity. Lipin 1 is a lipid phosphatase that is highly expressed in adipocytes and catalyzes the conversion of phosphatidic acid (PA) to diacylglycerol (DAG); the penultimate step in triglyceride synthesis. Adipose tissue lipin 1 expression positively correlates with insulin sensitivity in obese humans and mouse models. To test the role of adipose tissue lipin 1 in systemic insulin sensitivity, we generated mice lacking lipin 1 in adipocytes (Adn-Lpin1-/- mice). On normal chow, loss of lipin 1 in adipocytes modestly reduced white and brown adipose tissue mass, but did not affect glucose tolerance or insulin sensitivity. However, in response to 2 weeks high fat diet (HFD; 60% fat) feeding, Adn-Lpin1-/- mice remained lean, but developed hyperglycemia compared to littermate wild type (WT) mice. Lipidomic analyses demonstrated an increase in liver lipids (PA, DAG, and triglyceride) in HFD-fed Adn-Lpin1-/- mice compared to WT controls, whereas only PA accumulated in skeletal muscle. Plasma triglyceride concentration was not different, but free fatty acids and glycerol were significantly reduced in Adn-Lpin1-/- vs. WT mice fed HFD, suggesting that lipolysis may be decreased. Plasma adiponectin and resistin were significantly reduced in HFD-fed Adn-Lpin1-/- vs. WT control mice while leptin concentrations were not different. Lastly, with 5 weeks of HFD feeding, despite being leaner, Adn-Lpin1-/- mice became markedly hyperglycemic and glucose intolerant compared to littermate WT controls. Collectively, these data suggest that loss of lipin 1 in adipocytes leads to insulin resistance on HFD, likely due to an impaired capacity to store lipids appropriately in adipose tissue. Disclosure A.J. Lutkewitte: None. B.N. Finck: Advisory Panel; Self; Cirius Therapeutics. Stock/Shareholder; Self; Cirius Therapeutics.

Gluconeogenesis from amino acids contributes significantly to hepatic glucose production and hyperglycemia in diabetes. Alanine transaminases (ALT) catalyze the bi-directional interconversion of alanine to pyruvate with concomitant interconversion of α-ketoglutarate to glutamate. ALT1 is cytosolic while ALT2 is localized to the mitochondrial matrix. The mRNA expression and protein abundance of ALT1 and 2 was significantly increased in liver of db/db or diet induced obese mice compared to lean controls. The expression of ALT2 was also increased in obese humans compared to lean controls and was downregulated following bariatric surgery. The increase in hepatic ALT2 expression in diabetic mice was caused by endoplasmic reticulum stress and mediated by activating transcription factor 4 (ATF4). Indeed, inhibition of ATF4 reduced ALT2 expression in liver of db/db mice. Consistent with enhanced use of amino acids in gluconeogenesis, db/db and diet-induced obese mice had elevated blood glucose concentrations following IP administration of L-alanine or glutamine. ALT2 silencing in liver had no effect on glucose concentrations in lean mice, but alleviated alanine-induced hyperglycemia in db/db mice; likely by reducing the incorporation of alanine and/or glutamine into newly synthesized glucose. Plasma alanine tended to rise and glutamate significantly increased in db/db mice with ALT2 silencing whereas concentrations of the branched chain amino acids isoleucine, leucine, and valine decreased or tended to decrease. In summary, our results are consistent with a significant role for ALT2 in hepatic gluconeogenesis from amino acids and in the regulation of blood glucose levels in obesity and diabetes. Disclosure M.R. Martino: None. M. Gutierrez Aguilar: None. K.S. McCommis: None. J. Yoshino: None. B.N. Finck: Advisory Panel; Self; Cirius Therapeutics. Stock/Shareholder; Self; Cirius Therapeutics. Funding National Institutes of Health (R01DK117657)

The myocardium is metabolically flexible and can use fatty acids, glucose, lactate/pyruvate, ketones, or amino acids to fuel mechanical work. However, impaired metabolic flexibility is associated with cardiac dysfunction in conditions including diabetes and heart failure. The mitochondrial pyruvate carrier (MPC) is required for pyruvate metabolism and is composed of a hetero-oligomer of two proteins known as MPC1 and MPC2. Interestingly, MPC1 and MPC2 expression is downregulated in failing human hearts and in a mouse model of heart failure. Mice with cardiac-specific deletion of MPC2 (CS-MPC2-/-) exhibited loss of both MPC2 and MPC1 proteins and reduced pyruvate-stimulated mitochondrial respiration. CS-MPC2-/- mice exhibited normal cardiac size and function at 6-weeks old, but progressively developed cardiac dilation and contractile dysfunction thereafter. Feeding CS-MPC2-/- mice a ketogenic diet (KD) completely prevented or reversed the cardiac remodeling and dysfunction. Other diets with higher fat content and enough carbohydrate to limit ketosis also improved heart failure in CS-MPC2-/- mice, but direct ketone body provisioning provided only minor improvements in cardiac remodeling. Finally, KD was also able to prevent further remodeling in an ischemic, pressure-overload mouse model of heart failure. In conclusion, loss of mitochondrial pyruvate utilization leads to dilated cardiomyopathy that can be corrected by a ketogenic diet.

Adipocyte triglyceride storage provides a reservoir of energy that allows the organism to survive times of nutrient scarcity, but excessive adiposity has emerged as a health problem in many areas of the world. Monoacylglycerol acyltransferase (MGAT) acylates monoacylglycerol to produce diacylglycerol; the penultimate step in triglyceride synthesis. However, little is known about MGAT activity in adipocytes, which are believed to rely primarily on another pathway for triglyceride synthesis. We show that expression of the gene that encodes MGAT1 is robustly induced during adipocyte differentiation and that its expression is suppressed in fat of genetically-obese mice and metabolically-abnormal obese human subjects. Interestingly, MGAT1 expression is also reduced in physiologic contexts where lipolysis is high. Moreover, knockdown or knockout of MGAT1 in adipocytes leads to higher rates of basal adipocyte lipolysis. Collectively, these data suggest that MGAT1 activity may play a role in regulating basal adipocyte FFA retention.

Aims To examine the effects of a high-fat-diet (HFD) on monogenic neonatal diabetes, without the confounding effects of compensatory hyperinsulinaemia. Methods Mice expressing KATP channel gain-of-function (KATP -GOF) mutations, which models human neonatal diabetes, were fed an HFD. Results Surprisingly, KATP -GOF mice exhibited resistance to HFD-induced obesity, accompanied by markedly divergent blood glucose control, with some KATP -GOF mice showing persistent diabetes (KATP -GOF-non-remitter [NR] mice) and others showing remission of diabetes (KATP -GOF-remitter [R] mice). Compared with the severely diabetic and insulin-resistant KATP -GOF-NR mice, HFD-fed KATP -GOF-R mice had lower blood glucose, improved insulin sensitivity, and increased circulating plasma insulin and glucagon-like peptide-1 concentrations. Strikingly, while HFD-fed KATP -GOF-NR mice showed increased food intake and decreased physical activity, reduced whole body fat mass and increased plasma lipids, KATP -GOF-R mice showed similar features to those of control littermates. Importantly, KATP -GOF-R mice had restored insulin content and β-cell mass compared with the marked loss observed in both HFD-fed KATP -GOF-NR and chow-fed KATP -GOF mice. Conclusion Together, our results suggest that restriction of dietary carbohydrates and caloric replacement by fat can induce metabolic changes that are beneficial in reducing glucotoxicity and secondary consequences of diabetes in a mouse model of insulin-secretory deficiency.

The prevalence of obesity-associated nonalcoholic fatty liver disease has significantly increased over the past decade, and end-stage liver disease secondary to nonalcoholic steatohepatitis has become 1 of the most common indications for liver transplantation. This both increases the demand for organs and decreases the availability of donor livers deemed suitable for transplantation. Although in the past many steatotic livers were discarded due to concerns over enhanced susceptibility to ischemia/reperfusion injury (IRI) and organ failure, the discrepancy between supply and demand has resulted in increasing use of expanded criteria donor organs including steatotic livers. However, it remains controversial whether steatotic livers can be safely used for transplantation and how best to improve the performance of steatotic grafts. We aimed to evaluate the impact of diet-induced hepatic steatosis in a murine model of IRI. Using a diet of high trans-fat, fructose, and cholesterol (HTF-C) and a diet high in saturated fats, sucrose, and cholesterol (Western diet), we were able to establish models of mixed macrovesicular and microvesicular steatosis (HTF-C) and microvesicular steatosis (Western). We found that the presence of hepatic steatosis, whether it is predominantly macrovesicular or microvesicular, significantly worsens IRI as measured by plasma alanine aminotransferase levels and inflammatory cytokine concentration, and histological evaluation for necrosis. Additionally, we report on a novel finding in which hepatic IRI in the setting of steatosis results in the induction of the necroptosis factors, receptor interacting protein kinase (RIPK) 3, RIPK1, and mixed-lineage kinase domain-like. These data lay the groundwork for additional experimentation to test potential therapeutic approaches to limit IRI in steatotic livers by using a genetically tractable system. Liver Transplantation 24 908-921 2018 AASLD.

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. This article is protected by copyright. All rights reserved

Nonalcoholic fatty liver disease (NAFLD) encompasses a range of liver pathology ranging from simple steatosis to varying degrees of inflammation, hepatocyte injury and fibrosis. Without intervention it can progress to end-stage liver disease and hepatocellular carcinoma. Given its close association with obesity, the prevalence of NAFLD has increased dramatically worldwide. Currently, there are no FDA-approved medications for the treatment of NAFLD and although lifestyle modifications with appropriate diet and exercise have been shown to be beneficial, this has been difficult to achieve and sustain for the majority of patients. As such, the search for effective therapeutic agents is an active area of research. Peroxisome proliferator-activated receptors (PPARs) belong to a class of nuclear receptors. Because of their key role in the transcriptional regulation of glucose and lipid metabolism, PPAR ligands have been investigated as possible therapeutic agents for NAFLD. Here we review the current evidence from preclinical and clinical studies investigating the therapeutic potential of PPAR ligands for the treatment of this spectrum of liver disorders.

Modifying the entry of pyruvate into mitochondria may provide a unique approach to treat metabolic disease. The pharmacology of a new class of insulin sensitizers directed against a newly identified mitochondrial target may treat many aspects of nonalcoholic steatohepatitis, including fibrosis. This commentary suggests treating nonalcoholic steatohepatitis through a newly identified mechanism consistent with pathophysiology. (Hepatology Communications 2017;1:193-197).

Low 25-hydroxyvitamin D levels correlate with the prevalence of diabetes; however, the mechanisms remain uncertain. Here, we show that nutritional deprivation–responsive mechanisms regulate vitamin D metabolism. Both fasting and diabetes suppressed hepatic cytochrome P450 (CYP) 2R1, the main vitamin D 25-hydroxylase responsible for the first bioactivation step. Overexpression of coactivator peroxisome proliferator–activated receptor γ coactivator 1-α (PGC-1α), induced physiologically by fasting and pathologically in diabetes, resulted in dramatic downregulation of CYP2R1 in mouse hepatocytes in an estrogen-related receptor α (ERRα)–dependent manner. However, PGC-1α knockout did not prevent fasting-induced suppression of CYP2R1 in the liver, indicating that additional factors contribute to the CYP2R1 repression. Furthermore, glucocorticoid receptor (GR) activation repressed the liver CYP2R1, suggesting GR involvement in the regulation of CYP2R1. GR antagonist mifepristone partially prevented CYP2R1 repression during fasting, suggesting that glucocorticoids and GR contribute to the CYP2R1 repression during fasting. Moreover, fasting upregulated the vitamin D catabolizing CYP24A1 in the kidney through the PGC-1α-ERRα pathway. Our study uncovers a molecular mechanism for vitamin D deficiency in diabetes and reveals a novel negative feedback mechanism that controls crosstalk between energy homeostasis and the vitamin D pathway.

Cardiomyocyte proliferation accounts for the increase of cardiac muscle during fetal mammalian heart development. Shortly after birth, cardiomyocyte transits from hyperplasia to hypertrophic growth. Here, we have investigated the role of fatty acid β-oxidation in cardiomyocyte proliferation and hypertrophic growth during early postnatal life in mice. A transient wave of increased cell cycle activity of cardiomyocyte was observed between postnatal day 3 and 5, that proceeded as cardiomyocyte hypertrophic growth and maturation. Assessment of cardiomyocyte metabolism in neonatal mouse heart revealed a myocardial metabolic shift from glycolysis to fatty acid β-oxidation that coincided with the burst of cardiomyocyte cell cycle reactivation and hypertrophic growth. Inhibition of fatty acid β-oxidation metabolism in infant mouse heart delayed cardiomyocyte cell cycle exit, hypertrophic growth and maturation. By contrast, pharmacologic and genetic activation of PPARα, a major regulator of cardiac fatty acid metabolism, induced fatty acid β-oxidation and initially promoted cardiomyocyte proliferation rate in infant mice. As the cell cycle proceeded, activation of PPARα-mediated fatty acid β-oxidation promoted cardiomyocytes hypertrophic growth and maturation, which led to cell cycle exit. As a consequence, activation of PPARα-mediated fatty acid β-oxidation did not alter the total number of cardiomyocytes in infant mice. These findings indicate a unique role of fatty acid β-oxidation in regulating cardiomyocyte proliferation and hypertrophic growth in infant mice.

Overconsumption of fructose and other sugars has been linked to nonalcoholic fatty liver disease (NAFLD); however, the sugar-associated effects that lead to disease are poorly defined. In this issue of the JCI, Zhang and colleagues show that the carbohydrate response element-binding protein (ChREBP) coordinates an adaptive response to a high-fructose diet in mice and that loss of this transcription factor leads to hepatic inflammation and early signs of fibrosis. Intriguingly, ChREBP-dependent effects were due to an exaggerated activation of the proapoptotic arms of the endoplasmic reticulum stress response that is probably secondary to inappropriate derepression of cholesterol biosynthesis. These findings suggest that a previously unknown link exists between ChREBP and the regulation of cholesterol synthesis that affects liver injury.

Lipin 1 regulates glycerolipid homeostasis by acting as a phosphatidic acid phosphohydrolase (PAP) enzyme in the triglyceride-synthesis pathway and by regulating transcription factor activity. Mutations in human lipin 1 are a common cause of recurrent rhabdomyolysis in children. Mice with constitutive whole-body lipin 1 deficiency have been used to examine mechanisms connecting lipin 1 deficiency to myocyte injury. However, that mouse model is confounded by lipodystrophy not phenocopied in people. Herein, 2 muscle-specific mouse models were studied: 1) Lpin1 exon 3 and 4 deletion, resulting in a hypomorphic protein without PAP activity, but which preserved transcriptional coregulatory function; and 2) Lpin1 exon 7 deletion, resulting in total protein loss. In both models, skeletal muscles exhibited a chronic myopathy with ongoing muscle fiber necrosis and regeneration and accumulation of phosphatidic acid and, paradoxically, diacylglycerol. Additionally, lipin 1-deficient mice had abundant, but abnormal, mitochondria likely because of impaired autophagy. Finally, these mice exhibited increased plasma creatine kinase following exhaustive exercise when unfed. These data suggest that mice lacking lipin 1-mediated PAP activity in skeletal muscle may serve as a model for determining the mechanisms by which lipin 1 deficiency leads to myocyte injury and for testing potential therapeutic approaches.-Schweitzer, G. G., Collier, S. L., Chen, Z., McCommis, K. S., Pittman, S. K., Yoshino, J., Matkovich, S. J., Hsu, F.-F., Chrast, R., Eaton, J. M., Harris, T. E., Weihl, C. C., Finck, B. N. Loss of lipin 1-mediated phosphatidic acid phosphohydrolase activity in muscle leads to skeletal myopathy in mice.

The role of posttranscriptional metabolic gene regulatory programs in diabetes is not well understood. Here, we show that the RNA-binding protein tristetraprolin (TTP) is reduced in the livers of diabetic mice and humans and is transcriptionally induced in response to insulin treatment in murine livers in vitro and in vivo. Liver-specific Ttp-KO (lsTtp-KO) mice challenged with high-fat diet (HFD) have improved glucose tolerance and peripheral insulin sensitivity compared with littermate controls. Analysis of secreted hepatic factors demonstrated that fibroblast growth factor 21 (FGF21) is posttranscriptionally repressed by TTP. Consistent with increased FGF21, lsTtp-KO mice fed HFD have increased brown fat activation, peripheral tissue glucose uptake, and adiponectin production compared with littermate controls. Downregulation of hepatic Fgf21 via an adeno-associated virus-driven shRNA in mice fed HFD reverses the insulin-sensitizing effects of hepatic Ttp deletion. Thus, hepatic TTP posttranscriptionally regulates systemic insulin sensitivity in diabetes through liver-derived FGF21.

Thiazolidinediones (TZDs) enhance insulin sensitivity and have efficacy for treating nonalcoholic steatohepatitis (NASH). However, the use of TZDs, like rosiglitazone and pioglitazone, has been curtailed by significant side effects mediated via the peroxisome proliferator-activated receptor γ (PPARγ). We conducted a screen to identify compounds with insulin-sensitizing effects that did not contain a thiazolidinedione ring and act independently of PPARγ. A lead candidate, MSDC-5514, was selected for further study due to its ability to induce uncoupling protein 1 expression in brown adipocyte precursors and an inability to bind and activate PPARγ even at high concentrations. We gavaged diet-induced obese (DIO) mice with 30 mg/kg/day MSDC-5514 for six days. MSDC-5514 treatment did not affect body weight, but completely normalized glucose tolerance in insulin-resistant DIO mice. DIO mice exhibited elevated plasma insulin, free fatty acid, and triglyceride concentrations compared to lean mice, all of which were corrected by MSDC-5514 treatment. Liver diacylglycerol and triglyceride content, which were markedly increased in DIO mice compared to lean controls, were significantly decreased by MSDC-5514. The resolution of hepatic steatosis may be due to increased fatty acid oxidation since the expression of several genes encoding fatty acid oxidation enzymes was significantly elevated in livers from MSDC-5514 treated mice and directly by treatment of hepatocytes with MSDC-5514 in culture. Markers of liver injury and NASH, including plasma ALT and liver expression of markers of inflammation, stellate cell activation, and fibrosis were also attenuated by MSDC-5514 treatment. Although the molecular target of MSDC-5514 remains unclear, chemical-genetic screens are ongoing to identify the mechanism of action. In conclusion, these data suggest that MSDC-5514 may be a new PPARγ-independent insulin sensitizer with efficacy for treating diabetes and NASH. Disclosure Y. Chen: None. K. Chambers: None. W. McDonald: Stock/Shareholder; Self; Metabolic Solutions Development Company, LLC. B.N. Finck: Board Member; Self; Cirius Therapeutics. K.S. McCommis: None.

Numerous studies conducted on obese humans and various rodent models of obesity have identified a correlation between hepatic lipid content and the development of insulin resistance in liver and other tissues. Despite a large body of the literature on this topic, the cause and effect relationship between hepatic steatosis and insulin resistance remains controversial. If, as many believe, lipid aggregation in liver drives insulin resistance and other metabolic abnormalities, there are significant unanswered questions as to which lipid mediators are causative in this cascade. Several published papers have now correlated levels of diacylglycerol (DAG), the penultimate intermediate in triglyceride synthesis, with development of insulin resistance and have postulated that this occurs via activation of protein kinase C signaling. Although many studies have confirmed this relationship, many others have reported a disconnect between DAG content and insulin resistance. It has been postulated that differences in methods for DAG measurement, DAG compartmentalization within the cell, or fatty acid composition of the DAG may explain these discrepancies. The purpose of this review is to compare and contrast some of the relevant findings in this area and to discuss a number of unanswered questions regarding the relationship between DAG and insulin resistance.

Several recent studies have suggested that compounds known as endocrine-disrupting chemicals (EDCs) can promote obesity by serving as ligands for nuclear receptors, including the peroxisome proliferator-activated receptor γ (PPARγ) and the glucocorticoid receptor (GR). Thiazolidinedione insulin sensitizers, which act as ligands for PPARγ, also interact with and regulate the activity of the mitochondrial pyruvate carrier (MPC). We evaluated whether several EDCs might also affect MPC activity. Most of the EDCs evaluated did not acutely affect pyruvate metabolism. However, the putative endocrine disruptors tributyltin (TBT) and tolylfluanid (TF) acutely and markedly suppressed pyruvate metabolism in isolated mitochondria. Using mitochondria isolated from brown adipose tissue in mice with adipocyte-specific deletion of the MPC2 protein, we determined that the effect of TF on pyruvate metabolism required MPC2, whereas TBT did not. We attempted to determine whether the obesogenic effects of TF might involve MPC2 in adipose tissue. However, we were unable to replicate the published effects of TF on weight gain and adipose tissue gene expression in wild-type or fat-specific MPC2 knockout mice. Treatment with TF modestly enhanced adipogenic gene expression in vitro but had no effect on GR activation or phosphorylation in cultured cells. These data suggest that TF may affect mitochondrial pyruvate metabolism via the MPC complex but also call into question whether this compound affects GR activity and is obesogenic in mice.

SummaryCarrier-facilitated pyruvate transport across the inner mitochondrial membrane plays an essential role in anabolic and catabolic intermediary metabolism. Mitochondrial pyruvate carrier 2 (Mpc2) is believed to be a component of the complex that facilitates mitochondrial pyruvate import. Complete MPC2 deficiency resulted in embryonic lethality in mice. However, a second mouse line expressing an N-terminal truncated MPC2 protein (Mpc2Δ16) was viable but exhibited a reduced capacity for mitochondrial pyruvate oxidation. Metabolic studies demonstrated exaggerated blood lactate concentrations after pyruvate, glucose, or insulin challenge in Mpc2Δ16 mice. Additionally, compared with wild-type controls, Mpc2Δ16 mice exhibited normal insulin sensitivity but elevated blood glucose after bolus pyruvate or glucose injection. This was attributable to reduced glucose-stimulated insulin secretion and was corrected by sulfonylurea KATP channel inhibitor administration. Collectively, these data are consistent with a role for MPC2 in mitochondrial pyruvate import and suggest that Mpc2 deficiency results in defective pancreatic β cell glucose sensing.

Monoacylglycerol acyltransferase (MGAT) enzymes convert monoacylglycerol to diacylglycerol (DAG), a lipid that has been linked to the development of hepatic insulin resistance through activation of protein kinase C (PKC). The expression of genes that encode MGAT enzymes is induced in the livers of insulin-resistant human subjects with nonalcoholic fatty liver disease, but whether MGAT activation is causal of hepatic steatosis or insulin resistance is unknown. We show that the expression of Mogat1, which encodes MGAT1, and MGAT activity are also increased in diet-induced obese (DIO) and ob/obmice. To probe the metabolic effects of MGAT1 in the livers of obese mice, we administered antisense oligonucleotides (ASOs) against Mogat1 to DIO and ob/ob mice for 3 weeks. Knockdown of Mogat1 in liver, which reduced hepatic MGAT activity, did not affect hepatic triacylglycerol content and unexpectedly increased total DAG content. Mogat1 inhibition also increased both membrane and cytosolic compartment DAG levels. However, Mogat1 ASO treatment significantly improved glucose tolerance and hepatic insulin signaling in obese mice. In summary, inactivation of hepatic MGAT activity, which is markedly increased in obese mice, improved glucose tolerance and hepatic insulin signaling independent of changes in body weight, intrahepatic DAG and TAG content, and PKC signaling.

Nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum of metabolic abnormalities ranging from simple hepatic steatosis (accumulation of neutral lipid) to development of steatotic lesions, steatohepatitis, and cirrhosis. NAFLD is extremely prevalent in obese individuals and with the epidemic of obesity; nonalcoholic steatohepatitis (NASH) has become the most common cause of liver disease in the developed world. NASH is rapidly emerging as a prominent cause of liver failure and transplantation. Moreover, hepatic steatosis is tightly linked to risk of developing insulin resistance, diabetes, and cardiovascular disease. Abnormalities in hepatic lipid metabolism are part and parcel of the development of NAFLD and human genetic studies and work conducted in experimentally tractable systems have identified a number of enzymes involved in fat synthesis and degradation that are linked to NAFLD susceptibility as well as progression to NASH. The goal of this review is to summarize the current state of our knowledge on these pathways and focus on how they contribute to etiology of NAFLD and related metabolic diseases.

Background: GLUT8 is a facilitative fructose and glucose transporter expressed in liver. Results: GLUT8-deficient mice are resistant to fructose-induced fatty liver disease. Conclusion: Hexose transporters can mediate fructose-induced fatty liver disease. Significance: Hepatic hexose transporters represent a novel class of targets to prevent or modulate non-alcoholic fatty liver disease., Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the world, and it is thought to be the hepatic manifestation of the metabolic syndrome. Excess dietary fructose causes both metabolic syndrome and NAFLD in rodents and humans, but the pathogenic mechanisms of fructose-induced metabolic syndrome and NAFLD are poorly understood. GLUT8 (Slc2A8) is a facilitative glucose and fructose transporter that is highly expressed in liver, heart, and other oxidative tissues. We previously demonstrated that female mice lacking GLUT8 exhibit impaired first-pass hepatic fructose metabolism, suggesting that fructose transport into the hepatocyte, the primary site of fructose metabolism, is in part mediated by GLUT8. Here, we tested the hypothesis that GLUT8 is required for hepatocyte fructose uptake and for the development of fructose-induced NAFLD. We demonstrate that GLUT8 is a cell surface-localized transporter and that GLUT8 overexpression or GLUT8 shRNA-mediated gene silencing significantly induces and blocks radiolabeled fructose uptake in cultured hepatocytes. We further show diminished fructose uptake and de novo lipogenesis in fructose-challenged GLUT8-deficient hepatocytes. Finally, livers from long term high-fructose diet-fed GLUT8-deficient mice exhibited attenuated fructose-induced hepatic triglyceride and cholesterol accumulation without changes in hepatocyte insulin-stimulated Akt phosphorylation. GLUT8 is thus essential for hepatocyte fructose transport and fructose-induced macrosteatosis. Fructose delivery across the hepatocyte membrane is thus a proximal, modifiable disease mechanism that may be exploited to prevent NAFLD.

Lipin-1 regulates lipid metabolism by way of its function as an enzyme in the triglyceride synthesis pathway and as a transcriptional coregulatory protein and is highly up-regulated in alcoholic fatty liver disease. In the present study, using a liver-specific lipin-1-deficient (lipin-1LKO) mouse model, we aimed to investigate the functional role of lipin-1 in the development of alcoholic steatohepatitis and explore the underlying mechanisms. Alcoholic liver injury was achieved by pair feeding wild-type and lipin-1LKO mice with modified Lieber-DeCarli ethanol-containing low-fat diets for 4 weeks. Surprisingly, chronically ethanol-fed lipin-1LKO mice showed markedly greater hepatic triglyceride and cholesterol accumulation, and augmented elevation of serum liver enzymes accompanied by increased hepatic proinflammatory cytokine expression. Our studies further revealed that hepatic removal of lipin-1 in mice augmented ethanol-induced impairment of hepatic fatty acid oxidation and lipoprotein production, likely by way of deactivation of peroxisome proliferator-activated receptor γ coactivator-1alpha, a prominent transcriptional regulator of lipid metabolism. Conclusions: Liver-specific lipin-1 deficiency in mice exacerbates the development and progression of experimental alcohol-induced steatohepatitis. Pharmacological or nutritional modulation of hepatic lipin-1 may be beneficial for the prevention or treatment of human alcoholic fatty liver disease. (Hepatology 2013; 58:1953–1963)

It has been hypothesized that a greater decline in circulating branched-chain amino acids (BCAAs) after weight loss induced by Roux-en-Y gastric bypass (RYGB) surgery than after calorie restriction alone has independent effects on glucose homeostasis, possibly by decreased signaling through the mammalian target of rapamycin (mTOR). We evaluated plasma BCAAs and their C3 and C5 acylcarnitine metabolites, muscle mTOR phosphorylation, and insulin sensitivity (insulin-stimulated glucose Rd) in obese subjects before and after ∼20% weight loss induced by RYGB (n = 10, BMI 45.6 ± 6.7 kg/m2) or laparoscopic adjustable gastric banding (LAGB) (n = 10, BMI 46.5 ± 8.8 kg/m2). Weight loss increased insulin-stimulated glucose Rd by ∼55%, decreased total plasma BCAA and C3 and C5 acylcarnitine concentrations by 20–35%, and did not alter mTOR phosphorylation; no differences were detected between surgical groups (all P values for interaction >0.05). Insulin-stimulated glucose Rd correlated negatively with plasma BCAAs and with C3 and C5 acylcarnitine concentrations (r values −0.56 to −0.75, P < 0.05). These data demonstrate that weight loss induced by either LAGB or RYGB causes the same decline in circulating BCAAs and their C3 and C5 acylcarnitine metabolites. Plasma BCAA concentration is negatively associated with skeletal muscle insulin sensitivity, but the mechanism(s) responsible for this relationship is not known.

Lipin-1 is a phosphatidate phosphohydrolase (PAP) required for the generation of diacylglycerol during glycerolipid synthesis, and exhibits dual functions in the regulation of lipid metabolism. Lipin-1 has been implicated in the pathogenesis of alcoholic liver disease (ALD). In the present study, we assessed lipin-1 function in myeloid cells in ALD using a myeloid cell-specific lipin-1 knockout (mLipin-1KO) mouse model. Utilizing the Gao-binge ethanol feeding protocol, matched mLipin-1KO mice and littermate loxP control (WT) mice were pair-fed with either an ethanol-containing diet or an ethanol-free diet (control). Surprisingly, deletion of lipin-1 in myeloid cells dramatically attenuated liver inflammatory responses and ameliorated liver injury that would normally occur following the ethanol feeding protocol, but slightly exacerbated the ethanol-induced steatosis in mice. Mechanistically, myeloid cell-specific lipin-1 deficiency concomitantly increased the fat-derived adiponectin and ileum-derived fibroblast growth factor (FGF) 15. In concordance with concerted elevation of circulating adiponectin and FGF15, myeloid cell-specific lipin-1 deficiency diminished hepatic nuclear factor kappa B (NF-κB) activity, limited liver inflammatory responses, normalized serum levels of bile acids, and protected mice from liver damage after ethanol challenge. Our novel data demonstrate that myeloid cell-specific deletion of lipin-1 ameliorated inflammation and alcoholic hepatitis in mice via activation of endocrine adiponectin-FGF15 signaling.

Lipin 1 is a coregulator of DNA-bound transcription factors and a phosphatidic acid (PA) phosphatase (PAP) enzyme that catalyzes a critical step in the synthesis of glycerophospholipids. Lipin 1 is highly expressed in adipocytes, and constitutive loss of lipin 1 blocks adipocyte differentiation; however, the effects of Lpin1 deficiency in differentiated adipocytes are unknown. Here we report that adipocyte-specific Lpin1 gene recombination unexpectedly resulted in expression of a truncated lipin 1 protein lacking PAP activity but retaining transcriptional regulatory function. Loss of lipin 1-mediated PAP activity in adipocytes led to reduced glyceride synthesis and increased PA content. Characterization of the deficient mice also revealed that lipin 1 normally modulates cAMP-dependent signaling through protein kinase A to control lipolysis by metabolizing PA, which is an allosteric activator of phosphodiesterase 4 and the molecular target of rapamycin. Consistent with these findings, lipin 1 expression was significantly related to adipose tissue lipolytic rates and protein kinase A signaling in adipose tissue of obese human subjects. Taken together, our findings identify lipin 1 as a reciprocal regulator of triglyceride synthesis and hydrolysis in adipocytes, and suggest that regulation of lipolysis by lipin 1 is mediated by PA-dependent modulation of phosphodiesterase 4.

Currently approved thiazolidinediones (TZDs) are effective insulin-sensitizing drugs that may have efficacy for treatment of a variety of metabolic and inflammatory diseases, but their use is limited by side effects that are mediated through ectopic activation of the peroxisome proliferator-activated receptor γ (PPARγ). Emerging evidence suggests that the potent anti-diabetic efficacy of TZDs can be separated from the ability to serve as ligands for PPARγ. A novel TZD analog (MSDC-0602) with very low affinity for binding and activation of PPARγ was evaluated for its effects on insulin resistance in obese mice. MSDC-0602 treatment markedly improved several measures of multiorgan insulin sensitivity, adipose tissue inflammation, and hepatic metabolic derangements, including suppressing hepatic lipogenesis and gluconeogenesis. These beneficial effects were mediated at least in part via direct actions on hepatocytes and were preserved in hepatocytes from liver-specific PPARγ−/− mice, indicating that PPARγ was not required to suppress these pathways. In conclusion, the beneficial pharmacology exhibited by MSDC-0602 on insulin sensitivity suggests that PPARγ-sparing TZDs are effective for treatment of type 2 diabetes with reduced risk of PPARγ-mediated side effects. Background: Thiazolidinediones may have insulin-sensitizing effects independent of the nuclear receptor PPARγ. Results: A novel PPARγ-sparing thiazolidinedione ameliorated insulin resistance and inflammation in obese mice. Conclusion: The insulin-sensitizing effects of thiazolidinediones are separable from the ability to bind PPARγ. Significance: Identification of other molecular targets of thiazolidinediones may generate new therapeutics for treatment of insulin resistance and diabetes.

Intrahepatic lipid accumulation is extremely common in obese subjects and is associated with the development of insulin resistance and diabetes. Hepatic diacylglycerol and triacylglycerol synthesis predominantly occurs through acylation of glycerol-3-phosphate. However, an alternative pathway for synthesizing diacylglycerol from monoacylglycerol acyltransferases (MGAT) could also contribute to hepatic glyceride pools. MGAT activity and the expression of the three genes encoding MGAT enzymes (MOGAT1, MOGAT2, and MOGAT3) were determined in liver biopsies from obese human subjects before and after gastric bypass surgery. MOGAT expression was also assessed in liver of subjects with nonalcoholic fatty liver disease (NAFLD) or control livers. All MOGAT genes were expressed in liver, and hepatic MGAT activity was readily detectable in liver lysates. The hepatic expression of MOGAT3 was highly correlated with MGAT activity, whereas MOGAT1 and MOGAT2 expression was not, and knockdown of MOGAT3 expression attenuated MGAT activity in a liver-derived cell line. Marked weight loss following gastric bypass surgery was associated with a significant reduction in MOGAT2 and MOGAT3 expression, which were also overexpressed in NAFLD subjects. These data suggest that the MGAT pathway is active and dynamically regulated in human liver and could be an important target for pharmacologic intervention for the treatment of obesity-related insulin resistance and NAFLD.

To identify new gene regulatory pathways controlling skeletal muscle energy metabolism, comparative studies were conducted on muscle-specific transgenic mouse lines expressing the nuclear receptors peroxisome proliferator-activated receptor α (PPARα; muscle creatine kinase [MCK]-PPARα) or PPARβ/δ (MCK-PPARβ/δ). MCK-PPARβ/δ mice are known to have enhanced exercise performance, whereas MCK-PPARα mice perform at low levels. Transcriptional profiling revealed that the lactate dehydrogenase b (Ldhb)/Ldha gene expression ratio is increased in MCK-PPARβ/δ muscle, an isoenzyme shift that diverts pyruvate into the mitochondrion for the final steps of glucose oxidation. PPARβ/δ gain- and loss-of-function studies in skeletal myotubes demonstrated that PPARβ/δ, but not PPARα, interacts with the exercise-inducible kinase AMP-activated protein kinase (AMPK) to synergistically activate Ldhb gene transcription by cooperating with myocyte enhancer factor 2A (MEF2A) in a PPARβ/δ ligand-independent manner. MCK-PPARβ/δ muscle was shown to have high glycogen stores, increased levels of GLUT4, and augmented capacity for mitochondrial pyruvate oxidation, suggesting a broad reprogramming of glucose utilization pathways. Lastly, exercise studies demonstrated that MCK-PPARβ/δ mice persistently oxidized glucose compared with nontransgenic controls, while exhibiting supranormal performance. These results identify a transcriptional regulatory mechanism that increases capacity for muscle glucose utilization in a pattern that resembles the effects of exercise training.

Lipin family proteins (lipin 1, 2, and 3) are bifunctional intracellular proteins that regulate metabolism by acting as coregulators of DNA-bound transcription factors and also dephosphorylate phosphatidate to form diacylglycerol [phosphatidate phosphohydrolase activity] in the triglyceride synthesis pathway. Herein, we report that lipin 1 is enriched in heart and that hearts of mice lacking lipin 1 (fld mice) exhibit accumulation of phosphatidate. We also demonstrate that the expression of the gene encoding lipin 1 (Lpin1) is under the control of the estrogen-related receptors (ERRs) and their coactivator the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). PGC-1α, ERRα, or ERRγ overexpression increased Lpin1 transcription in cultured ventricular myocytes and the ERRs were associated with response elements in the first intron of the Lpin1 gene. Concomitant RNAi-mediated knockdown of ERRα and ERRγ abrogated the induction of lipin 1 expression by PGC-1α overexpression. Consistent with these data, 3-fold overexpression of PGC-1α in intact myocardium of transgenic mice increased cardiac lipin 1 and ERRα/γ expression. Similarly, injection of the β2-adrenergic agonist clenbuterol induced PGC-1α and lipin 1 expression, and the induction in lipin 1 after clenbuterol occurred in a PGC-1α-dependent manner. In contrast, expression of PGC-1α, ERRα, ERRγ, and lipin 1 was down-regulated in failing heart. Cardiac phosphatidic acid phosphohydrolase activity was also diminished, while cardiac phosphatidate content was increased, in failing heart. Collectively, these data suggest that lipin 1 is the principal lipin protein in the myocardium and is regulated in response to physiologic and pathologic stimuli that impact cardiac metabolism.

Fibroblast growth factor 21 (FGF21) is an endocrine hormone that is critical for regulation of intermediary metabolism, and it is also a potential drug target for treating diabetes and other metabolic diseases. Interest in FGF21 exploded with the discovery that pharmacological administration of FGF21 to diabetic rodents and primates increased insulin sensitivity, energy expenditure, and weight loss (1). Administration of the FGF21 analog, LY2405319, to obese humans also significantly reduced body weight and improved plasma metabolic profiles (2). These studies have spurred a number of drug development programs designed to enhance FGF21 activity as a therapeutic approach to increase insulin sensitivity and treat diabetes. FGF21 is expressed in and secreted by several tissues including liver, white adipose tissue, brown adipose tissue, striated muscle, heart, and pancreas. However, recent work has demonstrated that plasma FGF21 levels are derived primarily, if not completely, from the liver (3). FGF21 signals to specific tissues that express both the traditional FGF receptor, FGFR1, and a coreceptor called βKlotho. The physiological importance of FGF21 in regulating adaptive metabolic responses was demonstrated when complimentary articles showed that FGF21 was highly induced in liver by fasting through activation of the peroxisome proliferator–activated receptor α (4,5). Using multiple gain-of-function models, Inagaki et al. (4) showed that FGF21 produced by the liver acts as an endocrine hormone to coordinate adipose tissue lipolysis and stimulate ketogenesis, both critical responses to …

We previously reported that mice subjected to partial hepatectomy exhibit rapid development of hypoglycemia followed by transient accumulation of fat in the early regenerating liver. We also showed that disrupting these metabolic alterations results in impaired liver regeneration. The studies reported here were undertaken to further characterize and investigate the functional importance of changes in systemic adipose metabolism during normal liver regeneration. The results showed that a systemic catabolic response is induced in each of two distinct, commonly used experimental models of liver regeneration (partial hepatectomy and carbon tetrachloride treatment), and that this response occurs in proportion to the degree of induced hepatic insufficiency. Together, these observations suggest that catabolism of systemic adipose stores may be essential for normal liver regeneration. To test this possibility, we investigated the hepatic regenerative response in fatty liver dystrophy (fld) mice, which exhibit partial lipodystrophy and have diminished peripheral adipose stores. The results showed that the development of hypoglycemia and hepatic accumulation of fat was attenuated and liver regeneration was impaired following partial hepatectomy in these animals. The fld mice also exhibited increased hepatic p21 expression and diminished plasma levels of the adipose-derived hormones adiponectin and leptin, which have each been implicated as regulators of liver regeneration. Conclusion: These data suggest that the hypoglycemia that develops after partial hepatectomy induces systemic lipolysis followed by accumulation of fat derived from peripheral stores in the early regenerating liver, and that these events may be essential for initiation of normal liver regeneration. (HEPATOLOGY 2010)

OBJECTIVE Insulin resistance is commonly associated with obesity. Studies conducted in obese mouse models found that endoplasmic reticulum (ER) stress contributes to insulin resistance, and treatment with tauroursodeoxycholic acid (TUDCA), a bile acid derivative that acts as a chemical chaperone to enhance protein folding and ameliorate ER stress, increases insulin sensitivity. The purpose of this study was to determine the effect of TUDCA therapy on multiorgan insulin action and metabolic factors associated with insulin resistance in obese men and women. RESEARCH DESIGN AND METHODS Twenty obese subjects ([means ± SD] aged 48 ± 11 years, BMI 37 ± 4 kg/m2) were randomized to 4 weeks of treatment with TUDCA (1,750 mg/day) or placebo. A two-stage hyperinsulinemic-euglycemic clamp procedure in conjunction with stable isotopically labeled tracer infusions and muscle and adipose tissue biopsies were used to evaluate in vivo insulin sensitivity, cellular factors involved in insulin signaling, and cellular markers of ER stress. RESULTS Hepatic and muscle insulin sensitivity increased by ∼30% (P < 0.05) after treatment with TUDCA but did not change after placebo therapy. In addition, therapy with TUDCA, but not placebo, increased muscle insulin signaling (phosphorylated insulin receptor substrateTyr and AktSer473 levels) (P < 0.05). Markers of ER stress in muscle or adipose tissue did not change after treatment with either TUDCA or placebo. CONCLUSIONS These data demonstrate that TUDCA might be an effective pharmacological approach for treating insulin resistance. Additional studies are needed to evaluate the target cells and mechanisms responsible for this effect.

UCP2 -866GA (rs659366) has been implicated in cardiometabolic disease and represents a novel candidate gene for beta-blocker response, particularly among patients with diabetes. We assessed the function of -866GA and its role as a modifier of beta-blocker treatment outcomes by diabetes status in an acute coronary syndrome (ACS) cohort.ACS patients with genetic samples and 12 months of follow-up for cardiac rehospitalizations or death (n=468) were assessed. The influence of -866GA on beta-blocker treatment outcomes was evaluated in those with diabetes and without. To assess functional correlates of -866GA, we compared uncoupling protein 2 (UCP2) expression in the skeletal muscle of obese participants by genotype and compared the activity of UCP2 luciferase promoters with -866G and -866A alleles.An interaction between -866GA and beta-blocker treatment was found in individuals with diabetes (P=0.002) but not those without (P=0.79). Among G/G individuals with diabetes, discharge beta-blocker use was associated with an 80% reduction in cardiac rehospitalization (adjusted hazard ratio: 0.20; 95% confidence interval: 0.04-1.02). In contrast, among A-carrier patients with diabetes, there was an 11-fold increase in cardiac rehospitalizations with discharge beta-blocker therapy (adjusted hazard ratio: 11.75; 95% confidence interval: 1.28-108.2). Promoter activity assays showed that -866G had greater cyclic AMP response element binding protein-responsiveness compared with -866A, and compared with -866A carriers G/G individuals exhibited increased UCP2 expression in the skeletal muscle.We identified a significant interaction between -866GA and beta-blocker response among ACS patients with diabetes. Furthermore, -866G conferred greater gene transcriptional activity than -866A in cell lines and in obese patients. These findings may help us gain insight into the mechanisms underlying the beneficial and detrimental effects of beta-blockers in those with diabetes.

Lipid droplet proteins (LDPs) coat the surface of triglyceride-rich lipid droplets and regulate their formation and lipolysis. We profiled hepatic LDP expression in fatty liver dystrophic (fld) mice, a unique model of neonatal hepatic steatosis that predictably resolves between postnatal day 14 (P14) and P17. Western blotting revealed that perilipin-2/ADRP and perilipin-5/OXPAT were markedly increased in steatotic fld liver but returned to normal by P17. However, the changes in perilipin-2 and perilipin-5 protein content in fld mice were exaggerated compared with relatively modest increases in corresponding mRNAs encoding these proteins, a phenomenon likely mediated by increased protein stability. Conversely, cell death-inducing DFFA-like effector (Cide) family genes were strongly induced at the level of mRNA expression in steatotic fld mouse liver. Surprisingly, levels of peroxisome proliferator-activated receptor gamma, which is known to regulate Cide expression, were unchanged in fld mice. However, sterol-regulatory element binding protein 1 (SREBP-1) was activated in fld liver and CideA was revealed as a new direct target gene of SREBP-1. In summary, LDP content is markedly increased in liver of fld mice. However, whereas perilipin-2 and perilipin-5 levels are primarily regulated posttranslationally, Cide family mRNA expression is induced, suggesting that these families of LDP are controlled at different regulatory checkpoints.

The liver plays a crucial role in mobilizing energy during nutritional deprivation. During the early stages of fasting, hepatic glycogenolysis is a primary energy source. As fasting progresses and glycogen stores are depleted, hepatic gluconeogenesis and ketogenesis become major energy sources. Here, we show that fibroblast growth factor 21 (FGF21), a hormone that is induced in liver by fasting, induces hepatic expression of peroxisome proliferator-activated receptor gamma coactivator protein-1alpha (PGC-1alpha), a key transcriptional regulator of energy homeostasis, and causes corresponding increases in fatty acid oxidation, tricarboxylic acid cycle flux, and gluconeogenesis without increasing glycogenolysis. Mice lacking FGF21 fail to fully induce PGC-1alpha expression in response to a prolonged fast and have impaired gluconeogenesis and ketogenesis. These results reveal an unexpected relationship between FGF21 and PGC-1alpha and demonstrate an important role for FGF21 in coordinately regulating carbohydrate and fatty acid metabolism during the progression from fasting to starvation.

Objective— Lipin 1 controls fatty acid metabolism in the nucleus as a transcriptional regulator and in the cytosol as an enzyme catalyzing the penultimate step in phosphoglycerol triacylglyceride (TAG) synthesis. We sought to evaluate the effects of lipin 1 on hepatic TAG synthesis and secretion by gain-of-function and loss-of-function approaches. Methods and Results— Rates of TAG synthesis were not impaired in hepatocytes isolated from adult lipin 1–deficient ( fld ) mice and were actually increased in 14-day-old fld mice. Additionally, compared to littermate controls, VLDL-TAG secretion rates were markedly increased in fld mice of both ages. Lipin 1 overexpression did not alter TAG synthesis rates but significantly suppressed VLDL-TAG secretion. The lipin 1-mediated suppression of VLDL-TAG secretion was linked to the peptide motif mediating its transcriptional-regulatory effects. However, the expression of candidate genes required for VLDL assembly and secretion was unaltered by lipin 1 activation or deficiency. Finally, the hepatic expression of lipin 1 was diminished in obese insulin-resistant mice, whereas adenoviral-mediated overexpression of lipin 1 in liver of these mice inhibits VLDL-TAG secretion and improves hepatic insulin signaling. Conclusions— Collectively, these studies reveal new and unexpected effects of lipin 1 on hepatic TAG metabolism and obesity-related hepatic insulin resistance.

Diabetic cardiomyopathy is associated with abnormalities in glucose metabolism. We evaluated myocardial glucose metabolism in a rodent model of type 2 diabetes, namely the Zucker diabetic fatty (ZDF) rat, and validated PET measurements of glucose uptake against gene and protein expression of glucose transporters (GLUTs).Six lean and ZDF rats underwent small-animal PET at the age of 14 wk and at the age of 19 wk. The imaging protocol consisted of a 60-min dynamic acquisition with 18F-FDG (18.5-29.6 MBq). Dynamic images were reconstructed using filtered backprojection with a 2.5 zoom on the heart and 40 frames per imaging session. PET measurements of myocardial glucose uptake (MGUp) rate and utilization were determined with an input function derived by the hybrid image-blood-sampling algorithm on recovery-corrected anterolateral myocardial regions of interest. After the PET session at week 19 (W19), hearts were extracted for gene and protein expression analysis of GLUT-1 and GLUT-4. The dependence of MGUp on gene expression of GLUT-1 and GLUT-4 was characterized by multiple-regression analysis.MGUp in ZDF rats at both week 14 (W14) and W19 (P0.006) was significantly lower than MGUp in lean littermate control rats. Moreover, lean rats at W19 displayed significantly higher MGUp than they did at W14 (P = 0.007). Consistent with a diminished MGUp result, gene expression of GLUT-4 was significantly (P = 0.004) lower in ZDF rats. Finally, MGUp significantly (P = 0.0003) correlated with gene expression of GLUT-4.Using small-animal PET, we confirmed alterations in myocardial glucose utilization and validated PET measurement of MGUp against gene and protein expression of GLUTs in the diabetic heart of an animal model of type 2 diabetes.