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Single-nucleotide polymorphisms in the human juxtaposed with another zinc finger protein 1 ( JAZF1 ) gene have repeatedly been associated with both type 2 diabetes (T2D) and height in multiple genome-wide association studies (GWAS); however, the mechanism by which JAZF1 causes these traits is not yet known. To investigate the possible functional role of JAZF1 in growth and glucose metabolism in vivo, we generated Jazf1 knockout (KO) mice and examined body composition and insulin sensitivity both in young and adult mice by using 1 H-nuclear magnetic resonance and hyperinsulinemic-euglycemic clamp techniques. Plasma concentrations of insulin-like growth factor 1 (IGF-1) were reduced in both young and adult Jazf1 KO mice, and young Jazf1 KO mice were shorter in stature than age-matched wild-type mice. Young Jazf1 KO mice manifested reduced fat mass, whereas adult Jazf1 KO mice manifested increased fat mass and reductions in lean body mass associated with increased plasma growth hormone (GH) concentrations. Adult Jazf1 KO manifested muscle insulin resistance that was further exacerbated by high-fat diet feeding. Gene set enrichment analysis in Jazf1 KO liver identified the hepatocyte hepatic nuclear factor 4 alpha (HNF4α), which was decreased in Jazf1 KO liver and in JAZF1 knockdown cells. Moreover, GH-induced IGF-1 expression was inhibited by JAZF1 knockdown in human hepatocytes. Taken together these results demonstrate that reduction of JAZF1 leads to early growth retardation and late onset insulin resistance in vivo which may be mediated through alterations in the GH-IGF-1 axis and HNF4α.

Single-nucleotide polymorphisms in the human juxtaposed with another zinc finger protein 1 (

Microsomal triglyceride transfer protein (MTTP) deficiency results in a syndrome of hypolipidemia and accelerated NAFLD. Animal models of decreased hepatic MTTP activity have revealed an unexplained dissociation between hepatic steatosis and hepatic insulin resistance. Here, we performed comprehensive metabolic phenotyping of liver-specific MTTP knockout (L-Mttp(-/-)) mice and age-weight matched wild-type control mice. Young (10-12-week-old) L-Mttp(-/-) mice exhibited hepatic steatosis and increased DAG content; however, the increase in hepatic DAG content was partitioned to the lipid droplet and was not increased in the plasma membrane. Young L-Mttp(-/-) mice also manifested normal hepatic insulin sensitivity, as assessed by hyperinsulinemic-euglycemic clamps, no PKC epsilon activation, and normal hepatic insulin signaling from the insulin receptor through AKT Ser/Thr kinase. In contrast, aged (10-month-old) L-Mttp(-/-) mice exhibited glucose intolerance and hepatic insulin resistance along with an increase in hepatic plasma membrane sn-1,2-DAG content and PKC epsilon activation. Treatment with a functionally liver-targeted mitochondrial uncoupler protected the aged L-Mttp(-/-) mice against the development of hepatic steatosis, increased plasma membrane sn-1,2-DAG content, PKC epsilon activation, and hepatic insulin resistance. Furthermore, increased hepatic insulin sensitivity in the aged controlled-release mitochondrial protonophore-treated L-Mttp(-/-) mice was not associated with any reductions in hepatic ceramide content. Taken together, these data demonstrate that differences in the intracellular compartmentation of sn-1,2-DAGs in the lipid droplet versus plasma membrane explains the dissociation of NAFLD/lipid-induced hepatic insulin resistance in young L-Mttp(-/-) mice as well as the development of lipid-induced hepatic insulin resistance in aged L-Mttp(-/-) mice This work was supported by National Institutes of Health Grants R01 DK116774, R01 DK119968, R01 DK114793, R01 DK113984, K23 DK10287, P30 DK045735, DK121490, and HL137202 and the Veterans Health Administration Merit Review Awards I01 BX000901 and BX004113. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the U.S. Department of Veterans Affairs

Significance Hepatic glycogen synthesis plays a critical role in maintaining normal glucose homeostasis; however, the rate-controlling step regulating this process is unknown. Applying metabolic control analysis in vivo, we show that the regulation of insulin-stimulated hepatic glycogen synthesis under both normal and pathophysiological conditions of fatty liver-associated hepatic insulin resistance is controlled at the glucokinase (GCK) step through GCK translocation., Multiple insulin-regulated enzymes participate in hepatic glycogen synthesis, and the rate-controlling step responsible for insulin stimulation of glycogen synthesis is unknown. We demonstrate that glucokinase (GCK)-mediated glucose phosphorylation is the rate-controlling step in insulin-stimulated hepatic glycogen synthesis in vivo, by use of the somatostatin pancreatic clamp technique using [13C6]glucose with metabolic control analysis (MCA) in three rat models: 1) regular chow (RC)-fed male rats (control), 2) high fat diet (HFD)-fed rats, and 3) RC-fed rats with portal vein glucose delivery at a glucose infusion rate matched to the control. During hyperinsulinemia, hyperglycemia dose-dependently increased hepatic glycogen synthesis. At similar levels of hyperinsulinemia and hyperglycemia, HFD-fed rats exhibited a decrease and portal delivery rats exhibited an increase in hepatic glycogen synthesis via the direct pathway compared with controls. However, the strong correlation between liver glucose-6-phosphate concentration and net hepatic glycogen synthetic rate was nearly identical in these three groups, suggesting that the main difference between models is the activation of GCK. MCA yielded a high control coefficient for GCK in all three groups. We confirmed these findings in studies of hepatic GCK knockdown using an antisense oligonucleotide. Reduced liver glycogen synthesis in lipid-induced hepatic insulin resistance and increased glycogen synthesis during portal glucose infusion were explained by concordant changes in translocation of GCK. Taken together, these data indicate that the rate of insulin-stimulated hepatic glycogen synthesis is controlled chiefly through GCK translocation.

Mediators of Insulin Resistance Insulin resistance is a precursor to and accelerant of coexisting conditions such as type 2 diabetes, atherosclerosis, and nonalcoholic liver disease. A recent study...

The connection between adipose glucocorticoid action and whole-body metabolism is incompletely understood. Thus, we generated adipose tissue–specific glucocorticoid receptor–knockout (Ad-GcR(−/−)) mice to explore potential mechanisms. Ad-GcR(−/−) mice had a lower concentration of fasting plasma nonesterified fatty acids and less hepatic steatosis. This was associated with increased protein kinase B phosphorylation and increased hepatic glycogen synthesis after an oral glucose challenge. High-fat diet (HFD)–fed Ad-GcR(−/−) mice were protected against the development of hepatic steatosis and diacylglycerol-PKCε–induced impairments in hepatic insulin signaling. Under hyperinsulinemic-euglycemic conditions, hepatic insulin response was ∼10-fold higher in HFD-fed Ad-GcR(−/−) mice. Insulin-mediated suppression of adipose lipolysis was improved by 40% in Ad-GcR(−/−) mice. Adipose triglyceride lipase expression was decreased and insulin-mediated perilipin dephosphorylation was increased in Ad-GcR(−/−) mice. In metabolic cages, food intake decreased by 3 kcal/kg per hour in Ad-GcR(−/−) mice. Therefore, physiologic adipose glucocorticoid action appears to drive hepatic lipid accumulation during stressors such as fasting. The resultant hepatic insulin resistance prevents hepatic glycogen synthesis, preserving glucose for glucose-dependent organs. Absence of adipose glucocorticoid action attenuates HFD-induced hepatic insulin resistance; potential explanations for reduction in hepatic steatosis include reductions in adipose lipolysis and food intake.—Abulizi, A., Camporez, J.-P., Jurczak, M. J., Høyer, K. F., Zhang, D., Cline, G. W., Samuel, V. T., Shulman, G. I., Vatner, D. F. Adipose glucocorticoid action influences whole-body metabolism via modulation of hepatic insulin action.

CONTEXT: Excessive adipose glucocorticoid action is associated with insulin resistance, but the mechanisms linking adipose glucocorticoid action to insulin resistance are still debated. We hypothesized that insulin resistance from excess glucocorticoid action may be attributed in part to increased ectopic lipid deposition in liver. METHODS: We tested this hypothesis in the adipose specific 11β-Hydroxysteroid dehydrogenase-1 (HSD11B1) transgenic mouse, an established model of adipose glucocorticoid excess. Tissue specific insulin action was assessed by hyperinsulinemic-euglycemic clamps, hepatic lipid content was measured, hepatic insulin signaling was assessed by immunoblotting. The role of hepatic lipid content was further probed by administration of the functionally liver-targeted mitochondrial uncoupler, Controlled Release Mitochondrial Protonophore (CRMP). FINDINGS: High fat diet fed HSD11B1 transgenic mice developed more severe hepatic insulin resistance than littermate controls (endogenous suppression of hepatic glucose production was reduced by 3.8-fold, P < 0.05); this was reflected by decreased insulin-stimulated hepatic insulin receptor kinase tyrosine phosphorylation and AKT serine phosphorylation. Hepatic insulin resistance was associated with a 53% increase (P < 0.05) in hepatic triglyceride content, a 73% increase in diacylglycerol content (P < 0.01), and a 66% increase in PKCε translocation (P < 0.05). Hepatic insulin resistance was prevented with administration of CRMP by reversal of hepatic steatosis and prevention of hepatic diacylglycerol accumulation and PKCε activation. CONCLUSIONS: These findings are consistent with excess adipose glucocorticoid activity being a predisposing factor for the development of lipid (diacylglycerol-PKCε)-induced hepatic insulin resistance.

Nonalcoholic fatty liver disease is strongly associated with hepatic insulin resistance (HIR); however, the key lipid species and molecular mechanisms linking these conditions are widely debated. We developed a subcellular fractionation method to quantify diacylglycerol (DAG) stereoisomers and ceramides in the endoplasmic reticulum (ER), mitochondria, plasma membrane (PM), lipid droplets, and cytosol. Acute knockdown (KD) of diacylglycerol acyltransferase-2 in liver induced HIR in rats. This was due to PM sn-1,2-DAG accumulation, which promoted PKCϵ activation and insulin receptor kinase (IRK)-T1160 phosphorylation, resulting in decreased IRK-Y1162 phosphorylation. Liver PM sn-1,2-DAG content and IRK-T1160 phosphorylation were also higher in humans with HIR. In rats, liver-specific PKCϵ KD ameliorated high-fat diet-induced HIR by lowering IRK-T1160 phosphorylation, while liver-specific overexpression of constitutively active PKCϵ-induced HIR by promoting IRK-T1160 phosphorylation. These data identify PM sn-1,2-DAGs as the key pool of lipids that activate PKCϵ and that hepatic PKCϵ is both necessary and sufficient in mediating HIR.

Nonalcoholic fatty liver disease (NAFLD) is strongly associated with hepatic insulin resistance (HIR), however the key lipid species and molecular mechanisms linking these conditions are widely debated. In order to address these questions, we examined the metabolic impact of a liver-specific antisense oligonucleotide against PKCε and found that liver-specific PKCε knockdown ameliorated high-fat diet-induced HIR in rats, reflected by ∼2x increase in insulin-stimulated hepatic glycogen synthesis rate and improved insulin-mediated suppression of endogenous glucose production during a hyperinsulinemic-hyperglycemic (HH) clamp. This was associated with increased IRK Y1162 and Akt S473 phosphorylation and could be attributed to decreased PKCε-mediated IRK T1160 phosphorylation. Overexpressing a constitutively active PKCε (A159E) in the liver induced HIR in regular chow-fed rats, reflected by reduced insulin-stimulated glycogen synthesis rate during an HH clamp. This was associated with impaired IRK Y1162 and Akt S473 phosphorylation and could be attributed to increased PKCε-mediated IRK T1160 phosphorylation. We also developed a subcellular fractionation method to quantify diacylglycerol (DAG) stereoisomers and ceramides in the endoplasmic reticulum, mitochondria, plasma membrane (PM), lipid droplet and cytosol and applied this method to a cohort of obese human subjects with and without NAFLD and HIR. Liver PM sn-1,2 DAG content was ∼5x higher in HIR individuals with NAFLD compared to insulin-sensitive individuals without NAFLD, associated with ∼3x higher IRK T1160 phosphorylation, supporting the importance of PM sn-1,2 DAG-PKCε-IRK pT1160 pathway to mediate HIR in humans with NAFLD. In contrast there were no significant differences in ceramide content in any of the subcellular compartments. Conclusion: These data identify PM sn-1,2 DAGs as the key lipids that activate PKCε, and that hepatic PKCε is both necessary and sufficient in mediating HIR. Disclosure K. Lyu: None. D. Zhang: None. M. Kahn: None. M.R.S. Rodrigues: None. S. Hirabara: None. P. Luukkonen: None. S. Lee: None. S. Bhanot: None. J. Rinehart: None. N. Blume: Employee; Self; Novo Nordisk A/S. Stock/Shareholder; Self; Novo Nordisk A/S. M. Rasch: Employee; Self; Novo Nordisk A/S. Stock/Shareholder; Self; Novo Nordisk A/S. M.J. Serlie: None. J. Bogan: None. G. Cline: None. V. Samuel: None. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research & Development, LLC, Merck & Co., Inc. Advisory Panel; Spouse/Partner; Merck & Co., Inc. Consultant; Self; Novo Nordisk A/S. Consultant; Spouse/Partner; Novo Nordisk A/S. Other Relationship; Self; Gilead Sciences, Inc., iMetabolic Biopharma Corporation. Other Relationship; Spouse/Partner; iMetabolic Biopharma Corporation. Other Relationship; Self; Maze Therapeutics. Funding U.S. Public Health Service (R01DK116774, R01DK119968, R01DK113984, P30DK045735, R01 DK092661); U.S. Department of Veternas Affairs (I01BX000901)

Insulin-mediated suppression of white adipose tissue (WAT) lipolysis is an important anabolic function that is dysregulated in states of overnutrition. However, the mechanism of short-term high-fat diet (HFD)-induced WAT insulin resistance is poorly understood. Based on our recent studies we hypothesize that a short-term HFD causes WAT insulin resistance through increases in plasma membrane (PM) sn-1,2-diacylglycerols (DAG), which promotes protein kinase C-e (PKCe) activation to impair insulin signaling by phosphorylating insulin receptor (Insr) Thr1160. To test this hypothesis, we assessed WAT insulin action in 7-day HFD-fed versus regular chow diet-fed rats during a hyperinsulinemic-euglycemic clamp. HFD feeding caused WAT insulin resistance, reflected by reductions in both insulin-mediated WAT glucose uptake and suppression of WAT lipolysis. These changes were specifically associated with increased PM sn-1,2-diacylglycerol (DAG) content, increased PKCe activation and impaired insulin-stimulated InsrY1162 phosphorylation. In order to examine the role of InsrT1160 phosphorylation in mediating lipid-induced WAT insulin resistance, we examined these same parameters in short-term HFD-fed InsrT1150A knockin mice (mouse homolog for human Thr1160). Similar to the rat study HFD feeding induced WAT insulin resistance in WT control mice but failed to induce WAT insulin resistance in InsrT1150A mice. Taken together these data demonstrate that the PM sn-1,2-DAG - PKCe - InsrT1160 phosphorylation pathway plays an important role in mediating lipid-induced WAT insulin resistance and represents a potential therapeutic target to improve insulin sensitivity in WAT.

AIMS/HYPOTHESIS: Targeting regulators of adipose tissue lipoprotein lipase could enhance adipose lipid clearance, prevent ectopic lipid accumulation and consequently ameliorate insulin resistance and type 2 diabetes. Angiopoietin-like 8 (ANGPTL8) is an insulin-regulated lipoprotein lipase inhibitor strongly expressed in murine adipose tissue. However, Angptl8 knockout mice do not have improved insulin resistance. We hypothesised that pharmacological inhibition, using a second-generation antisense oligonucleotide (ASO) against Angptl8 in adult high-fat-fed rodents, would prevent ectopic lipid accumulation and insulin resistance by promoting adipose lipid uptake. METHODS: ANGPTL8 expression was assessed by quantitative PCR in omental adipose tissue of bariatric surgery patients. High-fat-fed Sprague Dawley rats and C57BL/6 mice were treated with ASO against Angptl8 and insulin sensitivity was assessed by hyperinsulinaemic–euglycaemic clamps in rats and glucose tolerance tests in mice. Factors mediating lipid-induced hepatic insulin resistance were assessed, including lipid content, protein kinase Cε (PKCε) activation and insulin-stimulated Akt phosphorylation. Rat adipose lipid uptake was assessed by mixed meal tolerance tests. Murine energy balance was assessed by indirect calorimetry. RESULTS: Omental fat ANGPTL8 mRNA expression is higher in obese individuals with fatty liver and insulin resistance compared with BMI-matched insulin-sensitive individuals. Angptl8 ASO prevented hepatic steatosis, PKCε activation and hepatic insulin resistance in high-fat-fed rats. Postprandial triacylglycerol uptake in white adipose tissue was increased in Angptl8 ASO-treated rats. Angptl8 ASO protected high-fat-fed mice from glucose intolerance. Although there was no change in net energy balance, Angptl8 ASO increased fat mass in high-fat-fed mice. CONCLUSIONS/INTERPRETATION: Disinhibition of adipose tissue lipoprotein lipase is a novel therapeutic modality to enhance adipose lipid uptake and treat non-alcoholic fatty liver disease and insulin resistance. In line with this, adipose ANGPTL8 is a candidate therapeutic target for these conditions.

NAFLD is closely linked with hepatic insulin resistance. Accumulation of hepatic diacylglycerol activates PKC-ε, impairing insulin receptor activation and insulin-stimulated glycogen synthesis. Peripheral insulin resistance indirectly influences hepatic glucose and lipid metabolism by increasing flux of substrates that promote lipogenesis (glucose and fatty acids) and gluconeogenesis (glycerol and fatty acid-derived acetyl-CoA, an allosteric activator of pyruvate carboxylase). Weight loss with diet or bariatric surgery effectively treats NAFLD, but drugs specifically approved for NAFLD are not available. Some new pharmacological strategies act broadly to alter energy balance or influence pathways that contribute to NAFLD (e.g., agonists for PPAR γ, PPAR α/δ, FXR and analogs for FGF-21, and GLP-1). Others specifically inhibit key enzymes involved in lipid synthesis (e.g., mitochondrial pyruvate carrier, acetyl-CoA carboxylase, stearoyl-CoA desaturase, and monoacyl- and diacyl-glycerol transferases). Finally, a novel class of liver-targeted mitochondrial uncoupling agents increases hepatocellular energy expenditure, reversing the metabolic and hepatic complications of NAFLD.

White adipose tissue (WAT), as an energy storage organ, plays a major role in regulating peripheral insulin resistance and hepatic gluconeogenesis, but the mechanism by which lipid induces insulin resistance in WAT is poorly understood. To examine the potential role of the diacylglycerol (DAG) - protein kinase Cε (PKCε) - insulin receptor (INSR) pathway in mediating lipid-induced WAT insulin resistance, we assessed insulin action in liver, muscle and WAT in 7-day high fat diet (HFD) and regular chow (RC) fed rats by hyperinsulinemic-euglycemic clamp (HEC) combined with [3-3H] glucose, [14C] deoxyglucose and [2H5]glycerol infusions. Using this approach, we found that HFD induced WAT insulin resistance as reflected by impaired suppression (∼20%, P Conclusion: The DAG-PKCε-INSR Thr1150pathway plays a major role in mediating lipid-induced WAT insulin resistance and represents a potential therapeutic target to improve insulin sensitivity in WAT. Disclosure D. Zhang: None. K. Lyu: None. J.D. Song: None. X. Li: None. R.J. Perry: Research Support; Self; AstraZeneca. V. Samuel: None. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research & Development, Merck & Co., Inc. Advisory Panel; Spouse/Partner; Merck & Co., Inc. Board Member; Self; Novo Nordisk A/S. Consultant; Self; Aegerion Pharmaceuticals, IMetabolic BioPharma Corporation, Longitude Capital, Nimbus Discovery, Inc., Staten Biotechnology B.V. Funding National Institutes of Health (R01DK116774)

The identity and subcellular localization of the bioactive lipid species that causes hepatic insulin resistance remain controversial. Though chronic diacylglycerol acyltransferase 2 (DGAT2) knockdown improves hepatic steatosis and insulin action, we hypothesized that acute DGAT2 knockdown would transiently increase hepatic diacylglycerol (DAG) content to specifically assess the role of hepatic DAG on hepatic insulin action. Regular chow-fed rats were treated with a single dose of DGAT2 antisense oligonucleotide and studied 2 days later. The content and subcellular localization of sn-1,2, sn-2,3 and sn-1,3 DAGs were assessed by LC-MS/MS in the endoplasmic reticulum (ER), mitochondrial (MIT), plasma membrane (PM), lipid droplet (LD) and cytosol (CYT) fractions. Acute knockdown of hepatic DGAT2 induced hepatic insulin resistance with impaired suppression of hepatic glucose production during a hyperinsulinemic-euglycemic clamp. Insulin receptor kinase (IRK) activation was impaired, as reflected by reduced insulin-stimulated IRK Y1162 phosphorylation. This was associated with increased PKCε activation, as reflected by increased translocation from CYT to membrane, and increased sn-1,2 DAG content specifically in the PM. In contrast, there were no changes in hepatic ceramide content or sn-2,3 and sn-1,3 DAGs in the five subcellular fractions tested (ER, MIT, PM, LD, CYT). We next infused [13C16] palmitate in 24-hr-fasted rats to track hepatic DAG synthesis in vivo and observed that sn-1,2 DAGs were the major (73%) product of re-esterification from exogenous fatty acids, compared to sn-2,3 (25%) and sn-1,3 (2%) DAGs. We also found that sn-1,2 and sn-2,3 DAGs were primarily synthesized in the ER before translocating to PM, whereas sn-1,3 DAGs were exclusively produced in the LD by lipolysis. Conclusion: These data demonstrate that sn-1,2 DAGs in the PM, derived from re-esterification of exogenous fatty acids, are sufficient to induce hepatic insulin resistance at the level of IRK. Disclosure K. Lyu: None. Y. Zhang: None. D. Zhang: None. M. Kahn: None. Y. Nozaki: None. S. Bhanot: None. J. Bogan: None. G. Cline: None. V. Samuel: None. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research & Development, Merck & Co., Inc. Advisory Panel; Spouse/Partner; Merck & Co., Inc. Board Member; Self; Novo Nordisk A/S. Consultant; Self; Aegerion Pharmaceuticals, IMetabolic BioPharma Corporation, Longitude Capital, Nimbus Discovery, Inc., Staten Biotechnology B.V. Funding National Institutes of Health (R01DK116774)

The ongoing debate regarding pathway selective hepatic insulin resistance (IR) focuses on potential insulin signaling branchpoints beyond which regulation of glucose metabolism is attenuated and regulation of de novo lipogenesis (DNL) remains intact. These models suggest that impaired insulin receptor kinase activity, proximal to any potential branchpoint, only plays a minor role in clinical hepatic IR. We compared insulin signaling in liver biopsies from human subjects with nonalcoholic fatty liver disease (NAFLD) and hepatic IR vs. weight matched controls, using glucose ingestion to drive insulin secretion and fructose ingestion as a low insulin comparator. Insulin signaling through phosphorylation of insulin receptor β, Akt, and GSK3β was suppressed in NAFLD (all P < 0.05). Insulin regulation of mTORC1 via PRAS40 phosphorylation was absent in NAFLD subjects; no differences were seen between controls and NAFLD subjects in AMPK dependent RAPTOR phosphorylation. Pathway selective IR implies SREBP1c is primarily responsible for DNL in the insulin resistant liver; we probed the transcriptional regulation of DNL by assessing the in vivo occupancy of lipogenic target gene promoters by SREBP1c and ChREBP using ChIP experiments in mice with diet-induced IR. Fasted-refed mice were refed with high fat diet (HFD) and 1% dextrose drinking water. Mice with hepatic IR (9 day HFD fed) or hepatic and peripheral IR (4 week HFD fed) were compared with more insulin sensitive 2 day HFD fed mice. SREBP1c was not bound to the Fasn promoter in insulin resistant livers, while ChREBP was bound to the Fasn promoter equally in all three groups of mice. Conclusions: There is a proximal block in insulin signaling in humans with hepatic IR and NAFLD, dissociating previously measured changes in DNL rates from insulin signaling. SREBP does not mediate DNL in insulin resistant mice while ChREBP action can explain the maintenance of the transcriptional program of lipogenesis. Disclosure D.F. Vatner: None. K.W. ter Horst: None. L. Goedeke: None. D. Zhang: None. V. Samuel: None. M.J. Serlie: Advisory Panel; Self; Fresenius Medical Care. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research & Development, Merck & Co., Inc. Advisory Panel; Spouse/Partner; Merck & Co., Inc. Board Member; Self; Novo Nordisk A/S. Consultant; Self; Aegerion Pharmaceuticals, IMetabolic BioPharma Corporation, Longitude Capital, Nimbus Discovery, Inc., Staten Biotechnology B.V. Funding National Institutes of Health

Introduction: Hypoglycemia is rare in patients without diabetes and requires investigation if Whipple’s triad is met. Case Description: Two patients were admitted to the same facility for workup of symptomatic hypoglycemia. Patient A is a 71-year-old man who self-presented due to concern for worsening cognition that was later found to correspond to blood glucose [BG] below 60 mg/dL. Patient B is a 70-year-old man who had a BG of 46 mg/dL on routine labs and was then fitted with a continuous glucose monitor [CGM] that reported nocturnal and post-prandial glucose values

Insulin resistance arises when the nutrient storage pathways evolved to maximize efficient energy utilization are exposed to chronic energy surplus. Ectopic lipid accumulation in liver and skeletal muscle triggers pathways that impair insulin signaling, leading to reduced muscle glucose uptake and decreased hepatic glycogen synthesis. Muscle insulin resistance, due to ectopic lipid, precedes liver insulin resistance and diverts ingested glucose to the liver, resulting in increased hepatic de novo lipogenesis and hyperlipidemia. Subsequent macrophage infiltration into white adipose tissue (WAT) leads to increased lipolysis, which further increases hepatic triglyceride synthesis and hyperlipidemia due to increased fatty acid esterification. Macrophage-induced WAT lipolysis also stimulates hepatic gluconeogenesis, promoting fasting and postprandial hyperglycemia through increased fatty acid delivery to the liver, which results in increased hepatic acetyl-CoA content, a potent activator of pyruvate carboxylase, and increased glycerol conversion to glucose. These substrate-regulated processes are mostly independent of insulin signaling in the liver but are dependent on insulin signaling in WAT, which becomes defective with inflammation. Therapies that decrease ectopic lipid storage and diminish macrophage-induced WAT lipolysis will reverse the root causes of type 2 diabetes.

Insulin resistance drives the development of type 2 diabetes (T2D). In liver, diacylglycerol (DAG) is a key mediator of lipid-induced insulin resistance. DAG activates protein kinase C ε (PKCε), which phosphorylates and inhibits the insulin receptor. In rats, a 3-day high-fat diet produces hepatic insulin resistance through this mechanism, and knockdown of hepatic PKCε protects against high-fat diet-induced hepatic insulin resistance. Here, we employed a systems-level approach to uncover additional signaling pathways involved in high-fat diet-induced hepatic insulin resistance. We used quantitative phosphoproteomics to map global in vivo changes in hepatic protein phosphorylation in chow-fed, high-fat–fed, and high-fat–fed with PKCε knockdown rats to distinguish the impact of lipid- and PKCε-induced protein phosphorylation. This was followed by a functional siRNA-based screen to determine which dynamically regulated phosphoproteins may be involved in canonical insulin signaling. Direct PKCε substrates were identified by motif analysis of phosphoproteomics data and validated using a large-scale in vitro kinase assay. These substrates included the p70S6K substrates RPS6 and IRS1, which suggested cross talk between PKCε and p70S6K in high-fat diet-induced hepatic insulin resistance. These results identify an expanded set of proteins through which PKCε may drive high-fat diet-induced hepatic insulin resistance that may direct new therapeutic approaches for T2D.

Insulin suppresses hepatic glucose production and increases hepatic de novo lipogenesis (DNL). Paradoxically, hepatic DNL remains elevated in insulin-resistant subjects, leading to the hypothesis that hepatic insulin resistance is pathway-selective. Prior studies of DNL in hepatic insulin resistance are complicated by confounders, such as use of markedly unphysiologic animal models or comparison of animals on different diets and thus different availability of DNL precursors. We measured DNL in InsrT1150A knockin (KI) mice, a strain protected from diacylglycerol-mediated hepatic insulin resistance, and in littermate controls. Diets were matched: 60% high fat diet (HFD) with 1% dextrose in drinking water. After two days of fat-feeding, before the development of significant insulin resistance, KI and WT mice displayed similar rates of DNL (KI 24.3% ± 3.6; WT 22.2% ± 3.5). After 9 days of HFD, when WT but not KI mice have developed hepatic insulin resistance, rates of DNL were reduced in WT mice but preserved in KI mice (KI 16.4% ± 2.4; WT 7.4% ± 1.3; P < 0.01). After 4 weeks of HFD, with the development of skeletal muscle resistance, rates of DNL increased in WT mice to reach those observed in KI mice (KI 19.3% ± 2.6; WT 15.9% ± 2.3). Consistent with the insulin-resistant phenotype, Srebp-1c cleavage was reduced in the setting of hepatic insulin resistance in WT mice; this effect was attenuated in KI mice. The Srebp-regulated lipogenic proteins Fasn and Scd1 were both reduced in 9d HFD fed livers regardless of genotype; at 4 weeks, protein abundance remained suppressed in WT mice but was restored in KI mice. Pklr, a protein associated with DNL but not regulated by Srebp, was unaffected by HFD-induced hepatic insulin resistance. Conclusion: Regulation of DNL is subject to lipid-induced hepatic insulin resistance, challenging the selective hepatic insulin resistance hypothesis. Increased DNL seen in insulin resistant subjects may be due to hyperinsulinemia and diversion of substrate from the insulin resistant periphery. Disclosure D.F. Vatner: None. M.C. Petersen: None. X. Li: None. J.C. Rogers: None. G. Cline: None. V. Samuel: None. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research & Development, Merck & Co., Inc., Novo Nordisk Inc.. Research Support; Self; Gilead Sciences, Inc..

Diacylglycerol (DAG) has been proposed to mediate lipid-induced hepatic insulin resistance. Accumulated hepatic DAG activates PKCε which phosphorylates insulin receptor kinase (IRK) at T1160 to inhibit IRK activation and cause insulin resistance. However, the importance of DAG in lipid-induced hepatic insulin resistance remains controversial. To test the physiological effect of acute DAG accumulation on hepatic insulin sensitivity, we used antisense oligonucleotide (ASO) to specifically inhibit hepatic diglyceride acyltransferase 2 (DGAT2) which catalyzes the conversion of DAG to triglyceride. Previously, chronic DGAT2 inhibition was shown to paradoxically decrease hepatic DAG content due to decreased SREBP-1c-mediated lipogenesis and improve hepatic insulin sensitivity. However, we hypothesized that acute DGAT2 inhibition might provide a narrow window to transiently increase hepatic DAG content and allow us to identify the association between DAG, PKCε activation/translocation and hepatic insulin resistance. As hypothesized, an acute (2-day) DGAT2 inhibition increased hepatic DAG content and PKCε activation (cytosol to membrane translocation). DGAT2 ASO-treated rats on regular chow diet displayed impaired insulin-mediated suppression of hepatic glucose production. This defect of hepatic insulin action was at the level of IRK as indicated by impaired insulin-stimulated IRK Y1158/1162 phosphorylation. Furthermore, measured by a novel LC-MS/MS method, hepatic sn-1,2 DAG, which is the DAG stereoisomer capable of activating PKCε, was increased in the subcellular membrane compartment with DGAT2 ASO treatment. Conclusion: These data support the importance of membrane sn-1,2 DAG in mediating lipid-induced hepatic insulin resistance through “increased membrane sn-1,2 DAG content -> PKCε activation/translocation -> increased IRK T1160 phosphorylation -> decreased IRK Y1158/1162 phosphorylation -> decreased IRK activity” in vivo. Disclosure K. Lyu: None. D. Zhang: None. Y. Nozaki: None. Y. Zhang: None. S. Bhanot: Employee; Self; Ionis Pharmaceuticals, Inc.. G. Cline: None. V. Samuel: None. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research & Development, Merck & Co., Inc., Novo Nordisk Inc.. Research Support; Self; Gilead Sciences, Inc..

Increased sucrose consumption is associated with rising rates of obesity and related conditions [e.g., insulin resistance, type 2 diabetes and nonalcoholic fatty liver disease (NAFLD)]. Ketohexokinase (KHK) catalyzes the first step of fructose metabolism and is highly expressed in liver, kidney and brain, though found in many tissues. Essential fructosuria (hepatic KHK deficiency) is a benign condition, suggesting that KHK is a viable drug target. Inhibition of hepatic KHK would block metabolism of fructose (50% of dietary sucrose) and improve numerous facets of metabolism. ASO’s potently decrease target expression in liver and WAT and we hypothesized that a novel KHK ASO would prevent NAFLD and insulin resistance in normal rats fed a low fat (12% calories from fat), sucrose containing diet (17% calories from fructose). KHK ASO decreased hepatic KHK mRNA and protein expression by 96 and 90%, respectively. KHK ASO decreased WAT mRNA expression by 65%. KHK ASO did not alter body mass. KHK ASO decreased fasting plasma glucose (102±2 vs. 94±2 mg/dL, P=0.01), fasting plasma triglyceride 60% (52±7 vs. 21±2 mg/dL P Conclusion: KHK ASO prevents NAFLD, decreases plasma triglyceride and improves insulin sensitivity. Disclosure D. Liu: None. J.A. Sterpka: None. D.F. Vatner: None. M. Bell: Employee; Self; Ionis Pharmaceuticals, Inc.. Employee; Spouse/Partner; Ionis Pharmaceuticals, Inc. S. Murray: Employee; Self; Ionis Pharmaceuticals, Inc. S. Bhanot: Employee; Self; Ionis Pharmaceuticals, Inc.. G. Cline: None. V. Samuel: None.

Reducing the expression of the Indy (I'm Not Dead Yet) gene in lower organisms extends life span by mechanisms resembling caloric restriction. Similarly, deletion of the mammalian homolog, mIndy (Slc13a5), encoding for a plasma membrane tricarboxylate transporter, protects from aging- and diet-induced adiposity and insulin resistance in mice. The organ specific contribution to this phenotype is unknown. We examined the impact of selective inducible hepatic knockdown of mIndy on whole body lipid and glucose metabolism using 2′-O-methoxyethyl chimeric anti-sense oligonucleotides (ASOs) in high-fat fed rats. 4-week treatment with 2′-O-methoxyethyl chimeric ASO reduced mIndy mRNA expression by 91% (P

In adipose and muscle cells, insulin stimulates the exocytic translocation of vesicles containing GLUT4, a glucose transporter, and insulin-regulated aminopeptidase (IRAP), a transmembrane aminopeptidase. A substrate of IRAP is vasopressin, which controls water homeostasis. The physiological importance of IRAP translocation to inactivate vasopressin remains uncertain. We previously showed that in skeletal muscle, insulin stimulates proteolytic processing of the GLUT4 retention protein, TUG, to promote GLUT4 translocation and glucose uptake. Here we show that TUG proteolysis also controls IRAP targeting and regulates vasopressin action in vivo. Transgenic mice with constitutive TUG proteolysis in muscle consumed much more water than wild-type control mice. The transgenic mice lost more body weight during water restriction, and the abundance of renal AQP2 water channels was reduced, implying that vasopressin activity is decreased. To compensate for accelerated vasopressin degradation, vasopressin secretion was increased, as assessed by the cosecreted protein copeptin. IRAP abundance was increased in T-tubule fractions of fasting transgenic mice, when compared with controls. Recombinant IRAP bound to TUG, and this interaction was mapped to a short peptide in IRAP that was previously shown to be critical for GLUT4 intracellular retention. In cultured 3T3-L1 adipocytes, IRAP was present in TUG-bound membranes and was released by insulin stimulation. Together with previous results, these data support a model in which TUG controls vesicle translocation by interacting with IRAP as well as GLUT4. Furthermore, the effect of IRAP to reduce vasopressin activity is a physiologically important consequence of vesicle translocation, which is coordinated with the stimulation of glucose uptake. Background: Insulin stimulates the exocytic translocation of vesicles containing GLUT4 glucose transporters and insulin-regulated aminopeptidase (IRAP). Results: Insulin acts through TUG proteins to control IRAP targeting, similar to GLUT4; the activity of vasopressin, an IRAP substrate, is reduced in mice with disrupted TUG action in muscle. Conclusion: TUG regulates vasopressin action. Significance: Exocytic translocation of vesicles in muscle coordinates vasopressin inactivation with glucose uptake.

ApoA5 has a critical role in the regulation of plasma TG concentrations. In order to determine whether ApoA5 also impacts ectopic lipid deposition in liver and skeletal muscle, as well as tissue insulin sensitivity, we treated mice with an antisense oligonucleotide (ASO) to decrease hepatic expression of ApoA5. ASO treatment reduced ApoA5 protein expression in liver by 60–70%. ApoA5 ASO-treated mice displayed approximately 3-fold higher plasma TG concentrations, which were associated with decreased plasma TG clearance. Furthermore, ApoA5 ASO-treated mice fed a high-fat diet (HFD) exhibited reduced liver and skeletal muscle TG uptake and reduced liver and muscle TG and diacylglycerol (DAG) content. HFD-fed ApoA5 ASO-treated mice were protected from HFD-induced insulin resistance, as assessed by hyperinsulinemic-euglycemic clamps. This protection could be attributed to increases in both hepatic and peripheral insulin responsiveness associated with decreased DAG activation of protein kinase C (PKC)-e and PKCθ in liver and muscle, respectively, and increased insulin-stimulated AKT2 pho­sphory­lation in these tissues. In summary, these studies demonstrate a novel role for ApoA5 as a modulator of susceptibility to diet-induced liver and muscle insulin resistance through regulation of ectopic lipid accumulation in liver and skeletal muscle.

Non-alcoholic fatty liver disease and its downstream sequelae, hepatic insulin resistance and type 2 diabetes, are rapidly growing epidemics, which lead to increased morbidity and mortality rates, and soaring health-care costs. Developing interventions requires a comprehensive understanding of the mechanisms by which excess hepatic lipid develops and causes hepatic insulin resistance and type 2 diabetes. Proposed mechanisms implicate various lipid species, inflammatory signalling and other cellular modifications. Studies in mice and humans have elucidated a key role for hepatic diacylglycerol activation of protein kinase Cε in triggering hepatic insulin resistance. Therapeutic approaches based on this mechanism could alleviate the related epidemics of non-alcoholic fatty liver disease and type 2 diabetes.

Metformin is considered to be one of the most effective therapeutics for treating type 2 diabetes because it specifically reduces hepatic gluconeogenesis without increasing insulin secretion, inducing weight gain or posing a risk of hypoglycaemia. For over half a century, this agent has been prescribed to patients with type 2 diabetes worldwide, yet the underlying mechanism by which metformin inhibits hepatic gluconeogenesis remains unknown. Here we show that metformin non-competitively inhibits the redox shuttle enzyme mitochondrial glycerophosphate dehydrogenase, resulting in an altered hepatocellular redox state, reduced conversion of lactate and glycerol to glucose, and decreased hepatic gluconeogenesis. Acute and chronic low-dose metformin treatment effectively reduced endogenous glucose production, while increasing cytosolic redox and decreasing mitochondrial redox states. Antisense oligonucleotide knockdown of hepatic mitochondrial glycerophosphate dehydrogenase in rats resulted in a phenotype akin to chronic metformin treatment, and abrogated metformin-mediated increases in cytosolic redox state, decreases in plasma glucose concentrations, and inhibition of endogenous glucose production. These findings were replicated in whole-body mitochondrial glycerophosphate dehydrogenase knockout mice. These results have significant implications for understanding the mechanism of metformin's blood glucose lowering effects and provide a new therapeutic target for type 2 diabetes.

Aerobic exercise increases muscle glucose and improves insulin action through numerous pathways, including activation of Ca(2+)/calmodulin-dependent protein kinases (CAMKs) and peroxisome proliferator γ coactivator 1α (PGC-1α). While overexpression of PGC-1α increases muscle mitochondrial content and oxidative type I fibres, it does not improve insulin action. Activation of CAMK4 also increases the content of type I muscle fibres, PGC-1α level and mitochondrial content. However, it remains unknown whether CAMK4 activation improves insulin action on glucose metabolism in vivo.The effects of CAMK4 activation on skeletal muscle insulin action were quantified using transgenic mice with a truncated and constitutively active form of CAMK4 (CAMK4([Symbol: see text])) in skeletal muscle. Tissue-specific insulin sensitivity was assessed in vivo using a hyperinsulinaemic-euglycaemic clamp and isotopic measurements of glucose metabolism.The rate of insulin-stimulated whole-body glucose uptake was increased by ∼25% in CAMK4([Symbol: see text]) mice. This was largely attributed to an increase of ∼60% in insulin-stimulated glucose uptake in the quadriceps, the largest hindlimb muscle. These changes were associated with improvements in insulin signalling, as reflected by increased phosphorylation of Akt and its substrates and an increase in the level of GLUT4 protein. In addition, there were extramuscular effects: CAMK4([Symbol: see text]) mice had improved hepatic and adipose insulin action. These pleiotropic effects were associated with increased levels of PGC-1α-related myokines in CAMK4([Symbol: see text]) skeletal muscle.Activation of CAMK4 enhances mitochondrial biogenesis in skeletal muscle while also coordinating improvements in whole-body insulin-mediated glucose.

Summary: Pyruvate kinase is an important enzyme in glycolysis and a key metabolic control point. We recently observed a pyruvate kinase liver isoform (PKL) phosphorylation site at S113 that correlates with insulin resistance in rats on a 3 day high-fat diet (HFD) and suggests additional control points for PKL activity. However, in contrast to the classical model of PKL regulation, neither authentically phosphorylated PKL at S12 nor S113 alone is sufficient to alter enzyme kinetics or structure. Instead, we show that cyclin-dependent kinases (CDKs) are activated by the HFD and responsible for PKL phosphorylation at position S113 in addition to other targets. These CDKs control PKL nuclear retention, alter cytosolic PKL activity, and ultimately influence glucose production. These results change our view of PKL regulation and highlight a previously unrecognized pathway of hepatic CDK activity and metabolic control points that may be important in insulin resistance and type 2 diabetes. : Gassaway et al. identify a diet-induced, cyclin-dependent kinase-regulated phosphorylation site at S113 on pyruvate kinase. Although they determine that neither phosphorylation of this site nor the canonical PKA-regulated S12 site directly impacts enzyme kinetics, they demonstrate that S113 phosphorylation alters pyruvate kinase subcellular localization and influences glucose production. Keywords: pyruvate kinase, cyclin-dependent kinase, metabolism, phosphorylation, hepatic glucose production, enzyme regulation, insulin resistance, sub-cellular localization, nuclear localization

Pigment epithelium-derived factor (PEDF), the protein product of the SERPINF1 gene, has been linked to distinct diseases involving adipose or bone tissue, the metabolic syndrome, and osteogenesis imperfecta (OI) type VI. Since mesenchymal stem cell (MSC) differentiation into adipocytes vs. osteoblasts can be regulated by specific factors, PEDF-directed dependency of murine and human MSCs was assessed. PEDF inhibited adipogenesis and promoted osteoblast differentiation of murine MSCs, osteoblast precursors, and human MSCs. Blockade of adipogenesis by PEDF suppressed peroxisome proliferator-activated receptor-γ (PPARγ), adiponectin, and other adipocyte markers by nearly 90% compared with control-treated cells (P50% compared with controls, illustrating its systemic role as a negative regulator of adipogenesis. Bones from KO mice demonstrated a reduction in mineral content recapitulating the OI type VI phenotype. These results demonstrate that the human diseases associated with PEDF reflect its ability to modulate MSC differentiation.—Gattu, A. K., Swenson, E. S., Iwakiri, Y., Samuel, V. T., Troiano, N., Berry, R., Church, C. D., Rodeheffer, M. S., Carpenter, T. O., Chung, C. Determination of mesenchymal stem cell fate by pigment epithelium-derived factor (PEDF) results in increased adiposity and reduced bone mineral content.

Liver-specific thyroid hormone receptor-β (TRβ)-specific agonists are potent lipid-lowering drugs that also hold promise for treating nonalcoholic fatty liver disease and hepatic insulin resistance. We investigated the effect of two TRβ agonists (GC-1 and KB-2115) in high-fat-fed male Sprague-Dawley rats treated for 10 days. GC-1 treatment reduced hepatic triglyceride content by 75%, but the rats developed fasting hyperglycemia and hyperinsulinemia, attributable to increased endogenous glucose production (EGP) and diminished hepatic insulin sensitivity. GC-1 also increased white adipose tissue lipolysis; the resulting increase in glycerol flux may have contributed to the increase in EGP. KB-2115, a more TRβ- and liver-specific thyromimetic, also prevented hepatic steatosis but did not induce fasting hyperglycemia, increase basal EGP rate, or diminish hepatic insulin sensitivity. Surprisingly, insulin-stimulated peripheral glucose disposal was diminished because of a decrease in insulin-stimulated skeletal muscle glucose uptake. Skeletal muscle insulin signaling was unaffected. Instead, KB-2115 treatment was associated with a decrease in GLUT4 protein content. Thus, although both GC-1 and KB-2115 potently treat hepatic steatosis in fat-fed rats, they each worsen insulin action via specific and discrete mechanisms. The development of future TRβ agonists must consider the potential adverse effects on insulin sensitivity.

Fibroblast growth factor 21 (FGF21) is a potent regulator of glucose and lipid metabolism and is currently being pursued as a therapeutic agent for insulin resistance and type 2 diabetes. However, the cellular mechanisms by which FGF21 modifies insulin action in vivo are unclear. To address this question, we assessed insulin action in regular chow– and high-fat diet (HFD)–fed wild-type mice chronically infused with FGF21 or vehicle. Here, we show that FGF21 administration results in improvements in both hepatic and peripheral insulin sensitivity in both regular chow– and HFD-fed mice. This improvement in insulin responsiveness in FGF21-treated HFD-fed mice was associated with decreased hepatocellular and myocellular diacylglycerol content and reduced protein kinase Cϵ activation in liver and protein kinase Cθ in skeletal muscle. In contrast, there were no effects of FGF21 on liver or muscle ceramide content. These effects may be attributed, in part, to increased energy expenditure in the liver and white adipose tissue. Taken together, these data provide a mechanism by which FGF21 protects mice from lipid-induced liver and muscle insulin resistance and support its development as a novel therapy for the treatment of nonalcoholic fatty liver disease, insulin resistance, and type 2 diabetes.

cAMP-responsive element-binding protein (CREB)-regulated transcription coactivator 2 (CRTC2) regulates transcription of gluconeogenic genes by specifying targets for the transcription factor CREB in response to glucagon. We used an antisense oligonucleotide directed against CRTC2 in both normal rodents and in rodent models of increased gluconeogenesis to better understand the role of CRTC2 in metabolic disease. In the context of severe hyperglycemia and elevated hepatic glucose production, CTRC2 knockdown (KD) improved glucose homeostasis by reducing endogenous glucose production. Interestingly, despite the known role of CRTC2 in coordinating gluconeogenic gene expression, CRTC2 KD in a rodent model of type 2 diabetes resulted in surprisingly little alteration of glucose production. However, CRTC2 KD animals had elevated circulating concentrations of glucagon and a ∼80% reduction in glucagon clearance. When this phenomenon was prevented with somatostatin or a glucagon-neutralizing antibody, endogenous glucose production was reduced by CRTC2 KD. Additionally, CRTC2 inhibition resulted in reduced expression of several glucagon-induced pyridoxal 5′-phosphate-dependent enzymes that convert amino acids to gluconeogenic intermediates, suggesting that it may control substrate availability as well as gluconeogenic gene expression. CRTC2 is an important regulator of gluconeogenesis with tremendous impact in models of elevated hepatic glucose production. Surprisingly, it is also part of a previously unidentified negative feedback loop that degrades glucagon and regulates amino acid metabolism to coordinately control glucose homeostasis in vivo. Background: CRTC2 translocates to the nucleus upon glucagon stimulation, yet its role in regulating blood glucose remains controversial. Results: CRTC2 promotes expression of enzymes that direct amino acids toward gluconeogenesis and is a key regulator of glucagon clearance. Conclusion: CRTC2 has antagonistic cell-autonomous and endocrine effects on glucose homeostasis. Significance: We present new insights into glucagon/CRTC2 physiology.

Genome-wide array studies have associated the patatin-like phospholipase domain-containing 3 (PNPLA3) gene polymorphisms with hepatic steatosis. However, it is unclear whether PNPLA3 functions as a lipase or a lipogenic enzyme and whether PNPLA3 is involved in the pathogenesis of hepatic insulin resistance. To address these questions we treated high-fat-fed rats with specific antisense oligonucleotides to decrease hepatic and adipose pnpla3 expression. Reducing pnpla3 expression prevented hepatic steatosis, which could be attributed to decreased fatty acid esterification measured by the incorporation of [U-13C]-palmitate into hepatic triglyceride. While the precursors for phosphatidic acid (PA) (long-chain fatty acyl-CoAs and lysophosphatidic acid [LPA]) were not decreased, we did observe an ∼20% reduction in the hepatic PA content, ∼35% reduction in the PA/LPA ratio, and ∼60%-70% reduction in transacylation activity at the level of acyl-CoA:1-acylglycerol-sn-3-phosphate acyltransferase. These changes were associated with an ∼50% reduction in hepatic diacylglycerol (DAG) content, an ∼80% reduction in hepatic protein kinase Cε activation, and increased hepatic insulin sensitivity, as reflected by a 2-fold greater suppression of endogenous glucose production during the hyperinsulinemic-euglycemic clamp. Finally, in humans, hepatic PNPLA3 messenger RNA (mRNA) expression was strongly correlated with hepatic triglyceride and DAG content, supporting a potential lipogenic role of PNPLA3 in humans. Conclusion: PNPLA3 may function primarily in a lipogenic capacity and inhibition of PNPLA3 may be a novel therapeutic approach for treatment of nonalcoholic fatty liver disease-associated hepatic insulin resistance. ((Hepatology 2013;57:1763-1772))

We explored the role of reactive oxygen species (ROS) in the pathogenesis of muscle insulin resistance. We assessed insulin action in vivo with a hyperinsulinemic-euglycemic clamp in mice expressing a mitochondrial-targeted catalase (MCAT) that were fed regular chow (RC) or a high-fat diet (HFD) or underwent an acute infusion of a lipid emulsion. RC-fed MCAT mice were similar to littermate wild-type (WT) mice. However, HFD-fed MCAT mice were protected from diet-induced insulin resistance. In contrast, an acute lipid infusion caused muscle insulin resistance in both MCAT and WT mice. ROS production was decreased in both HFD-fed and lipid-infused MCAT mice and cannot explain the divergent response in insulin action. MCAT mice had subtly increased energy expenditure and muscle fat oxidation with decreased intramuscular diacylglycerol (DAG) accumulation, protein kinase C-θ (PKCθ) activation, and impaired insulin signaling with HFD. In contrast, the insulin resistance with the acute lipid infusion was associated with increased muscle DAG content in both WT and MCAT mice. These studies suggest that altering muscle mitochondrial ROS production does not directly alter the development of lipid-induced insulin resistance. However, the altered energy balance in HFD-fed MCAT mice protected them from DAG accumulation, PKCθ activation, and impaired muscle insulin signaling.

Nonalcoholic fatty liver disease (NAFLD) is a risk factor for type 2 diabetes (T2D), but whether NAFLD plays a causal role in the pathogenesis of T2D is uncertain. One proposed mechanism linking NAFLD to hepatic insulin resistance involves diacylglycerol-mediated (DAG-mediated) activation of protein kinase C-e (PKCe) and the consequent inhibition of insulin receptor (INSR) kinase activity. However, the molecular mechanism underlying PKCe inhibition of INSR kinase activity is unknown. Here, we used mass spectrometry to identify the phosphorylation site Thr1160 as a PKCe substrate in the functionally critical INSR kinase activation loop. We hypothesized that Thr1160 phosphorylation impairs INSR kinase activity by destabilizing the active configuration of the INSR kinase, and our results confirmed this prediction by demonstrating severely impaired INSR kinase activity in phosphomimetic T1160E mutants. Conversely, the INSR T1160A mutant was not inhibited by PKCe in vitro. Furthermore, mice with a threonine-to-alanine mutation at the homologous residue Thr1150 (InsrT1150A mice) were protected from high fat diet-induced hepatic insulin resistance. InsrT1150A mice also displayed increased insulin signaling, suppression of hepatic glucose production, and increased hepatic glycogen synthesis compared with WT controls during hyperinsulinemic clamp studies. These data reveal a critical pathophysiological role for INSR Thr1160 phosphorylation and provide further mechanistic links between PKCe and INSR in mediating NAFLD-induced hepatic insulin resistance.

A main function of white adipose tissue is to release fatty acids from stored triacylglycerol for other tissues to use as an energy source. Whereas endocrine regulation of lipolysis has been extensively studied, autocrine and paracrine regulation is not well understood. Here we describe the role of the newly identified major adipocyte phospholipase A(2), AdPLA (encoded by Pla2g16, also called HREV107), in the regulation of lipolysis and adiposity. AdPLA-null mice have a markedly higher rate of lipolysis owing to increased cyclic AMP levels arising from the marked reduction in the amount of adipose prostaglandin E(2) that binds the Galpha(i)-coupled receptor, EP3. AdPLA-null mice have markedly reduced adipose tissue mass and triglyceride content but normal adipogenesis. They also have higher energy expenditure with increased fatty acid oxidation within adipocytes. AdPLA-deficient ob/ob mice remain hyperphagic but lean, with increased energy expenditure, yet have ectopic triglyceride storage and insulin resistance. AdPLA is a major regulator of adipocyte lipolysis and is crucial for the development of obesity.

A key sensor of cellular energy status, AMP-activated protein kinase (AMPK), interacts allosterically with AMP to maintain an active state. When active, AMPK triggers a metabolic switch, decreasing the activity of anabolic pathways and enhancing catabolic processes such as lipid oxidation to restore the energy balance. Unlike oxidative tissues, in which AMP is generated from adenylate kinase during states of high energy demand, the ornithine cycle enzyme argininosuccinate synthetase (ASS) is a principle site of AMP generation in the liver. Here we show that ASS regulates hepatic AMPK, revealing a central role for ureagenesis flux in the regulation of metabolism via AMPK. Treatment of primary rat hepatocytes with amino acids increased gluconeogenesis and ureagenesis and, despite nutrient excess, induced both AMPK and acetyl-CoA carboxylase (ACC) phosphorylation. Antisense oligonucleotide knockdown of hepatic ASS1 expression in vivo decreased liver AMPK activation, phosphorylation of ACC, and plasma β-hydroxybutyrate concentrations. Taken together these studies demonstrate that increased amino acid flux can activate AMPK through increased AMP generated by ASS, thus providing a novel link between protein catabolism, ureagenesis, and hepatic lipid metabolism.

Epidemiological studies link fructose consumption with metabolic disease, an association attributable in part to fructose-mediated lipogenesis. The mechanisms governing fructose-induced lipogenesis and disease remain debated. Acutely, fructose increases de novo lipogenesis through the efficient and uninhibited action of ketohexokinase and aldolase B which yields substrates for fatty-acid synthesis. Chronic fructose consumption further enhances the capacity for hepatic fructose metabolism by activating several key transcription factors (i.e., SREBP1c and ChREBP) which augment the expression of lipogenic enzymes, increasing lipogenesis and further compounding hypertriglyceridemia and hepatic steatosis. Hepatic insulin resistance develops from diacylglycerol–PKCɛ-mediated impairment of insulin signaling and possibly additional mechanisms. Initiatives that decrease fructose consumption and therapies that block fructose-mediated lipogenesis will be necessary to avert future metabolic pandemics.

Obesity is a significant risk factor for developing diabetes. Pigment epithelium-derived factor (PEDF) has been identified by experimental and clinical studies as both a causative and counter-regulatory factor in the metabolic syndrome. We set out to determine whether serum PEDF levels correlated with the degree of insulin resistance in morbidly obese patients. Sera from 53 patients who were evaluated prior to gastric bypass surgery were analyzed for PEDF levels using a commercial ELISA. None of the patients were on diabetes medications prior to enrollment. Baseline data included BMI, serum glucose and insulin, and homeostasis model assessment (HOMA) scores. Patients were stratified based on HOMA score and glucose levels into three groups: insulin sensitive (IS): HOMA 2 and glucose ≤126; and diabetes mellitus (DM): HOMA >2 and glucose >126. Pre- and post-gastric bypass sera from 12 patients were obtained for serial assessment of metabolic parameters and PEDF levels. PEDF secretion was assessed in primary human hepatocytes, HCC cells, and cultured adipocytes in the absence and presence of high glucose media. No significant differences in age, gender, and BMI were found among the three groups. PEDF levels were similar between IR patients and the other groups, but were significantly higher in DM compared to IS patients (p = 0.01). Serum PEDF in individual patients declined significantly after gastric bypass (p = 0.006). High glucose media led to significantly higher PEDF release by human hepatocytes in vitro (p = 0.016). These data demonstrate that serum PEDF concentrations better relate to insulin resistance than to adiposity and suggest that PEDF expression is closely linked to the development of insulin resistance.

Recent studies reveal a strong relationship between reduced mitochondrial content and insulin resistance in human skeletal muscle, although the underlying factors responsible for this association remain unknown. To address this question, we analyzed muscle biopsy samples from young, lean, insulin resistant (IR) offspring of parents with type 2 diabetes and control subjects by microarray analyses and found significant differences in expression of ∼512 probe pairs. We then screened these genes for their potential involvement in the regulation of mitochondrial biogenesis using RNA interference and found that mRNA and protein expression of lipoprotein lipase (LPL) in skeletal muscle was significantly decreased in the IR offspring and was associated with decreased mitochondrial density. Furthermore, we show that LPL knockdown in muscle cells decreased mitochondrial content by effectively decreasing fatty acid delivery and subsequent activation of peroxisome proliferator–activated receptor (PPAR)-δ. Taken together, these data suggest that decreased mitochondrial content in muscle of IR offspring may be due in part to reductions in LPL expression in skeletal muscle resulting in decreased PPAR-δ activation.

Body fat, insulin resistance, and type 2 diabetes are often linked together, but the molecular mechanisms that unify their association are poorly understood. Wnt signaling regulates adipogenesis, and its altered activity has been implicated in the pathogenesis of type 2 diabetes and metabolic syndrome. LRP6(+/-) mice on a high fat diet were protected against diet-induced obesity and hepatic and adipose tissue insulin resistance compared with their wild-type (WT) littermates. Brown adipose tissue insulin sensitivity and reduced adiposity of LRP6(+/-) mice were accounted for by diminished Wnt-dependent mTORC1 activity and enhanced expression of brown adipose tissue PGC1-α and UCP1. LRP6(+/-) mice also exhibited reduced endogenous hepatic glucose output, which was due to diminished FoxO1-dependent expression of the key gluconeogenic enzyme glucose-6-phosphatase (G6pase). In addition, in vivo and in vitro studies showed that loss of LRP6 allele is associated with increased leptin receptor expression, which is a likely cause of hepatic insulin sensitivity in LRP6(+/-) mice. Our study identifies LRP6 as a nutrient-sensitive regulator of body weight and glucose metabolism and as a potential target for pharmacological interventions in obesity and diabetes.