ABSTRACT: 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-? (PKC?) and the consequent inhibition of insulin receptor (INSR) kinase activity. However, the molecular mechanism underlying PKC? inhibition of INSR kinase activity is unknown. Here, we used mass spectrometry to identify the phosphorylation site Thr1160 as a PKC? 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 PKC? 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 PKC? and INSR in mediating NAFLD-induced hepatic insulin resistance.
Project description:Nonalcoholic fatty liver disease (NAFLD) affects one in three Americans and is a major predisposing condition for the metabolic syndrome and type 2 diabetes (T2D). We examined whether a functionally liver-targeted derivative of 2,4-dinitrophenol (DNP), DNP-methyl ether (DNPME), could safely decrease hypertriglyceridemia, NAFLD, and insulin resistance without systemic toxicities. Treatment with DNPME reversed hypertriglyceridemia, fatty liver, and whole-body insulin resistance in high-fat-fed rats and decreased hyperglycemia in a rat model of T2D with a wide therapeutic index. The reversal of liver and muscle insulin resistance was associated with reductions in tissue diacylglycerol content and reductions in protein kinase C epsilon (PKC?) and PKC? activity in liver and muscle, respectively. These results demonstrate that the beneficial effects of DNP on hypertriglyceridemia, fatty liver, and insulin resistance can be dissociated from systemic toxicities and suggest the potential utility of liver-targeted mitochondrial uncoupling agents for the treatment of hypertriglyceridemia, NAFLD, metabolic syndrome, and T2D.
Project description:Insulin resistance is a key driver of type 2 diabetes (T2D) and is characterized by defective insulin receptor (INSR) signalling. Although surface INSR downregulation is a well-established contributor to insulin resistance, the underlying molecular mechanisms remain obscure. Here we show that the E3 ubiquitin ligase MARCH1 impairs cellular insulin action by degrading cell surface INSR. Using a large-scale RNA interference screen, we identify MARCH1 as a negative regulator of INSR signalling. March1 loss-of-function enhances, and March1 overexpression impairs, hepatic insulin sensitivity in mice. MARCH1 ubiquitinates INSR to decrease cell surface INSR levels, but unlike other INSR ubiquitin ligases, MARCH1 acts in the basal state rather than after insulin stimulation. Thus, MARCH1 may help set the basal gain of insulin signalling. MARCH1 expression is increased in white adipose tissue of obese humans, suggesting that MARCH1 contributes to the pathophysiology of T2D and could be a new therapeutic target.
Project description:Mice overexpressing acylCoA:diacylglycerol (DAG) acyltransferase 2 in the liver (Liv-DGAT2) have been shown to have normal hepatic insulin responsiveness despite severe hepatic steatosis and increased hepatic triglyceride, diacylglycerol, and ceramide content, demonstrating a dissociation between hepatic steatosis and hepatic insulin resistance. This led us to reevaluate the role of DAG in causing hepatic insulin resistance in this mouse model of severe hepatic steatosis. Using hyperinsulinemic-euglycemic clamps, we studied insulin action in Liv-DGAT2 mice and their wild-type (WT) littermate controls. Here, we show that Liv-DGAT2 mice manifest severe hepatic insulin resistance as reflected by decreased suppression of endogenous glucose production (0.8 ± 41.8 vs. 87.7 ± 34.3% in WT mice, P < 0.01) during the clamps. Hepatic insulin resistance could be attributed to an almost 12-fold increase in hepatic DAG content (P < 0.01) resulting in a 3.6-fold increase in protein kinase C? (PKC?) activation (P < 0.01) and a subsequent 52% decrease in insulin-stimulated insulin receptor substrate 2 (IRS-2) tyrosine phosphorylation (P < 0.05), as well as a 64% decrease in fold increase pAkt/Akt ratio from basal conditions (P < 0.01). In contrast, hepatic insulin resistance in these mice was not associated with increased endoplasmic reticulum (ER) stress or inflammation. Importantly, hepatic insulin resistance in Liv-DGAT2 mice was independent of differences in body composition, energy expenditure, or food intake. In conclusion, these findings strengthen the link between hepatic steatosis and hepatic insulin resistance and support the hypothesis that DAG-induced PKC? activation plays a major role in nonalcoholic fatty liver disease (NAFLD)-associated hepatic insulin resistance.
Project description:Insulin resistance is associated with nonalcoholic fatty liver disease (NAFLD) and is a major factor in the pathogenesis of type 2 diabetes. The development of hepatic insulin resistance has been ascribed to multiple causes, including inflammation, endoplasmic reticulum (ER) stress, and accumulation of hepatocellular lipids in animal models of NAFLD. However, it is unknown whether these same cellular mechanisms link insulin resistance to hepatic steatosis in humans. To examine the cellular mechanisms that link hepatic steatosis to insulin resistance, we comprehensively assessed each of these pathways by using flash-frozen liver biopsies obtained from 37 obese, nondiabetic individuals and correlating key hepatic and plasma markers of inflammation, ER stress, and lipids with the homeostatic model assessment of insulin resistance index. We found that hepatic diacylglycerol (DAG) content in cytoplasmic lipid droplets was the best predictor of insulin resistance (R = 0.80, P < 0.001), and it was responsible for 64% of the variability in insulin sensitivity. Hepatic DAG content was also strongly correlated with activation of hepatic PKC? (R = 0.67, P < 0.001), which impairs insulin signaling. In contrast, there was no significant association between insulin resistance and other putative lipid metabolites or plasma or hepatic markers of inflammation. ER stress markers were only partly correlated with insulin resistance. In conclusion, these data show that hepatic DAG content in lipid droplets is the best predictor of insulin resistance in humans, and they support the hypothesis that NAFLD-associated hepatic insulin resistance is caused by an increase in hepatic DAG content, which results in activation of PKC?.
Project description:Insulin resistance (IR) and microRNAs (miRNAs), which regulate cell-to-cell communication between hepatocytes and hepatic stellate cells (HSCs), may intertwine in nonalcoholic fatty liver disease (NAFLD) pathogenesis. The aim of this study was to evaluate whether epigenetics and environmental factors interact to promote progressive NAFLD during IR. We examined the miRNA signature in insulin receptor haploinsufficient (InsR+/-) and wild-type (wt) HSCs by RNAseq (n = 4 per group). Then, we evaluated their impact in an IR-NASH (nonalcoholic steatohepatitis) model (InsR+/- mice fed standard or methionine choline deficient (MCD) diet, n = 10 per group) and in vitro. InsR+/- HSCs displayed 36 differentially expressed miRNAs (p < 0.05 vs. wt), whose expression was then analyzed in the liver of InsR+/- mice fed an MCD diet. We found that miR-101-3p negatively associated with both InsR+/- genotype and MCD (p < 0.05) and the histological spectrum of liver damage (p < 0.01). miR-101-3p was reduced in InsR+/- hepatocytes and HSCs and even more in InsR+/- cells exposed to insulin (0.33 µM) and fatty acids (0.25 mM), resembling the IR-NASH model. Conversely, insulin induced miR-101-3p expression in wt cells but not in InsR+/- ones (p < 0.05). In conclusion, IR combined with diet-induced liver injury favors miR-101-3p downregulation, which may promote progressive NAFLD through HSC and hepatocyte transdifferentiation and proliferation.
Project description:The pathophysiology of type 2 diabetes mellitus (T2D) is characterized by reduced or absent insulin receptor (INSR) responsiveness to its ligand, elevated hepatic glucose output and impaired glucose uptake in peripheral tissues, particularly skeletal muscle. Treatments to reduce hyperglycemia and reestablish normal insulin signaling are much sought after. Any agent which could be orally administered to restore INSR function, in an insulin-independent manner, would have major implications for the management of this global disease. We have discovered a non-peptidyl small molecule, adenosine, 5'-Se-methyl-5'-seleno-, 2',3'-diacetate [referred to as non-peptidyl compound #43 (NPC43)], which restores INSR signaling in the complete absence of insulin. Initial screening of numerous compounds in human HepG2 liver cells revealed that NPC43 significantly inhibited glucose production. The compound was potently anti-hyperglycemic and anti-hyperinsulinemic in vivo, in insulin-resistant T2D Leprdb/db mice, following either acute or chronic treatment by oral gavage and intraperitoneal injection, respectively. The compound acted at the level of INSR and activated it in both liver and skeletal muscle of Leprdb/db mice. In cell culture, the compound activated INSR in both liver and skeletal muscle cells; furthermore, it cooperated with insulin to depress glucose-6-phosphatase catalytic subunit (G6pc) expression and stimulate glucose uptake, respectively. Our results indicated that the compound directly interacted with INSR?, triggering appropriate phosphorylation and activation of the receptor and its downstream targets. Unlike insulin, NPC43 did not activate insulin-like growth factor 1 receptor in either liver or skeletal muscle. We believe this compound represents a potential oral and/or injectable insulin replacement therapy for diabetes and diseases associated with insulin resistance.
Project description:Hepatic lipid accumulation has been implicated in the development of insulin resistance, but translational evidence in humans is limited. We investigated the relationship between liver fat and tissue-specific insulin sensitivity in 133 obese subjects. Although the presence of hepatic steatosis in obese subjects was associated with hepatic, adipose tissue, and peripheral insulin resistance, we found that intrahepatic triglycerides were not strictly sufficient or essential for hepatic insulin resistance. Thus, to examine the molecular mechanisms that link hepatic steatosis to hepatic insulin resistance, we comprehensively analyzed liver biopsies from a subset of 29 subjects. Here, hepatic cytosolic diacylglycerol content, but not hepatic ceramide content, was increased in subjects with hepatic insulin resistance. Moreover, cytosolic diacylglycerols were strongly associated with hepatic PKC? activation, as reflected by PKC? translocation to the plasma membrane. These results demonstrate the relevance of hepatic diacylglycerol-induced PKC? activation in the pathogenesis of NAFLD-associated hepatic insulin resistance in humans.
Project description: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 11?-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 PKC? 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 PKC? activation. CONCLUSIONS:These findings are consistent with excess adipose glucocorticoid activity being a predisposing factor for the development of lipid (diacylglycerol-PKC?)-induced hepatic insulin resistance.
Project description:Met is the transmembrane tyrosine kinase cell surface receptor for hepatocyte growth factor (HGF) and is structurally related to the insulin receptor (INSR) tyrosine kinase. Here we report that the HGF-Met axis regulates metabolism by stimulating hepatic glucose uptake and suppressing hepatic glucose output. We show that Met is essential for an optimal hepatic insulin response by directly engaging INSR to form a Met-INSR hybrid complex, which culminates in a robust signal output. We also found that the HGF-Met system restores insulin responsiveness in a mouse model of insulin refractoriness. These results provide new insights into the molecular basis of hepatic insulin resistance and suggest that HGF may have therapeutic potential for type 2 diabetes in the clinical setting.
Project description:Obesity-related insulin resistance is associated with fatty liver, dyslipidemia, and low plasma adiponectin. Insulin resistance due to insulin receptor (INSR) dysfunction is associated with none of these, but when due to dysfunction of the downstream kinase AKT2 phenocopies obesity-related insulin resistance. We report 5 patients with SHORT syndrome and C-terminal mutations in PIK3R1, encoding the p85?/p55?/p50? subunits of PI3K, which act between INSR and AKT in insulin signaling. Four of 5 patients had extreme insulin resistance without dyslipidemia or hepatic steatosis. In 3 of these 4, plasma adiponectin was preserved, as in insulin receptor dysfunction. The fourth patient and her healthy mother had low plasma adiponectin associated with a potentially novel mutation, p.Asp231Ala, in adiponectin itself. Cells studied from one patient with the p.Tyr657X PIK3R1 mutation expressed abundant truncated PIK3R1 products and showed severely reduced insulin-stimulated association of mutant but not WT p85? with IRS1, but normal downstream signaling. In 3T3-L1 preadipocytes, mutant p85? overexpression attenuated insulin-induced AKT phosphorylation and adipocyte differentiation. Thus, PIK3R1 C-terminal mutations impair insulin signaling only in some cellular contexts and produce a subphenotype of insulin resistance resembling INSR dysfunction but unlike AKT2 dysfunction, implicating PI3K in the pathogenesis of key components of the metabolic syndrome.