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Introduction Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in industrialized nations (1). NAFLD is observed in nearly 100% of obese people with type 2 diabetes (T2D) [...], 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-ζ (PKCζ) and the consequent inhibition of insulin receptor (INSR) kinase activity. However, the molecular mechanism underlying PKCζinhibition of INSR kinase activity is unknown. Here, we used mass spectrometry to identify the phosphorylation site [Thr.sup.1160] as a PKCζ substrate in the functionally critical INSR kinase activation loop. We hypothesized that [Thr.sup.1160] 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 PKCζ in vitro. Furthermore, mice with a threonine-to-alanine mutation at the homologous residue [Thr.sup.1150] ([Insr.sup.T1150A] mice) were protected from high fat diet-induced hepatic insulin resistance. InsrTm0A 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 [Thr.sup.1160] phosphorylation and provide further mechanistic links between PKCζ and INSR in mediating NAFLD-induced hepatic insulin resistance.

Author(s): Alexander S. Banks [sup.1] , Fiona E. McAllister [sup.2] , João Paulo G. Camporez [sup.3] , Peter-James H. Zushin [sup.1] , Michael J. Jurczak [sup.3] , Dina Laznik-Bogoslavski [sup.4] [...], Blocking ERK/MAP kinases improves insulin sensitivity thorough a mechanism similar to the actions of the anti-diabetic thiazolidinediones drugs on PPAR[gamma]. Role of MEK/ERK pathway in glucose metabolism The nuclear receptor PPAR[gamma] is required for glucose metabolism and is a drug target in diabetes. Earlier work has suggested that PPAR[gamma] phosphorylation has an adverse effect on insulin sensitivity and that the kinase Cdk5 is the main kinase responsible for phosphorylation at this site. Now Bruce Spiegelman and colleagues report the paradoxical observation that insulin resistance is worsened in mice lacking Cdk5 in adipose tissue. The authors further show that Cdk5 suppresses the MEK/ERK signalling pathway that is central to the regulation of cell growth and proliferation, and that ERK directly phosphorylates PPAR[gamma]. MEK/ERK inhibitors can improve insulin resistance in obese wild-type and ob/ob mice. This work suggests that MEK/ERK inhibitors -- already approved for cancer treatment -- might also be effective against diabetes. Obesity-linked insulin resistance is a major precursor to the development of type 2 diabetes. Previous work has shown that phosphorylation of PPAR[gamma] (peroxisome proliferator-activated receptor [gamma]) at serine 273 by cyclin-dependent kinase 5 (Cdk5) stimulates diabetogenic gene expression in adipose tissues.sup.1. Inhibition of this modification is a key therapeutic mechanism for anti-diabetic drugs that bind PPAR[gamma], such as the thiazolidinediones and PPAR[gamma] partial agonists or non-agonists.sup.2. For a better understanding of the importance of this obesity-linked PPAR[gamma] phosphorylation, we created mice that ablated Cdk5 specifically in adipose tissues. These mice have both a paradoxical increase in PPAR[gamma] phosphorylation at serine 273 and worsened insulin resistance. Unbiased proteomic studies show that extracellular signal-regulated kinase (ERK) kinases are activated in these knockout animals. Here we show that ERK directly phosphorylates serine 273 of PPAR[gamma] in a robust manner and that Cdk5 suppresses ERKs through direct action on a novel site in MAP kinase/ERK kinase (MEK). Importantly, pharmacological inhibition of MEK and ERK markedly improves insulin resistance in both obese wild-type and ob/ob mice, and also completely reverses the deleterious effects of the Cdk5 ablation. These data show that an ERK/Cdk5 axis controls PPAR[gamma] function and suggest that MEK/ERK inhibitors may hold promise for the treatment of type 2 diabetes.

Obesity-linked insulin resistance is a major precursor to the development of type 2 diabetes. Previous work has shown that phosphorylation of PPARγ (peroxisome proliferator-activated receptor γ) at serine 273 by [...]

The prevalence of non-alcoholic fatty liver disease (NAFLD) ranges from 20-30% in the general population and up to 75-100% in obese individuals (1,2). NAFLD is considered one of the manifestations [...], Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome and the leading cause of chronic liver disease in the Western world. Twenty per cent of NAFLD individuals develop chronic hepatic inflammation (non-alcoholic steatohepatitis, NASH) associated with cirrhosis, portal hypertension and hepatocellular carcinoma, yet the causes of progression from NAFLD to NASH remain obscure. Here, we show that the NLRP6 and NLRP3 inflammasomes and the effector protein IL-18 negatively regulate NAFLD/NASH progression, as well as multiple aspects of metabolic syndrome via modulation of the gut microbiota. Different mouse models reveal that inflammasome-deficiency-associated changes in the configuration of the gut microbiota are associated with exacerbated hepatic steatosis and inflammation through influx of TLR4 and TLR9 agonists into the portal circulation, leading to enhanced hepatic tumour-necrosis factor (TNF)-α expression that drives NASH progression. Furthermore, co-housing of inflammasome-deficient mice with wild-type mice results in exacerbation of hepatic steatosis and obesity. Thus, altered interactions between the gut microbiota and the host, produced by defective NLRP3 and NLRP6 inflammasome sensing, may govern the rate of progression of multiple metabolic syndrome-associated abnormalities, highlighting the central role of the microbiota in the pathogenesis of heretofore seemingly unrelated systemic auto-inflammatory and metabolic disorders.

PPARγ is a member of the nuclear receptor family of transcription factors and is a dominant regulator of adipose cell differentiation and development (3,4). It is also the functioning receptor [...], PPARγ is the functioning receptor for the thiazolidinedione (TZD) class of antidiabetes drugs including rosiglitazone and pioglitazone (1). These drugs are full classical agonists for this nuclear receptor, but recent data have shown that many PPARγ-based drugs have a separate biochemical activity, blocking the obesity-linked phosphorylation of PPARγ by Cdk5 (ref. 2). Here we describe novel synthetic compounds that have a unique mode of binding to PPARγ,completely lack classical transcriptional agonism and block the Cdk5-mediated phosphorylation in cultured adipocytes and in insulin-resistant mice. Moreover, one such compound, SR1664, has potent antidiabetic activity while not causing the fluid retention and weight gain that are serious side effects of many of the PPARγ drugs. Unlike TZDs, SR1664 also does not interfere with bone formation in culture. These data illustrate that new classes of antidiabetes drugs can be developed by specifically targeting the Cdk5-mediated phosphorylation of PPARγ.

Low-carbohydrate, high-fat ketogenic diets (KD) have been suggested to be more effective in promoting weight loss than conventional caloric restriction, whereas their effect on hepatic glucose and lipid metabolism and the mechanisms by which they may promote weight loss remain controversial. The aim of this study was to explore the role of KD on liver and muscle insulin sensitivity, hepatic lipid metabolism, energy expenditure, and food intake. Using hyperinsulinemic-euglycemic clamps, we studied insulin action in mice fed a KD or regular chow (RC). Body composition was assessed by [sup.1]H magnetic resonance spectroscopy. Despite being 15% lighter (P < 0.001) than RC-fed mice because of a 17% increase in energy expenditure (P < 0.001), KD-fed mice manifested severe hepatic insulin resistance, as reflected by decreased suppression (0% vs. 100% in RC-fed mice, P < 0.01) of endogenous glucose production during the clamp. Hepatic insulin resistance could be attributed to a 350% increase in hepatic diacylglycerol content (P < 0.001), resulting in increased activation of PKC[epsilon] (P < 0.05) and decreased insulin receptor substrate-2 tyrosine phosphorylation (P < 0.01). Food intake was 56% (P < 0.001) lower in KD-fed mice, despite similar caloric intake, and could partly be attributed to a more than threefold increase (P < 0.05) in plasma N-acylphosphatidylethanolamine concentrations. In conclusion, despite preventing weight gain in mice, KD induces hepatic insulin resistance secondary to increased hepatic diacylglycerol content. Given the key role of nonalcoholic fatty liver disease in the development of type 2 diabetes and the widespread use of KD for the treatment of obesity, these results may have potentially important clinical implications. nonalcoholic fatty liver disease; weight loss; diacylglycerol; ceramide; protein kinase C[epsilon]; FGF21 doi: 10.1152/ajpendo.00361.2010.

Adipose tissue is a primary site for lipid storage containing trace amounts of glycogen. However, refeeding after a prolonged partial fast produces a marked transient spike in adipose glycogen, which dissipates in coordination with the initiation of lipid resynthesis. To further study the potential interplay between glycogen and lipid metabolism in adipose tissue, the aP2-PTG transgenic mouse line was utilized since it contains a 100- to 400-fold elevation of adipocyte glycogen levels that are mobilized upon fasting. To determine the fate of the released glucose 1-phosphate, a series of metabolic measurements were made. Basal and isoproterenol-stimulated lactate production in vitro was significantly increased in adipose tissue from transgenic animals. In parallel, basal and isoproterenol-induced release of nonesterified fatty acids (NEFAs) was significantly reduced in transgenic adipose tissue vs. control. Interestingly, glycerol release was unchanged between the genotypes, suggesting that enhanced triglyceride resynthesis was occurring in the transgenic tissue. Qualitatively similar results for NEFA and glycerol levels between wild-type and transgenic animals were obtained in vivo during fasting. Additionally, the physiological upregulation of the phosphoenolpyruvate carboxykinase cytosolic isoform (PEPCK-C) expression in adipose upon fasting was significantly blunted in transgenic mice. No changes in whole body metabolism were detected through indirect calorimetry. Yet weight loss following a weight gain/loss protocol was significantly impeded in the transgenic animals, indicating a further impairment in triglyceride mobilization. Cumulatively, these results support the notion that the adipocyte possesses a set point for glycogen, which is altered in response to nutritional cues, enabling the coordination of adipose glycogen turnover with lipid metabolism. glycogenolysis; fasting; lipolysis; protein phosphatase-1 doi: 10.1152/ajpendo.00741.2009.

Adipocytes express the rate-limiting enzymes required for glycogen metabolism and increase glycogen synthesis in response to insulin. However, the physiological function of adipocytic glycogen in vivo is unclear, clue in part to the low absolute levels and the apparent biophysical constraints of adipocyte morphology on glycogen accumulation. To further study the regulation of glycogen metabolism in adipose tissue, transgenic mice were generated that overexpressed the protein phosphatase-1 (PP1) glycogen-targeting subunit (PTG) driven by the adipocyte fatty acid binding protein (aP2) promoter. Exogenous PTG was detected in gonadal, perirenal, and brown fat depots, but it was not detected in any other tissue examined. PTG overexpression resulted in a modest redistribution of PPl to glycogen particles, corresponding to a threefold increase in the glycogen synthase activity ratio. Glycogen synthase protein levels were also increased twofold, resulting in a combined greater than sixfold enhancement of basal glycogen synthase specific activity. Adipocytic glycogen levels were increased 200- to 400-fold in transgenic animals, and this increase was maintained to 1 yr of age. In contrast, lipid metabolism in transgenic adipose tissue was not significantly altered, as assessed by lipogenic rates, weight gain on normal or high-fat diets, or circulating free fatty acid levels after a fast. However, circulating and adipocytic leptin levels were doubled in transgenic animals, whereas adiponectin expression was unchanged. Cumulatively, these data indicate that murine adipocytes are capable of storing far higher levels of glycogen than previously reported. Furthermore, these results were obtained by overexpression of an endogenous adipocytic protein, suggesting that mechanisms may exist in vivo to maintain adipocytic glycogen storage at a physiological set point. insulin; glycogen synthesis: lipogenesis; protein phosphatase-1; targeting subunit

Glycogen is the storage form of carbohydrate for virtually every organism from yeast to primates. Most mammalian tissues store glucose as glycogen, with the major depots located in muscle and liver. The French physiologist Claude Bernard first identified a starch-like substance in liver and muscle and coined the term glycogen, or 'sugar former,' in the 1850s. During the 150 years since its identification, researchers in the field of glycogen metabolism have made numerous discoveries that are now recognized as significant milestones in biochemistry and cell signaling. Even so, more questions remain, and studies continue to demonstrate the complexity of the regulation of glycogen metabolism. Under classical definitions, the functions of glycogen seem clear: muscle glycogen is degraded to generate ATP during increased energy demand, whereas hepatic glycogen is broken down for release of glucose into the bloodstream to supply other tissues. However, recent findings demonstrate that the roles of glycogen metabolism in energy sensing, integration of metabolic pathways, and coordination of cellular responses to hormonal stimuli are far more complex.

Author(s): Anila K. Madiraju [1, 2, 3]; Derek M. Erion [1, 2, 3]; Yasmeen Rahimi [1]; Xian-Man Zhang [1]; Demetrios T. Braddock [4]; Ronald A. Albright [4]; Brett J. Prigaro [...]