Dysregulated lipid metabolism and inflammation are linked to the development of insulin resistance in obesity, and the intracellular accumulation of the sphingolipid ceramide has been implicated in these processes. Here, we explored the role of circulating ceramide on the pathogenesis of insulin resistance. Ceramide transported in LDL is elevated in the plasma of obese patients with type 2 diabetes and correlated with insulin resistance but not with the degree of obesity. Treating cultured myotubes with LDL containing ceramide promoted ceramide accrual in cells and was accompanied by reduced insulin-stimulated glucose uptake, Akt phosphorylation, and GLUT4 translocation compared with LDL deficient in ceramide. LDL-ceramide induced a proinflammatory response in cultured macrophages via toll-like receptor–dependent and –independent mechanisms. Finally, infusing LDL-ceramide into lean mice reduced insulin-stimulated glucose uptake, and this was due to impaired insulin action specifically in skeletal muscle. These newly identified roles of LDL-ceramide suggest that strategies aimed at reducing hepatic ceramide production or reducing ceramide packaging into lipoproteins may improve skeletal muscle insulin action.

Pancreatic β-cells couple the oxidation of glucose to the secretion of insulin. Apart from the canonical KATP-dependent glucose-stimulated insulin secretion (GSIS), there are important KATP-independent mechanisms involving both anaplerosis and mitochondrial GTP (mtGTP). How mtGTP that is trapped within the mitochondrial matrix regulates the cytosolic calcium increases that drive GSIS remains a mystery. Here we have investigated whether the mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK-M) is the GTPase linking hydrolysis of mtGTP made by succinyl-CoA synthetase (SCS-GTP) to an anaplerotic pathway producing phosphoenolpyruvate (PEP). Although cytosolic PEPCK (PEPCK-C) is absent, PEPCK-M message and protein were detected in INS-1 832/13 cells, rat islets, and mouse islets. PEPCK enzymatic activity is half that of primary hepatocytes and is localized exclusively to the mitochondria. Novel 13C-labeling strategies in INS-1 832/13 cells and islets measured substantial contribution of PEPCK-M to the synthesis of PEP. As high as 30% of PEP in INS-1 832/13 cells and 41% of PEP in rat islets came from PEPCK-M. The contribution of PEPCK-M to overall PEP synthesis more than tripled with glucose stimulation. Silencing the PEPCK-M gene completely inhibited GSIS underscoring its central role in mitochondrial metabolism-mediated insulin secretion. Given that mtGTP synthesized by SCS-GTP is an indicator of TCA flux that is crucial for GSIS, PEPCK-M is a strong candidate to link mtGTP synthesis with insulin release through anaplerotic PEP cycling.

Summary Peroxisome proliferator-activated receptor-gamma coactivator 1 beta (PGC-1β) is known to be a transcriptional coactivator for SREBP-1, the master regulator of hepatic lipogenesis. Here we evaluated the role of PGC-1β in the pathogenesis of fructose-induced insulin resistance by using an antisense oligonucletoide (ASO) to knockdown PGC-1 β in liver and adipose tissue. PGC-1 β ASO improved the metabolic phenotype induced by fructose feeding by reducing expression of SREBP-1 and downstream lipogenic genes in liver. PGC-1 β ASO also reversed hepatic insulin resistance induced by fructose in both basal and insulin stimulated states. Furthermore, PGC-1β ASO increased insulin-stimulated whole body glucose disposal due to a threefold increase in glucose uptake in white adipose tissue. These data support an important role for PGC-1 β in the pathogenesis of fructose-induced insulin resistance and suggest that PGC-1 β inhibition may be a novel therapeutic target for treatment of NAFLD, hypertriglyceridemia and insulin resistance associated with increased de novo lipogenesis.

Hepatic gluconeogenesis is a major contributing factor to hyperglycemia in the fasting and postprandial states in type 2 diabetes mellitus (T2DM). Because Sirtuin 1 (SirT1) induces hepatic gluconeogenesis during fasting through the induction of phosphoenolpyruvate carboxylase kinase (PEPCK), fructose-1,6-bisphosphatase (FBPase), and glucose-6-phosphatase (G6Pase) gene transcription, we hypothesized that reducing SirT1, by using an antisense oligonucleotide (ASO), would decrease fasting hyperglycemia in a rat model of T2DM. SirT1 ASO lowered both fasting glucose concentration and hepatic glucose production in the T2DM rat model. Whole body insulin sensitivity was also increased in the SirT1 ASO treated rats as reflected by a 25% increase in the glucose infusion rate required to maintain euglycemia during the hyperinsulinemiceuglycemic clamp and could entirely be attributed to increased suppression of hepatic glucose production by insulin. The reduction in basal and clamped rates of glucose production could in turn be attributed to decreased expression of PEPCK, FBPase, and G6Pase due to increased acetylation of signal transducer and activator of transcription 3 (STAT3), forkhead box O1 (FOXO1), and peroxisome proliferator-activated receptor-␥ coactivator 1␣ (PGC-1␣), known substrates of SirT1. In addition to the effects on glucose metabolism, SirT1 ASO decreased plasma total cholesterol, which was attributed to increased cholesterol uptake and export from the liver. These results indicate that inhibition of hepatic SirT1 may be an attractive approach for treatment of T2DM. gluconeogenesis ͉ glucose 6 phosphatase ͉ phosphoenolpyruvate carboxykinase ͉ type 2 diabetes mellitus ͉ hepatic insulin resistance T ype 2 diabetes mellitus (T2DM) is associated with an increased rate of hepatic glucose production that contributes to fasting hyperglycemia (1-3). Specifically, increased endogenous glucose production can be accounted for by increased rates of hepatic gluconeogenesis (4). Inhibition of hepatic gluconeogenesis has been shown to improve fasting plasma glucose levels and decrease endogenous glucose production in T2DM patients (5). Furthermore, inhibition of transcriptional gluconeogenic activators, such as forkhead box O1 (FOXO1), and peroxisome proliferator-activated receptor-␥ coactivator 1␣ (PGC-1␣), in turn improved hepatic insulin resistance in rodent models of diabetes (6, 7). Therefore inhibitors of gluconeogenesis are potentially excellent targets for treatment of poorly controlled T2DM.Sirtuin1 (SirT1) is a NAD ϩ -dependent deacetylase activated in response to fasting and caloric restriction (8). In the ␤-cells of the pancreas, SirT1 has been shown to increase insulin secretion through repression of uncoupling protein 2 (UCP2) (9). In adipose tissue, SirT1 has been shown to inhibit adipogenesis and decrease lipolysis through inhibition of peroxisome proliferator-activated receptor-␥ (PPAR␥) (10). In liver tissue, SirT1 activates gluconeogenesis transcription through deacetylation of PGC-1␣, FOXO1, and signa...

Calorie restriction (CR) is neuroprotective in