We examined the hypothesis that insulin resistance in skeletal muscle promotes the development of atherogenic dyslipidemia, associated with the metabolic syndrome, by altering the distribution pattern of postprandial energy storage. Following ingestion of two high carbohydrate mixed meals, net muscle glycogen synthesis was reduced by Ϸ60% in young, lean, insulin-resistant subjects compared with a similar cohort of age-weight-body mass indexactivity-matched, insulin-sensitive, control subjects. In contrast, hepatic de novo lipogenesis and hepatic triglyceride synthesis were both increased by >2-fold in the insulin-resistant subjects. These changes were associated with a 60% increase in plasma triglyceride concentrations and an Ϸ20% reduction in plasma high-density lipoprotein concentrations but no differences in plasma concentrations of TNF-␣, IL-6, adiponectin, resistin, retinol binding protein-4, or intraabdominal fat volume. These data demonstrate that insulin resistance in skeletal muscle, due to decreased muscle glycogen synthesis, can promote atherogenic dyslipidemia by changing the pattern of ingested carbohydrate away from skeletal muscle glycogen synthesis into hepatic de novo lipogenesis, resulting in an increase in plasma triglyceride concentrations and a reduction in plasma high-density lipoprotein concentrations. Furthermore, insulin resistance in these subjects was independent of changes in the plasma concentrations of TNF-␣, IL-6, highmolecular-weight adiponectin, resistin, retinol binding protein-4, or intraabdominal obesity, suggesting that these factors do not play a primary role in causing insulin resistance in the early stages of the metabolic syndrome.type 2 diabetes ͉ nonalcoholic fatty liver disease ͉ adipocytokines ͉ abdominal obesity ͉ atherogenic dyslipidemia T he metabolic syndrome is characterized by a clustering of risk factors for cardiovascular disease that include insulin resistance, abdominal obesity, atherogenic dyslipidemia, hypertension, hyperuricemia, a prothrombotic state, and a proinflammatory state (1, 2). The metabolic syndrome is estimated to afflict Ͼ50 million Americans, and approximately half of all Americans are predisposed to it (2). Individuals with the metabolic syndrome are at increased risk for the development of coronary heart disease and other diseases related to plaque buildup in artery walls, such as stroke and peripheral vascular disease, as well as type 2 diabetes mellitus (T2DM).Abdominal obesity and insulin resistance have each been hypothesized to be the primary factors underlying the metabolic syndrome; however, the biologic mechanisms linking these and other metabolic risk factors associated with the metabolic syndrome are not fully understood and appear to be complex.In this study we examined the hypothesis that insulin resistance in skeletal muscle may promote the development of atherogenic dyslipidemia by diverting ingested carbohydrate away from muscle glycogen storage and into hepatic de novo lipogenesis, resulting in hypertriglyceridemia. To exam...

OBJECTIVEMacrophage recruitment to adipose tissue is a reproducible feature of obesity. However, the events that result in chemokine production and macrophage recruitment to adipose tissue during states of energetic excess are not clear. Sirtuin 1 (SirT1) is an essential nutrient-sensing histone deacetylase, which is increased by caloric restriction and reduced by overfeeding. We discovered that SirT1 depletion causes anorexia by stimulating production of inflammatory factors in white adipose tissue and thus posit that decreases in SirT1 link overnutrition and adipose tissue inflammation.RESEARCH DESIGN AND METHODSWe used antisense oligonucleotides to reduce SirT1 to levels similar to those seen during overnutrition and studied SirT1-overexpressing transgenic mice and fat-specific SirT1 knockout animals. Finally, we analyzed subcutaneous adipose tissue biopsies from two independent cohorts of human subjects.RESULTSWe found that inducible or genetic reduction of SirT1 in vivo causes macrophage recruitment to adipose tissue, whereas overexpression of SirT1 prevents adipose tissue macrophage accumulation caused by chronic high-fat feeding. We also found that SirT1 expression in human subcutaneous fat is inversely related to adipose tissue macrophage infiltration.CONCLUSIONSReduction of adipose tissue SirT1 expression, which leads to histone hyperacetylation and ectopic inflammatory gene expression, is identified as a key regulatory component of macrophage influx into adipose tissue during overnutrition in rodents and humans. Our results suggest that SirT1 regulates adipose tissue inflammation by controlling the gain of proinflammatory transcription in response to inducers such as fatty acids, hypoxia, and endoplasmic reticulum stress.

Possible involvement of the ␣1 isoform of 5ЈAMP-activated protein kinase in oxidative stressstimulated glucose transport in skeletal muscle. Am J Physiol Endocrinol Metab 287: E166 -E173, 2004. First published March 16, 2004 10.1152/ajpendo.00487.2003.-Recent studies have suggested that 5ЈAMP-activated protein kinase (AMPK) is activated in response to metabolic stresses, such as contraction, hypoxia, and the inhibition of oxidative phosphorylation, which leads to insulin-independent glucose transport in skeletal muscle. In the present study, we hypothesized that acute oxidative stress increases the rate of glucose transport via an AMPK-mediated mechanism. When rat epitrochlearis muscles were isolated and incubated in vitro in Krebs buffer containing the oxidative agent H2O2, AMPK␣1 activity increased in a time-and dose-dependent manner, whereas AMPK␣2 activity remained unchanged. The activation of AMPK␣1 was associated with phosphorylation of AMPK Thr 172 , suggesting that an upstream kinase is involved in the activation process. H2O2-induced AMPK␣1 activation was blocked in the presence of the antioxidant N-acetyl-L-cysteine (NAC), and H 2O2 significantly increased the ratio of oxidized glutathione to glutathione (GSSG/GSH) concentrations, a sensitive marker of oxidative stress. H2O2 did not cause an increase in the conventional parameters of AMPK activation, such as AMP and AMP/ATP. H2O2 increased 3-O-methyl-D-glucose transport, and this increase was partially, but significantly, blocked in the presence of NAC. Results were similar when the muscles were incubated in a superoxide-generating system using hypoxanthine and xanthine oxidase. Taken together, our data suggest that acute oxidative stress activates AMPK␣1 in skeletal muscle via an AMP-independent mechanism and leads to an increase in the rate of glucose transport, at least in part, via an AMPK␣1-mediated mechanism.

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...

A number of studies have demonstrated that insulin resistance in the skeletal muscle plays a pivotal role in the insulin resistance associated with obesity and type 2 diabetes. A decrease in GLUT4 translocation from the intracellular pool to the plasma membranes in skeletal muscles has been implicated as a possible cause of insulin resistance. Herein, we examined the effects of an insulin-sensitizing drug, troglitazone (TGZ), on glucose uptake and the translocation of GLUT4 in L6 myotubes. The prolonged exposure (24 h) of L6 myotubes to TGZ (10 ؊5 mol/l) caused a substantial increase in the 2-deoxy-[ 3 H]D-glucose (2-DG) uptake without changing the total amount of the glucose transporters GLUT4, GLUT1, and GLUT3. The TGZ-induced 2-DG uptake was completely abolished by cytochalasin-B (10 mol/l). The ability of TGZ to translocate GLUT4 from light microsomes to the crude plasma membranes was greater than that of insulin. Both cycloheximide treatment (3.5 ؋ 10 ؊6 mol/l) and the removal of TGZ by washing reversed the 2-DG uptake to the basal level. Moreover, insulin did not enhance the TGZ-induced 2-DG uptake additively. The TGZ-induced 2-DG uptake was only partially reversed by wortmannin to 80%, and TGZ did not change the expression and the phosphorylation of protein kinase B; the expression of protein kinase C (PKC)-, PKC-␤2, and PKC-; or 5AMP-activated protein kinase activity. ␣-Tocopherol, which has a molecular structure similar to that of TGZ, did not increase 2-DG uptake. We conclude that the glucose transport in L6 myotubes exposed to TGZ for 24 h is the result of an increased translocation of GLUT4. The present results imply that the effects of troglitazone on GLUT4 translocation may include a new mechanism for improving glucose transport in skeletal muscle.

Summary In patients with poorly controlled type 2 diabetes mellitus (T2DM), hepatic insulin resistance and increased gluconeogenesis contributes to fasting and postprandial hyperglycemia. Since CREB is a key regulator of gluconeogenic gene expression, we hypothesized that decreasing hepatic CREB expression would reduce fasting hyperglycemia in rodent models of T2DM. In order to test this hypothesis, we used a CREB-specific antisense oligonucleotide (ASO) to knock down CREB expression in liver. CREB ASO treatment dramatically reduced fasting plasma glucose concentrations in ZDF rats, ob/ob mice and a STZ-treated high-fat fed rat model of T2DM. Surprisingly, CREB ASO treatment also decreased plasma cholesterol and triglyceride concentrations, as well as hepatic triglyceride content due to decreases in hepatic lipogenesis. These results suggest that CREB is an attractive therapeutic target for correcting both hepatic insulin resistance and dyslipidemia associated with NAFLD and T2DM by down regulation of both lipogenic and gluconeogenic gene expression.

By 2030, nearly half of Americans will have nonalcoholic fatty liver disease. In part, this epidemic is fueled by the increasing consumption of caloric sweeteners coupled with an innate capacity to convert sugar into fat via hepatic de novo lipogenesis. In addition to serving as substrates, monosaccharides also increase the expression of key enzymes involved in de novo lipogenesis via the carbohydrate response element-binding protein (ChREBP). To determine whether ChREBP is a potential therapeutic target, we decreased hepatic expression of ChREBP with a specific antisense oligonucleotide (ASO) in male Sprague-Dawley rats fed either a high-fructose or high-fat diet. ChREBP ASO treatment decreased plasma triglyceride concentrations compared with control ASO treatment in both diet groups. The reduction was more pronounced in the fructose-fed group and attributed to decreased hepatic expression of ACC2, FAS, SCD1, and MTTP and a decrease in the rate of hepatic triglyceride secretion. This was associated with an increase in insulin-stimulated peripheral glucose uptake, as assessed by the hyperinsulinemic-euglycemic clamp. In contrast, ChREBP ASO did not alter hepatic lipid content or hepatic insulin sensitivity. Interestingly, fructose-fed rats treated with ChREBP ASO had increased plasma uric acid, alanine transaminase, and aspartate aminotransferase concentrations. This was associated with decreased expression of fructose aldolase and fructokinase, reminiscent of inherited disorders of fructose metabolism. In summary, these studies suggest that targeting ChREBP may prevent fructose-induced hypertriglyceridemia but without the improvements in hepatic steatosis and hepatic insulin responsiveness.