Type 2 diabetes mellitus (DM) is characterized by insulin resistance and pancreatic ␤ cell dysfunction. In high-risk subjects, the earliest detectable abnormality is insulin resistance in skeletal muscle. Impaired insulin-mediated signaling, gene expression, glycogen synthesis, and accumulation of intramyocellular triglycerides have all been linked with insulin resistance, but no specific defect responsible for insulin resistance and DM has been identified in humans. To identify genes potentially important in the pathogenesis of DM, we analyzed gene expression in skeletal muscle from healthy metabolically characterized nondiabetic (family history negative and positive for DM) and diabetic Mexican-American subjects. We demonstrate that insulin resistance and DM associate with reduced expression of multiple nuclear respiratory factor-1 (NRF-1)-dependent genes encoding key enzymes in oxidative metabolism and mitochondrial function. Although NRF-1 expression is decreased only in diabetic subjects, expression of both PPAR␥ coactivator 1-␣ and-␤ (PGC1-␣͞PPARGC1 and PGC1-␤͞PERC), coactivators of NRF-1 and PPAR␥-dependent transcription, is decreased in both diabetic subjects and family history-positive nondiabetic subjects. Decreased PGC1 expression may be responsible for decreased expression of NRF-dependent genes, leading to the metabolic disturbances characteristic of insulin resistance and DM.I nsulin resistance precedes and predicts the development of type 2 diabetes mellitus (DM) (1, 2). Defects in insulin signal transduction, gene expression, and muscle glycogen synthesis, and accumulation of intramyocellular triglycerides have all been identified as potential mediators of insulin resistance in high-risk individuals (1, 3-7). However, the molecular pathogenesis of DM remains unknown. Mouse data highlight the importance of glucose uptake into muscle but suggest a role for novel mechanisms, distinct from insulin signaling pathways (8). The importance of genetic risk factors is exemplified by the high concordance of DM in identical twins, the strong influence of family history and ethnicity on risk, and the identification of DNA sequence alterations in both rare and common forms of DM (9). Environmental factors, including obesity, inactivity, and aging, also play critical roles in DM risk. Because both genotype and environment converge to influence cellular function via gene and protein expression, we hypothesize that alterations in expression define a phenotype that parallels the metabolic evolution of DM and provides potential clues to pathogenesis. We used high-density oligonucleotide arrays to identify genes differentially expressed in skeletal muscle from nondiabetic and type 2 diabetic subjects. Because hyperglycemia per se can modulate expression, we also evaluated gene expression in insulin-resistant subjects at high risk for DM (''prediabetes'') on the basis of family history of DM and Mexican-American ethnicity (10). We demonstrate that prediabetic and diabetic muscle is characterized by decreased expressi...

In this proof-of-concept study, the administration of pioglitazone led to metabolic and histologic improvement in subjects with nonalcoholic steatohepatitis. Larger controlled trials of longer duration are warranted to assess the long-term clinical benefit of pioglitazone. (ClinicalTrials.gov number, NCT00227110 [ClinicalTrials.gov] .).

Increased intramyocellular lipid concentrations are thought to play a role in insulin resistance, but the precise nature of the lipid species that produce insulin resistance in human muscle are unknown. Ceramides, either generated via activation of sphingomyelinase or produced by de novo synthesis, induce insulin resistance in cultured cells by inhibitory effects on insulin signaling. The present study was undertaken to determine whether ceramides or other sphingolipids are increased in muscle from obese insulin-resistant subjects and to assess whether ceramide plays a role in the insulin resistance of Akt in human muscle. Lean insulinsensitive and obese insulin-resistant subjects (n ‫؍‬ 10 each) received euglycemic-hyperinsulinemic clamps with muscle biopsies basally and after 30, 45, or 60 min of insulin infusion. The rate of glucose infusion required to maintain euglycemia (reflecting glucose uptake) was reduced by >50%, as expected, in the obese subjects at each time point (P < 0.01). Under basal conditions, total muscle ceramide content was increased nearly twofold in the obese subjects (46 ؎ 9 vs. 25 ؎ 2 pmol/2 mg muscle, P < 0.05). All species of ceramides were increased similarly in the obese subjects; in contrast, no other sphingolipid was increased. Stimulation of Akt phosphorylation by insulin in the obese subjects was significantly reduced after 30 min (0.96 ؎ 0.11 vs. 1.84 ؎ 0.38 arbitrary units) or 45-60 min (0.68 ؎ 0.17 vs. 1.52 ؎ 0.26) of insulin infusion (P < 0.05 for both). Muscle ceramide content was significantly correlated with the plasma free fatty acid concentration (r ‫؍‬ 0.51, P < 0.05). We conclude that obesity is associated with increased intramyocellular ceramide content. This twofold increase in ceramide may be involved in the decrease in Akt phosphorylation observed after insulin infusion and could theoretically play a role in the reduced ability of insulin to stimulate glucose uptake in skeletal muscle from obese subjects.

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Acute elevations in free fatty acids (FFAs) stimulate insulin secretion, but prolonged lipid exposure impairs ␤-cell function in both in vitro studies and in vivo animal studies. In humans data are limited to short-term (<48 h) lipid infusion studies and have led to conflicting results. We examined insulin secretion and action during a 4-day lipid infusion in healthy normal glucose tolerant subjects with (FH؉ group, n ‫؍‬ 13) and without (control subjects, n ‫؍‬ 8) a family history of type 2 diabetes. Volunteers were admitted twice to the clinical research center and received, in random order, a lipid or saline infusion. On days 1 and 2, insulin and C-peptide concentration were measured as part of a metabolic profile after standardized mixed meals. Insulin secretion in response to glucose was assessed with a ؉125 mg/dl hyperglycemic clamp on day 3. On day 4, glucose turnover was measured with a euglycemic insulin clamp with [3-3 H]glucose. Day-long plasma FFA concentrations with lipid infusion were increased within the physiological range, to levels seen in type 2 diabetes (ϳ500 -800 mol/l). Lipid infusion had strikingly opposite effects on insulin secretion in the two groups. After mixed meals, day-long plasma C-peptide levels increased with lipid infusion in control subjects but decreased in the FH؉ group (؉28 vs. ؊30%, respectively, P < 0.01). During the hyperglycemic clamp, lipid infusion enhanced the insulin secretion rate (ISR) in control subjects but decreased it in the FH؉ group (first phase: ؉75 vs. ؊60%, P < 0.001; second phase: ؉25 vs. ؊35%, P < 0.04). When the ISR was adjusted for insulin resistance (ISR Rd ‫؍‬ ISR ، [1/R d ], where R d is the rate of insulin-stimulated glucose disposal), the inadequate ␤-cell response in the FH؉ group was even more evident. Although ISR Rd was not different between the two groups before lipid infusion, in the FH؉ group, lipid infusion reduced first-and second-phase ISR Rd to 25 and 42% of that in control subjects, respectively (both P < 0.001 vs. control subjects). Lipid infusion in the FH؉ group (but not in control subjects) also caused severe hepatic insulin resistance with an increase in basal endogenous glucose production (EGP), despite an elevation in fasting insulin levels, and impaired suppression of EGP to insulin. In summary, in individuals who are genetically predisposed to type 2 diabetes, a sustained physiological increase in plasma FFA impairs insulin secretion in response to mixed meals and to intravenous glucose, suggesting that in subjects at high risk of developing type 2 diabetes, ␤-cell lipotoxicity may play an important role in the progression from normal glucose tolerance to overt hyperglycemia.

The dose-response relationship between elevated plasma free fatty acid (FFA) levels and impaired insulin-mediated glucose disposal and insulin signaling was examined in 21 lean, healthy, normal glucose-tolerant subjects. Following a 4-h saline or Liposyn infusion at 30 (n ‫؍‬ 9), 60 (n ‫؍‬ 6), and 90 (n ‫؍‬ 6) ml/h, subjects received a 2-h euglycemic insulin (40 mU ⅐ m ؊2 ⅐ min ؊1 ) clamp. Basal plasma FFA concentration (ϳ440 mol/l) was increased to 695, 1,251, and 1,688 mol/l after 4 h of Liposyn infusion and resulted in a dose-dependent reduction in insulin-stimulated glucose disposal (R d ) by 22, 30, and 34%, respectively (all P < 0.05 vs. saline control). At the lowest lipid infusion rate (30 ml/h), insulin receptor and insulin receptor substrate (IRS)-1 tyrosine phosphorylation, phosphatidylinositol (PI) 3-kinase activity associated with IRS-1, and Akt serine phosphorylation were all significantly impaired (P < 0.05-0.01). The highest lipid infusion rate (90 ml/h) caused a further significant reduction in all insulin signaling events compared with the low-dose lipid infusion (P < 0.05-0.01) whereas the 60-ml/h lipid infusion caused an intermediate reduction in insulin signaling. However, about two-thirds of the maximal inhibition of insulin-stimulated glucose disposal already occurred at the rather modest increase in plasma FFA induced by the low-dose (30-ml/h) lipid infusion. Insulin-stimulated glucose disposal was inversely correlated with both the plasma FFA concentration after 4 h of lipid infusion (r ‫؍‬ ؊0.50, P ‫؍‬ 0.001) and the plasma FFA level during the last 30 min of the insulin clamp (r ‫؍‬ ؊0.54, P < 0.001). PI 3-kinase activity associated with IRS-1 correlated with insulin-stimulated glucose disposal (r ‫؍‬ 0.45, P < 0.01) and inversely with both the plasma FFA concentration after 4 h of lipid infusion (r ‫؍‬ ؊0.39, P ‫؍‬ 0.01) and during the last 30 min of the insulin clamp (r ‫؍‬ ؊0.43, P < 0.01). In summary, in skeletal muscle of lean, healthy subjects, a progressive increase in plasma FFA causes a dose-dependent inhibition of insulin-stimulated glucose disposal and insulin signaling. The inhibitory effect of plasma FFA was already significant following a rather modest increase in plasma FFA and develops at concentrations that are well within the physiological range (i.e., at plasma FFA levels observed in obesity and type 2 diabetes). Diabetes 54:1640 -1648, 2005

Aims/hypothesis. The recent discovery of two adiponectin receptors (AdipoR1 and AdipoR2) will improve our understanding of the molecular mechanisms underlying the insulin-sensitising effect of adiponectin. The aim of this study was to determine for the first time whether skeletal muscle AdipoR1 and/or AdipoR2 gene expression levels are associated with insulin resistance. Methods. Using RT-PCR and northern analysis we measured AdipoR1 and AdipoR2 gene expression in skeletal muscle from healthy Mexican Americans with normal glucose tolerance who had (n=8) or did not have (n=10) a family history of Type 2 diabetes. Results. Gene expression profiling indicated that the AdipoR1 and AdipoR2 isoforms are highly expressed in human skeletal muscle, unlike in mice where AdipoR2 expression was highest in the liver, and AdipoR1 was highest in skeletal muscle. In the study subjects, the expression levels of AdipoR1 (p=0.004) and AdipoR2 (p=0.04), as well as plasma adiponectin concentration (p=0.03) were lower in people with a family history of Type 2 diabetes than in those with no family history of the disease. Importantly, the expression levels of both receptors correlated positively with insulin sensitivity (r=0. 64, p=0.004 and r=0.47, p=0.048 respectively). Conclusions/interpretation. Collectively, these data indicate that both isoforms of the adiponectin receptor play a role in the insulin-sensitising effect of adiponectin.

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