Search

Objective: Forkhead box O (FoxO) transcription factors represent evolutionarily conserved targets of insulin signaling, regulating metabolism and cellular differentiation in response to changes in nutrient availability. Although the FoxO1 isoform is known to play a key role in adipogenesis, its physiological role in differentiated adipose tissue remains unclear.Research Design and Methods: In this study, we analyzed the phenotype of FoxO1 haploinsufficient mice to investigate the role of FoxO1 in high-fat diet-induced obesity and adipose tissue metabolism.Results: We showed that reduced FoxO1 expression protects mice against obesity-related insulin resistance with marked improvement not only in hepatic insulin sensitivity but also in skeletal muscle insulin action. FoxO1 haploinsufficiency also resulted in increased peroxisome proliferator-activated receptor (PPAR)gamma gene expression in adipose tissue, with enhanced expression of PPARgamma target genes known to influence metabolism. Moreover, treatment of mice with the PPARgamma agonist rosiglitazone caused a greater improvement in in vivo insulin sensitivity in FoxO1 haploinsufficient animals, including reductions in circulating proinflammatory cytokines.Conclusions: These findings indicate that FoxO1 proteins negatively regulate insulin action and that their effect may be explained, at least in part, by inhibition of PPARgamma function. [ABSTRACT FROM AUTHOR]

Objective: Many prevalent diseases of advanced societies, such as obesity-induced type 2 diabetes, are linked to indolent mononuclear cell-dependent inflammation. We previously proposed that blockade of alpha4 integrin signaling can inhibit inflammation while limiting mechanism-based toxicities of loss of alpha4 function. Thus, we hypothesized that mice bearing an alpha4(Y991A) mutation, which blocks signaling, would be protected from development of high-fat diet-induced insulin resistance.Research Design and Methods: Six- to eight-week-old wild-type and alpha4(Y991A) C57Bl/6 male mice were placed on either a high-fat diet that derived 60% calories from lipids or a chow diet. Metabolic testing was performed after 16-22 weeks of diet.Results: Alpha4(Y991A) mice were protected from development of high-fat diet-induced insulin resistance. This protection was conferred on wild-type mice by alpha4(Y991A) bone marrow transplantation. In the reverse experiment, wild-type bone marrow renders high-fat diet-fed alpha4(Y991A) acceptor animals insulin resistant. Furthermore, fat-fed alpha4(Y991A) mice showed a dramatic reduction of monocyte/macrophages in adipose tissue. This reduction was due to reduced monocyte/macrophage migration rather than reduced monocyte chemoattractant protein-1 production.Conclusions: Alpha4 integrins contribute to the development of HFD-induced insulin resistance by mediating the trafficking of monocytes into adipose tissue; hence, blockade of alpha4 integrin signaling can prevent the development of obesity-induced insulin resistance. [ABSTRACT FROM AUTHOR]

Troglitazone and metformin lower glucose levels in diabetic patients without increasing plasma insulin levels. We compared the insulin sparing actions of these two agents and their effects on insulin sensitivity and insulin secretion in 20 type 2 diabetic patients. To avoid the confounding effect of improved glycemic control on insulin action and secretion, patients were first rendered euglycemic with 4 weeks of continuous subcutaneous insulin infusion (CSII) before randomization to CSII plus troglitazone (n = 10) or CSII plus metformin (n = 10); euglycemia was maintained for another 6-7 weeks. Insulin sensitivity was assessed by a hyperinsulinemic-euglycemic clamp 1) at baseline, 2) after 4 weeks of CSII, and 3) after CSII plus either troglitazone or metformin. The 24-h glucose, insulin, and C-peptide profiles were performed on the day before the second and third glucose clamps. Good glycemic control was achieved with CSII alone and was maintained with CSII plus an oral agent (mean 24-h glucose: troglitazone, 6.2+/-0.6 mmol/l; metformin, 6.2 +/-0.3 mmol/l). Insulin requirements decreased 53% with troglitazone compared with CSII alone (48+/-4 vs. 102+/-13 U/day, P < 0.001), but only 31% with metformin (76+/-13 vs. 110+/-18 U/day, P < 0.005). The 24-h C-peptide profiles were similar. Normal fasting hepatic glucose output was maintained with both agents despite lower insulin levels than on CSII alone. Insulin sensitivity did not change significantly with CSII alone or with CSII plus metformin, but improved 29% with CSII plus troglitazone (P < 0.005 vs. CSII alone) and was then 45% higher than in the CSII plus metformin patients (P < 0.005). In conclusion, metformin has no effect on insulin-stimulated glucose disposal independent of glycemic control in type 2 diabetes. Troglitazone (600 mg/day) has greater insulin-sparing effects than metformin (1,700 mg/day) in CSII-treated euglycemic patients. This is probably explained by the peripheral tissue insulin-sensitizing effects of troglitazone. [ABSTRACT FROM AUTHOR]

The composition of the gastrointestinal microbiota and associated metabolites changes dramatically with diet and the development of obesity. Although many correlations have been described, specific mechanistic links between these changes and glucose homeostasis remain to be defined. Here we show that blood and intestinal levels of the microbiota-produced N- formyl peptide, formyl-methionyl-leucyl-phenylalanine, are elevated in high-fat diet-induced obese mice. Genetic or pharmacological inhibition of the N- formyl peptide receptor Fpr1 leads to increased insulin levels and improved glucose tolerance, dependent upon glucagon-like peptide 1. Obese Fpr1 knockout mice also display an altered microbiome, exemplifying the dynamic relationship between host metabolism and microbiota. Overall, we describe a new mechanism by which the gut microbiota can modulate glucose metabolism, providing a potential approach for the treatment of metabolic disease., (© 2019 by the American Diabetes Association.)

The intestinal microbiome can regulate host energy homeostasis and the development of metabolic disease. Here we identify GPR43, a receptor for bacterially produced short-chain fatty acids (SCFAs), as a modulator of microbiota-host interaction. β-Cell expression of GPR43 and serum levels of acetate, an endogenous SCFA, are increased with a high-fat diet (HFD). HFD-fed GPR43 knockout (KO) mice develop glucose intolerance due to a defect in insulin secretion. In vitro treatment of isolated murine islets, human islets, and Min6 cells with (S)-2-(4-chlorophenyl)-3,3-dimethyl-N-(5-phenylthiazol-2-yl)butanamide (PA), a specific agonist of GPR43, increased intracellular inositol triphosphate and Ca(2+) levels, and potentiated insulin secretion in a GPR43-, Gαq-, and phospholipase C-dependent manner. In addition, KO mice fed an HFD displayed reduced β-cell mass and expression of differentiation genes, and the treatment of Min6 cells with PA increased β-cell proliferation and gene expression. Together these findings identify GPR43 as a potential target for therapeutic intervention., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

The current dogma is that obesity-associated hepatic inflammation is due to increased Kupffer cell (KC) activation. However, recruited hepatic macrophages (RHMs) were recently shown to represent a sizable liver macrophage population in the context of obesity. Therefore, we assessed whether KCs and RHMs, or both, represent the major liver inflammatory cell type in obesity. We used a combination of in vivo macrophage tracking methodologies and adoptive transfer techniques in which KCs and RHMs are differentially labeled with fluorescent markers. With these approaches, the inflammatory phenotype of these distinct macrophage populations was determined under lean and obese conditions. In vivo macrophage tracking revealed an approximately sixfold higher number of RHMs in obese mice than in lean mice, whereas the number of KCs was comparable. In addition, RHMs comprised smaller size and immature, monocyte-derived cells compared with KCs. Furthermore, RHMs from obese mice were more inflamed and expressed higher levels of tumor necrosis factor-α and interleukin-6 than RHMs from lean mice. A comparison of the MCP-1/C-C chemokine receptor type 2 (CCR2) chemokine system between the two cell types showed that the ligand (MCP-1) is more highly expressed in KCs than in RHMs, whereas CCR2 expression is approximately fivefold greater in RHMs. We conclude that KCs can participate in obesity-induced inflammation by causing the recruitment of RHMs, which are distinct from KCs and are not precursors to KCs. These RHMs then enhance the severity of obesity-induced inflammation and hepatic insulin resistance., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Gastrointestinal bypass surgeries that result in rerouting and subsequent exclusion of nutrients from the duodenum appear to rapidly alleviate hyperglycemia and hyperinsulinemia independent of weight loss. While the mechanism(s) responsible for normalization of glucose homeostasis remains to be fully elucidated, this rapid normalization coupled with the well-known effects of vagal inputs into glucose homeostasis suggests a neurohormonally mediated mechanism. Our results show that duodenal bypass surgery on obese, insulin-resistant Zucker fa/fa rats restored insulin sensitivity in both liver and peripheral tissues independent of body weight. Restoration of normoglycemia was attributable to an enhancement in key insulin-signaling molecules, including insulin receptor substrate-2, and substrate metabolism through a multifaceted mechanism involving activation of AMP-activated protein kinase and downregulation of key regulatory genes involved in both lipid and glucose metabolism. Importantly, while central nervous system-derived vagal nerves were not essential for restoration of insulin sensitivity, rapid normalization in hepatic gluconeogenic capacity and basal hepatic glucose production required intact vagal innervation. Lastly, duodenal bypass surgery selectively altered the tissue concentration of intestinally derived glucoregulatory hormone peptides in a segment-specific manner. The present data highlight and support the significance of vagal inputs and intestinal hormone peptides toward normalization of glucose and lipid homeostasis after duodenal bypass surgery.

Obesity is associated with insulin resistance, a major risk factor for type 2 diabetes and cardiovascular disease. However, not all obese individuals are insulin resistant, which confounds our understanding of the mechanistic link between these conditions. We conducted transcriptome analyses on 835 obese subjects with mean BMI of 48.8, on which we have previously reported genetic associations of gene expression. Here, we selected ~320 nondiabetic (HbA(1c) <7.0) subjects and further stratified the cohort into insulin-resistant versus insulin-sensitive subgroups based on homeostasis model assessment-insulin resistance. An unsupervised informatics analysis revealed that immune response and inflammation-related genes were significantly downregulated in the omental adipose tissue of obese individuals with extreme insulin sensitivity and, to a much lesser extent, in subcutaneous adipose tissue. In contrast, genes related to β-oxidation and the citric acid cycle were relatively overexpressed in adipose of insulin-sensitive patients. These observations were verified by querying an independent cohort of our published dataset of 37 subjects whose subcutaneous adipose tissue was sampled before and after treatment with thiazolidinediones. Whereas the immune response and inflammation pathway genes were downregulated by thiazolidinedione treatment, β-oxidation and citric acid cycle genes were upregulated. This work highlights the critical role that omental adipose inflammatory pathways might play in the pathophysiology of insulin resistance, independent of body weight.

Macrophage-mediated inflammation is a key component of insulin resistance; however, the initial events of monocyte migration to become tissue macrophages remain poorly understood. We report a new method to quantitate in vivo macrophage tracking (i.e., blood monocytes from donor mice) labeled ex vivo with fluorescent PKH26 dye and injected into recipient mice. Labeled monocytes appear as adipose, liver, and splenic macrophages, peaking in 1-2 days. When CCR2 KO monocytes are injected into wild-type (WT) recipients, or WT monocytes given to MCP-1 KO recipients, adipose tissue macrophage (ATM) accumulation is reduced by ~40%, whereas hepatic macrophage content is decreased by ~80%. Using WT donor cells, ATM accumulation is several-fold greater in obese recipient mice compared with lean mice, regardless of the source of donor monocytes. After their appearance in adipose tissue, ATMs progressively polarize from the M2- to the M1-like state in obesity. In summary, the CCR2/MCP-1 system is a contributory factor to monocyte migration into adipose tissue and is the dominant signal controlling the appearance of recruited macrophages in the liver. Monocytes from obese mice are not programmed to become inflammatory ATMs but rather the increased proinflammatory ATM accumulation in obesity is in response to tissue signals.

Objective: Macrophage 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 Methods: We 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., Results: We 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., Conclusions: Reduction 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.

Increased accumulation of fatty acids and their derivatives can impair insulin-stimulated glucose disposal by skeletal muscle. To characterize the nature of the defects in lipid metabolism and to evaluate the effects of thiazolidinedione treatment, we analyzed the levels of triacylglycerol, long-chain fatty acyl-coA, malonyl-CoA, fatty acid oxidation, AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), malonyl-CoA decarboxylase, and fatty acid transport proteins in muscle biopsies from nondiabetic lean, obese, and type 2 subjects before and after an euglycemic-hyperinsulinemic clamp as well as pre-and post-3-month rosiglitazone treatment. We observed that low AMPK and high ACC activities resulted in elevation of malonyl-CoA levels and lower fatty acid oxidation rates. These conditions, along with the basal higher expression levels of fatty acid transporters, led accumulation of long-chain fatty acyl-coA and triacylglycerol in insulin-resistant muscle. During the insulin infusion, muscle fatty acid oxidation was reduced to a greater extent in the lean compared with the insulin-resistant subjects. In contrast, isolated muscle mitochondria from the type 2 subjects exhibited a greater rate of fatty acid oxidation compared with the lean group. All of these abnormalities in the type 2 diabetic group were reversed by rosiglitazone treatment. In conclusion, these studies have shown that elevated malonyl-CoA levels and decreased fatty acid oxidation are key abnormalities in insulin-resistant muscle, and, in type 2 diabetic patients, thiazolidinedione treatment can reverse these abnormalities.

Insulin resistance is predominantly characterized by decreased insulin-stimulated glucose uptake into skeletal muscle. In the current study, we have assessed various aspects of the phosphatidylinositol (PI) 3-kinase pathway in skeletal muscle biopsies obtained from normal, obese nondiabetic, and type 2 diabetic subjects, before and after a 5-h insulin infusion. We found a highly significant inverse correlation between in vivo insulin sensitivity (as measured by the glucose infusion rate) and increased protein expression of p85/55/50, protein kinase C (PKC)-theta activity, levels of pSer307 insulin receptor substrate (IRS)-1 and p-Jun NH2-terminal kinase (JNK)-1, and myosin heavy chain IIx fibers. Increased basal phosphorylation of Ser307 IRS-1 in the obese and type 2 diabetic subjects corresponds with decrease in insulin-stimulated IRS-1 tyrosine phosphorylation, PI 3-kinase activity, and insulin-induced activation of Akt and, more prominently, PKC-zeta/lambda. In summary, increased expression of the PI 3-kinase adaptor subunits p85/55/50, as well as increased activity of the proinflammatory kinases JNK-1, PKC-theta, and, to a lesser extent, inhibitor of kappaB kinase-beta, are associated with increased basal Ser307 IRS-1 phosphorylation and decreased PI 3-kinase activity and may follow a common pathway to attenuate in vivo insulin sensitivity in insulin-resistant subjects. These findings demonstrate interacting mechanisms that can lead to impaired insulin-stimulated PI 3-kinase activity in skeletal muscle from obese and type 2 diabetic subjects.

In this study, we investigated the chronic in vivo effect of adiponectin on insulin sensitivity and glucose metabolism by overexpressing the adiponectin protein in male Wistar rats using intravenous administration of an adenovirus (Adv-Adipo). Virally infected liver secreted adiponectin as high and low molecular weight complexes. After 7 days of physiological or supraphysiological hyperadiponectinemia, the animals displayed enhanced insulin sensitivity during the glucose tolerance and insulin tolerance tests. Glucose clamp studies performed at submaximal and maximal insulin infusion rates (4 and 25 mU x kg(-1) x min(-1), respectively) also demonstrated increased insulin sensitivity in Adv-Adipo animals, with the insulin-stimulated glucose disposal rate being increased by 20-67%. In contrast, insulin's effect on the suppression of hepatic glucose output and plasma free fatty acid levels was not enhanced in Adv-Adipo rats compared with controls, suggesting that high levels of adiponectin expression in the liver may lead to a local desensitization. Consistent with the clamp data, the activation of AMP-activated protein kinase was significantly enhanced in skeletal muscle (by 50%) but not in liver. One interesting finding was that in male Wistar rats, both AdipoR1 and AdipoR2 expression levels were higher in skeletal muscle than in liver, as it is the case in humans. These results indicate that chronic adiponectin treatment enhances insulin sensitivity and could serve as a therapy for human insulin resistance.

The mitochondrial Na(+)-Ca(2+) exchanger (mNCE) mediates efflux of Ca(2+) from mitochondria in exchange for influx of Na(+). We show that inhibition of the mNCE enhances mitochondrial oxidative metabolism and increases glucose-stimulated insulin secretion in rat islets and INS-1 cells. The benzothiazepine CGP37157 inhibited mNCE activity in INS-1 cells (50% inhibition at IC(50) = 1.5 micro mol/l) and increased the glucose-induced rise in mitochondrial Ca(2+) ([Ca(2+)](m)) 2.1 times. Cellular ATP content was increased by 13% in INS-1 cells and by 49% in rat islets by CGP37157 (1 micro mol/l). Krebs cycle flux was also stimulated by CGP37157 when glucose was present. Insulin secretion was increased in a glucose-dependent manner by CGP37157 in both INS-1 cells and islets. In islets, CGP37157 increased insulin secretion dose dependently (half-maximal efficacy at EC(50) = 0.06 micro mol/l) at 8 mmol/l glucose and shifted the glucose dose response curve to the left. In perifused islets, mNCE inhibition had no effect on insulin secretion at 2.8 mmol/l glucose but increased insulin secretion by 46% at 11 mmol/l glucose. The effects of CGP37157 could not be attributed to interactions with the plasma membrane sodium calcium exchanger, L-type calcium channels, ATP-sensitive K(+) channels, or [Ca(2+)](m) uniporter. In hyperglycemic clamp studies of Wistar rats, CGP37157 increased plasma insulin and C-peptide levels only during the hyperglycemic phase of the study. These results illustrate the potential utility of agents that affect mitochondrial metabolism as novel insulin secretagogues.

Despite extensive studies implicating tumor necrosis factor (TNF)-alpha as a contributing cause of insulin resistance, the mechanism(s) by which TNF-alpha alters energy metabolism in vivo and the tissue specificity of TNF-alpha action are unclear. Here, we investigated the effects of TNF-alpha infusion on gene expression and energy metabolism in adult rats. A 1-day TNF-alpha treatment decreased overall insulin sensitivity and caused a 70% increase (P = 0.005) in plasma levels of free fatty acids (FFAs) and a 46% decrease (P = 0.01) in ACRP30. A 4-day TNF-alpha infusion caused insulin resistance and significant elevation of plasma levels of FFAs and triglycerides and reduction of ACRP30. Plasma glucose concentration was not altered following TNF-alpha infusion for up to 4 days. As revealed by oligonucleotide microarrays, TNF-alpha evoked major and rapid changes in adipocyte gene expression, favoring FFA release and cytokine production, and fewer changes in liver gene expression, but favoring FFA and cholesterol synthesis and VLDL production. There was only a moderate repressive effect on skeletal muscle gene expression. We demonstrate that TNF-alpha antagonizes the actions of insulin, at least in part, through regulation of adipocyte gene expression including reduction in ACRP30 mRNA and induction of lipolysis resulting in increased plasma FFAs. TNF-alpha later alters systemic energy homeostasis that closely resembles the insulin resistance phenotype. Our data suggest that blockade of TNF-alpha action in adipose tissue may prevent TNF-alpha-induced insulin resistance in vivo.

The insulin-sensitizing effects of thiazolidinediones are thought to be mediated through peroxisome proliferator-activated receptor-gamma, a nuclear receptor that is highly abundant in adipose tissue. It has been reported that adipocytes secrete a variety of proteins, including tumor necrosis factor-alpha, resistin, plasminogen activator inhibitor-1, and adiponectin. Adiponectin is a fat cell-secreted protein that has been reported to increase fat oxidation and improve insulin sensitivity. Our aim was to study the effects of troglitazone on adiponectin levels in lean, obese, and diabetic subjects. Ten diabetic and 17 nondiabetic subjects (8 lean, BMI <27 kg/m(2) and 9 obese, BMI >27 kg/m(2)) participated in the study. All subjects underwent an 80 mU. m(-2). min(-1) hyperinsulinemic-euglycemic glucose clamp before and after 3 months' treatment with the thiazolidinedione (TZD) troglitazone (600 mg/day). Fasting plasma glucose significantly decreased in the diabetic group after 12 weeks of treatment compared with baseline (9.1 +/- 0.9 vs. 11.1 +/- 0.9 mmol/l, P < 0.005) but was unchanged in the lean and obese subjects. Fasting insulin for the entire group was significantly lower than baseline (P = 0.02) after treatment. At baseline, glucose disposal rate (R(d)) was lower in the diabetic subjects (3.4 +/- 0.5 mg. kg(-1). min(-1)) than in the lean (12.3 +/- 0.4) or obese subjects (6.7 +/- 0.7) (P < 0.001 for both) and was significantly improved in the diabetic and obese groups (P < 0.05) after treatment, and it remained unchanged in the lean subjects. Baseline adiponectin levels were significantly lower in the diabetic than the lean subjects (9.0 +/- 1.7 vs. 16.7 +/- 2.7 micro g/ml, P = 0.03) and rose uniformly in all subjects (12.2 +/- 2.3 vs. 25.7 +/- 2.6 micro g/ml, P < 10(-4)) after treatment, with no significant difference detected among the three groups. During the glucose clamps, adiponectin levels were suppressed below basal levels in all groups (10.2 +/- 2.3 vs. 12.2 +/- 2.3 micro g/ml, P < 0.01). Adiponectin levels correlated with R(d) (r = 0.46, P = 0.016) and HDL cholesterol levels (r = 0.59, P < 0.001) and negatively correlated with fasting insulin (r = -0.39, P = 0.042) and plasma triglyceride (r = -0.61, P < 0.001). Our findings show that TZD treatment increased adiponectin levels in all subjects, including normal subjects in which no other effects of TZDs are observed. Insulin also appears to suppress adiponectin levels. We have confirmed these results in normal rats. These findings suggest that adiponectin can be regulated by obesity, diabetes, TZDs, and insulin, and it may play a physiologic role in enhancing insulin sensitivity.

Elevation of plasma nonesterified fatty acid (NEFA) levels has been shown in various studies to induce peripheral tissue insulin resistance and impair the suppression of endogenous glucose production (EGP). These studies have been conducted predominantly in men. We compared the effects of elevated plasma NEFA levels on basal and insulin-stimulated glucose metabolism in 8 normal women (age 42 +/- 8 years [mean +/- SD], BMI 25 +/- 3 kg/m(2)) and 10 normal men (35 +/- 6 years, 24 +/- 3 kg/m(2)). Each subject underwent two 5-h 80 mU. m(-2). min(-1) hyperinsulinemic-euglycemic clamps with measurement of glucose kinetics (intravenous [3-(3)H]glucose) and substrate oxidation. Plasma NEFA levels were elevated in one study for 3 h before and during the clamp ( approximately 1 mmol/l in both groups) by infusion of 20% Intralipid (60 ml/h) and heparin (900 U/h). In the control studies, the men and women had similar insulin-stimulated glucose disposal rates (R(d)) and substrate oxidation rates. In the men, elevated NEFA levels decreased insulin-stimulated glucose R(d) during the final 40 min of the clamp by 23% (P < 0.001). By contrast, no significant change in glucose R(d) was found in the women (control 10.4 +/- 1.1, lipid study 9.9 +/- 1.3 mg. kg(-1). min(-1)). Glucose R(d) was also unchanged in six women studied at a lower insulin dose (40 mU. m(-2). min(-1)). During the last 40 min of the high-insulin dose clamps with elevated NEFA, glucose oxidation was decreased by 33% in the men (P < 0.001) and by 23% in the women (P < 0.02). Nonoxidative glucose R(d) at this time was decreased by 15% in the men (P = 0.02) but was not significantly affected in women. Basal EGP was unaffected by elevation of plasma NEFA levels in both groups. Suppression of EGP during the glucose clamps, however, was impaired. At the insulin infusion rate used, the magnitude of this defect was comparable in men and women. In summary, our findings suggest that although the effects on EGP appear comparable, the inhibitory effects of NEFA on peripheral tissue insulin sensitivity are observed in men but cannot be demonstrated in women.

The infusion of glucosamine causes insulin resistance, presumably by entering the hexosamine biosynthetic pathway; it has been proposed that this pathway plays a role in hyperglycemia-induced insulin resistance. This study was undertaken to determine if glucosamine infusion could influence exercise-stimulated glucose uptake. Male SD rats were infused with glucosamine at 0.1 mg x kg(-1) x min(-1) (low-GlcN group), 6.5 mg x kg(-1) x min(-1) (high-GlcN group), or saline (control group) for 6.5 h and exercised on a treadmill for 30 min (17 m/min) at the end of the infusion period. Glucosamine infusion caused a modest increase in basal glycemia in both experimental groups, with no change in tracer-determined basal glucose turnover. During exercise, glucose turnover increased approximately 2.2-fold from 46 +/- 2 to 101 +/- 5 pmol x kg(-1) x min(-1) in the control group. Glucose turnover increased to a lesser extent in the glucosamine groups and was limited to 88% of control in the low-GlcN group (47 +/- 2 to 90 +/- 3 pmol x kg(-1) x min(-1); P < 0.01) and 72% of control in the high-GlcN group (43 +/- 1 to 73 +/- 3 pmol kg(-1) 1 min(-1); P < 0.01). Similarly, the metabolic clearance rate (MCR) in the control group increased 72% from 6.1 +/- 0.2 to 10.5 +/- 0.7 ml kg(-1) x min(-1) in response to exercise. However, the increase in MCR was only 83% of control in the low-GlcN group (5.2 +/- 0.5 to 8.7 +/- 0.5 ml x kg(-1) x min(-1); P < 0.01) and 59% of control in the high-GlcN group (4.5 +/- 0.2 to 6.2 +/- 0.3 ml x kg(-1) x min(-1); P < 0.01). Neither glucosamine infusion nor exercise significantly affected plasma insulin or free fatty acid (FFA) concentrations. In conclusion, the infusion of glucosamine, which is known to cause insulin resistance, also impaired exercise-induced glucose uptake. This inhibition was independent of hyperglycemia and FFA levels.

Low plasma fibrinolytic activity in association with increased plasma plasminogen activator inhibitor 1 (PAI-1) levels has been linked to an increased risk of atherosclerosis in obesity and type 2 diabetes. We tested the hypothesis that troglitazone, which improves insulin sensitivity and lowers plasma insulin levels in insulin-resistant obese subjects and patients with type 2 diabetes, would also lower circulating PAI-1 antigen concentrations and activity. We assessed insulin sensitivity (5-h, 80 mU x m(-2) x min(-1) hyperinsulinemic-euglycemic clamp) and measured plasma PAI-1 antigen and activities and tissue plasminogen activator (tPA) in 14 patients with type 2 diabetes and 20 normal control subjects (10 lean, 10 obese) before and after 3 months of treatment with troglitazone (600 mg/day). At baseline, plasma PAI-1 antigen levels after an overnight fast were significantly higher in the obese (33.5 +/- 4.7 microg/l) and type 2 diabetic subjects (54.9 +/- 6.3 microg/l) than in the lean control subjects (16.3 +/- 3.2 microg/l; P < 0.01 and P < 0.001, respectively). Troglitazone decreased plasma PAI-1 antigen concentrations in the diabetic patients (36.8 +/- 5.0 microg/l; P < 0.001 vs. baseline), but the reduction in the obese subjects did not reach statistical significance (baseline, 33.5 +/- 4.7; after troglitazone, 25.6 +/- 5.2 microg/l). Changes in plasma PAI-1 activity paralleled those of PAI-1 antigen. The extent of the reduction in plasma PAI-1 antigen concentrations in the diabetic patients after troglitazone correlated with the reductions in fasting plasma insulin (r = 0.60, P < 0.05), nonesterified fatty acid (r = 0.63, P < 0.02), and glucose concentrations (r = 0.64, P < 0.02) but not with the improvement in glucose disposal rates during the glucose clamps. Three nonresponders to troglitazone with respect to effects on insulin sensitivity and fasting glucose and insulin levels also had no reduction in circulating PAI-1. In conclusion, troglitazone enhances fibrinolytic system activity in insulin-resistant type 2 diabetic patients. This effect appears to be intimately linked to its potential to lower plasma insulin levels and improve glycemic control through its peripheral tissue insulin-sensitizing effects.

Objective: To characterize metabolic effects of troglitazone in type 2 diabetic, obese, and lean subjects, and examine the effects of troglitazone 2-3 weeks after discontinuation., Research Design and Methods: Nine type 2 diabetic, nine obese, and nine lean subjects underwent baseline metabolic studies including an 8-h meal-tolerance test (MTT) and a 5-h glucose clamp. Subjects then received troglitazone (600 mg/day) for 12 weeks and subsequently had repeat metabolic studies. Diabetic subjects remained off hypoglycemic agents for 2-3 weeks and then underwent a 5-h glucose clamp., Results: In diabetic subjects, fasting plasma glucose was reduced (P<0.05) and insulin-stimulated glucose disposal (Rd) was enhanced by treatment (P<0.02). The area under the MTT 8-h plasma glucose curve declined with therapy (P<0.001), and its change was positively correlated with the improvement in Rd (r = 0.75, P<0.05). There was also a positive correlation between the change in fasting hepatic glucose output (HGO) and the change in fasting plasma glucose with treatment (r = 0.92, P<0.001). Discontinuation of therapy for 2-3 weeks did not significantly affect fasting plasma glucose or insulin-stimulated glucose Rd. In obese subjects, insulin-stimulated glucose Rd improved with therapy (P<0.001), allowing for maintenance of euglycemia by lower plasma insulin concentrations (P<0.05). In lean subjects, an increase in fasting HGO (P<0.001) and glucose clearance (P<0.01) was observed., Conclusions: Troglitazone lowers fasting and postprandial plasma glucose in type 2 diabetes by affecting both fasting HGO and peripheral insulin sensitivity. Its effects are evident 2-3 weeks after discontinuation. In obese subjects, its insulin sensitizing effects suggest a role for its use in the primary prevention of type 2 diabetes.

Troglitazone and metformin lower glucose levels in diabetic patients without increasing plasma insulin levels. We compared the insulin sparing actions of these two agents and their effects on insulin sensitivity and insulin secretion in 20 type 2 diabetic patients. To avoid the confounding effect of improved glycemic control on insulin action and secretion, patients were first rendered euglycemic with 4 weeks of continuous subcutaneous insulin infusion (CSII) before randomization to CSII plus troglitazone (n = 10) or CSII plus metformin (n = 10); euglycemia was maintained for another 6-7 weeks. Insulin sensitivity was assessed by a hyperinsulinemic-euglycemic clamp 1) at baseline, 2) after 4 weeks of CSII, and 3) after CSII plus either troglitazone or metformin. The 24-h glucose, insulin, and C-peptide profiles were performed on the day before the second and third glucose clamps. Good glycemic control was achieved with CSII alone and was maintained with CSII plus an oral agent (mean 24-h glucose: troglitazone, 6.2+/-0.6 mmol/l; metformin, 6.2 +/-0.3 mmol/l). Insulin requirements decreased 53% with troglitazone compared with CSII alone (48+/-4 vs. 102+/-13 U/day, P < 0.001), but only 31% with metformin (76+/-13 vs. 110+/-18 U/day, P < 0.005). The 24-h C-peptide profiles were similar. Normal fasting hepatic glucose output was maintained with both agents despite lower insulin levels than on CSII alone. Insulin sensitivity did not change significantly with CSII alone or with CSII plus metformin, but improved 29% with CSII plus troglitazone (P < 0.005 vs. CSII alone) and was then 45% higher than in the CSII plus metformin patients (P < 0.005). In conclusion, metformin has no effect on insulin-stimulated glucose disposal independent of glycemic control in type 2 diabetes. Troglitazone (600 mg/day) has greater insulin-sparing effects than metformin (1,700 mg/day) in CSII-treated euglycemic patients. This is probably explained by the peripheral tissue insulin-sensitizing effects of troglitazone.

Impaired muscle glucose phosphorylation to glucose-6-phosphate by hexokinases (HKs)-I and -II may contribute to insulin resistance in NIDDM and obesity. HK-II expression is regulated by insulin. We tested the hypothesis that basal and insulin-stimulated expression of HK-II is decreased in NIDDM and obese subjects. Skeletal muscle HK-I and HK-II activities were measured in seven lean and six obese normal subjects and eight patients with NIDDM before and at 3 and 5 h of a hyperinsulinemic (80 mU x m(-2) x min(-1)) euglycemic clamp. To assess whether changes in HK-II expression seen during a glucose clamp are likely to be physiologically relevant, we also measured HK-I and HK-II activity in 10 lean normal subjects before and after a high-carbohydrate meal. After an overnight fast, total HK, HK-I, and HK-II activities were similar in lean and obese control subjects; but HK-II was lower in NIDDM patients than in lean subjects (1.42 +/- 0.16 [SE] vs. 2.33 +/- 0.24 nmol x min(-1) x mg(-1) molecular weight, P < 0.05) and accounted for a lower proportion of total HK (33 +/- 3 vs. 47 +/- 3%, P < 0.025). HK-II (but not HK-I) activity increased during the clamp in lean and obese subjects by 34 and 36% after 3 h and by 14 and 22% after 5 h of hyperinsulinemia; no increase was found in the NIDDM patients. In the lean subjects, muscle HK-II activity also increased by 15% 4 h after the meal, from 2.47 +/- 0.19 basally to 2.86 +/- 0.28 nmol x min(-1) x mg(-1) protein (P < 0.05). During the clamps, muscle HK-II activity correlated with muscle citrate synthase activity in the normal subjects (r = 0.58, P < 0.05) but not in the NIDDM patients. A weak relationship was noted between muscle HK-II activity and glucose disposal rate at the end of the clamp when all three groups were combined (r = 0.49, P < 0.05). In summary, NIDDM patients have lower muscle HK-II activity basally and do not increase the activity of this enzyme in response to a 5-h insulin stimulus. This defect may contribute to their insulin resistance. In nondiabetic obese subjects, muscle HK-II expression and its regulation by insulin are normal.

Hyperglycemia can lead directly to a secondary state of insulin resistance or can worsen a preexisting insulin-resistant state. Troglitazone is an orally active hypoglycemic agent that has been shown to ameliorate insulin resistance and hyperinsulinemia in both diabetic animal models and NIDDM subjects. To determine whether this drug could prevent the development of hyperglycemia-induced insulin resistance and to investigate the mechanism by which this might occur, we studied troglitazone's effect on insulin action in rats made hyperglycemic or infused with glucosamine. Normal male SD rats were fed regular powdered diet with or without troglitazone as a food admixture (0.2%). After 2 weeks, rats were made hyperglycemic with glucose (52 mg x kg(-1) x min[-1]) and somatostatin (0.8 microg x kg(-1) x min[-1]) infusion or were infused with glucosamine (6.5 mg x kg(-1) x min[-1]) for 6.5 h. In vivo insulin action was measured by the hyperinsulinemic-euglycemic clamp technique at a submaximal (24 pmol x kg(-1) x min[-1]) or maximal (240 pmol x kg(-1) x min[-1]) insulin infusion rate. The infusion of glucose and somatostatin caused a pronounced rise in the plasma glucose concentration (19.8 +/- 0.6 mmol/l) compared with saline-infused animals (8.0 +/- 0.2 mmol/l; P < 0.001). Hyperglycemia resulted in insulin resistance, as evidenced by a marked reduction in the submaximal glucose disposal rate (GDR) (78 +/- 7 vs. 135 +/- 6 micromol x kg(-1) x min(-1); P < 0.01) and maximal GDR (141 +/- 9 vs. 237 +/- 6 micromol x kg(-1) x min(-1); P < 0.01) compared with the control group. Troglitazone treatment largely prevented the hyperglycemia-induced decline in submaximal (116 +/- 7 micromol x kg(-1) x min[-1]) and maximal GDR (209 +/- 9 micromol x kg(-1) x min(-1); P < 0.05). Glucosamine infusion also resulted in a marked reduction in the submaximal GDR (85 +/- 3 vs. 135 +/- 6 micromol x kg(-1) x min(-1); P < 0.01) and maximal GDR (137 +/- 14 vs. 237 +/- 6 micromol x kg(-1) x min(-1); P < 0.01) compared with the control group. In contrast to the results in the hyperglycemic animals, troglitazone treatment had no effect on glucosamine-induced insulin resistance. In summary, 1) in normal rats, experimental hyperglycemia, as well as glucosamine infusion, led to a marked state of peripheral and hepatic insulin resistance; 2) troglitazone treatment prevented the hyperglycemia-induced, but not the glucosamine-induced, insulin resistance; and 3) either troglitazone acts at one or more sites proximal to the entry of glucosamine into the hexosamine pathway, or the increased flux of glucose-derived products through the hexosamine pathway is not a major mechanism for the hyperglycemia-induced defect in insulin action in these animals.

Tumor necrosis factor (TNF)-alpha may play a role in the insulin resistance of obesity and NIDDM. Troglitazone is a new orally active hypoglycemic agent that has been shown to ameliorate insulin resistance and hyperinsulinemia in both diabetic animal models and NIDDM subjects. To determine whether this drug could prevent the development of TNF-alpha-induced insulin resistance, glucose turnover was assessed in rats infused with cytokine and pretreated with troglitazone. Normal male Sprague-Dawley rats were fed normal powdered food with or without troglitazone as a food admixture (0.2%). After approximately 10 days, rats were infused with TNF-alpha for 4-5 days, producing a plasma concentration of 632 +/- 30 pg/ml. In vivo insulin action was measured by the euglycemic-hyperinsulinemic clamp technique at a submaximal (24 micromol x kg[-1] x min[-1]) and maximal insulin infusion rate (240 micromol x kg[-1] x min[-1]). TNF-alpha infusion resulted in a pronounced reduction in submaximal insulin-stimulated glucose disposal rate (GDR) (97 +/- 10 vs. 141 +/- 4 micromol x kg[-1] x min[-1], P < 0.05), maximal GDR (175 +/- 8 vs. 267 +/- 6 micromol x kg[-1] x min[-1], P < 0.01), and in insulin receptor-tyrosine kinase activity (IR-TKA) (248 +/- 39 vs. 406 +/- 32 fmol ATP/fmol IR, P < 0.05). It also led to a marked increase in basal insulin (90 +/- 24 vs. 48 +/- 6 micromol/l, P < 0.05) and free fatty acid (FFA) concentration (2.56 +/- 0.76 vs. 0.87 +/- 0.13 mmol/l, P < 0.01). Troglitazone treatment completely prevented the TNF-alpha-induced decline in submaximal GDR (133 +/- 16 vs. 141 +/- 4 micromol x kg[-1] x min[-1], NS) and maximal GDR (271 +/- 19 vs. 267 +/- 6 micromol x kg[-1] x min[-1], NS). The hyperlipidemia was partially corrected by troglitazone (1.53 +/- 0.28 vs. 0.87 +/- 0.13 mmol/l, P < 0.05), while IR-TKA and insulin concentration remained unaffected by the drug. Troglitazone restores insulin action possibly by lowering the FFA concentration of the blood and/or by stimulating glucose uptake at an intracellular point distal to insulin receptor autophosphorylation in muscle. If TNF-alpha plays a role in the development of the obesity/NIDDM syndrome, troglitazone may prove useful in its treatment.

Impaired suppression of plasma nonesterified fatty acids (NEFAs) after glucose ingestion may contribute to glucose intolerance, but the mechanisms are unclear. Evidence that insulin inhibits hepatic glucose output (HGO), in part by suppressing plasma NEFA levels, suggests that impaired suppression of plasma NEFA after glucose ingestion would impair HGO suppression and increase the systemic delivery of glucose. To test this hypothesis, we studied glucose kinetics (constant intravenous [3-3H]glucose [0.4 microCi/min], oral [1-14C]glucose [100 microCi]), whole-body substrate oxidation, and leg glucose uptake in eight normal subjects (age, 39 +/- 9 years [mean +/- SD]; BMI, 24 +/- 2 kg/m2) in response to 75 g oral glucose on two occasions. In one study, plasma NEFAs were prevented from falling by infusion of 20% Liposyn (45 ml/h) and heparin (750 U/h). Plasma glucose rose more rapidly during lipid infusion (P < 0.05), and mean levels tended to be higher after 120 min (6.45 +/- 0.41 vs. 5.81 +/- 0.25 SE, 0.1 < P < 0.05, NS); peak glucose levels were similar. Total glucose appearance (Ra) was higher during lipid infusion due to a higher HGO (28.4 +/- 1.0 vs. 21.2 +/- 1.5 g over 4 h, P < 0.005). Total glucose disposal (Rd) was also higher (88 +/- 2 vs. 81 +/- 3 g in 4 h, P < 0.05). Plasma insulin rose more rapidly after glucose ingestion with lipid infusion, and leg glucose uptake was 33% higher (P < 0.05) during the 1st hour. During lipid infusion, subjects oxidized less glucose (47 +/- 3 vs. 55 +/- 2 g, P < 0.05) and more fat (7.1 +/- 0.8 vs. 3.9 +/- 0.9 g, P < 0.02). In summary, 1) impaired suppression of NEFAs after oral glucose impairs insulin's ability to suppress HGO, and 2) in normal subjects the greater insulin response compensates for the increased systemic glucose delivery by increasing peripheral glucose Rd.

To evaluate kinetic defects in insulin action, we performed time-course studies during hyperinsulinemic (120 mU x m(-2) x min(-1)) isoglycemic clamps in seven subjects with NIDDM (194 +/- 29 mg/dl) and in seven lean and seven obese nondiabetic subjects. The time course of whole-body glucose disposal rate (GDR), leg glucose uptake (LGU), hepatic glucose output (HGO), and muscle insulin receptor tyrosine kinase (IRTK) activation were measured. The obese and NIDDM subjects had marked delays in activation of GDR (T50 74 +/- 14 and 95 +/- 15 min, respectively, compared with 33 +/- 2 min in lean control subjects), arteriovenous glucose difference (T50 80 +/- 12 and 109 +/- 31 min compared with 30 +/- 3 min) and LGU (T50 89 +/- 25 and 98 +/- 27 min compared with 29 +/- 4 min). All three measurements reached normal levels in the NIDDM group after 4-5 h of insulin infusion. Although only a limited number of data points could be obtained from serial muscle biopsies, no delay in the rate of activation of IRTK was apparent in the obese and NIDDM groups. In conclusion, 1) in obese and NIDDM subjects, insulin-mediated GDR and LGU are delayed to a similar degree; 2) mass action normalizes GDR and LGU in NIDDM, but only after several hours of insulin infusion; and 3) The kinetic defect in NIDDM and obesity most likely involves intracellular loci distal to activation of the insulin receptor kinase.

Protein tyrosine phosphatase 1B (PTP1B) is a protein tyrosine phosphatase of unknown function, although increasing evidence supports a role for this phosphatase in insulin action. We have investigated the interaction of PTP1B with the insulin receptor using a PTP1B glutathione S-transferase (GST) fusion protein with a point mutation in the enzyme's catalytic domain. This fusion protein is catalytically inactive, but the phosphatase's phosphotyrosine binding site is maintained. The activated insulin receptor was precipitated from purified receptor preparations and whole-cell lysates by the inactive PTP1B-GST, demonstrating a direct association between the insulin receptor and PTP1B. A p120 of unknown identity was also precipitated from whole-cell lysates by the PTP1B fusion protein, but IRS-1 (pp185) was not. A catalytically inactive [35S]PTP1B-fusion protein bound directly to immobilized insulin receptor kinase domains and was displaced in a concentration-dependent manner. Finally, tyrosine-phosphorylated PTP1B was precipitated from whole-cell lysates by an anti-insulin receptor antibody after insulin stimulation. The site of interaction between PTP1B and the insulin receptor was studied using phosphopeptides modeled after the receptor's kinase domain, the NPXY domain, and the COOH-terminal. Each phosphopeptide inhibited the PTP1B-GST:insulin receptor interaction. Study of mutant insulin receptors demonstrated that activation of the kinase domain is necessary for the PTP1B:insulin receptor interaction, but receptors with deletion of the NPXY domain or of the COOH-terminal can still bind to the PTP1B-GST. We conclude that PTP1B can associate directly with the activated insulin receptor at multiple different phosphotyrosine sites and that dephosphorylation by PTP1B may play a significant role in insulin receptor signal transduction.

Leptin, the product of the ob gene, is a hormone secreted by adipocytes. Animals with mutations in the ob gene are obese and lose weight when given leptin, but little is known about the physiological role of leptin in humans. Obese subjects have higher concentrations of leptin than lean subjects, the strongest correlation being with percentage body fat. Thus, it appears that obese subjects are resistant to the effects of endogenously secreted leptin. We have also shown that insulin stimulates leptin production, chronically but not acutely, presumably through its trophic effect on adipocytes. Troglitazone is an insulin-sensitizing thiazolidinedione, which improves hepatic and skeletal muscle insulin resistance in NIDDM and obesity. This study was undertaken to investigate the effects of troglitazone on leptin production in vitro and in vivo. In the presence and absence of 100 nmol/l insulin and 10 umol/l troglitazone, 72-h primary cultures of isolated abdominal adipocytes were studied. Insulin led to an almost twofold increase in leptin in vitro, and this increase was completely abolished by coincubation with troglitazone. Incubation with troglitazone alone led to a 40% decrease in leptin production. In obese patients administered troglitazone 200 mg twice daily for 12 weeks, there was no significant change in fasting plasma leptin concentrations, despite a 40-50% reduction in fasting and postmeal plasma insulin concentrations. Troglitazone treatment led to a significant increase in insulin sensitivity, and there was a positive correlation between the change in insulin sensitivity and the change in plasma leptin concentration in these subjects. In conclusion, troglitazone treatment had no net effect on plasma leptin concentrations, possibly because of improvement in insulin sensitivity and reduction in plasma insulin concentrations.

To examine the kinetic steps in insulin's in vivo action, we have assessed the temporal relationship between arterial insulin, interstitial insulin, glucose disposal rate (GDR), and insulin receptor kinase (IRK) activity in muscle and between portal insulin, hepatic glucose production (HGP), and IRK activity in liver. Interstitial insulin, as measured by lymph-insulin concentration (muscle only), and IRK activity were used as independent methods to determine the arrival of insulin at its tissue site of action. Euglycemic clamps were conducted in seven mongrel dogs and consisted of an activation phase with a venous insulin infusion (7.2 nmol.kg-1.min-1, 100 min) and a deactivation phase. Liver and muscle biopsies were taken to assess IRK activity. Arterial, portal, and lymph insulin rose to 636 +/- 12, 558 +/- 18, and 402 +/- 24 pmol/l, respectively. GDR increased from 13.9 +/- 0.6 to 41.7 +/- 2.8, and HGP declined from 14.4 +/- 0.6 to 1.1 +/- 0.6 mumol.kg-1.min-1. Muscle and liver IRK activity increased significantly from 5.9 +/- 0.9 to 14.6 +/- 0.6 and 5.5 +/- 0.7 to 23.7 +/- 1.9 fmol P/fmol insulin receptor (IR), respectively. The time to half-maximum response (t1/2a) for stimulation of GDR (19.8 +/- 4.8 min) and suppression of HGP (21.5 +/- 3.7 min) were similar. The t1/2a for stimulation of GDR, muscle IRK, and rise in lymph insulin were not significantly different from one another and were all markedly greater than that for the approach to steady state of arterial insulin (2.3 +/- 1.2 min, P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Troglitazone is a new orally active hypoglycemic agent that has been shown to reduce insulin resistance and hyperinsulinemia in both diabetic animal models and non-insulin-dependent diabetes mellitus (NIDDM) subjects. To determine whether this drug could prevent the development of fructose-induced insulin resistance and related abnormalities, we studied the effects of troglitazone on the insulin resistance induced by fructose feeding in rats. Normal male Sprague-Dawley rats were fed a high-fructose diet for 3 weeks with and without troglitazone as a food admixture (0.2%) or were fed normal chow to serve as a control group. In vivo insulin resistnace was measured by the euglycemic hyperinsulinemic clamp technique at two different insulin infusion rates, 29 (submaximal stimulation) and 290 (maximal stimulation) pmol.kg-1.min-1. Fructose feeding markedly reduced submaximal glucose disposal rate (GDR) (113.8 +/- 8.3 vs. 176.0 +/- 5.6 mumol.kg-1.min-1, P < 0.05) and maximal GDR (255.9 +/- 5.6 vs. 313.6 +/- 10.5 mumol.kg-1.min-1, P < 0.05), reduced the suppressibility of submaximal hepatic glucose production (HGP; 45.5 +/- 5.0 vs. 11.7 +/- 5.0 mumol.kg-1.min-1, P < 0.05), and resulted in hypertriglyceridemia and hypertension. Troglitazone treatment completely restored the GDR (submaximal 158.2 +/- 5.6, maximal 305.3 +/- 6.1 mumol.kg-1.min-1) and submaximal HGP (9.4 +/- 2.8 mumol.kg-1.min-1) to control levels and also normalized the elevated plasma triglyceride concentration and systolic blood pressure levels in fructose-fed rats.(ABSTRACT TRUNCATED AT 250 WORDS)

During the infusion of insulin in vivo, the rate of activation of glucose disposal lags significantly behind the rate of increase in serum insulin levels. To determine whether this delay was related to transcapillary transport of insulin, we determined increments in serum insulin levels, glucose disposal rates (GDR), and insulin receptor (IR) kinase activity measured during continuous infusions of insulin (40 and 120 mU.m-2.min-1) administered to 8 nondiabetic males; similar studies were done at 1,200.m-2.min-1 in 2 of the subjects. Half-maximal insulin levels were achieved at a mean of 4.9 and 7.2 min during the 40 and 120 mU.m-2.min-1 clamps, respectively, with corresponding half-maximal GDR stimulation at a mean of 59 and 47 min. Unlike the rise in insulin levels, IR kinase activation was much slower with half-maximal activity occurring at approximately 40-60 min in the 2 clamps. Thus, the rise in serum insulin levels in each clamp was much faster than the increment in either kinase activity or glucose disposal. Insulin infusion increased both IR kinase and GDR maximally approximately 10-fold, with half-maximal stimulation at approximately 3,600 and approximately 700 pM, indicating spare kinase for glucose disposal. These results demonstrate that the delay in stimulation of glucose disposal by insulin is related to a rate-limiting step between the intravascular space and the cell-surface of skeletal muscle. This may involve delayed transendothelial transport of insulin.

Objective: To study the metabolic effects of a new oral antidiabetic agent, CS-045, in subjects with non-insulin-dependent diabetes mellitus (NIDDM)., Research Design and Methods: Eleven NIDDM subjects (mean age 59 yr and body mass index 32.3) were treated with 400 mg/day CS-045 for 6-12 wk. Patients were hospitalized before and at the end of the drug-treatment period for metabolic studies, including oral glucose tolerance test (OGTT), meal tolerance test (MTT), euglycemic glucose-clamp studies, and lipid analyses., Results: Eight subjects showed a marked clinical response to the drug, whereas 3 were nonresponders. The data were analyzed both for the total group and for the responders. Fasting plasma glucose (FPG) fell from 12.5 +/- 0.7 to 10.7 +/- 1.0 mM in the total group but fell more dramatically from 12.7 +/- 0.5 to 8.3 +/- 0.6 mM in the responder group. The area under the OGTT glucose curve improved by 17% in the total group and by 29% in the responders. The area under the MTT glucose curve improved by 38 and 52%, respectively. MTT levels of insulin, free fatty acids, and glucagon were significantly lower after treatment. Glucose disposal rates during glucose-clamp studies were increased in all subjects after CS-045 treatment. Mean increases were 63% at 120 mU.m-2.min-1 and 41% at 300 mU.m-2.min-1. Basal hepatic glucose production fell by 17% in the total group and by 28% in the responders., Conclusions: CS-045 improves insulin resistance, reduces insulinemia, lowers hepatic glucose production, and improves both fasting and postprandial glycemia in NIDDM subjects. CS-045 may represent a new therapeutic option for NIDDM.

In this study, we administered constant intravenous infusions of human islet amyloid polypeptide (hIAPP) to conscious dogs during euglycemic glucose-clamp studies. The doses of hIAPP used (5 and 50 pmol.kg-1.min-1) raised the circulating IAPP levels approximately 12- and 50-fold above basal levels, respectively. Studies were conducted at two different insulin infusion rates, resulting in steady-state plasma insulin levels of approximately 600 and 2800 pM. According to our results, the hIAPP infusions did not lead to any measurable change in the insulin-stimulated glucose disposal rate at either insulin infusion rate. Additionally, we observed no effect of IAPP on hepatic glucose production. Although we did not observe any effect of hIAPP on any of the aspects of glucose or insulin metabolism measured, we did find a consistent hypocalcemic effect of this peptide at the 50-pmol.kg-1.min-1 infusion rate. Shortly after the onset of hIAPP infusion, serum calcium levels fell by 10-15% and remained at these levels throughout the course of the hIAPP infusion. In summary, 1) infusion of hIAPP at doses of 5 or 50 pmol.kg-1.min-1 in conscious dogs raised the circulating IAPP level 12- to 50-fold above basal; 2) during these infusion studies, no effect of hIAPP was observed on any of the aspects of glucose or insulin homeostasis measured; 3) 50 pmol.kg-1.min-1 hIAPP lead to a prompt reduction in plasma calcium concentrations with intravenous administration.

Resistance to insulin action is a well-established feature of non-insulin-dependent diabetes mellitus (NIDDM) and is believed to contribute to the etiology of this condition. A strong genetic contribution to the etiology of NIDDM exists, and we previously identified an insulin-receptor gene restriction-fragment-length polymorphism (RFLP) associated with the NIDDM phenotype. In an attempt to elucidate whether structural defects in the insulin receptor could be a primary cause of insulin resistance in NIDDM, we analyzed the insulin-receptor cDNA sequence in a subject with NIDDM who is also homozygous for this RFLP. The insulin-receptor cDNA was sequenced with the polymerase chain reaction (PCR). mRNA from transformed lymphocytes was reverse transcribed and amplified with five overlapping sets of primers that span the coding sequence of both alpha- and beta-subunits. No difference was found in the predicted amino acid sequence of the subject's insulin receptor compared with the normal insulin receptor. At nucleotide positions 831 and 2247, the subject is heterozygous for silent nucleotide polymorphisms that do not affect the amino acid sequence. Exon 11 encodes a 12-amino acid insert in the alpha-subunit, which, due to alternate splicing, is not expressed in lymphocyte insulin-receptor mRNA. Consequently, exon 11 was amplified from genomic DNA by PCR; the sequence of exon 11 was found to be normal. In addition, when this patient's transformed lymphocytes were maintained in culture, no abnormalities in insulin binding were observed. We conclude that the insulin resistance seen in this NIDDM subject is not due to a structural alteration in the insulin receptor itself.(ABSTRACT TRUNCATED AT 250 WORDS)

The insulin receptor is a multifunctional protein encoded by a modular gene. Certain discrete domains within the insulin-receptor structure subserve specific functional properties. In some instances, these discrete domains are encoded by individual exons. This organizational model of the insulin receptor predicts the existence of divergent signaling pathways facilitating specific bioeffects. Some of these signaling pathways are shared with the closely related insulinlike growth factor I receptor (convergent pathways), whereas others are different (divergent). The concept of discrete functional domains also provides several mechanisms whereby inactive insulin receptors (no kinase activity) can inhibit the function of normal receptors. The ability of kinase-inactive insulin receptors to inhibit the signaling function of normal insulin receptors may be an operative mechanism in certain insulin-resistant states.

The kinetics of in vivo insulin-mediated glucose uptake in human obesity have not been previously studied. To examine this, we used the glucose-clamp technique to measure whole-body and leg muscle glucose uptake in seven lean and six obese men during hyperinsulinemia (approximately 2000 pM) at four glucose levels (approximately 4.5, approximately 8.3, approximately 13.5, and approximately 23.5 mM). To measure leg glucose uptake, the femoral artery and vein were catheterized, and blood flow was measured by thermodilution (leg glucose uptake = arteriovenous glucose difference x blood flow). With this approach, we found that rates of whole-body and leg glucose uptake were significantly lower in obese than in lean subjects at each glucose plateau. Leg blood flow rates increased from 4.3 +/- 0.4 to 6.5 +/- 0.8 dl/min over the range of glucose in lean subjects (P less than 0.05) but remained unchanged in obese subjects. The apparent maximal capacity (Vmax), based on whole-body and leg glucose uptake, was reduced in obese compared with lean subjects, but the apparent Km was similar in the lean and obese subjects (6-9 mM, NS). To assess the affinity of muscle for glucose extraction independent of changes in muscle plasma flow, we determined the mean half-maximal effective glucose concentration (EG50) and found it was similar in the lean and obese subjects (6.0 +/- 0.3 vs. 6.0 +/- 0.8 mM, NS). We conclude that 1) the kinetics of in vivo insulin-mediated glucose uptake in skeletal muscle in human obesity are characterized by reduced Vmax but normal Km; 2) the EG50 for insulin-mediated glucose extraction in skeletal muscle was 6 mM in both lean and obese subjects, consistent with a Km characteristic of the glucose-transport system; 3) obese subjects were unable to generate increases in blood flow in response to hyperglycemia under hyperinsulinemic conditions, and this contributed significantly to lower rates of leg and whole-body glucose uptake.

The kinetics of insulin-mediated glucose uptake (IMGU) and non-insulin-mediated glucose uptake (NIMGU) in humans have not been well defined. We used the glucose-clamp technique to measure rates of whole-body and leg muscle glucose uptake in six healthy lean men during hyperinsulinemia (approximately 460 pM) to study IMGU and during somatostatin-induced insulinopenia to study NIMGU at four glucose levels (4.5, 9, 12, and 21 mM). To measure leg glucose uptake, the femoral artery and vein were catheterized, and blood flow was measured by thermodilution (leg glucose uptake = arteriovenous glucose difference [A-VG] x blood flow). With this approach, we found that, during hyperinsulinemia, both whole-body and leg glucose uptake increased in a curvilinear fashion at every glucose level, the highest glucose uptake values obtained being 139 +/- 17 mumol.kg-1.min-1 and 3656 +/- 931 mumol.min-1.leg-1, respectively. Leg blood flow increased twofold from 6.0 +/- 1.7 to 11.7 +/- 3.1 dl/min (P less than 0.01) over the range of glucose and was correlated with whole-body glucose uptake (r = 0.55, P less than 0.005). Leg muscle glucose extraction, independent of changes in blood flow, which is reflected by the A-VG, saturated over the range of glucose (1.28 +/- 0.12, 2.22 +/- 0.30, 2.92 +/- 0.42, 3.02 +/- 0.41 mM, NS between last 2 values) with a half-maximal effective glucose concentration (EG50) of 5.3 +/- 0.4 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

During hyperinsulinemic glucose-clamp studies, intravenous infusion of calcitonin gene-related peptide (CGRP) in rats antagonized the ability of insulin to stimulate peripheral glucose disposal by 52% (196 +/- 7.2 vs. 105 +/- 10.5 mumol.kg-1.min-1, P less than 0.05) and to inhibit hepatic glucose output by 54% (P less than 0.01). CGRP also inhibited the in vitro effects of insulin to stimulate hexose uptake in cultured BC3H1 myocytes at all insulin concentrations studied. Amylin is a peptide isolated from amyloid deposits in pancreatic islets of type II (non-insulin-dependent) diabetic subjects, is present in normal beta-cells, and bears a striking homology to CGRP. When synthetic human amylin was infused during clamp studies, it inhibited the ability of insulin to stimulate glucose disposal by 56% (96.9 +/- 9.4 vs. 42.4 +/- 5.0 mumol.kg-1.min-1, P less than 0.05) and to suppress hepatic glucose output by 64%. Therefore, amylin and CGRP can cause insulin resistance in vivo and may be implicated in insulin-resistant states such as type II diabetes mellitus.

We identified a possible endogenous substrate (pp185) of the insulin-receptor kinase in human adipocytes by treating intact cells with insulin and immunoblotting the cellular extracts with polyclonal antiphosphotyrosine antibody. This 185,000-Mr protein was phosphorylated on tyrosine residues in response to insulin in both rat and human adipocytes. The time course of pp185 phosphorylation at 37 degrees C was rapid and corresponded closely to insulin-receptor autophosphorylation but preceded insulin-stimulated glucose transport. Unlike many growth factor receptors, including the insulin receptor, pp185 was not adsorbed to wheat-germ agglutinin. We found that pp185 phosphorylation occurred at 12 degrees C and that the phosphoprotein was associated with both cytoplasmic and membrane fractions at this temperature. Furthermore, pp185 phosphorylation was induced to the same extent as insulin by vanadate and hydrogen peroxide, compounds previously shown to mimic the biologic effects of insulin. In addition, dose-response analysis of insulin-stimulated glucose transport, receptor autophosphorylation, and pp185 phosphorylation resulted in ED50 values of 0.3, 12, and 12 ng/ml, respectively. These results demonstrate the magnitude of "spare" autophosphorylation and pp185 phosphorylation with respect to glucose transport stimulation in human adipocytes. To determine whether the insulin resistance characteristic of non-insulin-dependent diabetes mellitus (NIDDM) and obesity is associated with a defect in receptor autophosphorylation and/or endogenous substrate phosphorylation, we estimated the extent of beta-subunit and pp185 phosphorylation in adipocytes from NIDDM, obese, and healthy subjects. Although the efficiency of coupling between receptor activation and pp185 phosphorylation was normal in obesity and NIDDM, the capacity for insulin-receptor autophosphorylation was approximately 50% lower in NIDDM subjects compared with nondiabetic obese or lean subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Although sulfonylurea agents have been used in the clinical management of type II diabetes (non-insulin-dependent diabetes mellitus, NIDDM) for over two decades, the mechanisms responsible for their hypoglycemic action remain controversial. We have quantitated glycemic control, endogenous insulin secretion in response to mixed meals, adipocyte insulin binding, insulin-mediated peripheral glucose disposal, and basal hepatic glucose output in 17 type II diabetic subjects before and after 3 mo of therapy with the second-generation, sulfonylurea compound glyburide in an attempt to identify the factors responsible for the clinical response to the drug. In addition, 9 subjects were treated for an additional 15 mo to see if the response to the drug changed with time. The mean fasting serum glucose level fell from an initial value of 264 +/- 17 mg/dl to 178 +/- 16 mg/dl after 3 mo of drug therapy. Endogenous insulin secretion increased in all subjects, but the increase was most marked in those subjects who continued to exhibit fasting hyperglycemia (fasting serum glucose greater than 175 mg/dl) after 3 mo of therapy. Adipocyte insulin binding was unchanged after 3 mo of therapy, while the maximal rate of peripheral glucose disposal was increased by 23%, indicating enhancement of peripheral insulin action at a postreceptor site(s). Basal hepatic glucose output showed a significant correlation with the fasting serum glucose level both before and after therapy (r = 0.86, P less than 0.001) and fell from 141 +/- 12 mg/m2/min before therapy to 107 +/- 11 mg/m2/min after 3 mo of therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of small increases in plasma insulin on hepatic glucose production are incompletely understood. To partially elucidate this issue we have studied seven obese subjects with the euglycemic clamp technique with a low-dose insulin infusion rate of 15 mU X m-2 X min-1 over 3 h. Basal insulin levels were 24 +/- 7 microU/ml and increased to steady-state levels of 35 +/- 3 microU/ml during insulin infusion. Endogenous insulin secretion, quantitated by C-peptide measurements, decreased by 58% of the basal value after peripheral insulin infusion. Based on C-peptide measurements and the contribution of the peripheral insulin infusion to the circulating insulin concentrations, calculated portal insulin levels either decreased or remained unchanged during the clamp studies. Basal glucagon levels were 165 +/- 18 and did not change during the insulin infusion. The basal glucose disposal rate was 86 +/- 2 mg X m-2 X min-1 and did not increase significantly during the clamp studies. In contrast, hepatic glucose output (HGO) was suppressed by 82 +/- 5% of the basal value. In summary, in a group of insulin-resistant obese subjects, glucose-clamp studies were performed at peripheral insulin levels of 35 +/- 3 microU/ml; glucose disposal did not increase, whereas HGO was suppressed by 82%. At the same time, glucagon levels remained constant and estimated portal insulin levels either decreased or remained unchanged. These findings suggest that insulin can suppress HGO through indirect extrahepatic actions.

We studied the acute effects of oral ingestion of 50-g loads of dextrose, sucrose, and fructose on post-prandial serum glucose, insulin, and plasma glucagon responses in 9 normal subjects, 10 subjects with impaired glucose tolerance, and 17 non-insulin-dependent diabetic subjects. The response to each carbohydrate was quantified when the respective carbohydrate was given alone in a drink or when given in combination with protein and fat in a test meal. The data demonstrate that (1) fructose ingestion resulted in significantly lower serum glucose and insulin responses than did sucrose or dextrose ingestion in all study groups, either when given alone or in the test meal; (2) although fructose ingestion always led to the least glycemic response compared with the other hexoses, the serum glucose response to fructose was increased the more glucose intolerant the subject; (3) urinary glucose excretion during the 3 h after carbohydrate ingestion was greatest after dextrose and least after fructose in all groups. In conclusion, fructose ingestion results in markedly lower serum glucose and insulin responses and less glycosuria than either dextrose or sucrose, both when given alone or as a constituent in a test meal. However, as glucose tolerance worsens, an increasingly greater glycemic response to fructose is seen.

Insulin receptors on viable rat adipocytes were affinity-labeled using a biologically active and photosensitive analogue of insulin, 125I-B2(2-nitro, 4 azidophenylacetyl)-des-PheB1-insulin (125I-NAPA-DP-insulin). The radiolabeled proteins were identified by SDS polyacrylamide gel electrophoresis and autoradiography. Binding of 125I-NAPA-DP-insulin (40 ng/ml) to rat adipocytes at 16 degrees C, followed by photolysis, resulted in the specific labeling of essentially one protein with an apparent molecular weight of 430-450,000 daltons. When this radiolabeled protein was treated with dithiothreitol prior to electrophoresis, specific labeling occurred predominantly in a 125,000-dalton protein and to a lesser extent in a 90,000-dalton protein. In addition, there was a minimal amount of specific labeling of a 115,000-dalton protein. Under certain experimental conditions, the nonreduced form of the photoaffinity-labeled receptor appeared as a heterogeneous population of proteins having apparent molecular weights of 430,000, 350,000, and 270,000 daltons. Subsequent to photoaffinity labeling of insulin receptors at 16 degrees C, adipocytes were incubated at 37 degrees C for various periods of time to allow for internalization. This resulted in an initial rapid loss of radioactivity in the 430,000- and 125,000-dalton bands. At 60 min the amount of radioactivity in each of these bands was approximately 50% of that present before incubation at 37 degrees C and stayed constant for 120 min. A first-order plot of the decline in receptor-associated radioactivity was biphasic with the initial phase having a half-life of 1.4 h.(ABSTRACT TRUNCATED AT 250 WORDS)

A 53-y4-old male patient with insulin-resistant diabetes was found to have circulating inhibitors of both insulin and somatomedin-C binding. Serum obtained from the patient at the time of initial presentation inhibited 50% of both 125I-insulin and 125I-SM-C binding to IM-9 lymphocytes at dilutions of 1:150. Spontaneous improvement in the patient's diabetic state was associated with a simultaneous and equal decrease in the serum inhibitory titers for both radioligands. Scatchard analysis indicated that the observed serum-induced decrease in both insulin and SM-C binding was due to decreased receptor affinity, with no alteration in receptor number. The serum inhibitors of both insulin and SM-C binding were precipitated equally by Staph-A and also by 40% ammonium sulfate, suggesting they were immunoglobulins. The observation of naturally occurring autoantibodies against both the insulin and SM-C receptors suggests a structural homology between the two receptors.

We studied the effects of short-term (5 days) and long-term (2 wk) high carbohydrate (75%) feedings on insulin binding to isolated adipocytes and insulin sensitivity in vivo in normal subjects. Ingestion of the high carbohydrate diet led to daylong hyperinsulinemia in both short- and long-term groups. Insulin binding to isolated adipocytes was decreased in both groups; in the short-term groups this decrease in insulin binding was caused by a decrease in the receptor affinity, whereas in the long-term group it was caused by a decrease in receptor number. On the other hand, despite this decrease in insulin binding, total in vivo insulin sensitivity was markedly improved in both groups. In conclusion, (1) the short-term adaptive response of the insulin receptor is a decrease in binding affinity whereas the long-term response is a decrease in receptor number, (2) sustained and chronic hyperinsulinemia can lead to a decrease in the number of cellular insulin receptors, (3) high carbohydrate diets lead to a general increase in insulin's ability to promote glucose removal from plasma, and (4) the paradox of enhanced insulin sensitivity in the face of decreased insulin binding can be explained if high carbohydrate diets also lead to an increase in the activity of steps in glucose metabolism distal to the insulin receptor.