Background-Hyperglycemia impairs functional properties of cytosolic and nuclear proteins via O-linked glycosylation modification (O-GlcNAcylation). We studied the effects of O-GlcNAcylation on insulin signaling in human coronary artery endothelial cells. Methods and Results-O-GlcNAcylation impaired the metabolic branch of insulin signaling, ie, insulin receptor (IR) activation of the IR substrate (IRS)/phosphatidylinositol 3-kinase (PI3-K)/Akt, whereas it enhanced the mitogenic branch, ie, ERK-1/2 and p38 (mitogen-activated protein kinase). Both in vivo and in vitro phosphorylation of endothelial nitric oxide synthase (eNOS) by Akt were reduced by hyperglycemia and hexosamine activation. Insulin-induced eNOS activity in vivo was reduced by hyperglycemia and hexosamine activation, which was coupled to increased activation and expression of matrix metalloproteinase-2 and -9; these phenomena were reversed by inhibition of the hexosamine pathway. Finally, carotid plaques from type 2 diabetic patients showed increased endothelial O-GlcNAcylation with respect to nondiabetics. Conclusions-Our

Insulin receptor substrate (IRS) molecules are key mediators in insulin signaling and play a central role in maintaining basic cellular functions such as growth, survival, and metabolism. They act as docking proteins between the insulin receptor and a complex network of intracellular signaling molecules containing Src homology 2 (SH2) domains. Four members (IRS-1, IRS-2, IRS-3, IRS-4) of this family have been identified that differ as to tissue distribution, subcellular localization, developmental expression, binding to the insulin receptor, and interaction with SH2 domain-containing proteins. Results from targeted disruption of the IRS genes in mice have provided important clues to the functional differences among these related molecules, suggesting they play different and specific roles in vivo. The available data are consistent with the notion that IRS-1 and IRS-2 are not functionally interchangeable in tissues that are responsible for glucose production (liver), glucose uptake (skeletal muscle and adipose tissue), and insulin production (pancreatic beta cells). In fact, IRS-1 appears to have its major role in skeletal muscle whereas IRS-2 appears to regulate hepatic insulin action as well as pancreatic beta cell development and survival. By contrast, IRS-3 and IRS-4 genes appear to play a redundant role in the IRS signaling system. Defects in muscle IRS-1 expression and function have been reported in insulin-resistant states such as obesity and type 2 diabetes. Several polymorphisms in the IRS genes have been identified, but only the Gly-->Arg972 substitution of IRS-1, interacting with environmental factors, seems to have a pathogenic role in the development of type 2 diabetes. In contrast, polymorphisms of the other IRS genes do not appear to contribute to type 2 diabetes.

Type 2 diabetes is characterized by insulin resistance and inadequate insulin secretion. In the advanced stages of the disease, ␤-cell dysfunction worsens and insulin therapy may be necessary to achieve satisfactory metabolic control. Studies in autopsies found decreased ␤-cell mass in pancreas of people with type 2 diabetes. Apoptosis, a constitutive program of cell death modulated by the Bcl family genes, has been implicated in loss of ␤-cells in animal models of type 2 diabetes. In this study, we compared the effect of 5 days' culture in high glucose concentration (16.7 mmol/l) versus normal glucose levels (5.5 mmol/l) or hyperosmolar control (mannitol 11 mmol/l plus glucose 5 mmol/l) on the survival of human pancreatic islets. Apoptosis, analyzed by flow cytometry and electron and immunofluorescence microscopy, was increased in islets cultured in high glucose (HG5) as compared with normal glucose (NG5) or hyperosmolar control (NG5؉MAN5). We also analyzed by reverse transcriptase-polymerase chain reaction and Western blotting the expression of the Bcl family genes in human islets cultured in normal glucose or high glucose. The antiapoptotic gene Bcl-2 was unaffected by glucose change, whereas Bcl-xl was reduced upon treatment with HG5. On the other hand, proapoptotic genes Bad, Bid, and Bik were overexpressed in the islets maintained in HG5. To define the pancreatic localization of Bcl proteins, we performed confocal immunofluorescence analysis on human pancreas. Bad and Bid were specifically expressed in ␤-cells, and Bid was also expressed, although at low levels, in the exocrine pancreas. Bik and Bcl-xl were expressed in other endocrine islet cells as well as in the exocrine pancreas. These data suggest that in human islets, high glucose may modulate the balance of proapoptotic and antiapoptotic Bcl proteins toward apoptosis, thus favoring ␤-cell death.

Insulin-like growth factors promote myoblast differentiation through phosphoinositol 3-kinase and Akt signaling. Akt substrates required for myogenic differentiation are unknown. Forkhead transcription factors of the forkhead box gene, group O (Foxo) subfamily are phosphorylated in an insulin-responsive manner by phosphatidylinositol 3-kinase–dependent kinases. Phosphorylation leads to nuclear exclusion and inactivation. We show that a constitutively active Foxo1 mutant inhibits differentiation of C2C12 cells and prevents myotube differentiation induced by constitutively active Akt. In contrast, a transcriptionally inactive mutant Foxo1 partially rescues inhibition of C2C12 differentiation mediated by wortmannin, but not by rapamycin, and is able to induce aggregation-independent myogenic conversion of teratocarcinoma cells. Inhibition of Foxo expression by siRNA resulted in more efficient differentiation, associated with increased myosin expression. These observations indicate that Foxo proteins are key effectors of Akt-dependent myogenesis.

O besity is associated with metabolic and cardiovascular risk factors that include type 2 diabetes, hypertension, and dyslipidemia (1-4). A subset of obese subjects has been identified that appears to be protected from obesity-related metabolic abnormalities (5-11). These subjects, termed metabolically healthy but obese (MHO), are relatively insulin sensitive and have a rather favorable cardiovascular risk profile (5-11). Although the existence of MHO individuals has been recognized, only a few studies have examined in detail the metabolic characteristics associated with their protective profile (5-13). Whereas MHO individuals appear to have a more favorable cardiovascular risk profile than insulinresistant obese (IRO) individuals, they show early signs of atherosclerosis compared with lean subjects, which could not be explained by alterations in cardiovascular risk factors (12). Among the factors that may account for the early atherosclerosis, insulin-like growth factor (IGF)-1 is a plausible candidate because low plasma IGF-1 concentrations are associated with type 2 diabetes, insulin resistance (14 -16), and increased risk of coronary artery disease (17)(18)(19)(20)(21)(22). To further characterize the protective profile of MHO individuals, we compared clinical characteristics, including cardiovascular risk factors, plasma IGF-1 levels, and intima-media thickness (IMT) of the common carotid artery, of a group of MHO women from a cohort of nondiabetic Italian Caucasians with those of two age-matched groups comprising healthy nonobese or IRO women. RESEARCH DESIGN AND METHODS -The study group consisted of 73 nonobese (BMI Ͻ27 kg/m 2 ) and 80 obese (BMI Ͼ30 kg/m 2 ) women, recruited by announcements in the Universities of Rome and Catanzaro areas. The inclusion criteria included the following: aged 19 -48 years, absence of diabetes, and absence of known inflammatory disease or pathologies affecting glucose metabolism. A total of 68 women in the cohort used oral contraceptives, whereas the remaining had regular menses. Subjects underwent anthropometrical evaluation, a 75-g oral glucose tolerance test, and a euglycemic-hyperinsulinemic clamp, as previously described (23). Glucose disposal (M) was calculated as the mean rate of glucose infusion during the last 60 min of the examination and was expressed as milligrams per minute per kilogram fatfree mass (M FFM ). IMT of the common carotid artery was measured by highresolution B-mode ultrasound using an ATL-HDI 3000 system equipped with a 5-MHz linear array transducer, as previously described (24). The protocol was approved by the ethics committees, and informed written consent was obtained from all participants. The investigations were performed in accordance with the principles of the Declaration of Helsinki. Plasma insulin and IGF-1 concentrations were determined by a chemiluminescence-based assay. Relationships between variables were sought by stepwise multivariate linear regression analysis. RESULTS -Because insulin sensitivity is a continuous trait, there is n...

Activation of inflammatory pathways may contribute to the beginning and the progression of both atherosclerosis and type 2 diabetes. Here we report a novel interaction between insulin action and control of inflammation, resulting in glucose intolerance and vascular inflammation and amenable to therapeutic modulation. In insulin receptor heterozygous (Insr +/-) mice, we identified the deficiency of tissue inhibitor of metalloproteinase 3 (Timp3, an inhibitor of both TNF-α-converting enzyme [TACE] and MMPs) as a common bond between glucose intolerance and vascular inflammation. Among Insr +/-mice, those that develop diabetes have reduced Timp3 and increased TACE activity. Unchecked TACE activity causes an increase in levels of soluble TNF-α, which subsequently promotes diabetes and vascular inflammation. Double heterozygous Insr +/-Timp3 +/-mice develop mild hyperglycemia and hyperinsulinemia at 3 months and overt glucose intolerance and hyperinsulinemia at 6 months. A therapeutic role for Timp3/TACE modulation is supported by the observation that pharmacological inhibition of TACE led to marked reduction of hyperglycemia and vascular inflammation in Insr +/-diabetic mice, as well as by the observation of increased insulin sensitivity in Tace +/-mice compared with WT mice. Our results suggest that an interplay between reduced insulin action and unchecked TACE activity promotes diabetes and vascular inflammation.

Aims/hypothesis We examined the phenotype of individuals with impaired fasting glucose (IFG) and/or impaired glucose tolerance (IGT) with regard to insulin release and insulin resistance. Methods Non-diabetic offspring (n=874; mean age 40± 10.4 years; BMI 26.6±4.9 kg/m 2 ) of type 2 diabetic patients from five different European Centres (Denmark, Finland, Germany, Italy and Sweden) were examined with regard to insulin sensitivity (euglycaemic clamps), insulin release (IVGTT) and glucose tolerance (OGTT). The levels of glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP) were measured during the OGTT in 278 individuals. Results Normal glucose tolerance was found in 634 participants, while 110 had isolated IFG, 86 had isolated IGT and 44 had both IFG and IGT, i.e. about 28% had a form of reduced glucose tolerance. Participants with isolated IFG had lower glucose-corrected first-phase (0-10 min) and higher second-phase insulin release (10-60 min) during the IVGTT, while insulin sensitivity was reduced in all groups with abnormal glucose tolerance. Similarly, GLP-1 but not GIP levels were reduced in individuals with abnormal glucose tolerance. Conclusions/interpretation The primary mechanism leading to hyperglycaemia in participants with isolated IFG is likely to be impaired basal and first-phase insulin secretion, whereas in isolated IGT the primary mechanism leading to postglucose load hyperglycaemia is insulin resistance. Reduced GLP-1 levels were seen in all groups with abnormal glucose tolerance and were unrelated to the insulin release pattern during an IVGTT.

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