Marta Letizia Hribal

Defects of the insulin receptor substrate (IRS) system in human metabolic disorders

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, subcellu‐lar 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 β cells). In fact, IRS‐1 appears to have its major role in skeletal muscle whereas IRS‐2 appears to regulate he‐patic insulin action as well as pancreatic β 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 patho‐genic 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.—Sesti, G., Federici, M., Hribal, M. L., Lauro, D., Sbraccia, P., Lauro, R. Defects of the insulin receptor substrate (IRS) system in human metabolic disorders. FASEB J. 15, 2099–2111 (2001)

Insulin-Dependent Activation of Endothelial Nitric Oxide Synthase Is Impaired by O-Linked Glycosylation Modification of Signaling Proteins in Human Coronary Endothelial Cells

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 data show that hyperglycemia, through the hexosamine pathway, impairs activation of the IR/IRS/PI3-K/Akt pathway, resulting in deregulation of eNOS activity.

Regulation of insulin-like growth factor–dependent myoblast differentiation by Foxo forkhead transcription factors

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.

The Gly972-->Arg amino acid polymorphism in IRS-1 impairs insulin secretion in pancreatic beta cells.

Recent studies have identified several polymorphisms in the human insulin receptor substrate-1 (IRS-1) gene. The most prevalent IRS-1 variant, a Gly-->Arg change at the codon 972, has been reported to be increased in prevalence among patients with type 2 diabetes. Carriers of the Arg(972) substitution are characterized by lower fasting insulin and C-peptide levels compared with non-carriers, suggesting that the Arg(972) IRS-1 variant may contribute to impairment of insulin secretion. In this study, we stably overexpressed both wild-type IRS-1 (RIN-WT) and Arg(972) IRS-1 variant (RIN-Arg(972)) in RIN beta cells to investigate directly whether the polymorphism in codon 972 of IRS-1 impairs insulin secretion. The Arg(972) IRS-1 variant did not affect expression or function of endogenous IRS-2. RIN-WT showed a marked increase in both glucose- and insulin-stimulated tyrosine phosphorylation of IRS-1 compared with control RIN cells. The Arg(972) IRS-1 variant did not alter the extent of either glucose- or insulin-stimulated tyrosine phosphorylation of recombinant IRS-1. However, RIN-Arg(972) showed a significant decrease in binding of the p85 subunit of phosphatidylinositol-3-kinase (PI 3-kinase) with IRS-1, compared with RIN-WT. Compared with control RIN cells, insulin content was reduced to the same extent in RIN-WT or RIN-Arg(972) at both the protein and mRNA levels. Both glucose- and sulfonylurea-induced insulin secretion was increased in RIN-WT compared with control RIN cells. By contrast, RIN cells expressing Arg(972) IRS-1 exhibited a marked decrease in both glucose- and sulfonylurea-stimulated insulin secretion compared with RIN-WT. These data suggest that the insulin signaling pathway involving the IRS-1/PI 3-kinase may play an important role in the insulin secretory process in pancreatic beta cells. More importantly, the results suggest that the common Arg(972) IRS-1 polymorphism may impair glucose-stimulated insulin secretion, thus contributing to the relative insulin deficiency observed in carriers of this variant.

Timp3 deficiency in insulin receptor–haploinsufficient mice promotes diabetes and vascular inflammation via increased TNF-α

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-alpha-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-alpha, 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.

Insulin Secretion in Metabolically Obese, but Normal Weight, and in Metabolically Healthy but Obese Individuals

Metabolically obese but normal‐weight (MONW) individuals present metabolic disturbances typical of obese individuals. Additionally, metabolically healthy but obese (MHO) individuals have been identified who are relatively insulin sensitive and have a favorable cardiovascular risk profile. We compared insulin secretion patterns of MONW and MHO with those of two age‐matched groups comprising nonobese individuals or obese insulin‐resistant subjects, respectively. To this end, 110 nonobese subjects and 87 obese subjects were stratified into quartile based on their insulin‐stimulated glucose disposal (MFFM). Insulin secretion was estimated by acute insulin response (AIR) during an intravenous glucose‐tolerance test (IVGTT), and the disposition index was calculated as AIR × MFFM. We found that, as defined, MFFM was lower in MONW, who exhibited higher triglycerides, free‐fatty acid (FFA), and 2‐h postchallenge glucose levels compared to normal nonobese group. Insulin secretion was higher in MONW than in normal nonobese subjects, but disposition index was lower in MONW. Disposition index did not differ between MONW and insulin‐resistant obese. MFFM was higher in MHO who exhibited lower waist circumference, blood pressure (BP), triglycerides, FFA, insulin levels, and higher high‐density lipoprotein (HDL) cholesterol compared to insulin‐resistant obese. Insulin secretion did not differ between insulin‐resistant obese and MHO, but disposition index was lower in the former group. In conclusion, MONW and insulin‐resistant obese showed decreased compensatory insulin secretion compared to normal nonobese and MHO subjects, respectively. Because these subjects also exhibited a worse metabolic risk profile, these findings may account for their increased risk for type 2 diabetes.

Elevated one-hour post-load plasma glucose levels identifies subjects with normal glucose tolerance but early carotid atherosclerosis

OBJECTIVE To examine whether individuals with normal glucose tolerance (NGT), whose 1-h post-load plasma glucose is >or=155 mg/dl, or with impaired glucose tolerance (IGT) have an increased carotid intima-media thickness (IMT), as compared with NGT individuals with 1-h post-load plasma <155 mg/dl. METHODS Atherosclerosis risk factors, oral glucose tolerance test (OGTT), and ultrasound manual measurement of IMT were analyzed in 400 non-diabetic Caucasians. RESULTS As compared with individuals with a 1-h post-load plasma glucose <155 mg/dl, NGT individuals with a 1-h post-load plasma glucose >or=155 mg/dl exhibited higher hsCRP (2.0+/-1.5 vs. 1.5+/-1.0, P=0.008), and IMT (0.82+/-0.20 vs. 0.71+/-0.16; P=0.006), and lower insulin sensitivity (71+/-39 vs. 105+/-57; P<0.0001), and IGF-1 levels (214+/-88 vs. 176+/-49; P<0.03). No significant differences were observed in metabolic and cardiovascular risk factors between IGT and NGT subjects with a 1-h post-load glucose >or=155 mg/dl. Of the three glycemic parameters, 1-h and 2-h post-load glucose, but not fasting glucose, were significantly correlated with IMT. In a stepwise multivariate regression analysis in a model including age, gender, and a variety of atherosclerosis risk factors, the three variables that remained significantly associated with IMT were age (P<0.0001), BMI (P<0.0001), and 1-h post-load glucose (P=0.02) accounting for 20.2% of its variation. CONCLUSIONS NGT subjects with a 1-h post-load glucose >or=155 mg/dl have an atherogenic profile similar to IGT individuals. These data suggest that a cutoff point of 155 mg/dl for the 1-h post-load glucose during OGTT may be helpful in the identification of NGT subjects at increased risk for cardiovascular disease.

Role of the forkhead protein FoxO1 in beta cell compensation to insulin resistance

Diabetes is associated with defective beta cell function and altered beta cell mass. The mechanisms regulating beta cell mass and its adaptation to insulin resistance are unknown. It is unclear whether compensatory beta cell hyperplasia is achieved via proliferation of existing beta cells or neogenesis from progenitor cells embedded in duct epithelia. We have used transgenic mice expressing a mutant form of the forkhead-O1 transcription factor (FoxO1) in both pancreatic ductal and endocrine beta cells to assess the contribution of these 2 compartments to islet expansion. We show that the mutant FoxO1 transgene prevents beta cell replication in 2 models of beta cell hyperplasia, 1 due to peripheral insulin resistance (Insulin receptor transgenic knockouts) and 1 due to ectopic local expression of IGF2 (Elastase-IGF2 transgenics), without affecting insulin secretion. In contrast, we failed to detect a specific effect of the FoxO1 transgene on the number of periductal beta cells. We propose that beta cell compensation to insulin resistance is a proliferative response of existing beta cells to growth factor signaling and requires FoxO1 nuclear exclusion.

Role of transglutaminase 2 in glucose tolerance: knockout mice studies and a putative mutation in a MODY patient

Transglutaminase 2 (TGase 2) is a Ca+2‐ dependent enzyme that catalyzes both intracellular and extracellular cross‐linking reactions by transamidation of specific glutamine residues. TGase 2 is known to be involved in the membrane‐mediated events required for glucose‐stimulated insulin release from the pancreatic β cells. Here we show that targeted disruption of TGase 2 impairs glucose‐stimulated insulin secretion. TGase 2‐/‐mice show glucose intolerance after intraperitoneal glucose loading. TGase 2‐/‐mice manifest a tendency to develop hypoglycemia after administration of exogenous insulin as a consequence of enhanced insulin receptor substrate 2 (IRS‐2) phosphorylation. We suggest that the increased peripheral sensitivity to insulin partially compensates for the defective secretion in this animal model. TGase 2‐/‐mouse phenotype resembles that of the maturity‐onset diabetes of young (MODY) patients. In the course of screening for human TGase 2 gene in Italian subjects with the clinical features of MODY, we detected a missense mutation (N333S) in the active site of the enzyme. Collectively, these results identify TGase 2 as a potential candidate gene in type 2 diabetes.—Bernassola, F., Federici, M., Corazzari, M., Terrinoni, A., Hribal, M. L., De Laurenzi, V., Ranalli, M., Massa, O., Sesti, G., Mclean, W. H. I., Citro, G., Barbetti, F., Melino, G. Role of transglutaminase 2 in glucose tolerance: knockout mice studies and a putative mutation in a MODY patient. FASEB J. 16, 1371–1378 (2002)

Mice Heterozygous for Tumor Necrosis Factor-α Converting Enzyme Are Protected From Obesity-Induced Insulin Resistance and Diabetes

OBJECTIVE—Tumor necrosis factor (TNF)-α is known to affect insulin sensitivity, glucose, and lipid metabolism through alternative and redundant mechanisms at both translational and post-translational levels. TNF-α exerts its paracrine effects once the membrane-anchored form is shed and released from the cell membrane. TNF-α cleavage is regulated by TNF-α converting enzyme (TACE), which regulates the function of several transmembrane proteins, such as interleukin-6 receptor and epidermal growth factor receptor ligands. The role of TACE in high-fat diet (HFD)-induced obesity and its metabolic complications is unknown. RESEARCH DESIGN AND METHODS—To gain insights into the role of TACE in metabolic disorders, we used Tace+/− mice fed a standard or high-fat diet for 16 weeks. RESULTS—We observed that Tace+/− mice are relatively protected from obesity and insulin resistance compared with wild-type littermates. When fed an HFD, wild-type mice exhibited visceral obesity, increased free fatty acid and monocyte chemoattractant protein (MCP)1 levels, hypoadiponectinemia, glucose intolerance, and insulin resistance compared with Tace+/− mice. Interestingly, Tace+/− mice exhibited increased uncoupling protein-1 and GLUT4 expression in white adipose tissue. CONCLUSIONS—Our results suggest that modulation of TACE activity is a new pathway to be investigated for development of agents acting against obesity and its metabolic complications.