This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. OVERVIEW: 'Molecular Regulation of Muscle Glucose Metabolism in Man': The presence of insulin resistance in normoglycemic subjects with a strong family history of type 2 diabetes attests to its hereditary nature. The insulin receptor signaling system is a likely focus of insulin resistance. In type 2 diabetes, there are defects in insulin receptor signaling that include decreased insulin stimulation of insulin receptor and IRS-1 tyrosine phosphorylation and the association of PI 3-kinase with IRS-1. In diabetics one bout of exercise improves insulin receptor tyrosine phosphorylation but does not increase the association of IRS-1 with PI 3-kinase, suggesting that the latter abnormality may be intrinsic or hereditary. In normal glucose tolerant subjects with a family history of type 2 diabetes, insulin resistance is associated with a defect in insulin stimulated IRS-1 tyrosine phosphorylation and association of PI 3-kinase activity with IRS-1. In contrast, insulin-stimulated tyrosine phosphorylation of the insulin receptor is normal. Taking these findings together, we hypothesize that a defect in IRS-1 function in skeletal muscle plays a key role in insulin resistance and type 2 diabetes. Specifically, we propose: 1) To identify sites of serine/threonine phosphorylation of endogenous IRS-1 in human skeletal muscle in vivo. We will use recombinant IRS-1 proteins and synthetic peptides to identify candidate serine/threonine phosphorylation sites phosphorylated by known kinases or human skeletal muscle lysates and subcellular fractions. Sites will be identified by consensus sequence analysis, two-dimensional phosphopeptide mapping, MALDI-TOF and HPLC-ESI/MS/MS analysis. Based on these results, we will target the serine/threonine phosphorylation sites that are held in common by recombinant known kinases and human muscle lysates and fractions. Then we will identify endogenous IRS-1 phosphorylation sites by HPLC-ESI/MS/MS and MALDI-TOF analysis of immunoprecipitated IRS-1 from muscle biopsies taken from subjects basally and during insulin infusion (euglycemic hyperinsulinemic clamp). 2) To determine the role of serine/threonine phosphorylation of IRS-1 in insulin resistance. 3) To determine if common mutations in IRS-1 are associated with defects in IRS-1 function in nondiabetic Mexican American individuals who have a strong family history of type 2 diabetes and decreased insulin-stimulated association of IRS-1 with PI 3-kinase. 4) To use DNA microarray technology to search for genes that are abnormally expressed in normal glucose-tolerant subjects with a strong family history of type 2 diabetes. We will test the hypothesis that these subjects respond differently to experimental conditions that provoke insulin resistance. This protocol is one of seven experiments contained in the grant, 'Molecular Regulation of Muscle Glucose Metabolism in Man,' which has an overall hypothesis that 'a defect in IRS-1 function in skeletal muscle plays a key role in insulin resistance and T2DM and may be involved in a hereditary abnormality in the regulation of insulin signaling.' The goal of this study is to investigate the nature of biochemical and molecular abnormalities present in skeletal muscles of type 2 diabetics. Specifically, study 2a will investigate whether insulin resistance is associated with increased serine phosphorylation of IRS-1.
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