JCI - The Gly972→Arg amino acid polymorphism in IRS-1 impairs insulin secretion in pancreatic β cells

The Gly⁹⁷²→Arg amino acid polymorphism in IRS-1 impairs insulin secretion in pancreatic β cells

Ottavia Porzio, Massimo Federici, Marta Letizia Hribal, Davide Lauro, Domenico Accili, Renato Lauro, Patrizia Borboni, Giorgio Sesti

Published in Volume 104, Issue 3

J Clin Invest. 1999; 104(3):357–364 doi:10.1172/JCI5870

Abstract | Full text | PDF

R ecent 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 Arg⁹⁷² substitution are characterized by lower fasting insulin and C-peptide levels compared with non-carriers, suggesting that the Arg⁹⁷² IRS-1 variant may contribute to impairment of insulin secretion. In this study, we stably overexpressed both wild-type IRS-1 (RIN-WT) and Arg⁹⁷² IRS-1 variant (RIN-Arg⁹⁷²) in RIN β cells to investigate directly whether the polymorphism in codon 972 of IRS-1 impairs insulin secretion. The Arg⁹⁷² 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 Arg⁹⁷² IRS-1 variant did not alter the extent of either glucose- or insulin-stimulated tyrosine phosphorylation of recombinant IRS-1. However, RIN-Arg⁹⁷² 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-Arg⁹⁷² 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 Arg⁹⁷² 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 β cells. More importantly, the results suggest that the common Arg⁹⁷² IRS-1 polymorphism may impair glucose-stimulated insulin secretion, thus contributing to the relative insulin deficiency observed in carriers of this variant.

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Figure 2

Glucose- and insulin-stimulated tyrosine phosphorylation of IRS-1, and association with p85 subunit of PI 3-kinase in RIN β cells expressing wild-type IRS-1 and Arg⁹⁷² IRS-1. RIN-WT and RIN-Arg⁹⁷² cells were incubated in the presence or absence of 2.8 mM glucose for 0, 2, and 10 minutes, or in the presence or absence of 100 nM insulin for 2 minutes, at 37°C. The cells were then lysed, and equal amounts of total proteins were immunoprecipitated (IP) with anti–IRS-1 antibody, subjected to SDS-PAGE, and Western immunoblotted (WB) with either anti-phosphotyrosine antibody (top) or anti-p85 antibody (bottom). Proteins were detected by using enhanced chemiluminescence, and band densities were quantified by densitometry. The autoradiographs shown are representative of 3 independent experiments.