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

Abstract

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.

Figure 1

Quantitation of IRS-1 and IRS-2 expressed in RIN β cells. RIN cells were stably transfected with expression vectors for wild-type IRS-1 or Arg⁹⁷² IRS-1. The cells were lysed, and equal amount of proteins were immunoprecipitated (IP) with anti–IRS-1 or anti–IRS-2 antibody, separated by SDS-PAGE, transferred to PVDF membrane, and Western immunoblotted (WB) with either anti–IRS-1 (top) or anti–IRS-2 (bottom) antibody. Proteins were detected by using enhanced chemiluminescence, and band densities were quantified by densitometry. The autoradiographs shown are representative of 4 independent experiments.

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.

Figure 3

Glucose- and insulin-stimulated tyrosine phosphorylation of endogenous IRS-2, 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-2 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.

Figure 4

Insulin mRNA analysis. Total RNA was extracted from control RIN, RIN-WT, and RIN-Arg⁹⁷² and blotted onto nylon membranes. The blots were hybridized with a 450-kb mouse insulin cDNA labeled with [α-³²P]dCTP and were analyzed by an image-analyzer. The autoradiograph shown is representative of 3 independent experiments.

Figure 5

Time course of glucose-stimulated insulin secretion. Control RIN (diamonds), RIN-WT (squares), and RIN-Arg⁹⁷² (triangles) cells were incubated in the presence or absence of 2.8 mM glucose for the indicated periods of time. Aliquots of the supernatant were collected, and insulin concentration was determined by RIA. Results shown are representative of 3 independent experiments.

Figure 6

Fractional insulin secretion in response to glucose. Control RIN (diamonds), RIN-WT (squares), and RIN-Arg⁹⁷² (triangles) cells were incubated in the presence or absence of increasing concentrations of glucose. At the end of incubation, total cellular insulin content was extracted into acidified ethanol, and aliquots of the supernatant were collected. Insulin concentration in both cellular extracts and supernatant was determined by RIA. (a) Results are expressed as the amount of secreted insulin divided by the total insulin content. The data points are the mean ± SE of 5 independent experiments, each carried out in triplicate. (b) The same results are expressed as the percentage of fractional insulin secretion above basal. *P < 0.05, **P < 0.01.

Figure 7

Fractional insulin secretion in response to Glybenclamide. Control RIN, RIN-WT, and RIN-Arg⁹⁷² cells were incubated in glucose-free buffer in the presence or absence of increasing concentration of Glybenclamide. At the end of incubation, total cellular insulin content was extracted into acidified ethanol, and aliquots of the supernatant were collected. Insulin concentration in both cellular extracts and supernatant was determined by RIA. The amount of secreted insulin was normalized by the total insulin content. The results are expressed as the percentage of fractional insulin secretion above basal and are the mean ± SE of 4 independent experiments, each carried out in triplicate. *P < 0.05.