The common Arg 972 polymorphism in insulin receptor substrate‐1 causes apoptosis of human pancreatic islets

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Abstract
SPECIFIC AIMS Molecular scanning of the human IRS-1 gene revealed that diabetic and prediabetic carriers of a common polymorphism causing a Gly to Arg change at codon 972 (Arg972 IRS-1) are characterized by a low fasting plasma concentration of insulin and C peptide. We have directly addressed the effect of Arg972 IRS-1 variant in human pancreatic islet survival and characterized a downstream signaling pathway involved in anti-apoptotic effects of insulin in pancreatic β cells. PRINCIPAL FINDINGS 1. Survival of human pancreatic islets and RIN β cells expressing Arg972 IRS-1 To investigate directly whether the Arg972 IRS-1 variant affects islet cells survival, we analyzed pancreatic islets isolated from three donors heterozygous for the Arg972 and six donors carrying wild-type IRS-1. Immunofluorescence analysis revealed increased apoptosis induced by serum deprivation of insulin-positive β cells in islets from the carriers of Arg972 IRS-1 (Fig. 1A⤻ , lower panel) as compared with wild-type (Fig. 1A⤻ , upper panel). To confirm this finding quantitatively, we used flow cytometry to determine the number of hypodiploid events after propidium iodide staining of fixed cells. Under basal conditions, islets from carriers of Arg972 IRS-1 variant showed a twofold increase in the number of apoptotic cells as compared with wild-type (45±8% vs. 16±4%, respectively; PB⤻ ). Apoptosis induced by serum deprivation was also increased in islets from the carriers of Arg972 IRS-1 as compared with wild-type (65±12% vs. 21±5%, respectively; PB⤻ ). To determine whether insulin protects islets from apoptosis, we examined the effect of insulin on the survival of human islets subjected to serum deprivation. Addition of insulin at the time of serum deprivation was less effective at inhibiting apoptosis in islet cells from Arg972 IRS-1 carriers as compared with wild-type (57±15 vs. 13±2%, respectively; P≤0.006) (Fig. 1B⤻ ). We next compared IRS-1-associated PI 3-kinase activity in islet cells from carriers of Arg972 IRS-1 variant and wild-type carriers. Compared with wild-type carriers, PI 3-kinase activity associated with IRS-1 in islet cells from Arg972 IRS-1 carriers was decreased by 17% (PP972 IRS-1 variant, we used RIN β cell lines stably expressing equal amounts of either wild-type IRS-1 (RIN-WT) or Arg972 IRS-1 (RIN-Arg972). We performed flow cytometry using annexin V/PI double staining with fresh unfixed cells. The dot plot from RIN-WT cells did not show relevant positiveness to annexin V, whereas RIN-Arg972 cells showed marked positiveness to annexin V, which revealed higher apoptosis than RIN-WT cells (Fig. 1C⤻ ). Figure 1. Arg972-IRS-1 causes vulnerability to apoptosis in human islets and pancreatic β cells. A) Apoptosis in human islets from WT-IRS-1 and Arg972-IRS-1 donors is shown in green, and insulin-positive cells in red. B) Human islets from six WT-IRS-1 and three Arg972-IRS-1 donors were analyzed for apoptosis by propidium iodide under basal conditions (white bars), after serum deprivation (grey bars), and with the addition of 100 nM insulin to serum-deprived islets (hashed bars). C) Apoptotic population analyzed by annexin V/PI double staining in RIN-Arg972 and RIN-WT after serum deprivation. Download figure Download PowerPoint 2. PI-3-kinase activity and Akt phosphorylation, but not MAPK activation, are reduced in RIN β cells expressing wild-type IRS-1 or Arg972 IRS-1 The extent of basal and insulin-stimulated tyrosine phosphorylation of IRS-1 was similar in both RIN-WT and RIN-Arg972 cells (Fig. 2A⤻ ). As compared with RIN-WT cells, IRS-1-associated PI-3 kinase activity in RIN-Arg972 cells was reduced by 20% (P < 0.05) in the basal state and by 50% upon stimulation with insulin for 2 min, 10 min, or 20 min (P < 0.04) (Fig. 2A⤻ ). Basal Ser473-Akt phosphorylation was decreased by 20% in RIN-Arg972 compared with RIN-WT cells (P < 0.03) (Fig. 2A⤻ ). Insulin-induced Ser473-Akt phosphorylation was reduced in RIN-Arg972 by 50% (P < 0.001) (Fig. 2A⤻ ). Basal and insulin-stimulated MAPK phosphorylation did not differ between RIN-WT and RIN-Arg972 cells (data not shown). Figure 2. Basal and insulin-induced activation of IRS-1, PI 3-kinase, Akt, BAD, and caspase-9 pathway in RIN-WT and RIN-Arg972 cells. A) IRS-1 tyrosine phosphorylation, IRS-1-associated PI-3 kinase activity, Ser473-Akt phosphorylation, Ser136-BAD, and Ser112-BAD phosphorylation. B) BAD coprecipitation with Bcl-XL or 14-3-3 proteins. C) Caspase-9 and caspase-3 activities in the basal state and after serum deprivation. Data and images represent three independent experiments performed in duplicate. Download figure Download PowerPoint 3. BAD phosphorylation and association with Bcl-XL or 14–3-3 protein in RIN β cells expressing wild-type IRS-1 or Arg972 IRS-1 Compared with RIN-WT cells, BAD phosphorylation at Ser136 in RIN-Arg972 was decreased by 30% (P < 0.04) in the basal state, and by 50% upon stimulation with insulin (P < 0.004) (Fig....