Altered Expression of Insulin Signaling Proteins in Islets from T2DM Female Mice

Abstract

With development and progression of T2DM in KKAy mice, pancreatic islets alter their expression of GLUT2, insulin and insulin receptor. The study’s goal was to identify glucoregulatory proteins in the insulin receptor signaling pathway that were early markers of islet dysfunction after 4 weeks of uncontrolled hyperglycemia. Isolated islets were pooled from 2 groups of 12 week old mice: insulin-resistant, euglycemic KK mice and insulin-resistant, hyperglycemic KKAy T2DM mice. Samples were analyzed for 40 proteins using 221 antibodies in an insulin phospho antibody array. After 4 weeks of hyperglycemia in KKAy mice, many proteins were not altered as compared to KK mice (ratio: 0.94-1.02) including insulin receptor (Phospho-Tyr1355), FOXO1A (Phospho-Ser329), PI3-kinase p85-alpha (Phospho-Tyr607), ERK1/2, PP1-alpha, and AMPK1. The phosphorylation states of some proteins were altered including AKT1 (Phosphor-Ser124) (0.77) and IRS-1 (Phosphor-Ser636) (1.25). Three proteins were then selected for ELISA analysis in islets isolated from 4, 8 (onset of T2DM), and 12 week old mice: GLUT2, iNOS (1.41) and CBL (Phospho-Tyr700) (0.78), an E3 ubiquitin ligase. GLUT2 was lower in KKAy mice at 4 weeks (KK, 4.0±0.5; KKAy, 2.3±0.3, p<0.001), but not at 8 or 12 weeks, because GLUT2 decreased with age in both groups (12 weeks: KK, 1.0±0.1; KKAy, 1.4±0.1, p=0.31) [age*mouse: F(2,18)=11.104, p=0.001]. CBL-b was higher in KKAy mice (KK, 0.50±0.09; KKAy, 0.65±0.07) [F(1,17)=4.527, p=0.048] and decreased with age in both groups (12 week: KK, 0.30±0.02; KKAy, 0.45±0.13) [F(2,17)=12.231, p=0.001]. In contrast, iNOS was lower at 4 week (KK, 3.0±0.3; KKAy, 2.0±0.4; p=0.029), then higher at 8 and 12 week (KK, 0.8±0.4; KKAy, 2.1±0.2; p=0.007) with iNOS steady in KKAy mice [age*mouse: F(2,16)=10.902, p=0.001]. With short-term hyperglycemia in KKAy mice, many insulin signaling proteins were not altered as compared to diabetes susceptible KK mice. At onset on T2DM in KKAy mice, sustained iNOS and elevated CBL-b were early markers of islet dysfunction. Disclosure K.L. Sondgeroth: None. K. LePard: None.