Phosphoinositide 3-kinase regulatory subunit p85alpha suppresses insulin action via positive regulation of PTEN

Abstract

The phosphoinositide 3-kinase (PI3K) pathway is central to the metabolic actions of insulin on liver. Here, we show that mice with a liver-specific deletion of the p85alpha regulatory subunit of PI3K (L-Pik3r1KO) exhibit a paradoxical improvement of hepatic and peripheral insulin sensitivity. Although PI3K enzymatic activity is diminished in L-Pik3r1KO livers because of a reduced level of regulatory and catalytic subunits of PI3K, insulin-stimulated Akt activity is actually increased. This increased Akt activity correlates with increased phosphatidylinositol (3,4,5)-trisphosphate levels which are due, at least in part, to diminished activity of the (3,4,5)-trisphosphate phosphatase PTEN. Thus, the regulatory subunit p85alpha is a critical modulator of insulin sensitivity in vivo not only because of its effects on PI3K activation, but also as a regulator of PTEN activity.

Fig. 1.

Metabolic phenotype of L-Pik3r1KO mice. (A) Western blots for Pik3r1 gene products with an antibody against the N-terminal SH2 domain (pan-p85) in tissue lysates, as indicated, from control, heterozygous KO, and L-Pik3r1KO mice. Tissues were collected from mice after an overnight fast, and proteins were extracted and processed as described in Materials and Methods. Each lane represents lysates from a different mouse. (B and C) Fasted blood glucose and fasted serum insulin levels. (D and E) Serum triglycerides and serum nonesterified free fatty acid (FFA) levels from lox/lox or KO mice in the fasted state. (F) Glucose tolerance tests (2 g/kg, i.p.) were performed on mice after a 16-h fast, and blood samples were collected and glucose measured at the times indicated. All values are presented as mean ± SEM (n = 6–20). Open circles, lox/lox; filled circles, L-Pik3r1KO.

Fig. 2.

Hyperinsulinemic–euglycemic clamp analyses and gene expression changes in lox/lox (WT) and L-Pik3r1KO mice. We subjected male mice (n = 11) of the indicated genotype at 10–12 weeks of age to hyperinsulinemic–euglycemic clamp analysis and measured insulin suppression of HGP (A), glucose infusion rates (B), and in vivo ¹⁴C-deoxyglucose uptake in muscle (C) and epididymal fat tissue (D). (E and F) Weight of entire animals or of epididymal (WAT) and brown (BAT) fat pads. WAT and BAT are expressed as a percentage of body weight. (G) Quantitative RT-PCR analysis of mRNA levels in lox/lox and L-Pik3r1KO mice of phosphoenolpyruvate carboxykinase (Pck1), glucose-6-phosphatase (G6Pc), fructose-1,6-bisphosphatase (Fbp1), peroxisome proliferator-activated receptor (PPAR) γ coactivator 1α (Ppargc1), tribbles3 (trib3), and glucokinase (Gck1). n = 8 for each genotype for RT-PCR experiments. ∗, P < 0.05 vs. lox/lox mice.

Fig. 3.

Enhanced Akt activation in L-Pik3r1KO mice. (A) PI3K activity in IRS-1, IRS-2, and pTyr immunoprecipitates (bars represent mean ± SEM; n = 5; ∗, P < 0.05). (B and C) Western blot of p110α and p85α from p110α immunoprecipitates and pTyr and insulin receptor (IR) blots from insulin receptor (β-subunit) immunoprecipitates. (D) Ser-473 phosphorylation of Akt (Upper) and Akt kinase activity as measured from Akt immunoprecipitates by using Crosstide as a substrate (Lower). Bars represent mean ± SEM; n = 8; ∗, P < 0.05 vs. insulin-stimulated lox/lox mice.

Fig. 4.

Enhanced PIP₃ levels in L-Pik3r1KO mice due to decreased PTEN activity. (A) Immunofluorescent staining with a primary anti-PIP₃ antibody (IgM) and an anti-mouse secondary antibody conjugated to Alexa Fluor red and counterstained with DAPI. After an overnight fast, mice were injected with saline (time = 0) or 5 units of insulin for the indicated amount of time. Six mice of each genotype/treatment were fixed via cardiac perfusion of 10% buffered formalin in PBS. (B) Quantification of the immunofluorescence from PIP₃ staining. Representative slides were chosen from each mouse, and the fluorescence intensity was measured and analyzed with vh-h1a5 analyzer software (KEYENCE, Osaka, Japan). (C and D) Insulin-stimulated pTyr-associated PI3K activity and PTEN activity and PTEN protein levels in lox/lox or KO animals at the indicated time points after insulin stimulation. ∗, P < 0.05 vs. lox/lox after 5 min of insulin stimulation.