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PNAS 103 (32): 12093-12097

Copyright © 2006 by the National Academy of Sciences.

Phosphoinositide 3-kinase regulatory subunit p85{alpha} suppresses insulin action via positive regulation of PTEN

Cullen M. Taniguchi*, Thien T. Tran*, Tatsuya Kondo{dagger}, Ji Luo{ddagger},§, Kohjiro Ueki, Lewis C. Cantley{ddagger},§,||, and C. Ronald Kahn*,**

*Cellular and Molecular Physiology, Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215; {dagger}Department of Metabolic Medicine, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8555, Japan; {ddagger}Department of Systems Biology, Harvard Medical School, Boston, MA 02215; §Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA 02115; and Department of Metabolic Diseases, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan


Figure 1
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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.

 

Figure 2
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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) {gamma} coactivator 1{alpha} (Ppargc1), tribbles3 (trib3), and glucokinase (Gck1). n = 8 for each genotype for RT-PCR experiments. *, P < 0.05 vs. lox/lox mice.

 

Figure 3
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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 p110{alpha} and p85{alpha} from p110{alpha} 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.

 

Figure 4
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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.

 


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