Reduced expression of the murine p85alpha subunit of phosphoinositide 3-kinase improves insulin signaling and ameliorates diabetes

Abstract

A critical component of insulin action is the enzyme phosphoinositide (PI) 3-kinase. The major regulatory subunits of PI 3-kinase, p85alpha and its splice variants, are encoded by the Pik3r1 gene. Heterozygous disruption of Pik3r1 improves insulin signaling and glucose homeostasis in normal mice and mice made insulin-resistant by heterozygous deletion of the Insulin receptor and/or insulin receptor substrate-1 (IRS1) genes. Reduced expression of p85 modulates the molecular balance between this protein, the p110 catalytic subunit of PI 3-kinase, and the IRS proteins. Thus, despite the decrease in p85alpha, PI 3-kinase activation is normal, insulin-stimulated Akt activity is increased, and glucose tolerance and insulin sensitivity are improved. Furthermore, Pik3r1 heterozygosity protects mice with genetic insulin resistance from developing diabetes. These data suggest that regulation of p85alpha levels may provide a novel therapeutic target for the treatment of type 2 diabetes.

Figure 1

Creation of mutant mice. (a) Mice were bred and genotyped using PCR. The presence of the IR mutant allele results in a 240-bp product, while there is no product in the wild-type (WT) allele. The presence of the wild-type allele for the IRS-1 gene results in a 440-bp product; insertion of the neomycin cassette in the targeted allele results in a band of 1.25 kb. In the case of the Pik3r1 gene, the mutant allele results in a band at 600 bp, whereas the wild-type allele yields a 750-bp product. (b) Body weight of the various subgroups of mice was determined at the indicated ages. Results are expressed as mean ± SEM (n = 12–30 mice per genotype).

Figure 2

Signaling molecules involved in activation of PI 3-kinase by insulin. (a) Expression levels of Pik3r1 gene products were determined in lysates from liver (left) and skeletal muscle (right) by Western blotting with an anti-p85pan antibody (αp85pan). (b) Tyrosine phosphorylation of IRS proteins and association with p85 were determined using lysates from liver of the indicated animals. Proteins were immunoprecipitated with anti–IRS-1 antibody (αIRS-1) (top two panels) or anti–IRS-2 antibody (αIRS-2) (bottom two panels), and blotted with anti-phosphotyrosine antibody (αPY) or αp85pan. (c) To determine the molecular balance between p85 and p110, the liver lysates were subjected to three sequential rounds of immunodepletion using αp110, followed by Western blotting with αp110 (left top panel) or αp85pan (left bottom panel). The amount of the p85-p110 dimer and the p85 monomer was expressed as the ratio to the value of the amount of the p85 monomer in the wild-type cells. In the graph, each bar represents the ratio to the total p85 in wild-type cells (open bar, p85-p110 dimer; dotted bar, p85 monomer).

Figure 3

PI 3-kinase activation in liver and muscle of Pik3r1 mutant mice. Lysates from the liver of animals were immunoprecipitated with the indicated antibodies and subjected to a PI 3-kinase assay as described in Methods. PI 3-kinase activities associated with total regulatory subunit (αp85pan) (a), associated with p85α regulatory subunit (αp85α) (b), associated with the p110 catalytic subunit (αp110) (c), associated with tyrosine-phosphorylated proteins (αPY) (d), and associated with p85β regulatory subunit (αp85β) (e) were assessed. The upper panels shows representative PI 3-kinase assays, while each bar in the lower panels represents the mean ± SEM of the relative PI 3-kinase activity (% of unstimulated wild-type) calculated from at least three independent experiments. *P < 0.05, wild-type vs. p85^+/–. (f) Activation of PI 3-kinase in muscle. Lysates were immunoprecipitated with the p85pan antibody (αp85pan, top panel), tyrosine-phosphorylated proteins (αPY, middle panel), or the p85β regulatory subunit (αp85β, bottom panel), and subjected to PI 3-kinase assay as above.

Figure 4

Insulin-stimulated Akt activity in Pik3r1 mutant mice. Lysates from liver and muscle were subjected to Western blotting with αp-Akt (top panels). Akt activity was assessed following immunoprecipitation with an anti-Akt antibody in an immune complex kinase assay (bottom panels). The results are expressed as percent of unstimulated wild-type. Each bar represents the mean ± SEM of at least four independent experiments. *P < 0.05, wild-type vs. p85^+/– and IR/IRS-1^+/– vs. IR/IRS-1/p85^+/–.

Figure 5

Improved glucose homeostasis in the Pik3r1 mutant groups. Fasting blood glucose (a) and insulin levels (b), and random-fed blood glucose (c) and insulin levels (d), were determined in 6-month-old male mice of the indicated genotype. For random-fed glucose levels, a scattered plot is represented; bars represent the mean ± SEM (n = 12–30 mice per genotype). Glucose (e) and insulin (f) concentrations were determined during a glucose tolerance test (2 g/kg body weight, intraperitoneally) at the indicated time points in 6-month-old male mice of the indicated genotypes. Results are expressed as mean ± SEM (n = 12–30 mice). *P < 0.05, wild-type vs. p85^+/–; IR^+/– vs. IR/p85^+/–; IR/IRS-1^+/– vs. IR/IRS-1/p85^+/–.

Figure 6

Increased insulin sensitivity in the Pik3r1 mutant groups. (a) Insulin tolerance tests (1 U/kg, intraperitoneally) and (b) IGF-1 tolerance tests (1 mg/kg, intraperitoneally) were performed on 6-month-old male mice of the indicated genotypes. Results represent the blood glucose concentration as a percentage of the starting glucose value and are expressed as mean ± SEM (n = 12–30 mice per genotype). *P < 0.05, wild-type vs. p85^+/–; IR^+/– vs. IR/p85^+/–; IR/IRS-1^+/– vs. IR/IRS-1/p85^+/–. (c) Glucose transport activity in isolated skeletal muscle of the Pik3r1 mutant mice was estimated using 2-deoxyglucose uptake in isolated EDL and soleus muscles as described in Methods. Results are expressed as mean ± SEM (n = 4). *P < 0.05, wild-type vs. p85^+/–.