Specific roles of the p110alpha isoform of phosphatidylinsositol 3-kinase in hepatic insulin signaling and metabolic regulation

Abstract

The class I(A) phosphatidylinsositol 3-kinases (PI3Ks) form a critical node in the insulin metabolic pathway; however, the precise roles of the different isoforms of this enzyme remain elusive. Using tissue-specific gene inactivation, we demonstrate that p110alpha catalytic subunit of PI3K is a key mediator of insulin metabolic actions in the liver. Thus, deletion of p110alpha in liver results in markedly blunted insulin signaling with decreased generation of PIP(3) and loss of insulin activation of Akt, defects that could not be rescued by overexpression of p110beta. As a result, mice with hepatic knockout of p110alpha display reduced insulin sensitivity, impaired glucose tolerance, and increased gluconeogenesis, hypolipidemia, and hyperleptinemia. The diabetic syndrome induced by loss of p110alpha in liver did not respond to metformin treatment. Together, these data indicate that the p110alpha isoform of PI3K plays a fundamental role in insulin signaling and control of hepatic glucose and lipid metabolism.

Figure 1. Metabolic Phenotype of Mice with Acute Hepatic Loss of p110α

(A) Fed and fasted blood glucose levels in mice of the indicated genotypes were measured 12 days after adenovirus injection (n = 8–10). (B) Serum insulin levels in the fasted and random fed states were measured 2 weeks after adenovirus injection (n = 8–10). (C) Glucose tolerance tests (GTT) on mice of indicated genotypes 12 days after adenovirus injection (n = 9). (D) Insulin tolerance tests (ITT) on mice of indicated genotypes 2 weeks after adenovirus injection. Results represent blood glucose concentration as a percentage of starting value at zero time (n = 7–9). (E) Pyruvate challenge on mice of indicated genotypes 16 days after adenovirus injection (n = 7–8). (F) Quantitative RT-PCR analysis of mRNA levels of phosphoenolpyruvate carboxykinase (pck1), glucose 6-phosphatase (g6pc), fructose 1,6-bisphosphatase (fbp1), and hepatic nuclear factor-4α (hnf4α) from the fasting mouse livers 3 weeks after adenovirus injection.

Figure 2. Hepatic Insulin Signaling in Mice with Chronic Deletion of p110α

(A) Livers from control flox mice and mice with chronic deletion of p110α were fixated, and liver sections were stained with antibodies for PIP₃ lipids at 0′, 5′, and 15′ after vena cava insulin or saline injection. Data represent mean ± SEM (n = 4), *p < 0.05. (B) PI3K activity from liver lysates immunoprecipitated with phosphotyrosine (PY) or IRS-1 of fasted mice of indicated genotypes (n = 4–5), *p < 0.05. (C) Western blot of liver protein lysates from p110α flox and L-p110α KO mice treated with saline or insulin for 5′ through inferior vena cava injection. (D) Quantitative RT-PCR of p110α (pik3ca) gene expression in liver from mice injected with adenovirus expressing GFP, a wild-type p110α, or a kinase-dead (KD) mutant of p110α lacking the kinase domain. (E) Western blot of liver lysates prepared from p110α flox and L-p110α KO mice injected with control adenovirus (GFP) or adenovirus expressing a wild-type or a KD version of p110α. Mice were treated with either saline or insulin for 5′ through inferior vena cava injection. (*The p110α antibody does not recognize the truncated KD p110α.) (F) Western blot of liver protein lysates prepared from control flox and L-p110α KO mice subjected to tail vein injection with control adenovirus expressing GFP or adenovirus expressing p110β and treated with saline or insulin for 5′ through inferior vena cava injection. Data are representative of three separate experiments. (G) PI3K activity from liver lysates immunoprecipitated with phosphotyrosine (PY) or IRS-1 of fasted mice of indicated genotypes injected with adenovirus expressing GFP, a wild-type, or a KD version of p110α (n = 2–6).

Figure 3. Physiological Characteristics of Mice with Chronic Hepatic Deletion of p110α

(A) Whole-body weight. Open circles, p110α flox control mice; filled circles, L-p110α KO mice. Data represent mean ± SEM (n = 8–10), *p < 0.05. (B) Tissue weight. Data represent mean ± SEM (n = 8–10), *p < 0.05. (C) Activity level of male flox controls and L-p110α KO mice during 72 hr in metabolic chambers. The inset shows the area under the curve (AUC) for both genotypes. (D) Serum glucose levels of p110α flox and L-p110α KO mice at 10 and 18 weeks, random fed and fasted state. (E) Serum insulin levels of p110α flox and L-p110α KO mice at 10 and 18 weeks, random fed and fasted state. (F) Serum lipid levels of p110α flox and L-p110α KO mice at 8 and 18 weeks, random fed state.

Figure 4. Metabolic Phenotype of Mice with Chronic Deletion of p110α

(A) Mice were given an i.p. injection of 2 g dextrose/kg BW after an overnight fast. (B) Mice were given an i.p. injection of 1.25 U/kg BW insulin. Results represent blood glucose concentration as a percentage of starting value atzerotime. Areaunderthe curve (AUC) was calculated with baseline = 100% of basal. Open circles, p110α flox; filled circles, L-p110α KO. Data represent mean ± SEM (n = 8–10), *p < 0.05. (C and D) Insulin sensitivity quantified as glucose infusion rate (GIR) (C) and hepatic glucose production (HGP) (D) in response to insulin were measured by hyperinsulinemic-euglycemic clamp (n = 3–4), *p < 0.05. (E) Mice were treated with metformin for 3 weeks, and GTT was performed by i.p. injection of 2 g dextrose/kg BW after an overnight fast. Ob/ob mice were used as controls. The inset shows the AUC for untreated or metformin-treated ob/ob mice. (F) ITT was performed by i.p. injection of 1.25 U/kg BW insulin. Open circles, L-p110α KO or ob/ob mice treated without metformin; filled circles, L-p110α KO or ob/ob mice treated with 300 mg metformin/kg BW/day for 3 weeks. Data represent mean ± SEM (n = 3–4), *p < 0.05. (G) Serum glucose levels. Data represent mean ± SEM (n = 4).