Increased insulin action in SKIP heterozygous knockout mice

Abstract

Insulin controls glucose homeostasis and lipid metabolism, and insulin impairment plays a critical role in the pathogenesis of diabetes mellitus. Human skeletal muscle and kidney enriched inositol polyphosphate phosphatase (SKIP) is a member of the phosphatidylinositol 3,4,5-trisphosphate phosphatase family (T. Ijuin et al. J. Biol. Chem. 275:10870-10875, 2000; T. Ijuin and T. Takenawa, Mol. Cell. Biol. 23:1209-1220, 2003). Previous studies showed that SKIP negatively regulates insulin-induced phosphatidylinositol 3-kinase signaling (Ijuin and Takenawa, Mol. Cell. Biol. 23:1209-1220, 2003). We now have generated mice with a targeted mutation of the mouse ortholog of the human SKIP gene, Pps. Adult heterozygous Pps mutant mice show increased insulin sensitivity and reduced diet-induced obesity with increased Akt/protein kinase B (PKB) phosphorylation in skeletal muscle but not in adipose tissue. The insulin-induced uptake of 2-deoxyglucose into the isolated soleus muscle was significantly enhanced in Pps mutant mice. A hyperinsulinemic-euglycemic clamp study also revealed a significant increase in the rate of systemic glucose disposal in Pps mutant mice without any abnormalities in hepatic glucose production. Furthermore, in vitro knockdown studies in L6 myoblast cells revealed that reduction of SKIP expression level increased insulin-stimulated Akt/PKB phosphorylation and 2-deoxyglucose uptake. These results imply that SKIP regulates insulin signaling in skeletal muscle. Thus, SKIP may be a promising pharmacologic target for the treatment of insulin resistance and diabetes.

FIG. 1.

The Pps^Brdm1 allele and the phenotypic analysis of Pps^Brdm1/+ mice. (A) Schematic of the genomic Pps locus, the insertional targeting vector (pTVPps), and the targeted allele Pps^Brdm1. Restriction sites are as follows: B, BclI; R, EcoRI. Triangle, loxP; Neo, neomycin-resistance gene cassette; 5′-Hprt, nonfunctional 5′-Hprt cassette. The probes and diagnostic restriction fragments are indicated. (B) Southern blot genotyping of agouti offspring of chimeras was performed with EcoRI-digested tail DNA and probe A. Southern blot genotyping of embryonic day 7.5 embryos, generated by intercrossing of Pps^Brdm1/+ mice, was done with BclI-digested DNA and probe B. (C) Northern blot analysis of total RNA from quadriceps of wild-type and Pps^Brdm1/+ mice. Pps and Gapdh cDNA fragments were used as probes. (D) Western blot analysis of SKIP, SHIP2, and PTEN using quadriceps lysates of Pps^Brdm1/+ mice (n = 6) and wild-type mice (n = 6). Quantification of the Western blotting assay was analyzed by densitometry. WT, wild type.

FIG. 2.

SKIP mutant mice exhibited normal food intake and adiposity. (A) Growth curves of wild-type (n = 21) and Pps^Brdm1/+ (n = 27) mice fed a normal chow diet. (B) Food intake of wild-type (n = 12) and Pps^Brdm1/+ (n = 12) male mice fed a normal chow diet at 15 weeks of age. All the parameters are normalized by total body weight. (C) Feeding efficiency of wild-type (n = 6) and Pps^Brdm1/+ (n = 6) male mice fed a normal chow diet at 15 weeks of age. Feeding efficiency was calculated by gain in body weight normalized by food intake. (D) Percentage of quadriceps content isolated from wild-type mice (n = 18) and Pps^Brdm1/+ (n = 18) male mice at 15 weeks of age. All values are given as means ± standard errors of the means. WT, wild type.

FIG. 3.

Enhanced glucose homeostasis in Pps^Brdm1/+ mice. An insulin tolerance test (A) and an oral glucose tolerance test (B) of wild-type (n = 6) and Pps^Brdm1/+ (n = 6) male mice were performed. Filled columns indicate wild-type mice; open columns indicate Pps^Brdm1/+ mice. (C) Plasma insulin levels of wild-type (n = 3) and Pps^Brdm1/+ (n = 3) male mice in response to glucose injection. Glucose-induced insulin secretion was unchanged in Pps^Brdm1/+ mice. (D) Hyperinsulinemic-euglycemic clamp analysis in wild-type (n = 3) and Pps^Brdm1/+ (n = 3) mice from 17 weeks of age. Glucose infusion rate (GIR), rate of insulin-stimulated glucose disposal (Rd), and basal or insulin-induced hepatic glucose production (BHGP or IHGP, respectively) are indicated. (E and F) Clamp-based insulin-induced Akt/PKB phosphorylation in isolated soleus muscle (TA, for tibialis anterior) and EWAT in wild-type and Pps^Brdm1/+ mice. All values are given as means ± standard errors of the means. WT, wild type.

FIG. 4.

Enhanced insulin signaling in skeletal muscle of Pps^Brdm1/+ mice. (A) Phosphorylation of Akt/PKB in quadriceps in response to insulin. Mice were fasted overnight and injected intraperitoneally with insulin (1U/kg of body weight) for the indicated times. Levels of Akt/PKB phosphorylation on Ser-473 and Thr-308 were quantified, and results are shown in the graphs. (B and C) Activation of insulin signaling in quadriceps of Pps^Brdm1/+ mice. Phosphorylation of p70 S6 kinase (B) and GSK3β (C) are analyzed. (D) Enhanced insulin-induced glucose uptake in soleus muscle from Pps^Brdm1/+ (n = 6) male mice in comparison with wild-type (n = 6) mice at 12 weeks of age. (E) Tyrosine phosphorylation of insulin receptor was not changed between Pps^Brdm1/+ mice and wild-type mice. All values are given as means ± standard errors of the means. *, P < 0.05; **, P < 0.01. All values are normalized relative to basal phosphorylation of each protein in wild-type mice. WT, wild-type.

FIG. 5.

Pps^Brdm1/+ mice are resistant to diet-induced insulin resistance. (A) Western blot analysis of insulin-induced Akt/PKB phosphorylation on Thr-308 and Ser-473 in quadriceps isolated from Pps^Brdm1/+ mice fed a normal chow diet (ND) or an HFD. The quantifications of the Western blot analysis are shown. Phosphorylation levels were quantified and normalized by values for wild-type mice fed a normal diet. An insulin tolerance test (B) and an oral glucose tolerance test (C) were performed on wild-type (n = 6) and Pps^Brdm1/+ (n = 6) mice fed an HFD for 4 months. All values are given as means ± standard errors of the means. *, P < 0.05; **, P < 0.01. WT, wild type.

FIG. 6.

Enhanced insulin signaling by SKIP suppression in L6 myoblast cells. (A) Reduction of SKIP enhances insulin-induced Akt/PKB phosphorylation. L6 cells were transfected with control or SKIP siRNA and stimulated with insulin. Cells lysates were used for the detection for Akt/PKB and phosphorylated Ser-473 or Thr-308 of Akt/PKB. A time course of insulin-induced Akt phosphorylation is shown. (B) SKIP plays distinct roles from PTEN and SHIP2 in insulin signaling. L6 cells transfected with SKIP siRNA and PtdIns(3,4,5)P₃ phosphatases (PTEN, SHIP2, or SKIP) were stimulated with insulin for 20 min. Phosphorylation of Ser-473 and Thr-308 of Akt/PKB is shown. The enhanced effect of insulin on 2-deoxyglucose incorporation (C) and GLUT4 translocation (D) by reduction of SKIP is shown. Insulin-induced (100 nM) 2-deoxyglucose uptake and GLUT4 translocation are shown. All values are given as means ± standard errors of the means. GLUT4 reporter was expressed in L6 cells or L6 cells in conjunction with siRNA vectors. Ins, insulin.