p50alpha/p55alpha phosphoinositide 3-kinase knockout mice exhibit enhanced insulin sensitivity - PubMed (original) (raw)

p50alpha/p55alpha phosphoinositide 3-kinase knockout mice exhibit enhanced insulin sensitivity

Dong Chen et al. Mol Cell Biol. 2004 Jan.

Abstract

Class Ia phosphoinositide (PI) 3-kinases are heterodimers composed of a regulatory and a catalytic subunit and are essential for the metabolic actions of insulin. In addition to p85alpha and p85beta, insulin-sensitive tissues such as fat, muscle, and liver express the splice variants of the pik3r1 gene, p50alpha and p55alpha. To define the role of these variants, we have created mice with a deletion of p50alpha and p55alpha by using homologous recombination. These mice are viable, grow normally, and maintain normal blood glucose levels but have lower fasting insulin levels. Results of an insulin tolerance test indicate that p50alpha/p55alpha knockout mice have enhanced insulin sensitivity in vivo, and there is an increase in insulin-stimulated glucose transport in isolated extensor digitorum longus muscle tissues and adipocytes. In muscle, loss of p50alpha/p55alpha results in reduced levels of insulin-stimulated insulin receptor substrate 1 (IRS-1) and phosphotyrosine-associated PI 3-kinase but enhanced levels of IRS-2-associated PI 3-kinase and Akt activation, whereas in adipocytes levels of both insulin-stimulated PI 3-kinase and Akt are unchanged. Despite this, adipocytes of the knockout mice are smaller and have increased glucose uptake with altered glucose metabolic pathways. When treated with gold thioglucose, p50alpha/p55alpha knockout mice become hyperphagic like their wild-type littermates. However, they accumulate less fat and become mildly less hyperglycemic and markedly less hyperinsulinemic. Taken together, these data indicate that p50alpha and p55alpha play an important role in insulin signaling and action, especially in lipid and glucose metabolism.

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Figures

FIG. 1.

FIG. 1.

Gene targeting of the p50α/p55α locus. (A) Protein structures of the class Ia PI 3-kinase regulatory subunits. SH, Src homology; P, proline-rich region; BH, BCR homology. (B) Genomic structure of the mouse pik3r1 gene (top), targeting vector (middle), and targeted locus after homologous recombination (bottom). P, _Pst_I; E, Eco N1; Neo, neomycin cassette; TK, thymidine kinase. P1, P2, and P3 are primers for genotyping. (C) PCR genotyping of tail DNA identifying wild-type (+/+), p50α/p55α+/−, and p50α/p55α−/− mice.

FIG. 1.

FIG. 1.

Gene targeting of the p50α/p55α locus. (A) Protein structures of the class Ia PI 3-kinase regulatory subunits. SH, Src homology; P, proline-rich region; BH, BCR homology. (B) Genomic structure of the mouse pik3r1 gene (top), targeting vector (middle), and targeted locus after homologous recombination (bottom). P, _Pst_I; E, Eco N1; Neo, neomycin cassette; TK, thymidine kinase. P1, P2, and P3 are primers for genotyping. (C) PCR genotyping of tail DNA identifying wild-type (+/+), p50α/p55α+/−, and p50α/p55α−/− mice.

FIG. 1.

FIG. 1.

Gene targeting of the p50α/p55α locus. (A) Protein structures of the class Ia PI 3-kinase regulatory subunits. SH, Src homology; P, proline-rich region; BH, BCR homology. (B) Genomic structure of the mouse pik3r1 gene (top), targeting vector (middle), and targeted locus after homologous recombination (bottom). P, _Pst_I; E, Eco N1; Neo, neomycin cassette; TK, thymidine kinase. P1, P2, and P3 are primers for genotyping. (C) PCR genotyping of tail DNA identifying wild-type (+/+), p50α/p55α+/−, and p50α/p55α−/− mice.

FIG. 2.

FIG. 2.

Tissue expression of p50α and p55α in wild-type mice and evidence of deletion in p50α/p55α knockout mice. Isolated adipocytes (A), livers (B), muscle extracts (C), and a combination of equal amounts of these tissues (D) prepared from wild-type (W) and knockout (K) mice were subjected to Western immunoblotting (IB) with anti-p85α pan antisera (Upstate Biotechnology, Inc.) raised against the common N-SH2 domain of p85α, p55α, and p50α.

FIG. 3.

FIG. 3.

Increased in vivo insulin sensitivity in p50α/p55α knockout mice. (A) Glucose and insulin concentrations in randomly fed and fasting mice. Blood glucose (left panels) and insulin (right panels) concentrations were determined by tail bleeding in 5-month-old mice. Experimental groups consisted of 10 animals each. Values depict means ± standard errors of the means (SEM) (*, P < 0.05). WT, wild-type mice; KO, knockout mice. (B) Results of insulin tolerance tests (ITT) (left panel) and glucose tolerance tests (GTT) (right panel) for wild-type (WT) and p50α/p55α knockout (p50α/p55α KO) mice. Insulin and glucose tolerance tests were performed on 6-month-old mice with 0.75 U of insulin/kg of body weight and 2 g of glucose/kg of body weight, respectively. Each experimental group consisted of 10 animals. Values represent means ± SEM (**, P < 0.01).

FIG. 4.

FIG. 4.

Glucose transport in EDL muscles and isolated adipocytes. EDL muscle strips (A) and adipocytes (B) were isolated from wild-type (WT) and p50α/p55α−/− (KO) mice, and glucose transport activities were determined without (basal) or with insulin at the indicated concentrations. Four to six independent observations were made under each condition. Results shown are means ± SEM (**, P < 0.01).

FIG. 5.

FIG. 5.

Breakdown of glucose metabolism into different pathways by using isolated adipocytes. Metabolism of glucose into lactate (A), triglyceride (TG) (B), and CO2 (C) was measured in isolated adipocytes from 4-month-old wild-type (WT) and p50α/p55α−/− (KO) mice without (basal) or with 80 nM insulin. Four independent observations were made under each condition. Results shown are means ± SEM (**, P < 0.01).

FIG. 6.

FIG. 6.

Insulin signaling in muscles of wild-type (W) and p50α/p55α knockout (K) mice. Mice were injected with saline (−) or 5 U of insulin (+) via inferior venae cavae as described in Materials and Methods. (A) Muscle extracts were prepared and immunoprecipitated (IP) with anti-insulin receptor (IR; Santa Cruz), anti-IRS-1 (Santa Cruz), or anti-IRS-2 (Upstate Biotechnology) antibody. The immunoprecipitates were subjected to anti-PY (Transduction Laboratory) Western immunoblotting (IB). Ins, insulin. (B) Muscle extracts were immunoprecipitated with anti-PY (Santa Cruz), anti-IRS-1, or anti-IRS-2 antibody. The immunoprecipitates were subjected to anti-p85α pan (Upstate Biotechnology) Western immunoblotting. (C) Aliquots of the immunoprecipitates described in the legend to panel B were subjected to a PI 3-kinase assay. WT, wild-type mice; KO, knockout mice. (D) Muscle extracts were subjected to anti-phospho-Akt (Ser473) and anti-Akt (Cell Signaling Technology, Inc.) Western immunoblotting and an Akt in vitro kinase assay. The graphs in panels C and D depict the average activities in three to four animals of each genotype and condition. Results shown are means ± SEM (*, P < 0.05).

FIG. 7.

FIG. 7.

Epididymal (Epi) fat pad masses (n, 9 wild-type [WT] and 10 knockout [KO] mice) (A), total body triglyceride (TG) contents (n, 10 in each group) (B), serum leptin levels in fasting mice (n, 10 in each group) (C), and daily food intake (n, 10 wild-type and 6 knockout mice) (D) in 5-month-old wild-type and p50α/p55α knockout mice. All the measurements were normalized to body weights (BW) of individual mice, and the values are expressed as means ± SEM (*, P < 0.05; **, P < 0.01).

FIG. 8.

FIG. 8.

Lipid contents (n, nine in each group) (A), cell numbers (n, nine wild-type and eight knockout mice) (B), and hematoxylin and eosin (H&E) staining (C) of isolated adipocytes from 5-month-old wild-type (WT) and p50α/p55α knockout (KO) mice. Values in panels A and B are expressed as means ± SEM (**, P < 0.01). In panel C, each field is a representative hematoxylin-and-eosin-stained image from a separate animal of the indicated genotype.

FIG. 8.

FIG. 8.

Lipid contents (n, nine in each group) (A), cell numbers (n, nine wild-type and eight knockout mice) (B), and hematoxylin and eosin (H&E) staining (C) of isolated adipocytes from 5-month-old wild-type (WT) and p50α/p55α knockout (KO) mice. Values in panels A and B are expressed as means ± SEM (**, P < 0.01). In panel C, each field is a representative hematoxylin-and-eosin-stained image from a separate animal of the indicated genotype.

FIG. 9.

FIG. 9.

Effects of GTG on wild-type (WT) and p50α/p55α knockout (KO) mice. (A) Two-month-old wild-type (n = 9) and knockout (n = 9) mice were injected with 0.5 mg of GTG/g of body weight. Daily food intakes of GTG-treated wild-type and knockout mice were measured 6 weeks after injection. (B) Body weights of GTG-injected (n, nine in each group) and saline-injected control (n, six wild-type and nine knockout) mice were monitored for up to 9 weeks postinjection. (C and D) Glucose and insulin concentrations of fed mice were determined with GTG-injected mice and saline-injected control mice 8 weeks after GTG injection (**, P < 0.01). (E) Epididymal (Epi) fat pad masses normalized to body weights (BW) of the GTG-treated wild-type and knockout mice were determined 10 weeks after injection (*, P < 0.05). All values are expressed as means ± SEM.

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