Inactivation of class II PI3K-C2α induces leptin resistance, age-dependent insulin resistance and obesity in male mice - PubMed (original) (raw)

Inactivation of class II PI3K-C2α induces leptin resistance, age-dependent insulin resistance and obesity in male mice

Samira Alliouachene et al. Diabetologia. 2016 Jul.

Abstract

Aims/hypothesis: While the class I phosphoinositide 3-kinases (PI3Ks) are well-documented positive regulators of metabolism, the involvement of class II PI3K isoforms (PI3K-C2α, -C2β and -C2γ) in metabolic regulation is just emerging. Organismal inactivation of PI3K-C2β increases insulin signalling and sensitivity, whereas PI3K-C2γ inactivation has a negative metabolic impact. In contrast, the role of PI3K-C2α in organismal metabolism remains unexplored. In this study, we investigated whether kinase inactivation of PI3K-C2α affects glucose metabolism in mice.

Methods: We have generated and characterised a mouse line with a constitutive inactivating knock-in (KI) mutation in the kinase domain of the gene encoding PI3K-C2α (Pik3c2a).

Results: While homozygosity for kinase-dead PI3K-C2α was embryonic lethal, heterozygous PI3K-C2α KI mice were viable and fertile, with no significant histopathological findings. However, male heterozygous mice showed early onset leptin resistance, with a defect in leptin signalling in the hypothalamus, correlating with a mild, age-dependent obesity, insulin resistance and glucose intolerance. Insulin signalling was unaffected in insulin target tissues of PI3K-C2α KI mice, in contrast to previous reports in which downregulation of PI3K-C2α in cell lines was shown to dampen insulin signalling. Interestingly, no metabolic phenotypes were detected in female PI3K-C2α KI mice at any age.

Conclusions/interpretation: Our data uncover a sex-dependent role for PI3K-C2α in the modulation of hypothalamic leptin action and systemic glucose homeostasis.

Access to research materials: All reagents are available upon request.

Keywords: Food intake; Glucose homeostasis; Insulin; Insulin resistance; Knock-in leptin; Leptin resistance; Mouse gene targeting; Obesity; PI3K.

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Figures

Fig. 1

Generation and characterisation of C2αD1268A/WT KI mice. (a) Gene targeting strategy to introduce the D1268A mutation in the DFG motif in exon 24 of the Pik3c2a gene. The FRT-flanked cassette encoding the Pgk Neo selection marker was removed in vivo by breeding onto ACTB-Flp mice. (b) PI3K-C2α protein expression. Tissue homogenates were analysed by SDS–PAGE and immunoblotting using anti-PI3K-C2α antibody. (c) PI3K isoform expression in WT and C2αD1268A/WT cells and tissue. Each lane on the SDS–PAGE gel represents an independent mouse. Homogenates of MEFs or epididymal WAT from male mice were analysed by SDS–PAGE and immunoblotting using the indicated antibodies. (d) Lipid kinase activity associated with PI3K-C2α in WT and C2αD1268A/WT mice. Homogenates of MEFs or epididymal WAT from male mice were immunoprecipitated using PI3K-C2α antibody, and subjected to an in vitro PI3K activity assay. Results shown are pooled data from three independent experiments (each with 2–4 experimental replicates). *p < 0.05; ***p < 0.001

Fig. 2

Normal glucose homeostasis and insulin sensitivity in 12-week-old C2αD1268A/WT mice and hyperleptinaemia in 12-week-old male C2αD1268A/WT mice. Data represent mean ± SEM. (a–d) Fasted glycaemia in males (a) and in females (b) and fed glycaemia in males (c) and in females (d). n = 20–32 mice were used. (e–h) Plasma insulin levels under fasted condition in males (e) and in females (f) and under fed condition in males (g) and in females (h). n = 7–16 mice were used. (i–j) GTT. Results shown are pooled data from four or five independent experiments for male (i) and female (j) mice. The AUC is shown. n = 12–28 mice were used. (k, l) ITT. Results shown are pooled data from three independent experiments for male (k) and for female (l) mice. The AUC is shown. n = 8–13 mice were used. Solid line, WT; dashed line, C2αD1268A/WT. *p < 0.05

Fig. 3

Leptin resistance in male 12-week-old C2αD1268A/WT mice. Data represent mean ± SEM, except for (f) mean ± SD. (a) Cumulative food intake over a 28-day period in 12-week-old mice. n = 4/7 (WT/C2αD1268A/WT) mice were used. (b–e) Serum leptin levels in 12-week-old male (b) and female (c) mice and in 32-week-old male (d) and female (e) mice. n = 8–13 (WT/C2αD1268A/WT) mice were used. (f) Whole-body weight of 12-week-old mice. n = 6 mice were used. (g) Hypothalamic homogenates isolated from 12-week-old mice 30 min after i.p. injection of 2.5 mg/kg leptin were analysed by SDS–PAGE and immunoblotting using the indicated antibodies. Representative western blots are shown and quantification is based on pooled data from three independent experiments with 2–4 mice/condition/experiment. Each lane on the SDS–PAGE gel represents an independent mouse. (h) Defective functional response to exogenous leptin in 12-week-old mice. The data show food intake, before and after daily injection of vehicle or leptin. n = 5–7 mice were used. Solid line, WT; dashed line, C2αD1268A/WT. *p < 0.05; **p < 0.01

Fig. 4

Age-dependent adiposity in male 32-week-old C2αD1268A/WT mice. Data represent mean ± SEM. (a–e) Whole-body weight variation upon ageing of male mice at 5 weeks old (a), 9 weeks old (b), 13 weeks old (c), 21 weeks old (d) and 32 weeks old (e). (f–h) Organ weight to body weight ratios from 32-week-old mice. Epididymal WAT (f), gastrocnemius (g) and soleus (h). n = 6–8 mice were used. (i–k) Epididymal WAT histology from 32-week-old mice. HE staining of epididymal WAT sections is shown (i). Mean adipocyte areas (j) and adipocyte area distribution profiles (k) are also shown. Data are representative of epididymal WAT sections from individual mice. n = 9–12 mice were used. Scale bar: 20 μm. White bars, WT; black bars, C2αD1268A/WT. (l) Liver histology. HE staining and Oil Red O staining of liver sections. Data are representative of liver sections from n = 9–12 mice. Scale bar: 20 μm. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 5

Hyperglycaemia, glucose intolerance and insulin resistance in male 32-week-old C2αD1268A/WT mice. Data represent mean ± SEM. (a, b) Glycaemia under fed (a) and fasted (b) conditions of 32-week-old mice. n = 16/39 mice were used. (c, d) Plasma insulin levels under fed (c) and fasted (d) conditions of 32-week-old mice. n = 13/22 mice were used. (e) GTT showing glycaemia and (f) insulinaemia. Results shown are five independent experiments using mice from different litters. The AUC is shown. n = 14–31 (for glycaemia); n = 9–11 (for insulinaemia) mice were used. (g) ITT. Results shown are pooled data from three independent experiments using independent litters. The AUC is shown. n = 9–14 mice were used. (h) HOMA-IR index. n = 8–11 mice were used. (i) Insulin signalling in liver, muscle (gastrocnemius) and WAT of 32-week-old mice. Homogenates from mice injected i.p. with 0.75 U/kg insulin (30 min) were analysed by SDS–PAGE and immunoblotting using the indicated antibodies. Representative western blots are shown and quantification is based on pooled data from two independent experiments with 2–4 mice/condition/experiment. Each lane on the SDS–PAGE gel represents an independent mouse. Solid line, WT; dashed line, C2αD1268A/WT. *p < 0.05; **p < 0.01

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Inactivation of class II PI3K-C2α induces leptin resistance, age-dependent insulin resistance and obesity in male mice - PubMed (original) (raw)