PI3Kγ within a nonhematopoietic cell type negatively regulates diet-induced thermogenesis and promotes obesity and insulin resistance

Abstract

Obesity is associated with a chronic low-grade inflammation, and specific antiinflammatory interventions may be beneficial for the treatment of type 2 diabetes and other obesity-related diseases. The lipid kinase PI3Kγ is a central proinflammatory signal transducer that plays a major role in leukocyte chemotaxis, mast cell degranulation, and endothelial cell activation. It was also reported that PI3Kγ activity within hematopoietic cells plays an important role in obesity-induced inflammation and insulin resistance. Here, we show that protection from insulin resistance, metabolic inflammation, and fatty liver in mice lacking functional PI3Kγ is largely consequent to their leaner phenotype. We also show that this phenotype is largely based on decreased fat gain, despite normal caloric intake, consequent to increased energy expenditure. Furthermore, our data show that PI3Kγ action on diet-induced obesity depends on PI3Kγ activity within a nonhematopoietic compartment, where it promotes energetic efficiency for fat mass gain. We also show that metabolic modulation by PI3Kγ depends on its lipid kinase activity and might involve kinase-independent signaling. Thus, PI3Kγ is an unexpected but promising drug target for the treatment of obesity and its complications.

Fig. 1.

PI3Kγ ablation in mice leads to dramatic protection from diet-induced obesity and fatty liver. (A) Real-time qPCR analysis of PI3Kγ mRNA levels in tissues from lean mice, diet-induced obese mice (HFD), and genetically obese mice (ob/ob). (B) Immunoblot analysis of PI3Kγ in different adipose tissue pads from WT mice kept on standard chow diet. Protein extracts from WAT of PI3Kγ^−/− mice are loaded as control, and protein extracts from heart of WT mice are loaded for comparison. (C) Immunoblot analysis of PI3Kγ in epididymal adipose tissue of lean mice, HFD-induced obese mice, and ob/ob mice. (D) Densitometric quantification of the blots in C. (E) Immunoblot analysis of PI3Kγ in adipocytes and stromal vascular fractions (SVFs) of epididymal adipose tissue from diet-induced obese mice. (F) Growth curves of WT or PI3Kγ^−/− mice placed on HFD. The beginning of HFD treatment is indicated. (G) H&E staining of liver sections from WT or PI3Kγ^−/− mice placed on HFD. (H) Body composition of WT or PI3Kγ^−/− mice placed on HFD.

Fig. 2.

PI3Kγ^−/− mice placed on HFD display markedly improved glucose homeostasis, insulin sensitivity, and decreased adipose tissue inflammation. (A) GTT of WT and PI3Kγ^−/− mice placed on HFD. (B) ITT of WT and PI3Kγ^−/− mice on HFD. (C–I) Hyperinsulinemic euglycemic clamp analysis of WT and PI3Kγ^−/− mice on HFD. (C) Glucose infusion rate. (D) Glucose disposal rate. (E) Percent of suppression of hepatic glucose production. (F) 2DOG uptake in gastrocnemius or BAT. (G) In vivo rate of glycolysis and storage of glucose to glycogen and lipids. (H) Glucose infusion rates from C are plotted for each clamped mouse vs. their body weights. Glucose infusion rates not normalized per body mass are plotted for each clamped mouse vs. their body weights. (J) Real-time qPCR analysis of mRNA levels of inflammatory markers in WAT from WT and PI3Kγ^−/− mice placed on HFD.

Fig. 3.

PI3Kγ-mediated metabolic modulation operates within a nonhematopoietic compartment. (A) List of the radiation chimeras generated and experimental design. (B) Growth curves of the different radiation chimeras kept on HFD. (C) GTT of the radiation chimeric mice on HFD. (D) Insulin tolerance of the radiation chimeras on HFD. (E) H&E staining of liver sections from the different radiation chimeras on HFD. (F) MAC2 staining of WAT sections from the above-described chimeras, and (G) quantification of crown-like structures from F expressed as percentage of controls (WT + WT-BM). (H) Real-time qPCR analysis of mRNA levels of inflammatory markers in WATs from the different radiation chimeras placed on HFD. P values in B–D are for comparisons between WT + WT-BM and PI3Kγ^−/− + WT-BM.

Fig. 4.

Loss of PI3Kγ reduces weight gain efficiency on HFD and promotes adaptive thermogensis to HFD. (A–H) Energy balance study of WT or PI3Kγ^−/− mice placed on HFD for 6-wk: (A) growth curve, (B) fecal lipid content, (C) weekly weight gains, (D) 6-wk cumulative weight gain, (E) weekly food intakes, (F) 6-wk cumulative food intake, (G) weight gain efficiency (expressed as gained weight per food intake), and (H) average weight gain efficiency over 6 wk. (I and J) Indirect calorimetric analysis of WT and PI3Kγ^−/− mice during the transition from chow to HFD.

Fig. 5.

The metabolic phenotype of PI3Kγ^−/− mice is qualitatively conserved at thermoneutrality. (A) Growth curves of WT or PI3Kγ^−/− mice placed on HFD in a thermoneutral environment (30 °C). (B) H&E staining of liver sections from WT or PI3Kγ^−/− mice on HFD at 30 °C. (C) GTT of WT and PI3Kγ^−/− mice on HFD at 30 °C. (D) ITT of WT and PI3Kγ^−/− mice on HFD at 30 °C. (E) qPCR analysis of mRNA levels of inflammatory markers in epididimal adipose tissue from WT and PI3Kγ^−/− mice kept on HFD at 30 °C. Mice were 8 wk old when placed on HFD.

Fig. 6.

Specific loss of PI3Kγ kinase-dependent signaling is sufficient to recapitulate the metabolic phenotype of PI3Kγ^−/− mice. (A) Growth curves of WT or PI3Kγ kinase-dead mutants PI3Kγ^KD/KD mice kept on HFD. (B) H&E staining of liver sections from WT or PI3Kγ^KD/KD mice on HFD. (C) GTT of WT and PI3Kγ^KD/KD mice on HFD. (D) ITT of WT and PI3Kγ^KD/KD mice on HFD. (E) Real-time PCR analysis of mRNA levels of inflammatory markers in WAT from WT and PI3Kγ^KD/KD mice on HFD.

Fig. 7.

PI3Kγ metabolic modulation may implicate kinase-dependent and -independent pathways. (A) Immunoblot analysis of HSL phosphorylation and UCP-1 protein levels in BAT extracts from WT, PI3Kγ^−/−, and PI3Kγ^KD/KD mice in the fed state placed on HFD for 3 wk. (B) Densitometric quantification of the immunoblots in A. (C) Immunblot analysis of HSL phosphorylation in WAT. (D) Densitometric quantification of the immunoblots in C. (E and F) Our interpretation of PI3Kγ action in diet-induced obesity and insulin resistance is discussed. (E) PI3Kγ ablation protects from diet-induced insulin resistance by two mechanisms: (i) resistance to diet-induced obesity because of the lack of PI3Kγ in a nonhematopoietic compartment, which operates already from the first week of HFD and persists in time, and (ii) a direct effect of PI3Kγ ablation on leukocyte chemotaxis, a mechanism that is not recruited until mice reach the morbidly obese range. (F) Possible roles for PI3Kγ catalytic activity and kinase-independent scaffolding function in diet-induced obesity are discussed.

Fig. P1.

A model for the metabolic action of PI3Kγ in diet-induced obesity and insulin resistance. (A) PI3Kγ gene deletion protected mice from diet-induced insulin resistance by two mechanisms: (i) resistance to diet-induced obesity because of a lack of PI3Kγ activity in nonhematopoietic cells (this effect is detected in the first week of HFD and persists in time) and (ii) a direct effect of PI3Kγ gene deletion on the chemically directed movement of white blood cells (leukocyte chemotaxis), a mechanism that is not activated until mice reach the morbidly obese stage. (B) Possible roles of PI3Kγ lipid kinase activity and kinase-independent scaffolding signaling pathways in diet-induced obesity and insulin resistance are proposed.