mTOR complex 2 in adipose tissue negatively controls whole-body growth

Abstract

Mammalian target of rapamycin (mTOR), a highly conserved protein kinase that controls cell growth and metabolism in response to nutrients and growth factors, is found in 2 structurally and functionally distinct multiprotein complexes termed mTOR complex 1 (mTORC1) and mTORC2. mTORC2, which consists of rictor, mSIN1, mLST8, and mTOR, is activated by insulin/IGF1 and phosphorylates Ser-473 in the hydrophobic motif of Akt/PKB. Though the role of mTOR in single cells is relatively well characterized, the role of mTOR signaling in specific tissues and how this may contribute to overall body growth is poorly understood. To examine the role of mTORC2 in an individual tissue, we generated adipose-specific rictor knockout mice (rictor(ad-/-)). Rictor(ad-/-) mice are increased in body size due to an increase in size of nonadipose organs, including heart, kidney, spleen, and bone. Furthermore, rictor(ad-/-) mice have a disproportionately enlarged pancreas and are hyperinsulinemic, but glucose tolerant, and display elevated levels of insulin-like growth factor 1 (IGF1) and IGF1 binding protein 3 (IGFBP3). These effects are observed in mice on either a high-fat or a normal diet, but are generally more pronounced in mice on a high-fat diet. Our findings suggest that adipose tissue, in particular mTORC2 in adipose tissue, plays an unexpectedly central role in controlling whole-body growth.

Fig. 1.

Adipose-specific rictor knockout mice have increased body weight. (A) Adipose-specific rictor knockout mice (rictor^ad−/−) were generated using the Cre/LoxP system (see Methods). (B) Immunoblot showing knockout of rictor and impaired mTORC2 signaling in adipose tissue of 2 littermates. A short (Right) and long (Left) exposure are shown. Residual rictor protein in rictor^ad−/− in the long exposure is likely from stromal vascular cells in the adipose tissue that do not express aP2-Cre. (C) Weight curves of rictor^fl/fl (n = 16) and rictor^ad−/− (n = 14) male mice 8–18 weeks of age maintained on a chow diet. (D) Weight curves of rictor^fl/fl and rictor^ad−/− male mice fed an HFD for 10 weeks (n = 10 per genotype). (E) Body length determined by measuring nasal-to-anal distance. Mice were put on an HFD at the age of 8 weeks. Values in C–E are mean ± SEM. *P < 0.05; **P < 0.01, rictor^ad−/− vs. rictor^fl/fl.

Fig. 2.

Rictor^ad−/− mice have increased lean mass. (A and B) Lean mass (A) and fat mass (B) in rictor^fl/fl and rictor^ad−/− mice was determined by dexa scan analysis. Mice were maintained on a chow diet or HFD for 6 weeks (n = 6–8 per group). (C–J) Weight of individual organs. Individual organs were excised and weighed. Mice were maintained on a chow diet or HFD for 10 weeks. Bone mineral content was determined by dexa scan analysis as in A and B. Values in A–J are represented as mean ± SEM (n = 11–27 per genotype; n = 5 in F). *P < 0.05; **P < 0.01, rictor^ad−/− vs. rictor^fl/fl.

Fig. 3.

Rictor^ad−/− mice have unaltered adipose tissue morphology and increased hepatic steatosis. (A) Representative image of H&E stained sections of epididymal fat. (B) Rictor^ad−/− and rictor^fl/fl develop hepatic steatosis after HFD. Representative image of liver sections stained with Oil Red O and hematoxylin. (C) Quantification of liver triglycerides in mice fed a chow diet (n = 4 per group) or an HFD for 10 weeks (n = 16 per group).

Fig. 4.

Rictor^ad−/− mice are hyperinsulinemic. (A and B) Blood glucose from overnight fasted or fed mice on either a chow diet (A) or HFD for 10 weeks (B) (n = 10–15). (C and D) Blood insulin from overnight fasted or fed mice on either a chow diet (C) or HFD for 10 weeks (D) (n = 10). (E) Quantification of average islet area represented in arbitrary units (AU). n = 3–5 per group. (F) Representative image of an islet in rictor^fl/fl and rictor^ad−/− mice immunostained for insulin (red) and glucagon (green). Nuclei were stained with DAPI (blue). Images were taken at the same magnification, and islets are shown at the same scale. (G) Quantitative analysis of β-cell mass of rictor^fl/fl and rictor^ad−/− mice on chow diet.

Fig. 5.

Rictor^ad−/− mice have improved glucose tolerance after HFD. (A and B) Glucose tolerance tests in overnight-starved mice fed a chow diet (A) or an HFD for 10 weeks (B). Mice were injected with glucose (2 g/kg, i.p.), and blood glucose was subsequently measured at the indicated time points (n = 10–16). *P < 0.05, **P < 0.01, rictor^ad−/− vs. rictor^fl/fl. n = 9–10 per group. (C) Insulin sensitivity test in fed mice on a chow diet. Mice were injected with insulin (0.75 IU/kg, i.p.), and blood glucose was subsequently measured at the indicated time points (n = 10–11, P > 0.05). (D) Immunoblot of in vivo insulin-stimulated adipose tissue, muscle, and liver. Twenty-one-week-old mice fed a chow diet were starved overnight and anesthetized, followed by i.p. injection of saline or 150 mU/g body weight insulin. After 15 min adipose tissue, muscle and liver were removed and snap frozen. Lysates were run on an SDS-PAGE and immunoblotted for phosphorylated and total Akt, and phosphorylated and total GSK3. (E) Basal and insulin-stimulated glucose uptake was measured on isolated adipocytes from rictor^fl/fl and rictor^ad−/− mice on a chow diet. Glucose uptake was normalized to cell number, and data are shown in arbitrary units (n = 5).

Fig. 6.

Rictor^ad−/− mice have elevated levels of IGF1. (A) Blood IGF1 levels in mice fed a chow diet or HFD for 10 weeks. (B) IGF1 mRNA expression in adipose tissue (epididymal) and liver of mice fed a chow diet was determined by quantitative RT-PCR. IGF1 expression was normalized to Polr2a expression and is shown in arbitrary units (AU). (C and D) IGFBP3 (C) and GH (D) levels were determined in mice fed a chow diet or HFD for 10 weeks. Values in A–D are represented as mean ± SEM. *P < 0.05; **P < 0.01, rictor^ad−/− vs. rictor^fl/fl (n = 8–10). (E) Model of adipose mTORC2 regulating whole-body growth. Adipose mTORC2 negatively regulates IGF1 and insulin production by liver and pancreas, respectively, and thereby controls systemic growth and metabolism.