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Fat cell-specific ablation of rictor in mice impairs insulin-regulated fat cell and whole-body glucose and lipid metabolism.

Kumar A, Lawrence JC, Jung DY, Ko HJ, Keller SR, Kim JK, Magnuson MA, Harris TE - Diabetes (2010)

Bottom Line: Loss of rictor in fat cells prevents insulin-stimulated phosphorylation of Akt at S473, which, in turn, impairs the phosphorylation of downstream targets such as FoxO3a at T32 and AS160 at T642.However, glycogen synthase kinase-3beta phosphorylation at S9 is not affected.Furthermore, rictor- fat cells are unable to suppress lipolysis in response to insulin, leading to elevated circulating free fatty acids and glycerol.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Virginia Health System, Charlottesville, Virginia, USA. al4p@virginia.edu

ABSTRACT

Objective: Rictor is an essential component of mammalian target of rapamycin (mTOR) complex (mTORC) 2, a kinase that phosphorylates and activates Akt, an insulin signaling intermediary that regulates glucose and lipid metabolism in adipose tissue, skeletal muscle, and liver. To determine the physiological role of rictor/mTORC2 in insulin signaling and action in fat cells, we developed fat cell-specific rictor knockout (FRic(-/-)) mice.

Research design and methods: Insulin signaling and glucose and lipid metabolism were studied in FRic(-/-) fat cells. In vivo glucose metabolism was evaluated by hyperinsulinemic-euglycemic clamp.

Results: Loss of rictor in fat cells prevents insulin-stimulated phosphorylation of Akt at S473, which, in turn, impairs the phosphorylation of downstream targets such as FoxO3a at T32 and AS160 at T642. However, glycogen synthase kinase-3beta phosphorylation at S9 is not affected. The signaling defects in FRic(-/-) fat cells lead to impaired insulin-stimulated GLUT4 translocation to the plasma membrane and decreased glucose transport. Furthermore, rictor- fat cells are unable to suppress lipolysis in response to insulin, leading to elevated circulating free fatty acids and glycerol. These metabolic perturbations are likely to account for defects observed at the whole-body level of FRic(-/-) mice, including glucose intolerance, marked hyperinsulinemia, insulin resistance in skeletal muscle and liver, and hepatic steatosis.

Conclusions: Rictor/mTORC2 in fat cells plays an important role in whole-body energy homeostasis by mediating signaling necessary for the regulation of glucose and lipid metabolism in fat cells.

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Glucose and lipid metabolism and insulin signaling in skeletal muscles from FRic−/− mice. A: Insulin-stimulated glycogen synthesis in ex vivo–incubated soleus muscles (female, 3–5 months old, n = 4–5, *P < 0.02; **P < 0.0001) from FRic−/−mice using [U14C]-glucose. B: Insulin signaling in isolated EDL muscles from FRic+/+ and FRic−/− mice. Protein extracts prepared from EDL muscles stimulated without or with 20 mU of insulin for 30 min were immunoblotted with phospho-specific antibodies to the Y-972 residue of IR, the Y896 residue of IRS1, and the T308 and S473 residues of Akt (representative blot is shown, n = 3–4). C: In vivo total and phosphorylated IRS1 at S302. Protein extracts prepared from TA muscles of fed FRic+/+ and FRic−/− mice were immunoblotted using antibodies against phospho-S302 IRS1 (P-S302 IRS1), total IRS1, and actin (female, 3–5 months old, representative blots of two sets of FRic+/+ and FRic−/− mice labeled as I and II out of a total of eight sets). D: Triglyceride levels in skeletal muscle homogenates prepared from fed FRic+/+ and FRic−/− mice (3- to 5-month-old female, n = 5–6, *P < 0.04). Data shown are means ± SE. E: Protein extracts prepared from TA muscles of fed FRic+/+ and FRic−/− mice were immunoblotted for lipin1 and actin (3- to 5-month-old female mice, representative blots of two sets of FRic+/+ and FRic−/− mice labeled as I and II out of a total of eight sets). ins, insulin.
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Figure 5: Glucose and lipid metabolism and insulin signaling in skeletal muscles from FRic−/− mice. A: Insulin-stimulated glycogen synthesis in ex vivo–incubated soleus muscles (female, 3–5 months old, n = 4–5, *P < 0.02; **P < 0.0001) from FRic−/−mice using [U14C]-glucose. B: Insulin signaling in isolated EDL muscles from FRic+/+ and FRic−/− mice. Protein extracts prepared from EDL muscles stimulated without or with 20 mU of insulin for 30 min were immunoblotted with phospho-specific antibodies to the Y-972 residue of IR, the Y896 residue of IRS1, and the T308 and S473 residues of Akt (representative blot is shown, n = 3–4). C: In vivo total and phosphorylated IRS1 at S302. Protein extracts prepared from TA muscles of fed FRic+/+ and FRic−/− mice were immunoblotted using antibodies against phospho-S302 IRS1 (P-S302 IRS1), total IRS1, and actin (female, 3–5 months old, representative blots of two sets of FRic+/+ and FRic−/− mice labeled as I and II out of a total of eight sets). D: Triglyceride levels in skeletal muscle homogenates prepared from fed FRic+/+ and FRic−/− mice (3- to 5-month-old female, n = 5–6, *P < 0.04). Data shown are means ± SE. E: Protein extracts prepared from TA muscles of fed FRic+/+ and FRic−/− mice were immunoblotted for lipin1 and actin (3- to 5-month-old female mice, representative blots of two sets of FRic+/+ and FRic−/− mice labeled as I and II out of a total of eight sets). ins, insulin.

Mentions: Since fat cell–specific loss of rictor/mTORC2 decreased skeletal muscle glucose transport in response to insulin, we measured insulin-stimulated glycogen synthesis in isolated soleus and extensor digitorum longus (EDL) muscles. We found that insulin-stimulated glycogen synthesis was markedly reduced in both soleus (∼41% reduction, P < 0.0001) (Fig. 5A) and EDL (∼27% reduction, P < 0.0004) (supplemental Fig. 6) muscles from FRic−/− mice when compared with FRic+/+ mice. The isolated EDL muscle from FRic−/− mice showed normal insulin-stimulated phosphorylation of the IR at Y972. However, a marked reduction in insulin-stimulated phosphorylations of insulin receptor substrate (IRS) 1 at Y896 and of Akt at both T308 and S473 (Fig. 5B) was observed. Interestingly, basal phosphorylation of IRS1 at S302 in isolated EDL muscles from FRic−/− mice was slightly increased when compared with FRic+/+ mice (∼30%, n = 4, data not shown). In addition, we found a twofold increase (P < 0.03) in levels of IRS1 phosphorylated at S302 (Fig. 5C, top panel) and a 37% reduction (P < 0.04) in the total IRS1 (Fig. 5C, middle panel) levels in tibialis anterior (TA) muscles from FRic−/− mice.


Fat cell-specific ablation of rictor in mice impairs insulin-regulated fat cell and whole-body glucose and lipid metabolism.

Kumar A, Lawrence JC, Jung DY, Ko HJ, Keller SR, Kim JK, Magnuson MA, Harris TE - Diabetes (2010)

Glucose and lipid metabolism and insulin signaling in skeletal muscles from FRic−/− mice. A: Insulin-stimulated glycogen synthesis in ex vivo–incubated soleus muscles (female, 3–5 months old, n = 4–5, *P < 0.02; **P < 0.0001) from FRic−/−mice using [U14C]-glucose. B: Insulin signaling in isolated EDL muscles from FRic+/+ and FRic−/− mice. Protein extracts prepared from EDL muscles stimulated without or with 20 mU of insulin for 30 min were immunoblotted with phospho-specific antibodies to the Y-972 residue of IR, the Y896 residue of IRS1, and the T308 and S473 residues of Akt (representative blot is shown, n = 3–4). C: In vivo total and phosphorylated IRS1 at S302. Protein extracts prepared from TA muscles of fed FRic+/+ and FRic−/− mice were immunoblotted using antibodies against phospho-S302 IRS1 (P-S302 IRS1), total IRS1, and actin (female, 3–5 months old, representative blots of two sets of FRic+/+ and FRic−/− mice labeled as I and II out of a total of eight sets). D: Triglyceride levels in skeletal muscle homogenates prepared from fed FRic+/+ and FRic−/− mice (3- to 5-month-old female, n = 5–6, *P < 0.04). Data shown are means ± SE. E: Protein extracts prepared from TA muscles of fed FRic+/+ and FRic−/− mice were immunoblotted for lipin1 and actin (3- to 5-month-old female mice, representative blots of two sets of FRic+/+ and FRic−/− mice labeled as I and II out of a total of eight sets). ins, insulin.
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Related In: Results  -  Collection

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Figure 5: Glucose and lipid metabolism and insulin signaling in skeletal muscles from FRic−/− mice. A: Insulin-stimulated glycogen synthesis in ex vivo–incubated soleus muscles (female, 3–5 months old, n = 4–5, *P < 0.02; **P < 0.0001) from FRic−/−mice using [U14C]-glucose. B: Insulin signaling in isolated EDL muscles from FRic+/+ and FRic−/− mice. Protein extracts prepared from EDL muscles stimulated without or with 20 mU of insulin for 30 min were immunoblotted with phospho-specific antibodies to the Y-972 residue of IR, the Y896 residue of IRS1, and the T308 and S473 residues of Akt (representative blot is shown, n = 3–4). C: In vivo total and phosphorylated IRS1 at S302. Protein extracts prepared from TA muscles of fed FRic+/+ and FRic−/− mice were immunoblotted using antibodies against phospho-S302 IRS1 (P-S302 IRS1), total IRS1, and actin (female, 3–5 months old, representative blots of two sets of FRic+/+ and FRic−/− mice labeled as I and II out of a total of eight sets). D: Triglyceride levels in skeletal muscle homogenates prepared from fed FRic+/+ and FRic−/− mice (3- to 5-month-old female, n = 5–6, *P < 0.04). Data shown are means ± SE. E: Protein extracts prepared from TA muscles of fed FRic+/+ and FRic−/− mice were immunoblotted for lipin1 and actin (3- to 5-month-old female mice, representative blots of two sets of FRic+/+ and FRic−/− mice labeled as I and II out of a total of eight sets). ins, insulin.
Mentions: Since fat cell–specific loss of rictor/mTORC2 decreased skeletal muscle glucose transport in response to insulin, we measured insulin-stimulated glycogen synthesis in isolated soleus and extensor digitorum longus (EDL) muscles. We found that insulin-stimulated glycogen synthesis was markedly reduced in both soleus (∼41% reduction, P < 0.0001) (Fig. 5A) and EDL (∼27% reduction, P < 0.0004) (supplemental Fig. 6) muscles from FRic−/− mice when compared with FRic+/+ mice. The isolated EDL muscle from FRic−/− mice showed normal insulin-stimulated phosphorylation of the IR at Y972. However, a marked reduction in insulin-stimulated phosphorylations of insulin receptor substrate (IRS) 1 at Y896 and of Akt at both T308 and S473 (Fig. 5B) was observed. Interestingly, basal phosphorylation of IRS1 at S302 in isolated EDL muscles from FRic−/− mice was slightly increased when compared with FRic+/+ mice (∼30%, n = 4, data not shown). In addition, we found a twofold increase (P < 0.03) in levels of IRS1 phosphorylated at S302 (Fig. 5C, top panel) and a 37% reduction (P < 0.04) in the total IRS1 (Fig. 5C, middle panel) levels in tibialis anterior (TA) muscles from FRic−/− mice.

Bottom Line: Loss of rictor in fat cells prevents insulin-stimulated phosphorylation of Akt at S473, which, in turn, impairs the phosphorylation of downstream targets such as FoxO3a at T32 and AS160 at T642.However, glycogen synthase kinase-3beta phosphorylation at S9 is not affected.Furthermore, rictor- fat cells are unable to suppress lipolysis in response to insulin, leading to elevated circulating free fatty acids and glycerol.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Virginia Health System, Charlottesville, Virginia, USA. al4p@virginia.edu

ABSTRACT

Objective: Rictor is an essential component of mammalian target of rapamycin (mTOR) complex (mTORC) 2, a kinase that phosphorylates and activates Akt, an insulin signaling intermediary that regulates glucose and lipid metabolism in adipose tissue, skeletal muscle, and liver. To determine the physiological role of rictor/mTORC2 in insulin signaling and action in fat cells, we developed fat cell-specific rictor knockout (FRic(-/-)) mice.

Research design and methods: Insulin signaling and glucose and lipid metabolism were studied in FRic(-/-) fat cells. In vivo glucose metabolism was evaluated by hyperinsulinemic-euglycemic clamp.

Results: Loss of rictor in fat cells prevents insulin-stimulated phosphorylation of Akt at S473, which, in turn, impairs the phosphorylation of downstream targets such as FoxO3a at T32 and AS160 at T642. However, glycogen synthase kinase-3beta phosphorylation at S9 is not affected. The signaling defects in FRic(-/-) fat cells lead to impaired insulin-stimulated GLUT4 translocation to the plasma membrane and decreased glucose transport. Furthermore, rictor- fat cells are unable to suppress lipolysis in response to insulin, leading to elevated circulating free fatty acids and glycerol. These metabolic perturbations are likely to account for defects observed at the whole-body level of FRic(-/-) mice, including glucose intolerance, marked hyperinsulinemia, insulin resistance in skeletal muscle and liver, and hepatic steatosis.

Conclusions: Rictor/mTORC2 in fat cells plays an important role in whole-body energy homeostasis by mediating signaling necessary for the regulation of glucose and lipid metabolism in fat cells.

Show MeSH
Related in: MedlinePlus