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• W2018373960 abstract "Mammalian target of rapamycin complex 2 (mTORC2) phosphorylates and activates AGC kinase family members, including Akt, SGK1, and PKC, in response to insulin/IGF1. The liver is a key organ in insulin-mediated regulation of metabolism. To assess the role of hepatic mTORC2, we generated liver-specific rictor knockout (LiRiKO) mice. Fed LiRiKO mice displayed loss of Akt Ser473 phosphorylation and reduced glucokinase and SREBP1c activity in the liver, leading to constitutive gluconeogenesis, and impaired glycolysis and lipogenesis, suggesting that the mTORC2-deficient liver is unable to sense satiety. These liver-specific defects resulted in systemic hyperglycemia, hyperinsulinemia, and hypolipidemia. Expression of constitutively active Akt2 in mTORC2-deficient hepatocytes restored both glucose flux and lipogenesis, whereas glucokinase overexpression rescued glucose flux but not lipogenesis. Thus, mTORC2 regulates hepatic glucose and lipid metabolism via insulin-induced Akt signaling to control whole-body metabolic homeostasis. These findings have implications for emerging drug therapies that target mTORC2. Mammalian target of rapamycin complex 2 (mTORC2) phosphorylates and activates AGC kinase family members, including Akt, SGK1, and PKC, in response to insulin/IGF1. The liver is a key organ in insulin-mediated regulation of metabolism. To assess the role of hepatic mTORC2, we generated liver-specific rictor knockout (LiRiKO) mice. Fed LiRiKO mice displayed loss of Akt Ser473 phosphorylation and reduced glucokinase and SREBP1c activity in the liver, leading to constitutive gluconeogenesis, and impaired glycolysis and lipogenesis, suggesting that the mTORC2-deficient liver is unable to sense satiety. These liver-specific defects resulted in systemic hyperglycemia, hyperinsulinemia, and hypolipidemia. Expression of constitutively active Akt2 in mTORC2-deficient hepatocytes restored both glucose flux and lipogenesis, whereas glucokinase overexpression rescued glucose flux but not lipogenesis. Thus, mTORC2 regulates hepatic glucose and lipid metabolism via insulin-induced Akt signaling to control whole-body metabolic homeostasis. These findings have implications for emerging drug therapies that target mTORC2. Liver-specific disruption of mTORC2 leads to the development of diabetes Liver mTORC2 senses satiety to regulate gluconeogenesis, glycolysis and lipogenesis mTORC2-mediated Akt Ser473 phosphorylation is required for hepatic insulin action Glucose flux is not sufficient to restore lipogenesis in hepatocytes lacking mTORC2 Target of rapamycin (TOR) is a highly conserved protein kinase that controls cell growth and metabolism in response to nutrients, growth factors, and energy status. TOR exists in two structurally and functionally distinct complexes termed TOR complex 1 (TORC1) and TORC2 (Loewith et al., 2002Loewith R. Jacinto E. Wullschleger S. Lorberg A. Crespo J.L. Bonenfant D. Oppliger W. Jenoe P. Hall M.N. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control.Mol. Cell. 2002; 10: 457-468Abstract Full Text Full Text PDF PubMed Scopus (1457) Google Scholar). Mammalian TORC1 (mTORC1) contains mTOR, raptor, and mLST8 and phosphorylates a variety of substrates to control protein synthesis, ribosome biogenesis, autophagy, and other growth-related processes (Laplante and Sabatini, 2009Laplante M. Sabatini D.M. mTOR signaling at a glance.J. Cell Sci. 2009; 122: 3589-3594Crossref PubMed Scopus (1642) Google Scholar, Polak and Hall, 2009Polak P. Hall M.N. mTOR and the control of whole body metabolism.Curr. Opin. Cell Biol. 2009; 21: 209-218Crossref PubMed Scopus (248) Google Scholar, Russell et al., 2011Russell R.C. Fang C. Guan K.L. An emerging role for TOR signaling in mammalian tissue and stem cell physiology.Development. 2011; 138: 3343-3356Crossref PubMed Scopus (107) Google Scholar, Wullschleger et al., 2006Wullschleger S. Loewith R. Hall M.N. TOR signaling in growth and metabolism.Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4671) Google Scholar). The two best-characterized mTORC1 substrates are ribosomal protein S6 kinase (S6K) and eukaryotic initiation factor 4E-binding protein (4E-BP), both of which control protein synthesis. mTORC2 comprises mTOR, rictor, mSin1, mLST8, and PRR5 (also known as protor) and phosphorylates members of the AGC kinase family, including Akt (also known as PKB), SGK1, and PKC, via which mTORC2 controls cell survival, actin cytoskeleton organization, and other processes (Cybulski and Hall, 2009Cybulski N. Hall M.N. TOR complex 2: a signaling pathway of its own.Trends Biochem. Sci. 2009; 34: 620-627Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, García-Martínez and Alessi, 2008García-Martínez J.M. Alessi D.R. mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum- and glucocorticoid-induced protein kinase 1 (SGK1).Biochem. J. 2008; 416: 375-385Crossref PubMed Scopus (707) Google Scholar, Ikenoue et al., 2008Ikenoue T. Inoki K. Yang Q. Zhou X. Guan K.L. Essential function of TORC2 in PKC and Akt turn motif phosphorylation, maturation and signalling.EMBO J. 2008; 27: 1919-1931Crossref PubMed Scopus (500) Google Scholar, Jacinto et al., 2006Jacinto E. Facchinetti V. Liu D. Soto N. Wei S. Jung S.Y. Huang Q. Qin J. Su B. SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity.Cell. 2006; 127: 125-137Abstract Full Text Full Text PDF PubMed Scopus (1139) Google Scholar, Jacinto et al., 2004Jacinto E. Loewith R. Schmidt A. Lin S. Rüegg M.A. Hall A. Hall M.N. Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive.Nat. Cell Biol. 2004; 6: 1122-1128Crossref PubMed Scopus (1682) Google Scholar, Sarbassov et al., 2004Sarbassov D.D. Ali S.M. Kim D.H. Guertin D.A. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton.Curr. Biol. 2004; 14: 1296-1302Abstract Full Text Full Text PDF PubMed Scopus (2149) Google Scholar, Yang et al., 2006Yang Q. Inoki K. Ikenoue T. Guan K.L. Identification of Sin1 as an essential TORC2 component required for complex formation and kinase activity.Genes Dev. 2006; 20: 2820-2832Crossref PubMed Scopus (403) Google Scholar). By regulating a wide range of anabolic and catabolic processes, the mTOR complexes play a key role in growth, development, metabolism, and aging and are implicated in a variety of pathological states including cancer, obesity, and diabetes (Dazert and Hall, 2011Dazert E. Hall M.N. mTOR signaling in disease.Curr. Opin. Cell Biol. 2011; 23: 744-755Crossref PubMed Scopus (376) Google Scholar). Many studies with genetically modified animals indicate mTOR signaling plays a role in whole-animal metabolism and in the development of disease. Full-body knockout of any component of mTORC1 or mTORC2 causes embryonic lethality (Gangloff et al., 2004Gangloff Y.G. Mueller M. Dann S.G. Svoboda P. Sticker M. Spetz J.F. Um S.H. Brown E.J. Cereghini S. Thomas G. Kozma S.C. Disruption of the mouse mTOR gene leads to early postimplantation lethality and prohibits embryonic stem cell development.Mol. Cell. Biol. 2004; 24: 9508-9516Crossref PubMed Scopus (386) Google Scholar, Guertin et al., 2006Guertin D.A. Stevens D.M. Thoreen C.C. Burds A.A. Kalaany N.Y. Moffat J. Brown M. Fitzgerald K.J. Sabatini D.M. Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCalpha, but not S6K1.Dev. Cell. 2006; 11: 859-871Abstract Full Text Full Text PDF PubMed Scopus (1126) Google Scholar, Jacinto et al., 2006Jacinto E. Facchinetti V. Liu D. Soto N. Wei S. Jung S.Y. Huang Q. Qin J. Su B. SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity.Cell. 2006; 127: 125-137Abstract Full Text Full Text PDF PubMed Scopus (1139) Google Scholar, Murakami et al., 2004Murakami M. Ichisaka T. Maeda M. Oshiro N. Hara K. Edenhofer F. Kiyama H. Yonezawa K. Yamanaka S. mTOR is essential for growth and proliferation in early mouse embryos and embryonic stem cells.Mol. Cell. Biol. 2004; 24: 6710-6718Crossref PubMed Scopus (482) Google Scholar, Shiota et al., 2006Shiota C. Woo J.T. Lindner J. Shelton K.D. Magnuson M.A. Multiallelic disruption of the rictor gene in mice reveals that mTOR complex 2 is essential for fetal growth and viability.Dev. Cell. 2006; 11: 583-589Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, Yang et al., 2006Yang Q. Inoki K. Ikenoue T. Guan K.L. Identification of Sin1 as an essential TORC2 component required for complex formation and kinase activity.Genes Dev. 2006; 20: 2820-2832Crossref PubMed Scopus (403) Google Scholar). More recent studies have focused on mTOR function specifically in metabolic tissues, in large part due to these tissues being particularly sensitive to the three inputs that control mTOR (nutrients, insulin, and energy status). Conditional knockout of raptor (mTORC1) in skeletal muscle results in muscle dystrophy, glucose intolerance, and short lifespan, whereas knockout of rictor (mTORC2) in skeletal muscle confers little to no phenotype (Bentzinger et al., 2008Bentzinger C.F. Romanino K. Cloëtta D. Lin S. Mascarenhas J.B. Oliveri F. Xia J. Casanova E. Costa C.F. Brink M. et al.Skeletal muscle-specific ablation of raptor, but not of rictor, causes metabolic changes and results in muscle dystrophy.Cell Metab. 2008; 8: 411-424Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, Kumar et al., 2008Kumar A. Harris T.E. Keller S.R. Choi K.M. Magnuson M.A. Lawrence Jr., J.C. Muscle-specific deletion of rictor impairs insulin-stimulated glucose transport and enhances Basal glycogen synthase activity.Mol. Cell. Biol. 2008; 28: 61-70Crossref PubMed Scopus (175) Google Scholar). Adipose-specific raptor knockout mice display increased energy expenditure and resistance to diet-induced obesity (Polak et al., 2008Polak P. Cybulski N. Feige J.N. Auwerx J. Rüegg M.A. Hall M.N. Adipose-specific knockout of raptor results in lean mice with enhanced mitochondrial respiration.Cell Metab. 2008; 8: 399-410Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar). Adipose-specific rictor knockout mice are characterized by increased glucose metabolism and somatic growth due to high circulating levels of insulin and IGF1 (Cybulski et al., 2009Cybulski N. Polak P. Auwerx J. Rüegg M.A. Hall M.N. mTOR complex 2 in adipose tissue negatively controls whole-body growth.Proc. Natl. Acad. Sci. USA. 2009; 106: 9902-9907Crossref PubMed Scopus (142) Google Scholar, Kumar et al., 2010Kumar A. Lawrence Jr., J.C. Jung D.Y. Ko H.J. Keller S.R. Kim J.K. Magnuson M.A. Harris T.E. Fat cell-specific ablation of rictor in mice impairs insulin-regulated fat cell and whole-body glucose and lipid metabolism.Diabetes. 2010; 59: 1397-1406Crossref PubMed Scopus (202) Google Scholar). In podocytes, mTORC1 plays a role in the development of diabetic nephropathy, whereas mTORC2 appears to have a minor role (Gödel et al., 2011Gödel M. Hartleben B. Herbach N. Liu S. Zschiedrich S. Lu S. Debreczeni-Mór A. Lindenmeyer M.T. Rastaldi M.P. Hartleben G. et al.Role of mTOR in podocyte function and diabetic nephropathy in humans and mice.J. Clin. Invest. 2011; 121: 2197-2209Crossref PubMed Scopus (406) Google Scholar, Inoki et al., 2011Inoki K. Mori H. Wang J. Suzuki T. Hong S. Yoshida S. Blattner S.M. Ikenoue T. Rüegg M.A. Hall M.N. et al.mTORC1 activation in podocytes is a critical step in the development of diabetic nephropathy in mice.J. Clin. Invest. 2011; 121: 2181-2196Crossref PubMed Scopus (413) Google Scholar). Recent findings suggest that hepatic mTORC1 controls ketogenesis and possibly lipid metabolism (Kenerson et al., 2011Kenerson H.L. Yeh M.M. Yeung R.S. Tuberous sclerosis complex-1 deficiency attenuates diet-induced hepatic lipid accumulation.PLoS ONE. 2011; 6: e18075Crossref PubMed Scopus (54) Google Scholar, Sengupta et al., 2010Sengupta S. Peterson T.R. Laplante M. Oh S. Sabatini D.M. mTORC1 controls fasting-induced ketogenesis and its modulation by ageing.Nature. 2010; 468: 1100-1104Crossref PubMed Scopus (448) Google Scholar, Yecies et al., 2011Yecies J.L. Zhang H.H. Menon S. Liu S. Yecies D. Lipovsky A.I. Gorgun C. Kwiatkowski D.J. Hotamisligil G.S. Lee C.H. Manning B.D. Akt stimulates hepatic SREBP1c and lipogenesis through parallel mTORC1-dependent and independent pathways.Cell Metab. 2011; 14: 21-32Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). Together, the above studies suggest that the two mTOR complexes contribute to whole-body metabolic homeostasis via distinct roles in different metabolic tissues. However, the role of mTORC2 in the liver remains to be determined. The liver plays a central role in whole-body glucose and lipid homeostasis (Postic et al., 2004Postic C. Dentin R. Girard J. Role of the liver in the control of carbohydrate and lipid homeostasis.Diabetes Metab. 2004; 30: 398-408Abstract Full Text PDF PubMed Scopus (334) Google Scholar). In the fasted state, the liver maintains blood glucose levels by producing glucose via glycogen breakdown and via gluconeogenesis. In the postprandial state (i.e., satiety—the increased availability of glucose and insulin), the liver ceases to produce glucose and takes up excess circulating glucose to replenish glycogen and triglyceride (TG) stores. Insulin is the major hormone controlling the fasted to postprandial transition (Saltiel and Kahn, 2001Saltiel A.R. Kahn C.R. Insulin signalling and the regulation of glucose and lipid metabolism.Nature. 2001; 414: 799-806Crossref PubMed Scopus (3912) Google Scholar). In type 2 diabetes, hepatic insulin resistance leads to altered glucose metabolism and thereby hyperglycemia. Insulin signals through the PI3K-Akt pathway to inhibit gluconeogenesis and activate glycolysis and lipogenesis. Akt inhibits expression of gluconeogenic genes, by inhibiting FoxO (Puigserver et al., 2003Puigserver P. Rhee J. Donovan J. Walkey C.J. Yoon J.C. Oriente F. Kitamura Y. Altomonte J. Dong H. Accili D. Spiegelman B.M. Insulin-regulated hepatic gluconeogenesis through FOXO1-PGC-1alpha interaction.Nature. 2003; 423: 550-555Crossref PubMed Scopus (1174) Google Scholar), and induces glycolytic and lipogenic genes, by activating sterol regulatory element-binding protein 1c (SREBP1c) and glucokinase (GK). SREBP1c is a transcription factor that promotes expression of a number of lipogenic genes (Horton et al., 2002Horton J.D. Goldstein J.L. Brown M.S. SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver.J. Clin. Invest. 2002; 109: 1125-1131Crossref PubMed Scopus (3757) Google Scholar). GK, the rate-limiting enzyme of glycolysis in the liver, stimulates glycolysis and lipogenesis by enhancing glucose flux, including production of acetyl-CoA for lipid synthesis (Foufelle and Ferré, 2002Foufelle F. Ferré P. New perspectives in the regulation of hepatic glycolytic and lipogenic genes by insulin and glucose: a role for the transcription factor sterol regulatory element binding protein-1c.Biochem. J. 2002; 366: 377-391Crossref PubMed Scopus (404) Google Scholar). Furthermore, GK stimulates glycolysis and lipogenesis at the transcriptional level via the carbohydrate responsive element-binding protein (ChREBP) (Uyeda and Repa, 2006Uyeda K. Repa J.J. Carbohydrate response element binding protein, ChREBP, a transcription factor coupling hepatic glucose utilization and lipid synthesis.Cell Metab. 2006; 4: 107-110Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar). Thus, the combined action of insulin signaling and glucose flux regulates glucose and lipid metabolism in the liver. mTORC2 phosphorylates Ser473 in the so-called hydrophobic motif of Akt and thereby activates Akt toward some but not all substrates (Guertin et al., 2006Guertin D.A. Stevens D.M. Thoreen C.C. Burds A.A. Kalaany N.Y. Moffat J. Brown M. Fitzgerald K.J. Sabatini D.M. Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCalpha, but not S6K1.Dev. Cell. 2006; 11: 859-871Abstract Full Text Full Text PDF PubMed Scopus (1126) Google Scholar, Jacinto et al., 2006Jacinto E. Facchinetti V. Liu D. Soto N. Wei S. Jung S.Y. Huang Q. Qin J. Su B. SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity.Cell. 2006; 127: 125-137Abstract Full Text Full Text PDF PubMed Scopus (1139) Google Scholar, Sarbassov et al., 2005Sarbassov D.D. Guertin D.A. Ali S.M. Sabatini D.M. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex.Science. 2005; 307: 1098-1101Crossref PubMed Scopus (5223) Google Scholar, Shiota et al., 2006Shiota C. Woo J.T. Lindner J. Shelton K.D. Magnuson M.A. Multiallelic disruption of the rictor gene in mice reveals that mTOR complex 2 is essential for fetal growth and viability.Dev. Cell. 2006; 11: 583-589Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, Yang et al., 2006Yang Q. Inoki K. Ikenoue T. Guan K.L. Identification of Sin1 as an essential TORC2 component required for complex formation and kinase activity.Genes Dev. 2006; 20: 2820-2832Crossref PubMed Scopus (403) Google Scholar, Zinzalla et al., 2011Zinzalla V. Stracka D. Oppliger W. Hall M.N. Activation of mTORC2 by association with the ribosome.Cell. 2011; 144: 757-768Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar). To elucidate the role of mTORC2 in the liver in vivo, we generated liver-specific rictor knockout mice and investigated Akt signaling and glucose and lipid metabolism. We find that the mTORC2-deficient liver is unable to sense the state of satiety. Hepatic mTORC2 activates both Akt signaling and glucose flux to control glucose and lipid metabolism in the liver and thereby overall metabolic homeostasis. To investigate the role of mTORC2 in the liver, we generated mice lacking rictor, an essential and specific component of mTORC2, exclusively in the liver (see Experimental Procedures). Liver-specific rictor knockout (rictorfl/fl Alb-CreTg/0) mice (LiRiKO mice) were viable, born at the expected frequency for Mendelian inheritance, and showed normal fertility. In all subsequent experiments, littermates without the Cre transgene (rictorfl/fl) were used as controls. In LiRiKO mice, rictor protein was absent only in the liver (Figure S1A). To evaluate mTORC2 activity, we investigated the phosphorylation status of mTORC2 substrates in the liver of mice treated with insulin. mTORC2 phosphorylates the hydrophobic (Ser473) and turn (Thr450) motifs in Akt (Facchinetti et al., 2008Facchinetti V. Ouyang W. Wei H. Soto N. Lazorchak A. Gould C. Lowry C. Newton A.C. Mao Y. Miao R.Q. et al.The mammalian target of rapamycin complex 2 controls folding and stability of Akt and protein kinase C.EMBO J. 2008; 27: 1932-1943Crossref PubMed Scopus (410) Google Scholar, Ikenoue et al., 2008Ikenoue T. Inoki K. Yang Q. Zhou X. Guan K.L. Essential function of TORC2 in PKC and Akt turn motif phosphorylation, maturation and signalling.EMBO J. 2008; 27: 1919-1931Crossref PubMed Scopus (500) Google Scholar, Jacinto et al., 2006Jacinto E. Facchinetti V. Liu D. Soto N. Wei S. Jung S.Y. Huang Q. Qin J. Su B. SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity.Cell. 2006; 127: 125-137Abstract Full Text Full Text PDF PubMed Scopus (1139) Google Scholar, Sarbassov et al., 2005Sarbassov D.D. Guertin D.A. Ali S.M. Sabatini D.M. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex.Science. 2005; 307: 1098-1101Crossref PubMed Scopus (5223) Google Scholar, Yang et al., 2006Yang Q. Inoki K. Ikenoue T. Guan K.L. Identification of Sin1 as an essential TORC2 component required for complex formation and kinase activity.Genes Dev. 2006; 20: 2820-2832Crossref PubMed Scopus (403) Google Scholar). Akt phosphorylation at these two sites was significantly reduced in LiRiKO liver (Figure 1A ). Phosphorylation of Akt Thr308, a site in the catalytic loop phosphorylated by PDK1, was normal. mTORC2 also phosphorylates and thereby controls the stability and activity of PKCα and SGK1, respectively (García-Martínez and Alessi, 2008García-Martínez J.M. Alessi D.R. mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum- and glucocorticoid-induced protein kinase 1 (SGK1).Biochem. J. 2008; 416: 375-385Crossref PubMed Scopus (707) Google Scholar, Ikenoue et al., 2008Ikenoue T. Inoki K. Yang Q. Zhou X. Guan K.L. Essential function of TORC2 in PKC and Akt turn motif phosphorylation, maturation and signalling.EMBO J. 2008; 27: 1919-1931Crossref PubMed Scopus (500) Google Scholar). As expected, PKCα protein levels and phosphorylation of the SGK1 substrate NDRG1 were decreased in LiRiKO liver (Figure 1A). These observations confirm that both rictor protein and mTORC2 activity are absent in the liver of LiRiKO mice. We next examined systemic parameters of LiRiKO mice fed a normal (chow) or high-fat diet (HFD). On a chow diet, LiRiKO mice showed normal growth rates (Figure 1B) and normal body composition (Figure S1B). When fed a HFD for 10 weeks, LiRiKO mice were slightly lighter than controls, but this difference was not statistically significant (Figure S1C). After 20 weeks on a HFD, fat mass was significantly reduced (5%) in LiRiKO mice (Figure S1D). This was likely due to increased lipolysis and mitochondrial oxidation in adipose tissue of LiRiKO mice, as suggested by an increased level of plasma glycerol (Figure S1E) and increased expression of genes involved in lipolysis and mitochondrial oxidation in adipose tissue (Figure S1F). Analysis of plasma parameters revealed that plasma TG and cholesterol (total and HDL cholesterol) levels were significantly lower (Figure 1C), while plasma glucose (Figure 1D) and insulin (Figure 1E) levels were significantly higher, in both fasted and chow-fed LiRiKO mice. The same differences in blood parameters were observed with mice on a HFD (Figures S1G–S1I). The levels of ALT and AST were similar for the two genotypes (Figures S1J and S1K). Thus, deletion of mTORC2 in the liver leads to hyperglycemia, hyperinsulinemia, and hypolipidemia, suggesting that hepatic mTORC2 mediates metabolic homeostasis. The liver of LiRiKO mice displayed normal gross morphology (data not shown), and normal cell size and histopathology (Figure 1F). However, the weight of the liver of LiRiKO mice was 20% less compared to controls, for mice fed ad libitum either a chow or a HFD (Figures 1G and S1L). This difference was not observed in fasted mice (Figure S1M). Further analysis revealed that glycogen and TG levels in the liver of fed LiRiKO mice were significantly lower compared to controls (Figures 1F, 1H, 1I, and S1N). Thus, loss of hepatic mTORC2 results in a smaller liver with reduced glycogen and TG content in fed mice. LiRiKO mice developed mild hyperglycemia and hyperinsulinemia at a young age (6 weeks), indicating that impaired glucose homeostasis is an early effect of mTORC2 loss in the liver (Figures S2A and S2B). To evaluate further whole-body glucose homeostasis, we performed a glucose tolerance test (GTT) and an insulin tolerance test (ITT). In the GTT, LiRiKO mice exhibited significantly higher blood glucose levels before and after glucose administration (Figure 2A ). Plasma insulin levels were also significantly higher in LiRiKO mice (Figure 2B). The glucose intolerance became more pronounced in older (>8 months) LiRiKO mice (Figure S2C). In the ITT, when the mice were fasted for 6 hr before the experiment, LiRiKO mice showed normal insulin sensitivity (Figure 2C). In contrast, when the mice were fasted overnight, LiRiKO mice showed slightly but significantly decreased glucose clearance (Figure 2D). The fact that lower insulin sensitivity in LiRiKO mice was observed only after overnight fasting, when hepatic gluconeogenesis is more active, suggests that LiRiKO mice have hepatic insulin resistance but normal insulin sensitivity in other tissues. Consistent with this, insulin-induced activation of Akt was normal in both skeletal muscle and adipose tissue in LiRiKO mice (Figure 2E). However, older LiRiKO mice (>8 months) showed impaired glucose clearance even after 6 hr fasting (Figure S2E), suggesting that whole-body insulin sensitivity decreased with age as a consequence of chronic hyperinsulinemia. When fed a HFD, LiRiKO mice displayed glucose intolerance (Figure S2F) and moderate insulin resistance (Figure S2G) already at 14 weeks of age. Thus, LiRiKO mice develop diabetes (glucose intolerance, hyperinsulinemia, and insulin resistance), further suggesting that hepatic mTORC2 controls whole-body metabolic homeostasis. The above findings suggest that the primary defect of a hepatic mTORC2 deficiency may be insulin resistance in the liver. To investigate this further, we examined insulin signaling, in particular insulin-activated Akt signaling, in the liver. Previous studies have shown that mTORC2 (i.e., Akt Ser473 phosphorylation) appears to be necessary for Akt activity toward some but not all substrates (Guertin et al., 2006Guertin D.A. Stevens D.M. Thoreen C.C. Burds A.A. Kalaany N.Y. Moffat J. Brown M. Fitzgerald K.J. Sabatini D.M. Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCalpha, but not S6K1.Dev. Cell. 2006; 11: 859-871Abstract Full Text Full Text PDF PubMed Scopus (1126) Google Scholar, Jacinto et al., 2006Jacinto E. Facchinetti V. Liu D. Soto N. Wei S. Jung S.Y. Huang Q. Qin J. Su B. SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity.Cell. 2006; 127: 125-137Abstract Full Text Full Text PDF PubMed Scopus (1139) Google Scholar, Polak and Hall, 2006Polak P. Hall M.N. mTORC2 Caught in a SINful Akt.Dev. Cell. 2006; 11: 433-434Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, Sarbassov et al., 2005Sarbassov D.D. Guertin D.A. Ali S.M. Sabatini D.M. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex.Science. 2005; 307: 1098-1101Crossref PubMed Scopus (5223) Google Scholar, Shiota et al., 2006Shiota C. Woo J.T. Lindner J. Shelton K.D. Magnuson M.A. Multiallelic disruption of the rictor gene in mice reveals that mTOR complex 2 is essential for fetal growth and viability.Dev. Cell. 2006; 11: 583-589Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, Yang et al., 2006Yang Q. Inoki K. Ikenoue T. Guan K.L. Identification of Sin1 as an essential TORC2 component required for complex formation and kinase activity.Genes Dev. 2006; 20: 2820-2832Crossref PubMed Scopus (403) Google Scholar). We first analyzed the phosphorylation status of Akt and several Akt downstream effectors in insulin-stimulated primary hepatocytes. In control hepatocytes, insulin stimulated phosphorylation of Akt (Ser473 and Thr308), GSK3α/β (Ser21 and Ser9), FoxO1 (Thr24), S6K (Thr389), and S6 (Ser235/236) (Figure 3A ). In LiRiKO hepatocytes, insulin failed to stimulate Akt Ser473 phosphorylation whereas Thr308 phosphorylation was normal (Figure 3A), confirming our in vivo findings described above. The Akt substrates GSK3α/β and FoxO1 were significantly hypophosphorylated in insulin-treated LiRiKO hepatocytes (Figure 3A). Importantly, phosphorylation of the Akt downstream effectors S6K and S6 was normal, supporting the previous finding that Ser473 phosphorylation is not necessary for Akt to signal to mTORC1. Next, we examined Akt effectors in the liver of fasted and refed mice. Refeeding stimulated phosphorylation in control and LiRiKO livers in the same manner as observed in insulin-stimulated control and LiRiKO hepatocytes (Figure 3B). The only noteworthy exception in results obtained with hepatocytes versus livers is that Akt Thr308 was hyperphosphorylated in the liver of fasted and refed LiRiKO mice (Figure 3B). This is consistent with the hyperinsulinemia and increased tyrosine phosphorylation of the insulin receptor in LiRiKO mice (Figure 3B). Thus, hepatic mTORC2 is required for insulin-Akt signaling to FoxO1 and GSK3α/β, but not for insulin-Akt signaling to mTORC1. Furthermore, these findings confirm hepatic insulin resistance, albeit partial, in LiRiKO mice. How might the observed defect in hepatic insulin signaling lead to defects in metabolic homeostasis, e.g., hyperglycemia? As shown above, FoxO1 is hypophosphorylated in the liver of refed LiRiKO mice (Figure 3B). FoxO1 is a transcription factor that functions with the transcriptional coactivator PGC1α to induce gluconeogenic genes such as glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) in the fasted state. During the fasted to postprandial transition, FoxO1 is phosphorylated by Akt, which results in nuclear exclusion of FoxO1 and inihibition of gluconeogenesis (Matsumoto et al., 2007Matsumoto M. Pocai A. Rossetti L. Depinho R.A. Accili D. Impaired regulation of hepatic glucose production in mice lacking the forkhead transcription factor Foxo1 in liver.Cell Metab. 2007; 6: 208-216Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar, Puigserver et al., 2003Puigserver P. Rhee J. Donovan J. Walkey C.J. Yoon J.C. Oriente F. Kitamura Y. Altomonte J. Dong H. Accili D. Spiegelman B.M. Insulin-regulated hepatic gluconeogenesis through FOXO1-PGC-1alpha interaction.Nature. 2003; 423: 550-555Crossref PubMed Scopus (1174) Google Scholar). Consistent with hypophosphorylation of FoxO1 in the liver of LiRiKO mice, nuclear exclusion of FoxO1 in response to feeding was severely impaired (Figure 4A ). Furthermor" @default.
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