LETTERS Insulin modulates gluconeogenesis by inhibition of the coactivator TORC2 Renaud Dentin 1 *, Yi Liu 1 *, Seung-Hoi Koo 2 , Susan Hedrick 1 , Thomas Vargas 1 , Jose Heredia 1 , John Yates III 3 & Marc Montminy 1 During feeding, increases in circulating pancreatic insulin inhibit hepatic glucose output through the activation of the Ser/Thr kinase AKT and subsequent phosphorylation of the forkhead tran- scription factor FOXO1 (refs 1–3). Under fasting conditions, FOXO1 increases gluconeogenic gene expression in concert with the cAMP responsive coactivator TORC2 (refs 4–8). In response to pancreatic glucagon, TORC2 is de-phosphorylated at Ser 171 and transported to the nucleus, in which it stimulates the gluconeo- genic programme by binding to CREB. Here we show in mice that insulin inhibits gluconeogenic gene expression during re-feeding by promoting the phosphorylation and ubiquitin-dependent degradation of TORC2. Insulin disrupts TORC2 activity by induc- tion of the Ser/Thr kinase SIK2, which we show here undergoes AKT2-mediated phosphorylation at Ser 358. Activated SIK2 in turn stimulated the Ser 171 phosphorylation and cytoplasmic translocation of TORC2. Phosphorylated TORC2 was degraded by the 26S proteasome during re-feeding through an association with COP1, a substrate receptor for an E3 ligase complex that promoted TORC2 ubiquitination at Lys 628. Because TORC2 pro- tein levels and activity were increased in diabetes owing to a block in TORC2 phosphorylation, our results point to an important role for this pathway in the maintenance of glucose homeostasis. We monitored the activity of the TORC2 (also known as CRTC2) pathway in liver by in vivo imaging with a cAMP-responsive adenovirus—containing cAMP responsive elements (CREs) up- stream of a minimal promoter—CRE-luciferase reporter (Ad-CRE- Luc) 9 . Fasting increased hepatic Ad-CRE-Luc activity 20-fold over that of re-fed mice (Fig. 1a; Supplementary Figs 1, 2). Amounts of de-phosphorylated and therefore active TORC2 were correspond- ingly elevated in livers of fasted mice, whereas re-feeding promoted TORC2 re-phosphorylation and degradation (Fig. 1b; Supplemen- tary Fig. 2). RNA interference (RNAi)-mediated knockdown of TORC2 reduced Ad-CRE-Luc but not CREB-independent (Ad- Rsv(Rous sarcoma virus)-Luc) reporter activity in the liver (Fig. 1c; Supplementary Fig. 3) 8 . Moreover, gluconeogenic gene expression (Pepck/Pck2, G6Pase/G6pc) and fasting blood-glucose levels were decreased in Ad-TORC2i (adenovirus expressing TORC2 RNAi) mice compared to controls, indicating that TORC2 degradation during re-feeding may be sufficient to attenuate the gluconeogenic programme (Fig. 1d; Supplementary Fig. 4). Having seen that hepatic TORC2 protein levels are downregu- lated during re-feeding, we wondered whether TORC2 activity is increased in insulin resistance. Consistent with their elevations in gluconeogenic gene expression and blood glucose concentrations, db/db diabetic mice had higher levels of Ad-CRE-Luc reporter activity and TORC2 protein in the liver, and re-feeding did not trigger TORC2 phosphorylation or degradation (Fig. 1e, f; Supplementary Fig. 5). On the basis of their roles in regulating hepatic glucose produc- tion, glucagon and insulin would be expected to modulate TORC2 activity in the liver. Intraperitoneal administration of glucagon stimulated hepatic Ad-CRE-Luc activity in mice; these effects were reversed by subsequent exposure to insulin, which also promoted the phosphorylation and degradation of hepatic TORC2 in fasted mice (Fig. 1g, h). Indeed, sequential exposure of cultured primary hepa- tocytes to forskolin and insulin (FSK/INS) also triggered the dis- appearance of TORC2 protein, whereas insulin alone did not (Supplementary Fig. 6). Indicating a potential role for ubiquitin- dependent degradation, treatment with the proteasome inhibitor MG132 blocked effects of FSK/INS on TORC2. Notably, exposure to the tris-phosphate kinase (PIK3) inhibitor LY294002 also dis- rupted TORC2 degradation, pointing to the involvement of PIK3 or other components of the insulin pathway such as AKT (Supple- mentary Fig. 7). If insulin inhibits TORC2 activity by Ser 171 phosphorylation, then mutant TORC2(S171A) should be resistant to these effects. Supporting this idea, Ad-CRE-Luc activity, gluconeogenic gene expression, and blood-glucose concentrations were elevated in Ad- TORC2(S171A) mice relative to Ad-TORC2 mice during re-feeding (Fig. 2a; Supplementary Fig. 8). Correspondingly, Ad-TORC2 (S171A) was not degraded during re-feeding compared with wild- type Ad-TORC2 (Fig. 2b; Supplementary Fig. 9). Indeed, Ad- TORC2(S171A) was similarly active in primary hepatocytes exposed to FSK/INS (Supplementary Fig. 10). Previously, the salt inducible kinase 2 (SIK2; also known as SNF1LK2) has been found to inhibit TORC2 activity by promoting its Ser 171 phosphorylation 10 , prompting us to test the role of this kinase in the liver. RNAi-mediated knockdown of SIK2 (Ad-SIK2i) increased Ad-CRE-Luc activity during re-feeding compared with Ad-USi(unspecific RNAi) (Fig. 2c; Supplementary Fig. 11). TORC2 (Ser 171) phosphorylation was also disrupted in re-fed Ad-SIK2i mice, and TORC2 protein levels were consequently elevated (Fig. 2d). As a result, gluconeogenic gene expression and blood glu- cose concentrations were upregulated in Ad-SIK2i animals (Fig. 2c; Supplementary Figs 11, 12). Ad-SIK2i also increased TORC2 activity in primary hepatocytes; these effects were reversed by expression of an RNAi-resistant SIK2 (Supplementary Figs 13, 14). We noticed that SIK2 contains a single conserved AKT phosphor- ylation site (RQRRPS) at Ser 358. Indeed, exposure of HEK293T cells to insulin increased the phosphorylation of wild-type but not the SIK2(S358A) mutant, by western blot assay with phospho-AKT sub- strate antiserum and by metabolic labelling with inorganic 32 P; these *These authors contributed equally to this work. 1 Peptide Biology Laboratories, Salk Institute For Biological Studies, La Jolla, California 92037, USA. 2 Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, 300 Chunchun-dong, Jangan-gu, Suwon, 440-746, Gyeonggi-do, Korea. 3 The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA. Vol 449 | 20 September 2007 | doi:10.1038/nature06128 366 Nature ©2007 Publishing Group