Supplementary Figures: PDF format: 1.5MB Supplementary Figure Legends Supplementary Figure S1: Western blot assays of endogenous CREB, P-CREB, and TORC2 in liver extracts from mice 10 minutes after injection with glucagon, insulin, insulin+glucose, or insulin+glucagon, as indicated. Ser171 dephosphorylation of TORC2 in lanes 4 and 5 indicated by enhanced mobility relative to lanes 1-3. Supplementary Figure S2: Western blot assays of endogenous TORC2 and CREB in liver extracts from mice (n=2) following a 4 hour-fast or under ad libitium feeding. P- TORC2 and unphospho (TORC2) bands indicated. Supplementary Figure S3: Effect of FSK on P-CREB (P-CR) and CBP levels over gluconeogenic promoters by ChIP assay of rat FAO hepatocytes. Supplementary Figure S4: Effect of TORC2 and dominant negative A-CREB (AC) adenoviruses on induction of gluconeogenic G6Pase gene by FSK (10M, 2hr) in cultured primary rat hepatocytes. Data represent mean + s.e.m. Supplementary Figure S5: Glucose output from primary rat hepatocytes infected with Ad-TORC2 or control GFP virus and treated with FSK, FSK/INS, or INS alone, as indicated. Glucose levels in the medium were measured over a 4 hour collection period. Data represent mean + s.e.m. Supplementary Figure S6: Right, plasma insulin levels in Ad-TORC2 or control Ad- GFP injected mice under fasting conditions (24 hour fast; n=4). Data represent mean + s.e.m. (asterisk, p<0.01, t-test). Supplementary Figure S7: Effect of glucagon (GLU) treatment (3 hours) on SIK1 mRNA (top) and protein accumulation (bottom: 0, 0.5, 1, 3 hours) in primary rat hepatocytes. Effect of CHX on SIK1 protein levels shown. Supplementary Figure S8: Effect of co-transfected PKA, CREB dominant negative (A- CREB), or control (empty) expression vector on mSIK1(-425/+76) luciferase reporter activity in transiently transfected HepG2 hepatocytes. Data represent mean + s.e.m. Supplementary Figure S9: Effect of SIK1 RNAi adenovirus infection on levels of phospho (Ser171) TORC2 and unphosphorylated TORC2 in primary rat hepatocytes. Densitometric analysis of TORC2 bands in figure 4A. Ratio of unphospho TORC2/P- TORC2 in control (US) and Ad-SIK1 RNAi infected cells indicated. Supplementary Figure S10: Western blot analysis of HA-TORC2 immunoprecipitates showing effect of SIK1 over-expression on TORC2 and phospho (Ser171)TORC2 levels in hepa1c1c7 mouse hepatocytes infected with SIK1 plus wild-type or Ser171Ala mutant HA-tagged TORC2 expressing adenovirus. Supplementary Figure S11: Effect of SIK1 over-expression on activity of TORC2 (left) and PGC-1 (right) in HepG2 cells. Transient transfection assay of PEPCK (left) and AOX (right) luciferase reporter plasmids co-transfected with SIK1, TORC2, or PGC-1 expression vectors as indicated. Effect of FSK treatment (10M, 4 hours) shown. Data represent mean + s.e.m. Supplementary Figure S12: Circulating levels of insulin in SIK1, SIK2, or control GFP Adenovirus injected mice under fasting conditions (16 hour fast). Data represent mean + s.e.m. (asterisk, p<0.05, t-test). Supplementary Figure S13: Effect of Ad-SIK1, Ad-SIK2, and control Ad-GFP on fasting glucose levels in db/db diabetic mice (asterisk, p<0.05, t-test) (n=4). Supplementary Figure S14: SIK1 does not modulate insulin signaling in livers of diabetic db/db mice. Effect of Ad-SIK1 and Ad-SIK2 on AKT activation 10 minutes following acute IP insulin injection. Western blot analysis of liver extracts showing SIK1, SIK2, AKT, and phospho (Ser473)AKT (P-Akt) levels under control (PBS) and insulin injected conditions (top). Densitometric quantitation of p-Akt/Akt ratios shown below. Supplementary Figure S15: Q-PCR analysis showing effect of Ad-SIK1 on PGC-1 mRNA levels in primary rat hepatocytes expressing wild-type and (Ser171Ala) mutant Ad-TORC2. Supplementary Figure S16: TORC2 is a substrate for AMPK phosphorylation. In vitro phosphorylation of recombinant GST TORC2 (1-240) or control GST alone. Top, autoradiogram showing incorporation of -32P-ATP into TORC2. Bottom, Coomassie gel showing 52 kD GST-TORC2 (1-240) band. Stoichiometry of TORC2 phosphorylation by AMPK indicated (moles phosphate/mol GST TORC2/U AMPK). Supplementary Figure S17: AICAR disrupts dephosphorylation of TORC2 in response to fasting signals. Densitometric analysis of TORC2 Western blot assay in figure 5b showing ratio of unphosphorylated/phospho Ad-TORC2 in primary hepatocytes under control (DMS) or FSK stimulated conditions. Supplementary Figure S18: Effect of FSK and AICAR on CREB levels. Densitometric analysis of Western blot from figure 5b. Supplementary Figure S19: AMPK inhibits gluconeogenesis via Ser171 phosphorylation of TORC2. Effect of AICAR on expression of G6Pase gene in primary rat hepatocytes. Rescue with mutant (Ser171Ala) TORC2 adenovirus indicated. Treatment with AICAR and FSK shown. Data represent mean + s.e.m.
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