Regulation of Hepatic Gluconeogenesis by an ER-Bound Transcription Factor, CREBH

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1 Cell Metabolism, Volume 11 Supplemental Information Regulation of Hepatic Gluconeogenesis by an ER-Bound Transcription Factor, CREBH Min-Woo Lee, Dipanjan Chanda, Jianqi Yang, Hyunhee Oh, Su Sung Kim, Young-Sil Yoon, Sungpyo Hong, Keun-Gyu Park, In-Kyu Lee, Cheol Soo Choi, Richard W. Hanson, Hueng-Sik Choi, and Seung-Hoi Koo

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3 Figure S1. CREBH is transcriptionally induced by GR/PGC-1α A) Q-PCR analysis showing hepatic CREBH or gluconeogenic gene expression in mice under normal chow diet (NCD) or high fat diet (HFD) (**;P<0.01, t-test, n=3). B) Top, HNF-4α and GR binding sites on CREBH promoter. Bottom, Chromatin immunoprecipitation assay showing occupancy of HNF-4α and GR on CREBH promoter from control or dexamethasone-treated cells. C) Left, 5 - deletion analysis of human or mouse CREBH promoter using HepG2 cells exposing 100nM dexamethasone. Right, Luciferase assay using HepG2 cells transiently transfected with either wild type or GRE mutant luciferase CREBH promoter in the absence or in the presence of HNF-4α +/- dexamethasone. D) Luciferase assay using HepG2 cells transiently transfected with CREBH luciferase construct together with expression vectors for PGC-1α, AKT and/or SHP +/- 100 nm dexamethasone. E) Luciferase assay using HepG2 cells transiently transfected with CREBH luciferase construct together with expression vectors for HNF-4α, PGC-1α and/or SHP. F) Northern blot (left) or Q-PCR (right) analysis of CREBH and SHP expression using RNAs from rat primary hepatocytes exposed to Ad-GFP only, Ad-GFP plus 100nM dexamethasone, 100nM dexamethasone plus Ad-SHP or 100nM dexamethasone plus insulin (*;P<0.05 compared to control, # ;P<0.05 compared to dexamethasone).

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5 Figure S2. CREBH binding is critical in activating hepatic gluconeogenesis A) 16 hr-fasting body weight in mice expressing Ad-GFP or Ad-CREBH-N (n=5). B) Top, 16 hr-fasting insulin levels from wild type mice expressing Ad-GFP or Ad-CREBH-N (n=5-6). Bottom, A representative western blot analysis showing relative phosphorylation levels of CRTC2 in livers of mice injected with either Ad-GFP or Ad-CREBH-N adenovirus. C) Occupancy of CREBH on distinctive CREBHRE in gluconeogenic promoters. Top, Localization of CREBH response element (CREBHRE) on PEPCK-C or G6Pase promoter. Bottom, Chromatin immunoprecipitation assay showing occupancy of CREBH on PEPCK-C or G6Pase promoter. D) CREBH binds to PEPCK-C and G6Pase promoters. Electrophoretic mobility shift assay showing a binding of CREBH on PEPCK-C and G6Pase promoter elements. E) Luciferase assay using HepG2 cells transiently transfected with wild type or CREBHRE mutant PEPCK-C/G6Pase luciferase reporter construct exposed to 10 μm forskolin plus 100 nm dexamethasone (camp/dex) (n=3).

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7 Figure S3. CRTC2 is required for CREBH-mediated induction of hepatic gluconeogenic genes A) Live imaging of hepatic G6Pase-luciferase activity in response to feeding or fasting in C57BL/6 mice. Five days post Ad-WT G6Pase(-231/+57) LUC adenovirus injection, mice were fasted for 16 hr or fed and imaged following luciferin administration via IP injection. B) Top, Live imaging of hepatic G6Paseluciferase (Ad-WT G6Pase (-231/+57)-Luc) activity in response to CREBH-N or CREBH-FL in C57BL/6 mice. Five days post adenovirus injection, mice were imaged following luciferin administration via IP injection. Bottom, Western blot analysis showing protein levels of CREBH-N or CREBH-FL. C) A graphic representation of ROI as in Figure 3B (right; *,P<0.05, t-test, n=3). D) Coimmunoprecipitation assay showing the interaction between CREBH and CRTC2. Expression vector for flag-tagged CRTC2 together with empty HAvector, expression vector for HA-tagged CREB, or expression vector for HAtagged CREBH-N were cotransfected into 293T cells for 48 hr before being harvested. Immunoprecipitation was performed with either M2 (flag)-agarose (left) or HA-agarose (right) and blotted with antibodies against flag and HA. E) Co-immunoprecipitation assay showing endogenous interaction between CRTC2 and CREB or CREBH in either WT C57BL/6 or db/db mice. A representative western blot analysis was shown. F) Effects of ATF6 on CREBHmediated transcriptional activity. Luciferase assay using HepG2 cells transiently transfected with wild type, CRE mutant, or CREBHRE mutant G6Pase luciferase reporter construct exposed to 10 μm forskolin plus 100 nm dexamethasone (camp/dex) with or without CREBH-N/ATF6-N expression plasmids (n=3).

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9 Figure S4. Hepatic depletion of CREBH relieves fasting hyperglycemia A) A representative western blot analysis showing the protein levels of CRTC2, CREBH-N or HSP90 from livers of mice as in Figure 3E. B) Pyruvate challenge test using wild type mice injected with Ad-GFP + Ad-US, Ad-CREBH-N + Ad-US, or Ad-CREBH-N + Ad-CRTC2 RNAi. 2g/kg sodium pyruvate was intraperitoneally injected into each mice, from which glucose levels were measured at the designated time points (#; (Ad-CREBH-N + Ad-US) over (Ad- CREBH-N + Ad-CRTC2 RNAi), P<0.01, *; (Ad-GFP + Ad-US) over (Ad-CREBH- N + Ad-US), P<0.05, t-test, n=5-7). C) Glucose output assay showing effects of CRTC2 knockdown on CREBH-dependent glucose production in rat primary hepatocytes infected with Ad-GFP + Ad-US, Ad-CREBH-N + Ad-US, or Ad- CREBH-N + Ad-CRTC2 RNAi in the absence or in the presence of 10 μm Fsk for the final 8 hrs (**;P<0.01, t-test, n=3). D) Q-PCR analysis showing effect of adenoviruses as in S4C) on hepatic expression of G6Pase (top) or PEPCK-C (bottom) (**;P<0.01, t-test, n=3). E) Effects of CREBH knockdown on hepatic glucose metabolism. Top, Q-PCR analyses of PEPCK-C, G6Pase and CREBH expression using RNAs from rat primary hepatocytes infected with Ad CREBH shrna +/-100nM dexamethasone (*;P<0.05 compared to control, # ;P<0.01 compared dexamethasone). Bottom, Glucose output assay showing effects of CREBH knockdown on glucose production in rat primary hepatocytes (*,P<0.05, t-test, n=3). F) Q-PCR analysis showing effect of Ad-US or Ad-CREBH RNAi infection on hepatic expression of ER-stress genes in wild type mice (top, **;P<0.01, t-test, n=5) or db/db mice (bottom, **;P<0.01, t-test, n=5) fasted for 16 hr.

10 EXPERIMENTAL PROCEDURES Animal Experiments Male 8-week-old C57BL/6 mice or diabetic db/db mice (Charles River) were used. For high fat diet feeding, 60% calorie high fate diet (Research Diet) was fed on male 4-week-old C57BL/6 mice for 8-12 weeks. Delivery of recombinant adenovirus was performed via tail-vein injection. Sixteen hr fasting blood glucose levels were measured either 5 days (Ad-CREBH-N or Ad-CRTC2 RNAi) or 4 days (Ad-CREBH RNAi) post injection. Male 40-week-old Otsuka Long- Evans Tokushima fatty (OLETF) rats and their lean nondiabetic counterparts, Long-Evans Tokushima Otsuka (LETO) rats, were supplied by Otsuka Pharmaceutical (Tokushima, Japan). All procedures were approved by the Sungkyunkwan University School of Medicine Institutional Animal Care And Use Committee (IACUC). Plasmid and adenovirus G6pase (-231/+57) promoter was amplified from human genomic DNA and inserted into pgl4.12 vector. Sequences for CREBH full length (CREBH-FL) and active form (CREBH-N) were amplified from mouse cdna and inserted into flag tagged pcdna3 or GFP tagged pcdna3 vector. Ad-GFP, Ad-US control RNAi, and Ad CRTC2 RNAi were described previously (Koo et al., 2005; Koo et al., 2004). Ad-nuclear CREBH (CREBH-N), Ad-full length CREBH (CREBH-FL) and Ad-G6Pase-luc virus were generated via homologous recombination methods as described previously (Yoon et al., 2009b). Quantitative PCR Total RNA from either primary hepatocytes or liver tissue was extracted using easy spin RNA extraction kit (Intron). cdnas were generated by Superscript II enzyme (Invitrogen) and analyzed by quantitative PCR (Q-PCR) using a SYBR green PCR kit and Thermal Cycler DICE Real Time System TP800 (TAKARA). All data were normalized to ribosomal L32 expression. Western Blot Analyses Western blot analyses on μg of whole-cell extracts were performed as described previously (Ryu et al., 2009). GFP-HRP, M2 (flag) antibody were purchased from Sigma. Antibodies against HA and HSP90 were from Santa

11 Cruz Biotechnology and antibody against CREB was purchased from Cell Signaling Biotechnology. Mouse polyclonal antibody against CREBH was generated using amino acids 1 through 200 (Laboratory Animal Research Center, Sungkyunkwan University School of Medicine) and used exclusively for western blot analysis. For immunoprecipitation, CREBH antibody was purchased from Allelebiotechnology. Polyclonal antibodies against PGC-1α or CRTC2 were kindly provided by Prof. Marc Montminy (Koo et al., 2004; Screaton et al., 2004). M2 (flag)- agarose and HA- agarose were purchased from Sigma and used for co-immunoprecipitation studies. Glucose Output Assay After 40 h (CREBH-N) or 64 h (CREBH RNAi) infection of adenovirus in rat primary hepatocytes, media were replaced with Krebs-ringer buffer (115 mm NaCl, 5.9 mm KCl, 1.2 mm MgCl 2, 1.2 mm NaH 2 PO4, 2.5 mm CaCl 2, 25 mm NaHCO 3, ph 7.4) plus 10 mm lactate and 1 mm pyruvate. Eight hours later, the glucose level in the media was measured using a QuantiChromTM glucose assay kit (Bioassay Systems). Culture of Primary Hepatocytes Primary hepatocytes were prepared from g Sprague-Dawley rats or 20g C57BL/6 mice as described previously (Yoon et al., 2009a). Cells were plated in medium 199 (Media Tech or Sigma) with 10% FBS, 10 units/ml penicillin, 10ug/ml streptomycin and 10 mm dexamethasone. After attachment, cells were infected with various Adenoviruses and treated as indicated in the figure legends. Transient transfection Assays HepG2 hepatoma cells were maintained with Ham's F12 medium (Sigma) as described previously (Ryu et al., 2009). Each transient transfection was performed with 300 ng of luciferase construct, 50 ng of β-galactosidase plasmid, and ng of various expression constructs using Fugene 6 reagent (Roche) according to manufacturer s instruction. Measurement of Metabolites Tail vein-drawn blood was analyzed for glucose using an automatic glucose monitor (One Touch, Lifescan). Plasma insulin levels were measured using

12 commercial insulin assay ELISA kit (SHIBAYAGI). Optical In Vivo Imaging Mice were infected with Ad GFP, Ad CREBH-N or Ad CREBH-FL together with either Ad wild type G6Pase (-231/+57) luc or Ad CREBHRE mutant G6Pase (- 231/+57) luc. Five days post adenoviral injection, mice were fasted for 16 hr followed by 2 h refeeding. For imaging, mice were IP injected with 100 mg/kg sterile firefly D-luciferin (Golden Bio). Ten minutes later, mice were anaesthetized and imaged using the IVIS SPECTRUM imaging system (Xenogen) as described previously (Dentin et al., 2007). Hyperinsulinemic-Euglycemic Clamp Study Hyperinsulinemic-euglycemic clamp study was performed as descrbed previously with a slight modification (Choi et al., 2007; Ryu et al., 2009). Briefly, Indwelling catheters were placed into the right internal jugular vein seven days prior to the hyperinsulinemic-euglycemic clamp studies. After 18 hr fasting, [3-3 H] glucose was infused for 2 hr to assess the basal glucose turnover. Following the basal period, hyperinsulinemic-euglycemic clamp was conducted for 140 min with an infusion of human insulin (3.0mU/kg/min). Plasma glucose was immediately analyzed during the clamps by a glucose oxidase method (GM9 Analyzer; Analox Instruments; London), and 20% dextrose was infused to maintain plasma glucose at basal concentrations. To estimate insulin-stimulated whole-body glucose fluxes, [3-3 H]glucose was infused at a rate of 0.1 μci/min throughout the clamps, and 2-deoxy-D-[1-14 C]glucose (2-[ 14 C]DG; PerkinElmer) was injected as a bolus at the 85th minute of the clamp to estimate the rate of insulin-stimulated tissue glucose uptake. Rates of basal and insulin-stimulated whole-body glucose turnover were determined as the ratio of the [3-3 H]glucose infusion rate to the specific activity of plasma glucose at the end of the basal period and during the final 30 min of the clamp experiment, respectively. Hepatic glucose production was determined by subtracting the glucose infusion rate from the total glucose appearance rate. Statistical Analyses Values are expressed as mean ± standard deviation. Statistical significance was calculated using an unpaired two-tailed Student s t test with unequal variance. Differences were considered statistically significant at or under P<0.05.

13 REFERENCES Choi, C.S., Savage, D.B., Abu-Elheiga, L., Liu, Z.X., Kim, S., Kulkarni, A., Distefano, A., Hwang, Y.J., Reznick, R.M., Codella, R., Zhang, D., Cline, G.W., Wakil, S.J., and Shulman, G.I. (2007). Continuous fat oxidation in acetyl-coa carboxylase 2 knockout mice increases total energy expenditure, reduces fat mass, and improves insulin sensitivity. Proc Natl Acad Sci U S A 104, Dentin, R., Liu, Y., Koo, S.H., Hedrick, S., Vargas, T., Heredia, J., Yates, J., 3rd, and Montminy, M. (2007). Insulin modulates gluconeogenesis by inhibition of the coactivator TORC2. Nature 449, Koo, S.H., Flechner, L., Qi, L., Zhang, X., Screaton, R.A., Jeffries, S., Hedrick, S., Xu, W., Boussouar, F., Brindle, P., Takemori, H., and Montminy, M. (2005). The CREB coactivator TORC2 is a key regulator of fasting glucose metabolism. Nature 437, Koo, S.H., Satoh, H., Herzig, S., Lee, C.H., Hedrick, S., Kulkarni, R., Evans, R.M., Olefsky, J., and Montminy, M. (2004). PGC-1 promotes insulin resistance in liver through PPAR-alpha-dependent induction of TRB-3. Nat Med 10, Ryu, D., Oh, K.J., Jo, H.Y., Hedrick, S., Kim, Y.N., Hwang, Y.J., Park, T.S., Han, J.S., Choi, C.S., Montminy, M., and Koo, S.H. (2009). TORC2 regulates hepatic insulin signaling via a mammalian phosphatidic acid phosphatase, LIPIN1. Cell Metab 9, Screaton, R.A., Conkright, M.D., Katoh, Y., Best, J.L., Canettieri, G., Jeffries, S., Guzman, E., Niessen, S., Yates, J.R., 3rd, Takemori, H., Okamoto, M., and Montminy, M. (2004). The CREB coactivator TORC2 functions as a calciumand camp-sensitive coincidence detector. Cell 119, Yoon, Y.S., Ryu, D., Lee, M.W., Hong, S., and Koo, S.H. (2009a). Adiponectin and thiazolidinedione targets CRTC2 to regulate hepatic gluconeogenesis. Exp Mol Med 41, Yoon, Y.S., Seo, W.Y., Lee, M.W., Kim, S.T., and Koo, S.H. (2009b). Saltinducible kinase regulates hepatic lipogenesis by controlling SREBP-1c phosphorylation. J Biol Chem 284,