Volume 13, Issue 1, Pages 35-43 (January 2011) Cellular Energy Depletion Resets Whole-Body Energy by Promoting Coactivator- Mediated Dietary Fuel Absorption  Atul R. Chopra, Ramakrishna Kommagani, Pradip Saha, Jean-Francois Louet, Christina Salazar, Junghun Song, Jaewook Jeong, Milton Finegold, Benoit Viollet, Franco DeMayo, Lawrence Chan, David D. Moore, Bert W. O'Malley  Cell Metabolism  Volume 13, Issue 1, Pages 35-43 (January 2011) DOI: 10.1016/j.cmet.2010.12.001 Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 Whole-Body Ablation of SRC-2 Results in Dietary Fat Malabsorption (A–D) Food intake, fecal output, fecal triglycerides, and plasma triglycerides were measured in WT and SRC-2−/− mice (whole-body ablation) fed standard chow ad libitum for 24 hr following an overnight fast (n = 5 mice per group). Data are represented as mean + SEM. Unpaired Student's t test was used for evaluation of statistical significance. One asterisk indicates p < 0.05, two asterisks p < 0.01, and three asterisks p < 0.001. Cell Metabolism 2011 13, 35-43DOI: (10.1016/j.cmet.2010.12.001) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 Hepatic SRC-2 Modulates Dietary Fat Absorption in a BA-Dependent Manner (A–D) Food intake, fecal output, fecal triglycerides, and plasma triglycerides were measured in WT and SRC-2 LKO mice (liver-specific ablation) fed standard chow ad libitum for 24 hr following an overnight fast (n = 5 mice per group). (E and F) Plasma radioactivity and triglyceride levels were measured in chow-fed WT and SRC-2 LKO mice after injection of the lipase inhibitor Tyloxapol and gavage with olive oil containing 14C-trioleoylglycerol (n = 5 mice per group). Data are represented as mean + SEM. Two-way ANOVA with Bonferroni posttests to compare replicate means by row was used for evaluation of statistical significance. One asterisk indicates p < 0.05, two asterisks p < 0.01, and three asterisks p < 0.001. (G) Intestinal radioactivity levels were measured in WT and SRC-2 LKO mice 2 hr after gavage with olive oil containing 14C-trioleoylglycerol (n = 5 mice per group). Data are represented as mean + SEM. Two-way ANOVA with Bonferroni posttests to compare replicate means by row was used for evaluation of statistical significance. One asterisk indicates p < 0.05, two asterisks p < 0.01, and three asterisks p < 0.001. (H and I) Hepatic and biliary BA levels were measured in WT and SRC-2 LKO mice on standard chow and on 1% CA diet for 2 weeks. Bile was obtained from the gall bladder and is representative of intestinal BA levels (n = 4–5 mice per group). (J) Fecal triglyceride levels were measured in chow-fed WT and SRC-2 LKO mice upon exposure of mice to a high-fat diet (60% calories from fat) and a diet containing high fat plus 1% CA for 48 hr (n = 4–5 mice per group). Data are represented as mean + SEM. Unless otherwise indicated, unpaired Student's t test was used for evaluation of statistical significance. One asterisk indicates p < 0.05, two asterisks p < 0.01, and three asterisks p < 0.001. See also Figures S1 and S2. Cell Metabolism 2011 13, 35-43DOI: (10.1016/j.cmet.2010.12.001) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 SRC-2 Modulates Dietary Fat Absorption by Controlling the Expression of the BSEP (A) Expression of various BA transporter genes was measured via relative quantitation by quantitative PCR in the liver of WT and SRC-2 LKO mice that were exposed to standard chow or 1% CA for 2 weeks (n = 5–7 mice per group). Data are represented as mean + SEM. Unpaired Student's t test was used for evaluation of statistical significance. One asterisk indicates p < 0.05, two asterisks p < 0.01, and three asterisks p < 0.001. (B) BSEP protein expression was measured via western blot analysis in the liver of WT and SRC-2 LKO mice that were exposed to standard chow (n = 2 mice per group). GAPDH was used as a loading control. (C–F) SRC-2 LKO mice exposed to adenoviral BSEP (Ad. BSEP) were compared with SRC-2 LKO and WT mice exposed to empty adenovirus (Ad. Empty) using tail vein infusion. Eight days after virus infusion, hepatic BSEP expression, hepatic BA content, fecal triglyceride content, and plasma triglyceride content were measured (n = 6 mice per group). Statistical comparison was performed between WT (Ad. Empty) and SRC-2 LKO (Ad. Empty) and between SRC-2 LKO (Ad. Empty) and SRC-2 LKO (Ad. BSEP) groups. Data are represented as mean + SEM. One-way ANOVA with Tukey's multiple comparison test was used for evaluation of statistical significance. One asterisk indicates p < 0.05, two asterisks p < 0.01, and three asterisks p < 0.001. See also Figures S3 and S4. Cell Metabolism 2011 13, 35-43DOI: (10.1016/j.cmet.2010.12.001) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 SRC-2 Modulates BSEP Expression in a Cell-Autonomous Manner by Coactivating FXR (A) Expression of various BA transporter genes was measured via relative quantitation by quantitative PCR in primary hepatocytes exposed to DMSO or to CDCA from WT and SRC-2 LKO mice. Statistical comparison was performed between WT and SRC-2 KO PHs in each treatment group. (B) HepG2 liver hepatoma cells were transfected with a reporter-gene plasmid driven by the wild-type mouse BSEP promoter and the same promoter with a mutated FXRE motif, together with expression plasmids for SRC-2 and FXR, and exposed to CDCA. Reporter-gene levels were determined 48 hr after transfection. The empty vector (EV) value was fixed at 1, and the rest of the values are compared relative to that. (C) HepG2 liver hepatoma cells were transfected with a reporter-gene plasmid driven by the wild-type mouse BSEP promoter together with expression plasmids for WT SRC-2, SRC-2 mutant with AD1 deletion, SRC-2 mutant with AD2 deletion, and SRC-2 mutant with both AD1 and AD2 deletions and FXR and exposed to CDCA. Reporter-gene levels were determined 48 hr after transfection. The empty vector (EV) value was fixed at 1, and the rest of the values are compared relative to that. (D) In vivo ChIP assays were performed using liver tissue from WT mice with 150–200 bp amplicons flanking the region containing the FXRE motif of the BSEP promoter and an irrelevant region 3000 bp upstream of the transcription start site. SYBR Green quantitative PCR (normalized to input) was used to assess SRC-2 occupancy of the BSEP promoter, using two different antibodies that targeted different regions of SRC-2. Control antibody recognized mouse IgG. (E) In vivo ChIP assays were performed using liver tissue from WT and FXR knockout mice with primers flanking the region containing the FXRE motif of the BSEP promoter. SYBR Green quantitative PCR (normalized to input) was used to assess SRC-2 occupancy of the BSEP promoter, using an SRC-2-specific antibody. Control antibody recognized mouse IgG. Data are represented as mean + SEM. For all gene expression data, unpaired Student's t test was used for evaluation of statistical significance. One asterisk indicates p < 0.05, two asterisks p < 0.01, and three asterisks p < 0.001. See also Figure S5. Cell Metabolism 2011 13, 35-43DOI: (10.1016/j.cmet.2010.12.001) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 A Hepatocyte-Specific Screen Identifies AMPK as a Putative, Positive Modulator of BSEP Expression (A) BSEP expression was measured via relative quantitation by quantitative PCR in primary hepatocytes exposed to standard doses of the indicated agents for 24 hr. (B) BSEP expression was measured via relative quantitation by quantitative PCR in primary hepatocytes exposed to adenoviruses containing cDNAs representing either constitutively active AMPKα or dominant-negative AMPKα for 24 hr. Statistical comparison was made between the GFP group and either the constitutively active AMPKα or dominant-negative AMPKα groups. (C) BSEP expression was measured via relative quantitation by quantitative PCR in primary hepatocytes exposed to an adenovirus containing cDNA representing dominant-negative AMPKα and either vehicle or 1 mM AICAR for 18 hr. All gene expression data are represented as mean + SEM. Unpaired Student's t test was used for evaluation of statistical significance. One asterisk indicates p < 0.05, two asterisks p < 0.01, and three asterisks p < 0.001. Cell Metabolism 2011 13, 35-43DOI: (10.1016/j.cmet.2010.12.001) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 Hepatic AMPK Increases the Intrinsic Transcriptional Activity of SRC-2 and Drives It to the BSEP Promoter (A) HepG2 liver hepatoma cells were transfected with pG5Luc (five Gal4 binding sites driving luciferase expression) together with pBIND SRC-2 (SRC-2:Gal4 DNA binding domain fusion protein) and exposed to 1 mM AICAR. Luciferase levels were determined 48 hr after transfection. The vehicle value was fixed at 1, and the rest of the values are compared relative to that. (B) HepG2 liver hepatoma cells were transfected with pG5Luc together with pBIND SRC-2 and either empty vector (EV) or dominant-negative AMPKα2 (DnAMPK) and exposed to 0.3 mM, 0.6 mM, and 1 mM AICAR. Luciferase levels were determined 48 hr after transfection. The vehicle value was fixed at 1, and the rest of the values are compared relative to that. (C) HepG2 cell were treated with 1 mM AICAR for 30 min and subjected to immunoprecipitation with an AMPKα2-specific antibody. Immunoprecipitated samples were subjected to immunoblotting along with 10% input, as indicated. (D) Purified, full-length SRC-2 protein was subjected to in vitro phosphorylation with or without purified AMPK holoenzyme and AMP, as indicated. (E and F) Hepatic BSEP expression and BA content were measured via relative quantitation by quantitative PCR in the liver of WT and AMPKα1/α2 double knockout (AMPKα DKO) mice that were fasted for 24 hr (n = 7–8 mice per group). (G) Hepatic BSEP expression was measured via relative quantitation by quantitative PCR in the livers of mice that were exposed to either PBS or 0.5 mg/g BW AICAR for 12 hr and fed standard chow (n = 6 mice per group). (H) BSEP expression was measured via relative quantitation by quantitative PCR in primary hepatocytes from WT and SRC-2 LKO mice exposed to 1 mM AICAR for 18 hr. (I and J) ChIP assays were performed using HepG2 cells exposed to either vehicle or 1 mM AICAR for 30 min with primers flanking the region containing the FXRE motif of the BSEP promoter. SYBR Green quantitative PCR (normalized to input) was used to assess SRC-2 or AMPKα2 occupancy of the BSEP promoter upon ChIP, using an SRC-2- or AMPKα2-specific antibody. Control antibody recognized mouse IgG. All gene expression data are represented as mean + SEM. Unpaired Student's t test was used for evaluation of statistical significance. One asterisk indicates p < 0.05, two asterisks p < .01, and three asterisks p < 0.001. See also Figure S6. Cell Metabolism 2011 13, 35-43DOI: (10.1016/j.cmet.2010.12.001) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 7 Schematic Depicting the Cascade that Links Cellular Energy Depletion with Whole-Body Energy Repletion Cell Metabolism 2011 13, 35-43DOI: (10.1016/j.cmet.2010.12.001) Copyright © 2011 Elsevier Inc. Terms and Conditions