Volume 125, Issue 2, Pages 544-555 (August 2003) Farnesoid X receptor agonists suppress hepatic apolipoprotein CIII expression  Thierry Claudel, Yusuke Inoue, Olivier Barbier, Daniel Duran-Sandoval, Vladimir Kosykh, Jamila Fruchart, Jean-Charles Fruchart, Frank J Gonzalez, Bart Staels  Gastroenterology  Volume 125, Issue 2, Pages 544-555 (August 2003) DOI: 10.1016/S0016-5085(03)00896-5

Figure 1 Taurocholic acid (TCA) decreases serum triglyceride and apo CIII concentrations and liver apo CIII mRNA levels in wild-type, but not in FXR-deficient, mice. Wild-type (n = 4 per group) and FXR-deficient (n = 4 per group) mice were fed a diet containing TCA (0.5% wt/wt mixed in standard rodent chow) for 5 days. Control mice received standard rodent chow (CON). Triglyceride and apo CIII concentrations were measured in serum after a 4-hour fasting period as described in Materials and Methods. Values of serum triglycerides (A and D) and apo CIII (B and E) are expressed as means ± SD. (C and F) Mouse liver apo CIII mRNA levels were analyzed by real-time quantitative reverse-transcription PCR and expressed relative to control 28S RNA (∗∗∗P < 0.001, t test; NS, statistically nonsignificant). Gastroenterology 2003 125, 544-555DOI: (10.1016/S0016-5085(03)00896-5)

Figure 2 Bile acids decrease apo CIII mRNA levels in primary human hepatocytes and in human hepatoblastoma cells. (A and B) Primary human hepatocytes were incubated for 48 hours with TCA (30 and 100 μmol/L) or vehicle. (A) Human apo CIII mRNA was analyzed by Northern blot. 28S mRNA was measured as control. (B) Densitometric quantification of apo CIII/28S RNA levels. Values (means ± SD; n = 3) are expressed as a percentage of mRNA levels (∗P < 0.05; t test). (C and D) Human hepatoblastoma HepG2 cells were incubated for 48 hours with CDCA at the indicated concentrations or vehicle. (C) Human apo CIII mRNA was analyzed by Northern blot. 28S RNA was measured as control. (D) Densitometric quantification of apo CIII/28S mRNA levels. The experiment was repeated 3 times. Values (means ± SD; n = 3) are expressed as a percentage of mRNA levels in control cells (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; t test). Gastroenterology 2003 125, 544-555DOI: (10.1016/S0016-5085(03)00896-5)

Figure 3 A nonsteroidal FXR agonist decreases mRNA levels of human apo CIII in HepG2 cells. (A) HepG2 cells were incubated for 48 hours with GW4064 at 5 μmol/L or vehicle. (B) Densitometric quantification of apo CIII/28S RNA levels. The experiment was repeated 3 times. Values (means ± SD; n = 3) are expressed as percentages of mRNA levels in control cells (∗∗P < 0.01; t test). Gastroenterology 2003 125, 544-555DOI: (10.1016/S0016-5085(03)00896-5)

Figure 4 Bile acids and the nonsteroidal FXR agonist GW4064 down-regulate human apo CIII promoter activity via FXR. (A) HepG2 cells were transfected with the indicated human apo CIII promoter-driven luciferase reporter plasmids (1 μg) in the presence of either the pcDNA3 empty or FXR- and RXR-containing expression vectors (300 ng). Cells were subsequently treated with chenodeoxycholic acid (CDCA; 75 μmol/L) or vehicle for 36 hours. Values are expressed as percentage of control set at 100% normalized to internal control β-galactosidase activity as described in Materials and Methods (∗∗P < 0.01; t test). (B) HepG2 cells were transfected with the indicated human apo CIII promoter-driven luciferase reporter plasmid (1 μg) in the presence of either the pcDNA3 and pSG5 empty or FXR- and RXR-containing expression vectors (300 ng). Cells were subsequently treated with GW4064 (100 nmol/L) or vehicle for 36 hours. Values are expressed as percentage of control set at 100% normalized to internal control β-galactosidase activity as described in Materials and Methods (∗∗P < 0.01; t test). Transfections were performed in triplicate, and each experiment was repeated at least 3 times. Gastroenterology 2003 125, 544-555DOI: (10.1016/S0016-5085(03)00896-5)

Figure 5 Human apo CIII promoter activity is negatively regulated by FXR through the DR1 response element in the I4 site. (A) Mutations in bold were introduced in the human −1200/+24 apo CIII pGL3 vector. (B) HepG2 cells were transfected with the wild-type or FXRE site mutated human apo CIII promoter constructs (1 μg) in the presence of either the empty pcDNA3 or the FXR- and RXR-containing expression vectors (300 ng). Cells were subsequently treated with chenodeoxycholic acid (CDCA; 75 μmol/L) or vehicle for 36 hours (∗∗P < 0.01; t test). Transfections were performed in triplicate, and each experiment was repeated at least 3 times. Gastroenterology 2003 125, 544-555DOI: (10.1016/S0016-5085(03)00896-5)

Figure 6 The bile acid-activated FXR/RXR heterodimer replaces HNF4 on the FXRE site of the apo CIII gene promoter in HepG2 cells. Soluble chromatin was prepared from HepG2 cells treated with vehicle or CDCA (50 μmol/L) for 6 hours and immunoprecipitated with antibodies directed against HNF4 (aHNF4), FXR (aFXR), and RXR (aRXR) or with an anti-HA (aHA) antibody as a negative control. The immunoprecipitated DNA was amplified by using primer pairs covering the bile acid response element (−770/−647), a distal region of the apo CIII gene promoter (−559/−333), or the β-actin gene as a negative control. Gastroenterology 2003 125, 544-555DOI: (10.1016/S0016-5085(03)00896-5)

Figure 7 Bile acids decrease apo CIII mRNA levels in liver of HNF4α-deficient mice. (A) Northern blot analysis of apo CIII, HNF4α, and glyceraldehyde-3-phosphate dehydrogenase (phosphorylating; GAPDH) mRNA levels in HNF4α-deficient (KO) and wild-type (FLOX) mice fed standard rodent chow with or without cholic acid (CA) supplementation. (B) KO and FLOX mice (n = 3 per group) were fed a diet containing CA (0.4% wt/wt mixed in standard rodent chow) for 5 days. Control mice received standard rodent chow (CON). Mouse liver apo CIII mRNA levels were analyzed by real-time quantitative reverse-transcription PCR. Results were normalized to internal 28S control mRNA levels. Control wild-type and deficient mouse RNA levels were set at 100% (∗∗P < 0.01, ∗∗∗P < 0.001; t test). (C) FLOX mice (n = 3 per group) were fed a diet containing CA (0.4% wt/wt mixed in standard rodent chow) for 5 days. Liver HNF4α mRNA levels were analyzed by Northern blot and subsequently quantified. Data were normalized by using GAPDH mRNA as an internal control. Control RNA levels were set at 100%. Gastroenterology 2003 125, 544-555DOI: (10.1016/S0016-5085(03)00896-5)

Figure 8 Mechanism of bile acid regulation of apo CIII promoter activity. (A) Human apo CIII promoter activity in hepatocytes in the absence of bile acids. (B) Human apo CIII promoter activity in hepatocytes after FXR activation. Gastroenterology 2003 125, 544-555DOI: (10.1016/S0016-5085(03)00896-5)