Forkhead box transcription factor O1 inhibits cholesterol 7alpha-hydroxylase in human hepatocytes and in high fat diet-fed mice

Abstract

The conversion of cholesterol to bile acids is the major pathway for cholesterol catabolism. Bile acids are metabolic regulators of triglycerides and glucose metabolism in the liver. This study investigated the roles of FoxO1 in the regulation of cholesterol 7alpha-hydroxylase (CYP7A1) gene expression in primary human hepatocytes. Adenovirus-mediated expression of a phosphorylation defective and constitutively active form of FoxO1 (FoxO1-ADA) inhibited CYP7A1 mRNA expression and bile acid synthesis, while siRNA knockdown of FoxO1 resulted in a approximately 6-fold induction of CYP7A1 mRNA in human hepatocytes. Insulin caused rapid exclusion of FoxO1 from the nucleus and resulted in the induction of CYP7A1 mRNA expression, which was blocked by FoxO1-ADA. In high fat diet-fed mice, CYP7A1 mRNA expression was repressed and inversely correlated to increase hepatic FoxO1 mRNA expression and FoxO1 nuclear retention. In conclusion, our current study provides direct evidence that FoxO1 is a strong repressor of CYP7A1 gene expression and bile acid synthesis. Impaired regulation of FoxO1 may cause down-regulation of CYP7A1 gene expression and contribute to dyslipidemia in insulin resistance.

Figure 1. FoxO1-ADA inhibited CYP7A1 mRNA expression in primary human hepatocytes

Primary human hepatocytes prepared from seven different donor livers were infected with either adeno-EGFP or adeno-FoxO1-ADA at a MOI of 10. A. Twenty-four hr after infection mRNA expressions were measured by real-time PCR (n=7). Inset figure: FoxO1-ADA expression was detected by western blot with anti-HA antibody. B. Total bile acid amount was determined in primary human hepatocytes and culture media 48 hr after adeno-FoxO1-ADA infection (n=3). All results are expressed as mean ± S.D. The “*” indicates statistically significant, p< 0.05, Ad-FoxO1-ADA vs. Ad-EGFP.

Figure 2. FoxO1 interacted with HNF4αand repressed CYP7A1 mRNA expression

A. Upper panel: a diagram of the structure of wild type FoxO1 and FoxO1-Δ256. DBD:DNA binding domain; TA: transactivating domain. Lower panel: GST pull down assay of FoxO1 and HNF4αinteraction. Total cell lysates from HepG2 cells infected with adeno-FoxO1-ADA or adeno-FoxO1-Δ256 at MOI of 10 for 24 hr were used in GST pull-down assay. Ten % of total cell lysates were used as “Input”. FoxO1-ADA or FoxO1-detected by western blot with an anti-HA antibody. B. Chromatin immunoprecipitation assay of the effect of Ad-FoxO1-ADA (Ad-ADA) on FoxO1, HNF4 and PGC-1 association with CYP7A1 and PEPCK chromatin. Primary human hepatocytes were infected with adeno-FoxO1-ADA for 24 hr at an MOI of 10. ChIP assays were performed using antibodies against HA, HNF4 or PGC-1αas described in Material and Methods.

Figure 3. Knocking down endogenous FoxO1 induced CYP7A1 mRNA expression in primary human hepatocytes

Primary human hepatocytes were infected with either adeno-EGFP or adeno-siFoxO1 at MOI of 10 for 48 hr. FoxO1 mRNA (A) and CYP7A1 mRNA (B) were determined by real-time PCR (n=4). FoxO1 protein was determined by western blot with an anti-FoxO1 antibody (A, inset). Results are expressed as mean ±S.D. The “*” indicates statistically significant, p< 0.05, Ad-siFoxO1 vs. Ad-EGFP.

Figure 4. Constitutively active FoxO1 blocked insulin induction of CYP7A1

Human hepatocytes were infected with either adeno-EGFP or adeno-FoxO1-ADA at MOI of 10 for 24 hr in insulin-free media, followed by treatment with insulin (10 nM) for 2 hrs. CYP7A1 mRNA was determined by real-time PCR (n=5). Results are expressed as mean ± S.D. The “*” indicates statistically significant, p< 0.05, insulin vs. vehicle. The “**” indicates statistically significant, p< 0.05, Ad-FoxO1-ADA vs. AdEGFP.

Figure 5. Decreased expression of cyp7a1 mRNA in livers of western diet-fed mice

A. Quantitative real-time PCR was used to assay CYP7A1 and FoxO1 mRNA expressions in chow group (chow) or western diet group (WD) (n=3-4). Results are expressed as mean ± S.D. The “*” indicates statistically significant, p< 0.05, WD vs. chow. B. Immunoblot analysis of nuclear FoxO1 expression in mouse liver. Mouse livers from chow-fed and Western diet-fed (WD) mice (n =3-4) were pooled and nuclear extract was isolated for immunoblotting with antibodies against FoxO1 or Lamin B (negative control).