FIG 7.

Molecular mechanism by which CS and SULT2B1b inhibit HNF4α. (A) COS7 cells were transfected with FLAG-HNF4α. The cells were either cotransfected with SULT2B1b or treated with CS. The subcellular distribution of HNF4α was visualized by immunofluorescence staining with an anti-FLAG antibody (red). 4′,6-Diamidino-2-phenylindole (DAPI) (blue) was used for nuclear counterstaining. (B) Western blot analysis of HNF4α protein levels in nuclear and cytoplasmic fractions of COS7 cells transfected with an empty vector (Vec) or SULT2B1b. The purity of the nuclear and cytoplasmic fractions was confirmed by immunoblotting with an antibody against histone H3 (nuclear fraction marker) and an antibody against tubulin (cytoplasmic fraction marker). (C and D) The subcellular distribution of endogenous HNF4α in the livers of HFD-fed WT and TG mice was assessed by immunofluorescence (C) and Western blot analysis (D). (E) COS7 cells were transfected with FLAG-tagged WT HNF4α or the acetylation-resistant mutant HNF4α-M4. The cells were treated with a vehicle (Veh) or with CS. The subcellular distribution of HNF4α or HNF4α-M4 was visualized by immunofluorescence staining with an anti-FLAG antibody. (F) COS7 cells were transfected with FLAG-HNF4α (top) or FoxO1 (bottom). The cells were either cotransfected with SULT2B1b or treated with CS. Cell lysates were immunoprecipitated with an anti-FLAG (top) or anti-FoxO1 (bottom) antibody before being immunoblotted with antibodies against acetylated lysine and FLAG (top) or against acetylated lysine and FoxO1 (bottom). (G) Liver lysates were immunoprecipitated with an anti-HNF4α antibody before being immunoblotted with antibodies against acetylated lysine and HNF4α. (H) Relative expression of Acss mRNA in COS7 cells treated with CS or transfected with SULT2B1b (left) or in the livers of HFD-fed WT and TG mice (right). (I) Expression of Acss protein as measured by Western blotting. (J) Concentrations of acetyl-CoA in liver tissue. (K) Relative hepatic expression of Acss in fasted and refed mice. (L and M) Glucose production (L) and G6pase mRNA expression (M) in Hepa1-6 cells transfected with the indicated vectors and either left untreated or treated with 5 μM CS for 24 h. (N) Subcellular distribution of FLAG-HNF4α in COS7 cells transfected with the indicated vectors and either left untreated or treated with 5 μM CS for 24 h. HNF4α was visualized by immunofluorescence with an anti-FLAG antibody. Results are means ± SD for three independent experiments. *, P < 0.05; **, P < 0.01.