Fig 2: Involvement of Wnt/TCF7L2 signaling in the protective effect of CHS on islets β cell. βTC3 cells were treated with CHS and IHG or SHG, then the RNA was extracted by TRIZOL. The mRNA expression levels of Wnt2 (A), Wnt3a (B), Wnt4 (C), Wnt5a (D), and Wnt10b (E) were measured by RT-PCR. (F) The protein expression levels of Wnt3a was measured by western blotting after different treatments. The effects of CHS on β-catenin expression levels in the cytoplasm (G) and nuclear (H) were measured by western blotting. (I) The effects of CHS on protein expression levels of Axin-2, c-Myc, Naked-1 and P-GSK-3β. βTC3 cells were treated with CHS and XAV-939 (a Wnt/β-catenin antagonist, 10μM), and then subjected to IHG. (J) The protein expression levels of TCF7L2, cyclin D1, skp2, p53, p21 and GIPR were measured by western blotting. (K) Cell variability was measured by CCK8 assay. (L) Insulin secretion levels was measured by an insulin RIA kit. (M) BrdU positive ratio was calculated from the BrdU positive cell numbers vs total cell numbers. Data are representative of three independent experiments. ##P<0.01 vs. NG treatment group, **P<0.01 vs. IHG treatment group, ΔΔP<0.01 vs. scrb treatment group, &&P<0.01 vs.CHS treatment group.

Fig 3: CHS protected against cell cycle arrest of islet cells from IHG. (A) The βTC3 cell proliferation was measured by a BrdU labeling and detection kit. Inverted microscope (200×) images. (B) The cell cycle related proteins (Cyclin D1, Skp2, p21, andp27) were measured by western bolting. Data are representative of three independent experiments. ##P<0.01 vs. NG treatment group, **P<0.01 vs. SHG treatment group, ΔΔP<0.01 vs. IHG treatment group.

Fig 4: Effects of CHS on HBP1 expression in islets β cell. (A) Time dependent effects of CHS on the expression HBP1. (B) Dose dependent effects of CHS on the expression HBP1. **P<0.01 vs. 0h or 0mM treatment group. (C) Effects of CHS on HBP1 expression after IHG or SHG treatment. βTC3 cells were transfected with the empty vector pcDNA3 (scrb) or vectors encoding HA-tagged wild-type HBP1 (HBP1) at 30nM, and then cells were exposed to different treatments as indicated. (D) The protein expression levels of TCF7L2, GIPR, cyclinD1, Skp2, p53 and p21 were measured by western blotting. (E) Insulin secretion levels was measured by an insulin RIA kit. (F) Cell variability was measured by CCK8 assay. (G) BrdU positive ratio was calculated from the BrdU positive cell numbers vs total cell numbers. βTC3 cells were transfected with scrb and siHBP1 (30nM) for 24h, and then cells were exposed to different treatments as indicated. (H) The protein expression levels of GIPR, cyclinD1, and p53 were measured by western blotting. (I) Cell variability was measured by CCK8 assay. (J) Insulin secretion levels was measured by an insulin RIA kit. (K) BrdU positive ratio was calculated from the BrdU positive cell numbers vs total cell numbers. Data are representative of three independent experiments. ##P<0.01 vs. NG treatment group, ΔΔP<0.01 vs. IHG treatment group, &&P<0.01 vs.scrb treatment group.

Fig 5: Effect of ezetimibe (EZ) treatment on hepatic protein expression. (A) Western blot analysis of SKP2 in livers from the high fat chow (HF) and HF+EZ groups, and (B) densitometric quantification of SKP2/β-actin expression in livers from the HF+EZ (black bars) and HF (gray bars) groups. The data represent the means ± standard deviation (n=7). **P<0.01, vs. HF group.