Figure 1: Insulin regulates hepatic Bmal1 translocation and activity.

(a) Immunoblotting analysis of endogenous Bmal1, Clock, Per1 and Cry1 proteins in nuclear, cytosolic and total extracts of HepG2 cells treated with insulin (INS, 50 nM) or control vehicle for 30 min. (b) Immunostaining analysis of endogenous Bmal1 localization in primary hepatocytes in the presence or absence of LMB (20 ng ml⁻¹, 5 h pretreatment) or insulin (INS, 50 nM, 30 min). Bmal1 staining (left), DAPI staining (middle) to visualize nuclei, and merged together (right). A short purplish red line indicates a scale bar=50 μm. (c) Immunoblotting analysis of endogenous Bmal1 protein in nuclear, cytosolic and total extracts of primary hepatocytes treated with LMB (20 ng ml⁻¹, 5 h pretreatment) or insulin (INS, 50 nM, 30 min). Mice were fasted from ZT0 and injected intraperitoneally with insulin (INS, 2 U kg⁻¹) or normal saline at ZT6, and then animals were killed at indicated intervals (d–f, n=3). (d) Immunoblotting analysis of endogenous Bmal1 protein in pooled nuclear, cytosolic and total extracts of livers (top), and corresponding densitometry analysis of relative Bmal1 protein amounts were shown at the bottom; (e) ChIP analysis of the occupancy of Bmal1 on Dbp and Nr1d1 promoters in livers from mice collected at ZT6.5 or ZT12 (0.5 or 6 h post-injection, respectively); (f) quantitative PCR analysis of hepatic mRNA levels of Dbp and Nr1d1. (g) Quantitative PCR analysis of mRNA levels of Dbp in insulin-treated (INS, 50 nM, 1 h) primary hepatocytes from WT (+/+) or Bmal1 liver-specific knock-out (−/−) mice (n=3). Data are represented as mean ±s.e.m, statistical analyses were performed with a two-tailed unpaired Student’s t-test, *P<0.05, **P<0.01, ^#no significant difference.