Figure 1

Insulin acts as an acute entrainment signal in the cell-autonomous hepatic clock.

(a) Relative Per1, Per2 and Dec1 mRNA levels were determined by RT-PCR following addition of 50 nM insulin to the H4IIE and Rat-1 cell culture medium for 2 h. Each value was normalized to Gapdh. *p < 0.05 versus PBS-treated cells (Student's t-test). (b) Dose-dependent induction of Dec1 mRNA following 2-h insulin treatment of H4IIE and Rat-1 cells as determined by real-time RT-PCR. Each value was normalized to Gapdh. *p < 0.05 versus PBS-treated cells (Dunnett's test). (c) Relative Per1 and Per2 mRNA levels were determined by northern blotting in H4IIE cells for 30 min with inhibitors of proteins mediating the insulin signaling pathway (LY294002 and PD98059), followed by a 1-h treatment with 50 nM insulin. ApoE mRNA was used as an internal control. *p < 0.05 versus PBS- or insulin-treated cells (Student's t-test). (d) Temporal mRNA expression levels of Per2 and Dbp were determined by northern blotting following a shift of H4IIE cells to medium containing 50 nM insulin for 1 h at T 0, washing with PBS and incubation in serum-free medium. Three time independent experiments were performed.