Figure 8.

IGF-1 is a zeitgeber in temperature entraining cells. A and B, after 1 h of serum starvation mHypoA-2/10-Bmal-1-luc cells were treated with IGF-1 (100 nm) for 30 min at 37.0 °C, after which they were washed and placed into temperature-controlled incubators set either to 12 h 38.5/12 h 36.0 °C (A) or 12 h 36.0/12 h 38.5 °C in B for 48 h. Cells were sampled for luciferase activity in 4-h intervals. Data were normalized relative to time point-specific control cells kept at 37.0 °C. Asterisks indicate significant differences relative to basal at the given time point (two-way ANOVA). In the lower panels, the same data were analyzed by Circwave 1.4 using a 1-harmonic fit for each 24-h interval. All fits were significant (ZT12: 1st day basal r2 = 0.65, IGF-1 r2 = 0.79, 2nd day basal r2 = 0.51, IGF-1 r2 = 0.46; ZT0: 1st day basal r2 = 0.76, IGF-1 r2 = 0.56, 2nd day basal r2 = 0.51, IGF-1 r2 = 0.3, p < 0.001 for all). C, polar plots indicate the phase of maximum Bmal-1 expression on the circular axis and the amplitude of expression on the radial axis. On the outer circle pink indicates warm phase and blue shows the cold phase. The upper plot indicates the application of IGF-1 at ZT12 and the lower plot at ZT0. D, the phase shift of maximal Bmal-1 activity by IGF-1 application on day 1 and day 2 are shown. Asterisks indicate significant differences relative to basal (two-sample Student's t test) and hash marks refer to a difference relative to zero (one sample Student's t test). Data from six experiments were compiled and shown here as mean ± S.D.