FIG 3.

GsD activation stimulates the cAMP-CREB pathway in primary mouse hepatocytes. Primary hepatocytes were prepared from +/+ or GsD/+ mice that were injected with AAV-TBG-GFP or AAV-TBG-Cre 3 weeks earlier. Hepatocytes were treated with FSK (10 μM)-IBMX (18 μM), CNO (10 μM), or CNO (10 μM)-IBMX (18 μM). (A) cAMP levels after 15 or 30 min of the indicated treatments (means ± standard errors of the means; n = 3). (B) Western blots of phospho-CREB (S133), total CREB, CRTC2, HSP90, and GsD-GFP in hepatocytes treated for 15 min as indicated. Open arrowhead, phospho-CRTC2 (inactive); filled arrowhead, dephospho-CRTC2 (active). Duplicates are shown (quantified in Fig. S2A in the supplemental material). (C) Sik1 mRNA levels in hepatocytes treated as indicated for 1 h (means ± standard errors of the means; n = 3). (D) Luciferase activity in hepatocytes treated as indicated for 4 h (n = 3). #, significance versus untreated controls of the same genotype; *, significance for GFP-Cre within each treatment group. Data are representative of results from 2 or 3 independent experiments performed on cells harvested from separate animals. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (by t tests).