FGF-19 suppresses the stimulatory effect of insulin on fatty acid synthesis. Primary hepatocyte cultures were prepared, and rates of fatty acid synthesis were measured as described under “Experimental Procedures.” Fatty acid synthesis measurements were made during the last 3 h of a 24-h treatment period with insulin (50 nm) in the absence and presence of FGF-19 (50 ng/ml). Values are means ± S.E. of three experiments. The asterisk indicates that the mean is significantly (p ≤ 0.05) lower than that of cells incubated with insulin in the absence of FGF-19.

FGF-19 suppresses the stimulatory effect of the LXR agonist T0-901317 on the expression of GK, ACCα, and CYP7A1. Primary rat hepatocytes (A) and human HepG2 cells (B) were incubated in serum-free Waymouth's medium supplemented with or without FGF-19 (50 ng/ml), T0-901317 (6 μm), or T0-901317 plus FGF-19. After 24 h of treatment, total RNA was isolated, and the abundance of the indicated mRNAs was measured by quantitative real-time PCR. The level of mRNA in cells incubated with no additions (NA) was set at 1, and the other values were adjusted proportionately. Values are means ± five experiments. The asterisk indicates that the mean is significantly (p ≤ 0.05) lower than that of cells incubated with T0-901317 in the absence of FGF-19.

Time course of the effect of FGF-19 on the expression of SREBP-1c, GK, FAS, and ACCα. Primary hepatocyte cultures were prepared and incubated in Waymouth's medium containing insulin (A) or T0-901317 (B). At 36 h of incubation, FGF-19 (50 ng/ml) was added to the culture medium, and the incubation was continued for 0, 2, 6, and 12 h. Cells were harvested, and total cell extracts were prepared as described under “Experimental Procedures.” mSREBP-1 was measured by Western analysis. Top panels, Western analysis of mSREBP-1 from a representative experiment. Bottom panels, signals for mSREBP-1 were quantitated. In another set of plates treated in a similar manner, total RNA was isolated and the abundance of the mRNAs encoding SREBP-1c, GK, FAS, and ACCα was measured by quantitative real-time PCR. The level of mSREBP-1 protein and mRNA in cells treated with insulin for 36 h and FGF-19 for 0 h was set at 1, and the other values were adjusted proportionately. Values are means ± five experiments. An asterisk indicates that the mean is significantly (p ≤ 0.05) different compared with that of cells treated with insulin for the same time period.

Effect of FGF-19 on the expression of PGC-1β, PGC-1α, SHP, LXRα, and phosphorylated STAT3. A, primary hepatocyte cultures were prepared and incubated in Waymouth's medium containing insulin. At 36 h of incubation, FGF-19 (50 ng/ml) was added to the culture medium, and incubation was continued for 0, 2, 6, and 12 h. Cells were harvested, total RNA was isolated, and the abundance of the mRNAs encoding PGC-1β, PGC-1α, SHP, and LXRα was measured by quantitative real-time PCR. The level of mRNA in cells treated with insulin for 36 h and FGF-19 for 0 h was set at 1, and the other values were adjusted proportionately. Values are means ± four experiments. An asterisk indicates that the mean is significantly (p ≤ 0.05) different compared with that of cells treated with insulin for the same time period. B, primary hepatocyte cultures were prepared and incubated in Waymouth's medium. At 20 h of incubation, the medium was replaced with one of the same composition supplemented with or without FGF-19, insulin, or insulin plus FGF-19. After 24 h of treatment, total RNA was isolated, and the abundance of PGC-1β mRNA and SHP mRNA was measured by quantitative real-time PCR. The level of mRNA in cells incubated with no additions (NA) was set at 1, and the other values were adjusted proportionately. Values are means ± four experiments. An asterisk indicates that the mean is significantly (p ≤ 0.05) higher than that of any other treatment. Total cell extracts were prepared from hepatocytes treated as described in A. SHP protein (C) and phosphorylated STAT3 (Tyr⁷⁰⁵ and Ser⁷²⁷)(D) were measured by Western analysis. The data are representative of four independent experiments.

FGF-19 suppresses the stimulatory effect of insulin on the expression of lipogenic enzymes. A, primary hepatocyte cultures were prepared and incubated in Waymouth's medium. At 20 h of incubation, the medium was replaced with one of the same composition supplemented with or without FGF-19 (50 ng/ml), insulin (50 nm), or insulin plus FGF-19. After 24 h of treatment, total RNA was isolated, and the abundance of the indicated mRNAs was measured by quantitative real-time PCR. The level of mRNA in cells incubated with no additions (NA) was set at 1, and the other values were adjusted proportionately. Values are means ± five experiments. The asterisk indicates that the mean is significantly (p ≤ 0.05) lower than that of cells incubated with insulin in the absence of FGF-19. B, effect of different concentrations of FGF-19 (0 to 50 ng/ml) on the abundance of GK mRNA in hepatocytes incubated in the presence of insulin. The level of GK mRNA in cells incubated with insulin in the absence of FGF-19 was set at 100, and the other values were adjusted proportionately. C, the effect of FGF-19 on the concentration of ACCα, FAS, and ABCA1. Total cell extracts were prepared from hepatocytes incubated with or without FGF-19, insulin, or insulin plus FGF-19 for 48 h. The abundance of ACCα protein, FAS protein, and ABCA1 protein in cell extracts was measured by Western analysis. The data are representative of three independent experiments. D, effect of FGF-19 on the ability of glucose to stimulate l-PK expression. Hepatocytes were incubated in RPMI containing low (5 mm) or high (25 mm) glucose with or without FGF-19. After 24 h of treatment, total RNA was isolated, and the abundance of l-PK mRNA and GK mRNA was measured by quantitative real-time PCR. The level of mRNA in cells incubated with 5 mm glucose in the absence of FGF-19 was set at 1, and the other values were adjusted proportionately. Values are means ± S.E. of four experiments.

FGF-19 suppresses the stimulatory effect of T3 on the expression of GK, S14, and CYP7A1. Primary hepatocyte cultures were prepared and incubated in Waymouth's medium. At 20 h of incubation, the medium was replaced with one of the same composition supplemented with or without T3 (100 nm) or T3 plus FGF-19. After 24 h of treatment, total RNA was isolated, and the abundance of the indicated mRNAs was measured by quantitative real-time PCR. The level of mRNA in cells incubated with no additions (NA) was set at 1, and the other values were adjusted proportionately. Values are means ± four experiments. The asterisk indicates that the mean is significantly (p ≤ 0.05) lower than that of cells incubated with T3 in the absence of FGF-19.

Effect of FGF-19 on the activity of the signaling cascade mediating the stimulatory effect of insulin on the expression of lipogenic genes. Primary hepatocyte cultures were prepared and incubated in Waymouth's medium containing insulin. At 36 h of incubation, FGF-19 (50 ng/ml) was added to the culture medium, and the incubation was continued for 10 min, 1 h, 6 h, and 12 h. Cells were harvested, and total cell extracts were prepared as described under “Experimental Procedures.” A, Western analyses were performed using antibodies against phosphorylated Akt (Ser⁴⁷³), phosphorylated FoxO1 (Ser²⁵⁶), GSK-3α/β (Ser^21/9), and PKC λ/ζ (Thr^410/403) and total AKT, FoxO1, GSK-3α/β, and PKC λ/ζ. The data are representative of four independent experiments. B, PKC λ/ζ activity was measured as described under “Experimental Procedures.” PKC λ/ζ activity in cells incubated in the absence of insulin and FGF-19 for 36 h was set at 1, and the other values were adjusted proportionately. Values are means ± three experiments. An asterisk indicates that the mean is significantly (p ≤ 0.05) different compared with that of cells incubated in the absence of insulin and FGF-19 for the same time period. C, cell extracts were incubated with an antibody against IRS-1, and the resulting immunoprecipitates (IP) were subjected to Western blot analysis (WB) using antibodies against phosphotyrosine and IRS-1. The data are representative of three independent experiments.