FGF15/19 regulates hepatic glucose metabolism by inhibiting the CREB-PGC-1α pathway

Abstract

Regulation of hepatic carbohydrate homeostasis is crucial for maintaining energy balance in the face of fluctuating nutrient availability. Here, we show that the hormone fibroblast growth factor 15/19 (FGF15/19), which is released postprandially from the small intestine, inhibits hepatic gluconeogenesis, like insulin. However, unlike insulin, which peaks in serum 15 min after feeding, FGF15/19 expression peaks approximately 45 min later, when bile acid concentrations increase in the small intestine. FGF15/19 blocks the expression of genes involved in gluconeogenesis through a mechanism involving the dephosphorylation and inactivation of the transcription factor cAMP regulatory element-binding protein (CREB). This in turn blunts expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and other genes involved in hepatic metabolism. Overexpression of PGC-1α blocks the inhibitory effect of FGF15/19 on gluconeogenic gene expression. These results demonstrate that FGF15/19 works subsequent to insulin as a postprandial regulator of hepatic carbohydrate homeostasis.

Figure 1. FGF15/19 represses PGC-1α and gluconeogenic gene expression

(A) Hepatic gene expression measured by QPCR in mice treated with vehicle, FGF15 or FGF19 for 6 hr (n = 4/group). Mice were fasted during the treatment period. (B) Western blot analysis of PGC-1α in liver homogenates pooled from groups of 4 mice treated with vehicle, FGF15 or FGF19 as in (A). (C) Hepatic gene expression measured by QPCR in overnight fasted mice injected with FGF19 for the indicated times (n = 4/group). (D) Ileum Fgf15 mRNA and plasma insulin levels from fasted-refed mice at the indicated times after refeeding (n = 5/group). (E) Plasma glucose and glucagon levels from fasted-refed mice at the indicated times after refeeding (n = 5/group). (F) Quantification of hepatic phospho-Akt/total Akt, phospho-ERK1/2/total ERK1/2, and phospho-CREB/total CREB from fasted-refed mice at the indicated times after refeeding (n = 4/group). Data are shown as percent of maximal phosphorylation for each protein and represent the mean ± SEM. Different lowercase letters indicate statistical significance (a, P< 0.05; b, P< 0.01; c, P< 0.005; and d, P< 0.001 versus control). See also Fig. S1.

Figure 2. PGC-1α, but not SHP, is required for FGF15/19 regulation of metabolic gene expression

(A) Hepatic gene expression analyzed by QPCR in groups of PGC-1a^fl/fl mice administered control (Ad-Con) or Cre-expressing (Ad-Cre) adenovirus and subsequently administered vehicle or FGF19 for 6 hr (n = 5–6/group). Mice were fasted during the treatment period. (B) Hepatic gene expression analyzed by QPCR in groups of wild-type (WT) mice infected with Ad-Con or PGC-1α-expressing adenovirus (Ad-PGC-1α) and subsequently administered vehicle or FGF19 for 6 hr. Mice were fasted during the treatment period. Subgroups of the Ad-Con and Ad-PGC-1α groups were co-administered an FGF15-expressing adenovirus (Ad-FGF15). All mice were fasted during the last 6 hr of the experiment (n = 5–7/group). (C) Hepatic gene expression analyzed by QPCR in groups of WT and SHP-KO mice administered vehicle or FGF19 for 6 hr (n = 4/group). Mice were fasted during the treatment period. Data are presented as mean ± SEM (a, P< 0.05; b, P< 0.01; c, P< 0.005; d, P< 0.001).

Figure 3. FGF15 represses hepatic gluconeogenesis, TCA cycle flux and fatty acid oxidation

(A) Hepatic gene expression measured by QPCR in mice infected with control (Con) or FGF15-expressing adenovirus (Ad-FGF15) for 3 days and then fasted overnight (n = 5/group). (B) Metabolic pathway flux measured by NMR in perfused livers from mice infected with control or FGF15-expressing adenovirus and fasted as in (A) (n = 8/group). (C) Metabolic parameters in mice infected with control (Con) or FGF15-expressing adenovirus (Ad-FGF15) for 3 days and then fasted for 6 hr (n = 8/group). (D, E) Hepatic gene expression measured by QPCR in fed FGF15-knockout (KO) (D) or FGFR4-KO mice (E) or their wild-type (WT) counterparts (n = 6/group). (F, G) Plasma glucose (F) and mole percent (%) labeled glucose versus unlabeled glucose (G) following a labeled pyruvate/lactate challenge in WT and FGF15-KO mice (n = 5–6/group). (H–K) Plasma glucose and insulin concentrations in fasted-refed WT, FGF15-KO and FGFR4-KO mice at the indicated times after refeeding (n = 6/group). Data are presented as mean ± SEM (a, P< 0.05; b, P< 0.01; c, P< 0.005; d, P< 0.001). See also Fig. S2.

Figure 4. FGF15/19 signaling reduces CREB phosphorylation and activity

(A) Western blot analysis of total and phosphorylated FRS2α, ERK1/2, CREB, Akt, and FOXO1 in liver lysates from individual overnight fasted wild-type (WT) and FGFR4-knockout (KO) mice 30 min after treatment with vehicle or FGF19. β-Actin served as a loading control. (B–E) ChIP analysis of the cyclic AMP response elements (CRE) in the Pgc1α, G6pase, and Pepck promoters using CREB, CBP and PGC-1α antibodies as indicated and pooled liver lysates (three repeats/pool; n = 4/pool of each group). For (B), mice were administered saline or FGF19 for 1 hr and fasted during the treatment period. For (C–E), mice were injected with control or FGF15-expressing adenovirus for 3 days and killed after an overnight fast. (F) Images of luciferase activity in mice infected with a CRE-luciferase reporter (Ad-CRE-luc) or control adenovirus and subsequently treated with vehicle or FGF19 for 6 hr. Mice were fasted during the treatment period. (G) Quantified luciferase activity normalized to the number of virus particles per liver (n = 6/group). All data are presented as mean ± SEM (c, P< 0.005). See also Fig. S3.