PGC-1alpha negatively regulates hepatic FGF21 expression by modulating the heme/Rev-Erb(alpha) axis

Abstract

FGF21 is a hormone produced in liver and fat that dramatically improves peripheral insulin sensitivity and lipid metabolism. We show here that obese mice with genetically reduced levels of a key hepatic transcriptional coactivator, PGC-1alpha, have improved whole-body insulin sensitivity with increased levels of hepatic and circulating FGF21. Gain- and loss-of-function studies in primary mouse hepatocytes show that hepatic FGF21 levels are regulated by the expression of PGC-1alpha. Importantly, PGC-1alpha-mediated reduction of FGF21 expression is dependent on Rev-Erbalpha and the expression of ALAS-1. ALAS-1 is a PGC-1alpha target gene and the rate-limiting enzyme in the synthesis of heme, a ligand for Rev-Erbalpha. Modulation of intracellular heme levels mimics the effect of PGC-1alpha on FGF21 expression, and inhibition of heme biosynthesis completely abrogates the down-regulation of FGF21 in response to PGC-1alpha. Thus, PGC-1alpha can impact hepatic and systemic metabolism by regulating the levels of a nuclear receptor ligand.

Fig. 1.

High-fat fed LH mice exhibit improved glucose tolerance and insulin sensitivity. (A) Blood glucose levels in 16-week high fat diet (HFD)-fed WT or LH mice after an overnight fast (time 0) and at the indicated times after i.p. injection of glucose. *, P < 0.05 by two-way ANOVA and Bonferroni posthoc tests at each point (n = 16). (B) Circulating insulin levels in high-fat fed or overnight fasted mice. Glucose infusion rate (GIR), (C) whole body glucose turnover, glycolysis, and glycogen synthesis (D), and glucose uptake in muscle (E) and fat (F) during hyperinsulinemic/euglycemic clamp. Values are means ± SEM, n = 7–11 (*, P < 0.05). White bars, WT mice; black bars, LH mice.

Fig. 2.

PGC-1α negatively regulates hepatic FGF21 expression. (A) Hepatic mRNA levels in high-fat fed mice fasted for 24 h. (B) Circulating levels of FGF21 in 24-h fasted mice. Bars, means ± SEM. (n = 9), expressed relative to WT. mRNA expression in (C) epididymal WAT, (D) BAT (means ± SEM), and (E) primary hepatocytes from either WT, LH, or PGC-1α knockout mice (KO) (means ± SD). (F) mRNA expression in WT primary hepatocytes infected with increasing amounts of an adenovirus expressing PGC-1α (Ad-PGC-1α). Values are means ± SD of duplicates expressed relative to levels in cells infected with Ad-GFP. (G) Fasting time course of hepatic PGC-1α and FGF21 gene expression in wild-type mice. PGC-1α (*, P < 0.05) and FGF21 #, P < 0.05) mRNA levels were normalized to their corresponding controls (mice fed ad libitum) (mean ± SEM, n = 9).

Fig. 3.

Rev-Erbα decreases FGF21 expression in primary hepatocytes. (A) Alignment of human and mouse FGF21 promoter sequences. Capitalized letters correspond to consensus RORE half sites. mRNA expression levels in primary hepatocytes expressing either (B) PGC-1α, (C) Rev-Erbα, or GFP (control). Values are mean ± SD of triplicates expressed relative to GFP, representative of three individual experiments, *, P < 0.05.

Fig. 4.

Heme negatively regulates FGF21 expression via Rev-Erbα in primary hepatocytes. Gene expression (A and C) or ChIP analysis of the FGF21 promoter (B) in primary hepatocytes treated with either vehicle (media), 30 μM hemin (A and B), or (C) succinylacetone. (D) FGF21 mRNA expression after hemin treatment of primary hepatocytes expressing either shControl or shRev-Erbα. Values are means ± SD of triplicate values, representative of three independent experiments, *, P < 0.05.

Fig. 5.

Increased ALAS-1 decreases FGF21 expression. (A) Schematic representation of the possible role of PGC-1α in the regulation of FGF21 via increased Rev-Erbα expression and/or heme biosynthesis. mRNA expression levels in primary hepatocytes expressing either (B) PGC-1α or (C) HA-ALAS-1. Values are mean ± SD of triplicate values expressed relative to control virus (GFP) and are representative of two independent experiments, *, P < 0.05.

Fig. 6.

Heme biosynthesis is required for PGC-1α-mediated repression of FGF21. (A) Hepatic mRNA levels in high-fat fed mice after a 24-h fast. Bars, means ± SEM (n = 9–11) expressed relative to WT fasted values. mRNA expression in primary hepatocytes coinfected with either Ad-GFP or Ad-PGC-1α, and Ad-shControl or (B) Ad-shRev-Erbα, or (C) Ad-ALAS-1, as indicated. Values are relative to control (Ad-GFP + Ad-shControl). (D) mRNA levels in primary hepatocytes expressing either PGC-1α or GFP and treated with either vehicle or succinylacetone. Values are means ± SD of triplicate values, representative of two independent experiments, *, P < 0.05 compared to Ad-GFP, #, P < 0.05 compared to Ad-PGC-1α.