Fibroblast growth factor 21 induces glucose transporter-1 expression through activation of the serum response factor/Ets-like protein-1 in adipocytes

Abstract

Fibroblast growth factor 21 (FGF21) is a liver-secreted endocrine factor with multiple beneficial effects on obesity-related disorders. It enhances glucose uptake by inducing the expression of glucose transporter-1 (GLUT1) in adipocytes. Here we investigated the signaling pathways that mediate FGF21-induced GLUT1 expression and glucose uptake in vitro and in animals. Quantitative real-time PCR and a luciferase reporter assay showed that FGF21 induced GLUT1 expression through transcriptional activation. The truncation of the GLUT1 promoter from -3145 to -3105 bp, which contains two highly conserved serum response element (SRE) and E-Twenty Six (ETS) binding motif, dramatically decreased FGF21-induced promoter activity of the GLUT1 gene. A chromatin immunoprecipitation assay demonstrated that the transcription factors serum response factor (SRF) and Ets-like protein-1 (Elk-1) were recruited to the GLUT1 promoter upon FGF21 stimulation. The siRNA-mediated knockdown of either SRF or Elk-1 resulted in a marked attenuation in FGF21-induced GLUT1 expression and glucose uptake in adipocytes. In C57 lean mice, a single intravenous injection of FGF21 induced phosphorylation of Elk-1 at Ser(383) and SRF at Ser(103) and also led to the recruitment of Elk-1 and SRF to the GLUT1 promoter in epididymal fats. By contrast, such effects of in vivo FGF21 administration were blunted in high fat diet-induced obese mice. In conclusion, FGF21 induces GLUT1 expression and glucose uptake through sequential activation of ERK1/2 and SRF/Elk-1, which in turn triggers the transcriptional activation of GLUT1 in adipocytes. The impairment in this signaling pathway may contribute to FGF21 resistance in obese mice.

FIGURE 1.

FGF21 stimulates glucose uptake and GLUT1 transcription in 3T3-L1 adipocytes in an insulin-independent manner. A, 3T3-L1 adipocytes were treated with various concentrations of FGF21 for 24 h, and the glucose uptake level was determined using 2-deoxy-d-[³H]glucose as a tracer. B, the additive effect of FGF21 and insulin on glucose uptake in 3T3-L1 adipocytes is shown. Cells were treated with or without FGF21 (2 μg/ml) for 24 h and then stimulated with insulin (100 nm) for 30 min. C, shown is quantification of GLUT1 and GLUT4 mRNA expression by real-time PCR after cells were treated with various concentrations of FGF21 for 24 h. Data are expressed as -fold change relative to untreated cells. D, shown is the effect of the transcription inhibitor actinomycin D (ActD, 50 μm) on FGF21-induced GLUT1 expression in 3T3-L1 adipocytes. The mRNA level of GLUT1 was determined as in panel D. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus untreated controls (n = 4–6).

FIGURE 2.

ERK1/2 mediates FGF21-induced GLUT1 expression and glucose uptake in 3T3-L1 adipocytes. Cells were starved for 24 h in a serum-free medium followed by stimulation without or with various concentrations of FGF21 for 30 min. Phosphorylation of ERK1/2 at Thr²⁰²/Tyr²⁰⁴ (A), Akt at Ser⁴⁷³ (B), and AMPK at Thr¹⁷² (C) were analyzed by Western blot. The bar charts below each blot are quantitative analyses of their phosphorylation levels relative to untreated cells. In D–F, cells were preincubated with either PD98059 (PD, 25 μm) or AktI-1 (10 μm) for 1 h followed by treatment without or with 2 μg/ml FGF21 for 24 h. Glucose uptake and GLUT1 expression was quantified as in Fig. 1. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus untreated controls (n = 5–6).

FIGURE 3.

A 40-bp cis-element spanning −3145 to −3105 bp of the GLUT1 gene promoter mediates FGF21-induced gene transactivation in 3T3-L1 adipocytes. A, shown is a schematic presentation of the luciferase reporter vectors driven by 3.7-, 2.7-, and 1.7-kb GLUT1 promoter. B, relative luciferase activities in cells transfected with the reporter vectors are shown in A followed by stimulation with 2 μg/ml FGF21 for 24 h. C, the response of the progressively truncated GLUT1 promoter to FGF21 (2 μg/ml), as determined by the luciferase reporter assay as in (D), is shown. **, p < 0.01 versus untreated control in each group (n = 4–6).

FIGURE 4.

The putative SRE and ETS-binding sites within −3145 and −3105 of the promoter confer FGF21-induced transactivation of the GLUT1 gene. A, multiple sequence alignment of −3145 to −3105 bp of the GLUT1 promoter region from four mammalian species is shown. The DNA sequences inside the boxes are identical to the consensus SRE and ETS binding motifs respectively. B, shown is a schematic presentation of the luciferase reporter vectors driven by a 3.7-kb wild type GLUT1 promoter (3.7kb-luc) or mutant GLUT1 promoters bearing mutations within either SRE (3.7kbmu-luc(SRE)) or ETS-binding motif (3.7kbmu-luc(ETS)). The mutated nucleotides within each recognition motif are highlighted in bold. C, promoter activities of wild type and mutant promoter regions either at the basal or FGF21 stimulated condition are shown. **, p < 0.01; ***, p < 0.001 (n = 4).

FIGURE 5.

FGF21 induces the recruitment of endogenous Elk-1 and SRF to the GLUT1 gene promoter through ERK1/2. A ChIP assay was performed as described under “Experimental Procedures” to quantify the interaction between endogenous GLUT1 promoter with Elk-1 and SRF, respectively. 3T3-L1 adipocytes were preincubated without or with the ERK1/2 inhibitor PD98059 (PD, 25 μm) for 30 min and then treated with 2 μg/ml FGF21 for various periods. Chromatin was isolated and subjected to immunoprecipitation using antibodies specific to Elk-1or SRF or non-immune rabbit IgG as a negative control. The relative abundance of the GLUT1 promoter spanning −3182 and −3041 bp was quantified by real time PCR and expressed as fold of untreated control. *, p < 0.05; **, p < 0.01 versus the group precipitated with non-immune IgG (n = 4).

FIGURE 6.

Knockdown of either SRF or Elk-1 expression attenuates FGF21-induced transcriptional activation of the GLUT1 gene and glucose uptake in 3T3-L1 adipocytes. A and B, shown is a Western blot analysis for SRF and Elk-1 protein levels from cells transfected with siRNA specific to SRF (si-SRF), Elk-1 (si-Elk-1), and scrambled control (sc) for 72 h. The bar chart below is the densitometric quantification of the blot. C, shown is real-time PCR analysis for GLUT1 mRNA abundance in cells transfected with si-SRF, si-Elk-1, and scrambled control in response to stimulation with 2 μg/ml FGF21 for 24 h. D, glucose uptake was measured after treatment without or with 2 μg/ml FGF21 for 24 h. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (n = 4–5).

FIGURE 7.

The siRNA-mediated knockdown of β-klotho expression diminishes FGF21-evoked signaling events leading to GLUT1 expression and glucose uptake in 3T3-L1 adipocytes. Cells were transfected with siRNA specific to β-klotho (si-β-kl) or scrambled control (sc) for 48 h. A, the expression level of β-klotho was determined by real-time PCR. FGF21 (2 μg/ml)-induced glucose uptake (B), expression of GLUT1 gene (C), transactivation of the 3.7 kb GLUT1 promoter (D), and phosphorylation of ERK1/2 at Thr²⁰²/Tyr²⁰⁴ (E) was performed as in Fig. 2. **p < 0.01 (n = 4).

FIGURE 8.

FGF21-induced phosphorylation and recruitment of Elk-1 and SRF to the GLUT1 promoter is diminished in adipose tissue of diet-induced obese mice. A, epididymal fat pads were dissected from C57 lean mice or diet-induced obese mice at 30 min after receiving an intravenous injection of FGF21 (1.5 μg/100 g of body weight) or PBS as a negative control and then subjected to Western blot analysis to analyze the phosphorylation levels of Elk-1 at Ser³⁸³ and SRF at Ser¹⁰³. The bar chart (B) is the densitometry analysis for the relative phosphorylation levels of Elk-1 and SRF. C, chromatin was isolated from epididymal fats of lean or obese mice for different periods after receiving a single intravenous injection of FGF21, or PBS was subjected to immunoprecipitation using antibodies specific to Elk-1or SRF or non-immune rabbit IgG as a negative control. The relative abundance of the GLUT1 promoter spanning −3182 and −3041 bp was quantified by real time PCR and expressed as -fold of untreated control. D, the relative mRNA abundance of the GLUT1 gene in epididymal fat pads of lean or obese mice was determined by real-time PCR at 6 h after intravenous injection of FGF21. *, p < 0.05; **, p < 0.01 versus obese group treated with FGF21 at the same time points (n = 4–5).

FIGURE 9.

Schematic representation of the molecular pathway by which FGF21 induces GLUT1 expression through the activation of SRE/ETS signaling cascade in adipocyte. FGFR, FGF21 receptor.