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. 2014 Oct 24;289(43):29751-65.
doi: 10.1074/jbc.M114.565960. Epub 2014 Aug 28.

Fibroblast growth factor 21 is regulated by the IRE1α-XBP1 branch of the unfolded protein response and counteracts endoplasmic reticulum stress-induced hepatic steatosis

Shan Jiang  1 , Cheng Yan  2 , Qi-chen Fang  1 , Meng-le Shao  2 , Yong-liang Zhang  2 , Yang Liu  2 , Yi-ping Deng  2 , Bo Shan  2 , Jing-qi Liu  2 , Hua-ting Li  1 , Liu Yang  2 , Jian Zhou  3 , Zhi Dai  3 , Yong Liu  4 , Wei-ping Jia  5
Affiliations

Fibroblast growth factor 21 is regulated by the IRE1α-XBP1 branch of the unfolded protein response and counteracts endoplasmic reticulum stress-induced hepatic steatosis

Shan Jiang et al. J Biol Chem. .

Abstract

Endoplasmic reticulum (ER) stress activates the adaptive unfolded protein response (UPR) and represents a critical mechanism that underlies metabolic dysfunctions. Fibroblast growth factor 21 (FGF21), a hormone that is predominantly secreted by the liver, exerts a broad range of effects upon the metabolism of carbohydrates and lipids. Although increased circulating levels of FGF21 have been documented in animal models and human subjects with obesity and nonalcoholic fatty liver disease, the functional interconnections between metabolic ER stress and FGF21 are incompletely understood. Here, we report that increased ER stress along with the simultaneous elevation of FGF21 expression were associated with the occurrence of nonalcoholic fatty liver disease both in diet-induced obese mice and human patients. Intraperitoneal administration of the ER stressor tunicamycin in mice resulted in hepatic steatosis, accompanied by activation of the three canonical UPR branches and increased the expression of FGF21. Furthermore, the IRE1α-XBP1 pathway of the UPR could directly activate the transcriptional expression of Fgf21. Administration of recombinant FGF21 in mice alleviated tunicamycin-induced liver steatosis, in parallel with reduced eIF2α-ATF4-CHOP signaling. Taken together, these results suggest that FGF21 is an integral physiological component of the cellular UPR program, which exerts beneficial feedback effects upon lipid metabolism through counteracting ER stress.

Keywords: ER Stress; Fibroblast Growth Factor (FGF); Hepatocyte; Liver Metabolism; Unfolded Protein Response (UPR).

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Figures

FIGURE 1.
FIGURE 1.
Hepatic UPR activation and FGF21 expression in diet-induced obese mice. Male C57BL/6 mice at 6 weeks of age were maintained on a LFD (10% fat) or HFD (60% fat) for 16 weeks (n = 6 per group). A, body weight and hepatic levels of triglycerides (TG). B, serum levels of FGF21. Data in A and B are shown as the mean ± S.E. **, p < 0.01 by unpaired two-tailed t test. C, immunoblot analyses of the UPR markers, including phosphorylation of IRE1α (p-IRE1α) and eIF2α (p-eIF2α), and FGF21 protein abundance in whole liver lysates. Tubulin was used as the loading control, and representative results are shown for three individual mice per group. Ratios of p-IRE1α/IRE1α, p-eIF2α/eIF2α, and FGF21 protein levels were quantified by densitometry from the immunoblots. Relative FGF21 levels were normalized to tubulin. D, immunoblot analyses of the abundance of XBP1s, ATF4, and ATF6 proteins in liver nuclear extracts. Lamin A/C was used as the loading control. Relative levels of XBP1s, ATF4 and ATF6 are shown from densitometric quantification of the immunoblots after normalization to lamin A/C. E, assessment by quantitative real time RT-PCR of hepatic Xbp1 mRNA splicing, shown as the ratio of the spliced (s) to total (t) Xbp1 mRNA, along with the mRNA abundance of Atf4, Chop, Fgf21, and Fgf1. Data in C–E are presented as the mean ± S.E. (n = 6/group) relative to the LFD-fed control mice. *, p < 0.05; **, p < 0.01 by t test.
FIGURE 2.
FIGURE 2.
Hepatic UPR activation and FGF21 expression in human NAFLD patients. Human liver tissue samples were prepared from subjects without or with NAFLD (n = 5/group). A, immunoblot analyses of the UPR markers p-IRE1α and p-eIF2α and FGF21 protein abundance in whole liver lysates. Tubulin was used as the loading control, and representative results are shown for three individual subjects per group. Ratios of p-IRE1α/IRE1α and p-eIF2α/eIF2α and relative FGF21 levels were quantified. B, immunoblot analyses of the abundance of XBP1s, ATF4, and ATF6 proteins in liver nuclear extracts. Lamin A/C was used as the loading control. Results represent three individuals per group. Relative levels of XBP1s, ATF4, and ATF6 were quantified after normalization to lamin A/C. C, quantitative RT-PCR analyses of XBP1 mRNA splicing and the mRNA abundance of the indicated genes. All the results are shown as the mean ± S.E. (n = 5/group) relative to the control subjects. *, p < 0.05; **, p < 0.01 by t test.
FIGURE 3.
FIGURE 3.
ER stress results in increased expression of FGF21 in HepG2 cells. HepG2 cells were treated with tunicamycin (Tm, 10 μg/ml) or thapsigargin (Tg, 1 μm) for the indicated time intervals. A, ER stress markers and FGF21 protein abundance were analyzed by immunoblotting from whole cell lysates or nuclear extracts as indicated. Shown are representative results from three independent experiments. B and C, Xbp1 mRNA splicing (B) and the mRNA abundance of ATF4, CHOP, FGF21, and FGF1 (C) were determined by quantitative RT-PCR. Results were normalized to the values before Tm/thapsigargin treatment and are shown as the mean ± S.E. (n = 3 independent experiments). *, p < 0.05; **, p < 0.01 by one-way ANOVA. D, secreted FGF21 levels were measured from the culture medium, shown as the mean ± S.E. (n = 3 independent experiments). **, p < 0.01 by one-way ANOVA.
FIGURE 4.
FIGURE 4.
ER stress causes liver steatosis with up-regulated expression of hepatic FGF21 in mice. Male C57BL/6 mice at 12 weeks of age were treated for 24 h through intraperitoneal injection with PBS (vehicle) or Tm (1 mg/kg body weight). A, liver triglycerides were determined. B, serum levels of FGF21 were measured. Data are shown as the mean ± S.E. (n = 5/group). *, p < 0.05; **, p < 0.01 by t test. C, immunoblot analyses of the UPR markers, including p-IRE1α and p-eIF2α, and FGF21 protein abundance in whole liver lysates. Representative results are shown for three individual mice per group. Ratios of p-IRE1α/IRE1α and p-eIF2α/eIF2α as well as FGF21 protein levels were quantified. D, immunoblot analyses of nuclear XBP1s, ATF4, and ATF6 protein levels. Representative results are shown for three individual mice per group, and quantitation was done by normalization to lamin A/C. E, Xbp1 mRNA splicing and the mRNA abundance of Atf4, Chop, Fgf21, and Fgf1 were analyzed by quantitative RT-PCR. F, analysis by quantitative RT-PCR of the mRNA abundance of the FGF21 receptors Klb, Fgfr1, Fgfr2, Fgfr3, and Fgfr4. Data in C–F were normalized to the values of the vehicle control group and are presented as mean ± S.E. (n = 5/group). *, p < 0.05; **, p < 0.01 by t test.
FIGURE 5.
FIGURE 5.
ER stress induces the expression of FGF21 in kidney, white adipose tissue, and muscle. Mice were treated with PBS (vehicle) or Tm for 24 h. Xbp1 mRNA splicing and the mRNA abundance of Fgf21 and Fgf1 in kidney (A), fat tissue (B), and muscle (C) were determined by quantitative RT-PCR. Data were normalized to the values of the vehicle control group and are presented as mean ± S.E. (n = 5/group). *, p < 0.05; **, p < 0.01 by t test.
FIGURE 6.
FIGURE 6.
IRE1α is responsible for ER stress-induced expression of FGF21 in hepatocytes. A, male flox/flox or IRE1α null LKO mice were treated with tunicamycin (1 mg/kg body weight) for 8 h. Serum levels of FGF21 were measured and are shown as the mean ± S.E. (n = 3/group). B and C, primary hepatocytes from male flox/flox or LKO mice were treated for 6 h with dimethyl sulfoxide (DMSO), thapsigargin (Tg, 1 μm), or Tm (10 μg/ml). B, immunoblot analysis of IRE1α protein expression and quantitative RT-PCR analysis of Xbp1 mRNA splicing. C, analyses of the mRNA abundance of Atf4, Fgf21, and Fgf1. Data were normalized to values of DMSO-treated flox/flox hepatocytes and are shown as the mean ± S.E. (n = 3 independent experiments). **, p < 0.01 by two-way ANOVA. D and E, primary hepatocytes from male C57BL/6 mice were infected for 2 days with adenoviruses expressing EGFP or the wild-type (WT) or kinase-dead K599A mutant human IRE1α protein. D, immunoblot analysis of IRE1α and quantitative RT-PCR analysis of Xbp1 mRNA splicing. E, analyses of the mRNA abundance of the indicated genes. Data were normalized to values from Ad-EGFP control cells and are shown as the mean ± S.E. (n = 3 independent experiments). *, p < 0.05; **, p < 0.01 by one-way ANOVA.
FIGURE 7.
FIGURE 7.
XBP1s mediates ER stress-induced expression of FGF21 in hepatocytes. A and B, primary hepatocytes from male C57BL/6 mice were infected for 3 days with adenoviruses expressing a short hairpin (sh) RNA directed against LacZ (Ad-shLacZ) or XBP1 (Ad-shXBP1) before treatment for 6 h with DMSO, thapsigargin (Tg), or Tm. A, immunoblot analysis of XBP1s protein from nuclear extracts. B, analyses by quantitative RT-PCR of the mRNA abundance of the indicated genes. Results were normalized to values from DMSO-treated cells infected with Ad-shLacZ and are shown as the mean ± S.E. (n = 3 independent experiments). *, p < 0.05 by two-way ANOVA. C and D, primary hepatocytes were infected for 2 days with adenoviruses expressing EGFP or XPB1s protein. C, immunoblot analysis of XBP1s protein from cell lysates. D, quantitative RT-PCR analyses of the mRNA abundance of the indicated genes. Data were normalized to values from Ad-EGFP control cells and are shown as the mean ± S.E. (n = 3 independent experiments). **, p < 0.01 by t test.
FIGURE 8.
FIGURE 8.
XBP1s transactivates the transcriptional activity of the Fgf21 promoter. A, luciferase reporter assay. 293T cells were transfected with the Luc construct under the control of the mouse Fgf21 promoter that spans the region from −1983 to +5. Cells were then treated with DMSO, thapsigargin (Tg), or Tm for 6 h. Data were normalized to the DMSO control and are shown as the mean ± S.E. (n = 3 independent experiments). **, p < 0.01 by one-way ANOVA. B, sequence comparison of mouse, rat, and human Fgf21 promoter. Shown is the region that contains the signature XBP1s-binding element (ERSE) indicated by black boxes. C, luciferase reporter assay. 293T cells were co-transfected with the empty control vector or pCMV-XBP1s plasmid together with Luc constructs under the control of the mouse Fgf21 promoter (WT) or that with the CCACG element deleted (ΔCCACG). Data are shown as the mean ± S.E. (n = 3 independent experiments). **, p < 0.01 by two-way ANOVA. D, chromatin immunoprecipitation (IP) assay. Primary hepatocytes were infected for 2 days with adenoviruses expressing EGFP or XBP1s. ChIP was performed using control IgG or XBP1s antibody prior to PCR amplification of the indicated region (nucleotide −280 to −24) of the promoter. E, ChIP assay of extracts from 293T cells co-transfected with the empty control vector or pCMV-XBP1s plasmid together with the WT or ΔCCACG mutant reporter construct. ChIP was performed using anti-XBP1s antibody. F, ChIP assay of liver nuclear extracts from male C57BL/6 mice treated with PBS (vehicle) or Tm (1 mg/kg body weight) using control IgG or anti-XBP1s antibody. Quantitative PCR results are shown as the mean ± S.E. (n = 5/group). **, p < 0.01 by two-way ANOVA.
FIGURE 9.
FIGURE 9.
Recombinant FGF21 protein suppresses the eIF2α-ATF4-CHOP pathway during ER stress in hepatocytes. Primary hepatocytes were isolated from male C57BL/6 mice and treated with DMSO (vehicle), recombinant mouse FGF21 (1 μg/ml), Tm (10 μg/ml), or tunicamycin after pretreatment with FGF21. A, immunoblotting of p-IRE1α, p-eIF2α, IRE1α, and eIF2α. Tubulin was used as the loading control. Ratios of p-IRE1α/IRE1α and p-eIF2α/eIF2α were quantified (n = 3 independent experiments). B, quantitative RT-PCR analyses of Xbp1 mRNA splicing and the mRNA abundance of Atf4 and Chop (n = 3 independent experiments). C, immunoblotting of CHOP protein in nuclear extracts. Lamin A/C was used as the loading control. CHOP protein levels were quantified and normalized to lamin A/C (n = 3 independent experiments). Data were normalized to the values of the vehicle control group and are presented as mean ± S.E. **, p < 0.01 by two-way ANOVA.
FIGURE 10.
FIGURE 10.
Inhibition of ERK activation blunts the suppressive effect of FGF21 on the eIF2α-ATF4-CHOP pathway. Primary hepatocytes were treated with DMSO (vehicle), recombinant mouse FGF21 (1 μg/ml), Tm (10 μg/ml), or tunicamycin after pretreatment with FGF21 in the absence or presence of ERK inhibitor U0126 as indicated. A, immunoblotting of p-ERK, ERK, p-IRE1α, IRE1α, p-eIF2α, eIF2α, and CHOP. Tubulin was used as the loading control. B, ratios of p-eIF2α/eIF2α and CHOP protein levels were quantified (n = 3 independent experiments). C, quantitative RT-PCR analyses of Xbp1 mRNA splicing and the mRNA abundance of Atf4 and Chop (n = 3 independent experiments). Data were normalized to the values of vehicle control group and are shown as the mean ± S.E. *, p < 0.05; **, p < 0.01 by two-way ANOVA.
FIGURE 11.
FIGURE 11.
Administration of FGF21 reduces ER stress-induced hepatic steatosis in mice. Male C57BL/6 mice at 12 weeks of age were injected intraperitoneally with PBS (vehicle) or recombinant mouse FGF21 (1 mg/kg body weight) together with DMSO (vehicle) or Tm (1 mg/kg body weight) (n = 5/group). Mice were sacrificed at 24 h after treatment. A, liver TG contents and serum TG levels. B, immunoblot analysis of p-IRE1α, p-eIF2α, IRE1α, and eIF2α in whole liver lysates. GAPDH was used as the loading control. Representative results are shown for two individual mice per group. Ratios of p-IRE1α/IRE1α and p-eIF2α/eIF2α were quantified and are presented as the mean ± S.E. (n = 5/group). C, quantitative RT-PCR analyses of Xbp1 mRNA splicing and the mRNA abundance of Atf4 and Chop. Data are shown as the mean ± S.E. (n = 5/group). D, immunoblot analysis of matured SREBP1 protein (SREBP1-m) in whole liver lysates. Tubulin was used as the loading control. SREBP1-m levels were quantified and normalized to tubulin (n = 5/group). Data in B–D were normalized to the values of the vehicle control mice. *, p < 0.05; **, p < 0.01 by two-way ANOVA. E, schematic model. Dually controlled by XBP1s and ATF4, FGF21 acts in turn to suppress the eIF2α-ATF4-CHOP pathway and alleviate ER stress-induced hepatic steatosis.
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