Thyroid hormone regulates hepatic expression of fibroblast growth factor 21 in a PPARalpha-dependent manner

Abstract

Thyroid hormone has profound and diverse effects on liver metabolism. Here we show that tri-iodothyronine (T3) treatment in mice acutely and specifically induces hepatic expression of the metabolic regulator fibroblast growth factor 21 (FGF21). Mice treated with T3 showed a dose-dependent increase in hepatic FGF21 expression with significant induction at doses as low as 100 microg/kg. Time course studies determined that induction is seen as early as 4 h after treatment with a further increase in expression at 6 h after injection. As FGF21 expression is downstream of the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha), we treated PPARalpha knock-out mice with T3 and found no increase in expression, indicating that hepatic regulation of FGF21 by T3 in liver is via a PPARalpha-dependent mechanism. In contrast, in white adipose tissue, FGF21 expression was suppressed by T3 treatment, with other T3 targets unaffected. In cell culture studies with an FGF21 reporter construct, we determined that three transcription factors are required for induction of FGF21 expression: thyroid hormone receptor beta (TRbeta), retinoid X receptor (RXR), and PPARalpha. These findings indicate a novel regulatory pathway whereby T3 positively regulates hepatic FGF21 expression, presenting a novel therapeutic target for diseases such as non-alcoholic fatty liver disease.

Figures

FIGURE 1.

Panel 1, A, acute effects of T3 treatment in mice. T3 treatment of euthyroid male c57BL/6 mice via intraperitoneal injection causes a significant increase in FGF21 gene expression in the liver in a dose-dependent manner. AU, arbitrary units. B, increased FGF21 gene expression induced by T3 treatment in the liver is not significant at 2 h after injection (0.44 ± 0.12 versus 0.42 ± 0.10, n = 8 versus 8, NS); however, at 4 h after injection, expression is significantly elevated (0.44 ± 0.12 versus 1.30 ± 0.04, n = 8 versus 8, p = 0.021), and this increase in expression reaches maximal levels at 6 h (0.46 ± 0.119 versus 2.71 ± 0.22, n = 8 versus 8, p = <0.001). Panel 2, effects of T3 on hepatic PPARα targets in normal mice. To determine the extent to which PPARα target genes were induced by T3 treatment, we analyzed gene expression of several known hepatic PPARα target genes. Only expression of FGF21 (C) (PBS versus FGF21, 0.20 ± 0.06 versus 3.14 ± 1.22, 4 versus 4, p < 0.001) was significantly increased. No change between vehicle- and T3-treated mice was found for ABDL (D), ABCB4 (E), UCP2 (F), or CPT1a (G), indicating that in this paradigm, induction of FGF21 is unique among known PPARα targets. Panel 3, effects of T3 in PPARα-deficient mice. H, FGF21 is not induced in the liver of PPARα knock-out mice upon treatment with T3 using the maximal dose and optimum time point from our initial studies (WT versus PPARα KO, 3.14 ± 1.22 versus 0.17 ± 0.2, 4 versus 4, p < 0.001). Other T3-responsive genes such as SPOT14 (I) (WT versus PPARα KO, 1.07 ± 0.10 versus 1.48 ± 0.18,4 versus 4, p < 0.001) and G6Pase (J) (WT versus PPARα KO, 1.12 ± 0.20 versus 1.35 ± 0.40,4 versus 4, p < 0.001) show robust induction, indicating that PPARα is explicitly required for the induction of FGF21 expression by T3 in vivo. Error bars indicate mean ± S.E.

FIGURE 2.

Panel 1, effects of T3 on WAT gene expression of FGF21 and previously described T3 target genes. To examine the tissue specificity of the effects of T3, we examined WAT, which has previously been described as a key tissue for FGF21 action. A, FGF21 expression was significantly suppressed following treatment with 500 μg/kg of T3 at the 6-h time point (1.71 ± 0.54 versus 0.45 ± 0.06, n = 4 versus 4, p = 0.0041). AU, arbitrary units. However, expression of known T3 targets SPOT14 (B) (1.13 ± 0.25 versus 1.44 ± 0.19, n = 4 versus 4, NS), GLUT4 (C) (1.91 ± 0.55 versus 2.25 ± 1.10, n = 4 versus 4, NS), and FAS (D) was unaffected by the treatment. Suppression of FGF21 expression by T3 also occurred in a similar manner in WAT of PPARα KO mice (data not shown), suggesting a PPARα-independent mechanism of action. Panel 2, E, reconstitution of the T3 signaling pathway in 293T cells indicates that alongside the FGF21 reporter, only a combination of PPARα, TRβ, and RXR is sufficient for the induction of FGF21 by T3. Transfection with FGF21 reporter alone led to luciferase (Luc) accumulation; however, this effect was not altered by T3 treatment of the cells. The addition of PPARα and RXR increased the accumulation seen with reporter alone; however, as with the reporter, this effect was not changed by T3 treatment. Transfection with TRβ and RXR led to a slight reduction in expression when compared with reporter alone; however, this change was not significant and was not affected by T3 treatment. When cells were transfected with TRβ, RXR, PPARα, and the reporter construct, we did see a significant increase in luciferase accumulation. DR4, a T3-responsive element, is included as a positive control and was induced by T3 in an appropriate manner similar to that seen in previous experiments. To determine the requirement of the two known PPAR-response elements in the FGF21 promoter, 293T cells were transfected with PPARα, TRβ, and RXR along with FGF21 reporter constructs of varying lengths (F) (−1497, full length containing both PPREs, −977, containing only the proximal PPRE, and −66, which had neither PPRE present). Induction occurred as in our previous experiments using the full-length (−1497) promoter construct; however, when the distal PPRE was removed in the −977 construct, luciferase induction dropped significantly. All data are corrected for β-Gal expression to account for differences in transfection efficiency. Error bars indicate mean ± S.E.