Fibroblast growth factor-21 regulates PPARγ activity and the antidiabetic actions of thiazolidinediones

Abstract

Fibroblast growth factor-21 (FGF21) is a circulating hepatokine that beneficially affects carbohydrate and lipid metabolism. Here, we report that FGF21 is also an inducible, fed-state autocrine factor in adipose tissue that functions in a feed-forward loop to regulate the activity of peroxisome proliferator-activated receptor γ (PPARγ), a master transcriptional regulator of adipogenesis. FGF21 knockout (KO) mice display defects in PPARγ signaling including decreased body fat and attenuation of PPARγ-dependent gene expression. Moreover, FGF21-KO mice are refractory to both the beneficial insulin-sensitizing effects and the detrimental weight gain and edema side effects of the PPARγ agonist rosiglitazone. This loss of function in FGF21-KO mice is coincident with a marked increase in the sumoylation of PPARγ, which reduces its transcriptional activity. Adding back FGF21 prevents sumoylation and restores PPARγ activity. Collectively, these results reveal FGF21 as a key mediator of the physiologic and pharmacologic actions of PPARγ.

Figure 1. FGF21 is differentially regulated by PPARα and PPARγ agonists

(A–E) Two-month-old, male C57Bl/6 mice fed regular chow were treated for 17 hours with GW7647 (10 mg/kg), rosiglitazone (rosi; 10 mg/kg) or vehicle (1% methycellulose). Fgf21 mRNA in epididymal (e) WAT (A) and liver (B) was measured by RT-qPCR. C_t values are indicated. FGF21 protein concentrations in eWAT (C), liver (D) and plasma (E) were measured by ELISA. For (A–E), n=4/group. (F) Eight- to 10-week-old, male C57Bl/6 mice were food-entrained for 2 weeks by restricting their access to chow to a 4 hour period in the middle of the dark cycle. Tissues were collected at 4 hour intervals over a 24 hour period and Fgf21 mRNA was analyzed by RT-qPCR. Data were normalized to Fgf21 mRNA levels at their lowest point (16 hour for WAT, 4 hour for liver) and are double plotted (n=6/group). Error bars represent the mean ± SEM. a, p<0.05 vs vehicle; b, p<0.01 vs vehicle. See also Supplemental Figure S1.

Figure 2. FGF21-knockout mice have decreased fat mass and adipocyte size

(A–E) The mass of various body depots was measured in 2-month-old, male wild-type (WT) and FGF21-knockout (KO) mice fed regular chow. For (E), adipose tissue mass was measured and normalized to body weight for epididymal (e), subcutaneous (sc), mesenteric (m), retroperitoneal (rp) and brown adipose tissue (BAT) depots. For (A–D), n=11–12/group; for (E), n=6/group. (F) DNA content of eWAT pads was measured and normalized to body mass (n=4/group). (G) Representative hematoxylin and eosin-stained eWAT sections from WT and FGF21-KO mice. Scale bar = 100 μM. (H) Adipocyte size was measured using images of eWAT sections and ImageJ software (n>150 cells/group). Error bars represent the mean ± SEM. a, p<0.05 vs WT; b, p<0.01 vs WT; c, p<0.005. See also Supplemental Figure S2.

Figure 3. FGF21-knockout adipocytes have altered gene expression and lipid accumulation

(A, B) Stromal vascular fraction preadipocytes isolated from P4 wild-type (WT) and FGF21-knockout (KO) mice were differentiated in vitro over an 8 day period. FGF21-KO preadipocytes were differentiated in either the presence or absence of recombinant FGF21 (200 ng/ml). (A) Gene expression was measured by RT-qPCR. (B) Lipid accumulation was measured by oil red O staining. Representative oil red O-stained cells at day 8 of differentiation are shown. Error bars represent the mean ± SEM. a, p<0.05 vs WT; b, p<0.01 vs WT.

Figure 4. FGF21-knockout adipocytes have reduced PPARγ activity

(A–B) Stromal vascular fraction preadipocytes isolated from P4 wild-type (WT) and FGF21-knockout (KO) mice were differentiated for 8 days in the presence of 0.5 μM rosiglitazone (R), 0.5 μM rosiglitazone + 100 ng/ml FGF21 (R+F21) or vehicle (V). (A) Gene expression was measured by RT-qPCR. a, p<0.05 vs WT; b, p<0.01 vs WT. (B) Lipid accumulation was measured by oil red O staining. b, p<0.01; c, p<0.005. (C) Sumoylated and total PPARγ protein levels were measured in WT and FGF21-KO adipocytes differentiated for 8 days and treated with vehicle or FGF21 (200 ng/ml) for 4 hours prior to harvest. Sumoylated PPARγ was detected by immunoprecipitation with a SUMO1 antibody followed by western blot analysis with a PPARγ antibody. Phosphorylated and total PPARγ and β-actin were detected by western blot. Top panel, quantification by densitometry of sumoylated PPARγ normalized to total PPARγ is shown for an experiment performed in triplicate. a, p<0.05. Bottom panel, representative western blots are shown. (D) Sumoylated and total PPARγ protein levels were measured as in (C) in epididymal WAT extracts from 2- to 3-month-old male WT and FGF21-KO mice fed regular chow and killed in the fed state. Top panel, quantification by densitometry of sumoylated PPARγ normalized to total PPARγ for WT and KO mice (n=4/group). a, p<0.05. Bottom panel, western blots for pooled samples are shown. (E) Gene expression was measured by RT-qPCR in the epididymal WAT of 2- to 3-month-old, male WT and FGF21-KO mice killed during the fed state (n=6–7/group). a, p<0.05 vs WT; b, p<0.01 vs WT. (F) Flag-tagged PPARγ2, PPARγ2-K107R, PPARγ2-K395R or PPARγ2-K107R/K395R were introduced into primary FGF21-KO adipocytes by transfection and their sumoylation measured by immunoprecipitation with a Flag antibody followed by western blot analysis with either a SUMO1 or PPARγ antibody. Input levels of Flag-tagged PPARγ and the PPARγ mutants were determined by western blot analysis with a Flag antibody. Data were quantified by densitometry. a, p<0.05 vs WT. (G) Gene expression was measured by RT-qPCR in WT and FGF21-KO stromal vascular fraction preadipocytes transduced with lentiviruses expressing PPARγ2, PPARγ2-K107R, PPARγ2-K395R or GFP control and differentiated for 4 days. PPARγ2, PPARγ2-K107R, PPARγ2-K395R were expressed at comparable levels (Figure S3E). Data are plotted as relative mRNA expression in FGF21-KO adipocytes compared to WT adipocytes. a, p<0.05 vs GFP; b, p<0.01 vs GFP. Error bars represent the mean ± SEM. See also Supplemental Figure S3.

Figure 5. FGF21-knockout mice are refractory to rosiglitazone treatment

(A–P) Two- to 3-month-old male wild-type (WT) and FGF21-knockout (KO) mice were fed a high fat diet for 10 weeks. During the last two weeks, groups of mice were administered rosiglitazone (10 mg/kg) or vehicle (1% methylcellulose). The following parameters were measured: (A) Fgf21 mRNA in epididymal WAT by RT-qPCR; (B) FGF21 protein in epididymal WAT by ELISA; (C) Fgf21 mRNA in liver by RT-qPCR; (D) FGF21 protein in liver by ELISA; (E) Plasma FGF21 by ELISA; (F) Plasma glucose and insulin; (G) Plasma glucose concentrations for glucose tolerance tests in mice fasted for 8 hours; (H) Plasma glucose levels for insulin tolerance tests in mice fasted for 4 hours; (I) Plasma insulin concentrations during the glucose tolerance test; (J) Plasma non-esterified fatty acid concentrations; (K) Plasma triglyceride concentrations; (L) Hepatic triglyceride concentrations; (M) Body mass; (N) Fat mass; (O) Lean mass; (P) Fluid mass. For (A–E), n=5–6/group; for (F–I), n=13–16/group; for (J–P), n=5–6/group. For (A–E), a, p<0.05 vs WT, vehicle; b, p<0.01 vs WT, vehicle; for (F), a, p<0.05; b, p<0.01; c, p<0.005; for (G) and (H), left panels, a, p<0.05 vs WT, vehicle; b, p<0.01 vs WT, vehicle; for (G) and (H), right panels, a, p<0.05; for (I), a, p<0.05 vs vehicle; b, p<0.01 vs vehicle; * p<0.05 vs FGF21-KO; for (J–L), a, p<0.05; for (N–P) a, p<0.05 versus WT, vehicle; b, p<0.01 versus WT, vehicle; c, p<0.05 versus WT, rosiglitazone. Error bars represent the mean ± SEM. See also Supplemental Figure S4.

Figure 6. FGF21 is required for rosiglitazone effects in WAT

(A–E) Two- to 3-month-old wild-type (WT) and FGF21-knockout (KO) mice were fed a high fat diet for 10 weeks. During the last two weeks, groups of mice were administered rosiglitazone (10 mg/kg) or vehicle (1% methylcellulose). The following parameters were measured in epididymal WAT: (A) Adipocyte size was determined using ImageJ software (n>250 cells/group). a, p<0.05; b, p<0.01; c, p<0.001. (B) Sumoylated PPARγ was detected in epididymal WAT extracts by either immunoprecipitation with a SUMO1 antibody followed by western blot analysis with a PPARγ antibody or the reverse. Total PPARγ and β-actin were measured by western blot analysis. For each lane, WAT extracts were pooled from 4 mice. Numbers represent relative levels quantified by densitometry. (C) Microarray analysis resulted in the identification of 545 genes that were significantly changed in response to rosiglitazone treatment in epididymal WAT from either wild-type or FGF21-KO mice. A heat map of the data is shown. R, rosiglitazone. (D) Gene expression was measured by RT-qPCR (n=5–6/group). a, p<0.05 versus WT, vehicle. (E) Tnfa mRNA was measured by RT-qPCR (n=5–6/group). a, p<0.05 versus WT, vehicle. (F) Plasma adiponectin concentrations were measured by ELISA (n=11–14/group). c, p<0.001. Error bars represent the mean ± SEM. (G) Model of the PPARγ-FGF21 regulatory pathway in adipose tissue. See also Supplemental Figure S5.