Obesity Is a Fibroblast Growth Factor 21 (FGF21)-Resistant State ffolliott M. Fisher,1 Patricia C. Chui,1 Patrick J. Antonellis,1 Holly A. Bina,2 Alexei Kharitonenkov,2 Jeffrey S. Flier,1 and Eleftheria Maratos-Flier1 OBJECTIVE���Fibroblast growth factor 21 (FGF21) is a key mediator of fatty acid oxidation and lipid metabolism. Pharma- cological doses of FGF21 improve glucose tolerance, lower serum free fatty acids, and lead to weight loss in obese mice. Surprisingly, however, FGF21 levels are elevated in obese ob/ob and db/db mice and correlate positively with BMI in humans. However, the expected beneficial effects of endogenous FGF21 to increase glucose tolerance and reduce circulating triglycerides are absent in obesity. RESEARCH DESIGN AND METHODS���To test the hypothe- sis that obesity is a state of FGF21 resistance, we evaluated the response of obese mice to exogenous FGF21 administration. In doing this, we assessed the impact of diet-induced obesity on FGF21 signaling and resultant transcriptional events in the liver and white adipose tissue. We also analyzed the physiologic impact of FGF21 resistance by assessing serum parameters that are acutely regulated by FGF21. RESULTS���When obese mice are treated with FGF21, they display both a significantly attenuated signaling response as assessed by extracellular mitogen-activated protein kinase 1 and 2 (ERK1/2) phosphorylation as well as an impaired induction of FGF21 target genes, including cFos and EGR1. These effects were seen in both liver and fat. Similarly, changes in serum parameters such as the decline in glucose and free fatty acids are attenuated in FGF21-treated DIO mice. CONCLUSIONS���These data demonstrate that DIO mice have increased endogenous levels of FGF21 and respond poorly to exogenous FGF21. We therefore propose that obesity is an FGF21-resistant state. Diabetes 59:2781���2789, 2010 Fsuea ibroblast growth factor 21 (FGF21) has emerged as key mediator of the fasted state and contrib- utes to regulating lipolysis in white adipose tis- (WAT) (1���3) as well as increasing substrate utilization by increasing fatty acid oxidation in the liver (4). In addition, other studies have found that FGF21 increases insulin-independent glucose uptake in 3T3L1 adipocytes. Treatment of ob/ob mice with pharmacologic doses of FGF21 leads to improved glucose tolerance and reduced serum triglycerides (5). Subsequent studies have reported that chronic treatment of diet-induced obese (DIO) mice with FGF21 also leads to an improved meta- bolic profile (6,7). A similar effect has been reported in diabetic monkeys (8). Consistent with its actions on lipid oxidation in the liver and lipolysis in WAT, mice lacking FGF21 demonstrate a phenotype of mild obesity and an atypical response to feeding of a ketogenic diet (9). FGF21 binds to isoforms of FGF receptor 1, 2, 3, and 4 (10���12) in the presence of a critical co-receptor termed ��� Klotho.��� This leads to rapid dimerization and autophos- phorylation of the FGF receptor, which recruits and activates the ras/raf MAP kinase signaling cascade. This ultimately leads to activation of extracellular mitogen- activated protein kinase 1 and 2 (ERK1/2), which translo- cates to the nucleus and activates a subset of transcription factors. Part of this process is activation of transcription factors that regulate elements of the serum response, leading to induction of immediate early gene expression. It is now well established that exogenous treatment of FGF21 leads to a rapid induction of ERK1/2 phosphoryla- tion in adipose tissue depots (13���15). In addition, our lab and others (15) have found similar results in the liver. The wealth of data on this peptide suggests that FGF21 may be an excellent candidate molecule for therapeutic treatment of diabetes and cardiovascular disease associ- ated with obesity. It is therefore surprising that in obese states, which are typically associated with glucose intol- erance, serum FGF21 levels are high. In fact, in both rodent diet-induced obese (DIO) (16) and in genetically obese db/db (17) and ob/ob mice, FGF21 expression is increased in WAT and liver (18). In addition, in humans, circulating FGF21 levels were found to correlate positively with BMI (17,19). This increase in circulating levels is seen in the context of impaired glucose tolerance and increased accumulation of lipid in the liver. This suggests that, in the obese state, FGF21 fails to exert its expected effects on glucose homeostasis and lipid oxidation. Consistent with this, a recent article found that acute continuous infusion of FGF21 to control mice leads to reduced hepatic glucose output and increased insulin sensitivity while having no effect on obese ob/ob mice (20). These data have led us to hypothesize that obesity is an FGF21-resistant state. To test this hypothesis, we examined the effects of exogenous FGF21 on signal transduction and gene expression in the liver and WAT of DIO and lean mice. We found that DIO mice display a severely impaired signaling response to FGF21 and an attenuated effect to induce expression of target genes. In addition, low-dose FGF21 treatment was associated with an impaired im- provement in serum parameters in the DIO mice. These findings are consistent with obesity being an FGF21- resistant state. From the 1Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts and 2Lilly Research Lab- oratories, Indianapolis, Indiana. Corresponding author: Eleftheria Maratos-Flier, emaratos@bidmc.harvard. edu. Received 8 February 2010 and accepted 14 July 2010. Published ahead of print at http://diabetes.diabetesjournals.org on 3 August 2010. DOI: 10.2337/ db10-0193. �� 2010 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by -nc-nd/3.0/ for details. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ���advertisement��� in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ORIGINAL ARTICLE diabetes.diabetesjournals.org DIABETES, VOL. 59, NOVEMBER 2010 2781

obese 1.99 0.076 P 0.0001). Consistent with increased expression mRNA in both of these tissues, serum FGF21 levels were elevated in obese mice (Fig. 1C lean 604.2 62.85 pg/ml obese 2,315 269.7 pg/ml P 0.0001). FGF21 signaling is attenuated in liver and WAT of obese mice. FGF21 reduces circulating NEFAs and tri- glycerides and enhances glucose tolerance however, there were no apparent beneficial effects of the increased circu- lating levels of FGF21 in the obese animals. This is consistent with our hypothesis that obese mice are resis- tant to FGF21. To test this hypothesis, we assessed the ability of FGF21 to recruit FGF signaling pathways in the liver and WAT of obese mice. In this experiment, we used ERK1/2 phosphorylation as a reporter of FGF signaling. We found that at low doses (25 and 50 ng/g), there was a severe impairment in the ability of FGF21 to activate signaling pathways in obese mice (Fig. 2A) characterized by a reduced level of hepatic ERK1/2 phosphorylation. Because WAT is also a target of FGF21 action, we assessed ERK1/2 phosphorylation in perigonadal WAT (25, 50, and 100 ng/g) and found reduced phosphorylation of ERK1/2 in WAT of obese mice (Fig. 2B). Induction of immediate early gene expression down- stream of FGF21 signaling is impaired in obese mice. Phosphorylation of ERK1/2 leads to its translocation into the nucleus, where it phosphorylates and activates a subset of transcription factors. Among these are elements regulating the ���serum response��� that include rapid tran- scription of immediate early gene expression (21). To confirm FGF21 resistance, we used this as a secondary readout for FGF21 action in these tissues. In this experi- ment, we injected mice with FGF21 or intraperitoneal saline and assessed immediate genes Egr1 and cFos ex- pression in the liver and WAT. Egr1 mRNA expression was robustly increased in the liver of lean mice at the two higher doses (100 and 200 ng/g) (Fig. 3A). However, there was no effect of FGF21 administration on Egr1 expression in the liver of the obese mice. Consistent with this, we saw no increase in Egr1 protein expression in the liver of the obese mice, whereas a large increase in protein levels was seen in lean mice. Again, we saw a markedly attenuated response in WAT of obese mice, with little induction of Egr1 expression at any dose tested (Fig. 3B). This was also confirmed with Western blotting, where increases in Egr1 protein levels were only observed in the WAT from the lean mice. Induction of cFos mRNA expression was not as responsive to FGF21 treatment nevertheless, the effect seen in obese mice was attenuated to that seen in lean mice compared with obese mice (Fig. 3C). This was also the case in WAT (Fig. 3D). Impaired reduction in circulating glucose and NEFAs in response to low-dose FGF21 administration in obese mice. Administration of FGF21 leads to acute reduction in circulating glucose and NEFAs in mice. To see if this response is impaired in obese mice, we exam- ined the effect of a dose of 200 ng/g of FGF21 on these parameters. At this dose, FGF21 lowered circulating glu- cose levels by 17.4% in the lean mice (Fig. 4A saline 164.4 6.10 mg/dl FGF21 135.8 7.81 mg/dl P 0.0211). Although there was a slight reduction in the obese mice, this did not reach statistical significance (saline 167.6 10.58 mg/dl FGF21 153.7 4.98 mg/dl P NS). Because the ability of FGF21 to lower circulating glucose levels is thought to be due, in part, to increased expression of GLUT1 in adipose tissue, we assessed GLUT1 mRNA expression in the WAT of these mice (Fig. 4B). Although there was a small induction in GLUT1 expression in the obese mice (1.7-fold, P 0.0048), the response was far more prominent in the lean mice (2.8-fold, P 0.0003). In addition to lowering glucose, FGF21 acutely reduces circulating NEFAs���a phenomenon that was observed in this study. Serum NEFAs were reduced by FGF21 in the lean mice by 50% (Fig. 4C 0.51-fold, P 0.0068) in the obese mice, the effect was much smaller and levels were reduced by only 20% (0.79-fold, P 0.0371). FGF21 is thought to reduce NEFAs in part by attenuating lipolysis in WAT tissue (2). To see how this was affected in our model of FGF21 resistance, we looked at mRNA expression of lipolytic genes in WAT. Hormone-sensitive lipase (HSL) expression was significantly reduced by FGF21 in the lean mice (Fig. 4D 0.7-fold, P 0.0015) but not in the obese mice (0.9-fold, P NS). This was also the case with perilipin expression, which was far more suppressed in the FGF21-treated lean mice (Fig. 4E 0.71-fold, P 0.028) than in the obese mice (0.99-fold, P NS). Saline Saline Saline 25 25 50 50 100 100 Saline Saline 25 25 50 50 100 100 0 1 2 3 4 5 Lean Obese Lean Obese Obese Obese Obese Obese Obese Obese pERK1\2 ERK1\2 pERK1\2 ERK1\2 FGF21 Treatments ng/g FGF21 Treatments ng/g L O Lean Lean Lean 25ng/g 50ng/g 100ng/g Saline 25ng/g 50ng/g 100ng/g pERK/ERK pERK/ERK 0 1 2 3 L O Lean Lean Lean A B FIG. 2. FGF21 signaling is attenuated in liver and WAT of obese mice. To test whether FGF21 signaling is impaired in obese mice, we assessed FGF21-mediated ERK1/2 phosphorylation in the liver (A) and WAT (B). Results from every Western blot are shown in each case displaying both the phosphorylated form and total ERK1/2 expression. Above each blot, the densitometry is shown for all experimental regimes and is calculated as pERK/total ERK. Data are displayed as the mean SE. f.M. FISHER AND ASSOCIATES diabetes.diabetesjournals.org DIABETES, VOL. 59, NOVEMBER 2010 2783