SUMMARY
The mechanism by which pharmacologic administration of the hormone FGF21 increases energy expenditure to cause weight loss in obese animals is unknown. Here we report that FGF21 acts centrally to exert its effects on energy expenditure and body weight in obese mice. Using tissue-specific knockout mice, we show that βKlotho, the obligate co-receptor for FGF21, is required in the nervous system for these effects. FGF21 stimulates sympathetic nerve activity to brown adipose tissue through a mechanism that depends on the neuropeptide corticotropin-releasing factor. Our findings provide an unexpected mechanistic explanation for the strong pharmacologic effects of FGF21 on energy expenditure and weight loss in obese animals.
INTRODUCTION
FGF21 is a hormone expressed in liver, where it is induced by states of nutrient stress, including starvation and ketogenic or high carbohydrate diets, and the fibrate drugs. FGF21 is also expressed in white adipose tissue (WAT), where it is induced by fasting/refeeding regimens and the thiazolidinedione drugs, and in brown adipose tissue (BAT), where it is induced by cold (reviewed in (Potthoff et al., 2012)). In rodent models of obesity, FGF21 administration caused weight loss by increasing energy expenditure and improved insulin sensitivity and lipid parameters (Coskun et al., 2008; Kharitonenkov et al., 2005; Xu et al., 2009a; Xu et al., 2009b). Similar metabolic effects were seen in diabetic rhesus monkeys and patients with type 2 diabetes (Gaich et al., 2013; Kharitonenkov et al., 2007; Veniant et al., 2012b).
FGF21 acts through a cell surface receptor comprised of an FGF receptor (FGFR), with FGFR1c the preferred isoform, in complex with βKlotho (reviewed in (Potthoff et al., 2012)). While the FGFRs are broadly expressed, βKlotho is expressed in a more limited set of tissues, including WAT, BAT and liver (Fon Tacer et al., 2010). FGF21 mediates its pharmacologic effects on body weight and insulin sensitivity in part through WAT and BAT (Adams et al., 2012; Ding et al., 2012; Veniant et al., 2012a; Wu et al., 2011), where it induces uncoupling protein-1 (Ucp1) and uncoupled respiration (Fisher et al., 2012; Hondares et al., 2010) and the hormone adiponectin (Holland et al., 2013; Lin et al., 2013). FGF21 also acts on the nervous system. FGF21 is not expressed in the central nervous system (CNS) (Fon Tacer et al., 2010) but crosses the blood-brain barrier (Hsuchou et al., 2007) and is present in human cerebrospinal fluid (Tan et al., 2011). Intracerebroventricular (icv) injection of FGF21 increased metabolic rate and insulin sensitivity in rats (Sarruf et al., 2010). Within the nervous system, βKlotho is expressed in the suprachiasmatic nucleus (SCN) in the hypothalamus, and the area postrema, nucleus of the solitary tract and nodose ganglia, which although they are discrete anatomical nuclei, comprise the dorsal-vagal complex (DVC) (Bookout et al., 2013). Using lean mice specifically lacking βKlotho in the hypothalamus and/or the hindbrain, we showed that βKlotho in the hypothalamus is required for FGF21 to increase circulating ketone body and glucocorticoid concentrations, to suppress growth and female reproduction, and to modulate circadian behavior (Bookout et al., 2013; Owen et al., 2013). However, since these studies were all done in lean mice, they did not address whether the effects of FGF21 on energy expenditure require that it act on the nervous system. In this paper, we examine the contribution of the nervous system to the pharmacologic actions of FGF21 in the context of diet-induced obesity.
RESULTS AND DISCUSSION
FGF21 acts centrally to induce energy expenditure
Fgf21-transgenic (Tg) mice are resistant to weight gain (Ding et al., 2012; Kharitonenkov et al., 2005). To determine whether this effect involves FGF21 acting in the brain, we crossed KlbCamk2a mice, in which the βKlotho gene (Klb) is disrupted in both the hypothalamus (including the SCN) and DVC but not in adipose tissue and liver (Bookout et al., 2013), with Fgf21-Tg mice to generate four genotypes: Klbfl/fl, Klbfl/fl/Tg, KlbCamk2a and KlbCamk2a/Tg mice. As expected (Inagaki et al., 2008), Klbfl/fl/Tg mice weighed less than Klbfl/fl mice on the standard chow diet due to their smaller body size, and this effect was absent in KlbCamk2a/Tg mice (Figure 1A). When fed a high fat diet, Klbfl/fl/Tg mice gained less weight than Klbfl/fl and KlbCamk2a mice (Figure 1B). These effects of FGF21 overexpression were lost in the KlbCamk2a/Tg mice (Figure 1B). High fat diet-fed Klbfl/fl/Tg mice had reduced percent fat mass and plasma leptin concentrations and increased percent lean mass compared to the other three genotypes (Figure 1C; Table S1). In metabolic cage studies, the Klbfl/fl/Tg mice had significantly increased food consumption and energy expenditure with no change in physical activity (Figures 1D and E). This significant effect of FGF21 on energy expenditure was not observed in the same mice fed a regular chow diet (Figure 2E) suggesting that oxidative substrate was limiting. Together, these data demonstrate that in the context of nutritional surfeit, FGF21 acts on the nervous system to stimulate energy expenditure.
Figure 1. Klb expression in the nervous system is required for the effects of FGF21 on whole body energy expenditure.
(A) Body weight in chow-fed groups of Klbfl/fl, Klbfl/fl/Tg, KlbCamk2a and KlbCamk2a/Tg mice.
(B) Percent change in body weight in groups of mice fed a high fat diet (HFD). Body weights for the groups at the end of the study were (in grams): Klbfl/fl, 44±2.5; Klbfl/fl/Tg, 33±1.7; KlbCamk2a, 40±1.3; KlbCamk2a/Tg, 47±2.6.
(C) Body composition after 8 weeks on HFD.
(D) 24-hour food consumption (normalized to body weight) and physical activity on a HFD.
(E) Left panel, energy expenditure starting 24 hours after switching mice to the HFD. Right panel, quantification of 24-hour energy expenditure data for the same mice on either regular chow or HFD.
(A-C) were performed with mice housed at 22°-23°. (D, E) were performed using metabolic cages maintained at 21°-22°.
Data are shown as the mean ± S.E.M. n=8-13/group (A-C), n=6/group (D and E). *p<0.05 compared to control.
Figure 2. Klb expression in the nervous system is required for the effects of FGF21 on gene expression in high fat diet-fed mice.
(A-D) Gene expression was analysed by QPCR in (A) brown adipose tissue (BAT), (B) subcutaneous (sc) white adipose tissue (WAT), (C) epididymal (e) WAT, and (D) liver of Klbfl/fl, Klbfl/fl/Tg, KlbCamk2a and KlbCamk2a/Tg mice after 3 months on the high fat diet. QPCR cycle time values are shown for the group with highest expression for each gene. Data are shown as the mean ± S.E.M. n=8-13/group. *p<0.05 compared to control.
We further evaluated carbohydrate and lipid parameters in the high fat diet-fed Klbfl/fl, Klbfl/fl/Tg, KlbCamk2a and KlbCamk2a/Tg mice. Plasma glucose, insulin and cholesterol were all significantly lower in Klbfl/fl/Tg mice than in the other three genotypes, and plasma triglycerides showed a similar trend (Table S1). Likewise, hepatic triglyceride concentrations were significantly lower in Klbfl/fl/Tg mice than in the other genotypes and hepatic cholesterol concentrations trended lower (Table S1).
FGF21 acts centrally to induce thermogenic genes
We next compared gene expression in BAT, subcutaneous (sc) and epididymal (e) WAT and liver of Klbfl/fl, Klbfl/fl/Tg, KlbCamk2a and KlbCamk2a/Tg mice. As expected, Klb was unchanged in BAT, scWAT, eWAT and liver of KlbCamk2a compared to Klbfl/fl mice, although FGF21 overexpression increased Klb mRNA in eWAT in Klbfl/fl but not KlbCamk2a mice (Figure 2A-D). There were no differences among genotypes in Fgfr1c expression. In BAT, Ucp1, deiodinase 2 (Dio2) and elongation of very long chain fatty acids like 3 (Elovl3) were elevated in Klbfl/fl/Tg compared to Klbfl/fl mice, consistent with increased thermogenesis (Figure 2A). Interestingly, bone morphogenic protein 8b (Bmp8b), which is induced in BAT in response to cold or high fat diet feeding and sensitizes BAT to the thermogenic actions of norepinephrine (Whittle et al., 2012), was increased markedly in Klbfl/fl/Tg mice compared to Klbfl/fl controls (Figure 2A). In scWAT, Ucp1, peroxisome proliferator-activated receptor γ (Pparg), phosphoenolpyruvate carboxykinase (Pck1), adipocyte triacylglycerol lipase (Atgl), hormone sensitive lipase (Hsl), acetyl-CoA carboxylase α (Acaca), stearoyl-coenzyme A desaturase 1 (Scd1) and adiponectin (Adipoq) were increased in Klbfl/fl/Tg compared to Klbfl/fl controls (Figure 2B). The induction of Ucp1 is consistent with a recent report showing that FGF21 causes browning of WAT (Fisher et al., 2012). In eWAT, Pparg, PPARγ coactivator-1α (Pgc1a), Pck1, Atgl, Hsl, Acaca, Scd1 and Adipoq were increased in Klbfl/fl/Tg compared to Klbfl/fl mice (Figure 2C). All of these FGF21-dependent changes in gene expression were lost in KlbCamk2a/Tg mice (Figure 2A-C), demonstrating that βKlotho in the nervous system is important for FGF21 to exert many of its pharmacologic effects on gene expression in adipose tissue. Since FGF21 also acts directly on adipocytes to induce Ucp1 and other genes (Fisher et al., 2012; Hondares et al., 2010), their maximal induction likely involves cooperative effects of FGF21 on both adipose tissue and the nervous system. In liver, fatty acid synthase (Fasn), Scd1, Acaca, Cd36 and Elovl6 were decreased in Klbfl/fl/Tg compared to Klbfl/fl mice (Figure 2D). These inhibitory effects of FGF21 were also absent in KlbCamk2a/Tg mice (Figure 2D). A recent study showed that the effects of FGF21 on reducing triglyceride and cholesterol concentrations were lost in liver-specific insulin receptor knockout mice (Emanuelli et al., 2014). Similarly, the loss of FGF21 action in liver of KlbCamk2a/Tg mice may be secondary to the loss of its insulin-lowering action (Table S1). Expression of dual specificity phosphatase 4 (Dusp4), which inhibits ERK1/2, was increased in Klbfl/fl/Tg mice compared to Klbfl/fl mice in all three adipose tissue depots but not liver (Figures 2A-D). Dusp4 was still increased in KlbCamk2a/Tg compared to KlbCamk2a mice in BAT and eWAT (Figures 2A and C), reinforcing the notion that FGF21 can also act directly on adipose tissue.
FGF21 acts on the hypothalamus
To assess the relative contribution of βKlotho in the DVC versus the hypothalamus to the metabolic actions of FGF21, we crossed Fgf21-Tg mice with KlbPhox2b mice, in which Klb is disrupted in the DVC but not the hypothalamus (Bookout et al., 2013). Metabolic parameters were evaluated in HFD-fed Klbfl/fl, Klbfl/fl/Tg, KlbPhox2b and KlbPhox2b/Tg mice. In contrast to KlbCamk2a/Tg mice, KlbPhox2b/Tg mice were similar to Klbfl/fl/Tg mice with respect to weight gain, energy expenditure and plasma insulin and glucose concentrations, although the significance of the decrease in glucose was lost in the KlbPhox2b/Tg mice (Figure S1A). Likewise, KlbPhox2b/Tg and Klbfl/fl/Tg mice had comparable changes in the expression of Ucp1 in BAT (Figure S1B). However, the significance of Ucp1 induction in scWAT was lost in the KlbPhox2b/Tg mice (Figure S1B). Overall, these data support the importance of βKlotho in the hypothalamus and not the DVC for the central actions of FGF21.
Since Fgf21-Tg mice are chronically exposed to high levels of FGF21, we examined whether the metabolic effects of shorter-term exposure to recombinant FGF21 in DIO mice also require βKlotho in the nervous system. DIO Klbfl/fl and KlbCamk2a mice were administered FGF21 or vehicle for two weeks by osmotic minipump. Plasma FGF21 concentrations were 29±11 ng/ml and 36±13 ng/ml in the Klbfl/fl and KlbCamk2a mice, respectively. As expected, FGF21 decreased body weight, percent body fat and plasma insulin, glucose, cholesterol and leptin concentrations in DIO Klbfl/fl mice (Figure 3A). All of these effects of FGF21 were absent in the KlbCamk2a mice (Figure 3A). FGF21 administration also increased Ucp1 expression in BAT and scWAT in Klbfl/fl but not KlbCamk2a mice (Figure 3B). Overall, the pattern of gene expression in BAT, scWAT, eWAT and liver of Klbfl/fl and KlbCamk2a mice in response to recombinant FGF21 was similar to that seen with the FGF21 transgene, with nearly all of the effects of FGF21 lost in the KlbCamk2a background (Figure S2). Thus, βKlotho in the nervous system is required for the pharmacologic actions of FGF21 in DIO mice.
Figure 3. Klb expression in the nervous system is required for metabolic actions of recombinant FGF21 delivered by minipump.
(A, B) Groups of diet-induced obese mice were administered FGF21 (0.8 mg/kg/day) or vehicle by osmotic minipump for 2 weeks and evaluated for (A) percent change in body weight (day 1 vs day 14), body composition and plasma insulin, glucose, cholesterol and leptin concentrations (day 14) or (B) uncoupling protein-1 (Ucp1) gene expression in brown adipose tissue (BAT) and subcutaneous white adipose tissue (scWAT). For (A), body weights for the groups at the end of the study were (in grams): Klbfl/fl/vehicle, 34±1.3; Klbfl/fl/FGF21, 30±1.0; KlbCamk2a/vehicle, 36±1.6; KlbCamk2a/FGF21, 36±1.3.
Data are shown as the mean ± S.E.M. n=5-6/group. *p<0.05 compared to vehicle.
FGF21 induces sympathetic nerve activity
Since BAT-mediated energy expenditure is regulated by the sympathetic nervous system, we used multifiber sympathetic nerve recording to directly measure the effect of FGF21 on sympathetic nerve activity (SNA) subserving BAT in mice. Injection of FGF21 icv increased BAT SNA in a time and dose-dependent manner (Figure 4A and B). SNA was induced approximately 30 minutes after FGF21 administration and continued to increase over the 4-hour experiment. This effect was blocked by icv pre-treatment with PD173074 (Figure 4A and B), which inhibits the tyrosine kinase activities of FGFR1, FGFR2 and FGFR3 (Kunii et al., 2008; Mohammadi et al., 1998). Intravenous (iv) injection of FGF21 also increased BAT SNA, which was inhibited by icv pre-treatment with PD173074 (Figure 4C). The onset of SNA induction was slower after iv injection compared to icv injection as expected since FGF21 must cross the blood-brain barrier. The effect of FGF21 on SNA when administered peripherally was markedly attenuated in KlbCamk2a mice (Figure 4D). The residual effect of FGF21 on BAT SNA in the KlbCamk2a mice could be due to either incomplete knockout of βKlotho in the nervous system or FGF21 acting via other, unknown mechanisms. Regardless, the data in Figure 1E show that βKlotho in the nervous system is crucial for the effect of FGF21 on energy expenditure.
Figure 4. FGF21 acts centrally to stimulate brown adipose tissue sympathetic nerve activity.
(A) epresentative sympathetic nerve activity (SNA) recordings at baseline and 4 hours after intracerebroventricular (icv) administration of FGF21 (1 μg) or vehicle.
(B) Left panel, percent change in BAT SNA following icv injection of FGF21 (1 μg) or vehicle. Right panel, percent change in BAT SNA at 4 hours following icv injection of FGF21 at the indicated doses. Mice were pre-treated for 10 minutes with either vehicle or an FGF receptor inhibitor (PD173074, 25 μg) delivered icv as indicated.
(C) Left panel, percent change in BAT SNA following intravenous (iv) injection of vehicle (V) or FGF21 (1 mg/kg). Right panel, percent change in BAT SNA at 4 hours following iv injection of FGF21. Mice were pre-treated for 10 minutes with either PD173074 or vehicle delivered icv as indicated.
(D) Left panel, percent change in BAT SNA following iv injection of FGF21 (1 mg/kg) into Klbfl/fl or KlbCamk2a mice. Right panel, quantification of SNA data at the 4-hour time point.
(E) Crf mRNA levels in whole hypothalamus and plasma adrenocorticotropic hormone (ACTH) concentrations 3 hours after ip injection with either vehicle or FGF21 (1 mg/kg).
(F) Percent change in BAT SNA 4 hours after icv injection of FGF21 (1 μg). Mice were pre-treated for 10 minutes with either vehicle (saline, 2 μl) or α-helical CRF(9-41) (αhCRF(9-41); 6 μg) delivered icv as indicated.
Data are shown as the mean ± S.E.M. n=5-7/group. *p<0.05 compared to either vehicle (B,C,E) or Klbfl/fl (D) as determined by t-test. For (B-D, right hand panels) and (F), the percent change in BAT SNA was calculated based on the average of the final four time points relative to baseline. (G) odel for the effects of FGF21 on energy expenditure. FGF21 acts on the hypothalamus to induce corticotropin-releasing factor (CRF) and to stimulate sympathetic nerve activity (SNA), which in turn induces uncoupling protein 1 (UCP1) and lipolysis in brown adipose tissue (BAT). FGF21 also acts directly on BAT to stimulate glucose uptake and to mobilize oxidative substrate. These dual effects induce efficient energy expenditure. The model is based on this study and previous literature (Cannon and Nedergaard, 2004; Ding et al., 2012).
FGF21 actions require corticotropin-releasing factor
We previously showed that corticotropin-releasing factor (Crf) mRNA in hypothalamus and circulating corticosterone concentrations are increased in Fgf21-Tg mice (Bookout et al., 2013). Likewise, hypothalamic Crf mRNA was elevated 3 hours after intraperitoneal injection of FGF21, and there was a corresponding increase in plasma adrenocorticotropic hormone (ACTH) (Figure 4E). While ACTH concentrations were increased under these acute FGF21 treatment conditions, they were reduced after administration of FGF21 for two weeks and in Fgf21-Tg mice (Bookout et al., 2013), suggesting that chronic FGF21 exposure sensitizes the adrenal to ACTH. Indeed, there is evidence that the autonomic nervous system can modulate the sensitivity of the adrenal to ACTH (Kalsbeek et al., 2010). Since icv injection of CRF stimulates sympathetic outflow to BAT and thermogenesis in rats (Arase et al., 1988; Cerri and Morrison, 2006; LeFeuvre et al., 1987), we tested whether CRF contributes to the effects of FGF21 on BAT. Notably, icv injection of the CRF receptor antagonist, α-helical CRF(9-41) (Rivier et al., 1986), which inhibits both the CRF1 and CRF2 receptor subtypes, completely blocked the effect of FGF21 on SNA in BAT (Figure 4F). This finding that FGF21 action involves downstream effects on CRF likely explains the relatively slow onset of SNA in BAT in response to FGF21. In the hypothalamus, βKlotho is most highly expressed in the SCN whereas CRF is primarily localized in the paraventricular nucleus (PVN). Since the SCN is known to act on the PVN to regulate the circadian pattern of CRF and corticosterone release (Kalsbeek et al., 2010), FGF21 may mediate its effects on CRF indirectly via the SCN. However, the precise neuroanatomical relationship between βKlotho and CRF remains to be determined. Nevertheless, our findings suggest that FGF21 regulates both BAT SNA and corticosterone levels via its effects on CRF.
In summary, we demonstrate that FGF21 acts centrally to stimulate sympathetic outflow, energy expenditure and weight loss in DIO mice. We previously showed that the acute effects of FGF21 on whole-body insulin sensitivity and glucose uptake in BAT were lost in high fat diet-fed mice lacking βKlotho in adipose tissue (Ding et al., 2012). How do we reconcile these findings? In vivo, the thermogenic effects of UCP1 require both norepinephrine and oxidative substrate (Nedergaard et al., 2005). For example, while PPARγ agonists efficiently stimulate Ucp1 expression and lipid accumulation in both brown and white adipocytes, they do not increase thermogenesis. However, they potentiate the thermogenic actions of β-adrenergic receptor agonists (Foellmi-Adams et al., 1996; Sell et al., 2004; Thurlby et al., 1987). We suggest that FGF21 works similarly to this combination of PPARγ and β-adrenergic receptor agonists through a two-fold mechanism (Figure 4G). First, FGF21 acts on the nervous system to stimulate sympathetic outflow to BAT, which induces Ucp1 and lipolysis (Cannon and Nedergaard, 2004). FGF21 also promotes the browning of scWAT (Fisher et al., 2012), an effect that in vivo involves βKlotho in the nervous system. Second, FGF21 acts directly on BAT and scWAT to increase glucose uptake and substrate mobilization. Accordingly, we find that energy expenditure is markedly up-regulated in lean FGF21-Tg mice when they are switched to the high fat diet and additional substrate is made available for oxidation (Figure 1E). Thus, FGF21 regulates both the mobilization and uncoupled oxidation of substrate in BAT and browned WAT.
EXPERIMENTAL PROCEDURES
Mouse studies
Mouse strains have been described and are on mixed C57BL6J;129/Sv backgrounds (Bookout et al., 2013; Owen et al., 2013). Age-matched 3-7-month-old male littermates were used for all experiments and were fed either a standard chow (Harlan Teklad, TD.2916) or a high fat diet containing 60% fat (Research Diets, D12492i). For the minipump experiments, mice were maintained on the high fat diet for 12 weeks prior to initiating the experiment. Housing rooms were maintained between 22°C and 23°C. Metabolic cages studies were performed at 21°C-22°C. Indirect calorimetry using LabMaster metabolic cages (TSE Systems) was used to determine energy expenditure per gram of lean body mass. Energy expenditure was calculated as a function of O2 consumption and CO2 production according the formula: energy expenditure (Kcal/h) = ((3.941 × vO2(ml/h)) + (1.106 × vCO2(ml/h)))/1000. Body composition was measured using an EchoMRI-100 Body Composition Analyzer. All experiments were approved by the Institutional Animal Care and Research Advisory Committee of the University of Texas Southwestern Medical Center or the University of Iowa.
Materials
Recombinant human FGF21 was from Novo Nordisk (Måløv, Denmark). Subcutaneous osmotic pumps were from ALZET. PD173074 was from Sigma. α-Helical CRF(9-41) was from Phoenix Pharmaceuticals. The following kits were used to measure metabolic parameters: Glucose (Wako Chemicals Inc.), triglycerides (Wako Chemicals Inc.), cholesterol (Fisher Scientific), insulin (Crystal Chem Inc.), leptin (Linco) and adrenocorticotropic hormone. Liver triglyceride and cholesterol levels were measured as described (Zhang et al., 2012).
Real-time quantitative (Q) PCR analyses
Total RNA was extracted from liver using Stat 60 reagent (IsoTex Diagnostics, Inc.). For adipose tissue, RNeasy lipid tissue mini kits (Qiagen) were used. 1-2 micrograms of RNA from each sample was then used to generate cDNA (Invitrogen). QPCR was performed using SYBR green as described (Bookout et al., 2006).
Sympathetic nerve activity measurements
Measurement of sympathetic nerve activity to BAT was performed as described (Harlan et al., 2011; Lockie et al., 2012; Morgan and Rahmouni, 2010). Following the establishment of baseline values, mice were pretreated for 10 minutes with either vehicle (10 mM Na2HPO4, 2% (w/v) glycerol, pH 7.6), PD173074, or α-helical CRF(9-41) administered icv followed by icv or iv (jugular vein infusion) administration of vehicle (10 mM Na2HPO4, 2% (w/v) glycerol, pH 7.6) or recombinant FGF21. SNA was recorded for an additional 4 hours. All icv injection volumes were 2 μl. The dose of PD173074 used (25 μg), which had no effect on SNA on its own (Figure 4B), was based on a previous publication in which PD173074 inhibited FGF19 action (Morton et al., 2013). The dose of α-helical CRF(9-41) (6 μg) was based on a previous publication in which this CRF antagonist blocked SNA activity in rats (Correia et al., 2001).
Statistical Analyses
Statistical analyses were performed by 2-way ANOVA with post-hoc correction (GraphPad Prism) unless indicated otherwise. Data are presented as the mean ± SEM; p<0.05 was considered significant.
Supplementary Material
FGF21 acts directly on the nervous system to stimulate sympathetic nerve activity
The effects of FGF21 on sympathetic outflow require corticotropin-releasing factor
The central actions of FGF21 are required for it to cause weight loss
ACKNOWLEDGMENTS
We thank Yuan Zhang, Heather Lawrence, Kevin Vale and Sofya Perelman for technical assistance and Birgitte Andersen (Novo Nordisk) for providing human recombinant FGF21. This work was supported by National Institutes of Health grants R01DK067158 (S.A.K. and D.J.M.), 1F32DK098908 (K.C.C), GM007062 (A.L.B.) and HL084207 (K.R.), the Robert A. Welch Foundation (grant I-1558 to S.A.K. and grant I-1275 to D.J.M.), the American Heart Association (14EIA18860041 to K.R.) and the Howard Hughes Medical Institute (to K.C.C. and D.J.M.). X.D. is an employee and stockholder of NGM Biopharmaceuticals. The recombinant FGF21 was provided by Novo Nordisk.
Footnotes
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