The effects of FGF21 on taste preference require its action in the CNS

Fibroblast growth factor 21 (FGF21) is a hormone induced by various metabolic stresses, including ketogenic and high-carbohydrate diets, that regulates energy homeostasis. In humans, SNPs in and around the FGF21 gene have been associated with macronutrient preference, including carbohydrate, fat, and protein intake. Here we show that FGF21 administration markedly reduces sweet and alcohol preference in mice and sweet preference in cynomolgus monkeys. In mice, these effects require the FGF21 co-receptor β-Klotho in the central nervous system and correlate with reductions in dopamine concentrations in the nucleus accumbens. Since analogs of FGF21 are currently undergoing clinical evaluation for the treatment of obesity and type 2 diabetes, our findings raise the possibility that FGF21 administration could affect nutrient preference and other reward behaviors in humans.

In humans, SNPs in and around the FGF21 gene are associated with changes in macronutrient preference, including increases in carbohydrate consumption and decreases in fat and protein intake (). These findings raise the possibility of additional effects of FGF21 on the brain. In this report, we examine the effect of FGF21 on sweet and alcohol preference in mice and monkeys.

FGF21 acts through a cell-surface receptor composed of a conventional FGF receptor in complex with β-Klotho, a single-pass transmembrane protein (). FGF21 crosses the blood-brain barrier () and exerts many of its actions, including its effects on growth, female reproduction, and weight loss, by acting on its cognate receptor in the CNS (). Among its central actions, FGF21 induces corticotropin-releasing factor and suppresses arginine vasopressin expression in the hypothalamus ().

FGF21 maintains glucose homeostasis by mediating the cross talk between liver and brain during prolonged fasting.

FGF21 maintains glucose homeostasis by mediating the cross talk between liver and brain during prolonged fasting.

FGF21 is induced in liver in response to fasting and other forms of metabolic stress including ketogenic and high-carbohydrate diets (). FGF21 in turn regulates diverse aspects of energy homeostasis, including hepatic fatty acid oxidation and ketogenesis, circadian behavior, growth, and female reproduction (). Pharmacologically, FGF21 causes weight loss and improves insulin sensitivity in obese mice, monkeys, and humans (). Long-acting analogs of FGF21 are currently in clinical trials for the treatment of obesity and type 2 diabetes.

Response to carbohydrate and fat refeeding in the expression of genes involved in nutrient partitioning and metabolism: striking effects on fibroblast growth factor-21 induction.

Hepatic fibroblast growth factor 21 is regulated by PPARalpha and is a key mediator of hepatic lipid metabolism in ketotic states.

Results and Discussion

fl/fl) or mice specifically lacking β-Klotho in the CNS (KlbCamk2a), and two-bottle saccharin preference tests were performed. FGF21 strongly suppressed saccharin preference in Klbfl/fl mice but had no effect in KlbCamk2a mice ( Camporez et al., 2013 Camporez J.P.

Jornayvaz F.R.

Petersen M.C.

Pesta D.

Guigni B.A.

Serr J.

Zhang D.

Kahn M.

Samuel V.T.

Jurczak M.J.

Shulman G.I. Cellular mechanisms by which FGF21 improves insulin sensitivity in male mice. To determine whether FGF21 acts on the CNS to regulate sweet preference, we administered recombinant FGF21 or vehicle by osmotic minipump to groups of control mice with floxed β-Klotho alleles (Klb) or mice specifically lacking β-Klotho in the CNS (Klb), and two-bottle saccharin preference tests were performed. FGF21 strongly suppressed saccharin preference in Klbmice but had no effect in Klbmice ( Figure 1 C, Table S2 ). As reported (), administration of recombinant FGF21 increased total fluid intake, and this effect required β-Klotho in the CNS ( Table S2 ). In contrast, total fluid intake was unchanged in Tg(Fgf21) compared to control mice ( Table S1 ). The reason for this difference between Tg FGF21 overexpression and recombinant FGF21 administration on fluid intake is not known. Nevertheless, FGF21 decreased sweet preference in both contexts.

In additional two-bottle preference tests, FGF21 had no effect on preference for 1% sunflower oil (data not shown) or quinine ( Figure 1 D), indicating that FGF21 does not affect responses to fatty acids or bitter taste. FGF21 administration also had no effect on either tail-suspension or forced-swim tests, both standard measures of behavioral despair ( Figures S1 A and S1B). We conclude that FGF21 acts directly on the brain to regulate sweet preference without causing despair.

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Talukdar S. Pharmacokinetics (PK), pharmacodynamics (PD) and integrated PK/PD modeling of a novel long acting FGF21 clinical candidate PF-05231023 in diet-induced obese and leptin-deficient obese mice. Figure 2 A Stable FGF21 Analog Decreases Saccharin Preference in Mice and Monkeys Show full caption (A) Two-bottle preference assay with 0.1% saccharin in diet-induced obese mice administered either PF-05231023 (10 mg/kg) or vehicle on days 0, 3, 7, and 10. Data are shown as the mean ± SEM; n = 8/group. ∗∗p < 0.01, ∗∗∗p < 0.001 versus vehicle group. Pinheiro et al., 2013 Pinheiro, J., Bates, D., DebRoy, S., and Sarkar, D. (2013). nlme: linear and nonlinear mixed effects models. R package version 3.1-103. (R Foundation for Statistical Computing). http://CRAN.R-project.org/package=nlme. (B) Two-bottle preference assay with 0.2% saccharin in obese cynomolgus monkeys administered either PF-05231023 (n = 8; 10 mg/kg) or vehicle (n = 7) on days 1, 4, and 7. Data are presented as mean percentage change in saccharin water intake ± SEM for vehicle-treated (closed blue circles) and PF-05231023-treated (open red circles) monkeys. Solid lines are locally weighted scatterplot smoothing fits to the means of percent change. Mixed effect modeling fitted to these longitudinal data using R, version 3.1.2 (), showed a significant difference (p = 0.003) between groups. Number of days after first treatment, treatment type, and the interaction term between treatment groups and time were specified as fixed effects and monkey labels as a random effect. To determine whether FGF21 also affects sweet preference in primates, we analyzed saccharin preference in obese cynomolgus monkeys administered PF-05231023, a long-acting FGF21 analog consisting of two molecules of modified FGF21 linked by an antibody scaffold (). We first tested this analog in mice. PF-05231023 administration decreased saccharin preference to a degree similar to native FGF21, with maximal efficacy observed 3–5 days after dosing ( Figure 2 A). For the monkey study, PF-05231023 or vehicle was administered on days 1, 4, and 7 of the 3-week experiment. Notably, PF-05231023 administration significantly decreased saccharin preference in the monkeys ( Figure 2 B). The effect on saccharin intake in the monkeys was striking even within 1 day of receiving a single dose of the FGF21 analog, and the effect was sustained for several days after receiving the last dose. Thus, FGF21 also affects sweet preference in primates.

Fernstrom et al., 2012 Fernstrom J.D.

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Molinary S. Mechanisms for sweetness. +/− and Klb−/− mice, with the latter mice serving as a negative control. As expected ( Bookout et al., 2013 Bookout A.L.

de Groot M.H.

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Lawrence H.L.

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Kliewer S.A. FGF21 regulates metabolism and circadian behavior by acting on the nervous system. Liang et al., 2014 Liang Q.

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Xu A. FGF21 maintains glucose homeostasis by mediating the cross talk between liver and brain during prolonged fasting. +/− mice by quantitative PCR (qPCR) (+/− mice, albeit at relatively low levels, but not the PFC or CP (−/− control mice ( Figure 3 FGF21 Affects Dopamine Signaling Show full caption (A) β-Klotho (Klb) mRNA levels in the suprachiasmatic nucleus/paraventricular nucleus (SCN/PVN) region of the hypothalamus, ventral tegmental area (VTA), nucleus accumbens (NAc), medial prefrontal cortex (PFC), and caudate putamen of Klb+/− and Klb−/− mice (n = 6/group). Ct values are shown in the bars. ND, not detected. (B) Concentrations of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 3-methoxytyramine (3-MT) in the NAc of mice administered either vehicle or FGF21 for 2 weeks by osmotic minipump (n = 12/group). (C and D) mRNA levels of dopamine transporter (Slc6a3) or (D) catechol-O-methyl transferase (Comt) in VTA, NAc, and caudate putamen of mice administered either vehicle or FGF21 for 2 weeks by osmotic minipump (n = 7–8/group). Ct values are shown. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus control group by Student’s t test. See also Values are means ± SEM.p < 0.05,p < 0.01,p < 0.001 versus control group by Student’s t test. See also Figure S2 The neurotransmitter dopamine has a central role in regulating reward behavior, including sucrose and saccharin preference (). To examine whether FGF21 affects dopamine signaling, including the mesolimbic pathway, we first measured β-Klotho expression in the ventral tegmental area (VTA), nucleus accumbens (NAc), medial prefrontal cortex (PFC), and caudate putamen (CP) of Klband Klbmice, with the latter mice serving as a negative control. As expected (), Klb mRNA was detected in the suprachiasmatic nucleus/paraventricular (SCN/PVN) nucleus region of the hypothalamus in Klbmice by quantitative PCR (qPCR) ( Figure 3 A). Klb mRNA was also detected in VTA and NAc in Klbmice, albeit at relatively low levels, but not the PFC or CP ( Figure 3 A). As expected, Klb mRNA was not detected in any of the regions in the Klbcontrol mice ( Figure 3 A). Consistent with the qPCR data, Klb mRNA was detected by in situ hybridization in SCN and a small subset of cells in the VTA and NAc ( Figure S2 A). Expression of FGF receptor 1, which partners with β-Klotho to form the FGF21 receptor, was detected by qPCR in all of these brain regions ( Figure S2 B).

The FGF21 receptor expression data led us to examine whether FGF21 affects the levels of dopamine and its metabolites in NAc, which coordinates reward behaviors. Notably, FGF21 administration for 2 weeks significantly decreased dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 3-methoxytyramine (3-MT) concentrations ( Figure 3 B). FGF21 administration did not decrease dopamine, DOPAC, HVA, or 3-MT concentrations in the CP ( Figure S2 C). FGF21 administration also caused changes in the expression of dopamine-related genes, including an increase in the dopamine transporter in the NAc and CP and a decrease in catechol-O-methyl transferase in the VTA and CP ( Figures 3 C and 3D). FGF21 had little or no effect on the mRNA levels of tyrosine hydroxylase or dopamine receptor 1 in these brain regions ( Figures S2 D and S2E), nor did it change the levels of total and phosphorylated tyrosine hydroxylase in VTA ( Figure S2 F). Taken together, these data suggest that FGF21 may affect sweet preference via effects on dopamine signaling. However, additional experiments will be required to confirm this possibility and to determine the mechanism whereby FGF21 reduces dopamine concentrations.

Gonzales et al., 2004 Gonzales R.A.

Job M.O.

Doyon W.M. The role of mesolimbic dopamine in the development and maintenance of ethanol reinforcement. Figure 4 FGF21 Decreases Alcohol Preference Show full caption (A) Ethanol preference ratio in wild-type (WT) and Tg(Fgf21) mice at the indicated ethanol concentrations (n = 9/group). (B) Plasma ethanol concentrations in groups of WT and Tg(Fgf21) mice 1 or 3 hr after i.p. injection of ethanol (4 g/kg) (n = 4–5/group). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 versus control group by Student’s t test. See also Values are means ± SEM.p < 0.05,p < 0.01, andp < 0.001 versus control group by Student’s t test. See also Table S3 Since dopamine signaling impacts ethanol drinking behavior (), we examined whether FGF21 also affects alcohol preference. Groups of WT and Tg(Fgf21) mice were exposed stepwise to increasing concentrations of ethanol in a two-bottle preference assay. Tg(Fgf21) mice had a decreased ethanol preference ratio at the 4%, 8%, 12%, and 16% ethanol concentrations ( Figure 4 A, Table S3 ). In an ethanol bioavailability test, there was no difference between WT and Tg(Fgf21) mice in plasma ethanol concentrations at 1 and 3 hr after ethanol administration ( Figure 4 B). Thus, FGF21 suppresses ethanol preference without affecting its bioavailability.