Alcohol and ketogenic diets increase water consumption. Here, we show that the hormone FGF21 is required for this drinking response in mice. Circulating levels of FGF21 are increased by alcohol consumption in humans and by both alcohol and ketogenic diets in mice. Pharmacologic administration of FGF21 stimulates water drinking behavior in mice within 2 hr. Concordantly, mice lacking FGF21 fail to increase water intake in response to either alcohol or a ketogenic diet. The effect of FGF21 on drinking is mediated in part by SIM1-positive neurons of the hypothalamus and is inhibited by β-adrenergic receptor antagonists. Given that FGF21 also is known to suppress alcohol intake in favor of pure water, this work identifies FGF21 as a fundamental neurotropic hormone that governs water balance in response to specific nutrient stresses that can cause dehydration.

In this report, we show that FGF21 acts directly on the nervous system to markedly stimulate drinking in both pharmacologic and physiologic contexts, including in response to alcohol exposure and a ketogenic diet. These findings, together with the previous results from two-bottle preference studies (), show that FGF21 plays an important role in regulating water balance in response to specific nutritional stresses.

KLB is associated with alcohol drinking, and its gene product β-Klotho is necessary for FGF21 regulation of alcohol preference.

In human genome-wide association studies (GWASs), SNPs in and around the FGF21 and β-Klotho genes were linked to carbohydrate and alcohol consumption (). In two-bottle preference experiments performed with mice, pharmacologic administration of FGF21 suppressed intake of both alcohol and sweetened water by acting on the nervous system (). These results provided evidence for a feedback liver-brain endocrine pathway that limits alcohol and carbohydrate consumption. Interestingly, pharmacologic FGF21 administration also increased water consumption in mice (). However, these studies did not address whether endogenous FGF21 regulates water intake and, if so, its underlying mechanism of action.

KLB is associated with alcohol drinking, and its gene product β-Klotho is necessary for FGF21 regulation of alcohol preference.

KLB is associated with alcohol drinking, and its gene product β-Klotho is necessary for FGF21 regulation of alcohol preference.

FGF21 acts through a cell-surface receptor composed of a conventional fibroblast growth factor (FGF) receptor, FGFR1c, in complex with a single pass-transmembrane protein, β-Klotho (). In mice, FGFR1c and β-Klotho are co-expressed in only a few tissues, including white and brown adipose tissue and the hypothalamus and hindbrain (). FGF21 crosses the blood-brain barrier () and mediates its chronic pharmacologic effects on body weight and insulin sensitivity by acting on its receptor complex in neurons (). This, in turn, activates sympathetic nervous system outflow to both brown and white adipose tissue depots (), resulting in thermogenesis and weight loss. In humans, a long-acting FGF21 analog increased blood pressure and heart rate (), which may be a consequence of FGF21 inducing sympathetic nerve activity.

The hormone FGF21 is synthesized and secreted into the blood from liver in response to a wide variety of nutritional stresses, including starvation and low-protein diets, high-fat/low-carbohydrate ketogenic diets, simple sugars such as sucrose and fructose, and alcohol (). Among its myriad downstream actions, FGF21 regulates carbohydrate and lipid metabolism and energy expenditure. Pharmacologically, FGF21 causes weight loss and improves insulin sensitivity and lipid parameters in rodent and primate models of obesity (). In two recent clinical trials, long-acting FGF21 derivatives lowered body weight and insulin levels in obese subjects with type 2 diabetes ().

Results

To determine the kinetics with which FGF21 induces drinking, we performed experiments with a gustometer, which detects licks on a drinking tube. A significant increase in mouse licking behavior was seen 1.5 hr after a single intraperitoneal (i.p.) injection of FGF21 compared to vehicle, and this increased licking continued to rise for the entire 12 hr recording period of the experiment ( Figures 1 I and 1J; Figure S1 B). A corresponding increase in cumulative water intake was detected within 2 hr after FGF21 administration ( Figure 1 K). Metabolic cage experiments performed in parallel showed that the FGF21-induced increase in water intake was not secondary to changes in energy expenditure ( Figure S1 C).

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Maratos-Flier E. Hepatic fibroblast growth factor 21 is regulated by PPARalpha and is a key mediator of hepatic lipid metabolism in ketotic states. Figure 2 Liver-Derived FGF21 Is Required for Ketogenic Diet-Induced Drinking Show full caption (A) Daily water intake (mL/g-body weight) in wild-type (WT) and FGF21-knockout (F21 KO) mice fed either a normal chow diet (NCD) or a ketogenic diet (KD) (n = 8/group). The study was repeated independently three times with similar results. Values are means ± SEM. ∗∗p < 0.01 for WT NCD versus WT KD groups; #p < 0.05, ##p < 0.01, ###p < 0.001 for WT + KD versus KO + KD by two-way ANOVA with Tukey’s post hoc test. (B) Daily water intake in groups of WT and hepatocyte-specific FGF21-KO (Fgf21Alb) mice fed either a NCD (days 1 and 2) or a KD (days 3–19) (n = 6/group). Values are means ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 for Fgf21fl/fl NCD (on days 1 and 2) versus Fgf21fl/fl KD groups; #p < 0.05, ##p < 0.01, ###p < 0.001 for Fgf21fl/fl versus Fgf21alb by two-way ANOVA with Tukey’s post hoc test. (C) Total body water (left) and hydration ratios (right) were measured using an echo MRI in WT and FGF21-KO mice after 3 weeks on either a NCD or a KD (n = 7–8/group). In (A)–(C). values are means ± SEM. ∗∗p < 0.01 for NCD versus KD groups with the same genotype; #p < 0.05 for WT + KD versus KO + KD by two-way ANOVA with Tukey’s post hoc test. (D) Plasma osmolality in WT and FGF21-KO mice fed either a NCD (n = 6/group) or a KD (n = 7/group). Values are means ± SEM. (E) Systolic and diastolic blood pressure measurements in groups of WT and FGF21-KO mice fed either NCD or KD (n = 8/group). Measurements were done using a tail-cuff device over a 7 day period. Values are means ± SEM. ∗p < 0.05, ∗∗p < 0.01 for WT versus FGF21-KO groups by Student’s t test. (F and G) Hepatic Fgf21 mRNA (F) and plasma FGF21 concentrations (G) in WT and FGF21-KO mice fed a NCD or a KD. In (F), the Fgf21 quantitative real-time PCR cycle time (Ct) value for the WT/KD group is shown. Values are means ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001 for NCD versus KD groups by Student’s t test. N.D., not detected. See also Figure S2 To examine whether endogenous FGF21 induces drinking, we compared water consumption in groups of wild-type (WT) and whole-body FGF21-knockout (KO) mice fed either a normal chow or a ketogenic diet, which strongly induces FGF21 levels (). There was no difference in water intake between WT and FGF21-KO mice on the chow diet ( Figure 2 A). WT mice fed the ketogenic diet gradually increased water consumption until it plateaued approximately 2.5-fold higher after 8 days ( Figure 2 A). In marked contrast, FGF21-KO mice on the ketogenic diet significantly decreased their water intake ( Figure 2 A). While there was a trend toward increased food consumption in mice fed the ketogenic diet, there was no difference in food intake between the WT and FGF21-KO mice on either diet ( Figure S2 A). Mice selectively lacking FGF21 in hepatocytes showed this same decrease in water intake in response to the ketogenic diet as in mice with global FGF21 deletion ( Figure 2 B), demonstrating that FGF21 derived from liver is responsible for increased drinking. While the basis for decreased water consumption in KO mice fed ketogenic diet is not entirely clear, approximately 30% of it can be accounted for by increased water content in the ketogenic diet compared to normal chow diet. Consistent with their decreased water intake, whole-body FGF21-KO mice on the ketogenic diet had a decreased total body water level and hydration ratio compared to WT mice ( Figure 2 C), but no detectable change in plasma osmolality ( Figure 2 D), suggesting changes in total body water in proportion to solutes. We also measured blood pressure, since it can be affected by dehydration. While there was no difference between WT and FGF21-KO on the normal chow diet, FGF21-KO mice on the ketogenic diet mice had lower systolic and diastolic pressures ( Figure 2 E). As expected, FGF21 mRNA in liver and protein concentrations in blood were increased by the ketogenic diet ( Figures 2 F and 2G). Thus, endogenous FGF21 released from liver stimulates drinking.

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et al. Fibroblast growth factor 21 (FGF21) is robustly induced by ethanol and has a protective role in ethanol associated liver injury. Figure 3 FGF21 Is Required for Alcohol-Induced Drinking Show full caption (A) Plasma FGF21 levels in C57BL/6 mice given a single bolus of water or alcohol (3.5 g/kg) by oral gavage. Values are means ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001 for basal (n = 9) versus alcohol (n = 11) gavage groups at each time point by one-way ANOVA with Tukey’s post hoc test. (B) Plasma FGF21 levels in healthy volunteers who drank either alcohol in juice (2 mL/kg; n = 13) or the juice vehicle alone (n = 8) over a 30 min period. FGF21 concentrations were measured in plasma taken at the indicated times after the first drink. Values are means ± SEM. ∗∗∗p < 0.001 for vehicle versus alcohol groups at each time point by two-way ANOVA with Tukey’s post hoc test. (C) Daily water intake (mL/g-body weight) in wild-type (WT) and FGF21-knockout (KO) mice administered water or alcohol (3.5 g/kg/day) by oral gavage (n = 4 for water and 7–8 for alcohol groups). Values are means ± SEM. ∗p < 0.05, ∗∗p < 0.01 for WT + water versus WT + alcohol groups; #p < 0.05 for WT + alcohol versus KO + alcohol groups by two-way ANOVA with Tukey’s post hoc test. This study was repeated independently three times with similar results. (D and E) Hepatic Fgf21 mRNA (D) and plasma FGF21 levels (E) after 8 days of treatment. N.D., not detected. Values are means ± SEM. ∗∗p < 0.01 by Student’s t test. In (D), the Fgf21 QPCR Ct value for the WT/alcohol group is shown. (F) Plasma copeptin levels in WT and FGF21-KO mice after 8 days of treatment. Values are means ± SEM. ∗p < 0.05 for water versus alcohol groups by two-way ANOVA with Tukey’s post hoc test. See also Figure S2 Chronic alcohol consumption also induces circulating FGF21 concentrations in mice (). To determine whether FGF21 is induced by acute alcohol exposure, mice were administered a single dose of ethanol (31.5% v/v, 3.5 g/kg) by oral gavage, and plasma FGF21 concentrations were measured at 1, 2, and 4 hr. Plasma FGF21 concentrations were significantly elevated at 1 hr post treatment and declined after that ( Figure 3 A). To determine whether acute alcohol exposure likewise induces FGF21 in humans, we administered either alcohol (40.0% v/v, 2 mL/kg; n = 13) or the juice vehicle alone (n = 8) to healthy volunteers over a 30 min period and measured serum FGF21 concentrations hourly after the first drink. Alcohol—but not the juice vehicle—increased circulating FGF21 concentrations at the 2–4 hr time points to levels comparable to those achieved by ketogenic diet in mice ( Figure 3 B). These findings are consistent with a recent independent report in mice and humans (). Thus, acute alcohol exposure strongly induces FGF21 levels in both mice and humans.

Cicero, 1981 Cicero T.J. Neuroendocrinological effects of alcohol. We next examined whether endogenous FGF21 contributes to alcohol-induced increases in water consumption. Groups of WT and FGF21-KO mice were administered either alcohol (3.5 g/kg/day) or an equal volume of water by oral gavage once daily for 8 days with free access to water. From day 3, alcohol administration significantly increased water intake in WT mice ( Figure 3 C). Importantly, this effect was absent in FGF21-KO mice ( Figure 3 C). As expected, alcohol administration increased hepatic Fgf21 mRNA and plasma FGF21 levels in WT mice ( Figures 3 D and 3E). Moreover, alcohol significantly decreased plasma copeptin concentrations in WT mice ( Figure 3 F), which is consistent with the well-established inhibitory effect of alcohol on AVP release from the pituitary gland (). Alcohol also decreased circulating copeptin levels in FGF21-KO mice ( Figure 3 F). Taken together, these data show that alcohol stimulates drinking through an FGF21-dependent mechanism that appears to be independent of AVP levels.

We also examined whether FGF21 is regulated by high salt intake or water deprivation. Mice fed a high-salt diet had a striking increase in water consumption ( Figure S2 B) without changes in FGF21 mRNA levels in liver ( Figure S2 C) or protein levels in blood ( Figure S2 D). Thus, FGF21 does not contribute to salt-induced drinking. FGF21 mRNA ( Figure S2 E) and protein ( Figure S2 F) levels were also unchanged by 24 hr water deprivation, indicating that FGF21 is not induced by dehydration per se, at least in this time frame.

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Kliewer S.A. FGF21 regulates metabolism and circadian behavior by acting on the nervous system. fl/fl, but not KlbCamk2a, mice (fl/fl and KlbCamk2a mice (Camk2a mice. Alcohol exposure decreased plasma copeptin concentrations in both Klbfl/fl and KlbCamk2a mice ( Figure 4 FGF21 Acts on Neurons to Induce Drinking Show full caption (A) Cumulative water consumption in C57BL/6J mice after a single i.c.v. injection of FGF21 (1 μg) or vehicle (n = 8/group). Values are means ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001 for vehicle versus FGF21. (B) Daily water intake (mL/g-body weight) measured in control mice (Klbfl/fl) and neuron-specific β-Klotho-KO mice (KlbCamk2a) administered either vehicle (n = 4/group) or FGF21 (1 mg/kg/day, n = 5/group) by osmotic minipump. Values are means ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001 for Klbfl/fl + vehicle versus Klbfl/fl + FGF21 groups; #p < 0.05, ##p < 0.01 for Klbfl/fl + FGF21 versus KlbCamk2a + FGF21 groups by two-way ANOVA with Tukey’s post hoc test. This study was repeated independently three times with similar results. (C) Daily water intake (mL/g-body weight) in Klbfl/fl and KlbCamk2a mice fed a normal chow diet (NCD, n = 5/group) or ketogenic diet (KD, n = 7/group). Values are means ± SEM. ∗p < 0.05, ∗∗p < 0.01 for NCD versus KD groups for Klbfl/fl group; #p < 0.05, ##p < 0.01, ###p < 0.001 for Klbfl/fl + KD versus KlbCamk2a + KD by two-way ANOVA with Tukey’s post hoc test. This study was repeated independently three times with similar results. (D) Daily water intake (mL/g-body weight) in Klbfl/fl and KlbCamk2a mice administered either water or alcohol (3.5 g/kg/day) by gavage (n = 4 for water and 8 for alcohol group). Values are means ± SEM. ∗p < 0.05, ∗∗p < 0.01 for Klbfl/fl + water versus Klbfl/fl + alcohol gavage groups, ##p < 0.01 for Klbfl/fl + alcohol- versus KlbCamk2a + alcohol-treated group by two-way ANOVA with Tukey’s post hoc test. (E) Plasma copeptin levels in Klbfl/fl and KlbCamk2a mice after 8 days of water or alcohol administration. Values are means ± SEM. ∗p < 0.05 for water versus alcohol groups by two-way ANOVA with Tukey’s post hoc test. (F and G) c-Fos immunofluorescence staining in the indicated brain regions of C57BL/6J mice (F) or Klbfl/fl and KlbCamk2a mice (G) 2 hr after vehicle or FGF21 (1 mg/kg) administration by i.p. injection. c-Fos fluorescence is white. 3V, third ventricle. Scale bars, 50 μm in (F) and 100 μm in (G). (H) Cumulative water consumption in Klbfl/fl and KlbSim1 mice after a single i.p. injection of FGF21 (1 mg/kg, n = 7/group) or vehicle (n = 5/group). (I) Daily water intake (mL/g-body weight) measured in Klbfl/fl and KlbSim1 mice administered either vehicle or FGF21 (1 mg/kg/day) by osmotic minipump (n = 10∼12/group). Values are means ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 for vehicle versus FGF21 groups, #p < 0.05 for Klbfl/fl + FGF21 versus KlbSim1 + FGF21 groups by two-way ANOVA with Tukey’s post hoc test. (J) Daily water intake (mL/g-body weight) in groups of mice administered FGF21 or vehicle by osmotic minipump and either β-blockers or water vehicle by i.p. injection (n = 5 for water groups and 6 for β-blocker groups). Values are means ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 for vehicle- versus FGF21-treated groups; #p < 0.05, ##p < 0.01 for FGF21 + water versus FGF21 + β blockers by two-way ANOVA with Tukey’s post hoc test. (K) Daily water intake (mL/g-body weight) in KD-fed mice administered water or β-blockers by i.p. injection (n = 6/group for water and β-blocker treatment). Injection was started after 7 days on the KD. ∗p < 0.05, ∗∗p < 0.01 for water- versus β-blocker-treated groups with the same genotype; #p < 0.05, ###p < 0.001 for indicated groups by two-way ANOVA with Tukey’s post hoc test. See also Figure S3 FGF21 mediates many of its effects on metabolism and behavior by acting directly on its receptor in the nervous system (). In time course studies, direct intracerebroventricular (i.c.v.) injection of FGF21 into the brain increased water intake within 40 min ( Figure 4 A), which was faster than i.p. injection ( Figure 1 K), providing evidence that FGF21 can act centrally. To test whether peripheral FGF21 also stimulates drinking by acting on the nervous system, we used mice in which the β-Klotho gene (Klb), which encodes the obligate co-receptor for FGF21 (), was selectively eliminated in neurons (Klbmice) (). In pharmacologic studies, administration of recombinant FGF21 by osmotic minipump increased water consumption in control Klb, but not Klb, mice ( Figure 4 B). There was no difference in food intake between the Klband Klbmice ( Figure S3 A). Notably, the effect of both ketogenic diet ( Figure 4 C) and alcohol ( Figures 4 D) on water intake was also eliminated in the Klbmice. Alcohol exposure decreased plasma copeptin concentrations in both Klband Klbmice ( Figure 4 E), providing additional evidence that the effect of FGF21 on drinking is independent of AVP. Thus, endogenous FGF21 stimulates water consumption by acting directly on the nervous system.

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Kliewer S.A. FGF21 regulates metabolism and circadian behavior by acting on the nervous system. fl/fl and KlbPhox2b mice ( Within the brain, the drinking response is controlled by a number of different nuclei, including the subfornical organ (SFO), the organum vasculosum of the lamina terminalis (OVLT), and the median preoptic nucleus (MnPO), which together comprise the lamina terminalis in the forebrain; the area postrema (AP) and nucleus tractus solitarii (NTS), which are components of the dorsal-vagal complex in the hindbrain (); and the paraventricular nucleus (PVN), which mediates increased water consumption in response to direct injection by norepinephrine (). We previously showed that β-Klotho is expressed in the AP and NTS (). Using Klbmice, in which β-Klotho expression is disrupted in the dorsal-vagal complex (), we tested whether these hindbrain nuclei are involved in the FGF21-induced drinking response. In osmotic minipump experiments, FGF21 stimulated water intake equally well in control Klband Klbmice ( Figure S3 B). Thus, FGF21 does not appear to act through the dorsal-vagal complex to induce drinking.

fl/fl mice with Sim1-Cre mice, in which Cre expression is enriched in the PVN ( Balthasar et al., 2005 Balthasar N.

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et al. Divergence of melanocortin pathways in the control of food intake and energy expenditure. Sim1 mice based on in situ hybridization analysis (+. Notably, the effect of FGF21 on water intake during the first 12 hr after injection was lost in these KlbSim1 knockout mice (Sim1 mice (+ neurons most likely in the PVN. To further assess the contribution of the PVN to FGF21’s effect on drinking, we crossed Klbmice with Sim1-Cre mice, in which Cre expression is enriched in the PVN (). β-Klotho mRNA was reduced by ∼40% in the PVN of Klbmice based on in situ hybridization analysis ( Figure S3 E). The remaining β-Klotho expression may reflect either incomplete knockout of β-Klotho or its expression in cells that are not Sim1. Notably, the effect of FGF21 on water intake during the first 12 hr after injection was lost in these Klbknockout mice ( Figure 4 H). In longer-term osmotic minipump experiments, the effect of FGF21 on water intake was significantly blunted, but not absent, in Klbmice ( Figure 4 I). This remaining FGF21 activity could be due to either incomplete knockout of β-Klotho in the PVN or effects of FGF21 on other brain regions. We conclude that FGF21 mediates its effect on drinking in part by acting on Sim1neurons most likely in the PVN.