Figure 4. FGF21 acts on neurons to induce drinking.

(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 vs. FGF21.

(B) Daily water intake (ml/g·BW) measured in control mice (Klb^fl/fl) and neuron-specific β-Klotho-knockout mice (Klb^Camk2a) 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 Klb^fl/fl + vehicle vs. Klb^fl/fl + FGF21 groups; ^#p < 0.05, ^##p < 0.01 for Klb^fl/fl + FGF21 vs. Klb^Camk2a + 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·BW) in Klb^fl/fl and Klb^Camk2a 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 vs. KD groups for Klb^fl/fl group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 for Klb^fl/fl + KD vs. Klb^Camk2a + 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·BW) in Klb^fl/fl and Klb^Camk2a 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 Klb^fl/fl + water vs. Klb^fl/fl + alcohol gavage groups, ^##p < 0.01 for Klb^fl/fl + alcohol vs. Klb^Camk2a + alcohol treated group by two-way ANOVA with Tukey's post hoc test.

(E) Plasma copeptin levels in Klb^fl/fl and Klb^Camk2a mice after 8 days of water or alcohol administration. Values are means ± SEM. *p < 0.05 for water vs. alcohol groups by two-way ANOVA with Tukey's post hoc test.

(F, G) c-Fos immunofluorescence staining in the indicated brain regions of C57BL/6J mice (F) or Klb^fl/fl and Klb^Camk2a mice (G) 2 hours after vehicle or FGF21 (1 mg/kg) administration by i.p. injection. c-Fos fluorescence is white. 3V, third ventricle. Scale bar = 50 μm in (F) and 100 μm in (G).

(H) Cumulative water consumption in Klb^fl/fl and Klb^Sim1 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·BW) measured in Klb^fl/fl and Klb^Sim1 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 vs FGF21 groups, ^#p < 0.05 for Klb^fl/fl + FGF21 vs. Klb^Sim1 + FGF21 groups by two-way ANOVA with Tukey's post hoc test.

(J) Daily water intake (ml/g·BW) 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 vs. FGF21-treated groups; ^#p < 0.05, ^##p < 0.01 for FGF21 + water vs. FGF21 + β blockers by two-way ANOVA with Tukey's post hoc test.

(K) Daily water intake (ml/g·BW) 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 vs. 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.