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

Abstract

Excessive alcohol consumption is a major public health problem worldwide. Although drinking habits are known to be inherited, few genes have been identified that are robustly linked to alcohol drinking. We conducted a genome-wide association metaanalysis and replication study among >105,000 individuals of European ancestry and identified β-Klotho (KLB) as a locus associated with alcohol consumption (rs11940694; P = 9.2 × 10^-12). β-Klotho is an obligate coreceptor for the hormone FGF21, which is secreted from the liver and implicated in macronutrient preference in humans. We show that brain-specific β-Klotho KO mice have an increased alcohol preference and that FGF21 inhibits alcohol drinking by acting on the brain. These data suggest that a liver-brain endocrine axis may play an important role in the regulation of alcohol drinking behavior and provide a unique pharmacologic target for reducing alcohol consumption.

Keywords: FGF21; alcohol consumption; human; mouse model; β-Klotho.

Fig. 1.

Genome-wide association results of log grams per day alcohol in the Alcohol Genome-Wide Association (AlcGen) and Cohorts for Heart and Aging Research in Genomic Epidemiology Plus (CHARGE+) Consortia. (A) Manhattan plot showing the significance of the association (−log₁₀-transformed P value on the y axis) for each SNP at the chromosomal position shown on the x axis. The dotted line represents the genome-wide significance level at P = 5 × 10⁻⁸. The genes that were followed up are labeled. (B) Quantile–quantile plot comparing the expected P value on the x axis and the observed P value on the y axis (both were −log₁₀ transformed).

Fig. S1.

Forest plot for the association of rs11940694 in KLB with log grams per day alcohol in the discovery GWAS and replication cohorts. (A) rs11940694 in KLB in discovery GWAS cohorts. Discovery GWAS cohorts: the Alcohol Genome-Wide Association (AlcGen) Consortium: the Cohorte Lausannoise study (COLAUS), the Estonian Biobank Cohort (EGCUT), the European Prospective Investigation of Cancer–Norfolk study (EPIC-NORFOLK), the Erasmus Rucphen Family study (ERF), the Fenland study (FENLAND), the Older Finnish Twin Cohort_2 (FinnTwinOld_2), the Helsinki Birth Cohort Study (HBCS), the population-based Cooperative Health Research in the Region of Augsburg F3 Study (KORAF3), the population-based Cooperative Health Research in the Region of Augsburg F4 Study (KORAF4), the LifeLines Cohort Study & Biobank (LIFELINES), the London Life Sciences Prospective Population Study (LOLIPOP_EW_A, LOLIPOP_EW_P, and LOLIPOP_EW610), the Older Finnish Twin Cohort_3 (FinnTwinOld_3), the Netherlands Study of Depression and Anxiety (NESDA), the Northern Finland Birth Cohort 1966 (NFBC1996), the Netherlands Twin Register cohort (NTR), the Australian twin-family study of alcohol use disorder (OZALC), the Study of Health in Pomerania (SHIP), the TwinsUK study (TWINSUK), and the Cardiovascular Risk in Young Finns Study (YFS); and the Cohorts for Heart and Aging Research in Genomic Epidemiology Plus (CHARGE+) Consortium: the Age, Gene/Environment Susceptibility–Reykjavik (AGES-Reykjavik) study, the Cardiovascular Health Study (CHS), the Framingham Heart Study (FHS), the Health, Aging, and Body Composition (HABC), the Multi-Ethnic Study of Atherosclerosis (MESA), the Rotterdam Study (RS1, RS2, and RS3), and the Women's Genome Health Study (WGHS). In rs11940694, the coded allele was A, and the noncoded allele was G. The allele frequency for A was ∼0.42 in the entire sample. The beta/SE estimates were for A allele. (B) rs11940694 in KLB in discovery and replication cohorts. The coded allele was A, and the noncoded allele was G. The beta/SE estimates were for A allele.

Fig. S2.

Genome-wide association results of dichotomous alcohol in the Alcohol Genome-Wide Association (AlcGen) and Cohorts for Heart and Aging Research in Genomic Epidemiolog Plus (CHARGE+) consortia. (A) Manhattan plot showing the significance of the association (−log₁₀-transformed P value on the y axis) for each SNP at the chromosomal position shown on the x axis. The dotted line represents the genome-wide significance level at P = 5 × 10⁻⁸. The genes that were followed up are labeled. (B) Quantile–quantile plot comparing the expected P value on the x axis and the observed P value on the y axis (both were −log₁₀ transformed).

Fig. S3.

Illustration of common SNPs (minor allele frequency > 0.01) and LD structure in the genomic regions around the KLB gene. The target SNP rs11940694 is highlighted with the black background and indicated by a red arrow in the LD structure plot. LD is measured by r², and the darker the red color, the higher the r² value.

Fig. 2.

FGF21 reduces alcohol preference in mice by acting on β-Klotho in brain. (A) Alcohol preference ratios determined by two-bottle preference assays with water and the indicated ethanol concentrations for control (Klb^fl/fl) and brain-specific Klb^Camk2a mice administered either FGF21 (0.7 mg/kg per day) or vehicle (n = 10 per group). (B) Plasma ethanol and (C) FGF21 concentrations at the end of the 16% (vol/vol) ethanol step of the two-bottle assay. For A–C, ***P < 0.001 for Klb^fl/fl + vehicle vs. Klb^fl/fl + FGF21 groups; ^##P < 0.01 for Klb^fl/fl + FGF21 vs. Klb^Camk2a + FGF21 groups as determined by one-way ANOVA followed by Tukey's posttests; ^###P < 0.001 for Klb^fl/fl + FGF21 vs. Klb^Camk2a + FGF21 groups as determined by one-way ANOVA followed by Tukey's posttests. (D) Plasma ethanol concentrations 1 and 3 h after i.p. injection of 2 g/kg alcohol (n = 4 per each group). (E) Consumption of 16% (vol/vol) ethanol (grams per kilogram per day) and (F) alcohol preference ratios in two-bottle preferences assays performed with control (Klb^fl/fl) and brain-specific Klb^Camk2a mice. Alcohol preference was measured by volume of ethanol/total volume of fluid consumed (n = 13 per group). (G) Plasma ethanol concentrations 1 and 3 h after i.p. injection of 2 g/kg alcohol (n = 5 per group). Values are means ± SEM. For E and F, *P < 0.05; **P < 0.01.

Fig. 3.

Behavior tests in brain-specific Klb^Camk2a mice. Results from (A) novelty suppressed feeding, (B) elevated plus maze, and (C) open-field activity assays performed with control (Klb^fl/fl) and brain-specific Klb^Camk2a mice (n = 15 per each group). Values are the time (seconds) spent for each step of the assay.