FGF21 suppresses alcohol consumption through an amygdalo-striatal circuit

Abstract

Excessive alcohol consumption is a major health and social issue in our society. Pharmacologic administration of the endocrine hormone fibroblast growth factor 21 (FGF21) suppresses alcohol consumption through actions in the brain in rodents, and genome-wide association studies have identified single nucleotide polymorphisms in genes involved with FGF21 signaling as being associated with increased alcohol consumption in humans. However, the neural circuit(s) through which FGF21 signals to suppress alcohol consumption are unknown, as are its effects on alcohol consumption in higher organisms. Here, we demonstrate that administration of an FGF21 analog to alcohol-preferring non-human primates reduces alcohol intake by 50%. Further, we reveal that FGF21 suppresses alcohol consumption through a projection-specific subpopulation of KLB-expressing neurons in the basolateral amygdala. Our results illustrate how FGF21 suppresses alcohol consumption through a specific population of neurons in the brain and demonstrate its therapeutic potential in non-human primate models of excessive alcohol consumption.

Keywords: FGF21; alcohol; basolateral amygdala; betaklotho; brain; hepatokine; liver; nucleus accumbens.

Figure 1.. Endogenous and pharmacological FGF21 suppress alcohol consumption in rodents and non-human primates.

(A) Circulating levels of FGF21 in response to water or ethanol gavage (3.5g/kg) in male FGF21^fl/fl mice receiving either AAV-TBG-Con or AAV-TBG-Cre (n = 6–8 mice/group, one-way ANOVA, *=P<0.05 relative to mice receiving water gavage). (B) Ethanol (EtOH) consumption (g/day/kg) in male FGF21^fl/fl mice receiving either AAV-TBG-Con or AAV-TBG-Cre allowed ad libitum access to water and EtOH using a 2-bottle choice experimental design (n = 7 and 6/group, respectively, one-tailed unpaired t-test, *=P<0.05). (C) EtOH consumption (g/day/kg) in male FGF21 liver-specific knockout (FGF21 LivKO) mice and littermate controls allowed ad libitum access to water and EtOH using a 2-bottle choice experimental design (n = 8 and 9/group, respectively, one-tailed unpaired t-test, *=P<0.05). (D) EtOH consumption (g/day/kg) in wildtype (WT) C57BL/6J male mice treated with the indicated doses of FGF21 (vehicle or mg/kg) via daily intraperitoneal (IP) injection across different EtOH concentrations using a 2-bottle choice experimental design (n = 6 mice/group, 2-way ANOVA w/Holm-Sidak’s multiple comparisons test, *=P<0.05 relative to vehicle treated mice for both concentrations). (E) The percent (%) change in ethanol consumption (12%) in WT C57BL/6J male mice allowed ad libitum access to the Lieber-DeCarli liquid diet receiving daily injections of vehicle or FGF21 (IP, 1mg/kg, n = 12–13 mice/group, two-tailed unpaired t-test, *=P<0.05 relative to vehicle treated mice). (F) Percent (%) blood alcohol content following EtOH gavage (3.5 g/kg) in WT C57BL/6J male mice treated with vehicle or FGF21 (intraperitoneal (IP), 1 mg/kg) for three days prior to EtOH gavage (n = 5 mice/group). (G) EtOH consumption (g/day/kg) in WT C57BL/6J male mice treated with a single dose of the FGF21 analogue PF-05231023 (10 mg/kg, IP) or vehicle across different EtOH concentrations using a 2-bottle choice experimental design (n = 11 mice/group, 2-way ANOVA w/Holm-Sidak’s multiple comparisons test, *=P<0.05 relative to vehicle treated mice for both concentrations). (H-I) EtOH consumption (g/day/kg) in WT C57BL/6J male (H) and female (I) mice treated with vehicle or PF-05231023 using the intermittent access paradigm (n = 8 mice/group, 2-way ANOVA w/Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, *=P<0.05 when comparing to vehicle treated mice). (J) A schematic illustration of the experimental design used to assess effect of PF-05231023 on alcohol consumption in alcohol-preferring vervet monkeys in data presented in (K) and (L). Briefly, alcohol-preferring male vervet monkeys (n = 20) were given access to alcohol for 4 h a day for 9 days to establish baseline drinking behavior. After establishing a baseline, vervets were divided into either a placebo (n = 8) or treatment arm (n = 12). Those in the treatment arm received PF-05231023 at an initial dose of 1 mg/kg for 8 days, which was then increased to 2 mg/kg for an additional 8 days. A washout period in which no drug was administered was then performed over 8 days. (K-L) EtOH consumption (K) and water intake (L) in the indicated treatment groups (2-way ANOVA w/Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, *=P<0.05 when comparing to placebo treated animals). Data are presented as mean ± S.E.M. See also Figure S1 and Table S1.

Figure 2.. FGF21 signals to KLB expressing neurons in the basolateral amygdala to suppress alcohol consumption.

(A) A representative fluorescent image of tdTomato expressing cells in the basolateral amygdala (BLA) in brain slices generated from KLB-Cre mice 4 weeks following infection with PHP.eB-DIO-tdTomato. Scale bar = 200 μm. (B) Ethanol (EtOH) consumption (g/day/kg) in WT C57BL6/J mice treated with unilateral delivery of vehicle or FGF21 to the BLA via cannula (0.01 mg/kg) across different EtOH concentrations using a two-bottle choice experimental design (n = 9 and 11 mice/group, respectively). (C) EtOH consumption (g/day/kg) in WT C57BL6/J mice treated with unilateral delivery of vehicle or PF-05231023 to the BLA via cannula (0.01 mg/kg) across different EtOH concentrations using a two-bottle choice experimental design (n = 8 mice/group for both groups). (D) Schematic representation of the strategy used to delete KLB in the BLA of KLB^fl/fl mice through stereotactic delivery of AAV-hSyn-Cre-EGFP or AAV-hSyn-EGFP (as a control) to the BLA of KLB^fl/fl mice. Representative fluorescent image of viral targeting of AAV-hSyn-Cre- EGFP to the BLA of KLB^fl/fl mice. In the inset, relative Klb mRNA expression in the amygdala of KLB^fl/fl mice 4 weeks following delivery of either AAV-hSyn-EGFP or AAV-hSyn-Cre-EGFP is measured (n = 4 and 5 mice/group, respectively, one-tailed unpaired t-test, *=P<0.05). Scale bar = 500 μm. (E) EtOH consumption (g/day/kg) in KLB^fl/fl mice 4 weeks following injection of AAV-hSyn-EGFP (WT) or AAV-hSyn-Cre-EGFP (BLA^KLB-KO) into the BLA, treated daily with either vehicle or FGF21 (intraperitoneal (IP), 1 mg/kg) across different EtOH concentrations using a two-bottle experimental design (n = 6–9 mice/group). (F-H) Electrophysiological analysis of (F) resting membrane potential (RMP, mV), (G) membrane resistance (MΩ), and (H) action potential frequency (Hz) in tdTomato expressing neurons in KLB-Cre mice four weeks following retro-orbital delivery of PHP.eB-DIO-tdTomato before (aCSF) and after FGF21 application to acute brain slices (n = 16–30 neurons from at least 3 mice for each analysis, two-tailed paired t-test, *=P<0.05 relative to aCSF). (I) Single cell RNA sequencing of tdTomato expressing cells isolated from the BLA of KLB-Cre; Ai14-tdTomato mice and performed UMAP clustering to identify distinct cell types (RGC = radial glia cell, Nts = Neurotensin). (J) A violin plot representation of the expression of genes which encode the indicated cell-type markers across clusters in tdTomato⁺ cells isolated from the BLA of KLB-Cre;Ai14-tdTomato mice (RGC = radial glia cell, Nts = Neurotensin). (K-L) Gene ontology analysis of gene networks associated with (K) biological processes and (L) subcellular compartments significantly upregulated (P values on x-axis) in KLB⁺/Slc17a7⁺ cells isolated from mice treated with FGF21 (1 mg/kg) for 3 days relative to mice treated with vehicle. Data are presented as mean ± S.E.M. 2-way ANOVA w/Holm-Sidak’s multiple comparisons test was used for statistical analyses unless stated otherwise, *=P<0.05 relative to vehicle treated mice. See also Figure S2 and Table S2.

Figure 3.. FGF21 preferentially influences excitability of KLB^BLA→NAc projection neurons and D2 MSNs in the NAc.

(A) A schematic diagram illustrating how stereotactic delivery of AAV-DIO-mCherry to the basolateral amygdala (BLA) of KLB-Cre mice was used to identify downstream projection targets of KLB-expressing (KLB⁺) neurons in the BLA. (B) A schematic diagram illustrating how stereotactic delivery of AAVrg-DIO-mCherry to the nucleus accumbens (NAc) was used to confirm that KLB⁺ neurons in the BLA project to the NAc. (C) A schematic diagram illustrating the strategy used to distinguish between KLB⁺ neurons in the BLA which project to the NAc (KLB^BLA→NAc) and those that do not project to the NAc (KLB^BLA). (D-E) Electrophysiological analysis of latency to the first action potential spike correlated with current injection in (D) KLB^BLA and (E) KLB^BLA→NAc neurons in acute brain slices from mice treated with FGF21 (intraperitoneal (IP), 1 mg/kg) or vehicle for 3 days prior to recording (n = 7–12 neurons/group from at least 3 mice/group, 2-way ANOVA w/Holm-Sidak’s multiple comparisons test, *=P<0.05). (F) Representative traces of loose seal action current spikes for the data quantified in (G). (G) Firing frequency (Hz) in acute brain slices from mice treated with FGF21 (IP, 1 mg/kg) or vehicle for 3 days prior to recording (n = 25–52 neurons/group from at least 3 mice/group, 2-way ANOVA w/Sidak’s multiple comparisons test, *=P<0.05). (H) Representative traces of spontaneous miniature excitatory post-synaptic currents (sEPSCs) in D1- and putative D2-expressing medium spiny neurons (MSNs) in the NAc core in acute brain slices from mice treated with FGF21 (IP, 1 mg/kg) or vehicle for 3 days prior to recording. (I-J) sEPSC amplitude and frequency in D1-MSNs in the lateral NAc core in acute brain slices from vehicle and FGF21 treated mice (n = 12–14 neurons/group from at least 3 mice/group). (K-L) sEPSC amplitude and frequency in putative D2-MSNs in the lateral NAc core in acute brain slices from vehicle and FGF21 treated mice (n = 12–14 neurons/group from at least 3 mice/group, two-tailed unpaired t-test, *=P<0.05). Data are presented as mean ± S.E.M. See also Figure S3.

Figure 4.. KLB expression in BLA→NAc projection neurons is necessary and sufficient for suppression of alcohol consumption by FGF21 and PF-05231023.

(A) Schematic illustrating placement of a transcriptional blocking sequence (splice acceptor (orange) followed by 3x poly A tail sequence (yellow)) in the intronic region between the 1^st and 2^nd exon of Klb flanked by loxP sites to generate KLB loxTB mice. (B) Schematic illustrating the dual recombinase strategy used to return endogenous KLB expression specifically to BLA→NAc projecting neurons using an AAVrg-Flp expressing virus injected into the NAc and an AAV virus expressing a Flp-dependent HA-tagged version of Cre (AAV-fDIO-Cre-HA) injected into the BLA. (C-E) Ethanol (EtOH) consumption (g/day/kg) in wildtype mice (black, WT), KLB loxTB control mice (blue, KLB loxTB^Ctrl, KLB^loxTB/loxTB mice which received AAV-fDIO-Cre-HA in the BLA, but AAVrg-GFP in the NAc instead of AAVrg-Flp), and mice in which endogenous KLB expression was returned to BLA→NAc projecting neurons (green, KLB loxTB^BLA→NAc; KLB^loxTB/loxTB mice which received AAV-fDIO-Cre-HA in the BLA, and AAVrg-Flp in the NAc) during daily vehicle administration followed by daily FGF21 administration (IP, 1 mg/kg) as indicated by the brackets above the plots followed by a washout period. (F) Percent (%) change in EtOH consumption comparing FGF21 treatment period relative to the vehicle treated period for each group (n = 5 WT mice, 12 KLB loxTB^Ctrl, 9 KLB loxTB^BLA→NAc, one-way ANOVA w/Dunnett’s multiple comparison’s test, *=P<0.05 relative to WT mice). (G-I) EtOH consumption in WT, KLB loxTB^Ctrl mice, and KLB loxTB^BLA→NAc during daily vehicle administration followed by a single injection of PF-05231023 (IP, 10 mg/kg) as indicated by the brackets and arrow, respectively, above the plots. (J) Quantification of the percent (%) change in EtOH consumption comparing 1 day following PF-05231023 treatment relative to the vehicle treated period for each group (n = 5 WT mice, 12 KLB loxTB^Ctrl, 9 KLB loxTB^BLA→NAc, one-way ANOVA w/Dunnett’s multiple comparison’s test, *=P<0.05 relative to WT mice). (K-M) Sucrose consumption (mL/day) in WT, KLB loxTB^Ctrl mice, and KLB loxTB^BLA→NAc during daily vehicle administration followed by daily FGF21 administration (IP, 1mg/kg) as indicated by the brackets above the plots. (N) Quantification of the percent (%) change in sucrose consumption comparing FGF21 treatment period relative to the vehicle treated period for each group (n = 5 WT mice, 12 KLB loxTB^Ctrl, 9 KLB loxTB^BLA→NAc, one-way ANOVA w/Holm-Sidak’s multiple comparison’s test, *=P<0.05 for KLB loxTB^BLA→NAc mice relative to WT mice). Data are presented as mean ± S.E.M. See also Figure S3.