A Genetic Screen Identifies Hypothalamic Fgf15 as a Regulator of Glucagon Secretion

Abstract

The counterregulatory response to hypoglycemia, which restores normal blood glucose levels to ensure sufficient provision of glucose to the brain, is critical for survival. To discover underlying brain regulatory systems, we performed a genetic screen in recombinant inbred mice for quantitative trait loci (QTL) controlling glucagon secretion in response to neuroglucopenia. We identified a QTL on the distal part of chromosome 7 and combined this genetic information with transcriptomic analysis of hypothalami. This revealed Fgf15 as the strongest candidate to control the glucagon response. Fgf15 was expressed by neurons of the dorsomedial hypothalamus and the perifornical area. Intracerebroventricular injection of FGF19, the human ortholog of Fgf15, reduced activation by neuroglucopenia of dorsal vagal complex neurons, of the parasympathetic nerve, and lowered glucagon secretion. In contrast, silencing Fgf15 in the dorsomedial hypothalamus increased neuroglucopenia-induced glucagon secretion. These data identify hypothalamic Fgf15 as a regulator of glucagon secretion.

Keywords: FGF15; FGF19; QTL mapping; autonomic nervous system; dorsal vagal complex; genetic screen; glucagon secretion; glucose sensing; hypothalamus; neuroglucopenia.

Graphical abstract

Figure 1

A Locus on Chromosome 7 Controls Neuroglucopenia-Induced Glucagon Secretion (A) Distribution of the glucagon ratio (2DG/NaCl) phenotype in the 36 BXD strains (n = 4–14 for each strain). (B) Basal and neuroglucopenia-stimulated glucagonemia in the 36 BXD mouse strains. Each circle is the mean glucagonemia for each strain. (C) Scatterplot representing the absence of correlation between the glucagon ratio (2DG/NaCl) phenotype and the total pancreatic glucagon content in BXD mice. (D) Whole genome significant QTL on distal chromosome 7. The red line indicates the whole genome significative threshold (p ≤ 0.05). The blue line indicates the whole genome suggestive threshold (p ≤ 0.63). (E) Localization of the locus between with a LRS peak on marker rs2304086585.

Figure 2

Fgf15 Expression in Adult CNS (A–D) Expression level of Fgf15, Fgfr1c, Fgfr4, and Klotho beta in different brain structures and the ileum (n = 6–8). hypothalamus, Hy; hippocampus, Hip; thalamus, Th; cerebellum, Cb; brainstem, Bs; cortex, Cx; ileum, Il. (E–J) In situ hybridization detection of Fgf15 (red signal). (E–J) PeF; representative section of hypothalamus at bregma −1.94 mm (F); DMH (G); negative control in PeF using DapB probes (H); brainstem with central canal (CC) DMNX and nucleus of the solitary tract (NTS) (I); and illeal villi (J). The scale bar represents 50 μm. (K and L) Double in situ hybridization of Fgf15 (green dots) and NeuN (red dots) in PeF and DMH showing neuronal expression of Fgf15. The scale bar represents 10 μm. The data are shown as mean ± SEM.

Figure 3

Fgf19 and the Control of Glucagon Secretion (A) Glycemia of mice injected i.c.v. with aCSF or FGF19 60 min before i.p. injection (0 min) of 2DG (600 mg/kg) (n = 7). (B) Plasma glucagon 30 min after i.p. injection of saline or 2DG in mice that were treated 60 min before with i.c.v. injection of aCSF or FGF19 (n = 7). (C–E) Plasma insulin (C), corticosterone (D), and epinephrine (E) 30 min after i.p. injection of saline or 2DG in mice that were treated 60 min before with i.c.v. injection of aCSF or FGF19 (n = 9–10) (E). (F) Hypothalamic expression of Fgf15 in C57Bl6/J mice fed a NC or a HFD for 4 weeks (n = 6–8). (G) Glycemia in NC and HFD fed mice before and 30 min after i.p. injection of 2DG (n = 6–8). (H) Plasma glucagon in NC and HFD fed mice in the basal state and 30 min after 2DG injection (n = 6–8). Data are mean ± SEM. ^∗p ≤ 0.05 and ^∗∗∗p ≤ 0.001. Two-way ANOVA followed by Bonferroni post hoc test (A–E, G, and H). Student’s t test (F).

Figure 4

Fgf15 Silencing in the DMH Increases 2DG-Induced Glucagon Secretion (A) Silencing of Fgf15 expression after co-transfection of HEK293T cells of an Fg15-GFP cDNA with the selected Fgf15-specific shRNA encoding lentiviral vector (n = 3). (B) Quantification of Fgf15 protein expression in the same conditions as (A) (n = 3). (C) Western blot analysis of Fgf15 and β-actin expression quantitated in (B). (D) Western blot analysis of Fgf15 and β-actin expression in HEK293T cells infected with a control (scrambled) or an Fgf15-shRNA lentivirus and transfected with an Fgf15-GFP expression plasmid. (E) Example of the correct bilateral injection in the DMH of Fgf15-shRNA lentiviruses. The scale bar represents 100 μm. (F) Glycemia in mice injected in the DMH with control (scrambled) or Fgf15-shRNA lentiviruses before (0 min) and 30 min after i.p. injection of 2DG. missed: mice with incorrect Fgf15-shRNA encoding lentivirus injection sites. (n = 10–19). (G) Plasma glucagon levels 30 min after 2DG i.p. injections (n = 10–19). (H) Example of the correct bilateral injection in the PeF of Fgf15-shRNA lentiviruses. The scale bar represents 100 μm. (I) Glycemia in mice injected in the PeF with control (scrambled) or Fgf15-shRNA lentiviruses before and 30 min after i.p. injection of 2DG (n = 12). (J) Plasma glucagon levels 30 min after 2DG i.p. injections (n = 12). Data are mean ± SEM. ^∗p ≤ 0.05; ^∗∗p ≤ 0.01; and ^∗∗∗p ≤ 0.001. Student’s t test (A, B, and J). Two-way ANOVA followed by Bonferroni post hoc test (F and I). One-way ANOVA followed by Bonferroni post hoc test (G).

Figure 5

FGF19 Blunts 2DG-Induced Parasympathetic Nerve Activity (A) Parasympathetic nerve firing rate in the basal state and following i.p. 2DG injection in mice previously treated with i.c.v. aCSF or FGF19 (top: representative trace and bottom: quantification of the firing activity) (n = 6–7). (B) Sympathetic nerve firing rate in the basal state and following i.p. 2DG injection in mice previously treated with i.c.v. aCSF or FGF19 (top: representative trace and bottom: quantification of the firing activity) (n = 7). (C) Representative micrographs of pERK1/2 positive cells in the ARC 30 min after i.c.v. injection of aCSF or FGF19. (D) Quantification of pERK1/2 positive cells in the ARC 30 min after i.c.v. injection of aCSF or FGF19. (n = 5–7). (E) Representative micrographs of c-fos positive cells in NTS and DMNX at bregma −7.56 mm of mice that received i.c.v. aCSF and 60 min later i.p. NaCl (upper), i.c.v. aCSF and 60 min later i.p. 2DG (mid), or i.c.v FGF19 and 60 in later i.p. 2DG (lower). area postrema, AP; central canal, CC; nucleus of the solitary tract, NTS; DMNX. (F) Quantification of c-fos positive cells in NTS and DMNX of mice treated as in (E). The scale bar represents 50 μm. Two-way ANOVA followed by Bonferroni post hoc test (A and B). Student’s t test (D). One-way ANOVA followed by Bonferroni post hoc test (F). ^∗p ≤ 0.05; ^∗∗p ≤ 0.01; and ^∗∗∗p ≤ 0.001. Data are mean ± SEM.