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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus

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).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5120348&req=5

fig4: 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).

Mentions: To more directly determine whether the level of expression of Fgf15 in the hypothalamus regulates glucagon secretion, we developed recombinant lentiviruses to express a control or an Fgf15-specific short hairpin (sh)RNA. Three shRNAs were designed to target the Fgf15 sequence and were subcloned in lentiviral vectors. These were first tested in HEK293T cells co-transfected with an Fgf15-GFP expression plasmid. One vector induced efficient silencing of Fgf15 mRNA and protein expression (Figures 4A–4C). This was used to generate a recombinant lentivirus, which effectively silenced Fgf15 expression in transduction experiments (Figure 4D). This or a control lentivirus was then injected bilaterally in the DMH (Figure 4E). Two weeks later, the mice were injected i.p. with 2DG and plasma glucagon was measured 30 min later. The mice were then killed to verify the sites of virus injection. No difference in basal glycemia or in glycemic response to 2DG injection was observed between the two groups of mice (Figure 4F). However, neuroglucopenia-induced glucagon secretion was significantly increased (22%) in mice with correct bilateral DMH injection of the silencing lentivirus as compared to mice injected with the control lentivirus (Figure 4G) or mice injected with Fgf15-specific shRNA lentivirus outside of the DMH (Figure 4G, “missed”). Bilateral injection of the silencing lentivirus in the PeF (Figure 4H) did not impact glycemia before and 30 min after i.p. 2DG (Figure 4I), nor the neuroglucopenia-induced glucagon secretion (Figure 4H). Taken together, these data show that Fgf15 expression by DMH neurons has a negative impact on neuroglucopenia-induced glucagon secretion.


A Genetic Screen Identifies Hypothalamic Fgf15 as a Regulator of Glucagon Secretion
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).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120348&req=5

fig4: 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).
Mentions: To more directly determine whether the level of expression of Fgf15 in the hypothalamus regulates glucagon secretion, we developed recombinant lentiviruses to express a control or an Fgf15-specific short hairpin (sh)RNA. Three shRNAs were designed to target the Fgf15 sequence and were subcloned in lentiviral vectors. These were first tested in HEK293T cells co-transfected with an Fgf15-GFP expression plasmid. One vector induced efficient silencing of Fgf15 mRNA and protein expression (Figures 4A–4C). This was used to generate a recombinant lentivirus, which effectively silenced Fgf15 expression in transduction experiments (Figure 4D). This or a control lentivirus was then injected bilaterally in the DMH (Figure 4E). Two weeks later, the mice were injected i.p. with 2DG and plasma glucagon was measured 30 min later. The mice were then killed to verify the sites of virus injection. No difference in basal glycemia or in glycemic response to 2DG injection was observed between the two groups of mice (Figure 4F). However, neuroglucopenia-induced glucagon secretion was significantly increased (22%) in mice with correct bilateral DMH injection of the silencing lentivirus as compared to mice injected with the control lentivirus (Figure 4G) or mice injected with Fgf15-specific shRNA lentivirus outside of the DMH (Figure 4G, “missed”). Bilateral injection of the silencing lentivirus in the PeF (Figure 4H) did not impact glycemia before and 30 min after i.p. 2DG (Figure 4I), nor the neuroglucopenia-induced glucagon secretion (Figure 4H). Taken together, these data show that Fgf15 expression by DMH neurons has a negative impact on neuroglucopenia-induced glucagon secretion.

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus