EphA5-EphrinA5 interactions within the ventromedial hypothalamus influence counterregulatory hormone release and local glutamine/glutamate balance during hypoglycemia

Abstract

Activation of β-cell EphA5 receptors by its ligand ephrinA5 from adjacent β-cells has been reported to decrease insulin secretion during hypoglycemia. Given the similarities between islet and ventromedial hypothalamus (VMH) glucose sensing, we tested the hypothesis that the EphA5/ephrinA5 system might function within the VMH during hypoglycemia to stimulate counterregulatory hormone release as well. Counterregulatory responses and glutamine/glutamate concentrations in the VMH were assessed during a hyperinsulinemic-hypoglycemic glucose clamp study in chronically catheterized awake male Sprague-Dawley rats that received an acute VMH microinjection of ephrinA5-Fc, chronic VMH knockdown, or overexpression of ephrinA5 using an adenoassociated viral construct. Local stimulation of VMH EphA5 receptors by ephrinA5-Fc or ephrinA5 overexpression increased, whereas knockdown of VMH ephrinA5 reduced counterregulatory responses during hypoglycemia. Overexpression of VMH ephrinA5 transiently increased local glutamate concentrations, whereas ephrinA5 knockdown produced profound suppression of VMH interstitial fluid glutamine concentrations in the basal state and during hypoglycemia. Changes in ephrinA5/EphA5 interactions within the VMH, a key brain glucose-sensing region, act in concert with islets to restore glucose homeostasis during acute hypoglycemia, and its effect on counterregulation may be mediated by changes in glutamate/glutamine cycling.

FIG. 1.

Effect of acute stimulation of VMH EphA5 receptors with ephrin5A-Fc on glucose counterregulation during hypoglycemia in rats. The components include a schematic representation of the experimental protocol (A); plasma glucose (B); mean plasma insulin at 30, 60, and 90 min (C); glucose infusion rate (D); plasma glucagon (E); plasma epinephrine (F); and plasma norepinephrine during the hypoglycemic clamp study (G) (n = 12 for ephrin-Fc and n = 12 for ephrinA5-Fc). Data are presented as means ± SEM. Statistical analysis used mixed-model ANOVA with Bonferroni post hoc test: *P < 0.05, *** P < 0.001 vs. control-Fc.

FIG. 2.

Long-term effect of chronic changes in VMH ephrinA5 expression on glucose counterregulation during hypoglycemic clamp study. The components include schematic representation of the experimental protocol (A); plasma glucose (B); mean plasma insulin at 30, 60, and 90 min (C); glucose infusion rate (D); plasma glucagon (E); plasma epinephrine (F); and plasma norepinephrine during the hypoglycemic clamp (G) (n = 8 for control AAV, n = 7 for ephrinA5 downregulation AAV, and n = 7 for ephrinA5 overexpression AAV). Data are presented as means ± SEM. Statistical analysis used mixed-model ANOVA with Bonferroni post hoc test: *P < 0.05, **P < 0.01, ***P < 0.001 vs. control AAV.

FIG. 3.

A: Representative data show expression level of ephrinA5 in the VMH in rats after ∼14 days after VMH delivery of AAV constructs as determined by Western blood analysis. Tubulin served as the loading control. B: Relative VMH expression of ephrinA5 after VMH administration of control AAV (n = 6; white bars), ephrinA5 knockdown (n = 6; black bar), and ephrinA5 overexpression (n = 5; gray bar). Data are presented as means ± SEM. Statistical analysis used one-way ANOVA: *P < 0.05, ***P < 0.001 vs. control AAV.

FIG. 4.

Effect of ephrinA5 knockdown and overexpression on the levels of glutamate and glutamine in VMH interstitial fluid during hypoglycemia in rats given control AAV (n = 6), ephrin5A knockdown AAV (n = 9), or ephrinA5 over-expression AAV (n = 9). Collections of dialysis fluid were obtained over 20 min intervals beginning at −60 to −40 min and ending at 80–100 min. Data are expressed as means ± SEM. Statistical analysis used mixed-model ANOVA with Bonferroni post hoc test: *P < 0.05, **P < 0.01, *** P < 0.001 vs. control AAV.