Increased GABAergic tone in the ventromedial hypothalamus contributes to suppression of counterregulatory responses after antecedent hypoglycemia

Abstract

Objective: We have previously demonstrated that modulation of gamma-aminobutyric acid (GABA) inhibitory tone in the ventromedial hypothalamus (VMH), an important glucose-sensing region in the brain, modulates the magnitude of glucagon and sympathoadrenal responses to hypoglycemia. In the current study, we examined whether increased VMH GABAergic tone may contribute to suppression of counterregulatory responses after recurrent hypoglycemia.

Research design and methods: To test this hypothesis, we quantified expression of the GABA synthetic enzyme, glutamic acid decarboxylase (GAD), in the VMH of control and recurrently hypoglycemic rats. Subsequently, we used microdialysis and microinjection techniques to assess changes in VMH GABA levels and the effects of GABA(A) receptor blockade on counterregulatory responses to a standardized hypoglycemic stimulus.

Results: Quantitative RT-PCR and immunoblots in recurrently hypoglycemic animals revealed that GAD(65) mRNA and protein were increased 33 and 580%, respectively. Basal VMH GABA concentrations were more than threefold higher in recurrently hypoglycemic animals. Furthermore, whereas VMH GABA levels decreased in both control and recurrently hypoglycemic animals with the onset of hypoglycemia, the fall was not significant in recurrently hypoglycemic rats. During hypoglycemia, recurrently hypoglycemic rats exhibited a 49-63% reduction in glucagon and epinephrine release. These changes were reversed by delivery of a GABA(A) receptor antagonist to the VMH.

Conclusions: Our data suggest that recurrent hypoglycemia increases GABAergic inhibitory tone in the VMH and that this, in turn, suppresses glucagon and sympathoadrenal responses to subsequent bouts of acute hypoglycemia. Thus, hypoglycemia-associated autonomic failure may be due in part to a relative excess of the inhibitory neurotransmitter, GABA, within the VMH.

FIG. 1

Quantitative RT-PCR assessment of GAD₆₅, GAD₆₇, and GABA_A receptor (GABA_A-R) mRNA expression in the VMH of recurrently hypoglycemic animals (n = 6) compared with recurrent saline controls (n = 6) and expressed as fold change versus control levels. *P < 0.01 vs. controls; †P < 0.01 vs. controls.

FIG. 2

Representative images and densitometric analyses of immunoblots for GAD₆₅ (left panels) and GAD₆₇ (right panels) protein in the VMH (top) and cortex (bottom) of control (n = 4–7) and recurrently hypoglycemic (n = 4–7) animals. Data are represented as relative optical density expressed as a ratio over β-actin levels. Results are presented as means ± SE. *P < 0.001 vs. controls.

FIG. 3

Average extracellular GABA concentrations in microdialysate samples collected from control (n = 17) and recurrently hypoglycemic (RH; n = 17) animals during the baseline period (□) and during the 90-min hypoglycemic clamping period (■). *P < 0.02 vs. control basal. #P < 0.01 vs. control basal. §P < 0.01 vs. control hypoglycemia. †P < 0.04 vs. control basal. P = 0.36 for RH basal vs. RH hypoglycemia. Data are presented as means ± SE.

FIG. 4

Plasma glucose concentrations of normal saline controls (Control; n = 8), recurrently hypoglycemic animals (RH; n = 8), and recurrently hypoglycemic animals receiving the GABA_A receptor antagonist bicuculline methiodide (RH + BMI; n = 6) during the hyperinsulinemic-hypoglycemic glucose clamp.

FIG. 5

Average plasma insulin (A) concentrations and glucose infusion rates (B) during the final 90 min of the hypoglycemic clamp in normal saline controls (n = 8), recurrently hypoglycemic animals (RH; n = 8), and recurrently hypoglycemic animals receiving the GABA_A receptor antagonist bicuculline methiodide (RH + BMI; n = 6). Results are presented as means ± SE. *P < 0.05 vs. controls.

FIG. 6

Plasma glucagon (A) and epinephrine (B) responses during the hypoglycemic clamp in normal saline controls (n = 8), recurrently hypoglycemic animals (RH; n = 8), and recurrently hypoglycemic animals receiving the GABA_A receptor antagonist bicuculline methiodide (RH + BMI; n = 6). Results are presented as means ± SE. *P < 0.03 vs. controls. †P < 0.001 vs. controls.

FIG. 7

Peak plasma corticosterone concentrations during the hypoglycemic clamp in normal saline controls (n = 8), recurrently hypoglycemic animals (RH; n = 8), and recurrently hypoglycemic animals receiving the GABA_A receptor antagonist bicuculline methiodide (RH + BMI; n = 6). Results are presented as means ± SE.