Glucose prevents the fall in ventromedial hypothalamic GABA that is required for full activation of glucose counterregulatory responses during hypoglycemia

Abstract

Local delivery of glucose into a critical glucose-sensing region within the brain, the ventromedial hypothalamus (VMH), can suppress glucose counterregulatory responses to systemic hypoglycemia. Here, we investigated whether this suppression was accomplished through changes in GABA output in the VMH. Sprague-Dawley rats had catheters and guide cannulas implanted. Eight to ten days later, microdialysis-microinjection probes were inserted into the VMH, and they were dialyzed with varying concentrations of glucose from 0 to 100 mM. Two groups of rats were microdialyzed with 100 mM glucose and microinjected with either the K(ATP) channel opener diazoxide or a GABA(A) receptor antagonist. These animals were then subjected to a hyperinsulinemic-hypoglycemic glucose clamp. As expected, perfusion of glucose into the VMH suppressed the counterregulatory responses. Extracellular VMH GABA levels positively correlated with the concentration of glucose in the perfusate. In turn, extracellular GABA concentrations in the VMH were inversely related to the degree of counterregulatory hormone release. Of note, microinjection of either diazoxide or the GABA(A) receptor antagonist reversed the suppressive effects of VMH glucose delivery on counterregulatory responses. Some GABAergic neurons in the VMH respond to changes in local glucose concentration. Glucose in the VMH dose-dependently stimulates GABA release, and this in turn dose-dependently suppresses the glucagon and epinephrine responses to hypoglycemia. These data suggest that during hypoglycemia a decrease in glucose concentration within the VMH may provide an important signal that rapidly inactivates VMH GABAergic neurons, reducing inhibitory GABAergic tone, which in turn enhances the counterregulatory responses to hypoglycemia.

Fig. 1.

Simplified schema showing the potential mechanism by which glucose stimulates the release of GABA. Glucose enters GABAergic neurons in the ventromedial hypothalamus (VMH) through glucose transporters and is metabolized through glycolytic and oxidative pathways to generate ATP. An increase in ATP production then closes off ATP-sensitive potassium (K_ATP) channels, depolarizes the neuron, and allows for GABA release. Diazoxide (DZ) will open the K_ATP channel, hyperpolarize the neuron, and prevent GABA release. Bicuculline methiodide (BIC) acts on postsynaptic GABA_A receptors to block GABAergic inhibition.

Fig. 2.

Plasma glucose concentrations during baseline sampling period (t = −45–0 min) and during hypoglycemic clamp. Data are represented as means ± SE.

Fig. 3.

Average glucose infusion rates during the final 90 min of the hypoglycemic clamping period. Data are represented as means ± SE. *P < 0.05 vs. controls.

Fig. 4.

Peak plasma glucagon responses obtained during the hypoglycemic clamp. Data are shown as means ± SE. *P < 0.05 vs. controls; †P < 0.01 vs. controls; ¶P < 0.001 vs. 100 mM d-glucose.

Fig. 5.

Peak plasma epinephrine responses obtained during the hypoglycemic clamp. Data are shown as means ± SE. *P < 0.05 vs. controls; †P < 0.02 vs. controls; ¶P < 0.001 vs. 100 mM d-glucose.

Fig. 6.

Average extracellular GABA concentrations in the VMH at baseline (open bars) and during the 90-min hypoglycemic clamping period (filled bars), shown as means ± SE. *P < 0.03 vs. baseline; †P < 0.05 vs. baseline; ¶P < 0.001 vs. 100 mM d-glucose.

Fig. 7.

Representative hypothalamic sections showing immunofluorescent labeling for the type 1 sulfonylurea receptor (SUR1) subunit of the K_ATP channel in red (A) and in green fluorescent protein (GFP), which is expressed under the control of the GAD₆₅ promoter in green (B). ×100 magnification of the highlighted field within the VMH showing colabeling of the SUR1 subunit on GAD₆₅-GFP-positive neurons, as indicated by arrows (C).