Type II glucocorticoid receptor involvement in habituated activation of lateral hypothalamic area orexin-A-immunopositive neurons during recurring insulin-induced hypoglycemia

Neurons that synthesize the potent orexigenic neuropeptide, orexin-A (ORX-A) are confined to the lateral hypothalamic area (LHA) and adjacent structures, and project throughout the central neuroaxis to structures that govern central nervous system responses to energy imbalance. Insulin-induced hypoglycemia (IIH) upregulates prepro-orexin mRNA and Fos immunostaining of LHA ORX-A neurons. These neurons apparently become desensitized to this metabolic challenge, since both responses are diminished by recurrent insulin-induced hypoglycemia (RIIH). Recent studies implicate central type II glucocorticoid receptors (GR) in RIIH-associated glucose counterregulatory collapse and decline in Fos labeling of central metabolic loci, including the LHA. The present studies evaluated the role of GR in patterns of LHA ORX-A neuronal transcriptional activation during RIIH. Groups of adult male rats were injected subcutaneously with one or four doses of the intermediate-acting insulin, Humulin NPH, on as many days, or with diluent alone. Rats injected with four doses of insulin were pretreated by intracerebroventricular (icv) administration of the selective GR antagonist, CP-472555, or the vehicle, propylene glycol, prior to insulin administration on days 1–3. All animals were sacrificed by transcardial perfusion 2 h after injections on day 4. Processing of LHA tissue sections for dual-immunoperoxidase staining of ORX-A- and Fos-immunoreactivity (-ir) showed that colabeling of ORX-A neurons for Fos was increased by a single injection of NPH, whereas this genomic response was diminished by RIIH. Icv administration of CP-472555 during antecedent hypoglycemia prevented RIIH-associated reductions in Fos expression by these neurons. Antagonist treatment of diluent-injected controls did not alter mean numbers of ORX-A- plus Fos-ir neurons. Total numbers of ORX-A-immunopositive neurons were not different among treatment groups. These data demonstrate that precedent central GR blockade prevents adaptation of LHA ORX-A neuronal reactivity to RIIH. These results provide unique pharmacological evidence that hypoglycemic hypercorticosteronemia diminishes activation of this neurotransmitter phenotype in this critical metabolic structure to subsequent hypoglycemia via central GR-dependent mechanisms.