| Optimal clinical management of diabetes mellitus requires tight control of blood glucose levels, but iatrogenic hypoglycemia is an inescapable by-product of rigorous insulin treatment. Cellular energy is monitored in both the central nervous system (CNS) and periphery, including the hindbrain dorsal vagal complex (DVC). The counterregulatory responses to precedent hypoglycemia vary according to gender. In light of these evidences, the present studies investigated the hypothesis that recurring NPH-induced hypoglycemia may alter basal and/or hypoglycemic patterns of metabolic transducer, substrate transporter, and metabolic-sensitive catecholamine synthetic enzyme. We also investigated whether adaptation of these neurons, if any, are gender-specific and if so, to delineate the role of estrogen in these responses. Separate sets of male animals and ovariectomized female animals with or without estrogen replacement were utilized for addressing these hypotheses. To evaluate the gene profiles, we performed multi-transcriptional profiling of individual neurochemically-characterized DVC A2 neurons harvested, after single versus serial neutral protamine hagedorn (NPH) dosing, using a combinatory approach involving in situ immunocytochemistry, laser-catapult microdissection, and single cell quantitative real-time RT-PCR. We also investigated whether the mRNA and protein profiles deviate during similar conditions by analyzing levels of expression of those proteins in A2 cells. To achieve this aim, we developed a novel immunoblot technique with high sensitivity to detect protein expression in small-size pools of A2 neurons. We also evaluated the levels of phosphorylated adenosine 5'-monophosphate-activated protein kinase (pAMPK), a sensor of cellular energy which reflects the metabolic status of the cells, in A2 neurons under basal and hypoglycemic conditions. Results from our studies provide novel gender specific adaptation of metabolically sensitive genes to intermediate insulin induced hypoglycemia. The findings support the view that A2 neurons in males acclimate to recurrent insulin induced hypoglycemia, by adopting a new metabolic steady state, one that is characterized by energy deficiency and insensitivity to hypoglycemia. In contrast, A2 neurons in females maintain a metabolic balance, by upregulating substrate transporters, in response to chronic metabolic deficiency and estrogen is, in part, responsible for the maintenance of this metabolic equilibrium, may be by prioritizing the cellular requirements. |
