Effects of Estradiol on Glycemic and CNS Neuronal Activational Responses to Recurrent Insulin-Induced Hypoglycemia in the Ovariectomized Female Rat

Abstract

Recurrent insulin-induced hypoglycemia (RIIH) results in glucose counterregulatory dysfunction in men and male rodents. Intensified hypoglycemia in the latter coincides with diminished neuronal Fos expression in central metabolic regulatory structures, evidence that supports habituation of CNS-mediated compensatory motor outflow during re-exposure to this metabolic stress. In light of the evidence for counterregulatory resistance to precedent hypoglycemia in women, we utilized estradiol-treated ovariectomized (OVX) female rats to examine the hypothesis that this hormone regulates neural adaptability to recurring hypoglycemia. Groups of OVX rats were implanted with subcutaneous silastic capsules containing estradiol benzoate (E) or oil alone, and injected subcutaneously with one or four doses of the intermediate-acting insulin, Humulin NPH, one dose daily, or with diluent alone. Blood glucose levels were not altered by RIIH in E-implanted OVX animals, but were significantly decreased after four versus one insulin injection in the OVX+oil group. Mean numbers of Fos-immunoreactive (ir) neurons in the paraventricular nucleus hypothalamus (PVH), dorsomedial nucleus hypothalamus (DMH), and lateral hypothalamic area (LHA) were higher in both E- versus oil-implanted OVX rats injected with diluent only. Acute hypoglycemia significantly increased mean counts of Fos-ir-positive neurons in the PVH, DMH, and LHA, as well as the nucleus of the solitary tract (NTS) and area postrema (AP) in E- and oil-treated animals to an equivalent extent. OVX+E rats exhibited comparable numbers of Fos-positive neurons in the PVH, DMH, and LHA after one versus four insulin injections, whereas the numbers of labeled neurons in NTS and AP were increased or decreased, respectively, by RIIH. Oil-implanted OVX rats showed significantly diminished numbers of Fos-ir-positive neurons in each neural structure after repeated hypoglycemia. The present data demonstrate that estradiol sustains or enhances neuronal reactivity to recurring hypoglycemia in central metabolic structures, whereas hypoglycemic patterns of Fos expression in each site become habituated during RIIH in the absence of this steroid. The brain sites characterized here by estrogen-dependent maintenance of neuronal genomic reactivity to this substrate fuel imbalance may contain direct and/or indirect cellular targets for hormonal actions that prevent adaptation of CNS-controlled motor responses to this metabolic stress.

Impact of recurrent hypoglycemic stress on hindbrain A2 nerve cell energy metabolism and catecholamine biosynthesis: modulation by estradiol

“…This study did not affirm our prediction of parallelism of adaptation of A2 nerve cell DβH protein expression and DVC Fos immunolabeling (Nedungadi et al 2006) in both estradiol-and oil-implanted OVX rats. Rather, evidence for divergence of these two parameters, independent of estradiol status, detracts from our working assumption that hypoglycemia-induced DVC Fos immunoreactivity is a reliable indicator of metabolo-sensory neuron signaling.…”

“…Estradiol delivery to the ovariectomized (OVX) female rat caudal hindbrain sustains uniformity of glycemic responses to repeated insulin dosing while vehicle controls exhibit delayed recovery from recurring hypoglycemia (Nedungadi and Briski 2012). Moreover, estradiol prevents habituation of hypoglycemia-associated nerve cell Fos immunostaining in the DVC (Nedungadi et al 2006). Estradiol may thus prevent cellular energy state adjustments over serial hypoglycemic bouts or, alternatively, may mask acquired cellular energy instability by promoting signals of energy balance.…”

Effects of Estradiol on Acute and Recurrent Insulin-Induced Hypoglycemia-Associated Patterns of Arcuate Neuropeptide Y, Proopiomelanocortin, and Cocaine- and Amphetamine-Related Transcript Gene Expression in the Ovariectomized Rat

“…Beginning on day 1, the animals in each group were injected with an intermediate-release formulation of insulin or diluent according to the following schedule: (1) subcutaneous injection of diluent on days 1–4 (n = 6); (2) subcutaneous injection of diluent on days 1–3, followed by 12.5 U/kg Humulin NPH (NPH) on day 4 (n = 6); (3) subcutaneous injection of NPH on days 1–3, followed by diluent on day 4, or (4) subcutaneous injection of NPH on days 1–4 (n = 6). We have reported [24] that this dose of NPH decreases the blood glucose levels from approximately 120 to 40 mg/dl in both EB- and oil-implanted OVX rats and that glycemic and central neuronal activational responses to one versus repeated injections were equivalent in the presence of estrogen, whereas hypoglycemia was amplified and the patterns of CNS Fos immunolabeling were diminished in the absence of this hormone.…”

Sex Differences in Acclimation of Hypothalamic Arcuate Glucokinase Gene and Protein Expression to Repeated Intermediate Insulin Administration

“…Recent evidence that RIIH attenuates genomic reactivity of neurons located in key autonomic metabolic structures in the male but not female rat brain is consistent with a central neural component of this gender-specific response desensitization [7, 8]. We have developed an experimental model for RIIH based upon serial subcutaneous (sc) injection of the therapeutic intermediate-release insulin, neutral protamine Hagedorn (NPH), in order to replicate route of delivery, frequency of administration, and duration of action in the clinical setting.…”

“…The present studies utilized a model for recurrent insulin-induced hypoglycemia established and reported by our laboratory, which is characterized by exacerbated hypoglycemia, impaired glucagon and adrenomedullary catecholamine secretion, and attenuated CNS neuronal transcriptional activation in male rats [7]. In contrast, we have reported that female rats exposed to RIIH exhibit estrogen-dependent uniformity of glycemic and Fos protein responses to acute and recurring IIH [8]. For quantitative real-time RT-PCR analyses, animals of both genders were injected sc with the intermediate-release insulin formulation, humulin NPH (12.5 U/kg b.w.…”