Karen P. Briski

Recurrent insulin-induced hypoglycemia causes site-specific patterns of habituation or amplification of CNS neuronal genomic activation

Antecedent hypoglycemia is a primary factor in hypoglycemia-associated autonomic failure, a pathophysiological condition characterized by impaired glucose counterregulatory function. Conventional therapeutic strategies involving administration of intermediate dosage-release formulations of insulin in the management of insulin-dependent diabetes mellitus result in frequent iatrogenic hypoglycemia. This study investigated the neuroanatomical location, direction, and magnitude of CNS neuronal genomic activation by singular versus repeated induction of hypoglycemic bouts of greater than 6 h duration achieved by administration of the intermediate-acting insulin, humulin neutral protamine Hagedorn (NPH). Adult male rats injected subcutaneously with Humulin NPH exhibited robust immunolabeling for the nuclear transcription factor, Fos, in discrete telencephalic, diencephalic, midbrain, and caudal hindbrain loci in a pattern that was not identical to that described for regular insulin. Administration of four doses of insulin on as many days significantly diminished or extinguished Fos immunostaining within the parvocellular hypothalamic paraventricular nucleus, lateral hypothalamic area, dorsomedial hypothalamic nucleus, thalamic paraventricular nucleus, nucleus tractus solitarius, and area postrema, but did not modify labeling of other metabolic loci. However, numbers of Fos-immunoreactivity-positive magnocellular neurons in the hypothalamic paraventricular and supraoptic nuclei were significantly increased after the second and fourth insulin doses, relative to the single-dose group. Concurrent observations of exacerbated hypoglycemia and modified patterns of glucoregulatory hormone secretion after serial injections of intermediate-acting insulin suggest that central mechanisms governing compensatory endocrine responses, specifically glucagon, become habituated to repetitive hypoglycemia of extended duration. Resultant alterations in CNS-islet and -adrenomedullary output and hypothalamic-pituitary-adrenal activity may reflect diminished neuronal activation within one or more of the brain loci characterized here by nonuniform transcriptional activation. The current studies provide a neuroanatomical foundation for further investigation of the neurochemical phenotypes and interconnectivity of functionally adaptive neurons, underlying cellular and molecular mechanisms of diminished or enhanced activation, as well as the impact of these modified cellular responses on glucose counterregulation during administration of intermediate-acting insulin.

Hypothalamic orexin-A-immunpositive neurons express Fos in response to central glucopenia.

Reports that glucose antimetabolite treatment elicits hyperphagia and hyperglycemia suggest that decreased oxidation of this energy substrate elicits compensatory responses that enhance cellular fuel availability. Neurons in the lateral hypothalamic area (LHA) synthesize the orectic neuropeptide, orexin-A (ORX-A). The present study evaluated the functional responsiveness of orexinergic neurons to glucopenia by investigating whether these cells express the genomic regulatory protein, Fos, in response to glucoprivation. Adult male rats were sacrificed 2 h after i.p. (400 mg/kg) or intracerebroventricular (i.c.v.; 100 μg) administration of the antimetabolite, 2-deoxy-d-glucose (2DG) or saline. Sections through the LHA, from the level of the paraventricular nucleus (PVN) to the posterior hypothalamic area (PHA), were processed by dual-label immunocytochemistry for Fos- and OXY-A-immunoreactivity (-ir). Although orexinergic neurons expressed negligible Fos-ir following vehicle administration, dual-labeled ORX-A neurons were observed in the LHA, as well as the dorsomedial hypothalamic nucleus (DMN) and PHA, in both drug-treated groups. Bilateral cell counts from representative levels of the LHA, DMN, and PHA showed that in each structure, a greater proportion of ORX-A neurons were immunostained for Fos in response to systemic than following i.c.v. treatment with 2DG. These results provide evidence for the transcriptional activation of hypothalamic ORX-A neurons by diminished glucose availability, data that suggest that these cells may function within central pathways that govern adaptive responses to deficits of this substrate fuel. The findings also support the view that a proportion of this phenotypic population is responsive to glucoprivic stimuli of central origin.

Endogenous opiate involvement in acute and chronic stress-induced changes in plasma LH concentrations in the male rat

The present study was carried out to examine the possible role of the endogenous opioid peptides ( EOP s) on the pituitary luteinizing hormone (LH) response to both acute and chronic stress and to food deprivation. Thirty minutes after acute (2 min.) exposure to ether, plasma LH levels were elevated compared to controls; morphine (MOR) treatment prior to stress prevented this response. More prolonged etherization (15 minutes) significantly depressed circulating LH, whereas naltrexone ( NALT ), a specific opiate antagonist, reversed this decline. Immobilization for 8 hours resulted in a significant initial increase in LH release, followed by a decline toward baseline levels. Naltrexone treatment increased the magnitude of the acute LH rise, and attenuated the subsequent decrease in plasma LH. The effect of chronic stress on circulating LH was also examined. Plasma LH levels were depressed for 3 consecutive days following subcutaneous gauze pad implantation, whereas 3 daily NALT injections returned LH to control levels. Complete food deprivation for 5 days also resulted in a significant decline in circulating LH. Injection of NALT 3 times daily reversed this decline on days 2, 3 and 4 of treatment. These results support the hypothesis of a mediatory role for the EOP s in the effect of both chronic stress and food deprivation on LH release in the rat.

Effects of Estradiol on Glucoprivic Transactivation of Catecholaminergic Neurons in the Female Rat Caudal Brainstem

Hyperphagic and hypothalamic neuroendocrine responses to acute glucose deprivation are modified by the ovarian steroid estradiol (E). Observations of genomic activation of catecholaminergic (CA) neurons in the hindbrain lateral reticular nucleus, nucleus of the solitary tract, and area postrema (AP) by glucopenia support their potential function in pathways mediating regulatory effects of this metabolic challenge within the brain. Expression of E receptors by these cells suggests that their activity may be sensitive to steroid modulation during glucopenia. The present studies investigated the role of E on transcriptional activation of caudal brainstem CA neurons by the glucose antimetabolite, 2-deoxy-D-glucose (2DG). Ovariectomized rats were implanted with s.c. Silastic capsules containing E (30 or 250 µg/ml) or sesame oil, and injected i.p. 7 days later with 400 mg 2DG/kg or saline. Tyrosine hydroxylase (TH)-immunoreactive (-ir) neurons in the C1/A1, C2, C3, A2, A5, and A6 cell groups and AP were colabeled for Fos following antimetabolite administration, whereas vehicle injection resulted in negligible nuclear staining of these cells. With the exception of A2, A6, and AP cells, mean numbers of Fos- and TH-/Fos-ir-positive neurons in these brain sites did not differ between E- and sesame oil-implanted groups. Numbers of TH-positive A2 and A6 neurons that expressed Fos in response to 2DG were significantly greater in rats implanted with the high E dose vs. either the low steroid dose or sesame oil. These results show that the magnitude of cellular Fos labeling within discrete hindbrain CA neuron populations varies in accordance with circulating E levels. These findings suggest that E may exert potential modulatory effects on glucoprivic activation of the Fos stimulus/transcription cascade and consequent compensatory genomic responses within specific areas of the female rat caudal brainstem.

The Antiglucocorticoid, RU486, Attenuates Stress-Induced Decreases in Plasma-Luteinizing Hormone Concentrations in Male Rats

The present studies investigated the role of glucocorticoid receptors (GR) in the inhibitory effects of acute and chronic immobilization stress on pituitary luteinizing hormone (LH) release. Systemic administration of the GR antagonist, RU486, significantly attenuated the acute decline in circulating LH observed in intact male rats immobilized by encasement within paper cocoons. Whereas vehicle-injected controls exhibited a significant reduction in plasma LH between +1 and +5 h of stress, animals given subcutaneous (sc) injections of 2.5 mg RU846/kg did not exhibit a reduction in circulating LH until 4 h after initiation of stress. Plasma LH levels in the GR-treated group were significantly elevated compared to the vehicle controls between +1 and +3 h of stress. Repetitive exposure to the same stress stimulus 24 and 48 h later resulted in decreased plasma LH levels in the vehicle-treated rats, but not in the animals injected sc with RU486. Other studies showed that intracerebroventricular (icv) administration of RU486 (10.0 micrograms/rat) also blunted the inhibitory effects of acute and chronic immobilization stress on pituitary LH release. In experiments designed to evaluate whether activation of central GR can influence the magnitude and/or temporal characteristics of the LH secretory response to acute inhibitory stress, it was observed that animals pretreated by icv injection of the GR agonist, RU362, exhibited a greater reduction in plasma LH levels during the first hour of stress, as compared to rats pretreated with vehicle alone. In summary, the present findings that pharmacologic antagonism of GR attenuates acute and chronic stress-induced inhibition of circulating LH support a role for glucocorticoids in mechanisms underlying suppression of pituitary LH release during stress.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Inhibition of Pituitary LH Release in the Male Rat by the Glucocorticoid Agonist Decadron Phosphate

The present study was carried out to investigate the effects of acute administration of the soluble glucocorticoid receptor agonist, decadron phosphate (DEC, dexamethasone sodium phosphate), on pituitary luteinizing hormone (LH) release in intact adult male rats. DEC was administered intravenously to individual groups of animals at a dose of either 0.05 or 0.5 mg DEC/kg, and the magnitude and time course of drug-induced alterations in plasma LH concentrations were evaluated. DEC was observed to elicit a dose-proportionate decrease in plasma LH within hours after systemic injection. Both doses of DEC significantly reduced the magnitude of exogenous LH-releasing hormone (RH; 10 ng/100 g b.w.)-induced increases in plasma LH at time points coincident with drug-induced decreases in basal LH release. During in vitro perifusion of isolated anterior pituitary tissue fragments, the administration of DEC, via the perifusate, at a concentration of 1.0 microgram/ml had no impact upon basal LH release, but did effectively diminish LHRH-stimulated hormone release from perifused tissues. Intracerebroventricular injection of DEC also resulted in a decline in circulating LH. While intracerebroventricular (ICV) administration of 10 ng of DEC had no impact upon circulating LH, higher doses of 100 ng and 1.0 micrograms significantly depressed circulating LH levels in a dose-related manner. Both of these ICV doses were also found to diminish pituitary responsiveness to LHRH. Lastly, DEC was intravenously administered at a dose of 0.5 mg/kg to groups of orchidectomized and orchidectomized, testosterone (T)-treated male rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Habituation of insulin-induced hypoglycemic transcription activation of lateral hypothalamic orexin-A-containing neurons to recurring exposure

A CNS component of glucose counterregulatory collapse is supported by evidence for nonuniform genomic responsiveness of neurons in characterized central autonomic loci during recurring insulin-induced hypoglycemia (IIH). We have reported that exacerbated hypoglycemia and attenuated patterns of glucagon and epinephrine secretion in rats treated by daily sc injection of the intermediate-acting insulin formulation, Humulin NPH (NPH), are correlated with diminished immunodemonstrability of the AP-1 transcription factor, Fos, in several components of the central metabolic regulatory circuitry, including the lateral hypothalamic area (LHA). Neurons that synthesize the potent orexigenic peptide neurotransmitter, orexin-A, are restricted to the LHA and adjacent hypothalamic loci, and project throughout the central neuroaxis to structures that govern autonomic and behavioral motor output. Dual-label immunocytochemical and real-time RT-PCR techniques were utilized here to evaluate the functional status of this LHA phenotype during a single versus repetitive exposure to prolonged IIH. Tissue sections were collected at predetermined rostrocaudal levels of the LHA after acute or repeated NPH administration, and processed for nuclear Fos- and cytoplasmic orexin-A-immunoreactivity (-ir). Mean numbers of orexin-A-ir neurons were not different between treatment groups. Colabeling of these cells for Fos was increased relative to controls following a single injection of insulin, but numbers of Fos-ir-positive orexin-A neurons were significantly reduced after treatment with four versus one dose of insulin. Prepro-orexin mRNA levels in microdissected LHA tissue were upregulated during acute hypoglycemia, but were returned to control levels by repeated IIH. These data corroborate previous evidence that IIH is an activational stimulus for orexin-A-synthesizing neurons in the LHA, and further demonstrate that induction of cfos and prepro-orexin gene expression by acute hypoglycemia is attenuated by precedent exposure to hypoglycemia. The current results thus provide unique evidence for neurotransmitter-specific habituation of LHA neuronal sensitivity to IIH.

Stimulatory vs. inhibitory effects of acute stress on plasma LH : Differential effects of pretreatment with dexamethasone or the steroid receptor antagonist, RU 486

Acute stress elicits variable patterns of pituitary LH release in intact rats. While the pituitary-adrenal axis is capable of discrimination between stressors of graded intensity, the effects of variable glucocorticoid output on the direction and magnitude of LH release during stress remain unclear. The present studies compared the effects of a psychological stress and two different physical stressors on peripheral corticosterone (CORT) and LH concentrations. Plasma CORT levels were elevated during each stress, but this increase in hormone release was significantly greater in response to physical stress. This differential CORT sensitivity to psychological vs. physical stress was correlated with divergent patterns of pituitary LH release; novel environment (NE) stress resulted in a transient increase in plasma LH, whereas both physical stressors ultimately caused a reduction in circulating hormone levels. Pretreatment with the glucocorticoid receptor (GR) antagonist, RU 486, reversed physical stress-induced decreases in LH release, but did not further facilitate circulating LH during NE stress. Other studies showed that stimulation of GRs prior to stress with the potent ligand, dexamethasone (DEX), blunted the stimulatory effects of NE stress on circulating LH. Additional experiments investigated whether prolonged exposure to elevated glucocorticoid levels elicits adaptive responses from the hypothalamic-pituitary LH axis to acute stress. Chronic DEX administration resulted in a significant attenuation of the inhibitory LH response to acute immobilization, but had no impact upon the facilatory effects of NE stress on LH release. The current studies confirm previous reports of variation in the magnitude of CORT secretion elicited by stressors of different intensity, and provide new evidence that inhibitory patterns of pituitary LH release may be correlated with a high degree of activation of the pituitary-adrenal axis. Attenuation of the facilatory effects of novel environment stress on LH release by pretreatment with the GR agonist, DEX, suggests that GR-induced inhibition of LH requires occupation of GRs beyond that which occurs during this mild stressor. The present findings that stress-induced decreases in plasma LH are blunted by chronic glucocorticoid exposure support a role for glucocorticoid-dependent mechanisms in adaptation of GR-mediated inhibitory responses to stress.

Relation of gonadal hormones to differential LH response to naloxone in prepubertal male and female rats

Naloxone (NAL) has been shown to induce LH release in female but not in male rats 10-25 days of age. The purpose of this study was to examine the role of neonatal gonadal hormones on NAL-induced LH release in male and female rats 15, 25, and 35 days of age. On each of these days rats received a s.c. injection of either NAL (5 mg/kg) or physiological saline, and blood was collected 30 min later by decapitation. At 15 days of age, NAL induced LH release in intact and ovariectomized (OVX) female rats, and in male rats castrated (CAST) on the 1st day of life (neonate CAST males). Injection of 10 micrograms estradiol benzoate (EB) 24 h prior to NAL administration blocked NAL-induced LH release in these rats. NAL had no effect on LH release in 15- or 25-day-old intact and CAST male rats or in female rats given 2 mg testosterone propionate at 3 days of age (androgenized female rats). At 35 days of age, NAL induced LH release in intact, OVX, and OVX-EB treated female rats, and in neonate CAST and neonate CAST-EB treated male rats. NAL had no effect on serum LH levels in androgenized female rats. NAL induced LH release in intact and CAST 35-day-old male rats, but pretreatment with estrogen prevented NAL from eliciting LH release. These results indicate that neonatal exposure to androgen is responsible for the sex difference in the LH response to NAL observed in prepubertal male and female rats before 30 days of age.(ABSTRACT TRUNCATED AT 250 WORDS)

Site-specific induction of Fos immunoreactivity in preoptic and hypothalamic NADPH-positive neurons during glucoprivation.

Neuronal nitric oxide synthase, e.g. NADPH diaphorase (NADPH-d), catalyzes formation of the free radical, nitric oxide (NO), and occurs within brain structures that have functional significance for energy fuel homeostasis. The following studies examined whether populations of NADPH-d-positive neurons in the hypothalamus and nearby preoptic area express immunoreactivity for the nuclear transcription factor, Fos, in response to glucose substrate imbalance. Eight days after bilateral ovariectomy (OVX) and subcutaneous implantation of silastic capsules containing 30 µg estradiol benzoate/ml, female rats were injected i.p. with the glucose antimetabolite, 2-deoxy-D-glucose (2DG; 400 mg/kg), or the vehicle, saline. The animals were sacrificed by transcardial perfusion 2 h after these treatments. Sections at 150-µm intervals throughout preoptic area and anterior and tuberal regions of the hypothalamus were processed for dual cytoplasmic NADPH-d enzyme activity and nuclear Fos-immunoreactivity (-ir). The glucose antimetabolite elicited expression of nuclear Fos-ir by NADPH-d-positive neurons in several neural structures, including the medial preoptic area, median preoptic nucleus, anterior commissural, periventricular magnocellular supraoptic nucleus, paraventricular nucleus, and medial part of the bed nucleus of the stria terminalis. In contrast, the extensive populations of NADPH-d-positive neurons in the ventromedial hypothalamic nucleus and lateral hypothalamic area showed very little immunolabeling for Fos in response to glucoprivation. This demonstration of nuclear immunoreactivity for Fos suggests that cellular glucopenia elicits the transcriptional activation, via AP-1 regulatory sites, of multiple populations of hypothalamic neurons characterized by the functional capacity to generate NO, and thus that this gaseous neurotransmitter may fulfill a role(s) in central neural mechanisms governing regulation of compensatory motor responses to metabolic imbalance.