Role of dorsal vagal complex A2 noradrenergic neurons in hindbrain glucoprivic inhibition of the luteinizing hormone surge in the steroid-primed ovariectomized female rat: Effects of 5-thioglucose on A2 functional biomarker and AMPK activity

Abstract

Neuro-glucostasis is required for normal expression of the steroid positive-feedback - induced preovulatory pituitary luteinizing hormone (LH) surge, a critical element of female reproduction. Glucoprivic signals from the caudal hindbrain restrain this surge, but the cellular source of this stimulus is unclear. Norepinephrine (NE) exerts well-defined stimulatory effects on the reproductive neuroendocrine axis. Our studies show that medullary A2 noradrenergic neurons are both estrogen- and glucoprivic-sensitive. Here, we investigated the premise that the LH surge is inhibited by A2 cell reactivity to hindbrain glucopenia and diminished preoptic NE neurotransmission. Estradiol- and progesterone-primed ovariectomized (OVX) female rats were injected into the caudal fourth ventricle (CV4) with the glucose anti-metabolite, 5-thioglucose (5TG) or saline (SAL) prior to onset of the LH surge. Pretreatment by intra-CV4 delivery of the selective catecholamine neurotoxin, 6-OHDA, attenuated LH output, but prevented inhibition by 5TG. 5TG modified patterns of steroid feedback-associated Fos staining of A2, but not other medullary catecholamine cell groups. Intra-preoptic administration of the alpha1-adrenergic receptor agonist, methoxamine, elicited site-specific reversal of hindbrain glucoprivic suppression of gonadotropin-releasing hormone (GnRH) neuron Fos labeling and LH release. Western blotting of laser-microdissected A2 neurons revealed glucoprivic stimulation of Fos, but inhibition of the catecholamine synthetic enzyme, dopamine-β-hydroxylase; 5TG also diminished A2 estrogen receptor (ER)-α and progesterone receptor profiles, but augmented ER-β protein. Intriguingly, A2 AMPK activity was decreased in 5TG-treated rats, despite down-regulation of GLUT3 and no change in MCT2 protein expression. Rostral preoptic GnRH neurons also exhibited decreased AMPK activation simultaneous with apparent reduction of neuropeptide signaling to the pituitary. The present studies demonstrate that hindbrain glucoprivation inhibits the LH surge, in part, by reducing preoptic noradrenergic input, and furthermore implicate A2 neurons as a source of this altered signal. Results also suggest that AMPK sensor deactivation does not supersede the impact of pharmacological inhibition of glucose catabolism on A2 cell function nor afferent signaling of hindbrain glucopenia on GnRH neurons. Further studies are needed to determine if decreased AMPK activation in these cell populations reflect compensatory gain in positive energy balance and/or direct effects of estrogen on AMPK.

Hindbrain estrogen receptor-beta antagonism normalizes reproductive and counter-regulatory hormone secretion in hypoglycemic steroid-primed ovariectomized female rats

“…Alternatively, ERβ action upstream of AMPK during hypoglycemia to regulate AMPK reactivity to that condition would be consistent with outcomes showing PHTPP-mediated normalization of sensor activity in insulin-injected animals. A2 neurons provide inhibitory metabolic input to the GnRH-LH axis, as neurotoxin decimation of these cells abolishes hindbrain glucoprivic inhibition of the LH surge [Ibrahim and Briski, 2014]. PHTPP reversal of hypoglycemic suppression of LH implicates caudal hindbrain ERβ in metabolic restraint of the LH surge.…”

“…PHTPP reversal of hypoglycemic suppression of LH implicates caudal hindbrain ERβ in metabolic restraint of the LH surge. We presume that A2 neurons are a direct target for this ERβ action [Ibrahim and Briski, 2014], but cannot discount the possibility that this receptor may act through indirect mechanisms involving afferent input to A2 cells. Rat brain GnRH neurons that react to hindbrain metabolic signaling reside in the rPO [Briski and Sylvester, 1998].…”

“…Metabolic restraint of the LH surge involves diminished hindbrain NE signaling to the preoptic AVPV, as administration of the alpha-adrenergic receptor agonist methoxamine to that site reverses hindbrain glucoprivic suppression of GnRH neuron Fos immuno-labeling and pituitary LH release [Ibrahim and Briski, 2014]. Here, hypoglycemia elevated NE levels in the AVPV and other structures relevant to reproduction, e.g.…”

Role of estradiol in intrinsic hindbrain AMPK regulation of hypothalamic AMPK, metabolic neuropeptide, and norepinephrine activity and food intake in the female rat

“…sc-15318, Santa Cruz Biotechnol.) [Ibrahim and Briski, 2014; Ibrahim et al, 2015]. The housekeeping protein α-tubulin was probed with monoclonal antibodies (1:1,000; prod.…”

“…glucokinase, K ATP , and AMPK [Briski et al, 2009; Cherian and Briski, 2011; Ibrahim et al, 2013] suggests that these cells are substrates for estrogenic input to neural pathways that control energy homeostasis. Our studies show that estradiol controls A2 hindbrain and hypothalamic AMPK, hypothalamic metabolic neurotransmitter, and physiological and behavioral counter-regulatory responses to intra-caudal fourth ventricular administration of the AMP mimic 5-aminoimidazole-4-carboxamide-riboside (AICAR) in ovariectomized (OVX) female rats [Ibrahim et al, 2013; Alenazi et al, 2014; Ibrahim and Briski, 2014]. Outcomes of that work uniquely establish dependency of hindbrain-hypothalamic AMPK functional interaction on estrogen., as pAMPK expression was elevated in hypothalamic arcuate and parventricular nuclei, but diminished in the ventromedial hypothalamic nucleus in estradiol- versus oil-implanted OVX rats after hindbrain AMPK activation.…”