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

Abstract

Hindbrain dorsal vagal complex A2 noradrenergic signaling represses the pre-ovulatory luteinizing hormone (LH) surge in response to energy deficiency. Insulin-induced hypoglycemia augments A2 neuron adenosine 5′-monophosphate-activated protein kinase (AMPK) activity and estrogen receptor-beta (ERβ) expression, coincident with LH surge suppression. We hypothesized that ERβ is critical for hypoglycemia-associated patterns of LH secretion and norepinephrine (NE) activity in key reproduction-relevant forebrain structures. The neural mechanisms responsible for tight coupling of systemic energy balance and procreation remain unclear; here, we investigated whether ERβ-dependent hindbrain signals also control glucose counter-regulatory responses to hypoglycemia. Gonadal steroid-primed ovariectomized female rats were pretreated by caudal fourth ventricular administration of the ERβ antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP) or vehicle at LH surge onset before insulin injection. Western blot analysis of laser-microdissected A2 neurons revealed hypoglycemic augmentation of 5′-monophosphate-activated protein kinase activity and dopamine-β-hydroxylase protein expression; the latter response was attenuated by PHTPP pretreatment. PHTPP regularized LH release, but not preoptic GnRH-I precursor protein expression in insulin-injected rats, and reversed hypoglycemic stimulation of glucagon and corticosterone secretion. Hypoglycemia caused PHTPP-reversible changes in NE and prepro-kisspeptin protein content in the hypothalamic arcuate (ARH), but not anteroventral periventricular nucleus. Results provide novel evidence for ERβ-dependent caudal hindbrain regulation of LH and counter-regulatory hormone secretion during hypoglycemia. That inhibition of LH likely involves mechanisms at the axon terminal that impede GnRH neurotransmission. Data also show that caudal hindbrain ERβ exerts site-specific control of NE activity in forebrain projection sites during hypoglycemia, including the ARH where prepro-kisspeptin may be a target of this signaling.

Sex differences in forebrain estrogen receptor regulation of hypoglycemic patterns of counter-regulatory hormone secretion and ventromedial hypothalamic nucleus glucoregulatory neurotransmitter and astrocyte glycogen metabolic enzyme expression

“…This steroid replacement regimen yields approximate plasma E concentrations of 22 pg/ml [Briski et al, 2001], replicating circulating hormone levels characteristic of metestrus in 4-day cycling animals [Butcher et al, 1974]. At 08.45 hr on day 10, male rats were divided into four treatment groups, and injected to the LV with the vehicle dimethyl sulfoxide (V) (groups 1 and 2; n=6/group), the ERα antagonist MPP (10 µM/200 nL [Briski and Shrestha, 2016]; Tocris/Bio-Techne Corp., Minneapolis, MN) (group 3; n=6), or the ERβ antagonist PHTPP (10 µM/200 nL [Briski and Shrestha, 2016]; Tocris) (group 4; n=6) [Table 1]. At 9:00 hr on day 10, animals in group 1 were injected sc with sterile insulin diluent (V; Eli Lilly & Co., Indianapolis, IN); at the same time, groups 2–4 were treated by injection of neutral protamine Hagedorn insulin (INS; 10.0 U/kg bw ; Butler Schein Animal Health, Dublin, OH).…”

Hindbrain metabolic deficiency regulates ventromedial hypothalamic nucleus glycogen metabolism and glucose-regulatory signaling

“…On study day 1, animals were anesthetized with ketamine/xylazine (0.1mL/100 g bw ip, 90 mg ketamine: 10 mg xylazine/mL; Butler Schein Inc., Melville, NY), and implanted with a 26-gauge stainless steel cannula (prod no. C315G/ SPC; Plastics One, Inc., Roanoke, VA) aimed at the caudal fourth ventricle (CV4) (Briski and Shrestha, 2016). After surgery, rats were injected subcutaneously (sc) with ketoprofen (1 mg/kg bw) and intramuscularly with enrofloxacin (10 mg/0.1 mL), treated by topical application of 0.25% bupivacaine to closed incisions, then transferred to individual cages.…”

“…no. sc-15318; Santa Cruz Biotechnology, Santa Cruz, CA), as described (Ibrahim and Briski, 2014;Briski and Shrestha, 2016;Alenazi et al, 2016). Protein molecular weight markers were included in each Western blot analysis.…”

Hindbrain 5′-Adenosine Monophosphate-activated Protein Kinase Mediates Short-term Food Deprivation Inhibition of the Gonadotropin-releasing Hormone–Luteinizing Hormone Axis: Role of Nitric Oxide

“…Micropunch-dissected loci evaluated for FD-mediated changes in NE activity in low- versus high-E – replaced OVX female rats included structures [e.g. rostral preoptic area (rPO) and anteroventral periventricular (AVPV), medial preoptic (MPN), and hypothalamic arcuate (ARH) nuclei] previously reported to exhibit adjusted neurotransmitter content during hypoglycemic metabolic stress [Briski and Shrestha, 2016]. A related aim of the current project was to identify neurochemicals within discernible forebrain targets of FD-triggered NE signaling that may potentially link hindbrain AMPK with the GnRH-LH axis during this metabolic stress.…”