Estradiol regulates effects of hindbrain activator 5-aminoimidazole-4-carboxamide-riboside administration on hypothalamic adenosine 5′-monophosphate-activated protein kinase activity and metabolic neurotransmitter mRNA and protein expression

Abstract

Hindbrain adenosine 5'-monophosphate-activated protein kinase (AMPK) activation alters hypothalamic neuronal genomic activity in an estradiol (E)-dependent manner. This study examines the premise that E regulates metabolic effector neuron reactivity to hindbrain AMPK. Paraventricular (PVH), arcuate (ARH), and ventromedial (VMH) nuclei were micropunched from brains of E- or oil (O)-implanted ovariectomized female rats that had been injected, into the fourth ventricle, with the AMPK activator 5-aminoimidazole-4-carboxamide-riboside (AICAR; A) or saline (S) and analyzed by quantitative polymerase chain reaction and Western blotting for neurotransmitter mRNA and protein expression. PVH corticotrophin-releasing hormone gene and protein profiles were decreased in O/A and E/A animals. ARH pro-opiomelanocortin (POMC) mRNA and protein were both elevated in O/A but were diminished or unchanged, respectively, in E/A animals; ARH neuropeptide Y (NPY) transcription was inhibited in O/A and E/A animals, but neuropeptide content was augmented in E/A only. VMH SF-1 mRNA and protein were reduced in O and E animals. AICAR did not alter AMPK protein in any structure but elevated PVH (↑E), did not alter ARH, and decreased VMH (↓O,↓E) pAMPK. Results demonstrate hypothalamic metabolic neurotransmitter and AMPK reactivity to hindbrain AMPK activation, including E-dependent adjustments in POMC and NPY transcription and protein expression. Dissimilar POMC (↑O vs. ↔E) and NPY (↓O vs. ↑E) neuropeptide responses to caudal fourth ventricle AICAR indicate E regulation of hindbrain AMPK signaling and/or target receptivity, implying that ARH-controlled metabolic responses may differ in the presence vs. absence of E. Evidence for variable changes in hypothalamic AMPK activity resulting from hindbrain sensor manipulation suggests that individual (or region-based groups of) AMPK-expressing neuron populations are uniquely impacted by hindbrain AMPK.

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

“…Plasma was stored at −20°C. All animals used in this study exhibited cerebrospinal fluid reflex from the cannula tip on day 18; postmortem histological examination confirmed accuracy of cannula placements within the CV4 [Alenazi et al, 2014]. …”

“…The Palkovits micropunch technique was employed to dissect the rPO [+0.48 mm − 0.00 mm relative to bregm a; 0.51 mm needle diameter; Stoelting, Inc., Kiel, WI]; for each treatment group, lysate aliquots were combined from subjects to create four separate pools for Western blotting. Tissue lysate protein electrophoresis and transblotting was performed as described [Alenazi et al, 2014]. Membranes were blocked with TBS-T-BSA prior to overnight incubation at 4°C with rabbit primary anti-GnRH-I precursor (sc-32292; Santa Cruz Biotechnol.)…”

“…For both the AVPV and ARH, aliquots of tissue lysate were combined within treatment groups to create four sample pools for Western blot analysis of prepro-kisspeptin protein levels, using a goat polyclonal antiserum (sc-18134; Santa Cruz Biotechnol.). In addition to prepre-kisspeptin, ARH samples were also pooled within treatment groups for evaluation of POMC and NPY proteins using described methods [Alenazi et al, 2014]. …”

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

“…no. sc-8070) [evaluated in LHA], as described [Alenazi et al, 2014]. For each treatment group, pooled cDVC tissue was incubated with antisera against AMPK α1/2 , pAMPK α1/2, or the catecholamine biosynthetic enzyme dopamine-beta-hydroxylase (DβH; 1:500; 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.…”