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

Abstract

Hindbrain-derived stimuli restrain the gonadotropin-releasing hormone (GnRH)-pituitary luteinizing hormone (LH) reproductive neuroendocrine axis during energy insufficiency. Interruption of food intake, planned or unplanned, is emblematic of modern life. This study investigated the premise that the hindbrain energy sensor 5'-adenosine monophosphate-activated protein kinase (AMPK) inhibits reproductive neuroendocrine function in short term, e.g. 18-h food-deprived (FD) estradiol (E)-implanted ovariectomized female rats. Intra-caudal fourth ventricular administration of the AMPK inhibitor Compound C (Cc) reversed FD-induced inhibition of rostral preoptic (rPO) GnRH protein expression and LH release in animals given E to replicate proestrus (high-E dose-, but not metestrus (low-E dose)-stage plasma steroid levels. FD caused Cc-reversible augmentation or diminution of preoptic norepinephrine (NE) activity in high- versus low-E rats, respectively, and AMPK-independent reductions in hypothalamic NE accumulation in the latter. Nitric oxide (NO) and kisspeptin are key stimulatory signals for the preovulatory LH surge. Here, FD inhibited rPO neuronal nitric oxide synthase protein expression in high-, but not low-E-dosed animals. Lateral ventricular delivery of the NO donor 3-morpholinosydnonimine (SIN-1) reversed inhibitory GnRH and LH responses to FD in high-E rats, and normalized rPO Vglut2, anteroventral periventricular KiSS1, and dorsomedial hypothalamic RFRP-3 mRNA and/or protein profiles. Data show that FD curtails reproductive neuroendocrine outflow by hindbrain AMPK-dependent mechanisms in the presence of peak estrous cycle E levels. Results indicate that neural networks linking this sensor to GnRH neurons likely involve NO signaling, which may function upstream of one or more neurotransmitters identified here by SIN-1-reversible inhibitory responses to FD.

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

“…57401; Stoelting; Kiel, WI) were used to bilaterally dissect VMN tissue for immunoblotting (right-side VMN punches were collected into lysis buffer consisting of 2.0% sodium dodecyl sulfate, 0.05 M dithiothreitol, 10.0% glycerol, 1.0 mM EDTA, 60 mM Tris-HCl, pH 7.2) or for NE ELISA (left-side VMN punches were collected into 0.01 N HCl supplemented with 1.0 mM EDTA, 4.0 mM sodium metabisulfite). For each protein of interest, heat-denatured tissue aliquots from individual subjects were combined within each treatment group to create triplicate pools ahead of separation in Bio-Rad TGX 10-12% Stain-Free gels (Shakya et al, 2018). After electrophoresis, gels were activated (1 min) by UV light in a Bio-Rad ChemiDoc MP Imaging System (prod.…”

Norepinephrine regulation of ventromedial hypothalamic nucleus metabolic transmitter biomarker and astrocyte enzyme and receptor expression: Impact of 5′ AMP-activated protein kinase

“…VMN tissue was micropunch-dissected from 100 μm sections using 0.50 mm-diameter hollow kneedles (Stoelting, Inc., and collected into lysis buffer [2.0% sodium dodecyl sulfate (SDS), 0.05 M dithiothreitol, 10.0% glycerol, 1.0 mM EDTA, 60 mM Tris-HCl, pH 7.2] for heat denaturation. For each protein of interest, three separate tissue aliquot pools were created for each treatment group ahead of separation in BioRad TGX 10-12% stain-free gels [Shakya et al, 2018]. After electrophoresis, gels were activated for 1 min by UV light in a BioRad ChemiDoc TM Touch Imaging System prior to protein transfer (30 V, overnight at 4°C; Towbin buffer) to 0.45-μm PVDF membranes (ThermoFisherScientific; Waltham, MA).…”

Norepinephrine control of ventromedial hypothalamic nucleus glucoregulatory neurotransmitter expression in the female rat: Role of monocarboxylate transporter function

“…no. 161-0183, Bio-Rad Laboratories Inc., Hercules CA) [Shakya et al, 2018]. After electrophoresis, gels were activated for 1 min by UV light in a BioRad ChemiDoc TM Touch Imaging.…”