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

Abstract

The ventromedial hypothalamic energy sensor AMP-activated protein kinase (AMPK) maintains glucostasis via neurotransmitter signals that diminish [γ-aminobutyric acid] or enhance [nitric oxide] counter-regulation. Ventromedial hypothalamic nucleus (VMN) 'fuel-inhibited' neurons are sensitive to astrocyte-generated metabolic substrate stream. Norepinephrine (NE) regulates astrocyte glycogen metabolism in vitro, and hypoglycemia intensifies VMN NE activity in vivo. Current research investigated the premise that NE elicits AMPK-dependent adjustments in VMN astrocyte glycogen metabolic enzyme [glycogen synthase (GS); glycogen phosphorylase (GP)] and gluco-regulatory neuron biomarker [glutamate decarboxylase_65/67 (GAD); neuronal nitric oxide synthase (nNOS); SF-1] protein expression in male rats. We also examined whether VMN astrocytes are directly receptive to NE and if noradrenergic input regulates cellular sensitivity to the neuro-protective steroid estradiol. Intra-VMN NE correspondingly augmented or reduced VMN tissue GAD and nNOS protein despite no change in circulating glucose, data that imply that short-term exposure to NE promotes persistent improvement in VMN nerve cell energy stability. The AMPK inhibitor Compound C (Cc) normalized VMN nNOS, GS, and GP expression in NE-treated animals. NE caused AMPK-independent down-regulation of alpha₂-, alongside Cc-reversible augmentation of beta₁-adrenergic receptor protein profiles in laser-microdissected astrocytes. NE elicited divergent adjustments in astrocyte estrogen receptor-beta (AMPK-unrelated reduction) and GPR-30 (Cc-revocable increase) proteins. Outcomes implicate AMPK in noradrenergic diminution of VMN nitrergic metabolic-deficit signaling and astrocyte glycogen shunt activity. Differentiating NE effects on VMN astrocyte adrenergic and estrogen receptor variant expression suggest that noradrenergic regulation of glycogen metabolism may be mediated, in part, by one or more receptors characterized here by sensitivity to this catecholamine.

Keywords: Compound C; GPR-30; Glycogen synthase; Neuronal nitric oxide synthase; Norepinephrine; Ventromedial hypothalamic nucleus.

Figure 1.. Effects of the 5’-AMP-Activated Protein Kinase (AMPK) Inhibitor Compound C (Cc) on Norepinephrine (NE) Regulation of Ventromedial Hypothalamic Nucleus (VMN) Glutamate Decarboxylase_65/67 (GAD_65/67), Neuronal Nitric Oxide Synthase (nNOS), and Steroidogenic Factor-1 (SF-1) Protein Expressin in Adult Male Rats.

Groups of rats (n=5/treatment group) were pretreated by bilateral intra-VMN delivery of vehicle (NE/V; solid gray bars) or Cc (NE/Cc; diagonal-striped gray bars) prior to NE administration. Controls were given vehicle only (V/V; white bars) into the VMN. For each treatment group, three separate aliquot pools of micropunched VMN tissue were analyzed by Western blot for each of the following proteins: GAD_65/67 (Panel A), nNOS (Panel B), or SF-1 (Panel C). Bars depict mean normalized protein optical density (O.D.) measures ± S.E.M. **p<0.01; ***p<0.001.

Figure 2.. Effects of NE on VMN Glycogen Synthase (GS) and Glycogen Phosphorylase (GP) Protein Expression; Impact of Cc Pretreatment.

Bars depict mean normalized GS (Panel A) or GP (Panel B) protein O.D. measures ± S.E.M. for V/V (white bars), NE/V (solid gray bars), and NE/Cc (diagonal-striped gray bars) treatment groups. *p<0.05; **p<0.01.

Figure 3.. VMN Astrocyte Adrenoreceptor Protein Expression in NE-Treated Male Rats.

Astrocytes immunolabeled for the marker protein glial fibrillary acidic protein (GFAP) were harvested by laser-catapult microdssection from 10 μm-thick fresh frozen sections cut through the VMN. Alpha₁ adrenergic (α₁A-R; Panel A), alpha₂ adrenergic (α₂A-R; Panel B), and beta₁ adrenergic (β₁A-R; Panel C) receptor proteins were each analyzed by Western blot in triplicate pools of n=60 astrocyte lysates for each treatment group. Data depict mean normalized protein O.D. values ± S.E.M. for V/V (white bars), NE/V (solid gray bars), and NE/Cc (diagonal-striped gray bars) treatment groups. *p<0.05; ***p<0.001.

Figure 4.. NE Regulation of VMN Astrocyte Estrogen Receptor Protein Expression in Cc- Versus Vehicle-Pretreated Rats.

GFAP-immunopositive astrocyte lysates pools (n=60 cells per pool) were analyzed in triplicate, for each treatment group, for estrogen receptor-alpha (ERα; Panel A), estrogen receptor-beta (ERβ; Panel B), or GRP30 (Panel C). Data depict mean normalized protein O.D. values ± S.E.M. for V/V (white bars), NE/V (solid gray bars), and NE/Cc (diagonal-striped gray bars) treatment groups. *p<0.05; **p<0.01.

Figure 5.. Effects of Cc on Noradrenergic Regulation of VMN Astrocyte Phosphofructokinase/Liver (PFKL) and Monocarboxylate Transporter-1 (MCT1) Protein Expression.

GFAP-immunopositive astrocyte lysates pools (n=60 cells per pool) were analyzed in triplicate, for each treatment group, for PFKL (Panel A) and MCT1 (Panel B). Data depict mean normalized protein O.D. values ± S.E.M. for V/V (white bars), NE/V (solid gray bars), and NE/Cc (diagonal-striped gray bars) treatment groups. *p<0.05; **p<0.01.

Figure 6.. Western Blot Analysis of VMN GAD_65/67 and nNOS Nerve Cell Adrenergic Receptor Protein Expression.

VMN neurons labeled for GAD_65/67- or nNOS-immunoreactivity were individually removed by laser-catapult microdssection from 10 μm-thick sections cut from vehicle-injected control brains. For each nerve cell population, the presence of α₁A-R, α₂A-R,or β₁A-R protein was determined by Western blot analysis of separate triplicate pools of n=50 heat-denatured cell lysates. *p<0.05.

Figure 7.. PHTPP Alters Glucose, Luteinizing Hormone (LH), Glucagon, and Corticosterone Responses to Insulin-Induced Hypoglycemia.

Data depict mean glucose (Panel A), free fatty acid (Panel B), plasma glucagon (Panel C), and corticosterone (Panel D) levels ± S.E.M. for V/V (white bars), NE/V (solid gray bars), and NE/Cc (diagonal-striped gray bars) treatment groups. *p<0.05; **p<0.01.