Impact of caudal hindbrain glycogen metabolism on A2 noradrenergic neuron AMPK activation and ventromedial hypothalamic nucleus norepinephrine activity and glucoregulatory neurotransmitter marker protein expression

Abstract

The brain glycogen reserve is a source of oxidizable substrate fuel. Lactoprivic-sensitive hindbrain A2 noradrenergic neurons provide crucial metabolic-sensory input to downstream hypothalamic glucose-regulatory structures. Current research examined whether hindbrain glycogen fuel supply impacts A2 energy stability and governance of ventromedial hypothalamic nucleus (VMN) metabolic transmitter signaling. Male rats were injected into the caudal fourth ventricle (CV4) with the glycogen phosphorylase inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) prior to continuous intra-CV4 infusion of L-lactate or vehicle. Lactate reversed DAB suppression of A2 neuron AMPK protein and up-regulated phosphoAMPK profiles. A2 dopamine-β-hydroxylase expression was refractory to DAB, but elevated by DAB/lactate. Lactate normalized A2 estrogen receptor-alpha and GPER proteins and up-regulated estrogen receptor-beta levels in DAB-treated rats. VMN norepinephrine content was decreased by DAB, but partially restored by lactate. DAB caused lactate-reversible or -irreversible augmentation of VMN glycogen phosphorylase-brain (GPbb) and -muscle type (GPmm) variant profiles, and correspondingly up- or down-regulated VMN protein markers of glucose-stimulatory nitrergic and glucose-inhibitory γ-aminobutyric acid transmission. DAB did not alter plasma glucose, but suppressed or elevated circulating glucagon and corticosterone in that order. Results show that diminished hindbrain glycogen breakdown is communicated to the VMN, in part by NE signaling, to up-regulate VMN glycogen breakdown and trigger neurochemical signaling of energy imbalance in that site. DAB effects on GPmm, VMN glycogen content, and counter-regulatory hormone secretion were unabated by lactate infusion, suggesting that aside from substrate fuel provision rate, additional indicators of glycogen metabolism such as turnover rate may be monitored in the hindbrain.

Keywords: 5’-AMP-activated protein kinase; DAB; L-lactate; Neuronal nitric oxide synthase; Norepinephrine; Ventromedial hypothalamic nucleus.

Figure 1.. Laser-Catapult Microdissection of Immuno-Characterized Hindbrain Dorsal Vagal Complex A2 Noradrenergic.

A2 neurons located between 14.36 to 14.86 mm posterior to bregma were identified in situ for tyrosine hydroxylase (TH) immunoreactivity (-ir); pre-dissected TH-ir-positive neurons in Panel 1A are denoted by blue arrows. The area shown in Panel 1A was re-photographed after positioning of a continuous laser track (depicted in green) around individual TH-ir neurons [Panel 1B] and subsequent ejection of each cell by laser pulse [Panel 1C]. Note that this microdissection technique causes negligible destruction of surrounding tissue and minimal inclusion of adjacent tissue.

Figure 2.. Effects of Caudal Fourth Ventricular (CV4) Administration of the Glycogen Phosphorylase (GP Inhibitor 1,4-Ddideoxy-1,4-Imino-d-Arabinitol (DAB) on Dorsal Vagal Complex A2 Noradrenergic Neuron Energy Gauge, Catecholamine Biosynthetic Enzyme Marker, and Estrogen Receptor (ER) Variant Protein Expression: Impact of Exogenous L-Lactate Infusion.

Groups of adult male rats were treated by intra-CV4 administration of the vehicle artificial cerebrospinal fluid (aCSF; group 1) or DAB (groups 2 and 3) into the CV4, followed by continuous infusion of aCSF (groups 1 and 2) or L-lactate (group 3) into the same ventricle. Pooled A2 cell lysates from each treatment group were probed for 5’-AMP-Activated Protein Kinase (AMPK; Panel 2A), phosphoAMPK (pAMPK; Panel 2B), dopamine-beta-hydroxylase (DβH; Panel 2C), ER-alpha (ERα; Panel 2D), ER-beta (ERβ; Panel 2E), or G protein-coupled estrogen receptor-1 (Panel 2F) protein expression. Bars depict mean normalized protein optical density (O.D.) values ± S.E.M. for aCSF/aCSF (solid white bars), DAB/aCSF, and DAB/L-lactate treatment groups. *p<0.05; **p<0.01; ***p<0.001.

Figure 3.. Impact of Pharmacological Inhibition of Hindbrain GP With or Without Exogenous Lactate Infusion on Ventromedial Hypothalamic Nucleus Norepinephrine (NE) Content.

Bars show mean VMN tissue NE levels for aCSF/aCSF (solid white bars), DAB/aCSF (solid gray bars), and DAB/L-lactate (diagonal-striped gray bars) treatment groups. *p<0.05; **p<0.01; ***p<0.001.

Figure 4.. Regulation of VMN Glycogen Metabolic Enyzme Protein Expression and Glycogen Content by Hindbrain Glycogen Metabolic Status.

In Panels 4A-3C, bars depict mean normalized VMN glycogen synthase (GS; Panel 4A), glycogen phosphorylase-brain type (GPbb; Panel 4B), or glycogen phosphorylase GP-muscle type (GPmm; Panel 4C) protein O.D. measures ± S.E.M. for aCSF/aCSF (solid white bars), DAB/aCSF (slid gray bars), and DAB/L-lactate (diagonal-striped gray bars) treatment groups. Data in Panels 3D and 3E illustrate DAB effects with or without subsequent L-lactate infusion on mean VMN glycogen or glucose content, respectively, in the same treatment groups. *p<0.05; **p<0.01; ***p<0.001.

Figure 5.. Effects of Hindbrain GP Inhibition on VMN Gluco-stimulatory Nitrergic and Gluco-inhibitory γ-Aminobutyric Acid Neuron Marker Protein Expression; Impact of L-Lactate Infusion.

Bars in Panels 5A and 5B depict mean normalized VMN neuronal nitric oxide synthase (nNOS) or glutamate decarboxylase 65/67 (GAD) protein O.D. measures ± S.E.M. for aCSF/aCSF (solid white bars), DAB/aCSF (solid gray bars), and DAB/L-lactate (diagonal-striped gray bars) treatment groups. *p<0.05; **p<0.01; ***p<0.001.

Figure 6.. Effects of Caudal Fourth Ventricular DAB Administration on Circulating Glucose and Counter-Regulatory Hormone Concentrations in the Male Rat.

Data depict mean plasma glucose (Panel 6A), glucagon (Panel 6C), and corticosterone (Panel 6C) levels ± S.E.M. for aCSF/aCSF (solid white bars), DAB/aCSF (solid gray bars), and DAB/L-lactate (diagonal-striped gray bars) treatment groups. *p<0.05; **p<0.01; ***p<0.001.