Central Type II Glucocorticoid Receptor Regulation of Ventromedial Hypothalamic Nucleus Glycogen Metabolic Enzyme and Glucoregulatory Neurotransmitter Marker Protein Expression in the Male Rat

Abstract

The ventromedial hypothalamic nucleus (VMN) glucoregulatory neurotransmitters γ-aminobutyric acid (GABA) and nitric oxide (NO) signal adjustments in glycogen mobilization. Glucocorticoids control astrocyte glycogen metabolism in vitro. The classical (type II) glucocorticoid receptor (GR) is expressed in key brain structures that govern glucostasis, including the VMN. Current research addressed the hypothesis that forebrain GR regulation of VMN glycogen synthase (GS) and phosphorylase (GP) protein expression correlates with control of glucoregulatory transmission. Groups of male rats were pretreated by intracerebroventricular (icv) delivery of the GR antagonist RU486 or vehicle prior to insulin-induced hypoglycemia (IIH), or were pretreated icv with dexamethasone (DEX) or vehicle before subcutaneous insulin diluent injection. DEX increased VMN GS and norepinephrine-sensitive GP-muscle type (GPmm), but did not alter metabolic deficit-sensitive GP-brain type (GPbb) expression. RU486 enhanced GS and GPbb profiles during IIH. VMN astrocyte (MCT1) and neuronal (MCT2) monocarboxylate transporter profiles were up-regulated in euglycemic and hypoglycemic animals by DEX or RU486, respectively. Glutamate decarboxylase65/67 and neuronal nitric oxide synthase (nNOS) proteins were both increased by DEX, yet RU486 augmented hypoglycemic nNOS expression patterns. Results show that GR exert divergent effects on VMN GS, MCT1/2, and nNOS proteins during eu- (stimulatory) versus hypoglycemia (inhibitory); these findings imply that up-regulated NO transmission may reflect, in part, augmented glucose incorporation into glycogen and/or increased tissue lactate requirements. Data also provide novel evidence for metabolic state-dependent GR regulation of VMN GPmm and GPbb profiles; thus, GABA signaling of metabolic stability may reflect, in part, stimulus-specific glycogen breakdown during eu- versus hypoglycemia.

Keywords: Dexamethasone; Glucocorticoid receptor; Glycogen phosphorylase; Nitric oxide synthase; RU486; Ventromedial hypothalamic nucleus.

Figure 1:

Ventromedial Hypothalamic Nucleus (VMN) Micropunch Dissection. The rectangle at the bottom of the brain map (−2.85 mm posterior to bregma) in Panel A depicts the mediobasal hypothalamus wherein the VMN resides. The area within that rectangle is enlarged in Panel B to illustrate neuroanatomical relationships of the VMN with other mediobasal hypothalamic structures. The blue circle denotes the positioning of a 0.50 mm diameter circular micropunch tool over the center of the elliptical-shaped VMN, enabling sampling of tissue from dorsomedial, central, and ventrolateral divisions of the VMN. Abbreviations in Panel B: ARH: arcuate hypothalamic n.; DMHap: anterior and posterior part of dorsomedial hypothalamic n.; fx: fornix; LHA: lateral hypothalamic area; ME: median eminence; PVi: intermediate periventricular hypothalamic n.; VMHc,dm,vl: central/dorsomedial/ventormlateral parts of ventromedial hypothalamic n.; TU: tuberal n.; V3: third ventricle.

Figure 2:

Effects of Intracerebroventricular (icv) Administration of the Glucocorticoid Receptor (GR) Agonist Dexamethasone (DEX) or GR Antagonist RU486 on Ventromedial Hypothalamic Nucleus (VMN) Glycogen Metabolic Enzyme and Monocarboxylate Transporter Protein Expression in the Male Rat. Groups of male rats (n=4) were pretreated by intracerebroventricular (icv) delivery of the GR antagonist RU486 or vehicle (V) prior to subcutaneous (sc) insulin (INS) injection, or were pretreated icv with (DEX) or V before sc insulin diluent (V) injection. For each treatment group, three separate aliquot pools of micropunched VMN tissue were analyzed by Western blot for glycogen synthase (GS; Panel 2A), glycogen phosphorylase (GP)-muscle type (GPmm; Panel 2B), GP-brain type (GPbb; Panel 2C), astrocyte-specific monocarboxylate transporter-1 (MCT1; Panel 2D), or neuron-specific monocarboxylate transporter-2 (MCT2; Panel 2E) protein expression. Bars depict mean normalized protein optical density (O.D.) measures + S.E.M. for the following treatment groups: V/V (solid white bars). V/INS (solid light gray bars), RU486/INS (diagonal-striped light gray bars), and DEX/V (solid dark gray bars) treatment groups. *p<0.05; **p<0.01; ***p<0.001.

Figure 3:

Forebrain GR Regulation of VMN Protein Glucoregulatory Signaling Marker Protein Expression and 5′-AMP-Activated Protein Kinase (AMPK) Activity during Eu- and Hypoglycemia. Bars depict mean normalized VMN glutamate decarboxylase65/67 (GAD; Panel 3A), neuronal nitric oxide synthase nNOS (Panel 3B), AMPK (Panel 3C), or phosphoAMPK (pAMPK) (Panel 3D) protein O.D. measures + S.E.M. for V/V (solid white bars), V/INS (solid light gray bars), RU486/INS (diagonal-striped light gray bars), and DEX/V (solid dark gray bars) treatment groups. *p<0.05; **p<0.01; ***p<0.001.

Figure 4:

Effects of icv DEX or RU486 Administration on VMN Astrocyte Adrenergic Receptor (AR) Variant Protein Expression in the Male Rat. Pooled lysates of laser-microdissected VMN glial fibrillary acidic protein-immunopositive astrocytes from each treatment group were analyzed in triplicate for alpha1-AR (α1AR; Panel 4A), alpha2-AR (α2AR; Panel 4B), or beta1-adrenergic receptor (β1AR; Panel 4C) protein expression. Data depict mean protein O.D. measures + S.E.M. for V/V (solid white bars), V/INS (solid light gray bars), RU486/INS (diagonal-striped light gray bars), and DEX/V (solid dark gray bars) treatment groups. *p<0.05; **p<0.01; ***p<0.001.

Figure 5:

Forebrain GR Regulation of VMN Astrocyte Estrogen Receptor (ER) Variant Protein Expression in the Male Rat. Bars depict mean normalized ER-alpha (ERα) or ER-beta (Eβ) protein optical density (O.D.) values + S.E.M. for V/V (solid white bars), V/INS (solid light gray bars), RU486/INS (diagonal-striped light gray bars), and DEX/V (solid dark gray bars) treatment groups. *p<0.05; **p<0.01; ***p<0.001.

Figure 6:

Effects of icv DEX or RU486 Administration on VMN GR Protein Expression in the Male Rat. Bars illustrate mean normalized VMN GR O.D. measures + S.E.M. for V/V (solid white bars), V/INS (solid light gray bars), RU486/INS (diagonal-striped light gray bars), and DEX/V (solid dark gray bars) treatment groups. *p<0.05; **p<0.01; ***p<0.001.

Figure 7:

Effects of icv DEX or RU486 Administration on Circulating Glucose, Counter-Regulatory Hormone, and Free Fatty Acid (FFA) Levels in the Male Rat. Data show mean plasma glucose (Panel 7A), glucagon (Panel 7B), corticosterone (Panel 7C), and FFA (Panel 7D) concentrations + S.E.M. for V/V (solid white bars), V/INS (solid light gray bars), RU486/INS (diagonal-striped light gray bars), and DEX/V (solid dark gray bars) treatment groups. *p<0.05; **p<0.01; ***p<0.001.

Figure 8:

GR Regulation of VMN Astrocyte Receptor and Glycogen Metabolic Enzyme Protein Expression and Glucoregulatory Neuron Marker Protein Profiles during Eu- versus Hypoglycemia. A: Divergent effects of GR signaling on VMN astrocyte estrogen (ERα, ERβ) and adrenergic (α1AR, α2AR,β1AR) receptor protein profiles during eu- versus hypoglycemia [upper middle figure] suggest that GR result in dissimilar volume of estradiol and norepinephrine (NE) input to these glia during glucostasis versus dyshomeostasis. As shown in the lower middle figure, GR elicit metabolic state-dependent changes in astrocyte GS, GPmm, and GPbb levels; opposite changes in GS profiles suggest that GR correspondingly promote or inhibit VMN glycogen accumulation during eu- or hypoglycemia. Differential GR control of NE-sensitive GPmm versus AMP-sensitive GPbb expression may likely result in stimulus-specific VMN glycogen breakdown under distinctive metabolic conditions. Bottom left- (B.1) and right (B.2) -hand figures correspondingly depict effects of GR activation on gluco-stimulatory nitrergic (nNOS) and gluco-inhibitory GABAergic (GAD) neuron marker protein expression during eu- versus hypoglycemia. Outcomes infer that nNOS profiles may reflect, in part, up- or down-regulated glycogen synthesis alongside as-yet-undetermined effects of GR-mediated increases in euglycemic GPmm profiles versus GR suppression of GPbb expression during hypoglycemia. Divergent effects of GR activation on gluco-inhibitory GAD profiles may reflect increased NE-associated glycogen disassembly alone or in relation to glycogen buildup during eu- versus hypoglycemia.