Key Words: Glucocorticoid receptor; Ventromedial hypothalamic nucleus; RU486; Dexamethasone; Glycogen phosphorylase; Nitric oxide synthase
Adrenal glucocorticoid hormones act within the brain to regulate neural adaptation to recurring IIH . Glucocorticoids modulate norepinephrine (NE) control of astrocyte glycogen synthesis in vitro via classic/type II glucocorticoid receptor (GR) signaling . GR are one of two distinct glucocorticoid receptor populations in the brain, and differ from type I or corticosterone (CORT)-preferring mineral corticoid receptor (CR) concerning neuroanatomical distribution and ligand preferences [21-23]. CR are expressed exclusively in hippocampus and septum, and bind corticosterone with high affinity (Kd » 0.5nM), whereas GR occur throughout the brain, including the VMN and other hypothalamic glucoregulatory loci , and exhibit relatively greater affinity for synthetic ligands including dexamethasone (DEX; Kd » 1.5-2.0 nM) versus corticosterone (Kd » 2.5-5.0 nM). Current research utilized pharmacological, high-spatial resolution dissection, and Western blot techniques to investigate the premise that GR govern VMN glycogen metabolism and glucoregulatory transmitter signaling during euglycemia and/or hypoglycemia. Euglycemic adult male rats were injected into the lateral ventricle with DEX, whilst other animals were pretreated by intracerebroventricular (icv) delivery of the GR antagonist RU486 (mifepristone) [25, 26] prior to onset of IIH. VMN tissue obtained by micropunch dissection was analyzed by Western blot for protein markers of GABAergic (GAD) and nitrergic (nNOS) neuron function. Recent studies implicate NE and estradiol in regulation of VMN glycogen metabolism [27, 28]. Our studies show that VMN astrocytes express distinctive adrenoreceptor [alpha1-adrenoreceptor (α1AR), alpha2-AR (α2AR), and beta1-AR (β1AR)] and estrogen receptor [estrogen receptor-alpha (ERα), ER-beta (ERβ), and G protein-coupled estrogen receptor 1 (GPER)] proteins. In the present work, VMN tissue sections were also processed for in situ immunocytochemical identification of glial fibrillary acid protein(GFAP)-reactive astrocytes in advance of laser-catapult micro dissection (LCM) . Pure VMN astrocyte cell samples were analyzed by immunoblotting to examine whether GR regulate expression of one or more AR and ER protein profiles in these glia.
Adult male Sprague Dawley rats (3–4 months of age) were housed in individual cages under a 14 hr light/10 hr dark cycle (lights on at 05.00 h), and allowed ad-libitum access to standard laboratory chow (Harlan Teklad LM-485; Harlan industries, Madison, WI) and tap water. All surgical and experimental protocols were conducted in accordance with NIH guidelines for care and use of laboratory animals, under approval by the ULM Institutional Animal Care and Use Committee. On study day 1, animals were implanted with a PE-20 cannula directed to the left lateral ventricle (LV) [coordinates: 0.00 mm posterior to bregma, 1.50 mm lateral to midline, 5.0 mm ventral to skull surface; Mahmood et al., 2018] under ketamine/xylazine anesthesia (0.1 ml/100 g bw; 90 mg ketamine:10 mg xylazine/ml; Henry Schein Inc., Melville, NY). After surgery, rats were injected subcutaneously (sc) with ketoprofen (1 mg/kg bw) and intramuscularly with enrofloxacin (10 mg/kg bw), then transferred to individual cages. At 08.45 hr on study day 7, animals were divided into four treatment groups, and infused into the LV over a two minute period with vehicle (V; 2.0 μL; groups 1 and 2, n=4/group), the GR antagonist RU486 [25, 26] (10.0 μg/2.0 μL ; group 3, n=4), or dexamethasone (10 μg/2.0 μL , n=4). At 9:00 hr, rats in groups 1 and 4 received a sc injection of insulin diluent (V; Eli Lilly & Co., Indianapolis, IN), while groups 2 and 3 were injected sc with neutral protamine Hagedorn insulin (INS; 10.0 U/kg bw; Butler Schein Animal Health, Dublin, OH). Rats were sacrificed at 10:00 hr for brain tissue and trunk blood collection. Brains were snap-frozen in liquid nitrogen-cooled isopentane for storage at -80 °C. Plasma was stored at −20 °C.
VMN Micropunch Dissection and Western Blotting
Between -1.80 and -3-30 mm relative to bregma, alternating series of thin (10 μm-thick) and thick (100 μm-thick) frozen sections werecollected over the length of the VMN, overrepeating distances of 100 μm (n = 10 consecutive thin sections) and 200 μm (n = 2 consecutive thick sections), for LCM or Palkovits micropunch dissection, respectively. For each animal, micro punched VMN tissue samples were bilaterally removed from thick sections using calibrated hollow needle tools (prod. no. 57401; Stoelting Co., Wood Dale, IL; Figure 1), and transferred to lysis buffer (2% sodium dodecyl sulfate, 0.05 M dithiothreitol, 10% glycerol, 1 mM EDTA, 60 mM Tris-HCl, pH 7.2). In each treatment group, tissue lysate aliquots from individual subjects were combined to create three separate sample pools for each protein of interest. Sample proteins were separated in Bio-Rad Stain-Free 10% gradient acrylamide gels (Hercules, CA) after loading of approximately 25 μg protein per individual well, then transblotted to 0.45-μm PVDF-Plus membranes (Osmonics, Gloucester, MA) . Prior to transblotting, gels were UV light-activated (1 min) in a BioRad ChemiDoc TM Touch Imaging system. Membranes were blocked with Tris-buffered saline, pH7.4, containing 0.1% Tween-20 and 2% bovine serum albumin prior to incubation (48 hr) with primary antibodies. Proteins of interest were probed with polyclonal antisera raised in rabbit against glycogen synthase (GS; 1:2000; prod. no. 3893S; Cell Signaling Technology, Danvers, MA), glycogen phosphorylase-muscle type (GPmm; 1:2,000; prod. no NBP2-16689; Novus Biologicals, LLC, Centennial, CO.), glycogen phosphorylase-brain type (GPbb; 1:2,000; prod. no. NBP1-32799; Novus Biol.), MCT1 (1:2,000; prod. no. AB3540P; Millipore Sigma, Burlington, MA); MCT2 (1:2000; prod. no. sc-NBP1-87846; Novus Biol.); nNOS (1:1,000; prod. no. Nbp1-396B1; Novus Biol.), GAD (1:1,000; prod. no. ABN904; EMD Millipore, Billerica, MA), 5’-AMP-activated protein kinaseα1/2 (AMPK; 1:2000; prod. no. 2532S; Cell Signaling Technol.), phosphoAMPK-Thr 172 (pAMPK; 1:2000; prod. no. 2531S; Cell Signaling Technol.) or GR (1:6000, prod. no. 3660, Cell Signaling technol.). Membranes were incubated (1 hr) with horseradish peroxidase-labeled goat antirabbit secondary antibodies (1:5,000; prod. no. NEF812001EA; PerkinElmer, Waltham, MA), then exposed to SuperSignal West
VMN Astrocyte Laser-Catapult Microdissection and Western Blotting
Ten μm-thick frozen sections obtained at regular intervals over each VMN were mounted on PEN membrane-coated slides (Carl Zeiss Microscopy, LLC, White Plains, NY), and processed by avidinbiotin peroxidase immunocytochemistry to identify glial fibrillary acid protein (GFAP)-immunoreactive (-ir) astrocytes . Tissues were fixed with acetone (5 min), blocked with 1.5% normal horse serum (prod. no. S-2000, Vector Laboratories, Burlingame, CA), then incubated for 36-48 hr at 40C with mouse monoclonal antibodies against GFAP (prod. no. 3670S, Cell Signal. Technol.; 1:500). Sections were next incubated with biotinylated horse anti-mouse IgG secondary antibody, followed by ABC reagent (prod. no. PK-6101; Vector Lab.). GFAP-ir cells were visualized using Vector DAB peroxidase substrate kit reagents (prod. no. SK-4100; Vector Lab.), and collected individually into tissue lysis buffer using a Zeiss P.A.L.M. UV-A microlaser-IV system (Carl Zeiss Microscopy, LLC, Thornwood, NY). Each astrocyte protein of interest was evaluated by immunoblot in separate triplicate pools of n=60 astrocyte lysates for each treatment group. Proteins were detected with primary antisera raised in rabbit against alpha1A AR/ADRA1A (α1AR; 1:1000; prod. no. NB100- 78585; Novus Biol.); alpha2A AR/ADRA3A (α2AR; 1:1000; prod. no. NBP1-67832, Novus Biol.), beta1 AR/ADRB1 (β1AR; 1:1000; prod. no. NBP1-59007; Novus Biol.); ER-alpha/NR3A1 (ERα; 1:1000; prod. no. NB100-91756; Novus Biol.); or ER-beta/NR3A2 (ERβ; 1:1000; prod. no. NB120-3577; Novus Biol.). Protein O.D. measures obtained after chemiluminescent substrate incubation were normalized to total in-lane protein using Bio-Rad Stain-Free Imaging Technology, as described above.
Glucose and Counter-Regulatory Hormone Measurements
Plasma glucose levels were determined using an ACCUCHECK Aviva plus glucometer (Roche Diagnostic Corporation, Indianapolis, IN) . Plasma free fatty acid (FFA) concentrations were measured with Free Fatty Acid Quantitation Kit reagents (MAK044; Sigma Aldrich, St. Louis, Mo) . Plasma corticosterone (ADI-900-097; Enzo Life Sciences, Inc., Farmingdale, NY) and glucagon (EZGLU-30K, EMD Millipore, Billerica, MA) concentrations were determined using commercial ELISA kit reagents [29b].
Mean normalized tissue protein O.D. data and plasma glucose, glucagon, corticosterone, and FFA values were evaluated amongst treatment groups by one-way analysis of variance and Student Newman Keuls post-hoc test. Differences of p < 0.05 were considered significant.
Figure 3 illustrates effects of icv GR agonist or antagonist administration to eu- versus hypoglycemic rats, respectively, on VMN glucoregulatory neurotransmitter biomarker expression and AMPK energy sensor activity. In Panel 3A, results show that VMN GAD content was elevated in response to icv DEX [F(3,8) = 19.01, p = 0.0005], but was unaffected by IIH with or with RU486 pretreatment. VMN nNOS (Panel 3B) protein profiles
Data in Figure 4 show patterns of VMN astrocyte AR protein expression in euglycemic rats given vehicle or versus DEX icv and in hypoglycemic animals pretreated by icv administration of vehicle or RU486. Astrocyte α1AR [F(3,8) = 3.68, p = 0.04] and α2AR [F(3,8) = 4.15, p = 0.03] protein profiles were both refractory to DEX or IIH treatment (Panels 4A and 4B). Yet, hypoglycemic patterns of α2AR expression were amplified in RU486-pretreated animals [RU486/INS versus V/INS]. As shown in Panel 3C, DEX, but not IIH stimulated astrocyte β1AR content.
Figure 5 depicts effects of icv DEX or IIH after icv vehicle versus RU486 pretreatment on VMN astrocyte ERα (Panel 5A) and ERβ (Panel 5B) protein content. VMN astrocyte ERβ expression was increased in response to DEX administration [F(3,8) = 10.75, p = 0.001]. Astrocyte ERα expression was elevated in hypoglycemic versus euglycemic controls [V/INS versus V/V]; this profile did not differ between INS-injected groups pretreated with V versus RU486. VMN astrocyte ERβ expression was stimulated by DEX and amplified in hypoglycemic rats pretreated with RU486 [F(3,8) = 24.03, p < 0.0001].
Data in Figure 6 illustrate patterns of VMN GR protein expression after icv GR agonist or antagonist administration. Tissue GR protein content was significantly increased in response to DEX [F(3,8) = 15.02, p = 0.001]. GR profiles were unaffected by IIH, but augmented in RU486- vs. V-pretreated INS-injected rats Figure 7 presents plasma glucose, glucagon, corticosterone, and FFA responses to icv DEX or sc INS injection with or without icv RU486 pretreatment. As shown in Panel 7A, circulating glucose levels were significantly decreased in response to INS [V/INS versus V/V; F(3,12) = 138.50, p < 0.0001]. Mean glucose measures were not different among vehicle- versus RU486- pretreated hypoglycemic groups. Data also show that Intra-LV DEX administration did not alter plasma glucose levels. Plasma glucagon concentrations (Panel 7B) were significantly elevated in response to INS injection [V/INS versus V/V] [F(3,12) = 6.65, p = 0.009]; this secretory response to IIH was attenuated by RU486 pretreatment. However, plasma glucagon levels were refractory to icv DEX. Circulating corticosterone levels (Panel 7C) were also increased during hypoglycemia, but were unaffected by RU486 pretreatment [F(3,12) = 5.65, p = 0.02]. Icv DEX treatment likewise did not modify plasma corticosterone concentrations. Plasma FFA (Panel 7D) were significantly decreased in response to IIH; this response was not affected by RU486[F(3,12) = 14.88, p = 0.0008]. Icv DEX administration did not modify circulating FFA.
Data here show that GR increase VMN GS protein content during euglycemia, but conversely limit GS expression during hypoglycemia, inferring that GR may respectively stimulate or oppose glucose incorporation into VMN glycogen during glucostasis versus glucoprivation. Present outcomes do not clarify how neuro-glucopenia and/or associated cellular energy imbalance may mediate this directional switch in GR control of GS. Evidence that DEX causes concurrent up-regulation of VMN GR and GS supports the need to determine if the latter protein may be augmented, in part, in response to amplified GR signaling; however, the possibility that downstream GR-sensitive signal transduction pathway activity may also be enhanced by DEX should not be discounted. VMN GS protein content was refractory to IIH, but was significantly increased by GR antagonism during IIH. As the current experimental design did not involve icv administration of RU486 to controls, it remains unclear if and to what extent hypoglycemia-associated GR inhibition of GS may differ compared to euglycemia. In the event that ongoing research reveals that IIH augments GR suppression of GS, it could be presumed that this amplified negative stimulus may be counterbalanced by positive inputs of equivalent strength. Since current data show that VMN GR protein content was similar in V/V versus V/INS groups, it is plausible that direction (e.g. inhibition versus stimulation) of GR control of VMN GS expression may be established, in part, by absolute levels of GR expression. At the same time, the likelihood that hypoglycemia may regulate post-receptor signaling to shape GR regulation of GS merits consideration.
GP activity is controlled by serine phosphorylation and/or stimulatory (AMP) and inhibitory (glucose) allosteric effectors [34, 35]. Brain GP isoforms GPmm and GPbb show differential sensitivity to phosphorylation and AMP regulation . Phosphorylation fully activates GPmm, but not GPbb, which requires AMP binding for maximum activation . AMP exhibits greater binding affinity for GPbb versus GPmm, and reduces GPbb Km for glycogen . In brain, GPmm mediates noradrenergic stimulation of cortical astrocyte glycogenolysis in vitro, whereas GPbb mobilizes glycogen breakdown during energy deficiency . Present evidence that GR up-regulate VMN GPmm protein profiles during glucose stability, but impose an inhibitory tone on GPbb expression during glucoprivation suggests that GR may control physiological stimulus-specific effects on VMN glycogen metabolism. Effects of icv drug treatments on VMN GP variant enzyme activities were not assessed here due to lack of requisite analytical sensitivity pertaining to the small tissue sample volumes obtained here. Further studies are required to ascertain proportionate expression of GPmm versus GPbb in the VMN, and to investigate effects of isoform-specific GR regulation on net VMN glycogen mass under discrete metabolic conditions. There also remains the need to determine if DEX-associated up-regulation of VMN MCT1 and MCT2 protein expression is causally related to GR agonist stimulation of glycogen disassembly and associated conversion of liberated glucose to lactate for astrocyte-to-neuron trafficking.
VMN levels of GAD, the marker protein for gluco-inhibitory GABA signaling, were elevated in response to DEX, while hypoglycemic GAD profiles were unaffected by RU486 despite equivalent patterns of VMN GR protein expression in DEX/V versus RU486/ INS treatment groups. Potential mechanisms responsible for GABA insensitivity to GR during glucoprivation remain unclear, but could involve GR inactivation and/or suppression of downstream signaling under the latter conditions. Here, VMN GPmm and GAD proteins exhibited parallel up-regulation by DEX yet both proteins were insensitive to combinatory RU486 plus INS treatment, suggesting that GABAergic cues of metabolic stability may be an indicator of extent of stimulus-specific, e.g. NE-driven glycogen degradation. In contrast, VMN nNOS protein expression, an indicator of gluco-stimulatory NO release, was subject either to bi-directional, metabolic state-specific GR control. It would be informative to learn if measurable drug effects on nNOS profiles elicit or are a consequence of GS and/or MCT responses to those treatments. Findings that IIH did not suppress VMN GAD or elevate nNOS profiles concur with previous reports . Here, outcomes here provide unique proof that VMN AMPK activity is decreased one hour after initiation of hypoglycemia, reinforcing the notion that energy stability is maintained within this acute post-treatment interval and that moreover, GR mediate this reduction in sensor activity. Further studies are required to clarify whether decreased VMN pAMPK expression reflects, in part, reduced partitioning of glucose into the astrocyte glycogen shunt [40, 41] due to down-regulated GS
Recent studies implicate NE and estradiol in regulation of VMN glycogen metabolism [27, 28]. Current data show that GR enhance estradiol signaling to astrocytes via ERα and -β signaling during glucostasis, but attenuate ERβ-mediated input to this glia during glucoprivation. Outcomes also indicate that GR amplify NE input to astrocytes during euglycemia by up-regulating astrocyte β1AR protein content, but restrain noradrenergic stimulation of this glia during hypoglycemia through suppression of astrocyte α1- and α2AR expression. These data support the possibility that divergent GR regulation of VMN GS, MCT 1/2, and nNOS protein profiles during eu- versus hypoglycemia may be mediated by corresponding up-regulation of ERβ and β1AR and downregulation of ERβ and α1- and α2AR.
Outcomes document up-regulation of VMN GR by the GR agonist DEX, but show that this protein profile is refractory to IIH despite hypoglycemic corticosteronemia and that GR constrain VMN GR expression during IIH. These findings agree with recent reports that stress-associated glucocorticoid inhibition of GR involves diminished AMPK activity . Current studies do not shed light on whether possible differences in GR occupation and activation by DEX compared to that achieved by elevated corticosterone secretion during hypoglycemia may determine self-up- versus down-regulation of VMN GR. The experimental design here utilized an intraventricular route of administration of GR modulators to the brain to facilitate the study objective of activating or inhibiting activity of GR expressed in the VMN as well as other hypothalamic glucoregulatory loci that are functionally interconnected with the VMN. Thus, while observed drug effects on VMN GR protein expression imply that drug action occurs within that structure, there remains a likelihood that extra-VMN GR-sensitive neurotransmitters may mediate, to some degree, observed changes in VMN and systemic experimental endpoints. An objective of the current project was to investigate GR modulator effects on VMN astrocyte GR content. However, this protein was undetectable here in LCM-harvested astrocytes, suggesting that GR expression may fall below limits of analytical sensitivity, or that GR regulation of astrocyte protein expression may be mediated, in part, by chemical stimuli from glucocorticoidsensitive neurons or other glial cell types. Additional effort is required to determine if VMN GABA and/or nitrergic neurons express GR.
Results indicate that icv DEX administration did not significantly alter plasma glucose or counter-regulatory hormone levels despite observable effects of this treatment paradigm on VMN glucoregulatory transmitter marker protein expression. It is possible that concurrent augmentation of opposing glucostimulatory NO and -inhibitory GABA stimuli results in no net change in counter-regulatory outflow. Current evidence that INS suppression of glucose was unaffected by RU486 implies that hypoglycemic hypercorticosteronemia primarily attenuates glucose decrements [43-45] by action on GR that are not accessible to drugs delivered to the cerebral ventricular system. Evidence here that hypoglycemic glucagonemia was partially normalized by icv RU486 administration suggests that hypoglycemic patterns of corticosterone secretion act, in part, on periventricular substrates to limit glucagon outflow. As hypothalamic, specifically paraventricular hypothalamic nucleus GR are implicated in glucocorticoid negative feedback inhibition of the hypothalamic-pituitary-adrenal axis and function to limit the magnitude of corticosterone output during acute stress [46-48], our working premise was that RU486 would enhance corticosterone secretion in INS-injected rats. Yet, current data show that corticosterone levels did not differ between RU485- versus vehicle-pretreated hypoglycemic animals. It is possible that adjusted GR antagonist dosages might more effectively inhibit VMN GR signaling compared to the current treatment paradigm, and/or that analysis of corticosterone release at additional post- INS injection time points may document measurable RU486 effects on hormone secretion. Present evidence that neither DEX nor RU486 altered circulating FFA concentrations suggest that GR involved in regulating this non-glucose substrate fuel are located beyond diffusion range of the cerebroventricular system.
Previous studies in our laboratory utilized immunocytochemical methods to identify GR-immunoreactive neurons that undergo transcriptional activation in response to hypoglycemia . That approach was not used here as immunocytochemistry is an optimum approach for discernment of the cellular distribution of a protein(s) of interest, but relies upon visual discernment of possible treatment-associated differences in enzyme-derived reaction product or fluorophore-associated emitted light between treatment groups. Rather, Western blotting permits a semi-quantitative approach to be implemented for measurement of protein profiles within distinctive cell populations.
A limitation of the present project is the lack of inclusion of an additional treatment group involving RU486 administration to euglycemic animals. Investigation of GR antagonism effects during glucostasis balance could be expected to shed light on relative degree of control imposed by these receptors during glucostasis versus glucoprivation. There also remains a need for insight on how DEX may affect hypoglycemic patterns of protein expression described here. For example, it would be informative to learn if GR activation beyond that achieved by hypoglycemic hypercorticosteronemia. Ongoing studies seek to investigate these unresolved knowledge gaps.
The current experimental design closely adheres to a longstanding paradigm used in our laboratory, involving specific timing of systemic and intracranial drug therapies over a distinct morning time interval to evaluate concerning regulatory associations between hypothalamic neuroendocrine function and energy homeostasis, including GR involvement in neural mechanisms governing glucose counter-regulation . Nonetheless, in light of evidence for diurnal variation in GR activation by endogenous ligand , it should be noted that administration of GR agonists and antagonists at other time points over a 24 hour period may elicit outcomes that diverge from data presented here. Moreover, since only one time between drug delivery and tissue procurement was evaluated here, it is possible that response variables may show measurable differences in magnitude and/or direction at time points before or after that employed here.
In summary, current studies investigated effects of icv delivery of the GR agonist DEX or the GR antagonist RU486 on VMN glycogen metabolic enzyme and glucoregulatory transmitter biomarker protein expression in the male rat (Figure 8). Results show GR exert bi-directional regulatory effects on VMN GS, MCT1/2, and nNOS proteins during eu- (stimulatory) versus hypoglycemia (inhibitory), implying that up-regulated gluco-stimulatory NO transmission may reflect, in part, increased glucose incorporation into glycogen and/or augmented tissue lactate requirements. Data provide unique proof of metabolic state-dependent GR regulation of VMN NE-sensitive GPmm and AMP-sensitive GPbb profiles, and raise the prospect that gluco-inhibitory GABA signaling may reflect, in part, stimulus-specific glycogen breakdown alone or relative to rate of glycogen assembly.
Declaration of interestTop
α2AR: alpha2 adrenergic receptor
β1AR: beta1 adrenergic receptor
ERα: Estrogen receptor-alpha
ERβ: Estrogen receptor-beta
GABA: ϒ-aminobutyric acid
GAD: Glutamate decarboxylase65/67
GPbb: Glycogen phosphorylase-brain type
GPmm: Glycogen phosphorylase-muscle type
GPER: G protein-coupled estrogen receptor 1
GR: type II glucocorticoid receptor
GS: Glycogen synthase
IIH: Insulin-induced hypoglycemia
LV: Lateral ventricle
MCT1: Monocarboxylate transporter-1
MCT2: Monocarboxylate transporter-2
NO: Nitric oxide
nNOS: Neuronal nitric oxide synthase
VMN: Ventromedial hypothalamic nucleus
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