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. 2019 Oct 1:1720:146311.
doi: 10.1016/j.brainres.2019.146311. Epub 2019 Jun 29.

Sex-dimorphic estrogen receptor regulation of ventromedial hypothalamic nucleus glucoregulatory neuron adrenergic receptor expression in hypoglycemic male and female rats

M Main Uddin  1 , A S M Hasan Mahmood  1 , Mostafa M H Ibrahim  1 , Karen P Briski  2
Affiliations

Sex-dimorphic estrogen receptor regulation of ventromedial hypothalamic nucleus glucoregulatory neuron adrenergic receptor expression in hypoglycemic male and female rats

M Main Uddin et al. Brain Res. .

Abstract

The ventromedial hypothalamic nucleus (VMN) is a vital component of the neural circuitry that regulates glucostasis. Norepinephrine (NE) controls VMN gluco-inhibitory γ-aminobutyric acid (GABA) and gluco-stimulatory nitric oxide (NO) transmission. Sex-specific insulin-induced hypoglycemic (IIH) patterns of VMN GABA signaling are estrogen receptor-alpha (ERα)- and -beta (ERβ)-dependent. Current research utilized combinatory immunocytochemistry, laser-microdissection, and Western blot techniques in a pharmacological approach to address the hypothesis that ERα and/or -β mediate sex-dimorphic VMN GABAergic and/or nitrergic nerve cell receptivity to NE and estradiol during IIH. The impact of these ER on expression of the pyruvate recycling pathway marker proteins glutaminase (GLS) and malic enzyme-1 (ME-1) was also examined. Both VMN neuron populations express ERα, ERβ, and G protein-coupled estrogen receptor-1 (GPER), along with alpha1, alpha2, and beta1 adrenergic receptor (AR) proteins. NO neurons exhibited ERα/β-dependent (beta1 AR, GPER) and -independent (alpha1 AR) sex differences in receptor protein responses to hypoglycemia. Similarly, sex-dimorphic effects of IIH on alpha1 AR, alpha2 AR, and ERα profiles in GABA neurons involve ERα/β. These ERs also underlie divergent adjustments in gluco-regulatory nerve cell GLS and ME-1 protein expression in hypoglycemic males and females. Sex-specific nitrergic and GABAergic nerve cell sensitivity to NE and E, respectively, during IIH may contribute to sex-contingent patterns of neurotransmitter signaling.

Keywords: Glutamate decarboxylase(65/67); Glutaminase; Laser-catapult microdissection; MPP; Neuronal nitric oxide synthase; PHTPP.

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Figures

Figure 1.
Figure 1.. Laser-Catapult Microdissection of Immunolabeled Ventromedial Hypothalamic Nucleus (VMN) Nitrergic- or γ-Aminobutyric Acid (GABA) Neurons: Western Blot Confirmation of Accuracy of Immunocytochemical Identification of Neurotransmitter Phenotype.
VMN neurons were identified in situ for neuronal nitric oxide (nNOS)- [top row; Panel 1 A] or glutamate decarboxylase65/67 (GAD65/67)-[bottom row; Panel 2 A] immunoreactivity (-ir); representative nNOS- or GAD65/67-ir-positive neurons is indicated by green arrows. Areas shown in Panel 1 A and 2 A were re-photographed after positioning of a continuous laser track (depicted in blue) around a single nNOS-ir [Panel 1 B; green dashed arrow] or GAD65/67-ir neuron [Panel 2 B; green dashed arrow] and subsequent ejection of that cell by laser pulse [Panels 1 C and 2 C]. Note that this microdissection technique causes negligible destruction of surrounding tissue and minimal inclusion of adjacent tissue. Panels 1 D and 2 D show that nNOS or GAD65/67 protein is expressed in pure VMN nerve cell samples identified immunocytochemically for nNOS or GAD immunoreactivity, respectively.
Figure 2.
Figure 2.. Effects of Intracerebroventricular (icv) Administration of the ERα Antagonist 1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP) or the ERβ Antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP) on VMN Nitrergic Neuron Estrogen Receptor-Alpha (ERα), Estrogen Receptor-Beta (ERβ), and G Protein-Coupled Estrogen Receptor (GPER) Protein Expression in Insulin-Induced Hypoglycemic (IIH) Male versus Female Rats.
Pooled lysates of laser-catapult microdissected VMN nNOS-immunoreactive (-ir) neurons created for each treatment group were evaluated by Western blot for ERα [Panel A; male data at left, female data at right], ERβ [Panel B; male data at left, female data at right], and GPER [Panel C; male data at left, female data at right] protein expression in groups of vehicle-pretreated male or female rats injected subcutaneously (sc) with either vehicle (solid white bars; n=6 males, n=6 females) or neutral protamine Hagedorn insulin (INS; 10.0 U/kg bw; solid gray bars; n=6 males, n=6 females) and groups of INS-injected animals of either sex that were pretreated with MPP (diagonal-striped gray bars; n=6 males, n=6 females) or PHTPP (cross-hatched gray bars; n=6 males, n=6 females). Data depict mean normalized protein O.D. �� S.E.M. Data were analyzed by two-way ANOVA for sex versus treatment. *p <0.05; **p <0.01; ***p <0.001.
Figure 3.
Figure 3.. Effects of ERα or ERβ Blockade on VMN GABAergic Nerve Cell ERα, ERβ, and GPER Protein Expression in Hypoglycemic in Male versus Female Rats.
VMN GAD65/67-ir neurons were evaluated by Western blot for ERα [Panel A; male data at left, female data at right], ERβ [Panel B; male data at left, female data at right], and GPER [Panel C; male data at left, female data at right] protein expression in vehicle-pretreated euglycemic controls (solid white bars; n=6 males, n=6 females) controls and vehicle- (solid gray bars; n=6 males, n=6 females), MPP- (diagonal-striped bars; n=6 males, n=6 females), or PHTPP (cross-hatched gray bars; n=6 males, n=6 females)-pretreated INS (10.0 U/kg bw, sc) - injected animals. Data depict mean normalized protein O.D. measures ± S.E.M. Data were analyzed by two-way ANOVA for sex versus treatment. *p <0.05; **p <0.01; ***p <0.001.
Figure 4.
Figure 4.. Role of ERα and ERβ in Sex-Specific VMN Nitrergic Neuron Alpha1 Adrenergic Receptor (α1AR), Alpha2 AR (α2AR), and Beta1 AR (β1AR) Protein Responses to IIH.
Bars depict for each sex mean normalized nitrergic neuron α1AR [Panel A], α2AR [Panel B], and β1AR [Panel C] protein O.D. measures ± S.E.M. according to the following treatment groups: V/V (white bars; n=6 males, n=6 females), V/INS (10.0 U/kg bw, sc; solid gray bars; n=6 males, n=6 females), MPP/INS (diagonal-striped gray bars; n=6 males, n=6 females), and PHTPP/INS (cross-hatched gray bars; n=6 males, n=6 females). Data were analyzed by two-way ANOVA for sex versus treatment. *p<0.05; **p<0.01; ***p<0.001.
Figure 5.
Figure 5.. Impact of ERα or ERβ Antagonism on VMN GABAergic Nerve Cell α1AR, α2AR, and β1AR Protein Expression in Hypoglycemic Male versus Female Rats.
Data illustrate for each sex mean normalized GABAergic neuron α1AR [Panel A], α2AR [Panel B], and β1AR [Panel C] protein O.D. measures ± S.E.M. for groups of male and female rats treated as follows: V/V (white bars; n=6 males, n=6 females), V/INS (10.0 U/kg bw, sc; solid gray bars; n=6 males, n=6 females), MPP/INS (diagonalstriped gray bars; n=6 males, n=6 females), and PHTPP/INS (cross-hatched gray bars; n=6 males, n=6 females). Data were analyzed by two-way ANOVA for sex versus treatment. *p<0.05; **p<0.01; ***p<0.001.
Figure 6.
Figure 6.. Effects of MPP or PHTPP Pretreatment on VMN Nitrergic and GABAergic Neuron Monocarboxylate Transporter-2 (MCT2) Protein Expression in Hypoglycemic Male versus Female Rats.
Pooled lysates of nNOS- or GAD-ir neurons were created for each treatment group for Western blot analysis of MCT2 expression. Data show nitrergic [Panel A] and GABAergic [Panel B] neuron mean normalized MCT2 protein O.D. measures ± S.E.M. for groups of male (at left) and female (at right) animals treated as follows: V/V (n=6 males, n=6 females), V/INS (10.0 U/kg bw, sc; n=6 males, n=6 females), MPP/INS (n=6 males, n=6 females), and PHTPP/INS (n=6 males, n=6 females). Data were analyzed by two-way ANOVA for sex versus treatment. *p<0.05; **p<0.01; ***p<0.001.
Figure 7.
Figure 7.. Effects of MPP or PHTPP Pretreatment on VMN Nitrergic and GABAergic Neuron Glutaminase (GLS) and Malic Enzyme-1 (ME-1) Protein Expression in Hypoglycemic Male versus Female Rats.
Pooled lysates of nNOS- or GAD-ir neurons were created for each treatment group for Western blot analysis of GLS and ME-1. Data depict nitrergic nerve cell GLS [Panel A] and ME-1 [Panel B] and GABAergic neuron GLS [Panel C] and ME-1 [Panel D] profiles in groups of male (at left) and female (at right) rats treated by V/V (n=6 males, n=6 females), V/INS (10.0 U/kg bw, sc; n=6 males, n=6 females), MPP/INS (n=6 males, n=6 females), or PHTPP/INS (n=6 males, n=6 females). Bars indicate mean normalized protein O.D. measures ± S.E.M. Data were analyzed by two-way ANOVA for sex versus treatment. *p<0.05; **p<0.01; ***p<0.001.
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