Steroidogenic Factor-1 Regulation of Dorsomedial Ventromedial Hypothalamic Nucleus Ghrh Neuron Transmitter Marker and Estrogen Receptor Gene Expression in Male Rat

Abstract Ventromedial hypothalamic nucleus (VMN) growth hormone-releasing hormone (Ghrh) neurotransmission shapes counterregulatory hormone secretion. Dorsomedial VMN Ghrh neurons express the metabolic-sensitive transcription factor steroidogenic factor-1/NR5A1 (SF-1). In vivo SF-1 gene knockdown tools were used here to address the premise that in male rats, SF-1 may regulate basal and/or hypoglycemic patterns of Ghrh, co-transmitter biosynthetic enzyme, and estrogen receptor (ER) gene expression in these neurons. Single-cell multiplex qPCR analyses showed that SF-1 regulates basal profiles of mRNAs that encode Ghrh and protein markers for neurochemicals that suppress (γ-aminobutyric acid) or enhance (nitric oxide; glutamate) counterregulation. SF-1 siRNA pretreatment respectively exacerbated or blunted hypoglycemia-associated inhibition of glutamate decarboxylase67 (GAD67/GAD1) and -65 (GAD65/GAD2) transcripts. Hypoglycemia augmented or reduced nitric oxide synthase and glutaminase mRNAs, responses that were attenuated by SF-1 gene silencing. Ghrh and Ghrh receptor transcripts were correspondingly refractory to or increased by hypoglycemia, yet SF-1 knockdown decreased both gene profiles. Hypoglycemic inhibition of ER-alpha and G protein-coupled-ER gene expression was amplified by SF-1 siRNA pretreatment, whereas as ER-beta mRNA was amplified. SF-1 knockdown decreased (corticosterone) or elevated [glucagon, growth hormone (GH)] basal counterregulatory hormone profiles, but amplified hypoglycemic hypercorticosteronemia and -glucagonemia or prevented elevated GH release. Outcomes document SF-1 control of VMN Ghrh neuron counterregulatory neurotransmitter and ER gene transcription. SF-1 likely regulates Ghrh nerve cell receptivity to estradiol and release of distinctive neurochemicals during glucose homeostasis and systemic imbalance. VMN Ghrh neurons emerge as a likely substrate for SF-1 control of glucose counterregulation in the male rat.


Introduction
The brain maintains systemic glucose homeostasis through control of synchronized autonomic, neuroendocrine, and behavioral motor functions that govern glucose dietary intake, cellular uptake and utilization, de novo synthesis, and storage as glycogen.Final common control of this vital motor outflow is imposed by the hypothalamus, the hierarchic visceral motor center of the brain.The ventromedial hypothalamic nucleus (VMN), a bilateral component of the mediobasal hypothalamus, is a key sensory and integrative element of the glucose-regulatory circuitry that assimilates telencephalic, diencephalic, and brainstem loci (Chan & Sherwin, 2013;Tu et al., 2022;Watts & Donovan, 2010).The transcription factor steroidogenic factor-1 (SF-1; NR5A1) is uniquely expressed in the VMN, where it shapes establishment of VMN nerve cell population phenotypes (Davis et al., 2004;McClellan et al., 2006).SF-1 gene repression negatively affects VMN cytoarchitectural organization, culminating in thermogenic, metabolic, and reproductive dysfunction (Kim et al., 2009;2012;Zhao et al., 2008).SF-1 expression within the boundaries of the VMN is not ubiquitous, as it occurs in the dorsomedial (VMMdm) and central (VMNc), but not ventrolateral VMN (VMNvl) (Cheung et al., 2013;Kim et al., 2019).SF-1 participation in the neural control of bodily energy and glucose homeostasis is well documented (Choi et al., 2013;Garfield et al., 2014;Kim et al., 2011;Meek et al., 2016;Xu et al., 2011;Zhang et al., 2008).
Recent studies sought to characterize SF-1-expressing VMNdm neurons by neurochemical phenotype to facilitate understanding of how regulatory effects of this transcription factor are communicated to the neural glucostatic network (Sapkota et al., 2023).The neuropeptide growth hormone-releasing hormone (Ghrh; e.g., somatocrinin) is expressed in the VMNdm (Burgunder, 1991), where it may participate in nonhypophysiotropic mechanisms that stimulate pituitary growth hormone (GH) secretion (Frohman et al., 1968).Using combinative in situ immunocytochemistry/laser-catapult-microdissection/single-cell multiplex qPCR methodology within the context of a validated in vivo experimental model for insulin-induced hypoglycemia (IIH) (Paranjape & Briski, 2005), that work showed that VMNdm Ghrh neurons express hypoglycemia-sensitive SF-1 mRNA and that Ghrh neurotransmission is critical for optimal hypoglycemic induction of counterregulatory hormone outflow.Outcomes also established the co-presence in microdissected Ghrh nerve cell samples of gene transcripts that encode enzyme markers for several characterized counterregulation-enhancing or -constraining neurotransmitters, namely the labile gas nitric oxide (NO) and the amino acids c-aminobutyric acid (GABA) and glutamate (Glu).A correlated observation was that Ghrh neurons express Ghrh receptor (Ghrh-R), which evidently mediates neuromodulatory effects on those gene profiles.Co-transmission of neurochemicals of diverse chemical structure, spatial, and temporal profiles that impose distinctive control of counterregulatory hormone secretion supports the prospect that VMNdm Ghrh neurons supply complex, coordinated multi-modal input to the brain glucose-regulatory network.The current research investigated the hypothesis that in VMNdm Ghrh neurons in the male rat brain, SF-1 regulates expression patterns of genes that encode the neuropeptide transmitter Ghrh and protein markers for co-expressed neurochemicals.Here, adult male rats were pretreated by bilateral administration of self-delivering Accell TM SF-1 or control/scramble siRNA to the VMN before subcutaneous (sc) insulin or vehicle injection.Ghrh neurons were collected by laser-catapult-microdissection from the VMNdm of each subject across all treatment groups for single-cell multiplex qPCR analysis of Ghrh, neuronal nitric oxide synthase (nNOS), glutamate decarboxylase 67 (GAD1/GAD 67 ), glutamate decarboxylase 65 (GAD1/ GAD 65 ), and glutaminase (GLS) transcript profiles.
The ultra-sensitive energy gauge 5'-AMP-activated protein kinase (AMPK) undergoes activation via phosphorylation in reaction to increased cellular AMP/ ATP ratio (Hardie et al., 2012(Hardie et al., , 2016;;Hardie & Lin, 2017).Hypothalamic AMPK supplies vital cues on cell energy stability to neural pathways that govern bodily energy balance (L� opez, 2018;Pimentel et al., 2013;Xue & Kahn, 2006).Ventromedial hypothalamic AMPK is implicated in neural regulation of hypoglycemic patterns of counterregulatory hormone outflow (Han et al., 2005;McCrimmon et al., 2008).Brain cell glucopenia may be directly monitored by SF-1expressing VMN neurons as these neurons express AMPK protein, which undergoes increased phosphorylation in response to hypoglycemia (Ibrahim et al., 2020).The AMPK alpha catalytic subunit exists as two isoforms, e.g., Prkaa1/AMPK-alpha1 (AMPKa1) and Prkaa2/AMPK-alpha2 (AMPKa2).These variants are activated to a comparable extent when AMP levels rise, but each exhibits distinctive, dissimilar substrate specificity which likely results in dissimilar effects on cell function by phosphorylation of different target proteins (Woods et al., 1996).Current research investigated the hypothesis that VMNdm Ghrh neurons express transcripts that produce one or both AMPKalpha variants and that these gene profiles are subject to regulation by SF-1 under eu-and/or hypoglycemic conditions.

Animals
Adult male Sprague Dawley rats (250-300 g bw) were housed in shoe-box cages (2-3 per cage), under a 14hr light: 10-hr dark cycle; lights on at 05.00 hr.Before experimentation was initiated, animals were acclimated on a daily basis to handling.Animals had free access to standard laboratory chow and tap water.Study protocols and procedures were performed in conformity with the NIH Guide for Care and Use of Laboratory Animals, 8th Edition, under approval by the ULM Institutional Animal Care and Use Committee, approval No. 22SEPT-KPB-01.

Statistics
Mean normalized mRNA, glucose, and counterregulatory hormone values were analyzed across treatment groups by two-way analysis of variance and Student Newman Keuls post-hoc test.Differences of p < 0.05 were considered significant.In each figure, statistical differences between specific pairs of treatment groups are denoted as follows: � p < 0.05; �� p < 0.01; ��� p < 0.001.

Results
VMNdm Ghrh neurons express the transcription factor SF-1, which exerts well-documented regulatory effects on energy and glucose homeostasis.Current research employed siRNA reagents to repress VMN SF-1 gene expression in vivo to investigate the premise that in male rat Ghrh neurons, SF-1 controls eu-and/ or hypoglycemia-associated expression patterns of gene transcripts that produce counterregulatory neurotransmitter biosynthetic pathway and ER variant proteins.Individual VMNdm Ghrh neurons were obtained by in situ gene immunocytochemistry/lasercatapult-microdissection methods for single-cell multiplex single-cell qPCR analyses.The correlated study hypothesis that VMN SF-1 gene expression is crucial for optimal eu-and/or hypoglycemic patterns of corticosterone, glucagon, and GH secretion in this sex was also be addressed.
The neuropeptide Ghrh, lipid-permeable gas NO, and amino acid glutamate each have documented stimulatory effects on counterregulatory outflow, and are co-expressed in VMNdm Ghrh neurons.Figure 3 illustrates the effects of SF-1 gene knockdown on genes that encode Ghrh (Figure 3A) or the transmitter biosynthetic enzyme markers nNOS (Figure 3C) and GLS (Figure 3D).    Figure 6 depicts the effects of VMN SF-1 gene knockdown on circulating glucose and counterregulatory hormone concentrations in eu-and hypoglycemic male rats.As seen in Figure 6A

Discussion
The transcription factor SF-1, uniquely expressed in the VMN, acts to regulate systemic glucostasis.VMNdm Ghrh neurons express SF-1 mRNA (Sapkota et al., 2023).Here, in vivo gene silencing tools were used together with single-cell multiplex qPCR and ELISA methods to address the premise that SF-1 may govern Ghrh nerve cell counterregulatory neurotransmission and counterregulatory hormone secretion in the male rat.Study outcomes affirm SF-1 control of basal and hypoglycemic expression patterns of mRNAs that encode enzyme markers for NO, glutamate, and GABA.This transcription factor also shapes Ghrh neuron receptivity to Ghrh and estradiol.SF-1 up-regulates baseline ERa, ERb, and GPER gene expression in this cell type, but elicits distinctive adjustments in ER variant transcription during hypoglycemia.Results provide unique evidence that SF-1 governance of AMPKa isoform gene profiles shift from inhibitory-to-stimulatory during glucostasis versus imbalance.Data moreover substantiate VMN SF-1 regulation of circulating corticosterone, glucagon, and GH concentrations during eu-and hypoglycemia.Further research is warranted to establish whether, how, and where distinctive SF-1 -sensitive neurotransmitters characterized here may communicate sequelae of SF-1 transcriptional regulation to the neural glucostatic network.There also remains a need for insight on if and how signaling by individual ER variants may affect SF-1 control of Ghrh counterregulatory neurotransmission.
The overarching goal of the current research was to obtain proof-of-concept that genetic manipulation of VMN SF-1 gene expression affects VMNdm Ghrh neuron estrogen receptor variant, energy sensor, and/ or transmitter marker protein gene expression.Given the considerable quantity of work required to achieve this goal, we chose to begin by using a single sex, the male, as the experimental subject as this sex has been the default choice over decades of neuroscience research.Present outcomes provide solid justification for the expansion of this line of research through the application of the current experimental design in studies on female.
Present data corroborate earlier reports that Ghrh neurons are characterized by the co-presence of SF-1 transcripts with mRNAs that produce chemicallydiverse transmitters of documented impact on counterregulation, i.e., Ghrh, NO, glutamate, and GABA) (Sapkota et al., 2023).Findings here extend those observations with novel proof that SF-1 regulates basal and hypoglycemic expression patterns of these genes.Outcomes validate the efficacy of the present gene knockdown treatment paradigm, involving bilateral administration of gene-targeted or SCR siRNA to the VMN, for down-regulation of SF-1 gene and protein expression in VMNdm Ghrh neurons.The issue of whether additional neurotransmitter nerve populations in that discrete nuclear subdivision express SF-1 remains unanswered.It is noted that quantifiable experimental stimulus-induced changes in SF-1 mRNA profiles, such as hypoglycemia-associated decrements in SF-1 gene expression observed here, do not provide confirmation that Ghrh SF-1 protein undergoes changes of parallel magnitude.Hence, the possibility that SF-1 protein expression in this cell type may be refractory to hypoglycemia, despite decreased SF-1 transcription, should not be discounted.Observations here of diminished Ghrh nerve cell Ghrh mRNA profiles in SF-1 siRNA-pretreated, V-or INS-injected rats relative to SCR-pretreated controls infer that Ghrh neuropeptide signaling is upregulated by this transcription factor.Current data expand upon recent evidence that VMNdm Ghrh neurons express Ghrh-R gene transcripts with verification that this mRNA profile is augmented by SF-1 during eu-and hypoglycemia, and that this transcription factor may be a primary factor in hypoglycemic amplification of Ghrh-R gene expression.
In the brain, the conversion of glutamate to GABA is catalyzed by the biosynthetic enzyme GAD, which occurs as 67 kDa (GAD1/GAD 67 ) and 65 kDa (GAD2/ GAD 65 ) kDa size variants encoded by distinct genes.Prior and current data affirm that GAD1 and GAD2 transcripts are co-expressed in VMNdm Ghrh neurons.These molecular weight variants differ with regard to amino acid primary structure, neuronal subcellular distribution, and regulation.GAD1/GAD 67 expression is subject to transcriptional and posttranscriptional control, whereas GAD2/GAD 65 is controlled by transcriptional and kinetic mechanisms (Behar, 2009).Localization of GAD2/GAD 65 protein to axon terminals and vesicles versus intra-cytoplasmic GAD1/GAD 67 infers the existence of distinctive vesicular versus cytoplasmic GABA pools and a potential role for GAD2/GAD 65 in neurotransmission compared to GAD1/GAD 67 involvement in cellular metabolic functions (Martin & Barke, 1998; production and activity in this nerve cell type during hypoglycemia; data suggest that SF-1 may inhibit the former pool while stimulating the latter.The direction of SF-1 regulation of GAD2 transcription profiles is evidently dependent upon glucose status, as this control shifts from stimulatory-to-inhibitory during euversus hypoglycemia; elucidation of the mechanisms that mediate this directional switch will require further investigation. Previous studies showed that VMNdm Ghrh neurons express a critical enzyme component (i.e., GLS) of the biosynthetic pathway that manufactures the counterregulation-enhancing amino acid neurotransmitter glutamate (Sapkota et al., 2023).New results described here reveal that SF-1 suppresses GLS gene expression during eu-or hypoglycemia, and support the view that this transcription factor may mediate hypoglycemic inhibition of this gene profile.Ghrh neurons also release the diffusible counterregulationstimulating free radical NO, which shows that this cell type engages in both non-receptor-and receptormediated signaling to elevate circulating glucose levels.Data here show that SF-1 gene silencing down-regulated basal and hypoglycemic patterns of nNOS gene expression, denoting a positive impact of this transcription factor on NO release.Thus, SF-1 evidently imposes the opposite, i.e., inhibitory versus stimulatory effects on counterrregulatory-enhancing neurochemicals glutamine and NO produced by Ghrh neurons.Outstanding issues that remain to be addressed include identification of cellular targets and functional consequences of SF-1 -dependent release of glutamate versus NO from VMNdm Ghrh neurons for counterregulatory outflow.
Current studies provide unique proof that hypoglycemia-sensitive AMPKa1 and AMPKa2 gene transcripts are co-expressed in VMNdm Ghrh neurons.The presence of molecular machinery for energy screening infers that this nerve cell type likely utilizes intrinsic metabolic cues to shape neurochemical signaling.It remains to be determined if AMPK activity state controls production and release of all or a subset of co-expressed transmitters.Data disclose that SF-1 inhibits expression of both AMPKa variant mRNAs in Ghrh neurons during euglycemia, yet is paradoxically crucial for hypoglycemic up-regulation of these gene profiles.Further research is warranted to characterize mechanisms that achieve this evident glucose-dependent shift from SF-1 inhibitory-to-stimulatory control of AMPKa mRNA expression.While current outcomes support the view that SF-1 may control adaptation of Ghrh nerve cell energy sensory function to glucose imbalance, it should be noted that effects of SF-1 knockdown alone or in combination with hypoglycemia on phosphorylation state of either AMPKa variant were not evaluated here.Thus, insight on the direction and magnitude of SF-1 regulatory impact on AMPK activity during eu-or hypoglycemia will require additional effort.
Project results show that SF-1 up-regulates expression of nuclear and membrane ER variant genes in VMNdm Ghrh/SF-1 neurons, inferring that this transcription factor may exert a net positive impact on cellular receptivity to estradiol.It is noted that measured adjustments in ER variant mRNA levels should not be viewed as definitive proof of corresponding changes in receptor protein production.This notion will remain speculative until analytical methods of requisite sensitivity for quantification of these proteins in single-cell samples become available.Outcomes reported here confirm that Ghrh neurons acquired from male rats exhibit down-regulated ERa, ERb, and GPER gene profiles during hypoglycemia (Sapkota et al., 2023).While SF-1 evidently acts to antagonize hypoglycemic down-regulation of ERa and GPER gene expression, this transcription factor may coincidently inhibit ERb transcription.The mechanisms that underlie this glucose-dependent switch from stimulatory to inhibitory SF-1 control of Ghrh nerve cell ERb gene expression are not known.Additional research is also warranted to characterize the regulatory stimuli that impose inhibitory effects of hypoglycemia on Ghrh neuron ERa and GPER mRNA content.There is, above all, an urgent need to understand if SF-1dependent changes in ER variant-mediated estradiol signaling to Ghrh neurons during hypoglycemia are involved in neurotransmitter protein marker gene responses to glucose imbalance.
Application of in vivo SF-1 gene knockdown tools confirms a role for this VMN transcription factor in counterregulatory hormone release during hypoglycemia (Choi et al., 2013;Kim et al., 2012;Tong et al., 2007Tong et al., 2013)).Data presented here show that SF-1 imposes distinctive regulatory effects on basal circulating counterregulatory hormone profiles, as SF-1 siRNA reduced circulating corticosterone levels, yet elevated glucagon and GH concentrations.The direction of SF-1 control of corticosterone is seemingly glucose-dependent as hypoglycemic hypercorticosteronemia was seen to be enhanced by SF-1 gene silencing.An inverse scenario concerns SF-1 regulation of GH secretion; while SF-1 acts to inhibit GH secretion during euglycemia, this transcription factor evidently mediates hypoglycemic stimulation of this hormone profile.On the other hand, SF-1 has a consistently negative impact on glucagon release irrespective of systemic glucose status.Further research is required to identify the Ghrh nerve cell neurotransmitter(s) that impose SF-1-dependent control of individual counterregulatory hormones.Outcomes show that SF-1 gene knockdown did not modify plasma glucose levels after INS injection, despite measurable effects on circulating corticosterone, glucagon, and GH.As glucose measures were acquired at a single time point following INS treatment, the prospect that siRNA treatment might have affected glycemic profiles over some time interval prior to sacrifice cannot be discounted.It is reasonable to presume that circulating glucose concentrations may undergo dynamic change due to VMN SF-1 governance of counter-regulatory hormone release, or control of neural mechanisms governing hepatic gluconeogenic or glycogenolytic functions.Current results may thus provide a snapshot of a temporal phase during which plasma glucose levels are normalized after INS administration as an adaptive reaction to SF-1 -dependent actions that control contra-regulatory outflow.
Summary of project outcomes: Present data support the likely function of VMNdm Ghrh neurons as a cellular effector of SF-1 regulation of systemic glucostasis in the male rat.SF-1 regulates expression patterns of genes that encode the neuropeptide transmitter Ghrh as well as co-expressed counterregulation-inhibiting or -enhancing neurochemicals of distinctive chemical structure and spatial/temporal release profiles; these outcomes infer that SF-1 may coordinate complex, multi-modal neurotransmitter input to the brain glucostatic circuitry.Continuing research efforts intend to address the issue of whether such control involves AMPK-and/or ER-dependent mechanisms.Results provide novel evidence for SF-1 regulation of basal counterregulatory hormone secretion profiles in the male, documenting stimulation of corticosterone versus inhibition of glucagon and GH.This transcription factor also imposes distinctive control of these hormone profiles during hypoglycemia.Current research supports the need to characterize downstream cell targets of SF-1 -dependent neurotransmitter signaling and functional impact of this input on systems-level neural regulation of glucostasis.
TM 6.0.0 software.Bio-Rad Stain-Free gels contain a proprietary trihalo compound that is directly incorporated into the gel chemistry; this compound lacks inherent fluorescence, but renders in-gel proteins fluorescent upon UV photoactivation and thus measurable by O.D. Software sums all individual protein optical densities in a single lane, and relates that total protein O.D. value to target protein O.D. in the same lane, thereby deriving a normalized O.D. value.Each Western blot analysis employed precision plus protein molecular weight dual color standards (prod.no.161-0374, Bio-Rad).Our figures depict, as Y axis labels denote mean normalized O.D. measures.The formula used for normalization is the ratio of specific target protein O.D./total in-lane protein O.D.

Figure 1 .
Figure1.Effects of ventromedial hypothalamic nucleus steroidogenic factor-1 (SF-1) gene knockdown on dorsomedial VMN (VMNdm) growth hormone releasing hormone (Ghrh) neuron SF-1 mRNA and VMNdm SF-1 protein profiles in Eu-versus hypoglycemic male rats.Male rats were randomly assigned to treatment groups (n ¼ 6/group) wherein animals were pretreated by bilateral intra-VMN administration of SF-1 or control/scramble (SCR) siRNA seven days prior to subcutaneous (sc) injection of vehicle (V) or neutral protamine hagedorn insulin (INS; 10.0 U/kg bw).Individual brains were into alternating 10 or 100 lm-thick fresh frozen sections for laser-catapult-microdissection of individual Ghrh-immunopositive neurons or micropunch dissection of VMNdm tissue, respectively.Figure A depicts results of single-cell qPCR SF-1 mRNA analysis.Data are presented in box-and-whisker plot format, which displays the median, lower and upper quartiles, and lower and upper extremes of a data set.Plots depict mean normalized VMNdm Ghrh neuron SF-1 transcript measures for the following treatment groups: SCR siRNA/V (green box-and-whisker plots, n ¼ 12); SF-1 siRNA/V (purple box-and-whisker plots; n ¼ 12); SCR siRNA/INS (golden box-and-whisker plots; n ¼ 12; SF-1 siRNA/INS (blue box-and-whisker plots; male: n ¼ 12).For each treatment group, aliquots of micropunched VMNdm tissue obtained from each animal were combined to create triplicate sample pools for Western blot analysis of SF-1 protein.Figure B depicts mean SF-1 protein optical density values for the treatment groups described above.mRNA and protein data were analyzed by two-way ANOVA and Student-Neuman-keuls post-hoc test, using GraphPad prism, vol.8 software.Statistical differences between discrete pairs of treatment groups are denoted as follows: � p < 0.05; �� p < 0.01; ��� p < 0.001.

Figure 6 .
Figure 6.Effects of VMN SF-1 gene knockdown on plasma glucose and Counter-Regulatory hormone profiles in Eu-or hypoglycemic male rats.Plasma samples were obtained from groups of SF-1 or SCR siRNA-pretreated male rats one hour after sc injection of V or INS, and analyzed for glucose (A), corticosterone (B), glucagon (C), or growth hormone (D) concentrations.In each panel, individual treatment group data depict mean plasma concentrations ± S.E.M. for n ¼ 6 samples.Data were analyzed by two-way ANOVA and Student-Neuman-keuls post-hoc test, using GraphPad prism, vol.8 software.� p < 0.05, �� p < 0.01, ��� p < 0.001.