Identification of in vivo roles of ErbB4-JMa and its direct nuclear signaling using a novel isoform-specific knock out mouse.

Like all receptor tyrosine kinases (RTKs), ErbB4 signals through a canonical signaling involving phosphorylation cascades. However, ErbB4 can also signal through a non-canonical mechanism whereby the intracellular domain is released into the cytoplasm by regulated intramembrane proteolysis (RIP) and translocates to the nucleus where it regulates transcription. These different signaling mechanisms depend on the generation of alternative spliced isoforms, a RIP cleavable ErbB4-JMa and an uncleavable ErbB4-JMb. Non-canonical signaling by ErbB4-JMa has been implicated in the regulation of brain, heart, mammary gland, lung, and immune cell development. However, most studies on non-canonical ErbB4 signaling have been performed in vitro due to the lack of an adequate mouse model. We created an ErbB4-JMa specific knock out mouse and demonstrate that RIP-dependent, non-canonical signaling by ErbB4-JMa is required for the regulation of GFAP expression during cortical development. We also show that ErbB4-JMa signaling is not required for the development of the heart, mammary glands, sensory ganglia. Furthermore, we identify genes whose expression during cortical development is regulated by ErbB4, and show that the expression of three of them, CRYM and DBi, depend on ErbB4-JMa whereas WDFY1 relies on ErbB4-JMb. Thus, we provide the first animal model to directly study the roles of ErbB4-JMa and non-canonical ErbB4 signaling in vivo.

Effects of short-term food deprivation on catecholamine and metabolic-sensory biomarker gene expression in hindbrain A2 noradrenergic neurons projecting to the forebrain rostral preoptic area: Impact of negative versus positive estradiol feedback.

Hindbrain A2 noradrenergic neurons assimilate estrogenic and metabolic cues. In female mammals, negative- versus positive-feedback patterns of estradiol (E) secretion impose divergent regulation of the gonadotropin-releasing hormone (GnRH)-pituitary-gonadal (HPG) neuroendocrine axis. Current research used retrograde tracing, dual-label immunocytochemistry, single-cell laser-microdissection, and multiplex qPCR methods to address the premise that E feedback modes uniquely affect metabolic regulation of A2 neurons involved in HPG control. Ovariectomized female rats were given E replacement to replicate plasma hormone levels characteristic of positive (high-E dose) or negative (low-E dose) feedback. Animals were either full-fed (FF) or subjected to short-term, e.g., 18-h food deprivation (FD). After FF or FD, rostral preoptic area (rPO)-projecting A2 neurons were characterized by the presence or absence of nuclear glucokinase regulatory protein (nGKRP) immunostaining. FD augmented or suppressed mRNAs encoding the catecholamine enzyme dopamine-beta-hydroxylase (DßH) and the metabolic-sensory biomarker glucokinase (GCK), relative to FF controls, in nGKRP-immunoreactive (ir)-positive A2 neurons from low-E or high-E animals, respectively. Yet, these transcript profiles were unaffected by FD in nGKRP-ir-negative A2 neurons at either E dosage level. FD altered estrogen receptor (ER)-alpha and ATP-sensitive potassium channel subunit sulfonylurea receptor-1 gene expression in nGKRP-ir-positive neurons from low-E, but not high-E animals. Results provide novel evidence that distinct hindbrain A2 neuron populations exhibit altered versus unaffected transmission to the rPO during FD-associated metabolic imbalance, and that the direction of change in this noradrenergic input is controlled by E feedback mode. These A2 cell types are correspondingly distinguished by FD-sensitive or -insensitive GCK, which correlates with the presence versus absence of nGKRP-ir. Further studies are needed to determine how E signal volume regulates neurotransmitter and metabolic sensor responses to FD in GKRP-expressing A2 neurons.

Ventromedial hypothalamic nucleus glycogen regulation of metabolic-sensory neuron AMPK and neurotransmitter expression: role of lactate.

Astrocyte glycogen is dynamically remodeled during metabolic stability and provides oxidizable l-lactate equivalents during neuroglucopenia. Current research investigated the hypothesis that ventromedial hypothalamic nucleus (VMN) glycogen metabolism controls glucostimulatory nitric oxide (NO) and/or glucoinhibitory gamma-aminobutyric acid (GABA) neuron 5'-AMP-activated protein kinase (AMPK) and transmitter marker, e.g., neuronal nitric oxide synthase (nNOS), and glutamate decarboxylase65/67 (GAD) protein expression. Adult ovariectomized estradiol-implanted female rats were injected into the VMN with the glycogen phosphorylase inhibitor 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) before vehicle or l-lactate infusion. Western blot analysis of laser-catapult-microdissected nitrergic and GABAergic neurons showed that DAB caused lactate-reversible upregulation of nNOS and GAD proteins. DAB suppressed or increased total AMPK content of NO and GABA neurons, respectively, by lactate-independent mechanisms, but lactate prevented drug enhancement of pAMPK expression in nitrergic neurons. Inhibition of VMN glycogen disassembly caused divergent changes in counter-regulatory hormone, e.g. corticosterone (increased) and glucagon (decreased) secretion. Outcomes show that VMN glycogen metabolism controls local glucoregulatory transmission by means of lactate signal volume. Results implicate glycogen-derived lactate deficiency as a physiological stimulus of corticosterone release. Concurrent normalization of nitrergic neuron nNOS and pAMPK protein and corticosterone secretory response to DAB by lactate infers that the hypothalamic-pituitary-adrenal axis may be activated by VMN NO-mediated signals of cellular energy imbalance.

Norepinephrine Regulation of Ventromedial Hypothalamic Nucleus Astrocyte Glycogen Metabolism.

The catecholamine norepinephrine (NE) links hindbrain metabolic-sensory neurons with key glucostatic control structures in the brain, including the ventromedial hypothalamic nucleus (VMN). In the brain, the glycogen reserve is maintained within the astrocyte cell compartment as an alternative energy source to blood-derived glucose. VMN astrocytes are direct targets for metabolic stimulus-driven noradrenergic signaling due to their adrenergic receptor expression (AR). The current review discusses recent affirmative evidence that neuro-metabolic stability in the VMN may be shaped by NE influence on astrocyte glycogen metabolism and glycogen-derived substrate fuel supply. Noradrenergic modulation of estrogen receptor (ER) control of VMN glycogen phosphorylase (GP) isoform expression supports the interaction of catecholamine and estradiol signals in shaping the physiological stimulus-specific control of astrocyte glycogen mobilization. Sex-dimorphic NE control of glycogen synthase and GP brain versus muscle type proteins may be due, in part, to the dissimilar noradrenergic governance of astrocyte AR and ER variant profiles in males versus females. Forthcoming advances in the understanding of the molecular mechanistic framework for catecholamine stimulus integration with other regulatory inputs to VMN astrocytes will undoubtedly reveal useful new molecular targets in each sex for glycogen mediated defense of neuronal metabolic equilibrium during neuro-glucopenia.

HPLC-electrospray ionization-mass spectrometry optimization by high-performance design of experiments for astrocyte glutamine measurement.

The amino acid glutamine (Gln) is a likely source of energy in the brain during neuroglucopenia. Effects of glucose deficiency on astrocyte Gln homeostasis remain unclear, as analytical tools of requisite sensitivity for quantification of intracellular levels of this molecule are not currently available. Here, a primary hypothalamic astrocyte culture model was used in conjunction with design of experiments (DOE)-refined high-performance liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) methodology to investigate the hypothesis that glucoprivation alters astrocyte Gln content in a sex-specific manner. Critical mass spectrometric parameters for Gln derivative chromatographic response were identified by comparing the performance of central composite design, Box-Behnken design, and Optimal Design (OD)-A, -D, -I, -Distance, and -Modified Distance DOE models. The outcomes showed that the OD-A-generated response was superior relative to other design outcomes. Forecasted surface plot critical mass spectrometric parameters were maximized by OD-A, OD-Distance, and OD-Modified Distance designs. OD-A produced a high-performance method that yielded experimental run and forecasted surface plot maximal responses. Optimized mass spectrometric analysis of male versus female astrocyte Gln content provides novel evidence that glucoprivation significantly depletes this amino acid in female, but not in male, and that this sex-specific response may involve differential sensitivity to estrogen receptor signaling. This technological advance will facilitate efforts to ascertain how distinctive physiological and pathophysiological stimuli impact astrocyte Gln metabolism in each sex.

Sex-dimorphic Rostro-caudal Patterns of 5'-AMP-activated Protein Kinase Activation and Glucoregulatory Transmitter Marker Protein Expression in the Ventrolateral Ventromedial Hypothalamic Nucleus (VMNvl) in Hypoglycemic Male and Female Rats: Impact of Estradiol.

The ventromedial hypothalamic nucleus-ventrolateral part (VMNvl) is an estradiol-sensitive structure that controls sex-specific behavior. Electrical reactivity of VMNvl neurons to hypoglycemia infers that cellular energy stability is monitored there. Current research investigated the hypothesis that estradiol elicits sex-dimorphic patterns of VMNvl metabolic sensor activation and gluco-regulatory neurotransmission during hypoglycemia. Rostral-, middle-, and caudal-VMNvl tissue was separately micropunch-dissected from letrozole (Lz)- or vehicle-injected male and estradiol- or vehicle-implanted ovariectomized (OVX) female rats for Western blot analysis of total and phosphorylated 5'-AMP-activated protein kinase (AMPK) protein expression and gluco-stimulatory [neuronal nitric oxide synthase (nNOS); steroidogenic factor-1 (SF1) or -inhibitory (glutamate decarboxylase65/67 (GAD)] transmitter marker proteins after sc insulin (INS) or vehicle injection. In both sexes, hypoglycemic up-regulation of phosphoAMPK was estradiol-dependent in rostral and middle, but not caudal VMNvl. AMPK activity remained elevated after recovery from hypoglycemia over the rostro-caudal VMNvl in female, but only in the rostral segment in male. In each sex, hypoglycemia correspondingly augmented or suppressed nNOS profiles in rostral and middle versus caudal VMNvl; these segmental responses persisted longer in female. Rostral and middle segment SF1 protein was inhibited by estradiol-independent mechanisms in hypoglycemic males, but increased by estradiol-reliant mechanisms in female. After INS injection, GAD expression was inhibited in the male rostral VMNvl without estradiol involvement, but this hormone was required for broader suppression of this profile in the female. Neuroanatomical variability of VMNvl metabolic transmitter reactivity to hypoglycemia underscores the existence of functionally different subgroups in that structure. The regional distribution and estradiol sensitivity of hypoglycemia-sensitive VMNvl neurons of each neurochemical phenotype evidently vary between sexes.

Sex-dimorphic neuroestradiol regulation of ventromedial hypothalamic nucleus glucoregulatory transmitter and glycogen metabolism enzyme protein expression in the rat.

BACKGROUND: Ventromedial hypothalamic nucleus (VMN) gluco-regulatory transmission is subject to sex-specific control by estradiol. The VMN is characterized by high levels of aromatase expression. METHODS: The aromatase inhibitor letrozole (LZ) was used with high-resolution microdissection/Western blot techniques to address the hypothesis that neuroestradiol exerts sex-dimorphic control of VMN neuronal nitric oxide synthase (nNOS) and glutamate decarboxylase65/67 (GAD) protein expression. Glycogen metabolism impacts VMN nNOS and GAD profiles; here, LZ treatment effects on VMN glycogen synthase (GS) and phosphorylase brain- (GPbb; glucoprivic-sensitive) and muscle (GPmm; norepinephrine-sensitive) variant proteins were examined. RESULTS: VMN aromatase protein content was similar between sexes. Intracerebroventricular LZ infusion of testes-intact male and ovariectomized, estradiol-replaced female rats blocked insulin-induced hypoglycemic (IIH) up-regulation of this profile. LZ exerted sex-contingent effects on basal VMN nNOS and GAD expression, but blocked IIH-induced NO stimulation and GAD suppression in each sex. Sex-contingent LZ effects on basal and hypoglycemic patterns of GPbb and GPmm expression occurred at distinctive levels of the VMN. LZ correspondingly down- or up-regulated baseline pyruvate recycling pathway marker protein expression in males (glutaminase) and females (malic enzyme-1), and altered INS effects on those proteins. CONCLUSIONS: Results infer that neuroestradiol is required in each sex for optimal VMN metabolic transmitter signaling of hypoglycemic energy deficiency. Sex differences in VMN GP variant protein levels and sensitivity to aromatase may correlate with sex-dimorphic glycogen mobilization during this metabolic stress. Neuroestradiol may also exert sex-specific effects on glucogenic amino acid energy yield by actions on distinctive enzyme targets in each sex.

Ultra-High-Performance Liquid Chromatography-Electrospray Ionization-Mass Spectrometry for High-Neuroanatomical Resolution Quantification of Brain Estradiol Concentrations.

Ventromedial hypothalamic nucleus (VMN) control of glucostasis is estradiol (E-2)-dependent. E-2 regulation of VMN reactivity to hypoglycemia may involve changes in signal volume due to altered aromatase expression. Here, high-resolution micropunch dissection tools for isolation of segmental VMN tissue were used with Design of Experiments-refined uHPLC-electrospray ionization-mass spectrometry (LC-ESI-MS) methodology to investigate the premise that effects of acute and/or recurring hypoglycemia on VMN E-2 content are sex-dimorphic. Relationships among multiple independent mass spectrometric operational variables were assessed by Central Composite Design (CCD) to amplify E-2 chromatogram area. Combinations of spectrometric temperature and gas pressure variable combinations were screened by Akaike Information Criterion correction modeling. A Fibonacci Sequence design using CCD minimum and maximal variable limits produced a small-run model that replicated maximal response from CCD. E-2 chromatographic response was further enhanced by optimization of solid phase extraction and instrument source and collision-induced dissociation voltages. In male rats, acute and chronic hypoglycemia respectively elevated or diminished E-2 concentrations relative to baseline in both rostral and caudal VMN. However, females exhibited regional variability in tissue E-2 profiles during acute (increased, rostral VMN; no change, caudal VMN) and recurring (no change, rostral VMN; increased, caudal VMN) hypoglycemia. Outcomes demonstrate requisite LC-ESI-MS sensitivity for E-2 quantification in small-volume brain tissue samples acquired with high-neuroanatomical specificity. Current methodology will facilitate efforts to investigate physiological consequences of VMN rostro-caudal segment-specific acclimation of E-2 profiles to recurring hypoglycemia, including effects on gluco-regulatory function, in each sex.

Sex-dimorphic aromatase regulation of ventromedial hypothalamic nucleus glycogen content in euglycemic and insulin-induced hypoglycemic rats.

Estrogen receptors control hypothalamic astrocyte glycogen accumulation in vitro. Glycogen metabolism impacts metabolic transmitter signaling in the ventromedial hypothalamic nucleus (VMN), a key glucoregulatory structure. Aromatase, the enzyme that converts testosterone to estradiol, is expressed at high levels in the VMN. Here, the aromatase inhibitor letrozole (Lz) was used alongside high-resolution microdissection/UPHLC-electrospray ionization-mass spectrometric methods to determine if neuroestradiol imposes sex-specific control of VMN glycogen content during glucostasis and/or glucoprivation. Testes-intact male and estradiol-replaced ovariectomized female rats were pretreated by lateral ventricular letrozole (Lz) infusion prior to subcutaneous insulin (INS) injection. Vehicle-treated female controls exhibited higher VMN glycogen content compared to males. Lz increased VMN glycogen levels in males, not females. INS-induced hypoglycemia (IIH) elevated (males) or diminished (females) rostral VMN glycogen accumulation. Induction of IIH in Lz-pretreated animals reduced male VMN glycogen mass, but augmented content in females. Data provide novel evidence for regional variation, in both sexes, in glycogen reactivity to IIH. Results highlight sex-dimorphic neuroestradiol regulation of VMN glycogen amassment during glucostasis, e.g. inhibitory in males versus insignificant in females. Locally-generated estradiol is evidently involved in hypoglycemic enhancement of male VMN glycogen, but conversely limits glycogen content in hypoglycemic females. Further research is needed to characterize mechanisms that underlie the directional shift in aromatase regulation of VMN glycogen in eu- versus hypoglycemic male rats and gain in negative impact in hypoglycemic females.

Sex differences in glucoprivic regulation of glycogen metabolism in hypothalamic primary astrocyte cultures: Role of estrogen receptor signaling.

Hypoglycemia causes sex-reliant changes in hypothalamic astrocyte glycogen metabolism in vivo. The role of nuclear versus membrane astrocyte estrogen receptors (ER) in glucoprivic regulation of glycogen is unclear. Here, primary hypothalamic astrocyte cultures were treated with selective ER antagonists during glucoprivation to investigate the hypothesis that ER mediate sex-specific glycogen responses to glucoprivation. Results show that glucoprivic down-regulation of glycogen synthase expression is mediated by transmembrane G protein-coupled ER-1 (GPER) signaling in each sex and estrogen receptor (ER)-beta (ERß) activity in females. Glucoprivic inhibition of glycogen phosphorylase involves GPER and ERß in females, but ER-independent mechanisms in males. GPER, ERß, and ER-alpha (ERα) inhibit or stimulate AMPK protein expression in male versus female astrocytes, respectively. Glucoprivic augmentation of phospho-AMPK profiles in male glia was opposed by GPER activation, whereas GPER and ERß suppress this protein in females. Astrocyte ERα and GPER content was down-regulated in each sex during glucose deficiency, whereas ERß levels was unaltered (males) or increased (females). Glucoprivation correspondingly elevated or diminished male versus female astrocyte glycogen content; ER antagonism reversed this response in males, but not females. Results identify distinctive ER variants involved in sex-similar versus sex-specific astrocyte protein responses to withdrawal of this substrate fuel. Notably, glucoprivation elicits a directional switch or gain-of-effect of GPER and ERß on specific glial protein profiles. Outcomes infer that ERs are crucial for glucoprivic regulation of astrocyte glycogen accumulation in males. Alternatively, estradiol may act independently of ER signaling to disassemble this reserve in females.

Sex-dimorphic moderate hypoglycemia preconditioning effects on Hippocampal CA1 neuron bio-energetic and anti-oxidant function.

Hypoglycemia is a detrimental complication of rigorous management of type 1 diabetes mellitus. Moderate hypoglycemia (MH) preconditioning of male rats partially affords protection from loss of vulnerable brain neurons to severe hypoglycemia (SH). Current research investigated whether MH preconditioning exerts sex-dimorphic effects on hippocampal CA1 neuron bio-energetic and anti-oxidant responses to SH. SH up-regulated CA1 glucose or monocarboxylate transporter proteins in corresponding hypoglycemia-naïve male versus female rats; precedent MH amplified glucose transporter expression in SH irrespective of sex. Sex-differentiating SH effects on glycolytic and tricarboxylic pathway markers correlated with elevated tissue ATP content and diminished CA1 5'-AMP-activated protein kinase (AMPK) activation in females. MH-preconditioned suppression of mitochondrial energy pathway enzyme profiles and tissue ATP in SH rats coincided with amplified CA1 AMPK activity in both sexes. Anti-oxidative stress enzyme protein responses to SH were primarily sex-contingent; preconditioning amplified most of these profiles, yet exacerbated expression of lipid and protein oxidation markers in SH male and female rats, respectively. Results show that MH preconditioning abolishes female CA1 neuron neuroprotection of positive energy balance through SH, resulting in augmented CA1 AMPK activity and oxidative injury and diminished tissue ATP in hypoglycemia-conditioned versus naïve rats in each sex. It is unclear if SH elicits differential rates of CA1 neuronal destruction in the two sexes, or how MH may impact sex-specific cell loss. Further research is needed to determine if molecular mechanism(s) that maintain female CA1 neuron metabolic stability in the absence of MH preconditioning can be leveraged for therapeutic prevention of hypoglycemic nerve cell damage.

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

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-ß-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.

Norepinephrine Regulation of Ventromedial Hypothalamic Nucleus Metabolic-Sensory Neuron 5'-AMP-Activated Protein Kinase Activity: Impact of Estradiol.

The mediobasal hypothalamus (MBH) shapes the neural regulation of glucostasis by 5'-AMP-activated protein kinase (AMPK)-dependent mechanisms. Yet, the neurochemical identity and neuroanatomical distribution of MBH neurons that express glucoprivic-sensitive AMPK remain unclear. The neurotransmitters γ-aminobutyric acid (GABA) and nitric oxide (NO) act within the MBH to correspondingly inhibit or stimulate glucose counter-regulation. The current review highlights recent findings that GABA and NO, neurons located in the ventromedial hypothalamic nucleus (VMN), a distinct important element of the MBH, are direct targets of noradrenergic regulatory signaling, and thereby, likely operate under the control of hindbrain metabolic-sensory neurons. The ovarian hormone estradiol acts within the VMN to govern energy homeostasis. Discussed here is current evidence that estradiol regulates GABA and NO nerve cell receptivity to norepinephrine and moreover, controls the noradrenergic regulation of AMPK activity in each cell type. Future gains in insight on mechanisms underpinning estradiol's impact on neurotransmitter communication between the hindbrain and hypothalamic AMPKergic neurons are expected to disclose viable new molecular targets for the therapeutic simulation of hormonal enhancement of neuro-metabolic stability during circumstances of diminished endogenous estrogen secretion or glucose dysregulation.

Effects of acute versus recurrent insulin-induced hypoglycemia on ventromedial hypothalamic nucleus metabolic-sensory neuron AMPK activity: Impact of alpha1-adrenergic receptor signaling.

Mechanisms that underlie metabolic sensor acclimation to recurring insulin-induced hypoglycemia (RIIH) are unclear. Norepinephrine (NE) regulates ventromedial hypothalamic nucleus (VMN) gluco-stimulatory nitric oxide (NO) and gluco-inhibitory γ-aminobutryic acid (GABA) neuron signaling. Current research addressed the hypothesis that during RIIH, NE suppresses 5'-AMP-activated protein kinase (AMPK) reactivity in both populations and impedes counter-regulation. The brain is postulated to utilize non-glucose substrates, e.g. amino acids glutamine (Gln), glutamate (Glu), and aspartate (Asp), to produce energy during hypoglycemia. A correlated aim investigated whether NE controls pyruvate recycling pathway marker protein (glutaminase, GLT; malic enzyme, ME-1) expression in either metabolic-sensory cell population. Male rats were injected subcutaneously with vehicle or insulin on days 1-3, then pretreated on day 4 by intracerebroventricular delivery of the alpha1-adrenergic receptor (α1-AR) reverse-agonist prazocin (PRZ) or vehicle before final insulin therapy. PRZ prevented acute hypoglycemic augmentation of AMPK activation in each cell group. Antecedent hypoglycemic repression of sensor activity was reversed by PRZ in GABA neurons. During RIIH, nitrergic neurons exhibited α1-AR - dependent up-regulated GLT and α2-AR profiles, while GABA cells showed down-regulated α1-AR. LC-ESI-MS analysis documented a decline in VMN Glu, Gln, and Asp concentrations during acute hypoglycemia, and habituation of the former two profiles to RIIH. PRZ attenuated glucagon and corticosterone secretion during acute hypoglycemia, but reversed decrements in output of both hormones during RIIH. Results implicate adjustments in impact of α1-AR signaling in repressed VMN metabolic-sensory AMPK activation and counter-regulatory dysfunction during RIIH. Antecedent hypoglycemia may up-regulate NO neuron energy yield via α1-AR - mediated up-regulated pyruvate recycling.

Sex-specific estrogen regulation of hypothalamic astrocyte estrogen receptor expression and glycogen metabolism in rats.

Brain astrocytes are implicated in estrogenic neuroprotection against bio-energetic insults, which may involve their glycogen energy reserve. Forebrain estrogen receptors (ER)-alpha (ERα) and -beta (ERß) exert differential control of glycogen metabolic enzyme [glycogen synthase (GS); phosphorylase (GP)] expression in hypoglycemic male versus female rats. Studies were conducted using a rat hypothalamic astrocyte primary culture model along with selective ER agonists to investigate the premise that estradiol (E2) exerts sex-dimorphic control over astrocyte glycogen mass and metabolism. Female astrocyte GS and GP profiles are more sensitive to E2 stimulation than the male. E2 did not regulate expression of phospho-GS (inactive enzyme form) in either sex. Data also show that transmembrane G protein-coupled ER-1 (GPER) signaling is implicated in E2 control of GS profiles in each sex and alongside ERα, GP expression in females. E2 increases total 5'-AMP-activated protein kinase (AMPK) protein in female astrocytes, but stimulated pAMPK (active form) expression with equivalent potency via GPER in females and ERα in males. In female astrocytes, ERα protein was up-regulated at a lower E2 concentration and over a broader dosage range compared to males, whereas ERß was increased after exposure to 1-10 nM versus 100 pM E2 levels in females and males, respectively. GPER profiles were stimulated by E2 in female, but not male astrocytes. E2 increased astrocyte glycogen content in female, but not male astrocytes; selective ERß or ERα stimulation elevated glycogen levels in the female and male, respectively. Outcomes imply that dimorphic astrocyte ER and glycogen metabolic responses to E2 may reflect, in part, differential steroid induction of ER variant expression and/or regulation of post-receptor signaling in each sex.

Combinatory high-resolution microdissection/ultra performance liquid chromatographic-mass spectrometry approach for small tissue volume analysis of rat brain glycogen.

Cyto-architectural diversity of brain structures emphasizes need for analytical tools for discriminative investigation of distinctive neural structures. Glycogen is the major energy reserve in the brain. There is speculation that brain utilization of this fuel source may affect detection of hypoglycemia. To evaluate sex-specific regulation of glycogen mass and mobilization in the glucose-sensory ventromedial hypothalamic nucleus (VMN), current research coupled UHPLC-electrospray ionization mass spectrometric (LC-ESI-MS) analysis capabilities with novel derivatization protocols for high-sensitivity measurement of glucose and glycogen in small-volume neural tissue samples. This work also sought to demonstrate utility of pairing this approach with optimized Western blot methods for measurement of glycogen metabolic enzyme protein expression. Here, high-resolution micropunch dissection tools for discriminative isolation of VMN tissue were used in conjunction with newly developed glycogen analytical methods and an experimental treatment paradigm for intra-cranial hindbrain-targeted administration of estrogen receptor-alpha (ERα) or -beta (ERß) receptor antagonists to address the hypothesis that estradiol activates one or both hindbrain ER populations to exert sex-specific regulatory effects on VMN glycogen mass and hypoglycemia-associated mobilization. Outcomes validate a novel multi-analytical platform for investigation of in vivo sex-dimorphic regulation of glycogen metabolism in precisely-defined brain elements under conditions of energy balance versus imbalance. This combinatory approach will facilitate ongoing efforts to elucidate effects of acute versus chronic hypoglycemia on glycogen metabolism in characterized brain glucose-sensory loci and determine effects local glycogen mass and/or mobilization adaptions on sensory monitoring and signaling of recurring hypoglycemia in each sex.

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

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 (ERß)-dependent. Current research utilized combinatory immunocytochemistry, laser-microdissection, and Western blot techniques in a pharmacological approach to address the hypothesis that ERα and/or -ß 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, ERß, and G protein-coupled estrogen receptor-1 (GPER), along with alpha1, alpha2, and beta1 adrenergic receptor (AR) proteins. NO neurons exhibited ERα/ß-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/ß. 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.

Hindbrain Estrogen Receptor Regulation of Ventromedial Hypothalamic Glycogen Metabolism and Glucoregulatory Transmitter Expression in the Hypoglycemic Female Rat.

Neural substrates for estrogen regulation of glucose homeostasis remain unclear. Female rat dorsal vagal complex (DVC) A2 noradrenergic neurons are estrogen- and metabolic-sensitive. The ventromedial hypothalamic nucleus (VMN) is a key component of the brain network that governs counter-regulatory responses to insulin-induced hypoglycemia (IIH). Here, the selective estrogen receptor-alpha (ERα) or -beta (ERß) antagonists MPP and PHTPP were administered separately to the caudal fourth ventricle to address the premise that these hindbrain ER variants exert distinctive control of VMN reactivity to IIH in the female sex. Data show that ERα governs hypoglycemic patterns of VMN astrocyte glycogen metabolic enzyme, e.g. glycogen synthase and phosphorylase protein expression, whereas ERß mediates local glycogen breakdown. DVC ERs also regulate VMN neurotransmitter signaling of energy sufficiency [γ-aminobutyric acid] or deficiency [nitric oxide, steroidogenic factor-1] during IIH. Neither hindbrain ER mediates IIH-associated diminution of VMN norepinephrine (NE) content. Both ERs oppose hypoglycemic hyperglucagonemia, while ERß contributes to reduced corticosterone output. Outcomes reveal that input from the female hindbrain to the VMN is critical for energy reserve mobilization, metabolic transmitter signaling, and counter-regulatory hormone secretion during hypoglycemia, and that ERs control those cues. Evidence that VMN NE content is not controlled by hindbrain ERα or -ß implies that these receptors may regulate VMN function via NE-independent mechanisms, or alternatively, that other neurotransmitter signals to the VMN may control local substrate receptivity to NE.

Hindbrain estrogen receptor regulation of ventromedial hypothalamic glycogen metabolism and glucoregulatory transmitter expression in the hypoglycemic male rat.

Estrogen receptor-alpha (ERα) and -beta (ERß) occur in key elements of the brain gluco-homeostatic network in both sexes, including the hindbrain dorsal vagal complex (DVC), but the influence of distinct receptor populations on this critical function is unclear. The ventromedial hypothalamic nucleus (VMN) maintains glucose balance by integrating nutrient, endocrine, and neurochemical cues, including metabolic sensory information supplied by DVC A2 noradrenergic neurons. Current research utilized the selective ERα and ERß antagonists MPP and PHTPP to characterize effects of DVC ERs on VMN norepinephrine (NE) activity and metabolic neurotransmitter signaling in insulin-induced hypoglycemic (IIH) male rats. Data show that ERß inhibits VMN glycogen synthase and stimulates phosphorylase protein expression, while attenuating hypoglycemic augmentation of glycogen content. Furthermore, both ERs attenuate VMN glucose concentrations during IIH. Hypoglycemic up-regulation of nitric oxide (NO) and brain-derived neurotrophic factor (BDNF) signaling was correspondingly driven by ERα or -ß, whereas GABA and steroidogenic factor-1 were respectively suppressed independently of ER input or by ERß. IIH intensified VMN NE accumulation by ERß-dependent mechanisms, but did not alter NE levels in other gluco-regulatory loci. ERß amplified the magnitude of insulin-induced decline in blood glucose. Both ERs regulate corticosterone, but not glucagon secretion during IIH and oppose hypoglycemic diminution of circulating free fatty acids. These findings identify distinguishing versus common VMN functions targeted by DVC ERα and -ß. Sex differences in hypoglycemic VMN NE accumulation, glycogen metabolism, and transmitter signaling may involve, in part, discrepant regulatory involvement or differential magnitude of impact of these hindbrain ERs.

Norepinephrine regulation of ventromedial hypothalamic nucleus metabolic transmitter biomarker and astrocyte enzyme and receptor expression: Impact of 5' AMP-activated protein kinase.

The ventromedial hypothalamic energy sensor AMP-activated protein kinase (AMPK) maintains glucostasis via neurotransmitter signals that diminish [γ-aminobutyric acid] or enhance [nitric oxide] counter-regulation. Ventromedial hypothalamic nucleus (VMN) 'fuel-inhibited' neurons are sensitive to astrocyte-generated metabolic substrate stream. Norepinephrine (NE) regulates astrocyte glycogen metabolism in vitro, and hypoglycemia intensifies VMN NE activity in vivo. Current research investigated the premise that NE elicits AMPK-dependent adjustments in VMN astrocyte glycogen metabolic enzyme [glycogen synthase (GS); glycogen phosphorylase (GP)] and gluco-regulatory neuron biomarker [glutamate decarboxylase65/67 (GAD); neuronal nitric oxide synthase (nNOS); SF-1] protein expression in male rats. We also examined whether VMN astrocytes are directly receptive to NE and if noradrenergic input regulates cellular sensitivity to the neuro-protective steroid estradiol. Intra-VMN NE correspondingly augmented or reduced VMN tissue GAD and nNOS protein despite no change in circulating glucose, data that imply that short-term exposure to NE promotes persistent improvement in VMN nerve cell energy stability. The AMPK inhibitor Compound C (Cc) normalized VMN nNOS, GS, and GP expression in NE-treated animals. NE caused AMPK-independent down-regulation of alpha2-, alongside Cc-reversible augmentation of beta1-adrenergic receptor protein profiles in laser-microdissected astrocytes. NE elicited divergent adjustments in astrocyte estrogen receptor-beta (AMPK-unrelated reduction) and GPR-30 (Cc-revocable increase) proteins. Outcomes implicate AMPK in noradrenergic diminution of VMN nitrergic metabolic-deficit signaling and astrocyte glycogen shunt activity. Differentiating NE effects on VMN astrocyte adrenergic and estrogen receptor variant expression suggest that noradrenergic regulation of glycogen metabolism may be mediated, in part, by one or more receptors characterized here by sensitivity to this catecholamine.