Sex-dependent endozepinergic regulation of ventromedial hypothalamic nucleus glucose counter-regulatory neuron aromatase protein expression in the adult rat.

The hypothalamic brain cell types that produce estradiol from testosterone remain unclear. Aromatase inhibition affects ventromedial hypothalamic nucleus (VMN) glucose-stimulatory nitric oxide (NO) and glucose-inhibitory γ-aminobutyric acid (GABA) transmission during insulin (INS)-induced hypoglycemia (IIH). Pure GABA and NO nerve cell samples acquired by laser-catapult-microdissection from consecutive rostro-caudal segments of the VMN were analyzed by Western blot to investigate whether regional subpopulations of each cell type contain machinery for neuro-estradiol synthesis. Astrocyte endozepinergic signaling governs brain steroidogenesis. Pharmacological tools were used here to determine if the glio-peptide octadecaneuropeptide (ODN) controls aromatase expression in GABA and NO neurons during eu- and/or hypoglycemia. Intracerebroventricular administration of the ODN G-protein coupled-receptor antagonist cyclo(1-8)[DLeu5]OP (LV-1075) decreased (male) or enhanced (female) VMN GABAergic neuron aromatase expression, but increased or reduced this profile in nitrergic neurons in a region-specific manner in each sex. IIH suppressed aromatase levels in GABA neurons located in the middle segment of the male VMN or distributed throughout this nucleus in the female. This inhibitory response was altered by the ODN isoactive surrogate octapeptide (OP) in female, but was refractory to OP in male. NO neuron aromatase protein in hypoglycemic male (middle and caudal VMN) and female (rostral and caudal VMN) rats, but was normalized in OP- plus INS-treated rats of both sexes. Results provide novel evidence that VMN glucose-regulatory neurons may produce neuro-estradiol, and that the astrocyte endozepine transmitter ODN may impose sex-specific control of baseline and/or hypoglycemic patterns of aromatase expression in distinct subsets of nitrergic and GABAergic neurons in this neural structure.

Sex-Dimorphic Octadecaneuropeptide (ODN) Regulation of Ventromedial Hypothalamic Nucleus Glucoregulatory Neuron Function and Counterregulatory Hormone Secretion.

Central endozepinergic signaling is implicated in glucose homeostasis. Ventromedial hypothalamic nucleus (VMN) metabolic monitoring governs glucose counter-regulation. VMN glucose-stimulatory nitric oxide (NO) and glucose-inhibitory γ-aminobutyric acid (GABA) neurons express the energy gauge 5'-AMP-activated protein kinase (AMPK). Current research addresses the premise that the astrocyte glio-peptide octadecaneuropeptide (ODN) imposes sex-dimorphic control of metabolic sensor activity and neurotransmitter signaling in these neurons. The ODN G-protein coupled-receptor antagonist cyclo(1-8)[DLeu5]OP (LV-1075) was administered intracerebroventricularly (icv) to euglycemic rats of each sex; additional groups were pretreated icv with the ODN isoactive surrogate ODN11-18 (OP) before insulin-induced hypoglycemia. Western blotting of laser-catapult-microdissected VMN NO and GABA neurons showed that hypoglycemia caused OP-reversible augmentation of phospho-, e.g., activated AMPK and nitric oxide synthase (nNOS) expression in rostral (female) or middle (male) VMN segments or ODN-dependent suppression of nNOS in male caudal VMN. OP prevented hypoglycemic down-regulation of glutamate decarboxylase profiles in female rat rostral VMN, without affecting AMPK activity. LV-1075 treatment of male, not female rats elevated plasma glucagon and corticosterone concentrations. Moreover, OP attenuated hypoglycemia-associated augmentation of these hormones in males only. Results identify, for each sex, regional VMN metabolic transmitter signals that are subject to endozepinergic regulation. Directional shifts and gain-or-loss of ODN control during eu- versus hypoglycemia infer that VMN neuron receptivity to or post-receptor processing of this stimulus may be modulated by energy state. In male, counter-regulatory hormone secretion may be governed principally by ODN-sensitive neural pathways, whereas this endocrine outflow may be controlled by parallel, redundant ODN-dependent and -independent mechanisms in female.

Sex-dimorphic hindbrain lactate regulation of ventromedial hypothalamic nucleus glucoregulatory neuron 5'-AMP-activated protein kinase activity and transmitter marker protein expression.

BACKGROUND: The oxidizable glycolytic end-product L-lactate is a gauge of nerve cell metabolic fuel stability that metabolic-sensory hindbrain A2 noradrenergic neurons impart to the brain glucose-regulatory network. Current research investigated the premise that hindbrain lactate deficiency exerts sex-specific control of energy sensor and transmitter marker protein responses to hypoglycemia in ventromedial hypothalamic nucleus (VMN) glucose-regulatory nitrergic and γ-aminobutyric acid (GABA) neurons. METHODS: Nitric oxide synthase (nNOS)- or glutamate decarboxylase65/67 (GAD)-immunoreactive neurons were laser-catapult-microdissected from male and female rat VMN after subcutaneous insulin injection and caudal fourth ventricular L-lactate or vehicle infusion for Western blot protein analysis. RESULTS: Hindbrain lactate repletion reversed hypoglycemia-associated augmentation (males) or inhibition (females) of nitrergic neuron nNOS expression, and prevented up-regulation of phosphorylated AMPK 5'-AMP-activated protein kinase (pAMPK) expression in those neurons. Hypoglycemic suppression of GABAergic neuron GAD protein was averted by exogenous lactate over the rostro-caudal length of the male VMN and in the middle region of the female VMN. Lactate normalized GABA neuron pAMPK profiles in hypoglycemic male (caudal VMN) and female (all VMN segments) rats. Hypoglycemic patterns of norepinephrine (NE) signaling were lactate-dependent throughout the male VMN, but confined to the rostral and middle female VMN. CONCLUSIONS: Results document, in each sex, regional VMN glucose-regulatory transmitter responses to hypoglycemia that are controlled by hindbrain lactate status. Hindbrain metabolic-sensory regulation of hypoglycemia-correlated nitric oxide or GABA release may entail AMPK-dependent mechanisms in specific VMN rostro-caudal segments in each sex. Additional effort is required to examine the role of hindbrain lactoprivic-sensitive VMN neurotransmitters in lactate-mediated attenuation of hypoglycemic hyperglucagonemia and hypercorticosteronemia in male and female rats.

Effects of Ventromedial Hypothalamic Nucleus (VMN) Aromatase Gene Knockdown on VMN Glycogen Metabolism and Glucoregulatory Neurotransmission.

The enzyme aromatase is expressed at high levels in the ventromedial hypothalamic nucleus (VMN), a principal component of the brain gluco-regulatory network. Current research utilized selective gene knockdown tools to investigate the premise that VMN neuroestradiol controls glucostasis. Intra-VMN aromatase siRNA administration decreased baseline aromatase protein expression and tissue estradiol concentrations and either reversed or attenuated the hypoglycemic regulation of these profiles in a VMN segment-specific manner. Aromatase gene repression down-regulated protein biomarkers for gluco-stimulatory (nitric oxide; NO) and -inhibitory (gamma-aminobutyric acid; GABA) neurochemical transmitters. Insulin-induced hypoglycemia (IIH) up- or down-regulated neuronal nitric oxide synthase (nNOS) and glutamate decarboxylase65/67 (GAD), respectively, throughout the VMN. Interestingly, IIH caused divergent changes in tissue aromatase and estradiol levels in rostral (diminished) versus middle and caudal (elevated) VMN. Aromatase knockdown prevented hypoglycemic nNOS augmentation in VMN middle and caudal segments, but abolished the GAD inhibitory response to IIH throughout this nucleus. VMN nitrergic and GABAergic neurons monitor stimulus-specific glycogen breakdown. Here, glycogen synthase (GS) and phosphorylase brain- (GPbb; AMP-sensitive) and muscle- (GPmm; noradrenergic -responsive) type isoform responses to aromatase siRNA were evaluated. Aromatase repression reduced GPbb and GPmm content in euglycemic controls and prevented hypoglycemic regulation of GPmm but not GPbb expression while reversing glycogen accumulation. Aromatase siRNA elevated baseline glucagon and corticosterone secretion and abolished hypoglycemic hyperglucagonemia and hypercorticosteronemia. Outcomes document the involvement of VMN neuroestradiol signaling in brain control of glucose homeostasis. Aromatase regulation of VMN gluco-regulatory signaling of hypoglycemia-associated energy imbalance may entail, in part, control of GP variant-mediated glycogen disassembly.

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

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

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.

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.

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.

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.

Lateral but not Medial Hypothalamic AMPK Activation Occurs at the Hypoglycemic Nadir in Insulin-injected Male Rats: Impact of Caudal Dorsomedial Hindbrain Catecholamine Signaling.

The hypothalamic energy sensor adenosine 5'-monophosphate-activated protein kinase (AMPK), an important regulator of counter-regulatory responses to hypoglycemia, responds to pharmacological manipulation of hindbrain AMPK activity. Dorsomedial hindbrain A2 noradrenergic neurons express hypoglycemia-sensitive metabolo-sensory biomarkers, including AMPK. Here, adult male rats were pretreated by intra-caudal fourth ventricular administration of the selective neurotoxin 6-hydroxydopamine (6-OHDA) to determine if catecholamine signaling from the aforesaid site governs hypothalamic AMPK activation during insulin-induced hypoglycemia (IIH). Micropunched arcuate (ARH), ventromedial (VMH), paraventricular (PVH), dorsomedial (DMH) nuclei and lateral hypothalamic area (LHA) tissues were obtained at the neutral protamine Hagedorn insulin-induced hypoglycemic nadir, coincident with A2 AMPK activation, for Western blot analysis of AMPK, phospho-AMPK (pAMPK), and relevant metabolic neuropeptides. ARH, VMH, LHA, and DMH norepinephrine levels were altered according to insulin dose; 6-OHDA-mediated reversal of these responses was site-specific. IIH elevated LHA and reduced VMH pAMPK protein, profiles that were respectively unchanged or increased by 6-OHDA. PVH and ARH pAMPK was resistant to IIH, but augmented in ARH of neurotoxin- plus insulin-treated rats. ARH neuropeptide Y (NPY) and pro-opiomelanocortin (POMC) proteins were correspondingly increased or refractory to IIH; 6-OHDA pretreatment normalized NPY and elevated POMC expression after insulin injection. Results demonstrate site-specific bi-directional adjustments in hypothalamic AMPK reactivity to hypoglycemia. Intensification of ARH/VMH pAMPK by 6-OHDA implies dorsomedial hindbrain improvement of energy balance in those sites during IIH. Neurotoxin-mediated augmentation versus suppression of basal catabolic (ARH POMC/VMH steroidogenic factor-1) or IIH-associated anabolic (ARH NPY) neuropeptide profiles, respectively, may involve local AMPK-dependent against independent mechanisms.