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.

Norepinephrine (NE) directly regulates ventromedial hypothalamic nucleus (VMN) glucoregulatory neurons and also controls glycogen-derived fuel provision to those cells. VMN nitric oxide (NO) and γ-aminobutyric acid (GABA) neurons and astrocytes express estrogen receptor-alpha (ERα) and ER-beta (ERβ) proteins. Current research used selective ERα (1,3Bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride) or ERβ (4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol) antagonists to address the premise that these ERs govern basal and/or NE-associated patterns of VMN metabolic neuron signaling and astrocyte glycogen metabolism. Both ERs stimulate expression of the enzyme marker protein neuronal nitric oxide synthase, not glutamate decarboxylase65/67. NE inhibition or augmentation of neuronal nitric oxide synthase and glutamate decarboxylase65/67 profiles was ER-independent or -dependent, respectively. In both neuron types, VMN ERβ activity inhibited baseline alpha1- (α1-) and/or alpha2- (α2-)adrenergic receptor (AR) expression, but ERα and -β signaling was paradoxically crucial for noradrenergic upregulation of α2-AR. NE inhibited glycogen synthase expression and exerted opposite effects on VMN adenosine monophosphate-sensitive glycogen phosphorylase (GP)-brain type (stimulatory) versus NE-sensitive GP muscle (inhibitory) via ERα or -β activity. Results document unique ERα and ERβ actions on metabolic transmitter and AR protein expression in VMN nitrergic versus GABAergic neurons. ER effects varied in the presence versus absence of NE, indicating that both neuron types are substrates for estradiol and noradrenergic regulatory interaction. NE-dependent ER control of VMN GP variant expression implies that these signals also act on astrocytes to direct physiological stimulus-specific control of glycogen metabolism, which may in turn influence GABA transmission.

Ventromedial hypothalamic nucleus (VMN) glycogen metabolism affects local glucoregulatory signaling. The hindbrain metabolic-sensitive catecholamine (CA) neurotransmitter norepinephrine controls VMN glycogen phosphorylase (GP)-muscle (GPmm) and -brain (GPbb) type expression in male rats. Present studies addressed the premise that CA regulation of hypoglycemic patterns of VMN glycogen metabolic enzyme protein expression is sex-dimorphic, and that this signal is responsible for sex differences in acclimation of these profiles to recurrent insulin-induced hypoglycemia (RIIH). VMN tissue was acquired by micropunch-dissection from male and female rats pretreated by caudal fourth ventricular administration of the CA neurotoxin 6-hydroxydopamine (6OHDA) before single or serial insulin injection. 6-OHDA averted acute hypoglycemic inhibition of VMN glycogen synthase (GS) and augmentation of GPmm and GPbb protein expression in males, and prevented GPmm and -bb down-regulation in females. Males recovered from antecedent hypoglycemia (AH) exhibited neurotoxin-preventable diminution of baseline GS profiles, whereas acclimated GPmm and -bb expression in females occurred irrespective of pretreatment. RIIH did not alter VMN GS, GPmm, and GPbb expression in vehicle- or 6-OHDA-pretreated animals of either sex. VMN glycogen content was correspondingly unchanged or increased in males versus females following AH; 6-OHDA augmented glycogen mass in AH-exposed animals of both sexes. RIIH did not alter VMN glycogen accumulation in vehicle-pretreated rats of either sex, but diminished glycogen in neurotoxin-pretreated animals. AH suppresses baseline GS (CA-dependent) or GPmm/GPbb (CA-independent) expression in male and female rats, respectively, which corresponds with unaltered or augmented VMN glycogen content in those sexes. AH-associated loss of sex-distinctive CA-mediated enzyme protein sensitivity to hypoglycemia (male: GS, GPmm, GPbb; female: GPmm, Gpbb) may reflect, in part, VMN target desensitization to noradrenergic input.

Estrogen receptor-alpha (ERα) and -beta (ERβ) 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 ERβ 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 ERβ 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 -β, whereas GABA and steroidogenic factor-1 were respectively suppressed independently of ER input or by ERβ. IIH intensified VMN NE accumulation by ERβ-dependent mechanisms, but did not alter NE levels in other gluco-regulatory loci. ERβ 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 -β. 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.

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 (ERβ) 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 ERβ 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 ERβ 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 -β 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.

Astrocyte glycogen, the primary energy reserve in brain, undergoes continuous remodeling by glucose passage through the glycogen shunt prior to conversion to the oxidizable energy fuel L-lactate. Glucogenic amino acids (GAAs) are a potential non-glucose energy source during neuro-metabolic instability. Current research investigated whether diminished glycogen metabolism affects GAA homeostasis in astrocyte and/or nerve cell compartments. The glycogen phosphorylase (GP) inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) was injected into the ventromedial hypothalamic nucleus (VMN), a key metabolic-sensing structure, before vehicle or L-lactate infusion. Pure VMN astrocyte and metabolic-sensory neuron samples were obtained by combinatory immunocytochemistry/laser-catapult-microdissection for UHPLC-electrospray ionization-mass spectrometry (LC-ESI-MS) GAA analysis. DAB inhibition of VMN astrocyte aspartate and glutamine (Gln) levels was prevented or exacerbated, respectively, by lactate. VMN gluco-stimulatory nitric oxide (NO; neuronal nitric oxide synthase-immunoreactive (ir)-positive) and gluco-inhibitory γ-aminobutyric acid (GABA; glutamate decarboxylase65/67-ir-positive) neurons exhibited lactate-reversible asparate and glutamate augmentation by DAB, but dissimilar Gln responses to DAB. GP inhibition elevated NO and GABA nerve cell GABA content, but diminished astrocyte GABA; these responses were averted by lactate in neuron, but not astrocyte samples. Outcomes provide proof-of-principle of requisite LC-ESI-MS sensitivity for GAA measurement in specific brain cell populations. Results document divergent effects of decreased VMN glycogen breakdown on astrocyte versus neuron GAAs excepting Gln. Lactate-reversible DAB up-regulation of metabolic-sensory neuron GABA signaling may reflect compensatory nerve cell energy stabilization upon decline in astrocyte-derived metabolic fuel.