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.

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.

Glycogen phosphorylase isoform regulation of glucose and energy sensor expression in male versus female rat hypothalamic astrocyte primary cultures.

Astrocyte glycogen constitutes the primary energy fuel reserve in the brain. Current research investigated the novel premise that glycogen turnover governs astrocyte responsiveness to critical metabolic and neurotransmitter (norepinephrine) regulatory signals in a sex-dimorphic manner. Here, rat hypothalamic astrocyte glycogen phosphorylase (GP) gene expression was silenced by short-interfering RNA (siRNA) to investigate how glycogen metabolism controlled by GP-brain type (GPbb) or GP-muscle type (GPmm) activity affects glucose [glucose transporter-2 (GLUT2)] and energy [5'-AMP-activated protein kinase (AMPK)] sensor and adrenergic receptor (AR) proteins in each sex. Results show that in the presence of glucose, glycogen turnover is regulated by GPbb in the male or by GPmm in the female, yet in the absence of glucose, glycogen breakdown is controlled by GPbb in each sex. GLUT2 expression is governed by GPmm-mediated glycogen breakdown in glucose-supplied astrocytes of each sex, but glycogenolysis controls glucoprivic GLUT2 up-regulation in male only. GPbb-mediated glycogen disassembly causes divergent changes in total AMPK versus phosphoAMPK profiles in male. During glucoprivation, glycogenolysis up-regulates AMPK content in male astrocytes by GPbb- and GPmm-dependent mechanisms, whereas GPbb-mediated glycogen breakdown inhibits phosphoAMPK expression in female. GPbb and GPmm activity governs alpha2-AR and beta1-AR protein levels in male, but has no effect on these profiles in the female. Outcomes provide novel evidence for sex-specific glycogen regulation of glucose- and energy-sensory protein expression in hypothalamic astrocytes, and identify GP isoforms that mediate such control in each sex. Results also show that glycogen regulation of hypothalamic astrocyte receptivity to norepinephrine is male-specific. Further studies are needed to characterize the molecular mechanisms that underlie sex differences in glycogen control of astrocyte protein expression.

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.

Glycogen Phosphorylase Isoform Regulation of Ventromedial Hypothalamic Nucleus Gluco-Regulatory Neuron 5'-AMP-Activated Protein Kinase and Transmitter Marker Protein Expression.

Brain glycogen is remodeled during metabolic homeostasis and provides oxidizable L-lactate equivalents. Brain glycogen phosphorylase (GP)-brain (GPbb; AMP-sensitive) and -muscle (GPmm; norepinephrine-sensitive) type isoforms facilitate stimulus-specific control of glycogen disassembly. Here, a whole animal model involving stereotactic-targeted delivery of GPmm or GPbb siRNA to the ventromedial hypothalamic nucleus (VMN) was used to investigate the premise that these variants impose differential control of gluco-regulatory transmission. Intra-VMN GPmm or GPbb siRNA administration inhibited glutamate decarboxylate65/67 (GAD), a protein marker for the gluco-inhibitory transmitter γ--aminobutyric acid (GABA), in the caudal VMN. GPbb knockdown, respectively overturned or exacerbated hypoglycemia-associated GAD suppression in rostral and caudal VMN. GPmm siRNA caused a segment-specific reversal of hypoglycemic augmentation of the gluco-stimulatory transmitter indicator, neuronal nitric oxide synthase (nNOS). In both cell types, GP siRNA down-regulated 5'-AMP-activated protein kinase (AMPK) during euglycemia, but hypoglycemic suppression of AMPK was reversed by GPmm targeting. GP knockdown elevated baseline GABA neuron phosphoAMPK (pAMKP) content, and amplified hypoglycemic augmentation of pAMPK expression in each neuron type. GPbb knockdown increased corticosterone secretion in eu- and hypoglycemic rats. Outcomes validate efficacy of GP siRNA delivery for manipulation of glycogen breakdown in discrete brain structures in vivo, and document VMN GPbb control of local GPmm expression. Results document GPmm and/or -bb regulation of GABAergic and nitrergic transmission in discrete rostro-caudal VMN segments. Contrary effects of glycogenolysis on metabolic-sensory AMPK protein during eu- versus hypoglycemia may reflect energy state-specific astrocyte signaling. Amplifying effects of GPbb knockdown on hypoglycemic stimulation of pAMPK infer that glycogen mobilization by GPbb limits neuronal energy instability during hypoglycemia.

UHPLC-electrospray ionization-mass spectrometric analysis of brain cell-specific glucogenic and neurotransmitter amino acid content.

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.

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.

Hindbrain catecholamine regulation of ventromedial hypothalamic nucleus glycogen metabolism during acute versus recurring insulin-induced hypoglycemia in male versus female rat.

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.

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.

Sex differences in ventromedial hypothalamic nucleus glucoregulatory transmitter biomarker protein during recurring insulin-induced hypoglycemia.

Recurring insulin-induced hypoglycemia (RIIH) in males correlates with maladaptive glucose counter-regulatory collapse and acclimated expression of ventromedial hypothalamic nucleus (VMN) nitric oxide (NO) and γ-aminobutyric acid (GABA) metabolic transmitter biomarkers, e.g., neuronal nitric oxide synthase (nNOS) and glutamate decarboxylase65/67 (GAD). Hindbrain noradrenergic neurons innervate the VMN, where norepinephrine regulates nNOS and GAD expression. Current research investigated the hypothesis that antecedent hypoglycemia (AH) exposure causes sex-dimorphic habituation of VMN glucoregulatory biomarker proteins between and/or during serial hypoglycemic bouts, and that hindbrain catecholaminergic (CA) signaling may control sex-specific adaptation of one or more of these proteins. Data show that upon recovery from AH, females exhibit CA-mediated reductions in baseline VMN nNOS, GAD, steroidogenic factor-1 (SF-1), and brain-derived neurotrophic factor (BNDF) expression compared to euglycemic profiles. In males, however, AH caused 6-OHDA-insensitive suppression of only basal SF-1 levels in the VMN. VMN transmitter protein acclimation to RIIH was sex-contingent, as differential nNOS, GAD, SF-1, and BDNF responses to a single vs final bout of hypoglycemia occur in males, whereas females show acclimated reactivity of GAD and SF-1 only to renewed hypoglycemia. CA-mediated and -independent habituation of distinctive VMN protein profiles occurred in each sex. Further research is necessary to evaluate, in each sex, effects of altered baseline VMN metabolic neurotransmitter signals on glucose homeostasis as well as non-metabolic functions under the control of those neurochemicals. It would also be insightful to learn if and how sex-contingent habituation of VMN transmitter responses to hypoglycemia contribute to sex-dimorphic patterns of glucose counter-regulation during RIIH.

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.

Norepinephrine Regulation of Adrenergic Receptor Expression, 5' AMP-Activated Protein Kinase Activity, and Glycogen Metabolism and Mass in Male Versus Female Hypothalamic Primary Astrocyte Cultures.

Norepinephrine (NE) control of hypothalamic gluco-regulation involves astrocyte-derived energy fuel supply. In male rats, exogenous NE regulates astrocyte glycogen metabolic enzyme expression in vivo through 5'-AMP-activated protein kinase (AMPK)-dependent mechanisms. Current research utilized a rat hypothalamic astrocyte primary culture model to investigate the premise that NE imposes sex-specific direct control of AMPK activity and glycogen mass and metabolism in these glia. In male rats, NE down-regulation of pAMPK correlates with decreased CaMMKB and increased PP1 expression, whereas noradrenergic augmentation of female astrocyte pAMPK may not involve these upstream regulators. NE concentration is a critical determinant of control of hypothalamic astrocyte glycogen enzyme expression, but efficacy varies between sexes. Data show sex variations in glycogen synthase expression and glycogen phosphorylase-brain and -muscle type dose-responsiveness to NE. Narrow dose-dependent NE augmentation of astrocyte glycogen content during energy homeostasis infers that NE maintains, over a broad exposure range, constancy of glycogen content despite possible changes in turnover. In male rats, beta1- and beta2-adrenergic receptor (AR) profiles displayed bi-directional responses to increasing NE doses; female astrocytes exhibited diminished beta1-AR content at low dose exposure, but enhanced beta2-AR expression at high NE dosages. Thus, graded variations in noradrenergic stimulation may modulate astrocyte receptivity to NE in vivo. Sex dimorphic NE regulation of hypothalamic astrocyte AMPK activation and glycogen metabolism may be mediated, in part, by one or more ARs characterized here by divergent sensitivity to this transmitter.

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.