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 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.

High performance liquid chromatography-electrospray ionization mass spectrometric (LC-ESI-MS) methodology for analysis of amino acid energy substrates in microwave-fixed microdissected brain tissue.

Hypoglycemia deprives the brain of its primary energy source glucose. Reductions in whole-brain amino acid energy substrate levels suggest that these non-glucose fuels may be metabolized during glucose shortage. Recurring hypoglycemia can cause mal-adaptive impairment of glucose counter-regulation; yet, it is unclear if amplified reliance upon alternative metabolic substrates impedes detection of continuing neuro-glucopenia. This research aimed to develop high-sensitivity UHPLC-electrospray ionization mass spectrometric (LC-ESI-MS) methodology, for complementary use with high-neuroanatomical resolution microdissection tools, for measurement of glucogenic amino acid, e.g. glutamine (Gln), glutamate (Glu), and aspartate (Asp) content in the characterized glucose-sensing ventromedial hypothalamic nucleus (VMN) during acute versus chronic hypoglycemia. Results show that VMN tissue Gln, Glu, and Asp levels were significantly decreased during a single hypoglycemic episode, and that Gln and Asp measures were correspondingly normalized or further diminished during renewed hypoglycemia. Results provide proof-of-principle that LC-ESI-MS has requisite sensitivity for amino acid energy substrate quantification in distinctive brain gluco-regulatory structures under conditions of eu- versus hypoglycemia. This novel combinatory methodology will support ongoing efforts to determine how amino acid energy yield may impact VMN metabolic sensory function during persistent hypoglycemia.

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-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.

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.