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.

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.

Sex-specific acclimation of A2 noradrenergic neuron dopamine-ß-hydroxylase and estrogen receptor variant protein and 5'-AMP-Activated protein kinase reactivity to recurring hypoglycemia in rat.

Hindbrain estrogen receptors (ER) impose sex-dimorphic control of counter-regulatory hormone and hypothalamic glucoregulatory transmitter and glycogen metabolic responses to hypoglycemia. A2 noradrenergic neurons are estradiol- and metabolic-sensitive. Estradiol controls dopamine-beta-hydroxylase (DBH) protein habituation to recurrent insulin-induced hypoglycemia (RIIH) in females. Current research investigated the premise that sex-dimorphic patterns of A2 ER variant acclimation to RIIH correlate with differential A2 DBH and 5'-AMP-activated protein kinase (AMPK) adaptation to RIIH. A2 neurons were laser-catapult-microdissected from male and female rats after one or four insulin injections for Western blot analysis. A2 pAMPK and DBH levels were increased in males, but suppressed in females after single insulin dosing. ER-alpha (ERα) and -beta (ERß) protein profiles were unaffected or decreased by acute hypoglycemia in each sex, whereas G protein-linked ER-1 (GPER) reactivity varied by sex. Antecedent hypoglycemia diminished basal A2 ERα/GPER and elevated ERß content in each sex, yet reduced pAMPK and DBH levels in female rats only. Reintroduced hypoglycemia suppressed A2 ERß levels in each sex, but altered DBH (↓), ERα (↓), and GPER (↑) levels in males only. Data document sex differences in A2 DBH adaptation to RIIH, e.g. a shift from positive-to-negative response in males versus loss of negative reactivity in females, as well as attenuated AMPK activation in both sexes. Between hypoglycemic episodes, A2 neurons in each sex likely exhibit diminished sensitivity to ERα/GPER signaling, but heightened receptivity to ERß input. RIIH-induced changes in ERα and GPER expression in male but not female may contribute to DBH suppression (males) versus no change (females) relative to adapted baseline expression.

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 control of ventromedial hypothalamic nucleus glucoregulatory neurotransmitter expression in the female rat: Role of monocarboxylate transporter function.

The ventromedial hypothalamic nucleus (VMN) is a critical component of the neural circuitry that regulates glucostasis. Astrocyte glycogen is a vital reserve of glucose and its oxidizable metabolite L-lactate. In hypoglycemic female rats, estradiol-dependent augmentation of VMN glycogen phosphorylase (GP) protein requires hindbrain catecholamine input. Research here investigated the premise that norepinephrine (NE) regulation of VMN astrocyte metabolism shapes local glucoregulatory neurotransmitter signaling in this sex. Estradiol-implanted ovariectomized rats were pretreated by intra-VMN administration of the monocarboxylate transporter inhibitor alpha-cyano-4-hydroxy-cinnamic acid (4CIN) or vehicle before NE delivery to that site. NE caused 4CIN-reversible reduction or augmentation of VMN glycogen synthase and phosphorylase expression. 4CIN prevented NE stimulation of gluco-inhibitory (glutamate decarboxylase65/67) and suppression of gluco-stimulatory (neuronal nitric oxide synthase) neuron marker proteins. These outcomes imply that effects of noradrenergic stimulation of VMN astrocyte glycogen depletion on glucoregulatory transmitter signaling may be mediated, in part, by glycogen-derived substrate fuel provision. NE control of astrocyte glycogen metabolism may involve down-regulated adrenoreceptor (AR), e.g. alpha1 and alpha2, alongside amplified beta1 AR and estrogen receptor-beta signaling. Noradrenergic hypoglycemia was refractory to 4CIN, implying that additional NE-sensitive VMN glucoregulatory neurochemicals may be insensitive to monocarboxylate uptake. Augmentation of circulating free fatty acids by combinatory NE and 4CIN, but not NE alone implies that acute hypoglycemia induced here is an insufficient stimulus for mobilization of these fuels, but is adequate when paired with diminished brain monocarboxylate fuel availability.

Sex differences in forebrain estrogen receptor regulation of hypoglycemic patterns of counter-regulatory hormone secretion and ventromedial hypothalamic nucleus glucoregulatory neurotransmitter and astrocyte glycogen metabolic enzyme expression.

The female ventromedial hypothalamic nucleus (VMN) is a focal substrate for estradiol (E) regulation of energy balance, feeding, and body weight, but how E shapes VMN gluco-regulatory signaling in each sex is unclear. This study investigated the hypothesis that estrogen receptor-alpha (ERα) and/or -beta (ERß) control VMN signals that inhibit [γ-aminobutyric acid] or stimulate [nitric oxide, steroidogenic factor-1 (SF-1)] counter-regulation in a sex-dependent manner. VMN nitrergic neurons monitor astrocyte fuel provision; here, we examined how these ER regulate astrocyte glycogen metabolic enzyme, monocarboxylate transporter, and adrenoreceptor protein responses to insulin-induced hypoglycemia (IIH) in each sex. Testes-intact male and E-replaced ovariectomized female rats were pretreated by intracerebroventricular ERα antagonist (MPP) or ERß antagonist (PHTPP) administration before IIH. Data implicate both ER in hypoglycemic inhibition of neuronal nitric oxide synthase protein in each sex and up-regulation of glutamate decarboxylase65/67 and SF-1 expression in females. ERα and -ß enhance astrocyte AMPK and glycogen synthase expression and inhibit glycogen phosphorylase in hypoglycemic females, while ERß suppresses the same proteins in males. Differential VMN astrocyte protein responses to IIH may partially reflect ERα and -ß augmentation of ERß and down-regulation of alpha1, alpha2, and beta1 adrenoreceptor proteins in females, versus ERß repression of GPER and alpha2 adrenoreceptor profiles in males. MPP or PHTPP pretreatment blunted counter-regulatory hormone secretion in hypoglycemic males only, suggesting that in males one or more VMN neurotransmitters exhibiting sensitivity to forebrain ER may passively regulate this endocrine outflow, whereas female forebrain ERα and -ß are apparently uninvolved in these contra-regulatory responses.

Role of estradiol in intrinsic hindbrain AMPK regulation of hypothalamic AMPK, metabolic neuropeptide, and norepinephrine activity and food intake in the female rat.

This study addressed the hypothesis that dorsomedial hindbrain adenosine 5'-monophosphate-activated protein kinase (AMPK) imposes inherent estradiol-dependent control of hypothalamic AMPK, neuropeptide, and norepinephrine (NE) activity and feeding in the female rat. Estradiol (E)- or oil (O)-implanted ovariectomized rats were injected with the AMPK inhibitor compound c (Cc) or vehicle into the caudal fourth ventricle (CV4) prior to micropunch-dissection of individual hypothalamic metabolic loci or assessment of food intake. Cc decreased hindbrain dorsal vagal complex phosphoAMPK (pAMPK) in both E and O; tissue ATP levels were reduced by this treatment in O only. In E/Cc, pAMPK expression was diminished in the lateral hypothalamic area (LHA) and ventromedial (VMH) and paraventricular (PVH) nuclei; only PVH pAMPK was suppressed by this treatment in O/Cc. Cc decreased PVH corticotropin-releasing hormone and arcuate (ARH) proopiomelanocortin (POMC) and neuropeptide Y in O, but suppressed only POMC in E. O/Cc exhibited both augmented (PVH, VMH) and decreased (LHA, ARH) hypothalamic NE content, whereas Cc treatment of E elevated preoptic and dorsomedial hypothalamic nucleus NE. Cc completely or incompletely repressed feeding in E versus O, respectively. Results implicate dorsomedial hindbrain AMPK in physiological stimulus-induced feeding in females. Excepting POMC, hypothalamic neuropeptide responses to this sensor may be contingent on estrogen. Estradiol likely designates hypothalamic targets of altered NE signaling due to hindbrain AMPK activation. Divergent changes in NE content of hypothalamic loci in O/Cc uniquely demonstrate sensor-induced bimodal catecholamine signaling to those sites.

Hindbrain lactate regulates preoptic gonadotropin-releasing hormone (GnRH) neuron GnRH-I protein but not AMPK responses to hypoglycemia in the steroid-primed ovariectomized female rat.

Steroid positive-feedback activation of the gonadotropin-releasing hormone (GnRH)-pituitary luteinizing hormone (LH) neuroendocrine axis propagates the pre ovulatory LH surge, a crucial component of female reproduction. Our work shows that this key event is restrained by inhibitory metabolic input from hindbrain A2 noradrenergic neurons. GnRH neurons express the ultra-sensitive energy sensor adenosine 5'-monophosphate-activated protein kinase (AMPK); here, we investigated the hypothesis that GnRH nerve cell AMPK and peptide neurotransmitter responses to insulin-induced hypoglycemia are controlled by hindbrain lack of the oxidizable glycolytic end-product L-lactate. Data show that hypoglycemic inhibition of LH release in steroid-primed ovariectomized female rats was reversed by coincident caudal hindbrain lactate infusion. Western blot analyses of laser-microdissected A2 neurons demonstrate hypoglycemic augmentation [Fos, estrogen receptor-beta (ER-ß), phosphoAMPK (pAMPK)] and inhibition (dopamine-beta-hydroxylase, GLUT3, MCT2) of protein expression in these cells, responses that were normalized by insulin plus lactate treatment. Hypoglycemia diminished rostral preoptic GnRH nerve cell GnRH-I protein and pAMPK content; the former, but not the latter response was reversed by lactate. Results implicate caudal hindbrain lactoprivic signaling in hypoglycemia-induced suppression of the LH surge, demonstrating that lactate repletion of that site reverses decrements in A2 catecholamine biosynthetic enzyme and GnRH neuropeptide precursor protein expression. Lack of effect of lactate on hypoglycemic patterns of GnRH AMPK activity suggests that this sensor is uninvolved in metabolic-inhibition of positive-feedback-stimulated hypophysiotropic signaling to pituitary gonadotropes.

Dorsomedial hindbrain catecholamine regulation of hypothalamic astrocyte glycogen metabolic enzyme protein expression: Impact of estradiol.

The brain astrocyte glycogen reservoir is a vital energy reserve and, in the cerebral cortex, subject among other factors to noradrenergic control. The ovarian steroid estradiol potently stimulates nerve cell aerobic respiration, but its role in glial glycogen metabolism during energy homeostasis or mismatched substrate supply/demand is unclear. This study examined the premise that estradiol regulates hypothalamic astrocyte glycogen metabolic enzyme protein expression during normo- and hypoglycemia in vivo through dorsomedial hindbrain catecholamine (CA)-dependent mechanisms. Individual astrocytes identified in situ by glial fibrillary acidic protein immunolabeling were laser-microdissected from the ventromedial hypothalamic (VMH), arcuate hypothalamic (ARH), and paraventricular hypothalamic (PVH) nuclei and the lateral hypothalamic area (LHA) of estradiol (E)- or oil (O)-implanted ovariectomized (OVX) rats after insulin or vehicle injection, and pooled within each site. Stimulation [VMH, LHA] or suppression [PVH, ARH] of basal glycogen synthase (GS) protein expression by E was reversed in the former three sites by caudal fourth ventricular pretreatment with the CA neurotoxin 6-hydroxydopamine (6-OHDA). E diminished glycogen phosphorylase (GP) protein profiles by CA-dependent [VMH, PVH] or -independent mechanisms [LHA]. Insulin-induced hypoglycemia (IIH) increased GS expression in the PVH in OVX+E, but reduced this protein in the PVH, ARH, and LHA in OVX+O. Moreover, IIH augmented GP expression in the VMH, LHA, and ARH in OVX+E and in the ARH in OVX+O, responses that normalized by 6-OHDA. Results demonstrate site-specific effects of E on astrocyte glycogen metabolic enzyme expression in the female rat hypothalamus, and identify locations where dorsomedial hindbrain CA input is required for such action. Evidence that E correspondingly increases and reduces basal GS and GP in the VMH and LHA, but augments the latter protein during IIH suggests that E regulates glycogen content and turnover in these structures during glucose sufficiency and shortage.

Deferred feeding and body weight responses to short-term interruption of fuel acquisition: impact of estradiol.

Short-term abstinence from food intake, planned or unplanned, is unavoidable in modern life, but negatively correlated with appetite control and obesity. This study investigated the role of estradiol in feeding and body weight (BW) reactions to short-span cessation of feeding. During acute 1-6-h re-feeding, 12-h food-deprived (FD), estradiol benzoate (EB)-implanted ovariectomized rats ate less food and gained less weight than FD animals implanted with oil (O). Full fed (FF)- and FD-EB consumed equal amounts of food over 24 h, but weight gain was greater in the latter; 24-h food intake and BW gain in FD-O exceeded FD-EB. Caudal fourth ventricular administration of the AMPK activator AICAR increased dorsal vagal complex AMPK activity in FD-EB and FD-O, but elicited dissimilar adjustments in hypothalamic metabolic neuropeptide transmitter expression, while respectively enhancing or reducing acute re-feeding in these animals and reversing FD-O weight gain. Drug-treated FD-EB and FD-O exhibited respective feeding and weight gain increases between 6-24 h. AICAR enhanced 24-h consumption in FD-EB vs. FF-EB, but cumulative intake and BW gain were greater in AICAR-treated FD-O vs. FD-EB. Results show that estradiol limits acute re-feeding after short-term feeding suspension, but augments acute re-feeding when energy depletion coincides with suspended feeding. This compound metabolic stress exerts steroid-dependent effects during later resumption of circadian-induced feeding, for example, increased consumption vs. weight gain in the presence vs. absence of estradiol. These studies provide novel evidence that estrogen mitigates acute and post-acute adverse effects of disrupted fuel acquisition on energy balance.

Energy status determines hindbrain signal transduction pathway transcriptional reactivity to AMPK in the estradiol-treated ovariectomized female rat.

Dorsal vagal complex (DVC) AMPK regulation of food intake in the estradiol-treated ovariectomized (OVX) female rat is energy state-dependent. Here, RT-PCR array technology was used to identify estradiol-sensitive AMPK-regulated DVC signal transduction pathways that exhibit differential reactivity to sensor activation during energy balance versus imbalance. The AMP mimetic AICAR correspondingly reduced or stimulated cDVC phosphoAMPK (pAMPK) and estrogen receptor-beta (ERß) proteins in full-fed (F) versus 12-h food-deprived (D) estradiol-treated ovariectomized (OVX) rats, but elevated ER-alpha (ERα) in F only. Estradiol suppressed DVC ERß protein and hypoxia, NFκB, STAT3, STAT6, and Hedgehog signaling pathway marker genes against oil-implanted OVX controls. F+(A)ICAR and D+(S)aline groups each exhibited further inhibition of NFκB, STAT3, and Hedgehog pathway genes, and diminished PPAR, Notch, and STAT5 transcripts versus F+S. Conversely, genes in these six pathways were up-regulated by AICAR treatment of D. Results show that in this animal model, acute AMP augmentation or feeding cessation each inhibit both pAMPK and ERß expression, but in combination increase these protein profiles. pAMPK protein and DVC TNF (NFκB), SOCS3 (JAK/STAT), WNT6 (Hedgehog), and FABP1 (PPAR) mRNAs were down- or upregulated in parallel by AICAR in F versus D states, respectively. Further research is needed to determine the impact of ERß on opposing directionality of these responses, and to characterize the role of the aforementioned signaling pathways in hyperphagic responses in the female to AICAR-induced DVC AMPK activation during acute interruption of feeding.

Hindbrain medulla catecholamine cell group involvement in lactate-sensitive hypoglycemia-associated patterns of hypothalamic norepinephrine and epinephrine activity.

Cell-type compartmentation of glucose metabolism in the brain involves trafficking of the oxidizable glycolytic end product, l-lactate, by astrocytes to fuel neuronal mitochondrial aerobic respiration. Lactate availability within the hindbrain medulla is a monitored function that regulates systemic glucostasis as insulin-induced hypoglycemia (IIH) is exacerbated by lactate repletion of that brain region. A2 noradrenergic neurons are a plausible source of lactoprivic input to the neural gluco-regulatory circuit as caudal fourth ventricular (CV4) lactate infusion normalizes IIH-associated activation, e.g. phosphorylation of the high-sensitivity energy sensor, adenosine 5'-monophosphate-activated protein kinase (AMPK), in these cells. Here, we investigated the hypothesis that A2 neurons are unique among medullary catecholamine cells in directly screening lactate-derived energy. Adult male rats were injected with insulin or vehicle following initiation of continuous l-lactate infusion into the CV4. Two hours after injections, A1, C1, A2, and C2 neurons were collected by laser-microdissection for Western blot analysis of AMPKα1/2 and phosphoAMPKα1/2 proteins. Results show that AMPK is expressed in each cell group, but only a subset, e.g. A1, C1, and A2 neurons, exhibit increased sensor activity in response to IIH. Moreover, hindbrain lactate repletion reversed hypoglycemic augmentation of pAMPKα1/2 content in A2 and C1 but not A1 cells, and normalized hypothalamic norepinephrine and epinephrine content in a site-specific manner. The present evidence for discriminative reactivity of AMPK-expressing medullary catecholamine neurons to the screened energy substrate lactate implies that that lactoprivation is selectively signaled to the hypothalamus by A2 noradrenergic and C1 adrenergic cells.

Role of dorsal vagal complex A2 noradrenergic neurons in hindbrain glucoprivic inhibition of the luteinizing hormone surge in the steroid-primed ovariectomized female rat: effects of 5-thioglucose on A2 functional biomarker and AMPK activity.

Neuro-glucostasis is required for normal expression of the steroid positive-feedback-induced preovulatory pituitary luteinizing hormone (LH) surge, a critical element of female reproduction. Glucoprivic signals from the caudal hindbrain restrain this surge, but the cellular source of this stimulus is unclear. Norepinephrine (NE) exerts well-defined stimulatory effects on the reproductive neuroendocrine axis. Our studies show that medullary A2 noradrenergic neurons are both estrogen- and glucoprivic-sensitive. Here, we investigated the premise that the LH surge is inhibited by A2 cell reactivity to hindbrain glucopenia and diminished preoptic NE neurotransmission. Estradiol- and progesterone-primed ovariectomized (OVX) female rats were injected into the caudal fourth ventricle (CV4) with the glucose anti-metabolite, 5-thioglucose (5TG) or saline (SAL) prior to onset of the LH surge. Pretreatment by intra-CV4 delivery of the selective catecholamine neurotoxin, 6-OHDA, attenuated LH output, but prevented inhibition by 5TG. 5TG modified patterns of steroid feedback-associated Fos staining of A2, but not other medullary catecholamine cell groups. Intra-preoptic administration of the alpha1-adrenergic receptor agonist, methoxamine, elicited site-specific reversal of hindbrain glucoprivic suppression of gonadotropin-releasing hormone (GnRH) neuron Fos labeling and LH release. Western blotting of laser-microdissected A2 neurons revealed glucoprivic stimulation of Fos, but inhibition of the catecholamine synthetic enzyme, dopamine-ß-hydroxylase; 5TG also diminished A2 estrogen receptor (ER)-α and progesterone receptor profiles, but augmented ER-ß protein. Intriguingly, A2 AMPK activity was decreased in 5TG-treated rats, despite down-regulation of GLUT3 and no change in MCT2 protein expression. Rostral preoptic GnRH neurons also exhibited decreased AMPK activation simultaneous with apparent reduction of neuropeptide signaling to the pituitary. The present studies demonstrate that hindbrain glucoprivation inhibits the LH surge, in part, by reducing preoptic noradrenergic input, and furthermore implicate A2 neurons as a source of this altered signal. Results also suggest that AMPK sensor deactivation does not supersede the impact of pharmacological inhibition of glucose catabolism on A2 cell function nor afferent signaling of hindbrain glucopenia on GnRH neurons. Further studies are needed to determine if decreased AMPK activation in these cell populations reflect compensatory gain in positive energy balance and/or direct effects of estrogen on AMPK.

Effects of hypoglycaemia on neurotransmitter and hormone receptor gene expression in laser-dissected arcuate neuropeptide Y/agouti-related peptide neurones.

Arcuate neuropeptide Y (NPY)/agouti-related pepide (AgRP) neurones regulate energy homeostasis, and express the putative glucosensor, glucokinase (GCK). The present study performed multi-transcriptional profiling of these neurones to characterise NPY, AgRP and GCK gene expression during intermediate insulin-induced hypoglycaemia, and to determine whether these transcriptional responses acclimate to repeated insulin dosing. We also examined whether these neurones express insulin, glucocorticoid and oestrogen receptor gene transcripts, and whether the levels of these receptor mRNAs are modified by insulin-induced hypoglycaemia. Individual NPY-immunoreactive neurones were laser-microdissected from the caudal arcuate nucleus after single or serial dosing with neutral protamine Hagedorn insulin (NPH), and evaluated by quantitative real-time reverse transcriptase-polymerase chain reaction for the assessment of neurotransmitter and receptor gene expression. Mean NPY and AgRP mRNA in harvested NPY neurones was unchanged or augmented, respectively, by one NPH dose, although repeated NPH administration up-regulated NPY, whereas AgRP gene transcripts were down-regulated. NPH elicited divergent modifications in the ERalpha and ERbeta mRNA content of sampled neurones. ERalpha transcripts were amplified by both acute and chronic NPH-induced hypoglycaemia, whereas ERbeta gene expression was unaltered during a single bout, but suppressed during recurring hypoglycaemia. Glucocorticoid receptor (GR) mRNA levels were increased by a single insulin dose, but unaffected by serial NPH dosing. Insulin receptor-beta chain (InsRb) gene transcripts were insensitive to acute NPH-induced hypoglycaemia, but repeated NPH inhibited this gene transcript. Neither acute nor recurring hypoglycaemia modified GCK mRNA levels in caudal hypothalamic arcuate nucleus (ARH) NPY/AgRP neurones, but baseline GCK transcription was suppressed by the latter. This evidence for the habituation of hypoglycaemic patterns of InsRb, GR and ERbeta gene transcription to serial NPH dosing implies that such treatment may alter reactivity of caudal ARH NPY/AgRP neurones to receptor ligands, and supports the need to determine whether adaptive changes in neuronal sensitivity to insulin, corticosterone and/or oestrogen cause up- versus down-regulation of NPY and AgRP neurotransmission, respectively, by this caudal ARH subpopulation during chronic hypoglycaemia.

In situ coexpression of glucose and monocarboxylate transporter mRNAs in metabolic-sensitive caudal dorsal vagal complex catecholaminergic neurons: transcriptional reactivity to insulin-induced hypoglycemia and caudal hindbrain glucose or lactate repletion during insulin-induced hypoglycemia.

The neurochemical phenotype(s) of metabolic sensing neurons in the dorsal vagal complex (DVC) remains unclear. These studies utilized single-cell quantitative real-time RT-PCR, in conjunction with laser-catapult microdissection, to address the hypothesis that DVC A2 neurons express genes that encode the characterized metabolic transducers, e.g. glucokinase (GCK) and the energy-dependent potassium channel, K(ATP). Studies show that either glucose or lactate alters synaptic firing of DVC chemosensory neurons, and that delivery of the latter fuel into the caudal hindbrain amplifies insulin-induced hypoglycemia (IIH) and elevates neuronal glucose and monocarboxylate transporter, GCK, and sulfonylurea-1 mRNA in the DVC. We thus examined the additional premise that IIH modifies A2 substrate transporter and metabolic transducer gene profiles, and that such transcriptional responses may be reversed by exogenous lactate and/or glucose. Individual tyrosine hydroxylase (TH)-immunoreactive (-ir) A2 neurons were microdissected from the caudal DVC 2 h after injection of insulin or saline, and continuous caudal fourth ventricular (CV4) infusion of lactate, glucose, or artificial cerebrospinal fluid. The data show that IIH decreased MCT2, but elevated GLUT3, GLUT4, GCK, and SUR-1 transcripts in A2 neurons. Blood glucose levels in insulin-injected rats were further reduced by CV4 infusion of either lactate or glucose. Lactate plus insulin reversed hypoglycemic reductions in MCT2 mRNA and further augmented GLUT3 transcripts in A2 neurons, whereas glucose infusion in insulin-injected rats further increased GLUT3 and GCK gene profiles. The present results demonstrate that caudal DVC A2 neurons express molecular markers for metabolic sensing, and genes that encode glucose and monocarboxylate transporters. Evidence that IIH reduces A2 MCT2, but elevates GLUT3 and GLUT4 gene profiles suggests that glucose may be a primary energy source to these cells during hypoglycemia, while decreased lactate uptake, alone or relative to glucose uptake, may be a critical manifestation of systemic glucose deficiency at the cellular level. Findings that singular fuel repletion does not normalize hypoglycemic patterns of glucose transporter, GCK, or SUR-1 mRNA expression in A2 neurons imply that sufficient supply of both energy substrates is required for metabolic balance, and that cellular adaptation to the prevalence of either fuel may increase cellular dependence on glucose-specific metabolites or other products.

Adaptation of feeding and counter-regulatory hormone responses to intermediate insulin-induced hypoglycaemia in the ovariectomised female rat: effects of oestradiol.

Oestradiol regulates basal food intake and glucagon and corticosterone secretion, but its influence on these responses to acute and recurring hypoglycaemia remains unclear. The present study utilised an experimental model for repeated intermediate-acting insulin-induced hypoglycaemia that replicates the route of delivery, frequency of administration, and duration of insulin action in the clinical setting. Groups of ovariectomised (OVX) rats were implanted with s.c. capsules containing oestradiol benzoate (EB) or oil, and injected with one or four doses of Humulin neutral protamine Hagedorn (HN), on as many days, or diluent alone. Baseline feeding followed divergent trends in EB- versus oil-implanted animals over a 9-h period after final injections. Recurring HN-induced hypoglycaemia resulted in significantly greater baseline-corrected food intake in OVX + EB and OVX + oil groups, relative to acute hypoglycaemic hyperphagia. Although oestradiol did not modify net food consumption after single or serial HN doses, EB replacement maintained uniform feeding over time in each treatment paradigm. Baseline glucagon and corticosterone secretion was higher in EB- versus oil-treated OVX rats. Oestradiol prolonged acute hypoglycaemic glucagonemia, and increased the magnitude, but shortened the duration, of glucagon secretion during recurring hypoglycaemia. OVX + oil rats responded to both acute and recurring hypoglycaemia with elevated corticosterone secretion at a single time point, which was advanced from +6 to +4 h during recurrent insulin-induced hypoglycemia, whereas OVX + EB animals exhibited increased plasma hormone levels at both +4 and +6 h in response to each paradigm. Area-under-the curve analyses showed that total glucagon and corticosterone release was greater in EB- versus oil-implanted rats after both single and serial dosing with HN. These results demonstrate that repeated HN administration increases food intake in female rats via oestrogen-independent mechanisms, but that oestradiol preserves temporal patterns of hypoglycaemic hyperphagia. The data also reveal that normo- and hypoglyacemic glucagon and corticosterone secretion are enhanced in the presence of oestrogen. Further studies are necessary to identify the sites and cellular substrates that are responsible for this hormonal regulation of behavioural and endocrine responses to prolonged hypoglycaemia.

I.c.v. administration of the nonsteroidal glucocorticoid receptor antagonist, CP-472555, prevents exacerbated hypoglycemia during repeated insulin administration.

Hypoglycemia elicits an integrated array of CNS-mediated counterregulatory responses, including activation of the hypothalamic-pituitary-adrenal axis. The role of antecedent adrenocortical hypersecretion in impaired glucose counterregulation remains controversial. The present studies utilized the selective, nonsteroidal glucocorticoid receptor antagonist, CP-472555, as a pharmacological tool to investigate the hypothesis that hypoglycemic hypercorticosteronemia modulates CNS efferent autonomic and neuroendocrine motor responses to recurring insulin-induced hypoglycemia via glucocorticoid receptor-dependent mechanisms. Groups of adult male rats were injected s.c. with either one or four doses of the intermediate-acting insulin, Humulin neutral protamine Hagedorn (NPH), on as many days, while controls were injected with diluent alone. Animals injected with four doses of insulin were pretreated by i.c.v. administration of graded doses of the glucocorticoid receptor antagonist or vehicle alone prior to the first three doses of insulin. Repeated daily injection of NPH exacerbated hypoglycemia, attenuated patterns of glucagon and epinephrine secretion, and diminished neuronal transcriptional activation in discrete CNS metabolic loci, including the lateral hypothalamic area, dorsomedial hypothalamic nucleus, paraventricular hypothalamic nucleus, and nucleus of the solitary tract. While i.c.v. delivery of 25 or 100 ng doses of CP-472555 did not alter any of these parameters, animals treated with 500 ng exhibited circulating glucose, glucagon, and epinephrine levels that were similar to those in rats injected with one dose of insulin, as well as a reversal of recurring insulin-induced hypoglycemia-associated reductions in Fos immunolabeling in the lateral hypothalamic area, dorsomedial hypothalamic nucleus, and paraventricular hypothalamic nucleus. These results provide unique pharmacological evidence that antecedent activation of central glucocorticoid receptor is required for exacerbation of hypoglycemia during recurring insulin-induced hypoglycemia, and that these receptors mediate modulatory effects of hypoglycemic hypercorticosteronemia on autonomic efferent responses to recurring insulin-induced hypoglycemia. The data also suggest that neurons in central loci characterized here by antagonist-mediated overturn of recurring insulin-induced hypoglycemia-induced decreases in neuronal transcriptional activation may be direct or indirect substrates for this hormonal modulation action.

Effects of estradiol on glycemic and CNS neuronal activational responses to recurrent insulin-induced hypoglycemia in the ovariectomized female rat.

Recurrent insulin-induced hypoglycemia (RIIH) results in glucose counterregulatory dysfunction in men and male rodents. Intensified hypoglycemia in the latter coincides with diminished neuronal Fos expression in central metabolic regulatory structures, evidence that supports habituation of CNS-mediated compensatory motor outflow during re-exposure to this metabolic stress. In light of the evidence for counterregulatory resistance to precedent hypoglycemia in women, we utilized estradiol-treated ovariectomized (OVX) female rats to examine the hypothesis that this hormone regulates neural adaptability to recurring hypoglycemia. Groups of OVX rats were implanted with subcutaneous silastic capsules containing estradiol benzoate (E) or oil alone, and injected subcutaneously with one or four doses of the intermediate-acting insulin, Humulin NPH, one dose daily, or with diluent alone. Blood glucose levels were not altered by RIIH in E-implanted OVX animals, but were significantly decreased after four versus one insulin injection in the OVX+oil group. Mean numbers of Fos-immunoreactive (ir) neurons in the paraventricular nucleus hypothalamus (PVH), dorsomedial nucleus hypothalamus (DMH), and lateral hypothalamic area (LHA) were higher in both E- versus oil-implanted OVX rats injected with diluent only. Acute hypoglycemia significantly increased mean counts of Fos-ir-positive neurons in the PVH, DMH, and LHA, as well as the nucleus of the solitary tract (NTS) and area postrema (AP) in E- and oil-treated animals to an equivalent extent. OVX+E rats exhibited comparable numbers of Fos-positive neurons in the PVH, DMH, and LHA after one versus four insulin injections, whereas the numbers of labeled neurons in NTS and AP were increased or decreased, respectively, by RIIH. Oil-implanted OVX rats showed significantly diminished numbers of Fos-ir-positive neurons in each neural structure after repeated hypoglycemia. The present data demonstrate that estradiol sustains or enhances neuronal reactivity to recurring hypoglycemia in central metabolic structures, whereas hypoglycemic patterns of Fos expression in each site become habituated during RIIH in the absence of this steroid. The brain sites characterized here by estrogen-dependent maintenance of neuronal genomic reactivity to this substrate fuel imbalance may contain direct and/or indirect cellular targets for hormonal actions that prevent adaptation of CNS-controlled motor responses to this metabolic stress.

Effects of ultra-wideband electromagnetic pulses on pre-neoplastic mammary epithelial cell proliferation.

Electromagnetic ultra-wideband pulses (UWB) or nanopulses, are generated by a wide range of electronic devices used in communications and radar technology. However, the specific effects of nanopulse exposure on cell growth and function have not been extensively investigated. Here, studies have been conducted to determine the effects of prolonged exposure to non-ionizing, low to moderate intensity nanopulses on the growth of pre-neoplastic CL-S1 mammary epithelial cells in vitro. Cells were grown in culture and maintained in serum-free defined medium containing 10 ng/ml EGF and 10 microg/ml insulin as comitogens. Studies showed that 0.25-3.0 h exposure to nanopulses of 18 kV/m field intensity, 1 kHz repetition rate and 10 ns pulse width had no effect on CL-S1 cell growth or viability during the subsequent 72-h culture period. However, exposure to similar nanopulses for prolonged periods of time (4-6 h) resulted in a significant increase in cell proliferation, as compared to untreated controls. Additional studies showed that nanopulse exposure enhanced CL-S1 cell growth when cells were maintained in media containing only EGF, but had no effect on cells maintained in defined media that were mitogen-free or containing only insulin. Studies also showed that the growth-promoting effects of nanopulse exposure were associated with a relatively large increase in intracellular levels of phospho-MEK1 (active) and phospho-ERK1/2 (active) in these cells. These findings demonstrate that prolonged exposure to moderate levels of UWB enhanced EGF-dependent mitogenesis, and that this growth-promoting effect appears to be mediated by enhanced activation of the mitogen-activated protein kinase (MAPK) signalling pathway in pre-neoplastic CL-S1 mammary epithelial cells.