Neural substrates for estrogen regulation of glucose homeostasis remain unclear. Female rat dorsal vagal complex (DVC) A2 noradrenergic neurons are estrogen-and metabolic-sensitive. The ventromedial hypothalamic nucleus (VMN) is a key component of the brain network that governs counter-regulatory responses to insulin-induced hypoglycemia (IIH). Here, the selective estrogen receptor-alpha (ERα) or -beta (ERβ) antagonists MPP and PHTPP were administered separately to the caudal fourth ventricle to address the premise that these hindbrain ER variants exert distinctive control of VMN reactivity to IIH in the female sex. Data show that ERα governs hypoglycemic patterns of VMN astrocyte glycogen metabolic enzyme, e.g. glycogen synthase and phosphorylase protein expression, whereas ERβ mediates local glycogen breakdown. DVC ER 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 ER oppose hypoglycemic hyperglucagonemia, while ERβ contributes to reduced corticosterone output. Outcomes reveal that input from the female hindbrain to the VMN is critical for energy reserve mobilization, metabolic transmitter signaling, and counter-regulatory hormone secretion during hypoglycemia, and that ER control those cues. Evidence that VMN NE content is not controlled by hindbrain ERα or -β implies that these receptors may regulate VMN function via NE-independent mechanisms, or alternatively, that other neurotransmitter signals to the VMN may control local substrate receptivity to NE.

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Estrogen receptor-alpha (ERα) and -beta (ERβ) occur in key elements of the brain glucohomeostatic network in both sexes, including the hindbrain dorsal vagal complex (DVC), but the influence of distinct receptor populations on this critical function is unclear. The ventromedial hypothalamic nucleus (VMN) maintains glucose balance by integrating nutrient, endocrine, and neurochemical cues, including metabolic sensory information supplied by DVC A2 noradrenergic neurons. Current research utilized the selective ERα and ERβ antagonists MPP and PHTPP to characterize effects of DVC ERs on VMN norepinephrine (NE) activity and metabolic neurotransmitter signaling in insulin-induced hypoglycemic (IIH) male rats. Data show that ERβ inhibits VMN glycogen synthase and stimulates phosphorylase protein expression, while attenuating hypoglycemic augmentation of glycogen content. Furthermore, both ERs attenuate VMN glucose concentrations during IIH. Hypoglycemic up-regulation of nitric oxide (NO) and brain-derived neurotrophic factor (BDNF) signaling was correspondingly driven by ERα or -β, whereas GABA and steroidogenic factor-1 were respectively suppressed independently of ER input or by ERβ. IIH intensified VMN NE accumulation by ERβ-dependent mechanisms, but did not alter NE levels in other gluco-regulatory loci. ERβ amplified the magnitude of insulin-induced decline in blood glucose. Both ER regulate corticosterone, but not glucagon secretion during IIH and oppose hypoglycemic diminution of circulating free fatty acids. These findings identify distinguishing versus common VMN functions targeted by DVC ERα and -β. Sex differences in hypoglycemic VMN NE accumulation, glycogen metabolism, and transmitter signaling may involve, in part, discrepant regulatory involvement or differential magnitude of impact of these hindbrain ERs.

Norepinephrine (NE) directly regulates ventromedial hypothalamic nucleus (VMN) glucoregulatory neurons and also controls glycogen-derived fuel provision to those cells. VMN nitric oxide (NO) and c-aminobutyric acid (GABA) neurons and astrocytes express estrogen receptor-alpha (ERa) and ER-beta (ERb) proteins. Current research used selective ERa (1,3Bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride) or ERb (4-[2-phenyl-5,7bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol) antagonists to address the premise that these ERs govern basal and/or NE-associated patterns of VMN metabolic neuron signaling and astrocyte glycogen metabolism. Both ERs stimulate expression of the enzyme marker protein neuronal nitric oxide synthase, not glutamate decarboxylase 65/67. NE inhibition or augmentation of neuronal nitric oxide synthase and glutamate decarboxylase 65/67 profiles was ER-independent or-dependent, respectively. In both neuron types, VMN ERb activity inhibited baseline alpha1-(a 1-) and/or alpha2-(a 2-)adrenergic receptor (AR) expression, but ERa and-b signaling was paradoxically crucial for noradrenergic upregulation of a 2-AR. NE inhibited glycogen synthase expression and exerted opposite effects on VMN adenosine monophosphate-sensitive glycogen phosphorylase (GP)-brain type (stimulatory) versus NE-sensitive GP muscle (inhibitory) via ERa orb activity. Results document unique ERa and ERb actions on metabolic transmitter and AR protein expression in VMN nitrergic versus GABAergic neurons. ER effects varied in the presence versus absence of NE, indicating that both neuron types are substrates for estradiol and noradrenergic regulatory interaction. NE-dependent ER control of VMN GP variant expression implies that these signals also act on astrocytes to direct physiological stimulus-specific control of glycogen metabolism, which may in turn influence GABA transmission.

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Despite the apparent advantages for long-term treatment and local therapies against intestinal diseases, the oral delivery of nucleic acids has been challenging due to unfavorable physiological conditions for their stability. In this study, a novel nanodelivery system of PEG-PCL nanoparticles with encapsulated nucleic acids–mannosylated PEI (Man-PEI) complexes was developed for intestinal delivery. We complexed model nucleic acids with Man-PEI at the optimal N/P ratio of 20:1 for in vitro and in vivo analyses. Cells were transfected in vitro and analyzed for gene expression, receptor-mediated uptake, and PEG-PCL nanoparticles’ toxicity. We also evaluated the nucleic acid’s stability in the nanocarrier during formulation, and under simulated gastrointestinal environments or the presence of nucleases. Finally, we assessed the biodistribution for the PEG-PCL nanoparticles with encapsulated complexes and their ability to transfect intestinal cells in vivo. Nucleic acids complexed with Man-PEI were protected from degradation against nucleases. In comparison to the parent compound PEI, Man-PEI transfected the cells with an overall higher potency. Competition assay indicated receptor-mediated endocytosis promoted by mannose receptors. The PEG-PCL nanoparticles with Man-PEI/plasmid complexes indicated minimal cytotoxicity. The nanocarrier successfully protected the complexes in a simulated gastric fluid environment and released them in a simulated intestinal fluid environment, promoted by the presence of lipases. The oral administration of the PEG-PCL nanoparticles with encapsulated Man-PEI/plasmid complexes transfected intestinal cells with the plasmid in vivo, while presenting a time-dependent progression through the intestines. Conclusively, our carrier system can deliver genetic material to the GI tract and actively target mannose receptor overexpressing cells.

Astrocytes store glycogen as energy and promote neuro-metabolic stability through supply of oxidizable L-lactate. Whether lactate regulates ventromedial hypothalamic nucleus (VMN) glucostatic function as a metabolic volume transmitter is unknown. Current research investigated whether G-protein-coupled lactate receptor GPR81 controls astrocyte glycogen metabolism and glucose-regulatory neurotransmission in the ventrolateral VMN (VMNvl), where glucose-regulatory neurons reside. Female rats were pretreated by intra-VMN GPR81 or scramble siRNA infusion before insulin or vehicle injection. VMNvl cell or tissue samples were acquired by laser-catapult- or micropunch microdissection for Western blot protein or uHPLC-electrospray ionization-mass spectrometric glycogen analyses. Data show that GPR81 regulates eu- and/or hypoglycemic patterns of VMNvl astrocyte glycogen metabolic enzyme and 5'-AMP-activated protein kinase (AMPK) protein expression according to VMNvl segment. GPR81 inhibits baseline rostral and caudal VMNvl glycogen accumulation, but mediates glycogen breakdown in the former site during hypoglycemia. During euglycemia, GPR81 suppresses the transmitter marker neuronal nitric oxide synthase (nNOS) in rostral and caudal VMNvl nitrergic neurons, but stimulates (rostral VMNvl) or inhibits (caudal VMNvl) GABAergic neuron glutamate decarboxylase65/67 (GAD)protein. During hypoglycemia, GPR81 regulates AMPK activation in nitrergic and GABAergic neurons located in the rostral, but not caudal. VMNvl. VMN GPR81 knockdown amplified hypoglycemic hypercorticosteronemia, but not hyperglucagonemia. Results provide novel proof that VMNvl astrocyte and glucose-regulatory neurons express GPR81 protein. Data identify neuroanatomical subpopulations of VMNvl astrocytes and glucose-regulatory neurons that exhibit differential reactivity to GPR81 input. Heterogeneous GPR81 effects during eu- versus hypoglycemia infer that energy state may affect cellular sensitivity to or post-receptor processing of lactate transmitter signaling.

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