Rebound Feeding in the Wake of Short-Term Suspension of Food Intake Differs in the Presence of Estrous Cycle Peak versus Nadir Levels of Estradiol

BackgroundShort-term interruption of feeding is ordinary in modern life but negatively impacts appetite control and body weight. Estradiol (E) imposes long-term inhibitory tonus on food consumption; however, E influence on energy repletion secondary to food deprivation (FD) is unclear. This study investigated the hypothesis that E signal strength regulates hyperphagic responses to FD of varying duration.MethodsOvariectomized female rats were implanted with E-containing silastic capsules (30 [E-30] or 300 µg [E-300]/mL) to replicate plasma concentrations at cycle nadir versus peak levels.ResultsData show that food intake was increased equally in E-30 and E-300 rats after 12 hours of food deprivation (FD-12); yet, FD of 18 hours (FD-18) amplified refeeding by E-300 versus E-30. Caudal fourth ventricular administration of the 5′-monophosphate-activated protein kinase (AMPK) inhibitor compound C (Cc) did not modify FD-induced hyperphagia in E-30 (regardless of FD interval) or E-300 animals exposed to FD-12, but diminished refeeding after FD-18 in E-300 rats. Cc-reversible hyperglycemia occurred in refed FD-18 groups. Serum insulin was resistant to FD-12 plus refeeding, but was elevated by AMPK-dependent mechanisms in refed E-300 FD-18 rats; equivalent Cc-insensitive decrements in circulating leptin occurred in all FD groups.ConclusionCurrent results show that estrous cycle peak, but not baseline, E levels engage hindbrain AMPK signaling to intensify hyperphagia in response to prolongation of FD. Observations of hindbrain AMPK-dependent hyperglycemia, alongside elevated insulin secretion, in refed rats exposed to FD-18 implicate this sensor in insulin resistance mechanisms of glucose partitioning in response to this metabolic imbalance

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 (DβH) 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

Oral Delivery of Nucleic Acids with Passive and Active Targeting to the Intestinal Tissue Using Polymer-Based Nanocarriers

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

Diazepam Binding Inhibitor Control of Eu- and Hypoglycemic Patterns of Ventromedial Hypothalamic Nucleus Glucose-Regulatory Signaling

Pharmacological stimulation/antagonism of astrocyte glio-peptide octadecaneuropeptide signaling alters ventromedial hypothalamic nucleus (VMN) counterregulatory γ-aminobutyric acid (GABA) and nitric oxide transmission. The current research used newly developed capillary zone electrophoresis-mass spectrometry methods to investigate hypoglycemia effects on VMN octadecaneuropeptide content, along with gene knockdown tools to determine if octadecaneuropeptide signaling regulates these transmitters during eu- and/or hypoglycemia. Hypoglycemia caused dissimilar adjustments in the octadecaneuropeptide precursor, i.e., diazepam-binding-inhibitor and octadecaneuropeptide levels in dorsomedial versus ventrolateral VMN. Intra-VMN diazepam-binding-inhibitor siRNA administration decreased baseline 67 and 65 kDa glutamate decarboxylase mRNA levels in GABAergic neurons laser-microdissected from each location, but only affected hypoglycemic transcript expression in ventrolateral VMN. This knockdown therapy imposed dissimilar effects on eu- and hypoglycemic glucokinase and 5’-AMP-activated protein kinase-alpha1 (AMPKα1) and -alpha2 (AMPKα2) gene profiles in dorsomedial versus ventrolateral GABAergic neurons. Diazepam-binding-inhibitor gene silencing up-regulated baseline (dorsomedial) or hypoglycemic (ventrolateral) nitrergic neuron neuronal nitric oxide synthase mRNA profiles. Baseline nitrergic cell glucokinase mRNA was up- (ventrolateral) or down- (dorsomedial) regulated by diazepam-binding-inhibitor siRNA, but knockdown enhanced hypoglycemic profiles in both sites. Nitrergic nerve cell AMPKα1 and -α2 transcripts exhibited division-specific responses to this genetic manipulation during eu- and hypoglycemia. Results document the utility of capillary zone electrophoresis-mass spectrometric tools for quantification of ODN in small-volume brain tissue samples. Data show that hypoglycemia has dissimilar effects on ODN signaling in the two major neuroanatomical divisions of the VMN and that this glio-peptide imposes differential control of glucose-regulatory neurotransmission in the VMNdm versus VMNvl during eu- and hypoglycemia

Glucose Transporter-2 Regulation of Male versus Female Hypothalamic Astrocyte MAPK Expression and Activation: Impact of Glucose

The plasma membrane glucose transporter (GLUT)-2 is unique among GLUT family proteins in that it also functions as a glucose sensor. GLUT2 imposes sex-dimorphic control of hypothalamic astrocyte glucose storage and catabolism by unknown mechanisms. Mitogen-activated protein kinase (MAPK) signaling cascades operate within stress-sensitive signal transduction pathways. The present study employed an established primary astrocyte culture model and gene knockdown tools to investigate whether one or more of the three primary MAP kinase families are regulated by GLUT2. GLUT2 gene knockdown caused opposing adjustments in total ERK1/2 proteins in glucose-supplied male versus female astrocytes, augmenting or reducing the mean phosphorylated/total protein ratio for 44 and 42 kDa variants in these sexes. Glucose deprivation amplified this ratio for both ERK1/2 variants, albeit by a larger magnitude in males; GLUT2 siRNA exacerbated this stimulatory response in males only. Phosphorylated/total p38 MAPK protein ratios were up-regulated by GLUT2 knockdown in male, but not female astrocytes. Glucose-deprived astrocytes exhibited no change (male) or reduction (female) in this ratio after GLUT2 gene silencing. GLUT2 siRNA increased the phosphorylated/total protein ratio for 54 and 46 kDa SAPK/JNK proteins in each sex when glucose was present. However, glucose withdrawal suppressed (male) or amplified (female) these ratios, while GLUT2 knockdown attenuated these inverse responses. The results show that GLUT2 inhibits ERK1/2, p38, and SAPK/JNK MAPK activity in male astrocytes, but differentially stimulates and inhibits activity of these signaling pathways in female hypothalamic astrocytes. Glucoprivation induces divergent adjustments in astrocyte p38 MAPK and SAPK/JNK activities. The findings demonstrate a stimulatory role for GLUT2 in p38 MAPK activation in glucose-starved female astrocytes, but it can act as either an inhibitor or inducer of SAPK/JNK activation in glucose-deprived male versus female glial cells, respectively

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