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.

Sex-dependent endozepinergic regulation of ventromedial hypothalamic nucleus glucose counter-regulatory neuron aromatase protein expression in the adult rat.

The hypothalamic brain cell types that produce estradiol from testosterone remain unclear. Aromatase inhibition affects ventromedial hypothalamic nucleus (VMN) glucose-stimulatory nitric oxide (NO) and glucose-inhibitory γ-aminobutyric acid (GABA) transmission during insulin (INS)-induced hypoglycemia (IIH). Pure GABA and NO nerve cell samples acquired by laser-catapult-microdissection from consecutive rostro-caudal segments of the VMN were analyzed by Western blot to investigate whether regional subpopulations of each cell type contain machinery for neuro-estradiol synthesis. Astrocyte endozepinergic signaling governs brain steroidogenesis. Pharmacological tools were used here to determine if the glio-peptide octadecaneuropeptide (ODN) controls aromatase expression in GABA and NO neurons during eu- and/or hypoglycemia. Intracerebroventricular administration of the ODN G-protein coupled-receptor antagonist cyclo(1-8)[DLeu5]OP (LV-1075) decreased (male) or enhanced (female) VMN GABAergic neuron aromatase expression, but increased or reduced this profile in nitrergic neurons in a region-specific manner in each sex. IIH suppressed aromatase levels in GABA neurons located in the middle segment of the male VMN or distributed throughout this nucleus in the female. This inhibitory response was altered by the ODN isoactive surrogate octapeptide (OP) in female, but was refractory to OP in male. NO neuron aromatase protein in hypoglycemic male (middle and caudal VMN) and female (rostral and caudal VMN) rats, but was normalized in OP- plus INS-treated rats of both sexes. Results provide novel evidence that VMN glucose-regulatory neurons may produce neuro-estradiol, and that the astrocyte endozepine transmitter ODN may impose sex-specific control of baseline and/or hypoglycemic patterns of aromatase expression in distinct subsets of nitrergic and GABAergic neurons in this neural structure.

GHRH Neurons from the Ventromedial Hypothalamic Nucleus Provide Dynamic and Sex-Specific Input to the Brain Glucose-Regulatory Network.

The ventromedial hypothalamic nucleus (VMN) controls glucose counter-regulation, including pituitary growth hormone (GH) secretion. VMN neurons that express the transcription factor steroidogenic factor-1/NR5A1 (SF-1) participate in glucose homeostasis. Research utilized in vivo gene knockdown tools to determine if VMN growth hormone-releasing hormone (Ghrh) regulates hypoglycemic patterns of glucagon, corticosterone, and GH outflow according to sex. Intra-VMN Ghrh siRNA administration blunted hypoglycemic hypercorticosteronemia in each sex, but abolished elevated GH release in males only. Single-cell multiplex qPCR showed that dorsomedial VMN (VMNdm) Ghrh neurons express mRNAs encoding Ghrh, SF-1, and protein markers for glucose-inhibitory (γ-aminobutyric acid) or -stimulatory (nitric oxide; glutamate) neurotransmitters. Hypoglycemia decreased glutamate decarboxylase67 (GAD67) transcripts in male, not female VMNdm Ghrh/SF-1 neurons, a response that was refractory to Ghrh siRNA. Ghrh gene knockdown prevented, in each sex, hypoglycemic down-regulation of Ghrh/SF-1 nerve cell GAD65 transcription. Ghrh siRNA amplified hypoglycemia-associated up-regulation of Ghrh/SF-1 neuron nitric oxide synthase mRNA in male and female, without affecting glutaminase gene expression. Ghrh gene knockdown altered Ghrh/SF-1 neuron estrogen receptor-alpha (ERα) and ER-beta transcripts in hypoglycemic male, not female rats, but up-regulated GPR81 lactate receptor mRNA in both sexes. Outcomes infer that VMNdm Ghrh/SF-1 neurons may be an effector of SF-1 control of counter-regulation, and document Ghrh modulation of hypoglycemic patterns of glucose-regulatory neurotransmitter along with estradiol and lactate receptor gene transcription in these cells. Co-transmission of glucose-inhibitory and -stimulatory neurochemicals of diverse chemical structure, spatial, and temporal profiles may enable VMNdm Ghrh neurons to provide complex dynamic, sex-specific input to the brain glucose-regulatory network.

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. Current research 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 male; 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. Results show that GLUT2 inhibits ERK1/2, p38, and SAPK/JNK MAPK activity in male, 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 can act as either an inhibitor or inducer of SAPK/JNK activation in glucose-deprived male versus female glial cells, respectively.

Sex Dimorphic Glucose Transporter-2 Regulation of Hypothalamic Astrocyte Glucose and Energy Sensor Expression and Glycogen Metabolism.

The plasma membrane glucose transporter-2 (GLUT2) monitors brain cell uptake of the critical nutrient glucose, and functions within astrocytes of as-yet-unknown location to control glucose counter-regulation. Hypothalamic astrocyte-neuron metabolic coupling provides vital cues to the neural glucostatic network. Current research utilized an established hypothalamic primary astrocyte culture model along with gene knockdown tools to investigate whether GLUT2 imposes sex-specific regulation of glucose/energy sensor function and glycogen metabolism in this cell population. Data show that GLUT2 stimulates or inhibits glucokinase (GCK) expression in glucose-supplied versus -deprived male astrocytes, but does not control this protein in female. Astrocyte 5'-AMP-activated protein kinaseα1/2 (AMPK) protein is augmented by GLUT2 in each sex, but phosphoAMPKα1/2 is coincidently up- (male) or down- (female) regulated. GLUT2 effects on glycogen synthase (GS) diverges in the two sexes, but direction of this control is reversed by glucoprivation in each sex. GLUT2 increases (male) or decreases (female) glycogen phosphorylase-brain type (GPbb) protein during glucoprivation, yet simultaneously inhibits (male) or stimulates (female) GP-muscle type (GPmm) expression. Astrocyte glycogen accumulation is restrained by GLUT2 when glucose is present (male) or absent (both sexes). Outcomes disclose sex-dependent GLUT2 control of the astrocyte glycolytic pathway sensor GCK. Data show that glucose status determines GLUT2 regulation of GS (both sexes), GPbb (female), and GPmm (male), and that GLUT2 imposes opposite control of GS, GPbb, and GPmm profiles between sexes during glucoprivation. Ongoing studies aim to investigate molecular mechanisms underlying sex-dimorphic GLUT2 regulation of hypothalamic astrocyte metabolic-sensory and glycogen metabolic proteins, and to characterize effects of sex-specific astrocyte target protein responses to GLUT2 on glucose regulation.

p53 missense mutant G242A subverts natural killer cells in sheltering mouse breast cancer cells against immune rejection.

Cancer cells acquire immunoediting ability to evade immune surveillance and thus escape eradication. It is widely known that mutant proteins encoded from tumor suppressor TP53 exhibit gain-of-function in cancer cells, thereby promoting progression; however, how mutant p53 contributes to the sheltering of cancer cells from host anticancer immunity remains unclear. Herein, we report that murine p53 missense mutation G242A (corresponding to human G245A) suppresses the activation of host natural killer (NK) cells, thereby enabling breast cancer cells to avoid immune assault. We found that serial injection of EMT6 breast cancer cells that carry wild-type (wt) Trp53, like normal fibroblasts, promoted NK activity in mice, while SVTneg2 cells carrying Trp53 G242A+/+ mutation decreased NK cell numbers and increased CD8+ T lymphocyte numbers in spleen. Innate immunity based on NK cells and CD8 T cells was reduced in p53 mutant-carrying transgenic mice (Trp53 R172H/+, corresponding to human R175H/+). Further, upon co-culture with isolated NK cells, EMT6 cells substantively activated NK cells and proliferation thereof, increasing interferon-gamma (IFN-γ) production; however, SVTneg2 cells suppressed NK cell activation. Further mechanistic study elucidated that p53 can modulate expression by cancer cells of Mult-1 and H60a, which are activating and inhibitory ligands for NKG2D receptors of NK cells, respectively, to enhance immune surveillance against cancer. Our findings demonstrate that wt p53 is requisite for NK cell-based immune recognition and elimination of cancerous cells, and perhaps more importantly, that p53 missense mutant presence in cancer cells impairs NK cell-attributable responses, thus veiling cancerous cells from host immunity and enabling cancer progression.

Gb3-cSrc complex in glycosphingolipid-enriched microdomains contributes to the expression of p53 mutant protein and cancer drug resistance via ß-catenin-activated RNA methylation.

Glucosylceramide synthase (GCS) is a key enzyme catalyzing ceramide glycosylation to generate glucosylceramide (GlcCer), which in turn serves as the precursor for cells to produce glycosphingolipids (GSLs). In cell membranes, GSLs serve as essential components of GSL-enriched microdomains (GEMs) and mediate membrane functions and cell behaviors. Previous studies showed that ceramide glycosylation correlates with upregulated expression of p53 hotspot mutant R273H and cancer drug resistance. Yet, the underlying mechanisms remain elusive. We report herewith that globotriaosylceramide (Gb3) is associated with cSrc kinase in GEMs and plays a crucial role in modulating expression of p53 R273H mutant and drug resistance. Colon cancer cell lines, either WiDr homozygous for missense-mutated TP53 (R273H+/+) or SW48/TP53-Dox bearing heterozygous TP53 mutant (R273H/+), display drug resistance with increased ceramide glycosylation. Inhibition of GCS with Genz-161 (GENZ 667161) resensitized cells to apoptosis in these p53 mutant-carrying cancer cells. Genz-161 effectively inhibited GCS activity, and substantially suppressed the elevated Gb3 levels seen in GEMs of p53-mutant cells exposed to doxorubicin. Complex formation between Gb3 and cSrc in GEMs to activate ß-catenin was detected in both cultured cells and xenograft tumors. Suppression of ceramide glycosylation significantly decreased Gb3-cSrc in GEMs, ß-catenin, and methyltransferase-like 3 for m6A RNA methylation, thus altering pre-mRNA splicing, resulting in upregulated expression of wild-type p53 protein, but not mutants, in cells carrying p53 R273H. Altogether, increased Gb3-cSrc complex in GEMs of membranes in response to anticancer drug induced cell stress promotes expression of p53 mutant proteins and accordant cancer drug resistance.