Proopiomelanocortin Gene Expression Research Articles

Sex differences in intestinal morphology and increase in diencephalic neuropeptide Y gene expression in female but not male Pekin ducks exposed to chronic heat stress.

The impact of heat stress (HS) on production is intricately linked with feed intake. We investigated the effects of HS on intestines and diencephalic genes in Pekin ducks. One hundred and sixty adult ducks were allocated to two treatment rooms. The control room was maintained at 22°C and the HS room at 35°C for the first 10 h of the day then reduced to 29.5°C. After 3 weeks, 10 hens and 5 drakes were euthanized from each room and jejunum and ileum collected for histology. Brains were collected for gene expression analysis using qRT-PCR. Intestinal morphology data were analyzed with two-way ANOVA and diencephalic gene data were analyzed with Kruskal-Wallis test. There was an increase in villi width in the ileum (p = .0136) and jejunum (p = .0019) of HS hens compared to controls. HS drakes showed a higher crypt depth (CD) in the jejunum (p = .0198) compared to controls. There was an increase in crypt goblet cells (GC) count in the ileum (p = .0169) of HS drakes compared to HS hens. There was higher villi GC count (p = .07) in the jejunum of HS drakes compared to controls. There was an increase in the crypt GC density (p = .0054) in the ileum, not jejunum, of HS drakes compared to HS hens. Further, there were no differences in the proopiomelanocortin gene expression in either sex but there was an increase in the expression of neuropeptide Y (NPY) gene in HS hens (p = .031) only and a decrease in the corticotropin releasing hormone gene in the HS drakes (p = .037) compared to controls. These data show that there are sex differences in the effect of HS on gut morphology while the upregulation in NPY gene may suggest a role in mediating response to chronic HS.

Sirtuin1-Mediated Deacetylation of Hypothalamic TTF-1 Contributes to the Energy Deficiency Response.

TTF-1 stimulates appetite by regulating the expression of agouti-related peptide (AgRP) and proopiomelanocortin (POMC) genes in the hypothalamus of starving animals. However, the mechanism underlying TTF-1's response to decreased energy levels remains elusive. Here, we provide evidence that the NAD+-dependent deacetylase, sirtuin1 (Sirt1), activates TTF-1 in response to energy deficiency. Energy deficiency leads to a twofold increase in the expression of both Sirt1 and TTF-1, leading to the deacetylation of TTF-1 through the interaction between the two proteins. The activation of Sirt1, induced by energy deficiency or resveratrol treatment, leads to a significant increase in the deacetylation of TTF-1 and promotes its nuclear translocation. Conversely, the inhibition of Sirt1 prevents these Sirt1 effects. Notably, a point mutation in a lysine residue of TTF-1 significantly disrupts its deacetylation and thus nearly completely hinders its ability to regulate AgRP and POMC gene expression. These findings highlight the importance of energy-deficiency-induced deacetylation of TTF-1 in the control of AgRP and POMC gene expression.

Patterns of stress response to foreign eggs by a rejecter host of an obligate avian brood parasite.

One of the most effective defenses of avian hosts against obligate brood parasites is the ejection of parasitic eggs from the nests. Despite the clear fitness benefits of this behavior, individuals within so-called "egg-rejecter" host species still show substantial variation in their propensity to eliminate foreign eggs from the nest. We argue that this variation can be further understood by studying the physiological mechanisms of host responses to brood parasitic egg stimuli: independent lines of research increasingly support the hypothesis that stress-related physiological response to parasitic eggs may trigger egg rejection. The "stress-mediated egg rejection" hypothesis requires that hosts activate the stress-response when responding to parasitic egg stimuli. We tested this prediction by asking whether hosts showed differential stress response when exposed to host-like (mimetic) or parasite-like (non-mimetic) eggs. We experimentally parasitized incubating American robins Turdus migratorius, a robust egg-rejecter host to obligate brood parasitic brown-headed cowbirds Molothrus ater, with mimetic or non-mimetic model eggs. To assess the stress response, we measured the heart rate in incubating females immediately after experimental parasitism. We also measured plasma corticosterone and, in a subset of birds, used RNA-sequencing to analyze the expression of proopiomelanocortin (POMC), a precursor of adrenocorticotropic hormone, 2 h after experimental parasitism. We found that egg type had no effect on heart rate. Two hours following experimental parasitism, plasma corticosterone did not differ between the differently-colored model egg treatments or between rejecter and accepter females within the non-mimetic treatment. However, females exposed to non-mimetic eggs showed an upregulation of POMC gene expression (before FDR correction) in the pituitary compared with females treated with mimetic eggs. Our findings suggest that in an egg-rejecter host species, non-mimetic parasitic eggs may increase the activity of the stress-related hypothalamic-pituitary-adrenal axis compared with mimetic eggs, although the temporaldynamics of this response are not yet understood.

Roles of Ghrelin and Leptin in Body Mass Regulation under Food Restriction Based on the AMPK Pathway in the Red-Backed Vole, Eothenomys miletus, from Kunming and Dali Regions.

The phenotype plasticity of animals' physiological characteristics is an important survival strategy to cope with environmental changes, especially the change in climate factors. Small mammals that inhabit seasonally changing environments often face the stress of food shortage in winter. This study measured and compared the thermogenic characteristics and related physiological indicators in the adenosine-5'-monophosphate-activated protein kinase (AMPK) pathway in Eothenomys miletus between Kunming (KM, n = 18) and Dali (DL, n = 18) under food restriction and refeeding. The results showed that food restriction and the region have significant effects on body mass, the resting metabolic rate (RMR), hypothalamic neuropeptide gene expression, ghrelin levels in the stomach and serum, serum leptin level and the activity of AMPK, and malonyl CoA and carnitine palmitoyltransferase 1 (CPT-1) activity. Food restriction reduced the body mass, the gene expression of neuropeptide proopiomelanocortin (POMC), cocaine- and amphetamine-regulated transcription peptide (CART), and leptin level. However, the ghrelin concentration and AMPK activity increased. After refeeding, there was no difference in these physiological indexes between the food restriction and control groups. Moreover, the physiological indicators also showed regional differences, such as the body mass, POMC and CART gene expression, ghrelin concentration in the stomach and serum, and AMPK activity in DL changed more significantly. All these results showed that food restriction reduces energy metabolism in E. miletus. After refeeding, most of the relevant physiological indicators can return to the control level, indicating that E. miletus has strong phenotypic plasticity. Ghrelin, leptin, and the AMPK pathway play an important role in the energy metabolism of E. miletus under food restriction. Moreover, regional differences in physiological indicators under food restriction may be related to the different temperatures or food resources in different regions.

Transgenerational inheritance of fetal alcohol effects on proopiomelanocortin gene expression and methylation, cortisol response to stress, and anxiety-like behaviors in offspring for three generations in rats: Evidence for male germline transmission.

Previously it has been shown that fetal alcohol exposure increases the stress response partly due to lowering stress regulatory proopiomelanocortin (Pomc) gene expression in the hypothalamus via epigenetic mechanisms for multiple generations in mixed-breed rats. In this study we assess the induction of heritable epigenetic changes of Pomc-related variants by fetal alcohol exposure in isogenic Fischer 344 rats. Using transgenerational breeding models and fetal alcohol exposure procedures, we determined changes in hypothalamic Pomc gene expression and its methylation levels, plasma corticosterone hormone response to restraint stress, and anxiety-like behaviors using elevated plus maze tests in fetal alcohol-exposed offspring for multiple generations in isogenic Fischer rats. Fetal alcohol-exposed male and female rat offspring showed significant deficits in POMC neuronal functions with increased Pomc gene methylation and reduced expression. These changes in POMC neuronal functions were associated with increased plasma corticosterone response to restraint stress and increased anxiety-like behavior. These effects of fetal alcohol exposure persisted in the F1, F2, and F3 progeny of the male germline but not of the female germline. These data suggest that fetal alcohol exposure induces heritable changes in Pomc-related variants involving stress hyperresponsiveness and anxiety-like behaviors which perpetuate into subsequent generations through the male germline via epigenetic modifications.

Spexin Regulates Hypothalamic Leptin Action on Feeding Behavior.

Spexin (SPX) is a recently identified neuropeptide that is believed to play an important role in the regulation of energy homeostasis. Here, we describe a mediating function of SPX in hypothalamic leptin action. Intracerebroventricular (icv) SPX administration induced a decrease in food intake and body weight gain. SPX was found to be expressed in cells expressing leptin receptor ObRb in the mouse hypothalamus. In line with this finding, icv leptin injection increased SPX mRNA in the ObRb-positive cells of the hypothalamus, which was blocked by treatment with a STAT3 inhibitor. Leptin also increased STAT3 binding to the SPX promoter, as measured by chromatin immunoprecipitation assays. In vivo blockade of hypothalamic SPX biosynthesis with an antisense oligodeoxynucleotide (AS ODN) resulted in a diminished leptin effect on food intake and body weight. AS ODN reversed leptin’s effect on the proopiomelanocortin (POMC) mRNA expression and, moreover, decreased leptin-induced STAT3 binding to the POMC promoter sequence. These results suggest that SPX is involved in leptin’s action on POMC gene expression in the hypothalamus and impacts the anorexigenic effects of leptin.

Hypothalamic steroid receptor coactivator-2 regulates adaptations to fasting and overnutrition.

SUMMARYThe neuroendocrine system coordinates metabolic and behavioral adaptations to fasting, including reducing energy expenditure, promoting counterregulation, and suppressing satiation and anxiety to engage refeeding. Here, we show that steroid receptor coactivator-2 (SRC-2) in pro-opiomelanocortin (POMC) neurons is a key regulator of all these responses to fasting. POMC-specific deletion of SRC-2 enhances the basal excitability of POMC neurons; mutant mice fail to efficiently suppress energy expenditure during food deprivation. SRC-2 deficiency blunts electric responses of POMC neurons to glucose fluctuations, causing impaired counterregulation. When food becomes available, these mutant mice show insufficient refeeding associated with enhanced satiation and discoordination of anxiety and food-seeking behavior. SRC-2 coactivates Forkhead box protein O1 (FoxO1) to suppress POMC gene expression. POMC-specific deletion of SRC-2 protects mice from weight gain induced by an obesogenic diet feeding and/or FoxO1 overexpression. Collectively, we identify SRC-2 as a key molecule that coordinates multifaceted adaptive responses to food shortage.

The Ciji-Hua'ai-Baosheng II Formula Attenuates Chemotherapy-Induced Anorexia in Mice With H22 Hepatocellular Carcinoma.

Background: Ciji-Hua’ai-Baosheng II Formula (CHB-II-F) is a traditional Chinese medicine formula, which specifically targets different aspects of chemotherapy-induced adverse effects in patients with cancer. In our clinical application, CHB-II-F significantly alleviated chemotherapy-induced anorexia (loss of appetite) and improved the quality of life for patients with tumor during and after chemotherapy. However, the mechanism of CHB-II-F in alleviation of chemotherapy-induced anorexia remains to be further investigated.Aim of Study: To explore the therapeutic effect and mechanism of CHB-II-F on chemotherapy-induced anorexia in the mice model of H22 hepatoma.Materials and Methods: A total of 72 Kunming mice of SPF grade were inoculated subcutaneously with H22 hepatoma cells into the right anterior armpit of the mice. After 1 week of seeding, mice were injected intraperitoneally with a high dose of 5-fluorouracil (200 mg/kg 5-FU) to establish the model of chemotherapy. The mice were randomly divided into six groups: untreated group, 5-FU group, 5-FU plus Yangzheng Xiaoji capsule (YZXJC) group, and three groups of 5-FU plus different concentrations of CHB-II-F. All the mice in each group were treated for 14 days. The body weight, food intake, tumor volume, and tumor weight of mice were measured, and pathological examinations of tumor tissue, stomach, and duodenum were carried out. Expressions of serum Leptin, Neuropeptide Y (NPY), epidermal cell growth factor (EGF), Motilin (MTL), Orexin A (OXA), Gastrin (GAS), Ghrelin, Prostaglandin E2 (PGE2), and jejunum superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were examined. The protein and mRNA levels of proopiomelanocortin (POMC), Orexin receptor 1 (OX1R), neuropeptide Y (NPY), cocaine and amphetamine regulated transcript peptide (CART), Agouti gene-related protein (AgRP), Leptin receptor (Ob-R), and Ghrelin receptor (GHSR) were examined in hypothalamus, and the protein levels of substance P (SP) and 5-hydroxytryptamine (5-HT) in duodenum were measured.Results: The combination of CHB-II-F and 5-FU could enhance the inhibitory effect of 5-FU on tumor. The tumor inhibition rates of 5-FU group, YZXJC group, CHB-II-F(H) group, CHB-II-F(M) group, and CHB-II-F(L) group were 58.88, 28.08, 54.96, 37.69, and 28.61%, respectively. Compared with untreated group and 5-FU group, CHB-II-F significantly increased the body weight and food intake of tumor-bearing mice; increased the content of NPY, Orexin A, Ghrelin, GAS, MTL, EGF, and PGE2 in serum and the activity of SOD in jejunum; and decreased the content of Leptin in serum and the content of MDA in jejunum. Compared with untreated group and 5-FU group, CHB-II-F also enhanced the expression of OX1R, GHSR, NPY, and AgRP protein and gene and decreased the expression of Ob-R, POMC, and CART protein and gene in hypothalamus of mice, and the gene expression was consistent with the protein expression. In addition, CHB-II-F decreased the expression of 5-HT and SP protein in duodenum.Conclusion: In the murine model of H22 hepatocellular carcinoma (HCC) receiving chemotherapy, CHB-II-F enhances the inhibitory effect of 5-FU on tumor, significantly improves the pathological injury of gastrointestinal tract caused by chemotherapy, and regulates the secretion of gastrointestinal hormones. It may alleviate chemotherapy-induced anorexia by affecting appetite regulatory factors in the feeding area of hypothalamus central nervous system and peripheral appetite regulatory factors.

Differential Effects of Fkbp4 and Fkbp5 on Regulation of the Proopiomelanocortin Gene in Murine AtT-20 Corticotroph Cells.

The hypothalamic-pituitary-adrenal axis is stimulated in response to stress. When activated, it is suppressed by the negative feedback effect of glucocorticoids. Glucocorticoids directly inhibit proopiomelanocortin (Pomc) gene expression in the pituitary. Glucocorticoid signaling is mediated via glucocorticoid receptors, 11β-hydroxysteroid dehydrogenases, and the FK506-binding immunophilins, Fkbp4 and Fkbp5. Fkbp4 and Fkbp5 differentially regulate dynein interaction and nuclear translocation of the glucocorticoid receptor, resulting in modulation of the glucocorticoid action. Here, we explored the regulation of Fkbp4 and Fkbp5 genes and their proteins with dexamethasone, a major synthetic glucocorticoid drug, in murine AtT-20 corticotroph cells. To elucidate further roles of Fkbp4 and Fkbp5, we examined their effects on Pomc mRNA levels in corticotroph cells. Dexamethasone decreased Pomc mRNA levels as well as Fkpb4 mRNA levels in mouse corticotroph cells. Dexamethasone tended to decrease Fkbp4 protein levels, while it increased Fkpb5 mRNA and its protein levels. The dexamethasone-induced decreases in Pomc mRNA levels were partially canceled by Fkbp4 knockdown. Alternatively, Pomc mRNA levels were further decreased by Fkbp5 knockdown. Thus, Fkbp4 contributes to the negative feedback of glucocorticoids, and Fkbp5 reduces the efficiency of the glucocorticoid effect on Pomc gene expression in pituitary corticotroph cells.

Adenosine 5’-monophosphate induces hypothermia and alters gene expressions in the brain and liver of chicks

The adenosine A1 receptor is important for body temperature regulation in mammals; however, little is known about its function in avian species. In this study, we investigated the effects of the adenosine A1 receptor agonist and antagonist (adenosine 5′-monophosphate [5′-AMP] and 8 p-sulfophenyl theophylline [8-SPT], respectively) on thermoregulation in chickens. Male chicks were used in this study. After administration of 5′-AMP and 8-SPT, the rectal temperature, plasma metabolites, and gene expressions in the hypothalamus and liver were measured. The rectal temperature was reduced by peripheral administration of 5′-AMP, and the hypothermic effect of 5′-AMP was attenuated by central injection of 8-SPT in chicks. In the hypothalamus, the mRNA level of the agouti-related protein (AgRP) was increased by 5′-AMP administration, whereas it was suppressed by 8-SPT. The plasma levels of free fatty acid were elevated in 5′-AMP-treated chicks and that elevation was suppressed by the 8-SPT treatment. The gene expression of proopiomelanocortin in the hypothalamus was affected by 8-SPT. Nevertheless, the gene expressions of the thermoregulation-related genes, such as the thyrotropin-releasing hormone, were not affected by 5′-AMP and 8-SPT. Hepatic gene expressions related to lipid intake and metabolism were suppressed by 5′-AMP. However, the gene expression of the uncoupling protein was upregulated by 5′-AMP. Based on these results, birds, like mammals, will undergo adenosine A1 receptor-induced hypothermia. In conclusion, it is suggested that 5′-AMP-mediated hypothermia via the adenosine A1 receptor may affect the central melanocortin system and suppress hepatic lipid metabolism in chickens.

Sex-Determining Region Y Controls the Effects of Fetal Alcohol Exposure on Proopiomelanocortin Gene Expression.

Fetal alcohol exposure (FAE) causes various neurodevelopmental deficits in offspring, including reduced expression of the stress regulatory proopiomelanocortin (Pomc) gene and an elevated stress response for multiple generations via the male germline. Male germline-specific effects of FAE on the Pomc gene raises the question if the sex-determining region Y (SRY) may have a role in regulating Pomc gene expression. Using a transgenerational model of FAE in Fischer 344 rats, we determined the role of SRY in the regulation of the Pomc gene. FAEs, like on the Pomc gene, reduced Sry gene expression in sperm and the mediobasal hypothalamus (MBH) in male adult offspring. Fetal alcohol-induced inhibition of Sry gene expression was associated with increased Sry promoter DNA methylation. Additionally, fetal alcohol effects on the Sry gene persisted for three generations in the male germline but not in the female germline. Sry gene knockdown reduced the Pomc gene expression. Sry recruitment onto the Pomc promoter was found to be reduced in the hypothalamus of fetal alcohol-exposed rats compared to control rats. Pomc promoter luciferase activity was increased following Sry overexpression. A site-directed mutagenesis study revealed that SRY binding sites are required for POMC promoter transcription activity. Overall, these findings suggest that SRY plays a stimulatory role in the regulation of Pomc gene expression and may potentially contribute to the fetal alcohol-induced changes in the level of Pomc gene expression for multiple generations.

Expression and Role of Thyrotropin Receptors in Proopiomelanocortin-Producing Pituitary Cells.

Background: Thyrotropin (TSH) is well known as the hormone of the anterior pituitary thyrotrophs responsible for acting in the thyroid gland, where it stimulates synthesis and release of thyroid hormones through Gs and Gq/11 protein coupled TSH receptors (TSHRs). Methods: In this study, we examined whether the functional TSHRs are also expressed in cultured rat pituitary cells, using double immunocytochemistry, quantitative reverse transcription-polymerase chain reaction analysis, cAMP and hormone measurements, and single-cell calcium imaging. Results: Double immunocytochemistry revealed the expression of TSHRs in cultured corticotrophs and melanotrophs, in addition to previously identified receptors in folliculostellate cells. The functional coupling of these receptors to the Gq/11 signaling pathway was not observed, as demonstrated by the lack of TSH activation of IP3-dependent calcium mobilization in these cells when bathed in calcium-deficient medium. However, TSH increased cAMP production in a time- and concentration-dependent manner and facilitated calcium influx in single corticotrophs and melanotrophs, indicating their coupling to the Gs signaling pathway. Consistent with these findings, TSH stimulated adrenocorticotropin and β-endorphin release in male and female pituitary cells in a time- and concentration-dependent manner without affecting the expression of proopiomelanocortin gene. Conclusions: These results indicate that TSH is a potential paracrine modulator of anterior pituitary corticotrophs and melanotrophs, controlling the exocytotic but not the transcriptional pathway in a cAMP/calcium influx-dependent manner.

Decreased sensitivity to the anorectic effects of leptin in mice that lack a Pomc-specific neural enhancer.

Enhancer redundancy has been postulated to provide a buffer for gene expression against genetic and environmental perturbations. While work in Drosophila has identified functionally overlapping enhancers, work in mammalian models has been limited. Recently, we have identified two partially redundant enhancers, nPE1 and nPE2, that drive proopiomelanocortin gene expression in the hypothalamus. Here we demonstrate that deletion of nPE1 produces mild obesity while knockout of nPE2 has no discernible metabolic phenotypes. Additionally, we show that acute leptin administration has significant effects on nPE1 knockout mice, with food intake and body weight change significantly impacted by peripheral leptin treatment. nPE1 knockout mice became less responsive to leptin treatment over time as percent body weight change increased over 2 week exposure to peripheral leptin. Both Pomc and Agrp mRNA were not differentially affected by chronic leptin treatment however we did see a decrease in Pomc and Agrp mRNA in both nPE1 and nPE2 knockout calorie restricted mice as compared to calorie restricted PBS-treated WT mice. Collectively, these data suggest dynamic regulation of Pomc by nPE1 such that mice with nPE1 knockout become less responsive to the anorectic effects of leptin treatment over time. Our results also support our earlier findings in which nPE2 may only be critical in adult mice that lack nPE1, indicating that these neural enhancers work synergistically to influence metabolism.

The activation of G protein-coupled receptor 30 increases pro-opiomelanocortin gene expression through cAMP/PKA/NR4A pathway in mouse pituitary corticotroph AtT-20 cells

G protein-coupled receptor 30 (GPR30) signaling plays an important role in many regulatory pathways, such as gene expression, cell proliferation and migration. However, whether GPR30 is involved in transcription of the pro-opiomelanocortin (Pomc) gene in pituitary corticotroph cells is currently unknown. Here, we report that GPR30 signaling, activated by the GPR30 specific agonist G-1, increases Pomc expression in the mouse corticotroph cell line AtT-20. G-1 also increased nuclear receptor subfamily 4 group A member 1- and 2-dependent transcription activity and phosphorylation of cyclic adenosine monophosphate response element binding protein. Furthermore, protein kinase A inhibitors strongly attenuated G-1-mediated transactivation. The findings suggest that G-1 stimulates GPR30-mediated mechanisms via cyclic adenosine monophosphate/protein kinase A/nuclear receptor subfamily 4 group A members activity in the regulation of Pomc in corticotroph cells.

SAT-603 Growth Hormone-Releasing Hormone (GHRH) Antagonists Stimulate Feeding in Mice

Growth hormone-releasing hormone (GHRH) is a hypothalamic neuropeptide which stimulates the synthesis and secretion of growth hormone (GH) in pituitary gland. GHRH was also found to modulate food intake in mammals. MIA-690 is a synthetic GHRH antagonist of the Miami (MIA) series with potent antitumor effects. To date, its role in hypothalamic feeding modulation has not been evaluated. In the present study, we aimed to investigate the effects of chronic MIA-690 administration on feeding behavior, locomotor activity and hypothalamic dopamine (DA), norepinephrine (NE), serotonin (5-hydroxytriptamine, 5-HT), orexigenic peptides [agouti-related peptide (AgRP) and neuropeptide Y (NPY)] and anorexigenic peptides [cocaine and amphetamine-regulated transcript (CART) and proopiomelanocortin (POMC)] activity.Adult C57/BL6 mice were treated daily for 4 weeks by subcutaneous administration of (5 µg) MIA-690 or vehicle solution. Food intake and body weight were recorded every 4 days throughout the study. Immediately after the last injection, locomotor activity in the home cage was recorded, and thereafter animals were sacrificed. Visceral, subcutaneous and brown fat depots were quickly excised and weighed. Hypothalamus was also dissected for evaluating gene expression of AgRP, NPY, CART and POMC by real-time reverse transcription polymerase chain reaction. In addition, hypothalamic DA, NE and 5-HT levels were measured by high performance liquid chromatography (HPLC) coupled to electrochemical detection.Our findings show that administration of MIA-690 increased food intake and body weight, without affecting locomotor activity. No difference was observed in visceral, subcutaneous and brown fat mass in animals treated with MIA-690 or vehicle. As for neuromodulatory effects, a significant increase of AgRP gene expression and NE levels, along with a reduction of 5-HT levels were found after MIA-690 treatment. On the other hand, we did not observe any alteration in NPY, POMC and CART gene expression, as well as DA levels, following MIA-690 administration.In conclusion, chronic peripheral administration of MIA-690 could play an orexigenic role paralleled by increased body weight. The stimulation of feeding could be mediated, at least in part, by increased AgRP gene expression and NE levels and decreased 5-HT levels, in the hypothalamus.

Neuroprotective and Neuromodulatory Effects Induced by Cannabidiol and Cannabigerol in Rat Hypo-E22 cells and Isolated Hypothalamus.

Background: Cannabidiol (CBD) and cannabigerol (CBG) are non-psychotropic terpenophenols isolated from Cannabis sativa, which, besides their anti-inflammatory/antioxidant effects, are able to inhibit, the first, and to stimulate, the second, the appetite although there are no studies elucidating their role in the hypothalamic appetite-regulating network. Consequently, the aim of the present research is to investigate the role of CBD and CBG in regulating hypothalamic neuromodulators. Comparative evaluations between oxidative stress and food intake-modulating mediators were also performed. Methods: Rat hypothalamic Hypo-E22 cells and isolated tissues were exposed to either CBD or CBG, and the gene expressions of neuropeptide (NP)Y, pro-opiomelanocortin (POMC) and fatty acid amide hydrolase were assessed. In parallel, the influence of CBD on the synthesis and release of dopamine (DA), norepinephrine (NE), and serotonin (5-HT) was evaluated. The 3-hydroxykinurenine/kinurenic acid (3-HK/KA) ratio was also determined. Results: Both CBD and CBG inhibited NPY and POMC gene expression and decreased the 3-HK/KA ratio in the hypothalamus. The same compounds also reduced hypothalamic NE synthesis and DA release, whereas the sole CBD inhibited 5-HT synthesis. Conclusion: The CBD modulates hypothalamic neuromodulators consistently with its anorexigenic role, whereas the CBG effect on the same mediators suggests alternative mechanisms, possibly involving peripheral pathways.

Promoter Methylation Status in Pro-opiomelanocortin Does Not Contribute to Dyspigmentation in Hypertrophic Scar.

Burn injuries frequently result in hypertrophic scars (HTSs), specifically when excision and grafting are delayed due to limited resources or patient complications. In patient populations with dark baseline pigmentation, one symptom of HTS that often occurs is dyspigmentation. The mechanism behind dyspigmentation has not been explored, and, as such, prevention and treatment strategies for this morbidity are lacking. The mechanism by which cells make pigment is controlled at the apex of the pathway by pro-opiomelanocortin (POMC), which is cleaved to its products alpha-melanocyte-stimulating hormone (α-MSH) and adrenocorticotropin hormone (ACTH). α-MSH and ACTH secreted by keratinocytes bind to melanocortin 1 receptor (MC1R), expressed on melanocytes, to initiate melanogenesis. POMC protein expression is upregulated in hyperpigmented scar compared to hypopigmented scar by an unknown mechanism in a Duroc pig model of HTS. POMC RNA levels, as well as the POMC gene promoter methylation status were investigated as a possible mechanism. DNA was isolated from biopsies obtained from distinct areas of hyper- or hypopigmented scar and normal skin. DNA was bisulfite-converted, and amplified using two sets of primers to observe methylation patterns in two different CpG islands near the POMC promoter. Amplicons were then sequenced and methylation patterns were evaluated. POMC gene expression was significantly downregulated in hypopigmented scar compared to normal skin, consistent with previously reported protein expression levels. There were significant changes in methylation of the POMC promoter; however, none that would account for the development of hyper- or hypopigmentation. Future work will focus on other areas of POMC transcriptional regulation.

O-linked β-N-acetylglucosamine transferase is involved in pro-opiomelanocortin gene expression in mouse pituitary corticotroph AtT-20 cells

Glucocorticoids and glucocorticoid receptors (GRs) suppress pituitary pro-opiomelanocortin (Pomc) gene expression. O-linked β-N-acetylglucosamine (O-GlcNAc) modification, mediated by O-GlcNAc transferase (OGT), plays an important role during gene transcription. However, whether OGT is involved in the GR-mediated transrepression that occurs in pituitary corticotroph cells is currently unknown. Here, we report that OGT regulates Pomc expression in the mouse corticotroph cell line AtT-20. The overexpression of OGT has an additive effect on the GR-mediated negative transcription pathway. Both the knockdown of OGT by RNA interference and the use of a chemical OGT inhibitor abolished the repressive effects of Pomc expression induced by GRs. OGT inhibition leads to both the decreased recruitment of GRs and the increased recruitment of RNA polymerase II to the Pomc locus. O-GlcNAc modification is involved in the negative regulation of Pomc transcription in corticotroph cells. OGT may be a promising therapeutic target for the treatment of Cushing’s disease.

Activation of GPR40 produces mechanical antiallodynia via the spinal glial interleukin-10/\u03b2-endorphin pathway

BackgroundThe G protein-coupled receptor 40 (GPR40), broadly expressed in various tissues such as the spinal cord, exerts multiple physiological functions including pain regulation. This study aimed to elucidate the mechanisms underlying GPR40 activation-induced antinociception in neuropathic pain, particularly related to the spinal glial expression of IL-10 and subsequent β-endorphin.MethodsSpinal nerve ligation-induced neuropathic pain model was used in this study. β-Endorphin and IL-10 levels were measured in the spinal cord and cultured primary microglia, astrocytes, and neurons. Double immunofluorescence staining of β-endorphin with glial and neuronal cellular biomarkers was also detected in the spinal cord and cultured primary microglia, astrocytes, and neurons.ResultsGPR40 was expressed on microglia, astrocytes, and neurons in the spinal cords and upregulated by spinal nerve ligation. Intrathecal injection of the GPR40 agonist GW9508 dose-dependently attenuated mechanical allodynia and thermal hyperalgesia in neuropathic rats, with Emax values of 80% and 100% MPE and ED50 values of 6.7 and 5.4 μg, respectively. Its mechanical antiallodynia was blocked by the selective GPR40 antagonist GW1100 but not GPR120 antagonist AH7614. Intrathecal GW9508 significantly enhanced IL-10 and β-endorphin immunostaining in spinal microglia and astrocytes but not in neurons. GW9508 also markedly stimulated gene and protein expression of IL-10 and β-endorphin in cultured primary spinal microglia and astrocytes but not in neurons, originated from 1-day-old neonatal rats. The IL-10 antibody inhibited GW9508-stimulated gene expression of the β-endorphin precursor proopiomelanocortin (POMC) but not IL-10, whereas the β-endorphin antibody did not affect GW9508-stimulated IL-10 or POMC gene expression. GW9508 increased phosphorylation of mitogen-activated protein kinases (MAPKs) including p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), and its stimulatory effects on IL-10 and POMC expression were blocked by each MAPK isoform inhibitor. Spinal GW9508-induced mechanical antiallodynia was completely blocked by intrathecal minocycline, IL-10 neutralizing antibody, β-endorphin antiserum, and μ-opioid receptor-preferred antagonist naloxone.ConclusionsOur results illustrate that GPR40 activation produces antinociception via the spinal glial IL-10/β-endorphin antinociceptive pathway.

Age-dependent decline of hypothalamic HIF2α in response to insulin and its contribution to advanced age-associated metabolic disorders in mice

Hypoxia-inducible factor-2α (HIF2α) is a nuclear transcription factor that plays a critical role in cell survival including metabolic adaptation under hypoxia as well as normoxia, but whether HIF2α contributes to the control of whole-body metabolic balance is unclear. In this study, we found that the hypothalamic HIF2α protein level rapidly increases in young mice that are centrally stimulated with insulin. However, this insulin-induced HIF2α up-regulation is substantially attenuated in mice of advanced age. This attenuation is comparable with the effect of high-calorie feeding in young mice. Of note, unlike high-calorie feeding conditions, age-dependent HIF2α attenuation occurs without impaired activation of the hypothalamic IR/IRS-2/AKT/FOXO1 pathway in response to insulin. Molecular and physiological analyses revealed that hypothalamic HIF2α contributes to the action of central insulin in regulation of proopiomelanocortin (Pomc) gene expression and food intake. HIF2α knockout in POMC neurons led to age-dependent excess weight gain and fat increase, a phenotype that was associated with a mild degree of glucose intolerance and insulin resistance. In conclusion, hypothalamic HIF2α responds to insulin, and the up-regulation is involved in adaptive metabolic regulation as age increases, whereas impairment of HIF2α in the hypothalamus contributes to weight gain and glucose disorders in age-dependent manners.