Agouti-related Protein Gene Research Articles

Candidate orexigenic peptide hormone-related genes in yellowtail Seriola quinqueradiata: cloning and tissue distribution of two distinct agouti-related protein genes and response of five candidate appetite-related genes to fasting, fishmeal soluble fraction addition, and a fishmeal-based diet

Candidate orexigenic peptide hormone-related genes in yellowtail Seriola quinqueradiata: cloning and tissue distribution of two distinct agouti-related protein genes and response of five candidate appetite-related genes to fasting, fishmeal soluble fraction addition, and a fishmeal-based diet

Effects of stocking density on growth performance, digestive function and immune performance of Matou goats

The objective of this study was to examine the effects of different stocking densities on Matou goats. Thirty-six female Matou goats were randomly assigned into three experimental treatments including the low density (LD, 0.67 goat/m2), the medium density (MD, 1 goat/m2) and the high density (HD, 2 goats/m2) group. Compared with LD group, the average daily weight gain (ADG) of HD group decreased (p < .05). The length of duodenal and ileal villi in HD group was significantly lower than that in LD group (p < .05). The rumen pH and concentration of total volatile fatty acid (VFA), propionate butyrate and ammonia-nitrogen increased in HD group (p < .05). The acetate/propionate ratio was the highest in MD group (p < .05). The mRNA expression level of agouti-related protein gene (AgRP) in hypothalamus of LD was higher than that in HD group (p < 0.05). Proopiomelanocortin (POMC) mRNA expression level was the highest in MD group (p<.05) and the lowest in HD group (p < .05). An increase in growth hormone receptor (GHR), insulin-like growth factor 1 (IGF1) and claudin-1 mRNA expression level were noticed in HD group (p<.05). Compared with LD group, the serum total protein (TP), albumin (Alb), globulin (Glb) IgA and IgG levels in HD group decreased (p < .05). Glucocorticoid (GCS), β-endorphin (β-EP) and cortisol levels in serum were also significantly increased in HD group compared with LD group (p < .05). These results showed that stocking density affected rumen environment and high stocking density resulted in a decrease in growth, digestive and immune function of Matou goats.

Lipopolysaccharide inhibits hypothalamic Agouti-related protein gene expression via activating mechanistic target of rapamycin signaling in chicks

Lipopolysaccharide (LPS) induces profound anorexia in birds. However, the neuronal regulatory network underlying LPS-provoked anorexia is unclear. To determine whether any cross talk occurs among hypothalamic mechanistic target of rapamycin (mTOR) and LPS in the regulation of appetite, we performed an intracerebroventricular injection of rapamycin (an mTOR inhibitor) on LPS-treated chicks. The results indicate that peripheral administrations of LPS decreased the agouti-related protein (AgRP) mRNA level, but increased the phosphorylated mTOR and nuclear factor-кB (NF-кB) protein level. Blocking mTOR significantly attenuated LPS-induced anorexia, AgRP suppression, and p-NF-кB increase. Thus, the results suggest that LPS causes anorexia via the mTOR-AgRP signaling pathway, and mTOR signaling is also associated with the regulation of LPS in p-NF-кB.

Abstract P249: ICV Infusion Of AgRP Modulates Feeding Behavior And Energy Expenditure In Mice

A subset of Agouti-related protein (AgRP) neurons within the arcuate nucleus express angiotensin type 1a receptors (AT1A), and genetic ablation of AT1A in these cells disinhibits AgRP gene expression and attenuates energy expenditure (EE) in mice. To further understand the role of AgRP in metabolic control, and to establish relevant dosing schedules in this species, here we tested the effect of intracerebroventricular infusion (icv) of recombinant AgRP on metabolic functions in C57BL/6J male mice. First, we examined the effects of AgRP(82-132) (21 days, 1 or 10 nmol/d, icv) or artificial cerebrospinal fluid (aCSF) using metabolic cages. High dose AgRP reduced body mass (aCSF n=12, +0.8±0.6 vs 1 nmol/d n=14, +0.6±0.6 vs 10 nmol/d n=11, -1.5±0.5 p&lt;0.05 g/21d), without affecting food intake (15.1±1.2 vs 14.1±1.1 vs 16.3±1.1 kcal/d) or digestive efficiency (85.2±0.5 vs 84.5±0.7 vs 85.1±0.6 %), but a significant reduction in energy efficiency (+3.8±2.0 vs +2.7±2.8 vs -5.9±2.2 p&lt;0.05 mg/kcal) indicated increased total EE. Next, we examined the effect of AgRP(82-132) (14 days, 1 nmol/d, icv) using a multiplexed system (Promethion, Sable). AgRP had no effect on body mass (25.1±1.2, n=8 vs 26.7±0.5, n=8), overall body composition (by NMR), heat production (Weir, 24h: 0.485±0.015 vs 0.490±0.022 kcal/h), or respiratory exchange ratio (0.88±0.01 vs 0.89±0.01). AgRP increased total food intake (10.1±0.6, n=8 vs 11.9±0.5, n=8, kcal/d, p=0.03) through a synergistic effect on number of meals and median meal mass. We conclude that AgRP (1-10 nmol/d, 2-3 wk, icv) infusion causes subtle changes in feeding behavior without effect on digestive efficiency. In contrast, EE is paradoxically increased by AgRP when infused at a high dose (10 nmol/d). We postulate that these differences may reflect differential accessibility of the peptide to relevant feeding vs autonomic control regions of the hypothalamus when infused into the cerebral ventricles, and/or compensatory increases in EE secondary to changes in feeding behavior. Future studies to deconvolute the role of AgRP in the control of EE in mice will require site-specific delivery of the peptide to relevant target regions or manipulation of its receptor in those regions. Funding: HL134850, HL084207

SUN-LB007 The Mouse AgRP Gene Has Three Promoters: Differential Regulation by Nutritional States

Diabetes and obesity are metabolic indicators of an energy imbalance in the melanocortin system. Agouti related protein (AgRP) neurons in the arcuate nucleus of the hypothalamus regulate energy through the release of its neuropeptide- AgRP, along with Neuropeptide Y and GABA. AgRP acts on melanocortin 4 receptor (MC4-R) by stimulating appetite and decreasing energy expenditure. Overexpression of AgRP is known to contribute to the development of diabetes and obesity. Expression of the Agrp gene is regulated by weight loss, presumably via leptin. We found that the Ensembl database specifies that for the mouse Agrp gene there are three AgRP transcripts which differ in their 5’ untranslated regions and share the same coding exons. To determine if the AgRP mRNA isoforms are relevant for weight control, we collected tissue samples from adult mouse hypothalamus, and designed a quantitative PCR assay for each of the three AgRP mRNA isoforms. After establishing the presence of each isoform in the hypothalamus, we used these assays to determine the influence of fasting on the expression levels of the three different AgRP transcripts. We also sought to determine whether the sequences upstream of each of the three isoforms could function as promoters to drive gene expression. To determine if each upstream region can act as a promoter, we cloned the upstream regions into a reporter plasmid expressing FLAG-tagged red fluorescent protein. Each promoter construct was transfected into a neuronal cell line established from NPY/AgRP-GFP neurons. We found that all three AgRP mRNA isoforms are expressed in the adult mouse hypothalamus. While fasting caused an induction of the total AgRP mRNA (fed=1.00, fasted= 6.29; p<0.05), only AgRP-A mRNA was similarly induced by fasting (fed= 1.09, fasted=3.39 p<0.05). Surprisingly, AgRP-B and AgRP-C are not influenced by fasting. The transfection studies demonstrated that each of the upstream regions can drive expression of a reporter cassette, verified by immunofluorescence and western blot. These results indicate that studies of AgRP expression should be re-examined to take into account the complexity of Agrp gene expression. We believe that the identification of these three AgRP regulatory regions with differential expression could provide tools for understanding the regulation of Agrp gene expression by nutritional state and stress. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Plasma Agouti-Related Protein and Cortisol Levels in Cushing Disease: Evidence for the Regulation of Agouti-Related Protein by Glucocorticoids in Humans.

Glucocorticoids regulate energy balance, in part by stimulating the orexigenic neuropeptide agouti-related protein (AgRP). AgRP neurons express glucocorticoid receptors, and glucocorticoids have been shown to stimulate AgRP gene expression in rodents. We sought to determine whether there is a relationship between plasma AgRP and hypothalamic AgRP in rats and to evaluate the relationship between cortisol and plasma AgRP in humans. We retrospectively evaluated plasma AgRP levels prior to transsphenoidal surgery in 31 patients with Cushing disease (CD) vs 31 sex- and body mass index-matched controls from a separate study. We then prospectively measured plasma AgRP, before and 6 to 12 months after surgery, in a subgroup of 13 patients with CD. Plasma and hypothalamic AgRP were measured in adrenalectomized rats with and without corticosterone replacement. Plasma AgRP was stimulated by corticosterone in rats and correlated with hypothalamic AgRP expression. Plasma AgRP levels were higher in patients with CD than in controls (139 ± 12.3 vs 54.2 ± 3.1 pg/mL; P < 0.0001). Among patients with CD, mean 24-hour urine free cortisol (UFC) levels were 257 ± 39 μg/24 hours. Strong positive correlations were observed between plasma AgRP and UFC (r = 0.76; P < 0.0001). In 11 of 13 patients demonstrating surgical cure, AgRP decreased from 126 ± 20.6 to 62.5 ± 8.0 pg/mL (P < 0.05) postoperatively, in parallel with a decline in UFC. Plasma AgRP levels are elevated in CD, are tightly correlated with cortisol concentrations, and decline with surgical cure. These data support the regulation of AgRP by glucocorticoids in humans. AgRP's role as a potential biomarker and as a mediator of the adverse metabolic consequences of CD deserves further study.

Glucocorticoid positively regulate Agouti-related protein gene

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Angiotensin‐(1‐7)‐Mediated Increases in Energy Expenditure Appear Independent of the Hypothalamic Melanocortin System

Obesity is a major public health problem that greatly increases risk for cardiovascular disease and type II diabetes. Obesity represents an energy imbalance where the amount of calories consumed exceeds the amount of calories expended. This energy balance is in part controlled by activation of proopiomelanocortin (POMC) neurons or inhibition of agouti‐related protein (AgRP) neurons within the arcuate nucleus (ARC) of the hypothalamus, which activates downstream melanocortin pathways to produce satiety and increase energy expenditure. Our preliminary data show that the beneficial renin‐angiotensin system hormone angiotensin (Ang)‐(1‐7) may be an important contributor to energy balance, as it attenuates high fat diet (HFD)‐induced obesity in mice by browning subcutaneous white fat to enhance energy expenditure. In this study, we tested the hypothesis that the beneficial effects of Ang‐(1‐7) on energy expenditure may be due to actions on POMC or AgRP neurons in the ARC. To test this hypothesis, we first examined the ability of an acute subcutaneous injection of Ang‐(1‐7) to induce neuronal activation in the ARC, as measured by c‐fos gene expression, in chow fed mice. We found that Ang‐(1‐7) significantly increased the number of c‐fos positive cells within the ARC compared with saline (43±5 Ang‐(1‐7) vs. 19±3 saline; p=0.022; n=3 per group). We then performed chronic experiments in which eight‐week old male C57BL/6J mice were implanted with subcutaneous osmotic mini‐pumps to deliver saline or Ang‐(1‐7) [400 ng/kg/min]. Immediately following mini‐pump implantation, mice were placed on a 60% high fat diet (HFD) or matched control chow diet for 12 weeks. The groups of mice included in this chronic study were: (1) chow diet with saline infusion (n=5); (2) HFD with saline infusion (n=6); and (3) HFD with Ang‐(1‐7) infusion [n=9]. At the end of treatment, the hypothalamus from each mouse was collected to assess for gene expression of mas receptor, POMC, and AgRP using quantitative real‐time polymerase chain reaction methods. We found that HFD reduced gene expression for POMC (1.1±0.3 chow vs. 0.4±0.1 HFD; p=0.005) in the hypothalamus, with no significant effect on mas receptor (1.2±0.3 chow vs. 0.7±0.1 HFD; p=0.134) or AgRP (1.3±0.5 chow vs. 1.1±0.2 HFD; p=0.856). There was no effect of Ang‐(1‐7) on POMC (0.4±0.1; p=0.998), mas receptor (0.7±0.1; p=0.995) or AgRP (1.0±0.1; p=0.996) gene expression in HFD mice. These findings provide evidence that Ang‐(1‐7) can acutely induce activation of neurons in the ARC. Chronic Ang‐(1‐7) treatment was not able to restore obesity‐induced reductions in POMC gene expression, however, despite enhancing energy expenditure. Further studies are needed to evaluate if beneficial effects of Ang‐(1‐7) on energy balance involve changes in POMC neuronal activity, or melanocortin‐independent pathways.Support or Funding InformationThis study was supported by NIH grant HL122507.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Electroacupuncture Reduces Weight in Diet-Induced Obese Rats via Hypothalamic Tsc1 Promoter Demethylation and Inhibition of the Activity of mTORC1 Signaling Pathway.

Subject The study aimed to investigate the mechanism of electroacupuncture reducing weight via tuberous sclerosis complex 1 (Tsc1) promoter methylation, inhibiting the mammalian target of rapamycin complex 1 (mTORC1) pathway. Materials and Methods Male Sprague-Dawley rats were divided into chow-fed group (chow group) or high-fat diet group (HF group) for 14 weeks. The obesity rats in HF group were randomly divided into electroacupuncture group (EA group) and diet-induced obesity (DIO) group, which received EA stimulation on bilateral ST25, RN12, SP6, and ST36 for 4 weeks or no further treatment, respectively. Methylation of the Tsc1 gene promoter and expression of agouti-related protein (AgRP), neuropeptide Y (NPY), and proopiomelanocortin (PoMC) were detected at the 18th week. Results At week 18, weight, body fat, and the body fat rate in DIO group were significantly higher than those of the chow and EA group. Compared with the chow group, the DIO group had increased methylation of the Tsc1 gene promoter and expression of mTORC1, AgRP, and NPY gene and decreased PoMC in the hypothalamus; after EA, methylation of the Tsc1 gene promoter, mRNA, and protein of the mTORC1 and expression of AgRP and NPY gene decreased and PoMC increased significantly. Conclusions Our study could shed light on the potential pathway where EA exerts effects on the mechanism of EA treatment for obesity through the hypothalamic Tsc1 promoter demethylation and inhibition of the activity of mTORC1 signaling pathway.

Regulation of Agouti-Related Protein and Pro-Opiomelanocortin Gene Expression in the Avian Arcuate Nucleus.

The arcuate nucleus is generally conserved across vertebrate taxa in its neuroanatomy and neuropeptide expression. Gene expression of agouti-related protein (AGRP), neuropeptide Y (NPY), pro-opiomelanocortin (POMC), and cocaine- and amphetamine-regulated transcript (CART) has been established in the arcuate nucleus of several bird species and co-localization demonstrated for AGRP and NPY. The proteins encoded by these genes exert comparable effects on food intake in birds after central administration to those seen in other vertebrates, with AGRP and NPY being orexigenic and CART and α-melanocyte-stimulating hormone anorexigenic. We have focused on the measurement of arcuate nucleus AGRP and POMC expression in several avian models in relation to the regulation of energy balance, incubation, stress, and growth. AGRP mRNA and POMC mRNA are, respectively, up- and downregulated after energy deprivation and restriction. This suggests that coordinated changes in the activity of AGRP and POMC neurons help to drive the homeostatic response to replace depleted energy stores in birds as in other vertebrates. While AGRP and POMC expression are generally positively and negatively correlated with food intake, respectively, we review here situations in some avian models in which AGRP gene expression is dissociated from the level of food intake and may have an influence on growth independent of changes in appetite. This suggests the possibility that the central melanocortin system exerts more pleiotropic functions in birds. While the neuroanatomical arrangement of AGRP and POMC neurons and the sensitivity of their activity to nutritional state appear generally conserved with other vertebrates, detailed knowledge is lacking of the key nutritional feedback signals acting on the avian arcuate nucleus and there appear to be significant differences between birds and mammals. In particular, recently identified avian leptin genes show differences between bird species in their tissue expression patterns and appear less closely linked in their expression to nutritional state. It is presently uncertain how the regulation of the central melanocortin system in birds is brought about in the situation of the apparently reduced importance of leptin and ghrelin compared to mammals.

Evidence suggesting phosphodiesterase-3B regulation of NPY/AgRP gene expression in mHypoE-46 hypothalamic neurons

Hypothalamic neurons expressing neuropeptide Y (NPY) and agouti related-protein (AgRP) are critical regulators of feeding behavior and body weight, and transduce the action of many peripheral signals including leptin and insulin. However, intracellular signaling molecules involved in regulating NPY/AgRP neuronal activity are incompletely understood. Since phosphodiesterase-3B (PDE3B) mediates the hypothalamic action of leptin and insulin on feeding, and is expressed in NPY/AgRP neurons, PDE3B could play a significant role in regulating NPY/AgRP neuronal activity. To investigate the direct regulation of NPY/AgRP neuronal activity by PDE3B, we examined the effects of gain-of-function or reduced function of PDE3B on NPY/AgRP gene expression in a clonal hypothalamic neuronal cell line, mHypoE-46, which endogenously express NPY, AgRP and PDE3B. Overexpression of PDE3B in mHypoE-46 cells with transfection of pcDNA-3.1-PDE3B expression plasmid significantly decreased NPY and AgRP mRNA levels and p-CREB levels as compared to the control plasmid. For the PDE3B knockdown study, mHypoE-46 cells transfected with lentiviral PDE3BshRNAmir plasmid or non-silencing lentiviral shRNAmir control plasmid were selected with puromycin, and stably transfected cells were grown in culture for 48h. Results showed that PDE3BshRNAmir mediated knockdown of PDE3B mRNA and protein levels (∼60–70%) caused an increase in both NPY and AgRP gene expression and in p-CREB levels. Together, these results demonstrate a reciprocal change in NPY and AgRP gene expression following overexpression and knockdown of PDE3B, and suggest a significant role for PDE3B in the regulation of NPY/AgRP gene expression in mHypoE-46 hypothalamic neurons.

Characterization, tissue distribution and regulation of agouti-related protein (AgRP) in a cyprinid fish (Schizothorax prenanti)

Agouti-related protein (AgRP) is an important neuropeptide involved in the regulation of feeding in both mammals and fish. In this study, we have cloned the full-length cDNA sequence for AgRP in a cyprinid fish (Schizothorax prenanti). The AgRP gene, encoding 126-amino acids, was strongly expressed in the brain. The AgRP gene was detected in embryos at developmental stages. Further, its mRNA was detectable in unfertilized eggs. An experiment was conducted to determine the expression profile of AgRP during short-term and long-term fasting of the hypothalamus. The expression level of AgRP in unfed fish was significantly increased at 3 and 4h post-fasting than in fed fish but did not affect AgRP mRNA expression after 14days fasting. Overall, our results suggest that AgRP is a conserved peptide that might be involved in the regulation of short-term feeding and other physiological function in Schizothorax prenanti.

Polymorphisms of the pro-opiomelanocortin and agouti-related protein genes and their association with chicken production traits

Pro-opiomelanocortin (POMC) and agouti-related protein (AGRP) are hypothalamic neuropeptides that play a central role in the regulation of food intake and energy expenditure and for this reason the variations in the POMC and AGRP genes in chicken were examined by screening the DNA pools. Two silent cSNPs mutations in POMC gene and one silent cSNP mutation in AGRP gene were identified. PCR-restriction fragment length polymorphism (RFLP) was used to test the cSNPs c. C495T in the POMC and c. C9T in the AGRP gene in the F2 resource population of Gushi chicken crossed with Anak broiler. The association analysis on the polymorphisms of POMC, AGRP gene and production traits showed that the c. C495T mutation in the POMC gene was significantly linked to the pelvis breadth at 4 weeks of age (P = 0.035), body weight at 2 weeks of age (P = 0.013) and was highly significantly linked to the chest depth at 12 weeks of age (P = 0.006). The c. T9T genotype in the AGRP gene was associated with a low breast muscle water loss rate (P = 0.025), increased chest width at 12 weeks of age (P = 0.005) and body weight at 2 weeks of age (P = 0.036), a high slaughter rate (P = 0.049) and semi-evisceration weight (P = 0.019). These findings may have important implications for the molecular aspects of chicken breeding.

The effect of metformin on food intake and its potential role in hypothalamic regulation in obese diabetic rats

Metformin appears to be involved in altering energy expenditure and thermogenesis, and could affect hypothalamic feeding circuits. However, it is not clear whether metformin is able to cross the blood–brain barrier (BBB) to reach the hypothalamus and exert a direct effect on the central nervous system. Here we show the presence of metformin in cerebrospinal fluid (CSF) of diabetic rats administered orally with metformin which was confirmed by detecting the concentration of metformin with liquid chromatography-tandem mass spectrometry. Food intake of diabetic rats treated with metformin was reduced, and glucose homeostasis was gained. Expression of orexigenic peptides neuropeptide Y (NPY) and agouti-related protein (AgRP) decreased in the hypothalamus of metformin-treated diabetic rats, though anorexigenic peptides pro-opiomelanocortin (POMC) did not change significantly. The phosphorylation of signal transducer and activator of transcription 3 (STAT3) was increased but phosphorylated AMP-activated kinase (AMPK) was similar in the hypothalamus of metformin-treated diabetic rats. Our findings suggest that metformin may cross BBB and play a central mechanism on regulation of food intake in the hypothalamus. The anorexic effect of metformin may be mediated by inhibition of NPY and AgRP gene expression through the STAT3 signaling pathway.

Resveratrol, purified from the stem of Vitis coignetiae Pulliat, inhibits food intake in C57BL/6J Mice

Neuropeptide Y (NPY) and agouti-related protein (AgRP) have powerful stimulatory effects on food intake, which suggests that the downregulation of brain NPY or AgRP may help reduce obesity and diabetes by inhibiting food intake. To search for active compounds that inhibit NPY and AgRP expression, we made two luciferase reporter assay systems consisting of NPY and AgRP promoter-driven luciferase genes, together with the puromycin resistance gene, in a plasmid vector. Each plasmid was permanently transfected into N29-4 neuronal cells. Using the systems, resveratrol was purified from the stem of Vitis coignetiae Pulliat by activityguided fractionation. Resveratrol downregulated NPY and AgRP promoter-driven luciferase activity in a dose-dependent manner. The inhibitory concentrations (IC(50), 50% inhibition) of resveratrol against pNPY-luc and pAgRP-luc activities were 8.9 microM and 8.0 microM, respectively. Furthermore, one-time intraperitoneal injection of resveratrol (100 mg/kg) suppressed 20.0% and 17.2% of food intake during 24 and 48 h, respectively. These results indicated that resveratrol inhibited food intake, which may be related to the downregulation of NPY and AgRP gene expression.

Role of hypoleptinemia during cold adaptation in Brandt's voles (Lasiopodomys brandtii)

Brandt's voles Lasiopodomys brandtii exhibit large increases in nonshivering thermogenesis to cope with chronic cold exposure, resulting in compensatory hyperphagia and fat mobilization. These physiological events are accompanied by a remarkable reduction in serum leptin levels. However, the role of hypoleptinemia in cold adaptation in this species is still unknown. In the present study, we tested the hypothesis that hypoleptinemia contributes to increases in food intake and brown adipose tissue (BAT) thermogenesis by modifying hypothalamic neuropeptides in cold-exposed Brandt's voles. Adult male voles were transferred to 5 degrees C for 28 days. Accompanied by a decrease in serum leptin levels, hypothalamic agouti-related protein (AgRP) mRNA levels were significantly increased, but there were no changes in the long form of leptin receptor (Ob-Rb), suppressor of cytokine signaling 3 (SOCS3), neuropeptide Y (NPY) mRNA, proopiomelanocortin (POMC), and cocaine- and amphetamine-regulated peptide (CART) mRNA levels in the hypothalamus. When cold-exposed voles were returned to warm (23 degrees C) for 28 days, body mass, food intake, serum leptin, and AgRP mRNA were restored to control levels. Leptin administration in cold-exposed voles decreased food intake as well as hypothalamic AgRP mRNA levels. There were no significant effects of leptin administration on hypothalamic Ob-Rb, SOCS3, NPY, POMC, CART mRNA, and uncoupling protein 1 levels under cold conditions. These results suggest that hypoleptinemia partially contributes to cold-induced hyperphagia, which might involve the elevation of hypothalamic AgRP gene expression.

Control Elements in the Neighboring ATPase Gene Influence Spatiotemporal Expression of the Human Agouti-Related Protein

The agouti-related protein (AgRP) is an orexigenic peptide that plays a significant role in the regulation of energy balance. It is expressed in the hypothalamus, the adrenal glands, and the testis, but sequences determining its spatial and temporal expression have not been identified. Using an elaborate in vitro screening approach, we show here that two adjacent enhancers inside the first intron of the neighboring (1.4 kb downstream) ATPase gene ( ATP6V0D1) modulate the human AgRP promoter with profound spatiotemporal variation despite their diminutive sizes (221 and 231 nt). In transgenic mice, the proximal enhancer displayed specificity for the testis, tail, and ears, and the distal one for the testis, front feet, bone, heart, muscle, brain, spinal cord, and tongue, while dietary fat and overnight fasting had differential effects on enhancer activities. AgRP in the testis was localized to pachytene spermatocytes and in the tongue to epithelial cells. Comparative sequence analysis showed that the AgRP- ATP6V0D1 intergenic region is two times longer in humans than in mice and that the two enhancers are conserved in the rhesus monkey genome but not in the mouse genome. These data show that spatiotemporal expression of the human AgRP gene is influenced by diversified primate-specific intronic sequences in its neighboring ATP6V0D1 gene.

Enterostatin affects cyclic AMP and ERK signaling pathways to regulate Agouti-related Protein (AgRP) expression

Enterostatin, a gut–brain peptide, inhibits dietary fat intake in rats. The purpose of this study was to identify the intracellular signaling pathways that are responsive to enterostatin and that modulate the effects of enterostatin on the expression of Agouti-related protein (AgRP). We used the hypothalamic GT1-7 neuronal cell line to identify the effects of enterostatin on cyclic AMP and ERK signaling using conventional immunoassays or Western blots to assay the activity of these pathways. Enterostatin enhanced the level of cyclic AMP, PKA RIIβ and phospho-CREB and increased pERK levels in GT 1-7 cells. The effects on pERK were rapid (7.5 min) and dose-dependent. These signaling responses were blocked by an antibody to the enterostatin receptor (beta subunit of F1-ATPase), by the pERK inhibitor U0126 and by the P2Y receptor antagonist Suramin. Enterostatin showed a biphasic effect on AgRP mRNA, initially increasing but subsequently decreasing the levels. The cyclic AMP activator Sp-cAMP increased AgRP mRNA expression. Transfection of a wild type ERK construct reduced AgRP mRNA levels. Enterostatin inhibited expression of Krüppel-like factor 4 (KLF4), a transcriptional regulator of AgRP. KLF4 gene expression was increased by Sp-cAMP but decreased by wild-type ERK expression. U0126 blocked the effect of enterostatin on KLF4 expression. We conclude that enterostatin binding to its receptor activates the pERK pathway to inhibit AgRP gene expression but may enhance AgRP expression through activation of the cyclic AMP pathway. These pathways probably mediate the enterostatin inhibition of dietary fat intake.

Regulation of Agouti-Related Protein Messenger Ribonucleic Acid Transcription and Peptide Secretion by Acute and Chronic Inflammation

Agouti-related protein (AgRP) is an orexigenic neuropeptide produced by neurons in the hypothalamic arcuate nucleus (ARC) that is a key component of central neural circuits that control food intake and energy expenditure. Disorders in energy homeostasis, characterized by hypophagia and increased metabolic rate, frequently develop in animals with either acute or chronic diseases. Recently, studies have demonstrated that proopiomelanocortin-expressing neurons in the ARC are activated by the proinflammatory cytokine IL-1beta. In the current study, we sought to determine whether inflammatory processes regulate the expression of AgRP mRNA and to characterize the response of AgRP neurons to IL-1beta. Here, we show by real-time RT-PCR and in situ hybridization analysis that AgRP mRNA expression in rodents is increased in models of acute and chronic inflammation. AgRP neurons were found to express the type I IL-1 receptor, and the percentage of expression was significantly increased after peripheral administration of lipopolysaccharide. Furthermore, we demonstrate that IL-1beta inhibits the release of AgRP from hypothalamic explants. Collectively, these data indicate that proinflammatory signals decrease the secretion of AgRP while increasing the transcription of the AgRP gene. These observations suggest that AgRP neurons may participate with ARC proopiomelanocortin neurons in mediating the anorexic and metabolic responses to acute and chronic disease processes.

Central role of the melanocortin-4 receptors in appetite regulation after endotoxin1

Melanocortin-4 receptors (MC4R) are key factors in the depression of appetite during disease. This study was designed to determine the role of agouti-related protein (AgRP) in the effect of endotoxin (lipopolysaccharide, LPS) on appetite. Sheep received an intracerebroventricular injection of either saline or AgRP (0.5 nmol/kg of BW) 1 h before intravenous injection of either saline or LPS (0.6 microg/kg of BW) at time 0 and again at 4 h. Agouti-related protein prevented the reduction in feed intake due to LPS (P < 0.05). In a second experiment, AgRP gene expression was unaffected at 3 h and increased (P < 0.01) at 6 h after LPS. Immunohistochemical evidence indicated that there was an increase in the percentage of AgRP neurons with c-Fos immunoreactive nuclei 6 h after sheep were injected with LPS (P < 0.04) and a corresponding decrease in a-melanocyte-stimulating hormone neurons coexpressing c-Fos (P < 0.001). In situ hybridization provided evidence for an increase in AgRP gene expression and a decrease in proopiomelanocortin gene expression 6 h after LPS (P < 0.05). In a final experiment, physiological elevation of orexigenic agents by short-term fasting kept feed intake at the same level as controls, in spite of the presence of LPS, similar to the effects of AgRP in Exp. 1. The AgRP inhibition of the MC4R prevents appetite inhibition in response to LPS and well after LPS inhibition of feed intake, both AgRP and a-melanocyte-stimulating hormone may change in a pattern that favors appetite increases. These studies support the notion of the MC4R as a critical component of the mechanism for appetite suppression due to endotoxin.