Agouti-related Protein Expression Research Articles

1522-P: Identification of AgRP Cells in the Murine Hindbrain That Drive Feeding

The central melanocortin system is essential for the regulation of food intake and body weight in humans and rodents. Agouti-related protein (AgRP) is the sole orexigenic component of the central melanocortin system and is conserved across mammalian species. AgRP is currently known to express exclusively in the mediobasal hypothalamus, and hypothalamic AgRP-expressing neurons are essential for feeding regulation. Here we describe a previously unknown population of AgRP cells in the area postrema (AP) and the adjacent commissural nucleus of the solitary tract (cNTS) of the causal brainstem. AgRP was expressed in the embryonic AP, and hindbrain AgRP expression in adult mice was low under ad libitum fed condition but increased with food deprivation. AgRP cells in the AP and cNTS consisted of locally projecting neurons as well as tanycyte-like cells. In mice that lack hypothalamic AgRP neurons, diphtheria-toxin mediated ablation of AgRP cells led to anorexia and weight loss, whereas chemogenetic activation of AgRP neurons resulted in hyperphagia and weight gain. Moreover, focal transcranial photo-stimulation of AgRP cells in the AP and cNTS with a step-function opsin with ultra-high light sensitivity (SOUL) stimulated feeding in mice with or without hypothalamic AgRP neurons, suggesting that the hyperphagic effects of hindbrain AgRP neurons are independent of hypothalamic AgRP neurons. Collectively, our study indicates that the hindbrain has a balanced melanocortin system, complete with both orexigenic and anorexigenic components, and that AgRP neurons in the hindbrain drive feeding in adult animals. Disclosure T.P.Bachor: None. K.Attal: None. E.Vagena: None. F.Mifsud: None. V.Pham: None. M.Valdearcos-contreras: None. C.Vaisse: None. A.Xu: Advisory Panel; Pennington Biomedical Research Center, Research Support; Eli Lilly and Company. Funding National Institute of Diabetes and Digestive and Kidney Diseases (1P30DK098722-01A1, P30DK63720-06A1)

Loss of hypothalamic MCH decreases food intake in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is associated with impaired energy metabolism, including weight loss and decreased appetite which are negatively correlated with survival. Neural mechanisms underlying metabolic impairment in ALS remain unknown. ALS patients and presymptomatic gene carriers have early hypothalamic atrophy. The lateral hypothalamic area (LHA) controls metabolic homeostasis through the secretion of neuropeptides such as orexin/hypocretin and melanin-concentrating hormone (MCH). Here, we show loss of MCH-positive neurons in three mouse models of ALS based on SOD1 or FUS mutations. Supplementation with MCH (1.2 µg/d) through continuous intracerebroventricular delivery led to weight gain in male mutant Sod1G86R mice. MCH supplementation increased food intake, rescued expression of the key appetite-related neuropeptide AgRP (agouti-related protein) and modified respiratory exchange ratio, suggesting increased carbohydrate usage during the inactive phase. Importantly, we document pTDP-43 pathology and neurodegeneration in the LHA of sporadic ALS patients. Neuronal cell loss was associated with pTDP-43-positive inclusions and signs of neurodegeneration in MCH-positive neurons. These results suggest that hypothalamic MCH is lost in ALS and contributes to the metabolic changes, including weight loss and decreased appetite.

Impact of long-term fasting on the stomach-hypothalamus appetite regulating genes in Atlantic salmon postsmolts

Atlantic salmon will experience periods of fasting during its lifecycle. In nature, prolonged fasting periods occur owing to seasonal fluctuations in available feeds, migration or in relation to reproduction. In a culture setting, salmon is fasted mainly as part of planned operational handling prior to vaccination, delousing, transfer etc., and where fasting may last up to nine days. The mechanisms regulating the appetite during long-term fasting may vary among fish species. Here, we studied the impact of long-term fasting on neuro-endocrine regulation of appetite through the stomach-hypothalamic axis in Atlantic salmon post smolts (1.2 kg, ∼46 cm), reared in two experimental conditions (Fed and Fasted; triplicated tanks), and sampled after 4 weeks and 6 weeks of fasting. Fasted fish showed lower condition factor and hepatosomatic index at both sampling points compared to Fed group. In qPCR analysis, hypothalamic relative mRNA expression of agouti-related protein 1 (agrp1) was upregulated in fasted group at both sampling points. Among neuropeptide Y (npy) paralogs, only npya1 at 4 weeks was upregulated by fasting. As for cocaine- and amphetamine-regulated transcripts (cart), cart2a was elevated at 4 weeks, and cart2b at both 4 and 6 weeks in fasted group, while cart3a and cart4 showed no response to fasting. The pro-opiomelanocortin (pomc) a1, a2 and melanocortin-4 receptor (mc4r) a2 increased only after 6 weeks of fasting, while mc4rb1 did not respond to fasting. In stomach, 6 weeks of fasting resulted in a decrease of ghrelin1 (ghrl1), while expression of mboat4 was unaffected. The elevated levels of hypothalamic agrp1 and npya1 in fasted group support orexigenic roles for these neuropeptides. In addition, upregulation of cart2a, cart2b, pomca1 and pomca2 indicate that these play vital roles in appetite regulation and that fasting may halt and/or counteract hunger signals (agrp1 and npya1) to save energy from foraging search activities during catabolic conditions. Another possibility is that these neuropeptides play a role in fasting-induced stress. Based on the drop in mRNA expression of ghrl under catabolic conditions, we hypothesize that Ghrl might return as hunger signal once feed becomes available. We also propose that agrp1 is a potential appetite biomarker gene under feed deprived conditions.

Propionate stimulates the secretion of satiety hormones and reduces acute appetite in a cecal fistula pig model

Short-chain fatty acids (SCFA) can regulate appetite by stimulating the secretion of satiety hormones. However, the impact of short-chain fatty acid propionate on the release of gut satiety hormones and appetite regulation in pigs is not completely understood. In this study, 16 pigs were infused with saline or sodium propionate through a fistula in the caecum during a 28-day experimental period. We characterized the effects of propionate administration on peptide YY (PYY) and glucagon-like peptide 1 (GLP-1) secretion from colonic tissue, and investigated the role of propionate infusion on the expression of appetite-related genes in the colon and hypothalamus. Further, the direct impact of propionate administration on the expression of orexigenic neuropeptide agouti-related protein (AgRP) in hypothalamic N38 cells was also examined. The results showed that intra-cecal infusion of propionate reduced the short-term feed intake (P < 0.05) but not the long-term feed intake in pigs (P > 0.05). Propionate administration stimulated PYY and GLP-1 release from colon tissue in vivo and ex vivo (P < 0.05). It also upregulated PYY expression in the colonic mucosa (P < 0.05). Meanwhile, the GLP-1 and PYY levels in the blood were increased after intra-cecal infusion of propionate at d 28 (P < 0.05). Additionally, intra-cecal infusion of propionate upregulated the mRNA and protein expression of free fatty acid receptor 2/3 (FFAR2/FFAR3) in the colonic mucosa (P < 0.05). Propionate infusion also downregulated the orexigenic AgRP mRNA expression (P < 0.05) and upregulated the anorexigenic cocaine-and amphetamine-regulated transcript (CART) mRNA expression (P = 0.09) in the hypothalamus. Moreover, propionate administration directly downregulated AgRP expression in hypothalamic N38 cells in a dose-dependent manner (P < 0.05). Collectively, these findings demonstrated that cecal propionate stimulated colonic secretion of satiety hormones and suppressed appetite to reduce the short-term feed intake in pigs. This study highlights that microbial-derived propionate exerts an important role in regulating the physical functions of the host.

Feeding Rhythm-Induced Hypothalamic Agouti-Related Protein Elevation via Glucocorticoids Leads to Insulin Resistance in Skeletal Muscle.

Circadian phase shifts in peripheral clocks induced by changes in feeding rhythm often result in insulin resistance. However, whether the hypothalamic control system for energy metabolism is involved in the feeding rhythm-related development of insulin resistance is unknown. Here, we show the physiological significance and mechanism of the involvement of the agouti-related protein (AgRP) in evening feeding-associated alterations in insulin sensitivity. Evening feeding during the active dark period increased hypothalamic AgRP expression and skeletal muscle insulin resistance in mice. Inhibiting AgRP expression by administering an antisense oligo or a glucocorticoid receptor antagonist mitigated these effects. AgRP-producing neuron-specific glucocorticoid receptor-knockout (AgRP-GR-KO) mice had normal skeletal muscle insulin sensitivity even under evening feeding schedules. Hepatic vagotomy enhanced AgRP expression in the hypothalamus even during ad-lib feeding in wild-type mice but not in AgRP-GR-KO mice. The findings of this study indicate that feeding in the late active period may affect hypothalamic AgRP expression via glucocorticoids and induce skeletal muscle insulin resistance.

2027-P: Dietary Cholesterol and Neuropeptide Agouti Related Peptide Act in Concert to Regulate Locomotor Activity

In an ever-changing environment where food availability is highly unpredictable, the ability to maintain energy homeostasis requires proper sensing of dietary macronutrients, and subsequent adaptive adjustments in feeding behavior. Cholesterol is an important macronutrient, but cytotoxic in excess. Thus, cholesterol metabolism is under tight homeostatic regulation. Cholesterol is a major precursor of bile acids. After a meal, bile acids are released into the intestine and stimulate the secretion of fibroblast growth factor 15/19 (FGF-15/19). FGF-15/19 is an enterokine known to specifically suppress Agouti-related protein (AgRP), a neuropeptide of the central melanocortin system (CMS) that increases with fasting and promotes feeding and locomotor activity. Studies show that the CMS plays a modulatory role in hepatic cholesterol metabolism. However, the effects of dietary cholesterol on feeding behavior and energy homeostasis have remained largely unexplored. In this study, we found, surprisingly, that dietary cholesterol modulates expression of AgRP. Specific removal of cholesterol from the diets of wild type mice under ad-lib conditions leads to increased hypothalamic Agrp, but not Npy, mRNA expression. Furthermore, these mice also have low intestinal fGF-15mRNA expression. Cholesterol deficiency also leads to increased locomotor activities, an effect that is AgRP dependent as locomotor activity in AgRP knockout mice is unaffected by alterations in dietary cholesterol content. In wild type mice, fasting induces increased locomotor activity, which is widely thought to be a proxy for food seeking behavior. Notably, we observe dietary cholesterol content has no effect on ad-libfood intake, suggesting food seeking behaviors may be uncoupled from food intake. Taken together, these results suggest a novel paradigm where dietary cholesterol can act as a peripheral nutrient signal that can ultimately affect locomotor activity and possibly food seeking behavior. Disclosure A. Huang: None. M.T. Maier: None. A. Xu: None.

Sensing Glucose in the Central Melanocortin Circuits of Rainbow Trout: A Morphological Study

In mammals, glucosensing markers reside in brain areas known to play an important role in the control of food intake. The best characterized glucosensing mechanism is that dependent on glucokinase (GK) whose activation by increased levels of glucose leads in specific hypothalamic neurons to decreased or increased activity, ultimately leading to decreased food intake. In fish, evidence obtained in recent years suggested the presence of GK-like immunoreactive cells in different brain areas related to food intake control. However, it has not been established yet whether or not those neuronal populations having glucosensing capacity are the same that express the neuropeptides involved in the metabolic control of food intake. Therefore, we assessed through dual fluorescent in situ hybridization the possible expression of GK in the melanocortinergic neurons expressing proopiomelanocortin (POMC) or agouti-related protein (AGRP). POMC and AGRP expression localized exclusively in the rostral hypothalamus, in the ventral pole of the lateral tuberal nucleus, the homolog of the mammalian arcuate nucleus. Hypothalamic GK expression confined to the ependymal cells coating the ventral pole of the third ventricle but some expression level occurred in the AGRP neurons. GK expression seems to be absent in the hypothalamic POMC neurons. These results suggest that AGRP neurons might sense glucose directly through a mechanism involving GK. In contrast, POMC neurons would not directly respond to glucose through GK and would require presynaptic inputs to sense glucose. Ependymal cells could play a critical role relying glucose metabolic information to the central circuitry regulating food intake in fish, especially in POMC neurons.

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.

Chronic Stress Combined with a Fructose Diet Reduces Hypothalamic Insulin Signaling and Antioxidative Defense in Female Rats

Background: Increased fructose consumption and chronic exposure to stress have been associated with the development of obesity and insulin resistance. In the hypothalamus, a crossroad of stress responses and energy balance, insulin and glucocorticoids regulate the expression of orexigenic neuropeptides, neuropeptide Y (NPY) and agouti-related protein (AgRP), and anorexigenic neuropeptides, proopio­melanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART). Objectives: We investigated whether chronic stress and fructose diet disrupt these hormonal signaling pathways and appetite control in the hypothalamus, contributing to the development of insulin resistance and obesity. Potential roles of hypothalamic inflammation and oxidative stress in the development of insulin resistance were also analyzed. Methods: Insulin, glucocorticoid, and leptin signaling, expression of orexigenic and anorexigenic neuropeptides, and antioxidative and inflammatory statuses in the whole hypothalamus of fructose-fed female rats exposed to unpredictable stress for 9 weeks were analyzed using quantitative PCR and Western blotting. Results: Chronic stress combined with a fructose-enriched diet reduced protein content and stimulatory phosphorylation of Akt kinase, and elevated 11β-hydroxysteroid dehydrogenase 1 and glucocorticoid receptor expression, while alterations in appetite regulation (NPY, AgRP, POMC, CART, leptin receptor, and SOCS3 expression) were not observed. The expression of antioxidative defense enzymes (mitochondrial manganese superoxide dismutase 2, glutathione reductase, and catalase) and proinflammatory cytokines (IL-1β, IL-6, and TNFα) was reduced. Conclusions: Our results underline the combination of long-term stress exposure and fructose overconsumption as more detrimental for hypothalamic function than for either of the factors separately, as it enhanced glucocorticoid and impaired insulin signaling, antioxidative ­defense, and inflammatory responses of this homeostasis- regulating center.

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.

The effects of exercise on hypothalamic neurodegeneration of Alzheimer's disease mouse model.

Alzheimer’s disease is a neurodegenerative disorder that affects the central nervous system. In this study, we characterized and examined the early metabolic changes in the triple transgenic mouse AD model (3xtg-AD), and their relationship with the hypothalamus, a key regulator of metabolism in the central nervous system. We observed that the 3xtg-AD model exhibited significantly higher oxygen consumption as well as food intake before reported amyloid plaque formation, indicating that metabolic abnormalities occurred at early onset in the 3xtg-AD model compared with their counterparts. Analysis of gene expression in the hypothalamus indicated increased mRNA expression of inflammation- and apoptosis-related genes, as well as decreased gene expression of Agouti-related protein (AgRP) and Melanocortin 4 receptor (MC4R) at 12 weeks of age. Immunofluorescence analysis revealed that pro-opiomelanocortin (POMC) and NPY-expressing neurons decreased at 24 weeks in the 3xtg-AD model. Four weeks of voluntary exercise were sufficient to reverse the gene expression of inflammation and apoptotic markers in the hypothalamus, six weeks of exercise improved glucose metabolism, moreover, 8 weeks of voluntary exercise training attenuated apoptosis and augmented POMC and NPY-expressing neuronal populations in the hypothalamus compared to the control group. Our results indicated that early onset of metabolic abnormalities may contribute to the pathology of AD, which is associated with increased inflammation as well as decreased neuronal population and key neuropeptides in the hypothalamus. Furthermore, early intervention by voluntary exercise normalized hypothalamic inflammation and neurodegeneration as well as glucose metabolism in the 3xtg-AD model. The data, taken as a whole, suggests a hypothalamic-mediated mechanism where exercise prevents the progression of dementia and of Alzheimer’s disease.

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.

Roles of hypothalamic neuropeptide gene expression in body mass regulation in Eothenomys miletus (Mammalia: Rodentia: Cricetidae)

To investigate the roles of hypothalamic neuropeptide expression in body mass regulation in Eothenomys miletus, body mass, food intake, serum leptin levels and hypothalamic neuropeptide expression were measured in seasonal-acclimatized and lab-acclimated animals. The results showed that body mass, body fat mass and food intake showed seasonal variations. Serum leptin levels also appeared similar seasonal variation, showing a similar trend to body fat mass. Expression of hypothalamic neuropeptide Y (NPY) and agouti-related protein (AgRP) levels showed significant seasonal differences, while pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) expression showed no significant differences among seasons. Leptin had negative correlation with NPY and AgRP expression, positive correlation with POMC and CART expression among seasons. Cold and food restriction reduced body mass, body fat mass and serum leptin levels, and increased NPY and AgRP expression, but decreased POMC and CART expression. Photoperiods had no significant effect on these characters. Leptin was positively correlated with body fat mass, CART and POMC, and negatively correlated with NPY and AgRP under different temperatures, photoperiods and food quantity acclimation. All of these results showed that E. miletus reduced body mass and body fat mass, and increased food intake to survive in winter under seasonal changes. Temperature and food resources were the key factors influencing the body mass and energy metabolism in E. miletus. Leptin may play an important role in regulating body mass and energy metabolism by acting on hypothalamic neuropeptide expression in E. miletus.

PGC-1α expression in murine AgRP neurons regulates food intake and energy balance.

ObjectiveFood intake and whole-body energy homeostasis are controlled by agouti-related protein (AgRP) and pro-opiomelanocortin (POMC) neurons located in the arcuate nucleus of the hypothalamus. Key energy sensors, such as the AMP-activated protein kinase (AMPK) or sirtuin 1 (SIRT1), are essential in AgRP and POMC cells to ensure proper energy balance. In peripheral tissues, the transcriptional coactivator PGC-1α closely associates with these sensors to regulate cellular metabolism. The role of PGC-1α in the ARC nucleus, however, remains unknown.MethodsUsing AgRP and POMC neurons specific knockout (KO) mouse models we studied the consequences of PGC-1α deletion on metabolic parameters during fed and fasted states and on ghrelin and leptin responses. We also took advantage of an immortalized AgRP cell line to assess the impact of PGC-1α modulation on fasting induced AgRP expression.ResultsPGC-1α is dispensable for POMC functions in both fed and fasted states. In stark contrast, mice carrying a specific deletion of PGC-1α in AgRP neurons display increased adiposity concomitant with significantly lower body temperature and RER values during nighttime. In addition, the absence of PGC-1α in AgRP neurons reduces food intake in the fed and fasted states and alters the response to leptin. Finally, both in vivo and in an immortalized AgRP cell line, PGC-1α modulates AgRP expression induction upon fasting.ConclusionsCollectively, our results highlight a role for PGC-1α in the regulation of AgRP neuronal functions in the control of food intake and peripheral metabolism.

Food intake regulation during pregnancy and lactation in mice with reduced activity of the melanocortin system

Hypothalamic melanortin receptor (MCR) activation inhibits appetite. Neuropeptide Y (NPY) and Agouti Related Protein (AgRP) are coexpressed in some hypothalamic neurons and stimulate feeding, NPY via inhibition of MCR-expressing neurons, and AgRP via MCR4 antagonism. Mutation yellow at the mouse agouti locus (Ау) evokes MCR blockage and stimulates appetite in nulliparous females. The role of MCRs in food intake regulation during pregnancy and lactation is unclear. In this study we measured hypothalamic AgRP and NPY mRNA levels in virgin and mated C57Bl a/a (control) and Ау/a females on days 7, 13, 18 of pregnancy, 10, 21 of lactation, and after offspring separation, AgRP immunoreactivity in virgin and lactating females, and correlated gene expression with food intake (FI). Virgin Ау/a compared to a/a females had higher FI and lower AgRP expression. Pregnant Ау/a and a/a mice showed different patterns of food intake and neuropeptide expressions. NPY mRNA levels increased during pregnancy only in a/a mice, while AgRP mRNA levels increased in both genotypes being lower in Ау/a then in a/a mice. In lactating Ау/a and a/a mice, AgRP expression and NPY mRNA level were similar. AgRP expression was higher in lactating then in virgin Ау/a mice. The results obtained demonstrate that in nonbreeding female mice, MCR blockage is associated with AgRP expression inhibition which vanishes in lactation. In lactation, hyperphagia is independent of MCR blockage. In pregnancy, food intake regulation involves MCR signaling and activation of NPY and AgRP expression.

Human C-reactive protein impedes entry of leptin into the CNS and attenuates its physiological actions in the CNS.

Defective central leptin signalling and impaired leptin entry into the CNS (central nervous system) represent two important aspects of leptin resistance in obesity. In the present study, we tested whether circulating human CRP (C-reactive protein) not only diminishes signalling of leptin within the CNS, but also impedes this adipokine's access to the CNS. Peripheral infusion of human CRP together with co-infused human leptin was associated with significantly decreased leptin content in the CSF of ob/ob mice. Furthermore, following peripheral infusion of human leptin, the CSF (cerebrospinal fluid) concentration of leptin in transgenic mice overexpressing human CRP was sharply lower than that achieved in similarly infused wild-type mice. Administration of LPS (lipopolysaccharide) to human CRP-transgenic mice dramatically elevated the concentrations of human CRP in the CSF. The i.c.v. (intracerebroventricular) delivery of human CRP into the lateral ventricles of ob/ob mice blocked the satiety and weight-reducing actions of human leptin, but not those of mouse leptin. I.c.v. injection of human CRP abolished hypothalamic signalling by human leptin, and ameliorated the effects of leptin on the expression of NPY (neuropeptide Y), AgRP (Agouti-related protein), POMC (pro-opiomelanocortin) and SOCS-3 (suppressor of cytokine signalling 3). Human CRP can impede the access of leptin to the CNS, and elevation of human CRP within the CNS can have a negative impact on the physiological actions of leptin.

Linoleic acid and stearic acid elicit opposite effects on AgRP expression and secretion via TLR4-dependent signaling pathways in immortalized hypothalamic N38 cells

The regulation of food intake is a promising way to combat obesity. It has been implicated that various fatty acids exert different effects on food intake and body weight. However, the underlying mechanism remains poorly understood. The aim of the present study was to investigate the effects of linoleic acid (LA) and stearic acid (SA) on agouti-related protein (AgRP) expression and secretion in immortalized mouse hypothalamic N38 cells and to explore the likely underlying mechanisms. Our results demonstrated that LA inhibited, while SA stimulated AgRP expression and secretion of N38 cells in a dose-dependent manner. In addition, LA suppressed the protein expression of toll-like receptor 4 (TLR4), phosphorylation levels of JNK and IKKα/β, suggesting the inhibition of TLR4-dependent inflammation pathway. However, the above mentioned inhibitory effects of LA were eliminated by TLR4 agonist lipopolysaccharide (LPS). In contrast, SA promoted TLR4 protein expression and activated TLR4-dependent inflammation pathway, with elevated ratio of p-JNK/JNK. While TLR4 siRNA reversed the stimulatory effects of SA on AgRP expression and TLR4-dependent inflammation. Moreover, we found that TLR4 was also involved in LA-enhanced and SA-impaired leptin/insulin signal pathways in N38 cells. In conclusion, our findings indicated that LA elicited inhibitory while SA exerted stimulatory effects on AgRP expression and secretion via TLR4-dependent inflammation and leptin/insulin pathways in N38 cells. These data provided a better understanding of the mechanism underlying fatty acids-regulated food intake and suggested the potential role of long-chain unsaturated fatty acids such as LA in reducing food intake and treating obesity.

Maternal Triclosan consumption alters the appetite regulatory network on Wistar rat offspring and predispose to metabolic syndrome in the adulthood.

The objectives of this study were to evaluate the effects of maternal oral exposure to the antibacterial Triclosan (TCS) during gestation and lactation on the metabolic status of the adult offspring and on the expression of main genes controlling the appetite regulatory network. Pregnant rats were fed ad-libitum with ground food + TCS (1 mg/kg) from day 14 of gestation to day 20 of lactation (n=3) or ground food (n=3). After litter reduction, 12 males and 12 females born from the TCS exposed rats (TCS, n=24) or not (Control, n=24) were used to evaluate monthly body weight, food intake, plasma levels of cholesterol, glucose and triglycerides, and the hypothalamic mRNA expression of agouti-related protein (Agrp), neuropeptide Y (Npy) and propiomelanocortin (Pomc). Body weight for rats in the TCS group was 12.5% heavier for males at 4 months (p<0.001) and 19% heavier for females at 8 months (p=0.01). Food intake was significantly higher for rats in the TCS group at 5 months of age (p<0.01). Cholesterol and glucose levels were significantly higher for rats in the TCS group at 8 months (p<0.05). mRNA expression of Npy and Agrp were significantly increased in hypothalami of rats in the TCS group at 2 months for males or 8 months for females (p<0.05). In conclusion, low doses of oral TCS consumption by the pregnant and lactating dam increase the hypothalamic expression of the orexigenic neuropeptides Npy and Agrp in the offspring and alter their metabolic status during adulthood, resembling development of the metabolic syndrome.