Widespread expression of Agouti-related protein (AGRP) in the chicken: a possible involvement of AGRP in regulating peripheral melanocortin systems in the chicken

Agouti-related protein (AGRP) is a naturally occurring antagonist of melanocortin action. It is expressed mainly in the arcuate nucleus where it plays an important role in the hypothalamic control of feeding and energy homeostasis by antagonism of central melanocortin 4 receptors in mammals. Besides in the brain, the melanocortin 4 receptor is expressed in numerous peripheral tissues in the chicken. To examine whether or not the peripheral melanocortin 4 receptor signaling could be regulated by AGRP, we cloned and localized the expression of the AGRP gene in the chicken. The chicken AGRP gene was found to encode a 154 or 165 amino acid protein, depending on the usage of two alternative translation initiation sites. The coding sequence consisted of three exons, like that of mammalian species. The C-terminal cysteine-rich region of the predicted AGRP displayed high levels of identity to mammalian counterparts (78-84%) and all 10 cysteine residues conferring functional conformation of AGRP were conserved; however, other regions showed apparently no homology, suggesting that biological activities of AGRP are located in its C-terminal region. RT-PCR analysis detected the AGRP mRNA in all tissues examined: the brain, adrenal gland, heart, liver, spleen, gonads, kidney, uropygial gland, skeletal muscle and adipose tissues. Interestingly, the skin also expressed the AGRP mRNA, where Agouti, another melanocortin receptor antagonist regulating hair pigmentation, is expressed in rodents. Most of those AGRP-expressing tissues have been demonstrated to express melanocortin 4 receptors and/or other subtypes of melanocortin receptor whose mammalian counterparts can bind AGRP. These results imply the possibility that some peripheral melanocortin systems could be regulated by the functional interaction between melanocortins and AGRP at melanocortin receptors in the chicken.     

Feeding induced by cannabinoids is mediated independently of the melanocortin system

Background

Cannabinoids, the active components of marijuana, stimulate appetite, and cannabinoid receptor-1 (CB1-R) antagonists suppress appetite and promote weight loss. Little is known about how CB1-R antagonists affect the central neurocircuitry, specifically the melanocortin system that regulates energy balance.

Methodology/Principal Findings

Here, we show that peripherally administered CB1-R antagonist (AM251) or agonist equally suppressed or stimulated feeding respectively in A^y , which lack a functional melanocortin system, and wildtype mice, demonstrating that cannabinoid effects on feeding do not require melanocortin circuitry. CB1-R antagonist or agonist administered into the ventral tegmental area (VTA) equally suppressed or stimulated feeding respectively, in both genotypes. In addition, peripheral and central cannabinoid administration similarly induced c-Fos activation in brain sites suggesting mediation via motivational dopaminergic circuitry. Amperometry-detected increases in evoked dopamine (DA) release by the CB1-R antagonist in nucleus accumbens slices indicates that AM251 modulates DA release from VTA terminals.

Conclusions/Significance

Our results demonstrate that the effects of cannabinoids on energy balance are independent of hypothalamic melanocortin circuitry and is primarily driven by the reward system.     

Melanocortin activation of nucleus of the solitary tract avoids anorectic tachyphylaxis and induces prolonged weight loss

To examine the role of the brain stem melanocortin system in long-term energy regulation, we assessed the effects of overproduction of proopiomelanocortin (POMC) in the caudal brain stem of F344xBN rats with adult-onset obesity. Recombinant adeno-associated viral vector encoding POMC gene was delivered to the nucleus of solitary tract (NTS) in the hindbrain, and food intake, body weight, glucose and fat metabolism, brown adipose tissue thermogenesis, and mRNA levels of neuropeptides and melanocortin receptors were assessed. POMC delivery resulted in sustained reduction in food intake and body weight over 42 days and improved insulin sensitivity. At death, in recombinant adeno-associated viral vector-POMC-treated rats vs. control rats, alpha-melanocyte-stimulating hormone in NTS increased nearly 21-fold, whereas hypothalamic alpha-melanocyte-stimulating hormone remained unchanged. Visceral adiposity decreased by 37%; tissue triglyceride content diminished by 26% and 47% in liver and muscle, respectively; serum triglyceride and nonesterified fatty acids were reduced by 35% and 34%, respectively; phosphorylation of acetyl-CoA carboxylase was elevated by 63% in soleus muscle; brown adipose tissue uncoupling protein 1 increased by 30%; and melanocortin 3 receptor expression declined by 60%, whereas neuropeptide Y, agouti-related protein, and MC4 receptor mRNA levels were unchanged in the NTS. In conclusion, POMC overexpression in the NTS produces a characteristic unabated hypophagia that is uniquely different from the anorexic tachyphylaxis following POMC overexpression in the hypothalamus. The sustained anorectic response may result from absence of compensatory elements in the NTS, such as increased agouti-related protein expression, suggesting melanocortin activation of the brain stem may be a viable strategy to alleviate obesity.     

Brain stem melanocortinergic modulation of meal size and identification of hypothalamic POMC projections

Metabolic, cognitive, and environmental factors processed in the forebrain modulate food intake by changing the potency of direct controls of meal ingestion in the brain stem. Here, we behaviorally and anatomically test the role of the hypothalamic proopiomelanocortin (POMC) system in mediating some of these descending, indirect controls. Melanotan II (MTII), a stable melanocortin 4 receptor (MC4R) and melanocortin 3 receptor (MC3R) agonist injected into the fourth ventricle near the dorsal vagal complex, potently inhibited 14-h food intake by decreasing meal size but not meal frequency; SHU9119, an antagonist, increased food intake by selectively increasing meal size. Furthermore, MTII injected into the fourth ventricle increased and SHU9119 tended to decrease heart rate and body temperature measured telemetrically in freely moving rats. Numerous alpha-melanocyte-stimulating hormone-immunoreactive axons were in close anatomical apposition to nucleus tractus solitarius neurons showing c-Fos in response to gastric distension, expressing neurochemical phenotypes implicated in ingestive control, and projecting to brown adipose tissue. In retrograde tracing experiments, a small percentage of arcuate nucleus POMC neurons was found to project to the dorsal vagal complex. Thus melanocortin signaling in the brain stem is sufficient to alter food intake via changing the potency of satiety signals and to alter sympathetic outflow. Although the anatomical findings support the involvement of hypothalamomedullary POMC projections in mediating part of the descending, indirect signal, they do not rule out involvement of POMC neurons in the nucleus tractus solitarius in mediating part of the direct signal.     

Serotonin reciprocally regulates melanocortin neurons to modulate food intake

The neural pathways through which central serotonergic systems regulate food intake and body weight remain to be fully elucidated. We report that serotonin, via action at serotonin1B receptors (5-HT1BRs), modulates the endogenous release of both agonists and antagonists of the melanocortin receptors, which are a core component of the central circuitry controlling body weight homeostasis. We also show that serotonin-induced hypophagia requires downstream activation of melanocortin 4, but not melanocortin 3, receptors. These results identify a primary mechanism underlying the serotonergic regulation of energy balance and provide an example of a centrally derived signal that reciprocally regulates melanocortin receptor agonists and antagonists in a similar manner to peripheral adiposity signals.     

The Yellow Mouse Obesity Syndrome and Mechanisms of Agouti-Induced Obesity

The yellow mouse obesity syndrome is due to dominant mutations at the Agouti locus, which is characterized by obesity, hyperinsulinemia, insulin resistance, hyperglycemia, hyperleptinemia, increased linear growth, and yellow coat color. This syndrome is caused by ectopic expression of Agouti in multiple tissues. Mechanisms of Agouti action in obesity seem to involve, at least in part, competitive melanocortin antagonism. Both central and peripheral effects have been implicated in Agouti-induced obesity. An Agouti-Related Protein (AGRP) has been described recently. It has been shown to be expressed in mice hypothalamus and to act similarly to agouti as a potent antagonist to central melanocortin receptor MC4-R, suggesting that AGRP is an endogenous MC4-R ligand. Mice lacking MC4-R become hyperphagic and develop obesity, implying that agouti may lead to obesity by interfering with MC4-R signaling in the brain and consequently regulating food intake. Furthermore, food intake is inhibited by intracerebro-ventricular injection of a potent melanocortin agonist and was reversed by administration of an MC4-R antagonist. The direct cellular actions of Agouti include stimulation of fatty acid and triglyceride synthesis via a Ca²⁺-dependent mechanism. Agouti and insulin act in an additive manner to increase lipogenesis. This additive effect of agouti and insulin is demonstrated by the necessity of insulin in eliciting weight gain in transgenic mice expressing agouti specifically in adipose tissue. This suggests that agouti expression in adipose tissue combined with hyperinsulinemia may lead to increased adiposity. The roles of melanocortin receptors or agouti-specific receptor(s) in agouti regulation of adipocyte metabolism and other peripheral effects remain to be determined. In conclusion, both central and peripheral actions of agouti contribute to the yellow mouse obesity syndrome and this action is mediated at least in part by antagonism with melanocortin receptors and/or regulation of intracellular calcium.     

The regulation of food intake by selective stimulation of the type 3 melanocortin receptor (MC3R)

High levels of binding sites for melanocortin peptides exist within the arcuate nucleus, and a functional response to melanocortin peptides has been demonstrated in arcuate POMC neurons. Because the MC3R is thought to function as an inhibitory autoreceptor on POMC neurons, we reasoned that peripheral injections of MC3R-specific agonists would act within the arcuate nucleus to inhibit POMC neurons and thereby stimulate feeding. We demonstrate that the peptidergic MC3R agonist, d-Trp(8)-gamma-MSH, stimulates feeding via the MC3R when injected peripherally. These data provide the first evidence that feeding can be stimulated by peripheral injection of MC3R-specific agonists.     

Food entrainment modifies the c-Fos expression pattern in brain stem nuclei of rats

When food is restricted to a few hours daily, animals increase their locomotor activity 2-3 h before food access, which has been termed food anticipatory activity. Food entrainment has been linked to the expression of a circadian food-entrained oscillator (FEO) and the anatomic substrate of this oscillator seems to depend on diverse neural systems and peripheral organs. Previously, we have described a differential involvement of hypothalamic nuclei in the food-entrained process. For the food entrainment pathway, the communication between the gastrointestinal system and central nervous system is essential. The visceral synaptic input to the brain stem arrives at the dorsal vagal complex and is transmitted directly from the nucleus of the solitary tract (NST) or via the parabrachial nucleus (PBN) to hypothalamic nuclei and other areas of the forebrain. The present study aims to characterize the response of brain stem structures in food entrainment. The expression of c-Fos immunoreactivity (c-Fos-IR) was used to identify neuronal activation. Present data show an increased c-Fos-IR following meal time in all brain stem nuclei studied. Food-entrained temporal patterns did not persist under fasting conditions, indicating a direct dependence on feeding-elicited signals for this activation. Because NST and PBN exhibited a different and increased response from that expected after a regular meal, we suggest that food entrainment promotes ingestive adaptations that lead to a modified activation in these brain stem nuclei, e.g., stomach distension. Neural information provided by these nuclei to the brain may provide the essential entraining signal for FEO.     

Functional Heterogeneity of Arcuate Nucleus Pro-Opiomelanocortin Neurons: Implications for Diverging Melanocortin Pathways

Arcuate nucleus (ARC) pro-opiomelanocortin (POMC) neurons are essential regulators of food intake, energy expenditure, and glucose homeostasis. POMC neurons integrate several key metabolic signals that include neurotransmitters and hormones. The change in activity of POMC neurons is relayed to melanocortin receptors in distinct regions of the central nervous system. This review will summarize the role of leptin and serotonin receptors in regulating the activity of POMC neurons and provide a model in which different melanocortin pathways regulate energy and glucose homeostasis.     

Circumventing central leptin resistance: lessons from central leptin and POMC gene delivery

We identified that leptin resistance in aged-obese rats has both peripheral and central components. The central resistance is characterized by diminished hypothalamic leptin receptors and impaired leptin signal transduction. We developed a new model of leptin-induced leptin resistance in which application of the central leptin gene delivery produces unabated hypothalamic leptin over-expression. The chronic central elevation of leptin precipitates leptin resistance in young animals devoid of obesity and exacerbates it in mature or aged animals with obesity. Despite leptin resistance, our aged obese, DIO, and leptin-induced leptin resistant rats were fully responsive to central pharmacological melanocortin activation. We propose that the central leptin resistance resides between leptin receptor and melanocortin receptor activation. Our central POMC gene therapy overcame leptin resistance, producing weight and fat loss and improved insulin sensitivity in obese Zucker and aged rats. This success highlights the central melanocortin system as a useful drug target for combating obesity.     

Serotonergic pathways converge upon central melanocortin systems to regulate energy balance

Multiple lines of research provide compelling support for an important role for central serotonergic (5-hydroxytryptamine, 5-HT) and melanocortin pathways in the regulation of food intake and body weight. In this brief review, we outline data supporting a model in which serotonergic pathways affect energy balance, in part, by converging upon central melanocortin systems to stimulate the release of the endogenous melanocortin agonist, alpha-melanocyte stimulating hormone (alpha-MSH). Further, we review the neuroanatomical mapping of a downstream target of alpha-MSH, the melanocortin 4 receptor (MC4R), in the rodent brain. We propose that downstream activation of MC4R-expressing neurons substantially contributes to serotonin's effects on energy homeostasis.     

Differential expression of the melanocortin-4 receptor in male and female C57BL/6J mice

The melanocortin 4 receptor is a member of melanocortin receptors of G-protein-coupled receptors. By binding to melanocortin receptor agonists or antagonists, MC4R participates in the regulating of food intake, weight, energy homeostasis and sexual behavior. By activating the protein kinase A and leptin-melanocortin signalling pathways, MC4R mediates the amplification of signals from the hypothalamo–pituitary–adrenal and hypothalamo–pituitary–thyroid axes. This process permits peripheral information about the status of energy metabolism to be transmitted to the central nervous system. The hypothalamic nuclei then integrate these signals to evoke the appropriate reaction. We found that different sexes exhibited distinct metabolic regulation abilities, likely due to differences in these signalling pathways. MC4R plays a key role in coordinating the afferent messages from the peripheral and regulatory signals by controlling food intake and energy expenditure. To probe the disparities in metabolism and weight regulation between the sexes, we analyzed the expression of MC4R in different tissues from male and female mice by qRT-PCR and immunofluorescence. The results show that the expression of MC4R in brain and kidney is higher in female mice than in male mice, but in the livers, the result is opposition. Additionally, in both sexes, the expression of MC4R is higher in the brain than in the kidneys, and its expression in the liver is lowest, in males, the expression of MC4R in the testis is higher than that in the kidneys. These data show that the expression of MC4R exist different between sexes mice.     

Central melanocortin signaling restores skeletal muscle AMP-activated protein kinase phosphorylation in mice fed a high-fat diet

Little is known about the role of the central melanocortin system in the control of fuel metabolism in peripheral tissues. Skeletal muscle AMP-activated protein kinase (AMPK) is activated by leptin and serves as a master regulator of fatty acid beta-oxidation. To elucidate an unidentified role of the central melanocortin system in muscle AMPK regulation, we treated conscious, unrestrained mice intracerebroventricularly with the melanocortin agonist MT-II or the antagonist SHU9119. MT-II augmented phosphorylation of AMPK and its target acetyl-CoA carboxylase (ACC) independent of caloric intake. Conversely, AMPK/ACC phosphorylation by leptin was abrogated by the coadministration of SHU9119 or in KKA(y) mice, which centrally express endogenous melanocortin antagonist. Importantly, high-fat-diet-induced attenuation of AMPK/ACC phosphorylation in leptin-overexpressing transgenic mice was not reversed by central leptin but was markedly restored by MT-II. Our data provide evidence for the critical role of the central melanocortin system in the leptin-skeletal muscle AMPK axis and highlight the system as a therapeutic target in leptin resistance.     

Ghrelin acts on the dorsal vagal complex to stimulate pancreatic protein secretion

Ghrelin receptors are present in the central nervous system. We hypothesized that ghrelin released from the stomach acts as an endocrine substance and stimulates brain stem vagovagal circuitry to evoke pancreatic secretion. In an in vivo anesthetized rat model, an intravenous infusion of ghrelin at doses of 5, 10, and 25 nmol increased pancreatic protein secretion from a basal level of 125 +/- 6 to 186 +/- 8, 295 +/- 12, and 356 +/- 11 mg/h, respectively. Pretreatment with atropine or hexamethonium or an acute vagotomy, but not a perivagal application of capsaicin, completely abolished pancreatic protein secretion responses to ghrelin. In conscious rats, an intravenous infusion of ghrelin at a dose of 10 nmol resulted in a 2.2-fold increase in pancreatic protein secretion over basal volume. Selective ablation of the area postrema abolished pancreatic protein secretion stimulated by intravenous infusion of ghrelin but did not alter the increase in pancreatic protein secretion evoked by diversion of bile-pancreatic juice. Immunohistochemical staining showed a marked increase in the number of c-Fos-expressing neurons in the area postrema, nucleus of the solitary tract, and dorsal motor nucleus of the vagus after an intravenous infusion of ghrelin in sham-lesioned rats; selective ablation of the area postrema eliminated this increase. In conclusion, ghrelin stimulates pancreatic secretion via a vagal cholinergic efferent pathway. Circulating ghrelin gains access to the brain stem vagovagal circuitry via the area postrema, which represents the primary target on which peripheral ghrelin may act as an endocrine substance to stimulate pancreatic secretion.     

Losing the lust for life: a new role for an old feeding Peptide?

A recent paper in Nature (Lim et?al., 2012) describes the effects of melanocortin receptors in the nucleus accumbens. The studies connect a hypothalamic peptide system with brain reward centers and show effects on specific neuronal populations and behavioral components of mood.     

The distribution of alpha-melanocyte stimulating hormone (alpha-MSH) in the central nervous system of the rat: an immunohistochemical study. II. Lower brain stem

The distribution of immunoreactive alpha-melanocyte stimulating hormone (alpha-MSHI) in the rat lower brain stem was examined by indirect immunofluorescence or peroxidase- anti-peroxidase immunohistochemical method using an antiserum against synthetic alpha-MSH. The results confirmed the presence of alpha-MSHI fibers in the midbrain central gray matter and parabrachial area, and demonstrated a much more extensive distribution of these fibers in various parts of the lower brain stem areas previously thought not contain alpha-MSHI fibers. In addition, the commissural nucleus was identified as a new alpha-MSHI neurons-containing site. No alpha-MSHI neurons were seen in other regions of the rat lower brain stem.     

Neural control of the anorexia-cachexia syndrome

The anorexia-cachexia syndrome is a debilitating clinical condition characterizing the course of chronic diseases, which heavily impacts on patients' morbidity and quality of life, ultimately accelerating death. The pathogenesis is multifactorial and reflects the complexity and redundancy of the mechanisms controlling energy homeostasis under physiological conditions. Accumulating evidence indicates that, during disease, disturbances of the hypothalamic pathways controlling energy homeostasis occur, leading to profound metabolic changes in peripheral tissues. In particular, the hypothalamic melanocortin system does not respond appropriately to peripheral inputs, and its activity is diverted largely toward the promotion of catabolic stimuli (i.e., reduced energy intake, increased energy expenditure, possibly increased muscle proteolysis, and adipose tissue loss). Hypothalamic proinflammatory cytokines and serotonin, among other factors, are key in triggering hypothalamic resistance. These catabolic effects represent the central response to peripheral challenges (i.e., growing tumor, renal, cardiac failure, disrupted hepatic metabolism) that are likely sensed by the brain through the vagus nerve. Also, disease-induced changes in fatty acid oxidation within hypothalamic neurons may contribute to the dysfunction of the hypothalamic melanocortin system. Ultimately, sympathetic outflow mediates, at least in part, the metabolic changes in peripheral tissues. Other factors are likely involved in the pathogenesis of the anorexia-cachexia syndrome, and their role is currently being elucidated. However, available evidence shows that the constellation of symptoms characterizing this syndrome should be considered, at least in part, as different phenotypes of common neurochemical/metabolic alterations in the presence of a chronic inflammatory state.