On the origin of eating disorders: altered signaling between gut microbiota, adaptive immunity and the brain melanocortin system regulating feeding behavior.

Research in the field of gut microbiota - brain axis may contribute to clarifying the origin of anorexia nervosa and bulimia, the two principal forms of eating disorders (ED). The initial key findings in ED patients of plasma immunoglobulins (Ig) that react with α-melanocyte-stimulating hormone (α-MSH), a neuropeptide in the brain signaling satiety, have initiated further studies leading to the discovery of the origin of such autoantibodies and to the understanding their possible functional role. An anorexigenic bacterial protein Escherichia coli caseinolytic protease B was recently found to be responsible for the production of α-MSH-cross-reactive autoantibodies and this protein was also detected in human plasma. Another recent study revealed enhanced activation of appetite-regulating the melanocortin type 4 receptor by immune complexes withα-MSH. Taken together, these data serve to build a pathophysiological model of ED presented in this article.

Alpha-melanocyte stimulating hormone increases the activity of melanocortin-3 receptor-expressing neurons in the ventral tegmental area.

KEY POINTS: Alpha-melanocyte stimulating hormone (α-MSH) is an anorexigenic peptide. Injection of the α-MSH analog MTII into the ventral tegmental area (VTA) decreases food and sucrose intake and food reward. Melanocortin-3 receptors (MC3R) are highly expressed in the VTA, suggesting that the effects of intra-VTA α-MSH may be mediated by α-MSH changing the activity of MC3R-expressing VTA neurons. α-MSH increased the firing rate of MC3R VTA neurons in acute brain slices from mice, although it did not affect the firing rate of non-MC3R VTA neurons. The α-MSH induced increase in MC3R neuron firing rate is probably activity-dependent, and was independent of fast synaptic transmission and intracellular Ca2+ levels. These results help us to better understand how α-MSH acts in the VTA to affect feeding and other dopamine-dependent behaviours. ABSTRACT: The mesocorticolimbic dopamine system, the brain's reward system, regulates multiple behaviours, including food intake and food reward. There is substantial evidence that the melanocortin system of the hypothalamus, an important neural circuit controlling feeding and body weight, interacts with the mesocorticolimbic dopamine system to affect feeding, food reward and body weight. For example, melanocortin-3 receptors (MC3Rs) are expressed in the ventral tegmental area (VTA) and our laboratory previously showed that intra-VTA injection of the MC3R agonist, MTII, decreases home-cage food intake and operant responding for sucrose pellets. However, the cellular mechanisms underlying the effects of intra-VTA alpha-melanocyte stimulating hormone (α-MSH) on feeding and food reward are unknown. To determine how α-MSH acts in the VTA to affect feeding, we performed electrophysiological recordings in acute brain slices from mice expressing enhanced yellow fluorescent protein in MC3R neurons to test how α-MSH affects the activity of VTA MC3R neurons. α-MSH significantly increased the firing rate of VTA MC3R neurons without altering the activity of non-MC3R expressing VTA neurons. In addition, the α-MSH-induced increase in MC3R neuron activity was independent of fast synaptic transmission and intracellular Ca2+ levels. Finally, we show that the effect of α-MSH on MC3R neuron firing rate is probably activity-dependent. Overall, these studies provide an important advancement in the understanding of how α-MSH acts in the VTA to affect feeding and food reward.

Alcohol Withdrawal and Proopiomelanocortin Neuropeptides in an Animal Model of Alcohol Dependence.

BACKGROUND: Alcohol is one of the leading threats to health worldwide. Craving for alcohol makes abstinence a difficult challenge by maintaining alcohol dependence. Many studies suppose the hypothalamic-pituitary-adrenal axis, especially the proopiomelanocortin (POMC)-derived neuropeptides, to mediate craving during withdrawal in alcohol dependence. Evidence is available that the two POMC proteins, α-melanocyte-stimulating hormone (α-MSH) and ß-endorphin (ß-END) are altered by alcohol consumption and influence alcohol consumption, respectively. OBJECTIVES: We investigated the dynamics of α-MSH and ß-END during alcohol withdrawal and the influence of intraperitoneal administration of either α-MSH or ß-END in an established rodent model (Wistar rats) for alcohol dependence. RESULTS: After long-term alcohol self-administration over 12 months and repeated deprivation periods for 3 days, we found a significant decrease in α-MSH levels during withdrawal in rodents (p = 0.006) compared to controls, while ß-END levels remained unchanged. Treatment with intraperitoneally administered α-MSH and ß-END did not affect alcohol drinking behavior after deprivation. CONCLUSION: We demonstrate the effects of alcohol deprivation on α-MSH in alcohol-dependent rodents, which appear to mimic α-MSH alteration found after fasting periods during appetite regulation. Therefore, low α-MSH levels are a possible indicator for craving in alcohol-dependent individuals and hence would be a potential target for anti-craving treatment.

Angiotensin AT1A receptors on leptin receptor-expressing cells control resting metabolism.

Leptin contributes to the control of resting metabolic rate (RMR) and blood pressure (BP) through its actions in the arcuate nucleus (ARC). The renin-angiotensin system (RAS) and angiotensin AT1 receptors within the brain are also involved in the control of RMR and BP, but whether this regulation overlaps with leptin's actions is unclear. Here, we have demonstrated the selective requirement of the AT1A receptor in leptin-mediated control of RMR. We observed that AT1A receptors colocalized with leptin receptors (LEPRs) in the ARC. Cellular coexpression of AT1A and LEPR was almost exclusive to the ARC and occurred primarily within neurons expressing agouti-related peptide (AgRP). Mice lacking the AT1A receptor specifically in LEPR-expressing cells failed to show an increase in RMR in response to a high-fat diet and deoxycorticosterone acetate-salt (DOCA-salt) treatments, but BP control remained intact. Accordingly, loss of RMR control was recapitulated in mice lacking AT1A in AgRP-expressing cells. We conclude that angiotensin activates divergent mechanisms to control BP and RMR and that the brain RAS functions as a major integrator for RMR control through its actions at leptin-sensitive AgRP cells of the ARC.

The Role of Alpha-MSH as a Modulator of Ocular Immunobiology Exemplifies Mechanistic Differences between Melanocortins and Steroids.

Melanocortins are a highly conserved family of peptides and receptors that includes multiple proopiomelanocortin-derived peptides and five defined melanocortin receptors. The melanocortins have an important role in maintaining immune homeostasis and in suppressing inflammation. Within the healthy eye, the melanocortins have a central role in preventing inflammation and maintaining immune privilege. A central mediator of the anti-inflammatory activity is the non-steroidogenic melanocortin peptide alpha-melanocyte stimulating hormone. In this review we summarize the major findings of melanocortin regulation of ocular immunobiology with particular interest in the ability of melanocortin to induce immune tolerance and cytoprotection. The melanocortins have therapeutic potential because their mechanisms of action in regulating immunity are distinctly different from the actions of steroids.

The effect of physical exercise on orexigenic and anorexigenic peptides and its role on long-term feeding control.

Over the past decades, life-styles changing have led to exacerbated food and caloric intake and a reduction in energy expenditure. Obesity, main outcome of these changes, increases the risk for developing type 2 diabetes, cardiovascular disease and metabolic syndrome, the leading cause of death in adult and middle age population. Body weight and energy homeostasis are maintained via complex interactions between orexigenic and anorexigenic neuropeptides that take place predominantly in the hypothalamus. Overeating may disrupt the mechanisms of feeding control, by decreasing the expression of proopiomelanocortin (POMC) and α-melanocyte stimulating hormone (α-MSH) and increasing orexigenic neuropeptide Y (NPY) and agouti-related peptide (AgRP), which leads to a disturbance in appetite control and energy balance. Studies have shown that regular physical exercise might decrease body-weight, food intake and improve the metabolic profile, however until the currently there is no consensus about its effects on the expression of orexigenic/anorexigenic neuropeptides expression. Therefore, we propose that the type and length of physical exercise affect POMC/αMSH and NPY/AgRP systems differently and plays an important role in feeding behavior. Moreover, based on the present reports, we hypothesize that increased POMC/αMSH overcome NPY/AgRP expression decreasing food intake in long term physical exercise and that results in amelioration of several conditions related to overweight and obesity.

Role of the Paraventricular Nucleus of the Hypothalamus in the Sympathoexcitatory Effects of Leptin.

Leptin binds to receptors in multiple hypothalamic nuclei to increase sympathetic nerve activity; however, the neurocircuitry is unclear. Here, using anesthetized male Sprague-Dawley rats, we investigated the role of the paraventricular nucleus of the hypothalamus. Intracerebroventricular injection of leptin slowly increased lumbar sympathetic nerve activity (LSNA), heart rate, mean arterial pressure, and baroreflex control of LSNA and heart rate. Inhibition of the paraventricular nucleus with muscimol completely reversed leptin's effects. Blockade of paraventricular melanocortin 3/4 receptors with SHU9119 or ionotropic glutamate receptors with kynurenate, alone or together, each partially reversed the effects of leptin, implicating increased activation of glutamate and melanocortin 3/4 receptors. Conversely, although blockade of neuropeptide Y Y1 receptors in the paraventricular nucleus increased LSNA, mean arterial pressure, and heart rate, these responses were prevented by intracerebroventricular or arcuate nucleus injections of leptin, suggesting that, at least in part, leptin also increases sympathetic nerve activity by suppression of tonic neuropeptide Y inhibitory inputs from the arcuate nucleus. Injection of the melanocortin 3/4 receptor agonist melanotan-II into the paraventricular nucleus increased LSNA, mean arterial pressure, and heart rate only after blockade of neuropeptide Y Y1 receptors. Therefore, we conclude that leptin increases LSNA in part via increased glutamatergic and α-melanocyte-stimulating hormone drive of paraventricular sympathoexcitatory neurons, the latter of which requires simultaneous withdrawal of tonic neuropeptide Y inhibition.

Age-related alterations in the central thermoregulatory responsiveness to alpha-MSH.

Alpha-melanocyte-stimulating-hormone (alpha-MSH) is a neuropeptide that induces weight loss via its anorexigenic and hypermetabolic/hyperthermic effects. Two major public health problems of the human population involving energy balance (i.e. middle-aged obesity and aging cachexia) also appear in other mammals, therefore age-related regulatory alterations may also be assumed in the background. Previous studies demonstrated characteristic age-related shifts in the anorexigenic effects of centrally applied alpha-MSH with strong effects in young adult, diminished efficacy in middle-aged and very pronounced responsiveness in old rats. The present study aimed to investigate age-related changes in the acute central thermoregulatory responsiveness to an alpha-MSH injection in rats and to compare them with those of food intake-related responsiveness. Oxygen consumption (VO2), core (Tc) and tail skin temperatures (Ts, indicating heat loss) of male Wistar rats of different age groups (from 2 to 24 months of age), were recorded in an indirect calorimeter complemented by thermocouples upon intracerebroventricular alpha-MSH administration (0, 5 µg) at a slightly subthermoneutral environment (25-26 °C). Acute alpha-MSH-induced rises in VO2 and Tc were most pronounced in the young adult age-group. In these rats the hyperthemic effects were somewhat diminished by an activation of heat loss. Juvenile animals showed weaker hyperthermic responses, middle-aged rats none at all. Alpha-MSH-induced hyperthermia became significant again in old rats. Acute thermoregulatory (hypermetabolic/hyperthermic) responsiveness to alpha-MSH shows a distinct age-related pattern similar to that of acute anorexigenic responsiveness. Thus, our results may also contribute to the explanation of both middle-aged obesity and aging cachexia.

Responses of melanocytes and melanomacrophages of Eupemphix nattereri (Anura: Leiuperidae) to Nle4, D-Phe7-α-melanocyte stimulating hormone and lipopolysaccharides.

Melanocytes are found in various organs of ectothermic animals, playing a protective role against bacteria and free radicals. It is known that pigment cells from hematopoietic organs have immune functions. However, the role of visceral melanocytes is not well understood. Cutaneous melanocytes are responsive to α-melanocyte stimulating hormone (α-MSH), which is associated with the dispersion of melanin granules within melanocytes. α-MSH has also been reported to inhibit most forms of inflammatory responses by decreasing the pro-inflammatory cytokines and neutrophil migration. The present study evaluated the influence of an α-MSH analog (Nle(4), D-Phe(7)-α-MSH) and lipopolysaccharides (LPS) from Escherichia coli on the liver and testicular tissues of the anuran Eupemphix nattereri. The tested hypotheses were: (i) the pigmented area will increase following hormone and LPS administration, (ii) pre-treatment with α-MSH will decrease the number of mast cells, and (iii) the hormone will have protective effects against LPS-induced responses. We found that hormone administration did not change hepatic pigmentation, but increased testicular pigmentation. Testicular pigmentation quickly increased after LPS administration, whereas there was a late response in the liver. The response of enhanced pigmentation was delayed and the number of mast cells decreased in animals previously treated with the α-MSH analog when compared to the LPS group. Hemosiderin and lipofuscin were found in melanomacrophages, but not in testicular melanocytes. Although both the liver and the testes of E. nattereri have pigmented cells, these are distinct in morphology, embryonic origin, and pigmentary substances. These differences may be responsible for the different responses of these cells to the α-MSH analog and LPS administration.

Protective effect of alpha-melanocyte-stimulating hormone (α-MSH) on the recovery of ischemia/reperfusion (I/R)-induced retinal damage in a rat model.

The present study demonstrates capacity of α-MSH to augment recovery from ischemia/reperfusion (I/R)-induced retinal damage in vivo and correlation of its protective effects with expression of heme oxygenase-1 (HO-1). Used techniques include ocular ischemia and reperfusion, electroretinography, histology, electron microscopy, and molecular-biological techniques. The results demonstrate the α-MSH-mediated inhibition of I/R-induced functional deterioration of the retina. Outcomes suggest that the protective effects of α-MSH occur mainly through HO-1-dependent pathways but HO-1-independent mechanisms may also contribute to protection. The observation that post-ischemic treatment with α-MSH exhibits therapeutic efficacy in the same range as pre-ischemic treatment, is a novel result. This outcome suggests a highly conserved protective role for α-MSH as a major stress response mechanism--and offers the possibility for development of novel therapeutic strategies utilizing this hormone, in particular in treatment of conditions resulting from I/R injury, such as deterioration of retinal microcirculation. The merit of the study lies in the fact that I/R injury contribute significantly to the severity of retinopathies. However, currently there are no evidence-based treatments for retinal I/R injury available for clinical use. Our finding suggests that α-MSH may have a very wide range of uses in the prevention of I/R-mediated pathologies.

PGC-1 coactivators regulate MITF and the tanning response.

The production of pigment by melanocytes tans the skin and protects against skin cancers. UV-exposed keratinocytes secrete α-MSH, which then activates melanin formation in melanocytes by inducing the microphthalmia-associated transcription factor (MITF). We show that PPAR-γ coactivator (PGC)-1α and PGC-1ß are critical components of this melanogenic system in melanocytes. α-MSH signaling strongly induces PGC-1α expression and stabilizes both PGC-1α and PGC-1ß proteins. The PGC-1s in turn activate the MITF promoter, and their expression correlates strongly with that of MITF in human melanoma cell lines and biopsy specimens. Inhibition of PGC-1α and PGC-1ß blocks the α-MSH-mediated induction of MITF and melanogenic genes. Conversely, overexpression of PGC-1α induces pigment formation in cell culture and transgenic animals. Finally, polymorphism studies reveal expression quantitative trait loci in the PGC-1ß gene that correlate with tanning ability and protection from melanoma in humans. These data identify PGC-1 coactivators as regulators of human tanning.

A comparative study of the central effects of melanocortin peptides on food intake in broiler and layer chicks.

Broiler chicks eat more food than layer chicks. However, the causes of the difference in food intake in the neonatal period between these strains are not clear. In this study, we examined the involvement of proopiomelanocortin (POMC)-derived melanocortin peptides α-, ß- and γ-melanocyte-stimulating hormones (MSHs) in the difference in food intake between broiler and layer chicks. First, we compared the hypothalamic mRNA levels of POMC between these strains and found that there was no significant difference in these levels between broiler and layer chicks. Next, we examined the effects of central administration of MSHs on food intake in these strains. Central administration of α-MSH significantly suppressed food intake in both strains. Central administration of ß-MSH significantly suppressed food intake in layer chicks, but not in broiler chicks, while central administration of γ-MSH did not influence food intake in either strain. It is therefore likely that the absence of the anorexigenic effect of ß-MSH might be related to the increased food intake in broiler chicks.

Sexual behaviour and neuronal activation in the vomeronasal pathway and hypothalamus of food-deprived male rats.

As feeding and mating are mutually-exclusive goal-orientated behaviours, we investigated whether brief food deprivation would impair the display of sexual behaviour of male rats. Analysis of performance in a sexual incentive motivation test revealed that, similar to fed males, food-deprived males preferred spending time in the vicinity of receptive females rather than nonreceptive females. Despite this, food-deprived males were more likely to be slow to mate than normally-fed males, and a low dose of the satiety peptide α-melanocyte-stimulating-hormone attenuated the effect of hunger. Using Fos immunocytochemistry, we compared neuronal activity in the vomeronasal projection pathway in response to oestrous cues from receptive females between food-deprived and fed males. As in fed males, more Fos expression was seen in the rostral part of the bed nucleus of the stria terminalis and in the medial preoptic area in food-deprived males, confirming that food-deprived males can recognise and respond to female oestrous cues. However, although there was also an increase in Fos expression in the bed nucleus of the accessory tract and in the posteromedial amygdala in fed males, no increases were seen in these areas in food-deprived rats. We also found selective attenuation in the activation of lateral posterior paraventricular nucleus (lpPVN) oxytocin neurones in food-deprived males. Taken together, the data show that, although food-deprived males can still become sexually motivated, copulation is delayed, and this is accompanied by variations in neuronal activity in the vomeronasal projection pathway. We propose that, in hungry rats, the lpPVN oxytocin neurones (which project to the spinal cord and are involved in maintaining penile erection) facilitate the transition from motivation to intromission, and their lack of activation impairs intromission, and thus delays mating.

Melanocortin-induced PKA activation inhibits AMPK activity via ERK-1/2 and LKB-1 in hypothalamic GT1-7 cells.

α-Melanocyte-stimulating hormone (α-MSH)-induced activation of the melanocortin-4 receptor in hypothalamic neurons increases energy expenditure and inhibits food intake. Active hypothalamic AMP-activated protein kinase (AMPK) has recently been reported to enhance food intake, and in vivo experiments suggested that intrahypothalamic injection of melanocortins decreased food intake due to the inhibition of AMPK activity. However, it is not clear whether α-MSH affects AMPK via direct intracellular signaling cascades or if the release of paracrine factors is involved. Here, we used a murine, hypothalamic cell line (GT1-7 cells) and monitored AMPK phosphorylation at Thr(172), which has been suggested to increase AMPK activity. We found that α-MSH dephosphorylated AMPK at Thr(172) and consequently decreased phosphorylation of the established AMPK substrate acetyl-coenzyme A-carboxylase at Ser(79). Inhibitory effects of α-MSH on AMPK were blocked by specific inhibitors of protein kinase A (PKA) or ERK-1/2, pointing to an important role of both kinases in this process. Because α-MSH-induced activation of ERK-1/2 was blunted by PKA inhibitors, we propose that ERK-1/2 serves as a link between PKA and AMPK in GT1-7 cells. Furthermore, down-regulation of liver kinase B-1, but not inhibition of calcium-calmodulin-dependent kinase kinase-ß or TGFß-activated kinase-1 decreased basal phosphorylation of AMPK and its dephosphorylation induced by α-MSH. Thus, we propose that α-MSH inhibits AMPK activity via a linear pathway, including PKA, ERK-1/2, and liver kinase B-1 in GT1-7 cells. Given the importance of the melanocortin system in the formation of adipositas, detailed knowledge about this pathway might help to develop drugs targeting obesity.

Further evidence on acetylation-induced inhibition of the pigment-dispersing activity of α-melanocyte-stimulating hormone.

Our previous studies showed that in barfin flounder, α-melanocyte-stimulating hormone (α-MSH) stimulates pigment dispersion in xanthophores, while it shows negligible effects in melanophores. The present study was undertaken to evaluate whether these results are limited to barfin flounder by using Japanese flounder. Three subtypes of proopiomelanocortin gene encoding melanocortins (MCs) were expressed in the Japanese flounder pituitary, one of which was also expressed in the skin. Expression of melanocortin 5 receptor gene (Mc5r) was observed in isolated xanthophores, while that of Mc1r and Mc5r was found in melanophores. In the xanthophores of Japanese flounder skin, α-MSH as well as desacetyl (Des-Ac)-α-MSH and diacetyl (Di-Ac)-α-MSH exhibited dose-dependent pigment-dispersing activities, indicating that the signals of α-MSH-related peptides were mediated by MC5R. On the other hand, α-MSH did not stimulate pigment dispersion in melanophores, while Des-Ac-α-MSH and Di-Ac-α-MSH did, thus indicating that the expression of two different types of Mcr is related to the decrease in α-MSH activity. Thus, the molecular repertoire in MC system observed in Japanese flounder is similar to that in barfin flounder. Moreover, the relationship between the pigment-dispersing activities of α-MSH-related peptides and the expression of Mcr subtypes in xanthophores and melanophores were also similar between Japanese flounder and barfin flounder. Consequently, we hypothesize that inhibition of α-MSH activity could be due to the formation of heterodimers comprising MC1R and MC5R, often observed in G-protein-coupled receptors.

Intestinal inflammation influences α-MSH reactive autoantibodies: relevance to food intake and body weight.

Autoantibodies reacting with alpha-melanocyte-stimulating hormone (α-MSH), an anorexigenic neuropeptide, are involved in regulation of feeding. In this work we studied if intestinal inflammation (mucositis) may influence α-MSH autoantibodies production relevant to food intake and body weight. Mucositis and anorexia were produced in Sprague-Dawley rats by methotrexate (MTX, 2.5mg/kg/day, for three days, subcutaneously). Plasma levels of total IgG and of α-MSH autoantibodies were measured during and after MTX-induced mucositis and were compared with pair-fed and ad libitum-fed controls. Effects of intraperitoneal injections of rabbit anti-α-MSH IgG (3 or 10 µg/day/rat) on MTX-induced anorexia and on plasma α-MSH peptide concentration were separately studied. Here we show that in MTX rats, intestinal mucositis and anorexia were accompanied by decreased plasma levels of both total IgG and of α-MSH autoantibodies while refeeding was characterized by their elevated levels. In spite of similar food intake in MTX and pair-fed rats, recovery of body weight was delayed by at least 1 week in the MTX group. During refeeding and body weight deficit in MTX rats, α-MSH IgG autoantibody levels correlated negatively with food to water intake ratios. Injections of anti-α-MSH IgG induced a dose-dependent attenuation of food intake and body weight regain in MTX-treated rats accompanied by increased concentrations of α-MSH peptide which correlated positively with plasma levels of α-MSH autoantibodies. These data show that intestinal inflammation, independently from food restriction, affects general humoral immune response which may influence food intake and body weight control via modulation of α-MSH plasma concentration by α-MSH reactive autoantibodies.

Melanocortin 4 receptor activation induces brain-derived neurotrophic factor expression in rat astrocytes through cyclic AMP-protein kinase A pathway.

Melanocortin 4 receptors (MC4R) are mainly expressed in the brain. We previously showed that the anti-inflammatory action of α-melanocyte-stimulating hormone (α-MSH) in rat hypothalamus and in cultured astrocytes involved MC4R activation. However, MC4R mechanisms of action remain undetermined. Since brain-derived neurotrophic factor (BDNF) may be mediating MC4R hypothalamic anorexigenic actions, we determined melanocortin effects on BDNF expression in rat cultured astrocytes and certain mechanisms involved in MC4R signaling. α-MSH and its analogue NDP-MSH, induced production of cAMP in astrocytes. This effect was completely blocked by the MC4R antagonist, HS024. We found that NDP-MSH increased BDNF mRNA and protein levels in astrocytes. The effect of NDP-MSH on BDNF expression was abolished by the adenylate cyclase inhibitor SQ22536, and decreased by the PKA inhibitor Rp-cAMP. Since melanocortins are immunomodulators, we investigated their actions with bacterial lipopolysaccharide (LPS) and interferon-γ (IFN-γ) stimulus. Although both α-MSH and LPS+IFN-γ increased cAMP responding element binding protein (CREB) activation, LPS+IFN-γ did not modify BDNF expression. On the other hand, α-MSH did not modify basal or LPS+IFN-γ-induced nuclear factor-κB activation. Our results show for the first time that MC4R activation in astrocytes induces BDNF expression through cAMP-PKA-CREB pathway without involving NF-κB.

Inducible nitric oxide synthase (iNOS) and α-melanocyte-stimulating hormones of iNOS origin play important roles in the allergic reactions of atopic dermatitis in mice.

To elucidate the possible involvement of nitric oxide (NO) derived from inducible NO synthase (iNOS) in the pathogenesis of patients with allergic rhinitis, we used an animal model of atopic dermatitis (AD) induced by epicutaneous sensitization and analysed the differences in ear thickness, the frequency of scratching and plasma levels of ovalbumin-specific immunoglobulin E (OVA-IgE), transforming growth factor (TGF)-ß, tumor necrosis factor (TNF)-α, adrenocorticotropic hormone (ACTH) and α-melanocyte-stimulating hormone (α-MSH) between control and iNOS(-/-) mice. Eight-week-old control and iNOS(-/-) male C57BL/6j mice were sensitized three times with OVA antigen. Before and after the last skin sensitization, the number of scratching incidents and the thickness of the ear were examined, and the plasma levels of OVA-IgE, α-MSH, ACTH, TGF-ß and TNF-α were analysed by ELISA. Sensitization of mice with OVA resulted in increased plasma levels of OVA-IgE, α-MSH, ACTH, TGF-ß and TNF-α in control, but not in iNOS(-/-) mice. The administration of l-nitro-arginine-methyl ester (l-NAME) abolished all the above changes that occurred in the control mice. In addition, iNOS(-/-) mice given α-MSH exhibited a change similar to that seen in the control, whereas iNOS(-/-) mice given ACTH, TGF-ß or TNF-α did not demonstrate any changes. These results indicate that symptoms of AD such as scratching can be exacerbated by α-MSH, which is induced by iNOS-derived NO.

Functional studies on twenty novel naturally occurring melanocortin-4 receptor mutations.

The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor critically involved in regulating energy balance. MC4R activation results in decreased food intake and increased energy expenditure. Genetic and pharmacological studies demonstrated that the MC4R regulation of energy balance is conserved from fish to mammals. In humans, more than 150 naturally occurring mutations in the MC4R gene have been identified. Functional study of mutant MC4Rs is an important component in proving the causal link between MC4R mutation and obesity as well as the basis of personalized medicine. In this article, we studied 20 MC4R mutations that were either not characterized or not fully characterized. We showed that 11 mutants had decreased or absent cell surface expression. D126Y was defective in ligand binding. Three mutants were constitutively active but had decreased cell surface expression. Eleven mutants had decreased basal signaling, with two mutants defective only in this parameter, suggesting that impaired basal signaling might also be a cause of obesity. Five mutants had normal functions. In summary, we provided detailed functional data for further studies on identifying therapeutic approaches for personalized medicine to treat patients harboring these mutations.