Melanocortin MC Receptor Research Articles

P072 Melanocortin MC3 and MC5 receptors expression is different according to colonic disease activity in Crohn’s Disease and Ulcerative Colitis biopsies: results from an immunohistochemical observational study

Abstract Background MC3-R and MC5-R receptors belong to the complex melanocortin system, which has shown a strong immunomodulatory activity in several studies. Data on the role of these receptors in relation to inflammatory bowel disease are still missing and not fully elucidated. In this observational study we wanted to evaluate the expression profile of them in the colonic mucosa of Crohn’s Disease and Ulcerative Colitis patients both in the tracts directly affected by the disease activity and in the tracts spared. Methods We conducted on colonic biopsies of patients with histological diagnosis of inflammatory bowel disease immunohistochemical analyses in order to assess the degree of expression of MC3-R and MC5-R. Biopsies were performed on both the traits with macroscopic signs of inflammation and the macroscopically normal traits to assess whether the expression of the two receptors was different in relation to disease activity. Results In both Crohn’s disease and ulcerative colitis diseased tracts MC3-R (CD: +7.7 fold vs normal mucosa, P < 0.01; UC: +5.6 fold vs normal mucosa, P < 0.01) and MC5-R (CD: +5.5 fold vs normal mucosa, P < 0.01; UC: +8.1 fold vs normal mucosa, P < 0.01) were significantly increased compared to healthy mucosa. Conclusion MC3-R and MC5-R are expressed in the colon of patients with Crohn’s Disease and Ulcerative Colitis. In addition, expression is different with a higher degree of expression in the traits most affected by disease activity in both Crohn’s Disease and Ulcerative Colitis providing a potential use of these receptors in inflammatory bowel diseases pharmacology.

AB0068 NOVEL CHONDROPROTECTIVE AND ANTI-INFLAMMATORY EFFECTS OF THE SELECTIVE HUMAN MELANOCORTIN MC3 RECEPTOR AGONIST PG-990 ON SNAP ACTIVATED CHONDROCYTES

Background:Osteoarthritis (OA) is a degenerative joint disease that affects over 250 million people worldwide [1] with treatments focussing on the symptoms rather than the cause of the pathology [2, 3]. Thus, this degenerative joint disease requires novel treatment options [3, 4].Therefore, the melanocortin system [4] could provide a novel avenue to explore given its ability to exert anti-inflammatory effects and chondroprotection [5], although the receptor subtype involved is unclear.Objectives:This study aims to assess the chondroprotective and anti-inflammatory effects of the selective human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride and the selective human MC3 receptor agonist PG-990 on S-Nitroso-N-acetyl-DL-penicillamine (SNAP) activated chondrocytes.Methods:The human chondrocytic cell-line C-20/A4 was seeded at 25.0 x 106viable cells/ml (5 μl droplet was transferred into individual wells of a 96-well plate). Micromass cultures [6] were stimulated with SNAP (1.0 mM) and after 2h treated with Dexamethasone (1.0 μM), selective human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride (10.0 μg/ml) or selective human melanocortin MC3 receptor agonist PG-990 (10.0 μg/ml) for 6h. Cell viability was determined by MTT assay, Caspase -3 and -7 activity determined by Caspase-Glo 3/7 apoptosis assay. Glycosaminoglycan (GAG) content determined by alcian blue staining and anti-inflammatory heme-oxygenase-1 (HO-1) protein expression was determined by western blot. Data are expressed as Mean ±S.E.M ofn=4 samples repeated in triplicate. #p≤0.05vscontrol or *p≤0.05vsstimulus.Results:Cell viability analysis showed SNAP stimulation caused a maximal cell death of 23% (#p≤0.05), Dexamethasone, BMS-470539 dihydrochloride and PG-990 inhibited cell death by 2%, 98% and 129% respectively (*p≤0.05). SNAP stimulation caused a significant increase in Caspase -3 and -7 activity, which was inhibited by Dexamethasone, BMS-470539 dihydrochloride and PG-990 by 8%, 5% and 19% respectively (*p≤0.05). GAG content was significantly reduced by SNAP by 29% (#p≤0.05), which was inhibited by Dexamethasone, BMS-470539 dihydrochloride and PG-990 by 1%, 3% and 14% respectively (*p≤0.05). SNAP also caused a significant decrease in HO-1 protein expression, which was increased by Dexamethasone, BMS-470539 dihydrochloride and PG-990 by a 1.0-fold, 1.1-fold and 2.1-fold increase respectively (*p≤0.05).Conclusion:The selective human melanocortin MC3 receptor agonist PG-990 exhibited enhanced chondroprotection and modulation of inflammatory and tissue destructive mediators following SNAP activation compared to Dexamethasone and the selective human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride. This suggests that melanocortin peptides display enhanced chondroprotective and anti-inflammatory effects at the MC3 receptor sub-type in this cell line.

Melanocortin 3 receptor activation with [D-Trp8]-γ-MSH suppresses inflammation in apolipoprotein E deficient mice

The melanocortin MC1 and MC3 receptors elicit anti-inflammatory actions in leukocytes and activation of these receptors has been shown to alleviate arterial inflammation in experimental atherosclerosis. Thus, we aimed to investigate whether selective targeting of melanocortin MC3 receptor protects against atherosclerosis. Apolipoprotein E deficient (ApoE−/−) mice were fed high-fat diet for 12 weeks and randomly assigned to receive either vehicle (n = 11) or the selective melanocortin MC3 receptor agonist [D-Trp(8)]-gamma-melanocyte-stimulating hormone ([D-Trp8]-γ-MSH; 15 μg/day, n = 10) for the last 4 weeks. Lesion size as well as macrophage and collagen content in the aortic root plaques were determined. Furthermore, leukocyte counts in the blood and aorta and cytokine mRNA expression levels in the spleen, liver and aorta were quantified. No effect was observed in the body weight development or plasma cholesterol level between the two treatment groups. However, [D-Trp8]-γ-MSH treatment significantly reduced plasma levels of chemokine (C–C motif) ligands 2, 4 and 5. Likewise, cytokine and adhesion molecule expression levels were reduced in the spleen and liver of γ-MSH-treated mice, but not substantially in the aorta. In line with these findings, [D-Trp8]-γ-MSH treatment reduced leukocyte counts in the blood and aorta. Despite reduced inflammation, [D-Trp8]-γ-MSH did not change lesion size, macrophage content or collagen deposition of aortic root plaques. In conclusion, the findings indicate that selective activation of melanocortin MC3 receptor by [D-Trp8]-γ-MSH suppresses systemic and local inflammation and thereby also limits leukocyte accumulation in the aorta. However, the treatment was ineffective in reducing atherosclerotic plaque size.

Novel anti-inflammatory and chondroprotective effects of the human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride and human melanocortin MC3 receptor agonist PG-990 on lipopolysaccharide activated chondrocytes

Human melanocortin MC1 and MC3 receptors expressed on C-20/A4 chondrocytes exhibit chondroprotective and anti-inflammatory effects when activated by melanocortin peptides. Nearly 9 million people in the UK suffer from osteoarthritis, and bacterial infections play a role in its development. Here, we evaluate the effect of a panel of melanocortin peptides with different selectivity for human melanocortin MC1 (α-MSH, BMS-470539 dihydrochloride) and MC3 ([DTrp8]-γ-MSH, PG-990) receptors and C-terminal peptide α-MSH11-13(KPV), on inhibiting LPS-induced chondrocyte death, pro-inflammatory mediators and induction of anti-inflammatory proteins. C-20/A4 chondrocytes were treated with a panel of melanocortin peptides prophylactically and therapeutically in presence of LPS (0.1 μg/ml). The chondroprotective properties of these peptides determined by cell viability assay, RT-PCR, ELISA for detection of changes in inflammatory markers (IL-6, IL-8 and MMP-1, -3 and -13) and western blotting for expression of the anti-inflammatory protein heme-oxygenase-1. C-20/A4 expressed human melanocortin MC1 and MC3 receptors and melanocortin peptides elevated cAMP. LPS stimulation caused a reduction in C-20/A4 viability, attenuated by the human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride, and MC3 receptor agonists PG-990 and [DTrp8]-γ-MSH. Prophylactic and therapeutic regimes of [DTrp8]-γ-MSH significantly inhibited LPS-induced modulation of cartilage-damaging IL-6, IL-8, MMPs −1,-3 and −13 mediators both prophylactically and therapeutically, whilst human melanocortin MC1 and MC3 receptor agonists promoted an increase in HO-1 production. In the presence of LPS, activation of human melanocortin MC1 and MC3 receptors provided potent chondroprotection, upregulation of anti-inflammatory proteins and downregulation of inflammatory and proteolytic mediators involved in cartilage degradation, suggesting a new avenue for osteoarthritis treatment.

The MC4 receptor agonist RO27-3225 inhibits NLRP1-dependent neuronal pyroptosis via the ASK1/JNK/p38 MAPK pathway in a mouse model of intracerebral haemorrhage.

Inflammasome-mediated pyroptosis is an important neuronal cell death mechanism. Previous studies reported that activation of melanocortin MC4 receptor exerted neuroprotection in several neurological diseases. Here, we have investigated the role of MC4 receptor activation with RO27-3225 in suppressing neuronal pyroptosis after experimental intracerebral haemorrhage (ICH) and the underlying mechanism. One hundred and sixty-nine male CD1 mice were used. ICH was induced by injection of bacterial collagenase into the right-side basal ganglia. RO27-3225, a selective agonist of MC4 receptor, was injected intraperitoneally at 1hr after ICH. To elucidate the underlying mechanism, we used the specific MC4 receptor antagonist HS024 and NQDI-1, a specific inhibitor of the apoptosis signalling-regulating kinase 1 (ASK1). Neurological tests, Western blot, Fluoro-Jade C, TUNEL, and immunofluorescence staining were conducted. Expression of MC4 receptor and the NOD-like receptor family, pyrin domain containing 1 (NLRP1) inflammasome in brain were increased after ICH. RO27-3225 treatment decreased neuronal pyroptosis and neurobehavioural deficits at 24 and 72hr after ICH. RO27-3225 reduced the expression of p-ASK1, p-JNK, p-p38 MAPK, NLRP1 inflammasome, cleaved caspase-1, and IL-1β after ICH. HS024 pretreatment prevented the effects of RO27-3225. Similar to RO27-3225, NQDI-1 alone improved neurological functions and down-regulated ASK1/JNK/p38MAPK expression after ICH. RO27-3225 suppressed NLRP1-dependent neuronal pyroptosis and improved neurological function, possibly mediated by activation of MC4 receptor and inhibition of ASK1/JNK/p38 MAPK signalling pathways, after experimental ICH in mice. The MC4 receptor may be a promising therapeutic target for the management of ICH.

Knockdown of the transcript of ERK in the brain modulates hypothalamic neuropeptide-mediated appetite control in amphetamine-treated rats.

Amphetamine is a releaser of dopamine stored in synaptic terminals, which can suppress appetite by changing the expression levels of neuropeptide Y (NPY) and proopiomelanocortin (POMC) in the hypothalamus. This study explored whether ERKs are involved in appetite control mediated by cAMP response element binding protein (CREB), NPY and POMC in amphetamine-treated rats. Rats were given amphetamine for 4days, and changes in feeding behaviour and expression levels of phosphorylated-ERK (pERK), pCREB, NPY and melanocortin MC3 receptors were examined and compared. Following amphetamine treatment, food intake, body weight and NPY expression decreased, whereas the expression of pERK, pCREB, MC3 receptors and pCREB/DNA binding activity increased. In amphetamine-treated rats, both cerebral ERK knockdown and pretreatment with a peripheral dopamine receptor antagonist decreased NPY but increased pERK, pCREB and MC3 receptor expression. Moreover, the immunofluorescence of hypothalamic pERK increased following amphetamine treatment. These results suggest that ERK/CREB signalling participates in the effects mediated by dopamine receptor/NPY/POMC on appetite control in rats treated with amphetamine. These findings advance the knowledge on the involvement of ERK/CREB signalling in the reciprocal regulation by NPY and POMC of appetite after amphetamine treatment.

S.12.04 Melanocortin MC4 receptor agonists induce neuroprotection and neurogenesis in a transgenic mouse model of moderate Alzheimer's disease

S.12.04 Melanocortin MC4 receptor agonists induce neuroprotection and neurogenesis in a transgenic mouse model of moderate Alzheimer's disease

Transcriptional and Functional Characterization of the G Protein-Coupled Receptor Repertoire of Gastric Somatostatin Cells.

In the stomach, somatostatin (SST) acts as a general paracrine negative regulator of exocrine secretion of gastric acid and pepsinogen and endocrine secretion of gastrin, ghrelin, and histamine. Using reporter mice expressing red fluorescent protein (RFP) under control of the SST promotor, we have characterized the G protein-coupled receptors expressed in gastric Sst-RFP-positive cells and probed their effects on SST secretion in primary cell cultures. Surprisingly, besides SST, amylin and PYY were also highly enriched in the SST cells. Several receptors found to regulate SST secretion were highly expressed and/or enriched. 1) The metabolite receptors calcium-sensing receptor and free fatty acid receptor 4 (GPR120) functioned as positive and negative regulators, respectively. 2) Among the neurotransmitter receptors, adrenergic receptors α1a, α2a, α2b, and β1 were all highly expressed, with norepinephrine and isoproterenol acting as positive regulators. The muscarinic receptor M3 acted as a positive regulator, whereas M4 was conceivably a negative regulator. 3) Of the hormone receptors, the GLP-1 and GIP receptors, CCKb (stimulated by both CCK and gastrin) and surprisingly the melanocortin MC1 receptor were all positive regulators. 4) The neuropeptide receptors for calcitonin gene-related peptide, adrenomedullin, and vasoactive intestinal peptide acted as positive regulators, no effect was observed using galanin and nociceptin although transcripts for the corresponding receptors appeared highly expressed. 5) The SST receptors 1 and 2 functioned in an autocrine negative feedback loop. Thus, the article provides a comprehensive map of receptors through which SST secretion is regulated by hormones, neurotransmitters, neuropeptides and metabolites that act directly on the SST cells in the gastric mucosa.

NDP-α-MSH induces intense neurogenesis and cognitive recovery in Alzheimer transgenic mice through activation of melanocortin MC4 receptors.

Melanocortins exert neuroprotection in a variety of experimental neurodegenerative disorders, including Alzheimer's disease (AD). Further, in previous research we showed that these endogenous peptides stimulate neurogenesis in an acute neurodegenerative disorder such as ischemic stroke. In the present research, we investigated the potential neurogenic effect of melanocortins in AD using APPSwe transgenic mice (Tg2576). To this purpose, 24week-old animals were prepared for 5-bromo-2'-deoxyuridine (BrdU) labeling of proliferating cells on days 1-11 of the study. Treatment of Tg2576 mice with nanomolar doses of the melanocortin analog [Nle(4),D-Phe(7)]α-melanocyte-stimulating hormone (NDP-α-MSH), administered once daily from day 1 to 50, improved brain histology and cognitive functions relative to saline-treated Tg2576 animals. No signs of toxicity were observed. Immunohistochemical examination of the hippocampus at the end of the study (day 50) showed that NDP-α-MSH-treated Tg2576 mice had a greater number of BrdU immunoreactive cells colocalized with NeuN (an indicator of mature neurons) and Zif268 (an indicator of functionally integrated neurons) in the dentate gyrus, relative to saline-treated Tg2576 animals; no newly formed astrocytes were found. Animal pretreatment with the selective melanocortin MC4 receptor antagonist HS024 before each NDP-α-MSH administration prevented all the beneficial effects of the peptide. The present data indicate that MC4 receptor stimulation by a melanocortin prevents cognitive decline in experimental AD, this effect being associated not only with neuroprotection but also with an intense neurogenesis. MC4 receptor agonists could be innovative and safe candidates to counteract AD progression in humans.

Melanocortin MC4 receptor agonists alleviate brain damage in abdominal compartment syndrome in the rat

Intra-abdominal hypertension (IAH) is accompanied by high morbidity and mortality in surgical departments and ICUs. However, its specific pathophysiology is unclear. IAH not only leads to intra-abdominal tissue damage but also causes dysfunction in distal organs, such as the brain. In this study, we explore the protective effects of melanocortin 4 receptor agonists in IAH-induced brain injury. The IAH rat models were induced by hemorrhagic shock/resuscitation (with the mean arterial pressure (MAP) maintained at 30 mm Hg for 90 min followed by the reinfusion of the withdrawn blood with lactated Ringer's solution). Then, air was injected into the peritoneal cavity of the rats to maintain an intra-abdominal pressure of 20 mm Hg for 4 h. The effects of the melanocortin 4 receptor agonist RO27-3225 in alleviating the rats' IAH brain injuries were observed, which indicated that RO27-3225 could reduce brain edema, the expressions of the IL-1β and TNF-α inflammatory cytokines, the blood–brain barrier's permeability and the aquaporin4 (AQP4) and matrix metalloproteinase 9 (MMP9) levels. Moreover, the nicotinic acetylcholine receptor antagonist chlorisondamine and the selective melanocortin 4 receptor antagonist HS024 can negate the protective effects of the RO27-3225. The MC4R agonist can effectively reduce the intracerebral proinflammatory cytokine gene expression and alleviate the brain injury caused by blood–brain barrier damage following IAH.

Melanocortins protect against brain damage and counteract cognitive decline in a transgenic mouse model of moderate Alzheimer׳s disease

We previously reported that melanocortins induce neuroprotection in experimental acute and chronic neurodegenerative conditions, including Alzheimer׳s disease (AD) of mild severity. Here we investigated whether melanocortins afford neuroprotection and counteract cognitive decline in AD with a medium level of severity by using 24 week-old (at the start of the study) APPSwe transgenic mice (Tg2576). Saline-treated (days 1–50) control Tg2576 mice showed an impairment in spatial learning and memory, associated (at day 50, end of the study) with hippocampus at low levels of the synaptic activity-dependent gene Zif268, relevant brain changes such as cerebral cortex/hippocampus increased level of β-amyloid (Aβ) deposit, and neuronal loss, in comparison with wild-type animals. Treatment of Tg2576 mice (once daily at days 1–50) with a nanomolar dose of the melanocortin analog [Nle4,D-Phe7]α-melanocyte-stimulating hormone (NDP-α-MSH) reduced cerebral cortex/hippocampus level of Aβ deposit, decreased neuronal loss, increased hippocampus Zif268 expression and improved cognitive functions, relative to saline-treated Tg2576 mice. Pharmacological blockade of melanocortin MC4 receptors with the MC4 receptor antagonist HS024 prevented all favorable effects of NDP-α-MSH. Our data indicate that MC4 receptor-stimulating melanocortins are able to counteract cognitive decline in experimental AD of medium severity through induction of neuroprotection and improvement of synaptic transmission. After further studies, these agents could gain a role as disease modifying therapeutics for AD.

Involvement of hypothalamic PI3K–STAT3 signalling in regulating appetite suppression mediated by amphetamine

Appetite suppression induced by amphetamine has been attributed to its inhibition of neuropeptide Y (NPY) neurons and activation of pro-opiomelanocortin (POMC) neurons in the hypothalamus. This study examined whether STAT3 was involved in these actions of amphetamine. Rats were given amphetamine daily for 4 days. Changes in the expression of NPY, POMC, melanocortin MC3 receptors, PI3K and STAT3 in the hypothalamus were assessed by RT-PCR and Western blotting. Antisense oligonucleotides to STAT3 were also used. Expression of NPY decreased with a maximum effect day 2 of amphetamine treatment. Expression of POMC, MC3 receptors, PI3K and STAT3 increased with a maximum response on day 2. Moreover, phosphorylation of STAT3 and its DNA binding activity increased and was expressed in a similar pattern. Infusion (i.c.v.) of STAT3 antisense at 60 min before amphetamine treatment, partly blocked amphetamine-induced anorexia and modulated expression of NPY, POMC, MC3 receptors and PI3K, indicating the involvement of STAT3 in amphetamine-treated rats. Hypothalamic PI3K-STAT3 signalling participated in the regulation of NPY- and POMC-mediated appetite suppression. These findings may contribute to a better understanding of anorectic drugs.

Melanocortins protect against progression of Alzheimer's disease in triple-transgenic mice by targeting multiple pathophysiological pathways

Besides specific triggering causes, Alzheimer's disease (AD) involves pathophysiological pathways that are common to acute and chronic neurodegenerative disorders. Melanocortins induce neuroprotection in experimental acute neurodegenerative conditions, and low melanocortin levels have been found in occasional studies performed in AD-type dementia patients. Here we investigated the possible neuroprotective role of melanocortins in a chronic neurodegenerative disorder, AD, by using 12-week-old (at the start of the study) triple-transgenic (3xTg-AD) mice harboring human transgenes APPSwe, PS1M146V, and tauP301L. Treatment of 3xTg-AD mice, once daily until the end of the study (30 weeks of age), with the melanocortin analog [Nle4,D-Phe7]-α-melanocyte-stimulating hormone (NDP-α-MSH) reduced cerebral cortex/hippocampus phosphorylation/level of all AD-related biomarkers investigated (mediators of amyloid/tau cascade, oxidative/nitrosative stress, inflammation, apoptosis), decreased neuronal loss, induced over-expression of the synaptic activity–dependent gene Zif268, and improved cognitive functions, relative to saline-treated 3xTg-AD mice. Pharmacological blockade of melanocortin MC4 receptors prevented all neuroprotective effects of NDP-α-MSH. Our study identifies, for the first time, a class of drugs, MC4 receptor-stimulating melanocortins, that are able to counteract the progression of experimental AD by targeting pathophysiological mechanisms up- and down-stream of β-amyloid and tau. These data could have important clinical implications.

Repeated agouti related peptide (83–132) injections inhibit cocaine-induced locomotor sensitisation, but not via the nucleus accumbens

Drug addiction is a chronic relapsing brain disease for which many of the underlying neuronal mechanisms are yet to be unravelled. There seems to be an interaction between the melanocortin system and drugs of abuse. For instance, infusion of the melanocortin MC4 receptor antagonist SHU9119 (Ac-Nle-cyclo(-Asp-His-d-2-Nal-Arg-Trp-Lys)-NH2) into the nucleus accumbens results in conditioned place avoidance, reduces the amount of lever presses for cocaine and blocks development of cocaine-induced locomotor sensitisation.The aim of this study is to determine whether the induction of locomotor sensitisation to repeated cocaine is inhibited by the melanocortin MC4 receptor inverse agonist Agouti Related Peptide (AgRP83–132).Rats were sensitised to daily cocaine injections for 5 consecutive days and 30min prior to every daily cocaine injection, rats received an intracerebroventricular (i.c.v.) or intra nucleus accumbens injection with AgRP(83–132) or saline, to determine whether we could inhibit cocaine-induced locomotor sensitisation. We show that i.c.v. injections of AgRP(83–132) inhibit cocaine-induced locomotor sensitisation. This effect is not regulated via the nucleus accumbens, since injecting the melanocortin receptor inverse agonist AgRP(83–132) directly into the nucleus accumbens was unable to inhibit the cocaine-induced locomotor sensitisation. This implicates that the nucleus accumbens is an unlikely site to inhibit the induction of locomotor sensitisation via the melanocortin MC4 receptor. This is in contrast to other studies that show an effect of the melanocortin MC4 receptor antagonist SHU9119 on locomotor sensitisation when injected into the nucleus accumbens.

Up-regulation of the canonical Wnt-3A and Sonic hedgehog signaling underlies melanocortin-induced neurogenesis after cerebral ischemia

In experimental cerebral ischemia, melanocortin MC4 receptor agonists induce neuroprotection and neurogenesis with subsequent long-lasting functional recovery. Here we investigated the molecular mechanisms underlying melanocortin-induced neurogenesis. Gerbils were subjected to transient global cerebral ischemia, then they were treated every 12h, and until sacrifice, with 5-bromo-2′-deoxyuridine (BrdU; to label proliferating cells), and the melanocortin analog [Nle4,d-Phe7]α-melanocyte-stimulating hormone (NDP-α-MSH) or saline. NDP-α-MSH increased hippocampus dentate gyrus (DG) expression of Wnt-3A, β-catenin, Sonic hedgehog (Shh), Zif268, interleukin-10 (IL-10) and doublecortin (DCX), as detected at days 3, 6 and 10 after the ischemic insult. Further, an elevated number of BrdU immunoreactive cells was found at days 3 and 10, and an improved histological picture with reduced neuronal loss at day 10, associated with learning and memory recovery. Pharmacological blockade of the Wnt-3A/β-catenin and Shh pathways, as well as of melanocortin MC4 receptors, prevented all effects of NDP-α-MSH. These data indicate that, in experimental brain ischemia, treatment with melanocortins acting at MC4 receptors induces neural stem/progenitor cell proliferation in the DG by promptly and effectively triggering the canonical Wnt-3A/β-catenin and Shh signaling pathways. Activation of these pathways is associated with up-regulation of the repair factor Zif268 and the neurogenesis facilitating factor IL-10, and it seems to address mainly toward a neuronal fate, as indicated by the increase in DCX positive cells.

Protective effects of melanocortins on short-term changes in a rat model of traumatic brain injury*.

Treatment for traumatic brain injury remains elusive despite compelling evidence from animal models for a variety of therapeutic targets. Melanocortins have established neuroprotective effects against experimental ischemic stroke. We investigated whether melanocortin treatment of traumatic brain injury induces neuroprotection and promotes functional recovery. Randomized experiment. Research laboratory at a university hospital. Male Sprague-Dawley rats (n = 215). Experimental rat model of diffuse traumatic brain injury, the impact-acceleration model. Brain tissue nitrites, phosphorylation level of extracellular signal-regulated kinases, and c-jun N-terminal kinases; and expression of active caspase-3, tumor necrosis factor-α, BAX, and Bcl-2 as well as serum levels of interleukin-6, high mobility group box-1, interleukin-10, and brain histologic damage were evaluated 24 or 48 hrs after the insult. Sensorimotor orientation and limb use were evaluated at day 7 and learning and memory at days 23-30 after injury. Posttraumatic treatment every 12 hrs with the melanocortin analog [Nle, D-Phe]-α-melanocyte-stimulating hormone (starting 3 or 6 hrs after injury) inhibited traumatic brain injury-induced upregulation of nitric oxide synthesis, phosphorylation level of extracellular signal-regulated kinases, phosphorylation level of c-jun N-terminal kinases, and active caspase-3; reduced expressions/levels of tumor necrosis factor-α, BAX, interleukin-6, and high mobility group box-1; and increased those of Bcl-2 and interleukin-10. These molecular changes were associated with a reduction in brain tissue damage, as highlighted by histopathological findings and improved functional recovery. Pretreatment with the melanocortin MC4 receptor antagonist HS024 abated the positive effects of [Nle, D-Phe]-α-melanocyte-stimulating hormone. Our data indicate that melanocortins protect against traumatic brain injury, in a broad time window and through activation of MC4 receptors, by counteracting the main traumatic brain injury-related mechanisms of damage. These findings could have major clinical implications.

Melanocortin MC4 receptor agonists counteract late inflammatory and apoptotic responses and improve neuronal functionality after cerebral ischemia

Indirect evidence indicates that, in cerebral ischemia, melanocortins have neuroprotective effects likely mediated by MC₄ receptors. To gain direct insight into the role of melanocortin MC₄ receptors in ischemic stroke, we investigated the effects of a highly selective MC₄ receptor agonist. Gerbils were subjected to transient global cerebral ischemia by occluding both common carotid arteries for 10 min. In saline-treated stroke animals, an impairment in learning and memory occurred that, at day 11 after stroke, was associated with hippocampus up-regulation of tumor necrosis factor-α (TNF-α), BAX, activated extracellular signal-regulated kinases (ERK1/2), c-jun N-terminal kinases (JNK1/2) and caspase-3, down-regulation of Bcl-2, and neuronal loss. Treatment for 11days with the selective melanocortin MC₄ receptor agonist RO27-3225, as well as with the well known non-selective [Nle⁴,D-Phe⁷]α-melanocyte-stimulating hormone (NDP-α-MSH) as a reference non-selective melanocortin, counteracted the inflammatory and apoptotic responses, as indicated by the changes in TNF-α, BAX, ERK1/2, JNK1/2, caspase-3 and Bcl-2 protein expression. Furthermore, melanocortin treatment reduced neuronal loss and dose-dependently improved learning and memory. These positive effects were associated with overexpression of Zif268, an immediate early gene involved in injury repair, synaptic plasticity and memory formation. Pharmacological blockade of MC₄ receptors with the selective MC₄ receptor antagonist HS024 prevented all effects of RO27-3225 and NDP-α-MSH. These data give direct evidence that stimulation of MC₄ receptors affords neuroprotection and promotes functional recovery from stroke, by counteracting prolonged and/or recurrent inflammatory and apoptotic responses, and likely by triggering brain repair pathways.

Journal of Diabetes NEWS

Journal of Diabetes NEWS

Melanocortins protect against multiple organ dysfunction syndrome in mice.

Melanocortins reverse circulatory shock and improve survival by counteracting the systemic inflammatory response, and through the activation of the vagus nerve-mediated cholinergic anti-inflammatory pathway. To gain insight into the potential therapeutic value of melanocortins against multiple organ damage following systemic inflammatory response, here we investigated the effects of the melanocortin analogue [Nle⁴ D-Phe⁷]α-MSH (NDP-α-MSH) in a widely used murine model of multiple organ dysfunction syndrome (MODS). MODS was induced in mice by a single intraperitoneal injection of lipopolysaccharide followed, 6 days later (= day 0), by zymosan. After MODS or sham MODS induction, animals were randomized to receive intraperitoneally NDP-α-MSH (340 µg·kg⁻¹ day) or saline for up to 16 days. Additional groups of MODS mice were concomitantly treated with the melanocortin MC₄ receptor antagonist HS024, or the nicotinic acetylcholine receptor antagonist chlorisondamine, and NDP-α-MSH. At day 7, in the liver and lung NDP-α-MSH, significantly reduced mRNA expression of tumour necrosis factor-α (TNF-α), increased mRNA expression of interleukin-10 and improved the histological picture, as well as reduced TNF-α plasma levels; furthermore, NDP-α-MSH dose-dependently increased survival rate, as assessed throughout the 16 day observation period. HS024 and chlorisondamine prevented all the beneficial effects of NDP-α-MSH in MODS mice. These data indicate that NDP-α-MSH protects against experimental MODS by counteracting the systemic inflammatory response, probably through brain MC₄ receptor-triggered activation of the cholinergic anti-inflammatory pathway. These findings reveal previously undescribed effects of melanocortins and could have clinical relevance in the MODS setting.

Melanocortin activity in the amygdala influences alcohol intake

Melanocortins have been reported to affect alcohol intake through actions in the hypothalamus thought to be mediated by melanocortin MC4 receptors. Since these receptors are expressed in a number of amygdala regions, we have explored their role in the regulation of alcohol intake in both alcohol-preferring (P) and non-preferring (NP) rats. Injections were made at the border of the central amygdala nucleus and the basolateral amygdala. The MC3/MC4R agonist MTII reduced alcohol and food intake but increased water intake while the selective MC4R antagonist HS014 only increased food and water intake. The MC3/MC4R antagonist SHU9119 increased food and water but had little effect on alcohol intake. However, when the SHU9119 stimulation of food intake was prevented by pair-feeding, SHU9119 induced a large and prolonged decline in alcohol intake that was paralleled by an increase in water intake. These effects were only observed in P rats. We conclude that melanocortin activity in the amygdala can alter the selective preference for water and alcohol independent of effects on food intake.