Effects Of α-MSH Research Articles

The effects of α-MSH on epidermal γδ T cell activation and function

Abstract The hair follicle is a site of immune privilege that, when disrupted, results in the autoimmune disorder alopecia areata. Although there are a variety of cell types that reside near the hair follicle and may be involved in the initiation of alopecia areata, few studies have focused on γδ T cells. γδ T cells are resident in epithelial tissues where they normally play roles in inflammation and wound healing. Alpha-melanocyte-stimulating(α-MSH) is an immunoregulator locally produced within the hair follicle. α-MSH ensures the immune system is suppressed during anagen phase (growth phase) and restored during catagen phase (resting phase). The effects of α-MSH on epidermal γδ T cells via MC1R, one of five melanocortin receptors, is unknown. We therefore purified primary epidermal γδ T cells to investigate the immunomodulatory effects of α-MSH. Flow cytometry and the Luminex platform were used to compare changes in MC1R expression levels and cytokine production in response to α-MSH. Here we show that resting epidermal γδ T cells express MC1R, and TCR stimulation increases MC1R expression. Upregulation of MC1R expression is augmented further by stimulation with α-MSH. Epidermal γδ T cells significantly upregulate immunostimulatory cytokines when stimulated with anti-CD3, but cytokine production did not significantly change with the addition of α-MSH. Additional findings suggest that α-MSH instead plays a role in MC1R regulated pathways such as DNA repair and cell survival. This study begins to delineate the mechanisms by which α-MSH impacts γδ T cell function and leads to the disruption of immune privilege that occurs in alopecia areata.

Melanocortin-4 receptor regulation of reproductive function in black rockfish (Sebastes schlegelii).

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor with multiple functions in mammals. However, the functions of MC4R in fish have not been investigated extensively. The purpose of this study was to determine potential regulation of reproduction by the MC4R. We cloned the black rockfish MC4R and analyzed its tissue distribution and function. The results showed that black rockfish mc4r cDNA consisted of 981 nucleotides encoding a protein of 326 amino acids. The quantitative PCR data showed that mc4r mRNA was primarily expressed in the brain, gonad, stomach and intestine. In the brain, mc4r was found to be primarily located in the hypothalamus. Both α-MSH and β-MSH increased gnih expression and decreased sgnrh and cgnrh expression (P<0.05). α-MSH and β-MSH had opposite effects on kisspeptin expression. In contrast, α-MSH and β-MSH increased the expression of cyp11, cyp19, 3β-hsd and star. In summary, our study shows that MC4R in black rockfish might regulate reproductive function and that the effects of α-MSH and β-MSH might differ.

Альфа-меланоцитстимулирующий гормон как регулятор гипоталамо-гипофизарной оси

Alpha-melanocyte-stimulating hormone (α-MSH) has a wide range of biological activities and is produced in the pituitary, hypothalamus, brain stem, hippocampus and periphery. α-MSH and its receptors are critically involved in the regulation of energy balance and body weight. At the functional and neuroanatomical levels, there is a very close overlap between the regulation of energy balance and neuroendocrine stress response. The review focuses on the involvement of α-MSH in the regulation of the hypothalamic-pituitary (HP) axis. The effects of centrally administered α-MSH and its analogues, and the data obtained from genetically altered animals indicate that α-MSH from the arcuate nucleus both directly and indirectly, via the medial amygdala, activates the HP axis. Since the amygdala plays a crucial role in the integration of behavioral and neuroendocrine responses to stress, it is obvious that α-MSH is an important participant in this process. The role of α-MSH in the regulation of the HP axis by other limbic structures, such as the hippocampus and prefrontal cortex, remains unexplored. The melanocortin MC4R receptor plays the main role in the activation of the HP axis. However, some data indicate the possible involvement of MC3R or MC5R receptors. The well-known anti-inflammatory effects of α-MSH are consistent with its ability to attenuate neuroinflammation-induced activation of the HP axis. Like adrenocorticotropic hormone (ACTH), pituitary α-MSH is secreted into the bloodstream during the stress response. However, unlike ACTH, α-MSH secretion is stimulated by epinephrine, and not inhibited by glucocorticoids. The role of circulating α-MSH in the stress response remains unclear, but may involve glucocorticoid-independent negative regulation of the HP axis.

2009-P: Molecular Mechanisms of Obesity-Associated Vascular Vulnerability via Signals of Appetite Regulation

Background: Obesity is a risk factor of hypertension and abdominal aortic aneurysm (AAA). Melanocortin 4 receptor (MC4R) is activated by α-melanocyte-stimulating hormone (α-MSH) to mediate most of the anorectic effects of leptin. In humans, deficiency or mutation of MC4R is the most common monogenic form of obesity. However, it remains unknown whether MC4R signaling affects the development of obesity-associated hypertension, vascular vulnerability and aortic aneurysm. Methods and Results: MC4R-knockout (KO) mice fed a high-fat/high-sucrose diet (HF/HS) for 20 weeks displayed obesity associated with hyperleptinemia compared to HF/HS-fed wild type (WT) mice. MC4R-KO mice exhibited hypertension, increase of medial thickness and elastic lamina destruction, and elevation of inflammatory and fibrosis-related genes including TGF-β and osteopontin (OPN) in aortae. MC4R-KO mice showed higher incidence of angiotensin II (Ang II, 500 ng/kg/min)-induced AAA (73%) than WT mice (8%). MC4R-KO mice with ob/ob background exhibited lower incidence of Ang II-induced AAA (50%) than MC4R-KO mice, but higher than ob/ob mice (12.5%). Treatment of OPN-blocking antibody decreased Ang II-induced AAA incidence in HF/HS-fed MC4R-KO mice. In vascular smooth muscle cells (VSMCs), leptin (100 ng/ml) significantly upregulated OPN gene expression. α-MSH suppressed AngII-induced TGF-β gene expression in VSMCs, and LPS/IFN-γ-induced iNOS gene expression in peritoneal macrophages. However, these effects of α-MSH were abolished in primary macrophages from MC4R-KO mice and RAW264.7 cells pretreated with MC4R antagonist HS024. Conclusion: Our observation indicates that deficiency of MC4R promotes the vascular vulnerability in mice. It suggests that, not only hyperleptinemia, but also impairment of α-MSH-MC4R signaling in VSMCs and macrophages, may also promote the vascular vulnerability. Disclosure K. Mori: None. K. Tsuchiya: None. S. Nakamura: None. K. Shiba: None. Y. Miyachi: None. Y. Ogawa: None. K. Kitamura: None.

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

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. 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.

Combinatorial protective effects of α-melanocyte-stimulating hormone and carboxymethylcellulose on ocular surface in dry eye rat models

Background Reasearches showed that α-melanocyte-stimulating hormone (α-MSH) inhibits inflammation and ameliorates the ocular surface abnormalities in a scopolamine-induced dry eye rat model, and the managing effect of sodium carboxymethylcellulose (CMC) on dry eyes also has been determined.However, whether α-MSH can enhance the therapeutic effects of CMC remains to be investigated. Objective This study was to investigate the protective effects of α-MSH combined with CMC on ocular surface in a scopolamine-induced dry eye rat model. Methods Sixty clean female Wistar rats were randomly divided into normal control group, model control group, NaCl group, CMC group, α-MSH group and α-MSH+ CMC group, and 10 rats for each group.The dry eye models were established by subcutaneous injection of scopolamine hydrobromide at 9: 00, 12: 00, 15: 00 and 18: 00 per day for 28 days.0.9% NaCl solution, 1×10-3 mg/ml α-MSH solution, 0.5% CMC eye drop, and 1×10-3 mg/ml α-MSH+ 0.5% CMC solution were topically administered twice a day (8: 00, 17: 00) since the initial day of modeling according to grouping.Shirmer Ⅰ test (SⅠt), breakup time of tear film (BUT) and corneal fluorescence staining were performed before and 7, 14, 21, 28 days after the application of drugs.At 28 days following the administration of drugs, the eyeballs of the rats were collected.Hemotoxylin and eosin staining was employed to examine the morphology of corneas, and periodic acid schiff (PAS) staining was used to count the conjunctival goblet cells.This study protocol was approved by Experimental Animal Ethic Committee of Tianjin Medical University(SYXK 2009-0001), and the use and care of the rats complied with ARVO Statement. Results The SⅠt and BUT values were significantly reduced, and the corneal fluorescence staining scores were significantly increased over time following modeling in the model control group (all at P 0.05). At 7, 14 and 21 days after intervention, the SⅠt values were (4.800±0.789), (4.100±0.516) and (4.300±0.856)mm in the α-MSH+ CMC group, which were considerably higher than (2.875±0.719), (2.375±0.619) and (2.532±0.957)mm in the NaCl group (all at P<0.01). At 7 days after intervention, the BUT values were (4.938±1.843) seconds and (5.000±1.491) seconds in the α-MSH group and α-MSH+ CMC group, which were significantly higher than (3.250±1.000) seconds in the NaCl group (both at P<0.01). The corneal fluorescence staining scores in the CMC group, α-MSH group and α-MSH+ CMC group were significantly lower than that in the NaCl group, with the lowest score in the α-MSH+ CMC group (all at P<0.05). The thickening of corneal epithelial layer, corneal edema and arrangement disorder of corneal stroma were found in the model control group and NaCl group; while slight corneal edema and epithelial cell proliferation were exhibited in the α-MSH+ CMC group by hemotoxylin and eosin staining.PAS staining showed that the number of goblet cells was much more in the CMC group, α-MSH group and α-MSH+ CMC group than that in the model control group and NaCl group (all at P<0.01). Conclusions The sole application of α-MSH or CMC alleviates ocular surface damage and morphological abnormality to certain extent, and the combination of α-MSH and CMC generates more effective protection in comparison with sole administration of α-MSH or CMC.The early application of the drugs plays an improvement role in tear secretion and tear film stability in dry eyes. Key words: Dry eyes/drug therapeutic; Ocular surface; Protection; Sodium carboxymethylcellulose; α-Melanocyte-stimulating hormone; Disease models; Rats, Wistar

The melanocortin system regulates body pigmentation and social behaviour in a colour polymorphic cichlid fish.

The melanocortin system is a neuroendocrine system that regulates a range of physiological and behavioural processes. We examined the extent to which the melanocortin system simultaneously regulates colour and behaviour in the cichlid fish Astatotilapia burtoni We found that yellow males are more aggressive than blue males, in line with previous studies. We then found that exogenous α-melanocyte-stimulating hormone (α-MSH) increases yellowness of the body and dispersal of xanthophore pigments in both morphs. However, α-MSH had a morph-specific effect on aggression, with only blue males showing an increase in the rate of aggression. Exogenous agouti signalling peptide (ASIP), a melanocortin antagonist, did not affect coloration but reduced the rate of aggression in both colour morphs. Blue males had higher cortisol levels than yellow males. Neural gene expression of melanocortin receptors (mcr) and ligands was not differentially regulated between colour morphs. In the skin, however, mc1r and pro-opiomelanocortin (pomc) β were upregulated in blue males, while asip 1 was upregulated in yellow males. The effects of α-MSH on behaviour and body coloration, combined with morph-specific regulation of the stress response and the melanocortin system, suggest that the melanocortin system contributes to the polymorphism in behaviour and coloration in A. burtoni.

αMSH promotes preadipocyte proliferation by alleviating ER stress-induced leptin resistance and by activating Notch1 signal in mice

Alpha-melanocyte stimulating hormone (αMSH) has an important role in the regulation of body weight and energy expenditure. Nevertheless, the molecular mechanisms of circulating αMSH on preadipocyte proliferation remain elusive. We found αMSH was reduced by high fat diet (HFD) while leptin was elevated in adipose tissue. Leptin resistance and endoplasmic reticulum (ER) stress of adipose tissue were increased in obese mice. αMSH increased leptin sensitivity and alleviated ER stress along with increased p-STAT3 level and reduced SOCS3, GRP78, CHOP, ATF4, p27 and p53 levels. αMSH and leptin co-treatment alleviated ER stress through decreasing the levels of GRP78 and CHOP. Tunicamycin (TM) or thapsigargin (Tg) induced ER stress blunted leptin sensitivity and inhibited preadipocyte proliferation. αMSH and leptin co-treatment increased the cell number, augmented G1-S transition, elevated leptin sensitivity, and reduced ER stress; it also activated Notch1 signal and stimulated preadipocyte proliferation, whereas ER stress marker genes were decreased during this process. However, the effects of αMSH and leptin were blocked by the specific inhibitor of Notch1 signal. In summary, our data revealed αMSH enhanced leptin sensitivity and preadipocyte proliferation, meanwhile inhibited ER stress of preadipocytes by activating Notch1 signal.

Alleviating effects of α-melanocyte-stimulating hormone on retinal vessel leakage in diabetic rats

Background The primary pathogenic basis of diabetic retinopathy (DR) is microangiopathy and inflammatory procedure. Studies showed that α-melanocyte stimulating hormone (α-MSH) can play anti-oxidative stress and anti-apoptosis effects on retina via intravitreal injection and therefore mend blood-retinal barrier (BRB). However, experiment has not yet proved its mechanism. Objective This study was to investigate the protective effects of α-MSH on retinal vessel in diabetic rats. Methods Ninety clean SD rats were assigned to the normal control group, diabetes mellitus (DM) model group and α-MSH group. DM models were established by streptozocin (STZ) injection via tail vein in the rats of the DM model group and the α-MSH group, and sodium citrate buffer was injected in the same way in the normal control group. α-MSH of 3 μl (3.3 μg/μl) was intravitreally injected 1 week and 3 weeks after modeling in the rats of the α-MSH group, and normal saline solution was used in the same way in the normal control group; while no operation was performed in the DM model group. In the fifth week after modeling, Evans blue at the dose of 30 mg/ml (45 mg/kg) was injected via tail vein of rats, and retinal mount was prepared 2 hours later to examine the morphology of retinal vessels under the fluorescent microscope. The blood samples were collected from inner canthus vein for the assessment of leakage amount of Evans blue. The rat retinas were isolated after perfusion of sodium citrate buffer via the left ventricle for the observation of choroidal and retinal vessel ultrastructure by transmission electron microscopy (TEM). The relative expressions of intercellular cell adhesion molecule-1 (ICAM-1) mRNA, tumor necrosis factor-α (TNF-α), occluding mRNA and vascular endothelial growth factor (VEGF) mRNA were detected by quantitative real-time PCR. Results The body weight was lighter and the water intake was higher in the DM rats than those in the normal control rats. The blood glucose levels in the DM model group were (29.69±4.77) mmol/L at the third day and (24.64±2.72) mmol/L in the fifth week following the injection of STZ, respectively. An amount of Evans blue leaked from vessels in the rat retina of DM model group, but the retinal vessels were intact in the rats of the α-MSH group and the normal control group. The leakage amount was (10.04±8.18)μl/(g·h) in the DM model group, which was significantly more than that of (2.62±3.73)μl/(g·h) in the α-MSH group and (3.10±1.13)μl/(g·h) in the normal control group at the fifth week after modeling (P=0.035, 0.041). The ultrastructure of choriodal and retinal vessels was almost normal in the α-MSH group and the normal control group, but the swelling of choriodal vascular endothelial cells and vacuolar-like changes of the retinal pigment epithelium (RPE) were found in the DM model group. In addition, the relative expression levels of ICAM-1 mRNA and TNF-α mRNA were significantly elevated, but those of occludin mRNA were significantly declined in the DM model group compared with the α-MSH group and the normal control group (all at P<0.05). No significant difference was found in the relative expression level of VEGF mRNA among the three groups (F=0.791, P=0.466). Conclusions α-MSH ameliorates retinal vascular leakage in STZ-induced diabetic rats after intravitreal injection by improving ultrastructure of choroid and retinal vessels, down-regulating the expression of proinflammatory factors and up-regulating tight junction component in retina. Key words: α-Melanocyte-stimulating hormone; Diabetic retinopathy/therapy; Intercellular adhesion molecule-1; Tumor necrosis factor-α; Occludin; Vascular endothelial growth factor; Blood-retina barrier; Rats, Sprague Dawley

Central melanocortins regulate the motivation for sucrose reward.

The role of the melanocortin (MC) system in feeding behavior is well established. Food intake is potently suppressed by central infusion of the MC 3/4 receptor agonist α-melanocyte stimulating hormone (α-MSH), whereas the MC 3/4 receptor inverse-agonist Agouti Related Peptide (AGRP) has the opposite effect. MC receptors are widely expressed in both hypothalamic and extra-hypothalamic brain regions, including nuclei involved in food reward and motivation, such as the nucleus accumbens (NAc) and the ventral tegmental area. This suggests that MCs modulate motivational aspects of food intake. To test this hypothesis, rats were injected intracerebroventricularly with α-MSH or AGRP and their motivation for sucrose was tested under a progressive ratio schedule of reinforcement. Food motivated behavior was dose-dependently decreased by α-MSH. Conversely, AGRP increased responding for sucrose, an effect that was blocked by pretreatment with the dopamine receptor antagonist α-flupenthixol. In contrast to progressive ratio responding, free intake of sucrose remained unaltered upon α-MSH or AGRP infusion. In addition, we investigated whether the effects of α-MSH and AGRP on food motivation were mediated by the NAc shell. In situ hybridization of MC3 and MC4 receptor expression confirmed that the MC4 receptor was expressed throughout the NAc, and injection of α-MSH and AGRP into the NAc shell caused a decrease and an increase in motivation for sucrose, respectively. These data show that the motivation for palatable food is modulated by MC4 receptors in the NAc shell, and demonstrate cross-talk between the MC and dopamine system in the modulation of food motivation.

α-Melanocyte-stimulating hormone inhibits angiogenesis through attenuation of VEGF/VEGFR2 signaling pathway

BackgroundGene therapy of proopiomelanocortin, the precursor of α-melanocyte-stimulating hormone (α-MSH), suppresses the neovascularization in tumors. However, the roles of α-MSH in angiogenesis remain unclear. MethodsThe influence of α-MSH on angiogenesis was evaluated by ex vivo rat aorta and in vivo, including transgenic zebrafish and chicken chorioallantoic membrane (CAM) assays. The effect of α-MSH on proliferation, matrix metalloproteinase (MMP) secretion, migration and tube formation was examined using human umbilical vein endothelial cells (HUVECs). The expression of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) was investigated by quantitative RT-PCR, immunoblot and immunofluorescent analysis. Antibodies' neutralization was employed to dissect the receptor(s) transmitting α-MSH signaling. ResultsApplication of α-MSH potently suppressed the microvessels sprouting in organotypic aortic rings. Besides, α-MSH perturbed the vessels development in zebrafish and chicken embryos. α-MSH (0.01–10nM) inhibited the MMP-2 secretion, migration and tube formation of HUVECs without affecting proliferation. Mechanistic studies unveiled α-MSH decreased the VEGF expression and release in HUVECs. Besides, α-MSH downregulated the VEGFR2 expression at transcriptional and translational levels. Importantly, α-MSH attenuated the Akt phosphorylation, but enhanced the expression of PTEN, endogenous antagonist of PI3K/Akt signaling. Expression analysis and antibody neutralization revealed that MC1-R and MC2-R participated in α-MSH-induced blockage of migration and VEGF/VEGFR2/Akt signaling. However, VEGF supply failed to reverse the anti-angiogenic function of α-MSH. Conclusionsα-MSH inhibits the physiological angiogenesis by attenuating VEGF/VEGFR2/Akt signaling in endothelial cells. General significanceα-MSH is a potent angiogenesis inhibitor targeting at endothelial VEGF/VEGFR2 signaling, which may have potential for therapeutic application.

PReS-FINAL-2357: Effects of anti-melanocyte stimulating hormone in murine pristine-induced lupus

Alfa-melanocyte stimulating hormone (α-MSH) has a variety of biological functions such as downregulation of pro-inflammatory pathways, reduction of skin delayed-type hypersensitivity and blockage of leukocyte migration. Inhibition of experimental disease models development including inflammatory bowel disease and rheumatoid arthritis has been shown, however the immunomodulatory and anti-inflammatory effects of α-MSH on murine lupus remain undetermined.

Alpha-Melanocyte Stimulating Hormone (α-MSH) Is a Post-Caspase Suppressor of Apoptosis in RAW 264.7 Macrophages

The neuropeptide alpha-melanocyte stimulating hormone (α-MSH) is an important regulator of immune cell activity within the immunosuppressive ocular microenvironment. Its constitutive presence not only suppresses macrophage inflammatory activity, it also participates in retinal pigment epithelial cell (RPE) mediated activation of macrophages to function similar to myeloid suppressor cells. In addition, α-MSH promotes survival of the alternatively activated macrophages where without α-MSH RPE induce apoptosis in the macrophages, which is seen as increased TUNEL stained cells. Since there is little know about α-MSH as an anti-apoptotic factor, the effects of α-MSH on caspase activity, mitochondrial membrane potential, Bcl2 to BAX expression, along with TUNEL staining, and Annexin V binding were examined in RAW 264.7 macrophages under serum-starved conditions that trigger apoptosis. There was no effect of α-MSH on activated Caspase 9 and Caspase 3 while there was suppression of Caspase 8 activity. In addition, α-MSH did not improve mitochondrial membrane potential, change the ratio between Bcl-2 and BAX, nor reduce Annexin V binding. These results demonstrate that the diminution in TUNEL staining by α-MSH is through α-MSH mediating suppression of the apoptotic pathway that is post-Caspase 3, but before DNA fragmentation. Therefore, as α-MSH promotes the alternative activation of macrophages it also provides a survival signal, and the potential for the caspases to participate in non-apoptotic activities that can contribute to an immunosuppressive microenvironment.

Single Administration of Tripeptide α-MSH(11–13) Attenuates Brain Damage by Reduced Inflammation and Apoptosis after Experimental Traumatic Brain Injury in Mice

Following traumatic brain injury (TBI) neuroinflammatory processes promote neuronal cell loss. Alpha-melanocyte-stimulating hormone (α-MSH) is a neuropeptide with immunomodulatory properties, which may offer neuroprotection. Due to short half-life and pigmentary side-effects of α-MSH, the C-terminal tripeptide α-MSH(11–13) may be an anti-inflammatory alternative. The present study investigated the mRNA concentrations of the precursor hormone proopiomelanocortin (POMC) and of melanocortin receptors 1 and 4 (MC1R/MC4R) in naive mice and 15 min, 6, 12, 24, and 48 h after controlled cortical impact (CCI). Regulation of POMC and MC4R expression did not change after trauma, while MC1R levels increased over time with a 3-fold maximum at 12 h compared to naive brain tissue. The effect of α-MSH(11–13) on secondary lesion volume determined in cresyl violet stained sections (intraperitoneal injection 30 min after insult of 1 mg/kg α-MSH(11–13) or 0.9% NaCl) showed a considerable smaller trauma in α-MSH(11–13) injected mice. The expression of the inflammatory markers TNF-α and IL-1β as well as the total amount of Iba-1 positive cells were not reduced. However, cell branch counting of Iba-1 positive cells revealed a reduced activation of microglia. Furthermore, tripeptide injection reduced neuronal apoptosis analyzed by cleaved caspase-3 and NeuN staining. Based on the results single α-MSH(11–13) administration offers a promising neuroprotective property by modulation of inflammation and prevention of apoptosis after traumatic brain injury.

Effect of α-MSH on osteoclast formation

Raw264.7 cells were incubated with receptor activator of NF-kappa B ligand (RANKL) and α-melanocyte stimulating hormone(α-MSH) for6 d.The amount of osteoclast cells were counted by tartrate resistant acid phosphatase staining and the acid phosphatase activity was assayed.The expressions of 5 melanocortin receptors (MCR) in Raw264.7 cells were determined by RT-PCR.The results showed that the number of osteoclasts in RANKL +α-MSH group was significantly increased compared with RANKL group (P ＜ 0.05),but there was no osteoclast formation in α-MSH group.Compared with control group and α-MSH group,the acid phosphatase activities were significantly increased in RANKL group and α-MSH+RANKL group (P＜0.05).All five MCRs were expressed in the Raw264.7 cells shown by RT-PCR.These results suggest that α-MSH may promote osteoclasts formation through RANK signaling pathway. Key words: α-Melanocyte stimulating hormone; Osteoclast formation ; Melanocortin receptor

Chondroprotective and anti‐inflammatory role of melanocortin peptides in TNF‐α activated human C‐20/A4 chondrocytes

Melanocortin MC(1) and MC(3 ) receptors, mediate the anti-inflammatory effects of melanocortin peptides. Targeting these receptors could therefore lead to development of novel anti-inflammatory therapeutic agents. We investigated the expression of MC(1) and MC(3) receptors on chondrocytes and the role of α-melanocyte-stimulating hormone (α-MSH) and the selective MC(3) receptor agonist, [DTRP(8) ]-γ-MSH, in modulating production of inflammatory cytokines, tissue-destructive proteins and induction of apoptotic pathway(s) in the human chondrocytic C-20/A4 cells. Effects of α-MSH, [DTRP(8) ]-γ-MSH alone or in the presence of the MC(3/4) receptor antagonist, SHU9119, on TNF-α induced release of pro-inflammatory cytokines, MMPs, apoptotic pathway(s) and cell death in C-20/A4 chondrocytes were investigated, along with their effect on the release of the anti-inflammatory cytokine IL-10. C-20/A4 chondrocytes expressed functionally active MC(1,3) receptors. α-MSH and [DTRP(8) ]-γ-MSH treatment, for 30 min before TNF-α stimulation, provided a time-and-bell-shaped concentration-dependent decrease in pro-inflammatory cytokines (IL-1β, IL-6 and IL-8) release and increased release of the chondroprotective and anti-inflammatory cytokine, IL-10, whilst decreasing expression of MMP1, MMP3, MMP13 genes.α-MSH and [DTRP(8) ]-γ-MSH treatment also inhibited TNF-α-induced caspase-3/7 activation and chondrocyte death. The effects of [DTRP(8) ]-γ-MSH, but not α-MSH, were abolished by the MC(3/4) receptor antagonist, SHU9119. Activation of MC(1) /MC(3) receptors in C-20/A4 chondrocytes down-regulated production of pro-inflammatory cytokines and cartilage-destroying proteinases, inhibited initiation of apoptotic pathways and promoted release of chondroprotective and anti-inflammatory cytokines. Developing small molecule agonists to MC(1) /MC(3) receptors could be a viable approach for developing chondroprotective and anti-inflammatory therapies in rheumatoid and osteoarthritis.

Effects of α-MSH on ischemia/reperfusion injury in the rat sciatic nerve

Background:Ischemia/reperfusion (I/R) causes the production of toxic free radicals and leads to pathological changes in nerve tissue. We investigated the effect of alpha-melanocyte stimulating hormone (α-MSH) in a rat model for sciatic nerve I/R and discuss the possible cytoprotective and antioxidant mechanism of α-MSH against ischemic fiber degeneration.Methods:Experiments were performed using 42 adult male Wistar rats. Rats were divided into six experimental groups: control group, ischemia group, I/R groups, and α-MSH treated groups. Ischemia was produced by clamping of the femoral vessels. Immediately after ischemia that lasted 3 h, 75 μg/kg of α-MSH was administered subcutaneously before reperfusion and the tissue malondialdehyde (MDA) level was evaluated as an indicator of lipid peroxidation in groups with different reperfusion periods.Results:The reperfusion injury did not begin in the first hour of reperfusion after 3 h of ischemia, and MDA levels increased on the first day of reperfusion. During the first day, blood MDA levels were decreased in the α-MSH group compared to the control group. The tissue from animals pre-treated with α-MSH showed fewer morphological alterations. Myelin breakdown was significantly diminished after treatment with α-MSH, and the ultrastructural features of axons showed remarkable improvement. Two-way analysis of variance was used for comparing three or more groups. When a significant difference existed, the post-hoc multiple-comparison test was applied to demonstrate the differences.Conclusions:The results confirm that pre-treatment with α-MSH after ischemia protected the peripheral nerves against I/R injury.

Prolyl Carboxypeptidase Regulates Energy Expenditure and the Thyroid Axis

Hypothalamic α-melanocyte-stimulating hormone (α-MSH) plays a central role in regulating energy uptake and expenditure. Prolyl carboxypeptidase (PRCP), a protease expressed in the hypothalamus, is responsible for the degradation of α-MSH. PRCP null animals (PRCP(gt/gt) mice) display elevated α-MSH in the hypothalamus, lower body weight, and are protected from diet induced obesity. Here, we report that PRCP(gt/gt) mice have a significant decrease in fat mass, although an increase in lean mass was also observed. In agreement with low fat accumulation, reduced leptin levels were found. Consistent with the effect of α-MSH on energy metabolism, PRCP(gt/gt) mice had increased energy expenditure with elevated circulating thyroid hormone levels and brown adipose tissue uncoupling protein 1 mRNA levels compared with control mice when exposed to regular diet. TRH mRNA levels in the PVN were significantly higher in fed PRCP(gt/gt) animals compared with fed wild-type controls. Fasting significantly decreased TRH mRNA levels in both PRCP(gt/gt) and wild-type (WT) mice. However, TRH mRNA levels in fasted PRCP(gt/gt) animals were significantly higher than those of fasted WT mice. Refeeding analysis after fasting showed a reduced food intake in PRCP(gt/gt) compared with WT mice. Finally, TRH mRNA levels in T(3)-treated hypothyroid PRCP(gt/gt) mice showed a non significant reduction compared with those of hypothyroid PRCP(gt/gt) mice, supporting the impairment of the hypothalamo-pituitary-thyroid axis in PRCP(gt/gt) mice. All together, these data confirm that PRCP plays a role in the regulation of energy metabolism.

Modulation of basophil activity: A novel function of the neuropeptide α-melanocyte–stimulating hormone

Little is known about the effect of neuropeptides on basophils, which are important effector cells in immune and allergic responses. This study aimed at revealing the role of α-melanocyte-stimulating hormone (α-MSH) on basophil function. Expression of melanocortin receptors and proopiomelanocortin (POMC) was analyzed by means of RT-PCR, Western immunoblotting, fluorescence-activated cell sorting, and double-immunofluorescence analysis. Signal transduction studies included cyclic AMP and Ca(2+) mobilization assays. Basophil activity was assessed based on CD63 surface expression and cytokine release. MC-1R expression was detectable in basophils isolated from human peripheral blood, as well as in basophils within nasal tissue. In isolated basophils from human blood, truncated POMC transcripts were present, but there was no POMC protein. Treatment of basophils with α-MSH increased intracellular Ca(2+) but not cyclic AMP levels. α-MSH at physiologic doses potently suppressed basophil activation induced by N-formyl-methionyl-leucyl-phenylalanine, phorbol 12-myristate 13-acetate, or grass pollen allergen in whole blood of healthy or allergic subjects, respectively. The effect of α-MSH on basophil activation was MC-1R mediated (as shown by blockade with a peptide analogue of agouti-signaling protein) and imitated by adrenocorticotropic hormone but not elicited by the tripeptides KPV and KdPT, both of which lack the central pharmacophore of α-MSH. Moreover, α-MSH at physiologic doses significantly suppressed secretion of 3 proallergic cytokines, IL-4, IL-6, and IL-13, in basophils stimulated with anti-IgE, N-formyl-methionyl-leucyl-phenylalanine, or phorbol 12-myristate 13-acetate. Our findings highlight a novel functional activity of α-MSH, which acts as a natural antiallergic basophil-response modifier. These findings might point to novel therapeutic strategies in treating allergic diseases.

Anti-Candidaactivity of α-melanocyte-stimulating hormone (α-MSH) peptides

Previously, alpha-MSH, a 13-amino acid neuropeptide and the tripeptide alpha-MSH have been shown to possess potent antimicrobial properties. We performed microplate based growth inhibition assays on C. albicans with alpha-MSH, but could not observe any growth inhibiting effect. Repeating the originally published assay with different C. albicans strains, we detected a mild growth inhibiting effect of 100 microM alpha-MSH, but again no effect of alpha-MSH.