Melasolv induces melanosome autophagy to inhibit pigmentation in B16F1 cells.

The melanosome is a specialized membrane-bound organelle that is involved in melanin synthesis, storage, and transportation. In contrast to melanosome biogenesis, the processes underlying melanosome degradation remain largely unknown. Autophagy is a process that promotes degradation of intracellular components' cooperative process between autophagosomes and lysosomes, and its role for process of melanosome degradation remains unclear. Here, we assessed the regulation of autophagy and its contributions to depigmentation associated with Melasolv (3,4,5-trimethoxycinnamate thymol ester). B16F1 cells-treated with Melasolv suppressed the α-MSH-stimulated increase of melanin content and resulted in the activation of autophagy. However, introduction of bafilomycin A1 strongly suppressed melanosome degradation in Melasolv-treated cells. Furthermore, inhibition of autophagy by ATG5 resulted in significant suppression of Melasolv-mediated depigmentation in α-MSH-treated cells. Taken together, our results suggest that treatment with Melasolv inhibits skin pigmentation by promoting melanosome degradation via autophagy activation.

Antimelanogenesis Effects of Fungal Exopolysaccharides Prepared from Submerged Culture of Fomitopsis castanea Mycelia.

Fungal exopolysaccharides are important natural products having diverse biological functions. In this study, exopolysaccharides from Fomitopsis castanea mycelia (FEPS) were prepared, and the highest mushroom tyrosinase inhibitory activity was found. FEPS were prepared from cultivation broth by ethanol precipitation method. The extraction yield and protein concentration of FEPS were 213.1 mg/l and 0.03%, respectively. FEPS inhibited mushroom tyrosinase with the half maximal inhibitory concentration (IC50) of 16.5 mg/ml and dose-dependently inhibited cellular tyrosinase activity (63.9% at 50 µg/ml, and 83.3% at 100 µg/ml) in the cell-free extract of SK-MEL-5 human melanoma cell and α-melanocytestimulating hormone (α-MSH)-stimulated melanin formation in intact SK-MEL-5 human melanoma cell. The IC50 of FEPS against NO production from RAW264.7 macrophage cells was 42.8 ± 0.64 µg/ml. By in vivo study using a zebrafish model, exposure of FEPS at 400 µg/ml to dechorionated zebrafish embryos for 18 h decreased the pigment density, compared to that without FEPS-treated control.

SGK1/FOXO3 Signaling in Hypothalamic POMC Neurons Mediates Glucocorticoid-Increased Adiposity.

Although the central nervous system has been implicated in glucocorticoid-induced gain of fat mass, the underlying mechanisms are poorly understood. The aim of this study was to investigate the possible involvement of hypothalamic serum- and glucocorticoid-regulated kinase 1 (SGK1) in glucocorticoid-increased adiposity. It is well known that SGK1 expression is induced by acute glucocorticoid treatment, but it is interesting that we found its expression to be decreased in the arcuate nucleus of the hypothalamus, including proopiomelanocortin (POMC) neurons, following chronic dexamethasone (Dex) treatment. To study the role of SGK1 in POMC neurons, we produced mice that developed or experienced adult-onset SGK1 deletion in POMC neurons (PSKO). As observed in Dex-treated mice, PSKO mice exhibited increased adiposity and decreased energy expenditure. Mice overexpressing constitutively active SGK1 in POMC neurons consistently had the opposite phenotype and did not experience Dex-increased adiposity. Finally, Dex decreased hypothalamic α-melanocyte-stimulating hormone (α-MSH) content and its precursor Pomc expression via SGK1/FOXO3 signaling, and intracerebroventricular injection of α-MSH or adenovirus-mediated FOXO3 knockdown in the arcuate nucleus largely reversed the metabolic alterations in PSKO mice. These results demonstrate that POMC SGK1/FOXO3 signaling mediates glucocorticoid-increased adiposity, providing new insights into the mechanistic link between glucocorticoids and fat accumulation and important hints for possible treatment targets for obesity.

Alpha-melanocyte stimulating hormone modulates ethanol self-administration in posterior ventral tegmental area through melanocortin-4 receptors.

Although the role of alpha-melanocyte stimulating hormone (α-MSH) in alcohol seeking behaviour in rats has been demonstrated, the underlying mechanisms are not understood. Herein, we test the hypothesis that α-MSH might have a permissive effect in promoting the reward action of ethanol. Rats were implanted with cannulae targeted at the posterior ventral tegmental area (pVTA), because the site is sensitive to reinforcing effects of ethanol. These rats were trained to self-administer ethanol in standard two-lever (active/inactive) operant chamber test. Each active lever press resulted in self-administration of 100 nl of ethanol (100-300 mg%) containing solution. Over a period of 7 days, ethanol significantly increased the number of lever presses, which was considered as a measure of reward. Because ethanol at 200 mg% resulted in maximum number of lever presses (∼18-20 lever presses/30-minute session), the dose was employed in further studies. While prior administration of melanocortin (MC) agonists, α-MSH or [Nle4,D-Phe7]-alpha-MSH into pVTA, resulted in an 89% increase in lever presses, the response was attenuated following pre-treatment with MC4 receptors (MC4R) antagonist, HS014. In an immunohistochemical study, the brains of rats that were trained to self-infuse ethanol showed significantly increased α-MSH immunoreactivity in the nucleus accumbens shell, bed nucleus of stria terminalis and arcuate nucleus of the hypothalamus. In the pVTA, α-MSH fibres were found to run close to the dopamine cells, labelled with tyrosine hydroxylase antibodies. We suggest that α-MSH-MC4R system in the pVTA might be a part of the neuroadaptive mechanism underlying ethanol addiction.

Triterpene glycosides and other polar constituents of shea (Vitellaria paradoxa) kernels and their bioactivities.

The MeOH extract of defatted shea (Vitellaria paradoxa; Sapotaceae) kernels was investigated for its constituents, and fifteen oleanane-type triterpene acids and glycosides, two steroid glucosides, two pentane-2,4-diol glucosides, seven phenolic compounds, and three sugars, were isolated. The structures of five triterpene glycosides were elucidated on the basis of spectroscopic and chemical methods. Upon evaluation of the bioactivity of the isolated compounds, it was found that some or most of the compounds have potent or moderate inhibitory activities against the following: melanogenesis in B16 melanoma cells induced by α-melanocyte-stimulating hormone (α-MSH); generation of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, against Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-teradecanoylphorbol 13-acetate (TPA) in Raji cells; t TPA-induced inflammation in mice, and proliferation of one or more of HL-60, A549, AZ521, and SK-BR-3 human cancer cell lines, respectively. Western blot analysis established that paradoxoside E inhibits melanogenesis by regulation of expression of microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein-1 (TRP-1) and TRP-2. In addition, tieghemelin A was demonstrated to exhibit cytotoxic activity against A549 cells (IC50 13.5 µM) mainly due to induction of apoptosis by flow cytometry. The extract of defatted shea kernels and its constituents may be, therefore, valuable as potential antioxidant, anti-inflammatory, skin-whitening, chemopreventive, and anticancer agents.

Inhibition of melanogenesis and oxidation by protocatechuic acid from Origanum vulgare (oregano).

Antioxidant and antimelanogenesis activities of protocatechuic acid (1) from Origanum vulgare (oregano) were investigated. The antioxidative capacity of 1 was confirmed from its free-radical-scavenging activities, inhibition of lipid peroxidation, and suppression of reactive oxygen species in H(2)O(2)-induced BNLCL2 cells. The inhibition by 1 of tyrosinase and DOPA oxidase activity and melanin production was possibly related to the down-regulation of melanocortin-1 receptor, microphthalmia-associated transcription factor, tyrosinase, tyrosinase-related proteins-2, and tyrosinase-related proteins-1 expression in α-melanocyte-stimulating hormone-induced B16 cells. After a gel containing 1 was applied to mice, the values of L* slightly increased, and a* and erythema-melanin levels of skin were reduced by comparing the values of untreated control groups, indicating 1 can reduce melanin production. These results suggest that 1 may act as an effective quencher of oxidative attackers with antimelanogenesis properties.

Involvement of alpha-melanocyte stimulating hormone (alpha-MSH) in differential ethanol exposure and withdrawal related depression in rat: neuroanatomical-behavioral correlates.

We investigated the involvement of alpha-melanocyte stimulating hormone (alpha-MSH) following acute, chronic and withdrawal treatments of ethanol with reference to depression. The degree of depression was evaluated using Porsolt's forced swim test. While intracerebroventricular (i.c.v.) alpha-MSH (100-400 ng/rat) dose-dependently increased the immobility, opposite response was observed following administration of selective MC4 receptor antagonist HS014 (0.01-0.07 ng/rat, i.c.v.). The anti-immobility effect of acute ethanol (1-2 g/kg), injected via intra-peritoneal route (i.p.), was suppressed by central administration of alpha-MSH (100 ng/rat, i.c.v.), but was enhanced following pretreatment with HS014 (0.01 ng/rat, i.c.v.). Chronic ethanol resulted in increased immobility time, while further augmentation in immobility was noticed following ethanol withdrawal. However, concomitant HS014 (0.01 ng/rat, i.c.v.) treatment prevented tolerance as well as attenuated enhanced immobility in ethanol-withdrawn rats. Acute administration of HS014 (0.01-0.03 ng/rat, i.c.v.), at 24h post-withdrawal time point, also antagonized the ethanol withdrawal immobility in rats. The profile of alpha-MSH-immunoreactivity in the paraventricular (PVN), arcuate (ARC), paraventricular thalamic (PVT), dorsomedial hypothalamic-dorsal (DMNd) and -ventral (DMNv) nuclei, lateral hypothalamus (LH) and central nucleus of amygdala (CeA) was investigated with immunocytochemistry. Acute ethanol significantly reduced the alpha-MSH-immunoreactivity in the cells and fibers of ARC, and fibers in the PVN, DMNd, DMNv and CeA. While chronic ethanol treatment significantly increased the alpha-MSH-immunoreactivity as compared to the pair-fed control group, further augmentation was noticed following 24 h ethanol withdrawal. However, the alpha-MSH-immunoreactive profile in the PVT and LH did not respond. alpha-MSH in discrete areas may play a role in ethanol-induced antidepressant-like response and withdrawal-induced depression.

Oxidative stress via hydrogen peroxide affects proopiomelanocortin peptides directly in the epidermis of patients with vitiligo.

The human skin holds the capacity for autocrine processing of the proopiomelanocortin (POMC)-derived peptides. Recent data demonstrated the presence and functionality of ACTH, alpha- and beta-melanocyte-stimulating hormone (MSH), and beta-endorphin in the regulation of skin pigmentation, and a role has been put forward for alpha-MSH as an effective antioxidant. In patients with vitiligo, decreased epidermal POMC processing and low alpha-MSH levels were documented previously. These patients accumulate hydrogen peroxide (H2O2) in the 10(-3) M range in their epidermis. Therefore, we examined the involvement of H2O2 on POMC-derived peptides as possible targets for oxidation by this reactive oxygen species. To address this, we employed immunofluorescence labelling, dot blot analysis, Fourier transform Raman spectroscopy, functionality studies, and computer simulation of the peptide structures. We demonstrate H2O2-mediated oxidation of epidermal ACTH, alpha-MSH, and beta-endorphin in vitiligo owing to oxidation of methionine residues in the sequences of these peptides. Moreover, we show that oxidized beta-endorphin loses its function in the promotion of pigmentation in melanocytes. These changes are reversible upon the reduction of H2O2 levels by a pseudocatalase PC-KUS. Moreover, oxidation of alpha-MSH can be prevented by the formation of a 1:1 complex with the abundant cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin. Thus, using vitiligo, we demonstrate that H2O2 can affect pigmentation via epidermal POMC peptide redox homeostasis.

Feeding responses to a melanocortin agonist and antagonist in obesity induced by a palatable high-fat diet.

Hypothalamic melanocortins are critical for the control of food intake, and alterations in POMC mRNA have been described in genetic models of obesity. However, the time course of changes in brain transmitters over the development of dietary obesity is less clear. Therefore, we examined the effect of diet-induced obesity on hypothalamic alpha-MSH content and feeding responsiveness to synthetic melanocortins. Male Sprague-Dawley rats fed a high-fat cafeteria diet (30% fat) or chow (5% fat) for 4 or 12 weeks were implanted with intracerebroventricular cannulae and feeding responses to the MC3/4R agonist MTII (0.5 nmol) and the selective MC4R antagonist HS014 (0.8 nmol) were determined. MTII had a long-lasting inhibitory effect on food intake. Chronically overfed animals had a significantly exaggerated inhibitory feeding response 15 and 24 h after MTII injection and lost more body weight (15 +/- 3 g) compared to control rats (4 +/- 4 g; P < 0.05). Daytime administration of HS014 significantly increased food intake in all rats to the same extent (P < 0.05). No change in hypothalamic alpha-MSH content was observed after 2 or 12 weeks of high-fat diet. The observation of increased responsiveness to the melanocortin agonist, in the face of a high-fat diet, suggests melanocortin analogues may have potential for the pharmacological treatment of obesity.

Piperlonguminine from Piper longum with inhibitory effects on alpha-melanocyte-stimulating hormone-induced melanogenesis in melanoma B16 cells.

Skin hyperpigmentations such as melasma, freckles and senile lentigines can be subjectively treated by depigmenting agents. In our ongoing study to find melanogenesis inhibitors from natural sources, Piper longum L (fruits, Piperaceae) was discovered to have an inhibitory effect on alpha-melanocyte-stimulating hormone (alpha-MSH)-induced melanogenesis in melanoma B16 cells. Piperlonguminine has been identified as the melanogenesis inhibitor from P. longum by activity-guided extraction and isolation. The compound showed dose-dependent inhibitory effects with 85.1 +/- 4.9% inhibition at 25 microM, 62.1 +/- 6.1% at 12.5 microM, 36.4 +/- 4.6% at 6.3 microM and 18.4 +/- 5.1% at 3.1 microM on alpha-MSH-induced melanogenesis, showing an IC50 value of 9.6 microM. As a positive control, kojic acid exhibited an IC50 value of 44.6 microM on the melanogenesis. As to the mode of action, piperlonguminine showed an inhibitory effect on alpha-MSH-induced tyrosinase synthesis, documented by Western immunoblot analysis. However, piperlonguminine did not show an inhibitory effect on tyrosinase activity or a direct depigmenting effect of melanin.

Effects of anaesthesia and manual restraint on the plasma concentrations of pituitary and adrenocortical hormones in ferrets.

Two experiments were carried out to investigate the effect of sampling techniques on the plasma concentrations of pituitary and adrenocortical hormones in ferrets (Mustela putorius furo). In the first experiment blood was collected on two occasions from 29 ferrets which were either manually restrained or anaesthetised with isoflurane. In the second experiment eight intact ferrets were fitted with jugular catheters and blood was collected on four occasions, just before and as soon as possible after they had been manually restrained or anaesthetised with medetomidine or isoflurane; blood was also collected 10 and 30 minutes after the induction of anaesthesia. Medetomidine anaesthesia had no effect on the plasma concentrations of pituitary and adrenocortical hormones. Isoflurane anaesthesia resulted in a significant increase in the plasma concentration of alpha-melanocyte-stimulating hormone (alpha-MSH) directly after the induction of anaesthesia. Manual restraint resulted in a significant increase in the plasma concentrations of cortisol and adrenocorticotrophic hormone (ACTH) and a decrease in the plasma concentration of alpha-MSH.

Regulation of cellular alpha-MSH and beta-endorphin during stimulated secretion from intermediate pituitary cells: involvement of aspartyl and cysteine proteases in the control of cellular levels of alpha-MSH and beta-endorphin.

The regulation of cellular levels of alpha-melanocyte stimulating factor (alpha-MSH) and beta-endorphin in response to stimulated secretion from intermediate pituitary cells in primary culture was investigated in this study. Regulation of the cell content of alpha-MSH and beta-endorphin occurred in two phases consisting of (a) initial depletion of cellular levels of these peptide hormones during short-term secretion (3 h) induced by isoproterenol, forskolin, or phorbol myristate acetate (PMA) which was followed by (b) long-term (24 h) increases in cellular levels of alpha-MSH and beta-endorphin in response to stimulated secretion induced by isoproterenol and PMA. In short-term experiments (3 h), cellular levels of alpha-MSH and beta-endorphin were reduced by 30-50% during stimulated secretion of these peptide hormones by isoproterenol (agonist for the beta-adrenergic receptor), forskolin that activates protein kinase A (PKA), and PMA that activates protein kinase C (PKC). Moreover, dopamine inhibited isoproterenol-induced depletion of cellular alpha-MSH and beta-endorphin. During long-term incubation of cells (24 h) with isoproterenol, cellular alpha-MSH and beta-endorphin were increased to twice that of controls (unstimulated cells). Treatment with PMA for 24 h also increased cellular levels of alpha-MSH and beta-endorphin. Moreover, cellular levels of alpha-MSH and beta-endorphin were decreased during long-term treatment of cells with an aspartyl protease inhibitor, pepstatin A, and with the cysteine protease inhibitor E64c. These results implicate aspartyl and cysteine proteases in the cellular production of alpha-MSH and beta-endorphin that requires proteolytic processing of their common precursor proopiomelanocortin (POMC). These findings demonstrate the parallel regulation of cellular levels of alpha-MSH and beta-endorphin during their cosecretion, which may involve aspartyl and cysteine proteases in the metabolism of these peptide hormones.

Minocycline modulation of alpha-MSH production by keratinocytes in vitro.

The anti-inflammatory mechanisms of minocycline, an antibiotic used in the treatment of the inflammatory component of acne, are only partially understood. In addition to inflammation due to cytokines (IL-1, IL-6, TNF-alpha, etc.), recent studies have shown that neuropeptide-mediated neurogenic inflammation may play an important role in cutaneous inflammation. The purpose of this study was to investigate minocycline-induced modulation of cutaneous production of alpha-melanocyte-stimulating hormone (alpha-MSH), a neuropeptide with known anti-inflammatory activity. Two different skin models were used: explants of inflammatory skin and reconstituted skin, both incubated with minocycline at different concentrations and for different time periods. Epidermal production of alpha-MSH, as evaluated by immunofluorescence and immunoperoxidase techniques, showed increased expression in both models. This neuropeptide, which has an anti-inflammatory activity (notably through production of IL-10, antagonism of IL-1 and inhibition of the chemotaxis of polymorphonuclear leukocytes), thus plays a role in the anti-inflammatory action of minocycline.

Evidence for an interaction between alpha-MSH and opioids in the regulation of gonadotropin secretion in man.

Gonadotropin secretion is inhibited by the endogenous opioids and stimulated by their antagonist naloxone. LH secretion is stimulated by alpha-MSH, a tridecapeptide derived from the post-translational processing of POMC. The possibility that alpha-MSH interacts with the opioids, as suggested by the experimental evidence, was investigated in 7 normal males aged 24-29 through the performance of seven tests: naloxone (0.8 mg i.v. bolus, followed by infusion of 1.6 mg/h for 120'); alpha-MSH (2.5 mg i.v. bolus); naloxone + alpha-MSH (2.5 mg i.v. 15' after commencement of the naloxone infusion); naloxone + GnRH (100 micrograms i.v. 15' after commencement of the naloxone infusion); alpha-MSH + GnRH (respectively 2.5 mg and 100 micrograms at time 0), GnRH alone (100 micrograms at time 0), placebo (150 nmol/l NaCl solution). The LH AUCs during both naloxone (30.3 +/- 2.7 mIU/ml.min-1) and alpha-MSH test (32.9 +/- 4.6 mIU/ml.min-1) were significantly greater (p < 0.005) than that observed during placebo (16.9 +/- 3.6 mIU/ml.min-1). The LH AUC during alpha-MSH + naloxone (37.6 +/- 2.6 mIU/ml.min-1) was not significantly different from that recorded during their separate administration. GnRH injected alone, during the naloxone infusion and with alpha-MSH produced similar increases in LH, that were significantly higher than that observed during the other tests (AUCs: GnRH 89.4 +/- 10.6, GnRH + naloxone 100.5 +/- 9.1, GnRH + alpha-MSH 94.6 +/- 7.9 mIU/ml.min-1, p < 0.001). Significant increase in FSH (p < 0.001) was only observed during GnRH, GnRH + naloxone and GnRH + aMSH tests (AUCs: placebo 13.3 +/- 1.7; naloxone 14.7 +/- 2.5; alpha-MSH 15.5 +/- 2.3; alpha-MSH + naloxone 16.9 +/- 1.9; GnRH 19.1 +/- 1.1; GnRH + alpha-MSH 20.7 +/- 1.3; GnRH + naloxone 21.2 +/- 1.8 mIU/ml.min-1). These results are in line with the possibility of an interaction between alpha-MSH and the opioids in the regulation of gonadotropin secretion, perhaps with opposing effects on a final common pathway.

Differential effects of the D2 receptor agonist quinelorane on the secretion of prolactin and alpha-melanocyte-stimulating hormone.

The purpose of the present study was to determine the effects of quinelorane, a selective D2 receptor agonist, on concentrations of prolactin and alpha-melanocyte-stimulating hormone (alpha-MSH) in plasma of male rats. Quinelorane decreased plasma concentrations of prolactin but not of alpha-MSH, whereas, the D2 receptor antagonist raclopride increased plasma concentrations of both hormones. Quinelorane reversed the effects of raclopride on circulating levels of prolactin, but not alpha-MSH. The results of this study suggest that secretion of hormones from melanotrophs and lactotrophs is regulated by different subtypes of D2 receptors.

Dynamics of cyclic-AMP efflux in relation to alpha-MSH secretion from melanotrope cells of Xenopus laevis.

An important factor in regulating secretion from endocrine cells is the cytoplasmic concentration of cyclic-AMP. Many regulatory substances are known to either stimulate or inhibit the production of this second messenger through activation of their receptors. In the present study, we have monitored changes in cyclic-AMP efflux from melanotrope cells of Xenopus laevis in response to established neurochemical regulators of alpha-MSH secretion. In vitro superfusion of neurointermediate lobes allows for a dynamic recording of cyclic-AMP production in relation to hormone secretion. Unlike alpha-MSH secretion, the efflux of cyclic-AMP was not dependent on the concentration of extracellular calcium, indicating that hormone release and cyclic-AMP efflux are mediated by different mechanisms. The phosphodiesterase inhibitor IBMX and the adenylate cyclase activator forskolin stimulated cyclic-AMP efflux, but had no stimulatory effect on alpha-MSH release. This indicates that an increase in cyclic-AMP production in melanotrope cells is not necessarily accompanied by an increase in the rate of alpha-MSH release. Corticotropin-releasing factor stimulated cyclic-AMP efflux with dynamics similar to that induced by the amphibian peptide sauvagine. Dopamine and the GABAB receptor agonist baclofen both inhibited cyclic-AMP efflux and alpha-MSH release, with similar dynamics of inhibition and similar dose-response relationships. It is proposed that an inhibition of cyclic-AMP efflux is coupled to an inhibition of alpha-MSH secretion.