Melanocortin receptor-4 mediates the anorectic effect induced by the nucleus tractus solitarius injection of glucagon-like Peptide-2 in fasted rats.

Glucagon-like peptide-2 (GLP-2) is secreted from enteroendocrine L-type cells of the gut and also released from preproglucagonergic (PPG) neurons in the nucleus tractus solitarius (NTS) and adjacent medial reticular nucleus of the brain stem. The neurons in the NTS express GLP-2, and the neurons send extensive projections to the hypothalamus. Recent studies show that the intracerebroventricular administration of GLP-2 significantly suppresses food intake in animals and some evidence suggest that the melanocortin receptor-4 (MC4-R) signaling in the hypothalamus is required for intracerebroventricular GLP-2-mediated inhibition of feeding. There is proopiomelanocortin (POMC) positive neurons expressing MC4-R in the NTS. Suppression of MC4-R expressing neurons in the brain stem inhibits gastric emptying. In this study, we tested the effects of NTS GLP-2R activation and blockade on feeding behavior and evaluated the endogenous melanocortin system's role in the NTS in mediating effects of GLP-2 on feeding behavior in fed and fasted rats. Our results demonstrated that microinjection of GLP-2 into the NTS suppressed food intake in fasted-refeeding rats but did not affect food intake in free-feeding rats, and this inhibition was blocked by pretreatment of either Exendin (9-39) or SHU 9119, suggesting the GLP-2 system in the NTS exerts an inhibitory action on food intake. MC4-R mediates this action in the NTS.

Reduced MC4R signaling alters nociceptive thresholds associated with red hair.

Humans and mice with natural red hair have elevated basal pain thresholds and an increased sensitivity to opioid analgesics. We investigated the mechanisms responsible for higher nociceptive thresholds in red-haired mice resulting from a loss of melanocortin 1 receptor (MC1R) function and found that the increased thresholds are melanocyte dependent but melanin independent. MC1R loss of function decreases melanocytic proopiomelanocortin transcription and systemic melanocyte-stimulating hormone (MSH) levels in the plasma of red-haired (Mc1re/e ) mice. Decreased peripheral α-MSH derepresses the central opioid tone mediated by the opioid receptor OPRM1, resulting in increased nociceptive thresholds. We identified MC4R as the MSH-responsive receptor that opposes OPRM1 signaling and the periaqueductal gray area in the brainstem as a central area of opioid/melanocortin antagonism. This work highlights the physiologic role of melanocytic MC1R and circulating melanocortins in the regulation of nociception and provides a mechanistic framework for altered opioid signaling and pain sensitivity in red-haired individuals.

α-Melanocyte-Stimulating Hormone Triggers Melanogenesis Via Activation of the Aryl Hydrocarbon Receptor Pathway in B16F10 Mouse Melanoma Cells.

Melanin is a group of natural pigments that determines the human skin color and provides fundamental protection against the harmful impacts of physical and chemical stimuli. The aim of this study was to establish the regulatory role of aryl hydrocarbon receptor (AhR) in α-melanocyte-stimulating hormone (α-MSH) induced melanogenesis. In the present study, following knockdown of AhR, murine B16F10 cells were treated with α-MSH (200 nM) and tyrosinase activities, cellular melanin content, mRNA levels of several important genes involved in melanogenesis including AhR, CTNNB1, TYR2, and microphthalmia-associated transcription factor (MITF) were measured as endpoints. Exposure to α-MSH led to elevated expression of AhR, CTNNB1, MITF, and TYR in accordance with increased tyrosinase enzyme activity as well as a significant rise in the total melanin content. Our results suggest that AhR plays a regulatory role in α-MSH-stimulated melanogenesis.

Activation of the ARCPOMC→MeA Projection Reduces Food Intake.

Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARC) plays an essential role in the control of food intake and energy expenditure. Melanocortin-4 receptors (MC4Rs) are expressed in key areas that are implicated in regulating energy homeostasis. Although the importance of MC4Rs in the paraventricular hypothalamus (PVH) has been well documented, the role of MC4Rs in the medial amygdala (MeA) on feeding remains controversial. In this study, we specifically examine the role of a novel ARCPOMC→MeA neural circuit in the regulation of short-term food intake. To map a local melanocortinergic neural circuit, we use monosynaptic anterograde as well as retrograde viral tracers and perform double immunohistochemistry to determine the identity of the neurons receiving synaptic input from POMC neurons in the ARC. To investigate the role of the ARCPOMC→MeA projection on feeding, we optogenetically stimulate channelrhodopsin-2 (ChR2)-expressing POMC fibers in the MeA. Anterograde viral tracing studies reveal that ARC POMC neurons send axonal projections to estrogen receptor-α (ER-α)- and MC4R-expressing neurons in the MeA. Retrograde viral tracing experiments show that the neurons projecting to the MeA is located mainly in the lateral part of the ARC. Optogenetic stimulation of the ARCPOMC→MeA pathway reduces short-term food intake. This anorectic effect is blocked by treatment with the MC4R antagonist SHU9119. In addition to the melanocortinergic local circuits within the hypothalamus, this extrahypothalamic ARCPOMC→MeA neural circuit would play a role in regulating short-term food intake.

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.

Determination of the melanocortin-4 receptor structure identifies Ca2+ as a cofactor for ligand binding.

The melanocortin-4 receptor (MC4R) is involved in energy homeostasis and is an important drug target for syndromic obesity. We report the structure of the antagonist SHU9119-bound human MC4R at 2.8-angstrom resolution. Ca2+ is identified as a cofactor that is complexed with residues from both the receptor and peptide ligand. Extracellular Ca2+ increases the affinity and potency of the endogenous agonist α-melanocyte-stimulating hormone at the MC4R by 37- and 600-fold, respectively. The ability of the MC4R crystallized construct to couple to ion channel Kir7.1, while lacking cyclic adenosine monophosphate stimulation, highlights a heterotrimeric GTP-binding protein (G protein)-independent mechanism for this signaling modality. MC4R is revealed as a structurally divergent G protein-coupled receptor (GPCR), with more similarity to lipidic GPCRs than to the homologous peptidic GPCRs.

Sites and sources of sympathoexcitation in obese male rats: role of brain insulin.

In males, obesity increases sympathetic nerve activity (SNA), but the mechanisms are unclear. Here, we investigate insulin, via an action in the arcuate nucleus (ArcN), and downstream neuropathways, including melanocortin receptor 3/4 (MC3/4R) in the hypothalamic paraventricular nucleus (PVN) and dorsal medial hypothalamus (DMH). We studied conscious and α-chloralose-anesthetized Sprague-Dawley rats fed a high-fat diet, which causes obesity prone (OP) rats to accrue excess fat and obesity-resistant (OR) rats to maintain fat content, similar to rats fed a standard control (CON) diet. Nonspecific blockade of the ArcN with muscimol and specific blockade of ArcN insulin receptors (InsR) decreased lumbar SNA (LSNA), heart rate (HR), and mean arterial pressure (MAP) in OP, but not OR or CON, rats, indicating that insulin supports LSNA in obese males. In conscious rats, intracerebroventricular infusion of insulin increased MAP only in OP rats and also improved HR baroreflex function from subnormal to supranormal. The brain sensitization to insulin may elucidate how insulin can drive central SNA pathways when transport of insulin across the blood-brain barrier may be impaired. Blockade of PVN, but not DMH, MC3/4R with SHU9119 decreased LSNA, HR, and, MAP in OP, but not OR or CON, rats. Interestingly, nanoinjection of the MC3/4R agonist melanotan II (MTII) into the PVN increased LSNA only in OP rats, similar to PVN MTII-induced increases in LSNA in CON rats after blockade of sympathoinhibitory neuropeptide Y Y1 receptors. ArcN InsR expression was not increased in OP rats. Collectively, these data indicate that obesity increases SNA, in part via increased InsR signaling and downstream PVN MC3/4R.

Relay of peripheral oxytocin to central oxytocin neurons via vagal afferents for regulating feeding.

Oxytocin (Oxt), a neurohormone synthesized in the neurons of hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus induces milk-ejection and uterine contraction and regulates social behavior, stress responses, memory and food intake. Peripheral (intraperitoneal and subcutaneous) infusion of Oxt decreases food intake and body weight in obese animals via mechanisms involving vagal afferent nerves and in obese subjects when administered nasally. Peripherally injected and intracerebroventricularly injected Oxt inhibit food intake to similar extent and with similar time course. Thus, peripheral Oxt mimics the effects of central Oxt, however, underlying mechanisms are unclear. In the present study we explored whether intraperitoneal Oxt activates Oxt neurons in PVN via vagal afferents and whether this pathway is linked to inhibition of feeding. We here show that intraperitoneal Oxt injection induces c-Fos expression in PVN largely in Oxt neurons and inhibits food intake, and these effects are blunted by subdiaphragmatic vagotomy. The intraperitoneal Oxt-induced inhibition of food intake was blunted in Oxt KO mice, by intracerebroventricular injection of Oxt receptor antagonist, and by vagotomy. These results demonstrate that intraperitoneal Oxt injection activates PVN Oxt neurons via vagal afferent nerves, thereby inhibiting food intake. This vagal afferents-mediated Oxt's peripheral-to-central coupling may serve to promote satiety and possibly a series of neural functions of Oxt and to treat their disorders.

Replacement of Arg with Nle and modified D-Phe in the core sequence of MSHs, Ac-His-D-Phe-Arg-Trp-NH2, leads to hMC1R selectivity and pigmentation.

Melanoma skin cancer is the fastest growing cancer in the US [1]. A great need exists for improved formulations and mechanisms to prevent and protect human skin from cancers and other skin damage caused by sunlight exposure. Current efforts to prevent UV damage to human skin, which in many cases leads to melanoma and other skin cancers. The primordial melanocortin-1 receptor (MC1R) is involved in regulating skin pigmentation and hair color, which is a natural prevention from UV damage. The endogenous melanocortin agonists induce pigmentation and share a core pharmacophore sequence "His-Phe-Arg-Trp", and it was found that substitution of the Phe by D-Phe results in increasing melanocortin receptor potency. To improve the melanocortin 1 receptor (MC1R) selectivity a series of tetra-peptides with the moiety of Ac-Xaa-Yaa-Nle-Trp-NH2, and structural modifications to reduce electrostatic ligand-receptor interactions have been designed and synthesized. It is discovered that the tetrapeptide Ac-His-D-Phe(4-CF3)-Nle-Trp-NH2 resulted in a potent and selective hMC1R agonist at the hMC1R (EC50: 10 nM). Lizard anolis carolinensis pigmentation study shows very high potency in vivo. NMR studies revealed a reversed ß turn structure which led to the potency and selectivity towards the hMC1R.

Novel (1E,3E,5E)-1,6-bis(Substituted phenyl)hexa-1,3,5-triene Analogs Inhibit Melanogenesis in B16F10 Cells and Zebrafish.

The present study aimed to evaluate the anti-melanogenic activity of 1,6-diphenyl-1,3,5-hexatriene and its derivatives in B16F10 murine melanoma cells and zebrafish embryos. Twenty five (1E,3E,5E)-1,6-bis(substituted phenyl)hexa-1,3,5-triene analogs were synthesized and their non-cytotoxic effects were predictively analyzed using three-dimensional quantitative structure-activity relationship approach. Inhibitory activities of these synthetic compounds against melanin synthesis were determined by evaluating melanin content and melanogenic regulatory enzyme expression in B16F10 cells. The anti-melanogenic activity was verified by observing body pigmentation in zebrafishes treated with these compounds. Compound #2, #4, and #6 effectively decreased melanogenesis induced by α-melanocyte-stimulating hormone. In particular, compound #2 remarkably lowered the mRNA and protein expression levels of microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), and TYRP2 in B16F10 cells and substantially reduced skin pigmentation in the developed larvae of zebrafish. These findings suggest that compound #2 may be used as an anti-melanogenic agent for cosmetic purpose.

Design of MC1R Selective γ-MSH Analogues with Canonical Amino Acids Leads to Potency and Pigmentation.

Melanoma is a lethal form of skin cancer. Skin pigmentation, which is regulated by the melanocortin 1 receptor (MC1R), is an effective protection against melanoma. However, the endogenous MC1R agonists lack selectivity for the MC1R and thus can have side effects. The use of noncanonical amino acids in previous MC1R ligand development raises safety concerns. Here we report the development of the first potent and selective hMC1R agonist with only canonical amino acids. Using γ-MSH as a template, we developed a peptide, [Leu3, Leu7, Phe8]-γ-MSH-NH2 (compound 5), which is 16-fold selective for the hMC1R (EC50 = 4.5 nM) versus other melanocortin receptors. Conformational studies revealed a constrained conformation for this linear peptide. Molecular docking demonstrated a hydrophobic binding pocket for the melanocortin 1 receptor. In vivo pigmentation study shows high potency and short duration. [Leu3, Leu7, Phe8]-γ-MSH-NH2 is ideal for inducing short-term skin pigmentation without sun for melanoma prevention.

The thermogenic effect of nesfatin-1 requires recruitment of the melanocortin system.

Nesfatin-1 is a bioactive polypeptide expressed both in the brain and peripheral tissues and involved in the control of energy balance by reducing food intake. Central administration of nesfatin-1 significantly increases energy expenditure, as demonstrated by a higher dry heat loss; yet, the mechanisms underlying the thermogenic effect of central nesfatin-1 remain unknown. Therefore, in this study, we sought to investigate whether the increase in energy expenditure induced by nesfatin-1 is mediated by the central melanocortin pathway, which was previously reported to mediate central nesfatin-1´s effects on feeding and numerous other physiological functions. With the application of direct calorimetry, we found that intracerebroventricular nesfatin-1 (25 pmol) treatment increased dry heat loss and that this effect was fully blocked by simultaneous administration of an equimolar dose of the melanocortin 3/4 receptor antagonist, SHU9119. Interestingly, the nesfatin-1-induced increase in dry heat loss was positively correlated with body weight loss. In addition, as assessed with thermal imaging, intracerebroventricular nesfatin-1 (100 pmol) increased interscapular brown adipose tissue (iBAT) as well as tail temperature, suggesting increased heat production in the iBAT and heat dissipation over the tail surface. Finally, nesfatin-1 upregulated pro-opiomelanocortin and melanocortin 3 receptor mRNA expression in the hypothalamus, accompanied by a significant increase in iodothyronine deiodinase 2 and by a nonsignificant increase in uncoupling protein 1 and peroxisome proliferator-activated receptor gamma coactivator-1 alpha mRNA in the iBAT. Overall, we clearly demonstrate that nesfatin-1 requires the activation of the central melanocortin system to increase iBAT thermogenesis and, in turn, overall energy expenditure.

Chick subcutaneous and abdominal adipose tissue depots respond differently in lipolytic and adipogenic activity to α-melanocyte stimulating hormone (α-MSH).

In birds, α-MSH is anorexigenic, but effects on adipose tissue are unknown. Four day-old chicks were intraperitoneally injected with 0 (vehicle), 5, 10, or 50µg of α-MSH and subcutaneous and abdominal adipose tissue collected at 60min for RNA isolation (n=10). Plasma was collected post-euthanasia at 60 and 180min for measuring non-esterified fatty acids (NEFA) and α-MSH (n=10). Relative to the vehicle, food intake was reduced in the 50µg-treated group. Plasma NEFAs were greater in 10µg than vehicle-treated chicks at 3h. Plasma α-MSH was 3.06±0.57ng/ml. In subcutaneous tissue, melanocortin receptor 5 (MC5R) mRNA was increased in 10µg, MC2R and CCAAT-enhancer-binding protein ß (C/EBPß) mRNAs increased in 50µg, peroxisome proliferator-activated receptor γ and C/EBPα decreased in 5, 10 and 50µg, and Ki67 mRNA decreased in 50µg α-MSH-injected chicks, compared to vehicle-injected chicks. In abdominal tissue, adipose triglyceride lipase mRNA was greater in 10µg α-MSH- than vehicle-treated chicks. Cells isolated from abdominal fat that were treated with 10 and 100nM α-MSH for 4h expressed more MC5R and perilipin-1 than control cells (n=6). Cells that received 100nM α-MSH expressed more fatty acid binding protein 4 and comparative gene identification-58 mRNA than control cells. Glycerol-3-phosphate dehydrogenase (G3PDH) activity was greater in cells at 9days post-differentiation that were treated with 1 and 100nM α-MSH for 4h than in control cells (n=3). Results suggest that α-MSH increases lipolysis and reduces adipogenesis in adipose tissue.

Towards optimization of odonto/osteogenic bioengineering: in vitro comparison of simvastatin, sodium fluoride, melanocyte-stimulating hormone.

Tissue engineering has emerged as a potential therapeutic option for dental problems in recent years. One of the policies in tissue engineering is to use both scaffolds and additive factors for enhancing cell responses. This study aims to evaluate and compare the effect of three types of biofactors on poly-caprolactone-poly-ethylene glycol-poly caprolactone (PCL-PEG-PCL) nanofibrous scaffold on human dental pulp stem cell (hDPSCs) engineering. The PCL-PEG-PCL copolymer was synthesized with ring opening polymerization method, and its nanofiber scaffold was prepared by electrospinning method. Nanofibrous scaffold-seeded hDPSCs were treated with sodium fluoride (NaF), melanocyte-stimulating hormone (MSH), or simvastatin (SIM). Non-treated nanofiber seeded cells were utilized as control. The viability, biocompatibility, adhesion, proliferation rate, morphology, osteo/odontogenic potential, and the expression of tissue-specific genes were studied. The results showed that significant higher results demonstrated significant higher adhesive behavior, viability, alizarin red activity, and dentin specific gene expression in MSH- and SIM-treated cells (p < 0.05). This study is unique; in that, it compares the effects of different treatments for optimization of dental tissue engineering.

Effects of melanocortin-4 receptor agonists and antagonists on expression of genes related to reproduction in spotted scat, Scatophagus argus.

Melanocortin-4 receptor (Mc4r) function related to reproduction in fish has not been extensively investigated. Here, we report on gene expression changes by real-time PCR following treatment with Mc4r agonists and antagonists in the spotted scat (Scatophagus argus). Using in vitro incubated hypothalamus, the Mc4r nonselective agonist NDP-MSH ([Nle4, D-Phe7]-α-melanocyte stimulating hormone; 10-6 M) and selective agonist THIQ (N-[(3R)-1, 2, 3, 4-Tetrahydroisoquinolinium-3-ylcarbonyl]- (1R)-1-(4-chlorobenzyl)-2-[4-cyclohexyl-4-(1H-1,2,4-triazol-1-ylmethyl) piperidin-1-yl]-2-oxoethylamine; 10-7 M) significantly increased the expression of gnrh (Gonadotropin releasing hormone), while the Mc4r nonselective antagonist SHU9119 (Ac-Nle-[Asp-His-DPhe/DNal(2')-Arg-Trp-Lys]-NH2; 10-6 M) and selective antagonist Ipsen 5i (compound 5i synthesized in Ipsen Research Laboratories; 10-6 M) significantly inhibited gnrh expression after 3 h of incubation. In incubated pituitary tissue, NDP-MSH and THIQ significantly increased the expression of fshb (Follicle-stimulating hormone beta subunit) and lhb (Luteinizing hormone beta subunit), while SHU9119 and Ipsen 5i significantly decreased fshb and lhb expression after 3 h of incubation. During the in vivo experiment, THIQ (1 mg/kg bw) significantly increased gnrh expression in hypothalamic tissue, as well as the fshb and lhb expression in pituitary tissue 12 h after abdominal injection. Furthermore, Ipsen 5i (1 mg/kg bw) significantly inhibited gnrh expression in hypothalamic tissue, as well as fshb and lhb gene expression in pituitary tissue 12 h after abdominal injection. In summary, Mc4r singling appears to stimulate gnrh expression in the hypothalamus, thereby modulating the synthesis of Fsh and Lh in the pituitary. In addition, Mc4r also appears to directly regulate fshb and lhb levels in the pituitary in spotted scat. Our study suggests that Mc4r, through the hypothalamus and pituitary, participates in reproductive regulation in fish.

Preparation, cytotoxicity, and in vivo antitumor efficacy of 111In-labeled modular nanotransporters.

PURPOSE: Modular nanotransporters (MNTs) are a polyfunctional platform designed to achieve receptor-specific delivery of short-range therapeutics into the cell nucleus by receptor-mediated endocytosis, endosome escape, and targeted nuclear transport. This study evaluated the potential utility of the MNT platform in tandem with Auger electron emitting 111In for cancer therapy. METHODS: Three MNTs developed to target either melanocortin receptor-1 (MC1R), folate receptor (FR), or epidermal growth factor receptor (EGFR) that are overexpressed on cancer cells were modified with p-SCN-Bn-NOTA and then labeled with 111In in high specific activity. Cytotoxicity of the 111In-labeled MNTs was evaluated on cancer cell lines bearing the appropriate receptor target (FR: HeLa, SK-OV-3; EGFR: A431, U87MG.wtEGFR; and MC1R: B16-F1). In vivo micro-single-photon emission computed tomography/computed tomography imaging and antitumor efficacy studies were performed with intratumoral injection of MC1R-targeted 111In-labeled MNT in B16-F1 melanoma tumor-bearing mice. RESULTS: The three NOTA-MNT conjugates were labeled with a specific activity of 2.7 GBq/mg with nearly 100% yield, allowing use without subsequent purification. The cytotoxicity of 111In delivered by these MNTs was greatly enhanced on receptor-expressing cancer cells compared with 111In nontargeted control. In mice with B16-F1 tumors, prolonged retention of 111In by serial imaging and significant tumor growth delay (82% growth inhibition) were found. CONCLUSION: The specific in vitro cytotoxicity, prolonged tumor retention, and therapeutic efficacy of MC1R-targeted 111In-NOTA-MNT suggest that this Auger electron emitting conjugate warrants further evaluation as a locally delivered radiotherapeutic, such as for ocular melanoma brachytherapy. Moreover, the high cytotoxicity observed with FR- and EGFR-targeted 111In-NOTA-MNT suggests further applications of the MNT delivery strategy should be explored.

Orally Targeted Delivery of Tripeptide KPV via Hyaluronic Acid-Functionalized Nanoparticles Efficiently Alleviates Ulcerative Colitis.

Overcoming adverse effects and selectively delivering drug to target cells are two major challenges in the treatment of ulcerative colitis (UC). Lysine-proline-valine (KPV), a naturally occurring tripeptide, has been shown to attenuate the inflammatory responses of colonic cells. Here, we loaded KPV into hyaluronic acid (HA)-functionalized polymeric nanoparticles (NPs). The resultant HA-KPV-NPs had a desirable particle size (∼272.3 nm) and a slightly negative zeta potential (∼-5.3 mV). These NPs successfully mediated the targeted delivery of KPV to key UC therapy-related cells (colonic epithelial cells and macrophages). In addition, these KPV-loaded NPs appear to be nontoxic and biocompatible with intestinal cells. Intriguingly, we found that HA-KPV-NPs exert combined effects against UC by both accelerating mucosal healing and alleviating inflammation. Oral administration of HA-KPV-NPs encapsulated in a hydrogel (chitosan/alginate) exhibited a much stronger capacity to prevent mucosa damage and downregulate TNF-α, thus they showed a much better therapeutic efficacy against UC in a mouse model, compared with a KPV-NP/hydrogel system. These results collectively demonstrate that our HA-KPV-NP/hydrogel system has the capacity to release HA-KPV-NPs in the colonic lumen and that these NPs subsequently penetrate into colitis tissues and enable KPV to be internalized into target cells, thereby alleviating UC.

Effect of Melanotan-II on Brain Fos Immunoreactivity and Oxytocin Neuronal Activity and Secretion in Rats.

Melanocortins stimulate the central oxytocin systems that are involved in regulating social behaviours. Alterations in central oxytocin have been linked to neurological disorders such as autism, and melanocortins have been proposed for therapeutic treatment. In the present study, we investigated how systemic administration of melanotan-II (MT-II), a melanocortin agonist, affects oxytocin neuronal activity and secretion in rats. The results obtained show that i.v., but not intranasal, administration of MT-II markedly induced Fos expression in magnocellular neurones of the supraoptic (SON) and paraventricular nuclei (PVN) of the hypothalamus, and this response was attenuated by prior i.c.v. administration of the melanocortin antagonist, SHU-9119. Electrophysiological recordings from identified magnocellular neurones of the SON showed that i.v. administration of MT-II increased the firing rate in oxytocin neurones but did not trigger somatodendritic oxytocin release within the SON as measured by microdialysis. Our data suggest that, after i.v., but not intranasal, administration of MT-II, the activity of magnocellular neurones of the SON is increased. Because previous studies showed that SON oxytocin neurones are inhibited in response to direct application of melanocortin agonists, the actions of i.v. MT-II are likely to be mediated at least partly indirectly, possibly by activation of inputs from the caudal brainstem, where MT-II also increased Fos expression.

The serum/PDGF-dependent "melanogenic" role of the minute level of the oncogenic kinase PAK1 in melanoma cells proven by the highly sensitive kinase assay.

We previously demonstrated that the oncogenic kinase PAK4, which both melanomas and normal melanocytes express at a very high level, is essential for their melanogenesis. In the present study, using the highly sensitive "Macaroni-Western" (IP-ATP-Glo) kinase assay, we investigated the melanogenic potential of another oncogenic kinase PAK1, which melanoma (B16F10) cells express only at a very minute level. After transfecting melanoma cells with PAK1-shRNA for silencing PAK1 gene, melanin content, tyrosinase activity, and kinase activity of PAK1 were compared between the wild-type and transfectants. We found that (i) PAK1 is significantly activated by melanogenic hormones such as IBMX (3-isobutyl-1-methyl xanthine) and α-MSH (melanocyte-stimulating hormone), (ii) silencing the endogenous PAK1 gene in melanoma cells through PAK1-specific shRNA reduces both melanin content and tyrosinase activity in the presence of both serum and melanogenic hormones to the basal level, (iii) the exogenously added wild-type PAK1 in the melanoma cells boosts the α-MSH-inducible melanin level by several folds without affecting the basal, and (iv) α-MSH/IBMX-induced melanogenesis hardly takes place in the absence of either serum or PAK1, clearly indicating that PAK1 is essential mainly for serum- and α-MSH/IBMX-dependent melanogenesis, but not the basal, in melanoma cells. The outcome of this study might provide the first scientific basis for explaining why a wide variety of herbal PAK1-blockers such as CAPE (caffeic acid phenethyl ester), curcumin and shikonin in cosmetics are useful for skin-whitening.

Pituitary adenylate cyclase-activating polypeptide (PACAP) in the central nucleus of the amygdala induces anxiety via melanocortin receptors.

RATIONALE: Anxiety disorders are the most common mental disorders in the USA. Characterized by feelings of uncontrollable apprehension, they are accompanied by physical, affective, and behavioral symptoms. The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor PAC1 (PAC1R) are highly expressed in the central nucleus of the amygdala (CeA), and they have gained growing attention for their proposed role in mediating the body's response to stress. OBJECTIVES: The aim of this study was to evaluate the anxiogenic effects of PACAP in the CeA and its effects on the hypothalamic-pituitary-adrenal (HPA) axis. Furthermore, the mechanism of action of PACAP in the CeA was investigated. METHODS: PACAP was microinfused into the CeA of rats, and its effects in the elevated plus maze (EPM), the defensive withdrawal tests, and plasma corticosterone levels were evaluated. The ability of the melanocortin receptor antagonist SHU9119 to block PACAP effect in the EPM was assessed. RESULTS: Intra-CeA PACAP exerted a dose-dependent anxiogenic effect and activated the HPA axis. In contrast, PACAP microinfused into the basolateral nucleus of the amygdala (BlA) had no effect. Finally, the anxiogenic effect of intra-CeA PACAP was prevented by SHU9119. CONCLUSIONS: These data prove an anxiogenic role for the PACAP system of the CeA and reveal that the melanocortin receptor 4 (MC4R) system of CeA mediates these effects. Our data provide insights into this neuropeptide system as a mechanism for modulating the behavioral and endocrine response to stress and suggest that dysregulations of this system may contribute to the pathophysiology of anxiety-related disorders.