Construction of a Tandem Repeat Peptide Sequence with Pepsin Cutting Sites to Produce Recombinant α-Melanocyte-Stimulating Hormone.

The production of α-melanocyte-stimulating hormone (α-MSH), a peptide hormone composed of 13 amino acids, is attempted by recombinant expression using E. coli as the host. To achieve this aim, a synthetic gene containing eight tandem repeats of msh gene (8msh) was designed for ribosomal synthesis of 8 α-MSH. The merit of the strategy is to diminish the peptide toxicity against the host cell and to achieve a higher production yield. Pepsin cleavage sites are introduced between the peptides for enzymatic proteolysis to obtain the monomeric peptide of α-MSH. The constructed plasmid was transformed into different strains of E. coli hosts, and E. coli XL1-Blue with gene 8msh revealed the highest yield of 8 α-MSH. Although 8 α-MSH was fractionalized in the insoluble pellets after cell lysis, pepsin cleavage was able to produce soluble α-MSH peptide, as analyzed and confirmed by mass spectrometry and peptide activity assays. The production of α-MSH was quantified using HPLC with a yield of 42.9 mg/L of LB culture. This study demonstrates the feasibility of producing α-MSH using recombinant expression of tandem repeat gene. The production procedure involves minimal post-treatment and processing and can be scaled up for industrial application.

Forms of selenium in vitamin-mineral mixes differentially affect the expression of genes responsible for prolactin, ACTH, and α-MSH synthesis and mitochondrial dysfunction in pituitaries of steers grazing endophyte-infected tall fescue.

The goal of this study was to test the hypothesis that sodium selenite (inorganic Se, ISe), SEL-PLEX (organic forms of Se, OSe), vs. a 1:1 blend (MIX) of ISe and OSe in a basal vitamin-mineral (VM) mix would differentially alter pituitary transcriptome profiles in growing beef steers grazing an endophyte-infected tall fescue (E+) pasture. Predominately Angus steers (BW = 183 ± 34 kg) were randomly selected from fall-calving cows grazing E+ pasture and consuming VM mixes that contained 35 ppm Se as ISe, OSe, or MIX forms. Steers were weaned, depleted of Se for 98 d, and subjected to summer-long common grazing of a 10.1 ha E+ pasture containing 0.51 ppm ergot alkaloids. Steers were assigned (n = 8 per treatment) to the same Se-form treatments on which they were raised. Selenium treatments were administered by daily top-dressing 85 g of VM mix onto 0.23 kg soyhulls, using in-pasture Calan gates. As previously reported, serum prolactin was greater for MIX (52%) and OSe (59%) steers vs. ISe. Pituitaries were collected at slaughter and changes in global and selected mRNA expression patterns determined by microarray and real-time reverse transcription PCR analyses, respectively. The effects of Se treatment on relative gene expression were subjected to one-way ANOVA. The form of Se affected the expression of 542 annotated genes (P < 0.005). Integrated pathway analysis found a canonical pathway network between prolactin and pro-opiomelanocortin (POMC)/ACTH/α-melanocyte-stimulating hormone (α-MSH) synthesis-related proteins and that mitochondrial dysfunction was a top-affected canonical pathway. Targeted reverse transcription-PCR analysis found that the relative abundance of mRNA encoding prolactin and POMC/ACTH/α-MSH synthesis-related proteins was affected (P < 0.05) by the form of Se, as were (P ≤ 0.05) mitochondrial dysfunction-related proteins (CYB5A, FURIN, GPX4, and PSENEN). OSe steers appeared to have a greater prolactin synthesis capacity (more PRL mRNA) vs. ISe steers through decreased dopamine type two receptor signaling (more DRD2 mRNA), whereas MIX steers had a greater prolactin synthesis capacity (more PRL mRNA) and release potential by increasing thyrotropin-releasing hormone concentrations (less TRH receptor mRNA) than ISe steers. OSe steers also had a greater ACTH and α-MSH synthesis potential (more POMC, PCSK2, CPE, and PAM mRNA) than ISe steers. We conclude that form of Se in VM mixes altered expression of genes responsible for prolactin and POMC/ACTH/α-MSH synthesis, and mitochondrial function, in pituitaries of growing beef steers subjected to summer-long grazing an E+ pasture.

Exposure of pregnant mice to triclosan causes hyperphagic obesity of offspring via the hypermethylation of proopiomelanocortin promoter.

Triclosan (TCS), as a broad spectrum antibacterial agent, is commonly utilized in personal care and household products. Maternal urinary TCS level has been associated with changes in birth weight of infants. We in the present study investigated whether exposure of mice to 8 mg/kg TCS from gestational day (GD) 6 to GD14 alters prenatal and postnatal growth and development, and metabolic phenotypes in male and female offspring (TCS-offspring). Compared with control offspring, body weight in postnatal day (PND) 1 male or female TCS-offspring was reduced, but body weight gain was faster within postnatal 5 days. PND30 and PND60 TCS-offspring showed overweight with increases in visceral fat and adipocyte size. PND60 TCS-offspring displayed delayed glucose clearance and insulin resistance. PND30 TCS-offspring showed an increase in food intake without the changes in the oxygen consumption and respiratory exchange ratio (RER). The expression levels of proopiomelanocortin (POMC), α-melanocyte-stimulating hormone (α-MSH) and single-minded 1 (SIM1) in hypothalamus arcuate nucleus (ARC) and paraventricular nucleus (PVN), respectively, were significantly reduced in PND30 TCS-offspring compared to controls. The hypermethylation of CpG sites at the POMC promoter was observed in PND30 TCS-offspring, while the concentration of serum leptin was elevated and the level of STAT3 phosphorylation in ARC had no significant difference from control. This study demonstrates that TCS exposure during early/mid-gestation through the hypermethylation of the POMC promoter reduces the expression of anorexigenic neuropeptides to cause the postnatal hyperphagic obesity, leading to metabolic syndrome in adulthood.

LXR activation increases the expression of GnRH AND αMSH in the rat hypothalamus in vivo.

Liver X receptors (LXR) are important transcription factors involved in the regulation of carbohydrate and lipid metabolism. Recently, we described LXR receptors expression in the hypothalamus but their function in this brain area remains unknown. Here, we evaluated the function of LXR on the expression of factors produced in the hypothalamus in vitro and in vivo by Western blotting and immunocytochemistry. More precisely we studied the expression of GnRH and GHRH, αMSH and NPY in male Sprague-Dawley rats. The effects of two synthetic LXR agonists, T0901317 and GW3965, were first tested in vitro. Hypothalamic explants were treated with either T0901317 or GW3965 (10µM) for 2, 4, 6 and 8h. As a positive control the cholesterol ABCA1 and glucose GLUT2 transporters were used. No changes were observed in the expression of the factors evaluated in vitro. The effects of the LXR agonists were then tested in vivo. Rats were injected ICV into the third ventricle with either T0901317 or GW3965 (2.5µg/5µL ICV) and after 3.5h or 24h the hypothalami were dissected out and rapidly frozen for analysis. αMSH and GnRH expression was significantly increased after 3.5h of T0901317 treatment. Anterior/posterior hypothalamic ratio increases for αMSH expression and decreases for GnRH expression after 24h of LXR activation. Altogether these results show that LXR activation affects the expression of GnRH and αMSH, suggesting that LXR in the hypothalamus is capable of modulating hypothalamic responses related to appetite, sexual behavior and reproductive functions.

Ultraviolet B stimulates proopiomelanocortin signalling in the arcuate nucleus of the hypothalamus in mice.

We previously found that ultraviolet B (UVB) could stimulate the paraventricular nucleus (PVN) with activation the systemic hypothalamic-pituitary- adrenal (HPA) axis. To investigate whether UVB can also stimulate other hypothalamic nuclei, we tested its effect on the proopiomelanocortin (POMC) related signalling system in the arcuate nucleus (ARC) of female C57BL/6 and FVB albino mice. The shaved back skin of the mice was irradiated with either 100 or 400 mJ/cm2 of UVB. After 1, 3, 6 and 12 h, blood and hypothalamus were collected and processed for gene and protein expression, and measurement of α-MSH and ß-endorphin (ß-END) levels. An in situ immunohistochemical examination was performed for melanocortin receptor 4 (MC4R) and POMC-derived α-MSH. The expression of Pomc and MC4R mRNAs was stimulated, whereas that of AgRP was inhibited after exposure to UVB. It was accompanied by an increased number of both α-MSH- and MC4R-immunoreactive neurons in the ARC, and by increased levels of α-MSH and ß-END (both found in the hypothalamus and plasma). This surprising discovery of UVB stimulating the POMC system in the ARC, accompanied by the increased plasma levels of α-MSH and ß-END, paves the way for exciting areas of research on the communication between the skin and the brain, as well as is suggesting a new role for UVB in regulation of body metabolism.

Regulation of melanopsins and Per1 by α -MSH and melatonin in photosensitive Xenopus laevis melanophores.

α-MSH and light exert a dispersing effect on pigment granules of Xenopus laevis melanophores; however, the intracellular signaling pathways are different. Melatonin, a hormone that functions as an internal signal of darkness for the organism, has opposite effects, aggregating the melanin granules. Because light functions as an important synchronizing signal for circadian rhythms, we further investigated the effects of both hormones on genes related to the circadian system, namely, Per1 (one of the clock genes) and the melanopsins, Opn4x and Opn4m (photopigments). Per1 showed temporal oscillations, regardless of the presence of melatonin or α-MSH, which slightly inhibited its expression. Melatonin effects on melanopsins depend on the time of application: if applied in the photophase it dramatically decreased Opn4x and Opn4m expressions, and abolished their temporal oscillations, opposite to α-MSH, which increased the melanopsins' expressions. Our results demonstrate that unlike what has been reported for other peripheral clocks and cultured cells, medium changes or hormones do not play a major role in synchronizing the Xenopus melanophore population. This difference is probably due to the fact that X. laevis melanophores possess functional photopigments (melanopsins) that enable these cells to primarily respond to light, which triggers melanin dispersion and modulates gene expression.

Photoprotective and antioxidant effects of Rhubarb: inhibitory action on tyrosinase and tyrosine kinase activities and TNF-α, IL-1α and α-MSH production in human melanocytes.

BACKGROUND: Exposure to ultraviolet (UV) radiation causes various forms of acute and chronic skin damage, including immunosuppression, inflammation, premature aging and photodamage. Furthermore, it induces the generation of reactive oxygen species, produces proinflammatory cytokines and melanocyte-stimulating hormone (MSH) and increases tyrosinase activity. The aim of this study was to evaluate the potential photoprotective effects of Rheum rhaponticum L. rhizome extract on human UV-stimulated melanocytes. METHODS: The effects of Rheum rhaponticum rhizome extract on tyrosine kinase activity, and on interleukin-1α (IL-1α), tumour necrosis factor α (TNF-α), and α-MSH production in human epidermal melanocytes were evaluated under UV-stimulated and non-stimulated conditions. Antioxidant activity was evaluated by lipid peroxidation and 1,1-dyphenyl-2-picryl-hydrazyl (DPPH) assays, while anti-tyrosinase activity was evaluated by the mushroom tyrosinase method. RESULTS: Rheum rhaponticum L. rhizome extract showed in vitro antioxidant properties against lipid peroxidation, free radical scavenging and anti-tyrosinase activities, and inhibited the production of IL-1α, TNF-α, α-MSH, and tyrosine kinase activity in melanocytes subjected to UV radiation. CONCLUSIONS: These results support the inclusion of Rheum rhaponticum L. rhizome extract into cosmetic, sunscreen and skin care products for the prevention or reduction of photodamage.

Angiogenesis in the intermediate lobe of the pituitary gland alters its structure and function.

The pars distalis (PD) and the pars intermedia (PI) have the same embryonic origin, but their morphological and functional characteristics diverge during development. The PD is highly vascularized, whereas the highly innervated PI is essentially non-vascularized. Based on our previous finding that vascular endothelial growth factor-A (VEGF-A) is involved in vascularization of the rat PD, attempt was made to generate transgenic Xenopus expressing VEGF-A specifically in the melanotrope cells of the PI as a model system for studying the significance of vascularization or avascularization for the functional differentiation of the pituitary. The PI of the transgenic frogs, examined after metamorphosis, were distinctly vascularized but poorly innervated. The experimentally induced vascularization in the PI resulted in a marked increase in tissue volume and a decrease in the expression of both alpha-melanophore-stimulating hormone (α-MSH) and prohormone convertase 2, a cleavage enzyme essential for generating α-MSH. The transgenic animals had low plasma α-MSH concentrations and displayed incomplete adaptation to a black background. To our knowledge, this is the first report indicating that experimentally induced angiogenesis in the PI may bring about functional as well as structural alterations in this tissue.

Evidence for possible period 2 gene mediation of the effects of alcohol exposure during the postnatal period on genes associated with maintaining metabolic signaling in the mouse hypothalamus.

BACKGROUND: Animals exposed to alcohol during the developmental period develop circadian disturbances and metabolic problems that often persist during their adult period. In order to study whether alcohol and the circadian clock interact to alter metabolic signaling in the hypothalamus, we determined whether postnatal alcohol feeding in mice permanently alters metabolic sensing in the hypothalamus. Furthermore, we evaluated whether the effect of circadian disruption via Period 2 (Per2) gene mutation prevents alcohol's effects on metabolic signaling in the hypothalamus. METHODS: Per2 mutant and wild-type male and female mice of the same genetic background were given a milk formula containing ethanol (EtOH; 11.34% vol/vol) from postnatal day (PD) 2 to 7 and used for gene expression and peptide level determinations in the hypothalamus at PD7 and PD90. RESULTS: We report here that postnatal alcohol feeding reduces the expression of proopiomelanocortin (Pomc) gene and production of ß-endorphin and α-melanocyte stimulating hormone (α-MSH) in the hypothalamus that persists into adulthood. In addition, expressions of metabolic sensing genes in the hypothalamus were also reduced as a consequence of postnatal alcohol exposure. These effects were not sex-specific and were observed in both males and females. Mice carrying a mutation of the Per2 gene did not show any reductions in hypothalamic levels of Pomc and metabolic genes and ß-endorphin and α-MSH peptides following alcohol exposure. CONCLUSIONS: These data suggest that early-life exposure to alcohol alters metabolic sensing to the hypothalamus possibly via regulating Per2 gene and/or the cellular circadian clock mechanism.

NCKX5, a natural regulator of human skin colour variation, regulates the expression of key pigment genes MC1R and alpha-MSH and alters cholesterol homeostasis in normal human melanocytes.

Natural human skin colour is determined both by environmental exposure to ultraviolet light and through inherited genetic variation in a very limited number of genes. Variation of a non-synonymous single-nucleotide polymorphism (nsSNP; rs1426654) in the gene (SLC24A5) encoding the NCKX5 protein is associated with differences in constitutive skin colour in South Asians. The nsSNP encodes the substitution of alanine for threonine at residue 111 (A111T) near a transmembrane region required for exchanger activity, a region which is highly conserved across different species and between NCKX family members. We have shown that NCKX5 is located at the trans-Golgi network of melanocytes and functions as a potassium-dependent sodium-calcium exchanger. When heterologously expressed, the 111T variant of NCKX5 shows significantly lower exchanger activity than the A111 variant. We have postulated that lower exchanger activity causes the reduced melanogenesis and lighter skin in Thr111-positive individuals. We used gene expression microarrays with qPCR replication and validation to assess the impact of siRNA-mediated knockdown of SLC24A5 on the transcriptome of cultured normal human melanocytes (NHM). Very few genes associated with melanogenesis were altered at the transcript level except for MC1R, suggesting that SLC24A5 interacts with at least one well-characterized melanogenic signalling pathway. More surprisingly, the expression of a number of cholesterol homeostatic genes was altered after SLC24A5 knockdown, and the total cholesterol content of NHM was increased. Cholesterol has previously been identified as a potential melanogenic regulator, and our data imply that NCKX5 exchanger function influences natural variation in skin pigmentation via a novel, unknown mechanism affecting cellular sterol levels.

Inhibitory effect of homochlorcyclizine on melanogenesis in α-melanocyte stimulating hormone-stimulated mouse B16 melanoma cells.

The histamine receptor H1 antagonist homochlorcyclizine (HC) has been widely used as an antihistamine agent for the treatment of allergies. However, the effect of HC on skin pigmentation is not known. In the present study, we investigated the inhibitory effect of HC on melanogenesis in mouse B16 melanoma cells. Our results showed that HC inhibited melanogenesis in either α-melanocyte stimulating hormone (α-MSH)- or 3-isobutyl-1-methylxanthin (IBMX)-stimulated B16 cells in a dose-dependent manner. Despite the strong inhibition of melanogenesis by HC, it was surprisingly found that HC did not reduce either cellular or melanosomal tyrosinase activity in α-MSH-stimulated B16 cells. In addition, HC also did not directly inhibit either murine or mushroom tyrosinase activity in the cell-free system. Moreover, western blotting and reverse-transcription polymerase chain reaction (RT-PCR) analyses respectively confirmed that HC did not downregulate levels of tyrosinase protein and its mRNA in α-MSH-stimulated B16 cells. These results clearly demonstrated that HC inhibits melanogenesis of B16 cells by a mechanism other than reduction of the cellular tyrosinase activity. From the present study, HC was proven to be a good candidate as a skin-whitening agent for treatment of skin hyperpigmentation, and this generic drug might be suitable for use in combination with other depigmenting agents due to its unique inhibition mechanism.

Autophagy in hypothalamic AgRP neurons regulates food intake and energy balance.

Macroautophagy is a lysosomal degradative pathway that maintains cellular homeostasis by turning over cellular components. Here we demonstrate a role for autophagy in hypothalamic agouti-related peptide (AgRP) neurons in the regulation of food intake and energy balance. We show that starvation-induced hypothalamic autophagy mobilizes neuron-intrinsic lipids to generate endogenous free fatty acids, which in turn regulate AgRP levels. The functional consequences of inhibiting autophagy are the failure to upregulate AgRP in response to starvation, and constitutive increases in hypothalamic levels of pro-opiomelanocortin and its cleavage product α-melanocyte-stimulating hormone that typically contribute to a lean phenotype. We propose a conceptual framework for considering how autophagy-regulated lipid metabolism within hypothalamic neurons may modulate neuropeptide levels to have immediate effects on food intake, as well as long-term effects on energy homeostasis. Regulation of hypothalamic autophagy could become an effective intervention in conditions such as obesity and the metabolic syndrome.

Immunosuppressive activity of a novel peptide analog of α-melanocyte stimulating hormone (α-MSH) in experimental autoimmune uveitis.

Autoimmune uveitis is an inflammatory disorder of the eye that can lead to pain and vision loss. Steroids and immunosuppressive drugs are currently the only therapeutics for uveitis and have serious ocular and systemic toxicities. Therefore, safer alternative therapeutics are desired. Alpha-melanocyte stimulating hormone (α-MSH) is a neuropeptide that suppresses effector T cell functions, induces regulatory T cells and has beneficial effects in certain autoimmune and transplant models. A novel d-amino acid peptide analog of native α-MSH (dRI-α-MSH) was produced that was protected from protease digestion and had increased selectivity for the melanocortin-1 receptor. Systemic delivery of the dRI-α-MSH analog dramatically suppressed disease progression and retained retinal architecture in the experimental autoimmune uveitis (EAU) model. Local delivery by periorbital injection was equally effective. Importantly, treatment with the novel dRI-α-MSH analog suppressed uveitis with a similar magnitude to the corticosteroid, dexamethasone. Data indicate that the novel dRI-α-MSH analogs show anti-inflammatory activities and have potential therapeutic use in uveitis and other autoimmune diseases.

Neuropeptide delivery to the brain: a von Willebrand factor signal peptide to direct neuropeptide secretion.

BACKGROUND: Multiple neuropeptides, sometimes with opposing functions, can be produced from one precursor gene. To study the roles of the different neuropeptides encoded by one large precursor we developed a method to overexpress minigenes and establish local secretion. RESULTS: We fused the signal peptide from the Von Willebrand Factor (VWF) to a furin site followed by a processed form of the Agouti related protein (AgRP), AgRP(83-132) or alpha-melanocyte stimulating hormone. In vitro, these minigenes were secreted and biologically active. Additionally, the proteins of the minigenes were not transported into projections of primary neurons, thereby ensuring local release. In vivo administration of VWF-AgRP(83-132), using an adeno-associated viral vector as a delivery vehicle, into the paraventricular hypothalamus increased body weight and food intake of these rats compared to rats which received a control vector. CONCLUSIONS: This study demonstrated that removal of the N-terminal part of full length AgRP and addition of a VWF signal peptide is a successful strategy to deliver neuropeptide minigenes to the brain and establish local neuropeptide secretion.

Morphologic changes and the expression of alpha-melanocyte stimulating hormone and melanocortin-1 receptor in melasma lesions: a comparative study.

Melasma is a common acquired symmetrical hypermelanosis characterized by irregular light- to dark-brown macules on sun-exposed skin areas. The literature shows few studies on its physiopathogeny. However, changes in α-melanocyte stimulating hormone (α-MSH) secretion and melanocortin-1 receptor (MC1-R) expression may play a role to trigger this condition. Biopsies were taken from both melasma skin and adjacent perilesional normal skin of 44 patients. The biopsies were submitted for hematoxylin and eosin and Fontana-Masson staining and immunohistochemistry with Melan-A, α-MSH, and MC1-R, and processed for transmission electron microscopy. In some cases, they were submitted to MC1-R gene expression analysis by real-time polymerase chain reaction. Increased lymphohistiocytic infiltrate and solar elastosis, higher epidermal melanin were observed in melasma skin. Electron microscopy revealed a greater number of mature melanosomes in keratinocytes and melanocytes, and more prominent cytoplasmic organelles in melasma skin. There was no difference in melanocyte number between areas. However, melanocytes were larger and more dendritic in melasma skin. Immunohistochemistry with α-MSH and MC1-R showed significant labeling in melasmic epidermis but MC1-R messenger ribonucleic acid (RNAm) did not show significant quantitative difference between melasma and normal skin.

Expression of recombinant hybrid peptide hinnavin II/alpha-melanocyte-stimulating hormone in Escherichia coli: purification and characterization.

The increasing problem of antibiotic resistance among pathogenic bacteria requires novel strategies for the construction of multiple, joined genes of antimicrobial agents. The strategy used in this study involved synthesis of a cDNA-encoding hinnavin II/alpha-melanocyte-stimulating hormone (hin/MSH) hybrid peptide, which was cloned into the pET32a (+) vector to allow expression of the hybrid peptide as a fusion protein in Escherichia coli BL21 (DE3). The resulting expression of fusion protein Trx-hin/MSH could reach up to 20% of the total cell proteins. More than 50% of the target protein was in a soluble form. The target fusion protein from the soluble fraction, Trx-hin/MSH, was easily purified by Ni(2+)-chelating chromatography. Then, enterokinase cleavage effectively cleaved the Trx-hin/MSH to release the recombinant hin/MSH (rhin/MSH) hybrid peptide. After removing the contaminants, we purified the recombinant hybrid peptide to homogeneity by reversed-phase FPLC and obtained 210 mg of pure, active rhin/MSH from 800 ml of culture medium. Antimicrobial activity assay demonstrated that rhin/MSH had a broader spectrum of activity than did the parental hinnavin II or MSH against fungi and Gram-positive and Gram-negative bacteria. These results suggest an efficient method for producing high-level expression of various kinds of antimicrobial peptides that are toxic to the host, a reliable and simple method for producing different hybrid peptides for biological studies.

Electroacupuncture in the treatment of obesity.

Obesity is becoming one of the most common health problems in the world. Many other disorders, such as hypertension and diabetes are considered as the consequences of obesity. Since effective remedies are rare (only two drugs, Orlistat and Sibutramine, were officially approved by the US Food and Drug Administration for long-term obesity treatment so far), researchers are trying to discover new therapies for obesity, and acupuncture is among the most popular alternative approaches. To facilitate weight reduction, one can use manual acupuncture, electroacupuncture (EA) or transcutaneous electrical acupoint stimulation (TEAS). As the parameters of the EA or TEAS can be precisely characterized and the results are more or less reproducible, this review will focus on EA as a treatment modality for obesity. Results obtained in this laboratory in recent five years will be summarized in some detail.

Blood pressure regulation and cardiac autonomic control in mice overexpressing alpha- and gamma-melanocyte stimulating hormone.

Melanocyte stimulating hormones (MSH) derived from pro-opiomelanocortin have been demonstrated to participate in the central regulation of cardiovascular functions. The aim of the present study was to elucidate the chronic effects of increased melanocortin activation on blood pressure regulation and autonomic nervous system function. We adapted telemetry to transgenic mice overexpressing alpha- and gamma-MSH and measured blood pressure, heart rate and locomotor activity, and analyzed heart rate variability (HRV) in the frequency-domain as well as baroreflex function by the sequence technique. Transgenic (MSH-OE) mice had increased systolic blood pressure but their heart rate was similar to wild-type (WT) controls. The 24-h mean of systolic blood pressure was 132+/-7mmHg in MSH-OE and 113+/-4mmHg in WT mice. Locomotor activity was decreased in the MSH-OE mice. Furthermore, MSH-OE mice showed slower adaptation to mild environmental stress in terms of blood pressure changes. The low frequency (LF) power of HRV tended to be higher in MSH-OE mice compared to WT mice, without a difference in overall variability. The assessment of baroreflex function indicated enhanced baroreflex effectiveness and more frequent baroreflex operations in MSH-OE mice. Baseline heart rate, increased LF power of HRV and increased baroreflex activity may all reflect maintenance of baroreflex integrity and an increase in cardiac vagal activity to counteract the increased blood pressure. These results provide new evidence that long-term activation of the melanocortin system elevates blood pressure without increasing heart rate.

Multimeric alpha-MSH has increased efficacy to activate the melanocortin MC4 receptor.

alpha-Melanocyte stimulating hormone (alpha-MSH) has a relatively low affinity for the melanocortin MC4 receptor. Constructs of multimeric alpha-MSH varying from one to eight subunits were synthesized to test whether they displayed an improved ability to bind to and activate the human melanocortin MC4 receptor. alpha-MSH subunits were coupled by a flexible linker and placed in front of an IRES-eGFP sequence. Efficacy for activation of the melanocortin MC4 receptor increased with every extra subunit, resulting in a 100 fold lower EC50 value of alpha-MSH8 when compared with alpha-MSH1. Furthermore, supernatant of cells transfected with alpha-MSH8 proved to have an increased affinity to the melanocortin MC4 receptor when compared to cells transfected with the other multimers. Together, these data show that multimeric alpha-MSH has improved ability to activate the human melanocortin MC4 receptor in vitro.