Genetic deciphering of the antagonistic activities of the melanin-concentrating hormone and melanocortin pathways in skin pigmentation.

The genetic origin of human skin pigmentation remains an open question in biology. Several skin disorders and diseases originate from mutations in conserved pigmentation genes, including albinism, vitiligo, and melanoma. Teleosts possess the capacity to modify their pigmentation to adapt to their environmental background to avoid predators. This background adaptation occurs through melanosome aggregation (white background) or dispersion (black background) in melanocytes. These mechanisms are largely regulated by melanin-concentrating hormone (MCH) and α-melanocyte-stimulating hormone (α-MSH), two hypothalamic neuropeptides also involved in mammalian skin pigmentation. Despite evidence that the exogenous application of MCH peptides induces melanosome aggregation, it is not known if the MCH system is physiologically responsible for background adaptation. In zebrafish, we identify that MCH neurons target the pituitary gland-blood vessel portal and that endogenous MCH peptide expression regulates melanin concentration for background adaptation. We demonstrate that this effect is mediated by MCH receptor 2 (Mchr2) but not Mchr1a/b. mchr2 knock-out fish cannot adapt to a white background, providing the first genetic demonstration that MCH signaling is physiologically required to control skin pigmentation. mchr2 phenotype can be rescued in adult fish by knocking-out pomc, the gene coding for the precursor of α-MSH, demonstrating the relevance of the antagonistic activity between MCH and α-MSH in the control of melanosome organization. Interestingly, MCH receptor is also expressed in human melanocytes, thus a similar antagonistic activity regulating skin pigmentation may be conserved during evolution, and the dysregulation of these pathways is significant to our understanding of human skin disorders and cancers.

Cannabinoid CB1 receptor restrains accentuated activity of hypothalamic corticotropin-releasing factor and brainstem tyrosine hydroxylase neurons in endotoxemia-induced hypophagia in rats.

It is well known that endocannabinoids play an important role in the regulation of food intake and body weight. Endocannabinoids and cannabinoid receptors are found in the hypothalamus and brainstem, which are central areas involved in the control of food intake and energy expenditure. Activation of these areas is related to hypophagia observed during inflammatory stimulus. This study investigated the effects of cannabinoid (CB1) receptor blockade on lipopolysaccharide (LPS)-induced hypophagia. Male Wistar rats were pretreated with rimonabant (10 mg/kg, by gavage) or vehicle; 30 min later they received an injection of either LPS (100 µg/kg, intraperitoneal) or saline. Food intake, body weight, corticosterone response, CRF and CART mRNA expression, Fos-CRF and Fos-α-MSH immunoreactivity in the hypothalamus and Fos-tyrosine hydroxylase (TH) immunoreactivity in the brainstem were evaluated. LPS administration decreased food intake and body weight gain and increased plasma corticosterone levels and CRF mRNA expression in the PVN. We also observed an increase in Fos-CRF and Fos-TH double-labeled neurons after LPS injection in vehicle-pretreated rats, with no changes in CART mRNA or Fos-α-MSH immunoreactive neurons in the ARC. In saline-treated animals, rimonabant pretreatment decreased food intake and body weight gain but did not modify hormone response or Fos expression in the hypothalamus and brainstem compared with vehicle-pretreated rats. Rimonabant pretreatment potentiated LPS-induced hypophagia, body weight loss and Fos-CRF and Fos-TH expressing neurons. Rimonabant did not modify corticosterone, CRF mRNA or Fos-α-MSH responses in rats treated with LPS. These data suggest that the endocannabinoid system, mediated by CB1 receptors, modulates hypothalamic and brainstem circuitry underlying the hypophagic effect during endotoxemia to prevent an exaggerated food intake decrease. This article is part of a Special Issue entitled 'Central Control of Food Intake'.

Molecular engineering of short half-life small peptides (VIP, αMSH and γ3MSH) fused to latency-associated peptide results in improved anti-inflammatory therapeutics.

OBJECTIVE: To facilitate the targeting to inflammation sites of small anti-inflammatory peptides, with short half-lives, by fusion with the latency-associated peptide (LAP) of transforming growth factor ß1 through a cleavable matrix metalloproteinase (MMP) linker. This design improves efficacy, overcoming the limitations to their clinical use. METHODS: We generated latent forms of vasoactive intestinal peptide (VIP), α-melanocyte-stimulating hormone (MSH) and γ(3)MSH by fusion to LAP through an MMP cleavage site using recombinant DNA technology. The biological activities of these latent therapeutics were studied in vivo using monosodium urate (MSU)-induced peritonitis and collagen-induced arthritis (CIA) models. We assessed gene therapy and purified protein therapy. RESULTS: The recruitment of the polymorphonuclear cells induced by MSU injection into mouse peritoneal cavity was reduced by 35% with γ(3)MSH (1 nmol), whereas administration of a much lower dose of purified latent LAP-MMP-γ(3)MSH (0.03 nmol) attenuated leucocyte influx by 50%. Intramuscular gene delivery of plasmids coding LAP-MMP-VIP and LAP-MMP-αMSH at disease onset reduced the development of CIA compared with LAP-MMP, which does not contain any therapeutic moiety. Histological analysis confirmed a significantly lower degree of inflammation, bone and cartilage erosion in groups treated with LAP-MMP-VIP or LAP-MMP-αMSH. Antibody titres to collagen type II and inflammatory cytokine production were also reduced in these two groups. CONCLUSION: Incorporation of small anti-inflammatory peptides within the LAP shell and delivered as recombinant protein or through gene therapy can control inflammatory and arthritic disease. This platform delivery can be developed to control human arthritides and other autoimmune diseases.

Effect of morphine on proopiomelanocortin gene expression and peptide levels in the hypothalamus.

Opiates have been reported to suppress POMC in the medial basal hypothalamus (MBH) but studies have been complicated by the fact that acutely, in the rat, opiates stimulate corticosterone and inhibit gonadal steroid release, which could both affect POMC in brain. We have therefore examined POMC gene expression and peptide levels in the MBH of castrated rats after 10 days of treatment with subcutaneous morphine or placebo pellets and after pellet removal. POMC mRNA was measured by solution hybridization assay and beta-endorphin (beta-EP) and alpha-MSH were measured by RIA. In castrated male rats, the mean POMC mRNA concentration in the MBH was 1.67 +/- 0.11 pg/microgram RNA in the control animals and decreased to 1.17 +/- 0.11 pg/microgram RNA in the morphine-treated animals (P < 0.01). Similarly in castrated, estradiol replaced female rats, the mean POMC mRNA level in the MBH was 1.36 +/- 0.19 pg/microgram RNA and decreased to 0.82 +/- 0.08 pg/microgram RNA after morphine treatment (P < 0.05). beta-EP levels were not significantly different in either study. When castrated male rats were similarly morphine pelleted and killed either on day 10 or 2 days later after pellet removal, the mean POMC mRNA level again fell from 1.83 +/- 0.21 in the controls to 1.28 +/- 0.20 pg/microgram RNA after 10 days of morphine; 2 days after pellet removal levels remained suppressed at 0.80 +/- 0.08 pg/microgram RNA (P < 0.01). In this study the concentrations of beta-EP and alpha-MSH were both noted to decline in the MBH after morphine treatment (P < 0.05). When the forms of beta-EP in the MBH were characterized by HPLC, a decrease in the concentration of beta-EP was again seen after morphine but no significant differences in the pattern of beta-EP processing or in the relative amounts of beta-EP1-31 compared to beta-EP1-27 and beta-EP1-26 were noted in morphine-treated animals. There was also no significant effect of 10(-6)-10(-4) M morphine on basal or KCl-stimulated release of beta-EP or gamma 3-MSH release from the perifused rat hypothalamus in vitro. We conclude that morphine suppresses POMC gene expression in the hypothalamus of chronically treated male and female rats. Persistent changes were also noted during morphine withdrawal. In some cases this was accompanied by a fall in beta-EP peptide content. These effects were seen in castrated animals with and without sex steroid replacement and are thus independent of the effects of morphine on the pituitary-gonadal axis. These results show that opiate drugs modify endogenous opioid systems in the brain and provide further support for the hypothesis that such changes may contribute to mechanisms of opiate dependence and withdrawal.

Identification of proopiomelanocortin neurones in rat hypothalamus by in situ cDNA-mRNA hybridization.

Ardrenocorticotropic hormone (ACTH), beta-endorphin and the melanotropins (MSHs) are all derived from a single large precursor molecule, proopiomelanocortin (POMC) by individual processing through a series of co- and post-translational modifications. Although the primary site of synthesis is in the pituitary, POMC-derived peptides have been identified in various tissues, notably the brain (see refs 6, 7 for review). A major question concerning brain POMC is whether it is synthesized within the central nervous system (CNS) itself or whether it is taken up from plasma flowing in a retrograde fashion from the pituitary. POMC peptides have been detected immunohistochemically and biochemically in the medial basal hypothalamus, the amygdala and throughout the brain stem. POMC peptide-containing cell bodies have been identified only in two cell groups, however, principally in the periarcuate region of the hypothalamus and to a lesser extent in the nucleus of the tractus solitarius. These and other observations have suggested that POMC peptides are synthesized locally in the medial basal hypothalamus and reach other regions of the CNS by axonal transport. Civelli et al. identified POMC mRNAs in nucleic acid extracts of rat and bovine hypothalami by solution hybridization as well as Northern gel blot analysis, but because of the close proximity of the hypothalamus to the pituitary and the extremely low amounts of POMC mRNA being measured in the hypothalamus, the possibility of tissue contamination during dissection could not be ruled out. We report here the anatomical co-localization of POMC-related peptides and POMC-specific mRNAs to a single major cell group in the medial basal hypothalamus. The presence of POMC-specific mRNA in a POMC peptide-containing cell in the brain is strong support for POMC biosynthesis within brain tissue.