Proopiomelanocortin (POMC), the ACTH/melanocortin precursor, is secreted by human epidermal keratinocytes and melanocytes and stimulates melanogenesis.

Proopiomelanocortin (POMC) can be processed to ACTH and melanocortin peptides. However, processing is incomplete in some tissues, leading to POMC precursor release from cells. This study examined POMC processing in human skin and the effect of POMC on the melanocortin-1 receptor (MC-1R) and melanocyte regulation. POMC was secreted by both human epidermal keratinocytes (from 5 healthy donors) and matched epidermal melanocytes in culture. Much lower levels of alpha-MSH were secreted and only by the keratinocytes. Neither cell type released ACTH. Cell extracts contained significantly more ACTH than POMC, and alpha-MSH was detected only in keratinocytes. Nevertheless, the POMC processing components, prohormone convertases 1, 2 and regulatory protein 7B2, were detected in melanocytes and keratinocytes. In contrast, hair follicle melanocytes secreted both POMC and alpha-MSH, and this was enhanced in response to corticotrophin-releasing hormone (CRH) acting primarily through the CRH receptor 1. In cells stably transfected with the MC-1R, POMC stimulated cAMP, albeit with a lower potency than ACTH, alpha-MSH, and beta-MSH. POMC also increased melanogenesis and dendricity in human pigment cells. This release of POMC from skin cells and its functional activity at the MC-1R highlight the importance of POMC processing as a key regulatory event in the skin.

Neuropeptide processing and its impact on melanocortin pathways.

Proopiomelanocortin (POMC) is processed in an intracellular secretory pathway, primarily to enable release of ACTH from the pituitary and alpha-MSH from hypothalamic neurons and skin. However, processing is incomplete and unprocessed POMC is secreted from all three tissues. This review considers intracellular processing of neuronal POMC as a key checkpoint that controls flux through hypothalamic melanocortin receptor pathways. Regulation of the convertase, proprotein convertase (PC)-1/3, which cleaves POMC is likely to determine the extent of POMC processing. Reduced PC1/3 activity, in both humans and rodents, leads to reduced melanocortin signaling and hence obesity. In contrast to POMC, posttranslational processing of proagouti-related peptide, an endogenous melanocortin-4 receptor antagonist, is efficient and is unlikely to represent a regulatory checkpoint. Because POMC is fully processed to ACTH and MSH peptides in secretory vesicles, unprocessed POMC, which is released from cells, must exit via an unregulated constitutive pathway. Therefore, the targeting of POMC to secretory granules controls the extent of POMC cleavage. There is evidence that PC1/3 is involved in cleavage of POMC in the trans-Golgi network and regulation of trafficking to the secretory pathway, in which it subsequently cleaves POMC to the melanocortin peptides. This would suggest that alpha-MSH and beta-MSH may be subject to alternative sorting mechanisms, leading to heterogeneity in secretory granule content in POMC-producing cells. Overall, these studies implicate POMC processing as a key regulatory mechanism in the control of energy homeostasis.

Agouti-related protein is posttranslationally cleaved by proprotein convertase 1 to generate agouti-related protein (AGRP)83-132: interaction between AGRP83-132 and melanocortin receptors cannot be influenced by syndecan-3.

Agouti-related protein (AGRP) plays a key role in energy homeostasis. The carboxyl-terminal domain of AGRP acts as an endogenous antagonist of the melanocortin-4 receptor (MC4-R). It has been suggested that the amino-terminal domain of AGRP binds to syndecan-3, thereby modulating the effects of carboxyl-terminal AGRP at the MC4-R. This model assumes that AGRP is secreted as a full-length peptide. In this study we found that AGRP is processed intracellularly after Arg(79)-Glu(80)-Pro(81)-Arg(82). The processing site suggests cleavage by proprotein convertases (PCs). RNA interference and overexpression experiments showed that PC1/3 is primarily responsible for cleavage in vitro, although both PC2 and PC5/6A can also process AGRP. Dual in situ hybridization demonstrated that PC1/3 is expressed in AGRP neurons in the rat hypothalamus. Moreover, hypothalamic extracts from PC1-null mice contained 3.3-fold more unprocessed full-length AGRP, compared with wild-type mice, based on combined HPLC and RIA analysis, demonstrating that PC1/3 plays a role in AGRP cleavage in vivo. We also found that AGRP(83-132) is more potent an antagonist than full-length AGRP, based on cAMP reporter assays, suggesting that posttranslational cleavage is required to potentiate the effect of AGRP at the MC4-R. Because AGRP is cleaved into distinct amino-terminal and carboxyl-terminal peptides, we tested whether amino-terminal peptides modulate food intake. However, intracerebroventricular injection of rat AGRP(25-47) and AGRP(50-80) had no effect on body weight, food intake, or core body temperature. Because AGRP is cleaved before secretion, syndecan-3 must influence food intake independently of the MC4-R.

Agouti-related protein: more than a melanocortin-4 receptor antagonist?

It is well established that agouti-related protein (AGRP) can act as a competitive antagonist to proopiomelanocortin (POMC)-derived peptides at the melanocortin-4 receptor (MC4R), and that this homeostatic mechanism is important as a means of coordinating appetite with perceived metabolic requirement. However, there are clearly additional facets to the physiological role of AGRP, given that it is active in MC4R knockout mice and it has strikingly long-lasting effects on food intake, compared with MC4R agonists. In this review we focus on: (i) evidence that AGRP is more sensitive to perturbations in energy balance than POMC and is therefore the primary basis of melanocortinergic regulation. (ii) Evidence that the bioactive peptide AGRP83-132, acts by alternate mechanism(s) to elicit its long-term effects on food intake. (iii) Evidence that AGRP is post-translationally cleaved to generate AGRP83-132 and one or more N terminal peptides, which may have an important physiological role(s) that are independent of the melanocortin system. A clear understanding of how proAGRP processing is regulated, and the role of resultant peptides, may define additional therapeutic targets in the treatment of obesity.

Proopiomelanocortin-derived peptides in rat cerebrospinal fluid and hypothalamic extracts: evidence that secretion is regulated with respect to energy balance.

Regulation of proopiomelanocortin (POMC) is an important means of controlling the central melanocortin system. It has never been established whether the spectrum of POMC-derived peptides synthesized and secreted from the hypothalamus is altered in response to changes in energy homeostasis in vivo. To monitor secretion, we analyzed peptide content of rat cerebrospinal fluid. Strikingly, both the POMC precursor and ACTH were readily detected. Moreover, levels of both were lower in samples from obese Zucker rats (fa/fa) vs. lean Zucker rats (+/+, fa/+) and from fasted vs. fed rats, whereas alpha MSH could not be detected. POMC levels were also decreased in hypothalamic extracts from obese and fasted animals. In contrast, despite being the most predominant peptide in extracts, alpha MSH levels were not significantly changed in any of the rat models. The ratio of precursor to derived peptides in cerebrospinal fluid was significantly higher in obese vs. lean and fed vs. fasted rats, indicating that secretion of POMC-derived peptides is differentially down-regulated during negative energy balance. In contrast to peptide analysis, we found that POMC gene expression was not significantly decreased in fasted rat hypothalami. We conclude that regulation of peptide secretion is an important mechanism by which the POMC system is controlled.

Repeated administration of the anorectic factor prolactin-releasing peptide leads to tolerance to its effects on energy homeostasis.

Central administration of a single dose of prolactin-releasing peptide (PrRP) causes a reduction in both fast-induced and nocturnal food intake and body weight gain. The aim of this study was to examine the effect of repeated administration of PrRP on energy homeostasis, including a measure of the expression of the mitochondrial uncoupling protein-1 (UCP-1) in brown adipose tissue. Conscious, free-feeding animals received central injections of PrRP (4 nmol icv) or vehicle. A single injection at 1000 caused a sustained hyperthermia over the 4-h test period and an increase in the expression of UCP-1 mRNA. Repeated, twice daily injection caused a reduction in body weight gain greater than that seen in pair-fed animals for the first 48-72 h. After 72 h, the animals became refractory to the actions of PrRP. The pair-fed group showed a reduction in UCP-1 mRNA expression at 48 h, which was reversed by PrRP treatment. This study indicates that PrRP exerts its effects on energy homeostasis in the short-medium term by reducing food intake and increasing energy expenditure.

A missense mutation disrupting a dibasic prohormone processing site in pro-opiomelanocortin (POMC) increases susceptibility to early-onset obesity through a novel molecular mechanism.

The functional loss of both alleles of the human pro-opiomelanocortin (POMC) gene leads to a very rare syndrome of hypoadrenalism, red hair and early-onset obesity. In order to examine whether more subtle genetic variants in POMC might contribute to early-onset obesity, the coding region of the gene was sequenced in 262 Caucasian subjects with a history of severe obesity from childhood. Two children were found to be heterozygous for a missense mutation, R236G, which disrupts the dibasic cleavage site between beta melanocyte-stimulating hormone (beta-MSH) and beta-endorphin. Beta-TC3 cells transfected with the mutant POMC cDNA produced a mutant beta-MSH/beta-endorphin fusion protein. This fusion protein bound to the human melanocortin-4 receptor (hMC4R) with an affinity similar to its natural ligands, but had a markedly reduced ability to activate the receptor. This variant co-segregated with early-onset obesity over three generations in one family and was absent in 412 normal weight UK Caucasian controls. Combining the results in UK Caucasians with a new case-control study in French subjects and three previously published reports, mutations disrupting this processing site were present in 0.88% of subjects with early-onset obesity and 0.22% of normal-weight controls. These results suggest that the R236G mutation may confer an inherited susceptibility to obesity through the production of an aberrant fusion protein that has the capacity to interfere with central melanocortin signalling.