An indoline-derived compound that markedly reduces mouse body weight.

OBJECTIVE: We describe how a single intraperitoneal injection of an indoline-derived drug (SN 28127) reduced mouse body weight (25-45% loss) and adipose tissue mass (∼75%). METHODS AND RESULTS: The reductions in body weight peaked at ∼21-28 days post drug injection and were maintained throughout the study (160 days). The mice ate as much as vehicle-treated control mice. A more potent SN 28127 analog (SN 29220) reversed high-fat diet-induced obesity and type 2 diabetes in C57BL/6J mice on a high-fat diet. Insulin induced a sustained reduction in blood glucose in fasted SN 29220-treated mice compared with the vehicle-treated mice. All drug-treated mice exhibited a transient increase in water intake from ∼10 days post drug injection that lasted for ∼70 days. Following a single injection of (3)H-labeled SN 29220, radioactivity accumulated within 4 h in the liver, bile duct and ileum with little detected in the brain; within 1-2 days, most of the radioactivity was found in the pancreas, spleen, liver, bile duct, stomach, kidneys and white adipose tissue. High levels of glucose were detected in urine collected from SN 29220 but not vehicle-treated C57BL/6J mice at ∼60 days post injection, while fecal triacylglycerols and cholesterol were not different between SN 29220 and vehicle-treated mice. These data lead us to hypothesize that the hepatic system is the primary drug target. Genes involved in fatty acid synthesis (FASn, SCD1 and PPARγ) and appetite stimulation (AGRP) were upregulated at 160 days post drug treatment, indicative of adaptation to reduced body weight. CONCLUSION: We hypothesize that indoline-derived drug-induced chronic toxicity to the hepatic system leads to a reduction in white adipose tissue mass. The mice adapt to this drug-induced toxicity with reduced steady-state body weight. Understanding molecular mechanisms underlying these responses has potential to identify novel targets for prevention and treatment of obesity.

The cloning of a family of genes that encode the melanocortin receptors.

Melanocyte-stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH) regulate pigmentation and adrenal cortical function, respectively. These peptides also have a variety of biological activities in other areas, including the brain, the pituitary, and the immune system. A complete understanding of the biological activities of these hormones requires the isolation and characterization of their corresponding receptors. The murine and human MSH receptors (MSH-Rs) and a human ACTH receptor (ACTH-R) were cloned. These receptors define a subfamily of receptors coupled to guanine nucleotide-binding proteins that may include the cannabinoid receptor.

Influence of thyroidectomy and prolactin suppression on the growth of N-nitrosomethylurea-induced rat mammary carcinomas.

Mammary carcinomas were induced in female Sprague-Dawley rats with N-nitrosomethylurea. Thyroidectomy increased the serum prolactin and reduced serum growth hormone levels of 17 rats without affecting tumor growth. Pergolide mesylate, 80 micrograms twice daily for 7 days, suppressed the serum prolactin of another 17 animals; seven of 17 tumors continued to grow, four became static, and six (35%) underwent partial regression. Treatment with pergolide mesylate plus thyroidectomy reduced both serum prolactin and growth hormone in all of 14 rats, caused regression of ten of the 14 tumors (71%), while two became static, and two continued to grow. Five of the ten regressions were complete. Only the combined thyroidectomy-pergolide treatment group showed a significant difference in posttreatment surface area compared with the controls (p less than 0.001). Ovine growth hormone, 40 micrograms/hr delivered by s.c. osmotic minipumps for 7 days, stimulated regrowth of six of seven tumors undergoing regression in response to thyroidectomy plus pergolide; the other one became static. Thyroxine, 2 micrograms/100 g body weight, stimulated regrowth of the tumors in another six thyroidectomized rats despite continued suppression of prolactin by pergolide. Thus, regression of N-nitrosomethylurea-induced mammary tumors produced by thyroidectomy plus pergolide is due to the combined suppression of circulating growth hormone and prolactin.

Abnormal insulin-receptor down regulation and dissociation of down regulation from insulin biological action in cultured human tumor cells.

The sensitivity of insulin receptors to down regulation by insulin has been measured in cultured human tumor cells (breast tumor cell lines MCF-7, T-47D, and colon tumor cell line HCT-8). Insulin receptors on breast tumor cells were resistant to down regulation (15-17% maximum loss of insulin binding after 4 h exposure to 170 nM insulin). HCT-8 cells were sensitive to down regulation after 4 h exposure to 3.8 nM insulin, but the extent of down regulation then lessened at higher concentrations of insulin. This paradoxical behavior was associated with increasing affinity of insulin receptors for insulin following exposure to hormone. Insulin-stimulated [3H]leucine incorporation into protein was measured in parallel with studies of receptor regulation to assess the effect of preexposure of cells to insulin on cell metabolism. Maximum down regulation of receptors in all three types of tumor cell by prior exposure to insulin did not significantly alter the responsiveness of any of the cell lines to insulin. Thus insulin receptor down regulation is abnormal in these tumor lines compared with reported studies in normal cells, and this may contribute a metabolic advantage to these malignant cells over normal tissues.

Insulin receptor regulation in cultured human tumor cells.

Insulin binding to monolayer cell cultures of human fibroblasts, human colon carcinoma (HCT-8, HT-29), human breast carcinoma (MCF-7, T-47D), and melanoma (MM-96) was measured using 125I-insulin. Binding was time and temperature dependent in all cell lines, and only one cell line (MM-96) degraded 125I-insulin. High-affinity insulin-binding sites (Kd = 1.4 X 10(-9) M to 0.4 X 10(-10) M) were detected in all cell lines, and insulin-binding capacity ranged from 0.6 to 14 fmol/10(6) cells. Receptor down-regulation was studied by exposing cells to increasing concentrations of unlabeled insulin, dissociating surface-bound insulin and measuring residual receptors by 125I-insulin uptake. Exposure of tumor cells to greater than 10(-6) M insulin for 2 hr at 37 degrees led to a decrease in the number of insulin binding sites in MM-96 and colon cell lines only, with maximum down-regulation ranging from 58% (MM-96) to 88% (HCT-8) receptor loss. The decrease in insulin binding was due to a decreased number of receptors per cell with no change in affinity. Monolayers exposed to 1.7 X 10(-5) M unlabeled insulin for 7 hr at 37 degrees invariably showed greater than 50% receptor loss. However, monolayers exposed to 1.7 X 10(-8) M unlabeled insulin for 7 hr at 37 degrees showed less marked (0 to 39%) down-regulation. In comparison, human fibroblasts showed 57% receptor loss after exposure to 3.5 X 10(-9) M unlabeled insulin for 7 hr. Thus, markedly supraphysiological concentrations of insulin are required to down-regulate insulin receptors in tumor cell lines compared with normal cells. This suggests a tumor-associated resistance to receptor down-regulation.

Stimulation of tumors to synthesize tumor necrosis factor-alpha in situ using 5,6-dimethylxanthenone-4-acetic acid: a novel approach to cancer therapy.

The selective induction of tumor vascular collapse represents an exciting approach to cancer treatment. However, clinical evaluation of tumor necrosis factor-alpha (TNF), an agent that accomplishes this goal, has been limited by systemic toxicity, and clinical approaches using bacterial components to induce TNF production have also been disappointing. Our laboratory has developed synthetic low molecular weight inducers of TNF, including 5,6-dimethylxanthenone-4-acetic acid (DMXAA), as an alternative strategy. DMXAA induces rapid vascular collapse in transplantable murine tumors and induces TNF synthesis in vitro in both murine and human systems. We show here that the extent of DMXAA-induced TNF synthesis is greater in tumors than that in the spleen, liver, or serum. As shown by in situ hybridization studies of the murine Colon 38 tumor, DMXAA induced tumor as well as host cells to express TNF mRNA. The distribution of cells containing TNF mRNA in tumor tissues after DMXAA administration contrasted significantly with that obtained after lipopolysaccharide (LPS) treatment, although splenic and hepatic tissues showed a similar distribution of TNF mRNA-positive cells. In the Colon 38 tumor, the action of LPS was limited to host cells in the periphery of the vessels. DMXAA treatment induced 7-fold higher peak TNF levels in tumor than in serum. In contrast, LPS treatment induced 9-fold higher TNF levels in serum than in tumor. DMXAA induced 35-fold higher TNF activity in the Colon 38 tissue than did LPS. One ovarian, one squamous, and three melanoma human tumor xenografts implanted in athymic nude mice expressed TNF mRNA of human and murine origin in response to DMXAA, confirming that DMXAA can activate both host and tumor cells. The use of low molecular weight agents to induce TNF synthesis in situ in the tumor represents a novel approach to TNF-mediated therapy of cancers.

Molecular genetics of the ACTH and melanocyte-stimulating hormone receptors.

The related ACTH and melanocyte-stimulating hormone (MSH) receptors control adrenal steroidogenesis and pigmentation in response to an overlapping set of peptides derived from the proopiomelanocortin (POMC) molecule. The recent cloning of these receptors has already opened up a new understanding of their role in normal and pathologic functioning of the adrenal cortex, and of the process of pigmentation. The murine MSH receptor maps to a genetic locus called extension, a locus known since early in this century to control the relative amounts of the two major types of melanins: eumelanin and phaeomelanin. The highly variable pigmentation phenotypes resulting from different extension locus alleles are caused by structural mutations in the MSH receptor that alter the degree of its signal-transducing capacity. A mutation in the ACTH receptor in a patient with ACTH resistance has also recently been reported. It is likely that the etiology of this rare disease includes mutations that affect the functioning of the ACTH receptor.

Overproduction of the beta 1 form of protein kinase C enhances phorbol ester induction of glucose transporter mRNA.

Phorbol esters bind to and activate a family of Protein Kinase C (PKC) proteins, although the degree to which the various PKC forms mediate specific biological functions is unknown. We and others have previously shown that, after exposure to phorbol esters, cultured fibroblasts exhibit increased expression of the mRNA encoding the HepG2/brain glucose transporter (GT mRNA). We therefore studied phorbol ester regulation of GT mRNA in rat fibroblasts which do (R6-PKC3) or do not (R6-C1) stably overproduce a full-length cDNA encoding the PKC beta 1 isotype. When PKC beta 1 is overproduced in a cell that normally has undetectable levels, it is capable of increasing HepG2/brain GT mRNA in response to alpha-D-glucose phorbol 12-myristate 13-acetate (TPA). The level of GT mRNA is maximally induced 6 to 8-fold over basal levels 6 h after exposure to TPA (10 ng/ml) in R6-PKC3 cells that overproduce PKC beta 1, but only 2-fold over basal levels in the control R6-C1 cells. This TPA induced increase in the level of GT mRNA was observed as early as 1 h, peaked by 6-8 h and decreased markedly by 24 h in both cell types. The effect of PKC beta 1 on GT mRNA expression is probably mediated through enhancement of transcription, since the stability of GT mRNA was only minimally affected by TPA. Unlike the enhancement of TPA induced GT mRNA expression caused by overexpression of PKC beta 1, the responses of GT mRNA to calf serum, platelet-derived growth factor, epidermal growth factor, insulin or insulin-like growth factor-1 were the same in both cell types. After pretreatment with 1000 ng/ml TPA in 0.5% calf serum for 24 h, PKC activity was down-regulated and both R6-C1 and R6-PKC3 cells showed complete down-regulation of the GT mRNA responses to an additional treatment with 1000 ng/ml TPA. In contrast to the marked loss of responsiveness to TPA and PKC down-regulation, the responses of GT mRNA to serum, PDGF, EGF, insulin and IGF-1 were unaffected by down-regulation. Thus, our results provide direct evidence for both PKC-dependent and independent pathways regulating GT gene expression. Furthermore, it appears that the level of PKC beta 1 production, rather than down-stream signal transduction events, is the rate-limiting step in the pathway by which TPA induces an increase in GT mRNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Localization of the melanocortin-4 receptor (MC4-R) in neuroendocrine and autonomic control circuits in the brain.

POMC, the precursor of ACTH, MSH, and beta-endorphin peptides, is expressed in the pituitary and in two sites in the brain, in the arcuate nucleus of the hypothalamus and the commissural nucleus of the solitary tract of the brain stem. Little is known regarding the functions of melanocortin (ACTH and MSH) peptides in the brain. We report here the detailed neuroanatomical distribution of the MC4-R mRNA in the adult rat brain. The melanocortin 3 receptor (MC3-R), characterized previously, was found to be expressed in arcuate nucleus neurons and in a subset of their presumptive terminal fields but in few regions of the brainstem. The highly conserved MC4-R is much more widely expressed than MC3-R and is pharmacologically distinct. MC4-R mRNA was found in multiple sites in virtually every brain region, including the cortex, thalamus, hypothalamus, brainstem, and spinal cord. Unlike the MC3-R, MC4-R mRNA is found in both parvicellular and magnocellular neurons of the paraventricular nucleus of the hypothalamus, suggesting a role in the central control of pituitary function. MC4-R is also unique in its expression in numerous cortical and brainstem nuclei. Together, MC3-R and/or MC-4R mRNA are found in every nucleus reported to bind MSH in the adult rat brain and define neuronal circuitry known to be involved in the control of diverse neuroendocrine and autonomic functions. The high degree of conservation, distinct pharmacology, and unique neuronal distribution of the MC4 receptor suggest specific and complex roles for the melanocortin peptides in neuroendocrine and autonomic control.

Pro-Opiomelanocortin (POMC) Neurones, POMC-Derived Peptides, Melanocortin Receptors and Obesity: How Understanding of this System has Changed Over the Last Decade.

Following the cloning of the melanocortin receptor and agouti protein genes, a model was developed for the central melanocortin system with respect to the regulation of energy and glucose homeostasis. This model comprised leptin regulation of melanocortin peptides and agouti-related peptide (AgRP) produced from central pro-opiomelanocortin (POMC) and AgRP neurones, respectively, as well as AgRP competitive antagonism of melanocortin peptides activating melanocortin 4 receptor (MC4R) to Gαs and the cAMP signalling pathway. In the last decade, there have been paradigm shifts in our understanding of the central melanocortin system as a result of the application of advanced new technologies, including Cre-LoxP transgenic mouse technology, pharmacogenetics and optogenetics. During this period, our understanding of G protein coupled receptor signal transduction has also dramatically changed, such that these receptors are now known to exist in the plasma membrane oscillating between various inactive and active conformational states, and the active states signal through G protein-dependent and G protein-independent pathways. The present review focuses on evidence obtained over the past decade that has changed our understanding of POMC gene expression and regulation in the central nervous system, POMC and AgRP neuronal circuitry, neuroanatomical functions of melanocortin receptors, melanocortin 3 receptor (MC3R) and MC4R, and signal transduction through MC3R and MC4R.

Physiological consequences of ectopic agouti gene expression: the yellow obese mouse syndrome.

This review summarizes primary and downstream phenotypic manifestations, with emphasis on altered responsiveness to environmental stimuli, of dominant yellow mutations at the mouse agouti locus. Obvious effects include hyperinsulinemia, obesity, stimulation of somatic growth and tumorigenesis, and coat color. Downstream influences of hyperinsulinemia and obesity on the individual's physiology determine important components of the obese yellow agouti mouse syndrome. Collectively, the phenotypic aberrations described support the concept that identical genomes are expressed in a spectrum of physiological phenotypes that reflect the complex interdependence of gene-regulated physiological pathways and processes in the organism throughout extended, but temporally ordered, periods of fetal and neonatal development and aging. This summary identifies important areas for additional research and provides integrated information required for a systematic approach to the development of interventions for common adult human health problems.

Melanocortin receptor-mediated mobilization of intracellular free calcium in HEK293 cells.

Mouse melanocortin receptors, MC1-R, MC3-R, MC4-R, and MC5-R, when expressed in HEK293 cells and stimulated with either alpha-melanocyte-stimulating hormone (alpha-MSH) or desacetyl-alpha-MSH, mediate increases in intracellular free calcium concentration ([Ca(2+)](i)) with EC(50) values between 0.3 and 4.3 nM. The increase in [Ca(2+)](i) is cholera toxin sensitive and pertussis toxin insensitive. The mechanism involves calcium mobilization from intracellular stores without a transient rise in inositol trisphosphate. Mouse agouti protein (55 nM) is a competitive antagonist of alpha-MSH (6-fold) and desacetyl-alpha-MSH (8-fold), coupling the mMC1-R to increased [Ca(2+)](i). Agouti protein (55 nM) significantly increased the EC(50) for alpha-MSH (3-fold), and 550 nM agouti protein significantly increased the EC(50) for desacetyl-alpha-MSH (4-fold), coupling the mMC4-R to a rise in [Ca(2+)](i). However, agouti protein antagonism of the MC4-R may not be competitive since there was a trend for the maximum response to also increase. There was no significant antagonism of the MC3-R and MC5-R by agouti protein (55 nM). Understanding the physiological relevance of the transduction of a calcium signal by melanocortin peptides may be important for future development of therapeutic targeting of the melanocortin receptors.

Vanadate regulates glucose transporter (Glut-1) expression in NIH3T3 mouse fibroblasts.

Vanadate, the major oxidized form of the essential trace element vanadium, has rapid effects on glucose transport in vitro and more delayed effects on glucose transport in vivo. We addressed the question that one potential mechanism for the delayed effects of vanadate on glucose homeostasis could be altered expression of one or more of the genes encoding glucose transporters. To do this we studied vanadate regulation of Glut-1 and Glut-4 in NIH3T3 mouse fibroblasts. Vanadate (5-40 microM) induced cells to proliferate to higher cell densities, and in addition, 40 microM vanadate caused the cells to exhibit a transformed morphology. Glut-1 mRNA was maximally induced 4- to 5-fold over the control value after 6-h exposure to 30 microM vanadate. Unlike the response to serum and growth factors, the vanadate-induced increase in Glut-1 mRNA remained elevated over the control value in the presence of vanadate for 5 days. The vanadate effect was serum dependent and was fully reversible when vanadate was removed from the medium. In the absence of vanadate, the half-life of Glut-1 mRNA was 0.5-1 h, whereas after treatment for 5 h with 30 microM vanadate the half-life was increased to 1.5-2 h. Thus, mRNA stabilization accounts for at least a part of the increase in glucose transporter mRNA levels after vanadate treatment. Glut-4 mRNA was not detected in these cells in either the absence or presence of vanadate. While the importance of this increased Glut-1 gene expression for the vanadate effect on normalization of blood glucose in vivo remains to be determined, an association between vanadate-induced cell proliferation and transformed phenotype, and vanadate-induced Glut-1 mRNA in vitro has been made. Possible potential therapeutic use of vanadate for treatment of diabetes must, therefore, be viewed with caution.

Agouti antagonism of melanocortin-4 receptor: greater effect with desacetyl-alpha-melanocyte-stimulating hormone (MSH) than with alpha-MSH.

Desacetyl-alpha-MSH is more abundant than alpha-MSH in the brain, the fetus, human blood, and amniotic fluid, but there is little information on its ability to interact with melanocortin receptors. The aim of this study is to compare and contrast the ability of desacetyl-alpha-MSH and alpha-MSH to couple melanocortin receptors stably expressed in HEK293 cells, to the protein kinase A (PKA) signaling pathway. Desacetyl-alpha-MSH activated mouse MC1, MC3, MC4 and MC5 receptors with EC50s = 0.13, 0.96, 0.53, and 0.84 nM, and alpha-MSH activated these receptors with EC50s = 0.17, 0.88, 1.05, and 1.34 nM, respectively. Mouse agouti protein competitively antagonized alpha-MSH and desacetyl-alpha-MSH coupling to the MC1-R similarly. In contrast, mouse agouti protein antagonized desacetyl-alpha-MSH much more effectively and potently than alpha-MSH coupling the MC4-R to the PKA signaling pathway. Furthermore, mouse agouti protein (10 nM) significantly reduced (1.4-fold) the maximum response of mMC4-R to desacetyl-alpha-MSH and 100 nM mouse agouti significantly increased (4.8-fold) the EC50. Minimal antagonism of alpha-MSH coupling mMC4-R to the PKA signaling pathway was observed with 10 nM mouse agouti, whereas both 50 and 100 nM mouse agouti appeared to reduce the maximum reponse (1.1- and 1.3-fold, respectively) and increase the EC50 (2.5- and 3.4-fold respectively). Mouse agouti protein did not significantly antagonize either alpha-MSH or desacetyl-alpha-MSH coupling mouse MC3 and MC5 receptors. Understanding the similarities and differences in activation of melanocortin receptors by desacetyl-alpha-MSH and alpha-MSH will contribute to delineating the functional roles for these endogenous melanocortin peptides.

Melanocortin-4 receptor messenger RNA expression is up-regulated in the non-damaged striatum following unilateral hypoxic-ischaemic brain injury.

Melanocortin peptides (alpha-melanocyte-stimulating hormone, adrenocorticotropin and fragments thereof) have been shown to have numerous effects on the central nervous system, including recovery from nerve injury and retention of learned behaviour, but the mechanism of action of these peptides is unknown. A family of five melanocortin receptors have recently been discovered, two of which (melanocortin-3 and melanocortin-4 receptors) have been mapped in the rat brain. We have tested the hypothesis that the expression of one or more of the messenger RNAs for three melanocortin receptors (melanocortin-3, melanocortin-4 and melanocortin-5 receptors) would be altered in rat brain following unilateral transient hypoxic-ischaemic brain injury. In this study, using in situ hybridization, we show that melanocortin-4 receptor messenger RNA was up-regulated in the striatum in the non-damaged hemisphere within 24 h after severe hypoxic-ischaemic injury compared with control brains (P<0.05). In a small group of animals, this induction was not blocked by treatment with the anticonvulsant, carbamazepine. Expression of melanocortin-3 receptor messenger RNA in the brain was not altered in this hypoxic-ischaemic injury model and melanocortin-5 receptor messenger RNA was not detected in either control or hypoxic-ischaemic injured rat brains. We hypothesize that the up-regulation of melanocortin-4 receptor messenger RNA expression in the contralateral striatum may be involved in transfer of function to the uninjured hemisphere following unilateral brain injury.

Long-term alterations in body weight do not affect the expression of melanocortin receptor-3 and -4 mRNA in the ovine hypothalamus.

The pro-opiomelanocortin-derived peptides and the melanocortin receptors are implicated in various functions within the CNS including the regulation of food intake. In the present study, we used in situ hybridization, with specific 35S-labelled ovine riboprobes to map the expression of melanocortin receptor-3 (MC3-R) and -4 (MC4-R) mRNA in the diencephalon and brainstem of normal female sheep. Furthermore, we examined the effect of long-term alterations in energy balance on the distribution and expression of MC3-R and MC4-R mRNA in food-restricted and ad libitum-fed ovariectomized female sheep. The distribution of melanocortin receptors generally resembled that of the rat. A high number of MC3-R-labelled cells were seen in the ventral division of the lateral septum and the medial preoptic area. In the hypothalamus, a moderate number of MC3-R-labelled cells was observed in the lateral hypothalamic area while other nuclear groups had low to intermediate numbers of MC3-R-labelled cells. The distribution of MC4-R mRNA was generally similar to that of MC3-R mRNA in the septal/preoptic and hypothalamic regions, with a high number of labelled cells present in the intermediate division of the lateral septum. Within the hypothalamus, no MC4-R mRNA expression was observed in the arcuate nucleus. There was more widespread distribution of moderate to low numbers of MC4-R mRNA-expressing cells in the brainstem compared to that of MC3-R mRNA. Unlike findings in the rat, only a low number of cells expressed melanocortin receptor mRNA in the ovine hypothalamic nuclei associated with feeding behavior. The number of melanocortin receptor-labelled cells and the level of expression (silver grains/cell) in the hypothalamic feeding centers was similar in food-restricted and ad libitum-fed animals. These findings suggest that long-term alterations in metabolic status do not change the melanocortin receptor mRNA distribution and/or expression in the sheep hypothalamus.

The human melanocyte stimulating hormone receptor has evolved to become "super-sensitive" to melanocortin peptides.

Melanocyte-stimulating hormone (MSH) stimulates pigmentation in mammals by activating specific cell surface MSH receptors (MC1-Rs) on melanocytes. MC1-Rs on normal human melanocytes have been difficult to detect and characterise. The pharmacological characterisation of a cloned human MC1-R (hMC1-R) is reported here, and directly compared with that of a cloned mouse MC1-R (mMC1-R). The human and mouse MC1-Rs are equally sensitive (EC50 = 1-2 pM) to the super potent analogue of alpha-MSH, NDP-MSH. In contrast with the mMC1-R, the hMC1-R is also very sensitive to alpha-MSH (EC50 = 2 pM), ACTH (EC50 = 8 pM), and Lys gamma 3-MSH (EC50 < 10(-10) M). This suggests that in man, in contrast with rodents, both ACTH and Lys gamma 3-MSH may have physiological roles in pigmentation.

Obesity, diabetes and functions for proopiomelanocortin-derived peptides.

Melanocortin peptides (adrenocorticotropin (ACTH), alpha-,beta-, and gamma-melanocyte stimulating hormone (MSH), and fragments thereof) derived from proopiomelanocortin (POMC) have a diverse array of biological activities, many of which have yet to be fully elucidated. The recent cloning of a family of five distinct melanocortin receptors through which these peptides act has provided the tools to further our understanding of melanocortin peptide functions. Early work on melanocortin peptides focused on their roles in pigmentation, adrenocortical function, the immune, central and peripheral nervous systems. Although melanocortin peptides have long been known to affect lipolysis, characterisation of the melanocortin receptors has opened up several lines of evidence for important roles in the development of obesity, insulin resistance and type II diabetes. We present here a review of the current evidence for melanocortin peptides playing such a role, and based on this evidence, a model for melanocortin peptides and their receptors in maintaining energy balance.

ACTH induces up-regulation of ACTH receptor mRNA in mouse and human adrenocortical cell lines.

Corticotropin (ACTH) binds to specific receptors in the adrenal cortex and thereby regulates glucocorticoid and mineralocorticoid production. The number of ACTH binding sites on adrenocortical cells is increased by exposure of cells to activators of the cAMP pathway. The mechanism responsible for the increase in ACTH binding sites is not known. We therefore studied the levels of ACTH-R mRNA in mouse Y-1 and human NCI-H295 (H295) adrenocortical carcinoma cell lines. ACTH induced an increase in mouse ACTH-R mRNA in Y-1 cells that was time and dose dependent, increasing 6-fold over basal levels following exposure to 10(-8) M ACTH for 19-24 h. The amount of human ACTH-R mRNA in H295 cells increased 2-4-fold following a 24 h exposure to 10(-8) M ACTH, 1 mM dbcAMP, or 10(-5) M Forskolin. Treatment of H295 cells with angiotensin II (A-II) was found to dramatically increase the level of ACTH-R mRNA. These data indicate that regulation of ACTH-R mRNA levels is at least one mechanism by which ACTH and A-II elevate the number of ACTH binding sites in the adrenocortical cells.

Mouse melanocortin-4 receptor gene 5'-flanking region imparts cell specific expression in vitro.

Weight homeostasis is exquisitely sensitive to changes in the abundance of melanocortin-4 receptor (MC4-R). To begin to understand the factors that regulate MC4-R gene expression, we determined there are no introns in the gene, there are multiple starts of transcription, and a cluster of 3' ends. A series of MC4-R-luciferase gene reporter chimerics was developed and transfected into cell lines expressing (UMR106; GT1-7; HEK293) and not expressing (Neuro 2A) endogenous MC4-R mRNA. The longest construct, which includes approximately 3.3 kb 5'-flanking, 425 bp 5'-untranslated (UTR) and 1852 bp 3'-flanking, significantly increased luciferase reporter gene expression 24-, 13-, and 3-fold compared to pGL3-basic when expressed in HEK293, UMR106, and GT1-7 cells, respectively. Deletion analysis of mMC4-R 5'-flanking cDNA identified full mMC4-R promoter activity within 178 bp upstream of the major start of transcription. The mMC4-R gene structure and reporter chimerics provide a fundamental framework for the identification of specific factors regulating MC4-R gene expression.