Characterization of cell lines stably expressing human normal or mutated EGFP-tagged MC4R

The melanocortin receptor type 4 (MC4-R) is involved in food intake and represents a potential target for the treatment of some forms of obesity. The fluorescent protein EGFP was fused to the wild-type or mutated coding sequence of the human MC4-R. After transfection in HEK 293, clones stably expressing hMC4-R-EGFP were selected. Wild-type chimeric hMC4-R was well addressed to the cell membrane as demonstrated using confocal microscopy and displayed the same pharmacological characteristics as native hMC4R. NDP-alpha MSH induced a time-dependent internalization of MC4-R that was partially prevented by AgRP. The two mutated chimeric receptors studied here (CTCT-deleted and C271A) showed a high alteration of their response to ligand and were retained inside the cells. In conclusion, we have developed a model of clones stably expressing EGFP-tagged-hMC4-R. This is the only such model available to date and it provides a useful tool to follow the trafficking of MC4-R inside living cells.     

Antibodies raised against different extracellular loops of the melanocortin-3 receptor affect energy balance and autonomic function in rats

Melanocortin receptors (MCR) play an important role in the regulation of energy balance and autonomic function. In the present studies, we used active immunization against peptide sequences from the first and the third extracellular loop (EL1 and EL3) of the MC3R to generate selective antibodies (Abs) against this MCR subtype in rats. Immunization with the EL1 peptide resulted in Abs that enhanced the effects of the endogenous ligand α-melanocyte-stimulating hormone (α-MSH), whereas immunization with the EL3 peptide resulted in Abs acting as non-competitive antagonists. The phenotype of immunized rats chronically instrumented with telemetry transducers was studied under four different conditions: a high-fat diet was followed by standard lab chow, by fasting, and finally by an intraperitoneal injection of lipopolysaccharide (LPS). Under high-fat diet, food intake and body weight were higher in the EL3 than in the EL1 or the control group. Blood pressure was increased in EL3 rats and locomotor activity was reduced. Plasma concentrations of triglycerides, insulin, and leptin tended to rise in the EL3 group. After switching to standard lab chow, the EL1 group showed a small significant increase in blood pressure that was more pronounced and associated with an increase in heart rate during food restriction. No differences between the EL1 or the EL3 group were observed after LPS injection. These results show that immunization against the MC3R resulted in the production of Abs with positive or negative allosteric properties. The presence of such Abs induced small changes in metabolic and cardiovascular parameters.     

Antibodies raised against different extracellular loops of the melanocortin-3 receptor affect energy balance and autonomic function in rats

Melanocortin receptors (MCR) play an important role in the regulation of energy balance and autonomic function. In the present studies, we used active immunization against peptide sequences from the first and the third extracellular loop (EL1 and EL3) of the MC3R to generate selective antibodies (Abs) against this MCR subtype in rats. Immunization with the EL1 peptide resulted in Abs that enhanced the effects of the endogenous ligand α-melanocyte-stimulating hormone (α-MSH), whereas immunization with the EL3 peptide resulted in Abs acting as non-competitive antagonists. The phenotype of immunized rats chronically instrumented with telemetry transducers was studied under four different conditions: a high-fat diet was followed by standard lab chow, by fasting, and finally by an intraperitoneal injection of lipopolysaccharide (LPS). Under high-fat diet, food intake and body weight were higher in the EL3 than in the EL1 or the control group. Blood pressure was increased in EL3 rats and locomotor activity was reduced. Plasma concentrations of triglycerides, insulin, and leptin tended to rise in the EL3 group. After switching to standard lab chow, the EL1 group showed a small significant increase in blood pressure that was more pronounced and associated with an increase in heart rate during food restriction. No differences between the EL1 or the EL3 group were observed after LPS injection. These results show that immunization against the MC3R resulted in the production of Abs with positive or negative allosteric properties. The presence of such Abs induced small changes in metabolic and cardiovascular parameters.     

Long-term orexigenic effects of AgRP-(83---132) involve mechanisms other than melanocortin receptor blockade

Overexpression of agouti-related peptide (AgRP), an endogenous melanocortin (MC) 3 and 4 receptor antagonist (MC3/4-R), causes obesity. Exogenous AgRP-(83---132) increases food intake, but its duration and mode of action are unknown. We report herein that doses as low as 10 pmol can have a potent effect on food intake of rats over a 24-h period after intracerebroventricular injection. Additionally, a single third ventricular dose as low as 100 pmol in rats produces a robust increase in food intake that persists for an entire week. AgRP-(83---132) completely blocks the anorectic effect of MTII (MC3/4-R agonist), given simultaneously, consistent with a competitive antagonist action. However, when given 24 h prior to MTII, AgRP-(83---132) is ineffective at reversing the anorectic effects of the agonist. These results support a critical role of MC tone in limiting food intake and indicate that the orexigenic effects of AgRP-(83---132) are initially mediated by competitive antagonism at MC receptors but are sustained by alternate mechanisms.     

Downregulation of melanocortin-4 receptor during refeeding and its modulation by adrenalectomy in rats

Melanocortin system and corticotropin releasing hormone (CRH) are implicated in the control of feeding behavior. Besides its anorexigenic effect on food intake, CRH is one of the most important regulators of hypothalamic-pituitary-adrenal (HPA) axis activity. Therefore, there could be an interplay between HPA axis activity and melanocortin system. We investigated the expression of melanocortin-4 receptor (MC4-R) mRNA in the hypothalamus of rats after 14 days of food restriction or after a fasting-refeeding regimen, in sham or adrenalectomized rats. Male Wistar rats were subjected to free access to food or food ingestion restricted for 2 h a day (8-10 AM) during 14 d, when plasma corticosterone, ACTH, insulin, leptin concentrations, and MC4-R mRNA expression were determined before and after refeeding. Another set of rats was fasted for 48 h, followed by refeeding during 2 or 4 h on the seventh day after adrenalectomy (ADX) or sham surgery. On the day of the experiment, rats were anesthetized and perfused and the brain processed for MC4-R mRNA by in situ hybridization. Long-term reduction of food intake, either secondary to food restriction or adrenalectomy, reduced body weight gain and also leptin and insulin plasma concentrations. Food ingestion reduced MC4-R expression in the paraventricular nucleus in naive rats subjected to food restriction and also in sham rats fasted for 48 h. However, after ADX, MC4-R expression was not changed by refeeding. In conclusion, the present data indicate that MC4-R expression is downregulated by food ingestion and this response could be modulated by glucocorticoid withdrawal.     

Effects of selective modulation of the central melanocortin-3-receptor on food intake and hypothalamic POMC expression

Hypothalamic POMC neurons regulate energy balance via interactions with brain melanocortin receptors (MC-Rs). POMC neurons express the MC3-R which can function as an inhibitory autoreceptor in vitro. We now demonstrate that central activation of MC3-R with ICV infusion of the specific MC3-R agonist, [D-Trp(8)]-gamma-MSH, transiently suppresses hypothalamic Pomc expression and stimulates food intake in rats. Conversely, we also show that ICV infusion of a low dose of a selective MC3-R antagonist causes a transient decrease in feeding and weight gain. These data support a functional inhibitory role for the MC3-R on POMC neurons that leads to changes in food intake.     

C-terminal fragments of ACTH stimulate feeding in fasted rats.

Peptides derived from pro-opiomelanocortin, including alpha-MSH and ACTH, play important roles in the regulation of feeding. We investigated the central effect of ACTH 1-39 (ACTH) and peptides derived from the N-terminus (ACTH 1-10, Acetyl-ACTH 1-13-amide [alpha-MSH]) and C-terminus (ACTH 18-39 and ACTH 22-39) of this peptide on feeding in 16 hour-fasted or rats fed ad libitum. As expected, ACTH reduced feeding in fed and previously fasted rats, although this anorectic effect was more pronounced in fasted rats. The N-terminal-derived peptide alpha-MSH, but not ACTH 1-10, reduced cumulative food intake over 2 h after its injection intracerebroventricularly (icv) in 16 h-fasted, but not in fed rats. In contrast, the C-terminal fragments produced a long-lasting increase in feeding in fasted, but not in fed rats. The anorectic effects of N-terminal fragments of ACTH are recognised to be mediated via melanocortin MC4 receptors. However, the orexigenic effects of the C-terminal fragments do not appear to be conducted via MC4 receptors, since neither ACTH 18-39 nor ACTH 22-39 stimulated cAMP accumulation nor inhibited the ACTH-stimulated cAMP accumulation in HEK-293 cells transfected with the recombinant MC4 receptor.     

Pivotal roles of alpha-melanocyte-stimulating hormone and the melanocortin 4 receptor in leptin stimulation of prolactin secretion in rats

Leptin, the obese gene product, was reported to stimulate prolactin (PRL) secretion, but the neuroendocrine mechanism underlying this hormonal response is largely unknown. Thus, in this study we examined the involvement of several important PRL regulators in the leptin-induced PRL secretion in male rats. Compared with the values in normally fed rats, food deprivation for 3 days significantly decreased both PRL and leptin levels in the plasma. These changes were reverted to normal by a 3-day constant infusion of 75 microg/kg/day of leptin to the fasted rats, while 225 microg/kg/day of leptin further elevated both PRL and leptin levels. These four groups of animals were used for the following experiments. Results of dopamine and serotonin turnover studies in the brain and the pituitary indicated that neither of these biogenic amines plays a primary role in mediating leptin's effects on PRL. Repeated intracerebroventricular injections over 72 h of neutralizing antibodies against vasoactive intestinal peptide, PRL-releasing peptide, or beta-endorphin, did not significantly suppress the leptin actions. However, both the blockade of the melanocortin (MC) 4 receptor (R) and the immunoquenching of brain alpha-melanocyte-stimulating hormone (alpha-MSH) completely abolished the leptin-induced PRL release, and the stimulation of the MC4-R, but not the MC3-R, significantly elevated PRL levels in the fasted rats. These results suggest that alpha-MSH, a cleaved peptide from pro-opiomelanocortin of which synthesis is stimulated by leptin, may be the pivotal neuropeptide in the brain mediating the leptin's stimulatory influence on PRL secretion. It was also suggested that the MC4-R may be the primary subtype of the MC-Rs mediating this action of alpha-MSH.     

Early leptin response to a palatable diet predicts dietary obesity in rats: key role of melanocortin-4 receptors in the ventromedial hypothalamic nucleus

We have investigated whether interactions between leptin and hypothalamic melanocortin-4 receptors (MC4-Rs) determine individual susceptibility to dietary obesity in rats. Animals with relatively high plasma leptin levels 1 week after presentation of palatable food, before weight increased significantly, subsequently showed lower food intake and weight gain after 8 weeks of palatable feeding than those with low early leptin levels. The rats with lesser weight gain also showed significantly greater down-regulation of MC4-Rs, which mediate hypophagia, in specific hypothalamic areas, namely, the arcuate, dorsomedial, and ventromedial (VMH) nuclei and the median eminence. We suggest that this reflects enhanced receptor exposure to endogenous alpha-melanocyte-stimulating hormone, an appetite-suppressing peptide produced by hypothalamic proopiomelanocortin neurones. It is striking that plasma leptin levels at 1 week were inversely correlated with MC4-R density in the VMH, suggesting that this is a key site of leptin action. The early leptin response to palatable feeding may therefore "program" subsequent feeding behaviour and weight gain by regulating neurones that project selectively to the VMH.     

Paraventricular hypothalamic alpha-melanocyte-stimulating hormone and MTII reduce feeding without causing aversive effects

alpha-Melanocyte-stimulating hormone (alpha-MSH) appears to play a tonic inhibitory role in feeding and energy storage. MTII, a specific synthetic MC3-R/MC4-R agonist, has similar effects on feeding in rats. The current studies demonstrate that PVN administration of alpha-MSH or MTII decreases nocturnal and NPY-stimulated food intake without causing aversive effects. Co-administration with NPY of 600 pmol alpha-MSH or 1 pmol MTII into the PVN caused a significant decrease in NPY-induced feeding. PVN administration of MTII or alpha-MSH at doses effective to suppress feeding did not cause conditioned taste aversion (CTA). ICV administration of alpha-MSH, however, did cause weak CTA. These results indicate that the potent effects on feeding of MC3-R and MC4-R agonists when injected into the PVN are not due to aversive effects.     

Isolation and characterization of antagonist and agonist peptides to the human melanocortin 1 receptor

We identified a large number of peptide mimotopes of the adrenocorticotropic hormone (ACTH) and the alpha-melanocyte stimulating hormone (alpha-MSH) to analyze better the structure-function relationships of these hormones with the human MC1 receptor (hMC1R). We have investigated the use of phage-display technology to isolate specific peptides of this receptor by using three monoclonal anti-ACTH antibodies (mAbs). A library of 10(8) phage-peptides displaying randomized decapeptides was constructed and used to select phage-peptides that bind to mAbs. Forty-five phage-peptides have been isolated and from their amino acid sequences, we have identified two consensus sequences, EXFRWGKPA and WGXPVGKP, corresponding to the regions 5-13 and 9-16 of ACTH, respectively. A biological assay on cells expressing the hMC1-R was developed to determine the capacity of phage-peptides to stimulate the receptor. Only two phage-peptides showed detectable activity. Thirty-one peptides were synthesized to analyze their biological effect. We identified two weak agonists, EC50=16 and 11 microM, two strong agonists, EC50=25 and 14 nM and a partial antagonist, IC50=36 microM. This work confirmed the modulator agonist role of the regions 11-12 of alpha-MSH and ACTH, and the importance of the methionine residue at position 4 for the stimulation of the hMC1-R. We also identified analogues of the regions 8-17 of ACTH that exhibited a weak activator effect, and of one analogue of the N-terminal regions 1-9 of ACTH and alpha-MSH having a partial antagonist effect. These results may be useful in the development of potential agonists or antagonists of the hMC1R.     

Sustained orexigenic effect of Agouti related protein may be not mediated by the melanocortin 4 receptor

Intracerebroventricular (ICV) injection of Agouti related protein (AgRP), an endogenous melanocortin 3 and 4 receptor (MC3/4-R) antagonist, produces a prolonged increase in food intake. To clarify the roles of the MC3-R and MC4-R in AgRP-induced hyperphagia, the feeding effect of AgRP (83-132) was compared with that of the selective MC4-R antagonist, JKC-363 (cyclic [Mpr11, D-Nal14, Cys18, Asp22-NH2]-beta-MSH11-22). Single ICV administration of AgRP (83-132) increased food intake for 48 h whilst ICV JKC-363 increased food intake for 8h. An increase in body weight at 24 and 48 h was observed following AgRP (83-132) but not JKC-363 treatment. These data suggest that the sustained orexigenic action of AgRP (83-132) may not be through MC4-R antagonism.     

MTII attenuates ghrelin- and food deprivation-induced increases in food hoarding and food intake

Food deprivation triggers a constellation of physiological and behavioral changes including increases in peripherally-produced ghrelin and centrally-produced agouti-related protein (AgRP). Upon refeeding, food intake is increased in most species, however hamsters primarily increase food hoarding. Food deprivation-induced increases in food hoarding by Siberian hamsters are mimicked by peripheral ghrelin and central AgRP injections. Because food deprivation stimulates ghrelin as well as AgRP synthesis/release, food deprivation-induced increases in hoarding may be mediated by melanocortin 3 or 4 receptor (MC3/4-R) antagonism via AgRP, the MC3/4-R inverse agonist. Therefore, we asked: Can a MC3/4-R agonist block food deprivation- or ghrelin-induced increases in foraging, food hoarding and food intake? This was accomplished by injecting melanotan II (MTII), a synthetic MC3/4-R agonist, into the 3rd ventricle in food deprived, fed or peripheral ghrelin injected hamsters and housed in a running wheel-based food delivery foraging system. Three foraging conditions were used: a) no running wheel access, non-contingent food, b) running wheel access, non-contingent food or c) a foraging requirement for food (10 revolutions/pellet). Food deprivation was a more potent stimulator of foraging and hoarding than ghrelin. Concurrent injections of MTII completely blocked food deprivation- and ghrelin-induced increases in food intake and attenuated, but did not always completely block, food deprivation- and ghrelin-induced increases in food hoarding. Collectively, these data suggest that the MC3/4-R are involved in ghrelin- and food deprivation-induced increases in food intake, but other neurochemical systems, such as previously demonstrated with neuropeptide Y, also are involved in increases in food hoarding as well as foraging.     

MTII attenuates ghrelin- and food deprivation-induced increases in food hoarding and food intake

Food deprivation triggers a constellation of physiological and behavioral changes including increases in peripherally-produced ghrelin and centrally-produced agouti-related protein (AgRP). Upon refeeding, food intake is increased in most species, however hamsters primarily increase food hoarding. Food deprivation-induced increases in food hoarding by Siberian hamsters are mimicked by peripheral ghrelin and central AgRP injections. Because food deprivation stimulates ghrelin as well as AgRP synthesis/release, food deprivation-induced increases in hoarding may be mediated by melanocortin 3 or 4 receptor (MC3/4-R) antagonism via AgRP, the MC3/4-R inverse agonist. Therefore, we asked: Can a MC3/4-R agonist block food deprivation- or ghrelin-induced increases in foraging, food hoarding and food intake? This was accomplished by injecting melanotan II (MTII), a synthetic MC3/4-R agonist, into the 3rd ventricle in food deprived, fed or peripheral ghrelin injected hamsters and housed in a running wheel-based food delivery foraging system. Three foraging conditions were used: a) no running wheel access, non-contingent food, b) running wheel access, non-contingent food or c) a foraging requirement for food (10 revolutions/pellet). Food deprivation was a more potent stimulator of foraging and hoarding than ghrelin. Concurrent injections of MTII completely blocked food deprivation- and ghrelin-induced increases in food intake and attenuated, but did not always completely block, food deprivation- and ghrelin-induced increases in food hoarding. Collectively, these data suggest that the MC3/4-R are involved in ghrelin- and food deprivation-induced increases in food intake, but other neurochemical systems, such as previously demonstrated with neuropeptide Y, also are involved in increases in food hoarding as well as foraging.     

Intracellular localization of anti-tumor immune RNA.

Syngeneic spleen cells from normal, non-immune Fischer 344/N rats and allogeneic spleen cells from normal Wistar-Furth rats became cytotoxic, in vitro, to chemically induced Fischer rat sarcoma (MC3-R) target cells following incubation with xenogeneic Immune RNA (I-RNA) extracted from spleens of guinea pigs immunized with MC3-R tumor cells. I-RNA extracted from intact spleen cells or from the cytoplasmic fraction of spleen cells were equally active. RNA extracted from isolated spleen cell nuclei was inactive, as were all RNA fractions from spleen cells of nonspecifically immunized guinea pigs. Syngeneic I-RNA extracted from intact spleen cells or the cytoplasmic fraction of cells from spleens of Fischer rats bearing growing MC3-R transplants mediated cytotoxic reactions against MC3-R target cells when incubated with normal Fischer rat spleen cells. RNA from the nuclei of spleen cells of rats bearing MC3-R tumors was considerably less active. All RNA fractions from spleen cells of normal non-immune Fischer rats were inactive. The immunologically active component of xenogeneic and Syngeneic I-RNA, therefore, were found to be localized in the cytoplasm of specifically sensitized lymphoid cells.     

Neurotensin: effects of hypothalamic and intravenous injections on eating and drinking in rats

To investigate a role for the brain-gut peptide neurotensin (NT) in ingestive behavior, changes in food and water intake of food-deprived rats were examined following injection of NT into the paraventricular hypothalamic nucleus (PVN) or the mesenteric vein. Unilateral PVN NT (2.5, 5.0, 10.0 micrograms/0.3 microliter) produced substantial dose-dependent reductions in total food intake 0.5, 1, and 4 hr postinjection. In contrast, PVN NT had no effect on water intake and produced no change in grooming, rearing, sleeping, resting or locomotor activity. Bilateral PVN NT at a high dose (10.0 micrograms/side) suppressed consumption of solid or liquid diet in food-deprived rats, but did not affect water intake in water-deprived rats. This specificity is consistent with a role for CNS NT in feeding behavior. Intravenous NT (1-1000 pmole/kg/min for 30 min) did not specifically suppress food intake; however, low doses did increase water intake in food-deprived rats. These findings do not support a role for plasma NT in feeding, but do suggest that it may play a role in drinking behavior.     

Differential regulation of melanin-concentrating hormone and orexin genes in the agouti-related protein/melanocortin-4 receptor system

Agouti protein and agouti-related protein (AGRP) antagonize alpha-melanocyte-stimulating hormone that binds to and activates the melanocortin-4 receptor (MC4-R) in the hypothalamus, thereby stimulating food intake. Melanin-concentrating hormone (MCH) and orexin are orexigenic peptides that specifically are synthesized in the lateral hypothalamus. MCH gene expression was augmented in A(y)/a (agouti) mice which overexpress agouti protein, but orexin mRNA was not. AGRP administered intracerebroventricularly into wild-type rats augmented MCH but not orexin gene expression. Also, SHU9119, a peptidergic antagonist of MC4-R, increased only MCH mRNA. These findings indicate that interruption of signaling at MC4-R activates the MCH but not the orexin gene. The biosyntheses of MCH and orexin are regulated through different pathways.     

Structural insight into the role of the human melanocortin 3 receptor cysteine residues on receptor function

Melanocortin-3 receptor (MC3R), expressed in the hypothalamus and limbic systems of the brain, as well as by peripheral sites, plays an important role in the regulation of energy homeostasis and other physiological functions. Past work shows that MC3R-deficiency resulted in fat mass increase, feeding efficiency increase, hyperleptinemia and mild hyperinsulinemia in mice and human. MC3R belongs to G-protein coupled receptor (GPCR) family and many studies indicate that some cysteine residues in GPCR play key roles in maintaining receptor tertiary structure and function. In this study, we examined the role of cysteine residues in MC3R on receptor function. Human MC3R (hMC3R) has eighteen cysteine residues where they are located in the extracellular loops (ELs), the transmembrane domains (TMs) and the intracellular loops (ILs). We replaced these cysteines with serine and expressed these receptors in HEK-293 cells which lack endogenous MC3R. Our results indicate that five cysteines in eighteen of the hMC3R are important for hMC3R function. Mutations, C305S, C311S, and C313S in EL3, resulted in significant decrease in receptor expression and receptor function while two other mutations C115S and C162S in TM3 significantly decreased NDP-MSH binding affinity and potency. These results suggest that extracellular cysteine residue 305, 311 and 313 are crucial for receptor expression and the transmembrane cysteine residue, C115 and 162 are important for ligand binding and signaling. These findings provide important insights into the importance of cysteine residues of hMC3R on receptor tertiary structure and function.     

Effect of the melanocortin receptor stimulation or inhibition on ethanol intake in alcohol-preferring rats

It has been recently reported that acute intracerebroventricular injection of 1 nmol/rat of the non-selective melanocortin 3 and 4 receptor (MC3/4) agonist MTII reduces ethanol intake in female AA alcohol-preferring rats and alters opioid peptide levels in the ventral tegmental area of rats. To better understand the role of the MC system in the control of ethanol intake, we tested the acute and chronic effects of lateral ventricular (LV) injections of 0.01-1 nmol MTII, of 0.1-1 nmol of the MC3/4R receptor antagonist agouti related peptide (AgRP), and 0.1-0.5 nmol of the MC3/4R receptor antagonist SHU9119 on food, water, and 10% ethanol intake in Marchigian-Sardinian alcohol-preferring (msP) rats, which spontaneously ingest pharmacologically relevant quantities of ethanol both under short and long term access conditions. The data showed that with 2h/day ethanol access, LV MTII injections reduced intake of food and ethanol intakes. When food, water, and ethanol were available ad libitum and 0.01 nmol MTII was given by daily LV injection, however, ethanol intake was reduced for only the first 2 days, whereas food intake was reduced for all 5 days of treatment. Finally, acute LV injection of neither AgRP nor SHU9119 affected ethanol intake under ad libitum conditions, although both antagonists significantly increased food and water intake. In conclusion, these data fail to support a role for endogenous MC3/4R in the control of spontaneous ethanol intake in the msP rat. MC3/4R agonism, however, reduced ethanol intake in association with reduced food intake, suggesting that MTII might reduce nutrient-related controls of ethanol intake rather than, or in addition to, reward-related controls of ethanol intake.