Melanocortin-4 Receptor Blockade Research Articles

Sex differences in weight gain, blood pressure control, and responses to melanocortin-4 receptor antagonism in offspring from lean and obese parents.

We examined potential sex differences in appetite and blood pressure (BP) responses to melanocortin-4 receptor (MC4R) blockade in offspring from lean and obese parents. Offspring from normal (N) diet-fed parents were fed N (NN) or high-fat (H) diets (NH) from weaning until adulthood. Offspring from obese H diet-fed parents were also fed N (HN) or H diets (HH). Adult male and female offspring were implanted with BP telemetry probes and intracerebroventricular cannulas to infuse MC4R antagonist or vehicle. Infusion of the MC4R antagonist SHU-9119 (1 nmol/h) for 7 days caused larger increases in calorie intake and body weight in obese compared with lean offspring. In male offspring, HH and HN groups exhibited higher baseline BP compared with NN and NH, and HH showed a greater reduction in BP during SHU-9119 infusion. In female offspring, HH also showed higher baseline BP and greater reduction in BP during MC4R blockade. SHU-9119 reduced heart rate in all groups, but reductions were more pronounced in offspring from lean parents. Combined α and β-adrenergic blockade reduced BP more in male HH offspring compared with NN controls. Losartan reduced BP more in male NH, HN, and HH offspring compared with NN controls. Losartan and α- and β-adrenergic blockade reduced BP similarly in all female groups. These results suggest that endogenous MC4R activity contributes to elevated BP in obese offspring from obese parents. Our findings also indicate important sex differences in the mechanisms of BP control in male and female offspring of obese parents.

Hypertension Editors' Picks Preeclampsia.

Hypertension Editors' Picks Preeclampsia.

Melanocortin 4 receptor-mediated effects of amylin on thermogenesis and regulation of food intake.

Amylin, a pancreatic hormone cosecreted with insulin, exerts important anorexic and weight-loss effects. Melanocortin 4 receptor (MC4R) signalling plays a critical role in energy homeostasis; however, its role on amylin-dependent regulation of food intake and adaptive thermogenesis of interscapular brown adipose tissue (IBAT) are unclear. In this study, we examined the effects of amylin on food intake and thermogenesis on IBAT via the MC4R pathway in mice. Acute food consumption and thermogenesis in IBAT were measured in male wild-type (WT) and MC4R-deficient mice following intraperitoneal injection of amylin and SHU9119, an MC3R/4R antagonist, to determine the role of the central melanocortin system on the hypothalamus and IBAT. Amylin (50μg/kg) suppressed feeding and stimulated thermogenesis on IBAT via activation of the MC4R system in mice. Pharmacological blockade of MC4R using SHU9119 (50μg/kg) attenuated amylin-induced inhibition of feeding and stimulation of thermogenesis in IBAT. No changes were observed when SHU9119 was injected alone. Moreover, amylin significantly increased MC4R expression and c-Fos neuronal signals in the arcuate nucleus and significantly increased acetyl-CoA carboxylase (ACC) phosphorylation in the hypothalamus and IBAT and uncoupling protein-1 (UCP1) expression in the IBAT of WT mice via the MC4R pathway. The melanocortin system was involved in amylin-induced suppression of food intake and activation of thermogenesis in both the hypothalamus and IBAT via modulation of ACC phosphorylation and UCP1 expression.

Melanocortin-4 Receptor Deficiency Attenuates Placental Ischemia-Induced Hypertension in Pregnant Rats.

Preeclampsia is a pregnancy-specific disorder of new-onset hypertension linked to placental ischemia. While obesity is a major risk factor for preeclampsia, not all obese pregnant women develop pregnancy-induced hypertension or preeclampsia. Previously, we reported that placental ischemia-induced hypertension is dependent upon intact signaling of the sympathetic nervous system. Moreover, in various models of obesity, blockade of MC4R (melanocortin-4 receptor) signaling protects against the development of hypertension via suppression of the sympathetic nervous system. Less is known about this pathway during obese pregnancy. Although blockade of MC4R may lead to increased body weight during pregnancy, we tested the hypothesis that placental ischemia-induced hypertension is attenuated in obese MC4R-deficient pregnant rats. On gestational day 14, MC4R wild-type or heterozygous-deficient (MC4R-def) rats were subjected to chronic placental ischemia via the reduced uterine perfusion pressure procedure or Sham surgery then examined on gestational day 19. In Sham MC4R-def versus Sham wild-type pregnant rats, there was increased body weight, fat mass, and circulating leptin levels but they had similar fetus weights. Reduced uterine perfusion pressure reduced fetus weights in both strains. Reduced uterine perfusion pressure increased blood pressure in wild-type rats but this response was significantly attenuated in MC4R-def rats, although blood pressure was elevated in Sham MC4R-def over Sham wild-type. These data indicate that while obese MC4R-def pregnant rats have higher blood pressure during pregnancy, placental ischemia-induced hypertension is attenuated in obese MC4R-def pregnant rats. Thus, obese women with abnormal MC4R signaling may be less susceptible to the development of placental ischemia-induced hypertension.

Abstract P133: Melanocortin 4 Receptor is Required for the Effect of Testosterone Supplement on Metabolic Parameters and Blood Pressure

Melanocortin 4 receptor (MC4R) activation causes appetite suppression and sympathetic nervous system activation. Blockade of MC4R increases food intake and in some cases, decreases BP. MC4R has been implicated as playing a role in obesity and hypertension. Previously we observed that testosterone supplementation in male Zucker rats improved insulin resistance and the characteristics of metabolic syndrome, but increased BP. Similarly, testosterone increases BP in male SHR, and blockade of the MC4R reduces BP in both young and old male SHR. In the present study we tested the hypothesis that testosterone supplementation (T) requires activated MC4R to attenuate metabolic parameters, and to increase BP. Male obese MC4R -/- and Wistar Hannover rats (WT) were treated with T (MC4R-KO+T vs WT+T: 22 mg/10 mm silastic pellet) or placebo (empty pellets; MC4R-KO+P, and WT+P), beginning at 10 wks of age for 6 weeks. In WT animals, T reduced body weight by 13% compared to placebo (367 gr vs 432gr; p<0.05), but had no effect on food intake (WT+P: 19±7 gr/d vs WT+T: 16±5 gr/d, n=3, p:NS). In MC4R-KO rats, body weight and food intake were similar in both placebo and T rats (BW: 555±20 gr vs 526±18 gr, and FI: 26±1 gr vs 28±2; n=3, p:NS, respectively). T reduced fat mass (measured by ECHO-MRI ® ) in WT+T by 38% compared to placebo (50±2.9 gr vs 31±3.2 gr; p<0.05); however, there was no effect on fat mass in MC4R-KO + T (149±8 gr vs 147±11 gr; n=3, p:NS). Lean mass (by ECHO-MRI ® ) was not affected by T in either MC4R-KO+T or WT+T (p:NS). Finally, unlike our previous studies in obese Zucker and SHR males, T failed to affect mean arterial pressure or heart rate (by telemetry) in MC4R-KO+T rats compared to control MC4R-KO+P (MAP: 122±2 mmHg vs 122±1 mmHg; n=3, p:NS; HR:309±5 bpm vs 320±17 bpm; n=3, p:NS). These data suggest that the effect of T on metabolic parameters and BP may be mediated at least in part by MC4R since in the absence of active MC4R, T is not able to affect either body composition or BP. Also, these data gave a potential new insight into the mechanism by which T contributes to BP control and adipose tissue regulation. More studies are necessary to clarify the role of MC4R in mediating the effects of testosterone. Supported by NIH-R01HL66072, R01HL69194, PO1HL51971 (JFR), 14POST18640015 (ROM).

Melanocortin-4 receptor in the medial amygdala regulates emotional stress-induced anxiety-like behaviour, anorexia and corticosterone secretion

The central melanocortin system has been implicated in emotional stress-induced anxiety, anorexia and activation of the hypothalamo-pituitary-adrenal (HPA) axis. However, the underlying neural substrates have not been identified. The medial amygdala (MeA) is highly sensitive to emotional stress and expresses high levels of the melanocortin-4 receptor (MC4R). This study investigated the effects of activation and blockade of MC4R in the MeA on anxiety-like behaviour, food intake and corticosterone secretion. We demonstrate that MC4R-expressing neurons in the MeA were activated by acute restraint stress, as indicated by induction of c-fos mRNA expression. Infusion of a selective MC4R agonist into the MeA elicited anxiogenic-like effects in the elevated plus-maze test and decreased food intake. In contrast, local MeA infusion of SHU 9119, a MC4R antagonist, blocked restraint stress-induced anxiogenic and anorectic effects. Moreover, plasma corticosterone levels were increased by intra-MeA infusion of the MC4R agonist under non-stressed conditions and restraint stress-induced elevation of plasma corticosterone levels was attenuated by pretreatment with SHU 9119 in the MeA. Thus, stimulating MC4R in the MeA induces stress-like anxiogenic and anorectic effects as well as activation of the HPA axis, whereas antagonizing MC4R in this region blocks such effects induced by restraint stress. Together, our results implicate MC4R signalling in the MeA in behavioural and endocrine responses to stress.

Effect of periaqueductal gray melanocortin 4 receptor in pain facilitation and glial activation in rat model of chronic constriction injury

Objectives: Substantial evidence shows that spinal melanocortin 4 receptor (MC4R) may participate in regulation of central sensitization and chronic pain condition induced by peripheral nerve injury. Periaqueductal gray (PAG) is an important component of descending pain facilitatory system and takes part in spinal nociceptive information. This research will choose PAG to discuss the effect of MC4R in pain facilitation induced by chronic constriction injury (CCI) and further discuss its effect in glial activity and inflammatory factor levels in nerve injury.Methods: Behavior tests (von Frey test and hot-plate test), semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), and immunohistochemistry were used in this research.Results: PAG injection of HS014 (a selective inhibitor of MC4R), not only significantly reduced the established mechanical allodynia and thermal hyperalgesia, but also delayed the development of pain facilitation. Semi-quantitative RT-PCR analysis revealed that MC4R and proopiomelanocortin (POMC) expression in PAG was significantly increased after CCI, but agouti-related protein (AgRP) expression decreased. Immunohistochemistry analysis showed that protein levels of astrocytic marker (GFAP), microglial marker (OX-42), tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6 were significantly increased, but there was little change of the protein levels of IL-10 following CCI. Furthermore, blockade of MC4R decreased immunoreactivity of glia cells and protein levels of pro-inflammatory cytokines, and increased protein levels of anti-inflammatory cytokine IL-10 after CCI.Discussion: This research suggests that activation of MC4R in PAG after peripheral nerve injury participates in pain facilitation by regulating the glial activation and inflammatory cytokines secretion.

Protective effects of an anti‐melanocortin‐4 receptor scFv derivative in lipopolysaccharide‐induced cachexia in rats

BackgroundCachexia is a complex syndrome defined by weight loss due to an ongoing loss of skeletal muscle mass with or without loss of body fat. It is often associated with anorexia. Numerous results from experimental studies suggest that blockade of the melanocortin-4 receptor (MC4R) could be an effective treatment for anorexia and cachexia. In a previous study, we reported the basic pharmacological properties of a blocking anti-MC4R mAb 1E8a and its scFv derivative in vitro and in vivo.MethodsIn the present study, we further characterized the mode of action of the 1E8a scFv, evaluated its pharmacokinetic properties in mice, and assessed its therapeutic potential in a lipopolysaccharide (LPS)-induced cachexia model in rats.ResultsIn vitro, scFv enhanced the efficacy of the endogenous inverse agonist Agouti-related protein. After intravenous (i.v.) administration in mice, the scFv penetrated the blood–brain barrier (BBB) and reached its central sites of action: the scFv brain–serum concentration ratios increased up to 15-fold which suggests an active uptake into brain tissue. In telemetry experiments, i.v. administration of the scFv in rats was well tolerated and only induced slight cardiovascular effects consistent with MC4R blockade, i.e., a small decrease in mean arterial pressure and heart rate. In the model of LPS-induced anorexia, i.v. administration of scFv 1E8a prevented anorexia and loss of body weight. Moreover, it stimulated a myogenic response which may contribute to the preservation of muscle mass in cachexia.ConclusionThe pharmacological profile of scFv 1E8a suggests its potential value in the treatment of cachexia or anorexia.

The orally active melanocortin-4 receptor antagonist BL-6020/979: a promising candidate for the treatment of cancer cachexia

BackgroundUnder physiological conditions, the melanocortin system is a crucial part of the complex network regulating food intake and energy expenditure. In pathological states, like cachexia, these two parameters are deregulated, i.e., food intake is decreased and energy expenditure is increased—a vicious combination leading to catabolism. Agouti-related protein (AgRP), the endogenous antagonist at the melanocortin-4 receptor (MC-4R), was found to increase food intake and to reduce energy expenditure. This qualifies MC-4R blockade as an attractive mode of action for the treatment of cachexia. Based on this rationale, a novel series of small-molecule MC-4R antagonists was designed, from which the orally active compound BL-6020/979 (formerly known as SNT207979) emerged as the first promising development candidate showing encouraging pre-clinical efficacy and safety properties which are presented here.Methods and resultsBL-6020/979 is an orally available, selective and potent MC-4R antagonist with a drug-like profile. It increased food intake and decreased energy expenditure in healthy wild-type but not in MC-4R deficient mice. More importantly, it ameliorated cachexia-like symptoms in the murine C26 adenocarcinoma model; with an effect on body mass and body composition and on the expression of catabolic genes. Moreover, BL-6020/979 showed antidepressant-like properties in the chronic mild stress model in rats and exhibits a favorable safety profile.ConclusionThe properties of BL-6020/979 demonstrated in animal models and presented here make it a promising candidate suitable for further development towards a first-in-class treatment option for cachexia that potentially opens up the opportunity to treat two hallmarks of the disease, i.e., decreased food intake and increased energy expenditure, with one drug.

Antibodies as pharmacologic tools for studies on the regulation of energy balance

ObjectiveActive immunization in rats may serve several purposes: the production of a disease-like phenotype, the generation of pharmacologic tools, and the development of clinically useful therapies. We selected the melanocortin-4 receptor (MC4R) as a target because its blockade could provide a treatment for anorexia and cachexia. MethodsWe used a sequence of the N-terminal (NT) domain of the MC4R as an antigen. Rats immunized against the NT peptide produced specific MC4R antibodies (Abs) that were purified and characterized in vitro and in vivo. ResultsThe Abs acted as inverse agonists and reduced under basal conditions the production of cyclic adenosine monophosphate in HEK-293 cells expressing the human MC4R. Rats immunized against the NT peptide developed a phenotype consistent with hypothalamic MC4R blockade, i.e., increased food intake and body weight, liver and fat-pad weights, hepatic steatosis, and increased plasma triacylglycerols. With a high-fat diet, plasma insulin levels were significantly increased. In separate experiments an increase in food intake was observed after injection of purified MC4R Abs into the third ventricle. When lipopolysaccharide was administered in NT-immunized rats the reduction of food intake was partly prevented in this model of cytokine-induced anorexia. ConclusionOur results show that active immunization of rats against the MC4R resulted in the generation of specific Abs that stimulated food intake by acting as inverse agonists of the hypothalamic MC4R. Pharmacologically active monoclonal MC4R Abs could be the starting point for the development of novel treatments for patients with anorexia or cachexia.

Antibodies against the melanocortin-4 receptor act as inverse agonists in vitro and in vivo

Functionally active antibodies (Abs) against central G-protein-coupled receptors have not yet been reported. We selected the hypothalamic melanocortin-4 receptor (MC4-R) as a target because of its crucial role in the regulation of energy homeostasis. A 15 amino acid sequence of the N-terminal (NT) domain was used as an antigen. This peptide showed functional activity in surface plasmon resonance experiments and in studies on HEK-293 cells overexpressing the human MC4-R (hMC4-R). Rats immunized against the NT peptide produced specific antibodies, which were purified and characterized in vitro. In HEK-293 cells, rat anti-NT Abs showed specific immunofluorescence labeling of hMC4-R. They reduced the production of cAMP under basal conditions and after stimulation with a synthetic MC4-R agonist. Rats immunized against the NT peptide developed a phenotype consistent with MC4-R blockade, that is, increased food intake and body weight, increased liver and fat pad weight, and elevated plasma triglycerides. In a separate experiment in rats, an increase in food intake could be produced after injection of purified Abs into the third ventricle. Similar results were obtained in rats injected with anti-NT Abs raised in rabbits. Our data show for the first time that active immunization of rats against the NT sequence of the MC4-R results in specific Abs, which appear to stimulate food intake by acting as inverse agonists in the hypothalamus.

Peripheral Administration of a Melanocortin 4-Receptor Inverse Agonist Prevents Loss of Lean Body Mass in Tumor-Bearing Mice

Cachexia affects many different chronically ill patient populations, including those with cancer. It results in loss of body weight, particularly of lean body mass (LBM), and is estimated to be responsible for over 20% of all cancer-related deaths. Currently, available drugs are ineffective, and new therapies are urgently needed. Melanocortin 4-receptor (MC4-R) blockade has been shown recently to be effective in preventing cancer cachexia in rodent models. In the present study, we have tested a MC4-R blocker, ML00253764 [2-{2-[2-(5-bromo-2-methoxyphenyl)-ethyl]-3-fluorophenyl}-4,5-dihydro-1H-imidazolium hydrochloride] (Vos et al., 2004), in vitro and in vivo. In membranes of human embryonic kidney 293 cells expressing human MC4-R, ML00253764 displaced [Nle(4), d-Phe(7)]-alpha-melanocyte-stimulating hormone binding with an IC(50) of 0.32 microM. At concentrations above 1 microM, ML00253764 decreased cAMP accumulation (maximal reduction of -20%) indicative of inverse agonist activity. ML00253764 was administered twice daily (15 mg/kg s.c.) for 13 days to C57BL6 mice bearing s.c. Lewis lung carcinoma tumors. Food intake and body weight were measured, and body composition was assessed using magnetic resonance relaxometry. ML00253764 stimulated light-phase food intake relative to vehicle-treated controls (p < 0.05), although no effect was observed on 24-h food intake. During the 21 days of the experiment, the LBM of tumor vehicle-treated mice decreased (p < 0.05). In contrast, the tumor-bearing mice treated with ML00253764 maintained their LBM. These data support the view that MC4-R blockade may be a suitable approach for the treatment of cancer cachexia and that MC4-R inverse agonists may have potential as drug candidates.

Evaluation of the Role of Melanocortin 3 and 4 Receptors in Leptin-Stimulated and Spontaneous Growth Hormone Secretion in Rats

It has been reported that the melanocortin 4-receptor (MC4-R) may act downstream of leptin to mediate its effects on food intake and several neuroendocrine functions (the reproductive system, the hypothalamo-pituitary-thyroid axis, and prolactin secretion). However, no previous study examined whether MC4-R mediates leptin stimulatory actions on growth hormone (GH) secretion, or whether MC4-R signaling is involved in spontaneous pulsatile GH release in fed rats. Therefore in this study we examined the involvement of both MC3-R and MC4-R (the predominant MC-R subtypes expressed in the brain) in these two aspects of GH secretion in freely-moving male rats. In both fed and 3-day fasted rats, plasma GH levels were determined every 15 min over 5 h after single intracerebroventricular injections of the following substances or vehicle. Fasting diminished and leptin (0.3 nmol) reinstated the GH pulse amplitude without affecting the pulse frequency. Neither HS014 (1.0 nmol, a selective MC4-R antagonist) nor agouti-related peptide (1.0 nmol, a non-selective MC3/4-R antagonist) was effective in altering leptin-stimulated or spontaneous GH secretion. In addition, neither melanotan-II (1.0 nmol, a non-selective MC3/4-R agonist) nor γ₁-melanocyte-stimulating hormone (10 nmol, a selective MC3-R agonist) affected significantly GH release in fasted rats. We have previously demonstrated that stimulation or blockade of MC4-R, achieved by the same drug dosage as in this study, significantly affect luteinizing hormone and prolactin secretion in rats. The present results thus suggest that neither MC4-R nor MC3-R is involved in leptin-stimulated or spontaneous GH secretion, or at least that the level of MC4-R involvement in GH secretion is much lower than that in luteinizing hormone and prolactin release regulation.

Neuronal histamine regulates food intake, adiposity, and uncoupling protein expression in agouti yellow (A(y)/a) obese mice.

Hypothalamic neuronal histamine and its H(1) receptor (H(1)-R) form a part of the leptin-signaling pathway in the brain and have been shown to regulate body weight and adiposity in diabetic (db/db) and diet-induced obese mice by affecting food intake and uncoupling protein mRNA expression. The proopiomelanocortin (POMC) melanocortin-4 receptor (MC-4R) is also important for leptin signaling. The present study had two aims: first, to clarify the antiobesity action of neuronal histamine in agouti yellow (A(y)/a) obese mice, a model of obesity in which POMC/MC-4R signaling is disrupted by blockade of MC-4R and second, to investigate the functional relationship between neuronal histamine and POMC/MC-4R signaling. Central administration of histamine into the lateral cerebroventricle decreased cumulative food intake and body weight in A(y)/a obese mice. Histamine treatment also decreased mRNA expression of ob gene in epididymal white adipose tissue and up-regulated uncoupling protein 1 mRNA expression in brown adipose tissue. These effects were attenuated in A(y)/a obese mice with histamine H(1)-receptor (H(1)-R) knockout. Histamine treatment induced c-Fos-like immunoreactivity in both paraventricular and arcuate nucleus. There was no significant difference in histamine-induced c-Fos-like immunoreactivity in the hypothalamus between A(y)/a obese mice and lean littermates, indicating histamine signaling was not disrupted at the hypothalamic level in A(y)/a obese mice. These results suggest that neuronal histamine have an antiobese action, even in A(y)/a obese mice despite a deficiency in POMC/MC-4R signaling. In addition, it appears that the histamine H(1)-R signaling pathway may be independent or downstream of the POMC/MC-4R signaling.

Differential role of melanocortin receptor subtypes in cachexia.

Animals and humans respond to starvation with a complex neuroendocrine response that ultimately leads to an increase in appetite, a sparing of lean body mass (LBM) and burning of fat, and an overall decrease in basal metabolic rate. In contrast, cachexia is a pathological state of malnutrition associated with many infections and chronic diseases, wherein appetite is diminished concomitant with an increase in metabolic rate, and a relative wasting of LBM. In previous studies, we demonstrated that anorexia and weight loss in mouse cachexia models induced by lipopolysaccharide (LPS) administration and by tumor growth are ameliorated by central melanocortin-4 (MC4) receptor (MC4-R) blockade. In contrast to the results seen with MC4 blockade, melanocortin-3 (MC3) receptor knockout (MC3-RKO) mice show illness-induced anorexia and weight loss with LPS administration and with cytokine administration, and they have similar decreases in mobility. Both MC3-RKOs and MC4-RKOs have an intact corticosterone response and fever with LPS injection. In tumor models, we show that MC4-RKO mice resist the loss of LBM brought about by tumor growth, whereas MC3-RKO animals show enhanced tissue wasting. These data underscore the importance of central melanocortin signaling in weight homeostasis and demonstrate differential effects of MC3-R and MC4-R blockade on the development of cachexia.