Role Of Melanocortin Receptors Research Articles

Activation of the ARCPOMC→MeA Projection Reduces Food Intake.

Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARC) plays an essential role in the control of food intake and energy expenditure. Melanocortin-4 receptors (MC4Rs) are expressed in key areas that are implicated in regulating energy homeostasis. Although the importance of MC4Rs in the paraventricular hypothalamus (PVH) has been well documented, the role of MC4Rs in the medial amygdala (MeA) on feeding remains controversial. In this study, we specifically examine the role of a novel ARCPOMC→MeA neural circuit in the regulation of short-term food intake. To map a local melanocortinergic neural circuit, we use monosynaptic anterograde as well as retrograde viral tracers and perform double immunohistochemistry to determine the identity of the neurons receiving synaptic input from POMC neurons in the ARC. To investigate the role of the ARCPOMC→MeA projection on feeding, we optogenetically stimulate channelrhodopsin-2 (ChR2)-expressing POMC fibers in the MeA. Anterograde viral tracing studies reveal that ARC POMC neurons send axonal projections to estrogen receptor-α (ER-α)- and MC4R-expressing neurons in the MeA. Retrograde viral tracing experiments show that the neurons projecting to the MeA is located mainly in the lateral part of the ARC. Optogenetic stimulation of the ARCPOMC→MeA pathway reduces short-term food intake. This anorectic effect is blocked by treatment with the MC4R antagonist SHU9119. In addition to the melanocortinergic local circuits within the hypothalamus, this extrahypothalamic ARCPOMC→MeA neural circuit would play a role in regulating short-term food intake.

Abstract P213: Metabolic and Cardiovascular Effects of Melanocortin-4 Receptors in Leptin Receptor-expressing Neurons

Studies in both humans and rodents have clearly established a role for melanocortin-4 receptor (MC4R) in energy homeostasis, sympathetic and cardiovascular function. Both MC4R-deficient humans and rodents develop severe obesity while keeping the sympathetic tone and blood pressure below or within the normal range, indicating that MC4R is required for obesity-associated sympathetic overdrive and hypertension. Indeed, sympathetic overdrive and cardiovascular side effect have been a major problem in developing anti-obesity drugs that target MC4R. Thus, better understanding the neural substrates that mediate distinct metabolic and sympathetic actions of MC4R is needed to provide a more specific target to treat obesity and associated hypertension. We have recently mapped the pattern of co-expression of MC4R and leptin receptor (LepR) and found that, throughout entire mouse brain, these two metabolically important receptors are uniquely co-expressed only in neurons of two brain regions, including the lateral hypothalamic area (LHA) and the periaqueductal gray (PAG). To specifically test the role of MC4R in LepR-expressing neurons, we generated mice with MC4R expression only in LepR-positive neurons by breeding Cre-dependently “reactivatable” MC4R-null mice (MC4R-TB) to LepR-Cre mice. We found that specific re-expression of MC4Rs only in LepR-positive neurons partially suppresses body weight gain especially in female mice (MC4R-TB 31.73 ± 2.2g vs. MC4R-TB::LepR-Cre 27.97 ± 1.8 g, p=0.0013). Direct multi-fiber recording of renal nerve activity (RSNA), however, revealed that neither baseline activity nor MC4R agonist-induced RSNA is significantly altered by restoration of MC4R signaling in LepR-positive neurons (change of RSNA in MC4R-TB 8.844 ± 3.05 vs. MC4R-TB::LepR-Cre 11.95 ± 2.7, p>0.05). Our results suggest that MC4R signaling in leptin-responsive neurons is sufficient to suppress body weight in female mice, but seemingly has no effect on sympathetic traffic to the kidney. Further investigation of the effect of MC4Rs in LepR-positive neurons on blood pressure regulation is underway.

Food intake regulation during pregnancy and lactation in mice with reduced activity of the melanocortin system

Activation of melanocortin receptors (MCRs) in the hypothalamus inhibits appetite. Neuropeptide Y (NPY) and Agouti Related Protein (AgRP) are coexpressed in some hypothalamic neurons, and stimulate feeding: NPY, via the inhibition of MCR-expressing neurons; and AgRP, via the MCR4 antagonism. The yellow mutation at the mouse agouti locus (Ay) evokes MCR blockage and stimulates appetite in nonbreeding females. However, the role of MCRs in food intake regulation during pregnancy and lactation is unclear. In this study, we measured the Agrp and Npy mRNA levels in hypohalamus in virgin and breeding C57Bl a/a (control) and Ay/a females during pregnancy (on days 7, 13, 18), lactation (days 10 and 21), and after the separation of offspring, as well as AgRP immunoreactivity in virgin and lactating females, and correlated gene and protein expression with food intake. Virgin Ay/a females presented higher food intake compared to a/a controls, and showed lower Agrp mRNA and protein levels. Pregnant Ay/a and a/a mice differed in patterns of food intake and neuropeptide expression. Npy mRNA levels increased during pregnancy only in a/a mice, while Agrp mRNA levels increased in both genotypes, being lower in Ay/a mice. In lactating Ay/a and a/a mice, the Agrp and Npy mRNA levels were similar. The AgRP protein content was higher in lactating than in virgin Ay/a mice. The obtained results demonstrate that, the MCR blockage inhibits Agrp expression in nonbreeding female mice, but not in lactating mice. Thus, during pregnancy, food intake regulation involves MCR signaling and activation of the Npy and Agrp expression. However, in lactating dams, hyperphagia is independent of the MCR blockade.

Abstract P249: Melanocortin-4 Receptors in Cholinergic Preganglionic Neurons of the Hindbrain and Spinal Cord are Important in Mediating Cardiovascular Responses to Acute Stress

Previous studies suggest that melanocortin-4 receptor (MC4R) activation in areas outside of the hypothalamus may regulate appetite but the role of MC4R in specific neuronal populations of the hindbrain in regulating cardiovascular function are still unknown. We examined the impact of total body MC4R deficiency (LoxTB-MC4R mice) and restoration of MC4R specifically in the brainstem and/or spinal cord on blood pressure (BP) response to acute stress. We selectively rescued MC4R only in preganglionic parasympathetic neurons of the dorsal motor of the vagus (DMV) and in the nucleus tractus solitarius (NTS) (LoxTB-MC4R/Phox2B-cre mice, n=5) or in cholinergic preganglionic neurons of the hindbrain and spinal cord (LoxTB-MC4R/Chat-cre mice, n=4). Mice were implanted with telemetry probes for measurement of mean arterial pressure (MAP) and heart rate (HR). After a 10-day recovery period, MAP and HR were continuously measured for 30 minutes before, during and 30 minutes after an air jet stress test. Acute air jet stress significantly increased MAP by 33±3 in WT mice and 31±2 in LoxTB-MC4R/Chat-cre mice compared to only 18±4 or 20±3 mmHg in LoxTB-MC4R/Phox2B-cre mice and LoxTB-MC4R mice, respectively. HR was increased by 180±20, 110±20, 100±23, and 127±15 bpm in response to air jet stress in WT, LoxTB-MC4R, LoxTB-MC4R/Phox2B and LoxTB-MC4R/Chat-cre mice, respectively. These results indicate that MC4Rs in cholinergic preganglionic neurons of the hindbrain and spinal cord play a key role in the BP responses to acute stress. (NHLBI-PO1HL51971, NIGMS- P20GM104357, and AHA-SDG5680016)

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).

Pharmacologic analyses of four chicken melanocortin-4 receptor mutations

The melanocortin-4 receptor (MC4R) is a critical regulator of mammalian food intake and energy expenditure, with receptor activation resulting in decreased food intake and increased energy expenditure. Recently, studies on role of MC4R in regulation of food intake have been extended to other species, such as chicken. Functional study of mutant MC4Rs is important in proving the causal link between MC4R mutation and production traits. Herein, we cloned chicken MC4R (cMC4R) complementary DNA and generated 4 mutant cMC4Rs (Q18H, G21R, S76L, and L299P) by site-directed mutagenesis and measured their expression by flow cytometry. Pharmacologic characteristics were analyzed with binding and signaling assays using 3 agonists. We showed that G21R had decreased cell surface and total expression (P < 0.05), whereas the other 3 mutants had similar total and cell surface expression levels as wild-type cMC4R. The 4 mutants had either decreased (Q18H, G21R, S76L; P < 0.05) or no (L299P) binding to radiolabeled [Nle4, D-Phe7]-α-melanocyte-stimulating hormone (MSH). In signaling assays, Q18H was constitutively active. Q18H, G21R, and S76L had decreased responses to α-MSH stimulation (P < 0.05). L299P had decreased basal and ligand-stimulated signaling (P < 0.01). Nle4, D-Phe7-MSH was the most potent agonist for cMC4R and therefore would be better suited for further in vivo studies. We conclude that the cloned cMC4R was a functional receptor and provided detailed functional data for these mutations, contributing to a better understanding of cMC4R variants associated with production traits.

Melanocortin-4 receptor mutations paradoxically reduce preference for palatable foods

Haploinsufficiency of the melanocortin-4 receptor (MC4R) results in melanocortin obesity syndrome, the most common monogenic cause of severe early onset obesity in humans. The syndrome, which produces measurable hyperphagia, has focused attention on the role of MC4R in feeding behavior and macronutrient intake. Studies show that inhibition of MC4R signaling can acutely increase the consumption of high-fat foods. The current study examines the chronic feeding preferences of mice with deletion of one or both alleles of the MC4R to model the human syndrome. Using two-choice diet paradigms with high-fat or high-carbohydrate foods alongside normal chow, we show, paradoxically, that deletion of one allele has no effect, whereas deletion of both alleles of the MC4R actually decreases preference for palatable high-fat and high-sucrose foods, compared with wild-type mice. Nonetheless, we observed hyperphagic behavior from increased consumption of the low-fat standard chow when either heterozygous or homozygous mutant animals were presented with dietary variety. Thus, decreased MC4R signaling in melanocortin obesity syndrome consistently yields hyperphagia irrespective of the foods provided, but the hyperphagia appears driven by variety and/or novelty, rather than by a preference for high-fat or high-carbohydrate foodstuffs.

Hypothalamic mTORC1 Signaling Controls Sympathetic Nerve Activity and Arterial Pressure and Mediates Leptin Effects

The fundamental importance of the hypothalamus in the regulation of autonomic and cardiovascular functions is well established. However, the molecular processes involved are not well understood. Here, we show that the mammalian (or mechanistic) target of rapamycin (mTOR) signaling in the hypothalamus is tied to the activity of the sympathetic nervous system and to cardiovascular function. Modulation of mTOR complex 1 (mTORC1) signaling caused dramatic changes in sympathetic traffic, blood flow, and arterial pressure. Our data also demonstrate the importance of hypothalamic mTORC1 signaling in transducing the sympathetic and cardiovascular actions of leptin. Moreover, we show that the PI3K pathway links the leptin receptor to mTORC1 signaling and that changes in its activity impact sympathetic traffic and arterial pressure. These findings establish mTORC1 activity in the hypothalamus as a key determinant of sympathetic and cardiovascular regulation and suggest that dysregulated hypothalamic mTORC1 activity may influence the development of cardiovascular diseases.

Neuronostatin acts in brain to biphasically increase mean arterial pressure through sympatho-activation followed by vasopressin secretion: the role of melanocortin receptors

Neuronostatin is a recently described neuropeptide that is derived from the somatostatin preprohormone. We have shown previously that neuronostatin led to a biphasic, dose-related increase in mean arterial pressure when injected into the lateral cerebroventricle of adult, male rats. Because neuronostatin depolarized both magnocellular and parvocellular, paraventricular nucleus neurons in hypothalamic slice preparations, we hypothesized that neuronostatin elevated mean arterial pressure first by stimulating sympathetic nervous system activity followed by the release of a pressor hormone, specifically vasopressin. We found that the first phase of neuronostatin-induced increase in mean arterial pressure was reversed by pretreatment with phentolamine, indicating that phase 1 was, indeed, due to an increase in sympathetic activity. We also found that centrally injected neuronostatin led to a dose-related increase in vasopressin secretion in a time course consistent with the peak of the second phase. Furthermore, the second phase of arterial pressure elevation was reversed by pretreatment with a vasopressin 1 receptor antagonist, indicating that phase 2 was likely due to an increase in vasopressin secretion. We previously have shown that the anorexigenic and antidipsogenic effects of neuronostatin were reversed by pretreatment with the melanocortin 3/4 receptor antagonist, SHU9119, so we evaluated the ability of SHU9119 to reverse the effects of neuronostatin on MAP and vasopressin secretion. We found that SHU9119 abrogated the second phase of neuronostatin-induced increase in MAP and neuronostatin-induced vasopressin secretion, indicating that neuronostatin acts through the central melanocortin system to increase vasopressin release, ultimately leading to an elevation in MAP.

Role of Melanocortin Receptors in the Regulation of Gouty Inflammation

Gouty arthritis is a form of acute joint inflammation provoked by joint deposition of urate crystals. Although this acute pathology resolves after a few days, the marked degree of inflammation in the joint and--possibly more important to the patient--the excruciating pain it causes require proper therapeutic management. Often deemed a "poor sibling" of chronic joint pathologies such as rheumatoid arthritis and psoriatic arthritis, the increasing incidence of gout makes it a more palatable disease for novel drug discovery programs. This fact, associated with novel insights into the molecular mechanisms activated by the urate crystal deposition, is at the basis of new therapeutics under clinical development for gout, a valid example being the effective targeting of the proinflammatory cytokine interleukin-1. Here we briefly review the current status of antigout drug development and propose another target; our focus is on melanocortin receptor agonists as novel therapeutics for gout and inflammatory arthritides, a prototype of which, the adrenocorticotropic hormone, is already used in clinical settings.

Double Leptin and Melanocortin-4 Receptor Gene Mutations Have an Additive Effect on Fat Mass and Are Associated with Reduced Effects of Leptin on Weight Loss and Food Intake

Melanocortin-4 receptors (MC4Rs) are involved in the regulation of food intake, sympathetic nervous activity, and adrenal and thyroid function by leptin. The role of MC4Rs in regulating energy balance by leptin was investigated using double heterozygote or homozygous leptin (Lep(ob)) and Mc4r gene mutant mice. Double heterozygous or homozygous mutants were generated by crossing MC4R knockout (Mc4r-/-) mice, backcrossed onto C57BL/6J, with B6.V-Lep(ob) mice. Energy expenditure was measured using indirect calorimetry. The effect of leptin on food intake, weight loss, insulin, and corticosterone was compared for Lep(ob)/Lep(ob)Mc4r-/- mice and Lep(ob)/Lep(ob) mice. Double heterozygous and homozygous mutants exhibited an additive effect on fat mass. The 2-fold increase in body weight associated with severe obesity of Lep(ob)/Lep(ob) mice was associated with a significantly higher 24 h total and resting energy expenditure. The effect of obesity on energy expenditure was attenuated by 50% in Lep(ob)/Lep(ob) Mc4r+/- and Lep(ob)/Lep(ob) Mc4r-/- mice. Loss of MC4Rs did not affect basal food intake of Lep(ob)/Lep(ob) mice but was associated with partial leptin resistance in terms of food intake and weight loss. Leptin suppression of insulin and corticosterone in Lep(ob)/Lep(ob) mice were not significantly affected by Mc4r genotype. These results suggest a complex interaction between the Lep and Mc4r genes in energy homeostasis and suggest that MC4Rs retain significant anti-obesity function in the obese leptin-deficient state. Increased adiposity with double mutations may involve a reduction in energy expenditure. MC4Rs might have a modest role in the regulation of energy balance by exogenously administered leptin, primarily effecting food intake.

Evidence for direct actions of melanocortin peptides on bone metabolism

Expression of melanocortin-4 receptor (MC4R) mRNA in developing rat limb buds, teeth, and skull bone first indicated a possible role for MC4R in bone metabolism. We therefore investigated whether MC4R mRNA was expressed in the rat osteosarcoma UMR106.06 cell line and in primary rat osteoblast cells. Reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot analysis, and ribonuclease protection assay (RPA) were used to demonstrate MC4R mRNA expression in UMR106.06 and primary osteoblast cells. MC4R mRNA was found to be localized to the periosteum of mouse bone using in situ hybridization. We also used RT-PCR and rat specific MC2R and MC5R oligonucleotides to amplify the correct size DNA fragments for these melanocortin receptors from rat primary osteoblasts. In conclusion, melanocortin receptor expression in mouse periosteum and rat osteoblasts suggests a direct role for POMC derived peptides in bone development and bone metabolism.

Role of Melanocortin-4 Receptors in Mediating Renal Sympathoactivation to Leptin and Insulin

Central melanocortin signaling plays an important role in regulation of energy homeostasis by leptin and insulin. We investigated the interaction between leptin, insulin, and melanocortin-4 receptors (MC-4Rs) in the control of renal sympathetic nerve activity (RSNA) in mice. We compared the effects of intracerebroventricular (ICV) administration of leptin, insulin, MC-3/4R agonist (MTII), and corticotrophin-releasing factor (CRF) on RSNA in leptin receptor-deficient (db/db) mice, MC-4R knock-out mice, and their wild-type controls. ICV administration of leptin and MTII caused a significant and dose-dependent increase in RSNA in control mice. As expected, leptin had no significant effect on RSNA in the db/db mice. Interestingly, db/db mice exhibited markedly attenuated RSNA responses to ICV administration of MTII. However, the increase in RSNA induced by insulin and CRF was comparable between db/db and control mice. In the heterozygous and homozygous MC-4R knock-out mice, the RSNA response to MTII was attenuated and abolished, respectively. The RSNA response to ICV leptin and insulin was also attenuated and abolished in the heterozygous and homozygous MC-4R knock-out mice, respectively. In contrast, CRF induced a similar increase in RSNA in the MC-4R knock-out and wild-type mice. Our data demonstrate that in the absence of leptin receptors, the sympathoexcitatory effects of melanocortin system stimulation are attenuated. In addition, the renal sympathoexcitatory responses to leptin and insulin are dependent on the MC-4R, demonstrating an important role for the MC-4R in the regulation of renal sympathetic nerve outflow by leptin and insulin.

The role of melanocortin receptors in sexual behavior in female rats

Earlier data have indicated that α-MSH may play a role for sexual behavior in rats. In this study we investigated the effects of MSH peptides on sexual receptivity in ovariectomized-adrenalectomized female rats, pre-treated with benzoate of estradiol, in presence of vigorous male rats. The results show that α-MSH significantly increases lordosis behavior in female rats after injections into the ventromedial nucleus. Interestingly, we have for the first time shown that γ-MSH also causes significant increase in lordosis in female rats. Furthermore, we show that HS014, an antagonist for the central MC receptors, in dose dependent manner blocks the effect of α-MSH on lordosis. The results indicate that the effects of MSH peptides on female sexual behaviour are mediated through a specific MC receptor, which could be the MC3 receptor.

ACTH- and α-MSH-induced grooming, stretching, yawning and penile erection in male rats: Site of action in the brain and role of melanocortin receptors

The effect of adrenocorticotropin (ACTH)(1-24) and α-melanocyte stimulating hormone (α-MSH) on grooming, stretching, yawning and penile erection was studied after injection into different brain areas. Both peptides induce the above responses when injected into the hypothalamic periventricular region of the third ventricle. This region includes the paraventricular nucleus, the dorsomedial nucleus, the ventromedial nucleus and the anterior hypothalamic area. The minimal effective dose of both peptides was 0.5 μg and the maximal effect was seen with 2 μg, the highest dose tested. Irrespective of the injection site, grooming started 5–7 min after injection of either peptide, while stretching, yawning and penile erection started only after 15–35 min and lasted for 90–120 min. In contrast both peptides were ineffective when injected into the preoptic area, the caudate nucleus or the CA1 field of the hippocampus. Grooming, stretching and yawning, but not penile erection, were prevented by cyclic[AcCys 11, D-Nal 14, Cys 18, AspNH 2 22]-β-MSH (11-22) (HS014), a selective melanocortin 4 receptor antagonist, injected into the same periventricular area 10 min before of ACTH(1-24) or α-MSH. The results show that ACTH(1-24) and α-MSH act in the hypothalamic periventricular region to induce the above responses and that grooming, stretching and yawning, but not penile erection, are mediated by melanocortin 4 receptors.

Agouti regulation of intracellular calcium: role of melanocortin receptors.

Several dominant mutations at the murine agouti locus cause a syndrome of marked obesity and insulin resistance. We have recently reported that intracellular free Ca2+ concentration ([Ca2+]i) is elevated in viable yellow mice. Because [Ca2+]i has a key role in the pathogenesis of insulin resistance, obesity, and hypertension, the role of the purified agouti gene product in regulating [Ca2+]i was evaluated in a number of cell types. Purified murine agouti induced slow, sustained increases in [Ca2+]i in A7r5 vascular smooth muscle cells and 3T3-L1 adipocytes in a dose-dependent fashion. In L6 skeletal myocytes, agouti stimulated an increase in [Ca2+]i with an apparent concentration eliciting 50% of the maximal response (EC50) of 62 nM. This response was substantially inhibited by Ca2+ entry blockade with nitrendipine. To determine whether melanocortin receptors play a role in agouti regulation of [Ca2+]i, we examined the effect of melanocortin peptides and agouti in cells stably transfected with human melanocortin receptors. Human embryonic kidney cells (HEK-293 cells) transfected with either the human melanocortin 1 receptor (MC1R) or melanocortin 3 receptor responded to human agouti with slow, sustained increases in [Ca2+]i, whereas nontransfected HEK-293 cells with no melanocortin receptors did not respond to agouti. Dose-response curves in the MC1R line showed that agouti had an EC50 of 18 nM, which is comparable to that for agouti antagonism of (125)I-Nle,D-Phe-alpha-melanocyte-stimulating hormone binding in the same cell line. This direct effect of agouti on stimulating increases in [Ca2+]i suggests a potential mechanism for agouti-induced insulin resistance.