Optimized High-Yield Purification of Obesity-Associated Melanocortin 4 Receptor.

BACKGROUND: Obesity has emerged as a global public health challenge associated with increased risk of hyperlipidemia and hypertension. It contributes to high sympathetic activity and increased catecholamine levels. The hypothalamic melanocortin system is known to regulate the energy homeostasis. The role of melanocortin 4 receptor (MC4R) has been demonstrated pharmacologically and in animal studies, which showed that severe obesity in MC4R knockout mice was caused by increased food intake and decreased energy consumption. Over 70 multiple different mis- -sense and nonsense mutations in hMC4R have been found at a high frequency of 2-8% in severe early onset or hereditary obesity. The single amino acid variation (D90N) located in the second transmembrane domain (TM2) of MC4R results in accelerated growth and childhood onset obesity. Interestingly, the functional characterization of D90N hMC4R mutant TM2 (m-hMC4R-TM2) revealed normal cell surface expression and binding with agonist similar to the hMC4R wild-type TM2 (wt-hMC4R-TM2) but loss of signal transduction mediated via Gs/adenylyl cyclase activation. It is essential to delineate the three-dimensional structure of MC4Rs in order to elucidate their functional aspects. OBJECTIVE: In this study, we demonstrate the optimized expression and isolation of wt/m-hMC4R-TM2 proteins under different chemical cleavage reaction times and purification procedures via SDS precipitation. The solid-state NMR spectroscopy was carried out to study the structure of wt/m-hMC4R- TM2 protein in the anisotropic phospholipid bicelles. METHODS: The KSI-wt/m-hMC4R-TM2 fusion proteins developed in cell culture with LB medium. In order to isolate the expressed fusion protein from the cell, ultrasonication, Ni-NTA affinity chromatography, dialysis, and lyophilization techniques were used. Then, to obtain a protein with higher purity and higher yield, the CNBr chemical cleavage time was subdivided into 30 minutes, 1 h, 2 h, 3 h, and 4 h. Purification process was performed using FPLC, and 100 mM KCl and dialysis were used to remove the SDS. CD spectrometer, MALDI-TOF, solution-state NMR, and solid-state NMR were used to confirmed purity and structure of the wt/m-hMC4R-TM2. RESULTS: The precipitation method was used to remove the SDS bound to proteins as KCl-SDS. We optimized the 2 h cleavage reaction times for both wt-hMC4R-TM2 and m-hMC4R-TM2 depending on the purity based on mass spectra and 1H-15N HSQC spectra and the yield after final purification. The 1D 1H-15N CP (Cross polarization) solid-state NMR spectra suggest that the wt/m-hMC4R- TM2 undergo rotational diffusion around a perpendicular axis along the bilayer normal. CONCLUSION: We expressed wt/m-hMC4R-TM2 in E.coli and optimized the isolation and purification process, especially CNBr chemical cleavage time. The efficiency of KCl-SDS precipitation was confirmed via MALDI-TOF MS and the pure proteins obtained using this method were characterized by CD spectroscopy and solution-state NMR. The results of 1H-15N HSQC spectra in solution- state NMR also show the probability for structural studies. The 1D 1H-15N CP solid-state NMR spectra indicate that most of the residues in both the wt/m-hMC4R-TM2 peptides are integrated into the membrane.

Regulation of melanocortin-4-receptor (MC4R) expression by SNP rs17066842 is dependent on glucose concentration.

Melanocortin-4-receptor (MC4R) gene codes for a G-protein-coupled receptor that is highly expressed in the hypothalamus and involved in the regulation of appetite. Single-nucleotide polymorphisms (SNPs) in the MC4R gene region have been associated with obesity, type 2-diabetes (T2D) and with antipsychotic-induced weight gain. Of these, rs17066842 (G>A) in the MC4R promoter region is the top variant associated with obesity and diabetes. In this study, we investigated the effect of rs17066842 on MC4R expression at various glucose concentrations using reporter gene expression in the SH-SY5Y cell line and regulation of MC4R expression in human cerebral organoids. We observed that higher glucose concentrations significantly reduced MC4R mRNA expression in SH-SY5Y cells. In addition, at high glucose concentrations, the luciferase reporter plasmid containing the MC4R promoter insert with the G-allele of rs170066842 showed significantly reduced activity compared to the A-allele carrying plasmid. The immediate early gene product, early growth-response 1 (EGR-1), was identified to bind to the sequence containing the G-allele at rs17066842 but not to the A-allele-containing sequence. Interestingly, in human induced pluripotent stem cell (hiPSC)-derived cerebral organoids, we observed increased MC4R expression in response to high glucose exposure. These opposite observations might suggest that glucose regulation is complex and may be cell-specific. This study provides evidence that rs17066842 regulates MC4R gene expression through binding of EGR-1 and that this process is influenced by glucose concentration.

Ultraviolet B stimulates proopiomelanocortin signalling in the arcuate nucleus of the hypothalamus in mice.

We previously found that ultraviolet B (UVB) could stimulate the paraventricular nucleus (PVN) with activation the systemic hypothalamic-pituitary- adrenal (HPA) axis. To investigate whether UVB can also stimulate other hypothalamic nuclei, we tested its effect on the proopiomelanocortin (POMC) related signalling system in the arcuate nucleus (ARC) of female C57BL/6 and FVB albino mice. The shaved back skin of the mice was irradiated with either 100 or 400 mJ/cm2 of UVB. After 1, 3, 6 and 12 h, blood and hypothalamus were collected and processed for gene and protein expression, and measurement of α-MSH and ß-endorphin (ß-END) levels. An in situ immunohistochemical examination was performed for melanocortin receptor 4 (MC4R) and POMC-derived α-MSH. The expression of Pomc and MC4R mRNAs was stimulated, whereas that of AgRP was inhibited after exposure to UVB. It was accompanied by an increased number of both α-MSH- and MC4R-immunoreactive neurons in the ARC, and by increased levels of α-MSH and ß-END (both found in the hypothalamus and plasma). This surprising discovery of UVB stimulating the POMC system in the ARC, accompanied by the increased plasma levels of α-MSH and ß-END, paves the way for exciting areas of research on the communication between the skin and the brain, as well as is suggesting a new role for UVB in regulation of body metabolism.

Tankyrases regulate glucoregulatory mechanisms and somatic growth via the central melanocortin system in zebrafish larvae.

The central melanocortin system is a key regulator of energy homeostasis. Recent studies indicate that tankyrases (TNKSs), which poly(ADP-ribosyl)ate target proteins and direct them toward proteasomal degradation, affect overall metabolism, but the exact molecular mechanisms remain unclear. We used zebrafish larvae as a model to study the mechanisms by which TNKS1b, the zebrafish ortholog of mammalian TNKS1, regulates glucose homeostasis and somatic growth. In situ hybridization revealed that TNKS1b mRNA is prominently expressed in the hypothalamus and pituitary of the embryonic and larval brain. In the pituitary, TNKS1b is coexpressed with pro-opiomelanocortin a (pomca) gene in corticotropes and melanotropes. Knockdown of TNKS1b reduced the linear growth of the larvae, stimulated insulin gene and glucose transporter 4 protein, and suppressed gluconeogenic phosphoenolpyruvate carboxykinase 1 gene. This result indicates rapid glucose utilization and reduction of gluconeogenesis in TNKS1b-deficient larvae. Knockdown of TNKS1b down-regulated pomca expression and diminished α-melanocyte-stimulating hormone in the pars intermedia. Furthermore, down-regulation of TNKS1b suppressed the expression of melanocortin receptor 3 and increased the expression of melanocortin receptor 4. The collective data suggest that TNKS1b modulates glucoregulatory mechanisms and the somatic growth of zebrafish larvae via the central melanocortin system.

Maternal and post-weaning high-fat, high-sucrose diet modulates glucose homeostasis and hypothalamic POMC promoter methylation in mouse offspring.

Substantial evidence demonstrated that maternal dietary nutrients can significantly determine the susceptibility to developing metabolic disorders in the offspring. Therefore, we aimed to investigate the later-life effects of maternal and postweaning diets interaction on epigenetic modification of the central nervous system in the offspring. We examined the effects of dams fed a high-fat, high-sucrose (FS) diet during pregnancy and lactation and weaned to FS diet continuously until 32 weeks of age. Then, DNA methylation and gene expressions of hypothalamic proopiomelanocortin (POMC) and melanocortin receptor 4 (MC4R) were determined in the offspring. Offspring of FS diet had heavier body weight, impaired glucose tolerance, decreased insulin sensitivity and higher serum leptin level at 32-week age (p < 0.05). The expression of POMC and MC4R genes were significantly increased in offspring exposed to FS diet during gestation, lactation and into 32-week age (p < 0.05). Consistently, hypomethylation of POMC promoter in the hypothalamus occurred in the FS diet offspring (p < 0.05), compared with the C group. However, no methylation was detected of MC4R promoter in both the two groups. Furthermore, POMC-specific methylation (%) was negatively associated with glucose response to a glucose load (r = -0.273, p = 0.039). Maternal and post-weaning high-fat diet predisposes the offspring for obesity, glucose intolerance and insulin resistance in later life. Our findings can advance our thinking around the DNA methylation status of the promoter of the POMC and MC4R genes between long-term high-fat, high-sucrose diet and glucose homeostasis in mouse.

Co-expression of human agouti-related protein enhances expression and stability of human melanocortin-4 receptor.

G protein-coupled receptors (GPCRs) represent the largest family of transmembrane signaling proteins, and they are considered major targets of approximately half of all therapeutic agents. Human melanocortin-4 receptor (hMC4R) plays an important role in the control of energy homeostasis, and its mutants are directly related to severe human obesity. Here, we describe optimized protocols for the high-yield expression and purification of hMC4R that will accelerate structural study. Truncations of the N- and C-termini of hMC4R with T4 lysozyme (T4L) insertion increase the solubility as well as stability of the protein. Strikingly, co-expression of human mini-agouti-related protein (mini-AgRP) in Spodoptera frugiperda (Sf9) cells enables excellent stability of hMC4R. The protein yield in the human mini-AgRP co-expression system is increased by about 3-4 times compared to that of hMC4R alone. Data from analytical size exclusion chromatography (aSEC) and thermostability assay show that hMC4R becomes homogeneous and stable with a melting temperature of 58°C in the presence of human mini-AgRP.

Inhibiting neuropeptide Y Y1 receptor modulates melanocortin receptor- and NF-κB-mediated feeding behavior in phenylpropanolamine-treated rats.

Neuropeptide Y (NPY) and nuclear factor-kappa B (NF-κB) are involved in regulating anorexia elicited by phenylpropanolamine (PPA), a sympathomimetic drug. This study explored whether NPY Y1 receptor (Y1R) is involved in this process, and a potential role for the proopiomelanocortin system was identified. Rats were given PPA once a day for 4days. Changes in the hypothalamic expression of the NPY, Y1R, NF-κB, and melanocortin receptor 4 (MC4R) levels were assessed and compared. The results indicated that food intake and NPY expression decreased, with the largest reductions observed on Day 2 (approximately 50% and 45%, respectively), whereas NF-κB, MC4R, and Y1R increased, achieving maximums on Day 2 (160%, 200%, and 280%, respectively). To determine the role of Y1R, rats were pretreated with Y1R antisense or a Y1R antagonist via intracerebroventricular injection 1h before the daily PPA dose. Y1R knockdown and inhibition reduced PPA anorexia and partially restored the normal expression of NPY, MC4R, and NF-κB. The data suggest that hypothalamic Y1R participates in the appetite-suppression from PPA by regulating MC4R and NF-κB. The results of this study increase our understanding of the molecular mechanisms in PPA-induced anorexia.

Region-specific differences in brain melanocortin receptors in rats of the lean phenotype.

The brain melanocortin (MC) system is one of numerous overlapping systems regulating energy balance; it consists of peptides including α-melanocyte-stimulating hormone that act through melanocortin receptors (MCRs). Mutations and polymorphisms in MC3R and MC4R have been identified as one of the most common genetic contributors to obesity in human studies. Brain MC3R and MC4R are known to modulate energy expenditure (EE) and food intake, but much less is known regarding brain MC5R. To test the hypothesis that brain MC modulates physical activity (PA) and EE, we compared brain MCR profiles in rats that consistently show high versus low levels of 'spontaneous' daily PA. Compared with low-activity rats, high-activity rats show enhanced mRNA expression of MCRs in the brain, specifically of MC3R in the paraventricular nucleus (PVN), and MC4R and MC5R in the perifornical lateral hypothalamus. Next, we microinjected the MCR agonist melanotan II into the PVN region and measured PA and EE. Intra-PVN melanotan II induced a dose-dependent increase in PA and this effect was greater in high-activity rats compared with low-activity rats. These results indicate region-specific brain MCR expression in the heightened PA seen in association with high endurance capacity and identify promising targets in the brain MC system that may contribute to interindividual variability in energy balance.

Melanocortin-4 receptor expression in different classes of spinal and vagal primary afferent neurons in the mouse.

Melanocortin-4 receptor (MC4R) ligands are known to modulate nociception, but the site of action of MC4R signaling on nociception remains to be elucidated. The current study investigated MC4R expression in dorsal root ganglia (DRG) of the MC4R-GFP reporter mouse. Because MC4R is known to be expressed in vagal afferent neurons in the nodose ganglion (NG), we also systematically compared MC4R-expressing vagal and spinal afferent neurons. Abundant green fluorescent protein (GFP) immunoreactivity was found in about 45% of DRG neuronal profiles (at the mid-thoracic level), the majority being small-sized profiles. Immunohistochemistry combined with in situ hybridization confirmed that GFP was genuinely produced in MC4R-expressing neurons in the DRG. While a large number of GFP profiles in the DRG coexpressed Nav1.8 mRNA (84%) and bound isolectin B4 (72%), relatively few GFP profiles were positive for NF200 (16%) or CGRP (13%), suggesting preferential MC4R expression in C-fiber nonpeptidergic neurons. By contrast, GFP in the NG frequently colocalized with Nav1.8 mRNA (64%) and NF200 (29%), but only to a moderate extent with isolectin B4 (16%). Lastly, very few GFP profiles in the NG expressed CGRP (5%) or CART (4%). Together, our findings demonstrate variegated MC4R expression in different classes of vagal and spinal primary afferent neurons, and underscore the role of the melanocortin system in modulating nociceptive and nonnociceptive peripheral sensory modalities.

Melanocortin 4 receptor signaling in dopamine 1 receptor neurons is required for procedural memory learning.

It is now widely recognized that exposure to palatable foods engages reward circuits that promote over-eating and facilitate the development of obesity. While the melanocortin 4 receptor (MC4R) has previously been shown to regulate food intake and energy expenditure, little is known about its role in food reward. We demonstrate that MC4R is co-expressed with the dopamine 1 receptor (D1R) in the ventral striatum. While MC4R-null mice are hyperphagic and obese, they exhibit impairments in acquisition of operant responding for a high fat reinforcement. Restoration of MC4R signaling in D1R neurons normalizes procedural learning without affecting motivation to obtain high fat diet. MC4R signaling in D1R neurons is also required for learning in a non-food-reinforced version of the cued water maze. Finally, MC4R signaling in neostriatal slices increases phosphorylation of the Thr34 residue of DARPP-32, a protein phosphatase-1 inhibitor that regulates synaptic plasticity. These data identify a novel requirement for MC4R signaling in procedural memory learning.

Leptin resistance and desensitization of hypophagia during prolonged inflammatory challenge.

Acute exposure to bacterial lipopolysaccharide (LPS) is a potent inducer of immune response as well as hypophagia. Nevertheless, desensitization of responses to LPS occurs during long-term exposure to endotoxin. We induced endotoxin tolerance, injecting repeated (6LPS) LPS doses compared with single (1LPS) treatment. 1LPS, but not 6LPS group, showed decreased food intake and body weight, which was associated with an increased plasma leptin and higher mRNA expression of OB-Rb, MC4R, and SOCS3 in the hypothalamus. Hypophagia induced by 1LPS was associated with lower levels of 2-arachidonoylglycerol (2-AG), increased number of p-STAT3 neurons, and decreased AMP-activated protein kinase (AMPK) activity. Desensitization of hypophagia in the 6LPS group was related to high 2-AG, with no changes in p-STAT3 or increased p-AMPK. Leptin decreased food intake, body weight, 2-AG levels, and AMPK activity and enhanced p-STAT3 in control rats. However, leptin had no effects on 2-AG, p-STAT3, or p-AMPK in the 1LPS and 6LPS groups. Rats treated with HFD to induce leptin resistance showed neither hypophagia nor changes in p-STAT3 after 1LPS, suggesting that leptin and LPS recruit a common signaling pathway in the hypothalamus to modulate food intake reduction. Desensitization of hypophagia in response to repeated exposure to endotoxin is related to an inability of leptin to inhibit AMPK phosphorylation and 2-AG production and activate STAT3. SOCS3 is unlikely to underlie this resistance to leptin signaling in the endotoxin tolerance. The present model of prolonged inflammatory challenge may contribute to further investigations on mechanisms of leptin resistance.

Neuroanatomical distribution of the melanocortin-4 receptors in male and female rodent brain.

The melanocortin-4 receptor (MC4-R) plays a critical role in several physiological functions, from food intake, energy homeostasis, neuroendocrine and cardiovascular function, to sexual responses. The brain regions and the central neuronal pathways mediating the different actions of MC4-R remain largely unknown. We aimed to use immunocytochemistry using a specific antibody against rat MC4-R, to establish the detailed neuroanatomical distribution of MC4-R in brain slices of male and estrous female rats. We demonstrated that MC4-R-positive neurons were widely distributed in several brain regions including the cortex, thalamus, hypothalamus, and brainstem. In both male and female brains, MC4-R-positive cells were especially abundant in the hypothalamus, including the paraventricular hypothalamic nucleus, lateral septal nucleus, arcuate nucleus, supraoptic nucleus, medial preoptic area and lateral hypothalamic area. A moderate number of MC4-R-positive neurons were found in the piriform cortex, bed nucleus of the stria terminalis, medial and basolateral nuclei of amygdala, periaqueductal gray, red nucleus and raphe nucleus. A dimorphic sexual difference in the number of MC4-R-positive neurons was observed in some brain regions. In the medial preoptic area and arcuate nucleus, MC4-R-positive neurons were significantly more abundant in female than in males, whereas in the lateral hypothalamus the opposite proportion was observed. This is the first time the neuroanatomical distribution, and sex differences, of brain MC4-R localisation have been described. The distribution of MC4-R is consistent with the proposed roles of MC4-R-positive neurons and provides further information about the circuitry controlling food intake, energy balance and sexual responses in both males and females.

Neuropeptide delivery to the brain: a von Willebrand factor signal peptide to direct neuropeptide secretion.

BACKGROUND: Multiple neuropeptides, sometimes with opposing functions, can be produced from one precursor gene. To study the roles of the different neuropeptides encoded by one large precursor we developed a method to overexpress minigenes and establish local secretion. RESULTS: We fused the signal peptide from the Von Willebrand Factor (VWF) to a furin site followed by a processed form of the Agouti related protein (AgRP), AgRP(83-132) or alpha-melanocyte stimulating hormone. In vitro, these minigenes were secreted and biologically active. Additionally, the proteins of the minigenes were not transported into projections of primary neurons, thereby ensuring local release. In vivo administration of VWF-AgRP(83-132), using an adeno-associated viral vector as a delivery vehicle, into the paraventricular hypothalamus increased body weight and food intake of these rats compared to rats which received a control vector. CONCLUSIONS: This study demonstrated that removal of the N-terminal part of full length AgRP and addition of a VWF signal peptide is a successful strategy to deliver neuropeptide minigenes to the brain and establish local neuropeptide secretion.

Pharmacological characterization of 30 human melanocortin-4 receptor polymorphisms with the endogenous proopiomelanocortin-derived agonists, synthetic agonists, and the endogenous agouti-related protein antagonist.

The melanocortin-4 receptor (MC4R) is a G-protein-coupled receptor (GPCR) that is expressed in the central nervous system and has a role in regulating feeding behavior, obesity, energy homeostasis, male erectile response, and blood pressure. Since the report of the MC4R knockout mouse in 1997, the field has been searching for links between this genetic biomarker and human obesity and type 2 diabetes. More then 80 single nucleotide polymorphisms (SNPs) have been identified from human patients, both obese and nonobese controls. Many significant studies have been performed examining the pharmacological characteristics of these hMC4R SNPs in attempts to identify a molecular defects/insights that might link a genetic factor to the obese phenotype observed in patients possessing these mutations. Our laboratory has previously reported the pharmacological characterization of 40 of these polymorphic hMC4 receptors with multiple endogenous and synthetic ligands. The goal of the current study is to perform a similar comprehensive side-by-side characterization of 30 additional human hMC4R with single nucleotide polymorphisms using multiple endogenous agonists [alpha-, beta-, and gamma(2)-melanocyte stimulating hormones (MSH) and adrenocorticotropin (ACTH)], the antagonist agouti-related protein hAGRP(87-132), and synthetic agonists [NDP-MSH, MTII, and the tetrapeptide Ac-His-dPhe-Arg-Trp-NH(2) (JRH887-9)]. These in vitro data, in some cases, provide a putative molecular link between dysfunctional hMC4R's and human obesity. These 30 hMC4R SNPs include R7H, R18H, R18L, S36Y, P48S, V50M, F51L, E61K, I69T, D90N, S94R, G98R, I121T, A154D, Y157S, W174C, G181D, F202L, A219 V, I226T, G231S, G238D, N240S, C271R, S295P, P299L, E308K, I317V, L325F, and 750DelGA. All but the N240S hMC4R were identified in obese patients. Additionally, we have characterized a double I102T/V103I hMC4R. In addition to the pharmacological characterization, the hMC4R variants were evaluated for cell surface expression by flow cytometry. The F51L, I69T, and A219V hMC4Rs possessed full agonist activity and significantly decreased endogenous agonist ligand potency. At the E61K, D90N, Y157S, and C271R hMC4Rs, all agonist ligands examined were only partially efficacious in generating a maximal signaling response (partial agonists) and possessed significantly decreased endogenous agonist ligand potency. Only the A219V, G238D, and S295P hMC4Rs possessed significantly decreased AGRP(87-132) antagonist potency. These data provide new information for use in GPCR computational development as well as insights into MC4R structure ad function.

Postnatal Sim1 deficiency causes hyperphagic obesity and reduced Mc4r and oxytocin expression.

Single-minded 1 (SIM1) mutations are one of the few known causes of nonsyndromic monogenic obesity in both humans and mice. Although the role of Sim1 in the formation of the hypothalamus has been described, its postdevelopmental, physiological functions have not been well established. Here we demonstrate that postnatal CNS deficiency of Sim1 is sufficient to cause hyperphagic obesity. We conditionally deleted Sim1 after birth using CaMKII-Cre (alpha-calcium/calmodulin-dependent protein kinase II-Cre) lines to recombine a floxed Sim1 allele. Conditional Sim1 heterozygotes phenocopied germ line Sim1 heterozygotes, displaying hyperphagic obesity and increased length. We also generated viable conditional Sim1 homozygotes, demonstrating that adult Sim1 expression is not essential for mouse or neuron survival and revealing a dosage-dependent effect of Sim1 on obesity. Using stereological cell counting, we showed that the phenotype of both germ line heterozygotes and conditional Sim1 homozygotes was not attributable to global hypocellularity of the paraventricular nucleus (PVN) of the hypothalamus. We also used retrograde tract tracing to demonstrate that the PVN of germ line heterozygous mice projects normally to the dorsal vagal complex and the median eminence. Finally, we showed that conditional Sim1 homozygotes and germ line Sim1 heterozygotes exhibit a remarkable decrease in hypothalamic oxytocin (Oxt) and PVN melanocortin 4 receptor (Mc4r) mRNA. These results demonstrate that the role of Sim1 in feeding regulation is not limited to formation of the PVN or its projections and that the hyperphagic obesity in Sim1-deficient mice may be attributable to changes in the leptin-melanocortin-oxytocin pathway.

Melanocortin-4 receptor activation inhibits c-Jun N-terminal kinase activity and promotes insulin signaling.

The melanocortin system is crucial to regulation of energy homeostasis. The melanocortin receptor type 4 (MC4R) modulates insulin signaling via effects on c-Jun N-terminal kinase (JNK). The melanocortin agonist NDP-MSH dose-dependently inhibited JNK activity in HEK293 cells stably expressing the human MC4R; effects were reversed by melanocortin receptor antagonist. NDP-MSH time- and dose-dependently inhibited IRS-1(ser307) phosphorylation, effects also reversed by a specific melanocortin receptor antagonist. NDP-MSH augmented insulin-stimulated AKT phosphorylation in vitro. The melanocortin agonist melanotan II increased insulin-stimulated AKT phosphorylation in the rat hypothalamus in vivo. NDP-MSH increased insulin-stimulated glucose uptake in hypothalamic GT1-1 cells. The current study shows that the melanocortinergic system interacts with insulin signaling via novel effects on JNK activity.

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 fluoxetine administration on hypothalamic melanocortin system in obese Zucker rats.

The aim of the present work was to study the potential involvement of melanocortin system in the anorectic mechanism of fluoxetine, a selective serotonin reuptake inhibitors, in obese Zucker rats. Male obese Zucker (fa/fa) rats were administered fluoxetine (10 mg/kg; i.p.) daily for two weeks. The control group was given 0.9% NaCl solution. RT-PCR for pro-opiomelanocortin (POMC), Agouti gene related peptide (AgRP) and melanocortin receptor 4 (MC4-R) in the hypothalamus, as well as regional immunostaining for alpha-melanocyte stimulating hormone (alpha-MSH) and MC4-R were carried out. Fluoxetine administration increased POMC expression and reduced MC4-R expression in the hypothalamus, without changes in AgRP mRNA levels. Moreover, an increase in the numbers of alpha-MSH positively immunostained neural cells in the hypothalamic arcuate nucleus (ARC), as well as a significant decrease in the numbers of neural cells positively immunostained for MC4-R in the paraventricular nucleus (PVN), without changes in lateral hypothalamic area (LHA), were observed. These results suggest the involvement of alpha-MSH in central fluoxetine anorectic action.

Divergence of melanocortin pathways in the control of food intake and energy expenditure.

Activation of melanocortin-4-receptors (MC4Rs) reduces body fat stores by decreasing food intake and increasing energy expenditure. MC4Rs are expressed in multiple CNS sites, any number of which could mediate these effects. To identify the functionally relevant sites of MC4R expression, we generated a loxP-modified, null Mc4r allele (loxTB Mc4r) that can be reactivated by Cre-recombinase. Mice homozygous for the loxTB Mc4r allele do not express MC4Rs and are markedly obese. Restoration of MC4R expression in the paraventricular hypothalamus (PVH) and a subpopulation of amygdala neurons, using Sim1-Cre transgenic mice, prevented 60% of the obesity. Of note, increased food intake, typical of Mc4r null mice, was completely rescued while reduced energy expenditure was unaffected. These findings demonstrate that MC4Rs in the PVH and/or the amygdala control food intake but that MC4Rs elsewhere control energy expenditure. Disassociation of food intake and energy expenditure reveals unexpected divergence in melanocortin pathways controlling energy balance.