Type 1 melanocortin receptors in pro-opiomelanocortin-, vasopressin-, and oxytocin-immunopositive neurons in different areas of mouse brain.

Information on the localization of the Type 1 melanocortin receptors (MC1Rs) in different regions of the brain is very scarce. As a result, the role of MC1Rs in the functioning of brain neurons and in the central regulation of physiological functions has not been studied. This work aimed to study the expression and distribution of MС1Rs in different brain areas of female C57Bl/6J mice. Using real-time polymerase chain reaction, we demonstrated the Mс1R gene expression in the cerebral cortex, midbrain, hypothalamus, medulla oblongata, and hippocampus. Using an immunohistochemical approach, we showed the MС1R localization in neurons of the hypothalamic arcuate, paraventricular and supraoptic nuclei, nucleus tractus solitarius (NTS), dorsal hippocampus, substantia nigra, and cerebral cortex. Using double immunolabeling, the MC1Rs were visualized on the surface and in the bodies and outgrowths of pro-opiomelanocortin (POMC)-immunopositive neurons in the hypothalamic arcuate nucleus, NTS, hippocampal CA3 and CA1 regions, and cerebral cortex. Co-localization with POMC indicates that MC1R, like MC3R, is able to function as an autoreceptor. In the paraventricular and supraoptic nuclei, MC1Rs were visualized on the surface and in the cell bodies of vasopressin- and oxytocin-immunopositive neurons, indicating a relationship between hypothalamic MC1R signaling and vasopressin and oxytocin production. The data obtained indicate a wide distribution of MC1Rs in different areas of the mouse brain and their localization in POMC-, vasopressin- and oxytocin-immunopositive neurons, which may indicate the participation of MC1Rs in the control of many physiological processes in the central nervous system.

Identification of MRAP protein family as broad-spectrum GPCR modulators.

BACKGROUND: The melanocortin receptor accessory proteins (MRAP1 and MRAP2) are well-known endocrine regulators for the trafficking and signalling of all five melanocortin receptors (MC1R-MC5R). The observation of MRAP2 on regulating several non-melanocortin G protein-coupled receptors (GPCRs) has been sporadically reported, whereas other endogenous GPCR partners of the MRAP protein family are largely unknown. METHODS: Here, we performed single-cell transcriptome analysis and drew a fine GPCR blueprint and MRAPs-associated network of two major endocrine organs, the hypothalamus and adrenal gland at single-cell resolution. We also integrated multiple bulk RNA-seq profiles and single-cell datasets of human and mouse tissues, and narrowed down a list of 48 GPCRs with strong endogenous co-expression correlation with MRAPs. RESULTS: 36 and 46 metabolic-related GPCRs were consequently identified as novel interacting partners of MRAP1 or MRAP2, respectively. MRAPs exhibited protein-protein interactions and varying pharmacological properties on the surface translocation, constitutive activities and ligand-stimulated downstream signalling of these GPCRs. Knockdown of MRAP2 expression by hypothalamic administration of adeno-associated virus (AAV) packed shRNA stimulated body weight gain in mouse model. Co-injection of corticotropinreleasing factor (CRF), the agonist of corticotropin releasing hormone receptor 1 (CRHR1), suppressed feeding behaviour in a MRAP2-dependent manner. CONCLUSIONS: Collectively, our study has comprehensively elucidated the complex GPCR networks in two major endocrine organs and redefined the MRAP protein family as broad-spectrum GPCR modulators. MRAP proteins not only serve as a vital endocrine pivot on the regulation of global GPCR activities in vivo that could explain the composite physiological phenotypes of the MRAP2 null murine model but also provide us with new insights of the phenotyping investigation of GPCR-MRAP functional complexes.

Vasopressin neurons in the paraventricular hypothalamus promote wakefulness via lateral hypothalamic orexin neurons.

The sleep-wakefulness cycle is regulated by complicated neural networks that include many different populations of neurons throughout the brain. Arginine vasopressin neurons in the paraventricular nucleus of the hypothalamus (PVHAVP) regulate various physiological events and behaviors, such as body-fluid homeostasis, blood pressure, stress response, social interaction, and feeding. Changes in arousal level often accompany these PVHAVP-mediated adaptive responses. However, the contribution of PVHAVP neurons to sleep-wakefulness regulation has remained unknown. Here, we report the involvement of PVHAVP neurons in arousal promotion. Optogenetic stimulation of PVHAVP neurons rapidly induced transitions to wakefulness from both NREM and REM sleep. This arousal effect was dependent on AVP expression in these neurons. Similarly, chemogenetic activation of PVHAVP neurons increased wakefulness and reduced NREM and REM sleep, whereas chemogenetic inhibition of these neurons significantly reduced wakefulness and increased NREM sleep. We observed dense projections of PVHAVP neurons in the lateral hypothalamus with potential connections to orexin/hypocretin (LHOrx) neurons. Optogenetic stimulation of PVHAVP neuronal fibers in the LH immediately induced wakefulness, whereas blocking orexin receptors attenuated the arousal effect of PVHAVP neuronal activation drastically. Monosynaptic rabies-virus tracing revealed that PVHAVP neurons receive inputs from multiple brain regions involved in sleep-wakefulness regulation, as well as those involved in stress response and energy metabolism. Moreover, PVHAVP neurons mediated the arousal induced by novelty stress and a melanocortin receptor agonist melanotan-II. Thus, our data suggested that PVHAVP neurons promote wakefulness via LHOrx neurons in the basal sleep-wakefulness and some stressful conditions.

Determination of the Interaction and Pharmacological Modulation of MCHR1 Signaling by the C-Terminus of MRAP2 Protein.

Melanin concentrating hormone (MCH), an orexigenic neuropeptide, is primarily secreted by the hypothalamus and acts on its receptor, the melanin-concentrating hormone receptor 1 (MCHR1), to regulate appetite and energy homeostasis. The Melanocortin Receptor Accessory Protein 2 (MRAP2), a small single transmembrane protein broadly expressed in multiple tissues, has been defined as a vital endocrine modulator of five melanocortin receptors (MC1R-MC5R) and several other GPCRs in the regulation of central neuronal activities and peripheral energy balance. Here, we demonstrated the interaction between MRAP2 and MCHR1 by immunoprecipitation and bimolecular fluorescent assay and found that MRAP2 could inhibit MCHR1 signaling in vitro. A series of functional truncations of different regions further identified that the C-terminal domains of MRAP2 protein were required for the pharmacological modulation of intracellular Ca2+ coupled cascades and membrane transport. These findings elucidated the broad regulatory profile of MRAP2 protein in the central nervous system and may provide implications for the modulation of central MCHR1 function in vivo.

Aged Brains Express Less Melanocortin Receptors, Which Correlates with Age-Related Decline of Cognitive Functions.

Brain G-protein coupled receptors have been hypothesized to be potential targets for maintaining or restoring cognitive function in normal aged individuals or in patients with neurodegenerative disease. A number of recent reports suggest that activation of melanocortin receptors (MCRs) in the brain can significantly improve cognitive functions of normal rodents and of different rodent models of the Alzheimer's disease. However, the potential impact of normative aging on the expression of MCRs and their potential roles for modulating cognitive function remains to be elucidated. In the present study, we first investigated the expression of these receptors in six different brain regions of young (6 months) and aged (23 months) rats following assessment of their cognitive status. Correlation analysis was further performed to reveal potential contributions of MCR subtypes to spatial learning and memory. Our results revealed statistically significant correlations between the expression of several MCR subtypes in the frontal cortex/hypothalamus and the hippocampus regions and the rats' performance in spatial learning and memory only in the aged rats. These findings support the hypothesis that aging has a direct impact on the expression and function of MCRs, establishing MCRs as potential drug targets to alleviate aging-induced decline of cognitive function.

Transcriptomic analysis links diverse hypothalamic cell types to fibroblast growth factor 1-induced sustained diabetes remission.

In rodent models of type 2 diabetes (T2D), sustained remission of hyperglycemia can be induced by a single intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1), and the mediobasal hypothalamus (MBH) was recently implicated as the brain area responsible for this effect. To better understand the cellular response to FGF1 in the MBH, we sequenced >79,000 single-cell transcriptomes from the hypothalamus of diabetic Lepob/ob mice obtained on Days 1 and 5 after icv injection of either FGF1 or vehicle. A wide range of transcriptional responses to FGF1 was observed across diverse hypothalamic cell types, with glial cell types responding much more robustly than neurons at both time points. Tanycytes and ependymal cells were the most FGF1-responsive cell type at Day 1, but astrocytes and oligodendrocyte lineage cells subsequently became more responsive. Based on histochemical and ultrastructural evidence of enhanced cell-cell interactions between astrocytes and Agrp neurons (key components of the melanocortin system), we performed a series of studies showing that intact melanocortin signaling is required for the sustained antidiabetic action of FGF1. These data collectively suggest that hypothalamic glial cells are leading targets for the effects of FGF1 and that sustained diabetes remission is dependent on intact melanocortin signaling.

Hypothalamic mechanisms associated with neuropeptide K-induced anorexia in Japanese quail (Coturnix japonica).

Central administration of neuropeptide K (NPK), a 36-amino acid peptide, is associated with anorexigenic effects in rodents and chickens. The mechanisms underlying the potent anorexigenic effects of NPK are still poorly understood. Thus, the aim of the present study was to identify the hypothalamic nuclei and neuropeptides that mediate anorexic effects of NPK in 7 day-old Japanese quail (Coturnix japonica). After a 6 h fast, intracerebroventricular (ICV) injection of NPK decreased food and water intake for 180 min post-injection. Quail injected with NPK had more c-Fos immunoreactive cells in the arcuate nucleus (ARC), lateral hypothalamus, and paraventricular nucleus (PVN) compared to the birds that were injected with the vehicle. In the ARC of NPK-injected quail, there was decreased neuropeptide Y (NPY), NPY receptor sub-type 1, and agouti-related peptide mRNA, and increased CART, POMC, and neurokinin receptor 1 mRNA. NPK-injected quail expressed greater amounts of corticotropin-releasing factor (CRF), CRF receptor sub-type 2, melanocortin receptors 3 and 4, and urocortin 3 mRNA in the PVN. In conclusion, results provide insights into understanding NPK-induced changes in hypothalamic physiology and feeding behavior, and suggest that the anorexigenic effects of NPK involve the ARC and PVN, with increased CRF and melanocortin and reduced NPY signaling.

Expression and function of nesfatin-1 are altered by stage of the estrous cycle.

Nesfatin-1 is a peptide derived from the nucleobindin 2 (Nucb2) precursor protein that has been shown to exert potent effects on appetite and cardiovascular function in male animals. Sex hormones modulate the expression of Nucb2 in several species, including goldfish, mouse, and rat, and human studies have revealed differential expression based on male or female sex. We therefore hypothesized that the ability of nesfatin-1 to increase mean arterial pressure (MAP) would be influenced by stage of the estrous cycle. Indeed, we found that in cycling female Sprague-Dawley rats, nesfatin-1 induced an increase in MAP on diestrus, when both estrogen and progesterone levels are low but not on proestrus or estrus. The effect of nesfatin-1 on MAP was dependent on functional central melanocortin receptors, because the nesfatin-1-induced increase in MAP was abolished by pretreatment with the melanocortin 3/4 receptor antagonist, SHU9119. We previously reported that nesfatin-1 inhibited angiotensin II-induced water drinking in male rats but found no effect of nesfatin-1 in females in diestrus. However, nesfatin-1 enhanced angiotensin II-induced elevations in MAP in females in diestrus but had no effect on males. Finally, in agreement with previous reports, the expression of Nucb2 mRNA in hypothalamus was significantly reduced in female rats in proestrus compared with rats in diestrus. From these data we conclude that the function and expression of nesfatin-1 are modulated by sex hormone status. Further studies are required to determine the contributions of chromosomal sex and individual sex hormones to the cardiovascular effects of nesfatin-1.

Quantitative mass spectrometry for human melanocortin peptides in vitro and in vivo suggests prominent roles for ß-MSH and desacetyl α-MSH in energy homeostasis.

OBJECTIVE: The lack of pro-opiomelanocortin (POMC)-derived melanocortin peptides results in hypoadrenalism and severe obesity in both humans and rodents that is treatable with synthetic melanocortins. However, there are significant differences in POMC processing between humans and rodents, and little is known about the relative physiological importance of POMC products in the human brain. The aim of this study was to determine which POMC-derived peptides are present in the human brain, to establish their relative concentrations, and to test if their production is dynamically regulated. METHODS: We analysed both fresh post-mortem human hypothalamic tissue and hypothalamic neurons derived from human pluripotent stem cells (hPSCs) using liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine the sequence and quantify the production of hypothalamic neuropeptides, including those derived from POMC. RESULTS: In both in vitro and in vivo hypothalamic cells, LC-MS/MS revealed the sequence of hundreds of neuropeptides as a resource for the field. Although the existence of ß-melanocyte stimulating hormone (MSH) is controversial, we found that both this peptide and desacetyl α-MSH (d-α-MSH) were produced in considerable excess of acetylated α-MSH. In hPSC-derived hypothalamic neurons, these POMC derivatives were appropriately trafficked, secreted, and their production was significantly (P < 0.0001) increased in response to the hormone leptin. CONCLUSIONS: Our findings challenge the assumed pre-eminence of α-MSH and suggest that in humans, d-α-MSH and ß-MSH are likely to be the predominant physiological products acting on melanocortin receptors.

Pharmacological effect of human melanocortin-2 receptor accessory protein 2 variants on hypothalamic melanocortin receptors.

PURPOSE: Melanocortin-3 receptor (MC3R), melanocortin-4 receptor (MC4R), and a recently identified melanocortin-2 receptor accessory protein 2 (MRAP2), are highly expressed in hypothalamus and coordinately regulate energy homeostasis, but the single cellular transcriptome of melanocortin system remains unknown. Several infrequent MRAP2 variants are reported from severe obese human patients but the mechanisms on how they affect melanocortin signaling are unclear. METHODS: First, we performed in silico analysis of mouse hypothalamus RNA sequencing datasets at single-cell resolution from two independent studies. Next, we inspected the three-dimensional conformational alteration of three mutations on MRAP2 protein. Finally, the influence of MRAP2 variants on MC3R and MC4R signaling was analyzed in vitro. RESULTS: (1) We confirmed the actual co-expression of Mrap2 with Mc3r and Mc4r, and demonstrated more broad distribution of Mrap2-positive neuronal populations than Mc3r or Mc4r in mouse hypothalamus. (2) Compared with wild-type MRAP2, MRAP2N88Y, and MRAP2R125C showed impaired α-MSH-induced MC4R or MC3R stimulation. (3) MRAP2N88Yexhibited enhanced interaction with MC4R protein and its own. CONCLUSIONS: This is the first dedicated description of single-cell transcriptome signature of Mrap2, Mc3r, and Mc4r in the central nerve system and the first evidence describing the unique dimer formation, conformational change, and pharmacological effect of MRAP2 mutations on MC3R signaling.

Analyzing the effects of co-expression of chick (Gallus gallus) melanocortin receptors with either chick MRAP1 or MRAP2 in CHO cells on sensitivity to ACTH(1-24) or ACTH(1-13)NH2: Implications for the avian HPA axis and avian melanocortin circuits in the hypothalamus.

In order to better understand the roles that melanocortin receptors (cMCRs) and melanocortin-2 receptor accessory proteins (cMRAP1 and cMRAP2) play in the HPA axis and hypothalamus, adrenal gland and hypothalamus mRNA from 1day-old white leghorn chicks (Gallus gallus), were analyzed by real-time PCR. mRNA was also made for kidney, ovary, and liver. Mrap1 mRNA could be detected in adrenal tissue, but not in any of the other tissues, and mrap2 mRNA was also detected in the adrenal gland. Finally, all five melanocortin receptors mRNAs could be detected in the adrenal gland; mc2r and mc5r mRNAs were the most abundant. To evaluate any potential interactions between MRAP1 and the MCRs that may occur in adrenal cells, individual chick mcr cDNA constructs were transiently expressed in CHO cells either in the presence or absence of a chick mrap1 cDNA, and the transfected cells were stimulated with hACTH(1-24) at concentrations ranging from 10-13M to 10-6M. As expected, MC2R required co-expression with MRAP1 for functional expression; whereas, co-expression of cMC3R with cMRAP1 had no statistically significant effect on sensitivity to hACTH(1-24). However, co-expression of MC4R and MC5R with MRAP1, increased sensitivity for ACTH(1-24) by approximately 35 fold and 365 fold, respectively. However, co-expressing of cMRAP2 with these melanocortin receptors had no effect on sensitivity to hACTH(1-24). Since the real-time PCR analysis detected mrap2 mRNA and mc4r mRNA in the hypothalamus, the interaction between cMC4R and cMRAP2 with respect to sensitivity to ACTH(1-13)NH2 stimulation was also evaluated. However, no effect, either positive or negative, was observed. Finally, the highest levels of mc5r mRNA were detected in liver cells. This observation raises the possibility that in one-day old chicks, activation of the HPA axis may also involve a physiological response from liver cells.

The thermogenic effect of nesfatin-1 requires recruitment of the melanocortin system.

Nesfatin-1 is a bioactive polypeptide expressed both in the brain and peripheral tissues and involved in the control of energy balance by reducing food intake. Central administration of nesfatin-1 significantly increases energy expenditure, as demonstrated by a higher dry heat loss; yet, the mechanisms underlying the thermogenic effect of central nesfatin-1 remain unknown. Therefore, in this study, we sought to investigate whether the increase in energy expenditure induced by nesfatin-1 is mediated by the central melanocortin pathway, which was previously reported to mediate central nesfatin-1´s effects on feeding and numerous other physiological functions. With the application of direct calorimetry, we found that intracerebroventricular nesfatin-1 (25 pmol) treatment increased dry heat loss and that this effect was fully blocked by simultaneous administration of an equimolar dose of the melanocortin 3/4 receptor antagonist, SHU9119. Interestingly, the nesfatin-1-induced increase in dry heat loss was positively correlated with body weight loss. In addition, as assessed with thermal imaging, intracerebroventricular nesfatin-1 (100 pmol) increased interscapular brown adipose tissue (iBAT) as well as tail temperature, suggesting increased heat production in the iBAT and heat dissipation over the tail surface. Finally, nesfatin-1 upregulated pro-opiomelanocortin and melanocortin 3 receptor mRNA expression in the hypothalamus, accompanied by a significant increase in iodothyronine deiodinase 2 and by a nonsignificant increase in uncoupling protein 1 and peroxisome proliferator-activated receptor gamma coactivator-1 alpha mRNA in the iBAT. Overall, we clearly demonstrate that nesfatin-1 requires the activation of the central melanocortin system to increase iBAT thermogenesis and, in turn, overall energy expenditure.

Chronic intrahypothalamic rather than subcutaneous liraglutide treatment reduces body weight gain and stimulates the melanocortin receptor system.

BACKGROUND: The GLP-1 receptor agonist liraglutide is marketed for obesity treatment where it induces body weight reduction possibly via the hypothalamus, which regulates energy homeostasis. In animal studies, acute liraglutide treatment triggers satiety, weight loss and activates thermogenesis in adipose tissue. However, the precise mechanisms how liraglutide affects in particular chronic weight loss are still under investigation. OBJECTIVES: We aimed to evaluate whether chronic hypothalamic or chronic subcutaneous administration of liraglutide induces sustained weight loss through altered adipose tissue function and to what extent hypothalamic neuronal appetite regulators are involved in the liraglutide-induced weight loss in healthy lean rats on a normal diet. MATERIALS/METHODS: We continuously administered liraglutide either intrahypothalamically (10 µg per day) or subcutaneously (200 µg kg-1 per day) for 28 days to lean Sprague Dawley rats (n=8 each). We assessed changes in body weight, adipose tissue mass, adipocyte size and adipose tissue volume in the abdominal region by using micro-CT. We analyzed genetic expression patterns of browning, thermogenic and adipocyte differentiation regulators in adipose tissues as well as particular neuronal appetite regulators in the hypothalamus. RESULTS: Intrahypothalamic liraglutide administration induced an 8% body weight reduction at day 9 compared with the control group (P<0.01) and a 7% body weight loss at day 9 compared with subcutaneous liraglutide treatment (P<0.01), supported by a significant reduction in adipose tissue mass and volume with intrahypothalamic liraglutide administration (P<0.05). Our data show that chronic intrahypothalamic liraglutide treatment triggered an 18-fold induction of the hypothalamic mc4r gene (P<0.01) accompanied by a significant increase in circulating thyroxine (T4) levels (P<0.05). CONCLUSIONS: Chronic intrahypothalamic liraglutide administration resulted in a profound reduction in body weight and fat mass loss most likely mediated by the hypothalamic melanocortin system rather than by adipose tissue browning or improved thermogenesis.

Gene expression of pro-opiomelanocortin and melanocortin receptors is regulated in the hypothalamus and mesocorticolimbic system following nicotine administration.

Pro-opiomelanocortin (POMC)-derived peptides and their receptors have been shown to play important roles in natural and drug-induced reward and reinforcement. Reward process may involve the regulation of POMC gene expression and the gene expression of POMC-derived peptide receptors. The present study investigated the alterations observed in the transcript levels of POMC, melanocortin 3 (MC3R), melanocortin 4 (MC4R) and mu-opioid receptors (MOR) in the hypothalamus and mesocorticolimbic system during nicotine exposure. Rats were injected subcutaneously for 5days with one of the three doses (0.2, 0.4 or 0.6mg/kg/day, free base) of nicotine and were decapitated one hour after a challenge dose on the sixth day. mRNA levels of POMC in the hypothalamus, MC3R in the ventral tegmental area (VTA), MC4R and MOR in the medial prefrontal cortex (mPFC), nucleus accumbens, dorsal striatum, amygdala, lateral hypothalamic area and VTA were measured by quantitative real-time PCR. Our results showed that treatment with 0.6mg/kg/day nicotine upregulated POMC mRNA in the hypothalamus and MC4R mRNA in the mPFC. Additionally, all three nicotine doses increased MC3R mRNA expression in the VTA. On the other hand, none of the nicotine doses altered MOR mRNA levels in the mesocorticolimbic system and associated limbic structures. These results suggest that nicotine may enhance melanocortin signaling in the mesocorticolimbic system and this alteration may be an important mechanism mediating nicotine reward.

Ventromedial hypothalamic melanocortin receptor activation: regulation of activity energy expenditure and skeletal muscle thermogenesis.

KEY POINTS: The ventromedial hypothalamus (VMH) and the central melanocortin system both play vital roles in regulating energy balance by modulating energy intake and utilization. Recent evidence suggests that activation of the VMH alters skeletal muscle metabolism. We show that intra-VMH melanocortin receptor activation increases energy expenditure and physical activity, switches fuel utilization to fats, and lowers work efficiency such that excess calories are dissipated by skeletal muscle as heat. We also show that intra-VMH melanocortin receptor activation increases sympathetic nervous system outflow to skeletal muscle. Intra-VMH melanocortin receptor activation also induced significant changes in the expression of mediators of energy expenditure in muscle. These results support the role of melanocortin receptors in the VMH in the modulation of skeletal muscle metabolism. ABSTRACT: The ventromedial hypothalamus (VMH) and the brain melanocortin system both play vital roles in increasing energy expenditure (EE) and physical activity, decreasing appetite and modulating sympathetic nervous system (SNS) outflow. Because of recent evidence showing that VMH activation modulates skeletal muscle metabolism, we propose the existence of an axis between the VMH and skeletal muscle, modulated by brain melanocortins, modelled on the brain control of brown adipose tissue. Activation of melanocortin receptors in the VMH of rats using a non-specific agonist melanotan II (MTII), compared to vehicle, increased oxygen consumption and EE and decreased the respiratory exchange ratio. Intra-VMH MTII enhanced activity-related EE even when activity levels were held constant. MTII treatment increased gastrocnemius muscle heat dissipation during controlled activity, as well as in the home cage. Compared to vehicle-treated rats, rats with intra-VMH melanocortin receptor activation had higher skeletal muscle norepinephrine turnover, indicating an increased SNS drive to muscle. Lastly, intra-VMH MTII induced mRNA expression of muscle energetic mediators, whereas short-term changes at the protein level were primarily limited to phosphorylation events. These results support the hypothesis that melanocortin peptides act in the VMH to increase EE by lowering the economy of activity via the enhanced expression of mediators of EE in the periphery including skeletal muscle. The data are consistent with the role of melanocortins in the VMH in the modulation of skeletal muscle metabolism.

An in Vitro and in Vivo Investigation of Bivalent Ligands That Display Preferential Binding and Functional Activity for Different Melanocortin Receptor Homodimers.

Pharmacological probes for the melanocortin receptors have been utilized for studying various disease states including cancer, sexual function disorders, Alzheimer's disease, social disorders, cachexia, and obesity. This study focused on the design and synthesis of bivalent ligands to target melanocortin receptor homodimers. Lead ligands increased binding affinity by 14- to 25-fold and increased cAMP signaling potency by 3- to 5-fold compared to their monovalent counterparts. Unexpectedly, different bivalent ligands showed preferences for particular melanocortin receptor subtypes depending on the linker that connected the binding scaffolds, suggesting structural differences between the various dimer subtypes. Homobivalent compound 12 possessed a functional profile that was unique from its monovalent counterpart providing evidence of the discrete effects of bivalent ligands. Lead compound 7 significantly decreased feeding in mice after intracerebroventricular administration. To the best of our knowledge, this is the first report of a melanocortin bivalent ligand's in vivo physiological effects.

Role of Hypothalamic VGF in Energy Balance and Metabolic Adaption to Environmental Enrichment in Mice.

Environmental enrichment (EE), a housing condition providing complex physical, social, and cognitive stimulation, leads to improved metabolic health and resistance to diet-induced obesity and cancer. One underlying mechanism is the activation of the hypothalamic-sympathoneural-adipocyte axis with hypothalamic brain-derived neurotrophic factor (BDNF) as the key mediator. VGF, a peptide precursor particularly abundant in the hypothalamus, was up-regulated by EE. Overexpressing BDNF or acute injection of BDNF protein to the hypothalamus up-regulated VGF, whereas suppressing BDNF signaling down-regulated VGF expression. Moreover, hypothalamic VGF expression was regulated by leptin, melanocortin receptor agonist, and food deprivation mostly paralleled to BDNF expression. Recombinant adeno-associated virus-mediated gene transfer of Cre recombinase to floxed VGF mice specifically decreased VGF expression in the hypothalamus. In contrast to the lean and hypermetabolic phenotype of homozygous germline VGF knockout mice, specific knockdown of hypothalamic VGF in male adult mice led to increased adiposity, decreased core body temperature, reduced energy expenditure, and impaired glucose tolerance, as well as disturbance of molecular features of brown and white adipose tissues without effects on food intake. However, VGF knockdown failed to block the EE-induced BDNF up-regulation or decrease of adiposity indicating a minor role of VGF in the hypothalamic-sympathoneural-adipocyte axis. Taken together, our results suggest hypothalamic VGF responds to environmental demands and plays an important role in energy balance and glycemic control likely acting in the melanocortin pathway downstream of BDNF.

Central proopiomelanocortin but not neuropeptide Y mediates sympathoexcitation and hypertension in fat fed conscious rabbits.

OBJECTIVE: High-fat diet (HFD)-induced hypertension in rabbits is neurogenic because of the central sympathoexcitatory actions of leptin. Hypothalamic melanocortin and neuropeptide Y (NPY) neurons are recognized as the major signalling pathways through which leptin exerts its central effects. In this study, we assessed the effects of specific antagonists and agonists to melanocortin and NPY receptors on HFD-induced sympathoexcitation and hypertension. METHODS: Rabbits were instrumented with intracerebroventricular cannula, renal sympathetic nerve activity (RSNA) electrode, and blood pressure telemetry transmitter. RESULTS: After 3 weeks HFD (13.5% fat, n = 12) conscious rabbits had higher RSNA (+3.8  nu, P = 0.02), blood pressure (+8.6  mmHg, P < 0.001) and heart rate (+15  b/min, P = 0.01), and brain-derived neurotrophic factor levels in the hypothalamus compared with rabbits fed a control diet (4.2% fat, n = 11). Intracerebroventricular administration of the melanocortin receptor antagonist SHU9119 reduced RSNA (-2.7  nu) and blood pressure (-8.5  mmHg) in HFD but not control rabbits, thus reversing 100% of the hypertension and 70% of the sympathoexcitation induced by a HFD. By contrast, blocking central NPY Y1 receptors with BVD10 increased RSNA only in HFD rabbits. Intracerebroventricular α-melanocortin stimulating hormone increased RSNA and heart rate (P < 0.001) in HFD rabbits but had no effect in control rabbits. CONCLUSION: These findings suggest that obesity-induced hypertension and increased RSNA are dependent on the balance between greater activation of melanocortin signalling through melanocortin receptors and lesser activation of NPY sympathoinhibitory signalling. The amplification of the sympathoexcitatory effects of α-melanocortin stimulating hormone also indicates that the underlying mechanism is related to facilitation of leptin-melanocortin signalling, possibly involving chronic activation of brain-derived neurotrophic factor.

Chronic gastric electrical stimulation leads to weight loss via modulating multiple tissue neuropeptide Y, orexin, α-melanocyte-stimulating hormone and oxytocin in obese rats.

OBJECTIVES: Gastric electrical stimulation (GES) has great potential for the treatment of obesity. We investigated the impact of chronic GES on the alteration of adipose tissue and the regulation of neuropeptide Y (NPY), orexin (OX), α-melanocyte-stimulating hormone (α-MSH) and oxytocin (OXT), and their receptors in several tissues. MATERIAL AND METHODS: Most of the experiments included three groups of diet-induced obesity rats: (1) sham-GES (SGES); (2) GL-6mA (GES with 6 mA, 4 ms, 40 Hz, 2 s on, 3 s off at lesser curvature); and (3) SGES-PF (SGES rats receiving pair feeding to match the consumption of GL-6mA rats). Chronic GES was applied for 2 h every day for 4 weeks. During treatment with GES, food intake and body weight were monitored weekly. The alteration of epididymal fat weight, gastric emptying, and expression of peptides and their receptors in several tissues were determined. RESULTS: GL-6mA was more potent than SGES-PF in decreasing body weight gain, epididymal fat tissue weight, adipocyte size and gastric emptying. Chronic GES significantly altered NPY, OX, α-MSH and OXT and their receptors in the hypothalamus, adipose tissue and stomach. CONCLUSIONS: Chronic GES effectively leads to weight loss by reducing food intake, fat tissue weight and gastric emptying. NPY, α-MSH, orexin and OXT, and their receptors in the hypothalamus, adipose tissue and stomach appear to be involved in the anti-obesity effects of chronic GES.

Hypothalamic POMC Deficiency Improves Glucose Tolerance Despite Insulin Resistance by Increasing Glycosuria.

Hypothalamic proopiomelanocortin (POMC) is essential for the physiological regulation of energy balance; however, its role in glucose homeostasis remains less clear. We show that hypothalamic arcuate nucleus (Arc)POMC-deficient mice, which develop severe obesity and insulin resistance, unexpectedly exhibit improved glucose tolerance and remain protected from hyperglycemia. To explain these paradoxical phenotypes, we hypothesized that an insulin-independent pathway is responsible for the enhanced glucose tolerance. Indeed, the mutant mice demonstrated increased glucose effectiveness and exaggerated glycosuria relative to wild-type littermate controls at comparable blood glucose concentrations. Central administration of the melanocortin receptor agonist melanotan II in mutant mice reversed alterations in glucose tolerance and glycosuria, whereas, conversely, administration of the antagonist Agouti-related peptide (Agrp) to wild-type mice enhanced glucose tolerance. The glycosuria of ArcPOMC-deficient mice was due to decreased levels of renal GLUT 2 (rGLUT2) but not sodium-glucose cotransporter 2 and was associated with reduced renal catecholamine content. Epinephrine treatment abolished the genotype differences in glucose tolerance and rGLUT2 levels, suggesting that reduced renal sympathetic nervous system (SNS) activity is the underlying mechanism for the observed glycosuria and improved glucose tolerance in ArcPOMC-deficient mice. Therefore, the ArcPOMC-SNS-rGLUT2 axis is potentially an insulin-independent therapeutic target to control diabetes.