Differential control of metabolic and cardiovascular functions by melanocortin-4 receptors in proopiomelanocortin neurons.

We examined the role of melanocortin-4 receptors (MC4R) in proopiomelanocortin (Pomc) neurons in regulating metabolic and cardiovascular functions. Using Cre-loxP technology, we selectively rescued MC4R in Pomc neurons of mice with whole body MC4R deficiency (MC4R-Pomc-Cre mice). Body weight, food intake, and whole body oxygen consumption (Vo2) were determined daily, and blood pressure (BP), heart rate (HR), and body temperature were measured 24 h/day by telemetry. An intracerebroventricular cannula was placed in the right lateral ventricle for intracerebroventricular infusions. Littermate MC4R-deficient (LoxTB-MC4R) mice were used as controls. After control measurements, the MC4R antagonist (SHU-9119; 1 nmol/h) was infused intracerebroventricularly for 7 days. Compared with LoxTB-MC4R mice, MC4R-Pomc-Cre mice were less obese (47 ± 2 vs. 52 ± 2 g) and had increased energy expenditure (2,174 ± 98 vs. 1,990 ± 68 ml·kg⁻¹·min⁻¹), but food intake (4.4 ± 0.2 vs. 4.3 ± 0.3 g/day), BP (112 ± 1 vs. 109 ± 3 mmHg), and HR [557 ± 9 vs. 551 ± 14 beats per minute (bpm)] were similar between groups. Chronic SHU-9119 infusion increased food intake (4.2 ± 0.2 to 6.1 ± 0.5 g/day) and body weight (47 ± 2 to 52 ± 2 g) in MC4R-Pomc-Cre mice, while no changes were observed in LoxTB-MC4R mice. Chronic SHU-9119 infusion also increased BP and HR by 5 ± 1 mmHg and 60 ± 8 bpm in MC4R-Pomc-Cre mice without altering BP or HR in LoxTB-MC4R mice. These results indicate that MC4Rs in Pomc neurons are important for regulation of energy balance. In contrast, while activation of MC4R in Pomc neurons facilitates the BP response to acute stress, our data do not support a major role of MC4R in Pomc neurons in regulating baseline BP and HR.

Melanocortin 3/4 receptors in paraventricular nucleus modulate sympathetic outflow and blood pressure.

Central melanocortin 3/4 receptors (MC3/4Rs) are known to regulate energy balance. Activation of MC3/4Rs causes a greater increase in the firing activity of the PVN neurons in obese Zucker rats than in lean Zucker rats. The present study was undertaken to determine the roles of MC3/4Rs in the hypothalamic paraventricular nucleus (PVN) in modulating the sympathetic activity and blood pressure and its downstream pathway. Renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded in anaesthetized rats. Microinjection of the MC3/4R agonist melanotan II (MTII) into the PVN increased the RSNA and MAP. The MC3/4R antagonist agouti-related peptide (AgRP) or SHU9119 decreased the RSNA and MAP, but the MC4R antagonist HS024 had no significant effect on the RSNA and MAP. The effects of MTII were abolished by pretreatment of the PVN with AgRP, SHU9119, the adenylate cyclase inhibitor SQ22536 or the protein kinase A inhibitor Rp-cAMP, and substantially attenuated by HS024. Microinjection of SQ22536 alone into the PVN had no significant effect on the RSNA and MAP, but Rp-cAMP caused significant decreases in the RSNA and MAP. Furthermore, MTII increased the cAMP level in the PVN. These results indicate that activation of MC3/4Rs in the PVN increases the sympathetic outflow and blood pressure via the cAMP-protein kinase A pathway. Melanocortin 3 receptors in the PVN may exert a tonic excitatory effect on sympathetic activity.

Activation of the central melanocortin system contributes to the increased arterial pressure in obese Zucker rats.

We have previously demonstrated that leptin-mediated activation of the central nervous system (CNS) melanocortin system reduces appetite and increases sympathetic activity and blood pressure (BP). In the present study we examined whether endogenous melanocortin system activation, independent of leptin's actions, contributes to the regulation of BP and metabolic functions in obese Zucker rats, which have mutated leptin receptors. The long-term cardiovascular and metabolic effects of central melanocortin-3/4 receptor (MC3/4R) antagonism with SHU-9119 were assessed in lean (n = 6) and obese (n = 8) Zucker rats. BP and heart rate (HR) were measured 24-h/day by telemetry and an intracerebroventricular cannula was placed in the brain lateral ventricle. After stable control measurements, SHU-9119 was infused intracerebroventricularlly (1 nmol/h) for 10 days followed by a 10-day recovery period. Chronic CNS MC3/4R antagonism significantly increased food intake and body weight in lean (20 ± 1 to 45 ± 2 g and 373 ± 11 to 432 ± 14 g) and obese (25 ± 2 to 35 ± 2 g and 547 ± 10 to 604 ± 11 g) rats. No significant changes were observed in plasma glucose levels in lean or obese rats, whereas plasma leptin and insulin levels markedly increased in lean Zucker rats during CNS MC3/4R antagonism. Chronic SHU-9119 infusion in obese Zucker rats reduced mean arterial pressure (MAP) and HR by 6 ± 1 mmHg and 24 ± 5 beats/min, whereas in lean rats SHU-9119 infusion reduced HR by 31 ± 9 beats/min while causing only a transient decrease in MAP. These results suggest that in obese Zucker rats the CNS melanocortin system contributes to elevated BP independent of leptin receptor activation.

Agrp neurons mediate Sirt1's action on the melanocortin system and energy balance: roles for Sirt1 in neuronal firing and synaptic plasticity.

Sirt1 has been associated with various effects of calorie restriction, including an increase in lifespan. Here we show in mice that a central regulatory component in energy metabolism, the hypothalamic melanocortin system, is affected by Sirt1, which promotes the activity and connectivity of this system resulting in negative energy balance. In adult mice, the pharmacological inhibition of brain Sirt1 activity decreased Agrp neuronal activity and the inhibitory tone on the anorexigenic POMC neurons, as measured by the number of synaptic inputs to these neurons. When a Sirt1 inhibitor (EX-527) was injected either peripherally (i.p., 10 mg/kg) or directly into the brain (i.c.v., 1.5 nmol/mouse), it decreased both food intake during the dark cycle and ghrelin-induced food intake. This effect on feeding is mediated by upstream melanocortin receptors, because the MC4R antagonist, SHU9119, reversed Sirt1's effect on food intake. This action of Sirt1 required an appropriate shift in the mitochondrial redox state: in the absence of such an adaptation enabled by the mitochondrial protein, UCP2, Sirt1-induced cellular and behavioral responses were impaired. In accordance with the pharmacological results, the selective knock-out of Sirt1 in hypothalamic Agrp neurons through the use of Cre-Lox technology decreased electric responses of Agrp neurons to ghrelin and decreased food intake, leading to decreased lean mass, fat mass, and body weight. The present data indicate that Sirt1 has a central mode of action by acting on the NPY/Agrp neurons to affect body metabolism.

Drug-loaded nanoparticles targeted to the colon with polysaccharide hydrogel reduce colitis in a mouse model.

BACKGROUND & AIMS: One of the challenges to treating inflammatory bowel disease (IBD) is to target the site of inflammation. We engineered nanoparticles (NPs) to deliver an anti-inflammatory tripeptide Lys-Pro-Val (KPV) to the colon and assessed its therapeutic efficacy in a mouse model of colitis. METHODS: NPs were synthesized by double-emulsion/solvent evaporation. KPV was loaded into the NPs during the first emulsion of the synthesis process. To target KPV to the colon, loaded NPs (NP-KPV) were encapsulated into a polysaccharide gel containing 2 polymers: alginate and chitosan. The effect of KPV-loaded NPs on inflammatory parameters was determined in vitro as well as in the dextran sodium sulfate-induced colitis mouse model. RESULTS: NPs (400 nm) did not affect cell viability or barrier functions. A swelling degree study showed that alginate-chitosan hydrogel containing dextran-fluorescein isothiocyanate-labeled NPs collapsed in the colon. Once delivered, NPs quickly released KPV on or within the closed area of colonocytes. The inflammatory responses to lipopolysaccharide were reduced in Caco2-BBE (brush border enterocyte) cells exposed to NP-KPV compared with those exposed to NPs alone, in a dose-dependent fashion. Mice given dextran sodium sulfate (DSS) followed by NP-KPV were protected against inflammatory and histologic parameters, compared with mice given only DSS. CONCLUSIONS: Nanoparticles are a versatile drug delivery system that can overcome physiologic barriers and target anti-inflammatory agents such as the peptide KPV to inflamed areas. By using NPs, KPV can be delivered at a concentration that is 12,000-fold lower than that of KPV in free solution, but with similar therapeutic efficacy. Administration of encapsulated drug-loaded NPs is a novel therapeutic approach for IBD.

The central melanocortin system directly controls peripheral lipid metabolism.

Disruptions of the melanocortin signaling system have been linked to obesity. We investigated a possible role of the central nervous melanocortin system (CNS-Mcr) in the control of adiposity through effects on nutrient partitioning and cellular lipid metabolism independent of nutrient intake. We report that pharmacological inhibition of melanocortin receptors (Mcr) in rats and genetic disruption of Mc4r in mice directly and potently promoted lipid uptake, triglyceride synthesis, and fat accumulation in white adipose tissue (WAT), while increased CNS-Mcr signaling triggered lipid mobilization. These effects were independent of food intake and preceded changes in adiposity. In addition, decreased CNS-Mcr signaling promoted increased insulin sensitivity and glucose uptake in WAT while decreasing glucose utilization in muscle and brown adipose tissue. Such CNS control of peripheral nutrient partitioning depended on sympathetic nervous system function and was enhanced by synergistic effects on liver triglyceride synthesis. Our findings offer an explanation for enhanced adiposity resulting from decreased melanocortin signaling, even in the absence of hyperphagia, and are consistent with feeding-independent changes in substrate utilization as reflected by respiratory quotient, which is increased with chronic Mcr blockade in rodents and in humans with loss-of-function mutations in MC4R. We also reveal molecular underpinnings for direct control of the CNS-Mcr over lipid metabolism. These results suggest ways to design more efficient pharmacological methods for controlling adiposity.

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.

Ethanol preference in the rat as a function of photoperiod.

Ethanol-drinking was induced in laboratory rats that were maintained in total darkness. The established preference for ethanol was not reversed under conditions of constant illumination although a decrease in ethanol intake occurred. Administration of melatonin to rats maintained under "normal" photoperiods (9 hours of darkness during a 24-hour day) also induced ethanol-drinking.

Melatonin and abnormal movements induced by L-dopa in mice.

Melatonin has blocked adventitious movements induced by L-dopa in intact mice. It has reversed the adventitious turning to the right, and it has induced running to the left in mice receiving L-dopa after a lesion in the right caudate nucleus.

Opiate effects after adrenocorticotropin or beta-endorphin injection in the periaqueductal gray matter of rats.

Injections of adrenocorticotropic hormone (ACTH) into the periaqueductal gray matter of drug-naive rats resulted in a dose-dependent opiate abstinence syndrome characterized by fearful hyperreactivity and explosive motor behavior. Injecting shorter chains of ACTH caused attenuated forms of this behavior. Injections of beta-endorphin at this same site caused opposite behavior: sedative, analgestic, and catatonic. If the effects of morphine are mediated by two classes of receptor) and the other which is not stereospecific and naloxone-insensitive--the endogtor)--and the other which is not stereospecific and naloxone-insensitive the endogenous ligand of the second receptor may be ACTH. The neuropeptides ACTH and endorphin may be part of an integrated neuromodulatory system, and the opiate abstinence syndrome may be the result of an altered interaction between the two receptor systems.

Melanocortin peptides affect the motivation to feed in rainbow trout (Oncorhynchus mykiss).

In this study, we investigated the effects of one melanocortin receptor (MCR) agonist and two antagonists on food intake in juvenile rainbow trout. Baseline food intake was established prior to 1 microl intracerebroventricular injection (ICV) of the non-specific agonist MTII, the MC4R antagonist HS024 and the MC3/4R antagonist SHU9119 at concentrations of 0.3, 1 or 3 nM. Saline-injected fish and untreated fish served as controls. Changes in food intake were observed 1h after the ICV injections. Our results showed that treatment with MTII significantly decreased food intake at 3 nM compared to control, HS024 significantly increased food intake at 3 nM compared to control and saline-treated fish, and SHU9119 significantly increased food intake at 3 nM compared to saline-treated fish. In conclusion, our study provides further evidence, and hence strengthens the hypothesis, that MC4R participates in the control of energy balance in fish in the same manner as in mammals. Our findings that HS024 is more potent than SHU9119 in increasing food intake suggest that the effects of melanocortin on energy balance in rainbow trout are mainly regulated by activation of MC4R. Hence, HS024 seems an excellent tool as a MC4R antagonist in rainbow trout.

A derivative of the melanocortin receptor antagonist SHU9119 (PG932) increases food intake when administered peripherally.

Melanocortin receptors are considered promising candidates for the treatment of behavioral and metabolic disorders ranging from obesity to anorexia and cachexia. These experiments examined the response of mice to peripheral injections of two compounds. PG932 is a derivative of SHU9119 which is non-selective antagonist of melanocortin-3 and melanocortin-4 receptors (Mc3r and Mc4r). PG946 is a derivative of a hybrid of alpha- and beta-MSH, and is a moderately selective Mc3r antagonist. SHU9119 increases food intake when administered intracerebroventricularly but is without effect when injected into the periphery. In contrast, PG932 was found to be highly effective at stimulating food intake when administered peripherally by intraperitoneal injection. The orexigenic effect of PG932 required functional Mc4r, suggesting that inhibition of this receptor is involved in the stimulation of food intake. PG946 did not significantly affect on feeding behavior. PG932 is thus a useful new compound for studies examining the regulation of appetite and energy balance, and may also prove useful for the treatment of cachectic conditions.

Towards the therapeutic use of intranasal neuropeptide administration in metabolic and cognitive disorders.

The nose provides an effective way for delivering neuropeptides to the central nervous system, bypassing the blood-brain barrier and avoiding systemic side effects. Thereby intranasal neuropeptide administration enables the modulation of central nervous signaling pathways of body weight regulation and cognitive functions. Central nervous control of energy homeostasis is assumed to rely on hypothalamic neuropeptidergic pathways that are triggered by the peripheral adiposity signals insulin and leptin conveying the amount of body fat to the brain. Melanocortins, including alpha-melanocyte stimulating hormone (alpha-MSH), are essential for inducing anorexigenic/catabolic effects, i.e. for inhibiting caloric intake and increasing energy expenditure. Insulin, in addition to its function as an adiposity signal, also influences memory formation. Here we present a series of studies on the intranasal administration of MSH/ACTH4-10, a melanocortin receptor agonist, and of insulin. Prolonged administration of MSH/ACTH4-10 induced weight loss in normal-weight, but not in overweight humans. Intranasal insulin reduced body fat and improved memory functions in the absence of adverse peripheral side effects. Our results may contribute to the future development of therapeutic strategies in disorders like obesity and cognitive impairments that derive from dysfunctions of central nervous neuropeptidergic pathways.

Melanocortin-4 receptor activation stimulates hypothalamic brain-derived neurotrophic factor release to regulate food intake, body temperature and cardiovascular function.

In the present study, we aimed to investigate the neuromodulatory role played by hypothalamic brain-derived neurotrophic factor (BDNF) in the regulation of acute cardiovascular and feeding responses to melanocortin-4 receptor (MC4R) activation. In vitro, a selective MC4R agonist, MK1, stimulated BDNF release from isolated rat hypothalami and this effect was blocked by preincubation with the MC3/4R antagonist SHU-9119. In vivo, peripheral administration of MK1 decreased food intake in rats and this effect was blocked by pretreatment with an anti-BDNF antibody administered into the third ventricle. When anorexia was induced with the cannabinoid-1 receptor (CB1R) antagonist AM251, the anti-BDNF antibody did not prevent the reduction in food intake. Peripheral administration of MK1 also increased mean arterial pressure, heart rate and body temperature. These effects were prevented by pretreatment with the anti-BDNF antibody whereas the intracerebroventricular administration of BDNF caused changes similar to those of MK1. These findings demonstrate for the first time that activation of MC4R leads to an acute release of BDNF in the hypothalamus. This release is a prerequisite for MC4R-induced effects on appetite, body temperature and cardiovascular function. By contrast, CB1R antagonist-mediated anorexia is independent of the MC4R/BDNF pathway. Overall, these results show that BDNF is an important downstream mediator of the MC4R pathway.

Altered sucrose self-administration following injection of melanocortin receptor agonists and antagonists into the ventral tegmental area.

RATIONALE AND OBJECTIVES: Alpha-melanocyte stimulating hormone (αMSH) and agouti-related protein (AgRP) are antagonistic neuropeptides that play an important role in the control of feeding and body weight through their central actions on the melanocortin-3 and melanocortin-4 receptors. Increasing evidence indicates that αMSH and AgRP can interact with the mesolimbic dopamine system to regulate feeding as well as other behaviors. For example, we have shown previously that injection of melanocortin receptor agonists and antagonists into the ventral tegmental area (VTA) alters both normal home-cage feeding and the intake of sucrose solutions, but it remains unknown whether αMSH and AgRP can also act in the VTA to affect reward-related feeding. METHODS: We tested whether injection of the melanocortin receptor agonist, MTII, or the melanocortin receptor antagonist, SHU9119, directly into the VTA affected operant responding maintained by sucrose pellets in self-administration assays. RESULTS: Injection of MTII into the VTA decreased operant responding maintained by sucrose pellets on both fixed ratio and progressive ratio schedules of reinforcement, whereas SHU9119 increased operant responding under fixed ratio, but not progressive ratio schedules. MTII also increased and SHU9119 decreased 24-h home-cage food intake. CONCLUSIONS: This study demonstrates that αMSH and AgRP act in the VTA to affect sucrose self-administration. Thus, it adds critical information to the growing literature showing that in addition to their well-characterized role in controlling "need-based" feeding, αMSH and AgRP can also act on the mesolimbic dopamine system to control reward-related behavior.

Effect of Melanotan-II on Brain Fos Immunoreactivity and Oxytocin Neuronal Activity and Secretion in Rats.

Melanocortins stimulate the central oxytocin systems that are involved in regulating social behaviours. Alterations in central oxytocin have been linked to neurological disorders such as autism, and melanocortins have been proposed for therapeutic treatment. In the present study, we investigated how systemic administration of melanotan-II (MT-II), a melanocortin agonist, affects oxytocin neuronal activity and secretion in rats. The results obtained show that i.v., but not intranasal, administration of MT-II markedly induced Fos expression in magnocellular neurones of the supraoptic (SON) and paraventricular nuclei (PVN) of the hypothalamus, and this response was attenuated by prior i.c.v. administration of the melanocortin antagonist, SHU-9119. Electrophysiological recordings from identified magnocellular neurones of the SON showed that i.v. administration of MT-II increased the firing rate in oxytocin neurones but did not trigger somatodendritic oxytocin release within the SON as measured by microdialysis. Our data suggest that, after i.v., but not intranasal, administration of MT-II, the activity of magnocellular neurones of the SON is increased. Because previous studies showed that SON oxytocin neurones are inhibited in response to direct application of melanocortin agonists, the actions of i.v. MT-II are likely to be mediated at least partly indirectly, possibly by activation of inputs from the caudal brainstem, where MT-II also increased Fos expression.

Design of MC1R Selective γ-MSH Analogues with Canonical Amino Acids Leads to Potency and Pigmentation.

Melanoma is a lethal form of skin cancer. Skin pigmentation, which is regulated by the melanocortin 1 receptor (MC1R), is an effective protection against melanoma. However, the endogenous MC1R agonists lack selectivity for the MC1R and thus can have side effects. The use of noncanonical amino acids in previous MC1R ligand development raises safety concerns. Here we report the development of the first potent and selective hMC1R agonist with only canonical amino acids. Using γ-MSH as a template, we developed a peptide, [Leu3, Leu7, Phe8]-γ-MSH-NH2 (compound 5), which is 16-fold selective for the hMC1R (EC50 = 4.5 nM) versus other melanocortin receptors. Conformational studies revealed a constrained conformation for this linear peptide. Molecular docking demonstrated a hydrophobic binding pocket for the melanocortin 1 receptor. In vivo pigmentation study shows high potency and short duration. [Leu3, Leu7, Phe8]-γ-MSH-NH2 is ideal for inducing short-term skin pigmentation without sun for melanoma prevention.

Antagonism of the melanocortin system reduces cold and mechanical allodynia in mononeuropathic rats.

The presence of both pro-opiomelanocortin-derived peptides and melanocortin (MC) receptors in nociception-associated areas in the spinal cord suggests that, at the spinal level, the MC system might be involved in nociceptive transmission. In the present study, we demonstrate that a chronic constriction injury (CCI) to the rat sciatic nerve, a lesion that produces neuropathic pain, results in changes in the spinal cord MC system, as shown by an increased binding of (125)I-NDP-MSH to the dorsal horn. Furthermore, we investigated whether intrathecal administration (in the cisterna magna) of selective MC receptor ligands can affect the mechanical and cold allodynia associated with the CCI. Mechanical and cold allodynia were assessed by measuring withdrawal responses of the affected limb to von Frey filaments and withdrawal latencies upon immersion in a 4.5 degrees C water bath, respectively. We show that treatment with the MC receptor antagonist SHU9119 has a profound anti-allodynic effect, suggesting that the endogenous MC system has a tonic effect on nociception. In contrast, administration of the MC4 receptor agonists MTII and d-Tyr-MTII primarily increases the sensitivity to mechanical and cold stimulation. No antinociceptive action was observed after administration of the selective MC3 receptor agonist Nle-gamma-MSH. Together, our data suggest that the spinal cord MC system is involved in neuropathic pain and that the effects of MC receptor ligands on the responses to painful stimuli are exerted through the MC4 receptor. In conclusion, antagonism of the spinal melanocortin system might provide a new approach in the treatment of neuropathic pain.

Hypothalamic, metabolic, and behavioral responses to pharmacological inhibition of CNS melanocortin signaling in rats.

The CNS melanocortin (MC) system is implicated as a mediator of the central effects of leptin, and reduced activity of the CNS MC system promotes obesity in both rodents and humans. Because activation of CNS MC receptors has direct effects on autonomic outflow and metabolism, we hypothesized that food intake-independent mechanisms contribute to development of obesity induced by pharmacological blockade of MC receptors in the brain and that changes in hypothalamic neuropeptidergic systems known to regulate weight gain [i.e., corticotropin-releasing hormone (CRH), cocaine-amphetamine-related transcript (CART), proopiomelanocortin (POMC), and neuropeptide Y (NPY)] would trigger this effect. Relative to vehicle-treated controls, third intracerebroventricular (i3vt) administration of the MC receptor antagonist SHU9119 to rats for 11 d doubled food and water intake (toward the end of treatment) and increased body weight ( approximately 14%) and fat content ( approximately 90%), hepatic glycogen content ( approximately 40%), and plasma levels of cholesterol ( approximately 48%), insulin ( approximately 259%), glucagon ( approximately 80%), and leptin ( approximately 490%), whereas spontaneous locomotor activity and body temperature were reduced. Pair-feeding of i3vt SHU9119-treated animals to i3vt vehicle-treated controls normalized plasma levels of insulin, glucagon, and hepatic glycogen content, but only partially reversed the elevations of plasma cholesterol ( approximately 31%) and leptin ( approximately 104%) and body fat content ( approximately 27%). Reductions in body temperature and locomotor activity induced by i3vt SHU9119 were not reversed by pair feeding, but rather were more pronounced. None of the effects found can be explained by peripheral action of the compound. The obesity effects occurred despite a lack in neuropeptide expression responses in the neuroanatomical range selected across the arcuate (i.e., CART, POMC, and NPY) and paraventricular (i.e., CRH) hypothalamus. The results indicate that reduced activity of the CNS MC pathway promotes fat deposition via both food intake-dependent and -independent mechanisms.