Unabated anorexic and enhanced thermogenic responses to Melanotan 2 in diet-induced obese rats despite reduced melanocortin 3 and 4 receptor expression
G Li, Y Zhang

Abstract
The effects of the chronic activation of the central melanocortin (MC) system by melanotan II (MTII) were assessed in chow-fed (CH) and high-fat (HF) diet-induced obese (DIO) Sprague–Dawley rats. Six-day central infusion of MTII (1 nmol/day) reduced body weight and visceral adiposity compared with ad libitum-fed control and pairfed groups and markedly suppressed caloric intake in both CH and DIO rats. The anorexic response to MTII was similar in DIO relative to CH rats. MTII induced a sustained increase in oxygen consumption in DIO but a delayed response in CH rats. In both diet groups, MTII reduced serum insulin and cholesterol levels compared with controls. HF feeding increased brown adipose tissue (BAT) uncoupling protein 1 (UCP1) by over twofold, and UCP1 levels were further elevated in MTII-treated CH and DIO rats. MTII lowered acetyl-CoA carboxylase expression and prevented the reduction in muscle-type carnitine palmitoyltransferase I mRNA by pair-feeding in the muscle of DIO rats. Compared with CH controls, hypothalamic MC3 and MC4 receptor expression levels were reduced in DIO controls. This study has demonstrated that, despite reduced hypothalamic MC3/MC4 receptor expression, anorexic and thermogenic responses to MTII are unabated with an initial augmentation of energy expenditure in DIO versus CH rats. The HF induced up-regulation of UCP1 in BAT may contribute to the immediate increase in MTII-stimulated thermogenesis in DIO rats. MTII also increased fat catabolism in the muscle of DIO rats and improved glucose and cholesterol metabolism in both groups.

Introduction
Melanocortins (MCs) are bioactive peptides derived from pro-opiomelanocortin (POMC).* Among them, alpha-melanocyte stimulating hormone (a-MSH) is a major regulator of feeding and body weight via hypothalamic MC3 and 4 receptors (MC3R and MC4R). Central infusion of a-MSH or its synthetic agonists causes anorexia and weight loss, whereas infusion of MCR blockers or over-production of the endogenous MCR antagonist, agouti-related protein (AgRP), produces hyperphagia and obesity.* Knockout studies of the central MC3R and MC4R have identified these receptors as important players in energy homeostasis.* Targeted disruption of the MC4R gene leads to overfeeding and obesity, whereas MC3R knockouts over-accumulate fat with minimal changes in caloric intake. Deficiency in POMC also results in increased food intake and morbid obesity in both rodents and humans. Apparently, the central MC system has critical functions in the homeostatic regulation of body weight.

One of the hallmarks of obesity, whether it is genetic, diet induced or age related, is leptin resistance. Human obesity as well as many rodent models of obesity is accompanied by elevated serum leptin and leptin resistance, which becomes more pronounced with progressive degrees of obesity. Diet-induced obese (DIO) rodent models, characterized by hyperleptinemia and hyperinsulinemia, somewhat resemble the onset of human obesityand hence provide a valuable tool for investigating leptin resistance in humans. The nature of leptin resistance associated with DIO animals is not well understood. The blunted responsiveness to both endogenous and exogenous leptin has been, in part, attributed to defects in the blood–brain barrier transport system (peripheral resistance) and in leptin signal transduction in the hypothalamic leptin-responsive neurons (central resistance). A growing body of evidence suggests that the MC system is located downstream of the hypothalamic leptin-signaling pathway. Leptin activates POMC- and suppresses AgRP containing neurons of the ventrolateral and ventromedial arcuate nucleus respectively, resulting in an increase in the expression of POMC and a reduction in AgRP. It is possible that the central resistance is partially due to a failure of the leptin signal to activate POMC or suppress AgRP neurons so that the proper regulation of POMC and AgRP expression by leptin is lost. As a result, leptin-initiated MC activation is impaired. In support of this notion, a-MSH and a potent MC agonist, Melanotan II (MTII), as well as central POMC gene therapy were effective in obese Zucker rats with defective leptin receptor signaling and in DIO mice with leptin resistance. Chronic impairment in MC activation may generate hypersensitivity (or hyper-responsiveness) of the MC pathway to pharmacological MC stimulation, potentially through homeostatic up-regulation of MC3R and MC4Rs. This postulated hypersensitivity has been indicated in two studies, one demonstrating enhanced responses to MTII in obese Zucker rats and another reporting an acute enhanced anorexic response to a-MSH in leptin-resistant DIO rats. However, a recent study indicated that high-fat (HF) feeding actually decreased the anorexic effects of MTII, which could contribute to diet-induced obesity. Since most of these previous reports assessed the acute responses to MCs, one of our aims was to determine the chronic effects of central infusion of MTII in a DIO rat model with a normal genetic background. We also examined whether diet induced obesity alters the expression levels of hypothalamic MC3R and MC4R that may potentially mediate the differential response to MTII in DIO compared with lean animals. Furthermore, because of the scarcity of informationon how a-MSH influences energy expenditure and body metabolism (in contrast to plentiful information about the effect of a-MSH and its analogs on food intake), we assessed the thermogenic response as well as fat metabolism in brown adipose tissue (BAT) and skeletal muscle following central MTII administration in both lean and DIO rats.

To this end, we examined the effects of a 6-day central administration of leptin or MTII on energy balance, BAT thermogenesis, and indicators of skeletal muscle fat metabolism in chow-fed (CH) lean and DIO Sprague–Dawley rats. Food intake, body weight, adiposity, serum hormone and metabolite levels, oxygen consumption, BAT uncoupling protein 1 (UCP1) protein, expression of acetyl-CoA carboxylase (ACC) and muscle-type carnitine palmitoyltransferase palmitoyltransferase I (M-CPT I) in soleus muscle and MC3R and MC4R in the hypothalamus were measured.

Discussion
The present study assessed the effects of MTII, a potent MC3R/MC4R agonist, on several aspects of energy regulation in a DIO rodent model. After 10 weeks of HF feeding, 40% of the female Sprague–Dawley rats became obese and displayed leptin resistance to centrally infused leptin. However, subsequent central MTII infusion circumvented leptin resistance in these DIO rats, leading to suppressed food intake, reduced body weight, and visceral adiposity. Although in agreement with most of the earlier reports in that genetically obese and DIO animals respond robustly to a-MSH or MTII treatment, our study has provided an extension of the previous knowledge. First, despite a reduction in hypothalamic MC3R and MC4R expression levels in DIO rats, the animals responded to MTII administration with similar efficacy to that of CH rats. Moreover, an increase in the initial energy expenditure was evident as early as day 2 in DIO, but not apparent in CH rats until day 6. There is speculation that central MC receptor up-regulation might contribute to an enhanced anorexic response in DIO animals. For instance, enhanced responses to MTII in obese Zucker rats are linked to increased hypothalamic MC4R densities. Yet the same study also noted that MC4R densities in specific hypothalamic regions involved in energy regulation were actually diminished in DIO rats with a normal genetic background. Our data together with this report seem to argue against the homeostatic MC3R/MC4R up-regulation theory.

The present report also suggested that an increase in energy expenditure contributes to the loss of body weight and visceral adiposity following MTII treatment in both CH and DIO rats. Central MTII infusion markedly reduced body weight and visceral adiposity in obese DIO rats compared with their respective ad libitum-fed or pair-fed animals. Because pair-feeding accounts for changes due to reduced food intake, our observation is suggestive of a food intake-independent component in the fat-trimming effect of MTII. The elevation of oxygen consumption at day 2 in DIO and day 6 in CH and DIO rats during central MTII infusion further argues that an increase in energy expenditure is involved. Non-shivering thermogenesis in BAT represents an essential element in adaptive energy expenditure in rodents, and the UCP1 protein level is one indicator of the thermogenic status of BAT. A previous report indicated that animals treated with MTII had elevated levels of BAT UCP1 expression. Similarly, in the present study, MTII greatly enhanced UCP1 protein levels in BAT. This substantial increase in UCP1 may well be the mediator for the elevated thermogenesis following MTII treatment. The long-term HF feeding (10 weeks) also increased basal BAT UCP1 protein levels by more than twofold. This up-regulation of basal UCP1 in BAT may serve as one explanation for the immediate increase in the MTII induced energy expenditure (at day 2) in DIO rats.

Humans with excessive fat deposition in the body have a high risk of various obesity-related disorders such as type 2 diabetes, heart diseases, and stroke. MTII produced an impressive reduction in visceral adiposity in the present study, which was not matched by pair-feeding. Even though chronic caloric restriction has been shown to decrease visceral adiposity in rodents and humans, we did not observe a significant decrease in visceral fat mass in either CH or DIO animals pair-fed to MTII treatment. Such a discrepancy could result from the transient anorexic response to MTII. Unlike constant food restriction, pair-fed rats in our experiment were restricted to a small amount of food during the initial days of the experiment but then provided with much more food towards the later days of the treatment. This pair-feeding pattern resembles caloric restriction followed by partial re-feeding. Humans and animals under this kind of feeding pattern often undergo a greater weight gain and fat repletion. In our case, a significant loss in visceral adiposity was likely prevented by this variable pair-feeding. In contrast to pair-feeding, MTII treatment clearly reduced visceral adiposity in both CH and DIO groups in spite of the temporal change in food intake. Therefore, the food-independent effect of MTII, presumably the increased energy expenditure as reflected by both elevated oxygen consumption and BAT UCP1, plays a crucial part in fat catabolism.

MTII has been shown to increase the expression of liver CPT I in lean and obese Zucker rats as compared with their respective pair-fed controls. CPT I is a key enzyme in fat catabolism that controls the transfer of long-chain fatty acyl-CoA molecules into mitochondria where they are oxidized. Another important enzyme is ACC, the rate limiting enzyme for malonyl-CoA formation. Malonyl-CoA is an allosteric inhibitor of CPT I and, thus, a reduction in ACC is consistent with promotion of fat catabolism. In our study, MTII not only prevented the decrease in muscle CPT I expression associated with pair-feeding, but also reduced ACC mRNA in skeletal muscle in DIO rats. The simultaneous changes in the expression of ACC and M-CPT I indicate an overall increase in fatty acid oxidation in skeletal muscles, implying that the increased fat catabolism in muscle could be an additional factor in mediating the fat-reducing action of MTII. Considering the relatively small amount of BAT versus the large volume of skeletal muscles in humans, the MTII-evoked muscle fat metabolism seems to offer a much more promising target for any potential clinical application.

Finally, MTII also appeared to improve glucose and cholesterol metabolism and insulin sensitivity. Central MCR activation can reduce insulin release from the pancreas and enhance glucose metabolism. However, the results in obese animal models are controversial]
In summary, the present study has demonstrated that central MC activation by the MC3R/MC4R agonist MTII circumvents leptin resistance associated with DIO, resulting in a reduction in body mass and visceral adiposity. Despite reduced hypothalamic MC3R/MC4R expression, the anorexic and thermogenic responses to MTII are unabated with an even more rapid onset for the increase in energy expenditure in DIO versus CH rats. The HF-induced up-regulation of BAT UCP1 in DIO rats may account for this immediate increase in energy expenditure following central MTII infusion. Furthermore, MTII appears to increase fat catabolism in skeletal muscle, and improve glucose and cholesterol metabolism and insulin sensitivity in both CH and DIO rats. The hypophagia and/or increased energy expenditure are the likely mechanisms underlying these improvements.