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Clinuvel gets pre-approval win in Italy

Italy jumps the gun and 'approves' Clinuvel's photoprotective, afamelanotide, while it's still undergoing the regulatory process worldwide.


Italy's National Health System has bucked the European regulatory system and approved the use and reimbursement of Clinuvel's photoprotective drug, afamelanotide, prior to it gaining regulatory approval in Europe or the US.

This means patients who suffer from the rare disorder, erythropoietic protoporphyria (EPP) - for which there is no current treatment besides minimising exposure to sunlight - can be prescribed afamelanotide by Italian physicians, and the full cost will be reimbursed by Sistema Sanitario Nazionale, the Italian national health system.

Clinuvel has offered afamelanotide under 'compassionate use' in the past to patients following the end of clinical trials in Australia and the United Kingdom.

A similar arrangement was to be done in Italy, except Clinuvel arranged with the Italian regulators to reimburse the company for the compassionate use programme.

“Following the completion of the complex study of afamelanotide in Italian EPP patients, we continued to support their treatment through a compassionate use program," said Dr Philippe Wolgen, Clinuvel’s CEO.

"Unfortunately, the indefinite free supply of afamelanotide was not sustainable for Clinuvel as a small enterprise, and this approval by the Italian regulators not only makes the drug available to these trial patients, but also provides further incentive to the company to focus on orphan drug development.”

Italian law contains a provision to allow for the approval and reimbursement of drugs that treat conditions for which not alternative therapy exists, and when that drug is still in clinical development.

36 drugs have already been approved under this law since 1996, although afamelanotide is the first to be approved while under clinical investigation.

“Five years ago, Clinuvel made a decision to develop afamelanotide specifically for patient populations who were most severely and acutely affected by UV and light,” said Dr Philippe Wolgen, Clinuvel’s CEO.

“Today’s news comes as a welcome surprise but supports our choice to develop afamelanotide in these categories of patients.”

According to Clinuvel’s Chief Scientific Officer, Dr Hank Agersborg, this suggests physicians and patients are becoming more empowered in influencing drug development.

“We have seen a world-first in UV treatment: patients and physicians requesting regulators to allow access to the novel drug," he said.

[WHAT IS LEPTIN?]

As if insulin resistance wasn’t enough, now you can make room for Leptin resistance.

Leptin resistance is emerging as a leading cause of obesity, weight loss difficulty and age related weight gain. Under the category of things we didn’t know that we are starting to figure out about life long weight loss and weight management, it turns out that prolonged and chronic elevated leptin from being overweight and eating high density foods create leptin resistance, which in turns predisposes you towards weight gain, premature aging and obesity.

WHAT IS LEPTIN?
Leptin is one of the key hormones involved in hunger, metabolism and the control of how energy from fats and carbs get stored and utilized. Not coincidentally, it is derived from the Greek word ‘leptos’ meaning thin. Here are a few of the things that Leptin does

-Leptin is perhaps the key signaling hormone circulating in your blood that signals your hypothalamus you have eaten enough and need to start burning fat. In simplest terms, your hypothalamus is the control center for fullness and fat storage, and circulating leptin in your blood is the key signal for your hypothalamus in this regard.

-Leptin production by fat cells signals your hypothalamus that you need more or less body fat.

-The rate of production of leptin directly correlates with weight loss or weight gain. (Women have significantly higher circulating leptin than men)

WHAT IS LEPTIN RESISTANCE?
Leptin resistance is very similar conceptually to insulin resistance. With insulin resistance, chronic elevated levels of insulin make your muscle and fat cells more resistance to the action of insulin. Leptin resistance is very similar. Chronic elevated levels of leptin decrease the hypothalamus sensitivity to leptin.

To put it plainly, if your hypothalamus is resistant to leptin signaling, you tend to eat way too much. This also makes weight loss much more difficult because it makes dieting and restricting food something your body does not want to do.

What you need to understand about leptin resistance is that it is progressive. Once you become resistant to leptin, it leads to more eating, which leads to elevated leptin, which leads to more eating, which makes weight loss very difficult. This is why obesity research is now turning to leptin signaling as a potential causal agent in obesity. It’s that powerful a process.

HOW CAN YOU AVOID LEPTIN RESISTANCE?
There are a few basic things you can do to mitigate becoming leptin resistant.

AVOID HIGH DENSITY FOOD – Foods that are highly caloric promote elevated leptin. What do I mean by high-density food?  In short, foods that have a very high ratio of calories and energy to their total volume. Think processed foods – fried foods, cheesecake, pizza, concentrated sugar drinks.

If you think we are just rehashing old ground with the ‘crap foods to avoid’ list, you must consider your own hunger and appetite patterns.  If you have chronic appetite problems the thing we must now consider in light of the new information on leptin resistance is how this problem is progressive. It only gets worse and makes weight loss more difficult.

BALANCE YOUR FATS AND SUGARS – High sugar and low essential fat intake causes hunger. Good essential fat intake combined with heavy fibrous carbs for volume tends to make you full.

AVOID CHRONIC HUNGER VIA MORE MEALS: The current trend in the weight loss and medical community of medicating hunger is something I totally disagree with.  Hunger should be dealt with through food, not drugs. So much to do with hunger can be mediated with volume foods, good fats and frequent meals. This really becomes a tactical problem. The reality for most of us is too many tasks, too much pressure, and too little time to eat right. Fortunately, there are more solutions than ever. There are fantastic meal delivery services, healthier menus, better salads, meal replacements and weight loss meal services that are available to us. We will be putting together a directory of such services probably next month.

LOSE WEIGHT: While this seems like a catch-22, that’s exactly what leptin resistance is, a catch 22. The fact is, being overweight goes hand in hand with elevated leptin. You HAVE to lose the weight to really get control of this.

In summary, leptin resistance is no joke. It’s real and we want to avoid becoming leptin resistant at all costs.

[Abstract]

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, J T Wilsey and P J Scarpace

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: one study suggested that peripheral but not central MTII improved insulin resistance in DIO mice, whereas another reported that 3-day peripheral MTII administration had no effect on serum insulin in obese Zucker rats. In our present study, central MTII infusion resulted in significant reductions in serum insulin levels in CH and DIO rats and serum glucose levels in DIO rats, suggesting improved glucose metabolism and insulin sensitivity by MTII. Besides its impact on insulin and glucose, MTII also reduced total serum cholesterol levels in CH and DIO rats. How MTII lowers cholesterol is currently unknown. It is conceivable that, in addition to the cholesterol-reducing effect of hypophagia, insulin mediated stimulation of cholesterol synthesis diminishes following a fall in circulating insulin levels.

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.

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[Abstract:]

The Effect of the Melanocortin Agonist, MT-II, on the Defended Level of Body Adiposity

Abstract:  A wide range of experimental evidence implicates a critical role for melanocortin signaling in the control of food intake and body adiposity. Melanocortin receptor agonists such as MT-II potently reduce food intake and body weight, making such agonists potential therapeutics for obesity. The critical concept addressed by the present experiments is whether the homeostatic effects of melanocortin agonists directly regulate food intake or whether the effects on food intake are secondary, with the primary effects being the regulation of body weight and adiposity. To investigate this, we compared the effect of various doses of MT-II given via osmotic minipump for 28 d to alter food intake, body weight, and body fat in dietary-induced obese rats. In addition, before the implantation of the minipump, dietary-induced obese rats were weight reduced by differing amounts using varying levels of food restriction. The results show that in food-restricted rats, MT-II-treated rats consume significantly more calories than those receiving MT-II after ad libitum access to food. More importantly, regardless of the widely differing levels of body fat among the different dietary treatments employed, body fat at the end of the study was determined exclusively by the dose of MT-II, with MT-II-treated rats having less body fat than vehicle-treated rats. These experiments support the hypothesis that melanocortin signaling primarily regulates total body adiposity and that food intake is adjusted as necessary to achieve a specific level of body adiposity.

Introduction:  GORDON KENNEDY, in the 1950s, proposed that body adiposity is defended by the accurate matching of caloric intake to caloric expenditure over time (1). To do so requires that the central nervous system (CNS) have the ability to monitor the status of adipose stores and to use this information to adjust both caloric intake and energy expenditure. Convincing evidence indicates that hormones play this critical role as "adiposity signals" and provide negative feedback about how body fat stores are changing. One hormone hypothesized to be such an adiposity signal is the adipocyte-derived hormone, leptin. Leptin circulates in proportion to the amount of adipose tissue and crosses the blood-brain barrier where it interacts with a known receptor that is located in several regions of the CNS, including densely within the arcuate nucleus of the hypothalamus. CNS administration of leptin results in decreased food intake and weight loss, whereas genetic leptin deficiency and genetic leptin resistance results in increased food intake and greatly increased levels of body adiposity. Leptin may not be the only adiposity signal. Insulin may also function as an adiposity signal, inasmuch as it also circulates in proportion to body adipose mass. Like leptin, insulin crosses the blood-brain barrier where it interacts with its receptor that is expressed heavily within the arcuate nucleus of the hypothalamus. Most critically, CNS administration of insulin results in decreased food intake and body weight, whereas reduced CNS insulin action via central administration of insulin antibodies or by targeted genetic disruption of the CNS insulin receptor results in enhanced food intake and weight gain. Thus, diverse evidence points to both leptin and insulin as critical signals from the periphery to the CNS about the status of peripheral energy stores.

An important conceptual point is that adiposity signals influence food intake but do so to influence the amount of body adipose mass. That is to say, food intake is reduced by administration of these signals but only to achieve a lower body adipose mass. One piece of evidence in support of this hypothesis is that continuous infusion of either leptin or insulin does not result in sustained reductions in food intake. Rather, food intake is suppressed during active weight loss but returns to normal once a new level of body weight (and presumably body adiposity) is achieved. Thus, it would appear that once the animal has achieved a level of body adiposity that is appropriate to the degree of leptin or insulin signaling, leptin and insulin lose their ability to suppress food intake. A more direct test of the hypothesis that the degree of suppression of food intake elicited by an adiposity signal depends on the current level of body adiposity employs a paradigm first developed by Powley and Keesey. In this paradigm, animals are food restricted until they achieve a level of body weight equivalent or below that predicted to be achieved after exogenous administration of the signal. If the signal merely suppressed food intake, its effect should be the same in the weight-reduced state as it was in the ad libitum-fed state. Alternatively, if the signal is actually a component of an adiposity signaling pathway, then food intake after the infusion of the substance should be suppressed only in the ad libitum-fed condition and less so or not at all in the weight-reduced state. In the case of insulin, the data are quite clear. Insulin infusion in the weight-reduced state was much less effective at suppressing food intake than in the ad libitum-fed condition . Even more importantly, the ultimate level of body weight achieved to insulin administration was identical in both the weight-reduced and ad libitum-fed conditions. Such data strongly support the notion that insulin is an adiposity signal that influences food intake only to the extent that is necessary to achieve a new defended level of body adiposity.

Within the CNS, the control of energy balance is regulated by a complex neural network involving a number of systems both within and beyond the hypothalamus. Some of these systems are direct targets for the actions of adiposity signals. In particular, the central melanocortin system appears to be an important mediator of the actions of both leptin and insulin. The endogenous melanocortin agonist, {alpha}-MSH, is produced within neurons of the arcuate nucleus from the precursor peptide proopiomelanocortin. Exogenous administration of melanocortin agonists elicits a strong reduction in food intake and body weight and either pharmacological antagonists or genetic disruption of the melanocortin-4 receptor produces increased food intake and concomitant weight gain. These {alpha}-MSH-producing neurons express both leptin and insulin receptors, and both leptin and insulin stimulate expression of proopiomelanocortin, the precursor to {alpha}-MSH. Moreover, blockade of melanocortin receptors suppresses the ability of both insulin and leptin to suppress food intake and produce weight loss, supporting that both adiposity factors mediate their food intake effects by activating central melanocortin pathway.

Because the melanocortin system is critically regulated by the actions of adiposity signals, it is tempting to hypothesize that exogenous administration of melanocortin receptor agonists also alters the defended level of body adiposity, influencing food intake only secondarily to achieve a specific level of body adiposity. To test this hypothesis, we employed the Powley and Keesey strategy of comparing the ability of the stable {alpha}-MSH analog, MT-II, to suppress food intake and cause weight loss in rats at three different levels of body adiposity. If activity of the melanocortin system is tied to a specific level of energy intake directly, we would expect that previous food restriction should have little impact on the amount of calories consumed during MT-II treatment and this would be accompanied by differing levels of body adiposity. Alternatively, if the activity of the melanocortin system is tied to the defended level of body adiposity, the amount of calories consumed during MT-II treatment should be different between the ad libitum-fed and food-restricted conditions but they should work to achieve identical levels of body adiposity.

Results:  Consistent with the weight loss, both doses of MT-II suppressed food intake to a similar degree. The food intake suppression induced by either dose of MT-II was strongest during the first few days of infusion, with a slow return of food intake toward baseline levels (approximately 20 g/d) over the first 2-wk period. It is important to note that during the last 2 wk of the MT-II infusion, food intake had returned to the level of the vehicle-infused group despite the maintenance of the weight loss.  MT-II was more effective at suppressing food intake at the higher dose than at the lower dose, although as in the AD LIB setting, during the last 14 d of treatment there were no significant differences in daily food intake among the MT-II- and vehicle-treated rats.

A key aspect of these studies is the comparison of the effects of MT-II between the different dietary conditions. Although MT-II clearly suppressed food intake at all three levels of food restriction, the actual amount of calories consumed during MT-II treatment differed across the three levels of restriction. In the two-way ANOVA (dose x diet), the cumulative amount consumed either 1, 7, or 14 d after minipump implantation was significantly influenced by both the dose of MT-II (P < 0.0001) and the level of food restriction (P < 0.0001). On the first day, there was also a significant interaction between these two variables (P = 0.028) that was not apparent on cumulative intake after 7 or 14 d. The implication of this analysis is that, to predict the amount of food consumed, we would need to know both the degree of food restriction as well as the dose of MT-II delivered. This analysis also implies that the effect size of the MT-II on food intake is different across the dietary groups only on the first day but not on cumulative intake over 7 and 14 d. There was also a difference in the nature of the dose-effect curve across the three restriction levels. In AD LIB rats, there was no difference between the 0.3 and 3.0 dose of MT-II, although there was a difference in both the RES-30 and RES-60 conditions.

Most interesting is the comparison of the body weight achieved by the MT-II-treated groups. In the rats treated with 0.3 mg/kg·d dose of MT-II, both the food-restricted and ad libitum-fed rats achieved nearly identical body weights and body fats. Thus for this dose of MT-II, the starting point of the animal was not important in determining the final body fat. In the rats treated with the 3.0 mg/kg dose of MT-II, the data are similar. Both the 30 and 60% restriction groups treated with MT-II achieve identical body weights, but both groups trended for a lower body weight than the ad libitum-fed rats, although this trend did not achieve statistical significance. Body fats between all three treatments receiving the high dose of MT-II were clearly not different. Thus, the trend for lower body weight with higher dose of MT-II in food-restricted vs. ad libitum rats was driven not by differences in body fat, but instead was due to an enhanced lean mass effect in the ad libitum rats. This was probably a reflection of the lower lean mass attained during the food restriction period in nutritionally restricted rats, which was maintained during MT-II treatment. The results with body fat are exactly paralleled by those of plasma leptin levels. There was no difference between dietary groups in plasma leptin levels measured at the conclusion of the experiment, but plasma leptin levels are reduced in all of the MT-II-treated groups.

Body weights of MT-II-treated rats were not significantly different regardless of starting nutritive state of rats.  Interestingly, the majority of fat loss due to MT-II treatment was observed by d 14 of treatment, with only minimal further fat loss induced by an additional 14 d of treatment. This temporal pattern of fat loss corresponds with the temporal pattern of food intake suppression induced by MT-II in both food-restricted and ad libitum-feeding rats. Although food restriction induced a significant reduction of lean mass as mentioned previously, MT-II treatment failed to significantly affect lean mass when compared with vehicle-treated rats in either food-restricted or ad libitum conditions. In fact, lean mass loss due to food restriction in RES-60 rats appeared to recover over the subsequent 28 d of ad libitum feeding, even in MT-II-treated rats.

Discussion:  These data examine the effect of chronic administration of a melanocortin receptor agonist to influence food intake, body weight, and body composition. The first finding of these studies is that chronic sc infusion of MT-II is quite effective to produce reductions in food intake, body weight, and body fat even in rats made quite obese by prolonged exposure to a HF diet. This result agrees with other work using melanocortin agonists in both mouse and rat and separates that work from what has been generally reported with peripheral administration of leptin that appears to lose most of its efficacy in rats made obese after exposure to a HF diet.

The second finding of these studies is that several aspects of the effects of MT-II are altered by prior food restriction. MT-II-treated rats in either of the restricted groups consumed more than MT-II-treated rats that were ad libitum fed. However, this analysis does not take into account the different baseline intakes of the ad libitum and restricted groups. When one compares the effect size of MT-II to its vehicle-treated comparison group, our statistical analysis shows that the effect size is different only on the first day after minipump implantation but not on 7 or 14 d cumulative intake.

Importantly, the ability of MT-II to induce weight loss is much less evident in the restricted rats that weighed less at the beginning of the experiment than the ad libitum-fed rats. Thus, the effect of MT-II to induce body weight loss is highly dependent on the state at which the rats began the experiment. From one perspective this implies that MT-II was less effective in the restricted rats. In the case of the melanocortin system, activity at CNS melanocortin receptors is regulated not just by the presence of endogenous agonists but also by endogenous antagonists/inverse agonists. Agouti-related peptide (AgRP) is produced in arcuate nucleus cell bodies and is carefully regulated by peripheral adiposity signals such as leptin. Thus, when leptin levels are reduced, such as in the restricted groups in this experiment, hypothalamic AgRP levels should be elevated. Increased AgRP in the synapse would work to block melanocortin-4 receptors and decrease the ability of exogenous MT-II to inhibit food intake. Consequently, we can point to a straightforward hypothesis about why MT-II would be less effective to reduce food intake and induce weight loss in the restricted groups.

The greater number of calories consumed in restricted rats treated with MT-II stands in contrast to the effects on body fat. In this regard, the body fat levels achieved by rats treated with MT-II are similar regardless of whether the animal started the dosing regimen at a lower body fat because of food restriction. That is to say, in the animals whose body fat was reduced by enforced food restriction, the effect of MT-II was to maintain that body fat loss at a level identical to the body fat loss induced by the same dose of MT-II in ad libitum-fed rats with higher body fats.

This finding has two implications. The first is that melanocortin agonists that might be used to treat human obesity should be equally effective when used either to produce weight loss or to maintain weight loss already achieved. Thus, melanocortin agonists could be used to help individuals maintain weight loss that was achieved by other means and improve compliance to dietary regimens designed to maintain lost weight.

The second implication is more theoretical and concerns the parameters that are primarily regulated by the melanocortin system. To predict how much an animal will eat in these experiments you would need to know both the dose of MT-II it was receiving and the degree of prior food restriction. This stands in contrast to the degree of body adiposity. To predict the amount of body fat at the end of the experiment one would need to only know whether the animal received MT-II or vehicle, because prior restriction had no impact on the achieved level of body fat. Our conjecture is that this outcome supports the hypothesis that melanocortin signaling primarily regulates body adiposity. From this perspective, changes in food intake are simply a means to an end, and that end is the maintenance of a level of body fat consistent with the amount of melanocortin signaling. Further support for this hypothesis comes from the fact that, in MT-II-treated rats, food intake had returned to normal by d 14. By d 14, rats had already achieved a level of body fat that would be maintained through d 28. Consequently, the dissipating effect of MT-II on food consumption is not "tachyphalaxis" but rather is the result of animals now consuming the appropriate number of calories to maintain a specific level of adiposity consonant with their degree of melanocortin signaling. As a consequence, it might be more appropriate to term the melanocortin system as an effector pathway for regulating adiposity, rather than as an anorexigenic or satiety pathway per se.

[Results:]

Melanocortin activation of nucleus of the solitary tract avoids anorectic tachyphylaxis and induces prolonged weight loss

THE BRAIN MELANOCORTIN PATHWAY is a key leptin target in the central nervous system and plays an essential role in the homeostatic regulation of body weight. Melanocortins are peptides cleaved from a common precursor, proopiomelanocortin (POMC). Rodents with POMC deficiency and humans with POMC mutations are hyperphagic and obese. The contribution of the central melanocortin system on the regulation of food intake and body weight has been attributed primarily to hypothalamic POMC neurons in the arcuate nucleus (ARC), which produce alpha-melanocyte-stimulating hormone ({alpha}-MSH), the principal central melanocortin. {alpha}-MSH and its analog, melanotan II (MTII), inhibit food intake and enhance energy expenditure mainly through activation of melanocortin 3 (MC3R) and 4 (MC4R) receptors in the hypothalamus. However, the central melanocortin system is not limited to the hypothalamus. POMC neurons and {alpha}-MSH are both found within the commissural region of the nucleus of the solitary tract (NTS) in the brain stem where MC4Rs are also expressed. Thus melanocortin signaling within the NTS may contribute to, or even play a major role in, the overall central melanocortin system activity. Additionally, because hypothalamic POMC neurons project to the NTS, the NTS could serve as a site integrating the neuroendocrine and metabolic impact of POMC, both generated within the NTS and the ARC.

The role of the NTS POMC network in energy homeostasis is just beginning to be appreciated. The functions of melanocortin action in the NTS have been explored by acute administration of pharmacological agents into the fourth ventricle or the dorsal vagal complex. These studies indicated that MC4R agonists and antagonists affect food consumption in the caudal brain stem as potently as that in the hypothalamus. In addition, central infusion of MTII into either the third or fourth ventricle also increases brown adipose tissue (BAT) thermogenesis. Thus the brain stem melanocortin pathway is responsive to acute pharmacological melanocortin stimulation and likely participates in the regulation of energy balance, possibly in tandem with the melanocortin system in the hypothalamus. Furthermore, the NTS and other brain stem nuclei are generally assumed to respond to short-term signals that regulate meal initiation and termination, whereas those in the ARC and other areas of the hypothalamus predominately respond to long-term adiposity signals. However, it remains unknown how chronic activation of the melanocortin system in the caudal brain stem, including the NTS, will affect the overall energy homeostasis.

The F344xBN rats represent an established aging rodent model to investigate adult-onset obesity, which is characterized by a modest progression in body weight and visceral adiposity gain with age (20). Although the aged, obese F344xBN rats maintain their hypothalamic POMC expression with age (36), the induction of POMC by exogenous leptin is impaired, indicating an age-related leptin resistance (27). Our earlier study demonstrated that these animals, albeit leptin resistant, lost significant amounts of body fat and weight in response to MTII treatment or viral-mediated POMC gene delivery into the ARC. Anorexia induced by either treatment underlies one mechanism for the weight and fat loss. Unfortunately, the reduction in food intake attenuates within days or weeks after the initiation of either therapy, limiting long-term effectiveness of these modalities in treating the adult-onset obesity. The rapid attenuation of the anorexic response, or tachyphylaxis, may be a result of dwindling melanocortin receptor function due to reduced receptors and/or increased agouti-related protein (AgRP) antagonism. Melanocortin activation in the hypothalamus leads to both increased AgRP expression and reduced MC3R and MC4R expressions. However, these responses, especially the former, may be specific to the hypothalamus because AgRP mRNA expression is limited to the ARC. Thus it is unclear whether anorexic tachyphylaxis will occur after chronic POMC overexpression in the NTS.

To address these issues, recombinant adeno-associated virus (rAAV) vector encoding murine POMC (rAAV-POMC) was microinjected into the NTS, and the long-term consequences of this POMC gene delivery on energy balance, glucose and fat metabolism, BAT thermogenesis, and mRNA levels of neuropeptides and melanocortin receptors in either the NTS or ARC were assessed.

Results:  Food consumption and body weight. After rAAV-POMC administration into the NTS, food consumption decreased rapidly and became significantly different from control rats by day 3. Between days 3 and 7, the reduction in food intake reached a nadir, amounting to a 33% reduction compared with rats administered control vector. Starting at day 8, the anorexic response began to wane, yet food consumption remained diminished by >3 g/day throughout the duration of the experiment.


Food consumption (top) and body weight change (bottom) after rAAV-POMC (bullet) or rAAV-control ({circ}) delivery in aged obese rats. The vectors were injected at day 0. Values are means ± SE of 6 or 7 rats per group. P < 0.001 (difference from food intake and weight change with treatment by repeated-measures ANOVA).

Before vector delivery, average body weight of rAAV-POMC-treated rats was comparable to that of rAAV-control rats (585 ± 11 vs. 590 ± 6 g, respectively, at day 0). Immediately after vector delivery, both POMC and control rats lost ~20 g of body weight. This is what we normally observe after surgery in aged, obese rats. Whereas body weight of rAAV-control rats remained steady throughout the experimental period, there was a steady decrease in body weight during the first 30 days after rAAV-POMC gene delivery. After day 30, body weight stabilized in these rats despite the persistent reduction in food consumption. At the end of the experiment (day 42), rAAV-POMC rats had lost an average of 72 ± 6 g of body weight compared with 29 ± 5 g in control rats (P < 0.001).

Adiposity and serum leptin levels. The decrease in body weight with rAAV-POMC delivery was associated with diminished adiposity levels. Forty-two days after central POMC gene delivery, there was a >37% reduction in visceral adiposity, as reflected by the sum of the PWAT and retroperitoneal white adipose tissues in rAAV-POMC-treated vs. in control rats. In addition, epididymal adipose tissue was diminished by 31% with POMC overexpression. Given the difference in overall body weight, we also normalized the sum of the three fat depots to total body weight. By this calculation, visceral adiposity was also significantly reduced with POMC treatment relative to controls (3.52 ± 0.33% of total body weight vs. 4.89 ± 0.17%; P < 0.005). Serum leptin levels, another indicator of body fat mass, were 38% lower in the POMC group vs. controls.


Visceral adiposity and epididymal white adipose tissue (EWAT; top) and fasting serum leptin (bottom) 42 days after rAAV-POMC or rAAV-control delivery in aged obese rats. Visceral adiposity levels are represented by the sum of perirenal and retroperitoneal white adipose tissues (PWAT + RTWAT). Values are means ± SE of 6 or 7 rats per group. *P < 0.01 and **P < 0.005 (difference from POMC treatment compared with control by unpaired t-test).

Fasting insulin and glucose. Fasting glucose and insulin were determined on day 29 after POMC gene delivery. Whereas POMC treatment did not alter fasting glucose, fasting insulin levels were diminished by >60%. Calculation of the quantitative insulin sensitivity check index revealed that the rAAV-POMC-treated rats had increased insulin sensitivity.

Energy expenditure. Energy expenditure after rAAV-POMC was assessed as whole body oxygen consumption and UCP1 protein levels in BAT. Oxygen consumption was recorded at day 17 and day 25 after vector delivery. On both of these days, oxygen consumption, whether expressed as consumption per rat or normalized to body weight, was not different between rAAV-POMC-treated and control rats (data not shown). However, because food intake was still depressed in the rAAV-POMC-treated rats, it was possible that any POMC-induced increase in oxygen consumption was masked by the suppression in energy expenditure due to the diminished food intake. For this reason, we also assessed UCP1 protein levels in BAT at the termination of the experiment . Induction of UCP1 in BAT is an important marker for enhanced thermogenesis and thus energy expenditure in rodents. The activation of BAT by leptin or MTII is normally associated with an increase in UCP1 and a decline in BAT tissue mass due to the lipolysis associated with thermogenesis. In the present study, total BAT weight declined markedly with rAAV-POMC treatment, and UCP1 protein concentration was elevated by 30%. However, there was only a mild increase in total UCP1 protein per BAT.

Phosphorylation of ACC. Inactivation of ACC by phosphorylation is one indicator of augmented fatty acid oxidation and/or diminished fatty acid synthesis . We examined phosphorylation of ACC 42 days after POMC gene delivery in three tissues: soleus muscle, PWAT, and liver. In soleus muscle, POMC treatment increased ACC phosphorylation by 63%. On the contrary, in PWAT, phosphorylation of ACC was diminished by nearly 60%. This decrease in phosphorylated ACC was accompanied by a 40% decrease in total ACC (100 ± 11 vs. 63 ± 12 arbitrary units/mg protein, respectively, for POMC treatment and control; P = 0.047). In liver, phosphorylation of ACC was unchanged with POMC gene delivery.

Triglyceride and NEFA. At the termination of the experiment, we assessed triglyceride levels in serum, liver, and muscle as well as NEFA level in serum. Triglyceride levels were significantly diminished by 26% in liver and by 35% in serum. There was also a trend toward a decrease in triglyceride level in skeletal muscle without significance. Parallel to the decrease in serum triglyceride, NEFA serum level was diminished by 34%.

MC3R and MC4R are believed to be the predominant melanocortin receptors in the NTS that mediate the effects of POMC-derived {alpha}-MSH on the homeostatic regulation of body weight. Whereas the expression level of MC4R in NTS was unchanged, that of MC3R was reduced by nearly 60% in rAAV-POMC-treated compared with control rats.

Discussion: 
With the use of neural site-directed rAAV-mediated POMC gene delivery, POMC overexpression and increased {alpha}-MSH production were observed in the NTS area at 42 days after vector delivery. Conversely, neither POMC expression nor {alpha}-MSH levels were elevated in the hypothalamus.

The chronic POMC overexpression in the NTS causes persistent but moderate anorexia, a pattern in sharp contrast to pharmacological {alpha}-MSH or MTII administration into the third or MTII administration into the fourth ventricle, which induces a suppression in caloric intake for only a few days in either normal or dietary obese mice and rats. The sustained anorexic response in the present study is also considerably longer than the 20 days of anorexia observed after rAAV-POMC gene delivery targeted to the ARC of the hypothalamus in the age-matched rats of the same strain.

The mechanism of the rapid tachyphylaxis to melanocortin treatment in pharmacological studies is not clear but may involve agonist-mediated receptor internalization and/or elevated AgRP levels. MC3R and MC4R activation by {alpha}-MSH in the hypothalamus is subject to competitive suppression by the natural antagonist, AgRP. Because hypothalamic AgRP expression rises after either peripheral MTII application (1) or hypothalamic POMC gene delivery in young rats (our unpublished data), AgRP seems to be a good candidate for mediating the anorexic tachyphylaxis. Expression of AgRP mRNA is abundant in the ARC (2), and AgRP-containing neurons in the ARC project to other neurons in the hypothalamus and brain stem (34). However, immunohistochemical analysis identifies few AgRP-positive neurons in the NTS (2). In the present study, the expression of AgRP remains unchanged in both the NTS and ARC after POMC gene delivery into the NTS, and the basal level of AgRP in the NTS dwarfs that in the ARC. Thus the lack of AgRP antagonism in the NTS may be one factor preserving the anorectic response to POMC overexpression in the NTS. Another factor may involve MC3R and MC4R expressions. Our previous study with POMC gene delivery into the hypothalamus demonstrated diminished expression levels of MC3R and MC4R in the hypothalamus. On the contrary, only the expression level of MC3R was decreased in the present study, whereas MC4R expression level was unchanged. This absence of a downregulation of MC4R expression in the NTS may also contribute to the prolonged anorexia. The rAAV-mediated POMC overexpression in the NTS did result in the elevation of {alpha}-MSH peptide levels outside the NTS; therefore, the ectopic expression of POMC in the caudal brain might account for some of the responses observed.

POMC gene delivery into the NTS leads to a significant decrease in visceral adiposity and a sustained reduction in body weight in rats with adult-onset obesity. Body weight commenced to decline within days of rAAV-POMC gene delivery and continued for 30 days, after which body weight stabilized. The incongruent patterns of food intake and weight loss suggest factors other than just food intake contributed to the reduced body weight. The anorexic response displayed three phases: a peak response between days 3 to 8, a partial recovery period between days 8 and 14, and a prolonged, moderate anorexia between days 15 and 42. The body weight response, on the other hand, demonstrated a steady, almost linear decrease in body weight from day 3 to day 30, after which it stabilized. This discrepancy suggests that, in addition to the diminished food intake, increased energy expenditure may facilitate the weight and fat reduction. This notion is supported by elevated UCP1 protein levels. Albeit modest, the augmented UCP1 is indicative of an increase in BAT thermogenesis after POMC gene delivery to the NTS. Inconsistent with these observations is the lack of an increase in whole body oxygen consumption. Conclusive assessments of any increase in energy expenditure and any food-independent component of the body weight loss cannot be determined from these studies and would require the inclusion of a pair-fed group.

In addition to the reduction in body weight, there was a substantial decrease in adiposity levels and triglyceride levels and an apparent increase in fat oxidation in muscle. These may be a direct result of enhanced energy expenditure or a consequence of chronic anorexia or both. The evidence for augmented fat oxidation was an increase in phosphorylation of ACC, a key enzyme in regulation of fat oxidation in muscle. Phosphorylation of ACC inactivates the enzyme, thus reducing the synthesis of malonyl CoA. A reduction in the latter releases the inhibition of carnitine palmitoyl transferase-1, the activity of which is the rate-limiting step in muscle mitochondrial fat oxidation. Despite the apparent increase in fat oxidation in muscle, phosphorylation of ACC was not elevated in liver and was diminished in PWAT. The latter is inconsistent with what is observed after chronic leptin treatment in young, lean rats, which substantially increase fat oxidation within the fat tissue. The pattern observed in the present study, an increase in fat oxidation in muscle and reduced fat metabolism in fat tissue, seems to agree with what would be expected after chronic food restriction. In such situations, the stored fat would be used as necessary fuel (oxidation in muscle) and not for facilitated thermogenesis (oxidation in BAT or white adipose tissue). Conceivably, the chronic anorexia is the primary cause of the apparent increase in fat oxidation in muscle rather than an overt increase in energy expenditure.

The NTS POMC over-expression also improved insulin sensitivity. The aged F344xBN rats with adult-onset obesity normally demonstrate insulin resistance and glucose intolerance. The fasting insulin levels were substantially diminished in the POMC-treated rats, and the Quantitative Insulin Sensitivity Check Index indicated increased insulin sensitivity. These data are consistent with our previous report of improved glucose metabolism and insulin sensitivity after POMC gene delivery into the hypothalamus and in agreement with other findings that central melanocortin receptor activation suppresses insulin release from the pancreas and enhances glucose metabolism. The enhanced insulin sensitivity is probably due to the substantial decrease in visceral adiposity and muscle triglyceride content by the chronic POMC treatment and could be the result of the prolonged anorexia and/or some food-independent function(s) specific to POMC overexpression.

In conclusion, POMC gene delivery directed into the NTS suppresses food intake, reduces body weight and visceral adiposity, increases muscle fat oxidation and BAT UCP1 protein levels, lowers tissue triglyceride content, and improves insulin sensitivity in rats with adult-onset obesity. The unabated hypophagia, unique to POMC overexpression in the NTS compared with in the hypothalamus, suggests that the mechanisms leading to anorexic tachyphylaxis in response to melanocortin activation in the hypothalamus are lacking in the NTS. Therefore, rAAV-POMC gene delivery to the NTS appears more efficacious than comparable activation in the hypothalamus and is a new and viable strategy to combat adult-onset obesity in rodents.

[Melanocortinergic Activation by Melanotan II Inhibits Feeding and Increases Uncoupling Protein 1 Messenger Ribonucleic Acid in the Developing Rat]

Melanocortinergic Activation by Melanotan II Inhibits Feeding and Increases Uncoupling Protein 1 Messenger Ribonucleic Acid in the Developing Rat
Maria M. Glavas, Sandra E. Joachim, Shin J. Draper, M. Susan Smith and Kevin L. Grove.  April, 2007

Division of Neuroscience (M.M.G., S.E.J., S.J.D., M.S.S., K.L.G.), Oregon National Primate Research Center, Beaverton, Oregon 97006; and Department of Physiology and Pharmacology (M.S.S.), Oregon Health and Science University, Portland, Oregon 97209

Address all correspondence and requests for reprints to: Kevin L. Grove, Ph.D, Oregon National Primate Research Center, Oregon Health and Science University, 505 NW 185th Avenue, Beaverton, Oregon 97006.

Abstract:  The hypothalamic neurocircuitry that regulates energy homeostasis in adult rats is not fully developed until the third postnatal week. In particular, fibers from the hypothalamic arcuate nucleus, including both neuropeptide Y (NPY) and {alpha}-MSH fibers, do not begin to innervate downstream hypothalamic targets until the second postnatal week. However, {alpha}-MSH fibers from the brainstem and melanocortin receptors are present in the hypothalamus at birth. The present study investigated the melanocortin system in the early postnatal period by examining effects of the melanocortin receptor agonist melanotan II (MTII) on body weight, energy expenditure, and hypothalamic NPY expression. Rat pups were injected ip with MTII (3 mg/kg body weight) or saline on postnatal day (P) 5 to P6, P10–P11, or P15–P16 at 1700 and 0900 h and then killed at 1300 h. Stomach weight and brown adipose tissue uncoupling protein 1 mRNA were determined. In addition, we assessed central c-Fos activation 90 min after MTII administration and hypothalamic NPY mRNA after twice daily MTII administration from P5–P10 or P10–P15. MTII induced hypothalamic c-Fos activation as well as attenuating body weight gain in rat pups. Stomach weight was significantly decreased and uncoupling protein 1 mRNA was increased at all ages, indicating decreased food intake and increased energy expenditure, respectively. However, MTII had no effect on NPY mRNA levels in any hypothalamic region. These findings demonstrate that MTII can inhibit food intake and stimulate energy expenditure before the full development of hypothalamic feeding neurocircuitry. These effects do not appear to be mediated by changes in NPY expression.

Introduction:  IN THE ADULT rodent, the arcuate nucleus of the hypothalamus (ARH) plays a key role in body weight regulation, in part by responding to numerous peripheral signals of metabolic status, including leptin. These effects are mediated primarily by two populations of neurons, the orexigenic neuropeptide Y (NPY)/agouti-related protein (AgRP) neurons and the anorexigenic {alpha}-MSH neurons. These neuronal populations relay information to other hypothalamic sites that mediate food intake and energy expenditure, including the paraventricular nucleus of the hypothalamus (PVH), the dorsomedial nucleus of the hypothalamus (DMH), the perifornical region (PFR), and the lateral hypothalamic area (LHA), as well as the brainstem.

Although pathways mediating energy homeostasis during the early postnatal period are not well understood, the mechanisms appear to be less complex than in the adult. Importantly, the ARH neurocircuitry that regulates energy homeostasis in the adult rat is not fully developed at birth, such that ARH neurons do not begin to innervate downstream hypothalamic targets until postnatal day (P) 5 to P11. Before P6, ingestion appears to be mainly stimulated by dehydration, whereas the primary inhibitory signal is gastric distention. By P9–P12, rat pups begin responding to caloric signals; however, 2-deoxyglucose and insulin, which decrease available glucose, do not stimulate food intake until P25–P30. Furthermore, exogenous leptin has no effect on food intake during the first 3 wk of postnatal life, indicating functional immaturity of downstream hypothalamic pathways during the entire preweaning period. Peripheral metabolic and caloric signals thus appear to have a minimal role in food intake in the developing rat.

The early postnatal period is a time of rapid body growth and therefore high energy demands, suggesting a strong orexigenic drive or low anorexigenic signals. Although the major orexigenic neurocircuitry, i.e. the ARH NPY/AgRP neuronal projections, are not established in the early postnatal period, hypothalamic NPY content is abundant during this time. In fact, during development, NPY is expressed in a number of hypothalamic regions that typically do not show expression in adult rats. In the adult rat hypothalamus, NPY is expressed mainly in the ARH with an additional low level of expression in the central compact region of the DMH (DMHp). In addition to these regions, during development, there is a unique, transient expression of NPY in the noncompact zone of the DMH (DMHnc), the PFR, the LHA, and the PVH (8, 9). We hypothesize that these transient NPY populations drive food intake before the establishment of ARH feeding neurocircuitry and/or promotes the transition to independent ingestion. This transient NPY expression peaks at approximately P16 and subsequently declines to an adult-like expression by P30 (8, 9), suggesting the establishment of a tonic inhibitory signal that persists through adulthood. A likely candidate for this inhibitory signal is {alpha}-MSH. Evidence for this includes the induction of NPY expression in the DMHnc in specific models of reduced melanocortin signaling, including lactation, the melanocortin 4 receptor (MC4R) knockout mouse (11), and the agouti mouse (11). In addition, site-directed administration of the nonselective melanocortin receptor agonist melanotan II (MTII) greatly attenuates this NPY induction during lactation (12). The early postnatal period, before downstream innervation by arcuate melanocortinergic fibers, may similarly be considered a period of reduced melanocortin signaling, thereby providing a permissive environment for the novel NPY expression.

Orexigenic drive likely dominates under most conditions during development; however, anorexigenic mechanisms are not absent. Although the key anorexigenic pathway mediated by ARH {alpha}-MSH fibers is not fully established in the early postnatal period, {alpha}-MSH projections originating from the brainstem are widespread in the hypothalamus at birth, as are melanocortin receptors. Therefore, the components of a functional melanocortin system are present in rodent neonates. Because the projections of the endogenous melanocortin receptor antagonist AgRP, which originate solely from ARH NPY neurons, are not yet established during the early postnatal period, this would in fact suggest an enhanced capacity for {alpha}-MSH-mediated effects. To investigate the role of the melanocortin system in the developing rat, the present study used the melanocortin receptor agonist MTII to determine whether the melanocortin system can regulate food intake and energy expenditure during the early postnatal period. In addition, we investigated the ability of MTII to inhibit the transient hypothalamic NPY expression observed during the early postnatal period.

Materials and Methods: 
Animals
All animals were maintained under a 12-h light, 12-h dark (lights on at 0600 h) cycle and constant temperature (23 ± 2 C). Pregnant female rats were housed individually and checked for birth of pups every morning. The day of birth was considered P0, and litters were adjusted to eight male pups on P2. Adult male rats were housed individually. All animal procedures were approved by the Oregon National Primate Research Center Institutional Animal Care and Use Committee.

Drug preparation and administration
MTII (NeoMPS, San Diego, CA) was diluted in sterile saline and injected ip. Vehicle animals were injected with sterile saline. MTII was injected ip instead of icv because of possible confounding effects that would result from intracranial cannulation in suckling pups. In addition, previous studies have shown that ip MTII administration decreases food intake in adult rats. MTII was made fresh before use, and approximate volume of injection was 100 µl for pups and 250 µl for adult rats.

MTII dose response
To determine an appropriate dose of MTII, we examined the effects of ip MTII at various doses on body weight and food intake. Offspring of pregnant Wistar females (Charles River Laboratories, Wilmington, MA) were tested at P10–P11. Pups within each litter (five litters total) were randomly assigned to receive 0 (saline), 0.1, 3.0, or 10.0 mg/kg MTII (final n = 7–8 per dose). Before drug administration, the dam was removed from the home cage, and pups were weighed and then injected ip with either MTII or saline at 1700 h. Immediately after injections, the dam was returned to the home cage (dam absent <10 min). The following day, injections were repeated at 0900 h. Pups were reweighed and killed by decapitation at 1300 h. Stomachs were removed and weighed as an index of food intake. Subsequently, a dose of 3.0 mg/kg MTII was used in all additional studies.

c-Fos activation in response to peripheral MTII administration
There is some controversy in the literature as to whether MTII administered peripherally effectively crosses the blood-brain barrier (BBB). Therefore, we first assessed whether ip MTII administration would activate c-Fos in the CNS under our conditions. In addition, because BBB function develops progressively through to the fourth postnatal week, we examined whether the extent of central c-Fos activation was affected by age. Wistar rats (Simonsen Laboratories, Gilroy, CA) were tested during the early postnatal period (P6 or P15) or in adulthood (P90). For both P6 and P15 animals, two litters were used at each age, with n = 4 at P6 and n = 8 at P15. For adults, three saline-injected and four MTII-injected animals were assessed. To minimize any nonspecific stress effects of the treatment, adult rats were acclimatized for 7 d before testing by daily saline injections at 1100 h. Rat pups were not acclimatized before testing because of possible confounding effects of repeated handling and maternal separation and because preliminary investigation demonstrated that saline-injected naive pups exhibited minimal c-Fos activation. On the day of testing, adult rats and pups were injected ip with 3.0 mg/kg MTII or saline at 1100 h. For adult rats, food and water were removed at this time, and, for pups, the mother was removed and the cage was placed on a heating pad. Animals were killed with pentobarbital 90 min after injection and then perfused transcardially with saline, followed by ice-cold, phosphate-buffered 4% paraformaldehyde (pH 7.4). The brains were removed, postfixed in paraformaldehyde overnight, and then saturated in 25% sucrose. Brains were frozen in –40 C isopentane and stored at –80 C before immunohistochemical analysis for c-Fos (as described below).

Acute MTII administration: effects on food intake and energy expenditure in rat pups
Offspring of pregnant Wistar females (Charles River Laboratories) were tested on P5, P10, or P15. These ages were chosen to represent a spectrum of hypothalamic feeding neurocircuitry development. Pups within each litter (four to seven litters were tested per age) were randomly assigned to either the saline or MTII condition, with four pups per drug condition per litter (final n = 14–16). Before drug administration, the dam was removed from the home cage, and pups were weighed and then injected ip with either 3.0 mg/kg MTII or saline at 1700 h. Immediately after injections, the dam was returned to the home cage. The following day, injections were repeated at 0900 h. Pups were reweighed and killed by decapitation at 1300 h. Stomachs were removed and weighed as an index of food intake. Interscapular brown adipose tissue (BAT) was removed to a microcentrifuge tube containing a ribonuclease inactivator (RNAlater; Ambion, Austin, TX) and then stored at –20 C for analysis of uncoupling protein 1 (UCP1) (an index of BAT thermogenesis) mRNA by real-time PCR (as described below).

In a subset of the animals (two litters per age), pup behaviors were observed and quantified for 1 h after injection by an investigator blind to the treatment group. These behaviors included latency to feed, defined as the latency for an individual pup to attach to a nipple (measured in the morning only), number of yawns observed (measured in the evening only), and total time spent grooming (measured at P15, in the evening only).

Chronic MTII administration: effects on hypothalamic NPY mRNA in rat pups
To determine whether melanocortin receptor activation inhibits transient hypothalamic NPY expression, MTII was administered over 5 d at two different developmental stages. Offspring of pregnant Sprague Dawley females (Simonsen Laboratories) were randomly assigned to either the saline or MTII condition, with four pups per drug condition per litter. Two litters were tested per age group. Before drug administration, the dam was removed from the cage and returned on completion of injections. Pups were injected ip with MTII or saline twice daily (at 0900 and 1700 h) for 5 consecutive days, from P5 to P10 or P10 to P15, with the first injection at 1700 h and the last injection at 0900 h. Pups were weighed before each injection. On the final day (P10 or P15), pups were killed by decapitation at 1300 h. Brains were rapidly removed, frozen on powdered dry ice, and then stored at –80 C for NPY mRNA analysis by in situ hybridization (as described below), with six animals per group.

Immunohistochemistry for c-Fos
Immunohistochemistry was performed to detect c-Fos immunoreactivity as described previously. Briefly, perfused brains were sectioned (25 µm) on a microtome in a one-in-three coronal series through the extent of the hypothalamus and brainstem. Free-floating sections were rinsed in 0.05 M potassium PBS and then preincubated in blocking buffer, consisting of 0.05 M potassium PBS with 0.4% Triton X-100 and 2% donkey serum, for 30 min. Sections were then incubated with rabbit anti-c-Fos antibody (1:20,000; Santa Cruz Biotechnology, Santa Cruz, CA) in blocking buffer for 48 h. After incubation, the tissue was rinsed in 0.05 M potassium PBS, incubated in biotinylated donkey antirabbit IgG (1:600; Jackson ImmunoResearch, West Grove, PA) in potassium PBS with 0.4% Triton X-100 for 1 h and then washed and incubated in avidin-biotin solution (Vectastain; Vector Laboratories, Burlingame, CA) for 1 h. c-Fos immunoreactivity was visualized with 3,3'-diaminobenzidine enhanced with nickel chloride. Tissue sections were mounted on gelatin-coated glass slides, counterstained with cresyl violet, and coverslipped. c-Fos immunoreactive cells were counted under a light microscope, by an investigator blind to the treatment group, through the rostral to caudal extent of all regions of interest and expressed as the mean number of c-Fos-positive cells per section. Regions analyzed were anatomically matched across all animals.

RNA isolation and real-time RT-PCR analysis
Real-time RT-PCR was performed on BAT as described previously. Briefly, BAT was homogenized in 800 µl Trizol reagent (Invitrogen, Carlsbad, CA), and total cellular RNA was isolated according to the specifications of the manufacturer. Total RNA was further purified using the RNeasy Mini kit (Qiagen, Valencia, CA). The quality and concentration of the RNA were determined by measuring the absorbance at 260 and 280 nm, and RNA integrity was confirmed by bioanalysis (Agilent 2100 Bioanalyzer; Agilent Technologies, Santa Clara, CA). RNA samples (1 µg) were reverse transcribed using random hexamer primers (Promega, Madison, WI). The RT product was then diluted 1:100 for UCP1 PCR analysis. Reaction mixture (10 µl) consisted of 5 µl of TaqMan Universal PCR Master Mix, 300 nM specific target gene primers, 80 nM 18S rRNA gene primers, 250 nM specific probe, and 2 µl cDNA. Amplification was performed using the ABI/Prism 7700 Sequences Detector System (Applied Biosystems, Foster City, CA) with 2 min at 50 C, 10 min at 95 C and then 45 cycles each at 95 C for 15 sec and 60 C for 60 sec. The sequences of primers and probes used were as follows: UCP1 forward, TCC CTC AGG ATT GGC CTC TAC; UCP1 reverse, GTC ATC AAG CCA GCC GAG AT, UCP1 probe, 6-carboxyfluorescein-AACGCCTGCCTCTTTGGGAAGCAA-6-carboxytetramethylrhodamine.

In situ hybridization
Brains were sectioned (20 µm) on a cryostat in a one-in-four coronal series through the entire extent of the hypothalamus. NPY mRNA levels were determined in one series by in situ hybridization as described previously (8, 9). Briefly, a cRNA probe for NPY was transcribed from a 511-bp cDNA (obtained from Dr. S. L. Sabol, National Institutes of Health, Bethesda, MD) in which 25% of the UTP was 35S labeled (PerkinElmer, Wellesley, MA). Brain sections were fixed in phosphate-buffered 4% paraformaldehyde (pH 7.4) and treated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0). Sections were then rinsed in 2x sodium saline citrate (SSC), dehydrated through a graded series of alcohols, delipidated in chloroform, rehydrated through a second series of alcohols, and then air dried. The NPY probe (specific activity, 5–6 x 108 dpm/µg; saturating concentration, 0.3 µg/ml·kb) was diluted in hybridization buffer (50% formamide, 6.25% dextran sulfate, 0.7% Ficoll, and 0.7% polyvinylpyrolidone) and then the sections were exposed to the labeled probe overnight in a humidified chamber at 55 C. After incubation, the slides were washed in 4x SSC, ribonuclease A at 37 C and in 0.1x SSC at 60 C. Slides were then dehydrated through a graded series of alcohols and dried. For histological analysis of the distribution of NPY mRNA, slides were dipped in Kodak NTB emulsion (Eastman Kodak, Rochester, NY) diluted 1:1 in 600 mM ammonium acetate, placed in light-tight boxes containing desiccant, and stored at 4 C for 7 d. The slides were developed and counterstained with cresyl violet. To prevent interassay variability, sections from each age were run in separate assays, and no direct comparisons were made across ages.

Quantification of NPY mRNA
Images of silver grain distribution were captured under dark-field illumination using a CoolSNAPHQ CCD camera (Photometrics, Tucson, AZ) and analyzed using the MetaMorph Imaging system (Universal Imaging Corp., West Chester, PA). Silver grains were analyzed using a sampling box that encompassed the entire region of interest (ROI) and measured as the area occupied by silver grains within the ROI multiplied by the OD. Background labeling, determined using the same sampling box over an adjacent region that contained no NPY gene expression, was subtracted from this measurement. For the DMH, the sampling box encompassed both the central compact zone (DMHp) and the surrounding scattered neurons of the noncompact zone (DMHnc). To distinguish between these two regions, a second ROI was drawn to outline only the DMHp, and this measurement (minus its corresponding background measurement) was subtracted from the entire DMH measurement to produce a measure of the DMHnc. Measurements were taken bilaterally through the complete rostrocaudal extent of the ARH, DMH, and PFR. For data analysis, the brain sections were anatomically matched across animals from all groups, with equal numbers of sections sampled per animal. The mean value per region per animal was determined and used for statistical analysis.

Statistical analyses
To account for litter effects (because two to four pups per litter received the same treatment), data were analyzed using nested-design ANOVA for the factors of drug treatment and litter, with litter nested under treatment. For all data, individual ANOVAs were conducted at each age. Because c-Fos immunohistochemistry and NPY in situ hybridizations were conducted as different assays at each age, separate statistical analyses were conducted at each age for each hypothalamic region. Body weights for chronic MTII administration were analyzed by repeated-measures nested-design ANOVAs, with day treated as a repeated measure and litter nested under treatment. Any significant main or interaction effects were further analyzed by Newman-Keuls post hoc analysis. Statistical analyses were conducted using Statistica software (StatSoft, Tulsa, OK). In all cases in which significant litter effects were observed, the effect was attributable to differences among saline-injected animals or in the magnitude of response to MTII and not a difference in the direction of the response. All values are represented as the mean ± SEM. Statistical significance was defined as P < 0.05.

Results:
MTII dose response
In rat pups, stomach weight at the time of death was used as an index of food intake. Increasing doses of MTII resulted in significantly decreased stomach weight (Fig. 1AGo) at all doses (0.1, 3.0, and 10.0 mg/kg), as demonstrated by a significant main effect of dose (F(3,11) = 52.13, P < 0.0001), with saline > 0.1 mg/kg > 3.0 mg/kg = 10.0 mg/kg (P < 0.05). In addition, a significant litter effect was observed (F(16,11) = 2.88, P < 0.05). Body weight (Fig. 1BGo) showed a similar dose response effect (main effect of dose, F(3,11) = 76.65, P < 0.0001), with saline > 0.1 mg/kg > 3.0 mg/kg = 10.0 mg/kg (P < 0.005). A dose of 3.0 mg/kg MTII was used in all subsequent studies.


MTII dose response effects on body weight (A) and stomach weight (B). Rat pups (age P10–P11) were injected ip with MTII at 0.1, 3.0, or 10.0 mg/kg or saline vehicle at 1700 h and at 0900 h the following day and then were killed at 1300 h. *, P < 0.0001 vs. vehicle; **, P < 0.001 vs. vehicle and P < 0.01 vs. 0.1 mg/kg MTII dose. Values represent the mean ± SEM of seven to eight animals per group.

Effects of MTII on food intake and energy expenditure
Acute MTII administration resulted in significantly decreased stomach weight (Fig. 4Go) at all ages tested (P < 0.001), with significant main effects of treatment and litter at each age (P6 treatment effect, F(1,22) = 79.12, P < 0.0001; litter effect, F(6,22) = 12.97, P < 0.0001; P11 treatment effect, F(1,18) = 180.15, P < 0.0001; litter effect, F(12,18) = 5.39, P < 0.001; P16 treatment effect, F(1,24) = 126.143, P < 0.0001; litter effect, F(6,24) = 5.83, P < 0.001). In addition, MTII significantly attenuated body weight gain (Fig. 5Go) at P6 compared with saline, and P11 and P16 pups showed a loss in body weight (P < 0.001 compared with saline). Significant main effects of treatment were observed at all ages, and significant litter effects were observed at P11 and P16 (P6 treatment effect, F(1,22) = 81.63, P < 0.0001; P11 treatment effect, F(1,18) = 320.56, P < 0.0001; litter effect, F(12,18) = 3.53, P < 0.01; P16 treatment effect, F(1,24) = 313.38, P < 0.0001; litter effect, F(6,24) = 3.30, P < 0.05).

Effects of acute MTII administration on stomach weight in rat pups. Rat pups were injected ip with MTII (3.0 mg/kg) or saline at 1700 h and at 0900 h the following day and were then killed at 1300 h (age at time of death was P6, P11, or P16). **, P < 0.001 compared with saline. Values represent the mean ± SEM of 14–16 animals per group.


Body weight in response to acute MTII administration. MTII (3.0 mg/kg) or saline was administered ip at 1700 h and then 0900 h, and then the animals were killed at 1300 h. Values represent the change in weight from the time of the first injection until death 20 h later. Values represent the mean ± SEM of 14–16 animals per group. **, P < 0.001 compared with saline.

Chronic MTII administration from P5–P10 (Fig. 6AGo) or from P10–P15 (Fig. 6BGo) resulted in an attenuation of body weight gain on all days (P < 0.01) during the entire administration period, with a more pronounced attenuation in the P10–P15 group. In both age groups, the effect was primarily attributable to a decreased weight gain during the first 2 d. This is consistent with previous studies showing that the effects of MTII on body weight diminish over time. This was demonstrated by significant treatment x day interaction effects for both P5–P10 (F(4,48) = 3.03, P < 0.05) and P10–P15 (F(4,44) = 20.35, P < 0.0001) pups. In addition, there was a significant litter x day interaction effect in P10–P15 (F(8,44) = 22.90, P < 0.0001) pups only.

Body weight in response to chronic MTII administration. MTII (3.0 mg/kg) or saline was administered ip daily at 0900 and 1700 h from P5 to P10 (A) or P10 to P15 (B), and values represent the cumulative weight gain across this period (mean ± SEM of seven to eight animals per group). *, P < 0.01 compared with saline; **, P < 0.001.

MTII significantly increased BAT UCP1 mRNA levels (Fig. 7Go) in rat pups at all ages examined (P6, P11, and P16; P < 0.05), indicative of increased energy expenditure. This was demonstrated by significant main effects of treatment (P6, F(1,8) = 10.64, P < 0.05; P11, F(1,8) = 11.54, P < 0.01; P16, F(1,8) = 11.44, P < 0.01). The increase in UCP1 mRNA was more pronounced in older pups.


BAT thermogenesis in response to MTII. UCP1 mRNA was quantified by real-time PCR in BAT from rat pups at P6, P11, or P16 in response to acute MTII administration. MTII (3.0 mg/kg) or saline was administered ip at 1700 h and then 0900 h, and the animals were killed at 1300 h. Values represent the mean ± SEM of four saline and eight MTII animals per group. *, P < 0.05 compared with saline; **, P < 0.01.

Behavioral effects of MTII administration
As shown in Table 1Go, MTII significantly increased the latency to feed in P11 pups (main treatment effect, F(1,6) = 37.92, P < 0.001; litter effect, F(8,6) = 58.23, P < 0.0001), with a marginal increase (P = 0.065) in P16 pups and no significant effect in P6 pups. MTII also significantly increased the number of yawns (P < 0.0005) in animals at all ages (main treatment effects at P6, F(1,10) = 76.75, P < 0.0001; P11, F(1,6) = 67.47, P < 0.0001; P16, F(1,12) = 173.75, P < 0.0001). In addition, time spent grooming was significantly increased by MTII at P16 (main treatment effect, F(1,12) = 63.43, P < 0.0001).

Discussion:  The present studies demonstrate that, before the full maturation of central feeding neurocircuitry, melanocortin receptor activation via the agonist MTII can decrease food intake, increase sympathetic outflow, and subsequently attenuate body weight gain in rodent neonates as early as P5. Although both central and peripheral administration of MTII are known to reduce food intake in adult rodents, peripheral administration has been shown previously to produce limited or no central c-Fos activation in adult rats, whereas central administration shows abundant central activation. In contrast, we found significant central c-Fos activation in rat pups after peripheral MTII administration, with the greatest activation seen at P15. The same dose and route of MTII produced no central c-Fos activation in adult rats, confirming previous studies. These findings suggest that the BBB may be more permeable to MTII during development, allowing centrally mediated effects to be observed. In addition, the greater extent of c-Fos activation in P15 compared with P6 pups may reflect increased levels of melanocortin receptors and/or additional development of downstream pathways mediating melanocortin receptor activation with age. At P15, the most pronounced activation in the hypothalamus was observed in the PVH and VMH. Although PVH c-Fos activation has been shown previously in response to central MTII administration in adult rats, activation in the VMH has not been reported. Regions activated by MTII included numerous sites involved in energy homeostasis, namely the PVH, VMH, ARH, and solitary tract nucleus. All of these regions express MC4Rs, which have been shown to mediate MTII effects on food intake and metabolism. MTII also activates MC3 receptors, which are also expressed in the ARH and VMH; therefore, some of the c-Fos activation observed in these regions may have been mediated through this receptor subtype. It is also important to note that any c-Fos immunoreactivity observed could be the result of either direct MTII activation of a given region or an indirect activation via other central regions. It should be noted that, because melanocortin receptors are also expressed in peripheral tissue, it remains possible that some of the MTII effects observed may be mediated, in part, via these peripheral receptors.

Peripheral MTII administration (P5–P6, P10–P11, or P15–P16) significantly decreased stomach content weight, suggesting a decrease in milk intake. Rat pups also displayed an attenuated body weight gain that was most pronounced in P16 pups when there was in fact a loss in body weight. MTII administration resulted in a small but significant increased latency to feed, although only in P11 pups. In addition, MTII administration increased yawning (P5, P10, and P15) and time spent grooming (measured at P16 only) during the first hour after injection. Both yawning and grooming behaviors have been attributed previously to hypothalamic activation of MC4Rs, suggesting activation of central melanocortin pathways. As seen with acute administration, chronic MTII administration over 5 d (P5–P10 or P10–P15) also attenuated body weight gain in pups, with a greater effect in older pups. Although the effect on body weight was substantial after the first day of MTII administration, subsequent rate of body weight gain was similar between MTII and saline animals but remained at a lower level in the MTII group. A similar tachyphylactic response to chronic MTII administration has been observed in adult rodents and may be attributable in part to decreased circulating leptin levels or other secondary effects of reduced energy intake.

In the present studies, maternal milk provided the sole nutritional source for pups; consequently, the lower stomach content weight observed reflects an MTII-mediated inhibition of suckling and not necessarily adult-like feeding. Because suckling differs considerably from adult feeding behavior, a number of previous studies have used models of adult-like independent ingestion to study the ontogeny of food intake controls in pups. These studies have demonstrated that, before P6, food intake is primarily inhibited by gastric fill, and, by P9, independent ingestion can be inhibited by nutritive signals. In comparison, nutritive signals do not appear to inhibit suckling until at least P14. We, however, observed MTII-mediated inhibition of milk intake in suckling pups at all ages studied, from P6 to P16. This inhibition therefore does not appear to reflect the developmental progression of inhibitory ingestive controls but instead likely reflects activation of central melanocortin receptors that are already present at birth. Importantly, these studies demonstrate that, not only does MTII inhibit solid food intake in adult rats, but it can inhibit suckling-mediated milk intake as early as P6, a time when food intake is primarily mediated by gastric fill.

It is possible that, in the early postnatal period, vagal feedback can activate brainstem {alpha}-MSH neurons that project to the PVH even early in development. In addition to effects on food intake, MTII administration significantly increased BAT UCP1 mRNA levels in rat pups. Up-regulation of UCP1, which mediates BAT thermogenesis, is indicative of increased ß-adrenergic sympathetic outflow and subsequently increased energy expenditure. In adult rats, MTII has been shown to increase BAT UCP1 levels in response to central but not peripheral administration, indicating a centrally mediated mechanism. That we observed a significant increase in UCP1 mRNA in pups with peripheral MTII administration again suggests increased BBB permeability to MTII in rat pups and a central site of action. The neuroanatomical pathways mediating melanocortin effects on BAT thermogenesis are thought to involve PVH neurons that express melanocortin receptors. Intra-PVH MTII administration both increases oxygen consumption and inhibits food intake. We also demonstrated previously an increase in UCP1 mRNA levels in response to intra-DMH MTII administration in lactating rats. In addition, there appears to be an independent pathway in the caudal brainstem, as evidenced by elevated UCP1 mRNA in BAT after fourth ventricle MTII administration in chronic decerebrate rats. Because we observed MTII-induced c-Fos activation in both the hypothalamus and the brainstem in rat pups, the UCP1 activation and effects on food intake may have been mediated by either of these pathways.

In the adult, an increase in energy expenditure via BAT thermogenesis is predominantly used to maintain body weight homeostasis. Such a mechanism would at first glance appear to be detrimental to the developing rat, when appropriate energy utilization is critical to sustain rapid growth and development. However, under certain conditions, such as low ambient temperature, BAT thermogenesis may be critical for survival through defense of body temperature. Indeed, as early as 5 h after birth, rodent neonates can increase UCP1 levels in response to either cold or ß-adrenergic stimulation. Our findings suggest that sympathetic outflow to BAT, mediated through melanocortin receptor activation, is functional and responsive at birth. Increased energy expenditure through this mechanism, in addition to the decrease in food intake, likely both contributed to the effects that we observed of MTII on body weight.

Although our studies demonstrate that rat pups have the capacity for anorexigenic effects, orexigenic drive is expected to dominate during development to sustain rapid growth. We propose that the transient hypothalamic NPY expression (in the DMHnc, PFR, PVH, and LHA) observed during development may drive food intake in pups before the development of ARH projections. An orexigenic role for this population is suggested by adult rat models of reduced melanocortin signaling, including the lactating rat and the MC4R knockout mouse, which show a similar induction of NPY although limited to the DMHnc. We have shown previously that this DMH-NPY expression mediates hyperphagia in the lactating rat and is inhibited by MTII. We therefore hypothesized that the novel hypothalamic NPY induction during development similarly drives food intake and can be inhibited by MTII administration. However, we did not observe a significant MTII-induced reduction of NPY mRNA in any hypothalamic region. Although we have shown previously that MTII inhibits lactation-induced NPY expression in the DMHnc, these studies used MTII injection directly into the DMHnc, resulting in increased BAT UCP1 mRNA levels and decreased food intake. It is thus possible that the MTII effects we observed on food intake and energy expenditure in rat pups were also mediated in part through NPY neurons of the DMHnc. Although the lack of a decrease in DMHnc-NPY suggests that peripheral MTII administration may not have adequately penetrated the hypothalamus to down-regulate NPY expression, this seems unlikely because we saw robust c-Fos activation in the PVH. Another possibility is that competing mechanisms may have obscured any observable effects of MTII on NPY mRNA in the DMH. Alternately, a signal other than {alpha}-MSH may provide the primary inhibition of NPY expression in this region. Additional investigation is needed to determine whether {alpha}-MSH is the primary inhibitory signal, what the role of this transient NPY population is during development, and how the various regions involved (i.e. the DMHnc, PFR, LHA, and PVH) are related in function and regulation.

In summary, we demonstrated that, before the development of ARH projections, melanocortin receptor activation can inhibit food intake and increase energy expenditure. These effects were observed as early as P5, although the effectiveness of MTII was greater at P15, likely attributable to increased permeability of MTII and/or additional development of the melanocortin system.

[Bremelanotide]

People use all means so that their way of showing love for each other will not die down. Everywhere there are many people who start to envision that aphrodisiac is an effective way to keep their relationship working. Aphrodisiac is a term coined after Aphrodite, The Greek goddess of sensuality and love.  Now, to understand aphrodisiac more clearly, People who wants to achieve pleasure lean on this belief, because almost all of them experience a decrease in sexual desire at one point or another due to illness, disability, low self-esteem, obesity, medications, stress, malnutrition, recreational drugs, fatigue, past sexual experiences, and/or loss of interest in a partner, therefore they have to make sure that they can fulfill their function as a sexual partner by making sex more attainable and/or pleasurable.

Numerous ideas spawn making people believe that it can be found in certain foods, drinks or drugs. In fact, people belief stems not from the chemical compound of this aphrodisiac but mostly through the appearance or shape i.e many fruits and veggies were considered aphrodisiac due to its shape resembling genitalia. Foods like asparagus, carrots, bananas, and parsnips have phallic features and are said to have aphrodisiac effects because of their shape. Some aphrodisiac are believed to increase sexual drive due to its origin that displays or shows aggressiveness in behavior i.e tiger penis.

In Central America the sap of the red banana and cocoa is considered aphrodisiac. Cocoa which is made to chocos and drinks, Is a symbol of romance. Aztec and Mayans paid prostitutes in cocoa beans, while the nobles always drink or ate it before going to bed with a woman. Chocolates nowadays has been widely debated because of the chemical phenylethylamine, There are some evidence that support the theory that phenethylamine release in the brain is responsible in sexual attraction and arousal. Apparently, The chemical degrades quickly by the enzyme called MAO, so this chemical is unlikely to reach the brain in full concentration when taken orally.

You might be surprise with what you will find out, fruits like figs are considered the sexiest fruits because it is shaped like female sex organs. They believe that when a man open a fig and eat it in front of a female lover it can be considered a powerful erotic act. Others like Beetroot, Spinach, hemi-porous ragweed and salty water sometimes are used for sexual stimulation. Mamey sapote, tomatoes,truffles, strawberries, celery, ginkgobiloba, lettuce, Mamajuana known as Dominican Republic alcoholic sex drink, ginseng, oyster are also foods that can be considered as aphrodisiac.

Moreover, there are known substances that increase sexual desire such as testosterone supplements that will increase libido, especially for the individuals whose sex drive where reduce (e.g menopausal women or men over 60 years old) although when tested by other groups it didn’t fared well. Yohimbine is the main alkaloid of Yohimbe, referred to as a “weak MAO inhibitor” . Yohimbine is approve in the US, but it’s pharmaceutical preparations does not indicate that the drug is for treating impotence. It helps by increasing genital blood flow and both sexual sensitivity and excitation in some people. There are known side effects like rapid pulse, sweating, and anxiety reactions for people who are susceptible.



Bremelanotide, formerly known as PT-141, is undergoing clinical trials for the treatment of sexual arousal disorder and erectile dysfunction. It’s causative effects will benefit both men and women. Preliminary results have proven the efficacy of this drug, however development was briefly suspended because of its side effect which increase blood pressure, but in year 2009 Palatin, the company developing the drug, announced positive results (none of the previous heightened blood pressure effects were observed).

Saffrons primarily chemicals responsible for giving its color  known as crocin was tested in rats and demonstrated properties of aphrodisiac. Researchers agree that Alkyl Nitrites is known in promoting and enhancing sexual response. Stimulants affecting dopamine system like cocaine and amphetamines (e.g. methamphetamine, aka crystal meth) are associated with hyperarousal and hypersexuality and long-term used may impair sexual functioning. Viagra and Levitra, are not considered aphrodisiacs because they do not have any direct effect on the libido, although increased ability to attain an erection may be interpreted as increased sexual arousal by users of these drugs.

Other studies conducted by Chicago Smell and Taste treatment and Research Foundation shows that using cucumbers found out that women were aroused by the smell of it. But most importantly if you want to optimize your own sexual responsiveness for a better sexual function, always have a balanced diet. High cholesterol level can decrease sensitivity in the genitalia, pleasure drugs (e.g Alcohol, nicotine, coffee, and marijuana) reduce sexual vitality, while prescriptions like (e.g tranquilizers, anti-hypertensive drugs, particular beta-blockers, and hormones) and recreation drugs can decrease sexual desire.

It is quite overwhelming actually that many folks feel that they will be benefited from aphrodisiac.It is really an odd thing and so irrational but people have reasons. So, it sit well on their minds. The role of reason when it comes to this folks conclusion can not refute because they state that they based it with their own experience.

No one knows and no one can be certain that aphrodisiac really works because from scientific standpoint it is all a mere belief of the users and has no scientific evidence or whatsoever. Needless to say, The fulfillment of sex drive is really complicated, but If this were true and can change people course of their withering sex life by keeping the fire burning, I think we should just leave it to their own choice

It's a beautiful Sept day, Labor day weekend around the corner.  Applied sunscreen, but I figure I should also do some addt'l yard work.  This prompted the spontaneous 5mg MT-2 reconstitution.  

I took the 5mg MT-II out of the fridge.  The chilled vial of bacteriostatic water with it.

Figured a handful of low doses to place in the freezer would be nice

5mg MT-2 = 5,000mcg

desired dose:  250mcg

1ml (same as 100 unit) insulin syringes

2ml bact water to mix/reconstitute the 5mg MT-2 peptide (small vial holds 2.5ml maximum)

each 10 units on 20 syringes should yield approx 250mcg MT-2 to be dosed subq

leave a little bit of air in the barrel when stored in the freezer.  syringes thaw quick when removed

-ended up with 21 syringes/doses.  mission accomplished.

Everyone have a great weekend~!

[The first preparative solution phase synthesis of melanotan II]

The first preparative solution phase synthesis of melanotan II
2008
Abstract:  Melanotan II is a synthetic cyclic heptapeptide used to prevent a sunlight-induced skin cancer by stimulating the skin tanning process. In this paper we report the first solution phase synthesis of the title compound. The hexapeptide sequence has been assembled by [(2+2)+1+1] scheme. After removing the orthogonal protection, a carbodiimide mediated lactamization, involving the ε-amino group of lysine and γ-carboxy group of aspartic acid, led to a cyclic intermediate. Appending N-acetylnorleucine concluded the assembly of melanotan II molecule. Protection of the lateral groups in arginine and tryptophan was omitted for atom and step economy reasons. The total synthesis of melanotan II was accomplished in 12 steps with 2.6% overall yield, affording >90% pure peptide without using preparative chromatography.

Introduction:  Development of solid phase peptide synthesis methodology, recombinant techniques for expressing peptides and proteins in microorganisms, and most recently methods for producing peptides and proteins in transgenic animals and plants, have greatly increased the availability of peptide compounds. However, the classical solution phase approach still retains its usefulness, especially when performed on a large scale. Novel powerful solvent systems combined with special protection tactics allow for even proteins to be synthesized in solution. The 136-residue human pleiotrophin and the 238-residue Aequoria green fluorescent protein are examples of such syntheses. Most of the approved peptide pharmaceuticals are currently produced by chemical synthesis in solution including oxytocin, adrenocorticotropic hormone (ACTH), desmopressin, leuprolide, goserelin, and octreotide. There is no account of a solution phase synthesis of melanotan II. The present paper describes a classical approach to this important therapeutical heptapeptide in full detail. Special attention is paid to minimum orthogonal protection of lateral functional groups to achieve maximum atom and step economy.

α-Melanocyte stimulating hormone (α-MSH, α-melanotropin) is a linear tridecapeptide of the formula Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2, involved in the regulation of skin pigmentation. Production of this hormone is stimulated by irradiation of skin by the sun’s ultraviolet rays. The α-MSH triggers the skin tanning, a process in which skin tanning cells (melanocytes) produce skin tanning pigment (melanin). Since a tan is a body’s natural protection against the ultraviolet, stimulation of melanogenesis process with exogenic hormone prior to irradiation would be a good protection against the UV-induced skin cancer. Unfortunately, the native hormone, α-MSH, was found to be too unstable in vivo to be used as a therapeutical agent. Once the tetrapeptide Ac-His-Phe-Arg-Trp-NH2 was identified as a “message sequence” of melanotropin, responsible for minimal physiological action in the frog and lizard, a wide range of analogs was synthesized by the group of V. Hruby at University of Arizona and other research groups. Modification of the α-MSH structure, including replacement of the oxidizable L-methionine with isosteric L-norleucine, replacement of L-phenylalanine with its enantiomer D-phenylalanine, and locking of the linear peptide sequence in its biologically active conformation by lactamization of the lysine ε-amino group and glutamic acid γ-carboxy group, led to a cyclic pseudopeptide analog of α-MSH with good metabolic stability and exceptional activity, known as melanotan II.

Melanotan II structure:


Results and Discussion:  To date, several accounts of solid phase synthesis of peptide 2 have been reported. A representative one, reported by Hruby and co-workers, exploited p-methylbenzhydrylamine resin (pMBHA) as the solid support and tert-butyloxycarbonyl (Boc) tactics for temporary protection of α-amino groups. The ε-amino group of lysine and the γ-carboxy group of aspartic acid, involved in lactamization, were protected as the base-cleavable Fmoc amide and Fm ester respectively. After synthesizing the peptide chain and cleavage of the base-labile protecting groups, an efficient on-resin cyclization was performed using an excess of BOP as the coupling agent. Attaching of N-Boc-norleucine, removal of Boc, acetylation of norleucine amino group with acetic anhydride, HF mediated cleavage of the resulting peptide from the polymer support, and finally purification using RP-HPLC afforded the target compound 2 in 55–60% overall yield. The yield dropped to 30% when lactamization of the linear heptapeptide sequence, synthesized on the resin, was performed in DMF solution using DPPA/K2HPO4.

Despite the high overall yield in the described solid phase approach, it has several drawbacks for the scale-up process such as the application of the highly toxic and corrosive hydrogen fluoride for cleavage of the peptide from the resin, low loading (0.30–0.35 mmol/g of resin) proved necessary for successful on-resin cyclization, and the use of excess amounts of reagents (3-fold of DIC, 2.4-fold of HOBt, etc.) on each step.

Our plan for the solution phase synthesis of peptide 2 was based on the [(2+2)+1+1] scheme for assembly of the linear hexapeptide backbone using benzyloxycarbonyl (Z) group as the temporary protection for amino acids’ N-termini (Scheme 1). The presence of aspartic acid in the target molecule poses a potential problem, due to its susceptibility to base-catalyzed cyclization to aspartimide. Esterification of γ-carboxy group does not fully protect aspartic acid from this unwanted process. Aspartimides are known to readily racemize under basic conditions, and undergo ring-opening reactions with nucleophiles, leading to formation of a variety of by-products. Thus, attack of nucleophiles yields predominately β-aspartyl peptide derivatives. This deleterious side process usually takes place when the synthesis is based on Fmoc tactics, wherein large excess secondary amines are employed for deprotection. Bearing this in mind, we postponed appending aspartic acid to a late stage of the synthesis, used only equimolar quantities of N-methylmorpholine (NMM) when a base was required for a reaction, and avoided any base-cleavable protecting groups, such as Fmoc or formyl. Despite concerns that the indole nitrogen would be susceptible to attack by tert-butyl cation generated upon Boc-group cleavage, we found that protection of indole nitrogen in tryptophan could be omitted from this process without substantial deterioration of the product yield or purity. That allowed us to decrease the overall length of the synthesis by two steps. The ε-amino group of lysine and the γ-carboxy group of aspartic acid were protected as Boc amide and tert-butyl ester respectively. Thus, all the protecting groups we used were cleavable either under acidic or hydrogenolytic conditions, releasing only volatile by-products, and all the reagents used were relatively inexpensive. These two points are very advantageous for the preparative synthesis. Another feature of our synthetic plan was to keep the side-chain functionality of arginine unprotected. In order to suppress the nucleophilic nature of the guanidine group in arginine, it was deactivated as the monohydrochloride salt over the course of 4 steps, and then as trifluoroacetate for another 2 steps. A similar method for arginine deactivation had been applied earlier in the first solution-phase synthesis of ACTH.

Melanotan II synthesis:


The assembly of the melanotan II molecule was started by coupling of Nε-Boc-lysinamide with Nα-benzyloxycarbonyltryptophan pentafluorophenyl ester to yield dipeptide 4 (Scheme 1). Cleavage of the Z protecting group afforded dipeptide 5. Reaction of arginine with Nα -Z-D-phenylalanine pentafluorophenyl ester led to a protected dipeptide 6. Since arginine exists in DMF solution in zwitterionic form, no protection for highly basic guanidine group was required. The guanidine group of arginine was deactivated for the next 4 steps by adding an equivalent of HCl (dioxane solution) to dipeptide 6. The resulting salt was coupled with another dipeptide 5 using a combination of N,N′-dicyclohexylcarbodiimide (DCC) and N-hydroxynorbornene-2,3-dicarboximide (HONb) to yield a tetrapeptide product 7. The latter was subjected to catalytic hydrogenolysis leading to tetrapeptide salt 8 with an uncapped N-terminus. Hydrochloride of tetrapeptide 8 was coupled with His(Nα-Boc,NT-Z)-OPfp in methyl alcohol to produce pentapeptide 9. Further chain elongation was effected by de-blocking the N-terminus in pentapeptide 9 and coupling of the resulting product with t-butyl Nα-benzyloxycarbonylaspartate to give hexapeptide 10. One-step cleavage of all acid-labile groups with excess of trifluoroacetic acid (TFA) yielded the tris-trifluoroacetate salt of deprotected linear hexapeptide 11 with all prerequisites for cyclization. The cyclization step, involving the ε-amino group of lysine and γ-carboxy group of aspartic acid, was performed using an 8-fold excess of DCC as the coupling agent and 1-hydroxybenzotriazole (HOBt) as a racemization suppressant. The yield of the cyclized product 12  was 31%, very close to the reported 30% yield for solution-phase cyclization of a related linear heptapeptide obtained by solid phase peptide synthesis. The total synthesis of melanotan II was concluded by coupling N-acetylnorleucine to the cyclic hexapeptide using a combination of DCC and HONb (Scheme 1). The target compound 2  obtained was >90% pure by HPLC (UV). LC-MS analysis confirmed the identity of molecular masses and retention times for the synthesized product and melanotan II purchased from a commercial source. Electrospray injection mass spectrometry of both samples demonstrated two characteristic peaks with m/z 512 and 1024, corresponding to [M+2H]2+ and [M+H]+ respectively.

Conclusion: In conclusion, we have developed the first solution phase synthesis of the cyclic heptapeptide melanotan II in 2.6% overall yield for 12 steps. Full-scale optimization of the process is being investigated and the results will be reported in due course. 
The authors are grateful to Nikolay Uvarov for spectrometric characterization of compounds and Svetlana Andronova for HPLC analysis.

by:  Bunny
A few Random ideas for a few ladies/guys I work with on diet

No Complex Carbs until you’ve been up for 4 hours
No Complex Carbs 4 hours before bed

I am pretty basic and I don’t eat fish or cottage cheese, much fruit etc.

AM on empty – TRAIN/cardio
If I train in the PM as well, Meal 5 is usually another PWO shake, for 7 meals total… eating every 2-3 hours as needed to get my meals in with at least 1.5 – 2 hours spaced b/w the last 3 meals for the day, Meal 5,6,7

Example 1
Meal 1 – Protein (minimal to NO carb)
Meal 2 – Protein + Fat
Meal 3 – Protein + Carb
Meal 4 – Protein + Carb
Meal 5 – Protein + Veggie + Fat
Meal 6 – Protein + Fat (or just Fat, ANPB has Protein too & that works by itself, base this decision on hunger and macros for the day…)

Example 2
Meal 1 – Protein (minimal to NO carb)
Meal 2 – Protein + Carb (If Ive been up for 4 hours and am STARVING)
Meal 3 – Protein + Fat
Meal 4 – Protein + Carb
Meal 5 – Protein + Veggie + Fat
Meal 6 – Protein + Fat (or just Fat, ANPB has Protein too & that works by itself, base this decision on hunger and macros for the day…)

Example 3
Meal 1 – Protein (minimal to NO carb)
Meal 2 – Protein + Carb
Meal 3 – Protein + Carb
Meal 4 – Protein + Fat
Meal 5 – Protein + Veggie + Fat
Meal 6 – Protein + Fat (or just Fat, ANPB has Protein too & that works by itself, base this decision on hunger and macros for the day…)

Example 1:
7am Meal 1 – PWO Shake - No Carb/Minimal Carb Shake (Mine has 4 g)
10am Meal 2 – 4-6 oz Lean Meat + 1 tbsp ANBP (serving of nuts)
1pm Meal 3 – 4-6 oz Lean Meat + 3-4 oz sweet potato or 1/3 cup brown Rice (measured dry) + 1-2 cups green veggie
4pm Meal 4 – Same as 3
7pm Meal 5 – 4-6 oz Lean Meat, 1-2 Cups Green Veggie
10pm Meal 6 – 2 tbsp All Natural PB or 1 serving almonds/walnuts

Example 2:
7am Meal 1 – PWO Shake - No Carb/Minimal Carb Shake (Mine has 4 g)
10am Meal 2 – 4-6 oz Lean Meat + 3-4 oz sweet potato or 1/3 cup brown Rice (measured dry) + 1-2 cups green veggie
1pm Meal 3 –4-6 oz Lean Meat + 1 tbsp ANBP (serving of nuts)
4pm Meal 4 – Same as 2
7pm Meal 5 – 4-6 oz Lean Meat, 1-2 Cups Green Veggie
10pm Meal 6 – 2 tbsp All Natural PB or 1 serving almonds/walnuts

Example 3:
7am Meal 1 – PWO Shake - No Carb/Minimal Carb Shake (Mine has 4 g)
10am Meal 2 – 4-6 oz Lean Meat + 3-4 oz sweet potato or 1/3 cup brown Rice (measured dry) + 1-2 cups green veggie
1pm Meal 3 – Same as 2
4pm Meal 4 – 4-6 oz Lean Meat + 1 tbsp ANBP (serving of nuts)
7pm Meal 5 – 4-6 oz Lean Meat, 1-2 Cups Green Veggie
10pm Meal 6 – 2 tbsp All Natural PB or 1 serving almonds/walnuts