Main Research Findings

1) Administration of Melanotan II to adult male mice was found to effectively improve autism-like behavioral deficits.

2) Abnormalities in recognition memory and anxiety levels induced by a high-fat diet were reversed in response to treatment with Melanotan II.

 

Selected Data

1) The impact of Melanotan-II on autism-like features was studied by the research team of Minakova et al, utilizing C57BL/6J mice that were housed in standard laboratory conditions, characterized by a controlled 12-hour light/dark cycle and unrestricted access to food and water. The foundational aspect of the study involved the creation of a maternal immune activation (MIA) mouse model of autism. This was achieved by administering daily intraperitoneal injections of recombinant Interleukin-6 (IL-6) to pregnant C57BL/6J mice. The dosage was precisely set at 30 µg/kg, administered between embryonic days E12.5 and E16.5, a critical period and dosage regimen validated by prior research for its effectiveness in inducing a robust inflammatory response and subsequent autism-like phenotypes in offspring. Male offspring from these MIA-exposed mothers constituted the primary experimental group, while age-matched male C57BL/6J mice served as controls. The animals used in the study were consistently between four and six months of age, ensuring developmental consistency across experimental groups [1].

Melanotan-II (MT-II), a synthetic peptide known to mimic alpha-melanocyte-stimulating hormone (α-MSH) and act as a melanocortin receptor 4 (MC4R) agonist, was the central therapeutic agent explored. To prepare MT-II for administration, a stock solution of 5 mg/ml was initially dissolved in sterile water, which was then further diluted in a 0.9% saline solution immediately prior to use. A crucial aspect of the treatment methodology was the method of delivery: intracerebroventricular (ICV) infusion. This route was specifically chosen to ensure continuous and direct access of MT-II to the central nervous system, thereby maximizing its potential therapeutic effects. The ICV delivery was facilitated by surgically implanting a cannula connected to an Alzet micro-osmotic pump. Two different pump models were utilized: model 1007D for the MIA mice and model 1002D for the control C57BL/6J mice, tailored to the specific duration of infusion required for each experimental arm.

The surgical implantation of the cannula involved anesthetizing the mice with isoflurane. The cannula was then stereotaxically positioned into the left lateral ventricle using precise coordinates relative to Bregma: 0.20 mm posterior, 0.8 mm left, and 2.5 mm ventral. Once accurately placed, the cannula was secured to the skull with cyanoacrylate adhesive. To confirm the successful and correct placement of the cannula, a post-mortem histological examination was performed. This involved perfusing the brains with phosphate-buffered saline followed by 4% paraformaldehyde, sectioning them into eight-micrometer coronal slices, and visually confirming the cannula’s location within the left lateral ventricle [1].

For MIA mice, a continuous ICV infusion of 2.5 µg per day was administered over a seven-day period. This specific ICV dose was empirically derived from a systemic equivalent of 10 mg/kg, adjusted based on the approximate brain-to-body weight ratio of a mouse, approximately 1:100. Vehicle control groups received a continuous infusion of 0.9% sterile saline. To thoroughly assess any potential side effects associated with MT-II, such as changes in weight or general behavior, normal C57BL/6J mice underwent a longer, fourteen-day continuous ICV administration of MT-II at the same daily dose. This extended duration allowed for a more comprehensive evaluation of the peptide’s safety profile in a healthy control background [1].

A comprehensive battery of behavioral tests was employed to assess various autism-like characteristics and evaluate the efficacy of MT-II treatment. All behavioral testing sessions were conducted in a highly controlled environment: a noise-insulated, low-lit room, ensuring minimal external disturbances. Each session was video-taped, and subsequent analysis was performed blindly to prevent observer bias.

The behavioral tests included the self-grooming test that served as a measure of repetitive behavior. Next was the three-chamber test that was crucial for evaluating social behavior and was divided into two distinct phases.This was followed by phase 1, sociability that included a testing apparatus consisting of a 60 cm x 40 cm Plexiglas box with three interconnected chambers. After a ten-minute habituation period within the central chamber, the test mouse was presented with two choices in the side chambers: an unfamiliar, age-, strain-, and sex-matched conspecific mouse in one enclosure and a non-social plastic bottle cap (object) in the other. The time the test mouse spent interacting with each was recorded for ten minutes, and a sociability index was calculated to quantify its preference for social interaction. Immediately after phase 1, phase 2, social novelty was initiated. The object was replaced with a novel, unfamiliar conspecific mouse. The test mouse’s preference for the familiar versus the novel conspecific was then recorded for ten minutes, allowing for the calculation of a social novelty index, which reflects the mouse’s ability to discriminate between familiar and novel social stimuli [1].

Additionally, ultrasound vocalization (USV) testing was used to measure communication deficits. Adult male MIA mice were paired with estrous female C57BL/6J mice in a clean cage placed inside a noise-limiting chamber equipped with an ultrasonic microphone. Next, the open field test assessed exploratory behavior and provided insights into anxiety-like behavior and general locomotor activity. Finally, the marble burying test assessed for repetitive behavior. Mice were housed in a cage containing approximately five centimeters of wood-chip bedding. Twenty clean glass marbles were then placed equidistantly on the bedding. After a thirty-minute period, the number of marbles that were at least 50% covered by bedding was counted.

Beyond behavioral assessments, the study also delved into neurobiological changes through oxytocin receptor autoradiography. This technique was used to measure oxytocin receptor expression in specific brain regions of both adult male MIA mice and control C57BL/6J mice. Key areas analyzed included the prelimbic cortex, anterior olfactory nucleus, nucleus accumbens, lateral septal nucleus, bed nucleus of stria terminalis, ventral reuniens, paraventricular and ventromedial hypothalamic nuclei, hippocampal CA1 and CA2/3 regions, and the central amygdaloid nucleus. The procedure involved incubating 16-micrometer coronal brain sections with an iodinated ornithine vasotocin analog, [125I] OVT, to label oxytocin receptors. Non-specific binding was determined by including unlabeled oxytocin. Autoradiograms were then generated by exposing the sections to Biomax MR film, and the resulting digital images were quantitatively analyzed using Image J software, with five brains analyzed per group [1].

2) The research team of Wekwejt et al employed zebrafish, specifically 3-5 month old longfin phenotype individuals, as a model to investigate the impact of a short-term high-fat (HF) diet on neurobehavioral outcomes and the potential ameliorating effects of MT-II. Sixty-four naive zebrafish were acquired from a local retailer and equally divided into four experimental groups, each comprising 16 fish with an equal male to female ratio. These groups included: a high-fat diet group (HF), a high-fat diet with Melanotan-II treatment group (HF + MT-II), a control diet group (C), and a control diet with Melanotan-II treatment group (C + MT-II). All fish were housed in 10 L tanks under controlled conditions of constant aeration, mechanical filtration, 27 ± 2 °C water temperature, and a pH of 7.0 ± 0.2. An automated 14:10 hour light-dark cycle, incorporating 30-minute brightening and dimming periods, was maintained [2].

The dietary intervention spanned 20 days. Fish were fed twice daily with either a control diet or a high-fat diet. The control diet consisted solely of dry fish food, while the HF diet was prepared by mixing dry fish food with lard, making up 20% of the daily portion. Each tank received a 10 mg portion of their assigned diet per day. Food intake was meticulously measured visually on a 10-point scale by a blinded experimenter to avoid bias, assessing the proportion of food consumed. To condition the zebrafish, a red light was associated with feeding, leveraging existing reports on its efficacy in associative learning studies. This conditioning ensured that the fish linked the red light cue with a reward, a factor later utilized in behavioral testing to assess recognition memory.

Melanotan-II administration occurred after the initial 20-day feeding period. The peptide was prepared as a 5 mg/ml stock solution in sterile water, then further diluted to a final concentration of 20 µmol/L in tank water. MT-II was administered percutaneously by immersing the fish in tanks containing the peptide solution for two days. This non-invasive method was chosen to minimize stress associated with handling or injection, allowing fish to be subjected to the substance without additional confounding factors. Control groups during this phase received distilled water [2].

Following MT-II administration, comprehensive behavioral assessments were conducted on Days 21 and 22. Fish were acclimatized to the testing environments one day prior to each test. The Y-maze test, implemented on Day 21, measured exploratory activity, behavior, and recognition memory. The white plastic-coated Y-maze apparatus, featuring three equally sized arms, included one arm randomly marked with a red plastic coating. Fish were individually tested for one minute, three times, with a 3-hour resting period between trials. Parameters recorded via an overhead video camera and analyzed with motion tracking software included time spent in each arm, total distance traveled, swimming velocity, latency to enter the red arm, and frequency of choosing the red arm. The one-trial memory test was performed on Day 22 and assessed exploratory behavior and recognition memory using novel and familiar objects. Two 45 L aquariums, with outer layers covered in white plastic, were used. Zebrafish were placed for 10 minutes in a tank containing two identical LEGO figures. After a 10-minute familiarization phase and a break, one LEGO figure was replaced with a novel one. The time spent exploring both objects within a 5 cm radius was manually scored, and ratio indices were calculated to determine preference for novel versus familiar objects [2].

 

Discussion

1) The results of this study performed by Mirakova et al first established that male mice exposed to MIA during gestation exhibited significant autism-like behavioral deficits compared to control C57BL/6J mice. In the three-chamber test, MIA mice demonstrated a profound impairment in social interaction, as evidenced by a significantly lower sociability index score of 6.7 ± 1.3, compared to 29.5 ± 1.3 in control mice. This indicated a strong social indifference in the MIA group. Similarly, MIA mice showed deficits in social novelty, with an index score of 8.6 ± 2.2 compared to 17.5 ± 2.1 in controls, suggesting impaired social memory or preference for novel social stimuli. Communication was also severely affected, with MIA male mice producing significantly fewer USVs during male-female interactions, emitting only 101 ± 36 USVs compared to 523 ± 117 in control mice. Regarding repetitive behaviors, MIA mice displayed significantly increased grooming time of 153.3 ± 14.3 seconds compared to control grooming times of 105 ± 11.2 seconds. However, no significant difference was observed in marble burying behavior between MIA mice and control mice demonstrating 48.9 ± 6.3% buried and 50.2 ± 5.8% buried, respectively. In summary, MIA mice consistently exhibited core behavioral features associated with Autism Spectrum Disorder (ASD), including impaired social interaction, reduced communication, and increased repetitive behaviors like grooming [1].

Following the characterization of MIA mice, the study assessed the therapeutic potential of MT-II on these autism-like features. Continuous seven-day intraventricular administration of MT-II to MIA mice resulted in a significant improvement in sociability. The sociability index scores in MT-II-treated MIA mice increased dramatically from a pre-treatment baseline of 3.1 ± 1.2 to 26.3 ± 3.9 post-treatment, reaching levels comparable to those observed in control C57BL/6J mice. In contrast, vehicle-treated MIA mice showed no significant change in their sociability scores. However, MT-II treatment did not significantly impact social novelty scores in MIA mice. This finding suggests that MT-II effectively rescued the social interaction deficits but did not fully normalize social novelty processing in the MIA model [1].

The study also investigated the effects of MT-II administration on normal C57BL/6J mice to evaluate potential side effects and alterations in baseline social behaviors. A fourteen-day continuous intraventricular infusion of MT-II in normal C57BL/6J mice did not induce any significant changes in sociability or social novelty scores compared to vehicle-treated controls. This indicates that MT-II does not disrupt normal social behavioral metrics in healthy mice under the tested conditions. Furthermore, MT-II treatment in normal C57BL/6J mice did not significantly affect exploratory behavior in the open field test or repetitive behaviors as measured by the marble burying test. However, a notable side effect was observed: MT-II-treated normal C57BL/6J mice exhibited significant weight loss, with their average weight decreasing from a baseline of 30.9 ± 1.4 grams to 26.5 ± 1.7 grams after the 14-day treatment course, a change not seen in vehicle-treated controls.

 

Figure 1: Changes in A) sociability index, B) social novelty index, C) # USVs evoked, D) % marbles buried, E) grooming time in seconds between control mice and mice treated with MT-II

Finally, the study explored differences in oxytocin receptor expression in the brains of MIA mice compared to normal C57BL/6J mice. Oxytocin receptor binding site density was found to be significantly elevated in the anterior cingulate cortex of MIA male mice compared to normal C57BL/6J mice. No other significant differences in oxytocin receptor density were observed in other high-expression brain regions, suggesting a specific upregulation of oxytocin receptors in the anterior cingulate cortex of MIA mice. These findings collectively demonstrate that MT-II is an effective agent for improving specific autism-like behavioral deficits, particularly sociability, in the adult male MIA mouse model, without significantly altering normal social behaviors, though it does cause weight loss. The observed increase in oxytocin receptor expression in the anterior cingulate cortex of MIA mice suggests a potential neurobiological mechanism underlying these effects [1].

2) The results of the study performed by Wekwejt et al revealed several significant neurobehavioral changes in zebrafish exposed to a short-term HF diet and the subsequent ameliorative effects of MT-II treatment. Initially, the researchers assessed the basic physiological parameters related to the diet. It was found that a short-term HF diet did not significantly alter the feeding behavior or body weight of the zebrafish. Both control and HF diet groups consumed similar amounts of food. Furthermore, after 20 days of feeding, there were no significant differences in body weight between fish on the HF diet weighing 0.36 g ± 0.11 and those on the control diet weighing 0.33 g ± 0.099. This indicated that the observed behavioral changes were not primarily driven by overt obesity or differences in caloric intake, but rather by the quality of the diet itself [2].

A primary finding emerged from the Y-maze test, which evaluated recognition memory. Zebrafish fed the HF diet exhibited a significant impairment in their ability to recognize and prefer the red-colored arm, which had been previously conditioned with a food reward. Specifically, the HF group spent significantly less time in the red arm compared to the control group. This memory impairment was notably reversed by MT-II treatment. When HF diet-fed fish were treated with MT-II (HF + MT-II group), they spent significantly more time in the red arm compared to the untreated HF group. This suggests that MT-II effectively restored the recognition memory abilities that were compromised by the HF diet. Intriguingly, MT-II treatment in control diet-fed fish did not induce any adverse side effects or noticeable changes in their Y-maze behavior. The overall analysis of the Y-maze data indicated a significant interaction between diet and MT-II on the recognition abilities of zebrafish [2].

Figure 2: Changes in A) percentage of time spent in the red arm, B) percentage of time spent in the start arm, and C) distance swam in mm, between the zebrafish fed a control diet or those fed a HF diet.

Beyond memory, the Y-maze test also provided insights into the zebrafish’s propensity for exploration and anxiety-like behaviors. The HF diet significantly reduced the fish’s exploratory drive, as evidenced by the HF group spending a longer period in the start arm compared to the control group. This reduced exploration signifies a lower willingness to venture into new environments. However, MT-II administration partially reversed this effect. In HF + MT-II treated fish, the time spent in the start arm decreased, and their activity increased, making their exploration patterns comparable to those of the control groups. This indicates that MT-II enhanced the exploratory abilities of the HF diet-fed zebrafish, prompting them to leave the familiar start arm and choose either the white or red arms.

The study further highlighted that the HF diet evoked anxiety in zebrafish. In the Y-maze, the HF diet group spent most of their time in the familiar start arm and swam a slightly longer distance compared to the control group. This behavior was interpreted as an anxiety response to leaving the familiar, safe environment. Similar to its effects on exploration and memory, MT-II partially reversed this anxiety. HF + MT-II treated fish showed increased total distance traveled and a greater tendency to leave the start arm and choose the red arm, becoming more similar to control fish. Notably, neither the diet nor MT-II significantly affected swimming velocity, suggesting that the changes observed were behavioral rather than a result of altered motor function [2].

Finally, the one-trial memory test, which specifically assessed exploratory behavior and recognition memory using novel and familiar objects, further corroborated these findings. The exploration time in this test was dependent on both the HF diet and MT-II treatment. The HF diet group exhibited less exploration of both novel and familiar objects compared to the control group, suggesting a general reduction in exploratory behavior in an unfamiliar environment. Consistent with the Y-maze results, MT-II administration increased the object exploration time in the HF group, bringing it closer to the levels observed in the control diet group. However, no significant differences were found in the calculated ratio for novel versus familiar object exploration, indicating that while MT-II improved general exploration, it did not significantly enhance the fine-tuned recognition memory of specific objects in this particular test. This discrepancy might be attributable to methodological factors, such as the testing duration. Overall, the results strongly indicate that a short-term HF diet induces detrimental effects on memory, exploration, and anxiety in zebrafish, and MT-II treatment effectively reverses these negative neurobehavioral changes [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Minakova E, Lang J, Medel-Matus JS, et al. Melanotan-II reverses autistic features in a maternal immune activation mouse model of autism. PLoS One. 2019;14(1):e0210389. Published 2019 Jan 10. doi:10.1371/journal.pone.0210389

[2] Wekwejt P, Wojda U, Kiryk A. Melanotan-II reverses memory impairment induced by a short-term HF diet. Biomed Pharmacother. 2023;165:115129. doi:10.1016/j.biopha.2023.115129

What is Melanotan II?

Melanotan II is a synthetic peptide shown to have many positive effects on body composition and energy homeostasis. The compound has many similarities to the peptide hormone 𝛼-melanocyte-stimulating hormone. By mimicking the effects of melanocortin peptides, not only does the compound play a role in energy homeostasis, but it primarily aids in increasing pigmentation of the skin. Due to this action melanotan II is colloquially referred to as ‘the Barbie drug” [1]

Main Research Findings

1. When administered to juvenile rats, melanotan II elicits positive effects on the hypothalamic neurocircuitry system, as well as the homeostatic conditions of body weight, NPY expressions, and energy expenditure.

2. Melanotan II acts as an appetite suppressant; administration of the compound leads to a significant reduction in body weight without a drastic caloric deficit.

 

Selected Data

1. In a study conducted by Glavas et. Al, they examined the effects of melanotan II in juvenile rats on the hypothalamic neurocircuitry system and the resulting effects on homeostasis such as body weight, NPY expression, and energy expenditure. Juvenile rats were injected twice daily at 0900 and 1700 with 3 mg/kg of melanotan II either 5-6 days postnatal, 10-11 days postnatal, or 15-16 days postnatal. Following injection the researchers determined the effects on stomach weight and brown adipose tissue uncoupling protein 1. Additionally, the researchers examined the effects injection of melanotan II had on NPY mRNA expression after twice daily injections from postnatal days 5-10 and 10-15 as well as the effects the treatment had on the activation of the central c-Fos gene which was tested 90 minutes after injection of melanotan II [2].

2. The subjects used for this study were six-month-old male F344BN rats, originally obtained from the National Institute on Aging Colony at Charles River Laboratories. The rats were placed under typical laboratory conditions and given a week to acclimate, they were then split into groups to receive a “low dose” of 0.04 μg/day of melanotan II or a “high dose” of 1 μg/day. The testing period took place over the course of 40 days. Any changes in the rats body composition were measured by time-domain nuclear magnetic resonance (TD NMR), as well as through the collection of organ and muscle tissue [3].

 

Discussion

1. It was revealed that the hypothalamic neurocircuitry system does not develop until three weeks after birth. Furthermore, neuropeptide Y (NPY) and 𝛼-melanocyte-stimulating hormone (𝛼-MSH) fibers found in the hypothalamic arcuate nucleus do not send signals downstream until approximately two weeks after birth. However, in newborn rats the 𝛼-MSH fibers are present at birth as well as melanocortin receptors.

The study concluded that in newborn rats, the activation of the c-Fos gene worked to increase body weight, while at more advanced ages the treatment led to a decrease in body weight and an increase in uncoupling protein 1. This indicates that injection with melanotan II leads to a inhibition of food intake as well as an spike in energy expenditure in the rats before the hypothalamic neurocircuitry system is fully developed [2].

 

Figure 1: analysis of c-Fos positive cells in the hypothalamus

2. The research team reported that administration of melanotan II led to induced transient anorexia for approximately 5 days. It’s important to note that anorexia was promoted with the addition of voluntary wheel running (VWR). The subjects were then split into two groups in order to measure changes in food intake or body weight that could be potentially skewed by exercise. The study reported that there was no significant difference between the two groups.

After the initial reduction in appetite, the food intake of the experimental groups increased to the same amount as the rats in the control group, however, both the high dose and low dose groups were seen to have maintained a lower level of body weight despite the increase in calories. Additionally, both the melanotan II experimental groups were seen to have a lower level of adiposity than the rats in the control group. The research team came to the conclusion that supplementation of melanotan II can lead to a reduction in body mass without a drastic restriction in caloric intake [3].

 

Figure 2: changes in body mass and food intake based off the dosage of melanotan II given.

 

Conclusion

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

Citations

[1] Massy, Maddie. “What is Melanotan-II – the drug that the TGA urges consumers to avoid?” UNSW Newsroom, 30 January 2023, https://newsroom.unsw.edu.au/news/health/what-melanotan-ii-drug-tga-urges-consumers-avoid. Accessed 18 April 2023.

[2] Maria M. Glavas, Sandra E. Joachim, Shin J. Draper, M. Susan Smith, Kevin L. Grove, Melanocortinergic Activation by Melanotan II Inhibits Feeding and Increases Uncoupling Protein 1 Messenger Ribonucleic Acid in the Developing Rat, Endocrinology, Volume 148, Issue 7, 1 July 2007, Pages 3279–3287, https://doi.org/10.1210/en.2007-0184

[3] Côté I, Sakarya Y, Kirichenko N, Morgan D, Carter CS, Tümer N, Scarpace PJ. Activation of the central melanocortin system chronically reduces body mass without the necessity of long-term caloric restriction. Can J Physiol Pharmacol. 2017 Feb;95(2):206-214. doi: 10.1139/cjpp-2016-0290. Epub 2016 Oct 19. PMID: 28051332; PMCID: PMC5572812.