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. Author manuscript; available in PMC: 2018 Feb 1.
Published in final edited form as: Can J Physiol Pharmacol. 2016 Oct 19;95(2):206–214. doi: 10.1139/cjpp-2016-0290

Activation of the central melanocortin system chronically reduces body mass without the necessity of long-term caloric restriction

I Côté 1, Y Sakarya 2, N Kirichenko 3, D Morgan 4, CS Carter 5, N Tümer 6, PJ Scarpace 7
PMCID: PMC5572812  NIHMSID: NIHMS896559  PMID: 28051332

Abstract

Melanotan II (MTII) is a potent appetite suppressor that rapidly reduces body mass. Given the rapid loss of anorexic response upon chronic MTII treatment, most investigations have focused on the initial physiological adaptations. However, other evidence supports MTII as a long-term modulator of energy balance that remains to be established. Therefore, we examined the chronic effects of MTII on energy homeostasis. MTII (high or low dose) or artificial cerebrospinal fluid (aCSF) was infused into the lateral ventricle of the brain of 6-month-old F344BN rats (6–7/group) over 40 days. MTII suppressed appetite in a dose-dependent manner (P < 0.05). Although food intake promptly rose back to control level, body mass was persistently reduced in both MTII groups (P < 0.01). At day 40, both MTII groups displayed lower adiposity than the aCSF animals (P < 0.01). These results show that MTII chronically reduces body mass without the requirement of long-term caloric restriction. Our study proposes that food restriction helps initiate mass loss; however, combined with a secondary pharmacological approach preserving a negative energy balance state over time may help combat obesity.

Keywords: central melanocortin system, MTII, body mass, lean body mass, food intake

Introduction

The central melanocortin system is a critical regulator of energy homeostasis (Butler and Cone 2002; Lee et al. 2007; Li et al. 2004, 2005; Marks et al. 2002). Pharmacological and genetic studies have revealed pivotal roles for the central melanocortin pathway in the regulation of satiety and energy expenditure (Butler et al. 2000; Chen et al. 2000; Fan et al. 1997; Huszar et al. 1997; Krude et al. 1998; Mizuno and Mobbs 1999). The optimal function of the central melanocortin system requires 2 receptors expressed in the brain: the melanocortin receptors 3 and 4 (MC3/4 receptors) (Mountjoy 2010). These receptors modulate the activity of a broad neural network regulating appetite along with numerous metabolic pathways, including thermogenesis and lipolysis (Monge-Roffarello et al. 2014a, 2014b; Shrestha et al. 2010). The α-melanocyte-stimulating hormone (α-MSH) is the endogenous agonist ligand for the MC3/4 receptors. Given the very short half-life of α-MSH (~10 min), more stable synthetic melanocortinergic peptides have been employed to clarify the biological roles of MC3/4 receptor signalling (Lucas et al. 2015; Wallingford et al. 2009). One of the best suitable analogs is melanotan II (MTII), a nonselective agonist of MC receptors. When specifically administered in the brain, MTII reduces body mass and robustly suppresses appetite (Pierroz et al. 2002; Zhang et al. 2004). Indeed, central administration of MTII induces a consistent anorexic response ranging from 30% to 50% reduction in food intake, considered a primary mechanism of MTII-induced body mass loss (Blüher et al. 2004; Pierroz et al. 2002). However, energy consumption rises back to the pretreatment level within 2 to 5 days of MTII treatment (Lucas et al. 2015; Zhang et al. 2010a).

The MC3/4 receptors belong to the G protein-coupled receptor (GPCR) superfamily. Although most GPCRs are shown to quickly desensitize upon agonist exposure, the fate of activated MC3/4 receptors is unclear (Chuang et al. 1996; Ferguson 2001). Some investigators found that ligand binding to MC3/4 receptors triggers their degradation (Shinyama et al. 2003; Wachira et al. 2007). On the other hand, a receptor trafficking study showed that ligand-induced internalized MC4 receptors are recycled at the cell membrane within minutes and that ligands remain bound to the receptor over several endocytosis–exocytosis cycles (Molden et al. 2015). Thus, it is unclear how chronic in vivo MC3/4 receptor stimulation affects physiological responses over time.

The central melanocortin system has been proposed as a regulator of lean body mass (LBM) (Braun et al. 2012; Lucas et al. 2015). In fact, mice lacking the melanocortin 4 receptor gene (Mc4r) are resistant to LBM loss associated with tumour or renal failure (Cheung et al. 2005; Marks et al. 2001). Furthermore, inhibition of the MC4 receptor protects against LBM wasting under a wide range of catabolic conditions (Cheung et al. 2005; Joppa et al. 2007; Scarlett et al. 2010). Interestingly, Mc4r−/− mice have higher lean mass and muscle strength compared to their wild-type littermates (Braun et al. 2012). In agreement with these studies, chronic delivery of α-MSH microparticles decreased LBM by 5% after the first week of treatment (Lucas et al. 2015). Based on these findings, we postulate that chronic MTII treatment will decrease LBM.

We previously reported that central overexpression of the α-MSH precursor, proopiomelanocortin gene (Pomc), persistently reduced body mass in rats (Zhang et al. 2010b). Given that Pomc is the source of several active molecules also synthesized in the brain, including the antagonist of the MC receptors, γ-melanotropin, the contribution of central melanocortinergic activity to body mass loss could not be conclusively established.

The acute and short-term effects of MTII on feeding behaviours are extensively documented, but very little is known about the long-term physiological effects. To this end, we centrally infused MTII to determine the effectiveness of melanocortin system activation in long-term body mass and body composition regulation over the course of a 40-day treatment.

Materials and methods

Animals

Six-month-old male Fisher 344 × Brown Norway (F344BN) rats (n = 21), were obtained from the National Institute on Aging Colony at Charles River Laboratories (Wilmington, Massachusetts, USA). Adult F344BN rats were selected because they display more stable body mass under ad libitum access to food compared to other rat strains (Altun et al. 2007). This feature helps eliminate any mass gain artifact and to provide more reliable information on pharmacological modulation of energy homeostasis. Two animals were discarded from the study for postsurgical complications, hence the unequal sample size. Upon arrival, animals were housed individually under standard laboratory conditions (12 h/ light – 12 h dark cycle; 22 °C ± 2 °C). Following arrival, rats were allowed 1 week to acclimate to their new environment before beginning any experiment. Rats were fed a standard rodent chow (18% kcal from fat, 0% sucrose, 3.1 kcal/g; diet 2018, Harlan Teklad, Madison, Wisconsin, USA). Health status, body mass, and food intake were monitored daily throughout the duration of the study. All experimental protocols were approved by the University of Florida’s Animal Care and Use Committee, and were in compliance with the Guide for the Care and Use of Laboratory Animals.

Central melanotan II infusion

Rats were anesthetized with isoflurane (2%–3%) and administered the analgesics buprenorphine (0.025 mg/kg; SC) and carprofen (5 mg/kg; SC) daily starting immediately prior the surgery. All surgical procedures were performed using aseptic techniques. All animals were first infused with artificial cerebrospinal fluid (aCSF; NaCl 148 mmol/L, KCl 3 mmol/L, CaCl2–2H2O 1.5 mmol/L, MgCl2–6H2O 1.4 mmol/L, Na2HPO4 1.5 mmol/L, NaH2PO4 0.2 mmol/L) through a cannula implanted into the lateral ventricle using a stereotaxic device (1.3 mm posterior to bregma, 1.9 mm lateral to the midsagittal suture, and to a depth of 3.5 mm). The cannula was connected to an osmotic minipump (Durect Corporation, Cupertino, California, USA). Initially, all minipumps contained aCFS and these original minipumps were replaced twice. The first replacement was 14 days later, after a complete recovery from the surgery. The osmotic minipump was replaced through a small incision (1 cm) and fresh aCSF or MTII (0.04 μg/day or 1 μg/day diluted in aCSF; Genscript, New Jersey, USA) was administered for 40 days. The high dose was selected based on literature to induce a maximal response and the low dose was determined by a dose response curve (data not shown). To ensure MTII activity throughout the duration of the experiment, MTII was refreshed at day 14 by replacing all minipumps. Given that the maximum duration at which MTII was reported to be stable at 37 °C is 28 days (Jonsson et al. 2002), this second pump replacement was necessary. The day prior to the first pump replacement, before MTII treatment, animals were separated into treatment groups based on their body mass to obtain similar baseline values to be used for longitudinal comparisons. A successful implantation was confirmed by the initial hypophagia phase by MTII in all treated animals.

Determination of body composition using time-domain nuclear magnetic resonance

Body composition was determined using time-domain nuclear magnetic resonance (TD-NMR; Minispec, Bruker Optics, The Woodlands, Texas, USA). The MiniSpec quantifies 3 main components of body composition — fat mass (FM), free body fluid, and LBM — in grams by acquiring and analyzing TD-NMR signals from all protons in the sample area. Scans were acquired by placing the rats into a cylindrical restrainer that was inserted into the analyzer. The mean of 2 scans for each animal was used as the final value.

Tissue collection, harvesting, and preparation

Rats were euthanized 3–6 h after the end of their light cycle by thoracotomy and exsanguinated under anaesthesia (isoflurane, 3.5%). Several organs and tissues were removed and weighed (Mettler AE 163): fat depots (mesenteric, perirenal, epididymal, retro-peritoneal, and interscapular brown adipose tissue; iBAT) and muscles (soleus and tibialis anterior). Tissues were stored at −80 °C until the Western blot analyses were performed.

Western analyses

Protein lysates were separated on a SDS–PAGE gel and transferred to nitrocellulose membranes. Immunoreactivity was detected with ECL Prime (GE Healthcare, Piscataway, New Jersey, USA), scanned with a ChemiDoc XRS+ (BioRad, Hercules, California, USA), and quantified using ImageJ software. All values, including controls, were normalized to the mean of the aCSF group and reported as a percentage. Immunoreactivity was assessed with antibodies specific to uncoupling protein 1 (UCP1; Abcam, Cambridge, Massachusetts, USA). To estimate iBAT thermogenesis capacity, total UCP1 for iBAT was extrapolated from signal intensity divided by amount of protein (μg) loaded on the gel and subsequently multiplied by the total amount of protein (μg). To ascertain even loading across samples, beta-tubulin was also probed on the same blot (Abcam, Cambridge, Massachusetts, USA).

Voluntary physical activity

Based on our previous study (Li et al. 2005; Zhang et al. 2010a), we expected significant changes in body mass possibly through changes in energy expenditure. In an attempt to specify the effect of melanocortin system activation on spontaneous physical activity, rats were placed into cages equipped with Nalgene Activity Wheels (1.081 m/revolution; Fisher Scientific, Pittsburgh, Pennsylvania, USA) and allowed free access to the wheel for 3 days (days 20–22). Each wheel was equipped with a magnetic switch and counter. The number of revolutions was recorded daily and metres per day were calculated.

Grip strength

Forelimb grip strength was assessed with an automated grip strength meter (Columbus Instruments, Columbus, Ohio, USA). Each rat was allowed 3 trials. For each trial, animals were grasped by the tail and suspended above the device for 3 s. Subsequently, the rat was gently placed on the grip ring and allowed to grasp it with its forepaws. The rat’s body was then aligned horizontally, and quickly pulled by the tail until the forelimb grip was broken. The mean force in grams was calculated with an electronic pull strain gauge located directly on the grasping ring. Greatest force obtained from 3 trials was used as the maximal grip strength and values were also normalized to body mass in a separate analysis.

Statistical analyses

Results are presented as means ± standard error of the mean (SEM). One-way ANOVA with repeated measures for any longitudinal analyses and nonrepeated measures for all other comparisons were performed. When the main effect was significant (P < 0.05), a Tukey’s honestly significant difference test was applied to determine individual differences between means.

Results

Melanotan II transiently suppressed appetite

Consistent with our previous findings, central MTII administration induced a transient anorexia lasting 5 days (Fig. 1a). A second drop in food intake occurred at day 14 in all 3 groups due to surgery for the minipump replacement. We previously observed that Pomc gene overexpression in the brain enhanced voluntary wheel running (VWR) activity by 20% (Zhang et al. 2010b). To verify whether MTII would increase physical activity to the same extent, we measured running distance for 3 days. The introduction of VWR has been shown to evoke a robust anorexic response (Scarpace et al. 2012). To eliminate any exercise-induced anorexia bias, we performed 2 separate one-way ANOVA analyses (with repeated measures): one prior to VWR (days 0–19) and the second after wheel running (days 23–40). Prior to VWR assessment, rats infused with the high-dose MTII consumed significantly less food than those in the aCSF group (P < 0.05; Fig. 1a). However, no statistical difference was observed after wheel running. We also performed a separate one-way ANOVA with nonrepeated measures to compare daily food intake prior to MTII treatment (day 0), in the middle (day 19), and at the end (day 40) of the study. No significant difference in food consumption was reported for these 3 days (Figs. 2a–2c). Cumulative energy intake for the duration of the study was not affected by any treatment (data not shown).

Fig. 1.

Fig. 1

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Longitudinal changes in body mass and daily food intake. From day 20 to 22, rats were housed in voluntary wheel running (VWR) cages. aCSF (black circles), MTII low dose (grey circles), and MTII high dose (open circles). Two separate one-way ANOVA (with repeated measures) analyses were performed: before VWR (days 0–20) and after VWR (days 23–40). (a) Daily food consumption in grams during the course of the experiment. Prior to VWR, food intake was less in the high-dose MTII group (MTII high dose vs. aCSF: P < 0.05). After VWR, food intake was similar across groups. (b) Daily changes in body mass throughout the study. Prior to VWR, daily body mass changes were more variable in the high-dose MTII group (MTII high dose vs. aCSF: P < 0.05). After VWR, daily variations in body mass were similar across groups. (c) Evolution of body mass throughout the study. No statistical differences were found either before or after VWR. (d) Changes in body mass (delta body mass) from day 0 to day 40 relative to initial body mass. Prior to VWR, both MTII-treated groups were lower than the aCSF group (MTII high dose vs. aCSF: P < 0.01; MTII low dose vs. aCSF: P < 0.05). After VWR, both MTII-treated groups were also lower than the aCSF group (MTII high/low dose vs. aCSF: P < 0.05). Some error bars cannot be visualized due to overlapping symbols. All values represent the mean ± SEM of 6 rats per group (MTII high dose and aCSF) or 7 rats per group (MTII low dose).

Fig. 2.

Fig. 2

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Body mass, delta body mass, and daily food intake (in grams) at different time points. Daily food intake at (a) day 0 (prior to MTII treatment), (b) day 19 (prior to VWR), and (c) day 40 (last day of MTII treatment). Delta body mass at (d) day 19 and (e) day 40. Body mass at (f) day 0, (g) day 19, and (h) day 40. All values represent the mean ± SEM of 6 rats per group (MTII high dose and aCSF) or 7 rats per group (MTII low dose). *, P < 0.05; ***, P < 0.001.

Melanotan II induced a persistent reduction in body mass

Body mass prior to wheel running

At the beginning of the experiment, body mass did not differ across groups (Fig. 2f). Daily variations in body mass followed the same pattern as daily food intake suggesting that caloric intake might have been the primary mechanism for the initiation of body mass loss (Fig. 1b). Due to large variation in body mass within each group, longitudinal changes could only be detected looking at delta body mass values (Figs. 1c and 1d). Both MTII treatments favoured individual body mass loss (aCSF vs. MTII high dose: P < 0.01; aCSF vs. MTII low dose: P < 0.05). The higher dose of MTII had higher body mass loss than the lowest dose of MTII but this was not statistically significant. At day 19, just before transferring the animals into cages equipped with the VWR, only the high-dose group displayed significantly different delta body mass compared to the aCSF group (P < 0.05; Fig. 2d).

Body mass after wheel running

Cumulative changes in body mass (delta body mass) with MTII treatment remained statistically significant with both MTII treatment doses until the end of the study (aCSF vs. MTII high/low dose: P < 0.05; Fig. 1d). Even though body mass did not differ at day 0, day 19, or day 40 (Figs. 2f–2h), endpoint delta body mass was significantly altered by both MTII treatments (P < 0.001; Fig. 2e). There was no difference in daily changes in body mass across groups after day 23 (Fig. 1b). The MTII high dose was more effective than the low dose during the initial body mass loss from day 0 to day 19 (Fig. 2e); however, from day 19 to 40, the low-dose group regained less body mass (15 g) than the high-dose group (23 g) and the aCSF group (26 g), although no statistical difference was reported (separate statistical analysis using data in Fig. 1c).

The central melanocortin system: a novel regulator of LBM?

The TD-NMR analyses indicated that neither FM nor LBM differed between groups at any time point (Table 1). However, when longitudinal changes in individual rats were computed (absolute values relative to day 0), the high-dose MTII group displayed lower FM gain than the aCSF group (P < 0.05; Fig. 3a). Because longitudinal statistical analyses (one-way ANOVA with repeated measures) may be too stringent to detect all physiological responses, we performed a separate one-way ANOVA (nonrepeated measures) using endpoint values. This test showed that both MTII treatments resulted in lower FM gain than aCSF treatment (aCSF vs. MTII high dose: P < 0.01; aCSF vs. MTII low dose: P < 0.05; Fig. 3b). The same trend was observed for delta LBM (aCSF vs. MTII low/high dose: P < 0.001; Fig. 3d). Longitudinal statistical analysis of delta LBM showed a difference between rats treated with the high dose (aCSF vs. MTII high dose: P < 0.05; Fig. 3c), whereas separate endpoint values analysis indicated both MTII doses reduced LBM growth (aCSF vs. MTII high/low dose: P < 0.001; Fig. 3d).

Table 1.

Body composition measurements of fat mass (FM), lean body mass (LBM), and free body fluid.

FM
LBM
Fluid
g % g % g %
Day 0
 aCSF 86.5±3.2 23.4±0.3 211.5±5.4 57.4±0.3 30.2±0.9 8.2±0.0
 MTII low dose 82.3±3.7 22.5±0.3 211.2±9.2 57.6±0.3 29.5±1.2 8.1±0.0
 MTII high dose 85.6±3.9 23.0±0.4 216.4±9.7 58.0±0.4 30.2±1.3 8.1±0.1
Day 14
 aCSF 100.2±3.9 26.0±0.2 220.1±8.8 57.0±0.5 33.5±1.3 8.7±0.0
 MTII low dose 89.1±4.3 24.2±0.3 213.5±9.1 58.1±0.5 31.2±1.4 8.5±0.0
 MTII high dose 90.3±3.9 24.7±0.3 209.1±9.1 57.2±0.4 31.2±1.3 8.6±0.0
Day 25
 aCSF 93.3±3.6 24.6±0.2 213.6±7.6 56.3±0.2 31.5±1.1 8.3±0.1
 MTII low dose 86.8±3.5 24.1±0.5 207.6±10.1 57.6±0.6 30.0±1.3 8.3±0.1
 MTII high dose 82.8±4.5 23.3±0.4 202.7±9.5 57.2±0.2 29.1±1.4 8.2±0.0
Day 40
 aCSF 106.0±3.5 26.5±0.3 230.8±6.7 57.8±1.0 35.0±1.0 8.8±0.0
 MTII low dose 93.6±4.9 25.4±0.4 211.6±9.4 57.4±0.5 31.8±1.5 8.6±0.1
 MTII high dose 92.6±4.5 25.0±0.3 213.8±10.3 57.7±0.4 31.9±1.5 8.6±0.0
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Note: Values are means ± SEM of 6 animals/group (aCSF and MTII high dose) or 7 animals/group (MTII low dose). Measurements at day 40 were performed immediately prior to euthanasia.

Fig. 3.

Fig. 3

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Longitudinal analyses of changes in body composition. Values are individual changes in absolute fat mass (FM) or lean body mass (LBM) analyzed by TD-NMR. Values are changes (in grams) relative to day 0 and were compared by one-way ANOVA with repeated measures. aCSF (black circles), MTII low dose (grey circles), and MTII high dose (open circles). (a) Delta FM was lower for MTII high dose (MTII high dose vs. aCSF: P < 0.05). (b) Endpoint delta FM was significantly different with both MTII treatments (MTII low dose vs. aCSF: *P < 0.05, MTII high dose vs. aCSF: ***P < 0.001). (c) Delta LBM was lower in MTII high dose (MTII high dose vs. aCSF: P < 0.05). (d) Endpoint delta LBM was significantly lower with both MTII treatments (***P < 0.001: MTII high/low dose vs. aCSF). All values represent the mean ± SEM of 6 rats per group (MTII high dose and aCSF) or 7 rats per group (MTII low dose).

Although absolute FM did not differ between groups when measured by TD-NMR (Table 1), endpoint intraabdominal adiposity was lower in the MTII groups. Indeed, sum of intraabdominal fat pad mass was lowered by 35% and 55% in the low- and high-dose MTII groups, respectively, relative to the aCSF-treated rats (aCSF vs. MTII low dose: P < 0.01; aCSF vs. MTII high dose: P < 0.001; Fig. 4a). When individually analyzed, 3 fat depots (mesenteric, perirenal, and epididymal) followed this pattern (Table 2). Another finding was that MTII also targeted iBAT with the lowest mass in rats treated with high-dose MTII (aCSF vs. MTII high dose: P < 0.01; Fig. 4b). A similar trend was observed in rats treated with the low dose; however, differences did not reach significance. Although all groups displayed similar LBM (absolute values) when detected by TD-NMR, skeletal muscle tissue mass was lower by 30% in the high-dose MTII group compared to the aCSF group (P < 0.05; Fig. 4c). Nonetheless, skeletal muscle mass relative to body mass and grip strength remained unchanged (Table 3). Other than skeletal muscle, MTII treatment had no effect on the mass of other organs (Table 2).

Fig. 4.

Fig. 4

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Metabolic tissue mass of (a) intraabdominal fat pads, (b) interscapular brown adipose tissue (iBAT), and (c) skeletal muscles (tibialis anterior and soleus from the right leg). MTII-treated groups (low dose and high dose) significantly differed from aCSF (*P < 0.05, **P < 0.01, ***P < 0.001). iBAT mass was significantly lower in MTII high dose **P < 0.01: MTII high dose vs. aCSF, ††P < 0.01: MTII low dose vs. MTII high dose. All values represent the mean ± SEM of 6 rats per group (MTII high dose and aCSF) or 7 rats per group (MTII low dose).

Table 2.

Mass of lean and fat tissues.

aCSF MTII
low dose		 MTII
high dose
Brain (g) 1.93±0.03 2.00±0.04 2.00±0.03
Liver (g) 9.81±0.40 9.26±0.44 9.25±0.36
Kidneys (g) 2.29±0.08 2.32±0.09 2.35±0.13
Heart (g) 1.13±0.03 1.23±0.08 1.09±0.06
White adipose tissue (g)
 Mesenteric 3.56±0.30 1.98±0.19** 1.74±0.39***
 Epididymal 4.88±0.29 3.53±0.33** 2.99±0.43***
 Perirenal 1.10±0.09 0.63±0.10* 0.49±0.12***
 Retroperitoneal 3.51±0.23 1.93±0.22*** 1.75±0.34***
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Note: Values are means ± SEM. Mass of dissected fat pads and lean tissues after euthanasia. MTII-treated groups significantly differed from aCSF

*

P < 0.05,

**

P < 0.01,

***

P < 0.001.

Table 3.

Functional tests.

aCSF MTII
low dose		 MTII
high dose
Voluntary wheel running distance (m)
 Day 20 335±41 291±46 292±45
 Day 21 287±64 239±58 335±58
 Day 22 349±23 317±26 335±58
 Daily mean 306±38 287±47 330±22
 Total distance (day 20–22) 919±115 861±140 990±64
Grip strength
 Max force (g) 1.67±0.14 1.77±0.14 1.68±0.17
 Max force (g)/body mass (kg) 4.46±0.32 4.44±0.35 4.56±0.44
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Note: Values are means ± SEM of 6 animals/group (aCSF and MTII high dose) or 7 animals/group (MTII low dose). Measurement of grip strength was performed at day 40 immediately prior euthanasia.

Energy expenditure

To identify underlying mechanisms for the long-term effect of MTII on body mass, we assessed spontaneous physical activity via VWR distance and thermogenesis capacity. We employed VWR to estimate physical activity that could have contributed to maintaining a negative energy balance. Given that all 3 groups of rats displayed similar voluntary exercise volume (Table 3), physical activity could not be considered as a mechanism for long-term body mass loss in the current experiment. We then examined iBAT thermogenesis, a potential peripheral metabolic pathway known to be affected by short-term central MTII infusion. Total iBAT thermogenesis capacity was estimated based on total tissue content of UCP1 protein. Although both groups lost significant mass, only the high-dose MTII group displayed significantly higher iBAT thermogenic capacity compared to the aCSF group (3-fold increase; P < 0.01). The low-dose MTII group had 50% higher UCP1 protein expression, but this increase did not reach the level of significance (Fig. 5). The same pattern was observed when calculating UCP1 relative to beta-tubulin signal intensity (data not shown).

Fig. 5.

Fig. 5

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Interscapular brown adipose tissue uncoupling protein 1 (UCP1) content. Beta-tubulin image is displayed to show even loading across sample but was not used to calculate total UCP1 content. MTII-treated rats (high dose) significantly differed from aCSF (**P < 0.01). All values represent the mean ± SEM of 6 rats per group (MTII high dose and aCSF) or 7 rats per group (MTII low dose).

Discussion

Chronic central delivery of MTII transiently suppressed appetite, although persistently reduced body mass, indicating that the central melanocortin system may be a long-term regulator of energy homeostasis without the necessity of maintaining caloric restriction over time. Furthermore, we found that MTII decreased LBM within the first weeks of brain infusion, suggesting a role of the central melanocortin system in the regulation of LBM.

Currently, more than two-thirds of Americans are overweight with a body mass index (BMI) > 25, of which half are considered obese (BMI > 30). Because of obesity-associated health issues and morbidity, novel strategies to treat or prevent obesity have become a high priority. Feeding restriction is one of the most commonly employed methods to generate a negative energy balance and achieve body mass loss. However, a meta-analysis reported that more than 75% of the dieters regain their initial body mass within 5 years (Anderson et al. 2001). Therefore, alternative approaches to caloric restriction are attractive strategies to combat obesity. From this perspective, the present study uncovered the role of the melanocortin system for long-term body mass loss without the necessity of chronic caloric restriction. We found MTII transiently affects food intake and also supports long-term body mass loss, although its efficacy may decrease over time. This finding is consistent with our previous report that central delivery of the Pomc gene evoked a persistent body mass loss (Zhang et al. 2010b). However, it was unclear whether body mass changes were due to the release of α-MSH or other substances also derived from proopiomelanocortin polypeptide (POMC). The present study extends those findings by highlighting the potential of central melanocortin system activation on chronic body mass loss. Given the rapid attenuation of anorexia, we did not exclude that enhanced energy expenditure may have significantly contributed to the long-term melanocortin regulation of body mass. In support with this view, animals centrally administered MTII displayed exacerbated body mass loss compared to those pair-fed and treated with a vehicle solution (Raposinho et al. 2003). Furthermore, energy expenditure measured by oxygen consumption was significantly higher in mice centrally administered MTII compared to those pair-fed receiving vehicle (Pierroz et al. 2002). Future studies should address the long-term effects of MTII using pair-fed animals as a control group to verify whether MTII-associated mass loss is independent of the initial hypophagia.

Longitudinal analyses of body composition indicated that MTII disrupted both fat and lean mass growth. In agreement with our findings, a relationship between melanocortin signalling and LBM using a knockout model was recently proposed (Braun et al. 2012). Mice lacking the Mc4r gene exhibit greater lean mass levels than their wild-type littermates (Braun et al. 2012). Conversely, in the present study, MTII treatment suppressed LBM gain. This consequence appears to be a deleterious side effect. However, MTII treatment did not alter lean-to-FM ratio (data not shown), indicating that central delivery of MTII does not negatively alter body composition. However, muscle wasting is a concerning outcome. Nevertheless, skeletal muscle mass relative to body mass did not differ across groups. Future studies would be necessary to determine whether the effects of MTII on skeletal muscle mass would eventually result in lower muscle strength, metabolic defects, or other health issues.

Whereas endpoint FM measured by TD-NMR was similar across groups, rats treated with either the low or high dose of MTII had 40% smaller intraabdominal fat pads compared to animals administered aCSF. This discrepancy can be explained by the fact that TD-NMR measurements do not discriminate between lipids located in the adipose tissue and lipids located in other body compartments, such as cell membranes, ectopic tissues, or even within the intestinal lumen. The differences in intraabdominal fat pad mass suggest that MTII targets white adipose tissues. This interpretation is consistent with previous findings showing that central administration of MTII stimulates sympathetic drive to white adipose tissues (Brito et al. 2007). We also have previously reported that 30-day central administration of MTII was associated with a 13-fold enrichment in phosphorylated acetyl-CoA carboxylase in retroperitoneal and epididymal fat depots, indicative of reduced lipogenesis and (or) stimulated fatty acid oxidation (Zhang et al. 2010a). We postulated that the present MTII treatment resulted in comparable physiological responses; thus, enhanced fatty acid oxidation and diminished lipogenesis in white adipose tissues may have contributed to the reduction of intraabdominal adiposity.

Neural melanocortin system activation has been shown to reduce body mass and adiposity by promoting BAT thermogenesis (Zhang and Bi 2015). A knockout study has shown that the melanocortin regulation of BAT thermogenesis is, at least to a certain extent, mediated by the MC4 receptor (Voss-Andreae et al. 2007). We previously found that a 6-day central infusion of MTII improved iBAT thermogenesis capacity, estimated from tissue mass and UCP1 protein content (Li et al. 2004). In the present study, iBAT thermogenesis capacity was significantly higher following 40-day treatment with high-dose MTII; however, the higher UCP1 protein content did not reach statistical significance in the low-dose group. Another indicator of increased thermogenic activity is the reduction in iBAT mass that can be explained by triglyceride depletion and protein enrichment. In fact, animals treated with the high dose of MTII had twice as much iBAT protein as control animals (data not shown). Similar to iBAT UCP1, animals treated with the low dose displayed higher iBAT protein content than aCSF-infused animals, but the difference did not reach statistical difference. These data indicate that thermogenesis may have contributed to the higher mass loss in animals treated with the high dose and (or) a certain activation threshold may be required to enhance iBAT thermogenesis capacity. Given that the low-dose group also lost a significant amount of body mass, these data also suggest that additional mechanisms must have supported long-term mass loss.

At day 20, we introduced a novel VWR to evaluate whether a greater propensity to physical activity may also explain MTII-mediated mass loss. Although VWR does not delineate physical activity per se, a positive correlation between running distance and ambulatory activity has been established in untrained rats (Teske et al. 2014). Physical activity is another component of energy expenditure that plays a role in energy balance. We previously observed an increase in running distance after central delivery of the Pomc gene in the solitary tract. In addition, MC3/4 blockade by SHU9119 has been shown to reduce physical activity (Obici et al. 2015). Based on these findings, we anticipated higher physical activity volume in the MTII-treated groups. In contrast to our expectations, all groups displayed similar activity levels. It is possible that other substances synthesized from POMC were the underlying cause of increased activity in Pomc-overexpressing animals. On the other hand, there is a wide range of potential physiological compensations that make comparison difficult between a receptor agonist and antagonist. Other differences in experimental design, such as duration of the study, timing/duration of assessment, species, and rat strain, might also explain the lack of effect. Because VWR also activates the brain reward system (Novak et al. 2012), which could also be affected by MTII treatment, we do not reject the possibility that MTII may have an effect on physical activity level. A more accurate assessment of general activity (i.e., ambulatory activity) should be performed in future studies.

In summary, this study demonstrates that MTII chronically reduces body mass and intraabdominal adiposity without the necessity of maintaining low caloric intake. Given the very low success rate of dietary approaches, targeting additional pathways to changes in feeding behaviours would support body mass loss maintenance. From a clinical point of view, our study suggests that caloric restriction efficaciously initiates body mass loss despite normalization of food intake; however, a combination with secondary actions that preserve a negative energy balance state over time, such as targeting melanocortin receptors, may be a valuable tool to combat obesity. Given the complexity of targeting neural receptors, future studies in our lab will aim at examining whether long-term peripheral administration of MTII, a more translational approach for humans, would yield similar physiological responses. Additional experiments could also assess whether intermittent peripheral administration may extend MTII efficacy over a longer period of time.

Acknowledgments

This work was supported by a grant from the National Institutes of Health, USA (DK091710).

Footnotes

Conflict of interest

The authors declare that there is no conflict of interest associated with this work.

Contributor Information

I. Côté, Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL, USA

Y. Sakarya, Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL, USA; Geriatric Research, Education, and Clinical Center, North Florida/South Georgia Veterans Health System, Gainesville, FL, USA

N. Kirichenko, Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL, USA; Geriatric Research, Education, and Clinical Center, North Florida/South Georgia Veterans Health System, Gainesville, FL, USA

D. Morgan, Department of Psychiatry, University of Florida, Gainesville, FL, USA

C.S. Carter, Department of Aging and Geriatric Research, University of Florida, Gainesville, FL, USA

N. Tümer, Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL, USA; Geriatric Research, Education, and Clinical Center, North Florida/South Georgia Veterans Health System, Gainesville, FL, USA

P.J. Scarpace, Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL, USA

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