draft - tspace repository: home...draft 1 title: activation of the central melanocortin system...

Draft

Activation of the central melanocortin system chronically

reduces body weight without the necessity of long-term caloric restriction

Journal: Canadian Journal of Physiology and Pharmacology

Manuscript ID cjpp-2016-0290.R2

Manuscript Type: Article

Date Submitted by the Author: 21-Aug-2016

Complete List of Authors: Côté, Isabelle; University of Florida, Pharmacology and Therapeutics Sakarya, Yasemin; University of Florida, Pharmacology and Therapeutics Kirichenko, Nataliya; University of Florida, Pharmacology and Therapeutics; North Florida/ South Georgia Veterans , Geriatric Research, Education, and Clinical Center Morgan, Drake; University of Florida, Psychiatry Carter, Christy; University of Florida, Aging and Geriatric Research Tümer, Nihal; University of Florida, Pharmacology and Therapeutics; North Florida/ South Georgia Veterans , Geriatric Research, Education, and Clinical Center Scarpace, Philip; University of Florida, Pharmacology and Therapeutics

Keyword: Central melanocortin system, MTII, body weight, lean body mass, food intake

https://mc06.manuscriptcentral.com/cjpp-pubs

Canadian Journal of Physiology and Pharmacology

Draft

1

Title: Activation of the central melanocortin system chronically reduces body weight without the

necessity of long-term caloric restriction

Running title: Melanotan II persistently reduces BW

I Cote1, Y Sakarya1,4, N Kirichenko1,4, D Morgan2, CS Carter3, N Tümer1, 4 and PJ

Scarpace1

1 Department of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida

2 Department of Psychiatry, University of Florida College of Medicine, Gainesville

3 Department of Aging and Geriatric Research, University of Florida, Gainesville, Florida

4 Geriatric Research, Education, and Clinical Center, North Florida/ South Georgia Veterans

Health System, Gainesville, Florida

University of Florida

Department of Pharmacology and Therapeutics

1200 Newell Drive

Gainesville, FL 32610

Author to whom correspondence should be sent:

Philip J. Scarpace

[email protected]

Page 1 of 36



Draft

2

Abstract

Melanotan II (MTII) is a potent appetite suppressor that rapidly promotes body weight (BW)

loss. Given the rapid loss of anorexic response upon chronic MTII treatment, most investigations

focused on the initial physiological adaptations. However, other evidence supports MTII as a

long-term modulator of energy balance that yet 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, BW was

persistently reduced in MTII groups (P < 0.01). At day 40, both MTII groups displayed lower

adiposity than aCSF animals (P < 0.01). These results show that MTII chronically reduces BW

without the requirement of long-term caloric restriction. Our study proposes that food restriction

help initiate weight loss; however, combined with a secondary pharmacological approach

preserving a negative energy balance state over time may help combat obesity.

Résumé

Le Melanotan II (MTII) est un suppresseur d’appétit efficace induisant rapidement une perte de

poids. Étant donné l’atténuation rapide de la réponse anorexigène lors d’un traitement chronique,

les chercheurs ont focalisé davantage sur les réponses physiologiques initiales. D’autres données

indiquent pourtant que le MTII serait un régulateur de la balance énergétique à long terme, mais

cette hypothèse demeure à être validée. Par conséquent, nous avons examiné les effets

chroniques du MTII sur l’homéostasie énergétique. Le MTII (faible ou forte dose) ou du liquide

cérébrospinal artificiel (aCSF; placebo) a été infusé dans le ventricule latéral du cerveau de rats

F344BN âgés de 6 mois durant 40 jours. Le MTII a réduit l’appétit de manière dose-dépendante

Page 2 of 36



Draft

3

(P < 0.05). Bien que l’apport alimentaire soit rapidement remonté au même niveau que celui du

groupe contrôle, la perte de poids induite par le MTII a persisté tout au long de l’étude. Au 40e

jour, les deux groupes traités au MTII présentaient une adiposité abdominale plus faible que les

animaux du groupe contrôle (P < 0.01). Ces résultats montrent que le MTII diminue le poids

corporel à long terme sans la nécessité d’une restriction calorique chronique. Notre étude

propose la restriction alimentaire comme un moyen efficace d’initier la perte de poids, mais

combinée à une approche pharmacologique secondaire aidant à préserver une balance

énergétique négative s’avérerait une approche intéressante pour contrer l’obésité.

Key words

Central melanocortin system, MTII, BW, LBM, and food intake.

Mots-clés

Système de la mélanocortine centrale, MTII, poids corporel, masse maigre et apport alimentaire

Page 3 of 36



Draft

4

1. 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 two 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; Monge-Roffarello et al. 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 minutes),

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 analogues is the melanotan II (MTII), a non-selective agonist of MC receptors. When

specifically administered in the brain, MTII reduces body weight (BW) 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 BW loss (Bluher et al. 2004; Pierroz et al. 2002). However,

energy consumption rises back to the pre-treatment level within two to five days of MTII

treatment (Lucas et al. 2015; Zhang et al. 2010a).

The MC3/4 receptors belong to the G protein-coupled seven transmembrane receptor (GPCR)

superfamily. Although most GPCRs are shown to quickly desensitise upon agonist exposure, the

Page 4 of 36



Draft

5

fate of activated MC3/4 receptors is unclear (Chuang et al. 1996; Ferguson 2001). Some

investigators found that ligand binding to MC3/4 receptors trigger their degradation (Shinyama et

al. 2003; Wachira et al. 2007). On the other hand, a receptor trafficking study showed that

ligand-induced internalised 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 melanocortin receptor 4 gene (Mc4r)

are resistant to LBM loss associated with tumour or renal failure (Cheung et al. 2005; Marks et

al. 2001). Furthermore, inhibition of 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

micro particles decreased LBM by 5% upon the first week of treatment (Lucas et al. 2015).

Based on these findings, we postulated that chronic MTII treatment would decrease LBM.

We previously reported that central overexpression of the α-MSH precursor, pro-

opiomelanocortin gene (Pomc), persistently reduced BW in rats (Zhang et al. 2010b). Given that

Pomc is the source of several active molecules also synthesised in the brain, including the

antagonist of the MC receptors γ-melanotropin, the contribution of central melanocortinergic

activity to BW loss could not be conclusively established.

Page 5 of 36



Draft

6

While the acute and short-term effects of MTII on feeding behaviours are extensively

documented, 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 BW and body composition regulation over the course of a 40-day treatment.

Page 6 of 36



Draft

7

2. Materials and methods

2.1 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, MA,

USA). Adult F344BN rats were selected because they display more stable BW under ad libitum

access to food compared to other rat strains (Altun et al. 2007). This feature contributes to

eliminate any weight gain artefact and to provide more reliable information on pharmacological

modulation of energy homeostasis. Two animals have been discarded from the study for post-

surgical complications, hence the unequal sample size. Upon arrival, animals were housed

individually under standard laboratory conditions (12:12 light-dark cycle; 22° C ± 2° C).

Following arrival, rats were allowed one week to acclimate to their new environment before

beginning any experiment. Rats were fed a standard rodent chow (18% kcal from fat, no sucrose,

3.1 kcal/g, diet 2018; Harlan Teklad, Madison, WI, USA). Health status, BW, 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 in compliance

with the “Guide for the Care and Use of Laboratory Animals”.

2.2 Central MTII infusion

Rats were anaesthetised with isoflurane (2-3%) and administered the analgesics

Buprenorphine (0.025 mg kg−1; SC) and Carprofen (5 mg kg−1; SC) daily starting immediately

prior the surgery. All surgical procedures were performed using aseptic techniques. All animals

were first infused artificial cerebrospinal fluid (aCSF; NaCl 148 mM, KCl 3mM, CaCl2-

2H2O 1.5 mM, MgCl2-6H2O 1.4 mM, Na2HPO4 1.5 mM, NaH2PO4 0.2mM) through a cannula

implanted into the lateral ventricle using a stereotaxic device (1.3 mm posterior to bregma and

Page 7 of 36



Draft

8

1.9 mm lateral to the midsagittal suture and to a depth of 3.5 mm). The cannula was connected to

an osmotic mini-pump (Durect Corporation, Cupertino, CA). Initially all mini-pumps contained

aCFS and these original mini-pumps were replaced twice. The first replacement was fourteen

days later, after a complete recovery from the surgery. The osmotic mini-pump 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, NJ, 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 mini-pumps. Given that the maximum duration at which

MTII was reported to be stable at 37°C is 28 days, this second pump replacement was necessary

(Jonsson et al. 2002). The day prior to the first pump replacement, before MTII treatment,

animals were separated into treatment groups based on their BW in order to obtain similar

baseline values used for longitudinal comparisons. A successful implantation was confirmed by

the initial hypophagia phase by MTII in all treated animals.

2.3 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, TX, USA). The MiniSpec quantifies three

main components of body composition; fat mass (FM), free body fluid, and lean body mass

(LBM) in grams and % by acquiring and analysing 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 analyser. The average of two scans for each animal was used as the final value.

Page 8 of 36



Draft

9

2.4 Tissue Collection, Harvesting and preparation

Rats were killed 3-6 hours after the end of their light cycle by thoracotomy and

exsanguination under anaesthesia (Isoflurane; 3.5%). Several organs and tissues were removed

and weighed (Mettler AE 163): fat depots (mesenteric, perirenal, epididymal, retroperitoneal,

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. All other tissues

and plasma samples were also kept at −80 °C until analyses were performed

2.5 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, NJ,

USA), scanned with a ChemiDoc XRS+ (BioRad, Hercules, CA, USA) and quantified using

ImageJ software. All values, including controls, were normalised 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, MA). To estimate iBAT thermogenesis

capacity, total UCP1 for iBAT was extrapolated from signal intensity divided by the number of

µg of protein 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, MA).

2.6 Voluntary physical activity

Based on our previous study ((Li et al. 2005; Zhang et al. 2010a) we expected significant

changes in BW possibly through changes in energy expenditure. In 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, PA,

Page 9 of 36



Draft

10

USA) and allowed free access to the wheel for three days (Day 20-22). Each wheel was equipped

with a magnetic switch and counter. The number of revolutions was recorded daily and meters

per day calculated.

2.7 Grip Strength

Forelimb grip strength was assessed with an automated grip strength meter (Columbus

Instruments, Columbus, OH, USA). Each rat was allowed three trials. For each trial, animals

were grasped by the tail and suspended above the device for 3 seconds. 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 to the grasping

ring. Greatest force obtained from three trials was used as the maximal grip strength value and

values were also normalized to BW in a separate analysis.

2.8 Statistical Analyses

Results are presented as means ± standard error of the mean (SEM). One-way ANOVA with

repeated measures for any longitudinal analyses and non-repeated measures for all other

comparisons were performed. When the main effect was significant (P < 0.05), a Tukey’s honest

significant difference test was applied to determine individual differences between means.

Page 10 of 36



Draft

11

3. Results

3.1 MTII transiently suppressed appetite

Consistent with our previous findings, central MTII administration induced a transient

anorexia lasting five days (Fig. 1 a). A second drop in food intake occurred at day 14 in all three

groups due to surgery for the mini-pump 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 three 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 two separate one-way ANOVA analyses (with repeated measures): one prior

to VWR (day 0-20), and the second after wheel running (day 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. 1 a). However, no statistical difference was observed after wheel running.

We also performed separate one-way ANOVA with non-repeated 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 three days (Fig. 2 a-

c). Cumulative energy intake for the duration of the study was not affected by any treatment

(data not shown).

3.2 MTII induced a persistent weight loss

3.2.1 BW prior to wheel running

At the beginning of the experiment, BW was not different across groups (Fig. 2 f). Daily

variations in BW followed the same pattern as daily food intake suggesting that caloric intake

Page 11 of 36



Draft

12

might have been the primary mechanism for the initiation of BW loss (Fig. 1 b). Due to large

variation in BW within each group, longitudinal changes could only be detected looking at delta

BW values (Fig. 1 c, d). While both MTII treatments favored individual BW loss, the high dose

induced a more significant weight loss (P < 0.01; aCSF vs MTII high dose) than did the low dose

(P < 0.05: aCSF vs MTII low dose). Although MTII appeared to affect BW in a dose-dependent

manner, there was no statistical difference between MTII high dose and MTII low dose groups.

At day 19, just prior transferring the animals in cages equipped with the voluntary wheel

running, only the high dose group displayed significant different delta BW compared to the

aCSF group (P < 0.05; Fig. 2 d).

3.2.2 BW after wheel running

Cumulative changes in BW (delta BW) with MTII treatment remained statistically significant

with both MTII treatment doses until the end of the study, although the high and low dose

became equally significant (P < 0.05: aCSF vs MTII high/low dose; Fig. 1 d). Even though final

absolute BW did not differ across groups, end-point delta BW was significantly altered by both

MTII treatments (P < 0.001; Fig. 2 e). There was no difference in daily changes in BW across

groups after day 23 (Fig. 1 b). While MTII high dose was more effective than the low dose

during the initial BW loss from day 0 to day 19 (Fig. 2 e), MTII low dose group gained less

weight (15 g) than MTII high dose treated animals (23 g) and aCSF group (26 g), though no

statistical difference was reported (separate statistical analysis using data in Fig. 1 c).

Page 12 of 36



Draft

13

3.2.3 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 MTII high dose group displayed lower FM gain than the

aCSF group (P < 0.05; Fig. 3 a). 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 (non-repeated measures) using endpoint values. This test showed

that both MTII treatments resulted in lower FM growth than aCSF treatment (P < 0.01: aCSF vs

MTII high dose; P < 0.05: vs MTII low dose; Fig. 3 b). The same trend was observed for delta

LBM (P < 0.001: aCSF vs MTII low/high dose; Fig. 3 d). Longitudinal statistical analysis of

delta LBM only reported a difference between rats treated with the high dose (P < 0.05: aCSF vs

MTII high dose; Fig. 3 c) whereas separate endpoint values analysis exposed the lower LBM

growth upon both MTII treatments (P < 0.001: aCSF vs MTII high/low dose; Fig. 3 d).

While absolute FM did not differ between groups when measured by TD-NMR (Table 1),

end-point intra-abdominal adiposity was lower in MTII groups. Indeed, sum of intra-abdominal

fat pad weights was lowered by 35% and 55% in MTII low dose and high dose groups,

respectively, relative to aCSF treated rats (P < 0.01: aCSF vs MTII low dose, and P < 0.001:

aCSF vs MTII high dose; Fig. 4 a). When individually analysed, the three fat depots, mesenteric,

perirenal, and epididymal followed this pattern (Table 2). Only retroperitoneal fat pad difference

reached the same level of significance (P < 0.001) in both MTII groups. Another finding is that

MTII also targeted interscapular brown adipose tissue (iBAT) with the lowest mass in rats

treated with MTII high dose (P < 0.01: aCSF vs MTII high dose; Fig. 4 b). A similar trend was

observed in rats treated with the low dose, however; differences did not reach significance.

Page 13 of 36



Draft

14

Although all groups displayed similar LBM (absolute values) when detected by TD-NMR,

skeletal muscle tissue mass was lower by 30% in MTII high dose compared to the aCSF group

(P < 0.05; Fig. 4 c). Nonetheless, skeletal muscle mass relative to BW and grip strength

remained unchanged (Table 3). Other than skeletal muscle, MTII treatment had no effect on the

weight of other lean tissues (Table 2).

3.3 Energy expenditure

In attempt to identify underlying mechanisms for the long-term effect of MTII on BW, we

assessed spontaneous physical activity via voluntary wheel running (VWR) distance and

thermogenesis. We employed VWR to estimate physical activity that could have contributed to

maintain a negative energy balance. Given that all three groups of rats displayed similar

voluntary exercise volume (Table 3), physical activity could not be considered as a mechanism

for long-term BW 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 amount of weight, only the high dose group

displayed significantly higher iBAT thermogenic capacity compared to the aCSF group (3-fold

increase; P < 0.01). Whereas MTII low dose group had 50% higher UCP1 protein expression,

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).

Page 14 of 36



Draft

15

4. Discussion

Chronic central delivery of MTII transiently suppressed appetite, though persistently reduced

BW, 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 (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 BW loss. However, a meta-analysis reported that more than 75% of the

dieters regain their initial BW 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 BW

loss without the necessity of chronic caloric restriction. We found that although MTII transiently

affects food intake, this molecule supports long-term BW loss, though its efficacy may decrease

over time. This finding is consistent with our previous report in that central delivery of Pomc

gene evoked a persistent BW loss (Zhang et al. 2010b). However, it was unclear whether BW

changes were due to the release of α-MSH or other substances also derived from pro-

opiomelanocortin polypeptide (POMC). The present study extends those findings by highlighting

the potential of central melanocortin system activation on chronic BW 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 BW. In support with this

Page 15 of 36



Draft

16

view, animals centrally administered MTII displayed exacerbated BW 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 control group in

order to verify whether MTII-associated weight 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 Mc4r

gene exhibit greater lean mass level than their wild type littermates (Braun et al. 2012).

Conversely, in the present study, MTII treatment suppressed LBM gain. This consequence

appears a priori as 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 concerning outcome. Nevertheless, skeletal muscle

mass relative to BW was not different 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 end-point FM measured by TD-NMR was similar across groups, rats treated with

either the low or high dose of MTII had 40% smaller intra-abdominal 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 intra-abdominal fat pad weights suggest that MTII targets white

Page 16 of 36



Draft

17

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 (ACC) 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 intra-abdominal adiposity.

Neural melanocortin system activation has been shown to reduce BW and adiposity through

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 six-day central infusion of

MTII improved iBAT thermogenesis capacity, estimated from tissue weight and UCP1 protein

content (Li et al. 2004). In the present study, iBAT thermogenesis capacity was significantly

higher following 40-day MTII high dose treatment; 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 weight 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. This data indicates that thermogenesis may

have contributed the higher weight 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

Page 17 of 36



Draft

18

low dose group also lost a significant amount of BW, this data also suggests that additional

mechanisms must have supported long-term weight loss.

At day 20, we introduced a novel VWR to evaluate whether a greater propensity to

physical activity may also explain MTII-mediated weight 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 playing a role on energy balance. We previously

observed an increase in running distance after central delivery of Pomc gene in the solitary tract.

Besides, 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 MTII treated

groups. In contrast to our expectations, all groups displayed similar activity levels. It is possible

that other substances synthesised 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 making 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 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 BW and intra-

abdominal 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 BW loss maintenance. From a clinical point of view, our study

Page 18 of 36



Draft

19

suggests that caloric restriction efficaciously initiates BW loss despite normalization of food

intake, however, combination with secondary actions that preserve a negative energy balance

state over time, such as targeting melanocortin receptors, may constitute 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 human, 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).

Conflicts of interest

None to declare.

Page 19 of 36



Draft

20

References

Altun, M., Bergman, E., Edstrom, E., Johnson, H., and Ulfhake, B. 2007. Behavioral

impairments of the aging rat. Physiol. Behav. 92(5): 911-923. doi:

10.1016/j.physbeh.2007.06.017.

Anderson, J.W., Konz, E.C., Frederich, R.C., and Wood, C.L. 2001. Long-term weight-loss

maintenance: a meta-analysis of US studies. Am. J. Clin. Nutr. 74(5): 579-584.

Bluher, S., Ziotopoulou, M., Bullen, J.W., Jr., Moschos, S.J., Ungsunan, L., Kokkotou, E., et al.

2004. Responsiveness to peripherally administered melanocortins in lean and obese mice.

Diabetes, 53(1): 82-90.

Braun, T.P., Orwoll, B., Zhu, X., Levasseur, P.R., Szumowski, M., Nguyen, M.L., et al. 2012.

Regulation of lean mass, bone mass, and exercise tolerance by the central melanocortin system.

PLoS One, 7(7): e42183. doi: 10.1371/journal.pone.0042183.

Brito, M.N., Brito, N.A., Baro, D.J., Song, C.K., and Bartness, T.J. 2007. Differential activation

of the sympathetic innervation of adipose tissues by melanocortin receptor stimulation.

Endocrinology, 148(11): 5339-5347. doi: 10.1210/en.2007-0621.

Butler, A.A., and Cone, R.D. 2002. The melanocortin receptors: lessons from knockout models.

Neuropeptides, 36(2-3): 77-84.

Butler, A.A., Kesterson, R.A., Khong, K., Cullen, M.J., Pelleymounter, M.A., Dekoning, J., et al.

2000. A unique metabolic syndrome causes obesity in the melanocortin-3 receptor-deficient

mouse. Endocrinology, 141(9): 3518-3521. doi: 10.1210/endo.141.9.7791.

Chen, A.S., Marsh, D.J., Trumbauer, M.E., Frazier, E.G., Guan, X.M., Yu, H., et al. 2000.

Inactivation of the mouse melanocortin-3 receptor results in increased FM and reduced LBM.

Nat. Genet. 26(1): 97-102. doi: 10.1038/79254.

Page 20 of 36



Draft

21

Cheung, W., Yu, P.X., Little, B.M., Cone, R.D., Marks, D.L., and Mak, R.H. 2005. Role of

leptin and melanocortin signaling in uremia-associated cachexia. J. Clin. Invest. 115(6): 1659-

1665. doi: 10.1172/JCI22521.

Chuang, T.T., Iacovelli, L., Sallese, M., and De Blasi, A. 1996. G protein-coupled receptors:

heterologous regulation of homologous desensitization and its implications. Trends Pharmacol.

Sci. 17(11): 416-421.

Fan, W., Boston, B.A., Kesterson, R.A., Hruby, V.J., and Cone, R.D. 1997. Role of

melanocortinergic neurons in feeding and the agouti obesity syndrome. Nature, 385(6612): 165-

168. doi: 10.1038/385165a0.

Ferguson, S.S. 2001. Evolving concepts in G protein-coupled receptor endocytosis: the role in

receptor desensitization and signaling. Pharmacol. Rev. 53(1): 1-24.

Huszar, D., Lynch, C.A., Fairchild-Huntress, V., Dunmore, J.H., Fang, Q., Berkemeier, L.R., et

al. 1997. Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell,

88(1): 131-141.

Jonsson, L., Skarphedinsson, J.O., Skuladottir, G.V., Watanobe, H., and Schioth, H.B. 2002.

Food conversion is transiently affected during 4-week chronic administration of melanocortin

agonist and antagonist in rats. J. Endocrinol. 173(3): 517-523.

Joppa, M.A., Gogas, K.R., Foster, A.C., and Markison, S. 2007. Central infusion of the

melanocortin receptor antagonist agouti-related peptide (AgRP(83-132)) prevents cachexia-

related symptoms induced by radiation and colon-26 tumors in mice. Peptides, 28(3): 636-642.

doi: 10.1016/j.peptides.2006.11.021.

Page 21 of 36



Draft

22

Krude, H., Biebermann, H., Luck, W., Horn, R., Brabant, G., and Gruters, A. 1998. Severe early-

onset obesity, adrenal insufficiency and red hair pigmentation caused by POMC mutations in

humans. Nat. Genet. 19(2): 155-157. doi: 10.1038/509.

Lee, M., Kim, A., Chua, S.C., Jr., Obici, S., and Wardlaw, S.L. 2007. Transgenic MSH

overexpression attenuates the metabolic effects of a high-fat diet. Am. J. Physiol. Endocrinol.

Metab. 293(1): E121-131. doi: 10.1152/ajpendo.00555.2006.

Li, G., Zhang, Y., Wilsey, J.T., and Scarpace, P.J. 2004. Unabated anorexic and enhanced

thermogenic responses to melanotan II in diet-induced obese rats despite reduced melanocortin 3

and 4 receptor expression. J. Endocrinol. 182(1): 123-132.

Li, G., Zhang, Y., Wilsey, J.T., and Scarpace, P.J. 2005. Hypothalamic pro-opiomelanocortin

gene delivery ameliorates obesity and glucose intolerance in aged rats. Diabetologia, 48(11):

2376-2385. doi: 10.1007/s00125-005-1943-8.

Lucas, N., Legrand, R., Breton, J., Dechelotte, P., Edwards-Levy, F., and Fetissov, S.O. 2015.

Chronic delivery of alpha-melanocyte-stimulating hormone in rat hypothalamus using albumin-

alginate microparticles: effects on food intake and BW. Neuroscience, 290: 445-453. doi:

10.1016/j.neuroscience.2015.01.037.

Marks, D.L., Butler, A.A., and Cone, R.D. 2002. Melanocortin pathway: animal models of

obesity and disease. Ann. Endocrinol. (Paris) 63(2 Pt 1): 121-124.

Marks, D.L., Ling, N., and Cone, R.D. 2001. Role of the central melanocortin system in

cachexia. Cancer Res. 61(4): 1432-1438.

Mizuno, T.M., and Mobbs, C.V. 1999. Hypothalamic agouti-related protein messenger

ribonucleic acid is inhibited by leptin and stimulated by fasting. Endocrinology, 140(2): 814-817.

doi: 10.1210/endo.140.2.6491.

Page 22 of 36



Draft

23

Molden, B.M., Cooney, K.A., West, K., Van Der Ploeg, L.H., and Baldini, G. 2015. Temporal

cAMP signaling selectivity by natural and synthetic MC4R agonists. Mol. Endocrinol. 29(11):

1619–1633. doi: 10.1210/me.2015-1071.

Monge-Roffarello, B., Labbe, S.M., Lenglos, C., Caron, A., Lanfray, D., Samson, P., et al.

2014a. The medial preoptic nucleus as a site of the thermogenic and metabolic actions of

melanotan II in male rats. Am. J. Physiol. 307(2): R158-166. doi: 10.1152/ajpregu.00059.2014.

Monge-Roffarello, B., Labbe, S.M., Roy, M.C., Lemay, M.L., Coneggo, E., Samson, P., et al.

2014b. The PVH as a site of CB1-mediated stimulation of thermogenesis by MC4R agonism in

male rats. Endocrinology, 155(9): 3448-3458. doi: 10.1210/en.2013-2092.

Mountjoy, K.G. 2010. Distribution and function of melanocortin receptors within the brain. Adv

Exp. Med. Biol. 681: 29-48. doi: 10.1007/978-1-4419-6354-3_3.

Novak, C.M., Burghardt, P.R., and Levine, J.A. 2012. The use of a running wheel to measure

activity in rodents: relationship to energy balance, general activity, and reward. Neurosci.

Biobehav. Rev. 36(3): 1001-1014. doi: 10.1016/j.neubiorev.2011.12.012.

Obici, S., Magrisso, I.J., Ghazarian, A.S., Shirazian, A., Miller, J.R., Loyd, C.M., et al. 2015.

Moderate voluntary exercise attenuates the metabolic syndrome in melanocortin-4 receptor-

deficient rats showing central dopaminergic dysregulation. Mol. Metab. 4(10): 692-705. doi:

10.1016/j.molmet.2015.07.003.

Pierroz, D.D., Ziotopoulou, M., Ungsunan, L., Moschos, S., Flier, J.S., and Mantzoros, C.S.

2002. Effects of acute and chronic administration of the melanocortin agonist MTII in mice with

diet-induced obesity. Diabetes, 51(5): 1337-1345.

Raposinho, P.D., White, R.B., and Aubert, M.L. 2003. The melanocortin agonist Melanotan-II

reduces the orexigenic and adipogenic effects of neuropeptide Y (NPY) but does not affect the

Page 23 of 36



Draft

24

NPY-driven suppressive effects on the gonadotropic and somatotropic axes in the male rat. J.

Neuroendocrinol. 15(2): 173-181.

Scarlett, J.M., Bowe, D.D., Zhu, X., Batra, A.K., Grant, W.F., and Marks, D.L. 2010. Genetic

and pharmacologic blockade of central melanocortin signaling attenuates cardiac cachexia in

rodent models of heart failure. J. Endocrinol. 206(1): 121-130. doi: 10.1677/JOE-09-0397.

Scarpace, E.T., Matheny, M., Strehler, K.Y., Shapiro, A., Cheng, K.Y., Tumer, N., et al. 2012.

Simultaneous introduction of a novel high fat diet and wheel running induces anorexia. Physiol.

Behav. 105(4): 909-914. doi: 10.1016/j.physbeh.2011.11.011.

Shinyama, H., Masuzaki, H., Fang, H., and Flier, J.S. 2003. Regulation of melanocortin-4

receptor signaling: agonist-mediated desensitization and internalization. Endocrinology, 144(4):

1301-1314. doi: 10.1210/en.2002-220931.

Shrestha, Y.B., Vaughan, C.H., Smith, B.J., Jr., Song, C.K., Baro, D.J., and Bartness, T.J. 2010.

Central melanocortin stimulation increases phosphorylated perilipin A and hormone-sensitive

lipase in adipose tissues. Am. J. Physiol. 299(1): R140-149. doi: 10.1152/ajpregu.00535.2009.

Teske, J.A., Billington, C.J., and Kotz, C.M. 2014. Mechanisms underlying obesity resistance

associated with high spontaneous physical activity. Neuroscience, 256: 91-100. doi:

10.1016/j.neuroscience.2013.10.028.

Voss-Andreae, A., Murphy, J.G., Ellacott, K.L., Stuart, R.C., Nillni, E.A., Cone, R.D., et al.

2007. Role of the central melanocortin circuitry in adaptive thermogenesis of brown adipose

tissue. Endocrinology, 148(4): 1550-1560. doi: 10.1210/en.2006-1389.

Wachira, S.J., Guruswamy, B., Uradu, L., Hughes-Darden, C.A., and Denaro, F.J. 2007.

Activation and endocytic internalization of melanocortin 3 receptor in neuronal cells. Ann. N. Y.

Acad. Sci. 1096: 271-286. doi: 10.1196/annals.1397.093.

Page 24 of 36



Draft

25

Wallingford, N., Perroud, B., Gao, Q., Coppola, A., Gyengesi, E., Liu, Z.W., et al. 2009.

Prolylcarboxypeptidase regulates food intake by inactivating alpha-MSH in rodents. J. Clin.

Invest. 119(8): 2291-2303. doi: 10.1172/JCI37209.

Zhang, W., and Bi, S. 2015. Hypothalamic Regulation of Brown Adipose Tissue Thermogenesis

and Energy Homeostasis. Front Endocrinol (Lausanne) 6: 136. doi: 10.3389/fendo.2015.00136.

Zhang, Y., Collazo, R., Gao, Y., Li, G., and Scarpace, P.J. 2010a. Intermittent MTII application

evokes repeated anorexia and robust fat and weight loss. Peptides, 31(4): 639-643. doi:

10.1016/j.peptides.2009.12.019.

Zhang, Y., Matheny, M., Tumer, N., and Scarpace, P.J. 2004. Aged-obese rats exhibit robust

responses to a melanocortin agonist and antagonist despite leptin resistance. Neurobiol. Aging.

25(10): 1349-1360. doi: 10.1016/j.neurobiolaging.2004.02.012.

Zhang, Y., Rodrigues, E., Gao, Y.X., King, M., Cheng, K.Y., Erdos, B., et al. 2010b. Pro-

opiomelanocortin gene transfer to the nucleus of the solitary track but not arcuate nucleus

ameliorates chronic diet-induced obesity. Neuroscience, 169(4): 1662-1671. doi:

10.1016/j.neuroscience.2010.06.001.

Page 25 of 36



Draft

26

Table 1. Body composition measurements

FM (g)

FM (%)

LBM (g) LBM (%) Fluid (g)

Fluid (%)

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 ± 0.03 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 ± 0.03 MTII high dose 85.6 ± 3.9 23 ± 0.4 216.4 ± 9.7 58 ± 0.4 30.2 ± 1.3 8.1 ± 0.05

Day 14 aCSF 100.2 ± 3.9 26 ± 0.2 220.1 ± 8.8 57 ± 0.5 33.5 ± 1.3 8.7 ± 0.03 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 ± 0.02 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 ± 0.02 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 ± 0.05 MTII low dose 86.8 ± 3.5 24.1 ± 0.5 207.6 ± 10.1 57.6 ± 0.6 30 ± 1.3 8.3 ± 0.06 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 ± 0.04 Day 40 aCSF 106 ± 3.5 26.5 ± 0.3 230.8 ± 6.7 57.8 ± 1 35 ± 1 8.8 ± 0.04 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 ± 0.06 MTII high dose 92.6 ± 4.5 25 ± 0.3 213.8 ± 10.3 57.7 ± 0.4 31.9 ± 1.5 8.6 ± 0.04 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 sacrificing the rats.

Page 26 of 36



Draft

27

Table 2. Weight of lean and fat tissues

aCSF MTII low dose MTII high dose

Brain (g) 1.9 ± 0.03 2 ± 0.04 2 ± 0.03 Liver (g) 9.8 ± 0.4 9.3 ± 0.4 9.3 ± 0.4 Kidneys (g) 2.3 ± 0.1 2.3 ± 0.1 2.4 ± 0.1 Heart (g) 1.1 ± 0.03 1.2 ± 0.03 1.1 ± 0.09 iBAT (g) 0.4 ± 0.02 0.36 ± 0.01 0.29 ± 0.02 ** Mesenteric WAT (g) 3.6 ± 0.3 2 ± 0.2 ** 1.3 ± 0.4 *** Epididymal WAT (g) 4.9 ± 0.3 3.8 ± 0.3 * 2.6 ± 0.4 *** Perirenal WAT (g) 1.1 ± 0.1 0.7 ± 0.1 * 0.4 ± 0.05 ** Retroperitoneal WAT (g) 3.5 ± 0.2 2.1 ± 0.2 *** 1.4 ± 0.1 *** Mass of dissected fat pads and lean tissues after euthanasia.

MTII treated groups significantly differed from aCSF *** P < 0.001 ** P < 0.01 * P < 0.05.

Page 27 of 36



Draft

28

Table 3. Functional tests

aCSF MTII low dose MTII high dose

Voluntary wheel running distance

Day 20 (m) 335 ± 41 291 ± 46 292 ± 45 Day 21 (m) 287 ±64 239 ± 58 335 ± 58 Day 22 (m) 349 ± 23 317 ± 26 335 ± 58 Daily average (m) 306 ± 38 287 ± 47 330 ± 22 Total distance (Day 20-22; m) 919 ±115 861 ± 140 990 ± 64

Grip strength

Max force (g) Max force (g)/BW (kg)

1.67 ± 0.14 4.46 ± 0.32

1.77 ± 0.14 4.44 ± 0.35

1.68 ± 0.17 4.56 ± 0.44

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 to sacrificing the rat.

Page 28 of 36



Draft

29

Figure captions

Fig. 1 Longitudinal changes in BW and daily food intake From day 20 to 23, rats were housed

in voluntary wheel running cages (VWR). aCSF (black circles), MTII low dose (gray circles),

and MTII high dose (open circles). Two separate one-way ANOVA (with repeated measures)

analyses were performed, the first prior to VWR (day 0‒20), and a second after VWR (day 23‒

40). a: Daily food consumption in grams during the course of the experiment. Prior to VWR,

food intake was less in MTII high dose (P < 0.05: aCSF vs MTII high dose). After VWR, food

intake was similar across groups. b: Daily changes in BW throughout the study. Prior to VWR,

daily BW changes were different in MTII high dose group (P < 0.05: aCSF vs MTII high dose).

After VWR, daily variations in BW were similar across groups. c: Evolution of BW throughout

the study. No statistical differences were found either before or after VWR. d: changes in BW

from day 0 to day 40 relative to their initial BW. Prior to VWR, both MTII treated groups were

different than the aCSF group (P < 0.01: aCSF vs MTII high dose; P < 0.05: vs low dose). After

VWR, both MTII treated groups were also different than aCSF group (P < 0.05: aCSF vs MTII

high/low dose). 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 BW, delta BW and daily food intake at different time points

a, b, and c: daily food intake in g at day 0 (prior to MTII treatment), day 19 (prior to VWR), and

day 40 (last day of MTII treatment). d, and e: delta BW at day 19, and 40. f, g, and h: BW at day

Page 29 of 36



Draft

30

0, 19, and 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).

Fig. 3 Longitudinal analyses of changes in body composition Values are individual changes in

absolute FM or LBM analysed 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 (gray circles), and MTII high dose (open circles) a: Delta FM (delta FM) was different in

MTII high dose (P < 0.05: aCSF vs MTII high dose). b: Endpoint delta FM was significantly

different with MTII (*** P < 0.01: aCSF vs MTII high dose; & P < 0.05: aCSF vs MTII low

dose). c: Delta LBM (delta LBM) was different in MTII high dose (P < 0.05: aCSF vs MTII high

dose). d: Endpoint delta LBM was significantly different with both MTII treatment (P < 0.001:

aCSF vs MTII high/low 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).

Fig. 4 Metabolic tissue weights a: intra-abdominal fat pads. b: interscapular brown adipose

tissue c: skeletal muscles (TA and soleus from the right leg). MTII treated groups (low dose or

high dose) significantly differed from aCSF ** P < 0.01, * P < 0.05. 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. 5 iBAT UCP1 protein 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).

Page 30 of 36



Draft

31

Page 31 of 36



Draft

Longitudinal changes in BW and daily food intake Fig. 1

216x149mm (300 x 300 DPI)

Page 32 of 36



Draft

BW, delta BW and daily food intake at different time points Fig. 2

181x167mm (300 x 300 DPI)

Page 33 of 36



Draft

Longitudinal analyses of changes in body composition Fig. 3

217x162mm (300 x 300 DPI)

Page 34 of 36



Draft

Metabolic tissue weights

Fig. 4

264x460mm (300 x 300 DPI)

Page 35 of 36



Draft

iBAT UCP1 protein content

Fig. 5

145x125mm (300 x 300 DPI)

Page 36 of 36



draft - tspace repository: home...draft 1 title: activation of the central melanocortin system...

Documents