Objective: To examine the effects of a cafeteria diet and a chronic treatment with melanocortin agonist (MTII) on mature weight-stable female rats.
Research Methods and Procedures: Ex-breeder Chbb:Thom rats (350 to 400 g) were divided into two groups: highly palatable food (HPF) and normal rat chow (RC). Both groups had ab libitum access to rat chow. The HPF group had access to chocolate bars, cookies, cheese, and nuts (∼20 g/d). After 21 days, the rats in each group were then divided into control and treated groups. Mini-pumps delivering saline or MTII (1 mg/kg per day) for minimally 28 days were implanted. Oxygen consumption was measured for 17 days in a second group of rats implanted with mini-pumps containing MTII (1 mg/kg per day) or saline.
Results: HPF rats ate less (<50%) rat chow than RC rats. After 20 days, the HPF group had reached a plateau and weighed significantly more (p < 0.005) than the RC group (411.7 ± 9.3 g; n = 17 vs. 365.1 ± 9.4 g; n = 16). HPF rats and RC rats receiving MTII reduced their pellet intake and body weight in the initial 2 weeks of treatment (day 14, RC-saline: −1.6 ± 1.8 g; RC-MTII, −22.5 ± 3.7 g; HPF-saline, −7.1 ± 1.7 g; HPF-MTII, −30.7 ± 4.8 g). Subsequently, pellet intake returned to pre-implantation values, although body weights remained reduced in both HPF and RC groups. Oxygen consumption was increased in rats treated with MTII.
Discussion: This suggests that MTII initially reduced body weight by limiting food intake; however, maintenance of weight is most likely due to increased energy expenditure under conditions of normal and highly palatable diets in mature animals.
An abundance of high-calorie-dense food and labor-saving devices has contributed to an epidemic of obesity (1). Obesity presents a significant burden to the general health of developed and developing nations in terms of the growing number of affected people, the serious associated morbidity, and the lack of effective long-term therapeutic interventions (1, 2, 3). Fortunately, concurrently, the details of the mechanisms controlling energy balance are becoming elucidated. The melanocortinergic pathway has been demonstrated to play a key role in control of food intake and energy homeostasis (4). Disruptions within this pathway lead to obese phenotypes. The agouti mouse is hyperphagic, hyperinsulinemic, and obese (5, 6, 7). This phenotype is believed to result from ectopic expression of a protein that acts as a competitive antagonist for MC3 and MC4 receptors (8, 9, 10). Overexpression of an agouti homolog (AGRP or ART) also results in an obese mouse (4, 9). Mice lacking the MC4 receptor have an obese phenotype that mirrors the agouti mouse (11, 12). Polymorphisms and mutations in the MC4R are also associated with obesity in humans (13, 14, 15). The MC3R knockout mice have increased adipose tissue that is believed to arise not from increased food intake but from increased metabolic efficiency (16, 17). Mutations that prevent the generation ofα-MSH from pro-opiomelanocortin, the precursor that also gives rise to adrenocorticotropic hormone and several other peptides, also result in obesity in both humans and mice (18, 19, 20). Agonists for MC4/MC3 receptors have been shown to decrease food intake and increase energy expenditure in rodents (21, 22, 23, 24, 25, 26, 27). Antagonists, such as the peptide SHU9119, stimulate food intake (7, 28, 29, 30).
The nonselective agonist, melanocortin agonist (MTII), has been demonstrated to acutely reduce food intake and increase energy expenditure when administered both centrally as well as peripherally (7, 25, 28). The compound does not reduce food intake in mice lacking the MC4 receptor, suggesting that this receptor is necessary for functioning of the melanocortinergic pathway (31, 32). Interestingly, most of the studies have investigated relatively short-term actions of MTII in growing younger animals. We, therefore, wished to examine the chronic effects of stimulation of the melancortinergic pathway in mature animals, to eliminate the confounding variable of linear growth, with an environmental induced obesity.
Research Methods and Procedures
Adult (ex-breeder, ∼6 months) Chbb:Thom female rats weighing between 340 and 400 g were housed individually and maintained on a 12:12-hour dark–light cycle beginning at 9:00 am. Fed animals were weighed between 7:00 am and 9:00am before the start of the dark cycle. Tap water and standard laboratory chow (ECOSAN 9331; Eberle Nafag, Kaiseraugust, Switzerland) were available throughout the experiment. Additionally, one group received a supplemental diet of highly palatable food (HPF). The high-calorie food consisted of chocolate, cookies, and cheese. To increase interest in the diet, each day a different food was presented to the rats. After 3 weeks, the rats were anesthetized with sodium pentobarbital (60 mg/kg, intraperitoneally) for subcutaneous placement of the osmotic mini-pumps containing vehicle (0.9% NaCl) or MTII (1 mg/kg per day; Neosystem, Strasbourg, France). The mini-pumps (Model 2ML4; 2.5 μL/h for 28 days; Alzet, Cupertino, CA) delivered MTII at an average rate of 2.42 μL/h with an average filled volume of 2258.6 μL; therefore, they could theoretically deliver drugs up to 38 days, however, delivery is only guaranteed for 28 days. The pumps were preincubated for 12 hours in saline to begin the pumping before implantation into the rats. The rats were followed for 2 weeks after the end of the guaranteed 28-day delivery period. The stability of the MTII at 37 °C was examined by high-performance liquid chromatography (data not shown).
Fed rats were killed between 8:00 am and 11:00am by CO2 inhalation, and blood was collected through heart puncture and centrifuged. Plasma was stored for measurement of insulin, leptin, triglycerides, glycerol, and glucose. Plasma insulin and leptin levels were measured using ELISA kits(Murine-Leptin #EIA-21400 and Rat-Insulin #EIA-2943; DRG Instruments GmBH, Marburg, Germany). Triglyceride, glycerol, and glucose were measured enzymatically with commercial kits (Sigma 337-B triglyceride and glycerol; Sigma 115-A glucose) obtained from Sigma(Taufkirchen, Germany).
Oxygen consumption (Vo2) was determined in an in-house-constructed open-circuit respirometer. The system allows for 12 rats to be individually studied at one time. Vo2 and Vco2 are recorded on a computer every 5 minutes and expressed as milliliters per kilogram of metabolic body size per minute. Chow fed Chbb:Thom male rats weighing between 280 and 310 g were housed individually and maintained on a reverse 12:12-hour dark–light cycle beginning at 9:00 am. Tap water and standard laboratory chow (ECOSAN 9331; Eberle Nafag) were available throughout the experiment. The rats were followed for 17 days after mini-pump implantation. During the data analysis, it was determined that the Vo2 values substantially exceeded expectations. This was traced to the oxygen sensor's sensitivity to humidity. We have subsequently built a humidity sensor into the chambers to correct the Vo2 measurements. Based on measurements made after the sensors had been installed, a correction factor was used to establish the Vo2 values. Average coefficients of variation for oxygen measurements were 8.5% and 10.7% for MTII-treated rats and saline-treated rats, respectively.
Analyses were performed using either Student's t test or one-way ANOVA with GraphPad Prism (San Diego, CA). Post-tests were conducted with Newman–Keuls multiple comparison test. Values were considered significant at p ≤ 0.05. All values are presented as means ± SEM unless otherwise stated.
The ex-breeder rats responded to the HPF diet by significantly (p < 0.005) increasing their body weights in 3 weeks(day 20; 97.5 ± 0.4 vs. 109.2 ± 0.7% start weight for normal rat chow [RC] and HPF; p < 0.001, as determined by t test; Figure 1). Rats maintained on the HPF diet seemed to have achieved a plateau in their weight gain because their body mass did not significantly increase further. Rats receiving MTII had a pronounced weight loss in the first week of administration. This weight loss was maintained during administration of the agonist (day 49: control [RC] 98.9 ± 1.1; MTII [RC] 91.9 ± 1.0; control [HPF] 110.2 ± 1.5; MTII [HPF] 99.7 ± 0.9% start weight; except control[RC] vs. MTII [HPF] all columns were significantly different from another as determined by ANOVA; Figure 1). Interestingly, the weight loss extended beyond the guaranteed 28-day delivery expectations of the pump. This may be reflective on an extended action of MTII on energy homeostasis or a result of residual administration of MTII as within our mini-pump lot a theoretical maximal delivery time of ∼38 days could be expected. A slight trend toward an increase in body weight was seen in the HPF group starting at day 38 but was not apparent in the RC rats treated with MTII.
The distribution of the HPF in the wooden bedding of the cages prevented its measurement; however, weekly pellet consumption was determined. As expected, the rats having access to the HPF consumed less of the pellets (Figure 2). Weekly consumption was ∼150 g in the control group and 40 g in the HPF group. After mini-pump implantation, all rats reduced their food intake. As determined by one-way ANOVA, rats receiving MTII significantly reduced (p < 0.01) their intake more than rats receiving saline in the first week of treatment in both RC and HPF rats. This trend was weaker in the second week of treatment (p > 0.05) and was no longer apparent in the third and fourth weeks of treatment (Figure 2). Because the rats continued to have a reduced body weight in the face of similar nutrient intake, it suggested that an increase in energy expenditure was contributing to the weight maintenance.
To examine energy expenditure directly, a second group of chow-fed rats were implanted with mini-pumps and Vo2 was followed for 17 days (Figure 3). Vo2 consumption was similar between the two groups for the first 3 days, and then the MTII-treated group increased their Vo2 relative to the rats receiving saline. Interestingly, within both groups a relative increase in Vo2 was seen after 6 days(Figure 3A). The MTII-treated rats had increased Vo2 in both the light and dark phase. Additionally, Vco2 increased proportionately and no difference in the respiratory exchange ratio was seen between the treated and control groups.
Plasma leptin levels were significantly elevated in the rats placed on the HPF diet compared with control rats (Table 1).
Table 1. Plasma parameters of rats fed a highly palatable food (HPF) diet and normal rat chow (RC) treated with MT-II
Control (RC) Mean ± SEM n = 8
MT-II (RC) Mean ± SEM n = 8
Control (HPF) Mean ± SEM n = 9
MT-II (HPF) Mean ± SEM n = 8
Control, mini-pump infusion of saline; MT-II, mini-pump infusion of MT-II (1 mg/kg/d).
Groups were statistically analyzed with one-way ANOVA (PRISM). Post-tests were conducted with Newman–Keuls multiple comparison test(PRISM). Columns with the same symbol differ significantly(p < 0.05) from one another.
1.07 ± 0.18*
0.89 ± 0.15#
3.26 ± 0.81*#
1.93 ± 0.28
1.61 ± 0.18*
1.43 ± 0.22#
2.48 ± 0.26*#
1.94 ± 0.26
122.5 ± 4.3*
120.4 ± 3.7#
149.2 ± 12.9*#§
117.3 ± 5.8§
183.7 ± 13.4
179.6 ± 15.9
176.3 ± 23.2
131.6 ± 9.2
44.3 ± 5*
37.9 ± 4.7#
74.4 ± 9.8*#§
52.6 ± 4.6§
Leptin levels decreased in the HPF animals treated with MTII. Insulin and glucose were elevated in the control HPF group. MTII treatment normalized plasma glucose values and decreased slightly the insulin values. Assayed plasma triglyceride was not different between the groups. Plasma glycerol was also elevated the in the control HPF and tended to decrease on MTII treatment.
Here we report that mature female rats can become obese when fed a highly palatable diet. The rats increased their body weight by ∼10% and maintained this increased weight for an additional 6 weeks, suggesting that a new set-point had been pharmacologically defined (4). The hypothesis of a set-point is also defended by the control rats whose weight did not significantly differ during the course of the >60-day experiment. In choosing mature rats for these experiments, the potentially confounding variable of normal growth was eliminated. Females were chosen because they typically achieve a weight plateau earlier than males (data not shown). Our results indicated that MTII initially reduces body weight by limiting food intake and that the observed maintenance of weight loss is most likely due to increased energy expenditure.
Previously others have demonstrated that MTII reduces food intake in both rats and mice (9, 24, 25, 28). Indeed even in genetically obese models the administration of the compound promotes a negative energy balance (7). These previous studies have been shorter in duration. It was interesting that the action of MTII was of similar magnitude in both the normal-fed animals and the obese rats. This suggests that stimulation of the melanocortin system is independent of the initial starting parameters and that a particular degree of melanocortinergic stimulation sets the tonus for the energy homeostasis system. In this case the amount of MTII delivered would set the system to a body weight that is 10% to 12% lower than the endogenous system. That the high-fat rats were not less or more sensitive to MTII could suggest that a deregulation of melanocortin system is not present in these rats.
Although neither a glucose tolerance test nor an insulin tolerance test were performed, the plasma values of insulin and glucose suggest that the rats were or were becoming insulin-resistant. The high plasma leptin levels in the HPF control group are reflected by an increased fat mass. MTII reduced the leptin values, and coupled with a decrease in body weight would support a reduction in fat mass. Along with positive influence on body weight, MTII tended to improve the plasma parameters associated with impaired glucose homeostasis. It must also be noted that the values were measured minimally 4 days after the administration of the MTII would have been completed. The slight trend toward an increased body weight in the last 4 days of the experiment suggests that the MTII had ceased to function in the HPF rats.
The MC4R receptor is considered the main melanocortin receptor involved in mediating energy homeostasis (31, 32). Transgenic mice lacking the receptor are obese and fail to demonstrate an acute response to administration of MTII (31, 32). These two lines of evidence, supported by additional experiments, have until recently excluded a role of other melanocortin receptors playing a role in energy homeostasis. One of the additional lines of evidence excluding a role for the MC3R was the demonstration that in a specific rodent model of energy deregulation only changes in MC4 receptor mRNA were demonstrated, whereas the MC3R mRNA was unaltered (33). However, gene targeting of the MC3R suggests that it also can play a role in influencing body weight via regulation of metabolic efficiency (16).
Down-regulation of the MC4R in the obese state was seen as a response to increased agonistic signaling of α-MSH (i.e., desensitization) (33). In our model, the response of the HPF MTII rats would not suggest a significant desensitization of the MC4R system. Nevertheless, the RC group reached a plateau quicker than did the HPF group when treated with MTII; this could represent a difference in initial sensitivity of the system or a difference in response to reduced food intake. However, a lean animal would be less likely to resist weight loss than an animal with ample fat stores.
We noted that the effect of MTII on food intake was transient, lasting for only the first 2 weeks, but the effects on body weight persisted for 6 weeks. Our demonstration of increased Vo2 would suggest that increased energy expenditure was responsible for the maintenance of the reduced body weight. Because MTII is a mixed agonist, one hypothesis could be that initial signaling at the start of administration of MTII was predominately through the MC4R receptor, reducing food intake and concomitantly increasing energy expenditure (7, 28, 32). However, with continued treatment the MC4R became desensitized so that the weight maintenance effects were mediated though MC3R (32). Nevertheless, experiments with MC4R−/− mice clearly demonstrate that short-term effects of MTII require a MC4 receptor (17, 31). Chronic treatment of the MC3R−/− mice and chronic treatment with selective melanocortinergic agonists should help in addressing this question. More probably, similar to the distinct effects of leptin on energy homeostasis and reproduction (34), there could be two distinct pathways for MC4R signaling. Such integration and separation of the food intake pathway and the energy expenditure pathway would be advantageous in regulating energy balance.
In summary we have demonstrated that in a mature rodent model of obesity, chronic treatment with a melanocortin agonist results in sustained weight loss. The persistent effects of MTII on body weight reduction suggest that compounds acting at MC4R and MC3R could be effective therapeutic agents against obesity and its co-morbidity.
No outside funding/support was provided for this study. We thank S. Hecht, D. Nitz, and A. Wendl for their excellent technical assistance.