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Endocrinology Vol. 144, No. 4 1513-1523
Copyright © 2003 by The Endocrine Society

ARTICLE

Differential Role of Melanocortin Receptor Subtypes in Cachexia

Daniel L. Marks, Andrew A. Butler, Renn Turner, Gregor Brookhart and Roger D. Cone

Department of Pediatric Endocrinology (D.L.M., G.B.), Veterans Affairs Medical Center (R.T.), Vollum Institute (R.D.C.), Oregon Health and Science University, Portland, Oregon 97201; and Pennington Institute (A.A.B.), Baton Rouge, Louisiana 70808

Address all correspondence and requests for reprints to: Roger D. Cone, Vollum Institute Mailcode L474, Oregon Health Sciences University, 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97201. E-mail: cone{at}ohsu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Animals and humans respond to starvation with a complex neuroendocrine response that ultimately leads to an increase in appetite, a sparing of lean body mass (LBM) and burning of fat, and an overall decrease in basal metabolic rate. In contrast, cachexia is a pathological state of malnutrition associated with many infections and chronic diseases, wherein appetite is diminished concomitant with an increase in metabolic rate, and a relative wasting of LBM. In previous studies, we demonstrated that anorexia and weight loss in mouse cachexia models induced by lipopolysaccharide (LPS) administration and by tumor growth are ameliorated by central melanocortin-4 (MC4) receptor (MC4-R) blockade. In contrast to the results seen with MC4 blockade, melanocortin-3 (MC3) receptor knockout (MC3-RKO) mice show illness-induced anorexia and weight loss with LPS administration and with cytokine administration, and they have similar decreases in mobility. Both MC3-RKOs and MC4-RKOs have an intact corticosterone response and fever with LPS injection. In tumor models, we show that MC4-RKO mice resist the loss of LBM brought about by tumor growth, whereas MC3-RKO animals show enhanced tissue wasting. These data underscore the importance of central melanocortin signaling in weight homeostasis and demonstrate differential effects of MC3-R and MC4-R blockade on the development of cachexia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CACHEXIA, OR DISEASE-ASSOCIATED wasting, is a common occurrence in cancer and infectious disease. This devastating state of malnutrition is brought about by a synergistic combination of a dramatic decrease in appetite and an increase in metabolism of fat mass (FM) and lean body mass (LBM). The severity of cachexia in many illnesses is the primary determining factor in both quality of life and in eventual mortality (1, 2). There is currently no effective pharmaceutical treatment, and this disorder of energy homeostasis is poorly understood. This has led to intense investigation of cachexia and the proposal of numerous hypotheses regarding its etiology. At this point, most authors suggest that ILs and other cytokines may be the primary peripheral signal leading to cachexia. Several investigators have proposed that these factors are released during inflammation and malignancy and act on the central nervous system (CNS) to alter the release and function of a number of key neurotransmitters, thereby altering both appetite and metabolic rate (1, 3, 4, 5).

Support for this mechanism has been derived from experiments using a purified product found in the cell wall of gram-negative bacteria known generically as lipopolysaccharide (LPS). Injections of this compound reliably produces anorexia in experimental animals (6, 7), due in large part to the ability of LPS to potently stimulate the release of numerous cytokines from immune cells in the periphery and glia within the CNS (8, 9, 10, 11). Although cytokine receptors are found throughout the CNS and brain vasculature, it is plausible that the hypothalamus represents an important site of action of cytokines on feeding, metabolism, and other illness responses. In response to LPS injections, IL-1 expression is induced in circumventricular organs in the hypothalamus, and in hypothalamic glia and neurons (12, 13, 14, 15). Within the hypothalamus, attention has focused on the arcuate nucleus (ARC) because this nucleus plays a central role in feeding and metabolism and has ready access to the circulation due to its proximity to circumventricular structures (16). Several investigators have demonstrated that LPS injections cause a prompt and sustained up-regulation of IL-1 expression in this nucleus (12, 13, 14, 15). Furthermore, the receptor for IL-1 has been found in neurons located in this nucleus, as well as in hypothalamic nuclei known to receive innervation from neurons located in the arcuate (17, 18). Markers of neuronal activation, including the expression of c-fos and suppressor of cytokine signaling-3 (SOCS-3), are also seen in neurons within the ARC in response to cytokine administration, although the identity of these neurons is currently unknown (19, 20).

One candidate target for cytokine action in the ARC are the proopiomelanocortin (POMC) neurons (21). This propeptide precursor is cleaved into several peptides, including {alpha}- and {gamma}-MSH. {alpha}-MSH binds to central melanocortin receptors [including the type 4 melanocortin (MC4-R) receptor, MC4-R], where it is thought to produce a tonic inhibitory tone on food intake. Acute central administration of MC4-R agonists produces an inhibition of feeding and increased energy expenditure (22, 23), ultimately leading to a reduction in body weight (24, 25). Prolonged agonist administration leads to sustained loss of weight indicating that these receptors do not readily desensitize with prolonged stimulation (26, 27). In contrast, blocking melanocortin signaling causes animals to accumulate excess lean tissue mass and FM (22, 28, 29). Thus, activation of the MC4-R recapitulates the cardinal features of cachexia, leading to our hypothesis that POMC neurons transduce cachexigenic stimuli.

To test this hypothesis, we and others have sought out models of disease that are thought to be mediated, at least in part, by cytokines. Several studies have now shown that animals with pharmacologic or genetic blockade of MC4-R signaling are resistant to the anorexic and metabolic effects of LPS administration and also demonstrate decreased illness behavior after LPS exposure (30, 31), or central IL-1ß administration (32). In another recent study, Huang et al. (31) investigated the impact of central administration of {alpha}-MSH or a melanocortin receptor antagonist on LPS-induced anorexia and fever in rats. In this study, the investigators found a significant potentiation of the suppressive effects of LPS on food intake with administration of {alpha}-MSH, and a reversal of LPS-induced anorexia with antagonist administration. In general, it appears that LPS- and cytokine-induced fever is not altered by melanocortin antagonism, but all other features of cachexia tested so far are partially or completely reversed by this treatment (30, 32, 33). Recently, we have extended these results and demonstrated a role of melanocortin receptors in transducing the prolonged metabolic derangement observed in experimental cancer. At this point, it has been clearly established that hypophagia and carcass weight loss induced by tumor growth can be prevented and even partially reversed by blockade of the MC4-R (30, 34). In the present study, these results have been confirmed by serial dual energy x-ray absorbtometry (DEXA) scans, which also demonstrate that the tumor-bearing MC4-RKO animals continue to accumulate both LBM and FM in the face of tumor growth, whereas wild-type (WT) control animals lost both LBM and FM under identical conditions.

While the importance of the MC4-R in maintaining appropriate metabolic homeostasis is clear, the role of the melanocortin-3 (MC3) receptor (MC3-R) remains unclear. MC3-R knockout (MC3-RKO) mice have increased FM and reduced LBM and may in fact be slightly hypophagic relative to WT controls (35, 36). The effect of MC3-R deficiency on feeding and resting oxygen consumption is complex. Remarkably, despite the increased adiposity seen in the MC3-RKO mice, the animals do not exhibit increased food intake or weight gain, even on a high-fat chow that is known to induce hyperphagia in MC4-RKO animals (35, 37). It has also not been possible to demonstrate differences in resting basal or total metabolic rate in this model (35). However, MC3-RKO mice do show impairment in fatty acid oxidation, particularly when placed on a high-fat diet, and this may lead to both insulin resistance and increased body fat deposition (35, 36). It is also likely that reduced energy expenditure contributes to the obesity phenotype. Male MC3-RKO mice exhibit an approximately 50% reduction in wheel-running behavior compared with WT littermates and show obvious decreases in home cage activity as well (35, 36). In the present study, we demonstrate that MC3-RKO mice suffer enhanced cachexia with LPS challenge and with tumor growth. These data lend support to the hypothesis that the MC3-R functions, in part, to limit anorexigenic POMC neuronal activity by acting as an inhibitory autoreceptor on POMC neurons (38). We propose that the enhanced cachexia observed in the MC3-RKO animal results from a loss of this autoinhibitory function on POMC neurons, thereby increasing the overall stimulation of the MC4-R during illness.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
MC3-RKO mice and their WT controls were derived from the original C57BL/6Jx129 colony (35) maintained within the Vollum Institute that had been bred seven generations into the C57BL/6J strain and maintained as homozygous lines. MC4-RKO mice, described previously (29), were bred 10 generations into the same C57BL/6J strain. All mice were raised group housed in a 12-h light, 12-h dark cycle. For studies measuring food intake, mice were housed individually and food intake estimated by measuring the weight of powdered food remaining in feeding chambers designed to maximize spill capture. Mice were weaned at 21 d and allowed ad libitum access to powdered Laboratory Rodent Diet (Purina Mills, St. Louis, MO) that was weighed and replaced daily. In all studies, male animals aged 6–10 wk were used. In the tumor models, male animals, age 6 wk, were used at the start of each experiment. All studies were conducted according to the NIH Guide for the Care and Use of Laboratory Animal and approved by the Animal Care and Use Committee of the Oregon Health and Science University.

LPS administration
Each animal was handled daily for a minimum of a consecutive 5 d before the initiation of the experiment, simulating the restraint used during the injection of the compounds. LPS (Escherichia coli 055:B5, Sigma, St. Louis, MO) was dissolved in normal saline and administered ip. Knockout (KO) mice and littermate controls had basal feeding monitored for 2 d, and then during each 12-h period following an ip saline injection (1700 h) before injection of 100 µg/kg of LPS. For the MC3-RKO dose response curves, similar injections were performed with doses of 50, 100, and 200 µg/kg of LPS.

Tumor models
Lewis lung carcinoma cells were maintained as a primary culture in DMEM with 10% fetal bovine serum as recommended by the supplier (American Type Culture Collection, Manassas, VA). Lewis lung carcinoma tumor cells were harvested during exponential growth of the culture, washed in Hanks’ balanced salt solution, and 1 x 106 cells were injected sc into the upper flank of the mice. Sham-injected animals received an implant of a similar amount of heat-killed tumor cells. In all cases, the time of appearance of a tumor mass was noted in the log, and all experimental animals were found to have a palpable tumor within 5 d of the start of the experiment. At the time the mice were killed, tumors were dissected away from surrounding tissue and weighed. Gross examination of organs did not reveal the presence of any observable metastasis. Trunk blood was collected at the time they were killed for measurement of serum leptin with a rat leptin RIA kit (Linco Research, Inc., Manassas, VA).

Motor activity and feeding activity
Animals were housed individually in metabolic cages equipped with a running wheel (Mini-Mitter Co., Sunriver, OR). The metabolic cages allowed telemetric monitoring of circadian rhythms as assessed via multiple physiological parameters. The wheel revolutions were quantified by recording the magnetic switch closures of a magnet placed on the revolving wheel. Movement and body temperature were recorded via monitoring of telemetry from implants in the abdominal cavity of the experimental mice. These implants were placed under halothane anesthesia under sterile conditions, with 10 d of recovery time before the start of the experiments.

Body composition
Body composition was determined at the start and the end of the experiments by DEXA (PIXImus mouse densitometer, MEC Lunar Corp., Minster, OH). The instrument was calibrated at the start of each recording session with a murine calibration standard. In the first experiment, the results of the final DEXA scan were confirmed by standard chemical body composition analysis (39). All animals were fasted for 12 h before DEXA analysis to minimize the effect of ingested food on the DEXA analysis.

Indirect calorimetry
Oxygen consumption (VO2) and carbon dioxide production (VCO2) were simultaneously determined by indirect calorimetry (Oxymax, Columbus Instruments, Columbus, OH). Mice were housed in separate chambers at 24 ± 1 C. Mice were first acclimatized to the chambers for 2 d. Measurements were recorded for 4–8 h during the middle of the light cycle (1100–1600 h). Samples were recorded every 3 min with the room air reference taken every 30 min and the air flow to chambers 500 ml/min. Basal oxygen consumption was determined for individual curves as the average of the lowest plateau regions corresponding to resting periods. Total oxygen consumption was the result of all samples recorded corresponding to periods of movement as well as inactivity. The respiratory quotient was calculated as the molar ratio of VO2:VCO2.

Hormone analysis
Corticosterone was measured in triplicate with a standard commercial RIA kit (Linco Research, Inc., St. Charles, MO). For the corticosterone analysis, animals were injected with LPS (250 µg/kg) or saline control and blood was collected at 8 h post injection.

Statistical methods
Differences between feeding and activity curves in all experiments were analyzed by two-way, repeated measures ANOVA with time and treatment as the measured variables. Final tumor and body weights, and body composition by DEXA were analyzed by Student’s t test when two groups were included, or one-way ANOVA with post hoc analysis when three groups were included. Correlation between DEXA body composition and chemical carcass analysis was compared by linear regression. Data sets were analyzed for statistical significance using either the PRISM software package (GraphPad Software, Inc., San Diego, CA) for ANOVA with repeated measures, or in Excel (Microsoft Corp., Redmond, WA) using Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Normal fever and cortisol response to LPS in MCR-KO mice
Although MSH has been shown to have antipyretic properties mediated via central melanocortin receptors, other studies have provided evidence that blockade of the MC3-R and MC4-R does not affect the LPS-mediated febrile response (33, 40, 41). Furthermore, the MC3-RKO and MC4-RKO mice are not know to have gross defects in activation of the hypothalamic-pituitary-adrenal (HPA) axis and may actually be hypersensitive to certain stressors (36, 42). Thus, we hypothesized that both the MC3-RKO and the MC4-RKO animals would have normal fever and normal activation of the HPA axis in response to LPS. In the first study, groups of MC4-RKO and WT control animals (n = 10) were implanted with Mini-mitter transmitters and allowed to recover for 1 wk. The basal body temperature was then recorded for 48 h, with ip saline injections given at 1200 h on each day. On d 3, the groups of animals received LPS injections (250 µg/kg), and body temperature was observed for the following 24 h. No differences were seen between groups after saline treatment or after treatment with LPS (repeated measures ANOVA, P = 0.9). A comparison of average body temperature for 4 h after a saline injection or after LPS injection in MC4-RKO mice is shown in Fig. 1AGo. In a similar experiment, groups of 10 MC3-RKO mice and control WT mice were implanted with transmitters and allowed to recover for 1 wk. Saline was injected daily at 0800 h for a consecutive 2 d, and LPS (250 µg/kg) was injected at 0800 h on d 3. Again, no differences were observed between groups in body temperature either after saline injection, or after LPS injection. As with the MC4-RKO mice, the MC3-RKO mice had a significant increase in body temperature after LPS injection, and this increase was identical to that found in the WT controls. The average body temperature for the first 4 h after injection of saline or after LPS in MC3-RKO mice is shown in Fig. 1BGo. To measure baseline corticosterone, groups of 10 WT and 10 MC4-RKO animals were individually housed for 2 wk. During wk 2, the animals were handled daily in a fashion designed to mimic the handling required for ip injection. On the experimental day, animals were injected at 0800 h with saline (n = 3 per genotype), or LPS (250 µg/kg, n = 7 per genotype). After 4 h, animals were killed by quickly removing them from the housing room with decapitation occurring within 30 sec. No differences were observed between groups after saline injection (MC4-RKO 168 ± 36 ng/dl vs. WT 169 ± 43 ng/dl; n = 3, P = 0.98, Fig. 1CGo), or after LPS injection (MC4-RKO 810 ± 43 ng/dl vs. WT 699 ± 59 ng/dl; n = 7, P = 0.15, Fig. 1CGo). An identical study was performed on groups of 11 MC3-RKO mice and WT controls. In this case, a relatively small but significant increase in corticosterone was observed after saline injection in the MC3-RKO mice relative to the WT mice (MC3-RKO 275 ± 5 ng/dl vs. WT 121 ± 23 ng/dl; n = 6, P < 0.05, Fig. 1DGo), but there was no difference between groups after LPS injection (MC3-RKO 666 ± 60 ng/dl vs. WT 671 ± 68 ng/dl; n = 6, P = 0.98, Fig. 1DGo).



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  		Figure 1. Normal fever and activation of the HPA axis after LPS injection in MC3-RKO and MC4-RKO mice. A, Average body temperature in WT or MC4-RKO mice between 1200 and 1600 h on consecutive days, following injection of saline or LPS at 1200 h (*, P < 0.05). B, Average body temperature in MC3-RKO mice between 0800 and 1200 h on consecutive days following injection of saline or LPS at 0800 h (*, P < 0.05). C, Corticosterone release in response to saline or LPS injections (250 mcg/kg) are similar in MC4-RKO and WT control mice. D, Corticosterone release in response to LPS injection (250 mcg/kg) are similar in MC3-RKO and WT control mice. In response to saline injection, MC3-RKO mice show a significantly greater release of corticosterone than WT controls.

 
Normal starvation response in MC3-RKO mice
To demonstrate the specificity of our results with models of cachexia, we first demonstrated that MC3-RKO mice did not have gross disturbances in the metabolic response to fasting. MC3-RKO mice lost similar amounts of weight to WT animals with 24 h of fasting (KO 18.4 ± 0.8% vs. WT 17.7 ± 0.5, P = 0.5, Fig. 2AGo) and had similar rebound hyperphagia in the 24 h following the fast (24 h intake normalized to body surface area: KO 103 ± 4 g/kg0.75 vs. WT 115 ± 7 g/kg0.75, P = 0.2, not shown; percent of basal feeding KO 154 ± 11% vs. WT 156 ± 11%, P = 0.9, Fig. 2BGo). Furthermore, the MC3-RKO animals showed a normal decrease in V02 and a decrease in the respiratory quotient with fasting, indicating that they respond to this metabolic stress by decreasing metabolic rate and increasing fatty acid oxidation (Fig. 2Go, C and D). Indeed, after 3 d of restricting food intake to 5.1 Kcal/d, WT animals lost significantly more weight than MC3-RKO animals, both when measured as absolute weight loss, and as percentage of initial weight (total weight loss: KO 2.5 ± 0.2 g vs. WT 3.3 ± 0.2 g, P < 0.05; percent loss: KO 7.4 ± 0.7% vs. WT 10.4 ± 0.9%. P < 0.05, KO n = 8, WT n = 7). As with the shorter absolute fast, the MC3-RKO animals also showed a normal decrease in VO2 in response to the 3-d period of food restriction (% basal VO2 KO 86 ± 4% vs. WT 77 ± 4%, P = 0.11).



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  		Figure 2. Normal response to fasting in MC3-RKO mice. A, MC3-RKO and WT mice lose similar amounts of weight in response to a 24-h fast (NS, not significant; n = 10, P = 0.5). B, MC3-RKO and WT mice have normal rebound hyperphagia after fasting (NS, not significant; P = 0.9). C, Resting energy expenditure is decreased to a similar extent in fasting MC3-RKO and WT mice (P > 0.5 during both time intervals). D. MC3-RKO and WT mice have a similar decrease in respiratory quotient, indicating increased utilization of fats for fuel (ANOVA, P = 0.9).

 
MC3-RKO mice show enhanced cachexia with LPS challenge
Feeding and weight loss.
To test the role of the MC3-R in cachexia, MC3-RKO mice were tested for their response to LPS injections. Parameters monitored included food and water intake, wheel-running activity, and weight gain. Six-week-old male MC3-RKO mice were weight matched with WT littermate controls (KO 19.5 ± 0.5 vs. WT 20 ± 0.6, P = 0.5). Basal feeding and feeding after ip saline was not significantly different between groups (KO 4.0 ± 0.3 vs. WT 5.5 ± 0.2, P = 0.13), although there was a consistent trend toward relative hypophagia in MC3-KO animals, as reported previously (35). In response to 250 µg/kg of LPS, MC3-RKO animals showed enhanced anorexia when expressed both as total intake (24 h intake KO 1.16 ± 0.2 g/24 h vs. WT 1.75 ± 0.2 g/24 h, P < 0.05, n = 15, Fig. 3AGo) and as percent basal intake (KO 27 ± 4% vs. WT 43 ± 5%; n = 15, P < 0.05, Fig. 3BGo) in response to LPS. The MC3-RKO animals also lost more weight than WT controls over the 60-h experimental period (24-h weight change KO -2.1 ± 0.2 vs. WT -1.5 ± 0.1, n = 15, P < 0.05, Fig. 3CGo).



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  		Figure 3. Food intake and wheel-running behavior in response to LPS treatment in MC3-RKO mice. A and B, MC3-RKO mice have enhanced anorexia in response to LPS (250 mcg/kg) both when measured as absolute food intake (A), and as a percentage of basal intake (B; *, P < 0.05). C, MC3-RKO mice have increased weight loss after treatment with LPS (*, P < 0.05). D and E, MC3-RKO mice decrease wheel-running behavior after LPS to a similar extent as the WT animals, and show a similar recovery over the following 48 h. The dark phase is indicated by the black bars below the x-axis in D. Each data point represents a 60-min average for wheel turns. The MC3-RKO mice are in red, the MC3-RWT mice are in black.

 
Wheel running and home cage movement.
Wheel-running activity was less in the MC3-RKO animals at baseline, as previously reported (35) and was unchanged after saline injection (Fig. 3DGo). Groups of MC3-RKO and WT animals were injected with LPS (250 mcg/kg) and monitored as above. The MC3-RKO mice decreased wheel-running activity to the same degree as the WT animals (Fig. 3DGo). As a percentage of basal activity, there was no difference between groups during both 0–24 h and 24–48 h (percent basal running 0–24 h: MC3-RKO 6.8 ± 3.3% vs. WT 4.0 ± 0.6%, P = 0.4; percent basal running 0–48 h: MC3-RKO 23 ± 11% vs. WT 20 ± 7%, n = 5, P = 0.8, Fig. 3EGo).

Basal metabolic rate.
One of the cardinal features of cachexia is a lack of metabolic compensation for decreased food intake. We tested the metabolic response of both MC4-RKO and MC3-RKO mice to LPS with indirect calorimetry. We found that after injection of LPS, WT animals reproducibly increased their basal VO2 over the following 8 h by approximately 10% vs. saline. MC4-RKO mice did not have an increase in basal VO2 after LPS treatment (MC4-RKO 0 ± 2% vs. WT 10 ± 3%, n = 4, P < 0.05, Fig. 4AGo). In contrast, MC3-RKO animals show an increase in basal VO2 after LPS (MC3-RKO 11 ± 2% vs. WT 9 ± 2%, n = 13, P = 0.5, Fig. 4BGo).



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  		Figure 4. Resting energy expenditure in MC-RKO mice after LPS injection. A, MC3-RKO and WT mice have an increase in basal oxygen consumption after the injection of LPS. Data shown represent the percent change in average basal VO2 for the 8-h period after an LPS injection. The data for the LPS day are compared with the previous day during which the animals received saline.

 
Enhanced cachexia with IL-1ß injection
The results of our LPS studies could be due, at least in part, to impaired cytokine production. To test this hypothesis directly, we injected groups of MC3-RKO mice and littermate controls with recombinant murine IL-1ß. In this case, ip injection of 200 ng of IL-1ß resulted in a dramatic decrease in feeding and wheel-running behavior in both MC3-R KO and MC3-R WT animals (Fig. 5Go). Indeed, the decrease in feeding (as a percentage of consumption after a saline control injection) was significantly greater in the MC3-R KO animals than WT animals (MC3-RKO 32 ± 6% of saline vs. WT 48 ± 5% of saline, n = 10, P < 0.5 Fig. 5AGo), and the MC3-RKO animals lost more weight than WT controls as well (24-h weight loss: MC3-RKO 1.26 ± 0.1 g vs. WT 0.75 ± 0.1 g, n = 10, P < 0.1 Fig. 5BGo). Finally, there was a trend toward a greater decrease in wheel-running behavior as well (percent basal running 0–24 h: MC3-RKO 26 ± 9% vs. WT 39 ± 8%, n = 10, P = 0.3; percent basal running 0–48 h: MC3-RKO 74 ± 3% vs. WT 76 ± 13%, n = 10, P = 0.8, Fig. 5CGo).



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  		Figure 5. Enhanced cachexia in MC3-RKO mice in response to IL-1ß. A, MC3-RKO mice show a significantly greater decrease in food intake than WT controls in response to ip IL-1ß (200 ng ip; *, P < 0.05). B, MC3-RKO mice lose more weight over 24 h than WT control animals in response to IL-1ß injection (*, P < 0.01). C, MC3-RKO mice decrease wheel-running behavior after IL-1ß injection to a similar degree as WT mice.

 
Enhanced cachexia in tumor-bearing MC3-RKO mice
We next tested the response of MC3-RKO mice to the growth of a cachexigenic adenocarcinoma (43, 44). Parameters monitored included food and water intake, wheel-running activity, weight gain, and tumor mass. Animals were matched for weight at the start of the experiment (KO 26.4 ± 1.5 g, n = 29 vs. WT 25.5 ± 1.3 g, n = 25, P = 0.6) and baseline feeding was monitored for 3 d before any experimental manipulation. The average food intake over 3 d was used as a measure of basal feeding for each group. As previously reported, the MC3-RKO mice are slightly, but significantly hypophagic at baseline (KO 3.92 ± 0.09 g/d, n = 29 vs. WT 4.26 ± 0.13 g/d, n = 25, P < 0.05), but this difference was not significant when food intake was normalized to body surface area (P = 0.06). WT (n = 13) and KO (n = 17) were implanted with live tumor cells, whereas the remaining animals were implanted with heat-killed cells. The tumor-bearing mice began to show decreased 24-h feeding at d 6 post tumor implantation, coincident with the presence of a palpable tumor. The overall feeding curves were noticeably different from sham-implanted controls from that day onward, with the WT animals consuming 55 ± 10% of baseline levels and KO animals consuming 59 ± 11% of basal by the final day of the experiment (Fig. 6Go, ANOVA P < 0.0001 for treatment and group). The feeding curves for tumor-bearing WT and KO animals were not different when expressed as percentage of basal feeding (Fig. 6AGo) or as intake normalized to body surface area (Fig. 6BGo). MC3-RKO animals lost significantly more carcass weight than control animals during the course of tumor growth (tumor-bearing KO -2.1 ± 0.1 g vs. sham KO 0.9 ± 0.4 g; tumor-bearing WT 0.0 ± 0.3 g, sham WT 1.5 ± 0.4 g; Fig. 6CGo; ANOVA P < 0.0001; WT tumor vs. KO tumor P < 0.01 post hoc). The final tumor mass was not different between groups (KO 1.8 ± 0.3 g vs. WT 1.8 ± 0.2 g, Fig. 6DGo, P = 0.4).



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  		Figure 6. Anorexia and enhanced weight loss with tumor growth in MC3-RKO mice. A, Daily food consumption as a percentage of basal (average of 3 d before tumor implant) food intake during the final 6 d of tumor growth. MC3-RKO and WT mice show significant decreases in daily food intake from d 10 onward, and decrease their feeding to a similar degree by the end of the study. B, The feeding curves are similar when the food consumption is normalized to body surface area. C, MC3-RKO mice show enhanced carcass weight loss with tumor growth (*, P < 0.01; **, P < 0.001). D, MC3-RKO mice have normal tumor growth.

 
Body composition analysis of tumor-bearing MC-RKO mice.
The accuracy and reproducibility of our DEXA analysis were confirmed by chemical body composition analysis (39). The measurements of FM and LBM were tightly correlated between the two methods of analysis (r = 0.98). DEXA scans were performed on anesthetized tumor-bearing animals both before tumor implantation at and immediately after tumor dissection. Both WT and MC3-RKO mice lost FM and LBM during the course of the experiment, but the loss of lean tissues was much greater in the MC3-RKO mice than in their littermate controls (Fig. 7AGo, FM change: MC3R-WT sham 2.5 ± 0.7% vs. MC3-RKO sham 2.2 ± 0.6%, MC3R-WT tumor -2.5 ± 1.1%, MC3R-KO tumor -2.5 ± 0.5%; LBM change: MC3R-WT sham 13 ± 4% vs. MC3-RKO sham 11 ± 2%, MC3R-WT tumor 0.9 ± 1.2%, MC3R-KO tumor -11 ± 2%, ANOVA P < 0.0001, WT tumor vs. KO tumor P < 0.05 post hoc, Fig. 7BGo) MC4-RKO animals were studied in an identical series of experiments. In contrast to the MC3-RKO model, the MC4-RKO showed significant resistance to loss of body fat and lean tissue mass during the course of the experiment and were able to accumulate both types of tissue despite the presence of a growing tumor (FM change: MC4R-WT sham 4 ± 3% vs. MC4-RKO sham 19 ± 8%, MC4R-WT tumor -27 ± 4%, MC4R-KO tumor 20 ± 7%, Fig. 7CGo; LBM change: MC4R-WT sham 4 ± 2% vs. MC4-RKO Sham 19 ± 4%, MC4R-WT tumor -0.5 ± 1.4%, MC4R-KO tumor 9 ± 2%, ANOVA P < 0.0001, WT tumor vs. KO tumor P < 0.05 post hoc, Fig. 7DGo). The tumor resulted in loss of bone mineral density in all models studied, consistent with its known propensity to cause bone resorbtion and hypercalcemia of malignancy (data not shown; and Ref. 45).



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  		Figure 7. Body composition changes in tumorbearing MC3-RKO and MC4-RKO mice. A, MC3-RKO mice lose a similar amount of body fat (FM) as WT animals in response to tumor growth. MC3-RKO mice lose excess LBM in response to tumor growth. C, MC4-RKO mice continue to gain FM during tumor growth. D, MC4-RKO mice also accumulate LBM during tumor growth.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Within the last decade, our understanding of the endocrine and neural control of appetite, metabolism, and body weight has increased dramatically. In particular, we now understand that disorders of leptin feedback or hypothalamic melanocortin signaling can lead to pathological weight gain and diabetes in humans (46, 47, 48). Obviously, obesity is a major health challenge. However, other disorders affecting body weight are found in humans, and these may pose a more immediate threat to our survival, particularly during disease. This is most obvious in chronic diseases including renal failure, cystic fibrosis, acquired immune deficiency syndrome, and various forms of cancer that are often associated with disease-associated wasting or cachexia. Several studies have now demonstrated that the melanocortin system plays a key role in the development of this pathological state (30, 34, 49). Specifically, it has been established that hypophagia and carcass weight loss induced by tumor growth can be both reversed and prevented by administration of MC3/MC4 antagonists such as agouti-related peptide (AGRP) (30, 34). In each case, tumor growth was unaffected by the antagonist treatments, demonstrating the specificity and potential clinical utility of these results. The finding that central melanocortin blockade attenuates cancer cachexia has also been confirmed in MC4-RKO mice (30). MC4-RKO animals have normal feeding and growth even when bearing a carcinoma that produced classic cachexia in WT control animals. In the present study, serial DEXA scans have confirmed that the tumor-bearing MC4-RKO animals continue to accumulate both LBM and FM in the face of tumor growth, whereas WT animals lost both LBM and FM under identical conditions. This preservation of LBM provides critical evidence that the weight gain observed in these animals is not simply due to retention of water or fat, a feature that must be central to any cachexia therapy. The fact that these animals lost bone mass during the course of tumor growth is consistent with the known hypercalcemic effects of this tumor (45), an effect that is attributed to the action of a tumor-produced parathyroid-related peptide directly on bone. Indeed, this effect provides a demonstration that the tumor was growing and functioning normally in these animals during this experiment. Collectively, these data provide a compelling argument that the hypothalamic MC4-R is a critical component of the neural pathways that receive signals produced during illness and transduce these signals into the metabolic and behavioral outputs that define cachexia.

In contrast to the studies of the contribution of the MC4-R to cachexia, no previous studies have addressed the role of the MC3-R in this process. However, there are several reasons to believe that this receptor will be important in this process. First, the hypothalamic MC3-R is known to be expressed in the arcuate nucleus, and it has been proposed to act as an inhibitory autoreceptor on POMC neurons within this nucleus (50). Our hypothesis that peripheral signals produced during illness activate POMC neurons and thereby increase signaling at MC4 receptors predicts that the loss of an autoinhibitory brake on these neurons would enhance cachexia in our models. Second, MC3-RKO mice are known to be relatively hypophagic and to have decreased LBM at baseline, features that would be expected to put the animal at risk for the development of cachexia (35, 36). The fact that these animals also have increased FM may actually be a reflection of an inability to access this fuel depot efficiently during periods of metabolic stress, although this possibility has not been directly tested. Indeed, our current studies suggest that these animals can access storage fat as a fuel during a prolonged fast as reflected by a decrease in the RQ. Furthermore, their response to simple fasting is indistinguishable from that of WT animals in all parameters tested. In response to a more prolonged period of food restriction, the MC3-RKO animals actually lose less weight than WT controls, perhaps due to their increased feed efficiency (35, 36). Thus, observations regarding the response of the MC3-RKO mouse to cachexigenic stimuli are specific for this pathological state and are unlikely to simply reflect an inability to respond to decreased food intake.

In an initial series of experiments, we demonstrated that the MC3-RKO animals had significantly worsened anorexia and weight loss in response to LPS relative to WT control animals. Because the MC3-R is expressed on peritoneal macrophages and MC3-R agonists are thought to inhibit some aspects of the immune response, it is possible that this response to LPS is due to differences in the immune response to this compound (51). However, we have shown that cachexia in response to IL-1ß is also enhanced in MC3-RKO animals. This cytokine is known to produce all of the features of cachexia when chronically injected, and there is ample evidence that this cytokine exerts its effects, at least in part, via direct activation of cytokine receptors within the CNS (18, 52). Thus, these data argue that the MC3-R is directly involved in the behavioral response to cytokines. Perhaps the absence of the MC3-R, a putative inhibitory autoreceptor on POMC neurons, allows for enhanced release of {alpha}-MSH from these neurons with subsequent activation of the MC4-R (see below). These observations are strengthened by our data that show that the metabolic response to LPS is also altered in the KO models. Our previous data had shown that MC4-RKO animals resist the anorexia and decreased movement brought about by LPS administration (30). Here we have also demonstrated that the metabolic response to LPS is blocked in this animal, whereas the MC3-RKO mouse has a normal increase in VO2 in response to LPS. This increase in metabolic rate during a time of relative anorexia is a hallmark of cachexia and is likely to contribute to the enhanced weight loss observed in the MC3-RKO mouse in response to LPS. Although we have not yet performed a careful dose-response curve, in our current study the difference between WT and MC3-RKO animals in the effects of LPS and cytokines on feeding behavior and metabolism are modest. However, as discussed below, our data show that these differences can result in very large effects on the preservation of LBM during prolonged illness.

One of the most devastating features of cachexia is the severe depletion of LBM that leads not only to weakness and decreased quality of life but also to increased mortality in a number of disease states (1, 2). As stated above, deletion of the MC4-R can prevent the loss of LBM that occurs during tumor growth. Our present studies demonstrate that deletion of the MC3-R has the opposite effect, leading to a specific enhancement of loss of LBM in tumor-bearing animals. To our knowledge, this is the first demonstration of a specific enhancement of this feature of cachexia in any genetic model and indicates that the MC3-R may play a key role in limiting the loss of LBM that occurs during prolonged illness. In contrast, the loss of FM in this model was similar to that found in the tumor-bearing WT animals and is therefore likely to be due to the normal depletion of fat stores that occurs with prolonged periods of decreased food intake. This idea is supported by the observation that the MC4-RKO mouse, which does not decrease its feeding during tumor growth, is able to continue to accumulate FM during the experimental period. Our data also indicate that the increased feed efficiency observed in the MC3-RKO mouse at baseline is not observed during tumor growth (35, 36). It is likely that the small effect on feed efficiency is overwhelmed by the increased and unrestrained activation of the MC4-R during illness, but it is possible that the peripheral factors responsible for the development of cachexia act downstream of the MC3-R and are therefore capable of bypassing this feature of the MC3-RKO phenotype.

The mechanisms whereby the quantity and distribution of LBM and FM can be differentially regulated are numerous. We have previously shown that body mass depletion in tumor-bearing or LPS-treated mice can be reversed by central injections of AGRP (30), and others have demonstrated that the anorexia due to tumor growth or cytokine injection in rats can be reversed by central injections of another MC3/4 antagonist, SHU9119 (33, 34). Although these studies do not demonstrate a specific conservation of LBM, they do provide compelling evidence that melanocortin blockade prevents cachexia via a central rather than peripheral mechanism. One of the cardinal phenotypic features of the MC4-RKO mouse is an increase in LBM. This increase is also observed in mice with a genetic deletion of both the MC3-R and the MC4-R, and in mice overexpressing the MC3/4 antagonists agouti or AGRP (36, 53, 54, 55). Thus, we hypothesize that the enhanced loss of LBM observed in our cancer models is due to a prolonged increase in MC4-R signaling brought about by loss of the autoinhibitory MC3-R on POMC neurons. Collectively, our data are the first physiological evidence in support of the hypothesis that the MC3-R functions as an autoinhibitory receptor on POMC neurons, a function that may help to explain the decreased LBM and relative hypophagia seen in the MC3-RKO mouse (35, 36). However, it remains possible that the enhanced cachexia observed in this genetic model is unrelated to increased signaling at the MC4-R, but instead is attributable to other actions of the MC3-R either within the arcuate nucleus or at other sites of MC3-R expression within the brain such as the ventromedial hypothalamus, or at sites of expression outside of the CNS. Studies of the effects of specific central MC4-R blockade in cachexia in MC3-RKO mice may help to distinguish between these two models.

Our data demonstrating a grossly normal febrile response to LPS are consistent with previous experiments demonstrating that SHU9119 has no effect on IL-1ß-induced fever (33). However, the febrile response to cytokines is complex, and it is likely that some features of this response are altered in MC KO mice (32). A similar argument applies to our observations of corticosterone release in response to LPS. Our results are consistent with the apparently normal HPA axis in these models, but we cannot rule out important alterations in the overall duration and magnitude of HPA axis activation in response to our experimental manipulations (29, 36). Indeed, our own data demonstrate that the MC3-RKO animal has a relatively small, but significant increase in serum corticosterone in response to saline injection, implying a hypersensitivity to stressors in this animal model. These data are consistent with previously published results demonstrating enhanced stress-induced anorexia in mice with disrupted melanocortin signaling, but the contribution of the HPA axis to our data remain unresolved (42).

In summary, our studies have demonstrated a preservation of LBM in tumor-bearing MC4-RKO mice and have bolstered the idea that this receptor plays a critical role in producing all features of cachexia observed during illness. In contrast, blockade of the MC3-R enhances illness-induced cachexia and produces a dramatic and specific loss of LBM in tumor models. Collectively, our data suggest that the MC4-R represents a logical therapeutic target for treatment of cachexia observed in human diseases such as cancer, heart failure, Alzheimer’s disease, and AIDS. In contrast, dysfunction of the MC3-R confers an increased risk of developing cachexia implying that melanocortin antagonist therapy should be directed specifically at the MC4-R. The possibility that defects in MC3-R signaling lead to an enhanced susceptibility to weight loss in human disease remains an intriguing but unexplored possibility.


   Acknowledgments
 
The authors thank Katie Miles, Kathy Khong, and Wilmon Grant for their technical assistance.


   Footnotes
 
This work was supported by research grants from the NIH (DK-55819, DK-51730, HD-07497, and HD-33703).

Abbreviations: AGRP, Agouti-related protein; CNS, central nervous system; DEXA, dual-energy x-ray absorbtometry; FM, fat mass; HPA, hypothalamic-pituitary-adrenal; KO, knockout; LBM, lean body mass; LPS, lipopolysaccharide; MC3, melanocortin 3; MC3-R, melanocortin 3 receptor; MC3-RKO, MC3-R knockout; MC4, melanocortin-4; MC4-R, MC4 receptor; POMC, proopiomelanocortin; VCO2, carbon dioxide production; VO2, oxygen consumption; WT, wild-type.

Received October 23, 2002.

Accepted for publication December 12, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Tisdale MJ 1997 Biology of cachexia. J Natl Cancer Inst 89:1763–1773[Abstract/Free Full Text]
  2. Larkin M 1998 Thwarting the dwindling progression of cachexia. Lancet 351:1336[CrossRef][Medline]
  3. Plata-Salaman CR 1989 Immunomodulators and feeding regulation: a humoral link between the immune and nervous systems. Br Behav Immun 3: 193–213
  4. Plata-Salaman CR 1998 Brain mechanisms in cytokine-induced anorexia. Psychoneuroendocrinology 24:25–41
  5. Inui A 1999 Feeding and body-weight regulation by hypothalamic neuropeptides—mediation of the actions of leptin. Trends Neurosci 22:62–67[CrossRef][Medline]
  6. Baile CA, Naylor J, McLaughlin CL, Catanzaro CA 1981 Endotoxin-elicited fever and anorexia and elfazepam-stimulated feeding in sheep. Physiol Behav 27:271–277[CrossRef][Medline]
  7. Murray MJ, Murray AB 1979 Anorexia of infection as a mechanism of host defense. Am J Clin Nutr 32:593–596[Abstract/Free Full Text]
  8. Hillhouse EW, Mosley K 1993 Peripheral endotoxin induces hypothalamic immunoreactive interleukin-1ß in the rat. Br J Pharmacol 109:289–290[Medline]
  9. Van Dam AM, Bauer J, Tilders FJ, Berkenbosch F 1995 Endotoxin-induced appearance of immunoreactive interleukin-1ß in ramified microglia in rat brain: a light and electron microscopic study. Neuroscience 65:815–826[CrossRef][Medline]
  10. Cavaillon JM, Haeffner-Cavaillon N 1990 Signals involved in interleukin-1 synthesis and release by lipopolysaccharide-stimulated monocytes/macrophages. Cytokine 2:313–329[CrossRef][Medline]
  11. Molloy RG, Mannick JA, Rodrick ML 1993 Cytokines, sepsis and immunomodulation. Br J Surg 80:289–297[Medline]
  12. Laye S, Gheusi G, Cremona S, Combe C, Kelly K, Dantzer R, Parnet P 2000 Endogenous brain IL-1 mediates the response to peripheral LPS. Am J Phys Reg Integr Comp Physiol 279:R93–R98
  13. Gayle D, Ilyin SE, Flynn MC, Plata-Salaman CR 1998 Lipopolysaccharide (LPS)- and muramyl dipeptide (MDP)-induced anorexia during refeeding following acute fasting: characterization of brain cytokine and neuropeptide systems mRNAs. Brain Res 795:77–86[CrossRef][Medline]
  14. Eriksson C, Nobel S, Winblad B, Schultzberg M 2000 Expression of interleukin 1{alpha} and ß, and interleukin 1 receptor antagonist mRNA in the rat central nervous system after peripheral administration of lipopolysaccharides. Cytokine 12:423–431[CrossRef][Medline]
  15. Takao T, Hashimoto K, De Souza EB 1995 Modulation of interleukin-1 receptors in the neuro-endocrine-immune axis. Int J Dev Neurosci 13:167–178[CrossRef][Medline]
  16. Cone RD, Cowley MA, Butler AA, Fan W, Marks DL, Low MJ 2001 The arcuate nucleus as a conduit for diverse signals relevant to energy homeostasis. Int J Obes Relat Metab Disord 25(Suppl 5):S63–S67
  17. Erickson JC, Clegg KE, Palmiter RD 1996 Sensitivity to leptin and susceptibility to seizures of mice lacking neuropeptide Y. Nature 381:415–418[CrossRef][Medline]
  18. Yabuuchi K, Minami M, Katsumata S, Satoh M 1994 Localization of type-1 interleukin-1 receptor mRNA in the rat brain. Brain Res Mol Brain Res 27:27–36[Medline]
  19. Chang SL, Ren T, Zadina JE 1993 Interleukin-1 activation of Fos proto- oncogene protein in the rat hypothalamus. Brain Res 617:123–130[CrossRef][Medline]
  20. Elmquist JK, Scammell TE, Jacobsen CD, Saper CB 1996 Distribution of Fos-like immunoreactivity in the rat brain following intravenous lipopolysaccharide administration. J Comp Neurol 371:85–103[CrossRef][Medline]
  21. Jacobowitz DM, O’Donohue TL 1978 {alpha}-Melanocyte-stimulating hormone: immunohistochemical identification and mapping in neurons of rat brain. Proc Natl Acad Sci USA 75:6300–6304[Abstract/Free Full Text]
  22. Fan W, Boston BA, Kesterson RA, Hruby VJ, Cone RD 1997 Role of melanocortinergic neurons in feeding and the agouti obesity syndrome. Nature 385:165–168[CrossRef][Medline]
  23. Cowley MA, Pronchuk N, Fan W, Dinulescu DM, Colmers WF, Cone RD 1999 Integration of NPY, AGRP, and melanocortin signals in the paraventricular nucleus of the hypothalamus: evidence of a cellular basis for the adipostat. Neuron 24:155–163[CrossRef][Medline]
  24. Stair JN, Shu J, Camacho R, Murphy B, Hickey GJ, MacIntyre DE, Strack AM 1999 Feeding behavior in rats chronically treated with melanocortin agonist, MTII. Soc Neurosci Abstr 25:619
  25. Patterson TA, Hedde JR, She L, Newsome WP, Cornelius P 1999 Chronic infusion of a melanocortin receptor antagonist causes weight loss due to decreased food intake and increased metabolic rate. Soc Neurosci Abstr 25:618
  26. Hamilton BS, Doods HN 2002 Chronic application of MTII in a rat model of obesity results in sustained weight loss. Obes Res 10:182–187[Medline]
  27. Pierroz DD, Ziotopoulou M, Ungsunan L, Moschos S, Flier JS, Mantzoros CS 2002 Effects of acute and chronic administration of the melanocortin agonist MTII in mice with diet-induced obesity. Diabetes 51:1337–1345[Abstract/Free Full Text]
  28. Baran K, Preston E, Wilks D, Cooney GJ, Kraegen EW, Sainsbury A 2002 Chronic central melanocortin-4 receptor antagonism and central neuropeptide-Y infusion in rats produce increased adiposity by divergent pathways. Diabetes 51:152–158[Abstract/Free Full Text]
  29. Huszar D, Lynch CA, Fairchild-Huntress V, Dunmore JH, Fang Q, Berkemeier LR, Gu W, Kesterson RA, Boston BA, Cone RD, Smith FJ, Campfield LA, Burn P, Lee F 1997 Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell 88:131–141[CrossRef][Medline]
  30. Marks DL, Ling N, Cone RD 2001 Role of the central melanocortin system in cachexia. Cancer Res 61:1432–1438[Abstract/Free Full Text]
  31. Huang QH, Hruby VJ, Tatro JB 1999 Role of central melanocortins in endotoxin-induced anorexia. Am J Physiol 276:R864–R871
  32. Tatro JB, Huszar D, Fairchild-Huntress V, Huang QH 1999 Role of the melanocortin-4 receptor in thermoregulatory responses to central IL-1ß. Soc Neurosci Abstr 25:1558
  33. Lawrence CB, Rothwell NJ 2001 Anorexic but not pyrogenic actions of interleukin-1 are modulated by central melanocortin-3/4 receptors in the rat. J Neuroendocrinol 13:490–495[CrossRef][Medline]
  34. Wisse BE, Frayo RS, Schwartz MW, Cummings DE 2001 Reversal of cancer anorexia by blockade of central melanocortin receptors in rats. Endocrinology 142:3292–3301[Abstract/Free Full Text]
  35. Butler AA, Kesterson RA, Khong K, Cullen MJ, Pellymounter MA, Dekoning J, Baetscher M, Cone RD 2000 A unique metabolic syndrome causes obesity in the melanocortin-3 receptor-deficient mouse. Endocrinology 141:3518–3521[Abstract/Free Full Text]
  36. Chen AS, Marsh DJ, Trumbauer ME, Frazier EG, Guan XM, Yu H, Rosenblum CI, Vongs A, Feng Y, Cao L, Metzger JM, Strack AM, Camacho RE, Mellin TN, Nunes CN, Min W, Fisher J, Gopal-Truter S, MacIntyre DE, Chen HY, Van der Ploeg LH 2000 Inactivation of the mouse melanocortin-3 receptor results in increased fat mass and reduced lean body mass. Nat Genet 26:97–102[CrossRef][Medline]
  37. Butler AA, Marks DL, Fan W, Kuhn CM, Bartolome M, Cone RD 2001 Melanocortin-4 receptor is required for adaptive thermogenesis and appetite control in response to high-fat diet. Nature Neurosci 4:605–611[CrossRef][Medline]
  38. Bagnol T, Lu XY, Kaelin CB, Day HE, Ollmann M, Gantz I, Akil H, Barsh GS, Watson SJ 1999 The anatomy of an endogenous antagonist: relationship between agouti-related protein and proopiomelanocortin in brain. J Neurosci 19:RC26
  39. Leshner A, Litwin V, Squibb R 1972 A simple method for carcass analysis. Brain Res 9:281–282
  40. Huang Q-H, Entwistle ML, Alvaro JD, Duman RS, Hruby VJ, Tatro JB 1997 Antipyretic role of endogenous melanocortins mediated by central melanocortin receptors during endotoxin-induced fever. J Neurosci 17:3343–3351[Abstract/Free Full Text]
  41. Huang QH, Hruby VJ, Tatro JB 1998 Systemic {alpha}-MSH suppresses LPS fever via central melanocortin receptors independently of its suppression of corticosterone and IL-6 release. Am J Physiol 275:R524–R30
  42. De Souza J, Butler AA, Cone RD 2000 Disproportionate inhibition of feeding in A(y) mice by certain stressors: a cautionary note. Neuroendocrinology 72:126–132[CrossRef][Medline]
  43. Matthys P, Heremans H, Opdenakker G, Billiau A 1991 Anti-interferon-{gamma} antibody treatment, growth of Lewis lung tumors in mice and tumor-associated cachexia. Eur J Cancer 27:182–187[Medline]
  44. Llovera M, Garcia-Martinez C, Lopez-Soriano J, Agell N, Lopez-Soriano FJ, Garcia I, Argiles JM 1998 Protein turnover in skeletal muscle of tumor-bearing transgenic mice overexpressing the soluble TNF receptor-1. Cancer Lett 130:19–27[CrossRef][Medline]
  45. Maeda Y, Yamato H, Hirai T, Kobori N, Fujii T, Kobayashi Y, Saitoh K, Inoguchi E, Hakozaki M, Iijima H, Kodaira T, Konai Y, Katoh T, Yoshikumi C, Kawai Y, Nomoto K 1988 Antitumor and other effects of 24R, 25-dihydroxycholecalciferol in Lewis lung carcinoma causing abnormal calcium metabolism in tumor-bearing mice. Oncology 45:202–205[Medline]
  46. Montague CT, Farooqi IS, Whitehead JP, Soos MA, Rau H, Wareham NJ, Sewter CP, Digby JE, Mohammed SN, Hurst JA, Cheetham CH, Earley AR, Barnett AH, Prins JB, O’Rahilly S 1997 Congenital leptin deficiency is associated with severe early-onset obesity in humans. Nature 387:903–908[CrossRef][Medline]
  47. Krude H, Biebermann H, Luck W, Horn R, Brabant G, Gruters A 1998 Severe early-onset obesity, adrenal insufficiency and red hair pigmentation caused by POMC mutations in humans. Nat Genet 19:155–157[CrossRef][Medline]
  48. Clement K, Vaisse C, Lahlou N, Cabrol S, Pelloux V, Cassuto D, Gourmelen M, Dina C, Chambaz J, Lacorte JM, Basdevant A, Bougneres P, Lebouc Y, Froguel P, Guy-Grand B 1998 A mutation in the human leptin receptor gene causes obesity and pituitary dysfunction. Nature 392:398–401[CrossRef][Medline]
  49. Huang Q, Hruby VJ, Tatro JB 1999 Role of central melanocortins in endotoxin-induced anorexia. Am J Physiol 276:R864–R871
  50. Bagnol D, Lu XY, Kaelin CB, Day HE, Ollmann M, Gantz I, Akil H, Barsh GS, Watson SJ 1999 Anatomy of an endogenous antagonist: relationship between agouti-related protein and proopiomelanocortin in brain. J Neurosci 19:RC26
  51. Getting SJ, Flower RJ, Perretti M 1999 Agonism at melanocortin receptor type 3 on macrophages inhibits neutrophil influx. Inflamm Res 48(Suppl 2):S140–S141
  52. Masotto C, Caspani G, Grazia de Simoni M, Mengozzi M, Scatturin M, Sironi M, Carenzi A, Ghezzi P 1992 Evidence for a different sensitivity to various central effects of interleukin-1B in mice. Br Res Bull 28:161–165[CrossRef][Medline]
  53. Klebig ML, Wilkinson JE, Geisler JG, Woychik RP 1995 Ectopic expression of the agouti gene in transgenic mice causes obesity, features of type II diabetes, and yellow fur. Proc Natl Acad Sci USA 92:4728–4732[Abstract/Free Full Text]
  54. Ollmann MM, Wilson BD, Yang YK, Kerns JA, Chen Y, Gantz I, Barsh GS 1997 Antagonism of central melanocortin receptors in vitro and in vivo by agouti-related protein. Science 278:135–138[Abstract/Free Full Text]
  55. Yen TT, Gill AM, Frigeri LG, Barsh GS, Wolff GL 1994 Obesity, diabetes, and neoplasia in yellow A(vy)/- mice: ectopic expression of the agouti gene. FASEB J 8:479–488[Abstract]



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