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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-1124
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 2 1047-1054
Copyright © 2005 by The Endocrine Society

Circulating Melanin-Concentrating Hormone, Agouti-Related Protein, and {alpha}-Melanocyte-Stimulating Hormone Levels in Relation to Body Composition: Alterations in Response to Food Deprivation and Recombinant Human Leptin Administration

Alina Gavrila, Jean L. Chan, Lisa C. Miller, Kathleen Heist, Nikos Yiannakouris and Christos S. Mantzoros

Division of Endocrinology and Metabolism, Department of Internal Medicine, Beth Israel Deaconess Medical Center (A.G., J.L.C., K.H., C.S.M.), Boston, Massachusetts 02215; Department of Biostatistics, Harvard School of Public Health (L.C.M.), Boston, Massachusetts 02115; and Department of Nutrition and Home Economics and Ecology, Harokopio University (N.Y.), Athens, Greece

Address all correspondence and requests for reprints to: Dr. Christos S. Mantzoros, Division of Endocrinology and Metabolism, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Stoneman 816-820, Boston, Massachusetts 02215. E-mail: cmantzor{at}caregroup.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
We evaluated whether circulating levels of melanin-concentrating hormone (MCH), agouti-related protein (AGRP), and {alpha}-MSH could serve as useful markers of energy homeostasis in humans. We first assessed correlations of serum MCH, AGRP, and {alpha}-MSH with anthropometric, dietary, and hormonal variables in a cross-sectional study of 108 healthy humans. We then performed interventional studies to evaluate the effects of fasting and/or leptin administration. In eight healthy, normal weight men, we measured serum MCH, AGRP, and {alpha}-MSH levels at baseline, after 2 d of fasting alone (a low leptin state), and after 2 d of fasting with replacement dose recombinant methionyl human leptin (r-metHuLeptin) administration to normalize circulating leptin levels. In a separate group of five lean and five obese men, we measured MCH levels in response to increasing circulating leptin levels to the pharmacological range by administration of one r-metHuLeptin dose in the fed state. In the cross-sectional study, serum MCH levels were independently and positively associated with body mass index and fat mass and were higher in women than in men. Furthermore, in our interventional studies, fasting for 2 d significantly decreased leptin levels and increased serum MCH levels. Administration of replacement dose r-metHuLeptin during fasting prevented the fasting-induced increase in MCH levels, but administration of a pharmacological r-metHuLeptin dose in the fed state did not further alter MCH levels. Serum AGRP levels tended to change in directions similar to MCH, but this change was less pronounced and needs to be investigated in larger studies. In contrast, serum {alpha}-MSH levels did not correlate with body composition parameters, were not associated with caloric or macronutrient intake, and were not significantly affected by fasting or r-metHuLeptin administration. These findings suggest that serum MCH and possibly AGRP levels could serve as useful peripheral markers of changes in energy homeostasis and thus merit additional investigation.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
HYPOTHALAMIC EXPRESSION OF melanin-concentrating hormone (MCH) and agouti-related protein (AGRP), neuropeptides that increase food intake and decrease energy expenditure, is inhibited by leptin administration and induced by low leptin states, such as fasting, genetic leptin deficiency, or leptin resistance in rodents (1, 2, 3, 4, 5, 6). In contrast, hypothalamic expression of {alpha}-MSH, a neuropeptide that decreases food intake and increases energy expenditure, is induced by leptin administration and decreased in response to fasting and/or hypoleptinemic states (1, 2, 3, 7).

Although localization of MCH, AGRP, and {alpha}-MSH in the hypothalamus is similar in humans and rodents (8, 9, 10, 11, 12, 13, 14), hypothalamic expression of these neuropeptides cannot be directly measured in living human subjects. MCH, AGRP, and {alpha}-MSH are detectable in peripheral blood of rodents, however, and serum levels in rodents change in the same direction as in the hypothalamus (15, 16, 17, 18). Previous studies that evaluated plasma AGRP and {alpha}-MSH in humans have reported conflicting results with respect to their correlation with adiposity, serum leptin levels, and food intake or fasting (19, 20, 21). Predictors of serum MCH levels and whether these neuropeptides are affected by exogenous leptin administration in a manner that would be expected based on their physiological roles have not yet been investigated in humans.

We hypothesized that serum MCH, AGRP, and {alpha}-MSH levels would correlate with fat mass and would change in response to fasting and/or recombinant methionyl human leptin (r-metHuLeptin) administration in humans in directions similar to those previously demonstrated in animals. We first evaluated serum MCH, AGRP, and {alpha}-MSH levels in relation to anthropometric, hormonal, and dietary variables in a cross-sectional study of 108 healthy subjects. To elucidate the effect of fasting-induced hypoleptinemia and/or r-metHuLeptin administration at physiological replacement doses, we measured serum levels of these neuropeptides in healthy, normal weight men during a baseline fed state, a 2-d fasting study that reduced circulating leptin levels to 30% of baseline, and a 2-d fasting study with administration of replacement doses of r-metHuLeptin designed to normalize the fasting-induced decrease in leptin levels. Finally, in a separate group of lean and obese men, we evaluated whether MCH levels would be altered in response to increasing endogenous leptin to high physiological levels after r-metHuLeptin administration in the fed state.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Cross-sectional study

One hundred and eight healthy Greek subjects [age, 17.7 ± 1.7 (mean ± SD) yr; body mass index (BMI), 22.3 ± 3.6 kg/m2] were consecutively enrolled at Harokopio University (Athens, Greece). Blood samples for hormonal testing were collected after an overnight fast, and subjects completed a self-administered questionnaire on demographic data, general health, medications, as well as smoking, drinking, and exercise status. Women were not taking oral contraceptives at the time of the evaluation. Weight to the nearest 0.5 kg, height to the nearest 0.5 cm, waist circumference (WC), and hip circumference were measured, and BMI as well as waist to hip ratio (WHR) were determined. Fat body mass (in kilograms and percentage of body weight) and lean mass (kilograms) were calculated using bioelectrical impedance analysis (BIA) performed with a single frequency bioimpedance analyzer (model 101, RJL Systems, Mt. Clemens, MI) (22). We validated the BIA and anthropometric measurements in a separate study of 60 subjects who underwent a dual energy x-ray absorptiometry scan, as described previously (23). Thus, in our analysis we used BMI and fat mass calculated by BIA as markers of overall adiposity, and WC and WHR as markers of central obesity.

Dietary analysis was performed using 3-d food records for 2 consecutive weekdays and 1 weekend day. Estimates of energy and macro- and micronutrient consumption were generated as previously described (24). Dietary data were available for all except six subjects.

Interventional studies

Fasting with and without administration of physiological doses of r-metHuLeptin. Eight normal weight men (age, 23.4 ± 1.5 yr; BMI, 23.5 ± 1.7 kg/m2) were screened for any medical problems at Beth Israel Deaconess Medical Center (BIDMC) and were admitted to the General Clinical Research Center (GCRC) under three separate conditions: fed state, fasting with placebo administration, and fasting with r-metHuLeptin administration. The interval between admissions was no less than 7 wk, to allow for recovery of hematocrit, to ensure that subjects would return to their baseline weight at the beginning of each admission, and to avoid any potential long-term effects of r-metHuLeptin administration. All eight subjects completed the fed state and fasting/placebo admissions, and serum neuropeptide levels were available for five of six subjects (age, 22.0 ± 2.1 yr; BMI, 22.2 ± 0.8 kg/m2) who completed the fasting/r-metHuLeptin admission. We obtained similar results when analyzing the eight subjects who participated in at least two parts of the study (intention to treat analysis) as well as the five subjects who had complete data for all three study admissions (on treatment analysis), reported below.

Subjects were admitted the evening before study d 1, and fasting blood samples for hormonal measurements were obtained at 0800 h on d 1 and 3 of each admission. During the fed admission, subjects were placed on an isocaloric diet designed to keep weight stable, with four standardized meals per day, with 20% of calories from breakfast (0800 h), 35% from lunch (1300 h), 35% from dinner (1800 h), and 10% from a snack (2200 h). During both fasting admissions, subjects were allowed to drink only calorie-free and caffeine-free liquids for 2 d, and they received 500 mg NaCl, 40 mEq KCl, and a standard multivitamin with minerals daily. During the fasting/r-metHuLeptin admission, subjects received physiological doses of r-metHuLeptin by sc injection, designed to restore the fasting-induced decline in leptin levels to levels similar to those in the fed state. The daily dose of r-metHuLeptin was calculated based on previous pharmacokinetic studies (0.04 mg/kg on d 1 and 0.1 mg/kg on d 2, because leptin levels decrease further on the second day of fasting) and was divided into four equal doses, administered every 6 h. During the fasting/placebo admission, a buffer solution was administered sc every 6 h. The first dose of r-metHuLeptin or placebo was given on d 1 at 0800 h, after baseline blood samples were collected.

Administration of pharmacological dose of r-metHuLeptin in the fed state. Five healthy, normal weight men (age, 22.2 ± 2.0 yr; BMI, 22.0 ± 1.0 kg/m2) and five otherwise healthy obese men (age, 23.4 ± 3.4 yr; BMI, 32.0 ± 2.3 kg/m2) were admitted to the GCRC during the evening and received a 0.1 mg/kg dose of r-metHuLeptin the following morning, designed to achieve high physiological leptin levels. Fasting blood samples for leptin and MCH measurements were collected at 0800 h immediately before r-metHuLeptin administration and 12 h after the leptin dose (2000 h). Subjects received an isocaloric diet during the study, as described above.

All studies were approved by the institutional review board at BIDMC, and the cross-sectional study was also approved by the ethics committee at Harokopio University. All subjects (and their parents for the cross-sectional study) gave informed consent to participate. Clinical quality r-metHuLeptin was supplied by Amgen, Inc. (Thousand Oaks, CA), and administered under an Investigator-Initiated New Drug Application submitted to the FDA (submitted by C.S.M.).

Hormone measurements

Serum hormone levels were measured using commercially available RIAs, as follows: MCH, Phoenix Pharmaceuticals [Belmont, CA; sensitivity, 70 pg/ml; intraassay coefficient of variation (CV), 4.4%] (18); AGRP, Phoenix Pharmaceuticals (sensitivity, 17.41 pg/ml; intraassay CV, 4.4%) (19, 21, 25); {alpha}-MSH, Alpco Diagnostics (Windham, NH; sensitivity, 3 pmol/liter; intraassay CV, 11.8%) (20, 26); leptin, Linco Research, Inc. (St. Charles, MO; sensitivity, 0.5 ng/ml; intraassay CV, 8.3%); insulin, Diagnostic Systems Laboratories, Inc. [Webster, TX; sensitivity, 1.3 µIU/ml; intraassay CV, 8.3%; conversion factor (CF) from conventional units to Systeme International units, 7.175]; estradiol, Diagnostic Products Corp. (Los Angeles, CA; sensitivity, 8 pg/ml; intraassay CV, 4.3–7%; CF, 3.671); and free testosterone, Diagnostic Products Corp. (sensitivity, 0.15 pg/ml; intraassay CV, 8%; CF, 4.467). {alpha}-MSH was measured in sera from all 108 subjects who participated in the cross-sectional study; however, sera from only 90 and 76 subjects were available for AGRP and MCH measurements, respectively. To minimize variability, hormone concentrations were measured in one assay for all subjects participating in this study. Similarly, baseline and postintervention hormonal measurements were run in the same assay for each admission of the interventional studies. The MCH assay used herein has not been previously standardized for clinical use, and the effect of gender, race, or ethnic background on baseline levels is unknown.

Statistical analysis

We used SAS 8.2 general linear model procedure for the cross-sectional study and StatXact 4 to analyze the data from the interventional studies. For the cross-sectional study, we initially performed Pearson’s correlations between each of the three neuropeptides under consideration (MCH, AGRP, and {alpha}-MSH) and demographic, dietary, anthropometric, and hormonal parameters. We report Pearson’s r coefficients, which reflect the strength of an association in values between –1 and 1. A value of P < 0.05 was considered statistically significant in the cross-sectional study, although covariates with corresponding values of P < 0.10 were considered in other multivariate analysis. Based on these results, we then performed simple and multiple linear regression analyses, adjusting for the following potential confounders: age, gender, caffeine intake, smoking history, and current exercise and drinking status. For simple (bivariate) as well as multivariate regression, we report (unstd) ß coefficients, which reflect the strength of an association as the change in the dependent variable per unit change in the independent variable. We used logarithmic transformation of nonnormally distributed AGRP levels for all analyses. To examine the effect of nutritional parameters on peripheral concentrations of MCH, AGRP, and {alpha}-MSH while controlling for total energy intake, we used the residual method described by Willett and Stampfer (27). We also evaluated gender differences in serum MCH, AGRP, and {alpha}-MSH levels using two-tailed, independent-sample t tests, followed by linear regression analyses using the three neuropeptides as dependent variables and body composition parameters, leptin levels, and gender as independent variables.

We used nonparametric tests for the interventional studies, because the number of subjects was relatively small, and the data were not normally distributed. We performed exact Wilcoxon signed rank tests to evaluate changes in serum MCH, AGRP, {alpha}-MSH, and leptin levels and weight between d 1 and 3 of each intervention (feeding with isocaloric diet, fasting with placebo administration, and fasting with r-metHuLeptin administration) in the first interventional study. Based on Bonferroni correction for multiple comparisons, a value of P < 0.017 was considered statistically significant. We used Wilcoxon signed rank tests to evaluate changes in leptin and MCH levels from baseline to 12 h after administration of the r-metHuLeptin dose in the fed state in the second interventional study.

All statistical tests reported are two-tailed. The cross-sectional study (n = 108) had more than 80% power to detect associations with r ≥ 0.30 at the conventional {alpha} = 0.05 level. An interventional study with eight subjects studied under different experimental conditions would provide more than 80% power to detect changes in the mean effect estimates larger than 1.4 (one-tailed) or 1.24 (two-tailed) times the respective SD at the conventional {alpha} = 0.05 level and has proven to be of adequate size in terms of assessing changes of leptin levels and neuroendocrine variables in response to fasting and/or r-metHuLeptin administration (28, 29).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Cross-sectional study

Baseline characteristics of the study subjects are summarized in Table 1Go. Ninety-four percent of subjects were physically active, 88% did not consume alcohol, and 58% were nonsmokers at the time of evaluation. The mean ± SD serum MCH level for the entire study group was 97.8 ± 22.8 pg/ml, the mean AGRP level was 28.4 ± 16.7 pg/ml, and the mean {alpha}-MSH level was 10.5 ± 1.7 pmol/liter. Women had higher MCH levels than men (101.6 vs. 90.8 pg/ml; P = 0.048); however, this difference did not remain statistically significant after adjusting for body fat and/or leptin levels in multiple regression analysis. There were no gender-related differences in serum {alpha}-MSH or AGRP concentrations (Table 1Go). As expected, there were significant gender differences in caloric intake, body composition, and serum leptin, estradiol, and free testosterone levels (Table 1Go). Serum neuropeptide levels did not correlate with age, but these subjects were relatively young (age range, 14–26 yr). In addition, we did not find any correlation among any of the three neuropeptides of interest.


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  		TABLE 1. Cross-sectional study: baseline characteristics of the study participants

 
Association of serum MCH, AGRP, and {alpha}-MSH levels with body composition. We found a significant positive correlation of MCH levels with fat mass (r = 0.26; P = 0.02) and percent fat (r = 0.33; P = 0.003; Table 2Go and Fig. 1Go). The association between serum MCH levels and percent fat was significant in bivariate regression analysis (unadjusted unstd ß coefficient/unstd ß = 0.96; P < 0.01) and in multivariate regression analyses after adjusting for age, gender, caffeine intake, smoking history, as well as current exercise and drinking status (adjusted unstd ß = 1.12; 0.01 ≤ P < 0.05). The associations between MCH and BMI or lean mass gained significance after adjusting for the same potential confounders in multivariate analyses (adjusted unstd ß = 1.81 and adjusted unstd ß = 1.73, respectively; 0.01 ≤ P < 0.05). We also found a negative correlation between AGRP and lean mass (r = –0.23; P = 0.03; Table 2Go), which was significant after adjusting for age, but did not remain significant after controlling for gender and/or other study predictor variables. We found no correlation between serum levels of the three neuropeptides and central adiposity, as assessed by WC or WHR. In addition, there was no significant association between {alpha}-MSH levels and any body composition parameters (Table 2Go).


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  		TABLE 2. Cross-sectional study; bivariate analysis: Pearson correlation coefficients between serum MCH, AGRP, {alpha}-MSH levels, and body composition, hormonal, and dietary variables

 


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  		FIG. 1. Cross-sectional study. Correlations of serum leptin and MCH levels with body fat percentage. {diamondsuit}, Male; {circ}, female.

 
Association of serum MCH, AGRP, and {alpha}-MSH levels with hormone levels. There was a significant positive correlation between serum AGRP and estradiol levels in bivariate regression analysis (r = 0.33; P = 0.002; Table 2Go). This association remained significant after controlling for age, gender, and lifestyle variables (caffeine, smoking, drinking, and exercise) in addition to BMI (unstd ß = 0.004; P = 0.002), lean mass (unstd ß = 0.004; P = 0.003), or percent fat (unstd ß = 0.004; P = 0.002). The negative correlation between MCH and free testosterone (r = –0.20; P = 0.08; Table 2Go) became nonsignificant after controlling for the above-mentioned potential confounders in multivariate regression analysis (data not shown). Finally, we did not find significant associations between any of the three neuropeptides of interest and fasting insulin or leptin levels (Table 2Go).

Association of serum MCH, AGRP, and {alpha}-MSH levels with dietary parameters. The correlation between MCH levels and daily energy intake (r = –0.23; P = 0.056; Table 2Go) as well as caffeine intake (r = –0.22; P = 0.07; Table 2Go) became nonsignificant after controlling for age, gender, and body composition parameters. No significant correlations were found between {alpha}-MSH or AGRP levels and daily energy intake (Table 2Go). The significant correlation between MCH levels and daily protein intake (P = 0.02) disappeared after controlling for energy intake (P = 0.20). No other significant associations were observed between neuropeptide levels and macronutrient intake, either before or after controlling for energy intake.

Interventional studies

Fasting with and without administration of physiological doses of r-metHuLeptin. In healthy lean men, body weight did not change with isocaloric feeding (P = 0.64), but decreased by approximately 2 kg during both the fasting/placebo admission (P = 0.008) and the fasting/r-metHuLeptin admission (P = 0.06; Table 3Go). Serum leptin levels did not change in the fed state (P = 0.31), but decreased to 33.5% of baseline after fasting for 2 d (P = 0.008; Table 3Go). R-metHuLeptin administration in physiological doses prevented the fasting-induced decline in leptin levels, resulting in leptin levels within the physiological range for lean men, but slightly, although not significantly, higher than baseline levels in the fed state (target value; P = 0.06; Table 3Go).


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  		TABLE 3. Interventional study

 
Serum MCH levels did not change during the fed state (22.0 ± 5.5 vs. 20.7 ± 5.1 pg/ml; P = 0.26), but increased to 127% of baseline after subjects fasted for 2 d (20.1 ± 7.2 vs. 25.6 ± 10.0 pg/ml; P = 0.016; Table 3Go and Fig. 2Go). Specifically, seven of eight subjects showed an increase, and one subject showed no change in MCH levels during fasting. Importantly, administration of r-metHuLeptin in replacement doses prevented the fasting-induced increase in MCH levels (24.0 ± 12.2 vs. 20.5 ± 7.1 pg/ml; P = 0.63; Table 3Go and Fig. 2Go). In addition, the difference between changes in MCH levels during fasting alone vs. fasting with r-metHuLeptin administration was statistically significant (P = 0.04, by Mann-Whitney test).



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  		FIG. 2. Interventional study. Serum MCH levels expressed as percentage of the mean (±SE) baseline value (d 1). Fed, Two days of isocaloric feeding; Fasted, 2 d of fasting with placebo administration; Fasted + r-metHuLeptin, 2 d of fasting with r-metHuLeptin administration in physiological replacement doses. *, P < 0.017 vs. baseline (d 1).

 
AGRP levels tended to increase in the fasted state (P = 0.06), but did not change significantly from baseline when r-metHuLeptin was administered during fasting (P = 0.31; Table 3Go). There were no significant changes in {alpha}-MSH levels after 2 d of isocaloric feeding (P = 0.31), fasting alone (P = 0.68), or fasting with r-metHuLeptin administration (P = 0.50; Table 3Go).

Administration of pharmacological dose of r-metHuLeptin in the fed state. Serum leptin levels increased significantly by approximately 4-fold 12 h after a 0.1 mg/kg dose of r-metHuLeptin was administered in lean men (baseline, 2.1 ± 0.5 ng/ml; 12 h, 9.0 ± 2.5 ng/ml; P = 0.002) and obese men (baseline, 15.5 ± 8.6 ng/ml; 12 h, 54.0 ± 15.8 ng/ml; P = 0.002). Serum MCH levels did not change over this time frame, however (lean men baseline, 34.3 ± 6.4 µg/ml; 12 h, 39.4 ± 6.5 µg/ml; P = 0.29; obese men: baseline, 38.4 ± 6.2 µg/ml; 12 h, 37.8 ± 12.8 µg/ml; P = 0.89).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
We report for the first time that serum MCH levels, which are detectable in human sera, are independently and positively associated with BMI, fat mass, and lean mass in young healthy subjects and are higher in women than in men. Furthermore, MCH levels in normal weight individuals increase significantly in response to 2 d of fasting, but remain unchanged when r-metHuLeptin is administered during fasting to maintain serum leptin levels similar to levels observed in the fed state. In addition, serum MCH levels do not change in response to an increase in endogenous leptin levels to high physiological levels after exogenous r-metHuLeptin administration in the fed state. Serum AGRP levels also tended to increase with fasting, an effect that was not observed when r-metHuLeptin was administered during fasting. In contrast, circulating {alpha}-MSH levels were not associated with body composition parameters and did not change significantly in response to fasting for 2 d with or without r-metHuLeptin administration. Finally, serum levels of these three neuropeptides were not associated with total caloric and/or macronutrient intake.

In rodents, hypothalamic MCH expression is regulated by leptin and nutritional status, with increased MCH during starvation and in leptin-deficient (ob/ob) mice, whereas feeding and leptin replacement prevent the MCH mRNA rise in these paradigms (4). Furthermore, obese leptin receptor-deficient Zucker rats have elevated hypothalamic MCH mRNA levels and plasma MCH levels, suggesting that central and circulating MCH levels change in the same direction (17). In this study we show that serum MCH levels in humans change in response to fasting and r-metHuLeptin administration in replacement doses in the same direction as shown previously in rodents (4). It is also known that both fasting-induced hypoleptinemia and r-metHuLeptin replacement result in significant physiological changes in energy homeostasis and neuroendocrine function (28, 29). In contrast, r-metHuLeptin administration in the fed state to increase endogenous leptin levels to high physiological ranges does not alter MCH levels, nor does it change energy homeostasis (30). Therefore, all of these findings support the hypothesis that circulating MCH may serve as a peripheral marker of changes in energy balance.

MCH is synthesized in peripheral tissues, but in smaller amounts than in the hypothalamus, and peripherally derived MCH is larger than the fully processed, biologically active MCH found in the central nervous system of humans and rodents (31, 32, 33). Previous studies have shown that centrally produced MCH is secreted from hypothalamic neuron terminals (34) and crosses the blood-brain barrier (BBB) to reach the circulation (35, 36). To our knowledge, no previous study has evaluated whether MCH can be released from peripheral tissues into plasma. In addition, although hypothalamic MCH expression is regulated by leptin in rodents, MCH expression in peripheral organs of ob/ob mice was not altered by leptin administration (5). Taken together, these findings suggest that a significant proportion of circulating MCH may be of central origin, but this remains to be conclusively shown by future studies. Most importantly, serum MCH levels change in response to fasting and leptin administration in a manner consistent with changes in hypothalamic expression of MCH and, thus, leptin action.

Moreover, the positive association between MCH and fat mass detected in humans may reflect the fact that, similar to that in rodents (37, 38, 39), MCH increases food intake and/or decreases metabolism, thus resulting in increased adiposity. Given that serum MCH levels can be easily measured, these findings may have relevance for the use of this marker in the diagnosis and therapy of obese individuals. Future studies are needed to conclusively determine the exact role MCH plays in regulating energy homeostasis and fat mass in humans. In addition, other studies to accurately characterize the source(s) of serum MCH in humans and to assess whether circulating MCH may have effects of physiological importance in the periphery are required (40, 41). We also found that serum MCH levels are higher in women than in men, which can be largely explained by differences in body fat mass, although a minor gender effect independent of fat mass cannot be excluded. Additional larger studies are needed to accurately investigate this possibility.

Serum AGRP levels tended to increase after a 2-d fast, as previously reported in a study of 17 subjects (19). Similarly, central and peripheral AGRP change in the same direction after fasting and/or leptin administration in animals (6, 16). AGRP is secreted from hypothalamic neuron terminals (16) and crosses the BBB to reach the circulation (35, 42). Peripheral AGRP is expressed in significant amounts only in the adrenal cortex of humans and animals, where it probably plays a paracrine role (16, 43, 44, 45). Because bilateral adrenalectomy has no significant effect on serum AGRP levels in rats, the adrenal cortex is probably unlikely to be a major source of serum AGRP (16). Thus, serum AGRP levels may reflect central AGRP levels and represent another important peripheral marker of changes in energy balance, in addition to MCH. Based on data from a previously published study of 17 subjects (19), nine to 37 subjects are needed to provide more than 80% power to show a significant increase in AGRP levels of at least 73% after prolonging an overnight 9-h fast for 2 more hours. Our study, albeit slightly smaller (n = 8), evaluated a much longer period of food deprivation, which resulted in a highly significant decrease in leptin levels (P < 0.008) and showed a trend toward an increase in AGRP after a 2-d fast (P = 0.06). These findings merit additional investigation in larger studies using measurements of leptin and AGRP levels at several time points during prolonged food deprivation to determine whether fasting-induced hypoleptinemia results in increased AGRP levels and whether r-metHuLeptin administration decreases AGRP levels in humans.

Animal studies have reported similar changes in central and peripheral {alpha}-MSH levels in relation to fasting and/or leptin administration (7, 15), but our study failed to replicate these findings in humans. Similarly, Nam et al. (46) reported that {alpha}-MSH levels were similar in plasma and cerebrospinal fluid and did not change after weight loss in normal weight and obese subjects. Thus, regulation of serum {alpha}-MSH levels may be different in humans and animals. {alpha}-MSH is secreted from hypothalamic neuron terminals (47) and crosses the BBB (48, 49), but is also expressed in many peripheral tissues in rodents (50) and humans (51), where it may have various functions, such as modulation of inflammation (52, 53, 54). Because peripheral tissues may contribute significantly to circulating {alpha}-MSH (50, 52, 53, 54, 55, 56, 57, 58, 59), serum {alpha}-MSH levels may not accurately reflect hypothalamic {alpha}-MSH expression in humans.

Serum {alpha}-MSH and AGRP levels were similar in men and women, as previously reported (20, 21), and they were not significantly associated with body composition or leptin levels in our subjects, similar to the report by Shen et al. (19). However, a smaller Japanese study found that plasma {alpha}-MSH and AGRP levels were higher in obese subjects and correlated positively with each other and with body fat, and that plasma AGRP also correlated with leptin levels (20, 21). Whether the observed differences are due to more accurate effect estimates provided by the larger size of this study or to genetic differences in the studied populations needs to be additionally investigated.

In summary, we show that serum MCH levels are positively associated with fat mass and increase in response to fasting, but not when r-metHuLeptin is administered during fasting. We thus hypothesize that serum MCH and possibly AGRP levels may represent a useful peripheral marker of changes in energy homeostasis in humans. These findings could be of clinical importance, given the current availability of assays to measure serum levels of these neuropeptides and the ongoing efforts of pharmaceutical companies to develop agonists and/or antagonists of the MCH and AGRP/{alpha}-MSH systems for the treatment of obesity. Furthermore, the potential relationship of serum AGRP with alterations of energy homeostasis, including fasting and r-metHuLeptin administration, remains to be fully elucidated, and whether serum MCH levels can discriminate between leptin-sensitive and leptin-resistant subjects merits additional investigation.


   Acknowledgments
 
We thank the BIDMC GCRC staff for their assistance with nursing support, nutrition support, and specimen processing.


   Footnotes
 
This work was supported by NIDDK Grant DK-R01–58785, NIH Grant K30-HL-04095, NIH Grant MO1-RR-01032, as well as grants from Novartis and Amgen, Inc.

First Published Online November 16, 2004

Abbreviations: AGRP, Agouti-related protein; BBB, blood-brain barrier; BIA, bioelectrical impedance analysis; BMI, body mass index; CF, conversion factor; CV, coefficient of variation; MCH, melanin-concentrating hormone; r-metHuLeptin, recombinant methionyl human leptin; unstd, unstandardized; WC, waist circumference; WHR, waist to hip ratio.

Received June 15, 2004.

Accepted November 8, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Schwartz MW, Woods SC, Porte Jr D, Seeley RJ, Baskin DG 2000 Central nervous system control of food intake. Nature 404:661–671[Medline]
  2. Porte Jr D, Baskin DG, Schwartz MW 2002 Leptin and insulin action in the central nervous system. Nutr Rev 60:S20–S29
  3. Tritos NA, Maratos-Flier E 1999 Two important systems in energy homeostasis: melanocortins and melanin-concentrating hormone. Neuropeptides 33:339–349[CrossRef][Medline]
  4. Tritos NA, Mastaitis JW, Kokkotou E, Maratos-Flier E 2000 Characterization of melanin concentrating hormone and preproorexin expression in the murine hypothalamus. Brain Res 895:160–166
  5. Huang Q, Viale A, Picard F, Nahon JL, Richard D 1999 Effects of leptin on melanin-concentrating hormone expression in the brain of lean and obese Lepob/literepob mice. Neuroendocrinology 69:145–153[CrossRef][Medline]
  6. Ebihara K, Ogawa Y, Katsuura G, Numata Y, Masuzaki H, Satoh N, Tamaki M, Yoshioka T, Hayase M, Matsuoka N, Aizawa-Abe M, Yoshimasa Y, Nakao K 1999 Involvement of agouti-related protein, an endogenous antagonist of hypothalamic melanocortin receptor, in leptin action. Diabetes 48:2028–2033[Abstract]
  7. Schwartz MW, Seeley RJ, Woods SC, Weigle DS, Campfield LA, Burn P, Baskin DG 1997 Leptin increases hypothalamic pro-opiomelanocortin mRNA expression in the rostral arcuate nucleus. Diabetes 46:2119–2123[Abstract]
  8. Mihály E, Fekete C, Tatro JB, Liposits Z, Stopa EG, Lechan RM 2000 Hypophysiotropic thyrotropin-releasing hormone-synthesizing neurons in the human hypothalamus are innervated by neuropeptide Y, agouti-related protein, and {alpha}-melanocyte-stimulating hormone. J Clin Endocrinol Metab 85:2596–2603[Abstract/Free Full Text]
  9. Goldstone AP, Unmehopa UA, Bloom SR, Swaab DF 2002 Hypothalamic NPY and agouti-related protein are increased in human illness but not in Prader-Willi syndrome and other obese subjects. J Clin Endocrinol Metab 87:927–937[Abstract/Free Full Text]
  10. Bagnol D, Lu XY, Kaelin CB, Day HEW, Ollmann M, Grantz 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
  11. Haskell-Luevano C, Chen P, Li C, Chang K, Smith MS, Cameron JL, Cone RD 1999 Characterization of the neuroanatomical distribution of agouti-related protein immunoreactivity in the rhesus monkey and the rat. Endocrinology 140:1408–1415[Abstract/Free Full Text]
  12. Mouri T, Takahashi K, Kawauchi H, Sone M, Totsune K, Murakami O, Itoi K, Ohneda M, Sasano H, Sasano N 1993 Melanin-concentrating hormone in the human brain. Peptides 14:643–646[CrossRef][Medline]
  13. Takahashi K, Suzuki H, Totsune K, Murakami O, Satoh F, Sone M, Sasano H, Mouri T, Shibahara S 1995 Melanin-concentrating hormone in human and rat. Neuroendocrinology 61:493–498[Medline]
  14. Knigge KM, Baxter-Grillo D, Speciale J, Wagner J 1996 Melanotropic peptides in the mammalian brain: the melanin-concentrating hormone. Peptides 17:1063–1073[CrossRef][Medline]
  15. Forbes S, Bui S, Robinson BR, Hochgeshwender U, Brennan MB 2001 Integrated control of appetite and fat metabolism by the leptin-proopiomelanocortin pathway. Proc Natl Acad Sci USA 98:4233–4237[Abstract/Free Full Text]
  16. Li JY, Finniss S, Yang YK, Zeng Q, Qu SY, Barsh G, Dickinson C, Gantz I 2000 Agouti-related protein-like immunoreactivity: characterization of release from hypothalamic tissue and presence in serum. Endocrinology 141:1942–1950[Abstract/Free Full Text]
  17. Stricker-Kongrad A, Dimitrov T, Beck B 2001 Central and peripheral dysregulation of melanin-concentrating hormone in obese Zucker rats. Brain Res Mol Brain Res 92:43–48[Medline]
  18. Sun G, Tian Z, Murata T, Narita K, Honda K, Higuchi T 2004 Central and peripheral immunoreactivity of melanin-concentrating hormone in hypothalamic obese and lactating rats. J Neuroendocrinol 16:79–83[CrossRef][Medline]
  19. Shen CP, Wu KK, Shearman LP, Camacho R, Tota MR, Fong TM, Van der Ploeg LH 2002 Plasma agouti-related protein level: a possible correlation with fasted and fed states in humans and rats. J Neuroendocrinol 14:607–610[CrossRef][Medline]
  20. Katsuki A, Sumida Y, Murashima S, Furuta M, Araki-Sasaki R, Tsuchihashi K, Hori Y, Yano Y, Adachi Y 2000 Elevated plasma levels of {alpha}-melanocyte stimulating hormone ({alpha}-MSH) are correlated with insulin resistance in obese men. Int J Obes Relat Metab Disord 24:1260–1264[CrossRef][Medline]
  21. Katsuki A, Sumida Y, Gabazza EC, Murashima S, Tanaka T, Furuta M, Araki-Sasaki R, Hori Y, Nakatani K, Yano Y, Adachi Y 2001 Plasma levels of agouti-related protein are increased in obese men. J Clin Endocrinol Metab 86:1921–1924[Abstract/Free Full Text]
  22. Segal KR, Van Loan M, Fitzgerald PI, Hodgdon JA, Van Itallie TB 1988 Lean body mass estimation by bioelectrical impedance analysis: a four-site cross-validation study. Am J Clin Nutr 47:7–14[Abstract/Free Full Text]
  23. Gavrila A, Chan JL, Yiannakouris N, Kontogianni M, Miller LC, Orlova C, Mantzoros CS 2003 Serum adiponectin levels are inversely associated with overall and central fat distribution but are not directly regulated by acute fasting or leptin administration in humans: cross-sectional and interventional studies. J Clin Endocrinol Metab 88:4823–4831[Abstract/Free Full Text]
  24. Yannakoulia M, Yiannakouris N, Bluher S, Matalas AL, Klimis-Zacas D, Mantzoros CS 2003 Body fat mass and macronutrient intake in relation to circulating soluble leptin receptor, free leptin index, adiponectin, and resistin concentrations in healthy humans. J Clin Endocrinol Metab 88:1730–1736[Abstract/Free Full Text]
  25. Xue B, Zemel MB 2000 Relationship between human adipose tissue agouti and fatty acid synthase (FAS). J Nutr 130:2478–2481[Abstract/Free Full Text]
  26. Airaghi L, Garofalo L, Cutuli MG, Delgado R, Carlin A, Demitri MT, Badalamenti S, Graziani G, Lipton JM, Catania A 2000 Plasma concentrations of {alpha}-melanocyte-stimulating hormone are elevated in patients on chronic haemodialysis. Nephrol Dial Transplant 15:1212–1216[Abstract/Free Full Text]
  27. Willett W, Stampfer M 1998 Implications of total energy intake for epidemiologic analyses. In: Willett W, ed. Nutritional epidemiology. 2nd ed. New York: Oxford University Press; 273–301
  28. Chan JL, Heist K, DePaoli AM, Veldhuis JD, Mantzoros CS 2003 The role of falling leptin levels in the neuroendocrine and metabolic adaptation to short-term starvation in healthy men. J Clin Invest 111:1409–1421[CrossRef][Medline]
  29. Welt CK, Chan JL, Bullen J, Murphy R, Smith P, DePaoli AM, Karalis A, Mantzoros CS 2004 Recombinant human leptin administration in women with hypothalamic amenorrhea. N Engl J Med 351:987–997[Abstract/Free Full Text]
  30. Heymsfield SB, Greenberg AS, Fujioka K, Dixon RM, Kushner R, Hunt T, Lubina JA, Patane J, Self B, Hunt P, McCamish M 1999 Recombinant leptin for weight loss in obese and lean adults: a randomized, controlled, dose-escalation trial. JAMA 282:1568–1575[Abstract/Free Full Text]
  31. Hervieu G, Nahon JL 1995 Pro-melanin concentrating hormone messenger ribonucleic acid and peptides expression in peripheral tissues of the rat. Neuroendocrinology 61:348–364[Medline]
  32. Hervieu G, Volant K, Grishina O, Descroix-Vagne M, Nahon JL 1996 Similarities in cellular expression and functions of melanin-concentrating hormone and atrial natriuretic factor in the rat digestive tract. Endocrinology 137:561–571[Abstract]
  33. Viale A, Zhixing Y, Breton C, Pedeutour F, Coquerel A, Jordan D, Nahon JL 1997 The melanin-concentrating hormone gene in human: flanking region analysis, fine chromosome mapping, and tissue-specific expression. Brain Res Mol Brain Res 46:243–255[Medline]
  34. Parkes D, Vale W 1992 Secretion of melanin-concentrating hormone and neuropeptide-EI from cultured rat hypothalamic cells. Endocrinology 131:1826–1831[Abstract/Free Full Text]
  35. Kastin AJ, Pan W, Maness LM, Banks WA 1999 Peptides crossing the blood-brain barrier: some unusual observations. Brain Res 848:96–100[CrossRef][Medline]
  36. Kastin AJ, Akerstrom V, Hackler L, Zadina JE 2000 Phe13,Tyr19-melanin-concentrating hormone and blood-brain barrier: role of protein binding. J Neurochem 74:385–391[CrossRef][Medline]
  37. Ludwig DS, Tritos NA, Mastaitis JW, Kulkarni R, Kokkotou E, Elmquist J, Lowell B, Flier JS, Maratos-Flier E 2001 Melanin-concentrating hormone overexpression in transgenic mice leads to obesity and insulin resistance. J Clin Invest 107:379–386[Medline]
  38. Gomori A, Ishihara A, Ito M, Mashiko S, Matsushita H, Yumoto M, Ito M, Tanaka T, Tokita S, Moriya M, Iwaasa H, Kanatani A 2002 Chronic intracerebroventricular infusion of MCH causes obesity in mice. Am J Physiol 284:E583–E588
  39. Ito M, Gomori A, Ishihara A, Oda Z, Mashiko S, Matsushita H, Yumoto M, Ito M, Sano H, Tokita S, Moriya M, Iwaasa H, Kanatani A 2003 Characterization of MCH-mediated obesity in mice. Am J Physiol 284:E940–E945
  40. Bradley RL, Kokkotou EG, Maratos-Flier E, Cheatham B 2000 Melanin-concentrating hormone regulates leptin synthesis and secretion in rat adipocytes. Diabetes 49:1073–1077[Abstract]
  41. Hoogduijn MJ, Ancans J, Suzuki I, Estdale S, Thody AJ 2002 Melanin-concentrating hormone and its receptor are expressed and functional in human skin. Biochem Biophys Res Commun 296:698–701[CrossRef][Medline]
  42. Kastin AJ, Akerstrom V, Hackler L 2000 Agouti-related protein (83–132) aggregates and crosses the blood-brain barrier slowly. Metabolism 49:1444–1448[CrossRef][Medline]
  43. Shutter JR, Graham M, Kinsey AC, Scully S, Lthy R, Stark KL 1997 Hypothalamic expression of ART, a novel gene related to agouti, is up-regulated in obese and diabetic mutant mice. Genes Dev 11:593–602[Abstract/Free Full Text]
  44. Dhillo WS, Small CJ, Gardiner JV, Bewick GA, Whitworth EJ, Jethwa PH, Seal LJ, Ghatei MA, Hinson JP, Bloom SR 2003 Agouti-related protein has an inhibitory paracrine role in the rat adrenal gland. Biochem Biophys Res Commun 301:102–107[CrossRef][Medline]
  45. 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]
  46. Nam SY, Kratzsch J, Kim KW, Kim KR, Lim SK, Marcus C 2001 Cerebrospinal fluid and plasma concentrations of leptin, NPY, and {alpha}-MSH in obese women and their relationship to negative energy balance. J Clin Endocrinol Metab 86:4849–4853[Abstract/Free Full Text]
  47. Bunel DT, Delbende C, Blasquez C, Jégou S, Vaudry H 1990 Characterization of {alpha}-melanocyte-stimulating hormone ({alpha}-MSH)-like peptides in discrete regions of the rat brain. In vitro release of {alpha}-MSH from perifused hypothalamus and amygdala. Brain Res 513:299–307[CrossRef][Medline]
  48. De Rotte AA, Bouman HJ, Van Wimersma Greidanus TB 1980 Relationships between {alpha}-MSH levels in blood and in cerebrospinal fluid. Brain Res Bull 5:375–381[CrossRef][Medline]
  49. Wilson JF, Anderson S, Snook G, Llewellyn KD 1984 Quantification of the permeability of the blood-CSF barrier to {alpha}-MSH in the rat. Peptides 5:681–685[CrossRef][Medline]
  50. DeBold CR, Nicholson WE, Orth DN 1988 Immunoreactive proopiomelanocortin (POMC) peptides and POMC-like messenger ribonucleic acid are present in many rat non-pituitary tissues. Endocrinology 122:2648–2657[Abstract/Free Full Text]
  51. De Keyzer Y, Lenne F, Massias JF, Vieau D, Luton JP, Kahn A, Bertagna X 1990 Pituitary-like proopiomelanocortin transcripts in human Leydig cell tumors. J Clin Invest 86:871–877
  52. Norman D, Isidori AM, Frajese V, Caprio M, Chew SL, Grossman AB, Clark AJ, Michael Besser G, Fabbri A 2003 ACTH and {alpha}-MSH inhibit leptin expression and secretion in 3T3–L1 adipocytes: model for a central-peripheral melanocortin-leptin pathway. Mol Cell Endocrinol 200:99–109[CrossRef][Medline]
  53. Oktar BK, Alican I 2002 Modulation of the peripheral and central inflammatory responses by {alpha}-melanocyte stimulating hormone. Curr Protein Pept Sci 3:623–628[CrossRef][Medline]
  54. Moustafa M, Szabo M, Ghanem GE, Morandini R, Kemp EH, MacNeil S, Haycock JW 2002 Inhibition of tumor necrosis factor-{alpha} stimulated NF{kappa}B/p65 in human keratinocytes by {alpha}-melanocyte stimulating hormone and adrenocorticotropic hormone peptides. J Invest Dermatol 119:1244–1253[CrossRef][Medline]
  55. Blalock JE 1999 Proopiomelanocortin and the immune-neuroendocrine connection. Ann NY Acad Sci 885:161–172[CrossRef][Medline]
  56. Ichiyama T, Sato S, Okada K, Catania A, Lipton JM 2000 The neuroimmunomodulatory peptide {alpha}-MSH. Ann NY Acad Sci 917:221–226[Medline]
  57. Tsatmali M, Ancans J, Yukitake J, Thody AJ 2000 Skin POMC peptides: their actions at the human MC-1 receptor and roles in the tanning response. Pigment Cell Res 13(Suppl 8):125–129
  58. Luger TA, Brzoska T, Scholzen TE, Kalden DH, Sunderkotter C, Armstrong C, Ansel J 2000 The role of {alpha}-MSH as a modulator of cutaneous inflammation. Ann NY Acad Sci 917:232–238[Medline]
  59. Colombo G, Buffa R, Bardella MT, Garofalo L, Carlin A, Lipton JM, Catania A 2002–2003 Anti-inflammatory effects of {alpha}-melanocyte-stimulating hormone in celiac intestinal mucosa. Neuroimmunomodulation 10:208–216



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