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Plasma Levels of Agouti-Related Protein Are Increased in Obese Men

Third Department of Internal Medicine (A.K., Y.S., E.C.G., T.T., M.F., R.A.-S., Y.H., Y.Y., Y.A.), Department of Radiology (S.M.) and Department of Laboratory Medicine (K.N.), Mie University School of Medicine, Mie 514-8507, Japan

Address all correspondence and requests for reprints to: Akira Katsuki, M.D., Third Department of Internal Medicine, Mie University School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan. E-mail: katuki-a{at}-clin.medic.mie-u.ac.jp

	Abstract

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To investigate the relationship between peripheral blood levels of agouti-related protein (AGRP) and various parameters of obesity, we measured the plasma level of AGRP in 15 obese and 15 nonobese men and evaluated its relationship with body mass index (BMI), body fat weight, and visceral, sc, and total fat areas measured by computed tomography, fasting insulin levels, glucose infusion rate during an euglycemic hyperinsulinemic clamp study, serum leptin, and plasma -MSH.

Obese men had significantly higher plasma concentrations of AGRP than nonobese men (P < 0.01). Univariate analysis showed that the plasma levels of AGRP are proportionally correlated with BMI, body fat weight, and sc fat area in obese men (BMI: r = 0.732, P < 0.01; body fat weight: r = 0.603, P < 0.02; sc fat area: r = 0.668, P < 0.01) and in all men (BMI: r = 0.839, P < 0.0001; body fat weight: r = 0.818, P < 0.0001; sc fat area: r = 0.728, P < 0.0001). In all men, the plasma levels of AGRP were significantly correlated with the visceral fat area (r = 0.478, P < 0.01), total fat area (r = 0.655, P < 0.0001), fasting insulin level (r = 0.488, P < 0.01), glucose infusion rate (r = -0.564, P < 0.01), serum level of leptin (r = 0.661, P < 0.0001), and the plasma level of -MSH (r = 0.556, P < 0.01). In all subjects, multiple regression analysis showed that the plasma levels of AGRP are significantly (F = 15.522, r = 0.801, P < 0.03) correlated with the plasma levels of -MSH, independently from the total fat area. However, the correlation between plasma levels of AGRP and serum levels of leptin was found to be dependent on the total fat area.

In brief, these findings showed that the circulating levels of AGRP are increased in obese men and that they are correlated with various parameters of obesity. Although correlation does not prove causation, the results of this study suggest that peripheral AGRP may play a role in the pathogenesis of obesity.

	Introduction

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PREVIOUS STUDIES HAVE shown that the hypothalamic agouti-related protein (AGRP) regulates body weight via the melanocortin-4 receptor in mice, and that AGRP is present in the systemic circulation in rats (1, 2, 3, 4, 5, 6, 7). In chickens, AGRP messenger RNA has been detected in various tissues, including the brain, adrenal glands, skeletal muscle, and adipose tissue, suggesting the potential involvement of AGRP in the regulation of the peripheral melanocortin system (8).

In humans, AGRP has been detected also in the brain, adrenal glands, lung, and testis (9). However, the circulating levels of AGRP and the potential role of this protein in obesity have not been as yet reported.

In the present study, we measured the plasma levels of AGRP in obese and nonobese men to investigate the relationship between the plasma levels of AGRP and various parameters of obesity.

	Subjects and Methods

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Subjects

This study comprised 15 men with obesity [body mass index (BMI) 25.0 kg/m²] and 15 age-matched nonobese men (BMI < 25.0) (Table 1).

None of the subjects had diabetes mellitus, according to the diagnostic criteria of the American Diabetes Association and results from a 75-g oral glucose tolerance test (G 75; Trelan, Shimizu, Japan) (10).

None of the subjects were receiving any medication that could affect insulin levels or insulin sensitivity, and they were not under any exercise or dietary therapy before the beginning of this study.

Informed consent was obtained from all subjects before the beginning of the study.

Study protocol and methods

Body fat weight, body fat areas, insulin sensitivity, blood pressure, and several variables measured in blood samples were evaluated in all men. In all women, we evaluated blood pressure and several variables in blood samples. Venous blood was collected before breakfast, in the early morning, after overnight bed rest. After centrifugation, the plasma and serum samples were separated in small aliquots and then frozen at -70 C until use.

AGRP in plasma samples was measured using a commercially available RIA kit (AGRP (83–132)-NH2 (Human) RIA Kit; Phoenix, Mountain View, CA). Briefly, 100 µL of standard or plasma samples were added to tubes coated with primary antibody (rabbit antipeptide serum) and incubated at 4 C for 18 h. Thereafter, 100 µL ¹²⁵I labeled peptide were added and incubated at 4 C for an additional 18 h. One hundred microliters of goat antirabbit IgG serum and normal rabbit serum were added to each tube and incubated for 90 min at room temperature. After centrifugation at 3000 rpm for 20 min at 4 C, the supernatant radioactivity was counted. The values of plasma AGRP levels were then extrapolated from a curve drawn using standard concentrations of AGRP. This assay recognized both AGRP (83–132)-NH2 and AGRP C-NH2 and showed no significant cross-reactivity with, or interference by, other factors related to AGRP [leptin, orexin A, orexin B, neuropeptide Y, -MSH, melanin-concentrating hormone, and calcitonin gene related peptide (CGRP)]. The intra- and interassay coefficients of variation were 6.0 and 8.9%, respectively. To evaluate day-to-day variability, we also measured the peptide, 14 days later, but no significant changes were observed (obese men, 14.7 ± 1.7 vs. 14.2 ± 2.2 pmol/L; nonobese men, 5.1 ± 0.4 vs. 5.6 ± 0.4 pg/mL). Serum leptin levels were measured using a human leptin RIA kit (Linco Research, Inc., St. Charles, MO). The detection limit of this assay was 0.5 ng/mL, and the intra- and interassay coefficients of variation were 4.6 and 5.0%, respectively. Measurement of plasma -MSH was also done using an RIA kit (Eurodiagnostica, Malmo, Sweden). The detection limit of this assay was 3 pmol/L, and the intra- and interassay coefficients of variation were 11.8 and 13.0%, respectively. Blood glucose was measured by an automated enzymatic method, HbA1c (normal value, 4.3–5.8%) by high-performance liquid chromatography, and serum insulin was measured using an immunoradiometric assay kit (DAINABOT Corp., Tokyo, Japan). In addition, we measured blood pressure, in supine position, after a rest of 5 min.

Insulin resistance was evaluated by the euglycemic hyperinsulinemic clamp technique, using an artificial pancreas (STG-22; Nikkiso, Tokyo, Japan) (11). At 0800 h, a priming dose of insulin (Humulin R; Shionogi, Osaka, Japan) was administered during the initial 10 min, in a logarithmically decreasing manner, to rapidly raise serum insulin to the desired level (1200 pmol/L); this level was then maintained by continuous infusion of insulin at a rate of 13.44 pmol/kg·min for 120 min. The mean insulin level from 90–120 min after starting the clamp study was stable (obese group, 1186.45 ± 43.42 pmol/L; nonobese group, 1188.30 ± 54.76 pmol/L). Blood glucose was monitored continuously and maintained at the target clamp level (5.24 mmol/L) by infusing 10% glucose. The mean amount of glucose given during the last 30 min was defined as the glucose infusion rate (GIR), and it was used as a measure of peripheral insulin sensitivity.

Body fat weight was measured by bioelectric impedance using a TBF-101 (Tanita/Stellar Innovations, Inc., Tokyo, Japan).

Body fat area was evaluated by a previously described method (12). The total cross-sectional area and the intraabdominal visceral fat and sc fat areas were measured, in all subjects, by abdominal computed tomography scanning taken at the umbilical level. Any ip region having the same density as the sc fat layer was defined as a visceral fat area.

Statistical methods

Data were expressed as the mean ± SE. Comparisons between obese and nonobese subjects were done using the Mann-Whitney U test. Correlations were evaluated by univariate and multivariate analyses. All statistical analyses were performed with the StatView 4.0 software program (Abacus Concepts, Berkeley, CA) for the Macintosh. P < 0.05 was taken as statistically significant.

	Results

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The plasma concentrations of AGRP were significantly increased in obese men, compared with nonobese men (P < 0.01) (Fig. 1). The plasma concentrations of AGRP were proportionally correlated with BMI in obese (r = 0.732, P < 0.01) and all (obese and nonobese, r = 0.839, P < 0.0001) subjects (Fig. 2). The plasma levels of AGRP were significantly correlated with the body fat weight and sc fat area in obese men (body fat weight: r = 0.603, P < 0.02; sc fat area: r = 0.668, P < 0.01) and all men (body fat weight: r = 0.818, P < 0.0001; sc fat area: r = 0.728, P < 0.0001). The plasma levels of AGRP were significantly correlated with the visceral fat area (r = 0.478, P < 0.01), total fat area (r = 0.655, P < 0.0001), fasting insulin level (r = 0.488, P < 0.01), GIR (r = -0.564, P < 0.01), the serum level of leptin (r = 0.661, P < 0.0001, Fig. 3), and the plasma level of -MSH (r = 0.556, P < 0.01, Fig. 4) in all men. In all men, multiple regression analysis showed that the plasma levels of AGRP are significantly (F = 15.522, R = 0.801, P < 0.03) correlated with the plasma levels of -MSH, independently from the total fat area. However, the correlation between the plasma levels of AGRP and the serum levels of leptin was found to depend on the total fat area.

View larger version (12K): [in this window] [in a new window]   Figure 1. Plasma levels of AGRP in obese and nonobese men. Plasma levels of AGRP in obese men were significantly increased, compared with those in nonobese men (P < 0.01).

View larger version (14K): [in this window] [in a new window]   Figure 2. Correlation between the plasma levels of AGRP and BMI in all (obese and nonobese) men. A significant positive correlation was observed between the plasma levels of AGRP and BMI (y = 1.374x -25.435, r = 0.839, P < 0.0001). , Nonobese men; •, obese men.

View larger version (14K): [in this window] [in a new window]   Figure 3. Correlation between the plasma levels of AGRP and the serum levels of leptin in all (obese and nonobese) men. There was a significant correlation between the plasma levels of AGRP and the serum levels of leptin (y = 1.476x + 1.785, r = 0.661, P < 0.0001). , Nonobese men; •, obese men.

View larger version (14K): [in this window] [in a new window]   Figure 4. Correlation between the plasma levels of AGRP and -MSH in all (obese and nonobese) men. A significant positive correlation was observed between the plasma levels of AGRP and -MSH (y = 0.463x + 4.158, r = 0.556, P < 0.01). , Nonobese men; •, obese men.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The present study showed that the plasma levels of AGRP are significantly increased in obese men and that they are significantly correlated with various parameters of obesity. The mechanism by which AGRP increases in the systemic circulation of obese subjects and the cellular source of AGRP remain unknown.

Several studies have demonstrated that central AGRP and leptin are involved in feeding behavior (13, 14). AGRP is up-regulated by leptin deficiency and causes hyperphagia; an inverse correlation has been reported between AGRP and leptin in hypothalamus (15). In contrast to the central nervous system, a significant and positive correlation was observed between AGRP and leptin in the systemic circulation of all men.

In the present study, the plasma levels of AGRP were not significantly different between men and women. Based on these results, it may be inferred that testis is not the major source of the plasma levels of AGRP. Further studies must be carried out to clarify the mechanism of the increased circulating levels and the cellular source of AGRP in obese subjects.

Several studies have been recently reported regarding the peripheral actions of -MSH (16, 17, 18, 19, 20). Much attention has been particularly focused on the role of peripheral -MSH in obesity (21, 22). We previously reported that the circulating levels of -MSH are significantly increased in obese men and that they may influence leptin action via MC4R in the central nervous system (22). In accordance with this, the plasma levels of -MSH were positively correlated with BMI (r = 0.608, P < 0.05), fasting insulin levels (r = 0.561, P < 0.05), and visceral fat area (r = 0.606, P < 0.05), but negatively correlated with GIR (r = -0.584, P < 0.05) in obese men. Both AGRP and -MSH can bind to MC4R (23, 24); this may explain the significant correlation observed between the plasma levels of AGRP and -MSH in all men. The peripheral effect of AGRP have not been as yet defined, but it may influence leptin action or increase food intake in obesity.

In brief, the present study showed, for the first time, that the circulating level of AGRP is increased in obese subjects and that it is significantly correlated with several variables of obesity. These findings suggest that increased circulating levels of AGRP may be involved in the pathogenesis of obesity.

Received August 21, 2000.

Revised November 16, 2000.

Accepted November 27, 2000.

	References

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1. Li JY, Finniss S, Yang YK, et al. 2000 Agouti-related protein-like immunoreactivity: characterization of release from hypothalamic tissue and presence in serum. Endocrinology. 141:1942–1950.[Abstract/Free Full Text]
2. Dinulescu DM, Cone RD. 2000 Agouti and agouti-related protein: analogies and contrasts. J Biol Chem. 275:6695–6698.[Abstract/Free Full Text]
3. Kalra SP, Dube MG, Pu S, Xu B, Horvath TL, Kalra PS. 1999 Interacting appetite-regulating pathways in the hypothalamic regulation of body weight. Endocr Rev. 20:68–100.[Abstract/Free Full Text]
4. Loftus TM. 1999 An adipocyte-central nervous system regulatory loop in the control of adipose homeostasis. Semin Cell Dev Biol. 10:11–18.[CrossRef][Medline]
5. Marsh DJ, Hollopeter G, Huszar D, et al. 1999 Response of melanocortin-4 receptor-deficient mice to anorectic and orexigenic peptides. Nat Genet. 21:119–122.[CrossRef][Medline]
6. Graham M, Shutter JR, Sarmiento U, Sarosi I, Stark KL. 1997 Overexpression of AGRT leads to obesity in transgenic mice. Nat Genet. 17:273–274.[CrossRef][Medline]
7. Ollmann MM, Wilson BD, Yang Y-K, et al. 1997 Antagonism of central melanocortin receptors in vitro and in vivo by agouti-related protein. Science. 278:135–138.[Abstract/Free Full Text]
8. Takeuchi S, Teshigawara K, Takahashi S. 2000 Widespread expression of agouti-related protein (AGRP) in the chicken: a possible involvement of AGRP in regulating peripheral melanocortin systems in the chicken. Biochim Biophys Acta. 1496:261–269.[Medline]
9. Shutter JR, Graham M, Kinsey AC, Scully S, Luthy 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]
10. The Expert Committee on the Diagnosis and Classification of Diabetes Mellitus. 1997 Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus. Diabetes Care. 10:1183–1197.
11. DeFronzo RA, Tobin JD, Andres R. 1979 Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol. 237:E214–E223.
12. Tokunaga K, Matsuzawa Y, Ishikawa K, Tarui S. 1983 A novel technique for the determination of body fat by computed tomography. Int J Obes. 7:437–445.[Medline]
13. Friedman JM. 2000 Obesity in the new millennium. Nature. 404:632–634.[Medline]
14. Schwartz MW, Woods SC, Porte Jr D, Seeley RJ, Baskin DG. 2000 Central nervous system control of food intake. Nature. 404:661–671.[Medline]
15. Ebihara K, Ogawa Y, Katsuura G, et al. 1999 Involvement of agouti-related protein, an endogenous antagonist of hypothalamic melanocortin receptor, in leptin action. Diabetes. 48:2028–2033.[Abstract]
16. Shimizu H, Tanaka Y, Sato N, Mori M. 1995 -melanocyte-stimulating hormone (MSH) inhibits insulin secretion in HIT-T 15 cells. Peptides. 16:605–608.[CrossRef][Medline]
17. Lebovitz HE, Genuth S, Pooler K. 1966 Relationships between the structure and biological activities of corticotropin and related peptides: hyperglycemic action of N-acetylated corticotropin related peptides. Endocrinology. 79:635–642.[Medline]
18. Rajora N, Boccoli G, Burns D, Sharma S, Catania AP, Lipton JM. 1997 -MSH modulates local and circulating tumor necrosis factor- in experimental brain inflammation. J Neurosci. 17:2181–2186.[Abstract/Free Full Text]
19. Airaghi L, Capra R, Pravettoni G, et al. 1999 Elevated concentrations of plasma -melanocyte stimulating hormone are associated with reduced disease progression in HIV-infected patients. J Lab Clin Med. 133:309–315.[CrossRef][Medline]
20. Catania A, Airaghi L, Garofalo L, Cutuli M, Lipton JM. 1998 The neuropeptide -MSH in AIDS and other conditions in humans. Ann NY Acad Sci. 840:848–856.[CrossRef][Medline]
21. Yaswen L, Diehl N, Brennan MB, Hochgeschweder U. 1999 Obesity in the mouse model of pro-opiomelanocortin deficiency. Nat Med. 9:1066–1070.
22. Katsuki A, Sumida Y, Murashima S, et al. 2000 Elevated plasma levels of -melanocyte stimulating hormone (-MSH) are correlated with insulin resistance in obese men. Int J Obes. 24:1260–1264.
23. Yang Y, Dickinson CJ, Zeng Q, Li J-Y, Thompson DA, Gantz I. 1999 Contribution of melanocortin receptor exoloops to agouti-related protein binding. J Biol Chem. 274:14100–14106.[Abstract/Free Full Text]
24. Yang Y, Thompson DA, Dickinson CJ, et al. 1999 Characterization of agouti-related protein binding to melanocortin receptors. Mol Endocrinol. 13:148–155.[Abstract/Free Full Text]

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