Plasma Adiponectin Levels Are Not Associated ... - Wiley Online Library

Plasma Adiponectin Levels Are Not Associated with Fat Oxidation in Humans Norbert Stefan,* Barbora Vozarova,* Tohru Funahashi,† Yuji Matsuzawa,† Eric Ravussin,‡ Christian Weyer,* and P. Antonio Tataranni*

Abstract STEFAN, NORBERT, BARBORA VOZAROVA, TOHRU FUNAHASHI, YUJI MATSUZAWA, ERIC RAVUSSIN, CHRISTIAN WEYER, AND P. ANTONIO TATARANNI. Plasma adiponectin levels are not associated with fat oxidation in humans. Obes Res. 2002;10:1016 –1020. Objective: To test the hypothesis that low adiponectin is associated with low fat oxidation in humans. Research Methods and Procedures: We measured plasma adiponectin concentrations in 75 healthy, nondiabetic Pima Indians (age, 28 ⫾ 7 years; 55 men and 20 women; body fat, 29.7 ⫾ 7.5%) and 18 whites [(age, 33 ⫾ 8 years; 14 men and 4 women; body fat, 28.2 ⫾ 10.8% (means ⫾ SD)] whose body composition was measured by DXA and 24hour energy expenditure (24-hour EE) by a respiratory chamber. Respiratory quotient (an estimate of whole-body carbohydrate/lipid oxidation rate) was calculated over 24 hours (24-hour RQ). Results: Before correlational analyses, waist-to-thigh ratio (WTR) and percentage of body fat (PFAT) were adjusted for age, sex, and race; 24-hour EE was adjusted for fat mass and fat-free mass, and 24-hour RQ were adjusted for energy balance. Plasma adiponectin concentrations were negatively correlated with WTR (r ⫽ ⫺0.42, p ⬍ 0.0001) and PFAT (r ⫽ ⫺0.46, p ⬍ 0.0001). There was no correlation between plasma adiponectin concentrations and 24-hour RQ, (r ⫽ 0.09, p ⫽ 0.36) before or after adjustment for PFAT (r ⫽ 0.001, p ⫽ 0.99, respectively, partial correlation), and no correlation was found between plasma adiponectin concentrations and 24-hour EE (r ⫽ ⫺0.12, p ⫽ 0.27).

Received for review December 31, 2001. Accepted for publication in final form May 3, 2002. *Clinical Diabetes and Nutrition Section, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Phoenix, Arizona; †Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, Osaka, Japan; and ‡Pennington Biomedical Research Center, Baton Rouge, Louisiana. Address correspondence to Norbert Stefan, M.D., Clinical Diabetes and Nutrition Section, National Institutes of Health, 4212 North 16th Street, Rm. 5-41, Phoenix, AZ 85016. E-mail: [email protected] Copyright © 2002 NAASO

1016

OBESITY RESEARCH Vol. 10 No. 10 October 2002

Discussion: Our cross-sectional data do not suggest physiological concentrations of fasting plasma adiponectin play a role in the regulation of whole-body fat oxidation or energy expenditure in resting conditions. Whether administration of adiponectin to individuals with low levels of this hormone will increase their fat oxidation rates/energy expenditure remains to be established. Key words: respiratory quotient, adipocytokines, energy expenditure, ethnicity

Introduction Obesity is commonly associated with considerable morbidity, including hypertension, type 2 diabetes, and cardiovascular diseases (1,2). Adipose tissue not only serves as an energy storage organ but also secretes hormones and metabolites that are thought to regulate insulin sensitivity and energy metabolism (3,4). Adiponectin, the most abundant adipose-specific protein, has been found to be exclusively expressed in adipose tissue (5,6). It has been proposed that adiponectin has antiatherogenic effects (7,8), and plasma adiponectin concentrations were shown to be decreased in Japanese individuals with obesity (9) and type 2 diabetes (10). Low plasma adiponectin concentrations were shown to be associated with obesity and type 2 diabetes in Pima Indians and whites (11). In that study, the degree of plasma hypoadiponectinemia was more closely related to the degree of hyperinsulinemia and insulin resistance than to the degree of adiposity (11). However, in the absence of intervention studies of adiponectin administration to humans, little is known regarding the possible mechanisms, if any, of adiponectin action in vivo. Recent studies show that acute treatment of mice with the murine adiponectin homologue Acrp30 increases lipid oxidation rates in the muscle of these animals (12,13). To test the hypothesis that low adiponectin is associated with low fat oxidation in humans, we measured fasting

Adiponectin And Energy Metabolism, Stefan et al.

Table 1. Physical and metabolic characteristics of the study population (means ⫾ SD)

N (male/female) Age (year) Height (cm) Body weight (kg) BMI (kg/m2) Body fat (%) Fat mass (kg) Fat-free mass (kg) Waist-to-thigh ratio Adiponectin (␮g/ml) 24-hour EE (kcal/day) 24-hour RQ

All

Whites

Pima Indians

p

93 29 (19 to 44) 169 (151 to 187) 90.3 (49.5 to 147.7) 31.6 (19.6 to 49.5) 29.4 (11.8 to 47.0) 32.5 (8.4 to 65.1) 58.1 (29.6 to 85.5) 1.62 (1.33 to 1.92) 7.9 (3.1 to 19.8) 2314 (1359 to 3345) 0.86 (0.81 to 0.90)

18 (14/4) 33 ⫾ 8 172 ⫾ 10 95.2 ⫾ 25.5 32.2 ⫾ 9.1 28.2 ⫾ 10.8 33.7 ⫾ 20.0 61.7 ⫾ 11.7 1.55 ⫾ 0.12 10.5 ⫾ 4.5 2371 ⫾ 455 0.86 ⫾ 0.02

75 (55/20) 28 ⫾ 7 168 ⫾ 8 89.1 ⫾ 18.5 31.4 ⫾ 6.3 29.7 ⫾ 7.5 32.2 ⫾ 12.7 57.2 ⫾ 9.7 1.63 ⫾ 0.13 7.2 ⫾ 2.8 2301 ⫾ 346 0.85 ⫾ 0.02

⬍0.01 0.06 0.21 0.55 0.61 0.39 0.09 ⬍0.01 ⬍0.01 0.70 0.22

For comparison between the study populations, anthropometrics were adjusted for age and sex in general linear regression models. Adiponectin was additionally adjusted for percentage of body fat; 24-hour EE was adjusted for age, sex, fat mass, and fat-free mass, and 24-hour RQ was adjusted for age, sex, and energy balance. BMI, body mass index; EE, energy expenditure; RQ, respiratory quotient.

plasma adiponectin concentrations in Pima Indians and whites in whom we had also assessed 24-hour energy expenditure (24-hour EE) and respiratory quotient (24-hour RQ).

Research Methods and Procedures Subjects A total of 93 subjects [18 whites (14 men and 4 women) and 75 Pima Indians (55 men and 20 women); Table 1] who were participants in ongoing studies of the pathogenesis of obesity and type 2 diabetes were included in this analysis. These individuals represent a subgroup of a cohort in whom we studied the relationship between adiponectin and insulin sensitivity (11). All subjects were between 18 and 50 years of age, nonsmokers at the time of the study, nondiabetic (14), and healthy according to a physical examination and routine laboratory tests. Subjects were admitted for 8 to 10 days to the National Institutes of Health Clinical Research Unit in Phoenix, Arizona, where they were fed a weightmaintaining diet (50% of calories as carbohydrate, 30% as fat, and 20% as protein) and abstained from strenuous exercise. After at least 3 days on the diet, subjects underwent a series of tests for the assessment of body composition, glucose tolerance, insulin sensitivity, 24-hour EE, and 24-hour RQ (estimate of whole body carbohydrate/lipid oxidation rate). The protocol was approved by the Tribal Council of the Gila River Indian Community and by the Institutional Review Board of the National Institute of Di-

abetes and Digestive and Kidney Diseases, and all subjects provided written informed consent before participation. Methods Body composition was estimated by total body DXA (DPX-L; Lunar Corp., Madison, WI), with calculation of percentage of body fat, fat mass, and fat-free mass as described (15). Waist and thigh circumferences were measured at the level of the umbilicus and the gluteal fold in the supine and standing position, respectively, and the waistto-thigh ratio was calculated as an index of body fat distribution. After a 12-hour overnight fast, subjects underwent a 75-g oral glucose tolerance test. Baseline blood samples were drawn for the determination of fasting plasma glucose, insulin, and adiponectin concentrations. Plasma glucose concentrations were determined by the glucose oxidase method (Beckman Instruments, Fullerton, CA) and were also measured at 2 hours after glucose ingestion for assessment of glucose tolerance according to the 1985 World Health Organization diagnostic criteria (14). Plasma insulin concentrations were determined by an automated immunoassay (Access; Beckman Instruments). Blood samples for the measurement of fasting plasma adiponectin concentrations were drawn at 7:00 AM with prechilled syringes, transferred into prechilled EDTA tubes, and immediately placed on ice. All tubes were cold-centrifuged (⫹4 °C) within several minutes of collection and stored at ⫺70 °C until assayed at the Department of Internal Medicine and MolecOBESITY RESEARCH Vol. 10 No. 10 October 2002

1017


Table 2. Relationships between plasma adiponectin concentrations and selected anthropometrical and metabolic variables Whites (n ⴝ 18)

BMI Percentage of body fat Waist-to-thigh ratio 24-hour EE* 24-hour RQ†

Correlation coefficient

p

⫺0.72 ⫺0.73 ⫺0.36 ⫺0.30 0.27

⬍0.001 ⬍0.001 0.13 0.22 0.28

Pima Indians (n ⴝ 75)

Upper 95% Lower 95% confidence confidence Correlation interval interval coefficient ⫺0.38 ⫺0.40 ⫺0.71 0.19 0.65

⫺0.88 ⫺0.89 0.13 ⫺0.67 ⫺0.23

⫺0.37 ⫺0.34 ⫺0.37 ⫺0.09 0.04

p

Upper 95% Lower 95% confidence confidence interval interval

⬍0.001 0.003 0.001 0.47 0.71

⫺0.16 ⫺0.12 ⫺0.17 0.14 0.26

⫺0.55 ⫺0.53 ⫺0.55 ⫺0.31 ⫺0.19

* Adjusted for age, sex, race, fat mass, and fat-free mass. † Adjusted for age, sex, race, and energy balance. BMI, body mass index; EE, energy expenditure; RQ, respiratory quotient.

ular Sciences, Osaka University, Japan. Fasting plasma adiponectin concentrations were determined using a validated sandwich enzyme-linked immunosorbent assay employing an adiponectin-specific antibody (intra-assay and interassay coefficients of variation 3.3% and 7.4%, respectively). The measurement of energy expenditure and substrate oxidation in the respiratory chamber has previously been described (16). In brief, volunteers entered the chamber at 7:45 AM after an overnight fast and remained in it until 7:00 AM the following morning. Subjects were fed a standardized diet with the amount of calories reduced according to previously determined equations to achieve energy balance (17). The rate of energy expenditure was measured continuously, calculated for each 15-minute interval within the 23 hours in the chamber, and then extrapolated to 24 hours (24-hour EE). Energy balance was calculated by subtracting 24-hour EE from actual energy intake. The ratio of carbon dioxide production (VCO2) and oxygen consumption (VO2) was calculated for every 15-minute interval and averaged to obtain the 24-hour RQ. Statistical Analyses Statistical analyses were performed using the software of the SAS Institute (Cary, NC). Adiponectin concentrations were log-transformed (log10) to approximate a normal distribution. General linear regression models with adjustment for age and sex were used to compare anthropometric and metabolic characteristics between whites and Pima Indians. Multiple linear regression models were used to identify the determinants of 24-hour EE and 24-hour RQ. Before correlation analyses, 24-hour EE was adjusted for age, sex, race, fat mass, and fat-free mass, and 24-hour RQ was adjusted for age, sex, race, and energy balance. 1018


The relationship between plasma adiponectin concentrations adjusted for age, sex, and percentage of body fat with selected variables was examined in the whole group and in whites and Pima Indians separately. Partial correlation coefficients were used to account for a possible effect of percentage of body fat on the relation between rates of lipid oxidation and plasma adiponectin concentrations.

Results The anthropometric and metabolic characteristics of the entire study population are summarized in Table 1. Pima Indians were younger and had a higher waist-to-thigh ratio than whites. Pima Indians had lower plasma adiponectin concentrations than whites, even after adjustment for age, sex, and percentage of body fat. Table 2 shows the relationships between the plasma adiponectin concentrations and selected anthropometric and metabolic variables for Pima Indians and whites. Plasma adiponectin concentrations were negatively correlated with BMI, percentage of body fat, and waist-to-thigh ratio. Linear regression models after adjusting 24-hour RQ for age, sex, race, and energy balance and adjusting 24-hour EE for age, sex, race, fat mass, and fat-free mass, showed no correlation with plasma adiponectin concentrations, either with 24-hour RQ (R2 ⫽ 0.11, p ⫽ 0.34) or 24-hour EE (R2 ⫽ 0.81, p ⫽ 0.21). Using Pearson correlation coefficients, plasma adiponectin concentrations were not related to 24hour RQ (r ⫽ 0.09, p ⫽ 0.36; Figure 1) before or after adjustment for percentage of body fat (r ⫽ 0.001, p ⫽ 0.99, partial correlation) and rates of energy expenditure (r ⫽ ⫺0.12, p ⫽ 0.27; Figure 1). The correlations between the


Figure 1: (A) 24-hour respiratory quotient (RQ; adjusted for age, sex, race, and energy balance) and (B) 24-hour energy expenditure (EE; adjusted for age, sex, race, fat mass, and fat-free mass) in relationship with fasting plasma adiponectin concentrations in nondiabetic Pima Indians (F) and whites (E).

anthropometric measurements and plasma adiponectin concentrations were not different between the races (all p ⬎ 0.1 for the interaction of race ⫻ adiponectin).

In previous studies, plasma adiponectin concentrations were shown to be closely related to indices of obesity and type 2 diabetes (9 –10). More recently, it was reported that the administration of the mouse adiponectin homologue Acrp30 caused weight loss and increased fatty acid oxidation in mouse skeletal muscle. Administration of Acrp30 or its globular C-terminal domain decreased elevated levels of plasma free fatty acids (FFAs) that were induced either by the administration of a high-fat test meal or intravenous injection of Intralipid. This was accompanied by an increase in the lipid oxidation rate in muscle and resulted in lower plasma glucose levels without having a significant effect on insulin or glucagon levels. It was suggested that the decrease of plasma FFAs caused by accelerated FFA oxidation might have reduced their inhibitory effect on insulin signaling and thereby increased glucose uptake. In addition, chronic administration of adiponectin to mice consuming a high-fat/high-sucrose diet induced profound and sustained weight reduction, without an apparent effect on food intake (12). Moreover, administration of recombinant adiponectin to lipoatrophic mice reduced tissue triglyceride content in muscle and liver and reversed insulin resistance in those animals (13). In our study, there was no relationship between plasma adiponectin concentrations and lipid oxidation rates under resting conditions in humans. Because plasma adiponectin concentrations do not fluctuate during the day (10), one measurement of plasma adiponectin is representative of an individual’s average daily concentration. In animal studies, pharmacological doses of adiponectin were administered to overfed mice. Subjects who participated in our study, however, were on a weight-maintaining diet throughout the study. Therefore, it is possible that the effect of adiponectin on lipid oxidation and energy expenditure might be more important in the overfed state. In summary, these cross-sectional data in humans do not suggest a role of fasting plasma adiponectin concentrations in the regulation of whole-body fat oxidation or energy expenditure under resting conditions. Whether administration of adiponectin to individuals with low levels of this hormone will increase their fat oxidation rates and/or improve their insulin resistance remains to be established.

Acknowledgments Discussion In this study, we examined the relationships between plasma adiponectin concentrations and rates of 24-hour lipid oxidation and energy expenditure in humans. Ninetythree individuals were characterized for body composition and spent 1 day in a respiratory chamber. Fasting plasma adiponectin concentrations were not related to measurements of 24-hour carbohydrate/lipid oxidation rate or energy expenditure, either in the entire study population or in either ethnic subgroup.

T. Funahashi and Y. Matsuzawa were supported by the Japan Society for the Promotion of Science and Education (JSP-RFTF 97L00801) and a grant from Fuji Foundation for Protein Research. We gratefully acknowledge the help of the nursing and dietary staffs of the National Institutes of Health (NIH) metabolic unit for the care of the volunteers. We also thank the technical staff of the NIH Clinical Diabetes and Nutrition Section in Phoenix and of the Department of Internal Medicine and Molecular Sciences, Osaka University, Japan for assisting in the laboratory analyses. OBESITY RESEARCH Vol. 10 No. 10 October 2002

1019


Finally, we are grateful to the members and leaders of the Gila River Indian Community for their continuing cooperation in our studies. References 1. National Institutes of Health. Clinical Guidelines on the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults—The Evidence Report. Obes Res. 1998; 6(Suppl 2):51–209. 2. Pi-Sunyer FX. Health implications of obesity. Am J Clin Nutr. 1991;53:1595– 603. 3. Hotamisligil GS, Murray DL, Choy LN, Spiegelman BM. Tumor necrosis factor alpha inhibits signaling from the insulin receptor. Proc Natl Acad Sci USA. 1994;91:4854 –58. 4. Salbe AD, Nicolson M, Ravussin E. Total energy expenditure and the level of physical activity correlate with plasma leptin concentrations in five-year-old children. J Clin Invest. 1997;99:592–5. 5. Scherer PE, Williams S, Fogliano M, Baldini G, Lodish HF. A novel serum protein similar to C1q, produced exclusively in adipocytes. J Biol Chem. 1995;270:26746 –9. 6. Maeda K, Okubo K, Shimomura I, Funahashi T, Matsuzawa Y, Matsubara K. cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1 (adipose most abundant gene transcript 1). Biochem Biophys Res Commun. 1996;221:286 –9. 7. Ouchi N, Kihara S, Arita Y, et al. Novel modulator for endothelial adhesion molecules: adipocyte-derived plasma protein adiponectin. Circulation. 1999;100:2473– 6. 8. Ouchi N, Kihara S, Arita Y, et al. Adiponectin, an adipocyte-derived plasma protein, inhibits endothelial NF-kappaB signaling through a cAMP-dependent pathway. Circulation. 2000;102:1296 –301.

1020


9. Arita Y, Kihara S, Ouchi N, et al. Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity. Biochem Biophys Res Commun. 1999;257:79 – 83. 10. Hotta K, Funahashi T, Arita Y, et al. Plasma concentrations of a novel, adipose-specific protein, adiponectin, in type 2 diabetic patients. Arterioscler Thromb Vasc Biol. 2000;20: 1595–9. 11. Weyer C, Funahashi T, Tanaka S, et al. Hypoadiponectinemia in obesity and type 2 diabetes: close association with insulin resistance and hyperinsulinemia. J Clin Endocrinol Metab. 2001;86:1930 –5. 12. Fruebis J, Tsao TS, Javorschi S, et al. Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice. Proc Natl Acad Sci USA. 2001;98:2005–10. 13. Yamauchi T, Kamon J, Waki H, et al. The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. Nat Med. 2001;7:941– 6. 14. World Health Organization. Diabetes mellitus: report of a WHO study group. World Health Organization Technical Report Series 17. Geneva, Switzerland: World Health Organization; 1985, pp. 45–51. 15. Tataranni PA, Ravussin E. Use of dual-energy X-ray absorptiometry in obese individuals. Am J Clin Nutr. 1995;62: 730 – 4. 16. Ravussin E, Lillioja S, Anderson TE, Christin L, Bogardus C. Determinants of 24-hour energy expenditure in man. Methods and results using a respiratory chamber. J Clin Invest. 1986;78:1568 –78. 17. Abbott WG, Howard BV, Ruotolo G, Ravussin E. Energy expenditure in humans: effects of dietary fat and carbohydrate. Am J Physiol. 1990;258:347–51.