1 Department of Nutrition Sciences and Clinical Nutrition Research Center, University of Alabama at Birmingham, Birmingham, Alabama
2 General Clinical Research Center, University of Alabama at Birmingham, Birmingham, Alabama
3 Departments of Preventive Medicine and Physiology and Biophysics, University of Southern California, Los Angeles, California
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ABSTRACT |
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Adiponectin, a hormone secreted exclusively by adipose tissue, has gained much attention secondary to its close association with insulin sensitivity (Si) and obesity (1). Discovered in the mid-1990s by four different groups of researchers, it is also referred to as Acrp30, AdipoQ, ApM1, and GBP28 (25). Adiponectin circulates at relatively high levels in the serum (230 µg/ml range) as both a hexamer and a higher order complex (6). Research has indicated that adiponectin decreases serum glucose by inhibiting hepatic glucose production (79). Adiponectin has also been shown to increase skeletal muscle glucose uptake and fatty acid oxidation through phosphorylation and activation of 5'-AMPactivated protein kinase (10). In one study, adiponectin concentrations at one time predicted the change in Si over the subsequent 2-year period (11).
Although a positive relationship between adiponectin and Si has been well documented in adults (1115), little research has been done in children. The potential role of adiponectin in determining Si in children is of particular relevance with regard to ethnic differences in Si. It has been shown that African-American children and adolescents have lower Si than their Caucasian peers (1618); however, the physiological basis for this difference is not known. The possibility that ethnic differences in adiponectin production or action play a role has not been investigated.
Little is known about ethnic differences in adiponectin. Recently, it was shown that adiponectin was lower among African-American versus Caucasian females, but only among nonobese subjects (19). Another study found that African-American males had the lowest adiponectin concentrations among a group of African-American and Caucasian males and females aged 1221 years (20). In this study, no relationship was observed between adiponectin and estimated Si (estimated using the homeostasis model), after controlling for BMI or BMI percentile (20). The relationship between adiponectin and Si among African-American and Caucasian children has not been examined using robust measures of Si.
In addition to generalized obesity, body fat distribution appears to affect both Si and adiponectin concentrations. That central and visceral fat are inversely associated with Si is well established (2123). Considerably less is known about the relationship between fat distribution and adiponectin. Studies have found both central and visceral adipose tissue to be inversely associated with adiponectin concentrations (2426). Among healthy adults, visceral adipose tissue was independently related to adiponectin concentration after adjusting for measures of subcutaneous adipose tissue as well as other variables such as age, sex, fasting insulin, and glucose, homeostasis model assessment of insulin resistance, HDL cholesterol, and triglycerides (27,28). In contrast, among subjects with HIV, adiponectin was positively correlated with percent body fat, limb fat, and limb fattototal fat ratio (29). Little research exists on the relationship between peripheral fat and circulating adiponectin concentrations in healthy populations.
The purpose of this study, in a population of healthy African American and Caucasian children and adolescents, was to test the hypotheses that adiponectin would be 1) lower in African Americans as compared with Caucasians; 2) inversely related to central, but not peripheral, body fat; and 3) positively associated with Si. In addition, we determined whether baseline adiponectin would predict change in Si over 2 years.
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RESEARCH DESIGN AND METHODS |
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From the cohort of subjects followed in the parent study, a subset of subjects (n = 150) was selected for this current study. They consisted of African-American and Caucasian boys (39%) and girls (61%) with ages ranging from 5 to 16 years. Fifty percent of the boys and 41% of the girls were African American. Subjects for this current study were included based on the availability of Si and body composition data, as well as the availability of stored sera. Although not all measures were available on all subjects, for the cross-sectional component of the present study, there were at least 140 observations for any given variable, with a maximum of 150 observations for some variables, such as adiponectin, age, and BMI. For the longitudinal component of the present study, there were at least 88 observations for any given variable. Adiponectin concentrations were measured at the first visit only and were therefore not available at the 2-year follow-up visit. The Institutional Review Board at UAB approved this substudy.
Once enrolled, the children were scheduled for an overnight stay at the General Clinical Research Center. The children arrived during the late afternoon and were admitted, after which anthropometric measurements were obtained. At 1700, a computed tomography scan was performed by the Department of Radiology at UAB. The children were served a standardized dinner meal and snack consisting of 55% carbohydrate, 15% protein, and 30% fat, which was to be consumed before 2000. The children fasted, receiving only water and noncaffeinated, noncaloric beverages, from 2000 until testing the next morning. Two weeks after the General Clinical Research Center testing, children returned for body composition analysis by dual-energy X-ray absorptiometry, which was performed at the Department of Nutrition Sciences at UAB.
Tolbutamide-modified frequently sampled intravenous glucose tolerance test.
A frequently sampled intravenous glucose tolerance test was performed in the morning (0700) after an overnight fast to determine Si and the acute insulin response to glucose (AIRg) (16). Briefly, fasting blood samples were drawn for determination of glucose, insulin, lipid, and hormone concentrations. At time zero, glucose (25% dextrose; 11.4 g/m2) was given intravenously. Tolbutamide (125 mg/m2) was administered intravenously 20 min after glucose administration. A total of 18 additional blood samples were collected over a 3-h period. Values for glucose (Ektachem DT II System; Johnson & Johnson Clinical Diagnostics, Rochester, NY) and insulin (radioimmunoassay; Diagnostic Products, Los Angeles, CA) were obtained from the sera. These values were then entered into the MINMOD computer program (version 3.0; Richard N. Bergman, Department of Physiology and Biophysics, Keck School of Medicine, University of Southern California, Los Angeles, CA) to derive Si and AIRg.
Total body fat and abdominal fat.
Total body composition (fat mass and fat-free mass) was analyzed by dual-energy X-ray absorptiometry using the Lunar DPX-L densitometer (LUNAR Radiation, Madison, WI), which is described in detail elsewhere (33). As previously described (16), a computed tomography scan (HiLight/Advantage Scanner; General Electric, Milwaukee, WI) was used to measure visceral and subcutaneous abdominal fat. Briefly, a single slice (5-mm) scan taken at the level of the umbilicus was used for cross-sectional area analysis of intra-abdominal (visceral) adipose tissue (IAAT) and subcutaneous abdominal adipose tissue (SAAT).
Adiponectin assay.
After an overnight fast, three blood samples were collected during a 40-min period. The sera were separated, pooled, and stored at 85°C until needed for analysis. Adiponectin was measured using radioimmunoassay kits obtained from Linco Research (St. Charles, MO). The average intra-assay and interassay coefficients of variation at 50% bound were 9.23 and 13.3%, respectively. Manufacturers directions were followed for each assay, and all samples were assayed in duplicate.
Sample size considerations.
Preliminary power calculations, based on 100 children and adolescents, indicated that this study had adequate power to detect statistically significant correlations between measures of adiponectin, Si, and body composition of 0.28 (or greater) with 80% power and of 0.32 (or greater) with 90% power. Additional preliminary power calculations on racial and sex subgroups indicated that 50 children and adolescents would be needed to detect statistically significant correlations of 0.39 with 80% power and 0.45 with 90% power and that for the race-by-sex subgroup, 25 children and adolescents would be needed to detect statistically significant correlations of 0.54 and 0.60 with 80 and 90% power, respectively. Each of these calculations assumed a significance level of 5% and a two-tailed statistical test.
Statistical methods.
Descriptive statistics were computed for all variables of interest. Differences among the means for ethnic and sex subgroups were examined using a two-way ANOVA for endocrine, metabolic, anthropometric, and demographic variables. Adiponectin concentrations, Si, AIRg, fasting insulin, and testosterone concentrations were log10 transformed so that each of these variables followed an approximate normal distribution. Simple relationships between adiponectin and various metabolic and anthropometric variables were examined by using Pearson correlation analyses. Relationships among these variables were further quantified using multiple linear regression analyses. In particular, multiple linear regression models were developed for predicting serum adiponectin concentrations at baseline, for predicting Si at baseline, and also for predicting the change in Si over a 2-year period. Sex, race, and race-by-sex interaction were entered as covariates in the regression models for adiponectin, Si, and change in Si over a 2-year period. Testosterone and AIRg were tested as independent variables in individual models for baseline adiponectin because of their documented inhibitory effect on adiponectin expression and circulating concentrations (21,34,35). In addition, baseline measures of Si and adiponectin were included as independent variables in the model for predicting change in Si over a 2-year period, as were total fat mass, and Tanner stage at the 2-year follow-up visit. All statistical tests used a significance level of 5% and were two-tailed. Statistical analyses were performed using SAS (version 9.0; SAS Institute, Cary, NC).
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RESULTS |
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The number of subjects in each ethnic-sex subgroup is given in Table 1, along with baseline demographic and descriptive characteristics. Subjects ranged in age from 5 to 16 years, and the majority of subjects were Tanner stage I (n = 88; 59%). Results indicated that age and Si were significantly different between ethnic and sex groups, with African Americans being younger (P = 0.001) and having lower Si (P < 0.001) compared with Caucasians, and girls being older (P < 0.05) and having lower Si (P < 0.05) compared with boys. Additionally, measures of adiponectin were significantly lower in African Americans (P < 0.05), and measures of fasting insulin and AIRg were significantly higher in African Americans (P < 0.05 and P < 0.001, respectively). There was a trend toward significance for measures of IAAT among racial groups, with African Americans having less IAAT than Caucasians (0.05 < P < 0.10). A trend was observed for girls having more total fat and limb fat than boys (0.05 < P < 0.10 for both). The race-by-sex interaction term was not significant for any of the variables.
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Multiple linear regression model for dependent variable insulin sensitivity.
In multiple linear regression modeling, adiponectin was positively related to Si (P < 0.05; Table 5). Race, sex, and total fat were inversely related to Si (P < 0.001, P < 0.05, and P < 0.001, respectively). The model presented in Table 5 explained 50% of the variance in Si. In preliminary analysis, the race-by-sex interaction and total lean tissue mass were included in the model but were not significant predictors of Si and therefore were not included in the final model. Although fat distribution measures were tested in the model, they were not significant predictors of Si and did not alter the significance of other variables in the model. In addition, the model with fat distribution variables explained less of the variance (R2 = 0.49) than the model with total fat (R2 = 0.50). Therefore, the final model included only total fat.
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DISCUSSION |
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In the present study, lower adiponectin among African Americans was apparent after adjusting for potential confounding factors (age, sex, body fat distribution, and total testosterone). Because African Americans enter puberty at an earlier age and because previous studies have found a relationship between increased testosterone and decreased adiponectin concentrations (37,38), we reasoned that higher testosterone among African-American males may account for the lower adiponectin previously reported in this sexethnic subgroup (20). However, we did not observe a significant relationship between adiponectin and either Tanner stage or testosterone concentration. It is possible that the early pubertal status of our subjects resulted in concentrations of testosterone that were too low to affect adiponectin secretion.
Because African Americans have less IAAT but more subcutaneous fat and limb fat than Caucasians (21,22), we reasoned that ethnic differences in body fat distribution may account for ethnic differences in adiponectin. The relationship between body fat distribution and adiponectin has not been widely investigated. We found that trunk fat was inversely related to adiponectin after adjusting for limb fat, and that limb fat was positively related to adiponectin after controlling for central fat (both trunk fat and SAAT). To the best of our knowledge, this positive relationship between adiponectin and peripheral fat is a novel observation in a healthy population. However, such a positive relationship between adiponectin and peripheral fat has been observed in the HIV population (29).
Most of the literature indicates that increased adiposity, as assessed by either BMI or dual-energy X-ray absorptiometry, is inversely correlated with adiponectin. Of studies that examined fat distribution in adolescents, Bacha et al. (39) reported that a group of obese adolescents not only had lower adiponectin concentrations than their normal weight peers, but that the obese adolescents with high IAAT had significantly lower adiponectin concentrations than obese adolescents with low IAAT, suggesting that IAAT accumulation may result in lower adiponectin concentrations. In our study, we did not find IAAT to be independently related to adiponectin, as has been reported in adults (27,28); this may have been due to the younger age and developmental status of this group as well as lower IAAT among this population. IAAT also was not associated with Si in this cohort (16).
In the present study, the ethnic difference in adiponectin disappeared when the data were statistically adjusted for AIRg. This observation suggests that the higher AIRg typically observed in African Americans (16,36,40) may explain the lower adiponectin concentrations. Studies have shown that African-American children have higher AIRg compared with Caucasian children, independent of differences in Si, due to both increased insulin secretion as well as decreased hepatic insulin clearance (36,41). Thus, although both Caucasian and African-American subject populations exhibit characteristic "compensatory hyperinsulinemia" as demonstrated by an inverse relationship between Si and AIRg (42), at any given level of Si, African Americans have higher AIRg (41). Data have indicated that insulin suppresses adiponectin both in vitro and in vivo (34,35). Thus, higher AIRg among African Americans may lead to lower adiponectin secretion, which in turn may lead to lower Si.
The second aim of this study was to determine whether adiponectin would be an independent determinant of Si among children and adolescents after adjusting for confounding factors. We found that adiponectin was independently and positively related to Si. Several studies have shown similar findings in both children and adults (1214,43). The relationship between adiponectin and Si was independent of total body fat, sex, and race, all of which were independently related to Si. In this population, girls had lower Si than boys, independent of pubertal status and adiposity. The reason for lower Si among girls is not clear. As has previously been shown (16), total fat, rather than IAAT and/or SAAT, was the best predictor of Si among relatively young, healthy children.
Although African Americans had both lower Si and lower adiponectin, the lower adiponectin did not account for the ethnic difference in Si. Rather, both ethnicity and adiponectin made significant independent contributions to Si. The design of this study did not enable us to determine whether any of the variance in Si attributed to ethnicity was explained by lower adiponectin among African Americans. However, the significant association observed among adiponectin and Si is impetus for future research on the potential contribution of lower adiponectin among African Americans to impairment in glucose tolerance and risk for type 2 diabetes. This is especially true in light of recent findings suggesting that circulating adiponectin levels predict insulin resistance in certain adolescent populations (44,45). For example, Mexican children and adolescents with higher levels of adiponectin had decreased prevalence of type 2 diabetes (45).
The third aim of our study was to determine the relationship between baseline adiponectin and change in Si over a 2-year period. Yamamoto et al. (11) reported that baseline adiponectin was negatively related to changes in insulin and insulin resistance as measured by homeostasis model assessment in 590 Japanese men ages 3065 years. We were interested in determining whether similar findings would be observed in healthy children and adolescents. In a multiple linear regression model for Si at year 2, we adjusted for baseline Si, Tanner stage at follow-up visit, total fat at follow-up visit, sex, race, and age. We did not find that baseline adiponectin predicted change in Si over a 2-year period. Results did not differ if change in total fat was substituted for fat mass in the model.
Between-study differences may have been due to differences in methodology or to differences in the study population. Yamomoto et al. (11) used a surrogate measure of Si derived from fasting glucose and insulin, rather than a direct measure, such as the intravenous glucose tolerance test and minimal modeling used in the present study. Additionally, our population was comprised of both boys and girls and was, on average, younger than the population of Yamomoto et al. (11). The peripubertal status of our population may have influenced our results. Si decreases during the middle stages of puberty but recovers by the end of puberty (30). Perhaps the innate pubertal fluctuations in Si, along with the changes in other hormones related to growth and maturation, overshadowed any potential effect of adiponectin on Si. However, results did not differ if analyses were conducted separately for pre-/early pubertal subjects (Tanner stages I and II) and for mid-to-late pubertal subjects (Tanner stages IIIV).
Strengths of the study include the relatively large population size; robust measures of body composition, fat distribution, and Si; and statistical control for pubertal development via both Tanner stage and testosterone concentration. Limitations include the use of frozen, and in some cases previously thawed, sera, and missing measures on some subjects. Additionally, evidence suggests that the concentration or relative amount of the high versus low molecular weight form of adiponectin is more closely associated with insulin sensitivity than is the concentration of total adiponectin (46). We did not have measures of the high or low molecular weight forms of adiponectin; such measures are likely to provide greater insight into both ethnic differences in adiponectin and Si, and the longitudinal relationship between adiponectin and Si.
In conclusion, the major findings of this study are that adiponectin was lower among African-American versus Caucasian children and was positively related to Si. The lower adiponectin among African Americans was statistically explained by their higher AIRg. Among children, peripheral fat was significantly and positively related to adiponectin, whereas central fat was inversely related, suggesting that fat distribution should be considered when examining associations between measures of body composition and adiponectin. Future research is warranted on the potential contribution of lower adiponectin among African Americans to impairment in glucose tolerance and risk for type 2 diabetes.
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ACKNOWLEDGMENTS |
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The efforts of study coordinator Tena Hilario, laboratory staff Maryellen Williams and Cindy Zeng, and the participation of the subjects and their families are gratefully acknowledged.
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FOOTNOTES |
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Address correspondence and reprint requests to Barbara A. Gower, Department of Nutrition Sciences, University of Alabama at Birmingham, 429 Webb Building, 1675 University Blvd., Birmingham, AL 35294-3360. E-mail: bgower{at}uab.edu
Received for publication November 16, 2004 and accepted in revised form June 7, 2005
AIRg, acute insulin response to glucose; IAAT, intra-abdominal (visceral) adipose tissue; SAAT, subcutaneous abdominal adipose tissue; UAB, University of Alabama at Birmingham
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REFERENCES |
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