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Department of Pediatrics (R.W., S.E.T., S.C.), The Children’s and Adult Clinical Research Centers of Yale University School of Medicine (A.G., J.D.), Department of Internal Medicine (K.P.), and Howard Hughes Medical Institute (S.D., G.S.), New Haven, Connecticut 06520
Address all correspondence and requests for reprints to: Sonia Caprio, M.D., Department of Pediatrics, Yale University School of Medicine, 333 Cedar Street, P.O. Box 208064, New Haven, Connecticut 06520. E-mail: sonia.caprio{at}yale.edu.
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Introduction |
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(3)], its level is reduced in obesity (4). Of note, the decline in circulating adiponectin levels coincided with the onset of insulin resistance and the development of diabetes in monkeys (5). Using different measures of insulin sensitivity (M), several groups found that adiponectin gene expression and plasma levels correlated with the insulin-sensitive state in both rodents and obese adults (6, 7, 8). Administration of adiponectin to mice consuming a high-fat diet led to an improvement of M, which was associated with decreased muscle and liver triglyceride content and increased fat oxidation in muscle (9, 10). Thus, dysregulation in the synthesis and/or secretion of adiponectin from the adipose tissue may play a role in the pathogenesis of insulin resistance in both obesity and type 2 diabetes. Most studies regarding the link between adiponectin and insulin resistance have been performed in rodents, nonhuman primates, and adult subjects with obesity and type 2 diabetes. There is little information on circulating levels of adiponectin in pediatrics. Given that adolescent obesity profoundly affects insulin and glucose metabolism, we quantified this protein in a group of nonobese and obese adolescents and explored its relationships among total body fat, central adiposity, whole-body M (assessed by the insulin clamp technique), and muscle lipid content [determined by 1H-nuclear magnetic resonance (NMR) spectroscopy]. |
Subjects and Methods |
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Eight nonobese and 14 obese adolescents participated in the study. All subjects were otherwise in good health and had normal thyroid functions and were taking no medications. All obese adolescents had a body mass index (BMI) greater than 95th percentile specific for age and sex (11), whereas nonobese adolescents had a BMI between the 50th and 75th percentiles (11). Pubertal stage of development was similar in both groups as assessed by physical examination, according to the criteria of Tanner stage for breast development in girls and genital development in boys. In both groups Tanner stage ranged from III to IV. All obese adolescents were recruited from the Yale Pediatric Obesity Clinic. The nonobese adolescents were recruited from either the Yale Pediatric Obesity Clinic or Diabetes Clinic; therefore, they were siblings of obese or diabetic patients or had a parent with diabetes or obesity. Data on M and intramyocellular (IMCL) lipid content were reported previously on the eight nonobese adolescents and 6 of the 14 obese adolescents (12).
The nature and purpose of the study were carefully explained to both parents and every adolescent before written consent from parents and voluntary assent from the child were obtained. The study protocols were approved by the Human Investigation Committee of the Yale University School of Medicine.
Assessment of M: the euglycemic-hyperinsulinemic clamp
Euglycemic-hyperinsulinemic clamp.
In the morning at 0800 h, after an overnight fast of 10–12 h, total body M was measured by the euglycemic-hyperinsulinemic clamp during which time insulin was administered as a prime continuous infusion of 40 mU/m2·min for 120 min, as previously described (13). Two iv catheters were inserted before the clamp studies: one in an antecubital vein for administration of test substances and the other in a vein of the hand or distal forearm of the contralateral arm for blood sampling. The hand chosen for blood sampling was placed in a heated box (
65 C) to facilitate blood sampling and arterialize blood. Videotaped movies entertained the children and kept them relaxed before and during the infusion studies.
Assessment of im lipid content by in vivo 1H-NMR spectroscopy
Localized 1H-NMR spectra of the soleus muscle were acquired on a 2.1T Biospec system (Bruker Instruments, Inc., Billerica, MA) by using a coil assembly consisting of two circular hydrogen-1 coil loops (13-cm diameter each) arranged spatially to generate a quadrature field (14). During the measurements, the subjects remained supine, with the gastrocnemius-soleus complex of the right leg positioned within the homogeneous volume of the magnet. Scout images were acquired to position the volume of interest. Volumes of interest (typically 15 x 15 x 25 mm3) were centered within the soleus muscle and placed to avoid vascular structures and gross adipose tissue deposits. Localized shimming in the soleus muscle was performed using FASTERMAP (14) with typical line widths of approximately 10 Hz being obtained. Localized proton spectra were collected using a PRESS sequence with the following parameters: repetition time = 3 sec, echo time = 21.1 msec, 8192 data points over 5000 Hz spectral width, 128 scans. Signals in the time domain were multiplied by a Gaussian function before Fourier transformation and manual phase correction. 1H resonances were assigned to water and methyl-methylene of triglycerides from their chemical shift, in agreement with Schick et al. (15) and were line fitted using the Muc-Nuts-PPC software package (Acorn NMR, Inc., Livermore, CA). IMCL lipid content and extramyocellular (EMCL) lipid content were calculated from the peak areas of IMCL CH2 (methylene) and EMCL CH2, respectively, with respect to the water peak area and were corrected for T1 and T2 relaxation effects. IMCL and EMCL content were then expressed as a percentage of water content.
Anthropometric measurements, assessment of fat distribution, and total body composition. Weight (to the nearest 0.1 kg) and height (to the nearest 0.5 cm) were measured while the subjects were fasting and wearing only their undergarments. Magnetic resonance imaging (MRI) was used to assess directly intra-abdominal fat deposition, as previously described (16). This procedure was obtained in all nonobese control subjects and 10 obese subjects because four adolescents did not give their assent for this part of the study because of claustrophobia. Total body composition (fat mass and fat-free mass) was measured by dual-energy x-ray absorptiometry using a scanner (Hologic, Inc., Boston, MA). Dual-energy x-ray absorptiometry scans were performed and analyzed using pediatric software (version 1.5e; Hologic, Inc.).
Analytical procedures and calculations. Plasma glucose was measured by the glucose oxidase method (Glucose Analyzer II, Beckman Instruments, Fullerton, CA). Plasma insulin was determined by a double-antibody RIA (Linco RIA Laboratories, St. Charles, MO). Plasma adiponectin levels were determined by RIA kit (Linco Laboratories). The intra-assay and interassay variation was 3% and 10%, respectively.
Statistical analysis
All data are presented as mean ± SE. Comparisons between the obese and nonobese adolescents were performed using the Wilcoxon rank sum test for independent samples. Simple correlation analyses were performed using Spearman’s nonparametric rank correlation coefficient. Partial Spearman correlations were calculated to determine the relations of adiponectin with M, IMCL, and features of the metabolic syndrome and adjusting for measures of total and regional adiposity. For exploratory purposes, regression analysis with a forward selection procedure was used to determine the most statistically significant predictors of adiponectin levels and M. Where appropriate, independent variables were logarithmically transformed. For all analyses, differences were regarded as statistically significant if corresponding P values were 0.05 or less. All statistical analyses were performed using the SAS computer analysis program (version 6; SAS Institute, Inc., Cary, NC).
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Results |
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As reported in Table 1
, obese adolescents had significantly higher fasting insulin, free fatty acids (FFAs), and triglyceride levels than nonobese adolescents. All measures of obesity, including BMI, percentage total fat, intra-abdominal and sc fat (measured by MRI) as well as IMCL and EMCL lipid content (assessed by 1H-NMR spectroscopy) were greater in the obese adolescent group than in the nonobese group. Circulating plasma adiponectin levels were significantly lower in obese (9.2 ± 1.1 µg/ml) than nonobese adolescents (14.3 ± 1.6 µg/ml, P < 0.001) (Fig. 1). These differences remained significant after adjusting for gender (P < 0.002 for both IMCL and adiponectin).
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Relationships between adiponectin levels and features of insulin resistance
Plasma adiponectin concentrations were inversely related to percentage total body fat (r = -0.48, P < 0.05) but not with sc or visceral fat volume. Of particular interest, we found a strong inverse relationship between plasma adiponectin levels and IMCL lipid content (r = -0.73, P < 0.001), which persisted after controlling for percentage total fat and central adiposity (r = -0.63, P < 0.001) (Fig. 2). This relationship was stronger when tested only on the obese subjects (r = -0.78, P < 0.001) and nonsignificant when tested in the nonobese subjects. The correlation between adiponectin and EMCL lipid content was weaker (r = -0.50, P < 0.04) and became nonsignificant after controlling for percentage total fat (r = 0.2). As reported in obese adults (7), we found in our pediatric population that plasma adiponectin levels are positively related to M (r = 0.52, P < 0.02). This relationship was preserved when controlling for FFAs (r = 0.42, P = 0.05) yet was lost when controlling for triglycerides. Of particular note, we found that the relationship between adiponectin and M is completely lost after adjusting for IMCL lipid content (r = 0.13, P < 0.56). Likewise, the relationship between M and IMCL lipid content becomes insignificant after adjusting for adiponectin levels (r = -0.40, P < 0.07). Thus, the putative modulatory effects of adiponectin on M appear to be mediated via the accumulation of IMCL lipid content and plasma triglyceride levels in nonobese and obese adolescents.
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Using a multiple regression analysis with a forward selection procedure, we found that the plasma triglyceride levels and IMCL lipid content (both log transformed) were the most significant predictors, independent of each other, of plasma adiponectin levels. The log of triglyceride explained 52% of the variance in adiponectin levels (r2 = 0.52, P < 0.0002). Further addition of log IMCL to the model provided a better explanation (62%) of the variance of plasma adiponectin levels (r2 = 0.62, P < 0.0001). IMCL remained a good predictor of adiponectin when triglycerides were included in the model (r2 = 0.57, P = 0.03).
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Discussion |
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Further support of our findings originate from studies in obese mice that showed that physiologic replacement of adiponectin levels led to an increased expression of molecules involved in fatty acid transport and enzymes involved in ß-oxidation and uncoupling protein-2 (9). Targeted disruption of the adiponectin gene in mice was clearly associated with delayed clearance of FFAs, low levels of fatty acid transport protein 1 mRNA in muscle, and high levels of TNF mRNA levels in adipose tissue as well as increased plasma TNF levels (21). Of note, these knockout mice exhibited severe diet-induced insulin resistance with reduced insulin-receptor substrate 1-associated phosphatidylinositol 3-kinase (21). Moreover, these abnormalities were ameliorated by adenoviral production of adiponectin. Thus, this recent study strongly suggests that adiponectin enhances M by activating insulin-receptor substrate 1-associated phosphatidylinositol 3-kinase, accelerating FFA clearance by enhancing fatty acid transport protein 1 mRNA expression, and probably fatty acid oxidation in muscle, and decreasing TNF production (21). The presence of insulin resistance was also reported by Kubota et al. (22) in the F1 and F2 adipo knockout mice model fed a high-fat diet. In contrast to the above studies, the absence of insulin resistance was found by Ma et al. (23) in mice lacking adiponectin. The reason for the differences between the study by Ma et al. (23) and the other two groups are not easily explained but may be related to environmental factors or differences in genetic background. Studies performed by Freubis et al. (10) showed that administration of adiponectin in mice fed a high-fat sucrose meal significantly decreased the postprandial levels of FFAs and triglycerides. Hence, the reduced levels of circulating adiponectin found in obese subjects and animals might be responsible, in part, for the dyslipidemia that is frequently associated with obesity.
Consistent with these findings, our study also suggests a close, strong relationship between adiponectin and plasma triglyceride levels in a relatively small group of nonobese and obese adolescents. Indeed, we found that 52% of the variation in plasma adiponectin levels can be explained by triglyceride levels. The fact that the relationship between M and adiponectin loses significance after adjusting for plasma triglycerides (and not for FFAs) suggests that they both have a significant role as potential mediators of M. The relationship between triglycerides and adiponectin implies a possible interplay between these parameters, yet the cause-effect relationship is difficult to elucidate. The possible mechanisms underlying the link between adiponectin and triglyceride levels are not known and warrant study. A hypothetical mechanism might be that adiponectin affects the production of very low-density lipoprotein particles from the liver, thereby regulating serum triglyceride levels. In an insulin-resistant state, such as that associated with obesity and type 2 diabetes, low adiponectin levels may be responsible for the increased very low-density lipoprotein production, which may lead to hypertriglyceridemia.
It has been reported that adiponectin is influenced by gender in both animals and humans (24). However, studies in obese adult subjects (25) as well in obese children (26) found no effect of gender on adiponectin (25). Our results demonstrated no gender influence on differences between nonobese and obese children in IMCL content and adiponectin, yet the limited number of females in each group hinders interpretation of this finding.
There are reports in the literature of the effects of short-term exercise and diet changes on IMCL content (27). Our subjects were instructed to keep a balanced diet and refrain from physical exercise on the week before the studies. Although we do not have objective measures of their physical fitness, we assume that by adhering to these guidelines, the short-term effect of diet and activity is limited in this study.
Key questions that remain to be addressed are: What causes the reduction in adiponectin levels in obesity and type 2 diabetes, and what are the factors that regulate this protein? In conclusion, our study describes the impact of adolescent obesity on circulating adiponectin levels. Of note, we report for the first time a strong inverse relationship between adiponectin and IMCL lipid content, independent of total body fat and central obesity. The present data, albeit descriptive in nature, support the emerging concept that adiponectin may modulate M in part by regulating the accumulation of triglycerides in skeletal muscle tissue. The relationships described here in adolescents need to be evaluated in adult subjects as well.
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Acknowledgments |
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Footnotes |
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Abbreviations: BMI, Body mass index; EMCL, extramyocellular; FFA, free fatty acid; IMCL, intramyocellular; M, insulin sensitivity; MRI, magnetic resonance imaging; NMR, nuclear magnetic resonance.
Received November 6, 2002.
Accepted February 2, 2003.
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receptors in obesity and insulin resistance. J Intern Med 245:621–625[CrossRef][Medline]
a adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice. Proc Natl Acad Sci USA 98:2005–2010This article has been cited by other articles:
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