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Mature Subcutaneous and Visceral Adipocyte Concentrations of Adiponectin Are Highly Correlated in Prepubertal Children and Inversely Related to Body Mass Index Standard Deviation Score

Department of Paediatric Endocrinology and Metabolism (M.A.S., J.P.H.S., E.C.C.), Institute of Child Health, Royal Hospital for Children, Bristol BS2 8AE, United Kingdom; Clinical Sciences South Bristol (J.M.P.H., S.J.T., M.J.G.), University of Bristol, Bristol Royal Infirmary, Bristol BS2 8HW, United Kingdom; and Department of Exercise and Sport Science (C.E.H.S.), Manchester Metropolitan University Cheshire (Alsager Campus), Alsager ST7 2HL, United Kingdom

Address all correspondence and requests for reprints to: Dr. M. A. Sabin, Diabetes UK Clinical Training Fellow, Department of Pediatric Endocrinology and Metabolism, Institute of Child Health, Royal Hospital for Children, Upper Maudlin Street, Bristol BS2 8AE, United Kingdom. E-mail: mattsabin{at}doctors.org.uk.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Context: Adiponectin is an adipocyte-specific protein with insulin-sensitizing properties. Several studies have examined the expression of adiponectin mRNA or tissue/secreted protein levels in fat obtained from adults, but none has assessed tissue levels in childhood.

Patients: Paired subcutaneous (Sc) and visceral (V) fat samples were obtained from 12 normal-weight children.

Main Outcome Measures: Mature adipocytes were isolated and total adiponectin levels determined by ELISA. Insulin sensitivity and lipid parameters were assessed in fasting blood samples taken at the time of biopsy collection.

Results: A positive correlation was seen between the adiponectin concentration within the Sc and V mature adipocytes derived from each child (r = 0.924; P < 0.001). After logarithmic transformation of the Sc and V adiponectin concentrations (log-Sc and log-V) to render the data Gaussian, both log-Sc and log-V were found to be lower in those children with higher body mass index SD score (r = –0.621 and r = –0.357 respectively), although this reached statistical significance only in the Sc adipocytes (P = 0.03). Age was not related to either log-Sc or log-V adiponectin levels, although a significant negative association was seen with serum adiponectin (r = –0.589; P = 0.04). Log-Sc or log-V did not correlate with serum adiponectin concentrations, markers of insulin sensitivity, or circulating lipid levels.

Conclusions: These data indicate a relationship between total adiponectin levels in different tissue compartments, suggesting either some form of interaction or coregulation by systemic factors, possibly related to body size/fat mass. Serum concentrations of total adiponectin were inversely related to age but showed no relationship with either tissue levels or body mass index SD score.

	Introduction

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OBESITY IS ASSOCIATED with the development of insulin resistance, a process known to lead to impaired glucose tolerance and type II diabetes in susceptible individuals (1). Adipose tissue plays an important role in the regulation of energy homeostasis and metabolism by secreting numerous regulatory proteins, termed adipocytokines (2). One of these adipocytokines, termed adiponectin, has antiinflammatory, antiatherogenic and insulin-sensitizing actions, and although it is abundant in the circulation, its levels are reduced in obese individuals (3). Furthermore, although total adiposity negatively correlates with circulating adiponectin, the degree of visceral adiposity appears to be the only independent predictor of circulating adiponectin levels (4). Recent work has suggested that adiponectin is present as three main isomers, high, medium, and low molecular weight (HMW, MMW, LMW), and that HMW adiponectin constitutes the major component of intracellular adiponectin, whereas circulating adiponectin is predominantly of the LMW variety (2). This appears to be important because the ratio of HMW to total adiponectin appears to provide a better reflection of peripheral insulin sensitivity than total adiponectin alone, although the actual significance of the different isoforms remains unclear (5).

Adiponectin is thought to increase insulin sensitivity predominantly by an increase in fatty acid oxidation (by activation of AMP kinase) and inhibition of hepatic glucose production (2). However, although adiponectin levels are decreased in viscerally obese adults and children, the relationship between adiponectin protein expression in mature subcutaneous (Sc) and visceral (V) adipocytes in children has not been previously explored. Several studies have assessed adiponectin mRNA expression in adult human Sc and V fat samples, and these have found either no difference between the two compartments (6) or lower levels in the V adipocytes compared with the Sc (7, 8, 9). Interestingly, one of these studies also found a significant correlation between paired Sc and V adiponectin mRNA levels (6). Furthermore, in studies assessing mRNA expression in Sc and V adipocytes derived from adults over a wide body mass index (BMI) range, Sc levels appeared to decrease with increasing BMI, whereas V levels did not appear to change (10). We therefore sought to examine the adiponectin levels in paired Sc and V fat samples obtained from 12 normal-weight children to investigate whether there are inherent differences in adiponectin levels in different fat compartments from an early age as opposed to these differences developing in later life because of exposure to environmental factors or the differential accumulation of fat stores. Furthermore, we aimed to examine whether adiponectin protein levels in paired Sc and V fat samples were related to each other and whether they correlated with circulating adiponectin concentrations, homeostasis model assessment for insulin resistance (HOMA-R, a marker of peripheral insulin resistance) (11), circulating lipids, and/or the BMI SD score (SDS), which corrects the BMI for the age and sex of the child (12).

	Subjects and Methods

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Ethical approval was obtained from the United Bristol Healthcare Trust Local Research Ethics Committee. The families or guardians of normal-weight children admitted to the Bristol Royal Hospital for Children for routine surgery for nonseptic, nonmalignant conditions were approached. After full explanation and written consent, 5 ml of blood was taken at induction of anesthesia (fasting), and at the beginning of the operation, small pieces (0.2–0.5 g) of Sc and intraabdominal V fat were obtained. The fat samples were obtained by experienced pediatric surgeons with the Sc samples coming from directly under the skin and the V samples from fat located adjacent to the intraabdominal organs. Twelve paired biopsies (seven male) yielded sufficient mature adipocytes to analyze adiponectin levels. The median age (range) was 5.7 (1.1–9.9) yr, and the mean (range) BMI and BMI SDS were 17.2 (14.3–22.2) kg/m² and 0.55 (–1.64 to 2.13), respectively. Clinical details are shown in Table 1.

The biopsies were transported to the laboratory in sterile pots and the mature adipocytes isolated using a previously described method developed within our laboratory (14). Briefly, the adipose tissue was washed three times in Hank’s balanced salt solution, cut into 1-mm³ pieces, and digested with 10 ml of 1 mg/ml type II collagenase in Hank’s balanced salt solution for 60 min at 37 C. The mature adipocytes were separated from the stromal fraction by centrifugation at 80 x g for 3 min and were carefully removed to a sterile Eppendorf containing a matched packed cell volume of lysis buffer (10 mM Tris-Cl, 50 mM sodium chloride, 5 mM EDTA, 15 mM sodium pyrophosphate, 50 mM sodium fluoride, 100 µM sodium orthovanadate, and 1% Triton X-100 with freshly added 1% phenylmethylsulfonyl fluoride at the time of use). The mature adipocytes were lysed via repeated passaging through a 21-gauge needle and the lysates frozen at –20 C. Samples were batch processed, and adiponectin concentrations were determined using standard ELISA kits from Metachem Diagnostics, Inc. (Piddington Northampton, UK) (minimum detectable limit, 23.4 pg/ml). Each assay was performed in accordance with the manufacturer’s instructions, and adiponectin concentrations were related to the total protein in each sample (Pierce BCA protein assay; Pierce Biotechnology, Rockford, IL).

The fasting blood samples were analyzed for 1) adiponectin concentrations using the adiponectin ELISA kit by Metachem Diagnostics [intraassay coefficient of variation (CV), 4.3%; interassay CV 5.3%], 2) insulin using an ELISA method (DakoCytomation, Glostrup, Denmark; code no. K6219; intraassay CV, 2.0%; interassay CV, 3.9%; minimum detectable limit, 1 mIU/liter), 3) glucose using a standard hexokinase assay on an Olympus (Mellville, NY) automated analyzer, and 4) a standard lipid profile (cholesterol, high- and low-density lipoproteins, and triglyceride levels) using an Olympus Diagnostics System Group assay. HOMA-R was calculated as (insulin x glucose/22.5), as previously described (11).

Statistical analyses were by Student’s paired t test and Pearson’s correlation coefficient, with a P value < 0.05 being considered statistically significant.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
No gender differences in either circulating or tissue adiponectin concentrations were found in these prepubertal children and therefore the results from all 12 paired samples were analyzed together.

The adiponectin concentration within the mature Sc adipocyte lysates was positively associated with the paired V adiponectin concentration derived from each child (r = 0.924; P < 0.001) as shown in Fig. 1A. Overall within this group, there was no significant difference in the mean adiponectin concentration between the Sc and V mature adipocytes (mean ± SD was 238 ± 190 for Sc vs. 247 ± 177 ng/mg protein for V; P = 0.69).

View larger version (12K): [in this window] [in a new window]   FIG. 1. A, Paired Sc and V adiponectin levels from mature adipocyte lysates, demonstrating a positive correlation between the adiponectin levels within each fat compartment. Each point represents the paired Sc and V adiponectin level for one child. Pearson’s correlation coefficient r = 0.924; P < 0.001. B, Sc adiponectin concentration (ng/mg protein), plotted on a logarithmic scale, against BMI SDS demonstrating that children with higher BMI SDS have lower levels of adiponectin in the isolated Sc adipocytes. Pearson’s correlation coefficient r = –0.621; P = 0.03.

Both log-Sc and log-V were found to be lower in those children with higher BMI SDS (r = –0.621 and r = –0.357, respectively), although this reached statistical significance only in the Sc adipocytes (P = 0.03), as demonstrated in Fig. 1B.

No correlation was seen between the log-Sc or log-V values and the serum adiponectin level, lipid profile, fasting insulin, or HOMA-R after adjustment for age and sex. Likewise, no correlation was found between age-adjusted serum adiponectin levels and fasting insulin, HOMA-R, or components of a standard lipid profile (total cholesterol, high-density and low-density lipoproteins, and triglycerides).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
We have demonstrated a positive correlation between the adiponectin level in the Sc adipocytes with that in the corresponding V adipocytes from each child, supporting the data from adult studies that have assessed mRNA levels in paired Sc and V samples (6). This suggests either a degree of interaction between the two fat compartments or coregulation of adiponectin levels within adipocytes by other systemic factors. Overall, we found very similar levels of adiponectin protein concentrations in the paired Sc and V adipocytes within these prepubertal children, suggesting either that the differences observed in mRNA levels in adults are not translated to differences in protein production or that they develop later in life. Alternatively, large variations in total adiponectin between the two fat compartments may only be observed after the development of an abnormal body composition with an increased proportion of visceral fat.

Despite finding that increased age was associated with lower serum adiponectin concentrations, we were unable to show any significant age effect on adipose tissue adiponectin levels. Furthermore, we were unable to demonstrate any correlation between log-Sc or log-V adiponectin concentrations with corresponding serum adiponectin levels. These findings suggest that factors other than adipose tissue content, such as synthesis, secretion, clearance, and storage may be important in determining serum adiponectin concentrations. The current development of specific assays for HMW and LMW adiponectin may help to clarify the lack of concordance between tissue and serum total adiponectin levels.

Furthermore, we found that fat samples taken from children with higher BMI SDS, even within the nonobese range, had lower levels of adiponectin in both the Sc and V adipocytes compared with their leaner counterparts, although this negative correlation was significant only in the Sc adipocytes. This suggests that increasing BMI SDS in childhood is associated with reductions in Sc adiponectin content. This would be consistent with the findings in adults where adiponectin mRNA (10) and protein levels (8) were found to be lower in the Sc adipocytes derived from individuals with higher BMI. Furthermore, and in agreement with the findings from the former study, we were unable to show a significant decrease in V adiponectin concentrations with increasing BMI. However, it is unclear in the latter study whether the omission of any changes in omental adipocyte adiponectin concentrations with BMI was because of an absence of any significant differences or simply a lack of omental samples from their obese patients.

No correlation was observed between the serum adiponectin concentration and BMI SDS, fasting insulin, or HOMA-R in this group. Although previous studies have reported such associations, they have always incorporated a broader spectrum of clinical phenotypes in larger numbers of subjects. Similarly, we were unable to demonstrate any correlation between log-Sc and log-V levels with fasting insulin or HOMA-R, and (in 11 of the 12 children where fasting bloods were also analyzed for a standard lipid profile) there was no correlation with fasting lipids.

Very few children have routine operations for nonmalignant, nonseptic conditions, and therefore obtaining paired Sc and V adipocytes for this study proved difficult, limiting our sample size. Despite this constraint, we feel that our data raise some interesting points that require additional study. First, the significant association between the adiponectin levels within the Sc and V fat compartments suggests that, even in young children, a degree of interaction or coregulation of adiponectin tissue levels is present. Second, we have demonstrated that although serum adiponectin levels appear to decrease with increasing age, adiponectin levels within adipocytes appear to be more dependent upon an individual’s BMI SDS, especially within Sc fat tissue and even within a normal BMI range. The reasons for this are currently unclear. Third, we were unable to find any correlation between circulating adiponectin concentrations and tissue adiponectin levels in either the Sc or V fat samples, again suggesting that regulation of serum adiponectin levels is not solely dependent on adipose tissue levels. Additional work in this area is required if we are to fully understand the reasons for hypoadiponectinemia in viscerally obese individuals.

	Acknowledgments

 
We thank Dr. Janet Stone in the Clinical Chemistry Department at the Bristol Royal Infirmary for her assistance with this study, as well as Metachem Diagnostics, Inc., for the kind donation of the adiponectin ELISAkits, Pfizer Ltd. for initial running costs, and the United Bristol Healthcare Trust for donating pump-priming money through their Medical Research Committee and for providing indemnity for this work.

	Footnotes

 
M.S. is a Diabetes UK Clinical Training Fellow (BDA:RD 03/0002642).

First Published Online October 25, 2005

¹ C.E.H.S. and E.C.C. contributed equally to this work.

Abbreviations: BMI, Body mass index; HOMA-R, homeostasis model assessment for insulin resistance; HMW, high molecular weight; LMW, low molecular weight; Sc, subcutaneous; SDS, SD score; V, visceral.

Received July 14, 2005.

Accepted October 14, 2005.

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