Adipose Tissue Biology: Association With IR in Children
General characteristics of patients and samples of our Leipzig Childhood AT cohort are summarized in Table 1. Study participants in the lean and obese subgroups were not different with respect to sex distribution and pubertal stage, although obese children were older than lean children (Table 1).
We addressed the controversially discussed question whether fat accumulation is a result of hypertrophy and/or hyperplasia by evaluating potential associations of adipocyte size and total adipocyte number with AT accumulation. Compared with lean controls, adipocyte size and total adipocyte number were significantly increased in obese children by 17.2 and 164%, respectively (Table 1) and correlated with obesity-related parameters, such as BMI SDS (Fig. 1A and B) and AT mass (Fig. 1C and D). Both adipocyte size and total adipocyte number increased with age in the lean subgroup (Fig. 1E and F). The adipocyte size, but not adipocyte number, also correlated with age in the obese subgroup (Fig. 1E and F).
(Enlarge Image)
Figure 1.
Association of adipocyte cell size and number with age and fat mass. Mean adipocyte diameter and total number of adipocytes increase with BMI SDS (A and B) and AT mass (C and D). Adipocyte diameter was positively associated with age in lean and obese children (E), whereas only lean children showed a positive association between total number of adipocytes and age (F). Both adipocyte cell size (G) and adipocyte number (H) are increased in obese compared with lean children in all age groups from childhood (6–8 years) to early adulthood (16–19 years). Pearson correlation coefficient R and P value are given in each scatter plot. Significant P values (P < 0.05) are indicated in bold. Number of subjects in each age group is indicated in parentheses. Lean children are represented as open circles and obese as closed circles. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01.
For further analyses, we stratified children into age groups representing distinct stages of childhood development: 0–2 years (infancy), 3–5 years (early childhood), 6–8 years (prepubertal), 9–11 years (beginning of puberty), 12–15 years (puberty), and 16–19 years (adolescence). Adipocytes from obese children were larger than adipocytes from nonobese children in all age groups starting from the age of 6 years (Fig. 1G). In normal-weight children, adipocyte size increased from early childhood to adolescence and adulthood. In obese children, adipocyte size at 6–8 years was already significantly increased and then remained relatively constant until early adulthood, indicating that adipocyte size may reach a plateau at childhood age, which is higher in obese children (Fig. 1G). In all age groups, we observed an approximately twofold increase in total adipocyte number in obese compared with lean children (Fig. 1H). In both lean and obese children, adipocyte number appeared to plateau from 9–11 years onward, potentially indicating that individual adipocyte number is determined by this age. There were no significant sex differences in adipocyte size or number between lean girls and boys (data not shown).
In multiple regression analyses, we confirmed total adipocyte number and adipocyte size as independent predictors for AT mass accounting for 68 and 3% of waist circumference variability, respectively (Table 2). We selected waist circumference because, besides BMI, it is considered to be a good index of adiposity in children but is mathematically not directly related to variables in the model. Similar results were obtained for AT mass (Table 2).
The observed increase in adipocyte number may result from enhanced proliferation of adipogenic progenitor cells and subsequent differentiation into mature adipocytes. We therefore analyzed proliferation and differentiation potential of adherent cells of the SVF isolated from AT samples in vitro. The yield of obtained SVF cells was comparable between lean and obese children (9.7 ± 1.0 vs. 9.9 ± 1.4 × 10 SVF cells per g AT; P = 0.680) as was the percentage of adherent SVF cells (23.6 ± 6.6 vs. 21.2 ± 5.6%; P = 0.570).
The slope of cell number increase in cell culture appeared to be more steep in obese compared with lean children, leading to a fivefold higher cell number at day 10 postseeding in obese children (Fig. 2A). In line with this, SVF cell doubling time was accelerated in obese children (Table 1) and correlated negatively with BMI SDS (Fig. 2B). There was no association of SVF cell doubling time with age in the whole cohort (Fig. 2C) or in lean children only (R = 0.157; P = 0.547). Furthermore, SVF cell doubling time was not related to adipocyte size (Fig. 2D and Table 3).
(Enlarge Image)
Figure 2.
Obese children show enhanced proliferation of SVF cells, but no changes in the percentage of differentiated cells. (A) The number of SVF cells at day 10 after seeding was increased in obese children compared with normal-weight children. Adherent SVF cells were counted at days 2, 4, 6, 8, and 10 after seeding. Proliferation rate is expressed as fold change of the cell number counted at day 2 and is shown as mean ± SEM. Differences were analyzed by one-way ANOVA and post hoc Dunnett test. Number of samples is indicated in parentheses. (B) Doubling time of SVF cells was negatively correlated to BMI SDS. There was no association between SVF cell doubling time and age (C) or adipocyte diameter (D) in children. Pearson correlation coefficient R and P value are shown in each scatter plot. Adipocyte differentiation was determined in vitro by quantifying the percentage of differentiated adipocytes and Oil Red O absorbance (540 nm) 8 days after adipogenic induction. No significant differences in differentiation rate and in Oil Red O absorbance were observed between lean and obese children (E and F). In addition, no significant differences in the amount of released adiponectin were observed between lean and obese children (G). The number of differentiated cells was documented by Nile red/Hoechst double staining (H). Significant P values (P < 0.05) are indicated in bold. Lean children are represented as open circles and obese as closed circles. **P < 0.01.
The percentage of differentiated SVF cells was not different in obese compared with lean children (Table 1 and Fig. 2E) or in samples of small adipocytes compared with large adipocytes (Table 3) as documented by similar levels of Oil Red O absorbance (Fig. 2F) and adiponectin concentration in supernatants of the differentiated cells (Fig. 2G). Representative images of differentiated cells from a lean and an obese child are shown in Fig. 2H and Supplementary Fig. 2 http://diabetes.diabetesjournals.org/content/64/4/1249/suppl/DC1.
To assess inflammation in AT of children, we investigated the infiltration of macrophages into AT and the relationship with adipocyte size. The number of CD68 macrophages was doubled in obese children compared with lean children (Table 1), and there was a weak but significant positive correlation with BMI SDS (Fig. 3A) and age (Fig. 3B). When we restricted correlation analyses to lean children only, the association between macrophage number and age was lost (R = 0.177; P = 0.100). Similar results were obtained for CD68 expression (Table 1). As adipocyte hypertrophy is hypothesized to drive macrophage infiltration, we analyzed the relationship between adipocyte size and number of CD68 macrophages and confirmed a positive association (Fig. 3C). When we stratified AT samples in tertiles according to adipocyte size, we observed a threefold increase in macrophage number in samples containing large adipocytes compared with samples containing small adipocytes (Table 3).
(Enlarge Image)
Figure 3.
Macrophage infiltration is associated with obesity and adipocyte diameter. The number of AT macrophages positively correlated with BMI SDS (A), age (B), and cell size of adipocytes (C). Pearson correlation coefficient R and P value are shown in each scatter plot. Significant P values (P < 0.05) are indicated in bold. Lean children are represented as open circles and obese as closed circles. D: Representative images for macrophage infiltration at different tertiles of adipocyte size. CD68-positive cells are indicated by black arrows. CLS were identified by the typical arrangement of CD68-positive macrophages surrounding adipocytes.
We further documented enhanced AT inflammation in obese children by significantly increased presence of crown-like structures (CLS; CD68 macrophages surrounding an adipocyte) (Fig. 3D), which we found in almost half of the obese children but in less than 10% of the lean children (Table 1). In addition, the presence of CLS increased with adipocyte size (Table 3).
Next we analyzed the relation of macrophage infiltration in AT to inflammatory markers such as hs-CRP, TNF-α, or IL-6. We observed significantly increased hs-CRP serum levels in obese compared with lean children (Table 1). However, obese children did not show increased TNF-α or IL-6 serum levels nor TNF-α or IL-6 expression in AT (Table 1). In correlation analyses, macrophage number was not clearly associated with hs-CRP (R = 0.165; P = 0.085), TNF-α (R = −0.097; P = 0.312), or IL-6 (R = −0.001; P = 0.990) serum levels or TNF-α (R = 0.015; P = 0.860) and IL-6 (R = −0.067; P = 0.440) expression. Only hs-CRP serum levels showed a significant increase with increasing adipocyte size (Table 3).
Next we characterized metabolic function of adipocytes by assessing the lipolytic activity of isolated adipocytes. We observed a significant decrease in basal lipolytic activity in obese compared with lean children (Table 1 and Fig. 4A). Stimulation with the β-agonist isoproterenol led to a significant increase of lipolytic activity in adipocytes of both lean and obese children (Fig. 4A). However, there was no significant difference in the magnitude of isoproterenol-stimulated lipolytic activity between the two groups (Table 1). Basal lipolytic activity (Fig. 4B) but not isoproterenol-stimulated lipolysis (R = −0.184; P = 0.424) was negatively associated with BMI SDS. Moreover, basal lipolysis correlated negatively with adipocyte size (Fig. 4C and Table 3), which was, however, lost after adjustment for BMI SDS (R = −0.11; P = 0.643). Neither basal nor stimulated lipolytic activity changed with age in the whole cohort (Fig. 4D) or in the lean subgroup (R = −0.059; P = 0.863).
(Enlarge Image)
Figure 4.
Basal lipolytic activity of adipocytes is negatively associated with BMI SDS and adipocyte diameter. A: Isolated adipocytes of obese children (black bars) showed a reduced basal lipolysis capacity compared with lean children (white bars). Addition of 10 μmol/L isoproterenol stimulated lipolytic activity in lean and obese children, although no significant differences between adipocytes of lean and obese children could be observed. Data are presented as mean ± SEM. **P < 0.01. Basal lipolytic activity correlated negatively with BMI SDS (B) and adipocyte size (C), but not with age (D). Pearson correlation coefficient R and P values are shown in each scatter plot. Significant P values (P < 0.05) are indicated in bold. Lean children are represented as open circles and obese as closed circles.
Finally, we evaluated serum levels of the adipokines adiponectin and leptin for their association with obesity-related alterations in adipocyte biology.
As expected, we observed decreased adiponectin serum levels in obese compared with lean children and a negative association of adiponectin with BMI SDS (Table 1 and Fig. 5A) and age (R = −0.503; P < 0.001). Adiponectin levels did not differ between samples with small or large adipocytes (Table 3), nor did they show a correlation with adipocyte size (Fig. 5B) or number (R = −0.311; P = 0.875). However, we observed a negative association with macrophage infiltration (Fig. 5C), the presence of CLS (11.03 ± 0.83 vs. 5.34 ± 0.37 ng/mL; P < 0.001), and hs-CRP (R = −0.256; P = 0.003). On the other hand, adiponectin levels did not correlate with IL-6 serum levels (R = −0.042; P = 0.640) and IL-6 AT expression (R = 0.076; P = 0.391) nor with TNF-α expression (R = −0.042; P = 0.640) but with TNF-α serum levels (R = 0.316; P < 0.001).
(Enlarge Image)
Figure 5.
Association of serum adipokine levels and HOMA-IR with BMI SDS, adipocyte diameter, and macrophage infiltration. Adiponectin serum levels decrease with BMI SDS (A), whereas no association with adipocyte diameter was observed (B). Furthermore, we observed a negative association with macrophage infiltration (C). Serum leptin levels were positively associated with BMI SDS (D), adipocyte diameter (E), and macrophage infiltration (F). The insulin resistance marker HOMA-IR showed a positive correlation with BMI SDS (G), adipocyte size (H), and macrophage infiltration (I). Pearson correlation coefficient R and P values are given in each scatter plot. Significant P values (P < 0.05) are indicated in bold. Lean children are represented as open circles and obese as closed circles.
Serum leptin was positively correlated to the degree of obesity in children (Table 1 and Fig. 5D) and age (R = 0.477; P < 0.001). Furthermore, leptin levels significantly increased with adipocyte size (Fig. 5E and Table 3), number of macrophages (Fig. 5F), presence of CLS (9.99 ± 1.70 vs. 28.44 ± 4.65 ng/mL in children with or without CLS; P < 0.001), and hs-CRP serum levels (R = 0.591; P < 0.001). Adipocyte size was the strongest predictor for leptin levels in multivariate analyses (Table 2).
Similar to adiponectin, there were no correlations of serum leptin with circulating IL-6 (R = 0.185; P = 0.059) and TNF-α (R = −0.118; P = 0.231) nor with TNF-α expression in AT (R = 0.009; P = 0.920). In contrast to that, we observed a slightly negative association of serum leptin levels and IL-6 mRNA levels in AT (R = −0.195; P = 0.045).
Finally, we were interested in how obesity-associated alterations in AT biology relate to HOMA-IR as a clinical marker of insulin resistance. HOMA-IR levels showed a positive association with not only BMI SDS (Table 1 and Fig. 5G) and age (R = 0.634; P < 0.001), but also adipocyte size (Fig. 5H) and were fourfold higher in samples of patients containing large adipocytes compared with small adipocytes (Table 3). In addition to the association with adipocyte hypertrophy, HOMA-IR was related to macrophage infiltration (Fig. 5I) and increased in AT containing CLS (3.67 ± 0.41 vs. 1.34 ± 0.14; P < 0.001).
Even though there was a correlation of HOMA-IR with hs-CRP (R = 0.286; P = 0.001) and IL-6 (R = 0.220; P = 0.013) serum levels, we did not detect significant associations of HOMA-IR with AT expression of TNF-α (R = 0.071; P = 0.422) or IL-6 (R = −0.115; P = 0.194). Unexpectedly, we detected a negative association of HOMA-IR and TNF-α serum levels (R = −0.396; P < 0.001). In multivariate analyses, HOMA-IR was most strongly affected by adipocyte hypertrophy (Table 2).
Results
General characteristics of patients and samples of our Leipzig Childhood AT cohort are summarized in Table 1. Study participants in the lean and obese subgroups were not different with respect to sex distribution and pubertal stage, although obese children were older than lean children (Table 1).
Adipocyte Size and Number are Related to Accumulation of AT in Children
We addressed the controversially discussed question whether fat accumulation is a result of hypertrophy and/or hyperplasia by evaluating potential associations of adipocyte size and total adipocyte number with AT accumulation. Compared with lean controls, adipocyte size and total adipocyte number were significantly increased in obese children by 17.2 and 164%, respectively (Table 1) and correlated with obesity-related parameters, such as BMI SDS (Fig. 1A and B) and AT mass (Fig. 1C and D). Both adipocyte size and total adipocyte number increased with age in the lean subgroup (Fig. 1E and F). The adipocyte size, but not adipocyte number, also correlated with age in the obese subgroup (Fig. 1E and F).
(Enlarge Image)
Figure 1.
Association of adipocyte cell size and number with age and fat mass. Mean adipocyte diameter and total number of adipocytes increase with BMI SDS (A and B) and AT mass (C and D). Adipocyte diameter was positively associated with age in lean and obese children (E), whereas only lean children showed a positive association between total number of adipocytes and age (F). Both adipocyte cell size (G) and adipocyte number (H) are increased in obese compared with lean children in all age groups from childhood (6–8 years) to early adulthood (16–19 years). Pearson correlation coefficient R and P value are given in each scatter plot. Significant P values (P < 0.05) are indicated in bold. Number of subjects in each age group is indicated in parentheses. Lean children are represented as open circles and obese as closed circles. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01.
For further analyses, we stratified children into age groups representing distinct stages of childhood development: 0–2 years (infancy), 3–5 years (early childhood), 6–8 years (prepubertal), 9–11 years (beginning of puberty), 12–15 years (puberty), and 16–19 years (adolescence). Adipocytes from obese children were larger than adipocytes from nonobese children in all age groups starting from the age of 6 years (Fig. 1G). In normal-weight children, adipocyte size increased from early childhood to adolescence and adulthood. In obese children, adipocyte size at 6–8 years was already significantly increased and then remained relatively constant until early adulthood, indicating that adipocyte size may reach a plateau at childhood age, which is higher in obese children (Fig. 1G). In all age groups, we observed an approximately twofold increase in total adipocyte number in obese compared with lean children (Fig. 1H). In both lean and obese children, adipocyte number appeared to plateau from 9–11 years onward, potentially indicating that individual adipocyte number is determined by this age. There were no significant sex differences in adipocyte size or number between lean girls and boys (data not shown).
In multiple regression analyses, we confirmed total adipocyte number and adipocyte size as independent predictors for AT mass accounting for 68 and 3% of waist circumference variability, respectively (Table 2). We selected waist circumference because, besides BMI, it is considered to be a good index of adiposity in children but is mathematically not directly related to variables in the model. Similar results were obtained for AT mass (Table 2).
Proliferation but not Differentiation of SVF Cells is Enhanced in Obese Children
The observed increase in adipocyte number may result from enhanced proliferation of adipogenic progenitor cells and subsequent differentiation into mature adipocytes. We therefore analyzed proliferation and differentiation potential of adherent cells of the SVF isolated from AT samples in vitro. The yield of obtained SVF cells was comparable between lean and obese children (9.7 ± 1.0 vs. 9.9 ± 1.4 × 10 SVF cells per g AT; P = 0.680) as was the percentage of adherent SVF cells (23.6 ± 6.6 vs. 21.2 ± 5.6%; P = 0.570).
The slope of cell number increase in cell culture appeared to be more steep in obese compared with lean children, leading to a fivefold higher cell number at day 10 postseeding in obese children (Fig. 2A). In line with this, SVF cell doubling time was accelerated in obese children (Table 1) and correlated negatively with BMI SDS (Fig. 2B). There was no association of SVF cell doubling time with age in the whole cohort (Fig. 2C) or in lean children only (R = 0.157; P = 0.547). Furthermore, SVF cell doubling time was not related to adipocyte size (Fig. 2D and Table 3).
(Enlarge Image)
Figure 2.
Obese children show enhanced proliferation of SVF cells, but no changes in the percentage of differentiated cells. (A) The number of SVF cells at day 10 after seeding was increased in obese children compared with normal-weight children. Adherent SVF cells were counted at days 2, 4, 6, 8, and 10 after seeding. Proliferation rate is expressed as fold change of the cell number counted at day 2 and is shown as mean ± SEM. Differences were analyzed by one-way ANOVA and post hoc Dunnett test. Number of samples is indicated in parentheses. (B) Doubling time of SVF cells was negatively correlated to BMI SDS. There was no association between SVF cell doubling time and age (C) or adipocyte diameter (D) in children. Pearson correlation coefficient R and P value are shown in each scatter plot. Adipocyte differentiation was determined in vitro by quantifying the percentage of differentiated adipocytes and Oil Red O absorbance (540 nm) 8 days after adipogenic induction. No significant differences in differentiation rate and in Oil Red O absorbance were observed between lean and obese children (E and F). In addition, no significant differences in the amount of released adiponectin were observed between lean and obese children (G). The number of differentiated cells was documented by Nile red/Hoechst double staining (H). Significant P values (P < 0.05) are indicated in bold. Lean children are represented as open circles and obese as closed circles. **P < 0.01.
The percentage of differentiated SVF cells was not different in obese compared with lean children (Table 1 and Fig. 2E) or in samples of small adipocytes compared with large adipocytes (Table 3) as documented by similar levels of Oil Red O absorbance (Fig. 2F) and adiponectin concentration in supernatants of the differentiated cells (Fig. 2G). Representative images of differentiated cells from a lean and an obese child are shown in Fig. 2H and Supplementary Fig. 2 http://diabetes.diabetesjournals.org/content/64/4/1249/suppl/DC1.
Enhanced Macrophage Infiltration in AT of Obese Children
To assess inflammation in AT of children, we investigated the infiltration of macrophages into AT and the relationship with adipocyte size. The number of CD68 macrophages was doubled in obese children compared with lean children (Table 1), and there was a weak but significant positive correlation with BMI SDS (Fig. 3A) and age (Fig. 3B). When we restricted correlation analyses to lean children only, the association between macrophage number and age was lost (R = 0.177; P = 0.100). Similar results were obtained for CD68 expression (Table 1). As adipocyte hypertrophy is hypothesized to drive macrophage infiltration, we analyzed the relationship between adipocyte size and number of CD68 macrophages and confirmed a positive association (Fig. 3C). When we stratified AT samples in tertiles according to adipocyte size, we observed a threefold increase in macrophage number in samples containing large adipocytes compared with samples containing small adipocytes (Table 3).
(Enlarge Image)
Figure 3.
Macrophage infiltration is associated with obesity and adipocyte diameter. The number of AT macrophages positively correlated with BMI SDS (A), age (B), and cell size of adipocytes (C). Pearson correlation coefficient R and P value are shown in each scatter plot. Significant P values (P < 0.05) are indicated in bold. Lean children are represented as open circles and obese as closed circles. D: Representative images for macrophage infiltration at different tertiles of adipocyte size. CD68-positive cells are indicated by black arrows. CLS were identified by the typical arrangement of CD68-positive macrophages surrounding adipocytes.
We further documented enhanced AT inflammation in obese children by significantly increased presence of crown-like structures (CLS; CD68 macrophages surrounding an adipocyte) (Fig. 3D), which we found in almost half of the obese children but in less than 10% of the lean children (Table 1). In addition, the presence of CLS increased with adipocyte size (Table 3).
Next we analyzed the relation of macrophage infiltration in AT to inflammatory markers such as hs-CRP, TNF-α, or IL-6. We observed significantly increased hs-CRP serum levels in obese compared with lean children (Table 1). However, obese children did not show increased TNF-α or IL-6 serum levels nor TNF-α or IL-6 expression in AT (Table 1). In correlation analyses, macrophage number was not clearly associated with hs-CRP (R = 0.165; P = 0.085), TNF-α (R = −0.097; P = 0.312), or IL-6 (R = −0.001; P = 0.990) serum levels or TNF-α (R = 0.015; P = 0.860) and IL-6 (R = −0.067; P = 0.440) expression. Only hs-CRP serum levels showed a significant increase with increasing adipocyte size (Table 3).
Basal Lipolysis is Decreased in Adipocytes of Obese Children
Next we characterized metabolic function of adipocytes by assessing the lipolytic activity of isolated adipocytes. We observed a significant decrease in basal lipolytic activity in obese compared with lean children (Table 1 and Fig. 4A). Stimulation with the β-agonist isoproterenol led to a significant increase of lipolytic activity in adipocytes of both lean and obese children (Fig. 4A). However, there was no significant difference in the magnitude of isoproterenol-stimulated lipolytic activity between the two groups (Table 1). Basal lipolytic activity (Fig. 4B) but not isoproterenol-stimulated lipolysis (R = −0.184; P = 0.424) was negatively associated with BMI SDS. Moreover, basal lipolysis correlated negatively with adipocyte size (Fig. 4C and Table 3), which was, however, lost after adjustment for BMI SDS (R = −0.11; P = 0.643). Neither basal nor stimulated lipolytic activity changed with age in the whole cohort (Fig. 4D) or in the lean subgroup (R = −0.059; P = 0.863).
(Enlarge Image)
Figure 4.
Basal lipolytic activity of adipocytes is negatively associated with BMI SDS and adipocyte diameter. A: Isolated adipocytes of obese children (black bars) showed a reduced basal lipolysis capacity compared with lean children (white bars). Addition of 10 μmol/L isoproterenol stimulated lipolytic activity in lean and obese children, although no significant differences between adipocytes of lean and obese children could be observed. Data are presented as mean ± SEM. **P < 0.01. Basal lipolytic activity correlated negatively with BMI SDS (B) and adipocyte size (C), but not with age (D). Pearson correlation coefficient R and P values are shown in each scatter plot. Significant P values (P < 0.05) are indicated in bold. Lean children are represented as open circles and obese as closed circles.
Adipocyte Hypertrophy is Linked to Increased Leptin Serum Levels and Insulin Resistance
Finally, we evaluated serum levels of the adipokines adiponectin and leptin for their association with obesity-related alterations in adipocyte biology.
As expected, we observed decreased adiponectin serum levels in obese compared with lean children and a negative association of adiponectin with BMI SDS (Table 1 and Fig. 5A) and age (R = −0.503; P < 0.001). Adiponectin levels did not differ between samples with small or large adipocytes (Table 3), nor did they show a correlation with adipocyte size (Fig. 5B) or number (R = −0.311; P = 0.875). However, we observed a negative association with macrophage infiltration (Fig. 5C), the presence of CLS (11.03 ± 0.83 vs. 5.34 ± 0.37 ng/mL; P < 0.001), and hs-CRP (R = −0.256; P = 0.003). On the other hand, adiponectin levels did not correlate with IL-6 serum levels (R = −0.042; P = 0.640) and IL-6 AT expression (R = 0.076; P = 0.391) nor with TNF-α expression (R = −0.042; P = 0.640) but with TNF-α serum levels (R = 0.316; P < 0.001).
(Enlarge Image)
Figure 5.
Association of serum adipokine levels and HOMA-IR with BMI SDS, adipocyte diameter, and macrophage infiltration. Adiponectin serum levels decrease with BMI SDS (A), whereas no association with adipocyte diameter was observed (B). Furthermore, we observed a negative association with macrophage infiltration (C). Serum leptin levels were positively associated with BMI SDS (D), adipocyte diameter (E), and macrophage infiltration (F). The insulin resistance marker HOMA-IR showed a positive correlation with BMI SDS (G), adipocyte size (H), and macrophage infiltration (I). Pearson correlation coefficient R and P values are given in each scatter plot. Significant P values (P < 0.05) are indicated in bold. Lean children are represented as open circles and obese as closed circles.
Serum leptin was positively correlated to the degree of obesity in children (Table 1 and Fig. 5D) and age (R = 0.477; P < 0.001). Furthermore, leptin levels significantly increased with adipocyte size (Fig. 5E and Table 3), number of macrophages (Fig. 5F), presence of CLS (9.99 ± 1.70 vs. 28.44 ± 4.65 ng/mL in children with or without CLS; P < 0.001), and hs-CRP serum levels (R = 0.591; P < 0.001). Adipocyte size was the strongest predictor for leptin levels in multivariate analyses (Table 2).
Similar to adiponectin, there were no correlations of serum leptin with circulating IL-6 (R = 0.185; P = 0.059) and TNF-α (R = −0.118; P = 0.231) nor with TNF-α expression in AT (R = 0.009; P = 0.920). In contrast to that, we observed a slightly negative association of serum leptin levels and IL-6 mRNA levels in AT (R = −0.195; P = 0.045).
Finally, we were interested in how obesity-associated alterations in AT biology relate to HOMA-IR as a clinical marker of insulin resistance. HOMA-IR levels showed a positive association with not only BMI SDS (Table 1 and Fig. 5G) and age (R = 0.634; P < 0.001), but also adipocyte size (Fig. 5H) and were fourfold higher in samples of patients containing large adipocytes compared with small adipocytes (Table 3). In addition to the association with adipocyte hypertrophy, HOMA-IR was related to macrophage infiltration (Fig. 5I) and increased in AT containing CLS (3.67 ± 0.41 vs. 1.34 ± 0.14; P < 0.001).
Even though there was a correlation of HOMA-IR with hs-CRP (R = 0.286; P = 0.001) and IL-6 (R = 0.220; P = 0.013) serum levels, we did not detect significant associations of HOMA-IR with AT expression of TNF-α (R = 0.071; P = 0.422) or IL-6 (R = −0.115; P = 0.194). Unexpectedly, we detected a negative association of HOMA-IR and TNF-α serum levels (R = −0.396; P < 0.001). In multivariate analyses, HOMA-IR was most strongly affected by adipocyte hypertrophy (Table 2).