This study shows that, already in prepubertal age, obesity is linked with hyperleptinemia, impaired HMW adiponectin levels, as well as alterations of specific immune mediators with minor sex-associated differences. Notably, in this very young population, leptin, rather than HMW adiponectin, emerges as a sex-independent discrimination marker of adiposity degree, as well as a sex-related predictor of basal insulin resistance. Finally, also in prepubertal obesity, we confirm the lack of a universal upregulation of the immune response.
In obese children, persistent low-grade inflammation appears to increase the metabolic risk in later life. The importance to investigate adipose-derived mediators during the developmental stages of overweight and obesity allows to identify at what age and degree of adiposity these pathological events may occur. Our current work describes the combined associations of obesity, IR and sex on a cluster of adipokines in prepubertal children. The characterization of a wide panel of adipose-derived mediators, rather than measurement of single molecules only, appears of considerable importance in youth since current conventional risk factors may not be sensitive enough to detect high-risk individuals, and the prognostic information provided may be different to those in older obese subjects.
As yet, most of the studies on children analyzed a limited number of adipose-produced mediators, and comparative analyses for sex and pubertal stage were not always stratified over wide BMI ranges 
. Here we focused on prepubertal children in their early phase of fat gain, making confounding effects of co-morbidities or gonadal steroids unlikely. The first main finding was that, independently from sex, hyperleptinemia and, to a lesser extent, decreased HMW adiponectin, characterizes prepubertal obesity.
Among the adipokines, adiponectin and leptin are unique hormones because these pleiotropic mediators are almost exclusively secreted by the adipocytes, and their systemic levels reflect the degree of fat accrual in a reciprocal manner 
. Adiponectin, however, circulates in low- (LMW), middle- (MMW) and high-molecular weight (HMW) complexes, which exert different biological functions 
. In this regard, already in obese adolescents, the selective downregulation of the HMW multimers has been shown to reflect metabolic and vascular abnormalities better than total adiponectin levels 
. Prospectively, in adults, hypoadiponectinemia, decreased HMW adiponectin and increased leptin concentrations were differently reported as independent predictors of T2D development and, less consistently, of CHD events 
. Notably, in our study, adiposity-induced variations of leptin and, at least in female individuals, changes of HMW adiponectin concentrations, were of the same magnitude as those that predicted diabetes development in adulthood, suggesting an early effect of these molecules on the regulation of glucose homeostasis 
. Thus, leptin upregulation along with decreased HMW adiponectin subfractions may both have a relevant impact for future disease risk in this subset of obese, otherwise healthy, prepubertal children.
As long as at this age consistent effects of gonadal steroids appear unlikely, different reasons may underlie the dysfunctional profile of these adipokines. First, the lack of interconversion of the adiponectin complexes in the bloodstream highlights the importance of the adipocyte secretory pathway in determining the circulating pattern of adiponectin isoforms 
. In adult males, we have recently demonstrated that adipocyte enlargement and/or IR, rather than body fat mass or testosterone, appear prerequisites for the impaired cellular secretion of HMW adiponectin complexes 
. Accordingly, in prepubertal children, intracellular content of adiponectin in subcutaneous adipocytes was reported to be reduced with increasing BMIz-score 
. Our findings partly extend these results and implicate reduced HMW adiponectin and increased leptin levels as early indicators of adipocyte enlargement and cell dysfunction. The opposite associations between HMW and leptin with adiposity measures, along with the observation that BMIz-score
was the strongest determinant of both leptin and HMW multimers, partly support such a concept.
Second, since hyperinsulinemia or IR may either induce hyperleptinemia or promote the selective downregulation of the HMW adiponectin forms 
, a different degree of insulin sensitivity in the adipose cells may well have contributed to the impaired secretion of these adipokines. Accordingly, it has been demonstrated that total adiponectin concentrations are more closely related to variations in insulin-mediated glucose disposal than to fat mass expansion 
. In our study, we showed that the associations of fasting insulin () or HOMA-IR (supplementary Tables S3
) with HMW adiponectin were attenuated or became insignificant after adjustments for BMIz-score
or lipids, respectively. Thus, the association of HMW adiponectin with IR appears partly mediated by adiposity. In contrast, leptin, which was also largely explained by adiposity (), represented an independent and stronger predictor of the systemic IR as compared with HMW or the combinational effect of L/HMW (). The demonstration that in adipocytes adiponectin and leptin are secreted through distinct trafficking pathways 
, which appear modulated by insulin in different ways 
, may partly explain these findings, substantiating the hypothesis that derangements of glucose homeostasis develop at an early preclinical stage as a result of adipocyte dysfunction/enlargement 
. On the other hand, it has recently been postulated that, in obese prepubertal children, HMW adiponectin shows closer relationship with improvement of glucose homeostasis after weight loss, rather than with body fat content 
. Nonetheless, we have previously demonstrated that, in obese children and adolescents, the degree of hyperleptinemia was a reliable predictor of responsiveness to an educational program of weight excess reduction 
. It seems thus plausible that, in prepubertal age, leptin may provide prognostic information (ie, prediction of weight-excess reduction) different to that of HMW adiponectin (ie, improvement of glucose homeostasis after weight loss).
Third, although HMW adiponectin, leptin and inflammatory markers appear to some extent interdependent, the addition of leptin and sICAM-1 as “inflammation-related” confounders did not improve the prediction of HMW adiponectin, fostering the concept that, although mutually antagonistic, low-grade inflammation and HMW adiponectin may reflect two distinct features of the immune response, as previously postulated 
In adolescents, various and controversial studies addressed the sex-associated variability of total adiponectin and leptin 
, while information on HMW is scarce. In our cohort, when BMI-categorized groups were analyzed, a tendency toward a sex-dimorphic pattern for HMW adiponectin in lean individuals was found, whereas leptin allowed a sex-independent discrimination of adiposity degree. However, for both leptin and HMW adiponectin we did not find significant sex-by-BMI-group interaction, implicating the lack of consistent changes of the response variable when, at the same BMI category, sex varied. Even more, sex did not affect the prediction of BMIz-score
on HMW adiponectin () and leptin (supplementary Tables S5
). Hitherto, although two population-based surveys found higher total adiponectin levels in girls than in boys even in prepubertal age, consistent with our results other authors did not 
. These discrepancies may largely reflect the lack of comparisons across differentially categorized groups. Here, we adopted Cole's criteria 
and incorporated a broad spectrum of clinical phenotypes, making thus the occurrence of group misclassification unlikely. It is conceivable, and remains to be shown, that inherent characteristics of adipocyte cellular function as well as variations in molecular degradation or clearance rate may influence the levels of these mediators in lean prepubertal children 
On the other hand, sex represented an “effect modifier” in the interaction between leptin and basal IR. Our observation that leptin was an independent determinant of HOMA-IR in girls, but not in boys (), is however in line with the notion that, in adulthood, inflammation appears more strongly associated with IR and diabetes risk in females than in males 
. As far as in our study sex differences regarding body composition and systemic gonadal steroids levels (ie, testosterone) were not apparent, such sex-associated differences may have several explanations. Different degrees of adjustment in the multivariable models (ie, larger sex-differences according to the number of confounders) as well as chance (reduced sample size) might both have played a role. Moreover, although we can reasonably exclude the effect of ethnicity, the influence of different baseline risk factors of IR cannot be ruled out.
Turning to the other major finding of this study, childhood obesity is not characterized by a generalized immune activation, but rather a differential upregulation of specific chemokines/cytokines with minor sex differences 
. Obese girls, indeed, displayed higher IL-8, IL-18, MCP-1 and sICAM-1 levels, while in boys adiposity was linked with increased MIF concentrations. The lack of consistent sex-by-BMI-group interaction, as well as the absence of significant correlations between gonadal steroids and all of the above immune mediators (supplementary Table S1
), reasonably indicates that factors other than sex (ie., nutritional habits, physical activity) may partly explain these differences. Although no prior study has evaluated such a specific repertoire
of adipokines in prepubertal age, our data are of interest because these chemokines represent crucial immune mediators involved in macrophage infiltration into adipose tissue, which reasonably occurred also in our cohort of obese children 
. Moreover, as far as elevated concentrations of IL-8, MCP-1, IL-18 and MIF may precede the onset of T2D in adults 
, our data indicate that, in prepubertal age, the association between these immune mediators and HOMA-IR appears basically explained by adiposity (supplementary Tables S3
). Accordingly, in multivariate analysis, the inflammatory markers did neither affect the ability of leptin in predicting basal IR nor contributed significantly to variations in HOMA-IR (). On the other hand, the observation that the immune markers correlated directly with each other, also independently from individual's BMI (supplementary Table S4
), suggests that sources other than fat tissue may contribute to the elevated circulating levels of these chemokines.
Study limitations and Strengths
This study has some limitations that need to be considered. The cross-sectional nature of the survey does not allow conclusions to be drawn about the true prognostic values and the molecular mechanisms underlying the relationships between adipokines and IR. In addition, since in prepubertal age overweight and obesity may not necessarily track into adulthood, the prognostic relevance of these results should be further ascertained. However, obesity-linked variations of adipokines levels we found were almost comparable to those that predicted the risk of diabetes in adults 
, and the prognostic role of leptin also as a predictor of weight excess reduction was earlier demonstrated 
. Moreover, we did not characterize the middle- and low-molecular weight adiponectin oligomers, which may also have a differential distribution according to adiposity degree. On the other hand, the HMW multimers appear the major active forms in mediating the metabolic effects of adiponectin even in young children 
These constraints have to be weighed against the strengths of our survey which reside in the sample size, the representative nature of the population studied, the extensive characterization of the study participants, the simultaneous determination of multiple adipokines as well as the careful adjustment for various confounders using multivariable methods.
Conclusions and Perspectives
In conclusion, our findings demonstrate that, as early as in prepubertal age, adiposity exhibits an unfavourable pattern of adipokines with minor sex-associated differences. However, among the adipose-derived mediators, leptin can be envisioned as a sex-independent discrimination marker of adiposity degree and, at least in girls, a reliable indicator of the systemic insulin resistance as estimated by HOMA-IR. Therefore, in prepubertal children, hyperleptinemia may allow the early identification of “at-risk” individuals, providing important prognostic information in predicting the impairment of glucose homeostasis.