Early sex-dependent differences in metabolic profiles of overweight and adiposity in young children: a cross-sectional analysis

Obesity has become a hallmark of modern childhood with over 340 million children and adolescents with overweight or obesity worldwide [1, 2]. Its growing prevalence in children, including a disproportionate rise during the COVID-19 pandemic, likelihood of leading to lifetime obesity, and association with cardiometabolic risk factors are major concerns [3‐5].

Various methods are available for assessing obesity including body mass index (BMI), waist circumference, waist to height ratio, and body fat percentage estimated from sum of skin thickness or more quantitative measures (e.g., bioelectrical impedance analysis) [6]. While BMI standardized for age and sex remains the most commonly used metric of obesity in children, other adiposity measures of central body fat distribution such as waist circumference, and visceral fat are less investigated in children [7, 8]. In all cases, metabolite derived biomarkers hold promise in improved prediction of future cardiometabolic disease than weight or other traditional risk factors alone even at early life stages [9, 10].

State-of-the-art mass spectrometry (MS) or nuclear magnetic resonance (NMR) are major instrumental platforms used in metabolomics analysis of complex biological sample [11, 12]. As substrates or products of biochemical pathways, metabolites capture the output of upstream processes involving the genome, the transcriptome, and the proteome, as well as downstream changes in lifestyle, diet, and environmental exposures [13, 14].

A systematic review of metabolomic studies of obesity in children (age range of 0.25 to 18 years) in the past 15 years resulted in a total of 41 articles and reported an emerging metabolic profile of childhood obesity most consistent for branched-chain and aromatic amino acids [15]. However, the literature in young children (specifically < 6 years) is limited. Given that insulin resistance is usually only evident around puberty, as shown in the longitudinal study of the development of insulin resistance from age 5 to 16 years [16], studying overweight/adiposity during early childhood, e.g., at age 5 years, offers a novel window to investigate how early subclinical metabolic perturbations develop. Such investigations are valuable especially that childhood adiposity is a predictor of cardiometabolic health in adulthood. Likewise, no studies have researched sex-specific differences in early childhood despite the opportunity this could offer in understanding early pathophysiology and optimizing nutrition or growth rate intervention windows based on sex. We hypothesized that there would be sex-related differences in metabolic-adiposity profiles prior to and apart from puberty-related differences.

In the present study, we sought to (i) characterize serum metabolites associated with overweight/adiposity measures in 900 children at 5 years of age, (ii) investigate early sex-dependent differences, and (iii) validate our results in an independent cohort of 456 children of the same age.

We identified a robust metabolite phenotype that was associated with overweight and adiposity in young children. This panel is comprised of BCAAs and AAAs, glutamic acid, the glutamine/glutamic acid ratio, and threonine and was more evident in female children in a well-characterized cohort of 900 children and a replication cohort of 456 children at age 5 years. More direct measures of adiposity/fat distribution including waist circumference and sum of skinfold thickness yielded more significant associations than did z-BMI.

Our metabolomic phenotype findings are generally in agreement with prior obesity metabolome investigations in older children (7.7–11.1 years of age) despite the non-fasting status in our discovery cohort [31, 32]. Our study suggests that BCAAs in children do not primarily associate with postprandial interval [33] because the serum samples collected in CHILD were non-fasting—a finding that has also been made in adults [34, 35]. Our observation, that BCAAs concentration is associated with overweight and adiposity at 5 years of age, is most consistent with the literature as reported by at least 10 prior studies in children of varying age [15]. In studies with contrasting findings, BCAAs concentrations were lower in adolescents with obesity and type 2 diabetes compared with adolescents with both normal weight or with obesity [36] and were inversely associated with insulin resistance in a longitudinal follow-up of children (age 5–16) [16]. Of note, in our FAMILY replication cohort, we have observed significant negative associations of BCAAs with fasting serum glucose (data not shown). Furthermore, we have previously identified a distinct metabolomic phenotype associated with an aggregate metabolic syndrome risk score in the FAMILY cohort that did not include BCAAs [18]. Congruent with our current analysis, this metabolic syndrome signature included the aromatic amino acid tyrosine, threonine, alanine, and glutamine/glutamic acid ratio. These could be promising biomarkers of cardiometabolic disease risk in young children reflecting an aggregate of risk factors that includes but is not limited to excessive abdominal adiposity [18, 37].

Most studies in the literature have used z-BMI to classify childhood obesity, while other adiposity measures of central body fat distribution such as waist circumference, or skin fold thickness are infrequently measured or investigated among children. Using linear regression analysis, we have shown that metabolic differences can arise depending on whether an obesity or adiposity measure is used, with a greater number of serum metabolites being identified in relationship to adiposity rather than overweight/obesity. In accordance with research showing the independent and additive value of waist circumference metric in predicting morbidity [38], the approach we adopted by combining both z-BMI and waist circumference may serve as a useful approach for other researchers and future investigations. Few prior studies have investigated the variation of the overweight/adiposity metabolome in children by sex. Most studies included older children and youth where puberty changes become a major confounder [9, 10, 39‐42]. In our sex-stratified analysis, we observed that the metabolic profile associated with overweight and adiposity at age 5 years was mainly driven by the effects in girls, while boys showed mostly weaker and non-significant associations (albeit in the same direction). Saner et al. investigated sex and puberty-related metabolic differences related to adiposity in youth [mean age 11.9 (3.1 SD)] and found that tyrosine and phenylalanine were positively associated with z-BMI in girls only, consistent with our results, whereas in boys significant associations with leucine and isoleucine were seen post-puberty [9]. These sex-specific differences could be related to girls maturing earlier than boys but unlikely to be a direct result of sexual hormone maturation because adrenarche (even premature adrenarche) occurs at an older age than that of our cohort. Another hypothesis to explain our sex-related findings may be linked to differences in mitochondrial plasticity between boys and girls—a process of mitochondrial adaptation to normalize/correct metabolic impairments [36]. Further sex-specific analyses are necessary, ideally within longitudinal studies, to better understand the underpinning mechanisms and to test these hypotheses of sex- and puberty-related metabolomic associations with obesity and cardiometabolic disease.

Our study has several strengths which include its large sample size (1345 children) and high quality as defined by Handakas et al. [15]. In this systematic review of 41 metabolomic studies of childhood obesity, 66% of studies had a sample size < 200, and few were rated as high quality [15]. Furthermore, our study addresses the disparity in research of sex-based differences in metabolic changes in early childhood. Moreover, we included a validation of our findings in an independent cohort of children of the same age using the same validated metabolomics platform with rigorous quality control. Several limitations should also be considered. Serum samples were non-fasting in CHILD. Metabolome coverage was limited to polar ionic metabolites and did not include important circulating lipids, such as fatty acids and phospholipids [43, 44]; thus, complementing our analysis with other orthogonal platforms would be valuable. Finally, this is a cross-sectional evaluation and it is yet unknown if the overweight/adiposity metabolomic profiles are linked to future health outcomes.

Using metabolomics profiling of 1345 children, we have identified that BCAAs, AAAs, glutamic acid, and the glutamine/glutamic acid ratio are associated with overweight and adiposity in early childhood at age 5 years, and this relationship is more evident in girls than boys at age 5 years. The use of central adiposity measures, e.g., waist circumference, was optimal in defining this metabolomic pattern. Overall, our work underscores the value of high-throughput metabolomics in understanding disease pathways and the need for future sex-specific investigations to design future interventions to mitigate the obesity pandemic as early in life as possible.