All children and adolescents who visited the pediatric endocrinology clinic of Incheon St. Mary’s Hospital from December 2022 to November 2023 were screened for inclusion and exclusion criteria. Inclusion criteria were children and adolescents with obesity (defined as BMI ≥ 95th percentile for age and sex). Normal-weight subjects (defined as 15th ≤ BMI < 85th percentile for age and sex) were included as controls. Exclusion criteria were children with endocrine disorders (central precocious puberty, diabetes mellitus, growth hormone deficiency, idiopathic short stature, thyroid disease, rickets, Cushing disease), genetic disorders (Turner syndrome, Prader-Willi syndrome, congenital adrenal hypoplasia), and other chronic diseases (liver disease other than metabolic dysfunction-associated steatotic liver disease [MASLD], renal disease). Subjects whose parents refused to participate in the study or those from whom fasting blood samples were not obtained were excluded. Figure 1 shows the flowchart of inclusion and exclusion criteria for the study. Written informed consent was obtained from all participants and their parents. The study was approved by the Institutional Review Board of Incheon St. Mary’s Hospital (IRB number: OC22OISI0140).

Height was recorded to the nearest 0.1 cm using a Harpenden Stadiometer, and weight was recorded to the nearest 0.1 kg using an electronic scale. SDS for weight, height, and BMI were calculated according to age and sex using the reference data published in the 2017 Korean National Growth Charts (21). The pubertal stage was assessed by the pediatric endocrinologist according to Marshall and Tanner standards (22).

Venous blood samples were obtained in the morning after a 10-hour overnight fast. Serum samples were analyzed for HbA1c, glucose, insulin, total cholesterol, TG, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, aspartate transaminase (AST), alanine transaminase (ALT), uric acid, 25(OH)-vitamin D, and asprosin. In children with elevated ALT, measurements were taken every 3 months. If ALT was consistently more than twice the upper normal limit, further evaluation was conducted to exclude other causes of liver disease. Glycated hemoglobin (HbA1c) levels were measured using automated high-performance liquid chromatography (HLC-723 G7, Tosoh, Tokyo, Japan). Serum insulin levels were measured with Elecsys insulin Assay (Cobas e411 immunoassay analyzer; Roche Diagnostics, Mannheim, Germany). Serum glucose, total cholesterol, LDL cholesterol, HDL cholesterol, TG, AST, and ALT were tested using a Beckman Coulter AU5800 clinical chemistry analyzer and the manufacturer’s reagents (Beckman Coulter, Brea, CA, USA). Serum 25-hydroxy vitamin D was measured with a Siemens ADVIA Centaur^® vitamin D TOTAL immunoassay (Siemens Healthcare, Erlangen, Germany).

Blood samples for the measurement of plasma asprosin were collected using EDTA tubes and centrifuged at 2,000 × g for 10 minutes at room temperature. The isolated plasma samples were stored at -80°C until asprosin levels were measured. Plasma samples were diluted 1:2 with sample diluent, and asprosin levels were measured according to the manufacturer’s protocol using commercial ELISA kits (Cat. No. ab275108, ELISA sandwich kit, Abcam, USA). The manufacturer reported that the intra-assay coefficient of variation is 7.9% and the minimum detectable concentration is 0.92 ng/mL.

Dyslipidemia was defined as either or a combination of total cholesterol ≥200 mg/dL, LDL cholesterol ≥ 130 mg/dL, HDL cholesterol ≤40 mg/dL, and TG ≥150 mg/dL according to the Third Report of the National Cholesterol Education Program (23) and the American Diabetes Association (24). Insulin resistance was estimated from fasting glucose and insulin serum levels using the homeostasis model assessment of insulin resistance (HOMA-IR) as fasting insulin (µU/ml) × fasting glucose (mmol/L)/22.5. Insulin resistance was defined as a HOMA-IR >2.5 in pre-pubertal and >4.0 in pubertal subjects (25). The TyG index was calculated as ln (fasting triglycerides [mg/dL] × fasting plasma glucose [mg/dL]/2).

All analyses were performed using R statistical software (version 4.3.2, R Foundation for Statistical Computing, Vienna, Austria). Categorical variables are presented as numbers (%), and continuous variables are presented as mean ± standard deviation. Chi-square tests or Fisher’s exact tests were employed for categorical variables between two groups, as appropriate. The Student’s t-test and Mann-Whitney U test were utilized for continuous variables with normal and non-normal distributions, respectively. The normality of distribution was assessed using the Shapiro-Wilk test. The correlation between asprosin and continuous variables was examined using Pearson’s correlation and partial correlation analysis. Multiple linear regression was used to estimate the independent association between asprosin levels and clinical parameters. Age, sex, and variables that were statistically significant in simple linear regression were included in the multiple linear regression analysis. Mediation analysis was conducted to examine whether the association between BMI-SDS and HOMA-IR was mediated by asprosin. All p-values < 0.05 were considered statistically significant.

Table 1 shows the clinical and biochemical characteristics in the obese and normal weight groups. Of 109 children and adolescents, 62 (56.9%) were obese and 47 (43.1%) were normal weight ( Figure 1 ). The mean age was 9.6 years (median, 9.0; range, 6–14). Male sex was more common in the obese group than in the control group (59.7 vs. 31.9%; p = 0.007). Height SDS, glucose, insulin, HOMA-IR, total cholesterol, and ALT were higher in the obese group than in the control group. Insulin resistance was more common in the obese group than in the control group (58.1% vs. 2.1%; p < 0.001). Dyslipidemia was more common in the obese group than in the control group (59.7% vs. 36.2%; p = 0.03). There were no significant differences in systolic blood pressure, diastolic blood pressure, Tanner stage, HbA1c, TG, LDL cholesterol, HDL cholesterol, TG/HDL cholesterol ratio, TyG index, AST, uric acid, and 25(OH)-vitamin D levels.

Plasma asprosin levels did not differ between boys and girls (median 87.6 vs. 72.6; p = 0.14). Obese subjects had higher plasma asprosin levels than healthy subjects (mean 87.6 ± 26.2 vs. 69.3 ± 16.3 ng/mL; p = 0.001). This finding was not evident in boys (mean 87.8 ± 26.4 vs. 77.5 ± 17.6 ng/mL; p = 0.30), but it was evident in girls (mean 85.9 ± 26.4 vs. 63.9 ± 13.3 ng/mL; p = 0.002) ( Figure 2 ). Plasma asprosin levels were higher in the insulin resistance group than in the non-insulin resistance group (mean 98.6 ± 24.6 vs. 70.2 ± 17.7 ng/mL; p < 0.001). This finding was evident in both boys (mean 99.8 ± 24.5 vs. 72.6 ± 17.9 ng/mL; p < 0.001) and girls (mean 96.7 ± 25.7 vs. 67.8 ± 17.6 ng/mL; p < 0.001) ( Figure 3 ). There was no difference in plasma asprosin levels between subjects with and without dyslipidemia (mean 86.4 ± 29.0 vs. 78.4 ± 19.6 ng/mL; p = 0.14).

Table 2 shows the simple correlation analysis between plasma asprosin and clinical and biochemical parameters. Plasma asprosin levels were positively correlated with BMI SDS (r = 0.34; p = 0.002), HbA1c (r = 0.25; p = 0.02), fasting glucose (r = 0.33; p = 0.002), fasting insulin (r = 0.44; p < 0.001), HOMA-IR (r = 0.47; p < 0.001), and TyG index (r = 0.38; p < 0.001). Plasma asprosin levels were positively correlated with TG (r = 0.33; p = 0.003) and negatively correlated with HDL cholesterol (r = -0.29; p = 0.008). Plasma asprosin level was positively correlated with TG/HDL cholesterol ratio (r = 0.42; p < 0.001). The positive correlation between plasma asprosin and BMI SDS was evident in the insulin resistance group (r = 0.39; p = 0.02) but not in the non-insulin resistance group (r = 0.05; p = 0.76).

After adjusting for age and BMI SDS, glucose (r = 0.27; p = 0.02), insulin (r = 0.34; p = 0.002), HOMA-IR (r = 0.37; p < 0.001), TG (r = 0.27; p = 0.02), HDL cholesterol (r = -0.26; p = 0.02), TG/HDL cholesterol ratio (r = 0.37; p < 0.001), and TyG index (r = 0.31; p = 0.005) were significantly correlated with asprosin ( Table 3 ). When adjusting for age within the same sex, the correlation of asprosin with insulin, HOMA-IR, and the TG/HDL cholesterol ratio was evident in both boys and girls. The correlation of asprosin with glucose and TyG index was evident in boys but not in girls. In contrast, the correlation of asprosin with uric acid and 25(OH)-vitamin D was evident in girls but not in boys ( Table 3 ).

Table 4 shows multiple linear regression analysis between plasma asprosin levels and clinical parameters. Multiple linear regression analysis indicated that plasma asprosin levels were independently associated with HOMA-IR and TG/HDL cholesterol ratio (Adjusted R² = 0.37, p < 0.001). Mediating effects analysis was conducted to examine whether the association between BMI SDS and HOMA-IR was mediated by asprosin ( Figure 4 ). The size of the total effect of BMI SDS on HOMA-IR was estimated as β = 0.40 (where β is the standardized coefficient). The direct effect of BMI SDS on HOMA-IR was estimated as β = 0.27, accounting for 68% of the total effect. The indirect effect of BMI SDS on HOMA-IR was estimated as β = 0.13, accounting for 32% of the total effect. The results of the mediation analysis indicated that asprosin appeared to play a mediating role in the relationship between obesity and insulin resistance.