Copeptin and Nesfatin-1 are interesting interrelated biomarkers playing a role in the pathogenesis of insulin resistance in Chinese obese children

Background Recently, copeptin and nesfatin-1 have been identied as interesting novel peptides playing a role in the pathogenesis of obesity-related insulin resistance in adults, respectively. However, the relationship between them has not been elucidated; and their circulating levels in obese children have not been adequately studied. Therefore, the current study aimed to investigate whether their levels are altered in Chinese obese children and to study the correlation of these two peptides with each other and with insulin resistance and other biochemical parameters. Methods A total of 120 children were enrolled in this study, including 80 obese children and 40 lean controls. Anthropometric parameters and clinical data of all subjects were collected and circulating TNF-α, adiponectin, leptin, copeptin and nesfatin-1 levels were detected using enzyme-linked immunosorbent assays. Results Serum copeptin and nesfatin-1 levels were signicantly elevated in obese children and children with insulin resistance compared to control subjects. In addition, nesfatin-1 and copeptin levels were found to be signicantly positively correlated with one another in pearson’s correlation and partial correlation. More importantly, multiple regression analysis used nesfatin-1or copeptin as the dependent parameters and signicant correlation between nesfatin-1 and copeptin with each other and the associations between each of them with HOMA-IR were detected. Conclusion These ndings are novel and shed light on the possible interplay role of these two molecules in obesity-related insulin resistance. we and as independent predictors of in addition to and When using copeptin or nesfatin-1 as the dependent variable, in addition to BMI, DBP and HOMA-IR, copeptin and nesfatin-1 are signicantly associated with each other. The data indicates that these two interrelated molecules are reliable biomarkers of the presence of IR, and these molecules can be used as a tool in BMI, body mass index; DBP, diastolic blood pressure; FPG, fasting plasma glucose; HDL-C, high-density lipoproteincholesterol; HOMA-IR, homoeostasis model of insulin resistance; IR, Insulin resistance; LAR, leptin-to-adiponectin ratio; LDL-C, low-density lipoprotein; NUCB2, nucleobindin 2; SDS-BMI, BMI s.d. score; TC, totalcholesterol; TG, triglycerides; SBP, systolic blood pressure; TNF-α, tumour necrosis factor-α; WHtR, waist-height ratio; WC, Waist circumference; WHR, Waist-to-hip ratio.


Abstract
Background Recently, copeptin and nesfatin-1 have been identi ed as interesting novel peptides playing a role in the pathogenesis of obesity-related insulin resistance in adults, respectively. However, the relationship between them has not been elucidated; and their circulating levels in obese children have not been adequately studied. Therefore, the current study aimed to investigate whether their levels are altered in Chinese obese children and to study the correlation of these two peptides with each other and with insulin resistance and other biochemical parameters.
Methods A total of 120 children were enrolled in this study, including 80 obese children and 40 lean controls. Anthropometric parameters and clinical data of all subjects were collected and circulating TNFα, adiponectin, leptin, copeptin and nesfatin-1 levels were detected using enzyme-linked immunosorbent assays.
Results Serum copeptin and nesfatin-1 levels were signi cantly elevated in obese children and children with insulin resistance compared to control subjects. In addition, nesfatin-1 and copeptin levels were found to be signi cantly positively correlated with one another in pearson's correlation and partial correlation. More importantly, multiple regression analysis used nesfatin-1or copeptin as the dependent parameters and signi cant correlation between nesfatin-1 and copeptin with each other and the associations between each of them with HOMA-IR were detected.
Conclusion These ndings are novel and shed light on the possible interplay role of these two molecules in obesity-related insulin resistance.

Background
Over the past decade, Insulin resistance (IR) has gained special attention since it has been largely postulated as the fundamental aspect of the etiology of type 2 diabetes, metabolic syndrome, hypertension, atherosclerosis and polycystic ovarian disease [1]. Although insulin resistance in children was found in other diseases, such as polycystic ovary, obesity was still con rmed to be the major risk factor for the development of insulin resistance in children [2][3]. In a recent study conducted in American adolescents, more than 50% obese subjects have insulin resistance [4] and adiposity was considered to be the most important factor affecting insulin sensitivity [5]. However, mechanisms behind obesity development and progression to Insulin resistance have not been fully unraveled, and no speci c therapy has been identi ed yet.
Obesity is a complex disease involving a number of different peptides, and these peptides and their receptors control energy metabolism and participate in progression of obesity-related diseases. Copeptin, a 39-aminoacid glycopeptides, is a cleavage product of the C-terminal part of the preprovasopressin (pre-proAVP). It is released after stress situations and is correlated to AVP levels in plasma [6]. Beside the action of AVP exerts on BP including vasoconstriction, volume control, and direct cardiac effects, VP exerts major effects on glucose and lipid metabolism [7]. Clinical data has showed that elevated copeptin levels have been associated with increased risk of insulin resistance, type 2 diabetes, hypertension, hyperlipidemia, independently from obesity [7]. Nesfatin-1, a 82 aminoacids anorexigenic peptide, is produced from the nucleobindin2 (NUCB2)precursor, and is expressed in several tissues including neurons, pancreas, liver, adipose tissue and skeletal muscles [8]. Many studies have suggested that nesfatin-1 plays a role in modulating energy metabolism [9][10]. Recently, nesfatin-1 was proposed to contribute to the regulation of insulin secretion from pancreatic beta-cells [11][12].
Up to now, most of research on copeptin and nesfatin-1 have been conducted in animals and adult obese and type 1 and/or 2 diabetic patients [12][13][14]. To our knowledge, only a few studies investigate the relationship between metabolic parameters and serum copeptin and nesfatin-1 levels in obese children [15][16][17][18], and interrelation between copeptin, nesfatin-1and insulin resistance is scarce. The current study aimed to investigate circulating levels of copeptin and nesfatin-1 in Chinese obese children and to identify the correlation of these 2 interesting peptides with one another and between each of them with other biochemical parameters like insulin resistance, adiponectin, leptin, and anthropometric parameters.

Study population
For this study, we recruited 80 consecutive obese children with a body mass index (BMI) above the 95th percentile, who applied to our outpatient clinics at the Second A liated Hospital of Xi'an Jiaotong University with complaint of weight gain, and 40 non-obese controls with a BMI below the 85th percentile [19]. Children with previous diagnosis of endocrine disease (e.g., hypothyroidism and Cushing's disease), any syndrome associated with obesity (e.g., Prader-Willi and Laurence-Moon-Biedle syndromes), acute or chronic infectious disease, and/or a history of drug use were excluded from the study.

Anthropometric measurements
Weight and height were measured in all subjects wearing light clothing without shoes using a scale and a stadiometer with a sensitivity of 0.1kg and 0.1cm respectively. BMI was calculated as the body weight in kilograms divided by the square of height in meters. Waist and hip circumferences were measured using standard techniques to the nearest 0.1 cm. WHtR was calculated as the ratio of waist and hip circumferences. Systolic and diastolic blood pressures (SBP and DBP, respectively) were obtained using the oscillometric device OMRON705IT. Tanner stages were used to evaluate pubertal development [20]. A testicular volume of ≥4mL in boys and stage 2-5 of breast development in girls were considered to enter puberty.

De nition of IR
The homeostasis model assessment of insulin resistance was calculated using the following formula: HOMA-IR = fasting insulin (μU/ml) × fasting glucose (mmol/L)/22.5, Insulin resistance is de ned as values of HOMA index for prepubertal > 2.5 and pubertal > 4.0 [21].
Statistical analysis SPSS 19.0 statistical software was used for all analyses, and p < 0.05 (two-tailed) was considered statistically signi cant. Distribution of data was evaluated using the Kolmogorov-Smirnov test, and data was shown as the mean ± SD. Differences between obese and control groups were assessed using Student's t-test. Pearson's correlation and partial correlation were used to analyze the correlation between copeptin and nesfatin-1and metabolic parameters, anthropometric variables. Furthermore, multiple linear regressions analyses were calculated with HOMA-IR, copeptin or nesfatin-1 as the dependent variable adjusted for age, gender and pubertal stage, and with the independent variables including anthropometric variables, metabolic parameters, and some other clinical indicators.

Results
The clinical characteristics of subjects A total of 80 obese subjects (42 males, mean age: 10.9±2.1 yr) and 40 lean subjects (21 males, mean age: 11.03±1.9 yr) were included in this study. There were no differences of age, gender and pubertal stage distribution between lean and obese groups. Obese children present higher anthropometric indicators including BMI, SDS-BMI, weight, WHtR, WC and WHR. There were statistically signi cant differences between obese and lean subjects in terms of SBP, DBP, insulin, TG, TC, HOMA-IR, AST and ALT levels (p<0.05), whereas FPG, HDL-C levels were similar (p>0.05). Serum leptin, TNF-a, copeptin and nesfatin-1 concentrations and the leptin-to-adiponectin ratio (LAR) were signi cantly higher in obese children compared to controls, but serum levels of adiponectin was signi cantly lower in obese children compared to lean children (p<0.05) ( Table 1).

Serum adipokines and peptides concentrations in Subgroups
Adiponectin, leptin, LAR, TNF-a, copeptin and nesfatin-1 values were similar between male and female children ( Table 2). Pubertal children had signi cantly higher leptin, LAR, copeptin and nesfatin-1 levels when compared with prepubertal children (Table 3). However, comparisons of children regarding the presence of pubertal signs showed signi cant lower adiponectin compared with prepubertal children (p<0.05), (Table 3). Moreover, all participants were classi ed into insulin-resistant group (IR group) and non-insulin-resistant group (Non-IR group) according to their HOMA-IR. Children with IR had higher leptin, LAR, copeptin and nesfatin-1 than non-IR group. Serum TNF-a and adiponectin levels were similar in IR and non-IR groups ( Fig 1).

Correlations of copeptin or nesfatin-1 with clinical variables
We investigated a potential association of copeptin or nesfatin-1 with other clinical variables by Pearson's correlation and partial correlation in obsese children (Table 4). In subjects, copeptin was signi cantly positively correlated with weight, BMI, SDS-BMI, WC and WHtR ( p < 0.05), while there were no correlations with age and WHR ( p > 0.05). In addition, copeptin was positively correlated with insulin, HOMA-IR, blood pressure, HDL-C, leptin, LAR and nesfatin-1. Considering the effects of puberty and weight, we use puberty, age, gender and SDS-BMI as correction factors in the following statistics. After adjustment for the above indicators in partial correlation analysis, we found that HOMA-IR, DBP, HDL-C, leptin, LAR and nesfatin-1 were still signi cantly correlated with copeptin. After that, we determined a potential correlation between nesfatin-1 with other clinical variables in children. We observed associations between nesfatin-1 with weight, BMI, SDS-BMI, WC and WHR. Interestingly, we also found a positive correlation between nesfatin-1 and FPG, HOMA-IR, blood pressure, HDL-C, TNF-a, leptin, LAR and copeptin levels in the circulation, and this correlation is independent of puberty, age, gender and SDS-BMI (Table 4).
Multiple regression analysis to evaluate the association of HOMA-IR, copeptin or nesfatin-1 with other clinical variables in Chinese obese children In multiple linear regression analysis, HOMA-IR was used as the dependent variable and the factors associated with it, including SDS-BMI, WC, WHR, TG, FPG, ALT, SBP, DBP, leptin, LAR, copeptin and nesfatin-1 were used as the independent variables. We con rmed that LAR, copeptin and nesfatin-1 were independent predictors of Insulin resistance index in addition to SDS-BMI, WC and ALT, contributing 5%, 8% and 10% to the variability, respectively (Table 5).

Discussion
Insulin resistance has been largely postulated as the onset of metabolic impairment underlying obesity and metabolic syndrome [22]. As the incidence of childhood obesity rises, Insulin resistance has become the most important risk factor affecting children's health, so it becomes important to discover the early biomarker of IR in children and adolescents with obesity. Recent studies have found that peptides are related to appetite regulation, energy homeostasis, increased blood pressure, in ammation, and immune activity [23]. Therefore, we use the two peptides copeptin and nesfatin-1 as possible markers to predict insulin resistance and investigate the relationship between these two indicators and the insulin resistance index in obese children. Our data showed that copeptin and nesfatin-1 were signi cantly elevated in the circulation of obese children. In addition, copeptin and nesfatin-1 levels were found to be signi cantly positively correlated with each other and were signi cantly positively correlated with the insulin resistance index independent of puberty, age, gender and SDS-BMI. Moreover, in different multiple linear regression models, when using HOMA-IR as the dependent variable, we con rmed LAR, copeptin and nesfatin-1 as independent predictors of HOMA-IR in addition to SDS-BMI, WC and ALT. When using copeptin or nesfatin-1 as the dependent variable, in addition to BMI, DBP and HOMA-IR, copeptin and nesfatin-1 are signi cantly associated with each other. The data indicates that these two interrelated molecules are reliable biomarkers of the presence of IR, and these molecules can be used as a diagnostic tool in clinical practice.
Many hormonal peptides which have been found in hypothalamus and pituitary are involved in the regulation of satiety and play important roles in body weight homeostasis. In high-fat-fed mice, Ramanjaneya et al. have found that nesfatin-1 expression was signi cantly increased and play key roles in controlling food intake [24], but serum nesfatin-1 levels in patients with obesity are still controversial.

Ramanjaneya et al. demonstrated increased nesfatin-1 levels in obese subjects and showed signi cant positive correlation between BMI and nesfatin-1 levels [24]
, while Abaci et al. [25] found that nesfatin-1 levels decreased and observed a signi cant negative correlation between nesfatin-1 and BMI-SDS in obese subjects. Our data showed that serum nesfatin-1 levels were signi cantly higher in the obese children than controls. The reason of con icting results could be due to the different sample sizes included in these studies or other currently unde ned factors that may affect nesfatin-1. In addition, the present study that serum nesfatin-1 concentrations were strongly and positively correlated with WHR, but not correlated with WHtR. In contrast, serum copeptin concentrations were positively associated with WHtR, but not associated with WHR. WHR is a practical index of regional adipose tissue distribution and is correlated with the mass of all adipose tissue including abdominal subcutaneous and visceral fat, but WHR value did not account for the large variations in the level of abdominal visceral adipose tissues [26], whereas WHtR is a good predictor of intra-abdominal adipose tissue and has no correlation with subcutaneous adipose tissue [27]. This result would probably indicates that nesfatin-1 levels are closely related to regional adipose tissue, but copeptin levels are closely related to intra-abdominal adipose tissue.
We did not detect signi cant differences in nesfatin-1 levels between boys and girls. The nding in children ts well with studies in adults reporting no difference in nesfatin-1 concentrations between males and females [28]. Interestingly, we demonstrated that pubertal children had higher copeptin and nesfatin-1 levels than pre-pubertal children. Regulation during puberty is also well known for other adipokines [29], but the data about copeptin and nesfatin-1 levels in pubertal children are still absence. The underlying mechanism of this difference in prepubertal and pubertal children has not been speci cally investigated, and we speculate that this difference may be related to elevated hormone levels in adolescent.
Surprisingly, serum nesfatin-1 was found to be signi cantly positively correlated with TNF-a, which was not entirely due to obesity. TNF-a which secreted by adipose tissue was proven to affect insulin signaling and to modulate insulin resistance by various mechanisms, and to be a marker of systemic in ammation [30]. Ramanjaneya et al. observed in animal models of subcutaneous adipose tissue explants that nesfatin-1 production was signi cantly increased by TNF-α [24]. This observation revealed nesfatin-1is involved in the pathological process of in ammation and insulin resistance and the pathogenesis need further research to be elucidated. Although copeptin levels are elevated in in ammatory states in adults [31], we could not show a relationship between TNF-a and copeptin levels in obesity children.
In this study, obese children with insulin resistance had insigni cant higher nesfatin-1 and copeptin levels compared with obese children without insulin resistance. In correlation analysis, nesfatin-1 and copeptin were found to relate to plasma insulin and HOMA-IR, and this correlation is independent of puberty, age, gender and SDS-BMI. Multivariate linear regression analysis revealed that copeptin and nesfatin-1 can as independent predictors of Insulin resistance, contributing 8% and 10% to the variability, respectively. Our Interestingly, we detected signi cant associations between nesfatin-1 and copeptin with one another in pearson's correlation and partial correlation. More importantly, multiple regression analysis used nesfatin-1or copeptin as the dependent parameters and we did detect signi cant correlation between nesfatin-1 and copeptin with each other and the association between each of nesfatin-1 and copeptin with HOMA-IR. However, we did not detect any signi cant correlation with adiponectin or leptin. This nding suggests that nesfatin-1 and copeptin are interrelated peptides and play a role in obesity-related insulin resistance.
To the best of our knowledge, these ndings are novel and are shed light on the possible interplay between these two bioactive mediators for the rst time in children.
In summary, we detect that serum nesfatin-1 and copeptin concentrations are increased in obese children with insulin resistance. Furthermore we did a novel observation that nesfatin-1 and copeptin were signi cantly interrelated and associated with HOMA-IR. These ndings are shed light on the possible interplay role of these two molecules in obesity-related insulin resistance.

Availability of data and materials
The datasets used or analysed during the current study are available from the corresponding author upon reasonable request.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.