Differential association between S100A4 levels and insulin resistance in prepubertal children and adult subjects with clinically severe obesity

Summary Objectives S100A4 has been recently identified as an adipokine associated with insulin resistance (IR) in adult subjects with obesity. However, no data about its levels in children with obesity and only a few approaches regarding its potential mechanism of action have been reported. To obtain a deeper understanding of the role of S100A4 in obesity, (a) S100A4 levels were measured in prepubertal children and adult subjects with and without obesity and studied the relationship with IR and (b) the effects of S100A4 in cultured human adipocytes and vascular smooth muscle cells (VSMCs) were determined. Methods Sixty‐five children (50 with obesity, age 9.0 ±1.1 years and 15 normal weight, age 8.4 ±0.8 years) and fifty‐nine adults (43 with severe obesity, age 46 ±11 years and 16 normal weight, age 45 ±9 years) were included. Blood from children and adults and adipose tissue samples from adults were obtained and analysed. Human adipocytes and VSMC were incubated with S100A4 to evaluate their response to this adipokine. Results Circulating S100A4 levels were increased in both children (P = .002) and adults (P < .001) with obesity compared with their normal‐weight controls. In subjects with obesity, S100A4 levels were associated with homeostatic model assessment‐insulin resistance (HOMA‐IR) in adults (βstd = .42, P = .008) but not in children (βstd = .12, P = .356). Human adipocytes were not sensitive to S100A4, while incubation with this adipokine significantly reduced inflammatory markers in VSMC. Conclusions Our human data demonstrate that higher S100A4 levels are a marker of IR in adults with obesity but not in prepubertal children. Furthermore, the in vitro results suggest that S100A4 might exert an anti‐inflammatory effect. Further studies will be necessary to determine whether S100A4 can be a therapeutic target for obesity.


O R I G I N A L A R T I C L E
Naturvitenskapelige Universitet, Grant/Award Number: K-sted 651010-Project number 70084900-analyse numb; Norwegian University of Science and Technology, Grant/Award Number: K-sted 651010-project number 70084900-analyse number M50676; Fondo Europeo de Desarrollo Regional (FEDER) ±11 years and 16 normal weight, age 45 ±9 years) were included. Blood from children and adults and adipose tissue samples from adults were obtained and analysed.
Human adipocytes and VSMC were incubated with S100A4 to evaluate their response to this adipokine.
Results: Circulating S100A4 levels were increased in both children (P = .002) and adults (P < .001) with obesity compared with their normal-weight controls. In subjects with obesity, S100A4 levels were associated with homeostatic model assessmentinsulin resistance (HOMA-IR) in adults (βstd = .42, P = .008) but not in children (βstd = .12, P = .356). Human adipocytes were not sensitive to S100A4, while incubation with this adipokine significantly reduced inflammatory markers in VSMC.

Conclusions:
Our human data demonstrate that higher S100A4 levels are a marker of IR in adults with obesity but not in prepubertal children. Furthermore, the in vitro results suggest that S100A4 might exert an anti-inflammatory effect. Further studies will be necessary to determine whether S100A4 can be a therapeutic target for obesity.

| INTRODUCTION
According to updated World Health Organization (WHO) reports, overweight and obesity worldwide have almost tripled since 1975 1 .
In 2016, more than 1.9 billion adults (older than 18 years) were overweight, and out of these, 650 million were obese. The problem is not only increasing in adults but also in adolescent and children (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) years), where more than 340 million were either overweight or obese in 2016. 1 Hallmarks of the obesity state, including visceral fat accumulation and low-grade chronic inflammation, are associated with increased secretion of pro-inflammatory cytokines and the development of metabolic diseases including cardiovascular disease and type 2 diabetes (T2D). 2,3 The pro-inflammatory state plays an important role in insulin resistance, 2 but the link between obesity and the development of T2D is not well-characterized. It is known that white adipose tissue (WAT) has an endocrine role through the secretion of signalling molecules such as adipokines by adipocytes and other cell types present in adipose depots. These molecules participate in a hormonal network communicating several tissues and organs including muscle, liver, and brain. 4 Moreover, adipose tissue expansion occurring during obesity also leads to immune cell recruitment. Therefore, adipose tissue may be considered an immunological organ since these immune cells can secrete pro-inflammatory and anti-inflammatory cytokines affecting adipocyte biology, metabolism, and systemic inflammation, and being a key factor in insulin resistance development and obesity-related pathologies. 2,5 However, some patients with obesity show normal glucose homeostasis, 6 and previously unrecognized molecules could be involved in this situation. Moreover, browning of subcutaneous WAT (appearance of thermogenic beige/brite adipocytes in WAT depots) has been associated with protection against obesity and improvement of associated metabolic alterations. 7 Finally, angiogenesis is a key process required for adipogenesis in vivo and has an important role in inflammation-induced adipose tissue expansion. 8 In the normal weight state, adipose tissue is a well-vascularized tissue. 9 During the early stages of obesity development, angiogenic factors are overexpressed to facilitate healthy adipose tissue expansion and confer protection from metabolic insults. 10 Therefore, a better understanding of adipose tissue endocrine, immunological and angiogenic capability, and the identification of novel potential biomarkers and therapeutic targets to prevent and treat T2D is one of the goals in obesity field. S100A4 is a member of the S100 calcium-binding protein family and has been reported to be involved in controlling cell growth, differentiation, survival, and migration in different cell types, as well as in energy metabolism. 11,12 S100A4 was identified by Ebradlize and colleagues as a protein associated with tumour metastasis. 13 S100A4 binds to cytoskeletal proteins including F-actin 14 and nonmuscle myosin heavy chains. 15 S100A4 gene expression is elevated in activated macrophages. 16 A recent report has identified S100A4 as a cytokine secreted by adipose tissue mainly by precursors and immune cells, with a potential role as a marker of subcutaneous WAT dysfunction and insulin resistance in adult individuals with severe obesity. 17 Moreover, a study performed in mice postulates that S100A4 protects against diet-induced obesity and inflammatory state. 18 In this study, our hypothesis was that S100A4 would be elevated and directly correlated to insulin resistance in children with obesity as well as in adults. To address this hypothesis, S100A4 levels in children and adult patients with and without obesity were quantified and their relationship with insulin resistance was determined. Moreover, the effects of S100A4 in human adipocytes and vascular smooth muscle cells (VSMCs) were studied.

| Children study
Data from this cohort have been previously published, reporting the effects of lifestyle intervention on the metabolomic profiles in blood and urine. 19,20 In the present study, we quantified S100A4 levels in the blood samples to evaluate potential associations with glucose homeostasis. Briefly, children attending the Obesity Unit at the Sant Joan de Déu Hospital were recruited between January 2013 and December 2014. All parents signed the informed consent form. Inclusion criteria were (a) age from 7 to 10 years of age; (b) with obesity, defined as body mass index-standard deviation score (BMI-SDS) greater than 2 standard deviations for a given age and sex, using the WHO standards; and (c) prepubertal, defined as Tanner stage I breast development for girls and testicular volume less than 4 mL in boys.
Exclusion criteria included any form of endocrine obesity, major congenital or chronic disease, drug-induced obesity, use of drugs for weight loss, or involvement in another weight-loss programme. Fiftythree children with obesity were initially enrolled in the study; plasma samples were available from 50 of these subjects at the hospital's Biobank. Fifteen prepubertal normal-weight children (BMI-SDS between −1 and 1 standard deviations) between 7 and 10 years of age were also recruited for the study. Plasma samples from all subjects were obtained in the morning after an overnight fast by qualified personnel at the hospital.

| Adult subjects study
All participants gave their written informed consent before collecting clinical data. Forty-three patients (11 males and 32 females) with severe obesity were included in this study. Ten of them were diagnosed with T2D, and eight had pre-diabetes. All the patients were evaluated by the same endocrinologist (S. P.) following the institutional protocol for bariatric surgery between September 2012 and October

2015, according to criteria for bariatric surgery formulated in Spanish
Position Statement between Obesity, Endocrinology, Diabetes and Surgery Societies. 21 Inclusion criteria were (a) adults with BMI greater than 35 kg m −2 and (b) adults with BMI less than 26 kg m −2 and normal glucose levels (less than 100 mg dL −1 ). Exclusion criteria included any form of endocrine obesity, major congenital or chronic disease, druginduced obesity, use of drugs for weight loss, or involvement in another weight-loss programme. Demographic and clinical data, including age, history of diabetes, and hypertension were prospectively recorded in all subjects. Visceral adipose tissue was obtained from a subset of these patients (n = 16). These samples were collected during bariatric surgery and frozen at −80°C until they were used. Sixteen normal-weight healthy subjects were included as a control group to measure circulating levels of our molecule of interest. Plasma samples were collected after a 12-hour fasting and stored at −80°C in the Biobank for the Health Sciences Research Institute Germans Trias i Pujol Foundation.

| Circulating parameters
Glucose, insulin, and glycated haemoglobin (HbA1c) levels were measured in the certified core clinical laboratory at the hospital. The homeostatic model assessment-insulin resistance (HOMA-IR) was calculated. S100A4 circulating levels were measured using a human protein S100A4 ELISA kit (Cusabio Biotech Co, LTD, China). High molecular weight (HMW) adiponectin levels were measured using a human protein HMW-adiponectin ELISA kit (R&D Systems, China).

| Human aortic VSMCs
Human aortic VSMCs were obtained by using a modification of the explant technique and cultured as previously described. 22 Human VSMC were obtained from nonatherosclerotic aortas of hearts removed on occasion of transplant surgery at the Hospital de la Santa

| Gene expression
Visceral adipose tissue or cells were collected in liquid nitrogen. Total RNA was extracted using a column-affinity-based methodology (NucleoSpin RNA II; Macherey-Nagel, Duren, Germany). RNA yield was determined by spectrophotometry (NanoDrop). Five hundred nanograms of total RNA was retrotranscribed into cDNA using ran-

| Circulating S100A4 levels are elevated in both prepubertal children and adult subjects with obesity
Both prepubertal children and adults with obesity showed higher insulin resistance (estimated by HOMA-IR) compared with their normal-weight counterparts (Table 1). However, glucose and HbA1c levels in children with obesity were not altered compared with their normal-weight controls, while they were significantly elevated in adults with obesity (Table 1). Both children and adults with obesity showed higher triglyceride levels, lower HDL-cholesterol, and lower HMW-Adp concentration than normal-weight subjects (Table 1). Circulating S100A4 concentration was measured and significantly elevated levels in both prepubertal children (1.5-fold, P = .002) and adult subjects with obesity (2.2-fold, P < .001, Table 1) were found compared with normal-weight controls.

| S100A4 levels are associated with HOMA-IR in adults but not in prepubertal children
Then the association between circulating S100A4 levels and insulin resistance in the two different cohorts was tested. Given that obesity has a major impact on both S100A4 levels and insulin resistance, the focus of the analysis was on subjects with obesity to avoid this confounding factor. Circulating S100A4 levels were strongly correlated to HOMA-IR in adult subjects with obesity ( Figure 1A) and were maintained after adjusting for sex, age, and BMI (βstd = .42, P = .008, Table 2). However, no correlation between S100A4 and HOMA-IR was found in prepubertal children with obesity ( Figure 1B) before or after adjusting for sex, age, and BMI-SDS (βstd = .12, P = .356, Table 2). A modest trend towards a correlation with HMW-Adp was observed in children, without reaching statistical significance (βstd = .27, P = .061, Table 2).

| S100A4 gene expression in WAT is associated with S100A4 circulating levels and HOMA-IR in adult subjects with obesity
To evaluate the potential role of WAT in S100A4 secretion, S100A4 mRNA levels were measured in visceral WAT (vWAT) samples from adult patients with obesity. S100A4 expression in this tissue showed a positive correlation with its circulating levels and HOMA-IR ( Figure 1C,D).

| S100A4 exerts anti-inflammatory effects in vascular smooth-muscle cells in vitro
Since WAT seems to play a role in the secretion and function of S100A4, the aim was to evaluate the effects of this adipokine in two different cell types with a relevant role in adipose tissue expandability during obesity: adipocytes and VSMC. For these experiments, human adipocytes and VSMC were incubated with S100A4 for 24 hours

| DISCUSSION
To the best of our knowledge, this is the first time that S100A4 circulating levels are reported to be elevated in prepubertal children with obesity compared with normal weight healthy controls. As expected from previously published data (ref Arner), S100A4 levels were also elevated in our cohort of adult patients with severe obesity compared with normal-weight controls and showed a correlation with insulin resistance. Surprisingly, the association between S100A4 and insulin resistance was not present in children. S100A4 levels have been reported to be increased in human adults with obesity, being positively associated with sWAT dysfunction,  inflammation, and insulin resistance. 17 Other researchers have shown that S100A4 deficient mice were more susceptible to develop obesity and insulin resistance after a high-fat diet, suggesting a protective role for S100A4 in diet-induced obesity. 18 Furthermore, overexpressed S100A4 inhibited adipogenesis and decreased inflammatory markers in 3T3-L1 adipocytes. 18 Our results suggest a potential explanation for the controversial role of S100A4 in obesity and insulin resistance.
Our cohort of children with obesity showed hyperinsulinaemia, but this excess of insulin secretion was able to maintain normal glucose levels. Researchers have reported an induction of adiponectin by S100A4 in 3T3-L1 adipocytes and in mice. 18 Although there was no correlation between S100A4 and HMW-Adp, it is plausible that the elevated S100A4 levels may play a beneficial role during early states of obesity in an attempt to balance the hyperinsulinaemia and the potential development of insulin resistance in this population in which obesity is only at the beginning of eliciting metabolic dysfunction.
Moreover, our in vitro experiments demonstrate a beneficial direct effect of S100A4 on VSMC by reducing inflammatory markers. S100A4 is secreted by adipocyte precursors and immune cells 17 and, during obesity, both an alteration of adipogenesis and the recruitment of immune cells occur (reviewed in 24 ). In addition, new blood vessel formation (angiogenesis), including VSMC, is required for a correct FIGURE 3 Treatment with S100A4 reduces the expression of pro-inflammatory marker genes in human vascular smooth muscle cell (VSMC). Human VSMCs were exposed to S100A4 (37.5, 75, or 150 ng mL −1 ) or/and TNF-α for 24 h. (A-C) Expression of CCL2, IL6, and IL1β assessed by real-time PCR and normalized to GAPDH 24 h after incubation with S100A4 (37.5, 75, or 150 ng mL −1 ). (D-F) Expression of CCL2, IL6, and IL1β assessed by real-time PCR and normalized to GAPDH 24 h after incubation with TNF-α or TNF-α + S100A4 (75 or 150 ng mL −1 ). Values are shown as mean ± SEM (n = 5); one-way ANOVA was implemented to compare different treatments ( *** P < .001, ** P < .01, and * P < .05 vs vehicle; # P < .05 vs TNF-α) FIGURE 2 Treatment with S100A4 does not cause changes in human adipocytes. (A) Relative mRNA levels of different markers of adipogenesis, thermogenic capacity, and inflammation in human adipocytes treated with 150 or 750 ng mL −1 S100A4 for 24 hours or during differentiation, where indicated. Expression was normalized to 18S. (B) Representative optical microscopy images from beige adipocytes treated with 750 ng mL −1 S100A4 since day 1 of differentiation. ( * P < .05) relative to control. The bars represent means ± SEM adipose tissue expandability. 25 Moreover, inflammation and vascular dysfunction contribute to the development of obesity and insulin resistance. 26 Therefore, it is tempting to speculate that S100A4 secretion by immune cells infiltrated in adipose tissue during obesity acts on VSMC and thereby regulates the vascular tone, distribution of blood flow, as well as angiogenesis, inflammatory processes, and redox status. It is plausible that that this beneficial effect could contribute to improving the inflammatory state during the early stages of obesity development, delaying the development of adipose tissue dysfunction and insulin resistance.
In accordance with other researchers, 17 no direct effect of S100A4 on adipocytes was observed possibly due to the lack of specific receptors, suggesting that these cells are not a target for S100A4 action. In agreement with Arner et al, 17 who postulated that S100A4 is an adipokine, our data showed that S100A4 is expressed in WAT. In addition, S100A4 vWAT gene expression correlated with circulating levels, suggesting that vWAT is a source of circulating S100A4. Moreover, S100A4 mRNA and circulating levels positively correlated with glucose homeostasis parameters, suggesting a role of this adipokine in the metabolic status of these patients. An intriguing possibility is that a chronic release of this molecule in the obesity status could lead to an S100A4 resistant situation, in a similar way to what has been shown for other molecules including leptin and FGF21. 27,28 Further studies are warranted to determine the occurrence of an S100A4 resistant state in obesity.
The limitations of this study include the lack of adipose tissue samples in children to analyse the molecular mechanisms involved in the elevation of S100A4 and the lack of correlation with insulin resistance.
Moreover, HOMA-IR was used as a marker of insulin resistance due to the lack of a clamp assay. Another important limitation is that our vascular cellular model comes from aorta and not from adipose tissue vessels. In addition, longer and chronic S100A4 exposure would be desirable to evaluate the long-term effect of S100A4 in vitro. The strengths include the large cohort of plasma samples from prepubertal children with obesity and the availability of additional physiologic parameters allowing to identify the associations between S100A4 and other surrogates of metabolic state.
In conclusion, our human data demonstrate for the first time that S100A4 circulating levels are higher in children with obesity compared with normal-weight individuals but do not correlate with insulin resistance as in adult subjects. Moreover, our in vitro results suggest a potential anti-inflammatory effect of S100A4 in VSMC. Further studies will be necessary to determine whether S100A4 can be a therapeutic target for obesity and the development of insulin resistance associated with this condition.

AUTHOR CONTRIBUTIONS
Siri D. Taxerås Puig-Domingo contributed to study design and data interpretation.
Francesc Villarroya, Carles Lerin, and David Sánchez-Infantes contributed to study design, researched data, and wrote/reviewed/edited the manuscript. Each author listed in the manuscript has seen and approved the submission of this version of the manuscript and takes full responsibility for the contents.