
 childhood obesity is a serious and growing public health problem that has arisen over the past three decades .
the increasing occurrence of disorders such as type 2 diabetes during childhood is believed to be a consequence of this obesity epidemic .
in addition to several behavioral and dietary risk factors , genetic predisposition is an important factor in the pathogenesis of childhood obesity .
it is estimated that 4070% of adiposity variance can be explained by direct or indirect genetic factors .
growth arrest - specific 6 ( gas6 ) , cloned in 1988 and characterized in 1993 , is a secreted vitamin k - dependent protein present in the human circulatory system [ 4 , 5 ] .
initially , gas6 was shown to be upregulated in growth - arrested fibroblasts , suggesting that it plays a protective role in certain cellular stresses such as during apoptosis .
gas6 expression is widespread in many tissues , including immune cells , endothelial cells , vascular smooth muscle cells , and adipocytes [ 79 ] .
the protein is also a ligand for the tam ( tyro-3/axl / mer ) family tyrosine kinase receptor .
the gas6/tam system has been implicated in cell survival and proliferation , cell adhesion and migration , hemostasis , and inflammatory cytokine release [ 4 , 10 ] .
recently , the gas6/tam pathway was found to be involved in mediating adipocyte survival and proliferation in vitro [ 11 , 12 ] .
experiments with mice fed a high - fat diet indicated that overexpression of gas6 might enhance body - fat accumulation , but blocking gas6 signaling using an axl antagonist could reduce body - fat mass and body weight
. interestingly , transgenic animals that ectopically express the axl tyrosine kinase receptor also develop progressive obesity with elevated circulating proinflammatory cytokines and severe systemic insulin resistance .
this protein - array study also revealed higher levels of axl mrna in subcutaneous adipose tissue of obese humans than their lean control counterparts had .
this indicates that the axl receptor may be involved in the development of human obesity .
in addition , some studies in transgenic mice indicate that gas6/axl signaling might recruit macrophages and other immune cells into the adipose tissue resulting in the production and secretion of proinflammatory mediators .
this suggests that the gas6/axl signaling might play a role in the pathogenesis of obesity - associated systemic inflammation [ 8 , 16 , 17 ] .
recent studies have indicated that systemic inflammation , a hallmark of childhood and adult obesity , is a pivotal mechanism linking obesity to insulin resistance and type 2 diabetes [ 1821 ] . although gas6 gene polymorphisms are reported to be associated with stroke , acute coronary syndrome , and type 2 diabetes [ 2224 ] , to our knowledge , both gas6 and axl gene polymorphisms associated with childhood obesity have not yet been identified . in order to address this issue
, we conducted a community - based study to determine whether common variations in the gas6 and axl genes correlate with adiposity , systemic inflammation , insulin resistance among adolescents .
the taipei children heart study - iii was an epidemiological survey that investigated obesity and cardiovascular disease risk factors among adolescents in taipei city during 2006 . the sampling method and results
briefly , the survey included junior high school students in taipei city to collect a representative distribution of demographic , lifestyle , and biochemical characteristics to measure their risk for cardiovascular disease .
those with autoimmune diseases , cancers , or active infection and those taking medications known to interfere with insulin or glucose metabolism were excluded . excluding any missing data , 727 adolescents ( 358 boys and 369 girls )
the institutional review board of the tri - service general hospital approved these studies and obtained informed consent from both parents and adolescents .
all the participants completed a structured questionnaire detailing their gender , age , puberty development , and lifestyle characteristics ( including cigarette smoking , alcohol consumption , and physical activity ) . based on their responses
, the subjects were divided into young adolescents with history of smoking , those without , and those who currently smoke .
physical activity was divided into 5 levels based on amount of exercise per week : less than 1  h , 13  h , 35  h , 57  h , and over 7  h. survey questions concerning puberty onset included the development of the penis / testis and pubic hair for boys and development of breasts and pubic hair for girls .
body weight was measured of barefoot students wearing light indoor clothing and was rounded to the nearest 0.1  kg .
waist circumference ( wc ) was measured at the midway point between the inferior margin of the last rib and the crest of the ilium in a horizontal plane and was recorded to the nearest 0.1  cm .
body mass index ( bmi ) was calculated as weight in kilograms divided by the square of height in meters . to reduce extraneous variation between subjects , we collected blood samples from the students after 12  h fasting and who had consumed their usual diet for the previous 3 days .
children who had recently attended a holiday or family celebration were contacted for a blood sample several weeks after the event .
plasma glucose concentrations were determined by the glucose oxidase method by using the beckman glucose analyzer ii ( beckman instruments , fullerton , ca ) .
the intra- and interassay coefficients of variation ( cvs ) for glucose were 0.6% and 1.5% , respectively .
plasma insulin was measured using a commercial immunoradiometric kit ( biosource europe , nivelles , belgium ) .
serum levels for high - sensitivity c - reactive protein ( hscrp ) were measured using the tina - quant ( latex ) high - sensitivity assay ( roche , mannheim , germany ) .
serum il-6 concentrations were determined using a human high - sensitivity enzyme - linked immunosorbent assay ( elisa ) ( innotest , besancon , france ) .
serum tnf- was measured with the biotrak high - sensitivity human elisa kit from amersham biosciences ( buckinghamshire , uk ) .
insulin resistance was assessed using the homeostasis model assessment ( homa ) , in which the homa of insulin resistance ( homa - ir ) = fasting insulin ( u / ml )  fasting glucose ( mmol / l)/22.5 . gas6
protein concentration was measured using a sandwich elisa and a polyclonal mouse anti - human gas6 antibody ( r&d systems , lille , france ) as a catcher and a biotinylated goat antiserum as a detector ( r&d systems ) , using previously described methods .
the technique has been validated by food and drug administration guidelines in a previous study ( intra- and interassay cvs of 6.5% and 8.5% , resp . ;
mean recovery on 10 patients of 97% ; lower limit of quantification 0.26  ng / ml ) .
dna was isolated from buffy coats using the qiaamp dna blood kit and following the manufacturer 's instruction ( qiagen , valencia , ca , usa ) .
the qualities of isolated genomic dnas were quantified using agarose gel electrophoresis , and the quantities were determined using a spectrophotometer .
snps rs8191973 and rs8191974 in gas6 , as well as rs4802113 and rs2304232 in axl , were genotyped using a taqman assay with allele - specific probes on the abi prism 7900ht sequence detection system ( applied biosystems , foster city , ca , usa ) according to the standardized laboratory protocols .
descriptive results of continuous variables were expressed as means  sd . prior to statistical analysis , the normal distribution and homogeneity of the variables
were evaluated using the levene test for quality of variance , and the variables were then given a base logarithmic transformation if necessary .
the parameters homa - ir , triglycerides , hscrp , il-6 , and tnf- were analyzed and tested for significance using a log scale .
the studied adolescents were categorized into subgroups based on their gas6 rs8191973 genotype ( cc , cg , and gg ) , gas6 rs8191974 genotype ( gg , ga , and aa ) , axl rs4802113 genotype ( cc , ct , and tt ) , and axl rs2304232 ( aa , ag , and gg ) with gender specification . the differences between anthropometric and biochemistry data across genotypes were analyzed using a general linear model after adjusting for age , tanner stages , smoking status , drinking status , and physical activity .
chi - square tests were used to determine the genotype distributions for the hardy - weinberg equilibrium and to compare the proportions of abnormal anthropometric and biochemistry variables across genotypes .
we tested different genetic inheritance models , and a recessive model was applied in the final analyses for gas6 and axl . to determine whether the gas6 and axl snps are predictors of obesity and obesity - associated complications ,
logistic regression analysis was used to calculate the odds ratio ( or ) and 95% confidence interval ( ci ) for each genotype and combined genotypes
. a two - sided p - value of < 0.05 was considered statistically significant .
all statistical analyses were performed using pasw statistics 18.0 software ( spss inc . , chicago , il , usa ) .
in total , 727 adolescents ( 358 boys and 369 girls ) were included in this study .
the mean age of all adolescents in this study was 13.3 years ( range , 1215 ) and was similar between boys and girls .
in general , boys had higher bmi ( 22.3  4.0 versus 21.2  3.3  kg / m ) , wc ( 80.0  10.1 versus 75.1  8.1  cm ) , hscrp ( 0.9  1.3 versus 0.6  0.9 
mg / l ) , and glucose levels ( 93.8  6.3 versus 91.5  6.5  mg / dl ) than the girls ( all p < 0.001 ) .
however , girls had higher tanner stages ( 3.2  0.5 versus 3.0  0.4 ) than the boys ( p < 0.001 ) .
there was no statistically significant difference in the ages , tnf- , il-6 , and insulin levels , and homa - ir between the boys and girls .
the allele frequency for the least frequent allele in boys was 12.6 , 22.1 , 41.9 , and 29.7% , and 13.4 , 19.6 , 32.7 , and 33.5% in girls for the gas6 rs8191973 , gas6 rs8191974 , axl rs4802113 , and axl rs2304232 polymorphisms , respectively .
there was no significant difference in allele or genotype distribution between boys and girls at the 4 polymorphisms .
no statistically significant association between anthropometric characteristics , biochemistry data , and the gas6 rs8191973 genotypes was observed in the boys and girls ( table 1 ) .
however , there were significantly different hscrp levels between gg , ga , and aa genotypes of gas6 rs8191974 in boys , regardless of their age , tanner stages , smoking status , drinking status , or physical activity ( table 2 ) .
moreover , boys with the gg genotype of gas6 rs8191974 had significantly higher bmi , wc , and hscrp levels than those carrying the a allele .
the gas6 rs819174 genotypes were not significantly associated with any anthropometric characteristics and biochemistry in girls .
the p - values of all comparisons between anthropometric and biochemistry data across gas6 genotypes were presented in supplemental tables 1 and 2 available online at http://dx.doi.org/10.1155/2014/674069 .
in addition , the association between circulating gas6 protein levels and gas6 polymorphisms was investigated .
we found that the gas6 rs8191973 or rs8191974 genotypes were not significantly associated with circulating gas6 protein levels in both the sexes .
there were significantly different hscrp levels between tt , tc , and cc genotypes of axl rs4802113 in boys , independent of their age , tanner stages , smoking status , drinking status , or physical activity ( table 3 ) .
in addition , boys with the gg genotype of axl rs2304232 had significantly higher il-6 and insulin levels and increased homa - ir than those carrying the a allele ( table 4 ) .
however , in girls , axl rs4802113 or rs2304232 polymorphisms were not significantly associated with any anthropometric characteristics or biochemistry ( tables 3 and 4 ) .
the p - values of all comparisons between anthropometric and biochemistry data across axl genotypes were presented in supplemental tables 3 and 4 .
boys with the gg genotype of gas6 rs8191973 were 1.87-fold more likely to have higher hscrp levels than the c allele carriers . even after adjusting for age , tanner stage , smoking status , drinking status , and physical activity ,
a significant relationship between the gg genotype of gas6 rs8191973 and higher hscrp levels was still observed in boys ( table 5 ) .
moreover , boys with the gg genotype of gas6 rs8191974 exhibited a 1.40-fold greater risk for developing high bmi , a 1.58-fold greater risk for developing high wc , and a 2.68-fold greater risk to have higher il-6 levels than the a allele carriers . even after adjusting for all possible confounding factors including age , tanner stage , smoking / drinking status , and physical activity , the relationship between the gg genotype of gas6 rs8191974 , higher bmi / wc , and higher il-6 levels still remained significant in boys .
however , the axl rs4802113 or rs2304232 polymorphisms showed no significant association with abnormal adiposity , inflammatory markers , and homa - ir in boys or girls ( see supplemental table 5 ) .
logistic regression analyses were applied to evaluate whether the combination of the gas6 rs8191974 and rs8191973 polymorphisms is a stronger risk factor for high adiposity , inflammatory markers levels , and homa - ir than when alone .
the combined effects of the 2 gas6 gene polymorphisms in the risk of high bmi , wc , il-6 , and hscrp levels are shown in figure 1 .
after adjusting for the relevant confounding factors , we still observed that boys with the gg genotype of gas6 rs8191973 and the gg genotype of gas6 rs8191974 exhibited a 47-fold higher risk of high bmi , wc , il-6 , and hscrp levels than the individuals with both the c allele of the gas6 rs8191973 and the a allele of the gas6 rs8191974 did ( or = 4.92 , 95% ci : 1.0823.6 , p = 0.018 ; or = 4.18 , 95% ci : 1.0522.5 , p = 0.016 ; or = 4.08 , 95% ci : 1.0628.56 , p = 0.015 ; or = 7.22 , 95% ci : 1.4635.72 , p = 0.010 , resp . ) .
however , for girls , there was no statistically significant association between the combination of the gas6 rs8191974 and rs8191973 polymorphisms and abnormal variables .
in addition , we evaluated the combined effect of the gas6 rs8191973 or rs8191974 marker with axl gene polymorphisms and its association with risk of high adiposity , inflammatory marker , and homa - ir .
however , combinations of gas6 markers with axl gene polymorphisms were not found to be significantly associated with any abnormal variables in both boys and girls ( data not shown ) .
in this study , a strong association between gas6 and axl polymorphisms with body adiposity , systemic inflammation , and insulin resistance was identified among boys .
the risk of possessing high adiposity and inflammatory markers levels was higher in boys carrying the gg genotype with gas6 rs8191973 or rs8191974 than the noncarriers .
moreover , the combination of both gas6 polymorphisms had an additive effect on the development of obesity and obesity - associated inflammation in boys .
these data strongly suggest that gas6 and axl genes play a role in the pathogenesis of childhood obesity and its associated complications . 

gas6 was originally identified as a gene that is expressed in fibroblasts and increases with serum starvation and contact inhibition ; gas6 is also a potential growth factor for fibroblasts .
maquoi and colleagues demonstrated that when fed with a high - fat diet , gas6-deficient mice had significantly less fat than their wild - type counterparts .
the authors also reported the expression of gas6 and its 3 receptors ( tyro-3 , axl , mer ) in murine adipose tissues , thus suggesting that gas6 may act in an autocrine and/or paracrine manner to promote murine adipose tissue development .
previous experiments in transgenic mice demonstrate that gas6 might also induce obesity - associated inflammation via recruiting immune cells into the adipose tissue to producing and secreting proinflammatory cytokines [ 8 , 16 , 17 ] .
our recent clinical study found that circulating gas6 protein levels are associated with adiposity and inflammatory markers in overweight / obese adolescents . in this study , gas6 is further implicated as a candidate susceptibility gene for obesity and systemic inflammation .
however , the association between gas6 genotypes and circulating gas6 protein levels was not observed among adolescents .
we hypothesize that gas6 polymorphisms could affect the biology of the gas6 protein itself rather than its transcription or process rate , thus influencing adiposity regulation and systemic inflammation . to validate this ,
recent studies demonstrated that gas6/tam signaling is involved in releasing inflammatory cytokines ( such as il-6 and hscrp ) in diverse human diseases [ 23 , 31 , 32 ] .
in addition , the gas6/tam signaling is also known to be involved in several inflammation - related systems , including maturation of immune cells , endothelial activation , and immunoregulation .
our present study found that the gg genotype of gas6 rs8191973 and the gg genotype of gas6 rs8191974 are strongly associated with higher circulating il-6 and hscrp levels in boys .
therefore , the gas6 polymorphisms presumably influence gas6/tam signaling and could further activate inflammatory reactions and result in releasing circulating il-6 and hscrp .
however , the comprehensive effects of the gas6 polymorphisms in regulation of inflammatory cytokines still remain to be determined by more researches .
interestingly , a previous study found that the a allele or the aa genotype of gas6 rs8191974 is associated with decreased risk of stroke .
moreover , the a allele and the aa genotype are also thought to be related to a lower risk of developing acute coronary syndrome or type 2 diabetes , suggesting that this genotype exhibits protective activities against developing acute coronary syndrome and type 2 diabetes [ 23 , 24 ] .
we also observed similar results in those with the a allele or aa genotype of gas6 rs8191974 .
these subjects exhibited a lower risk for developing obesity and systemic inflammation than those with the gg genotypes .
together , these findings suggest that the gas6 rs8191974 polymorphisms play an important role in the development of obesity and obesity - associated complications ( e.g. , type 2 diabetes , cerebrovascular , and cardiovascular diseases ) .
the protective role of the aa genotype of gas6 rs8191974 against the developing childhood obesity and obesity - associated complications requires further study .
the axl protein is a membrane - bound receptor that belongs to the tam family of receptor tyrosine kinases .
axl exhibits the highest affinity for gas6 as compared to the other members of the tam family , whereas protein s predominantly binds mer and tyro-3 .
gas6/axl signaling has been shown to be involved in the pathogenesis of obesity and systemic inflammation [ 1315 ] .
however , our study demonstrates that axl polymorphisms are associated with systemic inflammation rather than childhood obesity .
moreover , the combination of gas6 and axl gene polymorphisms is not significantly associated with any variables in adiposity among adolescents .
our findings indicated that axl gene polymorphisms might not play a significant role in childhood obesity . recently ,
scroyen and colleagues   have published similar findings indicating that deficiency in a single axl receptor did not significantly affect adipogenesis or adipose tissue development in mice .
this is because an axl deficiency can be partially compensated by other tam family members ( tyro-3 and mer ) via gas6 interaction .
further studies are needed to investigate the effect of tyro-3 and mer receptors on the development of childhood obesity .
in addition , our present study also indicates that gender disparity exists regarding the effects of the gas6 polymorphisms on anthropometric characteristics and inflammatory markers .
we found no significant difference in genotype frequencies between boys and girls ; however , the effects of the gas6 polymorphisms , individually or combined , only manifest in boys .
the gas6 gene contains an estrogen response element in its promoter and is upregulated by estrogen via an activated estrogen receptor in mammary epithelial cells .
therefore , we hypothesized that the gender - specific effect of the gas6 polymorphisms on childhood obesity might be due to a disparity in sex hormone distributions .
this has been previously reported to be associated with gas6 expression and body composition [ 40 , 41 ] . despite these results , our study does have certain limitations .
first , this was a cross - sectional study , as such we might not be able to assess gas6 polymorphisms on weight dynamics and the development of obesity - associated complications throughout life .
second , because of the limitations of our questionnaire , we were not able to comprehensively estimate every adolescent 's daily intake .
the impact of dietary energy intake on genetic susceptibility also requires further investigation to better understand any confounding effect .
in conclusion , we indicate an association between the gas6 and axl polymorphisms with adiposity , circulating inflammatory markers , and insulin resistance of adolescents , especially in boys .
moreover , the gg genotype of gas6 rs8191973 or rs8191974 strongly correlates with susceptibility to develop obesity and systemic inflammation in boys .
nonetheless , these results together with those from studies in cellular and animal models encourage the study of the gas6/tam system in childhood obesity and its potential complications and further support the hypothesis that modulation of gas6 activity may indeed provide an important intervention point for future therapies .
 the present study was designed to explore the effects of gas6 and axl gene polymorphisms on adiposity , systemic inflammation , and insulin resistance in adolescents . 
 after multistage sampling from the data of the taipei children heart study - iii , we collected 358 boys and 369 girls with an average age of 13.3 years . 
 we genotyped the adolescents ' gas6 rs8191973 , gas6 rs8191974 , axl rs4802113 , and axl rs2304232 polymorphisms . 
 significantly higher body mass index ( bmi ) , waist circumference ( wc ) , and hscrp levels were found in boys with the gg genotype of gas6 rs8191974 than a allele carriers ; higher il-6 and insulin levels and increased homa - ir were found in boys with the gg genotype of axl rs2304232 than the a allele carriers . there was a significant difference in hscrp levels of boys with the tt , tc , and cc genotypes of axl rs4802113 . 
 boys with both the gg genotype of gas6 rs8191973 and the gg genotype of gas6 rs8191974 exhibited higher bmi , wc , il-6 , and hscrp levels than the boys carrying both the c allele of the gas6 rs8191973 and the a allele of the gas6 rs8191974 . in conclusion , 
 gas6 and axl polymorphisms are associated with adiposity , systemic inflammation , and insulin resistance in adolescents , especially in boys . 
1. Introduction 
2. Materials and Methods
3. Results
4. Discussion