Clinica Chimica Acta 441 (2015) 1–5

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Clinica Chimica Acta

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Gender differences in plasma growth arrest-specific protein 6 levels inadult subjects

Yi-Jen Hung a, Chien-Hsing Lee a, Yi-Shing Shieh b,c, Fone-Ching Hsiao a, Fu-Huang Lin d, Chang-Hsun Hsieh a,⁎a Division of Endocrinology and Metabolism, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwanb School of Dentistry, National Defense Medical Center, Taipei, Taiwanc Department of Oral Diagnosis and Pathology, Tri-Service General Hospital, Taipei, Taiwand School of Public Health, National Defense Medical Center, Taipei, Taiwan

⁎ Corresponding author at: Division of Endocrinology aInternal Medicine, Tri-Service General Hospital, No. 325, STaipei, Taiwan. Tel.: +886 2 87927182; fax: +886 2 8792

E-mail address: 10324@yahoo.com.tw (C.-H. Hsieh).

http://dx.doi.org/10.1016/j.cca.2014.12.0010009-8981/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 21 November 2013Received in revised form 3 November 2014Accepted 1 December 2014Available online 6 December 2014

Keywords:Growth arrest-specific protein 6EstradiolTestosterone and ages

Background:Growth-arrest-specific 6 (Gas6) is recognized as a secreted vitamin K-dependent protein, as it inter-acts with receptor tyrosine kinases of the TAM (Tyro-3, Axl, Mer) family. The plasma Gas6 are important to theinflammatory process, and are involved in diverse human diseases. Few studies have shown plasma Gas6concentration varies with genders. We determined whether plasma Gas6 concentrations are associated withsex hormones in both genders.Methods: A total of 589 adult subjects, including 361 male and 228 female were recruited. Plasma Gas6 concen-tration, biochemical, testosterone, estradiol (E2), and sex hormone-binding globulinwere assayed. The indices offree androgen (FAI) and free E2 (FEI) were calculated.Results: Significantly higher Gas6 concentrations were observed in adult male rather than female (P b 0.05). In

univariate regression analysis, plasma Gas6 concentrations were positively associated with FAI in male (β =0.167, P = 0.002) and both E2 and FEI in female (β = 0.384, P b 0.001 andβ = 0.292, P b 0.001, respectively).Otherwise, Gas6 concentrations were inversely associated with ages in both genders (β = −0.234, P b 0.001in male and β = −0.226, P = 0.001 in female, respectively). In multivariate regression analysis, only age inmale and E2 in female were independent variables to determine the plasma Gas6 concentrations (β = −0.231,P= 0.002 and β= 0.458, P= 0.001).Conclusions: Plasma Gas6 is associated with sex hormones in female and ages in male, indicating a potential role ofsex hormones and ages involving the Gas6/TAM system.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Growth-arrest-specific 6 (Gas6) is recognized as a secreted vitaminK-dependent protein, as it interacts with receptor tyrosine kinases ofthe TAM (Tyro-3, Axl, Mer) family via its C-terminal sex hormone bind-ing globulin (SHBG)-like domain [1]. Gas6 has the highest affinity forAxl among the TAM receptors and their binding can activate the Gas6/Ax1 pathway. Gas6 and soluble form of Axl (sAxl) are present in miceand human circulatory systems and circulate bound to each other in ahigh affinity complex [2]. Gas6 expression is widespread in manytissues, including immune cells, endothelial cells, vascular smoothmusclecells, and adipocytes [3–5]. The Gas6/TAM system has been implicated incell survival and proliferation, cell adhesion and migration, homeostasis,and inflammatory cytokine release [1,6]. The Gas6/TAM system has alsobeen implicated in mediating adipocyte survival and proliferation [7,8].

nd Metabolism, Department ofec. 2, Cheng-Gong Rd., Nei-Hu,7183.

Clinical studies recently indicate that plasma Gas6 concentrationscorrelatewith a number of inflammatorymarkers among adult patientswith systemic inflammatory diseases and cardiovascular disease [9–12].It is also represented as a surrogatemarker of disease activity of autoim-mune disorders [13,14]. In our previous studies, we demonstrate thatplasma Gas6 concentrations are associated with altered glucose toler-ance and inflammation in middle-aged adults [15]. It is also associatedwith obesity and its related chronic inflammation and metabolic com-plications in adolescents [16].

However, one study reported the presence of functional androgen-response elements (AREs) in theGAS6 promoter, and androgen receptorsignaling directly regulates Gas6 transcription,which leads to inhibitionof vascular calcification in vascular smooth muscle cells [17]. Clauseret al. discovered that plasma Gas6 concentration varies with gendersand is decreasedwith oral contraceptive use [18]. PlasmaGas6 concentra-tions in premenopausal women without oral contraception are signifi-cantly higher than in men, which may be related to up-regulation byestrogens and down-regulation by androgen [18]. Recently, we demon-strated that postmenopausal women have lower plasma Gas6 concentra-tions than premenopausal women. Endogenous estrogen concentrations

Table 1Anthropometric and biochemical data among adult subjects.

Male Female P

(n = 361) (n = 228)

Age (y) 46.7 ± 0.8 48.4 ± 1.0 NSBMI (kg/m2) 25.5 ± 0.2 25.2 ± 0.2 NSWaist (cm) 89.2 ± 0.5 85.1 ± 0.6 b0.001SBP (mm Hg) 131.5 ± 0.9 132.8 ± 1.1 NSDBP (mm Hg) 83.6 ± 0.6 83.8 ± 0.6 NSOGTT NS

FPG (mg/dl) 114.7 ± 1.8 112.7 ± 1.2 NS2 h BG (mg/dl) 197.1 ± 4.7 182.4 ± 4.2 b0.05

HbA1c (%) 6.1 ± 0.1 6.0 ± 0.1 NSDiabetes (%) 25.8 23.9 NSHDL-C (mg/dl) 48.1 ± 1.0 53.1 ± 1.2 b0.001TC (mg/dl) 189.0 ± 2.2 195.6 ± 2.6 NSLDL-C (mg/dl) 127.5 ± 2.6 132.5 ± 3.2 NSTriglyceride (mg/dl)a 141.7 ± 4.3 148.3 ± 8.9 NSCreatinine (mg/dl) 0.9 ± 0.0 0.8 ± 0.0 b0.001Testosterone (nmol/l) 17.8 ± 0.3 NDEstradiol (nmol/l) ND 56.2 ± 4.0FSH (mIU/ml) ND 39.7 ± 2.1SHBG (nmol/l)a 20.4 ± 0.6 36.7 ± 1.7 b0.001FAI 1.1 ± 0.0 NDFEI ND 9.1 ± 0.9Gas6 (ng/ml)a 22.8 ± 0.7 20.2 ± 0.6 b0.05

Data was showed as mean ± SEAbbreviations: ND: not done; NS: not significant; BMI: body mass index; SBP: systolicblood pressure; DBP: diastolic blood pressure; OGTT: oral glucose tolerance test; HDL-C:high deNSity lipoprotein-cholesterol; TC: total cholesterol; LDL-C: low density lipopro-tein-cholesterol; FSH: follicle-stimulating hormone; SHBG: sex hormone binding globulin;FAI: free androgen index; FEI: free estrogen index; Gas6: growth arrest-specific protein 6.

a The logarithms of these variables were used for the analysis.

2 Y.-J. Hung et al. / Clinica Chimica Acta 441 (2015) 1–5

are directly associated with plasma Gas6 concentrations in both pre- andpostmenopausal women [19]. Therefore, these results suggest thatplasmaGas6 concentrationsmight be regulated by sex hormones. Never-theless, till now, little is known of the clinical significance and associationbetween the Gas6/TAM system, sex hormones and its related bindingprotein particular in male subjects.

2. Materials and methods

2.1. Subjects and sample collection

A total of 589 apparent healthy adult subjects aged between 18 and80 y were recruited periodically from the outpatient clinics of Tri-Service General Hospital, Taipei, Taiwan from 2008 to 2011. The partic-ipants composed of adult male (n= 361) and female (n= 228). Othercriteria for inclusion into this study were as follows: body mass index(BMI) b35 kg/m2, absence of infection within the previous weeks, ab-sence of taking contraceptives, hormone replacement therapy, oral an-ticoagulants and anti-diabetic therapy, including oral hypoglycemicagents, insulin and glucagons-like peptide 1, and absence of malignanttumor history. Exclusion criteria included women who were pregnantor breast feeding; patients with impaired renal function (serum creati-nine≥1.5mg/dl); patients with abnormal serum aspartate aminotrans-ferase or alanine aminotransferase (2.5 times above the upper normalranges); patients with acute or chronic pancreatitis; patients with a his-tory of cerebrovascular accident, myocardial infarction or heart failure;patients with autoimmune disorders or psychiatric diseases, includingmood disorders and alcoholism; and patients taking concomitant drugssuch as beta-blockers, diuretics, cholestyramine or systemic steroids. A75 g oral glucose tolerance test (OGTT) was performed in all subjectsafter they had fasted for at least 10 h. According to AmericanDiabetes As-sociation criteria, type 2 diabetes was diagnosed with fasting glucose≥126 mg/dl or 2 h post-load glucose N200 mg/dl. The institutional re-view board of the Tri-Service General Hospital approved the protocoland all subjects gave written informed consent.

2.2. Analytic methods

After 10 h of fastening, blood samples were obtained to determineplasma glucose, creatinine, and lipid profiles. Serum total cholesterol,triglyceride, and low-density lipoprotein cholesterol (LDL-C) weremeasured using the dry, multilayer analytical slide method in the FujiDri-Chem 3000 analyzer (Fuji Photo Film Corp.). The intra-assay andinter-assay CVs for LDC-C were 0.8% and 2.5%, respectively. Serum con-centrations of high-density lipoprotein cholesterol (HDL-C) were deter-mined by an enzymatic cholesterol assay method after dextran sulfateprecipitation. The intra-assay and inter-assay CVs for HDL-C were 1.1%and 1.7%, respectively. The concentrations of HbA1c were evaluated bythe ion-exchange high pressure liquid chromatography (HPLC) method(Bio-Rad Variant II). The intra-assay and inter-assay CVs for HbA1cwere1.3% and 2.2%, respectively. Plasma glucose concentrations were deter-mined by the glucose oxidase method on a Beckman Glucose AnalyzerII (Beckman Instruments). The intra-assay and inter-assay CVs forglucose were 0.6% and 1.5%, respectively.

Plasma testosterone and estradiol (E2) were determined by theradioimmunoassay (BioSource Europe), with detection limit of0.05 ng/ml and 2 pg/ml, respectively. Intra-assay and inter-assay CVswere b7.0% for both methods. Sex hormone binding globulin (SHBG)was assayed by the immunoradiometric assay (ZenTech s.a.). Theintra-assay and inter-assay CVs for SHBGwere 2.9 and 4.6% respectively.All concentrations of the above biochemical variables were determinedin duplicate, and the values of the two samples were averaged. The freeandrogen index (FAI) was calculated, the molar ratio of total testoster-one/SHBG, which is highly correlated with free testosterone [20]. Simi-larly, the free E2 index (FEI), the molar ratio of E2/SHBGwas calculated,which is also correlated well with free E2 concentrations [20].

Plasma Gas6 protein was measured using DuoSet® ELISA Develop-ment kit (R&D Systems) contains the basic components required thedevelopment of sandwich ELISA to measure natural and recombinanthuman Gas6. For each plasma sample, 100 μl was directly transferredto the microtest strip wells of the ELISA plate coated with capture anti-body of mouse anti-human Gas6 and incubated for 2 h at room temper-ature. After three times of washing steps, the detection antibody wasadded, and the reaction mixture was incubated for 2 h at room temper-ature. Antibody binding was detected with streptavidin-conjugatedhorseradish peroxidase and developed with a substrate solution. Thereaction was stopped in adding stop solution, and optical density wasdetermined using a microplate reader set at 450 nm. The Gas6 concen-trationwas quantitated by a calibration curve using a human Gas6 stan-dard. Each plasma sample was assayed in duplicate according to theinstructions of themanufacturers, and the valueswerewithin the linearportion of the standard curve. The intra-assay and inter-assay CVs ofGas6 were 6.5 and 8.5% respectively, mean recovery on 10 patientswas 97%, and lower limit of quantification was 0.26 ng/ml).

2.3. Statistical methods

Descriptive results of continuous variables were expressed asmeans ± SEM. Before statistical analysis, normal distribution andhomogeneity of the variables were evaluated using Levene test for qual-ity of variance, and variables were then given a base logarithmic trans-formation if necessary. The parameters of triglyceride, testosterone, E2,SHBG, FAI, FEI and Gas6 were analyzed and tested for significance on alog scale. An unpaired t-test was applied to determine differences incontinuous variables between two groups. A univariate and multivari-ate linear regression analysis was employed with Gas6 as a dependentvariable and the other parameters as independent variables. One-wayANOVA was used to determine the trend between Gas6, sex hormonesand ages. A two-sided P-value b0.05 was considered statistically signif-icant. All statistical analyses were performed using SPSS Statistics 18.0software.

Fig. 1. Plasma Gas6 concentrations in subjects with adult male and female. The horizontallines represent themedian values in each group. Adultmale had significantly higher plasmaGas6 levels than female (P b 0.05).

3Y.-J. Hung et al. / Clinica Chimica Acta 441 (2015) 1–5

3. Results

Total of 589 subjects (361 male and 228 female) were enrolledfor analysis. As expected, there were significantly higher concentra-tion of blood creatinine and lower concentrations of HDL-C andSHBG (all P b 0.001) in adult male than female (Table 1). Other var-iables were compatible in both genders. Fig. 1 showed that signifi-cantly higher Gas6 concentrations were observed in adult male(median:19.3 ng/ml, range:3.8–98.0 ng/ml ng/ml) rather than female(median:18.2 ng/ml, range:5.7–47.8 ng/ml) (P b 0.05). In univariateregression analysis, plasma Gas6 concentrations were positively associ-ated with fasting, 2 h glucose during OGTT and FAI concentrations inmale (β = 0.167, P = 0.002) and both E2 and FEI in female (β =0.384, P b 0.001 andβ = 0.292, P b 0.001, respectively). Otherwise,Gas6 concentrations were inversely associated with ages in both gen-ders (β = −0.234, P b 0.001 in male and β = −0.226, P = 0.001 infemale, respectively). The association between Gas6 and fasting, 2 hglucose concentrations during OGTT and FAI in male, and age and FEIin female were abolished in multivariate regression analysis. Only agein male and E2 in female were independent variables to determinethe plasma Gas6 concentrations (β = −0.231, P = 0.002 and β =0.458, P = 0.001) (Table 2).

The ageswere divided into 5 categories and plasma testosterone, FAIand E2 and FEI concentrations were declined parallel with variousranges of ages in both genders, respectively (all P b 0.001 for trend).

Table 2Univariate and multivariate linear regression analysis for Gas6 as dependent variable among m

Male

Univariate Multivariate

β p β p

Age −0.234 b0.001 −0.231 0BMI 0.063 0.235 −0.073 0Waist 0.064 0.226 0.108 0FPG 0.144 0.006 0.007 02 hr BG 0.131 0.013 0.068 0HbA1c 0.077 0.155 0.027 0Cr 0.052 0.388 0.110 0Testosteronea 0.071 0.177 0.065 0FAIa 0.167 0.002 0.028 0Estradiola

FEIa

Abbreviation: FPG: fasting plasma glucose; 2 h BG: 2 h blood glucose; Cr: creatinine; FAI: freea The logarithms of these variables were used for the analysis.

Interestingly, plasma Gas6 concentrations also decreased parallel withages in both genders (both P b 0.001 for trend) (Figs. 2 & 3).

4. Discussion

Although the pleiotropic effects of Gas6/TAM signaling involved intriggering the systemic inflammation among diverse human diseasessuch as infection, acute coronary syndrome, type 2 diabetes and obesity[10,12,15]. The most important finding of our study is that adult malehad a higher concentration of circulating Gas6 protein than female,and its concentration was positively associated with sex hormoneconcentration and negatively with age in both genders. However, theassociation of the parameter of FAI in male and age in female withplasma Gas6 was abolished in multivariate regression analysis. Onlyage inmale and E2 concentrations in female were still present indepen-dent of adiposity, and several other parameters, such as glucose status,lipidemia, creatinine and SHBG. The actual reasons of these findingswere uncertain. We assumed that E2 might directly activate the EREof GAS6 gene to influence gene expression and protein production [21], which is more profound by E2 than by other contributors such asage, at least in female subjects in our study. In our recent study, bothage and E2 in postmenopausal and only E2 in premenopausal womenindependently determinate the concentrations of plasma Gas6 [19]. Itis perhaps E2 that plays a major role to regulate the GAS6 expressionin premenopausal status and age factor probably attenuates theinfluence of E2 in postmenopausal women. We believe that our findingindicated that ages and sex hormonesmay play a potential role in mod-ulating Gas6/TAM system to induce diverse clinical consequences.

Clauser et al. [18] have reported that plasma Gas6 concentration islower in male than in premenopausal healthy subjects without receiv-ing oral contraceptives, which is inconsistent with our result. The possi-ble reasons are only premenopausal healthy subjects and younger agesin both genders were recruited in their study. We found that premeno-pausal women have higher Gas6 concentration than those in postmen-opausal status in different study subjects [19]. However, in the presentstudy, we enrolled wide ranges of ages in women, including not onlypremenopausal, but also postmenopausal women. Hence, the concen-trations of Gas6 in male were higher than female subjects. Otherwise,greater different concentrations of Gas6 in their data were comparedto this present study, perhaps by using a different detection antibodyof the ELISA assays or different ages and ethnicity of study subjects. Intheir study, Gas6 concentrations in women with receiving oral contra-ceptive of ethinylestradiol and levonorgestrel significantly decreasedto similar concentrations in men. Their explanation of the decrease inGas6 is perhaps caused by an androgenic effect of levonorgestrel,instead of up-regulation of Gas6 by estrogens.

ale and female subjects.

Female

Univariate Multivariate

β p β p

.002 −0.226 0.001 −0.027 0.736

.547 0.050 0.450 −0.124 0.331

.371 0.102 0.124 0.088 0.505

.956 0.119 0.072 0.061 0.488

.537 0.015 0.825 −0.149 0.112

.787 0.114 0.094 0.090 0.263

.078 0.134 0.064 0.081 0.244

.351

.7140.384 b0.001 0.458 0.0010.292 b0.001 −0.048 0.706

androgen index; FEI: free estrogen index. bold values indicate significance

Fig. 2. Plasma Gas6, testosterone and free androgen index (FAI) levels were paralleldecline with various ranges of age in adult male (all P b 0.001 for trend).

Fig. 3. Plasma Gas6, estradiol and free estrogen index (FEI) levels were parallel declinewith various ranges of age in adult female (all P b 0.001 for trend).

4 Y.-J. Hung et al. / Clinica Chimica Acta 441 (2015) 1–5

Our result discovered that Gas6 concentrations were directly corre-lated with serum E2 concentrations, which was an independent factorfor attributing to Gas6 concentrations in women. GAS6 gene was com-posed of ERE in its promoter andwas up-regulated by estrogen via acti-vating estrogen receptor (ER) in mammary epithelial cells and ERpositive breast cancer cells [21]. The GAS6 gene expression in human

osteoblast cell line was also enhanced by E2 and was diminished by itsinhibitor,which indicates theGAS6 gene can beup-regulated by estrogen[22]. Thesemechanisms such as the signaling andmolecular target of theER may explain this finding. On the other hand, the premenopausalwomen on oral contraceptives and postmenopausal women on hor-mone replacement therapy which contain a variety of sex hormones

5Y.-J. Hung et al. / Clinica Chimica Acta 441 (2015) 1–5

are difficult to directly assess the endogenous estrogen status. In order toclarify the relationship between Gas6 and endogenous estrogen, ourparticipants did not take any medication influencing the sex hormonesmetabolism..

In adult male, FAI manifested weakly, but is significantly positivelycorrelated with Gas6 concentrations and its effect was abolished byother confounders such as age. It shows that androgen plays a minorrole to determine the Gas6 concentration than age factor. However, itis still inconclusive in the meaning of clinical consequence. Otherwise,most actions of androgen are mediated by the androgen receptor to ac-tivate transcription by binding to AREs in the promoter of GAS6 gene,which leads to inhibition of vascular smooth cell calcification [17].This potential mechanism can explain the situation of up-regulation ofGAS6 gene by androgen. In accordance to these accumulative evidences,sex hormone indeed can regulate and influence the expression of Gas6/TAM pathway to result in diverse human diseases. How the Gas6/TAMpathway is regulated by sex hormones in cellular concentrations,whether the activation of GAS6 gene by estrogen and androgen viaEREs and AREs in tissue-specific manners, is still inclusive because oflack of direct evidence of in-vitro studies.

In addition to sex hormone effect, age is also an independent deter-minant for the plasma Gas6 concentrations particular in adult male. Theactual reason of this observation is not clear. In our data, we foundthat plasma Gas6 concentrations were declined parallel with variousranges of ages in both genders and inversely independently associat-ed with age in adult male. Our explanation is the Gas6 has a numberof diverse functions and binds to its cognate receptor Axl and subse-quent Akt activation, resulting in increased cell survival, prolifera-tion, migration, adhesion and decreased apoptosis [6]. It also playsa pivotal role in inorganic phosphate-induced vascular smoothmuscle cell calcification through of the Gas6-mediated survival path-way [23]. Vascular calcification is often encountered in advancedatherosclerosis and is a common consequence of aging. Hence, wehypothesized that the expression of GAS6 gene and Gas6-inducedapoptosis might be attenuated as aging process. Whether Gas6/Ax1pathway has a potential property of anti-aging still needs moreevidences to elucidate.

Despite being purely observational, this study provides novel re-sults regarding the hormonal influence and ages on plasma Gas6with gender specification, in a human population not previously de-scribed. The weakness is that the correlations are relatively weak,but statistically significant and it is not possible to draw any conclu-sions related to the physiological meaning of the findings. Therefore,the observational correlations now reported are of questionablephysiological significance. Besides, our study did not recruit subjectsfrom a community-dwelling population, which is a better option forthis study. Although we did exclude the subjects with major system-ic disorders, subjects may have different factors influenced on theGas6 concentrations.

In summary, our study demonstrated that adult males havehigher circulating Gas6 concentrations than women. Plasma Gas6 isassociated with sex hormones in female and ages in male, indicatinga potential role of sex hormones and ages involving the Gas6/TAMsystem.

List of abbreviationsGas6 growth arrest-specific protein 6TAM Tyro-3, Axl, MerFSH follicle-stimulating hormoneSHBG sex hormone binding globulinE2 estradiolFAI free androgen indexFEI free estrogen indexSBP systolic blood pressure

DBP diastolic blood pressureOGTT oral glucose tolerance test;

Acknowledgments

Thisworkwas supported by research grants fromTri-Service GeneralHospital (TSGH-C102-118 & TSGH C103-007-S03), Taiwan. We aregrateful to Miss Wang for assistance with the laboratory assay.

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