Clinica Chimica Acta 445 (2015) 133–138
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Both diabetes and fetuin-A are independently associated with increased risk of arterial stiffness Horng-Yih Ou a, Feng-Hwa Lu b, Hung-Tsung Wu c, Hao-Chang Hung a, Jin-Shang Wu b, Yi-Ching Yang b, Chih-Jen Chang b,c,⁎ a b c
Department of Internal Medicine, National Cheng Kung University Medical College and Hospital, Taiwan Department of Family Medicine, National Cheng Kung University Medical College and Hospital, Taiwan Research Center of Herbal Medicine, New Drugs, and Nutritional Supplements, Research and Services Headquarters, National Cheng Kung University, Taiwan
a r t i c l e
i n f o
Article history: Received 12 August 2014 Received in revised form 24 March 2015 Accepted 24 March 2015 Available online 1 April 2015 Keywords: Fetuin-A Arterial stiffness Diabetes
a b s t r a c t Background: Arterial stiffness is a functional assessment of vascular damage caused by cardiovascular disease (CVD) risk factors. Fetuin-A is associated with subclinical CVD and incident or fatal CVD, with some modification of its effect occurring with the presence of diabetes. We investigated the impact of different glycemic statuses and serum fetuin-A levels on arterial stiffness. Methods: A total of 312 age- and sex-matched subjects with normal glucose tolerance (NGT), impaired fasting glucose (IFG), impaired glucose tolerance (IGT), and newly diagnosed diabetes (NDD) were recruited. Serum fetuin-A levels were measured, and arterial stiffness was assessed by brachial-ankle pulse-wave velocity (baPWV). Results: We found that the mean values of baPWV were 1533 ± 338, 1518 ± 353, 1589 ± 307, and 1690 ± 414 cm/s, and fetuin-A levels were 298 ± 69, 313 ± 67, 330 ± 86, and 342 ± 93 μg/ml, in subjects with NGT, IFG, IGT, and NDD, respectively (both p b 0.001, test for trend). NDD subjects had significantly higher baPWV and fetuin-A levels than those with NGT. Multiple linear regression analysis showed that age, fetuin-A, diabetes, hypertension, and hypertriglyceridemia are independently associated factors of baPWV after adjusting for cardiometabolic risk factors, HOMA-IR, and adiponectin. Conclusion: Both diabetes and fetuin-A levels are independently associated with arterial stiffness. Fetuin-A may further aggravate increased arterial stiffness in diabetes. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Fetuin-A is a glycoprotein produced exclusively in the liver and then secreted into the circulation in high concentrations [1]. Fetuin-A per se is an endogenous inhibitor of the insulin receptor tyrosine kinase in the liver and skeletal muscle [2], and is crucial in lipid-induced insulin resistance [3]. In animals, fetuin-A knockout mice showed improved insulin signaling, less adiposity, and lower free fatty acid, and were resistant to weight gain upon a high-fat diet compared with wild-type controls [4]. In human studies, fetuin-A is associated with various metabolic dysregulations. Serum fetuin-A concentrations correlate positively Abbreviations: ABI, ankle-brachial index; baPWV, brachial-ankle pulse wave velocity; HOMA, homeostasis model assessment; IFG, impaired fasting glucose; IGT, impaired glucose tolerance; IMT, intima-media thickness; IR, insulin resistance; NDD, newly diagnosed diabetes; NGT, normal glucose tolerance; PAD, peripheral arterial disease; TGF, transforming growth factor; WC, waist circumference. ⁎ Corresponding author at: Department of Family Medicine, National Cheng Kung University Hospital, 138, Sheng Li Road, Tainan 70403, Taiwan. Tel.: +886 6 235 3535x5210; fax: +886 6 275 4243. E-mail address: [email protected] (C.-J. Chang).
http://dx.doi.org/10.1016/j.cca.2015.03.030 0009-8981/© 2015 Elsevier B.V. All rights reserved.
with liver fat in nondiabetic individuals [5], and high fetuin-A concentrations are associated with subjects with metabolic syndrome [6], insulin resistance [7,8], impaired glucose tolerance, and diabetes [9]. In addition to its effect on insulin sensitivity, fetuin-A is also an important inhibitor of ectopic calcification acting on the systemic concentration [10]. It increases the blood solubility of calcium and phosphorus, and prevents spontaneous mineral precipitation in the vasculature [11]. Therefore, fetuin-A knockout mice develop severe calcification of various organs [10]. In human subjects with end-stage renal disease (ESRD), low fetuin-A concentrations are associated with the magnitude of valvular calcification in peritoneal dialysis patients [12], and a significant association between coronary calcification and fetuin-A deficiency has been shown in hemodialysis patients [13]. Many previous studies have suggested that the dual physiological functions of fetuin-A in inhibiting vascular calcification and insulin signaling may be critically important to cardiovascular outcomes. In the general population, fetuin-A is strongly associated with increased risk of myocardial infarction and ischemic stroke independent of standard risk factors [14]. In contrast, studies in ESRD subjects consistently showed that lower fetuin-A concentrations are associated with CVD
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events and all-cause mortality [15–17]. Interestingly, one recent prospective study also observed an association between fetuin-A concentrations and CVD mortality in the general population, while the direction of association differs by diabetes status [18]. Low fetuin-A concentrations predict greater risk for CVD mortality in non-diabetic subjects, but reduced risk of CVD death in those with diabetes. Furthermore, fetuin-A concentrations are also associated with various markers of subclinical CVD. Previous studies have indicated that fetuin-A concentrations are positively associated with carotid intima-media thickness (IMT) [19,20], but negatively related to coronary arterial calcification severity [21]. In addition, another studies demonstrated that serum fetuin-A concentrations are positively associated with arterial stiffness, a functional vascular property of atherosclerosis not associated with calcification, in healthy individuals independent of known atherogenic factors [22] and in diabetes [23], but inversely associated with baPWV in nondiabetic obese females [24]. However, whether the presence of glycemic dysregulations influences the association between fetuin-A and arterial stiffness remains unknown. On the basis of the existing literature, we hypothesized that both glycemic dysregulations and fetuin-A have independent effects on arterial stiffness. 2. Patients and methods The study protocol was approved by the Human Experiment and Ethics Committee of National Cheng Kung University Medical Center (NCKUMC), and all eligible subjects gave written informed consent prior to participation. From June 2007 to July 2009, all subjects who had been admitted for a physical checkup at the Preventive Health Center of NCKUMC were screened. All subjects who did not have a medical history of diabetes received a standard 75-g oral glucose tolerance test after a 10-h overnight fast. None of the women were pregnant. To avoid the confounding effects of age and sex, we selected subjects following this approach: The study subjects were classified into 4 groups according to ADA criteria in the order of their admission to the study: NGT, if fasting plasma glucose was b100 mg/dl and 2-h postload glucose was b140 mg/dl without a history of diabetes; IFG, if fasting plasma glucose was 100–125 mg/dl and 2-h postload glucose was b140 mg/dl; IGT, if fasting plasma glucose was b 100 mg/dl and 2-h postload glucose was 140–199 mg/dl; and NDD, if fasting plasma glucose was ≥ 126 mg/dl or 2-h postload glucose was ≥ 200 mg/dl. Each consecutive index NDD subject was matched to the first subject of the same gender in the other three groups from the list who had the same age. If an exact age match could not be found, then the first subject closest to the age of the index subject (within ± 1 year) was picked. After an overnight 12-h fast, all subjects received a blood test, including routine biochemistry, fasting plasma glucose, hemoglobin A1c (A1C), adiponectin, and fetuin-A. Wearing light indoor clothes, each subject's body height, weight, and waist circumference (WC) were measured. Waist circumference measurement was performed at the end of normal expiration in duplicate on bare skin midway between the lower rib margin and the iliac crest. For the blood pressure measurement, subjects were resting in a supine position in a quiet ambience, and measurements were obtained in a fasting state between 08:00 and 10:00 AM. Two blood pressure readings, separated by intervals of at least 5 min, were taken with an appropriate-sized cuff wrapped around the right upper arm using a DINAMAP vital sign monitor (model 1846SX; Critikon Inc.). Subjects with a systolic blood pressure of ≥140 mm Hg or diastolic blood pressure of ≥90 mm Hg were defined as having hypertension. Brachial-ankle pulse wave velocity (baPWV) was assessed by a noninvasive vascular screening device (BP-203RPE II; Colin Medical Technology) with 4 pneumatic pressure cuffs that simultaneously measured BP and pulse waves in bilateral brachial and tibial arteries after 5 min of bed rest. BaPWV was calculated as the distance traveled by
the pulse wave divided by the time taken to travel the distance [25]. Mean baPWV was used for the final analysis, because of a significant positive correlation between left baPWV and right baPWV (r = 0.968, p b 0.001). Blood glucose was measured by a hexokinase method (Roche Diagnostic GmbH, Mannheim, Germany). Serum total cholesterol, triglycerides, and high density lipoprotein cholesterol (HDL cholesterol) concentrations (Cobas C HDLC3, Roche Diagnostics GmbH) were determined in the central laboratory of NCKUMC with an autoanalyzer (Hitachi 747E). Low density lipoprotein cholesterol (LDL cholesterol) was calculated using the Friedewald formula. A1C was measured with a high performance liquid chromatographic method (intra-assay CV of 0.5%, inter-assay CV of 2.0%). Serum insulin was measured by immunoassay (Mercodia AB; intra-assay CV of 4%, inter-assay CV of 2.6%). Insulin resistance (IR) was estimated by using the homeostasis model assessment (HOMA-IR) index defined as [fasting insulin (μU/ml) × fasting plasma glucose (mmol/l)] / 22.5. Serum fetuin-A was determined by an enzyme-linked immunosorbent assay (ELISA) method (Biovendor Laboratory Medicine, Czech Republic; intra-assay CV of 2.7%, inter-assay CV of 3.2%). The determination of serum adiponectin was carried out using AssayMax Human Adiponectin (Acrp30) ELISA kits (AssayPro, St. Charles, Missouri; intra-assay CV of 2.5%, inter-assay CV of 6.5%). High-sensitive C-reactive protein (hsCRP) was measured with a highly-sensitive ELISA kit (Immunology Consultants Laboratory, Newberg, OR; intra-assay CV of 2.9%, interassay CV of 4.7%). Subjects with the following conditions or diseases were excluded: 1) alcohol consumption ≥ 20 g/day in the last year; 2) serum aspartate aminotransferase (AST) or alanine aminotransferase (ALT) concentrations N 2 times the normal limit; 3) a positive test for hepatitis B surface antigen, hepatitis C antibody, and other causes of liver disease; 4) serum creatinine N 1.6 mg/dl; 5) any acute or chronic inflammatory disease as determined by a leukocyte count of N10,000/mm 3 or clinical signs of infection; 6) overt peripheral arterial disease (PAD) defined by an abnormal ankle-brachial index (ABI b 0.9), or participants with ABI N 1.4 reflecting noncompressible arteries and amputation of either lower limb; 7) a history of coronary heart disease and stroke; 8) those taking medications influencing blood pressure, plasma glucose, and lipid profile; and 9) any other major diseases, including generalized inflammation or advanced malignant diseases contraindicating this study. 2.1. Statistical analysis SPSS software (version 17.0; SPSS) was used for statistical analysis. All normally distributed continuous variables were expressed as means ± SD. Study subjects were divided into 4 groups: NGT, IFG, IGT, and NDD. The continuous variables among groups were compared using ANOVA (analysis of variance) or a Kruskal–Wallis test when the distribution was not normal. Post hoc (Bonferroni method) tests were used to evaluate pairwise differences among the adjusted means of serum fetuin-A concentration and baPWV. Multiple linear regression analysis was conducted to identify variables that best predicted baPWV, of which the variable selection strategies were stepwise and backward. The independent variables included age, sex, IFG vs. NGT, IGT vs. NGT, NDD vs. NGT, fetuin-A, general obesity, hypertension, smoking, exercise, hypertriglyceridemia, low-HDL cholesterol, creatinine, adiponectin, hsCRP, and HOMA-IR. A p b 0.05 was considered statistically significant. 3. Results A total of 2161 subjects (out of 3167 subjects admitted for a physical checkup) receiving a standard 75-g oral glucose tolerance test were screened. Among them, 139 with any conditions listed in the exclusion criteria were excluded (NGT, 95/1672; IFG, 5/64; IGT, 27/326; NDD, 12/99). Finally, 312 subjects were selected according to the selection
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protocol described above (NGT, 87/1577; IFG, 51/59; IGT, 87/299; NDD, 87/87). Table 1 shows the clinical characteristics of the study subjects. There were significant differences in WC, BMI, systolic/diastolic blood pressure, fasting and 2-h postload plasma glucose, A1C, ALT, AST, hsCRP, adiponectin, triglyceride, LDL cholesterol, HDL cholesterol, LDL/ HDL-cholesterol ratio, and HOMA-IR among groups. Both baPWV and serum fetuin-A concentrations increase along with the severity of glycemic status (Fig. 1). The mean values of baPWV were 1533 ± 338, 1518 ± 353, 1589 ± 307, and 1690 ± 414 cm/s in subjects with NGT, IFG, IGT, and NDD, respectively (p b 0.001, test for trend) (Fig. 1B), and the serum fetuin-A concentrations were 298 ± 69, 313 ± 67, 330 ± 86, and 342 ± 93 μg/ml, respectively (p b 0.001, test for trend) (Fig. 1C). Post hoc tests showed that subjects with NDD, but not prediabetes, exhibited a significantly greater arterial stiffness (p = 0.035) and higher fetuin-A concentrations (p = 0.002) than NGT subjects. The results of simple regression analysis show that baPWV concentrations were positively related to age (p b 0.001) and hypertension (p b 0.001) in the whole subjects and in each glycemic group (Table 2). Hypertriglyceridemia, hsCRP, and fetuin-A were also positively associated with baPWV concentrations in NGT, IGT, and NDD subjects, respectively. Table 3 shows the results of multiple regression analysis. baPWV concentrations were positively associated with age (all 4 groups), hypertension (IFG, IGT, and NDD groups), hypertriglyceridemia (NGT group), and fetuin-A concentrations (NDD group). In the whole subjects, both stepwise and backward selection strategies showed a consistent model. No multicollinearity was detected among the independent variables. We found that age (p b 0.001), NDD vs. NGT (p = 0.040), fetuin-A (p = 0.030), hypertension (p b 0.001), and hypertriglyceridemia (p = 0.039) were independently associated factors of baPWV concentrations after adjustment for age, sex, obesity, smoking, exercise, lipid profiles, adiponectin, CRP, and HOMA-IR.
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4. Discussion We found that both diabetes and fetuin-A concentrations, but not prediabetes, are independently associated with baPWV concentrations after adjusting for cardiometabolic risk factors, HOMA-IR, and adiponectin. Our findings suggest that the elevated fetuin-A concentrations may further aggravate increased arterial stiffness in diabetic patients. The associations between fetuin-A and CVD in subjects without CKD are inconsistent in previous studies. In a case-cohort study based on the general population, fetuin-A concentrations are positively related to incident myocardial infarction and ischemic stroke irrespective of diabetes status [14]. Using the Mendelian randomization approach, the same group provided strong evidence to suggest the causal involvement of fetuin-A in the pathogenesis of CVD [26]. However, in another prospective study on CVD mortality in older community-dwelling individuals, the positive association of fatal CVD with fetuin-A was observed only in diabetic subjects. In contrast, in subjects without diabetes, low plasma fetuin-A concentrations are independently associated with increased risk of CVD mortality [18]. The authors suggested that these findings imply that the balance between protecting against vascular calcification and promoting insulin resistance/metabolic derangement may depend on the metabolic milieu or prior disease processes. The discrepancies among the above studies could be attributable to differences in the ages of the study subjects, outcome measures, and different methods used to diagnose diabetes. In addition, studies have also demonstrated that fetuin-A is an important predictor of subclinical CVD [19–21,27]. In nondiabetic subjects at risk of coronary artery disease (CAD) [19] or with known or clinically suspected CAD [27], high serum fetuin-A concentrations are positively associated with greater carotid IMT, an early anatomical sign of atherosclerosis [19], while negatively associated with calcified CAD [27]. In addition, Fiore et al. reported that fetuin-A concentrations are associated
Table 1 Clinical characteristics among study subjects with normal glucose tolerance (NGT), impaired fasting glucose (IFG), impaired glucose tolerance (IGT), and newly diagnosed diabetes (NDD).
n Age (years) Sex (F/M) Waist circumference (cm) BMI (kg/m2) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Fasting plasma glucose (mg/dl) 2-h post-load plasma glucose (mg/dl) A1C (%) GOT (U/l) GPT (U/l) hsCRP (mg/l) Creatinine (mg/dl) Triglyceride (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl) LDL/HDL-cholesterol ratio Fetuin-A (μg/ml) Adiponectin (μg/ml) HOMA-IR baPWV (cm/s) General obesity Central obesity Hypertension Low-HDL Hypertriglyceridemia Regular exercise Smoking Data are expressed as means ± SD or number (%). p values are by ANOVA or χ2 test. NS: not significant.
NGT
IFG
IGT
NDD
p
87 62 ± 12 39/48 78.9 ± 8.9 22.2 ± 2.6 121 ± 17 71 ± 10 85 ± 7 95 ± 23 5.7 ± 0.3 26 ± 8 22 ± 9 2.08 ± 3.29 0.8 ± 0.2 96 ± 37 118 ± 31 59 ± 19 2.2 ± 1.0 298 ± 69 15 ± 13 0.41 ± 0.28 1533 ± 338 12 (14) 19 (22) 12 (14) 16 (18) 8 (9) 4 (5) 6 (7)
51 57 ± 12 19/32 83.3 ± 7.5 24.2 ± 2.5 125 ± 18 74 ± 11 104 ± 5 104 ± 22 5.9 ± 0.4 27 ± 9 27 ± 19 3.00 ± 5.00 0.9 ± 0.2 105 ± 48 126 ± 35 56 ± 15 2.4 ± 0.9 313 ± 67 13 ± 9 0.81 ± 0.59 1518 ± 353 18 (35) 17 (33) 11 (22) 8 (16) 9 (18) 5 (10) 3 (6)
87 62 ± 12 34/53 81.8 ± 8.5 23.4 ± 2.9 129 ± 17 74 ± 10 89 ± 11 162 ± 17 5.8 ± 0.3 25 ± 8 23 ± 11 3.00 ± 5.02 0.9 ± 0.2 119 ± 62 120 ± 34 53 ± 14 2.4 ± 0.9 330 ± 86 11 ± 6 0.53 ± 0.48 1589 ± 307 27 (31) 26 (30) 18 (21) 23 (26) 18 (21) 7 (8) 4 (5)
87 61 ± 11 39/48 82.1 ± 8.9 23.2 ± 3.1 132 ± 19 76 ± 11 137 ± 59 264 ± 89 7.3 ± 2.1 30 ± 43 32 ± 54 5.06 ± 9.30 0.9 ± 0.2 140 ± 104 133 ± 39 53 ± 14 2.6 ± 0.9 342 ± 93 12 ± 11 0.94 ± 0.85 1690 ± 414 23 (26) 27 (31) 24 (28) 20 (23) 29 (33) 2 (2) 6 (7)
– NS NS 0.015 0.001 0.002 0.008 b0.001 b0.001 b0.001 NS NS 0.002 NS b0.001 0.035 0.020 0.047 0.002 NS b0.001 0.011 0.02 NS NS NS 0.001 NS NS
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baPWV (cm/sec)
A
B
2200
2000
1800
1600
1400
1200
1000
0
\\
\\
NGT
NGT
P < 0.001 test for trend.
450
C
400
350
300
250
200
0
P < 0.001 test for trend.
P = 0.035
P = 0.002
P = 0.032
IGT
IGT
Fetuin-A (µg/ml)
IFG
P = 0.047
IFG
Table 2 Simple regression analysis between brachial-ankle pulse wave velocity (baPWV) and clinical variables. NGT β Age (years) Sex, male vs. female General obesity IFG vs. NGT IGT vs. NGT NDD vs. NGT Fetuin-A (μg/ml) Hypertension, yes vs. no Regular exercise, yes vs. no Smoking, yes vs. no Low-HDL cholesterol, yes vs. no Hypertriglyceridemia, yes vs. no Creatinine (μmol/l) Adiponectin (μg/ml) hsCRP (mg/l) HOMA-IR
17.302⁎ −59.280 53.878 – – – −0.437 352.192⁎ −252.321 93.790 51.157 293.589† −1.851 3.124 19.377 190.119
IFG 95% CI
β
12.504–22.100 −204.262–85.702 −155.707–263.464
16.948⁎
−1.490–0.616 156.508–547.876 −593.615–88.973 −191.141–378.721 −135.353–237.667 51.233–535.945 −6.478–2.775 −2.653–8.900 −2.380–41.134 −61.989–442.227
81.271 −67.601 – – – −0.408 447.384⁎ −31.620 −153.198 −149.836 −93.524 2.927 11.526 8.705 −66.766
IGT 95% CI
β
9.313–24.583 −125.172–287.713 −276.894–141.692
11.499⁎
−1.953–1.136 239.664–655.104 −369.285–306.046 −577.827–271.431 −422.669–122.997 −355.641–168.593 −2.510–8.364 −0.146–22.907 −30.073–47.483 −242.377–108.845
−56.012 73.769 – – – −0.075 309.628⁎ −65.558 −71.873 87.084 −34.866 0.837 9.905 19.990† 86.713
NDD 95% CI
β
6.817–16.181 −190.227–78.203 −67.463–215.002
17.320⁎
−0.840–0.690 161.279–457.977 −306.878–175.762 −385.442–241.695 −60.829–234.996 −197.011–127.279 −3.276–4.949 −0.119–19.929 2.771–37.209 −50.822–224.248
3.978 38.450 -– – – 1.161† 409.159⁎ −324.724 −144.383 −150.443 −30.388 3.320 5.630 −0.032 35.576
All subjects 95% CI 10.417–24.222 −174.585–182.540 −162.749–239.649
0.234–2.088 232.105–586.212 −913.119–263.672 −493.450–204.684 −358.990–58.103 −218.654–157.878 −1.360–8.000 −2.338–13.599 −9.639–9.575 −69.378–140.529
β
95% CI 15.362⁎
−21.910 35.114 −86.205 −1.475 138.841# 0.416 388.528⁎ −150.747 −53.244 −13.189 41.978 1.626 5.045 6.783 60.672
NGT: normal glucose tolerance; IFG: impaired fasting glucose; IGT: impaired glucose tolerance; NDD: newly diagnosed diabetes; HDL, high density lipoprotein; triglyceride had been log transformed before analysis. Dependent variable: brachial-ankle pulse wave velocity (baPWV). † p b 0.05. # p b 0.01. ⁎ p b 0.001.
12.454–18.271 −103.255–59.435 −56.786–127.015 −194.394–21.984 −91.043–88.092 50.627–227.055 −0.071–0.903 299.692–477.364 −322.184–20.691 −221.093–114.606 −110.988–84.610 −57.380–141.336 −0.705–3.958 −0.128–8.955 −0.155–13.721 −3.680–125.023
H.-Y. Ou et al. / Clinica Chimica Acta 445 (2015) 133–138
P = NS
NDD
NDD
Fig. 1. Distribution of brachial-ankle pulse wave velocity (baPWV) and serum fetuin-A (A), and comparison of brachial-ankle pulse wave velocity (B) and serum fetuin-A (C) among subjects with normal glucose tolerance (NGT), impaired fasting glucose (IFG), impaired glucose tolerance (IGT), and newly diagnosed diabetes (NDD). Data are shown as mean ± SD. NS, not significant.
with IMT in patients with atherosclerosis of peripheral vessels (n = 90; 51% diabetes) after adjustment for diabetes and other cofounding variables [20]. Furthermore, in a population sample of community-dwelling subjects who were free of clinical CVD, Ix et al. observed that lower fetuin-A concentrations are independently associated with greater coronary artery calcification severity, but not carotid IMT [21]. Notably, no modifications of effects related to diabetes have been reported in
baPWV (cm/sec)
Fetuin-A (µg/mL)
0.237–1.961 119.236–486.954 −810.538–383.748 −406.799–237.824 −308.364–77.725 −84.125–287.003 −5.96–4.259 −2.603–14.104 −8.746–8.859 −139.575–57.498 −0.7–0.661 125.318–421.669 −330.886–93.526 −366.791–190.716 −136.3–170.934 −164.697–161.033 −4.023–5.002 −5.266–19.539 −2.259–30.319 −42.367–219.865 −1.177–1.745 138.359–617.19 −465.688–221.724 −235.159–659.839 −236.517–353.838 −325.059–192.049 −8.18–11.043 −1.773–20.056 −71.176–3.932 −158.235–149.671 −1.264–0.441 −108.938–300.581 −410.22–139.669 −199.024–274.191 −141.986–157.015 78.766–482.678 −7.745–2.115 −7.516–2.468 −16.01–24.145 −132.995–293.261
NGT: normal glucose tolerance; IFG: impaired fasting glucose; IGT: impaired glucose tolerance; NDD: newly diagnosed diabetes; HDL, high density lipoprotein; triglyceride had been log transformed before analysis. Dependent variable: brachial-ankle pulse wave velocity (baPWV). † p b 0.05. # p b 0.01. ⁎ p b 0.001.
10.367–16.489 −76.808–100.554 −72.902–85.895 −68.621–144.746 −69.064–109.472 4.726–194.635 0.045–0.860 177.832–348.061 −231.307–47.435 −161.533–118.143 −121.310–43.263 4.653–180.157 −1.843–3.107 −1.790–5.041 −5.642–6.137 −72.957–44.433 13.428⁎ 11.873 6.496 38.062 20.204 99.6814† 0.453† 262.947⁎ −91.936 −21.695 −39.023 92.405† 0.632 1.625 0.248 −14.262
β 95% CI
6.23–21.678 −121.173–269.363 −159.164–213.572
13.954⁎ 74.095 27.204 – – – 1.099† 303.095# −213.395 −84.488 −115.320 101.439 −0.850 5.750 0.056 −41.038
β 95% CI
1.523–12.925 −139.855–181.983 −115.88–155.154
7.224† 21.064 19.637 – – – −0.019 273.493⁎ −118.680 −88.037 17.317 −1.832 0.489 7.137 14.030 88.749
β 95% CI
5.937–27.318 −250.293–463.53 −141.613–243.083
16.628# 106.618 50.735 – – – 0.284 377.774# −121.982 212.340 58.660 −66.505 1.431 9.142 −33.622 −4.282
β
11.104–21.756 −139.792–176.468 −180.554–191.962
95% CI β
16.430⁎ 18.338 5.704 – – – −0411 95.822 −135.275 37.584 7.514 280.722# −2.815 −2.524 4.068 80.133
NDD IGT IFG NGT
Table 3 Multiple regression analysis between brachial-ankle pulse wave velocity (baPWV) and clinical variables.
Age (years) Sex, male vs. female General obesity IFG vs. NGT IGT vs. NGT NDD vs. NGT Fetuin-A (μg/ml) Hypertension, yes vs. no Regular exercise, yes vs. no Smoking, yes vs. no Low-HDL cholesterol, yes vs. no Hypertriglyceridemia, yes vs. no Creatinine (μmol/l) Adiponectin (μg/ml) hsCRP (mg/l) HOMA-IR
All subjects
95% CI
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the associations of fetuin-A concentrations and these measures of subclinical CVD [21]. The current study extends the relationship between fetuin-A and subclinical CVD to the early functional vascular changes of CVD. Recently, Jung et al. reported a positive correlation between fetuin-A and arterial stiffness in diabetic subjects [23], whereas Yang et al. showed a negative association in nondiabetic, obese women in a small-scaled study (n = 40) [24]. Here, we further find that the effect of fetuin-A on arterial stiffness is independent of age, hypertension, diabetes, insulin resistance, and traditional cardiometabolic risk factors. Arterial stiffness is a functional assessment of vascular damage caused by CVD risk factors. Increased arterial stiffness, which is usually measured by the PWV, causes increased cardiac afterload, a decrease in coronary perfusion, atherogenesis and/or microvascular damage [25]. PWV has thus been shown to be a predictor of future cardiovascular events in the general population [28], hypertensive subjects [29], and those with type 2 diabetes [30]. Increased baPWV is associated with older age, higher blood pressure, BMI, IGT, and diabetes [31]. In the current study, we found that NDD subjects had higher baPWV concentrations than NGT, IFG, and IGT subjects. Although the IFG group seems to have the lowest baPWV among 4 groups, the small difference did not reach statistical significance when compared with NGT subjects. Thus, in the final statistical analysis, only the differences of NDD vs. NGT and NDD vs. IFG were significant. The mechanisms leading to arterial stiffness are not the same among different CVD risk factors, and may involve the dysregulation of balance between extracellular matrix collagen and elastin, and deposition of glycoproteins and proteoglycans. Hypertension per se causes thinning, splitting and fragmentation of elastic fibers [25]. In diabetes, the formation of advanced glycation end-products (AGEs) on the arterial wall causes cross-linking of collagen molecules, which may lead to loss of collagen elasticity and a subsequent increase in arterial stiffness [32]. On the other hand, arterial stiffening with aging is associated with transforming growth factor-β1-related increases in adventitial collagen and reductions in medial elastin [33]. In addition to age, hypertension, and NDD, the current study also shows that fetuin-A is an independently associated factor of arterial stiffness. This result is consistent with that of Mori et al. in 141 nondiabetic subjects, showing that besides age, fetuin-A independently contributes to carotid artery stiffness [22]. Although both fetuin-A and baPWV increase due to glycemia, the results of this work show that fetuin-A and diabetes have independent associations with arterial stiffness in subjects with different glycemic statuses. While the mechanisms on how fetuin-A can regulate arterial stiffness remain unclear, Mori et al. postulated a plausible explanation of this [22]. As fetuin-A and type II TGFβ-receptors share a sequence homology at the major cytokine binding site, fetuin-A can inhibit type II receptor mediated signaling [34]. Therefore, by decreasing the type II/type I TGFβ-receptor ratio, vascular smooth muscle cells overproduce collagen and extracellular matrix [35], which leads to greater arterial stiffness [33]. This work has the following limitations. First, since this study used a cross-sectional design, it does not allow causal inferences between serum fetuin-A concentrations and the development of arterial stiffness. Second, arterial stiffness was assessed by brachial-ankle PWV in this study, but not the carotid–femoral PWV [35]. Although carotid–femoral PWV is still considered as the gold standard in measuring central arterial stiffness, a high concentration of skill and exposure of the inguinal region are required for its measurement, which limits its applicability in clinical practice. However, one previous study showed that baPWV correlates significantly with carotid–femoral PWV, and provides qualitatively similar information to that derived from central arterial stiffness [36]. Finally, the study subjects were confined to a Chinese population, and the findings may not be generalizable to other ethnicities. In conclusion, in this work we demonstrate that both baPWV and serum fetuin-A concentrations increase along with the severity of
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glycemic status. Subjects with NDD, but not prediabetes, exhibit significantly greater arterial stiffness and higher fetuin-A concentrations than NGT subjects. Diabetes and fetuin-A concentrations are associated with baPWV independent of age, sex, hypertension, hypertriglyceridemia, and other confounding factors. However, further studies are needed to investigate the possible mechanisms underlying the action of fetuin-A on vascular wall functioning. Acknowledgments This study was supported by grants from the National Science Council Taiwan (NSC-94-2314-B-006-114, NSC-95-2314-B-006-048, and NSC99-2314-B-006-049-MY3) and National Cheng Kung University Hospital (NCKUH-9501005, NCKUH-9702005, and NCKUH-9903034). References [1] Denecke B, Graber S, Schafer C, Heiss A, Woltje M, Jahnen-Dechent W. Tissue distribution and activity testing suggest a similar but not identical function of fetuin-B and fetuin-A. Biochem J 2003;376:135–45. [2] Srinivas PR, Wagner AS, Reddy LV, Deutsch DD, Leon MA, Goustin AS, et al. Serum alpha 2-HS-glycoprotein is an inhibitor of the human insulin receptor at the tyrosine kinase level. Mol Endocrinol 1993;7:1445–55. [3] Pal D, Dasgupta S, Kundu R, Maitra S, Das G, Mukhopadhyay S, et al. Fetuin-A acts as an endogenous ligand of TLR4 to promote lipid-induced insulin resistance. Nat Med 2012;18:1279–85. [4] Mathews ST, Singh GP, Ranalletta M, Cintron VJ, Qiang X, Goustin AS, et al. Improved insulin sensitivity and resistance to weight gain in mice null for the Ahsg gene. Diabetes 2002;51:2450–8. [5] Stefan N, Hennige AM, Staiger H, Machann J, Schick F, Krober SM, et al. Alpha2Heremans-Schmid glycoprotein/fetuin-A is associated with insulin resistance and fat accumulation in the liver in humans. Diabetes Care 2006;29:853–7. [6] Ix JH, Shlipak MG, Brandenburg VM, Ali S, Ketteler M, Whooley MA. Association between human fetuin-A and the metabolic syndrome: data from the Heart and Soul Study. Circulation 2006;113:1760–7. [7] Mori K, Emoto M, Yokoyama H, Araki T, Teramura M, Koyama H, et al. Association of serum fetuin-A with insulin resistance in type 2 diabetic and nondiabetic subjects. Diabetes Care 2006;29:468. [8] Ishibashi A, Ikeda Y, Ohguro T, Kumon Y, Yamanaka S, Takata H, et al. Serum fetuin-A is an independent marker of insulin resistance in Japanese men. J Atheroscler Thromb 2010;17:925–33. [9] Ou HY, Yang YC, Wu HT, Wu JS, Lu FH, Chang CJ. Serum fetuin-A concentrations are elevated in subjects with impaired glucose tolerance and newly diagnosed type 2 diabetes. Clin Endocrinol (Oxf) 2011;75:450–5. [10] Schafer C, Heiss A, Schwarz A, Westenfeld R, Ketteler M, Floege J, et al. The serum protein alpha 2-Heremans-Schmid glycoprotein/fetuin-A is a systemically acting inhibitor of ectopic calcification. J Clin Invest 2003;112:357–66. [11] Jahnen-Dechent W, Heiss A, Schäfer C, Ketteler M. Fetuin-A regulation of calcified matrix metabolism. Circ Res 2011;108:1494–509. [12] Wang A, Woo J, Lam C, Wang M, Chan I, Gao P, et al. Associations of serum fetuin-A with malnutrition, inflammation, atherosclerosis and valvular calcification syndrome and outcome in peritoneal dialysis patients. Nephrol Dial Transplant 2005; 20:1676–85. [13] Moe S, Reslerova M, Ketteler M, O'Neill K, Duan D, Koczman J, et al. Role of calcification inhibitors in the pathogenesis of vascular calcification in chronic kidney disease (CKD). Kidney Int 2005;67:2295–304. [14] Weikert C, Stefan N, Schulze MB, Pischon T, Berger K, Joost HG, et al. Plasma fetuin-a levels and the risk of myocardial infarction and ischemic stroke. Circulation 2008; 118:2555–62.
[15] Ketteler M, Bongartz P, Westenfeld R, Wildberger J, Mahnken A, Böhm R, et al. Association of low fetuin-A (AHSG) concentrations in serum with cardiovascular mortality in patients on dialysis: a cross-sectional study. Lancet 2003;361:827–33. [16] Stenvinkel P, Wang K, Qureshi A, Axelsson J, Pecoits-Filho R, Gao P, et al. Low fetuinA levels are associated with cardiovascular death: impact of variations in the gene encoding fetuin. Kidney Int 2005;67:2383–92. [17] Hermans M, Brandenburg V, Ketteler M, Kooman J, van der Sande F, Boeschoten E, et al. Association of serum fetuin-A levels with mortality in dialysis patients. Kidney Int 2007;72:202–7. [18] Laughlin G, Cummins K, Wassel C, Daniels L, Ix J. The association of fetuin-A with cardiovascular disease mortality in older community-dwelling adults: the Rancho Bernardo study. J Am Coll Cardiol 2012;59:1688–96. [19] Rittig K, Thamer C, Haupt A, Machann J, Peter A, Balletshofer B, et al. High plasma fetuin-A is associated with increased carotid intima-media thickness in a middleaged population. Atherosclerosis 2009;207:341–2. [20] Fiore C, Celotta G, Politi G, Di Pino L, Castelli Z, Mangiafico R, et al. Association of high alpha2-Heremans-Schmid glycoprotein/fetuin concentration in serum and intimamedia thickness in patients with atherosclerotic vascular disease and low bone mass. Atherosclerosis 2007;195:110–5. [21] Ix J, Barrett-Connor E, Wassel C, Cummins K, Bergstrom J, Daniels L, et al. The associations of fetuin-A with subclinical cardiovascular disease in community-dwelling persons: the Rancho Bernardo Study. J Am Coll Cardiol 2011;58:2372–9. [22] Mori K, Emoto M, Araki T, Yokoyama H, Teramura M, Lee E, et al. Association of serum fetuin-A with carotid arterial stiffness. Clin Endocrinol (Oxf) 2007;66: 246–50. [23] Jung CH, Kim BY, Kim CH, Kang SK, Jung SH, Mok JO. Associations of serum fetuin-A levels with insulin resistance and vascular complications in patients with type 2 diabetes. Diab Vasc Dis Res 2013;10:459–67. [24] Yang SJ, Hong HC, Choi HY, Yoo HJ, Cho GJ, Hwang TG, et al. Effects of a three-month combined exercise program on fibroblast growth factor 21 and fetuin-A levels and arterial stiffness in obese women. Clin Endocrinol (Oxf) 2011;75:464–9. [25] Tomiyama H, Yamashina A. Non-invasive vascular function tests: their pathophysiological background and clinical application. Circ J 2010;74:24–33. [26] Fisher E, Stefan N, Saar K, Drogan D, Schulze M, Fritsche A, et al. Association of AHSG gene polymorphisms with fetuin-A plasma levels and cardiovascular diseases in the EPIC-Potsdam study. Circ Cardiovasc Genet 2009;2:607–13. [27] Mori K, Ikari Y, Jono S, Emoto M, Shioi A, Koyama H, et al. Fetuin-A is associated with calcified coronary artery disease. Coron Artery Dis 2010;21:281–5. [28] Willum-Hansen T, Staessen J, Torp-Pedersen C, Rasmussen S, Thijs L, Ibsen H, et al. Prognostic value of aortic pulse wave velocity as index of arterial stiffness in the general population. Circulation 2006;113:664–70. [29] Boutouyrie P, Tropeano A, Asmar R, Gautier I, Benetos A, Lacolley P, et al. Aortic stiffness is an independent predictor of primary coronary events in hypertensive patients: a longitudinal study. Hypertension 2002;39:10–5. [30] Cruickshank K. Aortic pulse-wave velocity and its relationship to mortality in diabetes and glucose intolerance: an integrated index of vascular function? Circulation 2002;106:2085–90. [31] Li CH, Wu JS, Yang YC, Shih CC, Lu FH, Chang CJ. Increased arterial stiffness in subjects with impaired glucose tolerance and newly diagnosed diabetes but not isolated impaired fasting glucose. J Clin Endocrinol Metab 2012;97:E658-62. [32] Aronson D. Cross-linking of glycated collagen in the pathogenesis of arterial and myocardial stiffening of aging and diabetes. J Hypertens 2003;21:3–12. [33] Fleenor B, Marshall K, Durrant J, Lesniewski L, Seals D. Arterial stiffening with ageing is associated with transforming growth factor-β1-related changes in adventitial collagen: reversal by aerobic exercise. J Physiol 2010;588:3971–82. [34] Demetriou M, Binkert C, Sukhu B, Tenenbaum H, Dennis J. Fetuin/alpha2-HS glycoprotein is a transforming growth factor-beta type II receptor mimic and cytokine antagonist. J Biol Chem 1996;271:12755–61. [35] McCaffrey T, Consigli S, Du B, Falcone D, Sanborn T, Spokojny A, et al. Decreased type II/type I TGF-beta receptor ratio in cells derived from human atherosclerotic lesions. Conversion from an antiproliferative to profibrotic response to TGF-beta1. J Clin Invest 1995;96:2667–75. [36] Sugawara J, Hayashi K, Yokoi T, Cortez-Cooper MY, DeVan AE, Anton MA, et al. Brachial-ankle pulse wave velocity: an index of central arterial stiffness? J Hum Hypertens 2005;19:401–6.
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Both diabetes and fetuin-A are independently associated with increased risk of arterial stiffness Horng-Yih Ou a, Feng-Hwa Lu b, Hung-Tsung Wu c, Hao-Chang Hung a, Jin-Shang Wu b, Yi-Ching Yang b, Chih-Jen Chang b,c,⁎ a b c
Department of Internal Medicine, National Cheng Kung University Medical College and Hospital, Taiwan Department of Family Medicine, National Cheng Kung University Medical College and Hospital, Taiwan Research Center of Herbal Medicine, New Drugs, and Nutritional Supplements, Research and Services Headquarters, National Cheng Kung University, Taiwan
a r t i c l e
i n f o
Article history: Received 12 August 2014 Received in revised form 24 March 2015 Accepted 24 March 2015 Available online 1 April 2015 Keywords: Fetuin-A Arterial stiffness Diabetes
a b s t r a c t Background: Arterial stiffness is a functional assessment of vascular damage caused by cardiovascular disease (CVD) risk factors. Fetuin-A is associated with subclinical CVD and incident or fatal CVD, with some modification of its effect occurring with the presence of diabetes. We investigated the impact of different glycemic statuses and serum fetuin-A levels on arterial stiffness. Methods: A total of 312 age- and sex-matched subjects with normal glucose tolerance (NGT), impaired fasting glucose (IFG), impaired glucose tolerance (IGT), and newly diagnosed diabetes (NDD) were recruited. Serum fetuin-A levels were measured, and arterial stiffness was assessed by brachial-ankle pulse-wave velocity (baPWV). Results: We found that the mean values of baPWV were 1533 ± 338, 1518 ± 353, 1589 ± 307, and 1690 ± 414 cm/s, and fetuin-A levels were 298 ± 69, 313 ± 67, 330 ± 86, and 342 ± 93 μg/ml, in subjects with NGT, IFG, IGT, and NDD, respectively (both p b 0.001, test for trend). NDD subjects had significantly higher baPWV and fetuin-A levels than those with NGT. Multiple linear regression analysis showed that age, fetuin-A, diabetes, hypertension, and hypertriglyceridemia are independently associated factors of baPWV after adjusting for cardiometabolic risk factors, HOMA-IR, and adiponectin. Conclusion: Both diabetes and fetuin-A levels are independently associated with arterial stiffness. Fetuin-A may further aggravate increased arterial stiffness in diabetes. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Fetuin-A is a glycoprotein produced exclusively in the liver and then secreted into the circulation in high concentrations [1]. Fetuin-A per se is an endogenous inhibitor of the insulin receptor tyrosine kinase in the liver and skeletal muscle [2], and is crucial in lipid-induced insulin resistance [3]. In animals, fetuin-A knockout mice showed improved insulin signaling, less adiposity, and lower free fatty acid, and were resistant to weight gain upon a high-fat diet compared with wild-type controls [4]. In human studies, fetuin-A is associated with various metabolic dysregulations. Serum fetuin-A concentrations correlate positively Abbreviations: ABI, ankle-brachial index; baPWV, brachial-ankle pulse wave velocity; HOMA, homeostasis model assessment; IFG, impaired fasting glucose; IGT, impaired glucose tolerance; IMT, intima-media thickness; IR, insulin resistance; NDD, newly diagnosed diabetes; NGT, normal glucose tolerance; PAD, peripheral arterial disease; TGF, transforming growth factor; WC, waist circumference. ⁎ Corresponding author at: Department of Family Medicine, National Cheng Kung University Hospital, 138, Sheng Li Road, Tainan 70403, Taiwan. Tel.: +886 6 235 3535x5210; fax: +886 6 275 4243. E-mail address: [email protected] (C.-J. Chang).
http://dx.doi.org/10.1016/j.cca.2015.03.030 0009-8981/© 2015 Elsevier B.V. All rights reserved.
with liver fat in nondiabetic individuals [5], and high fetuin-A concentrations are associated with subjects with metabolic syndrome [6], insulin resistance [7,8], impaired glucose tolerance, and diabetes [9]. In addition to its effect on insulin sensitivity, fetuin-A is also an important inhibitor of ectopic calcification acting on the systemic concentration [10]. It increases the blood solubility of calcium and phosphorus, and prevents spontaneous mineral precipitation in the vasculature [11]. Therefore, fetuin-A knockout mice develop severe calcification of various organs [10]. In human subjects with end-stage renal disease (ESRD), low fetuin-A concentrations are associated with the magnitude of valvular calcification in peritoneal dialysis patients [12], and a significant association between coronary calcification and fetuin-A deficiency has been shown in hemodialysis patients [13]. Many previous studies have suggested that the dual physiological functions of fetuin-A in inhibiting vascular calcification and insulin signaling may be critically important to cardiovascular outcomes. In the general population, fetuin-A is strongly associated with increased risk of myocardial infarction and ischemic stroke independent of standard risk factors [14]. In contrast, studies in ESRD subjects consistently showed that lower fetuin-A concentrations are associated with CVD
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events and all-cause mortality [15–17]. Interestingly, one recent prospective study also observed an association between fetuin-A concentrations and CVD mortality in the general population, while the direction of association differs by diabetes status [18]. Low fetuin-A concentrations predict greater risk for CVD mortality in non-diabetic subjects, but reduced risk of CVD death in those with diabetes. Furthermore, fetuin-A concentrations are also associated with various markers of subclinical CVD. Previous studies have indicated that fetuin-A concentrations are positively associated with carotid intima-media thickness (IMT) [19,20], but negatively related to coronary arterial calcification severity [21]. In addition, another studies demonstrated that serum fetuin-A concentrations are positively associated with arterial stiffness, a functional vascular property of atherosclerosis not associated with calcification, in healthy individuals independent of known atherogenic factors [22] and in diabetes [23], but inversely associated with baPWV in nondiabetic obese females [24]. However, whether the presence of glycemic dysregulations influences the association between fetuin-A and arterial stiffness remains unknown. On the basis of the existing literature, we hypothesized that both glycemic dysregulations and fetuin-A have independent effects on arterial stiffness. 2. Patients and methods The study protocol was approved by the Human Experiment and Ethics Committee of National Cheng Kung University Medical Center (NCKUMC), and all eligible subjects gave written informed consent prior to participation. From June 2007 to July 2009, all subjects who had been admitted for a physical checkup at the Preventive Health Center of NCKUMC were screened. All subjects who did not have a medical history of diabetes received a standard 75-g oral glucose tolerance test after a 10-h overnight fast. None of the women were pregnant. To avoid the confounding effects of age and sex, we selected subjects following this approach: The study subjects were classified into 4 groups according to ADA criteria in the order of their admission to the study: NGT, if fasting plasma glucose was b100 mg/dl and 2-h postload glucose was b140 mg/dl without a history of diabetes; IFG, if fasting plasma glucose was 100–125 mg/dl and 2-h postload glucose was b140 mg/dl; IGT, if fasting plasma glucose was b 100 mg/dl and 2-h postload glucose was 140–199 mg/dl; and NDD, if fasting plasma glucose was ≥ 126 mg/dl or 2-h postload glucose was ≥ 200 mg/dl. Each consecutive index NDD subject was matched to the first subject of the same gender in the other three groups from the list who had the same age. If an exact age match could not be found, then the first subject closest to the age of the index subject (within ± 1 year) was picked. After an overnight 12-h fast, all subjects received a blood test, including routine biochemistry, fasting plasma glucose, hemoglobin A1c (A1C), adiponectin, and fetuin-A. Wearing light indoor clothes, each subject's body height, weight, and waist circumference (WC) were measured. Waist circumference measurement was performed at the end of normal expiration in duplicate on bare skin midway between the lower rib margin and the iliac crest. For the blood pressure measurement, subjects were resting in a supine position in a quiet ambience, and measurements were obtained in a fasting state between 08:00 and 10:00 AM. Two blood pressure readings, separated by intervals of at least 5 min, were taken with an appropriate-sized cuff wrapped around the right upper arm using a DINAMAP vital sign monitor (model 1846SX; Critikon Inc.). Subjects with a systolic blood pressure of ≥140 mm Hg or diastolic blood pressure of ≥90 mm Hg were defined as having hypertension. Brachial-ankle pulse wave velocity (baPWV) was assessed by a noninvasive vascular screening device (BP-203RPE II; Colin Medical Technology) with 4 pneumatic pressure cuffs that simultaneously measured BP and pulse waves in bilateral brachial and tibial arteries after 5 min of bed rest. BaPWV was calculated as the distance traveled by
the pulse wave divided by the time taken to travel the distance [25]. Mean baPWV was used for the final analysis, because of a significant positive correlation between left baPWV and right baPWV (r = 0.968, p b 0.001). Blood glucose was measured by a hexokinase method (Roche Diagnostic GmbH, Mannheim, Germany). Serum total cholesterol, triglycerides, and high density lipoprotein cholesterol (HDL cholesterol) concentrations (Cobas C HDLC3, Roche Diagnostics GmbH) were determined in the central laboratory of NCKUMC with an autoanalyzer (Hitachi 747E). Low density lipoprotein cholesterol (LDL cholesterol) was calculated using the Friedewald formula. A1C was measured with a high performance liquid chromatographic method (intra-assay CV of 0.5%, inter-assay CV of 2.0%). Serum insulin was measured by immunoassay (Mercodia AB; intra-assay CV of 4%, inter-assay CV of 2.6%). Insulin resistance (IR) was estimated by using the homeostasis model assessment (HOMA-IR) index defined as [fasting insulin (μU/ml) × fasting plasma glucose (mmol/l)] / 22.5. Serum fetuin-A was determined by an enzyme-linked immunosorbent assay (ELISA) method (Biovendor Laboratory Medicine, Czech Republic; intra-assay CV of 2.7%, inter-assay CV of 3.2%). The determination of serum adiponectin was carried out using AssayMax Human Adiponectin (Acrp30) ELISA kits (AssayPro, St. Charles, Missouri; intra-assay CV of 2.5%, inter-assay CV of 6.5%). High-sensitive C-reactive protein (hsCRP) was measured with a highly-sensitive ELISA kit (Immunology Consultants Laboratory, Newberg, OR; intra-assay CV of 2.9%, interassay CV of 4.7%). Subjects with the following conditions or diseases were excluded: 1) alcohol consumption ≥ 20 g/day in the last year; 2) serum aspartate aminotransferase (AST) or alanine aminotransferase (ALT) concentrations N 2 times the normal limit; 3) a positive test for hepatitis B surface antigen, hepatitis C antibody, and other causes of liver disease; 4) serum creatinine N 1.6 mg/dl; 5) any acute or chronic inflammatory disease as determined by a leukocyte count of N10,000/mm 3 or clinical signs of infection; 6) overt peripheral arterial disease (PAD) defined by an abnormal ankle-brachial index (ABI b 0.9), or participants with ABI N 1.4 reflecting noncompressible arteries and amputation of either lower limb; 7) a history of coronary heart disease and stroke; 8) those taking medications influencing blood pressure, plasma glucose, and lipid profile; and 9) any other major diseases, including generalized inflammation or advanced malignant diseases contraindicating this study. 2.1. Statistical analysis SPSS software (version 17.0; SPSS) was used for statistical analysis. All normally distributed continuous variables were expressed as means ± SD. Study subjects were divided into 4 groups: NGT, IFG, IGT, and NDD. The continuous variables among groups were compared using ANOVA (analysis of variance) or a Kruskal–Wallis test when the distribution was not normal. Post hoc (Bonferroni method) tests were used to evaluate pairwise differences among the adjusted means of serum fetuin-A concentration and baPWV. Multiple linear regression analysis was conducted to identify variables that best predicted baPWV, of which the variable selection strategies were stepwise and backward. The independent variables included age, sex, IFG vs. NGT, IGT vs. NGT, NDD vs. NGT, fetuin-A, general obesity, hypertension, smoking, exercise, hypertriglyceridemia, low-HDL cholesterol, creatinine, adiponectin, hsCRP, and HOMA-IR. A p b 0.05 was considered statistically significant. 3. Results A total of 2161 subjects (out of 3167 subjects admitted for a physical checkup) receiving a standard 75-g oral glucose tolerance test were screened. Among them, 139 with any conditions listed in the exclusion criteria were excluded (NGT, 95/1672; IFG, 5/64; IGT, 27/326; NDD, 12/99). Finally, 312 subjects were selected according to the selection
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protocol described above (NGT, 87/1577; IFG, 51/59; IGT, 87/299; NDD, 87/87). Table 1 shows the clinical characteristics of the study subjects. There were significant differences in WC, BMI, systolic/diastolic blood pressure, fasting and 2-h postload plasma glucose, A1C, ALT, AST, hsCRP, adiponectin, triglyceride, LDL cholesterol, HDL cholesterol, LDL/ HDL-cholesterol ratio, and HOMA-IR among groups. Both baPWV and serum fetuin-A concentrations increase along with the severity of glycemic status (Fig. 1). The mean values of baPWV were 1533 ± 338, 1518 ± 353, 1589 ± 307, and 1690 ± 414 cm/s in subjects with NGT, IFG, IGT, and NDD, respectively (p b 0.001, test for trend) (Fig. 1B), and the serum fetuin-A concentrations were 298 ± 69, 313 ± 67, 330 ± 86, and 342 ± 93 μg/ml, respectively (p b 0.001, test for trend) (Fig. 1C). Post hoc tests showed that subjects with NDD, but not prediabetes, exhibited a significantly greater arterial stiffness (p = 0.035) and higher fetuin-A concentrations (p = 0.002) than NGT subjects. The results of simple regression analysis show that baPWV concentrations were positively related to age (p b 0.001) and hypertension (p b 0.001) in the whole subjects and in each glycemic group (Table 2). Hypertriglyceridemia, hsCRP, and fetuin-A were also positively associated with baPWV concentrations in NGT, IGT, and NDD subjects, respectively. Table 3 shows the results of multiple regression analysis. baPWV concentrations were positively associated with age (all 4 groups), hypertension (IFG, IGT, and NDD groups), hypertriglyceridemia (NGT group), and fetuin-A concentrations (NDD group). In the whole subjects, both stepwise and backward selection strategies showed a consistent model. No multicollinearity was detected among the independent variables. We found that age (p b 0.001), NDD vs. NGT (p = 0.040), fetuin-A (p = 0.030), hypertension (p b 0.001), and hypertriglyceridemia (p = 0.039) were independently associated factors of baPWV concentrations after adjustment for age, sex, obesity, smoking, exercise, lipid profiles, adiponectin, CRP, and HOMA-IR.
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4. Discussion We found that both diabetes and fetuin-A concentrations, but not prediabetes, are independently associated with baPWV concentrations after adjusting for cardiometabolic risk factors, HOMA-IR, and adiponectin. Our findings suggest that the elevated fetuin-A concentrations may further aggravate increased arterial stiffness in diabetic patients. The associations between fetuin-A and CVD in subjects without CKD are inconsistent in previous studies. In a case-cohort study based on the general population, fetuin-A concentrations are positively related to incident myocardial infarction and ischemic stroke irrespective of diabetes status [14]. Using the Mendelian randomization approach, the same group provided strong evidence to suggest the causal involvement of fetuin-A in the pathogenesis of CVD [26]. However, in another prospective study on CVD mortality in older community-dwelling individuals, the positive association of fatal CVD with fetuin-A was observed only in diabetic subjects. In contrast, in subjects without diabetes, low plasma fetuin-A concentrations are independently associated with increased risk of CVD mortality [18]. The authors suggested that these findings imply that the balance between protecting against vascular calcification and promoting insulin resistance/metabolic derangement may depend on the metabolic milieu or prior disease processes. The discrepancies among the above studies could be attributable to differences in the ages of the study subjects, outcome measures, and different methods used to diagnose diabetes. In addition, studies have also demonstrated that fetuin-A is an important predictor of subclinical CVD [19–21,27]. In nondiabetic subjects at risk of coronary artery disease (CAD) [19] or with known or clinically suspected CAD [27], high serum fetuin-A concentrations are positively associated with greater carotid IMT, an early anatomical sign of atherosclerosis [19], while negatively associated with calcified CAD [27]. In addition, Fiore et al. reported that fetuin-A concentrations are associated
Table 1 Clinical characteristics among study subjects with normal glucose tolerance (NGT), impaired fasting glucose (IFG), impaired glucose tolerance (IGT), and newly diagnosed diabetes (NDD).
n Age (years) Sex (F/M) Waist circumference (cm) BMI (kg/m2) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Fasting plasma glucose (mg/dl) 2-h post-load plasma glucose (mg/dl) A1C (%) GOT (U/l) GPT (U/l) hsCRP (mg/l) Creatinine (mg/dl) Triglyceride (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl) LDL/HDL-cholesterol ratio Fetuin-A (μg/ml) Adiponectin (μg/ml) HOMA-IR baPWV (cm/s) General obesity Central obesity Hypertension Low-HDL Hypertriglyceridemia Regular exercise Smoking Data are expressed as means ± SD or number (%). p values are by ANOVA or χ2 test. NS: not significant.
NGT
IFG
IGT
NDD
p
87 62 ± 12 39/48 78.9 ± 8.9 22.2 ± 2.6 121 ± 17 71 ± 10 85 ± 7 95 ± 23 5.7 ± 0.3 26 ± 8 22 ± 9 2.08 ± 3.29 0.8 ± 0.2 96 ± 37 118 ± 31 59 ± 19 2.2 ± 1.0 298 ± 69 15 ± 13 0.41 ± 0.28 1533 ± 338 12 (14) 19 (22) 12 (14) 16 (18) 8 (9) 4 (5) 6 (7)
51 57 ± 12 19/32 83.3 ± 7.5 24.2 ± 2.5 125 ± 18 74 ± 11 104 ± 5 104 ± 22 5.9 ± 0.4 27 ± 9 27 ± 19 3.00 ± 5.00 0.9 ± 0.2 105 ± 48 126 ± 35 56 ± 15 2.4 ± 0.9 313 ± 67 13 ± 9 0.81 ± 0.59 1518 ± 353 18 (35) 17 (33) 11 (22) 8 (16) 9 (18) 5 (10) 3 (6)
87 62 ± 12 34/53 81.8 ± 8.5 23.4 ± 2.9 129 ± 17 74 ± 10 89 ± 11 162 ± 17 5.8 ± 0.3 25 ± 8 23 ± 11 3.00 ± 5.02 0.9 ± 0.2 119 ± 62 120 ± 34 53 ± 14 2.4 ± 0.9 330 ± 86 11 ± 6 0.53 ± 0.48 1589 ± 307 27 (31) 26 (30) 18 (21) 23 (26) 18 (21) 7 (8) 4 (5)
87 61 ± 11 39/48 82.1 ± 8.9 23.2 ± 3.1 132 ± 19 76 ± 11 137 ± 59 264 ± 89 7.3 ± 2.1 30 ± 43 32 ± 54 5.06 ± 9.30 0.9 ± 0.2 140 ± 104 133 ± 39 53 ± 14 2.6 ± 0.9 342 ± 93 12 ± 11 0.94 ± 0.85 1690 ± 414 23 (26) 27 (31) 24 (28) 20 (23) 29 (33) 2 (2) 6 (7)
– NS NS 0.015 0.001 0.002 0.008 b0.001 b0.001 b0.001 NS NS 0.002 NS b0.001 0.035 0.020 0.047 0.002 NS b0.001 0.011 0.02 NS NS NS 0.001 NS NS
136
baPWV (cm/sec)
A
B
2200
2000
1800
1600
1400
1200
1000
0
\\
\\
NGT
NGT
P < 0.001 test for trend.
450
C
400
350
300
250
200
0
P < 0.001 test for trend.
P = 0.035
P = 0.002
P = 0.032
IGT
IGT
Fetuin-A (µg/ml)
IFG
P = 0.047
IFG
Table 2 Simple regression analysis between brachial-ankle pulse wave velocity (baPWV) and clinical variables. NGT β Age (years) Sex, male vs. female General obesity IFG vs. NGT IGT vs. NGT NDD vs. NGT Fetuin-A (μg/ml) Hypertension, yes vs. no Regular exercise, yes vs. no Smoking, yes vs. no Low-HDL cholesterol, yes vs. no Hypertriglyceridemia, yes vs. no Creatinine (μmol/l) Adiponectin (μg/ml) hsCRP (mg/l) HOMA-IR
17.302⁎ −59.280 53.878 – – – −0.437 352.192⁎ −252.321 93.790 51.157 293.589† −1.851 3.124 19.377 190.119
IFG 95% CI
β
12.504–22.100 −204.262–85.702 −155.707–263.464
16.948⁎
−1.490–0.616 156.508–547.876 −593.615–88.973 −191.141–378.721 −135.353–237.667 51.233–535.945 −6.478–2.775 −2.653–8.900 −2.380–41.134 −61.989–442.227
81.271 −67.601 – – – −0.408 447.384⁎ −31.620 −153.198 −149.836 −93.524 2.927 11.526 8.705 −66.766
IGT 95% CI
β
9.313–24.583 −125.172–287.713 −276.894–141.692
11.499⁎
−1.953–1.136 239.664–655.104 −369.285–306.046 −577.827–271.431 −422.669–122.997 −355.641–168.593 −2.510–8.364 −0.146–22.907 −30.073–47.483 −242.377–108.845
−56.012 73.769 – – – −0.075 309.628⁎ −65.558 −71.873 87.084 −34.866 0.837 9.905 19.990† 86.713
NDD 95% CI
β
6.817–16.181 −190.227–78.203 −67.463–215.002
17.320⁎
−0.840–0.690 161.279–457.977 −306.878–175.762 −385.442–241.695 −60.829–234.996 −197.011–127.279 −3.276–4.949 −0.119–19.929 2.771–37.209 −50.822–224.248
3.978 38.450 -– – – 1.161† 409.159⁎ −324.724 −144.383 −150.443 −30.388 3.320 5.630 −0.032 35.576
All subjects 95% CI 10.417–24.222 −174.585–182.540 −162.749–239.649
0.234–2.088 232.105–586.212 −913.119–263.672 −493.450–204.684 −358.990–58.103 −218.654–157.878 −1.360–8.000 −2.338–13.599 −9.639–9.575 −69.378–140.529
β
95% CI 15.362⁎
−21.910 35.114 −86.205 −1.475 138.841# 0.416 388.528⁎ −150.747 −53.244 −13.189 41.978 1.626 5.045 6.783 60.672
NGT: normal glucose tolerance; IFG: impaired fasting glucose; IGT: impaired glucose tolerance; NDD: newly diagnosed diabetes; HDL, high density lipoprotein; triglyceride had been log transformed before analysis. Dependent variable: brachial-ankle pulse wave velocity (baPWV). † p b 0.05. # p b 0.01. ⁎ p b 0.001.
12.454–18.271 −103.255–59.435 −56.786–127.015 −194.394–21.984 −91.043–88.092 50.627–227.055 −0.071–0.903 299.692–477.364 −322.184–20.691 −221.093–114.606 −110.988–84.610 −57.380–141.336 −0.705–3.958 −0.128–8.955 −0.155–13.721 −3.680–125.023
H.-Y. Ou et al. / Clinica Chimica Acta 445 (2015) 133–138
P = NS
NDD
NDD
Fig. 1. Distribution of brachial-ankle pulse wave velocity (baPWV) and serum fetuin-A (A), and comparison of brachial-ankle pulse wave velocity (B) and serum fetuin-A (C) among subjects with normal glucose tolerance (NGT), impaired fasting glucose (IFG), impaired glucose tolerance (IGT), and newly diagnosed diabetes (NDD). Data are shown as mean ± SD. NS, not significant.
with IMT in patients with atherosclerosis of peripheral vessels (n = 90; 51% diabetes) after adjustment for diabetes and other cofounding variables [20]. Furthermore, in a population sample of community-dwelling subjects who were free of clinical CVD, Ix et al. observed that lower fetuin-A concentrations are independently associated with greater coronary artery calcification severity, but not carotid IMT [21]. Notably, no modifications of effects related to diabetes have been reported in
baPWV (cm/sec)
Fetuin-A (µg/mL)
0.237–1.961 119.236–486.954 −810.538–383.748 −406.799–237.824 −308.364–77.725 −84.125–287.003 −5.96–4.259 −2.603–14.104 −8.746–8.859 −139.575–57.498 −0.7–0.661 125.318–421.669 −330.886–93.526 −366.791–190.716 −136.3–170.934 −164.697–161.033 −4.023–5.002 −5.266–19.539 −2.259–30.319 −42.367–219.865 −1.177–1.745 138.359–617.19 −465.688–221.724 −235.159–659.839 −236.517–353.838 −325.059–192.049 −8.18–11.043 −1.773–20.056 −71.176–3.932 −158.235–149.671 −1.264–0.441 −108.938–300.581 −410.22–139.669 −199.024–274.191 −141.986–157.015 78.766–482.678 −7.745–2.115 −7.516–2.468 −16.01–24.145 −132.995–293.261
NGT: normal glucose tolerance; IFG: impaired fasting glucose; IGT: impaired glucose tolerance; NDD: newly diagnosed diabetes; HDL, high density lipoprotein; triglyceride had been log transformed before analysis. Dependent variable: brachial-ankle pulse wave velocity (baPWV). † p b 0.05. # p b 0.01. ⁎ p b 0.001.
10.367–16.489 −76.808–100.554 −72.902–85.895 −68.621–144.746 −69.064–109.472 4.726–194.635 0.045–0.860 177.832–348.061 −231.307–47.435 −161.533–118.143 −121.310–43.263 4.653–180.157 −1.843–3.107 −1.790–5.041 −5.642–6.137 −72.957–44.433 13.428⁎ 11.873 6.496 38.062 20.204 99.6814† 0.453† 262.947⁎ −91.936 −21.695 −39.023 92.405† 0.632 1.625 0.248 −14.262
β 95% CI
6.23–21.678 −121.173–269.363 −159.164–213.572
13.954⁎ 74.095 27.204 – – – 1.099† 303.095# −213.395 −84.488 −115.320 101.439 −0.850 5.750 0.056 −41.038
β 95% CI
1.523–12.925 −139.855–181.983 −115.88–155.154
7.224† 21.064 19.637 – – – −0.019 273.493⁎ −118.680 −88.037 17.317 −1.832 0.489 7.137 14.030 88.749
β 95% CI
5.937–27.318 −250.293–463.53 −141.613–243.083
16.628# 106.618 50.735 – – – 0.284 377.774# −121.982 212.340 58.660 −66.505 1.431 9.142 −33.622 −4.282
β
11.104–21.756 −139.792–176.468 −180.554–191.962
95% CI β
16.430⁎ 18.338 5.704 – – – −0411 95.822 −135.275 37.584 7.514 280.722# −2.815 −2.524 4.068 80.133
NDD IGT IFG NGT
Table 3 Multiple regression analysis between brachial-ankle pulse wave velocity (baPWV) and clinical variables.
Age (years) Sex, male vs. female General obesity IFG vs. NGT IGT vs. NGT NDD vs. NGT Fetuin-A (μg/ml) Hypertension, yes vs. no Regular exercise, yes vs. no Smoking, yes vs. no Low-HDL cholesterol, yes vs. no Hypertriglyceridemia, yes vs. no Creatinine (μmol/l) Adiponectin (μg/ml) hsCRP (mg/l) HOMA-IR
All subjects
95% CI
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the associations of fetuin-A concentrations and these measures of subclinical CVD [21]. The current study extends the relationship between fetuin-A and subclinical CVD to the early functional vascular changes of CVD. Recently, Jung et al. reported a positive correlation between fetuin-A and arterial stiffness in diabetic subjects [23], whereas Yang et al. showed a negative association in nondiabetic, obese women in a small-scaled study (n = 40) [24]. Here, we further find that the effect of fetuin-A on arterial stiffness is independent of age, hypertension, diabetes, insulin resistance, and traditional cardiometabolic risk factors. Arterial stiffness is a functional assessment of vascular damage caused by CVD risk factors. Increased arterial stiffness, which is usually measured by the PWV, causes increased cardiac afterload, a decrease in coronary perfusion, atherogenesis and/or microvascular damage [25]. PWV has thus been shown to be a predictor of future cardiovascular events in the general population [28], hypertensive subjects [29], and those with type 2 diabetes [30]. Increased baPWV is associated with older age, higher blood pressure, BMI, IGT, and diabetes [31]. In the current study, we found that NDD subjects had higher baPWV concentrations than NGT, IFG, and IGT subjects. Although the IFG group seems to have the lowest baPWV among 4 groups, the small difference did not reach statistical significance when compared with NGT subjects. Thus, in the final statistical analysis, only the differences of NDD vs. NGT and NDD vs. IFG were significant. The mechanisms leading to arterial stiffness are not the same among different CVD risk factors, and may involve the dysregulation of balance between extracellular matrix collagen and elastin, and deposition of glycoproteins and proteoglycans. Hypertension per se causes thinning, splitting and fragmentation of elastic fibers [25]. In diabetes, the formation of advanced glycation end-products (AGEs) on the arterial wall causes cross-linking of collagen molecules, which may lead to loss of collagen elasticity and a subsequent increase in arterial stiffness [32]. On the other hand, arterial stiffening with aging is associated with transforming growth factor-β1-related increases in adventitial collagen and reductions in medial elastin [33]. In addition to age, hypertension, and NDD, the current study also shows that fetuin-A is an independently associated factor of arterial stiffness. This result is consistent with that of Mori et al. in 141 nondiabetic subjects, showing that besides age, fetuin-A independently contributes to carotid artery stiffness [22]. Although both fetuin-A and baPWV increase due to glycemia, the results of this work show that fetuin-A and diabetes have independent associations with arterial stiffness in subjects with different glycemic statuses. While the mechanisms on how fetuin-A can regulate arterial stiffness remain unclear, Mori et al. postulated a plausible explanation of this [22]. As fetuin-A and type II TGFβ-receptors share a sequence homology at the major cytokine binding site, fetuin-A can inhibit type II receptor mediated signaling [34]. Therefore, by decreasing the type II/type I TGFβ-receptor ratio, vascular smooth muscle cells overproduce collagen and extracellular matrix [35], which leads to greater arterial stiffness [33]. This work has the following limitations. First, since this study used a cross-sectional design, it does not allow causal inferences between serum fetuin-A concentrations and the development of arterial stiffness. Second, arterial stiffness was assessed by brachial-ankle PWV in this study, but not the carotid–femoral PWV [35]. Although carotid–femoral PWV is still considered as the gold standard in measuring central arterial stiffness, a high concentration of skill and exposure of the inguinal region are required for its measurement, which limits its applicability in clinical practice. However, one previous study showed that baPWV correlates significantly with carotid–femoral PWV, and provides qualitatively similar information to that derived from central arterial stiffness [36]. Finally, the study subjects were confined to a Chinese population, and the findings may not be generalizable to other ethnicities. In conclusion, in this work we demonstrate that both baPWV and serum fetuin-A concentrations increase along with the severity of
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glycemic status. Subjects with NDD, but not prediabetes, exhibit significantly greater arterial stiffness and higher fetuin-A concentrations than NGT subjects. Diabetes and fetuin-A concentrations are associated with baPWV independent of age, sex, hypertension, hypertriglyceridemia, and other confounding factors. However, further studies are needed to investigate the possible mechanisms underlying the action of fetuin-A on vascular wall functioning. Acknowledgments This study was supported by grants from the National Science Council Taiwan (NSC-94-2314-B-006-114, NSC-95-2314-B-006-048, and NSC99-2314-B-006-049-MY3) and National Cheng Kung University Hospital (NCKUH-9501005, NCKUH-9702005, and NCKUH-9903034). References [1] Denecke B, Graber S, Schafer C, Heiss A, Woltje M, Jahnen-Dechent W. Tissue distribution and activity testing suggest a similar but not identical function of fetuin-B and fetuin-A. Biochem J 2003;376:135–45. [2] Srinivas PR, Wagner AS, Reddy LV, Deutsch DD, Leon MA, Goustin AS, et al. Serum alpha 2-HS-glycoprotein is an inhibitor of the human insulin receptor at the tyrosine kinase level. Mol Endocrinol 1993;7:1445–55. [3] Pal D, Dasgupta S, Kundu R, Maitra S, Das G, Mukhopadhyay S, et al. Fetuin-A acts as an endogenous ligand of TLR4 to promote lipid-induced insulin resistance. Nat Med 2012;18:1279–85. [4] Mathews ST, Singh GP, Ranalletta M, Cintron VJ, Qiang X, Goustin AS, et al. Improved insulin sensitivity and resistance to weight gain in mice null for the Ahsg gene. Diabetes 2002;51:2450–8. [5] Stefan N, Hennige AM, Staiger H, Machann J, Schick F, Krober SM, et al. Alpha2Heremans-Schmid glycoprotein/fetuin-A is associated with insulin resistance and fat accumulation in the liver in humans. Diabetes Care 2006;29:853–7. [6] Ix JH, Shlipak MG, Brandenburg VM, Ali S, Ketteler M, Whooley MA. Association between human fetuin-A and the metabolic syndrome: data from the Heart and Soul Study. Circulation 2006;113:1760–7. [7] Mori K, Emoto M, Yokoyama H, Araki T, Teramura M, Koyama H, et al. Association of serum fetuin-A with insulin resistance in type 2 diabetic and nondiabetic subjects. Diabetes Care 2006;29:468. [8] Ishibashi A, Ikeda Y, Ohguro T, Kumon Y, Yamanaka S, Takata H, et al. Serum fetuin-A is an independent marker of insulin resistance in Japanese men. J Atheroscler Thromb 2010;17:925–33. [9] Ou HY, Yang YC, Wu HT, Wu JS, Lu FH, Chang CJ. Serum fetuin-A concentrations are elevated in subjects with impaired glucose tolerance and newly diagnosed type 2 diabetes. Clin Endocrinol (Oxf) 2011;75:450–5. [10] Schafer C, Heiss A, Schwarz A, Westenfeld R, Ketteler M, Floege J, et al. The serum protein alpha 2-Heremans-Schmid glycoprotein/fetuin-A is a systemically acting inhibitor of ectopic calcification. J Clin Invest 2003;112:357–66. [11] Jahnen-Dechent W, Heiss A, Schäfer C, Ketteler M. Fetuin-A regulation of calcified matrix metabolism. Circ Res 2011;108:1494–509. [12] Wang A, Woo J, Lam C, Wang M, Chan I, Gao P, et al. Associations of serum fetuin-A with malnutrition, inflammation, atherosclerosis and valvular calcification syndrome and outcome in peritoneal dialysis patients. Nephrol Dial Transplant 2005; 20:1676–85. [13] Moe S, Reslerova M, Ketteler M, O'Neill K, Duan D, Koczman J, et al. Role of calcification inhibitors in the pathogenesis of vascular calcification in chronic kidney disease (CKD). Kidney Int 2005;67:2295–304. [14] Weikert C, Stefan N, Schulze MB, Pischon T, Berger K, Joost HG, et al. Plasma fetuin-a levels and the risk of myocardial infarction and ischemic stroke. Circulation 2008; 118:2555–62.
[15] Ketteler M, Bongartz P, Westenfeld R, Wildberger J, Mahnken A, Böhm R, et al. Association of low fetuin-A (AHSG) concentrations in serum with cardiovascular mortality in patients on dialysis: a cross-sectional study. Lancet 2003;361:827–33. [16] Stenvinkel P, Wang K, Qureshi A, Axelsson J, Pecoits-Filho R, Gao P, et al. Low fetuinA levels are associated with cardiovascular death: impact of variations in the gene encoding fetuin. Kidney Int 2005;67:2383–92. [17] Hermans M, Brandenburg V, Ketteler M, Kooman J, van der Sande F, Boeschoten E, et al. Association of serum fetuin-A levels with mortality in dialysis patients. Kidney Int 2007;72:202–7. [18] Laughlin G, Cummins K, Wassel C, Daniels L, Ix J. The association of fetuin-A with cardiovascular disease mortality in older community-dwelling adults: the Rancho Bernardo study. J Am Coll Cardiol 2012;59:1688–96. [19] Rittig K, Thamer C, Haupt A, Machann J, Peter A, Balletshofer B, et al. High plasma fetuin-A is associated with increased carotid intima-media thickness in a middleaged population. Atherosclerosis 2009;207:341–2. [20] Fiore C, Celotta G, Politi G, Di Pino L, Castelli Z, Mangiafico R, et al. Association of high alpha2-Heremans-Schmid glycoprotein/fetuin concentration in serum and intimamedia thickness in patients with atherosclerotic vascular disease and low bone mass. Atherosclerosis 2007;195:110–5. [21] Ix J, Barrett-Connor E, Wassel C, Cummins K, Bergstrom J, Daniels L, et al. The associations of fetuin-A with subclinical cardiovascular disease in community-dwelling persons: the Rancho Bernardo Study. J Am Coll Cardiol 2011;58:2372–9. [22] Mori K, Emoto M, Araki T, Yokoyama H, Teramura M, Lee E, et al. Association of serum fetuin-A with carotid arterial stiffness. Clin Endocrinol (Oxf) 2007;66: 246–50. [23] Jung CH, Kim BY, Kim CH, Kang SK, Jung SH, Mok JO. Associations of serum fetuin-A levels with insulin resistance and vascular complications in patients with type 2 diabetes. Diab Vasc Dis Res 2013;10:459–67. [24] Yang SJ, Hong HC, Choi HY, Yoo HJ, Cho GJ, Hwang TG, et al. Effects of a three-month combined exercise program on fibroblast growth factor 21 and fetuin-A levels and arterial stiffness in obese women. Clin Endocrinol (Oxf) 2011;75:464–9. [25] Tomiyama H, Yamashina A. Non-invasive vascular function tests: their pathophysiological background and clinical application. Circ J 2010;74:24–33. [26] Fisher E, Stefan N, Saar K, Drogan D, Schulze M, Fritsche A, et al. Association of AHSG gene polymorphisms with fetuin-A plasma levels and cardiovascular diseases in the EPIC-Potsdam study. Circ Cardiovasc Genet 2009;2:607–13. [27] Mori K, Ikari Y, Jono S, Emoto M, Shioi A, Koyama H, et al. Fetuin-A is associated with calcified coronary artery disease. Coron Artery Dis 2010;21:281–5. [28] Willum-Hansen T, Staessen J, Torp-Pedersen C, Rasmussen S, Thijs L, Ibsen H, et al. Prognostic value of aortic pulse wave velocity as index of arterial stiffness in the general population. Circulation 2006;113:664–70. [29] Boutouyrie P, Tropeano A, Asmar R, Gautier I, Benetos A, Lacolley P, et al. Aortic stiffness is an independent predictor of primary coronary events in hypertensive patients: a longitudinal study. Hypertension 2002;39:10–5. [30] Cruickshank K. Aortic pulse-wave velocity and its relationship to mortality in diabetes and glucose intolerance: an integrated index of vascular function? Circulation 2002;106:2085–90. [31] Li CH, Wu JS, Yang YC, Shih CC, Lu FH, Chang CJ. Increased arterial stiffness in subjects with impaired glucose tolerance and newly diagnosed diabetes but not isolated impaired fasting glucose. J Clin Endocrinol Metab 2012;97:E658-62. [32] Aronson D. Cross-linking of glycated collagen in the pathogenesis of arterial and myocardial stiffening of aging and diabetes. J Hypertens 2003;21:3–12. [33] Fleenor B, Marshall K, Durrant J, Lesniewski L, Seals D. Arterial stiffening with ageing is associated with transforming growth factor-β1-related changes in adventitial collagen: reversal by aerobic exercise. J Physiol 2010;588:3971–82. [34] Demetriou M, Binkert C, Sukhu B, Tenenbaum H, Dennis J. Fetuin/alpha2-HS glycoprotein is a transforming growth factor-beta type II receptor mimic and cytokine antagonist. J Biol Chem 1996;271:12755–61. [35] McCaffrey T, Consigli S, Du B, Falcone D, Sanborn T, Spokojny A, et al. Decreased type II/type I TGF-beta receptor ratio in cells derived from human atherosclerotic lesions. Conversion from an antiproliferative to profibrotic response to TGF-beta1. J Clin Invest 1995;96:2667–75. [36] Sugawara J, Hayashi K, Yokoi T, Cortez-Cooper MY, DeVan AE, Anton MA, et al. Brachial-ankle pulse wave velocity: an index of central arterial stiffness? J Hum Hypertens 2005;19:401–6.
