total adiponectin ratio correlates with coronary artery disease in Japanese type 2 diabetics

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Available online at www.sciencedirect.com

Metabolism www.metabolismjournal.com

High serum C1q-adiponectin/total adiponectin ratio correlates with coronary artery disease in Japanese type 2 diabetics Ayumu Hirata a , Ken Kishida a,⁎, Hideaki Nakatsuji a , Hironori Kobayashi b , Tohru Funahashi a, c , Iichiro Shimomura a a b c

Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan Department of Research and Development, Diagnostic Division, Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan Department of Metabolism and Atherosclerosis, Graduate School of Medicine, Osaka University, Osaka, Japan

A R T I C LE I N FO Article history:

AB S T R A C T Objective. Adiponectin, an adipocyte-derived protein, has potential antiatherogenic

Received 23 August 2012

properties. Low levels of serum total-adiponectin (Total-APN) correlate with diabetes and

Accepted 18 October 2012

coronary artery disease (CAD). Adiponectin and C1q form a protein complex in blood, and serum C1q-binding adiponectin (C1q-APN) can be measured. We investigated the

Keywords: Adiponectin

correlation between C1q-APN and CAD in patients with type 2 diabetes mellitus (T2DM). Methods. The study subjects were 107 outpatients with T2DM who underwent evaluation

C1q

for CAD. Blood C1q, Total-APN, high-molecular weight-adiponectin (HMW-APN) and C1q-

C1q-binding adiponectin

APN were measured by enzyme-linked immunosorbent assays.

Coronary artery disease

Results. Serum levels of C1q-APN/Total-APN ratio were higher in patients diagnosed with CAD (10.47 ± 0.59, mean ± SEM, n = 54) than those without CAD (8.88 ± 0.60, n = 53, p = 0.0482). Age- and sex-adjusted logistic regression analysis identified serum C1q-APN/Total-APN ratio and hypertension as significant and independent determinants of CAD. A high serum C1q-APN/Total-APN ratio was associated with 3.965-fold increase in CAD prevalence. Conclusions. High serum C1q-APN/Total-APN ratio correlates with CAD in T2DM. © 2013 Elsevier Inc. All rights reserved.

1.

Introduction

Adiponectin is an adipocyte-derived plasma protein [1] present abundantly in injured arteries [2,3] and atherosclerotic lesions [4]. It has anti-atherogenic and insulin-sensitizing properties [5]. Previous studies demonstrated a close relationship between low circulating levels of total-adiponectin (Total-APN, i.e., hypoadiponectinemia, < 4 μg/mL) and type 2 diabetes mellitus (T2DM) [6] and coronary artery disease (CAD) [7–10]. Adiponectin circulates in blood mainly in three forms: trimer, hexamer, and high-molecular weight (HMW)

form [11]. However, the main form of blood adiponectin remains to be elucidated. We recently reported that adiponectin forms a protein-complex with C1q in human blood, and introduced measurement of human serum C1q-binding adiponectin (C1q-APN), and serum C1q-APN/Total-APN ratio as a novel marker of the metabolic syndrome [12]. We also reported recently that serum C1q-APN/Total-APN ratio correlates with polyvascular lesions detected by vascular ultrasonography [13]. The activated complement system plays a role in atherosclerosis [14–17]. These results suggest that adiponectin may have a protective role in activated complement

Abbreviations: CAD, coronary artery disease; C1q-APN, C1q-binding adiponectin; ELISA, enzyme-linked immunosorbent assay; eVFA, estimated visceral fat area; HMW, high-molecular weight; HMW-APN, high-molecular weight-adiponectin; MDCT, multi-detector row computed tomography; Total-APN, total-adiponectin; T2DM, type 2 diabetes mellitus. ⁎ Corresponding author. Tel.: +81 6 6879 3732; fax: + 81 6 6879 3739. E-mail address: [email protected] (K. Kishida). 0026-0495/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.metabol.2012.10.011

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system in the development of atherosclerosis. What proportion of tissue damage-defensive adiponectin forms protein complex with tissue damage-offensive C1q remains unclear. Analysis of this adiponectin complex, in addition to TotalAPN, is likely to enhance our understanding of the pathogenesis of CAD. The aim of the present study was to determine the role of serum C1q-APN in the pathogenesis of CAD in T2DM patients.

2.

Patients and methods

2.1.

Participants

The study (ADMIT study; UMIN 000002271) subjects were 107 consecutive Japanese patients with T2DM, who visited the outpatient clinic of “Diabetes & Metabolic Station”, Osaka University Hospital, between September 2009 and September 2011. Patients treated with pioglitazone, which is known to increase serum levels of Total-APN and C1q-APN in T2DM [18,19] and those with renal dysfunction [estimated glomerular filtration rate (eGFR) < 30 mL/min] [20] were excluded from the study. The Medical Ethics Committee of Osaka University approved the study. Each participant gave a written informed consent.

2.2.

579

Brinkman index represented the daily number of cigarettes × years of smoking. The eGFR was calculated using the simplified Modification of Diet in Renal Disease (MDRD) equation, modified by the appropriate coefficient for Japanese populations by gender, as described previously [23].

2.3.

Definition of CAD

The definition of CAD has been published elsewhere [22]. Briefly, CAD was defined as follows: 1) intracoronary lesion identified as atherosclerotic stenosis of at least one segment of a major coronary artery, which was a candidate for revascularization, evaluated with multi-detector row computed tomography (MDCT) or coronary angiography, according to the guidelines of the American Heart Association(n = 47/54), or 2) horizontal or down-sloping ST-segment depression (≥ 1 mm) on stress electrocardiography, such as ergometry or double master's two-step test, or ischemic lesion on myocardial scintigraphy defined as perfusion defect of > 5% of the total left ventricular area in the bull's-eye map (n = 7/54). These patients did not undergo MDCT or coronary angiography due to renal insufficiency or allergic reaction to the contrast medium. Two experienced invasive cardiologists evaluated the above features of CAD. Patients with negative results by either of the above tests were considered to be CAD-free.

Anthropometry and laboratory tests 2.4.

Anthropometric variables [height, weight and waist circumference (WC)] were measured in the standing position. Visceral fat area was estimated by the bioelectrical impedance analysis method (eVFA), as reported previously by our group [21]. Systolic and diastolic blood pressures (SBP, DBP) were measured with a standard mercury sphygmomanometer on the left or right arm in the supine position after at least 10-min rest. Venous blood samples were collected in the morning after overnight fast for measurements of serum creatinine, lipids, glucose, and HbA1c (Japan Diabetes Society [JDS]). The value of HbA1c (%) was estimated as the National Glycohemoglobin Standardization Program (NGSP) equivalent value (%), calculated by the formula HbA1c (%) = HbA1c (JDS, %) + 0.4%. For the purpose of the present study, serum samples that were obtained at baseline from each participant were stored promptly at − 20 °C. After thawing the samples, serum levels of Total-APN and high-molecular weight-adiponectin (HMWAPN) were measured by enzyme-linked immunosorbent assay (ELISA) (Human adiponectin ELISA kit, Human HMWadiponectin ELISA kit, Otsuka Pharmaceutical Co. Tokushima, Japan) [1,11]. Serum levels of C1q-APN and C1q were measured by our handmade ELISA, as reported previously by our group [12]. The intra- and inter-coefficients of variation (CV) for C1q-APN ELISA are below 4.6% and 6.7%, respectively. The intra- and inter-CV for C1q ELISA are below 4.6% and 5.0%, respectively. Urine albumin–creatinine ratio (UACR) was calculated from a single spot urine specimen collected in the morning. Diabetic nephropathy (≥ stage 2), diabetic retinopathy (≥simple) and diabetic peripheral neuropathy were diagnosed according to the criteria defined in our previous study [22].

Statistical analysis

Data are presented as mean ± SEM. Data of the CAD (+) and CAD (−) groups were compared by the Student's t-test or the Mann–Whitney test. Differences in frequencies were examined by the χ2 test. Relationships between two continuous variables were analyzed using scatter plots and Pearson's correlation coefficient. The correlations between CAD and various serum adiponectin parameters were first analyzed by simple regression analysis (Model; non-adjusted, Model; ageand sex-adjusted). The age- and sex-adjusted odds ratios (ORs) are presented together with 95% confidence intervals (95% CI). The receiver operating characteristic (ROC) curve analysis was performed to determine the appropriate cutoff value for each adiponectin parameter and C1q in identifying subjects with CAD. In all cases, p values < 0.05 were considered statistically significant. All analyses were performed with the JMP Statistical Discovery Software 9.0 (SAS Institute, Cary, NC) or the Statistical Package for Social Sciences (version 11.0.1 J; SPSS, Chicago, IL).

3.

Results

3.1.

Characteristics of T2DM patients

Table 1 summarizes the characteristics of the study subjects. C1q-APN correlated significantly and positively with TotalAPN in all, males and females (Fig. 1A). There was no significant correlation between C1q-APN and C1q in all, males and females (Fig. 1B). C1q-APN correlated significantly and negatively with Total-APN in all and females, but not in males (Fig. 1C). C1q-APN/Total-APN correlated significantly

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Table 1 – Baseline characteristics of diabetic subjects enrolled in the present study.

Age, years Sex [males/females] Body mass index, kg/m2 Waist circumference, cm (males) Waist circumference, cm (females) eVFA, cm2 (males) eVFA, cm2 (females) Smoking (none-/ex-/current-smoker) Brinkman index Systolic blood pressure, mmHg Diastolic blood pressure, mmHg Duration of diabetes mellitus, years Diabetic neuropathy Diabetic retinopathy (NDR/SDR/PDR) Diabetic nephropathy Hypertension Dyslipidemia Metabolic Syndrome Past history (CAD/CVD) Fasting blood glucose, mg/dL Hemoglobin A1c (NGSP), % UACR, mg/g creatinine Estimated glomerular filtration rate, mL/min Total cholesterol, mg/dL Low-density lipoprotein-cholesterol, mg/dL Triglyceride, mg/dL High-density lipoprotein-cholesterol, mg/dL Serum APN, μg/mL (all) APN, μg/mL (males) APN, μg/mL (females) Serum HMW-APN, μg/mL (all) HMW-APN, μg/mL (males) HMW-APN, μg/mL (females) Serum C1q-APN, U/mL (all) C1q-APN, U/mL (males) C1q-APN, U/mL (females) Serum C1q, μg/mL (all) C1q, μg/mL (males) C1q, μg/mL (females) Serum HWM-APN/Total-APN (all) HWM-APN/Total-APN (males) HWM-APN/Total-APN (females) Serum C1q-APN/Total-APN (all) C1q-APN/Total-APN (males) C1q-APN/Total-APN (females) Serum C1q-APN/HMW-APN (all) C1q-APN/HMW-APN (males) C1q-APN/HMWl-APN (females) Serum Total-APN/C1q (all) Total-APN/C1q (males) Total-APN/C1q (females) Serum C1q-APN/C1q (all) C1q-APN/C1q (males) C1q-APN/C1q (females)

CAD (−) group

CAD (+) group

p value

63 ± 1 (40–80) n = 53 [24/29] 24.6 ± 0.4 (19.8–32.7) 90.1 ± 1.3 (78–107) 85.5 ± 1.2 (71–100) 150.1 ± 9.2 (75–238) 91.2 ± 7.2 (40–208) n = 35/13/5 440 ± 81 (0–1800) 132 ± 4 (103–171) 77 ± 1 (53–100) 9.9 ± 1.3 (1–30) n = 10 n = 47/4/2 n = 13 n = 22 n = 37 n = 16 n = 31/2 132 ± 3 (89–260) 7.1 ± 0.1 (5.9–10.6) 188.1 ± 111.0 (0.5–3958) 69.6 ± 2.32 (34.4–114.0) 196 ± 5 (138–256) 112 ± 4 (66–153) 141 ± 7 (49–396) 56 ± 2 (31–108) 9.4 ± 0.6 (2.8–29.7) 8.1 ± 0.8 (2.8–21.5) 10.5 ± 1.0 (3.7–29.7) 7.4 ± 0.8 (0.7–44.5) 6.0 ± 1.0 (1.4–28.6) 8.6 ± 1.4 (0.7–44.5) 68.3 ± 3.5 (31.9–159.1) 64.9 ± 5.4 (31.9–159.1) 71.2 ± 4.4 (36.1–127.0) 52.2 ± 1.4 (35.0–81.8) 48.8 ± 2.0 (35.0–62.7) 55.0 ± 1.9 (36.9–81.8) 0.72 ± 0.05 (0.07–3.21) 0.64 ± 0.24 (0.24–1.33) 0.79 ± 0.10 (0.07–3.21) 8.88 ± 0.60 (2.73–22.90) 9.33 ± 0.81 (2.74–18.40) 8.51 ± 0.80 (2.73–22.90) 15.81 ± 2.03 (0.83–67.30) 16.27 ± 3.14 (2.92–42.22) 15.43 ± 2.65 (0.83–67.30) 7.73 ± 0.77 (1.25–18.80) 7.98 ± 0.89 (2.66–18.80) 7.52 ± 0.90 (1.25–18.70) 1.34 ± 0.07 (0.59–2.92) 1.34 ± 0.13 (0.71–2.46) 1.35 ± 0.09 (0.59–2.92)

69 ± 1 (52–85) n = 54 [38/16] 25.1 ± 0.5 (19.1–36.4) 90.1 ± 1.3 (65–120.5) 85.5 ± 1.2 (70–109.5) 145.4 ± 7.3 (50–234) 107.2 ± 9.5 (58–202) n = 35/12/7 717 ± 80 (0–2760) 132 ± 4 (105–180) 75 ± 1 (48–111) 14.7 ± 1.3 (3–36) n = 21 n = 36/6/12 n = 25 n = 42 n = 44 n = 40 n = 31/2 132 ± 3 (83–221) 7.3 ± 0.1 (5.4–10.2) 232.7 ± 102.4 (2.1–4268) 67.4 ± 2.30 (34.4–114.0) 191 ± 5 (120–335) 110 ± 4 (57–236) 141 ± 7 (41–420) 51 ± 2 (35–84) 7.2 ± 0.6 (1.9–20.7) 7.0 ± 0.7 (1.9–20.7) 7.6 ± 1.3 (3.3–13.1) 5.5 ± 0.8 (0.4–16.4) 5.2 ± 0.8 (0.4–16.4) 6.1 ± 1.9 (1.3–14.0) 64.5 ± 3.4 (35.8–147.1) 61.5 ± 4.3 (35.8–108.3) 71.7 ± 6.0 (36.3–147.1) 54.0 ± 1.4 (37.4–99.1) 53.6 ± 1.6 (37.4–99.1) 54.9 ± 2.5 (41.2–65.7) 0.71 ± 0.05 (0.18–1.88) 0.71 ± 0.36 (0.18–1.88) 0.71 ± 0.26 (0.28–1.14) 10.47 ± 0.59 (4.55–21.80) 10.47 ± 0.79(4.55–21.80) 10.48 ± 1.02 (4.72–18.80) 19.16 ± 2.05 (3.14–148.7) 19.76 ± 2.53(9.35–148.7) 17.67 ± 3.68 (3.14–48.02) 10.06 ± 0.77 (1.86–29.90) 10.54 ± 1.16 (1.86–29.9) 8.96 ± 1.29 (4.49–19.80) 1.20 ± 0.07 (0.50–2.46) 1.16 ± 0.08 (0.50–2.46) 1.30 ± 0.10 (0.81–2.39)

0.0162a 0.0086 0.4124a 0.8013 0.0946 0.6911a 0.1864a 0.8336 0.0168a 0.9668a 0.3754 0.0191a 0.0268 0.0199 0.0186 <0.0001 0.1093 <0.0001 0.0001 0.9668a 0.4263 0.0154a 0.5816 0.4714 0.7244 0.4185a 0.0551 0.0784a 0.2972a 0.0901a 0.3940a 0.5579a 0.2952a 0.4422a 0.6252a 0.9432a 0.3614a 0.0717a 0.9830a 0.8574a 0.4026a 0.5868a 0.0482a 0.3413a 0.0753a 0.3954a 0.9060a 0.3930a 0.0637a 0.2232a 0.2358a 0.3258a 0.2249a 0.8869a

Data are mean ± SEM (range), or number of subjects analyzed. Data of the CAD (+) and CAD (−) groups were compared by the Student's t-test or the Mann–Whitney testa. Differences in frequencies were examined by the χ2 test. P values <0.05 were considered statistically significant (bold typeface). eVFA: estimated visceral fat area, UACR: urinary albumin–creatinine ratio, NDR: non diabetic retinopathy, SDR; simple diabetic retinopathy, PDR; proliferative diabetic retinopathy, CAD: coronary artery disease, CVD: cerebrovascular disease, APN; adiponectin, HMW; high-molecular weight.

and negatively with Total-APN in all, males and females (Fig. 1D). There were no significant differences in serum TotalAPN, HMW-APN, C1q-APN and C1q levels between the CAD (−)

(n = 53) and CAD (+) (n = 54) groups. The C1q-APN/Total-APN ratio was significantly higher in the CAD (+) group (10.47 ± 0.59) than CAD (−) group (8.88 ± 0.60, p = 0.0482).

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Total; r=0.51 p<0.001 Male; r=0.50 p<0.001 Female ; r=0.49 p<0.001

100

Male Female

60 40

0

A

10

20

B

Total-APN (µg/mL)

Total; r=-0.26 p=0.007 Male; p=0.112 Female ; r=-0.42 p=0.004

100

0

30

50

200

100

C1q-APN (U/mL)

Total; r=-0.65 p<0.001 Male; r=-0.62 p<0.001 Female ; r=-0.69 p<0.001

30

C1q-APN/Total-APN

0

C1q (µg/mL)

80

20

0

20

10

0

0 0

C

Total; p=0.1826 Male; p=0.5154 Female; p=0.3669

100

C1q (µg/mL)

C1q-APN (U/mL)

200

10

20

30

Total-APN (µg/mL)

0

D

10

20

30

Total-APN (µg/mL)

Fig. 1 – Correlations between total adiponectin (Total-APN), C1q-binding adiponectin (C1q-APN), C1q and C1q-APN/Total-APN. Pearson's correlation coefficient was used to examine the relationship between Total-APN and C1q-APN (A), C1q and C1q-APN (B), Total-APN and C1q (C), Total-APN and C1q-APN/Total-APN (D). Males (closed circle), Females (open circle).

3.2. Simple and multivariate logistic regression analyses of CAD Simple logistic regression analysis was used to evaluate the relationship between CAD and various serum adiponectin parameters (Table 2). Age, sex, hypertension, C1q, C1q-APN/ Total-APN and Total-APN/C1q correlated significantly with CAD (Model 1; no adjustment). Serum level of Total-APN differs between males and females [24]. Simple linear regression analysis showed that age- and sex-adjusted C1qAPN/Total-APN ratio as well as hypertension correlated significantly with CAD. Multiple regression analysis identified C1q-APN/Total-APN ratio and hypertension as significant determinants of CAD in T2DM (Table 2).

3.3.

Odds ratio of each adiponectin parameter for CAD

Next, to investigate the role of each serum adiponectin parameter on CAD, subjects were divided into three groups, tertile of each adiponectin parameter. The age- and sexadjusted ORs and 95% CI values for CAD in the first (Total-APN category 1) and second (Total-APN category 2) tertiles were 2.296 (95% CI, 0.819–6.705) and 1.374 (0.493–3.861), respectively, compared with the third tertile set at 1.0 (Total-APN category 3) (Fig. 2A). Data for HMW-APN were similar (Fig. 2B). The age- and sex-adjusted OR values for CAD in the second (C1q-APN category 2) and third (C1q-APN category 3) tertiles were 1.064 (0.379–3.021) and 1.035 (0.369–2.883), respectively,

Table 2 – Correlations between CAD and each adiponectin parameter. Model 1: no Model 2: age- & Model 3: adjustment sex-adjusted Multivariate

Age Sex (Male) Body mass index eVFA Dyslipidemia Hypertension Smoking (current) Total-APN HMW-APN C1q-APN C1q HMW-APN/ Total-APN C1q-APN/ Total-APN C1q-APN/ HMW-APN Total-APN/C1q C1q-APN/C1q

p

p

0.0075 0.0082 0.4094

0.2163

0.1744 0.1074 0.0001 0.6265

0.5166 0.0578 0.0016 0.5115

0.1036 0.4113 0.5351 0.0073 0.4306

0.0610 0.1501 0.3241 0.1344 0.6766

0.0410

0.0452

0.1105

0.0852

0.0131 0.2107

0.0501 0.2973

p -

0.0030

0.0463

Parameters with p < 0.05 in Model 2 were subsequently entered into multiple regression analysis as significant and independent variables (bold typeface). Abbreviations as in Table 1.

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1.9 to 5.6 (4.2)

2

5.6 to 9.3 (6.9)

3

9.3 to 29.7 (12.1) 0

1

10

0.4 to 3.0 (2.2)

2

3.1 to 6.4 (4.5)

p=0.7029 1.221 (0.435-3.424) p=0.7250

n=18/35

1

tertile

n=17/35

31.9 to 54.1 (44.0) p=0.9056 54.2 to 71.2 (61.1)

0

n=19/36

1.064 (0.379-3.021) p=0.9464 1.035 (0.369-2.883)

2

7.40 to 11.15 (8.96)

n=19/35 10

p=0.0160 for trend n=14/36 p=0.6085 n=17/36 1.309 (0.466-3.727) p=0.0106 3.965 (1.3699-12.372) n=23/35 0

1

1

1

10

2

p=0.7050

5.43 to 9.55 (7.66)

C

5

n=13/35

1.25 to 5.34 (3.87) 1.219 (0.436-3.470) p=0.0949 2.398 (0.860-6.963)

3 9.78 to 29.90 (13.47)

n=18/36

0

1

n=20/36 n=21/36

5

C1q-APN/C1q

C1q (µg/mL) tertile

2.73 to 7.36 (6.10)

1

n=15/36

Total-APN/C1q

3 71.3 to 159.1 (90.3)

D

1

B

5

C1q-APN (U/mL)

C

0

3 11.31 to 22.90 (13.53)

n=17/36

6.5 to 44.5 (10.8) 0

2

n=20/36

p=0.1618 0.55 to 0.79 (0.65) 0.4847 (0.169-1.334) p=0.7758 0.80 to 3.21 (0.99) 0.860 (0.302-2.431)

A

n=19/36

1.199 (0.434-3.340)

1

3

tertile

tertile

1

B

2

Subjects with CAD/Total

0.07 to 0.54 (0.43)

C1q-APN/Total-APN

HMW-APN (µg/mL)

3

1

tertile

A

HWM-APN/Total-APN tertile

1

Subjects with CAD/Total p=0.1144 2.296 (0.819-6.705) n=21/36 p=0.3320 n=19/35 1.374 (0.493-3.861) n=14/36

1

35.0 to 48.9 (43.3)

2

49.0 to 57.3 (52.2)

3

57.4 to 99.1 (61.9) range (median) 0

p=0.0528 2.744 (0.988-7.996) p=0.1745 2.074 (0.725-6.207) 1

n=17/36 n=19/36

1 tertile

tertile

Total-APN (µg/mL)

n=18/35 10

age- and sex-adjusted OR values for CAD (95% CI)

Fig. 2 – Age- and sex-adjusted odds ratio of total adiponectin (Total-APN), high-molecular weight-adiponectin (HMW-APN), C1q-binding adiponectin (C1q-APN), and C1q categories for coronary artery disease (CAD). Transverse bars indicate the odds ratio (OR) (solid diamonds) and 95% confidence intervals (95% CI) (lines). P value compared with category 1 or 3.

compared with the first tertile set at 1.0 (C1q-APN category 1) (Fig. 2C). Data of C1q were similar (Fig. 2D). Total-APN, HMWAPN and C1q-APN did not correlate with CAD. We also investigated various serum adiponectin parameters including the C1q-APN/Total-APN ratio. The age- and sexadjusted OR values for CAD in the second (C1q-APN/Total-APN category 2) and third (C1q-APN/Total-APN category 3) tertiles were 0.763 (0.268–2.146) and 3.965 (1.3699–12.372, p = 0.0106), respectively, compared with the first tertile set at 1.0 (C1qAPN/Total-APN category 1) (Fig. 3B) (p = 0.0160 for trend). On the other hand, all other adiponectin parameters (HWM-APN/ Total-APN, Total-APN/C1q, C1q-APN/C1q) did not correlate with CAD (Fig. 3A, C, D). The results indicate that serum C1qAPN/Total-APN ratio seems to be a useful biomarker for CAD.

3.4. ROC curve analysis of various serum adiponectin parameters and C1q for identifying subjects with CAD Table 3 shows the ROC curves for various serum adiponectin parameters and C1q for detecting CAD. Among all the

D

0.50 to 0.95 (0.82)

n=20/36 p=0.9538

2

0.96 to 1.45 (1.23)

3

1.46 to 2.92 (1.74) range (median)

n=17/36

1.030 (0.372-2.826) p=0.7812 1.156 (0.412-3.239) 0

1

n=17/35 5

age- and sex-adjusted OR values for CAD (95% CI)

Fig. 3 – Age- and sex-adjusted odds ratio of HMW-APN/ Total-APN, C1q-APN/Total-APN, Total-APN/C1q and C1q-APN/C1q categories for coronary artery disease (CAD). Transverse bars indicate the odds ratio (OR) (solid diamonds) and 95% confidence intervals (95% CI) (lines). P value compared with category 1.

Table 3 – Receiver operating characteristic (ROC) curve analysis for the value of various serum adiponectin parameters and C1q in the diagnosis of CAD. AUC

95% CI lower upper

Total-APN C1q-APN/Total-APN Total-APN/C1q HMW-APN C1q/HMW-APN C1q-APN/C1q C1q-APN/HMW-APN C1q C1q-APN HMW-APN/Total-APN

0.598 0.611 0.604 0.548 0.552 0.562 0.551 0.560 0.525 0.494

0.499 0.504 0.496 0.437 0.441 0.450 0.440 0.450 0.414 0.395

0.717 0.718 0.713 0.660 0.662 0.672 0.662 0.670 0.635 0.618

cut-off p value value 11.50 7.76 4.98 2.18 27.74 1.19 20.72 49.01 47.22 0.56

0.0131 0.0415 0.0423 0.0955 0.1105 0.1223 0.2383 0.3547 0.4366 0.8557

This table shows alignment of higher AUC from the top (all). P values with <0.05 were considered statistically significant (bold typeface). Abbreviations as in Table 1. AUC; area under the specificity–sensitivity curve, 95% CI; 95% confidence intervals.

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Table 4 – Diagnostic accuracy of cutoff values of Total-APN and C1q-APN/Total-APN ratio for CAD (% of CAD = 50.5%) from the ROC curve analysis (Table 3). Total-APN (cutoff = 11.50) Sensitivity (%) Specificity (%) False positive rate (%) False negative rate (%) Likelihood ratio for a positive finding Likelihood ratio for a negative finding Odds ratio for a positive finding Positive predictive value (%) Negative predictive value (%)

87.0 30.8 30.4 43.3 1.257

C1q-APN/TotalAPN (cutoff = 7.76)

(79.3–93.0) (22.8–37.0) (11.6–49.2) (32.7–57.5) (1.027–1.476)

75.9 (64.5–87.3) 50.9 (37.4–64.4) 32.2 (2.79–19.8) 38.1 (17.9–47.0) 1.548(1.075–5.832)

0.421 (0.189–0.907)

0.473 (0.275–0.793)

2.984 (1.133–7.824)

3.013 (1.094–8.244)

56.6 (51.6–60.5)

69.0 (60.7–76.7)

69.6 (51.5–83.6)

55.0 (37.5–71.0)

adiponectin parameters tested in this study, the significant marker of CAD was serum Total-APN (AUC 0.598 [95% CI, 0.499–0.717], p = 0.0131), C1q-APN/Total-APN ratio (AUC 0.611 [95% CI, 0.504–0.718], p = 0.0415) and C1q-APN/C1q ratio (AUC 0.604 [95% CI, 0.496–0.713], p = 0.0423).

3.5. Diagnostic performance of Total-APN and C1q-APN/ Total-APN ratio for CAD First, we analyzed the diagnostic value for cut-off value of Total-APN (< 11.50 μg/mL) and C1q-APN/Total-APN (≥ 7.76) from the ROC curve analysis analyzed in Table 3. Table 4 lists the sensitivity, specificity, false positive and negative values, likelihood ratio for positive and negative findings, positive predictive values, and negative predictive values for the above parameters. In this study, the proportion of patients with CAD was 50.5% (n = 54/107). The specificity values of Total-APN (< 11.50 μg/mL) and C1q-APN/Total-APN ratio ≥ 7.76 were 30.8% and 50.9%, respectively. From odds ratio analysis, hypoadiponectinemia (TotalAPN < 4 μg/mL) has been described as a candidate risk factor for CAD [8]. Next, we assessed the diagnostic value for cutoff values of Total-APN (<4.2 μg/mL; Fig. 2A, category 1 median) and C1q-APN/Total-APN (≥ 13.53; Fig. 3B, category 3 median) from age- and sex-adjusted odds ratio analysis (Figs. 2 and 3) for CAD in Table 5. Although the sensitivity was low, the specificity values of Total-APN (< 4.20 μg/mL) and C1q-APN/ Total-APN ratio ≥13.53 were 83.3% and 88.7%, respectively. All other parameters that describe diagnostic performance were better for the C1q-APN/Total-APN ratio ≥13.53 than for TotalAPN < 4.2 μg/mL (Hypoadiponectinemia).

4.

Discussion

The major finding of the present study was that high serum C1q-APN/Total-APN ratio, but not C1q-APN, correlates with CAD, in T2DM patients. High serum C1q-APN/Total-APN ratio was associated with 4-fold increase in CAD prevalence, independent of other CAD risk factors. Thus, high serum

Table 5 – Diagnostic accuracy of cutoff values of Total-APN (< 4.20 μg/mL; Fig. 2A, category 1 median) and C1q-APN/ Total-APN ratio (≥ 13.53; Fig. 3B, category 3 median) for CAD (% of CAD = 50.5%) from age- and sex-adjusted odds ratio. Total-APN (cutoff = 4.20) Sensitivity (%) 17.0 (10.3–23.6) Specificity (%) 83.3 (76.8–89.9) False positive rate (%) 16.9 (6.87–27.0) False negative rate (%) 83.3 (73.3–107.6) Likelihood ratio for a 1.019 (0.444–2.335) positive finding Likelihood ratio for a 0.996 (0.850–1.168) negative finding Odds ratio for a 1.023 (0.381–2.748) positive finding Positive predictive 50.0 (30.4–69.6) value (%) Negative predictive 50.6 (46.6–54.5) value (%)

C1q-APN/Total-APN (cutoff = 13.53) 27.8 88.7 11.3 72.2 2.454

(20.3–33.3) (81.1–94.3) (2.79–19.8) (60.2–94.9) (1.075–5.832)

0.814 (0.708–0.983) 3.013 (1.094–8.244) 71.4 (52.3–85.6) 54.7 (50.0–58.1)

C1q-APN/Total-APN ratio (≥13.5) can be considered an important risk factor for CAD, in addition to low serum levels of Total-APN (< 4 μg/mL), hypoadiponectinemia, in T2DM. A proposed relative cutoff value from the ROC curve analysis with low specificity should be read with caution (Table 4). On the other hand, an absolute cutoff value from age- and sex-adjusted odds ratio showed high specificity (Table 5). These results suggested that the cutoff values of Total-APN and C1q-APN/Total-APN ratio for CAD should be based on an absolute value from the odds ratio analysis, rather than a relative value calculated from the ROC curve analysis, in T2DM subjects. Taken together, the data suggest that not only quantitative change in adiponectin (Total-APN) but also qualitative change (C1q-APN/Total-APN ratio), may influence the etiopathology of CAD. A large-scale prospective study of the general population is required to confirm these findings. However, to date, the precise values for serum C1q-APN cannot be measured, because the proportion of blood adiponectin that forms protein complex with C1q remains unclear. The underlying pathology of atherosclerotic diseases, including CAD, is chronic inflammation of the arterial wall. This process encompasses an imbalance of lipid metabolism and maladaptive immune response [25]. The complement system is part of the immune system in atherosclerosis [14– 17]. C1q interacts with complement cell-surface receptor to promote phagocytosis and local pro-inflammatory response [26]. C1q also activates canonical Wnt signaling [27]. Adiponectin shows structural homology to collagen VIII, X and complement factor C1q [2]. Adiponectin is also known to inhibit inflammatory reactions and protects against cardiovascular diseases [28]. We reported that C1q and C3 deposits in the joints murine collagen-induced arthritis were significantly suppressed by adenovirus–adiponectin treatment, compared with adenovirus–β-galactosides, using immunohistochemical staining [29], and that adiponectin binds to C1q in human blood using co-immunoprecipitation experiments [12]. We recently reported that serum C1q-APN/Total-APN can be potentially used to investigate the pathophysiological significance of polyvascular atherosclerosis using systemic

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vascular ultrasonography in T2DM patients [13]. This is the first report that describes the relation of serum C1q-APN/ Total-APN ratio with CAD in T2DM. These results suggest that adiponectin seems to have a protective role in activated complement system in the development of atherosclerosis. Further studies are needed to clarify the role of adiponectin and C1q network in atherosclerogenesis. The present study has several limitations. First, this is a cross-sectional study, making it difficult to establish a cause–effect relationship. Further prospective studies should be conducted in the future to analyze this relationship. Second, all patients in this study were Japanese and any differences from other ethnicities are unknown. Third, there is bias in single center trials. Fourth, all subjects had T2DM. Our other study demonstrates that C1q-APN/Total-APN ratio correlates significantly with CAD in male patients with T2DM and non-DM male patients (unpublished data), as well as in T2DM patients (Table 2). Fifth, serum level of C1qAPN/Total-APN was 9.77 ± 1.51 (mean ± SEM) in male subjects without T2DM and CAD (unpublished data). However, to date, the proportion of circulating adiponectin that forms protein complex with C1q remains unclear. Therefore, normal level of C1q-APN/Total-APN remains unclear. Sixth, the study population included 107 Japanese subjects (males/ females; 62/45). The results of analysis of 62 male subjects were similar to those of all subjects (n = 107) (data not shown). However, the present study could not obtain similar results for analysis of 45 females. Number of female subjects is small (n <50) and limited. Finally, the number of patients was relatively small. In the present study, based on 80% power to detect statistically significant correlation between each adiponectin parameter and CAD (p = 0.05; two-sided, α = 0.05, 1−β = 0.8), a sample size of at least 48 patients in each group (total sample size = 96) was required, based on data presented in previous reports [17]. Further multicenter studies that include larger samples of subjects, especially those of other races, are needed. In conclusion, the present study demonstrated that high serum C1q-APN/Total-APN ratio correlates with CAD in T2DM. Serum C1q-APN/Total-APN ratio is a promising target in attempts to reduce the morbidity and mortality of atherosclerotic disease.

Author contributions AH and KK researched and analyzed the data. KK also participated in the concept and design of the study, interpretation of data and reviewed/edited the manuscript. HK analyzed the data. HN recruited the patients and collected the data. TF and IS contributed to the discussion and wrote the manuscript. All authors read and approved the final version of the manuscript.

Funding This research was supported in part by a Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area) "Molecular Basis and Disorders of

Control of Appetite and Fat Accumulation" (#22126008, to T.F. and K.K.), and Osaka University's academia–industry collaboration policy position between Osaka University and Otsuka Pharmaceutical Co., Ltd.

Acknowledgments We thank all staff of the “Diabetes & Metabolic Station” for the excellent medical care, and Messrs Shigeo Takahashi, Suguru Akamatsu, and Tetsuya Oda for the statistical advice and helpful discussion, and Messrs Hideaki Tanaka and Tohru Hadama and Mrs. Miyuki Nakamura for the excellent technical assistance.

Conflict of interests KK, TF and IS are promotional speakers for Otsuka Pharmaceutical Co., Ltd. TF is a member of the “Department of Metabolism and Atherosclerosis”, a sponsored course endowed by Kowa Co. Ltd.. The company has a scientific officer who oversees the program. All other authors declare no competing interests. Human serum C1q-binding adiponectin complex assay is under patent application in Japan.

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