M E T A B O L I S M C L I N I C A L A N D E X P E R I M E N T A L 6 2 ( 2 0 1 3 ) 6 7 7 – 6 8 5

Ava i l ab l e on l i ne a t www.sc i enced i r ec t . com

Metabolismwww.metabo l i sm jou rna l . com

Variants of the Adiponectin Gene and Diabetic MicrovascularComplications in Patients with Type 2 Diabetes

Eun Yeong Choea, Hye Jin Wangb, Obin Kwona, Kwang Joon Kima, Bum Seok Kimd,Byung-Wan Leea, Chul Woo Ahna, b, c, Bong Soo Chaa, b, c, Hyun Chul Leea, b, c,Eun Seok Kanga, b, c,⁎, Christos S. Mantzoros e, f

a Division of Endocrinology and Metabolism, Department of Internal Medicine, Yonsei University College of Medicine, Seoul 120-752, Koreab Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Koreac Institute of Endocrine Research, Yonsei University College of Medicine, Seoul 120-752, Koread Division of Nephrology, Department of Internal Medicine, Yonsei University College of Medicine, Seoul 120-752, Koreae Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center,Harvard Medical School, Boston, MA 02215, USAf Department of Environmental Health, Harvard School of Public Health, Boston, MA 02215, USA

A R T I C L E I N F O

Abbreviations: ADIPOQ, adiponectin gene;disease; SNP, single nucleotide polymorphlipoprotein cholesterol; BUN, blood urea nitrohigh-sensitivity C-reactive protein; HbA1c, hepressure; DBP, diastolic blood pressure; FPG,ratio; eGFR, estimated glomerular filtration raurine microalbumin; CAOD, coronary artery⁎ Corresponding author.Department of Intern

752, Korea. Tel.: +82-2-2228-1968; fax: +82-2-E-mail address: edgo@yuhs.ac (E.S. Kang)

0026-0495/$ – see front matter © 2013 Elsevihttp://dx.doi.org/10.1016/j.metabol.2012.11.00

A B S T R A C T

Article history:Received 7 August 2012Accepted 16 November 2012

Objective. The aim of this study was to examine the association between commonpolymorphisms of the adiponectin gene (ADIPOQ) and microvascular complications inpatients with type 2 diabetes mellitus (T2DM).

Research design and methods. Rs2241766 and rs1501299 of ADIPOQ were genotyped in 708patients with T2DM. Fundus photography, nerve conducting velocity, and urine analysiswere performed to check for the presence of microvascular complications including diabeticnephropathy, retinopathy and neuropathy.

Results. The prevalence of diabetic nephropathy tended to be different according tors2241766 genotype (p=0.057) and the GG genotype of rs2241766 was associated withdiabetic nephropathy [urine albumin/creatinine ratio (UACR) greater than 30 mg/g] afteradjusting for age, sex, body mass index, duration of diabetes, HDL-cholesterol, smokingstatus, and blood pressure (odds ratio=1.96; 95% confidence interval=1.01–3.82, p=0.049).Also, the G allele of rs2241766 demonstrated a trend to be associated with an increase inUACR (p=0.087). Rs2241766 genotype was not associated with diabetic retinopathy (p=0.955) and neuropathy (p=0.104) or any diabetic microvascular complications (p=0.104).There was no significant association between the rs1501299 genotype of ADIPOQ and theprevalence of diabetic retinopathy and neuropathy or any diabetic microvascularcomplications even after adjustment.

Keywords:AdiponectinGeneType 2 diabetes mellitusMicrovascular complications

T2DM, type 2 diabetes mellitus; UACR, urine albumin/creatinine ratio; CVD, cardiovascularism; TG, triglycerides; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-densitygen; Cr, creatinine; AST, aspartate aminotransferase; ALT, alanine aminotransferase; hsCRP,moglobin A1c; NCV, Nerve conducting velocity; IQR, interquartile ranges; SBP, systolic bloodfasting plasma glucose; PPG, 2-h postprandial plasma glucose; OR, odds ratio; HR, hazardte; ARB, angiotensin II receptor blocker; ACEI, angiotensin converting enzyme inhibitor; UA,occlusive disease; PAOD, peripheral artery occlusive disease.al Medicine, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-Ku, Seoul, 120-393-6884..

er Inc. All rights reserved.5

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Conclusion. These data suggest that the GG genotype at rs2241766 is implicated in thepathogenesis of risk for diabetic nephropathy defined as UACR greater than 30 mg/day inpatients with T2DM.

© 2013 Elsevier Inc. All rights reserved.

1. Introduction

The incidence of microvascular complications is increasingamong the growing number of patients suffering from type 2diabetes (T2DM) [1–3]. The prevalence of overall microvascularcomplication is 30% to 40% at the time of diagnosis [4,2], andalthough itmay not affect the overall survival, it contributes tothe patient's quality of life [5,6]. Also, diabetic microvascularcomplications are associated with increased risk for cardio-vascular disease (CVD) [7], and diabetic patients with CVDhave been shown to have higher mortality and morbiditycompared to nondiabetic subjects [8,9]. Microvascular com-plications are the results of multiple causative factors [10,11].Genetic susceptibility is one of the reasons proven in previousstudies [12–14].

Adiponectin is an adipokine that plays an important rolein the regulation of insulin action, glucose and lipidmetabolism, and exerts anti-atherogenic and anti-inflam-matory effects [15,16]. Previous studies have shown thatabnormal serum adiponectin levels are associated withobesity, insulin resistance, T2DM, CVD and nephropathy[12,13,15,17,18]. A strong relationship has been reportedbetween the chromosomal region encompassing the adipo-nectin gene (ADIPOQ) and cardiovascular risk factors [19,20].Also, two single nucleotide polymorphisms (SNPs) at theADIPOQ locus, rs2241766 (T45G) and rs1501299 (G276T), havebeen reported to be associated with T2DM and macrovas-cular complications [15,16,21–23]. However, the associationbetween the ADIPOQ variations and diabetic microvascularcomplications has not been well studied. Lawrence Wu et al.reported the ADIPOQ, rs266729 is associated with diabeticnephropathy [24]. Bostrom et al. evaluated the 24 SNP ofADIPOQ and reported the association between rs182052 anddiabetic nephropathy [25]. Rudofsky et al. reported that therewas no association of T94G polymorphism (rs2241766) inADIPOQ with diabetic complications in group of patientswith combined T1DM and T2DM [26]. However, in thatprevious study, the diagnostic criteria of diabetic nephrop-athy was overestimated (microalbumin≥20 mg/L), and con-founding factors such as any drug effects had not been takeninto consideration.

In this study, we examined the association betweenrs2241766 and rs1501299 in ADIPOQ with diabetic microvas-cular complications in 708 patients with T2DM.

2. Material and methods

2.1. Study subjects

A total of 708 Korean patients with T2DM were recruited fromthe outpatient clinic of the Severance Hospital DiabetesCenter, Yonsei University Medical Center, from June 2003 to

September 2005 and closely followed up until these days.Diabetes was defined according to the American DiabetesAssociation diagnostic criteria [27]. The diabetes durationfrom diagnosis date was calculated and the presence ofmicrovascular and macrovascular complication of recentyear was evaluated. Exclusion criteria were as follows: 1)type 1 diabetes mellitus, or history of ketoacidosis, 2) positivefor glutamic acid decarboxylase antibody, 3) current diag-nosed malignancy, 4) posttransplantation status, 5) severekidney disease (GFR<30 mL/min/1.73 m2), 6) severe liverdisease (Child–Pugh class C), 7) had a recent ischemic event,8) thyroid function abnormalities, or 9) corticosteroid use. Thestudy protocol was approved by the Institutional ReviewBoard of Yonsei University College of Medicine. All patientsprovided informed consent prior to participating in this study.

2.2. Genetic analysis

Genotyping ofADIPOQ rs2241766 (T45G) and rs1501299 (G276T)was performed as previously reported [28,29]. Genomic DNAwas extracted from leukocytes in whole blood [28]. Thegenotyping was analyzed by a single base primer extensionassay using a SNaPShot assay kit according to the manufac-turer's recommendations (ABI, Foster City, CA) [29]. Resultswere analyzed using the Gene Mapper software (ABI).

2.3. Clinical data and biochemical measurements

After 8 h of fasting, blood samples were obtained from theenrolled patient and stored at −70 °C for subsequent assays.Plasma triglycerides (TG), total cholesterol, high-densitylipoprotein cholesterol (HDL-C), blood urea nitrogen (BUN),creatinine (Cr), aspartate aminotransferase (AST), and ala-nine aminotransferase (ALT) levels were assayed using aroutine Hitachi 7600 autoanalyzer (Hitachi InstrumentsService, Tokyo, Japan). Low-density lipoprotein (LDL) choles-terol levels were calculated using the Friedewald formula[30]. High-sensitivity C-reactive protein (hsCRP) was mea-sured as previously described [31]. Plasma glucose wasmeasured using the glucose oxidase method. HemoglobinA1c (HbA1c) values were determined by high-performanceliquid chromatography. Insulin concentrations were mea-sured using a radioimmunoassay kit (IRMA kit; DAINABOT,Tokyo, Japan). Plasma adiponectin concentrations weremeasured using commercially available enzyme-linked im-munosorbent assay kits (B-Bridge International, San Jose,CA). The intra- and inter-assay coefficients of variation were3.3% and 7.4%, respectively.

2.4. Diagnosis of diabetic nephropathy

Urinary albumin/creatinine ratio (UACR) was calculated usingresults of urine microanalysis results (Hitachi 7180, Tokyo,

Table 1 – Baseline clinical and biochemical characteristics of the subjects.

rs2241766 P rs1501299 P

TT(346) TG(306) GG(56) GG(351) GT(297) TT(60)

Sex (M/F) 146/200 133/173 28/28 .55 142/209 133/164 32/28 .14Age (y) 62.1±10.2 61.8±9.3 60.9±8.2 .68 61.7±9.3 62.0±10.1 62.3±9.7 .89Age at dx (y) 50.4±10.2 29.8±9.9 29.4±8.7 .67 49.9±9.7 50.1±10.3 50.8±9.6 .78Duration (y) 11.7±7.6 12.0±6.3 11.5±6.7 .79 11.8±6.8 11.8±7.3 11.5±8.2 .93BMI (kg/m2) 24.7±3.3 24.7±3.3 24.6±2.9 .96 24.8±3.3 24.6±3.1 25.1±3.3 .45SBP (mmHg) 127.1±14 127.3±16.1 128.3±11.6 .87 127.7±16.1 127.1±13.6 126.0±12.2 .67DBP (mmHg) 78.4±2.0 78.6±7.3 78.6±6.5 .91 78.7±7.2 78.2±7.3 78.6±6.7 .73HTN (%) 192(55.7) 164(53.6) 35(62.5) .46 190(54.3) 167(56.2) 34(56.7) .86FPG (mmol/L) 7.14±2.0 7.44±2.1 7.5±2.2 .15 7.4±2.1 7.3±0.4 6.7±1.6 .02⁎

2hPPG (mmol/L) 11.3±3.9 11.2±3.9 12.4±5.4 .11 11.5±4.3 11.3±3.8 10.5±3.9 .17HbA1c (%) 7.3±1.2 7.4±1.2 7.5±1.1 .51 7.4±1.2 7.3±1.2 7.2±1.0 .24HOMA_B 17.3±1.3 29.7±11.1 15.3±2.5 .42 18.3±1.9 27.9±11.6 21.0±11.6 .63HOMA_IR 3.0±3.0 3.4±4.5 2.5±1.8 .27 2.9±3.0 3.3±4.2 3.5±3.6 .38Adiponectin (μg/mL) 9.8±8.5 10.2±8.8 10.5±11.5 .87 9.6±8.3 10.7±9.6 9.3±8.6 .31hsCRP (mg/L) 1.2±0.1 1.3±0.1 1.3±0.2 .98 1.4±0.1 1.3±0.1 1.3±0.3 .29Total cholesterol (mmol/L) 4.6±0.9 4.6±0.8 4.6 ±0.9 .96 4.7±0.9 4.6±0.8 4.5±0.8 .41TG (mmol/L) 1.7±1.1 1.7±2.0 1.9±1.1 .33 1.8±1.2 1.6±1.0 1.7±1.0 .23HDL-C (mmol/L) 1.3±0.3 1.3±0.3 1.3±0.7 .57 2.5±0.3 2.6±0.7 2.5±0.8 .89LDL-C (mmol/L) 2.5±0.8 2.5±0.7 2.4±0.7 .57 2.5±0.8 2.6±0.7 2.5±0.8 .89UACR (mg/g) 17.0(8.7–47.8) 16.1(7.9–40.2) 24.4(12.7–152.4) .27 20.2(9.7–53.7) 14.7(7.3–45.2) 15.7(9.3–31.2) .6624 h urine (mg/d) 14.3(6.8–65.3) 17.2(7.8–100.3) 24.9(10.1–110.3) .96 16.2(7.9–81.2) 16.7(7.3–92.5) 20.0(6.4–60.6) .36Insulin use (%) 55(16) 45(14.9) 9(14.3) .89 60(17.1) 37(12.6) 11(18.3) .23OAD use (%) 287(83.4) 263(85.9) 48(85.7) .66 296(84.6) 249(84.1) 53(88.3) .71Statin use (%) 91(26.5) 75(24.6) 11(19.6) .53 91(26) 73(24.7) 13(21.7) .76ARB use (%) 102(29.7) 77(25.3) 18(32.1) .37 103(29.5) 80(27.1) 14(23.3) .56ACEI use (%) 43(12.5) 42(13.8) 8(13.2) .86 46(13.2) 40(13.6) 7(11.7) .93

One way ANOVA was performed. Data presented as Mean±SD, dx; diagnosis of diabetes, y; years, SBP; systolic blood pressure, DBP; diastolicblood pressure, FPG; fasting plasma glucose, 2hPPG; 2 h postprandial glucose, hsCRP; high-sensitive C-reactive protein, TG; triglyceride, HDL-C;high-density lipoprotein cholesterol, LDL-C; low-density lipoprotein cholesterol, UACR; urine albumin/creatinine ratio, ARB; angiotensin IIreceptor blocker, ACEI; angiotensin converting enzyme inhibitor.

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Japan). The presence of diabetic nephropathy was defined asUACR of 30 mg/g or more. If spot urine microalbumin was notmeasured, nephropathy was diagnosed in patients with 24 hurine microalbumin levels of greater than 30 mg/day.

2.5. Diagnosis of diabetic retinopathy

A six-field fundus photography was performed as screeningtest to examine the retina. The results were analyzed by anophthalmologist. The presence of diabetic retinopathy wasdefined as the existence of either proliferative or non-proliferative retinal disease or with a previous history oflaser photocoagulation therapy.

Table 2 – Prevalence of microvascular complication according t

N (%) Nephropathy P Retinopathy

No Yes No Yes

rs2241766TT 216(63.5) 124(36.5) .057 213(61.6) 111(43.2) .9TG 204(66.7) 95(31) 194(63.4) 96(31.4)GG 28(51.9) 26(48.1) 35(62.5) 18(32.1)

rs1501299GG 219(62.4) 123(35) .916 222(63.2) 109(31.1) .2GT 189(63.6) 102(34.2) 178(59.9) 102(34.3)TT 40(66.7) 20(33.3) 40(66.7) 20(33.3)

χ2 test was performed. N (%) was described. Any; patients having any of

2.6. Diagnosis of diabetic neuropathy

Nerve conducting velocity (NCV) was performed to confirmthe presence of neuropathy. Abnormalities in sensory ormotor neuronal velocity were defined as neuropathy. If NCVhad not been performed, the presence of neuropathy wasdetermined according to the existence of symptoms or historyof medication alleviating symptoms of diabetic neuropathy.

2.7. Statistical analysis

Data are shown as means±standard deviations or medianswith interquartile ranges (IQR), when variables were not

o genotype.

P Neuropathy P Any P

No Yes No Yes

55 213(61.6) 94(27.2) .621 136(47.1) 210(50.1) .104199(65.0) 80(26.1) 136(47.1) 170(40.6)31(55.4) 17(30.4) 17(5.9) 39(9.3)

33 210(59.8) 104(29.6) .237 139(48.1) 212(50.6) .707190(64.0) 73(24.6) 123(42.6) 174(41.5)43(71.7) 14(23.3) 27(9.3) 33(7.9)

microvascular complications.

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normally distributed. The normal distribution of each variablewas examined using the Kolmogorov–Smirnov test. Thedifferences among the genotypes were evaluated using a

Any of microvascular complication

Nephropathy

Retinopathy

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p=0.116

p=0.219

p=0.723

C

B

A

Fig. 1 – Proportion rate of diabetic nephropathy (A), retinopathy (according to rs2241766 and rs1501299 genotype.

one-way analysis of variance test, survival analysis, Cox'sregression, t-test, or χ2 test as appropriate. Multiple linearregressions were used to assess differences in the amount of

rs1501299

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p=0.446

p=0.746

p=0.864

B) and neuropathy (C) according to rs2241766 genotype. (D)

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p=0.208 p=0.402

D

Fig. 1 – (continued)

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microalbuminuria between genotypes. Differences in geno-type distribution between subjects with andwithout nephrop-athy were assessed by χ2 test and t-test. Genetic risk ofnephropathy was assessed using logistic regression methods.P-values were adjusted for multiple comparisons using theBonferroni method. A P-value of less than 0.05 was considered

Table 3 – Baseline characteristics of patients with andwithout nephropathy.

Nephropathy

Yes No P

Age (years) 63.4±9.3 61.1±9.8 0.020Sex (M/F) 104/141 195/253 0.784Diabetes duration (years) 13.2±7.7 11.1±6.8 0.001UACR (mg/g) 94.3(45.3–275.8) 10.2(6.7–17.7) 0.001

24 h UA (mg/day) 150.5(53.7–516.0) 8.6(6.3–15.0) 0.001BMI (kg/m2) 25.4 ±3.6 24.4 ±2.9 0.001SBP (mmHg) 130.1±15.4 125.7±14.1 0.001DBP (mmHg) 79.0±7.6 78.2±6.9 0.130FPG (mmol/L) 7.4±2.3 7.3±2.0 0.4802hPPG (mmol/L) 11.9±4.5 11.1±3.8 0.017HbA1c (%) 7.5±1.3 7.3±1.1 0.023Adiponectin (μg/mL) 10.3±8.9 10.0±9.0 0.793hsCRP (mg/L) 1.4±0.1 1.2±0.1 0.130Total cholesterol (mmol/L) 4.7 ±0.9 4.6±0.8 0.483TG (mmol/L) 1.9±1.3 1.6±1.0 0.001HDL-C (mmol/L) 1.3±0.3 1.3±0.3 0.059LDL-C (mmol/L) 2.5±0.8 2.6±0.8 0.308History of CAOD (%) 26(10.65) 35(7.8) 0.214Presence of stroke (%) 26(10.5) 35(7.8) 0.214Presence of PAOD (%) 12(4.9) 8(1.8) 0.019

T test and χ2 test were performed. Data presented as mean±SD, ormedian (IQR), M; male, F; female, UACR; urine microalbumin/creatinine ratio, UA; urine microalbumin, SBP; systolic bloodpressure, DBP; diastolic blood pressure, FPG; fasting plasmaglucose, 2hPPG; 2 h postprandial glucose, hsCRP; high-sensitiveC-reactive protein, TG; triglyceride, HDL-C; high-densitylipoprotein cholesterol, LDL-C; low-density lipoproteincholesterol, CAOD; coronary artery occlusive disease, PAOD;peripheral artery occlusive disease.

to be statistically significant and that of less than 0.1 wasconsidered to be borderline significant. All calculations andstatistical analyses were performed using SPSS softwareversion 18 (SPSS, Chicago, IL).

3. Results

The clinical and biochemical characteristics of the enrolledpatients according to ADIPOQ genotype at rs2241766 andrs1501299 are shown in Table 1. Subjects with the rs1501299TT genotype showed a lower fasting plasma glucose level thanthose with the other genotypes (P=0.019). Otherwise, therewere no significant differences in age, duration of diabetesafter diagnosis, BMI, HbA1c, hsCRP, adiponectin, lipid profilesand medication between the rs2241766 and rs1501299 geno-types (Table 1).

Overall, the prevalence of diabetic nephropathy, retinopa-thy and neuropathy was 34.6%, 31.8%, and 27%, respectively.The patients with any of and two or more microvascularcomplications of nephropathy, retinopathy and neuropathywere 419 (59.2%) and 189 (26.7%), respectively. Neitherrs2241766 nor rs1501299 genotype was nominally associatedwith the prevalence of microvascular complications (Table 2).

Table 4 – Adiponectin genotype and its association withdiabetic nephropathy risk.

SNP Genotype OR(95% CI) Adjusted OR (95% CI)

rs2241766 TT 1 –TG 0.81(0.58–1.13) 0.91(0.63–1.32)GG 1.62(0.91–2.88) 1.96(1.01–3.82)

rs1501299 GG 1 –GT 0.96(0.69–1.33) 1.01(0.70–1.47)TT 0.89(0.49–1.59) 0.73(0.36–1.48)

Adjustments for age, sex, BMI, duration of diabetes, TG, HbA1c,smoking status, PAOD and SBP were performed. Common alleleswere used to reference genotype.

Table 5 – The median duration of microvascular complications for each genotype.

SNP Genotype Nephropathy P Retinopathy P Neuropathy P

rs2241766 TT 19.4 0.219 19.9 0.723 21.0 0.208TG 19.9 19.9 21.7GG 17.0 18.1 18.2

rs1501299 GG 19.5 0.746 19.8 0.446 21.2 0.402GT 19.3 19.8 21.2TT 19.3 18.9 26.3

Survival analysis was done. Year was described.

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The association between the prevalence of diabetic nephrop-athy and the genotypes was borderline significant, at 48.1%(26/56) in the GG, 31% (95/306) in the TG, and 36.5% (124/346) inthe TT genotype of rs2241766 (p=0.057). The prevalence ofproliferative and non-proliferative retinopathy was 24% and7.9%, respectively, which was not significantly differentbetween the groups.

The incidence of any diabetic microvascular complica-tions, diabetic nephropathy, retinopathy and neuropathyaccording to the rs2241766 and rs1501299 genotype was notsignificantly different (Fig. 1). The median duration ofdevelopment of microvascular complications for each geno-type is shown in Table 5. There was no statistically significantdifference in the median duration according to genotypes.However, any of microvascular complications trended tooccur earlier in GG genotype of rs2241766 compared to othergenotypes by 2 to 3 years (Hazard Ratio=1.28, 95% confidenceinterval [95% CI]=0.91–1.79, p=0.080). Regarding rs1501299genotype, there was no difference in the time of onset ofmicrovascular complications.

3.1. ADIPOQ genotype and diabetic nephropathy

The clinical and biochemical characteristics of subjects withand without nephropathy are shown in Table 3. Patients withDN were older and had a longer duration of diabetes, highersystolic blood pressure (SBP), higher 2-h postprandial plasmaglucose (PPG) levels, higher mean HbA1c, higher TG and lowerHDL cholesterol levels compared to patients without nephrop-athy. Patientswith diabetic nephropathy also showed a higherprevalence of peripheral artery occlusive disease (Table 3).However, adiponectin levelswerenot statistically significantlydifferent between patients with and without nephropathy.Although there was no statistically significant difference,patients with the rs2241766 GG genotype showed a tendencyof increased risk of diabetic nephropathy (Odds Ratio [OR]=1.62; 95% CI=0.91–2.88). After adjusting for age, sex, BMI,duration of diabetes, HbA1c, TG, current smoking status, PAODstatus and SBP, subjects carrying the rs2241766 GG genotypewere at a significantly higher risk of diabetic nephropathy(OR=1.96; 95% CI=1.01–3.82, p=0049) compared to carriers ofthe TT genotype (Table 4), while subjects with the TG showedno significant difference. There was no significant differencein the risk for diabetic nephropathy for rs1501299 afteradjusting for confounding factors (Table 4).

UACR was slightly increased in the subjects carryingthe G allele compared with those carrying the Tallele, with the difference reaching borderline significance(p=0.087, Table S1).

3.2. ADIPOQ genotype and diabetic retinopathy

The characteristics of diabetic retinopathy were analyzed(Table S2). Patients with retinopathywere older and also had alonger duration of diabetes, higher levels of SBP, DBP, 2-h PPG,mean HbA1c, and adiponectin as well as a lower levels of LDLcholesterol than subjects without retinopathy. Individualswith diabetic retinopathy also showed a higher prevalence ofhypertension and CAOD (Table S2). There was no significantstatistical difference in the risk of diabetic retinopathyaccording to the genotypes, even after adjusting for thepotential confounding variables.

3.3. ADIPOQ genotype and diabetic neuropathy

The clinical and biochemical characteristics of subjectswith diabetic neuropathy were similar to those withdiabetic nephropathy and retinopathy (Table S3). Significantdifference in age, duration of diabetes, SBP, 2-h PPG, andmean HbA1c was observed between the subjects with andwithout diabetic neuropathy. In addition, subjects withneuropathy showed a higher prevalence of hypertensionand PAOD (Table S3). However, rs2241766 and rs1501299were not significantly associated with the prevalence ofdiabetic neuropathy.

4. Discussion

We evaluated the association between two common poly-morphisms (rs2241766 and rs1501299) of the adiponectingene previously known to be associated with CVD [22,23,32]in relation to the prevalence of microvascular complications(nephropathy, retinopathy and neuropathy), as well as theduration of onset of such complication. We found thatrs2241766 GG genotype is associated with a higher risk ofnephropathy compared to the TT genotype after adjustingfor confounding variables. The prevalence of retinopathy orneuropathy was not associated with the rs2241766 geno-type. In addition, the prevalence of all microvascularcomplications was not significantly different among thegenotype of rs1501299.

Clinically, microvascular complications are diagnosedthrough a variety of methods. Diabetic nephropathy, retinop-athy, and neuropathy were defined as urinary albumin/creatinine ratio (UACR) of 30 mg/g or greater, presence ofproliferative and non-proliferative retinopathy retinal diseasedefined as with or without neovascularization at six fieldfundoscopy, and the presence of abnormal nerve conducting

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velocity (NCV) or subjective neuropathic pain, respectively.Diabetic microvascular complications are thought to beprimarily associated with chronic inflammation and cytokineas TNF-a, IL-6 and highly sensitive-CRP due to metabolicabnormality [33], as well as insulin resistance. Metabolicabnormalities could be affected by genetic susceptibility[12,13,24,34–36]. Adiponectin protects the vasculature throughits anti-inflammatory properties [37–39]. Therefore, we eval-uated the association of common SNP of adiponectin genewith the results of UACR, NCV and fundus examinations inT2DM patients.

The ADIPOQ polymorphisms rs2241766 and rs1501299 arecommon SNPs (MAF>20%) among Korean patients with T2DMand have been reported to be associated with obesity, insulinresistance, diabetes mellitus, glucose and lipid metabolism,and inflammation [15,21]. Although there have been severalstudies on the association between macrovascular complica-tions and the genetic polymorphisms of ADIPOQ [16,23,40],few studies have been performed on the relationship betweenthe genetic variants and microvascular complications[25,26,36]. We found that GG genotype of rs2241766 may beassociated with increased UACR and the risk of diabeticnephropathy. We previously reported that GG genotype ofrs2241766 associated with the presence of carotid arteryplaque and may contribute to atherosclerosis linked withmacrovascular complication [32]. As shown in this study, theprevalence of PAOD was higher in subjects with nephropathyand neuropathy than in subjects without the disease (p=0.019and 0.041, respectively) in the present data. We found thatcertain SNPs in the adiponectin gene may play a role in thedevelopment of macrovascular complications as well asmicrovascular complications.

Adiponectin protects the vasculature through its pleiotro-pic actions on endothelial cells, endothelial progenitor cells,smooth muscle cells and macrophages [37]. Adiponectinmodulates the inflammatory response of endothelial cells byreducing TNF-a production [38], and inhibits the proliferationand migration of smooth muscle cells to prevent vascularremodeling [39]. Its vasculoprotective effects prevent theendothelial injury and dysfunction [37]. The role of polymor-phism of the adiponectin gene has not been clarified. It seemsto play a role in vascular damage by interfering with anti-inflammatory and anti-diabetic effects of adiponectin. It maycontribute to worsening or acceleration of microvascularcomplications [41]. In our results, the median time of onsetof diabetic nephropathywas shorter by 2 years in GG genotypeof rs2241766 compared to other genotypes, although thestrength of the association was borderline significant. Also,additional research is needed to prove the genetic effect onthe development vascular complications.

The pathogenesis of diabetic nephropathy is comprisedof metabolic, hemodynamic and genetic factors [13,42].These factors have been shown to lead toward elevatedinflammatory cytokines, TGF-β, VEGF, and oxidative stress,as well as increases in the vascular permeability of albuminin the glomeruli [42]. Many studies have revealed thatgenetic factors contribute in the development of diabeticnephropathy. Lawrence et al. reported ADIPOQ rs266729,ENPP1 rs1044498, GHSR rs490683, PPARγ rs1801282 andTCF7L2 rs7903146 to be associated with the risk of nephrop-

athy [24]. Jaziri et al. reported that rs2241766 G allele ofADIPOQ is associated with higher incidence of diabeticnephropathy [12].

Considering that rs2241766 is a silent mutation for Gly15,it is possible that this SNP inactivates the gene by influenc-ing transcript activity; on the other hand, this SNP may belinked with other functional SNPs. We could not find asignificant difference in adiponectin level between thegenotypes, even after adjustment (Table 1). Therefore,further studies are required to discern the vasculoprotectivefunction of the genetic variants of adiponectin. Althoughseveral studies have shown the rs1501299 G allele to beassociated with CVD, no significant association with diabeticnephropathy could be found. This may reflect an ethnicdifference or reflect a difference in genetic determinantsbetween CVD and nephropathy.

Our study has several limitations. First, we only usedUACR as the diagnostic criteria to define diabetic nephrop-athy, and did not use creatinine clearance or age adjustedeGFR. However, the diagnosis criteria met the definition fordiabetic microalbuminuria, which is a traditional definitionof the outcome of interest. Second, subjects with abnormalfundus photography were referred to the ophthalmologist.However, not all photographic abnormalities exactly corre-sponded to diagnosis with diabetic retinopathy. Thus, thefinal diagnosis of retinopathy was determined by ophthal-mologists who were blinded to the goals of this study andthus no bias was introduced. Third, neuropathy was diag-nosed by NCV or in case of missing NCV data by the presenceof symptoms or use of medications specific for neuropathy.However, these criteria could have overlooked the subjectswith neuropathy despite normal NCV and minor symptoms,requiring no medication.

In summary, this study suggests that the GG genotype atrs2241766 may be associated with the presence of micro-albuminuria and may contribute to diabetic nephropathy intype 2 diabetes mellitus patients. Retinopathy or neuropathywas not associated with the adiponectin rs2241766 genotype.In addition, no microvascular complications were associatedwith the ADIPOQ rs1501299 genotype.

In conclusion, the GG genotype at rs2241766 may play arole in the development both of macrovascular and micro-vascular complications in T2DM.

Author contributions

EY Choe participated in the design and conduct of the study,data collection and analysis, data interpretation, and manu-script writing; ES Kang participated in the design and conductof the study, data collection and analysis, data interpretation,and manuscript writing; HJ Wang and BS Kim participated indata interpretation, and manuscript writing; KJ Kim and BWLee participated in the data interpretation, and manuscriptwriting; CW Ahn participated in the data interpretation, andmanuscript writing; BS Cha participated in the data interpre-tation, and manuscript writing; HC Lee participated in theconduct of the study, and data collection; CS Mantzorosparticipated in the design of the study, data interpretation,and manuscript writing.

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Acknowledgment

This work was supported by a faculty research grant ofYonsei University College of Medicine (6-2011-0119, 6-2011-0085), a grant from the Department of Internal Medicine,Yonsei University College of Medicine (7-2008-0148), and theNational Research Foundation of Korea Grant funded by theKorean Government (MEST, Basic Research Promotion Fund;NRF-2012000891).

Conflict of interest

The authors declare that there is no duality of interestassociated with this manuscript.

R E F E R E N C E S

[1] Whiting DR, Guariguata L, Weil C, et al. IDF diabetes atlas:global estimates of the prevalence of diabetes for 2011 and2030. Diabetes Res Clin Pract 2011;94:311–21.

[2] Kim JH, Kim DJ, Jang HC, et al. Epidemiology of micro- andmacrovascular complications of type 2 diabetes in Korea.Diabetes Metab J 2011;35:571–7.

[3] Guariguata L, Whiting D, Weil C, et al. The InternationalDiabetes Federation diabetes atlas methodology forestimating global and national prevalence of diabetes inadults. Diabetes Res Clin Pract 2011;94:322–32.

[4] UK Prospective Diabetes Study (UKPDS) Group. Intensiveblood-glucose control with sulphonylureas or insulincompared with conventional treatment and risk ofcomplications in patients with type 2 diabetes (UKPDS 33).Lancet 1998;352:837–53.

[5] Newman DJ, Mattock MB, Dawnay AB, et al. Systematicreview on urine albumin testing for early detection of diabeticcomplications. Health Technol Assess 2005;9:iii-vi, xiii-163.

[6] Yoshida S, Hirai M, Suzuki S, et al. Neuropathy is associatedwith depression independently of health-related quality oflife in Japanese patients with diabetes. Psychiatry ClinNeurosci 2009;63:65–72.

[7] Rosenson RS, Fioretto P, Dodson PM. Does microvasculardisease predict macrovascular events in type 2 diabetes?Atherosclerosis 2011;218:13–8.

[8] Estacio RO, Dale RA, Schrier R, et al. Relation of reduction inurinary albumin excretion to ten-year cardiovascularmortality in patients with type 2 diabetes and systemichypertension. Am J Cardiol 2012.

[9] Kannel WB, McGee DL. Diabetes and cardiovascular disease.The Framingham study. JAMA 1979;241:2035–8.

[10] Raman R, Gupta A, Krishna S, et al. Prevalence and riskfactors for diabetic microvascular complications in newlydiagnosed type II diabetes mellitus. Sankara NethralayaDiabetic Retinopathy Epidemiology And Molecular GeneticStudy (SN-DREAMS, report 27). J Diabetes Complications2012;26:123–8.

[11] Adler AI, Stratton IM, Neil HA, et al. Association of systolicblood pressure with macrovascular and microvascularcomplications of type 2 diabetes (UKPDS 36): prospectiveobservational study. BMJ 2000;321:412–9.

[12] Jaziri R, Aubert R, Roussel R, et al. Association of ADIPOQgenetic variants and plasma adiponectin isoforms with therisk of incident renal events in type 2 diabetes. Nephrol DialTransplant 2010;25:2231–7.

[13] Freedman BI, Bostrom M, Daeihagh P, et al. Genetic factors indiabetic nephropathy. Clin J Am Soc Nephrol 2007;2:1306–16.

[14] Mehrab-Mohseni M, Tabatabaei-Malazy O, Hasani-Ranjbar S,et al. Endothelial nitric oxide synthase VNTR (intron 4 a/b)polymorphism association with type 2 diabetes and itschronic complications. Diabetes Res Clin Pract 2011;91:348–52.

[15] Menzaghi C, Ercolino T, Di Paola R, et al. A haplotype at theadiponectin locus is associated with obesity and other featuresof the insulin resistance syndrome. Diabetes 2002;51:2306–12.

[16] Qi L, Li T, Rimm E, et al. The +276 polymorphism of the APM1gene, plasma adiponectin concentration, and cardiovascularrisk in diabetic men. Diabetes 2005;54:1607–10.

[17] Savopoulos C, Michalakis K, Apostolopoulou M, et al.Adipokines and stroke: a review of the literature. Maturitas2011;70:322–7.

[18] Apostolopoulou M, Savopoulos C, Michalakis K, et al. Age,weight and obesity. Maturitas 2012;71:115–9.

[19] Francke S, Manraj M, Lacquemant C, et al. A genome-widescan for coronary heart disease suggests in Indo-Mauritians asusceptibility locus on chromosome 16p13 and replicateslinkage with the metabolic syndrome on 3q27. Hum MolGenet 2001;10:2751–65.

[20] Kissebah AH, Sonnenberg GE, Myklebust J, et al. Quantitativetrait loci on chromosomes 3 and 17 influence phenotypes ofthe metabolic syndrome. Proc Natl Acad Sci U S A 2000;97:14478–83.

[21] Hara K, Boutin P, Mori Y, et al. Genetic variation in the geneencoding adiponectin is associated with an increased risk oftype 2 diabetes in the Japanese population. Diabetes 2002;51:536–40.

[22] Lacquemant C, Froguel P, Lobbens S, et al. The adiponectingene SNP+45 is associated with coronary artery disease intype 2 (non-insulin-dependent) diabetesmellitus. Diabet Med2004;21:776–81.

[23] Jang Y, Lee JH, Kim OY, et al. The SNP276G>T polymorphismin the adiponectin (ACDC) gene is more strongly associatedwith insulin resistance and cardiovascular disease risk thanSNP45T>G in nonobese/nondiabetic Korean menindependent of abdominal adiposity and circulating plasmaadiponectin. Metabolism 2006;55:59–66.

[24] Wu LS, Hsieh CH, Pei D, et al. Association and interactionanalyses of genetic variants in ADIPOQ, ENPP1, GHSR,PPARgamma and TCF7L2 genes for diabetic nephropathy in aTaiwanese population with type 2 diabetes. Nephrol DialTransplant 2009;24:3360–6.

[25] Bostrom MA, Freedman BI, Langefeld CD, et al. Association ofadiponectin gene polymorphisms with type 2 diabetes in anAfrican American population enriched for nephropathy.Diabetes 2009;58:499–504.

[26] Rudofsky Jr G, Schlimme M, Schlotterer A, et al. Noassociation of the 94T/G polymorphism in the adiponectingene with diabetic complications. Diabetes Obes Metab2005;7:455–9.

[27] Diagnosis and classification of diabetes mellitus. DiabetesCare 2012;35(Suppl 1):S64–71.

[28] Kang ES, Park SY, Kim HJ, et al. The influence of adiponectingene polymorphism on the rosiglitazone response in patientswith type 2 diabetes. Diabetes Care 2005;28:1139–44.

[29] Kang ES, Cha BS, Kim HJ, et al. The 11482G >A polymorphismin the perilipin gene is associated with weight gain withrosiglitazone treatment in type 2 diabetes. Diabetes Care2006;29:1320–4.

[30] Friedewald WT, Levy RI, Fredrickson DS. Estimation of theconcentration of low-density lipoprotein cholesterol inplasma, without use of the preparative ultracentrifuge. ClinChem 1972;18:499–502.

[31] Kang ES, Kim HJ, Ahn CW, et al. Relationship of serum highsensitivity C-reactive protein to metabolic syndrome and

685M E T A B O L I S M C L I N I C A L A N D E X P E R I M E N T A L 6 2 ( 2 0 1 3 ) 6 7 7 – 6 8 5

microvascular complications in type 2 diabetes. Diabetes ResClin Pract 2005;69:151–9.

[32] Kim SH, Kang ES, Hur KY, et al. Adiponectin genepolymorphism 45T>G is associated with carotid arteryplaques in patients with type 2 diabetes mellitus. Metabolism2008;57:274–9.

[33] Vincent AM, Callaghan BC, Smith AL, et al. Diabeticneuropathy: cellular mechanisms as therapeutic targets. NatRev Neurol 2011;7:573–83.

[34] Ukinc K, Ersoz HO, Karahan C, et al. Methyltetrahydrofolatereductase C677T gene mutation and hyperhomocysteinemiaas a novel risk factor for diabetic nephropathy. Endocrine2009;36:255–61.

[35] Costa V, Ciccodicola A. Is PPARG the key gene in diabeticretinopathy? Br J Pharmacol 2012;165:1–3.

[36] Zietz B, Buechler C, Kobuch K, et al. Serum levels ofadiponectin are associated with diabetic retinopathy andwith adiponectin gene mutations in Caucasian patients withdiabetes mellitus type 2. Exp Clin Endocrinol Diabetes2008;116:532–6.

[37] Li FY, Cheng KK, Lam KS, et al. Cross-talk between adiposetissue and vasculature: role of adiponectin. Acta Physiol (Oxf)2011;203:167–80.

[38] Ouchi N, Kihara S, Arita Y, et al. Novel modulator forendothelial adhesion molecules: adipocyte-derived plasmaprotein adiponectin. Circulation 1999;100:2473–6.

[39] Arita Y, Kihara S, Ouchi N, et al. Adipocyte-derived plasmaprotein adiponectin acts as a platelet-derived growthfactor-BB-binding protein and regulates growthfactor-induced common postreceptor signal in vascularsmooth muscle cell. Circulation 2002;105:2893–8.

[40] Qi L, Doria A, Manson JE, et al. Adiponectin genetic variability,plasma adiponectin, and cardiovascular risk in patients withtype 2 diabetes. Diabetes 2006;55:1512–6.

[41] Rivero A, Mora C, Muros M, et al. Pathogenic perspectives forthe role of inflammation in diabetic nephropathy. Clin Sci(Lond) 2009;116:479–92.

[42] Soldatos G, Cooper ME. Diabetic nephropathy: importantpathophysiologic mechanisms. Diabetes Res Clin Pract2008;82(Suppl 1):S75–9.