Association of adiponectin gene polymorphisms with an elevated risk of diabetic peripheral neuropathy in type 2 diabetes patients

Journal of Diabetes and Its Complications 29 (2015) 887–892

Contents lists available at ScienceDirect

Journal of Diabetes and Its Complications journal homepage: WWW.JDCJOURNAL.COM

Association of adiponectin gene polymorphisms with an elevated risk of diabetic peripheral neuropathy in type 2 diabetes patients Zhi-Yong Ji a, Hai-Feng Li a, Yu Lei a, Yan-Wei Rao a, Zeng-Xian Tan b, Huai-Jun Liu c, Gen-Dong Yao d, Bo Hou e, Ming-Li Sun a,⁎ a

Department of Emergency, First Affiliated Hospital of Jilin University, Changchun130031, P. R. China Department of Intervention, Handan Central Hospital, Handan 056001, P. R. China c Department of Radiology, the Second Hospital of Hebei Medical University, Shi Jiazhuang 050050, P. R. China d Department of Function, Handan Central Hospital, Handan 056001, P. R. China e Department of Computed Tomography, Handan Central Hospital, Handan 056001, P. R. China b

a r t i c l e

i n f o

Article history: Received 2 March 2015 Received in revised form 8 June 2015 Accepted 14 June 2015 Available online 19 June 2015 Keywords: Type 2 diabetes mellitus Diabetic peripheral neuropathy Fasting plasma glucose Glycosylated hemoglobin Single nucleotide polymorphism

a b s t r a c t Objective: In this study, we examined the association between two adiponectin (ADPN) gene polymorphisms, + 45 T/G and + 276G/T, and susceptibility to diabetic peripheral neuropathy (DPN) in type 2 diabetes mellitus (T2DM) patients. Methods: A total of 180 T2DM patients were enrolled in this study and assigned to two groups: DPN group (n = 90) and non-DPN (NDPN) group (n = 90). In addition, 90 healthy subjects were chosen as healthy normal control (NC). The plasma level of ADPN was quantified by ELISA method and polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) was used for genotype analysis of the two ADPN polymorphisms, +45 T/G (rs2241766) and + 276G/T (rs1501299), in all the study subjects. Statistical analysis of data was performed with SPSS version 20.0 software. Results: Serum levels of ADPN were markedly reduced in the DPN group compared to NDPN and NC groups (all P b 0.05). The frequencies of TT, TG and GG genotypes and the T and G alleles of T45G and G276T polymorphisms in DPN group were significantly different than the NDPN group (all P b 0.05). Notably, T45G and G276T polymorphisms were associated with significantly reduced plasma levels of ADPN in DPN and NDPN groups, compared to the NC group (P b 0.001). Significant difference in ADPN plasma levels were also observed between TT, TG and GG genotypes of T45G and G276T polymorphisms. Our results indicate that the T allele in + 45 T/G and + 276G/T polymorphisms is correlated with an elevated risk of DPN in T2DM patients. Haplotype analysis showed that GG and GT haplotypes showed a negative relationship with DPN, while TG haplotype positively correlated with risk of DPN in T2DM patients (all P b 0.05). Conclusion: Our results show that T45G and G276T polymorphisms of ADPN are associated with a significantly elevated risk of DPN in T2DM patients, likely by down-regulating ADPN serum level. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Peripheral neuropathy is a frequent complication in type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) patients (Davies et al., 2006). Diabetic peripheral neuropathy (DPN) is a serious global health problem because diabetic patients sustain extensive nerve damage and peripheral nerve dysfunction, without any overt symptoms, and DPN is often diagnosed in these patients by excluding other potential causes (He et al., 2014; Kolla et al., 2009). In Conflict of interest: none. ⁎ Corresponding author at: Department of Emergency, First Affiliated Hospital of Jilin University, No.456 Jilinda Road, Erdao District, Changchun 130031, P. R. China. Tel./fax: + 86 18237372897. E-mail addresses: [email protected] (H-J. Liu), [email protected] (M.-L. Sun). http://dx.doi.org/10.1016/j.jdiacomp.2015.06.008 1056-8727/© 2015 Elsevier Inc. All rights reserved.

western countries, DPN exhibits high incidence rates in diabetics at 60%–90% each year, with no gender differences. The incidence of distal sensory neuropathy is 4% in first the five years following the diabetes onset, but is as high as 20% within the next twenty years (Allen et al., 2014; Raafat & Samy, 2014). The fatality rate of DPN is 44% within 2.5 years and 56% in 5 years, with half the deaths resulting from renal failure and the other half attributed to sudden respiratory circulation arrest and hypoglycemia (Ponirakis et al., 2014; Razazian et al., 2014). The underlying factors in DPN progression include hyperglycemia, metabolic disorders, vascular injury, neurotrophic factor deficiency, abnormal cytokine release, oxidative stress and immune factors, with hyperglycemia as the most prominent factor leading to DPN (Basol et al., 2013; Funnell, 2014; Ziegler et al., 2014). Not surprisingly, almost 50% of T2DM patients are diagnosed with DPN and chronically suffer agonizing pain caused by peripheral nerve damage. The diabetes

888

Z.-Y. Ji et al. / Journal of Diabetes and Its Complications 29 (2015) 887–892

control and complications trial showed that rigid glycemic control effectively decreased or halted the progression of DPN and 35% patients successfully achieved rigid glycemic control within ten years (Deng et al., 2014; Jensen et al., 2014; Tang et al., 2012). In this context, adiponectin (ADPN) is a hormone secreted by fat cells and plays an important role in DPN because ADPN regulates lipid and carbohydrate metabolism (Jeong et al., 2012; Otsuka et al., 2006; Tuttolomondo et al., 2010). ADPN circulates at high concentrations in healthy human plasma and participates in anti-inflammation, insulin sensitivity and cardioprotective mechanisms (Ando et al., 2013; Jeong et al., 2012; Siitonen et al., 2011). ADPN protein is encoded by the ADPN gene, which is mapped to chromosome 3q27, a susceptibility locus for metabolic syndrome and diabetes. The human ADPN spans 17 kb and consists of three exons and two introns (Hu et al., 1996; Scherer et al., 1995). ADPN is also the major adipokine in plasma, constituting approximately 0.01% of the total plasma protein (Sandy An et al., 2013). ADPN decreases insulin resistance by promoting glucose uptake in skeletal muscles, enhances fatty-acid oxidation in the liver and skeletal muscles, reduces triglyceride levels and inhibits gluconeogenesis and glucose output (Abdelgadir et al., 2013; Yadav et al., 2013). Importantly, plasma ADPN levels negatively correlate with insulin sensitivity and reduced plasma ADPN level can lead to insulin resistance and accelerate diabetes progression (Abdelgadir et al., 2013; Han et al., 2011; Sandy An et al., 2013). Single-nucleotide polymorphisms (SNPs) in ADPN gene are critical to human physiology because plasma levels of ADPN are dramatically influenced by these polymorphisms, among which + 45 T/G in exon 2 and + 276G/T in intron 2 increase insulin resistance and body weight by reducing ADPN expression levels (Al-Daghri et al., 2011; Han et al., 2011). Curiously, the + 45 T/G variant is associated with increased morbidity in T2DM patients from China and Japan and the + 276G/T variant is linked with higher morbidity inT2DM patients from Poland and Japan (Esteghamati et al., 2012; Mtiraoui et al., 2012). Previous studies presented a strong evidence of a high contribution of heredity factors in altering ADPN plasma levels, with the influence by genetic factors ranging between 30% and 70%, and these factors are tightly associated with elevated risk of diabetes (Chung et al., 2011; Murea et al., 2012). Although a few previous studies examined the association between ADPN polymorphisms and diabetes (Al-Daghri et al., 2011; Udomsinprasert et al., 2012), the limited data available and the contradictory results from these studies prompted us to undertake a systematic analysis of two ADPN polymorphisms (+ 45 T/G and + 276G/T variants) for their affect on the plasma ADPN levels and risk of DPN in T2DM patients. In this study, our aim was to develop reliable tools to identify T2DM patients at high-risk for DPN. 2. Materials and methods 2.1. Ethics statement The study was approved by the Institutional Review Board of First Affiliated Hospital of Jilin University. The written informed consent was obtained from each eligible patient and the entire study was performed based on the Declaration of Helsinki. 2.2. Subjects and experiment group The study was conducted as a case–control design. A total of 180 T2DM patients were enrolled at the First Affiliated Hospital of Jilin University between May 2012 and August 2013. All T2DM patients were diagnosed based on the 1999 WHO criteria: fasting plasma glucose (FPG) ≥ 7.0 mmol/L or 2-h PG ≥ 11.1 mmol/L (Gabir et al., 2000). Inclusion criteria for DPN patients were: clinical manifestation with acroparesthesia or motor nerve involvement; reduced degree of deep and superficial sensation; reduced sensory nerve conduction

velocity (SCV) and motor nerve conduction velocity (MNCV). Eligible T2DM patients were assigned into two groups: DPN group and non-DPN (NDPN) group. DPN group included 90 patients (46 males and 44 females), with a mean age of 54.1 ± 5.6 years, and NDPN group consisted of 90 patients (50 males and 40 females), with a mean age of 54.9 ± 5.1 years. In addition, ninety healthy subjects (40 males and 50 females), with a mean age of 53.5 ± 5.0 years, were chosen as healthy normal control (NC). 2.3. Baseline data collection Data related to baseline characteristics of the study subjects were collected and recorded, including gender, age, body mass index (BMI), diabetes duration and waist-to-hip ratio (WHR). Waist circumference (waistline) was measured at the midpoint between iliac crest and costal margin at the mid-axillary line and hipline was measured from left greater trochanter marker and right greater trochanter marker. Glycosylated hemoglobin (HbA1c) was measured by high performance liquid chromatography (HPLC, Bio-Rad Laboratories, Hercules, CA, USA). Fasting plasma glucose (FPG) was measured by glucose oxidase test (using assay kit from Shanghai Kehua Bio-Engineering Co., Ltd). Total cholesterol (TC) and triglyceride (TG) were measured by standard enzyme-based colorimetric assays and the assay kits for TC and TG were obtained from Yantai Ausbio Biochemical Engineering Co., Ltd. High density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were measured by direct detection methods (using assays kit from Daiichi Pure Chemicals Co., Ltd.). 2.4. ADPN level After overnight fasting, forearm venous blood samples (2 × 5 ml) were collected through venipuncture from all T2DM patients and healthy controls. The blood was centrifuged at 2700 rpm for 10 min at room temperature and the supernatant was collected and placed in EDTA containing tubes and stored at − 80 °C until further use. Total plasma ADPN concentration was measured using Enzyme-linked immunosorbent assay (ELISA) with components from ADPN detection kit (Innogent Company, Shenzhen). 2.5. Detection of ADPN gene + 45 T/G and + 276G/T polymorphisms Genomic DNA from whole blood samples was extracted using whole blood genomic DNA extraction kit (Shanghai Sangon, SK8224), according to manufacturer’s instructions. Laboratory personnel were blinded to the case–control status of the subjects. ADPN + 45 T/G (rs2241766) and + 276G/T (rs1501299) were genotyped by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP). Primer sequences for ADPN + 45 T/G (rs2241766) and + 276G/T (rs1501299) are shown in Table 1. PCR amplification was performed in a total volume of 20 μl reaction mixture containing: genomic DNA (4.0 μl), Taq DNA polymerase (5 U/μl) (Shanghai Sangon, SK2492), 10 × PCR buffer (2.0 μl), 10 mmol/L dNTP Mix (0.6 μl), 25 mmol/L MgCl2 (1.8 μl), upstream primer and downstream primer (per 0.5 μl) (20 μmol/L, Shanghai Sangon), and double distilled water (10.3 μl). PCR conditions were: predenaturing (94 °C for 2 min), denaturing (94 °C for 40 s), annealing (60.5 °C for 60 s), Table 1 Primer sequences for ADPN +45 T/G (rs2241766) and +276G/T (rs1501299). Primer

Sequences

G276T

F: 5'-CTCCTACACTGATATAAACTATATGAAT-3 R: 5'-AATGTACTGGGAATAGGGATGA-3' F: 5'-CTCCCTGTGTCTAGGCCTTAC-3' R: 5'-TAGAAGTAGACTCTGCTGAGATG-3'

+45T/G F: forward; R: reverse.

Z.-Y. Ji et al. / Journal of Diabetes and Its Complications 29 (2015) 887–892

and elongation (72 °C for 40 s), amplified for 35 cycles and final elongation at 72 °C for 10 min. Each sample was analyzed by PCR at least three times. After completion of the + PCR, 5 μl of PCR products were analyzed by agarose gel electrophoresis and the results were recorded using gel-imaging system (Kodak). PCR products were digested overnight at 37 °C with Eco88I for + 45 T/G (rs2241766) and BsmI for + 276G/T (rs1501299) and separated on 2% agarose gel. To confirm the genotyping results, randomly selected PCR samples were examined by DNA sequencing. 2.6. Statistical analyses Continuous variables with normal distribution were expressed as mean ± standard deviation (SD). The t-test was used to measure differences between two groups of continuous variables, while One-way analysis of variance (ANOVA) was used to test the differences among three groups of continuous variables. Categorical data were presented with frequency counts or ratios, and analyzed by chi-square test. Gene counting was employed to estimate the genotype/allele frequencies and departure from Hardy–Weinberg equilibrium (HWE) was detected in healthy controls and T2DM patients separately. The association between SNPs and T2DM was assessed by χ 2 tests. Haplotypes at + 45 T/G and + 276G/T of ADPN gene were analyzed in DPN patients and normal controls using SHEsis software platform (http://analysis.bio-x.cn). Adjusted odds ratios (ORs) with 95% confidence intervals (95% CIs) for T2DM were calculated by logistic regression. All statistical analyses were performed using SPSS program version 20.0 (SPSS Inc., Chicago, IL). Statistical significance was set at P b 0.05.

889

were markedly decreased, compared with NC group (all P b 0.05). In addition, compared with NDPN group, plasma ADPN level in DPN group was dramatically reduced, and this reduction was statistically significant (P b 0.05). 3.2. Distribution of + 45 T/G and + 276 G/T polymorphisms of the ADPN gene The distribution of T45G and G276T polymorphisms in the three groups is shown in Table 3. The genotype distributions of T45G and G276T SNPs in the NC group were in agreement with HWE (T45G: χ 2 = 1.27, P = 0.260; G276T: χ 2 = 0.002, P = 0.965). The frequencies of TT, TG and GG genotypes of T45G SNP in the DPN group were significantly different between NDPN group and NC group (all P b 0.05), while the frequencies of TT, TG and GG genotypes of T45G SNP did not exhibit a statistically significant difference between NDPN group and NC group (all P N 0.05). Compared with NDPN and NC groups, DPN patients displayed lower frequency of TG and GG genotypes of T45G SNP and the TT genotype at a higher frequency (all P b 0.05). Additionally, the frequencies of T and G alleles of + 45 T/G polymorphism in the DPN group were markedly different between NDPN and NC groups (all P b 0.05). With respect to + 276 G/ T polymorphism, we found no significant difference in the frequencies of TT, TG and GG genotypes between NDPN group and NC group (P N 0.05), while the frequencies of T and G alleles displayed a marked difference between NDPN group and NC group (P b 0.05). Further, the frequencies of TT, TG and GG genotypes and T and G alleles of 276 G/T SNP in DPN group were significantly different compared withNDPN and NC groups (all P b 0.05).

3. Results 3.3. Effect of ADPN gene SNPs on ADPN levels 3.1. Study subjects The baseline characteristics of the study subjects in DPN group, NDPN group and NC group are shown in Table 2. FPG and HbA1C levels were significantly increased in T2DM patients compared with normal controls, while HDL-C level decreased in T2DM patients compared to NC group (all P b 0.05). However, no significant differences were detected in age, gender, BMI, WHR, TC, TG and LDL-C among the three groups (all P N 0.05). Additionally, there was also no significant difference in the disease course between DPN and NDPN groups (P N 0.05). Compared with NC group, FPG and HbA1c levels were significantly increased in DPN group and NDPN group (all P b 0.05) and HDL-C level decreased in both DPN and NDPN patients (all P b 0.05). The plasma ADPN levels in both DPN and NDPN groups

Next, we sought to understand the relationship between T45G and G276T SNPs and the circulating ADPN levels in plasma. Plasma ADPN levels associated with T45G and G276T were significantly reduced in the DPN and NDPN groups, compared to the NC group, irrespective of the SNP (P b 0.001), as shown in Fig. 1. In addition, significant differences in plasma ADPN levels were observed among the TT, TG and GG genotypes of T45G polymorphism (all P b 0.001) and the plasma ADPN level in carriers of TT genotype was significantly lower than carriers with TG and GG genotypes (both P b 0.05). There were significant differences in ADPN plasma levels among TT, TG and GG genotypes of G276T polymorphism (all P b 0.001) and ADPN plasma level in carriers with TT genotype was also significantly lower than carriers with GG and GT genotypes (both P b 0. 05).

Table 2 Baseline characteristics of the DPN group (patients with diabetic peripheral neuropathy), NDPN group (patients with non-diabetic peripheral neuropathy) and NC group (normal control). Parameters

NC (n = 90)

NDPN (n = 90)

P⁎

DPN (n = 90)

P#

P&

Male/Female Age (years) Course of disease (years) BMI (kg/m2) WHR TC (mmol/L) TG (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L) FPG (mmol/L) HbA1c (%) ADPN serum level

40/50 53.5 ± 5.0 – 24.5 ± 2.8 0.93 ± 0.06 4.88 ± 0.82 2.01 ± 1.37 1.46 ± 0.36 2.78 ± 0.71 5.6 ± 1.4 5.2 ± 0.6 11.4 ± 3.0

50/40 54.9 ± 5.1 10.8 ± 5.8 24.4 ± 2.5 0.94 ± 0.09 4.96 ± 2.74 2.07 ± 0.97 1.12 ± 0.49 2.90 ± 0.88 13.8 ± 4.8 7.9 ± 1.2 7.4 ± 2.1

0.136 0.065 – 0.801 0.382 0.791 0.735 b0.001 0.315 b0.001 b0.001 b0.001

46/44 54.1 ± 5.6 11.8 ± 6.3 24.2 ± 2.1 0.95 ± 0.08 4.78 ± 1.09 2.04 ± 1.45 1.18 ± 0.34 2.73 ± 0.82 14.6 ± 5.0 8.1 ± 1.6 7.2 ± 1.3

0.371 0.449 – 0.417 0.059 0.488 0.887 b0.001 0.662 b0.001 b0.001 b0.001

0.550 0.318 0.269 0.562 0.432 0.563 0.871 0.341 0.182 0.275 0.344 0.022

DPN: diabetic peripheral neuropathy; NDPN: non-diabetic peripheral neuropathy; NC: normal control group; BMI: body mass index; WHR: waist-to-hip ratio; TC: total cholesterol; TG: total triglyceride; HDL-C: high density lipoprotein cholesterol; LDL-C: low density lipoprotein cholesterol; HbA1c: glycosylated hemoglobin; FPG: fasting plasma glucose; ADPN: adiponectin. *, NDPN group compared with NC group, P b 0.05; #, DPN group compared with NC group, P b 0.05; &, DPN group compared with NDPN group, P b 0.05.

890

Z.-Y. Ji et al. / Journal of Diabetes and Its Complications 29 (2015) 887–892

Table 3 Genotypes and allele frequencies of ADPN gene, +45 T/G (rs2241766) and +276 G/T (rs1501299) polymorphisms, in patients with diabetic peripheral neuropathy (DPN group), non-diabetic peripheral neuropathy patients (NDPN group) and normal healthy controls (NC group) (n/%).

+45T/G

+276G/T

Genotypes/Allele

NC (n = 90)

NDPN (n = 90)

P⁎

DPN (n = 90)

P#

P&

TT TG GG T G TT TG GG T G

45 (50.0%) 34 (37.8%) 11 (12.2%) 124 (68.9%) 56 (31.1%) 47 (52.2) 37 (41.1%) 6 (6.7%) 131 (72.8%) 49 (27.2%)

44 26 20 114 66 38 36 16 112 68

0.158

73 (81.1%) 12 (13.3%) 5 (5.6%) 158 (87.8%) 22 (12.2%) 70 (77.8%) 18 (20.0%) 2 (2.22%) 158 (87.8%) 22 (12.2%)

b0.001

b0.001

b0.001

b0.001

0.001

b0.001

b0.001

b0.001

(48.9%) (28.9%) (22.2%) (63.3%) (36.7%) (42.2%) (40.0%) (27.8%) (62.2%) (37.8%)

0.316 0.064

0.043

ADPN: adiponectin; DPN: diabetic peripheral neuropathy; NDPN: non-diabetic peripheral neuropathy; NC: normal control group. *, NDPN group compared with NC group, P b 0.05; #, DPN group compared with NC group, P b 0.05; &, DPN group compared with NDPN group, P b 0.05.

3.4. Associations between SNPs in ADPN gene and DPN in T2DM patients We tested the associations of T45G and G276T SNPs with DPN risk in T2DM patients. The frequency of TT genotype of + 45 T/G polymorphism in DPN group was significant higher than the NC group, suggesting that TT genotype may be a high risk factor for DPN in T2DM patients (OR = 3.569, 95%CI = 1.164–10.95) (Table 4). Further, the frequency of T allele of + 45 T/G and + 276G/T polymorphisms in DPN group was also significantly higher than the NC group, suggesting that the T allele confers significantly higher risk of DPN in T2DM patients (+ 45 T/G: OR = 3.243, 95%CI = 1.878– 5.602; + 276G/T: OR = 3.243, 95%CI = 1.878–5.602), as shown in Table 5. 3.5. Haplotypes analysis of ADPN gene + 45 T/G and + 276G/T in DPN patients and normal healthy controls Haplotypes at positions + 45 T/G and + 276G/T in ADPN gene in DPN patients and normal controls are shown in Table 6. Haplotypes of the SNPs were analyzed by SHEsis software. The results showed that the frequencies of three haplotypes (TG, GG and GT) in ADPN gene displayed significant differences between DPN group and NC group (all P b 0.05). Further, the frequencies of GG and GT haplotypes in DPN group were significantly lower than NC group, while the frequency of TG haplotype in the DPN group was markedly higher than the NC group (all P b 0.05). 4. Discussion ADPN serves as a protective factor in preventing diabetes progression by suppressing inflammatory responses and increasing insulin sensitivity (Otsuka et al., 2006; Semple et al., 2006). Several

SNPs of ADPN may influence T2DM, but ADPN polymorphisms SNP45 (+ 45 T/G, rs2241766) and SNP276 (+ 276G/T, rs1501299) are the two most prominent polymorphisms influencing the disease progression (Esteghamati et al., 2012; Yu et al., 2011). Our results suggest that + 45 T/G (rs2241766) and + 276G/T (rs1501299) confer dramatically increased risk of DPN in T2DM patients by down-regulating ADPN expression, resulting in significantly reduced circulating ADPN plasma levels. In addition, the frequencies of GG and GT haplotypes in DPN group were also significantly lower than the NC group, while the frequency of TG haplotype in the DPN group was markedly higher than NC group. These results suggest that GG and GT haplotypes are negatively correlated with the risk of DPN, while TG haplotype is positively correlated with DPN risk in T2DM patients. The T45G polymorphism in ADPN gene is located in exon 2 near the exon–intron junction, influencing mRNA splicing or mRNA stability (Al-Daghri et al., 2011; Han et al., 2011), resulting in decreased ADPN expression and elevating DPN risk in T2DM patients (Biswas et al., 2011; Low et al., 2011). The +276G/T polymorphism in ADPN gene is a mutation in the intron 2, but a previous study showed a strong linkage disequilibrium between SNP 276 and G90S missense mutation (Kacso et al., 2012), indicating that altered pre-mRNA splicing may result in lower ADPN expression. The SNP 276 in ADPN gene is also tightly linked with the SNP within the promoter region and reduces mRNA levels by altering pre-mRNA splicing (Li et al., 2011). Several variants within this region have remarkable effects on T2DM development and insulin sensitivity, indicating the significance of ADPN gene polymorphisms in T2DM and DPN disease progression (Huang et al., 2010). On the other hand, the + 45 T/G (rs2241766) and + 276G/T (rs1501299) polymorphisms within exon 2 and intron 2, respectively, are in a linkage disequilibrium block (Menzaghi et al., 2007). The two polymorphisms may also be in linkage disequilibrium with other functional gene loci that alter ADPN synthesis or its ability to

Fig. 1. Associations of ADPN gene, +45 T/G (rs2241766) and +276 G/T (rs1501299) polymorphism, with the ADPN serum levels among the patients with diabetic peripheral neuropathy (DPN group), non-diabetic peripheral neuropathy patients (NDPN group) and normal healthy controls (NC group). Note: *, compared with NC group, P b 0.05; #, compared with GG genotype, P b 0.05.

Z.-Y. Ji et al. / Journal of Diabetes and Its Complications 29 (2015) 887–892 Table 4 Associations of ADPN +45 T/G (rs2241766) polymorphism with the risk of diabetic peripheral neuropathy in type 2 diabetes patients. Genotypes

DPN

NC

GG TG TT G T

5 (5.6%) 12 (13.3%) 73 (81.1%) 22 (12.2%) 158 (87.8%)

11 (12.2%) 34 (37.8%) 45 (50.0%) 56 (31.1%) 124 (68.9%)

P value 0.690 0.020 b0.001

OR Ref. 0.777 3.569 Ref. 3.243

95%CI

1.878–5.602

DPN: diabetic peripheral neuropathy; NC: normal control group, OR: odds radio; 95%CI: 95% confidence intervals; Ref.: reference.

polymerize, severely altering its biological activity (Waki et al., 2003). Therefore, further studies are needed to confirm the direct role of the two polymorphisms in DPN progression in T2DM patients. Our study also showed that ADPN plasma levels were significantly decreased in T2DM patients with DPN, and SNP45 and SNP276 were strongly linked with reduced ADPN serum levels. ADPN plays a crucial protective role against inflammation and atherosclerosis (Goldstein et al., 2009; Gustafson, 2010; Kassab & Piwowar, 2012; Luo et al., 2010). ADPN protects against inflammation by polarizing macrophages towards anti-inflammatory phenotype, leading to wound-healing and resolution of inflammation. ADPN also regulates a variety of receptor-mediated signaling pathways in several cell types to influence metabolic pathways. Therefore, down-regulation of ADPN serum level is intimately linked with chronic inflammation and metabolic disturbances (Ohashi et al., 2010). ADPN also inhibits production of inflammatory cytokine secretion from monocyte-derived macrophages, prevents adhesion of monocytes to endothelial cells and suppresses macrophage-to-foam cell transformation (Halvatsiotis et al., 2010). In addition, ADPN reduces the intracellular levels of cholesteryl ester in human macrophages by inhibiting class A scavenger receptor (sR-A) expression (Ouchi et al., 2011). Our data showed that serum levels of ADPN in DPN group were significantly decreased compared to the NDPN group, which was consistent with previous studies (Esteghamati et al., 2012; Li et al., 2011; Sun et al., 2008). Compared to the serum levels of other cytokines, ADPN level is more than 1000-fold higher in normal healthy individuals, indicating the significant importance of maintaining high circulating levels of ADPN in plasma. ADPN activates AMPK and p38MAPK signaling pathways and reduced plasma levels of ADPN interfere with normal functioning of these important pathways, promoting diabetic phenotype (Zhang et al., 2011). Altered lipid regulation, as a result of low plasma ADPN levels, leads to accumulation of fat metabolites in Schwann cells, which impacts peripheral nerve functions in diabetic patients (Askwith et al., 2009). In this context, a previous study noted that SNP45 and SNP276 are commonly associated with T2DM and insulin resistance (Huang et al., 2010). Lee et al., also suspected that these polymorphisms lower plasma ADPN levels. Our direct results presented in this study strongly support these previous conclusions (Lee et al., 2013).

Table 5 Associations of ADPN +276G/T (rs1501299) polymorphism with the risk of diabetic peripheral neuropathy in type 2 diabetes patients. Genotypes

DPN

NC

GG TG TT G T

2 (2.22%) 18 (20.0%) 70 (77.8%) 22 (12.2%) 158 (87.8%)

6 (6.7%) 37 (41.1%) 47 (52.2%) 49 (27.2%) 131 (72.8%)

P value 0.661 0.054 b0.001

OR Ref. 1.459 4.468 Ref. 2.686

Table 6 Haplotypes analysis of ADPN gene +45 T/G and +276G/T in patients with diabetic peripheral neuropathy (DPN group) and normal healthy controls (NC group). Haplotypes +45 T/G +276G/T

0.224–2.698 1.164–10.95

95%CI 0.267–7.965 0.864–23.10 1.544–4.674

DPN: diabetic peripheral neuropathy; NC: normal control group, OR: odds radio; 95%CI: 95% confidence intervals; Ref.: reference.

891

T T G G

G T G T

DPN (n = 90)

NC χ2 (n = 90)

P value

69 10 10 1

45 17 20 8

0.000 0.144 0.046 0.017

13.78 2.135 4.000 5.731

OR

95%CI Lower Upper limit limit

0.304 0.161 1.863 0.802 2.286 1.002 8.683 1.062

0.577 4.330 5.212 70.97

DPN: diabetic peripheral neuropathy; NC: normal control group, OR: odds radio; 95%CI: 95% confidence intervals.

Our study also has several limitations. First, the genotype distribution in ADPN gene may be influenced by ethnicity. In our study, only Chinese subjects were included due to geographical restrictions and we detected the genotypes and alleles of ADPN polymorphisms among Chinese populations, which may have resulted in subject selection bias. Thus, it is essential to perform related studies to confirm the associations of ADPN polymorphisms with DPN risk in T2DM patients in other ethnicities. Second, the genetic studies might be affected by random variation due to small sample size, therefore, further studies should be conducted to evaluate ADPN polymorphisms with DPN risk in T2DM patients with a larger sample size to achieve a more accurate and systematic outcome. Third, the observed changes in ADPN serum levels may also have been affected by other influencing factors such as BMI, WHR, glycemic, HDL-C and insulin resistance index. In this study, only the association between ADPN gene polymorphisms and ADPN serum level was examined. Thus, further studies are required to confirm our results. Finally, the affect of ADPN polymorphisms in conferring an increased risk of DPN may be significantly influenced by polymorphisms in other related genes, and this may have influenced the results in our study. In conclusion, our results provide evidence that two ADPN gene polymorphisms, T45G and G276T, confer high risk of susceptibility to DPN in T2DM patients as a result of loss of protective effects of ADPN caused by reduced plasma ADPN levels. Our results support the use of these two polymorphisms as biomarkers for early identification of high-risk T2DM patients and recommend appropriate clinical intervention to halt DPN progression in these patients. Acknowledgments We would like to acknowledge our instructors who gave us lots of valuable advices. We also thank the reviewers for their precious comments on this paper. References Abdelgadir, M., Karlsson, A. F., Berglund, L., & Berne, C. (2013). Low serum adiponectin concentrations are associated with insulin sensitivity independent of obesity in Sudanese subjects with type 2 diabetes mellitus. Diabetology and Metabolic Syndrome, 5(1), 15. http://dx.doi.org/10.1186/1758-5996-5-15 [PMID: 23497407]. Al-Daghri, N. M., Al-Attas, O. S., Alokail, M. S., Alkharfy, K. M, & Hussain, T. (2011). Adiponectin gene variants and the risk of coronary artery disease in patients with type 2 diabetes. Molecular Biology Reports, 38(6), 3703–3708. Allen, R., Sharma, U., & Barlas, S. (2014). Clinical experience with desvenlafaxine in treatment of pain associated with diabetic peripheral neuropathy. The Journal of Pain Research, 7, 339–351. Ando, T., Ishikawa, T., Takagi, T., Imamoto, E., Kishimoto, E., Okajima, A., et al. (2013). Impact of Helicobacter pylori eradication on circulating adiponectin in humans. Helicobacter, 18(2), 158–164. Askwith, T., Zeng, W., Eggo, M. C., & Stevens, M. J. (2009). Oxidative stress and dysregulation of the taurine transporter in high-glucose-exposed human Schwann cells: Implications for pathogenesis of diabetic neuropathy. American Journal of Physiology. Endocrinology and Metabolism, 297(3), E620–E628. Basol, N., Inanir, A., Yigit, S., Karakus, N., & Kaya, S. U. (2013). High association of IL-4 gene intron 3 VNTR polymorphism with diabetic peripheral neuropathy. Journal of Molecular Neuroscience, 51(2), 437–441.

892

Z.-Y. Ji et al. / Journal of Diabetes and Its Complications 29 (2015) 887–892

Biswas, D., Vettriselvi, V., Choudhury, J., & Jothimalar, R. (2011). Adiponectin gene polymorphism and its association with type 2 diabetes mellitus. Indian Journal of Clinical Biochemistry, 26(2), 172–177. Chung, C. M., Lin, T. H., Chen, J. W., Leu, H. B., Yang, H. C., Ho, H. Y., et al. (2011). A genome-wide association study reveals a quantitative trait locus of adiponectin on CDH13 that predicts cardiometabolic outcomes. Diabetes, 60(9), 2417–2423. Davies, M., Brophy, S., Williams, R., & Taylor, A. (2006). The prevalence, severity, and impact of painful diabetic peripheral neuropathy in type 2 diabetes. Diabetes Care, 29(7), 1518–1522. Deng, W., Dong, X., Zhang, Y., Jiang, Y., Lu, D., Wu, Q., et al. (2014). Transcutaneous oxygen pressure (TcPO2): A novel diagnostic tool for peripheral neuropathy in type 2 diabetes patients. Diabetes Research and Clinical Practice, 105(3), 336–343. Esteghamati, A., Mansournia, N., Nakhjavani, M., Mansournia, M. A., Nikzamir, A., & Abbasi, M. (2012). Association of +45(T/G) and +276(G/T) polymorphisms in the adiponectin gene with coronary artery disease in a population of Iranian patients with type 2 diabetes. Molecular Biology Reports, 39(4), 3791–3797. Funnell, M. M. (2014). Managing the pain of diabetic peripheral neuropathy. Nursing, 44(7), 64–65. Gabir, M. M., Hanson, R. L., Dabelea, D., Imperatore, G., Roumain, J., Bennett, P. H., et al. (2000). The 1997 American Diabetes Association and 1999 World Health Organization criteria for hyperglycemia in the diagnosis and prediction of diabetes. Diabetes Care, 23(8), 1108–1112. Goldstein, B. J., Scalia, R. G., Ma, X. L., et al. (2009). Protective vascular and myocardial effects of adiponectin. Nature Clinical Practice. Cardiovascular Medicine, 6(1), 27–35. Gustafson, B. (2010). Adipose tissue, inflammation and atherosclerosis. Journal of Atherosclerosis and Thrombosis, 17(4), 332–341. Halvatsiotis, I., Tsiotra, P. C., Ikonomidis, I., Kollias, A., Mitrou, P., Maratou, E., et al. (2010). Genetic variation in the adiponectin receptor 2 (ADIPOR2) gene is associated with coronary artery disease and increased ADIPOR2 expression in peripheral monocytes. Cardiovascular Diabetology, 9, 10-1186. Han, L. Y., Wu, Q. H., Jiao, M. L., Hao, Y. H., Liang, L. B., Gao, L. J., et al. (2011). Associations between single-nucleotide polymorphisms (+ 45 T N G, + 276G N T, −11377C N G, −11391G N A) of adiponectin gene and type 2 diabetes mellitus: A systematic review and meta-analysis. Diabetologia, 54(9), 2303–2314. He, S., Pan, Y. N., & Zhang, Z. Y. (2014). Comment on Xu et al.: Meta-analysis of methylcobalamin alone and in combination with lipoic acid in patients with diabetic peripheral neuropathy. Diabetes Research and Clinical Practice, 105(3), e9. Hu, E., Liang, P., & Spiegelman, B. M. (1996). AdipoQ is a novel adipose-specific gene dysregulated in obesity. Journal of Biological Chemistry, 271(18), 10697–10703. Huang, M. C., Wang, T. N., Lee, K. T., Wu, Y. J., Tu, H. P., Liu, C. S., et al. (2010). Adiponectin gene SNP276 variants and central obesity confer risks for hyperglycemia in indigenous Taiwanese. Kaohsiung Journal of Medical Sciences, 26(5), 227–236. Jensen, V. F., Molck, A. M., Bogh, I. B., & Lykkesfeldt, J. (2014). Effect of insulin-induced hypoglycaemia on the peripheral nervous system: Focus on adaptive mechanisms, pathogenesis and histopathological changes. Journal of Neuroendocrinology, 26(8), 482–496. Jeong, H. G., Min, B. J., Lim, S., Kim, T. H., Lee, J. J., Park, J. H., et al. (2012). Plasma adiponectin elevation in elderly individuals with subsyndromal depression. Psychoneuroendocrinology, 37(7), 948–955. Kacso, I. M., Farcas, M. F., Pop, I. V., Bondor, C. I., Potra, A. R., & Moldovan, D. (2012). 276G N T polymorphism of the ADIPOQ gene influences plasma adiponectin in type 2 diabetes patients but is not predictive for presence of type 2 diabetes in a Caucasian cohort from Romania. Maedica (Buchar), 7(4), 271–276. Kassab, A., & Piwowar, A. (2012). Cell oxidant stress delivery and cell dysfunction onset in type 2 diabetes. Biochimie, 94(9), 1837–1848. Kolla, V. K., Madhavi, G., Pulla Reddy, B., Srikanth Babu, B. M., Yashovanthi, J., Valluri, V. L., et al. (2009). Association of tumor necrosis factor alpha, interferon gamma and interleukin 10 gene polymorphisms with peripheral neuropathy in South Indian patients with type 2 diabetes. Cytokine, 47(3), 173–177. Lee, K. Y., Kang, H. S., & Shin, Y. A. (2013). Exercise improves adiponectin concentrations irrespective of the adiponectin gene polymorphisms SNP45 and the SNP276 in obese Korean women. Gene, 516(2), 271–276. Li, Y., Li, X., Shi, L., Yang, M., Yang, Y., Tao, W., et al. (2011). Association of adiponectin SNP + 45 and SNP + 276 with type 2 diabetes in Han Chinese populations: A meta-analysis of 26 case–control studies. PLoS ONE, 6(5), e19686. Low, C. F., Mohd Tohit, E. R., Chong, P. P., & Idris, F. (2011). Adiponectin SNP45TG is associated with gestational diabetes mellitus. Archives of Gynecology and Obstetrics, 283(6), 1255–1260. Luo, N., Liu, J., Chung, B. H., Yang, Q., Klein, R. L., Garvey, W. T., et al. (2010). Macrophage adiponectin expression improves insulin sensitivity and protects against inflammation and atherosclerosis. Diabetes, 59(4), 791–799. Menzaghi, C., Trischitta, V., & Doria, A. (2007). Genetic influences of adiponectin on insulin resistance, type 2 diabetes, and cardiovascular disease. Diabetes, 56(5), 1198–1209.

Mtiraoui, N., Ezzidi, I., Turki, A., Chaieb, A., Mahjoub, T., & Almawi, W. Y. (2012). Singlenucleotide polymorphisms and haplotypes in the adiponectin gene contribute to the genetic risk for type 2 diabetes in Tunisian Arabs. Diabetes Research and Clinical Practice, 97(2), 290–297. Murea, M., Ma, L., & Freedman, B. I. (2012). Genetic and environmental factors associated with type 2 diabetes and diabetic vascular complications. The Review of Diabetic Studies, 9(1), 6–22. Ohashi, K., Parker, J. L., Ouchi, N., Higuchi, A., Vita, J. A., Gokce, N., et al. (2010). Adiponectin promotes macrophage polarization toward an anti-inflammatory phenotype. Journal of Biological Chemistry, 285(9), 6153–6160. Otsuka, F., Sugiyama, S., Kojima, S., Maruyoshi, H., Funahashi, T., Matsui, K., et al. (2006). Plasma adiponectin levels are associated with coronary lesion complexity in men with coronary artery disease. Journal of the American College of Cardiology, 48(6), 1155–1162. Ouchi, N., Parker, J. L., Lugus, J. J., & Walsh, K. (2011). Adipokines in inflammation and metabolic disease. Nature Reviews Immunology, 11(2), 85–97. Ponirakis, G., Petropoulos, I. N., Fadavi, H., Alam, U., Asghar, O., Marshall, A., et al. (2014). The diagnostic accuracy of Neuropad for assessing large and small fibre diabetic neuropathy. Diabetic Medicine, 31(12), 1673–1680. Raafat, K., & Samy, W. (2014). Amelioration of diabetes and painful diabetic neuropathy by Punica granatum L. extract and its spray dried biopolymeric dispersions. Evidence-based Complementary and Alternative Medicine, 180495. http://dx.doi.org/ 10.1155/2014/180495 [PMID: 24982685]. Razazian, N., Baziyar, M., Moradian, N., Afshari, D., Bostani, A., & Mahmoodi, M. (2014). Evaluation of the efficacy and safety of pregabalin, venlafaxine, and carbamazepine in patients with painful diabetic peripheral neuropathy. A randomized, doubleblind trial. Neurosciences (Riyadh), 19(3), 192–198. Sandy An, S., Palmer, N. D., Hanley, A. J., Ziegler, J. T., Mark Brown, W., Freedman, B. I., et al. (2013). Genetic analysis of adiponectin variation and its association with type 2 diabetes in African Americans. Obesity (Silver Spring), 21(12), E721–E729. Scherer, P. E., Williams, S., Fogliano, M., Baldini, G., & Lodish, H. F. (1995). A novel serum protein similar to C1q, produced exclusively in adipocytes. Journal of Biological Chemistry, 270(45), 26746–26749. Semple, R. K., Soos, M. A., Luan, J., Mitchell, C. S., Wilson, J. C., Gurnell, M., et al. (2006). Elevated plasma adiponectin in humans with genetically defective insulin receptors. Journal of Clinical Endocrinology and Metabolism, 91(8), 3219–3223. Siitonen, N., Pulkkinen, L., Lindstrom, J., Kolehmainen, M., Eriksson, J. G., Venojarvi, M., et al. (2011). Association of ADIPOQ gene variants with body weight, type 2 diabetes and serum adiponectin concentrations: The Finnish Diabetes Prevention Study. BMC Medical Genetics, 12, 5. http://dx.doi.org/10.1186/1471-2350-12-5 [PMID: 21219602]. Sun, H., Gong, Z. C., Yin, J. Y., Liu, H. L., Liu, Y. Z., Guo, Z. W., et al. (2008). The association of adiponectin allele 45 T/G and -11377C/G polymorphisms with type 2 diabetes and rosiglitazone response in Chinese patients. British Journal of Clinical Pharmacology, 65(6), 917–926. Tang, T. S., Prior, S. L., Li, K. W., Ireland, H. A., Bain, S. C., Hurel, S. J., et al. (2012). Association between the rs1050450 glutathione peroxidase-1 (C N T) gene variant and peripheral neuropathy in two independent samples of subjects with diabetes mellitus. Nutrition, Metabolism, and Cardiovascular Diseases, 22(5), 417–425. Tuttolomondo, A., La Placa, S., Di Raimondo, D., Bellia, C., Caruso, A., Lo Sasso, B., et al. (2010). Adiponectin, resistin and IL-6 plasma levels in subjects with diabetic foot and possible correlations with clinical variables and cardiovascular co-morbidity. Cardiovascular Diabetology, 9, 50. http://dx.doi.org/10.1186/1475-2840-9-50 [PMID: 20836881]. Udomsinprasert, W., Tencomnao, T., Honsawek, S., Anomasiri, W., Vejchapipat, P., Chongsrisawat, V., et al. (2012). +276 G/T single nucleotide polymorphism of the adiponectin gene is associated with the susceptibility to biliary atresia. World Journal of Pediatrics, 8(4), 328–334. Waki, H., Yamauchi, T., Kamon, J., Ito, Y., Uchida, S., Kita, S., et al. (2003). Impaired multimerization of human adiponectin mutants associated with diabetes. Molecular structure and multimer formation of adiponectin. Journal of Biological Chemistry, 278(41), 40352–40363. Yadav, A., Kataria, M. A., Saini, V., & Yadav, A. (2013). Role of leptin and adiponectin in insulin resistance. Clinica Chimica Acta, 417, 80–84. Yu, A. R., Xin, H. W., Wu, X. C., Fan, X., Liu, H. M., Li, G., et al. (2011). Adiponectin gene polymorphisms are associated with posttransplantation diabetes mellitus in Chinese renal allograft recipients. Transplantation Proceedings, 43(5), 1607–1611. Zhang, D., Guo, M., Zhang, W., & Lu, X. Y. (2011). Adiponectin stimulates proliferation of adult hippocampal neural stem/progenitor cells through activation of p38 mitogen-activated protein kinase (p38MAPK)/glycogen synthase kinase 3beta (GSK-3beta)/beta-catenin signaling cascade. Journal of Biological Chemistry, 286(52), 44913–44920. Ziegler, D., Buchholz, S., Sohr, C., Nourooz-Zadeh, J., & Roden, M. (2014). Oxidative stress predicts progression of peripheral and cardiac autonomic nerve dysfunction over 6 years in diabetic patients. Acta Diabetologica, 5(1), 65–72.