In the present study, a total of 570 T2DM patients, aged 20 years and older (mean age, 63.6±11.5; 51.4% male individuals) were enrolled at China Medical University Hospital (Taichung, Taiwan) between August 2014 and July 2015. All patients met the diagnostic criteria (19) for T2DM with the exclusion criteria those without T2DM. To determine the prevalence of polymorphism in these patients, genotype frequency data of 1,700 healthy controls was downloaded from the Taiwan Biobank (https://taiwanview.twbiobank.org.tw/) (case no: TWBR10509-02; control no: TWBR10309-001) (9). In addition, to determine the association between polymorphism and clinical features in T2DM patients, the clinical features, including age, BMI, hemoglobin A1c, blood urea nitrogen, creatinine, uric acid, total calcium, phosphorus, parathyroid hormone (PTH), albumin, cholesterol, triglycerides, low-density lipoprotein-C and alanine aminotransferase (ALT) were also downloaded from the Taiwan Biobank. The present study obtained the rs1062048 single-nucleotide polymorphism (SNP) at chromosome region 8q22.3 in AZIN1 (Fig. 1) from the National Center for Biotechnology Information's SNP database (http://www.ncbi.nlm.nih.gov/snp). The SNPs in the T2DM and control groups were then compared. Tag SNPs were selected using the Tagger function (http://software.broadinstitute.org/mpg/tagger/server.html) with the additional criteria: ii) A threshold minor allele frequency in the HapMap phase 3; and ii) Han Chinese in Beijing, China (CHB) + Japanese in Tokyo, Japan (JPT) population of 0.05 for ‘tag SNPs’. Chi-square tests were used to calculate odds ratios and P-values. All study protocols were approved by the Ethical Committee of China Medical University Hospital (approval no. CMUH103-REC2-071). Informed consent was obtained from all analyzed patients.

A total 24 male background BKS.Cg-Dock 7^m+/+ Lepr^db/JNarl mice (age, 4 weeks old) were purchased from the National Laboratory Animal Center. Mice with the +Dock7^m/+Dock7^m genotype constituted the control group (n=12) and those with the +Lepr^db/+Lepr^db genotype constituted the T2DM group (n=12). The diabetic mice averaged 62±3 g in body weight at the time of the studies and their control littermates averaged 20±1 g. The animals were housed in individual cages and provided with lab chow ad libitum (Lab Diet 5k52; Purina) in a room at 22–25°C, with sufficient water, a relative humidity of 50–70%, and a 12-h light/dark cycle. The present study involved animal experiments and considered the 3R principles of ‘Replace’, ‘Reduce’ and ‘Refine’ to optimize experimental design (20). According to the information from Jackson Laboratory (JAX stock #000642) (21), the mouse model exhibited elevated blood sugar at 4–8 weeks and mortality by 10 months of age. The mice in the present study were divided into six groups (four mice per group), namely three control groups of mice aged 4 (early stage), 16 (middle stage) and 32 (late stage) weeks, and three T2DM groups of mice aged 4 (early stage), 16 (middle stage) and 32 (late stage) weeks. Mice were sacrificed and the liver tissue was obtained at the scheduled time. Sample went through the methodology of flash freezing (22), which provided excellent specimen integrity and a wide array of options for tissue analysis, including extraction of RNA and proteins in the present research. The present study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of China Medical University (IACUC permit no. 2106-221).

The RNeasy Mini kit (Qiagen, Inc.) was used for total RNA isolation from the liver tissues of the control and T2DM mice, and the Superscript First-Strand Synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc.) was used for complementary DNA transcription, as previously described (23). Briefly, the cDNA synthesis reaction protocol including reverse transcription step, 30 min at 42°C and RT inactivation step, 1 min at 95°C. To study gene expression, qPCR was performed using the ABI ViiA™ 7 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) and TaqMan™ MGB (minor groove binder)-NFQ (nonfluorescent quencher) Universal ProbeLibrary (Roche Diagnostics) probes, as previously described (24). The primer sequences were as follows: Mus musculus AZIN1 (NM_018745.5) forward, 5′-TGCTAAGAAAGTTGTTGAAAATGATAA-3′; murine AZIN1 reverse, 5′-CTGGCTCATCACTCCCATTT-3′ with UPL probe #6 (Roche, cat. no. 04685032001); mouse GAPDH (M32599.1) forward, 5′-GAGCCAAACGGGTCATCA-3′; and murine GAPDH reverse, 5′-CATATTTCTCGTGGTTCACACC-3′ with UPL probe #29 (Roche, cat. no. 04687612001). PCR amplification conditions were: Initial denaturation at 95°C for 5 min, followed by 25 cycles of 95°C for 10 sec, 56°C for 10 sec and 72°C for 20 sec, with a final extension at 72°C for 5 min. The target gene expression levels were normalized to mouse GAPDH expression. The relative quantification gene expression of AZIN1 was determined using the 2^−ΔΔCq method (25). The assay was run in triplicate for each case to allow for assessment of technical variability. To account for PCR amplification of contaminating genomic DNA, a control without reverse transcription was included. To improve the accuracy of RT-qPCR for quantification, amplifications were performed in triplicate for each RNA sample.

In the present study, an anti-AZIN1 (cat. no. orb154904; Biorbyt, Ltd.) polyclonal anti-rabbit antibodies were used to detect AZIN1 in the WB analysis. Samples were separated by SDS-PAGE on a 12.5% gel. WB was performed as described previously (26). Frozen liver tissue samples were homogenized with three volumes of 10 mM ice cold phosphate buffer (pH 7.0), containing 1 mM ethylenediaminetetraacetic acid, 0.25 M sucrose, 1 mM sodium azide and 0.1 mM phenylmethylsulfonyl fluoride. Samples were centrifuged at 20,000 × g for 30 min at 4°C. The protein concentration was measured using a bicinchoninic acid assay (Pierce; Thermo Fisher Scientific, Inc.). The tissue lysate was obtained through denaturing electrophoresis by SDS-PAGE on a 12.5% with 60 mg protein loaded per lane, electrotransferred to a polyvinylidene difluoride membrane, the blots was incubated with blocking buffer (1X PBS and 5% nonfat dry milk) for 1 h at room temperature and then probed with primary anti-AZIN1 antibodies (1:1,000; cat. no. orb154904; Biorbyt, Ltd.) overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibody (1:5,000; cat. no. GTX213110; GeneTex) for 1 hour at RT. To control equal loading of total protein in all lanes, blots were stained with mouse anti-β-actin antibody (1:5,000; cat. no. ab8226; Abcam). The bands were visualized using an enhanced chemiluminescence kit (GE Healthcare) according to the manufacturer's protocol. Finally, the blots were visualized using an ImageQuant™ LAS 4000 system (GE Healthcare) (27).

AZIN1 protein expression was determined using IHC analysis of paraffin-embedded liver sections. Anti-AZIN1 IHC staining was conducted using the LAB-SA Detection System (cat. no. 85-8943; Invitrogen; Thermo Fisher Scientific, Inc.). IHC was performed as described previously (28). Paraffin tissue sections (5 µm thick) were dewaxed, treated with proteinase K enzyme and incubated in 3% hydrogen peroxide for 10 min at room temperature to block endogenous peroxidase activity. After being washed in PBS (pH 7.6) for 5 min, the sections were incubated in 0.1% Triton X in PBS with primary anti-AZIN1 antibody (cat. no. orb154904; 1:200 dilution; Biorbyt, Ltd.) at 4°C for overnight followed by staining with secondary rabbit anti-rat antibody conjugated with horseradish peroxidase (1× reagent B; cat. no. 1454284A; Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 20 min. The immunocomplexes were visualized after treatment with 3,3′-diaminobenzidine (cat. no. 00-2014; Invitrogen; Thermo Fisher Scientific, Inc.) solution for 5 min at room temperature. The sections were then washed with PBS (pH 7.6) for further processing (29–31). The AZIN1 protein expression was defined using light microscopy (Leica DM 1000 LED Lab; cat. no. 10052-384; Leica Microsystems, Inc.) at a magnification of ×100 or ×400.

Statistically significant differences in allele/genotype frequencies of AZIN1 SNP (rs1062048) between the T2DM and control groups were determined using the χ² test. Odds ratios were calculated from the genotypic frequency and allelic frequency at 95% CI for the AZIN1 SNP (rs1062048). Statistical analysis was performed using SPSS software (version 11; SPSS, Inc.). Data from three independent experiments are expressed as the mean ± SE. Statistical comparisons between the T2DM and control groups were performed using Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Table I presents the allelic and genotypic frequencies of the rs1062048 AZIN1 gene polymorphism distribution in patients with T2DM and controls. It was observed that the T allele in the rs1062048 polymorphism was higher in the patients with T2DM (87.6%; 999/1,140) than in the controls (85.2%; 2,897/3,400). The frequencies of the TT genotype at rs1062048 differed between the control group (72.3%; 1229/1700) and patients with T2DM (77.4%; 441/570). Genotype frequencies were significantly different between T2DM and control groups (P=0.039). The data suggested that the T allele and TT genotype at rs1062048 SNP are risk factors for T2DM.

The control (+Dock7^m/+Dock7^m) and T2DM (+Lepr^db/+Lepr^db) mice were sacrificed at 4, 16 and 32 weeks of age, and liver tissue RNA was extracted for RT-qPCR. The qPCR data in Fig. 2 shows AZIN1 gene expression in the liver tissues of mice aged 4, 16 and 32 weeks. AZIN1 gene expression in the liver tissues of mice in the T2DM group was significantly lower than that in the control group (P<0.05; Fig. 2). These results suggested that downregulation of AZIN1 gene expression occurred in the T2DM mice.

Liver tissues were homogenized and 60 µg protein was used in WB analysis using an anti-AZIN1 antibody and β-actin as a control. Representative blots are shown in Fig. 3A. Samples from the control mice are in lanes 1, 3 and 5 and those from the T2DM mice are in lanes 2, 4 and 6 at 4, 16 and 32 weeks, respectively. Compared with T2DM mice, no matter which stage, the AZIN1 protein expression level was always higher in the control group. In addition, the protein expression levels of AZIN1 were significantly lower in early stage (4th week) and late stage (32nd week) T2DM mouse liver samples when compared with that in control samples (Fig. 3B).

In Fig. 4, the IHC staining revealed AZIN1 protein expression in the liver tissues at 4, 16, and 32 weeks of age. AZN1 protein expression in the liver tissues of the T2DM group was lower than that in the control group (Figs. 3 and 4). The results suggested that downregulation of AZIN1 protein expression occurred in T2DM mice.

There were 246 patients with T2DM developing into T2DN in the cohort of the present study. A comparison between the clinical features of the patients with T2DN with and without the TT genotype at rs1062048 SNP is detailed in Table II. The two patient groups did not differ significantly in most of terms. However, the levels of hemoglobin A1c, blood urea nitrogen, creatinine, total calcium and triglycerides in the patients with T2DN with and without the TT genotype at rs1062048 SNP were higher than the normal values. In addition, the laboratory tests did not reveal differences between the TT genotype and non-TT genotype groups at rs1062048 SNP in the AZIN1 gene. In addition, a significantly higher level of parathyroid hormone (PTH) was observed in the TT group (202.7±176.62) than in the non-TT group (41.0±8.91; P=0.009; Table II).