A total of 40 patients, newly diagnosed with T2D and 40 age and gender-matched healthy control individuals (HC) were recruited at Zhongnan Hospital of Wuhan University (Wuhan, China) between October 2012 and February 2013. The patients with T2D were diagnosed, according to the diagnostic criteria of the American Diabetes Association 2003 (25). Individuals were excluded if they had a history of hypoglycemia, ketoacidosis, hypertension, cardiovascular disease, liver disease, any autoimmune disease or a recent infectious disease. Written informed consent was obtained from each individual involved, and the experimental procedure was approved by the Ethics Committee of Zhongnan Hospital of Wuhan University (Approval no. 2012012).

The body weight, height and body mass index of each individual subject were measured. Individual subjects were fasted for at least 12 h, and median cubital venous blood samples (4 ml per patient) were obtained. The concentrations of fasting plasma glucose (FPG), total triglycemia (TG), total cholesterol (TC), high density lipoprotein (HDL), low density lipoprotein, (LDL) as well as the levels of plasma alanine aminotransferase (ALT) were measured by scatter turbidimetry using a Siemens Special Protein Analysis instrument (Siemens Healthcare Diagnostics Products, GmbH, Marburg, Germany). The plasma concentrations of fasting insulin (FINS) were measured by chemiluminescent immunoassay, using the Insulin (Human) CLIA kit, according to manufacturer's instructions (Abnova, Walnut, CA, USA). The values of homeostasis model assessment of insulin resistance (HOMA-IR) in individual subjects were calculated, according to the formula: Fasting insulin (microU/L) x fasting glucose (nmol/l)/22.5 (26).

Fasting blood samples were obtained from individual subjects and their PBMCs were prepared by density gradient centrifugation using Ficoll-Hypaque (PAA Laboratories GmbH, Pasching, Austria). PBMCs were used to analyze the levels of the Nurr1 mRNA transcript using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and Nurr1 protein expression and GSK-3β phosphorylation using western blot analysis.

In addition, a quantity of the PBMCs (3×10⁵/ml) were stimulated with, or without, 250 or 500 µM palmitic acid, or with 16.7 or 33.3 mM glucose (Sigma-Aldrich, St Louis, MO, USA) in 10% fetal bovine serum (FBS) RPMI-1640 (Invitrogen Life Technologies, Carlsbad, CA, USA) at 37°C for 45 min and 24 h, respectively. The supernatants were then harvested for analysis of cytokines, and the cells were collected for the extraction of RNA and proteins for further analyses.

The levels of the Nurr1 mRNA transcripts relative to GAPDH in individual PBMC samples were determined using RT-qPCR. Briefly, total RNA was isolated from the PBMCs using TRIzol reagent (Invitrogen Life Technologies), and reverse-transcribed into cDNA using a RevertAid™ First Strand cDNA Synthesis kit (Fermentas, Pittsburgh, PA, USA), according to the manufacturer's instructions. The qPCR was performed using SYBR Green PCR Master Mix and specific primers on a 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA), and 40 ng cDNA per sample was used for the template. The following primer sequences were used: Nurr1 forward 5′-CCTTGTGTTCAGGCGCAGTAT-3′ and reverse 5′-GTGGCAGTGATTTCAGTGTTGGT-3′ (158 bp); TNF-α forward 5′-GCCAGCTCCCTCTATTTATG-3′ and reverse 5′-TGGTCACCAAATCAGCATTG-3′ for TNF-α (272 bp), and GAPDH forward 5′-GGC TGAGAACCGGAAGCTTGTCAT-3′ and reverse 5′-CAGCCT TCTCCATGCTGGTGGTGAAGA-3′ for (314 bp). The amplification was performed at 95°C for 5 min followed by 40 cycles of 94°C for 15 sec, 55°C for 20 sec, 72°C for 20 sec, followed by 72°C for 7 min. The levels of mRNA transcripts were analyzed using the 2^−ΔΔCt method (27).

The collected PBMC samples were lyzed in radioimmunoprecipitation assay buffer containing 1% Triton X-100, 50 mM KCl, 25 mM Hepes (pH 7.8), 10 µg/ml leupeptin, 20 µg/ml aprotinin, 125 µM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride and 1 mM sodium orthovanadate (Sigma-Aldrich), and centrifuged at 12,000 x g for 15 min at 4°C. Following quantification of the protein concentrations using a bicinchoninic acid assay (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), the individual cell lysates (40 µg/lane) were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% fat-free dry-milk in Tris-buffered saline with 0.1% Tween 20 and incubated with the following antibodies: Mouse monoclonal anti-Nurr1 (cat. no. ab54366; 1:1,000; Abcam, Cambridge, MA, USA), rabbit polyclonal anti-pho-GSK-3β (cat. no. ab75745; 1:1,000; Abcam), goat polyclonal anti-GAPDH (cat. no. sc-20356; 1:10,00; Santa Cruz Biotechnology, Inc.) and rabbit polyclonal anti-β-actin (cat. no. sc1616R; 1:1,000; Santa Cruz Biotechnology, Inc.) overnight at 4°C, respectively. Following washing twice, the bound antibodies were detected using horseradish peroxidase-conjugated anti-rabbit (cat. no. sc-2030; 1:3,000; Santa Cruz Biotechnology, Inc.), or anti-mouse (cat. no. sc-2005; 1:3,000; Santa Cruz Biotechnology, Inc.)at room temperature for 1 h, and visualized using enhanced chemiluminescence (Santa Cruz Biotechnology, Inc.). Purified mouse, or rabbit immunoglobulin G were used as negative controls. The levels of targeting proteins relative to β-actin or GAPDH were determined using Alpha EaseFC 4.0 (Alpha Innotech, San Leandro, CA, USA).

The levels of IL-6 and TNFα in individual plasma samples were determined using specific ELISA kits (Boster Statems, Inc., Wuhan, China), according to the manufacturer's instructions. The limitations of detection for plasma IL-6 and TNFα were 1.8 pg/ml, and 13.3 pg/ml, respectively.

Data are presented as the median (range) or the mean ± standard deviation. Differences between groups were analyzed using Student's t-test for normally distributed data or using a Wilcoxon signed-rank test for skewed data. The differences in the category data between these two groups were analyzed using a χ²-square test. The potential association between variants was analyzed using Spearman's rank correlation coefficient. All statistical analyses were performed using the SPSS statistics 16.0 software package. P<0.05 was considered to indicate a statistically significant difference.

To understand the potential regulation of the expression Nurr1 on chronic inflammation during the development of T2D, 40 patients with newly diagnosed T2D and 40 age- and gender-matched HC were recruited to the present study. As shown in Table I, no significant differences were apparent in the distribution of age and gender or BMI between the T2D patients and HC group. The levels of FPG and FINS, and the HOMA IR values in the T2D patients were significantly higher, as compared with that in the HC group (P<0.01 for all), demonstrating hyperglycemia and compensated increased secretion of insulin and the development of insulin resistance in the T2D patients. Furthermore, although no significant difference was observed in the levels of plasma levels of TC and LDL-c between the patients and HC group, significantly higher levels of plasma TG and lower levels of HDL-c were detected in the T2D patients (P<0.01 for all), indicating dysfunctional lipid metabolism in T2D patients.

Further analysis revealed that the plasma levels of IL-6 and TNFα in the T2D patients were significantly higher than those in the HC group (P<0.01; Fig. 1A and B). Notably, the levels of IL-6 and TNFα were positively correlated with the values of HOMA-IR in the T2D patients (r=0.427: P=0.008 and r=0.39; P=0.015, respectively), as shown in Fig. 1C and D. In addition, PBMCs were isolated from the HC and T2D patients, and were stimulated with PMA/ionomycin in vitro, followed by assessment of the relative expression levels of IL-6 and TNFα using western blot analysis. The results revealed that the relative expression levels of IL-6 and TNFα in the PBMCs from the T2D patients were significantly higher than those in the PBMCs from the HC group (1.08±0.11, vs. 0.49±0.14 for IL-6; P<0.01 and 1.14±0.11, vs. 0.56±0.09 for TNFα; P<0.01). These data suggested that high levels of pro-inflammatory cytokines were present in patients with T2D and may be associated with the development of insulin resistance during the development of T2D.

Nurr1 can downregulate inflammation and pro-inflammatory cytokine production, which depends on activity of GSK-3β in astrocytes and microglial cells (11,19,21). To understand the molecular mechanisms underlying the role of Nurr1 in the development of chronic inflammation, the present study analyzed the relative expression levels of Nurr1 in the PBMCs from the patients with T2D and the HC group using RT-qPCR and western blot analysis. As shown in Fig. 2A, the relative levels of Nurr1 mRNA transcripts in the T2D patients were significantly lower, compared with those in the HC group (P=0.012). The western blot analysis indicated that the relative protein levels of Nurr1 in the PBMCs from the T2D patients were also significantly lower, compared with those in the HC (P=0.0188; Fig. 2B). GSK-3β is crucial for the recruitment of Nurr1 to the promoter of pro-inflammatory cytokines in immunocompetent cells, and the phosphorylation of GSK-3β at tyrosine-216 can enhance its activity (19). To understand the potential role of GSK-3β phosphorylation in regulating the Nurr1-mediated inhibition of cytokine production in human immunocompetent cells, the present study analyzed the relative levels of GSK-3β tyrosine-216 phosphorylation in the PBMCs from the T2D patients and HC group. The relative levels of GSK-3β tyrosine-216 phosphorylation in the PBMCs from the T2D patients were significantly lower than those in the HC (Fig. 2C), which may have contributed to the higher levels of pro-inflammatory cytokines in the T2D patients. Therefore, the decreased expression of Nurr1 and phosphorylation of GSK-3β tyrosine-216 may be associated with the development of chronic inflammation and T2D.

To understand the clinical significance of the results, the present study analyzed the association between the relative levels of Nurr1 mRNA transcripts in the PBMCs, the levels of plasma cytokines and insulin, and the degree of insulin resistance in the T2D patients. As shown in Fig. 3, the relative levels of Nurr1 mRNA transcripts in the PBMCs were positively correlated with the plasma levels of HDL-c (r=0.45, P=0.005), but negatively correlated with the plasma levels of IL-6 (r=−0.353; P=0.030), TNF-α (−0.426; P=0.008), FINS (r=−0.485; P=0.002) and HOMA-IR (r=−0.424; P=0.008). Therefore, decreased expression levels of Nurr1 were inversely correlated with the levels of plasma pro-inflammatory cytokines and the degree of insulin resistance in the T2D patients.

Previous studies have demonstrated that high levels of glucose and saturated fatty acids can enhance the activity of NF-kB and promote the production of TNF-α in human PBMCs (28–30). To further understand the role of hyperglycemia and hyperlipidemia in the development of chronic inflammation, the present study examined the impact of different concentrations of glucose and palmitic acid on the expression levels of Nurr1 in the PBMCs from the HC group. As shown in Fig. 4, treatment with 16.7 mmol/l glucose significantly reduced the expression levels of Nurr1 in the PBMCs, compared with the untreated PBMCs. Treatment with a higher concentration of glucose further reduced the expression of Nurr1 in the PBMCs. In addition, treatment with a lower dose of glucose for 45 min reduced the expression levels of Nurr1 by almost 30%, and treatment with the same dose of glucose decreased the expression levels of Nurr1 by 57% for 24 h. Notably, a similar pattern of reduced levels of Nurr1 were observed in the PBMCs following treatment with different concentrations of palmitic acid for varying time-periods in vitro. Therefore, high levels of glucose or palmitic acid inhibited the expression of Nurr1 in the PBMCs in a dose- and time-dependent manner.