H₂-dichlorodihydroflurescein diacetate (DCFDA) was obtained from Molecular Probes Inc. (Eugene, OR, USA). pGEM-T Easy Vector, pGL3 Luciferase Reporter Vector and CMV-driven β-galactosidase expression vector were from Promega (Madison, WI, USA), and Top-PfuTM DNA polymerase from Bio-online (Seoul, Korea). Anti-mouse Pdx1 antibody was obtained from Developmental Studies Hybridoma Bank (Iowa City, IA, USA). Western blotting and luminal reagents were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). INS-1 mice beta cells were donated by Professor Kyu-Jang Won at Yeungnam University. Pdx1 plasmids fused with insulin promoter, Pdx1 promoter plasmid, and GFP were donated by Professor Myung-Jun Kim at the Catholic University College of Medicine.

INS-1 cells were cultured in RPMI-1640 culture medium containing 10% FBS, 11.1 mM glucose and 2 mM L-glutamine at 37℃ with 5% CO₂. After the cells were cultured in six-well plates at 70-80% confluency, they were incubated in the absence of FBS for 16 hours. Then, 30 mM glucose, 0.25 mM palmitate, and 1% BSA were added to the medium for 48 hours. Some experiments involved pretreatment with N-acetyl-L-cysteine (NAC), an antioxidant, or SP600125, a JNK inhibitor.

In order to measure the intracellular production of ROS, cells were exposed to 25 µM H₂-DCFDA for 2 hours. Fluorescently stained cells were subjected to fluorescence-activated cell sorting (FACS).

RNA extraction, cDNA synthesis and PCR were performed using commercial kits. The primers were designed based on the sequences of the mouse genes (for insulin, forward: 5'-TCTTCTACACACCCATGTCCC-3'; reverse: 5'-GGTGCAGCACTGATCCAC-3'; for Pdx1, forward: 5'-CGGACATCTCCCCATACG-3'; reverse: 5'-AAAGGGAGATGAACGCGG-3'). Cyclophillin was used as the control gene (forward: 5'-AACCCCACCGTGTTCTTC-3'; reverse: 5'-TGCCTTCTTTCACCTTCCC-3'). Semi-quantitative analysis of the PCR products was done.

Extraction of nuclear proteins was done as reported previously [17]. The cells were removed by a cell lifter and centrifuged at 1,500 g at 4℃ for 5 minutes. Then, they were resuspended in 300 µL buffer solution A (10 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, 0.5 mM PMSF) to a final concentration of 0.6%. Nonidet P-40 was then added, and the cells were placed on ice for 15 minutes to destroy the cytoplasmic membranes. After the precipitation, the samples were centrifuged at 12,000 g for 30 seconds, resuspended in 100 µL hypertonic buffer solution B (20 mM HEPES, pH 7.9, 25% [v/v] glycerol, 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 1 mM DTT, 0.5 mM PMSF) and incubated on ice for 30 minutes before being centrifuged again at 12,000 g for 30 minutes at 4℃. All samples were stored at -80℃ for use when needed.

Western blot analysis was done as reported previously [18]. Cells were incubated in RIPA buffer (25 mM Tris-HCl, pH 7.4, 0.1% SDS, 0.1% Triton X-100, 1% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na₃VO₄, 1 mM PMSF, 10 µg/mL aprotinin and 5 µg/mL leucopeptin) and then centrifuged at 10,000 g for 30 minutes at 4℃. After electrophoresis in 10% SDS-polyacrylamide gel, the proteins were transferred to a nitrocellulose membrane in a transfer tank filled with transfer buffer (20% methanol, 25 mM Tris, and 192 mM glycine) for 2 hours at 70 V. In order to block the nonspecific binding, the nitrocellulose membrane was incubated with 5% skim milk in TTBS (20 mM Tris-HCl, pH 7.4, 500 mM NaCl, and 0.1% Tween 20) for 1 hour. Pdx1 antibody (0.5 µg/mL) was added at 4℃ for 16 hours. Then, a peroxidase-conjugated secondary antibody (0.5 µg/mL) was added at room temperature for 1 hour. Equal loading and transfer of samples were verified by the band intensity of β-tubulin.

We measured the activity of each promoter by performing co-transfection of insulin promoter plasmid or Pdx1 promoter plasmid and pCMV/β-gal plasmid. INS-1 cells were cultured at a density of 2 × 10⁵ cells/mL in 12-well plates for 24 hours. The DNA was mixed with OPTI-MEM medium and incubated for 15 minutes at room temperature. The mixture of 50 µL OPTI-MEM medium and 2 µL FuGENE6 was added and incubated for 30 minutes. The mixture of DNA and FuGENE6 was added to the cultured cells and incubated for 16 hours. After washing the cells with PBS, 30 mM glucose and 0.25 mM palmitate were incubated for 48 hours. Cells were washed with PBS and dissolved with 1 × reporter lysis buffer and repeatedly frozen and thawed. The samples were centrifuged at 12,000 g for 10 minutes at 4℃, and then the supernatant was extracted. The activities of luciferase and β-galactosidase were each measured using a luminometer (TD20/20; Turner Designs Instrument, Sunnyvale, CA, USA) and a MR700 microplate reader (Dynatech Laboratories Inc., Chantilly, VA, USA). Transfection efficiencies were normalized by a ratio of luciferase activity to β-galactosidase activity.

To investigate the intracellular nucleo-cytoplasmic translocation of Pdx1 in beta cells, Pdx1 plasmid fused with GFP was transiently transfected with FuGENE6 transfection solution. After INS-1 cells were cultured at a concentration of 2 × 10⁵ cells/mL in six-well plates for 24 hours, DNA (1 µg) was mixed with 100 µL OPTI-MEM medium and incubated for 15 minutes. Then, a mixture of 100 µL OPTI-MEM medium and 6 µL FuGENE6 solution was added and incubated for 30 minutes at room temperature. After the prepared mixture of DNA and FuGENE6 solution was added to the cultured cells, they were incubated for 16 hours. The cells were washed with PBS, 30 mM glucose and 0.25 mM palmitate were added and then the cells were cultured for 48 hours. After incubation, cells were fixed in 4% PFA, and GFP antibodies (1:100) were used to stain the cells. The translocate of Pdx1 in the cells was observed using confocal microscopy.

The experimental results are presented as the average ± standard deviation, and the comparison between experimental groups was analyzed using Prism software (GraphPad Software, San Diego, CA, USA) and one-way ANOVA. A Bonferroni multiple range t-test was used for the post test. Statistical significance was set at P < 0.05.

To investigate how high levels of glucose and palmitate affect to the expression of insulin and PDX-1 genes, we treated INS-1 cells with 30 mM glucose and 0.25 mM palmitate both separately and combined. After 48 hours, the expression of insulin gene was not significantly changed and expression of Pdx1 gene was decreased by high levels of glucose and palmitate. The combined treatment had a greater inhibitory effect on gene expression than did the single treatment (Fig. 1A). Also, to investigate whether ROS was affected to expression of insulin and PDX-1 gene, hydrogen peroxide was treated to INS-1 cells. The expression of insulin gene was not significantly changed and expression of Pdx1 gene was decreased in concentration dependent manner (Fig. 1B).

To investigate whether high levels of glucose and palmitate induced ROS generation in INS-1 cells, we exposed INS-1 cells to high levels of glucose and palmitate for 48 hours and then, treated with H₂-DCFDA for two hours, we measured intracellular ROS using FACS. We were known that the high levels of glucose and palmitate increased the generation of ROS in INS-1 cells. We also observed partial inhibition of ROS generation by treatment with the antioxidant NAC (Fig. 2).

To investigate whether high levels of glucose and palmitate affect to the activities of the insulin and Pdx1 promoters, we performed promoter luciferase assay. The high levels of glucose and palmitate induced a significant decrease in the promoter activities of both genes. Also, we observed that the antioxidant NAC prevented the decrease in promoter activity. Therefore, these results suggest that ROS is involved in the decrease of insulin and Pdx1 gene expressions induced by high levels of glucose and palmitate (Fig. 3).

The decrease in the expression of Pdx1 protein in response to high levels of glucose and palmitate was confirmed using Western blot analysis (Fig. 4A). The decrease in Pdx1 protein expression was not prevented by NAC or JNK inhibitor (SP6 00125). However, decrease of Pdx1 protein in the nucleus was observed, and this decrease was prevented by treatment with NAC or SP600125 (Fig. 4B). To confirm these results, we infected beta cells with a GFP: Pdx1 plasmid and observed the nucleo-cytoplasmic translocation of Pdx1 by high levels of glucose and palmitate. Through confocal microscopy, we observed that Pdx1 proteins are presented to the cytoplasm by high levels of glucose and palmitate (Fig. 4C).