All general reagents for cell culture were purchased from GIBCO, USA. Nifedipine, hoschst 33342 and DAPI were from Sigma-Aldrich, USA. The fluorescence dyes Fluo-4/AM were from Invitrogen, USA. Insulin ELISA kit were from Millipore. Rabbit anti-GAPDH, phosphor-eIF2α, eIF2α, caspase 3 and insulin antibodies were purchased from Cell signaling technology, rabbit anti-CHOP (GADD153) antibody were from Santa Cruz Biotechnology. Peroxidase-conjugated Goat anti-rabbit IgG was purchased from Jackson Immuno Research.

Rat insulinoma cell line INS-1 was obtained from American type culture collection (ATCC). INS-1 cells were cultured in RPMI-1640 medium containing 10% (vv-l) fetal bovine serum (FBS), 5.5 mM glucose, 10 mM HEPES, 100 units/mL penicillin, 100 μg/mL streptomycin and 50 μM β-mercaptoethanol at 37 °C and 5% CO₂ condition. Before the co-treatment with glucose at different concentration and nifedipine, cells were precultured in low-glucose condition (5.5 mM) overnight. In each glucose concentration, cells were incubated with or without 10 μM nifedipine for indicated time.

INS-1 cells were seeded in 96-well plates (10, 000 cells per well) and treated as described above. After 24 h cultured, cell viability was determined by using a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay described previously [26]. The results were shown as relative optical density.

Apoptotic cells are evaluated by Hoechst 33342 staining. The nuclear of cells are stained by Hoechst 33342 and show blue fluorescence. Compared with normal cells, the nuclei of apoptotic cells have highly condensed chromatin which could be visualized by fluorescence microscopy.

Cells were cultured on coverglasses in 12-well plates. After 24 h treatment, the apoptotic cells were stained by tunel staining kit following its protocol, the apoptotic cells were stained by green fluorescence, and all cells were marked with blue fluorescence using DAPI. The apoptotic ratio was calculated as tunnel-positive cells divided by total cell number.

INS-1 cells were treated as described above, and then cells were lysed by protein extraction kit (Beyotime, CN) according to its protocol. Western blot was performed as previously described [27], the following primary antibodies were used: phosphor-eIF2α (dilutions 1:1000), eIF2α (1:1000), CHOP (1:200), caspase 3 (1:500), GAPDH (1:5000).

Calcium mobilization assay was performed as described in [28]. In acute calcium influx determination, cells were stimulated by 33.3 mM glucose for 1 min in the presence of 0, 1, 3, 10 μM nifedipine. For long-term experiment, after 6 or 24 h treatment, the medium was removed and cells were pretreated with medium without glucose for 1 h, then INS-1 cells were stimulated with 33.3 mM glucose to measure [Ca²⁺]_i.

INS-1 cells were seeded into 96-well plates (30,000 cells per well). After the treatment, cells were fixed for 2 h in 4% paraformaldehyde and then incubated with insulin antibody as described [29], the insulin content in INS-1 cells was captured by fluorescence microscope. In the end of treatment, cells were washed twice with Krebs buffer (118 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 4.2 mM NaHCO₃, 2 mM CaCl₂), then the cells were incubated with 100 μL Krebs buffer containing 2.5 mM glucose for 30 min. Subsequently, wells were washed twice again and incubated with 20 mM glucose Krebs buffer instead. After 1 h incubation, the supernatant from each well were collected, following, the concentration of insulin was measured by rat insulin ELISA kit according to the manufacturer’s instructions (Millipore).

Data were expressed as the mean ± SD. Two-tailed t test was performed and a value of p < 0.05 was considered significant.

High concentration of glucose has been demonstrated to be toxic to cultured β-cells [5–7]. In a concentration of 33.3 mM, glucose inhibited the cell viability compared with 5.5 mM glucose treated group (Figure 1A). However, 10 μM nifedipine suppressed this process significantly, which had no effect on 5.5 mM glucose cultured group (Figure 1A). In addition, nifedipine could not promote cell viability in normal glucose concentration (11.1 mM), but the value of its viability was approximately two times more than other groups (data not shown).

The reduction of cell viability under high glucose-treatment could be involved in increased cell apoptotsis. To determine the apoptotic rate of treated INS-1 cells, Hoechst 33342 staining was performed. The cells incubated with 33.3 mM glucose showed less live cells and highly condensed chromatin in nuclei (Figure 1B). However, 10 μM nifedipine obviously decreased the apoptotic ratio in comparison with high glucose treated cells (Figure 1B). Furthermore, the result of tunel staining indicated that nifedipine prevented high glucose-induced INS-1 cell apoptosis (Figure 1C). Taken together, it suggested that inhibition of calcium influx by l-type-Ca²⁺-channel antagonist could provide benefit to prevent high glucose-induced β-cell apoptosis.

Pancreatic β-cell generates a highly developed ER for Ca²⁺ stores, insulin biosynthesis and secretion [18,30,31]. Thus, high glucose-induced β-cell apoptosis could be closely related to the breakdown of Ca²⁺ homeostasis, which was able to trigger ER stress [32]. To identify whether nifedipine protected INS-1 cells from high glucose-induced apoptosis via ER stress, the expression levels of some proteins involved in ER stress pathway were analyzed by western blot. As depicted in Figure 2, high glucose caused an elevated expression level of p-eIF2α, CHOP and caspase 3 proteins after 24 h treatment. However, these enhanced ER stress markers were reduced by nifedipine (Figure 2), suggesting that l-type-Ca²⁺-channel antagonists could suppress the high glucose-induced ER stress in INS-1 β-cells line.

Chronic hyperglycemia is thought to be important in the pathogenesis of T2D [33,34]. Under physiological conditions, an increase in [Ca²⁺]_i in β-cells is of crucial importance in coupling the GSIS [35,36]. Thus, the l-type-Ca²⁺-channel antagonist, nifedipine, might inhibit the sustained increase in [Ca²⁺]_i. We had investigated the effect of nifedipine on the calcium influx in INS-1 β-cells. Nifedipine could dose-dependently inhibit calcium influx in INS-1 cells (Figure 3A). When cells were treated for 6 h, high glucose promoted the activation of calcium influx and GSIS function (Figure 3B,D). However, in long-term incubation, the function of calcium influx was impaired by high glucose, which was protected by nifedipine (Figure 3B). Similarly, high glucose-impaired insulin content and GSIS in INS-1 cells were also protected by nifedipine (Figure 3C,D). It seemed that intracellular calcium homeostasis played an important role in insulin secretion and synthesis. Insulin release in pancreatic β-cells was through the modulation of calcium influx, but the accumulation of excessive calcium would damage cells function, eventually leading to the β-cells apoptosis. Thus, nifedipine could partially prevent high glucose-induced INS-1 cell dysfunction through the regulation of adequate calcium levels. However, the abrogation of l-type-Ca²⁺-channel activation did not completely prevent INS-1 cells from high glucose-induced apoptosis, suggesting that this event may not be totally dependent on calcium homeostasis.