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Abstract
Background
The characteristic feature of pancreatic β cells is highly developed endoplasmic reticulum (ER) due to a heavy engagement in insulin secretion. The ER serves several important function, including post-translational modification, folding, and assembly of newly synthesized secretory proteins, and its proper function is essential to cell survival. Various stress conditions can interfere with ER function. Pancreatic β cells may be particularly vulnerable to ER stress that causes to impair insulin biosynthesis and β cell survival through apoptosis. Glucagon like peptide-1 (GLP-1) is a new drug for treatment of type 2 diabetes and has effects on stimulation of insulin secretion and β cell preservation. Also, it may have an antiapoptotic effect on β cells, but detailed mechanisms are not proven. Therefore, we investigated the protective mechanism of GLP-1 in β cells through ER stress response induced by 2-deoxy-D-glucose (2DG).
Methods
For induction of the ER stress, HIT-T15 cells (hamster β cell line) were treated with 2DG (10 mM). Apoptosis was evaluated with MTT assay, hoechst 33342 staining and Annexin/PI flow cytometry. Expression of ER stress-related molecules was determined by real-time PCR or western blot. For blocking ER stress, we pretreated HIT-T15 cells with exendin-4 (Ex-4; GLP-1 receptor agonist) for 1 hour before stress induction.
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Fig. 1.
2DG-induced apoptosis in HIT-T15 cells. After exposure to 10 mM 2DG, HIT-T15 cells apoptosis increased by time. A. Cells viability was measured with the MTT assay. B. HIT-T15 cells were exposed to 2DG (10 mM) for 48 hours. Apoptotic nuclei was stained with hoechst 33342 and examined by fluorescence microscope. Photographs were taken using a blue filter at a magnification of ×400. C. Apoptotic cells were measured by FACS analysis after Annexin V/PI staining. Data are shown as the means ± S.E. of six independent experiments. ***significant vs. control cells (P < 0.001).
Fig. 2.
ER chaperone (GRP78 and GRP94) and CHOP significantly increased by 2DG (ER stress). A, B. Expression levels of GRP78, GRP94 and CHOP were examined by real time PCR and densitometry analysis. Data were expressed as the rates to the expression levels to GAPDH in the same sample. GAPDH used for loading control. C. Western blotting of GRP78, GRP94 and CHOP, -actin used for loading control. D. XBP-1 mRNA splicing was determined by RT-PCR. Unsplicing (u) and splicing (s) XBP-1 mRNA products are indicated. Data are shown as the means ± S.E. of three independent experiments. * P < 0.05. ** P < 0.01. *** P < 0.001.
Fig. 3.
Effects of the exendin-4 (Ex-4) on ER stress-induced ER chaperone (GRP78 and GRP94) and CHOP. HIT-T15 cells were pretreated with Ex-4. After 1 hour, HIT-T15 cells were treated with 2DG (10 mM) for 48 hours. A. After treated of 2DG, effects of the Ex-4 on GRP78, 94 and CHOP were determined by western blot. -actin used for loading control. CHOP expression levels were detected by densitometry analysis. B. Changed-expression levels of phospho-eIF2 and eIF2 were evaluated by western blot and densitometry analysis. C. Effects of the Ex-4 on XBP-1 mRNA splicing were detected by RT-PCR. Data are shown as the means ± S.E. of three independent experiments. ** P < 0.01. *** P < 0.001.
Fig. 4.
Effects of the exendin-4 (Ex-4) on ER stress-induced apoptosis. HIT-T15 cells were pretreated Ex-4 for 1 hour before stress induction. A. After treated of 2DG (10 mM, 48 hours), effects of the Ex-4 on cell viability were measured by MTT assay. B. 2DG (10 mM, 48 hours)-induced apoptotic nuclei reduced via Ex-4. Fixed cells were stained with hoechst 33342 and examined by fluorescence microscope. Photographs were taken using a blue filter at a magnification of ×400. C. Flow cytometric analysis of apoptosis of HIT-T15 cells exposed to 72 hours. Apoptotic cells were measured by FACS analysis after Annexin V/PI staining. Data are shown as the means ± S.E. of six independent experiments. ***significant vs. control cells (P < 0.001).
Fig. 5.
Effects of the exendin-4 (Ex-4) on insulin secretion. HIT-T15 cells were pretreated with Ex-4. After 1 hour, HIT-T15 cells were treated with 2DG (10 mM) for 48 hours. Expression levels of insulin2 mRNA were examined by real time PCR. Data were expressed as the rates to the expression levels to GAPDH in the same sample. GAPDH used for loading control. Data are shown as the means ± S.E. of four independent experiments. ** P < 0.01.
Table 1.
Real-time PCR primer sequence
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