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Cholesterol induces pancreatic β cell apoptosis through oxidative stress pathway

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Cell Stress and Chaperones Aims and scope

Abstract

Type 2 diabetes is often associated with high blood cholesterol. Here, we investigated the effect of cholesterol loading on MIN6 cells derived from pancreatic β cells. Exposure of MIN6 cells to cholesterol-induced apoptosis in time- and dose-dependent manner. Treatment with methyl-β-cyclodextrin that removes cholesterol from plasma membrane prevented the cells from cholesterol-induced apoptosis. Western blot analysis revealed that the levels of phosphorylated-p38 mitogen-activated protein kinase (P-p38 MAPK) and c-Jun N-terminal kinases (P-JNK) were significantly increased after the cholesterol loading, suggesting that the stress-activated protein kinase signaling was stimulated. A specific p38 inhibitor rescued MIN6 cells from cholesterol-induced apoptosis, while JNK inhibitor failed, suggesting the importance of activation of p38 MAPK signaling in response to cholesterol. The expression of Bip and CHOP, the endoplasmic reticulum (ER) stress markers, remained unaffected, indicating that the ER stress may not be involved in the cytotoxicity of cholesterol on the ΜΙΝ6 cells. The intracellular concentration of reactive oxygen species measured by use of 2′,7′-dichlorofluorescin diacetate was significantly increased after cholesterol loading, demonstrating the induced apoptosis was mediated through oxidative stress. Addition of reduced form of glutathione in the medium rescued MIN6 cells from apoptosis induced by cholesterol loading. Taken together, these results demonstrate that the free cholesterol loading can induce apoptosis of MIN6 cells mediated by oxidative stress and the activation of p38 MAPK signaling.

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Acknowledgment

This work was supported by grants from National Natural Science Foundation of China to QC (No.30800575) and Natural Science Foundation of Liaoning Provincial Education Board (No. 2009A304).

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Authors and Affiliations

  1. School of Life Science, Liaoning University, Shenyang, 110036, China

    Xiuli Lu, Jianli Liu, Fangfang Hou, Zhenqing Liu & Xiangyu Cao

  2. College of Life and Health Science, Chubu University, Aichi, 487–8501, Japan

    Hisao Seo

  3. Institute of Basic Medical Sciences, Shenyang Medical College, Shenyang, 110034, China

    Bing Gao

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  1. Xiuli Lu
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  2. Jianli Liu
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  3. Fangfang Hou
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  6. Hisao Seo
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Correspondence to Xiuli Lu or Bing Gao.

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X Lu and J Liu contributed equally to this work.

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Supplemental Figure 1

Low doses of CD does not induce cytotoxicity in MIN6 cells. The MIN6 cells were exposed to CD with indicated concentrations for 24 h. The cell images were obtained with a phase-contrast microscope at 24 h after the exposure. Bar 25 μm (Supplemental Figure 1a). The numbers of adherent cells were expressed as percentage of the number of control cells (Supplemental Figure 1b). Mean ± SD (n = 3). *p < 0.05 vs. the levels of control (PDF 342 kb)

Supplemental Figure 2

The cholesterol loading impairs glucose-induced insulin secretion. MIN6 cells were exposed to cholesterol for 24 h in the growth medium. After washing with KRB buffer containing 3 mmol/l of glucose, the cells were incubated with KRB buffer for 1 h and then transferred to the medium containing 20 mmol/l of glucose. At the end of the 1-h stimulation period, the amount of insulin release in the spent medium was determined by ELISA insulin kit, according to the manufacturer instructions (PDF 342 kb)

Supplemental Figure 3

MIN6 cells were exposed to 250 times diluted CLC in the growth medium for 24 h. The cells then were harvested and counted. The total lipids were extracted from cells and subjected to the intracellular cholesterol measurement. Mean ± SD (n = 3). *p < 0.05 vs. the levels of control (PDF 342 kb)

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Lu, X., Liu, J., Hou, F. et al. Cholesterol induces pancreatic β cell apoptosis through oxidative stress pathway. Cell Stress and Chaperones 16, 539–548 (2011). https://doi.org/10.1007/s12192-011-0265-7

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  • DOI: https://doi.org/10.1007/s12192-011-0265-7

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