Endoplasmic Reticulum Stress Links Oxidative Stress to Impaired Pancreatic Beta-Cell Function Caused by Human Oxidized LDL

Abstract

Elevated plasma concentration of the pro-atherogenic oxidized low density lipoprotein cholesterol (LDL) triggers adverse effects in pancreatic beta-cells and is associated with type 2 diabetes. Here, we investigated whether the endoplasmic reticulum (ER) stress is a key player coupling oxidative stress to beta-cell dysfunction and death elicited by human oxidized LDL. We found that human oxidized LDL activates ER stress as evidenced by the activation of the inositol requiring 1α, and the elevated expression of both DDIT3 (also called CHOP) and DNAJC3 (also called P58IPK) ER stress markers in isolated human islets and the mouse insulin secreting MIN6 cells. Silencing of Chop and inhibition of ER stress markers by the chemical chaperone phenyl butyric acid (PBA) prevented cell death caused by oxidized LDL. Finally, we found that oxidative stress accounts for activation of ER stress markers induced by oxidized LDL. Induction of Chop/CHOP and p58IPK/P58IPK by oxidized LDL was mimicked by hydrogen peroxide and was blocked by co-treatment with the N-acetylcystein antioxidant. As a conclusion, the harmful effects of oxidized LDL in beta-cells requires ER stress activation in a manner that involves oxidative stress. This mechanism may account for impaired beta-cell function in diabetes and can be reversed by antioxidant treatment.

Fig 1. Activation of ER stress by human oxidized LDL.

(a) Western blotting analysis comparing changes in PERK, eIF2 and Ire1α and their phosphorylated forms (p). Total proteins were prepared from MIN6 cells cultured with 2 mmol/l cholesterol oxidized LDL (oxLDL) for the indicated times and 1 μmol/l thapsigargin (Thaps) for 6 h. The α-tubulin protein served as loading control. The figure is a representative experiment out of three. Measurement of CHOP/Chop, P58IPK/p58IPK and ATF4/Atf4 mRNA levels in (b) and (d) MIN6 and (c) and (e) isolated human islets cells cultured with oxidized LDL. The mRNA level was quantified by quantitative real-time PCR in MIN6 or isolated human islets cells cultured for 48 h with vehicle (V), native (nLDL) or oxidized LDL (oxLDL). The PBA chemical chaperone 2.5 mmol/l were added in the cells cultured with oxidized LDL (oxLDL, filled bar). For d) and e), cells were cultured with oxidized LDL plus 1 mmol/l cholesterol HDL (filled bar). The mRNA level was normalized against the housekeeping acidic ribosomal phosphoprotein P0 gene (RPLP0/Rplp0) and the expression levels from cells cultured with vehicle were set to 100%. Data are the mean of ± SEM of 3 independent experiments performed in triplicate (***, P<0.001; **, P<0.01).

Fig 2. Effects of BPA on apoptosis evoked by human oxidized LDL.

Measurement of apoptotic cells in (a) MIN6 and (b) isolated human islets. MIN6 cells were cultured with vehicle (V), native LDL (nLDL) or oxidized LDL (oxLDL) 2 mmol/l cholesterol with or without PBA 2.5 mmol/l (filled bar) for 72 h. The fraction of cells undergoing apoptosis was determined by scoring the percentage of cells displaying pyknotic nuclei. Data are the mean of ± SEM of 3 independent experiments (***, P<0.001; *, P<0.05). Quantification of the anti-apoptotic Bcl2/BCL2 and Ib1/IB1 mRNA levels in (c) MIN6 and (d) isolated human islets. The mRNA level of the two genes was quantified by quantitative real-time PCR and was normalized against the Rplp0/RPLP0. The expression levels from cells cultured with vehicle were set to 100%. Data are the mean of ± SEM of 3 independent experiments performed in triplicate (***, P<0.001; *, P<0.05).

Fig 3. Effects of Chop silencing in apoptosis caused by human oxidized LDL.

MIN6 cells were transfected with a control RNA si-GFP duplex (Ctrl, open bar) or with siCHOP (filled bar). Thereafter, the cells were cultured for 72 h with vehicle (V) or oxidized LDL (oxLDL) 2 mmol/l cholesterol. The fraction of cells undergoing apoptosis was determined by scoring the percentage of cells displaying pyknotic nuclei. Data are the mean of ± SEM of 3 independent experiments performed in triplicate (**, P<0.01).

Fig 4. Effects of 4-BPA chemical chaperone on the loss of insulin expression caused by human oxidized LDL.

The preproinsulin mRNA was quantified in MIN6 cells (a) and (b) human islets. Cells were exposed to vehicle (V), human native LDL (nLDL) or oxidized LDL (oxLDL) 2 mmol/l cholesterol, in the presence or absence of PBA 2.5 mmol/l (filled bars) for 48 h. The mRNA levels were normalized against the Rplp0/RPLP0 and the expression levels from cells cultured with vehicle were set to 100%. Data are the mean of ± SEM of 3 independent experiments performed in triplicate (*, P<0.05).

Fig 5. Effects of 4-BPA chemical chaperone on the induction of Icer/ICER mRNA evoked by human oxidized LDL.

Quantification of Icer/ICER mRNA levels in response to vehicle (V), human native LDL (nLDL) or oxidized LDL (oxLDL) 2 mmol/l cholesterol in (a) MIN6 and (b) human islets. The expression levels from total islets or cells cultured with vehicle were set to 100%. Data are the mean of ± SEM of 3 independent experiments performed in triplicate.

Fig 6. Role of oxidative stress on the ER stress markers expression induced by human oxidized LDL.

Expression of CHOP/Chop and P58IPK/p58IPK in (a) MIN6 and (b) human islets cells exposed to hydrogen peroxide (H₂O₂) 150 μM at indicated times. The expression of the two genes was quantified in (c) MIN6 cells or (d) human islets that were co-incubated with vehicle (V), human native LDL (nLDL) or oxidized LDL (oxLDL) 2 mmol/l cholesterol supplemented by either DMSO (control, open bar) or N-acetylcystein (NAC, filled bar) 1 mmol/l for MIN6 and 10 mmol/l for human islets cells. The results were normalized against Rplp0/RPLP0 and the expression levels from cells cultured with vehicle were set to 100%. Data are the mean ± SEM of 3 independent experiments performed in triplicate (***, P<0.001; **, P<0.01).

Fig 7. Schematic representation of mechanism coupling oxidized LDL to beta cell dysfunction and death.