Effect of endoplasmic reticulum (ER) stress inducer thapsigargin on the expression of extracellular-superoxide dismutase in mouse 3T3-L1 adipocytes

Abstract

Endoplasmic reticulum stress is related to metabolic disorders, including atherosclerosis and type 2 diabetes. It is known that inflammatory adipocytokines and oxidative stress are increased, while anti-inflammatory adipocytokines such as adiponectin are decreased in adipocytes during above conditions. Extracellular-superoxide dismutase is an anti-inflammatory enzyme that protects cells from oxidative stress. Because plasma extracellular-superoxide dismutase levels in type 2 diabetes patients were inversely related to the body mass index and homeostasis model assessment-insulin resistance index, it is speculated that the regulation of extracellular-superoxide dismutase might lead to the suppression of metabolic disorders. Here, we observed the reduction of extracellular-superoxide dismutase and adiponectin in 3T3-L1 adipocytes treated with thapsigargin, an endoplasmic reticulum stress inducer. Interestingly, tunicamycin, another endoplasmic reticulum stress inducer, did not decrease the expression of extracellular-superoxide dismutase in spite of the induction of glucose regulated protein kinase 78 kDa, an endoplasmic reticulum stress marker. Moreover, eukaryotic translation initiation factor 2α signaling cascade plays a pivotal role in the reduction of extracellular-superoxide dismutase in 3T3-L1 adipocytes during endoplasmic reticulum stress conditions.

Keywords: endoplasmic reticulum stress; eukaryotic translation initiation factor 2α; extracellular-superoxide dismutase; obesity; reactive oxygen species.

Fig. 1

Thapsigargin but not tunicamycin decreases the expression of EC-SOD. (A) 3T3-L1 adipocytes were treated with 1 µM thapsigargin (Tg) or 1 µg/mL tunicamycin (Tu) for 24 h. (B) Adipocytes were treated with the indicated concentrations of Tg for 24 h or 1 µM Tg for the indicated time. After the cells had been treated, RT-PCR was carried out. Data were normalized using GAPDH levels (**p<0.01 vs vehicle or untreated cells, ^##p<0.01 vs Tu-treated cells).

Fig. 2

Involvement of calcium ion in the thapsigargin-triggered reduction of EC-SOD. (A) 3T3-L1 adipocytes were treated with 1 µM A23187 (A) for 24 h. (B) Cells were pretreated with (+) or without (−) 20 µM BAPTA-AM for 1 h, and then treated with 1 µM thapsigargin (Tg) for 24 h. After the cells had been treated, RT-PCR was carried out. Data were normalized using GAPDH levels (**p<0.01 vs vehicle (V), ^##p<0.01 vs Tg-treated cells).

Fig. 3

Involvement of ER stress mediators, eIF2α, XBP-1 and ATF6, in the thapsigargin-triggered reduction of EC-SOD. (A) 3T3-L1 adipocytes were treated with 1 µM thapsigargin (Tg) or 1 µg/mL tunicamycin (Tu) for 1 h (A, D) or 24 h (B, C). After the cells had been treated, Western blotting (A, D) and RT-PCR (B, C) were carried out. RT-PCR data (B) were normalized using GADPH levels (**p<0.01 vs vehicle (V), ^##p<0.01 vs Tu-treated cells).

Fig. 4

Involvement of eIF2α signaling in the thapsigargin-triggered reduction of EC-SOD. 3T3-L1 adipocytes were pretreated with (+) or without (−) 15 µM salubrinal (Sal) for 1 h, and then treated with 1 µM thapsigargin (Tg) or 1 µg/mL tunicamycin (Tu) for 24 h. After the cells had been treated, RT-PCR (A, B) was carried out. RT-PCR data (A) were normalized using GAPDH levels (**p<0.01 vs vehicle, ^##p<0.01 vs Tg-treated cells, ^††p<0.01 vs Tu-treated cells).