4μ8C Inhibits Insulin Secretion Independent of IRE1α RNase Activity

IRE1 alpha plays an important role in the unfolded protein response (UPR), which is activated by the accumulation of unfolded proteins in the endoplasmic reticulum. 4 mu 8C, a well-known inhibitor of IRE1a RNase activity, is commonly used to analyze IRE1 alpha function during ER stress in cultured mammalian cells. However, the off-target effects of 4 mu 8C remain elusive. Pancreatic beta-cells synthesize a large amount of insulin in response to high glucose stimulation, and IRE1a plays an important role in insulin secretion from pancreatic beta-cells. Here, to analyze the role of IRE1a in pancreatic beta-cells, we examined insulin secretion after 4 mu 8C treatment. Although 4 mu 8C inhibited insulin secretion within 2 hr, neither insulin synthesis nor maturation was inhibited by 4 mu 8C under the same conditions. This result prompted us to examine the precise effects of 4 mu 8C on insulin secretion in pancreatic beta-cells. Unexpectedly, with just 5 min of treatment, 4 mu 8C blocked insulin secretion in cultured pancreatic beta-cells as well as in pancreatic islets. Furthermore, insulin secretion was prevented by 4 mu 8C, even in pancreatic beta-cells lacking the IRE1 alpha RNase domain, suggesting that 4 mu 8C blocked the late stage of the insulin secretory process, independent of the IRE1 alpha-XBP1 pathway. Our results indicate that 4 mu 8C has an off-target effect on insulin secretion in pancreatic beta-cells. These findings inform the researchers in the field that the use of 4 mu 8C requires the special consideration for the future studies.