Endoplasmic reticulum (ER) health is an important determinant of pancreatic beta cell function or failure. Adaptive ER stress leads to expansion of beta cell number through proliferation, but decompensated ER stress results in beta cell loss and contributes to both T1D and T2D. Therefore, understanding the mechanisms of ER-stress-induced beta cell death could help identify novel strategies for the prevention or treatment of diabetes. Glucose responsive protein GRP78/HSPA5 is a key ER chaperone. Loss of GRP78 causes cell death in many cell types; including, in our own prior work, the beta cell. To study the mechanism by which GRP78 loss leads to beta cell death we devised an ex vivo model of GRP78 knockdown by transducing Grp78-flox mouse islets with adenovirus expressing Cre recombinase. To study human islets, we used adenovirus expressing shRNA against GRP78. Using these tools, we successfully reduced GRP78 RNA and protein in primary mouse and human islet cells. Loss of GRP78 strongly activated the ATF6, IRE1 and PERK pathways of the unfolded protein response (UPR). GRP78 knockdown decreased beta cell number due to increased cell death as measured by Annexin V and TUNEL staining. Probing the mechanisms of cell loss, we found that loss of GRP78 activated multiple death-related pathways, notably autophagy and apoptosis; cell death effectors Chop, caspase 9 and cleaved caspase 3 were increased. Additionally, GRP78 deletion increased stress kinase JNK (c-Jun-N-terminal kinase) which has been implicated in cellular proliferation as well as cell death. Inhibition of JNK kinase using JNK-IN-8 inhibitor during GRP78 loss conditions decreased phosphorylation of JNK and rescued beta cells from cell death as measured with Annexin V and TUNEL. Therefore, we conclude that ER stress related cell death induced by loss of GRP78 (ER stress) involves JNK activation, and that modulation of JNK under certain circumstances may help maintain beta cell mass to protect against diabetes.
R.B. Sharma: None. C.O. Darko: None. B. Gablaski: None. A.S. Lee: None. L.C. Alonso: Consultant; Self; Fairbanks Pharmaceuticals Inc. Research Support; Self; American Diabetes Association.
National Institute of Diabetes and Digestive and Kidney Diseases (5R01DK113300-02)