Grp78 Is Critical for Beta-Cell Maturation and Survival

Abstract

Endoplasmic reticulum (ER) stress causes beta cell loss and dysfunction in T1D and T2D. Glucose regulated protein 78 (Grp78) is an abundant, widely expressed ER chaperone with functions including peptide translocation and folding, calcium storage, and regulating activity of the Perk, Ire1 and Atf6 unfolded protein response (UPR) pathways. Whole-body Grp78 deletion in mice is lethal at E3.5 days. Despite its critical importance, the impact of Grp78 deletion in beta cells has not yet been reported. Mice lacking both copies of Grp78 in beta cells (Grp78flox x Ins1CreThor) developed insulin-deficient diabetes by 2 weeks of age; heterozygotes remained normal. Islet architecture was disturbed in KO pups, with fewer beta cells than alpha cells (14.8±1.7% beta cells-KO, 70.5±3.8%-WT), reduced beta cell mass (13.5±1.7mg-KO, 69.7±6.4mg-WT), and abnormal cellular arrangement, with alpha and beta cells interspersed throughout islets. Reduced beta cell number was likely due to increased cell death; TUNEL+ beta cells were increased early, by 2 weeks of age (2.6±0.4%-KO, 0.1±0.0%-WT), whereas proliferation was not reduced until 4 weeks. Intriguingly, when BG was still normal, an increase in bi-hormonal glucagon/insulin cells was observed at 3-5 days (7.1±0.6%-KO, 3.0±0.2%-WT) and 2 weeks (7.7±0.6% in KO, 2.2±0.2% in WT) of age, suggesting defective fetal maturation, de-differentiation or trans-differentiation. Pdx1 staining was heterogeneously lost in insulin+ cells. Acute deletion of Grp78 in ex vivo mouse islet cells, human islet cells and the human EndoC-BH1 beta cell line strongly reduced Grp78 RNA/protein, induced compensatory chaperone Grp94, and activated all three UPR pathways. Grp78 knockdown increased beta cell death (AnnexinV staining, Chop and Txnip) and caused loss of differentiation markers Pdx1, Nkx6.1, Mafa and Neurod1, and induction of Ngn3, a fetal beta cell marker. In summary, Grp78 is critically important for beta cell survival and plays an unexpected role in maintaining beta cell differentiation markers. Disclosure R.B. Sharma: None. X. Zheng: None. B. Gablaski: None. J.K. Kim: None. A.S. Lee: None. L.C. Alonso: None.