Objective
Activating transcription factor 4 (ATF4) is a transcriptional regulator of the unfolded protein response and integrated stress response (ISR) that promote the restoration of normal endoplasmic reticulum (ER) function. Previous reports demonstrated that dysregulation of the ISR showed development of severe diabetes. However, the contribution of ATF4 to pancreatic beta cells remains poorly understood. In this study, we aimed to analyze the effect of ISR enhancer Sephin1 and ATF4-deficient beta cells for clarifying the role of ATF4 in beta cells under ER stress conditions.

Methods
To examine the role of ATF4 in vivo, ISR enhancer Sephin1 (5 mg/kg body weight, p.o.) was administered daily for 21 days to Akita mice. We also established beta cell-specific Atf4 knockout (βAtf4-KO) mice that were further crossed with Akita mice. These mice were analyzed for characteristics of diabetes, beta cell function and morphology of the islets. To identify the downstream factors of ATF4 in beta cells, the islets of βAtf4-KO mice were subjected to cDNA microarray analyses. To examine the transcriptional regulation by ATF4, we also performed in situ PCR analysis of pancreatic sections from mice and ChIP-qPCR analysis in CT215 beta cells.

Results
Administration of the ISR enhancer Sephin1 improved glucose metabolism in Akita mice. Sephin1 also increased the insulin-immunopositive area and ATF4 expression in the pancreatic islets. Akita/βAtf4-KO mice exhibited dramatically exacerbated diabetes as shown by hyperglycemia in their early age as well as a remarkable short life span owing to diabetic ketoacidosis. Moreover, the islets of Akita/βAtf4-KO mice presented increased numbers of cells stained for glucagon, somatostatin, and pancreatic polypeptide and increased expression of aldehyde dehydrogenase 1 family member 3, a marker of dedifferentiation. Using microarray analysis, we identified atonal BHLH transcription factor 8 (ATOH8) as a downstream factor of ATF4. Deletion of ATF4 in beta cells showed reduced Atoh8 expression and increased expressions of undifferentiated markers, Nanog and Pou5f1. Atoh8 expression was also abolished in the islets of Akita/βAtf4-KO mice.

Conclusions
We conclude that transcriptional regulation by ATF4 maintains beta cell identity via ISR modulation. This mechanism provides a promising target for the treatment of diabetes.