ATF4-mediated transcriptional regulation protects against β-cell loss during endoplasmic reticulum stress in a mouse model

Keisuke Kitakaze,Miho Oyadomari,Jun Zhang,Yoshimasa Hamada,Yasuhiro Takenouchi,Kazuhito Tsuboi,Mai Inagaki,Masanori Tachikawa,Yoshio Fujitani,Yasuo Okamoto,Seiichi Oyadomari

doi:10.1016/j.molmet.2021.101338

Keisuke Kitakaze, Miho Oyadomari + Show 9 more

Open Access

https://doi.org/10.1016/j.molmet.2021.101338

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Abstract

ObjectiveActivating 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 led to development of severe diabetes. However, the contribution of ATF4 to pancreatic β-cells remains poorly understood. In this study, we aimed to analyze the effect of ISR enhancer Sephin1 and ATF4-deficient β-cells to clarify the role of ATF4 in β-cells under ER stress conditions. MethodsTo 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 β-cell–specific Atf4 knockout (βAtf4-KO) mice that were further crossed with Akita mice. These mice were analyzed for characteristics of diabetes, β-cell function, and morphology of the islets. To identify the downstream factors of ATF4 in β-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 of CT215 β-cells. ResultsAdministration 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, shown by hyperglycemia at an early age, as well as a remarkably short lifespan 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 β-cells showed reduced Atoh8 expression and increased expression of undifferentiated markers, Nanog and Pou5f1. Atoh8 expression was also abolished in the islets of Akita/βAtf4-KO mice. ConclusionsWe conclude that transcriptional regulation by ATF4 maintains β-cell identity via ISR modulation. This mechanism provides a promising target for the treatment of diabetes.

Highlights

In the prediabetic stage, insulin resistance is compensated by increased insulin secretion that maintains normal blood glucose levels.a further increase in insulin resistance exposes b-cells to endoplasmic reticulum (ER) stress because of high insulin demand [1e3]
In CT215 b-cells, thapsigargin (TG) treatment enhanced the protein levels of phosphorylated eukaryotic initiation factor-2a (eIF2a) and Activating transcription factor 4 (ATF4) (Figure 1AeC) and the mRNA levels of ATF4 downstream factors, eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), C/EBP homologous protein (CHOP), and PPP1R15A (Figure 1D). The levels of these proteins and mRNAs were enhanced by treatment with Sephin1 for 18 h (Figure 1BeD), while mRNA levels of spliced X-box binding protein 1 (XBP1s) and heat shock protein family A member 5 (HSPA5), ATF4-independent unfolded protein response (UPR) factors, showed no significant difference between the Sephin1-and vehicle-treated cells
We identified atonal BHLH transcription factor 8 (ATOH8) as a downstream factor of ATF4 and showed that its downregulation enhances the expression of undifferentiated markers in b-cells (Figure 7)

Summary

Introduction

Insulin resistance is compensated by increased insulin secretion that maintains normal blood glucose levels.a further increase in insulin resistance exposes b-cells to endoplasmic reticulum (ER) stress because of high insulin demand [1e3]. Insulin resistance is compensated by increased insulin secretion that maintains normal blood glucose levels. A further increase in insulin resistance exposes b-cells to endoplasmic reticulum (ER) stress because of high insulin demand [1e. ER stress is induced in the b-cells of type 2 diabetic animals [4], causing b-cell failure and apoptosis. Prolonged ER stress leads to the development of diabetes in several animal models, including Akita insulin-misfolded [5] and Wolfram syndrome mice [6] as well as humans [7, 8]. To adapt to ER stress, cells have specific stress response mechanisms such as unfolded protein response (UPR) [3] and integrated stress response (ISR) [9].

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