The integrated stress response (ISR) is a cytoprotective pathway initiated upon phosphorylation of the eukaryotic translation initiation factor 2 (eIF2α) residue designated serine-51, which is critical for translational control in response to various stress conditions. Four eIF2α kinases, namely heme-regulated inhibitor (HRI), protein kinase R (PKR), PKR-like endoplasmic reticulum kinase, (PERK) and general control non-depressible 2 (GCN2), have been identified thus far, and they are known to be activated by heme depletion, viral infection, endoplasmic reticulum stress, and amino acid starvation, respectively. Because eIF2α is phosphorylated under various stress conditions, the existence of an additional eIF2α kinase has been suggested. To validate the existence of the unidentified eIF2α kinase, we constructed an eIF2α kinase quadruple knockout cells (4KO cells) in which the four known eIF2α kinase genes were deleted using the CRISPR/Cas9-mediated genome editing. Phosphorylation of eIF2α was completely abolished in the 4KO cells by various stress stimulations. Our data suggests that the four known eIF2α kinases are sufficient for ISR and that there are no additional eIF2α kinases in vertebrates.

The eukaryotic translation initiation factor 2a (eIF2α) phosphorylation‐dependent integrated stress response (ISR), a component of the unfolded protein response, has long been known to regulate intermediary metabolism, but the details are poorly worked out. We report that profiling of mRNAs of transgenic mice harboring a ligand‐activated skeletal muscle–specific derivative of the eIF2α protein kinase R‐like ER kinase revealed the expected up‐regulation of genes involved in amino acid biosynthesis and transport but also uncovered the induced expression and secretion of a myokine, fibroblast growth factor 21 (FGF21), that stimulates energy consumption and prevents obesity. The link between the ISR and FGF21 expression was further reinforced by the identification of a small‐molecule ISR activator that promoted Fgf21 expression in cell‐based screens and by implication of the ISR‐inducible activating transcription factor 4 in the process. Our findings establish that eIF2α phosphorylation regulates not only cell‐autonomous proteostasis and amino acid metabolism, but also affects non‐cell‐autonomous metabolic regulation by induced expression of a potent myokine.—Miyake, M., Nomura, A., Ogura, A., Takehana, K., Kitahara, Y., Takahara, K., Tsugawa, K., Miyamoto, C., Miura, N., Sato, R., Kurahashi, K., Harding, H. P., Oyadomari, M., Ron, D., Oyadomari, S. Skeletal muscle‐specific eukaryotic translation initiation factor 2a phosphorylation controls amino acid metabolism and fibroblast growth factor 21‐mediated non‐cell‐autonomous energy metabolism. FASEB J. 30, 798–812 (2016). www.fasebj.org

The effects of imeglimin, a novel anti-diabetes agent, on β-cell function remain unclear. Here, we unveiled the impact of imeglimin on β-cell survival. Treatment with imeglimin augmented mitochondrial function, enhanced insulin secretion, promoted β-cell proliferation, and improved β-cell survival in mouse islets. Imeglimin upregulated the expression of endoplasmic reticulum (ER)-related molecules including Chop (Ddit3), Gadd34 (Ppp1r15a), Atf3, and Sdf2l1, and decreased eIF2α phosphorylation, after treatment with thapsigargin, and restored global protein synthesis in β-cells under ER stress. Imeglimin failed to protect ER stress-induced β-cell apoptosis in CHOP-deficient islets or in the presence of GADD34 inhibitor. Treatment with imeglimin showed a significant decrease in the number of apoptotic β-cells and increased β-cell mass in Akita mice. Imeglimin also protected against β-cell apoptosis in both human islets and human pluripotent stem cell (hPSC)-derived β-like cells. Taken together, imeglimin modulates ER homeostasis pathway, which results in the prevention of β-cell apoptosis both in vitro and in vivo.

The eIF2α phosphorylation-dependent integrated stress response (ISR) is a signaling pathway that maintains homeostasis in mammalian cells exposed to various stresses. Here, ISR activation in adipocytes improves obesity and diabetes by regulating appetite in a non-cell-autonomous manner. Adipocyte-specific ISR activation using transgenic mice decreases body weight and improves glucose tolerance and obesity induced by a high-fat diet (HFD) via preferential inhibition of HFD intake. The transcriptome analysis of ISR-activated adipose tissue reveals that growth differentiation factor 15 (GDF15) expression is induced by the ISR through the direct regulation of the transcription factors ATF4 and DDIT3. Deficiency in the GDF15 receptor GFRAL abolishes the adipocyte ISR-dependent preferential inhibition of HFD intake and the anti-obesity effects. Pharmacologically, 10(E), 12(Z)-octadecadienoic acid induces ISR-dependent GDF15 expression in adipocytes and decreases the intake of the HFD. Based on our findings the specific activation of the ISR in adipocytes controls the non-cell-autonomous regulation of appetite.

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 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. 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 β-cell–specific Atf4 knockout (βAtf 4-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. 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, shown by hyperglycemia at an early age, as well as a remarkably short lifespan owing to diabetic ketoacidosis. Moreover, the islets of Akita/βAtf 4-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/βAtf 4-KO mice. We conclude that transcriptional regulation by ATF4 maintains β-cell identity via ISR modulation. This mechanism provides a promising target for the treatment of diabetes.