24 research outputs found
Dephosphorylation of Translation Initiation Factor 2Ī± Enhances Glucose Tolerance and Attenuates Hepatosteatosis in Mice
SummaryThe molecular mechanisms linking the stress of unfolded proteins in the endoplasmic reticulum (ER stress) to glucose intolerance in obese animals are poorly understood. In this study, enforced expression of a translation initiation factor 2Ī± (eIF2Ī±)-specific phosphatase, GADD34, was used to selectively compromise signaling in the eIF2(Ī±P)-dependent arm of the ER unfolded protein response in liver of transgenic mice. The transgene resulted in lower liver glycogen levels and susceptibility to fasting hypoglycemia in lean mice and glucose tolerance and diminished hepatosteatosis in animals fed a high-fat diet. Attenuated eIF2(Ī±P) correlated with lower expression of the adipogenic nuclear receptor PPARĪ³ and its upstream regulators, the transcription factors C/EBPĪ± and C/EBPĪ², in transgenic mouse liver, whereas eIF2Ī± phosphorylation promoted C/EBP translation in cultured cells and primary hepatocytes. These observations suggest that eIF2(Ī±P)-mediated translation of key hepatic transcriptional regulators of intermediary metabolism contributes to the detrimental consequences of nutrient excess
Integrated stress response of vertebrates is regulated by four eIF2Ī± kinases
The integrated stress response (ISR) is a cytoprotective pathway initiated upon phosphorylation of the eukaryotic translation initiation factor 2 (eIF2Ī±) residue designated serine-51, which is critical for translational control in response to various stress conditions. Four eIF2Ī± 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 eIF2Ī± is phosphorylated under various stress conditions, the existence of an additional eIF2Ī± kinase has been suggested. To validate the existence of the unidentified eIF2Ī± kinase, we constructed an eIF2Ī± kinase quadruple knockout cells (4KO cells) in which the four known eIF2Ī± kinase genes were deleted using the CRISPR/Cas9-mediated genome editing. Phosphorylation of eIF2Ī± was completely abolished in the 4KO cells by various stress stimulations. Our data suggests that the four known eIF2Ī± kinases are sufficient for ISR and that there are no additional eIF2Ī± kinases in vertebrates
Integrated stress response of vertebrates is regulated by four eIF2Ī± kinases
The integrated stress response (ISR) is a cytoprotective pathway initiated upon phosphorylation of the eukaryotic translation initiation factor 2 (eIF2Ī±) residue designated serine-51, which is critical for translational control in response to various stress conditions. Four eIF2Ī± 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 eIF2Ī± is phosphorylated under various stress conditions, the existence of an additional eIF2Ī± kinase has been suggested. To validate the existence of the unidentified eIF2Ī± kinase, we constructed an eIF2Ī± kinase quadruple knockout cells (4KO cells) in which the four known eIF2Ī± kinase genes were deleted using the CRISPR/Cas9-mediated genome editing. Phosphorylation of eIF2Ī± was completely abolished in the 4KO cells by various stress stimulations. Our data suggests that the four known eIF2Ī± kinases are sufficient for ISR and that there are no additional eIF2Ī± kinases in vertebrates
Integrated stress response regulates GDF15 secretion from adipocytes, preferentially suppresses appetite for a high-fat diet and improves obesity
The eIF2Ī± 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
nsPEFs induce the ISR via ROS-mediated HRI activation
The integrated stress response (ISR) is one of the most important cytoprotective mechanisms and is integrated by phosphorylation of the Ī± subunit of eukaryotic translation initiation factor 2 (eIF2Ī±). Four eIF2Ī± kinases, heme-regulated inhibitor (HRI), double-stranded RNA-dependent protein kinase (PKR), PKR-like endoplasmic reticulum kinase (PERK), and general control nonderepressible 2 (GCN2), are activated in response to several stress conditions. We previously reported that nanosecond pulsed electric fields (nsPEFs) are a potential therapeutic tool for ISR activation. In this study, we examined which eIF2Ī± kinase is activated by nsPEF treatment. To assess the responsible eIF2Ī± kinase, we used previously established eIF2Ī± kinase quadruple knockout (4KO) and single eIF2Ī± kinase-rescued 4KO mouse embryonic fibroblast (MEF) cells. nsPEFs 70 ns in duration with 30 kV/cm electric fields caused eIF2Ī± phosphorylation in wild-type (WT) MEF cells. On the other hand, nsPEF-induced eIF2Ī± phosphorylation was completely abolished in 4KO MEF cells and was recovered by HRI overexpression. CM-H2DCFDA staining showed that nsPEFs generated reactive oxygen species (ROS), which activated HRI. nsPEF-induced eIF2Ī± phosphorylation was blocked by treatment with the ROS scavenger N-acetyl-L-cysteine (NAC). Our results indicate that the eIF2Ī± kinase HRI is responsible for nsPEF-induced ISR activation and is activated by nsPEF-generated ROS
A novel mouse model of muscle wasting
Background
Formation of 43S and 48S preinitiation complexes plays an important role in muscle protein synthesis. There is no muscle-wasting mouse model caused by a repressed 43S preinitiation complex assembly.
Objective
The aim of the present study was to develop a convenient mouse model of skeletal muscle wasting with repressed 43S preinitiation complex assembly.
Material and methods
A ligand-activatable PERK derivative Fv2E-PERK causes the phosphorylation of eukaryotic initiation factor 2Ī± (eIF2Ī±), which inhibits 43S preinitiation complex assembly. Thus, muscle atrophic phenotypes, intracellular signaling pathways, and intracellular free amino acid profiles were investigated in human skeletal muscle Ī±-actin (HSA) promoter-driven Fv2E-PERK transgenic (Tg) mice.
Results
HSA-Fv2E-PERK Tg mice treated with the artificial dimerizer AP20187 phosphorylates eIF2Ī± in skeletal muscles and leads to severe muscle atrophy within a few days of ligand injection. Muscle atrophy was accompanied by a counter regulatory activation of mTORC1 signaling. Moreover, intracellular free amino acid levels were distinctively altered in the skeletal muscles of HSA-Fv2E-PERK Tg mice.
Conclusions
As a novel model of muscle wasting, HSA-Fv2E-PERK Tg mice provide a convenient tool for studying the pathogenesis of muscle loss and for assessing putative therapeutics
PERK-mediated translational control is required for collagen secretion in chondrocytes
As chondrocytes are highly secretory and they experience a variety of stresses, physiological unfolded protein response (UPR) signalling is essential for extracellular matrix (ECM) secretion and chondrogenesis. In the three branches of the UPR pathway, PERK governs the translational attenuation and transcriptional upregulation of amino acid and redox metabolism and induction of apoptosis. It was previously demonstrated that a defect of the PERK branch of the UPR signalling pathway causes the accumulation of unfolded proteins, leading to cell death without perturbing endoplasmic reticulum (ER)-to-Golgi transport in pancreatic Ī² cells. However, little is known about the role of PERK in chondrocytes. In this study, we found that PERK signalling is activated in chondrocytes, and inhibition of PERK reduces collagen secretion despite causing excessive collagen synthesis in the ER. Perkā/ā mice displayed reduced collagen in articular cartilage but no differences in chondrocyte proliferation or apoptosis compared to the findings in wild-type mice. PERK inhibition increases misfolded protein levels in the ER, which largely hinder ER-to-Golgi transport. These results suggest that the translational control mediated by PERK is a critical determinant of ECM secretion in chondrocytes
IRE1Ī² Inhibits Chylomicron Production by Selectively Degrading MTP mRNA
SummaryMicrosomal triglyceride transfer protein (MTP) is needed to assemble chylomicrons in the endoplasmic reticulum (ER) of enterocytes. We explored the role of an ER stress protein, inositol-requiring enzyme 1Ī² (IRE1Ī²), in regulating this process. High-cholesterol and high-fat diets decreased intestinal IRE1Ī² mRNA in wild-type mice. Ire1bā/ā mice fed high-cholesterol and high-fat diets developed more pronounced hyperlipidemia because these mice secreted more chylomicrons and expressed more intestinal MTP, though not hepatic MTP, than wild-type mice did. Chylomicron secretion and MTP expression also were increased in primary enterocytes isolated from cholesterol-fed Ire1bā/ā mice. There was no correlation between ER stress and MTP expression. Instead, cell culture studies revealed that IRE1Ī², but not its ubiquitous homolog IRE1Ī±, decreased MTP mRNA through increased posttranscriptional degradation. Conversely, knockdown of IRE1Ī² enhanced MTP expression. These studies show that IRE1Ī² plays a role in regulating MTP and in chylomicron production
ISR-DEPENDENT METABOLIC REGULATION
The eukaryotic translation initiation factor 2Ī± (eIF2Ī±) phosphorylationādependent 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 ligandāactivated skeletal muscleāspecific derivative of the eIF2Ī± protein kinase Rālike ER kinase revealed the expected upāregulation 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 smallāmolecule ISR activator that promoted Fgf21 expression in cellābased screens and by implication of the ISRāinducible activating transcription factor 4 in the process. Our findings establish that eIF2Ī± phosphorylation regulates not only cellāautonomous proteostasis and amino acid metabolism, but also affects nonācellāautonomous metabolic regulation by induced expression of a potent myokine.āMiyake, 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 muscleāspecific eukaryotic translation initiation factor 2Ī± phosphorylation controls amino acid metabolism and fibroblast growth factor 21āmediated nonācellāautonomous energy metabolism
Skeletal muscleāspecific eukaryotic translation initiation factor 2Ī± phosphorylation controls amino acid metabolism and fibroblast growth factor 21āmediated nonācell-autonomous energy metabolism
The eukaryotic translation initiation factor 2Ī± (eIF2Ī±) 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 eIF2Ī± 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 eIF2Ī± 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.Ministry of Education, Culture, Sports, Science and Culture (MEXT) of Japan
Inoue Foundation for Science
Mitsubishi Foundation
Uehara Memorial Foundation
Naito Foundation
Cell Science Research Foundation
Takeda Science Foundation
Sankyo Foundation
Ono Medical Research Foundation
Mochida Memorial Foundation
Ube Foundation
Kowa Life Science Foundation
Suzuken Memorial Foundation
Kanae Foundation
Japan Diabetes Foundation
Japan Society for Promotion of Science (JSPS)
EU FP7. Grant Number: 277713
Wellcome Trust. Grant Number: 084812/Z/08/