Summary The unfolded protein response (UPR) is linked to metabolic dysfunction, yet it is not known how ER disruption might influence metabolic pathways. Using a multilayered genetic approach, we find that mice with genetic ablations of either ER stress sensing pathways (ATF6α, eIF2α, IRE1α), or of ER quality control (p58IPK), share a common dysregulated response to ER stress that includes the development of microvesicular steatosis. The rescue of ER protein processing capacity by the combined action of UPR pathways during stress prevents the suppression of a subset of metabolic transcription factors that regulate lipid homeostasis. This suppression occurs in part by unresolved ER stress perpetuating expression of the transcriptional repressor CHOP. As a consequence, metabolic gene expression networks are directly responsive to ER homeostasis. These results reveal an unanticipated direct link between ER homeostasis and the transcriptional regulation of metabolism and suggest mechanisms by which ER stress might underlie microvesicular steatosis.

The endoplasmic reticulum (ER) is the cellular organelle responsible for protein folding and assembly, lipid and sterol biosynthesis, and calcium storage. The unfolded protein response (UPR) is an adaptive intracellular stress response to accumulation of unfolded or misfolded proteins in the ER. In this study, we show that the most conserved UPR sensor inositol‐requiring enzyme 1 α (IRE1α), an ER transmembrane protein kinase/endoribonuclease, is required to maintain hepatic lipid homeostasis under ER stress conditions through repressing hepatic lipid accumulation and maintaining lipoprotein secretion. To elucidate physiological roles of IRE1α‐mediated signalling in the liver, we generated hepatocyte‐specific Ire1α‐null mice by utilizing an albumin promoter‐controlled Cre recombinase‐mediated deletion. Deletion of Ire1α caused defective induction of genes encoding functions in ER‐to‐Golgi protein transport, oxidative protein folding, and ER‐associated degradation (ERAD) of misfolded proteins, and led to selective induction of pro‐apoptotic UPR trans‐activators. We show that IRE1α is required to maintain the secretion efficiency of selective proteins. In the absence of ER stress, mice with hepatocyte‐specific Ire1α deletion displayed modest hepatosteatosis that became profound after induction of ER stress. Further investigation revealed that IRE1α represses expression of key metabolic transcriptional regulators, including CCAAT/enhancer‐binding protein (C/EBP) β, C/EBPδ, peroxisome proliferator‐activated receptor γ (PPARγ), and enzymes involved in triglyceride biosynthesis. IRE1α was also found to be required for efficient secretion of apolipoproteins upon disruption of ER homeostasis. Consistent with a role for IRE1α in preventing intracellular lipid accumulation, mice with hepatocyte‐specific deletion of Ire1α developed severe hepatic steatosis after treatment with an ER stress‐inducing anti‐cancer drug Bortezomib, upon expression of a misfolding‐prone human blood clotting factor VIII, or after partial hepatectomy. The identification of IRE1α as a key regulator to prevent hepatic steatosis provides novel insights into ER stress mechanisms in fatty liver diseases associated with toxic liver injuries.

Summary The unfolded protein response (UPR) is a signaling pathway required to maintain endoplasmic reticulum (ER) homeostasis and hepatic lipid metabolism. Here, we identify an essential role for the inositol-requiring transmembrane kinase/endoribonuclease 1α (IRE1α)-X-box binding protein 1 (XBP1) arm of the UPR in regulation of hepatic very low-density lipoprotein (VLDL) assembly and secretion. Hepatocyte-specific deletion of Ire1α reduces lipid partitioning into the ER lumen and impairs the assembly of triglyceride (TG)-rich VLDL, but does not affect TG synthesis, de novo lipogenesis, or the synthesis or secretion of apolipoprotein B (apoB). The defect in VLDL assembly is, at least in part, due to decreased microsomal triglyceride-transfer protein (MTP) activity resulting from reduced protein disulfide isomerase (PDI) expression. Collectively, our findings reveal a key role for the IRE1α-XBP1s-PDI axis in linking ER homeostasis with regulation of VLDL production and hepatic lipid homeostasis that may provide a therapeutic target for disorders of lipid metabolism.

Although glucose uniquely stimulates proinsulin biosynthesis in β cells, surprisingly little is known of the underlying mechanism(s). Here, we demonstrate that glucose activates the unfolded protein response transducer inositol-requiring enzyme 1 alpha (IRE1α) to initiate X-box-binding protein 1 (Xbp1) mRNA splicing in adult primary β cells. Using mRNA sequencing (mRNA-Seq), we show that unconventional Xbp1 mRNA splicing is required to increase and decrease the expression of several hundred mRNAs encoding functions that expand the protein secretory capacity for increased insulin production and protect from oxidative damage, respectively. At 2 wk after tamoxifen-mediated Ire1α deletion, mice develop hyperglycemia and hypoinsulinemia, due to defective β cell function that was exacerbated upon feeding and glucose stimulation. Although previous reports suggest IRE1α degrades insulin mRNAs, Ire1α deletion did not alter insulin mRNA expression either in the presence or absence of glucose stimulation. Instead, β cell failure upon Ire1α deletion was primarily due to reduced proinsulin mRNA translation primarily because of defective glucose-stimulated induction of a dozen genes required for the signal recognition particle (SRP), SRP receptors, the translocon, the signal peptidase complex, and over 100 other genes with many other intracellular functions. In contrast, Ire1α deletion in β cells increased the expression of over 300 mRNAs encoding functions that cause inflammation and oxidative stress, yet only a few of these accumulated during high glucose. Antioxidant treatment significantly reduced glucose intolerance and markers of inflammation and oxidative stress in mice with β cell-specific Ire1α deletion. The results demonstrate that glucose activates IRE1α-mediated Xbp1 splicing to expand the secretory capacity of the β cell for increased proinsulin synthesis and to limit oxidative stress that leads to β cell failure.

Extracellular membrane vesicles (EVs) function as vehicles of intercellular communication, but how the biomaterials they carry reach the target site in recipient cells is an open question. We report that subdomains of Rab7+ late endosomes and nuclear envelope invaginations come together to create a sub-nuclear compartment, where biomaterials associated with CD9+ EVs are delivered. EV-derived biomaterials were also found in the nuclei of host cells. The inhibition of nuclear import and export pathways abrogated the nuclear localization of EV-derived biomaterials or led to their accumulation therein, respectively, suggesting that their translocation is dependent on nuclear pores. Nuclear envelope invagination-associated late endosomes were observed in ex vivo biopsies in both breast carcinoma and associated stromal cells. The transcriptome of stromal cells exposed to cancer cell-derived CD9+ EVs revealed that the regulation of eleven genes, notably those involved in inflammation, relies on the nuclear translocation of EV-derived biomaterials. Our findings uncover a new cellular pathway used by EVs to reach nuclear compartment.

Diabetes is an epidemic of worldwide proportions caused by β-cell failure. Nutrient fluctuations and insulin resistance drive β-cells to synthesize insulin beyond their capacity for protein folding and secretion and thereby activate the unfolded protein response (UPR), an adaptive signalling pathway to promote cell survival upon accumulation of unfolded protein in the endoplasmic reticulum (ER). Protein kinase-like endoplasmic reticulum kinase (PERK) signals one component of the UPR through phosphorylation of eukaryotic initiation factor 2 on the α-subunit (eIF2α) to attenuate protein synthesis, thereby reducing the biosynthetic burden. β-Cells uniquely require PERK-mediated phosphorylation of eIF2α to preserve cell function. Unabated protein synthesis in β-cells is sufficient to initiate a cascade of events, including oxidative stress, that are characteristic of β-cell failure observed in type 2 diabetes. In contrast to acute adaptive UPR activation, chronic activation increases expression of the proapoptotic transcription factor CAAT/enhancer-binding protein homologous protein (CHOP). Chop deletion in insulin-resistant mice profoundly increases β-cell mass and prevents β-cell failure to forestall the progression of diabetes. The findings suggest an unprecedented link by which protein synthesis and/or misfolding in the ER causes oxidative stress and should encourage the development of novel strategies to treat diabetes.

Proinsulin misfolding in the endoplasmic reticulum (ER) initiates a cell death response, although the mechanism(s) remains unknown. To provide insight into how protein misfolding may cause β-cell failure, we analyzed mice with the deletion of P58IPK/DnajC3, an ER luminal co-chaperone. P58IPK−/− mice become diabetic as a result of decreased β-cell function and mass accompanied by induction of oxidative stress and cell death. Treatment with a chemical chaperone, as well as deletion of Chop, improved β-cell function and ameliorated the diabetic phenotype in P58IPK−/− mice, suggesting P58IPK deletion causes β-cell death through ER stress. Significantly, a diet of chow supplemented with antioxidant dramatically and rapidly restored β-cell function in P58IPK−/− mice and corrected abnormal localization of MafA, a critical transcription factor for β-cell function. Antioxidant feeding also preserved β-cell function in Akita mice that express mutant misfolded proinsulin. Therefore defective protein folding in the β-cell causes oxidative stress as an essential proximal signal required for apoptosis in response to ER stress. Remarkably, these findings demonstrate that antioxidant feeding restores cell function upon deletion of an ER molecular chaperone. Therefore antioxidant or chemical chaperone treatment may be a promising therapeutic approach for type 2 diabetes.

IRE1α, the most conserved transducer of the unfolded protein response, plays critical roles in many biological processes and cell fate decisions. Reporting in Science, Upton et al. (2012) broadened our understanding of IRE1a as a cell-death executioner, showing that upon ER stress, IRE1α degrades microRNAs to promote translation of caspase-2.