Splice variants of mitofusin 2 shape the endoplasmic reticulum and tether it to mitochondria.

In eukaryotic cells, different organelles interact at membrane contact sites stabilized by tethers. Mitochondrial mitofusin 2 (MFN2) acts as a membrane tether that interacts with an unknown partner on the endoplasmic reticulum (ER). In this work, we identified the MFN2 splice variant ERMIT2 as the ER tethering partner of MFN2. Splicing of MFN2 produced ERMIT2 and ERMIN2, two ER-specific variants. ERMIN2 regulated ER morphology, whereas ERMIT2 localized at the ER-mitochondria interface and interacted with mitochondrial mitofusins to tether ER and mitochondria. This tethering allowed efficient mitochondrial calcium ion uptake and phospholipid transfer. Expression of ERMIT2 ameliorated the ER stress, inflammation, and fibrosis typical of liver-specific Mfn2 knockout mice. Thus, ER-specific MFN2 variants display entirely extramitochondrial MFN2 functions involved in interorganellar tethering and liver metabolic activities.

Transcriptional profiles in the chicken ductus arteriosus during hatching.

The ductus arteriosus, an essential embryonic blood vessel between the pulmonary artery and the descending aorta, constricts after birth or hatching and eventually closes to terminate embryonic circulation. Chicken embryos have two long ductus arteriosi, which anatomically differ from mammal ductus arteriosus. Each long ductus arteriosus is divided into two parts: the pulmonary artery-sided and descending aorta-sided ductus arteriosi. Although the pulmonary artery-sided and descending aorta-sided ductus arteriosi have distinct functional characteristics, such as oxygen responsiveness, the difference in their transcriptional profiles has not been investigated. We performed a DNA microarray analysis (GSE 120116 at NCBI GEO) with pooled tissues from the chicken pulmonary artery-sided ductus arteriosus, descending aorta-sided ductus arteriosus, and aorta at the internal pipping stage. Although several known ductus arteriosus-dominant genes such as tfap2b were highly expressed in the pulmonary artery-sided ductus arteriosus, we newly found genes that were dominantly expressed in the chicken pulmonary artery-sided ductus arteriosus. Interestingly, cluster analysis showed that the expression pattern of the pulmonary artery-sided ductus arteriosus was closer to that of the descending aorta-sided ductus arteriosus than that of the aorta, whereas the morphology of the descending aorta-sided ductus arteriosus was closer to that of the aorta than that of the pulmonary artery-sided ductus arteriosus. Subsequent pathway analysis with DAVID bioinformatics resources revealed that the pulmonary artery-sided ductus arteriosus showed enhanced expression of the genes involved in melanogenesis and tyrosine metabolism compared with the descending aorta-sided ductus arteriosus, suggesting that tyrosinase and the related genes play an important role in the proper differentiation of neural crest-derived cells during vascular remodeling in the ductus arteriosus. In conclusion, the transcription profiles of the chicken ductus arteriosus provide new insights for investigating the mechanism of ductus arteriosus closure.

Loss of hypoxia inducible factor-1α aggravates γδ T-cell-mediated inflammation during acetaminophen-induced liver injury.

Acetaminophen (APAP)-induced liver injury is closely associated with acute hepatic inflammation. Hypoxia-inducible factor-1 (HIF-1) is activated during immunological processes and regulates gene expressions in various types of immune cells. Although HIF-1 controls the differentiation and functions of conventional T cells in chronic inflammation, the pathological importance of HIF-1 in innate-like T cells during acute inflammation remains unknown. Here, we investigated the role of HIF-1 in innate-like γδ T cells during APAP-induced acute liver injury. In response to APAP administration, T-cell-specific Hif-1α gene knockout mice sustained severe liver damage compared to wild-type control mice but without any impacts on the initial hepatic insult. This severe liver damage was accompanied by excessive neutrophil infiltration into the liver, increased serum interleukin (IL)-17A levels, and increased hepatic expressions of C-X-C chemokine ligand (Cxcl) 1 and Cxcl2. Neutrophil depletion and IL-17A neutralization completely abolished the aggravated phenotypes in T-cell-specific Hif-1α gene knockout mice. Loss of the Hif-1α gene enhanced the aberrant accumulation of IL-17A-producing innate-like γδ T cells in the affected liver with no apparent effects on their IL-17A-producing ability. Adoptive transfer of Hif-1α-deficient splenic γδ T cells into recombination activating gene 2 (Rag2)-deficient mice aggravated APAP-induced liver injury with increased neutrophil accumulation in the liver compared to that of wild-type γδ T cells. Furthermore, Hif-1α-deficient γδ T cells selectively showed aberrantly enhanced migratory ability. This ability was totally abolished by treatment with the mitochondrial adenosine triphosphate synthase inhibitor oligomycin. Conclusion: Deletion of Hif-1α gene in T cells aggravates APAP-induced acute inflammatory responses by enhancing aberrant innate-like γδ T-cell recruitment, thereby increasing excessive neutrophil infiltration into the liver. (Hepatology Communications 2018;2:571-581).

Disruption of the mitochondria-associated ER membrane (MAM) plays a central role in palmitic acid-induced insulin resistance.

The mitochondria-associated ER membrane (MAM) is a specialized subdomain of ER that physically connects with mitochondria. Although disruption of inter-organellar crosstalk via the MAM impairs cellular homeostasis, its pathological significance in insulin resistance in type 2 diabetes mellitus remains unclear. Here, we reveal the importance of reduced MAM formation in the induction of fatty acid-evoked insulin resistance in hepatocytes. Palmitic acid (PA) repressed insulin-stimulated Akt phosphorylation in HepG2 cells within 12h. Treatment with an inhibitor of the ER stress response failed to restore PA-mediated suppression of Akt activation. Mitochondrial reactive oxygen species (ROS) production did not increase in PA-treated cells. Even short-term exposure (3h) to PA reduced the calcium flux from ER to mitochondria, followed by a significant decrease in MAM contact area, suggesting that PA suppressed the functional interaction between ER and mitochondria. Forced expression of mitofusin-2, a critical component of the MAM, partially restored MAM contact area and ameliorated the PA-elicited suppression of insulin sensitivity with Ser473 phosphorylation of Akt selectively improved. These results suggest that loss of proximity between ER and mitochondria, but not perturbation of homeostasis in the two organelles individually, plays crucial roles in PA-evoked Akt inactivation in hepatic insulin resistance.

Type I neuregulin1α is a novel local mediator to suppress hepatic gluconeogenesis in mice.

Neuregulin1 is an epidermal growth factor (EGF)-like domain-containing protein that has multiple isoforms and functions as a local mediator in the control of various cellular functions. Here we show that type I isoform of neuregulin1 with an α-type EGF-like domain (Nrg1α) is the major isoform in mouse liver and regulates hepatic glucose production. Forced expression of Nrg1α in mouse liver enhanced systemic glucose disposal and decreased hepatic glucose production with reduced fasting blood glucose levels. Nuclear forkhead box protein O1 (FoxO1) and its downstream targets, PEPCK and G6Pase, were suppressed in liver and isolated hepatocytes by Nrg1α overexpression. In contrast, silencing of Nrg1α enhanced glucose production with increased PEPCK and G6Pase expressions in cAMP/dexamethasone-stimulated hepatocytes. Mechanistically, the recombinant α-type EGF-like domain of NRG1α (rNRG1α) stimulated the ERBB3 signalling pathway in hepatocytes, resulting in decreased nuclear FoxO1 accumulation via activation of both the AKT and ERK pathways. In addition, acute treatment with rNRG1α also suppressed elevation of blood glucose levels after both glucose and pyruvate challenge. Although a liver-specific deletion of Nrg1 gene in mice showed little effect on systemic glucose metabolism, these results suggest that NRG1α have a novel regulatory function in hepatic gluconeogenesis by regulating the ERBB3-AKT/ERK-FoxO1 cascade.

Apoptosis induction associated with the ER stress response through up-regulation of JNK in HeLa cells by gambogic acid.

BACKGROUND: Gambogic acid (GA) was extracted from the dried yellow resin of gamboge (Garcinia hanburyi) which is traditionally used as a coloring material for painting and cloth dying. Gamboge has been also used as a folk medicine for an internal purgative and externally infected wound. We focused on the mechanisms of apoptosis induction by GA through the unfold protein response (ER stress) in HeLa cells. METHODS: The cytotoxic effect of GA against HeLa cells was determined by trypan blue exclusion assay. Markers of ER stress such as XBP-1, GRP78, CHOP, GADD34 and ERdj4 were analyzed by RT-PCR and Real-time RT-PCR. Cell morphological changes and apoptotic proteins were performed by Hoechst33342 staining and Western blotting technique. RESULTS: Our results indicated a time- and dose-dependent decrease of cell viability by GA. The ER stress induction is determined by the up-regulation of spliced XBP1 mRNA and activated GRP78, CHOP, GADD34 and ERdj4 expression. GA also induced cell morphological changes such as nuclear condensation, membrane blebbing and apoptotic body in Hela cells. Apoptosis cell death detected by increased DR5, caspase-8, -9, and -3 expression as well as increased cleaved-PARP, while decreased Bcl-2 upon GA treatment. In addition, phosphorylated JNK was up-regulated but phosphorylated ERK was down-regulated after exposure to GA. CONCLUSIONS: These results suggest that GA induce apoptosis associated with the ER stress response through up-regulation of p-JNK and down-regulation of p-ERK in HeLa cells.

WITHDRAWN: Loss of hepatic HIF-1α accelerates lipid accumulation by inhibiting peroxisomal fatty acid oxidation in nonalcoholic fatty liver disease.

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Hypoxia and fatty liver.

The liver is a central organ that metabolizes excessive nutrients for storage in the form of glycogen and lipids and supplies energy-producing substrates to the peripheral tissues to maintain their function, even under starved conditions. These processes require a considerable amount of oxygen, which causes a steep oxygen gradient throughout the hepatic lobules. Alcohol consumption and/or excessive food intake can alter the hepatic metabolic balance drastically, which can precipitate fatty liver disease, a major cause of chronic liver diseases worldwide, ranging from simple steatosis, through steatohepatitis and hepatic fibrosis, to liver cirrhosis. Altered hepatic metabolism and tissue remodeling in fatty liver disease further disrupt hepatic oxygen homeostasis, resulting in severe liver hypoxia. As master regulators of adaptive responses to hypoxic stress, hypoxia-inducible factors (HIFs) modulate various cellular and organ functions, including erythropoiesis, angiogenesis, metabolic demand, and cell survival, by activating their target genes during fetal development and also in many disease conditions such as cancer, heart failure, and diabetes. In the past decade, it has become clear that HIFs serve as key factors in the regulation of lipid metabolism and fatty liver formation. This review discusses the molecular mechanisms by which hypoxia and HIFs regulate lipid metabolism in the development and progression of fatty liver disease.

Establishment of a new detection system for the dimerization of IRE1α by BiFC assay.

We developed a new detection system for the activation of an endoplasmic reticulum (ER) stress sensor, inositol requiring kinase 1 α (IRE1α), by evaluating dimerization of it by bimolecular fluorescence complementation (BiFC) assay. By detecting the fluorescence derived from the reconstituted cerulean, this assay system enabled us to distinguish the activation behaviors of IRE1α as to ER stress-inducing compounds.

Comparative analysis of the expression patterns of UPR-target genes caused by UPR-inducing compounds.

Endoplasmic reticulum (ER) stress, due to an accumulation of unfolded proteins in the ER, leads to a process known as the unfolded protein response (UPR). Since the several compounds used to induce UPR have different modes of action, their mechanisms of protein accumulation are thought to be different, but it is unclear whether these compounds can upregulate UPR target genes with similar kinetics. Hence, we sought to compare the expression patterns of nine UPR target genes induced by seven UPR-inducing compounds. Hierarchical clustering analysis revealed that the expression patterns of the UPR target genes induced by the seven compounds were classified into two clusters; cluster A (thapsigargin, tunicamycin, 2-deoxyglucose, and dithiothreitol) and cluster B (brefeldin A, monensin, and eeyarestatin I). Thus, this study suggests the existence of at least two types of UPR target gene expression profiles, which depend on the mode of action of the compounds.