Decreased prereceptorial glucocorticoid activating capacity in starvation due to an oxidative shift of pyridine nucleotides in the endoplasmic reticulum

Redox state of pyridine nucleotides of the endoplasmic reticulum (ER) lumen was determined in different nutritional conditions. NADPH-dependent cortisone reduction and NADP⁺-dependent cortisol oxidation were measured in rat liver microsomes, by utilizing the luminal 11β-hydroxysteroid dehydrogenase type 1 activity. Cortisone reduction decreased, while cortisol oxidation increased during onward starvation, showing that the luminal NADPH/NADP⁺ ratio was substantially decreased. Cortisone or metyrapone addition caused a smaller decrease in NADPH fluorescence in microsomes from starved rats. The results demonstrate that nutrient supply is mirrored by the redox state of ER luminal pyridine nucleotides.     

The glucose-6-phosphate transport is not mediated by a glucose-6-phosphate/phosphate exchange in liver microsomes

A phosphate-linked antiporter activity of the glucose-6-phosphate transporter (G6PT) has been recently described in liposomes including the reconstituded transporter protein. We directly investigated the mechanism of glucose-6-phosphate (G6P) transport in rat liver microsomal vesicles. Pre-loading with inorganic phosphate (Pi) did not stimulate G6P or Pi microsomal inward transport. Pi efflux from pre-loaded microsomes could not be enhanced by G6P or Pi addition. Rapid G6P or Pi influx was registered by light-scattering in microsomes not containing G6P or Pi. The G6PT inhibitor, S3483, blocked G6P transport irrespectively of experimental conditions. We conclude that hepatic G6PT functions as an uniporter.     

Oxidative protein folding in the plant endoplasmic reticulum

For most of the proteins synthesized in the endoplasmic reticulum (ER), disulfide bond formation accompanies protein folding in a process called oxidative folding. Oxidative folding is catalyzed by a number of enzymes, including the family of protein disulfide isomerases (PDIs), as well as other proteins that supply oxidizing equivalents to PDI family proteins, like ER oxidoreductin 1 (Ero1). Oxidative protein folding in the ER is a basic vital function, and understanding its molecular mechanism is critical for the application of plants as protein production tools. Here, I review the recent research and progress related to the enzymes involved in oxidative folding in the plant ER. Firstly, nine groups of plant PDI family proteins are introduced. Next, the enzymatic properties of plant Ero1 are described. Finally, the cooperative folding by multiple PDI family proteins and Ero1 is described.     

Heterotrimeric G protein subunits differentially respond to endoplasmic reticulum stress in Arabidopsis

Canonical heterotrimeric G proteins in eukaryotes are major components that localize at plasma membrane and transmit extracellular stimuli into the cell. Genome of a seed plant Arabidopsis thaliana encodes at least one Gα (GPA1), one Gβ (AGB1), and 3 Gγ (AGG1, AGG2 and AGG3) subunits. The loss-of-function mutations of G protein subunit(s) cause multiple defects in development as well as biotic and abiotic stress responses. However, it remains elusive how these subunits differentially express these defects. Here, we report that Arabidopsis heterotrimeric G protein subunits differentially respond to the endoplasmic reticulum (ER) stress. An isolated homozygous mutant of AGB1, agb1-3, was more sensitive to the tunicamycin-induced ER stress compared to the wild type and the other loss-of-function mutants of G protein subunits. Moreover, ER stress responsive genes were highly expressed in the agb1-3 plant. Our results indicate that AGB1 positively contributes to ER stress tolerance in Arabidopsis.     

Endoplasmic reticulum: a metabolic compartment

Several biochemical reactions and processes of cell biology are compartmentalized in the endoplasmic reticulum (ER). The view that the ER membrane is basically a scaffold for ER proteins, which is permeable to small molecules, is inconsistent with recent findings. The luminal micro-environment is characteristically different from the cytosol; its protein and glutathione thiols are remarkably more oxidized, and it contains a separate pyridine nucleotide pool. The substrate specificity and activity of certain luminal enzymes are dependent on selective transport of possible substrates and co-factors from the cytosol. Abundant biochemical, pharmacological, clinical and genetic data indicate that the barrier function of the lipid bilayer and specific transport activities in the membrane make the ER a separate metabolic compartment.     

Characterization of sulfate transport in the hepatic endoplasmic reticulum

The transport of sulfate ion across the endoplasmic reticulum membrane was investigated using rapid filtration and light scattering assays. We found a protein-mediated, bi-directional, low-affinity, and high-capacity, facilitative sulfate transport in rat liver microsomes, which could be inhibited by the prototypical anion transport inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. It was resistant to various phosphate transport inhibitors and was not influenced by high concentration of phosphate or pyrophosphate, which is contradictory to involvement of phosphate transporters. It was sensitive to S3483 that has been reported to inhibit the glucose 6-phosphate transporter (G6PT), but the weak competition between sulfate and glucose 6-phosphate did not confirm the participation of this transporter. Moreover, the comparison of the activity and S3483 sensitivity of sulfate transport in microsomes prepared from G6PT-overexpressing or wild type COS-7 cells did not show any significant difference. Our results indicate that sulfate fluxes in the endoplasmic reticulum are mediated by a novel, S3483-sensitive transport pathway(s).     

Bufotalin-induced apoptosis in osteoblastoma cells is associated with endoplasmic reticulum stress activation

The search for novel and more efficient chemo-agents against malignant osteoblastoma is important. In this study, we examined the potential anti-osteoblastoma function of bufotalin, and studied the underlying mechanisms. Our results showed that bufotalin induced osteoblastoma cell death and apoptosis in dose- and time-dependent manners. Further, bufotalin induced endoplasmic reticulum (ER) stress activation in osteoblastoma cells, the latter was detected by the induction of C/EBP homologous protein (CHOP), phosphorylation of inositol-requiring enzyme 1 (IRE1) and PKR-like endoplasmic reticulum kinase (PERK), as well as caspase-12 activation. Conversely, the ER stress inhibitor salubrinal, the caspase-12 inhibitor z-ATAD-fmk as well as CHOP depletion by shRNA significantly inhibited bufotalin-induced osteoblastoma cell death and apoptosis. Finally, by using a mice xenograft model, we demonstrated that bufotalin inhibited U2OS osteoblastoma cell growth in vivo. In summary, our results suggest that ER stress contributes to bufotalin-induced apoptosis in osteoblastoma cells. Bufotalin might be investigated as a novel anti-osteoblastoma agent.     

Autophagy inhibition induces podocyte apoptosis by activating the pro-apoptotic pathway of endoplasmic reticulum stress

Podocyte apoptosis is a major factor inducing podocyte depletion that predicts the progressive course of glomerulosclerosis. However, the molecular mechanisms underlying podocyte apoptosis are still not well understood. Autophagy is a lysosomal degradation system involving the degradation and recycling of obsolete, damaged, or harmful cytoplasmic materials and organelles. Recent advances in the understanding of the molecular processes contributing to autophagy have provided insight into the relationship between autophagy and apoptosis. However, their cross-talk remains largely obscure until now. Here, we found that podocytes both in vivo and in vitro always exhibited high basal levels of autophagy, whereas autophagy inhibition could induce podocyte apoptosis, suggesting the pro-survival role of autophagy in podocytes. Besides, we found that autophagy inhibition by 3-methyladenine (3-MA) could induce the activation of endoplasmic reticulum stress even without any external stimulations, whereas knockdown of CHOP could effectively improve podocyte apoptosis and down-regulated expression of slit-diaphragm proteins induced by autophagy inhibition. Collectively, this study demonstrated that autophagy might act as a crucial regulatory mechanism of apoptotic cell death by modulating the balance between the pro-survival pathway and the pro-apoptotic pathway of endoplasmic reticulum stress, which might provide a novel mechanistic insight into the interface between autophagy and apoptosis in the progression of podocyte injury.     

Inhibition of hepatic glucose 6-phosphatase system by the green tea flavanol epigallocatechin gallate

Effect of 5-100 microM epigallocatechin gallate (EGCG) on hepatic glucose 6-phosphatase (G6Pase) system was investigated. EGCG inhibited G6Pase in intact but not in permeabilized rat liver microsomes, suggesting the interference with the transport. However, EGCG did not hinder microsomal glucose 6-phosphate (G6P) uptake. Instead, it increased the accumulation of radioactivity after the addition of [(14)C]G6P, presumably due to a slower release of [(14)C]glucose, the product of luminal hydrolysis. Indeed, EGCG was found to inhibit microsomal glucose efflux. Since G6Pase activity is depressed by glucose in a concentration-dependent manner, we concluded that EGCG inhibits G6Pase through an elevated luminal glucose level.     

Germ cell-specific gene 1 targets testis-specific poly(A) polymerase to the endoplasmic reticulum through protein-protein interactions

Testis-specific poly(A) polymerase (TPAP) is a cytoplasmic poly(A) polymerase that is highly expressed in round spermatids. We identified germ cell-specific gene 1 (GSG1) as a TPAP interaction partner protein using yeast two-hybrid and coimmunoprecipitation assays. Subcellular fractionation analysis showed that GSG1 is exclusively localized in the endoplasmic reticulum (ER) of mouse testis where TPAP is also present. In NIH3T3 cells cotransfected with TPAP and GSG1, both proteins colocalize in the ER. Moreover, expression of GSG1 stimulates TPAP targeting to the ER, suggesting that interactions between the two proteins lead to the redistribution of TPAP from the cytosol to the ER.     

Effect of Liraglutide on endoplasmic reticulum stress in diabetes

Endoplasmic reticulum (ER) stress is associated with the development of diabetes. The present study sought to investigate the effect of Liraglutide, a glucagon like peptide 1 analogue, on ER stress in β-cells. We found that Liraglutide protected the pancreatic INS-1 cells from thapsigargin-induced ER stress and the ER stress associated cell apoptosis, mainly by suppressing the PERK and IRE1 pathways. We further tested the effects of Liraglutide in the Akita mouse, an ER-stress induced type 1 diabetes model. After administration of Liraglutide for 8 weeks, p-eIF2α and p-JNK were significantly decreased in the pancreas of the Akita mouse, while the treatment showed no significant impact on the levels of insulin of INS-cells. Taken together, our findings suggest that Liraglutide may protect pancreatic cells from ER stress and its related cell death.     

Evidence for proprotein convertase activity in the endoplasmic reticulum/early Golgi

Processing of precursor proteins by the proprotein convertases is thought to occur mainly in the trans-Golgi network or post-Golgi compartments. Such cleavage is inhibited by the prosegment of the convertases. During our studies of the use of the inhibitory prosegment of PC1, we noticed that a construct containing the prosegment fused to the C-terminal secretory granule sorting domain was cleaved in the endoplasmic reticulum (ER) at a pair of basic residues, best recognized by furin and PC7. This was further confirmed when this construct was fused at the C-terminus with a KDEL ER-retention signal. This suggests that the convertases could cleave some substrates within the ER, possibly by displacing the inhibitory prosegment associated with them.     

Crossing the divide--transport between the endoplasmic reticulum and Golgi apparatus in plants

The transport of proteins between the endoplasmic reticulum (ER) and the Golgi apparatus in plants is an exciting and constantly expanding topic, which has attracted much attention in recent years. The study of protein transport within the secretory pathway is a relatively new field, dating back to the 1970s for mammalian cells and considerably later for plants. This may explain why COPI- and COPII-mediated transport between the ER and the Golgi in plants is only now becoming clear, while the existence of these pathways in other organisms is relatively well documented. We summarize current knowledge of these protein transport routes, as well as highlighting key differences between those of plant systems and those of mammals and yeast. These differences have necessitated the study of plant-specific aspects of protein transport in the early secretory pathway, and this review discusses recent developments in this area. Advances in live-cell-imaging technology have allowed the observation of protein movement in vivo, giving a new insight into many of the processes involved in vesicle formation and protein trafficking. The use of these new technologies has been combined with more traditional methods, such as protein biochemistry and electron microscopy, to increase our understanding of the transport routes in the cell.     

Glucose exposure pattern determines glucagon-like peptide 1 receptor expression and signaling through endoplasmic reticulum stress in rat insulinoma cells

Repeated fluctuation in plasma glucose levels, as well as chronic hyperglycemia, is an important phenomenon frequently observed in diabetic patients. Recently, several studies have reported that glucose fluctuation, compared to chronic hyperglycemia, mediates more adverse effects due to induced oxidative and/or endoplasmic reticulum (ER) stress. In type 2 diabetes, stimulation of insulin secretion by glucagon-like peptide-1 (GLP-1) has been found to be reduced, and the results of recent studies have shown that the expression of the GLP-1 receptor (GLP-1R) is reduced by chronic hyperglycemia. However, GLP-1R signaling in glucose fluctuation has not been elucidated clearly. In this study, we hypothesized that intermittent high glucose (IHG) conditions also reduced GLP-1-mediated cellular signaling via reduction in GLP-1R expression. To evaluate this hypothesis, rat insulinoma cells (INS-1) were exposed for 72 h to either sustained high glucose (SHG) conditions (30 mM glucose) or IHG conditions (11 and 30 mM glucose, alternating every 12 h). In comparison to both the SHG and control groups, IHG conditions induced a more significant impairment of insulin release and calcium influx in response to 1 nM GLP-1 treatment. In addition, the activity of caspase 3/7 as well as the gene expression of binding protein (Bip) and C/EBP homologous protein (CHOP), molecular markers of ER stress, was significantly higher in IHG-treated cells than in SHG-treated cells. Interestingly, the expression level of GLP-1R was significantly lower under IHG conditions than under SHG conditions. Collectively, these findings indicated that glucose fluctuation reduces GLP-1R expression through ER stress more profoundly than sustained hyperglycemia, which may contribute to the diminished response of GLP-1.     

Inhibition of anion permeability of sarcoplasmic reticulum vesicles by stilbene derivatives and the identification of an inhibitor-binding protein

The permeability of sarcoplasmic reticulum vesicles to sulfate ions was inhibited by diisothiocyano-1,2-diphenylethane-2,2′-disulfonic acid (H₂DIDS), which is a potent inhibitor of anion permeability in red blood cell membrane. The amount of H₂DIDS bound to the vesicles was determined by using [³H]-H₂DIDS. Apparent half inhibition of sulfate permeation was observed on the binding of 2.5 μmol/g protein. SDS-polyacrylamide gel electrophoresis of the vesicles treated with [³H]H₂DIDS showed that about 10% of the total bound H₂DIDS corresponds to a 100 000-dalton protein, but the remaining 90% to non-protein components. The content of the H₂DIDS-binding protein was about 0.5 μmol/g protein. These results suggest that the H₂DIDS-binding protein is different from the calcium pump protein and is possibly an anion transport system similar to band 3 in red blood cell membrane.     

Apelin alleviates diabetes-associated endoplasmic reticulum stress in the pancreas of Akita mice

Apelin, a newly identified bioactive adipokine, has been found to play important roles in multiple diseases, including diabetes, hypertension and cardiovascular diseases with unclear molecular mechanisms. The present study aimed to investigate the effects of apelin on endoplasmic reticulum (ER) stress in the pancreas of Akita mice, a well-established type 1 diabetic model. Apelin-13 (400 pmol/kg) was injected from tail vein for 10 weeks. The physiological characters of experimental animals were evaluated, pancreatic islet morphology and insulin content were assessed by immunohistochemistry, and ER stress markers in the pancreas were examined by Western blots. Our results indicate apelin treatment significantly ameliorates diabetes-induced reduction in pancreatic islet mass and insulin content. Further studies suggested apelin treatment alleviates ER stress by inhibiting the diabetes induced up-regulation of PERK and IRE1α and chaperones (GRP78, calnexin and Hsp70) levels in Akita mice. We also demonstrated that apelin treatment normalizes the diabetes induced alteration of AKT and ERK activations in the pancreas of Akita mice. Taken together, these results suggest a novel physiological role of apelin in alleviating ER stress in the pancreas of type 1 diabetes.     

Involvement of TR3/Nur77 translocation to the endoplasmic reticulum in ER stress-induced apoptosis

Nuclear orphan receptor TR3/Nur77/NGFI-B is a novel apoptotic effector protein that initiates apoptosis largely by translocating from the nucleus to the mitochondria, causing the release of cytochrome c. However, it is possible that TR3 translocates to other organelles. The present study was designed to determine the intracellular localization of TR3 following CD437-induced nucleocytoplasmic translocation and the mechanisms involved in TR3-induced apoptosis. In human neuroblastoma SK-N-SH cells and human esophageal squamous carcinoma EC109 and EC9706 cells, 5 microM CD437 induced translocation of TR3 to the endoplasmic reticulum (ER). This distribution was confirmed by immunofluorescence analysis, subcellular fractionation analysis and coimmunoprecipitation analysis. The translocated TR3 interacted with ER-targeting Bcl-2; initiated an early release of Ca(2+) from ER; resulted in ER stress and induced apoptosis through ER-specific caspase-4 activation, together with induction of mitochondrial stress and subsequent activation of caspase-9. Our results identified a novel distribution of TR3 in the ER and defined two parallel mitochondrial- and ER-based pathways that ultimately result in apoptotic cell death.