Modulation of palmitate-induced endoplasmic reticulum stress and apoptosis in pancreatic β-cells by stearoyl-CoA desaturase and Elovl6

Induction of endoplasmic reticulum (ER) stress and apoptosis by elevated exogenous saturated fatty acids (FAs) plays a role in the pathogenesis of β-cell dysfunction and loss of islet mass in type 2 diabetes. Regulation of monounsaturated FA (MUFA) synthesis through FA desaturases and elongases may alter the susceptibility of β-cells to saturated FA-induced ER stress and apoptosis. Herein, stearoyl-CoA desaturase (SCD)1 and SCD2 mRNA expression were shown to be induced in islets from prediabetic hyperinsulinemic Zucker diabetic fatty (ZDF) rats, whereas SCD1, SCD2, and fatty acid elongase 6 (Elovl6) mRNA levels were markedly reduced in diabetic ZDF rat islets. Knockdown of SCD in INS-1 β-cells decreased desaturation of palmitate to MUFA, lowered FA partitioning into complex neutral lipids, and increased palmitate-induced ER stress and apoptosis. Overexpression of SCD2 increased desaturation of palmitate to MUFA and attenuated palmitate-induced ER stress and apoptosis. Knockdown of Elovl6 limited palmitate elongation to stearate, increasing palmitoleate production and attenuating palmitate-induced ER stress and apoptosis, whereas overexpression of Elovl6 increased palmitate elongation to stearate and palmitate-induced ER stress and apoptosis. Overall, these data support the hypothesis that enhanced MUFA synthesis via upregulation of SCD2 activity can protect β-cells from elevated saturated FAs, as occurs in prediabetic states. Overt type 2 diabetes is associated with diminished islet expression of SCD and Elovl6, and this can disrupt desaturation of saturated FAs to MUFAs, rendering β-cells more susceptible to saturated FA-induced ER stress and apoptosis.     

Acacetin antagonized lipotoxicity in pancreatic β-cells via ameliorating oxidative stress and endoplasmic reticulum stress

Molecular Biology Reports - During the pathogenesis and progression of diabetes, lipotoxicity is a major threat to the function and survival of pancreatic β-cells. To battle against the...     

Action of protein disulfide isomerase on proinsulin exit from endoplasmic reticulum of pancreatic β-cells

For insulin synthesis, the proinsulin precursor is translated at the endoplasmic reticulum (ER), folds to include its three native disulfide bonds, and is exported to secretory granules for processing and secretion. Protein disulfide isomerase (PDI) has long been assumed to assist proinsulin in this process. Herein we have examined the effect of PDI knockdown (PDI-KD) in β-cells. The data establish that upon PDI-KD, oxidation of proinsulin to form native disulfide bonds is unimpaired and in fact enhanced. This is accompanied by improved proinsulin exit from the ER and increased total insulin secretion, with no evidence of ER stress. We provide evidence for direct physical interaction between PDI and proinsulin in the ER of pancreatic β-cells, in a manner requiring the catalytic activity of PDI. In β-cells after PDI-KD, enhanced export is selective for proinsulin over other secretory proteins, but the same effect is observed for recombinant proinsulin trafficking upon PDI-KD in heterologous cells. We hypothesize that PDI exhibits unfoldase activity for proinsulin, increasing retention of proinsulin within the ER of pancreatic β-cells.     

A role for aberrant protein palmitoylation in FFA-induced ER stress and β-cell death

Exposure of insulin-producing cells to elevated levels of the free fatty acid (FFA) palmitate results in the loss of β-cell function and induction of apoptosis. The induction of endoplasmic reticulum (ER) stress is one mechanism proposed to be responsible for the loss of β-cell viability in response to palmitate treatment; however, the pathways responsible for the induction of ER stress by palmitate have yet to be determined. Protein palmitoylation is a major posttranslational modification that regulates protein localization, stability, and activity. Defects in, or dysregulation of, protein palmitoylation could be one mechanism by which palmitate may induce ER stress in β-cells. The purpose of this study was to evaluate the hypothesis that palmitate-induced ER stress and β-cell toxicity are mediated by excess or aberrant protein palmitoylation. In a concentration-dependent fashion, palmitate treatment of RINm5F cells results in a loss of viability. Similar to palmitate, stearate also induces a concentration-related loss of RINm5F cell viability, while the monounsaturated fatty acids, such as palmoleate and oleate, are not toxic to RINm5F cells. 2-Bromopalmitate (2BrP), a classical inhibitor of protein palmitoylation that has been extensively used as an inhibitor of G protein-coupled receptor signaling, attenuates palmitate-induced RINm5F cell death in a concentration-dependent manner. The protective effects of 2BrP are associated with the inhibition of [(3)H]palmitate incorporation into RINm5F cell protein. Furthermore, 2BrP does not inhibit, but appears to enhance, the oxidation of palmitate. The induction of ER stress in response to palmitate treatment and the activation of caspase activity are attenuated by 2BrP. Consistent with protective effects on insulinoma cells, 2BrP also attenuates the inhibitory actions of prolonged palmitate treatment on insulin secretion by isolated rat islets. These studies support a role for aberrant protein palmitoylation as a mechanism by which palmitate enhances ER stress activation and causes the loss of insulinoma cell viability.     

Inhibition of GPR40 protects MIN6 β cells from palmitate-induced ER stress and apoptosis

Chronic exposure to elevated concentration of free fatty acids (FFA) has been verified to induce endoplasmic reticulum (ER) stress, which leads to pancreatic β-cell apoptosis. As one of the medium and long chain FFA receptors, GPR40 is highly expressed in pancreatic β cells, mediates both acute and chronic effects of FFA on β-cell function, but the role of GPR40 in FFA-induced β-cell apoptosis remains unclear. In this study, we investigated the possible effects of GPR40 in palmitate-induced MIN6 β-cell apoptosis, and found that DC260126, a novel small molecular antagonist of GPR40, could protect MIN6 β cells from palmitate-induced ER stress and apoptosis. Similar results were observed in GPR40-deficient MIN6 cells, indicating that palmitate-induced β-cell apoptosis is at least partially dependent on ER stress pathway via GRP40.     

Influence of SelS gene silence on β-Mercaptoethanol-mediated endoplasmic reticulum stress and cell apoptosis in HepG2 cells

Selenoprotein S (SelS), a transmembrane selenoprotein, may be related to the response of endoplasmic reticulum (ER) stress. In this report, the influence of selenite supplementation and SelS gene silence on β-mercaptoethanol (β-ME)-mediated ER stress and cell apoptosis in HepG2 cells were examined. The results showed that SelS protein expression was markedly increased by 10 mM β-ME and 100 nM sodium selenite in HepG2 cells. GRP78 protein level was significantly increased after treatment with 10 mM β-ME in HepG2 cells, which suggested that β-ME was also an ER stress inducer. Meanwhile, β-ME (10 mM) was found to induce cell apoptosis, which was alleviated obviously when cells were pretreated with 100 nM selenite before exposure to β-ME. Moreover, the suppression of SelS gene by siRNA could aggravate HepG2 cell apoptosis induced by β-ME significantly. In conclusion, these results suggested that β-ME, also an ER stress agent, could induce cell apoptosis, and SelS may play an important role in protecting cells from apoptosis induced by ER stress in HepG2 cells.     

Phosphatidylethanolamines modified by γ-ketoaldehyde (γKA) induce endoplasmic reticulum stress and endothelial activation

Peroxidation of plasma lipoproteins has been implicated in the endothelial cell activation and monocyte adhesion that initiate atherosclerosis, but the exact mechanisms underlying this activation remain unclear. Lipid peroxidation generates lipid aldehydes, including the γ-ketoaldehydes (γKA), also termed isoketals or isolevuglandins, that readily modify the amine headgroup of phosphatidylethanolamine (PE). We hypothesized that aldehyde modification of PE could mediate some of the proinflammatory effects of lipid peroxidation. We found that PE modified by γKA (γKA-PE) induced THP-1 monocyte adhesion to human umbilical cord endothelial cells. γKA-PE also induced expression of adhesion molecules and increased MCP-1 and IL-8 mRNA in human umbilical cord endothelial cells. To determine the structural requirements for γKA-PE activity, we tested several related compounds. PE modified by 4-oxo-pentanal induced THP-1 adhesion, but N-glutaroyl-PE and C(18:0)N-acyl-PE did not, suggesting that an N-pyrrole moiety was essential for cellular activity. As the N-pyrrole headgroup might distort the membrane, we tested the effect of the pyrrole-PEs on membrane parameters. γKA-PE and 4-oxo-pentanal significantly reduced the temperature for the liquid crystalline to hexagonal phase transition in artificial bilayers, suggesting that these pyrrole-PE markedly altered membrane curvature. Additionally, fluorescently labeled γKA-PE rapidly internalized to the endoplasmic reticulum (ER); γKA-PE induced C/EBP homologous protein CHOP and BiP expression and p38 MAPK activity, and inhibitors of ER stress reduced γKA-PE-induced C/EBP homologous protein CHOP and BiP expression as well as EC activation, consistent with γKA-PE inducing ER stress responses that have been previously linked to inflammatory chemokine expression. Thus, γKA-PE is a potential mediator of the inflammation induced by lipid peroxidation.     

A2a adenosine receptor agonist improves endoplasmic reticulum stress in MIN6 cell line through protein kinase A/ protein kinase B/ Cyclic adenosine monophosphate response element-binding protein/ and Growth Arrest And DNA-Damage-Inducible 34/ eukaryotic Initiation Factor 2α pathways

Endoplasmic reticulum (ER) stress is one of the main molecular events underlying pancreatic beta cell (PBC) failure, apoptosis, and a decrease in insulin secretion. Recent studies have highlighted the fundamental role of A2a adenosine receptor (A2aR) in potentiation of insulin secretion and proliferation of PBCs. However, possible protective effects of A2aR signaling against ER stress have not been elucidated yet. Thus, in the present study, we aimed to investigate the effects of A2aR activation in MIN6 beta cells undergoing tunicamycin (TM)-mediated ER stress. A2aR expression and activity were evaluated using real-time polymerase chain reaction and measurement of the cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), phospho-protein kinase B or Akt (p-Akt)/Akt, and phospho-Cyclic adenosine monophosphate response element-binding protein/CREB levels in response to a specific agonist (CGS 21680). Survival and proliferation in TM and CGS 21680 cotreated cells were evaluated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), annexin V–fluorescein isothiocyanate (FITC)/propidium iodide staining, colony formation, and 5-bromo-2′-deoxyuridine (Brdu) assays. In addition, the effects of A2aR stimulation on insulin secretion were evaluated using the enzyme-linked immunosorbent assay. B-cell lymphoma 2 (Bcl-2), phospho-eukaryotic Initiation Factor 2α (p-eIF2α)/eIF2α, growth arrest and DNA-damage-inducible 34 (GADD34), X-box binding protein 1 (XBP-1), spliced X-box binding protein 1 (XBP-1s), immunoglobulin heavy-chain-binding protein (BIP), and CCAAT-enhancer-binding protein homologous protein (CHOP) levels were evaluated using western blotting. Our results showed a decrease in A2aR expression and p-Akt/Akt and p-CREB/CREB levels in TM-pretreated cells. We also mentioned that CGS 21680 effectively increased cell survival, proliferation, and insulin secretion in TM-treated cells. The antiapoptotic effects were possibly mediated through Bcl-2 upregulation. Our western blotting results indicated that A2aR effectively downregulated p-eIF2α/eIF2α, XBP-1, XBP-1s, BIP, and CHOP levels, whereas GADD34 was upregulated. Altogether, the present study revealed that A2aR signaling through PKA/Akt/CREB mediators alleviated TM cytotoxicity effects in MIN6 beta cells. Thus, the stimulation of this receptor was seen as a new approach to control ER stress in the PBC cells.     

Modulation of endoplasmic reticulum (ER) stress-induced autophagy by C/EBP homologous protein (CHOP) and inositol-requiring enzyme 1α (IRE1α) in human colon cancer cells

To explore the relationship between UPR and autophagy in intestinal epithelial cells, we investigated whether autophagy was induced by endoplasmic reticulum (ER) stress in colon cancer cell lines. We demonstrated that autophagy was induced by ER stress in HT29, SW480, and Caco-2 cells. In these cells, inositol-requiring enzyme1α (IRE1α) and C/EBP homologous protein (CHOP) were involved in the ER stress–autophagy pathway, and CHOP was a regulator of IRE1α protein expression. Our findings suggest that CHOP promotes IRE1α and autophagy especially in ER stress conditions. This study will provide important insights into the disclosure of the ER stress–autophagy pathway.     

ERO1α-dependent endoplasmic reticulum–mitochondrial calcium flux contributes to ER stress and mitochondrial permeabilization by procaspase-activating compound-1 (PAC-1)

Procaspase-activating compound-1 (PAC-1) is the first direct caspase-activating compound discovered; using an in vitro cell-free system of caspase activation. Subsequently, this compound was shown to induce apoptosis in a variety of cancer cells with promising in vivo antitumor activity in canine lymphoma model. Recently, we have reported its ability to kill drug-resistant, Bcl-2/Bcl-xL overexpressing and Bax/Bak-deficient cells despite the essential requirement of mitochondrial cytochrome c (cyt. c) release for caspase activation, indicating that the key molecular targets of PAC-1 in cancer cells are yet to be identified. Here, we have identified Ero1α-dependent endoplasmic reticulum (ER) calcium leakage to mitochondria through mitochondria-associated ER membranes (MAM) and ER luminal hyper-oxidation as the critical events of PAC-1-mediated cell death. PAC-1 treatment upregulated Ero1α in multiple cell lines, whereas silencing of Ero1α significantly inhibited calcium release from ER and cell death. Loss of ER calcium and hyper-oxidation of ER lumen by Ero1α collectively triggered ER stress. Upregulation of GRP78 and splicing of X-box-binding protein 1 (XBP1) mRNA in multiple cancer cells suggested ER stress as the general event triggered by PAC-1. XBP1 mRNA splicing and GRP78 upregulation confirmed ER stress even in Bax/Bak double knockout and PAC-1-resistant Apaf-1-knockout cells, indicating an induction of ER stress-mediated mitochondrial apoptosis by PAC-1. Furthermore, we identified BH3-only protein p53 upregulated modulator of apoptosis (PUMA) as the key molecular link that orchestrates overwhelmed ER stress to mitochondria-mediated apoptosis, involving mitochondrial reactive oxygen species, in a p53-independent manner. Silencing of PUMA in cancer cells effectively reduced cyt. c release and cell death by PAC-1.     

Action of multiple endoplasmic reticulum chaperon-like proteins is required for proper folding and polarized localization of Kre6 protein essential in yeast cell wall β-1,6-glucan synthesis

Saccharomyces cerevisiae Kre6 is a type II membrane protein essential for cell wall β-1,6-glucan synthesis. Recently we reported that the majority of Kre6 is in the endoplasmic reticulum (ER), but a significant portion of Kre6 is found in the plasma membrane of buds, and this polarized appearance of Kre6 is required for β-1,6-glucan synthesis. An essential membrane protein, Keg1, and ER chaperon Rot1 bind to Kre6. In this study we found that in mutant keg1-1 cells, accumulation of Kre6 at the buds is diminished, binding of Kre6 to Keg1 is decreased, and Kre6 becomes susceptible to ER-associated degradation (ERAD), which suggests Keg1 participates in folding and transport of Kre6. All mutants of the calnexin cycle member homologues (cwh41, rot2, kre5, and cne1) showed defects in β-1,6-glucan synthesis, although the calnexin chaperon system is considered not functional in yeast. We found synthetic defects between them and keg1-1, and Cne1 co-immunoprecipitated with Keg1 and Kre6. A stronger binding of Cne1 to Kre6 was detected when two glucosidases (Cwh41 and Rot2) that remove glucose on N-glycan were functional. Skn1, a Kre6 homologue, was not detected by immunofluorescence in the wild type yeast, but in kre6Δ cells it became detectable and behaved like Kre6. In conclusion, the action of multiple ER chaperon-like proteins is required for proper folding and localization of Kre6 and probably Skn1 to function in β-1,6-glucan synthesis.     

Cdc37/Hsp90 protein-mediated regulation of IRE1α protein activity in endoplasmic reticulum stress response and insulin synthesis in INS-1 cells

IRE1α is an endoplasmic reticulum (ER) localized signaling molecule critical for unfolded protein response. During ER stress, IRE1α activation is induced by oligomerization and autophosphorylation in its cytosolic domain, a process triggered by dissociation of an ER luminal chaperone, binding immunoglobulin-protein (BiP), from IRE1α. In addition, inhibition of a cytosolic chaperone protein Hsp90 also induces IRE1α oligomerization and activation in the absence of an ER stressor. Here, we report that the Hsp90 cochaperone Cdc37 directly interacts with IRE1α through a highly conserved cytosolic motif of IRE1α. Cdc37 knockdown or disruption of Cdc37 interaction with IRE1α significantly increased basal IRE1α activity. In INS-1 cells, Hsp90 inhibition and disruption of IRE1α-Cdc37 interaction both induced an ER stress response and impaired insulin synthesis and secretion. These data suggest that Cdc37-mediated direct interaction between Hsp90/Cdc37 and an IRE1α cytosolic motif is important to maintain basal IRE1α activity and contributes to normal protein homeostasis and unfolded protein response under physiological stimulation.     

Cyclic AMP signaling stimulates proteasome degradation of thioredoxin interacting protein (TxNIP) in pancreatic β-cells

Thioredoxin interacting protein (TxNIP) functions as an effector of glucotoxicity in pancreatic β-cells. Exendin-4 (Ex-4), a long-term effective GLP-1 receptor agonist, reduces TxNIP level in pancreatic β-cells. Mechanisms underlying this reduction, however, remain largely unknown. We show here that Ex-4, 8-bromo-cAMP, the cAMP promoting agent forskolin, as well as activators of protein kinase A (PKA) and exchange protein activated by cAMP (Epac), all attenuated the effect of high glucose (20 mM) on TxNIP level in the pancreatic β-cell line Ins-1. Forskolin and Ex-4 also reduced TxNIP level in cultured primary rat islets. This repressive effect is at least partially mediated via stimulating proteasome-dependent TxNIP degradation, since the proteasomal inhibitor MG132, but not the lysosomal inhibitor chloroquine, significantly blocked the repressive effect of forskolin. Furthermore, forskolin enhanced TxNIP ubiquitination. Both PKA inhibition and Epac inhibition partially blocked the repressive effect of forskolin on TxNIP level. In addition, forskolin and Ex-4 protected Ins-1 cells from high glucose-induced apoptotic activity, assessed by measuring caspase 3 activity. Finally, knockdown of TxNIP expression led to reduced caspase 3 expression levels and blunted response to forskolin treatment. We suggest that proteasome-dependent TxNIP degradation is a novel mechanism by which Ex-4-cAMP signaling protects pancreatic β cells.     

A role for protein kinase C in the regulation of membrane fluidity and Ca²(+) flux at the endoplasmic reticulum and plasma membranes of HEK293 and Jurkat cells

Protein kinase C (PKC) plays a prominent role in the regulation of a variety of cellular functions, including Ca²⁺ signalling. In HEK293 and Jurkat cells, the Ca²⁺ release and Ca²⁺ uptake stimulated by several different activators were attenuated by activation of PKC with phorbol myristate acetate (PMA) or 1-oleoyl-2-acetyl-sn-glycerol (OAG) and potentiated by PKC inhibition with Gö6983 or knockdown of PKCα or PKCβ using shRNA. Immunostaining and Western blotting analyses revealed that PKCα and PKCβII accumulated at the plasma membrane (PM) and that these isoforms, along with PKCβI, also translocated to the endoplasmic reticulum (ER) upon activation with PMA. Measurements of membrane fluidity showed that, like the cell membrane stabilizers bovine serum albumin (BSA) and ursodeoxycholate (UDCA), PMA and OAG significantly reduced the fluidity of both the PM and ER membranes; these effects were blocked in PKC-knockdown cells. Interestingly, both BSA and UDCA inhibited the Ca²⁺ responses to agonists to the same extent as PMA, whereas Tween 20, which increases membrane fluidity, raised the internal Ca²⁺ concentration. Thus, activation of PKC induces both translocation of PKC to the PM and ER membranes and downregulation of membrane fluidity, thereby negatively modulating Ca²⁺ flux.