A critical role for eukaryotic elongation factor 1A-1 in lipotoxic cell death

The deleterious consequences of fatty acid (FA) and neutral lipid accumulation in nonadipose tissues, such as the heart, contribute to the pathogenesis of type 2 diabetes. To elucidate mechanisms of FA-induced cell death, or lipotoxicity, we generated Chinese hamster ovary (CHO) cell mutants resistant to palmitate-induced death and isolated a clone with disruption of eukaryotic elongation factor (eEF) 1A-1. eEF1A-1 involvement in lipotoxicity was confirmed in H9c2 cardiomyoblasts, in which small interfering RNA-mediated knockdown also conferred palmitate resistance. In wild-type CHO and H9c2 cells, palmitate increased reactive oxygen species and induced endoplasmic reticulum (ER) stress, changes accompanied by increased eEF1A-1 expression. Disruption of eEF1A-1 expression rendered these cells resistant to hydrogen peroxide- and ER stress-induced death, indicating that eEF1A-1 plays a critical role in the cell death response to these stressors downstream of lipid overload. Disruption of eEF1A-1 also resulted in actin cytoskeleton defects under basal conditions and in response to palmitate, suggesting that eEF1A-1 mediates lipotoxic cell death, secondary to oxidative and ER stress, by regulating cytoskeletal changes critical for this process. Furthermore, our observations of oxidative stress, ER stress, and induction of eEF1A-1 expression in a mouse model of lipotoxic cardiomyopathy implicate this cellular response in the pathophysiology of metabolic disease.     

The eukaryotic translation elongation factor 1A regulation of actin stress fibers is important for infectious RSV production

Cellular protein eukaryotic translation elongation factor 1A (eEF1A) is an actin binding protein that plays a role in the formation of filamentous actin (F-actin) bundles. F-Actin regulates multiple stages of respiratory syncytial virus (RSV) replication including assembly and budding. Our previous study demonstrated that eEF1A knock-down significantly reduced RSV replication. Here we investigated if the eEF1A function in actin bundle formation was important for RSV replication and release. To investigate this, eEF1A function was impaired in HEp-2 cells by either knock-down of eEF1A with siRNA, or treatment with an eEF1A inhibitor, didemnin B (Did B). Cell staining and confocal microscopy analysis showed that both eEF1A knock-down and treatment with Did B resulted in disruption of cellular stress fiber formation and elevated accumulation of F-actin near the plasma membrane. When treated cells were then infected with RSV, there was also reduced formation of virus-induced cellular filopodia. Did B treatment, similarly to eEF1A knock-down, reduced the release of infectious RSV, but unlike eEF1A knock-down, did not significantly affect RSV genome replication. The lower infectious virus production in Did B treated cells also reduced RSV-induced cell death. In conclusion, the cellular factor eEF1A plays an important role in the regulation of F-actin stress fiber formation required for RSV assembly and release.     

Cellular Inhibitor of Apoptosis Protein-1 (cIAP1) Plays a Critical Role in β-Cell Survival under Endoplasmic Reticulum Stress: PROMOTING UBIQUITINATION AND DEGRADATION OF C/EBP HOMOLOGOUS PROTEIN (CHOP)

Lipotoxicity in pancreatic β-cells, arising from excess free fatty acid-induced endoplasmic reticulum (ER) stress response, has been recognized as a key pathogenic factor causing loss of β-cell mass and contributing to type 2 diabetes. However, how the adaptive ER stress response causes cell death remains enigmatic. We report herein a critical role of cellular inhibitor of apoptosis protein-1 (cIAP1) in controlling β-cell survival under ER stress. While both palmitate and palmitoleate induced an overt ER stress response, lipotoxicity was only observed in β-cells exposed to palmitate but not palmitoleate. Interestingly, cells treated with palmitoleate exerted a sustainable level of cIAP1, whereas the protein quickly degraded following palmitate treatment. Enforced overexpression of cIAP1 prevented palmitate-induced cell death. In contrast, siRNA-mediated knockdown of cIAP1 in β-cells or knock-out of cIap1 in mouse embryonic fibroblasts not only increased palmitate-induced apoptosis, but also committed cells to death in response to the nontoxic palmitoleate treatment. Of importance, we found that cIAP1 functions as an E3 ubiquitin ligase promoting ubiquitination and degradation of C/EBP homologous protein (CHOP), a key mediator of ER stress-induced cell death. These findings define a novel mechanism for β-cell survival under ER stress and help to identify targets for therapeutic intervention against lipotoxicity in β-cells.     

The TLR4‐IRE1α pathway activation contributes to palmitate‐elicited lipotoxicity in hepatocytes

Lipotoxicity induced by saturated fatty acids (SFAs) plays a pathological role in the development of non‐alcoholic fatty liver disease (NAFLD); however, the exact mechanism(s) remain to be clearly elucidated. Toll‐like receptor (TLR) 4 plays a fundamental role in activating the innate immune system. Intriguingly, hepatocytes express TLR4 and machinery for TLR4 signalling pathway. That liver‐specific TLR4 knockout mice are protective against diet‐induced NAFLD suggests that hepatocyte TLR4 signalling pathway plays an important role in NAFLD pathogenesis. Herein, using cultured hepatocytes, we sought to directly examine the role of TLR4 signalling pathway in palmitate‐elicited hepatotoxicity and to elucidate underlying mechanism(s). Our data reveal that palmitate exposure up‐regulates TLR4 expression at both mRNA and protein levels in hepatocytes, which are associated with NF‐κB activation. The inhibition of TLR4 signalling pathway through both pharmacological and genetic approaches abolished palmitate‐induced cell death, suggesting that TLR4 signalling pathway activation contributes to palmitate‐induced hepatotoxicity. Mechanistic investigations demonstrate that inositol‐requiring enzyme 1α (IRE1α), one of three major signal transduction pathways activated during endoplasmic reticulum (ER) stress, is the downstream target of palmitate‐elicited TLR4 activation and mechanistically implicated in TLR4 activation‐triggered cell death in response to palmitate exposure. Collectively, our data identify that the TLR4‐IRE1α pathway activation contributes to palmitate‐elicited lipotoxicity in hepatocytes. Our findings suggest that targeting TLR4‐IRE1α pathway can be a potential therapeutic choice for the treatment of NAFLD as well as other metabolic disorders, with lipotoxicity being the principal pathomechanism.     

Mouse translation elongation factor eEF1A-2 interacts with Prdx-I to protect cells against apoptotic death induced by oxidative stress

eEF1A-1 and eEF1A-2 are two isoforms of translation elongation factor eEF1A. In adult mammalian tissues, isoform eEF1A-1 is present in all tissues except neurons, cardiomyocytes, and myotubes, where its isoform, eEF1A-2, is the only form expressed. Both forms of eEF1A have been characterized to function in the protein elongation step of translation, and eEF1A-1 is shown to possess additional non-canonical roles in actin binding/bundling, microtubule bundling/severing, and cellular transformation processes. To study whether eEF1A-2 has similar non-canonical functions, we carried out a yeast two-hybrid screening using a full sequence of mouse eEF1A-2 as bait. A total of 78 hits, representing 23 proteins, were identified and validated to be true positives. We have focused on the protein with the highest frequency of hits, peroxiredoxin I (Prdx-I), for in-depth study of its functional implication for eEF1A-2. Here we show that Prdx-I coimmunoprecipitates with eEF1A-2 from extracts of both cultured cells and mouse tissues expressing this protein, but it does not do so with its isoform, eEF1A-1, even though the latter is abundantly present. We also report that an eEF1A-2 and Prdx-I double transfectant increases resistance to peroxide-induced cell death as high as 1 mM peroxide treatment, significantly higher than do single transfectants with either gene alone; this protection is correlated with reduced activation of caspases 3 and 8, and with increased expression of pro-survival factor Akt. Thus, our results suggest that eEF1A-2 interacts with Prdx-I to functionally provide cells with extraordinary resistance to oxidative stress-induced cell death.     

Saturated fatty acids induce endoplasmic reticulum stress and apoptosis independently of ceramide in liver cells

Accumulation of lipids in nonadipose tissues can lead to cell dysfunction and cell death, a phenomenon known as lipotoxicity. However, the signaling pathways and mechanisms linking lipid accumulation to cell death are poorly understood. The present study examined the hypothesis that saturated fatty acids disrupt endoplasmic reticulum (ER) homeostasis and promote apoptosis in liver cells via accumulation of ceramide. H4IIE liver cells were exposed to varying concentrations of saturated (palmitate or stearate) or unsaturated (oleate or linoleate) fatty acids. ER homeostasis was monitored using markers of the ER stress response pathway, including phosphorylation of IRE1alpha and eIF2alpha, splicing of XBP1 mRNA, and expression of molecular chaperone (e.g., GRP78) and proapoptotic (CCAAT/enhancer-binding protein homologous protein) genes. Apoptosis was monitored using caspase activity and DNA laddering. Palmitate and stearate induced ER stress, caspase activity, and DNA laddering. Inhibition of caspase activation prevented DNA laddering. Unsaturated fatty acids did not induce ER stress or apoptosis. Saturated fatty acids increased ceramide concentration; however, inhibition of de novo ceramide synthesis did not prevent saturated fatty acid-induced ER stress and apoptosis. Unsaturated fatty acids rescued palmitate-induced ER stress and apoptosis. These data demonstrate that saturated fatty acids disrupt ER homeostasis and induce apoptosis in liver cells via mechanisms that do not involve ceramide accumulation.     

Alteration of Endoplasmic Reticulum Lipid Rafts Contributes to Lipotoxicity in Pancreatic β-Cells

Chronic saturated fatty acid exposure causes β-cell apoptosis and, thus, contributes to type 2 diabetes. Although endoplasmic reticulum (ER) stress and reduced ER-to-Golgi protein trafficking have been implicated, the exact mechanisms whereby saturated fatty acids trigger β-cell death remain elusive. Using mass spectroscopic lipidomics and subcellular fractionation, we demonstrate that palmitate pretreatment of MIN6 β-cells promoted ER remodeling of both phospholipids and sphingolipids, but only the latter was causally linked to lipotoxic ER stress. Thus, overexpression of glucosylceramide synthase, previously shown to protect against defective protein trafficking and ER stress, partially reversed lipotoxic reductions in ER sphingomyelin (SM) content and aggregation of ER lipid rafts, as visualized using Erlin1-GFP. Using both lipidomics and a sterol response element reporter assay, we confirmed that free cholesterol in the ER was also reciprocally modulated by chronic palmitate and glucosylceramide synthase overexpression. This is consistent with the known coregulation and association of SM and free cholesterol in lipid rafts. Inhibition of SM hydrolysis partially protected against ATF4/C/EBP homology protein induction because of palmitate. Our results suggest that loss of SM in the ER is a key event for initiating β-cell lipotoxicity, which leads to disruption of ER lipid rafts, perturbation of protein trafficking, and initiation of ER stress.     

Characterization of elongation factor-1A (eEF1A-1) and eEF1A-2/S1 protein expression in normal and wasted mice

The eEF1Alpha-2 gene (S1) encodes a tissue-specific isoform of peptide elongation factor-1A (eEF1A-1); its mRNA is expressed only in brain, heart, and skeletal muscle, tissues dominated by terminally differentiated, long-lived cells. Homozygous mutant mice exhibit muscle wasting and neurodegeneration, resulting in death around postnatal day 28. eEF1Alpha-2/S1 protein shares 92% identity with eEF1A-1; because specific antibodies for each were not available previously, it was difficult to study the developmental expression patterns of these two peptide elongation factors 1A in wasted and wild-type mice. We generated a peptide-derived antiserum that recognizes the eEF1Alpha-2/S1 isoform and does not cross-react with eEF1A-1. We characterized the expression profiles of eEF1A-1 and eEF1A-2/S1 during development in wild-type (+/+), heterozygous (+/wst), and homozygous (wst/wst) mice. In wild-type and heterozygous animals, eEF1A-2/S1 protein is present only in brain, heart, and muscle; the onset of its expression coincides with a concomitant decrease in the eEF1A-1 protein level. In wasted mutant tissues, even though eEF1A-2/S1 protein is absent, the scheduled decline of eEF1A-1 occurs nonetheless during postnatal development, as it does in wild-type counterparts. In the brain of adult wild-type mice, the eEF1A-2/S1 isoform is localized in neurons, whereas eEF1A-1 is found in non-neuronal cells. In neurons prior to postnatal day 7, eEF1A-1 is the major isoform, but it is later replaced by eEF1A-2/S1, which by postnatal day 14 is the only isoform present. The postdevelopmental appearance of eEF1A-2/S1 protein and the decline in eEF1A-1 expression in brain, heart, and muscle suggest that eEF1A-2/S1 is the adult form of peptide elongation factor, whereas its sister is the embryonic isoform, in these tissues. The absence of eEF1A-2/S1, as well as the on-schedule development-dependent disappearance of its sister gene, eEF1A, in wst/wst mice may result in loss of protein synthesis ability, which may account for the numerous defects and ultimate fatality seen in these mice.     

Peptide elongation factor eEF1A-2/S1 expression in cultured differentiated myotubes and its protective effect against caspase-3-mediated apoptosis.

Peptide elongation factor eEF1A-2/S1, which shares 92% homology with eEF1A-1/EF-1alpha, is exclusively expressed in brain, heart, and skeletal muscle. In these tissues, eEF1A-2/S1 is the only type 1A elongation factor expressed in adulthood because a transition from eEF1A-1/EF-1alpha to eEF1A-2/S1 occurs in early postnatal development. In this article, we report that the expression of eEF1A-2/S1 protein is activated upon myogenic differentiation. Furthermore, we show that upon serum deprivation-induced apoptosis, eEF1A-2/S1 protein disappears and is replaced by its homolog eEF1A-1/EF-1alpha in dying myotubes; cell death is characterized by the activation of caspase-3. In addition, we show that the continuous expression of eEF1A-2/S1 resulting from adenoviral gene transfer protects differentiated myotubes from apoptosis by delaying their death, thus suggesting a prosurvival function for eEF1A-2/S1 in skeletal muscle. In contrast, myotube death is accelerated by the introduction of the homologous gene, eEF1A-1/EF-1alpha, whereas cells transfected with antisense eEF1A-1/EF-1alpha are protected from apoptosis. These results demonstrate that the two sister genes, eEF1A-1/EF-1alpha and eEF1A-2/S1, regulate myotube survival with the former exerting prodeath activity and the latter a prosurvival effect.     

Overexpression of translation elongation factor 1A affects the organization and function of the actin cytoskeleton in yeast

The translation elongation factor 1 complex (eEF1) plays a central role in protein synthesis, delivering aminoacyl-tRNAs to the elongating ribosome. The eEF1A subunit, a classic G-protein, also performs roles aside from protein synthesis. The overexpression of either eEF1A or eEF1B alpha, the catalytic subunit of the guanine nucleotide exchange factor, in Saccharomyces cerevisiae results in effects on cell growth. Here we demonstrate that overexpression of either factor does not affect the levels of the other subunit or the rate or accuracy of protein synthesis. Instead, the major effects in vivo appear to be at the level of cell morphology and budding. eEF1A overexpression results in dosage-dependent reduced budding and altered actin distribution and cellular morphology. In addition, the effects of excess eEF1A in actin mutant strains show synthetic growth defects, establishing a genetic connection between the two proteins. As the ability of eEF1A to bind and bundle actin is conserved in yeast, these results link the established ability of eEF1A to bind and bundle actin in vitro with nontranslational roles for the protein in vivo.     

Enhanced synthesis of saturated phospholipids is associated with ER stress and lipotoxicity in palmitate treated hepatic cells

High levels of saturated FAs (SFAs) are acutely toxic to a variety of cell types, including hepatocytes, and have been associated with diseases such as type 2 diabetes and nonalcoholic fatty liver disease. SFA accumulation has been previously shown to degrade endoplasmic reticulum (ER) function leading to other manifestations of the lipoapoptotic cascade. We hypothesized that dysfunctional phospholipid (PL) metabolism is an initiating factor in this ER stress response. Treatment of either primary hepatocytes or H4IIEC3 cells with the SFA palmitate resulted in dramatic dilation of the ER membrane, coinciding with other markers of organelle dysfunction. This was accompanied by increased de novo glycerolipid synthesis, significant elevation of dipalmitoyl phosphatidic acid, diacylglycerol, and total PL content in H4IIEC3 cells. Supplementation with oleate (OA) reversed these markers of palmitate (PA)-induced lipotoxicity. OA/PA cotreatment modulated the distribution of PA between lipid classes, increasing the flux toward triacylglycerols while reducing its incorporation into PLs. Similar trends were demonstrated in both primary hepatocytes and the H4IIEC3 hepatoma cell line. Overall, these findings suggest that modifying the FA composition of structural PLs can protect hepatocytes from PA-induced ER stress and associated lipotoxicity.     

The eukaryotic translation elongation Factor 1Bgamma has a non-guanine nucleotide exchange factor role in protein metabolism

The turnover of damaged proteins is critical to cell survival during stressful conditions such as heat shock or oxidative stress. The accumulation of misfolded proteins in the endoplasmic reticulum (ER) is toxic to cells. Therefore these proteins must be efficiently exported from the ER and degraded by the proteasome or the vacuole. Previously it was shown that the loss of eukaryotic elongation factor 1Bγ (eEF1Bγ) from the yeast Saccharomyces cerevisiae results in resistance to oxidative stress. Strains lacking eEF1Bγ show severe defects in protein turnover during conditions of oxidative stress. Furthermore, these strains accumulate a greater amount of oxidized proteins, which correlates with changes in heat shock chaperones. These strains show severe defects in vacuole morphology and defects related to the maturation of carboxypeptidase Y that is not dependent on the catalytic subunit of the eEF1B complex as a guanine nucleotide exchange factor. Finally, eEF1Bγ co-immunoprecipitates with an essential component of ER-Golgi transport vesicles. Taken together, these results support a broader protein metabolism role for eEF1Bγ.     

1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125) induces phosphorylation of eukaryotic elongation factor-2 (eEF2): a cautionary note on the anticancer mechanism of an eEF2 kinase inhibitor

Eukaryotic elongation factor-2 kinase (eEF2K) relays growth and stress signals to protein synthesis through phosphorylation and inactivation of eukaryotic elongation factor 2 (eEF2). 1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125) is a widely accepted inhibitor of mammalian eEF2K and an efficacious anti-proliferation agent against different cancer cells. It implied that eEF2K could be an efficacious anticancer target. However, eEF2K siRNA was ineffective against cancer cells including those sensitive to NH125. To test if pharmacological intervention differs from siRNA interference, we identified a highly selective small molecule eEF2K inhibitor A-484954. Like siRNA, A-484954 had little effect on cancer cell growth. We carefully examined the effect of NH125 and A-484954 on phosphorylation of eEF2, the known cellular substrate of eEF2K. Surprisingly, NH125 increased eEF2 phosphorylation, whereas A-484954 inhibited the phosphorylation as expected for an eEF2K inhibitor. Both A-484954 and eEF2K siRNA inhibited eEF2K and reduced eEF2 phosphorylation with little effect on cancer cell growth. These data demonstrated clearly that the anticancer activity of NH125 was more correlated with induction of eEF2 phosphorylation than inhibition of eEF2K. Actually, induction of eEF2 phosphorylation was reported to correlate with inhibition of cancer cell growth. We compared several known inducers of eEF2 phosphorylation including AMPK activators and an mTOR inhibitor. Interestingly, stronger induction of eEF2 phosphorylation correlated with more effective growth inhibition. We also explored signal transduction pathways leading to NH125-induced eEF2 phosphorylation. Preliminary data suggested that NH125-induced eEF2 phosphorylation was likely mediated through multiple pathways. These observations identified an opportunity for a new multipathway approach to anticancer therapies.     

Mechanisms of lysophosphatidylcholine-induced hepatocyte lipoapoptosis

Isolated hepatocytes undergo lipoapoptosis, a feature of hepatic lipotoxicity, on treatment with saturated free fatty acids (FFA) such as palmitate (PA). However, it is unknown if palmitate is directly toxic to hepatocytes or if its toxicity is indirect via the generation of lipid metabolites such as lysophosphatidylcholine (LPC). PA-mediated hepatocyte lipoapoptosis is associated with endoplasmic reticulum (ER) stress, c-Jun NH(2)-terminal kinase (JNK) activation, and a JNK-dependent upregulation of the potent proapoptotic BH3-only protein PUMA (p53 upregulated modulator of apoptosis). Our aim was to determine which of these mechanisms of lipotoxicity are activated by PA-derived LPC. We employed Huh-7 cells and isolated murine and human primary hepatocytes. Intracellular LPC concentrations increase linearly as a function of the exogenous, extracellular PA, stearate, or LPC concentration. Incubation of Huh-7 cells or primary hepatocytes with LPC induced cell death by apoptosis in a concentration-dependent manner. Substituting LPC for PA resulted in caspase-dependent cell death that was accompanied by activating phosphorylation of JNK with c-Jun phosphorylation and an increase in PUMA expression. LPC also induced ER stress as manifest by eIF2α phosphorylation and CAAT/enhancer binding homologous protein (CHOP) induction. LPC cytotoxicity was attenuated by pharmacological inhibition of JNK or glycogen synthase kinase-3 (GSK-3). Similarly, short-hairpin RNA (shRNA)-targeted knockdown of CHOP protected Huh-7 cells against LPC-induced toxicity. The LPC-induced PUMA upregulation was prevented by JNK inhibition or shRNA-targeted knockdown of CHOP. Finally, genetic deficiency of PUMA rendered murine hepatocytes resistant to LPC-induced apoptosis. We concluded that LPC-induced lipoapoptosis is dependent on mechanisms largely indistinguishable from PA. These data suggest that FFA-mediated cytotoxicity is indirect via the generation of the toxic metabolite, LPC.     

Nerve growth factor specifically stimulates translation of eukaryotic elongation factor 1A-1 (eEF1A-1) mRNA by recruitment to polyribosomes in PC12 cells

During postnatal brain development the level of peptide elongation factor-1A (eEF1A-1) expression declines and that of the highly homologous isoform, eEF1A-2, increases in neurons. eEF1A-1 is implicated in cytoskeletal interactions, tumorigenesis, differentiation, and the absence of eEF1A-2 is implicated in neurodegeneration in the mouse mutant, wasted. The translation of eEF1A-1 mRNA is up-regulated via mitogenic stimulation. However, it is not known if eEF1A-1 mRNA translation is regulated by neurotrophins or if its synthesis is differentially regulated than that of the neuronal isoform, eEF1A-2. Regulated translation of these factors by neurotrophins, particularly by the Trk class of neurotrophin receptors, would implicate them in differentiation, survival, and neuronal plasticity. In this study, we investigated the effect of nerve growth factor (NGF) stimulation on the synthesis of eEF1A-1 and eEF1A-2. We found that NGF stimulation causes a preferential synthesis of eEF1A-1 over eEF1A-2 in PC12 cells. We analyzed the co-sedimentation of eEF1A-1 mRNA with polyribosome fractions in sucrose gradients, and found that NGF stimulation enriched the presence of eEF1A-1 mRNA in polyribosomes, indicating that the translation of eEF1A-1 mRNA is regulated by NGF. Inhibitors of phosphatidylinositol 3-kinase (LY 294002), mammalian target of rapamycin (rapamycin), and the NGF receptor, TrkA (K-252a), but not of mitogen-activated protein kinase (PD 98059), prevented the recruitment of eEF1A-1 mRNA to polyribosomes. The mobilization of eEF1A-1 mRNA to polyribosomes was rapamycin-sensitive in both proliferating and differentiated PC12 cells, indicating the importance of this pathway during differentiation. Our data shows that after growth factor withdrawal, an NGF-signaling pathway stimulates eEF1A-1 mRNA translation in proliferating and differentiated PC12 cells. Therefore, eEF1A-1 mRNA is a specific translational target of TrkA signaling.     

Elongation factor 1beta is an actin-binding protein

A 17 kDa polypeptide found in association with actin in cellular extracts of Dictyostelium discoideum was identified as a proteolytic fragment of eEF1beta. Antibody elicited against the 17 kDa protein reacted with a single 29 kDa polypeptide in Dictyostelium, indicating that the 17 kDa peptide arises from degradation of a larger precursor. The cDNA isolated from a Dictyostelium library using this antibody as a probe encodes Dictyostelium elongation factor 1beta. Amino acid degradation of the 17 kDa protein fragment confirmed the identity of the protein as eEF1beta. Direct interaction of eEF1beta with actin in vitro was further demonstrated in mixtures of actin with the 17 kDa protein fragment of Dictyostelium eEF1beta, recombinant preparations of Dictyostelium eEF1beta expressed in Escherichia coli, and the intact eEF1betagamma complex purified from wheat germ. Localization of eEF1beta in Dictyostelium by immunofluorescence microscopy reveals both diffuse cytoplasmic staining, and some concentration in the cortical and hyaline cytoplasm. The results support the existence of physical and functional interactions of the translation apparatus with the cytoskeleton, and suggest that eEF1beta may function in a dual role both to promote the elongation phase of protein synthesis, and to interact with cytoplasmic actin.     

Disruption of endoplasmic reticulum structure and integrity in lipotoxic cell death

Cell dysfunction and death induced by lipid accumulation in nonadipose tissues, or lipotoxicity, may contribute to the pathogenesis of obesity and type 2 diabetes. However, the mechanisms leading to lipotoxic cell death are poorly understood. We recently reported that, in Chinese hamster ovary (CHO) cells and in H9c2 cardiomyoblasts, lipid overload induced by incubation with 500 muM palmitate leads to intracellular accumulation of reactive oxygen species, which subsequently induce endoplasmic reticulum (ER) stress and cell death. Here, we show that palmitate also impairs ER function through a more direct mechanism. Palmitate was rapidly incorporated into saturated phospholipid and triglyceride species in microsomal membranes of CHO cells. The resulting membrane remodeling was associated with dramatic dilatation of the ER and redistribution of protein-folding chaperones to the cytosol within 5 h, indicating compromised ER membrane integrity. Increasing beta-oxidation, through the activation of AMP-activated protein kinase, decreased palmitate incorporation into microsomes, decreased the escape of chaperones to the cytosol, and decreased subsequent caspase activation and cell death. Thus, palmitate rapidly increases the saturated lipid content of the ER, leading to compromised ER morphology and integrity, suggesting that impairment of the structure and function of this organelle is involved in the cellular response to fatty acid overload.