A Critical Role for Eukaryotic Elongation Factor 1A-1 in Lipotoxic Cell Death

Abstract

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. INTRODUCTIONThe increased prevalence of obesity worldwide has contributed to the emergence of a disease cluster, the metabolic syndrome, that includes insulin resistance, type 2 diabetes, and cardiovascular disease. Elevated serum triglyceride and fatty acid (FA) levels associated with this syndrome contribute to lipid accumulation in many nonadipose tissues, including the heart. This inappropriate accumulation of excess lipid can lead to cellular dysfunction and cell death-a process called lipotoxicity (Unger, 2003).Lipotoxic cardiomyocyte death has been proposed to play a central role in heart failure associated with diabetes and obesity in animal models and in humans. Although FAs are the principal source of energy for cardiomyocytes, high serum triglyceride and FA levels result in FA uptake by the heart that exceeds the anabolic and catabolic needs of the tissue. Triglyceride accumulation in cardiomyocytes of leptin-or leptin receptor-deficient obese diabetic animal models is associated with cardiomyocyte apoptosis (Zhou et al., 2000) and contractile dysfunction (Zhou et al., 2000;Aasum et al., 2002;Christoffersen et al., 2003), suggesting that lipotoxic cell death in the heart may be important in the genesis of diabetic cardiomyopathy. Recently, similar observations were reported in patients with metabolic syndrome and nonischemic heart failure (Sharma et al., 2004). Consistent with this apparent cardiac lipotoxicity, cardiomyocyte-specific increases in FA uptake in mice with cardiac-restricted overexpression of long-chain acyl-CoA synthetase 1 (ACS1), li...

RNASET2 is required for ROS propagation during oxidative stress-mediated cell death

“…To identify genes that are critical for the cellular response to lipotoxicity, we performed a genetic screen in Chinese hamster ovary (CHO) cells using the ROSAβgeo retroviral promoter trap to generate mutants, as previously described. 24 The promoterless proviral β-galactosidase-neomycin cassette confers antibiotic resistance only when it integrates downstream of an actively transcribed RNA polymerase II promoter, resulting in a fusion transcript that contains host and viral neomycin phosphotransferase sequences. CHO mutants were selected by growth in G418 and subsequently challenged by growth in media supplemented with 500 μM palmitate (palm) for 48 h, which has been shown to cause lipotoxic cell death in wild type (WT) cells (Figure 1a).…”

“…ROS is an important signaling intermediate during lipotoxicity, given that pre-treatment with antioxidants decreases palm-induced ROS and cell death. 24,31 The observation that treatment of cells with more direct inducers of oxidative stress, such as hydrogen peroxide, also causes cytoplasmic snoRNA accumulation suggests that ROS may be an upstream signal for the redistribution of snoRNAs. 22 Because cells haploinsufficient for RNASET2 fail to accumulate the rpL13a snoRNAs in the cytoplasm, but have no apparent defect in the generation of these snoRNAs (that is, nuclear levels are unchanged), we tested whether haploinsufficiency of RNASET2 modulated the generation of ROS in response to palm.…”

“…CHO cells were transduced with vesicular stomatitis virus G protein-pseudotyped murine retrovirus containing the ROSAβgeo retroviral promoter trap at a low multiplicity of infection, as previously described. 24 Retroviral insertion into the genome was assessed by Southern blotting. The gene disrupted by retroviral insertion was identified by 5′ RACE using an oligonucleotide tag and ROSAβgeo sequences (SMART RACE cDNA amplification kit; Clonetech, Mountain View, CA, USA).…”

Palmitate modulates intracellular signaling, induces endoplasmic reticulum stress, and causes apoptosis in mouse 3T3-L1 and rat primary preadipocytes

“…Palmitate has been shown to induce apoptosis in several cell types (4,5,30) but to enhance adipogenesis in preadipocytes maintained in a differentiation cocktail (18). To test its effects on preadipocytes in the absence of adipogenic inducers, we treated 3T3-L1 preadipocytes with palmitate in normal growth medium.…”

“…Learning how these conditions affect preadipocytes is important for understanding the mechanism of adipose dysfunction in obesity and type 2 diabetes. As the first step toward this goal, we tested the hypothesis that fatty acids can induce cellular "stress" in preadipocytes, as they do in several other cell types (5,24,30,39). We also tested how different fatty acids act on preadipocytes.…”