Excess lipid accumulation in non-adipose tissues is associated with insulin resistance, pancreatic ␤-cell apoptosis and heart failure. Here, we demonstrate in cultured cells that the relative toxicity of two common dietary long chain fatty acids is related to channeling of these lipids to distinct cellular metabolic fates. Oleic acid supplementation leads to triglyceride accumulation and is well tolerated, whereas excess palmitic acid is poorly incorporated into triglyceride and causes apoptosis. Unsaturated fatty acids rescue palmitate-induced apoptosis by channeling palmitate into triglyceride pools and away from pathways leading to apoptosis. Moreover, in the setting of impaired triglyceride synthesis, oleate induces lipotoxicity. Our findings support a model of cellular lipid metabolism in which unsaturated fatty acids serve a protective function against lipotoxicity though promotion of triglyceride accumulation.

Cytotoxic accumulation of long chain fatty acids has been proposed to play an important role in the pathogenesis of diabetes mellitus and heart disease. To explore the mechanism of cellular lipotoxicity, we cultured Chinese hamster ovary cells in the presence of media supplemented with fatty acid. The saturated fatty acid palmitate, but not the monounsaturated fatty acid oleate, induced programmed cell death as determined by annexin V positivity, caspase 3 activity, and DNA laddering. De novo ceramide synthesis increased 2.4-fold with palmitate supplementation; however, this was not required for palmitate-induced apoptosis. Neither biochemical nor genetic inhibition of de novo ceramide synthesis arrested apoptosis in Chinese hamster ovary cells in response to palmitate supplementation. Rather, our data suggest that palmitate-induced apoptosis occurs through the generation of reactive oxygen species. Fluorescence of an oxidant-sensitive probe was increased 3.5-fold with palmitate supplementation indicating that production of reactive intermediates increased. In addition, palmitate-induced apoptosis was blocked by pyrrolidine dithiocarbamate and 4,5-dihydroxy-1,3-benzene-disulfonic acid, two compounds that scavenge reactive intermediates. These studies suggest that generation of reactive oxygen species, independent of ceramide synthesis, is important for the lipotoxic response and may contribute to the pathogenesis of diseases involving intracellular lipid accumulation.

Although neutral lipid storage droplets are ubiquitous in eukaryotic cells, very little is known about how their synthesis and turnover are controlled. Adipocyte differentiation-related protein (ADRP; also known as adipophilin) is found on the surface of lipid droplets in most mammalian cell types. To learn how ADRP affects lipid storage, we stably expressed the protein in human embryonic kidney 293 (HEK 293) cells, which express little endogenous ADRP. As expected, ADRP was targeted to the surface of lipid droplets and caused an increase in triacylglycerol (TAG) mass under both basal and oleate-supplemented conditions. At least part of the increased mass resulted from a 50% decrease in the rate of TAG hydrolysis in ADRPexpressing cells. Furthermore, ADRP expression increased the fraction of total cellular TAG that was stored in lipid droplets. ADRP expression induced a striking decrease in the association of adipose triglyceride lipase (ATGL) and mannose-6-phosphate receptor tail-interacting protein of 47 kDa with lipid droplets and also decreased the lipid droplet association of several other unknown proteins. Transient expression of ADRP in two other cell lines also reduced the lipid droplet association of catalytically inactive ATGL. We conclude that the reduced lipid droplet association of ATGL and/or other lipases may explain the decrease in TAG turnover observed in ADRP-expressing HEK 293 cells. Eukaryotes store lipid in cytosolic lipid droplets, which consist of neutral lipid cores surrounded by phospholipid monolayers (1-3). In mammals, lipid droplets are most abundant in adipose tissue, where stored triacylglycerol (TAG) provides the primary energy reserve for the organism. Lipid droplets in steroidogenic cells contain cholesteryl esters used in the synthesis of steroid hormones. Most other mammalian cells contain smaller lipid droplets, whose function remains unclear. They may serve as local energy reserves or sources of lipid for membrane synthesis. Furthermore, they may protect cells from the harmful effects of excess lipid accumulation by sequestering toxic lipid species away from pathways that lead to cell death (4, 5).Mechanisms controlling the synthesis and turnover of lipid droplets are only partially understood. According to one model of lipid droplet biogenesis, newly synthesized neutral lipids accumulate inside the endoplasmic reticulum membrane, forming a disk that eventually buds into the cytoplasm surrounded by an endoplasmic reticulumderived phospholipid monolayer (2, 6). Conversely, lipid droplet turnover occurs via the hydrolysis of stored neutral lipids by cytosolic lipases. Much of what we know about the regulation of lipolysis stems from studies in adipocytes. In response to hormone stimulation, protein kinase A phosphorylates two key substrates: hormone-sensitive lipase (HSL) (7) and perilipins (8, 9). Phosphorylation of HSL stimulates both its activity and its association with lipid droplets, in a manner that depends on perilipins.Perilipins regulate TAG hydrolysis in two...

SUMMARY Lipotoxicity is a metabolic stress response implicated in the pathogenesis of diabetes complications and has been shown to involve lipid-induced oxidative stress. To elucidate the molecular mechanisms of lipotoxicity, we used retroviral promoter trap mutagenesis to isolate a cell line that is resistant to lipotoxic and oxidative stress. We show that loss of three box C/D small nucleolar RNAs (snoRNAs) encoded in the ribosomal protein L13a (rpL13a) locus is sufficient to confer resistance to lipotoxic and oxidative stress in vitro and prevents the propagation of oxidative stress in vivo. Our results provide evidence for a previously unappreciated, non-canonical role for box C/D snoRNAs as regulators of metabolic stress response pathways in mammalian cells.

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...

Most eukaryotic cells can store excess lipid in cytosolic lipid droplets. This unit discusses techniques for the visualization of lipid droplets and associated proteins in cultured mammalian cells. Protocols for the detection of lipid droplets with nile red and BODIPY 493/503 are included. The differences in the spectral properties of these two lipophilic dyes and advantages of each are discussed. The best method for combining visualization of intracellular lipid droplets with indirect immunofluorescent detection of lipid droplet–associated proteins is described. Techniques for sample fixation and permeabilization must be chosen carefully to avoid alterations to lipid droplet morphology. Immunofluorescent detection of adipophilin, a broadly expressed, lipid droplet–associated protein, widely used as a marker for lipid droplet accumulation, is presented as an example. Finally, a simple protocol for enhancing lipid droplet accumulation through supplementation with excess fatty acid is included.

The murine fatty acid transport protein (FATP1) was identified in an expression cloning screen for proteins that facilitate transport of fatty acids across the plasma membranes of mammalian cells. Hydropathy analysis of this protein suggests a model in which FATP1 has multiple membrane-spanning domains. To test this model, we inserted a hemagglutinin epitope tag at the amino terminus or a FLAG tag at the carboxyl terminus of the FATP1 cDNA and expressed these constructs in NIH 3T3 cells. Both tagged constructs produce proteins of the expected molecular masses and are functional in fatty acid import assays. Indirect immunofluorescence studies with selective permeabilization conditions and protease protection studies of sealed membrane vesicles from cells expressing epitope-tagged FATP1 were performed. These experiments show that the extreme amino terminus of tagged FATP1 is oriented toward the extracellular space, whereas the carboxyl terminus faces the cytosol. Additionally, enhanced green fluorescent protein fusion constructs containing predicted membrane-associated or soluble portions of FATP1 were expressed in Cos7 cells and analyzed by immunofluorescence and subcellular fractionation. These experiments demonstrate that amino acids 1-51, 52-100, and 101-190 contain signals for integral association with the membrane, whereas residues 258 -313 and 314 -475 are only peripherally membrane-associated. Amino acid residues 191-257 and 476 -646 do not direct membrane association and likely face the cytosol. Taken together, these data support a model of FATP1 as a polytopic membrane protein with at least one transmembrane and multiple membrane-associated domains. This study provides the first experimental evidence for topology of a member of the family of plasma membrane fatty acid transport proteins.

In obesity and diabetes, an imbalance in fatty acid uptake and fatty acid utilization leads to excess accumulation of lipid in non-adipose tissues. This lipid overload is associated with cellular dysfunction and cell death, which contribute to organ failure, a phenomenon termed lipotoxicity. To elucidate the molecular mechanism of lipid-mediated cell death, we generated and characterized a mutant Chinese hamster ovary cell line that is resistant to palmitate-induced cell death. In this mutant, random insertion of a retroviral promoter trap has disrupted the gene for the non-coding RNA, growth arrested DNA-damage inducible gene 7 (gadd7). Here we report that gadd7 is induced by lipotoxic stress in a reactive oxygen species (ROS)-dependent fashion and is necessary for both lipid-and general oxidative stressmediated cell death. Depletion of gadd7 by mutagenesis or short hairpin RNA knockdown significantly reduces lipid and nonlipid induced ROS. Furthermore, depletion of gadd7 delays and diminishes ROS-induced endoplasmic reticulum stress. Together these data are the first to implicate a non-coding RNA in a feed-forward loop with oxidative stress and its induction of the endoplasmic reticulum stress response.Cellular homeostasis can be perturbed by a myriad of stimuli, including metabolic imbalance, oxidative stress, and aberrant protein folding. In response to such stressors, cells induce specific molecular pathways that commonly involve activation of signaling cascades or alterations in gene expression (1, 2). These responses enable cells to adapt to relatively modest stress and regain homeostasis. However, if the stress is extreme or prolonged, cells are unable to re-establish homeostasis and in turn, activate pathways that result in cell death.In obesity and diabetes, high serum triglycerides and free fatty acids (FFAs) 2 lead to excess accumulation of lipid in nonadipose tissues. This lipid accumulation is associated with cellular dysfunction and cell death, which contribute to organ failure, a phenomenon termed lipotoxicity (3). Evidence from human studies implicates lipotoxicity in heart failure associated with obesity and diabetes by showing a link between cardiomyocyte lipid accumulation and heart muscle dysfunction (4 -6). In rodent models of diabetes and in several transgenic mouse models, increased cardiac fatty acid uptake and oxidation and/or cardiomyocyte lipid accumulation is associated with heart failure (7-12). Similarly, lipid accumulation in the pancreas, kidney, and liver in obesity and diabetes is associated with organ dysfunction (13-15). Furthermore, end-organ damage in diabetes and obesity is associated with oxidative and endoplasmic reticulum (ER) stress that may be related in part to lipotoxicity, because perturbation of lipid metabolism alone can lead to these responses (11, 16 -22).Studies from our laboratory and others show that lipotoxicity can be modeled in established cell lines by supplementation of culture media with pathophysiological concentrations of the saturated FFA, palmitate...