Excess fatty acid accumulation in non-adipose tissues is a hallmark of metabolic disease. When elevated lipid levels exceed the cell’s capacity to store or utilize fatty acids, a lipotoxic-response is elicited, characterized by destruction of organelle membranes, activation of stress pathways, and apoptosis. This review focuses on the mechanisms by which lipid overload causes non-adipose cell death and contributes to the pathogenesis of obesity and diabetes.

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.

Fragile X mental retardation 1 (Fmr1) is a highly conserved gene with major roles in CNS structure and function. Its product, the RNA-binding protein FMRP, is believed to regulate translation of specific transcripts at postsynaptic sites in an activity-dependent manner. Hence, Fmr1 is central to the molecular mechanisms of synaptic plasticity required for normal neuronal maturation and cognitive ability. Mutations in its Drosophila ortholog, dfmr1, produce phenotypes of brain interneurons and axon terminals at the neuromuscular junction, as well as behavioral defects of circadian rhythms and courtship. We hypothesized that dfmr1 mutations would disrupt morphology of the mushroom bodies (MBs), highly plastic brain regions essential for many forms of learning and memory. We found developmental defects of MB lobe morphogenesis, of which the most common is a failure of ␤ lobes to stop at the brain midline. A similar recessive ␤-lobe midline-crossing phenotype has been previously reported in the memory mutant linotte. The dfmr1 MB defects are highly sensitive to genetic background, which is reminiscent of mammalian fragile-X phenotypes. Mutations of dfmr1 also interact with one or more third-chromosome loci to promote ␣/␤-lobe maturation. These data further support the use of the Drosophila model system for study of hereditary cognitive disorders of humans.

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...

Accumulation of excess lipid in nonadipose tissues is associated with oxidative stress and organ dysfunction and plays an important role in diabetic complications. To elucidate molecular events critical for lipotoxicity, we used retroviral promoter trap mutagenesis to generate mutant Chinese hamster ovary cell lines resistant to lipotoxic and oxidative stress. A previous report of a mutant from this screen demonstrated that under lipotoxic conditions, small nucleolar RNAs (snoRNAs) in the rpL13a gene accumulate in the cytosol and serve as critical mediators of lipotoxic cell death. We now report a novel, independent mutant in which a single provirus disrupted one allele of the gene encoding the spliceosomal protein SmD3, creating a model of haploinsufficiency. We show that snoRNA expression and the abundance of snoRNA-containing intron lariats are decreased in SmD3 mutant cells, even though haploinsufficiency of SmD3 supports pre-mRNA splicing. The mechanism through which SmD3 regulates the expression of intronic snoRNAs likely involves effects of SmD3 on the levels of small nuclear RNAs (snRNAs) U4 and U5. Our data implicate SmD3 as a critical determinant in the processing of intronic noncoding RNAs in general and as an upstream mediator of metabolic stress response pathways through the regulation of snoRNA expression. Elevations in serum triglycerides and free fatty acids (FA) play an important role in the pathogenesis of diabetic complications. Under physiological conditions, mammalian adipose cells internalize and store large quantities of lipid. However, under pathophysiological conditions, accumulation of fatty acids in nonadipose tissues causes cell dysfunction and cell death that lead to impaired organ function (43). This phenomenon, known as lipotoxicity, contributes to the pathogenesis of heart failure, renal dysfunction, steatohepatitis, and progressive pancreatic insufficiency (1,17,37,38).In vitro models in which the medium of cultured cells is supplemented with excess fatty acid have been used to probe metabolic and signaling pathways involved in the cellular response to lipid overload. In a time-and dose-dependent manner, longchain saturated fatty acids induce apoptosis in a variety of cell types (6,7,21,24,45), and this response is enhanced by high glucose (8). Although lipid overload in nonadipose cells is initially buffered by cytoprotective triglyceride stores (20, 23), when the limited capacity for neutral lipid storage in nonadipose cells is exceeded, excess saturated fatty acids initiate several cellular stress response pathways. Fatty acid-induced endoplasmic reticulum stress can result in reactive oxygen species (ROS) generation (40). Independently, oxidative stress is induced in a variety of cell types through activation of NADPH oxidase, mitochondrial dysfunction due to remodeling of organelle membranes, and excessive cycles of oxidative phosphorylation (16,31,41). Administration of antioxidants to cultured cells and animal models of lipotoxicity mitigate against lipotoxic cell death (4...