Palmitate-induced Apoptosis Can Occur through a Ceramide-independent Pathway

Abstract

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.

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

“…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). 18 Herein, we focus on a novel, independently isolated mutant, the 2B1 cell line.…”

“…For lipotoxicity experiments, medium was supplemented with 500 μM palm complexed to BSA at a 2 : 1 molar ratio, as described previously. 18 For ROS induction, medium was supplemented with the indicated concentrations of hydrogen peroxide or menadione.…”

“…[14][15][16][17] The importance of oxidative stress in the pathophysiological response to substrate excess is underscored by observation that treatment with chemical antioxidants and overexpression of ROS-scavenging enzymes mitigates against lipotoxic cell death and against diabetic complications in animal models. [18][19][20][21] To identify critical mediators of lipotoxic cell death, our laboratory has focused on characterizing genes identified through a loss-of-function genetic screen in mammalian fibroblasts. We found that cells become resistant to death from lipotoxic and generalized oxidative stress stimuli upon disruption of small nucleolar RNAs (snoRNAs) encoded within the ribosomal protein L13a (rpL13a) locus or disruption of expression of a splicosomal protein necessary for production of these non-coding RNAs from intron lariats.…”

Cardiovascular consequences of obesity and targets for treatment

“…This process has also been implicated in the death of the insulinproducing pancreatic beta cells, which may also contribute to the development of type 2 diabetes. Studies in animal models also demonstrate toxic effects of elevated FA levels on beta cells (and other nonadipocytes) via a pathway known as "lipotoxicity" [23,24]. These pathways provide intriguing targets for future therapeutic interventions, in addition to the more traditional weight loss (discussed below) for the amelioration of obesity-related diabetes.…”

“…Although much of this excess lipid may be stored in a relatively neutral form such as triglycerides, some of the FAs that enter the cell may contribute to apoptosis, via lipotoxicity [23]. Studies in animal models show that lipotoxicity may occur via ceramide-dependent or -independent pathways [23,24]. In these animal models of lipotoxicity, the myocardial metabolic abnormalities precede cardiac dysfunction and are thought to contribute to it [23,55,57].…”