Triglyceride accumulation protects against fatty acid-induced lipotoxicity - PubMed (original) (raw)

Triglyceride accumulation protects against fatty acid-induced lipotoxicity

Laura L Listenberger et al. Proc Natl Acad Sci U S A. 2003.

Abstract

Excess lipid accumulation in non-adipose tissues is associated with insulin resistance, pancreatic beta-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.

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Figures

Figure 1

Figure 1

Cosupplementation with oleate prevents palmitate-induced apoptosis. (A) DNA laddering was assessed in CHO cells after supplementation with the indicated FAs for 26 h. Each sample is representative of three independent experiments. (B) Cells were supplemented with the indicated concentrations of palmitate and oleate for 14 h followed by C-2938 loading. C-2938 fluorescence was determined by flow cytometry and is indicative of the oxidation of C-2938 by intracellular reactive intermediates. The bar graph displays median C-2938 fluorescence of 104 cells normalized to unsupplemented CHO cells. Each bar represents the average median fluorescence of three or four independent experiments ± SE. (C) Ceramide levels were determined by mass spectrometry after 6 h of FA supplementation. The bar graph displays mean ± SE ceramide content per mg of cellular protein, measured in three independent experiments.

Figure 2

Figure 2

Oleate supplementation promotes neutral lipid storage. (A) CHO cells were supplemented with media alone (Aa) or media containing 500 μM palmitate (Ab), 200 μM oleate (Ac), or 500 μM palmitate + 200 μM oleate (Ad) for 6 h. Cells were fixed with paraformaldehyde and stained with Nile red as a marker for neutral lipid. Confocal images are of a single section. Scale bar = 10 μm. (B) Incorporation of exogenous [14C]palmitate into triglyceride was measured 6 h after FA supplementation as indicated. Bar graph displays palmitate incorporation per μg of cellular protein. Data are the average ± SE for 6–12 samples from three independent experiments.

Figure 3

Figure 3

Increased FA desaturase activity correlates with triglyceride storage. Data are represented by filled bars are for CHO cells, open bars for 25RA cells, and shaded bars for SCD cells. Desaturase activity (A) of equivalent quantities of microsomal proteins from CHO, 25RA, and SCD-overexpressing cells was determined (mean ± SE, CHO n = 8, 25RA n = 8; SCD n = 10). Total triglyceride (nmol triglyceride per mg of protein, B) and accumulation of palmitate into triglyceride (nmol palmitate per mg of protein, C) were measured by mass spectrometry after 6 h of FA supplementation. Data displayed in bar graphs are the average of three separate experiments ± SE.

Figure 4

Figure 4

25RA and SCD cells are resistant to palmitate-induced apoptosis. (A and B) DNA laddering was assessed in CHO and 25RA (A) or SCD (B) cells after supplementation with 500 μM palmitate (palm) for 0, 18, 24, and 28 h (A) or 28 h (B). The data are representative of three independent experiments. (C) Caspase 3 activity was measured in cell lysates collected after 24 h of FA supplementation. Data are expressed as the average fold increase over untreated cells ± SE. For each sample, n = 9 from three independent experiments.

Figure 5

Figure 5

Lipotoxicity in _Dgat1_−/− cells. (A) Wild-type (Aa_–_Ac) and _Dgat1_−/− (Ad_–_Af) mouse embryonic fibroblasts were incubated with media alone (Aa and Ad) or media supplemented with 1 mM palmitate (Ab and Ae) or 1 mM oleate (Ac and Af) for 12 h. Triglyceride accumulation was detected by Nile red staining. Bar = 20 μm. (B) Wild-type and _Dgat1_−/− fibroblasts were supplemented with 1 mM palmitate or 1 mM oleate for 46 h. Cell death was assessed by PI staining and flow cytometry. For each sample, fluorescence of 104 cells was assessed, and the percentage of cells with PI fluorescence was determined. The bar graph displays the median percentage of PI-positive cells from three independent measurements ± SE.

Figure 6

Figure 6

Mechanism for preventing lipotoxicity though triglyceride storage. (A) The long chain saturated FA palmitate induces apoptosis in CHO cells though a mechanism involving reactive intermediate (ROS) and ceramide generation. In the absence of additional signals, palmitate is poorly incorporated into cellular triglyceride pools. (B) The presence of unsaturated FAs promotes channeling of palmitate toward triglyceride storage, thus sequestering palmitate away from pathways leading to cell death. Unsaturated FAs provided as media supplements (though cosupplementation with oleate) or generated though the action of cellular desaturase enzymes (e.g., SCD) show this effect.

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