Apoptosis is associated with triacylglycerol accumulation in Jurkat T-cells

Abstract

Magnetic resonance spectroscopy is increasingly used as a non-invasive method to investigate apoptosis. Apoptosis was induced in Jurkat T-cells by Fas mAb. (1)H magnetic resonance spectra of live cells showed an increase in methylene signal as well as methylene/methyl ratio of fatty acid side chains at 5 and 24 h following induction of apoptosis. To explain this observation, (1)H magnetic resonance spectra of cell extracts were investigated. These demonstrated a 70.0+/-7.0%, 114.0+/-8.0% and 90.0+/-5.0% increase in the concentration of triacylglycerols following 3, 5 and 7 h of Fas mAb treatment (P<0.05). Confocal microscopy images of cells stained with the lipophilic dye Nile Red demonstrated the presence of lipid droplets in the cell cytoplasm. Quantification of the stained lipids by flow cytometry showed a good correlation with the magnetic resonance results (P > or =0.05 at 3, 5 and 7 h). (31)P magnetic resonance spectra showed a drop in phosphatidylcholine content of apoptosing cells, indicating that alteration in phosphatidylcholine metabolism could be the source of triacylglycerol accumulation during apoptosis. In summary, apoptosis is associated with an early accumulation of mobile triacylglycerols mostly in the form of cytoplasmic lipid droplets. This is reflected in an increase in the methylene/methyl ratio which could be detected by magnetic resonance spectroscopy.

Figure 1

Cell cycle analysis by FACS of (A) Control, (B) Fas mAb treated (100 ng ml⁻¹) Jurkat T-cells as a function of time. The presence of hypodiploid apoptotic cells is indicated by build up of the sub-G1 population. Analysis of mitochondrial ΔΨ_m of (C) Control, (D) Fas mAb treated (100 ng ml⁻¹, 1 h) Jurkat T-cells by DiOC₆(3) uptake. N=normal cells, strong green (DiOC₆) log fluorescence (GR-FL), weak red (PI) log fluorescence (Red-FL); Ap=apoptotic cells, weak green (DiOC₆) log fluorescence (GR-FL), weak red (PI) log fluorescence (Red-FL).

Figure 2

(A) ¹H MR spectra of control (bottom) and 24 h Fas mAb treated (100 ng ml⁻¹) (top) intact Jurkat T-cells, (B) An expanded region of the ¹H MR spectra showing the increase in CH₃ and CH₂ resonances following induction of apoptosis, (C) An example of the deconvolution carried out to quantify changes in the CH₂ peak. Spectra are the result of 128 scans plotted with line broadening of 0.1 Hz and are acquired using similar cell numbers for control and treated cells (residual acetone @ 2.22 p.p.m. was removed from plots).

Figure 3

(A) ¹H MR spectrum of the lipid fraction of Jurkat T-cell extracts, (B) An expanded region of the CPMG ¹H MR spectra of the lipid fractions of control and Fas mAb treated (100 ng ml⁻¹) Jurkat T-cell extracts. This illustrates the increase in the triacylglycerol (TAG)-specific peak around 4.3 p.p.m. Spectra are the result of 128 scans plotted with line broadening of 1 Hz.

Figure 4

(A) Confocal microscopy images of control and Fas mAb treated (100 ng ml⁻¹) Jurkat T-cells co-stained with the lipophilic dye Nile Red and PI. The yellow represents Nile Red stained neutral lipid droplets, examples are indicated by full arrowheads. Examples of apoptotic bodies are indicated by open arrowheads. (B) Yellow/Green log fluorescence (Yel/GR-FL) in Nile Red stained Jurkat T-cells following treatment with Fas mAb (100 ng ml⁻¹) as a function of time. This illustrates the build up of neutral lipids in treated cells as detected by FACS.

Figure 5

(A) ¹H decoupled ³¹P MR spectrum of the lipid fractions of Jurkat T-cell extracts (1=cardiolipin, 2=plasmalogen phosphatidylethanolamine, 3=phosphatidylethanolamine, 4=phosphatidylserine, 5=sphingomyelin, 6=phosphatidylinisitol, 7=plasmalogen phosphatidylcholine, 8=phosphatidylcholine). Spectrum is the result of 1280 scans plotted with line broadening of 0.1 Hz. (B) Time course for the changes in phospholipid content of Jurkat T-cells showing the drop in phosphatidylcholine levels following Fas mAb treatment (100 ng ml⁻¹) (* P<0.01).