Characterization of Lipid Droplets by Evaluating Neutral Lipid and Phospholipid Contents in Peroxisome-Deficient Cell Lines
1. Department of Human Genetics, McGill University-Montreal Children’s Hospital Research Institute, Montreal, QC, Canada; 2. Department of Biochemistry and Molecular Biology, Broad Center for Regenerative Medicine and Stem Cell Research, University of Southern California, Los Angeles, CA, USA
Introduction. Peroxisomes and lipid droplets (LDs) are distinct subcellular organelles that play an integral role in regulation of intracellular lipid homeostasis. While the main function of LDs is storage of excess lipids as a source of energy for cellular growth and metabolic activities, they also play a vital role in prolongation of cell survival during stress conditions like starvation. LDs accumulate in C. elegans with defective peroxisomal β oxidation (MAOC-1/DHS-28/DAF-22 genes) and defective peroxisome assembly (PRX10, the ortholog of the human PEX10 gene). LD accumulation was also observed in liver tissue from mice with defective peroxisome assembly (Pex11α). Thus peroxisomes are likely to play a role in regulating lipid droplets. Here we evaluate LDs in mammalian cells with peroxisomal dysfunction.
Methods. We studied LD numbers by their content of either neutral lipids or phospholipids in normal Chinese Hamster Ovary (CHO) cells and CHO cells with PEX2 and AGPS null alleles; the former representing deficiency in peroxisome assembly and the latter in a single peroxisome metabolic pathway that synthesizes a unique ether phospholipid class, plasmalogens. We compared LDs in wild type (WT) and mutant CHO cells, under normal and starvation conditions, by immunofluorescence using green neutral lipid and red phospholipid specific staining (LipidTOX™).
Results. Thus far, we found that the number of LDs assessed by neutral lipid content was similar in all 3 cell lines in normal conditions. In contrast, under starvation conditions, we observed a significant reduction in LDs in WT and AGPS null cells, but not in PEX2 null cells. Taken together, this study suggests that normal peroxisome assembly is critical for mobilizing neutral lipids from LDs during starvation. This study highlights another pathway of intracellular organelle communication between peroxisomes and LDs, and helps us to understand pathophysiology in the peroxisomal biogenesis disorders.