Fatty acids liberated from high-density lipoprotein phospholipids by endothelial-derived lipase are incorporated into lipids in HepG2 cells.

We previously reported that endothelial-derived lipase (EDL) efficiently hydrolyses high-density-lipoprotein-derived phosphatidycholine (HDL-PC). In the present study, we assessed the ability of EDL to supply HepG2 cells with non-esterified fatty acids (NEFA) liberated from HDL-phospholipids. For this purpose, HepG2 cells infected with adenovirus encoding human EDL (EDL-Ad), or with control beta-galactosidase-expressing adenovirus (LacZ-Ad), were incubated with (14)C-HDL-PC. The analysis of the cellular lipids by TLC revealed that EDL overexpression led to an increase in the amount of cellular (14)C-lipids, whereby the label was mainly incorporated into phospholipids and triacylglycerols (TAG). Cells expressing mutant enzymically inactive EDL (MUT-EDL-Ad) contained similar amounts of (14)C-TAG but higher amounts of (14)C-phosphatidylcholine (PC) compared with LacZ-Ad-infected cells. The co-expression of CD36 augmented the EDL-mediated accumulation of (14)C-lipids in HEK-293 cells. The quadrupole MS analysis of the cellular lipids revealed an increased content of PC and TAG in EDL-expressing HepG2 cells compared with MUT-EDL-Ad-expressing and control cells. However, the MUT-EDL-Ad-expressing cells contained more PC than control cells. Additionally, EDL overexpression led to a 2-fold decrease in the amount of fatty acid synthase mRNA and, in turn, a slightly, but significantly, decreased rate of fatty acid (FA) synthesis in HepG2 cells. In the present study, we show for the first time that EDL efficiently supplies HepG2 cells with NEFA derived from HDL-PL, thus affecting cellular lipid composition and FA synthesis.

Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis.

Hormone-sensitive lipase (HSL) is expressed predominantly in white and brown adipose tissue where it is believed to play a crucial role in the lipolysis of stored triglycerides (TG), thereby providing the body with energy substrate in the form of free fatty acids (FFA). From in vitro assays, HSL is known to hydrolyze TG, diglycerides (DG), cholesteryl esters, and retinyl esters. In the current study we have generated HSL knock-out mice and demonstrate three lines of evidence that HSL is instrumental in the catabolism of DG in vivo. First, HSL deficiency in mice causes the accumulation of DG in white adipose tissue, brown adipose tissue, skeletal muscle, cardiac muscle, and testis. Second, when tissue extracts were used in an in vitro lipase assay, a reduced FFA release and the accumulation of DG was observed in HSL knock-out mice which did not occur when tissue extracts from control mice were used. Third, in vitro lipolysis experiments with HSL-deficient fat pads demonstrated that the isoproterenol-stimulated release of FFA was decreased and DG accumulated intracellularly resulting in the essential absence of the isoproterenol-stimulated glycerol formation typically observed in control fat pads. Additionally, the absence of HSL in white adipose tissue caused a shift of the fatty acid composition of the TG moiety toward increased long chain fatty acids implying a substrate specificity of the enzyme in vivo. From these in vivo results we conclude that HSL is the rate-limiting enzyme for the cellular catabolism of DG in adipose tissue and muscle.