Analysis of lipid uptake and processing in cultured cells = Lipid-Aufnahme und Stoffwechsel in kultivierten Zellen

Abstract

To characterize lipid uptake pathways in cultured cells, uptake and metabolism of different isotope-labeled lipid probes were investigated in human skin fibroblasts, a mouse macrophage-like cell line (RAW264.7), human hepatocellular carcinoma cells (HepG2 cells) and a human squamous carcinoma cell line (A431 cells). As lipid probes, we used different fatty acids, cholesterol, a cholesteryl ester, a triacylglycerol, and the phospholipids phosphatidylcholine and sphingomyelin. The uptake and metabolism of exogenously added lipid probes differed with cell type, lipid structure, and mode of delivery. Cationic amphiphilic drugs (CADs) are widely used drugs that are known to interfere with lipid metabolism and to induce phospholipidosis in human patients. We investigated the influence of the desipramine, imipramine, chlorpromazine, chloroquine, and FTY720 as representative CADs on uptake and processing of the phospholipid probes. Desipramine was found to have drastic and cell-type specific effects on FA processing. Lipid processing was also impaired in a genetic phospholipidosis, Niemann-Pick disease, type A.<br /><strong>Fatty acids</strong>: to study the uptake and processing of C-18 fatty acids in cultured cells, uptake and incorporation of four FA probes in membrane phospholipids and in nonpolar lipids were monitored. We used FA probes that differed in the degree of unsaturation: stearic acid (18:0), oleic acid (18:1,ω-9), linoleic acid (18:2, ω-6), and linolenic acid (18:3, ω-3). These FA were applied in complex with bovine serum albumin (BSA) to the four different types of cultured cells. Significant differences were found between uptake and metabolism of these fatty acids, when fatty acid class and cell type were varied. FA uptake by fibroblasts and macrophages was highest with 18:1, and lowest with 18:3, and 18:0, respectively. Uptake by A431 cells and HepG2 cells was lowest with 18:0, and highest with 18:2 and 18:3, respectively. In macrophages, stearic acid and oleic acid are predominantly incorporated into nonpolar lipid droplet (LD) lipids, while linoleic and linolenic acid are predominantly incorporated into polar lipids. Also in HepG2 cells, the level of FAs incorporated into polar lipids was much greater for unsaturated FAs than for saturated FAs. In fibroblasts, only a minor incorporation of FAs into neutral lipids and a major incorporation into polar lipids were observed. In A431 cells, 18:2 was best incorporated into neutral lipids, followed by 18:3, 18:1, and 18:0. The impact of a cationic amphiphilic drug (CAD, FIASMA = functional inhibitor of acid sphingomyelinase), desipramine, on this process was also analyzed. Treatment with desipramine caused a tremendous reduction of FA-incorporation into triacylglycerols of macrophages and A431 cells, but only a slight decrease in HepG2 cells. Fibroblasts showed an unexpected increase in the incorporation of FAs into triacylglycerol (TAG) and diacylglycerol (DAG). We also measured the uptake of [³H]desipramine by different types of cells, which was lowest in fibroblasts.<br /><strong>Cholesterol, cholesteryloleate, and triolein</strong>: to characterize the uptake pathways for these lipids, we investigated the effect of different lipid delivery methods. The exogenous lipid probes were applied to the four different types of cultured cells either in complex with bovine serum albumin (BSA), or as components of low density lipoprotein (LDL) particles. Significant differences in uptake and metabolism after application of these two methods were found. When incorporated into LDL, uptake of cholesterol, cholesterol ester, and triacylglycerol was 2-4-fold higher than when delivered by BSA. Furthermore, the uptake of cholesterol presented as BSA-complexes was best for A431 cells, while uptake of the other lipids presented as LDL- and BSA-complexes were higher in fibroblasts than the other cell types. Also the metabolic incorporation of cholesterol and oleate derived from cholesterol ester and triacylglycerol was higher. These findings indicate that LDL-associated lipid is incorporated into cultured cells via a pathway that differs significantly from that of BSA-lipid.<br /><strong>Cholesterol and phosphatidylcholine processing in Niemann-Pick disease, type A</strong>: to investigate the role of Niemann-Pick disease, type A (NPA), one of the lysosomal storage diseases, on the processing of [¹⁴C]cholesterol and [¹⁴C]phosphatidylcholine, we applied the methods mentioned above to human fibroblasts and to fibroblasts from patients with NPA disease. Incubation with LDL-associated [¹⁴C]phosphatidylcholine and LDL-associated [¹⁴C]cholesterol show reduced processing of [¹⁴C]phosphatidylcholine and [¹⁴C]cholesterol by 25, and 21%, respectively. This study indicates that in NPA disease, also nutrient delivery via the endolysosomal system is impaired.<br /><strong>Phosphatidylcholine (PC) and sphingomyelin (SM) processing in drug-treated cells</strong>: to investigate the influence of cationic amphiphilic drugs on uptake and processing of exogenously added choline-containing phospholipids, the influence of five cationic amphiphilic drugs, desipramine (DMI), imipramine (IM), chlorpromazine (CPZ), chloroquine (CQ), and fingolimod (FTY720), were studied. The lipid probes were delivered as components of LDL-particles, and the metabolic fate of their isotope-labeled fatty acid moieties was monitored in the four different cell types mentioned before. Concentrations of 10μM had slightly to no apparent effect on [¹⁴C]-SM and [¹⁴C]-PC processing for all CADs tested. Profound changes were observed when CADs were administered in high concentration (20μM, and 40μM). In macrophages, all investigated drugs lead to an impaired processing of SM and PC. Incorporation of the fatty acids released from PC and SM into diacylglycerols, triacylglycerols, and glycerophospholipids was drastically reduced in the presence of 20μM of the drugs. Furthermore, each cell type showed a characteristic neutral lipid and phospholipid pattern. The effect of the investigated CADs on SM and PC processing in terms of pmol per μg cell protein depend on the concentration of the investigated CADs, the cell type, and the identity of the FA in lipid probes. For example, 20μM FTY720 caused a drastically reduced incorporation of sphingomyelin-derived stearic acid into triacylglycerols in macrophages, fibroblasts and A431 cells, but not in HepG2 cells. While 20μM FTY720 caused a slightly reduced incorporation of phosphatidylcholine-derived palmitic acid into triacylglycerols in macrophages. Therefore, our method is able to detect metabolic steps that are affected in the presence of CADs and to predict the potential of CADs to induce phospholipidosis.