Abstract
Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester (CE) and triglyceride (TG) between lipoproteins in plasma. However, short term suppression of CETP biosynthesis in cells alters cellular cholesterol homeostasis, demonstrating an intracellular role for CETP as well. The consequences of chronic CETP deficiency in lipid-storing cells normally expressing CETP have not been reported. Here, SW872 adipocytes stably expressing antisense CETP cDNA and synthesizing 20% of normal CETP were created. CETP-deficient cells had 4-fold more CE but an approximately 3-fold decrease in cholesterol biosynthesis. This phenotype of cholesterol overload is consistent with the observed 45% reduction in low density lipoprotein receptor and 2.5-fold increase in ABCA1 levels. However, cholesterol mass in CETP-deficient adipocytes was actually reduced. Strikingly, CETP-deficient adipocytes stored <50% of normal TG, principally reflecting reduced synthesis. The hydrolysis of cellular CE and TG in CETP-deficient cells was reduced by >50%, although hydrolase/lipase activity was increased 3-fold. Notably, the incorporation of recently synthesized CE and TG into lipid storage droplets in CETP-deficient cells was just 40% of control, suggesting that these lipids are inefficiently transported to droplets where the hydrolase/lipase resides. The capacity of cellular CETP to transport CE and TG into storage droplets was directly demonstrated in vitro. Overall, chronic CETP deficiency disrupts lipid homeostasis and compromises the TG storage function of adipocytes. Inefficient CETP-mediated translocation of CE and TG from the endoplasmic reticulum to their site of storage may partially explain these defects. These studies in adipocytic cells strongly support a novel role for CETP in intracellular lipid transport and storage.
Highlights
Cholesteryl ester transfer protein (CETP)1 [1] mediates the transfer of cholesteryl ester (CE) and triglyceride (TG) among plasma lipoproteins [2,3,4]
Confluent cultures of cells were assayed for CETP expression by determining CETP activity and mass (Western blot) in 48-hr conditioned medium collected in the absence of serum
Selection of CETP-deficient clones - A 549 bp fragment of CETP cDNA was inserted in reverse orientation into pcDNA3, transfected into SW872 adipocytic cells, and stable transfectants selected with Geneticin
Summary
Materials - The human lipocarcinoma cell line, SW872 (HTB-92), was purchased from American Type Culture Collection (Manassas, VA). To determine the distribution of recently synthesized lipids among cellular fractions, cells in 100 mm dishes were grown in 5 ml media containing 10% FBS and 2 Ci [14C]acetate for 3 days before being homogenized and fractionated by the sucrose gradient procedure above. Interorganelle lipid transfer assay - Confluent CETP-deficient cells were incubated with DMEM/F12 2% FBS containing 100 M 3H-oleic acid-BSA for 48 hours. Neutral cholesteryl ester hydrolase assay - To quantify cellular neutral CE hydrolase activity, confluent cells, incubated with media with or without 10% FBS, were washed with PBS suspended in cold buffer containing 25 mM TrisHCl pH 7.4, 1 mM EDTA, 20% glycerol and protease inhibitor cocktail, and sonicated 2 x 10 seconds using a probe sonicator at low power setting. The reaction mixture was extracted using a mixture of methanol/chloroform/benzene (2.4:2:1) (v/v), and the 14C content of the aqueous phase was determined by liquid scintillation counting
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