Cholesteryl Ester Transfer Protein Biosynthesis and Cellular Cholesterol Homeostasis Are Tightly Interconnected

Abstract

Cholesteryl ester transfer protein (CETP) mediates triglyceride and cholesteryl ester (CE) transfer between lipoproteins, and its activity is strongly modulated by dietary cholesterol. To better understand the regulation of CETP synthesis and the relationship between CETP levels and cellular lipid metabolism, we selected the SW872 adipocytic cell line as a model. These cells secrete CETP in a time-dependent manner at levels exceeding those observed for Caco-2 or HepG2 cells. The addition of LDL, 25OH-cholesterol, oleic acid, or acetylated LDL to SW872 cells increased CETP secretion (activity and mass) up to 6-fold. In contrast, CETP production was decreased by almost 60% after treatment with lipoprotein-deficient serum or ␤-cyclodextrin. These effects, which were paralleled by changes in CETP mRNA, show that CETP biosynthesis in SW872 cells directly correlates with cellular lipid status. To investigate a possible, reciprocal relationship between CETP expression and cellular lipid homeostasis, CETP biosynthesis in SW872 cells was suppressed with CETP antisense oligonucleotides. Antisense oligonucleotides reduced CETP secretion (activity and mass) by 60% compared with sensetreated cells. When CETP synthesis was suppressed for 24 h, triglyceride synthesis was unchanged, but cholesterol biosynthesis was reduced by 20%, and acetate incorporation into CE increased 31%. After 3 days of suppressed CETP synthesis, acetate incorporation into the CE pool increased 3-fold over control. This mirrored a similar increase in CE mass. The efflux of free cholesterol to HDL was the same in sense and antisensetreated cells; however, HDL-induced CE hydrolysis in antisense-treated cells was diminished 2-fold even though neutral CE hydrolase activity was unchanged. Thus, CETP-compromised SW872 cells display a phenotype characterized by inefficient mobilization of CE stores leading to CE accumulation. These results strongly suggest that CETP expression levels contribute to normal cholesterol homeostasis in adipocytic cells.Overall, these studies demonstrate that lipid homeostasis and CETP expression are tightly coupled.

Overexpression of full-length cholesteryl ester transfer protein in SW872 cells reduces lipid accumulation

“…In hypertriglyceridemic mice, CETP expression normalizes subcutaneous adipose depots and visceral adipocyte size ( 16 ). And in humans, a CETP gene variant that affects the coding sequence of both full-length and exon 9-deleted CETP is associated with increased adiposity following long-term overfeeding ( 17 ).Our initial studies in intracellular CETP, which used a molecular approach that reduced both full-length and exon 9-deleted CETP expression, identifi ed multiple aberrations in lipid metabolism and storage ( 13,14 ). In that Abstract Cells produce two cholesteryl ester transfer protein (CETP) isoforms, full-length and a shorter variant produced by alternative splicing.…”

“…Our initial studies in intracellular CETP, which used a molecular approach that reduced both full-length and exon 9-deleted CETP expression, identifi ed multiple aberrations in lipid metabolism and storage ( 13,14 ). In that Abstract Cells produce two cholesteryl ester transfer protein (CETP) isoforms, full-length and a shorter variant produced by alternative splicing.…”

“…Cells were lysed by passage through a 26-gauge needle, centrifuged at 1,000 g for 10 min, and the supernatant (25 µg protein) fractionated by electrophoresis. After transfer to polyvinylidene difl uoride membranes, CETP was detected by TP2 antibody ( 13 ). Clones overexpressing CETP were designated as CETP + cells.…”

“…Clones were tested for CETP expression by measuring CETP activity in 48 h conditioned medium collected in the absence of serum ( 3,13 ). Volumes of conditioned media assayed were normalized by the cell count per well.…”

Biochemical evidence for energy-independent flippase activity in bovine epididymal sperm membranes: an insight into membrane biogenesis

“…However, the demand for new PLs during differentiation exceeds the storage capacity, indicating that spermatozoa might have the ability to synthesize PLs on-site. Spermatozoal lysates were reported to synthesize cholesterol from acetate (Cross 1998, Izem & Morton 2001, and ejaculated spermatozoa were shown to synthesize ether lipids that are usually abundant in sperm membranes (Jones 1997). Spermatozoa were able to incorporate radiolabeled precursors of palmitic acid, glycerol, choline, and arachidonic acid into DAG and phosphatidylcholine (PC), indicating that they possess active PL-synthesizing capability (Neill & Masters 1972, Vasquez & Roldan 1997.…”