Possible Role for Intracellular Cholesteryl Ester Transfer Protein in Adipocyte Lipid Metabolism and Storage

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 ϳ3-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. Cholesteryl ester transfer protein (CETP)2 (1) mediates the transfer of cholesteryl ester (CE) and triglyceride (TG) among plasma lipoproteins (2-4). CETP is a member of the lipid transfer/lipopolysaccharide-binding protein gene family that is composed of highly hydrophobic proteins involved in lipid binding and transport (5, 6). Two CETP transcripts exist, a 1.6-kb transcript produced from the full-length, 16-exon CETP gene and a 1.4-kb transcript lacking exon 9 (180 nucleotides) derived by alternative splicing (7). mRNAs for both variants are present in all human tissues expressing CETP. When compared with the full-length form, which accounts for essentially all plasma CETP, exon 9-deleted CETP is poorly secreted, and its function is unknown (7,8). Exon 9-deleted CETP retains the amino acids that constitute the binding sites for TG and CE (residues 470 -475) (9, 10) and the bulk of the putative lipoprotein-binding region (11,12). Absence of a portion of this binding region in exon 9-deleted CETP may explain its poor interaction with HDL (8) and may lead to a variant with different substrate preference or affinity.In addition to its abundant hepatic expression, CETP is synthesized by many tissues, such as adipose, adrenal ...

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

“…Cells defi cient in CETP have impaired TG storage capacity ( 14 ). To examine the impact of CETP overexpression on TG storage, cells were incubated with 200 µM 3 H-oleate/BSA for 48 h. Surprisingly, all clones overexpressing CETP had reduced ability to accumulate TG.…”

“…Diacylglycerol acyltransferase (DGAT) activity was measured using [ ( 21 ). TG in the extract was isolated by thin layer chromatography in a developing system of hexanes:diethyl ether:acetic acid (70:30:1), and its 14 C content determined by scintillation counting.…”

“…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.…”

“…CETP-defi cient SW872 cells, prepared as previously described ( 14 ), were used in select experiments.…”