Cholesteryl ester transfer protein biosynthesis and cellular cholesterol homeostasis are tightly interconnected

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 beta-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 sense-treated 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 antisense-treated 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.     

Defective triglyceride biosynthesis in CETP-deficient SW872 cells

We previously reported that reducing the expression of cholesteryl ester transfer protein (CETP) disrupts cholesterol homeostasis in SW872 cells and causes an ∼50% reduction in TG. The causes of this reduced TG content, investigated here, could not be attributed to changes in the differentiation status of CETP-deficient cells, nor was there evidence of endoplasmic reticulum (ER) stress. In short-term studies, the total flux of oleate through the TG biosynthetic pathway was not altered in CETP-deficient cells, although mRNA levels of some pathway enzymes were different. However, the conversion of diglyceride (DG) to TG was impaired. In longer-term studies, newly synthesized TG was not effectively transported to lipid droplets, yet this lipid did not accumulate in the ER, apparently due to elevated lipase activity in this organelle. DG, shown to be a novel CETP substrate, was also inefficiently transferred to lipid droplets. This may reduce TG synthesis on droplets by resident diacylglycerol acyltransferase. Overall, these data suggest that the decreased TG content of CETP-deficient cells arises from the reduced conversion of DG to TG in the ER and/or on the lipid droplet surface, and enhanced TG degradation in the ER due to its ineffective transport from this organelle.     

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

Cells produce two cholesteryl ester transfer protein (CETP) isoforms, full-length and a shorter variant produced by alternative splicing. Blocking synthesis of both isoforms disrupts lipid metabolism and storage. To further define the role of CETP in cellular lipid metabolism, we stably overexpressed full-length CETP in SW872 cells. These CETP⁺ cells had several-fold higher intracellular CETP and accumulated 50% less TG due to a 26% decrease in TG synthesis and 2.5-fold higher TG turnover rate. Reduced TG synthesis was due to decreased fatty acid uptake and impaired conversion of diglyceride to TG even though diacylglycerol acyltransferase activity was normal. Sterol-regulatory element binding protein 1 mRNA levels were normal, and although PPARγ expression was reduced, the expression of several of its target genes including adipocyte triglyceride lipase, FASN, and APOE was normal. CETP⁺ cells contained smaller lipid droplets, consistent with their higher levels of perilipin protein family (PLIN) 3 compared with PLIN1 and PLIN2. Intracellular CETP was mostly associated with the endoplasmic reticulum, although CETP near lipid droplets poorly colocalized with this membrane. A small pool of CETP resided in the cytoplasm, and a subfraction coisolated with lipid droplets. These data show that overexpression of full-length CETP disrupts lipid homeostasis resulting in the formation of smaller, more metabolically active lipid droplets.     

Remodeling of HDL by CETP in vivo and by CETP and hepatic lipase in vitro results in enhanced uptake of HDL CE by cells expressing scavenger receptor B-I.

The transport of HDL cholesteryl esters (CE) from plasma to the liver involves a direct uptake pathway, mediated by hepatic scavenger receptor B-I (SR-BI), and an indirect pathway, involving the exchange of HDL CE for triglycerides (TG) of TG-rich lipoproteins by cholesteryl ester transfer protein (CETP). We carried out HDL CE turnover studies in mice expressing human CETP and/or human lecithin:cholesterol acyltransferase (LCAT) transgenes on a background of human apoA-I expression. The fractional clearance of HDL CE by the liver was delayed by LCAT transgene, while the CETP transgene increased it. However, there was no incremental transfer of HDL CE radioactivity to the TG-rich lipoprotein fraction in mice expressing CETP, suggesting increased direct removal of HDL CE in the liver. To evaluate the possibility that this might be mediated by SR-BI, HDL isolated from plasma of the different groups of transgenic mice was incubated with SR-BI transfected or control CHO cells. HDL isolated from mice expressing CETP showed a 2- to 4-fold increase in SR-BI-mediated HDL CE uptake, compared to HDL from mice lacking CETP. The addition of pure CETP to HDL in cell culture did not lead to increased selective uptake of HDL CE by cells. However, when human HDL was enriched with TG by incubation with TG-rich lipoproteins in the presence of CETP, then treated with hepatic lipase, there was a significant enhancement of HDL CE uptake. Thus, the remodeling of human HDL by CETP, involving CE;-TG interchange, followed by the action of hepatic lipase (HL), leads to the enhanced uptake of HDL CE by cellular SR-BI.These observations suggest that in animals such as humans in which both the selective uptake and CETP pathways are active, the two pathways could operate in a synergistic fashion to enhance reverse cholesterol transport.     

Cytoskeleton disruption in J774 macrophages: Consequences for lipid droplet formation and cholesterol flux

Macrophages store excess unesterified cholesterol (free, FC) in the form of cholesteryl ester (CE) in cytoplasmic lipid droplets. The hydrolysis of droplet-CE in peripheral foam cells is critical to HDL-promoted reverse cholesterol transport because it represents the first step in cellular cholesterol clearance, as only FC is effluxed from cells to HDL. Cytoplasmic lipid droplets move within the cell utilizing the cytoskeletal network, but, little is known about the influence of the cytoskeleton on lipid droplet formation. To understand this role we employed cytochalasin D (cyt.D) to promote actin depolymerization in J774 macrophages. Incubating J774 with acetylated LDL creates foam cells having a 4-fold increase in cellular cholesterol content (30-40% cholesterol present as cholesteryl ester (CE)) in cytoplasmic droplets. Lipid droplets formed in the presence of cyt.D are smaller in diameter. CE-deposition and -hydrolysis are decreased when cells are cholesterol-enriched in the presence of cyt.D or latrunculin A, another cytoskeleton disrupting agent. However, when lipid droplets formed in the presence of cyt.D are isolated and incubated with an exogenous CE hydrolase, the CE is more rapidly metabolized compared to droplets from control cells. This is apparently due to the smaller size and altered lipid composition of the droplets formed in the presence of cyt.D. Cytoskeletal proteins found on CE droplets influence droplet lipid composition and maturation in model foam cells. In J774 macrophages, cytoskeletal proteins are apparently involved in facilitating the interaction of lipid droplets and a cytosolic neutral CE hydrolase and may play a role in foam cell formation. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Triglyceride depletion in THP-1 cells alters cholesteryl ester physical state and cholesterol efflux

To study macrophage lipid droplet composition and the effects of TG on cholesteryl ester (CE) physical state, hydrolysis, and cholesterol efflux, a technique was developed to remove the majority of accumulated TG with minimal effect on CE content. THP-1 macrophages were incubated with acetylated LDL, and the accumulated TG was depleted by incubation with the acyl-CoA synthetase inhibitor triacsin D in the presence of albumin. Before TG removal, all cellular lipid droplets were isotropic as determined by polarizing light microscopy. When the TG concentration was reduced, anisotropic lipid droplets were visible, indicating a change in physical state, and suggesting that TG and CE originally accumulated in mixed lipid droplets. This change in physical state of lipid droplets was associated with slower rates of CE hydrolysis and cholesterol efflux. Although lipid droplets within the same cell had a similar physical state after TG depletion, there was considerable variability among cells in the physical state of their lipid droplets.In conclusion, THP-1 macrophages store accumulated CE and TG in mixed droplets, and the proportion of CE to TG varies among cells. Reducing accumulated TG altered CE physical state, which in turn affected hydrolysis of CE and cholesterol efflux.     

Redistribution of macrophage cholesteryl ester hydrolase from cytoplasm to lipid droplets upon lipid loading

Hydrolysis of intracellular cholesteryl esters (CEs) represents the first step in the removal of cholesterol from lipid-laden foam cells associated with atherosclerotic lesions. Neutral cholesteryl ester hydrolase (CEH) catalyzes this reaction, and we recently cloned the cDNA for the human macrophage CEH and demonstrated increased mobilization of intracellular CE droplets by CEH overexpression. The present study was undertaken to test the hypothesis that for CE hydrolysis, CEH must become associated with the surface of the cytoplasmic lipid droplets. Our data show the redistribution of CEH from cytosol to lipid droplets upon lipid loading of human THP-1 macrophages. Depletion of triacylglycerol (TG) by incubation with the acyl-CoA synthetase inhibitor Triacsin D had no effect on CEH association with the lipid droplets, suggesting that CEH associates with mixed (CE + TG) as well as TG-depleted CE droplets. However, CEH had 2.5-fold higher activity when mixed droplets were used as substrate in an in vitro assay, consistent with the reported higher cholesterol efflux from cells containing mixed isotropic droplets. Perilipin as well as adipophilin, two lipid droplet-associated proteins, were also present on the lipid droplets in THP-1 macrophages. In conclusion, CEH associates with its intracellular substrate (lipid droplets) and hydrolyzes CE more efficiently from mixed droplets.     

Effects of LDL enriched with different dietary fatty acids on cholesteryl ester accumulation and turnover in THP-1 macrophages

LDL enriched with either saturated, monounsaturated, n-6 polyunsaturated, or n-3 polyunsaturated fatty acids were used to study the effects of dietary fatty acids on macrophage cholesteryl ester (CE) accumulation, physical state, hydrolysis, and cholesterol efflux. Incubation of THP-1 macrophages with acetylated LDL (AcLDL) from each of the four diet groups resulted in both CE and triglyceride (TG) accumulation, in addition to alterations of cellular CE, TG, and phospholipid fatty acyl compositions reflective of the individual LDLs. Incubation with monounsaturated LDL resulted in significantly higher total and CE accumulation when compared with the other groups. After TG depletion, intracellular anisotropic lipid droplets were visible in all four groups, with 71% of the cells incubated with monounsaturated AcLDL containing anisotropic lipid droplets, compared with 30% of cells incubated with n-3 AcLDL. These physical state differences translated into higher rates of both CE hydrolysis and cholesterol efflux in the n-3 group. These data suggest that monounsaturated fatty acids may enhance atherosclerosis by increasing both cholesterol delivery to macrophage foam cells and the percentage of anisotropic lipid droplets, while n-3 PUFAs decrease atherosclerosis by creating more fluid cellular CE droplets that accelerate the rate of CE hydrolysis and the efflux of cholesterol from the cell.     

High-Density Lipoprotein (HDL) Particle Subpopulations in Heterozygous Cholesteryl Ester Transfer Protein (CETP) Deficiency: Maintenance of Antioxidative Activity

Cholesteryl ester transfer protein (CETP) deficiency causes elevated high-density lipoprotein-cholesterol (HDL-C) levels; its impact on HDL functionality however remains elusive. We compared functional and compositional properties of HDL derived from 9 Caucasian heterozygous CETP mutation carriers (splice-site mutation in intron 7 resulting in premature truncation) with those of 9 age- and sex-matched normolipidemic family controls. As expected, HDL-C levels were increased 1.5-fold, and CETP mass and activity were decreased by −31% and −38% respectively, in carriers versus non-carriers. HDL particles from carriers were enriched in CE (up to +19%, p<0.05) and depleted of triglycerides (TG; up to −54%, p<0.01), resulting in a reduced TG/CE ratio (up to 2.5-fold, p<0.01). In parallel, the apoA-I content was increased in HDL from carriers (up to +22%, p<0.05). Both the total HDL fraction and small, dense HDL3 particles from CETP-deficient subjects displayed normal antioxidative activity by attenuating low-density lipoprotein oxidation with similar efficacy on a particle mass basis as compared to control HDL3. Consistent with these data, circulating levels of systemic biomarkers of oxidative stress (8-isoprostanes) were similar between the two groups. These findings support the contention that HDL functionality is maintained in heterozygous CETP deficiency despite modifications in lipid and protein composition.     

Imaging of lipid biosynthesis: how a neutral lipid enters lipid droplets

The biosynthesis and storage of triglyceride (TG) is an important cellular process conserved from yeast to man. Most mammalian cells accumulate TG in lipid droplets, most prominent in adipocytes, which are specialized to store large amounts of the TG over long periods. In this study, we followed TG biosynthesis and targeting by fluorescence imaging in living 3T3-L1 adipocytes and COS7 fibroblasts. Key findings were (i) not only TG but also its direct metabolic precursor diacylglycerol, DG, accumulates on lipid droplets; (ii) the essential enzyme diacylglycerol acyltransferase 2 associates specifically with lipid droplets where it catalyzes the conversion of DG to TG and (iii) individual lipid droplets within one cell acquire TG at very different rates, suggesting unequal access to the biosynthetic machinery. We conclude that at least part of TG biosynthesis takes place in the immediate vicinity of lipid droplets. In vitro assays on purified lipid droplets show that this fraction of the biosynthetic TG is directly inserted into the growing droplet.     

Mobilization of cytoplasmic CE droplets by overexpression of human macrophage cholesteryl ester hydrolase

The obligatory first step in the removal of cholesterol from foam cells is the hydrolysis of stored cholesteryl esters (CEs) to release free cholesterol (FC). Neutral cholesteryl ester hydrolase (CEH) catalyzes this hydrolysis, and limiting levels of CEH could play a role in determining the susceptibility to atherosclerosis. We have recently reported the first identification and cloning of cDNA for human macrophage CEH. In the present study, we tested the hypothesis that systematically varied levels of overexpression of human macrophage CEH results in a proportional degree of reduction in cellular CE content in a cell system with known and reproducible amounts of CE accumulation. CEH expression was confirmed by demonstrating the presence of CEH mRNA and protein with an increase in CEH activity. A significant reduction in intracellular lipid droplets was observed in CEH-expressing cells, together with a decrease in cellular CE mass and a 2-fold increase in FC efflux. These results demonstrate that when human macrophage CEH is expressed in lipid-laden cells, hydrolysis and mobilization of CE (stored as lipid droplets) occur. These data establish the possibility that increased CE hydrolysis, mediated by CEH up-regulation, could represent an important mechanism to reduce the cholesterol burden of foam cells.     

Cholesteryl ester transfer proteins from different species do not have equivalent activities

Site-specific changes in the amino acid composition of human cholesteryl ester transfer protein (CETP) modify its preference for triglyceride (TG) versus cholesteryl ester (CE) as substrate. CETP homologs are found in many species but little is known about their activity. Here, we examined the lipid transfer properties of CETP species with 80–96% amino acid identity to human CETP. TG/CE transfer ratios for recombinant rabbit, monkey, and hamster CETPs were 1.40-, 1.44-, and 6.08-fold higher than human CETP, respectively. In transfer assays between VLDL and HDL, net transfers of CE into VLDL by human and monkey CETPs were offset by equimolar net transfers of TG toward HDL. For hamster CETP this process was not equimolar but resulted in a net flow of lipid (TG) into HDL. When assayed for the ability to transfer lipid to an acceptor particle lacking CE and TG, monkey and hamster CETPs were most effective, although all CETP species were able to promote this one-way movement of neutral lipid. We conclude that CETPs from human, monkey, rabbit, and hamster are not functionally equivalent. Most unique was hamster CETP, which strongly prefers TG as a substrate and promotes the net flow of lipid from VLDL to HDL.     

Molecular biology and pathophysiological aspects of plasma cholesteryl ester transfer protein

Plasma cholesteryl ester transfer protein (CETP) facilitates the transfer of cholesteryl ester (CE) from high density lipoprotein (HDL) to apolipoprotein B-containing lipoproteins. Since CETP regulates the plasma levels of HDL cholesterol and the size of HDL particles, CETP is considered to be a key protein in reverse cholesterol transport, a protective system against atherosclerosis. CETP, as well as plasma phospholipid transfer protein, belongs to members of the lipid transfer/lipopolysaccharide-binding protein (LBP) gene family, which also includes the lipopolysaccharide-binding protein (LBP) and bactericidal/permeability-increasing protein. Although these four proteins possess different physiological functions, they share marked biochemical and structural similarities. The importance of plasma CETP in lipoprotein metabolism was demonstrated by the discovery of CETP-deficient subjects with a marked hyperalphalipoproteinemia (HALP). Two common mutations in the CETP gene, intron 14 splicing defect and exon 15 missense mutation (D442G), have been identified in Japanese HALP patients with CETP deficiency. The deficiency of CETP causes various abnormalities in the concentration, composition, and functions of both HDL and low density lipoprotein. Although the pathophysiological significance of CETP in terms of atherosclerosis has been controversial, the in vitro experiments showed that large CE-rich HDL particles in CETP deficiency are defective in cholesterol efflux. Epidemiological studies in Japanese-Americans and in the Omagari area where HALP subjects with the intron 14 splicing defect of CETP gene are markedly frequent, have shown an increased incidence of coronary atherosclerosis in CETP-deficient patients. The current review will focus on the recent findings on the molecular biology and pathophysiological aspects of plasma CETP, a key protein in reverse cholesterol transport.     

Role of cholesteryl ester transfer protein in selective uptake of high density lipoprotein cholesteryl esters by adipocytes

Previous reports attributed cholesteryl ester transfer protein (CETP)-mediated HDL cholesteryl ester (CE) selective uptake to the CETP-mediated transfer of CE from HDL to newly secreted apolipoprotein B-containing lipoproteins, which are then internalized by the LDL receptor (LDL-R). CETP has also been implicated in the remodeling of HDL, which renders it a better substrate for selective uptake by scavenger receptor class B type I (SR-BI). However, CETP-mediated selective uptake of HDL3-derived CE was not diminished in LDL-R null adipocytes, SR-BI null adipocytes, or in the presence of the receptor-associated protein. We found that monensin treatment or energy depletion of the SW872 liposarcoma cells with 2-deoxyglucose and NaN3 had no effect on CETP-mediated selective uptake, demonstrating that endocytosis is not required. This is supported by data indicating that CETP transfers CE into a compartment from which it can be extracted by unlabeled HDL. CETP could also mediate the selective uptake of HDL3-derived triacylglycerol (TG) and phospholipid (PL). The CETP-specific kinetics for TG and CE uptake were similar, and both reached saturation at approximately 5 microg/ml HDL. In contrast, CETP-specific PL uptake did not attain saturation at 5 microg/ml HDL and was approximately 6-fold greater than the uptake of CE. We propose two possible mechanisms to account for the role of CETP in selective uptake.     

Modification of CETP function by changing its substrate preference: a new paradigm for CETP drug design

We previously determined that hamster cholesteryl ester transfer protein (CETP), unlike human CETP, promotes a novel one-way transfer of TG from VLDL to HDL, causing HDL to gain lipid. We hypothesize that this nonreciprocal lipid transfer activity arises from the usually high TG/cholesteryl ester (CE) substrate preference of hamster CETP. Consistent with this, we report here that ∼25% of the total lipid transfer promoted by the human Q199A CETP mutant, which prefers TG as substrate, is nonreciprocal transfer. Other human CETP mutants with TG/CE substrate preferences higher or lower than wild-type also possess nonreciprocal lipid transfer activity. Mutants with high TG/CE substrate preference promote the nonreciprocal lipid transfer of TG from VLDL to HDL, but mutants with low TG/CE substrate preference promote the nonreciprocal lipid transfer of CE, not TG, and this lipid flow is in the reverse direction (from HDL to VLDL). Anti-CETP TP2 antibody alters the TG/CE substrate preference of CETP and also changes the extent of nonreciprocal lipid transfer, showing the potential for externally acting agents to modify the transfer properties of CETP. Overall, these data show that the lipid transfer properties of CETP can be manipulated. Function-altering pharmaceuticals may offer a novel approach to modify CETP activity and achieve specific modifications in lipoprotein metabolism.     

Perilipin A increases triacylglycerol storage by decreasing the rate of triacylglycerol hydrolysis

The perilipins are the most abundant proteins at the surfaces of lipid droplets in adipocytes and are also found in steroidogenic cells. To investigate perilipin function, perilipin A, the predominant isoform, was ectopically expressed in fibroblastic 3T3-L1 pre-adipocytes that normally lack the perilipins. In control cells, fluorescent staining of neutral lipids with Bodipy 493/503 showed a few minute and widely dispersed lipid droplets, while in cells stably expressing perilipin A, the lipid droplets were more numerous and tightly clustered in one or two regions of the cytoplasm. Immunofluorescence microscopy revealed that the ectopic perilipin A localized to the surfaces of the tiny clustered lipid droplets; subcellular fractionation of the cells using sucrose gradients confirmed that the perilipin A localized exclusively to lipid droplets. Cells expressing perilipin A stored 6-30-fold more triacylglycerol than control cells due to reduced lipolysis of triacylglycerol stores. The lipolysis of stored triacylglycerol was 5 times slower in lipid-loaded cells expressing perilipin A than in lipid-loaded control cells, when triacylglycerol synthesis was blocked with 6 microm triacsin C. This stabilization of triacylglycerol was not due to the suppression of triacylglycerol lipase activity by the expression of perilipin A. We conclude that perilipin A increases the triacylglycerol content of cells by forming a barrier that reduces the access of soluble lipases to stored lipids, thus inhibiting triacylglycerol hydrolysis. These studies suggest that perilipin A plays a major role in the regulation of triacylglycerol storage and lipolysis in adipocytes.     

Mechanism of cholesteryl ester transfer protein inhibition by a neutralizing monoclonal antibody and mapping of the monoclonal antibody epitope

The plasma cholesteryl ester transfer protein (CETP, Mr 74,000) has a binding site for neutral lipid which can readily equilibrate with lipoprotein cholesteryl esters or triglycerides. Recently, a monoclonal antibody (TP2) was obtained which neutralizes the cholesteryl ester (CE) and triglyceride (TG) transfer activities of the CETP. In this report, the epitope of the inhibitory monoclonal antibody has been localized to a hydrophobic 26-amino acid sequence at the COOH terminus of CETP. The Fab fragments of TP2 caused partial (50%) inhibition of CE transfer and complete inhibition of TG transfer by the CETP. Similarly, the Fab fragments inhibited (37%) the binding of CE to the CETP and abolished the binding of TG to the CETP. Surprisingly, the TP2 Fab was also found to enhance the binding of CETP to plasma lipoproteins and to phospholipid vesicles. In conclusion, the TP2 monoclonal antibody inhibits lipid transfer by blocking the uptake of lipid by CETP. The COOH-terminal epitope may be in or near the neutral lipid binding site. Occupancy of this site by TP2 Fab fragments or by neutral lipid may result in a conformational change of CETP causing enhanced binding to lipoproteins or vesicles.     

Lipid transfer inhibitor protein defines the participation of high density lipoprotein subfractions in lipid transfer reactions mediated by cholesterol ester transfer protein (CETP)

Cholesterol ester transfer protein (CETP) moves triglyceride (TG) and cholesteryl ester (CE) between lipoproteins. CETP has no apparent preference for high (HDL) or low (LDL) density lipoprotein as lipid donor to very low density lipoprotein (VLDL), and the preference for HDL observed in plasma is due to suppression of LDL transfers by lipid transfer inhibitor protein (LTIP). Given the heterogeneity of HDL, and a demonstrated ability of HDL subfractions to bind LTIP, we examined whether LTIP might also control CETP-facilitated lipid flux among HDL subfractions. CETP-mediated CE transfers from [3H]CE VLDL to various lipoproteins, combined on an equal phospholipid basis, ranged 2-fold and followed the order: HDL3 > LDL > HDL2. LTIP inhibited VLDL to HDL2 transfer at one-half the rate of VLDL to LDL. In contrast, VLDL to HDL3 transfer was stimulated, resulting in a CETP preference for HDL3 that was 3-fold greater than that for LDL or HDL2. Long-term mass transfer experiments confirmed these findings and further established that the previously observed stimulation of CETP activity on HDL by LTIP is due solely to its stimulation of transfer activity on HDL3. TG enrichment of HDL2, which occurs during the HDL cycle, inhibited CETP activity by approximately 2-fold and LTIP activity was blocked almost completely. This suggests that LTIP keeps lipid transfer activity on HDL2 low and constant regardless of its TG enrichment status. Overall, these results show that LTIP tailors CETP-mediated remodeling of HDL3 and HDL2 particles in subclass-specific ways, strongly implicating LTIP as a regulator of HDL metabolism.     

An in vitro model to study adipose differentiation in serum-free medium

Adipose differentiation was studied in a teratoma-derived fibroadipogenic cell line (1246) cultured in serum-free medium. The addition of dexamethasone and 1-methyl-3-isobutylxanthine to the serum-free medium induced confluent 1246 cells to differentiate into adipocyte-like cells as evidenced by triglyceride accumulation and increased levels of lipolytic enzyme activities. Hormone-sensitive lipase activity measured 5 days after the addition of dexamethasone and 1-methyl-3-isobutylxanthine increased 17-fold and was activated by cAMP-dependent protein kinase. Neutral diglyceride lipase, monoglyceride lipase, and cholesterol ester hydrolase specific activities increased 23-, 75-, and 73-fold, respectively. Among these three activities, only cholesterol ester hydrolase was activated by cAMP-dependent protein kinase. Differentiated 1246 cells expressed receptors to lipolytic hormones as shown by the stimulation of glycerol release by epinephrine (8.6-fold), glucagon (2.2-fold), and adrenocorticotrophic hormone (5.5-fold). Heparin treatment of 1246 cells in serum-free medium resulted in the release of lipoprotein lipase activity into the culture medium. Thus, 1246 cells can serve as a model for the study of adipose differentiation under defined culture conditions since they are capable of growth and survival in the absence of serum while retaining their ability to differentiate into adipocytes.     

Adipose tissue-specific CETP expression in mice: impact on plasma lipoprotein metabolism

Adipose tissue appears to be a highly conserved site of cholesteryl ester transfer protein (CETP) expression across species. To investigate the impact of adipose CETP expression on lipid metabolism, we created adipose tissue-specific CETP transgenic (CETPTg) mice. CETP mRNA is predominantly expressed in adipose tissue. Plasma CETP mass and activity are readily detectable in CETPTg mice but not in controls. Plasma lipoprotein analysis shows marked reductions in HDL cholesterol and phospholipids, increases non-HDL lipids, decreases apolipoprotein A-I (apoA-I), and increases apoB. Unexpectedly, CETPTg adipocytes are significantly smaller than those in control mice (44%), triglyceride and cholesterol in adipose tissue were significantly decreased compared with controls (50% and 37%, respectively), and phospholipids showed no significant changes. To study the mechanism, we measured peroxisome proliferator-activated receptor gamma, sterol-regulatory element binding protein-1c, LPL, and hormone-sensitive lipase (HSL) in aP2-CETPTg adipose tissue and controls and found that, except for HSL, all mRNA levels are significantly decreased in the transgenic mice compared with controls (26, 33, and 22%). In conclusion, adipose tissue CETP makes a major contribution to CETP in the circulation, reduces HDL, and increases non-HDL cholesterol levels. Moreover, adipose tissue CETP expression changes triglyceride and cholesterol content and the size of adipocytes.