Modification of the N-terminal cysteine of plasma cholesteryl ester transfer protein selectively inhibits triglyceride transfer activity

151

119

The mechanism by which cholesteryl ester transfer protein (CETP) activity affects HDL metabolism was investigated using agents that selectively target CETP (dalcetrapib, torcetrapib, anacetrapib). In contrast with torcetrapib and anacetrapib, dalcetrapib requires cysteine 13 to decrease CETP activity, measured as transfer of cholesteryl ester (CE) from HDL to LDL, and does not affect transfer of CE from HDL3 to HDL2. Only dalcetrapib induced a conformational change in CETP, when added to human plasma in vitro, also observed in vivo and correlated with CETP activity. CETP-induced pre-β-HDL formation in vitro in human plasma was unchanged by dalcetrapib ≤3 µM and increased at 10 µM. A dose-dependent inhibition of pre-β-HDL formation by torcetrapib and anacetrapib (0.1 to 10 µM) suggested that dalcetrapib modulates CETP activity. In hamsters injected with [3H]cholesterol-labeled autologous macrophages, and given dalcetrapib (100 mg twice daily), torcetrapib [30 mg once daily (QD)], or anacetrapib (30 mg QD), only dalcetrapib significantly increased fecal elimination of both [3H]neutral sterols and [3H]bile acids, whereas all compounds increased plasma HDL-[3H]cholesterol. These data suggest that modulation of CETP activity by dalcetrapib does not inhibit CETP-induced pre-β-HDL formation, which may be required to increase reverse cholesterol transport.

Section: Discussionmentioning

confidence: 99%

Modulating cholesteryl ester transfer protein activity maintains efficient pre-β-HDL formation and increases reverse cholesterol transport

Niesor

Magg

Ogawa

et al. 2010

151

119

“…Thimerosal and other mercurial agents derivatize CETP cysteines. Because these cysteines are not necessary for CETP activity ( 45 ), the effect of these compounds on CETP activity likely results from alterations in its conformation. The partial inhibition of DG compared with TG is likely due to its smaller molecular size, making it less sensitive to small conformational changes in CETP structure.…”

Section: Cellular Tg Synthesis and Movementmentioning

confidence: 99%

Defective triglyceride biosynthesis in CETP-deficient SW872 cells

Greene

Izem

Morton

2015

with adipose tissue CETP mRNA levels in both man and hamster ( 6,7 ), showing the importance of this tissue in both lipid storage and the modulation of intravascular lipid metabolism.While most CETP is secreted by cells, a portion is retained ( 8-10 ). Much of the intracellular CETP pool is associated with the endoplasmic reticulum (ER), with increased concentrations near lipid droplets ( 8,11 ). Both cytoplasmic and lipid-droplet pools of CETP have been observed as well ( 11 ). Although the mechanism for CETP release into the cytoplasm is unknown, the escape of other ER luminal proteins to the cytoplasm has been reported (12)(13)(14).In addition to its role in plasma lipoprotein metabolism, multiple studies suggest that CETP has other functions. Cell-associated CETP promotes CE uptake from HDL, stimulates the cell's ability to effl ux cholesterol, and infl uences the storage of CE in cells (15)(16)(17)(18)(19). In addition to altering cellular cholesterol metabolism, both overexpression and under-expression of CETP in SW872 cells, a human cell line commonly used as an adipocyte model, reduce the capacity of these cells to store TG ( 10, 11 ). A role for CETP in adipocyte lipid storage is further supported by observations in transgenic mice. Adipose tissue-specifi c expression of human CETP in mice results in smaller adipocytes containing less TG and cholesterol, and signifi cantly reduces the expression of key lipogenic genes ( 20 ). In hypertriglyceridemic mice, CETP expression normalizes subcutaneous adipose depots and visceral adipocyte size ( 21 ). And in humans, a CETP gene variant that affects the coding sequence of CETP is associated with increased adiposity following long-term overfeeding ( 22 ).In this study, we have examined pathways involved in TG homeostasis in order to understand the metabolic Abstract We previously reported that reducing the expression of cholesteryl ester transfer protein (CETP) disrupts cholesterol homeostasis in SW872 cells and causes an ‫ف‬ 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-defi cient cells, nor was there evidence of endoplasmic reticulum (ER) stress. In short-term studies, the total fl ux of oleate through the TG biosynthetic pathway was not altered in CETP-defi cient 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 ineffi ciently 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-defi cient cells arises from the reduced conversion of DG to TG in the ER and/or on the lipid droplet surface, and enhanced ...

“…This is noteworthy because some pharmacologic CETP inhibitors, such as dalcetrapib, block CETP by derivatizing cysteine ( 32 ). Although thimerosal and dalcetrapib likely react with different cysteines ( 23,32 ), our data raise the C-CE LDL (10 g cholesterol) and either PC-cholesterol liposomes containing 0.5 mol% CE and 0.5% mol% TG (CE+TG liposomes) or PC-cholesterol liposomes containing no CE or TG (null liposomes) as acceptor (750 nmol PC). CETP was incubated with buffer, anti-CETP (TP2, 5.5 g) or thimerosal (thim, 1.9 mM), for 1 h at room temperature prior to initiating the transfer reaction.…”

Section: Discussionmentioning

confidence: 69%

“…Assuming these compounds act by similar mechanisms, inhibition appears to result from derivatization of the N-terminal cysteine residue ( 23 ). Because rabbit, The indicated CETP was preincubated with 1.9 mM thimerosal then added to assays containing 3 H-TG, 14 C-CE LDL, and unlabeled HDL as acceptor.…”

Section: Net Ce and Tg Transfer Between Lipoproteinsmentioning

confidence: 99%

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

Morton

Izem

2014

This article is available online at http://www.jlr.org homologs in more than 20 species. In species where CETP activity has been experimentally verifi ed, this is typically based solely on the presence of CE transfer activity in plasma. Mutagenesis studies have shown that minor changes in the structure of human CETP can have profound effects on its ability to bind lipoproteins or selectively alter its ability to utilize TG and/or CE as a transfer substrate ( 5, 6 ). Given this, it seems likely that CETP from common experimental models such as hamster and rabbit, which have only ‫ف‬ 80% identity to human CETP at the amino acid level, may have unique lipid transfer properties.The purpose of this study was to characterize the lipid transfer properties of CETP from three species (monkey, rabbit, and hamster) and compare them to human CETP. We have determined the relative preference of each CETP for CE and TG as a substrate and assessed their abilities to promote net TG and CE transfer between lipoproteins. Such processes are central to CETP's role in facilitating lipoprotein metabolism. Additionally, because there is growing evidence that intracellular CETP infl uences cellular lipid metabolism ( 7-9 ), perhaps by facilitating the formation of lipid droplets ( 10 ), we investigated the capacity of all four CETP species to mediate net unidirectional lipid transfer. We show species-specifi c differences in the lipid transfer properties of CETPs in terms of their substrate preference and their ability to promote net lipid transfer between lipoproteins, and between lipoproteins and model membrane surfaces. METHODS MaterialsMouse monoclonal antibody to human CETP (TP2) was purchased from the Ottawa Heart Institute (Ottawa, Ontario, Canada). Cholesteryl ester (CE) transfer activity was fi rst observed in human plasma by Nichols and Smith ( 1 ). Zilversmit, Hughes, and Balmer ( 2 ) were the fi rst to identify a plasma protein responsible for this activity, and subsequent study showed that this protein, now called cholesteryl ester transfer protein (CETP), is responsible for all CE and triglyceride (TG) transfer activity in human plasma ( 3,4 ). Although absent in the plasma of common laboratory animals such as mice and rats, genome sequencing has identifi ed CETP