Evaluation of CETP activity in vivo under non-steady-state conditions: influence of anacetrapib on HDL-TG flux

Studies in lipoprotein kinetics almost exclusively rely on steady-state approaches to modeling. Herein, we have used a non-steady-state experimental design to examine the role of cholesteryl ester transfer protein (CETP) in mediating HDL-TG flux in vivo in rhesus macaques, and therefore, we developed an alternative strategy to model the data. Two isotopomers ([2H11] and [13C18]) of oleic acid were administered (orally and intravenously, respectively) to serve as precursors for labeling TGs in apoB-containing lipoproteins. The flux of a specific TG (52:2) from these donor lipoproteins to HDL was used as the measure of CETP activity; calculations are also presented to estimate total HDL-TG flux. Based on our data, we estimate that the peak total postprandial TG flux to HDL via CETP is ∼13 mg·h−1·kg−1 and show that this transfer was inhibited by 97% following anacetrapib treatment. Collectively, these data demonstrate that HDL TG flux can be used as a measure of CETP activity in vivo. The fact that the donor lipoproteins can be labeled in situ using well-established stable isotope tracer techniques suggests ways to measure this activity for native lipoproteins in free-living subjects under any physiological conditions. Studies in lipoprotein kinetics almost exclusively rely on steady-state approaches to modeling. Herein, we have used a non-steady-state experimental design to examine the role of cholesteryl ester transfer protein (CETP) in mediating HDL-TG flux in vivo in rhesus macaques, and therefore, we developed an alternative strategy to model the data. Two isotopomers ([2H11] and [13C18]) of oleic acid were administered (orally and intravenously, respectively) to serve as precursors for labeling TGs in apoB-containing lipoproteins. The flux of a specific TG (52:2) from these donor lipoproteins to HDL was used as the measure of CETP activity; calculations are also presented to estimate total HDL-TG flux. Based on our data, we estimate that the peak total postprandial TG flux to HDL via CETP is ∼13 mg·h−1·kg−1 and show that this transfer was inhibited by 97% following anacetrapib treatment. Collectively, these data demonstrate that HDL TG flux can be used as a measure of CETP activity in vivo. The fact that the donor lipoproteins can be labeled in situ using well-established stable isotope tracer techniques suggests ways to measure this activity for native lipoproteins in free-living subjects under any physiological conditions. Nichols and Smith (1Nichols A.V. Smith L. Effect of very low-density lipoproteins on lipid transfer in incubated serum.J. Lipid Res. 1965; 6: 206-210Abstract Full Text PDF PubMed Google Scholar) were the first to observe that cholesteryl esters (CEs) could be transferred from HDL to apoB-containing lipoproteins in exchange for TG in incubated human serum, a finding later confirmed by Hopkins and Barter (2Hopkins G.J. Barter P.J. Transfers of esterified cholesterol and triglyceride between high density and very low density lipoproteins: in vitro studies of rabbits and humans.Metabolism. 1980; 29: 546-550Abstract Full Text PDF PubMed Scopus (37) Google Scholar). Subsequent investigations by Morton et al. (3Morton R.E. Zilversmit D.B. Purification and characterization of lipid transfer protein (s) from human lipoprotein-deficient plasma.J. Lipid Res. 1982; 23: 1058-1067Abstract Full Text PDF PubMed Google Scholar, 4Morton R.E. Zilversmit D.B. Inter-relationship of lipids transferred by the lipid-transfer protein isolated from human lipoprotein-deficient plasma.J. Biol. Chem. 1983; 258: 11751-11757Abstract Full Text PDF PubMed Google Scholar) identified a single lipid transfer protein in human plasma that was capable of carrying out this apparent heterolipid exchange and that is known today as cholesteryl ester transfer protein (CETP). In these and other reports, the exchange of HDL-CE for apoB-lipoprotein-TG was evaluated in vitro using lipoproteins containing radiolabeled CE and/or TG. Using this approach, the effects of donor and acceptor lipid composition (4Morton R.E. Zilversmit D.B. Inter-relationship of lipids transferred by the lipid-transfer protein isolated from human lipoprotein-deficient plasma.J. Biol. Chem. 1983; 258: 11751-11757Abstract Full Text PDF PubMed Google Scholar), hypercholesterolemia (5Son Y.S. Zilversmit D.B. Increased lipid transfer activities in hyperlipidemic rabbit plasma.Arterioscler. Thromb. Vasc. Biol. 1986; 6: 345-351Crossref Google Scholar), and many other parameters on CETP-mediated neutral lipid exchange have been assessed. The CETP-mediated exchange of neutral lipids between lipoproteins in vivo has also been studied (6Lally J.I. Barter P.J. The in vivo metabolism of esterified cholesterol in the plasma high-density lipoproteins of rabbits.J. Lab. Clin. Med. 1979; 93: 570-582PubMed Google Scholar, 7Nestel P.J. Reardon M. Billington T. In vivo transfer of cholesteryl esters from high density lipoproteins to very low density lipoproteins in man.Biochim. Biophys. Acta. 1979; 573: 403-407Crossref PubMed Scopus (41) Google Scholar, 8Ha Y.C. Calvert G.D. McIntosh G.H. Barter P.J. A physiologic role for the esterified cholesterol transfer protein: in vivo studies in rabbits and pigs.Metabolism. 1981; 30: 380-383Abstract Full Text PDF PubMed Scopus (26) Google Scholar, 9Schwartz C.C. Vlahcevic Z. Halloran L.G. Swell L. An in vivo evaluation in man of the transfer of esterified cholesterol between lipoproteins and into the liver and bile.Biochim. Biophys. Acta. 1981; 663: 143-162Crossref PubMed Scopus (46) Google Scholar). In such cases, the experimental protocol typically involves injection of lipoprotein particles, radiolabeled with CE either in vitro or in vivo, into subjects followed by collection of blood samples for further analysis. Transfer activity is then evaluated by measuring the loss of enrichment from the donor particle and increase in enrichment of acceptor particles that were isolated from the postinjection blood samples. We hypothesized that the movement of TG from apoB lipoproteins to HDL could serve as a quantifiable activity measurement in vivo that would not require prior isolation and reinjection of donor particles. As noted, although the protein is commonly referred to as “cholesteryl ester transfer protein,” the transfer of TG also occurs; this should allow an experimental protocol to be designed based on well-characterized methods for labeling TGs in free-living subjects. Lipoprotein TG kinetics have been studied for at least 30 years using both radioisotopes and stable isotopes to label the fatty acid precursors or the glycerol backbone. In the majority of cases, the goals of such studies have been determination of the flux (mass per unit time) of TG in specific lipoprotein compartments at one or more physiological steady states (10Grundy S.M. Mok H.Y.I. Zech L. Steinberg D. Berman M. Transport of very low density lipoprotein triglycerides in varying degrees of obesity and hypertriglyceridemia.J. Clin. Invest. 1979; 63: 1274Crossref PubMed Scopus (185) Google Scholar, 11Marsh J.B. Lipoprotein metabolism in obesity and diabetes: insights from stable isotope kinetic studies in humans.Nutr. Rev. 2003; 61: 363-375Crossref PubMed Scopus (36) Google Scholar, 12Parks E.J. Krauss R.M. Christiansen M.P. Neese R.A. Hellerstein M.K. Effects of a low-fat, high-carbohydrate diet on VLDL-triglyceride assembly, production, and clearance.J. Clin. Invest. 1999; 104: 1087-1096Crossref PubMed Scopus (260) Google Scholar, 13Patterson B.W. Mittendorfer B. Elias N. Satyanarayana R. Klein S. Use of stable isotopically labeled tracers to measure very low density lipoprotein-triglyceride turnover.J. Lipid Res. 2002; 43: 223-233Abstract Full Text Full Text PDF PubMed Google Scholar). Using these methods as a starting point, we previously demonstrated the techniques necessary to characterize TG transfer between lipoprotein compartments in vivo using C57Bl/6 wild-type mice (which lack CETP) and natural flanking region (NFR)-CETP-transgenic mice (14McLaren D.G. Wang S-P. Stout S.J. Xie D. Miller P.L. Mendoza V. Rosa R. Castro-Perez J. Previs S.F. Johns D.G. Tracking fatty acid kinetics in distinct lipoprotein fractions in vivo: a novel high-throughput approach for studying dyslipidemia in rodent models.J. Lipid Res. 2013; 54: 276-281Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Clear differences in the kinetics of TG appearance in HDL were observed between these two extreme conditions (i.e., in the absence and presence of CETP). The goals of the present study were 2-fold: 1) to develop and test a mathematical model to describe HDL-TG flux as a measure of neutral lipid exchange between particles in a non-steady state and 2) to determine whether this approach would be useful to assess pharmacological inhibition of CETP activity in vivo. In order to achieve these goals, we sought to develop the simplest possible model that allows in vivo quantification of CETP-mediated TG flux and to use this to measure inhibition by anacetrapib. While development of a more complex model to account for other neutral lipid exchanges could be pursued following similar techniques, we specifically constrained our interest to measuring HDL-TG flux in order to establish a clear proof of concept on which future studies could be based. We selected rhesus macaques as the experimental model for these studies based on the facts that they endogenously express CETP and are frequently used as a translational model for pharmaceutical research. To as as possible for we designed a protocol was with or the anacetrapib. have previously that of CE transfer are in the postprandial state M. R. ester flux from HDL to is in in the postprandial Lipid Res. 2002; 43: Full Text Full Text PDF PubMed Scopus Google Scholar) and under conditions of T. S. R. L. are of cholesterol and HDL in human plasma.Arterioscler. Thromb. Vasc. Biol. PubMed Scopus Google Scholar, D. of cholesteryl ester transfer from high density lipoproteins to lipoproteins Clin. Invest. 1986; PubMed Scopus Google Scholar, R. M. transfer of cholesteryl esters in of cholesteryl ester transfer Clin. Invest. PubMed Scopus Google Scholar). that the differences in HDL-TG flux between and subjects would under similar conditions. We to study TG transfer kinetics following of an lipid and a of which isotopomers of oleic and to label lipoprotein a hyperlipidemic non-steady approaches to non-steady-state flux calculations been based on R. of and of on N. PubMed Scopus Google Scholar) that are to in many experimental of model and for J. Google Scholar, of calculations in steady and non-steady states and to J. Google Scholar) and that could have our mathematical in kinetic E.J. of plasma fatty acid in Thromb. Vasc. Biol. PubMed Scopus Google Scholar) have these calculations and we sought to these methods to a model capable of TG from plasma and to plasma HDL and the effects of the CETP anacetrapib on these were in by the for and of experimental were by the and Use and were in with the for the and Use of rhesus macaques in from to years and with an of were used in A design was used as were with the of tracer study were with either or of anacetrapib in for were the been composition by the of tracer study and following the the were transferred to and were and with 30 were with or of anacetrapib in on later at were with of a of two and which also the of oleic acid at a of for a of in the is in were a of which the of oleic acid at a of for a of isotopomers of oleic acid were from the to and following blood was via at and and and and were with for the and blood samples were on and at for at 30 of was and into containing and for analysis. were isolated from plasma by following the of et al. M. The cholesterol hypercholesterolemia in J. PubMed Scopus Google Scholar). of plasma was to and with of a were at in a using a for 30 at The of was as and at prior to analysis. was used to and HDL from a of plasma using containing the isolated lipoprotein fractions were and in for lipid as previously (14McLaren D.G. Wang S-P. Stout S.J. Xie D. Miller P.L. Mendoza V. Rosa R. Castro-Perez J. Previs S.F. Johns D.G. Tracking fatty acid kinetics in distinct lipoprotein fractions in vivo: a novel high-throughput approach for studying dyslipidemia in rodent models.J. Lipid Res. 2013; 54: 276-281Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). The of isotopomers of and were in lipoprotein using an that has been D.G. Stout Wang S-P. Mendoza V. Castro-Perez Miller P.L. et use of oleic acid and to study of plasma triglycerides in vivo: and experimental Chem. 2013; PubMed Scopus Google Scholar). is that we not observe any of the that could have been by of both the and oleic acid isotopomers into a single of The total cholesterol and TG of plasma were using the lipid composition of distinct lipoprotein fractions was by as previously S. D. R. Mendoza V. Rosa R. N. Castro-Perez et of lipoprotein and lipid for human and plasma Res. PubMed Scopus Google Scholar). CETP activity was using a transfer to the of transferred from to HDL by CETP M. D. M. characterization of cholesteryl ester transfer protein Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). of CETP protein in plasma were by R.A. for plasma cholesteryl ester transfer protein 1999; PubMed Scopus Google Scholar) and are in The of anacetrapib in plasma were by using methods similar to that have been previously S. M.P. and of a novel of the cholesteryl ester transfer in and rhesus PubMed Scopus Google are in the data, and of the are presented in on the of CETP on CETP as a protein and that TG transfer was by a exchange with more P.J. ester transfer protein a novel for HDL and Thromb. Vasc. Biol. 2003; 23: PubMed Scopus Google Scholar) transfer by the of CETP as a L. S. D. M. Krauss R.M. et of transfer between lipoproteins by cholesteryl ester transfer Chem. Biol. PubMed Scopus Google Scholar) lipoprotein is to cholesteryl ester transfer Lipid Res. Full Text PDF PubMed Google Scholar) complex model and with the exchange The of the is two neutral lipid a for that data that transfer of neutral lipid from apoB-containing particles to HDL is more and we this concept in the model presented CETP is as the for of TG to is possible to a of on the of CETP-mediated the and frequently of plasma 1) 2) and were as to the total of isotopomers the are in a non-steady with were as per and were in both and anacetrapib of the The were used as by both the tracer and data as for CETP-mediated transfer were based on kinetic and parameters were by these to account for the and both in the absence and in the presence of anacetrapib. Use of the to CETP-mediated the apoB lipoprotein the of such is in the experimental data and presented to the model as CETP for transfer to is to that for this study a single for of plasma was the the can be to other TG our of total CETP-mediated TG are based on the known of in the total TG A is an that the flux (mass per unit time) a as a of the and of that the flux of TG from to HDL as a the flux CETP can be as in 1) is the flux of from to is a the for transfer presented by neutral lipids other fractions were as of in the lipoprotein by total of neutral lipid in the lipoprotein and were as for the of this is the of CETP-mediated neutral lipid is the or of for this is the for the other is the plasma of CETP and is the anacetrapib inhibition In the of a non-steady-state physiological the be of the in with the postprandial based on the flux of a is a with The for the apoB lipoproteins for the of the CETP of in these is to the that neutral lipids in the lipoprotein are to be the CETP were by the data with for lipoprotein lipid composition T. S. R. L. are of cholesterol and HDL in human plasma.Arterioscler. Thromb. Vasc. Biol. PubMed Scopus Google Scholar, cholesteryl ester transfer and lipoprotein composition in Lipid Res. Full Text PDF PubMed Google Scholar). and TGs other were to with for into the CETP were using of for CE and for TG. Using these of were as by the of TG that is for and HDL are and per of neutral et al. M. Barter P.J. of high density lipoproteins by transfer Lipid Res. Full Text Full Text PDF PubMed Google Scholar) and have CETP-mediated transfer of TG from to we this in the The for is the of TG that is J. J. J. composition and in and Chem. Scopus Google Scholar). were for and neutral lipid transfer the CETP were experimental and data were and were to of in for and using the In are using a and the A in Scopus Google Scholar). was with a that the particle and J. A using particle of the on and in Scholar). on a with a 30 particles with or in The a of between and that are and the The in this study is the of by the of the measurement The was for as of the The in selected by particle then as the starting for using the for Scopus Google Scholar) to allow and on a with Scopus (37) Google Scholar). for parameters were from differences were evaluated using a were used to test for of the model for were as the of the for on plasma lipids from both studies are in with anacetrapib in this rhesus model HDL cholesterol by with the effects of anacetrapib in the effects on cholesterol were in the rhesus observed in on D. D. and of the cholesteryl ester transfer protein anacetrapib as and with in J. PubMed Scopus Google Scholar). In to the effects on HDL and we observed in plasma TGs in rhesus with anacetrapib. The was in HDL-TG and this is with transfer from lipoproteins such as and to inhibition of The enrichment for in lipoprotein labeled with the and are in The that the in the and fractions were both in the HDL fractions were following anacetrapib that the effects of anacetrapib on HDL-TG flux could be using a strategy based on either or of the of this we to develop our mathematical model based on both the and data in order to the of the data CETP-mediated TG experimental data for and in and are in plasma were to plasma using a plasma of R.A. S. plasma total blood and in the rhesus J. PubMed Scopus Google Scholar), and were for by from to the using the for total neutral lipid transfer via CETP on lipoprotein we the that was from or was to account for the experimental data. The alternative that are the was also degrees of and the data as for the two by which is the of in the to the that the of CETP a single that not on which apoB-containing lipoprotein is to the and of the CETP both lipoprotein as by et al. L. S. D. M. Krauss R.M. et of transfer between lipoproteins by cholesteryl ester transfer Chem. Biol. PubMed Scopus Google Scholar), is that with neutral lipids is of the of the To test this and for HDL-TG TG flux from HDL to apoB would be constrained to be to one We not this in the present study was to be any of the other possible that the of from the presence of TG in the The simplest we have that is with data in data is the single The for this model is in for CETP-mediated transfer to HDL are as for 1) for 2) for for is the inhibition are as in and The are as any of the or The is TG from HDL by other is as a and this is presented in the data. was out as in and the for the parameters for with the for in are the for the in are the of of these are for of which that this could not be identified in of the and for the neutral in a The for was to be which is the plasma of anacetrapib in this study is in with and human M. D. M. characterization of cholesteryl ester transfer protein Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar), which to of and these the in of the data and and in The in the at point, and the the model using the methods used for the for the are in A for CETP-mediated TG transfer in vivo is an of these in are in for the following the test and lipid for at the postprandial on and are and in in the flux from to HDL is TG flux from to HDL with the estimate that P.J. ester transfer protein a novel for HDL and Thromb. Vasc. Biol. 2003; 23: PubMed Scopus Google Scholar). the of transfer is in the presence of anacetrapib can be as possible to estimate the in vivo of using the plasma of CETP of plasma and as that of CETP with transfer of neutral lipid per in flux for in a The of this study was of and for the in vivo flux of CETP-mediated TG transfer from of the apoB-containing lipoproteins to the in demonstrate that this is should be that this is possible the and were and In the of tracer for the of or these is as for non-steady-state such as the study we that the or be and The for CETP-mediated neutral lipid transfer lipoproteins in a of based on the for CETP-mediated transfer to the neutral lipid composition and HDL neutral lipid in the between and In the we have the and The have been presented as and the as the and are flux on the neutral lipid of the starting not account for apoB-containing lipoproteins with HDL for the of could this of the in the of neutral lipid exchanges the apoB lipoproteins. the experimental data not TG from was to the of CETP-mediated transfer from and from The to these two on 1) the and tracer data and 2) the absence of labeled in TG. The of the flux has on the transfer of or the data in the presence of anacetrapib the could not be in the flux was the for TG is to be to that the for either or and the of is are with the that is a CETP with native lipoproteins. are in to the of in et al. S. of human low-density lipoprotein by Biophys. Acta. PubMed Scopus Google Scholar) demonstrated exchange of human plasma CE for TG incubated with in the presence of and In vitro exchange of and lipids between human high density lipoproteins and an 1982; Full Text PDF PubMed Scopus Google Scholar) also that TG could be for human plasma HDL CE in that was a acceptor which was not in our differences between has been in vitro and our in vivo data are not at In our in vivo the with native lipoproteins for transfer of TG to HDL via a to the of our has not been in is in the presented as The and inhibition and were both to be lipoprotein that transfer from to HDL is to anacetrapib inhibition that of the lipoprotein is a of anacetrapib the we out the that a for CETP or that this be a The could be with an experimental protocol that not is of interest to from the that was the of this study to estimate the total flux of CETP-mediated TG transfer from apoB-containing lipoproteins to flux is and typically of total plasma we estimate that postprandial total TG transfer flux as the of a is TG per per anacetrapib present at a plasma of peak total TG transfer flux is TG per per to a 97% in neutral lipid transfer using this in vivo evaluated using the more in vitro approach M. D. M. characterization of cholesteryl ester transfer protein Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar), CETP activity was to be by both a on neutral lipid transfer following inhibition of CETP with anacetrapib in the data that the in vivo approach measuring HDL-TG flux a for differences in for are presented in The model we have presented a for in vivo CETP-mediated TG transfer from apoB-containing lipoproteins to We this serve as a useful for future of the model and for future development are as of tracer data in apoB lipoproteins: In this the tracer data in apoB lipoproteins were used as with to describe the CETP-mediated flux of neutral lipid to which was the of our While the use of is B.W. Mittendorfer B. Elias N. Satyanarayana R. Klein S. Use of stable isotopically labeled tracers to measure very low density lipoprotein-triglyceride turnover.J. Lipid Res. 2002; 43: 223-233Abstract Full Text Full Text PDF PubMed Google Scholar, R. model of the 54: PubMed Scopus Google Scholar) is a clear to the model to describe CETP-mediated flux between the apoB lipoproteins and/or to as an alternative to of The in first of the for the lipoproteins these is that and are for CETP-mediated transfer are and the two that are would be to these in of rhesus and under experimental In studies in which an tracer is in the absence of containing a tracer is administered in the absence of a be useful in further CETP-mediated transfer from and particles in vivo. As with any the one presented be to be our model very the experimental data, other could be that as of the model presented in a of steady-state and non-steady-state tracer kinetic is could the effects of and pharmacological on the flux of tracers such as glycerol could be used to whether the model can account for the data studies in lipid and lipoprotein kinetics have almost exclusively on experimental that are constrained in order to a steady-state and of and The model we have presented a from these and are that we are 1) We by a model for studying CETP-mediated lipid which of the CETP the CETP for and lipid and inhibition by anacetrapib. model is the and are 2) We and the labeled and of in lipoprotein to use both of the data to test our model and both 1) and 2) a and non-steady-state that the of with the of tracers to in from in The a novel in vivo for measuring the flux of neutral lipid plasma and to our this the first on the of using HDL-TG flux as a measure for CETP activity in vivo.