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W2078563212 abstract "Fluorescent sterols, dehydroergosterol and NBD-cholesterol, were used to examine high density lipoprotein-mediated cholesterol uptake and intracellular targeting in L-cell fibroblasts. The uptake, but not esterification or targeting to lipid droplets, of these sterols differed >100-fold, suggesting significant differences in uptake pathways. NBD-cholesterol uptake kinetics and lipoprotein specificity reflected high density lipoprotein-mediated sterol uptake via the scavenger receptor B1. Fluorescence energy transfer showed an average intermolecular distance of 26 Å between the two fluorescent sterols in L-cells. Indirect immunofluorescence revealed that both fluorescent sterols localized to L-cell lipid droplets, the surface of which contained adipose differentiation-related protein. This lipid droplet-specific protein specifically bound NBD-cholesterol with high affinity (K d = 2 nm) at a single site. Thus, NBD-cholesterol and dehydroergosterol were useful fluorescent probes of sterol uptake and intracellular sterol targeting. NBD-cholesterol more selectively probed high density lipoprotein-mediated uptake and rapid intracellular targeting of sterol to lipid droplets. Targeting of sterol to lipid droplets was correlated with the presence of adipose differentiation related protein, a lipid droplet-specific protein shown for the first time to bind unesterified sterol with high affinity. Fluorescent sterols, dehydroergosterol and NBD-cholesterol, were used to examine high density lipoprotein-mediated cholesterol uptake and intracellular targeting in L-cell fibroblasts. The uptake, but not esterification or targeting to lipid droplets, of these sterols differed >100-fold, suggesting significant differences in uptake pathways. NBD-cholesterol uptake kinetics and lipoprotein specificity reflected high density lipoprotein-mediated sterol uptake via the scavenger receptor B1. Fluorescence energy transfer showed an average intermolecular distance of 26 Å between the two fluorescent sterols in L-cells. Indirect immunofluorescence revealed that both fluorescent sterols localized to L-cell lipid droplets, the surface of which contained adipose differentiation-related protein. This lipid droplet-specific protein specifically bound NBD-cholesterol with high affinity (K d = 2 nm) at a single site. Thus, NBD-cholesterol and dehydroergosterol were useful fluorescent probes of sterol uptake and intracellular sterol targeting. NBD-cholesterol more selectively probed high density lipoprotein-mediated uptake and rapid intracellular targeting of sterol to lipid droplets. Targeting of sterol to lipid droplets was correlated with the presence of adipose differentiation related protein, a lipid droplet-specific protein shown for the first time to bind unesterified sterol with high affinity. low density lipoprotein high density lipoprotein very low density lipoprotein scavenger receptor BI dehydroergosterol NBD-cholesterol, 22-(N-7-nitrobenz-2-oxa-1,3-diazo-4-yl)-amino-23,24-bisnor-5-cholen-3β-ol)) fluorescence resonance energy transfer multiphoton laser scanning microscopy laser scanning confocal microscopy adipose differentiation-related protein fetal bovine serum high performance liquid chromatography bovine serum albumin Because of cholesterol's dual role in both normal cell function and the pathobiology of atherosclerosis, it is essential to resolve the mechanisms whereby exogenous cholesterol is taken up and distributed within the cell (reviewed in Refs. 1.Schroeder F. Woodford J.K. Kavecansky J. Wood W.G. Joiner C. Mol. Membr. Biol. 1995; 12: 113-119Crossref PubMed Scopus (111) Google Scholar, 2.Schroeder F. Frolov A.A. Murphy E.J. Atshaves B.P. Jefferson J.R. Pu L. Wood W.G. Foxworth W.B. Kier A.B. Proc. Soc. Exp. Biol. Med. 1996; 213: 150-177Crossref PubMed Scopus (132) Google Scholar, 3.Fielding C.J. Fielding P.E. J. Lipid. Res. 1997; 38: 1503-1521Abstract Full Text PDF PubMed Google Scholar). Unesterified cholesterol uptake shares some, but not all, aspects of cholesterol ester uptake. Unesterified cholesterol enters the cell either by the slower LDL1 receptor mediated pathway wherein it leaves the LDL endocytosed within clathrin-coated vesicles prior to vesicle fusion at the lysosome (for review, see Ref.3.Fielding C.J. Fielding P.E. J. Lipid. Res. 1997; 38: 1503-1521Abstract Full Text PDF PubMed Google Scholar) or by the rapid “alternate” HDL receptor pathway (3.Fielding C.J. Fielding P.E. J. Lipid. Res. 1997; 38: 1503-1521Abstract Full Text PDF PubMed Google Scholar, 5.Stangl H. Cao G. Wyne K.L. Hobbs H.H. J. Biol. Chem. 1998; 273: 31002-31008Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). However, most attention has focused on the HDL-mediated cholesterol efflux from rather than uptake of cholesterol into the cell (for review, see Ref. 4.Smart E.J. van der Westhuyzen D.R. Chang T.Y. Freeman D.A. Intracellular Cholesterol Trafficking. Kluwer Academic Publishers, Boston1998: 253-272Crossref Google Scholar). Almost nothing is known regarding the uptake and intracellular targeting of unesterified cholesterol via the HDL receptor pathway nor has the process been directly visualized.Fluorescent cholesterol analogs represent an opportunity for real-time monitoring of the rapid, HDL-mediated uptake of unesterified sterol uptake and movement in living cells. The main requirement for choice of an appropriate fluorescent cholesterol analogue is that it should mimic the behavior of cholesterol. Unfortunately, the functional properties of many fluorescent sterol analogues do not closely resemble those of cholesterol (for review, see Refs. 6.Schroeder F. Prog. Lipid Res. 1984; 23: 97-113Crossref PubMed Scopus (95) Google Scholar, 7.Schroeder F. Roodyn D.B. Subcellular Biochemistry. Plenum Press, New York1985: 51-101Google Scholar, 8.Schroeder F. Nemecz G. Esfahani M. Swaney J. Advances in Cholesterol Research. Telford Press, Caldwell, NJ1990: 47-87Google Scholar, 9.Nemecz G. Fontaine R.N. Schroeder F. Biochim. Biophys. Acta. 1988; 943: 511-521Crossref PubMed Scopus (60) Google Scholar). The advent of dehydroergosterol (DHE), whose structure closely resembles that of cholesterol, represents a major advance for examining the structure of lipoproteins (10.Schroeder F. Goh E.H. Heimberg M. FEBS Lett. 1979; 97: 233-236Crossref PubMed Scopus (22) Google Scholar, 11.Schroeder F. Goh E.H. Heimberg M. J. Biol. Chem. 1979; 254: 2456-2463Abstract Full Text PDF PubMed Google Scholar) and membranes (12.Hale J.E. Schroeder F. Eur. J. Biochem. 1982; 122: 649-661Crossref PubMed Scopus (107) Google Scholar). DHE is a naturally occurring sterol where it comprises >20% of sterols in certain animals (yeast and sponge) and can replace up to 85% of cultured L-cell fibroblast cholesterol without altering cell growth, membrane function, or membrane lipid composition (for review, see Ref. 6.Schroeder F. Prog. Lipid Res. 1984; 23: 97-113Crossref PubMed Scopus (95) Google Scholar). Furthermore, DHE codistributes with cholesterol in model (2.Schroeder F. Frolov A.A. Murphy E.J. Atshaves B.P. Jefferson J.R. Pu L. Wood W.G. Foxworth W.B. Kier A.B. Proc. Soc. Exp. Biol. Med. 1996; 213: 150-177Crossref PubMed Scopus (132) Google Scholar, 6.Schroeder F. Prog. Lipid Res. 1984; 23: 97-113Crossref PubMed Scopus (95) Google Scholar, 13.Schroeder F. Nemecz G. Wood W.G. Joiner C. Morrot G. Ayraut-Jarrier M. Devaux P.F. Biochim. Biophys. Acta. 1991; 1066: 183-192Crossref PubMed Scopus (123) Google Scholar, 14.Smutzer G. Crawford B.F. Yeagle P.L. Biochim. Biophys. Acta. 1986; 862: 361-371Crossref PubMed Scopus (69) Google Scholar, 15.Schroeder F. Barenholz Y. Gratton E. Thompson T.E. Biochemistry. 1987; 26: 2441-2448Crossref PubMed Scopus (90) Google Scholar) and biological membranes (2.Schroeder F. Frolov A.A. Murphy E.J. Atshaves B.P. Jefferson J.R. Pu L. Wood W.G. Foxworth W.B. Kier A.B. Proc. Soc. Exp. Biol. Med. 1996; 213: 150-177Crossref PubMed Scopus (132) Google Scholar, 16.Schroeder F. Frolov A. Schoer J. Gallegos A. Atshaves B.P. Stolowich N.J. Scott A.I. Kier A.B. Chang T.Y. Freeman D.A. Intracellular Cholesterol Trafficking. Kluwer Academic Publishers, Boston1998: 213-234Crossref Google Scholar, 17.Wood W.G. Schroeder F. Avdulov N.A. Chochina S.V. Igbavboa U. Lipids. 1999; 34: 225-234Crossref PubMed Scopus (106) Google Scholar, 18.Frolov A. Woodford J.K. Murphy E.J. Billheimer J.T. Schroeder F. J. Biol. Chem. 1996; 271: 16075-16083Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 19.Frolov A.A. Woodford J.K. Murphy E.J. Billheimer J.T. Schroeder F. J. Lipid. Res. 1996; 37: 1862-1874Abstract Full Text PDF PubMed Google Scholar), desorbs from membranes with very similar kinetics as does cholesterol, and both cholesterol and DHE are esterified in L-cells (2.Schroeder F. Frolov A.A. Murphy E.J. Atshaves B.P. Jefferson J.R. Pu L. Wood W.G. Foxworth W.B. Kier A.B. Proc. Soc. Exp. Biol. Med. 1996; 213: 150-177Crossref PubMed Scopus (132) Google Scholar). Finally, DHE readily incorporates into rat, rabbit, or human VLDL, LDL, and HDL either in vitro (10.Schroeder F. Goh E.H. Heimberg M. FEBS Lett. 1979; 97: 233-236Crossref PubMed Scopus (22) Google Scholar, 11.Schroeder F. Goh E.H. Heimberg M. J. Biol. Chem. 1979; 254: 2456-2463Abstract Full Text PDF PubMed Google Scholar,20.Smith R.J.M. Green C. Biochem. J. 1974; 137: 413-415Crossref PubMed Scopus (25) Google Scholar, 21.Bergeron R.J. Scott J. Anal. Chem. 1982; 119: 128-134Google Scholar) or in vivo (21.Bergeron R.J. Scott J. Anal. Chem. 1982; 119: 128-134Google Scholar, 22.Bergeron R.J. Scott J. J. Lipid Res. 1982; 23: 391-404Abstract Full Text PDF PubMed Google Scholar) to reflect the organization of cholesterol and its interactions with apoproteins therein.Despite the more than two decades wherein DHE was used to determine lipoprotein and membrane structure, only recently were confocal laser scanning microscopy (16.Schroeder F. Frolov A. Schoer J. Gallegos A. Atshaves B.P. Stolowich N.J. Scott A.I. Kier A.B. Chang T.Y. Freeman D.A. Intracellular Cholesterol Trafficking. Kluwer Academic Publishers, Boston1998: 213-234Crossref Google Scholar) and conventional fluorescence microscopy (23.Mukherjee S. Zha X. Tabas I. Maxfield F.R. Biophys. J. 1998; 75: 1915-1925Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar) used to directly visualize intracellular DHE distribution in living cells. It was concluded that DHE colocalized with cholesterol in living cells (23.Mukherjee S. Zha X. Tabas I. Maxfield F.R. Biophys. J. 1998; 75: 1915-1925Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). As both these studies pointed out, however, DHE has very significant disadvantages for use in conventional and confocal microscopy in that DHE requires excitation in the UV region (325 nm) of the spectrum where it becomes severely photobleached.In the present investigation the disadvantages of UV excitation for DHE were overcome through the use of multiphoton excitation and multiphoton laser scanning microscopy (MLSM). Furthermore, NBD-cholesterol (NBD-chol) proved to be an alternate fluorescent cholesterol analog to visualize HDL-mediated uptake and intracellular targeting of unesterified cholesterol in L-cell fibroblasts. NBD-chol is absorbed by the intestine of hamsters fed NBD-chol as well as by cultured Caco-2 cells (24.Sparrow C.P. Patel S. Baffic J. Chao Y.-S. Hernandez M. Lam M.-H. Montenegro J. Wright S.D. Detmers P.A. J. Lipid Res. 1999; 40: 1747-1757Abstract Full Text Full Text PDF PubMed Google Scholar). In contrast to DHE, the literature with regard to NBD-chol esterification is more complex. NBD-chol is esterified in vitro by acyl-CoA cholesterol acyltransferase of hamster intestinal microsomes (24.Sparrow C.P. Patel S. Baffic J. Chao Y.-S. Hernandez M. Lam M.-H. Montenegro J. Wright S.D. Detmers P.A. J. Lipid Res. 1999; 40: 1747-1757Abstract Full Text Full Text PDF PubMed Google Scholar), but not rat liver microsomes (25.Billheimer J.T. Gillies P.J. Esfahani M. Swaney J.B. Advances in Cholesterol Research. The Telford Press, Caldwell, NJ1990: 7-45Google Scholar). Furthermore, NBD-chol is esterified in hamster intestine and cultured Caco-2 intestinal cells (24.Sparrow C.P. Patel S. Baffic J. Chao Y.-S. Hernandez M. Lam M.-H. Montenegro J. Wright S.D. Detmers P.A. J. Lipid Res. 1999; 40: 1747-1757Abstract Full Text Full Text PDF PubMed Google Scholar).Based on the above findings, the fluorescence properties of DHE and NBD-chol as well as cultured L-cell fibroblasts, a cell line that grows in serum-free medium (26.Schroeder F. Perlmutter J.F. Glaser M. Vagelos P.R. J. Biol. Chem. 1976; 251: 6739-6746Abstract Full Text PDF PubMed Google Scholar), were used to (i) compare the uptake and esterification of these sterols in the same cell type, (ii) determine if uptake of either fluorescent sterol was characteristic of HDL receptor mediated uptake, (iii) resolve the specificity of intracellular targeting of fluorescent sterol, and (iv) begin to determine the molecular basis for trafficking of cholesterol to lipid droplets.DISCUSSIONAlthough LDL receptor-mediated lipid metabolism has been characterized in depth over the past two decades, much less is known regarding the role of HDL in unesterified cholesterol dynamics, especially in living cells (for review, see Refs. 5.Stangl H. Cao G. Wyne K.L. Hobbs H.H. J. Biol. Chem. 1998; 273: 31002-31008Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 51.Krieger M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4077-4080Crossref PubMed Scopus (114) Google Scholar, and 52.Fielding C.J. Bist A. Fielding P.E. Chang T.Y. Freeman D.A. Intracellular Cholesterol Trafficking. Kluwer Academic Publishers, Boston1998: 273-288Crossref Google Scholar). In contrast to LDL receptor-mediated lipid uptake, this “alternate” pathway utilizes a completely different receptor (SRB1 receptor instead of LDL receptor), has a different apoprotein specificity (binds HDL as well as LDL and VLDL), is mediated through a different plasma membrane microdomain (caveolae instead of clathrin-coated pits), does not internalize (endocytose) whole lipoprotein particles, and takes up lipids in the order: cholesterol esters ≫ phosphatidylserine > phosphatidylcholine = phosphatidylinositol > sphingomyelin (53.Rodrigueza W.V. Thuanhnai S.T. Temel R.E. Lund-Katz S. Phillips M.C. Williams D.L. J. Biol. Chem. 1999; 274: 20344-20350Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Although HDL mediates cholesterol ester uptake in nonplacental steroidogenic tissues as well as cultured cells (for review, see Refs. 4.Smart E.J. van der Westhuyzen D.R. Chang T.Y. Freeman D.A. Intracellular Cholesterol Trafficking. Kluwer Academic Publishers, Boston1998: 253-272Crossref Google Scholar and 54.Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (1986) Google Scholar) only recently was this visualized with fluorescent sterol ester (55.Reaven E. Tsai L. Azhar S. J. Biol. Chem. 1996; 271: 16208-16217Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). In contrast, while it is generally accepted that HDL mediates rapid efflux of cellular unesterified cholesterol, a process termed “reverse cholesterol transport,” very little is actually known of HDLs role in uptake and intracellular targeting of unesterified cholesterol, especially in living cells (for review, see Refs. 5.Stangl H. Cao G. Wyne K.L. Hobbs H.H. J. Biol. Chem. 1998; 273: 31002-31008Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar and 54.Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (1986) Google Scholar). The results of the present investigation provided several new insights on HDL-mediated uptake on unesterified sterol uptake.First, comparison of the extent as well as t 12 of uptake for the fluorescent sterols, DHE and NBD-chol, to that of [3H]cholesterol suggested that the two fluorescent sterols may selectively probe different uptake pathways. Although the uptake and half-time for maximal uptake of the fluorescent DHE resembled that of [3H]cholesterol, NBD-chol uptake was >100-fold more rapid, but >100-fold less efficient. Recent data showed that NBD-chol uptake and flux through hamster intestine as well as NBD-chol uptake in Caco-2 cells is also markedly faster than that of radiolabeled cholesterol (24.Sparrow C.P. Patel S. Baffic J. Chao Y.-S. Hernandez M. Lam M.-H. Montenegro J. Wright S.D. Detmers P.A. J. Lipid Res. 1999; 40: 1747-1757Abstract Full Text Full Text PDF PubMed Google Scholar). Although it was suggested that these differences are due to lower affinity of NBD-chol for a cholesterol transporter or decreased solubility of NBD-chol, the present data suggested otherwise. NBD-chol has very high affinity (nm) for lipid-binding proteins such as ADRP (present data) and sterol carrier protein-2 (16.Schroeder F. Frolov A. Schoer J. Gallegos A. Atshaves B.P. Stolowich N.J. Scott A.I. Kier A.B. Chang T.Y. Freeman D.A. Intracellular Cholesterol Trafficking. Kluwer Academic Publishers, Boston1998: 213-234Crossref Google Scholar, 56.Stolowich N. Frolov A. Petrescu A. Scott A.I. Billheimer J.T. Schroeder F. J. Biol. Chem. 1999; 274: 35425-35433Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Likewise, the critical micellar concentration of NBD-chol differs only 2-fold from that of DHE and cholesterol (27.Fischer R.T. Cowlen M.S. Dempsey M.E. Schroeder F. Biochemistry. 1985; 24: 3322-3331Crossref PubMed Scopus (66) Google Scholar, 57.Avdulov N.A. Chochina S.V. Igbavboa U. Warden C.H. Schroeder F. Wood W.G. Biochim. Biophys. Acta. 1999; 1437: 37-45Crossref PubMed Scopus (63) Google Scholar). The two major receptor-mediated sterol transport pathways differ markedly in speed: slow (15–45 min) LDL receptor-mediated (endocytic) uptake (for review, see Ref. 52.Fielding C.J. Bist A. Fielding P.E. Chang T.Y. Freeman D.A. Intracellular Cholesterol Trafficking. Kluwer Academic Publishers, Boston1998: 273-288Crossref Google Scholar); fast (1 min) HDL receptor-mediated efflux of cholesterol (for review, see Ref. 4.Smart E.J. van der Westhuyzen D.R. Chang T.Y. Freeman D.A. Intracellular Cholesterol Trafficking. Kluwer Academic Publishers, Boston1998: 253-272Crossref Google Scholar). These data, taken together with the lipoprotein (HDLversus LDL and VLDL) specificity of NBD-chol uptake shown herein and earlier for esterified cholesterol (for review, see Refs. 5.Stangl H. Cao G. Wyne K.L. Hobbs H.H. J. Biol. Chem. 1998; 273: 31002-31008Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar,53.Rodrigueza W.V. Thuanhnai S.T. Temel R.E. Lund-Katz S. Phillips M.C. Williams D.L. J. Biol. Chem. 1999; 274: 20344-20350Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 54.Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (1986) Google Scholar, 55.Reaven E. Tsai L. Azhar S. J. Biol. Chem. 1996; 271: 16208-16217Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, and 58.Acton S.L. Scherer P.E. Lodish H.F. Krieger M. J. Biol. Chem. 1994; 269: 21003-21009Abstract Full Text PDF PubMed Google Scholar) as well as the effects of filipin on NBD-chol uptake shown herein and on cholesterol ester uptake elsewhere (for review, see Refs. 47.Schnitzer J.E. Allard J. Oh P. Am. J. Physiol. 1995; 268: H48-H55Crossref PubMed Google Scholar, 48.Schnitzer J.E. Liu J. Oh P. J. Biol. Chem. 1995; 270: 14399-14404Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, and 55.Reaven E. Tsai L. Azhar S. J. Biol. Chem. 1996; 271: 16208-16217Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) were consistent with the uptake of the two fluorescent, unesterified sterols being mediated, at least in part, by different mechanism(s).Second, once internalized the DHE and NBD-chol appear to follow similar pathways to be esterified as compared with [3H]cholesterol. At equimolar concentrations these sterols differed less than <2-fold in esterification. L-cell esterification of DHE confirms an earlier study (2.Schroeder F. Frolov A.A. Murphy E.J. Atshaves B.P. Jefferson J.R. Pu L. Wood W.G. Foxworth W.B. Kier A.B. Proc. Soc. Exp. Biol. Med. 1996; 213: 150-177Crossref PubMed Scopus (132) Google Scholar) while that of NBD-chol clarifies a controversy in the literature (see Introduction). NBD-chol is esterified in vivo by hamster intestine and Caco-2 cells, derived from intestine, as well as in vitro by intestinal microsomes (24.Sparrow C.P. Patel S. Baffic J. Chao Y.-S. Hernandez M. Lam M.-H. Montenegro J. Wright S.D. Detmers P.A. J. Lipid Res. 1999; 40: 1747-1757Abstract Full Text Full Text PDF PubMed Google Scholar). In contrast, rat liver microsomes did not esterify NBD-chol (25.Billheimer J.T. Gillies P.J. Esfahani M. Swaney J.B. Advances in Cholesterol Research. The Telford Press, Caldwell, NJ1990: 7-45Google Scholar). The different observations may be due to differences in the substrate specificities of the two known acyl-CoA cholesterol acyltransferases (1 or 2). Acyl-CoA:cholesterolO-acyltransferase 1 is absent from intestinal cells while liver contains both acyl-CoA:cholesterol O-acyltransferase 1 and 2 (59.Yu C. Chen J. Lin S. Liu J. Chang C.C.Y. Chang T.-Y. J. Biol. Chem. 1999; 274: 36139-36145Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar).Third, once internalized the fluorescent sterols rapidly target to lipid droplets. LSCM and MLSM imaging, colocalization with Nile Red, and FRET all indicated specific targeting of the HDL-mediated unesterified sterol uptake to lipid droplets. This process was specific for the HDL-mediated uptake of unesterified (shown herein) and esterified sterols, but not DiI (55.Reaven E. Tsai L. Azhar S. J. Biol. Chem. 1996; 271: 16208-16217Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The speed (<30 s) of HDL-mediated NBD-chol targeting to lipid droplets was much faster than that of HDL-mediated uptake of BODIPY-cholesterol ester, 5 min (55.Reaven E. Tsai L. Azhar S. J. Biol. Chem. 1996; 271: 16208-16217Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). However, this very rapid (<30 s) intracellular transfer of unesterified NBD-chol was similar to that of endogenously synthesized cholesterol from the endoplasmic reticulum to the cell surface, 1 min (for review, see Ref. 4.Smart E.J. van der Westhuyzen D.R. Chang T.Y. Freeman D.A. Intracellular Cholesterol Trafficking. Kluwer Academic Publishers, Boston1998: 253-272Crossref Google Scholar), and implied a nonvesicular pathway(s). Several candidate intracellular cholesterol-binding proteins (sterol carrier protein-2, caveolin-1, steroidogenic acute regulatory protein, etc.) for such a nonvesicular mechanism have been proposed (for review, see Refs. 4.Smart E.J. van der Westhuyzen D.R. Chang T.Y. Freeman D.A. Intracellular Cholesterol Trafficking. Kluwer Academic Publishers, Boston1998: 253-272Crossref Google Scholar and 16.Schroeder F. Frolov A. Schoer J. Gallegos A. Atshaves B.P. Stolowich N.J. Scott A.I. Kier A.B. Chang T.Y. Freeman D.A. Intracellular Cholesterol Trafficking. Kluwer Academic Publishers, Boston1998: 213-234Crossref Google Scholar).Fourth, the specific targeting of unesterified cholesterol to lipid droplets may be mediated, at least in part by ADRP, a lipid droplet specific protein (49.Jiang H.P. Serrero G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7856-7860Crossref PubMed Scopus (226) Google Scholar). ADRP in L-cells was localized on the surface of lipid droplets as in other cell types (43.Brasaemle D.L. Barber T. Wolins N. Serrero G. Blanchette-Mackie E.J. Londos C. J. Lipid Res. 1997; 38: 2249-2263Abstract Full Text PDF PubMed Google Scholar). More important, indirect immunofluorescence revealed that the ADRP was highly colocalized with NBD-chol in the lipid droplet. Finally, ADRP specifically bound NBD-chol with high affinity (K d = 2 nm), suggesting that this colocalization was due at least in part to direct binding of NBD-chol ADRP. This affinity was in the same range of that of other known sterol-binding proteins, e.g. sterol carrier protein-2 with K d = 6–11 nm(16.Schroeder F. Frolov A. Schoer J. Gallegos A. Atshaves B.P. Stolowich N.J. Scott A.I. Kier A.B. Chang T.Y. Freeman D.A. Intracellular Cholesterol Trafficking. Kluwer Academic Publishers, Boston1998: 213-234Crossref Google Scholar, 57.Avdulov N.A. Chochina S.V. Igbavboa U. Warden C.H. Schroeder F. Wood W.G. Biochim. Biophys. Acta. 1999; 1437: 37-45Crossref PubMed Scopus (63) Google Scholar). Since ADRP has been shown to also bind fatty acid (50.Gao J. Serrero G. J. Biol. Chem. 1999; 274: 16825-16830Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar),2 this would indicate that ADRP can bind several types of lipid substrates, similar to sterol carrier protein-2 (30.Schroeder F. Myers-Payne S.C. Billheimer J.T. Wood W.G. Biochemistry. 1995; 34: 11919-11927Crossref PubMed Scopus (97) Google Scholar, 56.Stolowich N. Frolov A. Petrescu A. Scott A.I. Billheimer J.T. Schroeder F. J. Biol. Chem. 1999; 274: 35425-35433Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 57.Avdulov N.A. Chochina S.V. Igbavboa U. Warden C.H. Schroeder F. Wood W.G. Biochim. Biophys. Acta. 1999; 1437: 37-45Crossref PubMed Scopus (63) Google Scholar,60.Frolov A. Cho T.H. Billheimer J.T. Schroeder F. J. Biol. Chem. 1996; 271: 31878-31884Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 61.Colles S.M. Woodford J.K. Moncecchi D. Myers-Payne S.C. McLean L.R. Billheimer J.T. Schroeder F. Lipids. 1995; 30: 795-804Crossref PubMed Scopus (61) Google Scholar, 62.Stolowich N.J. Frolov A. Atshaves B.P. Murphy E. Jolly C.A. Billheimer J.T. Scott A.I. Schroeder F. Biochemistry. 1997; 36: 1719-1729Crossref PubMed Scopus (62) Google Scholar).In summary, the data presented herein were consistent with DHE and NBD-chol uptake preferentially taking place by different pathways,e.g. LDL receptor endocytic versus HDL receptor molecular transfer via caveolae. Furthermore, both DHE and NBD-chol demonstrated specific targeting of unesterified sterol to lipid droplets, a process that was very rapid and potentially mediated by ADRP, a lipid-binding protein specific to droplets. Thus, these data provided basic new observations contributing to our understanding of HDL receptor-mediated uptake of unesterified cholesterol, its intracellular dynamics, and its targeting to lipid droplets, an organelle about which very little is known (43.Brasaemle D.L. Barber T. Wolins N. Serrero G. Blanchette-Mackie E.J. Londos C. J. Lipid Res. 1997; 38: 2249-2263Abstract Full Text PDF PubMed Google Scholar, 45.Londos C. Brasaemle D.L. Schultz C.J. Segrest J. Kimmel A.R. Cell Dev. Biol. 1999; 10: 51-58Crossref PubMed Scopus (365) Google Scholar, 49.Jiang H.P. Serrero G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7856-7860Crossref PubMed Scopus (226) Google Scholar). Because of cholesterol's dual role in both normal cell function and the pathobiology of atherosclerosis, it is essential to resolve the mechanisms whereby exogenous cholesterol is taken up and distributed within the cell (reviewed in Refs. 1.Schroeder F. Woodford J.K. Kavecansky J. Wood W.G. Joiner C. Mol. Membr. Biol. 1995; 12: 113-119Crossref PubMed Scopus (111) Google Scholar, 2.Schroeder F. Frolov A.A. Murphy E.J. Atshaves B.P. Jefferson J.R. Pu L. Wood W.G. Foxworth W.B. Kier A.B. Proc. Soc. Exp. Biol. Med. 1996; 213: 150-177Crossref PubMed Scopus (132) Google Scholar, 3.Fielding C.J. Fielding P.E. J. Lipid. 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