Uptake of long-chain fatty acids in HepG2 cells involves caveolae

We investigated the role of caveolae in uptake and intracellular trafficking of long chain fatty acids (LCFA) in HepG2 human hepatoma cells. The uptake of [3H]oleic acid and [3H]stearic acid into HepG2 cells was measured by radioactive assays and internalization of the non-metabolizable fluorescent fatty acid 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] (12-NBD) stearate into single HepG2 cells was semi-quantitatively assessed by laser scanning microscopy. The initial rate of [3H]oleic acid uptake (V0) in HepG2 cells exhibited saturable transport kinetics with increasing concentrations of free oleic acid (Vmax 854 ± 46 pmol mg protein−1 min−1, Km 100 ± 14 nmol/l). While inhibition of clathrin coated pits did not influence LCFA uptake in HepG2, inhibition of caveolae formation by filipin III, cyclodextrin, and caveolin-1 antisense oligonucleotides resulted in reduction of [3H]oleic acid uptake by 54%, 45%, and 23%, respectively. Furthermore, filipin III inhibited the uptake of [3H]stearic acid and its fluorescent derivative 12-NBD stearate by 44% and 50%, respectively. Transfection studies with α-caveolin-1/cyanofluorescent protein chimeras showed significant colocalization of caveolae and internalized 12-NBD stearate.In conclusion, these data suggest a significant role for caveolae mediated uptake and intracellular trafficking of LCFA in HepG2 cells. We investigated the role of caveolae in uptake and intracellular trafficking of long chain fatty acids (LCFA) in HepG2 human hepatoma cells. The uptake of [3H]oleic acid and [3H]stearic acid into HepG2 cells was measured by radioactive assays and internalization of the non-metabolizable fluorescent fatty acid 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] (12-NBD) stearate into single HepG2 cells was semi-quantitatively assessed by laser scanning microscopy. The initial rate of [3H]oleic acid uptake (V0) in HepG2 cells exhibited saturable transport kinetics with increasing concentrations of free oleic acid (Vmax 854 ± 46 pmol mg protein−1 min−1, Km 100 ± 14 nmol/l). While inhibition of clathrin coated pits did not influence LCFA uptake in HepG2, inhibition of caveolae formation by filipin III, cyclodextrin, and caveolin-1 antisense oligonucleotides resulted in reduction of [3H]oleic acid uptake by 54%, 45%, and 23%, respectively. Furthermore, filipin III inhibited the uptake of [3H]stearic acid and its fluorescent derivative 12-NBD stearate by 44% and 50%, respectively. Transfection studies with α-caveolin-1/cyanofluorescent protein chimeras showed significant colocalization of caveolae and internalized 12-NBD stearate. In conclusion, these data suggest a significant role for caveolae mediated uptake and intracellular trafficking of LCFA in HepG2 cells. Long-chain fatty acids (LCFAs) serve a number of important biological functions as energy substrates (1Distel R.J. Robinson G.S. Spiegelman B.M. Fatty acid regulation of gene expression.J. Biol. Chem. 1992; 267: 5937-5941Google Scholar), as precursors for glyco- and phospholipid components of cell membranes (2Amri W-Z. Ailhaud G. Grimaldi P.A. Fatty acids as signal transducing molecules: involvement in the differentiation of preadipose to adipose cells.J. Lipid Res. 1994; 35: 930-937Google Scholar), as biological mediators like eicosanoids (3Noy N. Zakim D. Physical chemical basis for the uptake of organic compounds by cells.in: Tavoloni N. Berk P.D. Hepatic Transport and Bile Secretion. Raven Press, New York1993: 313-335Google Scholar), and as mediators of cellular processes (e.g., gene expression and growth regulation) (1Distel R.J. Robinson G.S. Spiegelman B.M. Fatty acid regulation of gene expression.J. Biol. Chem. 1992; 267: 5937-5941Google Scholar, 2Amri W-Z. Ailhaud G. Grimaldi P.A. Fatty acids as signal transducing molecules: involvement in the differentiation of preadipose to adipose cells.J. Lipid Res. 1994; 35: 930-937Google Scholar). These multiple roles suggest that careful regulation of cellular uptake and utilization of LCFA is essential to cellular homeostasis. However, the general issue of cellular LCFA uptake was controversial until today. Passage of LCFA across cell membranes has traditionally been considered to occur by passive diffusion through the membrane bilayer, with the transmembrane gradient being the driving force for the direction of net movement. Studies in model membrane systems (4Hamilton J.A. Kamp F. How are free fatty acids transported in membranes? Is it by proteins or by free diffusion through the lipids?.Diabetes. 1999; 48: 2255-2269Google Scholar, 5Kleinfeld A.M. Lipid phase fatty acid flip-flop, is it fast enough for cellular transport?.J. Membr. Biol. 2000; 175: 79-86Google Scholar, 6Zakim D. Thermodynamics of fatty acid transfer.J. Membr. Biol. 2000; 176: 101-109Google Scholar) have shown that the protonized LCFA species can cross a protein-free phospholipid bilayer rapidly by a flip-flop mechanism. The diffusion hypothesis is challenged by the identification of a number of membrane-associated fatty acid binding proteins (7Stremmel W. Berk P.D. Hepatocellular influx of [14C] oleate reflects membrane transport rather than intracellular metabolism or binding.Proc. Natl. Acad. Sci. USA. 1986; 83: 3086-3090Google Scholar, 8Stremmel W. Strohmeyer G. Borchard F. Kochwa S. Berk P.D. Isolation and characterization of a fatty acid binding protein in rat liver plasma membranes.Proc. Natl. Acad. Sci. USA. 1985; 82: 4-8Google Scholar, 9Sorrentino D. Berk P.D. Free fatty acids and the sinusoidal plasma membrane: Concepts, trends and controversies.in: Tavoloni N. Berk P.D. Hepatic Transport and Bile Secretion. Raven Press, New York1993: 197-210Google Scholar, 10Trigatti B.L. Anderson R.G.W. Gerber G.E. Identification of caveolin-1 as a fatty acid binding protein.Biochem. Biophys. Res. Commun. 1999; 255: 34-39Google Scholar, 11Abumrad N.A. El-Maghrabi M.R. Amri E.Z. Lopez E. Grimaldi P.A. Cloning of rat adipocyte membrane-protein implicated in binding or transport of long chain fatty acids that is induced during preadipocyte differentiation: homology with human CD 36.J. Biol. Chem. 1993; 268: 17665-17668Google Scholar) that might be involved in facilitated fatty acid uptake: fatty acid translocase (FAT/CD36), the fatty acid transport protein (FATP) family, plasma membrane fatty acid binding protein (FABPpm), and caveolin-1. The latter is the 22 kDa major structural protein of caveolae. Since caveolin-1 was shown to have a fatty acid binding site that binds fatty acids saturably with high affinity (10Trigatti B.L. Anderson R.G.W. Gerber G.E. Identification of caveolin-1 as a fatty acid binding protein.Biochem. Biophys. Res. Commun. 1999; 255: 34-39Google Scholar), we investigated involvement of caveolin-1 and caveolae in LCFA uptake and transport in HepG2 cells. Caveolae are 50–100 nm flask-shaped invaginations of the plasma membrane that are biochemically characterized by their enrichment in cholesterol, glycolipids, and caveolin. They participate in several crucial cellular functions such as signal transduction (12Lisanti M.P. Scherer P.E. Tang Z-L. Sargiacomo M. Caveolae, caveolin and caveolin-rich membrane domains: a signalling hypothesis.Trends Cell Biol. 1994; 4: 231-235Google Scholar), regulation of glucose uptake (13Ros-Baró A. Lopez-Iglesias C. Peiro S. Bellido D. Palacin M. Zorzano A. Camps M. Lipid rafts are required for GLUT4 internalizationin adipose cells.Proc. Natl. Acad. Sci. USA. 2001; 98: 12050-12055Google Scholar), cholesterol transport (14Fielding P.E. Fielding C.J. Intracellular transport of low density lipoprotein derived free cholesterol begins at clathrin-coated pits and terminates at cell surface caveolae.Biochemistry. 1996; 35: 14932-14938Google Scholar), potocytosis (15Anderson R.G.W. Potocytosis of small molecules and ions by caveolae.Trends Cell Biol. 1993; 3: 69-72Google Scholar), and fluid-phase and receptor-mediated endocytosis (16Schnitzer J.E. Oh P. Pinney E. Allard J. Filipin III-sensitive caveolae-mediated transport in endothelium: reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules.J. Cell Biol. 1994; 127: 1217-1232Google Scholar, 17Parton R.G. Joggerst B. Simons K. Regulated internalization of caveolae.J. Cell Biol. 1994; 127: 1199-1215Google Scholar). Several studies have provided evidence for the internalization and trafficking of different ligands through caveolae to specific target sites, e.g., endosomes (18Pol A. Lu A. Pons M. Peiro S. Enrich C. Epidermal growth factor-mediated caveolin recruitment to early endosomes and MAPK activation.J. Biol. Chem. 2000; 275: 30566-30572Google Scholar), or the Golgi apparatus (19Conrad P.A. Smart E.J. Ying Y.S. Anderson R.G.W. Bloom G.S. Caveolin cycles between plasma membrane caveolae and the Golgi complex by microtubule-dependent and microtubule-independent steps.J. Cell Biol. 1995; 131: 1421-1433Google Scholar). Recently, it was reported that in the hepatocyte, caveolae are mainly localized to the sinusoidal blood-facing plasma membrane (20Pol A. Calvo M. Lu A. Enrich C. The “early-sorting” endocytic compartment of rat hepatocytes is involved in the intracellular pathway of caveolin-1 (VIP-21).Hepatology. 1999; 29: 1848-1857Google Scholar, 21Calvo M. Enrich C. Biochemical analysis of a caveolae-enriched plasma membrane fraction from rat liver.Electrophoresis. 2000; 21: 3386-3395Google Scholar) representing dynamic structures that cycle between the cell surface and the endocytic compartment (20Pol A. Calvo M. Lu A. Enrich C. The “early-sorting” endocytic compartment of rat hepatocytes is involved in the intracellular pathway of caveolin-1 (VIP-21).Hepatology. 1999; 29: 1848-1857Google Scholar). Research on the role of caveolae in transport processes has been significantly facilitated by the finding that this pathway can be selectively inhibited by cholesterol depletion (16Schnitzer J.E. Oh P. Pinney E. Allard J. Filipin III-sensitive caveolae-mediated transport in endothelium: reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules.J. Cell Biol. 1994; 127: 1217-1232Google Scholar) and by modulation of caveolin-1 expression (22Engelman J.A. Wycoff C.C. Yasuhara S. Song K.S. Okamoto T. Lisanti M.P. Recombinant expression of caveolin-1 in cells Biol. Chem. Scholar, A.M. E. Simons K. R.G. formation of caveolae in by expression of Natl. Acad. Sci. USA. 1995; Scholar). The involvement of caveolae in LCFA transport has not been on in on and caveolae inhibition studies as as the of LCFA transport suggest that in HepG2 caveolae be in LCFA uptake and intracellular We HepG2 cells in as this cell has been shown to be a model to fatty acid uptake and metabolism M. D. Berk P.D. fatty acid uptake and plasma membrane expression and of in HepG2 Scholar, A. Lipid in HepG2 and Lipid Res. 1994; 35: Scholar). acid and from [3H]oleic acid and [3H]stearic acid from was from was from Fatty cyclodextrin, filipin III, and oleic acid and stearate from from was from The caveolin-1 was from The was from the HepG2 cells to in with The [3H]oleic acid uptake assays as W. Strohmeyer G. Berk P.D. Hepatocellular uptake of oleate is energy and inhibited by to a plasma membrane fatty acid binding Natl. Acad. Sci. USA. 1986; 83: Scholar) HepG2 cell of [3H]oleic acid with measured of oleic acid in a at different from to of the with cell in a at The uptake was by of the by of of The was and the by in was to the and of the for protein and was the of The in a The concentrations of fatty acids in [3H]oleic oleic from to by the of and P.A. of in plasma acid as Scholar), the for the oleic complex reported by J.E. of free fatty acid binding to by of Scholar). with was as by J.E. F. J. and of and in cells.J. Biol. Chem. Scholar) for or filipin of cells. The of [3H]oleic acid into cellular was of [3H]oleic with HepG2 as Cell in of The and of to The phase was a of in of and to The was acid by of was into a for of laser scanning has been by in C. W. analysis of fatty acid J. 1995; Scholar). uptake studies in and the and stearate concentrations the stearate 12-NBD stearate and stearate. for at in filipin III filipin the on the of the and with 12-NBD stearate at a rate of HepG2 cells with a by microscopy. The was to the and a was with the nm of the laser for uptake studies with 12-NBD stearate. The was by a a nm to nm and The in intracellular by cellular 12-NBD stearate uptake was as with being the of The in a of the was the in to a of 12-NBD stearate antisense oligonucleotides to human caveolin-1 and antisense oligonucleotides to human clathrin have been and by The as Transfection of oligonucleotides was and of to the of intracellular uptake of antisense into HepG2 cells was by caveolin-1 and clathrin oligonucleotides in of HepG2 cells with antisense oligonucleotides was for at the reduction of caveolin-1 or clathrin protein by antisense was assessed by of cell protein on and as HepG2 cells in and and The protein was by the of cell protein by and to was to and chain a at a of and a from binding was the fluorescent protein was provided by of was from this and to protein the Transfection of the was and of as by the HepG2 cells and with or with 12-NBD stearate stearate by for with with a a and fluorescent HepG2 cells that protein in and in The was for at a of at by with The was for at in was by a are as ± was to for significant and to the of free oleic acid on the rate of initial uptake and to the model that the that HepG2 cells the of hepatocytes with to LCFA we uptake of [3H]oleic acid at concentrations of free oleic acid in the The oleic acid was its by of a of and increasing concentrations of [3H]oleic The initial rate of uptake was by from uptake measured the initial The are 854 ± 46 pmol mg protein−1 and Km 100 ± 14 is to the kinetics of initial [3H]oleic acid uptake by hepatocytes W. Strohmeyer G. Berk P.D. Hepatocellular uptake of oleate is energy and inhibited by to a plasma membrane fatty acid binding Natl. Acad. Sci. USA. 1986; 83: Scholar). the HepG2 cell model for LCFA cells with different transport and to influence LCFA transport in a HepG2 cells with of and [3H]oleic acid for with or reduced [3H]oleic acid uptake to ± and ± of of the in rat and a of cellular transport reduced the uptake of [3H]oleic acid to ± and ± of not inhibition is with reported of LCFA uptake by human hepatocytes (7Stremmel W. Berk P.D. Hepatocellular influx of [14C] oleate reflects membrane transport rather than intracellular metabolism or binding.Proc. Natl. Acad. Sci. USA. 1986; 83: 3086-3090Google Scholar, W. Strohmeyer G. Berk P.D. Hepatocellular uptake of oleate is energy and inhibited by to a plasma membrane fatty acid binding Natl. Acad. Sci. USA. 1986; 83: Scholar). these that HepG2 cells the of hepatocytes with to LCFA is a crucial required to the structural of caveolae. of cells to filipin III, a cholesterol from the plasma of caveolae (16Schnitzer J.E. Oh P. Pinney E. Allard J. Filipin III-sensitive caveolae-mediated transport in endothelium: reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules.J. Cell Biol. 1994; 127: 1217-1232Google Scholar, J.E. C. Ying Anderson R.G.W. a protein of caveolae membrane 1992; Scholar). We investigated the of filipin III on LCFA uptake in HepG2 cells. of with filipin III [3H]oleic acid was significantly reduced was increasing that filipin III with a uptake While in cells the of [3H]oleic acid uptake was filipin III resulted in a characterized by a the uptake of [3H]oleic acid was inhibited by and of that filipin III uptake of we investigated the uptake of [3H]stearic acid filipin III and a 44% reduction of ± pmol mg protein−1 in the filipin cells with ± pmol mg protein−1 in the In the of filipin III on LCFA uptake was in the uptake phase with the initial uptake phase we to the filipin III LCFA uptake by radioactive analysis of intracellular that ± of the [3H]oleic acid was to and complex ± was as free oleic acid was are ± of to of (7Stremmel W. Berk P.D. Hepatocellular influx of [14C] oleate reflects membrane transport rather than intracellular metabolism or binding.Proc. Natl. Acad. Sci. USA. 1986; 83: 3086-3090Google Scholar), the intracellular of not influx of is the net of and is that filipin III might its on intracellular LCFA by with LCFA metabolism (e.g., by for of internalized LCFA of the cells. However, in the of filipin III of ± of the [3H]oleic acid and ± as are ± of as HepG2 cells. of we assessed the uptake of 12-NBD a fluorescent LCFA derivative that is into Hepatic transport of a fluorescent stearate driving in rat J. Scholar, The cellular fatty acid binding of and Scholar). analysis showed that 12-NBD stearate was not of with HepG2 cells not The uptake of 12-NBD stearate the as the uptake of LCFA C. W. analysis of fatty acid J. 1995; Scholar). C. W. analysis of fatty acid J. 1995; Scholar) a of and the uptake of 12-NBD stearate by hepatocytes on a We this to the of in a of in a single cell as 12-NBD stearate and acid by a of is in The in cell the cellular influx of 12-NBD stearate. However, of the of the the of a with filipin III, the uptake of 12-NBD stearate was inhibited by a as [3H]oleic acid and [3H]stearic acid We the of the of filipin III on cells. HepG2 cell with filipin III for and for in cell to membrane HepG2 through the [3H]oleic acid that this resulted in of [3H]oleic acid uptake in cells. We the of cholesterol depletion on LCFA uptake by of HepG2 cells with cyclodextrin, to selectively caveolae by cholesterol from the plasma membrane cholesterol mediated by of and of Biol. Chem. 1996; Scholar, S. M. P. depletion caveolae and signalling for not for protein Biol. Chem. 2001; Scholar). with for a reduction of [3H]oleic acid depletion not internalization of ligands clathrin-coated pits (16Schnitzer J.E. Oh P. Pinney E. Allard J. Filipin III-sensitive caveolae-mediated transport in endothelium: reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules.J. Cell Biol. 1994; 127: 1217-1232Google Scholar) or in filipin III the of is by clathrin-coated B. Robinson and Cell Biol. Scholar). clathrin-coated pits is selectively reduced by depletion of intracellular or with K. B. and of and Cell Biol. 1993; Scholar). The uptake of [3H]oleic acid in HepG2 cells was not inhibited these In the suggest that caveolae not clathrin-coated are in the uptake of [3H]oleic acid by HepG2 cells. expression is important for the formation of caveolae (22Engelman J.A. Wycoff C.C. Yasuhara S. Song K.S. Okamoto T. Lisanti M.P. Recombinant expression of caveolin-1 in cells Biol. Chem. Scholar, A.M. E. Simons K. R.G. formation of caveolae in by expression of Natl. Acad. Sci. USA. 1995; Scholar). In to of we HepG2 cells with antisense oligonucleotides to human caveolin-1. resulted in a reduction of cellular caveolin-1 protein as assessed by and scanning to a reduction of [3H]oleic acid uptake ± pmol mg protein−1 and ± pmol mg protein−1 in cells with caveolin-1 antisense and are ± of inhibition is than that by filipin III and However, the reduction of caveolin-1 protein to 50%, a of caveolae might have been in these the of oleic acid uptake inhibition by antisense with the The as and the caveolin-1 protein with [3H]oleic acid with antisense to clathrin resulted in a reduction of cellular clathrin did not in inhibition of [3H]oleic acid uptake ± pmol mg protein−1 in HepG2 cells with clathrin antisense ± pmol mg protein−1 in the intracellular trafficking and of we chimeras into HepG2 cells. was as small single in the caveolin-1 in cells was by to caveolin-1 it showed a with the in cells with structures than representing caveolin-1 Transfection of resulted in of the and the involvement of caveolin-1 in intracellular trafficking of 12-NBD cells with with a of 12-NBD stearate and stearate for in the of at 12-NBD caveolae showed a high of colocalization 12-NBD stearate of intracellular membranes and of the fatty acids in and significantly the plasma of and 12-NBD stearate HepG2 cells with with 12-NBD stearate for and for analysis by the of 12-NBD stearate and a high of of and 12-NBD intracellular small and a caveolin-1 cell the and the fluorescent is the fluorescent the of the and a HepG2 cell that was with 12-NBD stearate for caveolin-1 While is specific fluorescent the fluorescent is the for In the of the fluorescent systems was a caveolin-1 cell the and the fluorescent is the fluorescent the of the a HepG2 cell that was with 12-NBD stearate for caveolin-1 While is specific fluorescent the fluorescent is the for the with 12-NBD stearate was for of in the of these structures was as assessed by the fluorescent However, 12-NBD stearate is not it is to the We that 12-NBD stearate is not internalized into is with the membrane of the and transport are important functions for are to that has a role in and different molecules from the and to In this we for the data of caveolae to the uptake and intracellular trafficking of LCFA in HepG2 cells. Furthermore, we evidence that HepG2 cells a and model to LCFA uptake processes in The evidence is on and caveolae inhibition studies as as the of LCFA binding to intracellular caveolae is the important of caveolae required to structural of these membrane to (e.g., filipin III and cholesterol from the plasma of caveolae and of in caveolae (16Schnitzer J.E. Oh P. Pinney E. Allard J. Filipin III-sensitive caveolae-mediated transport in endothelium: reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules.J. Cell Biol. 1994; 127: 1217-1232Google Scholar, J.E. C. Ying Anderson R.G.W. a protein of caveolae membrane 1992; Scholar, cholesterol mediated by of and of Biol. Chem. 1996; Scholar, D. R.G. of caveolin and caveolae by cholesterol in cells.J. Lipid Res. Scholar). inhibition with filipin III and suggest a caveolae-mediated LCFA uptake pathway in HepG2 cells that is in the uptake acid was significantly reduced by inhibition of caveolae formation by antisense to caveolin-1. The inhibition of the of the non-metabolizable fatty acid derivative 12-NBD stearate uptake by filipin III to a as the inhibition of fatty In filipin III did not LCFA metabolism in HepG2 cells. that filipin inhibition of LCFA uptake is not to of filipin III with LCFA and 12-NBD stearate to be a for LCFA uptake internalization by HepG2 12-NBD stearate significantly with the of evidence a role of caveolae for LCFA uptake and transport in In to clathrin-coated not to a role in this as depletion of intracellular or inhibition of [3H]oleic acid uptake by HepG2 cells. specific might caveolae of LCFA uptake and Caveolae a specific of membrane proteins that can and transport molecules like fatty acids and a protein that is involved in fatty acid uptake N.A. El-Maghrabi M.R. Amri E.Z. Lopez E. Grimaldi P.A. Cloning of rat adipocyte membrane-protein implicated in binding or transport of long chain fatty acids that is induced during preadipocyte differentiation: homology with human CD 36.J. Biol. Chem. 1993; 268: 17665-17668Google Scholar), and scavenger a that uptake of cholesterol in caveolae J. B. A. Smart E.J. Anderson R.G. S. M. a high density lipoprotein that is and fatty and with plasma membrane caveolae.J. Biol. Chem. Scholar, T. T. The expression of scavenger and caveolin-1 in and liver Res. Scholar). caveolin-1 can saturably fatty acids with high affinity (10Trigatti B.L. Anderson R.G.W. Gerber G.E. Identification of caveolin-1 as a fatty acid binding protein.Biochem. Biophys. Res. Commun. 1999; 255: 34-39Google Scholar, G.E. D. B.L. Identification of high affinity fatty proteins a fatty 1993; Scholar) and has a in the regulation of influx and of cholesterol (14Fielding P.E. Fielding C.J. Intracellular transport of low density lipoprotein derived free cholesterol begins at clathrin-coated pits and terminates at cell surface caveolae.Biochemistry. 1996; 35: 14932-14938Google Scholar, P.A. Smart E.J. Ying Y.S. Anderson R.G.W. Bloom G.S. Caveolin cycles between plasma membrane caveolae and the Golgi complex by microtubule-dependent and microtubule-independent steps.J. Cell Biol. 1995; 131: 1421-1433Google Scholar, D. R.G. of caveolin and caveolae by cholesterol in cells.J. Lipid Res. Scholar, E.J. Ying Y.S. P.A. Anderson R.G.W. Caveolin from caveolae to the Golgi apparatus in to cholesterol Cell Biol. 1994; 127: Scholar). from specific membrane the of cholesterol and into caveolae a to the membrane that it from the of the phospholipid membrane E.J. J.A. Scherer P.E. Okamoto T. Lisanti M.P. and signal Biol. 1999; Scholar, K. E. rafts in cell Scholar). is not this flip-flop of Gerber by and Fatty acid in that LCFA into the caveolae The of the the is in of the LCFA by into the plasma membrane and passive diffusion across the However, these are and the of caveolae-mediated uptake of LCFA data studies that LCFA influx in HepG2 The initial uptake of LCFA of is inhibited by and a protein is to and However, the filipin III pathway with and of the uptake In the are and controversial data on the and of caveolae mediated by J. A. endocytosis of a pathway to the Cell Biol. 2001; 3: Scholar) showed that was internalized by caveolae and transported into with a of is not or a specific binding protein can influence uptake by that the caveolae from the plasma membrane as by J. A. endocytosis of a pathway to the Cell Biol. 2001; 3: Scholar). on we that at of LCFA uptake in HepG2 a filipin III in the of and a caveolae-mediated that The of LCFA caveolae with uptake be derived from data filipin III might not caveolae as reported by and (18Pol A. Lu A. Pons M. Peiro S. Enrich C. Epidermal growth factor-mediated caveolin recruitment to early endosomes and MAPK activation.J. Biol. Chem. 2000; 275: 30566-30572Google Scholar) growth and filipin III pathway of caveolae internalization in from a role of caveolae for LCFA data suggest the involvement of caveolae in intracellular LCFA The of fatty acids in the of the specific intracellular trafficking to LCFA to for studies suggest that a significant of internalized 12-NBD stearate was localized to caveolae of of the fluorescent to have on from caveolae intracellular like Recently, and The the of fatty acids in 2001; Scholar) investigated intracellular trafficking of 12-NBD stearate in hepatocytes by a laser and that transport of 12-NBD stearate by diffusion with evidence for They binding of 12-NBD stearate to membranes and that fatty acid binding protein trafficking by the of fatty acids to However, their did not the fraction of and have this transport pathway in In conclusion, data a role of caveolae for cellular uptake and intracellular trafficking of The of LCFA in the membrane The for and for We are to of for the of was by the the of to and and by a to protein fatty acid translocase fatty acid binding protein fatty acid transport protein fatty acids laser scanning 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]