Adipose Differentiation Related Protein (ADRP) Expressed in Transfected COS-7 Cells Selectively Stimulates Long Chain Fatty Acid Uptake
1999; Elsevier BV; Volume: 274; Issue: 24 Linguagem: Inglês
10.1074/jbc.274.24.16825
ISSN1083-351X
Autores Tópico(s)Lipid metabolism and biosynthesis
ResumoAdipose differentiation related protein (ADRP) is a 50-kDa novel protein cloned from a mouse 1246 adipocyte cDNA library, rapidly induced during adipocyte differentiation. We have examined ADRP function, and we show here that ADRP facilitates fatty acid uptake in COS cells transfected with ADRP cDNA. We demonstrate that uptake of long chain fatty acids was significantly stimulated in a time-dependent fashion in ADRP-expressing COS-7 cells compared with empty vector-transfected control cells. Oleic acid uptake velocity increased significantly in a dose-dependent manner in ADRP-expressing COS-7 cells compared with control cells. The transport K m was 0.051 μm, andV max was 57.97 pmol/105 cells/min in ADRP-expressing cells, and K m was 0.093 μm and V max was 20.13 pmol/105 cells/min in control cells. The oleate uptake measured at 4 °C was only 10% that at 37 °C. ADRP also stimulated uptake of palmitate and arachidonate but had no effect on uptake of medium chain fatty acid such as octanoic acid and glucose. These data suggest that ADRP specifically enhances uptake of long chain fatty acids by increasing the initial rate of uptake and provide novel information about ADRP function as a saturable transport component for long chain fatty acids. Adipose differentiation related protein (ADRP) is a 50-kDa novel protein cloned from a mouse 1246 adipocyte cDNA library, rapidly induced during adipocyte differentiation. We have examined ADRP function, and we show here that ADRP facilitates fatty acid uptake in COS cells transfected with ADRP cDNA. We demonstrate that uptake of long chain fatty acids was significantly stimulated in a time-dependent fashion in ADRP-expressing COS-7 cells compared with empty vector-transfected control cells. Oleic acid uptake velocity increased significantly in a dose-dependent manner in ADRP-expressing COS-7 cells compared with control cells. The transport K m was 0.051 μm, andV max was 57.97 pmol/105 cells/min in ADRP-expressing cells, and K m was 0.093 μm and V max was 20.13 pmol/105 cells/min in control cells. The oleate uptake measured at 4 °C was only 10% that at 37 °C. ADRP also stimulated uptake of palmitate and arachidonate but had no effect on uptake of medium chain fatty acid such as octanoic acid and glucose. These data suggest that ADRP specifically enhances uptake of long chain fatty acids by increasing the initial rate of uptake and provide novel information about ADRP function as a saturable transport component for long chain fatty acids. Long chain non-esterified free fatty acids (FA) 1The abbreviations used are: FA, fatty acids; ADRP, adipose differentiation related protein; BSA, fatty acid-free bovine serum albumin; DME-F12, 1 to 1 mixture of Dulbecco's modified Eagle's and Ham's F12 media; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; KRP, Krebs-Ringer phosphate buffer; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; FBS, fetal bovine serum; FAT, fatty acid translocase; FATP, fatty acid transport protein; FABPpm, plasma membrane fatty acid-binding protein 1The abbreviations used are: FA, fatty acids; ADRP, adipose differentiation related protein; BSA, fatty acid-free bovine serum albumin; DME-F12, 1 to 1 mixture of Dulbecco's modified Eagle's and Ham's F12 media; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; KRP, Krebs-Ringer phosphate buffer; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; FBS, fetal bovine serum; FAT, fatty acid translocase; FATP, fatty acid transport protein; FABPpm, plasma membrane fatty acid-binding protein and their derivatives have multiple functions as either essential components of membrane, efficient sources of energy, or important effective molecules that regulate metabolism and mediate gene expression (1Distel R.J. Robinson G.S. Spiegelman B.M. J. Biol. Chem. 1992; 267: 5937-5941Abstract Full Text PDF PubMed Google Scholar, 2Amri E.-Z Ailhaud G. Grimaldi P.A. J. Lipid Res. 1994; 35: 930-937Abstract Full Text PDF PubMed Google Scholar). Adipose tissue is the main source of lipids and fatty acids in the body where they play key roles in the regulation of energy balance in mammals. Proteins involved in FA and triglyceride synthesis and accumulation as well as utilization of exogenous lipid are induced during adipocyte differentiation (3Spiegelman B.M. Cook K.S. Hunt C.R. Prog. Clin. Biol. Res. 1986; 226: 445-454PubMed Google Scholar, 4MacDougald O.A. Lane M.D. Annu. Rev. Biochem. 1995; 64: 345-373Crossref PubMed Scopus (934) Google Scholar). Increase of enzymatic activities regulating lipogenesis and lipolysis is also ongoing at this stage. These multiple roles of FA suggest that careful regulation of cellular aspects of FA metabolism including cellular uptake in liver, fat, cardiac and skeletal muscles, and other organs is essential. The mechanism by which long chain free FA enter the cells is not completely understood. It has long been postulated that the movement of long chain FA across the cell membrane is invariably passive (5DeGrella R.F. Light R.J. J. Biol. Chem. 1980; 255: 9731-9738Abstract Full Text PDF PubMed Google Scholar, 6DeGrella R.F. Light R.J. J. Biol. Chem. 1980; 255: 9739-9745Abstract Full Text PDF PubMed Google Scholar). Studies (5DeGrella R.F. Light R.J. J. Biol. Chem. 1980; 255: 9731-9738Abstract Full Text PDF PubMed Google Scholar, 6DeGrella R.F. Light R.J. J. Biol. Chem. 1980; 255: 9739-9745Abstract Full Text PDF PubMed Google Scholar) have suggested that FA penetrate cardiac myocytes by a passive unregulated mechanism rather than by a specific facilitated process and that saturation of an intracellular metabolism step is the cause for apparent saturation of uptake in other studies. Others (7Noy N. Donnelly T.M. Zakim D. Biochemistry. 1986; 25: 2013-2021Crossref PubMed Scopus (105) Google Scholar) have also shown that the entry of FA into hepatocytes reflected their passive partitioning into the lipid component of the cell membrane. However, recent observations indicated that at least a portion of FA uptake might occur by a carrier-mediated transport system (8Stremmel W. Berk P.D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3086-3090Crossref PubMed Scopus (128) Google Scholar, 9Stremmel W. Strohmeyer G. Berk P.D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3584-3588Crossref PubMed Scopus (186) Google Scholar). Studies with liver, fat cells, cardiac tissue, and skeletal myocytes have shown that free FA uptake exhibited many of the kinetic properties of a facilitated process, namely saturation, trans-stimulation, cis-inhibition, stereospecificity, and counter transport (8Stremmel W. Berk P.D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3086-3090Crossref PubMed Scopus (128) Google Scholar, 9Stremmel W. Strohmeyer G. Berk P.D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3584-3588Crossref PubMed Scopus (186) Google Scholar, 10Schwieterman W. Sorrentino D. Potter B.J. Rand J. Kiang C.L. Stump D. Berk P.D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 359-363Crossref PubMed Scopus (154) Google Scholar, 11Sorrentino D. Stump D. Potter B.J. Robinson R.B. White R. Kiang C.L. Berk P.D. J. Clin. Invest. 1988; 82: 928-935Crossref PubMed Scopus (155) Google Scholar, 12Stump D.D. Nunes R.M. Sorrentino D. Berk P.D. J. Hepatol. 1992; 16: 304-315Abstract Full Text PDF PubMed Scopus (42) Google Scholar). Several studies have indicated that FA uptake by adipocytes was phloretin-inhibitable and blocked by antibody raised against specific binding proteins (10Schwieterman W. Sorrentino D. Potter B.J. Rand J. Kiang C.L. Stump D. Berk P.D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 359-363Crossref PubMed Scopus (154) Google Scholar, 13Abumrad N.A. Perkins R.C. Park J.H. Park C.R. J. Biol. Chem. 1981; 256: 9183-9191Abstract Full Text PDF PubMed Google Scholar). These features could not be explained by diffusion. It is then possible to assume that facilitated and passive uptake processes both occur simultaneously in cells (12Stump D.D. Nunes R.M. Sorrentino D. Berk P.D. J. Hepatol. 1992; 16: 304-315Abstract Full Text PDF PubMed Scopus (42) Google Scholar), with the facilitated transport being the predominant process. In support of these findings, five putative mammalian free FA transporters have been identified so far in the plasma membrane of several tissues (14Stremmel W. Strohmeyer G. Kochwa S. Berk P.D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4-8Crossref PubMed Scopus (289) Google Scholar, 15Abumrad N.A. el-Maghrabi M.R. Amri E.Z. Lopez E. Grimaldi P.A. J. Biol. Chem. 1993; 268: 17665-17668Abstract Full Text PDF PubMed Google Scholar, 16Schaffer J.E. Lodish H.F. Cell. 1994; 79: 427-436Abstract Full Text PDF PubMed Scopus (740) Google Scholar, 17Trigatti B.L. Mangroo D. Gerber G.E. J. Biol. Chem. 1991; 266: 22621-22625Abstract Full Text PDF PubMed Google Scholar, 18Fujii S. Kawaguchi H. Yasuda H. Lipids. 1987; 22: 544-546Crossref PubMed Scopus (33) Google Scholar). cDNA clones have been isolated for three of them, specifically plasma membrane fatty acid-binding protein (FABPpm) (14Stremmel W. Strohmeyer G. Kochwa S. Berk P.D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4-8Crossref PubMed Scopus (289) Google Scholar), fatty acid translocase (FAT) (15Abumrad N.A. el-Maghrabi M.R. Amri E.Z. Lopez E. Grimaldi P.A. J. Biol. Chem. 1993; 268: 17665-17668Abstract Full Text PDF PubMed Google Scholar), and fatty acid transport protein (FATP) (16Schaffer J.E. Lodish H.F. Cell. 1994; 79: 427-436Abstract Full Text PDF PubMed Scopus (740) Google Scholar). In addition, small molecular weight cytosolic fatty acid-binding proteins have been characterized, cloned, and extensively studied (for review see Ref. 19Schroeder F. Jolly C.A. Cho T.H. Frolov A. Chem. Phys. Lipids. 1998; 92: 1-25Crossref PubMed Scopus (115) Google Scholar). Adipose differentiation related protein (ADRP) is a novel 50-kDa protein originally cloned by differential hybridization from a cDNA library of differentiated mouse 1246 adipocytes (20Jiang H.P. Serrero G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7856-7860Crossref PubMed Scopus (226) Google Scholar). The 1.7-kilobase pair ADRP mRNA was induced 50–100-fold a few hours after the onset of adipose differentiation in 1246 cells, thus making ADRP an early marker of the adipose differentiation program (20Jiang H.P. Serrero G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7856-7860Crossref PubMed Scopus (226) Google Scholar). It has been shown that ADRP mRNA was expressed at high levels in adipose tissue (20Jiang H.P. Serrero G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7856-7860Crossref PubMed Scopus (226) Google Scholar) and also in many different types of cells and tissues where lipids were accumulated or synthesized, although at lower levels than in adipocytes (21Brasaemle D.L. Barber T. Wolins N.E. Serrero G. Blanchette-Mackie E.J. Londos C. J. Lipid Res. 1997; 11: 2249-2263Abstract Full Text PDF Google Scholar). Sequencing of ADRP did not provide any information about its possible function in adipocytes. Immunolocalization studies done at different times during the adipose differentiation program in 1246 and 3T3-L1 adipocytes indicated that ADRP was localized in the vicinity of the plasma membrane in cells that started to differentiate and was found on the surface of lipid droplets in mature adipocytes (20Jiang H.P. Serrero G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7856-7860Crossref PubMed Scopus (226) Google Scholar, 21Brasaemle D.L. Barber T. Wolins N.E. Serrero G. Blanchette-Mackie E.J. Londos C. J. Lipid Res. 1997; 11: 2249-2263Abstract Full Text PDF Google Scholar). Moreover, ADRP expression was found to be induced in the liver of mice treated with the carnitine palmitoyltransferase I inhibitor, etomoxir, which caused neutral lipid accumulation in the organ (22Steiner S. Wahl D. Magold B.L.K. Robison R. Raymackers L. Metheus L. Anderson N.L. Cordier A. Biochem. Biophys. Res. Commun. 1996; 218: 777-782Crossref PubMed Scopus (62) Google Scholar). These various studies suggested that ADRP might be involved in the formation or stabilization of lipid droplets in adipocytes. As a first step to investigate this hypothesis, we have examined here whether ADRP was involved in FA uptake using ADRP-transfected COS-7 cells as an experimental model system. The results presented in this paper show that ADRP plays a role in facilitated FA transport in COS-7 cells expressing the protein. COS-7 cells (CRL 1651, American Type Culture Collection, Manassas, VA) were cultivated in DME-F12 medium (1:1 mixture of Dulbecco's modified Eagle's medium and Ham's nutrient F12 medium) supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Inc.) in T-75-cm2 plates. Cell stocks were cultivated in these conditions until nearly confluent and subcultured at 1:20 dilution or plated for an experiment. For expression into COS-7 cells, ADRP cDNA was cloned into the mammalian expression vector pcDNA3 (Invitrogen, Carlsbad, CA). Mouse ADRP cDNA fragment containing the 1.3-kilobase pair ADRP open reading frame was cloned in the sense orientation in XbaI and BamHI sites of pcDNA3. Expression of green fluorescent protein (GFP, CLONTECH, Palo Alto, CA) was used to monitor transfection efficiency. GFP plasmid DNA was co-transfected into COS-7 cells with pcDNA3 empty vector (control cells) or pcDNA3-ADRP vector plasmid DNA. Transient transfection of plasmid DNA into COS-7 cells was carried out by the DEAE-dextran method (23Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley-Interscience, New York1993: 9.2.1-9.2.6Google Scholar) established for COS cells. Briefly, 2 ml of a solution containing plasmid DNA and DEAE-dextran was prepared by mixing 0.2 ml of DEAE-dextran at 10 mg/ml, 5 pmol (about 10 μg) of plasmid DNA for each vector into 1.8 ml of phosphate-buffered saline (PBS) and incubated for 1 h at 37 °C with COS-7 cells cultivated in T-75-cm2 flasks. After 1 h, the cells were treated with 100 μm chloroquine for 4 h at 37 °C in serum-free culture medium followed by treatment with 10% dimethyl sulfoxide for 2 min. Cells were then washed twice with culture medium and then cultivated in DME/F12 medium supplemented with 10% FBS for 48 h for maximal ADRP expression in the transfected cells. Control cells were co-transfected with empty vector pcDNA3 and with GFP plasmid DNA using a similar method. Transfection efficiency was determined by counting the number of cells expressing GFP (co-transfected with either ADRP-pcDNA3 or with pcDNA3 empty vector) when compared with the total number of cells counted with a hemocytometer. ADRP protein expression in transfected COS cells was examined in cell lysates prepared from transfected COS-7 cells by Western blot analysis using rabbit anti-ADRP antibody. Briefly, cells were washed once with PBS and lysed in 1 ml of SDS sample buffer (62.5 mmTris-HCl, pH 6.8, 2% SDS, 10% glycerol) without β-mercaptoethanol and bromphenol blue (all chemicals were from Bio-Rad). The cell lysate was then sonicated at 40%, 20-watt output for 10 s using a Vibra Cell sonicator (Sonics & Materials Inc., Danbury, CT), and centrifuged at 10,000 × g for 10 min, and the supernatant was collected. The protein concentration of cell lysate was measured by using micro-BCA protein assay reagent kit (Pierce). After adding 1/10 volume of 10× loading dye (50% 2-Me, 1% bromphenol blue), equal amounts of protein (20 μg) from ADRP- and empty vector-transfected COS-7 cells were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate on a 10% gel followed by Western blot analysis to measure ADRP expression in both cell types. Conditions for SDS-PAGE and Western blot analysis were similar to the ones described previously to measure ADRP expression in adipocytes (24Ye H. Serrero G. Biochem. J. 1998; 330: 803-809Crossref PubMed Scopus (26) Google Scholar). COS-7 cells were plated in chamber slides (Becton Dickinson) in DME/F12 medium supplemented with 10% FBS. Transfection of ADRP or empty vector plasmid DNA was carried out as described above. After 48 h, cells were washed with PBS, fixed with 2% paraformaldehyde, and permeabilized with 0.2% Triton X-100 using a previously described method (16Schaffer J.E. Lodish H.F. Cell. 1994; 79: 427-436Abstract Full Text PDF PubMed Scopus (740) Google Scholar) followed by several washes with PBS. Cells were incubated with affinity purified anti-ADRP antibody for 1 h at room temperature followed by incubation with FITC-conjugated goat anti-rabbit secondary antibody (Bio-Rad). After extensive washing with PBS, slides were mounted in buffered glycerol solution and examined with an Olympus BX40 fluorescence microscope equipped with an Olympus camera. [9,10-3H]Oleic acid (1.2 μCi/nmol) (NEN Life Science Products) and unlabeled oleic acid sodium salts were dissolved in 10 ml of water at 40 °C to give a concentration of about 320 μm (48 μCi/ml). When the solution was completely clear after ∼10 min, fatty acid-free BSA (Sigma) from a concentrated stock solution (20%) was added with gentle mixing to obtain a final concentration of 80 μm BSA and an oleic acid/BSA molar ratio of 4.0. For the assays, an aliquot of the stock solution of FA/BSA (320 μm) was diluted 1:8 with PBS containing or not containing additional fatty acid-free BSA to obtain the final desired concentrations of oleic acid (40 μm) and of fatty acid-free BSA (10, 13, 20, 40, and 80 μm) and oleic acid/BSA molar ratios of 4.0, 3.0, 2.0, 1.0, and 0.5, respectively. The final concentration of fatty acids in the assay was 20 μm [3H]oleic acid (3 μCi/ml) with various concentrations of fatty acid-free BSA. The unbound oleic acid concentration in the presence of BSA was determined from the oleate/BSA molar ratio according to the calculation of Abumrad et al.(13Abumrad N.A. Perkins R.C. Park J.H. Park C.R. J. Biol. Chem. 1981; 256: 9183-9191Abstract Full Text PDF PubMed Google Scholar), using the association constants of Spector and Fletcher (25Spector A.A. Fletcher J.E. Dietschy J.M. Gotto A.M. Ontko J.A. Disturbances in Lipid and Lipoprotein Metabolism. Williams & Wilkins, Baltimore, MD1978: 229-249Google Scholar) and the model of stepwise equilibrium developed by Klotz et al.(26Klotz I.M. Walker F.M. Pivan R.B. J. Am. Chem. Soc. 1946; 68: 1486-1490Crossref PubMed Scopus (397) Google Scholar). [9,10-3H]Palmitic acid (43 μCi/nmol), [5,6,8,11,12,14,15-3H]arachidonic acid (222 μCi/nmol), and [1-14C]octanoic acid (55 μCi/μmol) (NEN Life Science Products) were diluted to 40 μm with a fatty acid-free BSA concentration of 10 μm. The final working concentration in the tubes was 20 μm[3H]palmitic acid (3.1 μCi/ml), [3H]arachidonic acid (1.3 μCi/ml), and [14C]octanoic acid (12 nCi/ml), respectively, with 5 μm fatty acid-free BSA. Transfected COS-7 cells were resuspended in PBS (5 × 105 cells/ml) in 15 ml of polypropylene centrifuge tubes. 200-μl aliquots of cell suspension (105 cells) were placed in 5-ml polypropylene centrifuge tubes. Cell suspensions in PBS were preincubated for 5 min at 37 °C in a shaking water bath. An equal volume of 2-fold concentrated stock fatty acid/BSA solutions was added to each tube to perform fatty acid uptake. At specific intervals, uptake was stopped by adding 5 ml of ice-cold PBS containing 0.1% BSA and 200 μm phloretin (wash solution) into each assay tube. Then the solutions and cell suspensions were filtered through GF/C filters (Whatman, Maidstone, UK) which had been presaturated with 15 ml of 0.1% BSA in PBS. Cells retained by filters were rapidly washed 3 times with 5 ml of cold wash solution. Filters were soaked overnight in 10 ml of scintillation mixture prior to counting with a liquid scintillation counter. Nonspecific fatty acid adsorbed to filters lacking cells was routinely measured and subtracted from experimental values. Background radioactivity representing isotope trapped extracellularly and bound nonspecifically by the cells was measured from zero time incubation determined by adding stop solution to the cells before adding radiolabeled fatty acid-BSA complex. Fatty acid uptake data were normalized with the transfection efficiency that had been determined by counting the number of fluorescent cells expressing GFP compared with the total cell number. Since non-transfected cells could also uptake fatty acid, it was necessary to remove their contribution to the total FA uptake in order to determine the contribution due to the expression of ADRP in the cells. To achieve this, the uptake of FA by non-transfected cells was determined by multiplying the value of total FA uptake in non-transfected control cells by the percentage of non-transfected cells (100% − transfection efficiency). Non-transfected control cells were treated similarly to transfected cells but without adding plasmid DNA of vectors used for transfection. The value for the remaining FA uptake contributed by the transfected cells was obtained by subtracting the nontransfected cells uptake from the total uptake measured experimentally. Then the remaining FA uptake of transfected cells was normalized to the transfection efficiency. The final amount of uptake was expressed as pmol per 105 transfected cells. The distribution of labeled oleate in intracellular lipids in ADRP-transfected or empty vector-transfected COS-7 cells was examined after uptake was performed. Uptake was carried out as described above. At various time points after uptake was stopped, cells from triplicate samples (6 × 105 cells) were washed by centrifugation. Lipids were immediately extracted by the method of Bligh and Dyer (27Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42411) Google Scholar) from cells resuspended in 0.8 ml of PBS. Extracted lipids were resuspended in 100 μl of CHCl3/MeOH (2:1) and separated by thin layer chromatography (TLC) on a silica gel G plate (Analtech, Newark, DE) in the solvent mixture of hexane/ether/formic acid (60:40:1). Triolein, diolein, mono-olein, oleic acid, and 1-palmityl,2-oleyl lecithin (Sigma) were used as standards for the TLC. Lipid spots were visualized by iodine vapors and immediately scraped with a razor blade into scintillation vials to count radioactivity incorporated into intracellular lipids in ADRP and empty vector-transfected COS-7 cells. ADRP and empty vector-transfected COS-7 cells were transferred from T-75 flasks to 24-well plates, 24 h after transfection. After incubation at 37 °C for another 24 h, media were changed to DME/F12 without FBS. Cells were incubated at 37 °C for 3 h. The cells were then washed in Krebs-Ringer phosphate (KRP) buffer (137 mm NaCl, 4.7 mm KCl, 0.4 mm MgCl2, 1 mm CaCl2, 10 mmNa2HPO4/NaH2PO4 buffer, pH 7.3, 0.2% BSA) and incubated in KRP buffer for 2 h prior to performing the uptake. 200 μm2-[14C]deoxyglucose (1.6 μCi/ml) (NEN Life Science Products) was freshly added into KRP buffer. After 10, 20, and 30 min incubation at 37 °C, cells were washed 2 times with ice-cold KRP buffer and then dissolved in 200 μl of 0.1 m NaOH. 5 ml of scintillation mixture was added into each tube for scintillation counting. All experiments were repeated at least three times. The data presented here are expressed as mean ± S.D. Differences between experimental groups were evaluated with two-tailed Student's t tests. Differences were considered significant if p < 0.05. To test whether ADRP promoted FA transport, the pcDNA3 expression vector containing ADRP cDNA insert was transiently transfected into COS-7 cells. Plasmid DNA from empty pcDNA3 vector was transfected into COS-7 cells as control. Cells were harvested 48 h after transfection, and cell lysates were prepared as described under "Experimental Procedures," in order to measure ADRP protein expression by Western blot analysis. As shown in Fig. 1, ADRP protein expression was very low in empty vector-transfected COS-7 cells. In contrast, cells transfected with the ADRP-pCDNA3 expression vector expressed high levels of ADRP protein equivalent to the one in 1246 adipocytes known to express high level of ADRP (19Schroeder F. Jolly C.A. Cho T.H. Frolov A. Chem. Phys. Lipids. 1998; 92: 1-25Crossref PubMed Scopus (115) Google Scholar, 24Ye H. Serrero G. Biochem. J. 1998; 330: 803-809Crossref PubMed Scopus (26) Google Scholar) and used as positive controls. These results demonstrated that ADRP protein was effectively expressed in COS-7 cells and that the transfected COS-7 cells could be used as a model of ADRP protein overexpression to examine the function of ADRP in mammalian cells. Localization of ADRP in transfected COS-7 cells was examined by immunofluorescence staining of fixed cells with anti-ADRP antibody followed by FITC-conjugated goat anti-rabbit secondary antibody. Fluorescence microscopy revealed a specific pattern of staining at the cell periphery in ADRP-transfected COS-7 cells (Fig.2 top panel) which was not observed in empty vector-transfected COS-7 cells (Fig. 2 bottom panel) or in cells incubated with preimmune IgG used as negative control (data not shown). Additional immunostaining was also found associated with the nucleus in ADRP-transfected cells as well as in control cells although with a lesser degree. These data show that ADRP is preferentially found associated with the plasma membrane in the transfected COS-7 cells. Assays of oleic acid transport were performed in control and in ADRP-expressing COS-7 cells. [3H]Oleate at a final concentration of 20 μm corresponding to an oleate/BSA molar ratio of 4:1 was incubated with 105 cells at 37 °C from 15 s to 30 min. Uptake was stopped by adding 5 ml of an ice-cold stop solution containing 200 μm phloretin and 0.1% BSA in PBS. Since the stop solution removed surface-bound [3H]oleate while blocking efflux from the cells of oleate already internalized (8Stremmel W. Berk P.D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3086-3090Crossref PubMed Scopus (128) Google Scholar), accurate quantitation of cumulative cellular oleate uptake could be obtained after stop solution treatment. The [3H]oleate uptake measured as the total [3H]oleate accumulated in ADRP or empty vector-transfected cells was normalized to the transfection efficiency as described under "Experimental Procedures." Fig. 3 showed the time course of oleate uptake in transfected COS-7 cells determined at 2.30 μm unbound oleic acid with an oleate/BSA ratio of 4:1 (mol/mol). The time course was biphasic with a fast early phase linear for up to 0.5 min (Fig. 3 A) followed by a slower phase (Fig.3 B) both for control and ADRP-transfected cells. In the later slower phase, the oleate uptake rate decreased although the cells continued to accumulate oleate. ADRP increased [3H]oleate uptake by about 3-fold over the uptake in control cells at all time points measured. Each of the two phases were significantly faster in ADRP-transfected cells than in control cells (p < 0.01 in each case). By 30 min, ADRP-transfected COS-7 cells had incorporated 401.0 ± 57.6 pmol/105 cells and control cells 194.6 ± 42.6 pmol/105 cells, respectively. These values were significantly different from each other (p< 0.01). The initial uptake rate determined by the slope of the linear portion of the uptake curve over the initial 0.5-min period (Fig.2 A) increased from 21.15 ± 0.46 pmol/105cells/min in control cells to 58.01 ± 9.48 pmol/105cells/min in ADRP-transfected cells (p < 0.01). Accurate quantitative measure of influx velocity could be obtained by measuring the initial uptake rate (8Stremmel W. Berk P.D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3086-3090Crossref PubMed Scopus (128) Google Scholar). These data indicated that ADRP promoted [3H]oleate uptake in COS-7 cells by increasing the influx velocity of oleic acid. The distribution of the label in intracellular lipids in ADRP and control COS-7 cells at different time points of the uptake reaction was examined. For this purpose, cell-associated lipids were extracted by the Bligh-Dyer method and analyzed by thin layer chromatography as described under "Experimental Procedures." Initially, the majority of the label was found in the free fatty acid pool. At 5 min, 35% of the label was found in the free fatty acids with 31% in the phospholipids and the rest in the neutral lipids. At 15 min, less than 10% of the label was found associated with free fatty acids, whereas 30% of the label was still found in the phospholipids and 60% in the neutral lipid pools. The same distribution of label over time was observed in empty vector-transfected cells indicating that ADRP did not change the distribution of label in the intracellular lipids but simply increased the level of uptake of fatty acids. The kinetics of [3H]oleic acid uptake by ADRP was assessed by examining oleate uptake over a 15-s period within a range of unbound oleic acid concentrations from 0.043 to 2.30 μm corresponding to a molar ratio of oleate/BSA of 0.5 to 4, respectively. As shown in Fig. 4, the uptake in control and ADRP-transfected cells was saturable. [3H]Oleate uptake velocity in control cells and ADRP-transfected cells reached a plateau at concentrations above 0.11 μm unbound oleic acid. After a 15-s incubation, the [3H]oleic acid uptake velocity in ADRP-transfected COS-7 cells was significantly higher than that of control cells at this range of oleic acid concentrations (p < 0.01). From a double-reciprocal plot of these data, the transportK m of ADRP was determined to be 0.051 μm and V max was 57.97 pmol/105 cells/min, whereas K m was 0.093 μm and V max was 20.13 pmol/105 cells/min in control cells. These results suggested that ADRP had the function of facilitating the uptake of fatty acid by increasing fatty acid uptake velocity. Uptake of [3H]oleate was also examined at
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