Sterol carrier protein-2 expression increases NBD-stearate uptake and cytoplasmic diffusion in L cells

Eric J. Murphy

Department of Physiology and Pharmacology, Texas A & M University, College Station, Texas 77843-4466

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The effects of sterol carrier protein-2 (SCP-2) expression on fatty acid uptake and cytoplasmic diffusion were determined using L cell fibroblasts transfected with cDNA encoding either the 15- or 13.2-kDa form of SCP-2. Cis-parinarate and 12-N-methyl-(7-nitrobenz-2-oxa-1,3-diazol)aminostearate (NBD-stearate) were used as nonesterifiable fluorescent fatty acid probes. NBD-stearate and cis-parinarate uptake was rapid and saturable. In 15-kDa SCP-2-expressing cells, the extent of NBD-stearate and cis-parinarate uptake was increased 1.4- and 1.2-fold, respectively, compared with control. In the 13.2-kDa SCP-2-expressing cells, the extent of NBD-stearate and cis-parinarate uptake was increased 1.3- and 1.1-fold, respectively, compared with control cells. NBD-stearate cytoplasmic diffusion was increased 1.5-fold in 15-kDa SCP-2-expressing cells, but not in 13.2-kDa SCP-2-expressing cells, compared with control cells. After incubation with NBD-stearate for 30 min at 37°C, fluorescence imaging indicated that NBD-stearate was localized primarily in lipid droplets in all cell lines. These results suggest that SCP-2 may be involved not only in fatty acid uptake but also in intracellular fatty acid trafficking.

fluorescence microscopy; fluorescence recovery after photobleaching

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

STEROL CARRIER PROTEINS (SCP) are thought to mediate intracellular cholesterol transfer and metabolism. These small ubiquitous intracellular proteins are encoded by a single gene with two initiation sites (19, 24). Translation products are found as 15- and 58-kDa proteins (15, 19, 20, 32). The 15-kDa protein contains a 20-amino acid presequence that has homology to the intracellular targeting sequence for mitochondria (15, 24). After translation, this presequence is rapidly cleaved to form the 13.2-kDa SCP-2 (13, 25). Each of these proteins shares the putative COOH-terminal tripeptide peroxisomal targeting sequence Ala-Lys-Leu (32).

Immunocytochemical analysis is consistent with the localization of the 58-kDa protein exclusively in the peroxisomal matrix (29, 30). Although immunocytochemical analysis indicates that the 13.2-kDa SCP-2 is predominantly localized with the peroxisome (10), this association appears to be on the cytoplasmic side of the peroxisomal membrane (30). The 13.2-kDa SCP-2 has also been immunocytochemically localized to mitochondria (3, 10), endoplasmic reticulum (10), and cytosol (10, 27). Because of the rapid cleavage of the 20-amino acid presequence from the 15-kDa protein, this protein is rarely detected by immunoblotting (13, 31). Consequently, the 15-kDa protein has not been localized within the cellular milieu.

Although many studies in vitro suggest a role for SCP-2 in cholesterol transfer and metabolism, a similar role for fatty acid transfer has not been proposed. SCP-2 binds cholesterol (2, 7), sterols (2), and phospholipids (7) and mediates cholesterol transfer between both model and natural membranes (6, 7). It was initially thought that SCP-2 did not interact with fatty acids in vitro (22); however, a more recent study clearly demonstrates that SCP-2 binds fatty acids in vitro with a high affinity similar to that seen for fatty acid binding proteins (FABP) (23, 26).

Because the simple association of a ligand with a protein does not necessarily correlate with protein function, it is not known whether fatty acid binding by SCP-2 reflects a functional role. Therefore, the role of SCP-2 in fatty acid uptake was examined using L cell fibroblasts transfected with the cDNA coding for either the 13.2- or 15-kDa SCP-2 (14). Fatty acid uptake was assessed using 12-N-methyl-(7-nitrobenz-2-oxa-1,3-diazol)aminostearate (NBD-stearate) and cis-parinarate as fluorescent fatty acid probes coupled with a steady-state fluorescence stirred cell assay (16, 21). In addition, NBD-stearate uptake was determined using kinetic analysis of digital fluorescent images, cytoplasmic diffusion was measured by fluorescence recovery after photobleaching (FRAP), and intracellular localization was examined using fluorescence deconvolution imaging. It is reported for the first time that 15-kDa SCP-2 expression increased both fatty acid uptake and fatty acid cytoplasmic diffusion in intact cells.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cells. Murine L cells (L arpt-tk-) previously transfected with the cDNA encoding the 15-kDa or 13.2-kDa forms of SCP-2 were used in this study (14). These two cell lines both contain the 13.2-kDa form of SCP-2, consistent with the reported rapid cleavage of the 20-amino acid presequence (13, 25). The protein levels expressed as a percentage of total cytosolic protein are 0.030% and 0.036% for the 15- and 13.2-kDa SCP-2-expressing cells, respectively (14). These levels are within the physiological range found in most tissue (28). Control and transfected L cell clones were grown to confluence in Higuchi medium containing 10% fetal bovine serum (Hyclone, Logan, UT) (9). For digital fluorescence imaging experiments, cells were either seeded onto 35-mm tissue culture plates (Falcon, Becton-Dickinson, Lincoln Park, NJ) at a density of 50,000 cells/ml or onto Lab-Tek chambered cover glass slides (Nunc, Naperville, IL) at a density of 25,000 cells/ml and used after 2 days. For the steady-state fluorescence stirred cell assay, cells were harvested from confluent monolayer cultures after several washes with PBS to remove culture medium. Cells were scraped from the flask using a rubber policeman and suspended in PBS to a final concentration of 300,000 cells/assay. For all assays, cell viability was assessed using a combination of trypan blue exclusion (with viability >95%), phase-contrast microscopy, and probe loading.

Steady-state fluorescence stirred cell assay. Fluorescent fatty acid uptake was examined using cis-parinarate (16, 21) or NBD-stearate as fatty acid probes. Both probes were purchased from Molecular Probes (Eugene, OR). Assays were performed using 300,000 cells per assay suspended in PBS in a total volume of 2 ml. To minimize cellular damage, cells were stored at 4°C and used for up to 4 h without loss of viability. Before the addition of the fluorescent fatty acid probe, the cells were warmed to 37°C for 7 min. Cells were continuously stirred using a Bel-Art cell spin bar (Fisher Scientific, Pittsburgh, PA), with the temperature maintained at 37°C. Cis-parinarate or NBD-stearate were dissolved in ethanol (95% vol/vol). The fluorescent fatty acid was added directly to the cuvette containing the cells within the sample compartment through a septum in the sample compartment lid using a 2-µl syringe (Hamilton, Reno, NV). The final concentrations for NBD-stearate and cis-parinarate in the assay were 0.5 and 2 µM, respectively. The final ethanol concentration in the assay did not exceed 0.1%. For each cell line, all measurements were repeated four to six times on the same batch of cells to avoid variability due to cell counts. These experimental results were confirmed using a separate set of nonsister cultures. To limit instrumental drift, all measurements were done on the same day.

Fluorescence intensity was measured using a photon-counting fluorimeter (ISS Instruments, Champaign, IL). For cis-parinarate, the excitation wavelength was set at 325 nm, with a 4-nm spectral slit width, and emission was measured through a KV 389 low fluorescence cutoff filter (Schott Glass Technologies, Duryea, PA). For NBD-stearate, the excitation wavelength was set at 466 nm, with a 4-nm spectral slit width, and emission was measured through a 530-nm interference filter (Oriel, Stratford, CT).

Laser cytometry. Kinetic analysis of NBD-stearate uptake was determined in individual cells using an ACAS 570 (Meridian Instruments, Okemos, MI). This instrument is equipped with a 5-W argon ion laser (Coherent, Sunnyvale, CA) and an IM-T inverted epifluorescence microscope (Olympus, Lake Success, NY) equipped with an x-y motorized stage and photomultiplier tube fluorescence detection. Laser power was set at 100 mW, with the excitation beam (488 nm) passing through a 1% neutral density filter. Scan strength was set at 3% (0.03 mW), and emission was measured at 530 nm. Before all studies, the instrument was optimized to generate the maximum signal while minimizing photobleaching. For all experiments, the stage was maintained at room temperature.

The kinetic uptake parameters for NBD-stearate were determined in control cells using 0.5, 1.0, and 4.0 µM NBD-stearate. Before the experiment, the L cells were washed two times with physiological buffer to remove trace culture medium. This buffer contained (in mM) 1.8 CaCl2, 5.0 KCl, 0.9 KH2PO4, 1.0 Na2HPO4, 0.6 MgSO4 septahydrate, 6.0 glucose, 138 NaCl, and 10.0 HEPES. Physiological buffer (1 ml) was added, and the cells were cooled to 4°C for 20 min. A concentrated stock solution of NBD-stearate in ethanol was diluted in physiological buffer (1 ml) and mixed to give working stock solutions of 1.0, 2.0, and 8.0 µM. After 20 min, the cell dishes were placed onto the stage, an area containing five or six viable cells was located, focus was established, and NBD-stearate working stock solution was added. The final ethanol concentration was maintained at <0.1% during the experiment. For these experiments, the number of cells analyzed (n) was 15 for each concentration. These cells were imaged from three separate sister cultures.

On the basis of these kinetic uptake experiments in L cells, 1.0 µM NBD-stearate was used to determine the extent of NBD-stearate uptake in the SCP-2-expressing cells using the same protocol as described previously. Note that cooling the cells before incubation with the probe had no effect on either the cytoplasmic diffusion coefficient or lateral membrane mobility measurements relative to values obtained when the cells were maintained at 37°C. Furthermore, although maintaining the cells at 4°C before probe loading decreased the fatty acid uptake rate, the cells did not exhibit biphasic uptake kinetics. Thus these two parameters were independent of the preincubation temperature, suggesting that the cells maintained at either 4 or 37°C had the same membrane biophysical state at the time the cytoplasmic diffusion coefficient and membrane mobility were measured. To minimize instrument variability, uptake and cytoplasmic diffusion were measured in control and SCP-2-expressing cells on the same day. For NBD-stearate uptake, n was 28-32 and represented cells imaged from six separate sister cultures.

Cytoplasmic diffusion. FRAP was used to determine the effects of SCP-2 expression on cytoplasmic NBD-stearate diffusion by quantifying the cytoplasmic NBD-stearate diffusion coefficient by a previously established method (12). NBD-stearate (1 µM) was loaded for 15 min at room temperature and followed by removal of the incubation medium, and the cells were washed with physiological buffer to remove noninternalized NBD-stearate. Lipid droplets at the cell periphery were avoided, and an area between the nucleus and the edge of the cell was photobleached with a 15-ms blast by 1 mW of laser power (beam radius set at 1.3 µm) to effectively bleach 80-90% of the NBD-stearate in that region. Recovery of fluorescence into this region was monitored over time using digital fluorescence imaging; the diffusion coefficient was calculated using a software algorithm for flat cells provided by the ACAS manufacturer. For the diffusion experiment, n was 28-32 and represented cells imaged from six separate sister cultures.

Intracellular localization. Subcellular localization of NBD-stearate was performed using a fluorescence deconvolution imaging workstation (CELLscan, Scanalytics, Billerica, MA). This workstation was equipped with a Zeiss Axiovert 135TV inverted epifluorescence microscope (Zeiss, Thornwood, NY) fitted with a 100-W mercury lamp. Digital images were acquired using wide-field illumination, a charge-coupled device camera, and piezoelectric z-axis control of the objective lens for image collection in a "through focus" series of 45 focal planes at 0.5-µm increments. To reduce photobleaching, the cells were exposed to the light source for 0.1 s and exposure was regulated by a computer-controlled shutter. Three-dimensional imaging and fluorescence deconvolution algorithms, which use an acquired point-spread function to vector out-of-focus fluorescence to the point of origin in the specimen image, were done using CELLscan software. The point-spread function was measured for the deconvolution algorithm by taking 0.125-µm z-axis intervals of 0.19-µm-diameter fluorescent latex beads (Molecular Probes).

To determine NBD-stearate intracellular localization, two different probes were used, each of which had specific loading requirements. Nile red (Molecular Probes) was used as a stain that primarily detects neutral lipid droplets (5, 8). Cells in chambered cover glass slides were rinsed two times with physiological buffer and incubated with Nile red (1.0 µM) at 37°C for 1 min. After this incubation, the cells were rinsed two times to remove excess stain, 1 ml of physiological buffer was added, fluorescence imaging was done using excitation at 551 nm, and emission was monitored at 630 nm. Rhodamine 123 was used to visualize mitochondria (1). Procedures for rhodamine 123 staining were the same as those for Nile red, except the cells were incubated with rhodamine 123 (5 µg/ml) at 37°C for 20 min. Fluorescence images were obtained after rinsing of cells to remove excess probe, using an excitation wavelength of 488 nm with emission monitored at 530 nm. NBD-stearate localization was performed by incubating cells with the probe at a concentration of 4 µM at 37°C for 30 min. Note that these incubation conditions are substantially different from those used in the NBD-stearate uptake and cytoplasmic diffusion experiments. All other procedures were done as described. NBD-stearate fluorescent images were obtained after removal of excess probe by rinsing the cells, using an excitation wavelength of 488 nm with emission monitored at 530 nm. Because NBD-stearate and Nile red have overlapping excitation and emission spectra, colocalization studies using these two probes were not possible. However, colocalization studies were done using rhodamine 123 and either Nile red or NBD-stearate.

Statistics. Statistical analysis was done using Instat 2 statistical software package (Graphpad, San Diego, CA), using one-way ANOVA combined with a Newman-Keuls multicomparison post hoc test. Statistical significance was defined as P < 0.05. For fluorescence imaging experiments, values represent means ± SE with n equal to 28-32, except for the concentration-dependent uptake experiments, where n was 15. For steady-state fluorescence stirred cell experiments, values represent means ± SE, with n equal to 4-6.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Steady-state fluorescent stirred cell assay. The effects of SCP-2 expression on fatty acid uptake were examined using steady-state fluorescence coupled with a stirred cell assay. Cis-parinarate is a nonesterified, naturally occurring fluorescent fatty acid that lacks any bulky fluorophores and is taken up in a manner similar to radiolabeled oleate. Thus this cis-parinaric acid uptake assay measures fatty acid uptake in the absence of appreciable esterification into glycerolipids (16, 21). In the stirred cell assay, cis-parinarate uptake rapidly reached saturation in control, 15-kDa, and 13.2-kDa SCP-2-expressing cells (Fig. 1A). The extent of cis-parinarate uptake was modestly increased 1.2- and 1.1-fold in 15-kDa and 13.2-kDa SCP-2-expressing cells, respectively, compared with control cells (Fig. 1A, Table 1).


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Fig. 1.   Cis-parinarate (2.0 µM, A) and 12-N-methyl-(7-nitrobenz-2-oxa-1,3-diazol)aminostearate (NBD-stearate) (0.5 µM, B) uptake into control, 13.2-kDa, and 15-kDa sterol carrier protein-2 (SCP-2)-expressing cells. Cell concentration was 300,000 cells per assay, with a final volume of 2 ml. Cuvette temperature was maintained at 37°C. Excitation and emission parameters were as stated in MATERIALS AND METHODS. Values are means ± SE; n = 4-6.

                              
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Table 1.   Effects of SCP-2 expression on NBD-stearate and cis-parinarate uptake

Although the stirred cell fluorescence fatty acid uptake assay measures fatty acid uptake, it does not readily resolve fatty acid internalization from intercalation into the plasma membrane. To avoid this problem, fluorescence digital imaging of single cells was used to quantify the extent of fatty acid uptake and deconvolution imaging of single cells was used to determine fatty acid localization. Because the ACAS laser cytometer lacked the necessary excitation wavelength for cis-parinarate, NBD-stearate was used. To confirm that NBD-stearate could serve as an acceptable probe for fatty acid uptake and internalization, NBD-stearate uptake was first determined using the stirred cell uptake assay. Similar to cis-parinarate, NBD-stearate uptake rapidly reached saturation in control, 13.2-kDa, and 15-kDa SCP-2-expressing cells (Fig. 1B). Maximal NBD-stearate uptake was increased 1.4- and 1.3-fold in the 15-kDa and 13.2-kDa SCP-2-expressing cells, respectively, compared with control cells (Fig. 1B and Table 1). Thus, with the use of two different fluorescent fatty acid probes, the steady-state fluorescence stirred cell assay confirmed that the 15-kDa SCP-2-expressing cells and, to a lesser extent, the 13.2-kDa SCP-2-expressing cells, had increased fatty acid uptake.

Laser cytometry. The concentration dependence of NBD-stearate uptake kinetics was analyzed in L cell monolayer cultures using the ACAS laser cytometer (Fig. 2). NBD-stearate uptake was concentration dependent. The rate constant for 1.0 µM NBD-stearate uptake was slightly more than double the rate constant for 0.5 µM NBD-stearate, even though both concentrations had similar maximal fluorescence values (Table 2 and Fig. 2). Subsequently, the 1.0 µM concentration reached a plateau much faster than the 0.5 µM concentration (Fig. 2). L cells exposed to 4 µM NBD-stearate had a higher rate constant; however, this rate constant was not fourfold greater than the constant at 1.0 µM NBD-stearate (Table 2). These observations suggest that the uptake mechanisms had reached saturation at 4 µM and that the increased fluorescence represents elevated NBD-stearate levels resulting from an increase in nonspecific localization into the cellular membrane. On the basis of these results, 1.0 µM NBD-stearate was selected as the concentration used to determine the maximal uptake and cytoplasmic diffusion rate in control, 13.2-kDa, and 15-kDa SCP-2-expressing cells.


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Fig. 2.   NBD-stearate uptake into L cell fibroblasts determined using fluorescence microscopy. Concentrations used were 0.5 (bullet ), 1.0 (black-triangle), and 4.0 µM (). Microscope stage was maintained at room temperature, and cells were kept for 20 min at 4°C until just before addition of NBD-stearate. Experiments were all done on same day. Values are means ± SE; n = 15.

                              
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Table 2.   NBD-stearate uptake in L cell fibroblasts

The effects of SCP-2 expression on the extent of NBD-stearate uptake were also determined by laser cytometry. L cells that expressed either the 15-kDa or 13.2-kDa form of SCP-2 had significantly greater (P < 0.05) maximal NBD-stearate fluorescence intensity values than control L cells (Table 3). The extent of NBD-stearate uptake in the 15-kDa and 13.2-kDa SCP-2-expressing cells was increased 1.4- and 1.2-fold, respectively, compared with control L cells. These values compared favorably to those obtained with the stirred cell fluorescence uptake assay.

                              
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Table 3.   Effects of SCP-2 expression on NBD-stearate uptake and diffusion

Cytoplasmic diffusion. Although expression of either the 13.2-kDa or 15-kDa SCP-2 increased the extent of NBD-stearate uptake, the effect of these proteins on cytoplasmic NBD-stearate diffusion is unknown. To determine this effect, NBD-stearate cytoplasmic diffusion was measured at room temperature using FRAP. The 15-kDa SCP-2-expressing cells had a significant (P < 0.01) 1.5-fold increase in the cytoplasmic diffusion rate compared with control and 13.2-kDa SCP-2-expressing cells. Although the 13.2-kDa SCP-2-expressing cells had significantly increased maximal NBD-stearate fluorescence, the NBD-stearate diffusion rate was not significantly altered compared with control cells as determined by FRAP (Table 3). Hence expression of only the 15-kDa SCP-2 in L cells increased both the maximal NBD-stearate uptake and its cytoplasmic diffusion rate.

Intracellular localization. The intracellular distribution of NBD-stearate was evaluated by optical sectioning and fluorescence deconvolution. After incubation with NBD-stearate (4 µM) for 30 min at 37°C, the NBD-stearate was localized primarily in intracellular membranes and lipid droplets, whereas staining was excluded from the nucleus in all three cell lines (Fig. 3). Note that these conditions are different from those used in the uptake and cytoplasmic diffusion experiments. Furthermore, both NBD-stearate and Nile red appeared to localize in similar round, highly fluorescent structures that had a similar cellular distribution near the periphery of the cell (Fig. 4). Rhodamine 123, a mitochondrial stain, did not localize in the same pattern as either NBD-stearate or Nile red, indicating that these round structures were indeed not mitochondrial in origin (Fig. 3). Colocalization experiments showed that neither NBD-stearate nor Nile red was localized with rhodamine 123 (data not shown). Hence NBD-stearate was internalized into the cell and found localized primarily in lipid droplets and intracellular membranes.


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Fig. 3.   NBD-stearate intracellular localization using CELLscan system. Left, control cells; middle, 13.2-kDa SCP-2-expressing cells; right, 15-kDa SCP-2-expressing cells. Note increased fluorescence in lipid droplets, indicating localization of NBD-stearate.


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Fig. 4.   Intracellular localization of NBD-stearate in L cell fibroblasts done using CELLscan system. Left, NBD-stearate; middle, Nile red; right, rhodamine 123. Note similar location and distribution of Nile red and NBD-stearate staining vesicular structures, identified as lipid droplets. Also note different pattern for NBD-stearate and rhodamine 123 localization, indicating NBD-stearate is not found in mitochondria.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Although many studies in vitro are consistent with a role for SCP-2 in sterol transfer (7), a similar role for SCP-2 in fatty acid uptake and transfer has not been demonstrated. In part, a role for SCP-2 in fatty acid uptake and intracellular transfer has not been elucidated because an earlier report suggested SCP-2 did not specifically bind fatty acids in the Lipidex competition assay (22). Using a fluorescent fatty acid assay not requiring competition with Lipidex resin to separate bound from free ligand, SCP-2 binds fatty acids in a 1:1 molar ratio, with a Kd in the submicromolar range (23). Recently, this stoichiometry was confirmed using nuclear magnetic resonance with 13C-labeled stearic and oleic acid (26). The Kd values for SCP-2 are very similar to those reported for liver (L)- and intestinal (I)-FABP, using a similar fluorescent fatty acid assay (18). Furthermore, this study showed that solvents such as those used in the earlier study (22) disrupted the interaction of fatty acids with SCP-2 (23). Thus the binding of fatty acids to SCP-2 suggests that SCP-2 may mediate cellular fatty acid uptake and/or intracellular transfer. To ascertain whether SCP-2 expression can stimulate fatty acid uptake, studies were performed using L cell fibroblasts that were stably transfected with cDNA for either the 15 or 13.2-kDa SCP-2 (14).

Fatty acid uptake was assessed using both single-cell fluorescence digital imaging of anchored cells and a steady-state fluorescence stirred cell assay of cells in suspension. The steady-state fluorescence stirred cell assay was previously used to reproducibly determine cis-parinarate uptake into L cells transfected with I-FABP (21) or L-FABP (16), and it was found that the effect of these proteins produced no effect on uptake (21) or produced a modest increase in fatty acid uptake (16) of a similar magnitude to that reported here for SCP-2. Because cis-parinarate is a naturally occurring fatty acid that is poorly esterified (<3% in 30 min) in L cells, fatty acid uptake can be measured in the absence of esterification (16, 21). Similarly, NBD-stearate is also poorly esterified (<5%) during incubation times similar to the times reported herein with the L cell lines (4). Results from the steady-state fluorescence stirred cell assay (Table 1) are quantitatively similar to those obtained using fluorescence microscopy (Table 3) and support the validity of using NBD-stearate in single- cell fluorescence studies to determine cellular NBD-stearate uptake.

Compared with control cells, fatty acid uptake was enhanced to a greater extent in 15-kDa SCP-2-expressing cells than in the 13.2-kDa SCP-2-expressing cells. The 15-kDa SCP-2-expressing cells had a 1.4-fold increase in NBD-stearate uptake and a 1.2-fold increase in cis-parinarate uptake. The 13.2-kDa SCP-2-expressing cells had a 1.3-fold increase in NBD-stearate uptake and a 1.1-fold increase in cis-parinarate uptake. These differences in uptake between the two probes may pertain to either intrinsic fluorophore differences or to differences in partitioning of these two different probes into the plasma membrane. The magnitude of increased fatty acid uptake in 15-kDa SCP-2 cells compared with control cells is similar to that observed in L cells transfected with L-FABP. In L-FABP-expressing cells, cis-parinarate uptake is increased 1.3-fold compared with control cells (16, 21). Furthermore, [3H]oleic acid uptake was enhanced 1.6-fold in 15-kDa and 13.2-kDa SCP-2-expressing cells relative to control cells (unpublished results). This increase is similar to that reported for L-FABP-expressing cells (16), suggesting that not only does SCP-2 expression increase fatty acid uptake, but it also stimulates fatty acid esterification, apparently primarily into neutral lipids (17). This observation provides further evidence that SCP-2 may function as a ubiquitous FABP.

Although the increase in fatty acid uptake compared with controls was nearly the same for the SCP-2 and L-FABP-expressing cells, there was an 11- to 13-fold difference in the amount of transfected protein expressed in these cells (14, 17). The 15-kDa SCP-2-expressing cells contained 0.03% SCP-2 compared with 0.4% L-FABP in the L-FABP-expressing cells. Furthermore, because L-FABP has a stoichiometry of 2:1, there is an even greater difference based on the number of potential fatty acid binding sites. Hence, the increase in fatty acid uptake in the 15-kDa SCP-2-expressing cells suggests that SCP-2, a ubiquitous protein, has a potentially significant role in facilitating cellular fatty acid uptake and diffusion.

Cytoplasmic diffusion rates can be measured using several different methods, including FRAP (12). Amphipathic molecules such as NBD-stearate have an appreciably slower cytoplasmic diffusion rate compared with diffusion in an aqueous solution (12). This is because the cytoplasmic diffusion rates for amphipathic molecules are reduced even further by interaction of the amphipathic molecules with the cytoplasmic matrix (11, 12). The interaction of molecules with the cytoplasmic matrix may account for the reported 6.5- to 100-fold slower cytoplasmic diffusion rates compared with rates in an aqueous environment devoid of any structural components capable of interacting with the molecule of interest (11, 12).

In this light, the effects of SCP-2 expression on NBD-stearate cytoplasmic diffusion were studied. Expression of 15-kDa SCP-2 enhanced cytoplasmic diffusion 1.5-fold, whereas 13.2-kDa SCP-2 expression did not significantly enhance cytoplasmic diffusion compared with control cells. A similar increase was reported for NBD-stearate cytoplasmic diffusion in hepatocytes derived from female compared with male rats (12). In these cells, a 1.7-fold increase in cytoplasmic diffusion was attributed to an increase in L-FABP levels in the female-derived hepatocytes compared with male-derived hepatocytes. It was suggested that in these hepatocytes, L-FABP reduces the binding of NBD-stearate to immobile cytoplasmic membranes, thereby increasing the NBD-stearate present in the cytoplasm in the protein-bound form (12). Our results indicate that the expression of 15-kDa SCP-2 in L cell fibroblasts results in a similar increase in the cytoplasmic diffusion rate, consistent with SCP-2 acting to increase the amount of protein-bound NBD-stearate in the cytoplasm.

The lack of an increase in cytoplasmic diffusion rate and a limited increase in fatty acid uptake in 13.2-kDa SCP-2-expressing cells may in part reflect a less effective localization of the 13.2-kDa SCP-2 within the cell. A similar lack of effectiveness in cholesterol uptake was seen in transfected L cells expressing the 13.2-kDa form of SCP-2 (14). The gene for SCP-2 encodes a 15-kDa protein, not the 13.2-kDa mature form of SCP-2 that is seen in tissues (15, 24, 32). Western blot analysis showed that both the transfected cells contained only the 13.2-kDa protein product, although the 15-kDa SCP-2-expressing cells contain the entire cDNA sequence, including the 20-amino acid presequence (14). This presequence is rapidly cleaved in situ (13, 25), and a similar rapid posttranslational processing is seen in the transfected cells (14). Because this 20-amino acid presequence has homology to the mitochondrial targeting sequence (15, 24), it may be important for proper intracellular protein localization and function. The lack of this presequence in the protein expressed from the cDNA encoding for the 13.2-kDa form of SCP-2 may account for the decreased ability of this protein to facilitate NBD-stearate uptake and cytoplasmic diffusion, respectively.

NBD-stearate was internalized and primarily incorporated into lipid droplets. Nile red preferentially stains lipid droplets (5, 8) and was localized into similar round vesicular structures near the periphery of the cell. These vesicles were very similar in both size and intracellular distribution to those stained by NBD-stearate, suggesting that NBD-stearate was localized in lipid droplets. Rhodamine 123, a dye used to identify mitochondria, did not colocalize with either NBD-stearate or Nile red, indicating that the round vesicular structures were not mitochondria. This localization study confirmed the absence of NBD-stearate from the nucleus (12) and confirmed that NBD-stearate was internalized into the cells and confined to distinct intracellular regions.

In summary, these results show that SCP-2 expression stimulated fatty acid uptake in an intact cell. SCP-2 expression stimulated an increase in fatty acid uptake similar to the increase seen in L-FABP-expressing L cells (16, 21), even though the L-FABP-expressing cells had 13-fold higher levels of L-FABP relative to the SCP-2 levels in the 15-kDa SCP-2-expressing cells. The 15-kDa SCP-2-expressing cells had a 1.4-fold increase in NBD-stearate uptake and a 1.5-fold increase in the cytoplasmic diffusion rate compared with control cells, indicating that this protein increased fatty acid uptake and diffusion. Similar but smaller changes in these two parameters in 13.2-kDa SCP-2-expressing cells suggests that the 20-amino acid presequence found in the 15-kDa SCP-2 is required for proper protein localization and function. Similar effects were found in cholesterol trafficking and cholesteryl ester formation in these two transfected cell lines (14, 17). Hence, these results showed that SCP-2 can facilitate fatty acid uptake and diffusion in transfected L cells, suggesting a new role for the ubiquitously distributed SCP-2 in cellular fatty acid uptake.

    ACKNOWLEDGEMENTS

I thank Cindy Murphy for typed preparation of this manuscript.

    FOOTNOTES

Address for reprint requests: E. J. Murphy, Laboratory of Neuroscience, National Institute on Aging, National Institutes of Health, Bldg. 10, Rm. 6C-103, 9000 Rockville Pike, Bethesda, MD 20892-1582.

Received 17 March 1997; accepted in final form 10 April 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Gastroint Liver Physiol 275(2):G237-G243
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