Cellular differentiation and I-FABP protein expression modulate fatty acid uptake and diffusion

Barbara P. Atshaves1, William B. Foxworth2, Andrey Frolov1, John B. Roths2, Ann B. Kier2, Betty K. Oetama3, Jorge A. Piedrahita3, and Friedhelm Schroeder1

Departments of 1 Physiology and Pharmacology, 2 Pathobiology, and 3 Anatomy and Public Health, Texas Veterinary Medical Center, Texas A&M University, College Station, Texas 77843-4466

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The effect of cellular differentiation on fatty acid uptake and intracellular diffusion was examined in transfected pluripotent mouse embryonic stem (ES) cells stably expressing intestinal fatty acid binding protein (I-FABP). Control ES cells, whether differentiated or undifferentiated, did not express I-FABP. The initial rate and maximal uptake of the fluorescent fatty acid, 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-octadecanoic acid (NBD-stearic acid), was measured in single cells by kinetic digital fluorescence imaging. I-FABP expression in undifferentiated ES cells increased the initial rate and maximal uptake of NBD-stearic acid 1.7- and 1.6-fold, respectively, as well as increased its effective intracellular diffusion constant (Deff) 1.8-fold as measured by the fluorescence recovery after photobleaching technique. In contrast, ES cell differentiation decreased I-FABP expression up to 3-fold and decreased the NBD-stearic acid initial rate of uptake, maximal uptake, and Deff by 10-, 4.7-, and 2-fold, respectively. There were no significant differences in these parameters between the differentiated control and differentiated I-FABP-expressing ES cell lines. In summary, differentiation and expression of I-FABP oppositely modulated NBD-stearic acid uptake parameters and intracellular diffusion in ES cells.

embryonic stem cells; intracellular localization; laser cytometry; fluorescence recovery after photobleaching; intestinal fatty acid binding protein

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

ALTHOUGH CELLULAR UPTAKE AND cytoplasmic diffusion of hydrophilic molecules have been extensively studied, relatively little is known regarding cellular uptake and diffusion of molecules such as fatty acids. Studies of cellular fatty acid uptake not performed on single cells do not resolve fatty acid binding to the cell from internalization and cytoplasmic processes. Likewise, measurements of fatty acid diffusion are complicated by factors such as a 15- to 20-fold higher viscosity of cytoplasm than aqueous buffer (22, 30), the presence of cytoplasmic fatty acid binding proteins (5, 9, 19, 21), and extensive (as much as 95%) binding of probe to intracellular membranes (17, 53).

The effect of differentiation on fatty acid uptake is complex. For example, fatty acid uptake is increased as much as 30-fold in differentiated 3T3 adipocytes compared with 3T3 preadipocyte fibroblasts (3, 49, 54). However, it is unclear whether this increase is due to a 10-fold differentiation-induced increase in the cytosolic adipocyte fatty acid binding protein (49) or to an 80-fold differentiation-induced increase of plasma membrane fatty acid binding protein(s) (2, 36, 54). The intracellular fatty acid binding proteins have long been postulated to enhance fatty acid uptake into the cell (6, 50); however, the discovery of plasma membrane fatty acid binding proteins brought into question the proposed role for cytosolic fatty acid binding proteins in stimulating cellular influx of fatty acids (16, 31, 36). However, data from model systems indicate that liver fatty acid binding protein (L-FABP) stimulates oleate flux threefold through a lipid-water interface (50). Only recently has direct evidence supporting this hypothesis been presented for intact cells. The fluorescent fatty acid uptake in cell suspension assays showed that L-FABP expression in transfected L cells enhances fluorescent fatty acid uptake 1.5-fold (29, 33, 34, 37). Unfortunately, the cell suspension uptake assay does not readily distinguish between uptake into the cell surface membrane vs. internalization of fatty acid.

One mechanism whereby a cytosolic fatty acid binding protein may stimulate fatty acid uptake is by enhancing intracellular diffusion of the fatty acid. Although it has also been proposed that cytosolic fatty acid binding proteins stimulate intracellular fatty acid transport, direct evidence has been difficult to obtain (6, 40). Most evidence has been obtained in vitro with model systems (20, 27, 40, 48). For example, fatty acid binding proteins stimulated fatty acid diffusion as much as sixfold when added to isolated muscle cytosol deficient in fatty acid binding protein (41). In contrast, permeabilization of human hepatocytes and removal of 90% of cytosolic proteins decreased the intracellular effective diffusion constant (Deff) of 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-octadecanoic acid (NBD-stearic acid) nearly 20-fold compared with control values. Hepatocytes from female rats express more L-FABP and have 65% faster effective intracellular diffusion of NBD-stearic acid than hepatocytes from male rats (25). However, direct interpretation of the latter is complicated by hepatocyte plasma membrane fatty acid binding protein(s) from female rats having a 56% lower Michaelis-Menten constant (higher affinity) for fatty acid (39).

The primary purpose of the present investigation was to examine fatty acid uptake and intracellular diffusion with single cell imaging techniques. Embryonic stem (ES) cells provided a simple model system for examining the effects of differentiation on fatty acid uptake and intracellular diffusion in stably transfected ES cell clones expressing intestinal fatty acid binding protein (I-FABP).

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. Lab-Tek chambered coverglass slides were purchased from Fisher Scientific (Pittsburgh, PA); gelatin, retinoic acid, penicillin, and streptomycin were purchased from Sigma Chemical (St. Louis, MO); ES cell-qualified serum, Dulbecco's modified Eagle's medium (DMEM), ESGRO murine leukemia inhibitory factor (LIF), and L-glutamine were obtained from GIBCO Life Technologies (Gaithersburg, MD); and NBD-stearic acid and nile red were from Molecular Probes (Eugene, OR). All reagents used were of the highest grade available and were cell culture tested as necessary.

Undifferentiated ES cell culture. Mouse ES cells of the E14 line (15) were cultured on STO fibroblasts in DMEM buffered with 2.2 g/l sodium bicarbonate. The medium was supplemented with L-glutamine (2 mM), ES cell-qualified serum (15%), 2-mercaptoethanol (0.1 mM), penicillin (50 IU/ml), streptomycin (50 IU/ml), and the recombinant murine cytokine LIF (1,000 IU/ml). To preserve the undifferentiated state and maintain a proliferating population, the continued presence of LIF was necessary (1).

For fluorescence imaging experiments, all cells were seeded at a density of 50,000 cells/chamber onto gelatinized (0.1% gelatin) Lab-Tek chambered coverglass slides (Nunc, Naperville, IL). To ensure cells were in a monolayer, the samples were analyzed within 20 h of seeding. Only viable cells, as determined by trypan blue exclusion and probe loading, were studied. In the undifferentiated state, ES cells exhibited a rounded morphology.

ES cell differentiation. ES cells are nontransformed, pluripotent cells, which, if grown to high density, will spontaneously differentiate to many cell types (51). Controlled differentiation of ES cells was accomplished by adding retinoic acid to the culture medium (45). This resulted in conversion of undifferentiated ES cells (rounded "adipocyte-like" morphology) to a population of differentiated ES cells with "fibroblast-like" morphology. Differentiation was induced by seeding 500,000 cells onto a 10-cm2 gelatinized dish in medium without LIF but with retinoic acid added to a final concentration of 0.67 µM. For imaging studies, cells were grown for 5 days and then seeded at a density of 50,000 cells/chamber on gelatinized chambered slides to form a layer of differentiated fibroblast-like cells. The cells were cultured for 15-20 h before analysis.

I-FABP expression vector and electroporation into ES cells. Plasmid pBA25-I contains the entire coding region of a rat I-FABP cDNA cloned into the eukaryotic vector pcDNA3 (Invitrogen, San Diego, CA). The cDNA was cloned downstream from the human cytomegalovirus promoter and upstream from the bovine growth hormone polyadenylation and splice sequences. A 426-base pair BamH I fragment containing the rat I-FABP cDNA was isolated from pDPMT-I (32) and cloned into the unique BamH I site of pcDNA3. After they were screened, one plasmid containing the insert in the correct orientation was designated as pBA25-I. In addition to I-FABP, plasmid pBA25-I contains the neomycin gene upstream from the simian virus 40 promoter. The mock-transfected control ES cell line survived selection after transfection but contained no I-FABP DNA. To prepare for transfection, the plasmid DNA was isolated on a large scale with Qiagen Maxi-prep columns (Qiagen, Chatsworth, CA), linearized with Bgl II, phenol extracted, precipitated with ethanol, and dissolved in Milli-Q water (Millipore, Bedford, MA) to a concentration of 1 µg/µl. Approximately 15-20 µg of linearized DNA were used per electroporation experiment. Linearized DNA was added to ES cells (1 × 106 cells) resuspended in 500 µl of culture medium. After 5 min, electroporation was performed with a Bio-Rad gene pulser (Bio-Rad, Hercules, CA) at the following settings: 300 V, 250 µF. The electroporated cells were seeded onto STO fibroblasts in 10-cm2 plates. After 24 h, culture medium was replaced with medium containing 500 µg/ml G418 supplemented with LIF (1,000 IU/ml). Resistant clones were selected after 7-9 days. Sixteen colonies were picked and cultured in 24-well dishes. When confluent, they were expanded to six-well dishes and then to 10-cm2 plates for further analysis.

Polymerase chain reaction screen. Genomic DNA was isolated from each clone at the six-well stage of the clonal expansion and used to screen the clones for the presence of plasmid pBA25-I DNA. Primers were made (Integrated DNA Technologies, Coralville, IA) corresponding to DNA in the vector pCDNA3 promoter region (5'-CGC-GAT-GTA-CGG-GCC AGA-TAT-ACG-3') and the multiple cloning region (5'-GCT-CTA-GCA-TTT-AGG-TGA-CAC-3'). Each polymerase chain reaction (PCR) experiment included 1 µg of genomic DNA and was cycled 35 times as follows: 95°C for 45 s, 55°C for 30 s, and 72°C for 90 s. PCR products were run on a 1% agarose gel and visualized by ethidium bromide staining.

Western blotting. Expression of I-FABP in electroporated ES cell clones containing the plasmid pBA25-I was analyzed by quantitative Western blotting (see Fig. 3A). Cells were washed in phosphate-buffered saline, trypsinized, and centrifuged. The cell pellets were resuspended in lysing buffer [10 mM tris(hydroxymethyl)aminomethane (Tris) · HCl, 100 mM NaCl, 1 µM leupeptin, 2 µM pepstatin, and 100 µg/ml phenylmethylsulfonyl fluoride] and sonicated in pulse mode four times for 20 s over 30 min at 4°C. The samples were centrifuged at 10,000 g for 20 min at 4°C. Exact protein concentrations of the resulting supernatants were determined by the Bradford method (8). Protein samples (20 µg of supernatant) were run on tricine gels for high resolution of the low-molecular-mass proteins in the 10- to 20-kDa range. To avoid differential loss in loading low amounts of I-FABP standards (0.3-1.8 ng I-FABP) in the Western gels, L cell fibroblasts expressing a known amount of I-FABP (32, 33) were used as standards for I-FABP protein quantitation. The protein was transferred to 0.45-µm nitrocellulose paper (Sigma Chemical) by electroblotting in a continuous buffer system at 0.8 mA/cm2 for 4 h. The Phototope-HRP Western blot detection kit (New England Biolabs, Beverly, MA) with a polyclonal-monospecific rabbit anti-intestinal fatty acid binding protein antiserum (1:1,000 dilution) was used to detect I-FABP expression in the protein samples in which the blocking buffer was 5% casein in Tris-buffered saline with 0.1% Tween 20. The blot was further developed according to product literature. To determine if other binding proteins were affected by differentiation or expression of I-FABP in the ES cells, Western blot analysis was performed on the clones using antibodies against L-FABP, acyl CoA binding protein (ACBP), and sterol carrier protein 2 (SCP-2) (see Fig. 3, B-D). Protein samples (30, 10, and 50 µg of supernatant) were loaded on tricine gels with the corresponding protein standards for quantitation (L-FABP, ACBP, and SCP-2). The gels were run as described above with the following exception: anti-rabbit immunoglobulin G alkaline phosphatase conjugate and Sigma Fast 5-bromo-4-chloro-3-indolyphosphate p-toluidine salt/nitro blue tetrazolium tablets (Sigma Chemical) were used for blot development. The polyclonal-monospecific rabbit antibodies (1:1,000 dilution) made against I-FABP, L-FABP, ACBP, and SCP-2 were developed for use in Dr. Schroeder's laboratory.

Proteins were quantitated by densitometric analysis following image acquisition using a single-chip charge-coupled device video camera and a computer workstation (IS-500 system from Alpha Innotech, San Leandro, CA). Image files were analyzed (mean 8-bit gray scale density) on a Power Macintosh workstation using NIH Image, a program written by W. Rasband and available by anonymous FTP from zippy.nimh.nih.gov.

Lipid determination. All glassware was washed with sulfuric acid-chromate before use. Cells from three 10-cm2 plates were combined to make one sample (n = 1). Total triglyceride and phospholipid content were determined by the method of Marzo et al. (26). Protein concentrations were determined by the method of Bradford (8). The lipids were separated by thin-layer chromatography on silica gel g plates (Analtech, Newark, DE) using the following solvent system: petroleum ether-diethyl ether-methanol-acetic acid (90:7:2:0.5).

Laser cytometry. Single cell NBD-stearic acid uptake was measured by laser cytometry using an ACAS Ultima (Meridian Instruments, Okemos, MI) equipped with two photomultiplier tubes (PMT) for fluorescence detection, a 5-W argon-ion laser (Coherent, Sunnyvale, CA), an X-Y motorized stage, and an Olympus IM-T inverted epifluorescence microscope (Olympus, Lake Success, NY). The laser power was set at 100 mW, with the excitation beam (488 nm) passing through a 1% neutral density filter. Emission was measured at 530 nm with the PMT voltage gain set at 3% (0.03 mW). All studies were performed at room temperature and optimized to ensure maximum fluorescence intensity with minimal photobleaching.

NBD-stearic acid kinetic uptake studies were performed in mock-transfected control and transfected cell lines with increasing concentrations of NBD-stearate ranging from 0 to 1.0 µM. This also allowed selection of NBD-stearic acid probe concentrations at nonsaturating uptake levels. Because there was no significant difference between untransfected control ES cells and mock-transfected control ES cells in the uptake experiments, data for both were combined and used as the control. The total area of the cell showing increased fluorescence over time was measured and used to gauge the rate of uptake of NBD-stearate into the cell. The maximum capacity (fluorescence intensity) values (Fmax) were obtained by fitting the saturation curves to a rectangular hyperbola with the equation: I = Fmax · t/(B + t), where I is fluorescence intensity, B is a constant, and t is time. Fmax and initial rate parameters were determined at 0.4 µM NBD-stearate. To determine the initial rates, the linear regions (0-50 s) of the uptake curves were fit to the linear equation y = mx + B, where m is the slope that corresponds to the initial rate. Alternatively, for a short period of time, the uptake rate can be determined using the ratio Fmax/B from the equation describing the rectangular hyperbola.

To perform the uptake experiments, the cells were removed from the 37°C CO2 incubator and washed at 24°C with preequilibrated Puck's buffer [in mM: 1.0 Na2HPO4, 0.9 H2PO4, 5 KCl, 1.8 CaCl2, 0.6 MgSO4 · 7H20, 6.0 glucose, 138 NaCl, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)] to remove serum-containing medium. Puck's buffer (0.5 ml) at 24°C was then added to the cells, the chamber slides were placed on the microscope stage, and the cells were allowed to complete equilibration at room temperature. An area containing two to five cells was then located and scanned for background fluorescence. NBD-stearate was added to give a final concentration of 0.4 µM in 1 ml of buffer with ethanol concentrations kept below 0.1%. Digital fluorescence imaging was then performed to observe single cell NBD-stearic acid uptake.

Although it was not possible to simultaneously determine the level of expression of I-FABP as well as NBD-stearic acid uptake in each individual cell on the coverslip, the G418 clonal selection procedure ensured a homogeneous population of cells expressing I-FABP. Those clones not expressing I-FABP did not survive G418 selection. Clones I-1 and I-7 were checked for I-FABP expression by Western blot analysis before preparing the chamber slides for NBD-stearic acid uptake. When NBD-stearic acid uptake was measured, the population of cells in each clone expressing I-FABP was measured by random sampling of cells from different areas of the coverslip. When the NBD-stearic acid uptake was plotted for each cell individually, each cell from the I-FABP overexpressing population had increased NBD-stearic acid uptake (see RESULTS). In addition, data from these samplings showed statistically significant differences between the clones that correlated with the I-FABP expression levels previously determined. This ensured that the cells on the slide represented the whole population.

Intracellular localization. To determine the subcellular localization of NBD-stearic acid, digital images were acquired using an MRC-1024 laser scanning confocal imaging system with LaserSharp image software (Bio-Rad, Hercules, CA) equipped with three PMT for fluorescence detection in separate channels, a 15-mW krypton-argon laser (American Laser, Salt Lake City, UT) with a 5-mW output measured at the microscope stage, and an Olympus IX70 inverted epifluorescence microscope using a PlanApo ×60 oil immersion objective with a numerical aperture of 1.4. Cells were exposed to the light source for a minimal amount of time to reduce photobleaching. To determine if NBD-stearic acid localized in the lipid droplets, the ES cells (undifferentiated, differentiated, clones, and controls) were incubated with nile red, a neutral lipid stain, in the presence of NBD-stearic acid in a series of colocalization experiments. Cells, plated in chambered slides at a density of 50,000 cells/well, were rinsed in room-temperature Puck's medium and incubated with NBD-stearic acid (0.4 µM) and nile red (0.5 µM) for 5 min at room temperature. After incubation, individual cells were chosen and fluorescence imaging was performed using 488-nm excitation with a 522/532 band-width filter for NBD-stearic acid (green channel, PMT1) and 568-nm excitation with 598-nm band-path emission filter for nile red (red channel, PMT2). The confocal images derived from the green and red channels were then combined. Organelles wherein NBD-stearic acid and the lipid stain, nile red, colocalized appeared as orange [red and green are additive and yield orange in red, green, blue (RGB) color space].

Fluorescence recovery after photobleaching. The intracellular diffusional mobility of NBD-stearic acid, expressed as Deff, was first described by Luxon and Weisiger (24). Deff values for control and transfected cell lines were determined by fluorescence recovery after photobleaching (FRAP) using established methods (24, 25). Briefly, the cells were washed twice with Puck's buffer at 24°C as above and 0.5 ml of buffer was added to the chamber slides. The cells were loaded with NBD-stearate at a final concentration of 0.4 µM in 1 ml buffer at room temperature. After a 5-min incubation, the cells were washed with buffer to remove unincorporated NBD-stearate, placed on the stage, and scanned to find an area with two to five fluorescent-labeled cells. An area between the cell membrane and nucleus was photobleached with a 15-ms blast by 1 mW of laser power (beam radius set at 1.3 µm) to give a 70-90% bleach of NBD-stearate. In choosing sites for laser photobleaching, areas that included lipid droplets, the nucleus, and other organelles were avoided. Digital fluorescence imaging was used to monitor fluorescence recovery of NBD-stearate into the area. The Deff was calculated from the photobleach recovery curves using the analysis software provided by the manufacturer, Meridian Instruments.

Statistics. All values are expressed as means ± SE, with n and P indicated in the RESULTS. Statistical analyses were performed using Student's t-test (GraphPad Prism, San Diego, CA). Values with P < 0.05 were considered statistically significant.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

I-FABP expression modulates NBD-stearic acid uptake parameters in ES cells. To study the effect of I-FABP expression on fatty acid uptake and intracellular diffusion in an undifferentiated and differentiated cell system, ES cells were stably transfected with cDNA (Fig. 1) encoding rat I-FABP. After transfection with linearized plasmid pBA25-I and selection in G418, several stable cell lines survived and were expanded for further analysis by PCR and Western blot analysis. For the PCR analysis, primers were designed to detect the presence of plasmid pBA25-I. Several clones produced the expected PCR product, and two of these, I-1 and I-7, are shown in Fig. 2 along with the untransfected control ES cell line. A positive control consisting of plasmid pBA25-I digested with Bgl II and Xho I is shown in lane 1 in the PCR gel. Clones positive for the PCR screen were examined by Western blot analysis with only two (I-1 and I-7) showing I-FABP protein expression (Fig. 3A). Expression levels of I-FABP in I-1, I-7, and the untransfected ES cell line in both the undifferentiated and differentiated cells were quantitated by densitometric analysis of the Western blot (Fig. 3A). Calibration of I-FABP quantitation was performed by comparison to a cell lysate expressing known quantities of I-FABP in mouse L fibroblasts (see MATERIALS AND METHODS) and analyzed on the same Western blot (Fig. 3A). The levels of I-FABP expression in the cell lines studied were 0.024 and 0.05 ng/µg protein for undifferentiated clones I-1 and I-7, respectively. Neither the untransfected-undifferentiated nor the untransfected-differentiated control ES cell line expressed detectable amounts of I-FABP. Differentiation decreased the level of I-FABP expression in clones I-1 and I-7 by 1.5- to 3-fold (to 0.016 and 0.014 ng/µg protein), respectively.


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Fig. 1.   Linear map of plasmid pBA25-I. Entire coding region of rat intestinal fatty acid binding protein (I-FABP) cDNA was cloned into the unique BamH I restriction site of vector pcDNA3 to place the insert between the human cytomegalovirus promoter (PCMV) and the bovine growth hormone polyadenylation (BGHpA) and splice sequences. Arrows indicate positions of the 5'-start and 3'-halt primers used for the polymerase chain reaction (PCR) analysis described in Fig. 2. ori, Origin.


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Fig. 2.   PCR analysis of DNA isolated from embryonic stem (ES) cells. Cells were transfected with cDNA encoding for I-FABP as described in MATERIALS AND METHODS. DNA isolated from the transfected clones or control ES cells was used as a template in the PCR analysis with the samples run on a 1% agarose gel and visualized by ethidium bromide staining. Lane 1, positive control [1330 base pairs (bp)] resulting from plasmid pBA25-I digested with Bgl II and Xho I; lane 2, molecular weight markers; lane 3, negative PCR control generated from using DNA isolated from untransfected ES cells; and lanes 4 and 5, PCR products using DNA isolated from ES cells expressing I-FABP clones I-1, and I-7, respectively.


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Fig. 3.   Western blot analysis of ES cells expressing I-FABP in undifferentiated (undif) and differentiated (dif) cells. Protein lysates were run on 16% tricine gels, loaded as follows: lane 1, ES cell clone I-1 (undif); lane 2, I-7 (undif); lane 3, ES cell control (undif); lane 4, I-1 (dif); lane 5, I-7 (dif); and lane 6, ES cell control (dif). Remaining lanes were used to generate a standard curve for quantitation of the following proteins: 0.3-1.8 ng of I-FABP (A), 0.3-1.0 ng liver fatty acid binding protein (L-FABP; B), 2-10 ng acyl CoA binding protein (ACBP; C), and 0.3-0.9 ng sterol carrier protein 2 (SCP-2; D).

In some differentiating cell systems (e.g., intestinal epithelial cells), I-FABP is coexpressed with another fatty acid binding protein, L-FABP. More important, enhanced expression of I-FABP in such cells resulted in altered expression of L-FABP (4). Therefore, it was necessary to determine if I-FABP expression in the stably transfected ES cell clones was an effect compensated by altered L-FABP expression. To resolve this possibility, L-FABP expression was measured in transfected and control ES cell lines in both the undifferentiated and differentiated states. In addition, the level of expression of two other fatty acid and/or fatty acyl CoA binding proteins, SCP-2 and ACBP, was determined in other Western blots run in parallel (Fig. 3, B-D). Levels of L-FABP were not detectable in any cell line. ACBP, which binds fatty acyl CoAs but not fatty acids, was present at high levels in all cell lines (representing 0.017% of the total protein) but was unaffected by I-FABP expression. The expression of SCP-2 in all samples was very low (0.001% of total protein), near the limit of detection. To resolve whether this low level of SCP-2 immunoreactivity might be attributable to cross-reactivity with other proteins of similar molecular weight, Northern blot analysis was performed wherein it was observed that SCP-2 mRNA was barely above levels of detectability in all cell lines compared with the high amounts of SCP-2 mRNA observed in mouse liver (data not shown).

Intracellular distribution of NBD-stearic acid in ES cells. As pointed out in the introduction, most previous determinations of fatty acid uptake with cultured cells utilized fluorescent or radiolabeled fatty acids with cells in suspension or on a monolayer where there was no distinction between internalized vs. plasma membrane adsorbed fatty acid. Therefore, fluorescence confocal imaging of single cells was undertaken to examine fatty acid intracellular distribution in ES cells.

The intracellular distribution of NBD-stearate at maximal uptake (see Fig. 4) was examined in both undifferentiated and differentiated ES cells (Fig. 4). Undifferentiated ES cells had a rounded, adipocyte-like morphology (Fig. 4, A-C), whereas differentiated ES cells had a fibroblast-like morphology (Fig. 4D). During the uptake experiments, it was observed that, whereas cells in both the undifferentiated and the differentiated state had NBD-stearic acid localized throughout the cell, very little NBD-stearic acid fluorescence appeared associated with the cell surface membrane or in the nuclear region. Instead, the NBD-stearic acid appeared within the cell interior and highly localized/accumulated in intense cytoplasmic staining regions believed to be lipid droplets. To determine if the latter were the case, a series of colocalization experiments were performed to ascertain if NBD-stearic acid and nile red colocalized. Nile red, known to selectively stain lipid droplets (10-13), was coincubated with NBD-stearic acid in chambered slides containing undifferentiated and differentiated control and transfected ES cells. Colocalization experiments were performed on an MRC-1024 laser scanning confocal imaging system in which the fluorescence image from nile red (red channel) was superimposed on the image for NBD-stearic acid (green channel) to give an orange color when both dyes were colocalized. It was clear that both NBD-stearic acid and nile red had very similar intracellular localization patterns. This result was consistent with NBD-stearate preferentially accumulating in lipid droplets.


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Fig. 4.   Intracellular distribution of 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-octadecanoic acid (NBD-stearic acid) and nile red in undifferentiated and differentiated ES cells. Pseudo-coloring was used to show the colocalization patterns in which the 8-bit gray scale (intensity) look-up table, resulting from confocal image acquisition from individual red- and green-specific photomultiplier tube channels, was reassigned linear red and green pseudo-color palettes, respectively. A 24-bit red, green, blue (RGB) image was created from the red plus green plus blue (null) channels. Red and green are additive in RGB color space, yielding orange, thus representing the colocalization of red- and green-specific fluorescence. An ES cell (undifferentiated) stained with nile red (red, A) and NBD-stearate (green, B) were combined to yield orange (C) in which both probes are colocalized. A similar experiment was performed with a differentiated ES cell (D). Arrows in A-C point to areas within the cell where stain is quickly localized to lipid droplets.

If the decrease in NBD-stearic acid uptake parameters in the differentiated vs. undifferentiated ES cells (see Fig. 6) were due to a gradient effect, then a decrease in lipids comprising the neutral lipid droplet should be observed. However, this was not the case. Neutral lipid content was quantitated in the undifferentiated and differentiated cell lines by extraction of cellular lipids, chromatographic separation, and quantitation of triglyceride, cholesterol ester, total neutral lipid, and total phospholipid compositions (Table 1). A twofold increase, rather than a decrease, was observed in total neutral lipids per milligram protein in differentiated vs. undifferentiated ES cells. This differentiation-induced increase in the total neutral lipids was primarily in the triglycerides, which increased nearly 4-fold compared with the cholesteryl esters, which increased 1.4-fold. A 1.8-fold increase was also observed for phospholipids, which provide neutral lipid droplet surface components as well as intracellular membrane lipid matrices. Likewise, a gradient effect was also not observed in the differentiated vs. undifferentiated I-FABP-expressing ES cells (see Fig. 6, Table 1). I-FABP expression and differentiation effects on specific neutral lipid classes were distinct. I-FABP expression in undifferentiated cells increased neutral lipid per milligram protein by 18-fold (Table 1): 6.6-fold increased triglycerides and 19.5-fold increased cholesteryl esters. In addition, a 5.8-fold increase was observed in phospholipids per milligram protein, respectively, when the undifferentiated I-FABP expression clone (clone I-7) was compared with the undifferentiated ES cell control. In the differentiated cells, neutral lipid per milligram protein increased only 2.9-fold: 2-fold increased triglycerides and 3-fold increased cholesteryl esters. In contrast, the total phospholipid per milligram protein remained unchanged (compare differentiated ES cell control with differentiated clone I-7). These results suggest that I-FABP expression and differentiation in the absence of I-FABP expression enhanced neutral lipid and phospholipid production in the undifferentiated cells, whereas differentiation concomitant with I-FABP expression dramatically attenuated and eliminated, respectively, the effects of I-FABP on neutral lipid and total phospholipid content. As observed for the non-I-FABP-expressing cells, differentiation affected the cholesteryl esters much more than the triglycerides. These changes did not support a gradient effect as accounting for differences in NBD-stearic acid uptake.

                              
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Table 1.   Lipid composition of undifferentiated and differentiated ES cells expressing I-FABP compared with control cell line

Concentration dependence of NBD-stearic acid uptake in control ES cells. To optimize conditions for determining effects of differentiation and I-FABP expression on NBD-stearic acid uptake, it was first necessary to establish the NBD-stearic acid concentration dependence of fluorescence intensity in ES cells. The initial rate of NBD-stearic acid fluorescence increase (Fig. 5A) and the maximal fluorescence increase (Fig. 5B) were linear at low NBD-stearic acid concentrations. At concentrations >0.4 µM NBD-stearic acid, the NBD-stearic acid initial rate (Fig. 5A) and maximal uptake (Fig. 5B) deviated from linearity. In ES cells, the NBD-stearic acid concentration at which the uptake parameters were well below saturation and at which adequate signals for all uptake studies (as well as FRAP, see below) were presented was 0.4 µM. Therefore, this NBD-stearic acid concentration was used for all subsequent studies on NBD-stearic acid uptake, localization, and diffusion.


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Fig. 5.   Initial rate and maximal capacity (Fmax) of NBD-stearic acid uptake for ES cells. Top: initial rate at several concentration ranging from 0.1 to 1.0 µM was determined as described in MATERIALS AND METHODS to obtain the optimal concentration for uptake studies. Data are expressed as means ± SE. Bottom: Fmax for NBD-stearic acid was determined as described in MATERIALS AND METHODS. Data are expressed as means ± SE.

Effect of ES cell differentiation on uptake and internalization of NBD-stearic acid. Differentiation of control ES cell lines dramatically inhibited NBD-stearic acid uptake and internalization. The initial rate of NBD-stearic acid uptake decreased 6.9-fold (Fig. 6A), whereas maximal uptake of NBD-stearic acid concomitantly decreased 3.1-fold (Fig. 6B). These results suggest that differentiation of ES cells from rounded to fibroblastic morphology decreased fluorescent NBD-stearic acid incorporation into the cell.


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Fig. 6.   Initial rates and Fmax of NBD-stearic acid uptake for clones I-1, I-7, and the control line in the undifferentiated and differentiated state. Top: initial rate data were obtained from fatty acid uptake curves from the slope of the linear region (10-40 s). Values are means ± SE, n = 7-27. * Significance, P < 0.0002 compared with the control. Bottom: Fmax values of NBD-stearic acid uptake data were obtained from the fatty acid uptake curves fitted to a rectangular hyperbola where I = Fmax · t/(B + t), where t is time, B is a constant, and I is the intensity. Values are means ± SE, n = 7-27. * Significance, P < 0.007 compared with the control.

Concentration dependence of NBD-stearic acid uptake in transfected ES cells overexpressing I-FABP. To establish optimal NBD-stearic acid concentration for uptake and intracellular diffusion studies in I-FABP-expressing cells, fluorescence vs. concentration curves were established as described above for the control ES cells. Similar to observations with control ES cells, at low concentrations, NBD-stearic acid uptake in clone I-1 ES cells expressing I-FABP was linearly dependent on concentration (Fig. 7). At higher NBD-stearic acid levels, both the initial rate (Fig. 7A) and the maximal uptake (Fig. 7B) of NBD-stearic acid deviated from linearity in the I-FABP-expressing ES cells. As observed for the control ES cells (Fig. 5, A and B), in the I-FABP-expressing cells, neither uptake parameter was saturated even at 1 µM NBD-stearic acid. These studies allowed establishment of conditions for measuring NBD-stearic acid uptake wherein selfquenching and associated artifactual differences in NBD-stearic acid quantum yield were avoided. Fluorescence quenching of NBD-stearic acid resulting from a high probe concentration was avoided by obtaining all data under nonsaturating, linear conditions (Figs. 5 and 7). Furthermore, the uniformity of the microenvironments in which the NBD-stearic acid was localized in each clone was determined from the emission spectra of the probe located in a suspension of control, mock-transfected, and I-FABP-expressing ES cells. In all cases, the emission maximum was at 532 nm, which corresponded to a dielectric constant of 2-4 (38). Thus the cell-associated NBD-stearic acid was localized in a very hydrophobic environment, as found in membranes and lipidic particles. Consequently, it is unlikely that differences in NBD-stearic acid uptake parameters between the control, mock-transfected, and I-FABP ES cell clones (see below) were due to dissimilar microenvironments in which the NBD-stearic acid probe differed significantly in quantum yield.


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Fig. 7.   Initial rate and Fmax of NBD-stearic acid uptake for clone I-1 expressing I-FABP. Top: initial rate values were determined at several concentrations from 0.4 to 1 µM to determine the optimal concentration for uptake studies. Solid line represents a spline curve in which the most probable connection of the points is made taking into account SE values. Individual values are means ± SE. Bottom: Fmax for NBD-stearic acid for clone I-1 expressing I-FABP was determined as described in MATERIALS AND METHODS. Values are means ± SE.

NBD-stearic acid uptake in I-FABP-expressing ES cells. Digital fluorescence imaging of single cells was utilized to determine the effects of I-FABP expression and differentiation in ES cells. The ability to facilitate movement of the fatty acid probe NBD-stearate through and within the cell was measured to gauge the effects of I-FABP expression. To directly compare NBD-stearic acid uptake parameters between the I-FABP-expressing and control ES cells, the same nonsaturating NBD-stearate concentration (0.4 µM) was selected. In the undifferentiated state, the maximum NBD-stearate uptake was increased 1.5- and 1.6-fold, respectively (P < 0.0002, n = 7-33), for I-FABP-expressing I-1 and I-7 cells over the control (Fig. 6B). This observation was consistent with Fmax not being saturated in I-FABP-expressing cells even at 1 µM NBD-stearic acid (Fig. 7A). The initial rates of uptake for both of the I-FABP-expressing ES cell lines (I-1 and I-7) were 1.6- and 1.7-fold greater (P < 0.0001, n = 7-36) than controls (Fig. 6A). This observation was likewise consistent with initial rate of uptake of I-FABP-expressing cells not being saturated even at 1 µM NBD-stearic acid (Fig. 7A).

Effect of differentiation on NBD-stearic acid uptake in transfected ES cells expressing I-FABP. In the differentiated ES cells expressing I-FABP, Fmax (Fig. 6B) and initial rates (Fig. 6A) were not significantly different from the control. When Fmax and initial rate values were compared between the undifferentiated and differentiated I-FABP-expressing clones, a respective 4.7- and 10-fold decrease on differentiation was observed (P < 0.0001, n = 12 and 13, respectively). These results indicated that, whereas higher expression of I-FABP in undifferentiated ES cells increased the capacity and initial rate of uptake for fatty acids over the control, differentiation caused a substantial decrease in both parameters. Differentiation negated the effect of I-FABP expression and caused the maximal uptake and initial rate of uptake to decrease such that there was no significant difference between the cell lines. Overall, ES cell differentiation depressed the ability to facilitate internalization and intracellular movement of fatty acids.

Effect of ES cell differentiation on Deff of NBD-stearic acid. The Deff of NBD-stearic acid in undifferentiated and differentiated ES cells was measured by FRAP. ES cells were incubated with NBD-stearic acid at a nonsaturating concentration (0.4 µM). After a 5-min incubation, when the probe was completely equilibrated within the ES cells, the intensity of NBD-stearic acid was measured across a single ES cell. Each cell was then subjected to a short laser "blast" that bleached 70-90% of the signal. The recovery of NBD-stearate fluorescence intensity (Fig. 8) at the center of the bleached beam was then analyzed as a function of time as described in MATERIALS AND METHODS to obtain the Deff. The Deff (Fig. 9) for the undifferentiated control cell line was 0.83 ± 0.23 × 10-9 cm2/s (n = 17), which correlated closely with the differentiated value of 0.81 ± 0.06 × 10-9 cm2/s (n = 18). Differentiation of the control cell line did not decrease NBD-stearic acid intracellular diffusion.


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Fig. 8.   Representative reciprocal fit of the fluorescence recovery curve for undifferentiated ES cells using NBD-stearate as the fatty acid fluorescent probe. Points indicate fluorescence intensity at the center of bleach vs. time. An effective intracellular diffusion constant (Deff) of 0.58 × 10-9 cm2/s with an initial bleach of 89%, a 77% recovery after photobleaching, and a mobile fraction (52) of 0.74 were found for this representative experiment.


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Fig. 9.   Deff at 0.4 µM for I-1, I-7, and the control cell lines in the undifferentiated and differentiated state was determined using fluorescence recovery after photobleaching as described in MATERIALS AND METHODS. Values are means ± SE, n = 10-20. * Significance, P < 0.01. ** Significance, P < 0.0002. Both are compared with the control.

Effect of I-FABP expression in undifferentiated ES cells on Deff of NBD-stearic acid. The NBD-stearate Deff was increased 1.3- and 1.8-fold (P < 0.01, n = 10-20), respectively, in the undifferentiated I-1 and I-7 clones compared with the undifferentiated ES cell control (Fig. 9). When the two clones were compared, a significant difference in Deff was observed (P < 0.01). The increase in Deff correlated with the level of I-FABP expression. These results indicate that expression of I-FABP in the cells enhanced intracellular movement of NBD-stearate.

Effect of differentiation on Deff of NBD-stearic acid in I-FABP-expressing ES cells. Differentiation of I-FABP-expressing ES cell clones reversed the effects on intracellular diffusion of NBD-stearic acid observed in undifferentiated I-FABP-expressing ES cells. The NBD-stearate Deff values for differentiated I-1 and I-7 ES cell clones were not significantly different from each other or from the control cell line (Fig. 9). However, comparing undifferentiated clones expressing I-FABP to differentiated clones revealed a 1.6- and 2.1-fold decrease for I-1 and I-7, respectively. Thus fatty acid movement within I-FABP-expressing cells appeared enhanced in undifferentiated cells but was suppressed in differentiated cells.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Despite the multiple regulatory functions of fatty acids, very little is known regarding the role(s) that cell differentiation and cytoplasmic fatty acid binding proteins have in regulating fatty acid uptake and/or intracellular diffusion in intact cells. Although some reports examined radiolabeled fatty acid uptake and metabolism in differentiated adipocytes (3, 49) and intestinal Caco-2 cells grown on oriented filters (4, 47), the use of radiolabeled fatty acid did not readily resolve binding to the plasma membrane from alterations due to intracellular fatty acid binding protein expression, intracellular membrane binding sites, intracellular diffusion, or metabolism. As pointed out in the introduction, each of these parameters can influence cellular fatty acid uptake.

In the present study, advantage was taken of several technologies to fully explore the effect(s) of cellular differentiation on fatty acid uptake and intracellular diffusion. First, ES cells, widely used in transgenic research, were chosen as the model system because they can be readily induced to differentiate into morphologically distinct fibroblast-like cells (45). Second, NBD-stearic acid was used as a nonmetabolizable fatty acid (7) to discriminate between fatty acid uptake/binding into the plasma membrane vs. intracellular metabolism of fatty acid. Third, single cell fluorescence imaging allowed examination of fatty acid internalization vs. simply surface binding, a complication present in most cell suspension fluorescence assays (28, 29, 33-35, 37). Fourth, as shown in the present work, ES cells did not express detectable levels of the major cytoplasmic fatty acid binding proteins normally found in intestine (I-FABP, L-FABP) or of an unrelated fatty acid binding protein ubiquitous in all cells examined, (SCP-2) (38, 42). ES cells, therefore, provide a model for transfection with cDNA encoding I-FABP and measuring the effects of I-FABP expression on fatty acid uptake as well as the effects of cellular differentiation on this process.

As shown herein, NBD-stearic acid was taken up with a half time of <1 min (near that observed for radiolabeled fatty acids in a variety of cell systems), translocated throughout the cell, and accumulated in intracellular structures that colocalized with nile red (stain for lipid droplets/particles). Furthermore, I-FABP dramatically increased neutral lipid storage in undifferentiated ES cells and, to a lesser extent, the differentiated cells. The latter correlated with decreased uptake, diffusion, and I-FABP expression levels observed in the differentiated cells.

Differentiation of ES cells dramatically inhibited NBD-stearic acid uptake, measured by single cell fluorescence imaging, with the effect being greatest for differentiated I-FABP-expressing cells. In contrast, in another differentiating system, insulin induced differentiation of 3T3 preadipocytes (fibroblast-like) into adipocytes (round/spherical) and concomitantly enhanced fatty acid uptake 36-fold (14, 54), which is consistent with the expression of cytosolic adipocyte fatty acid binding protein in the differentiated 3T3 cells (49). Although this change appears opposite to that observed herein with differentiated ES cells, in both systems the cells with fibroblast-like morphology (preadipocytes and differentiated ES cells) had lower fatty acid uptake.

A postulated function of cytosolic fatty acid binding proteins is to stimulate fatty acid uptake/translocation into the cell. In cell suspension assays using cis-parinaric acid uptake, very different results were obtained with transfected L cells overexpressing L-FABP vs. I-FABP. Both cell lines have the same fibroblast morphology as differentiated ES cells, yet L cells overexpressing L-FABP showed enhanced cis-parinaric acid uptake (29, 33), whereas I-FABP-overexpressing L cells did not (32, 33). This correlates with the data observed in this work with differentiated ES cells. However, the cell suspension assay used in these studies did not readily discriminate between cis-parinaric acid incorporation into the cell surface membrane vs. internalization. Likewise, although secretion of radiolabeled triglyceride by Caco-2 cells growing on filters was stimulated in transfected Caco-2 cells expressing I-FABP expression (4), these methods did not differentiate between effects of I-FABP on triglyceride formation vs. uptake, internalization or diffusion. In contrast, single cell imaging studies presented herein showed that I-FABP expression in undifferentiated ES cells stimulated the NBD-stearic acid initial rate and maximal uptake 1.7- and 1.6-fold, respectively. The effects of I-FABP expression in the undifferentiated ES cells (rounded morphology) were negated on differentiation to the fibroblast-like morphology. The latter observations are consistent with those observed with I-FABP-expressing L-cells, which also exhibit the fibroblast morphology (32-34).

With regard to intracellular mobility, I-FABP expression in undifferentiated transfected ES cells increased the Deff as much as 1.8-fold. These results provide some of the first evidence that cytosolic I-FABP can stimulate intracellular mobility of a fatty acid in intact cells in which only a minor genetic change has been introduced. Although the magnitude of this effect may appear modest, it must be considered that the Deff actually represents an average diffusion constant of NBD-stearic acid in both the cytoplasmic and membrane-bound intracellular compartments (25). A microsomal-aqueous buffer partitioning assay revealed that more than 95% of added fatty acid was associated with the membranes (18). Thus Deff values could underestimate the actual effect of I-FABP expression on the cytoplasmic diffusion component. It should be noted that the enhanced Deff in I-FABP-expressing cells is also consistent with earlier work of others showing 1.65-fold-enhanced NBD-stearic acid Deff in female (higher L-FABP expression) vs. male hepatocytes (25). However, the Deff values obtained for the different types of ES cells (0.8-1.6 × 10-9 cm2/s) were significantly three- to sixfold slower than those obtained for hepatocytes from female rat livers (4.78-5.23 × 10-9 cm2/s). This observation is consistent with the much higher expression of L-FABP (near 3% of cytosolic protein) in hepatocytes compared with the I-FABP expressed in ES cells described herein and further supports a role for cytoplasmic fatty acid binding protein expression enhancing cytoplasmic diffusion of fatty acids. Finally, it should be noted that the faster cytoplasmic diffusion of fatty acids observed herein and by others is not specific for the fatty acid binding protein family. Other proteins that bind fatty acids, e.g., albumin, also enhance intracellular diffusion when present inside the cell (23). Liver expresses significant amounts of albumin, which could also account for the faster diffusion of fatty acids described above. In this study, differentiation decreased I-FABP expression up to threefold and negated the effects of I-FABP expression on Deff.

The goal of this work was to examine the role of I-FABP in fatty acid movement and utilization in ES cells. In the undifferentiated cells, increased I-FABP expression correlated with an increase in fatty acid uptake and diffusion. In contrast, differentiation abolished effects of I-FABP expression on these parameters. Several mechanisms may account for this effect. First, we speculate this differentiation-dependent effect was due to the up to threefold downregulation of I-FABP expression in the differentiated I-FABP-expressing cells. Second, differentiation did not affect expression levels of several other cytoplasmic fatty acid and/or fatty acyl CoA binding proteins (SCP-2, L-FABP, and ACBP). Third, whereas differentiation of ES cells from rounded adipocyte morphology to fibroblast-like morphology is consistent with a downregulation of fatty acyl CoA synthase activity, the lipid composition data were not entirely consistent with this accounting for the abolition of I-FABP expression effects on fatty acid uptake/diffusion. A role for fatty acyl CoA synthase in stimulating fatty acid uptake was first established by Shaeffer and Lodish (36). However, I-FABP did not directly affect the activity of this enzyme (17). In addition, downregulation of fatty acyl CoA might be expected to result in reduced formation of triglycerides, phospholipids, and cholesteryl esters. However, the data show that, whereas phospholipids were decreased in the differentiated ES cells expressing I-FABP, triglyceride and cholesteryl ester levels were still two- and threefold higher than differentiated control ES cells. Fourth, loss of transport activity may have occurred by downregulation of the plasma membrane fatty acid binding protein, transporter, or translocase. Several of these proteins have been demonstrated in other cell types (2, 43, 44, 46). However, which one or more of these plasma membrane fatty acid transporters is expressed and/or regulated by differentiation in ES cells remains to be determined. In any case, differences in plasma membrane transporter expression could alter the NBD-stearic acid uptake parameters but not its intracellular mobility.

In summary, with the ES cell clones, differentiation markedly inhibited fatty acid uptake and reduced the intracellular mobility of fatty acids. The expression of I-FABP in transfected ES cells showed for the first time that cytosolic I-FABP can enhance fatty acid uptake and intracellular mobility in single, undifferentiated ES cells. Furthermore, differentiation reversed/negated these differences in I-FABP-expressing cells compared with control ES cells. We speculate that the factor most contributing to the latter effect was the up to threefold decrease in I-FABP levels in the differentiated I-FABP-expressing ES cells. Finally, expression of I-FABP dramatically increased production of both polar and neutral lipids. Differentiation attenuated but did not abolish this effect of I-FABP, consistent with the decreased uptake, diffusion, and I-FABP expression levels observed in the differentiated cells.

    ACKNOWLEDGEMENTS

We thank Amy Boedeker, Anca Petrescu, and Rolla Barhoumi for their excellent technical assistance.

    FOOTNOTES

This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-41402.

Address for reprint requests: F. Schroeder, Dept. of Physiology and Pharmacology, Texas A&M Univ., TVMC, College Station, TX 77843-4466.

Received 25 June 1997; accepted in final form 10 November 1997.

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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Cell Physiol 274(3):C633-C644
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