Cytoplasmic transport of fatty acids in rat enterocytes: role of binding to fatty acid-binding protein

Bruce A. Luxon and Michael T. Milliano

Division of Gastroenterology and Hepatology, Department of Internal Medicine, St. Louis University Health Sciences Center, St. Louis, Missouri 63110-0250


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The intracellular movement of fatty acids is thought to be facilitated through codiffusion with fatty acid-binding protein (FABP). This facilitation may occur by decreasing binding to immobile membranes, leading to faster cytoplasmic diffusion. The aims of this study were to measure the intracellular transport of 12-N-methyl-(7-nitrobenzo-2-oxa-1,3-diazol)aminostearate (NBD-stearate) in villus rat enterocytes and to determine 1) the mechanism of its cytoplasmic transport and 2) if its transport rate correlated with the known variation of FABP binding capacity along the length of the small intestine. Two-dimensional laser photobleaching was used to measure the movement of a fluorescent fatty acid NBD-stearate in enterocytes isolated from different segments of rat intestine. The fraction of NBD-stearate found in the cytostol of enterocytes was determined by differential centrifugation. Cytoplasmic transport of NBD-stearate occurred solely by diffusion and not by convection. Diffusion was homogeneous (nondirectional), consistent with isotropic diffusion. The diffusion rate varied with location along the intestine, correlating with the local FABP concentration and measured cytosolic binding. We conclude that cytoplasmic proteins like FABP promote the intracellular transport of fatty acids by enhancing their diffusive flux. We suggest that facilitation is not specific for a particular cell type but occurs in a variety of cells that transport fatty acids and may contain different types of FABP.

fluorescence recovery after photobleaching; liver; NBD-stearate


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

LOW MOLECULAR WEIGHT cytosolic proteins capable of binding hydrophobic molecules like fatty acids, bile acids, and bilirubin have been studied in hepatocytes, enterocytes, and myocytes for nearly two decades (3, 10). These proteins have been well characterized with regard to their binding affinity for a variety of ligands (16, 20, 22), tissue localization (2, 4, 10, 19), and subcellular distribution (24). However, despite an enormous body of literature, their precise function remains an enigma (3, 10). It has been hypothesized that the intracellular transport of hydrophobic molecules like fatty acids is facilitated by binding to a group of these proteins called fatty acid-binding proteins (FABPs). This transport is thought to occur by simple diffusion, and indirect evidence has suggested that FABP promotes cytoplasmic transport by increasing the amount of fatty acid that is mobile (bound to soluble proteins) and decreasing the amount that is relatively immobile (bound to fixed cytoplasmic membranes). Direct experimental evidence supporting a role for FABP in the intracellular transport of fatty acids, however, is lacking because of the difficulty of measuring intracellular flux rates in intact living cells.

We previously used the technique of fluorescence recovery after photobleaching (FRAP) to measure the intracellular transport of a fluorescent fatty acid, 12-N-methyl-(7-nitrobenzo-2-oxa-1,3-diazol)aminostearate (NBD-stearate), in isolated rat hepatocytes from male and female rats (12, 14). We determined that transport occurred solely by simple diffusion and not by convection. We also noted that the diffusion rate was much slower than expected for diffusion in free water of either NBD-stearate itself or the NBD-stearate-FABP complex. We hypothesized that this marked retardation of the observed diffusion rate of NBD-stearate occurred because of extensive binding of the fatty acid to immobile membranes. In separate experiments, we displaced NBD-stearate from FABP by using a binding competitor, alpha -bromo-palmitate. If NBD-stearate was displaced from FABP, its observed diffusion rate decreased in proportion to the shift in the partition of NBD-stearate from cytosol to membranes (12). This finding supported the hypothesis that binding to FABP promotes the intracellular movement of fatty acids by decreasing the amount of fatty acid bound to immobile membranes.

The current studies were designed to determine if facilitation of the intracellular transport of fatty acids by FABP is specific for liver cells or if it occurs in other cells that contain FABP. To address this issue, we used laser photobleaching to measure the diffusion rate of the fluorescent fatty acid NBD-stearate in enterocytes isolated from the small intestine of rats. Because both the concentration and specific type of FABP change with location along the small intestine (5, 17, 19), this system represents a unique opportunity to study the tissue specificity of the facilitated codiffusion of fatty acids with FABP. To take advantage of this naturally occurring gradient of FABP expression, we measured the diffusion rate of NBD-stearate in villus cells isolated from the duodenum, jejunum, and ileum and correlated the observed diffusion rate of NBD-stearate with its degree of cytosolic binding.

The results of these experiments show that the diffusion rate of NBD-stearate as determined by laser photobleaching varied along the length of the small intestine, correlating with both the local FABP concentration and the measured partition of NBD-stearate between cytosol and immobile membranes. We suggest that these data indicate that intracellular binding proteins like FABP enhance the diffusive flux of fatty acids by modifying their partition between membrane and aqueous phases. These data provide further evidence that the FABPs serve as important carriers of hydrophobic molecules in a variety of tissues. These proteins increase the mobility of a variety of amphipathic and hydrophobic molecules, such as long-chain fatty acids, by decreasing their binding to membranes. In this manner they can increase the overall utilization of these important metabolic substrates by helping to shuttle hydrophobic molecules through the cytoplasm.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Sources of materials. BSA (essentially fatty acid free, product A6003) was from Sigma (St. Louis, MO). NBD-stearate was from Molecular Probes (Eugene, OR). Culture chambers and coverslips for laser photobleaching experiments were from Lab-Tek (product 4802, Los Altos, CA). Monoclonal antibodies to FABP were obtained from Research Diagnostic (product numbers RDI-FABP-10E1 and RDI-FABP-9F3, Flanders, NJ). All other chemicals and reagents were of the highest grade commercially available.

Cell isolation. Intestines from male Sprague-Dawley rats (55-60 days old) were separated into proximal (15 cm from the pylorus), middle, and distal (15 cm from the cecum) segments. Enterocytes were isolated with the use of the nonenzymatic method of Rowling as modified by Meddings et al. (15). Briefly, the intestine was flushed with the use of PBS containing 1 mM dithiothreitol (DTT) (product number D5652, Sigma Chemical) and then everted on a 4-mm-diameter glass rod. It was filled with citrate buffer (in mM: 96 NaCl, 27 sodium citrate, 5.6 KH2PO4, and 1.5 KCl, pH 7.3), and both ends were tied. The loop was then placed in a tube containing isolation buffer (PBS with 1.5 mM EDTA, 0.5 mM DTT). The intestine segments were gently shaken by hand inversion for 15 min at 37°C. Cells from the villus tips were then collected from the buffer by gentle centrifugation at 500 g. Midvillus cells were removed after an additional 20 min of shaking. Crypt cells were collected by gently scraping the remaining mucosa after both villus tip and midvillus cells were removed. Cells were maintained at 37°C under an atmosphere of 95% air-5% CO2 in PBS (pH 7.4) until used for laser photobleaching or binding experiments (~60 min). Viability was assessed by trypan blue exclusion after isolation and immediately before binding or photobleaching studies. Preparations were discarded if less than 85% of the cells excluded the dye.

Characterization of enterocytes. Cells released from the villus and crypt regions of each segment of intestine were identified by the alkaline phosphatase activity in the cell isolates. Cell samples were disrupted for 30 s with a sonicating homogenizer (Cole Parmer, Niles, IL). The alkaline phosphatase activity was determined with the use of a commercial kit (product number 245-10, Sigma Chemical). To account for the different number of cells isolated in the various sections, all measurements were normalized to total protein content as determined by the method of Bradford (6).

Measurement of FABP in enterocytes. To determine the amount of FABP, we used a commercially available IgG monoclonal antibody that detects both the intestinal and liver forms of FABP (25). Enterocytes were isolated as described above and were disrupted by sonication. The cytosolic fraction was prepared by centrifugation at 105,000 g for 60 min. The total FABP concentration was measured by Western blotting after loading 10 µg of total cell protein per lane. Bands of FABP (mol mass ~14 kDa) were detected by enhanced chemiluminescence (Amersham Life Sciences, Buckinghamshire, UK).

The FABP concentration was also measured by a sandwich ELISA on identical aliquots of enterocyte cytosol placed in 96-well ELISA plates (no. 1 565-136, Nunc Immuno). Two monoclonal antibodies recognizing the different epitopes of intestinal or liver FABP were used. Fluorescent signals from the detection antibody were measured in a microplate reader (CytoFluor II, PerSeptive Biosystems, Framingham, MA). Results were expressed as micrograms of FABP (either intestine or liver) per milligram of total cell protein.

Measurement of soluble fraction of NBD-stearate in enterocytes. To determine the partition of NBD-stearate between the aqueous cytosol and membranes, enterocytes were suspended in Krebs bicarbonate buffer (1-2 × 106 cells/ml) containing 1 g/dl BSA and 25 µM NBD-stearate for 15 min. The cells were separated from the incubation media by low-speed centrifugation (1,000 g) for 5 min; the cell pellet was washed twice with Krebs buffer to remove BSA and extracellular NBD-stearate and was resuspended in 10 ml of PBS (10 mM NaH2PO4 and 150 mM NaCl, pH 7.4) at 4°C. The cells were disrupted by homogenization for 15 s. The fluorescence was measured in 1 ml of the cell homogenate. The cytosolic fraction was prepared by centrifugation at 105,000 g for 60 min, and the fluorescence was measured in the supernatant. For all fluorescence measurements, care was taken to avoid spurious photobleaching or autoquenching. The fraction of NBD-stearate in the cytosol was expressed as the ratio of the fluorescence in the cytosolic supernatant to that initially present in the cell homogenate (12, 14).

Laser photobleaching. FRAP was used to measure the cytoplasmic movement of NBD-stearate in intestinal cells 1 h after isolation. Enterocytes were incubated in Krebs buffer containing 1 g/dl BSA and 25 µM NBD-stearate for 15 min at 37°C. After incubation, photobleaching experiments were performed as previously described (12-14) with the use of a custom-designed video laser photobleaching apparatus. A brief pulse of high-intensity laser illumination was used to irreversibly bleach the NBD-stearate molecules in the selected area. The bleach-beam radius was nominally set at 1.3 µm and was calculated in each experiment as described previously (12-14). Following the bleaching pulse, the recovery of fluorescence in a rectangle of observation was detected at time intervals of 0.1-0.5 s for a total of 30-60 s. All fluorescence data, including 5-10 prescans before the bleach pulse, were stored on magnetic disks for subsequent analysis.

Photobleaching data analysis. The cytoplasmic transport rate, expressed as an effective diffusion constant, and the mobile fraction (Fm) of NBD-stearate were determined by a two-step fitting procedure as previously described (9, 11-14, 27). Briefly, the fluorescence curves recorded after bleaching were normalized by expressing the fluorescence present at each point in the observation rectangle as a fraction of that present before the bleach. Each normalized postbleach scan was fitted to a two-dimensional Gaussian function, determining the parameters of the Gaussian distribution by nonlinear least-squares regression (9, 12, 14). Estimates of the diffusion rate constants in the x- and y-coordinate planes (Dx and Dy) and Fm were obtained by a second nonlinear least-squares fit of the center point of the Gaussian curve as a function of time (9, 11-14, 27). Convection was detected if the center of the bleach point moved in the xy plane.

Statistical methods. Results of multiple measurements are expressed as means ± SE. The unpaired Student's t-test was used to determine the significance of differences between experimental groups.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Characterization of enterocytes. The isolation procedure effectively separated villus cells from those generated in the crypts based on morphological and enzymatic features. Villus cells had at least an eightfold enrichment in alkaline phosphatase activity compared with crypt cells isolated from the same intestinal segment (7, 26). Morphologically, cells from the villus region were characterized as large sheets of epithelia with distinct brush borders. Cells from the crypt region showed classic tubular organization and markedly reduced alkaline phosphatase activity.

Quality of fluorescence recovery curve fits. For each bleach study, Dx, Dy, and Fm were estimated by nonlinear regression. A representative data set and its regression curve are shown in Fig. 1. A total of 119 curves were recorded: 27, 47, and 45 in villus cells from the duodenum, jejunum, and ileum, respectively. The average coefficient of determination (R2) for these fits was 0.95, with a range of 0.91-0.99. The width of the bleach beam, estimated as previously described (12, 14), was 1.35 ± 0.15 µm, consistent with the nominal value of 1.30 µm. Experiments in which the beam width deviated more than 20% from this value were excluded from further analysis. Besides estimating the individual values of diffusion constants and mobile fraction, the fitting algorithm returned their asymptotic standard deviations determined from the information matrix at convergence. The average ratios of the standard deviation to the parameter estimate were 0.12 and 0.03 for diffusion constants and mobile fraction, respectively. The type of cell (proximal, middle, or distal small bowel) did not affect the quality of the fits judged by either the coefficients of determination or the standard deviations in the estimates, although the estimates of the individual parameters changed (see Fig. 2).


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Fig. 1.   Fluorescence recovery curve recorded in villus enterocyte from duodenum. Circles represent individual data points from a typical experiment. Solid line is least-squares fit of data to fluorescence recovery model described in text. The estimated diffusion constant was 2.8 ± 0.2 × 10-9 cm2/s. The mobile fraction was 0.63 ± 0.08. Results are means ± SE.

Convection and nonhomogeneous diffusion. The technique of laser photobleaching measures the overall movement of fluorescent molecules into the area of observation. This movement could occur by a variety of processes, including simple diffusion, anisotropic diffusion, convection (flow of cytoplasm), or a combination of processes (14, 27). Our data analysis algorithm is designed to detect the presence of convection as it locates the minimum of each curve at each time point. However, no convection was detected in any of the recovery curves. The algorithm also detects nonhomogeneous (anisotropic) diffusion, that is, diffusion that proceeds at different rates in different directions. For these studies we measured the diffusion rate of NBD-stearate along two perpendicular lines intersecting at the bleach point. Estimates of the diffusion rate of NBD-stearate were not dependent on direction, with the two directional estimates always being within 11% of each other. Values reported here are therefore the mean of the individual values in the x and y planes, denoted as Deff. Finally, the diffusion rate did not depend upon location within an enterocyte as long as the area of observation did not include the nucleus.

Diffusion and cytoplasmic binding of NBD-stearate and FABP concentration. The cytoplasmic transport of NBD-stearate occurred solely by diffusion in enterocytes with a Deff between 2.07 ± 0.27 × 10-9 (proximal segment) and 4.19 ± 0.34 × 10-9 cm2 · s-1 (middle segment), depending on the location from which the enterocyte had been obtained (Fig. 2). The percentage of NBD-stearate found in the cytosol also varied with the enterocyte position along the length of the intestine, ranging from 2.83 ± 0.53% in the duodenum to 4.83 ± 0.75% in the middle segment (n = 8 for each location) (Fig. 3). The concentration of total FABP (both liver and intestinal) varied along the length of the intestine as previously described (1, 3, 19). Concentrations of total FABP measured by ELISA and Western blotting were similar, although the ELISA assay was more reproducible. Jejunum had approximately twice as much total FABP (37 ± 9 µg/mg total protein) compared with duodenum (16 ± 4 µg/mg total protein) or ileum (17 ± 6 µg/mg total cytosolic protein) as measured by ELISA. The distribution of liver FABP was not homogeneous as previously shown with duodenum and jejunum, having significantly more compared with ileum (duodenum 10 ± 4; jejunum 22 ± 4; ileum 6 ± 4 µg/mg total protein) (1). Intestinal FABP was more homogeneously distributed (duodenum 8 ± 5; jejunum 15 ± 5; ileum 11 ± 5 µg/mg total protein) (3, 19). There was good correlation between the observed diffusion rate and the fraction of NBD-stearate found in cytosol (r = 0.93, P < 0.01) (Fig. 4).


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Fig. 2.   Observed diffusion rate of NBD-stearate varied with position. Diffusion was fastest in enterocytes isolated from middle segment (P < 0.01, middle vs. proximal; P < 0.05, distal vs. middle). Transport by convection was not observed. Results are means ± SE; n = 27, 47, and 45 in villus cells from proximal, middle, and distal segments, respectively.



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Fig. 3.   Percentage of NBD-stearate (NBDS) found in cytosol varied with position. Fraction of NBD-stearate found in cytosol was determined by differential centrifugation as described in MATERIALS AND METHODS. Proximal and distal enterocytes had significantly lower cytosolic fraction (P < 0.05, proximal compared with middle segment). Results are means ± SE; n = 8.



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Fig. 4.   Relationship between observed diffusion rate of NBD-stearate and extent of cytosolic binding. Results are means ± SE from data shown in Figs. 2 and 3. Line represents least-squares regression with correlation coefficient = 0.925.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Since FABPs were first isolated in enterocytes nearly 20 years ago, the family of FABPs has been well characterized with regard to their structure and regulation. The precise function that this class of small proteins plays in fatty acid utilization remains unclear. Previously we had reported measurements of the cytoplasmic diffusion rate of NBD-stearate in male and female rat hepatocytes and postulated that FABP enhanced this transport (12, 14). The current studies were designed to further define the role that small binding proteins like FABP may play in modulating the intracellular transport of hydrophobic molecules. We wanted to determine the mechanism for fatty acid transport in enterocytes and to see whether the enhanced codiffusion of fatty acids with FABP was specific for hepatocytes or if other cell types that contain FABP would show the same phenomenon. Finally, we wanted to take advantage of the differences in FABP type and concentration along the length of the intestine to determine if a relationship existed among the transport rate, degree of cytosolic binding of fatty acids, and FABP concentration as suggested by numerous theoretical models (14, 23).

The small intestine is highly differentiated along its length as far as its ability to absorb various nutrients, including sugars, amino acids, and lipids (4, 8). This local specialization extends to the FABPs, with FABPs being most abundant in the jejunum and relatively less abundant in duodenum and ileum. Adding to this complexity is the fact that the small intestine contains both liver FABP (L-FABP) and intestinal FABP (I-FABP) (5, 17). Immunohistochemical staining suggests that only the enterocytes at the tips of the intestinal villi contain FABP, with crypt cells having virtually no FABP (1, 19). Most of the regional differences in total FABP concentration are due to changes in L-FABP (19) since I-FABP levels do not change as dramatically (1, 3). Our measurements of both the total FABP and the L-FABP and I-FABP isoforms confirm these previous findings.

These results represent direct measurements of the intracellular movement of a fatty acid in enterocytes. Consistent with previous work done in hepatocytes, we found that this transport occurred solely by simple diffusion. From an analysis of the two-dimensional fluorescent recovery curves, we were unable to detect the presence of convection. Based on simulation analysis as well as direct experimental evidence, our data analysis algorithm is able to detect convection velocities as slow as 0.05 µm/min (unpublished observations). We are confident therefore that in enterocytes the transport of NBD-stearate occurs by simple diffusion. We were also unable to detect any variation in the diffusion rate based on the position within the enterocyte.

We have investigated the role of FABP in promoting the intracellular diffusion of fatty acids in isolated enterocytes. We found that this transport occurs solely by isotropic diffusion, that is, fatty acid movement is nondirectional. In the intact small intestine, enterocytes are polarized, with the overall transport of fatty acids and triglycerides being directed from the gut lumen to the blood. Fatty acid transport in the cytoplasm may not be as directed; the reason is that the enzymes for esterification (located in the smooth endoplasmic reticulum) are fairly evenly distributed throughout the enterocyte cytosol. FABP may act as a shuttle for long-chain fatty acids from the luminal plasma membrane to the endoplasmic reticulum, where they are esterified to form triglycerides. Extrapolation of our results with the use of isolated enterocytes that have lost their polarity to enterocytes in intact intestine must be done with caution. However, we are not aware of any experimental techniques that would allow us to make these types of measurements in intact polarized epithelium.

Enterocytes had relatively slow cytoplasmic transport of NBD-stearate. In villus enterocytes from the jejunum, the fastest average diffusion rate was 4.2 ± 0.3 × 10-9 cm2/s. This rate is nearly two orders of magnitude slower than would be expected for NBD-stearate or the FABP molecule in free water. Based on their molecular weights, the predicted diffusion rate of the NBD-stearate-FABP complex in water at 37°C is 1.4 × 10-6 cm2/s (14). As we suggested earlier, this slow observed diffusion rate of NBD-stearate is consistent with extensive but reversible binding of the fluorescent probe to immobile membranes (12, 14). Others have shown that this binding is reversible both in vitro and in vivo (21, 22). The degree to which the observed diffusion rate of NBD-stearate is diminished relative to the rate of the NBD-stearate-FABP complex indicates the extent of reversible binding to immobile membranes (14). Based on the diffusion rate observed in jejunum (4 × 10-9 cm2/s), we expect that only 5.1% of the NBD-stearate is in the cytosol and freely diffusible (either bound to FABP or free). This is in good agreement with our observed cytosolic fraction of 4.8%.

Because multiple proteins are involved in cytosolic binding of fatty acids, we have chosen to measure the fraction of fatty acid found in the cytosol as well as measuring the particular protein concentrations. Because there are slight differences in the binding affinities between liver and intestinal FABP, the relationship between protein concentration and bound concentration of fatty acid is complicated. Our assay therefore is a functional one that reflects the fraction of fatty acid that we believe is available for rapid diffusion (12, 14). The location-dependent concentration changes in FABP (either I-FABP or L-FABP) were associated with an increase in the measured diffusion rate of NBD-stearate in direct proportion to the extent of binding. Thus there is a constant relationship between the "cytosolic" fraction of NBD-stearate, the concentration of total FABP, and the observed diffusion rate. This relationship presumes that the concentration of cytosolic membranes in binding sites is very large compared with those on FABP and the total concentration of NBD-stearate (i.e., membranes represent an infinite sink). In the design of our experiments we attempted to keep the concentration of NBD-stearate small to allow first-order kinetics. Modifying the concentration of NBD-stearate over a 100-fold range (1-100 µM) did not change the observed diffusion rates or the mobile fraction (data not shown).

We postulate that cytoplasmic binding proteins stimulate transport of fatty acids by limiting their binding to immobile membranes. We suggest that the diffusional flux of fatty acids occurs by movement of the protein-bound pool. It is this pool that has the greater mobility (compared with the membrane-bound pool) and the greater concentration (compared with the unbound aqueous pool). We propose that all three of these pools are in rapid equilibrium (14). We base this hypothesis on the observation that nearly 60% of the NBD-stearate was mobile in enterocytes, yet less than 5% of the total cytoplasmic NBD-stearate was found in the cytosolic phase. Rapid exchange between these pools is also supported by direct measurement of the "on" and "off " rates for the interaction among fatty acids, membranes, and both hepatic and intestinal FABP (21, 22). Based on previous work (1, 10, 14, 22), this equilibrium appears to be modulated by the concentration of cytoplasmic binding proteins.

It is possible that the observed rate of NBD-stearate movement was also influenced by slow exchange between membrane-bound, FABP-bound, and free NBD-stearate. Slow exchange between the protein-bound and one or more membrane-bound pools would lead to multiple pools of fluorescent probes that may not be in instantaneous equilibrium. As suggested by Kaufman and Jain (11), the equations used to analyze the fluorescent recovery curves can be modified to account for this process. Even a model containing a single pool of membrane-bound NBD-stearate out of equilibrium with free NBD-stearate leads to partial differential equations that are nonlinear and cannot be solved analytically. However, they can be solved numerically (11). Using our data, we were unable to detect deviance from a single membrane-bound pool with instantaneous equilibrium.

During the time of observation of the photobleaching experiments (30-90 s), ~60% of the NBD-stearate was mobile. The remaining 40% was "permanently bleached" and did not exchange with "unbleached" NBD-stearate. Our experiments do not address what this pool represents. A possibility includes a deeper pool of membrane-bound NBD-stearate that undergoes slow exchange with other pools. We loaded the enterocytes with NBD-stearate for ~15 min, allowing considerably more time for the probe to be incorporated into membranes that are relatively inaccessible. Photobleaching studies using isolated hepatocytes demonstrated that 15% of NBD-stearate was not mobile over 90-120 s (14). It is not clear why the immobile fraction of NBD-stearate would be different in hepatocytes compared with enterocytes. Further work will be needed to identify what this immobile pool represents.

In summary we have used the technique of laser photobleaching to measure the cytoplasmic transport of a fluorescent fatty acid analog, NBD-stearate, in enterocytes isolated from different segments of the small intestine. We conclude from these experiments that binding proteins like FABP enhance the diffusive flux by modifying the partition of fatty acids between membrane and aqueous phases. We suggest that this facilitation is not specific for a particular FABP or cell type but may occur in a variety of cells that contain different types of FABPs. The extent of the phenomenon seems to depend solely on the increase in the fatty acid's aqueous concentration. The data obtained in these experiments suggest that small binding proteins like FABP increase the intracellular transport of amphipathic molecules by decreasing their binding to immobile cytoplasmic membranes and may increase the overall transport and utilization rates of these important metabolic substrates.


    ACKNOWLEDGEMENTS

B. A. Luxon is a recipient of the Mary Richards Liver Scholar Award from the American Liver Foundation. Awards to B. A. Luxon from the Glaxo Institute of Digestive Health, the Whitaker Foundation for Biomedical Research, and the National Institute of Diabetes and Digestive and Kidney Diseases (DK-46922) funded portions of this research.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: B. A. Luxon, P.O. Box 15250, Division of Gastroenterology and Hepatology, St. Louis Univ. Health Sciences Center, 3635 Vista Ave. at Grand Blvd., St. Louis, MO 63110-0250 (E-mail: luxonba{at}slu.edu).

Received 16 June 1998; accepted in final form 26 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastroint Liver Physiol 277(2):G361-G366
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