ROBRT K. OcKNER and JoAN A. MANNING From the Department of Medicine, University of California School of Medicine, San Francisco, California 94143

A B S T R A C T Fatty acid binding protein (FABP) is a protein of 12,000 mol wt found in cytosol of intestinal mucosa and other tissues, which exhibits high affinity for long chain fatty acids. It has been suggested that FABP (which may comprise a group of closely related proteins of 12,000 mol wt) participates in cellular fatty acid transport and metabolism. Although earlier findings were consistent with this concept, the present studies were designed to examine its physiological function more directly. Everted jejunal sacs were incubated in mixed fatty acid-monoglyceride-bile acid micelles, in the presence or absence of equimolar concentrations of either of two compounds which inhibit oleate binding to FABP: flavaspidic acid-N-methyl-glucaminate and a-bromopalmitate. Oleate uptake, mucosal morphology, and oxidation of ["C]acetate remained unaffected by these agents, but oleate incorporation into triglyceride was inhibited by 62-64% after 4 min. The inhibition by flavaspidic acid was reversible with higher oleate concentrations. The effect of these compounds on enzymes of triglyceride biosynthesis was examined in intestinal microsomes. Neither flavaspidic acid nor a-bromopalmitate inhibited acyl CoA: monoglyceride acyl-transferase. Fatty acid: coenzyme A ligase activity was significantly enhanced in the presence of partially purified FABP, probably reflecting a physical effect on the fatty acid substrate or on the formation of the enzyme-substrate complex. Activity of the enzyme in the presence of 0.1 mM oleate was only modestly inhibited by equimolar Dr. Ockner was the recipient of Research Career Development Award AM-36586 from the National Institutes of


Received for publiecation 16 June 1975 and in revised form 4 May 1976.


flavaspidic acid and a-bromopalmitate, and this effect was blunted or prevented by FABP. We conclude that in everted gut sacs, inhibition of triglyceride synthesis by flavaspidic acid and a-bromopalmitate could not be explained as an effect on fatty acid uptake or on esterifying enzymes in the endoplasmic reticulum, but rather can be interpreted as reflecting inhibition of fatty acid binding to FABP. These findings lend further support to the concept that FABP participates in cellular fatty acid transport and metabolism. It is also possible that FABP, by effecting an intracellular compartmentalization of fatty acids and acyl CoA, may play a broader role in cellular lipid metabolism.

INTRODUCTION The cytosol of rat and human intestinal mucosa, liver, myocardium, kidney, and adipose tissue contains one or more proteins of 12,000-mol wt designated fatty acid binding protein (FABP)' which exhibit high affinity for long chain fatty acids (1-3). Hepatic FABP appears closely related to the Z protein of Levi et al. (4), also shown to bind long chain fatty acids (5). We proposed that intestinal FABP participates in the intracellular transport of fatty acid, and that its counterparts in other tissues play a similar role (1). Recently, the isolation of FABP from rat intestine was described (2), as was the preparation of a specific rabbit antiserum. FABP was quantified in intestinal mucosa by radial immunodiffusion, and was localized chiefly in the cytosol. Its tissue concentration, expressed per milligram of soluble protein, per gram of tissue, and per gram of DNA, was significantly greater in villi than

'Abbreviations binding protein;

used in this paper: FABP, fatty acid retardation factor.


The Journal of Clinical Investigation Volume 58 September 1976 .632-641

in crypts, and in mucosa from proximal and mid-jejunoileum than from distal. Mucosal FABP concentrations were significantly higher in animals maintained on high fat diets than in those on low fat diets. After intraluminal administration, [3H]palmitate was recovered in the FABP fraction of mucosal cytosol. These findings were consistent with a role for FABP in cellular fatty acid transport, but because the evidence was indirect, additional experimental approaches were required. Fatty acid uptake by the intestine and other cells is passive; fatty acids appear to interact with plasma membrane lipids, and no specific receptors have been demonstrated (6-10). It was postulated that FABP facilitates the desorption of fatty acid from the inner aspect of the membrane, and that the fatty acid-FABP complex diffuses to the endoplasmic reticulum where the fatty acid is activated to its coenzyme A thioester. If this concept is correct, agents which inhibit the binding of fatty acids to FABP should, thereby, inhibit activation and esterification in everted gut sacs, but should have a lesser effect on initial uptake. Two compounds which inhibit binding of fatty acid to hepatic Z protein are flavaspidic acid-N-methyl-glucaminate' and a-bromopalmitate (11). The latter was shown by earlier investigators to inhibit fatty acid oxidation in perfused heart muscle (12) and isolated hepatocytes (13); the oxidation of medium chain fatty acid was not affected (13), while that of glucose was enhanced (12). In 1971, Mehadevan and Sauer concluded that a-bromopalmitate exerted its effect by competing for an as yet unrecognized cellular transport system for long chain fatty acids (13). In the present studies, flavaspidic acid and a-bromopalmitate, structurally dissimilar compounds which are not known to be metabolized in the intestine, were shown to inhibit the binding of oleate to partially purified intestinal FABP, and were employed with preparations of rat intestine in experiments designed to test the proposed function of this protein. The results provide additional support for the hypothesis, and also suggest that FABP may play a broader role in cellular fatty acid metabolism. Portions of these studies were included in a preliminary communication (3).

METHODS Materials. [14C] Oleic acid,

["C] sodium acetate, and

["C]palmitoyl coenzyme A were purchased from New England Nuclear, Boston, Mass. Sephadex G-25 was obtained from Pharmacia Fine Chemicals, Inc., Piscataway, N. J., and a-bromopalmitic acid from ICN K&K Laboratories Inc., Plainview, N. Y. Flavaspidic acid-N-methyl-glucaminate was generously provided by Dr. A. Aho, Turku, Fin-

'Used interchangeably throughout the text with flavaspidic acid.

land. Unlabeled oleic acid, glycerol-1-monooleate, and sodium taurocholate were purchased from Calbiochem, San Diego, Calif. Egg lecithin and defatted bovine serum albumin were purchased from Schwarz/Mann, Div., Becton, Dickinson & Co., Orangeburg, N. Y.; the albumin was delipidated further by the method of Chen (14). Egg lecithin and glycerol monooleate were purified by silicic acid chromatography (15). Tris buffer, unlabeled palmitoyl coenzyme A, ATP, coenzyme A, and dithiothreitol were obtained from Sigma Chemical Co., St. Louis, Mo. Hydroxylamine hydrochloride was purchased from Matheson Coleman & Bell, East Rutherford, N. J. Male Sprague-Dawley rats, 300350 g, maintained on standard laboratory chow (Feedstuffs Processing Company, San Francisco, Calif.) were used in all experiments.

Partial purification of intestinal FABP and in vitro bindinig studies. Intestinal FABP was partially purified as the fraction of 12,000 mol wt from intestinal mucosal 105,000-g supernate on Sephadex G-50, as previously described (1). Three batches of this material, the "FABP fraction," contained 23.1±2.6% (SEM) FABP, as determined by quantitative radial immunodiffusion (2). For semiquantitative estimates of binding of ["C]oleate and flavaspidic acid, ligands dissolved in 8 ,l methyl ethyl ketone or in 50 Al 50% propylene glycol were added to 1.0 ml FABP fraction (0.5 mg/ml) and were subjected to nonequilibrium Sephadex G-25 column gel filtration (1). Studies of everted jejunal sacs. Studies were performed as described and standardized previously (16). Sacs were incubated for up to 4 min in mixed micellar solutions of oleic acid, glycerol monooleate, and sodium taurocholate, with or without flavaspidic acid or a-bromopalmitate, in KrebsRinger bicarbonate buffer containing 5 mM glucose, with no calcium or magnesium. Values for "uptake" were not corrected f or adsorbed incubation medium. Although this could overestimate uptake at 1 and 2 min, Sallee et al. (17) showed that at 4 min, correction for adherent fluid in studies with long chain fatty acid in mixed micelles was not essential. In the experiments in which conversion of ["C] oleate, ['4C]acetate, and ["C]glucose to "CO2 was measured, sacs were incubated under identical conditions in flasks fitted with a center well. At the conclusion of the incubation, 0.4 ml Hydroxide of Hyamine (Packard Instrument Co., Inc., Downers Grove, Ill.) was added to the center well, and the contents of the flasks were acidified with 0.2 ml of 8 M sulfuric acid. "CO2 was collected during an additional 30min incubation, and the contents of the center well were assayed for radioactivity. In some experiments, incorporation of ["C] glucose into the glycerol and fatty acid moieties of mucosal phospholipids and triglycerides was measured. Sacs incubated for 4 min in mixed micellar solutions, with or without flavaspidic acid or a-bromopalmitate, were examined histologically. No significant differences were observed, consistent with the earlier observation that flavaspidic acid was not toxic to liver tissue (18). Jejunal microsomes were prepared by a modification of the method of Rodgers et al. (19). Intestinal mucosa was homogenized in a teflon-glass Potter-Elvejhm homogenizer in 10 vol of 0.28 M mannitol and 50 mM EDTA in 0.01 M Na2HPO4, pH 7.4, which contained 0.75% defatted bovine albumin. Addition of albumin to the homogenizing medium was found to diminish the microsomal FFA concentration (ordinarily approximately 0.3 Amol/mg microsomal protein) by 80-90% (unpublished observations). Microsomal enzyme assays. Fatty acyl coenzyme A: monoglyceride acyltransferase activity was measured by a modification of the method of Rodgers (20). Incubations

Fatty Acid Binding Protein and Intestinal Absorption


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FIGURE 1 Effect of flavaspidic acid and a-bromopalmitate on [14C] oleate binding to jejunal FABP. [1"C] Oleate in 8 Al methyl ethyl ketone was added to 0.52 mg FABP fraction (approximately 6.4 nmol FABP in this preparation) in 1.0 ml 0.154 M KCI in 0.01 M KH2PO4, pH 7.4, with or without 10 nmol flavaspidic acid or a-bromopalmitate. The mixture was applied to Sephadex G-25 columns, 0.9 X 21 cm, 4°C. Oleate binding was calculated from the radioactivity eluting with the void volume. contained 0.02 mM ["C] palmitoyl coenzyme A, 0.08 mM glycerol monooleate and 0.08 mg microsomal protein in 66.7 mM Tris buffer, and 0.67% albumin; final volume was 0.3 ml. Incubations were for 2 min at 300 C, at which time the reaction was terminated by the addition of methanol and chloroform. Enzyme activity, approximately linear to 2 min and from 0.02 to 0.06 mM palmitoyl coenzyme A, was expressed as nanomoles of palmitate incorporated into diglycerides and triglycerides, determined by thin-layer chromatography of the lipid extract. Fatty acid: coenzyme A ligase was measured by a modification of the method of Rodgers et al. (21), employing the hydroxamate trapping technique of Kornberg and Pricer (22). Approximately 1.0 mg microsomal protein was incubated with substrate and inhibitors in a final 2-ml volume of 0.1 M Tris buffer, pH 7.4, containing 10.0 mM ATP, 10 mM MgC12, 5 mM KF, 0.2 mM coenzyme A, 1.04 mM dithiothreitol, 0.75 M hydroxylamine hydrochloride, and up to 2% (vol/vol) ethanol. Incubations were carried out for 15 min (during which time the reaction was approximately linear), at 37°C, and were terminated by the addition of 2 ml 95%o ethanol followed by 0.5 ml 6 N HCI. Hydroxamates were extracted from the precipitate with petroleum ether and dried under Na; color was developed with the Hill reagent (23) and read at 520 nm. Hydroxamate formation was calculated on the basis of molar absorbancy, E = 1,000 (22). Analytical procedures. Lipids were extracted by the method of Folch et al. (24). Lipid classes were separated by thin-layer chromatography on 0.25-mm silica gel H, and identified as described previously (16). Appropriate zones were scraped directly into counting vials and assayed for radioactivity, or were eluted for subsequent quantification. Flavaspidic by its bright in the above it moved with


acid-N-methyl-glucaminate, readily identified yellow color, was chromatographically pure thin-layer chromatography system, in which a retardation factor (Rr) of 0.11. This mate-

R. K. Ockner and 1. A. Manning

rial was completely extracted into the chloroform phase of the extraction system of Folch et al. (24), and analysis of everted sacs incubated in flavaspidic acid solutions indicated that all pigmented material so extracted moved with an Rr identical to that of the native compound, suggesting that it was not metabolized. No flavaspidic acid was detected in the aqueous phase. Advantage was taken of these properties of the compound, and its absorbancy peak at 350 nm to develop an assay for tissue flavaspidic acid concentration. Gut sacs were extracted by the method of Folch et al., and the lipid extract analyzed by thin-layer chromatography. The flavaspidic acid spot was identified, scraped, and eluted with chloroform: methanol (2: 1). Absorbancy was compared with a standard curve (flavaspidic acid in chloroform: methanol) and was corrected for a silica gel blank. The assay was linear over a wide range (0.001-0.1 ,&mol/ml); recovery from the silica gel was approximately 90%. Radioactivity was assayed in a Beckman LS-250 liquid scintillation system (Beckman Instruments, Inc., Fullerton, Calif.). Aqueous samples were solubilized in a 10%o solution of Biosolv BBS-3 (Beckman Instruments, Inc.) in Liquifluor (New England Nuclear) and toluene. Statistical methods. Significance of differences among experimental groups was determined by paired or unpaired t tests (25).

RESULTS Effect of flavaspidic acid and a-bromopalmitate on binding of oleate to FABP in vitro. Binding of [14C]oleate to partially purified intestinal FABP fraction was estimated by gel chromatography (Methods). The significant and reversible inhibition of binding by flavaspidic acid and a-bromopalmitate are apparent (Fig. 1). In other experiments, binding of flavaspidic acid to FABP was inhibited to a similar extent by equimolar oleate. These observations are consistent with the findings of Mishkin et al. (11) in studies of hepatic Z protein fraction. TABLE I

Rev,ersibility of Oleate Binding to FABP: Effect of Albumin [14C]Oleate recovered in G-50 eluate

Applied cpm eluting with Protein(s) applied to G-50 column FABP fraction (0.26 mg)

+buffer FABP fraction (0.26 mg)

+albumin (0.26 mg)

Total percent FABP



13.4 (12.8, 13.9) 10.1 (10.1, 10.1)


12.8, 13.9

70.0 (71.2, 68.8)

81.3, 78.9

['4C]Oleate was combined with FABP fraction and passed through Sephadex G-25 as described in the legend to Fig. 2. The FABP-oleate complex appearing in the void volume (0.17 mg protein/ml) was mixed with an equal volume (1.5 ml) of buffer or defatted albumin in buffer (0.17 mg/ml) and allowed to stand at 20°C for 15 min. The mixture (containing 3.37 nmol oleate) was then applied to a Sephadex G-50 column (2.5 X 37 cm, 4°C, 3.6-ml fractions) and counts per minute eluting with albumin and FABP were determined. Results are expressed as percent of applied counts per minute recovered. (Approximate molar quantities applied to G-50: oleate, 3.4 nmol; FABP -4 nmol; albumin -4 nmol.)

Reversibility of ["C]oleate to FABP is shown in Table I. ["C]Oleate-FABP complex was prepared by gel chromatography, mixed with an equal volume of buffer or with defatted albumin in buffer, and re-chromatographed on Sephadex G-50, thus permitting separation of FABP from albumin. It can be seen that most of the ["C]oleate-FABP complex dissociated during G-50 chromatography, whether or not albumin was present. Significantly, although total recovery of ["C]oleate was greatly increased in the presence of albumin, this protein had little effect on the retention of ["C]oleate by FABP (13.4 vs. 10.1%), despite the fact that there were almost certainly many more unoccupied binding sites on albumin than on FABP under these conditions. Effect of flavaspidic acid and a-bromopalmitate on fatty acid uptake and utilization by everted jejunal sacs. As shown in Fig. 2, neither flavaspidic acid nor a-bromopalmitate significantly affected oleate uptake. In contrast, both compounds markedly inhibited incorporation of oleate into triglyceride (Fig. 3). Incorporation into other esterified lipids (phospholipids, diglycerides, and cholesterol ester) also was inhibited, but more variably and to a lesser degree (Table II). Since these other lipids together account only for approximately 10% of the oleate esterified, and only 5-6% of mucosal uptake, the significance of changes in these minor fractions is difficult to interpret. Because of the overall decrease in esterification, however, ["C]oleate accumulated in the mucosa (Fig. 4), suggesting that little if any fatty acid was diverted into oxidative pathways. In fact, oxidation of [1-"C]oleate to "CO also was significantly inhibited (Table III), although this minor pathway accounted o







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Fatty acid binding protein. Role in esterification of absorbed long chain fatty acid in rat intestine.

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