AMERICAN
JOURNAL
OP PHYSIOLOGY
Vol. 228, No. 6, June 1975.
Printed
in U.S.A.
Z protein
in hepatic uptake and esterification of long-chain fatty acids SEYMOUR MISHKIN, LAWRENCE STEIN, GERALD FLEISCHNER, ZENAIDA GATMAITAN, AXD IRWIN M. ARIAS Diuisz’on of Gastroenterology-cliivar Disease, Department of Medicine,und Liver Reseurch Center, Albert Einstein College of Medicine, Bronx, New York, 10461; and Gastrointestinal Research Laboratory, ~McGill University Clinic, Department of Medicine, Royal Victoria Hospital, Montreal, Canada
MISHKIN, SEYMOUR, LAWRENCE STEIN,GERALD FLEISCHNER, ZENAXDA GATMAITAN, AND IRWIN bf. ARIAS. Z protein in hepatic uptake and esteriJication of long-chain fatty acids. Am. J. Physiol. 228(6) : 1634-164-O. 1975-Fatty acid radioactivity was bound to 2 protein in liver after administration of [3H]oleate to rats or to a perfused rat liver preparation. Pretreatment with flavaspidic acid (340 pmol/kg), a potent inhibitor of fatty acid binding to hepatic 2
protein
in
vitro, effectively reduced oleate radioactivity
bound to 2
by 90.2 + 4.3 y0 and 85.0 =t 6.27;’ in the intact rat and perfused liver, respectively. In spite of this effect, pretreatment of rats with flavaspidic acid did not alter plasma clearance, hepatic uptake, and esterification of C3H]oleate. In contrast, in the perfused liver preparation, infusion of flavaspidic acid (340 pmol/kg) or bromosulphalein (360 pmol./kg) increased uptake of [3H]oleate at least twofold, and oleate esterification was decreased by 15-30 54. These results suggest that the binding of long-chain fatty acids to 2 protein is not an obligatory step in their uptake by the liver and that 2 protein may be involved in fatty acid esterification.
flavaspidic
acid
THE HEPATIC UPTAKE of free fatty acids (FFA) is extremely rapid (2, 6, 11) and involves their transfer from albumin through the plasma membrane. According to the classical theory of Overton, FFA dissolves in the oil phase of the membrane (19). Recent studies suggest that membrane binding sites, possibly involving lipoproteins, may play a role in FFA uptake (10, 24-28). Cytoplasmic proteins may also be involved in the hepatic uptake and intracellular transport of FFA (18, 20). 2 protein is a cytoplasmic protein of 12,000 daltons present in liver, myocardium, intestinal rnucosa, and other tissues, has a high afinity for long-chain fatty acids in vitro (18, 20, 2 l), and has been implicated in the hepatic uptake of bilirubin, sulfobromophthalein (BP), and various other organic anions (15, 17). Based on binding studies in vitro, it was proposed that 2 is important in the uptake and intracellular translocation of long-chain fatty acids by intestine and other tissues (20, 21). To test this hypothesis, we studied the binding of fatty acid following intravenous administration to 2 protein in vivo. Following administration of flavaspidic acid, which inhibits fatty acid binding to Z protein (17), the hepatic uptake and esterification of fatty acid were measured to determine whether binding of longchain fatty acids to 2 protein is obligatory in these processes.
METHODS
3H-Labeled oleic acid was purchased from New England Nuclear Corporation (Boston, Mass.). The stated purity of oleic acid (99 76) was confirmed by gas-liquid and thinlayer chromatography and was rechecked immediately prior to use. Bovine and rat serum albumin fraction V as well as delipidated bovine serum albumin were obtained from Pentex, Inc., Kankakee, Ill. Flavaspidic acid-nmethylglucaminatel was obtained from Dr. Esa Ah& Turku, Finland, and sodium sulfobromophthalein was obtained from Hynson, Westcott & Dunning, Inc., Baltimore, Md. Unlabeled o 1eic acid was obtained from Sigma Chemical Company, St. Louis, MO. In viuo experiments. Male Sprague-Dawley rats, 225-300 g (Marland F arms, Peekskill, N. YJ were fed Purina rat chow, given water ad libitum, and used in all experiments. The effect of flavaspidic acid on the plasma disappearance of [3H]oleate was studied in 48 rats under light ether anesthesia. Twenty-four rats received an infusion of Aavaspidic acid (340 pm/kg) made up in 1.5 ml of neutral sodium phosphate buffer (300 mosmol, pH 7.4, 37 “C) administered through a femoral vein catheter at a constant rate over a 15-min period. Twenty-four rats received a comparable infusion of buffer alone and served as controls. Five minutes later, each rat was rapidly injected intravenously with 20 PCi of a tracer amount of C3H]oleate in 0.5 ml of 3 g/ 100 ml bovine serum albumin. At precise minute intervals for 8 min following intravenous injection of labeled oleate, blood samples were obtained from the tail vein in heparinized capillary tubes. At least four samples were obtained from each rat. The tubes were centrifuged, 50 ~1 of plasma were removed by micropipette, by counting in a liquid quots of plasma were
and radioactivity scintillation
extracted (211 by volume) and subjected raphy to determine distribution and esterified fatty acid fractions. Using a similar protocol, hepatic I Flavaspidic acid i s a number of tained from male fern. Most have the A is butyrylfilicinic acid and B is various glucinol. Intravenous administration nate salt which, for brevity, is referred
was
determined
system. Random aliwith chloroform-methanol to thin-layer chromatogof 3H radioactivity in free accumulation
of radio-
phloroglucinol derivatives obgeneral formula A-CI-L-I3 where butvryl derivatives of phloroinvolved the n-rnethylglucamito as flavaspidic acid in the text.
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-PHYSIOLOGICAL
ROLE
OF
2 PROTEIN
.activity after administration of [3H]oleate was determined in an additional 20 Aavaspidic acid-treated and 20 control rats in which plasma-disappearance studies were not performed. In these rats, the liver was removed from groups of two to four rats 1, 2, 5, 10, and 15 min after injection of -oleate, perfused through the hepatic veins with 5 ml isotonic saline, and weighed. A 33 Lil (wt/vol) homogenate was prepared in 0.25 M sucrose, 0.01 M phosphate buffer (pH 7) using an iced all-glass Potter-Elvehjem homogenizer. Radioactivity was determined in an aliquot of liver homogenate and was calculated as percent administered dose in the total liver. Another group of rats (6 animals) received similar intravenous infusion of Aavaspidic acid via a femoral vein catheter over a 15-min period and six control animals resceived 1.5 ml of neutral phosphate buffer during the same period. Five minutes later, rats were infused for 2.5 min with unlabeled oleate (100 pmol/kg) in 0.5 ml of 3 g/ 100 ml bovine serum albumin (pH 7.4). The infusion of unlabeled oleate was omitted in two experiments, and in two other rats oleate was infused as described at a dose of 40 pmol/kg. In six other studies, delipidated bovine serum albumin, rat serum albumin, or rat serum was used to bind [3H]oleate instead of bovine serum albumin. Immediately after infusion of unlabeled oleate, 20 &i of a tracer amount of [3H]oleate in 0.5 ml of 3 g/ 100 ml bovine serum albumin were injected intravenously as a pulse. Other rats received 20 &i of [3H]oleate immediately following infusion of Aavaspidic acid without administration of unlabeled oleate. Additional rats received flavaspidic acid (340 pmol/kg) for 15 min simultaneously with unlabeled oleate (100 which was infused during the latter 5 min, and wwkg), 20 &i[3H]oleate were administered immediately on conclusion of the flavaspidic acid-oleate infusion. Exactly 2 or 5 min after injection of [3H]oleate, each rat was killed, and the liyer was removed, weighed, perfused with iced saline, and homogenized in an all-glass Potter-Elvehjem homogenizer (6 upward and downward strokes). The homogenate, a 33 Y& (wt/vol) sus p ension in 0.25 M sucrose, 0.01 M phosphate bufler (pH 7), was spun at 12,000 X g for 20 min at 4°C. The resulting supernatant was decanted and respun at 110,000 X g for 90 min at 4°C to yield a microsomal pellet and a particulate-free solution henceforth referred to as liver supernatant. Liver perfusion experiments. Because possible extrahepatic effects of flavaspidic acid have not been studied, an analogous experimental model was designed using in situ liver perfusion. In these experiments, the same amounts of flavaspidic acid were infused but over a shorter period; 1 min as opposed to 15 min. in the experiments in vivo. Furthermore, oleate was administered over a I-min period instead of 2.5 min as in the intact animals. These modifications were introduced to demonstrate better any inhibitory effect of Aavaspidic acid on hepatic uptake or esterification of [3H]oleate. Male Sprague-Dawley rats, 225-300 g (Quebec Breeders, Laprairie, Quebec), fed ad libitum were anesthetized with ether. The portal vein and right ventricle were exposed and cannulated. The liver in situ was washed free of blood with 100 ml of neutral phosphate bufler (300 mosmol, pH 7.4, 37°C) infused through the portal vein cannula at the rate of
1635 15 ml/min by means of a Holter peristaltic pump (model RL-175, Brent Surgical Ltd., Toronto, Canada). The drainoff through the ventricular catheter was discarded in all cases. Subsequently, either 15 ml of neutral phosphate buffer alone or buffer containing flavaspidic acid (340 pmol/kg) was infused over a 1-min period. BSP in varying concentrations (90-720 pmol/kg) was substituted for flavaspidic acid in three experiments. Phosphate buffer (15 ml) was then infused in each case prior to administration of 15 ml of a solution of [3H]oleate (60 pmol) containing 3 g/ 100 ml bovine serum albumin in neutral phosphate buffer. The liver was perfused with 100 ml of Krebs-Ringer-phosphate buffer (25 ml/ min) prior to removal and homogenization as described above. The resultant homogenate was centrifuged at 110,000 X g for 90 min to yield liver supernatant. The possibility that flavaspidic acid is overtly toxic to the liver is unlikely because electron microscope studies revealed no significant alterations in hepatic ultrastructure following flavaspidic acid administration, and oxygen uptake by liver perfused at a similar flow rate was unaffected during a ZO-min perfusion with flavaspidic acid (340 ,umol/kg). Sephadex chromatogruphy of liver supernutant and identz$cation of Z fmtein. Aliquots of liver supernatant equivalent to 2 g of liver were chromatographed on Sephadex gel G-100 columns (2.5 x 43 cm) as previously described (15). Reproducibility of the amount of r3H]oleate bound by 2 fraction after elution of supernate from Sephadex G-100 was investigated by performing repeated studies on four columns. Four times lO-g mol of [sH]oleate was added to aliquots of supernate from 0.5 to 2 g of rat liver containing 40 mg protein per gram. The mixture was eluted as described. Micromoles [“H]oleate per milligram protein were determined in the peak tubes in the 2 fraction, and micromoles of [“Hloleate in each fraction per gram liver were calculated by triangulation from the area under each peak ((peak oleate concentration-base-line oleate concentration) X volume per tube X number of tubes X 0.5). This method was reproducible within 95 % when columns of equal size and constant amounts of protein were used. Buffer pH and lnolarity, elution flow rate, and temperature were kept constant. 3H-Labeled oleate (4 X 10mg mol) added to the Sephadex columns in the absence of supernate protein was not eluted in the 2 fraction; approximately 5% appeared in later samples, These results indicate that Sephadex has an appreciable affinity for oleate and that determinations of binding of fatty acid to 2 protein by Sephadex chromatography give falsely low values (18). Nevertheless, we utilized this method because 2 protein has not been purified in sufricient quantity for binding studies by classical techniques and because results are of comparative value when gel filtration is performed under standardized, well-controlled circumstances. We examined for the presence of 2 protein in the following manner: a) The mobility of protein in the 2 fraction was compared with that of purified 2 protein using neutral sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (16 and unpublished data). b) Twenty-five milligrams of BSP (dissolved in 50 ~1 of normal saline) and 4 X 10mg mol of [“H]oleate (dissolved in 50 ~1 of propylene glycol dioxane 2 : 1 vol/vol) were added
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1636
MISHKIN
simultaneously to aliquots of supernatant representing 2 g of liver. The mixture was equilibrated for 30 min at 4°C and subjected to upward-flow chromatography on Sephadex G-l 00 (2.5 X 43 cm) as described previously (15). c) Five-milliliter aliquots of 2 fraction obtained by Sephadex G-l 00 chromatography of supernatant harvested from a 33 % liver homogenate (wt/vol) were added to tubes containing 5 ml of isotonic saline, monospecific antirat 2 IgG (unpublished data) or nonspecific rabbit IgG as previously described (8). Each mixture was stirred separately at room temperature for 1 h and at 4°C for 48 h, after which it was centrifuged at 18,000 X g for 30 min and any precipitates were separated from supernatant fractions. Five milligrams BSP were added to each of the three supernatants, each of the mixtures was rechromatographed on Sephadex G-100 columns and BSP binding was determined as described earlier (15). d) 3H-Labeled oleate (4 X 10mg mol. dissolved in 50 ~1 of propylene glycol dioxane 2 : 1 vol/vol), and flavaspidic acid (4.5 x 1o-g mol. dissolved in 50 ~1 of neutral phosphate buffer) were added simultaneously to aliquots of liver supernatant representing 2 g of liver. The mixture after equilibrating for 30 min at 4°C was subjected to upward-flow chromatography on Sephadex G-100 (2.5 X 43 cm) as described (17). The distribution of flavaspidic acid among the different liver supernatant proteins after Sephadex chromatography was determined by spectrophotometric analysis at 295 mpm. Analvtical methods. Total radioactivity in plasma, liver homogenate, microsomal, and supernatan t fractions was determ ined by cou nting 1.0-m 1 aliquots of each in 12 ml of Bray’s solu tion (3 ). Quench correction was by external as well as standardization. Each subcellul ar liver fraction proteins contain ing signifiplasma and eluted supernatant cant radioactivity were extracted with chloroform-methanol (2: 1 by volume) (9). In excess of 90 % of radioactivity was recovered in the chloroform phase. Aliquots of the lipid
11
2
4 MINUTES
6
0
FIG. 1. Disappearance of 113H]oleate in control (--) and Aavaspidie acid-treated rats ( -----). Each point represents means + SE of 3-5 observations. As described more fully under METHODS, rats received Tither an intravenous infusion for 15 min of neutral phosphate buffer or in buffer prior to intravenous injection of flavaspidic acid (340 pm/kg) 20 &i of a tracer amount: of C3H]oleate in 0.5 ml of 3 g/100 ml bovine serum albumin. Blood was serially obtained from a tail vein, and radioactivity in 50 ~1 aliquots of plasma was determined in a liquid scintillation system.
ET
AL.
extracts were subjected to thin-layer chromatography to determine the distribution of 3H radioactivity among free and esterified fatty acid fractions. Thin-layer chromatography was performed on standard glass plates (20 cm x 20 cm) coated with Silica Gel G (E, Merck Company, Darmstadt, Germany). The solvent system consisted of rz-hexanediethyl ether-acetic acid-methanol (90 120 12 : 3 by volume) (4). Lipid fractions were identified by comparison with simultaneously run standards (Hormel Institute). The fractions were visualized with the use of iodine vapor, and the corresponding silica gel as well as appropriate standards were scraped directly into counting vials containing 12 ml of a scintillation solution made up of 4 g of Omnifluor (New England Nuclear Corporation, Boston, Mass.) dissolved in 1 liter of toluene. From 85 to 95 % of radioactivity applied to thin-layer chrom a tographs was revealed in the free and ester&d fatty acid fractions. Absolute quantitation of the different lipid fractions was not performed in these experiments except for determination of serum FF,\ concentration by the method of Dole (5). RESULTS
In vivo experiments. The half-time for the plasma disappearance of [3H]01eate in control rats (58.4 =t 2.1 s) was not significantly altered by flavaspidic acid pretreatment (Fig. 1). Hepatic accumulation of radioactivity after administration of a tracer dose of [3H]oleate increased rapidly within 5 min after intravenous injection. Two minutes after injection, the hepatic uptake of radioactivity was 60.4 & 5.6 % of the administered dose in control rats and 58.7 & 6.2 % of the administered dose in flavaspidic acid-treated rats. Hepatic uptake of radioactivity increased more slowly thereafter and at 15 min, 82.3 =t 7.6 % of adlninistered radioactivity was in the liver in control rats, and 80.4 & 8.2 % of administered radioactivity was in the li\Ter in flavaspidic acid-treated rats. Thin-layer chromatography of lipid extracts of plasma obtained within 8 nk after intravenous injection revealed that in excess of 97 % of radioactivity was as free fatty acid. In rats killed 2 or 5 min following an intravenous pulse of r3H]oleate, recovery of administered radioactivity in the various experiments is shown in Table 1. Administration of Aavaspidic acid with or without prior or simultaneous infusion of unlabeled oleate, irrespective of the use of delipidated bovine serum albumin, rat serum, or rat serum albumin to bind [“Hloleate, and regardless of a 5-min interval between administration of flavaspidic acid and C3H]oleate, had no significant effect on recovery of administered radioactivity in liver when compared with results of similar studies performed in control rats (U > 0.5 in all pairs of studies). Ir some experiments, unlabeled oleic acid was infused to illcrease the serum concentration of fatty acid in the hope of saturating mechanisms involved in the uptake and metabolisms of fatty acid. The serum free fatty acid concentration following infusion of 100 pmol/kg oleic acid was 3-4 mM, approximately twice that achieved in the fasting rat in vivo (1). It was felt that any inhibitory effects of flavaspidic acid on hepatic uptake and esterification of oleic acid would be best detected under these conditions. Table 1 also presents the percentage of injected radio-
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PHYSIOLOGICAL
ROLE
OF
2
1637
PROTEIN
TABLE 1. Recovery of radioactivity in her homogenate, microsomal, and supernatant fractions following administration of C3H]oleate with and without prior or simultaneous administration of unlabeled &ate and/ur jlavaspidic acid -_p---^_Ip.. Recovery of [3H]Oleate Radioactivity Liver, y0 Administered Dose
2 min _--i)
Control
2)
Flavaspidic oleate 0 pm/kg 40 12) 100 (2)
3)
Flavaspidic acid and pm/kg) prior to bound to various Delipidated bovine Rat serum (2) Rat serum albumin
4)
t3H]oleate after
5)
Simultaneous labeled
Values details.
unlabeled infusion protein serum
and
(2)
are
infusion of flavaspidic oleate, and r3H]oleate means
+ SE.
Number
5 min
Recovered
in
r0 Administered Supernatant
$1 icrosomal
14.2
&
15.4, 17.2, 16.2
15.8, 16.8, 15.4,
1.1
25.0
=t
4.0
16.2 15.8 zt 3.2
24.3, 22.3, 23.5
25.1 24.8 zt 2.6
16.1 17.2 X6,4
25.1, 22.1, 21.9,
23.6 23.8 24.6
3.9
zk 0.2
0.73
dr
0.2
unlabeled
oleate (100 of [3H]oleate preparations: albumin (2)
(20 p C) 1 a d ministered flavaspidic acid infusion
in
Radioactivity at 2 min, Dose
--.
(6) acid pretreatment in various dosage (2)
--s) [aH]Oleate Liver Fraction
immediately
acid,
un-
3.8, 3.6 4.1, 4.0 4.9 dz 0.8
4.7, 4.4, 4.6,
4.5 4.9 4.8
0.71, 0.63, 0.62
0.68 0.64 + 0.2
0.59, 0.64, 0.69,
0.63 0.70 0.71
15.8
+
2.7
24.1
+
2.3
4.1
*
0.7
0.68
It
16.8
*
3.3
25.1
It
3.9
4.4
zk 0.9
0.71
=t: 0.3
0.3
(4) of rats
in each
group
are
given
in parentheses.
activity recovered in liver microsomal and supernatant fractions of rats killed 2 min. after an intravenous tracer dose of C3H]oleate in the various experimental conditions. In control rats, the percentage of injected radioactivity recovered in liver microsomal and supernatant fractions was 3.9 & 0.2 ?i and 0.73 & 0.2 %, respectively. Flavaspidic acid administration under each experimental condition failed to alter this distribution at statistically significant (P < 0.05) levels. The remainder of radioactivity found in totalliver homogenates was recovered in the pellet obtained after initial centrifugation at 12,000 X g for 20 min; the identity of 3H-labeled lipids in this pellet was not determined. The percentage esterification of oleate radioactivity in these experiments was also determined. The proportion of radioactivity present as free fatty acid was 95.1 L& 2.0 % in plasma, 52.3 2 3.3 % in liver homogenate, 23.3 & 2.9 % in liver microsomes, and 60.2 h 6.4 70 in liver supernatant of control rats; comparable results after flavaspidic acid pretreatment were 96.3 & 1.8 70, 55.1 & 3.6%, 22.9 & 3-O%, and 68.3 =t 6.2 70, respectively and not significantly different (P > 0.5). Identical experiments in which O-40 pmol of unlabeled oleate per kilogram were infused or C3H]oleate was infused immediately after Aavaspidic acid, yielded results which were not significantly different (P > 0.5). A representative elution pattern on Sephadex G-100 of radioactivity bound to liver supernatant proteins 2 min after administration of [3H]oleate in vivo is illustrated in Fig. 2* In all experiments, following chromatography of liver supernatants, radioactivity appeared in three regions: the first zone of radioactivity, labeled A, corresponds to a heterogenous high molecular weight region which contains proteins with molecular weights in excess of 60,000, including albumin and the sterol carrier protein (23). The remain-
Table
refers
2.5r
to
experimental
group.
See
text
for
further
A
15
20
25 30 TUBE NUMBER
35
40
FIG. 2. Effect of flavaspidic acid infusion in vivo on binding of [3H]oleate radioactivity to proteins in liver supernatant after chromatography on Sephadex G-100. Results shown were obtained from a single experiment and are representative. Rats were infused with flavaspidic acid (340 pm/kg) in neutral phosphate buffer for 15 min; 5 min later, unlabeled oleate (100 pmol/kg) in 0.5 ml of 3 g/100 ml bovine serum albumin was infused for 2.5 min. Immediately thereafter 20 PC1 of a tracer amount of C3H]oleate in 0.5 ml of a 3 g/100 ml bovine serum albumin were injected intravenously as a pulse. Control rats were treated identically, except neutral phosphate buffer was infused in place of flavaspidic acid. Exactly 2 min after injection of [3H]oleate, rats were killed and the liver was removed, perfused, homogenized, centrifuged at 100,000 X g, and the supernatant was chromatographed on Sephadex G-100. Radioactivity was determined in each aliquot eluted. Further details are provided under METHODS.
ing two zones of radioactivity correspond to the location of Y (ligandin) and 2 proteins (15). The presence of 2 protein in the 2 fraction of the liver supernates was established by the following observations : a) electrophoresis of 2 fraction on neutral SDS acrylamide gel revealed several protein bands including one with the same electrophoretic mobility as purified 2 protein (unpublished data) ; b) on Sephadex gel filtration of liver supernatant, BSP and fatty acid binding occurred in identical fractions (18) ; c) anti-Z-IgG, but not control IgG, precipi-
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1638 TABLE
MISHKIN 2.
estarij%ation .-~~
Effect ofjhvasfiidic acid on uptake and of [3H]oleate by perfused rat liver -_...--_ ~“-.-1 Total
Uptake
Total
-EsterificaI
ion
-.--Percent of in jetted dose
Homogenate Control (4) Flavaspidic acid (4) Sufwnatant Control
FIavaspidic
5.93+0.38
Percent of uptake
15.58&3.14
3.55*0.23 9.35zt1.89
68.Ozk2.04 19.5+2.63
0.45+0.02 0.47*0.01
0.28+0.01
22.Oh2.35
pm01
2.42&0.21 1.68~1~0.13
0. K&o.01
acid Values
pm01
0.06+0.01
-are
means
&
SE.
Numbers
----__. -of animals
used
are
given
in
parentheses.
tated 75-85 % of protein-bound 3BSP or oleic acid in 2 fraction; and d) flavaspidic acid, previously shown to displace BSP binding from 2 protein (17, 18), inhibited fatty acid binding (Fig. 2). In liver supernatant from control rats, 9.0 s+ 1.6 % of radioactivity was recovered in fraction A, whereas 18.3 & 5.0 % was present in this region after rats were pretreated with flavaspidic acid (Fig. 2). Thin-layer chromatography revealed that over 70 ci; of 3H labeling in this fraction was in the form of esterified fatty acids. The appearance of radioactivity in Y fraction was variable in all experimental conditions and could not be satisfactorily quantitated or compared. In control rats, 3.4 & 0.4 % of supernatant radioactivity was associated with 2, whereas only 0.1 & 0.2 % was recovered on 2 in the supernatant of Aavaspidic acidtreated animals. Treatment with flavaspidic acid, regardless of infusion of unlabeled oleate or timing of isotope administrations, inhibited binding of oleate radioactivity to 2 by 94.2 & 4.3 $0 (range 87-100 %). Mean recovery of supernatant radioactivity after Sephadex chromatography was 16.3 =t 3.4 % and 23.8 =t: 2.9% in control and flavaspidic acid-treated rats, respectively. The low recovery of supernatant radioactivity reflects the affinity of Sephadex for free fatty acids (11). Increased recovery of radioactivity in supernatant after flavaspidic acid administration resulted from the greater proportion of radioactivity, largely as esterified fatty acid, associated with the high molecular weight protein region (Fig 2). In control rats, 60.0 =cI: 1.4 % of radioactivity associated with 2 was free fatty acid; 15.1 & 3.1 % was monoglyceride & phospholipid, 22.0 + 1.1 % was diglyceride + cholesterol, and 4.2 & 1.8 5: was triglyceride + cholesterol esters. In two experiments, addition of anti-Z-IgG, but not control IgG, resulted in precipitation of 75-80 % of radioactivity in each of these lipid classes. This observation suggests that lipid other than FFA can be bound to 2 protein in vivo and is consistent with previous observations that cholesterol and triglyceride bind poorly, if at all, to 2 fraction in vitro (15). Radioactivity associated with 2 fraction in flavaspidic acid-treated rats was too low to permit meaningful lipid fractionation. Spectrophotometric analysis of the eluted fractions relatively localized flavaspidic acid to 2 protein. Quantitative
ET
AL.
immunoprecipitation of 2 protein (unpublished data), densitometry of stained polyacrylamide gels after electrophoresis (23), and radioimmunoassay revealed that equal amounts of 2 protein were eluted in the 2 region from supernatants derived from control and flavaspidic acidtreated rats. 2 protein was not detected in other supernatant fractions by immunoprecipitation or SDS polyacrylamide gel electrophoresis after administration of Aavaspidic acid. Liver perfusion experiments. The concentration of oleate in the perfusion media was 304 mM, the level at which hepatic uptake is maximal in vitro (13). In these experiments, both uptake and esterification of C3H]oleate were significantly aflected by pretreatment with Aavaspidic acid (Table 2). Although administration of Aavaspidic acid almost tripled the proportion and absil ute hepatic- uptake of [3H]oleate, of radioactivity present in liver homogenate as amount esterified fatty acid was significantly reduced. Total radioactivity in the supernatant fraction was unchanged by flavaspidic acid; however, the proportion esterified was significantly reduced. A representative elution pattern on Sephadex G- 100 of supernatant proteins is presented in Fig. 3. As in experiments in vivo, protein-bound radioactivity was localized to with flavaspidic three regions : A, Y, and 2. Pretreatment acid reduced the proportion of supernatant radioactivity associated with 2 by 88.0 & 6.2 % (range 83-99 5-i). The distribution of radioactivity between free and esterified fatty acids for each protein region was not different from results in experiments in vivo (P > .05 for all fractions). The results of identical experiments in which BSP (90was infused in place of flavaspidic acid are 720 pmol/kg) shown in Table 3. The hepatic uptake of [3H]oleate was increased following infusion of BSP in concentrations of 360 and 720 pmol/kg, and both the percentage and absolute esterification of C3H]oleate by liver homogenate decreased. The changes in hepatic supernatant content of total
O-0
%-OLEIC
W
3H-OLElC
2.c n
+ FLAVASPIDIC
wI 2 g 10
N & 5 E 0 ii
0 15
20
25 TUBE
30
35
Q
40
NUMBER
FTG. 3. Effect of Aavaspidic acid on binding of rW]oleate radioactivity to proteins in liver supernatant in vitro after chromatography on Sephadex G-100. Results shown were obtained from a single experiment and are representative. The 4 X lOA mol of [“Hjoleate was mixed with aliquots of rat liver supernatant obtained from 2 g of rat liver and eluted from Sephadex gel G-100 columns (2.5 x 43 cln) as previously described (15). Further details are provided under METHODS.
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PHYSIOLOGICAL TABLE
3.
ROLE
OF
1639
2 PROTEIN
Efect of BSP on ubtake and esterij%ation by fierf used rat liver -__-_--^- -- ^.--
of [3H]oleate
--
Total
Uptake
Per cent of injected dose
pm01
Total
Esteriflcstion
Percent of uptake
I 1
pm01
HOT?lOgeI
nafe
0
90 360 720
I 4 1 1 ’ 1
5.9310.38*!3*55f0.23* ‘3.75 6.25 12.30 7.40 12.40 ;7.45
68 .OztZ .04* 2.42zkO.21* 50.3 1.88 21.4 1 .60 19.7 1.45
Supernatant
I
0 90
I
4
360 720
I
l
* Values
! I
J. 1
0.45+0.02*Oo27+0.02* 0.47 0.28 0.78 0.47 0.87 0.52
are means
45.8&1,25*00.1~30.01* 45 . 9 0.16 32.7 IO.15 IO.16 29.6 b
+ SE.
and esterified oleate were qualitatively similar but less pronounced. The inhibition of oleate binding to 2 following infusion of 90, 360, and 720 pmol/kg of BSP was 25.6, 77.3, and 78.4 %, respectively. DISCUSSION
Within 2 min after injection of [3H]oleate into the fcnlural or portal vein of the rat, radioactivity is associated with hepatic 2 protein as well as other hepatic cytoplaslnic proteins. In the intact rat and in the perfused liver, pretreatment with flavaspidic acid inhibited binding of fatty acid radioactivity to 2 protein as previously described in vitro (18). In intact rats, flavaspidic acid administration increased radioactivity, associated with high molecular weight fraction of liver supernatant (Fig. 2). This radioactivity was largely in esterified fatty acid, which may be bound to the sterol carrier protein (23) or lipoproteins released from intracellular organelles during homogenization. In spite of quantitative differences in the binding pattern of oleatc radioactivity to hepatic cytoplasmic proteins, flavaspidic acid consistently reduced radioactivity associated with 2 protein by approximately 90 %. Despite this effect, hepatic uptake of r3H]oleate was unchanged in the intact animal and increased in the perfused liver preparation. In the perfused liver preparation, a quantitatively similar increase in uptake of [3H]oleate was observed following infusion of BSP which also displaces fatty acids from 2 (16). These results indicate that binding of fatty acids to 2 protein is not obligatory in the hepatic uptake of long-chain fatty acids. Even if 2 protein is a plasma-membrane component, it is unlikely to be an important mediator of fatty acid uptake. Binding of r3H]oleate by a preparation rich in liver plasma membranes was not inhibited by simultaneous incubation with flavaspidic acid (unpublished observations). The mechanism by which hepatic uptake of fatty acids by the perfused liver is increased after flavaspidic acid or BSP administration is not clear. Absence of endogcnous albumin and other plasma proteins in the perfused liver may enhance hepatic uptake of free fatty acids, although, as stated under METHODS, the experiments were designed to
eliminate this possibility Liver perfusion at 15 ml/min in situ may theoretically result in sinusoidal perfusion of a greater number of parenchymal liver cells than occurs in viva. Regardless of these considerations, the important observation is that administration of flavaspidic acid or BSP desnite increased hepatic reduced olea te csterification, uptake of oleate (Tables 2, 3). Possible cflects on fatty acid oxidation were not studied. To minimize PO tential effects of A avaspidic acid on oleate binding to seru 1n album In, severa studies w *ere performed in vivo and with the perfused liver preparation in which infusion of flavaspidic acid preceded administration of oleate bound to albumin. These procedural modifications had no significant effect on olea te up take or esterification (Table l)- In addition, circular dichroism studies of the major high-affinity site of organic anion binding to bovine or rat serum albumin did not reveal colnpetition between flavaspidic acid and oleate at the concentrations used in the present experilnents (16). Because flavaspidic acid did not manifest over hepatotoxicity, reduction in fatty acid cs teriiicatio n n-i he -perfused liver after its administration raises several possibilities. Flavaspidic acid may inhibit microsomal esterifying enzymes, or reduced binding of fatty acid radioactivity to 2 protein may be responsible. Either possibility suggests that 2 protein may be involved in fatty acid intermediary meta holism. This latter possibility is supported by the requircment of an unidentified SUKKrna tant protein (7) for free fatty acid activation to coenzyme A thioesters by rat liver mitochondria. In addition, fatty acid CoA derivatives have a high affinity for 2 protein in vitro (21). CYBromopalmitin inhibits activation of long-chain fatty acids by rat liver preparations and also inhibits binding of palmityl-CoA to 2 protein (19). BSP infusion in the perfused liver in situ produced effects on oleate uptake and esterification similar to those observed with flavaspidic acid (Tables 2, 3). For example, infusion of 720 pm01 BSP/kg enhanced hepatic uptake of oleate by 209 %I above control values, reduced fatty acid esterification by 59 ‘g), and inhibited oleate binding to 2 protein by 78 % (Table 3). The effect of BSP on hepatic uptake and esterification of oleate in vivo was not investigated. In the perfused liver, BSP presumably acts in a manner similar to flavaspidic acid in enhancing hepatic uptake of oleate as well as reducing esterification. BSP inhibits the activity of several hepatic microsomal enzymes in vitro including uridine diphosphatc glucuronyl transferase, cytochrome P-450, and Inixed-function oxidase (12 and unpublished observations) and may have a similar cflect on microsomal fatty acid estcrifying enzymes. In other studies, flavaspidic acid reduced plasma disappearance and hepatic uptake of BSP in association with displacement of the dye from 2 but not Y fraction (15, 17). These observations indicate that 2 protein and ligandin (Y protein) may function as organic anion binding proteins. Although 2 protein is not obligatory in fatty acid uptake by the liver, it may be important in fatty acid esterification. WC thank Dr. Keith Henley (U nivcrsity of Michigan) for determinations of hepatic oxygen uptake in the presence of Aavaspidic acid, and Dr. Huntington Sheldon (McGill University) for electron microscopic studies.
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I640
MISHKIN
This resealch was supported by Public I-kalth Service Grants AM 02019, 05384, 10880, and 17702 and the Medical Research council of Canada Grant MA 4658. Parts of this work were presented at the annual meeting of the kimerican Association for the Study of Liver Disease, Chicago, Ill., November 2, 1972, and have been published in abstract form (17).
S. Mishkin is a Scholar of the Medical G. Fleischner is the recipient of AM 70228 from the National Institute Digestive Diseases. Received
for
publication
22 July
ET
AL.
Research Council of Canada. Clinical Investigation Award of Arthritis, Metabolism, and
1974.
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