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The effect of bile salts on the formation and hydrolysis of cholesterol esters by rat liver enzymes
137
Biochimica et Biophysics Acta, 487 (1977) @ Elsevier/North-Holland Biomedical Press
137-144
BBA 56970
THE EFFECT OF BILE SALTS ON THE FORMATION AND HYDROLYSIS OF CHOLESTEROL ESTERS BY RAT LIVER ENZYMES
V.J. NEELON
* and L. LACK
Department of Physiology and Pharmacology, Durham, N.C. 27710 (U.S.A.) (Received
November
Box 3185, Duke University Medical Center,
lst, 1976)
Summary To determine the effects of different bile salts on the enzymic esterification of cholesterol and the hydrolysis of cholesterol esters rat liver homogenates and rat liver microsomes were incubated with varying amounts of different bile salts. Bile salts inhibited the formation of radioactive cholesterol esters in incubations of either rat liver homogenates or rat liver microsomes containing [ “C]cholesterol. Chenodeoxycholate, glycochenodeoxycholate and taurochenodeoxycholate were more potent inhibitors than their comparable cholate analogues. Bile salts stimulated the hydrolysis of cholesterol esters when incubations were carried out with the liver homogenates. The dihydroxy bile salts were again more potent in this regard than the trihydroxylated bile salts. When the effects of bile salts on cholesterol ester hydrolysis were studied in in vitro incubations of hepatic microsomes a biphasic mode of action was observed. In the absence of Na’ or K’ bile salts stimulated the hydrolysis of cholesterol oleate. However, following the addition of either Na’ or K’ to the microsomal incubations, bile salts caused an inhibition of cholesterol ester hydrolysis. Since cholesterol esterification was also inhibited under these conditions a direct inhibitory effect (not attributable to enhanced hydrolase activity) of the bile salts on the formation of cholesterol esters by the microsomes was established. Furthermore, this inhibition takes place at the transacylation step involving the fatty acyl-CoA ester and the sterol. These results suggest that bile salts can significantly alter the cholesterol-cholesterol ester profile in the liver, and furthermore, that these effects may be influenced by small changes in the intracellular environment in the region where these reactions occur.
* Present address: Department of Physiology. Chapel Hill, N.C. 27514. U.S.A.
The
University
of
North
Carolina
at Chapel
Hill,
138
Introduction Cholesterol ester formation in rat liver is an ATP-CoA-dependent enzymatic transfer of a fatty acyl group from a fatty acyl-CoA ester to the free hydroxyl group of cholesterol (EC 2.3.1). The enzymes are particulate, have a pH optimum around 7, and activity can be demonstrated in both mitochondrial and microsomal fractions [ 11. The requirements for ATP and CoA can be replaced by using preformed fatty acyl coenzyme A esters in the incubation mixture. Cholesterol ester-hydrolyzing enzymes in rat liver (sterol ester hydrolases, EC 3.1.1.13) have been located in various subcellular fractions, the bulk of the activity reported by Deykin and Goodman [2] as being present in the soluble fraction. No cofactors are required for the reaction and the pH optimum was reported to be between 6 and 7. Limited information is available on the effects of bile salts on the formation and hydrolysis of cholesterol esters in liver. Goodman et al. [l] reported that bot.h taurocholate and glycocholate caused more than 70% inhibition of esterification at 1 mg/ml (2 pmol). Cholic acid was reported to inhibit esterification at 10e6 M in rat liver homogenates [3]. Stokke [4], studying the effects of an acid cholesterol esterase found in human liver on labelled plasma cholesterol esters, reported that hydrolysis was slightly stimulated in the presence of low concentrations of sodium taurocholate (0.1 mg/ml) while concentrations of 1 mg/ml were inhibitory to hydrolysis. In order to evaluate in more detail the effects of different bile salts on the enzymatic esterification of cholesterol and hydrolysis of cholesterol esters, experiments were undertaken using bile salts synthesized and purified in this laboratory. The following paper reports the findings of in vitro studies of cholesterol ester formation and hydrolysis, using rat liver homogenates and microsomes, in the presence of varying amounts of different bile salts. Evidence is also presented demonstrating that the observed inhibition of microsomal esterification by bile salts is a direct one and not attributable to enhanced esterase activity. In addition, the present study provides information that the effects of bile salts on hydrolytic activity can be reversed by altering the ionic conditions of the reaction media. Materials and Methods The conjugated bile salts were prepared by the previously described procedure of Lack et al. [5] and purified by the reverse phase chromatographic procedure of Norman [6]. The [4-14C]cholesterol isotopes of cholesterol and cholesterol oleate (New England Nuclear) were checked for purity by thinlayer chromatography. The cholesterol oleate was purified before each use by silica gel thin-layer chromatography using hexane/ethyl ether/acetic acid (80 : 20 : 1 v/v). Human serum albumin (Sigma Co.) was defatted by the method of Chen [7]. Rat liver homogenates and microsomal preparations, as well as the conditions for incubation, developed from the methods of Stokke [4], Deykin and Goodman [2] and Goodman et al. [l]. Male Sprague-Dawley rats (200-500 g) were killed by decapitation, the liver excised, and washed with ice-cold 0.25 M
139
sucrose or 0.1 M potassium phosphate buffer (pH 7.4). The livers were homogenized in 2.5 ml of buffer or sucrose per g of liver pulp. Serial centrifugations at 2000 X g (30 min), 10 000 X g (30 min), and 104 000 X g (60 min) were performed to obtain the microsomal pellet, which was washed once and centrifuged again at high speed for 1 h. The microsomes were resuspended in a volume of media equal to the S-10 supernatant and divided into aliquots and frozen at -70°C until used. The crude homogenate, S-6, was prepared by homogenization in 0.25 M sucrose and centrifugation at 6000 X g for 30 min and the supernatant removed and frozen in aliquots at -70°C for later use. The incubation procedure for evaluation of cholesterol esterification and hydrolysis was carried out at least in duplicate for 60 min in air at 37°C in an agitating water bath. Depending on the effect being examined, the flasks contained specified amounts of the following compounds: ATP, CoA, defatted human serum albumin, potassium phosphate buffer (pH 7.4 or 6.1), potassium palmitate, [4-‘4C]cholesterol or cholesterol oleate, homogenate equivalent to 20 mg of whole liver or microsomes (0.2-l mg protein/ml), and specific bile salts. Control flasks contained boiled homogenate or microsomes. Incubations were terminated by additions of chloroform/methanol (2 : 1, v/v). The cholesterol and cholesterol esters were extracted as described by Goodman [ 81. Cholesterol and cholesterol esters were then separated by thin-layer chromatography on silica gel. The solvent system used was hexane/ethyl ether/acetic acid (80 : 20 : 1, v/v). The plates were scraped in l-cm divisions into vials and the radioactivity determined by liquid scintillation spectrometry to assay for relative amounts of free and esterified cholesterol. Protein determinations were done by the method of Lowry et al. [9]. Results
Whole homogenates in sucrose, S-6 Fig. 1 shows that the esterification of cholesterol by homogenates of rat liver is sensitive to the presence of free fatty acids. Potassium palmitate (0.1-l pmol per incubation) stimulated esterification (4-5s) when added to incubations containing defatted human serum albumin. Increasing concentrations of fatty acids, beyond a critical mol ratio of free fatty acid to human serum albumin, or removal of the human serum albumin resulted in inhibition. The extent of inhibition was proportional to the amount of free fatty acid added. When CoA or ATP was omitted from the incubation mixture esterification was inhibited greater than 90%. Substitution of palmityl-CoA for ATP and CoA restored the ability of the preparation to esterify cholesterol. The addition of conjugated and unconjugated bile salts also inhibited esterification (Fig. 2). The dihydroxy bile salt, sodium taurochenodeoxycholate, inhibited esterification by nearly 90% at 0.5 pmol/ml while the trihydroxy compound, sodium taurocholate, resulted in less than 50% inhibition at the same concentration. The same inhibition by bile salts was still observed when palmityl-CoA was substituted for ATP and CoA. The pattern of inhibition by bile salts was not altered by the addition of free fatty acid. The hydrolytic activity of the S-6 homogenate was examined to determine whether reduced incorporation of total radioactivity into the cholesterol ester
140
Potossium Polmhde
(pmoles
j
Fig. 1. Effect of potassium palmitate on the enzyme esterification of cholesterol in whole homogenates (S-6) of rat liver. The basic incubation mixture contained: crude homogenate (S-6). 2.5 mg protein; ATP, 25 pmol; CoA, 1 !~mol: potassium palmitate, 0.75 pmol; human serum albumin. (free of fatty acid), 12 mg; potassium phosphate buffer, PH 7.4. 120 ~mol: [ 14Clcholesterol. 0.02 PCi, spec. act. 58 Ci/mol. Modifications of potassium palmitate are as indicated. The total volume was 2.0 ml. Incubations were carried out at 37OC for 1 h. , representsan individual incubation. n, represents the average value of the incubation at the specified concentrations of fatty acid. The values at 0.5 and 1.0 are significantly different from the value at 0 (P < 0.002 for 0.5 ~mol/ml: P < 0.015 for 1.0 /.~mol/ml: using the Student’s t-test.
fraction in the presence of bile salts can be attributed to the enhanced hydrolysis of this fraction by cholesterol esterase. Hydrolysis of cholesterol oleate by this preparation requires no cofactors and, in 60 min, resulted in an average of 30% hydrolysis of the added substrate. Hydrolysis was stimulated by the addition of bile salts. A 60% increase over the controls was observed with 0.25 ~mol/ml of taurochenodeoxycholate, while at 0.5 pmol/ml the hydrolysis was 90% greater than control values. The trihydroxylated bile salt, taurocholate, also stimulated the hydrolysis of cholesterol oleate but to a lesser extent than that observed for taurochenodeoxycholate (73% stimulation at 0.5 pmol/ml).
Microsomal In order
025
preparations to clarify
05
whether
IO pmoles BileSalt per ml
the
effect
of bile salts on esterification
was
20
Fig. 2. Effect <,f various bile salts on the en/.ymic esterification of cholesterol by S-6 homogenates of rat liver. Conditions are the same as thwe described as basic incubation mixture in Fig. 1. Each point is the average of two incubatir)ns. TC, taurocholate. GC, glycocholate; TCDC. taurochrnodeoxyrholate: CDC. chenodeoxycholate; GCDC, glycochrnodeoxycholatr.
141
primarily that of inhibition of ester formation rather than stimulation of ester hydrolysis, studies were undertaken with microsomes. Microsomes prepared in potassium phosphate buffer showed similar properties with respect to esterification as did the S-6 homogenate. Table I shows both control levels of esterification and the effect on this process of bile salts. The results were similar to those observed with crude homogenates. Esterification was inhibited by the addition of bile salts, and the dihydroxylated bile salts again were more effective than the trihydroxylated bile salts. Hydrolysis of cholesterol oleate by these microsomal preparations was much more variable than had been seen with the S-6 homogenate. Values ranged between 12 and 40% with an average value for 12 experiments of 24.5%, an average value higher than previously reported for microsomal catalysed hydrolysis [l]. In contrast to the stimulation of hydrolysis seen in the S-6 homogenate, bile salts inhibited hydrolysis in the microsomal preparation (Fig. 3). This inhibitiory effect occurred under incubation conditions identical to those required to inhibit esterification (Table I). The percent inhibition was similar for the trihydroxy taurocholate and the dihydroxy taurochenodeoxycholate at low concentrations. When the concentration was increased to 0.5 pmol/ml taurochenodeoxycholate was slightly more effective as an inhibitor than taurocholate. Thus in the microsomal preparation bile salts were inhibitory to both esterification and hydrolysis while in the S-6 fraction bile salts inhibited esterification but stimulated hydrolysis. Effects
of ions, pH, and bile salts
In an attempt to explain the difference observed, microsomes were prepared in 0.25 M sucrose rather than potassium phosphate buffer. Cholesterol ester hydrolysis by microsomes prepared in sucrose is shown in Table II. In the presence of potassium phosphate buffer, pH 7.4, (100 E.tmol/ml) hydrolysis tended to be low (average 10%). If the buffer pH was reduced to pH 6.1, or if the potassium buffer was absent, the control hydrolysis increased to 36 and 28.3% of added substrate, respectively. Table II also shows the effect of adding sodium taurochenodeoxycholate under these incubation conditions. In the presence of 100 pmol/ml potassium phosphate buffer at either pH 7.4 or 6.1, the addition of taurochenodeoxycholate (0.5 pmol/ml) inhibited hydrolysis by
TABLE
I
ESTERIFICATION
OF
SIUM
PHOSPHATE
BUFFER
Each
3 ml
Potassium ,umol;
incubation phosphate
[ 14Clcholesterol,
Bile salt
[14ClCHOLESTEROL
contained buffer. 0.02
1 mg
PH 7.4, &i.
of
300
spec.
BY
Amount
microsomal
pmol;
act.
RAT
CoA,
58 Ci/mol. added
LIVER
protein; 0.3
pmol;
MICROSOMES
human ATP,
Incubations Number
serum 6 gmol;
were of
PREPARED
for
albumin
IN POTAS-
(defatted),
potassium
3 mg;
palmitate.
0.075
1 h at 37OC.
Esterification
Inhibition
Olmol)
experiments
average
(%)
NOIW
-
6
13.2
0
Taurocholata
1
2
8.2
38
Taurochenodeoxycholate
1
2
2.0
85
Glycochenodeoxycholate
1
2
2.5
81
(a)
142
I+) bile solt i 0 5 pmoteslntl I
,
017
033 05 &mole Bde Salt per ml
f
-
,
25 50 too pmoles Potosstum Phosphole Buffer /ml
f
150 (pH6 I)
Fig. 3. The effects of bile salts on the enzymic hydrolysis of cholesterol oleatc by rat liver microsomes prepared in potassium phosphate buffer. Conditions are those described in Table I, except that [ ’ 4Clcholesterol oleate. 0.02 PCi. spec. act. 52 Ci/mol, was substituted for the cholesterol. Each point represents the average of two incubations. j), incubations with taurocholate. 0, those with taurochenodeoxycholate. Fig. 4. The effects of K+ on the hydrolysis of cholesterol oleate by rat liver microsomes in the presence and absence of taurochenodeoxycholate. Each point is the average of two incubations. Each incubation mixture contained: microsomal protein, 1 mg; human serum albumin, 3 mg; [ 14CIcholesterol oleate. 0.02 @i. spec. act. 52 Ci/mol. Final volume was 2.0 ml. Incubations were for 1 h at 37’C. The microsomes were prepared in sucrose media as described in Materials and Methods.
over 70%. In contrast, the presence of the bile salt in the absence of potassium phosphate buffer results in a 3-fold stimulation of hydrolysis. Fig. 4 demonstrates the effects on hydrolysis of increasing potassium phosphate in the presence of taurochenodeoxycholate (0.5 ~mol/ml). This inhibitory effect can
TABLE
II
EFFECT OF pH, POTASSIUM PHOSPHATE AND TAUROCHENODEOXYCHOLATE ON CHOLESTEROL ESTER HYDROLYSIS BY MICROSOMES WHICH WERE PREPARED KN SUCROSE Each 2 ml incubation contained: microsomal protein, 1 mg; human serum albumin (defatted). cholesterol oleate, 0.02 nCi, spec. act. 52 Ci/mol. incubations were for 1 h at 37OC. Expt.
No.
ph of incubations
Potassium phosphate wmollml)
Taurochenodeoxycholate (&mol/ml)
3 ma: 1’ 4C1-
Hydrolysis
Control
(%I
(%)
A
3 3
1.4 7.4
100 100
0.5
10.3 2.2
100 22
B
2 2
6.1 6.1
100 100
0.5
36.0 10.4
100 29
C
2 4
6.0 6.0
-
0.5
28.3 65.2
100 231
143
also be produced by substituting KC1 or NaCl for potassium phosphate. In addition, it would also appear from the data in Fig. 4 that potassium phosphate alone may have some stimulatory effect on hydrolysis although the level of this enhancement was much less than that seen with bile acids in the absence of buffer. Discussion Cholesterol ester formation in rat liver is an ATP-CoA-dependent process which, in both the crude homogenate and the microsomal preparation, consistently resulted in about 12-16s ester formation from added cholesterol substrate. The stimulation of the ester formation at low concentrations of free fatty acids, followed by inhibition at greater concentrations and the relation to human serum albumin concentrations has previously been observed and discussed by Goodman and coworkers [ 11. Ability to stimulate ester formation with added palmitic acid rather than only with oleate, as previously reported by Goodman et al. [ 11, may reflect the differences in available endogenous substrate in these preparations. The findings from these in vitro studies suggest that bile acids which are synthesized in the liver and are present there during their enterohepatic circulation may have real effects on cholesterol ester formation and hydrolysis. Both the trihydroxy bile salts (cholic acid and its glycine and taurine conjugates) and the dihydroxy bile salts (chenodeoxycholic acid and its glycine and taurine conjugates) inhibited the biosynthesis of cholesterol ester. In this regard the chenodeoxycholic acid compounds were more potent than the cholate compounds. That this inhibition includes a direct effect on the enzymes involved in esterification rather than only the stimulation of hydrolysis is suggested by the inhibition of ester formation observed in those incubations of microsomes under conditions where hydrolysis was also inhibited. The difference in inhibition between the dihydroxy compounds and the trihydroxy salts might be explained also by a difference in binding capacity of albumin for the tvo kinds of bile acids. However, this explanation seems unlikely with respect to estefication since experiments in which human serum albumin concentration varied did not seem to significantly alter the dose vs. response curve for taurochenodeoxycholate inhibition of ester formation. Hydrolysis of cholesterol esters by hepatic cholesterol esterase appears to be a process much more susceptible to changes in its chemical environment. In the S-6 homogenate, where the concentration of added potassium was approx. 60 E.tmol/ml, the addition of bile salts stimulated the hydrolysis of cholesterol esters. This stimulation increased with increasing bile salts concentrations up to 1 pmol/ml and again the dihydroxy bile salts appeared more effective than the trihydroxy bile salts. In contrast to these findings with the S-6 preparations, hydrolysis of cholesterol esters by microsomes show a biphasic pattern with respect to the presence of bile salts. Depending on the concentration of K+, bile salts can either enhance or inhibit esterase acitvity. The stimulation of microsomal esterase activity by bile salts was observed at concentrations of K’ below 25 pmol/ml. At higher concentrations bile salts elicited an inhibitory effect. This ability of bile salts to effect either stimulation
144
or inhibition of the cholesterol esterase would suggest that the cholesterolcholesterol ester profile in the liver could be significantly influenced by small changes in the intracellular environment in the region where these reactions occur. Acknowledgements This work was supported by research grant AM-09582 from the National Institutes of Health, and in part by grant (RR-30) from the General Clinical Research Centers Program for the Division of Research Sources, National Institutes of Health. Virginia J. Neelon was the recipient of a postdoctoral fellowship award from the Division of Nursing, D.H.E.W, U.S.P.H.S. (No. 5F04NU-27,137-06 during the tenure of this study. References Goodman,
D.S.,
Deykin,
D. and
Takeuchi,
K.T. L.,
Norman, Chen.
D.S.
Dorrity, A.
R.F.
Yamamura,
(1972)
(1967) D.S.
Academic
Press. O.H.,
Y.
Chem.
J. Biol. (1969) New
in
(1973)
Chem.
J.T.
J. Biol.
Chem.
237.
Atherosclerosis Acta
and
Stand.
270,
Singletary,
Chem.
239,
1335
3649
17,
211
156 G.D.
(1973)
J. Lipid
Res.
14,
367
7, 1413
242.
Methods
T. (1964)
J. Biol.
Biophys.
Walker,
Acta
Shiratori. (1962)
Biochim. F.O..
(1953)
Goodman, Lowry,
D. and
N. and
Stokke, Lack,
Deykin, Goodman,
173 in
Enzymology
(Clayton,
R.B.,
ed.),
Vol.
XV,
Chap.
22.
York
Rosebrough,
N.J.,
Fan,
A.L.
and
Randall,
R.J.
(1951)
J. Biol.
Chem.
193,
265-275
P. 522.
Biochimica et Biophysics Acta, 487 (1977) @ Elsevier/North-Holland Biomedical Press
137-144
BBA 56970
THE EFFECT OF BILE SALTS ON THE FORMATION AND HYDROLYSIS OF CHOLESTEROL ESTERS BY RAT LIVER ENZYMES
V.J. NEELON
* and L. LACK
Department of Physiology and Pharmacology, Durham, N.C. 27710 (U.S.A.) (Received
November
Box 3185, Duke University Medical Center,
lst, 1976)
Summary To determine the effects of different bile salts on the enzymic esterification of cholesterol and the hydrolysis of cholesterol esters rat liver homogenates and rat liver microsomes were incubated with varying amounts of different bile salts. Bile salts inhibited the formation of radioactive cholesterol esters in incubations of either rat liver homogenates or rat liver microsomes containing [ “C]cholesterol. Chenodeoxycholate, glycochenodeoxycholate and taurochenodeoxycholate were more potent inhibitors than their comparable cholate analogues. Bile salts stimulated the hydrolysis of cholesterol esters when incubations were carried out with the liver homogenates. The dihydroxy bile salts were again more potent in this regard than the trihydroxylated bile salts. When the effects of bile salts on cholesterol ester hydrolysis were studied in in vitro incubations of hepatic microsomes a biphasic mode of action was observed. In the absence of Na’ or K’ bile salts stimulated the hydrolysis of cholesterol oleate. However, following the addition of either Na’ or K’ to the microsomal incubations, bile salts caused an inhibition of cholesterol ester hydrolysis. Since cholesterol esterification was also inhibited under these conditions a direct inhibitory effect (not attributable to enhanced hydrolase activity) of the bile salts on the formation of cholesterol esters by the microsomes was established. Furthermore, this inhibition takes place at the transacylation step involving the fatty acyl-CoA ester and the sterol. These results suggest that bile salts can significantly alter the cholesterol-cholesterol ester profile in the liver, and furthermore, that these effects may be influenced by small changes in the intracellular environment in the region where these reactions occur.
* Present address: Department of Physiology. Chapel Hill, N.C. 27514. U.S.A.
The
University
of
North
Carolina
at Chapel
Hill,
138
Introduction Cholesterol ester formation in rat liver is an ATP-CoA-dependent enzymatic transfer of a fatty acyl group from a fatty acyl-CoA ester to the free hydroxyl group of cholesterol (EC 2.3.1). The enzymes are particulate, have a pH optimum around 7, and activity can be demonstrated in both mitochondrial and microsomal fractions [ 11. The requirements for ATP and CoA can be replaced by using preformed fatty acyl coenzyme A esters in the incubation mixture. Cholesterol ester-hydrolyzing enzymes in rat liver (sterol ester hydrolases, EC 3.1.1.13) have been located in various subcellular fractions, the bulk of the activity reported by Deykin and Goodman [2] as being present in the soluble fraction. No cofactors are required for the reaction and the pH optimum was reported to be between 6 and 7. Limited information is available on the effects of bile salts on the formation and hydrolysis of cholesterol esters in liver. Goodman et al. [l] reported that bot.h taurocholate and glycocholate caused more than 70% inhibition of esterification at 1 mg/ml (2 pmol). Cholic acid was reported to inhibit esterification at 10e6 M in rat liver homogenates [3]. Stokke [4], studying the effects of an acid cholesterol esterase found in human liver on labelled plasma cholesterol esters, reported that hydrolysis was slightly stimulated in the presence of low concentrations of sodium taurocholate (0.1 mg/ml) while concentrations of 1 mg/ml were inhibitory to hydrolysis. In order to evaluate in more detail the effects of different bile salts on the enzymatic esterification of cholesterol and hydrolysis of cholesterol esters, experiments were undertaken using bile salts synthesized and purified in this laboratory. The following paper reports the findings of in vitro studies of cholesterol ester formation and hydrolysis, using rat liver homogenates and microsomes, in the presence of varying amounts of different bile salts. Evidence is also presented demonstrating that the observed inhibition of microsomal esterification by bile salts is a direct one and not attributable to enhanced esterase activity. In addition, the present study provides information that the effects of bile salts on hydrolytic activity can be reversed by altering the ionic conditions of the reaction media. Materials and Methods The conjugated bile salts were prepared by the previously described procedure of Lack et al. [5] and purified by the reverse phase chromatographic procedure of Norman [6]. The [4-14C]cholesterol isotopes of cholesterol and cholesterol oleate (New England Nuclear) were checked for purity by thinlayer chromatography. The cholesterol oleate was purified before each use by silica gel thin-layer chromatography using hexane/ethyl ether/acetic acid (80 : 20 : 1 v/v). Human serum albumin (Sigma Co.) was defatted by the method of Chen [7]. Rat liver homogenates and microsomal preparations, as well as the conditions for incubation, developed from the methods of Stokke [4], Deykin and Goodman [2] and Goodman et al. [l]. Male Sprague-Dawley rats (200-500 g) were killed by decapitation, the liver excised, and washed with ice-cold 0.25 M
139
sucrose or 0.1 M potassium phosphate buffer (pH 7.4). The livers were homogenized in 2.5 ml of buffer or sucrose per g of liver pulp. Serial centrifugations at 2000 X g (30 min), 10 000 X g (30 min), and 104 000 X g (60 min) were performed to obtain the microsomal pellet, which was washed once and centrifuged again at high speed for 1 h. The microsomes were resuspended in a volume of media equal to the S-10 supernatant and divided into aliquots and frozen at -70°C until used. The crude homogenate, S-6, was prepared by homogenization in 0.25 M sucrose and centrifugation at 6000 X g for 30 min and the supernatant removed and frozen in aliquots at -70°C for later use. The incubation procedure for evaluation of cholesterol esterification and hydrolysis was carried out at least in duplicate for 60 min in air at 37°C in an agitating water bath. Depending on the effect being examined, the flasks contained specified amounts of the following compounds: ATP, CoA, defatted human serum albumin, potassium phosphate buffer (pH 7.4 or 6.1), potassium palmitate, [4-‘4C]cholesterol or cholesterol oleate, homogenate equivalent to 20 mg of whole liver or microsomes (0.2-l mg protein/ml), and specific bile salts. Control flasks contained boiled homogenate or microsomes. Incubations were terminated by additions of chloroform/methanol (2 : 1, v/v). The cholesterol and cholesterol esters were extracted as described by Goodman [ 81. Cholesterol and cholesterol esters were then separated by thin-layer chromatography on silica gel. The solvent system used was hexane/ethyl ether/acetic acid (80 : 20 : 1, v/v). The plates were scraped in l-cm divisions into vials and the radioactivity determined by liquid scintillation spectrometry to assay for relative amounts of free and esterified cholesterol. Protein determinations were done by the method of Lowry et al. [9]. Results
Whole homogenates in sucrose, S-6 Fig. 1 shows that the esterification of cholesterol by homogenates of rat liver is sensitive to the presence of free fatty acids. Potassium palmitate (0.1-l pmol per incubation) stimulated esterification (4-5s) when added to incubations containing defatted human serum albumin. Increasing concentrations of fatty acids, beyond a critical mol ratio of free fatty acid to human serum albumin, or removal of the human serum albumin resulted in inhibition. The extent of inhibition was proportional to the amount of free fatty acid added. When CoA or ATP was omitted from the incubation mixture esterification was inhibited greater than 90%. Substitution of palmityl-CoA for ATP and CoA restored the ability of the preparation to esterify cholesterol. The addition of conjugated and unconjugated bile salts also inhibited esterification (Fig. 2). The dihydroxy bile salt, sodium taurochenodeoxycholate, inhibited esterification by nearly 90% at 0.5 pmol/ml while the trihydroxy compound, sodium taurocholate, resulted in less than 50% inhibition at the same concentration. The same inhibition by bile salts was still observed when palmityl-CoA was substituted for ATP and CoA. The pattern of inhibition by bile salts was not altered by the addition of free fatty acid. The hydrolytic activity of the S-6 homogenate was examined to determine whether reduced incorporation of total radioactivity into the cholesterol ester
140
Potossium Polmhde
(pmoles
j
Fig. 1. Effect of potassium palmitate on the enzyme esterification of cholesterol in whole homogenates (S-6) of rat liver. The basic incubation mixture contained: crude homogenate (S-6). 2.5 mg protein; ATP, 25 pmol; CoA, 1 !~mol: potassium palmitate, 0.75 pmol; human serum albumin. (free of fatty acid), 12 mg; potassium phosphate buffer, PH 7.4. 120 ~mol: [ 14Clcholesterol. 0.02 PCi, spec. act. 58 Ci/mol. Modifications of potassium palmitate are as indicated. The total volume was 2.0 ml. Incubations were carried out at 37OC for 1 h. , representsan individual incubation. n, represents the average value of the incubation at the specified concentrations of fatty acid. The values at 0.5 and 1.0 are significantly different from the value at 0 (P < 0.002 for 0.5 ~mol/ml: P < 0.015 for 1.0 /.~mol/ml: using the Student’s t-test.
fraction in the presence of bile salts can be attributed to the enhanced hydrolysis of this fraction by cholesterol esterase. Hydrolysis of cholesterol oleate by this preparation requires no cofactors and, in 60 min, resulted in an average of 30% hydrolysis of the added substrate. Hydrolysis was stimulated by the addition of bile salts. A 60% increase over the controls was observed with 0.25 ~mol/ml of taurochenodeoxycholate, while at 0.5 pmol/ml the hydrolysis was 90% greater than control values. The trihydroxylated bile salt, taurocholate, also stimulated the hydrolysis of cholesterol oleate but to a lesser extent than that observed for taurochenodeoxycholate (73% stimulation at 0.5 pmol/ml).
Microsomal In order
025
preparations to clarify
05
whether
IO pmoles BileSalt per ml
the
effect
of bile salts on esterification
was
20
Fig. 2. Effect <,f various bile salts on the en/.ymic esterification of cholesterol by S-6 homogenates of rat liver. Conditions are the same as thwe described as basic incubation mixture in Fig. 1. Each point is the average of two incubatir)ns. TC, taurocholate. GC, glycocholate; TCDC. taurochrnodeoxyrholate: CDC. chenodeoxycholate; GCDC, glycochrnodeoxycholatr.
141
primarily that of inhibition of ester formation rather than stimulation of ester hydrolysis, studies were undertaken with microsomes. Microsomes prepared in potassium phosphate buffer showed similar properties with respect to esterification as did the S-6 homogenate. Table I shows both control levels of esterification and the effect on this process of bile salts. The results were similar to those observed with crude homogenates. Esterification was inhibited by the addition of bile salts, and the dihydroxylated bile salts again were more effective than the trihydroxylated bile salts. Hydrolysis of cholesterol oleate by these microsomal preparations was much more variable than had been seen with the S-6 homogenate. Values ranged between 12 and 40% with an average value for 12 experiments of 24.5%, an average value higher than previously reported for microsomal catalysed hydrolysis [l]. In contrast to the stimulation of hydrolysis seen in the S-6 homogenate, bile salts inhibited hydrolysis in the microsomal preparation (Fig. 3). This inhibitiory effect occurred under incubation conditions identical to those required to inhibit esterification (Table I). The percent inhibition was similar for the trihydroxy taurocholate and the dihydroxy taurochenodeoxycholate at low concentrations. When the concentration was increased to 0.5 pmol/ml taurochenodeoxycholate was slightly more effective as an inhibitor than taurocholate. Thus in the microsomal preparation bile salts were inhibitory to both esterification and hydrolysis while in the S-6 fraction bile salts inhibited esterification but stimulated hydrolysis. Effects
of ions, pH, and bile salts
In an attempt to explain the difference observed, microsomes were prepared in 0.25 M sucrose rather than potassium phosphate buffer. Cholesterol ester hydrolysis by microsomes prepared in sucrose is shown in Table II. In the presence of potassium phosphate buffer, pH 7.4, (100 E.tmol/ml) hydrolysis tended to be low (average 10%). If the buffer pH was reduced to pH 6.1, or if the potassium buffer was absent, the control hydrolysis increased to 36 and 28.3% of added substrate, respectively. Table II also shows the effect of adding sodium taurochenodeoxycholate under these incubation conditions. In the presence of 100 pmol/ml potassium phosphate buffer at either pH 7.4 or 6.1, the addition of taurochenodeoxycholate (0.5 pmol/ml) inhibited hydrolysis by
TABLE
I
ESTERIFICATION
OF
SIUM
PHOSPHATE
BUFFER
Each
3 ml
Potassium ,umol;
incubation phosphate
[ 14Clcholesterol,
Bile salt
[14ClCHOLESTEROL
contained buffer. 0.02
1 mg
PH 7.4, &i.
of
300
spec.
BY
Amount
microsomal
pmol;
act.
RAT
CoA,
58 Ci/mol. added
LIVER
protein; 0.3
pmol;
MICROSOMES
human ATP,
Incubations Number
serum 6 gmol;
were of
PREPARED
for
albumin
IN POTAS-
(defatted),
potassium
3 mg;
palmitate.
0.075
1 h at 37OC.
Esterification
Inhibition
Olmol)
experiments
average
(%)
NOIW
-
6
13.2
0
Taurocholata
1
2
8.2
38
Taurochenodeoxycholate
1
2
2.0
85
Glycochenodeoxycholate
1
2
2.5
81
(a)
142
I+) bile solt i 0 5 pmoteslntl I
,
017
033 05 &mole Bde Salt per ml
f
-
,
25 50 too pmoles Potosstum Phosphole Buffer /ml
f
150 (pH6 I)
Fig. 3. The effects of bile salts on the enzymic hydrolysis of cholesterol oleatc by rat liver microsomes prepared in potassium phosphate buffer. Conditions are those described in Table I, except that [ ’ 4Clcholesterol oleate. 0.02 PCi. spec. act. 52 Ci/mol, was substituted for the cholesterol. Each point represents the average of two incubations. j), incubations with taurocholate. 0, those with taurochenodeoxycholate. Fig. 4. The effects of K+ on the hydrolysis of cholesterol oleate by rat liver microsomes in the presence and absence of taurochenodeoxycholate. Each point is the average of two incubations. Each incubation mixture contained: microsomal protein, 1 mg; human serum albumin, 3 mg; [ 14CIcholesterol oleate. 0.02 @i. spec. act. 52 Ci/mol. Final volume was 2.0 ml. Incubations were for 1 h at 37’C. The microsomes were prepared in sucrose media as described in Materials and Methods.
over 70%. In contrast, the presence of the bile salt in the absence of potassium phosphate buffer results in a 3-fold stimulation of hydrolysis. Fig. 4 demonstrates the effects on hydrolysis of increasing potassium phosphate in the presence of taurochenodeoxycholate (0.5 ~mol/ml). This inhibitory effect can
TABLE
II
EFFECT OF pH, POTASSIUM PHOSPHATE AND TAUROCHENODEOXYCHOLATE ON CHOLESTEROL ESTER HYDROLYSIS BY MICROSOMES WHICH WERE PREPARED KN SUCROSE Each 2 ml incubation contained: microsomal protein, 1 mg; human serum albumin (defatted). cholesterol oleate, 0.02 nCi, spec. act. 52 Ci/mol. incubations were for 1 h at 37OC. Expt.
No.
ph of incubations
Potassium phosphate wmollml)
Taurochenodeoxycholate (&mol/ml)
3 ma: 1’ 4C1-
Hydrolysis
Control
(%I
(%)
A
3 3
1.4 7.4
100 100
0.5
10.3 2.2
100 22
B
2 2
6.1 6.1
100 100
0.5
36.0 10.4
100 29
C
2 4
6.0 6.0
-
0.5
28.3 65.2
100 231
143
also be produced by substituting KC1 or NaCl for potassium phosphate. In addition, it would also appear from the data in Fig. 4 that potassium phosphate alone may have some stimulatory effect on hydrolysis although the level of this enhancement was much less than that seen with bile acids in the absence of buffer. Discussion Cholesterol ester formation in rat liver is an ATP-CoA-dependent process which, in both the crude homogenate and the microsomal preparation, consistently resulted in about 12-16s ester formation from added cholesterol substrate. The stimulation of the ester formation at low concentrations of free fatty acids, followed by inhibition at greater concentrations and the relation to human serum albumin concentrations has previously been observed and discussed by Goodman and coworkers [ 11. Ability to stimulate ester formation with added palmitic acid rather than only with oleate, as previously reported by Goodman et al. [ 11, may reflect the differences in available endogenous substrate in these preparations. The findings from these in vitro studies suggest that bile acids which are synthesized in the liver and are present there during their enterohepatic circulation may have real effects on cholesterol ester formation and hydrolysis. Both the trihydroxy bile salts (cholic acid and its glycine and taurine conjugates) and the dihydroxy bile salts (chenodeoxycholic acid and its glycine and taurine conjugates) inhibited the biosynthesis of cholesterol ester. In this regard the chenodeoxycholic acid compounds were more potent than the cholate compounds. That this inhibition includes a direct effect on the enzymes involved in esterification rather than only the stimulation of hydrolysis is suggested by the inhibition of ester formation observed in those incubations of microsomes under conditions where hydrolysis was also inhibited. The difference in inhibition between the dihydroxy compounds and the trihydroxy salts might be explained also by a difference in binding capacity of albumin for the tvo kinds of bile acids. However, this explanation seems unlikely with respect to estefication since experiments in which human serum albumin concentration varied did not seem to significantly alter the dose vs. response curve for taurochenodeoxycholate inhibition of ester formation. Hydrolysis of cholesterol esters by hepatic cholesterol esterase appears to be a process much more susceptible to changes in its chemical environment. In the S-6 homogenate, where the concentration of added potassium was approx. 60 E.tmol/ml, the addition of bile salts stimulated the hydrolysis of cholesterol esters. This stimulation increased with increasing bile salts concentrations up to 1 pmol/ml and again the dihydroxy bile salts appeared more effective than the trihydroxy bile salts. In contrast to these findings with the S-6 preparations, hydrolysis of cholesterol esters by microsomes show a biphasic pattern with respect to the presence of bile salts. Depending on the concentration of K+, bile salts can either enhance or inhibit esterase acitvity. The stimulation of microsomal esterase activity by bile salts was observed at concentrations of K’ below 25 pmol/ml. At higher concentrations bile salts elicited an inhibitory effect. This ability of bile salts to effect either stimulation
144
or inhibition of the cholesterol esterase would suggest that the cholesterolcholesterol ester profile in the liver could be significantly influenced by small changes in the intracellular environment in the region where these reactions occur. Acknowledgements This work was supported by research grant AM-09582 from the National Institutes of Health, and in part by grant (RR-30) from the General Clinical Research Centers Program for the Division of Research Sources, National Institutes of Health. Virginia J. Neelon was the recipient of a postdoctoral fellowship award from the Division of Nursing, D.H.E.W, U.S.P.H.S. (No. 5F04NU-27,137-06 during the tenure of this study. References Goodman,
D.S.,
Deykin,
D. and
Takeuchi,
K.T. L.,
Norman, Chen.
D.S.
Dorrity, A.
R.F.
Yamamura,
(1972)
(1967) D.S.
Academic
Press. O.H.,
Y.
Chem.
J. Biol. (1969) New
in
(1973)
Chem.
J.T.
J. Biol.
Chem.
237.
Atherosclerosis Acta
and
Stand.
270,
Singletary,
Chem.
239,
1335
3649
17,
211
156 G.D.
(1973)
J. Lipid
Res.
14,
367
7, 1413
242.
Methods
T. (1964)
J. Biol.
Biophys.
Walker,
Acta
Shiratori. (1962)
Biochim. F.O..
(1953)
Goodman, Lowry,
D. and
N. and
Stokke, Lack,
Deykin, Goodman,
173 in
Enzymology
(Clayton,
R.B.,
ed.),
Vol.
XV,
Chap.
22.
York
Rosebrough,
N.J.,
Fan,
A.L.
and
Randall,
R.J.
(1951)
J. Biol.
Chem.
193,
265-275
P. 522.