Biochimica Elsevier
BBALIP
413
et Biophysrca Aera. 1042 (1990) 413-416
BBA Report
50286
The effect of chylomicron remnants on bile acid synthesis in cultured rat hepatocytes Richard
P. Fry, Peter A. Mayes, Keith E. Suckling
Deparrment of Veterinaq
Busrc Sciences. The Rqval Veterinuy Welwy (Received
Key words:
Bile acid synthesis;
Cholesterol;
’
and Kathleen
M. Botham
College, London and ’ Smrth, Kline and French Research Ltd. (U.K.)
13 September
Chylomicron
1989)
remnant:
Chylomicron;
(Cultured
rat hepatocyte)
effect of chylomicron remnants on bile acid synthesis in isolated rat hepatocytes in monolayer cultures was investigated. Production of bile acids by the cells in the presence of chylomicron remnants at a cholesterol concentration of 7.8-9 nmol/ml was increased by approx. 75% after 17 h and 25% after 24 h incubation. Similar concentrations of cholesterol added to the cells in the form of chylomicrons had no significant effect on bile acid synthesis. These results suggest that cholesterol taken up in chylomicron remnants may be an important source of substrate for bile acid synthesis. The
Conversion to bile acids is a major route for the removal of cholesterol from the body [1,2]. Feeding rats cholesterol has been shown to increase bile acid synthesis both in vivo [3-61 and in cultured rat hepatocytes in vitro [7,8], suggesting that dietary cholesterol may have an important role in the supply of substrate for bile acid production. Cholesterol from the diet reaches the liver mainly in the form of chylomicron remnants, which are formed from the larger, triacylglycerol-rich chylomicrons by the action of lipoprotein lipase in extrahepatic capillary beds [9]. The remnant particles are then rapidly taken up by the liver [9-111 accompanied by extensive hydrolysis of cholesteryl ester [12]. Previous work in our laboratory [13] and that of Davis and co-workers [14] has shown that a high-density lipoprotein (HDL) fraction prepared from rat plasma increases bile acid synthesis in rat hepatocytes in monolayer culture. Davis et al. [7] have also obtained similar results with a lipoprotein fraction of d < 1.02 g/ml isolated from the plasma of cholesterol-fed rats. This fraction may contain some chylomicron remnants in addition to very-low-density lipoprotein (VLDL) and low-density lipoproteins (LDL). but because of the very rapid clearance of the remnant particles by the liver [15], a preparation substantially enriched in chylomicron remnants can only be prepared from chylomicrons incubated with lipoprotein lipase in vitro, or using functionally hepatectomised rats.
Correspondence: K.M. Botham. Department ences, The Royal Veterinary College, Royal NW1 OTU. U.K. 0005-2760/90/$03.50
of Veterinary Basic SciCollege Street. London.
$3 1990 Elsevier Science Publishers
B.V. (Biomedical
In the present study we have investigated the effect of chylomicron remnants, prepared after injectoin of chylomicrons into functionally hepatectomised rats, on bile acid synthesis in isolated rat hepatocytes in monolayer culture. The effect of the parent chylomicrons has also been studied for comparison. Male Wistar rats weighing 250-400 g maintained on a commercial pellet diet and allowed access to food and water ad libitum were used. After an oral dose of 0.5 ml of corn oil, rats were anaesthetised with Nembutal. the thoracic ducts cannulated and lymph collected as described previously [12]. Chylomicron remnants were prepared from the serum of functionally hepatectomised rats after injection of 1 ml lymph into an ileolumbar vein [12]. Hepatocytes were isolated and maintained in monolayer culture as before [16]. After adhesion of the cells to plastic dishes. the medium containing fetal calf serum was replaced with serum-free medium. Lipoproteins were added at this stage when required at a concentration of 7.8-9 nmol of cholesterol/ml. Conjugated cholic, chenodeoxycholic and ,&muricholic acids in the cell and medium samples were determined by radioimmunoassay [17-191. These three bile acids have been shown to represent about 95% of the total bile acid synthesised by isolated rat liver cells [16.20]. Bile acid synthesis was calculated by subtracting the amount found associated with the cells at time zero from the sum of the amounts found in the cell and medium samples after incubation. Proteins were determined by the method of Lowry et al. [21] and triacylglycerols and cholesterol were estimated enzymatically [22,23]. Significance limits were calculated using Student’s t-test. Division)
414 The triacylglycerol : cholesterol molar ratios found in the chylomicron and chylomicron remnant preparations used were 13.1 + 1.0 : 1 and 3.3 + 0.4 : 1, respectively. These ratios are consistent with those described previously [ 191. The absolute rate of bile acid synthesis in the hepatocytes without added lipoprotein was linear over 24 h, but showed a large variation from preparation to preparation, for example, total bile synthesis (cholic + chenodeoxycholic + ,kmuricholic acid) ranged from 0.06-0.24 nmol/mg protein per h, corresponding to 12-43 nmol/g liver per h. This is somewhat lower than the estimated rate of bile acid production by the rat liver in vivo (56-205 nmol/g liver per h) [24], but is comparable to values reported previously for hepatocytes in monolayer culture [8,16]. Despite the wide variation in the absolute rates of bile acid synthesis, the changes caused by the addition of lipoprotein to the cell 3
I
Percentage changes in bile mid synthesis in rut hepatocytes UI monolq~er culture caused by chylomcrons and chylomicron remnunts Hepatocytes isolated and maintained m culture as described in the text were incubated in the absence or presence of chylomwons or chylomicron-remnants (7.8-9.0 nmol cholesterol/ml in both cases). Bile acids were determined by radioimmunoassay. Data are expressed as a percentage of the values found in the absence of added lipoprotein and are the mean from fwe rats? S.E. At time zero the absolute values (nmol/mg protein) were 1.02 i 0.50 (total bile acid). 0.16 + 0.08 (cholic acid) and 0.86 i 0.21 (/3-muricholic acid). Significance limits compared to the values found with chylomicrons: ‘. P c 0.001: h> PiO.01. Bile acid
Bile acid synthesis
Total bile acid Conjugated cholic acid Conjugated ,0muricholic
A
1
TABLE
acid
(% control
value)
+ chylomicrons
+ chylomicron
17 h
24 h
17 h
109.7*4.4
109.Ok3.6
175.4k4.1
110.0+4.4
108.8*2.4
174.6k4.4’
109.6k
remnants 24 h
” 125.952.2
h
120.8k2.3
h
4.4 110.1 k 3.X 177.0 i_ 5.2 ’ 126.3 k 2.5 h
..L)----------o
time t h 1 Fig. 1. The effect of chylomicrons and chylomicron remnants on bile acid synthesis in rat hepatocytes in monolayer culture. Hepatocytes isolated and maintained as described in the text were incubated in the A) or presence of chylomicrons (O---O), or absence (Achylomicron remnants (o- - -0) (7.8-9 nmol cholesterol/ml in both cases). Bile acids were determined by radioimmunoassay. The experiment shown is typical of five performed. Each point is the mean of duplicate samples. (A) Total bile acid synthesis; (B) conjugated cholic acid synthesis; (C) conjugated fi-muricholic acid synthesis.
cultures were very consistent. For this reason, the absolute values for a typical experiment are shown in Fig. 1, while the aggregate data from five experiments are given in Table I as percentages of the values found in the absence of chylomicrons or chylomicron remnants. When hepatocyte monolayers were incubated in the presence of chylomicron remnants, the total amount of bile acid synthesised was significantly increased (about 75% after 17 h and 26% after 24 h) (Fig. 1A. Table I) compared with that in control cells. Similar increases were seen in the production of conjugated cholic and ,&muricholic acids (Fig. lB,C, Table I). Conjugated chenodeoxycholic acid synthesis, however, represented less than 1% of the total bile acid formed and was not significantly affected by the addition of chylomicron remnants. The amount of cholesterol required to bring about the observed increases (7.889.0 nmol/ml) is at least one order of magnitude lower than that needed to produce similar rises when the cholesterol is added as HDL or other lipoprotein fractions (110-930 nmol/ml) [8,13,14]. Furthermore, the changes can be seen without the need to maximise bile acid synthesis by feeding rats a bile acid sequestrant [13], or to prepare the cells in the presence of trypsin inhibitor [14]. Bile acid production in the chylomicron remnanttreated cells slowed down considerably between 17 and 24 h, leading to a fall in the observed increment over the control hepatocytes (Fig. 1, Table I). Clearly it is possible that this decrease from the maximum rate of synthesis may occur at a time earlier than 17 h, however, in a separate experiment using hepatocytes cultured for 5 h, we were unable to detect any significant
415 difference in the amount of bile acid synthesised by chylomicron remnant-treated as compared to control monolayers. It seems unlikely, therefore, that the maximum effect of chylomicron remnants occurs very much earlier than 17 h. Evidence from previous work with isolated hepatocytes has suggested that, in the absence of an extracellular source of cholesterol, the main source of substrate for bile acid production is newly synthesised cholesterol [25]. Davis et al. [8], however, found that a lipoprotein fraction of d < 1.02 g/ml isolated from the plasma of cholesterol fed rats decreased cholesterol synthesis in cultured hepatocytes by about 65% and chylomicron remnants have also been shown to have this effect in isolated hepatocytes in suspension and in the perfused liver [26]. It is possible, therefore, that in our experiments the small amount of added chylomicron remnant cholesterol is largely exhausted after a certain time, leaving bile acid production dependent on cholesterol synthesis, which is down-regulated compared to the control cells. When cholesterol was added to the hepatocyte monolayers in the form of chylomicrons at a similar concentration to that used in the experiments with chylomicron remnants there was no significant increase either in total bile acid synthesis, or in the amounts of the individual bile acids formed (Figs. 1 A-C, Table I). This is consistent with a number of earlier studies which have shown that isolated hepatocytes internalise and degrade chylomicron remnants at a faster rate than chylomicrons [27-301. The stimulatory effect of HDL on bile acid synthesis in cultured rat hepatocytes reported by Davis et al. [14] was only seen when the cells were isolated in the presence of soy bean trypsin inhibitor, suggesting that surface receptors for HDL are protected under these conditions. In our experiments, however, inclusion of the inhibitor in the collagenase perfusion buffer at a concentration similar to that used by Davis et al. (133 pg/ml) during the preparation of the hepatocytes did not affect total bile acid synthesis in the presence or absence of chylomicrons or chylomicron remnants (Fig. 2). Production of the individual bile acids measured was also unchanged (data not shown). Chylomicron remnants are responsible for the transport of most of the cholesterol of dietary origin to the liver. Our results show that this cholesterol is very much more effective in stimulating bile acid synthesis in isolated rat hepatocytes than any other lipoprotein fraction so far tested. These findings support the idea that the amount of cholesterol in the diet has a role in the regulation of bile acid synthesis by providing an important source of substrate via chylomicron remnants. Recently, however, BjGrkhem and Akerlund have reported that the rate-limiting enzyme for bile acid synthesis, cholesterol 7a_hydroxylase, is saturated with
200
6 1
4
A
El
150
control
remnants
> 3
100
8 ap
50
n Y
.
. trvwin _. inhibitor
+ trvpsin inhktor
Fig. 2. The effect of chylomicrons and chylomicron remnants on bile acid synthesis in rat hepatocytes prepared in the presence of trypsin inhibitor. Hepatocytes were isolated in the presence or absence of soy bean trypsin inhibitor (133 pg/ml) in the collagenase perfusion buffer. Monolayers were maintained as described in the text and were incubated without (0) or with chylomicrons (o), or chylomicron remnants (m). Bile acids were determined by radioimmunoassay. Data are expressed as a percentage of the values found in the absence of added lipoprotein and are the mean from four rats rt S. E.
substrate under most experimental conditions, and is therefore, unlikely to be influenced by changes in cholesterol availability [31]. Although another study has suggested that this is not the case [32], we cannot rule out the possibility that the effect of chylomicron remnants on bile acid synthesis seen in our experiments is caused by a mechanism other than increased substrate supply. R.P.F. was in receipt of a SERC CASE studentship sponsored by Smith, Kline and French Research Ltd. Part of this work was supported by a grant from the British Heart Foundation. References 1 Siperstein, M.D., Jayko, M.E., Chaikoff, I.L. and Dauben, W.G. (1952) Proc. Sot. Exp. Biol. Med. 81, 720-729. 2 Bergstrom, S. and Norman, A. (1953) Proc. Sot. Exp. Biol. Med. 83, 71-74. 3 Wilson, J.D. (1964) J. Lipid Res. 5, 409-417. 4 Beher, W.T., Casazza, K.K., Beher, M.E., Filus, A.M. and Bertasius, J.C. (1970) Proc. Sot. Exp. Biol. Med. 134, 595-602. 5 Raicht, R.F., Cohen, B.I., Shefer, S. and Mosbach, E.H. (1975) B&him. Biophys. Acta 388, 374-384. 6 Math& D. and Chevallier, F. (1979) J. Nutr. 109, 2076-2083. 7 Sutton, CM. and Botham, K.M. (1989) B&him. Biophys. Acta 1001, 210-217. 8 Davis, R.A., Hyde, P.M., Kuan, J.C.W., Malone-McNeal, M. and Archambault-Schexnayder, J. (1983) J. Biol. Chem. 258, 3661-3667. 9 Redgrave, T.G. (1970) J. Clin. Invest. 49, 465-471. 10 Noel, S.P., Dolphin, P.J. and Rubinstein, D. (1975) B&hem. Biophys. Res. Commun. 63, 764-712. 11 Felts, J.M., Itakura, H. and Crane, R.T. (1975) Biochem. Biophys. Res. Commun. 66, 1467-1475. 12 Gardner, R.S. and Mayes, P.A. (1978) Biochem. J. 170, 47-55. 13 Ford, R.P., Botham, K.M., Suckling, K.E. and Boyd, G.S. (1985) FEBS Lett. 179, 177-180. 14 Mackinnon, A.M., Drevon, CA., Sand, T.M. and Davis, R.A. (1987) J. Lipid Res. 28, 847-856.
416 15 Redgrave, T.G. (1983) Int. Rev. Physiol. 28, 103-130. 16 Ford, R.P., Botham, K.M., Suckling, K.E. and Boyd, G.S. (1985) Biochim. Biophys. Acta 836, 185-191. 17 Beckett, G.J., Hunter, W.M. and Percy-Robb, I.W. (1978) Clin. Chim. Acta 88, 257-266. 18 Beckett, G.J., Corrie, J.E.T. and Percy-Robb, I.W. (1979) Clin. Chim. Acta 93, 145-150. 19 Botham, K.M., Boyd, G.S., Williamson, D. and Beckett, G.J. (1983) FEBS Lett. 151, 19-21. 20 Kempen, H.J.. Van Holstein, M.P. and DeLange, J. (1983) J. Lipid Res. 23, 823-830. 21 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 22 Wahlefeld, A.W. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.), pp. 1831-1835, Academic Press, New York.
23 Seidel, J. (1981) J. Clin. Chem. Clin. Biochem. 19, 838. 24 Whiting, M.J. and Edwards, A.M. (1979) J. Lipid Res. 20.914-918. 25 Kempen, H.J., Van Holstein, M. and DeLange. J. (1983) J. Lipid Res. 24, 316-323. 26 Lakshmanan, M.R., Muesing, R.A. and LaRosa, J.C. (1981) J. Biol. Chem. 256, 3037-3043. 27 Nilsson, A. (1977) Biochem. J. 162, 367-377. 28 Floren, C.H. and Nilsson. A. (1977) Biochem. J. 168, 483-494. 29 Floren, C.H. and Nilsson, A. (1977) Biochem. Biophys. Res. Commun. 74, 520-528. 30 Floren. C.H. and Nilsson. A. (1978) Biochem. J. 174. 827-83X. 31 Bjiirkhem, 1. and Akerlund, J.E. (1988) J. Lipid Res. 29, 136-143. 32 Shefer, S., Cheng, F.M., Hauser. A.. Batta, K. and Salen. G. (1981) J. Lipid Res. 22, 532-536.
BBALIP
413
et Biophysrca Aera. 1042 (1990) 413-416
BBA Report
50286
The effect of chylomicron remnants on bile acid synthesis in cultured rat hepatocytes Richard
P. Fry, Peter A. Mayes, Keith E. Suckling
Deparrment of Veterinaq
Busrc Sciences. The Rqval Veterinuy Welwy (Received
Key words:
Bile acid synthesis;
Cholesterol;
’
and Kathleen
M. Botham
College, London and ’ Smrth, Kline and French Research Ltd. (U.K.)
13 September
Chylomicron
1989)
remnant:
Chylomicron;
(Cultured
rat hepatocyte)
effect of chylomicron remnants on bile acid synthesis in isolated rat hepatocytes in monolayer cultures was investigated. Production of bile acids by the cells in the presence of chylomicron remnants at a cholesterol concentration of 7.8-9 nmol/ml was increased by approx. 75% after 17 h and 25% after 24 h incubation. Similar concentrations of cholesterol added to the cells in the form of chylomicrons had no significant effect on bile acid synthesis. These results suggest that cholesterol taken up in chylomicron remnants may be an important source of substrate for bile acid synthesis. The
Conversion to bile acids is a major route for the removal of cholesterol from the body [1,2]. Feeding rats cholesterol has been shown to increase bile acid synthesis both in vivo [3-61 and in cultured rat hepatocytes in vitro [7,8], suggesting that dietary cholesterol may have an important role in the supply of substrate for bile acid production. Cholesterol from the diet reaches the liver mainly in the form of chylomicron remnants, which are formed from the larger, triacylglycerol-rich chylomicrons by the action of lipoprotein lipase in extrahepatic capillary beds [9]. The remnant particles are then rapidly taken up by the liver [9-111 accompanied by extensive hydrolysis of cholesteryl ester [12]. Previous work in our laboratory [13] and that of Davis and co-workers [14] has shown that a high-density lipoprotein (HDL) fraction prepared from rat plasma increases bile acid synthesis in rat hepatocytes in monolayer culture. Davis et al. [7] have also obtained similar results with a lipoprotein fraction of d < 1.02 g/ml isolated from the plasma of cholesterol-fed rats. This fraction may contain some chylomicron remnants in addition to very-low-density lipoprotein (VLDL) and low-density lipoproteins (LDL). but because of the very rapid clearance of the remnant particles by the liver [15], a preparation substantially enriched in chylomicron remnants can only be prepared from chylomicrons incubated with lipoprotein lipase in vitro, or using functionally hepatectomised rats.
Correspondence: K.M. Botham. Department ences, The Royal Veterinary College, Royal NW1 OTU. U.K. 0005-2760/90/$03.50
of Veterinary Basic SciCollege Street. London.
$3 1990 Elsevier Science Publishers
B.V. (Biomedical
In the present study we have investigated the effect of chylomicron remnants, prepared after injectoin of chylomicrons into functionally hepatectomised rats, on bile acid synthesis in isolated rat hepatocytes in monolayer culture. The effect of the parent chylomicrons has also been studied for comparison. Male Wistar rats weighing 250-400 g maintained on a commercial pellet diet and allowed access to food and water ad libitum were used. After an oral dose of 0.5 ml of corn oil, rats were anaesthetised with Nembutal. the thoracic ducts cannulated and lymph collected as described previously [12]. Chylomicron remnants were prepared from the serum of functionally hepatectomised rats after injection of 1 ml lymph into an ileolumbar vein [12]. Hepatocytes were isolated and maintained in monolayer culture as before [16]. After adhesion of the cells to plastic dishes. the medium containing fetal calf serum was replaced with serum-free medium. Lipoproteins were added at this stage when required at a concentration of 7.8-9 nmol of cholesterol/ml. Conjugated cholic, chenodeoxycholic and ,&muricholic acids in the cell and medium samples were determined by radioimmunoassay [17-191. These three bile acids have been shown to represent about 95% of the total bile acid synthesised by isolated rat liver cells [16.20]. Bile acid synthesis was calculated by subtracting the amount found associated with the cells at time zero from the sum of the amounts found in the cell and medium samples after incubation. Proteins were determined by the method of Lowry et al. [21] and triacylglycerols and cholesterol were estimated enzymatically [22,23]. Significance limits were calculated using Student’s t-test. Division)
414 The triacylglycerol : cholesterol molar ratios found in the chylomicron and chylomicron remnant preparations used were 13.1 + 1.0 : 1 and 3.3 + 0.4 : 1, respectively. These ratios are consistent with those described previously [ 191. The absolute rate of bile acid synthesis in the hepatocytes without added lipoprotein was linear over 24 h, but showed a large variation from preparation to preparation, for example, total bile synthesis (cholic + chenodeoxycholic + ,kmuricholic acid) ranged from 0.06-0.24 nmol/mg protein per h, corresponding to 12-43 nmol/g liver per h. This is somewhat lower than the estimated rate of bile acid production by the rat liver in vivo (56-205 nmol/g liver per h) [24], but is comparable to values reported previously for hepatocytes in monolayer culture [8,16]. Despite the wide variation in the absolute rates of bile acid synthesis, the changes caused by the addition of lipoprotein to the cell 3
I
Percentage changes in bile mid synthesis in rut hepatocytes UI monolq~er culture caused by chylomcrons and chylomicron remnunts Hepatocytes isolated and maintained m culture as described in the text were incubated in the absence or presence of chylomwons or chylomicron-remnants (7.8-9.0 nmol cholesterol/ml in both cases). Bile acids were determined by radioimmunoassay. Data are expressed as a percentage of the values found in the absence of added lipoprotein and are the mean from fwe rats? S.E. At time zero the absolute values (nmol/mg protein) were 1.02 i 0.50 (total bile acid). 0.16 + 0.08 (cholic acid) and 0.86 i 0.21 (/3-muricholic acid). Significance limits compared to the values found with chylomicrons: ‘. P c 0.001: h> PiO.01. Bile acid
Bile acid synthesis
Total bile acid Conjugated cholic acid Conjugated ,0muricholic
A
1
TABLE
acid
(% control
value)
+ chylomicrons
+ chylomicron
17 h
24 h
17 h
109.7*4.4
109.Ok3.6
175.4k4.1
110.0+4.4
108.8*2.4
174.6k4.4’
109.6k
remnants 24 h
” 125.952.2
h
120.8k2.3
h
4.4 110.1 k 3.X 177.0 i_ 5.2 ’ 126.3 k 2.5 h
..L)----------o
time t h 1 Fig. 1. The effect of chylomicrons and chylomicron remnants on bile acid synthesis in rat hepatocytes in monolayer culture. Hepatocytes isolated and maintained as described in the text were incubated in the A) or presence of chylomicrons (O---O), or absence (Achylomicron remnants (o- - -0) (7.8-9 nmol cholesterol/ml in both cases). Bile acids were determined by radioimmunoassay. The experiment shown is typical of five performed. Each point is the mean of duplicate samples. (A) Total bile acid synthesis; (B) conjugated cholic acid synthesis; (C) conjugated fi-muricholic acid synthesis.
cultures were very consistent. For this reason, the absolute values for a typical experiment are shown in Fig. 1, while the aggregate data from five experiments are given in Table I as percentages of the values found in the absence of chylomicrons or chylomicron remnants. When hepatocyte monolayers were incubated in the presence of chylomicron remnants, the total amount of bile acid synthesised was significantly increased (about 75% after 17 h and 26% after 24 h) (Fig. 1A. Table I) compared with that in control cells. Similar increases were seen in the production of conjugated cholic and ,&muricholic acids (Fig. lB,C, Table I). Conjugated chenodeoxycholic acid synthesis, however, represented less than 1% of the total bile acid formed and was not significantly affected by the addition of chylomicron remnants. The amount of cholesterol required to bring about the observed increases (7.889.0 nmol/ml) is at least one order of magnitude lower than that needed to produce similar rises when the cholesterol is added as HDL or other lipoprotein fractions (110-930 nmol/ml) [8,13,14]. Furthermore, the changes can be seen without the need to maximise bile acid synthesis by feeding rats a bile acid sequestrant [13], or to prepare the cells in the presence of trypsin inhibitor [14]. Bile acid production in the chylomicron remnanttreated cells slowed down considerably between 17 and 24 h, leading to a fall in the observed increment over the control hepatocytes (Fig. 1, Table I). Clearly it is possible that this decrease from the maximum rate of synthesis may occur at a time earlier than 17 h, however, in a separate experiment using hepatocytes cultured for 5 h, we were unable to detect any significant
415 difference in the amount of bile acid synthesised by chylomicron remnant-treated as compared to control monolayers. It seems unlikely, therefore, that the maximum effect of chylomicron remnants occurs very much earlier than 17 h. Evidence from previous work with isolated hepatocytes has suggested that, in the absence of an extracellular source of cholesterol, the main source of substrate for bile acid production is newly synthesised cholesterol [25]. Davis et al. [8], however, found that a lipoprotein fraction of d < 1.02 g/ml isolated from the plasma of cholesterol fed rats decreased cholesterol synthesis in cultured hepatocytes by about 65% and chylomicron remnants have also been shown to have this effect in isolated hepatocytes in suspension and in the perfused liver [26]. It is possible, therefore, that in our experiments the small amount of added chylomicron remnant cholesterol is largely exhausted after a certain time, leaving bile acid production dependent on cholesterol synthesis, which is down-regulated compared to the control cells. When cholesterol was added to the hepatocyte monolayers in the form of chylomicrons at a similar concentration to that used in the experiments with chylomicron remnants there was no significant increase either in total bile acid synthesis, or in the amounts of the individual bile acids formed (Figs. 1 A-C, Table I). This is consistent with a number of earlier studies which have shown that isolated hepatocytes internalise and degrade chylomicron remnants at a faster rate than chylomicrons [27-301. The stimulatory effect of HDL on bile acid synthesis in cultured rat hepatocytes reported by Davis et al. [14] was only seen when the cells were isolated in the presence of soy bean trypsin inhibitor, suggesting that surface receptors for HDL are protected under these conditions. In our experiments, however, inclusion of the inhibitor in the collagenase perfusion buffer at a concentration similar to that used by Davis et al. (133 pg/ml) during the preparation of the hepatocytes did not affect total bile acid synthesis in the presence or absence of chylomicrons or chylomicron remnants (Fig. 2). Production of the individual bile acids measured was also unchanged (data not shown). Chylomicron remnants are responsible for the transport of most of the cholesterol of dietary origin to the liver. Our results show that this cholesterol is very much more effective in stimulating bile acid synthesis in isolated rat hepatocytes than any other lipoprotein fraction so far tested. These findings support the idea that the amount of cholesterol in the diet has a role in the regulation of bile acid synthesis by providing an important source of substrate via chylomicron remnants. Recently, however, BjGrkhem and Akerlund have reported that the rate-limiting enzyme for bile acid synthesis, cholesterol 7a_hydroxylase, is saturated with
200
6 1
4
A
El
150
control
remnants
> 3
100
8 ap
50
n Y
.
. trvwin _. inhibitor
+ trvpsin inhktor
Fig. 2. The effect of chylomicrons and chylomicron remnants on bile acid synthesis in rat hepatocytes prepared in the presence of trypsin inhibitor. Hepatocytes were isolated in the presence or absence of soy bean trypsin inhibitor (133 pg/ml) in the collagenase perfusion buffer. Monolayers were maintained as described in the text and were incubated without (0) or with chylomicrons (o), or chylomicron remnants (m). Bile acids were determined by radioimmunoassay. Data are expressed as a percentage of the values found in the absence of added lipoprotein and are the mean from four rats rt S. E.
substrate under most experimental conditions, and is therefore, unlikely to be influenced by changes in cholesterol availability [31]. Although another study has suggested that this is not the case [32], we cannot rule out the possibility that the effect of chylomicron remnants on bile acid synthesis seen in our experiments is caused by a mechanism other than increased substrate supply. R.P.F. was in receipt of a SERC CASE studentship sponsored by Smith, Kline and French Research Ltd. Part of this work was supported by a grant from the British Heart Foundation. References 1 Siperstein, M.D., Jayko, M.E., Chaikoff, I.L. and Dauben, W.G. (1952) Proc. Sot. Exp. Biol. Med. 81, 720-729. 2 Bergstrom, S. and Norman, A. (1953) Proc. Sot. Exp. Biol. Med. 83, 71-74. 3 Wilson, J.D. (1964) J. Lipid Res. 5, 409-417. 4 Beher, W.T., Casazza, K.K., Beher, M.E., Filus, A.M. and Bertasius, J.C. (1970) Proc. Sot. Exp. Biol. Med. 134, 595-602. 5 Raicht, R.F., Cohen, B.I., Shefer, S. and Mosbach, E.H. (1975) B&him. Biophys. Acta 388, 374-384. 6 Math& D. and Chevallier, F. (1979) J. Nutr. 109, 2076-2083. 7 Sutton, CM. and Botham, K.M. (1989) B&him. Biophys. Acta 1001, 210-217. 8 Davis, R.A., Hyde, P.M., Kuan, J.C.W., Malone-McNeal, M. and Archambault-Schexnayder, J. (1983) J. Biol. Chem. 258, 3661-3667. 9 Redgrave, T.G. (1970) J. Clin. Invest. 49, 465-471. 10 Noel, S.P., Dolphin, P.J. and Rubinstein, D. (1975) B&hem. Biophys. Res. Commun. 63, 764-712. 11 Felts, J.M., Itakura, H. and Crane, R.T. (1975) Biochem. Biophys. Res. Commun. 66, 1467-1475. 12 Gardner, R.S. and Mayes, P.A. (1978) Biochem. J. 170, 47-55. 13 Ford, R.P., Botham, K.M., Suckling, K.E. and Boyd, G.S. (1985) FEBS Lett. 179, 177-180. 14 Mackinnon, A.M., Drevon, CA., Sand, T.M. and Davis, R.A. (1987) J. Lipid Res. 28, 847-856.
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