THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 241, No. 20, Issue of October 25, PP. 4722-4730, 1966

Printed in U.S.A.

The Inhibition of Sterol Biosynthesis in Rat

Liver Homogenates by Bile*

(Received for publication, March 14, 1966)

JAMES W. OGILVIE$ AND BARRY H. KAPLAN~

From the Department of Physiological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21605

SUMMARY

The incorporation in vitro of acetate-l-14C into digitonin- precipitable sterols in rat liver homogenates is markedly inhibited by small amounts of rat bile. The incorporation in vitro of mevalonate-2-14C into digitonin-precipitable sterols in the same enzyme system is much less sensitive to bile, suggesting that the major site of action of bile in suppressing sterol biosynthesis from acetate is at a pre-mevalonate step in the biosynthetic pathway. Fasting, which is known to suppress the rate of hepatic cholesterol biosynthesis from acetate in the rat, results in a X-fold increase in the inhibitory activity of bile. A major part of the inhibitory activity appears to be associated with the protein fraction of bile. This inhibitory protein fraction of bile has been partially purified and characterized. An approximate molecular weight of 19,000 has been determined for the inhibitory protein from the results of gel titration and sucrose density gradient centrifugation experiments.

The possible role of cholesterol in the etiology of atherosclerosis has focused considerable attention on the physiological control mechanisms involved in the regulation of cholesterol metabolism. The major pathway for the catabolism of cholesterol, its conver- sion to bile acids, appears to be regulated by a negative feedback control mechanism (1). The biosynthesis of cholesterol also appears to be subject to some type of control; however, the mechanism (or mechanisms) by which cholesterol synthesis is regulated is not clearly defined at the present time.

The rates of cholesterol biosynthesis from acetate in vitro and in &JO are highly dependent upon the physiological and nutri- tional states of the animal. For example, the rate of hepatic cholesterol synthesis in the rat is increased several fold above normal by x-irradiation, Triton injection, or removal of bile from the enterohepatic circulation via a biliary fistula (Z-5), and it is

* This investigation was supported by Research Grant H4977 from The National Institutes of Health, United States Public Health Service.

$ Established Investigator of the Heart Association of Mary- land.

5 Edward J. Noble Foundation Fellow, 1958 to 1962; Henry Strong Denison Scholar, 1961 to 1962.

dramatically reduced, to rates far below normal, by fasting or cholesterol feeding (6-10). Although the latter observation suggests that cholesterol may control its own rate of biosynthesis by a negative feedback mechanism, the inhibition of cholesterol biosynthesis in vitro by added cholesterol has not as yet been demonstrated (11). Furthermore, even the relationship between hepatic cholesterol levels and the rate of hepatic cholesterol syn- thesis does not appear to be simple and straightforward (12-17).

A detailed study of the alteration of cholesterol biosynthetic rates by some of the methods cited above has been made by Bucher et al. (18). They found that, under all experimental conditions investigated, a pre-mevalonate enzymatic reaction in the cholesterol biosynthetic pathway exhibited the greatest alterations in rate. In addition, they obtained evidence sug- gesting that these agencies, which alter the rate of cholesterol biosynthesis, may operate by affecting the same pre-mevalonate reaction. The results of later investigations by Bucher, Overath, and Lynen (19) strongly implicate the /3-hydroxy-P-methyl- glutaryl coenzyme A reductase step as the rate-limiting reaction upon which the control is exerted. These findings have been confirmed and extended by Siperstein (ll), Siperstein and Guest (14), and Siperstein and Fagan (20, 21).

The fact that such diverse factors as x-irradiation, Triton in- jection, and biliary drainage can so similarly affect the biosyn- thetic pathway suggested that the regulation of this pathway might be mediated by an inhibitory substance with a concentra- tion or activity in the liver that was decreased by the above agencies. This working hypothesis led us to investigate the ef- fect of added rat bile on the rate of sterol biosynthesis in rat liver homogenates. The results of this investigation are de- scribed in this communication.

EXPERIMENTAL PROCEDURE

Materials

Carworth Farms (CFN strain) and Sprague-Dawley male albino rats were used throughout this investigation. No differ- ences were noted between these two strains. ATP, NAD, and NADP were obtained from Sigma. Fructose 1,6-diphosphate monobarium salt was a product of Mann. A solution of the potassium salt of fructose 1,6-diphosphate was prepared by precipitation of the barium with K&SO, and neutralization of the supernatant fluid with KOH. Sodium acetate-1-14C (specific activity, 2.0 mC per mmole) was obtained from New England

4722

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Issue of October 25, 1966 J. W. O&vie and B. H. Kaplan 4723

Nuclear. Mevalonic acid-2-14C (specific activity, 0.82 mC per mmole) was obtained from Isotopes Specialties. Sephadex G-25, G-100, and G-200 were obtained from Pharmacia. Selectacel reagent grade ion exchange DEAE-cellulose was obtained from Schleicher and Schuell and purified by the procedure of Peterson and Sober (22).

Bile Fistula Preparation

Animals used for cannulation of the bile duct weighed from 150 to 300 g. The bile was obtained by the insertion of a poly- ethylene cannula (Clay-Adams, Intramedic PE 10) into the proximal one-third of the common bile duct while the rat was under sodium pentobarbital anesthesia. The cannula was brought to the outside and inserted through a rubber cap into a serum bottle taped to the animal’s back. In this manner, 10 to 12 ml of bile were collected per 24 hours per rat. These animals could be maintained for 4 to 5 days on regular rat chow, with 0.9% NaCl in their water reservoirs.

Preparation of Rat Liver Homogenates

The rat liver homogenates (1 g of liver per 2.5 ml of medium) were prepared in a loosely fitting stainless steel homogenizer in Bucher’s medium (23) to which ethylenediaminetetraacetic acid had been added to a final concentration of 0.2 mM. The super- natant fraction after centrifugation at 500 X g for 10 min was used as the whole homogenate enzyme preparation. The super- natant fraction after centrifugation of the whole homogenate enzyme preparation at 10,000 x g for 20 min was used as the microsomal plus soluble fraction of rat liver homogenate.

Assay Procedure

Erlenmeyer flasks (25-ml) were used as incubation vessels and all incubations were carried out aerobically at 37” for 1 hour with gentle shaking. Each incubation flask contained 2.0 ml of homogenate, 2.0 bmoles of ATP, 5.0 pmoles of NAD, 40.0 pmoles of fructose 1,6-diphosphate (potassium salt), and either 10.0 pmoles of potassium acetate-lJ4C (specific activity, 0.04 PC per pmole) or 10.0 pmoles of mevalonic acid-2-W (specific activity, 0.005 PC per pmole). The total volume of the incubation mixture ranged from 2.57 to 2.82 ml, depending upon the volumes of any other additions made; however, in any given series of experiments, all volumes were adjusted to the same amount by the addition of Bucher’s medium. After in- cubation, 2.0 ml of diethylene glycol and two pellets of KOH were added to each flask and the contents of each flask were saponified in an autoclave for 20 min at 15 psi. pressure.

After saponification, 5.0 ml of 50% ethanol were added to each flask and the nonsaponifiable lipids were isolated from each saponified incubation mixture by extraction with three lo-ml portions of petroleum ether. The combined petroleum ether extract of each incubation mixture was washed once with 15 ml of dilute KHCOa and twice with 10 ml of HzO, dried over an- hydrous Na2S04, filtered, and then taken to near dryness on a steam bath. The remaining solvent was removed from each with a jet of Nz. The resulting residues of nonsaponifiable lipids were each dissolved in 2.0 ml of hot 95% ethanol. The ethanol solutions were transferred to Wml centrifuge tubes, and 0.2-ml aliquots of each were removed and were plated in planchets for determination of the radioactivity in the nonsaponifiable lipids. Sterol digitonides were prepared from each of the remaining 1.8ml portions of the alcohol solutions by adding 1.0 ml of a

hot 1% digitonin solution in 90% ethanol to each and then gently heating to boiling on a steam bath with stirring. After the solutions were allowed to stand overnight at room tempera- ture in a sealed container, the sterol digitonides were isolated by centrifugation. The sterol digitonide pellets were each washed twice with l.O-ml quantities of hot water, twice with l.O-ml quantities of acetone at 0”, and once with 1.0 ml of ether at 0”. The washed sterol digitonide pellets were each suspended in approximately 0.25 ml of ether and were transferred to weighed planchets. After evaporation of the ether suspensions to dry- ness at room temperature, the planchets containing the sterol digitonides were evacuated in a vacuum desiccator at an oil pump for 10 min. The planchets were then reweighed and the radio- activity determined. The weight of the sterol digitonides in each planchet was typically between 0.5 and 0.8 mg.

The fatty acid fraction from each saponified incubation mix- ture was isolated from the alkaline aqueous phase after the ex- traction of the nonsaponifiable lipids by acidifying this alkaline phase with 6 N HCl until acid to Congo red paper and extracting with three lo-ml portions of petroleum ether. The combined petroleum ether extract of each aqueous phase was washed once with 15 ml of dilute acetic acid and twice with 10 ml of water. The washed petroleum ether extracts were dried over Na2S04, filtered, and taken to near dryness on a steam bath. The re- maining solvent was removed with a jet of Nt. The resulting fatty acid residues were each dissolved in 1.0 ml of ethanol-ether (l:l, v/v), and a 0.3.ml aliquot was removed and plated in a weighed planchet. After evaporation of the solvent, the planchets were placed in a vacuum desiccator and the desiccator was evacuated at an oil pump for 10 min. The planchets were then reweighed and the radioactivity of the acids was determined. The weight of the fatty acids in each planchet was typically about 1 mg.

All radioactivity measurements were made in an Atomic Ac- cessories model FC-73 windowless gas flow counter with an efficiency of approximately 45%. Samples of 1 mg or less were plated in aluminum planchets 20 mm in diameter; consequently, the correction for self-absorption was negligible. The samples were routinely counted until at least 1000 counts above back- ground were obtained. Specific activities in terms of counts per min per mg were obtained for each sample from the counts per min corrected for background and from the weight of the plated sample. Experiments were either performed in duplicate or repeated several times with different homogenate prepara- tions. The individual values obtained in duplicate experiments usually were within +5% of the mean value. The percentage inhibition produced by an inhibitor in the incorporation of ace- tate-1-W or mevalonate-2-14C into nonsaponifiable lipids was calculated as follows.

(cpm in control) - (cpm in presence of inhibitor) x loo cpm in control

The percentage inhibition produced by an inhibitor in the in- corporation of acetate-1-14C or mevalonate-2-14C into digitonin- precipitable sterols was calculated as follows.

(specific activity in control) - (specific activity in presence of inhibitor)

specific activity in control x loo

The percentage inhibition produced by an inhibitor in the in- corporation of acetate-1-r4C or mevalonate-2-14C into the fatty acid fraction was calculated as follows.

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4724 Inhibition of Sterol Biosynthesis by Bile Vol. 241, No. 20

(specific activity in control) - (specific activity in presence of inhibitor)

specific activity in control x 100

Acetone Fractionation of Bile

Cold bile was mixed with acetone at 0” in a ratio of 1: 13 (v/v) and stirred gently for 20 min in an ice bath. The acetone-in- soluble material was isolated by centrifugation at approximately 1500 X g in the cold room. After the supernatant fluid was decanted, the remaining acetone was removed from the pellet at 0” by a stream of Nz. The pellet and all solid material ad- hering to the walls of the original reaction vessel were dissolved in 0.01 M potassium phosphate buffer, pH 7.0, and the resulting solution was designated the acetone-insoluble fraction. The supernatant fluid from the above centrifugation was flash-evap- orated to dryness at 40” in a vacuum. The residue was dissolved in 0.01 M potassium phosphate buffer (pH 7.0) and the resulting solution was designated the acetone-soluble fraction.

(NH&S04 Fractionation of Acetone-insoluble Fraction of Bile

Fractionation of the acetone-insoluble fraction of bile into a fraction insoluble in 0 to 30% (NH&SO4 and a fraction insoluble in 30 to 60% (NH&SO4 was carried out at 0” by the slow addi- tion of solid (NH&S04. These (NH&SOh-precipitated frac- tions were desalted by passage through a jacketed Sephadex G-25 column maintained at 4”.

DEAE-cellulose Ion Exchange Chromatography

The desalted 30 to 60% (NH&SO(-precipitated fraction was placed on a jacketed DEAE-cellulose ion exchange column main- tained at 4” which had been packed as a slurry in 0.01 M phos- phate buffer, pH 7.0, under 1 p.s.i. of air pressure. The column was eluted with a linear salt gradient formed by mixing 0.01 M

potassium phosphate buffer, pH 7.0, with an equal volume of 1.0 M KC1 in 0.01 M phosphate buffer, pH 7.0. It was found that the desalted 30 to 60% (NH&S04 fraction in a quantity up to the equivalent of 50 ml of bile could be chromatographed on a column 1 x 25 cm. The fractions were collected in a refrigerated fraction collector maintained at 4”, and the elution pattern was determined by measuring the absorbance of each fraction at 280 rnp. The protein components of individual fractions or com- bined fractions were isolated by precipitation with (NH&S04. The resulting precipitates were dissolved in 0.01 M phosphate buffer pH 7.0, and any contaminating (NH&SO4 was removed by passage through a Sephadex G-25 column. These DEAE- cellulose fractions appeared quite stable to storage when frozen.

Approximate protein concentrations of DEAE-cellulose frac- tions were routinely estimated from the absorbance of the frac- tions at 280 rnp in l-cm cells (24). Det$erminations of protein concentrations in three different samples of DEAE-cellulose Fraction IV by the microbiuret method (25) indicated that the relationship between the protein concentration, in milligrams of protein per ml, and its absorbance at 280 rnp was 0.65.

Determination of Molecular Size of Fraction IV

Sephadex G-f?00 Gel Filtration-A Sephadex G-200 column, 2.2 x 40 cm (bed volume, 119 ml), was prepared and calibrated as described by Rogers, Hellerman, and Thompson (26). A 0.1 M sodium pyrophosphate solution, adjusted to pH 8.5 with 2 M

H2S04, was used as the eluting buffer (I’/2 = 0.9 M). A l.O-ml sample of Fraction IV (A;;: 0.945; A,,,,:A,,, = 1.69) in the

above pyrophosphate buffer was placed on the column and eluted at 24” with the same buffer. The elution pattern was determined by monitoring the ultraviolet absorption of the eluate at 220 rnk with a Vanguard recording spectrophotometer. The flow rate of the column was 5.4 ml per hour.

Sephadex G-100 Gel Filtration-A column, 25 X 1 cm, of Sephadex G-100 in 0.1 M potassium phosphate buffer (pH 7.0) was prepared in a jacketed column maintained at 4”. The void volume of the column was determined by use of dilute solutions of ferritin or tobacco mosaic virus. The effective pore radius of the column was determined by the method described by Ackers (27), with the use of ovalbumin. A 0.2.~~ sample of Fraction IV in 0.1 M phosphate buffer, pH 7.0, was placed on the column and the column was eluted with the same buffer. The effluent peak positions were determined by a double beam ultraviolet column monitor recording at 254 m/*. Three effluent peaks were ob- served, with the second peak representing material with a calcu- lated Stokes radius of 1.83 mp. In order to determine which of these three effluent peaks represented the inhibitory material, a larger sample of Fraction IV, 0.9 cc (equivalent to that isolated from 18 ml of whole bile), was placed on a Sephadex G-100 col- umn 19 x 1 cm, prepared and eluted as described above. The effluent peaks were collected and 0.4.ml aliquots of the eluate containing each of the peaks were assayed directly in a whole homogenate for inhibitory activity toward acetate-1-14C in- corporation into digitonin-precipitable sterols. Only the second effluent peak (average Stokes radius, 1.83 mp) was found to contain significant biological activity. The absorbance at 280 rnp of the fraction of eluate containing this active second peak was found to be 0.230 (l-cm cell).

Sucrose Density Gradient Centriyugation of Fraction IV

The sedimentation behavior of Fraction IV in a linear 5 to 20% (w/v) sucrose gradient was investigated by the method described by Martin and Ames (28). Solutions of bovine serum mercaptal- bumin and papain were used as standards. A O.lO-ml sample of Fraction IV (equivalent to the amount isolated from 10 ml of whole bile) and O.lO-ml samples of the two standards were layered on top of separate centrifuge tubes containing 4.6 ml of linear 5 to 20% sucrose gradients in 0.05 M Tris, pH 7.5, at 0”. The tubes were placed in a precooled Spinco SW39L rotor and centrifuged at 37,000 rpm for 16 hours in a Spinco model L centrifuge set at -18”. Fractions of 10 drops each were col- lected by piercing the bottom of the tubes with a needle. The absorbance at 280 rnp of each fraction was determined with a Beckman DU spectrophotometer equipped with a microcell at- tachment. The lo-drop fractions from the tube containing Fraction IV were then combined into six fractions, as shown in Fig. 3, for biological assay.

Disc Electrophoresis

Electrophoretic analyses of 0.2-ml samples of bile, Fractions I, and IV, by disc electrophoresis on polyacrylamide gel columns, were carried out in tubes of 5.0-mm internal diameter by the procedure described by Davis (29) with two modifications: the K,Fe(CN)G was omitted from the lower gel, and the samples were applied as 30% sucrose solutions. The samples were sub- jected to a constant current of 2.5 ma per tube for 90 min with cooling by an ice bath. The protein bands were fixed and stained with a 0.25% solution of naphthol blue black dye in 7% acetic acid.

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Issue of October 25, 1966 J. W. Ogilvie and B. H. Kaplan 4725

TABLE I

Effect of bile on incorporation of acetic acid-i-IT and mevalonic acid-d-W into nonsaponijiable lipids, sterols, and fatty acid

fraction by rat liver homogenate

Homogenate preparation, assay conditions, isolation proce- dures, and calculation of percentage inhibition are described under “Experimental Procedure.” 1-alues presented are the means of duplicate experiments.

RESULTS

The addition of small quantities of rat bile (less than 1 y0 of the daily output of a bile fistula rat) to a 2.75.ml enzyme assay sys- tem consisting of a liver homogenate derived from 1 g of rat liver has been found to inhibit markedly the rate of incorporation of acetate-1-r4C into nonsaponifiable lipids and digitonin-precipita- ble sterols. The effects of increasing amounts of added bile on the incorporation of acetate-PC into nonsaponifiable lipids and sterol digitonides are presented in Figs. 1 A and B. The effects of the same quantities of bile on the incorporation of acetate-1-i4C into fatty acids in the same enzyme system are presented in Fig. 1C. Although the effect of added bile on nonsaponifiable lipid and sterol biosynthesis is clearly and invariably inhibitory, the effect of added bile on fatty acid synthesis is so variable that it is impossible to attach any significance to the inhibition of fatty acid synthesis represented by the data presented in Fig. 1C.

Any given sample of bile has been found to produce dosage curves similar in shape to those presented in Fig. 1; however, some samples of bile have been found to be considerably more inhibitory than others. In the course of this work there have been four periods of several months’ duration when the in- hibitory activity in the bile was approximately one-fourth of that depicted in Fig. 1. During two of these periods, the rats used for cannulation of the bile duct and for preparation of the liver homogenate were suffering from a respiratory disease. Gross and microscopic studies of the rats during these periods revealed typical lesions in the nasal passages, eustachian tubes, middle ear, and lungs. The results presented will not include those ob- tained with the less active bile.

Bile has been found to be much more inhibitory to the incorpo- ration of acetate-l-W than to the incorporation of mevalonate- 2-r4C into digitonin-precipitable sterols; therefore, the major site of action of bile in the inhibition of sterol biosynthesis would appear to be at one or more of the pre-mevalonate reactions in the biosynthetic pathway. The results of a typical experiment on the effect of bile on the incorporation of isotopically labeled

-7 i Inhibition ipecific activity F

1

t

Total ctivity ir

non- saponili- ble lipids

2411 231

540 95

1043 138

5280 955

-

1

_-

-

Substrate NIXI- Lponifi able

lipids

%

69

20

%

78

8 140

-

Acetate- 1-W

Acetate- 1-W

Mevalo- nate-2- 1%

Mevalo- nate-2- 14C

None

Bile, 0.1 ml

None

Bile, 0. ml

TABLE II

Effect of dialysis on inhibitory properties of bile

Homogenate preparation, assay conditions, isolation pro- cedures, and calculation of percentage inhibition are described under “Experimental Procedure.” Substrate in all cases was acetate-l-X!. Values presented are the means of duplicate ex- periments. -

I Specific activity Inhibition

Additions

Total activity in

nonsaponifi- able lipids

cPm,‘mg

914 198 5 92

315 345

746 123

%

99

61

26

None. . 4825 Bile, 0.15 ml. 50 Dialyzed bile,

0.15 ml.. 1885 Concentrated di-

alysate, 0.30 mla. 3575

A.NONSAPONIFIABLE 0 LIPIDS

C.FATTY ACIDS

z 30 0 ,40

- a The 0.30-ml concentrated dialysate contains the dialyzable

constituents of 0.30 ml of bile. m50 5 - 60

s 70 acetate and mevalonate into digitonin-precipitable sterols are presented in Table I. The specific activity of the acetate-1-r4C used in this experiment was 8-fold greater than the specific activity of the mevalonic acid-2-r4C; thus, the rate of sterol bio- synthesis from mevalonate was much greater than the rate from acetate in this experiment.

The data presented in Table II indicate that the inhibitory ac- tivity of bile is associated for the most part with the nondialyza- ble components. In this experiment, 10 ml of bile were dialyzed at 4” for a total of 38 hours against three 200-ml volumes of distilled water. The combined dialysate was flash evaporated to dryness at less than 40’ in a vacuum, and the resulting residue was dissolved in 10 ml of water. The resulting solution was designated concentrated dialysate. Although some loss of total

80

90

100 1 ’ ’ I I I I I I I I I 0 ,025 .05 0.10 0 025 05 0.10 0 .025.05 0.10

ML. BILE ADDED

FIG. 1. Effect of added bile on the incorporation of acetate-l- 14C into nonsaponifiable lipids (A), digitonin-precipitable sterols (B), and fatty acids (C) in rat liver homogenates. Each point represents an average value obtained from 4, 8, and 12 different samples of bile at the 0.025-, 0.05-, and O.lO-ml levels, respectively. The vertical lines through each point represent fl S.E.M. Assay conditions and method of calculation of percentage inhibition are described in “Experimental Procedure.”

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4726 Inhibition of Sterol Biosynthesis by Bile Vol. 241, No. 20

TABLE III

Effect of bile on acetate-l J4C incorporation into sterols by microsomal plus soluble fraction of rat liver

Preparation of microsomal plus soluble fraction of rat liver homogenate, assay conditions, isolation procedures, and calcula- tion of percentage inhibition are described under “Experimental Procedure.” Values presented are the means of duplicate experi-

in mitochondria (30). The data of Table III also indicate that when this enzyme system is fortified for fatty acid synthesis by the addition of NADP and citrate, the effect of added bile on sterol and fatty acid synthesis is still very similar to its effect in the absence of added NADP and citrate, even though the rate of sterol biosynthesis is decreased and the rate of fatty acid syn- thesis is increased under these experimental conditions.

If the inhibitor of sterol biosynthesis which is present in bile is the mediator in the regulation of cholesterol biosynthesis, the concentration or activity of this inhibitor should be sensitive to the various physiological states which produce alterations in the rate of cholesterol biosynthesis. Fasting is one method com- monly used to produce a decreased rate of synthesis of cholesterol from acetate; therefore, it might be expected that the concentra- tion or activity of the inhibitor would be greater in the fasted animal than in the fed animal. The effect on the inhibitory activ- ity of the bile produced by fasting the animal for 24 to 48 hours prior to cannulation of the bile duct can be seen in Table IV. In each experiment two animals of the same age, sex, and weight were paired, and one was fasted for 24 to 48 hours while the other was maintained on a normal diet. The bile ducts of both animals were then cannulated under identical conditions, and the first 0.2 to 0.3 ml of bile which flowed from the cannula of each was collected for assay in a normal homogenate. The data presented in Table IV strongly suggest that the inhibitory activity of the initial sample of bile isolated from the fasted animal was greater than the inhibitory activity of the initial sample of bile isolated from the paired fed animal (t value, 3.55; p < 0.01). This increase in inhibitory activity of the bile from the fasted animal cannot be a reflection of changes in the concentrations of all biliary constituents since the dry weights of the bile did not differ greatly. This period of fasting is sufficient to block almost completely the synthesis of cholesterol from acetate in homog- enates prepared from the livers of these fasted animals.

The data in Table V show that fractionation of bile into ace-

ments.

Specific activity Inhibition

::2

%

29

47

Total activity in

nonsaponifi- able lipids

Additions (0.05 ml) L;Tg- sg lipids tonider

% %

81 92

56 89

@% 1870

350

450

200

c@dmg

1765 80 145 57

252 1145

27 606

None............ Bile NADP, lo-* M;

citrate, 1 M; MgClz, 1 M.

NADP, 1O-2 M;

citrate, 1 M; MgClz, 1 M; bile

TABLE IV Effect of bile from fasted and fed rats on incorporation

of acetate-l-% into sterols

Fed animals were maintained on regular diet prior to cannula- tion of the common bile duct. Fasted animals were deprived of all food for 24 to 48 hours prior to cannulation of the common bile duct. The first 0.2 to 0.3 ml of bile that flowed from the cannula of each animal was used in the assay. Homogenate preparation, assay conditions, isolation procedures, and calcula- tion of percentage inhibition are described under “Experimental Procedure.”

7 Inhibition” Dry weight of bile

Experiment

Fed Fasted Fed Fasted

% %

58 88

19 92 0 55

36 38 3 72

42 45 54 61 39 57 30 63

45 41 41 49 48 47 42 52 38 43 46 50 42 48 45 47

Average 31.2b 63.4* 43.4 47.1

TABLE V

Properties and e$ect of acetone-insoluble and acetone-soluble fractions of bile on incorporation of acetate-l-i%

into sterols

Isolation of bile fractions, homogenate preparation, assay con- ditions, isolation procedures, and calculation of percentage in- hibition are described under “Experimental Procedure.” All additions are expressed in volumes equivalent to the volume of bile from which that quantity was isolated.

Additions

Inhibition of acetate-

I

1-X incorporation into

Dry weight

a Based on the decrease in specific activity of the sterol digitonides produced by the addition of 0.05 ml of bile isolated from a fasted animal or 0.05 ml of bile isolated from a fed an- imal.

*t = 3.55; p < 0.01.

.~ % 0

68 100

% 0

57 99 71

27.8

8.6

None. . . . . . . . . . Bile, 0.05 ml . Bile, 0.10 ml. . . . . . . . . . Acetone-insoluble fraction, 0.1 ml. Acetone-insoluble fraction, heated 4

min at loo”, 0.1 ml.. . . . Acetone-soluble fraction, 0.1 ml. Acetone-soluble fraction, 0.2 ml. . . Acetone-soluble fraction, heated 4

min at loo’, 0.2 ml.. _. .

activity was observed in this experiment, the dialyzed bile still contained the greater part of the inhibitory activity.

In Table III it can be seen that bile also inhibits sterol biosyn- thesis in an enzyme system consisting of the microsomal and soluble fractions of a liver homogenate. Therefore, it appears unlikely that the mode of action of bile involves the extraction of the inhibitory material which has been reported to be present

83

8 13 8 36

84 91

81 90

18.8

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Issue of October 25, 1966 J. W. Ogilvie and B. H. Kaplan 4727

tone-soluble and acetone-insoluble components resulted in the recovery in the acetone-insoluble fraction of most of the inhibi- tory activity but only one-third of the solids. It can also be seen in Table V that the inhibitory activities of the acetone- insoluble and the acetone-soluble fractions were associated with heat-labile and heat-stable components, respectively.

Since most of the inhibitory activity of bile was recovered in the acetone-insoluble fraction, purification of this fraction was undertaken. It was found that the material inhibitory to sterol biosynthesis in this fraction was precipitated between 30 and 60% saturation with (NH&S04. Further purification of this 30 to 60% (NH&Sob-precipitated fraction was achieved by gel filtration on Sephadex G-25 and by ion exchange chromatography on a DEAE-cellulose column with the use of a linear KC1 gradient for elution. A typical elution profile from a DEAE-cellulose column is presented in Fig. 2. The column eluate fractions in this experiment were combined into five fractions (I to V) as shown in Fig. 2, and (NH&SO4 was added to each of these fractions to 60% saturation. The resulting precipitates were isolated by centrifugation and redissolved in lo+ M phosphate buffer, pH 7.0. As indicated in Fig. 2, when these fractions were assayed for inhibition of sterol biosynthesis in a liver homogenate, the inhibitory activity was typically found in two fractions, I and IV. Fraction I, the first ultraviolet-absorbing fraction from the DEAE-cellulose column, corresponds to that material which is not adsorbed or is only weakly adsorbed by the DEAE-cellulose. Although this ultraviolet-absorbing fraction was always obtained, the amount of inhibitory material present in Fraction I was variable from one chromatographic separation to another,

2.0-

l.O-

0.8-

0.6-

0.4-

0.2-

I II III El P COMBIN;;MFBW$TION

FIG. 2. Elution profile of the 30 to 60% (NH4)BOa precipitate of the acetone-insoluble fraction of bile from a DEAE-cellulose column. The open bars indicate the absorbance at 280 rn* of each fraction of the column eluate. The stippled bars represent the percentage inhibition in the incorporation of acetate-l-l% into digitonin-precipitable sterols produced by the addition of an aliquot of each combined fraction (I to V) of the column eluate to a rat liver homogenate. See the text for details.

TABLE VI

Fractionation of rat bile

Isolation of bile fractions, homogenate preparation, assay conditions, isolation procedures, and calculation of percentage inhibition are described under “Experimental Procedure.” All additions are expressed in volumes equivalent to the volume of bile from which that quantity was isolated.

Additions Dry weight

None.................... Bile, 0.1 ml.. Acetone insoluble frac-

tion, 0.075 ml.. 3@607, (NH,)&30a pre-

cipitate, 0.1 ml. Sephadex G-25 eluate,

0.1 ml . . . ..___._.... DEAE-cellulose Fraction

IV, 0.2 ml..

-

w/ml

22.6

8.9

1.28

1.45

1.50

NOtI- saponifi- ble lipid:

% 0

97

Sk-01 igitonides

%

0 99

36 56

28 53

54 64

65 75

nhibition of acetate- l-lpC incorporation

into

ranging from a small amount up to an amount greater than that present in Fraction IV. On the other hand, Fraction IV was consistently inhibitory, and usually contained the major fraction of the inhibitory activity present in the acetone-insoluble frac- tion. Fraction I was colorless, and its spectrum was similar to that of a simple protein. The absorption spectrum of Fraction IV was also similar to that of a simple protein; however, a con- centrated solution of Fraction IV was faintly yellow in color, because of the presence of a weak but broad absorption band in the 380 rnl.c region. The yellow color was possibly the result of pigment adsorption to the proteins of this fraction since the adjacent fractions, III and V, were also the same color. Typical results on the amount of inhibition produced by each fraction in the several stages of purification outlined above are presented in Table VI. All volumes denoted in Table VI are equivalent to the volume of whole bile from which that fraction was isolated. It is apparent from the data in this table that in this experiment the recovery of inhibitory activity in Fraction IV was somewhat less than 50% of that present in whole bile. Yields of inhibitory activity isolated in Fraction IV as high as 50% of that present in whole bile have been obtained by these procedures.

As would be expected from the elution profile in Fig. 2, Fraction IV was not homogeneous. Experimental data indicating the presence of two major protein components and several trace protein components were obtained by disc electrophoresis. However, even at this level of purity, Fraction IV inhibited the rate of incorporation of acetate into digitonin-precipitable sterols by more than 500/, when present in the incubation mix- ture at a concentration of 15 to 20 pg of protein per ml.

Table VII summarizes the effects of Fraction IV on acetate- 1J4C and mevalonate-2-14C incorporation into nonsaponifiable lipids, digitonin-precipitable sterols, and fatty acids. With one exception, these effects are similar to those of whole bile. The exception is the increase in the specific activity and the total radioactivity incorporated into the fatty acid fraction from acetate-1-14C when Fraction IV is present. Although this

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4728 Inhibition of Sterol Biosynthesis by Bile

TABLE VII

Vol. 241, No. 20

Effect of DEAE-cellulose Fraction IV on incorporation of acetate-i-l46 and mevalonate-2J4C into nonsaponifiable lipids, sterols, and fatty acid fraction by rat liver homogenate

Isolation of bile fractions, homogenate preparation, assay conditions, isolation procedures, and calculation of percentage inhibition are described under “Experimental Procedure.” Values presented are the means of duplicate experiments.

Additions Substrate

None DEAE-cellulose Fraction IV, 0.2 ml None DEAE-cellulose Fraction IV, 0.2 ml

0.6

,0.5

a 0.3 I

A

6 0.4 8

Acetate-l-i% Acetate-l-i% Mevalonate-2-l% Mevalonate-2-i%

2 0.3 0

E 0.2 a

0. I

0

1; B

1‘

5 IO 15 20 25 30

FRACTION NUMBER

FIG. 3. Sucrose density gradient centrifugation patterns for bovine serum mercaptalbumin (A), papain (B), and DEAE- cellulose Fraction IV (C). The open bars indicate the absorbance of each lo-drop fraction collected from the bottoms of the centri- fuge tubes. The stippled bars represent the percentage inhibition in the incorporation of acetate-l-l% into digitonin-precipitable sterols produced by the addition of a 0.4-ml aliquot of each of the combined fractions to a rat liver homogenate. See the text for details.

increase was consistently observed, no obvious correlation be- tween the radioactivity lost from the nonsaponifiable lipid fraction and the increase in radioactivity of the fatty acid fraction was detected. The nature of the acids responsible for the in- crease in radioactivity of the fatty acid fraction in the presence of Fraction IV has not yet been investigated.

The results of a sucrose density gradient centrifugation study of Fraction IV are presented in Fig. 3. Based on an 820,~ value of 4.3 S for bovine serum mercaptalbumin as a standard (31), the SZQ,~ of the inhibitory component of Fraction IV was calcu- lated to be 2.6 S; based on an ~20,~ value of 2.42 S for papain as a

Total activity Specific activity Inhibition

JonsaPonifi- Fatty acids di,$yn$les Fatty acids Nonsaponifi- stero1 able lipids able lipids digitonides 22

___~ cm @m/mE 9% % %

2195 ’ 580 1336 149 855 877 600 236 61 55 0

5270 1218 1127 383 4585 1313 1227 385 13 0 0

standard (32), the s20,W value of the inhibitor was calculated to be 2.4 S. Stokes radii of 1.78 rnp and 1.83 rnh for the inhibitory component of Fraction IV were estimated from the elution volume of the inhibitor on Sephadex G-200 and Sephadex G-100 calibrated molecular sieve columns, respectively (Fig. 4). The Sephadex G-200 column was operated at 24”, and the elution volume was based solely on the appearance of a 220 mp-absorbing peak. The Sephadex G-100 column was operated at 4” to min- imize denaturation of the inhibitor during the experiment, and the elution volume was based on the appearance of inhibitory activity in the eluate as determined by a biological assay, as well as on the appearance of an ultraviolet-absorbing peak. A diffusion coefficient, D, of 12.0 x lo-’ cm2 per set was calculated from the estimated Stokes radius of 1.78 rnN according to the Stokes-Einstein equation (33),

P 70 s

80

0 50 100 % BED VOLUME

FIG. 4. Elution patterns for DEAE-cellulose Fraction IV from Sephadex G-200 at 24” (A) and from Sephadex G-100 at 4” (B). Percentage bed volume = (elution volume/gel bed volume) X 100. The curves represent percentage transmittance at 220 rnp (A) and relative percentage transmittance at 254 rnp (B). The bars represent the percentage inhibition in the incorporation of ace- tate-l-% into digitonin-precipitable sterols produced by the addition of a 0.4-ml aliquot of the eluate volume designated by the position of the bar on the abscissa.

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Issue of October 25, 1966 J. W. Ogilvie and B. H. Kaplan 4729

where k is the Boltzmann constant, T the absolute temperature, 7 the solvent viscosity, and CZZ~,~ the Stokes radius. From a diffusion coefficient of 12.0 X 10-r cm2 per set, an szo,w of 2.4 S, and an assumed partial specific volume ($ of 0.74, an approxi- mate molecular weight (M) of 19,000 was calculated for the inhibitory component of Fraction IV from the equation

M= RTs

D(1 - tip)

The acetone-soluble fraction of bile should contain the low molecular weight components of bile. Since this fraction was somewhat inhibitory to acetate incorporation into digitonin- precipitable sterols, several low molecular weight fractions of bile were isolated for study. The bile salts, the major low molecular weight components of bile, were isolated by the pro- cedure of Harold and Chaikoff (34), and were found to produce an 11% inhibition in the incorporation of acetate-1-14C into digitonin-precipitable sterols when the quantity of bile salts isolated from 0.2 ml of bile was added to the assay system. Thus, the bile salts appear to be responsible for only a part of the inhibition produced by the low molecular weight components of bile. The greater part of the inhibitory activity in the dry residue of the acetone-soluble fraction is soluble in a chloroform- methanol (4: 1, v/v) mixture. Preliminary experiments in- volving the chromatographic separation of the components of the acetone-soluble fraction on silicic acid-Celite columns suggest that the inhibition produced by the acetone-soluble fraction is really a summation of the inhibitory activities of at least three different constituents of this fraction.

DISCUSSION

Of the several fractions of bile which are inhibitory to the incorporation of acetate into digitonin-precipitable sterols, the low molecular weight protein fraction described above is a particularly interesting one since it may contain a possible physiologically important modulator of cholesterol biosynthesis. Not only is this protein fraction responsible for an appreciable amount of the inhibition produced by whole bile, but it also produces a type of inhibition which is compatible with the ex- perimental observations on the regulation of cholesterologenesis in the rat. For example, it produces a much greater inhibition of the incorporation of acetic acid than of mevalonic acid into digitonin-precipitable sterols. Furthermore, it does not suppress the rate of incorporation of acetic acid into the fatty acid fraction. These observations are consistent with the hypothesis that its site of action is at an enzymatic step in the biosynthetic pathway between acetic acid and mevalonic acid, but after the point at which the fatty acid biosynthetic pathway branches off. The rather high specific activity of this protein fraction, inhibitory at less than lo-” M even in its present state of purity, is also com- patible with its being of some physiological significance in the regulation of cholesterologenesis.

Additional support for the presence in bile of a physiologically important modulator for the control of cholesterol biosynthesis is provided by the observations that removal of bile from the enterohepatic circulation leads to an increased rate of hepatic and intestinal cholesterol biosynthesis (4, 5, 35) and that bile from the fasted rat is more inhibitory than bile from the fed

animal to sterol biosynthesis. The observation by Dietschy and Siperstein (35) on the effect of bile on intestinal cholesterol synthesis is particularly pertinent, since they report that intes- tinal cholesterol synthesis is insensitive to fasting and cholesterol feeding but is inhibited by the infusion of bile into the small intestine.

On the other hand, the observation by Fredrickson et al. (36), that bile duct ligation produces an increased rate of cholesterol biosynthesis in the rat, is somewhat difficult to reconcile with the idea that bile contains a physiologically important inhibitor of cholesterol biosynthesis. A part of the increased rate of choles- terol biosynthesis produced by bile duct ligation can be accounted for by the increased rate of intestinal cholesterologenesis (35). Moreover, since it has been demonstrated that intravenous in- jection of the detergent Triton is capable of overcoming the inhibition of hepatic cholesterologenesis produced by cholesterol feeding or fasting (3), it is possible that other detergents, such as bile salts, when present in elevated concentrations could have a similar action. Thus, the effect of bile duct ligation on choles- terol biosynthesis does not at present appear to constitute suffi- cient evidence to demand abandonment of the hypothesis that bile may contain a physiologically important modulator of cholesterol biosynthesis.

Inhibitory effects of other naturally occurring substances on cholesterol biosynthesis have been observed. Siperstein and Fagan (21) have reported that a cholesterol-lipoprotein complex present in the plasma of cholesterol-fed chickens inhibits cho- lesterol biosynthesis when injected into a mouse. On the other hand, they report that the plasma of chickens with stilbestrol- induced hypercholesterolemia has only a slight effect on choles- terol biosynthesis in the mouse. Beher and Baker (37) have reported the inhibition of cholesterologenesis in rats maintained on a diet containing 0.5% cholic acid. Fimognari and Rodwell (38) have demonstrated the inhibition of mevalonic acid syn- thesis from acetic acid in rat liver homogenates by cholic, de- oxycholic, taurocholic, and taurodeoxycholic acids. Some of the latter compounds may account for a part of the inhibition in sterol biosynthesis observed upon the addition of the acetone- soluble fraction of bile to a liver homogenate. Migicovsky (30) has reported the isolation from rat liver mitochondria of an inhibitor of cholesterol biosynthesis which appears to be an electrodialyzable component associated with a protein. Wood and Migicovsky have also reported the inhibition in vitro of cholesterol biosynthesis by fatty acids (39) and by rat sera (40).

Thus, cholesterol biosynthesis from acetic acid, particularly when measured in vitro, appears to be extremely sensitive to the presence of a number of different naturally occurring compounds. At the present time, no experimental data exist which con- clusively implicate any of these compounds, including the protein fraction of bile reported in this communication, in the regulatory mechanism. Until such direct evidence is forthcoming, an evaluation of the physiological significance of the inhibitions produced by these various compounds is not possible. However, the close similarities cited above between the response in vitro of the hepatic sterol biosynthetic pathway to the inhibitory pro- tein fraction of bile and the response in viva of the same pathway to such diverse factors as fasting, creation of a biliary fistula, and cholesterol feeding are certainly compatible with and suggestive of a regulatory role for this inhibitory component of bile.

AclcnowledgmentsThe authors thank Mrs. Ann Decker,

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4730 Inhibition of Xterol Biosynthesis by Bile Vol. 241, No. 20

Mr. S. Grzybowski, Mr. J. Goss, and Mr. R. Rudolph for their 19. BUCHER, N. L. R., OVERATH, P., AND LYNEN, F., Biochim.

valuable technical assistance. They are also indebted to Drs. K. Biophys. Acta, 40, 491 (1960).

Rogers and G. Ackers for their hels and advice in the gel filtration 20. SIPERSTEIN, M. D., AND FAGAN, V. M., J. Biol. Chem., 241,

experiments, to Dr. A. H. Phillips for his help and advice in the 21, density gradient centrifugation experiments, and to Dr. P. Hart and Mr. James Sightler for their help in the early stages of this work. 22.

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James W. Ogilvie and Barry H. KaplanThe Inhibition of Sterol Biosynthesis in Rat Liver Homogenates by Bile

1966, 241:4722-4730.J. Biol. Chem. 

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