The effect of starvation and cholesterol feeding on intestinal cholesterol synthesis in the rat

546 BIOCHIMICA ET BIOPHYSICA ACTA

EEA55637

THE EFFECT OF STARVATION AND CHOLESTEROL FEEDING ON

INTESTINAL CHOLESTEROL SYNTHESIS IN THE RAT”

MITCHELL N. CATEN (with the assistance of MARSHA BLACK)

Department of Biochemistry, Aycrst Laboratories, Montreal, Quebec (Canada)

(Received August 11th, 1969)

SUMMARY

I. Hepatic and intestinal cholesterol synthesis were compared by measuring

the incorporation of jQ4C]acetate or of simultaneously incubated [z-%]acetate and

[3H]mevalonate into cholesterol by liver homogenates and intestinal sections pre-

pared from rats subjected to various dietary regimens. Fasting for 24 h suppressed

both hepatic and intestinal cholesterol synthesis, increased liver cholesterol and

phospholipids, and lowered liver triglyceride content. Dietary cholesterol suppressed

hepatic, but did not alter intestinal cholesterol formation, while the bile acid seques-

tering resin cholestyramine produced dose-dependent increases of both hepatic and

intestinal cholesterogenesis. z. Rats fed 2% cholesterol could be given up to 0.5% cholestyramine without

affecting the uptake of added [4-14C]cholesterol. The enhancement in cholesterol

synthesis by 0.5% cholestyramine was reversed by 2% cholesterol, i.e., in the intes-

tine, cholesterol synthesis proceeded as in those from normal rats while it was practi-

cally arrested in the liver. The results show that when the inhibitory activity of bile

acids is masked with cholestyramine, dietary cholesterol alters the bile acid pool

in such a manner so as to suppress intestinal cholesterol synthesis.

INTRODUCTION

GOULD’ in 1951 was the first to demonstrate that the addition of cholesterol

to the diet of rats produces a marked depression of hepatic cholesterol synthesis. This finding was elaborated and confirmed by numerous investigatorsz-6. This action

is referred to as the “negative feedback control of cholesterol biosynthesis” and

affects the conversion of /3-hydroxy-fl-methylglutaryl coenzyme A (HMG-CoA) to mevalonic acids-s. The suppression of hepatic cholesterogenesis on fasting5,*“-12 may

also be due to a reduction in the activity of HMG-CoA reductase9913, although the

Abbreviation used : HMG-CoA, B-hyd roxy-,!-methylglutaryl coenzyme A.

* Presented in part at the Canadian Federation of Biological Societies Meeting, June 14, 1968 (Proc. Can. Federation Biol. Sm., II (1968) 141).

Bzochim. Biophys. Acta, 187 (1969) 546-554

INTESTINAL CHOLESTEROL SYNTHESIS 547

release of an inhibitor from liver mitochondria14 and lowered glycogen 1eveW have also been implicated.

Most mammalian tissues are capable of cholesterol synthesi9-‘*. Except for the liver, the intestine has the greatest capacity for the synthesis of cholesterol18 and this biosynthesized cholesterol contributes to the circulating cholesterol po01~@~~~. Since dietary cholesterol has little21 or no3p17y18 inhibitory effect on intestinal cholesterogenesis, it has been suggested that the “negative feedback” control is absent in the intestine. DIETSCHY AND SIPERSTEIN 17~18 have reported that starvation also has no effect on intestinal cholesterol synthesis, but other workers have observed a depression of synthesis after prolonged fasting11122.

Thus, although the pathways of intestinal and hepatic cholesterol synthesis are similarz3, the controlling mechanisms appear to be different. BEHER et aLz4 origi- nally proposed that the bile acids control hepatic cholesterol synthesis: cholesterol suppresses the conversion of HMG-CoA to mevalonic acid and the bile acids suppress the conversion of cholesterol to bile acids. In viva, bile acids inhibitZ51Z8, while biliary drainage increases the rate of hepatic cholesterol synthesiF. Bile acids affect intestinal cholesterol synthesis in a similar manner 11~21*28. However, the more recent con- sensus is that the effect of bile acids on intestinal cholesterol formation is a direct one, while that on the liver is indirect. Thus, DIETSCHY~~ has concluded that the rate of hepatic sterol synthesis is regulated by exogenous cholesterol while intestinal sterol synthesis is controlled by bile acids, the major end product of the catabolism of

cholesterol. The question thus arises as to why the bile acids can suppress both hepatic

and intestinal cholesterogenesis while cholesterol, which is converted in the liver to bile acids, suppresses only hepatic cholesterol synthesis and has no observed effect on the intestine. In the following experiments, this problem was studied with the aid of cholestyramine, a nonabsorbable anion-exchange resin which sequesters bile acids in the gut, resulting in enhanced hepati? and intestina12* cholesterol synthesis and an increased rate of bile acid turnover 3o. The use of cholestyramine as an experimental tool provided a means to deplete a portion of the bile acid pool, and thus mask the inhibitory effects of bile acids.

In the present report, various aspects of starvation and cholesterol feeding have been examined in rats in order to study more closely the differences and similarities between hepatic and intestinal cholesterol synthesis.

MATERIALS AND METHODS

Animals Male albino rats (Charles River), weighing 120-140 g, were kept under observa-

tion for 7 days prior to each experiment. Only animals with normal food intake and weight gain were used. Dietary regimens comprised Purina Laboratory Chow supplemented as indicated.

Measurement of hepatic and intestinal cholesterogenesis Rats were decapitated and livers and intestines were immersed in ice-cold iso-

tonic saline. Intestines (distal 20 cm) were flushed thoroughly with ice-cold Krebs- Ringer bicarbonate buffer (pH 7.4), sections were incubated for I h at 37’ in buffer

Biochim. Biophys. Acta, 187 (1969) 546-554

548 M. N. CAYEN

containing jz-%]acetate alone or simultaneously with /,“H]mevalonate, and cholesterol was isolated as its 5,6-dibromo derivative as described previouslyz3. Liver homogenates were incubated for I h at 37’ in potassium phosphate buffer (pH 7.4) containing labeled precursors and appropriate cofactors and the cholesterogenic activity was measuredsr.

Measurement of lipid levels Total cholesterol levels were determined by the method of ZLATKI~ ef ~1.32 as

modified for the autoanalyzer (Method Np-24). PhosI~holipids were determined by the semi-automated method of KRAML~~, and triglycerides were measured by a semi-automated method using the manual extraction and saponification steps of LAURELL~~ followed by automated determination of glycerol with chromotropic acid3S. Total nitrogen in liver homogenates was determined by the Kjeldahl digestion

procedure as adapted for the autoanalyzer~5.

Source of compounds Sodium 12-l%]acetate, 13H]acetate and DL-[3H]mevalolactone were purchased

from the Radiochemical Centre, Amersham. The mevalolactone was treated with dilute methanolic KOH and neutralized to obtain free DL-mevalonic acid. Cholesterol, used as carrier and for feeding, was purified via its 5,6-dibromo derivativez?. Choles- tyramine (Cuemid) was kindly supplied by Dr. J. Mailloux of Merck, Sharp and Dohme of Canada, Montreal.

EXPERIMENTAL

Rats were deprived of food at 9: oo a.m., fasted for 24 h and decapitated. The incorporation of simultaneously incubated [G%]acetate and [3H]mevalonate into cholesterolbyintestinalsectionsandliverhomogenates wasdetermined. Serumandliver lipid levels were measured. Theresults (Table I) show that the fast produced a marked decrease in acetate incorporation into neutral lipids and cholesterol in both the liver and intestine; the extent of suppression was similar in both tissues (98 and 99::, respectively). The low incorporation of mevalonate into neutral lipids and cholesterol by the intestine in control animals has also been observed in a previous studyz3 and this may reflect limited uptake of mevalonate to the site of intestinal cholesterol synthesis. Since the 1 %]cholesterol in the intestinal sections from control rats con- tained only a few counts above background, the observed lack of j3H]mevalonate incorporation cannot be ascribed with certainty to fasting.

In order to eliminate the possibility that fasting increased the acetate (or acetyl-CoA) pool size and thus diluted the added radioactivity, liver homogenates and intestinal sections from fed and fasted rats were incubated with [V4C]acetate with and without unlabeled acetate added to yield a concentration 200 times greater than that of labeled acetate. The depression of /2-“CJacetate incorl~oration into neutral lipids by fasting was virtually identical in the presence and absence of unlabeled acetate in both liver and intestinal preparations.

The effect of fasting on serum and liver lipids is presented in Table II. Serum and liver cholesterol was elevated, while liver triglycerides were markedly depressed.


TABLE I

EFFECT OF STARVATION ON INTESTINAL AND HEPATIC CHOLESTEROGENESIS i?t Vi&‘0

Rats were fasted for 24 h, decapitated, and livers and intestines immersed in ice-cold saline. The ilea (distal 20 cm) were cut away and flushed with roe ml of ice-cold Krebs-Ringer bicarbonate buffer (pH 7.4) containing 2 mg/ml glucose, which had been gassed with O,-CO, (g5:5, v/v). The ilea were ever-ted, cut into sections 5 mm in length and weighed. Incubations were carried out in 5 ml of buffer containing 5 PC (0.02 pmole) [2-r*C]acetate and 0.5 @UC (0.01 pmole) [3H]mevalonate for I h at 37” in an atmosphere of O,-CO, (95: 5, v/v). The liver homogenate incubation medium was comprised of 1.5 ml of homogenate (in 0.1 M potassium phosphate buffer (pH 7.4) containing 0.05 M MgCl, and 0.03 M nicotin- amide), 2 yC (0.08 pmole) [2J*C/acetate (0.2 ml), 2.5 pm&s ATP, z {amoles glucose &phosphate, I

pmole NADP+ and I ymole NAD+ to make a total volume of 2.2 ml. The homogenates were incubated for I h at 37’ in an atmosphere of O,-CO, (95:5, v/v). In both preparations, enzymatic activity was terminated with 12 pellets of KOH. Ethanol (4 ml), water (2 ml) and carrier cholesterol (50 mg) were added, the suspension was heated for I h at 75-80’ and the neutral lipids were extracted with light petroleum (b.p. 30-50”). Cholesterol was isolated, purified and counted as 5,6-dibromocholestan-3fl-olz3. Samples were dissolved in 15 ml of Liquifluor solution (4 g/l z,5-diphenyloxazole and 50 mg/l r,4-bis- (5-phenoxazolyl-z)benzene), and the radioactivity was measured in a Packard Tri-Carb liquid scintillation counter, Model 3375. Data are presented as mean _t SE. for 9 rats per group. _..~~ -.- f;rozsp Intestine (disint. lmin per g tiss2ce) Liver (c&s&. jmin per mg

jz-‘q4cetate [3H]Meualonate nitroge+z) ~__ ____.. _ -__ Neutral lipids Cholesterol Neutral Cholesterol [@ClAcetate

lipids Neutral Cholesterol lipids

Control 225500 + 24130 21780 & 2856 7921 + 796 (100 & 46) ‘79-1 f 268 1002 * 206

Starved I7000 + 1400 464 f 82 3398 + 432 321 I63 4*r Depression 92%” 98%* 57%* azy** 99% *

* P < 0.01.

Liver phospholipids were slightly higher. No significant changes were observed in serum phospholipids and triglycerides.

Efect of cholestyramine on intestinal and hepatic cholesterogenesis and on cholesterol levels in the rat

These experiments were performed in order to establish a basis for studies on the effect of dietary cholesterol on intestinal cholesterogenesis in cholestyramine- treated rats. In the first experiment, rats were fed Purina chow containing 2% cholestyramine for 7 days, intestinal sections and liver homogenates were prepared and the incorporation of [@*C]acetate into cholesterol was determined. The results in Table III show that 2% cholestyramine increased cholesterol synthesis r/-fold in the liver and z-fold in the intestine.

TABLE II

EFFECT OF STARVATION ON SERUM AND LIVER LIPIDS

Rats were fasted for 24 h and lipid levels were measured. Data are presented as mean f S.E. for r2 rats per group.

.____ .- GoUp Cholesterol Phosphokipid P

(mg/100 m1) Triglyceride glycerol

(mg/roo ml) (mgjroo ml) .-..__ Serum Liver Serum Liver Serzcm Liver

--____ ~-~- Control 87 + 3.3 272 * 6.5 5.2 * o.rg 122 + 2.2 Starved

7-5 i 0.83 55.2 i 2.9 102 f 4.3** 413 i r5.6** 4.8 -& 0.22 138 If; 3.8* 8.7 + 0.60 34.7 _t 1.7**

____.- --_ * P < 0.01.

** P < 0.001.

BiocLim. Biaphys. Acta, 187 (1969) 546-554

550 M. N. CAYEN

TABLE III

EFFECT OF 2% DIETARY CHOLESTYRAMINE ON INTESTINAL AND HEPATIC CHOLESTEROGENESIS

For 7 days rats were fed Purina chow containing 2% cholestyramine. Intestinal sections were incubated with 4.97 ,uC (0.13 pmole) [z-l%]acetate and liver homogenates with I.97 ,uC (0.05 pmole) [z-“Clacetate. Experimental conditions were the same as those described in Table I. Data are presented as mean & S.E. for 9 rats per group.

G%X@

Control Cholestyramine

* P < 0.01. ** P < 0.001.

Intestine (disint./min per g tissue) Liver (disint. /min pev mg nitrogen)

Neutral li$ids Cholesterol Neutral lipids Cholesterol

106200 + 19020 5588 5 I224 I794 1’- 268 I002 ,E 206

247300 rt 3oo4o* 3I370 * 5060** I2330 & 1444** 7260 _Ir 846**

TABLE IV

EFFECT OF DIFFERENT DIETARY LEVELS OF CHOLESTYRAMINE OX HEPATIC CHOLESTEROGENESIS

Rats were fed Purina chow supplemented with different levels of cholestyramine for 7 days. Liver homogenates were incubated simultaneously with 2.1 PC (0.05 pmole) [2-l’C]acetate and 0.5 ,uC (0.01 pmole) [3H]mevalonate and the rate of cholesterogenesis was measured. Data are presented as mean .I: S.E. for 7 rats per group.

Group Disint. /min per mg nitrogen Cholesterol

[~+~C]Acetate i3H]Mevalonate (mg/100 ml)

ti&ral lipids Cholesterol Neutral lipids Cholesterol Livev Serum

Control 6838 + 1012 IO96 t 358 47990 + 1352 12150~~1122 242 ~1 3 63 k5 0.02 o/0 cholestyr-

amine 6443 I- 856 1190 4 282 48960 _$_ ‘727 15630 -’ 1936 252 ~1: 8 67 -1: 2

o. I o/0 cholestyramine 6 839 4: 646 1454.t216 47390 i 1278 19350&1150*** 253+8 68 ;: 3

0.5’$/~ cholestyramine 10640 + 1023* 4968 & 1200** 43 830 5 2414 25540 + 1196*** 248 :‘: IO 70 1k 3

2.0% cholestyramine 36630 +3784*** 23030 + 1936*** 39600 & 1477*** 29960 & 784*** 227 :k 6% 74 :t 3

* P < 0.05. ** P < 0.01.

*** P < 0.001.

Experiments were performed to determine the effects of different doses of cholestyramine on hepatic cholesterol synthesis. For 7 days rats were fed diets containing 0.02, 0.1, 0.5 or 2.0% cholestyramine, and liver homogenates were incubated simultaneously with [z+C]acetate and [3H]mevalonate. The results are presented in Table IV. In this instance, 2% cholestyramine produced a zo-fold increase in the incorporation into cholesterol of acetate and a 2.5-fold increase of mevalonate; the incorporation into neutral lipids of acetate was increased 6-fold, while that of mevalonate was slightly decreased. Thus, the primary site of increased cholesterol synthesis by cholestyramine was between acetate and mevalonate, with a secondary site after the formation of neutral lipids. The increase in cholesterogenesis was dose-dependent. The lowest dose at which a statistically significant increase in cholesterol synthesis from acetate was observed was at 0.5% cholestyramine, while 0.1% of the resin produced a significant increase in cholesterol synthesis from mevalonate. In contrast to the results presented in Table III, z”& cholestyramine lowered liver cholesterol in this experiment; serum cholesterol was unchanged.


INTESTINAL CHOLESTEROL SYNTHESIS 5s

E$ect of dietary cholesterol on intestinal and hepatic cholesterogenesis in normal and

cholestyramine-fed rats Since cholestyramine binds bile acids in the gut and since bile acids are re-

quired for the absorption of cholesterol, cholestyramine fed to rats on dietary cholesterol may conceivably depress cholesterol absorption. It was therefore necessary to determine the maximal dose of cholestyramine which would not affect cholesterol absorption in cholesterol-fed rats. Rats were fed for 7 days 2% cholesterol containing 0.15 ,& [‘Klcholesterol per g chow and different levels of cholestyramine. Animals were decapitated, livers were homogenized in and extracted with ethanol-ether (3:r, v/v), and the 14C content of the extract was determined. The radioactivity in this extract would retlect the amount of cholesterol absorbed and taken up by the liver. The results are presented in Table V. It was found that the liver 14C content was virtually identical in control animals and those fed 0.1 and 0.5% cholestyramine. The l*C content of the livers of rats fed 2% cholestyramine was approximately one- quarter that of controls. Thus, rats could be fed up to 0.5% cholestyramine without interfering with cholesterol absorption.

TABLE V

UPTAKE OF CHOLESTEROL IN CHOLESTYRAMINE-FED RATS

Rats were fed for 7 days Purina chow supplemented with 2% cholesterol containing o.15,uC i4-‘4C] cholesterol per g chow and different levels of cholestyramine. Animals were decapitated, livers were homogenized in and extracted with ethanol-diethyl ether (3 :I v/v) and the ‘*C content was measured. Data are presented as mean + S.E. for 5 rats per group.

GWW@

Control o. I T/o cholestyramine 0.5% cholestyramine 2.07& cholestyramine

* P<O.OOI.

Total disint. /win in extract Disint. /min per g liver

1070000 * 14700 Io52oo k 3740 1020000 * 58800 107500 * 10140

114.5 ooo zt 143 3oo 120200 5 13500 250000 * 5g200* 3’500 + 725o*

In the final experiment, rats were divided into four groups and fed the following diets for 7 days: (I) Purina chow; (2) 2% cholesterol; (3) 0.5% cholestyramine; and (4) 2% cholesterol plus 0.5% cholestyramine. Animals were decapitated, and the incorporation of [3H]acetate into cholesterol was measured in intestinal sections and liver homogenates. Cholesterol, phospholipid and triglyceride levels were determined. The results are presented in Tables VI and VII. As reported previously, dietary cholesterol suppressed hepaticl+ and did not significantly alter intestina13,1?p18 cholesterogenesis, while 0.5% cholestyramine increased both hepatiP and intestina12*,30 cholesterol synthesis. Hepatic and intestinal cholesterogenesis in rats fed both cholesterol and cholestyramine was virtually the same as that from rats fed cholesterol alone, i.e., the rate was completely suppressed in the liver and was not significantly different from untreated in the intestine. Cholesterol feeding increased serum and liver cholesterol while cholestyramine had no effect on these parameters. None of the treatments altered the cholesterol level in the intestine. Cholestyramine slightly increased serum phospholipids and had no effect on liver triglycerides.

DISCUSSION

The results of this study show that fasting for 24 h markedly depressed both


552 M. N. CAYEN

TABLE VI

EFFECT OF DIETARY CHOLESTEROL ON CHOLESTYRAMINE-INDUCED INCREASE IS INTESTINAL AND

HEPATIC CHOLESTEROGENESIS

l?or 7 days rats were fed Purina chow supplemented with 2% cholesterol, 0.50/b cholestyraminc, or 201; cholesterol together with 0.5% cholestyramine. Animals were decapitated, intestinal sections were incubated with 3.5 ,uC (0.017 ,umole) [3H]acetate and liver homogenates with 1.6 ,uC (o.008 ,rLmole) [3H]acetate and the rate of cholesterogenesis was measured. Data are presented as mean

1 S.E. for 8 rats per group.

G&& Intestine (disint.lmin per g tzssue) Liver (duint. lmin per mg nitrogen)

hreutral lipids Cholesterol Neutral lipids Cholestwol

Untreated 33280 & 7800 1248 + 418 522 I: 90 262 _I: 60 2% cholesterol 58800 sr 983o** 1153 + 251** 109 ~2 8t 6-I: 2t 0.5oj cholestyramine XI 410 1; 10060*** 5702 -!I 762t 1184 + 191*** 642 t 106*** 296 cholesterol+o.50/6

cholestyramine* 62 090 I_ 9 180** 1580 & 292t 202 & 417 24 I 9t

* Differences in this group compared with 0.5% cholestyramine. * * Differences not significant.

*** P < 0.01.

t P < 0.001.

TABLE VII

EFFECT OF DIETARY CHOLESTYRAMINE WITH AND WITHOUT CHOLESTEROL ON LIPID LEVELS IN RATS

Animals used were those described for Table VI. ._

GYOUp Cholesterol (mg/~oo ml)

Livev Serum

Phospholipid P Triglyceride

Intestine (+%lIoo mr) glycerol

Liver Serum (mgl100 ml)

Controls 250 zk 9 87 :k 3 245 & 4 129 * 2 5.5 -I_ 0.1 56 + 3 2 o,0 cholesterol 795 + 61t 122 i 4t 260 IL 6 r43 + 3*** 6.9 $ 0.3*** 210 _t 137

0.5”/; cholestyramine 265 zk 6 90 * 5 249 5 ro I34 ~: 2 6.2 -k 0.3** 55 ‘3 2:/o cholesterol+0.5~~

cholestyramine* 485 + 74*** 118 + 8** 261 + 7 141 + I** 6.9 3: 0.3 154 > 32**

* Differences in this group compared with 0.5% cholestyramine. ** P < 0.05.

*** P < 0.01.

t P < 0.001.

hepatic and intestinal cholesterol synthesis in the rat. Thus, not only is the pathway of intestinal cholesterogenesis similar to that of the liver23, but the response to fasting is similar in both organs. The depression of hepatic cholesterogenesis by fasting is a well-documented phenomenon 5y10p12, but the effect of fasting on sterol formation in

extrahepatic tissues has not been clearly established. HUTCHENS et al.ll and JANSEX

et aJ22 reported that starvation suppressed extrahepatic cholesterol synthesis, but not to the same degree as in the liver. However, DIETSCHY AND SIPERSTEIN~~~~* were unable to demonstrate that the rate of intestinal sterol formation was altered by fasting. In the present studies, fasting for 24 h increased the concentration of serum and liver cholesterol and liver phospholipids and markedly suppressed liver triglycerides. Although much has been reported on the effects of starvation on lipid levels (especially sterols), literature comparisons are difficult due to such variations as species, sex, age and length of fast. Thus no attempt will be made to compare these data with those in the literature. It is, however, generally agreed that fasting produces elevated liver sterol concentrations (e.g., ref. II).



The reports that dietary cholestyramine increases hepatic? and intestinalZ8 cholesterogenesis were confirmed in this study. Dietary cholesterol reversed the cholestyramine-induced increase in both hepatic and intestinal cholesterogenesis”. The mechanisms involved may be described as follows.

Dietary cholesterol is absorbed with the aid of bile acids but does not alter the rate of intestinal cholesterol synthesis as it passes through the intestinal wall. Choles- terol is transported to the liver where it is partly degraded to bile acids. Bile acids or some bile acid precursor control the rate of hepatic cholesterol synthesisZ4. Cholesterol added to the diet results in a net increase in bile acid formation and excretion38, suppressed hepatic cholesterol synthesis, and a slight increase in the bile acid concentration in the intestinal wa112*~**.

The enhancing effect of cholestyramine on hepatic cholesterogenesis followed a dose-response relationship (Table IV). Simultaneous feeding of 2% cholesterol and 0.5% cholestyramine produced in the liver the same effect on cholesterol synthesis as did 2% cholesterol alone; it is proposed that hepatic bile acids formed from cholesterol of dietary origin reached the intestine in sufficient quantity to overcome the loss of bile acids due to cholestyramine binding. This is supported by the observation that cholestyramine lowered cholesterol-induced elevation in liver cholesterol levels (Table VII); however, the concentration of liver cholesterol in rats fed cholesterol and cholestyramine was still higher than that of untreated animals, resulting in a suppression of hepatic cholesterol synthesis. In the intestine, it is proposed that dietary cholesterol offset cholestyramine-induced increase in cholesterogenesis ac- cording to the following mechanisms. Dietary cholesterol is converted to bile acids in the liver where it suppressed cholesterol formation in situ. In order for these newly formed bile acids to suppress intestinal cholesterogenesis, they must first reach the circulation and thus become part of the enterohepatic pool of bile acids. Dietary cholesterol does produce increased bile acid excretion38, but any increase in bile acid pool size under these conditions may not be sufficient to alter intestinal cholesterol synthesis. The use of 0.5% cholestyramine provided a means to sequester only a portion of the circulating bile acids (Table IV). In animals fed cholesterol and cholestyramine, the sequestered bile acids would be replaced by bile acids synthesized from cholesterol of dietary origin. This is reflected by the observation that dietary cholesterol reversed cholestyramine-induced increase in intestinal cholesterol synthesis.

These studies show that when the inhibitory effect of bile acids is masked with cholestyramine, dietary cholesterol alters the bile acid pool in such a manner so as to suppress intestinal cholesterol synthesis. The data thus support the concept of DIETSCHY~~ that bile acids regulate intestinal cholesterol synthesis. In view of the major contribution of cholesterol synthesized in the intestinal wall to the circulating

* Cholestyramine binds bile acids which are required for the absorption of cholesterol. However, the dose of chclestyramine used in this experiment (0.5%) was sufficiently large to produce an increase in both hepatic and intestinal cholesterol synthesis (Table VI) without affecting the amount of dietary cholesterol absorbed and taken up by the liver (Table V). ** Thus, on a theoretical basis, cholesterol feeding should produce at least some depression of intestinal cholesterogenesis. Although the experiment outlined in this report and those of others (refs. 3, 17, 18) have not shown any such effect, DIETSCHY 21 has recently reported that when rats were fed I .5’-JJ0 cholesterol for I week a very slight but significant decrease in intestinal cholesterol synthesis took place.


554 M. N. CAYEN

cholesterol pool in man18~20, this effect on intestinal cholesterol formation may be of importance in man consuming a diet which contains bile acid precipitants or adsor- bents, such as lignin3g.

ACKNOWLEDGMENTS

The author wishes to thank Dr. D. Dvornik for helpful discussions during the course of this study, Dr. J. G. Rochefort for treating the experimental animals and Dr. A. Boudreau and his staff for measuring the lipid levels. This work was supported in part by the National Research Council of Canada Industrial Research Program.

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Biochim. Biophys. Acta. 187 (1969) 546-554

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The effect of starvation and cholesterol feeding on intestinal cholesterol synthesis in the rat