The hypocholesterolemic effect of sulfur-substituted fatty acid analogues in rats fed a high carbohydrate diet

Biochimica et Biophysics Acta, 1167 (1993) 175-181 0 1993 Elsevier Science Publishers B.V. All rights reserved

BBALIP

175 000%2760/93/$06.00

54144

The hypocholesterolemic effect of sulfur-substituted fatty acid analogues in rats fed a high carbohydrate diet Jon Skorve and Rolf K. Berge Laboratory of Clinical Biochemistv,

University of Bergen, Haukeland Sykehus, Bergen (Norway) (Received

Key words:

Hypocholesterolemic

7 August

fatty acid analogue;

1992)

Lipogenic

enzyme;

ACAT;

VLDL; (Rat)

fatty acid analogues have been administered to rats fed a high carbohydrate diet, and the effect on plasma and hepatic lipid metabolism was investigated. Two of the analogues studied, 3-thiadicarboxylic acid and tetradecylthioacetic acid, reduced the plasma cholesterol level significantly, whereas the effect on plasma triacylglycerol level was only marginal. 3-Thiadicarboxylic acid was the most potent, decreasing the cholesterol level faster and at a lower dose than tetradecylthioacetic acid. The relative effects on plasma cholesterol and triacylglycerol levels were different from what have been observed in rats fed a conventional pellet diet. Tetradecylthiopropionic acid had no hypocholesterolemic effect. The activities of three lipogenic enzymes: ATP-citrate lyase, acetyl-CoA carboxylase and fatty acid synthase was measured. The two hypocholesterolemic analogues reduced the activities of these enzymes in a coordinated manner. The enzyme activities was found to correlate with the the plasma cholesterol level, indicating a coordinated regulation of these enzymes and cholesterol synthesis or secretion. The effect on two enzymes involved in cholesterol metabolism was also studied. The activity of acyl-CoA:cholesterol acyltransferase (ACAT) was reduced by the two hypocholesterolemic analogues, in contrast to the rate-limiting enzyme in cholesterol Sulfur-substituted

biosynthesis, HMG-CoA reductase, which tended to increase. The cholesterol lowering effect of 3-thiadicarboxylic acid and tetradecylthioacetic acid can probably be ascribed to diminished cholesterol synthesis due to a reduced availability of acetyl-CoA. A reduction

in the esterification

of hepatic

cholesterol

may be a contributing

factor.

Introduction

oxidation. It has hypolipidemic effect only when fed to rats in relatively high doses [2,4].

Sulfur-substituted fatty acid analogues have been shown to have hypotriglyceridemic and hypocholesterolemic properties when fed to rats [l-3]. 3,14-Di-

The exact mechanisms behind the hypolipidemic effect of these analogues are still not completely understood. Lipogenesis may potentially be one of the pathways that are affected. Lipogenesis is subject to hormonal and nutritional regulation mainly effected through changes in mRNA transcription rates and mRNA stability, but also through variations in metabolite concentrations (acetyl-CoA, malonyl-CoA) [5-91. We were interested in examining the effect of the substituted fatty acid analogues on lipogenesis, and if this eventually could explain some of their hypolipidemic effects. To this end we have used rats fed a high carbohydrate diet. It is well known that feeding rats a diet high in carbohydrates and low in fat increases de novo fatty acid synthesis, esterification of fatty acids to TG and synthesis and secretion of VLDL [5,10-151. It has been proposed that lipogenesis and VLDL synthesis and secretion might be coordinatedly regulated [14]. Inhibitors of ACAT are known to have hypolipidemic effects [16]. The hypocholesterolemic effect of ACAT inhibitors has been ascribed to decreased absorption and secretion of dietary cholesterol due to

thiahexadecanedioic acid (trivial name 3-thiadicarboxylic acid) (HOOC-CH,-S-(CH &-SCH ,-COOH) and tetradecylthioacetic acid (CH3-(CH,),,-S-CH,-COOH) are in addition strong peroxisome proliferators, the dicarboxylic acid being the most potent. The hypotriglyceridemic effect has been partly ascribed to increased mitochondrial fatty acid oxidation, reduced availability of free fatty acids and diminished triacylglycerol biosynthesis [3]. Tetradecylthiopropionic acid (CH ,-(CH 2),3-S-CH ,-CH ,-COOH) is a weak peroxisome proliferator, and inhibit the mitochondrial p-

Correspondence to: J. Skorve, Laboratory of Clinical Biochemistry, Haukeland Sykehus, 5021 Bergen, Norway. Abbreviations: VLDL, very-low-density lipoprotein; ACAT, acyCoA:cholesterol acyltransferase; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; DAG, sn-1,2-diacylglycerol; MEDICA, p,p’methyl-substituted cy,w-dicarboxylic acids.

176 inhibition of intestinal ACAT activity [17-191. The importance of hepatic ACAT activity for plasma cholesterol level is still obscure. When an ACAT inhibitor [58-0351 was added to HepG2 cells in culture, the synthesis of triacylglycerols was increased, while cholesterol ester synthesis and apo B secretion was decreased [20]. This may indicate that ACAT is implicated in the regulation of VLDL synthesis and secretion, and we have therefore examined the effect of the fatty acid analogues on this enzyme activity too. Materials

and Methods

fat) and 20% glucose in the drinking water. The fatty acid analogues were suspended in 0.5% sodium carboxymethyl cellulose and administered by gastric incubation in a volume of 0.7-l ml once a day. The control animal groups received only sodium carboxymethyl cellulose. All animals had free acess to water and food. Dietary intake and the increse in body weights of all animal groups were similar. At the end of the experiments rats were lightly anesthesized and cardiac puncture was performed. The livers were removed and immediately chilled on ice and weighed. In time-course studies, blood samples (0.4 ml) were collected from the vena saphena minor at 2-3 day intervals.

Chemicals and drugs

3-Thiadicarboxylic acid, tetradecylthioacetic acid, tetradecylthiopropionic acid were prepared as earlier described [21]. Na[‘4C]HC0, (0,l mCi/mmol), [l“C]acetyl-CoA (60 mCi/mmol), [l-‘4Cloleoyl-CoA (53 mCi/mmol), 3-hydroxy-3-methyl[3-‘4C]glutaryl-CoA (55 mCi/mmol), [y-“‘P]ATP (3000 Ci/mmol) and sn1,2-diacylglycerol assay reagents system were purchased from Amersham, England. Nonesterified Fatty Acid kit was purchased from Wako Chemicals, Germany. All other chemicals were obtained from common commercial sources and were of reagent grade.

Preparation of total homogenate and cellular fractions

Animals and treatments

Analytical methods

Male Wistar rats from Mollegaard Breeding Laboratory, Ejby, Denmark, weighing 150-200 g, were housed individually in metal wire cages in a room maintained at 12 h light-dark cycles and a constant temperature of 20 + 3°C. The animals were acclimatized for at least 5 days under these conditions before the start of the experiments. The animals were fasted for 48 h before they were fed a high carbohydrate diet consisting of white bread (containing 45% starch, 7% protein, 2.8%

The activity of ATP-citrate lyase was measured in the cytosolic fraction as described by Bar-Tana et al. [22]. Acetyl-CoA carboxylase was measured in the cytosolic fraction as fixation of 14C0, from NaH14C0, as described by Tanabe et al. [23] with minor modifications. Fatty acid synthase was measured in the cytosolic fraction as described by Roncari [241 with some modifications. 300 PM NADPH was used instead of a NADPH generating system, and [t4Clacetyl-CoA was

TABLE

The livers from individual rats were homogenized in ice-cold sucrose medium (0.25 M sucrose in 10 mM Hepes buffer (pH 7.4) and 2 mM EDTA). The postnuclear fraction was used for further analysis by differential centrifugation. Samples from three animals were pooled and mitochondrial-enriched, peroxisome-enriched, microsomal and cytosolic fractions were isolated [l]. Variation in the response from animal to animal was estimated separately for selected enzymes and lipids in the group of control animals.

I

Hepatic leclels of triacylglycerols, cholesterol, phospholipids and sn - 1,2-diacylglycerol Rats were fed fatty acid analogues for 7 days, The tabulated values (pmol/g liver) represents means f S.D. for nine control animals and three animals in each experimental group. Means in a column with different superscripts are significantly different from each other (P < 0.05). ND: not determined. Dose

Treatment

Triacylglycerols

Cholesterol

Phospholipids

DAG

8,24rt 8,02+ 7,77+ 11,22+ 724+ 9:oo; 604+ > 4,31* 11,76+ 12,61 k 39,25 +

2,62 + 2,81+ 2,45 + 1,44 + 0,62 + 2,15 f 2,58 I 2,03 f 350 k 3,31 i 3.17+

12,18+0,9 11,66+ 1,7 12,85 + 1,2 16,83 k 1,0 18,84 f 0,8 13,88*0,7 17,04f 1,2 16,05f1,2 12,00 k 0,8 13,54 f 0,5 15,02 f 2,3

ND 0,42 k 0,18 0,48 k 0,06 0,61+ 0,08 0,43 zk0,04 0,69 k 0,08 0,44 f 0.05 0,38 + 0,05 0,59 + 0,lO 0,52 + 0,08 0,74 f 0,38

mg/day per kg body weight Control Palmitic

acid

3-Thiadicarboxylic Tetradecylthioacetic

Tetradecylthiopropionic

acid acid

acid

0 150 400 150 400 150 250 400 150 400 800

15 b.c.d 2,3 b,c.d 1,l b,c 1,l d 15b.c 019 Gd 13”.h 2 1,4?! 2,4 d 1,7 ’ 13,3 ’

0,30 0,25 0,30 0,75 0,06 ’ 0,80 0,33 0,72 0,06 a 0,70 1,05

h ’ h h

r.k l.k k

’ k

’ i ‘,k i.k i.k

177 used as the radioactive isotope instead of [14C]malonylCoA. The assay was started by the addition of preincubated enzyme fraction (100 pg) and run for 6 min at 37°C. Acyl-CoA : cholesterol acyltransferase was measured in the microsmal fraction mainly as described by Field et al. [18]. The assay was run for 3 min at 37°C. HMG-CoA reductase was measured in the microsomal fraction as described by Angelin et al. [25] with modifications. The assay was started by adding [3-‘4C]HMGCoA (60 PM) and run for 30 min at 37°C. sn-1-2-Diacylglycerol was quantitated enzymatically (DAG kinase) using a commercially available kit (Amersham). Plasma-free fatty acids were determined by an enzymatic calorimetric method (WAKO Nefa 0. Lipid analyses were carried out by the Monotest cholesterol and phospholipids enzymatic kits, Boehringer-Mannheim, Germany and the Biopdk triacylglycerol enzymatic kit, Biotrol, Paris, France. Statistical analysis was by Student’s t-test and P < 0.05 was taken to be statistically significant. Results When rats are fed a high carbohydrate, low-fat diet the synthesis of fatty acids and triacylglycerols, as well as the VLDL output from the liver, increases. In rats fed this diet for 7 days and which in addition received palmitic acid, there were no changes in the hepatic levels (Table I) nor in the plasma levels of lipids (Table II, Fig. 1). When the rats received 3-thiadicarboxylic acid, however, the hepatic (Table I> and the plasma cholesterol level was decreased in a dose-(Fig. 1) and time- (Fig. 2) dependent manner compared to control and palmitic acid-treated rats. The hepatic triacylglycerol (Table I) and plasma triacylglycerol (Table II) levels were unchanged in comparison to palmitic acidfed rats at equal doses. The hepatic phospholipids, TABLE

1,s

, I

1.6 1.4 1.2 1.0 -

0.6 0.4 0.2 0.0

-I

0

50

150

100

200

250

3w

350

400

Dose ( mg / day / kg body weight) Fig. 1. Effect of palmitic acid COO), 3-thiadicarboxylic acid A) and tetradecylthioacetic acid (0 -0) on the plasma (Acholesterol level in rats intubated with different doses of the compounds for 7 days. The data are the meansfS.D. for nine control animals and three animals in each experimental group. * P < 0,05 between rats fed palmitic acid and fatty acid analogues.

however, were increased (Table I>. Repeated administration of the monocarboxylic acid, tetradecylthioacetic acid, decreased the level of triacylglycerols in the liver (Table I), but not the plasma concentration (Table II) compared to palmitic acid feeding. The hepatic phospholipid content was increased by tetradecylthioacetic acid treatment too (Table I). Moreover, Table I and Table II shows that the amount of hepatic triacylglycerols and diacylglycerols as well as the plasma triacylglycerol level were significantly lower in rats fed a high dose of 3-thia fatty acids compared to rats fed a low dose. The lowest level of plasma triacylglycerols was found in tetradecylthioacetic acid-treated rats, probably reflecting the relatively low level of hepatic triacylglycerols in these rats. It is noteworthy that the relative changes of hepatic and plasma triacylglycerols

II

Plasma levels of triacylglycerols and free fatty acids Rats were fed fatty acid analogues for 7 days. The tabulated values (~mol/ml) represents meansrf:S.D. for nine control animals and three animals in each experimental group. Means in a column with different superscripts are significantly different from each other (P < 0.05). ND: not determined. Treatment

Control Palmitic

acid

3-Thiadicarboxylic Tetradecylthioacetic

Tetradecylthiopropionic

acid acid

acid

Dose mg/day per kg body weight

Plasma triacylglycerols

Plasma nonesterified fatty acids

Plasma phospholipids

0 150 400 150 400 150 250 400 150 400 800

0,49 * 0,15 0,52 f 0,15 0,40 * 0,12 0,58 k 0,07 0,44 * 0,lO 0,56+0,10 0,33 f 0,07 0,31+0,10 0,72 + 0,lO 0,48 f 0,22 0,64 f 0,45

0,52 k 0,09 0,72 f 0,08 0,59 + 0,08 0,60+0,13 0,36*0,11 0,27 rt 0,05 ND 0,32 + 0,14 0,28 + 0,07 0,52 + 0,14 ND

1,37 * 0,lO 1,26 k 0,20 1,32+.0,13 1,00+0,11 0,76 + 0,12 1,27 f 0,07 0,95 f 0,14 0,72 + 0,14 1,47+0,18 1,36 + 0,06 0,98 + 0.27

0 a,b a,b b a,b b a = b a,b a,b

d,e e d,e d,e c*d ’ c,d ’ d,e

f f,g

f I&h h

* Gh h

f f f.g,h

178 with these two analogues were different from what have been observed in pellet-fed rats, where 3-thiadicarboxylic acid gave the largest decrease in hepatic triacylglycerols [l-2]. The dose needed to obtain this effect seems to be higher in the glucose-fed rats than in rats fed a standard pellet diet. In pellet-fed rats we have observed a 40-50% reduction in the plasma levels of both triacylglycerols and cholesterol, even at a dose of 150 mg/day per kg [2]. In contrast, in the present experiments the plasma triacylglycerol level was only marginally effected, while plasma cholesterol level was reduced by more than 50% in rats administered 3-thiadicarboxylic acid and tetradecylthioacetic acid (Fig. 1). 3-thiadicarboxylic acid was most potent, reducing the cholesterol level after a shorter time of feeding and at a lower dose than tetradecylthioacetic acid. In rats treated with these two analogues the plasma-free fatty acids was decreased with the exception of the lowest dose (150 mg/day per kg) with 3-thiadicarboxylic acid. Tetradecylthiopropionic acid, which we have previously reported to produce fatty liver in pellet-fed rats [2-41, resulted in accumulation of hepatic cholesterol and triacylglycerols, especially at the highest dose (800 mg/day per kg) used (Table I>. The dose needed to obtain this effect seems to be higher than in rats fed a standard pellet. The plasma concentrations of triacylglycerols and cholesterol were only marginally affected (data not shown). The plasma-free fatty acid level was decreased in rats fed a low dose of tetradecylthiopropionic acid. This effect was, however, not seen at higher doses.

________---

h .C

.z ii

.O 5

4

.E

B f

loo-

75-

50 -

25 -

O-1

c

,D____-__---__--___-a

125-

25

_N

1

01

0

.’

50 Dose

30

,

I

Fig. 2. Plasma cholesterol level as a function of days of feeding. Rats were intubated once a day with palmitic acid (Oo), 3-thiadicarboxylic acid ( A ~ A) or tetradecylthioacetic acid (*0) (400 mg/day per kg body weight). The data are the means + S.D. for five animals (see Materials and Methods). * I’< 0.05 between rats fed palmitic acid and fatty acid analogues.

/’

100 (mg

150

/day I

200

kg body

250

300

350

400

weight)

Fig. 3. Changes in the relative specific activities of ATP-citrate lyase (A), acetyl-CoA carboxylase (B) and fatty acid synthase CC). Rats were intubated with different doses of palmitic acid CO-o), n ), 3-thiadicarboxylic acid tetradecylthiopropionic acid ( n A) or tetradecylthioacetic acid (o0). The data rep(Aresents the means of three animals in each experimental group (see Materials and Methods). Control values (lOO%i) for ATP-citrate lyase, acetyl-CoA carboxylase and fatty acid synthase were 17.85+ 1.14; 26.6 k 1.44 and 0.5 + 0.07 nmol/min per my protein, respectively.

Effect on lipogenic enzymes The different fatty acid analogues had a quantitatively similar effect on the activities of the three lipogenic enzymes studied (Fig. 3). Feeding 3-thiadicarboxylic acid and tetradecylthioacetic acid reduced the activities of ATP-citrate lyase, acetyl-CoA carboxylase and fatty acid synthase. Here too it is apparent that 3-thiadicarboxylic acid is more potent than tetradecylthioacetic acid at the lowest dose administered (Fig. 31, resembling the effects on the plasma cholesterol level. It is further noted that the 3-thia fatty acids had a much stronger effect on these enzyme activities than

179

Plasma

cholesterol

210

136

12

018

(pmol /

ml)

Fig. 4. Correlation between plasma cholesterol and citrate lyase activity after feeding rats different doses with sulfur substituted fatty acid analogues (r = 0.90; P < 0.0001). Rats were intubated with 3-thiadicarboxylic acid (a), tetradecylthibacetic acid (e) and tetradecylthiopropionic acid (01 for 7 days.

tetradecylthiopropionic acid. From Fig. 3 it is apparent that the three enzymes are all regulated in a coordinated manner in rats fed fatty acid analogues. It is noteworthy that the activities of these lipogenic enzymes changes in parallel with the plasma cholesterol level. Fig. 4 shows a rather strong correlation between the plasma cholesterol level and the activity of citrate lyase (r = 0,901. A similar correlation was found for the acetyl-CoA carboxylase activity (not shown). Enzymes in cholesterol metabolism As the main effect of the sulfur-substituted fatty acids in this study was a reduction in the plasma cholesterol level, we have examined two key enzymes involved in cholesterol metabolism. HMG-CoA reductase is regarded as the rate-limiting enzyme in cholesterol biosynthesis. In 3-thiadicarboxylic acid and teTABLE

III

Relative specific activities of HMG-CoA reductase and ACAT Rats were fed fatty acid analogues for 7 days. The data represents the means of three animals in each experimental group (see Materials and Methods). The specific activities (100%) for nine control animals were 71k 19 and 325+26 pmol/min per mg protein for HMG-CoA reductase and ACAT, respectively. Treatment

Palmitic

acid

3-Thiadicarboxylicacid Tetradecylthioacetic acid Tetradecylthiopropionic acid

Dose mg/day per kg body weight

HMG-CoA reductase

ACAT

150 400 1.50 400 150 250 400 150 400 800

164 137 59 155 91 122 132 87 242 124

122 116 66 56 76 41 58 119 90 91

tradecylthioacetic acid treated rats the HMG-CoA reductase activity was decreased compared to palmitic acid-fed rats at the lowest dose (1.50 mg/day per kg), while the activity was only marginally affected in rats fed a dose of 400 mg/day per kg (Table III>. In contrast, the hepatic ACAT activity decreased in a dose-dependent manner in rats treated with 3-thiadicarboxylic acid and tetradecylthioacetic acid, but not with tetradecylthiopropionic acid (Table III). The response was comparable to the effects on the lipogenic enzymes and to the changes in the plasma cholesterol level. However, the correlation between ACAT activity and plasma cholesterol (r = 0,61) (not shown) was not as strong as for citrate lyase and acetyl CoA-carboxylase. Discussion

Several factors may affect the synthesis and the composition of VLDL particles in the liver. De novo synthesis of fatty acids, synthesis of triacylglycerol, cholesterol and apo B and cholesterol esterification can all potentially be of importance in the regulation of VLDL metabolism. In pellet-fed rats, 3-thiadicarboxylic acid and tetradecylthioacetic acid decreased both triacylglycerol and cholesterol plasma levels [l-2]. The hypotriglyceridemic effect has partly been ascribed to reduced availability of free fatty acids and diminished triacylglycerol biosynthesis 131. In the present study, where the lipogenic flux is increased due to the high carbohydrate intake, the 3-thia fatty acids decreased the plasma cholesterol level by more than 50% (Fig. l), while the effect on the plasma triacylglycerol level was remarkably smaller. Under these conditions the activities of the three lipogenic enzymes examined were reduced by up to 50% (Fig. 3). In pellet-fed rats where the activities of these enzymes are lower, we have observed that 3-thiadicarboxylic acid and tetradecylthioacetic acid have much smaller effects on these enzyme activities (unpublished data). Based on these results it can be inferred that changes in de novo fatty acid synthesis does not correlate with the plasma triacylglycerol level either in pellet-fed or glucose-fed rats. Tetradecylthioacetic acid, and to a lesser extent 3-thiadicarboxylic acid, decreased nonesterified fatty acids in the plasma, possibly indicating reduced peripheral hydrolysis of triacylglycerols. Nonesterified fatty acids were also decreased in pellet-fed rats intubated with 3-thiadicarboxylic acid and tetradecylthioacetic acid [3]. In the hyperlipogenic rats, however, there seems to be no correlation between the levels of nonesterified fatty acids in plasma and the synthesis and secretion of triacylglycerols from the liver. It is striking that tetradecylthioacetic acid seems to be more potent than 3-thiadicarboxylic acid in affecting

the hepatic and plasma triacylglycerol levels (Table 1 and 21. The amount of hepatic triacylglycerols and diacylglycerols is also decreased with increasing doses, indicating that the effect on triacylglycerol metabolism may be dependent on the dose administered. When lipogenesis and triacylglycerol synthesis are increased, higher doses of fatty acid analogues may be needed to get an effect on these parameters. This is in contrast to what we have observed in pellet-fed rats where 3thiadicarboxylic acid was more potent than tetradecylthioacetic acid in reducing hepatic triacylglycerols, at all doses tested [2]. We have previously shown that tetradecylthiopropionic acid inhibited the mitochondrial /?-oxidation and resulted in the development of fatty liver in pellet-fed rats [4]. In the glucose-fed rats we see again that higher doses (800 mg/day per kg) than reported earlier, seems to be necessary to produce fatty liver. This may be explained by the fact that the mitochondrial p-oxidation is not inhibited at a dose of 400 mg/day per kg or below (unpublished observation). The increased amount of hepatic phospholipids seen in 3-thiadicarboxylic acid and tetradecylthioacetic acid-treated rats (Table I) is probably due to the proliferation of peroxisomes observed in the same rats [l]. The three lipogenic enzymes examined in this study, ATP-citrate lyase, acetyl-CoA carboxylase and fatty acid synthase are all regulated in a coordinated manner after feeding rats fatty acid analogues (Fig. 3). This kind of regulation is often seen with these lipogenic enzymes, and several others, for instance during fasting and refeeding [5]. It is striking that the plasma cholesterol level changes in parallel with the activities of these enzymes. There is a relative strong correlation between the activities of acetyl-CoA carboxylase, ATP-citrate lyase and plasma cholesterol (Fig. 41. The reason for this is not obvious. The product of the citrate lyase reaction is acetyl-CoA which is the starting point for the synthesis of both fatty acids and cholesterol. If the flux of acetyl-CoA in the liver changes in the same direction as the citrate lyase activity, this may indicate that the availability of acetyl-CoA limits cholesterol synthesis under these feeding conditions. It has been reported that inhibitors of ATP citrate-lyase like ( - )-hydroxycitrate inhibited cholesterogenesis to a greater extent than lipogenesis and that the plasma cholesterol level was decreased significantly. At the same time the activity of HMG-CoA reductase was increased [26]. A differential sensitivity for inhibition of cholesterogenesis and lipogenesis was also observed in MEDICA treated rats [22]. The fraction of acetylCoA that is channeled into cholesterogenesis is small compared to what goes into lipogenesis [271. It is conceivable that in hyperlipogenic rats cholesterol synthesis is more sensitive to variations in the acetyl-CoA level than fatty acid synthesis. Reduced activity of

citrate lyase may thus result in decreased cholesterol biosynthesis without appreciably affecting fatty acid synthesis. At present our knowledge regarding the regulation of VLDL secretion from the liver is rather limited. Feeding a high carbohydrate diet results in increased lipogenesis and also in increased VLDL secretion. It has been proposed that these two phenomenons may be coordinately regulated [14]. If so, one should expect decreased VLDL secretion when lipogenesis is inhibited. As already mentioned the esterification of hepatic cholesterol may be involved in the regulation of VLDL secretion. The rate of cholesterol esterification is dependent on the supply of substrates (cholesterol) and the activity of ACAT. ACAT is located both in rat liver and in the intestine. The intestinal ACAT activity is probably involved in the absorption and esterification of intestinal cholesterol and the secretion of cholesterol ester into the lymphatics [16]. Both enzyme activities may thus potentially influence the level of plasma cholesterol. The data presented shows that the hepatic HMGCoA reductase activity does not change in parallel with the plasma cholesterol level, suggesting that this enzyme may not account for the hypocholesterolemic effect. Contrary to this the hepatic ACAT activity in 3-thiadicarboxylic acid and tetradecylthioacetic acidtreated rats is reduced to an extent comparable to the reduction in the lipogenic enzyme activities. This may point to a coordinated regulation of ACAT and the three lipogenic enzymes. The hypocholesterolemic effect of 3-thiadicarboxylic acid and tetradecylthioacetic acid may thus be ascribed to diminished cholesterogenesis due to reduced availability of acetyl-CoA. A reduction in the esterification of hepatic cholesterol might be a contributing factor. It is conceivable that this could change the composition of the VLDL particles secreted from the liver. To what extent the rate of VLDL secretion might be affected by feeding rats 3-thia fatty acids will be a matter for future studies. One should however, also consider the possibility that intestinal ACAT activity could be affected. This might result in diminished absorption of intestinal cholesterol and decreased secretion of cholesterol ester from intestinal epithelial cells. Acknowledgements

The authors are grateful to Mr. Svein Kruger, Mr. Terje ‘Bjorndal and Ms. Randi Sandvik for excellent technical assistance. The work was supported by the Norwegian Council on Cardiovascular Diseases (NCCD), Odd Fellow Vitenskapelige Forskningsfond and Nordisk Insulinfond. J. Skorve is a Research Fellow of NCCD.

181 References

1 Berge, R.K., Aarsland, A., Kryvi, H., Bremer, J. and Aarsaether, N. (1989) Biochem. Pharmacol. 38, 3969-3979. 2 Aarsland, A., Aarsaether, N., Bremer, J. and Berge, R.K. (19891 J. Lipid Res. 30, 1711-1718. 3 Skorve, J., Asiedu, D., Rustan, A.C., Drevon, C.A., Al-Shurbaji, A. and Berge, R.K. (1990) J. Lipid Res. 31, 1627-1635. 4 Asiedu, D., Aarsland, A., Skorve, J., Svardal, A.M. and Berge, R.K. (1990) Biochim. Biophys. Acta 1044, 211-221. 5 Iritani, N. (1992) Eur. J. Biochem. 205, 433-442. 6 Fukuda, H., Katsurada, A. and Iritani, N. (1992) J. Biochem. 111, 25-30. 7 Swierczynski, J., Mitchell, D.A., Reinhold, D.S., Salati, L.M., Stapleton, S.R., Klautky, S.A., Struve, A.E. and Goodridge, A.G. (1991) J. Biol. Chem. 266, 17459-17466. 8 Siddiqui, U.A., Goldflam, T. and Goodridge, A.G. (1981) J. Biol. Chem. 256, 4544-4550. 9 Sun, J.D. and Holten, D. (1978) J. Biol. Chem. 253, 6832-6836. IO Gibson, D.M., Lyons, R.T., Scott, D.F. and Muto, Y. (1972) in Advances in enzyme regulation (Weber, G., ed.), Vol. 10, pp. 187-204, Pergamon Press, Oxford. II Gibbons, G.F. (1990) Biochem. J. 268, 1-13. 12 Gibbons, G.F. and Burnham, F.J. (1991) Biochem. J. 275, 87-92.

13 Waddell, M. and Fallon, H.J. (1973) J. Clin. Invest. 52, 2725-2731. 14 Boogaerts, J.R., Malone-McNeal, M., Archambault-Schexnayder, J. and Davis, R.A. (1984) Am. J. Physiol. 246, E77-E83. 15 Yamamoto, M., Yamamoto, I., Tanaka, Y. and Ontko, J.A. (1987) J. Lipid Res. 28, 1156-1165. 16 Sliskovic, D.R. and White, A.D. (1991) TiPS 12, 194-199. S., Simons, L.A., Chang, S., Roach, P.D. and 17 Balasubramaniam, Nestel, P.J. (19901 Atherosclerosis 82, 1-5. 18 Field, F.J., Albright, E. and Mathur, S. (1991) Lipids 26, l-8. 19 Suckling, K.E. and Stange, E.F. (1985) J. Lipid Res. 26, 647-671. K.M., Yasruel, Z., Rodriguez, M.A., Vas, D. and 20 Cianflone, Sniderman, A.D. (1990) J. Lipid Res. 31. 2045-2055. N. (1990) 21 Aarsland, A., Berge, R.K., Bremer, J. and Aarsaether, Biochim. Biophys. Acta 1033, 176-183. J., Rose-Kahn, G. and Srebnik, M. (1985) J. Biol. 22 Bar-Tana, Chem. 260, 840448410. S., Hashimoto, T., Ogiwara, H., Nikawa, 23 Tanabe, T., Nakanishi, J.-I. and Numa, S. (1981) Methods Enzymol. 71, 5-16. 24 Roncari, D.A.K. (19811 Methods Enzymol. 71, 73-79. K., Liljeqvist, L., Nilsell, K. and Heller, 25 Angelin, B., Einarsson, R.A. (1984) J. Lipid Res. 25, 1159-1166. T.A., Havekes, L.M., Pearce, N.J. and Groot, P.H.E. 26 Berkhout, (1990) Biochem. J. 272, 181-186. 27 McGarry, J.D., Takabayashi, Y. and Foster, D.W. (1978) J. Biol. Chem. 253, 8294-8300.