Gender-related effects of chronic ethanol ingestion on rat hepatic acyl-CoA: cholesterol acyltransferase

66

Biochimicu et Bioph_vsicu Actcr 879 (1985) 66-72 Elsevier

BBA 52389

Gender-related effects of chronic ethanol ingestion on rat hepatic acyl-CoA : cholesterol acyltransferase Sam Hashimoto u Research

a,h, Bernadine

J. Wisnieskie

’ and Howard Wong a

Senuce, Veterans Administration Wudsworth Me&cd Center. Los Angeles. CA 90073, Depurtments h Medicine and ’ Microhiologv and The Moleculur Biologv Institute, Unmersr
Key words:

Acyl-CoA

: cholesterol

acyltransferase;

of

22 April 1986)

Sex difference;

Ethanol;

Membrane

fluidity;

(Rat liver)

The influence of chronic ethanol ingestion on hepatic acyl-CoA: cholesterol acyltransferase activity was investigated to determine the relationship between alcohol intake and cholesterol ester accumulation. Rats were given nutritionally complete liquid diets supplemented with 6.3% ethanol or an isocaloric equivalent of dextrin-maltose for 5 weeks. During this period, the hepatic acyl-CoA : cholesterol acyltransferase activity of ethanol-fed male rats remained constant, whereas the same activity in pair-fed controls as well as chow-fed rats exhibited a 30% decrease in activity. Unlike alcohol-fed male rats, the hepatic acyl-CoA:cholesterol acyltransferase activity of female rats decreased by approximately 30% by the fifth week of ethanol ingestion. Despite the fact that the gender of the animals led to disparate levels of acyl-CoA:cholesterol acyltransferase activity in response to ethanol ingestion, similar levels of cholesteryl ester accumulation were observed. The altered levels of acyl-CoA : cholesterol acyltransferase activity caused no significant change in the cholesterol concentration, cholesterol/phospholipid ratio, phospholipid fatty acid composition, or the membrane fluidity of the hepatic microsomes. We conclude that the altered hepatic acyl-CoA : cholesterol acyltransferase activity of ethanol-fed female rats cannot be directly responsible for ethanol-induced accumulation of cholesteryl esters.

Introduction There is little doubt that cholesterol esterification, catalyzed by acyl-CoA : cholesterol acyltransferase (EC 1.1.1.34), is modulated by the concentration of cholesterol in the hepatic endoplasmic reticulum [l-4]. The mechanisms by which cholesterol esterification is modulated in the absence of a bulk change in membrane cholesterol are less obvious. For example, it has been noted that acyl-CoA : cholesterol acyltransferase is Correspondence address: Veterans Administration Wadsworth Medical Center, Building 114. Room 113. Los Angeles, CA 90072. U.S.A. 0005.2760/X6/$03.50

Q 1986 Elsevier Science Publishers

activated by certain ATP-dependent processes [3,5-lo], by increases in the phospholipid unsaturated fatty acid content [8-lo], by 25-hydroxycholesterol [111, and by preincubating microsomes before assaying acyl-CoA : cholesterol acyltransferase activity, and is suppressed by progesterone [12]. The possibility of a direct link between cholesteryl ester metabolism and its accumulation in the livers of ethanol-fed rats was postulated by several investigators [13-151. But it has not yet been established that the accumulation of cholesteryl ester is actually associated with an increased synthesis of cholesteryl ester. Augmentation [14,16] as well as suppression [17] of acyl-CoA : cholesterol

B.V. (Biomedical

Division)

67

acyltransferase activity has been noted in response to alcohol diet. These conflicting observations may be related to the gender of the experimental animals employed in the different studies. Modulation of cholesterol-esterifying activity in hepatic microsomes of ethanol-fed rats has been reported to occur in the absence of changes (compared to controls) in the bulk concentration of membrane cholesterol and in phospholipid fatty acid composition [14]. This result suggests that an ethanol diet may in some way affect the enzyme’s accessibility to cholesterol. We have considered the possibility that chronic ethanol ingestion might influence the distribution of cholesterol between membrane pools or the accessibility of cholesterol substrate to the enzyme. Hence, we analyzed the membrane lipid composition and fluidity of microsomes isolated from the livers of ethanol-fed and control rats. We also investigated the effect of chronic ethanol ingestion on hepatic acylCoA : cholesterol acyltransferase activity as a function of gender and duration of ethanol ingestion. Materials and Methods Treatment of rats Wistar rats were fed a Lieber liquid diet (BioNJ) containing 6.3% serve Inc., Frenchtown, ethanol [18]. Their pair-fed controls received a caloric equivalent of ethanol as dextrin-maltose. A third group of rats were fed a chow pellet diet ad libitum. The lipid contents of the liquid diet and chow diet represented 35 and 7.7% of the total calories, respectively. Ten animals per diet group were killed weekly for five weeks. Rats were decapitated after a 16 h fast to minimize blood ethanol concentration at the time of killing. The livers were minced and the pulp was diluted with 4 vol. of 0.3 M sucrose and homogenized with a motorized pestle. The post-mitochondrial supernatant was obtained by centrifugation of the homogenate at 12000 x g for 20 min. The microsomes were resuspended in 0.1 M Tris buffer (pH 7.4) to a protein concentration of 5 mg/ml. Assay for cholesterol-esterifying activity The conditions of the assay were optimized as to the microsomal protein and palmitoyl-CoA

concentrations and the time of incubation [19]. Microsomes, 75 pg protein, were added to 200 ~1 of 0.1 M Tris buffer (pH 7.4) containing 0.1% defatted bovine serum albumin and 36 PM [l“C]palmitoyl-CoA (specific activity 15 ~Ci/~mol) and incubated for 8 min at 37OC. The reaction was stopped by the addition of methanol. The microsomal lipid was isolated by the procedure of Bligh and Dyer [20] and fractionated by thin-layer chromatography on silica gel G using 10% diethyl ether/l% acetic acid/89% petroleum ether (b.p. 60-11OOC). Recovery of cholesteryl ester was approx. 95%. The area corresponding to the position of cholesteryl ester was scraped into a counting vial, the powder was suspended in Handifluor (Mallinckrodt)/water (10 : 4, v/v) and assayed for radioactivity in a liquid scintillation counter. Phospholipid fatty acid analysis Microsomal phospholipids were separated from the neutral lipids on a chromatographic column (5 mm x 10 cm) containing 1.6 g of silicic acid (Unisil, Clarkson Chemical Co., Williamsport, PA). Neutral lipids were eluted with 10 ml chloroform/ methanol (2: 1) and with 30 ml methanol. The phospholipid fractions were pooled and concentrated under nitrogen at 60 o C. Phospholipids were saponified and the fatty acids recovered by petroleum ether extraction of the acidified aqueous phase [21]. The fatty acids were methylated with diazomethane and analyzed by gas-liquid chromatography on a 10% SP2330 column (Supelco, Bellefonte, PA). Analytical methods Phospholipid, triacylglycerol and protein were measured in homogenates and microsomes by the procedures of Chen et al. [22], Newman et al. [23], and Lowry et al. [24], respectively. Free and esterified cholesterol were quantified by the procedure of Rude1 and Morris [25] after separation of these substances from other lipids by column chromatography on 0.8 g of silicic acid. The cholesteryl ester was eluted form the column with 1% diethyl ether in petroleum ether, and free cholesterol with chloroform. Membrane fluidity The freedom of motion

of a fatty

acid mem-

68

Results

brane probe 5-nitroxide stearate (Syva Co., Palo Alto, CA) was measured by electron spin resonance spectroscopy - the 5-nitroxide group refers to the 4’,4’-dimethyl-N-oxyloxazolidine ring. Hepatic microsomes were labeled with 5-nitroxide stearate by incubating microsomes (1 mg protein in 0.2 ml Tris buffer (pH 7.4)) in a centrifuge tube coated with spin-label probe (30 min at room temperature). The molar ratio of probe to microsomal phospholipid was 1: 133. The microsomes were collected by centrifugation and withdrawn into a capillary tube. After sealing, the capillary tube was inserted in a Varian E-104 Century Series spectrometer Spectra were recorded at 37OC. The field was centered at 3258 Gauss with a microwave frequency of 9.18 GHz. The order parameter (S) was calculated from the equation of Sauerheber and co-workers [26].

TABLE

The effects of chronic ethanol ingestion on male rats are shown in Table I. The data on the chow-fed animals are presented in the text. The weight gain of ethanol-fed rats was comparable to those of the pair-fed controls during the 5 week feeding. As compared to the chow-fed animals, the weight gain was 84%, probably because of restricted caloric intake. The liver weight of the ethanol-fed rat (expressed as percent of body weight) was greater than that of the control after the second week of feeding (3.2 vs. 2.6%). Such enlargements of the liver have been attributed to an increase in protein, lipid and water content

v71. Hepatic concentrations of esterified (2.8 pg/mg protein) and triacylglycerol

cholesterol (14.8 pg/

I

CHRONIC PROTEIN,

EFFECT OF CONTROL AND ETHANOL-CONTAINING TRIACYLGLYCEROL AND CHOLESTERYL ESTER

DIETS ON RAT WEIGHT

AND LIVER CONTENT

OF

Rats were given a nutritionally complete liquid diet containing 6.3% (v/v) ethanol for 5 weeks. The pair-fed controls were given the same diet exceptfor the substitution of dextrin-maltose for ethanol. Rats were killed after an overnight fast. Analyses were performed on liver homogenates. Values are mean? S.E.. n = 10. a Comparison between ethanol and control groups. Weeks on diet 1 Body weight (g): Control Ethanol

203 202

Liver (S body weight): Control Ethanol P value a Liver protein Control Ethanol P value

(mg/lOO

Triacylglycerol Control Ethanol P value

(pg/mg

Cholesteryl Control Ethanol P value

2

k *

2.9? 2.g*

5 3

0.1 0.2

g body weight): 546 i64 501 i15

ester (pg/mg

liver protein): 60 k 5 43 *22

liver protein): 10.9& 2.3 12.9* 3.2

3

230 227

+ 9 + 8

2.6i 2.95

547 596

0.1 0.2

+57 +25

42 ? 6 97 +19 < 0.05

12.5? 9.3*

2.6 2.1

235 213

4

& 8 * 7

2.7+ 3.4* < 0.01

0.2 0.1

261 262

5

i f

2.4i 3.3* < 0.01

9 9

271 252

& 6 k 9

0.1 0.1

2.55 3.2* i 0.01

0.1 0.1

497 +40 644 *3X i 0.05

492 ~35 638 +41 < 0.05

513 i53 689 513 < 0.01

41 *24 206 i24 < 0.01

73 *19 219 ?52 < 0.05

270 *40 < 0.01

12.6-t 27.6 i < 0.05

g.5* 23.9+ < 0.01

9.7+ 19.1+ < 0.01

1.2 2.2

3.0 4.2

2.2 0.5

69

rats, ethanol appeared to prevent the natural tendency toward lower acyl-CoA : cholesterol acyltransferase activity, a tendency for which we have no immediate explanation. Parameters that may influence acyl-CoA : cholesterol acyltransferase activity were systematically investigated. The microsomal cholesterol concentrations and the cholesterol/ phospholipid ratios were the same in the ethanol-fed animals and the controls (Table II). The data indicated that the acyl-CoA : cholesterol acyltransferase of the controls had di~nished access to membrane cholesterol substrate after the third week of the experiment, despite the absence of any change in the microsomal membrane cholesterol content and phospholipid fatty acid composition (Table III). The order parameters (S), a measure of membrane fluidity, was determined from the electron spin resonance spectrum of 5-nitroxide stearate incorporated into the microsome membrane. These measurements were done in a separate experiment in which rats were fed ethanol, control and chow diets for 5 weeks. The S values (mean + S.E., n = 4) for the three diet groups were 0.570 + 0.009, 0.578 ? 0.009 and 0.570 + 0.009, respectively, showing no change as a function of diet. The data indicated that if membrane fluidity did exert an effect on the accessibility of the cholesterol sub-

mg protein) in chow-fed rats were unchanged during the 5 weeks of the experiment. Feeding the liquid control diet increased the esterified cholesterol and triacylglycerol concentrations approximately 3.8- and 5.3-fold, respectively, after the third week. Greater increases were observed with the ethanol-cont~ning diet; esterified cholesterol and triacylglycerol concentrations increased on the average 9.1- and 15.5-fold, respectively, compared to the concentrations in the chow-fed rats. Accumulation of these lipids in the livers of the controls fed on the liquid diet may be due to the higher lipid content of the liquid diet as compared to the chow diet (35.0 vs. 7.7%) [28], and to mild starvation, since these animals had experienced not only a somewhat restricted caloric intake but also a 16-h fast before they were killed. Fasting tends to increase triacylglycerol accumulation 1291. Since the cholesteryl ester component of hepatic tissue is derived from synthesis, the activity of acyl-CoA : cholesterol acyltransferase was measured in microsomal preparations isolated from the livers of these animals (Table II), The cholesterol-este~fying activity of ethanol-fed rats remained unchanged during the 5 week period, while the activity of this enzyme in the controls decreased after 3 weeks on the diet. Since a similar decreasing trend was observed in the chow-fed

TABLE

II

INFLUENCE OF A DIET CONTAINING ETHANOL TIONS OF LIPID COMPONENTS OF MICROSOMES

ON CHOLESTEROL-ESTERIFYING

ACTIVITY

AND CONCENTRA-

Rats were treated as described in Table I. Microsomes were isolated from rat liver homogenates as described in Methods. Synthesis of cholesteryl ester was measured by the inco~oration of label from [l-‘4C]palmitoyl-CoA into cholesteryl ester. Free cholesterol was separated from esterified cholesterol by silica gel column chromatography. Free cholesterol and phospholipid phosphorus were quantitated calorimetrically. Values are mean * S.E., )t = 10. h P < 0.05: comparison between control and ethanol groups. Weeks on diet 1 Cholesteryl Control Ethanol Free cholesterol Control Ethanol

2

ester formed (pmol/min 511 513 (

4

5

i33 i28

556 505

+26 +29

502 500

i29 * 40

399 520

i31 +41 h

401 531

i 26 i43h

pg/mg protein)

248 22.4

Ratio of free cholesterol Control Ethanol

3

per mg protein)

& 1.2 i 2.5

17.3 21.3

to phospholipid

(w/w)

0.066 i 0.003 0.063 * 0.003

+ 1.4 & 1.7

0.054i 0.063 i

0.009 0.003

20.2 19.5

f i

0.065 i 0.057 *

1.2 1.4

0.004 0.003

21.9 20.3

rt 3.5 + 1.4

0.068 + 0.010 0.062 + 0.004

21.0 19.6

i k

1.3 1.3

0.05g * 0.004 0.056 + 0.003

70

TABLE

ethanol-fed males, this enzyme activity of ethanolfed female rats was suppressed by 30% by the end of the 4th week. Thus, the direction of modulation of acyl-CoA : cholesterol acyltransferase activity in ethanol-fed animals is strongly dependent upon the sex of the animal. The observed suppression of acyl-CoA : cholesterol acyltransferase activity was not associated with any change in the membrane concentration of cholesterol or in the cholesterol/ phospholipid ratio.

III

INFLUENCE OF CONTROL AND ETHANOL-CONTAINING DIETS ON PHOSPHOLIPID FATTY ACID COMPOSITION OF RAT HEPATIC MICROSOMES Phospholipids were isolated from microsomal lipids by silica gel column chromatography. Phospholipid fatty acids were released by saponification and acidified. The methyl esters were prepared by diazomethane and analyzed by gas-liquid chromatogrpahy on 10% SP-2330. Values are mean It SE, n = 4. Chain-length

: double bonds

16:0 18:0 IS:1 IS:2 20:4 2216

Control

Ethanol

17.2 + 0.6 31.3k1.3 9.6 + 0.6 9.8 + 0.5 30.3 IO.9 1.8+_0.8

15.8 * 1.2 31.9* 1.3 11.3kO.6 11.2iO.9 28.2& 1.4 1.6kO.4

Discussion An apparent increase in acyl-CoA : cholesterol acyltransferase activity after 4 weeks or longer on an ethanol diet has been noted by other investigators [14] but this has not been a consistent observation. In a study carried out with female rats, the cholesterol-esterifying actiivty was shown to be suppressed [17]. Our observations are in general agreement with both reports, and resolve the controversy by showing that the direction of modulation of acyl-CoA : cholesterol acyltransferase activity depends upon the gender of the animal. Since hepatic acyl-CoA : cholesterol acyltransferase activity was not influenced by the gender of the control animals, it would appear that chronic ethanol ingestion may affect the normal metabolism of the gonadal hormones in a manner which subsequently modulates acylCoA : cholesterol acyltransferase activity. Alcoholrelated disturbances of plasma hormone levels have been observed in humans: testosterone levels decreased in men [30], and progesterone and

s&ate it was not correlated with any notable alteration in the order of the membrane hydrocarbon domain. Contrary to our own findings, ethanol consumption has been reported to suppress cholesterol-esterifying activity [17]. Since female rats had been used in the previous studies, we felt it was important to examine the possibility of a sex-related response to alcohol intake. When female rats were subjected to the ethanol-containing and control diets, we found that the acylCoA : cholesterol acyltransferase activity of the female controls (Table IV) was similar to that of the male controls even during the 4th and 5th week of feeding (Table II). However, unlike the

TABLE

IV

INFLUENCE MICROSOMES

OF CHRONIC ETHANOL FROM FEMALE RATS

INGESTION

ON

CHOLESTEROL-ESTERIFYING

ACTIVITY

OF

HEPATIC

Female rats were given a diet supplemented with ethanol or dextrin-maltose for 4 wekes and killed. The treatment of the rats and livers was the same as described in Tables I and II. Values are mean+ SE. Number of rats is given in parentheses. ’ P < 0.01; comparison between control and ethanol group. Standard error of the difference was weighted according to sample size n. Control Cholesteryl ester formed (pmol/min per mg protein) Cholesterol

pg/mg

( protein) Ratio of free cholesterol to phospholipid (w/w)

373 20.2 0.055*

Ethanol +16 + 0.9 0.003

(IQa (4) (4)

25s 19.2 0,056~

+37 k 0.5 0.001

(14) (5) (5)

71

estradiol levels increased in women [31]. Among these hormones, progesterone had the greatest effect on cholesterol ester synthesis in cultured fibroblasts [32]. Whether the observed increases in plasma progesterone levels are sufficient to suppress the acyl-CoA : cholesterol acyltransferase activity of the hepatic endoplasmic reticulum is unknown. Nonetheless, such observations would be of acylconsistent with the suppression CoA : cholesterol acyltransferase activity in ethanol-fed female rats that we have uncovered in the course of this study. The effects of ethanol ingestion were not manifested in changes in the microsomal membrane usually associated with modulation of acylCoA : cholesterol acyltransferase activity - cholesterol concentration [l-4], unsaturation of phospholipid fatty acids [g-lo], and membrane fluidity [9]. Since these measurements represent averages obtained from heterogeneous pools of microsomal membranes, it might still be possible that ethanol directly or indirectly provokes subtle changes in local membrane domains and that this affects the enzyme’s accessibility to cholesterol. But we think that this is highly unlikely because ethanol feeding would be expected to have a unidirectional effect on a membrane, either increasing or decreasing substrate availability, and what we observed was a sex-dependent outcome. Clearly, other factors must be involved. The order parameter of the hepatic microsomes of ethanol-fed rats did not change, confirming what might be anticiated by the lack of change in the determinants of membrane fluidity [33] with ethanol feeding, i.e., cholesterol concentration, phospholipid fatty acid composition and cholesterol/phospholipid ratio. Unlike microsomes [34] and mitochondria [35], liver plasma membranes displayed less order (an increase in fluidity) after chronic ethanol feeding [36]. This has been attributed to loss of plasma membrane cholesterol due to enhanced cholesterol esterification [36] and to increased biliary excretion of cholesterol as bile acids [37]. This study and others [13-151 have shown that cholesteryl ester accumulates in the livers of chronic ethanol-fed male and female rats but, as we have demonstrated here, the increase in content of cholesteryl ester is not correlated with

enhanced acyl-CoA : cholesterol acyltransferase activity. The lack of correlation may be due in part to our assay protocol, which is designed to measure acessibility of endogenous cholesterol substrate in the microsomal membrane at saturating concentrations of acyl-CoA. Cholesterol esterification in vivo may be controlled by the concentration of acyl-CoA. If so, then we would expect hepatic acyl-CoA concentrations to be higher in the ethanol-fed male and lower in the female rats than in the controls. This is not the case [38,39]. Other factors are obviously involved. One factor that may turn out to be significant in terms of understanding the basis of the gender dependence is the alcohol-induced suppression of cholesteryl ester hydrolysis that occurs in female rats [17]. Considering the greater risk that human females face with respect to alcohol-related ascitic cirrhosis, and the earlier onset [40], it is important to understand the mechanisms by which genderdependent effects arise. Acknowledgements This research was supported in part by VA Medical Research Funds and by U.S. Public Health Service Grant GM-2240. We thank Mrs. Loretta Berg for her valuable technical assistance. References Hashimoto, S., Drevon. C.A., Weinstein, D.B., Bemett J.S., Dayton, S. and Steinberg, D. (1983) Biochim. Biophys. Acta 754, 126-133 Synouri-Vrettakou, S. and Mitropoulos, K.A. (1983) Eur. J. Biochem. 133, 299-307

6 7 8 9 10

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