121

Biochimica et Biophysics Acta, 572 (1979) @ Elsevier~No~h-Holland Biomedical Press

121-131

BBA 57290

THE EFFECT OF ETHANOL ON GLYCEROLIPID BIOSYNTHESIS BY PRIMARY MONOLAYER CULTURES OF ADULT RAT HEPATOCYTES

CRAIG

K. WOOD and ROBERT

G. LAMB

Departments of Pharmacology and Medicine, Medical College of Virginia, Richmond, 23298 (U.S.A.) (Received

*

VA

June 2nd, 1978)

Key words: Ethanol effect; Glycolipid biosynthesis; (Hepatocyte)

Summary

This study evaluates the effects of ethanol exposure on glyeerolipid production and release by hepatocyte monolayers. Glycerolipid formation from [1,314C]glycerol was increased in monolayers exposed to ethanol (l-50 mM) for 6 h. Monolayers exposed to 1 mM ethanol for 24 h also exhibited a rise in glycerolipid formation from either fl,3-14C]glycerol or [l-‘4C]palmitate; however, higher ethanol concentrations produced a dose dependent decrease in glycerolipid formation. Glycerolipids released into the medium by monolayers declined after all periods of ethanol exposure. The effects of ethanol on the enzymatic reactions involved in glycerolipid biosynthesis were determined in homogenates prepared from monolayers exposed to ethanol. Two enzymes, phosphatidate phosphohydrol~e and glycerol kinase, exhibited ethanol-induced alterations in enzyme activity; however, only glycerol kinase activity correlated well with monolayer glycerolipid formation, These ethanol-induced alterations in enzyme activities and glycerolipid biosynthesis were reduced by simultaneously exposing monolayers to pyrazole or cydoheximide.

Introduction Ethanol intake in man [l--5] and experimental animals [1,5-S] elevates hepatic and serum glycerolipid levels. The relationship between ethanol dependent changes in hepatic glycerolipid content and Errol-induced a&erations in hepatocyte membrane structure and function is not clear. However, glycerolipids are integral components of hepatocyte membranes and are essen* A preliminary report of this research was presented on April 4, 1977 in Chicago, IL, at the annual meeting of the Federation of American Societies for Experimental Biology.

122

tial for various hepatic functions including: the proliferation and repair of intracellular membranes; the formation of very low density lipoproteins and biliary phospholipids. The ethanol associated elevation in heptic glycerolipid levels is attributed to an increased capacity of hepatic glycerolipid formation [ 1, 6-261 since hepatic fatty acid [12-191 and sn-glycerol3-phosphate [ l,S-111 content rise and the liver’s enzymatic capacity of glycerolipid production increases [ 21-251. The present study utilizes adult rat hepatocytes in primary monolayer culture, to determine whether ethanol or some metabolite of ethanol causes these alterations in hepatic enzyme activity and glycerolipid content. Methods separation and treatment of monolayer Hepatocyte monolayers were prepared from adult male opine-Dawley rats (200-250 g) as previously described [27]. Unless specified, aqueous solutions of ethanol and other agents were added to the monolayer culture medium in a total vol. of 0.1 ml, 2 h after the freshly isolated hepatocytes had been plated. In some studies, an aqueous solution of pyrazole was added to the culture medium to produce a medium concentration of 1 mM, immediately prior to the addition of ethanol, Control monolayers were treated with 0.1 ml water. All concentrations refer to the initial medium concentrations. Glycerolipid formation by monolayers The formation of glycerolipids from [ 1,3-*4C]glycerol and their release into the culture medium was measured after monolayers had been exposed to ethanol and other reagents for various time periods, as previously described [ 271. The incorporation of [ l-14C]palmitate into glycerolipids was measured in a manner similar to that outlined for [1,3-14C,]glycerol incorporation except that 1.0 mM [1-14C]palmitate (0.2 &Ji) and 0.5 mM glycerol were substituted for 1.0 mM palmitoleate and 0.5 mM [1,3-‘4C]glycerol (0.4 &i), respectively. The time course of cellular glycerolipid formation and release into the medium from [I-14C]palmitate during a 90 min incubation period was identical to that described previously for labeled glycerol incorporation [ 271. Incorporation of [ 1,3-“C Iglycerol and [ 1-r4C]palmitate into cellular and medium glycerolipids was used as a measure of glycerolipid formation and release, respectively. Enzyme assays Glycerol kinase and sn-glycerol 3-phosphate acyltransferase activity was measured in monolayer homogenates as previously described [27]. Phosphatidate phosphohydrolase activity was estimated by determining the ratio of neutral lipid [diacylglycerol (10-30%) + triacylglycerol (70-90%)] to phospho~pid ~lysophosphatidate (lo-20%) + phosphatida~ (80-90%)] formed during the incubation used to measure sn-glycerol-3-phosphate acyltransferase activity [ 271. Although this technique for determining phosphatidate phosphohydrolase activity is indirect, it seems to be reliable [24,28,29]. Choline phosphotransferase activity was estimated by incubating cell homogenates [O.l--O.Z mg protein] with 0.06 mM methyl[14~]cytidine diphos-

123

phocholine (0.02 @i), 0.5 mM ammonium palmitate, 2.5 mM sn-glycerol 3-phosphate 2.1 mM ATP, 0.6 mM dithiothreitol, 0.03 mM coenzyme A, 3.2 mM MgC&, 16.0 mM Tris-HCl (pH 8.3) and 1.25 mg albumin in a total vol. of 0.43 ml. Reactions were initiated by the addition of homogenate, incubated for 60 min at 37°C with shaking and terminated by adding 3 ml CHC13/CH30H (1 : 2, v/v). Lipids were extracted and identified as described previously [27, 281. The formation of phosphatidylcholine under these conditions was linear with respect to incubation time and the amount of homogenate protein, and was not altered by the addition of diacylglycerol (0.3-0.8 mM) dispersed in water. Determination of medium ethanol concentrations The enzymatic method of Bonnichsen [30] was used to measure ethanol concentrations at various time periods after ethanol had been added to hepatocyte monolayers. Aliquots (0.04 ml) of cell medium were added to a solution (3 ml) containing 75 mM semicarbazide - HCl, 21 mM glycine (pH 8.8), 15.0 fl NAD and 0.2 mg yeast alcohol dehydrogenase (200 IU/mg). Reduced NAD was measured spectrophotometrically at 340 nm (light path, 1.0 cm). Ethanol levels in the aliquots were determined by comparison to known standards (O-15 mM) prepared from absolute ethanol. Results Fig. 1 shows the incorporation of [1,3-14C]glycero1 into triacylglycerol and phospholipids by hepatocyte monolayers exposed to l-50 mM ethanol for 6 h (section A) and 24 h (secion B). Triacylglycerol formation by monolayers exposed to ethanol for 6 h increased in a dose dependent manner, reaching a maximum at 5 mM (Fig. 1A). Glycerol incorporation into phospholipids was modestly but significantly (P< 0.05, level of significance from control) increased after monolayers had been exposed to 5 mM ethanol for 6 h. After 24 h of exposure to 1 mM ethanol, triacylglycerol formation was increased 55% and phospholipid formation 28%. Higher concentrations of ethanol caused a marked, dose dependent decrease in glycerol incorporation into triacylglycerol and phospholipids (Fig. 1B). The ethanol dependent reduction in [1,3-‘4C]glycerol incorporation into glycerolipids by monolayers could be attributed to an ethanol-induced rise in cellular glycerol 3-phosphate content [ 1,8-111, which would reduce the specific activity of labeled glycerol 3-phosphate. To exclude this possibility the glycerol 3-phosphate content of monolayers exposed to 5 and 10 mM ethanol for 24 h was measured and found to be unaltered. Furthermore, the results shown in Table I indicates that [1-14C]paImitate incorporation into glycerolipids in monolayer-s exposed to ethanol, fructose and glucose closely paralleled that of [1,3-14C]glycerol. Data in Fig. 2 indicate also that glycerolipid formation from [l-‘4C]palmitate and [1,3-14C]glycerol by monolayers exposed to ethanol (O-35 mM) for 24 h is similar. These observations (Fig. 2, Table I) suggest that ethanol-induced changes in monolayer glycerolipid formation are probably not the result of alterations in intracellular glycerol 3-phosphate content.

124 A T L

TG

u

\

d 5

I 5

8

i;lfi& 2.5 5

g

PL T

P . 0.08 Y ‘a ”

li

‘E

‘.,

10

35

10

35

PL

P c 0.04

0

2.5

5

mM Ethanol Fig. 1. The effect of ethanol on the incorporation of [1,3-14C]glycerol into triacylglycerol (TG) and phospholipid (PL) by hepatocyte monolayers. 2 h after freshly isolated hepatocytes had been plated, ethanol was added to produce the indicated medium concentration, 6 (section A) or 24 (section B) h following ethanol addition, the incorporation of [1,3- l4C]glycerol Each point represents the mean nmol glycerol incorporated/mg protein in triplicate from 5-8 separate experiments. lP < 0.01, "P G 0.001. TABLE

into glycerollpids was measured. per mln f S.E. for values obtained

I

THE INFLUENCE Ik]GLYCEROL

OF VARIOUS AGENTS OR [l-l’+C]PALMITATE

ON THE FORMATION OF GLYCEROLIPIDS BY HEPATOCYTE MONOLAYERS

FROM

[1,3-

2 h after freshly isolated hepatocytes had been plated, agents were added to produce the indicated medium concentration. After monolayers had been exposed to the appropriate agents for 24 h. glycerolipid biosynthesis was measured. Each point represents the mean + S.E. of 3-5 separate experiments in which each determination was made from 3 separate plates. Results are expressed as nmol incorporatedlmg protein per min. Addltons

[lJ4C]Palmitate

[1,3-14C]Glycerol

(mW Triacylglycerol None Ethanol (10) Glucose (20) Fructose (1)

0.187 0.011 0.194 0.192

t f f f

0.014 0.001 0.001 0.008

Phosphollpid

**

0.074 0.005 0.084 0.086

+ + f +

0.005 0.002 0.005 0.003

* P < 0.10 level of significance from control (none). ** P 6 0.001 level of significance from control (none).

Phospholipid

Trlacylglycerol

**

0.430 0.021 0.346 0.338

+ f f f

0.002 0.003 0.002 0.009

**

0.086 0.006 0.052 0.060

f f t ?

0.015 0.001 0.006 0.008

** *

125

I

PL

L ..

..

1

2.5

6

mM

10

36

ETHANOL

into triacylglycerol (TG) and phosFig. 2. The effect of ethanol on the incorporation of [l- 14CIpahnitate pholipid (PL) by hepatocyte monolayers. 2 h after freshly isolated hepatocytes had been plated, ethanol was added to produce the indicated medium concentration. 24 h after the addition of ethanol, the incorporation of [I-14Clpalmitate into glycerolipids wass measured. Each point represents the mean nmol palmitate incorporated/mg protein per min i S.E. for values obtained in triplicate from 3 separate experiments. lP G 0.01. lOP < 0.001.

Ethanol dependent alterations in the activity of hepatic enzymes which regulate glycerolipid biosynthesis, may be an alternative explanation of the ethanolinduced changes in glycerolipid formation. This possibility is supported by the results in Fig. 3A which demonstrates that glycerol kinase activity is increased in homogenates of monolayers exposed to ethanol (l-50 mM) for 6 h. After 24 h of exposure (Fig. 3B) 1 mM ethanol elevated, while higher ethanol concentrations caused a dose dependent decrease in glycerol kinase activity. These changes in glycerol kinase activity correlated well with the rate of glycerol or palmitate incorporation into glycerolipids by the intact monolayers (Fig. 1 and 2). Monolayers exposed to ethanol for 6 or 24 h, did not exhibit significant alterations in sn-glycerol-3-phosphate acyltransferase activity (Fig. 3C, D). However, ethanol exposure for 6 and 24 h increased the incorporation of [sn1,3-14C,]glycerol 3-phosphate into neutral lipid by monolayer homogenates. The ethanol-induced rise in neutral lipid (NL) biosynthesis occurred at the expense of phospholipid (PL, primarily phosphatidate) and was reflected as an increase in the NL/PL ratio (Fig. 3E, F Table II). Previous studies [24,28,29] suggest that the NL/PL ratio represents an indirect but reliable indicator of hepatic phosphatidate phosphohydrolase activity. These results suggest that hepatocyte monolayers exposed to ethanol exhibit a rise in phosphatidate phosphohydrolase activity. Table II shows the influence of pyrazole on the ethanol dependent altera-

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126

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0

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Fig. 3. The effect of ethanol on the activity of glycerol kinase, sn-glycerol-3-phosphate acyltransferase, choline phosphotransferase, and on the NL/PL ratio. 2 h after freshly isolated hepatocytes had been plated, ethanol was added to produce the indicated medium concentration. 6 (A. C. E, G) or 24 (B, D. F. H) h after the addition of ethanol, the monolayers were homogenized and the appropriate enzyme a&ivity and the NL/PL ratio was determined in monolayer homogenates as described under Methods. Each value represents the mean f S.E. determined in triplicate from 3 separate plates in 5-6 separate exPerimerits. lP 4 0.05, loP < 0.001.

tions in the formation and release of glycerolipids by hepatocyte monolayers. Pyrazole blocked both the ethanol-induced increases and decreases in glycerolipid formation as well as the ethanol dependent decline in monolayer glycerolipid release. Ethanol appears to reduce triacylglycerol release more than that of phospholipid. Pyrazole, as shown in Table III, also blocked the ethanolinduced alterations in glycerol kinase activity and rise in NL/PL which is an indirect measure of phosphatidate phosphohydrolase activity.

127 TABLE II THE EFFECT OF ETHANOL AND PYRAZOLE CEROLIPIDS BY HEPATOCYTE MONOLAYERS

ON THE FORMATION

AND RELEASE

OF GLY-

2 h after the freshly isolated hepatocytes had been plated, ethanol was added to produce the indicated medium concentration. Pyrazole was added immediately prior to the addition of ethanol to produce the indicated medium concentration. The formation of triacylglycerol and phosphohpid from [1.3-14C21glycerol and the release of glycerolipids into the medium was determined, Each value represents the mean % control ?r SE. (nmol incorporated/mg protein per mg) of values obtained from S-8 separate exPerimerits in which determinations were made from 3 separate plates. Control values for ceIIuIar and medium triacylglycerol and phospholipid were 0.282, 0.074. 0.0066, 0.0075 for the 6-h, and 0.187. 0.050. 0.0036.0.0032 for the 24-h exposure periods, respectively. Additions (mMI

6 hours None Ethanol (1) Ethanol (10) Pyrazole (1) Ethanol (10) + pyrazole (1) 24 hours None Ethanol (1) Ethanol (10) Pyrazole (1) Ethanol (1) + pyrazole (1) Ethanol (10) + pyrazole (1)

CeII

Media

Triacylglycerol

lOOi: 12ort 13l.k 102 f ill?:

4 5* 5** 9 4

100f 8 155 t 12 ** 6!: l** 92C 4 1Olf: 7 982 2

Phospholipid

Triacylglycerol

lOOk 100% 108f 105+ 1032

lOO+ 59+ 262 1022 111 f

1 1 5 8 1

100 + 10 128? 5 * 8-1 2+* 106k 7 99? 5 822 7

Phospholipid

8 si** 2** 9 4

lOO+ 8 332 8** 3+ 1** 94 + 13 85 r 10 83f 3

lOOf Slfr 51f 105+ 103 +

1 2 8** 8 2

1002 6 85 + 11 22+ 3** 106? 9 102 r 5 842 4

* P $ 0.01 level of significance from control (none). ** P < 0.001 level of significance from control (none).

Table III also shows the influence of cycloheximide (10m5M) on the NL/PL ratio and the activities of sn-glycerol-3-phosphate acyltransferase and glycerol kinase measured in homogenates of control and ethanol-exposed monolayers. Cycloheximide was added to the monolayer incubation medium 2 h before ethanol and then the appropriate monolayers were incubated for an additional 6 or 24 h. The results indicate that cycloheximide did not prevent the rise in glycerol kinase activity of monolayers exposed to ethanol for 6 h. However, cycloheximide reduced the decline in glycerol kinase activity and the rise in the NL/PL ratio of monolayers exposed to 10 mM ethanol for 24 h. The pyrazole experiments suggest that ethanol metabolites alter the incorporatio of [ 1,3-14C]glycerol into glycerolipids and their release into the medium by hepatocyte monolayers. Therefore, we studied the effects of acetate (l20 mM) and acetaldehyde (l-10 mM) exposure (24 h) on monolayer glycerolipid formation and release, Acetate had no effect on these processes but acetaldehyde (Table IV) reduced glycerolipid release (l-10 mM) and formation (10 mM). These acetaldehyde effects’ on monolayer triacylglycerol and phospholipid biosynthesis and release are similar to those observed in monolayers exposed to ethanol (Table III). However, the level of acetaldehyde which produces these effects is greater than that which accumulates intracellul~ly during monolayer ethanol metabolism. This discrepancy is attributed to a rapid

128 TABLE

III

THE INFLUENCE OF PYRAZOLE AND CYCLOHEXIMIDE ON THE ETHANOL-INDUCED ALTERATIONS IN THE ACTIVITY OF GLYCEROL KINASE, sn-GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE AND THE ALTERATIONS IN THE NL/PL RATIO Cycloheximide was added to the culture medium to produce the indicated concentration, 2 h after freshly isolated hepatocytes had been plated. Ethanol was added to produce the indicated medium concentration 2 h following the addition of cycloheximide. Pyrazole was added immediately prior to the addition of ethanol. 6 or 24 h following the addition of ethanol, the monolayers were homogenized and the activity of glycerol kinase (GK), sn-glycerol-3.phosphate acyltransferase (GPAT) and the NL/PL ratio was determined. Each value represents the mean % control f S.E. from at least 5 experiments in which enzyme activity and the NL/PL ratio was measured in triplicate from monolayer homogenates prepared from 2-3 plates. Control values for glycerol kinase and sn-glycerol-3-phosphate acyltransferase after 6 and 24 h exposure periods were 3.28, 0.529 and 2.01, 0.237 nmol incorporated/mg protein per min, respectively. Control values for NL/PL ratio after 6 and 24 h exposure periods were 0.246 and 0.110, respectively. Additions

GK

GPAT

NL/PL

(mM) 6 hours None Ethanol (10) Pyrazole (1) Cycloheximide Ethanol (10) Ethanol (10) 24 hours None Ethanol (10) Pyrazole (1) Cycloheximide Ethanol (10) Ethanol (10)

(0.01) + pyrazole (1) + cycloheximide

(0.01) + pyrazole (1) + cycloheximide

(0.01)

lOO? 178? 101 + 107 ? 110* 166 ?

8 3 * 8 8 5 4 *

lOO?ll 96f 9 94 f 14 115t 8 91 r 11 108? 5

1OOf 9 102 r 7 95 f 12 93+ 4 1052 6 106 t 8

6

(0.01)

100 t 14*3* 95 ? 100f 90 + 75 f

100 * 10 78? 6 91? 15 96? 4 82 r 10 78 t 12

loo? 9 259r 7 87 r 10 109f 3 84f 12 178f 2 **

6 2 7 7 **

* P Q 0.001 level of significance from control (none). ** P Q 0.01 level of significance from ethanol (10 mM).

TABLE

IV

EFFECT OF ACETALDEHYDE CYTE MONOLAYERS

ON GLYCEROLIPID

FORMATION

AND

RELEASE

BY

HEPATO-

2 h after freshly isolated hepatocytes had been plated, acetaldehyde was added to produce the indicated medium concentration. 24 h later the formation and release of glycerol&ids from [1.3-14C21glycerol was determined. Each point represents the mean % control ? S.E. for values obtained in triplicate from 3 separate experiments. Control values for triacylglycerol and phospholipid formation and release were 0.189. 0.068,0.0068, and 0.0071 moles glycerol incorporated/mg protein per min. respectively. Concn.

Cell

Media

(mM)

0.0 1.0 2.5 5.0 10.0 * P < 0.001

Trlacylglycerol

Phospholipid

Triacylglycerol

Phospholipid

100 ? 86 + 101+ 98 f 19*4*

100 f 10 97t 4 110 2 7 94 + 10 22*5*

100 i: 8 90 + 6 31 t: 8 18+2* 8+1*

100 + 6 94 f 5 25f:l* 28f2* 20?:2*

7 7 4 4

level of significance

from control

0.0.

129

TABLE THE

V

EFFECT

OF INCUBATION

TION OF ETHANOL 2 h after

freshly

TIME.

PYRAZOLE

IN THE CULTURE

isolated

hepatocytes

MEDIUM

AND CYCLOHEXIMIDE OF HEPATOCYTE

had been plated,

cycloheximide

ON THE CONCENTRA-

MONOLAYERS was added to-produce

the indicated

medium concentration. 2 h after the addition of cycloheximide, ethanol was added to produce the indicated medium concentration. In some experiments pyrazole was added to produce the indicated medium concentration, immediately prior to the addition of ethanol. 0, 6, 12, and 24 h following the addition of ethanol, the concentration of ethanol in the culture medium was determined. Each point represents the mean ethanol concentration (mM) + S.E. of determinations made in duplicate from 3 plates in 3 separate experiments. Hours after ethanol

Additions

addition

(mM) 0 Ethanol (10) Ethanol (10) Ethanol (10) Etahnol(10); * P < 0.01 (1 mM). ** P G 0.001 (1 mM).

+ cycloheximide + pyrarole (1) no cells

(0.01)

9.97 10.01 10.00 9.98

c 0.02 k 0.01 ?r 0.01 + 0.01

5.98 6.23 7.29 7.67

24

12

6 i + + +

0.18 0.21 0.11 0.16

* *

3.82? 0.17 3.92 t 0.09 5.11 t 0.12 5.43 i 0.12

** **

1.78 1.85 2.81 3.04

f + + ?

0.03 0.10 0.09 0.10

** ** **

level of significance

from control

(ethanol

(10 mM); no cells) or ethanol

(10 mM) + pyrazole

level of significance

from control

(ethanol

(10 mM): no cells) or ethanol

(10 mM) + pyrazole

evaporation of acetaldehyde under our experimental conditions. Table V shows the influence of incubation time on ethanol concentrations in the culture medium of hepatocyte monolayers. The rate of ethanol evaporation was determined with plates which contained no hepatocytes. Ethanol levels in the medium were inversely proportional to the incubation period, indicating that ethanol evaporation occurred throughout the 24 h exposure period. The concentration of ethanol in the medium of monolayers exposed to ethanol or ethanol + cycloheximide for various time periods was similar, but significantly lower, than that of plates which contained no hepatocytes. Ethanol metabolism by hepatocyte monolayers during the initial 12 h incubation period was estimated to be 3.6 f 0.2 mol/g wet weight cells per min. This rate of ethanol oxidation agrees well with values reported in studies with intact rat [31--331 and isolated rat hepatocytes [34-361. Table IV also illustrates the effect of pyrazole on the medium ethanol concentration of monolayers incubated for various time periods. Monolayers simultaneously exposed to ethanol and pyrazole eliminated ethanol at rates which were not significantly different than those exhibited by control plates (no cells). These results suggest that pyrazole but not cycloheximide reduces monolayer ethanol oxidation. Discussion Ethanol intake in man [l--5] and experimental animals [ 1,5-81 causes a rise in hepatic glycerolipid content which is associated with an increase in hepatic triacylglycerol and phospholipid biosynthesis [ 1,6-261. Obviously, an ethanol dependent rise in glycerolipid substrates would contribute to an increase in glycerolipid formation provided their normal intracellular content was below that required for maximum enzyme activity. Alternatively, ethanol may increase the formation of glycerolipids by elevating the liver’s enzymatic capa-

130

city of glycerolipid biosynthesis. This latter concept is supported by recent studies in vitro which demonstrate that ethanol induces a rise in hepatic phosphatidate phosphohydrolase [21-241, sn-glycerol-3-phosphate acyltransferase 1251 and glycerol kinase [24,26] activities. The mechanism(s) by which ethanol alters glycerolipid formation and the activity of these liver enzymes is unclear. Presumably, hepatocyte monolayers can be used to resolve these questions since this preparation exhibits many morphological [37] and functional [ 38-401 characteristics of normal rat liver for prolonged periods including the ability to synthesize and secrete glycerolipids [27]. Our studies indicate that hepatocyte monolayers exposed to ethanol for 6 h exhibit a rise in glycerolipid formation. These findings agree well with previous studies in vitro utilizing isolated hepatocytes [ 171 and liver slices [ 15,16,20]. In contrast, monolayers exposed to ethanol concentrations greater than 1 mM for 24 h showed a marked depression in triacylglycerol and phospholipid biosynthesis. This latter observation is not the result of an ethanol dependent rise in hepatocyte glycerol 3-phosphate content and may be a response to ethanol that alters cellular processes [ 44,451. This interesting possibility is supported by the knowledge that glycerolipids play an integral role in the structure and function of hepatocyte membranes. Ethanol-induced alterations in monolayer glycerolipid formation were accompanied by changes in the activity of glycerol kinase and phosphatidate phosphohydrolase. Glycerol kinase activity correlated well with ethanoldependent changes in monolayer glycerolipid formation from either labeled glycerol or palmitate. In contrast, phosphatidate phosphohydrolase activity did not correlate well with the incorporation of [1,3-14Cz]glycero1 into monolayer glycerolipids. These observations conflict with other studies in which ethanolinduced alterations in phosphatidate phosphohydrolase activity measured in vitro, correlated well with the rate of glycerolipid formation measured in vivo [21,22,24]. This discrepancy is difficult to resolve and may indicate that hepatocyte monolayers represent a unique situation. Nevertheless, recent evidence suggests that glycerol may provide a major source of hepatic glycerideglycerol [27]. If this is true, then glycerol uptake or glycerol kinase may participate in the regulation of hepatic glycerolipid formation. The oxidation of ethanol by hepatocyte monolayers was inhibited by pyrazole but not cycloheximide. However, both agents reduced the ethanol-induced changes in glycerol kinase and phosphatidate phosphohydrolase (NL/PL) activity. These results suggest that ethanol related changes in monolayer glycerolipid formation involve protein synthesis and ethanol oxidation. The latter hypothesis is supported by the observation that monolayers exposed to acetaldehyde (l-10 mM, Table IV) exhibited alterations in glycerolipid biosynthesis and release which resembled those induced by ethanol (Fig. 1, Table II). Ethanol-induced increases in serum glycerolipid content [l-8,21,41,42] are usually attributed to a rise in hepatic glycerolipid formation [1,6--8,10,12,21, 231 and secretion or a reduction in serum lipoprotein clearance [1,3,5,43]. However, monolayers exposed to ethanol for 6 or 24 h exhibited a marked decrease in glycerolipid release. The reason for this discrepancy is not clear but may be related to the observation that glycerolipids released by monolayers are not representative of lipoproteins secreted by the liver [27].

131

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The effect of ethanol on glycerolipid biosynthesis by primary monolayer cultures of adult rat hepatocytes.

121 Biochimica et Biophysics Acta, 572 (1979) @ Elsevier~No~h-Holland Biomedical Press 121-131 BBA 57290 THE EFFECT OF ETHANOL ON GLYCEROLIPID BIO...
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