Vol. 15, No. 2 March/April 1991
0145-6008/91/1502-0184$3.00/0 ALCOHOLISM: CLINICAL AND EXPERIMENTAL
RESEARCH
The Metabolism of Ethyl Esters of Fatty Acids in Adipose Tissue of Rats Chronically Exposed to Ethanol Giovanni DePergola, Christer Kjellstrom, Cecilia Holm, Nils Conradi, Per Pettersson, and Per Bjorntorp
been demonstrated in several tissues, including adipose tissue The presence of EEFA in adipose tissue is of potential interest for at least two reasons. First, adipose tissue is a tissue that lends itself to repeated sampling. Second, adipose tissue has the highest content of free fatty acids compared with any other tissue. This would provide high substrate levels for ethyl fatty acid ester synthase to esterify fatty acids to ethanol. Third, hydrophobic compounds formed in, or taken up by adipose tissue will presumably be stored in the large mass of adipocyte triacylglycerols. These esters have a long half-life,' and any compound with a similar pathway for removal would presumably have an equally prolonged retention time in adipocytes. Theoretically, this would be the case with EEFA in adipose tissue, which therefore were thought to be a potentially useful marker for ethanol consumption, comparably easy to follow by adipose tissue biopsies. With this background we decided to study the potential usefulness of EEFA and the synthase activity in adipose tissue after fully controlled exposure to ethanol during HE QUANTITATIVELY most important pathway different periods of time in the rat, as well as after different for disposal of circulating ethanol is through oxidation periods of abstinence from ethanol. Furthermore, in order via alcohol dehydrogenase. This enzyme is, however, pres- to study the half-life of EEFA, labeled ethanol was given and labeled EEFA concentrations followed. Finally, the 'ent only in a few tissues, notably the liver. Other tissues relative hydrolysis rate of EEFA and triacylglycerol by the have a limited capacity to metabolize ethanol. One rehormone-sensitive lipase of adipose tissue was determined cently discovered pathway is through the esterification of to elucidate the major rate-limiting step for intracellular ethanol to fatty acid with formation of ethyl esters. This lipid hydrolysis in adipocytes. The results show retention enzyme activity, fatty acid ethyl ester synthase, as well as of EEFA in adipose tissue and that the synthase activity is retention of the product, the ethyl ester of fatty acids increased by long-term ethanol exposure. A preliminary (EEFA), have been found in most tissues and have recently report has been presented.' been studied in some detail in heart, brain, and white blood cells.'-' In a limited number of post-mortem samMATERIAL AND METHODS ples in humans both EEFA and the enzyme have also The concentration of ethyl esters of fatty acids as well as the activity of the enzyme synthesizing these esters (fatty acid ethyl ester synthase) were determined in adipose tissue of rats ingesting ethanol (9-16 g/kg body weight/day) for different periods of time. After 10 and 17 weeks of ethanol exposure about 300 nmol of ethyl esters of oleic, palmitic, stearic, and linoleic acids were found per gram adipose tissue. The ethyl esters disappeared after 1 week of abstinence. Closer analyses, using radioactive ethanol, revealed a half-life of the esters of less than 24 hr. The bulk of the esters was found in a membrane preparation of isolated adipocytes. Hormonesensitive lipase hydrolyzed emulsified ethyl oleate as efficiently as that of trioleoylglycerol, but in mixed ethyl oleate/trioleoyl glycerol particles the hydrolysis of ethyl oleate was slower, suggesting a decreased accessibility. Synthase activity was found in adipose tissue from rats not exposed to ethanol. It doubled after 10 and 17 weeks of ethanol and decreased with a half-life of at least a week after abstinence. It was concluded that ethyl esters of fatty acids are formed in rat adipose tissue as previously shown in other tissues. They seem to be stored mainly in membranous parts of the adipocytes. Synthase activity is induced by ethanol. The elevated activity has a longer half-life, and may be useful as an indicator of alcohol abuse. Key Words: Ethanol, Ethyl Esters, Fatty Acids, Synthase, Adipose Tissue.
3 9 5
T
Animals From the Clinica Medica HI, University of Bari (G.D.); Department of Medicine I and the Wallenberg Laboratory (P.P., P.B.): Department of Pathology I, (C.K., N.C.), University of Goteborg: and Department of Medical and Physiological Chemistry (C.H.), University of Lund, Sweden. Received for publication February 26, 1990: accepted September 18, 1990 Financial support was given by The Bank of Sweden Tercentary Foundation, the Swedish Medical Research Council (Grant No. 3363 to P. Beljrage, and 07121 to C.K. and N.C.). Reprint requests: Professor Per Bjorntorp, Department of Medicine I, Sahlgren 3 Hospital, University of Goteborg, 413 45 Goteborg, Sweden. Copyright 0 1991 by The Research Society on Alcoholism. 104
Male rats of a Sprague-Dawleystrain were used. A total of 1g pairs of two littermates each. They were given a normal commercial laboratory diet (5% fat, 55% carbohydrate, 22.5% protein, vitamins and minerals, EWOS, Sodertiilje, Sweden), and tap water ad libitum until they were 35 days old, at which time they were housed individually in plastic cages in a room at 22°C temperature, 60% humidity, and a 12/12 hr light/dark cycle. Animals were given an ethanol-containing liquid dietlo in drinking tubes as the only source of food and water. They were pairfed: one rat was given an ethanol formula (AE), the other, the control (C), a formula in which the ethanol had been isocaloncally replaced with dextrinmaltose. The introduction of ethanol was gradual and on the fourth day the final concentration of 50 g ethanol/liter formula was achieved, sustaining a daily ethanol intake of 9 to 16 g/kg body weight. Changes Alcohol Clin Exp Res, Vol 15, No 2, 1991: pp 184-189
185
FATTY ACID ETHYL ESTERS IN ADIPOSE TISSUE
in body weight during exposition are presented in Fig. 1. The rat given alcohol received its diet ad libitum, the corresponding littermate was fed the same isocaloric amount of the control diet the following day. For eight of the AE rats, the ethanol-exposure lasted for 10 weeks after which the animals were either killed directly, without prior ethanol withdrawal ( n = 4), or were given a diet with a reduced ethanol content for 3 days and then an ethanol-free diet for I week, ( n = 4), before they were killed. Four AE rats were exposed for I I weeks and then given a diet with a reduced ethanol content for 3 days, and an ethanol-free diet for 4 weeks before they were killed. The last group with six AE rats was exposed for 17 weeks and killed without ethanol withdrawal.
Determination of EEFA Concentration This was performed in principle as described previously for rabbit myocardium.' One g adipose tissue was homogenized in a glass homogenizer in 1 ml acetone and centrifuged (1000 X g, 10 min) at room temperature. One hundred microliters of the acetone phase were used for thin layer chromatography (TLC). This was performed on glass plates (20 X 20 cm), on which a 0.5-mm layer of silicic acid was placed, dried, and activated at 80°C for 3 hr. The liquid phase was petroleum ether:diethyl ether:acetic acid (755: I ) . Before use, the plates were cleaned by development in the same solvent mixture. The sample was applied as a 5-cm long band, surrounded by lipid standards including routinely 40.0 pg ethyl oleate. The standard spots were visualized by iodine with the sample covered by thin plastic film. The following Rf values were obtained: phospholipids, 0-0.1, fatty acids, cholesterol, mono- and diacylglycerols, 0.1 to 0.15; triacylglycerols, 0.15 to 0.25; EEFA, 0.45 to 0.55; cholesterol esters, 0.60 to 0.70. Recovery of added ethyl oleate was examined by addition of ethyl oleate to varying amounts of extracted adipose tissue triglycerides by extraction of the ethyl oleate region on the plate with acetone and quantitation of ester bonds." It was ascertained that cholesterol esters did not interfere with this reaction by separate analyses of cholesterol. Recovery of ethyl oleate exceeded 95% up to 200 mg of added triacylglycerides. With higher concentrations, trailing of triacylglycerols was visible on the TLC, and recovery of ethyl oleate was not complete. After this test 100 mg of adipose tissue was routinely used for analyses. With this amount of tissue the ethyl ester of margaric acid (C17) was used as internal standard for the whole procedure. The recovery of this acid was about 70%, and losses corrected for in the calculations. The amount of ester bonds in the EEFA region of the TLC was proportional to extracted adipose tissue up to amounts of 200 mg. In order to determine the biological half life of EEFA 200 to 300 g male rats were given 3 ml 90% ethanol with 50 pCi of (1-14C) ethanol orally. Radioactivity was determined after 4 hr in different lipids of adipose tissue extracts on TLC. As seen in Table 1, label was found in
several lipid fractions including EEFA, but not in cholesterol esters. Total radioactivity in half of the lipid extract subjected to TLC was counted directly. The other half was saponified in 2 ml ethanol and 0.5 ml of 5 N KOH at 37°C for 1 hr. This was then evaporated to dryness under nitrogen, redissolved in 1 ml ethanol, and radioactivity again counted.' The difference in counts was then compared with that found in the EEFA spot of the TLC and found to be 90% ( n = 4). Therefore, for the determination of the half-life of EEFA, the radioactivity lost after saponification and evaporation was taken as EEFA radioactivity. The area on TLC corresponding to the EEFA standards, from the top of the triacylglycerol spot to about Rf value 0.7, was scraped off and eluted with acetone for gas liquid chromatography (GLC) on a PerkinElmer, Sigma 300, instrument, with a capillary column (SP-1000) operated at 80°C to 220°C. Peaks were detected and quantitated by a flame ionization detector and a computerized integrator. Retention times for the ethyl esters of palmitic (C16), margaric (C17), stearic (CIS), oleic ((218: I), and linoleic (18:2) acids were determined by injection of standards and were found to be 12.24, 14.33, 16.63, 17.16, and 18.13 min, respectively, reproducible within less than +0.03 min. Enzyme Assay This was camed out in principle as described for rat myocardium' modified for the purpose of determination in adipose tissue. One hundred milligrams of adipose tissue were homogenized. The tubes were centrifuged (1000 X g, 10 min) at room temperature, and the infranatant (between the pellet and the fat cake) was collected. From this phase, 100 pl were taken for protein determination and 100 pl were added to the enzyme assay tubes. The total volume of the reaction was 170 p1, with the following final concentrations: 1 mM ethanol, 0.9 mM non-labeled oleic acid, 0.4 pCi I-14C-oleic acid, 68 mM phosphate buffer, pH 7.4. Every sample was run in duplicate, and followed by a blank containing 100 pl boiled cytosol in phosphate buffer. Incubations were camed out in closed glass tubes at 37"C, for 1 hr. The reaction was stopped by adding 2 ml cold acetone, containing ethyl(lH)oleate as internal yield standard ( 5 X lo5 cpm). After evaporation to dryness under nitrogen, 300 pl acetone were added and applied on TLC as described above. The EEFA spot was identified by standards, scraped off, eluted in acetone, and counted in an LKB (Wallac, Rackbeta) Liquid Scintillator Counter. Losses were corrected for by quench correction and by use of the internal standard. Results were expressed as nmoles per hour and mg protein. Protein was determined according to Lowry et a1." The assay conditions for the enzyme activity were tested to determine non-limiting substrate concentrations of both ethanol and oleic acid. Ethanol concentrations up to 1 mM increased the formation of EEFA while at 2 mM no further increase was seen (Fig. 2). Activity in the presence of 1 mM ethanol increased with increasing concentrations of
weight (g) 500
1
400
Fig. 1. Body weight of rats exposed to alcohol (AE) and controls (C) during a 17-week period. (n = 6).
300
200
100
! 5
I
i
15
25 age (weeks)
DEPERGOLA ET AL.
186 Table 1. Distribution of Label in Rat Epididymal Adipose Tissue Lipids after Administration of ''C-Ethanol Radioactivity (% of total) (n = 4)
Fraction
a,
a
.-cn
v)
c.
*
27 3 32 f 1 41 k 2 0
Phospholipids Cholesterol, free fatty acids and acylglycerols EEFA Cholesterol esters Mean k SEM
A
3
0 P .-U Q
w
h
0
E
rn
Y
n
1
40 -I
0
L
f Q
20 40
60
80100120140
minutes
a
.-+cn. v)
Fig. 4. The dependence of synthesis of ethyl oleate on time. Ethanol concentration, 1 mM, oleic acid concentration, 0 9 mM Other conditions as in Material and Methods section One representative experiments out of two performed
Q v)
0
.
***
0
Y
10
!
,
0
.
I
1
.
,
2
1
.
3
4
Ethanol (mM) Fig. 2. The dependence of synthesis of ethyl oleate on ethanol concentration Oleic acid concentration 0 9 mM Other conditions as in Materlal and Methods section One representative experiment out of three performed
50
1
40: 30
/--
Control
10
010
low
17W
Fig. 5. Ethyl ester synthase activity in rats exposed to ethanol for 10 or 17 weeks and in controls. Mean k SEM. " ' p < 0.001.
.
I
I
1
1
2
3
Oleic acid (mM) Fig. 3. The dependence of synthesis of ethyl oleate on oleic acid concentration. Ethanol concentration 1 mM. Other conditions as in Material and Methods section. Four individual experiments.
oleic acid up to about 1 mM (Fig. 3). Guided by these experiments, concentrations of I mM ethanol and 0.9 mM oleic acid were selected for further assays. Under these conditions activity was linear with time up to 60 min and then levelled off (Fig. 4). With an assay time of 60 min activity was proportional to the amount of added adipose tissue up to about 200 mg of adipose tissue (not shown). Amounts of about 100 mg tissue were used. Isolation of adipocytes with collagenase and preparation of fat cell ghosts were performed as previously described."
Lipid Ester Hydrolysis with Hormone-Sensitive Lipase Lipids used for preparing substrates were synthesized by Dr. Lennart Krabisch, Department of Medical and Physiological Chemistry, University of Lund. Briefly, tri(3H)oleoylglyceroI was synthesized and purified as describedI4 to at least 99% purity. Ethyl esters of ('H)oleic acid (Amersham, England) or unlabeled oleic acid were synthesized by adding
oleic acid chloride to ethanol and letting it react overnight at room temperature with constant ~tirring.'~ The mixture was then extracted twice with petroleum ether and the petroleum ether phases washed with 0.01 M NaOH, followed by water. The purity of the ethyl oleate was 98.4% by TLC. Alternatively, to obtain an ethyl ester of (3H)oleic acid with a very high specific activity, acetyl chloride was added directly to the (3H)oleic acid, which is obtained in ethanol from the manufacturer (Amersham, England). The mixture was allowed to react for 3 hr at room temperature and was then extracted as described above. The purity of this ethyl oleate was 98.4% by TLC. Substrates of trioleoylglycerol,ethyl oleate, and mixtures of these were obtained by sonication in 0.1 M potassium phosphate, pH 7.0. Pure ethyl oleate substrates were made 5 mM, whereas pure trioleoylglycerol substrates and substrates containing a mixture of ethyl oleate and trioleoylglycerol were made 1.5 mM. For stabilization of the emulsions, phospholipids (phosphatidylcholine and inositol 3:l [w/w]) were included at a concentration of 0.15 mg/ml.14 After sonication, defatted bovine serum albumin was added to a final concentration of 0.2 mg/ml. Hormonesensitive lipase was purified from rat epididymal adipose tissue to approximately 5% protein purity.'6 Hormone-sensitive lipase activity was measured, at non-inhibitory detergent concentrations, at 37°C over 30 min. Labeled free oleic acid was isolated with a simple one-step liquidliquid partition system."
FATTY ACID ETHYL ESTERS IN ADIPOSE TISSUE
187
Chemicals
1-14Cokic acid (S.A. 58 mCi/mmol)and I-14C ethanol (54.1mCi/ mmol) were from Amersham International (Buckinghamshire, England) and 9,10-3H (H)oleic acid (S.A. 10 Ci/mmol) from New England Nuclear (Boston, MA). All the lipid standards purchased were of highest commercially available quality. Ethyl ester of ('H)oleic acid used as TLC standard was synthesized in ethanol with gas from concentrated hydrochloric acid for 20 min.18 Ethanol was then evaporated under nitrogen, lipids redissolved in acetone and ethyl oleate purified by repeated TLC to less than 5% contamination with free oIeic acid.
are seen in Table 3. The rates of hydrolysis with the two substrates were comparable, and hydrolysis of trioleoylglycerol did not Seem to be influenced by the presence of 1% or 20% ethyl oleate. H ~when (3Hlethyl ~ ~oleate and ~ trioleoylglycerol was mixed in the same emulsion particles the Of Oleate seemed to decrease than accounted for by the dilution of the labeled substrate (third part of Table 3).
Contents of EEFA in Membranes and Triacylglycerols of RESULTS
A dipocytes
EEFA Concentrations As seen in Table 2, no EEFA was found in the control rats, which were not given ethanol. In rats exposed to ethanol for 10 and 17 weeks EEFA were found in all rats. EEFA of oleic, palmitic, stearic, and linoleic acids were identifiable. The proportion between these esters were 5 I k 5,24 k 3, 16 f 4, and 9 & 3% (Means + SEM, n = 10) of total, respectively, in all rats exposed to ethanol until examination. The absolute values of the sum of these concentrations in 100 mg adipose tissue is used for further presentation of results. No further increase of EEFA concentration after 17 weeks of ethanol was seen in comparison with 10 weeks. One week after exposure to ethanol had ended, no EEFA were found, and this was essentially true also for rats free from ethanol during 4 weeks after 1 I weeks of exposure. EEFA Half Life The results of measurements of EEFA concentrations suggested that the half-life of EEFA esters in rat adipose tissue is shorter than 1 week. T o analyse this more accurately, labeled ethanol was used as described in the Methods section. Rats were given radioactive ethanol, and the values in adipose tissue of evaporated ethanol radioactivity 4 hr after administration were taken as comparison for similar measurements after another 16 hr which was found to be 34% to 40% of original ( n = 3). After 1 week no radioactivity remained ( n = 2). Hydrolysis of Ethyl Oleate and Trioleoylglycerol by Isolated Hormone-Sensitive Lipase The results of hydrolysis experiments by hormone-sensitive lipase of emulsified ethyl oleate or trioleoylglycerol Table 2. EEFA in Adipose Tissue of Rats Exposed to Ethanol Length of ethanol-exposure withdrawal (weeks)
+
0 (n = 4) 10 (n = 4) 10+1(n=4) 11 + 4 ( n = 4 ) 17 (n = 6) Mean c SEM
(nmol/s)
0 310 f 30 0 10k1 340 f 38
The bulk of triacylglycerol was separated from the membranous parts of a d i p ~ c y t e s and ' ~ EEFA concentration determined in these two fractions. In two experiments the membrane fraction contained 80% and 84% of total EEFA.
Enzyme Activity for EEFA Synthesis Fig. 6 shows the EEFA synthase activity in rats exposed to ethanol for different periods of time. Control rats showed a rather high activity. This increased to almost double after 10 weeks of alcohol exposure. After 17 weeks of ethanol no further increase seemed to have occurred compared with 10 weeks. After 1 week of abstinence, the activity was still elevated to more than 50% above that found in controls, but after 1 1 weeks of ethanol exposure and 4 weeks abstinence the activity was back to that of control rats. DISCUSSION
This study shows that EEFA are formed in adipose tissue of ethanol exposed rats. This has previously been shown in post-mortem samples of human adipose t i ~ s u e . ~ Table 3. Ethyl Oleate Hydrolysis by Hormone-SensitivesLipase Experiment 2 (nmol 3H-fatty acids/min/ml)
Experiment 1 (nmol 3H-fatty acids/min/ml) Tri(3H)oleoylglyceroI Eth~l(~H)oleate Trioleoylglycerol hydrolase activity (nmol 3Hfatty acids/min/ml) 1YO oleate 99% tri(3H)oleoylglyceroI 20% ethyl oleate 80% tri(3H)oleoylglyceroI
+
+
563 541
435 537
539
Trioleoylglycerol hydrolase activity (nmol 3H-fatty acid/min/ml) 515
507
366
Pure ethyl oleate, pure trioleoylglycerol, and different mixtures of these were used as substrates for hormone-sensitive lipase as described in "Materials and Methods." The mixed substrates are given as molar percent of each component. Results are mean of five determinations. Ethyl oleate hydrolase activity (nmol 3H-fatty acids/ml) (numbers within parentheses are the theoretical values for dilution of ethyl(3H)oleatewith unlabeled ethyl oleate). 100% eth~l(~H)oleate 608 20% eth~l(~H)oleate 80% trioleoylglycerol 99 (122) 5% eth~l(~H)oleate 95% trioleoylglycerol 22 (30) 1% eth~l(~H)oleate + 99% trioleoylglyceroi 4.3 (6.1) 0.25% eth~l(~H)oleate99.75% trioleoylglycerol 0.94 (1.5) 0.1 YOethyl(3H)~leate 99.9% trioleoylglycerol 0.26 (0.6)
+ +
+ +
~
DEPERGOLA ET AL.
***
EEFA in adipose tissue was actually less than 24 hr. This is in agreement with recent findings in the rabbit.3 Such a ** -r short half-life of EEFA suggests that they are not turning over concomitantly with the adipocyte triacylglycerol, the -r half-life of which is much longer in the rat. Instead, they might form a specific pool in adipocytes. Recently accumulated triacylglycerols seem to form such a pool with a short half-life, remaining only for a limited period of time.21The radioactive triacylglycerolformed after administration of 14C-ethanol actually had a much longer halflife than that of the EEFA in the experiments performed here. Although this was not studied in detail, it was probably of the order of at least a week. This observation indicates that the EEFA have indeed a shorter half-life than the bulk of triacylglycerols in the adipocyte. 10 - l l w 10 - l l w Control 10-11 w Adipocyte triacylglycerols are hydrolyzed by the hor1W (abst 1 4W labst) mone-sensitive lipase to provide the FFA that are mobiFig. 6. Ethyl ester synthase activity in rats exposed to ethanol during 10 to 11 lized from the cell. This lipase was shown to hydrolyze weeks and then given no ethanol for 1 and 4 weeks and controls. Mean f SEM. " ' p < 0.001, * * p< 0.01, comparisons with controls. trioleoylglycerol equally well in the absence or presence of ethyl oleate. However, in mixed emulsions ethyl oleate The synthesis was linear with time with concentrations of seemed to be hydrolysed less efficiently, particularly when fatty acids found physiologically in adipose tissue," and present in the concentrations that could maximally be expected in vivo in the triacylglycerol droplets of adipose of ethanol found in blood during social drinking. tissue. This might mean that ethyl oleate is comparably The rats exposed to controlled ethanol consumption less available for hydrolysis in such mixed emulsion parwere also shown to form EEFA, which were found in their ticles and therefore would be expected to have a longer adipose tissue. Esters were found of oleic, palmitic, stearic, half-life than triacylglycerols under such conditions. Other and linoleic acids, the main fatty acids present in rat mechanisms must, hence, explain the shorter half-life of adipose tissue, and in the approximate proportion to the EEFA than triacylglycerols in the adipocyte. concentrations of these fatty acids.20Total EEFA concenOne remaining possibility is that EEFA are retained in tration in adipose tissue was not higher after 17 weeks of a pool outside the lipid droplet such as in a membranous ethanol ingestion than after 10 weeks, suggesting that a compartment of the cell. Findings of synthase activity in limit of EEFA retention in adipose tissue had been reached the brain4 indicate that much of it is located in plasma as at 10 weeks or before. The enzyme activity determined well as mitochondria1 membranes. It has been suggested simultaneously demonstrated an unimpaired capacity for that EEFA may negatively affect mitochondria and that formation of new EEFA at 17 in comparison with 10 this mechanism can explain some aspects of alcoholic weeks. This suggests a limited turn-over time. When altissue damage.2 Experiments separating a membranous cohol ingestion had stopped, essentially all EEFA disapfraction from adipocyte lipids showed that accumulation peared from adipose tissue within 1 week. of EEFA in adipocyte membranes seems indeed to occur. A closer study of the half-life of EEFA in rat adipose Ethanol is also incorporated into another lipid compotissue utilized labeled ethanol to obtain labeled EEFA. nent of cellular membranes, viz. phosphatidyl ethanol. However, other adipose tissue lipids were labeled as well, necessitating separation of adipose tissue lipids by TLC. The half-life of this compound seems, however, to be of Because of the relatively small synthesis of labeled EEFA the order of 3 to 4 hr.22The half-life of the EEFA is thus in relation to the bulk of triacylglycerol separated on TLC, longer by a factor of 4 to 5. It is notable that rats never exposed to ethanol have it was necessary to administer larger amounts of radioacquite a considerable activity of the EEFA synthase in tivity to determine EEFA concentration by this procedure. adipose tissue. With ethanol ingestion the enzyme activity Instead an indirect procedure was therefore developed, was clearly increasing, and this induction seemed to have where volatile radioactive constituents of saponified lipids reached maximal values at 10 weeks or earlier. When could be quantitated after removal by evaporation. Apt alcohol exposure had ended the activity was again decreasparently, only ethanol is removed by this procedure, because this method turned out to agree with the direct ing back to that of controls. The rate of this decrease was determination of radioactive EEFA after chromatographic fairly slow, with half-maximal values of activity after about separation. This method might therefore be useful in 1 week. This study thus has shown that EEFA are formed in studies in humans where exposure to radioactivity has to adipose tissue of rats consuming ethanol. Their half-life is be limited. Utilizing this method it was found that the half-life of longer than that of ethanol in the circulation, and they are
i
FATTY ACID ETHYL ESTERS IN ADIPOSE TISSUE
retained in the adipocyte with a half-life of less than 24 hr probably in a membranous compartment of the cell. It may well be that the storage is different after a longer period of ethanol exposure. The induction of elevated activity of the EEFA synthase seems to persist longer and might well be a potentially useful tool for diagnosis of alcohol abuse. In contrast to alcohol dehydrogenase in the liver, another enzyme induced by alcohol, the EEFA synthase activity of adipose tissue is readily measurable in samples, obtainable by atraumatic needle biopsies. ACKNOWLEDGMENTS Margareta Medberg is acknowledged for expert GLC analyses. We thank Per Belfrage for fruitful discussions and constructive criticism of this manuscript.
REFERENCES 1. Lange LG, Bergmann SR, Sobel BE: Identification of fatty acid ethyl esters as products of rabbit myocardial ethanol metabolism. J Biol Chem 256:12968-12973, 1981 2. Lange LG, Sobel BE: Mitochondria1 dysfunction induced by fatty acid ethyl esters, myocardial metabolites of ethanol. J Clin Invest 72:2428, 1983 3. Laposata EA, Sherrer DE, Lange LG: Fatty acid ethyl esters in adipose tissue. Arch Pathol Lab Med 113:762-766, 1989 4. Laposata EA, Sherrer DE, Magow C, Lange LG: Metabolism of ethanol by human brain to fatty acid ethyl esters. J Biol Chem 262:46534657, 1987 5. Laposata EA, Lange LG: Presence of nonoxidative ethanol metabolism in human organs commonly damaged by ethanol abuse. Science 23 11497-499, 1986 6. Mogelson S, Lange LG: Nonoxidative ethanol metabolism in rabbit myocardium: Purification to homogeneity of fatty acyl ester synthase. Biochemistry 23:4075-408 1 , 1984 7. Wright M, Bieser KJ, Kinnunen PM, Lange LG: Nonoxidative ethanol metabolism in human leucocytes. Detection of fatty acid ethyl
189
ester synthase activity. Biochem Biophys Res Commun 142:979-985, 1987 8. M i n n P, Rebuffk-Scrive M, Smith U, Bjorntorp P: Glucose uptake in human adipose tissue. Metabolism 36: 1154-1 160, 1987 9. DePergola G, Kjellstrom C, Conradi N, Bjorntorp P: Chronic ethanol exposure and ethyl esters of fatty acids in adipose tissue of rats. 5th Eur Lipoprotein Symposium, Padua, 1989 (abstr) 10. Lieber CS, DeCarli LM: The feeding of alcohol in liquid diets: Two decades of applications and 1982 update. Alcohol: Clin Exp Res 6~523-531, 1982 1 1. Rapport MR, Alonzo N Photometric determination of fatty acid ester groups in phospholipids. J Biol Chem 193:265-275, 1951 12. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275, 1951 13. Mauriege P, DePergola G , Berlan M, LaFontan M: Human fat cell 0-agonist receptors: 0-agonist dependent lipolytic responses and characterization of 0-adrenergic binding sites in human fat cell membranes with highly selective p-,antagonists. J Lipid Res 29:587-601, 1988 14. Tornqvist H, Krabisch L, Belfrage P Rapid assay for hormonesensitive lipase activity of adipose tissue. J Lipid Res 13:424-426, 1972 15. Krabisch L, Borgstrom B: A note on the preparation of small quantities of labelled fatty acid chlorides. J Lipid Res 4:357, 1963 16. Nilsson S, Belfrage P: Purification of hormone-sensitive lipase.by high-performance on exchange chromatography. Ann Biochem 158:399407, 1986 17. Belfrage P, Vaughan M: Simple liquid-liquid partition system for isolation of labelled oleic acid from mixtures with glycerides. J Lipid Res 10~341-344, 1969 18. Smith, HA: Kinetics of the catalyzed esterification of normal aliphatic acids in methyl alcohol. J Am Chem SOC61:254-261, 1939 19. Vaughan M, Steinberg D: Effects of hormones on lipolysis and esterification of free fatty acids during incubation of adipose tissue in vitro. J Lipid Res 4:193-199, 1963 20. Hirsch J, Farquhar JW, Ahrens EH, Peterson ML, Stoffel W: Studies of adipose tissue in man. A microtechnic for sampling and analysis. Am J Clin Nutr 8:499-5 11, 1960 2 1. Ekstedt B, Olivecrona T Uptake and release of fatty acids by rat adipose tissue. Last in-first out. Lipids 52358-860, 1970 22. Alling C, Gustavsson L, Anggird E An abnormal phospholipid in rat organs after ethanol treatment. FEBS Lett 152:24-28, 1983
0145-6008/91/1502-0184$3.00/0 ALCOHOLISM: CLINICAL AND EXPERIMENTAL
RESEARCH
The Metabolism of Ethyl Esters of Fatty Acids in Adipose Tissue of Rats Chronically Exposed to Ethanol Giovanni DePergola, Christer Kjellstrom, Cecilia Holm, Nils Conradi, Per Pettersson, and Per Bjorntorp
been demonstrated in several tissues, including adipose tissue The presence of EEFA in adipose tissue is of potential interest for at least two reasons. First, adipose tissue is a tissue that lends itself to repeated sampling. Second, adipose tissue has the highest content of free fatty acids compared with any other tissue. This would provide high substrate levels for ethyl fatty acid ester synthase to esterify fatty acids to ethanol. Third, hydrophobic compounds formed in, or taken up by adipose tissue will presumably be stored in the large mass of adipocyte triacylglycerols. These esters have a long half-life,' and any compound with a similar pathway for removal would presumably have an equally prolonged retention time in adipocytes. Theoretically, this would be the case with EEFA in adipose tissue, which therefore were thought to be a potentially useful marker for ethanol consumption, comparably easy to follow by adipose tissue biopsies. With this background we decided to study the potential usefulness of EEFA and the synthase activity in adipose tissue after fully controlled exposure to ethanol during HE QUANTITATIVELY most important pathway different periods of time in the rat, as well as after different for disposal of circulating ethanol is through oxidation periods of abstinence from ethanol. Furthermore, in order via alcohol dehydrogenase. This enzyme is, however, pres- to study the half-life of EEFA, labeled ethanol was given and labeled EEFA concentrations followed. Finally, the 'ent only in a few tissues, notably the liver. Other tissues relative hydrolysis rate of EEFA and triacylglycerol by the have a limited capacity to metabolize ethanol. One rehormone-sensitive lipase of adipose tissue was determined cently discovered pathway is through the esterification of to elucidate the major rate-limiting step for intracellular ethanol to fatty acid with formation of ethyl esters. This lipid hydrolysis in adipocytes. The results show retention enzyme activity, fatty acid ethyl ester synthase, as well as of EEFA in adipose tissue and that the synthase activity is retention of the product, the ethyl ester of fatty acids increased by long-term ethanol exposure. A preliminary (EEFA), have been found in most tissues and have recently report has been presented.' been studied in some detail in heart, brain, and white blood cells.'-' In a limited number of post-mortem samMATERIAL AND METHODS ples in humans both EEFA and the enzyme have also The concentration of ethyl esters of fatty acids as well as the activity of the enzyme synthesizing these esters (fatty acid ethyl ester synthase) were determined in adipose tissue of rats ingesting ethanol (9-16 g/kg body weight/day) for different periods of time. After 10 and 17 weeks of ethanol exposure about 300 nmol of ethyl esters of oleic, palmitic, stearic, and linoleic acids were found per gram adipose tissue. The ethyl esters disappeared after 1 week of abstinence. Closer analyses, using radioactive ethanol, revealed a half-life of the esters of less than 24 hr. The bulk of the esters was found in a membrane preparation of isolated adipocytes. Hormonesensitive lipase hydrolyzed emulsified ethyl oleate as efficiently as that of trioleoylglycerol, but in mixed ethyl oleate/trioleoyl glycerol particles the hydrolysis of ethyl oleate was slower, suggesting a decreased accessibility. Synthase activity was found in adipose tissue from rats not exposed to ethanol. It doubled after 10 and 17 weeks of ethanol and decreased with a half-life of at least a week after abstinence. It was concluded that ethyl esters of fatty acids are formed in rat adipose tissue as previously shown in other tissues. They seem to be stored mainly in membranous parts of the adipocytes. Synthase activity is induced by ethanol. The elevated activity has a longer half-life, and may be useful as an indicator of alcohol abuse. Key Words: Ethanol, Ethyl Esters, Fatty Acids, Synthase, Adipose Tissue.
3 9 5
T
Animals From the Clinica Medica HI, University of Bari (G.D.); Department of Medicine I and the Wallenberg Laboratory (P.P., P.B.): Department of Pathology I, (C.K., N.C.), University of Goteborg: and Department of Medical and Physiological Chemistry (C.H.), University of Lund, Sweden. Received for publication February 26, 1990: accepted September 18, 1990 Financial support was given by The Bank of Sweden Tercentary Foundation, the Swedish Medical Research Council (Grant No. 3363 to P. Beljrage, and 07121 to C.K. and N.C.). Reprint requests: Professor Per Bjorntorp, Department of Medicine I, Sahlgren 3 Hospital, University of Goteborg, 413 45 Goteborg, Sweden. Copyright 0 1991 by The Research Society on Alcoholism. 104
Male rats of a Sprague-Dawleystrain were used. A total of 1g pairs of two littermates each. They were given a normal commercial laboratory diet (5% fat, 55% carbohydrate, 22.5% protein, vitamins and minerals, EWOS, Sodertiilje, Sweden), and tap water ad libitum until they were 35 days old, at which time they were housed individually in plastic cages in a room at 22°C temperature, 60% humidity, and a 12/12 hr light/dark cycle. Animals were given an ethanol-containing liquid dietlo in drinking tubes as the only source of food and water. They were pairfed: one rat was given an ethanol formula (AE), the other, the control (C), a formula in which the ethanol had been isocaloncally replaced with dextrinmaltose. The introduction of ethanol was gradual and on the fourth day the final concentration of 50 g ethanol/liter formula was achieved, sustaining a daily ethanol intake of 9 to 16 g/kg body weight. Changes Alcohol Clin Exp Res, Vol 15, No 2, 1991: pp 184-189
185
FATTY ACID ETHYL ESTERS IN ADIPOSE TISSUE
in body weight during exposition are presented in Fig. 1. The rat given alcohol received its diet ad libitum, the corresponding littermate was fed the same isocaloric amount of the control diet the following day. For eight of the AE rats, the ethanol-exposure lasted for 10 weeks after which the animals were either killed directly, without prior ethanol withdrawal ( n = 4), or were given a diet with a reduced ethanol content for 3 days and then an ethanol-free diet for I week, ( n = 4), before they were killed. Four AE rats were exposed for I I weeks and then given a diet with a reduced ethanol content for 3 days, and an ethanol-free diet for 4 weeks before they were killed. The last group with six AE rats was exposed for 17 weeks and killed without ethanol withdrawal.
Determination of EEFA Concentration This was performed in principle as described previously for rabbit myocardium.' One g adipose tissue was homogenized in a glass homogenizer in 1 ml acetone and centrifuged (1000 X g, 10 min) at room temperature. One hundred microliters of the acetone phase were used for thin layer chromatography (TLC). This was performed on glass plates (20 X 20 cm), on which a 0.5-mm layer of silicic acid was placed, dried, and activated at 80°C for 3 hr. The liquid phase was petroleum ether:diethyl ether:acetic acid (755: I ) . Before use, the plates were cleaned by development in the same solvent mixture. The sample was applied as a 5-cm long band, surrounded by lipid standards including routinely 40.0 pg ethyl oleate. The standard spots were visualized by iodine with the sample covered by thin plastic film. The following Rf values were obtained: phospholipids, 0-0.1, fatty acids, cholesterol, mono- and diacylglycerols, 0.1 to 0.15; triacylglycerols, 0.15 to 0.25; EEFA, 0.45 to 0.55; cholesterol esters, 0.60 to 0.70. Recovery of added ethyl oleate was examined by addition of ethyl oleate to varying amounts of extracted adipose tissue triglycerides by extraction of the ethyl oleate region on the plate with acetone and quantitation of ester bonds." It was ascertained that cholesterol esters did not interfere with this reaction by separate analyses of cholesterol. Recovery of ethyl oleate exceeded 95% up to 200 mg of added triacylglycerides. With higher concentrations, trailing of triacylglycerols was visible on the TLC, and recovery of ethyl oleate was not complete. After this test 100 mg of adipose tissue was routinely used for analyses. With this amount of tissue the ethyl ester of margaric acid (C17) was used as internal standard for the whole procedure. The recovery of this acid was about 70%, and losses corrected for in the calculations. The amount of ester bonds in the EEFA region of the TLC was proportional to extracted adipose tissue up to amounts of 200 mg. In order to determine the biological half life of EEFA 200 to 300 g male rats were given 3 ml 90% ethanol with 50 pCi of (1-14C) ethanol orally. Radioactivity was determined after 4 hr in different lipids of adipose tissue extracts on TLC. As seen in Table 1, label was found in
several lipid fractions including EEFA, but not in cholesterol esters. Total radioactivity in half of the lipid extract subjected to TLC was counted directly. The other half was saponified in 2 ml ethanol and 0.5 ml of 5 N KOH at 37°C for 1 hr. This was then evaporated to dryness under nitrogen, redissolved in 1 ml ethanol, and radioactivity again counted.' The difference in counts was then compared with that found in the EEFA spot of the TLC and found to be 90% ( n = 4). Therefore, for the determination of the half-life of EEFA, the radioactivity lost after saponification and evaporation was taken as EEFA radioactivity. The area on TLC corresponding to the EEFA standards, from the top of the triacylglycerol spot to about Rf value 0.7, was scraped off and eluted with acetone for gas liquid chromatography (GLC) on a PerkinElmer, Sigma 300, instrument, with a capillary column (SP-1000) operated at 80°C to 220°C. Peaks were detected and quantitated by a flame ionization detector and a computerized integrator. Retention times for the ethyl esters of palmitic (C16), margaric (C17), stearic (CIS), oleic ((218: I), and linoleic (18:2) acids were determined by injection of standards and were found to be 12.24, 14.33, 16.63, 17.16, and 18.13 min, respectively, reproducible within less than +0.03 min. Enzyme Assay This was camed out in principle as described for rat myocardium' modified for the purpose of determination in adipose tissue. One hundred milligrams of adipose tissue were homogenized. The tubes were centrifuged (1000 X g, 10 min) at room temperature, and the infranatant (between the pellet and the fat cake) was collected. From this phase, 100 pl were taken for protein determination and 100 pl were added to the enzyme assay tubes. The total volume of the reaction was 170 p1, with the following final concentrations: 1 mM ethanol, 0.9 mM non-labeled oleic acid, 0.4 pCi I-14C-oleic acid, 68 mM phosphate buffer, pH 7.4. Every sample was run in duplicate, and followed by a blank containing 100 pl boiled cytosol in phosphate buffer. Incubations were camed out in closed glass tubes at 37"C, for 1 hr. The reaction was stopped by adding 2 ml cold acetone, containing ethyl(lH)oleate as internal yield standard ( 5 X lo5 cpm). After evaporation to dryness under nitrogen, 300 pl acetone were added and applied on TLC as described above. The EEFA spot was identified by standards, scraped off, eluted in acetone, and counted in an LKB (Wallac, Rackbeta) Liquid Scintillator Counter. Losses were corrected for by quench correction and by use of the internal standard. Results were expressed as nmoles per hour and mg protein. Protein was determined according to Lowry et a1." The assay conditions for the enzyme activity were tested to determine non-limiting substrate concentrations of both ethanol and oleic acid. Ethanol concentrations up to 1 mM increased the formation of EEFA while at 2 mM no further increase was seen (Fig. 2). Activity in the presence of 1 mM ethanol increased with increasing concentrations of
weight (g) 500
1
400
Fig. 1. Body weight of rats exposed to alcohol (AE) and controls (C) during a 17-week period. (n = 6).
300
200
100
! 5
I
i
15
25 age (weeks)
DEPERGOLA ET AL.
186 Table 1. Distribution of Label in Rat Epididymal Adipose Tissue Lipids after Administration of ''C-Ethanol Radioactivity (% of total) (n = 4)
Fraction
a,
a
.-cn
v)
c.
*
27 3 32 f 1 41 k 2 0
Phospholipids Cholesterol, free fatty acids and acylglycerols EEFA Cholesterol esters Mean k SEM
A
3
0 P .-U Q
w
h
0
E
rn
Y
n
1
40 -I
0
L
f Q
20 40
60
80100120140
minutes
a
.-+cn. v)
Fig. 4. The dependence of synthesis of ethyl oleate on time. Ethanol concentration, 1 mM, oleic acid concentration, 0 9 mM Other conditions as in Material and Methods section One representative experiments out of two performed
Q v)
0
.
***
0
Y
10
!
,
0
.
I
1
.
,
2
1
.
3
4
Ethanol (mM) Fig. 2. The dependence of synthesis of ethyl oleate on ethanol concentration Oleic acid concentration 0 9 mM Other conditions as in Materlal and Methods section One representative experiment out of three performed
50
1
40: 30
/--
Control
10
010
low
17W
Fig. 5. Ethyl ester synthase activity in rats exposed to ethanol for 10 or 17 weeks and in controls. Mean k SEM. " ' p < 0.001.
.
I
I
1
1
2
3
Oleic acid (mM) Fig. 3. The dependence of synthesis of ethyl oleate on oleic acid concentration. Ethanol concentration 1 mM. Other conditions as in Material and Methods section. Four individual experiments.
oleic acid up to about 1 mM (Fig. 3). Guided by these experiments, concentrations of I mM ethanol and 0.9 mM oleic acid were selected for further assays. Under these conditions activity was linear with time up to 60 min and then levelled off (Fig. 4). With an assay time of 60 min activity was proportional to the amount of added adipose tissue up to about 200 mg of adipose tissue (not shown). Amounts of about 100 mg tissue were used. Isolation of adipocytes with collagenase and preparation of fat cell ghosts were performed as previously described."
Lipid Ester Hydrolysis with Hormone-Sensitive Lipase Lipids used for preparing substrates were synthesized by Dr. Lennart Krabisch, Department of Medical and Physiological Chemistry, University of Lund. Briefly, tri(3H)oleoylglyceroI was synthesized and purified as describedI4 to at least 99% purity. Ethyl esters of ('H)oleic acid (Amersham, England) or unlabeled oleic acid were synthesized by adding
oleic acid chloride to ethanol and letting it react overnight at room temperature with constant ~tirring.'~ The mixture was then extracted twice with petroleum ether and the petroleum ether phases washed with 0.01 M NaOH, followed by water. The purity of the ethyl oleate was 98.4% by TLC. Alternatively, to obtain an ethyl ester of (3H)oleic acid with a very high specific activity, acetyl chloride was added directly to the (3H)oleic acid, which is obtained in ethanol from the manufacturer (Amersham, England). The mixture was allowed to react for 3 hr at room temperature and was then extracted as described above. The purity of this ethyl oleate was 98.4% by TLC. Substrates of trioleoylglycerol,ethyl oleate, and mixtures of these were obtained by sonication in 0.1 M potassium phosphate, pH 7.0. Pure ethyl oleate substrates were made 5 mM, whereas pure trioleoylglycerol substrates and substrates containing a mixture of ethyl oleate and trioleoylglycerol were made 1.5 mM. For stabilization of the emulsions, phospholipids (phosphatidylcholine and inositol 3:l [w/w]) were included at a concentration of 0.15 mg/ml.14 After sonication, defatted bovine serum albumin was added to a final concentration of 0.2 mg/ml. Hormonesensitive lipase was purified from rat epididymal adipose tissue to approximately 5% protein purity.'6 Hormone-sensitive lipase activity was measured, at non-inhibitory detergent concentrations, at 37°C over 30 min. Labeled free oleic acid was isolated with a simple one-step liquidliquid partition system."
FATTY ACID ETHYL ESTERS IN ADIPOSE TISSUE
187
Chemicals
1-14Cokic acid (S.A. 58 mCi/mmol)and I-14C ethanol (54.1mCi/ mmol) were from Amersham International (Buckinghamshire, England) and 9,10-3H (H)oleic acid (S.A. 10 Ci/mmol) from New England Nuclear (Boston, MA). All the lipid standards purchased were of highest commercially available quality. Ethyl ester of ('H)oleic acid used as TLC standard was synthesized in ethanol with gas from concentrated hydrochloric acid for 20 min.18 Ethanol was then evaporated under nitrogen, lipids redissolved in acetone and ethyl oleate purified by repeated TLC to less than 5% contamination with free oIeic acid.
are seen in Table 3. The rates of hydrolysis with the two substrates were comparable, and hydrolysis of trioleoylglycerol did not Seem to be influenced by the presence of 1% or 20% ethyl oleate. H ~when (3Hlethyl ~ ~oleate and ~ trioleoylglycerol was mixed in the same emulsion particles the Of Oleate seemed to decrease than accounted for by the dilution of the labeled substrate (third part of Table 3).
Contents of EEFA in Membranes and Triacylglycerols of RESULTS
A dipocytes
EEFA Concentrations As seen in Table 2, no EEFA was found in the control rats, which were not given ethanol. In rats exposed to ethanol for 10 and 17 weeks EEFA were found in all rats. EEFA of oleic, palmitic, stearic, and linoleic acids were identifiable. The proportion between these esters were 5 I k 5,24 k 3, 16 f 4, and 9 & 3% (Means + SEM, n = 10) of total, respectively, in all rats exposed to ethanol until examination. The absolute values of the sum of these concentrations in 100 mg adipose tissue is used for further presentation of results. No further increase of EEFA concentration after 17 weeks of ethanol was seen in comparison with 10 weeks. One week after exposure to ethanol had ended, no EEFA were found, and this was essentially true also for rats free from ethanol during 4 weeks after 1 I weeks of exposure. EEFA Half Life The results of measurements of EEFA concentrations suggested that the half-life of EEFA esters in rat adipose tissue is shorter than 1 week. T o analyse this more accurately, labeled ethanol was used as described in the Methods section. Rats were given radioactive ethanol, and the values in adipose tissue of evaporated ethanol radioactivity 4 hr after administration were taken as comparison for similar measurements after another 16 hr which was found to be 34% to 40% of original ( n = 3). After 1 week no radioactivity remained ( n = 2). Hydrolysis of Ethyl Oleate and Trioleoylglycerol by Isolated Hormone-Sensitive Lipase The results of hydrolysis experiments by hormone-sensitive lipase of emulsified ethyl oleate or trioleoylglycerol Table 2. EEFA in Adipose Tissue of Rats Exposed to Ethanol Length of ethanol-exposure withdrawal (weeks)
+
0 (n = 4) 10 (n = 4) 10+1(n=4) 11 + 4 ( n = 4 ) 17 (n = 6) Mean c SEM
(nmol/s)
0 310 f 30 0 10k1 340 f 38
The bulk of triacylglycerol was separated from the membranous parts of a d i p ~ c y t e s and ' ~ EEFA concentration determined in these two fractions. In two experiments the membrane fraction contained 80% and 84% of total EEFA.
Enzyme Activity for EEFA Synthesis Fig. 6 shows the EEFA synthase activity in rats exposed to ethanol for different periods of time. Control rats showed a rather high activity. This increased to almost double after 10 weeks of alcohol exposure. After 17 weeks of ethanol no further increase seemed to have occurred compared with 10 weeks. After 1 week of abstinence, the activity was still elevated to more than 50% above that found in controls, but after 1 1 weeks of ethanol exposure and 4 weeks abstinence the activity was back to that of control rats. DISCUSSION
This study shows that EEFA are formed in adipose tissue of ethanol exposed rats. This has previously been shown in post-mortem samples of human adipose t i ~ s u e . ~ Table 3. Ethyl Oleate Hydrolysis by Hormone-SensitivesLipase Experiment 2 (nmol 3H-fatty acids/min/ml)
Experiment 1 (nmol 3H-fatty acids/min/ml) Tri(3H)oleoylglyceroI Eth~l(~H)oleate Trioleoylglycerol hydrolase activity (nmol 3Hfatty acids/min/ml) 1YO oleate 99% tri(3H)oleoylglyceroI 20% ethyl oleate 80% tri(3H)oleoylglyceroI
+
+
563 541
435 537
539
Trioleoylglycerol hydrolase activity (nmol 3H-fatty acid/min/ml) 515
507
366
Pure ethyl oleate, pure trioleoylglycerol, and different mixtures of these were used as substrates for hormone-sensitive lipase as described in "Materials and Methods." The mixed substrates are given as molar percent of each component. Results are mean of five determinations. Ethyl oleate hydrolase activity (nmol 3H-fatty acids/ml) (numbers within parentheses are the theoretical values for dilution of ethyl(3H)oleatewith unlabeled ethyl oleate). 100% eth~l(~H)oleate 608 20% eth~l(~H)oleate 80% trioleoylglycerol 99 (122) 5% eth~l(~H)oleate 95% trioleoylglycerol 22 (30) 1% eth~l(~H)oleate + 99% trioleoylglyceroi 4.3 (6.1) 0.25% eth~l(~H)oleate99.75% trioleoylglycerol 0.94 (1.5) 0.1 YOethyl(3H)~leate 99.9% trioleoylglycerol 0.26 (0.6)
+ +
+ +
~
DEPERGOLA ET AL.
***
EEFA in adipose tissue was actually less than 24 hr. This is in agreement with recent findings in the rabbit.3 Such a ** -r short half-life of EEFA suggests that they are not turning over concomitantly with the adipocyte triacylglycerol, the -r half-life of which is much longer in the rat. Instead, they might form a specific pool in adipocytes. Recently accumulated triacylglycerols seem to form such a pool with a short half-life, remaining only for a limited period of time.21The radioactive triacylglycerolformed after administration of 14C-ethanol actually had a much longer halflife than that of the EEFA in the experiments performed here. Although this was not studied in detail, it was probably of the order of at least a week. This observation indicates that the EEFA have indeed a shorter half-life than the bulk of triacylglycerols in the adipocyte. 10 - l l w 10 - l l w Control 10-11 w Adipocyte triacylglycerols are hydrolyzed by the hor1W (abst 1 4W labst) mone-sensitive lipase to provide the FFA that are mobiFig. 6. Ethyl ester synthase activity in rats exposed to ethanol during 10 to 11 lized from the cell. This lipase was shown to hydrolyze weeks and then given no ethanol for 1 and 4 weeks and controls. Mean f SEM. " ' p < 0.001, * * p< 0.01, comparisons with controls. trioleoylglycerol equally well in the absence or presence of ethyl oleate. However, in mixed emulsions ethyl oleate The synthesis was linear with time with concentrations of seemed to be hydrolysed less efficiently, particularly when fatty acids found physiologically in adipose tissue," and present in the concentrations that could maximally be expected in vivo in the triacylglycerol droplets of adipose of ethanol found in blood during social drinking. tissue. This might mean that ethyl oleate is comparably The rats exposed to controlled ethanol consumption less available for hydrolysis in such mixed emulsion parwere also shown to form EEFA, which were found in their ticles and therefore would be expected to have a longer adipose tissue. Esters were found of oleic, palmitic, stearic, half-life than triacylglycerols under such conditions. Other and linoleic acids, the main fatty acids present in rat mechanisms must, hence, explain the shorter half-life of adipose tissue, and in the approximate proportion to the EEFA than triacylglycerols in the adipocyte. concentrations of these fatty acids.20Total EEFA concenOne remaining possibility is that EEFA are retained in tration in adipose tissue was not higher after 17 weeks of a pool outside the lipid droplet such as in a membranous ethanol ingestion than after 10 weeks, suggesting that a compartment of the cell. Findings of synthase activity in limit of EEFA retention in adipose tissue had been reached the brain4 indicate that much of it is located in plasma as at 10 weeks or before. The enzyme activity determined well as mitochondria1 membranes. It has been suggested simultaneously demonstrated an unimpaired capacity for that EEFA may negatively affect mitochondria and that formation of new EEFA at 17 in comparison with 10 this mechanism can explain some aspects of alcoholic weeks. This suggests a limited turn-over time. When altissue damage.2 Experiments separating a membranous cohol ingestion had stopped, essentially all EEFA disapfraction from adipocyte lipids showed that accumulation peared from adipose tissue within 1 week. of EEFA in adipocyte membranes seems indeed to occur. A closer study of the half-life of EEFA in rat adipose Ethanol is also incorporated into another lipid compotissue utilized labeled ethanol to obtain labeled EEFA. nent of cellular membranes, viz. phosphatidyl ethanol. However, other adipose tissue lipids were labeled as well, necessitating separation of adipose tissue lipids by TLC. The half-life of this compound seems, however, to be of Because of the relatively small synthesis of labeled EEFA the order of 3 to 4 hr.22The half-life of the EEFA is thus in relation to the bulk of triacylglycerol separated on TLC, longer by a factor of 4 to 5. It is notable that rats never exposed to ethanol have it was necessary to administer larger amounts of radioacquite a considerable activity of the EEFA synthase in tivity to determine EEFA concentration by this procedure. adipose tissue. With ethanol ingestion the enzyme activity Instead an indirect procedure was therefore developed, was clearly increasing, and this induction seemed to have where volatile radioactive constituents of saponified lipids reached maximal values at 10 weeks or earlier. When could be quantitated after removal by evaporation. Apt alcohol exposure had ended the activity was again decreasparently, only ethanol is removed by this procedure, because this method turned out to agree with the direct ing back to that of controls. The rate of this decrease was determination of radioactive EEFA after chromatographic fairly slow, with half-maximal values of activity after about separation. This method might therefore be useful in 1 week. This study thus has shown that EEFA are formed in studies in humans where exposure to radioactivity has to adipose tissue of rats consuming ethanol. Their half-life is be limited. Utilizing this method it was found that the half-life of longer than that of ethanol in the circulation, and they are
i
FATTY ACID ETHYL ESTERS IN ADIPOSE TISSUE
retained in the adipocyte with a half-life of less than 24 hr probably in a membranous compartment of the cell. It may well be that the storage is different after a longer period of ethanol exposure. The induction of elevated activity of the EEFA synthase seems to persist longer and might well be a potentially useful tool for diagnosis of alcohol abuse. In contrast to alcohol dehydrogenase in the liver, another enzyme induced by alcohol, the EEFA synthase activity of adipose tissue is readily measurable in samples, obtainable by atraumatic needle biopsies. ACKNOWLEDGMENTS Margareta Medberg is acknowledged for expert GLC analyses. We thank Per Belfrage for fruitful discussions and constructive criticism of this manuscript.
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ester synthase activity. Biochem Biophys Res Commun 142:979-985, 1987 8. M i n n P, Rebuffk-Scrive M, Smith U, Bjorntorp P: Glucose uptake in human adipose tissue. Metabolism 36: 1154-1 160, 1987 9. DePergola G, Kjellstrom C, Conradi N, Bjorntorp P: Chronic ethanol exposure and ethyl esters of fatty acids in adipose tissue of rats. 5th Eur Lipoprotein Symposium, Padua, 1989 (abstr) 10. Lieber CS, DeCarli LM: The feeding of alcohol in liquid diets: Two decades of applications and 1982 update. Alcohol: Clin Exp Res 6~523-531, 1982 1 1. Rapport MR, Alonzo N Photometric determination of fatty acid ester groups in phospholipids. J Biol Chem 193:265-275, 1951 12. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275, 1951 13. Mauriege P, DePergola G , Berlan M, LaFontan M: Human fat cell 0-agonist receptors: 0-agonist dependent lipolytic responses and characterization of 0-adrenergic binding sites in human fat cell membranes with highly selective p-,antagonists. J Lipid Res 29:587-601, 1988 14. Tornqvist H, Krabisch L, Belfrage P Rapid assay for hormonesensitive lipase activity of adipose tissue. J Lipid Res 13:424-426, 1972 15. Krabisch L, Borgstrom B: A note on the preparation of small quantities of labelled fatty acid chlorides. J Lipid Res 4:357, 1963 16. Nilsson S, Belfrage P: Purification of hormone-sensitive lipase.by high-performance on exchange chromatography. Ann Biochem 158:399407, 1986 17. Belfrage P, Vaughan M: Simple liquid-liquid partition system for isolation of labelled oleic acid from mixtures with glycerides. J Lipid Res 10~341-344, 1969 18. Smith, HA: Kinetics of the catalyzed esterification of normal aliphatic acids in methyl alcohol. J Am Chem SOC61:254-261, 1939 19. Vaughan M, Steinberg D: Effects of hormones on lipolysis and esterification of free fatty acids during incubation of adipose tissue in vitro. J Lipid Res 4:193-199, 1963 20. Hirsch J, Farquhar JW, Ahrens EH, Peterson ML, Stoffel W: Studies of adipose tissue in man. A microtechnic for sampling and analysis. Am J Clin Nutr 8:499-5 11, 1960 2 1. Ekstedt B, Olivecrona T Uptake and release of fatty acids by rat adipose tissue. Last in-first out. Lipids 52358-860, 1970 22. Alling C, Gustavsson L, Anggird E An abnormal phospholipid in rat organs after ethanol treatment. FEBS Lett 152:24-28, 1983
