Effects of ethanol on the remodeling of neutral lipids and phospholipids in brain mitochondria and microsomes

Neurochemistry International 50 (2007) 858–865 www.elsevier.com/locate/neuint

Effects of ethanol on the remodeling of neutral lipids and phospholipids in brain mitochondria and microsomes Marı´a P. Carrasco, Jose´ M. Jime´nez-Lo´pez, Josefa L. Segovia, Carmen Marco * Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Granada, Av. Fuentenueva s/n, Granada 18001, Spain Received 12 February 2007; accepted 21 February 2007 Available online 3 March 2007

Abstract We have analyzed the effects of ethanol in vitro on the remodeling of neutral lipids and phospholipids in mitochondria and microsomes isolated from chick brain. We used three different fatty acyl-CoAs of similar chain lengths but different degrees of unsaturation. Our results demonstrate the existence of active mechanisms for acyl-CoA transfer into neutral lipids and phospholipids in both mitochondria and microsomes. The profile of fatty acid incorporation was clearly different according to the membrane and lipid fraction in question. Thus, in mitochondrial lipids, the remodeling processes showed a clear preference for the saturated fatty acid whilst the polyunsaturated one was the preferred substrate for microsomal lipid acylation. With regard to the effects of ethanol in vitro, we were able to demonstrate that exposure of the membrane to ethanol led to an increase in the incorporation of polyunsaturated fatty acid into triacylglycerol (TG) in both mitochondria and microsomes, indicating that it directly stimulates the acylation of diacylglycerol (DG) to give TG. This effect may then contribute to the widely reported stimulation of TG biosynthesis in cases of both acute and chronic ethanol ingestion. It is noteworthy that the exposure of microsomes to ethanol in vitro also stimulated the incorporation of oleoyl-CoA into the aminophospholipids phosphatidylethanolamine (PE) and phosphatidylserine (PS). We also demonstrate that both mitochondria and microsomes synthesize fatty acid ethyl esters (FAEEs) from fatty acyl-CoA, although there is a clear difference in preference for the fatty acid used as substrate in the esterification of the alcohol. Thus, mitochondria were capable of forming FAEEs from the polyunsaturated fatty acid whilst in microsomes the saturated fatty acid was the preferred substrate. In both types of membrane, FAEE production was lowest with the monounsaturated fatty acyl-CoA. # 2007 Elsevier Ltd. All rights reserved. Keywords: Ethanol; Lipid remodeling; Neutral lipids; Phospholipids; Brain mitochondria; Brain microsomes

1. Introduction A great body of experimental evidence exists to demonstrate that ethanol exerts its pharmacological effects by modulating the function of many membrane components such as those of intracellular signal transduction pathways (Nagy, 2004). It has also been suggested that the composition of the lipids and degree of phospholipid unsaturation may play a role in the modulation of membrane-protein functions (Litman and

Abbreviations: ACAT, acyl-CoA:cholesterol acyltransferase; DGAT, acylCoA:diacylglycerol acyltransferase; AEAT, acyl-CoA:ethanol acyltransferase; DG, diacylglycerol; CE, esterified cholesterol; FAEE, fatty acid ethyl ester; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin; TG, triacylglycerol * Corresponding author. Tel.: +34 958 243248; fax: +34 958 249945. E-mail address: [email protected] (C. Marco). 0197-0186/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2007.02.007

Mitchell, 1996). The effects upon biomembranes of xenobiotics such as ethanol are influenced among other factors by the physico-chemical structure of the membrane itself and therefore by the chemical composition of its lipid matrix. In previous publications, we have described significant changes in the composition of neutral lipid and phospholipid acyl groups after both chronic (Carrasco et al., 1996a; Sa´nchezAmate et al., 1992) and acute (Carrasco et al., 1996b; Sa´nchezAmate et al., 1992; Marco et al., 1986a) ethanol consumption, and the consequent alterations in the properties of brain and liver membranes. Furthermore, there is general agreement that some of the effects of ethanol in vitro are brought about by its very physical presence, which disorders the acyl chains of the phospholipids in the hydrophobic core of cell membranes and thus tends to fluidize and/or expand the membranes (Patra et al., 2006). Little doubt remains nowadays that the membrane responds to this initial ethanol effect by modifying its chemical

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composition in order to counter these actions, leading to the phenomenon known as membrane adaptation. Thus, changes in the physico-chemical properties of the membrane induced by ethanol in vitro may in turn affect processes linked to the biomembranes such as transport, signal transmission and enzyme activities. We have reported in the past that the exposure of chick-liver microsomes and mitochondria to ethanol in vitro affects enzyme activities involved in electronic transport systems (Sa´nchez-Amate et al., 1995). We have also demonstrated in a recent paper that the exposure of rat-liver microsomes to ethanol specifically alters the synthesis and acylation of aminophospholipids (Carrasco et al., 2006). In general, acylation reactions contribute to the establishment and/or continuous remodeling of lipid components and thereby to the membrane function. In spite of the fact that chronic alcohol consumption is known to have adverse effects on the central nervous system (Akbar et al., 2006), no studies have yet been made into the initial effects produced by ethanol on the remodeling of brain-membrane lipids. Therefore, we have undertaken a study into the effects of ethanol on the incorporation of fatty acids from exogenous acyl-CoA into the different lipids in mitochondria and microsomes isolated from chick brains. To analyze differences in the use of these fatty acids for the esterification of phospholipids and neutral lipids we chose three fatty acids with similar chain lengths but differing in their degree of unsaturation. 2. Materials and methods

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Pellets containing mitochondria or microsomes were resuspended in 0.32 M sucrose and used without storage. NADPH cytochrome c reductase was used as a marker enzyme for microsomes whilst cytochrome oxidase was used to characterize the mitochondrial fraction. Assays with the marker enzymes of plasma membrane (50 -nucleotidase), lysosomes (acid phosphatase) and myelin (20 ,30 -cyclic nucleotide 30 -phosphohydrolase) demonstrated that contamination of the mitochondria and microsomes by other membranes was less than 10%.

2.4. Synthesis of acyl-CoAs The different fatty acyl-CoAs were prepared following the method of Taylor et al. (1990) with minor modifications. Briefly, 18 mCi of fatty acid in ethanol was dried under nitrogen. Following this, 0.7 ml of the following components were added at the concentrations indicated: 0.1% (w/v) Triton X-100, 5 mM CoA, 10 mM ATP, 1 mM DTT, 10 mM MgCl2, 100 mM MOPS-NaOH (pH 7.5). The mixture was sonicated for 5 min at 4 8C. Subsequently, 0.3 ml of acylCoA synthetase from Pseudomonas (1 mg/ml) was added to give a final reaction volume of 1 ml. The reaction mixture was stirred for 2 h with a micro-stir bar under a nitrogen atmosphere at 35 8C. After incubation, the reaction mixture was applied to a Sep-Pak C18 column (Waters, Madrid, Spain) that had been washed with 3-column volume of HPLCgrade methanol and equilibrated with 3 volumes of 100 mM MOPS-NaOH (pH 7.5). Following the application of the sample, the column was washed with 0.5 ml of 100 mM MOPS-NaOH (pH 7.5) and 1 ml of 1:1methanol/water. [1-14C]acyl-CoAs were eluted with 20 ml of methanol and the solvent removed in a rotary evaporator at 30 8C. The residue was redissolved in 1 ml of methanol, flushed with nitrogen and stored at 30 8C. Purity of the acyl-CoAs was determined by TLC using a mixture of nbutanol/water/acetic acid (50:30:20 by volume) as solvent. Recovery was always higher than 90%.

2.5. Effects of ethanol in vitro upon the incorporation of fatty acids into neutral lipids and phospholipids

2.1. Materials All radiolabeled compounds were supplied by American Radiolabeled Chemicals (St. Louis, MO, USA). Thin-layer chromatography (TLC) plates came from Sigma–Aldrich (Madrid, Spain). All other reagents were of analytical grade.

2.2. Animals Newborn, male, White Leghorn chicks were supplied by the Scientific Instrumentation Centre of the University of Granada (Spain) and fed ad libitum on a commercial diet (Sanders A-00) in a chamber with a 12-h light:12-h dark cycle and a constant temperature of 31 8C. The chicks were deprived of food for 12 h before brain mitochondria and microsomes were isolated.

2.3. Preparation of mitochondria and microsomes The chicks were killed by decapitation and their brains immediately removed, weighed, minced and homogenized in 5 volumes of 0.32 M sucrose with a Potter-Elvehjem homogenizer. After centrifugation at 800  g for 10 min, the supernatant was recentrifuged at 16,000  g for 20 min. The resulting pellet, corresponding to the mitochondrial fraction, was resuspended in 0.32 M sucrose. To further purify the mitochondrial fraction, samples of this suspension were layered over a discontinuous density gradient of 0.8, 1 and 1.2 M sucrose and centrifuged at 63,500  g for 2 h. High purity mitochondria were collected in the pellet of this last centrifugation. Microsomes were obtained following the procedure of Marco et al. (1986b) with minor modifications. Briefly, after decapitation, the chick brains were removed immediately and homogenized in 3 volumes of 50 mM potassium phosphate buffer (pH 7.4) containing 30 mM EDTA, 250 mM NaCl and 1 mM DTT. The homogenate was centrifuged at 5000  g for 15 min and the supernatant centrifuged at 15,000  g for 15 min. Microsomes were sedimented by centrifuging the 15,000 supernatant fraction at 105,000  g for 60 min.

To analyze the acylation processes of neutral lipids and phospholipids in mitochondria and microsomes isolated from chick brains, we followed the procedure of Marco et al. (1986b) using [1-14C]stearoyl-CoA, [1-14C]oleoylCoA and 11,14,17-[1-14C]eicosatrienoyl-CoA as exogenous substrates. Briefly, the incubation mixture consisted of 50 ml of mitochondrial or microsomal suspension (5 mg/ml) in 100 mM potassium phosphate buffer (pH 7.4), 2 mM DTT and 1.2 mg of free fatty acid, bovine-serum albumin. After 5 min preincubation at 37 8C in the presence or absence of 100 mM ethanol, the enzyme reaction was initiated by adding 20 nmol of the corresponding [1-14C]acyl-CoA derivative (600,000 dpm) to give a final volume of 150 ml. Reactions were terminated after 10 min by the addition of 4 ml of chloroform/methanol (2:1 by volume). In the acylation assays, [1,2-3H]cholesteryl oleate (10,000 dpm) and 1,2dipalmitoyl-3-phosphatidyl-[N-methyl-3H]choline (150,000 dpm) were used as internal standards for neutral lipids and phospholipids, respectively.

2.6. Lipid extraction and analysis After incubation, the lipids were extracted from the membranes with chloroform/methanol (2:1 by volume) according to Folch et al. (1957). The solvent was evaporated under nitrogen. The neutral lipids were separated by TLC using a mixture of hexane/diethylether/glacial acetic acid (70:30:1 by volume) as solvent. The main phospholipids were separated by TLC using a solvent of chloroform/methanol/acetic acid/water (60:50:1:4 by volume). The spots were rendered visible by exposure to iodine vapor. Radiometric measurements of scraped lipid spots were made by liquid scintillation using a Beckman 6000-TA counter (Madrid, Spain).

2.7. Other analyses Protein was estimated by the method of Lowry et al. (1951) using bovine albumin as standard. The results are expressed as the mean  S.E.M. for three

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different experiments. Statistical comparisons were made with Student’s t-test using the SPSS 9.0 program. Values of P  0.05 were considered to be statistically significant.

3. Results 3.1. Acylation of neutral lipids: effects of ethanol Mitochondria and microsomes isolated from chick brains were incubated at 37 8C for 10 min with 0.13 mM of radioactive stearoyl, oleoyl and eicosatrienoyl-CoA in the presence or absence of 100 mM ethanol. The results set out in Table 1 indicate that the pattern of incorporation of the different fatty acids into neutral lipids in the control membranes differed according to the fatty acyl-CoA derivative used in the assay system. Thus, in both membranes, diacylglycerol (DG) tended to exhibit higher acylation activity when oleoyl-CoA was used as substrate. Furthermore, the much lower incorporation of fatty acyl-CoA into triacylglycerol (TG) in mitochondria than in microsomes is also noteworthy. Of equal interest is the fact that in both membranes the uptake of oleate into TG was markedly lower than it was for the other fatty acyl-CoA derivatives. As far as esterified cholesterol (CE) synthesis is concerned, our results demonstrate that the activity of this process was higher in microsomes than in mitochondria, and that there was a clear specificity for cholesterol esterification from stearoylCoA in mitochondria. The effects of ethanol on the acylation of neutral lipids in both mitochondria and microsomes (Table 2) were significantly different as far as the incorporation of the various fatty acids is concerned. It can be seen that ethanol altered the acylation of mitochondrial lipids more than it did microsomal lipids. Thus, the presence of alcohol in mitochondria induced an increase in the uptake of polyunsaturated fatty acids into TG, DG and free cholesterol to produce the esterified form. The incorporation of stearate into CE was also stimulated, whereas it was inhibited in TG. In microsomes, however, ethanol exclusively altered the acylation of TG since a higher incorporation of the three fatty acids was found in this neutral lipid. Table 1 Incorporation of the different fatty acyl-CoAs into neutral lipids of chick-brain mitochondria and microsomes DG

TG

CE

Mitochondria Stearoyl-CoA Oleoyl-CoA Eicosatrienoyl-CoA

0.585  0.018 0.687  0.074 0.333  0.018

0.085  0.004 0.026  0.006 0.061  0.003

0.028  0.001 0.007  0.001 0.010  0.001

Microsomes Stearoyl-CoA Oleoyl-CoA Eicosatrienoyl-CoA

0.267  0.012 0.703  0.018 0.206  0.006

0.247  0.035 0.069  0.002 0.658  0.065

0.038  0.005 0.033  0.001 0.035  0.006

The incorporation of fatty acyl-CoAs into neutral lipids was determined as described in Section 2. Results are expressed as nmol/min  mg protein and are the mean  S.E.M. of four determinations.

Table 2 Effects of ethanol in vitro upon the acylation of neutral lipids from chick-brain mitochondria and microsomes DG

TG

CE

Mitochondria Stearoyl-CoA Control Ethanol

0.585  0.018 0.609  0.017

0.085  0.004 0.043  0.009b

0.028  0.001 0.060  0.002a

Oleoyl-CoA Control Ethanol

0.687  0.074 0.512  0.032

0.026  0.006 0.024  0.001

0.007  0.001 0.011  0.004

0.061  0.003 0.082  0.002b

0.010  0.001 0.019  0.001a

Eicosatrienoyl-CoA Control 0.333  0.018 Ethanol 0.399  0.005c Microsomes Stearoyl-CoA Control Ethanol

0.267  0.012 0.262  0.010

0.247  0.035 0.290  0.005c

0.038  0.005 0.054  0.006

Oleoyl-CoA Control Ethanol

0.703  0.018 0.689  0.018

0.069  0.002 0.082  0.004c

0.033  0.001 0.029  0.002

Eicosatrienoyl-CoA Control 0.206  0.006 Ethanol 0.226  0.008

0.658  0.065 0.786  0.046c

0.035  0.006 0.029  0.003

The incorporation of fatty acyl-CoAs into neutral lipids was determined as described in Section 2. Results are expressed as nmol/min  mg protein and are the mean  S.E.M. of four determinations. a P < 0.01 as compared to control values. b P < 0.02 as compared to control values. c P < 0.05 as compared to control values.

3.2. Acylation of phospholipids: effects of ethanol Table 3 shows the results of the incorporation of fatty acids into the different phospholipids of control mitochondria and microsomes after incubation with the different fatty acyl-CoA derivatives. The major glycerophospholipid, phosphatidylcholine (PC) was a primary site for the incorporation of the radiolabeled fatty acids into both subcellular membranes, whilst sphingomyelin (SM) exhibited the lowest uptake of fatty acids, according to the low turnover rates of this phospholipid. Acylation patterns for PC indicate a preference for stearoyl-CoA and eicosatrienoylCoA whereas it was quantitatively lower with oleoyl-CoA. The profiles of fatty acid incorporation for the quantitatively minor phospholipid phosphatidylserine (PS) indicate no specificity for any fatty acid, although acylation was higher in microsomes than in mitochondria. The results of the effects of 100 mM ethanol on the acylation process in brain microsomes are set out in Table 4, where it can be seen that ethanol specifically increased the acylation of aminophospholipids phosphatidylethanolamine (PE) and PS exclusively from oleoyl-CoA. The alteration by ethanol of the acylation of PS is of great interest since it is the major acidic phospholipid reported to be involved in neuron signaling. In mitochondria exposed to ethanol (Table 5), the acylation of phosphatidylinositol (PI) was only altered when the saturated

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Table 3 Incorporation of the different fatty acyl-CoAs into phospholipids from chick-brain mitochondria and microsomes SM

PC

PS

PI

PE

Mitochondria Stearoyl-CoA Oleoyl-CoA Eicosatrienoyl-CoA

0.012  0.003 0.003  0.001 0.011  0.002

0.845  0.031 0.277  0.013 0.742  0.062

0.068  0.002 0.046  0.007 0.041  0.007

0.117  0.006 0.063  0.006 0.009  0.001

0.433  0.014 0.285  0.029 0.134  0.016

Microsomes Stearoyl-CoA Oleoyl-CoA Eicosatrienoyl-CoA

0.006  0.001 0.005  0.001 0.018  0.002

0.405  0.065 0.143  0.020 2.332  0.377

0.061  0.004 0.189  0.007 0.120  0.014

0.041  0.002 0.153  0.011 0.267  0.016

0.099  0.009 0.230  0.015 0.157  0.015

The incorporation of fatty acyl-CoAs into phospholipids was determined as described in Section 2. Results are expressed as nmol/min  mg protein and are the mean  S.E.M. of four determinations. Table 4 Effects of ethanol in vitro upon the acylation of phospholipids from chick-brain microsomes SM

PC

PS

PI

PE

Stearoyl-CoA Control Ethanol

0.006  0.001 0.005  0.001

0.405  0.065 0.405  0.058

0.061  0.004 0.059  0.005

0.041  0.002 0.040  0.004

0.099  0.009 0.101  0.012

Oleoyl-CoA Control Ethanol

0.005  0.001 0.010  0.004

0.143  0.020 0.191  0.026

0.189  0.007 0.250  0.017a

0.153  0.011 0.178  0.009

0.230  0.015 0.322  0.029b

Eicosatrienoyl-CoA Control Ethanol

0.018  0.002 0.012  0.002

2.332  0.377 2.072  0.279

0.120  0.014 0.142  0.011

0.267  0.016 0.299  0.014

0.157  0.015 0.143  0.019

The incorporation of fatty acyl-CoAs into phospholipids was determined as described in Section 2. Results are expressed as nmol/min  mg protein and are the mean  S.E.M. of four determinations. a P < 0.02 as compared to control values. b P < 0.05 as compared to control values.

acyl-CoA was used as substrate. As can be seen, ethanol caused a significant decrease in the incorporation of stearate. 3.3. Synthesis of fatty acid ethyl esters We also analyzed the synthesis of fatty acid ethyl esters (FAEEs) from the different acyl-CoA derivatives in both chickbrain mitochondria and microsomes. To this end, we exposed the membranes to different quantities of ethanol and quantified the incorporation of the different radiolabeled fatty acyl-CoAs

into the corresponding FAEE. The results are shown in Figs. 1 and 2 where it can be seen that both mitochondria and microsomes have a high capacity to use the fatty acyl-CoA to esterify ethanol. Furthermore, the formation of FAEE was proportional to the quantity of ethanol within the range of 100– 400 mM. The preference for the fatty acyl-CoAs was clearly different between mitochondria and microsomes. Thus, in mitochondria, the synthesis of FAEE reached its maximum when the polyunsaturated fatty acid was the substrate for the esterification reaction whilst microsomes exhibited a clear

Table 5 Effects of ethanol in vitro upon the acylation of phospholipids from chick-brain mitochondria SM

PC

PS

PI

PE

Stearoyl-CoA Control Ethanol

0.012  0.003 0.008  0.001

0.845  0.031 0.812  0.018

0.068  0.002 0.063  0.005

0.117  0.006 0.085  0.005a

0.433  0.014 0.443  0.010

Oleoyl-CoA Control Ethanol

0.003  0.001 0.005  0.001

0.277  0.013 0.246  0.004

0.046  0.007 0.042  0.002

0.063  0.006 0.064  0.003

0.285  0.029 0.315  0.007

Eicosatrienoyl-CoA Control Ethanol

0.011  0.002 0.007  0.001

0.742  0.062 0.832  0.011

0.041  0.007 0.060  0.009

0.009  0.001 0.011  0.000

0.134  0.016 0.150  0.003

The incorporation of fatty acyl-CoAs into phospholipids was determined as described in Section 2. Results are expressed as nmol/min  mg protein and are the mean  S.E.M. of four determinations. a P < 0.01 as compared to control values.

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Fig. 1. Fatty acid ethyl ester synthesis from different fatty acyl-CoAs in chickbrain mitochondria. Membranes were incubated with [1-14C]stearoyl-CoA, [1-14C]oleoyl-CoA and 11,14,17-[1-14C]eicosatrienoyl-CoA as exogenous substrates for 10 min at 37 8C in the absence and presence of 100 and 400 mM ethanol. Lipid extraction and analysis were carried out as described in Section 2.

exogenous substrates, all of which must be activated by the corresponding synthetase. To bypass the CoA activation step and thus forestall any limitation that acyl-CoA synthetase might exert on the incorporation of fatty acids into membrane lipids we used fatty acyl-CoA derivatives previously synthesized in our laboratory, as described in Section 2. To analyze the effects of ethanol 100 mM on the acylation processes involved in the remodeling of neutral lipids and phospholipids in mitochondria and microsomes isolated from chick brains, we used stearoyl-CoA, oleoyl-CoA and eicosatrienoyl-CoA to compare the specificity of the incorporation processes of these fatty acids. We (Sa´nchez-Amate et al., 1991; Carrasco et al., 2006) and other researchers (Cassagne et al., 1991; MacQuarrie et al., 1993; Juguelin et al., 1996) have previously demonstrated the existence of significant acylation activity in the absence of any exogenous acceptor in various different membranes. Because we introduced no exogenous lipid acceptor we could simulate quite closely the physiological situation in which fatty acid remodeling of the membrane lipids was limited by the availability of endogenous substrates. 4.1. Effects of ethanol upon acylation of neutral lipids

Fig. 2. Fatty acid ethyl ester synthesis from different fatty acyl-CoAs in chickbrain microsomes. Membranes were incubated with [1-14C]stearoyl-CoA, [1-14C]oleoyl-CoA and 11,14,17-[1-14C]eicosatrienoyl-CoA as exogenous substrates for 10 min at 37 8C in the absence and presence of 100 and 400 mM ethanol. Lipid extraction and analysis were carried out as described in Section 2.

preference for the saturated one. It is remarkable, however, that in both membranes the synthesis of these neutral lipids was at its lowest when the monounsaturated fatty acyl-CoA oleoylCoA was the substrate used in the esterification reaction. 4. Discussion It is well established that one primary route by which the fatty acid composition of membrane lipids is modulated is the deacylation–reacylation of phospholipids, or Lands pathway (reviewed by Yamashita et al., 1997). In vivo the synthesis of fatty acyl-CoA substrates of the reacylation processes requires acyl-CoA synthetase activity. Several other authors (Baker and Chang, 1983; Lin et al., 1988a) have studied the incorporation of fatty acids into membrane phospholipids by using them as

In the light of the fact that we had little recourse to data concerning the deacylation–reacylation processes of lipids in brain membranes, we firstly analyzed the extent of incorporation of the different fatty acyl-CoAs into neutral lipids of control mitochondria and microsomes (Table 1). With regard to the synthesis of CE, as we have previously shown (Marco et al., 1986b), the incorporation of fatty acyl-CoAs into CE is a direct measure of acyl-CoA:cholesterol acyltransferase (ACAT) activity. As can be seen, in chick-brain microsomes this enzyme activity showed the same specificity for all three fatty acids assayed. These results contrast with the higher affinity that liver microsomal ACAT shows for oleoyl-CoA (Chang et al., 1997). Although no similar cholesterol-esterifying activity has been described in mitochondria, our results demonstrate that in chick-brain mitochondria significant quantities of CE are synthesized from stearoyl-CoA but not from oleoyl-CoA or eicosatrienoyl-CoA, thus suggesting the presence of an ACAT activity that preferentially uses the saturated fatty acid derivative for the esterification processes. With regard to the incorporation of fatty acids into TG, our results demonstrate that the activity of the acylation process depends upon the degree of saturation of the fatty acid present and suggest the following pattern of acyl-CoA:diacylglycerol acyltransferase (DGAT) specificity for the different substrates used: 20:3 > 18:0 > 18:1. The profiles of incorporation into mitochondria also indicate a similar pattern, with a clearly lower affinity for oleoyl-CoA. The specificity of fatty acid incorporation into TG, particularly with regard to the degree of acyl-chain unsaturation, appears to vary according to the cell species and membrane in question (Lerique et al., 1994). Our results agree with those of other authors who have also demonstrated that brain DGAT shows high affinity for polyunsaturated fatty acids (Baker and Chang, 1983) and that relatively high levels of a seemingly arachidonate-specific

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DGAT activity have been reported in purified plasma membranes from rat-brain cortex (Lin et al., 1988a). In fact the fatty acid profile of TG in the brain is quite different from that in blood plasma. Fatty acids in brain TG are rich in 20:4 and 22:6 but contain only a low quantity of 18:0 (Cunnane and Chen, 1992), whereas in blood they are high in 18:2 but contain little polyunsaturated fatty acid (Spector, 2001). Although TG levels are quite low in brain membranes (0.2–0.5%), its specific profile in acyl groups indicate that in brain membranes TG may be engaged in some specific functions. In any case, the high rates of acylation found in our study indicate a rapid turnover of the acyl chains, suggesting that TG might be one of the most active lipids in the brain. As described in Section 3, 100 mM ethanol increased the incorporation of fatty acyl-CoA (except for stearoyl-CoA in mitochondria) into TG in both mitochondria and microsomes. Previous results obtained in our laboratory have also shown that long-term ethanol administration results in an increase in the incorporation of oleoyl-CoA into TG in brain and liver microsomes (Sa´nchez-Amate et al., 1991). Several studies carried out into erytrocytes from alcoholic subjects (Lerique et al., 1991; Gastaldi et al., 1991) have demonstrated that chronic alcoholism is associated with increases in the turnover rates of the acyl moieties of TG. A great deal of information exists concerning the accumulation of TG in the liver after both acute and chronic ethanol ingestion (Carrasco et al., 2001; Eaton et al., 1997). In spite of the fact that this accumulation has been put down to an increase in the intracellular redox-state due to ethanol metabolism via alcohol dehydrogenase, our data demonstrate that ethanol also directly affects microsomal DGAT, thus contributing to the stimulation of TG biosynthesis. 4.2. Effects of ethanol upon acylation of phospholipids Although experimental evidence does exist to describe phospholipid deacylation–reacylation processes, no consistent data has been obtained as yet to describe the characteristics and specificity of the enzymes involved in the remodeling of brain phospholipids. The profiles of incorporation of different fatty acids into individual phospholipids of control mitochondria and microsomes obtained in our work indicate that in neither phospholipid was the pattern of substrate preference consistent with the composition of the acyl groups in these membrane lipids. This has also been observed by other authors (Lin et al., 1988b) and suggests the involvement in the remodeling of phospholipids of both acyltransferase activities and other different mechanisms (e.g. base-exchange), which simultaneously contribute in vivo to the establishment of the specific fatty acid profile of membrane phospholipids. The distribution profiles in the phospholipid fractions of stearoyl-CoA, oleoyl-CoA and eicosatrienoyl-CA in microsomes and mitochondria indicate that, as might be expected, PC, the main phospholipid, exhibited the highest levels of fatty acid incorporation into both membranes, although the relatively low PC acylation observed with oleoyl-CoA and the clearly higher incorporation, especially into microsomes, when the fatty acid used was eicosatrienoyl-CoA are also noteworthy. Of

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similar importance is the fact that we found considerable differences in PI acylation in both membranes. In microsomes, for example, the incorporation of polyunsaturated fatty acid into PI was quite high, as opposed to that in mitochondria, in which incorporation diminished concomitantly with the unsaturation of the exogenous fatty acid. Our results agree with those of other authors who describe the high capacity of brain membranes to incorporate polyunsaturated fatty acids into PI and PC due to the presence of lysophosphatidylcholine and lysophosphatidylinositol acyltransferase activities, which have a high affinity for polyunsaturated fatty acid (Ross and Kish, 1994). This specificity might be put down to the brain’s need for a potential store of polyunsaturated fatty acids, which may be released from membranes in response to several stimuli. In mitochondria, on the other hand, the enzymes involved in the fatty acid remodeling of PC and PI show no special preference for polyunsaturated fatty acids. Finally, it is also noteworthy that both microsomal PE and PS showed higher acylation rates when oleoyl-CoA was used as exogenous donor. Thus, the results of our work with mitochondria show for the first time the existence of active mechanisms for acyl transfer from acyl-CoA derivatives to various polar and neutral lipids. In mitochondria, the remodeling mechanisms of neutral lipid and phospholipids seem to exhibit a marked preference for saturated fatty acids, contrary to the behaviour of microsomes, which present a higher affinity for polyunsaturated fatty acids. This suggests the involvement of different isoenzymatic activities in mitochondria and microsomes. With regard to the effects of ethanol on the phospholipid acylation of brain membranes, previous results obtained in our laboratory have demonstrated that chronic ethanol treatment specifically alters the turnover of phospholipids and TG acyl moieties in chick-liver and chick-brain microsomes (Sa´nchezAmate et al., 1991). Similarly, several studies have described the influence of alcoholism in reacylation processes in erythrocytes (Verine et al., 1991). Nevertheless, to the best of our knowledge, our results are the first to provide information concerning the initial effects of ethanol upon lipid acylation in brain membranes. As can be seen, the incubation of brain microsomes in the presence of 100 mM ethanol specifically stimulated the incorporation of oleoyl-CoA into the aminophospholipids PE and PS. Acylation of the other phospholipids, including PC, the main one present, remained unaltered. In a recent study (Carrasco et al., 2006), we demonstrated that ethanol in vitro alters the biosynthesis of aminophospholipids and their intramembrane transport in ratliver microsomes in different ways depending upon the fatty acid used in the acylation process. We have also demonstrated that chick-brain and hepatic microsomes subject to long-term ethanol treatment show a higher capacity to take up oleate into aminophospholipids than do control microsomes (Sa´nchezAmate et al., 1991). Thus, our results suggest that in microsomal membranes alcohol specifically interferes with the metabolism of aminophospholipids, lipid components that are involved in crucial and specific functions in biomembranes. Interestingly, after incubating mitochondria with 100 mM ethanol, only PI, a minor phospholipid, showed any specific

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alcohol-induced decrease in stearate incorporation, whilst the acylation of the other phospholipids remained unaltered. Although we can find no data on bird species, it is well known that mammalian cells in vivo maintain a remarkably consistent and simple molecular species composition of membrane PI, dominated by a single species containing stearate at the sn-1 position and arachidonate at sn-2. The mechanisms whereby cells acquire and maintain this PI composition are still not fully understood but our results indicate that ethanol causes an alteration in the remodeling of PI in mitochondria, probably due to an inhibition of the corresponding acyltransferase activity that uses stearate specifically as substrate. In the light of studies into isolated mitochondria, which have shown that proton leakage through the mitochondrial inner membrane correlates with the fatty acid composition of its phospholipids (Piquet et al., 2004), this ethanol-induced alteration may have repercussions upon the mitochondrial functions. 4.3. Synthesis of fatty acid ethyl esters One further aim of this study was to analyze the synthesis of FAEEs by chick-brain mitochondria and microsomes. This pathway is important in the metabolism of ethanol, particularly in tissues such as the brain, which have limited capacity to oxidize it via alcohol dehydrogenase. FAEEs have been shown to be mediators of ethanol-induced cell injury and may be an important factor in alcohol-induced organ damage (Laposata et al., 2002). In fact this neutral lipid does exert disordering effects on membranes, thus affecting such processes as the inhibition of protein synthesis and cell division in intact human hepatoblastoma cells (Doyle et al., 1996), the uncoupling of oxidative phosphorylation in isolated mitochondria (Lange and Sobel, 1983) and an increase in the fragility of isolated lysosomes (Haber et al., 1993). Interestingly, the organs most frequently damaged by ethanol abuse such as the pancreas, liver, heart and brain contain the highest levels of fatty acyl esters after ethanol abuse and thence their presence in the blood and tissues is a marker of ethanol intake in adults (Best and Laposata, 2003) and in neonates (Caprara et al., 2006). We have investigated the activity of FAEEs synthesis in both mitochondria and microsomes by analyzing the different fatty acyl-CoAs. Our results demonstrate that this non-oxidative ethanol metabolic pathway exists in both subcellular membranes and that its activity differs according to the degree of unsaturation of the fatty acyl chain. Thus, the synthesis of FAEEs increases concomitantly with an increase in ethanol from 100 to 400 mM and in both mitochondria and microsomes the synthesis of FAEEs was lowest when the monounsaturated fatty acyl-CoA was used as substrate. The enzyme system responsible for the formation of FAEEs seems, on the other hand, to have high specificity for polyunsaturated fatty acids in mitochondria whilst in microsomes ethyl stearate was the main FAEE synthesized. Little is known about the formation of FAEEs, although their synthesis has been described as being catalyzed by a variety of well-characterized enzymes (reviewed by Best and Laposata, 2003). Nowadays, however, the formation of FAEEs in vivo is generally attributed to the activity of two main enzymes: FAEE

synthase, an enzyme that uses free fatty acids as substrate (Lange, 1982) and acyl-CoA:ethanol acyltransferase (AEAT) (Diczfalusy et al., 1999). Since the ratio of acyl-CoA/free fatty acid is high under normal conditions, AEAT is probably the most important enzyme in FAEE synthesis. Our results confirm that microsomes can catalyze the synthesis of FAEEs from acyl-CoA and ethanol and suggest that this subcellular membrane shows low specificity for the monounsaturated fatty acyl-CoA. This low synthesis of ethyl oleate from chickbrain microsomes does not coincide with that described in human mononuclear cells, in which the only FAEE formed after exposure to ethanol was ethyl oleate (Alhomsi et al., 2006) or HepG2 cells, which show a preference for the synthesis of ethyl palmitate and ethyl oleate (Dan and Laposata, 1997), the two fatty acyl esters found in plasma after ethanol ingestion. Other authors have also described the existence of FAEE synthase activity in a crude membrane preparation from mouse brains (Zheng and Hungund, 1998). This membrane fraction showed preference for the polyunsaturated fatty acid for FAEE synthesis over the monounsaturated ones, although the authors did not analyze the specificity of this reaction for the saturated fatty acid. To our knowledge, no studies have been published to date concerning the synthesis of FAEEs by mitochondria, although Lange and Sobel (1983) have demonstrated that 72% of ethyl esters synthesized within the cell bind to mitochondria isolated from intact tissue incubated with ethanol. Our data demonstrate that mitochondria have the capacity to synthesize FAEEs from fatty acyl-CoA and ethanol and that AEAT activity seems also to be present in cell compartments other than microsomes. In addition, both synthesizing ethyl ester activities have different affinities for fatty acyl-CoAs: the mitochondrial activity uses preferably the polyunsaturated fatty acyl-CoA whilst the microsomal system shows higher affinity for the saturated exogenous fatty acyl-CoA. The results in mitochondria agree with the high amounts of polyunsaturated fatty ethyl esters found in the brains of individuals who died from ethanol intoxication (Refaai et al., 2003). Thus, the composition of FAEEs accumulated in different subcellular membranes may differ according to the membrane in question and this process may underlie the different effects of ethanol upon the various cell organelles. Acknowledgements Financial support for this work was provided by the Feder funds and the Ministerio de Ciencia y Tecnologı´a (Spain) under grant BMC2003-05886, by the Instituto de Salud Carlos III (Spain) under grant PI061268 and by the Junta de Andalucı´a. We thank Dr. J. Trout for his help in the correction of the manuscript. References Akbar, M., Baick, J., Calderon, F., Wen, Z., Kim, H.Y., 2006. Ethanol promotes neuronal apoptosis by inhibiting phosphatidylserine accumulation. J. Neurosci. Res. 83, 432–440.

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