Dietary ethanol stimulates the activity of phosphatidylcholine-specific phospholipase D and the formation of phosphatidylethanol in Drosophila melanogaster larvae

Insect Biochem. Molec. BioL Vol. 23, No. 6, pp. 749-755, 1993

0965-1748/93 $6.00 + 0.00 Copyright © 1993 Pergamon Press Ltd

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Dietary Ethanol Stimulates the Activity of Phosphatidylcholine-specific Phospholipase D and the Formation of Phosphatidylethanol in Drosophila melanogaster Larvae ROBERT R. MILLER JR,*t JONATHAN W. YATES,* BILLY W. GEER*:~ Received 30 June 1992; revised and accepted 29 December 1992

When administered in the diet to third instar Drosophila melanogaster larvae, short chain primary alcohols reduce phosphatidyleholine (PC) levels. The ethanol-induced reductions in larval PC may be in part due to an increase in the activity of PC-specific phospholipase D (PC-specific PLD, EC 3.1.4.4). PC-specific PLD not only hydrolyzes PC, but it also apparently catalyzes the formation of phosphatidylethanol. PC-specific PLD activity was also stimulated by 200 mM ethanol, methanol, isopropanol, n-butanol, and n-propanol. In vitro studies indicated that Drosophila PC-specific PLD activities were enhanced by submicromolar concentrations of Ca 2+ and by GTP-yS. In vivo studies utilizing [t4C]lyso-palmitoyl phosphatidylcholine indicated that dietary ethanol promoted the flux of label into triacylglycerol, 1, 2 diacylglycerol, and fatty acid ethyl esters, while the label in PC decreased. Choline Phosphatidylcholine PC-specific PLD Phosphatidylethanol ance Signal transduction Alcohol

INTRODUCTION Alcoholics suffering from ethanol-induced liver damage typically display abnormally low levels of phosphatidylcholine (PC) (Ailing et al., 1980). This ethanol-induced reduction in PC is not specific to vertebrates. Dietary ethanol and other short-chain primary alcohols increased the incorporation of label from [14C]glucose into larval phosphatidylethanolamine (PE), and decreased the incorporation of label into PC in Drosophila melanogaster larvae (Geer et al., 1986, 1991; Miller et al., 1992). Alcohol induced changes in PE and PC were not correlated with superior ethanol tolerance (Geer et al., 1991; Miller et al., 1993a). Ethanol-induced PC reductions and PE increases were seen in ethanol-tolerant and intolerant strains (Miller et al., 1993a), and alcohol*Department of Biology,Knox College,Galesburg, IL 61401, U.S.A. tPresent address: Department of Biology,Grand View College, Des Moines, IA 50316-1599,U.S.A. :~Author for correspondence. (1) Phosphatidylcholine

PC-specific PLC

Ethanol-induced

Ethanol toler-

induced reductions in PC levels may reflect cell damage. Larvae raised on choline-deficient media were more susceptible to toxic effects of ethanol, and exhibited a low level of PC accumulation (Geer et al., 1986; Tilghman and Geer, 1988). In an investigation to find out how dietary ethanol reduces PC levels we found that dietary ethanol inhibits the uptake of choline into D. melanogaster larvae (Miller et al., 1993a). The inhibition of choline uptake may not account for all of the ethanol-induced reductions in larval PC, because the incorporation of dietary [14C]choline was not significantly correlated to ethanolinfluenced PC levels (Miller et al., 1993a). The activities of phospholipases may be associated with PC levels in D. melanogaster. In vertebrates, PC-specific PLD (PLD, EC 3.1.4.4) and PC-specific PLC (PLC, EC 3.1.4.3) have been shown to hydrolyze PC, reduce intracellular levels of PC, and promote elevated levels of 1, 2 diacylglycerol (1, 2 DAG) (Besterman et al., 1986; Exton, 1990; Welsch and Schmeichel, 1991). The synthesis of 1, 2 DAG by PC hydrolysis is illustrated as follows:

, Phosphorylcholine + 1,2 Diacylglycerol

(2) Phosphatidylcholine Pc-sp~ificPLO Phosphatidate + Choline Phosphatidate Phosphohydrolase , 1,2 Diacylglycerol 749

750

ROBERT R. MILLER JR et al.

mixtures of 85/~1 contained 10/~1 substrate (341 mM 1,2-dipalmitoyl-sn-glycerol-3-phosphoryl [3H]choline (c. 6500DPMs per nmol), 40#1 tissue homoge~te (100-200 #g of protein), and 35/~I 40 mM Hepes bttlfer, pH 7; 0.1 mM MgCI2; 0.4% Triton X-100, and concentrations of GTPF S [guanosine 5'-(3-O-thio) triphosphate ranging from 0 to 400mM. Either CaC12 (0-1.0/tM], MATERIALS AND METHODS ethylene glycol bis-(fl-aminoethyl ether) N, N, N', N',tetraacetic acid (EGTA) (0-50 nM), or EDTA Chemicals (0-50 nM) were also present. Reaction mixtures were 1,2-dipalmitoyl-sn-glycerol-3-phosphoryl [3-H]choline incubated for 30 min at 22°C. Reactions were stopped by (sp. act. 31 Ci-mmol), lysopalmitoyl [1-14C]phosthe addition of 50~tl of ice cold 1% acetic acid in phatidylcholine (sp. act. 57mCi/mmol), and 25mCi methanol. [32P]orthophosphate salt (H3 PO4in water) were obtained GTP-TS was used because it is known to be an from New England Nuclear (Boston, Mass). Phos- activator of G-proteins and PC-specific PLD (Bocckino phatidylethanol was purchased from Avanti Polar et al., 1987). Exogenous CaC12, NaCI, EDTA, and Lipids (Alabaster, Ala), and tissue solubilizer was ob- EGTA were employed because in a variety o f systems, tained from Beckman Instruments (Fullerton, Calif.) the activities of PC-specific PLC (Roberts and Dennis, (BTS-450) and from New England Nuclear (Boston, 1989) and PLD (Kanfer, 1989) are enhanced by Ca 2÷. Mass) (Solvable). All other chemicals were purchased Taki and Kanfer (1979) found that concentrations of from Sigma Chemical (St Louis, Mo). CaC12 ranging to 5/~M stimulated the catalytic activity of rat brain-derived PC-specific PLD. Animals and culture conditions After the reactions were terminated, unhydrolyzed Third instar Canton-S larvae were employed in this lipids were removed through the addition of 0.5 ml study. To avoid incorporation of microbial-derived fatty chloroform-methanol (2:1, v/v) (Folch et al., 1957). The acids, Canton-S larvae and flies were axenically cultured radioactive choline and phosphorylcholine that were on sterile, defined media (Geer et al., 1985). Although liberated appeared in the upper, aqueous phases and wild type strains do not require exogenous fatty acids for were separated on silica gel thin layer chromatography growth, larvae readily incorporate exogenous fatty acids (TLC) plates using a solvent mixture of methanol, 0.6% into lipids (Keith, 1966, 1967; Geer and PeriUe, 1977; NaCI, and concentrated ammonium hydroxide (50: 50: 5 Geer et al., 1986). Prior to these studies, the Canton-S v/v/v). Choline (Rf = 0.244 _ 0.008) and phosphorylstrain had been axenically maintained on defined choline (Rf = 0.437 ___0.002) TLC plates were visualized medium for more than 8 yr. In all experiments, 4-day on TLC plates with DragendortFs reagent and molybpost-hatch (third instar) larvae were transferred to sterile denum blue (Sigma Chemical, St Louis, Mo) and were defined medium with 14.6mM (0.5% (w/v)) sucrose identified by co-migration with standards. Choline and supplemented with various concentrations of either phosphorylcholine were removed from the TLC plates methanol, ethanol, butanol, n-propanol, or isopropanol. and placed into scintillation vials containing 0.5 m l of Dietary ethanol concentrations in test diets varied from tissue solubilizer. After 1 h incubation at 600C, 10 ml of 0 to 925 mM. Methanol, n-butanol, n-propanol, and scintillation cocktail was added and radioactivity moniisopropanol were tested at 200 mM concentrations. Lar- tored in a Beckman LS 6000 IC liquid scintillation vae were incubated at 22.5°C for 15 h light:9h dark counter using the auto-DPM computer program (Beckphotoperiods at 45% r.h. At 6 days post-hatch, third man instruments, Fullerton, Calif.). PC-specific PLC instar larvae were removed from the test media for and PLD activities were linear over a 30 min incubation biochemical analysis. time in a reaction mixture with 100-200 #g of homogenate protein. The determination of PC-specific phospholipase C and D The formation of phosphatidylethanol activities PC-specific PLD catalyzes a transphosphatidylation The effects of ethanol on Drosophila-PC-specific PLC and PLD activities were determined by a modification of reaction where the choline head group is cleaved from the assay of Qian and Drewes (1989). 40 Canton-S larvae PC and replaced with ethanol. The product of this per sample were homogenized in 0.4 ml 40 mM Hepes reaction is phosphatidylethanol (Ailing et aL, 1984; Sale ( N - 2 - hydroxypiperazine - N' - 2 - ethanesulfonic acid) et al., 1989). This reaction has been observed in REF52 buffer, pH 7.0; 0.1 mM MgCI2; 0.07mM PTU; 0.2mM fibroblasts (Cabot et al., 1988, 1989). H-60 granulocytes DTT and were centrifuged at 15,000g for 15 min. Ali- (Pai et al., 1988a, b; Tettenborn and Mueller, 1988), quots were removed for enzyme analysis and protein hepatocytes (Bocckino et aL, 1987; Qian and Drewes, determination (Bradford, 1976). To measure the hydro- 1989), vasular endothelial ceils (Martin, 1988; Martin lytic activities of PLC and PLD, 1,2-di-palmitoyl-sn- and Michaelis, 1988), neuronal cells (Martinson et al., glycerol-3-phosphoryl [3H]choline (c. 6500DPMs per 1989; Lavie and Liscovitch, 1990; Gustavsson and nmol) was used as the exogenous substrate. Reaction Hansson, 1990), and lymphocytes (Pai et al., 1987;

In the current study we examined the affects of dietary alcohols on the activities of PC-specific phospholipase D and/or PC-specific phospholipase C and on the formation of phosphatidylethanol in D. melanogaster larvae.

ALCOHOL-INDUCED PC-SPECIFIC PLD ACTIVITY Mueller et al., 1988; Agwu et al., 1991). PC-specific PLD has been suggested as a marker for human alcoholism (Mueller et al., 1988). To validate the previously described PC-specific PLD assay, the in vivo formation of phosphatidylethanol was monitored in third instar Canton-S larvae. 4-day posthatch larvae were transferred to sterile defined medium containing 14.6 mM (0.5% (w/v)) sucrose and 25 mCi [32p]orthophosphate salt (c. 165,000DPMs/#I food). Dietary ethanol concentrations ranged from 0 to 925 mM. After 48 h of exposure, 50 larvae were collected per sample and homogenized in 400 #1 10 mM potassium phosphate buffer, pH7.2; 2 m M Na2H2EDTA (disodium ethylenediamine-tetraacetic acid); 0.2mM dithiothreitol; and 0.07 mM phenylthiourea and centrifuged for 15 min at 10,000g. Aliquots were removed and assayed for protein (Bradford, 1976). Lipids were extracted from the homogenates by the method of Folch et al. (1957) and were fractionated on activated silica gel TLC plates using the short bed/continuous-development TLC system of Welsch and Schmeichel (1991). Lipids were visualized by spraying the plates with 10% CuSO4 in 10% H3PO 4 and charring the plates at 100°C for 20 min. [32p]phosphatidylethanol spots were identified by their co-migration with nonlabelled phosphatidylethanol and counted for the incorporation of label.

Phospholipids were fractionated by TLC (Gilfillan et al., 1983). Neutral lipids were fractionated on activated silica gel TLC plates using hexane diethyl ether-acetic acid (85:15:2 v/v/v). Fatty acid ethyl esters (FAEE), triacylglycerol (TAG), free fatty acids (FFA), 1, 2 DAG, and 1, 3 diacylglycerol (1, 3 DAG) were resolved by this system. Lipid spots were visualized by either 12 staining or acid charring and identified with appropriate standards. The lipids were eluted with 500/~1 of either 2:1 chloroform-methanol (v/v) for phospholipids or 5:1 chloroform-methanol (v/v) for neutral lipids, and the radioactivity determined by liquid scintillation methods (Geer and Downing, 1972). Statistical methods

Analyses were carried out using the Statview 512+ program (Brain Power, Calabasas, Calif.) and analysis of variance and the t-test as described by Snedecor and Cochran (1980). RESULTS The effects of ethanol on PC-spcific P L C and PLD Dietary ethanol exhibited biphasic control over the activities of PC-specific PLD and PLC [Fig. I(A)]. Low to moderate levels of dietary ethanol caused increased enzymatic activities, but at higher concentrations,

The production of 1, 2 diacylglycerol by PC-specific PLD

In vertebrate cell types, the degradation of phosphatidylcholine by PC-specific PLD catalyzes the formation of choline and phosphatidic acid. Phosphatidic acid is converted into 1,2diacylglycerol by phosphatidate phosphohydrolase (PAPase). Thus, the degradation of PC by PC-specific PLD promotes elevated levels of 1, 2 D A G and is argued to be a form of signal transduction (Besterman et al., 1987; Billah et al., 1989; Exton, 1990; Welsch and Schmeichel, 1991). This suggests that the ethanol-induced stimulation of Drosophila PCspecific PLD may promote the formation of 1, 2 DAG levels. To test this possibility, a modification of the technique of Billah et al. (1989) was employed. Surface-sterilized embryos were transferred to the sterile, defined media (Geer et al., 1986) containing 29.2 mM [1% sucrose (w/v)] and 500 #M [t4C]lysopalmitoyl phosphatidylcholine [(L-l-[Palmitolyl-l-14C]phosphatidylcholine)] (c. 80,500 DPMs/ml of media). After a 4 day pulse of label, larvae were chased by removing the larvae from labeled media and placing the larvae onto unlabeled media containing 14.6mM (0.5%) sucrose supplemented with ethanol in concentrations ranging to 925 mM. After 48 h of exposure, 50 larvae per sample were collected and homogenized in 400/~1 10 mM potassium phosphate buffer, pH 7.2; 2 mM Na2H2EDTA; 0.2 mM dithiothreitol; and 0.07 mM phenylthiourea and centrifuged for 15 min at 10,000 g. Aliquots were removed and assayed for protein (Bradford, 1976). Lipids were then extracted from the homogenates by the method of Folch et al. (1957).

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FIGURE 1. The effect of dietary ethanol on the activities of PCspecific PLD and PC-specificPLC (A) and the in vivo formation of phosphatidylethanol (B) in third instar Canton-S larvae. Means+ SD are shown. Ethanol-fed groups significantlydifferedfrom ethanol-free diets at: 1, P ~<0.05 and 2, P ~<0.0001.

752

ROBERT R. MILLER JR et al.

PC-specific PLD and PLC activities were near control levels. When larvae were fed an ethanol-free diet, PCspecific PLD activity was 0.154 +0.057nmol of PC consumed/min/mg of protein, while PC-specific PLC activity was 0.070+0.033nmol of PC consumed/ min/mg of protein. PC-specific PLD activity increased c. 4-fold in larvae fed 400 mM ethanol, while PC-specific PLC activity increased by only 2.5-fold. However, in larvae fed 925 mM ethanol, PC-specific PLC and PLC activities were close to control levels. Thus, although low to moderate levels of dietary ethanol stimulated the activities of PC-specific PLD and PLC, high concentrations of dietary ethanol impaired activity. The ability of low-moderate concentrations of dietary alcohol to enhance PC-specific PLC and PLD activities were not ethanol-specific. 200mM methanol, isopropanol, butanol, and n-propanol all increased the activities of PC-specific PLD and PLC (Table 1). Thus, the stimulation of PC-specific PLC and PLD activities is shared by a number of short chain alcohols. The in vivo synthesis of phosphatidylethanol Dietary ethanol promoted the formation of phosphatidylethanol in wild type larvae [Fig. I(B)]. Phosphatidylethanol formation was biphasic where low to moderate levels of dietary ethanol caused a 2-5-fold increase in labeled phosphatidylethanol levels. However, at high concentrations of dietary ethanol, the incorporation of label into phosphatidylethanol decreased. No significant differences were seen when comparing larvae fed an ethanol-free diet to larvae fed a 925 mM ethanol diet. Nonetheless, larvae fed 200, 400, and 600 mM ethanol diets all exhibited enhanced accumulation of label into phosphatidylethanol fractions as compared to larvae fed an ethanol-free diet (F = 93.763; d.f. = 4, 25; P ~<0.0001). Because PC-specific PLD catalyzes the hydrolysis of PC and the transphosphatidylation reaction that forms phosphatidylethanol (Alling et al., 1984; Sale et aL, 1989), these two reactions should be correTABLE I. The effects of dietary alcohols on relative PC-specific PLD and PLC activities in Canton-S larvae Diets No alcohol 200 m M ethanol 200 m M methanol 200 mM isopropanol 200 mM n-butanol 200 m M n-propanol

Relative PLC activity

Relative PLD activity

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F I G U R E 2. The effect of Ca 2+ (m) and GTP-TS (B) on the activities of PC-specific PLD and PLC in third instar Canton-S larvae. Means + SD are shown. Significant differences were observed in the PC-specific PLD activities (B) between the GTP-yS-treated and GTPyS-free supernatants at: 1, P ~< 0.02 and 2, P ~<0.002.

lated [Fig. 1(A) and (B)]. When PC-PLD activities were correlated to endogenous levels of PC (Miller et al., 1993a) a Pearson product-moment correlation coefficient of -0.550 was observed (F = 14.74; d.f. = 1, 34; P < 0.0005). When PC-PLD activities were correlated to phosphatidylethanol formation, a Pearson product-moment correlation coefficient of 0.657 was observed (F = 18.955; d.f. = 1, 25; P ~<0.0002). The effect of Ca e÷ on PC-specific PLD The in vitro activity o f PC-specific PLD was stimulated by high levels of Ca 2÷ [Fig. 2(A)]: F = 53.169; d.f.=3,25; P~<0.0001), especially Ca 2+ levels of 700-800 nM. PC-specific PLC was influenced to a much lesser extent by Ca 2÷. Although activities of PC-specific PLD were stimulated by Ca 2÷, the removal of available Ca 2+ by the addition of EGTA failed to eliminate all of the activities, indicating that Drosophila-PC-specific PLD and PLC activities are not solely dependent on C a 2+"

Data presented as m e a n s _ S D where n = 8 - 1 3 observations per experimental group. Relative enzymatic activity was calculated by dividing the enzymatic activity of individual samples by the mean activity of the control group. All data was subjected to 1-way analysis of variance and a t-test. All o f the test groups that were fed an alcohol differed from the group that was fed an alcohol-free diet at the * ~<0.005 level according to a t-test.

To test whether Drosophila-PLD and PLC requires divalent ions for activity, PC-specific PLD and PLC activities were measured in the presence and absence of 20 mM EDTA. In the presence of 20 mM EDTA, PCspecific PLD activity was 0.128_+0.027nmol of PC consumed/min/mg protein, but without EDTA, PCspecific PLD activity was 0.131 _+0.030 nmol of PC

ALCOHOL-INDUCED PC-SPECIFIC PLD ACTIVITY

consumed/min/mg protein. This difference was insignificant (F = 0.056, d.f. = 1, 14, P = 0.82). Consequently, the in vitro activity of Drosophila PC-specific PLD can be stimulated by Ca 2÷, but does not require divalent cations for activity. In contrast, the in vitro activity of Drosophila-PLC required divalent ions to be active [Fig. 2(A)]. In the presence of 20 mM EDTA, PC-specific PLC activity was 0.021+0.012nmol of PC consumed/min/mg protein; whereas, the PC-specific PLC activity was 0.065 + 0 . 0 2 6 n m o l of PC consumed/min/mg protein without EDTA. This difference was significant (ANOVA, F = 17.98, d.f. = 1, 14, P = 0.0008) and indicated that PC-specific PLC is more dependent on divalent cations than PC-specific-PLD.

The effect of GTP-TS on PC-specific PLD Although the enzymatic activity of PC-specific PLC was not stimulated by GTP-7-S (F = 1.868; d.f. = 4, 20; P = 0.1555), exogenous GTP-v S significantly enhanced the activity of PC-specific PLD (F = 6.299; d.f. = 4, 20; P = 0.0019) [Fig. 2(B)]. PC-specific PLD activity significantly increased in the presence of 50mM GTP-TS (t = 3.024; d.f. = 8; P ~<0.01). Since GTP-vS is a known activator of G-proteins, the activity of Drosophila PCspecific PLD may be influenced by G-proteins.

The hydrolysis of PC and lyso PC and the formation of neutral lipids When Canton-S embryos were pulse-chased with lysopalmitoyl [l-~4C]phosphatidylcholine, a significant amount of label was found in the PC and lyso-PC pools at 6 days post-hatch. When chased with ethanol-free media, c. 2.5 times more label was found in the PC pool than in the lyso PC pool (Fecpool= 223.709_ 19.693 DPM/ mg protein; F~ysoPC= 90.381 _ 8.298 DPM/mg protein). When chased with ethanol-containing media, the incorporation of label significantly decreased into both PC and lyso PC (Fig. 3) (Fpcpool= 151.116; d . f . = 4 , 2 0 ; P ~<0.0001; FlysoPC = 38.539; d.f. = 4, 20; P ~<0.0001).

250

200 , t ~

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FIGURE 3. The effect of dietary ethanol on the incorporation of label from lyso-l-[palmitolyl-lJ4C]phosphatidylcholine into phosphatidylcholine (PC) and lysophosphatidylcholine (lyso-PC) in third instar Canton-S larvae. Means __+SD are shown. Ethanol-fed groups significantly differed from ethanol-free diets at: 1, P~<0.01 and 2, P ~<0.0001.

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FIGURE 4. The effect of dietary ethanol on the incorporation of label from lyso-l-[palmitolyl-lJ4C]-phosphatidylcholine into neutral lipids in third instar Canton-S larvae. (A) shows the accumulation of label in the triacylglycerols (TAG) and (B) shows the accumulation of label in 1, 2 diacylglycerol (1, 2 DAG) and 1, 3 diacylglycerol (1, 3 DAG). Means _ SD are shown. Ethanol-fed groups significantly differed from groups fed ethanol-free diets at: 1, P ~<0.001.

The 2-2.5-fold decrease in labeled PC and lyso PC is correlated with the enhanced hydrolytic activity of PCspecific PLD (labeled PC vs PC-specific PLD activity: r = 0.734; F = 24.470; d.f. = 1, 21;P ~< 0.0001) (labeled lyso PC vs PC-specific PLD activity: r = 0 . 6 5 0 ; F = 15.360; d.f. = 1, 21; P ~<0.0001). The ethanol-induced decrease in labeled PC also correlates with previous data which shows that ethanol promotes a decrease in the total PC pool (Miller et al., 1993a) (r = 0.833; F = 49.775; d.f. = 1, 22; P ~<0.0001). Ethanol-containing diets promoted a loss of label in PC and the lyso PC, and ethanol-containing diets increased the incorporation of label into different neutral lipids. TAG was the most heavily labeled lipid. When chased with ethanol-free media, approximately twice as much label was found in TAG [Fig. 4(A)] as compared to PC (Fig. 3) and approximately five times as much label was found in TAG was seen as compared to the lyso PC pool (Fig. 3). When chased with ethanol-containing media, a 2-4-fold increase in the incorporation of label TAG was observed as compared to larvae chased with ethanol-free media [Fig. 4(A)] (F = 27.173; d.f. = 4, 29; P ~<0.0001). Ethanol-induced incorporation of label into other neutral lipids was lower, but it was observed in the 1, 2 D A G pool (F = 12.300; d.f. = 4, 29; P ~<0.0001),

754

ROBERT R. MILLER JR et al.

1, 3 DAG (F = 11.453; d.f. = 4, 29; P ~<0.0001) [Fig. 4(B)], and FAEE [F = 29.075; d.f. = 4, 29; P ~<0.0001) [Fig. 5(B)]. When chased with unlabeled media containing 200mM ethanol, a 2-fold increase in label in 1, 2 DAG [Fig. 4(B)] and FAEE (Fig. 5) was observed when compared to larvae chased with ethanol-free media (1,2DAG: t = 5.598; d.f. = 11; P ~<0.0001) (FAEE: t = 6.095; d.f. = 11; P ~<0.0001). Incorporation of label into 1, 2 DAG plateaued when chased with unlabeled media containing > 200 mM ethanol. Although the incorporation of label into FAEE increased when larvae were fed unlabeled diets containing 200 to 400 mM ethanol, reduced incorporation was observed when larvae were fed unlabeled media with 600 or 925 mM ethanol (Fig. 5). Thus, the incorporation of label into FAEE appears to be biphasic (Fig. 5) and was associated with the incorporation of label into FFA (r = 0.718; F = 34.064; d.f. = 1, 32). The FFA level exhibited a reciprocal relation to the FAEE level; when one increased, the other decreased. A 1-way ANOVA indicated that the ethanol-induced changes in the incorporation of label into FFA was significant ( F = 52.372; d.f. = 4, 29; P ~<0.0001). Increased incorporation of label into 1, 3 DAG was only observed in larvae chased with unlabeled media containing 925mM ethanol [Fig. 4(B)] (t = 5.938; d.f.= 10; P~<0.0001). Increased levels of labeled 1, 3 DAG correlated with increased levels of labeled FFA (r =0.572; F = 15.529; d.f. = 1, 32; P ~<0.0004). The incorporation of label into 1, 3 DAG was not significantly correlated with the incorporation of label into other neutral lipids. DISCUSSION

Two systems that may constitute minor pathways for ethanol elimination in Drosophila larvae were evident in this study: (1) the formation of FAEE and (2) the formation of phosphatidylethanol. Both pathways have previously been described in ethanol-stressed vertebrates 120 100

2

"~ 40

0 0

200

400 600 800 mM Dietary Ethanol

1000

FIGURE 5. The effect of dietary ethanol on the incorportion of label from lyso-l-[palmitolyl-1-14C]phosphatidylcholine into free fatty acids (FFA) and fatty acid ethyl esters (FAEE) of third instar Canton-S larvae. M e a n s + SD are shown. Ethanol-fed groups of larvae significantly differed from ethanol-free diets at: 1, P ~<0.01 and 2, P ~<0.0001.

(Mueller et al., 1988; Bora and Lange, 1991). The observation of these pathways in Drosophila indicates that the pathways are evolutionarily conserved. By labeling the FAEE pool with [14C]ethanol, we found that FAEE formation is increased when the FFA pool is increased in larvae by dietary stearic acid (R. R. Miller Jr, unpubl.). Nonetheless, FAEE formation is a very minor pathway since larvae normally possess a very small FFA pool (Heinstra et al., 1990). The physiological consequences of phosphatidylethanol formation and FAEE formation are unknown. Dietary alcohols also stimulate the activity of PCspecific PLD. The activation of this enzyme not only catalyzes the formation of phosphatidylethanol (Ailing et al., 1984; Sale et al., 1989), but also promotes the ethanol-induced hydrolysis of PC. Multiple mechanisms apparently exist for the alcohol-induced decrease in larval PC (Geer et al., 1991; Miller et al., 1991, 1993a). These apparently include the ethanol-induced inhibition of choline uptake by larvae and the enhanced activity by PC-specific PLD. It has been demonstrated in other systems that PCspecific PLD activity is affected by a variety of molecules that include Ca2+-mobilizing hormones, dibutyryl cAMP, cAMP, theophylline, forskolin, prostaglandin E2, and GTP-y S. This implies that PC-specific PLD may be regulated by G-proteins. PC hydrolysis by PC-specific PLD and/or PLC may lead to the formation of diacyglycerols and subsequent mobilization of Ca 2+. This sequence of events may be part of a signal transduction system (Besterman et al., 1987; Billah et al., 1989; Exton, 1990; Welsch and Schmeichel, 1991). In the present study we demonstrated in vivo that dietary alcohols stimulate PC-PLD activity. Our in vitro studies indicate that GTP-y S and C a 2+ stimulate Drosophila-PC-specific PLD. By using L-l-[Palmitolyl-l-14C]phosphatidyl choline to label the PC and lyso-PC pools, we also demonstrated that dietary ethanol promotes the accumulation of label in the 1, 2 DAG pool. The flow of label from the PC and lyso-PC pools into the 1, 2 D A G pool suggests that a PC-mediated form of signal transduction exists in alcohol-stressed Drosophila larvae. Our recent observations in D. melanogaster suggest that ethanol-induced signal transduction is associated with cell damage (Geer et al., 1989; Miller et al., 1993a, b). This agrees with Hock and Rubin's (1990) conclusion that an ethanol-induced form of signal transduction exists in the vertebrate liver a n d that signal transduction is associated with ethanol-toxicity. Alcohol tolerance in D. melanogaster larvae and other organisms may be dependent on the capacity of cells to avoid this mode of signal transduction.

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