ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 228, No. 1, January, pp. 185-196, 1984 The Effect of Hypolipidemic Drugs WY14643 and DH990, and Lysophospholipids on the Metabolism of Oleate in Plants JUAN SANCHEZ’ AND PAUL K. STUMPF2 Department of Biochemistry and Biophysics, University of Cal@rnia, Davis, California 95616 Received July 5, 1983, and in revised form August 30, 1983 The effects of the addition of hypolipidemic drugs and 1-acylglycerolipids on the metabolism of oleate in plants have been studied in vivo and in vitro. Using aged potato slices with [‘*C]oleate as a precursor, it was found that these drugs markedly inhibited both the incorporation into complex lipids and the desaturation of oleate to linoleate. Moreover, in vitro experiments, carried out with microsomes prepared from developing safflower seeds and [‘4C]oleate or [14C]oleoyl-CoA as precursors, confirmed the inhibitory effect of the drugs on oleate desaturation, and showed that while WY14643 mainly affected oleoyl thiokinase activity, DH990 exerted its strongest effect on the formation of PL, indicating that the mode of action of these two drugs in safflower microsomes is essentially different. Addition of LPC or LPE stimulated the incorporation of ra- diolabeled precursor into PC and PE, respectively, as well as the desaturation of oleate to linoleate when [14C]oleoyl-CoA was the precursor. The evidence obtained suggests that oleoyl-PE, as well as oleoyl-PC, should be considered as a possible substrate for oleate desaturation in plants. Gurr et aL (1) were the first to present evidence that oleoyl-PC3 was the imme- diate substrate of the oleate desaturase in plants. Subsequent data provided further evidence to support these initial observa- tions [see (2-4) for reviews]. These exper- iments showed that the formation of oleoyl-PC occurred before the desaturation of 18:1, and that the product of the desat- uration, 18:2, appeared first esterified to PC. Nevertheless, the solubilization and purification of the Al2 desaturase, and the subsequent employment of suitable sub- strates, has not been carried out as yet. * Present address: ARGO Plant Cell Research In- stitute, 6560 Trinity Co&t, Dublin. Calif. 94568. ’ To whom correspondence should be addressed. ’ Abbreviations used: BSA, bovine serum albumin; GLC, gas-liquid chromatography; TLC, thin-layer chromatography; PC, phosphatidyl choline; PE, phosphatidylethanolamine; L-, lyso; FFA, free fatty acid, TG, triglyceride; PL, polar lipid, DG, diazoglyc- eride; PCS, phase-combining system; PG, phospho- glycerol. In order to gain more information con- cerning the correlation between formation of oleoyl-PC and desaturation of the oleoyl moiety, we have studied (a) the effect of lysophospholipids on PC formation and desaturation; and (b) the effect of two hy- polipidemic drugs, WY14643 ([4-chloro(2,3- xylidino)-2-pyrimidinylthiolacetic acid) and DH990 (2-[3,5-di-(t-butyl-4-hydroxy- phenyl)thio]hexanoic acid), which have been recently reported to inhibit the for- mation of PC from oleoyl-CoA and LPC in rat liver microsomes (5). The results of experiments carried out both in vivo and in vitro, with [‘*C]oleate and [14C]oleoyl-CoA as precursors, in the absence and presence of these two drugs are presented in this paper. MATERIALS AND METHODS Mutti Potato (Solarium tuberasum) tubers were purchased in the local market. Developing safflower seeds (Carthamus tinetorius var. Portugal) were har- vested from the field and stored at -20°C until used. 185 0003-9861/84 $3.00 Copyright 0 lSS4 by Academic Press, Inc. All rights of reproduction in any form reserved.
Transcript
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 228, No. 1, January, pp. 185-196, 1984
The Effect of Hypolipidemic Drugs WY14643 and DH990, and Lysophospholipids on the Metabolism of Oleate in Plants
JUAN SANCHEZ’ AND PAUL K. STUMPF2
Department of Biochemistry and Biophysics, University of Cal@rnia, Davis, California 95616
Received July 5, 1983, and in revised form August 30, 1983
The effects of the addition of hypolipidemic drugs and 1-acylglycerolipids on the metabolism of oleate in plants have been studied in vivo and in vitro. Using aged potato slices with [‘*C]oleate as a precursor, it was found that these drugs markedly inhibited both the incorporation into complex lipids and the desaturation of oleate to linoleate. Moreover, in vitro experiments, carried out with microsomes prepared from developing safflower seeds and [‘4C]oleate or [14C]oleoyl-CoA as precursors, confirmed the inhibitory effect of the drugs on oleate desaturation, and showed that while WY14643 mainly affected oleoyl thiokinase activity, DH990 exerted its strongest effect on the formation of PL, indicating that the mode of action of these two drugs in safflower microsomes is essentially different. Addition of LPC or LPE stimulated the incorporation of ra- diolabeled precursor into PC and PE, respectively, as well as the desaturation of oleate to linoleate when [14C]oleoyl-CoA was the precursor. The evidence obtained suggests that oleoyl-PE, as well as oleoyl-PC, should be considered as a possible substrate for oleate desaturation in plants.
Gurr et aL (1) were the first to present evidence that oleoyl-PC3 was the imme- diate substrate of the oleate desaturase in plants. Subsequent data provided further evidence to support these initial observa- tions [see (2-4) for reviews]. These exper- iments showed that the formation of oleoyl-PC occurred before the desaturation of 18:1, and that the product of the desat- uration, 18:2, appeared first esterified to PC. Nevertheless, the solubilization and purification of the Al2 desaturase, and the subsequent employment of suitable sub- strates, has not been carried out as yet.
In order to gain more information con- cerning the correlation between formation of oleoyl-PC and desaturation of the oleoyl moiety, we have studied (a) the effect of lysophospholipids on PC formation and desaturation; and (b) the effect of two hy- polipidemic drugs, WY14643 ([4-chloro(2,3- xylidino)-2-pyrimidinylthiolacetic acid) and DH990 (2-[3,5-di-(t-butyl-4-hydroxy- phenyl)thio]hexanoic acid), which have been recently reported to inhibit the for- mation of PC from oleoyl-CoA and LPC in rat liver microsomes (5).
The results of experiments carried out both in vivo and in vitro, with [‘*C]oleate and [14C]oleoyl-CoA as precursors, in the absence and presence of these two drugs are presented in this paper.
MATERIALS AND METHODS
Mutti Potato (Solarium tuberasum) tubers were purchased in the local market. Developing safflower seeds (Carthamus tinetorius var. Portugal) were har- vested from the field and stored at -20°C until used.
185 0003-9861/84 $3.00 Copyright 0 lSS4 by Academic Press, Inc. All rights of reproduction in any form reserved.
186 SANCHEZ AND STUMPF
[1-“‘CjOleic acid (57 mCi/mmol) and [1-‘“Cjoleoyl-CoA (60 mCi/mmol) were obtained from Amersham Searle. DH990 was obtained from Dr. J. Heeg, Merrell Dow Pharmaceuticals Inc., Indianapolis, Indiana, and WY14643 was a kind gift from Dr. W. J. Baumann, The Hormel Institute, Austin, Minnesota. All other reagents were from Sigma.
Preparation of aged potato slices and so&lower mi- cro.somca Disks (1 cm diameter X 0.1 cm) were pre- pared from potato tubers. After rinsing thoroughly with distilled water, they were aged in 0.1 mM CaS04 for 16 h in the dark at room temperature and with constant shaking. Safflower microsomes were pre- pared from 2 to 3 g of cotyledons after removing seed coats. The tissue was ground with a mortar and pestle in 8 ml of 0.1 M Tris/HCl, pH 7.5, containing 0.33 M sorbitol, 1 mM EDTA, 1 mM MgClr, 1 mM pmercap- toethanol, and 0.1% BSA. After filtering through Mir- acloth, the residues were reground three more times with 6 ml of the same buffer. The whole filtrate was centrifuged at 18,OOOg for 20 min and the pellet dis- carded. The supernatant was carefully filtered through Miracloth to remove the fat layer and centrifuged at 105,OOOg for 1 h. Microsomal pellets were routinely resuspended in 2 to 3 ml of incubation buffer, con- sisting of 0.1 M potassium phosphate, pH 7.2, con- taining 10 mM MgCl, and 0.1% BSA, and immediately used in incubations.
Incubations In experiments carried out in viva with aged potato slices, 0.5 g of tissue was incubated in 0.5 ml of 0.1 M potassium phosphate, pH 7.2, buffer containing 0.5 &i of ammonium [1-i4C]oleate and, where indicated, 2.5 rnbi of DH990 (which had been neutralized beforehand with KOH) or 2.5 rnbi of WY14643. Incubations were carried out at room tem- perature for 3 h and with constant shaking.
Safflower microsomes were incubated in 0.25 ml of the incubation buffer described above containing 0.05 pmol of NADH and the equivalent to approximately 0.5 mg of microsomal protein. When [“Cjoleate was the precursor, 1 pmol of ATP and 0.1 pmol of CoA were added to the incubation medium. Incubations were carried out at room temperature with constant shaking and, unless otherwise stated, for 1 h.
And~sis. At the end of the incubations carried out with aged potato slices, the remaining radiolabeled precursor was removed with a Pasteur pipet, and the tissue was then rinsed twice with 1 ml of distilled water. Two milliliters of isopropanol was added into each tube and the reaction mixtures were kept at 60°C for 20 min. After cooling the tubes, 3 ml of hexane was added and the tissue was homogenized with a Teflon homogenizer to extract lipids according to Hara and Radin (6). Residues were reextracted with 5 ml of hexane-isopropanol (32). The mixture was filtered and, after separation into two phases by addition of l/2 vol of 6.7% NarSO,, aliquots were taken from both layers to determine radioactivity by liquid
scintillation. The aqueous layer was saponified, after addition of 60% KOH to make a final concentration of 6%, by heating at 75°C for 30 min. After acidifi- cation, fatty acids were extracted with chloroform and, after blowing down the solvent under nitrogen in the hood, methylated with 2.5% H.#O, in methanol- benzene (95:5) at 75°C for 20 min. Methyl esters were extracted with petroleum ether and analyzed by radio- GLC. Aliquots were taken from the organic phases and methyl esters were prepared in the same way for fatty acid analysis. Lipids in the organic layer were separated by TLC on silica gel plates, using pe- troleum ether-diethyl ether-acetic acid (60:90:3) as the first solvent, followed by chloroform-methanol- water (65:25:4) as the second solvent run up to the fatty acid band. Standards of PC, PE, and FFA were cochromatographed, and they were visualized by io- dine staining. Radioactive lipids were located with a Packard radiochromatogram scanner (Model 7201). Radioactive bands were scrapped and silica gel was put into counting vials containing 1 ml of methanol- water (l:l), and radioactivity was counted with PCS- xylene (21). In those experiments carried out in vitro with safflower microsomes, aliquots of 25 nl (one- tenth) were taken and saponified with 1 ml of 6% KOH in methanol-water (1:l) in the presence of 200 pg of heptadecanoic acid. Saponification and subse- quent preparation of methyl esters for fatty acid analysis were performed as described above. To the remaining incubation mixture (225 pl), 4 ml of chlo- roform-methanol (21) was added and, after vortexing to make a single phase, acyl derivatives were separated in two layers after addition of 0.8 ml of 0.7% NaCl; the aqueous phase contained acyl-CoAs, whereas lipids partitioned into the lower organic layer (7). Lipids were chromatographed on silica gel plates as described before, and radioactive bands were scrapped either for determination of radioactivity by liquid scintil- lation or analysis of fatty acids by radio-GLC. In the second case, lipids, adsorbed on the silica gel, were submitted to saponification in 2 ml of 6% KOH in methanol-water (1:l) in the presence of 500 pg of (corn) oil. After acidification, fatty acids were ex- tracted twice with 4 ml of petroleum ether, and methyl esters were prepared as described above.
Protein was determined according to Lowry et aL (8) in the presence of 0.1% sodium deoxycholate and with BSA as standard.
RESULTS
Effect of Drugs In Viva
Aged potato slices were chosen to study the effect of DH990 and WY14643 on the metabolism of [14C]oleate because this sys- tem had a remarkable capacity to synthe- size linoleic acid as the major product from
OLEATE METABOLISM IN PLANTS 187
[14C]acetate (9), as well as desaturate ex- ogenously added [14C]oleate (10). It was confirmed that aged potato-tuber tissues incorporated radioactivity from [14C]oleate into several complex lipids (Table I), mainly PC and TG. Considerable conver- sion of oleate to linoleate was also ob- served.
On addition of hypolipidemic drugs, a significant (WY14643), or dramatic (DH990) inhibition of those activities was observed. The effectiveness of these two drugs in inhibiting incorporation into PC correlated well with the results of Par- thasarathy et al. (5), who reported that DH990 was a more effective inhibitor of the biosynthesis of PC than WY14643. Moreover, the conversion of oleate to lin- oleic acid was completely blocked by DH990.
Effect of Hypolipidemic Drugs and Lyse PLs on the Metabolism of [14CjOleate by Saflower Miorosomes
Since these hypolipidemic drugs affected the metabolism of [14C]oleate in vivo, it was of interest to determine if they had the same effects in vitro. Safllower microsomes were selected as experimental material since much is known about the Al2 desatu- rase of this system (11-13).
Microsomes isolated from developing safflower seeds convert [‘4C]oleate to [‘4C]oleoyl-CoA when incubated in the presence of ATP and CoA (Table II). How-
ever, little incorporation of radioactivity into complex lipids was obtained under these conditions. Furthermore, no desat- uration to linoleate was observed in those experiments carried out under the same conditions and in the presence of NADH (results not shown). These results differ from those described by Murphy et aZ. (7), who observed desaturation of [14C]oleate in microsomes from pea leaves. However, we also observed that addition of CoA and ATP inhibited both desaturation and in- corporation of [14C]oleoyl-CoA into PC and PE in safflower microsomes (Sanchez and Stumpf, unpublished), and this inhibition possibly originated by dilution of the ra- diolabeled substrate, due to the formation of acyl-CoAs from endogenous fatty acids in the presence of CoA and ATP (14).
Addition of WY14643 and DH990 re- sulted in a severe decrease of the radio- activity into the acyl-CoA fraction, sug- gesting inhibition of acyl-CoA synthetase. WY14643 was the most effective inhibitor. Although incorporation of label into com- plex lipids was impaired by the addition of both drugs, the effect was not as pro- nounced as the in vivo experiment de- scribed above (Table I).
Since addition of lyso-PL greatly stim- ulates acyltransferase activity in safflower microsomes (15), LPC and LPE were added to the incubation medium. Addition of LPC promoted a considerable increase (over 30- fold) in the radioactivity accumulated in PC, with a concomitant decrease in both
TABLE I
EFFECT OF HYPOLIPIDEMIC DRUGS ON THE METABOLISM OF [‘4C]O~~~~~ IN Vrvo BY AGED POTATO SLICES
Treatment
Total incorporation
(nmol [“C]oleate)
TOM Percentage distribution of the radiolabel
desaturation (% “C-18:2) PC PE DG FFA TG Aq Phase Others
Note. Aged potato slices (0.5 g) were incubated with [“Cjoleate for 3 h at room temperature. Lipids were extracted with hexane-isopropanol (3:2). After separation of two phases by addition of 6.7% Na$O,, lipids in the organic layer were separated by TLC.
‘WY = WY14643; DH = DH990. ’ t = traces.
133 SANCHEZ AND STUMPF
TABLE II
EFFECT OF HYPOLIPIDEYIC DRUGS AND LYSOPHOSPHOLIPIDS ON THE METABOLISM
OF [‘%]OLEATE IN SAFFLOWER MICROSOMES
Percentage distribution of the radiolabel (&SD)
Treatment Acyl-CoAs PC PE DG FA TG
Control 2.5 mM WY@ 2.5 mM DH” 50 LPC pM 50 PM LPE LPC + WY LPE + WY LPC + DH LPE + DH
Note. ATP (4 mM) and CoA (0.4 mM) were present in the incubation medium. a WY = WY14643; DH = DH990. b t = traces.
acyl-CoAs and FFA. Addition of LPE pro- duced a 1Cfold increase in the incorpo- ration of [14C]oleate into PE, and this increase was almost matched by a de- crease of the radioactivity found as FFA (Table II).
The effect of the drugs on acyl-CoA syn- thetase activity was confirmed when they were tested in the presence of LPC or LPE. Interestingly, the decrease in radioactivity measured as acyl-CoAs in those experi- ments carried out in the presence of WY14643 (20.7 to 1.2 for LPC and 42.5 to 8.6 for LPE) can account for the decrease in the incorporation into PC (58.0 to 16.2) and PE (16.6 to 4.2), indicating that WY14643 mainly affects acyl-CoA synthe- tase activity. A parallel increase in 14C- labeled fatty acid was also observed. In experiments carried out with DH990, the inhibition of the incorporation of [14C]ole- ate into PC (58.0 to 1.2) and PE (16.6 to traces) did not match with the correspond- ing changes of radioactivity in the acyl- CoA fraction (20.7 to 24.5 for LPC and 42.5 to 21.7 for LPE). This result suggests that DH990 may affect an acyltransferase ac- tivity. In summary, both drugs markedly lower the incorporation of [‘*C]oleate into PC and PE in safflower microsomes.
Eflect of Hypolipidemic Drugs and Lys/so- PC on the Metabolism of [14CjOleoyl- CoA in SaBower Microsomes
Microsomes prepared from developing safflower seeds have a very active oleate desaturase (ll-13,16), especially when the precursor is added as oleoyl-CoA.
The effect of increasing concentrations of DH990 and WY14643 on the desatura- tion of [14C]oleoyl-CoA and distribution of label among different acyl fractions is shown in Table III. In agreement with the results obtained from the in vivo experi- ment with aged potato disks (Table I), it is found that DH990 severely inhibited de- saturation of oleoyl-CoA, whereas the in- hibition by WY14643 was not as pro- nounced. Furthermore, DH990 inhibited the incorporation of label into complex lip- ids. Of interest, the transfer of [‘4C]oleoyl- CoA to PE was more susceptible to the effect of the drug. Surprisingly, WY14643 did not inhibit incorporation into PC; on the contrary, a stimulation of the accu- mulation of radioactivity into PC was found; incorporation into PE and TG was clearly inhibited by this drug. In summary, DH990 inhibited incorporation of [14C]oleoyl-Coa into PE with a parallel de-
OLEATE METABOLISM IN PLANTS 189
TABLE III
EFFECT OF THE CONCENTRATION OF HYPOLIPIDEMIC DRUGS ON THE DESATURATION OF [i4C]O~~o~-CoA AND
DISTRIBUTION OF THE LABEL AMONG DIFFERENT Acn FRACTIONS IN SAFFLOWER MICROSOMES
Note. Experiments were carried out as described under Materials and Methods and in duplicate. “DH = DH990; WY = WY14643. b t = traces.
cline in the conversion of [‘4C]oleoyl to [‘4C]linoleoyl moiety. The effect was far less with the PC fractions.
The effect of increasing concentrations of lysophospholipids on the desaturation and distribution of [14C]oleoyl-CoA among different acyl lipids was also examined (Table IV). A stimulation of Al2 desaturase activity was found when lysophospholipids were added at low concentrations, but with LPC, higher concentrations actually in- hibited the desaturation of the precursor;
complete inhibition of the activity was ob- served at a concentration of 1 mM LPC (results not shown). However, this inhib- itory effect was not seen at high concen- trations of LPE. It is worth noting that addition of LPE increased the total for- mation of 14C-18:2 although incorporation of radiolabeled precursor into PC was im- paired. This result suggested that oleoyl- PE may also be an efficient substrate for the Al2 desaturase (to be discussed later). On the other hand, stimulation of the in-
TABLE IV
EFFECT OF INCREASING CONCENTRATIONS OF LYSO-PC AND LYSO-PE ON THE METABOLISM OF [“CIOLEOYL-COA
Treatment Total Desaturation
(% of “C-1&2 + SD) Acyl-CoAs
Percentage distribution of the radiolabel
PC PE Other PLs la
Control 26.3 f 0.8 14 30 (35) 20 (36) 4 32
10 PM LPC 33.1 f 0.5 11 49 (36) 5 (42) 4 32
50 pu LPC 36.4 14 57 (35) 2 W) 7 20
500 LPC NM 16.7 + 1.0 12 6'7 (19) 1 (nd)” 9 11
10 CM LPE 31.6 f 1.9 10 24 w 34 (39) 6 27
50 film
LPE 30.1 f 0.4 11 17 (2% 42 (36) 4 26
500 WM LPE 33.1 + 2.0 14 13 (26) 44 (42) 8 21
Note. Figures in parentheses express percentages of “C-18:2 in PC and PE, determined after separation of these lipids by TLC.
“nd = not determined.
190 SANCHEZ AND STUMPF
TABLE V
EFFECT OF LYSOPHOSPHOLIPIDS AND HYPOLIPIDEMIC DRUGS ON THE METABOLISM OF [~%]OLEO~L.-COA IN SAFFLOWER MICROSOMES
Note Data are means of several independent experiments (number indicated in parentheses) carried out in duplicate or triplicate.
‘WY = WY14643; DH = DH990.
corporation into PC and PE on addition of LPC and LPE, respectively, is in the line of the results shown in Table II.
The effect of the addition of lyso-PL and drugs on the metabolism of [14C]oleoyl-CoA is shown in Table V. It will be seen that the inhibitory effects of DH990 on the de- saturation of the [14C]oleoyl moiety and its incorporation into complex lipids could not be reversed by addition of lyso-PLs in con- firmation of the results obtained in viva (Table I) and in vitro using [14C]oleate (Ta- ble II). These results are also in line with the conclusion that DH990 strongly inhib- its lyso-PC acyltransferase, as reported by Parthasarathy et al. (5), and lyso-PE acyl- transferase. On the other hand, WY14643 showed no effect on incorporation of label into PC contrary to the results reported previously (5) with liver microsomes. However, the inhibitory effect of this drug on the desaturase activity, as well as on the incorporation of radioactivity into PE and TG, was clearly observed although the magnitude of the effect was not as dramatic as it was with DH990.
To study more extensively the effect of hypolipidemic drugs and lysophospholipids on the metabolism of [‘4C]oleoyl-CoA by safflower microsomes, a time-course ex-
periment was carried out. In good agree- ment with the results discussed previously, it was found that addition of LPC and LPE stimulated the accumulation of radioac- tivity into PC and PE, respectively, whereas the hypolipidemic drugs mainly affected incorporation into PE, without in- hibiting the incorporation into PC (Table VI). It should be pointed out that in these experiments one-tenth the usual normal DH990 concentration (0.25 mM) was used because 2.5 mM generally inhibited both PC and PE. As indicated previously, it is of interest that addition of LPE produced a stimulation in the formation of 14C-l&2 quantitatively similar to that induced on addition of LPC, despite the fact that in- corporation of radiolabeled precursor into PC was significantly reduced. Once again, oleoyl-PE may also be an effective sub- strate for the Al2 desaturase.
This finding prompted us to determine the percentage of 14C-18:2 in PC and PE. The results of these analyses are shown in Table VII. It was found in the control ex- periment that a significant proportion of 14C-18:2 accumulated also in PE, even after short incubation periods, and similar re- sults were found in the time course carried out in the presence of LPE, where most of
Note. No “C-18:2 was found in the aqueous phase after partition with chloroform-methanol (2:l). ‘t = trace. * nm = not measured because of low’% content. ‘a = determined together with “other” lipids. d WY = WY14643; DH = DH990.
the radiolabeled 18:2 was found to be es- terified to PE. Although these results could be interpreted in terms of a rapid exchange of acyl residues, diacylglycerol moieties, or bases between PC and PE, oleate may also be desaturated when esterified to either PC or PE. No 14C-18:2 was found in the aqueous phase of any experiment which supports the view that oleoyl-CoA was not a substrate for the oleate desaturase.
Of further interest, both drugs inhibited total desaturation of [‘4C]oleoyl moiety in PE although incorporation of [‘4C]oleoyl- CoA into PC was not inhibited. These re- sults suggest that these drugs may affect
oleate desaturase in addition to inhibiting [14C]oleoyl-CoA incorporation into PL. Moreover, severe inhibition was found in the percentage of 18:2 accumulation into PC in those experiments carried out in the presence of hypolipidemic drugs, in spite of the fact that incorporation of radioac- tivity into this lipid was not found to be decreased by the presence of drugs (2.5 mM for WY14643) and 0.25 mM for DH990).
Since hypolipidemic drugs and lyso- phospholipids showed antagonistic effects on the desaturation of [14C]oleoyl-CoA, it was of interest to study the kinetic effect of both compounds. It was found that when
OLEATE METABOLISM IN PLANTS 193
LPC and WY14643 were present together in the incubation medium, the percentages of desaturation obtained were those theo- retically expected for the two compounds acting independently and in opposite di- rections and on the desaturase itself (Fig. 1); both in the absence and in the presence of the drug, addition of LPC induced an approximate 1.5-fold stimulation in the percentages of desaturation obtained after 20 min of incubation when the reaction seemed to reach a plateau, and this trend was maintained up to 1 h. The results sug- gested that WY14643 inhibited the desat- uration of the [14C]oleoyl PC formed from endogenous membrane lipids by exchange reactions but had no effect on the “C-18:1 PC formed when LPC was added as the precursor.
The changes in the distribution of ra- dioactivity into different acyl fractions upon addition of WY14643 and LPC are shown in Fig. 2. The decrease in the rate of utilization of [14C]oleoyl-CoA by addition of WY14643 is in complete agreement with
TIME (min)
FIG. 1. Effect of addition of WY14643 and lyso-PC on the desaturation of [‘%]oleoyl-CoA by safflower microsomes. The concentration of WY14643 was 2.5 mM, and lyso-PC was added at a final concentration of 50 PM.
the results shown in Table VI, as well as the opposite effect promoted by addition of LPC (Fig. 2a). Interestingly, WY14643 did not show any inhibitory effect on the incorporation of radioactivity into PC, nei- ther in the presence nor in the absence of LPC (Fig. 2b), supporting the conclusion that this drug does not affect the incor- poration into PC. However, the presence of the drug inhibited the incorporation in all the other complex lipids, mainly PE (Fig. 2~) and also DG and TG (Figs. 2d and 2f).
Although WY14643 does not affect the incorporation of radiolabeled [‘4C]oleoyl- CoA into PC, it is now generally assumed that oleoyl-PL is the immediate substrate of the oleate desaturase in both animal and plant tissues. The question then arises as to why WY14643 inhibits the desatu- ration of [‘4C]oleoyl PC when [‘4C]oleoyl- CoA is the initial substrate. The evidence indicates that there are at least four en- zymatic activities involved in the substrate desaturation of oleate in plants: (a) the acyl-CoA synthetase; (b) the acyltransfer- ase which provides the substrate for the desaturase, presumably oleoyl-PC, (c) the cytochrome-electron transport chain, which transfers electrons from NADH to the desaturase (perhaps including cyto- chrome b5 and NADH:cyt b5 reductase); and finally (d) the oleate desaturase itself. If NADH reversed the effect of the drug, then the drug might block the electron transport system. When NADH was added to the in- cubation medium, no significant effect was found with concentrations of NADH of up to 10 mrvr, compared with the standard 2 mM concentration (results not shown).
Table VIII shows the results of another experiment designed to clarify the mode of action of hypolipidemic drugs on the oleate desaturase. After a preincubation of [14C]oleoyl-CoA for 15 min in the pres- ence or absence of NADH or drugs, further additions were made and the incubation was allowed to proceed for 30 more min. It was found that oleate desaturation was severely inhibited under conditions in which PC and PE were already charged with [‘4C]oleoyl moiety (Experiments 2, 3,
194 SANCHEZ AND STUMPF
e ,- o-
: -------- ,-.a------ “-.-_ --O
I1
I j
-------- .
awl-CoAs 1:
PC
!: II
; 0 .
PE DC
TG
TIME (min)
FIG. 2. Effect of addition of WY14643 and lyso-PC on the distribution of radioactivity from [‘4C]oleoyl-CoA into different acyl fractions. Control (0); 2.5 mM WY14643 (0); 50 pM lyso-PC (m); 2.5 mM WY14643 and 50 pM lyso-PC (El).
7, and 8). These results suggest that both drugs may also affect a component of the NADH - Oz - desaturation system.
DISCUSSION
The results presented here indicate that hypolipidemic drugs WY14543 and DH990 inhibit several of the activities reported to be associated with membranes in the plant cell, as has already been described for microsomal membranes in liver micro- somes (5).
As indicated in Fig. 3, several enzymatic activities are directly involved in the for- mation of linoleate, the most abundant acyl unit in nonphotosynthetic membranes. These enzymes include acyl-CoA synthe- tase, LPC-acyltransferase, Al2 desaturase, and an electron carrier system. All of these are membrane-bound enzymes.
Both drugs appear to be multifunctional in that they block one or more of the com- ponents involved in the conversion of oleate to linoleate in safflower microsomes, as well as in potato slices. DH990 appears to in-
OLEATE METABOLISM IN PLANTS
hibit both a lyso-PE and a lyso-PC acyl- transferase, as well as a possible exchange system which permits the incorporation of oleoyl-CoA into either PC or PE (17). In addition as indicated in Table VIII, DH990 has a direct effect on the desaturation re- action itself.
On the other hand, WY14643 appears to block strongly the acyl-CoA synthetase re- sponsible for the activation of oleic acid to oleoyl-CoA, has no effect on the lyso PC acyltransferase but partially blocks the lyso PE acyltransferase system, and also has a direct effect on the Al2 desaturase. Ki data for these drugs have not been de- termined since the nature of the substrates and the membrane proteins are so poorly defined that Ki determination have little value. However, the concentration of DH990 that will give 50% of the Al2 de- saturase is approximately 0.3 mM and for WY14643 approximately 1.3 mM.
The data obtained for Table IV dem- onstrate that it is possible to perturb markedly the levels of PC and PE in mi- crosomal systems by supplying lyso-PC and lyso-PE to microsomes in the presence of [14C]oleoyl-CoA. With both lyso-PC and lyso-PE, a modest increase in oleate - lin- oleate conversion is noted. Of interest is the suppression of PE synthesis when lyso- PC is the acceptor and the suppression of PC synthesis when lyso-PE is the final ac- ceptor. Of additional interest are the data of Fig. 1. In this figure, the desaturation of oleoyl-CoA to linoleoyl moiety is ex- amined as a function of time and the ab- sence or presence of LPC and WY14643. When endogenous PC (of the microsomal preparation) served as the acceptor of oleoyl-CoA, a marked inhibition of Al2 de- saturate was observed when WY14643 was introduced to the reaction mixture. When lyso-PC was added to the reaction mix- ture, a large increase in PC synthesis oc- curred which was depressed by WY14643. However, the ratio of control/control + WY14643 and LPC/LPC + WY14643 was 1.5 in both cases, suggesting that the LPC system was insensitive to WY14643 when- ever the endogenous PC system was sen- sitive to WY14643 in the Al2 desaturation reaction. Other interpretations are, of
.1. Y* 8% XF. w
L2 ++ exxxxxxx annnnnnn =44*44-4-4-t zzzzzzzz I I I I I +++
196 SANCHEZ AND STIJMPF
NADHz
NAD+
FIG. 3. Summary of the effects of lypolipidemic drugs on the enzymes involved in the metabolism of oleate. A double slash indicates inhibition.
course, possible but this proposal is suggestive of two different types of PCs in membrane lipids.
Finally, the data clearly show that in addition to PC, PE must be considered as a site for Al2 desaturation as shown in Ta- ble VII as well as other data presented here.
Final definition of the Al2 desaturation must await the solubilization and purifi- cation of the Al2 desaturase and its utili- zation of precisely defined substrates. However, the use of these drugs have re- vealed new facets in the complex problem of Al2 desaturation.
ACKNOWLEDGMENTS
Dr. J. Shnchez wishes to thank the Fundacion Juan March (Spain) for his travel grant. We also wish to thank Professor Wolfgang Baumann (Hormel Insti- tute) for WY14643 and to Dr. J. Heeg (Merreil-Dow) for DH990.
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