Biochimica et Biophysics Acta, 1165 (1992) 189-193 0 1992 Elsevier Science Publishers B.V. All rights reserved
BBALIP
189 0005-2760/92/%05.00
54057
In vivo phospholipid modification induces changes in microsomal ASdesaturase activity Alicia I. Leikin and Rodolfo R. Brenner Institute de Investigaciones Bioqui’micas de La Plata (INIBIOLP), CONICET-UNLP, La Plata (Argentina)
(Revised
Key words:
Microsomal
membrane;
(Received manuscript
A5 Desaturase;
Facultad de Ciencias Midicas,
6 April 1992) received 22 June 1992)
Phospholipid
composition;
Arachidonic
acid; Fluidity;
(Rat liver)
The phospholipid composition of rat-liver microsomes was modified by feeding weaning rats a choline-free diet. After 21 days, the phosphatidylcholine content decreased with a concomitant increment of phosphatidylserine and cholesterol. The bulk fluidity
of the membrane decreased. Under these conditions, the A5-desaturase activity was diminished as well as the arachidonic-acid content of the membrane
lipids.
Introduction Fatty-acid desaturases are key amphipathic enzymes that regulate polyunsaturated fatty-acid biosynthesis [l]. A5 Desaturase leads to the production of arachidonic acid (20 : 4 )2 - 6) from eicosatrienoic acid (20 : 3 n - 6), which is not only incorporated into membrane phospholipids but is also transformed into eicosanoids [2]. The behaviour of microsomal desaturases may be strongly influenced by the physical state of the membrane [3]. It has been reported that the rigidization of the membrane by sterol incorporation in vitro or in vivo with no changes in the phospholipid composition leads to an enhanced response of A5-desaturase activity [4,5]. We will refer here to ‘rigidization’ as the increase in the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5_hexatriene, and to ‘fluidity’ as the decrease in values of the same parameters. However, when the rigidization of the membrane goes along with changes in the phospholipid composition, the enzyme activity decreases [6]. It is not clear, yet, which of the following factors: the sterol content, the phospholipid polar head distribution or the particular sterol/ phospholipid or phospholipid/ phospholipid ratio, is
Correspondence to: AI. Leikin, Instituto de Investigaciones Bioquimicas de La Plata (INIBIOLP), CONICET-UNLP, Fact&ad de Ciencias Medicas, 60 y 120, 1900-La Plats, Argentina. Abbreviations: C, cholesterol; P, total phosphorus; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; SM, sphingomyelin; DPH, 1,6-diphenyl-1,3,5hexatriene.
the parameter that contributes to the regulation of the fatty-acid desaturase activity. The aim of this work was to elucidate the influence of other membrane phospholipid modification, achieved by dietary means, on the A5-desaturase activity. It was found that the reduction of the membrane phosphatidylcholine content together with the increment of the cholesterol/ phosphatidylcholine ratio led to a lower enzymatic activity in a rigidized bilayer environment. Materials and Methods Adenosine, cycloleucine, ATP, CoA, NADH, Nacetyl cysteine and florisil were provided by Sigma Chemical (St. Louis, MO), 1,6-diphenyl-1,3,5hexatriene DPH (98%) was from Aldrich (Wisconsin). Silica-gel H was from Merck (Darmstadt). Corn oil was provided by Refinerias de Maiz, Buenos Aires, Argentina. Vitamins were donated by Abbott Laboratories, Buenos Aires, Argentina. Solvents and reagentgrade chemicals were from Carlo Erba, Argentina. [l-‘4C]Eicosa-8,11,14-trienoic acid (54.9 mCi/mmol) was provided by New England Nuclear, Argentina. It was 98-99% radiochemically pure. The packing for gas-liquid chromatography columns was from Supelco, Supelco Park, Bellefonte (PA). Animals and Diets. Male Wistar rats weighing 25-30 g were separated after weaning into two groups of six animals each. Experimental diets and tap water were provided ad libitum. The control diet was composed of 64% starch, 23% casein and 13% corn oil plus 2% vitamins and 4% minerals according to Rogers and
190 Harper [7]. The test diet was the same as the control without the addition of choline chloride to the vitamin mixture. After 21 days of dietary treatment, animals in the experimental group received O-20 prnol freshly prepared periodate-oxidized adenosine [S] in a saline solution per g rat-weight administered by intraperitoneal injection and two more injections at a dose of 0.10 pmol/g each were administered at 24-h intervals. Cycloleucine (a methionine analogue) was also included in the treatment at a dose of 7.75 mmol/kg in a saline solution injected on the first day. Animals in the control group received only saline. The animals were killed on the day following the last injection. Livers were quickly excised and placed in homogenizing solution (1: 3, w/v). This solution contained 0.25 M sucrose, 62 mM phosphate buffers (pH 7.01, 0.15 M KC1 and 1.5 mM N-acetyl-cysteine. Microsomes were obtained by differential ultracentrifugation at 110 000 x g (Beckman ultracentrifuge) as described elsewhere [9]. The protein concentration was measured according to Lowry et al. [lo] as modified by Markwell et al. [l l] with bovine serum albumin as standard. ASDesaturase assay. The A5-desaturase activity was studied by measuring the conversion of [l- 14C]20: 3 (n - 6) to [1-‘4C]20: 4 (n - 6) for 15 min in the presence of 1.3 mg/ml of microsomal protein and substrate concentration of 33 PM. The assay was carried out in a final volume of 1.5 ml of an incubating solution and under conditions already described [6]. The reaction products were converted to methyl esters [12] and analyzed by radio-chromatography in an Acromat CG100 apparatus equipped with a proportional counter and using the liquid phase as described before. Lipid analysis. Lipids were extracted from microsomes according to the procedure of Folch et al. 1131. The total amount was calculated after aliquot evaporation to a constant weight. Cholesterol was determined according to Huang et al. [14] and phosphorus by the method of Chen et al. [15]. Polar lipids were separated by thin-layer chromatography (TLC) on silica-gel H plates with florisil. They were developed with a double-solvent system constituted by CHCl,/ CH,OH/ NH,/ H,O (70 : 25 : 3.5 : 1.5) followed by CHCl,/CH,OH/ CH,COOH/H,O (80 : 10 : 2 : 0.75) [16]. Fatty acids of total lipids or phospholipids scrapped from TLC plates were analyzed by gas-liquid chromatography (GLC) in a Hewlett-Packard 5840-A apparatus after esterification with methanol [61. A 10% SP-2330 column packed on Chromosorb WAW was employed. The temperature was programmed to obtain a linear increase of 3”C/min from 140 to 220°C. The chromatographic peaks were tentatively identified by comparison of the retention times with those of standards chromatographed under the same conditions. Fluorescence anisotropy measurements. The steadystate fluorescence anisotropy of 1,6-diphenyl-1,3,5-
hexatriene added to a microsomal suspension under conditions already described [6] was measured in an Aminco Bowman spectrofluorometer equipped with two glan polarizers. Appropriate blanks were prepared and their values were substracted from the samples. The fluorescence intensities were measured at 32S-nm excitation, 460-nm emission wavelengths and at 37°C. A 2.0 M NaNO, solution was placed between the emission monochromator and the photo-multiplier as a cut-off filter for wavelengths lower than 390 nm. The steady-state fluorescence anisotropy (r,), that measures the range and rate of the rotation of the probe. was calculated as follows: I r\ =
I
- GI i +2a,
where I,, and I, are the fluorescence intensities parallel and perpendicular, respectively, to the plane of polarization of the excitation beam, and G is a correcting factor arising from parallel diffraction anomalies introduced by the monochromator [16]. Results A5-Desaturase acticity after choline-free diet
The effect of a choline-free diet on A5-desaturase activity was studied by the conversion of eicosatrienoic acid (20: 3) [8,11,14] into arachidonic acid (20: 4) [5,8,11,14]. The results are presented in Table I. After 21 days of diet and the injection of the methylation inhibitor, the A5-desaturase activity was 25% lower for treated than for control animals, thus indicating a decrease in the production of arachidonic acid. Effect of a choline-free composition
diet on microsomal
rat-liuer
Table II shows the effect of the choline-free diet on the lipid composition of the microsomes. The total cholesterol increased slightly and, among phospholipids, phosphatidylcholine content decreased, whereas that of phosphatidylserine increased significantly. These variations are also expressed as the interlipid relationships, the most remarkable one being the increase of C/PC ratio of about 45%. The PC/PE ratio also decreased. This decrease of the membrane PC
TABLE
I
d5-Desaturase actir~ity in liter microsomes after a choline-free dier (pm01 20: 4 n - 6 produced /min per mg microsomal proteinl Control
Choline-free
388.20 f 17.00
312.71 k 25.02 *
Results are the meanfS.D. of six animals. control using Student’s I-test. * P < 0.02.
Data
are compared
to
191 TABLE IV
TABLE II Microsomal lipid composition after choline-free diet
Lipid a
Control
Choline-free
Cholesterol (0 Total phospholipids (P) Phospholipid composition PC PE PI PS SM Interlipid relationship C/P C/PC PC/PE
189.25 + 11.00 558.05 k 36.01
211.50* 15.10 489.23 k 40.02
302.40 + 20.21 122.21+ 8.90 80.91+ 6.52 14.83 + 1.02 38.31 f 2.52
234.12 + 18.50 * * 129.60+ 10.31 70.81 k 3.70 43.90& 3.20 *** 25.42 k 2.40
a
0.34* 0.04 0.62+ 0.07 2.47* 0.34
Fluorescence anisotropy of DPH in microsomes after choline-free diet
(nmol/ mg lipid).
rF
Control
Choline-free
0.1440 + 0.0028
0.1599 + 0.0040 *
Results are the mean+S.D. of six animals. Data are compared to control using Student’s t-test. * P < 0.01.
The fatty-acid composition of microsomal total lipids and the main phospholipids, PC and PE, is presented in Table III. Some important differences were observed in the total lipid fatty-acid composition. These were the significant decrease of arachidonic acid 20 : 4 (n - 6), the product of the A5 desaturation of eicosatrienoic acid and the significant decrease of docosahexaenoic acid 22 : 6 (n - 31, the product of A5 desaturation and elongation of 20: 4 (n - 3). The 20: 4 + 22: 4/18 : 2 ratio also decreased due to the 20 : 4 and 22: 4 reduction. In PC, a similar trend was observed, but the changes were more striking. The decreases of 20 : 4 (n - 6) and 22 : 6 (n - 3) were very significant. These changes were well expressed as a diminished 20 : 4 + 22 : 4/18 : 2 ratio and the decreased unsaturation index. Regarding PE, the most significant variations were at 22 : 4 (n - 6) and 22 : 6 (n - 3) reduction and at the decreased index.
0.43+ 0.07 0.90* 0.13 * 1.80+ 0.28
Other lipids such as triacylglycerols or cholesterol esters were not considered. Results are the mean+ S.D. of six animals analyzed separately. Data are compared to control using the Student’s t-test. * P < 0.05; ** P < 0.02; *** P < 0.01.
content is due not only to the lack of dietary choline but also to the methylation inhibitors that contribute to impair completely the PC synthesis from PE. The periodate-oxidated adenosine alters the adenosine susceptible to methylation and increases the content of sadenosine homocysteine. The cycloleucine, in turn, competes with methionine, thus eliminating the source of methyl groups. Therefore, the two main pathways described for the de-novo biosynthesis of PC (1) the incorporation of choline into PC, and (2) the stepwise methylation of PE, are abolished.
Steady-state jluorescence anisotropy of DPH
The DPH fluorescence anisotropy increased in ratliver microsomes after 21 days of choline-free diet reaching values of 0.1599 (Table IV).
TABLE III Fatty-acid composition of microsomal lipids after a choline-free diet (mol%)
Fatty acids a
Total lipids
16:0 16:l 18:0 18:l 18:2 18:3(n -6) 20:4(n
-6)
22:4(n -6) 22:4(n-3)or22:5(n-6) 22:tin -3) 20:4(n -6)+22:4(n 18:2(n -6) U.I.
-6)
PC
PE
control
choline-free
control
choline-free
control
choline-free
9.15 + 0.90 0.40 + 0.00 19.10 + 0.20 8.01+ 0.10 12.7OkO.11 0.18kO.02 29.30 + 0.90 4.80 + 0.25 5.6OkO.32 7.03+ 1.01
10.80 + 0.80 0.30 f 0.00 20.41 f 0.20 7.40 &-0.10 12.31+ 0.10 0.15 + 0.04 25.21+ 0.80 * * 5.21+ 0.30 4.01 f 0.32 4.52f0.51 **
20.51 f 0.91 1.15 +0.07 21.44 + 0.08 8.76 + 0.54 16.30 + 0.98 0.20 f 0.06 23.54& 1.12 1.19+0.04 2.24 + 0.08 2.21& 0.06
23.30 + 0.50 1.14+0.08 19.85 k3.10 9.31 f 0.80 14.30 + 1.52 0.43 + 0.04 14.11+0.90 *** 1.10+0.31 1.46kO.15 0.29 f 0.02
18.90 + 0.97 0.90 f 0.01 28.33 + 0.28 8.18& 1.22 10.06+0.81 0.15 f 0.00 21.27+2.18 2.68 + 0.36 3.89 f 0.55 3.21 f 0.53
23.82 + 6.2 1.10~0.02 29.04 + 0.01 10.24+5.21 10.18+ 1.01 0.25 + 0.09 19.90+ 1.50 0.51kO.15 *** 3.01+ 0.50 1.09 + 0.43 * *
2.6850.11
2.47+0.10
1.52*0.1
1.06-10.19
2.38 f0.44
2.28 f 0.36
2.47 + 0.28
2.20+ 0.20
1.69kO.18
1.26+0.10 **
1.65 kO.15
1.35 fO.10
a Other minor fatty acids complete 100%. Results are the mean+S.D. of six animals. Datum is compared to control by using Student’s t-test. * P < 0.1; ** P < 0.01; *** P < 0.005. U.I., unsaturation index (E~,x, /FA) where n1 = number of double bonds in each fatty acid, x1 = mol of each fatty acid. FA, total mol of fatty acids.
192 Discussion
The lipid composition of biological membranes can be altered by dietary means [lS-201. The membrane composition is of fundamental importance since it is linked to several physiological and biochemical parameters, like the membrane-bound enzyme activity, among others [21]. These enzymes require an optimal lipid environment for the full expression of their functional activity [22]. A5 Desaturase converts fatty acids of 20 carbons into more unsaturated higher homologs. It represents a second regulatory step after the A6 desaturase in the regulation of polyunsaturated fatty-acid biosynthesis and bio-transformation of ingested essential fatty acids. A5 Desaturase is a component of an electron-transport system [23,241. The lipid bilayer of mammal endoplasmic reticulum is sufficiently fluid to allow rotational and lateral diffusion of these proteins 1251.Cons~quently~ contact between the different components of the system may depend on the fluidity of the surrounding lipid environment [3,4]. Based on different in-vitro and in-vivo membrane-lipid modifications [3-61, we have previously proposed that A5 desaturase would be regulated by the physical status of the membrane, exclusively in the absence of phospholipid changes. Thus, the rigidization of the membrane after cholesterol [4] or phytosterol incorporation in vitro and in vivo, respectively [S] leads to an increased AS-desaturase activity. Consequently, there is a higher arachidonic acid content in the phospholipids with no changes in their head polar group distribution. In turn, the rigidization of the membrane after cholesterol incorporation in vivo takes place together with an increase in the C/PC and PC/PE ratios and a lower A5-desaturase activity and arachidonic-acid content in the phospholipids [6]. The results obtained following a choline-free diet indicate several changes in the microsomal membrane. The decrease in the rotational mobility of the bulk lipids expressed by the higher value of rs of DPH (Table IV) may be attributed to the higher C/PC ratio, the lower PC/PE ratio (Table II) and/or the lower polyunsaturated fatty-acid content of the lipids in the bilayer. Under these particuIar conditions, the A5-desaturase activity decreased (Table I) and, in consequence, their desaturated fatty-acid products also decreased. The most important observation is that the enzymatic activity is diminished in a rigidized membrane, but in a different way than after the in-vivo cholesterol incorporation; the PC/PE ratio is now diminished. Therefore, the common features in both cases are the decreased rotational mobility of the lipids and the increase C/PC ratio. However, the lower ~uidity of the membrane should not be related to the lower AS-desaturase activity since a similar bilayer behaviour after chofesterol or phytosterol incorporation in vitro and in vivo, respec-
tively, induced the opposite enzymatic response. It is aIso important to ruIe out the absolute increment of cholesterol as responsible for the enzyme changes [26,271 for its incorporation in vivo and in vitro evoked opposite responses. On the basis of these results. the hypothesis postulating that AS desaturase responds to viscotropic variations only in the absence of phospholipids changes is reinforced. The C/PC ratio and specifically the PC would play a key role in this rcgulation. It is likely that AS desaturasc interacts with PC molecules of the membrane and its activity may he altered by variations of C/PC ratio. Further cxpcrimcnts are necessary to test this hypothesis. Acknowledgements
This work was supported by grants from CONICET. Argentina. The technical assistance of Mrs. Laura Hernandez and the participation of the undergraduated student, Alejandro Schamun, in the dietary trcatment of the animals, are gratefully acknowledged. References 1 Brenner, R.R. (1977) Adv. Expl. Med. Biol. 83. 85-101. 2 Samuelson, B. (1981) Prog. Lipid Res. 20, 23-X). 3 Garda, H.A. and Brenner, R.R. (3984) Biochim. Biophys. Acta 769, 160- 170. 4 Garda, H.A. and Brenner, R.R. (lY85) Biochim. Biophys. Acta 819, 4s-54. S Leikin, A.I. and Brenner, R.R. f1989) Biochim. Biophys. Acta IOOS. 187-191. h Leikin, A.]. and Brenner, R.R. (198’7) Biochim. Biophys. Acta 922, 244-303. 7 Rogers, R.R. and Harper, A.E. tl9h5) J. Nutr. 87. Zh?-273. X Boyle, 0. and Dean. W. (1982) Biochim. Biophys. Acta 6x8. 667-670. 9 Catalri, A.. Nervi, A.M. and Brenner, R.R. (1475) J. Riol. Chem. 250. 748 l-7488. IO Lowry. O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (IS51 1J. Biol. Chem. 193, 265-275. t I Markwell. M.A.K., Haas, S.M., Bieher, L.L and Tolhcrt, N.J. f 1978) Anal. Biochem. 87, 206-21 I. 12 Havel, Z. and Djerassi, C. (1980) Lipids 15, 694-696. 13 Folch, J., Lees, M. and Sloane-Stanley, G.A. (1957) J. Biol. Chem. 226, 497-509. 14 Huang, T.C., Chen, C.P., Wefler. V. and Raftery, J. (lOhI) Anal. Chem. 33, 1405-1406. 15 Chen. P.S.. Torihara. T.Y. and Warner, H. (19%) Anal. Chem. 28, 17X- 1758. 16 Ascott. J. and Heaven, R.S. (1968) J. Chromatogr. 35. 297-300. 17 Burton, J., Litman, J. and Barenholz. Y. (1982) Methods Enzymol. 81, 678-685. 18 Wahler, K.W.J. (1983) Proc. Nutr. Sot. 42, 273-287. 19 Stub& CD. and Smith, AD. (1984) Biochim. Biophys. Acta 770, 89- 173. M.. Field. C., Hargraves, K., Morson, L.. and Zsig20 Clandinin, mend, ( 198% Can. J. Physiol. Pharmacol. 63, S46-556. 21 Sun, G.Y. and Sun, A.Y. (1974) J. Neurochem. 22, 15-18. 22 Shinitzky, M. (19X4) Physiology of Membrane Fluidity. pp. I-5 I. CRC Press, Boca Raton. FL.
23 Enoch, H.C., Catal& A. and Strittmatter, P. (1976) J. Biol. Chem. 251, 5095-5103. 24 Oshino, N., Imai, Y. and Sato, R. (1966) Biochim. Biophys. Acta 128, 13-28. 25 Brenner, R.R. (1984) Prog. Lipid Res. 23, 69-96.
26 Silonis, J.R., McMullen, D.A., Saley, N., Lost, P.C. and Hayes, Griffith, 0. (1984) Biochemistry 23, 538-547. 27 Regenass-Klotz, M. and Heiniger, H. (1983) Can. J. Biochem. Cell. Biol. 62, 94-99.
BBALIP
189 0005-2760/92/%05.00
54057
In vivo phospholipid modification induces changes in microsomal ASdesaturase activity Alicia I. Leikin and Rodolfo R. Brenner Institute de Investigaciones Bioqui’micas de La Plata (INIBIOLP), CONICET-UNLP, La Plata (Argentina)
(Revised
Key words:
Microsomal
membrane;
(Received manuscript
A5 Desaturase;
Facultad de Ciencias Midicas,
6 April 1992) received 22 June 1992)
Phospholipid
composition;
Arachidonic
acid; Fluidity;
(Rat liver)
The phospholipid composition of rat-liver microsomes was modified by feeding weaning rats a choline-free diet. After 21 days, the phosphatidylcholine content decreased with a concomitant increment of phosphatidylserine and cholesterol. The bulk fluidity
of the membrane decreased. Under these conditions, the A5-desaturase activity was diminished as well as the arachidonic-acid content of the membrane
lipids.
Introduction Fatty-acid desaturases are key amphipathic enzymes that regulate polyunsaturated fatty-acid biosynthesis [l]. A5 Desaturase leads to the production of arachidonic acid (20 : 4 )2 - 6) from eicosatrienoic acid (20 : 3 n - 6), which is not only incorporated into membrane phospholipids but is also transformed into eicosanoids [2]. The behaviour of microsomal desaturases may be strongly influenced by the physical state of the membrane [3]. It has been reported that the rigidization of the membrane by sterol incorporation in vitro or in vivo with no changes in the phospholipid composition leads to an enhanced response of A5-desaturase activity [4,5]. We will refer here to ‘rigidization’ as the increase in the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5_hexatriene, and to ‘fluidity’ as the decrease in values of the same parameters. However, when the rigidization of the membrane goes along with changes in the phospholipid composition, the enzyme activity decreases [6]. It is not clear, yet, which of the following factors: the sterol content, the phospholipid polar head distribution or the particular sterol/ phospholipid or phospholipid/ phospholipid ratio, is
Correspondence to: AI. Leikin, Instituto de Investigaciones Bioquimicas de La Plata (INIBIOLP), CONICET-UNLP, Fact&ad de Ciencias Medicas, 60 y 120, 1900-La Plats, Argentina. Abbreviations: C, cholesterol; P, total phosphorus; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; SM, sphingomyelin; DPH, 1,6-diphenyl-1,3,5hexatriene.
the parameter that contributes to the regulation of the fatty-acid desaturase activity. The aim of this work was to elucidate the influence of other membrane phospholipid modification, achieved by dietary means, on the A5-desaturase activity. It was found that the reduction of the membrane phosphatidylcholine content together with the increment of the cholesterol/ phosphatidylcholine ratio led to a lower enzymatic activity in a rigidized bilayer environment. Materials and Methods Adenosine, cycloleucine, ATP, CoA, NADH, Nacetyl cysteine and florisil were provided by Sigma Chemical (St. Louis, MO), 1,6-diphenyl-1,3,5hexatriene DPH (98%) was from Aldrich (Wisconsin). Silica-gel H was from Merck (Darmstadt). Corn oil was provided by Refinerias de Maiz, Buenos Aires, Argentina. Vitamins were donated by Abbott Laboratories, Buenos Aires, Argentina. Solvents and reagentgrade chemicals were from Carlo Erba, Argentina. [l-‘4C]Eicosa-8,11,14-trienoic acid (54.9 mCi/mmol) was provided by New England Nuclear, Argentina. It was 98-99% radiochemically pure. The packing for gas-liquid chromatography columns was from Supelco, Supelco Park, Bellefonte (PA). Animals and Diets. Male Wistar rats weighing 25-30 g were separated after weaning into two groups of six animals each. Experimental diets and tap water were provided ad libitum. The control diet was composed of 64% starch, 23% casein and 13% corn oil plus 2% vitamins and 4% minerals according to Rogers and
190 Harper [7]. The test diet was the same as the control without the addition of choline chloride to the vitamin mixture. After 21 days of dietary treatment, animals in the experimental group received O-20 prnol freshly prepared periodate-oxidized adenosine [S] in a saline solution per g rat-weight administered by intraperitoneal injection and two more injections at a dose of 0.10 pmol/g each were administered at 24-h intervals. Cycloleucine (a methionine analogue) was also included in the treatment at a dose of 7.75 mmol/kg in a saline solution injected on the first day. Animals in the control group received only saline. The animals were killed on the day following the last injection. Livers were quickly excised and placed in homogenizing solution (1: 3, w/v). This solution contained 0.25 M sucrose, 62 mM phosphate buffers (pH 7.01, 0.15 M KC1 and 1.5 mM N-acetyl-cysteine. Microsomes were obtained by differential ultracentrifugation at 110 000 x g (Beckman ultracentrifuge) as described elsewhere [9]. The protein concentration was measured according to Lowry et al. [lo] as modified by Markwell et al. [l l] with bovine serum albumin as standard. ASDesaturase assay. The A5-desaturase activity was studied by measuring the conversion of [l- 14C]20: 3 (n - 6) to [1-‘4C]20: 4 (n - 6) for 15 min in the presence of 1.3 mg/ml of microsomal protein and substrate concentration of 33 PM. The assay was carried out in a final volume of 1.5 ml of an incubating solution and under conditions already described [6]. The reaction products were converted to methyl esters [12] and analyzed by radio-chromatography in an Acromat CG100 apparatus equipped with a proportional counter and using the liquid phase as described before. Lipid analysis. Lipids were extracted from microsomes according to the procedure of Folch et al. 1131. The total amount was calculated after aliquot evaporation to a constant weight. Cholesterol was determined according to Huang et al. [14] and phosphorus by the method of Chen et al. [15]. Polar lipids were separated by thin-layer chromatography (TLC) on silica-gel H plates with florisil. They were developed with a double-solvent system constituted by CHCl,/ CH,OH/ NH,/ H,O (70 : 25 : 3.5 : 1.5) followed by CHCl,/CH,OH/ CH,COOH/H,O (80 : 10 : 2 : 0.75) [16]. Fatty acids of total lipids or phospholipids scrapped from TLC plates were analyzed by gas-liquid chromatography (GLC) in a Hewlett-Packard 5840-A apparatus after esterification with methanol [61. A 10% SP-2330 column packed on Chromosorb WAW was employed. The temperature was programmed to obtain a linear increase of 3”C/min from 140 to 220°C. The chromatographic peaks were tentatively identified by comparison of the retention times with those of standards chromatographed under the same conditions. Fluorescence anisotropy measurements. The steadystate fluorescence anisotropy of 1,6-diphenyl-1,3,5-
hexatriene added to a microsomal suspension under conditions already described [6] was measured in an Aminco Bowman spectrofluorometer equipped with two glan polarizers. Appropriate blanks were prepared and their values were substracted from the samples. The fluorescence intensities were measured at 32S-nm excitation, 460-nm emission wavelengths and at 37°C. A 2.0 M NaNO, solution was placed between the emission monochromator and the photo-multiplier as a cut-off filter for wavelengths lower than 390 nm. The steady-state fluorescence anisotropy (r,), that measures the range and rate of the rotation of the probe. was calculated as follows: I r\ =
I
- GI i +2a,
where I,, and I, are the fluorescence intensities parallel and perpendicular, respectively, to the plane of polarization of the excitation beam, and G is a correcting factor arising from parallel diffraction anomalies introduced by the monochromator [16]. Results A5-Desaturase acticity after choline-free diet
The effect of a choline-free diet on A5-desaturase activity was studied by the conversion of eicosatrienoic acid (20: 3) [8,11,14] into arachidonic acid (20: 4) [5,8,11,14]. The results are presented in Table I. After 21 days of diet and the injection of the methylation inhibitor, the A5-desaturase activity was 25% lower for treated than for control animals, thus indicating a decrease in the production of arachidonic acid. Effect of a choline-free composition
diet on microsomal
rat-liuer
Table II shows the effect of the choline-free diet on the lipid composition of the microsomes. The total cholesterol increased slightly and, among phospholipids, phosphatidylcholine content decreased, whereas that of phosphatidylserine increased significantly. These variations are also expressed as the interlipid relationships, the most remarkable one being the increase of C/PC ratio of about 45%. The PC/PE ratio also decreased. This decrease of the membrane PC
TABLE
I
d5-Desaturase actir~ity in liter microsomes after a choline-free dier (pm01 20: 4 n - 6 produced /min per mg microsomal proteinl Control
Choline-free
388.20 f 17.00
312.71 k 25.02 *
Results are the meanfS.D. of six animals. control using Student’s I-test. * P < 0.02.
Data
are compared
to
191 TABLE IV
TABLE II Microsomal lipid composition after choline-free diet
Lipid a
Control
Choline-free
Cholesterol (0 Total phospholipids (P) Phospholipid composition PC PE PI PS SM Interlipid relationship C/P C/PC PC/PE
189.25 + 11.00 558.05 k 36.01
211.50* 15.10 489.23 k 40.02
302.40 + 20.21 122.21+ 8.90 80.91+ 6.52 14.83 + 1.02 38.31 f 2.52
234.12 + 18.50 * * 129.60+ 10.31 70.81 k 3.70 43.90& 3.20 *** 25.42 k 2.40
a
0.34* 0.04 0.62+ 0.07 2.47* 0.34
Fluorescence anisotropy of DPH in microsomes after choline-free diet
(nmol/ mg lipid).
rF
Control
Choline-free
0.1440 + 0.0028
0.1599 + 0.0040 *
Results are the mean+S.D. of six animals. Data are compared to control using Student’s t-test. * P < 0.01.
The fatty-acid composition of microsomal total lipids and the main phospholipids, PC and PE, is presented in Table III. Some important differences were observed in the total lipid fatty-acid composition. These were the significant decrease of arachidonic acid 20 : 4 (n - 6), the product of the A5 desaturation of eicosatrienoic acid and the significant decrease of docosahexaenoic acid 22 : 6 (n - 31, the product of A5 desaturation and elongation of 20: 4 (n - 3). The 20: 4 + 22: 4/18 : 2 ratio also decreased due to the 20 : 4 and 22: 4 reduction. In PC, a similar trend was observed, but the changes were more striking. The decreases of 20 : 4 (n - 6) and 22 : 6 (n - 3) were very significant. These changes were well expressed as a diminished 20 : 4 + 22 : 4/18 : 2 ratio and the decreased unsaturation index. Regarding PE, the most significant variations were at 22 : 4 (n - 6) and 22 : 6 (n - 3) reduction and at the decreased index.
0.43+ 0.07 0.90* 0.13 * 1.80+ 0.28
Other lipids such as triacylglycerols or cholesterol esters were not considered. Results are the mean+ S.D. of six animals analyzed separately. Data are compared to control using the Student’s t-test. * P < 0.05; ** P < 0.02; *** P < 0.01.
content is due not only to the lack of dietary choline but also to the methylation inhibitors that contribute to impair completely the PC synthesis from PE. The periodate-oxidated adenosine alters the adenosine susceptible to methylation and increases the content of sadenosine homocysteine. The cycloleucine, in turn, competes with methionine, thus eliminating the source of methyl groups. Therefore, the two main pathways described for the de-novo biosynthesis of PC (1) the incorporation of choline into PC, and (2) the stepwise methylation of PE, are abolished.
Steady-state jluorescence anisotropy of DPH
The DPH fluorescence anisotropy increased in ratliver microsomes after 21 days of choline-free diet reaching values of 0.1599 (Table IV).
TABLE III Fatty-acid composition of microsomal lipids after a choline-free diet (mol%)
Fatty acids a
Total lipids
16:0 16:l 18:0 18:l 18:2 18:3(n -6) 20:4(n
-6)
22:4(n -6) 22:4(n-3)or22:5(n-6) 22:tin -3) 20:4(n -6)+22:4(n 18:2(n -6) U.I.
-6)
PC
PE
control
choline-free
control
choline-free
control
choline-free
9.15 + 0.90 0.40 + 0.00 19.10 + 0.20 8.01+ 0.10 12.7OkO.11 0.18kO.02 29.30 + 0.90 4.80 + 0.25 5.6OkO.32 7.03+ 1.01
10.80 + 0.80 0.30 f 0.00 20.41 f 0.20 7.40 &-0.10 12.31+ 0.10 0.15 + 0.04 25.21+ 0.80 * * 5.21+ 0.30 4.01 f 0.32 4.52f0.51 **
20.51 f 0.91 1.15 +0.07 21.44 + 0.08 8.76 + 0.54 16.30 + 0.98 0.20 f 0.06 23.54& 1.12 1.19+0.04 2.24 + 0.08 2.21& 0.06
23.30 + 0.50 1.14+0.08 19.85 k3.10 9.31 f 0.80 14.30 + 1.52 0.43 + 0.04 14.11+0.90 *** 1.10+0.31 1.46kO.15 0.29 f 0.02
18.90 + 0.97 0.90 f 0.01 28.33 + 0.28 8.18& 1.22 10.06+0.81 0.15 f 0.00 21.27+2.18 2.68 + 0.36 3.89 f 0.55 3.21 f 0.53
23.82 + 6.2 1.10~0.02 29.04 + 0.01 10.24+5.21 10.18+ 1.01 0.25 + 0.09 19.90+ 1.50 0.51kO.15 *** 3.01+ 0.50 1.09 + 0.43 * *
2.6850.11
2.47+0.10
1.52*0.1
1.06-10.19
2.38 f0.44
2.28 f 0.36
2.47 + 0.28
2.20+ 0.20
1.69kO.18
1.26+0.10 **
1.65 kO.15
1.35 fO.10
a Other minor fatty acids complete 100%. Results are the mean+S.D. of six animals. Datum is compared to control by using Student’s t-test. * P < 0.1; ** P < 0.01; *** P < 0.005. U.I., unsaturation index (E~,x, /FA) where n1 = number of double bonds in each fatty acid, x1 = mol of each fatty acid. FA, total mol of fatty acids.
192 Discussion
The lipid composition of biological membranes can be altered by dietary means [lS-201. The membrane composition is of fundamental importance since it is linked to several physiological and biochemical parameters, like the membrane-bound enzyme activity, among others [21]. These enzymes require an optimal lipid environment for the full expression of their functional activity [22]. A5 Desaturase converts fatty acids of 20 carbons into more unsaturated higher homologs. It represents a second regulatory step after the A6 desaturase in the regulation of polyunsaturated fatty-acid biosynthesis and bio-transformation of ingested essential fatty acids. A5 Desaturase is a component of an electron-transport system [23,241. The lipid bilayer of mammal endoplasmic reticulum is sufficiently fluid to allow rotational and lateral diffusion of these proteins 1251.Cons~quently~ contact between the different components of the system may depend on the fluidity of the surrounding lipid environment [3,4]. Based on different in-vitro and in-vivo membrane-lipid modifications [3-61, we have previously proposed that A5 desaturase would be regulated by the physical status of the membrane, exclusively in the absence of phospholipid changes. Thus, the rigidization of the membrane after cholesterol [4] or phytosterol incorporation in vitro and in vivo, respectively [S] leads to an increased AS-desaturase activity. Consequently, there is a higher arachidonic acid content in the phospholipids with no changes in their head polar group distribution. In turn, the rigidization of the membrane after cholesterol incorporation in vivo takes place together with an increase in the C/PC and PC/PE ratios and a lower A5-desaturase activity and arachidonic-acid content in the phospholipids [6]. The results obtained following a choline-free diet indicate several changes in the microsomal membrane. The decrease in the rotational mobility of the bulk lipids expressed by the higher value of rs of DPH (Table IV) may be attributed to the higher C/PC ratio, the lower PC/PE ratio (Table II) and/or the lower polyunsaturated fatty-acid content of the lipids in the bilayer. Under these particuIar conditions, the A5-desaturase activity decreased (Table I) and, in consequence, their desaturated fatty-acid products also decreased. The most important observation is that the enzymatic activity is diminished in a rigidized membrane, but in a different way than after the in-vivo cholesterol incorporation; the PC/PE ratio is now diminished. Therefore, the common features in both cases are the decreased rotational mobility of the lipids and the increase C/PC ratio. However, the lower ~uidity of the membrane should not be related to the lower AS-desaturase activity since a similar bilayer behaviour after chofesterol or phytosterol incorporation in vitro and in vivo, respec-
tively, induced the opposite enzymatic response. It is aIso important to ruIe out the absolute increment of cholesterol as responsible for the enzyme changes [26,271 for its incorporation in vivo and in vitro evoked opposite responses. On the basis of these results. the hypothesis postulating that AS desaturase responds to viscotropic variations only in the absence of phospholipids changes is reinforced. The C/PC ratio and specifically the PC would play a key role in this rcgulation. It is likely that AS desaturasc interacts with PC molecules of the membrane and its activity may he altered by variations of C/PC ratio. Further cxpcrimcnts are necessary to test this hypothesis. Acknowledgements
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