High de novo synthesis of glycerolipids compared to deacylation-reacylation in rat liver microsomes

Biochimica

et Biophysics

Elsevier Biomedical

Acta, 712 (1982)

605-615

605

Press

BBA 51194

HIGH DE NOVO SYNTHESIS DEACYLATION-REACYLATION PAUL

L. FOX and DONALD

Diuision

of Nutritional

University,

(Received

Ithaca,

March

TO

B. ZILVERSMIT

Sciences

NY 14853

OF GLYCEROLIPIDS COMPARED IN RAT LIVER MICROSOMES

and Section

of Biochemistry,

Molecular

and

Cell Biology,

Division

of Biological

Sciences,

Cornell

(U.S.A.)

18th, 1982)

Key words: Phosphatidylcholine;

Triacylglycerol;

Glycerolipid

synthesis; Acylation;

(Rat liver microsome)

A microsomal system characterized by high flux through the entire de novo pathway from glycerol phosphate to phosphatidylcholine and triacylglycerol has been developed. Optimum synthesis of phosphatidylcholine requires CDPcholine, Mg *+ , KC1 and a palmitoyl-CoA-generating system containing palmitic acid, ATP and CoA. Incorporation of [‘4C]glycerol phosphate into phosphatidylcholine is greater than its incorporation into triacylglycerol at all levels of added palmitate, but the phosphatidylcholine/triacylglycerol synthesis ratio decreases as palmitate is increased. Phosphatidylcholine synthesis from glycerol phosphate is stimulated more by palmitate than by other saturated fatty acids; phosphatidylcholine synthesis increases with increasing unsaturation of the added fatty acids. The ratio of incorporation of [3H]palmitate to [14C]glycerol phosphate was determined for phosphatidic acid, diacylglycerol, phosphatidylcholine and triacylglycerol. This ratio is approximately 2 for all diacylglycerolipids and 3 for triacylglycerol. In our system, incorporation of palmitate into microsomal glycerolipid proceeds primarily by the de novo pathway, with minimal fatty acid recycling via deacylation-reacylation.

Introduction

fatty acid distribution is due to deacylation and subsequent reacylation of pre-existing glycerolipids. There is some evidence for random acylation of glycerol phosphate to phosphatidic acid [6], and the acyltransferases responsible for glycerolipid modification by deacylation-reacylation possess a specificity consistent with non-random distribution of fatty acids [7,8]. The relative importance of the de novo and deacylation-reacylation pathways has been assessed by more quantitative means. In such experiments the amount of fatty acid incorporated into glycerolipid by each pathway was determined. Scherphof and Van Deenen [9] examined the uptake into glycerolipid of [‘4C]palmitic acid and [3H]glycerol phosphate in rat liver mitochondria and microsomes. By comparison of the 3H/‘4C ratio of glycerolipids to that of phosphatidic acid,

The asymmetric distribution of fatty acids in the glycerolipids of mammalian tissue has been well documented [l]. This non-random distribution has been ascribed to the specificity of the acyltransferases of the de novo pathway of glycerolipid synthesis. Specificity of glycerol phosphate acyltransferase for saturated fatty acids and of I-acylglycerol phosphate acyltransferase for unsaturated fatty acids has been shown in vivo [2,3] and with purified enzymes [4,5]. On the other hand, Lands and Hart [6] proposed that fatty acids are incorporated into phosphatidic acid randomly by the de novo pathway and that non-random Abbreviations: EGTA, ethyleneglycol-bis (P-aminoethyl ether)N, N’-tetraacetic acid; Hepes, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid. 0005/2760/82/0000-0000/$02.75

0 1982 Elsevier

Biomedical

Press

606

they concluded that essentially all pahnitic acid entered phosphatidylcholine, phosphatidylethanola~ne and triacylglycerol by deacylationreacylation and that de novo synthesis was responsible for incorporation of fatty acid into diacylglycerol only. Van Golde et al. [IO] and Sarzala et al. [ 1I] reached similar conclusions with several saturated and unsaturated fatty acids added to both rat liver microsomes and homogenates. In rat liver slices, however, considerable incorporation of fatty acid by de novo synthesis of phosphatidylcholine and phosphatidylethanolamine was observed [lo]. Similarly, Stein and Stein [ 121 observed a low deacylation of phospholipid by intact rat liver. Van Golde et al. [ 101proposed that the conflicting results from liver slice and microsome experiments were due either to impairment of de novo synthesis or to accumulation of lysophospholipids during homogenization or cell fractionation. Evidence for impaired de novo synthesis in microsomes is widespread; the principal product of the pathway is a function of microsomal preparation and incubation conditions. Lysophosphatidic acid [ 13,141 and phosphatidic acid [4,9,15,16 ] have been suggested as end products of the glycerol phosphate biosynthetic pathway; there are reports of significant accumulation of label into diacylglycerol [ 17,181 and, on addition of a cytosolic fraction, into triacylglycerol [ 19,201. Formation of phosphatidylcholine or phosphatidylethanolamine from glycerol phosphate is generally reported to be low and also cytosol-dependent [21]. During the course of our investigations on the regulation of de novo glycerolipid synthesis, we developed a microsomal system optimized for phosphatidylcholine synthesis. It is the purpose of this report to describe and characterize this system and quantitate the relative importance of fatty acid entry into microsomal glycerolipid by the de novo and deacylation-reacylation pathways. Materials and Methods Materials

An 80 mM solution of rat-glycerol phosphate (Sigma Chemical Co., St. Louis, MO) was prepared containing L-[U-14~]glycerol phosphate (New England Nuclear, Boston MA), at a specific

activity of 0.05 mCi/mmol, and stored at -20°C. [9,10-3H]Palmitic acid (Amersham Corp., Arlington Heights IL) was purified by thin-layer chromatography in hexane/diethyl ether/acetic acid (50: 50: 1, v/v) to a radiopurity of at least 99%. A neutral, homogeneous solution of 2 mM [3H]palmitate was prepared by combination of purified [3H]palmitate, unlabeled palmitate (Sigma} and 20 mg bovine serum albumin/ml (fraction V, fatty acid-free, Miles Laboratories, Elkhart, IN) at a specific activity of 1.25 mCi/mmol, in 5 mM Tris-HCl (pH 7.0) by an adaptation of the procedure of Laurel1 [22]. Laurie, heptadecanoic, stearic (Eastman Organic Chemicals, Rochester, NY), myristic (Applied Science Laboratories, State College, PA), oleic (Sigma) and linoleic (General Biochemicals, Chagrin Falls, OH) acids were prepared as albumin solutions and stored at 4°C. Solutions of palmitoyl-CoA, ATP, CoA and CDPcholine (Sigma) were prepared in 5 mM Tris-HCl (pH 7.0) and stored at -20°C. 1-Oleoyl glycerophosphate, pig liver diacylglycerol, triolein (Serdary Research Laboratories, London, Ontario), phosphatidic acid (Lipid Products, South Nutfield, U.K.) and cholesteryl ester (Sigma) were used as thin-layer chromatography standards. All aqueous solutions contained 0.02% NaN, to prevent bacterial growth.

Microsomes were prepared from the livers of male rats (250-400g) fasted overnight. For each preparation, one to three rats were anesthetized in a CO, atmosphere and decapitated. The livers were removed, minced, and washed in ice-cold buffer containing 150 mM KCl/5 mM Hepes/O.S mM EDTA, pH 7.0. Substitution of sucrose for KC1 resulted in consistently lower phosphatidylcholine synthesis. A 30% homogenate was prepared in the same buffer by 5-6 passes at approximately 300 rpm in a Potter-EIvehjem homogenizer. The suspension was centrifuged twice at 13 000 X g for 7 min to remove nudei, mitochondria, lysosomes, intact cells and debris. Microsomes were pelleted by centrifugation at 1050~ X g for 60 min. The pellets, without the small underlying viscous, high-density layer, were returned to the homogenizer and resuspended, working the teflon pestle by hand. Microsomes

607

were resedimented by centrifugation at 105000 X g for 30 min and resuspended in the same buffer to a final protein concentration of 25-35 mg/ml. All procedures were done at 0-4°C. Microsomes were quickly frozen in approximately 0.05-ml aliquots pipetted directly into liquid N,. Slow freezing caused considerable decrease in phosphatidylcholine synthesis from glycerol phosphate, but storage of quick-frozen microsomes for up to six months at -70°C had little effect on synthesis. Microsomal protein was measured by the biuret method with bovine serum albumin as standard [23]. Analytical methods Glycerolipids were separated from radioactively labeled precursors by a modification of the chloroform/methanol extraction procedure described by Folch et al. [24]. All washes contained 0.15 M HCl, which is necessary for quantitative recovery of phosphatidic acid and, especially, lysophosphatidic acid. Aliquots of the lower phases were dried in glass vials under a stream of N, and applied to silica gel 60 thin-layer chromatography plates (E. Merck, Darmstadt, F.R.G.) with four O.l-ml rinses of chloroform/methanol/water (25 : 15 : 2, v/v). Preliminary tests showed that, unlike less polar solvents, this solvent system completely solubilized all acidic phospholipids. Phospholipids were separated by development of the plate to approximately 2/3 its length in chloroform/acetone/ methanol/acetic acid/water (6 : 8 : 2 : 2 : 1, v/v) [25]. For separation of neutral lipids, the plate was dried under N, and redeveloped in the same direction to the top in hexane/diethyl ether/acetic acid (50 : 50 : 1, v/v). In experiments with [3H]palmitate, triacylglycerol was separated from cholesteryl ester with hexane/diethyl ether/acetic acid (80:20: 1, v/v). Chromatographed lipids were visualized in an I, chamber and spots compared to authentic lipid standards. Silica gel scrapings were transferred to 20-ml scintillation vials and dispersed in 1 ml H,O by sonication for lo-20 s in a Branson HD-50 (Heat Systems, Melville, NY) bath sonicator. In preliminary tests, this procedure was shown to amphiphilic and neutral elute the acidic, glycerolipids completely from silica gel. Radioactivity was measured in a Beckman LS8100 liquid

scintillation counter with 10 ml ACS (Amersham) as scintillant. Most samples were counted to a standard deviation of under 2%; samples with low count rates (i.e., lysophospholipids and early time points for other lipids) were counted to a standard deviation of under 5%. Greater than 95% of the radioactivity applied to the thin-layer plate was recovered after chromatography. Results Incorporation of [‘4C]glycerof phosphate into glycerolipid Fig. 1 shows a time course of incorporation of [‘4C]glycerol phosphate into the metabolites of de novo glycerolipid synthesis. The data are typical of several replications. Lysophosphatidic acid quickly reached a plateau, which was maintained for several hours. Label rapidly increased in phosphatidic acid for lo-60 min (depending on the microsomal preparation), followed by a gradual decline. The decline was not due to exhaustion of substrates or cofactors since their addition during the incubation had little effect on shape of the curves. The level of radioactivity in diacylglycerol reached a maximum after about 1 h, followed by a slow decline. After a short lag period, phosphatidylcholine accumulated label linearly for l-2 h, then continued at a lower rate for more than 3 h; incorporation of label into triacylglycerol and phosphatidylethanolamine was considerably lower than into phosphatidylcholine. The measured incorporation of [‘4C]glycerol phosphate into lysophosphatidic acid, although small, may be exaggerated because the thin-layer chromatography system did not completely separate phosphatidylserine and phosphatidylinositol from lysophosphatidic acid. Labeling of the contaminants should be low, however, as phosphatidylserine and phosphatidylinositol precursors were not included in the incubation medium. Moreover, analysis of thin-layer chromatograms by scraping narrow bands showed that most of the label was present in lysophosphatidic acid. When microsomes were incubated under the same conditions but with palmitoyl-CoA instead of with the palmitoyl-CoA-generating system (palmitate, ATP, CoA), far less [‘4C]glycerol phosphate was incorporated into all lipids (TableI).

60 TIME

120

180

(mid

Fig. 1. De novo glycerolipid biosynthesis. Microsomes (0.4 mg protein) were pre-incubated 10 min at 37°C in 150 mM KCl, 3 mM MgCl,, 50 mM Hepes, 0.1 mM EGTA, 10 mM cysteine, 3.5 mM ATP, 0.167 mM CoA, 0.333 mM CDPcholine, 0.133 mM pahnitate and 1.33 mg bovine serum albumin/ml (pH 7.0). Incubation was started by addition of 0.2 PCi L[‘4C]glycerol phosphate in rat-glycerol phosphate to a final concentration of 3.33 mM L-glycerol phosphate in a total incubation volume of 0.6 ml. All calculations are based on the specific activity of [‘4C]glycerol phosphate (GP), ignoring the D form. The reaction was terminated at times shown by addition of 11.4 ml chloroform/methanol (2 : 1, v/v). Incorporation into glycerolipid is shown as nmol/mg microsomal protein. 0, Lysophosphatidic acid; X, phosphatidic acid; D, diacylglycerol; 0, phosphatidylcholine; n , triacylglycerol; -. , phosphatidylethanolamine.

The relatively high accumulation of label into phosphatidic acid compared to phosphatidylcholine and triacylglycerol is due to the short incubation period. It is well known that fatty acyl-CoA inhibits membrane-bound enzyme activities by a detergent-like action. Experiments were done to separate this cause of low incorporation of label from a second possibility - that exogenously added pahnitoyl-CoA is a poor substrate. In an experiment in which equimolar levels of palmitate (plus ATP and CoA) and palmitoyl-CoA were present, low levels of incorporation of label from glycerol phosphate were observed. This argues for inhibition via enzyme inactivation by palmitoylCoA.

The incubation conditions described in Fig. 1 result from optimization of phosphatidylcholine synthesis by varying KCl, Mg*+, CoA, ATP and CDPcholine concentrations and the palmitate/ bovine serum albumin ratio. The incorporation of label from glycerol phosphate into the various metabolites as a function of Mg*+ concentration is shown in Fig. 2. In the absence of added Mg*+ , 85% of the lipid synthesized was phosphatidic acid. The optimum Mg*+ concentration for phosphatidylcholine synthesis was 3 mM, whereas that for triacylglycerol was at most 1 mM. The former was chosen for all subsequent experiments. The dependence of glycerolipid synthesis on fatty acid chain length and degree of saturation is shown in Fig. 3. Stimulation of incorporation of glycerol phosphate into phosphatidylcholine was considerably higher with palmitate than with other saturated fatty acids. There was also a direct correlation of phosphatidylcholine synthesis with degree of fatty acid unsaturation. Triacylglycerol synthesis was much less dependent on fatty acid length and unsaturation. The incorporation of [‘4C]glycerol phosphate into phosphatidylcholine and triacylglycerol and the molar ratio of incorporation as a function of exogenous palmitate are shown in Fig. 4. Although phosphatidylcholine synthesis at all levels of

TABLE I INCORPORATION OF [‘4C]GLYCEROL PHOSPHATE INTO GLYCEROLIPID WITH PALMITOYL-CoA AND A PALMITOYL-CoA-GENERATING SYSTEM. All tubes were incubated as described in Fig. 1 for 30 min. Results with a palmitoyl-CoA-generating system (0.133 mM palmitate, 3.5 mM ATP, 0.167 mM CoA) are compared to results with 0.133 mM palmitoyl-CoA. Results are expressed as mean *S.D. of duplicate incubations. Values are nmol/mg microsomal protein. Lipid

PalmitoylCoA

Palmitoyl-CoAGenerating system

Lysophosphatidic acid Phosphatidic acid Diacylglycerol Phosphatidylcholine Triacylglycerol

0.71 eo.22 5.12*0.21 1.311-0.05 1.90r0.06 0.65-fO.12

2.29*0.02 44.0 -c 2.5 14.7-cO.6 18.322.2 6.81 kO.78

609

/

P

i0.. I

,O.. I

d !O..> I:

Q-

18 -iS: [Mg*+l (mM) Fig. 2. Incorporation of [‘4C]glycerol phosphate (GP) into glycerolipid as function of [Mg2+ 1. Microsomes were incubated as in Fig. 1, for I h. 0, Lysophosphatidic acid; X, phosphatidic acid; A, diacylglycerol; Cl, phosphatidylcholine; m, triacylglycerol.

palmitate was greater than triacylglycerol synthesis, the relative amount of phosphatidylcholine formed decreased at increasing palmitate concentrations. Incorporation of [I4Clglycerol phosphate into glycerolipid at three temperatures is shown in Fig. 5. The amount of label in phosphatidylcholine was highest at 37°C. At lower temperatures there was greater accumulation of labeled total lipid, with phosphatidic acid the principal product. In some experiments, microsomes were pre-incubated at several temperatures for 2 h without glycerol phosphate. Upon subsequent incubation at 37°C with [‘4C]glycerol phosphate, incorporation of label into total glycerolipid was decreased to a greater extent by pre-incubation at 37°C than at lower temperatures. The loss of synthetic activity was considerably less in KC1 buffer than in sucrose, but still amounted to approximately 60% and is probably responsible for the lower total lipid formation at high temperatures, as well as the failure to achieve a steady-state level of phosphatidic acid in the time course shown in Fig. 1.

_-a

1 18: 2

FATTY ACID LENGTH Fig. 3. Dependence of glycerolipid synthesis on fatty acid chain length and unsaturation. Microsomes were. incubated as in Fig. 1 for 120 min. Laurie, myristic, palmitic, heptadecanoic, stearic, oleic and linoleic acids were added as albumin solutions to a final concentration of 0.133 mM fatty acid and 1.33 mg albumin/ml. 0, Lysophosphatidic acid; X, phosphatidic acid; A, diacylglycerol; 0, phosphatidylcholine; fl , triacylglycerol. GP. glycerol phosphate.

Relative incorporation of [jH] p~~rni~ate and [‘4C]gIycerol phosphate into microsomal glycerolipids The molar ratio of fatty acid to glycerol phosphate incorporation is a measure of the relative importance of the de novo and deacylation-reacylation pathways. A molar ratio of 2 in ‘a diacylglycerolipid implies that synthesis is solely via the de novo pathway. A ratio greater than 2 indicates incorporation of labeled fatty acid into unlabeled glycerolipid by deacylation-reacylation. In several preliminary studies, the ratios for phosphatidic acid, diacylglycerol and phosphatidylcholine were all found to be significantly less than 2. This unexpected result can be explained by dilution of labeled fatty acid by microsomal fatty acid (and its CoA ester). To minimize the effect of an endogenous pool, higher concentrations of [3H]palmitate were added. The results are shown in Fig. 6A. At the highest palmitate concentration,

610

0.0

II'4 0.8

1.2 0.0

0.4

0.8

1.2

PALMITATE (mM)

PALMITATE (mM> Fig. 4. Incorporation of ji4C]glycerol phosphate (GP) into phosphatidylcholine (PC) and triacylgycerol (TG) as function of exogenous palmitate. Microsomes were incubated 2 h in the absence of palm&ate and at three concentrations of exogenous pahnitate: 0.133, 0.4 and 1.2 mM. Conditions were the same as in Fig. I except that fatty acid was added after the IO min pre-incubation. 0, Phosphatidylcholine; m, triacylglycerol; 0, phosphatidylcholine/triacylglycerol.

150

-,

P

D %

100

‘;’

2 4 50

E t

3‘

Lo

0 TEMPERATURE ("0 Fig. 5. Incorporation of glycerolipid as a function incubated as in Fig. I for 0, Lysophosphatidic acid A, diacylglycerol (DG); triacylglycerol (TG).

[‘4C]glycerol phosphate (GP) into of temperature. Microsomes were 60 min at the temperatures shown. (LPA); X, phosphatidic acid (PA): 0, phosphatidylcholine (PC); W,

Fig. 6. Molar ratio of ‘H-labeled fatty acid (FA)/[‘4C]glycerol phosphate (GP) incorporation into glycerolipid as a function of added ~3H]pal~tate. A, Tubes wereincubated as in Fig. 1, for 120 min, except for addition of [3H]palmitate after 10 min pre-incubation; B, fatty acid incorporation into glycerolipid is measured by esterification of [3H]palmitate and corrected for endogenous fatty acid as described in text. X, Phosphatidic acid; a, diacylglycerol; Cl, phosphatidylcholine; n , triacylglycerol.

the f3H]palmitate/[ r4C]glycerol phosphate ratio was approximately 2 for diacylglycerol, phosphatidic acid and phosphatidylcholine and 3 for triacylglycerol. Furthermore, there appears to be a flattening of the curves, consistent with asymptotic convergence at higher palmitate levels. The quantity of endogenous microsomal fatty acid can be determined indirectly by calculating the fatty acid esterification from [‘4C]glycerol phosphate incorporation into various lipids in the absence of exogenous palmitate. This calculation assumes complete and exclusive esterification of free fatty acid with glycerol phosphate during the incubation period. This requirement is satisfied, because after incubation for 2 h with low levels of j3H]palmitate, all of the label is esterified with glycerol phosphate. The calculated level of endogenous fatty acid is 72.6 * 2.9 nmol/mg microsomal protein. This is considerably larger than the 15-28 nmol/mg as determined chemically 1261; the difference probably represents fatty acid released from fatty acyl esters during incubation.

611

TABLE

II

RATIO

OF FATTY

ACID/GLYCEROL

PHOSPHATE

INCORPORATION

INTO GLYCEROLIPID

Tubes were incubated as described in Fig. 1, for 120 min, except for addition of [3H]palmitate (and unlabeled linoleate in Expt. 3) after 10 min pre-incubation. For Expts. 1 and 2, fatty acid incorporation into glycerolipid is measured by esterification of [‘Hlpalmitate and corrected for endogenous fatty acid. For Expt. 3, fatty acid incorporation is corrected for added linoleate as well as endogenous fatty acid. Results of Expt. 1 are expressed as mean2S.D. in duplicate incubations. Expt.

Exogenous fatty acid (mM) C,,:,

C18:2

Fatty acid/glycerol Phosphatidic

phosphate

incorporation

(mol/mol)

Diacylglycerol

Phosphatidylcholine

Triacylglycerol

Phosphatidylethanolamine

3.41 3.45 3.21

2.68 2.41 2.38

acid 0.133

0

1.6lk.03

1.91 k.05

2.19k.01

a b

0.133 0.400

C

1.200

0 0 0

1.90 1.92 2.00

2.08 2.04 2.19

2.34 2.14 2.12

a b

0.133 0.133

0 0.133

2.15 1.74

C

0.133

0.267

1.85

1 L

3

The amount of fatty acid incorporated into each lipid was calculated from the specific activity of the added fatty acid, corrected for the amount of endogenous fatty acid generated during the incubation. The corrected results appear in Fig. 6B, in which all curves are more horizontal. Table II shows three experiments in which the results were corrected for endogenous fatty acids. The molar ratio of incorporation is less than, or approximately equal to, 2 for phosphatidic acid, diand phosphatidylcholine, slightly acylglycerol, greater than 2 for phosphatidylethanolamine and slightly greater than 3 for triacylglycerol. It can be argued that the high level of saturated fatty acid in these experiments results in formation of molecular species which are largely absent from normal rat liver microsomes. Therefore, we examined the effect of unlabeled linoleate on the ratio of incorporation of [3H]palmitate to [ I4 Clglycerol phosphate in phosphatidylcholine. Linoleate competes with palmitate for incorporation into phosphatidylcholine since the ratio of 3H/‘4C was found to be inversely related to linoleate concentration. The molar ratio of fatty acid to glycerol phosphate incorporated into phosphatidylcholine was, therefore, calculated based on the total non-esterified fatty acid in the medium

(i.e., the sum of [3H]palmitate, linoleate dogenous fatty acid). After this correction the ratio remains approximately 2 (Table

0

120

60 TIME

and enis made, II, Expt.

180

(mid

Fig. 7. Time course of molar ratio of ‘H-labeled fatty acid/[‘4C]glycerol phosphate incorporation into glycerolipid. Tubes were incubated as described in Fig. 6. X, Phosphatidic acid; A, diacylglycerol; 0, phosphatidylcholine; W, triacylglycerol. GP, glycerol phosphate.

612

A time course of the ratio of incorporation of [‘Hlpalmitate to [‘4C]glycerol phosphate into glycerolipid is shown in Fig. 7. At the earliest time examined, 15 min, all diacylglycerolipids have initial ratios of approximately 2 and triacylglycerol approximately 3. These ratios all decrease at later times.

dence

for a Mg2+ -independent

hydrolase

as well

WI.

Discussion

The effects of fatty acid length, saturation and concentration on microsomal glycerolipid synthesis agree in general with results of more physiological studies. Optimum stimulation of phosphatidylcholine synthesis by C,, fatty acids has been reported in isolated rat hepatocytes [29,30]. We find that phosphatidylcholine synthesis is more sensitive than triacylglycerol synthesis to fatty acid

In our experiments the end products of microsomal glycerolipid synthesis from glycerol phosphate are phosphatidylcholine and triacylglycerol; because CDPethanolamine was not included, low levels of phosphatidylethanolamine are formed. The formation of phosphatidylcholine from labeled glycerol phosphate is very high compared to results by others with rat liver microsomes. Possmayer et al. [ 171 report less than 1% of total lipid radioactivity in phosphatidylcholine after 30 min, whereas we find approximately 25%. Mitchell et al. [21] incubated microsomes with labeled glycerol phosphate under conditions in which phosphatidic acid is the principal product. After a second 20-min incubation in a medium containing CDPcholine, they reported synthesis of 0.31 nmol phosphatidylcholine/mg microsomal protein. An increase to 0.97 nmol phosphatidylcholine/mg in the presence of a cytosolic fraction led them to postulate a requirement for phosphatidic acid phosphohydrolase (EC 3.1.3.4) in phosphatidylcholine synthesis. We consistently observed synthesis of lo- 12 nmol phosphatidylcholine/mg after a 20 min incubation in the absence of cytosol, and found only a slight increase upon addition of cytosol or partially purified phosphatidic acid phosphohydrolase (currently under investigation). The high level of incorporation of glycerol phosphate into phosphatidylcholine and triacylglycerol results from optimization of several parameters. The increased synthesis resulting from substitution of the palmitoyl-CoA-generating system for palmitoyl-CoA is consistent with its stimulation of triacylglycerol synthesis in microsomes from intestinal mucosa [19]. The requirement for Mg2+ is not unexpected, as microsomal phosphatidic acid phosphohydrolase has been shown to [27], although there is evibe Mg 2+-dependent

chain length and degree of unsaturation. This is consistent with a specificity of cholinephosphotransferase (EC 2.7.8.2) for particular diacylglycerol species. This conclusion does not agree with previous reports of a low selectivity of cholinephosphotransferase [31,32]. It is not likely that the relation of phosphatidylcholine synthesis to fatty acid chain length and unsaturation is due to the specificity of fatty acyl-CoA synthetase or to the partitioning of fatty acids between microsomes and albumin complexes, as there is no such relation with triacylglycerol synthesis. Modification of the ratio of phospholipid synthesis to triacylglycerol synthesis by fatty acid concentration has been reported in liver slices [33] and liver cells [34]. Our results show that regulation at the branchpoint from diacylglycerol to phosphatidylcholine and triacylglycerol is also expressed by isolated microsomes. The molar ratio, R, of incorporation into lipid of [3H]palmitate to [‘4C]glycerol phosphate is a function of de novo synthesis and deacylation-reacylation, i.e.: R = (fatty acid incorporated de novo (mol) + fatty acid incorporated by deacylation-reacylation (mol))/glycerol phosphate incorporated (mol). Assuming n mol of fatty acid per mol of glycerolipid, then by substitution and rearrangement, the ratio of fatty acid incorporated by de novo synthesis to deacylation-reacylation is: fatty acid incorporated de novo (mol)/fatty acid incorporated by deacylation-reacylation (mol) = n/( R - n).Applying this equation to the data in Table II, Expt. 2, we calculate the amount of fatty acid incorporated by de novo synthesis is 20.0, 10.0, 8.1 and 3.9 times that incorporated by deacylation-reacylation for diacylglycerol, phosphatidylcholine, triacylglycerol, and phosphatidylethanolamine, respectively. This calculation cannot be made for phosphatidic acid since R in yields a negative

613

TABLE POOL

III SIZES OF NEWLY

FORMED

AND PRE-EXISTING

LIPIDS

IN MICROSOMES

Microsomes were incubated with glycerol phosphate for 120 min at three palmitate levels, as described in Fig. 4. Pool sizes of newly Pool sizes of pre-existing synthesized lipids are calculated from lipid 14C divided by the specific activity of [‘4C]glycerol phosphate. lipids are literature values.Values are nmol/mg microsomal protein. Lipid

Newly formed

Phosphatidic acid Diacylglycerol Phosphatidylcholine

37.0-222.4 8.8- 47.7

Phosphatidylethanolamine Triacylglycerol

40.92.811.6-

lipid

63.5 10.1 46.4

number. These results clearly indicate relatively low deacylation-reacylation of all tested lipids, with the possible exception of phosphatidylethanolamine, which may reflect the absence of CDPethanolarnine in our system. A second interpretation of Table II is obtained by dividing the ratio R for each lipid by R for phosphatidic acid. If no deacylation-reacylation of lipid products beyond phosphatidic acid occurs, this ratio should be the same for all species. We calculate R dmcylglycerol/R phosphatidic acid was ‘. l8 and 1.08 in Expts. 1 and 2, respectively. This result is in close agreement with the range of values reported by others for palmitate as added fatty acid, 1.07- 1.34 [9- 111. These authors, however, report quotients of 20-80, 15-30, and 8.7-30, for phosphatidylcholine, triacylglycerol and phosphatidylethanolamine, respectively. Our values for these lipids, 1.13-1.36, 1.74, and 1.29, respectively, are considerably lower, confirming our conclusion on the relative importance of de novo synthesis. There is a small but monotonic and reproducible increase of R along the de novo pathway to phosphatidylcholine (Table II). This is consistent with a low level of fatty acid recycling at each successive step. This relationship between R and position in the pathway is affirmed by the results of the time-course experiment in which this relation holds at all time points (Fig. 7). The relative importance of the de novo and deacylation-reacylation pathways is relatively independent of the incubation period. The slow decrease of R values is probably due to progressive dilution of [3H]palmitate by unlabeled fatty acid produced during the incubation.

Pre-existing 3-10 12- 20 265-400 108 46

lipid

Ref. 17,35 36,37 37,38 38 26

The effect of pool size of endogenous glycerolipids on the determination of deacylationreacylation merits discussion. If the microsomal pool of a lipid is small compared to the amount of the lipid synthesized via the de novo pathway, then deacylation-reacylation may not be readily detected. This is so, because recycling of fatty acid in newly synthesized glycerolipid does not alter the relative abundance of labeled fatty acid per mol of glycerolipid. Only acylation of pre-existing lipid would increase the amount of labeled fatty acid esterified. The amount of phosphatidic acid formed by de novo synthesis is an order of magnitude greater than that of endogenous phosphatidic acid (Table III). It follows that the presence of deacylation-reacylation of phosphatidic acid cannot be determined from our data. On the other hand, the amounts of diacylglycerol and triacylglycerol formed during our experiments, by the de novo pathway, are nearly equal to the amounts present in unincubated microsomes. The phosphatidylcholine formed is much less than the endogenous pool of this lipid. Therefore, deacylation-reacylation of diacylglycerol, triacylglycerol and phosphatidylcholine should be readily measurable. We conclude that in our microsomal system, modification of diacylglycerol, phosphatidylcholine and triacylglycerol by deacylation-reacylation with palmitate exists, but is at a low level compared to de novo synthesis. Our approach differs from previous investigations in several respects. Scherphof and Van Deenen [9], Van Golde et al. [lo] and Sarzala et al. [ 1 l] compared the incorporation of labeled fatty acid to labeled glycerol phosphate as a ratio of

radioactivities. They compared this ratio in various glycerolipids to that of phosphatidic acid, which was assumed to be synthesized solely by the de novo pathway. This assumption is reasonable, as the extremely small pool size of phosphatidic acid [ 17,351 implies rapid turnover and low probability for modification by deacylation-reacylation. In our studies we have used the molar ratio of incorporation, which provides an additional comparison: it not only relates deacylation-reacylation of various glycerolipids to that of phosphatidic acid, but also compares it to the theoretical value. The critical difference between this work and previous work is the high flux through the entire de novo pathway. The great importance of the deacylation-reacylation pathway for phosphatidylcholine and phosphatidylethanolamine modification reported previously is possibly explained by low de novo synthesis of these lipids. The low synthesis may be due to the absence of CDPcholine and CDPethanolamine and their precursors. This, however, does not explain the analogous result for triacylglycerol. It is probably not due to lack of phosphatidate phosphohydrolase activity in the microsomes because there is a relatively large de novo synthesis of diacylglycerol. These conflicting results may be due to differences in microsomal preparation, substrate and cofactor concentrations, or incubation conditions. An additional investigation of the problem in triacylglycerol synthesis was reported by Tzur and Shapiro [39]. They measured incorporation of two labeled forms of phosphatidic acid, 1,2diacyl[‘4C]glycerol phosphate and 1,2-[‘4C]diacylglycerol phosphate, into diacylglycerol and triacylglycerol, concluding that 60% of diacylglycerol and O-10% of triacylglycerol are formed by the de novo pathway. These results, especially that for triacylglycerol, are in contrast to our own. An unusual aspect of our microsomal system is the large amount of fully saturated glycerolipid formed. At the highest palmitate level used in our study, approximately 400 nmol of lipid were newly synthesized per mg of microsomal protein. This is close to the amount of lipid present in unincubated microsomes. It is apparent that there is a gross compositional change in these membranes. This may prove useful in studies of membrane activity as a function of lipid composition or fluidity.

It should be recognized that these studies were conducted on microsomes optimized for phosphatidylcholine synthesis, and that during the optimization procedure we did not assess deacylation-reacylation. A requirement for Ca2+ by microsomal phospholipases A, and A, has been described by Newkirk and Waite [40], but a conflicting stimulation by EDTA has also been reported [41]. Others have shown that Mg*+ is nearly as efficient as Ca2+ [42] and KC more efficient [16] at stimulating microsomal phospholipase A activities. The differences between our results and those of others regarding deacylation-reacylation cannot be ascribed to Ca’+, since in none of the studies was Ca2+ added. Previous in vitro studies have shown that several types of fatty acids are incorporated into glycerolipids primarily by deacylation-reacylation. In vivo studies, on the other hand, have emphasized the importance of de novo synthesis for the incorporation of all but the longest saturated fatty acids (e.g., stearate) and highly unsaturated fatty acids (e.g., arachidonate) [43,44]. Our in vitro results similarly stress the relative importance of the de novo pathway for the incorporation of palmitate into microsomal glycerolipids. Acknowledgements This investigation was supported by Research Grant HL 10940 from the National Heart, Lung, and Blood Institute, U.S. Public Health Service. D.B.Z. is a Career Investigator of the American Heart Association. P.L.F. was supported in part by N.I.H. Training Grant 2 T32 GM07273. We wish to thank Mark Nigogosyan for valuable technical assistance. References Holub, B.J. and Kuksis, A. (1978) Adv. Lipid Res. 16, 1-125 Akesson, B., Elovson, J. and Arvidson, G. (1970) Biochim. Biophys. Acta 218, 44-56 Akesson, B. (1970) B&him. Biophys. Acta 218, 57-70 Yamashita, S. and Numa, S. (1972) Eur. J. B&hem. 31, 565-573 Yamashita, S., Hosaka, K. and Numa, S. (1973) Eur. J. Biochem. 38, 25-31 Lands, W.E.M. and Hart, P. (1964) J. Lipid Res. 5, 81-87

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