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Biochimica et Biophyrica Acta, 380 (1976) 245-266 @ Elaevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

BBA 56561

DISTRIBUTION OF FATTY ACIDS INCORPORATED INTO TRIACYLGLYCEROLS BY MICROSOME/CYTOSOL PREPARATIONS FROM ADIPOSE TISSUE

GILBERT B. STOKES*, LINDA W. PGTEAT and SAMUEL B. TGVE

Deportment of Biochemistry, North Carolina State University, Raleigh, N.C. 27607 (U.S.A.)

(Received July 3Oth, 1974)

Microsome/cyt.osol preparations from adipose tissue of the mouse, pig, rat, and chicken and from pig liver synthesize triacylglycerols containing a fatty acid distribution consistent with that in their respective fats. Since the incor- poration of fatty acids depends on the presence of glycerol phosphate and no loss of tritium occurs during the incorporation of sn-[2-j H] glycerol 3-phos- phate into triacylglycerols, it would appear that the specific distribution is a property of the transacylases of the glycerophosphate pathway. The apparent Michaelis constant for sn-glycerol 3-phosphate, measured either by sn-glycerol 3-phosphate or palmitate incorporation, averaged 1.4 * lo-’ M for both the mouse and pig adipose tissue enzyme systems.

Introduction

There are three metabolic pathways that could lead to the synthesis of a triacylglycerol by adipose tissue. All of these involve enzymes that are mem- brane bound and thus far have not been extensively purified. The first of these is the glycerophosphate pathway in which sn-glycerol 3-phosphate is doubly acylated to form a phosphatidic acid [ 11. Hydrolysis of the phosphate then occurs [2,3] followed by acylation of the dia&ylglycerol to form the triacyl derivative [4]. The second pathway involves the acylation of a monoacylgly- cerol to form first the diacylglycerol and then the triacylglycerol [6]. In the third pathway, dihydroxyacetone phosphate is acylated [6], cind the carbonyl group is subsequently reduced to yield a l-acyl-en-glycerol 3-phosphate [7]. This product is then acylated at the 2-position to form a phosphatidic acid and

‘* Pmsent addrem: Detmrtment of Eiochemhtw md Biophydcs, University of Cdifomla. Lhvis. Calif. 95616. U.S.A.

246

from this point on the pathway follows the glycerophosphate pathway. The non-random distribution of fatty acids within a triacylglycerol molecule might presumably result from the specificity of the enzymes from one or more of these biosynthetic pathways.

The positional distribution of fatty acids in natural triacylglycerols is not random. There is a tendency for unsaturated fatty acids to occupy the middle position and for saturated fatty acids to be esterified to one or both of the outer positions [8]. The pig and some of its relatives [9,10] provide an excep- tion to this pattern in that an overwhelming percentage of the palmitic acid is esterified at the a-position. This unusual distribution of palmitic acid is found in the triacylglycerols from all tissues of the pig examined except the liver, where the fatty acid distribution conforms to the general pattern of other organisms [ll] . When structural analyses were extended to include the princi- pal phospholipids from pig kidney and adipose tissue, unsaturated fatty acids rather than palmitic acid were found in the 2-position [12,13]. The fatty acid distribution of the pig lipids is therefore of particular interest since the gly- cerophosphate pathway, with 1,2diacyl-sn-glycerol serving as a common inter- mediate for both triacylglycerol and phospholipid biosynthesis [14], is con- sidered to be the major pathway for the synthesis of both phospholipids and triacylglycerols.

In this paper, we present results which show that the triacylglycerols synthesized by subcellular preparations from adipose tissue of several species, including the pig, show a fatty acid distribution which resembles that of their natural triacylglycerols.

Experimental procedure

Substrates and reagents The 1-l 4 C-labeled fatty acids, [l-l 4 C] palmitoyl-CoA, [l-i 4 C] stearyl-

CoA, sn- [ IJ-’ 4 C Iglycerol-3-P, and the [2-3H]glycerol were purchased from New England Nuclear. The purity of the fatty acids was greater than 90% as judged by reversed-phase [16] and argentation thin-layer chromatography [16]. The sn-[2-3 H] glycerol-3-P was prepared essentially as described by Bublitz and Kennedy [17] by means of glycerokinase obtained from Boehrin- ger-Mannheim. The identity of the product was confirmed by the reduction of NAD’ in the presence of glycerolphosphate dehydrogenase that was also ob- tained from Boehringer-Mannheim and by paper chromatography [18].

Non-radioactive fatty acids and other lipids were obtained from commer- cial sources. Dithiothreitol, mc-glycerol-l-P, the disodium salt of EDTA, ATP, and reduced glutathione were products of Sigma Chemical Co. The CoA and non-radioactive acyl-CoA preparations were obtained from P-L Biochemicals, and the pancreatic lipase (steapsin) was obtained from Nutritional Biochemicals Corp. All other chemicals were of reagent grade and were obtained from com- mercial sources.

Prepamtion of microsomekytosol fractions As soon as possible after sacrifice, samples of pig perinephric adipose

tissue, chicken mesenteric adipose tissue, and rat epididymal fat pads were

247

chilled in ice-cold 5 mM Tris/maleate buffer, pH 7.3, containing 0.26 M sucrose, 1 mM EDTA, and 1 'mM dithiothreitol. Pig liver samples were chilled with ice. Mouse epididymal fat pads were chilled in either Tris/maleate/sucrose buffer or in 0.15 M KCl. Preliminary experiments showed that the microsome/ cytosol fractions prepared with either buffer gave the same results. The rat and mouse tissues were homogenized in 2-4 vol. of the chilling buffer in a Teflon/ glass Potter-Elvehjem homogenizer and then centrifuged at 600-700 X g for 10-15 min. Without disrupting the congealed fat, the infranatant liquid was aspirated into a chilled syringe equipped with a 4-inch 17gauge needle. The other adipose tissue samples were chopped into cubes of about 1 cm and homo- genized in 2 vol. of Tris/maleat.e/sucrose buffer by means of a Sorvall Omni- mixer. The homogenates were then filtered through four layers. of cheesecloth, centrifuged at 700 X g for 10 min, and the supematant fraction filtered again through cheesecloth. Liver was handled as the adipose tissue except the homo- genization was carried out with 4 vol. of Tris/maleat.e/sucrose buffer.

All enzyme solutions were then centrifuged at 15 000 X g for 15 min to remove the larger organelles. The mouse microsome/cytosol fraction was de- canted from the pellet, and the others were filtered through Schleicher and Schuell (Keene, New Hampshire) 404 rapid-flow filter paper.

Measurement of glyceride formation The typical assay mixture contained 70 pmol of either phosphate or gly-

cylglycine buffer, pH 7.0, 20 pmol of sodium or potassium fluoride, 6 pmol MgClz , 10 pmol ATP, 0.3 pmol CoA, 2.5 pmol GSH, 0.1-0.2 pmol 1-l 4 C- labeled fatty acid, and 0.8 pmol rat-glycerol-l-P containing about 0.5 pCi ~n-[2-~ H] glycerol-3-P. Acylglycerol synthesis was initiated by the addition of 1.5-2 ml of a microsome/cytosol fraction of the appropriate tissue. The final volume was 2.8-3 ml. Incubation was carried out with shaking at 20°C for most mouse preparations and at 37°C for the rat, chicken, pig, and the rest of the mouse preparations. In earlier experiments, the lower temperature appeared to give a more linear rate of substrate incorporation, but this was subsequently shown not to be true. Studies comparing both temperatures showed no dif- ferences in the distribution of the fatty acids incorporated. Following various times up to 20 min, samples were removed and the lipids extracted by either the method of Dole [ 191 or Bligh and Dyer [20] after the addition of 1 mg of olive oil as carrier.. When the former procedure was used, the triacylglycerols were obtained by column chromatography of the extract on acid-washed Florisil [21] after prior removal of the free fatty acids by washing the heptane layer with alkaline ethanol [22] . Lipids extracted by the Bligh and Dyer proce- dure were separated into classes by thin-layer chromatography on O.&mm Supelcosil 12A (Supelco) plates. The lipids were visualized by a brief exposure to iodine vapors after development with heptane/isopropyl ether/acetic acid (7 : 6 : 1, v/v). The appropriate areas were scraped and eluted by successive extraction with chloroform/methanol (4 : 1, v/v) and diethyl ether. Lipids were counted in 10 ml of toluene containing 0.04 g of Omnifluor (New England Nuclear) by a Packard liquid scintillation spectrometer. Corrections for back- ground, quench, double label, and efficiency were made by means of a com- puter program.

248

hsitional distribution of fatty acids incorpomted into triacylglycerols Two samples were removed from the isolated triacylglycerols; one of these

was subjected to ester analysis [23] and the other retained for radioactivity measurement. The solvent was removed from the remainder of the triacylgly- cerol fraction by a stream of nitrogen. and the lipids hydrolyzed by pancreatic lipase by either the method of Mattson and Beck [24] or by a modification of the semi-micro procedure of Luddy et al. [25] . The lipids were extracted and the monoacylglycerols isolated either by chromatography on Florisil or by thin-layer chromatography. The radioactivity in and the ester value of the isolated monoacylglycerols was measured. The incorporation in the 2-position of the triacylglycerols was calculated from the specific activity (dpm/pmol of ester) of the monoacylglycerols and the triacylglycerols.

Results

Cofactor requirements for triacyigiycerol formation The cofactor requirements for triacylglycerol formation by the mouse and

pig microsome/cytosol preparations was studied by the incorporation of [l-l 4 C] palmitate and sn-[2-3 H] glycerol-3-P into neutral lipids. For both spe- cies, neither labeled substrate was incorporated in the absence of ATP, Mg’*, and CoA (Table I). Although incorporation of palm&ate was nearly eliminated by the omission of sn-glycerol-3-P, in both species the incorporation of sn-gly-

TABLE I

COMPONENTS REQUIRED FOR THE SYNTHESIS OF GLYCERIDES BY THE MICROSOME/ CYTOSOL FRACTION OF MOUSE AND PIG ADIPOSE TISSUE

ulth mouse l dlpow tluue fractions. the complste reaction mixture contelned 35 moI of phosphete buffer @H 7.0). 10 pmd of NxF. 3 pmol of MgCl2. 5 pm01 of ATP. 0.15 wmol of CoA. 1.25 pm01 of GSH. 0.0s pmol of potualum [l- 14C]palmItate, xnd 0.4 umol o! mc-glpcerol-1Q conteining 4 * 10s dpm m-[%3H]glyceroA-3-P in P volume of 0.5 ml. The reaction wu,sterted by the xdditlon of 1 ml of P mlcrorome/cytorol prepentlon. Wlth the pig xdlpoee tlnue fraction. the complete reection mixture contelned double the above xmounte of phoephxte buffer, ATP. MgCl2, end mcJycerol-19 ln a volume of 1.6 ml. Tbla reectlon mixture dao contained 12 lmol of GSH. 20 ~01 of KF. 6 mg of delipideted bovine serum aIbumln. 0.23 pmol of CoA. and 0.28 pmol of potuelum [1-14Clpelmitete. The reectlon wax xterted by the addltlon of 1.6 ml of the mlcroaome/cytorol frection. Incubation wee carried out at 20°C for the mouee prepuetlon end et 37’C for the pig preperetion. After 20 mln, the llpide were extactcd with heptane end weehed with elkaline ethanol. The radloxctlve neutral UDidr In the wuhed heptxne were counted. The complste mouse ayetern Incorporated 18 nmol of m_plycaol-84 end 21 n&01 of pxlmltete and the complete pl# system incorporeted 16 nmol of snOyc.erol-3-P end 7 nmol of pelmitxte.

Omhwion

None ATP I&c12 CoA NaF or KF GSH Glycerol-P Pllmltete Enzyme

Glycerol-P incorporxtion (96) ------

Mouse Plr

100 100 1 0 9 19

16 19 106 88

95 - - -

112 sa 1 -

Palmitate incorporation (%) ._

MOUW Pir _

100 100 2 7 9 29

11 21 97 82 82 -

4 17 - - 1 -

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cerol-3-P showed only a slight dependence on the addition of palmitate. This finding, coupled with the requirement for ATP, Mg”, and CoA, indicated that the level of endogenous fatty acids in the enzyme preparation was sufficient for triacylglycerol synthesis. Subsequent studies, particularly with the mouse, showed the response to addition of fatty acids to be a highly variable feature with different preparations. The omission of NaF or GSH from the reaction mixture did not materially affect triacylglycerol formation from either labeled substrate.

Effect of protein level on triacylglycerol formation With the mouse preparations, the rate of incorporation of both palmitate

and sn-glycerol-3-P into triacylglycerols was directly proportional to the amount of protein added to the incubation mixture. The time course of incor- poration for both palmitate and snglycerol-3-P was linear during the 20-min assay period at all levels of protein used. Similar observations were made with the microsome/cytosol preparation from the pig.

Acylglycerol formation The acylglycerols were separated into four fractions by thin-layer chro-

matography following incubation of the mouse preparation with ~n-[2-~ H] gly- cerol-3-P and one of four ’ 4 C-labeled fatty acids, and the pig preparation with sn- [ 2-3 H] glycerol-3-P and [ 1 -’ 4 C] palmitate. They were the phospholipid frac- tion, which remained at the origin on the chromatoplates and included phos- phatidic acids, the monoacylglycerol, diacylglycerol, and triacylglycerol frac- tions. In each case, whether measured by I 4 C or tritium incorporation, the majority of the radioactivity was found in the triacylglycerol fraction (Fig. 1). With the mouse, the sn-[ 2-j H] glycerol-3-P incorporation was similar for all of the fatty acid cosubstrates tested. The incorporation of the four fatty acids into the triacylglycerols varied widely.

As will be shown in a subsequent publication, the labeled fatty acids do not mix with the bulk of the endogenous free fatty acids in the microsome- cytosol preparation. Although the absence of a fatty acid requirement indicates that a pool of endogenous fatty acids is active in triacylglycerol synthesis, it is too small to have materially affected the specific activity of the fatty acid substrates. When saturated fatty acids were the labeled substrates, the ratio of fatty acid incorporation to sn-glycerol-3-P incorporation indicated that the sn-glycerol-3-P was acylated by endogenous fatty acids. In contrast, when un- saturated fatty acids were the labeled substrates, the fatty acid incorporation was more than three times the incorporation of sn-glycerol-3-P; and therefore, endogenous glycerides must have been esterified. With the pig preparation, the acylglycerols formed from labeled palmitate and sn-glycerol-3-P were both quantitatively and qualitatively similar to that observed with the mouse prepa- rations.

The dihydroxyacetone phosphate pathway and the esterification of sn-gly- cerol-3-P

The esterification of radioactive snglycerol-3-P and the increased incor- poration of labeled fatty acids into phosphatidic acid and neutral lipids in

260

% 16:O 100

MOUSE

;;;p 13 17 IO 6 12 72 954 25 31

18:O PIG 16~0

Fig. 1. DiaMbution of labeled fatty acids (FA) and sn%lycerol-3-P (GP) between triacylglycerolr (TG). tic~lgl~cerob (DG), monoacyklycerols (MG). and phospholipida (PL). The reaction mixturea contained 10 pmol of ATP. 6 timol of MgCl2.0.3 pmol of CoA. 2.5 Nmol of GSH. 20pmol of NaF. and 0.8 )rmol of mc-glycerol-l-P labeled with sn-[ 2-3HI glycerol-3-P. In addition. mouse adipose tiaue prepars,Uon~ ware incubated with 70 Pm01 of dycylglycine buffer (PH 7.0) and 0.1 ~01 of l-14C-labcled fatty acid. The flasks containing the pig preparation contained 200 Nmol phosphate buffer (pH 7.0). 5.6 mg d&pi&ted bovine serum albumin. and 0.18 Mmol potassium [l- 14Cl~almltate. The mouse pmpumtiona were incu- bated at 20°C for 16 min. and the pig preeparatio~~ were incubated at 37’C for 18 min. Lipidr wsre extracted with chloroform/methanol and separated into the glyceride clues by thin-layer chromato- graphy. Spoti were visualized by briefly staining with iodine vapors and &raped into counting viab.

response to added sn-glycerol-3-P have been tacitly assumed to indicate the activity of the acyltransferases of the glycerophosphate pathway [26-281. This assumption need not be valid, however, since alternate routes of esterifica- tion could yield results similar to those expected of the glycerophosphate pathway. In the presence of glycerolphosphate dehydrogenase, it would be possible for some of the sn-glycerol-34 to be converted to dihydroxyacetone phosphate and then incorporated into triacylglycerols via the dihydroxy- acetone phosphate pathway. Such a sequence could account for the low incor- poration of 8n-[2-’ H] glycerol-3-P relative to [l-l 4 C] oleate and [I-’ 4 C] lino- leate by the mouse enzyme system since the tritium would be lost during the oxidation of sn-glycerol-3-P.

To test this supposition, microsome/cyt.osol preparations from both mouse and pig adipose tissue were incubated with a mixture of 8n-[2-3 H] gly- cerol-3-P and sn-[U-l 4 Clglycerol-3-P. The pig enzyme preparation was in- cubated without an exogenous fatty acid cosubstrate, whereas microsome/ cytosol fraction from the mouSe wa8 incubated with or without non-labeled palmitate or oleate. Since in all incubations the incorporation of each labeled sn-glycerol-3-P into total lipid wa8 the same (Table II), it is presumed that in these systems sn-glycerol-3-P does not serve as a precursor for the dihydroxy- acetone phosphate pathway.

After this work was completed, two reports appeared indicating that a tritium isotope effect of from 2- [29] to &fold [30] occurred during the oxidation of 8?~-[%’ H] glycerol-34 to dihydroxyacetone phosphate. The mag- nitude of this isotope effect makes it difficult to asses8 the relative contribu-

261

TABLE II

INCORPORATION OF m-[2-3H] GLYCEROG3-P AND m-[U-l 4C] GLYCEROL-3-P

The glycerol phosphate in each fla8k wu labeled with both m-[2-3Hl~~cerol-3-P and m-W-‘4C13~- o~ol-3-P. The ume incubation ~ondittona u described in Table I were used except that for the moue preparation. glycylglyctne buffer replaced the phosphate and the !lask wu incubated either with or without 0.06 nmol of palmitate or oleate. With the pig preparation. 150 pmol of Mcine/KOH buffer (PH 7.3) replaced the phosphate, 13 pmol of dithiothnitol replaced the GSH. and palmitate was not added.

Preparation

MOuM Mours MOW

Pir

Fatty Acid

-

None palmitate Oleate None

Glycerol-P incorporation (nmol)

Tritium 1%

12.3 12.6 13.2 14.9 14.0 13.6

2.1 2.5

tions of the glycerol phosphate and dihydroxyacetone phosphate pathways using this experimental approach. If the ’ 4 C-labeled dihydroxyacetone phos- phate produced by the oxidation of doubly-labeled srtglycerol-3-P was metabo- lized to non-lipid components (chloroform-insoluble), the ratio of tritium to 1 4 C would be greater in the lipid products than in the substrate initially. Indeed, this has been observed by several investigators working with intact cells or mitochondria [ 30-321. If the only metabolic fate of the ’ 4 C-labeled dihy- droxyacetone phosphate was acylation, then the, tritium to ’ 4 C ratio of the lipids should decrease. The ratio would remain constant only if the loss of l 4 C to dihydroxyacetone phosphate and non-lipid metabolites balanced the net loss of tritium in the oxidation of sn-[2-’ H] glycerol-3-P or if, as seems more plausi- ble, the oxidation reaction was trivial.

Therefore, in the studies described, the dependence of acylglycerol syn- thesis on sn-glycerol-3-P would imply direct acylation of snglycerol-3-P by the acyltransferases of the glycerophosphate pathway.

Effect of level of sn-glycerol-3-P With both the mouse and the pig preparations, the incorporation of sndy-

cerol-3-P and palm&ate were similarly affected by a variation in the level of me-glycerol-1-P. Assuming that the inactive isomer did not affect the results, the apparent Michaelis constants, calculated by a modification of the computer program of Cleland [33], were 0.9 f 0.4 * 10e4 M and 1.0 f 0.3 - 10e4 M for the mouse system as measured by the incorporation of sn-[ 2-j H] glycerol&P and [l-l4 C] palmitate, respectively. For the p@, these values were 1.5 f 0.4 - 10e4 M based on ~n-[2-~ H] glycerol-3-P incorporation and 2.2 f 0.4 - 10e4 M based on palmitate incorporation. Similar values were obtained with each sub- strate and for both species, and these are in agreement with the value of 2 - 10V4 M for a partially purified glycerolphosphate acyltransferase from rat liver [34] and slightly lower than the apparent Michaelis constants of 5 - 10m4 M [34] and 7 - 10m4 M [35] reported for rat liver microsomes.

Fbsitional distribution of fatty acids in the triacylglycerols formed Studies on the fatty acid distribution in triacylglycerols isolated from

252

5-PALMITATE

4-

win) (mln)

Fig. 2. Podtlonal distribution of fatty acidr in triacylalycerolr. The incubation mtrtum contained 70 /mml of pho&ate buffer (PH 7.0). 20 ~mol of NaF. 6 pal of M~Cl2.10 pmol of ATP, 0.3 pm01 of CoA. 2.6 ~01 of GSH. 0.8 jimal of mcJyce?ol-14’ labeled with m-[23H]3ycerol-a+ and 0.2 fimol of the potaadum ult of the indicated fatty acid labeled with 14C in the cuboxyl group. The dpm 14C added we: ualmitati. 2.2 * lo5 ; oleate. 2.6 * 10s ; starate. 2.9 * lo5 ; and linoleate. 1.3 * 10s. The reaction w” lfuted by the addition of 2 ml of a microaome/c~torol wexwation of mouaa adipose tbue contdnh# about 3 mp protein. Flaal volume wu 3.0 ml, and O&III ail~uotr w- ramowd at 6-&n intends after incubation at 2O’C. Llukb weae extracted with hsptuw and the trlacyl&ycezol~ isolated by column chromatography and the monoacylglycerol iaolatad dmilarly following hydrolydn by P-@-a* w. 0. the incorporation in the average of tbe two outer poaitloas calculated from the total trhcylglycerolr minus that in the 2-podtion; 0, the incorporation in the 2qodtlon. Data am the avomgc of three aperlmenw.

mice have Bhown that the majority of the linoleate was esterified at the 2-posi- tion, moat of the palm&ate and &ear-ate was found at the 1,8positiono, and a tendency for more oleate to be eeterified at the 2-position than the 1,3posi- tiona [36]. Pancreatic lipase treatment of the labeled triacylglycerols produced by incubation with a mouse microsome/cytosol fraction, followed by isolation of the monoacylglycerok, showed that the distribution of palmitate, stearate, oleate, and linoleate (Fig. 2) resembled that expected of the intact mouse triacylglycerols. In these experiments, the total incorporation of palmitate, oleate, and linoleate wa8 eimik. However, the level of stearate incorporation was only about one-fifth that of the other fatty acide. Evidence that this difference lies at the level of the acyl-CoA synthetaae rather than the acyltrans- fera8es is provided by two observations. First, etearyl-CoA is incorporated into triacylglycerols by these preparation8 a8 efficiently a8 palmitoyl-CoA (Fig. 3). Second, studies on the epecificity of long-chain fatty acyl-CoA eynthetase8 have shown that 8tearate is not a8 active a 8ub&nte a8 the 0the.r fatty acid8 [37].

The distribution of palmitate and oleate in the tziacylglycerob formed by microsome/cytosol preparations from other epecies was inve&-i@ed. The pal- mitate was ester&d principally at the 1,3po8ition when preparations of rat adipose tissue, chicken adipose tiesue, and moue adipose tissue, a8 well aa pig liver, were incubated in the preeence of me-glycerol-1-P (Table III). Oleate was distributed more uniformly. In contrast, the preparation8 of pig adipose tissues

263

Acyl CoA (n 1~701)

Fig. 3. Lncoxwretton of pelmttoyl-CoA (0) end eteewlCoA (0) into neutral liuids by mkroeomelcytoeol prepenttonr of edipoa tiesue. The incubetion mixtum conteined 70 pmol of glycylglycine buffer @H 7.0). 6 prnd of MgCl2. 0.8 flmol of mc+lycerol-1-P. end the indicated emountn of either pebnRoyl-CoA

lebeled wtth 3.3 * lo5 dpm [l- 14CIpebnttoyl~oA or eteuyl-CoA lebeled with 4.4 . lo5 dpm [l-14C1- rLeuyl-CoA in l fineI volume of 1 ml. The reectton xv- stuted by the eddttion of 2 ml of l mkroaome/ cytorol prepuetton of moue edtpoee We. After ‘6 min at 2O’C. eliquota wea8 removed end the neutrel lipida were ertrected with hexene end counted ea deacrtbed in Experlmentel Procedure.

ester&d palmitate principally at the 2.position of the triacylglycerols and oleate at one or both of the outer positions.

As observed previously (Table I) with microsome/cytosol preparations of pig and mouse adipose tissue, the presence of glycerol phosphate markedly stimulated the incorporation of palmitate. The data of Table III show that these results extend to palmitate for adipose tissue preparations from rat and

TABLE III

THE INCORPORATION AND DISTRIBUTION OF PALMITATE AND OLEATE IN TRIACYLGLY- CEROLS SYNTHESIZED BY MICROSOME-CYTOSOL PREPARATIONS FROM SEVERAL ANIMAL SPECIES IN RESPONSE TO an-GLYCEROL 3-P

The complete reectlon mixture contelned 19 ~01 of Trtcine buffex (pH 7.3). 19 pmol of poteadum phoephete. 4 wd of KF. 36 jimol of NQOL. 3 gmol of ATP. 8 wmol of M&12.0.1 jimal of CoA. 0.2 ~01 of dithlothreltol. SO nmol of [9.10-3H]palmttic l cid. end 60 am01 of [1-14C]oleic l cid in e volume of 0.8 ml. Wheze indiceted, 3 pmol of mcilycerol-1-P wm else included. The tncubetion opu started by eddttton of 1.5 ml of the epproprtete mk.roeome/cytoeol preperetion. After 20 mtn et 37’C. the ltpida wem extrected with hexene end the trlecy~ycaols tsoleted by chrometogrephy on Florldl. The pod- Uond dlatrlbutton of pelmitete end oleete wn detexmhad u dsrcribed in ExperimmW Procedure.

Tirue source mc-Glycerol-l-P Pabnitete (nmol inc0rD0reted) Oleate (Ml01 incorpo-

mted)

TOW 2-Podtion Totel 2.Podtton

plr edimne plr adip- PLIlker PIa liver Ret l dtvosc

Rat edtmee Mouec l dlgoee Moue edlpoet chicken edlpoee chicken ailDow

+ 18.8 3.6

+ 16.2 - 3.0 + 10.4

I.4 + 33.7 - 16 + 6.1

1.8

10.8 26.4 21 0.1 2.3 0.0 1.8 10.6 3.6 0.1 1.7 0.1 1.4 18.0 5.6 0.0 2.3 0.0 3.4 41.3 16.8 0.0 2.3 0.1 0.7 16.7 4.3 0.0 6.3 0.1

254

chicken as well as pig liver. Oleate incorporation was also greatly increased by glycerol phosphate. However, in the absence of glycerol phosphate, more than 96% of both palmitate and oleate were esterified at the primary hydroxyl groups of the triacylglycerols (Table III).

Discussion

The requirements for the synthesis of triacylglycerols by microsome/ cytosol preparations from the mouse and pig parallel the observations made by other investigators with liver microsomal preparations [ 381 and defatted dipose tissue homogenates [39]. It is probable that endogenous fatty acids in the enzyme preparation served as the acyl donors for triacylglycerol synthesis after activation to the acyECoA esters since the incorporation of labeled sn-gly- cerol-3-P was observed in the absence of added fatty acids, but only if ATP, CoA, and Mg2+ were present. These findings further indicate that the tissue preparations from adipose tissue, as in the case of liver [ 381 and brain [ 401, do not contain a pool of endogenous fatty acyl-CoA esters.

Furthermore, the results obtained under our assay conditions indicate that the major pathway for triacylglycerol biosynthesis is the glycerophosphate pathway. The monoacylglycerol pathway was of minor quantitative signifi- cance as inferred by the dependence of sn-glycerol-3-P for palmitate incorpora- tion. In these preparations, the dihydroxyacetone phosphate pathway did not contribute significantly to triacylglyoerol biosynthesis either since the ratio of ’ 4 C to tritium in the total lipid products was the same as in the substrate labeled with both sn-[ U-’ 4 C ] glycerol-3-P and sn-[2-3 H] glycerol-3-P.

It is well established that the fatty acyl substituents of glycerolipids do not occur randomly. This is true for the triacylglycerols as well as the phospho- lipids. Studies with intact animals [41] , liver slices [27] , and epididymal fat pads [42] all indicate that this specificity is imparted at the time the lipids are synthesized, presumably via the glycerophosphate pathway. Most of the workers who have used cell-free preparations to probe the origin of this assy- metry of fatty acid distribution in animal glycerides have used liver microsomes as the source of the transacylase enzymes and have isolated phosphatidic acid as the end product. The success of these experiments has been mixed; several groups of investigators [27,43-451 were unable to show preference for either the l-position or 2-position of glycerol phosphate using acyl-CoA esters as substrates, whereas others [46,47], using free fatty acids as substrates, did obtain the expected specificity.

When the incorporation of labeled palmitate and oleate into triacylgly- cerols was studied using microsome/cytosol preparations from rat and chicken adipose tissue and pig liver, a response to sn-glycerol-3-P, similar to that shown for mouse and pig adipose tissue preparations, was again observed. That is, sn-glycerol-3-P was required for the incorporation of fatty acids in the 2-posi- tion. Thus, although a low rate of incorporation of fatty acids was observed in the absence of sn-glycerol-3-P the acids were esterified to one or both of the primary hydroxyl groups of the glycerol moiety. Moreover, when the incor- poration of fatty acids into triacylglycerols in the presence of sn-glycerol-3-P was corrected for the synthesis in its absence, the distribution of palmitate and

255

TABLE IV

DIETRIBUTION OF PALYITATE AND OLEATE IN THE 2-POSITION OF TRXACYLGLYCEROLS

FROM VARIOUS SPECIES CORRECTED FOR SYNTHESIS IN THE ABSENCE OF GLYCEROL PHOSPHATE

The data ehown am thoee derived from the difference in incorpoation of p&nit&e and oleate in the presence and ebmce of glycerol phosphate ehown in Table IV.

Tlseue P&&ate (96 in 2-position) Oleate ($6 in flpodtion) Reference for expected

valuu

Expected Obeewed Expected Obeerved

p&t adipore 82 82 6 9 11 Pig Mer 22 14 40 40 11 Ret edimee 14 8 35 36 52

Mouse edipoee 21 10 38 40 36 Chlcken adipose 23 21 39 a6 52

oleate was strikingly similar to that reported for the fat extracted from the respective tissue8 (Table IV).

These data imply that, with all tissues examined, the acyl donor specific- ity of the microsomal acyltransferases of the glycerophosphate pathway, mea- cured by the sn-glycerol-3-Pdependent incorporation of palmitate and oleate, can account for the structure of the respective tissues’ triacylglycerols.

Thus far, none of the transacylases of the glycerophosphate pathway have been highly purified, and only recently have the two transacylases involved in phosphatidate synthesis been resolved [4S] . Even though partially purified sn-glycerol-3-P acyltransferases from rat liver mitochondria [40] and micro- sornes show a preference for palmitoyl-CoA over oleoyl-CoA or linoleoyl-CoA, the l-acyl-sn-glycerol-3-P acyltransferase from these source8 showed no prefer- ence for acylation at the 2-position. Thus, these result8 are not in accord with the findings reported herein.

Although study of the glyceride-synthesizing systems is simplified by the use of acyECoA sub8trates, these have usually been added to the incubation mixtures at levels considerably in excess of the critical micelle concentration of 3-4 PM [60]. Thus, the u8e of an acyl-CoA may introduce artifacts because of detergent effects or by kinetic effects of an intermediate at an abnormally high level [ 511. Moreover, these preparations contain active ‘acyl-CoA hydrolyases, and the stimulation often observed upon the addition of ATP may indicate reactivation of the fatty acid substrate. Finally, the use of acyl-CoA esters ignores the possibility that the acyl-CoA synthetase may be part of a multi- enzyme complex involved in glyceride synthesis. In this connection, Abou-Issa and Cleland [38] observed that the rate of incorporation of palmitate in the presence of ATP and CoA was faster than the rate of incorporation of palmi- toyl-CoA and suggested that free fatty acid8 rather than acyl-CoA substrate8 are the normal ‘substrates for glyceride synthesis by microsomal enzyme systems.

Acknowledgment8

This is a contribution from the Department of Biochemistry, School of Agriculture and Life Science8 and School of Physical and Mathematical

256

Sciences, North Carolina State University, Raleigh, N.C. Paper No. 4432 of the Journal Series of the North Carolina State Agricultural Experiment Station, Raleigh, N.C. 27607 U.S.A. This work was supported in part by grant No. AM-02483 from the National Institutes of Health.

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