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Biochimica et Biophysica Acta, 1126 0992) 327-336 327 © t992 ElsevierScience Pubtishers B.V. All righls reserved0005-2760/92/$05.00 BBALIP 53942 Diacylglycerol metabolism in neonatal rat liver: characterization of cytosolic diacylglycerol lipase activity and its activation by monoalkylglycerols Tian Xia end Rosalind A. Coleman Departments of Nutrition and ~ediatdcs. Schools of l~blic Heahh and Medicine. U~.h'ersityof North Carolina.Chapel Hill. Chapel Hill, NC (U-¢.4) (Received 16 October 1991) Key words: Diacylglyccrok Olyccmtipid,Ether lipid~Monoacylglyccmk Lipid topogcnesis; Lipase Diacylglycerol tipasc (glycerol ester hydrolase, EC 3,1.1.3) activities were investigated in subcellular fractions from neonatal and adult rat liver in order to determine whether one or more different lipases might provide the substrate for the developmentally expressed, activity nmnoacylglyccrol acyltransfer~se. The assay for diacylglycerol lipase examined the hydrolysis of sn.l-stea- royl,2-[14C]olcoylglycerol to labeled monoaeylglycerol and fatty acid. Highest specific activities were found in lysosomes (pH 4,8) and ¢ytesol and microsomcs (pH 8), The specific activity from plasma membrane from adult liver was 5,8-fo[~ higher than the corresponding activity in the neonate, in other fractions, however, no developmental differences were observed in activity or distribution, h~ both lysosomes and eytosol, 75 to 90% of the labeled product was monoacylglycerol, suggesting that these fractions contained relatively little monoacylglycerol lipase acti,Aty. In contrast, 80% of the labeled product from microsomes was fatty acid, suggest;ng the presence of monoacylglyccrol lipase in this fraction. Analysis of the reaction products strongly suggested th?~. the lysosomal and cytosolic diacy!glycerol lipase activities hydrolyzed the acyl-gvoup at the sn-| position. The effects of serum and NaCI on diacylglycerol lipase from each of the subeellular fractions dh'fcr,:d hum those effects routinely observed on lipoprotein iipase and hepatic lipase, suggesting that the hepatic diacylgly¢crol lipase activities were not second functions of these triacylglycer~l lipascs, Cytomlic diacylglycerol lipasc activity from neonatal liver and adult liver was characterized, The apparent K., for t-stearoyl,2.oleoylglycerol was t 15 #M. I here was no preference for a diacylglycerol with arachidonate in the sn-2 position. Bovine serum a~bumin stimulated the activity, whereas dithiothreito!, N eth;,Im~lei;nide, and ATP inhibited the activity. Both sn-l(3)- and 2-monooieylglycerol ethers stimulated cytosolic diacylglycerol lipase activity 2-3-fold. The correspond- ing amide analogs stimulated 28 to 85%. monooleoylglycerol itself had little effect, and I-alkyl- or l-acyl-lysophosphatidylcholine inhibited the activity. These data provide the first characterization of hepatic su~ellular lipase activities from neonatal and adult rat liver and suggest that independent diacylglycerol and monoaeylglyeerol lipase activities are present in microsomal membranes and that the micro:.omal and cytosolic diacylglyccrol lipase activities may describe an ambipathic e.nzyme. The data also suggest possible cellular rcg~datlon by monoalkylglyccrois. Introduction Diacylglycerol has two distinct functions in cells. It is a central intermediat~ in the synthesi,~ of the major glycerolipids (triacylglyccrol, phosphatidylcholine, and phosphatid,~,!ethano!amine), and i, aim functions as a second messenger. As a glyceroli[ id intermediate, dia- cylglycerol arises via the sequent al acylation of glyc. erol 3*phosphate and the action of phosphatidate phos- Correspondence:R.A. Coleman,Department of Nutrition. 2203Me- Gavran.Or©enberg Hall, School of Public:HealtL. CB# 1400, Uni- v©rsity of ~orth C,.~_-!ina dt Chapel Hill, Chapel Hill, NC 27599-7400, USA, phatase [1]. As a second messenger, diacylglycerol is formed when plasma membrane phospbatidylinositol or phosphatidylcholine is hydrolyzeh!, bya phospho- lipasc C that has bccn activated b~ an cxtracellular signal [2]. The synthesis of diacFIglyccrol is more com- plex in neonatal liver than in adult liver and other tissues, because an additional synthetic pathway is pre- sent from monoacylglycerol [3]. The microsomal activ- ity, monoacylglycerol acyltransfcrase (EC 2,3A,22), that defines this alternate route is 700-fokl higher in the neonate than in the adult and may cot~,titute the major route fur the de novo synthesis of diat.yigiycerol in the neonate, The pathway may function to ensur: the synthesis of diacylglycerol for membrane phosphalipid biogenesis azad to synthesize triacylglycerol for VLDL

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Page 1: Diacylglycerol metabolism in neonatal rat liver: characterization of cytosolic diacylglycerol lipase activity and its activation by monoalkylglycerols

Biochimica et Biophysica Acta, 1126 0992) 327-336 327 © t992 Elsevier Science Pubtishers B.V. All righls reserved 0005-2760/92/$05.00

BBALIP 53942

Diacylglycerol metabolism in neonatal rat liver: characterization of cytosolic diacylglycerol lipase activity and its activation

by monoalkylglycerols

T i a n X ia e n d R o s a l i n d A. C o l e m a n

Departments of Nutrition and ~ediatdcs. Schools of l~blic Heahh and Medicine. U~.h'ersity of North Carolina.Chapel Hill. Chapel Hill, NC (U-¢.4)

(Received 16 October 1991)

Key words: Diacylglyccrok Olyccmtipid, Ether lipid~ Monoacylglyccmk Lipid topogcnesis; Lipase

Diacylglycerol tipasc (glycerol ester hydrolase, EC 3,1.1.3) activities were investigated in subcellular fractions from neonatal and adult rat liver in order to determine whether one or more different lipases might provide the substrate for the developmentally expressed, activity nmnoacylglyccrol acyltransfer~se. The assay for diacylglycerol lipase examined the hydrolysis of sn.l-stea- royl,2-[14C]olcoylglycerol to labeled monoaeylglycerol and fatty acid. Highest specific activities were found in lysosomes (pH 4,8) and ¢ytesol and microsomcs (pH 8), The specific activity from plasma membrane from adult liver was 5,8-fo[~ higher than the corresponding activity in the neonate, in other fractions, however, no developmental differences were observed in activity or distribution, h~ both lysosomes and eytosol, 75 to 90% of the labeled product was monoacylglycerol, suggesting that these fractions contained relatively little monoacylglycerol lipase acti,Aty. In contrast, 80% of the labeled product from microsomes was fatty acid, suggest;ng the presence of monoacylglyccrol lipase in this fraction. Analysis of the reaction products strongly suggested th?~. the lysosomal and cytosolic diacy!glycerol lipase activities hydrolyzed the acyl-gvoup at the sn-| position. The effects of serum and NaCI on diacylglycerol lipase from each of the subeellular fractions dh'fcr,:d hum those effects routinely observed on lipoprotein iipase and hepatic lipase, suggesting that the hepatic diacylgly¢crol lipase activities were not second functions of these triacylglycer~l lipascs, Cytomlic diacylglycerol lipasc activity from neonatal liver and adult liver was characterized, The apparent K., for t-stearoyl,2.oleoylglycerol was t 15 #M. I here was no preference for a diacylglycerol with arachidonate in the sn-2 position. Bovine serum a~bumin stimulated the activity, whereas dithiothreito!, N eth;,Im~lei;nide, and ATP inhibited the activity. Both sn-l(3)- and 2-monooieylglycerol ethers stimulated cytosolic diacylglycerol lipase activity 2-3-fold. The correspond- ing amide analogs stimulated 28 to 85%. monooleoylglycerol itself had little effect, and I-alkyl- or l-acyl-lysophosphatidylcholine inhibited the activity. These data provide the first characterization of hepatic su~ellular lipase activities from neonatal and adult rat liver and suggest that independent diacylglycerol and monoaeylglyeerol lipase activities are present in microsomal membranes and that the micro:.omal and cytosolic diacylglyccrol lipase activities may describe an ambipathic e.nzyme. The data also suggest possible cellular rcg~datlon by monoalkylglyccrois.

Introduction

Diacylglycerol has two distinct functions in cells. It is a central intermediat~ in the synthesi,~ of the major glycerolipids (triacylglyccrol, phosphatidylcholine, and phosphatid,~,!ethano!amine), and i, a im functions as a second messenger. As a glyceroli[ id intermediate, dia- cylglycerol arises via the sequent al acylation of glyc. erol 3*phosphate and the action of phosphatidate phos-

Correspondence: R.A. Coleman, Department of Nutrition. 2203 Me- Gavran.Or©enberg Hall, School of Public: HealtL. CB# 1400, Uni- v©rsity of ~orth C,.~_-!ina dt Chapel Hill, Chapel Hill, NC 27599-7400, USA,

phatase [1]. As a second messenger, diacylglycerol is formed when plasma membrane phospbatidylinositol or phosphatidylcholine is hydrolyzeh!, b y a phospho- lipasc C that has bccn activated b~ an cxtracellular signal [2]. The synthesis of diacFIglyccrol is more com- plex in neonatal liver than in adult liver and other tissues, because an additional synthetic pathway is pre- sent from monoacylglycerol [3]. The microsomal activ- ity, monoacylglycerol acyltransfcrase (EC 2,3A,22), that defines this alternate route is 700-fokl higher in the neonate than in the adult and may cot~,titute the major route fur the de novo synthesis of diat.yigiycerol in the neonate, The pathway may function to ensur: the synthesis of diacylglycerol for membrane phosphalipid biogenesis azad to synthesize triacylglycerol for VLDL

Page 2: Diacylglycerol metabolism in neonatal rat liver: characterization of cytosolic diacylglycerol lipase activity and its activation by monoalkylglycerols

328

production. Additionally, monoacylglycerol acyltrans- ferase together with diacylglycerol lipase could provide the type of substrate cycle that is frequently associated with metabolic control points [4].

The source of the monoacylglycerol substrate for neona!al fi~oiioac~'!glycerol acyltransferase is unclear. Monoacylglycerol is unlikely to be derived directly from lipoprotein tr~acylglyceroi because both hepatic lipase [5] and a plasma lipase associated with lipoprctein particles [6] are extremely active. Instead, the mono- acylglycerol substrate is more likely to originate from the hydrolysis of intraccllular triacylglycerol. Three in- dependent routes are possible. One involves the action of lysosomal lipases which hydrolyze the triacylglycerol that has entered cells via the receptor.mediated endo- cytosis of lipoproteins. The second route involve~ the partial hydrolysis of triacylglycerol that has been ftored in the numerous cytosolic droplets of the neonatal rat liver. Iv each case, the monoacylgly.,:erol that. is formed might function as a substrate for tl,~ ~o,oacylglyceroi acyltransferase activity. Alternatively, monoacylglycerol could arise from the hydrolysis of diacylglycerol that has been released during phospholipid turnover after hydrolysis by activated phospholipase C.

In order to gain ,a better understanding of the diacylglycerol lipases (glycerol ester hydrolase, EC 3.1.1.3) that might be functioning in neonatal liver, we investigated the subcellular distribution of diacyi- glycerol lipases and determined whether any hepatic diacylglycerol lipase e, ctivities were expressed at high levels in the neonate ir~ a pattern similar to that which has been observed for ~he monoacylglycerol acyltrans- ferase activity. We also investigated whether the pres- ence of a moaoacylglycc, rol lipase activity would limit the accumulation of the monoacylglyceroi substrate for subsequent reacylatior by the monoacyiglyceroi acyl- transferase. This stucLy represents the first charac- terization of diao/Igtycero[ lipase activity from neonatal rat liver and compares the neonatal activit~ with that in the adult.

Experimental procedures

Materials Glycerol [9,10-3H]trioleate, [9,10-3H]pelmitate, and

[5,6,8,9,11,12,14,15- 3H]arat, hidonate were from New England Nuclear. [1-~4C]Oleate was from Amershar,1. sn-l-Stearoyl 2.oieoylglycerol, sn-l-st~aroyl 2-arachi- donoylglycerol, sn-2- and l(3).monooleoylglycerols, sn- 2- and l(3)-monooleylglycerol ethers, sn-l,2-dioleoyl- glycerol, 1-stearoyl, 2.1ysophosphatidylcholine and l-al- kyl,2-1y~phosphatidylcholine (LPAF) (from beef heart) were from Serdary Research Laboratories. Heparin sodium sail (from porcine inteftinal mucosa), oleic acid, Coenz3~ne A, ATP, phospholipase C (from Bacil. lus cereus), r¢~:tuced glutathione, bovine serum albumin

(essentially fatty acid free), and p-nitrophenol standard solution were from Sigma. Sodium diO:hylbarbiturate was from Fisher Scientific. Sodium sulfite, sodium thio. sulfate, and l-amino-2-naphthol-4-sulfonie acid were from Mai[inckrodt. Silica gel G plates were from Anal- tech. [3H]Palmitoyl-CoA was synthesized enzymatically [7]. The sn-i(3)- and ~n-2-mu,Lt~l~ylglycetol amides were synthesized as previously described [8]. L-~'toscint was from ICH Biomedicals.

Methods Preparation of subcellular fractions. SubceUular frac-

tions were prepared from livers of 300-350 g adult female Zivic.MiUer-Sprague Dawley rats fed Purina rat chow ad lib or from I 1-day-old suckling rats. The rats were on a 12 h light cycle and preparations were begun between 8:00-9:00 a.m. Previously described methods were modified to combine percoll gradient and differ- ential centrifugation [9-11]. Briefly, after one adtdt or 9-12 ll-day-old suckling rats were decapitated, the livers were removed, immediately flushed with ice-cold 0.9% NaCi, weighed, minced and homogenized in cold STM buffer (0.25 M sucrose, 5 mM Tris-HCI ~pH 7.4) and 1 mM MgC! z) with ten up and down strokes using a D~unce homogenizer (B-type) to yield a 10% ho- mogenate (g/v). The homogenate was filtered through four layers of moistened gauze and then centrifuged at 280 ;< g for 5 rain. The supernatent was saved and the pellet was rehomogenized with three strokes in STM buff~.~ (usip~; about one-half of the initial volume) and recer.~trifuged at 280 × g for 5 rain. The combined supernatants were centrifuged at 1500 ×g for 10 rain and the resultint~ supernatant was removed and saved ir~ an ice bath. The pellet w~s rehomogenized with thr~,e strokes in STE-I buffer (0.25 M sucrose, 10 mM Tris-i-tCI (pH 7.4) and 2 mM EGTA), 1-2 ml per g of liver. Each 1 ml of homogenate was then mixed with 3.5 ml of percoll, 0.5 m[ of STE-2 buffer (2 M sucrose, 80 mM Tris-HC! (pH "/,4) and 8 mM EGTA) and 16 ml of STE.1 buffer, and centrifuged at 10000 × 8 for 15 mi~. Bands were observed at the surface and the bottom of the tube. To remove the percoll, the top band (plasma membrane) was washed in 5 vol. of NaCI buffer (0.15 M NaCi, 10 mM Tris.HCI (pH 7.4) and 1 mM EGTA) and the bottom band (mi'~chondria) was washed in 5 vol. of STE-I buffer by centrifuging at I0000 ×g for 2 rain. Each pellet was res~,~st~ended in a small amount of the appropriat,; buffer. To prepare lysosema[, microsc, mal and cytosolic fractions, the 1500 ×g supernatant was centrifuged at 10000×g for 15 rain. The pellet (lysosomes) was resuspended in STM buffer and the supernatant was fur,.her c~ntrifuged at 23000 ×g for 60 rain. The resulting pello! was re!~- mogenized in STM buffer and centrifuged at 100000 x g for I it. This microsomal pellet was resuspended in STM buffer.

Page 3: Diacylglycerol metabolism in neonatal rat liver: characterization of cytosolic diacylglycerol lipase activity and its activation by monoalkylglycerols

329

The following marker enzymes were assayed in each fraction. 5'-nucleotidase and phosphodiesterase I [121 for plasma membrane, diacyiglycerol acyltransferase r q , ~ l t,,~J for endoplasmic reticul,m, and acid phosphatase [14] for lysosomes. The protein content was determined by the method of Lowry et al. I15] using crystalline

. | L . ~ ' . _ . * . IL . . _ _ t ' . . . . . k,,,,:-,,,,,,,,,,,..~?.r::m a,uUmht a~ t,,c .¢z©actzuc si'~lldard. Preparation of l.stearoyl,2-[ ~4 C]oleoyl.sn-glyceroL l-

Stearoyl,2-[t4C]oleoyl-phosphatidylchohne was en~- matically synthesized by rat liver microsomes from l- stearoyl,2-1ysophosphatidylcholine and [1- ~4C]oieic acid [161. The radiochemical purity of the [14C]-phospha- tidylcholine product was greater than 99% and no detectable [t4C]oleic acid was observed by thin-layer chromatography on silica gel G plates, developed in chloroform/methanol/water (65 : 25" 4, v/v), The [14C]phosphatidylcholine was stable for at least 4 months at -20°C In order to minimize acyl migration to 1,3-diacytglycerol, the 1-stearoyl,2-[t4C]oleoyl-sn - glycerol, was freshly prepared just before use by modi- fying a previously described method [17], Briefly, ["C]phosphatidylcholine in 40% methanol/CHCI s was dried under a stream of nitrogen and then suspended by sonication in 1 ml of 30 rnM sodium barbital buffer (pH 7.4) containing 0.12 M NaCi and 1 mM ZaSO4. After adding phospholipase C (approx. 0.4 U/nmol pi~osphatidylcholine), the reactioa mixture was incu- bated at YPC for 10 rain. I ml of ethyl ethe- was added to stop the reaction and extract the diacyigiycerol. The lower phase was extracted twice with 1 mi of ethyl ether. The combined ether extracts were evaporated under N 2 and the white residue was extracted twice with 1 ml of heptane. Uniabeied i-stearoyl,2- oleoylglycerol was added to achieve a specific activity of 2500-3000 cpm/nmol, and the diacylglyeerol wes dissolved in acetune :o yield the appropriate concen- tration. The radiochemical nurity of [t4C]diacylglycerol was greater than 98%. Thin-layer chromatography on silica gel G plates developed with heptane/isopropyl- ether/acetic acid (60: 40: 4, v/v) showed that less than 5% of the diacyigiycerol was the 1,3-stereoisomer. l-

r! 3 Stearo3,,2-[ tt]arachidonoylglycerol was synthesized from l-stearoyl,2-1ysophosphatidylcholine and [5,6,8,9,1 s 1,12,14,15-'H]araehidonate by a similar method. The radiochemical purity of l-stearoyl,2[3H] arachidonoylphosphatidylcholine was 99.4%. No free 1 " 3 1 L I 1 , . ~ , , ~ L . % . t - - _ : .~ _ ~. , L - , . . . . ,,,,,v,,,~, ,.,,L, was detected.

Diacylglycerol lipase assay. Diacylglycerol iipase ac- tivity was determined by measuring the formation of labeled monoacylglyccrol and fatty acid from l-stea-

14 royl,2-[ C]oleoyigIycerol. The standard reaction mix- ture contained 0.25 M Tris-HCi (pH 8.0), I m g / m l

I bcMnc ~rum a,bumiit, ~ mM MgClz, I0 to 50 ~L~ 14 protein, and 120/.tM l-stearoyl,2-[ C]oleoylglycerol in

a final volume of 200/ti. The reaction was started by ac~d[ng the diacylglvcemt a' , • . __...;sso,,~,eu in 5 or iO p.I ,ace-

tene. After 15 rain at room temperature, the reaction was stopped by adding 1.5 ml isopropanol/heptane/ water (80: 20: 2; v/v/. Then 1.0 mi of heptanc and 0.5 mI of water were added to separate the phases. A 200 /zl aliquot of the upper phase was counted and the remainder of the upper phase was analyzed by thin layer chromatography developed in heptane/isopropyl ether/acetic acid (60; 40: 4, v/v), Blanks contained all components of the reaction mixture except protein. During a 15 rain assay, the percent of labeled sn-l,3- diacylglyceroI increased from 1% to 6% at pH 4.8 and to 13-16% at pH 8. To determine the relative amounts of sn-l(3)- and sn-2-monoacylglycerol products, the products were analyzed by thin-layer chromatography on 3% borate-impregnated silica gel G plates devel- oped in CHCI3 / acetone/ methanol / acetic acid (90: 5: 2: 0.5, v/v).

Results and Discussion

Subcellular localization of diacylglycerol lipase activities In order to localize the diacylg[ycerol lipase(s) of rat

liver and to determine whether liver contains one or more diacylglycerol lipases that are distinct from the plasma membrane hepatic lipase, we obtained subeel- lular fractions from both adult and suckling rat livers by percoli gradient and differential centrifugation (Ta- ble I). Hepatic subcellular fractions were purified to a similar extent in additional preparations from both adr,,~t and [1-day-old rats. Microsomes were cleanly separated from plasma membrane to avoid hepatic lipase activity, which can hydrolyze diacylglycerol, In liw, r from l l-day-old suckling rats, 5'-nucleotidase ac. tivi:,V was enriched 16.2-fold in the plasma membrane

TABI.E I Loc~.l~:ation of marker enzyme acti¢iti~ in hepatic subceUular free. tions #ore II day.old suckli~ rats Marker enzyme activities were assayed as described in the Experi. ment.-.l procedm'es and expressed as relative specific activity using the highest activity as 100%, The specific activity of 5'.nueleozidas¢ (plasma membrane) was 1079 nmol phosphate released/min per mg protein. The specific activities of diacylglycerol acy[transferas¢ (STM-washed microsomes) and acid phosphatase (lymsomes) were 15,2 nmol triacyl$1ycerot synthesized/rain per mg and 120 nmol phosphate released/rain per me, respectively. This result represents one subeeliular preparation. Similar results were obtained from two other liver preparations from adult and I i,day-old tats,

Fraction Relative specific activity (% of maximum)

5'.nucleotidase acid phosphatase DGAT"

Homogenate 0.17 26.66 28.95 Plasma membrane 100.00 11.54 8.88 Mi~luyJui~.', i i.i2 33. i 5 ITS.00 Lysosomes 2.38 100,00 23.03 Cytosol 0,00 8.2,~ 7,89

DGAT. diacylglycerol acyltran,~ferase.

Page 4: Diacylglycerol metabolism in neonatal rat liver: characterization of cytosolic diacylglycerol lipase activity and its activation by monoalkylglycerols

330

TABLE 11

Diecylglycerol lit,use actidties in di.iferent subcelhdar fractions at pH 8.0 and pH 4.8

The diacylglycer.t lipase was assayed as de~ribcd in Experimental procedures excep~ that the Tris-HCI buffer was replaced by t buffer coW.aining acetic acid (62.5 raM}. Mcs (62.5 raM) and Tris (125 raM} which was adjusted to pH 8.0 ot 4.8.

nmol producl/min per mg protein

pH 8.0 pH 4.8

MAG " FA" MAG FA

Homogenate 0.154 0.614 0.112 0,046 Plasma membrane 0.153 0.245 o ~ 7 0.042 Microsomes 0.198 1.514 0.624 0,234 Lysosomes 0.035 0.658 0.788 0.356 L~,tosol 0.697 0.240 0.1~'~4 0.004

a MAG, monoacylglycerol; FA, fatty acid.

fraction, and diacylglyccrol acyltransferase activity was enriched 3.5.fold in the microsomal fraction. When phosphodiesterase ! was used as an additional plasma membrane marker, its enrichment in the plasma mem- brane fraction paralled that of 5'-nucleotidase (data not shown). Neither the plasma membrane nor the microsomal fraction was enriched for the alternate marker enzyme activity, indicating that virtually no cross-contamination was present in these two fractions and strengly supporting the conclusion that micro- somes contain an independent diacylglycerol lipase ac- tivity. In addition, diacyiglyccrol lipase activities were identified in cytosol, lysosomes, and plasma membrane.

A t t",.~lt| The lysosomal fraction had a .,.~- . . . . cniichmcat 6f acid phosphatase and the cytosolic fraction was virtu- ally free of all membrane marker activities.

Because two pH optima, a, 4.8 and at 8,0, were observed in a total particulate preparation from suck- ling rat liver, dia~lglycerol lipase activity was assayed at these oH optima in each subcellular fraction (Table I1). As expected in liver from 1 l-day-old suckling rats, the major pH 4.8 activity was found in the lysosomal fraction. At pH 8.0, diacylglycerol iipase activities were predominantl~ associated with microsomes and cytosol. Although microsomes contain,~d the highest specific activity, most activity was recovered in the cytosol because of its hi!~h protein concentration. The total activity recovered in plasma membrane was low be- cause vf the low protein cot|teal. The pH 8.0 activity in the ly~somal fraction is likely to have rc.,ulted from cross.contaminal~on with microsomes (Table !). Al- though the pH 4.8 activity in microsomes may also be the result of cross-contamir~ation, triacylglycero!, diacytglyeerol, cholesterol ester., and retinol ester hy. dro!ase activities ,~'ith low oH optima have been re- po~cd in micro:~),-nes or in endocyfic vesicles that fractionate with micmsomes [18-20]. The subcellular

distribution and specific activities of the diacylglycerol lipascs in livers from i l-day-old rats and adult rats were virtually identical (data not shown).

Analysis of reaction products With the sn-l-stearoyl,2-[14C]oleoylglycerol sub-

stLate, two ,adiotabeled products, monooleoylglycerol and oleic acid, were released. However, the distribu- tion of these two products in the various fractions differed greatly (Table II). In microsomes (pH 8) more than 80% of the product was fatty acid. This finding, suggested that either monoacylglycerol [ipase activity strongly predominated or that hydrolysis occured first at the sn-2 position, a preference hitherto undescribed for a diacylglycerol [ipase (see discussion in Ref. 21). In cytosol (pH 8) and lysosomes (pH 4.8), however, more than 75% of the total product was mor~oacyiglycerol, suggesting that the an-1 position was hyd~olyzed before the sn-2 position. Analysis of the monoacylglycerol ~tereoisomcrs by thin-layer chromatography strength- ened this interpretation. At pH 4.8 (lysosomes), little aeyl migration occurred and 93% of the radiolabeled monoacylglycerol product was the sn-2 isomer, con- firming that the sn.l bond was: hydrolyzed first. Since diacyl- and monoacylglycerois undergo enhanced acyl migration at alkaline pH [22], it was not surprising at pH 8 to find that 5.5% of the labeled monoacylglycerol product from cytosol was the sn-2 isomer and 45% was the se-l(3) isomer, consistant with acyl migration of the sn-2.[~4C]oleoyl moiety to the sn-l(3) position after the sn-l-stearoyl group had been hydrolyzed. If diacyl- glycerol lipase had been specific for sn-2 acyl groups, ,~,i~ would have expec:~d '.o find '.!-:t :he major prod- uct was labeled fatty acid; and if, on the other hand, the acyl moieties of the diacylglycerol substrate had migrated first, one would have ob:ained approx, equal amounts of labeled monc~acylglycerol and fatty acid. The relative distribution of the radiolabeled products a~so suggested that diacylglyccrol and monoacylglycerol l;p',se actNitics were independent, the latter being mainly located in microsomes, but not in ~tosol or lysosomes. This hypothesis was supported by the fol- lowing study of washed microsomes,

Release of diacylglycerol lij•se activity from mtcrosomes In order to determine whether diacyiglycerol lipase

activity could be released from microsomes, microso- real membranes were incubated with buffers, o." with heparin at 37°C for IC rain. The pellets and super. natants were recollected by centrifugation, and diacyl- glycerol Iipase activity was measured (Fig. 1). After the washes, 90 to 115% of the total protein was recovered. ]he total diacygglycerol lipase activit,~cs r,~covered from T:!~-t-iCI, ~TM buffer and heparh~-wash,d microsomes were 121, 124 and !b3%, respectively. The Tris-HC! wash released 41% of the protekl and 5?% v[ the

Page 5: Diacylglycerol metabolism in neonatal rat liver: characterization of cytosolic diacylglycerol lipase activity and its activation by monoalkylglycerols

331 ,..[ 3.S

3,0 [ ] Fm~ l~tl

i - F ] iA(}

I= 2.0 HIIteRo$ Ttql8

1.0

0.1

0 |O 1101 J Ill IQi

8TM p

0 | 1 t1§

I ll

HEPARIN

I 18 100

PROTEIN (%) Fig. 1. Diacylglycerol lipase activ;,y from washed microsomes. Non- STM-washed microsomes (Micror,,* (!.7 mg protein in 0.3 ml) from suckling rat livers were incubated ~ith the indicated buffers at 37°C as follows: Initial activity, no addition; "iris, 1,5 ml 0.1 M Tris-HCi (pH 8.5); STM, 1,5 ml STM buffer; Heparin, 1.5 ml STM buffer containing 8.8 U of heparin. After a 10 min incubation, the prepara- tions were centrifuged at 100000x g for 30 rain. The supernatants (S) and pellets (P) were separated, and the pellets were resuspended in 1.0 ml of the appropriate buffer. To n',easure the initial activity, microsomes were incubated for tO min at 37C before assay. Diacyl- glycerol lipase activities were assaye~l as described under Ex~rimcn- tal Procedures. The data represent the average of duplicate samples.

diacylglycerol iipase activity, and the STM buffer re. leased 20% of the protein and 18% of the diacyl- glycerol lipase activity. Heparin released about 30% of the protein together with 69% of the microsomal diacylglycerol lipase activity. After incubation and re- lease by heparin, the total activity recovered also in- creased 63%, suggesting either that heparin had acti- vated the diacylglycerol iipase or that heparin had separated the diacylglycerol lipase from an inhibitcL To test the direct effect of heparin on diacylglycerol iipase activity, microsom~l and cytosolic fractions were assayed in the presence of 1 U heparin/ 6 Atg of cytosolic protein. No change in diacylglycerol lipase activity was observed.

The distribution of the monoacylglycerol and fatty acid products differed in the supernatants and washed mict'osomes. In tl=e microsomes, both at the start and after repelleting, more than 80% of the product was fatty acid, ~uggesting that a monoacylglycerol lipase was present in this fraction and tightly bound to the membrane. In the supernatants, however, monooleoyi- glycerol comprised more than 90% of the total prod- t, ct. Since almost 70% of the microsomal diacylglycerol lipase activity could be readily displaced by a brief incubation with buffer or heparir,, much, but not all, of the activity appeared ~o b~ Ioo~e!y bound to ~he micro= soma! membrane. The micrommal and cytosolic diacyl- glycero! lipas, e activities may represent two forms of an ambip~t.hic en-%~e that moves between the two cellu-

lar compartments. Regulated movement to a specific membrane occurs during the activation of protein ki- na~¢ C r,v~l L,..n as w~]l as two other enzymes of giyccrolipid metabolism, phosphatidate phosphatase [24] and phos- phocholil~e cyt;@!yltransferase [25].

Because higher amounts uf ilcparin (i U /~g pro- tein) strongly inhibit microsomal diacylglycerol lipase from bovine brain [26], microsomcs and cytosol were also assayed with ! U heparin/0.13 to 1.3/tg protein. The mierosomal activity was unaffected by the higher hcparin concentrations (data not shown), in cytosol, the activity was not affected by 1 U heparin/ 1.3 pg protein, but at 1 U hcparin/0.13/tg protein, diacyl- glycerol lipase activity decreased 40%.

Because heparin has a negative charge, it is thought to bind proteins that contain a linear arrangement of positively charged amino acids [27]. The ability of hep- arin to enhance the release of diacyiglycerol lioase from microsomes suggests that agarose-heparin may be as useful a ligand for the purification of hepatic diacyl- glycerol lipas¢ as it is with the membrane-bound diacyl- glycerol lipase from bovine brain [26].

Ontogeny of diacylglycerol lipase One function of hepatic diacylglyceroi lipase activity

might be to provide the monoacylglycerol substrate for the developmentally expressed monoacylglycerol acyl- transferase. In order to determine whether the on- togcny of monoacylglycerol acy[transferase and diacyl- glycerol lipase was similar, we studied liver subceilular fractions from adult and ]l-day-old rats, since monoa- cylglycerol acyltransferase activity peaks between the eighth and twelfth postnatal days [3]. The specific activities of the diacylglycerol lipases were similar in microsomes, iysosomes and cyto~! from the young and adult rats (data not shown), tn plasma membrane, however, diacylglycerol lipase specific activity was 5.8- fold higher (2,3 nmol/min per rag) in the adult than the suckling rats (0.4 nmol/min per rag). These data indicate that none of the hepatic diacylglycerol lipases exhibits the ontogenic pattern expressed by the monoa- cylglycerol acyltransferase ~ctivity.

Dependences of diacylglyc.erol lipase activity Diacylglycerol lipase activity was measured with re-

spect to time and to protein concentration in cytosol from suckling rat liver (Fig. 2A and B). Although the total diacyiglycerol iipase activity and the formation of mo,oacylglycerol were not prol~ortional to time ¢r protein, fatty acid release was directly proportional tn both the reaction time and to the protein concentra- tion. This result is consistent with the use of the monoacylglyccrol product as a substrate tb~ a cyt,-~oiic monoa~!glycero] lipase specific for the sn-2 postion or with the progressive mlt;rafion of the awl group fol- lowed by the slower hydrolysis of the resultin~o sn-l-

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332

,-o4[ A 0.2

g;

o S 10 15 20 25

TIME (rain)

B 20

$_ • 1

O 20 40 60 80 PROTEIN (pg)

Fig. 2. Dependences of dia~.~lglyc~tol lipa~e activity. Cyto~lic diacyl- glycerol lipase activity from I l-day-hid rqls was mea.~ured as de- scribed under Experimental Procedures al the indicated (A) times and (B) protein concentrations. The time dependence employed 50.6 /=g protein. The symbols represept the formal[on of monc~de~ylglyc- erol (o). and fatty acid ( I ) , Each point represents the mean±

standard deviation from triplicate assays.

monoa~dgtycerol by diacyiglycerol lipase. Although di- rect proportionality of total diacylglycerol lipase eoul~d be obtained with protein concmarations lower than 10 pg an~ with reaction times shorter that, 5 rain, routin~:. measurement of initial rates was precluded by lhu relatively low end,me activity.

Diao,lglycerol dependencies The dependence of diacylglycerol iipase activity on

its substrate showed saturation kinetics with an appar- ent K m of 115 #M (Fig. 3). The release of arachi- donate from the sn-2 position of glycerolipids is of interest because v,-r ,,.,,,,.. put~:miai metavu:,~,, . . . . of ~ J~cifi- donate via the lipoxygenase and cyclooxygenase path- ways. Therefore, we compared the activity of diacyl- ~,iycerol iipase on diacylglyccrol substratcs d~at had either radiolabeled oleate or arachidonate at the sn-2 positiun (Table l|l). No difference was obselved in the re!ease of either monoacylglycerol or the labeled fatty actd. -[his observation suggested that, unlike ti~e activ- ity from platelets [21], i.he cytosolic diacylglycerol lipase i.~ not specific for diacylglycerol with arachidonate in the sn-2 position.

soo •

S soo

..=

400

200 ~ 0.00 0.02 0 ,04 0.00 / I /OtACYLGL¥cEnI~. ~M) ] i , = , , ~ , - ~ .

50 100 1 SO 200 2S0

sn-I,2-DIACYLGLYCEROL (pM)

Fig. 3. Dependence of diacylglycerol lipase on substrata concentra- tion. Cylo~lic diacylglycerol lipase activity from I l-day-old rats was measured during a 15 rain incubation using 33.7 ~.g protein as described under Experimental procedures, The data represent the f.rmatiop of monooleoylglycerol plus oleic acid. Each point repre- sents the average of triplicate assays. The inset represents a double- reciprocal plot according to the method of Lineweaver and Burk, subjected to computer-assisted least-squares analysis [28]. Th~ re-

gression c¢~fficicnl was 0.99.

In order to characterize the cytosolic diacylglycerol lipase and to determine whether the activities from ad~dt and suckling rats had similar or different charac- teristics, each fraction wa~ assayed in the presence of compounds that had been previously reported to affect diacylglycerol lipase activity in other tissues (Table IV). This characterization indicates that the activities from adult and suckling rats share several properties. Both were strongly stimulated in lh¢ presence of bovine serum albumin and were inhibited similarly by both dithiothreitol and N-ethylmaleimidc, suggesting the ,aresence of critical reduced as wei i -~s-uxidizcd sulfhydryl bonds. The different effects of ATP, how. ever, suggest that the diacyl~lycerol lipases from adult

TABLE Ill

Compari.~'on o/ diao'lglyceml lipase aclirity with I.stearoyl,2. / l~Chdeo)'lg~yceml mid I-stearoyl,2./~H/amtttMonoy;glycerol

Cyto:~'~l from ll-day-old rats was incubated with each of the two substrates as :~escribcd under E:~perimental procedures. Each re.m- her represents the average of duplicate assays.

Protein DAG lipase a,:tivity (pmol/min)

~'~'g) l.stcaroyl.2-arachidono:d- l-stear~;yl,2-oleoyl- glyceml glycerol

MAG " FA :' MAG FA

2i.4 t7.3 2.79 18.78 2.02 42,7 i6.93 ZOO 2n,44 7.2"/ ~.1 ~5.91 I 1.74 35.96 ll).tJ5

" MAG, m~t, ucyiglyLerol, i, FA, fatty ac;d

Page 7: Diacylglycerol metabolism in neonatal rat liver: characterization of cytosolic diacylglycerol lipase activity and its activation by monoalkylglycerols

333

and suckling rat liver might be similar, but distinct, isoe.zymes.

Relationship between diacyl~lycernl l;4:ase and preciously described hepatic triacylglycerol lipases

Since triacylglycerol lipases can hydro!yze diacyl- glycerol, and because both hepatic and lipoprotein lipase activities have been reported in neonatal rat liver [29], we tested the effects of serum, NaCI, and protamine to determine whether these known modera- tors of lipoprotein lipase and hepatic lipase would affect the diacyiglycerol lipase(s) in subcellular frac- tions from rat liver (Table V). NaCI, which inhibits lipoprotein lipase 80 to 90% [30,31], actually increased the hepatic plasma membrane activity from adult rats and the cytosolic activity from both adult and suckling rats, and had little effect on microsomal activity from either adult or suckling rat liver ~if'rum, which is required to fully activate lipoprotein lipase [31], in- creased the cytosolic activity only 50% in adult liver, and either had no effect or decreased the activities from other fl:;ctions, suggesting that the diacylglycerol lipase from these fractions was not a second function of lipoprotein lipase. Protamine, which inhibits hepatic lipase more than 90% [31,32], only partially inhibited the cytosolic and microsomat diacylglycerol lipase activ- ities, and had no effect on plasma membrane activity. Thus, all three compounds either had no effect on the hepatic diacylglycerol lipases or their effects differed from those normally expected for hepatic or lipopro- tein lipase activities *. Taken as a whole, these results suggest that the microsomal and cytosolic diacyl- glycerol Iip~,,ses are not second functions of either

TABLE IV

Del~'Itdt'ttces of CytOsOliC diuo'lgly"erol lipa.~e actirity front adtdt ut;d i i.day.old stickling rat lirer

Assay conditions Percent of control activity

Adult I l-day-old

Complete system ~' I(H) 10(} - BSA 49 63 - MgCI 2 98 106 -f- ATP. |[) mM IIH) ~I + NaF, 5 mM 82 94 + DTT, 2 mM 67 84

5 mM 48 63 + NEM. S mM tO 26

" The complete syslem, containing 0,25 M Tris-HCI (pit 8.0), I m e / ml BSA, 8 mM MeCh, 10 to 50 pg protein, and 120 p.M I-stea- =uyi,:,[~aC]~!co~,!~';'c-rel i.-. :; fi~.':! vcht."a,,: cf 2~ ,. !, is de.~ribed under Exl~rimental procedures. Specific activities for the com. p!ete .%,;:tern were !.03 and !.3~ nmol/m!.~ per rng f,;r .%'te':c[ fi'om i I.day-old and adult rats, respectively. The relative activities rep- resent the averages of duplicate determinations from u,c to three iadepcnaent ex~'rim,'n!';, ~SA, ,bovine serum albumin: Err, dithiothreitol; NEM. N-uthylmaleimide.

TABLE V

Effi'ct of NaCI, hmmm .wmm, trod pn~lanlinc on diao'lglyccrol lif~L~¢ acticity

Prolein from each source was pre-incubat.~d with the putative effec. tar for i.O h at 23°C before diacylglycerol lipase activity was assayed as described under Ex,~rimental procedm¢:.~. Control preparations wcrc incubated without any addition. The data are rei~trted as the aver;lees of one or Iwo independent ex~rim~nts, each performed with duplicate samples.

Subcellular fraclkm Percent of control activity

NaCI serum pmtamine ( I .0M) (25%) (I.6 mg/2lE!plassay)

L-~'losol adnl! 1 ! ~ 15l 37 ! l .day.old 175 I l I 56

Plasma membrane adult 138 82 99 I 1-day-old 85 63 i01

Microsomes adult 94 50 h7 I l-day-old 79 43 4f~

hepatic or lipoprotein lipase. The varying responses of the microsomal and plasma membrane activities to serum and to protamine (Table V), suggest that the activities may be isoenzymes or that the responses may have resulted from the differences in interaction be- tween ~h¢ substrate and enzyme in the presence of membranes of varying composition,

To compare the diacylglycerol and triacylglycerol lipase activities directly. ['~H]triolein was employed as a substrate under conditions otherwise identkal to those used to measure diacylglycerol lipase activity. In each fraction, d[acylglycerol lipase activity was 6-9-fold higher than the triacylglycerol lipase activity measured in the same fractions (Table VI), indicating that the diacylglycerol activity wedomiitated.

Effec; of subslrate analogs on diacylglycerol Rpase actic- ity

It has been previously shown that I-monooleoyl- glycerol elevates diacylglyccrol levels in both basal and stimulated platelets wlt.hou: apparently affecting diacyiglycerol kinase [33], and that it inhibits by 30% the metabolism of dioctanoyiglycerol to water-sclublc metabolites [34]. Since this finding was consistant with the direct inhibition of diacyiglycerol lip~e, we tested the effects of l-monoacylglycerol and several of its analog~ ,:'n hepatic diacylglycero[ lipase activity ('Fable

* Under similar assay conditions using tri['~H]oleoylglyceroi ~ d rat adipose tissue fur cake-free homogenates, IS~. serum increased tria~.3"lglyccrol lipase activity 3-fldd and 1.0 M NaCI inhibited triacylglycerol lipase activity 84r,~,

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334

TABLE Vl

Compariso~ o[ diat3'lglycerol arid triao'lglycerol lipases in ilepatic subcellular fractions [r~'m l l-day.old suckling rats

Enzyme activity was measured using either l-stearyl,2-il4C]oleoyi - sn-glyeero! or glycerol [g, lO--~H]trioleate, under otherwise identical conditions. The d;a~lgb'ccrol product rcpre~nts the nmol of mono[l'lC}oleaie plus [l~Cloleaie formed/tin per mg protein: for iriacylglyeem], the product represents 0,33x the ['XH]olcaie hydro- i~zed/~in per mg pr~'.-i~. DAG, diacylglyccrol: To, G. tria~l- glycerol.

Entree Specific ac'ivity a source (nmol sub: irate hydrolyzed/mg per t in)

DAG iipase TAG iipase DAG Iipa~%/ TAG Iipase b

Plasma membrane 0.406 0.066 6.2

Microsomes 2.579 0.281 9.2 Cylusol 1.234 0.193 6.4

" The data are the average of duplicate assays. h Ratio of the Iipas¢ activities.

VII), Neither sn-1(3)- or 2-moziooteoy'~glycerol ~ior sii- 1,2-dioleylglyceroi ether altered the cytosolic diacyl- glycerol lipase activity. However, diacylglyeerol lipase activity increased with the addition of sn-l(3)- and 2-monooleylglycerol antides or sn-!(3)- and 2-mono- oleylglycerol ethers. Activation of diacylglycerol lipase activity depended upon the concentration of the

TABLE VII

E[/ect of sn.l C i ) . arid 2.monooleoylg6"cerots arid their arnide arid emer analogs an diacylglycerol lipase arririty from licer t')'tvsol of I i-day-e!d and adult rats

Addition Cont.:ntration Percent of control activity "

adult ! l-day-old

!-Me 250/zM I 1O 76 2-Me 250 ,~M I It 127

I-Me amide 250 ~.M 128 129 2-Me amide 250 ,a M 185 145

I-Me ether ~ 0 # M 204 lf~6 2-Me ether 250 pM 145 259

1,2-DO ether 125 pM 76 97 LPAF 50 p g/ml nd 57

LPC 50/~g/ml nd 42

a The lipid additions were dispersed in acctnne~ bringing the final concentration of acetone to 10'7~ in the assay mixture. Control iietivities were 0,28 and 0.24 nmol/min ~ r mg protein from l l-day-old and adult cytosol, respectively. Me, monooleoyiglyc- erol: DO, dioleylglycerot: [PAF. |-alkyl.2-1ysophosphalidvlcholine: LPC, lysopbosphatidyicholine; nd, not delermined. The data are plotted as the averages of duplicate a.c.~ays, except for the 1 l-day-old rat data on the monoal~lglycerols which awrage three indepen- dent e'~pe~rlmeni~: each peff~;'rficd in d-p!i,:.!~ie.

'l A r

Q n"

• , ~ , . ~ _ _ _ _ ~ , , ~ . _ _ t

0 100 200 300 400 500

pM sn-l-Monooleylglycerol

[B

i | 0 100 200 300 400 500

pM sn-2-Monooteylglycer¢ll Fig. 4. Effect of monoalk,/Igl!,'cerols on cytosolic diacylglycerol lipase activity from ! l-day-ola rats. Diacylgly,'erol lipas¢ activity was teas. ured in the presence of either (A) I-monooleoylglyccml ether or (B) 2-mononlc~lglycerol ether added in 5 # | acelone at the concentra- tion indicated. The control reaction was performed in the presence of 5 pl acetone alone. The symbols repre~nt ~he formation of monoolenylglycerol (¢), oteic acid ( r: ) and monooleoytglyccrol+ ol¢ic acid (o). The specific activity (mean:l:S.D.)of lhree independent controls was 0.46+0.04 nmol/min per mg protein. Each data point obtained with 10 to 1(141 #M monoalkylglyeeroi is the average of two observations: each point above 100 ~M is the average of two inde-

pcndeut experiments, each performed in duplicate.

mont, alkylglycerols (Fig. 4). Maximal effects were ob- served at 125 gM and higher concentrations did not produce a furtkcr increase. Although the tota~ specific activity increased in the presence of the monoai~tglyc- erois, the small amount of fatty acid released de- creased further by approx. 50%. This observation sug- gests that the monoalk'ylglycerols stimulate diacyl. glycerol lipase activity but may inhibit the migration of the sn-2 acyl group to the sn-l(3) position where it would be accessable to diacylglycerol lipase. An alter. nate possibility is that cytosol contains a low monoacyl- glycerol lipase activity that is inhibited by the

I h. monoal.'ylg.ycerols. If the latter possibili~' z: true, the observation supports the conclusion that the complete hydrolysis of diacylglycerol to glycerol and fatty acid is cate,!yzed by two independent enzymes. Because i.

Page 9: Diacylglycerol metabolism in neonatal rat liver: characterization of cytosolic diacylglycerol lipase activity and its activation by monoalkylglycerols

monoalk3,1glycerol might arise from the metabohsm of platelet activating factor (PAF), we also te,ste,! the effect of iysoPAF and its acyl analog l?~aphc,~p~,,~ id?!. choline (Table VII). Both compounds inhibited, rather than stimulated, diacylglyeerol lipase activity from ~uckling liver.

If monoalkylglycerols play a physiological role in regulating diacylglycerol lipase activity, they are likely to arise after the initial ~teps o.~ ether lipid synthesis in peroxisomes or during the metabolism of platelet acti- vating factor or other ether pilosphol[pids. Although the ether linkage of the best.studied a!kylglycerols is at the sn-1 po~itiou, sn-2-1inkages have been identified in glycerolipids from heart and brain [35]. Ether lipids are thought to play a role in regulating diacylglyccrol metabolism and protein kinase C activation [36] and monoalkylglycerols are competitive inhibitors of the committed step of the glyceroI-P pathway [37] and consequently inhibit the synthesis of phosphatidic acid, diacylglycerol, and triacylglycerol [38]. It remains to be established whether *he ether lipid metabolites play physiological roles in ce!lular metabolism, but it should be noted that monoalkytglycerols and alkyllysophos- pholipids inhibit the growth of several types of cultured cells [39-42], possibly through their actions on glycero- lipid and diacylglycerol metabolism.

Final Discussion

Diacylglycerol iipase activity from liver has received attention in only a single report in which phospho. lipase C was used to generate the diacylglycerol sub- strate from phosphatidylcholine directly in the assay [43]. Although both diacylglycerol and monoacylglyc. erol lipase activities appeared to be present in eytosol, the diacylglycerol substrate may have undergone acyl- migration after its generation in phosphatidylcholine vesicle~. Using subcellular fractions )hat v, ere less en- riched ia their ,,espective marker cn~mc activities ~han those reported here, these investigators found that the highest specific activity of diacylglycerol lipase was ip lysosomes, but that 50% of the total cellular activity was in the cytosol.

in non-hepatic sources of diacylglycerol lipase activ- ity, microsom~l or membrane locations were reported in human fetal membranes [44]. h ima.,, platelets [21], rat heart [30], and bovine brain [26]. In fetal mem- branes and platelets, the activity preferentially hydro- lyzed the sn-I position and more arachidonate than oleate was hydrolyzed fr,.~m the sn.2 postion indicatir, g a preference for diacylglycerols with arachidonate iin the sn-2 position. None of the diacylglycerol lipases has been. extensively characterized; inhibition has been var- iously repc~ted after in..,'ubafion with N.e[hylmaleimlde [21], phenylmethylsulfonyl fluoride [21,49], NaCi [30], NaF [30,44], protamine [30], Triton X-100 [21,30] and

33:;

heparin [26]. ATP has been reported both to inhibit [26] and to have no effect [30].

~; ?!:m:~I~ t:'"'.:~:t r'embranes [21] and fetal mem- branes [44] r;,vnoacylglycerol lipase activity is more than 10-fold higher than diacylglycerol lipase; thus, fatty acid is the major product of the diacylglycerol lipases previously described in nonhenatic tissues. In contrast, we found that liver subcellular fractions vary in their relative predominence el monoae~,igiycerol and diacytglycerol lipases. The monoa~,lglycerol lipase ac- tivity it, hepatic microsomes may be comprised of one or more microsomal carboxyesterases whici, hydrolyze monoacylglycerol [45-47].

Perinatally, the rat undergoes major changes in lipid metabolism as the percentage of calories derived from lipid increases from less than 10% in the fetus to 70% during the suckling period, and then decreases to about 15% after weaning [48,49]. Coacommitant with these changes in dietary lipid composition, are dramatic changes in many hepatic enzymes of lipid metabolism, including fatty acid synthase and HMG-CoA reductase activities [32,50], enzymes required for the/3-oxidation of fatty acids [51,52], the microsomal enzymes of phos. phatidylcholine and triacylglycerol synthesis {13], and the liver-specific monoacylglycerol acy!transferase ac- tivity [31.

We studied hepatic diacylglycerol lipase activity, in part, to answer two questions re;ated to the develop- mentally-expressed monoacylglycerol acyih'ansferase activity: (i) can a diacylglycerol lipase associated with one or more subcellular fractions provide the sn-2- monoacylglyeerol sabstrate for monoacylglycerol acyl- transferase; and (ii) is there a diacylglycerol lipase activity that is also expressed developmentally. Since long-chain monoacylglycerols do not enter the portal vein (Coleman, R,A. and Ney, K., unpublished data), and since hepatic [5] and plasma [6] lipases hydrolyze monoacylglycerols readily, it seemed unlikely that large amounts of monoacylgIycerol would originate outside the hepatocyte. Instead, we hypothesized that monoa- cylglycerol may be produced from the partial hydrolysis of either the chylomicron remnant triacylglycerol that enters lysosomes, or the triacylglycerol stored in the numerous cytosolic lipid droplets witl~in neonatal hep- atocytes [5~]. In either ca~, to be a sub~trate tor monoacylglycerol acyltransferase, monoacyigfycerol would need to translocate to the endop~asmc reticu- lure. Our data suggest that sn-2-monoacylglycerol could accumulate after the hydrolysis of diacylglycerol in botll lysoso~:~es and cytosol. The c, bse~vation that the cytosolic and microsomal diaeylglycerol iipast" activities were expressed similarly in suckling,: and adult rats suggests that the activity may function, not merely to metabolize lipid droplets, but also to llydrolyze diacyl- glycerol released during phospholipid tm~lover and second messenger stimulation.

Page 10: Diacylglycerol metabolism in neonatal rat liver: characterization of cytosolic diacylglycerol lipase activity and its activation by monoalkylglycerols

336

Acb_n.ow!edgemen!

This work was suppor ted by grant H D 19068 f rom

the Nat ional Inst i tutes o f Health.

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