46

BBALIP

Biochlmica

et Biophysics

Acta. 1046 (1990) 46-56 Elsevier

53460

The effect of monostearoylglycerol on the metabolism of chylomicron-like lipid emulsions injected intravenously in rats B-C. Mortimer

‘, W.J. Simmonds

‘, S.J. Cockman

I Department of Physiolog), and ’ Department of Organic Chemistv, (Received

Key words:

Lipid metabolism;

15 February

2, R.V. Stick 2 and T.G. Redgrave University

of Western

Australia,



Nedlands (Australia)

1990)

Monostearylglycerol;

Chylomicron:

(Rat)

In rats, remnant particles derived from chylomicron-like emulsions containing 1,3-dioleoyl-2-stearoylglycerol (OSO) are removed from plasma more slowly than remnants derived from triolein emulsions. The effect associated with a saturated acyl chain at the glycerol 2position could be reproduced by incorporating 2-stearoylglycerol (MS) in a triolein emulsion. When MS solubilized with rat albumin or in plasma was injected before the injection of a triolein emulsion, clearance of the triolein emulsion was unchanged. The metabolic fate of MS, monitored with “C-labelled MS, was similar whether incorporated in triacylglycerol emulsion or injected independently. More than 95% of MS had disappeared from the circulation by 5 min after the injection and the radioactivity was found in liver, spleen, muscle and adipose tissue. Some MS label appeared in plasma triacylglycerol. Remnants made in vitro by incubating triolein or OS0 emulsions with post-heparin plasma showed no differences in their disappearance from plasma. With OS0 emulsion, the in vitro remnants were found to contain more MS than remnants made in vivo in hepatectomized rats. Simultaneous injections of mixtures containing OS0 and triolein emulsions, or triolein emulsions with and without MS, each labelled with either [ 3HJcholesteryl oleate or [ 14C]cholesteryl oleate showed consistently slower remnant removal and decreased liver uptake of the emulsions containing OS0 or MS. Affinity columns and immunodiffusion all indicated that there was no difference in the amounts of apolipoprotein E associated with OS0 or triolein particles. The protein spectra of in vivo remnants derived from OS0 and triolein emulsion were also similar when examined by SDS-PAGE and isoelectric focusing gels. Our results show that the effects due to OS0 or MS are mediated by the presence of MS in the emulsion particle surface, while indirect effects expressed in plasma or liver are excluded. The precise mechanism of the effect remains to be established, but it does not correlate with measurable changes in the spectra of apolipoproteins associated with the emulsion remnants.

Introduction Triacylglycerol-phospholipid emulsions containing cholesteryl ester and free cholesterol can be prepared to resemble closely lymph chylomicrons in size and lipid composition [l]. These emulsions are metabolised in the same way as natural chylomicrons when injected intravenously in conscious rats [2]. When the triacylglycerol is triolein, lipolysis of triacylglycerol to form remnant particles and uptake of remnants by liver are comparable with natural chylomicrons [3]. The metabolism of

Abbreviations: MS, 2-stearoylglycerol; EYPC, egg yolk phosphatidylcholine; OSO, 1,3-dioleoyl-2-stearoylglycerol; EDTA, ethylenediaminetetraacetic acid; PBS, phosphate-buffered saline; VLDL, verylow-density lipoproteins; LDL, low-density lipoproteins; HDL, highdensity lipoproteins; SDS, sodium dodecyl sulfate. Correspondence: sity of Western

T.G. Redgrave, Department of Physiology, Australia, Nedlands 6009, Australia.

0005-2760/90/$03.50

0 1990 Elsevier Science Publishers

Univer-

B.V. (Biomedical

the artificial chylomicron particles can be modified by controlled alterations in the contents of cholesterol [4], phospholipid [5], or in the triacylglycerol. With regard to triacylglycerol content, for both natural and artificial chylomicrons clearance of the particles from plasma was slower when the triacylglycerol was rich in 1,3-dioleyl-2-stearoylglycerol [6]. The same effects were observed when the saturated monoacylglycerol, 2-stearoylglycerol (MS) or l-stearoylglycerol was incorporated into triolein emulsions, but not when the unsaturated monoacylglycerol, l-oleoylglycerol, or the saturated fatty acid, stearic acid, were substituted for monostearoylglycerols [7]. These results suggested that a slow clearance of triacylglycerol-rich particles was mediated by monostearoylglycerol, whether generated in the OS0 emulsion particles by lipolysis or separately incorporated in a triolein emulsion. Monoacylglycerol is freely transferable from the surface of triacylglycerol-rich emulsion to plasma albumin or other acceptors in plasma [S] and so in this Division)

47 study we investigated (i) if the injection of MS solubilized in plasma or in serum albumin affected the clearance of triolein emulsion; (ii) if the slow clearance of OS0 remnant particles was an effect of MS on the properties of the particle surface or was an indirect effect due to uptake of MS by hepatic cells. We also compare the metabolic fate of radiolabelled MS complexed with albumin, into orated in chylomicron-like % particles, or liberated from C-labelled OS0 by lipolysis. Methods Synthesis of glycerides

Unlabelled OS0 and 2-stearoylglycerol were prepared as previously reported [6,7]. For the synthesis of 1,3-dioleoyl-2-[l-‘4C]stearoylglycerol ([‘4C]OSO), the acylation of 1,3-dioleoylglycerol was used instead of the acylation of 2-stearoylglycerol as reported earlier [6]. The diacylglycerol was obtained by the reduction of the acylated dihydroxyacetone with sodium borohydride in aqueous tetrahydrofuran at 0-5°C according to Bentley and McCrae [9]. Radioactive 2-[1-‘4C]stearoylglycerol ([14C]MS) was obtained by the hydrolysis of [‘4C]OS0 by pancreatic lipase followed by thin-layer chromatographic separation of the pure monoacylglycerol. 5-10 mg [‘4C]OS0 (20-30 PCi) was dried under N, and emulsified in 1 ml 30 mM sodium taurocholate (Koch Light Lab) solution in buffered saline (pH 8) containing 20 mM calcium chloride. The mixture was incubated with 1 ml of reconstituted freezed-dried pancreatic juice (2 mg, obtained by cannulation of rat pancreatic duct after ligation of the common bile duct) at 37°C for 20 min. Lipolytic products were extracted into ethanol/diethyl ether/petroleum ether (1: 1: 1, v/v) and separated by thin-layer chromatography in chloroform/ acetone (75 : 25, v/v) mixture. The monoacylglycerol band was scraped and eluted with chloroform/ methanol (2 : 1, v/v) mixture. [ 3H]Cholesteryl oleate was prepared by reacting cholesterol and [9,10(n)- 3H]oleic acid, in the presence of DMAP and DCC [lo]. Final purity was 99% by thin-layer chromatography (TLC). Preparation of emulsions

Mixtures of pure lipids were emulsified by sonication in dilute NaCl solutions. Triolein, cholesteryl oleate and cholesterol (Nu Chek Prep, Elysian, MN), egg phosphatidy1 choline (Lipid Products, Surrey, U.K.) and [‘4C]triolein (Amersham, Sydney) were each greater than 99% pure by TLC. Lipids were dispensed from stock solutions into vials. Solvents were evaporated under a stream of N, before overnight vacuum desiccation to eliminate residual solvent traces. Then 100 mg of a lipid mixture was sonicated in 8.5 ml of 10 mM Hepes (pH 7.4) in 0.15 M NaCl solution using a Vibra Cell Disrupter (Danbury, U.S.A.) as previously described [6,7].

Injection studies

Emulsions were injected into the bloodstream of conscious rats for measurements of the plasma disappearance rates and the organ distributions of the injected radiolabels. Non-fasted male albino Wistar rats weighing 250 + 20 g obtained from the Animal Resources Centre (Willeton, WA) were anesthetized with diethyl ether. A saline-filled Teflon cannula 0.76 mm o.d. x 0.33 mm i.d. (Small Parts, Miami, FL) was inserted through the left common carotid artery so that the tip was located in the aortic arch and a venous cannula 0.8 mm o.d. x 0.5 mm i.d. (Dural, NSW, Aust.) was inserted near the junction of the left jugular and subclavian veins. The tip was advanced to lie in the superior vena cava. Heparin was not used, but clotting was prevented by treatment of the tubing with Siliclad (Becton Dickinson and Co., Parsippany, NJ) before use. After surgery, the animals recovered from the anesthesia in individual restraint cages for 2-4 h before the injection study commenced. The emulsions were injected as a bolus of approx. 3 mg lipid in a volume of approx. 0.5 ml, into the venous cannula. Blood samples of 0.35 ml were then taken at 3 and 5 or 3, 5, 8 and 12 min. Each withdrawal was replaced with an equal volume of 0.15 M NaCl. Following the final blood sample a fatal dose of nembutal was injected. The liver and spleen were excised for extraction of radioactive lipid from minced whole spleen and from minced 1 g pieces of weighed liver. Lipids were extracted into 30 ml of chloroform/ methanol (2 : 1 v/v) mixture then aliquots were taken, the solvent was evaporated and radioactivity was measured by liquid scintillation spectrometry. Radioactivity in plasma was measured, without extraction, in 150 ~1 aliquots using Readysolv EP (Beckman). Plasma clearance kinetics were computed from exponential curves fitted by least squares procedures to samples taken during the first 12 min alter injection. Preparation of remnants

In vivo remnants were prepared in donor rats anesthetized with ether plus nembutal (30-40 mg/kg, intraperitoneally). The abdomen was opened. The liver and other viscera were excluded from the circulation by ligation of the celiac and inferior mesenteric arteries and the portal vein, with ligatures at the recta-sigmoid and esophago-gastric junctions to exclude porto-systemic anastomoses. About 1 ml of emulsion containing approx. 6-10 mg of total lipid was injected into a cannula in the jugular vein. 15 min later the animal was exsanguinated by heart puncture. A second group of donor rats was treated with 4-aminopyrazolopyrimidine 18 h prior to surgery to inhibit the release of endogenous VLDL [ll]. These rats were exsanguinated 30 min after injection of emulsion. The blood was mixed with EDTA (final concentration 4 mM) as anticoagulant, and 5,5’-dithiobis(2-nitrobenzoic acid) (final concentra-

48

tion 2 mM) to inhibit lecithin: cholesterol acyltransferase activity. Plasma was collected after low speed centrifugation. For remnant isolation 0.14 g/ml KBr was added to increase the density to 1.10 g/ml. 4 ml aliquots were layered under preformed step-density gradients made up with NaCl solutions as previously described [6]. After centrifugation in the Beckman SW41 rotor at 20°C for 2 h at 40000 rpm the layer of remnants floating at the top of the tube was aspirated for subsequent analysis. In vitro remnants were prepared according to Lenich and Ross with slight modification [12]. 3 ml of emulsion (4-6 mg total lipid/ml) was mixed with 5 ml Tris-HCl, (0.2 M, pH 8) containing 10% bovine serum albumin (w/v) and incubated at 37°C with 0.45 ml post-heparin plasma from hepatectomized rats until the initial turbidity of the mixture had cleared. Solid KBr (0.14 g/ml) was then added to the mixture and the remnants were harvested by gradient centrifugation as described above. Protein purification

Rat apolipoprotein E-rich lipoproteins (VLDL, LDL and HDLl) were prepared from EDTA plasma by adjusting the density to 1.063 with NaBr and centrifugation for 16 h at 35000 rpm in a SW 41 rotor. The supernatant containing the lipoproteins were aspirated and delipidated according to Holmquist and Carlson [13]. Specifically, the lipoproteins were mixed with an equal volume of isopropanol at room temperature and centrifuged at 18000 t-pm (IEC, B-20) for 30 min. The precipitate was discarded while the supernatant was mixed with an equal volume of ethyl acetate. The lower aqueous layer was dialyzed against four changes of 10 mM ammonium bicarbonate solution and lyophilised. Apolipoprotein E was separated from the protein mixture by SDS-gel electrophoresis according to Laemmli [14] with some modification. A 3-mm thick vertical slab gel was prepared with 15% polyacrylamide and layered with a 4% stacking gel. 6-8 mg of the apolipoproteins were dissolved in 1 ml of 0.0625 M Tris-HCI (pH 6.8) 2% (w/v) SDS, 10% (v/v) glycerol and 0.65% dithiothreitol and applied to each gel. The protein bands were visualized in 0.25 M KC1 solution [15]. The apolipoprotein E band was cut out and eluted from the gel with 10 mM ammonium bicarbonate, dialyzed against 10 mM ammonium bicarbonate to remove KC1 and then lyophilized. SDS was assayed by titration with 10 M KOH [16]. If SDS was detected the protein was passed through an Amberlite CG-400 resin (Sigma) according to Brysk et al [16]. The purity of apolipoprotein E was assessed by analytical SDS-gel electrophoresis [14]. Rat albumin was prepared by trichloroacetic acid/ethanol precipitation from the plasma according to Michael u71. Antibody to rat apolipoprotein E 500 pg of purified apolipoprotein

E suspended in 0.5

ml of saline was emulsified with an equal volume of complete Freund’s adjuvant before intra-muscular injection into a rabbit. At subsequent intervals of 2-4 weeks boosters of 100-300 pg were injected at the same sites. 7 d after the final injection, rabbits were bled and the serum was frozen at -20°C. Partially purified antibody fraction was prepared by DEAE Affi-gel Blue (Bio-Rad) according to the manufacturer’s instruction. Antisera or antibody fractions were reacted against purified apolipoprotein E, rat plasma and rat VLDL fractions on Ouchterlony double immunodiffusion plates for 2 d at 4°C. Affinity

chromatography

The purified antibody was oxidized for 1 h with sodium m-periodate (final concentration, 2 mg/ml) at room temperature. Excess sodium periodate was removed by a Sephadex G-25 (Pharmacia) desalting column and the oxidized antibody was coupled to Affi-Gel Hydrazide gel (Bio-Rad) in a capped tube rotating end over end at room temperature overnight. The gel was packed into a 1.0 x 10 cm column and washed with 0.5 M NaCl in PBS (pH 7.4) and stored in PBS containing 0.02% sodium azide. Prior to use, the column was washed with 5-10 bed volumes of PBS and 2-4 bed volumes of 3 M NaSCN, 5 mM Tris-HCl (pH 7.2) and then re-equilibrated with PBS [18]. Plasma or remnants were applied to the column and allowed to penetrate to 2/3 of the total gel bed. The column was then left at 4’C overnight. Unbound protein was eluted from the affinity column with lo-12 bed volumes of PBS. Bound proteins were eluted with 3 M NaSCN, 5 mM Tris-HCl (pH 7.2) [18]. The proteins of both the retained and unretained fractions were analyzed by analytical SDSgel electrophoresis [14]. Heparin-Sepharose affinity columns were prepared according to Wiesweiller [19]. 1.0 g of heparin-Sepharose CL 6B (Pharmacia) was swollen in distilled water overnight and packed into a column with a bed volume of 4.0 ml and a void volume of 0.96 ml. The column was equilibrated with 2 mM sodium phosphate (pH 7.4). Samples were loaded in the same buffer and allowed to penetrate to about 2/3 of the gel bed and either eluted immediately with 0.64 M NaCl in 2 mM sodium phosphate (pH 7.4) or left at 4°C overnight and then eluted. Isoelectric focusing

Lipoprotein samples in saline were delipidated using a 1: 1 (v/v) mixture of 2-propanol and I-pentanol and the aqueous phase was solubilized in sample buffer containing 10 mM Tris-HCL, 10 mM dithiothreitol and 8 M urea [20]. Electrofocusing in vertical slab gels was done according to Warnick et al [20] with some modification. 1.0 g of acrylamide, 27 mg of N, N’-methylenebisacrylamide, 9.6 g of urea, 1.0 ml carrier ampholyte (40%, Pharmacia LKB) (pH 4-6) was made up to 20 ml

49 with double distilled water. 20 ~1 of N, N, N’, N ‘-tetramethyethylene diamine and 200 ~1 of fresh 10 mg/ml ammonium persulphate were mixed with this solution before pouring the slab. The gel was prefocused for 1-2 h at 100 volts in a prefocusing buffer containing 2.4 g urea, 0.1 ml carrier ampholyte, 6 mg Tris and 7.8 mg of dithiothreitol in 10 ml double distilled water. The upper buffer (catholyte) was 20 mM sodium hydroxide and anolyte was 10 mM phosphoric acid. The gels were focused overnight at 250 V and then at 1000 V for 2 h, then fixed in 12.5% trichloroacetic acid for 1 h, stained in Coomassie blue G 250 overnight and destained in water. Chemical analysis

Lipid phosphorus was measured by a modified Bartlett [21] procedure. Triacylglycerol and monoacylglycerol, free and esterified cholesterol were assayed on lipid extracts, after separation by thin-layer chromatography. Triacylglycerol and monoacylglycerol were quantified as glycerol by chromotropic acid [22]; free and esterified cholesterol by the o-phthalaldehyde procedure [23] after saponification of the separated bands. Proteins were measured by a fluorescamine assay according to Tajima et al [24]. For protein analysis, remnant suspensions were concentrated by ultrafiltration and applied to SDS-PAGE without delipidation. Bands were stained by Coomassie blue R-250 (Bio-Rad) and quantified by laser densitometry (LKB, Bromma, Sweden).

Results Effect of 2-stearoylglycerol on the metabolism of triolein emulsion The uptake of radiolabelled emulsion lipid by liver

and spleen 5 min alter injection is compared in Table IA and IB. Table IA is from previous work [7] showing the much lower liver recovery of cholesteryl oleate from triolein emulsion incorporating MS compared with controls (triolein). Table IB shows the results from groups of rats given triolein emulsion after previously receiving a dose of 2-stearoylglycerol in either rat serum albumin, rat whole plasma or in bovine serum albumin solution. In group 1, 60 pg of MS solubilized in 0.5 ml 10% rat albumin solution was injected immediately before 0.5 ml labelled emulsion. As a control, the second group was injected with rat albumin solution only followed by labelled emulsion. In the third group, 45 pg of MS solubilized in 1 ml rat EDTA plasma was injected immediately before 0.5 ml labelled emulsion and a fourth group, the control, received 1 ml plasma without MS followed by emulsion. A fifth group received 180 pg of MS solubilized in 0.5 ml 10% bovine serum albumin immediately before labelled emulsion while in a sixth, no MS was added to the bovine serum albumin solution. Compared with the appropriate controls (plasma or albumin only) the addition of MS did not significantly reduce liver uptake of labelled cholesteryl oleate or triacylglycerol. The prior injection of plasma or al-

TABLE I Effect of monostearoylglycerol on plasma removal and organ uptake of injected triolein emulsions

Triolein emulsions labelled with radioactive triolein and cholesteryl oleate were injected into unanesthetized rats and blood was taken 3 and 5 min later for measurement of radioactivities remaining in the plasma. Lipid radioactivities in the liver and spleen were measured 5 min after injection. In A, the effect of monostearoylglycerol (MS) incorporated into the triolein emulsion before injection is compared with control injections of emulsion only. In B, groups of rats received emulsion injected immediately after intravenous injection of (i) vehicle only, (ii) 60 pg MS in 10% rat serum albumin solution; (iii) vehicle only, (iv) 45 pg MS in rat EDTA plasma; (v) vehicle only; (vi) 180 pg MS in 10% bovine serum albumin (BSA) solution. Results are means f S.E., (n) = number of rats in each experimental group. Radioactivity recovered (W of injected dose) cholesteryl oleate liver A. Effect of MS incorporated into emulsion No MS (6) 53.4k2.66 With MS (9) a 21.1 f 2.99 B. Effect of pre-injected MS Rat albumin only (4) Rat albumin + MS (3) Plasma only (4) Plasma + MS (4) BSA only (8) BSA + MS (4)

43.1 f 5.12 45.0f0.76 d 34.8 f 6.80 26.3 f 1.24 26.5 f 6.33 28.4k5.35

triolein spleen

liver

spleen

1.62kO.35 b 2.61 f 0.30

20.5k1.34 19.0 & 2.59

0.93 f 0.23 b 2.21* 0.28

0.8 f0.13 0.62 ztO.03

13.6f0.33 14.5 f 1.27

0.7 kO.16 0.44*0.04

d 14.2 f 1.57 9.0*2.03

0.64 f 0.03 ’ 0.42 f 0.06

d 13.6k2.31 13.1 f 1.61

0.78 kO.11 0.89 kO.12

1.00f0.09 b 0.58 f0.05 0.98 f 0.16 1.11 kO.98

b Significantly different from the matched control group, P < 0.001, by Student’s t-test. P < 0.01, compared with matched control group. i P c. 0.05, compared with matched control group. Differs from the uninjected controls (row l), P < 0.05, by Student’s t-test.

50 TABLE

II

Removalfromplasma and organ uptake of radiolabelled 2-stearoylglycerol

incorporated

in triolern emulsions

In A, triolein emulsions containing 2-stearoylglycerol (MS, about 1% of total lipid, 35 pg/O.5 ml) and labelled with [t4C]MS and [sH]cholesteryl oleate were injected into unanesthetized rats and blood samples taken at various time intervals. Lipid radioactivities in the organs were measured 12 min after injection. Results are means+S.E. and (n) = 5. In B, 60 pg of MS Iabelled with [14C]MS was solubilized in 0.5 ml 10% bovine serum albumin and injected into unanesthetized rats. The removal from plasma and organ uptake of radioactivity were studied for 5 min. Results are the average of duplicate experiments in two rats. Radioactivity

recovered

(W of injected

dose)

plasma Time (mm): A. MS in emulsion ]14CI

[‘HI B. MS solubilized

l14Cl

3

5

8

Removal from plasma (fractional clearance rate) (nun-‘)

liver

spleen

1.4+0.1

26.5 4 1.8

0.43 f 0.1

0.153_+0.01

21.2k3.7

46.5 f 0.1

0.88 f 0.06

0.083 f 0.01

35.5

0.55

12

5.6 f 0.8

3.3 f 0.5

1.9*0.1

44.2 * 5.0

37.8 f 4.2

28.6 + 4.4

in bovine serum albumin 7.0 3.7

bumin reduced uptake of labelled emulsion lipids below that for emulsion alone (Table IA) and increased the variability, perhaps by hemodynamic effects or plasma volume expansion. The metabolism of radiolabelled 2-stearoylglycerol

The metabolism was investigated of [14C]MS incorporated into a triolein emulsion before injection in rats. The emulsion contained 82% triolein, cholesterol l%, EYPC 13.5% cholesteryl oleate 3% and 0.5-18 of 2stearoylglycerol by weight. The rats were each given 0.5 ml emulsion and killed at 12 min for detailed study of the tissue distribution of MS and the fractional clearance rate of the particle core cholesteryl oleate label, as shown in Table II. After injection, more than 96% of labeled MS disappeared from the plasma within 5 min although removal of cholesteryl oleate was slower. Despite the almost complete disappearance of MS label from plasma within 5 min, about 40% of the original injected cholesteryl oleate remained at this time indicating a rapid dissociation of the two labels. Similarly, 96% of the MS-label disappeared from plasma by 5 min when MS solubilized in bovine serum albumin was injected. Table III shows the distribution of labelled MS and cholesteryl oleate in the plasma lipids at various times after injection. In the injected emulsion 98% of radioactivity was in MS. At 3 min after injection, 71% of the residual label was in free fatty acid, presumably due to the hydrolysis of MS. There was also a steady increase of 14C associated with diacylglycerol and triacylglycerol, apparently by trans-esterification of acyl groups. It will be noted (Table II) that over 98% of injected MS had disappeared within 15 min. In contrast, labeled emulsion cholesteryl oleate was still found as cholesteryl oleate 15 min after injection to the extent of 98% compared with 98.5% in the original emulsion.

When MS (35 pg) was incorporated into a triolein emulsion, most of the label recovered in tissues 12 min after injection was in liver (26.5%) and skeletal muscle (18.5%) with a small amount (2.8%) of the injected dose in adipose tissue and 0.4% in spleen (Table Iv>. In other sampled tissues (kidneys, heart adrenal, thymus, lung and bone marrow) an aggregate of only 2% of injected label was recovered and 1.4% remained in plasma. In contrast, liver uptake of emulsion cholesteryl oleate was TABLE

III

Distribution of ‘“C and ‘H in plasma after injection of triolein emulsion incorporated with 2-stearoylglycerol Triolein emulsion containing 2-stearoylglycerol (MS, about 1% of total lipid, 70 pg/mI) labelled with [i4C]MS and [‘H]cholesteryl oleate was injected into an unanesthetized rat and blood samples collected at various time intervals. Plasma was separated from the blood cells instantly and delivered into a stopped glass tube containing 7.5 ml of chloroform/methanol (2 : 1, v/v) mixture. The lipid extract was plated on thin-layer chromatographic plates, developed in hexane/ether/formic acid (90 : 60 : 4, v/v) and the radioactivity measured in the scraped bands. Injected emulsion

Time (mm):

Distribution of label (% of total radioactivity) plasma 3

5

12

15

t4C-distribution Origin Fatty acid Monoacylglycerol Diacylglycerol Triacylglycerol

0.8 0 98.1 0.5 0.6

2.3 71.0 10.1 9.8 6.8

4.0 56.3 5.2 14.2 20.4

4.7 39.3 16.1 26.4 15.5

6.3 28.0 10.1 28.0 27.5

3H-distribution Origin Fatty acid Monoacylglycerol Cholesterol Cholesteryl oleate

0.2 0 0 1.4 98.5

0.3 1.5 0.1 1.0 97.1

0.4 1.4 0.2 1.0 97.0

0.2 0.7 0 0.5 98.6

0.3 0.9 0.2 0.6 98.0

51 TABLE

IV

Distribution of ‘C and -‘H in various tissues after injection of 2-stearoylglycerol in triolein emulsion or in bovine serum albumin In A, 1 ml each of triolein emulsion incorporating 70 ug MS and labelled with [14C]MS and [3H]cholesteryl oleate was injected intraveneously into unanesthetized rats and in (B) 2 rats each received 60 pg MS labelled with [14C]MS only and solubilized in 0.5 ml 10% bovine serum albumin. After 12 min (A), or 5 min (B), the rats were killed for measurements of radioactivities in various tissues. Lipids were extracted by chloroform/methanol (2: 1, v/v) mixture and the radioactivity measured by liquid scintillation counter. Total body fat was calculated as 0.078 of the body weight and muscle as 0.42 of body weight. Results are mean* SE. and n = number of rats. Organ

Isotopes

uptake

A. MS incorporated ]14Cl

in emulsion 2.85fl.l

13W

3.9 f1.2

B. MS solubilized ]14Cl

of radioactivity

(I% of injected

muscle

fat

(n = 5) 18.5k1.5 5.1 f 0.4

in bovine serum albumin (n = 2) 7.2 k1.1 25.5 f 1.2

dose)

liver

spleen

26.5j11.8

0.4 +0.1

46.5 f 6.7

0.9 *0.1

35.5 +0.6

0.55 Ito.

higher, consistent with the results reported earlier [7], whereas muscle uptake was lower. Injection of MS solubilized in bovine serum albumin showed a tissue distribution at 5 min after injection similar to MS in emulsion, with higher uptake in muscle and liver. Lipolysis of OS0 emulsion The above experiments supported the hypothesis that clearance of remnants from OS0 particles was impeded by the presence of a small concentration of MS on the particles. The lipolysis of OS0 during remnant formation and the release of lipolytic products was further investigated both in vivo and in vitro. Emulsion doubly-labelled with [14C]stearic acid at the glycerol 2-position and with [3H]cholesteryl oleate was injected intravenously in two functionally hepatectomized rats. After 15 min the rats were sacrificed by exsangination. In one rat, remnant particles were isolated by ultracentrifugation. Analysis of the remnants revealed that

TABLE

plasma

1.4 +0.1 22.2

*3.7

3.75 f 0.1

other

total

2.0

51.6+2.8

2.3

80.9 f 2.5

N.D.

72.5

50% of the original 14C radioactivity remained as triacylglycerol and there was very little radioactivity in monoacylglycerol (0.16%). About 3% of the total radioactivity was found in free fatty acid and diacylglycerol and 47% of the 14C was unaccounted for. This was probably explained by the presence of the albumin and other acceptors in vivo, which picked up the lipolytic products as soon as they were produced. In the second rat the whole plasma was analyzed without isolating the remnant particle fraction. At 15 min 90% of the injected cholesteryl oleate label remained in the plasma but only 70% of OS0 label, of which less than 0.5% was present as MS. Doubly-labelled emulsion was incubated for 90 min with post-heparin plasma from a functionally hepatectomized rat and then remnant particles were isolated by ultracentrifugation. The distribution of 14C in remnant lipid showed that 30.3% of the original OS0 remained as triacylglycerol, 7.2% was found as monoacylglycerol

V

Comparison of plasma removal of in vivo remnants with in vitro remnants In vivo remnants were prepared by the intravenous injection of [‘H]cholesteryl oleate-labelled triolein (000) or OS0 emulsions into functionally hepatectomized rats. After circulation for 15 min, blood plasma from the donor rats was injected into recipient rats without further purification. In vitro remnants were made by incubation of emulsion with post-heparin plasma from hepatectomized rats. Results are means f S.E. Remnants

n

Removal from plasma (fractional clearance rate) (mm’)

Organ uptake (W of injected

000 OS0

a 0.200 *0.017 0.065 kO.015

a 78.9 f 2.30 61.9 f 5.32

000 OS0

0.085 f 0.006 0.075 f 0.017

66.2 f 3.06 54.3 * 1.40

liver

of radioactivity dose)

at 12 min

SDleerl

In vivo 14 10

0.81*0.14 1.32kO.13

In vitro 5 5

’ Significantly different from other means by the Newman-Keuls not significantly different.

method

after analysis

of variance,

1.4 *0.53 84.4 *0.40

with P -z 0.001 for each column.

Other means

52 and 2.3% as free fatty acid and diacylglycerol. Relative to [3H]cholesteryl oleate content, remnant particles had 60% of [‘4C]stearoyl label less than emulsion particles, presumably due to binding of lipolytic products by albumin in solution. Labelled monoacylglycerol associated with the remnants was 45-times more than with the in vivo remnants. Remnants prepared in vitro from triolein and OS0 emulsions were injected into conscious rats. Table V shows that fractional clearance rates of [3H]cholesteryl oleate were not significantly different, with values of 0.085 and 0.075 for triolein and OS0 remnants, respectively. In contrast, OS0 remnants prepared in donor hepatectomized rats were cleared from plasma and taken up by the liver more slowly than remnants derived from a triolein emulsion [6]. The hepatic uptake was less, 54% for OS0 in vitro remnants compared with 66% for triolein remnants, while spleen uptake was higher.

*

o! 0

[‘4C]-C0

in holein+ MS

. , . , . , . , . , . , 2

4

6

6

10

12

Time (min) Fig. 1. The effect of 2-stearoylglycerol (MS) on the removal from plasma of radiolabelled cholesteryl oleate (CO) after simultaneous injection of triolein emulsions with and without MS. The cholesteryl oleate was labeled with ‘H in the triolein emulsion and 14C in the triolein emulsions containing MS.

Plasma clearance of triolein and OS0 emulsion injected simultaneously in rats As a further test of the site and mechanism of the effect due to OSO, triolein emulsion labelled with [i4C]cholesteryl oleate and OS0 emulsion with [3H]cholesteryl oleate were mixed in 1: 1 proportion, then the mixture was injected intravenously as described in Methods. Results from eight rats (Table VI) show that the fractional clearance rate of 3H-label tracing the disappearance of OS0 remnant, was consistently slower than that of 14C-label, tracing the triolein remnant. In every case, more 14C-label was taken up by the liver, while less was recovered in the spleen.

Similar effects were observed when MS was incorporated into the triolein emulsion with [‘Hlcholesteryl oleate label and then mixed with [‘4C]cholesteryl oleate labelled triolein emulsion. Clearance (Fig. 1) following the injection of the mixture again showed a slower fractional clearance rate associated with [ 3H]cholesteryl oleate. When the isotopes associated with cholesteryl oleate in the two emulsions were reversed, the clearance of the triolein remnants remained faster, thus eliminating any possible misinterpretation due to isotope effects. Persistence when mixed with other particles showed that slow uptake was a property of OS0 remnants themselves.

TABLE VI Cholesteryl oleate clearance after simultaneous

A mixture intravenously Rats

injections of emulsions

containing [ 3H]cholesteryl oleate-labelled and the removal from plasma measured

OS0 emulsion and [14C]cholesteryl oleate-labelled triolein (000) emulsion was injected over 12 min. The rats were then lolled for measurements of radioactivities in liver and spleen.

Removal from plasma (fractional clearance rate) (mu-‘)

Organ

uptake

of lipid radioactivities

liver

(% of injected

dose)

spleen

OS0

000

OS0

000

OS0

13Hl

f4c1

ISHI

t4c1

13Hl

K:

1 2 3 4 5 6 7 8

0.102 0.106 0.065 0.085 0.127 0.081 0.121 0.081

0.130 0.165 0.083 0.159 0.183 0.177 0.212 0.147

52.1 64.0 40.3 50.3 56.7 55.9 64.8 54.9

62.0 15.6 49.5 69.9 68.1 15.2 77.2 16.5

1.3 0.9 1.4 3.0 2.0 2.1 2.7 1.6

0.8 0.7 1.0 0.7 1.1 0.8 2.2 0.7

Mean S.E.

0.096 0.008

0.157 0.014

54.9 2.75

69.2 3.37

1.9 0.24

1.0 0.18

a Two-tailed

< 0.001

< 0.001

Pa probability

after t-test for paired

observations.

< 0.01

53 Affinity columns and immunoprecipitation Apolipoprotein E associated with in vivo remnants harvested from a mixture of triolein and OS0 emulsion with differently labelled cholesteryl oleate was assessed by an anti-apolipoprotein E affinity column and a heparin-Sepharose affinity column. An emulsion mixture containing triolein particles labelled with [14C]cholesteryl oleate and [ 3H]cholesteryl oleate-labelled OS0 particles was injected intravenously into a hepatectomized rat. Aliquots of remnant-rich plasma were applied to an anti-apolipoprotein E immunoaffinity column and on to a heparin-Sepharose column. In addition, remnants from the same plasma were purified by density gradient ultracentrifugation and then applied to the immunoaffinity column. Preliminary experiments had shown that the amounts loaded were within the binding capacity of the columns. The radioactivity measured in the fractions from the immunoaffinity column (Fig. 2) showed that 10-18s of the remnants bound during elution with saline. Bound remnants were released by elution with thiocyanate. In every case triolein and OS0 remnants were bound to the same extent. Controls were done by applying aliquots of the intact emulsion mixture to the column, with or without apolipoprotein E. In the absence of apolipoprotein E, more that 99% of the emulsion radioactivity was recovered in the unbound fraction. When about 3 pg of apolipoprotein E was added to the emulsion mixture, only 3% of radioactivity was bound by the column; again there was no difference between triolein and OS0 particles. Heparin-Sepharose affinity columns retained less radioactivity than the immunoaffinity columns. With different aliquots of plasma loaded, 2-10% (results not shown) of the radiolabels were detected from the bound fraction with again no difference between OS0 and triolein particles.

0

q

A

B

OSObound OOObound

C

Fig. 2. Remnant binding to anti-apolipoprotein E affinity columns. In A and B, 25 and 50 ~1, respectively, of remnant-rich plasma obtained from donor hepatectomized rats were applied to the column; in C, 25 pl remnants purified by ultracentrifugation from remnant-rich plasma were applied. The unbound remnants were eluted with PBS and the bound remnants with 3 M NaSCN (pH 7.2) followed by PBS. The fraction bound is shown as a percentage of the total (bound+ unbound) radioactivity eluted from the column.

92 kD66 kD-

Alb

45 kD-

AIV

31 kD-

21 kD-

14 kDABC

DE

FG

Fig. 3. The proteins associated with purified remnants and the bound and unbound fractions of the remnants after elution from affinity columns. Proteins were dialyzed against 5 mM ammonium bicarbonate, lyophilized and then dissolved in SDS sample buffer before separation on 15% SDS-PAGE slab gels. Gels were stained with Coomassie blue IWO overnight and destained in 5% acetic acid. A, molecular weight standard; B, triolein remnants; C, OS0 remnants; D unbound fraction of triolein remnants; E, unbound fraction of OS0 remnants; F, bound fraction of triolein remnants; G, bound fraction of OS0 remnants.

The amounts of apolipoprotein E associated with labelled remnants were also examined by the Mancini immunodiffusion technique. 5 ~1 aliquots of purified remnants in various dilutions were pipetted in the wells of an agarose gel containing 5% anti-apolipoprotein E antiserum and incubated in a moist chamber at 4°C for 3 d. The gel was washed free of unprecipitated proteins and salts in distilled water and after staining with Biebrich Scarlet [25] the precipitation rings were cut from the gel and dissolved in 200 ~1 of tissue solubiliser (Amersham) before the addition of scintillant. The radioactivity measured by liquid scintillation counting showed that the proportion of the two isotopes precipitated were similar for OS0 and triolein remnants. The protein associated with the purified triolein and OS0 remnants harvested from hepatectomized rats and the fractions that were bound and unbound by the immunoaffinity column were analyzed by SDS gel electrophoresis. There was also no difference in the overall spectrum of apolipoproteins when the remnants harvested separately from triolein and OS0 emulsions were examined by SDS-gel electrophoresis and isoelectric focusing gels. The original purified OS0 remnants contained less of a protein similar in mobility to serum albumin than the triolein remnants (Fig. 3B, C) [6]. Both the unbound (Fig. 3D, E) and bound fractions (F, G) contained some apolipoprotein E. There were also bands corresponding to apolipoprotein A-IV in the unbound fractions of triolein and OS0 remnants.

54

PH 6.0

C-II

C-Ill-0

C-Ill-3

4.5

4.0 A

B

Fig. 4. Isoelectric focusing patterns of remnants prepared in hepatectomized donors and purified by gradient ultracentrifugation. A, remnants obtained from triolein emulsion and B, remnants obtained from OS0 emulsion.

In order to decrease the contamination of remnants by endogenous VLDL [ll], rats were treated with 4aminopyrazolopyrimidine (30 mg/kg) 18 h before hepatectomy and the injection of triolein and OS0 emulsions. Remnants harvested from plasma recovered 30 min after injection were analyzed for proteins by SDS electrophoresis. The pattern of apolipoproteins associated with triolein and OS0 remnants were similar to those obtained from untreated rats.

Isoelectric focusing The isoelectric focusing

pattern of apolipoproteins associated with in vivo triolein and OS0 remnants was examined by isoelectric focusing on a slab gel containing pH 4-6 gradient. As shown Fig. 4, there were no differences in the amounts and isoforms of apolipoproteins E or C in the two remnants. Quantification by densitometric scanning failed to show any differences between the two remnants.

Table IA showed that when MS was incorporated into the triolein emulsion before injection, recovery of cholesteryl oleate label in liver was less than 50% of the control triolein emulsion without MS, but triacylglycerol label in the liver was unaffected, as described previously [7], In an attempt to establish whether the effect of MS was exerted directly on the emulsion or indirectly after dissociation of MS from the emulsion, MS was injected prior to the emulsion. As shown in Table IB, interpretation was complicated because con-

trol injection of either rat plasma or of bovine serum albumin significantly decreased the amount of cholesteryl oleate radioactivity recovered in the liver, for reasons so far not known. However, MS solubilized in these vehicles had no significant effect in further decreasing uptake by the liver. Therefore it seems that the effect of MS was exerted directly, due to its association with the emulsion. The delayed remnant clearance observed previously, indicated by the slow disappearance of labelled cholesteryl oleate, was confirmed by simultaneous injection of a mixture of triolein emulsion labelled with [14C]cholesteryl oleate and OS0 emulsion labelled with [ 3H]cholesteryl oleate. Mixtures of triolein emulsion and MS incorporated in a triolein emulsion, each labelled with either r4C or 3H-labelled cholesteryl oleate were also studied. The results were all consistent with a slower clearance of remnants when particles contained OS0 or MS. The fractional clearance rates of OS0 emulsions or triolein emulsions incorporating MS were found to be consistently slower than those of the control triolein emulsion. To see how the mode of introduction to the circulation affected the handling of the MS itself, the fate of 2-[1-‘4C]stearoylglycerol ([14C]MS) was compared after injection in bovine serum albumin or in triolein emulsion particles. The results in Table II shows that in either case nearly all the labeled MS was cleared from the plasma within a few minutes. By 3 min about 93-94% of the MS had left the plasma. The fractional clearance rate of the remaining small amount in the plasma at subsequent time intervals gave a value of 0.15 mm-‘. The bulk of the MS was probably immediately transferred to albumin and subsequently removed from circulation. Of the small amount remaining in the plasma, the distribution of radioactivity in lipids at different time points shown in Table III suggest that MS in the emulsion particles was readily hydrolyzed and the acyl group trans-esterified with plasma glycerides. In contrast with the redistribution of the labelled acyl groups of MS, the [3H]cholesteryl oleate was recovered almost quantitatively, indicating its stability as a label of the core of the particles during remnant formation and uptake. The fractional clearance rates of [3H]cholesteryl oleate (Table III) were comparable to previous data [7]. The use of [3H]cholesteryl oleate label emphasizes the divergence in the handling of MS and particle remnants. For example after 12 min, as Table IV shows, 46.5% of cholesteryl oleate label were recovered in the liver compared with about 27% of MS. On the other hand about 5.1% of the cholesteryl oleate label was present in total body muscle (possibly trapped with capillaries) whereas 3-times more MS label was extracted from the same muscle samples. The pattern of

55 recovery was similar in the presence or absence of emulsion. This suggests that uptake of MS label by tissue was a separate process from uptake of remnant particles. The data suggest that the effect of MS whether added to the emulsion particle or generated by lipolysis reflected changes on the surface of the particle remnant. The amphipathic nature of monoacylglycerols would favor their location at the surface of an emulsion particle. The MS may rapidly transfer from the particle surface via the aqueous phase of plasma into the tissues, explaining the low recoveries obtained in the plasma compartment. The effect of OS0 in emulsion was evident in the presence of less than 1% (total lipid) MS. If expressed as a mole fraction of lipids in the remnant surface, 5-15s of molecules could be MS. Analysis of exogenous particle remnants was made difficult by the presence of contaminating endogenous particles of similar composition and size and by possible artefacts of isolation procedures. To avoid some of these problems chylomicron-like particles were prepared in which the core cholesteryl oleate was labelled with 3H and in which all the triacylglycerol was 1,3-dioleoyl-2-[1I4 Clstearoylglycerol. These doubly labelled particles were then used to study remnant formation in vitro and in vivo and to study the fate of the doubly labelled remnants after intravenous injection. OS0 remnants were cleared more slowly from plasma and taken up less by liver than triolein remnants [6]. The differences did not achieve statistical significance for remnants prepared in vitro, despite the higher MS content of in vitro remnants. Triolein remnants made in vitro were cleared more slowly in recipient rats than those prepared in donor hepatectomized rats (fractional clearance rates, 0.085 compared with 0.2, Table V). On the other hand OS0 remnants whether made in vitro or in vivo were cleared somewhat similarly by the recipient rats (0.075 compared with 0.065). Possibly, some of the apolipoproteins dissociate from the remnant particles during ultracentrifugation [26], hence making them less effective ligands for the liver remnant receptors. Previous studies have shown that the lipid compositions of remnants derived from triolein and OS0 emulsions were similar [6]. In the present study results from affinity columns (Fig. 2) and immunodiffusion suggested that there were similar amounts of apolipoprotein E in both the triolein and OS0 remnants. SDS-gel electrophoresis and isoelectric focusing gel showed that the general pattern of the apolipoproteins associated with the two remnants were also similar (Figs. 3 and 4). In the SDS-gel electrophoresis apolipoprotein E was detected in all fractions, including those not bound by the antibody affinity column (Fig. 3, F, G). Binding of apolipoprotein E to lipoproteins may cause changes in conformation preventing recognition tissue

by the antibody [18]. Evans et al [27] also found that not all apolipoprotein E-containing lipoproteins were bound by heparin-Sepharose columns. In conclusion, there is still no clear explanation of the slow clearance from the circulation of remnants derived from chylomicron-like particles in which all or most of the triacylglycerol is OSO. The presence of small smount of MS on remnants is implicated, but how this affects hepatic uptake is unclear. Remnant apolipoprotein spectra and binding to affinity columns are not demonstrably altered. The present study has eliminated the possibility of effects exerted by MS at sites other than the lipid particle surface. Effects of small amounts of MS on the conformation of apolipoproteins present on the surface remain to be explored. Acknowledgements This work is supported by the Arnold Yeldham and Mary Raine Medical Research Foundation and a grant from the National Health and Medical Research Council. BCM is a Raine Postdoctoral Fellow. We thank Dr. R.G.H. Morgan for providing the rat pancreatic juice, and M. Callow and M. Kenrick for expert technical assistance. References 1 Miller, K.W. and Small, D.M. (1982) J. Colloid Interface. Sci. 89, 466-478. 2 Redgrave, T.G. and Maranhao, R.C. (1985) B&him. Biophys. Acta 835, 104-112. 3 Redgrave, T.G., Kodali, D.R. and Small, D.M. (1988) J. Biol. Chem. 63, 5118-5123. 4 Redgrave, T.G., Vassiliou, G.G. and Callow, M.J. (1987) B&him. Biophys. Acta 921, 154-157. 5 Lenzo, N.P, Martins, I., Mortimer, B-C. and Redgrave, T.G. (1988) B&him. Biophys. Acta 960, 111-118. 6 Mortimer, B-C., Simmonds, W.J., Joll, C.A., Stick, R.V. and Redgrave, T.G. (1988) J. Lipid Res. 29, 713-720. 7 Mortimer, B-C, Simmonds, W.J., Joll, C.A., Stick, R.V. and Redgrave, T.G. (1989) Biochim. Biophys. Acta, 1002, 359-364. 8 Scow, R.O., Desnuelle, P. and Verger, R. (1979) J. Biol. Chem. 254, 6456-6463. 9 Bentley, P.H. and McCrae, W. (1970) J. Org. Chem. 35.2082-2083. 10 Kodali, D.R., Atkinson, D., Redgrave, T.G. and Small, D.M. (1984) J. Am. Oil Chem. Sot. 61,1078-1084. 11 Shiff, T.S., Roheim, P.S. and Eder, H.A. (1971) J. Lipid Res. 12, 596-603. 12 Lenich, C.M. and Ross, A.C. (1987) J. Lipid Res. 28, 183-194. 13 Holmquist, L. and Carlson, K. (1977) B&him. Biophys. Acta, 493,400~409. 14 Laemmli, U.K. (1970) Nature 227, 680-685. 15 Hager, D.A. and Burgess, R.R. (1980) Anal. B&hem., 109.76-86. 16 Brysk, M.M., Barlow, E., Bell T., Rajaraman, S. and Stach, R.W. (1988) Preparative B&hem. 18, 217-225. 17 Michael, S.E. (1962) B&hem. J. 82, 212-218. 18 Krul, E.S., Tiianen, J. and Schonfeld, G. (1988) J. Lipid Res. 29, 1309-1325. 19 Wiesweiller, P. (1988) J. Chromatogr. 425, 169-174.

56 20 Warnick, G.R., Mayfie~d, J., Albers, J. and Hazard, W.R. (1979) Clin. Chem. 25, 279-284. 21 Bartlett, G.R. (1959) J. Biol. Chem. 234, 466-468. 22 Carlson, L.A. (1963) J. Atheroscler. Res. 3, 334-336. 23 Zlatkis, A. and Zak, B. (1969) Anal. B&hem. 29, 143-148. 24 Tajima, S., Yokoyama, S., and Yamamoto, A. (1983) J. Biol. Chem. 258, 10073-10082.

25 Crowlf, A.J. and Cline, L.J. (1977) J. Immun Methods 17, 379-381. 26 Fainaru. M., Have& R.J. and Imaizumi, H. (1977) Biochim. Biophys. Acta 490, 144-145. 27 Evans, A.J., Huff, M.W. and Wolfe. B.M. (1989) J. Lipid Res. 30, 1691-1701.

The effect of monostearoylglycerol on the metabolism of chylomicron-like lipid emulsions injected intravenously in rats.

In rats, remnant particles derived from chylomicron-like emulsions containing 1,3-dioleoyl-2-stearoylglycerol (OSO) are removed from plasma more slowl...
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