Báo cáo Y học: Structural and compositional changes in very low density lipoprotein triacylglycerols during basal lipolysis potx

Structural and compositional changes in very low density lipoprotein triacylglycerols during basal lipolysis Jyrki J. A ˚ gren 1,2 , Amir Ravandi 1 , Arnis Kuksis 1 and George Steiner 3 1 Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada; 2 Department of Physiology, University of Kuopio, Finland; 3 Department of Medicine and Physiology, The Toronto Hospital (General Division), Toronto, Ontario, Canada Triacylglycerols secreted by liver and carried by very low density lipoprotein (VLDL) are hydrolysed in circulation by lipoprotein and hepatic lipases. These enzymes have been shown to have positional and fatty acid specificity in vitro.If there were specificity in basal lipolysis in vivo, triacylglycerol compositions ofcirculating and newly secreted VLDL would be different. To study this we compared the composition of normal fasting VLDL triacylglycerol of Wistar rats to that obtained after blocking lipolysis by Triton WR1339, which increased plasma VLDL triacylglycerol concentration about 4.7-fold in 2 h. Analyses of molecular species of sn-1,2- and sn-2,3-diacylglycerol moieties and stereospecific triacylglyc- erol analysis revealed major differences between the groups in the VLDL triacylglycerol composition. In nontreated rats, the proportion of 16:0 was higher and that of 18:2n-6 lower in the sn-1 position. The proportion of 14:0 was lower in all positions and that of 18:0 was lower in the sn-1 and sn-3 positions in nontreated rats whereas the proportions of 20:4n-6, 20:5n-3, 22:5n-3 and 22:6n-3 were higher in the sn-1 and lower in the sn-2 position. These results suggest that the fatty acid of the sn-1 position is the most decisive factor in determining the sensitivity for hydrolysis of the triacylglyc- erol. In addition, triacylglycerol species with highly unsat- urated fatty acids in the sn-2 position also favoured hydrolysis. The in vivo substrate specificity followed only partly that obtained in in vitro studies indicating that the nature of molecular association of fatty acids in natural triacylglycerol affects its susceptibility to lipolysis. To con- clude, our results indicate that preferential basal lipolysis leads to major structural differences between circulating and newly secreted VLDL triacylglycerol. These differences extend beyond those anticipated from analysis of total fatty acids and constitute a previously unrecognized feature of VLDL triacylglycerol metabolism. Keywords: diacylglycerols; enantiomers; hydrolysis; stereo- specific analysis; reverse isomers. 1 Very low density lipoprotein (VLDL) secreted from liver is the major carrier of triacylglycerols in the fasting state and its assembly, secretion and hydrolysis have been extensively studied [1–3]. It has been generally assumed that the triacylglycerol composition of circulating VLDL resembles that of the VLDL newly secreted by the liver, although very few studies have examined the effects of basal lipolysis on circulating VLDL. VLDL triacylglycerols are hydrolysed by lipoprotein and hepatic lipases [4]. These enzymes have been shown to have positional and fatty acid specificity in vitro [5,6]. However, most studies concerning substrate specificity have been performed with human or bovine milk lipoprotein lipase using chylomicrons or synthetic triacylglycerols, including alkyl ethers, as substrates [5–8]. The properties of human and bovine milk lipoprotein lipase may differ [9] as may the properties of milk and endothelial lipoprotein lipase [10]. Because the biosynthesis of intestinal chylomicron and hepatic triacylglycerols proceed along different routes [11], structurally dissimilar triacylglycerols would have been subject to endogenous lipolysis during clearance of post- prandial chylomicron triacylglycerols and of VLDL triacyl- glycerols further complicating the interpretation of earlier results. Previous studies [12–14] have shown that Triton WR1339 (a nonionic detergent) blocks lipolysis by inhibition of lipoprotein and hepatic lipases, which leads to accumulation of VLDL triacylglycerols. In one study [15], the fatty acid composition of serum lipids was shown to differ between control serum and serum collected 6 h after Triton injection. However, only the major fatty acids were measured in serum total triacylglycerols, while the fatty acids of liver triacylglycerols were not determined. As Triton WR1339 has been shown to disturb lysosomal lipolysis in liver [16], it is possible that prolonged treatment had affected the fatty acid composition directed by the liver to the VLDL assembly. Short-term injections of Triton WR1339 have given no indication of ill effects [17–19]. The present study was carried out to confirm in vivo the nonrandomness of the basal lipolysis demonstrated in vitro and to establish the extent to which the newly secreted VLDL triacylglycerol is modified during circulation. We limited treatment with Triton WR1339 to 2 h, which did not induce changes in the fatty acid composition of liver triacylglycerol. The results showed significant differences in the VLDL triacylglycerol composition between control and Triton-treated animals. Correspondence to J. A ˚ gren, Department of Physiology, University of Kuopio, P.O.B. 1627, FIN70211 Kuopio, Finland. Fax: +358 17 163112, Tel.: +358 17 163091, E-mail: Jyrki.Agren@uku.fi Abbreviations: VLDL, very low density lipoprotein; NEU, naphthyl ethyl urethane. (Received 18 September 2002, accepted 31 October 2002) Eur. J. Biochem. 269, 6223–6232 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03341.x MATERIALS AND METHODS Animal procedures 2 Male Wistar rats weighing 300–350 g and maintained on standard chow and 12-h light/dark cycle (07.00, 19.00 hours) were used (n ¼ 10). In the experimental day food was removed at 08.00 hours and rats were anesthetized with Somnatol (50 mgÆkg )1 ) at 12.00 hours. A cannula was inserted into femoral vein and Triton WR1339 (600 mgÆkg )1 ) or saline was injected (12.30–13.00 hours). Two hours after injection blood was drawn and rats were killed by heart puncture. The blood samples were taken into 5% EDTA and plasma was immediately separated by centrifu- gation. Plasma was overlaid with a NaCl solution (d ¼ 1.006 gÆmL )1 ) and VLDL was separated by ultracen- trifugation at 37 000 r.p.m. in a 70.1 Ti rotor (Beckman) for 16 h at 16 °C. Livers were removed and stored at )70 °C. All experiments were performed under protocols approved by the Animal Care Committee and the University of Toronto. Separation of triacylglycerols Lipids from VLDL and liver samples were extracted with chloroform/methanol (2:1, v/v) [20]. Triacylglycerols were separated by TLC on silica gel H plates using heptane/ isopropyl ether/acetic acid (60 : 40 : 4, v/v/v) as the devel- oping solvent. The triacylglycerol band was scraped off, extracted in chloroform/methanol (2 : 1) and stored in chloroform at )20 °C. Analysis of fatty acid methyl esters Triacylglycerols were subjected to acidic methanolysis using 6% H 2 SO 4 in methanol at 80 °C for 2 h. The fatty acid methyl esters were extracted with hexane and analysed by capillary gas chromatograph (HP 5880, Hewlett-Packard) using a 15-m SP-2380 column (0.25 mm i.d., 0.20 lmfilm thickness). Plasma VLDL neutral lipid profile Neutral lipids were separated from total lipid extract using a silica Sep-Pak column (Waters) [21] and their profile was determined with capillary GC [22]. Samples were silylated with pyridine/trimethylchlorosilane (1 : 1) and extracted with hexane before injection into 8 M HP-5 column (Hewlett-Packard; 0.30 mm i.d., 0.25 lm film thickness). The oven temperature was programmed from 40 to 150 °C at 30 °CÆmin )1 and to 340 °Cat10°CÆmin )1 . Preparation of diacylglycerols and their naphthyl ethyl urethane (NEU) derivatives Plasma VLDL and liver triacylglycerols were partially deacylated to sn-1,2, sn-2,3 and sn-1,3 diacylglycerols by Grignard reaction and products were immediately deriva- tized [23,24]. Diacylglycerols were dissolved in dry toluene (0.3 mL) and (R)-(–)-1-(1-naphthyl)ethyl isocyanate (10 lL) and 4-pyrrolidinopyrridine (4 mg) were added. The mixture was heated at 50 °C overnight. After evaporation of solvents with nitrogen stream the reaction products were dissolved in methanol/water (95 : 5) and applied to Sep-Pak C18 column (Waters), which had been solvated with the same solvent. Further 15 mL of this solvent was passed through the column and NEU derivatives were then eluted with acetone (10 mL). HPLC/ESI/MS of diacylglycerols The sn-1,2 and sn-2,3-diacylglycerols were separated [24] and analysed as diastereomeric NEU derivatives with a Waters 550 HPLC connected through a Waters 990 photodiode array detector to a Hewlett-Packard 5989A quadrupole mass spectrometer equipped with a nebulizer- assisted electrospray interface. Two normal phase silica gel columns (Supelcosil LC-Si, 5 lm, 25 cm · 4.6 mm i.d., Supelco Inc., Bellefonte, PA, USA) in series were used and 0.37% isopropanol in hexane was used as a mobile phase at a flow rate of 0.7 mLÆmin )1 . Positive chemical ionization was obtained by postcolumn addition of chloroform/ methanol/30% ammonium hydroxide (75 : 24.5 : 0.5, v/v) at 0.6 mLÆmin )1 . The capillary exit voltage was 220 V and the mass range scanned was m/z 500–720. The relative proportions of diacylglycerol species were calculated from the areas of the single ion plots obtained from the mass spectra. NEU derivatives of VLDL diacylglycerols were collected after HPLC separation. A sufficient amount of sample for fatty acid analyses was obtained from three Triton-treated and two nontreated rats. Almost complete separation of sn-1,2 and sn-2,3-diacyl- glycerol was obtained as their R-forms of NEU derivatives. However, a part of 38 and 40 acyl carbon sn-1,2-diacyl- glycerol eluted concurrently with sn-2,3-diacylglycerol. For example, 16:1–22:6 sn-1,2-diacylglycerol was separated into two peaks (indicating a separation on the basis fatty acid location in diacylglycerol), and the first one eluted with the sn-2,3-diacylglycerol fraction. There was, however, overlap with the corresponding sn-2,3-diacylglycerol species only in few cases and these values were corrected using the results from the runs of (S)-form NEU derivatives. Also sn-1,3 diacylglycerols were separated but they were not used for calculations because this fraction has been reported to be readily contaminated by isomerization [25]. Stereospecific analysis and calculations The stereospecific positional distribution of the fatty acids was determined by calculation from the fatty acid compo- sition of total triacylglycerols and the sn-1,2- and sn-2,3- diacylglycerols recovered from a HPLC separation as described by Yang and Kuksis [26]. The molecular associ- ation of the fatty acids and reverse isomer content of the triacylglycerol and the derived sn-1,2- and sn-2,3-diacyl- glycerols were determined by calculation on the basis of the knowledge of the fatty acid composition of the sn-1, sn-2 and sn-3 positions of the acylglycerols, assuming 1-random, 2-random and 3-random distribution [27]. The molecular associations give the exact pairs of fatty acids in individual diacylglycerol and the exact triplets of fatty acids in individual triacylglycerol. Statistics The values have been expressed as mean ± SD. The Mann–Whitney U-test was used for comparisons of groups. 6224 J. J. A ˚ gren et al. (Eur. J. Biochem. 269) Ó FEBS 2002 RESULTS Effect of Triton WR1339 on plasma and VLDL triacyl- glycerol levels Plasma and VLDL triacylglycerol concentrations were 4.8 ± 0.8 and 4.4 ± 0.9 mmolÆL )1 in Triton-treated and 1.4 ± 0.2 and 0.9 ± 0.2 mmolÆL )1 in nontreated rats, respectively. This shows that blocking lipolysis by Triton WR1339 increased plasma and VLDL triacylglycerol levels about 3.5 and 4.7 times in 2 h, respectively. On the presumption that the VLDL triacylglycerol concentration in Triton-treated rats was the same as in nontreated rats before injection it could be estimated that VLDL contained at least 80% unmodified triacylglycerol in the Triton-treated group. This percentage could be also somewhat higher if there has been any removal of VLDL particles during the treatment. The very small amount of VLDL diacylglycerol (0.2 ± 0.0% of neutral lipids) in Triton-treated rats indi- cate also a minor contribution of modified VLDL. Prelimi- nary studies showed linear increase of plasma triacylglycerol concentration at least for 4 h after Triton injection. However, 4-h treatment was found to affect the fatty acid composition of liver triacylglycerols, and a similar tendency was observed in overnight fasted rats with 2-h Triton treatment. To avoid this effect on liver triacylgly- cerols, and possibly on secreted triacylglycerol composition, a shorter food deprivation period was used in the present study. The increase of VLDL triacylglycerol concentration was identical to that seen in overnight fasted rats and also the neutral lipid profiles of VLDL fractions were similar indicating that chylomicrons did not contribute to the VLDL fraction. Neutral lipid profile of VLDL There were statistically significant differences in the neutral lipid profile between Triton-treated and nontreated rats (Table 1). The relative amounts of free cholesterol, choles- terol esters and diacylglycerols were greater and those of total triacylglycerols were smaller in nontreated rats. In addition, the proportions of 50 and 54 acyl carbon triacylglycerols were lower and those of 56 and 58 acyl carbon triacylglycerols were higher in nontreated compared with Triton-treated rats. Fatty acid composition of VLDL and liver The proportions of saturated fatty acids in the total VLDL triacylglycerols were lower in nontreated rats, with the exception of slightly higher proportion of 16:0 (Table 2). The levels of 18:1n-9, 18:2n-6 and 20:4n-6 were similar in both groups whereas the proportions of 16:1n-7, 18:1n-7 and 18:3n-3 were lower, and those of 20:3n-6, 20:5n-3, 22:5n-3 and 22:6n-3 were higher in nontreated rats. There were no significant differences in the liver triacylglycerol fatty acid composition between the groups although two rats in the Triton-treated group with lower proportions of polyunsaturated fatty acids caused some differences in the mean values. These rats did not differ, however, from the other Triton-treated rats in their VLDL fatty acid compo- sition. Molecular species of sn -1,2 and sn -2,3-diacylglycerols There were statistically significant differences between the groups in about half of the measured sn-1,2- and sn-2,3- diacylglycerol species (Table 3). In nontreated rats there was less 16:0–16:1 and 16:1–16:1 in both sn-1,2- and sn-2,3- diacylglycerols and less 18:0–18:1 and 18:1–18:2 in the sn-1,2-diacylglycerols and 16:0–18:2 and 16:1–18:2 in the sn-2,3-diacylglycerols whereas the proportion of 16:0–18:1 was higher in the sn-1,2-diacylglycerols. In addition, the proportions of 16:0–20:4 and 16:0–20:5 were lower and those of diacylglycerol species with a combination of 18- and 22-acyl carbon fatty acids were higher in both sn-1,2- and sn-2,3-diacylglycerols in nontreated rats. A small amount of 20:5–22:6 was also found in the sn-2,3-diacyl- glycerols and its level was higher in nontreated rats. There were not significant differences in the sn-1,2 and sn-2,3-diacylglycerol composition of liver triacylglycerols between the groups. Compared with VLDL the proportions of 16:0–16:0, 16:0–18:0 and 16:0–18:1 were higher and those of 16:0–18:2, 16:0–20:4 and 16:0–20:5 were lower in the liver sn-1,2-diacylglycerol (Fig. 1A). In the sn-2,3-diacylglycerols the proportions of 16:0–18:0, 16:0–18:1 and 18:0–18:1 were higher and those of 16:0–20:4 and most species containing 22:6n-3 were lower in the liver than in the VLDL triacylglycerols (Fig. 1B). Positional distribution of VLDL triacylglycerol fatty acids Positional analyses of VLDL triacylglycerol fatty acids showed that the lower proportions of most saturated fatty acids (14:0, 15:0, 17:0 and 18:0) in nontreated rats were mainly caused by their lower proportions in the positions sn-1 and sn-3 (Table 4). In contrast, the proportion of major saturated fatty acid, 16:0, was higher in the sn-1 position in nontreated rats. The proportions of major 20- and 22-acyl carbon fatty acids were higher in the sn-1 position and lower in the sn-2 position in nontreated rats. Table 1. Composition of plasma VLDL neutral lipids in Triton-treated and nontreated rats. Neutral Lipids were separated from total lipid extracts by solid phase extraction and analysed by GLC. Results are expressed as mass percentages and are mean ± SD for each group of five rats. Triton-treated Nontreated Cholesterol 4.5 ± 0.4 4.0 ± 0.3 Cholesterol esters 2.2 ± 0.4 3.6 ± 0.5 c C16 0.6 ± 0.1 1.0 ± 0.1 b C18 1.6 ± 0.2 2.6 ± 0.3 c Diacylglycerols 0.2 ± 0.0 1.2 ± 0.4 c Triacylglycerols 93.1 ± 0.5 91.1 ± 0.7 c C48 (+ C49) 3.2 ± 0.8 2.9 ± 0.5 C50 (+ C51) 13.2 ± 1.3 11.0 ± 0.6 b C52 33.8 ± 1.3 33.5 ± 1.2 C54 20.6 ± 0.7 18.4 ± 0.7 c C56 17.2 ± 0.9 19.2 ± 0.9 b C58 5.1 ± 0.5 6.1 ± 0.5 c Statistical comparison between the groups: a P < 0.05; b P < 0.02; c P < 0.01. Ó FEBS 2002 Modification of VLDL triacylglycerols by lipolysis (Eur. J. Biochem. 269) 6225 The proportions of some major VLDL triacylglycerol species and their reverse isomers, calculated on the basis of stereospecific positional distribution of fatty acids, are presented in Table 5. In nontreated rats, the proportions of most triacylglycerol species with 50 or fewer acyl carbons or containing 18:0 were lower. Otherwise the fatty acid in the sn-1 position seemed to have greatest influence on hydro- lysis. The proportions of triacylglycerol species with 18:2 were mostly lower, and those with 16:0 were higher in nontreated rats whereas there were not much difference in the species with 18:1 in the sn-1 position. In addition, the proportions of triacylglycerol species with 20:4, 20:5, 22:5 or 22:6 in the sn-1 position were higher in nontreated rats. DISCUSSION Effect of Triton WR1339 on lipoprotein metabolism The effect of Triton WR1339 on VLDL secretion rates has been extensively investigated [17] and the developed meth- odology applied in recent studies [18,19] assessing the effects of genetic manipulation of the secretion of apoB-48 and apoB-100 containing VLDL. In the present study we investigated the effect of basal lipolysis on plasma VLDL triacylglycerols by comparing the composition of VLDL triacylglycerols under normal physiological conditions to that obtained after Triton WR1339 treatment. In addition to blocking triacylglycerol lipolysis by inhibition of lipo- protein and hepatic lipases [12,14], Triton WR1339 has also other effects on plasma and liver lipid metabolism. It has been reported to incorporate preferentially into the high density lipoprotein particles displacing especially apolipo- protein A-I [28]. Apolipoprotein B or lipids were not displaced from low density lipoprotein (LDL) particles indicating that neither should there be marked association of Triton to newly secreted VLDL particles containing mainly apolipoprotein B. Triton accumulates also in liver lysosomes disturbing the lysosomal hydrolysis of VLDL [16]. This may change the fatty acid composition of secreted VLDL due to possible depletion of cellular triacylglycerol stores and increased use of phospholipids as a source of triacylglycerol fatty acids [29] during prolonged Triton treatment. To minimize these effects we used only a 2 h treatment and were able to obtain VLDL fraction with 4.7 times greater triacylglycerols concentration than in non- treated rats. Although the possibility of unknown side- effects of Triton treatment cannot be ruled out, none of the known effects of Triton WR1339 gives reason to suppose that it had affected the composition of accumulated VLDL triacylglycerols in circulation. Furthermore, similar fatty acid compositions of liver triacylglycerols and phospholi- pids (data not shown) in both groups indicate that the composition of secreted VLDL triacylglycerols was not affected by Triton WR1339 during the treatment. The effect of lipolysis on plasma VLDL neutral lipids in nontreated rats was evident from a reduced triacylglycerol and increased diacylglycerol proportion. The higher pro- portion of cholesterol esters in nontreated rats could also result from the removal of triacylglycerols. However, the proportion of free cholesterol was not higher in nontreated rats indicating that the ratio cholesterol ester : cholesterol in VLDL is modified in the circulation and that Triton treatment affects these events also. It was shown earlier that lecithin cholestrol acyltransferase activity is decreased as Triton WR1339 displaces apolipoprotein A-I in the high density lipoprotein particles [28] but its other possible effects on cholesterol metabolism or transfer are unknown. The greater proportion of 54 and 56 acyl carbon triacylglycerols, Table 2. Fatty acid composition of VLDL and liver triacylglycerols. Fatty acid methyl esters were prepared from VLDL and liver triacylglycerols by acidic methanolysis and analysed by GLC. Results are expressed as mole percentages and are mean ± SD for five rats per group. Triton-treated VLDL Liver Triton-treated Nontreated Triton-treated Nontreated 14:0 1.5 ± 0.3 1.0 ± 0.1 c 1.4 ± 0.2 1.3 ± 0.2 15:0 0.8 ± 0.1 0.6 ± 0.1 c 0.8 ± 0.2 0.7 ± 0.1 16:0 27.0 ± 0.4 27.8 ± 0.4 a 31.2 ± 2.8 29.5 ± 1.7 17:0 0.5 ± 0.1 0.4 ± 0.0 c 0.6 ± 0.1 0.5 ± 0.1 18:0 3.5 ± 0.7 2.5 ± 0.3 a 3.3 ± 0.6 3.6 ± 0.7 16:1n-9 0.6 ± 0.1 0.5 ± 0.1 0.7 ± 0.1 0.7 ± 0.2 16:1n-7 2.8 ± 0.3 2.0 ± 0.5 a 2.8 ± 0.8 1.8 ± 0.6 18:1n-9 24.8 ± 1.0 24.9 ± 0.7 26.1 ± 1.6 23.9 ± 1.5 18:1n-7 3.7 ± 0.1 3.2 ± 0.3 c 4.7 ± 0.4 3.7 ± 0.8 20:1n-9 + 11 0.4 ± 0.1 0.4 ± 0.1 0.4 ± 0.1 0.3 ± 0.1 18:2n-6 21.6 ± 1.0 21.8 ± 1.3 17.6 ± 1.7 20.5 ± 2.1 18:3n-3 1.3 ± 0.1 1.0 ± 0.1 c 0.8 ± 0.1 0.9 ± 0.2 20:2n-6 0.3 ± 0.1 0.4 ± 0.0 0.4 ± 0.1 0.4 ± 0.0 20:3n-6 0.4 ± 0.1 0.7 ± 0.2 a 0.4 ± 0.1 0.5 ± 0.1 20:4n-6 1.7 ± 0.1 1.7 ± 0.2 1.6 ± 0.2 2.0 ± 0.4 20:5n-3 1.5 ± 0.1 1.7 ± 0.1 a 0.8 ± 0.2 1.2 ± 0.4 22:4n-6 0.4 ± 0.1 0.5 ± 0.2 0.4 ± 0.1 0.5 ± 0.1 22:5n-3 1.8 ± 0.2 2.2 ± 0.3 a 1.6 ± 0.5 2.2 ± 0.6 22:6n-3 5.3 ± 0.4 6.7 ± 1.1 b 4.3 ± 1.5 5.6 ± 0.8 Statistical comparison between the groups: a P < 0.05; b P < 0.02; c P < 0.01. 6226 J. J. A ˚ gren et al. (Eur. J. Biochem. 269) Ó FEBS 2002 containing most of the 20- and 22-carbon fatty acids in plasma VLDL [30] of nontreated rats corresponds with the differences observed in the fatty acid and enantiomeric diacylglycerol composition. Specificity of endogenous lipases Lipoprotein and hepatic lipases hydrolyse VLDL triacyl- glycerols and they have been shown to have positional specificity for the sn-1 ester of triacylglycerol [5,31,32]. Using purified human milk lipoprotein lipase and a synthetic triacylglycerol mixture [5], the relative order of fatty acid release was 18:1 > 18:3 > 18:2 > 14:0 > 16 > 0 > 18:0. The observation of preferential hydrolysis of 18:2n-6 over 16:0 in the sn-1 position as well as the lower proportions of 14:0 and 18:3n-3 in the VLDL triacylglycerol of nontreated rats are in accordance with these results. On the other hand, the proportion of 18:0, which would have been expected to be most resistant to hydrolysis, was lower in nontreated rats. Furthermore, no preference for 18:1n-9 was seen in the present study. This discrepancy could be due to the fact that in the in vitro studies monoacid triacylglycerols were used, e.g. tristearoyl- and trioleoylglycerol, whereas natural triacylglycerols would have a more varied fatty acid distribution. The results obtained indicate that the accessi- bility of triacylglycerol to lipase is determined by both the nature of the fatty acid and its position and association with other fatty acids in the triacylglycerol molecule. In keeping with the above discussion, greater differences were found between the treated and nontreated groups of Table 3. The proportions of sn-1,2 and sn-2,3 diacylglycerols from plasma VLDL triacylglycerols. NEU derivatives of diacylglycerols were prepared after partial deacylation of plasma VLDL triacylglycerols and they were separated and analysed by HPLC/MS as described in Methods. Results are expressed as mean ± SD for five rats per group. C:DB Diacylglycerol species sn-1,2 sn-2,3 Triton-treated Nontreated Triton-treated Nontreated 30:0 14:0–16:0 0.8 ± 0.3 0.4 ± 0.2 a 0.3 ± 0.1 0.3 ± 0.1 30:1 14:0–16:1 0.6 ± 0.2 0.4 ± 0.1 0.3 ± 0.1 0.2 ± 0.1 32:0 16:0–16:0 4.8 ± 0.9 4.6 ± 0.3 2.5 ± 0.5 2.1 ± 0.4 32:1 16:0–16:1 4.9 ± 0.5 3.6 ± 0.2 c 2.5 ± 0.3 2.0 ± 0.3 a 32:2 16:1–16:1 2.3 ± 0.3 1.3 ± 0.1 c 1.1 ± 0.2 0.7 ± 0.1 b 33:0 16:0–17:0 + 15:0–18:0 0.6 ± 0.2 0.6 ± 0.1 0.3 ± 0.1 0.3 ± 0.1 33:1 16:1–17:0 + 15:0–18:1 1.4 ± 0.2 1.1 ± 0.1 a 0.6 ± 0.1 0.5 ± 0.1 34:0 16:0–18:0 2.2 ± 0.2 2.2 ± 0.7 1.6 ± 0.1 1.3 ± 0.2 34:1 16:0–18:1 + 16:1–18:0 19.3 ± 0.9 22.3 ± 0.8 c 7.0 ± 0.4 6.5 ± 0.5 34:2 16:0–18:2 + 16:1–18:1 21.7 ± 1.1 22.4 ± 0.5 9.4 ± 0.5 7.9 ± 0.5 c 34:3 16:1–18:2 + 16:0–18:3 6.5 ± 0.8 7.0 ± 0.9 4.5 ± 0.4 3.6 ± 0.2 c 34:4 16:1–18:3 + 14:0–20:4 0.8 ± 0.1 0.4 ± 0.1 c 0.6 ± 0.1 0.3 ± 0.0 c 35:1 17:0–18:1 + 15:0–20:1 0.9 ± 0.0 1.1 ± 0.1 b 0.4 ± 0.1 0.6 ± 0.2 35:2 17:0–18:2 + 15:0–20:2 1.2 ± 0.1 1.2 ± 0.1 0.9 ± 0.1 0.9 ± 0.2 35:3 17:0–18:3 + 15:0–20:3 0.5 ± 0.0 0.7 ± 0.1 c 0.4 ± 0.2 0.6 ± 0.2 36:0 18:0–18:0 0.5 ± 0.1 0.4 ± 0.1 0.5 ± 0.1 0.4 ± 0.1 36:1 18:0–18:1 2.6 ± 0.2 2.1 ± 0.1 c 2.3 ± 0.3 2.1 ± 0.4 36:2 18:1–18:1 + 18:0-18-2 7.4 ± 0.3 6.2 ± 0.7 13.0 ± 1.2 13.4 ± 1.0 36:3 18:1–18:2 8.8 ± 0.3 7.4 ± 0.3 c 16.0 ± 1.6 16.7 ± 0.7 36:4 18:2–18:2 + 18:1–18:3 2.7 ± 0.3 2.3 ± 0.2 4.1 ± 0.4 4.1 ± 0.6 36:4 16:0–20:4 1.7 ± 0.3 1.1 ± 0.1 c 5.1 ± 0.3 4.2 ± 0.3 c 36:5 16:0–20:5 0.9 ± 0.3 0.5 ± 0.2 a 2.2 ± 0.1 1.8 ± 0.1 c 38:2 18:0–20:2 + 18:1–20:1 0.5 ± 0.1 0.7 ± 0.2 0.9 ± 0.1 1.2 ± 0.2 a 38:3 18:0–20:3 + 18:1–20:2 +18:2–20:1 0.8 ± 0.1 1.1 ± 0.3 1.4 ± 0.1 2.0 ± 0.3 c 38:4 16:0–22:4 0.6 ± 0.3 0.5 ± 0.1 0.8 ± 0.3 1.1 ± 0.1 a 38:4 18:0–20:4 + 18:1–20:3 +18:2–20:2 0.6 ± 0.1 0.9 ± 0.1 c 1.3 ± 0.1 2.0 ± 0.1 c 38:5 16:0–22:5 + 16:1–22:4 0.6 ± 0.2 0.8 ± 0.1 1.8 ± 0.5 2.2 ± 0.2 38:5 18:0–20:5 + 18:1–20:4 +18:2–20:3 0.6 ± 0.1 0.9 ± 0.1 c 0.9 ± 0.1 1.2 ± 0.1 b 38:6 16:0–22:6 + 16:1–22:5 0.9 ± 0.3 0.9 ± 0.1 2.1 ± 0.3 2.0 ± 0.3 38:6 18:1–20:5 + 18:2–20:4 0.3 ± 0.1 0.4 ± 0.1 b 0.4 ± 0.1 0.6 ± 0.2 38:7 16:1–22:6 0.4 ± 0.1 0.6 ± 0.1 0.9 ± 0.2 0.7 ± 0.1 40:4 18:0–22:4 0.0 ± 0.0 0.1 ± 0.1 c 0.2 ± 0.1 0.3 ± 0.1 40:5 18:0–22:5 + 18:1–22:4 0.1 ± 0.0 0.3 ± 0.1 c 1.1 ± 0.1 1.3 ± 0.3 40:6 18:0–22:6 + 18:1–22:5 0.3 ± 0.1 0.8 ± 0.2 c 3.1 ± 0.3 3.7 ± 0.2 c 40:7 18:1–22:6 + 18:2–22:5 0.7 ± 0.2 1.4 ± 0.2 c 5.3 ± 0.8 6.2 ± 0.4 a 40:8 18:2–22:6 0.4 ± 0.1 1.0 ± 0.1 c 3.3 ± 0.4 3.9 ± 0.4 a 40:9 18:3–22:6 0.1 ± 0.1 0.3 ± 0.1 c 0.8 ± 0.1 0.8 ± 0.1 42:11 20:5–22:6 ND ND 0.1 ± 0.0 0.3 ± 0.1 c Statistical comparison between the groups: a P < 0.05; b P < 0.02; c P < 0.01. Ó FEBS 2002 Modification of VLDL triacylglycerols by lipolysis (Eur. J. Biochem. 269) 6227 animals in the sn-1,2-diacylglycerol moieties of VLDL triacylglycerols in the present study. Stereospecific analysis and calculations of molecular associations of fatty acids indicated that expressly the fatty acid in the sn-1 position affected the susceptibility to hydrolysis. The comparison of the proportions of triacylglycerol species like 16:0–18:1– 18:2, 16:0–18:2–18:1, 16:0–18:2–18:2 and 18:2–18:1–18:1 and their reverse isomers suggest that from these triacyl- glycerols, 18:2n-6 is the most readily hydrolysed major fatty acid in the sn-1 position, while 16:0 is the most resistant. Differences in the calculated diacylglycerol species between the groups support also this conclusion. Fig. 2A shows measured and calculated proportions of 16:0–18:2 (+ 16:1– 18:1) in the sn-1,2-diacylglycerols. In addition, the propor- tions of reverse isomers (16:0–18:2 and 18:2–16:0) have been presented. It could be seen that measured and calculated proportions were very similar and that there was no difference between the groups. There were, however, clearly more 16:0–18:2 and less 18:2–16:0 in nontreated rats. Similar examination of 18:1–18:2 indicates that its smaller proportion in nontreated rats was due to differences in 18:2– 18:1 whereas the values for 18:1–18:2 were about the same (Fig. 2B). These findings demonstrate that the modification of circulating VLDL triacylglycerols by basal lipolysis is only partly revealed by the analysis of fatty acid composi- tion because of the uncertainty of the origin of each fatty acid. Previous work has reported that the polyunsaturated 20- and 22-acyl carbon fatty acids of human chylomicrons are released by bovine milk lipoprotein lipase more slowly than the shorter chain fatty acids [7], and that 22:6n-3 is released more readily than 20:4n-6 or 20:5n-3 [7,33]. On the other hand, human chylomicrons enriched with polyunsaturated fatty acids were hydrolysed faster by human milk lipopro- tein lipase than chylomicrons containing more saturated fatty acids [34]. In the present study, triacylglycerol species containing highly unsaturated fatty acids were also differ- entially affected by lipolysis. In the sn-1,2 diacylglycerol the Fig. 1. Proportions of selected sn-1,2-diacylglycerol (A) and sn-2,3- diacylglycerol (B) species derived from VLDL and liver triacylglycerols of Triton-treated rats. NEU derivatives of sn-1,2- and sn-2,3-diacyl- glycerols derived from VLDL and liver triacylglycerols were analysed by HPLC/MS as described in Materials and methods. Results are expressed as mean ± SD. Statistical comparison between VLDL and liver: *P <0.05. Table 4. Positional distribution of fatty acids in plasma VLDL triacylglycerols. Fatty acid compositions of sn-1,2- and sn-2,3-diacylglycerols recovered from the HPLC separation were determined from three Triton-treated and two nontreated rats. Stereospecific positional distribution of fatty acids was calculated from diacylglycerol and total triacylglycerol fatty acid compositions. Results are expressed as mean ± SD. Triton-treated Nontreated sn-1 sn-2 sn-3 sn-1 sn-2 sn-3 14:0 2.4 ± 0.7 0.7 ± 0.2 1.7 ± 0.6 1.5 ± 0.5 0.4 ± 0.1 1.0 ± 0.3 15:0 1.2 ± 0.3 0.7 ± 0.2 0.4 ± 0.1 0.8 ± 0.4 0.5 ± 0.4 0.5 ± 0.2 16:0 51.4 ± 3.9 18.8 ± 3.3 10.8 ± 3.0 57.6 ± 2.2 16.8 ± 2.7 8.8 ± 1.2 17:0 0.7 ± 0.2 0.6 ± 0.1 0.3 ± 0.2 0.4 ± 0.2 0.7 ± 0.2 0.1 ± 0.0 18:0 4.5 ± 1.3 3.6 ± 1.7 2.9 ± 0.9 1.9 ± 1.1 3.6 ± 0.3 1.5 ± 0.3 16:1n-9 0.3 ± 0.2 1.2 ± 0.3 0.2 ± 0.2 0.6 ± 0.2 0.6 ± 0.2 0.4 ± 0.1 16:1n-7 4.2 ± 0.3 0.4 ± 0.3 3.3 ± 0.3 2.6 ± 0.2 0.5 ± 0.1 4.0 ± 0.7 18:1n-9 12.4 ± 0.6 31.0 ± 0.8 30.1 ± 2.7 11.9 ± 2.8 34.0 ± 0.8 30.4 ± 1.6 18:1n-7 4.0 ± 0.3 0.9 ± 0.4 6.4 ± 0.4 3.8 ± 0.5 0.3 ± 0.1 5.9 ± 0.1 20:1n-9 + 11 0.1 ± 0.1 0.6 ± 0.4 0.6 ± 0.3 ) 0.2 ± 0.1 1.1 ± 0.2 0.2 ± 0.2 18.2n-6 15.8 ± 2.7 33.0 ± 1.9 17.9 ± 1.1 10.7 ± 3.3 34.8 ± 2.5 18.6 ± 0.4 18:3n-3 0.6 ± 0.3 2.0 ± 0.8 1.5 ± 0.6 0.6 ± 0.1 2.1 ± 0.3 0.4 ± 0.5 20:2n-6 0.1 ± 0.0 0.5 ± 0.3 0.4 ± 0.1 0.0 ± 0.1 0.6 ± 0.2 0.6 ± 0.0 20:3n-6 0.1 ± 0.0 0.6 ± 0.2 0.5 ± 0.2 0.5 ± 0.1 0.2 ± 0.2 1.1 ± 0.3 20:4n-6 0.0 ± 0.1 2.6 ± 0.6 2.4 ± 0.8 0.2 ± 0.1 2.0 ± 0.3 2.5 ± 0.3 20:5n-3 0.4 ± 0.2 1.2 ± 0.2 2.8 ± 0.4 1.3 ± 0.1 0.3 ± 0.1 3.8 ± 0.2 22:4n-6 0.0 ± 0.2 0.6 ± 0.4 0.4 ± 0.2 ) 0.2 ± 0.5 0.9 ± 0.2 0.6 ± 0.2 22:5n-3 0.4 ± 0.2 0.5 ± 0.4 4.4 ± 0.7 1.7 ± 0.3 0.2 ± 0.1 4.7 ± 0.1 22:6n-3 1.3 ± 0.6 0.8 ± 0.5 13.0 ± 0.2 4.5 ± 0.3 0.4 ± 0.1 14.8 ± 0.9 6228 J. J. A ˚ gren et al. (Eur. J. Biochem. 269) Ó FEBS 2002 content of 16:0–20:4 and 16:0–20:5 was lower in nontreated than in treated rats, whereas the content of species with the combination of 18 and 20 or 22-acyl carbon fatty acid were higher. Positional analysis revealed higher proportions of major 20- and 22-acyl carbon fatty acids in the sn-1 position and lower proportions in the sn-2 position in nontreated rats. These results indicate that the presence of both 20- and 22-acyl carbon polyunsaturated fatty acids in the sn-1, and possibly also in the sn-3, but not in the sn-2 position of VLDL triacylglycerol retards the hydrolysis. This would explain the divergent changes in the diacylglycerol moieties containing these fatty acids, and possibly also the differences found in the studies with VLDL and chylomicrons as the positional distribution of fatty acids is not similar in these lipoproteins. VLDL triacylglycerol is derived from liver cytosolic triacylglycerol through hydrolysis and reesterifica- tion, and possibly by lipolysis from cellular phospholipids [29,30,35] The major source of chylomicron triacylglycerol is the 2-monoacylglycerol pathway in which original dietary fatty acids are retained in the sn-2 position, e.g. 22:6n-3 in fish oils [26]. This means that, in addition to difference between VLDL and chylomicrons, there could be also substantial differences in the distribution of polyunsaturated fatty acids within chylomicrons depending on the relative contribution of endogenous and dietary fatty acids and on the nature of dietary lipids. Contrary to retarding hydrolysis when located in the sn-1 position of VLDL triacylglycerol, 20- and 22-acyl carbon fatty acids in the sn-2 position seemed to advance lipolysis. It could be speculated that these fatty acids affect the structure of triacylglycerol molecule in a way that facilitates the action of lipolytic enzymes. It is also possible that hepatic lipase has a specific role in hydrolysis of triacylgly- cerol species with these fatty acids. It has been shown that the hydrolysis of 20:4 containing triacylglycerol and diacylglycerol was slower in rat postheparin plasma when hepatic lipase was inhibited [36]. Hepatic lipase is capable of hydrolysing fatty acids from the sn-1 position of phospho- lipids, and so it may have some preference also for triacylglycerol species with a phospholipid-like combination of fatty acids in the sn-1 and sn-2 positions. In other studies, polyunsaturated long chain fatty acids containing a double bond at carbons 4 and 5 were observed to be relatively resistant to hydrolysis by pancreatic lipase in vitro [37] although such resistance was not seen in vivo [38]. Structural differences between liver and VLDL triacylglycerols Compositions of sn-1,2 and sn-2,3-diacylglycerol moieties of liver triacylglycerols resembled those of VLDL triacylgly- cerols but there were also some clear differences (data not shown). It has been suggested that major part of the stored liver triacylglycerol is hydrolysed, mostly to sn-1,2-diacyl- glycerol, and reesterified before entering VLDL triacylgly- cerol [30,35]. This would expose especially the sn-3 position to modifications. In Triton-treated rats sn-2,3-diacylglycer- ols from VLDL triacylglycerols contained less saturated and monounsaturated 34- and 36-acyl carbon species and more species with highly unsaturated fatty acids, especially 22:6n-3, than sn-2,3-diacylglycerols from liver triacylglycer- ols. This indicates substitution of 20- and 22-carbon polyun- saturated fatty acids for 16:0 and 18:1 when liver tria- cylglycerol is used in VLDL triacylglycerol synthesis. There were differences also in the sn-1,2-diacylglycerols between VLDL and liver triacylglycerol in Triton-treated rats suggesting that liver triacylglycerols are partly hydrolysed to sn-2,3-diacylglycerol when used for VLDL triacylglycerol Table 5. Content of major calculated molecular species and their reverse isomers in VLDL triacylglycerols. The molecular associations of fatty acids and reverse isomer content of triacylglycerols were calculated from the compositions of fatty acids in the sn-1, sn-2 and sn-3 positions of triacylglycerols. Written isomer Reverse isomer Triton-treated Nontreated Triton-treated Nontreated 16:0–16:0–18:1 3.53 3.51 0.34 0.23 16:0–18:1–16:0 1.77 1.74 16:0–18:1–18:1 5.98 7.17 0.57 0.47 16:0–18:2–16:0 1.83 1.76 18:0–18:1–18:1 0.52 0.24 0.15 0.02 16:0–18:1–18:2 2.94 3.67 0.54 0.32 16:0–18:2–18:1 6.19 7.28 0.59 0.48 18:1–18:1–18:1 1.93 1.95 16:0–18:2–18:2 3.04 3.72 0.56 0.32 18:1–18:2–18:1 2.00 1.98 18:2–18:1–18:1 1.84 1.33 0.95 1.00 16:0–16:0–22:6 1.26 1.43 0.03 0.07 16:0–18:1–22:6 2.13 2.92 0.04 0.14 18:1–16:0–22:6 0.41 0.39 0.09 0.27 16:0–18:2–22:6 2.21 2.97 0.05 0.14 18:2–16:0–22:6 0.39 0.27 0.04 0.14 18:1–18:1–22:6 0.69 0.80 0.15 0.56 18:1–18:2–22:6 0.71 0.81 0.16 0.57 18:2–18:1–22:6 0.66 0.54 0.07 0.29 18:2–18:2–22:6 0.68 0.55 0.08 0.29 Ó FEBS 2002 Modification of VLDL triacylglycerols by lipolysis (Eur. J. Biochem. 269) 6229 synthesis. It has been found earlier that 10% of liver-free diacylglycerol is sn-2,3-diacylglycerol [30]. Another possible source for these differences is the use of phospholipids for VLDL triacylglycerol synthesis [29]. As liver phospholipids contain more polyunsaturated fatty acids, and especially 20:4n-6, than triacylglycerols, this would be consistent with the lower level of saturated species and higher content of 16:0–20:4 in the sn-1,2-diacylglycerols of VLDL triacylgly- cerols. Physiological significance of nonrandom lipolysis of VLDL The nonrandom lipolysis may have significant meaning for the trafficking of fatty acids. The lower proportions of 14:0, 15:0, 17:0, 18:0, 16:1n-7 and 18:1n-7 in circulating VLDL triacylglycerols could indicate that they were directed towards oxidation. With the exception of 18:0, this would be in accordance with their lower proportions in other lipid fractions. The preferential liberation of 18:2n-6 from the sn-1 position may also have importance in the regulation of its utilization. The behavior of 20- and 22-acyl carbon polyunsaturated fatty acids differs from that of other fatty acids as well as from each other. The levels of 20:5n-3 are more rapidly changed by dietary modification than those of 22:6n-3 and in plasma it is directed more efficiently to phospholipids whereas 22:6n-3 prefers triacylglycerols [39]. On the other hand, 22:6n-3 is better preserved and its amount is relatively high in plasma and tissue lipids even when dietary supply is low [39]. In the present study, 22:5n-3 and 22:6n-3 showed clear resistance to hydrolysis when locatedinthesn-1 and sn-3 positions. This may serve as a mechanism to preserve and direct the utilization of these fatty acids. It was shown previously that 20:4, 20:5 and 22:6 accumulated in diacylglycerols and monoacylglycerols when chylomicrons were incubated with lipoprotein lipase [7]. Thus, the decrease of 20:4n-6, 20:5n-3, 22:5n-3 and 22:6n-3 in the sn-2 position observed in our study could be also a consequence of hydrolysis only in the primary position leaving them to diacylglycerols and monoacylglycerols; the direction of these glycerols may contribute to the divergent distribution of polyunsaturated fatty acids. In view of the marked difference in the composition of the triacylglycerols of nascent and circulating VLDL, some speculation would seem to be justified about the physiolo- gical significance of their nonrandom lipolysis. The general preferential hydrolysis of the unsaturated triacylglycerols may be related to their higher solubility. However, a retrieval of the essential fatty acids, which may have facilitated the VLDL secretion, could also be involved. The preferential attack on the sn-1-position may serve to avoid flooding of the lipoprotein and cell membrane surfaces with sn-1,2-diacyl- glycerols, which may promote glycerolipid resynthesis as well as compromise the sn-1,2-diacylglycerol signalling pathway. Furthermore, formation of sn-2,3-diacylglycerols may pre- vent their accumulation on the lipoprotein surfaces because of stereochemical incompatibility. Other hypotheses could be advanced about a preferential release of the saturated and monounsaturated fatty acids for the purposes of oxidation as well as about special metabolic roles of specific molecular species of diacylglycerols or triacylglycerols. In conclusion, the results of this study show that the basal lipolysis causes significant modifications in the fatty acid Fig. 2. Measured and calculated proportions of (A) 16:0–18:2 and (B) 18:1–18:2 in the sn-1,2-diacylglycerol moieties of VLDL triacylglycerols in Triton-treated and nontreated rats. Measured proportions were obtained from HPLC/MS analyses of sn-1,2-diacylglycerols and cal- culated proportions from the calculation of molecular association of fatty acids. The fatty acid in the sn-1 position is on the left in the abbreviated notation of molecular species. The first two columns in A (measured and calculated 16:0–18:2 + 18:2–16:0) contain also 16:1– 18:1 + 18:1–16:1. Statistical comparison between the groups (mea- sured proportions): *P <0.01. 6230 J. J. A ˚ gren et al. (Eur. J. Biochem. 269) Ó FEBS 2002 distribution of circulating VLDL triacylglycerols. Triacyl- glycerol species containing 16:0 or 20–22-acyl carbon polyunsaturated fatty acids in the sn-1 position were more resistant to hydrolysis than those with 18:2 in this position. In contrast, triacylglycerol species with 20–22-acyl carbon polyunsaturated fatty acids in the sn-2 position were hydrolysed more readily than those with other fatty acids in this position. 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