The molecular associ-ation ofthe fatty acids and reverse isomer content ofthe triacylglycerol and the derived sn-1,2- and sn-2,3-diacyl-glycerols were determined by calculation on the ba
Trang 1Structural and compositional changes in very low density lipoprotein triacylglycerols during basal lipolysis
Jyrki J A˚gren1,2, Amir Ravandi1, Arnis Kuksis1and George Steiner3
1
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada;2Department of Physiology, University of Kuopio, Finland;3Department 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 of16:0 was higher and that of18:2n-6 lower in
the sn-1 position The proportion of14:0 was lower in all
positions and that of18:0 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 ofmolecular association offatty 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
1Very low density lipoprotein (VLDL) secreted from liver is
the major carrier oftriacylglycerols 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 ofcirculating VLDL resembles
that ofthe 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 ofhuman
and bovine milk lipoprotein lipase may differ [9] as may the
properties ofmilk and endothelial lipoprotein lipase [10]
Because the biosynthesis ofintestinal chylomicron and
hepatic triacylglycerols proceed along different routes [11],
structurally dissimilar triacylglycerols would have been subject to endogenous lipolysis during clearance ofpost-prandial chylomicron triacylglycerols and ofVLDL 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 ofVLDL 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 ofTriton WR1339 have given no indication ofill effects [17–19]
The present study was carried out to confirm in vivo the nonrandomness ofthe 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 ofPhysiology,
University ofKuopio, 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)
Trang 2M A T E R I A L S A N D M E T H O D S
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 (n¼ 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
(d¼ 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 16C Livers were removed and stored at)70 C All
experiments were performed under protocols approved by
the Animal Care Committee and the University ofToronto
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% H2SO4 in methanol at 80C 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 lm film
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 150C
at 30CÆmin)1and to 340C at 10 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 50C 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 ofthis 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 of0.7 mLÆmin)1 Positive chemical ionization was obtained by postcolumn addition ofchloroform/ 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 ofdiacylglycerol species were calculated from the areas ofthe single ion plots obtained from the mass spectra NEU derivatives ofVLDL 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 of38 and 40 acyl carbon sn-1,2-diacyl-glycerol eluted concurrently with sn-2,3-diacylsn-1,2-diacyl-glycerol 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 ofthe fatty acids was determined by calculation from the fatty acid compo-sition oftotal 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 ofthe fatty acids and reverse isomer content ofthe triacylglycerol and the derived sn-1,2- and sn-2,3-diacyl-glycerols were determined by calculation on the basis ofthe knowledge ofthe fatty acid composition ofthe sn-1, sn-2 and sn-3 positions ofthe acylglycerols, assuming 1-random, 2-random and 3-random distribution [27] The molecular associations give the exact pairs offatty acids in individual diacylglycerol and the exact triplets offatty acids in individual triacylglycerol
Statistics The values have been expressed as mean ± SD The Mann–Whitney U-test was used for comparisons of groups
Trang 3R E S U L T S
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)1in 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 ofVLDL particles during the
treatment The very small amount ofVLDL diacylglycerol
(0.2 ± 0.0% ofneutral lipids) in Triton-treated rats
indi-cate also a minor contribution ofmodified VLDL
Prelimi-nary studies showed linear increase ofplasma triacylglycerol
concentration at least for 4 h after Triton injection
However, 4-h treatment was found to affect the fatty
acid composition ofliver 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 ofVLDL triacylglycerol concentration
was identical to that seen in overnight fasted rats and also
the neutral lipid profiles ofVLDL 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 offree cholesterol,
choles-terol esters and diacylglycerols were greater and those of total triacylglycerols were smaller in nontreated rats In addition, the proportions of50 and 54 acyl carbon triacylglycerols were lower and those of56 and 58 acyl carbon triacylglycerols were higher in nontreated compared with Triton-treated rats
Fatty acid composition of VLDL and liver The proportions ofsaturated fatty acids in the total VLDL triacylglycerols were lower in nontreated rats, with the exception ofslightly higher proportion of16:0 (Table 2) The levels of18:1n-9, 18:2n-6 and 20:4n-6 were similar in both groups whereas the proportions of16:1n-7, 18:1n-7 and 18:3n-3 were lower, and those of20:3n-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 ofsn -1,2 and sn -2,3-diacylglycerols There were statistically significant differences between the groups in about halfofthe 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 of16:0–18:1 was higher in the sn-1,2-diacylglycerols In addition, the proportions of16:0–20:4 and 16:0–20:5 were lower and those ofdiacylglycerol 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 of20:5–22:6 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 ofliver triacylglycerols between the groups Compared with VLDL the proportions of16:0–16:0, 16:0–18:0 and 16:0–18:1 were higher and those of16:0–18:2, 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 of16:0–18:0, 16:0–18:1 and 18:0–18:1 were higher and those of16:0–20:4 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 ofVLDL triacylglycerol fatty acids showed that the lower proportions ofmost 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 ofmajor saturated fatty acid, 16:0, was higher in the sn-1 position in nontreated rats The proportions ofmajor 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.
Statistical comparison between the groups:aP < 0.05;bP < 0.02;
c
P < 0.01.
Trang 4The proportions ofsome major VLDL triacylglycerol
species and their reverse isomers, calculated on the basis of
stereospecific positional distribution offatty 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 oftriacylglycerol 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 oftriacylglycerol species with 20:4, 20:5, 22:5 or
22:6 in the sn-1 position were higher in nontreated rats
D I S C U S S I O N
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
ofgenetic manipulation ofthe secretion ofapoB-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 ofVLDL
triacylglycerols under normal physiological conditions to
that obtained after Triton WR1339 treatment In addition
to blocking triacylglycerol lipolysis by inhibition
oflipo-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] Apolipoapolipo-protein B or lipids were not
displaced from low density lipoprotein (LDL) particles
indicating that neither should there be marked association ofTriton to newly secreted VLDL particles containing mainly apolipoprotein B Triton accumulates also in liver lysosomes disturbing the lysosomal hydrolysis ofVLDL [16] This may change the fatty acid composition of secreted VLDL due to possible depletion ofcellular triacylglycerol stores and increased use ofphospholipids 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 ofunknown side-effects ofTriton treatment cannot be ruled out, none ofthe 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 ofliver triacylglycerols and phospholi-pids (data not shown) in both groups indicate that the composition ofsecreted 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 ofcholesterol esters in nontreated rats could also result from the removal of triacylglycerols However, the proportion offree 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 of54 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
Statistical comparison between the groups:aP < 0.05;bP < 0.02;cP < 0.01.
Trang 5containing most ofthe 20- and 22-carbon fatty acids in
plasma VLDL [30] ofnontreated 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 oftriacylglycerol [5,31,32] Using
purified human milk lipoprotein lipase and a synthetic
triacylglycerol mixture [5], the relative order offatty acid
release was 18:1 > 18:3 > 18:2 > 14:0 > 16 > 0 > 18:0
The observation ofpreferential hydrolysis of18:2n-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 ofnontreated rats are in accordance with these results On the other hand, the proportion of18:0, 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 oftriacylglycerol to lipase is determined by both the nature ofthe 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-2,3diacylglycerols from plasma VLDL triacylglycerols NEU derivatives ofdiacylglycerols 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.
Statistical comparison between the groups:aP < 0.05;bP < 0.02;cP < 0.01.
Trang 6animals in the sn-1,2-diacylglycerol moieties ofVLDL
triacylglycerols in the present study Stereospecific analysis
and calculations ofmolecular associations offatty acids
indicated that expressly the fatty acid in the sn-1 position affected the susceptibility to hydrolysis The comparison of the proportions oftriacylglycerol 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 of16:0–18:2 (+ 16:1– 18:1) in the sn-1,2-diacylglycerols In addition, the propor-tions ofreverse 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 of18:1–18:2 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 ofcirculating VLDL triacylglycerols by basal lipolysis is only partly revealed by the analysis offatty acid composi-tion because ofthe uncertainty ofthe origin ofeach 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 triacylsn-2,3-diacyl-glycerols 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.
Trang 7content of16:0–20:4 and 16:0–20:5 was lower in nontreated
than in treated rats, whereas the content ofspecies with the
combination of18 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 ofboth 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 offatty 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 ofchylomicron 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 ofendogenous and dietary fatty acids and on
the nature ofdietary lipids
Contrary to retarding hydrolysis when located in the sn-1
position ofVLDL 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 oftriacylglycerol molecule in a way that facilitates
the action oflipolytic enzymes It is also possible that
hepatic lipase has a specific role in hydrolysis
oftriacylgly-cerol species with these fatty acids It has been shown
that the hydrolysis of20:4 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 ofphospho-lipids, and so it may have some preference also for triacylglycerol species with a phospholipid-like combination offatty 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 ofVLDL triacylgly-cerols but there were also some clear differences (data not shown) It has been suggested that major part ofthe 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 triacylglycersn-2,3-diacylglycer-ols 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 of20- and 22-carbon polyun-saturated fatty acids for 16:0 and 18:1 when liver tria-cylglycerol is used in VLDL triatria-cylglycerol 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 offatty acids and reverse isomer content oftriacylglycerols were calculated f rom the compositions off atty acids in the sn-1, sn-2 and sn-3 positions of triacylglycerols.
Trang 8synthesis 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 ofsaturated species and higher content of 16:0–20:4 in the sn-1,2-diacylglycerols ofVLDL 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 of18:0, 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 of20- 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 located in the sn-1 and sn-3 positions This may serve as a mechanism to preserve and direct the utilization ofthese 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 of20:4n-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 ofhydrolysis only in the primary position leaving them to diacylglycerols and monoacylglycerols; the direction ofthese glycerols may contribute to the divergent distribution ofpolyunsaturated fatty acids
In view ofthe marked difference in the composition ofthe triacylglycerols ofnascent and circulating VLDL, some speculation would seem to be justified about the physiolo-gical significance oftheir nonrandom lipolysis The general preferential hydrolysis of the unsaturated triacylglycerols may be related to their higher solubility However, a retrieval ofthe 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 ofthe 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 ofstereochemical 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 ofspecific molecular species ofdiacylglycerols or triacylglycerols
In conclusion, the results ofthis 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 ofmolecular 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.
Trang 9distribution ofcirculating 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 There were also differences in the
triacyl-glycerol composition ofnewly secreted VLDL
triacylgly-cerols and liver triacylglytriacylgly-cerols, which could be explained as
resulting from a hydrolysis–reesterification processes of
triacylglycerols and/or involvement ofphospholipids in the
VLDL triacylglycerol formation
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