Nuffield Department of Anaesthetics, University of Oxford, Radcliffe Infirmary, Oxford, Oxfordshire OX2 6HE, United Kingdom
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ABSTRACT |
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The effect of endotoxin on myocardial utilization of very low density lipoprotein (VLDL) triacylglycerol (TAG) was studied. VLDL was prepared by rat liver perfusion and tested as substrate in the isolated working rat heart. Both liver and heart donor rats were pretreated in vivo with endotoxin or vehicle (control). VLDL-TAG synthesized by endotoxin-pretreated livers was assimilated and oxidized at an increased rate by hearts compared with control VLDL-TAG, regardless of the cardiac endotoxic status, with increased cardiac mechanical performance (cardiac output, hydraulic work). There was no change in incorporation of labeled VLDL lipids into myocardial tissue lipids. Lipoprotein lipase (LPL) activity was increased in endotoxin-pretreated hearts, and after perfusion with "endotoxic" VLDL, there was a tendency for translocation of LPL from tissue-residual to heparin-releasable compartments, but these changes were modest. Analysis of the VLDL composition showed that endotoxin-pretreated livers produced apolipoprotein (apo)-B48 VLDL with decreased particle size (and hence TAG content), but apo-B100 VLDL was unchanged. Oleate content of VLDL was increased, but there was no difference in apo-C or apo-E content. These results suggest that VLDL-TAG produced during sepsis/endotoxinemia may be destined for utilization by the heart as energy substrate. However, the mechanism for its increased efficacy is uncertain.
lipoprotein lipase
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INTRODUCTION |
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SYSTEMIC INFLAMMATION IS ASSOCIATED with a wide variety
of physiological and biochemical changes, including increased
concentrations of plasma triacylglycerols (TAG), principally in the
form of very low density lipoprotein (VLDL) (12). The etiology of this
effect is uncertain, but in the case of systemic infection (sepsis) it is likely that endotoxin [lipopolysaccharide (LPS)] derived
from the cell wall of invading bacteria stimulates the host immune system to produce excessive amounts of proinflammatory cytokines such
as tumor necrosis factor- (TNF-
), interleukin-1
(IL-1
), and
other inflammatory mediators such as platelet activating factor. These
paracrine hormones enter the circulation and, acting as endocrine
signals, cause a wide variety of apparently deleterious effects,
including alterations of lipid metabolism (8, 10). Some of these
effects are undoubtedly pathological and in extreme form cause tissue
dysfunction and eventually death. However, the possibility remains that
a coordinated host response operates, at least in the early phase of
pathogen invasion, which may be part of a host survival strategy.
Sepsis, endotoxin, and proinflammatory mediators all cause increased
plasma TAG concentrations experimentally, and this occurs by two
mechanisms, 1) increased hepatic production of TAG (by
increased de novo lipogenesis and fatty acid esterification) with
increased VLDL synthesis and secretion (7, 13, 14) and 2)
decreased uptake of VLDL-TAG by certain peripheral tissues normally
associated with plasma TAG clearance [by decreased activity of
lipoprotein lipase (EC 3.1.1.34; LPL) (20, 21), the rate-limiting enzyme responsible for hydrolysis of TAG in circulating
lipoproteins], e.g., adipose tissue and skeletal muscle. The
composition of the excess VLDL seen in systemic inflammatory states is
also probably abnormal, both in terms of lipid and apolipoprotein
content (22).
The teleology of the hypertriacylglycerolemia is also uncertain; the
process is energetically expensive, and this would seem counterproductive at times of metabolic stress. One suggestion is that
increased VLDL binds and sequesters endotoxin, facilitating its
disposal and decreasing infective lethality; evidence exists for this
in rodents (28). Another possibility is that the increased TAG within
the VLDL produced by the liver (and denied access to storage and other
"nonessential" tissues) is destined for utilization by tissues
with increased energy requirement during sepsis by a redirection
mechanism based on TAG availability through lipoprotein lipase (LPL)
activity. Given the intimate relationship between the signaling systems
of the immune response (e.g., cytokines), still poorly understood, and
the regulation of intermediary (substrate) metabolism (12), it is
possible that part of a host response during systemic inflammation may
involve concerted redirection of substrates from nonessential to
essential tissue depots, as occurs physiologically in conditions such
as fasting and lactation (39). Heart work and cardiac output (CO) are
increased during early phase sepsis/endotoxinemia (26), despite
decreased myocardial contractility (26). Cardiac energy status is
preserved (6, 31), and endotoxin administered to rats in vivo increases
myocardial metabolic rate, with increased peak systolic pressure and
maximal positive dP/dt, while uncoupling coronary flow from
cardiac metabolism (31). However, endotoxin can cause myocardial
depression directly (34) or via TNF- (34), but myofilament
Ca2+ responsiveness of rat ventricular myocytes remains
intact (29), and IL-1 released by endotoxin itself attenuates the
endotoxin depression of cardiac contractility (40). The heart is a
quantitatively important consumer of TAG fatty acid as energetic
substrate with high LPL activity (30), and incorporation of fatty acid
into intracellular TAG (and phospholipids) is increased after endotoxin treatment in dog myocytes (23); therefore, the heart is a putative destination of this excess VLDL-TAG. Furthermore, endotoxin directly causes coronary vasodilatation (3) and induces myocardial nitric oxide
synthase (34), and this would increase substrate delivery in the
working heart preparation in vitro in which afterload (coronary perfusion pressure) is fixed. To test this hypothesis, rat VLDL was
synthesized in both normal and endotoxin-exposed livers and tested as
substrate for the working rat heart, again under control and endotoxic conditions.
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MATERIALS AND METHODS |
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The investigation was performed in accordance with the Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986 published by HMSO, London, UK.
Animals. Male Wistar rats were fed ad libitum a chow diet [comprising by weight ~52% carbohydrate, 21% protein, and 4% fat; the residue was nondigestible material (Special Diet Services, Witham, Essex, UK)] and free access to drinking water and were maintained at an ambient temperature of 20 ± 2°C with a 12:12-h light-dark cycle (light from 0730).
Hearts from two groups (250-350 g, fed ad libitum) were studied: 1) rats injected with endotoxin (from Escherichia coli serotype 055:B5) 100 µg/kg body wt, and 2) rats injected with 0.9% (wt/vol) NaCl (control). Both groups were injected intraperitoneally 15 h before experimentation. Livers from two groups (400-500 g, fasted for 24 h before experimentation) were perfused to prepare rat VLDL: 1) rats injected with endotoxin (from E. coli serotype 055:B5) 100 µg/kg body wt, and 2) rats injected with 0.9% (wt/vol) NaCl (control). Both groups were injected intraperitoneally 15 h before experimentation. Rectal temperature was checked by digital thermometer before organ preparation.Chemicals. [9,10-3H(N)]oleic acid and glycerol tri[9,10-3H(N)]oleate were obtained from Amersham International, (Amersham, Buckinghamshire, UK); Waymouth's medium was purchased from GIBCO BRL, Life Technologies, Paisley, UK; other biochemicals were obtained from Sigma Chemical, Poole, Dorset, UK.
Preparation of lipid substrates. 3H-labeled sodium oleate (specific activity 485 mCi/mmol) was prebound to fatty acid- and endotoxin-free BSA (37) and added to liver perfusates. In some experiments, oleate (prepared as above) was added to the perfusate of some hearts to give a final concentration of 1.1 mM [nonesterified fatty acid (NEFA) group].
3H-labeled triolein in the form of rat VLDL was prepared by an extended rat liver perfusion technique under aseptic conditions. Rats were anesthetized with intraperitoneal pentobarbitone sodium (60 mg/kg body wt), and the portal vein and thoracic inferior vena cava were rapidly cannulated; the abdominal inferior vena cava was ligated; heparin was not used. The liver was perfused in situ for 8 h with a recirculating solution comprising Waymouth's synthetic tissue culture medium supplemented with amino acids (glutamine, serine, and alanine) and glucose. Washed red cells were added to give a final hematocrit of 10% (vol/vol), and the perfusate was gassed with 95% O2-5% CO2 (vol/vol) at 37°C; [3H]oleate prebound to fatty acid-free albumin (see above) was added to the perfusate before liver perfusion (1.0 mM) and subsequently also infused into the perfusate for the first 4 h of the perfusion to maintain the circulating NEFA concentration at ~0.4 mM for the first half of the procedure. By the end of the perfusion, circulating oleate concentration was undetectable. Perfusate PO2, PCO2, pH, HCOAnalysis of VLDL composition. VLDL from control and endotoxic liver perfusions was analyzed, together with "native" VLDL obtained from plasma by venesection of 24-h-starved rats. Plasma was centrifuged as liver perfusate (above) to obtain the density <1.006 g/ml fraction. VLDL lipid content was determined with commercial kits for NEFA, TAG, cholesterol and cholesterol ester, and phospholipid (Boehringer Mannheim, Lewes, Sussex, UK). Fatty acid composition of VLDL lipid esters was determined as described in Ref. 15. Briefly, VLDL lipids were extracted by the method of Folch, separated by TLC, transesterified with toluene/H2SO4 in methanol, and analyzed by gas chromatography using free/esterified heptadecanoate as internal standards. Apolipoprotein (apo) composition was analyzed after VLDL delipidation (ethanol/diethyl ether) by denaturing SDS-PAGE on a 3/20% gradient and after that by Coomassie brilliant blue staining and densitometry; bovine albumin was co-run as a standard together with a molecular weight ladder. Apolipoprotein bands were analyzed by optical density, and the apo-B band was quantified against the albumin standard [corrected for differential albumin and apolipoprotein-B chromogenicity (41)]; other apoprotein bands were then expressed as a proportion of the apo-B band.
Isolated perfused working heart preparation. All experiments were commenced between 1100 and 1200. Hearts were perfused through the left atrium (anterograde) in "working" mode by the method of Taegtmeyer et al. (35). Fed rats were anesthetized with intraperitoneal pentobarbitone sodium (60 mg/kg body wt). The heart was rapidly excised and briefly placed in ice-cold Krebs-Henseleit bicarbonate saline. It was then cannulated via the aorta (<2 min from excision) and perfused retrogradely through the coronary arteries in Langendorff mode while lung, mediastinal, and pericardiac brown adipose tissue was excised, right pulmonary arteriotomy was performed, and the left atrium was separately cannulated, after which the apparatus was switched to working mode, and cardiac perfusion was maintained through the left atrium. A recirculating Krebs-Henseleit bicarbonate buffer solution containing 1.3 mM CaCl2 and 10 mM glucose and 2.5% (wt/vol) endotoxin- and fatty acid-free BSA was filtered through a 5-µm cellulose nitrate filter (Millipore, Bedford, MA) and gassed with 95% O2-5% CO2 at 37°C. The first 50 ml of coronary effluent were discarded to free the circuit of blood cells. Afterload was maintained at 100 cmH2O and preload (atrial filling pressure) at 15 cmH2O. After an initial 15-min stabilization period, VLDL was added slowly (2 min) to the reservoir (at t = 0). Peak systolic pressure (PSP) and heart rate (HR) were measured by calibrated pressure transducer (Druck, Groby, Leicestershire, UK) connected to a side arm of the aortic cannula. Aortic flow rate (AFR) was measured by a timed collection of perfusate ejected through the aortic line, and coronary flow rate (CFR) was measured by a timed collection of perfusate effluent dripping from the heart. Measurements were made at t = 0 and at 10-min intervals for 60 min. CO was calculated as (CFR + AFR). Rate-pressure product (RPP) was calculated as (HR × PSP). Hydraulic work (HW) was calculated as (CO × mean aortic pressure / heart wet wt). After the final measurements at 60 min, 5 IU/ml heparin (Leo Laboratories, Princes Risborough, Buckinghamshire, UK) were added to the perfusate, and after a further 2 min, the heart was rapidly excised, freeze-clamped in light alloy tongs cooled in liquid nitrogen, and weighed. A duplicate sample of the postheparin perfusate was also frozen in liquid nitrogen.
Measurement of lipid oxidation rate. TAG oxidation rate was estimated by measuring 3H2O production in the perfusate from [3H]triolein, as described (11); at 10-min intervals, aliquots of perfusate (1.0 ml) were removed and subjected to Folch lipid extraction with chloroform-methanol (2:1, vol/vol) and water (37). An aliquot of the water phase was removed and counted for radioactivity.
TAG utilization rate (disappearance from the perfusate) was measured by assay of TAG in the organic infranatant phase of the Folch extracts of the timed perfusate aliquots after evaporation of the chloroform and resolubilization with ethanol by means of an enzymatic colorimetric assay test kit (see above).Incorporation of exogenous lipid into myocardial lipid. Myocardial 3H-labeled lipid content was estimated by grinding frozen myocardium to powder under liquid N2 and extracting the lipids from an aliquot with chloroform-methanol (Folch). After repeated washing, the lipids were resolubilized in chloroform and were separated by TLC by use of a hexane-diethyl ether-acetic acid system (16) with standards co-run. 3H radioactivity was measured in the various lipid bands after visualization with rhodamine 6G under ultraviolet light.
LPL activity.
LPL activity was estimated in duplicate samples by using a
3H-labeled triolein substrate emulsion containing starved
rat serum as a source of apolipoprotein-CII to maximize LPL detection
(25). The serum was pretreated by heating to 56°C to inactivate
nonspecific plasma lipases. Radioactivity in evolved fatty acids was
counted after extraction in methanol-chloroform-heptane.
Heparin-releasable LPL activity was measured by adding postheparin
perfusate taken at 62 min directly in the above assay system without
modification (expressed as nmol fatty acid
released · min1 · g
wet wt of heart
1). Tissue residual LPL
activity was measured in acetone-diethyl ether-dried tissue powders
ground from the working hearts frozen in liquid nitrogen. A duplicate
sample of frozen heart tissue was weighed, dried down with
acetone-ether in parallel with the samples, and reweighed to correct
expression of activity (from nmol fatty acid
released · min
1 · mg
of acetone dried powder
1 to nmol fatty
acid
released · min
1 · g
wet wt of heart
1). Total LPL activity in
these experiments was heparin-releasable + residual LPL activities
(nmol fatty acid
released · min
1 · g
wet wt of heart
1).
Statistics. Results are expressed as mean values ± SE. Statistical analysis was performed by one-way ANOVA for repeated measurements and Tukey's test, or by Student's t-test with Bonferroni correction for multiple comparisons where appropriate. Statistical significance was set at P < 0.05.
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RESULTS |
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Uptake of TAG by perfused working hearts was significantly greater from
"endotoxic" VLDL (derived from endotoxin-exposed rat livers) than
from control VLDL, regardless of previous exposure of the heart itself
to endotoxin (Fig. 1). In the course of the 60-min experiment, this represented removal of up to ~1 mg of VLDL-TAG from the perfusate, ~3% of the total available circulating TAG. The possibility that endotoxin itself bound to the VLDL particle might be responsible for the effects of "endotoxic" VLDL was
investigated by preincubating control VLDL with endotoxin. When added
to control hearts, a similar TAG uptake to control VLDL was observed
(Fig. 1), suggesting that particle-associated endotoxin was not
involved.
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Metabolic fate of assimilated [3H]TAG was
examined by measuring oxidation and deposition of tissue
3H-labeled lipids. As with TAG uptake (Fig. 1), TAG
oxidation rate was greater from endotoxic VLDL than from control VLDL
(with or without preincubation with endotoxin), again regardless of
heart endotoxic status. This was significant in the case of endotoxic VLDL perfusing hearts from animals previously injected with endotoxin (Fig. 2). However, there was no significant
difference in accumulation of 3H-labeled tissue lipids (in
any class examined by TLC) from 3H-labeled VLDL during the
course of the experiment in any group studied (Table
1). Utilization of TAG expressed as TAG
oxidation plus total tissue 3H-labeled lipid accumulation
(assuming no other metabolic fate of TAG taken up from the perfusate by
the heart) showed a similar pattern to TAG oxidation, i.e., increased
TAG utilization from endotoxic VLDL, significant in the case of hearts
from endotoxic animals (data not shown), a reflection on the expected
relatively greater contribution of oxidation (>70%; Fig. 2) compared
with tissue pool deposition in heart in the working state. The
proportion of VLDL-TAG oxidized was greater from endotoxic VLDL than
from control VLDL (Fig. 2). Assuming that all TAG in the VLDL was
triolein and that only the TAG (triolein) component of the prepared
VLDL was radiolabeled, this gives a recovery of ~60% for TAG uptake (Fig. 1) vs. TAG utilization in both control and endotoxic groups.
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Cardiac mechanical performance was measured in terms of flow and
pressure. At conditions of fixed preload and afterload, perfusion of
endotoxic hearts with endotoxic VLDL improved cardiac performance by up
to ~100% compared with control VLDL, whereas control hearts had
significantly increased hydraulic work when perfused with endotoxic
VLDL (Fig. 3).
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Cardiac LPL activity was measured in the perfusate at the end of the
60-min perfusion period after heparin administration (heparin
releasable, corresponding to the physiologically active endothelial
portion) and in the heart tissue itself (tissue residual). In addition,
a group of (control) rat hearts were perfused with NEFA, and LPL
activity was similarly measured for comparison (Fig. 4). There was no difference in cardiac LPL
activity between control hearts perfused with NEFA and control VLDL
(± preincubation with endotoxin); however, hearts from
endotoxin-exposed rats had significantly greater LPL activity than
VLDL-perfused control hearts (Fig. 4). Perfusion of the heart with
endotoxic VLDL (VLDL-E; Fig. 4) was associated with a significant
translocation of LPL activity from tissue-residual to
heparin-releasable (endothelial) compartments (P < 0.05), an
effect not observed in hearts perfused with control VLDL or NEFA.
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VLDL both from liver perfusions (as used in heart perfusion
experiments) and from native whole rat plasma (obtained by aortic puncture for comparison) in control and endotoxin-exposed sources was
examined for apolipoprotein and lipid content.
Apolipoproteins derived from a given mass of VLDL-TAG (100 µg) were loaded onto the gel for analysis. Because TAG-rich
lipoproteins such as VLDL contain one copy of apo-B per particle and
TAG is the predominant lipid, this method has the advantage of
permitting limited estimation of particle size. Unlike human VLDL, rat
hepatic VLDL may contain either apo-B100 or the spliced version
apo-B48, and both were therefore examined (Fig.
5). VLDL from liver perfusions had less apo-B (both apo-B48 and apo-B100) per unit mass of TAG than native rat
plasma VLDL (P < 0.05; Fig. 5), implying that VLDL particles produced by liver perfusion in vitro are larger than occur in vivo.
However, endotoxin treatment increased apo-B48 content of the VLDL
relative to TAG in both liver perfusion and native plasma experiments
(P < 0.05; Fig. 5), suggesting that endotoxic apo-B48 VLDL
particles were smaller than corresponding controls. Other major
apolipoproteins are present in variable amounts in VLDL and were
expressed as a proportion of combined apo-B100 + apo-B48 to
correct for particle numbers (Fig. 6).
Apolipoprotein-H was present in relatively high copy number in liver
perfusion-derived VLDL compared with native VLDL, regardless of
endotoxin exposure, whereas endotoxin exposure tended to decrease VLDL
content of apo-E and apo-C (the latter representing combined apo-CII
and CIII), regardless of VLDL origin. This was significant in the case
of apo-C in endotoxic native VLDL compared with control native VLDL
(Fig. 6). VLDL lipid content was expressed as a proportion of total
lipid content; as expected, TAG was found to be the predominant lipid
class present (Fig. 7). Cholesterol content
was increased in VLDL from endotoxic liver perfusions but was not found
in VLDL from endotoxic rat plasma, and the increased cholesterol ester content of native plasma was not seen in liver perfusion VLDL (Fig. 7).
When fatty acid content of VLDL lipids was examined, liver
perfusion-derived VLDL contained more 18:19 (oleate) in
TAG than native VLDL (livers perfused with oleic acid), and this was
further increased by endotoxin exposure, but no other differences in
fatty acid distribution were noted (Fig. 8).
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DISCUSSION |
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A striking feature of sepsis and endotoxinemia is the associated hypertriacylglycerolemia resulting from increased circulating VLDL. Because this occurs by two distinct mechanisms, increased (hepatic) production (7, 14, 21) and decreased peripheral uptake by certain tissues (20), and because both these effects are mediated by inflammatory mediators of the host immune response (9, 13), a teleological function seems likely. Heart is a candidate tissue destination, because cardiac work is increased in the early hyperdynamic phase of sepsis/endotoxinemia (26), and lipids are favored metabolic fuels for the myocardium (24, 30).
A method of producing radiolabeled rat VLDL by perfusing rat liver was
necessarily developed to generate sufficient VLDL for subsequent rat
whole heart perfusions; even with the increased circulating VLDL in
sepsis/endotoxinemia, isolation of native VLDL from rats in vivo for
subsequent heart perfusions was quantitatively impractical. After 8 h
of perfusion, livers remained functional with perfusate flow 10
ml/min, bile flow
1.0 µl/h, and perfusate pH >7.26 (if no
HCO
3 additions were made), with K+ <6 mM and an unchanged lactate-to-pyruvate ratio (data
not shown). VLDL-TAG production was linear, with control livers
producing 13 ± 2 mg TAG/h (n = 8) and endotoxic
livers producing 8 ± 1 mg TAG/h (n = 6; not
significant). Total recovery of
[3H]oleate (oxidized to
3H2O, 3H-labeled ketogenesis,
deposition as tissue 3H-labeled lipids, and
VLDL-3H-labeled lipid production) was >90% with 64% of
[3H]oleate recovery in
VLDL-[3H]TAG (data not shown). The VLDL
produced by liver perfusion was examined for lipid and apolipoprotein
composition; it contained more TAG per particle (i.e., per apo-B, hence
was larger) than native VLDL. This effect has been observed by others
in liver perfusion experiments with unsaturated fatty acids (33, 38) and is presumably a reflection of ongoing hydrolysis of the native VLDL
particle in vivo; relative distribution of lipid classes in the VLDL
particle agrees well with this work (38). The fatty acid composition of
VLDL from the two sources examined differed markedly. As expected, the
predominant fatty acid species (>50%) in VLDL-TAG from liver
perfusions was 18:1
9 (i.e., oleate), derived from the
perfusate; a smaller amount of fatty acid (16:0,
18:2
9,12) was derived from endogenous hepatic stores.
Fatty acid species composition in native VLDL-TAG reflected the wider
range of fatty acids available for VLDL synthesis in
vivo with chow diet. VLDL produced by control liver perfusions appeared
to have a normal complement of apo-E and -C compared with control
native VLDL (relative to apo-B). However, in both liver
perfusion-derived VLDL and native VLDL, previous exposure of donor
animal to endotoxin in vivo produced a modest but significant change in
composition and/or size of the apo-B48 VLDL subtype (smaller particles)
with a resulting decrease in overall VLDL particle. Therefore the
(smaller) apo-B48 VLDL particles produced after endotoxin treatment
carried less TAG. Endotoxin treatment also altered the fatty acid
distribution profile of the VLDL: endotoxic VLDL contained
significantly more (perfusate-derived) oleate than control VLDL (Fig.
8). The mechanism for this effect was not pursued but could be due to
increased uptake of exogenous fatty acid relative to endogenous fatty
acid utilization by the endotoxic liver, or decreased oxidation of the
perfusate fatty acid compared with intrahepatic sources. Endotoxin is
known to affect hepatic lipogenesis and VLDL synthesis in vivo (14),
but contributions of specific fatty acids to this process have not been
previously investigated. Endotoxin also stimulates hepatic
glycogenolysis in perfused rat liver in vitro as a paracrine effect
involving eicosanoids (4), but previous endotoxin treatment of rats in
vivo did not stimulate TAG output or ketogenesis in subsequent liver
perfusion experiments (5).
Endotoxic VLDL was found to be a better substrate for isolated working
hearts than control VLDL in that it was oxidized more rapidly and
supported greater cardiac work; however, the reasons for this were not
immediately apparent. Furthermore, despite taking up more endotoxic
VLDL than control VLDL, control hearts (Fig. 1) did not oxidize (Fig.
2) or incorporate into tissue lipids (Table 1) a similarly greater
proportion of label. The fate of this [3H]TAG
is uncertain. It is possible that some water-soluble products (e.g.,
ketone bodies) were produced (unlikely, given that the heart is not a
ketogenic organ), and endotoxin (and/or LPL) may have mediated
lipoprotein binding/sequestration to the perfusion glassware, but the
most plausible explanation is that the 3H2O
technique underestimates the oxidation rate, especially at higher rates
(11). The efficacy of VLDL as substrate for working myocardium is
poorly documented, because a methodology for generating sufficient
lipoprotein for testing in whole organ systems has not been available.
Using a system similar to the present study, we recently attempted to
quantify cardiac utilization of VLDL (37). VLDL-triolein was utilized
at a rate comparable to chylomicron-triolein but did not support
cardiac mechanical function as efficiently and was oxidized at only
about one-half the rate of NEFA (36, 37). However, utilization of
VLDL-TAG is not insignificant, because a physiological suppression of
VLDL-TAG utilization and oxidation occurs during lactation, when demand
for circulating TAG by the mammary gland is very high, a mechanism
mediated by suppression of cardiac LPL activity and thus also of
chylomicron-TAG utilization, and VLDL-TAG utilization increases again
on litter weaning (37). In the present experiments, differences in
VLDL-TAG utilization were accompanied by only minor changes in cardiac LPL activity. The regulation of cardiac LPL is not completely understood, and alterations in the enzyme activity in this site are
modest compared with changes observed elsewhere (e.g., white adipose
tissue and mammary gland) after pathophysiological/endocrine signaling
(2). We and others previously found increased LPL activity in rat
hearts after endotoxin treatment (36), in polymicrobial sepsis (32),
and in guinea pig hearts after burn injury (1); however, decreased
heart LPL activity in vivo has been reported after high-dose (30
mg/kg) endotoxin treatment for relatively prolonged periods (
24 h)
without change in heart LPL mRNA (17). Endotoxin administered to
perfused rat hearts in vitro decreases myocardial LPL activity, but
only in protein-free perfusate; addition of albumin to the perfusate
abolished the inhibitory effect of LPS (18). Experimental Gram-negative
sepsis decreases myocardial LPL activity in fasted but not fed rats
(21). It is likely that nutritional status, heart work, and/or VLDL
itself may be important physiological regulators of LPL activity in
heart in vivo, but the mechanism for this is unknown. The
finding that perfusion with endotoxic VLDL was associated with
increased translocation of enzyme from inactive tissue-residual depot
to the physiologically active endothelial (heparin-releasable) site may
partly explain the increased TAG assimilation (and hence oxidation)
observed in endotoxic VLDL compared with control VLDL, but the changes are very modest. LPL was measured only at the end of the 60-min perfusion period; at the beginning of the perfusion, the control heart
groups should have identical LPL status, as should the endotoxic heart
groups, but despite this, there is a significant difference in TAG
uptake between the control heart groups and the two endotoxic heart
groups after the perfusion (Fig. 1). Therefore, if LPL is involved, it
must change after VLDL exposure during the perfusion. This is feasible,
because lipoproteins bind LPL and can both remove and replenish
endothelial enzyme. LPL is a notoriously difficult enzyme to assay
reliably, with large interassay variability, and it is possible that
subtle but important shifts were underrepresented by the current data
despite batching of assay samples. However, some workers would argue
that TAG uptake/utilization itself is the most accurate and
physiological assessment of functional LPL activity.
Another possibility is that endotoxic VLDL is a better substrate for
LPL by virtue of a superior structural composition, i.e., of lipid
and/or apolipoprotein. Evidence suggests that large TAG-rich lipoprotein particles are better substrates for LPL than smaller relatively TAG-poor particles; thus chylomicrons outcompete VLDL for
TAG hydrolysis by virtue of their larger size and greater TAG mass. In
the present study, both control and endotoxic VLDL, as generated by
liver perfusion in vitro, were large particles compared with native
VLDL; however, endotoxin exposure made apo-B48 particles smaller, each
particle carrying less TAG but contributing more to the apolipoprotein
pool. The relative efficacy of apo-B48 and apo-B100 VLDL as substrate
for cardiac LPL is not known, but it is possible that apo-B100 VLDL is
more readily assimilated; the smaller size of the endotoxic apo-B48
VLDL would suggest that it is a poorer substrate for LPL. The relevance
of this to the human situation is questionable because human liver
lacks apo-B editing function, and all human VLDL, therefore, contains
apo-B100. Differences in apo-CII/apo-CIII content may also explain
different LPL substrate efficacy, because apo-CII is an obligatory
cofactor and activator of LPL, and apo-CIII is an inhibitor. In the
present studies, endotoxin treatment tended to decrease apo-C content of VLDL. It was not possible to distinguish the apo-C subtypes, because
they have similar electrophoretic mobilities and are codetected, but
apo-CII and apo-CIII have reciprocal ratios in disease states such as
diabetes (27), and because apo-CIII is the predominant subtype, the
decreased apo-C may therefore represent less apo-CIII inhibition of
LPL. Endotoxin treatment also increased 18:19 fatty acid
content of liver perfusion VLDL, an effect not previously noted. It is
possible that a higher oleate-triolein content renders the VLDL a
better substrate for the heart, and this mechanism may be distal to the
action of LPL hydrolysis (i.e., intracardiomyocyte). However, the
relative efficacy of different TAG-derived fatty acid species as
cardiac substrates is unknown. The presence of inflammatory hepatic
acute phase proteins or modified (e.g., oxidized) lipids in VLDL after
endotoxin exposure was not examined in the present study.
A putative portal of entry for VLDL-TAG into the cardiac cell is the VLDL/apo-E receptor (19); however, because no significant differences in apo-E were detected between endotoxic and control VLDL, this argues against a role for this route in the present situation.
Therefore, despite the suggestion that the effect of endotoxin on VLDL utilization by the working rat heart is mediated through the VLDL particle rather than through an effect on the myocardium, it has not been possible to define the mechanism. The possibility remains that a complex interplay of several factors may be involved, and these require further elucidation.
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ACKNOWLEDGEMENTS |
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We are grateful to the Wellcome Trust and the British Journal of Anaesthesia for financial support.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. D. Evans, Nuffield Department of Anaesthetics, University of Oxford, Radcliffe Infirmary, Woodstock Road, Oxford, Oxfordshire OX2 6HE, United Kingdom (E-mail: rhys.evans{at}nda.ox.ac.uk).
Received 5 April 1999; accepted in final form 6 December 1999.
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