Role of endotoxin in the hypermetabolic state after acute
ethanol exposure
Chantal A.
Rivera,
Blair U.
Bradford,
Vitor
Seabra, and
Ronald G.
Thurman
Laboratory of Hepatobiology and Toxicology, Department of
Pharmacology, The University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina 27599
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ABSTRACT |
This study investigated the role of endotoxin
in the hypermetabolic state or swift increase in alcohol metabolism
(SIAM) due to acute ethanol exposure. Female Sprague-Dawley rats
(100-120 g) were given ethanol (5 g/kg) by gavage. Endotoxin
measured in plasma from portal blood was not detectable in
saline-treated controls; however, 90 min after ethanol, endotoxin was
increased to 85 ± 14 pg/ml, and endotoxin clearance was diminished
by ~50%. Oxygen uptake in perfused livers was increased 48% by
ethanol, and production of PGE2 by
isolated Kupffer cells was increased similarly. These effects were
blunted by elimination of gram-negative bacteria and endotoxin with
antibiotics before ethanol administration. To reproduce ethanol-induced
endotoxemia, endotoxin was infused via the mesenteric vein at a rate of
2 ng · kg
1 · h
1.
Endotoxin mimicked the effect of ethanol on oxygen uptake. The specific
Kupffer cell toxicant GdCl3
completely prevented increases in oxygen uptake due to endotoxin. These
findings demonstrate that endotoxin plays a pivotal role in SIAM, most
likely by stimulating eicosanoid release from Kupffer cells.
Kupffer cells; liver; antibiotics; eicosanoids; gadolinium chloride
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INTRODUCTION |
ENDOTOXIN IS A cell wall component of gram-negative
bacteria that is cleared from the systemic circulation largely by
Kupffer cells, the resident macrophages of the liver. Kupffer cells are activated by endotoxin to release toxic free radicals and cytokines that may cause injury to the surrounding parenchyma (11). For example,
findings using an experimental model of chronic ethanol exposure
demonstrated that destruction of Kupffer cells diminished radical
formation and blunted liver injury (1). It is well known that a
positive correlation exists between the extent of hepatic injury due to
chronic ethanol exposure and blood levels of endotoxin in rats and
humans (16, 20). Moreover, treatment of rats with agents that minimize
gram-negative bacteria and endotoxin abolished alcohol-induced liver
injury (2). Taken together, these findings support the hypothesis that
Kupffer cells mediate many of the toxic effects of alcohol on the liver
and endotoxin is the likely stimulus for Kupffer cell activation.
Previous work has shown that acute ethanol exposure causes a swift
increase in alcohol metabolism (SIAM) characterized by increases in
oxygen uptake and ethanol metabolism along with decreased rates of
glycolysis (32). Alterations of the hepatic metabolic state have been
attributed to the release of mediators such as prostaglandins from
activated Kupffer cells (8, 23). In support of this idea, prior
destruction of Kupffer cells prevented the hypermetabolic state due to
ethanol (5). However, whether endotoxin is a stimulus for Kupffer cell
activation during acute ethanol exposure is not known. Therefore, the
present study was conducted to test the hypothesis that acute ethanol
exposure increases circulating endotoxin to levels sufficient to
activate Kupffer cells and induce a hypermetabolic state in the liver.
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METHODS |
Animal treatment. Ethanol (5 g/kg) or
an equal volume of saline was administered by gavage to female
Sprague-Dawley rats (100-120 g). One experimental group was
treated with the antibiotics polymixin B (150 mg · kg
1 · day
1)
and neomycin (450 mg · kg
1 · day
1)
by gavage and in the drinking water for 5 days before ethanol to
eliminate gram-negative bacteria and endotoxin (2). Blood ethanol
levels were determined from the concentration of ethanol in breath
measured by gas chromatography at 15-min intervals (14). After 2.5 h,
livers of saline- or ethanol-treated rats were perfused via the portal
vein with Krebs-Henseleit buffer (pH 7.4, 37°C) saturated with 95%
oxygen-5% carbon dioxide in a hemoglobin-free, nonrecirculating system
(25). The superior hepatic vena cava was also cannulated, and oxygen
uptake was measured in the effluent perfusate as it flowed past a
Clark-type oxygen electrode. Ethanol was infused in the portal vein at
a final concentration of 2 mM; ethanol metabolism was determined from
the difference between the influent and effluent concentrations by
standard enzymatic procedures (4). All rats used in this study received
humane care in accordance with institutional guidelines.
Endotoxin measurement. To measure
endotoxin, rats were anesthetized, and heparinized blood samples (50 U/ml blood) were collected from the portal vein 30, 60, 90, 120, or 150 min after administration of saline or ethanol
(n = 5 rats/time point). Blood was
centrifuged at 150 g for 10 min, and
platelet-rich plasma was stored at
80°C. Just before assay,
plasma samples were diluted 1:10 and heated to 75°C for 10 min to
denature endotoxin-binding proteins that interfere with the assay (12).
Tubes used for sample collection, storage, and assay preparation were
borosilicate glass heated to 200°C overnight to destroy endotoxin.
Endotoxin was measured kinetically using a chromogenic test based on
the limulus amebocyte lysate assay (BioWhittaker). Pyrogen-free water
and pooled normal rat plasma were used as controls. The concentration
of endotoxin in each sample was calculated from a standard curve
prepared for each assay.
Endotoxin clearance. Two hours after
treatment of rats with saline or ethanol, blood samples were drawn from
the tail vein to establish basal endotoxin values. Endotoxin (3 µg/kg
lipopolysaccharide; Escherichia coli
serotype 0111:B4; Sigma Chemical, St. Louis, MO) suspended in
pyrogen-free saline was administered via the tail vein. Blood samples
were collected from the inferior vena cava 2, 4, 7, and 10 min after
injection of endotoxin, and clearance was measured in platelet-rich
plasma as described above.
Endotoxin infusion. Endotoxin was
infused via the portal vein according to the method of Arita et al.
(3). Briefly, rats were anesthetized, and the mesenteric vein was
cannulated with siliconized PE-10 tubing that was advanced to the level
of the portal vein. The cannula was attached to an infusion pump via PE-60 tubing. Endotoxin or an equal volume of pyrogen-free saline (vehicle control) was infused at a rate of 2 ng · kg
1 · h
1
for 90 min to approximate levels of endotoxin observed during acute
ethanol exposure. After a 30-min recovery period, livers were perfused,
and oxygen uptake was measured. In one experimental series, rats were
treated with the specific Kupffer cell toxicant GdCl3 (10 mg/kg) 24 h before the
infusion of endotoxin. GdCl3 was
dissolved in acidic saline and administered via the tail vein.
Gut permeability. Gut permeability was
measured in isolated intestinal segments using horseradish peroxidase
(HRP; 40,000 molecular weight), as described previously (6). Briefly,
4-cm segments of ileum were everted, filled with 1 ml of Tris buffer (125 mM NaCl, 10 mM fructose, and 30 mM Tris; pH 7.5), and ligated at
both ends. The filled gut segments were incubated in Tris buffer containing 40 mg/ml HRP. After 45 min, gut sacs were removed and blotted lightly to eliminate excess HRP, and the contents (~750 µl)
of each sac were collected carefully using a 1-ml syringe. HRP activity
in the contents of each sac was determined spectrophotometrically from
the rate of oxidation of pyrogallol as described elsewhere (10).
Prostaglandin measurement. Ethanol (5 g/kg) or an equal volume of saline was administered by gavage to
control (untreated) and antibiotic-treated rats (see Animal
treatment). Two hours later, Kupffer cells were isolated by
collagenase digestion and differential centrifugation as described
previously (22). The nonparenchymal cell fraction was centrifuged
through a Percoll gradient at 1,000 g
for 15 min. Viability determined by trypan blue exclusion was >90%.
Cells were seeded on 60-mm culture dishes, and culture medium was
exchanged after 1 h to remove nonadherent cells. Kupffer cells were
incubated in RPMI-1640 supplemented with 10% fetal bovine serum, 100 U/ml penicillin G, and 100 µg/ml streptomycin sulfate. After 0, 2, or
4 h, the supernatant was collected and stored at
80°C for
later determination of PGE2 by
radioimmunoassay (29).
Statistical analysis. Results were
analyzed using two-way ANOVA, one-way ANOVA, or Mann-Whitney's
rank-sum test as appropriate. P < 0.05 was selected before the study to reflect significance.
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RESULTS |
Ethanol increases circulating
endotoxin. After a bolus dose of ethanol, blood ethanol
levels increased rapidly, reached a maximum value of 215 ± 12 mg/100 ml after 1 h, and declined steadily over the next 3 h as
expected (Fig.
1A).
Endotoxin was measured in blood samples taken from the portal vein
using the limulus lysate assay as described in METHODS and
was undetectable in rats treated with saline. However, there was a
marked transient increase in portal blood endotoxin levels beginning
~30 min after ethanol administration. Endotoxin reached a peak value
of 85 ± 14 pg/ml after 90 min and declined to 9 ± 9 pg/ml after
~3 h (Fig. 1B). Endotoxin reached
maximal levels just before the peak of oxygen uptake (Fig.
1C).

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Fig. 1.
Time course of blood ethanol and endotoxin levels and oxygen uptake
after acute ethanol administration. A:
rats were given 5 g/kg ethanol ig, and the concentration of ethanol was
determined by gas chromatography at 30-min intervals for 3 h. Blood
ethanol values were calculated from the concentration in breath. In
separate experiments, blood samples were collected directly from the
portal vein before and 30, 60, 90, 120, or 180 min after ethanol or
saline. B: endotoxin was measured in
platelet-rich plasma using the limulus amebocyte lysate assay;
n = 5 rats at each time point.
C: effect of ethanol on oxygen uptake.
Data were modified from Yuki and Thurman (32). Rats were given a 5 g/kg
dose of ethanol, and livers were perfused at times indicated. Mean ± SE is given; n = at least 5 rats/time point.
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Antibiotics prevent the hypermetabolic state induced
by ethanol. To index the hypermetabolic state, oxygen
uptake and ethanol metabolism were measured in livers isolated from
rats 2.5 h after treatment with saline or ethanol. Representative
traces of hepatic oxygen uptake are shown in Fig.
2. Oxygen uptake reached steady-state levels after ~10 min of perfusion and was 128 ± 10 µmol · g
1 · h
1
in livers from saline-treated rats; antibiotics did not alter basal
rates of oxygen uptake (130 ± 13 µmol · g
1 · h
1).
Ethanol increased the rate of hepatic oxygen uptake to 190 ± 16 µmol · g
1 · h
1
as expected (Fig.
3A). In
rats treated with antibiotics for 5 days before given ethanol, however,
oxygen uptake only reached 139 ± 6 µmol · g
1 · h
1
(Fig. 3A). After the infusion of
ethanol directly in the perfused liver, the rate of ethanol elimination
was 37 ± 5 and 47 ± 3 µmol · g
1 · h
1
in saline- and antibiotic-treated rats, respectively (Fig.
3B). The rate of ethanol elimination
was increased significantly in livers of ethanol-treated rats to 68 ± 5 µmol · g
1 · h
1,
an effect largely blocked by antibiotics (48 ± 3 µmol · g
1 · h
1).

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Fig. 2.
Representative traces of hepatic oxygen consumption by isolated
perfused liver. Typical traces of oxygen uptake by perfused livers of
rats treated with saline, ethanol, or ethanol + antibiotics are shown.
Traces from livers of rats treated with saline + antibiotics were
similar to saline controls (data not shown). Oxygen uptake was measured
continuously in the effluent perfusate using a Clark-type oxygen
electrode.
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Fig. 3.
Antibiotics blunt the stimulation in oxygen and ethanol uptake due to
acute ethanol (EtOH) exposure. Rats were given polymixin B and neomycin
as described in METHODS. Oxygen uptake
(A) and ethanol metabolism
(B) were measured 2.5 h after 5 g/kg
ethanol as described in METHODS;
n = 6 rats;
* P < 0.01 compared with
control; # P < 0.05 compared
with ethanol. Data were analyzed using 2-way ANOVA with Bonferroni's
post hoc test.
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Effect of ethanol on PGE2 production by
isolated Kupffer cells.
In a previous study, it was shown that the release of
PGE2, which stimulated hepatocyte
oxygen uptake, was increased in Kupffer cells isolated from rats after
chronic ethanol exposure (23). To determine if antibiotics block
PGE2 production due to acute ethanol, Kupffer cells were isolated from control or antibiotic-treated rats 2 h after treatment with saline or ethanol. The time course of
PGE2 production by isolated
Kupffer cells is shown in Fig. 4. Kupffer
cells from saline-treated rats produced 30 ± 6 and 47 ± 3 pg/ml
after 2 or 4 h, respectively. Treatment of rats with ethanol 2 h before
Kupffer cell isolation increased
PGE2 production significantly,
whereas antibiotics blunted the ethanol-stimulated release of
PGE2 to control levels.

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Fig. 4.
Effect of ethanol on PGE2
production by isolated Kupffer cells. Kupffer cells were isolated 2 h
after saline or ethanol treatment and were cultured for up to 4 h.
PGE2 was measured in the culture
medium by radioimmunoassay; n = 5 rats; * P < 0.05 using 2-way
ANOVA with Bonferroni's post hoc test.
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Treatment of rats with endotoxin in vivo stimulates
hepatic oxygen uptake. To mimic mild endotoxemia caused
by ethanol, endotoxin was infused in the portal vein at a rate of 2 ng · kg
1 · h
1
for 90 min. The total volume administered over the 90-min infusion was
negligible (130 µl). Samples of portal blood were collected immediately after infusion and were analyzed for endotoxin. Levels of
endotoxin were 145 ± 91 pg/ml and were not significantly different from levels measured 90 min after ethanol (85 ± 14). Oxygen uptake in livers isolated from rats infused with pyrogen-free saline was 98 ± 4 µmol · g
1 · h
1
(Fig. 5), whereas low-dose endotoxin
infusion significantly increased oxygen uptake to 234 ± 32 µmol · g
1 · h
1.
To determine if increases in hepatic oxygen uptake due to endotoxin are
dependent on Kupffer cells, one experimental group was treated with the
specific Kupffer cell toxicant
GdCl3 24 h before giving ethanol.
Treatment of rats with GdCl3
completely prevented the increase in oxygen uptake due to exogenous
endotoxin infusion (112 ± 12 µmol · g
1 · h
1).

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Fig. 5.
Effect of in vivo endotoxin infusion on hepatic oxygen uptake by the
isolated perfused liver. Endotoxin (2 ng · kg 1 · h 1)
or an equal volume of pyrogen-free saline was infused directly in the
portal vein for 90 min. One experimental group was treated with
GdCl3 before infusion as described
in METHODS. Livers were perfused, and oxygen uptake was
measured; n = 5 rats;
* P < 0.05 compared with
saline; # P < 0.05 compared
with the endotoxin group using Kruskal-Wallis 1-way ANOVA.
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Ethanol diminishes endotoxin
clearance. In an attempt to determine the cause(s) of
increases in circulating endotoxin, gut permeability to HRP was
evaluated. In contrast to more commonly used small-molecular
weight-markers, HRP approximates the movement of large molecules such
as endotoxin and has been shown to penetrate the mucosal barrier via
both transcellular and paracellular pathways (28). In the present
study, the amount of HRP in gut sacs of saline-treated control rats
after the 45-min incubation period was 1.7 ± 0.3 U/ml. There was no
significant increase in movement of HRP across the intestinal mucosa
after ethanol treatment (1.5 ± 0.3 U/ml). Because HRP is much
smaller than endotoxin, it is unlikely that ethanol increases the
absorption of endotoxin. Therefore, the effect of ethanol on the rate
of clearance of an injected dose of endotoxin was evaluated. Because a
high degree of variability in rates of clearance was observed when rats
were injected with endotoxin at levels achieved after ethanol
administration, slightly larger amounts were given to produce more
consistent results. Peak blood endotoxin levels were 30 ± 2 ng/ml
in saline-treated rats and declined to 14 ± 3 ng/ml within 10 min
(Fig.
6A).
Ethanol significantly increased peak endotoxin to 49 ± 4 ng/ml, and
the rate of endotoxin clearance was blunted by ~50% (Fig. 6,
A and B). Accordingly, the area under the
endotoxin elimination curve was increased approximately twofold by
ethanol (Fig. 6C).

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Fig. 6.
Effect of ethanol on endotoxin clearance. Two hours after saline or
ethanol treatment, endotoxin (3 µg/kg) was administered to rats via
the tail vein. Rats were anesthetized, and blood samples were collected
from the vena cava after 2, 4, 7, and 10 min. Endotoxin was measured in
plasma as described in METHODS. Data are presented as means ± SE of at least 4 observations.
A: time course of endotoxin clearance.
* P < 0.05 using 2-way
repeated-measures ANOVA. B: linear
regression of clearance data from A.
Slopes of the linear regression of saline and ethanol data are 0.07 and
0.03, respectively. C: average area
under the curve; n = 5 rats;
* P < 0.05 using
Mann-Whitney's rank-sum test.
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DISCUSSION |
Acute ethanol induces mild
endotoxemia. It is well known that endotoxemia is
associated with Kupffer cell activation and liver pathology due to
chronic ethanol exposure (2, 16, 20). The purpose of the present study
was to investigate the role of endotoxin in SIAM after acute ethanol
exposure. Induction of transient mild endotoxemia was observed after a
single dose of ethanol (Fig. 1). Endotoxin concentrations in portal
blood reached peak levels before maximal induction of SIAM, which
occurs ~2.5 h after ethanol (Fig. 3,
B and
C). Elimination of gram-negative
bacteria and endotoxin prevented the hypermetabolic state (Fig. 3).
Moreover, the effect of ethanol on oxygen uptake was mimicked by
administering endotoxin at blood levels of endotoxin achieved after
ethanol treatment (Fig. 5). Although the participation of other
mediators such as catecholamines cannot be ruled out, these findings
provide evidence that endotoxemia plays a pivotal role in the
hypermetabolic state caused by ethanol.
Endotoxin mimics the effects of acute ethanol
exposure. It is well known that endotoxemia resulting
from burns, trauma, and sepsis is associated with a hypermetabolic
state in humans (9, 24). Arita et al. (3) reported that endotoxemia
significantly increased the resting metabolic expenditure of guinea
pigs. However, these findings do not reflect the effects of ethanol,
since levels of endotoxin used were as much as 1,000-fold higher than
would be expected after acute ethanol exposure (Fig.
1B). Here, a model of intravenous
infusion was employed to create mild endotoxemia. Blood levels of
endotoxin achieved as a result of infusion were comparable to levels
observed after treatment of rats with ethanol for similar times (Fig.
1). Infusion of endotoxin in this manner stimulated a Kupffer
cell-dependent hypermetabolic state (Fig. 5). These findings
demonstrate that even very mild endotoxemia is sufficient to increase
hepatic oxygen uptake.
Mechanism of ethanol-induced
endotoxemia. Endotoxin resides in the lower gut where
gram-negative bacteria predominate. Under pathological conditions that
compromise gut mucosal integrity, endotoxin can escape into the portal
blood system where it is cleared largely by Kupffer cells, which are
strategically placed along the sinusoids (31); endothelial cells also
clear endotoxin to a lesser extent (27). Removal and catabolism of
endotoxin by macrophages are primarily achieved through phagocytosis
via scavenger receptors as reviewed elsewhere (15). Phagocytosis by
Kupffer cells in vitro is inhibited by the addition of ethanol directly
to the culture medium (18). Moreover, acute tolerance to tumor necrosis
factor-
production by isolated Kupffer cells caused by ethanol was
blocked by antibiotics. Treatment with ethanol in vivo reduced the rate
of endotoxin clearance of high levels of endotoxin in rats and septic
mice (13, 21). Results presented here support the findings that acute
ethanol blunts endotoxin clearance (Fig. 6). Because no alteration in
ileal permeability to HRP was noted, data presented here support the
idea that the observed accumulation of endotoxin in portal blood is
most likely due to decreased phagocytosis by macrophages. However,
changes in permeability of the lower gut where endotoxin predominates cannot be ruled out.
Kupffer cell participation in the hypermetabolic state
is mediated by endotoxin. Destruction of Kupffer cells
with GdCl3 largely blocked SIAM in
a previous study (5), demonstrating that the hypermetabolic state is
Kupffer cell dependent. However, the mechanism of Kupffer cell
activation during ethanol exposure has not been identified. As
demonstrated in Fig. 5, SIAM can be mimicked with low-dose endotoxin
infusion; this phenomenon is also blocked by destruction of Kupffer
cells. Because antibiotics also blunted SIAM, it is concluded that
endotoxin is responsible for activating Kupffer cells during acute
ethanol exposure.
Endotoxin binding to CD14 receptors on Kupffer cells is known to be a
potent activator of these cells. For endotoxin to be recognized by
CD14, it must first form a complex with lipopolysaccharide-binding protein (LBP; see Ref. 26). Recent studies demonstrated that ethanol
exposure upregulated serum LBP levels after only 2 h; however, there
was no change in Kupffer cell CD14 expression (17). Because acute
ethanol decreases endotoxin clearance, endotoxin increases in blood and
binds to LBP. Kupffer cell activation by the endotoxin-LBP complex
binding to CD14 is the most likely outcome. The consequence of the
interaction of endotoxin with Kupffer cells via CD14 is the release of
free radicals, cytokines, and eicosanoids (30). Eicosanoids play an
important role in stimulating hepatic metabolism during endotoxemia (7,
8, 24). One of the major eicosanoids produced is
PGE2, which is responsible for the
stimulation of hepatic respiration after ethanol exposure (23).
Eicosanoids are primarily synthesized by Kupffer cells (66%) but also
by hepatic endothelial cells (22%) and to a lesser extent by
parenchymal cells (16%; see Ref. 19). Elimination of gram-negative
bacteria and endotoxin from the gut before ethanol exposure prevented
the production of PGE2 by isolated
Kupffer cells (Fig. 4) and blunted the hypermetabolic state (Fig. 3),
supporting the hypothesis that endotoxin activates Kupffer cells to
release eicosanoids that trigger SIAM.
Working hypothesis. The proposed role
of endotoxin and Kupffer cells in SIAM is summarized in Fig.
7. It is concluded that acute ethanol
administration rapidly diminishes the phagocytic ability of Kupffer
cells, probably by inhibiting scavenger receptor function. This results
in higher circulating levels of endotoxin, which complex with LBP.
Elevated endotoxin-LBP complexes then bind to CD14 receptors on Kupffer
cells and trigger the release of mediators such as
PGE2, which stimulate hepatic
oxygen consumption and ethanol metabolism. This study provides the
first direct evidence that endotoxemia plays a critical role in the
hypermetabolic state after acute ethanol exposure and supports the
hypothesis that endotoxin mediates many of the hepatic effects of
ethanol.

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Fig. 7.
Proposed mechanism of the effect of alcohol and endotoxin on Kupffer
cell activity. Data demonstrate that endotoxin (Etx) clearance,
probably via the scavenger pathway, is diminished by alcohol, leading
to a transient increase in blood endotoxin levels. Ethanol also
increases lipopolysaccharide-binding protein (LBP), an opsonin
necessary for endotoxin binding to CD14 receptors. Enhanced Kupffer
cell activation via endotoxin-CD14 complexes is a likely result of
these phenomena. It is well known that endotoxin-LBP binding to CD14
causes an increase in intracellular calcium in Kupffer cells, an
important second messenger in the production of mediators such as
PGE2. This occurs largely via
voltage-dependent calcium channels (VDCC). Here, acute ethanol
stimulated PGE2 production by
isolated Kupffer cells, an effect blocked by elimination of
gram-negative bacteria and endotoxin with antibiotics. Importantly,
findings reported here provide direct evidence that the hepatic
hypermetabolic state (swift increase in alcohol metabolism) caused by
ethanol exposure is mediated by endotoxin and involves the release of
factors such as PGE2, which is
known to stimulate oxygen uptake in hepatocytes.
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ACKNOWLEDGEMENTS |
This work was supported, in part, by National Institute of Alcohol
Abuse and Alcoholism Grant AA-03624.
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FOOTNOTES |
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: R. G. Thurman, Mary Ellen Jones Bldg. CB#
7365, Chapel Hill, NC 27599-7365.
Received 21 April 1998; accepted in final form 7 August 1998.
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