Medium-chain triglycerides inhibit free radical formation and
TNF-
production in rats given enteral ethanol
Hiroshi
Kono1,
Nobuyuki
Enomoto1,
Henry D.
Connor1,
Michael D.
Wheeler1,
Blair U.
Bradford1,
Chantal A.
Rivera1,
Maria B.
Kadiiska2,
Ronald P.
Mason2, and
Ronald G.
Thurman1
1 Laboratory of Hepatobiology and Toxicology,
Department of Pharmacology, University of North Carolina at Chapel
Hill, Chapel Hill 27599-7365; and 2 Laboratory
of Pharmacology and Chemistry, National Institute of Environmental
Health Sciences, Research Triangle Park, North Carolina
27709
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ABSTRACT |
This study determined whether free radical formation by
the liver, tumor necrosis factor (TNF)-
production by isolated
Kupffer cells, and plasma endotoxin are affected by dietary saturated fat. Rats were fed enteral ethanol and corn oil (E-CO) or
medium-chain triglycerides (E-MCT) and control rats received corn oil
(C-CO) or medium-chain triglycerides (C-MCT) for 2 wk. E-CO rats
developed moderate fatty infiltration and slight inflammation; however, E-MCT prevented liver injury. Serum aspartate aminotransferase levels,
gut permeability, and plasma endotoxin doubled with E-CO but were
blunted ~50% with E-MCT. In Kupffer cells from E-CO rats, intracellular calcium was elevated by lipopolysaccharide (LPS) in a
dose-dependent manner. In cells from E-MCT rats, increases were blunted
by ~40-50% at all concentrations of LPS. The LPS-induced increase in TNF-
production by Kupffer cells was dose dependent and
was blunted by 40% by MCT. E-CO increased radical adducts and was
reduced ~50% by MCT. MCT prevent early alcohol-induced liver injury,
in part, by inhibition of free radical formation and TNF-
production
by inhibition of endotoxin-mediated activation of Kupffer cells.
tumor necrosis factor-
; intracellular calcium; free radicals; alcohol
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INTRODUCTION |
THE ESTABLISHMENT of a continuous intragastric in vivo
enteral feeding protocol in the rat by Tsukamoto and French (17, 46)
was a major development in alcohol research. With this model, not only
is steatosis observed, which is characteristic of several animal
models, but inflammation and necrosis also occur in ~2-4 wk and
fibrosis begins to develop in 8-10 wk. Inactivation of Kupffer
cells with GdCl3 prevented early alcohol-induced liver injury (1), diminished hypoxia (4), and prevented free radical formation (28). Furthermore, intestinal sterilization with antibiotics (2) or lactobacillus feeding (34) diminished endotoxin and minimized
liver injury. Moreover, treatment with antibody to tumor necrosis
factor (TNF)-
prevented early alcohol-induced liver injury in the
Tsukamoto-French model (24). Alcohol-induced liver injury was also
prevented in TNF receptor-1 knockout mice given enteral ethanol
intragastrically (50). These results are consistent with the hypothesis
that Kupffer cells activated by gut-derived endotoxin play an important
role in the mechanism of alcohol-induced liver injury by producing
TNF-
(43).
Activated Kupffer cells produce mediators, including inflammatory
cytokines, eicosanoids, proteases, and oxygen radicals. Indeed, plasma
levels of TNF-
(6), interleukin (IL)-1 (32), and IL-6 were increased
in patients with severe alcoholic hepatitis, and values correlated with
the clinical course of the disease (22). Calcium is essential for
activation of Kupffer cells (26), which contain voltage-dependent
Ca2+ channels (21), and they are easier to activate after
chronic ethanol treatment (19). Moreover, ethanol causes both tolerance and sensitization of Kupffer cells (15). On the basis of sensitivity to
antibiotics, it was concluded that both of these phenomena were caused
by gut-derived endotoxin and that sensitization involves increases in
the endotoxin receptor CD14 (15).
The type of dietary fat is important in the pathogenesis of alcoholic
liver injury (35). It is known that saturated fat prevents early
alcohol-induced liver injury (37); however, the mechanisms remain
unclear. Medium-chain triglycerides (saturated fat) decreased TNF-
mRNA expression in the liver and prevented liver injury in the
Tsukamoto-French model (38). Accordingly, the purpose of this study was
to determine whether plasma endotoxin levels, TNF-
production by
isolated Kupffer cells, and free radical formation by the liver were
affected by dietary saturated fat. Preliminary accounts of this work
have appeared elsewhere (29).
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MATERIALS AND METHODS |
Animals and diets.
Male Wistar rats (275-300 g, n = 6) were fed enteral
ethanol (10-14
g · kg
1 · day
1)
and a diet (190 kcal · kg body
wt
1 · day
1)
containing either corn oil (unsaturated fat) or medium-chain triglycerides (saturated fat) without supplemental essential fatty acids continuously for up to 2 wk via intragastric feeding using the
enteral protocol developed by Tsukamoto and French (17, 46). Control
rats were fed a high-fat diet containing corn oil or medium-chain
triglycerides without ethanol. A liquid diet described first by
Thompson and Reitz (42) and supplemented with lipotropes as described
by Morimoto et al. was used (33). It contained corn oil or medium-chain
triglycerides as fat (37% of total calories), protein (23%),
carbohydrate (5%), minerals and vitamins, plus ethanol (35-40%
of total calories) or isocaloric maltose-dextrin (control diet) as
described elsewhere (45).
Clinical chemistry.
Ethanol concentration in urine, which is representative of blood
alcohol levels (5), was measured daily. Rats were housed in metabolic
cages that separated urine from feces, and urine was collected over 24 h in bottles containing mineral oil to prevent evaporation. Each day at
9:00 AM, urine collection bottles were changed and a 1-ml sample was
stored at
20°C in a microtube for later analysis. Ethanol
concentration was determined by measuring absorbance at 366 nm
resulting from the reduction of NAD+ to NADH by alcohol
dehydrogenase (7).
Blood was collected via the tail vein once a week and centrifuged.
Serum was stored at
20°C in a microtube until assayed for
aspartate aminotransferase (AST) by standard enzymatic procedures (7).
Endotoxin assay.
For measurement of plasma endotoxin, blood was collected via the portal
vein in pyrogen-free heparinized syringes and centrifuged at 1,200 rpm
for 10 min. Plasma was stored at
20°C in pyrogen-free glass
tubes until measurement of endotoxin using a Limulus amebocyte lysate test kit (Kinetic-QCL, BioWhittaker, Walkersville, MD; Ref. 31).
Pathological evaluation.
After 2 wk of ethanol treatment, liver and gut sections from the small
or large intestine near the cecum were formalin fixed, embedded in
paraffin, and stained with hematoxylin-eosin to assess steatosis,
inflammation, and necrosis. Liver pathology was scored as described by
Nanji et al. (35): steatosis (percentage of liver cells containing
fat): <25% = 1+, <50% = 2+, <75% = 3+, 75%> = 4+;
inflammation and necrosis: one focus per low-power field = 1+, two or
more foci = 2+. Pathology was scored in a blinded manner by one of the
authors and by an outside expert in rodent liver pathology.
Alcohol metabolism.
Ethanol-containing diets were removed immediately before measurement.
Rats were forced to breathe into a closed, heated chamber (37°C)
for 20 s, and 1 ml of breath was collected with a gas-tight syringe.
Concentration of ethanol in breath was determined by gas
chromatography, and rates of alcohol metabolism were calculated from
linear decreases in blood alcohol concentration per unit time using
Widmark's formula as described elsewhere (18).
Gut permeability.
Gut permeability was measured in isolated intestinal segments as
described previously (11). Briefly, 8-cm segments of the intestine were
removed, rinsed with ice-cold saline, inverted, filled with 1 ml of
Tris buffer (in mM: 125 NaCl, 10 fructose, and 30 Tris; pH 7.5), and
ligated at both ends. The filled gut segments were incubated in Tris
buffer containing 0.4 mg/ml horseradish peroxidase (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 (12).
Kupffer cell preparation and culture.
Ethanol was removed 24 h before isolation because Kupffer cells from
rats treated with ethanol acutely for 24 h were sensitized to LPS (15).
Kupffer cells were isolated by collagenase digestion and differential
centrifugation using Percoll (Pharmacia, Uppsala, Sweden) as described
elsewhere, with slight modifications (39). Briefly, the liver was
perfused through the portal vein with Ca2+- and
Mg2+-free Hanks' balanced salt solution (HBSS) at 37°C
for 5 min. Subsequently, the liver was perfused with HBSS containing
0.025% collagenase IV (Sigma Chemical, St. Louis. MO) at 37°C for
5 min. After the liver was digested, it was excised and cut into small pieces and placed in collagenase buffer. The suspension was filtered through nylon gauze, and the filtrate was centrifuged at 450 g for 10 min at 4°C. Cell pellets were resuspended in buffer,
parenchymal cells were removed by centrifugation at 50 g for 3 min, and the nonparenchymal cell fraction was washed twice with buffer.
Cells were centrifuged on a density cushion of Percoll at 1,000 g for 15 min, and the Kupffer cell fraction was collected and
washed with buffer again. Viability of cells determined from trypan
blue exclusion was >90%. Cells were seeded onto 25-mm glass
coverslips and cultured in DMEM (GIBCO-Life Technologies, Grand Island,
NY) supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, and antibiotics (100 U/ml of penicillin G and 100 µg/ml of streptomycin sulfate) at 37°C with 5% CO2. Nonadherent cells were
removed after 1 h by replacing the culture medium. All adherent cells
phagocytosed latex beads, indicating that they were Kupffer cells.
Cells were cultured 24 h before experiments.
Measurement of intracellular calcium.
Intracellular calcium concentration
([Ca2+]i) was measured
fluorometrically using the Ca2+ indicator dye fura 2 and a
microspectrofluorometer (Photon Technology International, South
Brunswick, NJ) interfaced with an inverted microscope (Diaphoto, Nikon,
Japan). Kupffer cells were incubated in modified Hanks' buffer (in mM:
115 NaCl, 5 KCl, 0.3 Na2HPO4, 5.6 glucose, 0.8 MgSO4, 1.26 CaCl2, and 15 HEPES, pH 7.4),
containing 5 µM fura 2-AM (Molecular Probes, Eugene, OR) and 0.03%
Pluronic F127 (BASF Wyandotte, Wyandotte, MI) at room temperature for
60 min. Coverslips plated with Kupffer cells were rinsed and placed in
chambers with buffer at room temperature. Changes in fluorescence intensity of fura 2 at excitation wavelengths of 340 and 380 nm and
emission at 510 nm were monitored in individual Kupffer cells. Each
value was corrected by subtracting the system dark noise and
autofluorescence, assessed by quenching fura 2 fluorescence with
Mn2+ as described previously (15).
[Ca2+]i was determined from the
equation
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where
Fo/Fs is the ratio of fluorescence intensities
evoked by 380-nm light from fura 2 pentapotassium salt loaded in cells using a buffer containing 3 mM EGTA and 1 µM ionomycin
([Ca2+]min) or 10 mM
Ca2+ and 1 µM ionomycin
([Ca2+]max); R is the ratio of
fluorescence intensities at excitation wavelengths of 340 and 380 nm;
and Rmax and Rmin are values of R at
[Ca2+]max and
[Ca2+]min, respectively. The values
of these constants were determined at the end of each experiment, and a
dissociation constant (Kd) of 135 nM was used (20).
TNF-
production by Kupffer cells.
Kupffer cells were seeded onto 24-well plates and cultured in DMEM
supplemented with 10% FBS and antibiotics at 37°C in the presence
of 5% CO2. Cells were incubated with fresh medium
containing LPS (10-10,000 ng/ml supplemented with 5% rat serum)
for an additional 4 h. Medium was collected and kept at
80°C
until assay. TNF-
in the culture medium was measured using an ELISA
kit (Genzyme, Cambridge, MA), and data were corrected for dilution.
Kupffer cell preparation.
Kupffer cells were isolated by collagenase digestion and differential
centrifugation using Percoll as described elsewhere, with slight
modifications (39). Cells were plated on plastic culture dishes and
cultured in RPMI 1640 medium (GIBCO-Life Technologies) supplemented
with 25 mM HEPES, 10% FBS and antibiotics (100 U/ml of penicillin G
and 100 µg/ml of streptomycin sulfate). After 1 h of incubation,
Kupffer cells were scraped with a sterile cell scraper and pelleted by
centrifugation at 500 g for 7 min. Cell pellets were
resuspended in 250 µl of suspension buffer with Triton X-100,
agitated for 15 min at 4°C, and centrifuged at 12,000 g for
10 min at 4°C. The supernatant was removed, and the pellet was
resuspended in suspension buffer. Protein was stored at
20°C for subsequent Western blotting.
Western blotting for CD14.
Extracted proteins (50 µg) were separated by 10% SDS-PAGE and
transferred to polyvinylidene fluoride membranes. Membranes were
blocked by Tris-buffered saline-Tween 20 containing 5% skim milk and
probed with mouse anti-rat ED9 monoclonal antibody (Serotec, Oxford,
UK), followed by HRP-conjugated secondary antibody as appropriate.
Membranes were incubated with a chemiluminescence substrate (ECL
reagent, Amersham Life Science, Amersham, UK) and exposed to X-OMAT
films (Eastman Kodak, Rochester, NY).
Collection of bile and free radical detection.
Ethanol concentration in the breath was analyzed by gas chromatography
to verify that levels were between 200 and 250 mg/dl when experiments
were initiated (18). The rat was anesthetized with pentobarbital
sodium (75 mg/kg), the abdomen was opened, and the spin trap
-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN, 1 g/kg) was administered intravenously. The proximal bile duct was
cannulated with a small length of PE-10 tubing, and bile samples were
collected at 30-min intervals for 3 h into 35 µl of 0.5 mM Desferal
(deferoxamine mesylate) to prevent ex vivo radical formation. Samples were stored at
80°C until analysis of free radical
adducts by electron spin resonance (ESR) spectroscopy (28). Samples were thawed and placed in a quartz flat cell, and ESR spectra were
obtained using a Varian E-109 spectrometer equipped with a TM110
cavity. Instrument conditions were as follows: 20-mW microwave power,
1.0-G modulation amplitude, 80-G scan width, 16-min scan, and 1-s time
constant. Data were collected with an IBM-type computer interfaced to a
spectrometer. Simulations and double integrations of spectra to
determine amplitude were carried out with a computer program (14).
Statistics.
Data are expressed as mean values ± SE. ANOVA or Student's
t-test was used for determination of statistical significance
as appropriate. For comparison of pathological scores, the Mann-Whitney rank-sum test was used. A P value <0.05 was selected before
the study as the level of significance.
 |
RESULTS |
Body weight.
To allow for full recovery from surgery, diets were initiated 1 wk
after surgery. No complications were observed by feeding medium-chain
triglyceride for 2 wk. The mean body weight gain of rats infused with a
high-fat control diet containing corn oil without ethanol was 5.2 ± 0.4 g/day, whereas rats receiving medium-chain triglycerides grew at a
rate of 5.0 ± 0.2 g/day, as expected. The mean body weight gain of
rats fed an ethanol-containing diet was 4.9 ± 0.4 (corn oil) and 4.6 ± 0.2 (medium-chain triglycerides) g/day. There were no significant
differences in body weight gain between control and ethanol-treated groups.
Ethanol concentrations in urine and ethanol metabolism.
Daily urine alcohol concentrations in rats fed ethanol with corn
oil and medium-chain triglycerides are depicted in Fig.
1. As reported previously by several groups
(1, 36, 44), alcohol levels fluctuate in a cyclic pattern from zero to
>300 mg/dl for unknown reasons. Similar patterns were observed
here in rats fed corn oil and medium-chain triglycerides. There were no
significant differences in the cyclic pattern and mean urine alcohol
concentrations between rats fed corn oil (135 ± 6 mg/dl) and
medium-chain triglycerides (140 ± 11 mg/dl).

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Fig. 1.
Representative plot of daily urine alcohol concentrations of
ethanol-fed rats. Urine alcohol concentrations were measured daily as
described in MATERIALS AND METHODS. Data represent means ± SE (n = 6 rats).
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After 2 wk of ethanol, the rate of ethanol elimination was 6.5 ± 0.4 mmol · kg
1 · h
1
in rats fed corn oil and 6.4 ± 0.7 mmol · kg
1 · h
1
in rats fed medium-chain triglycerides. There were no significant differences between groups.
Serum transaminase levels and endotoxin.
In control groups, serum AST levels were ~45 IU/l after 2 wk. Enteral
ethanol with corn oil caused an approximately twofold increase over
control (Fig. 2). In contrast, values were
blunted significantly by ~50% by dietary medium-chain triglycerides.

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Fig. 2.
Effect of chronic ethanol and lipid type on serum aspartate
aminotransferase (AST) levels. Blood samples were collected at 2 wk,
and AST was measured as described in MATERIALS AND METHODS.
Data represent means ± SE (n = 6 rats). CO, corn oil; MCT,
medium-chain triglycerides. *P < 0.05 compared with rats fed
corn oil without ethanol, #P < 0.05 compared with rats fed
corn oil with ethanol by ANOVA and Bonferroni's post hoc test.
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Plasma endotoxin levels were <10 pg/ml in control groups after 2 wk.
In rats fed corn oil with ethanol, however, values were increased
significantly to 89 ± 20 pg/ml (n = 6). In contrast, this
increase was blunted significantly >50% by dietary medium-chain triglycerides (29 ± 3 pg/ml, P < 0.05).
Gut permeability and pathology.
In control groups, gut permeability to HRP was ~50 U/l after 2 wk of the high-fat diet. Enteral ethanol with corn oil caused an
approximately twofold increase over control values. This increase was
significantly blunted to the same values as those for control dietary
medium-chain triglycerides (Fig. 3).

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Fig. 3.
Effect of chronic ethanol and lipid type on gut permeability. Gut
permeability was measured as described in MATERIALS AND
METHODS. Data represent means ± SE (n = 4 rats). HRP,
horseradish peroxidase. *P < 0.05 compared with rats fed corn
oil without ethanol and #P < 0.05 compared with rats fed corn
oil with ethanol by ANOVA and Bonferroni's post hoc test.
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No pathological changes were observed in gut sections from rats fed a
high-fat control diet with corn oil or medium-chain triglycerides for 2 wk (Fig. 4, A and B). In
contrast, destructive structure of the mucosal layer, hemorrhagic
changes, and infiltrating inflammatory cells were observed in
intestinal sections from rats fed ethanol with corn oil (Fig.
4C). Dietary medium-chain triglycerides prevented these
pathological changes nearly completely (Fig. 4D). Hepatic
pathology is summarized in Fig. 5.
Histology was normal in rats fed corn oil or medium-chain triglycerides
without ethanol. In contrast, rats fed corn oil with ethanol developed
moderate fatty infiltration and slight inflammation after 2 wk. On the other hand, rats fed medium-chain triglycerides with ethanol had essentially no liver injury, confirming earlier work (36).

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Fig. 4.
Effect of chronic ethanol and lipid type on gut histology. Gut sections
from rats given high-fat control diet (A: corn oil; B:
medium-chain triglycerides) and high-fat ethanol-containing diet
(C: corn oil; D: medium-chain triglycerides) are shown.
Representative photomicrographs are shown. Original magnification,
×200.
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Fig. 5.
Effect of chronic ethanol and lipid type on hepatic pathology score.
Pathology was scored as described in MATERIALS AND METHODS.
Steatosis and inflammation are shown individually. Data represent means ± SE (n = 6 rats). *P < 0.05 compared with rats fed
corn oil without ethanol and #P < 0.05 compared with rats fed
corn oil with ethanol by Mann-Whitney rank-sum test.
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Effect of chronic ethanol and lipid type on LPS-induced increases in
[Ca2+]i
in isolated Kupffer cells.
Kupffer cells contain voltage-dependent calcium channels (21), and
[Ca2+]i plays an important role in
activation of Kupffer cells (13). Accordingly,
[Ca2+]i in isolated Kupffer cells
was measured. After addition of LPS (10 µg/ml) to Kupffer cells
from rats fed corn oil with ethanol for 2 wk,
[Ca2+]i increased rapidly from
basal values around 10 nM to a maximal values of 338 nM within 60 s
(Fig. 6). This increase was blunted ~50% in cells from rats fed medium-chain triglycerides with ethanol.

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Fig. 6.
Effect of chronic ethanol and lipid type on lipopolysaccharide
(LPS)-induced increase in intracellular Ca2+ concentration
([Ca2+]i) in isolated Kupffer
cells. [Ca2+]i in isolated Kupffer
cells from rats fed ethanol with corn oil or medium-chain triglycerides
was assessed fluorometrically using fura 2 as described in
MATERIALS AND METHODS. Representative traces are shown.
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In cultured Kupffer cells from rats fed corn oil with ethanol, the
increase in [Ca2+]i caused by LPS
was dose dependent, with maximal responses observed with 10 µg/ml LPS
(Fig. 7). In cells from rats fed
medium-chain triglycerides with ethanol, however, the increase in
[Ca2+]i caused by LPS was blunted
by ~50% at all concentrations studied.

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Fig. 7.
Effect of chronic ethanol and lipid type on dose response to
LPS-induced increase in [Ca2+]i in
isolated Kupffer cells. [Ca2+]i in
isolated Kupffer cells was measured fluorometrically using fura 2 as
described in MATERIALS AND METHODS. Changes in
[Ca2+]i after addition of various
doses of LPS (10 ng/ml to 10 µg/ml, supplemented with 5% rat serum)
are plotted. Data represent means ± SE (n = 6 rats).
*P < 0.05 compared with rats fed corn oil with ethanol by
ANOVA and Bonferroni's post hoc test.
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Effect of chronic ethanol and lipid type on TNF-
production by isolated Kupffer cells.
In isolated Kupffer cells from rats fed corn oil with ethanol, TNF-
production caused by LPS was dose dependent, with maximal responses
observed with 10 µg/ml LPS (Fig. 8). In
cells from rats fed medium-chain triglycerides with ethanol, however,
values were blunted significantly by ~40% at the two higher
concentrations studied.

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Fig. 8.
Effect of chronic ethanol and lipid type on dose-response to
LPS-induced tumor necrosis factor- (TNF- ) production in isolated
Kupffer cells. TNF- was measured in culture medium using an ELISA as
described in MATERIALS AND METHODS. Kupffer cells were
isolated and cultured in 24-well culture plates. After 24 h of
incubation, changes in TNF- production at 4 h after addition of
various doses of LPS supplemented with 5% rat serum are plotted. Data
represent means ± SE (n = 4 rats). *P < 0.05 compared with rats fed corn oil with ethanol by ANOVA and Bonferroni's
post hoc test.
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Effects of chronic ethanol and lipid type on free radical formation.
Radical adducts were barely detectable in bile from rats fed an
ethanol-free, high-fat control diet in both groups (data not shown).
After enteral ethanol for 2 wk, however, POBN radical adducts were
about twofold greater in corn oil-treated than in medium-chain
triglyceride-treated rats (Fig. 9).
Computer simulations of these spectra are shown in Fig. 9. Coupling
constants were aN = 15.70 G and
aH
= 2.72 G. Average
ESR signal intensity was measured as the double integral of the two low-field peaks of each spectrum (Fig.
10). Although ethanol treatment significantly increased the intensity of these signals in both groups,
the average intensity from rats fed corn oil was about twofold greater
than values from rats fed medium-chain triglycerides.

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Fig. 9.
Effect of chronic ethanol and lipid type on electron spin resonance
(ESR) spectra in bile. Rats were fed enteral ethanol with corn oil or
medium-chain triglycerides for 2 wk. After POBN (1 g/kg, intravenously)
injection, bile was collected into Desferal (deferoxamine mesylate, 0.5 mM) and analysis of ESR spectra was performed as described in
MATERIALS AND METHODS. Representative ESR spectra are
shown.
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Fig. 10.
Effect of chronic ethanol and lipid type on average radical adduct
signal intensity from bile. Conditions were the same as for Fig. 7. ESR
signal intensity was quantitated as double integral of peaks from bile
samples and was averaged for rats treated as described in
MATERIALS AND METHODS. Data represent means ± SE
(n = 6 rats). *P < 0.05 compared with rats
fed corn oil with ethanol by ANOVA and Bonferroni's post hoc test.
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Effects of chronic ethanol on CD14 expression on the Kupffer cell.
After 2 wk of a high-fat control diet with corn oil or medium-chain
triglycerides, expression of the endotoxin receptor CD14 on Kupffer
cells was low; however, enteral ethanol with corn oil increased CD14
expression significantly about threefold (Fig. 11). These increases were blunted
significantly to a value similar to that of controls by dietary
medium-chain triglycerides.

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Fig. 11.
Effect of chronic ethanol and lipid type on CD14 expression on the
Kupffer cell. Protein extracts (50 µg) from isolated Kupffer cells
were analyzed by Western blotting using mouse anti-rat ED-9 antibody.
A: specific bands for CD14 (55 kDa) are shown. B:
densitometric analysis of CD14 was carried out by image analysis as
described in MATERIALS AND METHODS. Data represent means ± SE (n = 4 rats). *P < 0.05 compared
with rats fed corn oil without ethanol and #P < 0.05 compared
with rats fed corn oil with ethanol by ANOVA and Bonferroni's post hoc
test.
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 |
DISCUSSION |
Effect of dietary medium-chain triglycerides on plasma endotoxin
levels.
Gram-negative bacterial species are a major source of endotoxin in the
gut microflora (9). Alcohol modifies gut flora (10) and increases gut
permeability to normally nonabsorbed substances, leading to an increase
of blood endotoxin levels (8). Indeed, gut permeability was increased
after 2 wk of ethanol in this study (Fig. 3). Blood endotoxin levels
began to increase after 2-4 wk of ethanol treatment in the
Tsukamoto-French model, and a good correlation between blood endotoxin
and pathology has been observed (2). Previous work has shown that
intestinal sterilization with antibiotics (2) and suppression of
endotoxin production with lactobacillus feeding (34) minimized early
alcohol-induced liver injury in the Tsukamoto-French model.
Furthermore, destruction of Kupffer cells by GdCl3
prevented liver injury (1). These data are consistent with the
hypothesis that Kupffer cells activated by gut-derived endotoxin are
involved in the mechanism of early alcohol-induced liver injury (43).
Nutrition and dietary factors are important in the pathogenesis of
alcohol-induced liver disease (16). It is known that saturated fat
prevents early alcohol-induced liver injury; however, mechanisms have
remained unclear (35, 38). It has been reported that medium-chain
triglycerides change brush-border structure in the small intestine (48)
and may affect gut permeability or gut microflora. Indeed, the increase
of gut permeability caused by enteral ethanol was blunted significantly
by medium-chain triglycerides (Fig. 3). Furthermore, dietary
medium-chain triglycerides prevented injury of the intestine caused by
enteral ethanol with corn oil (Fig. 4). Importantly, medium-chain
triglycerides blunted the increase in plasma endotoxin caused by
alcohol by ~50% in this study. Thus it is concluded that
medium-chain triglycerides blunted alcohol-induced liver injury by
preventing increases in plasma endotoxin levels.
Role of endotoxin and Kupffer cells in early alcohol-induced liver
injury.
The major target of endotoxin is the Kupffer cell (30). Endotoxin
activates Kupffer cells via the endotoxin receptor CD14, which is on
the plasma membrane in Kupffer cells. Activated Kupffer cells release
mediators, such as cytokines, eicosanoids, and free radicals, which
induce liver injury. Because destruction of Kupffer cells by
GdCl3 prevented liver injury in the Tsukamoto-French model
(1), it was proposed that activation of Kupffer cells by endotoxin was
responsible for alcohol-induced liver injury. CD14 is upregulated
in Kupffer cells from rats given acute and enteral ethanol (15, 41).
Furthermore, medium-chain triglyceride feeding for 2 wk blunted
CD14 expression on the Kupffer cell (Fig. 11), confirming previous work
by Su et al. (41). Moreover, medium-chain triglycerides significantly
blunted increases in intracellular Ca2+ caused by LPS in
isolated Kupffer cells (Figs. 6 and 7). Thus medium-chain triglycerides
blunt the response of Kupffer cells to endotoxin, in part, by
inhibition of expression of the endotoxin receptor CD14.
Ca2+ is essential for activation of Kupffer cells (26),
which contain voltage-dependent Ca2+ channels (21).
Nimodipine, a dihydropyridine-type Ca2+ channel antagonist,
prevented alcoholic liver injury in the Tsukamoto-French model (25).
These data suggest that [Ca2+]i
plays an important role in mechanisms of alcohol-induced liver injury.
A transient increase of [Ca2+]i is
required for LPS-induced expression of TNF-
(49), which plays a
pivotal role in the inflammatory cytokine cascade (30). TNF-
stimulates endothelial cells to synthesize adhesion molecules, such as
intercellular adhesion molecule 1 (ICAM-1), that induce infiltration of
neutrophils in the liver and cause microcirculatory disturbances
leading to liver injury (51). Indeed, ethanol increased TNF-
mRNA
and ICAM-1 expression in the liver in the Tsukamoto-French model (23).
Furthermore, anti-TNF-
antibody reduced inflammatory cell
infiltration and necrosis in the liver in this model (24). Moreover,
early alcohol-induced liver injury was diminished in TNF receptor-1
knockout mice treated with enteral ethanol (50). Thus evidence
continues to accumulate indicating that TNF-
plays an important role
in alcohol-induced liver injury. Indeed, Nanji et al. (38) reported
that medium-chain triglycerides decreased TNF-
mRNA expression in
the liver in the Tsukamoto-French model. Here, the increase of TNF-
production in isolated Kupffer cells from rats fed ethanol was
decreased significantly by medium-chain triglycerides (Fig. 8). Thus
medium-chain triglycerides are most likely protective by reducing
TNF-
production.
Role of hypoxia and free radicals.
Alcohol causes a hypermetabolic state that could cause hypoxia in the
liver (47). Moreover, Kupffer cells from rats given ethanol produce
eicosanoids that stimulate parenchymal cell oxygen metabolism, leading
to hypoxia and free radical formation (40). Indeed, hypoxia at the
tissue level detected with 2-nitroimidazole hypoxia markers and free
radicals in bile detected by spin trapping and ESR are hallmarks of the
Tsukamoto-French model (3). Moreover, destruction of Kupffer cells with
GdCl3 diminished free radical formation and prevented liver
injury in this model. In the present study, free radical formation was
blunted significantly by dietary medium-chain triglycerides (Figs. 9
and 10). These data are consistent with the hypothesis that hypoxia
after ethanol and free radical formation by Kupffer cells are involved
in early alcohol-induced liver injury.
In conclusion, it is proposed that medium-chain triglycerides prevent
early alcoholic liver injury by inhibition of free radical formation in
the liver and TNF-
production caused by endotoxin-mediated activation of Kupffer cells. This likely involves effects on both the
gut and the cell membrane structure of Kupffer cells, leading to both
decreased plasma endotoxin levels and blunted responsiveness of Kupffer
cells to gut-derived endotoxin (see Fig.
12).

View larger version (25K):
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|
Fig. 12.
Scheme depicting working hypothesis for effect of medium-chain
triglycerides on early alcohol-induced liver injury. Endotoxin
activates Kupffer cells to release inflammatory mediators such as
TNF- , eicosanoids, and free radicals. Increase of endotoxin levels
after ethanol exposure is blunted by medium-chain triglycerides. It is
possible that medium-chain triglycerides affect microflora in the gut,
gut permeability to endotoxin, or endotoxin clearance. Moreover,
because medium-chain triglycerides blunt increase in
[Ca2+]i and production of TNF-
caused by LPS in isolated Kupffer cells, it is likely that medium-chain
triglycerides also alter signaling cascade triggered by LPS binding to
receptors on Kupffer cells, possibly by altering membrane structure.
|
|
 |
ACKNOWLEDGEMENTS |
This work was supported, in part, by grants from the National
Institute of Alcohol Abuse and Alcoholism.
 |
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 and other correspondence: B. U. Bradford,
Lab. of Hepatobiology and Toxicology, Dept. of Pharmacology, CB#7365,
Mary Ellen Jones Bldg., Univ. of North Carolina at Chapel Hill, Chapel
Hill, NC 27599-7365 (E-mail: beub{at}med.unc.edu).
Received 16 February 1999; accepted in final form 7 October 1999.
 |
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