Fatty liver vulnerability to endotoxin-induced
damage despite NF-
B induction and inhibited caspase 3 activation
Shiqi
Yang,
Huizhi
Lin, and
Anna Mae
Diehl
Department of Medicine, The Johns Hopkins University, Baltimore,
Maryland 21205
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ABSTRACT |
Fatty livers
are sensitive to lipopolysaccharide (LPS) damage. This study tests the
hypothesis that this vulnerability occurs because protective,
antiapoptotic mechanisms are not upregulated appropriately.
Genetically obese, leptin-deficient ob/ob mice, a model for
nonalcoholic fatty liver disease, and their lean litter mates were
treated with a small dose of LPS. General measures of liver injury,
early (i.e., cytochrome c release) and late (i.e., activation of caspase 3) events that occur during hepatocyte
apoptosis, and various aspects of the signal transduction
pathways that induce nuclear factor-
B (NF-
B) and several of its
antiapoptotic transcriptional targets (e.g., inducible nitric oxide
synthase, bfl-1, and bcl-xL) were compared. Within 0.5-6 h after
LPS exposure, cytochrome c begins to accumulate in the
cytosol of normal livers, and procaspase 3 cleavage increases.
Coincident with these events, kinases (e.g., AKT and Erk-1 and -2) that
result in the degradation of inhibitor
-B are activated; NF-
B
activity is induced, and NF-
B-regulated gene products accumulate.
Throughout this period, there is negligible histological evidence of
liver damage, and serum alanine aminotransferase values barely increase
over baseline values. Although ob/ob livers have significant
histological liver injury and 11-fold greater serum alanine
aminotransferase values than those of lean mice by 6 h post-LPS,
they exhibit greater activation of AKT and Erk, more profound
reductions in inhibitor
-B, enhanced activation of NF-
B, and
greater induction of NF-
B-regulated genes. Consistent with this
heightened antiapoptotic response, increases in cytochrome c and procaspase 3 cleavage products are inhibited. Together
with evidence that ob/ob hepatocytes have a reduced ATP content and undergo increased lysis after in vitro exposure to tumor necrosis factor-
, these findings suggest that fatty livers are sensitive to
LPS damage because of vulnerability to necrosis, rather than because of apoptosis.
nuclear factor-
B; obesity; nonalcoholic fatty liver
disease
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INTRODUCTION |
FATTY LIVER IS
THE EARLIEST, and most common, form of both alcoholic liver
disease and nonalcoholic fatty liver disease (NAFLD) (44,
45). Inflammation and hepatocyte death (i.e., hepatitis) are
inconspicuous in fatty livers and, hence, the prognosis of hepatic
steatosis is generally benign (49, 66). However, fatty livers are unusually vulnerable to injury from various causes, and when
hepatitis develops, the probability of eventual, liver-related morbidity and mortality increase dramatically (46, 52,
57). Thus the transition from steatosis to steatohepatitis is an
important, rate-limiting step in the progression of both alcoholic
liver disease and NAFLD. To clarify the mechanisms involved in this process, our laboratory has been studying genetically obese ob/ob mice.
Ob/ob mice have a spontaneous mutation in the ob gene that prevents synthesis of the satiety hormone leptin (8).
Because ob/ob mice are also type 2 diabetic and hypertriglyceridemic
(18, 59), these animals have three of the major risk
factors for NAFLD in humans (1, 57). Also, similar to the
fatty livers of obese or alcoholic humans (50), ob/ob
fatty livers are unusually vulnerable to injury from lipopolysaccharide
(LPS) endotoxin (70). Treatment of ob/ob mice with small
doses of LPS that are relatively innocuous to their lean littermates
produces severe steatohepatitis within 24 h, providing a
convenient model to investigate the mechanisms that mediate the early
stages of NAFLD.
LPS liver injury requires the proinflammatory cytokine tumor necrosis
factor-
(TNF-
) (47). TNF-
kills cells by
activating caspases that cause apoptosis (3, 24,
25). However, healthy hepatocytes normally are not killed by
TNF-
because, when they are exposed to TNF-
, they activate
antiapoptotic transcription factors, such as nuclear factor-
B
(NF-
B) (68). NF-
B, in turn, upregulates the
synthesis of antiapoptotic members of the bcl-2 family that prevent
the release of cytochrome c from mitochondria. In addition,
NF-
B increases the transcription of genes that encode protective
enzymes, such as inducible nitric oxide synthase (iNOS) (20,
30), whose products prevent the activation of the
proapoptotic caspases, including caspase 3 (7, 48), a
major effector of TNF-
-mediated hepatocyte apoptosis
(28). Hence, fatty livers might be more vulnerable to
cytokine-mediated liver injury because the normal induction of TNF-
survival pathways is inhibited, permitting the unrestrained activation
of TNF-
's apoptotic pathways. To test this hypothesis,
we treated lean and ob/ob mice with a small dose of LPS and then
compared the degree of liver damage and activation of various
components in the anti- and proapoptotic pathways in the livers of
the two groups of mice. Consistent with earlier reports (17,
70), ob/ob livers develop significantly more damage than normal
livers after LPS exposure. However, our other findings were unexpected
and demonstrate that, after LPS, ob/ob livers exhibit superinduction of
antiapoptotic kinases, NF-
B, and NF-
B target genes
(including the antiapoptotic bcl-2-related proteins bfl-1 and
bcl-xL and iNOS). This heightened antiapoptotic response is associated with a reduced cytosolic accumulation
of cytochrome c and inhibited caspase 3 activation.
Subsequent experiments with isolated hepatocytes from control and fatty
livers demonstrated that fatty hepatocytes are ATP depleted and undergo
more lysis when exposed to TNF-
in vitro. Together, the results of
these in vivo and in vitro studies refute our original hypothesis that fatty livers are vulnerable to apoptosis. Rather, our findings are more consistent with the possibility that hepatic steatosis increases cellular vulnerability to necrosis.
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MATERIALS AND METHODS |
Animal studies.
Adult (8- to 10-wk-old) male ob/ob C57BL-6 mice and their lean
littermates were purchased from Jackson Laboratories (Bar Harbor, ME).
Animals were permitted ad libitum access to standard pellet chow and
were housed in temperature- and light-controlled animal facilities. On
the day of the experiment, 12 ob/ob mice and 12 lean mice were injected
with LPS (12 µg/mouse ip), and then 4 mice/group were killed at
various time points (0.5, 1, or 6 h after LPS). These time points
were selected for evaluation to monitor the early responses to LPS
administration because our earlier work had shown that this dose of LPS
significantly increased liver damage and mortality in ob/ob mice
12-24 h after treatment (70). Serum activity of
alanine aminotransferase (ALT) and histological evidence of liver
injury were evaluated in each mouse to verify that the expected
differences in liver damage were reproduced in the present experiment.
An additional 4 ob/ob and 4 lean untreated mice were killed to compare
basal (time 0) differences in the treatment end points. At
death, liver and sera were freeze clamped in liquid nitrogen and stored
at
80°C until analysis.
Liver protein isolation and immunoblot analysis.
Liver tissue from each mouse was used to obtain whole liver protein,
cytosolic protein, and nuclear protein. Whole liver homogenates were
prepared by homogenizing frozen tissue in homogenization buffer (50 mM
HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 10% glycerol, 5 mM EGTA, 1% Triton X-100) on ice for 10 min, followed by
centrifugation in a microfuge for 15 min. Cytosolic proteins were
isolated from the liver homogenates by ultracentrifugation at 55,000 rpm for 80 min in a TL-100 rotor (Beckman Instruments). Nuclear
proteins were prepared from the same tissues according to the method of Lavery and Schibler (35), as our laboratory described
(41). Proteins were isolated from ob/ob and lean mouse
liver tissues concurrently, and the protein concentration in each
extract was determined with dye-binding assays using reagents from
Pierce Chemical (Rockford, IL). To evaluate variations in the
expression of specific proteins, immunoblot analysis was performed.
Liver proteins in Laemmli sample buffer were separated by
electrophoresis on SDS-polyacrylamide gels and transferred to nylon
membranes. After a brief incubation with 5% low-fat milk to block
nonspecific binding, membranes were exposed overnight to specific
antisera at 4°C. Next the membranes were washed and exposed to
secondary, peroxidase-conjugated antisera, and antigens were visualized
by enhanced chemiluminescence using reagents from Amersham (Arlington Heights, IL). For immunoblot analysis, primary antisera were used at
the following concentrations: iNOS (Transduction Laboratories, Lexington, KY) 1:4,000; NF-
B p50 and p65 (Santa Cruz Biotechnology, Santa Cruz, CA) 1:1,000; inhibitor
B (I
-B)
and I
-B
(Santa Cruz Biotechnology) 1:500; total mitogen-activating protein
kinase (MAPK), phosphorylated p42, and p44 MAPK (Cell Signaling
Technology) 1:500; total and phospho-AKT (Cell Signaling Technology)
1:500; cytochrome c (Santa Cruz Biotechnology) 1:1,000; and
caspase 3 (Pharmingen, San Diego, CA) 1:1,000.
Electrophoretic mobility shift analysis (EMSA) for NF-
B.
Nuclear extracts (10 µg/assay) from individual mice in each group
were used for EMSA. EMSAs were done with double-stranded oligonucleotide fragments that contain the core 
-binding motif from the mouse
-light chain enhancer element (62).
Probe preparation and EMSA were performed according to the methods
described previously (71). One microliter antisera to
NF-
B p65 or p50 (Santa Cruz Biotechnology) or nonimmune sera were
used in supershift EMSA assays to verify that the DNA binding complexes
contained NF-
B.
RNA isolation and RT-PCR analysis.
Total liver RNA was isolated from each mouse according to the methods
of Chomczynski and Sacchi (10) as described previously (41). RNA was quantitated by spectroscopy, and its quality
was evaluated by agarose gel electrophoresis and subsequent ethidium bromide staining. iNOS transcripts in 3 µg total RNA were amplified in a Perkin-Elmer thermocycler using the primers
5'-GAGAGATCCGATTTAGAGTCT-3' and 5'-GCAGATTCT GCTGGGATTCA-3'
(29) and semiquantitative RT techniques described
previously (55, 56, 71). To control for potential
sample variation,
-actin expression was evaluated in the same RNA
aliquots. The expression of two antiapoptotic bcl-2-related genes,
bfl-1 and bcl-xL, was evaluated by RNAase protection assays by using
reagents from Pharmingen (San Diego, CA) according to the
manufacturers' procedures that permit concurrent quantitation of
transcripts for these genes as well as a housekeeping gene,
glyceraldehyde-3-phosphate dehydrogenase, in each 10-µg RNA
sample. All products were resolved by electrophoresis on 1.2% agarose
gels and quantified by phosphoimage analysis using ImageQuant software
(Molecular Dynamics, Sunnyvale, CA).
Hepatocyte isolation and cell culture.
As we have described (13), in situ perfusion of livers
with collagenase, followed by differential centrifugation of the cellular suspensions, was used to isolate hepatocytes from an additional six ob/ob and six lean mice. To evaluate the yield and
viability of each preparation, an aliquot of cells was stained with
trypan blue and counted in a hemocytometer. In each assay, an equal
number (2 × 105) of freshly isolated hepatocytes from
each group of mice was evaluated for ATP content by using luciferase
assays with reagents from Sigma Chemical (St. Louis, MO). All assays
were performed in triplicate and included a concurrently run standard
curve, according to the manufacturer's specifications. Results are
expressed as picograms ATP per cell. Other hepatocytes suspended in
serum-free DMEM with insulin (15 µg/ml), transferrin (15 µg/ml),
and selenium (0.55 ng/ml) (ITS) were seeded at a density of 1 × 106 cells/ml onto plastic tissue culture plates (Falcon,
Becton Dickenson Labs, Franklin Lakes, NJ) that had been coated with
type I rat tail collagen (Collagen, Palo Alto, CA). After 2 h, the
medium was replaced with fresh DMEM plus ITS that contained actinomycin D (0.4 µmol/ml) and varying amounts of murine recombinant TNF-
(0, 12.5, 50, 200, or 800 pmol/ml) (Sigma Chemical). After overnight incubation in these media, the medium was harvested for measurement of
lactate dehydrogenase (LDH) as described (13); the
cells were stained with propidium iodide, harvested, and analyzed by flow cytometry.
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RESULTS |
Increased LPS-liver damage in fatty livers.
Consistent with previous reports from our laboratory (70)
and another group (17), ob/ob mice develop significantly
greater liver damage than lean mice do after LPS treatment (Fig.
1). After this small dose of LPS, a few
foci of damaged hepatocytes occur in lean mice, but serum ALT values
barely increase above baseline values. In contrast, after exposure to
the same dose of LPS, ob/ob mice exhibit many, large foci of liver
injury, and ALT values are more than 11-fold greater than in the lean
mice by the 6-h time point. Because LPS-induced liver injury requires
TNF-
(67, 69) and TNF-
kills hepatocytes when their
ability to induce antiapoptotic protective responses is inhibited
(37), we next evaluated the possibility that
antiapoptotic defenses are inhibited in ob/ob livers, leading to a
greater induction of proapoptotic signals in response to LPS.

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Fig. 1.
Lipopolysaccharide (LPS)-induced liver damage in lean and ob/ob
mice. A: photomicrographs of hematoxylin- and eosin-stained
liver sections from representative lean (left) and ob/ob
(right) mice 6 h after ip injection with LPS (12 µg/mouse). Final magnification 200X. Arrow on the section from the
lean mouse indicates typical condensed, darkly stained nuclei in an
apoptotic, binucleated hepatocyte. Note that there are many
apoptotic hepatocytes on this section. Brackets on the ob/ob liver
section draw attention to a focal area of hypereosinophilic, swollen
hepatocytes with surrounding inflammatory cell infiltration adjacent to
a portal tract. B: serum alanine aminotransferase (ALT)
activity in lean and ob/ob mice at various time points after LPS
treatment.
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Early and late apoptotic events are decreased in fatty livers.
Release of mitochondrial cytochrome c is an early event in
TNF-
-induced apoptosis (24). In the cytosol,
cytochrome c is thought to promote the eventual activation
of effector caspases, including caspase 3, that are required for
TNF-
-mediated apoptotic cell death in hepatocytes
(28). To evaluate the possibility that increased
accumulation of cytochrome c might contribute to the
enhanced toxicity of LPS in ob/ob livers, cytochrome c
content was assessed by immunoblot analysis of cytosolic and whole
liver homogenates from lean and ob/ob livers at various time points. The content of cytochrome c in whole liver homogenates is
similar in the two groups of mice and remains generally stable after
LPS treatment. However, contrary to our expectations, the cytosolic content of cytochrome c increases more in lean livers than
in ob/ob livers after LPS exposure (Fig.
2). The latter finding suggests that
downstream consequences of cytochrome c release, such as the
cleavage of procaspase 3 to its enzymatically active products, might
also differ in the two groups. As shown in Fig.
3, before LPS exposure, 32-kDa procaspase
3 can be identified easily in whole liver homogenates from
both lean and ob/ob mice, but neither of the groups contains many of
the enzymatically active 11- and 14-kDa cleavage products of procaspase
3. After LPS exposure, procaspase 3 cleavage products accumulate in the
liver homogenates from lean mice but cannot be detected in homogenates
from ob/ob mice. Thus, consistent with the observation that cytosolic
accumulation of cytochrome c is inhibited after LPS exposure
in ob/ob livers, the activation of more direct effectors of
apoptosis, such as caspase 3, is also inhibited in the ob/ob
group.

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Fig. 2.
Effect of LPS treatment on cytochrome c
content in hepatic cytosol and whole liver homogenates. Top:
representative immunoblots. Each lane contains either cytosolic protein
(50 µg) or whole liver protein (40 µg) from the same mouse.
Different mice were killed to obtain protein for each of the different
time points. The immunoblot analysis was repeated with extracts from 4 different mice/group at each time point. Signal intensity was evaluated
by densitometry, and the expression of cytosolic/whole liver cytochrome
c was determined in each extract. Graph summarizes the data
(means ± SE) from 4 mice/group per time point.
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Fig. 3.
Variations in caspase 3 after LPS treatment. Whole liver
homogenates were prepared from 16 lean and 16 ob/ob mice. Immunoblot
analysis was performed to evaluate the expression of procaspase 3 (p38
and p32) and activated caspase 3 (p17 and p11). A representative
immunoblot is shown (50 µg protein/lane). Identical results were
obtained when the experiment was repeated with extracts from other
mice. In total, caspase 3 expression was evaluated in 4 mice/group per
time point. Active forms of caspase 3 (p17 or p11) were never detected
in extracts from the LPS-treated ob/ob mice.
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Greater induction of NF-
B, an antiapoptotic transcription
factor, in fatty livers.
If early and late apoptotic events are, indeed, less prominent
after LPS treatment in ob/ob livers than in lean livers, then ob/ob
livers might induce a more robust antiapoptotic defense than lean
livers when challenged by LPS. To evaluate this possibility, we first
compared the induction of NF-
B, a critical antiapoptotic transcription factor (68), in the two groups. LPS
treatment led to a slight increase in NF-
B DNA binding activity in
lean livers. However, this induction was greater and more sustained in
the ob/ob group (Fig. 4). Increased
NF-
B DNA binding activity results when p65 and p50 NF-
B subunits
accumulate in nuclei after being released from inhibitory binding
proteins, I-
B
and -
, that retain NF-
B in the cytosol
(4). Hence, post-LPS increases in NF-
B binding activity
are expected to be associated with increases in the nuclear content of
the NF-
B p50 and p65 subunits. As expected, the nuclear content of
both NF-
B isoforms increases in lean mice after LPS exposure. Also,
consistent with the greater induction of NF-
B binding activity in
ob/ob livers, the nuclear content of NF-
B p50 and p65 is greater in
ob/ob livers than in lean livers after LPS (Fig.
5). The release of NF-
B subunits from
cytosolic I-
B
and -
occurs when the I-
Bs become
phosphorylated and targeted for proteosomal degradation
(42). The greater accumulation of NF-
B subunits in
ob/ob liver nuclei after LPS exposure suggests that I-
B degradation
might be enhanced in these livers. Indeed, immunoblot analysis of
cytosolic extracts from the two groups of mice at various time points
after LPS treatment confirms that suspicion, i.e., the cytosolic
contents of I-
B
and I-
B
are decreased in ob/ob
livers compared with lean livers (Fig.
6). I-
B degradation follows
phosphorylation by IKKB, a kinase complex that is a target for both
MAPK and AKT kinases (2, 6, 61). Initiation of the MAPK
cascade activates Erk-1 (p42 MAPK) and Erk-2 (p44 MAPK) and leads to
phosphorylation of the IKKB complex. As shown in Fig.
7, transient MAPK activation occurs in
the livers of lean mice after LPS exposure, and this response is
enhanced in ob/ob livers, as evidenced by the greater hepatic
accumulation of phosphorylated Erks in the latter group. The IKKB
complex can also be activated by AKT (29, 58, 63, 75). AKT
activation occurs in both lean and ob/ob livers after LPS exposure,
but, similar to MAPK activation, AKT activation after LPS is greater in
the ob/ob group (Fig. 8). Thus ob/ob mice
exhibit greater activation of two different kinase cascades that are
known to mediate the eventual degradation of I-
B, greater nuclear
translocation of NF-
B, and enhanced induction of NF-
B DNA binding
activity.

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Fig. 4.
Nuclear factor (NF)- B DNA binding activity in the
livers of LPS-treated mice. Nuclear proteins were isolated from 12 lean
and 12 ob/ob mice (4 mice/group per time point), and NF- B binding
activity was evaluated by electrophoretic mobility sift analysis
(EMSA). Each EMSA used nuclear extracts (10 µg/lane) from 2 mice/group per time point. The results of a representative EMSA are
shown. Identical results were obtained when the experiment was repeated
with extracts from the remaining 2 mice/group per time point.
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Fig. 5.
Variations in nuclear NF- B p50 and p65 accumulation after LPS
treatment. Nuclear extracts were prepared from the livers of 16 lean
and 16 ob/ob mice and evaluated by immunoblot analysis (10 µg/lane).
A total of 4 immunoblots were generated, each of which evaluated liver
nuclear extract from a different mouse/group at each time point.
Representative immunoblots are shown. NF- B expression was quantified
by phosphoimager, and the results on each blot were normalized to the
NF- B expression in the extract from the lean control mouse at
time 0 on the same blot. The graphs summarize the results
from all 4 mice/group per time point.
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Fig. 6.
Variations in cytosolic inhibitor B (I- B)- and I- B
accumulation after LPS treatment. Cytosolic extracts were prepared from
the livers of 16 lean and 16 ob/ob mice and evaluated by immunoblot
analysis (50 µg/lane). A total of 4 immunoblots was generated, each
of which evaluated liver cytosol from a different mouse/group at each
time point. Representative immunoblots are shown. I- B
expression was quantified by phosphoimager, and the results on each
blot were normalized to the I- B expression in the extract from
the lean control mouse at time 0 on the same blot. The
graphs summarize the results from all 4 mice/group per time point.
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Fig. 7.
Variations in mitogen-activated protein kinase (MAPK)
activity after LPS treatment. Whole liver homogenates were prepared
from the livers of 16 lean and 16 ob/ob mice and evaluated by
immunoblot analysis (50 µg/lane). A total of 4 immunoblots was
generated, each of which evaluated liver extracts from a different
mouse/group at each time point. Each blot was probed sequentially for
activated MAPK expression (using antisera specific for phospho-p42 and
phospho-p44 MAPK) and then for total MAPK expression (using a different
antisera described in METHODS). A representative immunoblot
is shown and demonstrates that total MAPK expression is relatively
constant across treatment groups and at various time points, confirming
the lane-lane equivalency of protein loading. Activated MAPK expression
(phospho-p42 + phospho-p44) was quantified by a phosphoimager, and
the results on each blot were normalized to the expression of
phospho-MAPK in the extract from the lean control mouse at time
0. The graph summarizes the results from 4 mice/group per time
point.
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Fig. 8.
Variations in AKT activity after LPS treatment. Whole
liver homogenates were prepared from the livers of 16 lean and 16 ob/ob
mice and evaluated by immunoblot analysis (50 µg/lane). A total of 4 immunoblots were generated, each of which evaluated liver extracts from
a different mouse/group at each time point. Each blot was probed
sequentially for activated AKT expression (using antisera specific for
phospho-AKT) and then for total AKT expression (using a different
antisera described in METHODS). A representative immunoblot
is shown, demonstrating phospho-AKT (top) and total AKT
(bottom) in the same extracts. Activated AKT expression
(phospho-AKT/total AKT) was quantified by a phosphoimager, and the
results on each blot were normalized to the expression of activated AKT
in the extract from the lean control mouse at time 0. The
graph summarizes the results from 4 mice/group per time point.
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Increased accumulation of certain NF-
B-regulated gene products
in fatty livers.
The enhanced activation of NF-
B in LPS-treated ob/ob livers is
expected to promote increased induction of NF-
B target genes, such
as bfl-1 and bcl-xL, antiapoptotic members of the bcl-2 family (64, 76). To evaluate this possibility, total mRNA was
obtained from lean and ob/ob livers before and after LPS treatment, and steady-state transcript levels were assessed by RNAase protection analysis. LPS induces bfl-1 and bcl-xL expression in both lean and
ob/ob livers. However, after LPS exposure, significantly more of these
transcripts accumulate in the ob/ob livers (Fig.
9), a finding that is consistent with the
greater activation of NF-
B in the ob/ob group. iNOS is another
transcriptional target for NF-
B (30). To determine
whether the enhanced NF-
B activity in ob/ob livers is also
accompanied by an increased induction of iNOS after LPS exposure, iNOS
mRNA levels in lean and ob/ob mice were compared by RT-PCR. Although a
slight delay in iNOS mRNA accumulation occurs in the ob/ob group, by
6 h after LPS treatment the levels of iNOS mRNA are actually
greater in ob/ob mice than controls (Fig.
10). iNOS is known to protect
hepatocytes from the toxic actions of TNF-
by preventing the
activation of caspase 3 (31). Thus we wondered whether
increases in iNOS protein content resulted from the accumulation of
iNOS mRNAs and might account for our earlier finding that caspase 3 activation is inhibited in ob/ob livers. The latter does not appear to
be true because, after LPS exposure, the hepatic induction of iNOS
protein is severely inhibited in ob/ob mice compared with lean controls
(Fig. 11). On the other hand, the
posttranscriptional mechanisms that reduce the content of iNOS protein
in ob/ob livers might play an indirect role in enhancing NF-
B
activation in ob/ob livers because NO, the product of iNOS, is known to
inhibit NF-
B activation by inducing and stabilizing I-
B
(53).

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Fig. 9.
Hepatic expression of bfl-1 and bcl-xL mRNA before and after LPS
treatment. Total RNA was extracted from the livers of 4 lean and 4 ob/ob mice before and 6 h after LPS treatment. Antiapoptotic
gene expression was evaluated by RNAase protection assay as detailed in
METHODS. This procedure permits concurrent evaluation of
bfl-1, bcl-xL, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a
housekeeping gene, in each 10-µg RNA sample. Top: result
of a representative autoradiograph that contains RNA from 2 different
mice/group at each time point. This experiment was repeated with RNA
from another 2 mice/group per time point and the cumulative results
from all 4 mice/group at each time point are graphed.
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Fig. 10.
Accumulation of inducible nitric oxide synthase (iNOS)
transcripts in the liver after LPS treatment. Total liver RNA was
isolated from 12 lean and 12 ob/ob mice (4 mice/group per time point)
and RT-PCR was performed to evaluate differences in iNOS mRNA
accumulation. The RT-PCR experiment was repeated 3 times with each
assay, using liver RNA from a different lean or ob/ob mouse at each
time point. A representative Southern blot is shown. The graph
summarizes the results from all 24 mice (4 mice/group per time
point).
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Fig. 11.
Variations in the hepatic expression of iNOS protein
after LPS treatment. Immunoblot analysis was performed using whole
liver homogenates (40 µg protein/lane). A representative immunoblot
that contains protein from a different lean or ob/ob mouse at each time
point is illustrated. In total, 4 immunoblots were generated using
liver proteins from 16 lean and 16 ob/ob mice (4 mice/group at each of
the time points shown), and iNOS expression was quantified by
phosphoimager analysis. On each blot, all results were normalized to
iNOS expression in the lean mouse extracts at time 0. The
graph demonstrates mean ± SE iNOS expression in 4 mice from each
group at each time point.
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ATP depletion and increased TNF-induced lysis in fatty hepatocytes.
The previously cited evidence demonstrates that LPS induces
significantly greater liver damage in ob/ob mice with fatty livers than
in lean mice with normal livers. However, the enhanced vulnerability of
fatty livers to LPS-mediated injury is not easily explained by an
increased activation of proapoptotic pathways. Rather, ob/ob mice
with fatty livers exhibit an enhanced activation of several different
antiapoptotic mechanisms and decreased activation of proapoptotic responses after LPS exposure. Thus, although it
seems that apoptosis accounts for LPS-induced liver damage in
normal mice, some other process is more likely to be responsible for the LPS-related liver damage that develops in ob/ob mice.
Ob/ob mice with fatty livers are known to have abnormal hepatic
mitochondria (72). Given the importance of oxidative
phosphorylation in cellular ATP homeostasis (22, 33), it
is conceivable that ob/ob mitochondrial abnormalities reduce the
hepatocyte ATP content, thereby promoting necrosis while inhibiting
apoptosis (33, 74). ATP depletion compromises the
activity of ATP-dependent transporters that maintain cell volume and
membrane integrity. Extreme ATP depletion results in cell death by
lysis (i.e., necrosis) (33). Hepatocytes were isolated
from ob/ob and lean livers so that ATP content and membrane integrity
could be compared. Hepatocytes isolated from ob/ob livers have a lower
ATP content than hepatocytes from lean livers (Fig.
12A). In addition, when lean
and fatty hepatocytes are treated with various doses of TNF-
in
vitro, fatty hepatocytes release significantly more LDH into the medium
(Fig. 12B), consistent with the concept that the ob/ob cells
are dying by lysis. In contrast, under these experimental conditions,
normal hepatocytes undergo apoptosis (37). By
using the flow cytometry technique that Galle (19)
recommends as a measure of apoptosis, this apoptotic
response was verified in our experiments by the appearance of a new
peak to the left of the G0-G1 fraction after
normal cells are treated with TNF-
-actinomycin D. Although
exhibiting the expected apoptotic response to these in vitro
conditions, normal hepatocytes do not release more LDH into the culture
medium (Fig. 12B). Thus the combined data from our in vivo
and in vitro studies fail to support our original hypothesis that fatty
hepatocytes are vulnerable to LPS-mediated damage because of
unconstrained apoptotic responses. Rather, our findings are more
consistent with the concept that LPS injures normal livers by causing
hepatocyte apoptosis but damages fatty livers by causing
excessive hepatocyte necrosis.

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|
Fig. 12.
Hepatocyte ATP stores and lactate dehydrogenase (LDH)
release. A: freshly isolated hepatocytes (2 × 105 cells/assay) pooled from 6 lean mice and 6 ob/ob mice
were evaluated for ATP content using luciferase assays as described in
METHODS. Each assay was done in triplicate. Mean ± SE
results from the 2 groups of mice are shown. B: hepatocytes
from the same preparations were seeded onto collagen-coated dishes at a
density of 1 × 106 cells/ml and cultured overnight in
serum-free medium containing actinomycin D + varying amounts (0, 12.5, 50, 200, or 800 pmol/ml) of murine recombinant TNF- . Medium
was collected, and the LDH concentration was measured. For each group,
the concentration of LDH in the medium was normalized to the LDH
concentration in the medium of ob/ob or lean hepatocyte cultures that
had not been treated with TNF- . Results are expressed % of the
untreated control in the same experiment.
|
|
 |
DISCUSSION |
Hepatic steatosis is one of the most common forms of liver disease
in the United States and other industrialized countries. The prevalence
of this condition in the general population parallels the prevalence of
the major risk factors for fatty liver disease (i.e., obesity,
hyperinsulinemic insulin resistance, and habitual alcohol consumption)
in the adults living in these countries (5, 51, 57).
Hepatic steatosis itself is thought to be a relatively benign condition
because it is seldom associated with liver-related morbidity and
mortality (66). On the other hand, steatosis significantly potentiates the severity of liver damage that is caused by other agents, such as drugs or infections (12, 26, 54). In
addition, some patients with NAFLD develop steatohepatitis (hepatic
steatosis with inflammation and hepatocyte death) spontaneously (i.e.,
without any obvious superimposed insult). After this occurs, the risk and rate of progression to cirrhosis, with consequent liver-related morbidity and mortality, increases significantly (46). To
improve the prognosis of patients with fatty livers, it is important to understand why hepatic steatosis increases the risk for more serious liver disease.
Because fatty livers are unusually vulnerable to injury from endotoxin
(17, 70), which requires the proinflammatory cytokine, TNF-
, to kill hepatocytes (47), we speculated that
hepatic steatosis might inhibit the mechanisms that normally protect
hepatocytes from TNF-induced apoptosis. Quite unexpectedly, our
results demonstrate that hepatocytes in fatty livers actually
superinduce the promitogenic kinases, p42 and p44 MAPK, and the
antiapoptotic kinase, AKT, when challenged by a hepatotoxic dose of
LPS. Consistent with the enhanced activation of these upstream kinases
that phosphorylate (and activate) components of the IKKB complex
(2, 58, 75), the cytosolic content of the NF-
B
inhibitors I-
B
and I-
B
is more reduced and the nuclear
accumulation of NF-
B subunits and their DNA binding activities is
greater and more sustained in ob/ob mice than in lean mice after LPS
treatment. Subsequent induction of three NF-
B target genes, bfl-1,
bcl-xL (64, 76), and iNOS (30), is also
increased in ob/ob livers. Given that bfl-1 and bcl-xL inhibit
TNF-
-initiated apoptosis by limiting the release of
mitochondrial cytochrome c (15, 32, 64, 73), it
is conceivable that the enhanced expression of these two factors contributes to the reduced cytosolic cytochrome c content of
ob/ob livers at 1.5 and 6 h after LPS exposure. Increased iNOS
activity is also known to protect hepatocytes from TNF-
-mediated
apoptosis (7, 23, 34). The mechanisms for this
protective effect of iNOS appear to involve the ability of its product,
NO, to inhibit the activation of effector caspases, such as caspase 3 (31, 40). However, the latter action cannot explain the
reduced activation of caspase 3 that occurs in ob/ob livers after LPS
exposure because iNOS protein levels are reduced (rather than
increased) in ob/ob livers. Indeed, the discordance between iNOS mRNA
and protein levels in the livers of LPS-treated ob/ob mice suggests
that posttranscriptional mechanisms play an important role in limiting
iNOS activity in fatty livers. Interestingly, inhibition of iNOS can
also inhibit apoptosis in some settings (39), and
this response has been attributed to the fact that decreases in NO
production promote the degradation of I-
B
, which, in turn, leads
to the activation of NF-
B (53). In addition to
significant reductions in iNOS protein content in ob/ob livers, the
cytosolic accumulation of I-
B
after LPS exposure is
also inhibited in ob/ob livers, suggesting that decreases in iNOS
activity might also contribute to the enhanced activation of NF-
B in
ob/ob mice. Therefore, our analysis of several key regulatory factors
for cellular apoptosis demonstrates that ob/ob livers respond
to a low dose of LPS with a greater induction of antiapoptotic
responses and less induction of proapoptotic responses than lean mice.
Given these results, it is reasonable to expect that the livers of lean
mice will exhibit more apoptosis than those of ob/ob mice after
LPS treatment. However, the latter is difficult to prove with certainty
because virtually all of the markers for apoptosis are
nonspecific and, particularly when applied to liver tissue, cannot
reliably distinguish apoptosis from necrosis (19). There is no doubt that ob/ob mice with fatty livers are more vulnerable to LPS-induced liver injury than lean mice with normal livers. This
fact has been reported previously (17, 70) and is
confirmed in the present studies. However, the present work
evaluates earlier time points after LPS exposure and shows that the
evolution of hepatic injury temporally correlates with the
apoptotic response in lean mice. In ob/ob mice, severe liver damage
also develops during this time period. However, unlike lean mice, ob/ob
mice accumulate only small amounts of cytosolic cytochrome c
and do not activate caspase 3 after LPS treatment. These findings imply either that hepatocyte apoptosis is occurring via caspase
3-independent mechanisms or that the hepatocyte death results from
necrosis in ob/ob animals. Because our studies did not evaluate the
activation of caspases other than caspase 3, we cannot comment on the
first possibility. However, the results of our in vitro hepatocyte
culture experiments strongly support a role for hepatocyte necrosis in the ob/ob LPS response. Hepatocytes from ob/ob mice have a reduced content of ATP, and it is well established that decreased ATP inhibits
apoptosis while promoting necrosis (16, 33, 38, 74). In addition, during in vitro conditions that reproducibly induce apoptosis in normal hepatocytes (37), LDH
does not accumulate in the medium of normal hepatocyte cultures but
increases significantly in cultures of ob/ob hepatocytes. This finding
suggests that normal and ob/ob hepatocytes die by different mechanisms
after exposure to TNF-
, with normal hepatocytes dying by
apoptosis but fatty hepatocytes dying from lysis (i.e., necrosis).
Our studies did not evaluate which of the multiple hormonal, metabolic,
and immunologic abnormalities of ob/ob mice might mediate the unusual
response of ob/ob hepatocytes to LPS-induced cytokines. Because leptin
deficiency is the primary genetic abnormality of these mice (8,
18) and leptin is known to be immunomodulatory (21, 27,
36, 43, 60), it is likely that leptin deficiency contributes, in
some way, to the LPS sensitivity of ob/ob hepatocytes. Consistent with
this concept, other groups have already reported that treatment with
supplemental leptin protects ob/ob mice from LPS toxicity
(17). However, the hepatoprotective effects of leptin are
likely to be mediated indirectly because adult hepatocytes are not
known to express ObRb, the signaling-competent leptin receptor
(65). In earlier papers, our laboratory suggested that the
leptin-deficient state leads to abnormalities in the hepatic microenvironment that subject hepatocytes to apoptotic stress and
speculated that adaptation to this apoptotic stress increases hepatocyte vulnerability to necrosis (9, 11, 14). The
results from these most recent studies lend additional support to that concept. Careful inspection of the time 0 (i.e., pre-LPS)
data demonstrates that ob/ob livers have somewhat increased expression of bfl-1 and phospho-MAPK and decreased expression of iNOS protein, I-
B, and activated caspase 3 isoforms, suggesting that an
antiapoptotic response has already been initiated in these livers.
Despite this, when challenged with a dose of LPS that induces only a
small amount of hepatocyte apoptosis in normal livers, ob/ob
livers develop extensive damage coincident with supranormal induction
of antiapoptotic responses. Moreover, during the evolution of this
liver damage, ob/ob livers do not activate caspase 3, a key effector of
apoptosis in hepatocytes (28). Regardless of the
terminology that one chooses to describe this end result of this
process, there is no question that extensive LPS-mediated liver damage
occurs in fatty livers despite increased induction of the same
mechanisms that are known to constrain apoptosis in normal
livers. Because antiapoptotic mechanisms fail to protect fatty
hepatocytes from LPS-induced death, it is conceivable that these cells
are not being killed by apoptosis. Indeed, the data support the
possibility that fatty livers have an enhanced vulnerability to necrosis.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: A. M. Diehl, 912 Ross Bldg., The Johns Hopkins Univ., 720 Rutland St., Baltimore, MD 21205 (E-mail: amdiehl{at}welch.jhu.edu).
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. Section 1734 solely to indicate this fact.
Received 27 November 2000; accepted in final form 20 February 2001.
 |
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