Fatty liver vulnerability to endotoxin-induced damage despite NF-kappa 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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-kappa B (NF-kappa 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 kappa -B are activated; NF-kappa B activity is induced, and NF-kappa 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 kappa -B, enhanced activation of NF-kappa B, and greater induction of NF-kappa 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-alpha , these findings suggest that fatty livers are sensitive to LPS damage because of vulnerability to necrosis, rather than because of apoptosis.

nuclear factor-kappa B; obesity; nonalcoholic fatty liver disease


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha (TNF-alpha ) (47). TNF-alpha kills cells by activating caspases that cause apoptosis (3, 24, 25). However, healthy hepatocytes normally are not killed by TNF-alpha because, when they are exposed to TNF-alpha , they activate antiapoptotic transcription factors, such as nuclear factor-kappa B (NF-kappa B) (68). NF-kappa 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-kappa 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-alpha -mediated hepatocyte apoptosis (28). Hence, fatty livers might be more vulnerable to cytokine-mediated liver injury because the normal induction of TNF-alpha survival pathways is inhibited, permitting the unrestrained activation of TNF-alpha '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-kappa B, and NF-kappa 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-alpha 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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-kappa B p50 and p65 (Santa Cruz Biotechnology, Santa Cruz, CA) 1:1,000; inhibitor kappa B (Ikappa -B) alpha  and Ikappa -Bbeta (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-kappa 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 kappa beta -binding motif from the mouse kappa -light chain enhancer element (62). Probe preparation and EMSA were performed according to the methods described previously (71). One microliter antisera to NF-kappa 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-kappa 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, beta -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-alpha (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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha (67, 69) and TNF-alpha 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.

Early and late apoptotic events are decreased in fatty livers. Release of mitochondrial cytochrome c is an early event in TNF-alpha -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-alpha -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.

Greater induction of NF-kappa 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-kappa B, a critical antiapoptotic transcription factor (68), in the two groups. LPS treatment led to a slight increase in NF-kappa B DNA binding activity in lean livers. However, this induction was greater and more sustained in the ob/ob group (Fig. 4). Increased NF-kappa B DNA binding activity results when p65 and p50 NF-kappa B subunits accumulate in nuclei after being released from inhibitory binding proteins, I-kappa Balpha and -beta , that retain NF-kappa B in the cytosol (4). Hence, post-LPS increases in NF-kappa B binding activity are expected to be associated with increases in the nuclear content of the NF-kappa B p50 and p65 subunits. As expected, the nuclear content of both NF-kappa B isoforms increases in lean mice after LPS exposure. Also, consistent with the greater induction of NF-kappa B binding activity in ob/ob livers, the nuclear content of NF-kappa B p50 and p65 is greater in ob/ob livers than in lean livers after LPS (Fig. 5). The release of NF-kappa B subunits from cytosolic I-kappa Balpha and -beta occurs when the I-kappa Bs become phosphorylated and targeted for proteosomal degradation (42). The greater accumulation of NF-kappa B subunits in ob/ob liver nuclei after LPS exposure suggests that I-kappa 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-kappa Balpha and I-kappa Bbeta are decreased in ob/ob livers compared with lean livers (Fig. 6). I-kappa 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-kappa B, greater nuclear translocation of NF-kappa B, and enhanced induction of NF-kappa B DNA binding activity.


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Fig. 4.   Nuclear factor (NF)-kappa 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-kappa 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-kappa 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-kappa B expression was quantified by phosphoimager, and the results on each blot were normalized to the NF-kappa 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 kappa B (I-kappa B)-alpha and I-kappa Bbeta 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-kappa B expression was quantified by phosphoimager, and the results on each blot were normalized to the I-kappa 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.

Increased accumulation of certain NF-kappa B-regulated gene products in fatty livers. The enhanced activation of NF-kappa B in LPS-treated ob/ob livers is expected to promote increased induction of NF-kappa 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-kappa B in the ob/ob group. iNOS is another transcriptional target for NF-kappa B (30). To determine whether the enhanced NF-kappa 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-alpha 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-kappa B activation in ob/ob livers because NO, the product of iNOS, is known to inhibit NF-kappa B activation by inducing and stabilizing I-kappa Balpha (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.

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-alpha 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-alpha -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-alpha . 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-alpha . Results are expressed % of the untreated control in the same experiment.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha , 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-kappa B inhibitors I-kappa Balpha and I-kappa Bbeta is more reduced and the nuclear accumulation of NF-kappa 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-kappa 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-alpha -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-alpha -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-kappa Balpha , which, in turn, leads to the activation of NF-kappa B (53). In addition to significant reductions in iNOS protein content in ob/ob livers, the cytosolic accumulation of I-kappa Balpha 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-kappa 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-alpha , 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-kappa 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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