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Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis and varying degrees of necroinflammation. Although chronic oxidative stress, inflammatory cytokines, and insulin resistance have been implicated in the pathogenesis of NAFLD, the mechanisms that underlie the initiation and progression of this disease remain unknown. c-Jun N-terminal kinase (JNK) is activated by oxidants and cytokines and regulates hepatocellular injury and insulin resistance, suggesting that this kinase may mediate the development of steatohepatitis. The presence and function of JNK activation were therefore examined in the murine methionine- and choline-deficient (MCD) diet model of steatohepatitis. Activation of hepatic JNK, c-Jun, and AP-1 signaling occurred in parallel with the development of steatohepatitis in MCD diet–fed mice. Investigations in jnk1 and jnk2 knockout mice demonstrated that jnk1, but not jnk2, was critical for MCD diet–induced JNK activation. JNK promoted the development of steatohepatitis as MCD diet–fed jnk1 null mice had significantly reduced levels of hepatic triglyceride accumulation, inflammation, lipid peroxidation, liver injury, and apoptosis compared with wild-type and jnk2 −/− mice. Ablation of jnk1 led to an increase in serum adiponectin but had no effect on serum levels of tumor necrosis factor-α. In conclusion, JNK1 is responsible for JNK activation that promotes the development of steatohepatitis in the MCD diet model. These findings also provide additional support for the critical mechanistic involvement of JNK1 overactivation in conditions associated with insulin resistance and the metabolic syndrome. (HEPATOLOGY 2006;43:163–172.)
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The most common liver disease in Western countries is nonalcoholic fatty liver disease (NAFLD), which encompasses a spectrum from simple steatosis to steatosis combined with varying degrees of necroinflammation and fibrosis.1, 2 The initial hepatic lipid accumulation and subsequent progression to cellular injury and inflammation termed nonalcoholic steatohepatitis (NASH) are thought to occur through distinct mechanisms.3 NAFLD is frequently associated with obesity, dyslipidemia, and insulin resistance, a group of disorders that constitute the metabolic syndrome.4 Although these conditions may predispose individuals to the development of NAFLD, the molecular mechanisms that underlie hepatic fat accumulation and trigger the hepatocyte injury and cell death of NASH are unknown.
There is considerable interest in delineating the factors that initiate the onset of necroinflammation because the existence of steatosis by itself is insufficient to cause chronic liver disease. A factor that may trigger the progression to actual cell injury in the setting of a fatty liver is oxidant stress from increased expression of the prooxidant cytochrome P450 isoform 2E1 (CYP2E1),5, 6 or the generation of excessive reactive oxygen species from mitochondrial dysfunction.7, 8 In addition, modulators of the inflammatory and immune responses, such as adiponectin and tumor necrosis factor-α (TNF), may regulate steatotic liver injury.9 TNF promotes insulin resistance and becomes a hepatotoxin when hepatocytes are sensitized to its cytotoxicity by certain factors, including overexpression of cytochrome P450 2E1.10 Adiponectin may reduce liver steatosis and/or injury because of its antilipogenic, anti-inflammatory, and insulin-sensitizing properties.11–13 Serum adiponectin levels are decreased in obesity and insulin-resistant states and correlate inversely with necroinflammatory activity in human NAFLD.14
Intracellular signaling pathways are likely to be critical regulators of both the hepatic accumulation of lipid and the hepatocyte's injury response in the setting of steatosis. Recently activation of the mitogen-activated protein kinase c-Jun N-terminal kinase (JNK) has been implicated in the development of obesity and insulin resistance,15 two prominent risk factors for NAFLD that may act to promote steatosis. Other investigations have implicated JNK activation in the regulation of liver injury.16 JNK is encoded for by three genes, each of which is alternatively spliced to yield α and β forms of both a p54 and p46 protein.17 In most cells, including hepatocytes, only two of the genes, jnk1 and jnk2, are expressed.17 JNK expression has been implicated in hepatocyte injury mediated by TNF,18, 19 ischemia reperfusion,20 hepatitis virus,21 and bile acids.22 The mechanism(s) of this effect is unknown, although it is clear that, whereas transient JNK activation may be beneficial to the hepatocyte, sustained JNK activity triggers cell death.18 Although the differential effects of jnk1 and jnk2 generated isoforms in liver injury are largely unknown, recent studies in cultured hepatocytes have demonstrated that jnk1 promotes, and jnk2 attenuates hepatocyte injury from toxic bile acids.22 Whether JNK activation occurs in vivo in chronic liver injuries such as NASH is unknown. We have previously demonstrated that in vitro chronic hepatocyte overexpression of CYP2E1 as occurs in NASH causes sustained JNK activation that promotes insulin resistance.10, 23 These findings combined suggest that JNK activation may occur in NAFLD and modulate the pathophysiology of this disease.
To examine JNK involvement in NAFLD, studies were performed in a methionine-and choline-deficient (MCD) diet–induced model of steatohepatitis that mimics some of the features of human NAFLD, including the development of steatohepatitis, CYP2E1 overexpression, increased lipid peroxidation, and hepatic insulin resistance.23, 24 Our studies demonstrate that sustained JNK activation occurs with the development of steatohepatitis in this model. Additional studies in jnk1 and jnk2 knockout mice indicate that the jnk1 but not jnk2 gene is critical for hepatic JNK activity in vivo. Ablation of jnk1 also significantly attenuated the development of steatohepatitis. These findings define a critical mechanistic function for jnk1 in experimental steatohepatitis.
Male and female wild-type C57BL/6, jnk1 −/−25 and jnk2 −/−26 mice (Jackson Laboratory, Bar Harbor, ME) were maintained under 12-hour light/dark cycles with unlimited access to food and water. Genotypes were confirmed by PCR with established primers.25, 26 Both knockout mice had been backcrossed onto a C57BL/6 background for more than six generations. Mice were fed the MCD diet or a corresponding control diet, supplemented with 300 mg DL-methionine and 200 mg choline per 100 g of diet (ICN Biomedicals Inc., Costa Mesa, CA). To accustom the animals to the high sucrose diet, 6-week-old animals were initially placed on the control diet for 2 weeks. Animals were then randomly assigned to an additional 4 weeks of either control diet or MCD diet. At weekly intervals, serum was obtained by retro-orbital bleed and the mice were sacrificed for the removal of liver tissue. All studies were approved by the Animal Care and Use Committee of the Albert Einstein College of Medicine and followed the National Institutes of Health guidelines on the care and use of animals.
Liver specimens were fixed in 10% neutral formalin and 6-μm sections stained with hematoxylin and eosin. Tissue sections were examined in a blinded fashion by a single pathologist and graded for steatosis and inflammation. The degree of steatosis in each specimen was determined by assessing the overall percentage of liver parenchyma containing lipid vacuoles with 0 = none, 1 = mild (<30%), 2 = moderate (30%–60%), and 3 = marked (>60%). Inflammation was graded by the presence or absence of inflammatory cells with 0 = absent, 1 = minimal or focal occasional single clusters of inflammatory cells present in a few microscopic fields, 2 = mild inflammation, 3 = moderate inflammation, and 4 = marked inflammation.
Hepatic Triglyceride Content.
The triglyceride content of liver tissue was determined following an extraction in a 2:1 chloroform:methanol mixture containing 0.05% sulfuric acid at −20°C for 16 hours. Triglyceride measurements were performed using a commercial kit according to the manufacturer's instructions (Roche Diagnostics, Indianapolis, IN).
As a reflection of hepatic levels of lipid peroxidation, malondialdehyde (MDA) levels were determined by colorimetric assay (EMD Biosciences, La Jolla, CA). Fifty mg of liver tissue were homogenized in 20 mmol/L Tris-HCl, pH 7.4, and 500 μmol/L 3,5-Di-tert-butyl-4-hydroxytoluene, and the colorimetric reaction carried out accordingly to the manufacturer's instructions.
Commercial kits were used to measure serum alanine aminotransferase (ALT) (TECO Diagnostics, Anaheim, CA), adiponectin (Linco Research Inc., St. Charles, MO), and TNF (BD Biosciences, San Diego, CA) levels. Adiponectin measurements were performed at the same time of day in all experiments.
Terminal Deoxynucleotide Transferase-Mediated Deoxyuridine Triphosphate Nick End-Labeling Assay.
The numbers of apoptotic cells in liver sections were determined by terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay with a commercial kit (Promega, Madison, WI). Tissue sections were prepared by deparaffination in xylene followed by gradual rehydration with decreasing concentrations of ethanol. The TUNEL assay was then performed according to the manufacturer's instructions. Under light microscopy the numbers of TUNEL positive cells were counted per high-powered field (400× magnification) with 20 random fields counted per liver.
All numerical results are expressed as mean ±SE and represent data from a minimum of three independent experiments. Calculations were made with Sigma Plot 2000 (SPSS Science, Chicago, IL). Western and RT-PCR signals were quantitated using a FluorChem densitometer (Alpha Innotech, San Leonardo, CA).
MCD Diet–Induced Steatohepatitis Is Associated With Activation of JNK, c-Jun, and AP-1.
The mitogen-activated protein kinase JNK is a critical modulator of the liver's response to injurious stress.16 JNK also regulates the development of obesity and insulin resistance,15 the two main risk factors for NAFLD. These facts suggest that JNK may modulate the development of steatohepatitis. To assess whether hepatic JNK activation occurs in steatohepatitis, JNK activity was examined in C57BL/6 mice placed on a high carbohydrate diet lacking methionine and choline (MCD diet) for 4 weeks. As previously reported,23, 24, 27 animals fed this diet developed steatosis, inflammation and hepatocellular injury within 1 week that was sustained over the 4-week course of the experiments (data not shown). JNK activity was compared between control diet and MCD diet–fed animals by an in vitro kinase assay employing c-Jun as the substrate. JNK activity, as reflected by levels of phospho-c-Jun on immunoblots, was increased 7 fold within 1 week of the start of the MCD diet and remained elevated over the 4-week course of the experiment (Fig. 1A). Levels of total c-Jun were equivalent among samples, confirming the equal loading of reaction product (Fig. 1A). JNK activation therefore occurred early in the development of MCD diet–induced steatohepatitis and was sustained once the disease was established.
As further evidence of JNK activation, levels of active, phosphorylated JNK were examined in MCD diet–fed animals by Western blot analysis. Increased levels of phosphorylated p54 and p46 JNK were detected from 1 to 4 weeks after the start of the MCD diet by immunoblotting (Fig. 1B). A slight increase in JNK phosphorylation was also observed in control diet–fed animals after 2 weeks despite their lack of increased JNK activity. Still phospho-JNK levels in MCD diet–fed mice were always at least 2-fold greater than those in control diet–fed animals (Fig. 1B). Levels of total JNK were unchanged over time by either diet (Fig. 1B).
Downstream effects of JNK activation are partially mediated through phosphorylation and activation of c-Jun. In parallel with the increases in JNK activity and phosphorylation, levels of phosphorylated c-Jun were increased 3 to 6 fold in animals fed MCD diet over the 4-week time course. c-Jun expression is regulated transcriptionally as well as by posttranslational phosphorylation, and levels of total c-Jun protein increased 3 to 5 fold in animals fed MCD diet over 4 weeks (Fig. 1B). Control diet–fed animals also had a slight increase in expression of total c-Jun that did not reach statistical significance (Fig. 1B).
c-Jun is a critical subunit of the transcription factor AP-1 (activator protein-1). Elevated levels of total and activated c-Jun may therefore lead to increased AP-1 activation in MCD diet–fed mice. Analysis of levels of AP-1 DNA binding by EMSA revealed a marked increase in hepatic AP-1 binding in mice fed the MCD diet compared with mice on control diet (Fig. 1C). In total these data indicate that induction of steatohepatitis by the MCD diet was associated with a significant and sustained activation of the JNK/c-Jun/AP-1 pathway.
Steatohepatitis-Associated JNK Activation Is Dependent on jnk1 But Not jnk2 Expression.
The hepatic p54 and p46 JNK isoforms are the products of expression of the jnk1 and jnk2 genes.16 Recent investigations in nonhepatic models have indicated that there may be functional differences between the JNK isoforms expressed by these two genes in terms of their effect on JNK activity.28 To determine whether jnk1 or jnk2 was responsible for the increase in hepatic JNK activity in steatohepatitis, the degree of JNK activation was compared in wild-type and jnk1 and jnk2 knockout mice fed the control and MCD diets. Western blot analysis of phosphorylated and total JNK was performed in animals fed control or MCD diet. As demonstrated previously, phosphorylation of JNK increased in wild-type mice after 4 weeks of MCD diet (Fig. 2A). Ablation of the jnk1 gene predominantly decreased hepatic expression of the total p46 JNK isoform, and slightly reduced amounts of the p54 isoform (Fig 2A), consistent with reported findings principally of a decrease in p46 JNK in other organs of these mice.25, 28 Levels of phosphorylated JNK were reduced by 70% in jnk1 knockout mice compared with wild-type mice (Fig. 2B). Mice lacking the jnk2 gene on control diet had predominantly reduced levels of the p54 JNK isoform (Fig. 2A), consistent with prior findings that p54 JNK is primarily expressed by jnk2.26, 28 Interestingly, in response to MCD diet levels of p54 JNK increased in jnk2 −/− livers, suggesting that jnk1 expression was induced and partially compensated for the absence of jnk2. Nonetheless, in jnk2 −/− mice, exclusive phosphorylation of the p46 isoform occurred in mice fed the MCD diet to a level that was increased 9 fold over control diet–fed jnk2 −/− mice (Fig. 2A). Thus, based on both activity assays and immunoblots for the active, phosphorylated forms of JNK, the level of JNK activation in response to the MCD diet was significantly reduced in jnk1 but not jnk2 knockout animals.
The net effect of altered JNK isoform expression and phosphorylation in the jnk knockout animals on hepatic JNK activity was determined by in vitro kinase assay. Jnk1 null animals on either control or MCD diet for 4 weeks had virtually no detectable JNK activity (Fig. 2C). In contrast, jnk2 knockout animals had an increased level of JNK activity on control diet, which was further increased with the MCD diet compared with wild-type mice (Fig. 2C). JNK activity in MCD diet–fed mice was therefore dependent completely on jnk1 gene expression while the loss of jnk2 increased JNK activity. In parallel with the absence of JNK activation in jnk1 knockout mice, c-Jun phosphorylation was reduced by 50% to 70% in these animals on either diet compared with wild-type mice (Fig. 2A). Levels of total c-Jun were also decreased in control diet and MCD diet–fed jnk1 −/− mice (Fig. 2A).
JNK affects the function of it downstream effector c-Jun by phosphorylating sites that increase the AP-1 transcriptional activity of c-Jun. Levels of c-Jun gene expression are regulated by AP-1 and therefore also JNK dependent. The effect of jnk1 ablation on the MCD diet–induced increase in AP-1 DNA binding was examined. AP-1 DNA binding was increased in all three types of mice fed the MCD diet (Fig. 2D). However, levels of AP-1 DNA binding were decreased 30% in jnk1 −/− animals as compared with wild-type and jnk2 −/− mice.
MCD Diet–fed jnk1 −/− Mice Have Improved Histology.
The marked decrease in JNK activation in MCD diet–fed jnk1 null mice made these animals an appropriate model for a study of the effects of the absence of JNK activity on the development of steatohepatitis. Wild-type, jnk1 −/− and jnk2 −/− mice all gained weight on control diet over the 4-week course of the experiment, although the increase in jnk2 null mice was significantly greater (Table 1). All three mouse strains lost similar amounts of weight on the MCD diet (Table 1). The ratio of liver weight to body weight, a determinant of hepatomegaly, was significantly greater in wild-type and jnk2 −/− mice than in jnk1 −/− mice fed the MCD diet (Table 1).
Table 1. Change in Body Weights and Liver-to-Body Weight Ratios of Wild-Type (WT), jnk1 −/− and jnk2 −/− Mice Fed a Control (Con) or Methionine- and Choline-Deficient (MCD) Diet for 4 Weeks*
*Change in body weight is expressed as the gain (+) or loss (−) in weight in grams over the 4 weeks of diet administration. Liver weight is expressed as a percentage of body weight.
There is a statistically significant difference in this value from wild-type mice on control diet or §from wild-type and jnk2 −/− mice on MCD diet (all P < .02).
Histological analysis revealed minimal steatosis and no inflammation in all three types of mice fed the control diet (Fig. 3A–C). MCD diet–fed mice developed macrovesicular and microvesicular steatosis and inflammatory aggregates of lymphocytes and neutrophils (Fig. 3D–F). However, jnk1 −/− mice on the MCD diet developed markedly reduced hepatic steatosis and inflammation (Fig. 3E). This impression was confirmed by blinded histological grading of liver sections. The grade of steatosis was approximately 2-fold greater in wild-type and jnk2 knockout mice than in jnk1 null mice (Fig. 4A). Jnk1 −/− mice had a statistically significant decrease in steatosis on control diet as well. Similarly the inflammation grade in jnk1 knockout mice was reduced 45% compared with that in wild-type and jnk2 knockout mice (Fig. 4B). Thus, although wild-type and jnk1 and jnk2 knockout mice all developed steatosis and inflammation on the MCD diet, the degree of steatosis and inflammation was significantly reduced in animals lacking jnk1.
Triglyceride Content, Proinflammatory Gene Expression and Levels of Lipid Peroxidation Are Decreased in jnk1 Knockout Mice Fed the MCD Diet.
To further validate the histological differences in steatosis observed among the three mouse strains on MCD diet, hepatic triglyceride content was determined. Triglyceride content among the three strains of mice fed the control diet was equivalent (Fig. 5A). A 5-fold increase in hepatic triglyceride content occurred after 4 weeks of MCD diet in wild-type and jnk2 knockout mice (Fig. 5A). In contrast, consistent with the histological grading, the increase in hepatic triglyceride content in jnk1 knockout mice was significantly decreased by 60% compared with the levels in wild-type and jnk2 null mice (Fig. 5A).
The decreased histological evidence of inflammation in jnk1 −/− mice was further supported by RT-PCR analysis of mRNA expression of two proinflammatory genes, ICAM-1 and MCP-1. Both genes have previously been demonstrated to be upregulated in parallel with the development of MCD diet–induced steatohepatitis.29 Similar to findings of Leclercq et al.,29 the MCD diet resulted in marked increases in ICAM-1 (intracellular adhesion molecule-1) and MCP-1 (monocyte chemoattractant protein-1) mRNA expression in wild-type mice (Fig. 5B). Both genes were upregulated in jnk1 −/− mice but the levels of ICAM-1 and MCP-1 mRNA in these mice were significantly decreased by 48% and 65%, respectively, compared with wild-type mice (Fig. 5C). Levels of both mRNAs were decreased in jnk2 −/− mice compared with wild-type mice but the changes were not statistically significant.
MCD diet–induced experimental steatohepatitis and human NASH are associated with increased oxidative stress.7, 24 The generation of excessive reactive oxygen species leads to the nonenzymatic formation of lipid hydroperoxides that cause cellular injury through a modification of cellular macromolecules. To additionally assess the function of jnk1 and jnk2 expression in steatohepatitis, hepatic levels of MDA, a product of lipid peroxidation, and serum ALT, a marker of hepatocellular injury, were determined in MCD diet–fed mice. MDA levels increased 2 fold in wild-type and jnk2 knockout mice on MCD diet compared with control diet (Fig. 5D). However, the increase in MDA levels in jnk1 −/− mice was significantly decreased by 25% compared with wild-type and jnk2 knockout mice (Fig. 5D). MDA levels did not differ significantly among control diet–fed animals.
Liver Injury and Cell Death Are Decreased in the Absence of jnk1.
To examine whether reductions in steatosis, inflammation, and oxidative stress translated into a decrease in hepatocyte injury, levels of serum ALT and hepatic apoptosis were compared in the three types of mice. In accordance with previously published observations,24, 27 ALT levels increased 15 fold in MCD diet–fed wild-type mice (Fig. 6A). While jnk2 −/− mice developed a similar increase in serum ALT as wild-type mice, this increase was significantly reduced by 40% in jnk1 knockout mice (Fig. 6A).
It has been suggested that hepatocyte injury in steatohepatitis results in hepatocyte apoptosis.30 Consistent with this concept, the number of TUNEL-positive cells increased 10 to 20 fold in the livers of MCD diet–fed wild-type and jnk2 −/− mice (Fig. 6B). In contrast, the number of apoptotic cells in MCD diet–fed jnk1 −/− mice was only 27% of that in wild-type animals (Fig. 6B). Thus, the absence of jnk1 ultimately led to a significant decrease in hepatocellular injury and death.
Loss of jnk1 Affects Serum Levels of Adiponectin But Not TNF.
Recently the importance of inflammatory modulators in the development of steatohepatitis has been recognized.9 Changes in levels of both adiponectin and TNF have been linked to the development of insulin resistance, obesity, and liver injury; an imbalance in these factors could potentially contribute to the initiation and progression of steatohepatitis.12, 31 To address this possibility, levels of adiponectin and TNF were examined in MCD diet–fed mice. Serum adiponectin levels were equivalent in wild-type and jnk2 knockout mice and unchanged by administration of the MCD diet (Fig. 7A). However, whereas levels in jnk1 −/− mice on control diet were similar, levels in MCD diet–fed jnk1 −/− animals were increased 50% over those in wild-type and jnk2 −/− mice. No significant differences in the levels of serum TNF were observed among control diet and MCD diet–fed wild-type, jnk1 and jnk2 knockout mice (Fig. 7B). Thus, jnk1 ablation led to the potentially protective effect of increasing serum adiponectin but failed to affect levels of TNF.
In cultured cells, activation of JNK by a variety of environmental stresses is known to regulate critical cellular responses ranging from proliferation to apoptosis.17, 32 The differential effects of JNK1 and JNK2 in vitro have just begun to be delineated with specific effects ascribed to both isoforms.22, 33–35 The nature of JNK activation and function of the different JNK isoforms in whole animals is less clear. The present investigations demonstrate increased activation of the hepatic JNK signaling cascade in an in vivo model of chronic steatohepatitis induced by the MCD diet. This conclusion is based on findings of increased levels of hepatic JNK activity, phospho-JNK, total and phospho-c-Jun, and AP-1 DNA binding in MCD diet–fed animals. JNK activation occurred at the initial 1-week time point examined in this model, and continued throughout the 4-week course of the study, providing the first evidence that sustained JNK activation is associated with chronic liver disease. Increased JNK activity has been reported previously in obese mice, although the increase in JNK activation in liver was slight compared with other organs such as fat and muscle.15 It is possible that JNK activation in our model occurred with the onset of fat accumulation or at a later time during the development of steatohepatitis. The present studies could not distinguish between these two possibilities because steatohepatitis occurs shortly after the onset of significant steatosis in the MCD diet model. One factor that may cause sustained activation of JNK is the oxidative stress associated with this model.24 Overexpression of the prooxidant enzyme CYP2E1 as occurs in NASH is sufficient to cause sustained overactivation of JNK in hepatocytes.10
The prolonged nature of JNK activation in MCD diet–fed mice has important implications for the finding that JNK promoted liver injury in this model. A change in the duration of JNK activation from a transient to sustained event is thought to convert JNK to a pro-death signal as exemplified by the ability of NF-κB inactivation to trigger prolonged JNK activation that sensitizes hepatocytes to death from TNF.18, 19 The JNK activation that was sustained for weeks in the MCD diet–fed mice may have similarly triggered a cell death response in hepatocytes in vivo. Recently JNK overactivation was associated with immune-mediated liver injury from concanavalin A.36 In contrast to our findings, this form of acute liver injury was decreased in both jnk1 −/− and jnk2 −/− mice.37 In our study, selective loss of jnk1 but not jnk2 inhibited the development of steatohepatitis. This finding may be explained by the fact that JNK kinase activity as determined by phosphorylation of c-Jun was lost in jnk1 −/− mice but unaffected in jnk2 −/− mice. Levels of both total and phospho-c-Jun were reduced in MCD diet–fed jnk1 −/− mice consistent with the decrease in JNK activity. In parallel with these findings, AP-1 DNA binding was also decreased. The ability of JNK1 to promote steatohepatitis may be mediated through kinase activity that leads to c-Jun phosphorylation, the phosphorylation of other proteins,37 or an effect totally unrelated to its kinase activity. The present investigations cannot differentiate among these possibilities. Our studies are consistent with, but not proof of, the conclusion that the classic c-Jun kinase activity of JNK promoted steatohepatitis because only loss of the specific JNK isoform responsible for c-Jun kinase activity resulted in reduced disease. This finding supports in vitro studies in non-hepatic cells demonstrating that the JNK1 but not JNK2 isoform is responsible for c-Jun kinase activity and that loss of JNK2 may act to further increase JNK activation.35 In our studies jnk2 −/− mice also had increased JNK activity although it did not translate into an increase in steady-state levels of phosphorylated c-Jun. The protective effect of loss of JNK1 but not JNK2 is also consistent with in vitro hepatocyte studies that first demonstrated a selective protective effect of JNK1 but not JNK2.22 A determination of whether JNK1 but not JNK2 always serves as an injurious signaling pathway in the hepatocyte, or the roles of the two isoforms vary depending on the injurious stimulus, requires further investigation into other forms of in vivo liver injury.
The development of both hepatic steatosis and injury was significantly but not completely inhibited by loss of JNK1. The partial nature of the response indicates that either JNK-independent mechanisms contribute to this process or that JNK2 is capable of partially replacing the function of JNK1. JNK1 activation could serve as either of the two “hits” that culminate in steatohepatitis.3 First, JNK1 may promote the lipid accumulation that leads to the initial steatosis as loss of JNK1 significantly reduced the development of steatosis in the MCD diet model. A primary reduction in hepatic lipid accumulation should translate into a secondary decrease in liver injury because lipid serves as a substrate that fuels the oxidant stress that causes cellular injury. JNK1 may promote the development of steatosis through its ability to induce insulin resistance, as MCD diet–induced steatohepatitis is associated with JNK-dependent hepatic insulin resistance.23 Alternatively, JNK1 may have some as yet undescribed effect on lipid synthesis or oxidation. Second, JNK1 activation could act as the second “hit” in the development of steatohepatitis by acting to initiate the process of cellular injury. The JNK/c-Jun pathway mediates hepatocyte injury from the cytokine TNF and oxidants.10, 18, 19, 38 TNF and oxidative stress have both been implicated in induction of the cellular injury of NASH,24, 31 suggesting that JNK1 activation may promote hepatocellular death from these factors in this disease. The fact that endoplasmic reticulum stress induces insulin resistance and a JNK-dependent apoptosis39, 40 suggests that JNK1 may promote steatohepatitis through activation of ER stress. However, hepatic ER stress does not appear to occur in the MCD diet model as there is no associated increase in protein levels of the ER stress markers BiP and GADD153 in the liver (data not shown). Finally, it is possible that JNK1 activation may act as the first or second hit, not through its direct effects in the liver but through extrahepatic effects on inflammatory or immunomodulatory factors such as cytokines or adipokines. Loss of jnk1 failed to alter serum TNF levels but did significantly increase adiponectin levels as previously reported in obese mice.12, 41 Adiponectin levels are inversely correlated with disease activity in human NASH,14 and adiponectin administration prevents experimental NASH.12, 41 Thus, it is possible that increased adiponectin levels in jnk1−/− mice may have inhibited the development of steatohepatitis. Against this possibility is the fact that the degree of MCD diet–induced steatohepatitis is equivalent in wild-type and adiponectin knockout animals (Czaja, MJ, unpublished data). To completely exclude an effect of increased adiponectin levels in this model, however, the effects of adiponectin supplementation on MCD diet–fed wild-type mice would have to be investigated.
These investigations demonstrate that sustained JNK activation occurs in chronic steatohepatitis and promotes both the steatosis and liver injury that comprise NASH. Therefore, JNK not only modulates acute states of injury and cell death but also chronic conditions of tissue injury as well. Further studies will have to address whether anti-JNK treatment is effective in reducing already-established steatohepatitis. However, these findings together with other recent studies demonstrating the potential therapeutic benefit of JNK inhibitors in diabetes,42 suggest that JNK inhibition may have widespread benefit in the treatment of complications of the insulin resistant state.