Hepatology highlights
Authors: Neil Kaplowitz
Journal: Hepatology (2004)

Gap junctions allow chemicals to move from one hepatocyte to the next. Asamoto et al. examined the role of gap junctions in hepatotoxicity in transgenic rats expressing a dominant-negative mutant connexin 32 (major gap junction protein in liver) under the control of the albumin promoter. High expression of the transgene led to a marked decrease in endogenous gap junctions as determined by both structural and functional analyses. Transgenic animals were strikingly protected against severe centrilobular necrosis induced by galactosamine (Fig. 1a) and CCl4 (Fig. 1c) versus wild type (Fig. 1b and 1d, respectively). Thus, in this short report a dominant negative connexin abrogated normal junctional plaque formation in liver and provided support for the innocent-bystander mode of collateral damage in which toxic factors are transferred from one cell to its neighbor, spreading the damage. It will be interesting to learn if specific metabolites of CCl4 or galactosamine versus nonspecific molecules that participate downstream in cell injury (e.g., caspases, calpains) are transferred and if chemical inhibitors of gap junctions can be identified as hepatoprotective. (See HEPATOLOGY 2004;40:205–210.) Although much evidence suggests a link between death receptors (tumor necrosis factor receptor 1 [TNF-R1] and Fas) and reactive oxygen species (ROS), the postreceptor signaling pathway, the source of ROS and their importance is not completely known. Ding et al. examined the role of Bid, a pro-apoptotic Bcl-2 family member, using hepatocytes from bid-deficient and wild-type mice. Cells were treated with TNF/actinomycin D or anti-Fas. An SOD mimetic (MnTBAP) and vitamin E analog (Trolox) protected against apoptosis, caspase 3,8,9 activation, and ROS production. In Bid-deficient cells no increase in ROS was detected. A pancaspase inhibitor blocked ROS, Bid activation, and apoptosis. ROS (green) production co-localized (yellow) with mitochondria (red) (Fig. 1). FLICE-inhibitory protein (FLIP), a caspase 8 inhibitor, was rapidly degraded in a ROS-dependent fashion. FLIP degradation was not seen in Bid-deficient cells. In isolated mitochondria, cytochrome c release, cristae reorganization, and lipid peroxidation in response to recombinant tBid was inhibited by antioxidants. This work helps clarify the sequence of events after death receptor ligation: Caspase 8 is activated to a sufficient extent to cleave Bid, which then acts on mitochondria to promote ROS, which leads to FLIP degradation, allowing more caspase 8 activation in a feedback self-amplification loop. This event then leads to more Bid cleavage, cytochrome c release, and ROS production, sufficient to irreversibly promote apoptosis. This work provides an important paradigm to be probed in future studies to assess the site on the electron transport chain affected by Bid (or Bax), the contribution of mitochondrial lipid peroxidation, the mechanism of FLIP degradation, and the applicability of these findings to the in vivo situation. Another interesting caveat needing more detailed study is the apparent importance of superoxide relative to H2O2. (See HEPATOLOGY 2004;40:403–413.) Exposure to increased free fatty acids (FFA) exerts cellular effects (lipotoxicity), which may contribute to nonalcoholic fatty liver disease (NAFLD). Feldstein et al. examined the effects of FFA on the liver in a variety of in vitro and in vivo conditions. In HepG2 cells loaded with FFA, Bax translocated to lysosomes, lysosomal cathepsin B was released, and apoptosis occurred. Using hepatocytes from wild-type mice, FFA exposure increased tumor necrosis factor (TNF) expression while cathepsin B knockout cells did not show this response, thus linking cathepsin B to TNF expression (via nuclear factor κB [NF-κB]). Using liver sections from patients with NAFLD, cathepsin B was found to have redistributed from liposomes to cytosol. When fed a high-carbohydrate diet, the cathepsin B knockout mice did not develop fatty liver or increased TNF expression as seen in wild-type mice. Treatment with a cathepsin inhibitor reproduced this finding. TNF-receptor 1 (R1)−/− mice were also protected against diet-induced fatty liver. Diet-induced obesity was not altered in cathepsin B and TNF-R1−/− mice, while the liver was spared of steatosis. This work thus demonstrates that exposure to FFA causes Bax translocation to mitochondria, which may lead to release of cathepsin B. The cathepsin B release is followed by NF-κB activation and increased TNF expression. TNF then leads to NAFLD. The mechanism for NF-κB activation and the cellular source of TNF in NAFLD need to be clarified in future studies. Also, it would be of interest to know whether lysosomal cathepsin B is released in response to diet in TNF-R1−/− mice, and whether the fatty liver is required for this to occur. Although the interplay of events described in this report is complex and may feed back and forth, cathepsin B clearly emerges as a potential therapeutic target. (See HEPATOLOGY 2004;40:185–194.) Fatty livers are more vulnerable to inflammatory stress. This fact is exemplified by the sensitivity of leptin-deficient ob/ob mice to lipopolysaccharide (LPS)-induced liver injury, which correlates with an underlying reduction in natural killer T (NKT) cells, liver lymphocytes that play a key role in the innate immune response. Norepinephrine (NE) levels are deceased in ob/ob mice, which might lead to dysregulation of NKT cells. Li et al. examined the role of NKT cells in sensitivity to LPS. They confirmed that ob/ob mice developed thymic atrophy, but with sparing of NKT cells, whereas hepatic NKT cell population declined, suggesting that extrathymic factors account for the latter. NKT cells express adrenoreceptors, and α-adrenoreceptor blockade or chemical sympathectomy in wild-type mice lead to decreased hepatic NKT cells. Dopamine β-hydroxylase−/− mice, which cannot produce NE, also exhibited decreased hepatic NKT cells that could be reversed by NE supplementation. NE also reversed NKT depletion in ob/ob mice by decreasing NKT cell apoptosis while stimulating interleukin (IL) 4 production. Interestingly, CD1d−/− mice, which are also NKT deficient, exhibited a moderate increased sensitivity to LPS-induced liver injury. Furthermore, treatment of ob/ob mice with NE returned basal serum alanine aminotransferase to near normal. This study suggests that leptin deficiency disrupts the adreno-hypothalamic regulation of NE, and the lack of NE then leads to loss of NKT cells, which disrupts cytokine balance (decreased interleukin 4) in favor of a Th1 proinflammatory state. The effect of NE on liver histology, fatty liver, and insulin resistance needs to be examined, and the site of tumor necrosis factor action (NKT cell apoptosis, etc.) needs to be defined. In addition, the status of NKT cells in other models of NAFLD with increased leptin needs to be addressed to see if this work can be extrapolated. Nevertheless, this study is a fascinating demonstration of the effect of neurohumoral dysregulation on the innate immune system and the susceptibility to a hepatotoxic response to an LPS-induced proinflammatory stimulus. (See HEPATOLOGY 2004;40:434–441.) Adiponectin is a cytokine expressed in adipose tissue, but inversely to the body mass index. Interestingly, adiponectin-R2 receptors are found mainly in liver. Masaki et al. examined the role of adiponectin in lipopolysaccharide (LPS)/galactosamine-induced liver injury. More severe liver injury and greater lethality was observed in KK-Ay obese mice versus lean controls. Adiponectin levels were lower in serum and adipose tissue of obese mice. Pretreatment with adiponectin significantly attenuated the effects of LPS/galactosamine in obese mice in conjunction with inhibition of the LPS-induced upregulation of tumor necrosis factor (TNF). Peritoneal macrophages from the obese mice pretreated with adiponectin in vitro exhibited diminished TNF production in response to LPS. When TNF treatment replaced LPS in vivo, adiponectin offered less protection. This work adds to the evidence that obesity and fatty liver sensitize to TNF-induced hepatotoxicity and suggests that lower levels of adiponectin contribute to this phenomenon. Administration of adiponectin suppresses TNF production. The mechanism for this effect may be related to upregulation of peroxisome proliferator-activated receptor α, but this theory requires more investigation. (See HEPATOLOGY 2004;40:177–184.) Transcription factors control the inflammatory response: nuclear factor κB (NF-κB) promotes inflammatory gene expression, whereas peroxisome proliferator-activated receptor (PPAR) α and PPARγ inhibit NF-κB transactivation. Romics et al. tested the hypothesis that increased endotoxin sensitivity in fatty liver disease is due to a proinflammatory imbalance in the activity of these transcription factors. Ob/ob leptin-deficient obese and wild-type lean mice were given acute doses of endotoxin (lipopolysaccharide [LPS]), alcohol, or both. Either LPS or alcohol-induced higher serum tumor necrosis factor and interleukin (IL) 6 in obese mice. In lean mice, alcohol attenuated the cytokine response to LPS but failed to do so in obese mice. Hepatic messenger RNA (mRNA) expression of IL-12, IL-1 and IL-1Ra were increased and IL-10 decreased in ob/ob mice. The response of mRNA to LPS was inhibited by ethanol in lean but not obese mice. These findings correlated with histological inflammatory infiltration in response to LPS: attenuated by ethanol in lean livers, and aggravated by ethanol in obese livers. An examination of transcription factors revealed parallel findings with respect to NF-κB: enhanced nuclear p50p65 and p50p50 in obese versus lean livers in response to LPS, and selective suppression of p50p65 by ethanol only in lean livers (Fig. 1). Thus, not only is NF-κB enhanced in obese liver in response to LPS, but also the increase in transcriptionally active p50p65 is greater than the inactive and inhibitory p50p50 component, and the p50p65 activation is not inhibited by ethanol. The investigators then examined the relationship of NF-κB results to changes in PPARs. Peroxisome proliferator response element (PPRE) binding activity increased markedly in ob/ob nuclear extracts in response to LPS or ethanol, whereas there was a decrease in lean extracts. Alcohol decreased LPS-induced PPRE binding in ob/ob extracts and increased it in lean liver extracts. Nuclear PPARα decreased in obese mice treated with ethanol + LPS, whereas LPS alone decreased PPARα and PPARγ in lean mice, which was reversed by cotreatment with ethanol. Overall, this work confirms the increased sensitivity of LPS treated ob/ob mice to LPS-induced NF-κB activation and hepatic inflammation. Striking differences in response of obese and lean mice to addition of ethanol to LPS provide evidence for dysregulation of inflammation in ob/ob mice. The investigators suggest that the antiinflammatory effects of PPARs may be defective in obese livers. Decreased PPARα activation could contribute to increased NF-κB activation and decreased inhibitory κB-α expression. The investigators show clear differences in PPAR responses of obese and lean mice, but more work is required to prove that these responses account for sensitivity of fatty liver to LPS and ethanol. In addition, the cell-type specificity of the responses will need to be sorted out to better understand the significance of transcriptional dysregulation. (See HEPATOLOGY 2004;40:376–385.)