Hepatology highlights
Authors: Neil Kaplowitz
Journal: Hepatology (2005)

JAG1 (Jagged 1 ligand) and Notch 1-4 (receptors) are involved in organogenesis and mutations in the former are associated with Alagille's syndrome (paucity of interlobular bile ducts). Croquelois et al. assessed the role of Notch 1 in liver development. To circumvent embryonic lethality of Notch 1 KO they used inducible inactivation with a “loxed” Notch 1 strain crossed with MxCre strain which is activated by interferon-alpha induced viral infection (mimicked by injection of double stranded polyiosinic–polycytidylic acid shortly after birth). This approach efficiently inactivated the Notch 1 gene in hepatocytes and bile duct epithelial cells. This lead to the development of an enlarged nodular liver (See Fig.) with characteristic histological picture of nodular regenerative hyperplasia (NRH) without vascular abnormalities, accompanied by increased BrdU-labeling of hepatocytes. However, following partial hepatectomy, Notch 1 KO mice regenerated liver more slowly than wild type but this was because the remaining liver was larger to begin with after a fixed % resection. Interestingly, no paucity of bile ducts was observed and intrahepatic bile duct proliferation after bile duct ligation was not altered in Notch 1 KO mice. This work suggests that Notch 1 alone is not the critical receptor for Jagged 1 on bile ducts. However, absence of Notch 1 causes striking spontaneous proliferation of hepatocytes and a picture of NRH. Thus Notch 1 is an inhibitor of hepatocyte proliferation. An intriguing possibility is that Notch 1 functions as a tumor suppressor; proliferation in KO mice might predispose to HCC. In addition, Notch 1 may be a signal for sustaining normal hepatocytes in Go. What happens to Jagged-1 and Notch 1 expression in normal liver regeneration? How does the presence or absence of Notch1 affect the expression of cytokines and other growth factors? Finally, these findings demonstrate that NRH may occur independent of the widely accepted vascular mechanism. (See HEPATOLOGY 2005; 41:487-496) Keratin 8/18 mutations have been identified as a risk factor for the development of liver disease. These mutations increased the susceptibility to various toxic insults. Zhou et al. addressed possible mechanisms for this predisposition using transgenic mice expressing normal and mutated cytokeratin 18, which exhibit cytoplasmic keratin filament disruption. Using gene expression profiling with a twofold cutoff, several genes in the methionine–GSH metabolism pathway were affected: SAH hydroxylase and cysteine sulfinic acid decarboxylase (csad). Hepatic GSH and cysteine levels were unchanged but modest decreases (15%-22%) in SAMe and SAH were observed. Interestingly, malondialdehyde and protein carbonyl levels were significantly increased, reflecting oxidative stress, in the mice expressing mutated K18. These animals have previously been shown to exhibit greater susceptibility to oxidative stress. The authors suggest that the changes in csad and SAMe and SAH under basal conditions prime the liver to respond to increased oxidative stress with an inadequate antioxidant response. Although the findings indicate that oxidative stress is increased under basal conditions, the mechanism needs further explanation: role of inflammation, mitochondrial abnormalities. Furthermore, the changes in the antioxidant system are generally modest and of uncertain importance. Further work is needed to address the responsiveness of the various components of this system to an imposed stress. Is the mild chronic hepatitis seen in this model prevented by antioxidants? (See HEPATOLOGY 2005; 41:517-525) Two orphan nuclear receptors, PXR and CAR, have been shown to regulate the bilirubin-detoxifying pathway. Transgenic mice that over express either PXR or CAR show increased bilirubin clearance. However the relative contribution of these two transcription factors is unclear. To get at this equation, Saini et al. used single or double knockout mice. Single knockouts showed normal serum bilirubin levels. However, surprisingly PXR-KO mice were resistant to hyperbilirubinemia induced by bilirubin infusion (BR) or hemolysis (PHZ) but CAR-KO and double KO were not (see Fig.). PXR-KO (but not CAR-KO and double KO) mice exhibited increased expression of oatp-4, ugt1A1, gstA2 and mrp2, the components of the pathway for uptake, conjugation and excretion of bilirubin. Conditional overexpression of either PXR or CAR activated bilirubin clearance. Thus, both the loss and activation of PXR led to resistance to hyperbilirubinemia. To elucidate the mechanism, studies were performed using cells with an mrp2- reporter cotransfected with PXR or CAR. CAR as expected increased ligand-independent reporter expression but PXR suppressed basal reporter expression, which was overcome by addition of a PXR ligand. Ligand-free PXR also suppressed ligand-stimulated CAR induced reporter expression. Ligand-free PXR disrupted the interaction of CAR and transcriptional coactivator (steroid receptor coactivator-1). This elegant work demonstrates that ligand-free PXR suppresses CAR, a transcription factor with constitutive activity in the absence of ligand, by interfering with coactivator recruitment to the CAR transcriptional apparatus. Increased bilirubin clearance in PXR-null mice, therefore, is explained by derepression of CAR. On the other hand, the increase in bilirubin clearance in response to PXR ligand activation is due to a combination of derepression of CAR and direct activation of detoxifying genes. Since double KO mice did not exhibit increased sensitivity to hyperbilirubinemia, other pathways must exist aside from PXR and CAR. (See HEPATOLOGY 2005;41:497-505) Amiodarone, benzbromarone and benzarone are three hepatotoxic chemicals, which share a benzofuran moiety. Kaufmann et al. compared the effects of these three structurally related toxins on mitochondrial function and HepG2 cell and rat hepatocyte viability. All three drugs depolarized and uncoupled mitochondria and inhibited β-oxidation of fatty acid and electron transport in intact cells in a dose dependent fashion accompanied by oxidative stress. The disturbed mitochondrial function lead to permeability transition, cytochrome c release and apoptosis and necrosis. These features were partially inhibited by ascorbate confirming an important role for oxidative stress in inducing cell death. The benzofuran ring along was not toxic indicating the importance of side chain substituents. Pure uncouplers, such as dinitrophenol, also did not have these effects. These carefully conducted, detailed studies illustrate how a combination of uncoupling (increased respiration) and inhibition of electron transport are a potentially lethal combination. The importance of inhibition of β-oxidation, which appeared to be an independent effect of these drugs, is uncertain but could aggravate ATP depletion and modulate the balance of apoptosis versus necrosis. A few additional aspects merit further investigation. Benzbromarone was much more potent than the other two drugs. Does this relate to the extent of mitochondrial accumulation of these drugs? Also, amiodarone is a weak base, which has been suggested to be important in its toxic effect on mitochondrial membrane potential, but the other two drugs lack a nitrogen in the side chains. Thus, the precise mechanism for the effects of these substituted benzofurans on mitochondria remains to be determined. (See HEPATOLOGY 2005;41:925-935) Primary biliary cirrhosis is accompanied by mitochondrial autoantibodies and auto reactive T cells directed at the pyruvate dehydrogenase complex (PDC), which reflects loss of tolerance. Robe et al. investigated the mechanism for auto reactive T cells to PDC. The authors confirmed their previous work demonstrating that mice sensitized to bovine-PDC develop B-cell sensitization and cross-reacting autoantibodies but no T-cell response, whereas cosensitization to both bovine and mouse PDC resulted in breakdown of T cell tolerance. They then showed that injection of splenic B-cells from bovine PDC sensitized animals, when pulsed with self m-PDC, developed T-cell sensitization to self-PDC as measured by in vitro proliferation of splenocytes from treated mice exposed to self-PDC. In contrast, transfer of purified IgG from bovine-PDC immunized mice exhibiting autoantibody to mouse-PDC did not induce T-cell sensitization upon exposure to self-PDC. These results support the hypothesis that binding of self-PDC to reactive B-cell surface Ig receptors may lead to uptake and presentation of self-derived epitopes. Thus, sensitized B-cells may act as antigen presenting cells, breaking T-cell tolerance. It is likely that the T-cell response leads to attack on the interlobular bile ducts. What accounts for initial B-cell sensitization and autoantibody production? Suggestions include cross-reaction to bacterial PDC or chemically modified PDC. What is the source of self-PDC needed to drive breakdown of T-cell tolerance? Suggestions include localized infection leading to necrosis (release of PDC) or apoptosis (surface targeting of PDC). Finally, do these mice exhibiting B- and T-cell sensitization to self-PDC develop interlobular duct destruction or can they be induced to do so? (See HEPATOLOGY 2005;41:1106-1112) An apical Na+ dependent bile acid transporter (ASBT) is present in the ileum where it governs the bile acid reabsorption step of the enterohepatic circulation. ASBT is also expressed in cholangiocytes where its functional significance in vivo is not well understood. Alpini et al. exploited the secretin-induced membrane retrieval of ASBT to assess its functional role. In isolated cholangiocytes, secretin induced a near doubling of taurocholate (TC) uptake capacity, which was prevented by colchicine pretreatment. This is consistent with microtubule dependent cell membrane insertion of ASBT recruited from an intracellular pool in response to secretin. Subcellular fractionation and Western blotting of ASBT and immunohistochemistry of ASBT in vivo (see Fig.) directly confirmed this interpretation. In vivo studies in bile fistula rats demonstrated that secretin pretreatment tripled TC-stimulated choleresis accompanied by increased biliary cholesterol and phospholipid output along with slower biliary transit time of TC (time to recover 50% of radiolabeled bolus). Colchicine pretreatment in the absence of secretin administration had no effect on these parameters but abrogated the effects of secretin, including the secretin-induced delayed biliary transit of TC. This work provides rather convincing evidence that, by inducing membrane insertion of ASBT in cholangiocytes, more bile acid flux through ductal reabsorptive cholehepatic shunting slows biliary TC transit through recirculation and thus amplifies bile acid-induced choleresis and biliary lipid secretion (more bang for your buck). However, the evidence for cholehepatic shunting is indirect and, as suggested by the authors, repetition of these studies in ASBT-KO mice would help to verify the current interpretation. (See HEPATOLOGY 2005; 41:1037-1045)