John L. Gollan

Tumor necrosis factor–alpha decreases hepatocyte bile salt uptake and mediates endotoxin-induced cholestasis

Tumor necrosis factor–alpha (TNF,α), a cytokine that is produced in a variety of inflammatory diseases associated with cholestasis, is believed to be the primary mediator of the systemic effects of endotoxin. Thus, we have investigated the role of TNFα in the pathogenesis of endotoxin‐induced cholestasis in intact animals, and in the uptake of taurocholate by cultured hepatocytes. Male Sprague‐Dawley rats received either intravenous (IV) endotoxin (7.5 mg/kg) or monoclonal anti‐TNFα antibody followed by endotoxin. Basal bile flow and bile salt excretion were measured for a 2‐hour period, after which all animals received an IV bolus of taurocholate (10 μmol/100 g body weight). Endotoxin decreased basal bile flow by 41% and bile salt stimulated bile flow by 38% (n = 12; P < .01). Basal bile salt excretion was decreased 86% after endotoxin administration. Passive immunization with anti‐TNFα antibody blocked this endotoxin‐associated cholestasis. In addition, rat hepatocytes were isolated and cultured in the presence of either endotoxin (10 μg/mL) or TNFα (100 ng/mL) for 24 hours. These primary hepatocyte cultures exhibited a dose‐ and timedependent, noncompetitive, inhibition of taurocholate uptake. We postulate that TNFα is an important mediator of the cholestasis of sepsis. (HEPATOLOGY 1995; 22:1273–1278.).

Enhanced activity of the free radical producing enzyme xanthine oxidase in hypoxic rat liver. Regulation and pathophysiologic significance.

It has been widely proposed that conversion of xanthine dehydrogenase (XDH) to its free radical-producing form, xanthine oxidase (XOD), underlies ischemic/reperfusion injury, although the relationship of this conversion to hypoxia and its physiologic control have not been defined. This study details the time course and control of this enzymatic interconversion. In a functionally intact, isolated perfused rat liver model, mean % XOD activity increased as a function of both the duration (25 to 45% in 3 h) and degree (r = 0.97) of hypoxia. This process was markedly accelerated in ischemic liver by an overnight fast (45 vs. 30% at 2 h), and by imposing a short period of in vivo ischemia (cardiopulmonary arrest 72%). Moreover, only under these conditions was there a significant rise in the XOD activity due to the conformationally altered XDH molecule (XODc, 18%), as well as concomitant morphologic injury. Neither circulating white blood cells nor thrombosis appeared to contribute to the effects of in vivo ischemia on enzyme conversion. Thus, it is apparent that conversion to the free radical-producing state, with high levels of XOD activity and concurrent cellular injury, can be achieved during a relatively short period of hypoxia under certain well-defined physiologic conditions, in a time course consistent with its purported role in modulating reperfusion injury. These data also suggest that the premorbid condition of organ donors (e.g., nutritional status and relative state of hypoxia) is important in achieving optimal organ preservation.

A Unique Multifunctional Transporter Translocates Estradiol-17β-Glucuronide in Rat Liver Microsomal Vesicles

A wide array of drugs, xenobiotics, and endogenous compounds undergo detoxification by conjugation with glucuronic acid in the liver via the action of UDP-glucuronosyltransferases. The mechanism whereby glucuronides, generated by this enzyme system in the lumen of the endoplasmic reticulum (ER), are exported to the cytosol prior to excretion is unknown. We examined this process in purified rat liver microsomes using a rapid filtration technique and [3H]estradiol-17β-d-glucuronide ([3H]E217βG) as model substrate. Time-dependent uptake of intact [3H]E217βG was observed and shrinkage of ER vesicles by raffinose lowered the steady-state level of [3H]E217βG accumulation. In addition, rapid efflux of [3H]E217βG from rat liver microsomal vesicles suggested that the transport process is bidirectional. Microsomal uptake was saturable with an apparentKm and V max of 3.29 ± 0.58 μm and 0.19 ± 0.02 nmol·min−1·mg protein−1, respectively. Transport of [3H]E217βG was inhibited by the anion transport inhibitors 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid and probenecid. Specificity of the transport process was investigated by studying thecis-inhibitory effect of anionic metabolites, as well as substrates of the plasma membrane multidrug resistance-associated proteins on the uptake of [3H]E217βG. Collectively, these data are indicative of a novel multifunctional and bidirectional protein carrier for E217βG and other anionic compounds in the hepatic ER. This intracellular membrane transporter may contribute to the phenomenon of multidrug resistance.

Influence of glutathione S-transferase B (ligandin) on the intermembrane transfer of bilirubin. Implications for the intracellular transport of nonsubstrate ligands in hepatocytes.

To examine the hypothesis that glutathione S-transferases (GST) play an important role in the hepatocellular transport of hydrophobic organic anions, the kinetics of the spontaneous transfer of unconjugated bilirubin between membrane vesicles and rat liver glutathione S-transferase B (ligandin) was studied, using stopped-flow fluorometry. Bilirubin transfer from glutathione S-transferase B to phosphatidylcholine vesicles was best described by a single exponential function, with a rate constant of 8.0 +/- 0.7 s-1 (+/- SD) at 25 degrees C. The variations in transfer rate with respect to acceptor phospholipid concentration provide strong evidence for aqueous diffusion of free bilirubin. This finding was verified using rhodamine-labeled microsomal membranes as acceptors. Bilirubin transfer from phospholipid vesicles to GST also exhibited diffusional kinetics. Thermodynamic parameters for bilirubin dissociation from GST were similar to those for human serum albumin. The rate of bilirubin transfer from rat liver basolateral plasma membranes to acceptor vesicles in the presence of glutathione S-transferase B declined asymptotically with increasing GST concentration. These data suggest that glutathione S-transferase B does not function as an intracellular bilirubin transporter, although expression of this protein may serve to regulate the delivery of bilirubin, and other nonsubstrate ligands, to sites of metabolism within the cell.

Primary Biliary Cirrhosis: New Therapeutic Directions

The immunologic mechanisms responsible for the development of primary biliary cirrhosis (PBC) remain poorly defined, although recent investigations have provided new clues as to the role of cellular membrane proteins such as the mitochondrial autoantigens and intercellular adhesion molecules. Additionally, new therapeutic agents have become available that markedly enhance the prospect for medical management of both hepatic and extrahepatic manifestations of PBC. Definitive therapy with ursodeoxycholic acid and/or methotrexate, and symptomatic relief of pruritus with rifampicin or metronidazole may become standard in the years ahead. The results of their use to date in the treatment of PBC are detailed in this review. Successful therapy of hepatic osteodystrophy associated with PBC has yet to be achieved, although early data suggest a role for ursodeoxycholic acid, estrogen, calcium, and vitamin D in the management of this debilitating problem. Orthotopic liver transplantation continues to be successful for the management of advanced disease (greater than 70% 5-year survival) and will remain an essential therapeutic tool until definitive medical therapy for PBC becomes available.

Characterization of UDP-glucuronic acid transport in rat liver microsomal vesicles with photoaffinity analogs

The endoplasmic reticulum (ER) of rat liver contains several well characterized UDP-glucuronosyltransferases (UGTs), membrane-bound proteins of 50-54 kDa, and also less well identified UDP-glucosyltransferases, with nucleotide binding sites located on the lumenal surface. There is evidence that the substrates for these enzymes, UDP-glucuronic acid (UDP-GlcUA) and UDP-glucose (UDP-Glc), biosynthesized in the cytosol, are transported into the lumen of the ER via unknown mechanisms, the characteristics of which are poorly defined. A new approach for the study of the transport process has been devised using two active-site directed photoaffinity analogs, [beta-32P]5-azido-UDP-GlcUA and [beta-32P]5-azido-UDP-Glc. Photoincorporation of these probes into the lumenally oriented UGTs of intact rat liver microsomal vesicles was used as an indicator of transport. In intact vesicles, [32P]5N3UDP-GlcUA was efficiently incorporated into UGTs in a time, temperature and concentration dependent manner. In contrast, [32P]5N3UDP-Glc apparently was not transported effectively; maximal photolabeling of the 50-54 kDa proteins by this probe was dependent on detergent disruption of the vesicles. Vesicular uptake of and subsequent photolabeling of the 50-54 kDa proteins by [32P]5N3UDP-GlcUA were inhibited by UDP-GlcUA and 5N3UDP-GlcUA while UDP-Glc, 5N3UDP-Glc, UDP-xylose and UDP-N-acetylglucosamine were less inhibitory, suggesting a high degree of specificity for the uptake/photolabeling process. The anionic transport inhibitors DIDS and SITS inhibited [32P]5N3UDP-GlcUA photoincorporation into UGTs in intact vesicles, but also inhibited photolabeling of these and other enzymes in detergent disrupted vesicles. These data suggest the presence in rat liver microsomal vesicles of a specific, carrier-mediated transport process for UDP-GlcUA which is distinct from the mechanism of UDP-Glc transport.

Hepatocellular expression of glucose-6-phosphatase is unaltered during hepatic regeneration

Gluconeogenesis and glycogenolysis are essential hepatic functions required for glucose homeostasis. During the initial phase of hepatic regeneration, the immediate-early genes (IEG) are rapidly expressed, and the IEG RL-1 encodes for glucose-6-phosphatase (G-6- Pase). G-6- Pase is a microsomal enzyme essential for gluconeogenesis and glycogenolysis. This study employs a partial-hepatectomy model to examine the expression and activity of G-6- Pase. After partial hepatectomy, rat hepatic G-6- Pase gene expression is transcriptionally regulated, and mRNA levels are increased ≈30-fold. However, in contrast to this rapid gene induction, microsomal enzyme activity is unchanged after partial hepatectomy. Western blotting demonstrates that microsomal G-6- Pase protein expression is also unchanged after partial hepatectomy, and similar results are also noted in whole liver homogenate. Thus, despite marked induction in gene expression of the IEG G-6- Pase after partial hepatectomy, protein expression and enzyme activity remain unchanged. These data indicate that, although this hepatocyte IEG is transcriptionally regulated, the physiologically important level of regulation is posttranscriptional. This highlights the importance of correlating gene expression of IEG with protein expression and physiological function.Gluconeogenesis and glycogenolysis are essential hepatic functions required for glucose homeostasis. During the initial phase of hepatic regeneration, the immediate-early genes (IEG) are rapidly expressed, and the IEG RL-1 encodes for glucose-6-phosphatase (G-6-Pase). G-6-Pase is a microsomal enzyme essential for gluconeogenesis and glycogenolysis. This study employs a partial-hepatectomy model to examine the expression and activity of G-6-Pase. After partial hepatectomy, rat hepatic G-6-Pase gene expression is transcriptionally regulated, and mRNA levels are increased approximately 30-fold. However, in contrast to this rapid gene induction, microsomal enzyme activity is unchanged after partial hepatectomy. Western blotting demonstrates that microsomal G-6-Pase protein expression is also unchanged after partial hepatectomy, and similar results are also noted in whole liver homogenate. Thus, despite marked induction in gene expression of the IEG G-6-Pase after partial hepatectomy, protein expression and enzyme activity remain unchanged. These data indicate that, although this hepatocyte IEG is transcriptionally regulated, the physiologically important level of regulation is posttranscriptional. This highlights the importance of correlating gene expression of IEG with protein expression and physiological function.

Copper Metabolism and Wilson’s Disease

Wilson’s disease, or hepatolenticular degeneration, is an inherited disorder of copper metabolism, characterized by decreased biliary copper excretion. Copper accumulation and deposition in a variety of organ systems results in progressive tissue injury and leads inexorably to death unless appropriate therapy is initiated. Kinnear Wilson first defined the familial syndrome of progressive lenticular degeneration associated with cirrhosis of the liver in 1912. Many of his original clinical observations remain valid, although considerable advances have since been made with respect to an understanding of the pathogenetic basis, the natural history, and the treatment of the disorder. This article will attempt to review the current knowledge of Wilson’s disease, highlighting some of the recent advances and controversies.

Cholestatic diseases and mechanisms of cholestasis

Primary biliary cirrhosis and primary sclerosing cholangitis are distinct hepatobiliary disorders that are both characterized by bile duct injury, the development of cirrhosis, and, in many cases, liver failure, often necessitating transplantation. Research continues to focus on the pathogenesis of these disorders and on the efficacy and mechanism of action of ursodeoxycholic acid, which currently is the therapy of choice. The association of primary biliary cirrhosis with HLA-DR8 was confirmed, the involvement of both activated B-as well as T-cell populations has been documented, and the T-cell response to the pyruvate dehydrogenase complex has been shown to be blocked by antibodies to HLA class II antigens. B cells were found in significant numbers within the portal tracts of affected livers, and were shown to be maximally stimulated to produce specific antimitochondrial antibodies. It was proposed that antigen recognition may be triggered through a process involving immunoglobulin A secretion into bile, whereas another report confirmed significant cross-reactivity with bacterial antigens. Research into the mechanism of action of ursodeoxycholic acid demonstrated a direct cytoprotective effect against toxic bile acids, as well as effects on interleukin-2 production and on bile acid pools. In primary sclerosing cholangitis, the role of neutrophil cytoplasmic antibodies was further evaluated, and the benefits and timing of liver transplantation were analyzed. Other cholestatic disorders investigated include drug-induced jaundice, cholestasis of pregnancy, cystic fibrosis, and Carolis disease, as well as the role of ursodeoxycholic acid and other therapeutic modalities.

Apoptosis induced by Fas signaling does not alter hepatic hepcidin expression

AIM To determine the regulation of human hepcidin (HAMP) and mouse hepcidin (hepcidin-1 and hepcidin-2) gene expression in the liver by apoptosis using in vivo and in vitro experimental models. METHODS For the induction of the extrinsic apoptotic pathway, HepG2 cells were treated with various concentrations of CH11, an activating antibody for human Fas receptor, for 12 h. Male C57BL/6NCR and C57BL/6J strains of mice were injected intraperitoneally with sublethal doses of an activating antibody for mouse Fas receptor, Jo2. The mice were anesthetized and sacrificed 1 or 6 h after the injection. The level of apoptosis was quantified by caspase-3 activity assay. Liver injury was assessed by measuring the levels of ALT/AST enzymes in the serum. The acute phase reaction in the liver was examined by determining the expression levels of IL-6 and SAA3 genes by SYBR green quantitative real-time PCR (qPCR). The phosphorylation of transcription factors, Stat3, Smad4 and NF-κB was determined by western blotting. Hepcidin gene expression was determined by Taqman qPCR. The binding of transcription factors to hepcidin-1 promoter was studied using chromatin immunoprecipitation (ChIP) assays. RESULTS The treatment of HepG2 cells with CH11 induced apoptosis, as shown by the significant activation of caspase-3 (P < 0.001), but did not cause any significant changes in HAMP expression. Short-term (1 h) Jo2 treatment (0.2 μg/g b.w.) neither induced apoptosis and acute phase reaction nor altered mRNA expression of mouse hepcidin-1 in the livers of C57BL/6NCR mice. In contrast, 6 h after Jo2 injection, the livers of C57BL/6NCR mice exhibited a significant level of apoptosis (P < 0.001) and an increase in SAA3 (P < 0.023) and IL-6 (P < 0.005) expression in the liver. However, mRNA expression of hepcidin-1 in the liver was not significantly altered. Despite the Jo2-induced phosphorylation of Stat3, no occupancy of hepcidin-1 promoter by Stat3 was observed, as shown by ChIP assays. Compared to C57BL/6NCR mice, Jo2 treatment (0.2 μg/g b.w.) of C57BL/6J strain mice for 6 h induced a more prominent activation of apoptosis, liver injury and acute phase reaction. Similar to C57BL/6NCR mice, the level of liver hepcidin-1 mRNA expression in the livers of C57BL/6J mice injected with a sublethal dose of Jo2 (0.2 μg/g b.w.) remained unchanged. The injection of C57BL/6J mice with a higher dose of Jo2 (0.32 μg/g b.w.) did not also alter hepatic hepcidin expression. CONCLUSION Our findings suggest that human or mouse hepcidin gene expression is not regulated by apoptosis induced via Fas receptor activation in the liver.