Kazuyoshi Kon

Translocation of iron from lysosomes into mitochondria is a key event during oxidative stress-induced hepatocellular injury†

Iron overload exacerbates various liver diseases. In hepatocytes, a portion of non‐heme iron is sequestered in lysosomes and endosomes. The precise mechanisms by which lysosomal iron participates in hepatocellular injury remain uncertain. Here, our aim was to determine the role of intracellular movement of chelatable iron in oxidative stress‐induced killing to cultured hepatocytes from C3Heb mice and Sprague‐Dawley rats. Mitochondrial polarization and chelatable iron were visualized by confocal microscopy of tetramethylrhodamine methylester (TMRM) and quenching of calcein, respectively. Cell viability and hydroperoxide formation (a measure of lipid peroxidation) were measured fluorometrically using propidium iodide and chloromethyl dihydrodichlorofluorescein, respectively. After collapse of lysosomal/endosomal acidic pH gradients with bafilomycin (50 nM), an inhibitor of the vacuolar proton‐pumping adenosine triphosphatase, cytosolic calcein fluorescence became quenched. Deferoxamine mesylate and starch‐deferoxamine (1 mM) prevented bafilomycin‐induced calcein quenching, indicating that bafilomycin induced release of chelatable iron from lysosomes/endosomes. Bafilomycin also quenched calcein fluorescence in mitochondria, which was blocked by 20 μM Ru360, an inhibitor of the mitochondrial calcium uniporter, consistent with mitochondrial iron uptake by the uniporter. Bafilomycin alone was not sufficient to induce mitochondrial depolarization and cell killing, but in the presence of low‐dose tert‐butylhydroperoxide (25 μM), bafilomycin enhanced hydroperoxide generation, leading to mitochondrial depolarization and subsequent cell death. Conclusion: Taken together, the results are consistent with the conclusion that bafilomycin induces release of chelatable iron from lysosomes/endosomes, which is taken up by mitochondria. Oxidative stress and chelatable iron thus act as two “hits” synergistically promoting toxic radical formation, mitochondrial dysfunction, and cell death. This pathway of intracellular iron translocation is a potential therapeutic target against oxidative stress–mediated hepatotoxicity. (HEPATOLOGY 2008.)

Role of adipocytokines in hepatic fibrogenesis

Obesity and insulin resistance are the key factors for progression of hepatic fibrosis in various chronic liver diseases including non‐alcoholic steatohepatitis (NASH). Recently it has been shown that leptin plays a pivotal role in development of hepatic fibrosis. Leptin promotes hepatic fibrogenesis through upregulation of transforming growth factor‐β in Kupffer cells and sinusoidal endothelial cells. Further, leptin facilitates proliferation and prevents apoptosis of hepatic stellate cells. There is a paradox, however, in that ob/ob mice and Zucker rats, which are the obese and diabetic strains, had minimal profibrogenic responses in the liver, most likely because they lack leptin and its receptors. To establish a more clinically relevant model to study the mechanism of fibrogenesis under steatohepatitis, fatty changes and profibrogenic responses in the liver caused by methionine–choline deficiency (MCD) were investigated in the KK‐Ay mouse, which is an obese and diabetic strain. KK‐Ay mice developed more severe hepatic steatosis, inflammation and fibrosis induced by an MCD diet as compared to C57Bl/6 controls. Importantly, KK‐Ay mice lack physiological upregulation of adiponectin levels, suggesting that adiponectin plays a pivotal role not only in regulation of insulin sensitivity but also in modulation of inflammatory and profibrogenic responses in dietary steatohepatitis. Collectively, these findings support the hypothesis that the balance of adipocytokine expression is a key regulator for the progression of hepatic fibrosis in the setting of steatohepatitis.

Lysosomal Iron Mobilization and Induction of the Mitochondrial Permeability Transition in Acetaminophen-Induced Toxicity to Mouse Hepatocytes

Acetaminophen induces the mitochondrial permeability transition (MPT) in hepatocytes. Reactive oxygen species (ROS) trigger the MPT and play an important role in AAP-induced hepatocellular injury. Because iron is a catalyst for ROS formation, our aim was to investigate the role of chelatable iron in MPT-dependent acetaminophen toxicity to mouse hepatocytes. Hepatocytes were isolated from fasted male C3Heb/FeJ mice. Necrotic cell killing was determined by propidium iodide fluorometry. Mitochondrial membrane potential was visualized by confocal microscopy of tetramethylrhodamine methylester. Chelatable ferrous ion was monitored by calcein quenching, and 70 kDa rhodamine-dextran was used to visualize lysosomes. Cell killing after acetaminophen (10mM) was delayed and decreased by more than half after 6 h by 1mM desferal or 1mM starch-desferal. In a cell-free system, ferrous but not ferric iron quenched calcein fluorescence, an effect reversed by dipyridyl, a membrane-permeable iron chelator. In hepatocytes loaded with calcein, intracellular calcein fluorescence decreased progressively beginning about 4 h after acetaminophen. Mitochondria then depolarized after about 6 h. Dipyridyl (20mM) dequenched calcein fluorescence. Desferal and starch-desferal conjugate prevented acetaminophen-induced calcein quenching and mitochondrial depolarization. As calcein fluorescence became quenched, lysosomes disappeared, consistent with release of iron from ruptured lysosomes. In conclusion, an increase of cytosolic chelatable ferrous iron occurs during acetaminophen hepatotoxicity, which triggers the MPT and cell killing. Disrupted lysosomes are the likely source of iron, and chelation of this iron decreases acetaminophen toxicity to hepatocytes.

Abnormality of autophagic function and cathepsin expression in the liver from patients with non‐alcoholic fatty liver disease

Recent evidences indicate that hepatic steatosis suppresses autophagic proteolysis. The present study evaluated the correlation between autophagic function and cathepsin expression in the liver from patients with non‐alcoholic fatty liver disease (NAFLD).

Role of apoptosis in acetaminophen hepatotoxicity

Acetaminophen overdose causes liver injury by mechanisms involving glutathione depletion, oxidative stress and mitochondrial dysfunction. The role of apoptosis in acetaminophen‐induced cell killing is still controversial. Here, our aim was to evaluate the mitochondrial permeability transition (MPT) as a key factor in acetaminophen‐induced necrotic and apoptotic killing of primary cultured mouse hepatocytes. Acetaminophen (10 μmol/L) induced necrotic killing in approximately 50% of hepatocytes after 6 h and cyclosporin A (CsA), MPT inhibitor, temporarily decreased necrotic killing after 6 h, but cytoprotection was lost after 16 h. Confocal microscopy revealed mitochondrial depolarization and inner membrane permeabilization at approximately 4.5 h after acetaminophen. CsA delayed these changes indicative of the MPT to about 11 h after acetaminophen. TUNEL labeling and caspase 3 activation also increased after acetaminophen. Fructose (20 mmol/L, an ATP‐generating glycolytic substrate) plus glycine (5 mmol/L, a membrane stabilizing amino acid) prevented nearly all necrotic cell killing but paradoxically increased apoptosis. In conclusion, acetaminophen induces the MPT and ATP‐depletion‐dependent necrosis or caspase‐dependent apoptosis as determined, in part, by ATP availability from glycolysis.

CD1d-restricted natural killer T cells contribute to hepatic inflammation and fibrogenesis in mice

BACKGROUND & AIMS Several lines of evidence suggest that innate immunity plays a key role in hepatic fibrogenesis. To clarify the role of natural killer (NK) T cells in hepatic inflammation and fibrogenesis, we here investigated xenobiotics-induced liver injury and subsequent fibrogenesis in mice lacking mature NKT cells caused by genetic disruption of the CD1d molecule. METHODS Male CD1d-knockout (KO) and wild-type (WT) mice were given repeated intraperitoneal injections of thioacetamide (TAA, 3times/week; 0.1-0.2mg/g BW) for up to 9 weeks, or a single intraperitoneal injection of CCl(4) (1 μl/g). Liver histology was evaluated, and expression levels of cytokines and matrix-related genes in the liver were quantitatively measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS Mortality following repeated injections of TAA was prevented almost completely in CD1d-KO mice. TAA-induced inflammatory responses and hepatocellular damage were markedly ameliorated in CD1d-KO mice. TAA-induced expression of smooth muscle α-actin (SMA) and transforming growth factor (TGF)β1 mRNA in the liver were also prevented largely in CD1d-KO mice. In fact, CD1d-KO mice developed minimal hepatic fibrosis after 9-weeks of administration of TAA, which caused overt bridging fibrosis in WT mice. Indeed, TAA-induced increases in α1(I)procollagen (COL1A1) and tissue inhibitor of matrix metalloproteinase (TIMP)-1 mRNA were blunted significantly in CD1d-KO mice. Similarly, acute CCl(4)-induced hepatic injury and subsequent profibrogenic responses were also reduced significantly in CD1d-KO mice. CONCLUSIONS These findings clearly indicated that CD1d-restricted NKT cells contribute to xenobiotics-induced hepatic inflammation, hepatocellular damage, and subsequent profibrogenic responses in the liver.

Pioglitazone promotes survival and prevents hepatic regeneration failure after partial hepatectomy in obese and diabetic KK‐Ay mice

Pathogenesis of metabolic syndrome–related nonalcoholic steatohepatitis (NASH) involves abnormal tissue‐repairing responses in the liver. We investigated the effect of pioglitazone, a thiazolidinedione derivative (TZD), on hepatic regenerative responses in obese, diabetic KK‐Ay mice. Male KK‐Ay mice 9 weeks after birth underwent two‐thirds partial hepatectomy (PH) after repeated intragastric injections of pioglitazone (25 mg/kg) for 5 days. Almost half of the KK‐Ay mice died within 48 hours of PH;however, mortality was completely prevented in mice pretreated with pioglitazone. In KK‐Ay mice, bromodeoxyuridine (BrdU) incorporation to hepatocyte nuclei 48 hours after PH reached only 1%; however, pioglitazone pretreatment significantly increased BrdU‐positive cells to 8%. Cyclin D1 was barely detectable in KK‐Ay mice within 48 hours after PH. In contrast, overt expression of cyclin D1 was observed 24 hours after PH in KK‐Ay mice pretreated with pioglitazone. Hepatic tumor necrosis factor alpha (TNF‐α) messenger RNA (mRNA) was tremendously increased 1 hour after PH in KK‐Ay mice, the levels reaching ninefold over C57Bl/6 given PH, whereas pioglitazone blunted this increase by almost three‐fourths. Pioglitazone normalized hypoadiponectinemia in KK‐Ay mice almost completely. Serum interleukin (IL)‐6 and leptin levels were elevated extensively 24 hours after PH in KK‐Ay mice, whereas the levels were largely decreased in KK‐Ay mice given pioglitazone. Indeed, pioglitazone prevented aberrant increases in signal transducers and activators of transcription (STAT)3 phosphorylation and suppressor of cytokine signaling (SOCS)‐3 mRNA in the liver in KK‐Ay mice. Conclusion: These findings indicated that pioglitazone improved hepatic regeneration failure in KK‐Ay mice. The mechanism underlying the effect of pioglitazone on regeneration failure most likely involves normalization of expression pattern of adipokines and subsequent cytokine responses during the early stage of PH. (HEPATOLOGY 2009.)

Ursolic acid ameliorates hepatic fibrosis in the rat by specific induction of apoptosis in hepatic stellate cells.

BACKGROUND & AIMS Specific induction of cell death in activated hepatic stellate cells (HSCs) is a promising therapeutic strategy for hepatic fibrosis. In this study, we evaluated the cell-killing effect of ursolic acid (UA), a pentacyclic triterpenoid, in activated HSCs both in vitro and in vivo. METHODS Culture-activated rat HSCs were treated with UA (0-40μM), and the mechanisms of cell death were evaluated. The cell killing effect of UA on activated HSCs in rats chronically treated with thioacetamide (TAA) was detected by dual staining of TdT-mediated dUTP nick-end labeling (TUNEL) and smooth muscle α-actin (αSMA) immunohistochemistry, and resolution of hepatic fibrosis was evaluated. Further, the protective effects of UA on progression of hepatic fibrosis caused by TAA and bile duct ligation (BDL) were evaluated. RESULTS UA induced apoptotic cell death in culture-activated HSCs, but not in isolated hepatocytes and quiescent HSCs. Mitochodrial permeability transition (MPT) preceded the cleavage of caspase-3 and -9 following UA treatment. UA also decreased phosphorylation levels of Akt, and diminished nuclear localization of NFκB in these cells. In rats pretreated with TAA for 6weeks, a single injection of UA induced remarkable increases in TUNEL- and αSMA-dual-positive cells in 24h, and significant regression of hepatic fibrosis within 48h. Moreover, UA ameliorated hepatic fibrogenesis caused by both chronic TAA administration and BDL. CONCLUSIONS UA ameliorated experimental hepatic fibrosis most likely through specific induction of apoptosis in activated HSCs. It is therefore postulated that UA is a potential therapeutic reagent for resolution of hepatic fibrosis.

Innate immune responses involving natural killer and natural killer T cells promote liver regeneration after partial hepatectomy in mice

To clarify the roles of innate immune cells in liver regeneration, here, we investigated the alteration in regenerative responses after partial hepatectomy (PH) under selective depletion of natural killer (NK) and/or NKT cells. Male, wild-type (WT; C57Bl/6), and CD1d-knockout (KO) mice were injected with anti-NK1.1 or anti-asialo ganglio-N-tetraosylceramide (GM1) antibody and then underwent the 70% PH. Regenerative responses after PH were evaluated, and hepatic expression levels of cytokines and growth factors were measured by real-time RT-PCR and ELISA. Phosphorylation of STAT3 was detected by Western blotting. Depletion of both NK and NKT cells with an anti-NK1.1 antibody in WT mice caused drastic decreases in bromodeoxyuridine uptake, expression of proliferating cell nuclear antigen, and cyclin D1, 48 h after PH. In mice given NK1.1 antibody, increases in hepatic TNF-α, IL-6/phospho-STAT3, and hepatocyte growth factor (HGF) levels following PH were also blunted significantly, whereas IFN-γ mRNA levels were not different. CD1d-KO mice per se showed normal liver regeneration; however, pretreatment with an antiasialo GM1 antibody to CD1d-KO mice, resulting in depletion of both NK and NKT cells, also blunted regenerative responses. Collectively, these observations clearly indicated that depletion of both NK and NKT cells by two different ways results in impaired liver regeneration. NK and NKT cells most likely upregulate TNF-α, IL-6/STAT3, and HGF in a coordinate fashion, thus promoting normal regenerative responses in the liver.

Exposure to a high total dosage of glucocorticoids produces non-alcoholic steatohepatits.

To the Editor: Non-alcoholic steatohepatitis (NASH) is a disease involving histological changes similar to those in alcoholic liver disease in patients without alcohol abuse. NASH occasionally progresses to liver cirrhosis, and is commonly associated with obesity, diabetes and hyperlipidemia. As for druginduced NASH, Farrell, and Stavitz and Sanyal reported that glucocorticoids induce NASH, based on a case report published in 1977 describing NASH in a patient with systemic lupus erythematosus (SLE) who had received long-term glucocorticoid therapy. After that case report, two further case reports describing the occurrence of NASH in SLE patients were published. In those cases the duration of corticosteroid therapy differed, that is, short-term in one and long-term in another. Therefore, it became important to clarify the relationship between the duration of glucocorticoid therapy (or total dosage of administered glucocorticoids) and the occurrence of NASH in SLE. Our previous study of hepatic lesions in 52 SLE cases indicated that exposure to a high total dosage of glucocorticoids is related to the occurrence of severe fatty liver, but we did not address NASH in that study. Therefore, the present study examined relationships between the administration of glucocorticoids and the occurrence of NASH in two groups of 48 SLE cases (31 autopsy cases and 17 liver biopsy cases), consisting of the low total dosage group (14 cases; total dosage of prednisolone <3 g, duration of prednisolone administration 2 months-1 year) and the high total dosage group (34 cases; total dosage of prednisolone >10 g, duration of prednisolone administration 2–20 years). The diagnosis of SLE was based on the criteria of the American College of Rheumatology for SLE. NASH was present in two patients in the high total dosage group, producing a 6% incidence of NASH in that group (Table 1). The two patients with NASH had neither obesity, diabetes nor hyperlipidemia. In one of those two patients with NASH, the liver had severe steatosis, ballooning of many hepatocytes and many Mallory bodies, associated with focal perisinusoidal fibrosis and focal portal fibrosis histologically (Fig. 1). The histological diagnosis of NASH was grade 3 and stage 2. In the other patient with NASH, the liver had severe steatosis, ballooning of many hepatocytes and focal perisinusoidal fibrosis histologically. The histological diagnosis of NASH in that patient was grade 2 and stage 1. In addition, severe steatosis was present only in the high total dosage group (Table 1). The present study concluded that exposure to a high total dosage of glucocorticoids induces NASH because in the low total dosage group there was neither NASH nor severe steatosis. Also, severe steatosis occurred in the high total dosage group, and two patients with NASH belonged to the high total dosage group and they had severe steatosis. Moreover, they had not major factors for the occurrence of NASH, such as obesity, diabetes mellitus or hyperlipidemia. In addition, severe steatosis frequently occurs in NASH. The mechanism of glucocorticoid-induced steatosis and steatohepatitis is considered as follows. The glucocorticoids inhibit mitochondrial b oxidation, and decreases hepatic triglyceride secretion from the liver, which result in steatosis. Induction of lipid peroxidation in the liver by the repeated administration of glucocorticoids also produces steatosis. The mechanism by which steatosis evolves into steatohepatitis in some patients receiving glucocorticoids remained unexplored, but probably involves an exacerbating pathogenetic mechanism underlying NASH. We therefore think that exposure to a high total dosage of glucocorticoid may produce exacerbation of the pathogenetic mechanism underlying NASH. However, more studies are needed to confirm the mechanism of glucocorticoidinduced NASH.