Kumiko Arai

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.

Sodium 4-phenylbutyrate prevents murine dietary steatohepatitis caused by trans-fatty acid plus fructose.

Excess consumption of trans-fatty acid could increase the risk of non-alcoholic steatohepatitis (NASH); however, treatment targeting trans-fatty acid-induced NASH has not been examined. Here we focused on the influence of trans-fatty acid intake on endoplasmic reticulum (ER) stress in hepatocytes, so we investigated the effect of the chemical chaperone 4-phenylbutyric acid (PBA), on trans-fatty acid-caused steatohepatitis using diabetic KK-Ay mice. Elaidic acid (EA, trans-fatty acid) alone did not cause definitive liver injury. In contrast, EA plus low-dose fructose induced extensive apoptosis in hepatocytes with severe fat accumulation. EA plus fructose significantly increased ER stress markers such as glucose-regulated protein 78 (GRP78), eukaryotic initiation factor 2α (eIF2α) and phosphorylated c-jun N-terminal kinase (JNK), while PBA significantly reduced this response. In vitro, EA promoted expression of GRP78 and phosphorylation of eIF2α in primary-cultured hepatocytes. EA also increased hepatocellular susceptibility to low-dose tert-butyl hydroperoxide. Treatment with PBA significantly reduced these responses. In conclusion, EA potentiates susceptibly to non-hazardous dose of fructose, and increases ER and oxidative stress. PBA improved steatohepatitis induced by EA plus fructose through amelioration of ER stress. Therefore, ER stress-targeted therapy using a chemical chaperone is a promising novel strategy for trans-fatty acid-induced steatohepatitis.

Glycine prevents metabolic steatohepatitis in diabetic KK-Ay mice through modulation of hepatic innate immunity

Strategies for prevention and treatment of nonalcoholic steatohepatitis remain to be established. We evaluated the effect of glycine on metabolic steatohepatitis in genetically obese, diabetic KK-Ay mice. Male KK-Ay mice were fed a diet containing 5% glycine for 4 wk, and liver pathology was evaluated. Hepatic mRNA levels for lipid-regulating molecules, cytokines/chemokines, and macrophage M1/M2 markers were determined by real-time RT-PCR. Hepatic expression of natural killer (NK) T cells was analyzed by flow cytometry. Body weight gain was significantly blunted and development of hepatic steatosis and inflammatory infiltration were remarkably prevented in mice fed the glycine-containing diet compared with controls. Indeed, hepatic induction levels of molecules related to lipogenesis were largely blunted in the glycine diet-fed mice. Elevations of hepatic mRNA levels for TNFα and chemokine (C-C motif) ligand 2 were also remarkably blunted in the glycine diet-fed mice. Furthermore, suppression of hepatic NK T cells was reversed in glycine diet-fed KK-Ay mice, and basal hepatic expression levels of NK T cell-derived cytokines, such as IL-4 and IL-13, were increased. Moreover, hepatic mRNA levels of arginase-1, a marker of macrophage M2 transformation, were significantly increased in glycine diet-fed mice. In addition, dietary glycine improved glucose tolerance and hyperinsulinemia in KK-Ay mice. These observations clearly indicate that glycine prevents maturity-onset obesity and metabolic steatohepatitis in genetically diabetic KK-Ay mice. The underlying mechanisms most likely include normalization of hepatic innate immune responses involving NK T cells and M2 transformation of Kupffer cells. It is proposed that glycine is a promising immunonutrient for prevention and treatment of metabolic syndrome-related nonalcoholic steatohepatitis.

L-carnitine prevents metabolic steatohepatitis in obese diabetic KK-A(y) mice.

Pharmacological treatment for metabolic syndrome‐related non‐alcoholic steatohepatitis has not been established. We investigated the effect of L‐carnitine, an essential substance for β‐oxidation, on metabolic steatohepatitis in mice.

Tu1818 The Chemical Chaperon 4-Phenylbutyrate Inhibits Liver Fibrosis and Tumorgenesis in High-Fat Diet With N-Acetyl-ß-D-Glucosaminedase Inhibitor-Induced NASH Model Mice

A S L D A b st ra ct s and liver cholesterol levels were measured. We compared gene expression profiles of liver specimen from MCD diet fed mice with those from MCS diet fed mice, and examined the effect of exendin-4 on gene expression profiles in the liver on MCD diet fed mice, using Agilent whole mouse genome oligo microarrays (Agilent Technologies). We also performed a cholesterol biosynthesis pathway analysis significantly affected transcripts of the liver. Results: While serum cholesterol levels were significantly decreased by MCD diet, exendin4 had a tendency to decrease serum cholesterol levels. On the other hand, hepatic cholesterol levels were significantly increased by MCD diet compared with those by MCS diet. Exendin4 significantly decreased MCD diet-induced increase of hepatic cholesterol levels compared with saline. Transcripts for 6476 mRNAs differed in expression patterns > 2 fold between MCS diet and MCD diet with saline groups. Furthermore, transcripts for 7740 mRNAs differed in expression patterns > 2 fold between MCD diet with saline groups and MCD diet with exendin-4. The pathway of gene expressions most impacted by MCD diet was cholesterol biosynthesis. MCD diet up-regulated cholesterol biosynthesis signaling. In contrast, exendin-4 down-regulated cholesterol biosynthesis signaling. Conclusions: Exendin4 inhibited MCD diet-induced increase of hepatic cholesterol levels through the inhibition of cholesterol biosynthesis. These results indicate that exendin-4 may decrease hepatic cholesterol resulting in the attenuation of hepatic inflammation in NASH model.

High Fat Diet Increases Susceptibility to Hypoxia-Induced Zone 3-Dominant Hepatocyte Apoptosis in Mice

PCR), real time PCR, chromatin immunoprecipitation (ChIP) assay, and immunoblot analysis. Results: Cellular TG content was increased when HepG2 were cultured in the presence of oleic acid, and this increase was prevented by guggulsterone treatment. Guggulsterone prevented LXRα agonist (T0901317) induced activation of SREBP-1c, inhibited its mRNA and protein induction, thereby resulting in the down-regulation of SREBP-1c target genes. This inhibitory effect of guggulsterone was blocked by the protein kinase C (PKC) inhibitor. Moreover, guggulsterone enhanced LXRα phosphorylation at threonine residue and decreased phosphorylation at serine residue, which caused impaired DNA binding activity, leading to inhibition of LXRα transactivation and expression. Conclusion: These results show that guggulsterone prevents LXRα mediated SREBP-1c dependent hepatic steatosis through PKC dependent pathway, and suggests that guggulsterone can therapeutically be used to treat nonalcoholic fatty liver disease.