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Aim 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. Methods Male KK-Ay mice were fed a high-fat diet (HFD) for 8 weeks, with supplementation of L-carnitine (1.25 mg/mL) in drinking water for the latter 4 weeks. Results Serum total carnitine levels were decreased following HFD feeding, whereas the levels were reversed almost completely by L-carnitine supplementation. In mice given L-carnitine, exacerbation of hepatic steatosis and hepatocyte apoptosis was markedly prevented even though HFD feeding was continued. Body weight gain, as well as hyperlipidemia, hyperglycemia, and hyperinsulinemia, following HFD feeding were also significantly prevented in mice given L-carnitine. High-fat diet feeding elevated hepatic expression levels of carnitine palmitoyltransferase 1A mRNA; however, production of β-hydroxybutyrate in the liver was not affected by HFD alone. In contrast, L-carnitine treatment significantly increased hepatic β-hydroxybutyrate contents in HFD-fed mice. L-carnitine also blunted HFD induction in sterol regulatory element binding protein-1c mRNA in the liver. Furthermore, L-carnitine inhibited HFD-induced serine phosphorylation of insulin receptor substrate-1 in the liver. L-carnitine decreased hepatic free fatty acid content in 1 week, with morphological improvement of swollen mitochondria in hepatocytes, and increases in hepatic adenosine 5'-triphosphate content. Conclusions L-carnitine ameliorates steatohepatitis in KK-Ay mice fed an HFD, most likely through facilitating mitochondrial β-oxidation, normalizing insulin signals, and inhibiting de novo lipogenesis in the liver. It is therefore postulated that supplementation of L-carnitine is a promising approach for prevention and treatment of metabolic syndrome-related non-alcoholic steatohepatitis. [ABSTRACT FROM AUTHOR]

Aim: To evaluate the role of natural killer (NK)T cells in the pathogenesis of non-alcoholic steatohepatitis (NASH), here we investigated the expression and function of hepatic NKT cells in KK-Ay mice, an animal model of metabolic syndrome. Methods: Male, 8-week-old KK-Ay and C57Bl/6 mice were fed a high-fat (HF) diet for 4 weeks. Some mice were given daily intragastric injections of pioglitazone for 5 days prior to or after dietary treatment. Results: In untreated KK-Ay mice, the percentages of NKT cells in liver mononucleolar cells were nearly one-third of those in C57Bl/6 controls. Elevations in interleukin (IL)-4 and interferon (IFN)-γ mRNA in the liver after a single injection of α-galactosylceramide (GalCer) were blunted in KK-Ay mice largely. Percentages of NKT cells, as well as GalCer-induced increases in IL-4 mRNA, were blunted significantly in both strains after HF diet feeding for 4 weeks. Interestingly, KK-Ay mice pretreated with pioglitazone showed significant increases in NKT cell proportion, and GalCer-induced increases in IL-4 and IFN-γ mRNA were also enhanced by pioglitazone. In KK-Ay mice, the percentages of annexin V positive NKT cells were nearly 2.5-fold higher than those in C57Bl/6 controls; however, pioglitazone decreased annexin V positive cells significantly. Moreover, pioglitazone increased NKT cell fraction in KK-Ay mice even after HF diet feeding. Conclusion: KK-Ay mice exhibit proportional and functional alterations in hepatic NKT cells in close relation with the development of steatohepatitis, and it is postulated that pioglitazone improves steatohepatitis in part through restoration of hepatic NKT cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Despite pathophysiological similarities to alcoholic liver disease, susceptibility to acetaminophen hepatotoxicity in metabolic syndrome-related nonalcoholic steatohepatitis (NASH) has not been well elucidated. In this study, therefore, we investigated acetaminophen-induced liver injury in KK-Ay mice, an animal model of metabolic syndrome. Twelve-week-old male KK-Ay and C57B1/6 mice were injected intraperitoneally with 300 or 600 mg/kg acetaminophen, and euthanized 6 h later. Liver histology was assessed, and hepatic expression of 4-hydroxy-2-nonenal was detected by immunohistochemistry. Levels of reduced glutathione were determined spectrophotometrically. Phosphorylation of c-Jun NH2-terminal kinase (JNK) was analyzed by Western blotting. Hepatocytes were isolated from both strains by collagenase perfusion, and cell death and oxidative stress were measured fluorometrically by use of propidium iodide and 5-(and-6)-chloromethyl-2'7'-dichlorodihydrofluorescein diacetate acetyl ester, respectively. Acetaminophen induced more severe necrosis and apoptosis of hepatocytes in KK-Ay mice than in C57B1/6 mice and significantly increased serum alanine aminotransferase levels in KK-Ay mice. Acetaminophen-induction of 4-hydroxy-2-nonenal in the liver was potentiated, whereas the levels of reduced glutathione in liver were lower in KK-Ay mice. Acetaminophen-induced phosphorylation of JNK in the liver was also enhanced in KK-Ay mice. Exposure to 20 µM tert-butyl hydroperoxide did not kill hepatocytes isolated from C57B1/6 mice but induced cell death and higher oxidative stress in hepatocytes from KK-Ay mice. These results demonstrated that acetaminophen toxicity is increased in diabetic KK-Ay mice mainly due to enhanced oxidative stress in hepatocytes, suggesting that metabolic syndrome-related steatohepatitis is an exacerbating factor for acetaminophen-induced liver injury. [ABSTRACT FROM AUTHOR]

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 9weeks, or a single intraperitoneal injection of CCl4 (1μl/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 CCl4-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. [ABSTRACT FROM AUTHOR]