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The increased production of reactive oxygen species (ROS) has been postulated to play a key role in the progression of nonalcoholic fatty liver disease (NAFLD). However, the source of ROS and mechanisms underlying the development of NAFLD have yet to be established. We observed a significant up-regulation of a minor isoform of NADPH oxidase, NOX1, in the liver of nonalcoholic steatohepatitis (NASH) patients as well as of mice fed a high-fat and high-cholesterol (HFC) diet for 8 weeks. In mice deficient in Nox1 (Nox1KO), increased levels of serum alanine aminotransferase and hepatic cleaved caspase-3 demonstrated in HFC diet-fed wild-type mice (WT) were significantly attenuated. Concomitantly, increased protein nitrotyrosine adducts, a marker of peroxynitrite-induced injury detected in hepatic sinusoids of WT, were significantly suppressed in Nox1KO. The expression of NOX1 mRNA was much higher in the fractions of enriched liver sinusoidal endothelial cells (LSECs) than in those of hepatocytes. In primary cultured LSECs, palmitic acid (PA) up-regulated the mRNA level of NOX1, but not of NOX2 or NOX4. The production of nitric oxide by LSECs was significantly attenuated by PA-treatment in WT but not in Nox1KO. When the in vitro relaxation of TWNT1, a cell line that originated from hepatic stellate cells, was assessed by the gel contraction assay, the relaxation of stellate cells induced by LSECs was attenuated by PA treatment. In contrast, the relaxation effect of LSECs was preserved in cells isolated from Nox1KO. Taken together, the up-regulation of NOX1 in LSECs may elicit peroxynitrite-mediated cellular injury and impaired hepatic microcirculation through the reduced bioavailability of nitric oxide. ROS derived from NOX1 may therefore constitute a critical component in the progression of NAFLD.

Background & aimsReactive oxidative species (ROS) are believed to be involved in the progression of nonalcoholic steatohepatitis (NASH). However, little is known about the sources of ROS in hepatocytes or their role in disease progression. We studied the effects of nicotinamide adenine dinucleotide phosphate reduced oxidase 4 (NOX4) in liver tissues from patients with NASH and mice with steatohepatitis.MethodsLiver biopsy samples were obtained from 5 patients with NASH, as well as 4 patients with simple steatosis and 5 patients without steatosis (controls) from the University of California, Davis Cancer Center Biorepository. Mice with hepatocyte-specific deletion of NOX4 (NOX4(hepKO)) and NOX4(floxp+/+) C57BL/6 mice (controls) were given fast-food diets (supplemented with high-fructose corn syrup) or choline-deficient l-amino acid defined diets to induce steatohepatitis, or control diets, for 20 weeks. A separate group of mice were given the NOX4 inhibitor (GKT137831). Liver tissues were collected and immunoblot analyses were performed determine levels of NOX4, markers of inflammation and fibrosis, double-stranded RNA-activated protein kinase, and phospho-eIF-2α kinase-mediated stress signaling pathways. We performed hyperinsulinemic-euglycemic clamp studies and immunoprecipitation analyses to determine the oxidation and phosphatase activity of PP1C.ResultsLevels of NOX4 were increased in patients with NASH compared with controls. Hepatocyte-specific deletion of NOX4 reduced oxidative stress, lipid peroxidation, and liver fibrosis in mice with diet-induced steatohepatitis. A small molecule inhibitor of NOX4 reduced liver inflammation and fibrosis and increased insulin sensitivity in mice with diet-induced steatohepatitis. In primary hepatocytes, NOX4 reduced the activity of the phosphatase PP1C, prolonging activation of double-stranded RNA-activated protein kinase and phosphorylation of extracellular signal-regulated kinase-mediated stress signaling. Mice with hepatocyte-specific deletion of NOX4 and mice given GKT137831 had increased insulin sensitivity.ConclusionsNOX4 regulates oxidative stress in the liver and its levels are increased in patients with NASH and mice with diet-induced steatohepatitis. Inhibitors of NOX4 reduce liver inflammation and fibrosis and increase insulin sensitivity, and might be developed for treatment of NASH.

Background: Subacute myelo‐optico‐neuropathy (SMON) is a neurological disorder associated with the administration of clioquinol, particularly at very high doses. Although clioquinol has been used worldwide, there was an outbreak of SMON in the 1950s–1970s in which the majority of cases were in Japan, prompting speculation that the unique genetic background of the Japanese population may have contributed to the development of SMON. Recently, a possible association between loss‐of‐function polymorphisms in NQO1 and the development of SMON has been reported. In this study, we analyzed the relationship between NQO1 polymorphisms and SMON in Japan. Methods: We analyzed 125 Japanese patients with SMON. NQO1 loss‐of‐function polymorphisms (rs1800566, rs10517, rs689452, and rs689456) were evaluated. The allele frequency distribution of each polymorphism was compared between the patients and the healthy Japanese individuals (Human Genomic Variation Database and Integrative Japanese Genome Variation Database), as well as our in‐house healthy controls. Results: The frequencies of the loss‐of‐function NQO1 alleles in patients with SMON and the normal control group did not differ significantly. Conclusion: We conclude that known NQO1 polymorphisms are not associated with the development of SMON. [ABSTRACT FROM AUTHOR]

Fever is elicited by exogenous pyrogens, such as lipopolysaccharide (LPS). This stimulates the production of cytokines such as interluekin (IL)-1Beta. Aspirin-like anti-inflammatory drugs can suppress fever, and they can inhibit prostaglandin biosysthensis. Mice lacking prostaglandin E(sub2) receptor subtypes were generated, and only mice lacking EP(sub3) did not show a febrile response to PGE(sub2) and to either Il-1Beta or LPS.

In situ hybridization techniques on murine gastrointestinal tract were utilized to determine the cellular distribution of prostaglandin E2 (PGE2) receptor messenger RNAs (mRNAs). In situ hybridization of PGE receptors mRNAs indicated the presence of EP1, EP2 and EP3 PGE receptor subtypes in the distinct regions of the murine gastrointestinal tract. The presence of PGE receptors indicated the role of PGE2 in the modulation of gastric and intestinal activity via PGE receptor subtypes.

Background: Subacute myelo‐optico‐neuropathy (SMON) is a severe neurological disorder associated with clioquinol administration, which frequently occurred in Japan during the 1950s and 1960s. The unique genetic background of the Japanese population is considered to be strongly involved in the development of this neurological disease. Recently, genetic variants of ABCC4 (OMIM: 605250) and ABCC11 (OMIM: 607040), which are particularly common in the Japanese population, were suggested as possible genetic susceptibility factors for the development of SMON. Methods: We analyzed 125 Japanese SMON patients who provided consent for this study. Patient DNA was collected from peripheral blood, and genetic analysis was performed for ABCC4 rs3765534 (c.2268G>A, p.Glu857Lys) and ABCC11 rs17822931 (c.538G>A, p.Gly180Arg) polymorphisms using the Sanger sequencing method and/or TaqMan PCR method. The frequency distribution of each polymorphism was compared with that in healthy Japanese people recorded in two genomic databases (Human Genomic Variation Database and Integrative Japanese Genome Variation Database), and each genotype was compared with the clinical features of patients. Results: The frequencies of ABCC4 rs3765334 and ABCC11 rs17822931 polymorphisms in SMON patients and healthy Japanese people were not significantly different in the multifaceted analysis. Conclusion: We conclude that the ABCC4 rs3765334 and ABCC11 rs17822931 polymorphisms are not associated with the development of SMON. [ABSTRACT FROM AUTHOR]