Deficiency of eNOS exacerbates early-stage NAFLD pathogenesis by changing the fat distribution.

BACKGROUND: Although many factors and molecules that are closely associated with non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH) have been reported, the role of endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) in the pathogenesis of NAFLD/NASH remains unclear. We therefore investigated the role of eNOS-derived NO in NAFLD pathogenesis using systemic eNOS-knockout mice fed a high-fat diet. METHODS: eNOS-knockout and wild-type mice were fed a basal diet or a high-fat diet for 12 weeks. Lipid accumulation and inflammation were evaluated in the liver, and various factors that are closely associated with NAFLD/NASH and hepatic tissue blood flow were analyzed. RESULTS: Lipid accumulation and inflammation were more extensive in the liver and lipid accumulation was less extensive in the visceral fat tissue in eNOS-knockout mice, compared with wild-type mice, after 12 weeks of being fed a high-fat diet. While systemic insulin resistance was comparable between the eNOS-knockout and wild-type mice fed a high-fat diet, hepatic tissue blood flow was significantly suppressed in the eNOS-knockout mice, compared with the wild-type mice, in mice fed a high-fat diet. The microsomal triglyceride transfer protein activity was down-regulated in eNOS-knockout mice, compared with wild-type mice, in mice fed a high-fat diet. CONCLUSIONS: A deficiency of eNOS-derived NO may exacerbate the early-stage of NASH pathogenesis by changing the fat distribution in a mouse model via the regulation of hepatic tissue blood flow.

Deficiency of iNOS-derived NO accelerates lipid accumulation-independent liver fibrosis in non-alcoholic steatohepatitis mouse model.

BACKGROUND: Although many of the factors and molecules closely associated with non-alcoholic steatohepatitis (NASH) have been reported, the role of inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) on the progression of NASH remains unclear. We therefore investigated the role of iNOS-derived NO in NASH pathogenesis with a long-term follow-up study using systemic iNOS-knockout mice under high-fat diet (HFD) conditions. METHODS: iNOS-knockout and wild-type mice were fed a basal or HFD for 10 or 48 weeks. Lipid accumulation, fibrosis, and inflammation were evaluated, and various factors and molecules closely associated with NASH were analyzed. RESULTS: Marked fibrosis and inflammation (indicators of NASH) were observed in the livers of iNOS-knockout mice compared to wild-type mice after 48 weeks of a HFD; however, lipid accumulation in iNOS-knockout mice livers was less than in the wild-type. Increased expressions of various cytokines that are transcriptionally controlled by NF-kB in iNOS-deficient mice livers were observed during HFD conditions. CONCLUSIONS: iNOS-derived NO may play a protective role against the progression to NASH during an HFD by preventing fibrosis and inflammation, which are mediated by NF-kB activation in Kupffer cells. A lack of iNOS-derived NO accelerates progression to NASH without excessive lipid accumulation.

Exposure to High Doses of Lipopolysaccharide during Ovalbumin Sensitization Prevents the Development of Allergic Th2 Responses to a Dietary Antigen.

Food allergies are driven by aberrant T helper (Th) 2 cells. Lipopolysaccharide (LPS) influences the development of Th2-mediated diseases, but its role in food allergy and tolerance remains unclear. To address this issue, we established mouse models presenting allergic or tolerant responses to ovalbumin (OVA). Mice sensitized with crude OVA developed Th2 responses including acute diarrhea, increases in serum OVA-specific IgE, dominant production of serum OVA-specific IgG1, increases in Th2-type cytokines and proliferation of mast cells in duodenal and colonic tissues. Sensitization of mice with crude OVA and LPS abrogated Th2-type responses observed in allergic mice. The level of OVA-specific proliferation in mesenteric lymph node CD4(+) T cells was comparable in allergic and tolerant mice, indicating that the tolerance is not caused by anergy and apoptosis of antigen-primed T cells. Expression of Th1- and Th2-type cytokines was suppressed in whole spleen cells and/or purified spleen CD4(+) T cells of tolerant mice, indicating that the tolerance was not caused by the shift from Th2 to Th1. On the other hand, interleukin (IL)-10, a regulatory cytokine produced by regulatory T cells, was upregulated in whole spleen cells and purified spleen CD4(+) T cells of tolerant mice. Furthermore, spleen CD4(+) T cells from tolerant mice suppressed the growth of CD4(+) T cells from DO11.10 mice in co-culture. These results indicate that tolerance is induced in allergic mice by simultaneous exposure to LPS during sensitization with OVA and that a population of T cells producing IL-10 plays an important role in the tolerance induction.

Unfolded protein response pathways regulate Hepatitis C virus replication via modulation of autophagy.

BACKGROUND: Hepatitis C virus (HCV) induces endoplasmic reticulum (ER) stress which, in turn, activates the unfolding protein response (UPR). UPR activates three distinct signalling pathways. Additionally, UPR induces autophagy (UPR-autophagy pathways). On the other hand, it has become clear that some positive-single-strand RNA viruses utilize autophagy. Some groups have used the siRNA silencing approach to show that autophagy is required for HCV RNA replication. However, the mechanism of induction of the UPR-autophagy pathways remain unclear in the cells with HCV. METHOD AND RESULTS: we used a genome-length HCV RNA (strain O of genotype 1b) replication system (OR6) in hepatoma cells (HuH-7-derived OR6 cells). As control, we used OR6c cells from which the HCV genome had been removed by treatment with interferon-α. The UPR-autophagy pathways were activated to a greater degree in the OR6 cells as compared to the OR6c cells. Rapamycin, mTOR-independent autophagy inducer, activated HCV replication in the OR6 cells. On the other hand, HCV replication in the cells was inhibited by 3-methyladenine (3-MA), which is an inhibitor of autophagy. Salubrinal (Eukaryotic Initiation Factor 2(eIF2)-alpha phosphatase inhibitor), 3-ethoxy-5, 6-dibromosalicylaldehyde (X-box binding protein-1 (XBP-1) splicing inhibitor) and sp600125 (c-Jun N-terminal kinases (JNK) inhibitor) inhibited HCV replication and autophagy. Additionally, HCV replication and autophagy were inhibited more strongly by combination of these inhibitors. CONCLUSION: Our results suggest that UPR-autophagy pathways exert an influence on HCV replication. Therefore, control these pathways may serve as a novel therapeutic strategy against replication of HCV.

Evaluation of the Liver Fibrosis Index calculated by using real-time tissue elastography for the non-invasive assessment of liver fibrosis in chronic liver diseases.

AIM: A rapid and non-invasive method of detecting fibrosis in patients with chronic liver diseases is of major clinical interest. The purpose of this study was to comparatively investigate the effectiveness of the Liver Fibrosis Index (LF Index) calculated using real-time tissue elastography (RTE) in patients with non-alcoholic fatty liver disease (NAFLD) and patients with chronic hepatitis C (CHC). METHODS: Twenty-seven patients with biopsy-proven NAFLD and 93 patients with biopsy-proven CHC were included. They underwent transient elastography (TE), serum liver fibrosis marker testing and RTE to calculate the LF Index. RESULTS: The LF Index showed a stepwise increase with increasing histological severity of fibrosis in CHC patients (P = 0.0102), whereas no significant correlation of the LF Index with the histological severity of liver fibrosis in NAFLD patients (P = 0.852). There was a significant correlation between the LF Index and liver stiffness measured by TE in CHC patients (r = 0.319, P = 0.0009). On the other hand, no such correlation was observed in NAFLD patients. While in CHC patients, the LF Index was correlated with the FIB-4 index, no such correlation was observed in NAFLD patients. CONCLUSION: The LF Index calculated by RTE is effective for assessment of liver fibrosis in patients with CHC. On the other hand, it is not useful in patients with NAFLD. This is the first study to compare the clinical usefulness of RTE as non-invasive assessment of liver fibrosis between CHC and NAFLD. Further investigations are required to refine statistical assessment of RTE.

Hepatic triglyceride lipase plays an essential role in changing the lipid metabolism in genotype 1b hepatitis C virus replicon cells and hepatitis C patients.

AIM: Recently, several studies have shown the existence of associations between lipoprotein profiles and hepatitis C virus (HCV), although only a limited amount of information is available about the mechanisms underlying the changes in the lipoprotein profiles associated with HCV. In this study, we investigated the association between lipoprotein profile, classified according to the particle size, and lipoprotein metabolism. METHODS: We used four kinds of cells for this experiment; full-length genome HCV RNA replicon cells (OR6), sub-genomic HCV RNA replicon cells (sO), and OR6c cells and sOc cells, which were the same cell lines treated with interferon-α. The triglyceride (TG) levels in the lipoprotein subclasses of the culture medium were measured by high-performance liquid chromatography. The mRNA expression levels of several molecules associated with lipoprotein metabolism were measured in the OR6, OR6c, sO and sOc cells. To confirm some of the results obtained using the in vitro system, liver biopsy samples obtained from the patients were also examined. RESULTS: The content of TG in the large low-density lipoprotein (LDL) and medium LDL in the culture medium was increased only in the OR6 cells. The hepatic triglyceride lipase (HTGL) mRNA expression levels were lower in the OR6 cells than in the OR6c cells (P < 0.01). Examination of the HTGL expression levels in the patients' livers revealed a decrease in HTGL expression in the chronic hepatitis C liver as compared with that in the chronic hepatitis B or non-alcoholic steatohepatitis liver (P < 0.01). CONCLUSION: We showed that HCV inhibits HTGL production in hepatocytes, inducing a change of the lipoprotein profile.

Semaphorin 4D, a lymphocyte semaphorin, enhances tumor cell motility through binding its receptor, plexinB1, in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumor, for which the development of new biomarkers and therapeutic targets has become critical. The main cause of poor prognosis in PDAC patients is the high invasive and metastatic potential of the cancer. In the present study, we report a new signaling pathway that was found to mediate the enhanced tumor cell motility in pancreatic cancer. Semaphorin 4D (Sema4D) is a ligand known to be expressed on different cell types, and has been reported to be involved in the regulation of immune functions, epithelial morphogenesis, and tumor growth and metastasis. In this study, we revealed for the first time that the cancer tissue cells expressing Sema4D in PDAC are tumor-infiltrating lymphocytes. The overexpression of Sema4D and of its receptor, plexinB1, was found to be significantly correlated with clinical factors, such as lymph node metastasis, distant metastasis, and poor prognosis in patients with PDAC. Through in vitro analysis, we demonstrated that Sema4D can potentiate the invasiveness of pancreatic cancer cells and we identified the downstream molecules. The binding of Sema4D to plexinB1 induced small GTPase Ras homolog gene family, member A activation and resulted in the phosphorylation of MAPK and Akt. In addition, in terms of potential therapeutic application, we clearly demonstrated that the enhanced-cell invasiveness induced by Sema4D could be inhibited by knockdown of plexinB1, suggesting that blockade of plexinB1 might diminish the invasive potential of pancreatic cancer cells. Our findings provide new insight into possible prognostic biomarkers and therapeutic targets in PDAC patients.

Novel findings for the development of drug therapy for various liver diseases: Liver microsomal triglyceride transfer protein activator may be a possible therapeutic agent in non-alcoholic steatohepatitis.

The factors involved in the progression of non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH) are not fully understood and thus it is urgently needed to elucidate these factors. Steatosis is not causal in the development of NASH, but rather it sensitizes the liver to the damaging effects of second hits such that stressors innocuous to a healthy liver lead to the development of NASH in the steatotic liver. In the previous study, most of the hepatic lipid metabolite profiles were similar in the NAFL and NASH groups. However, very-low-density lipoprotein (VLDL) synthesis, especially hepatic microsomal triglyceride transfer protein (MTP) mRNA expression, was impaired in the NASH group. Moreover, NASH showed significantly higher incidence of minor alley appearance compared with NAFL, indicating the possibility of association between NASH pathogenesis and decreased congenital MTP activity. MTP is one of the enzymes that transfer triglycerides to nascent apolipoprotein B, producing VLDL and removing lipid from the hepatocyte. A growing body of literature suggests that the measurement of hepatic MTP expression may be helpful for diagnosis; and moreover, hepatic MTP activator may be a possible therapeutic agent for the treatment of NASH.

Long-term combination therapy of ezetimibe and acarbose for non-alcoholic fatty liver disease.

BACKGROUND/AIMS: Non-alcoholic fatty liver disease (NAFLD) is a hepatic manifestation of metabolic syndrome that is closely associated with multiple factors such as obesity, hyperlipidemia, type 2 diabetes mellitus and hypertension, making it difficult to treat NAFLD effectively using any monotherapy available to date. In this study, we propose a novel combination therapy for NAFLD comprising ezetimibe (EZ), a cholesterol absorption inhibitor, and acarbose (AC), an alpha-glucosidase inhibitor. METHODS: C57BL/6J mice were divided into five treatment groups as follows: basal diet (BD), high-fat diet (HFD) only, HFD with EZ (5mg/kg/day), HFD with AC (100mg/kg/day), and HFD with both EZ and AC for 24 weeks. RESULTS: Long-term combination therapy with EZ and AC significantly reduced steatosis, inflammation and fibrosis in the liver, compared with long-term monotherapy with either drug, in an HFD-induced NAFLD mouse model; the combination therapy also significantly increased the expression of microsomal triglyceride transfer protein (MTP) and peroxisome proliferators-activated receptor-alpha1 (PPAR-alpha1) in the liver, compared with either monotherapy, which may have led to the improvement in lipid metabolic disorder seen in this model. CONCLUSIONS: Combination therapy with EZ and AC for 24 weeks improved the histopathological findings in a mouse model of NAFLD.

Appearance of apoptotic cells and granular cells in the silkworm midgut lumen during larval-pupal ecdysis.

To study midgut degradation and programmed cell death, we performed methyl green-pyronin staining and Giemsa staining of the midgut of silkworms during metamorphosis. Midgut epithelial cells underwent pyknosis and cytoplasmic shrinkage on the second day of spinning. In the prepupal stage, all midgut epithelial cells desquamated into the midgut lumen, rapidly forming apoptotic bodies. The number of apoptotic bodies in the midgut decreased rapidly from the prepupal stage to the third day of the pupal stage. DNA fragmentation at the time of apoptotic body formation was confirmed by the comet assay. In the midgut lumen from the prepupal stage to the first through third days of the pupal stage in which apoptotic bodies were observed, granular cells were present. Their morphology was similar to that in the body fluid and, during the pupal stage, intracellular granules increased in size and number with time, giving the appearance of a foamy cell. In this stage, numerous granular cells were observed under the basement membrane of the midgut, and phagocytosed apoptotic bodies were seen within granular cells in the midgut lumen. Granular cells may be actively involved in the clearance of apoptotic bodies from the midgut during larval-pupal ecdysis.

Oral choline tolerance test as a novel noninvasive method for predicting nonalcoholic steatohepatitis.

BACKGROUND: Although therapeutic intervention for nonalcoholic steatohepatitis (NASH) at an early stage is important owing to the progressive nature of the disease, diagnosis using noninvasive methods remains difficult. We previously demonstrated NASH specific impairment of choline metabolism and the use of fasting plasma free choline (fCh) levels for NASH diagnosis. Here, we investigated the utility of an oral choline tolerance test (OCTT), based on disordered choline metabolism, as a novel noninvasive method for NASH diagnosis. METHODS: Sixty-five patients with biopsy proven nonalcoholic fatty liver disease (NAFLD) and 17 healthy controls were enrolled. Blood samples were obtained from all subjects five times during the OCTT (before and 1, 2, 3, and 4 h after oral loading with 260 mg choline). RESULTS: Four-hour fCh levels after oral loading choline were markedly increased in NASH patients, compared with non-NASH subjects. For detecting NASH, compared with non-NASH subjects, the area under the curve for 4-h fCh levels was 0.829 on receiver operating characteristic (ROC) analysis. The cut-off level for NASH diagnosis was ≥0.16 mg/dL, and the sensitivity, specificity, positive predictive value, and negative predictive value were 80.1, 82.6, 78.4, and 84.4 %, respectively. Moreover, 4-h fCh levels were significantly associated with the disease activity based on NAFLD activity score in patients with NAFLD. CONCLUSIONS: Four-hour fCh levels obtained by an OCTT reflect a NASH specific disorder of choline metabolism, suggesting that the OCTT is a novel and useful noninvasive method for diagnosing NASH at an early stage with sufficient accuracy for clinical practice.

Two-colored fluorescence correlation spectroscopy screening for LC3-P62 interaction inhibitors.

The fluorescence correlation spectroscopy (FCS)-based competitive binding assay to screen for protein-protein interaction inhibitors is a highly sensitive method as compared with the fluorescent polarization assay used conventionally. However, the FCS assay identifies many false-positive compounds, which requires specifically designed orthogonal screenings. A two-colored application of the FCS-based screening was newly developed, and inhibitors of a protein-protein interaction, involving selective autophagy, were selected. We focused on the interaction of LC3 with the adaptor protein p62, because the interaction is crucial to degrade the specific target proteins recruited by p62. First, about 10,000 compounds were subjected to the FCS-based competitive assay using a TAMRA-labeled p62-derived probe, and 29 hit compounds were selected. Next, the obtained hits were evaluated by the second FCS assay, using an Alexa647-labeled p62-derived probe to remove the false-positive compounds, and six hit compounds inhibited the interaction. Finally, we tested all 29 compounds by surface plasmon resonance-based competitive binding assay to evaluate their inhibition of the LC3-p62 interaction and selected two inhibitors with IC50 values less than 2 µM. The two-colored FCS-based screening was shown to be effective to screen for protein-protein interaction inhibitors.

Soluble CD14 levels reflect liver inflammation in patients with nonalcoholic steatohepatitis.

BACKGROUND AIMS: Liver inflammation is a risk factor for the progression of nonalcoholic fatty liver disease (NAFLD). However, the diagnosis of liver inflammation is very difficult and invasive liver biopsy is still the only method to reliably detect liver inflammation. We previously reported that overexpression of CD14 in Kupffer cells may trigger the progression to nonalcoholic steatohepatitis (NASH) via liver inflammation following hyper-reactivity to low-dose lipopolysaccharide. Therefore, the aim of this study was to investigate the relationship between soluble type of CD14 (sCD14) and histological features in patients with NAFLD. METHODS: Our cohort consisted of 113 patients with liver biopsy-confirmed NAFLD and 21 age-matched healthy controls. Serum sCD14 levels were measured by an enzyme-linked immunosorbent assay. RESULTS: Serum sCD14 levels were significantly associated with diagnosis of NASH and the area under the receiver operator characteristic curve (AUROC) to distinguish between not NASH and NASH was 0.802. Moreover, serum sCD14 levels were significantly associated with the disease activity based on NAFLD activity score and hepatic CD14 mRNA expression, which is correlated with membrane CD14 (mCD14) expression, in patients with NAFLD. In multiple regression analysis, the serum sCD14 levels were independently associated with liver inflammation. The AUROC to distinguish between mild and severe liver inflammation in patients with NAFLD was 0.752. CONCLUSIONS: We found that serum sCD14 levels increased significantly with increasing liver inflammation grade in patients with NAFLD, reflecting increased hepatic CD14 expression. Serum sCD14 is a promising tool to predict the worsening of liver inflammation, and may offer a potential biomarker for evaluation of therapeutic effects in NAFLD.

Peroxisome Proliferator-Activated Receptor Gamma Exacerbates Concanavalin A-Induced Liver Injury via Suppressing the Translocation of NF-κB into the Nucleus.

Peroxisome proliferator-activated receptor-γ (PPARγ) has been reported to reduce inflammation and attenuate fibrosis in the liver. In this study, we investigated the effects of PPARγ on the liver injury induced by 20 mg/kg Concanavalin A (Con A). The mice were administered one of the three types of PPARγ ligands (pioglitazone, ciglitazone, and troglitazone) for 1 week, and the serum alanine aminotransferase (ALT) levels at 20 h after Con A injection were significantly elevated in the PPARγ ligand-treated mice. Furthermore, the serum ALT levels after Con A injection in the PPARγ hetero-knock-out mice (PPARγ(+/-) mice) were lower than those in the wild-type mice (WT mice). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) revealed extensive liver damage induced by Con A in the pioglitazone-treated mice. Electrophoresis mobility shift assay (EMSA) revealed that activation of translocation of nuclear factor- (NF-) κB, which is a suppressor of apoptosis, in the nucleus of the hepatocytes was suppressed in the pioglitazone-treated mice after Con A injection. In this study, we showed that PPARγ exacerbated Con A-induced liver injury via suppressing the translocation of NF-κB into the nucleus, thereby inhibiting the suppression of liver cell apoptosis.

Hyperresponsivity to low-dose endotoxin during progression to nonalcoholic steatohepatitis is regulated by leptin-mediated signaling.

Although bacterial endotoxin, such as lipopolysaccharide (LPS), plays a key role in the pathogenesis of nonalcoholic steatohepatitis (NASH), detailed mechanisms of this pathogenesis remain unclear. Here, we demonstrate that upregulation of CD14 by leptin-mediated signaling is critical to hyperreactivity against endotoxin during NASH progression. Upregulation of CD14 in Kupffer cells and hyperreactivity against low-dose LPS were observed in high-fat diet (HFD)-induced steatosis mice, but not chow-fed-control mice. Hyperresponsivity against low-dose LPS led to accelerated NASH progression, including liver inflammation and fibrosis. Administering leptin in chow-fed mice caused increased hepatic expression of CD14 via STAT3 signaling, resulting in hyperreactivity against low-dose LPS without steatosis. In contrast, a marked decrease in hepatic CD14 expression was observed in leptin-deficient ob/ob mice, despite severe steatosis. Our results indicate that obesity-induced leptin plays a crucial role in NASH progression via enhanced responsivity to endotoxin, and we propose a mechanism of bacteria-mediated progression of NASH.

Plasma free choline is a novel non-invasive biomarker for early-stage non-alcoholic steatohepatitis: A multi-center validation study.

AIM: Choline is a dietary component that is crucial for normal cellular function. Choline is predominantly absorbed from the small intestine and completely metabolized in the liver. We recently demonstrated that free choline (fCh) levels in blood reflect the level of phosphatidylcholine synthesis in the liver and is correlated with the onset of non-alcoholic steatohepatitis (NASH). Our aim here was to validate the utility of this biomarker for NASH diagnosis. METHODS: Our cohort consisted of 110 patients with biopsy proven non-alcoholic fatty liver disease (NAFLD) from four centers across Japan and 25 age-matched healthy controls. Plasma fCh levels were measured using high-performance liquid chromatography. RESULTS: Patients with diagnosed or borderline NASH had significantly increased plasma fCh levels when compared with control subjects, or patients not diagnosed with NASH. Interestingly, an association between plasma fCh levels and expression of microsomal triglyceride transfer protein, which catalyzes the transfer of triglyceride, was reflected in the markedly negative correlation between these two variables in patients with NAFLD. Moreover, the grade of liver steatosis and fibrosis stage increased with increasing plasma fCh levels (P < 0.05). The area under the receiver-operating characteristic (ROC) curves for NASH, including borderline diagnosis, was 0.811. Additionally, the areas under the ROC for fibrosis stage were 0.816 for >stage 1, 0.805 for >stage 2, 0.809 for >stage 3 and 0.818 for >stage 4. CONCLUSION: Plasma fCh levels are closely related to the grade of liver steatosis and fibrosis, and predict NASH severity. Plasma fCh levels are therefore a potential diagnostic marker for early-stage NASH in clinical practice.