Development Of Steatohepatitis In Mice Research Articles

Induction of the Lipid Droplet Formation Genes in Steatohepatitis Mice by Embryo/Postnatal Nutrient Environment Is Associated with Histone Acetylation around the Genes.

Recently, we have demonstrated that mice, cultured embryos in α-minimum essential medium (αMEM) and subsequent fed a high-fat, high-sugar diet, developed steatohepatitis. In this study, we investigated using these samples whether the expression of lipid droplet formation genes in the liver is higher in MEM mice, whether these expressions are regulated by histone acetylation, writers/readers of histone acetylation, and the transcriptional factors of endoplasmic reticulum stress. Mice were produced by two-cell embryos in αMEM or standard potassium simplex-optimized medium (control) in vitro for 48 h, and implanted into an oviduct for spontaneous delivery. MEM and control-mice were fed a high-fat, high-sugar diet for 18 wk, and then liver samples were collected and analyzed by histology, qRT-PCR, and chromatin immunoprecipitation assay. Gene expression of Cidea, Cidec, and Plin4 were higher in MEM mice and histone H3K9 acetylation, BRD4, and CBP were higher in MEM mice than in control mice around those genes. However, the binding of endoplasmic reticulum stress-related transcription factors (ATF4, CHOP and C/EBPα) around those genes in the liver, was not clearly differed between MEM mice and control mice. The increased expression of Cidea, Cidec and Plin4 in the liver, accompanied by the development of steatohepatitis in mice induced is positively associated with increased histone H3K9 acetylation and CBP and BRD4 binding around these genes.

HPS protects the liver against steatosis, cell death, inflammation, and fibrosis in mice with steatohepatitis.

Hepassocin (HPS) is a hepatokine associated with metabolic regulation and development of non-alcoholic steatohepatitis (NASH). However, previous reports on HPS are controversial and its true function is not yet understood. Here, we demonstrated that hepatic HPS expression levels were upregulated in short-term feeding and downregulated in long-term feeding in high-fat diet (HFD)- and methionine- and choline-deficient (MCD) diet-fed mice, as well as in genetically obese (ob/ob) mice. HFD- and MCD-induced hepatic steatosis, inflammation, apoptosis, and fibrosis were more pronounced in HPS knockout mice than in the wild-type mice. Moreover, HPS depletion aggravated HFD-induced insulin resistance. By contrast, HPS administration improved MCD- or HFD-induced liver phenotypes and insulin resistance in HPS knockout and wild-type mice. Mechanistic studies revealed that MCD-induced hepatic oxidative stress was significantly increased by HPS deficiency and could be attenuated by HPS administration. Furthermore, palmitic acid-induced lipid accumulation and oxidative stress were exclusively enhanced in HPS knockout hepatocytes and diminished by HPS cotreatment. These data suggest that HPS ameliorates NASH in mice, at least in part, by inhibiting the oxidative stress. HPS expression levels are downregulated in human fatty liver tissues, suggesting that it may play an important protective role in NASH. Collectively, our findings provide clear genetic evidence that HPS has beneficial effects on the development of steatohepatitis in mice and suggest that upregulating HPS signaling may represent an effective treatment strategy for NASH.

Role of XBP1 in regulating the progression of non-alcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH) is associated with the dysregulation of lipid metabolism and hepatic inflammation, though the underlying mechanisms remain unclear. We aimed to investigate the role of X-box binding protein-1 (XBP1) in the progression of NASH. Human liver tissues obtained from patients with NASH and controls were used to assess XBP1 expression. NASH models were developed in hepatocyte-specific Xbp1 knockout (Xbp1ΔHep), macrophage-specific Xbp1 knockout (Xbp1ΔMf), macrophage-specific Nlrp3 knockout, and wild-type (Xbp1FL/FL or Nlrp3FL/FL) mice fed with a high-fat diet for 26 weeks or a methionine/choline-deficient diet for 6 weeks. The expression of XBP1 was significantly upregulated in liver samples from patients with NASH. Hepatocyte-specific Xbp1 deficiency inhibited the development of steatohepatitis in mice fed the high-fat or methionine/choline-deficient diets. Meanwhile, macrophage-specific Xbp1 knockout mice developed less severe steatohepatitis and fibrosis than wild-type Xbp1FL/FL mice in response to the high-fat or methionine/choline-deficient diets. Macrophage-specific Xbp1 knockout mice showed M2 anti-inflammatory polarization. Xbp1-deleted macrophages reduced steatohepatitis by decreasing the expression of NLRP3 and secretion of pro-inflammatory cytokines, which mediate M2 macrophage polarization in macrophage-specific Xbp1 knockout mice. Steatohepatitis was less severe in macrophage-specific Nlrp3 knockout mice than in wild-type Nlrp3FL/FL mice. Xbp1-deleted macrophages prevented hepatic stellate cell activation by decreasing expression of TGF-β1. Less fibrotic changes were observed in macrophage-specific Xbp1 knockout mice than in wild-type Xbp1FL/FL mice. Inhibition of XBP1 suppressed the development of NASH. XBP1 regulates the development of NASH. XBP1 inhibitors protect against steatohepatitis. Thus, XBP1 is a potential target for the treatment of NASH. XBP1 is a transcription factor that is upregulated in liver tissues of patients with non-alcoholic steatohepatitis (NASH). Conditional knockout of Xbp1 in hepatocytes resulted in decreased lipid accumulation in mice, while genetic deletion of Xbp1 in macrophages ameliorated nutritional steatohepatitis and fibrosis in mice. Pharmacological inhibition of XBP1 protects against steatohepatitis and fibrosis, highlighting a promising therapeutic strategy for NASH.

TNAP: A New Multitask Enzyme in Energy Metabolism.

Tissue-nonspecific alkaline phosphatase (TNAP) is mainly known for its necessary role in skeletal and dental mineralization, which relies on the hydrolysis of the mineralization inhibitor inorganic pyrophosphate (PPi). Mutations in the gene encoding TNAP leading to severe hypophosphatasia result in strongly reduced mineralization and perinatal death. Fortunately, the relatively recent development of a recombinant TNAP with a bone anchor has allowed to correct the bone defects and prolong the life of affected babies and children. Researches on TNAP must however not be slowed down, because accumulating evidence indicates that TNAP activation in individuals with metabolic syndrome (MetS) is associated with enhanced cardiovascular mortality, presumably in relation with cardiovascular calcification. On the other hand, TNAP appears to be necessary to prevent the development of steatohepatitis in mice, suggesting that TNAP plays protective roles. The aim of the present review is to highlight the known or suspected functions of TNAP in energy metabolism that may be associated with the development of MetS. The location of TNAP in liver and its function in bile excretion, lipopolysaccharide (LPS) detoxification and fatty acid transport will be presented. The expression and function of TNAP in adipocyte differentiation and thermogenesis will also be discussed. Given that TNAP is a tissue- and substrate-nonspecific phosphatase, we believe that it exerts several crucial pathophysiological functions that are just beginning to be discovered.

Murine CD103+ dendritic cells protect against steatosis progression towards steatohepatitis

Non-alcoholic fatty liver (NAFL) is the hepatic consequence of metabolic syndrome and can progress to non-alcoholic steatohepatitis (NASH). The identification of molecular and cellular factors that determine the progression of NASH and lead to irreversible hepatocellular damage are crucial. Dendritic cells (DCs) represent a heterogeneous cell population among which CD103+ DCs play a significant role in immunity and tolerance. We aimed to clarify the role of this DC subset in the pathomechanism of NASH. Steatosis progression towards steatohepatitis was analysed using multicolor FACS analyses, cytokine and qPCR array in high sucrose diet (HSD) and methionine and choline deficient diet (MCD) fed wild-type and basic leucine zipper transcription factor, ATF-Like-3 (Batf3) deficient animals, which lack CD103+ DCs (classical type-1 DC, cDC1s). Metabolic challenge of Batf3-/- animals resulted in the progression of steatosis towards steatohepatitis, manifesting by an increased influx of inflammatory cells into the liver and elevated inflammatory cytokine production of myeloid cells upon innate stimuli. However, the lack of cDC1s did not affect cellular apoptosis and fibrosis progression but altered genes involved in lipid metabolism. The adoptive transfer of CD103+ cDC1s to Batf3 deficient animals reversed these observed changes and more importantly could attenuate cellular damage and inflammation in established murine steatohepatitis. Here, we have identified the murine CD103+ cDC1s as a protective DC subtype that influences the pro-anti-inflammatory balance and protects the liver from metabolic damage. As guardians of liver integrity, they play a key role in the inflammatory process during the development of steatohepatitis in mice. Non-alcoholic fatty liver (NAFL) is the hepatic consequence of metabolic syndrome and can lead to non-alcoholic steatohepatitis (NASH). The current study demonstrated that a specific murine dendritic cell subtype possesses a potent regulatory role to influence the inflammatory milieu of the liver in this process.

Hepatocyte Nicotinamide Adenine Dinucleotide Phosphate Reduced Oxidase 4 Regulates Stress Signaling, Fibrosis, and Insulin Sensitivity During Development of Steatohepatitis in Mice

Reactive 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. Liver 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. Levels 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. NOX4 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.

THU0230 Bezafitrate for Methotorexate-Induced Acute Liver Injur\\y

BackgroundMethotrexate (MTX) is an anchor drug widely used in rheumatoid arthritis (RA). Higher dosage of MTX leads to more favorable outcome and its dose-dependent toxicity, therefore has gained much more...

Inflammatory Signals shift from adipose to liver during high fat feeding and influence the development of steatohepatitis in mice

BackgroundObesity and inflammation are highly integrated processes in the pathogenesis of insulin resistance, diabetes, dyslipidemia, and non-alcoholic fatty liver disease. Molecular mechanisms underlying inflammatory events during high fat diet-induced obesity are poorly defined in mouse models of obesity. This work investigated gene activation signals integral to the temporal development of obesity.MethodsGene expression analysis in multiple organs from obese mice was done with Taqman Low Density Array (TLDA) using a panel of 92 genes representing cell markers, cytokines, chemokines, metabolic, and activation genes. Mice were monitored for systemic changes characteristic of the disease, including hyperinsulinemia, body weight, and liver enzymes. Liver steatosis and fibrosis as well as cellular infiltrates in liver and adipose tissues were analyzed by histology and immunohistochemistry.ResultsObese C57BL/6 mice were fed with high fat and cholesterol diet (HFC) for 6, 16 and 26 weeks. Here we report that the mRNA levels of macrophage and inflammation associated genes were strongly upregulated at different time points in adipose tissues (6-16 weeks) and liver (16-26 weeks), after the start of HFC feeding. CD11b+ and CD11c+ macrophages highly infiltrated HFC liver at 16 and 26 weeks. We found clear evidence that signals for IL-1β, IL1RN, TNF-α and TGFβ-1 are present in both adipose and liver tissues and that these are linked to the development of inflammation and insulin resistance in the HFC-fed mice.ConclusionsMacrophage infiltration accompanied by severe inflammation and metabolic changes occurred in both adipose and liver tissues with a temporal shift in these signals depending upon the duration of HFC feeding. The evidences of gene expression profile, elevated serum alanine aminotransferase, and histological data support a progression towards nonalcoholic fatty liver disease and steatohepatitis in these HFC-fed mice within the time frame of 26 weeks.

74 Toll-Like Receptor (TLR) 2 in Kupffer Cells Plays a Crucial Role in the Development of Steatohepatitis in Mice

Background: Gut-derived bacterial products are candidates for a promoting factor to progress non-alcoholic steatohepatitis (NASH). Toll-like receptors are pattern recognition receptors that sense microbial products such as LPS. We have previously shown that TLR9, a receptor for bacterial DNA, plays an important role in the development of NASH. However, little information on TLR2, a receptor for Gram-positive bacterial products such as peptidoglycan, is available in that process. In addition, the role of TLR2 in Kupffer cells, hepatic macrophages, remain unknown. Materials and Methods: WT and TLR2 KO were fed choline-deficient (CDAA) or Choline-supplemented control (CSAA) diet for 22 weeks. Steatosis, inflammation and fibrosis were assessed by histological examination, quantitative real-time PCR and ELISA. To examine the role of Kupffer cells in the development of steatohepatitis, WT mice were injected clodronate to deplete Kupffer cells, followed by CDAA diet feeding for 2 weeks. To further explore whether Kupffer cells mediate TLR2-dependent inflammation and fibrosis, we generated TLR2-chimeric mice using a combination of Kupffer cell depletion, irradiation and bone-marrow transplantation, followed by CDAA diet feeding for 22 weeks. Results: After 22 weeks of CDAA diet feeding, WT mice showed typical steatohepatitis characterized by severe steatosis, inflammatory cell infiltration and hepatocyte ballooning as well as perisinusoidal fibrosis. In contrast, TLR2 KOmice exhibited less inflammatory cell infiltration and hepatocyte ballooning. NAFLD activity score in TLR2KO mice was significantly lower than that in WT mice. In addition, TLR2KO mice showed a significant reduction in fibrosis compared with WT mice. In Kupffer cells-depleted WT mice, diet-induced steatohepatitis and fibrosis were significantly suppressed compared with WT mice without Kupffer cell depletion. In chimeric mice, full reconstitution of Kupffer cells was achieved, but hepatic stellate cells were not replaced by bone marrow-derived cells. WT mice containing Tlr2deficient bone marrow showed suppressed steatohepatitis and fibrosis, whereas Tlr2-deficient mice containing wild-type bone marrow showed advanced steatohepatitis and fibrosis. Conclusion: TLR2 expression in Kupffer cells plays a crucial role in CDAA diet-induced steatohepatitis and fibrosis in mice.

Effects of bezafibrate, PPAR pan-agonist, and GW501516, PPARδ agonist, on development of steatohepatitis in mice fed a methionine- and choline-deficient diet

We evaluated the effects of bezafibrate, a peroxisome proliferator-activated receptor (PPAR) pan-agonist, and GW501516, a PPARδ agonist, on mice fed a methionine- and choline-deficient (MCD) diet, a model of non-alcholic steatohepatitis (NASH), to investigate (a) the efficacy of bezafibrate against non-alcholic steatohepatitis and (b) the relation between non-alcholic steatohepatitis and the functional role of PPARδ. Bezafibrate (50 or 100 mg/kg/day) and GW501516 (10 mg/kg/day) were administered by gavage once a day for 5 weeks. Hepatic lipid contents, plasma triglyceride, high density lipoprotein (HDL)-cholesterol and alanine aminotransferase (ALT) concentrations were evaluated, as were histopathological changes in the liver and hepatic mRNA expression levels. Bezafibrate and GW501516 inhibited the MCD-diet-induced elevations of hepatic triglyceride and thiobarbituric acid-reactants contents and the histopathological increases in fatty droplets within hepatocytes, liver inflammation and number of activated hepatic stellate cells. In this model, bezafibrate and GW501516 increased the levels of hepatic mRNAs associated with fatty acid β-oxidation [acyl-CoA oxidase (ACO), carnitine palmitoyltransferase-1 (CPT-1), liver-fatty acid binding protein (L-FABP) and peroxisomal ketothiolase], and reduced the levels of those associated with inflammatory cytokines or chemokine [transforming growth factor (TGF)-β1, interleukin (IL)-6, IL-1β, monocyte chemoattractant protein (MCP)-1, tumor necrosis factor (TNF) α and nuclear factor (NF)-κB1]. In addition, bezafibrate characteristically reduced the elevation in the level of plasma ALT, but enhanced that in plasma adiponectin and increased the mRNA expression levels of its receptors (adiponectin receptors 1 and 2). These results suggest that (a) bezafibrate (especially) and GW501516 might improve hepatic steatosis via an improvement in fatty acid β-oxidation and a direct prevention of inflammation, (b) treatment with a PPARδ agonist might improve non-alcholic steatohepatitis, (c) bezafibrate may improve non-alcholic steatohepatitis via activation not only of PPARα but also of PPARδ, because bezafibrate is a PPAR pan-agonist.

Jnk1 but not jnk2 promotes the development of steatohepatitis in mice

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis and varying degrees of necroinflammation. Although chronic oxidative stress, inflammatory cytokines, and insulin resistance have been implicated in the pathogenesis of NAFLD, the mechanisms that underlie the initiation and progression of this disease remain unknown. c-Jun N-terminal kinase (JNK) is activated by oxidants and cytokines and regulates hepatocellular injury and insulin resistance, suggesting that this kinase may mediate the development of steatohepatitis. The presence and function of JNK activation were therefore examined in the murine methionine- and choline-deficient (MCD) diet model of steatohepatitis. Activation of hepatic JNK, c-Jun, and AP-1 signaling occurred in parallel with the development of steatohepatitis in MCD diet-fed mice. Investigations in jnk1 and jnk2 knockout mice demonstrated that jnk1, but not jnk2, was critical for MCD diet-induced JNK activation. JNK promoted the development of steatohepatitis as MCD diet-fed jnk1 null mice had significantly reduced levels of hepatic triglyceride accumulation, inflammation, lipid peroxidation, liver injury, and apoptosis compared with wild-type and jnk2 -/- mice. Ablation of jnk1 led to an increase in serum adiponectin but had no effect on serum levels of tumor necrosis factor-alpha. In conclusion, JNK1 is responsible for JNK activation that promotes the development of steatohepatitis in the MCD diet model. These findings also provide additional support for the critical mechanistic involvement of JNK1 overactivation in conditions associated with insulin resistance and the metabolic syndrome.