Growth arrest and DNA damage-inducible 45α protects against nonalcoholic steatohepatitis induced by methionine- and choline-deficient diet.

Growth arrest and DNA damage-inducible 45 α (Gadd45α) is a stress-inducible protein that plays an important role in cell survival/death and DNA repair, but its contribution to the development of nonalcoholic steatohepatitis (NASH) has not been investigated. C57BL/6 Gadd45a-null and wild-type (WT) mice were treated with a methionine and choline-deficient diet (MCD) for eight weeks and phenotypic changes examined. Gadd45a-null mice had more severe hepatic inflammation and fibrosis, higher levels of mRNAs encoding pro-inflammatory, pro-fibrotic, and pro-apoptotic proteins, and greater oxidative and endoplasmic reticulum (ER) stress compared with WT mice. Indeed, Gadd45a mRNA was induced in response to ER stress in primary hepatocytes. Lipidomic analysis of NASH livers demonstrated decreased triacylglycerol (TG) in MCD-treated Gadd45a-null mice, which was associated with increased mRNAs encoding phospholipase D (Pld1/2), phosphatidic acid phosphatase type 2A, and choline/ethanolamine phosphotransferase 1 (Cept1), involved in the phosphatidylcholine-phosphatidic acid-diacylglycerol cycle, and decreased mRNAs encoding fatty acid (FA)-binding protein 1 (Fabp1) and FA transport protein 5. Treatment of cultured primary hepatocytes with tumor necrosis factor α, transforming growth factor β, and hydrogen peroxide led to the corresponding induction of Fabp1, Pld1/2, and Cept1 mRNAs. Collectively, Gadd45α plays protective roles against MCD-induced NASH likely due to attenuating cellular stress and ensuing inflammatory signaling. These results also suggest an interconnection between hepatocyte injury, inflammation and disrupted glycerophospholipid/FA metabolism that yields a possible mechanism for decreased TG accumulation with NASH progression (i.e., "burned-out" NASH).

Tanaka N ，Takahashi S ，Hu X ，Lu Y ，Fujimori N ，Golla S ，Fang ZZ ，Aoyama T ，Krausz KW ，Gonzalez FJ ... -  《-》

Role of fibroblast growth factor 21 in the early stage of NASH induced by methionine- and choline-deficient diet.

Fibroblast growth factor 21 (FGF21) is a modulator of energy homeostasis and is increased in human nonalcoholic liver disease (NAFLD) and after feeding of methionine- and choline-deficient diet (MCD), a conventional inducer of murine nonalcoholic steatohepatitis (NASH). However, the significance of FGF21 induction in the occurrence of MCD-induced NASH remains undetermined. C57BL/6J Fgf21-null and wild-type mice were treated with MCD for 1 week. Hepatic Fgf21 mRNA was increased early after commencing MCD treatment independent of peroxisome proliferator-activated receptor (PPAR) α and farnesoid X receptor. While no significant differences in white adipose lipolysis were seen in both genotypes, hepatic triglyceride (TG) contents were increased in Fgf21-null mice, likely due to the up-regulation of genes encoding CD36 and phosphatidic acid phosphatase 2a/2c, involved in fatty acid (FA) uptake and diacylglycerol synthesis, respectively, and suppression of increased mRNAs encoding carnitine palmitoyl-CoA transferase 1α, PPARγ coactivator 1α, and adipose TG lipase, which are associated with lipid clearance in the liver. The MCD-treated Fgf21-null mice showed increased hepatic endoplasmic reticulum (ER) stress. Exposure of primary hepatocytes to palmitic acid elevated the mRNA levels encoding DNA damage-inducible transcript 3, an indicator of ER stress, and FGF21 in a PPARα-independent manner, suggesting that lipid-induced ER stress can enhance hepatic FGF21 expression. Collectively, FGF21 is elevated in the early stage of MCD-induced NASH likely to minimize hepatic lipid accumulation and ensuing ER stress. These results provide a possible mechanism on how FGF21 is increased in NAFLD/NASH.

Tanaka N ，Takahashi S ，Zhang Y ，Krausz KW ，Smith PB ，Patterson AD ，Gonzalez FJ ... -  《-》

Elevation of liver endoplasmic reticulum stress in a modified choline-deficient l-amino acid-defined diet-fed non-alcoholic steatohepatitis mouse model.

Endoplasmic reticulum (ER) stress caused by accumulation of misfolded proteins is observed in several kinds of diseases. Since ER stress is reported to be involved in the progression of non-alcoholic steatohepatitis (NASH), highly sensitive and simple measurement methods are required for research into developing novel therapy for NASH. To investigate the involvement of ER stress in NASH pathogenesis in a mouse model, an assay for liver ER stress was developed using ER stress activated indicator-luciferase (ERAI-Luc) mice. To establish the assay method for detection of ER stress in the liver, tunicamycin (TM) (0.3 mg/kg i. p.) was administered to ERAI-Luc mice, and the luciferase activity was measured in ex vivo and in vivo. To evaluate ER stress in the NASH model, ERAI-Luc mice were fed a modified choline-deficient l-amino acid-defined (mCDAA) diet for 14 weeks. After measurement of ER stress by luminescence imaging, levels of liver lipids and pro-fibrotic and pro-inflammatory gene expression were measured as NASH-related indexes. In non-invasive whole-body imaging, TM elevated luciferase activity in the liver, induced by activation of ER stress. The highest luminescence in the liver was confirmed by ex vivo imaging of isolated tissues. In parallel with progression of NASH, elevated luminescence induced by ER stress in liver was observed in mCDAA diet-fed ERAI-Luc mice. Luciferase activity was significantly and positively correlated to levels of triglyceride and free cholesterol in the liver, as well as to the mRNA expression of type 1 collagen α1 chain and tumor necrosis factor α. These data indicated that the use of ERAI-Luc mice was effective in the detection of ER stress in the liver. Moreover, the NASH model using ERAI-Luc mice can be a useful tool to clarify the role of ER stress in pathogenesis of NASH and to evaluate effects of drugs targeted against ER stress.

Muraki Y ，Makita Y ，Yamasaki M ，Amano Y ，Matsuo T ... -  《-》

Epigallocatechin gallate attenuated non-alcoholic steatohepatitis induced by methionine- and choline-deficient diet.

Nonalcoholic fatty liver disease (NAFLD) and its progressive form, nonalcoholic steatohepatitis (NASH), are the most common causes of chronic liver disease. In this study, we evaluated the effects of Epigallocatechin gallate (EGCG) on methionine- and choline-deficient (MCD) diet-induced NASH. Our data showed that EGCG significantly prevented MCD diet-induced liver and body weight loss. Histological analysis showed that EGCG inhibited MCD diet-induced steatohepatitis including fat accumulation and inflammatory cells infiltration. Biochemical analysis data showed that EGCG significantly reduced the elevation of plasma ALT and AST levels but increased plasma triglyceride and cholesterol contents. However, EGCG significantly inhibited hepatic triglyceride and cholesterol content in MCD diet fed mice. Consistent with histology results, EGCG treatment significantly inhibited MCD diet-induced IL-1β, IL-6, TNF-α and MCP-1 mRNA expression. As an antioxidant, EGCG treatment significant inhibited hepatic MDA contents and increased hepatic SOD contents. In addition, transforming growth factor (TGF)-β, collagen I-α1, tissue inhibitor of metalloproteinase 1 (TIMP-1) and α-smooth muscle actin (SMA) mRNA expression, which are markers of hepatic fibrosis, were markedly inhibited by EGCG treatment. Western blot data showed that EGCG inhibited Smad2 and Smad3 phosphorylation in the liver and LX-2 cells which were involved in TGF-β-induced pathway. Taken together, EGCG attenuated NASH induced by MCD diet associated with ameliorating fibrosis, oxidative stress, and hepatic inflammation. Our results indicate that EGCG has beneficial roles in the development of MCD diet-induced NASH.

Ding Y ，Sun X ，Chen Y ，Deng Y ，Qian K ... -  《-》

Caspase-1-mediated regulation of fibrogenesis in diet-induced steatohepatitis.

Dixon LJ ，Berk M ，Thapaliya S ，Papouchado BG ，Feldstein AE ... -  《-》