Trehalose alleviates oxidative stress-mediated liver injury and Mallor-Denk body formation via activating autophagy in mice.

Autophagy is a degradation pathway for long-lived cytoplasmic proteins or damaged organelles and also for many aggregate-prone and disease-causing proteins. Endoplasmic reticulum (ER) stress and oxidative stress are associated with the pathophysiology of various liver diseases. These stresses induce the accumulation of abnormal proteins, Mallory-Denk body (MDB) formation and apoptosis in hepatocytes. A disaccharide trehalose had been reported to induce autophagy and decrease aggregate-prone proteins and cytotoxicity in neurodegenerative disease models. But the effects of trehalose in hepatocytes have not been fully understood. We examined the effect of trehalose on autophagy, ER stress and oxidative stress-mediated cytotoxicity and MDB formation in hepatocytes using mice model with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) treatment for 3 months. We administered trehalose by intraperitoneal injection of water containing 10% trehalose (0.02 mg/g body weight) every other day for 3 months. Our results demonstrated that trehalose induced autophagy and reduced ER stress, oxidative stress, MDB formation and apoptosis in hepatocytes of DDC-fed mice by Western blotting and immunostaining analyses. Electron microscopy revealed that trehalose induced autolysosome formation, which located is close to the MDBs. Thus, our findings suggest that trehalose can become a therapeutic agent for oxidative stress-related liver diseases via activating autophagy.

Ufmylation and FATylation pathways are downregulated in human alcoholic and nonalcoholic steatohepatitis, and mice fed DDC, where Mallory-Denk bodies (MDBs) form.

We previously reported the mechanisms involved in the formation of Mallory-Denk bodies (MDBs) in mice fed DDC. To further provide clinical evidence as to how ubiquitin-like protein (Ubls) modification, gene transcript expression in Ufmylation and FATylation were investigated in human archived formalin-fixed, paraffin-embedded (FFPE) liver biopsies and frozen liver sections from DDC re-fed mice were used. Real-time PCR analysis showed that all Ufmylation molecules (Ufm1, Uba5, Ufc1, Ufl1 and UfSPs) were significantly downregulated, both in DDC re-fed mice livers and patients' livers where MDBs had formed, indicating that gene transcript changes were limited to MDB-forming livers where the protein quality control system was downregulated. FAT10 and subunits of the immunoproteasome (LMP2 and LMP7) were both upregulated as previously shown. An approximate 176- and 5-fold upregulation (respectively) of FAT10 was observed in the DDC re-fed mice liver and in the livers of human alcoholic hepatitis with MDBs present, implying that there was an important role played by this gene. The FAT10-specific E1 and E2 enzymes Uba6 and USE1, however, were found to be downregulated both in patients' livers and in the liver of DDC re-fed mice. Interestedly, the downregulation of mRNA levels was proportionate to MDB abundance in the liver tissues. Our results show the first systematic demonstration of transcript regulation of Ufmylation and FATylation in the liver of patients who form MDBs, where protein quality control is downregulated. This was also shown in the livers of DDC re-fed mice where MDBs had formed.

Transition between acute and chronic hepatotoxicity in mice is associated with impaired energy metabolism and induction of mitochondrial heme oxygenase-1.

The formation of protein inclusions is frequently associated with chronic metabolic diseases. In mice, short-term intoxication with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) leads to hepatocellular damage indicated by elevated serum liver enzyme activities, whereas only minor morphological changes are observed. Conversely, chronic administration of DDC for several weeks results in severe morphological damage, characterized by hepatocellular ballooning, disruption of the intermediate filament cytoskeleton, and formation of Mallory-Denk bodies consisting predominantly of misfolded keratins, Sqstm1/p62, and heat shock proteins. To evaluate the mechanistic underpinnings for this dichotomy we dissected the time-course of DDC intoxication for up to 10 weeks. We determined body weight change, serum liver enzyme activities, morphologic alterations, induction of antioxidant response (heme oxygenase-1, HO-1), oxidative damage and ATP content in livers as well as respiration, oxidative damage and the presence and activity of HO-1 in endoplasmic reticulum and mitochondria (mtHO-1). Elevated serum liver enzyme activity and oxidative liver damage were already present at early intoxication stages without further subsequent increase. After 2 weeks of intoxication, mice had transiently lost 9% of their body weight, liver ATP-content was reduced to 58% of controls, succinate-driven respiration was uncoupled from ATP-production and antioxidant response was associated with the appearance of catalytically active mtHO-1. Oxidative damage was associated with both acute and chronic DDC toxicity whereas the onset of chronic intoxication was specifically associated with mitochondrial dysfunction which was maximal after 2 weeks of intoxication. At this transition stage, adaptive responses involving mtHO-1 were induced, indirectly leading to improved respiration and preventing further drop of ATP levels. Our observations clearly demonstrate principally different mechanisms for acute and chronic toxic damage.

Mallory-Denk bodies form when EZH2/H3K27me3 fails to methylate DNA in the nuclei of human and mice liver cells.

EZH2/H3K27me3 and polycomb group complex (PcG) play a major role in regulating global gene expression including tumor suppressor genes. EZH2 is linked to cell cycle regulated EZH2 phosphorylation by CDK1, a mitotic kinase which increases in arrested mitosis compared to S phase. CDK1 phosphorylation of EZH2 accelerates the degradation of pEZH2. Phospho-EZH2 is subjected to ubiquitination. The half-like of pEZH2 is shorter when compared to total EZH2. In the present study, pEZH2 was found concentrated together with ubiquitin in the Mallory-Denk bodies (MDB) that were formed in hepatocytes in the livers of drug primed mice refed DDC and humans with alcoholic hepatitis or hepatocellular carcinoma. The cells that formed MDBs in the mice livers studied were associated with a growth advantage and a high proliferative index. However, the livers from patients with alcoholic hepatitis showed evidence of cell cycle arrest where PCNA, cyclin D1 and p27 positive nuclei were numerous but Ki-67 positive nuclei were scarce. It is concluded that MDB formation is linked to the cell cycle and global gene expression (i.e. loss of gene silencing) through its association with the regulation of the polycomb group PRC2/EZH2/H3K27me3 complex.

Genetic background effects of keratin 8 and 18 in a DDC-induced hepatotoxicity and Mallory-Denk body formation mouse model.

Keratin 8 (K8) and keratin 18 (K18) form the major hepatocyte cytoskeleton. We investigated the impact of genetic loss of either K8 or K18 on liver homeostasis under toxic stress with the hypothesis that K8 and K18 exert different functions. krt8⁻/⁻ and krt18⁻/⁻ mice crossed into the same 129-ola genetic background were treated by acute and chronic administration of 3,5-diethoxy-carbonyl-1,4-dihydrocollidine (DDC). In acutely DDC-intoxicated mice, macrovesicular steatosis was more pronounced in krt8⁻/⁻ and krt18⁻/⁻ compared with wild-type (wt) animals. Mallory-Denk bodies (MDBs) appeared in krt18⁻/⁻ mice already at an early stage of intoxication in contrast to krt8⁻/⁻ mice that did not display MDB formation when fed with DDC. Keratin-deficient mice displayed significantly lower numbers of apoptotic hepatocytes than wt animals. krt8⁻/⁻, krt18⁻/⁻ and control mice displayed comparable cell proliferation rates. Chronically DDC-intoxicated krt18⁻/⁻ and wt mice showed a similarly increased degree of steatohepatitis with hepatocyte ballooning and MDB formation. In krt8⁻/⁻ mice, steatosis was less, ballooning, and MDBs were absent. krt18⁻/⁻ mice developed MDBs whereas krt8⁻/⁻ mice on the same genetic background did not, highlighting the significance of different structural properties of keratins. They are independent of the genetic background as an intrinsic factor. By contrast, toxicity effects may depend on the genetic background. krt8⁻/⁻ and krt18⁻/⁻ mice on the same genetic background show similar sensitivity to DDC intoxication and almost resemble wt animals regarding survival, degree of porphyria, liver-to-body weight ratio, serum bilirubin and liver enzyme levels. This stands in contrast to previous work where krt8⁻/⁻ and krt18⁻/⁻ mice on different genetic backgrounds were investigated.

Changes in IL12A methylation pattern in livers from mice fed DDC.

Mallory-Denk body (MDB) formation is a component of alcoholic and non alcoholic hepatitis. Proteins of the TLR pathway were shown to be involved in the formation of MDBs, in mice fed DDC. TLR genes are upregulated and SAMe supplementation prevents this up regulation and prevented the formation of MDBs. DNA of livers from control mice, from mice fed DDC 10weeks, refed 1week with DDC and with DDC+SAMe were extracted and used to study the methylation pattern of genes involves in the TLR pathway. A PCR array was used to analyze it. Using PCR arrays for the mouse TLR pathway,24 genes were found whose expression of IL12A was regulated by the methylation of its gene. DDC fed for 10weeks reduced the methylation of the IL12A gene expression. This expression was also reduced when DDC was refed. However, when SAMe was fed, the intermediate level methylation of IL12A was up regulated to the intermediate level and the methylation of the promoter decreased compared to DDC refeeding or DDC 10weeks. IL12A is known to induce the production of IFNg by NK and L(T). We showed in a previous publication that IFNg is one of the major cytokines involved in the induction of MDB formation. The low expression of IL12A associated with the intermediate methylation of its promoter could explain one step in the mechanism which leads to the formation of MDBs.