Investigations on IMP2-2 and Kupffer cells in steatohepatitis

Alcoholic and non-alcoholic steatohepatitis (ASH and NASH) represent risk factors for the development of hepatocellular carcinoma. The prevalence of ASH and NASH is strongly increasing worldwide. Within this work, different mechanisms responsible for steatohepatitis disease progression were elucidated in murine models. The insulin-like growth factor 2 (IGF2) mRNA binding protein (IMP) p62/IMP2-2 was shown to promote progenitor or dedifferentiated cell populations in a model of NASH and thereby amplify fibrosis. In diet-induced steatohepatitis, epigenetic alterations strongly affected genetic regions playing a role in lipid metabolism and liver morphology. Depletion of Kupffer cells, the resident macrophages of the liver, induced liver damage and attenuated hepatic accumulation of storage lipids, while hepatotoxic lipids were incorporated. Taken together, this work provides evidence that p62 promotes the appearance of undifferentiated or dedifferentiated cells and thereby disease progression and furthermore that macrophages are crucial in hepatic lipid homeostasis and protection of lipotoxicity.Die alkoholische und nicht-alkoholische Steatohepatitis (ASH und NASH) stellen Risikofaktoren für die Entwicklung eines hepatozellulären Karzinoms dar. Die Verbreitung von ASH und NASH nimmt weltweit stark zu. Innerhalb dieser Arbeit wurden verschiedene Mechanismen in Mausmodellen aufgeklärt, die für das Fortschreiten der Steatohepatitis Erkrankung verantwortlich sind. Es wurde gezeigt, dass das Insulin-ähnliche Wachstumsfaktor 2 (IGF2) mRNA bindende Protein (IMP) p62/IMP2-2 das Auftreten hepatischer Progenitorzellen oder dedifferenzierter Zellpopulationen in einem NASH Modell fördert und dadurch eine Fibrose begünstigt. In Diät-induzierter Steatohepatitis beeinflussten epigenetische Veränderungen genetische Regionen, die eine Rolle im Lipidstoffwechsel und in der Morphologie der Leber spielen. Die Depletion von Kupffer-Zellen, den Makrophagen der Leber, rief Leberschäden hervor und verringerte die Menge an Speicherlipiden in der Leber, während hepatotoxische Lipide eingelagert wurden. Zusammengefasst bietet diese Arbeit Hinweise darauf, dass p62 das Auftreten von undifferenzierten oder dedifferenzierten Zellen und damit den Krankheitsverlauf fördert und dass Makrophagen von entscheidender Bedeutung in der Lipidhomöostase der Leber und im Schutz vor Lipotoxizität sind

Kupffer cells are protective in alcoholic steatosis

Massive accumulation of lipids is a characteristic of alcoholic liver disease. Excess of hepatic fat activates Kupffer cells (KCs), which affect disease progression. Yet, KCs contribute to the resolution and advancement of liver injury. Aim of the present study was to evaluate the effect of KC depletion on markers of liver injury and the hepatic lipidome in liver steatosis (Lieber-DeCarli diet, LDC, female mice, mixed C57BL/6J and DBA/2J background). LDC increased the number of dead hepatocytes without changing the mRNA levels of inflammatory cytokines in the liver. Animals fed LDC accumulated elevated levels of almost all lipid classes. KC ablation normalized phosphatidylcholine and phosphatidylinositol levels in LDC livers, but had no effect in the controls. A modest decline of trigylceride and diglyceride levels upon KC loss was observed in both groups. Serum aminotransferases and hepatic ceramide were elevated in all animals upon KC depletion, and in particular, cytotoxic very long-chain ceramides increased in the LDC livers. Meta-biclustering revealed that eight lipid species occurred in more than 40% of the biclusters, and four of them were very long-chain ceramides. KC loss was further associated with excess free cholesterol levels in LDC livers. Expression of inflammatory cytokines did, however, not increase in parallel. In summary, the current study described a function of KCs in hepatic ceramide and cholesterol metabolism in an animal model of LDC liver steatosis. High abundance of cytotoxic ceramides and free cholesterol predispose the liver to disease progression suggesting a protective role of KCs in alcoholic liver diseases

IGF2 mRNA Binding Protein 2 Transgenic Mice Are More Prone to Develop a Ductular Reaction and to Progress Toward Cirrhosis

The insulin-like growth factor 2 (IGF2) mRNA binding proteins (IMPs/IGF2BPs) IMP1 and 3 are regarded as oncofetal proteins, whereas the hepatic IMP2 expression in adults is controversially discussed. The splice variant IMP2-2/p62 promotes steatohepatitis and hepatocellular carcinoma. Aim of this study was to clarify whether IMP2 is expressed in the adult liver and influences progression toward cirrhosis. IMP2 was expressed at higher levels in embryonic compared to adult tissues as quantified in embryonic, newborn, and adult C57BL/6J mouse livers and suggested by analysis of publicly available human data. In an IMP2-2 transgenic mouse model microarray and qPCR analyses revealed increased expression of liver progenitor cell (LPC) markers Bex1, Prom1, Spp1, and Cdh1 indicating a de-differentiated liver cell phenotype. Induction of these LPC markers was confirmed in human cirrhotic tissue datasets. The LPC marker SPP1 has been described to play a major role in fibrogenesis. Thus, DNA methylation was investigated in order to decipher the regulatory mechanism of Spp1 induction. In IMP2-2 transgenic mouse livers single CpG sites were differentially methylated, as quantified by amplicon sequencing, whereas human HCC samples of a human publicly available dataset showed promoter hypomethylation. In order to study the impact of IMP2 on fibrogenesis in the context of steatohepatitis wild-type or IMP2-2 transgenic mice were fed either a methionine-choline deficient (MCD) or a control diet for 2-12 weeks. MCD-fed IMP2-2 transgenic mice showed a higher incidence of ductular reaction (DR), accompanied by hepatic stellate cell activation, extracellular matrix (ECM) deposition, and induction of the LPC markers Spp1, Cdh1, and Afp suggesting the occurrence of de-differentiated cells in transgenic livers. In human cirrhotic samples IMP2 overexpression correlated with LPC marker and ECM component expression. Progression of liver disease was induced by combined MCD and diethylnitrosamine (DEN) treatment. Combined MCD-DEN treatment resulted in shorter survival of IMP2-2 transgenic compared to wild-type mice. Only IMP2-2 transgenic livers progressed to cirrhosis, which was accompanied by strong DR. In conclusion, IMP2 is an oncofetal protein in the liver that promotes DR characterized by de-differentiated cells toward steatohepatitis-associated cirrhosis development with poor survival

Susceptibility of Different Mouse Wild Type Strains to Develop Diet-Induced NAFLD/AFLD-Associated Liver Disease.

Although non-alcoholic and alcoholic fatty liver disease have been intensively studied, concerning pathophysiological mechanisms are still incompletely understood. This may be due to the use of different animal models and resulting model-associated variation. Therefore, this study aimed to compare three frequently used wild type mouse strains in their susceptibility to develop diet-induced features of non-alcoholic/alcoholic fatty liver disease. Fatty liver disease associated clinical, biochemical, and histological features in C57BL/6, CD-1, and 129Sv WT mice were induced by (i) high-fat diet feeding, (ii) ethanol feeding only, and (iii) the combination of high-fat diet and ethanol feeding. Hepatic and subcutaneous adipose lipid profiles were compared in CD-1 and 129Sv mice. Additionally hepatic fatty acid composition was determined in 129Sv mice. In C57BL/6 mice dietary regimens resulted in heterogeneous hepatic responses, ranging from pronounced steatosis and inflammation to a lack of any features of fatty liver disease. Liver-related serum biochemistry showed high deviations within the regimen groups. CD-1 mice did not exhibit significant changes in metabolic and liver markers and developed no significant steatosis or inflammation as a response to dietary regimens. Although 129Sv mice showed no weight gain, this strain achieved most consistent features of fatty liver disease, apparent from concentration alterations of liver-related serum biochemistry as well as moderate steatosis and inflammation as a result of all dietary regimens. Furthermore, the hepatic lipid profile as well as the fatty acid composition of 129Sv mice were considerably altered, upon feeding the different dietary regimens. Accordingly, diet-induced non-alcoholic/alcoholic fatty liver disease is most consistently promoted in 129Sv mice compared to C57BL/6 and CD-1 mice. As a conclusion, this study demonstrates the importance of genetic background of used mouse strains for modeling diet-induced non-alcoholic/alcoholic fatty liver disease

Kupffer cells are protective in alcoholic steatosis

Massive accumulation of lipids is a characteristic of alcoholic liver disease. Excess of hepatic fat activates Kupffer cells (KCs), which affect disease progression. Yet, KCs contribute to the resolution and advancement of liver injury. Aim of the present study was to evaluate the effect of KC depletion on markers of liver injury and the hepatic lipidome in liver steatosis (Lieber-DeCarli diet, LDC, female mice, mixed C57BL/6J and DBA/2J background). LDC increased the number of dead hepatocytes without changing the mRNA levels of inflammatory cytokines in the liver. Animals fed LDC accumulated elevated levels of almost all lipid classes. KC ablation normalized phosphatidylcholine and phosphatidylinositol levels in LDC livers, but had no effect in the controls. A modest decline of trigylceride and diglyceride levels upon KC loss was observed in both groups. Serum aminotransferases and hepatic ceramide were elevated in all animals upon KC depletion, and in particular, cytotoxic very long-chain ceramides increased in the LDC livers. Meta-biclustering revealed that eight lipid species occurred in more than 40% of the biclusters, and four of them were very long-chain ceramides. KC loss was further associated with excess free cholesterol levels in LDC livers. Expression of inflammatory cytokines did, however, not increase in parallel. In summary, the current study described a function of KCs in hepatic ceramide and cholesterol metabolism in an animal model of LDC liver steatosis. High abundance of cytotoxic ceramides and free cholesterol predispose the liver to disease progression suggesting a protective role of KCs in alcoholic liver diseases

Determination of hepatic lipid profile of regimen treated CD-1 and 129Sv mice.

<p>Hepatic lipid content was determined out of livers from regimen fed CD-1 and 129Sv mice by a semi-quantitative chromatographic method and is shown as relative amount compared to untreated counterparts. (A) TG levels were increased in both mouse strains, with elevated hepatic TG storage in 129Sv mice compared to CD-1 mice. (B) Free cholesterol was only altered in some of the regimen treated 129Sv mice, including decreased free cholesterol in feeding group EtOH 14 weeks and increased free cholesterol in HF + EtOH fed for 7 and 9 weeks. (C) CE as well as (D) CER levels were increased in all feeding groups of either strain, with higher CE content in 129Sv mice compared to CD-1 mice. (E) PC levels were not altered in CD-1 mice, while HF feeding for 7 weeks caused elevated PC content and HF + EtOH feeding for 7 weeks caused decreased PC levels. (F) PE content was similar in both strains, with no alterations between untreated and regimen treated mice. Values are given as means with standard deviations and relevant significant differences are marked by an asterisk (Mann-Whitney U test, <i>P</i> values of <0.05).</p

FA composition of regimen treated 129Sv mice livers.

<p>FA were determined out of livers from regimen fed 129Sv mice by GC-MS. (A) Total hepatic cholesterol was significantly increased by feeding EtOH for 12 and 16 weeks. (B) Sum of FA was elevated in mice fed with EtOH for 16 weeks as well as in mice fed with HF + EtOH for 7 and 9 weeks. (C) Saturated FA and (D) unsaturated FA were increased in 129Sv mice fed with EtOH for 14 and 16 weeks. (E) MUFA elevated due to feeding EtOH for 16 weeks and HF + EtOH for 7 and 9 weeks respectively. (F) PUFA were only increased in 129Sv mice either receiving HF or HF + EtOH. (G) Ratios of saturated to unsaturated and (H) C18 to C16 FA were not affected by dietary regimens, while (I) ratio of n-6 to n-3 FA was increased due to HF and HF + EtOH feeding at all time points. Shown are medians with interquartile ranges and relevant significant differences are marked by an asterisk (Kruskal-Wallis followed by Dunns test of selected pairs of columns, <i>P</i> values of <0.05).</p

Medians and interquartile ranges of histopathological features of steatosis, ballooning, and inflammation as well as NAS of regimen treated C57BL/6, CD-1, and 129Sv mice.

<p>Medians and interquartile ranges of histopathological features of steatosis, ballooning, and inflammation as well as NAS of regimen treated C57BL/6, CD-1, and 129Sv mice.</p

Measurement of inflammation markers IL-6, TNFα, and MCP-1.

<p>Inflammation markers were determined out of liver homogenates of all livers of the respective mouse strains and dietary groups. IL-6, TNFα, and MCP-1 levels of (A) C57BL/6 mice, (B) CD-1 mice, and (C) 129Sv mice, indicating inflammatory response in C57BL/6 and 129Sv mice by decreased hepatic IL-6 and increased hepatic TNFα and MCP-1 concentrations due to regimen feeding. Given are medians with interquartile ranges and relevant significant differences are marked by an asterisk (Kruskal-Wallis followed by Dunns test of selected pairs of columns, <i>P</i> values of <0.05).</p

Weight increase and adipose to body weight ratio of different dietary groups and mouse strains.

<p>(A-C) Weight increase of HF, EtOH, and HF + EtOH groups and different mouse strains was monitored twice a week, resulting in regimen induced weight increase in C57BL/6 and CD-1 but not in 129Sv mice. (D-F) Adipose to body weight ratios of respective strains and dietary groups, showing increased subcutaneous as well as total adipose to body weight ratio in C57BL/6 and 129Sv mice, while CD-1 mice exhibited only minor changes due to HF, EtOH, and HF + EtOH feeding. At the end of each experiment, subcutaneous as well as visceral adipose tissue was dissected, weighted and subcutaneous adipose, visceral adipose, and total adipose to body weight ratios were calculated. Shown are medians with interquartile ranges and relevant significant differences are marked by an asterisk (Kruskal-Wallis followed by Dunns test of selected pairs of columns, <i>P</i> values of <0.05).</p