The interaction of morphogenic pathways in hepatic metabolism under consideration of liver dimorphism

The liver is a multifunctional organ that regulates important processes such as lipid, glucose and xenobiotic metabolism. Most of the hepatic functions are implemented in the hepatocytes, which make up the largest cellular proportion of the liver. Various signaling pathways are required for the liver to perform its diverse metabolic tasks, two of them are the Hedgehog (HH) and mechanistic target of rapamycin (mTOR) signaling pathways. Dysregulation of these signaling pathways leads to various liver diseases, such as non-alcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC). In order to develop appropriate treatments for these disorders, it is important to understand the molecular interactions of the various signaling pathways involved in their development and progression. However, another aspect is particularly important in the treatment of hepatic diseases - the sexual dimorphism of the liver. A great variety of genes are differentially expressed in the liver of males and females. Signaling pathways affected by hepatic sexual dimorphism include lipid and drug metabolism. Although the influence of sex-specific dimorphism in the liver on diseases and drug doses is well known, there are currently only a few sex-specific treatment concepts. In order to incorporate this aspect in the development of new and more effective treatments, it is important to understand the molecular mechanism and characteristics of sexual dimorphism in model systems. Primary hepatocytes are often used for in vitro analysis of pharmaceutical studies. However, little is known about the development of sexual dimorphism during the culture of primary cells. To facilitate the development of novel sex-specific treatment approaches, the present thesis investigated the interaction of the HH and mTOR signaling pathways in hepatocytes, as well as the development of sexual dimorphism during the culture of primary hepatocytes for up to 96 h. Male and female mice with a hepatocyte-specific knock-out (KO) of the HH signaling were used to examine a possible interaction of HH and mTOR signaling. The repression of the HH signaling cascade leads to reduced mRNA expression of molecules involved in mTOR Signaling as well as decreased mitochondrial adenosine triphosphate (ATP) production in hepatocytes from female KO mice. In contrast, hepatocytes from male KO mice showed reduced phosphorylation of mTOR molecules. Furthermore, an impaired autophagy was detected in primary hepatocytes from both sexes. Consequently, the hepatocyte-specific HH-KO leads to decreased mTOR activity in males and females. However, the KO shows different points of action depending on the sex of the mice. To investigate the crosstalk of the two pathways in more detail, primary hepatocytes from male and female C57BL/6N mice were incubated with the HH inhibitor cyclopamine and the mTOR inhibitors rapamycin and Torin. Thereby, a synergistic effect of cyclopamine and rapamycin could be demonstrated on the repression of the mTOR signaling cascade and mitochondrial ATP production. Although primary hepatocytes have been isolated from male and female mice for all experiments, the further development of the sexual dimorphism of the hepatocytes in monolayer-cultivation is unclear. Therefore, cells from male and female C57BL/6N mice were isolated to conduct transcriptome, proteome and metabolome studies directly after isolation and after 24, 48, 72 and 96 h of cultivation. It was striking that the expression pattern of sexually differentiated genes developed differently during cell culture compared to the translation pattern of sexually differentiated proteins. While mRNA expression showed a large shift after 24 h, there was a feminization of the translation pattern at the protein level throughout the cultivation period. Furthermore, the study revealed a change in the sex-specific gene expression of xenobiotic metabolism with a significant decrease of female-specific expression of Cytochrome P450 (Cyp) 2b13 and Cyp2b9. Since no sex hormones or growth factors were added to the cell culture, a reduction in sex differences during the time of cell culture was to be expected. Instead, we found an increase in sex-specific gene expression for some signaling pathways, such as serotonin and melatonin degradation. For the expression of genes related to androgen signaling, beta-oxidation, hepatic steatosis and amino acid degradation, even a reversal of the sex-specific gene expression could be shown. In conclusion, this investigation reveals a connection between HH and mTOR signaling in hepatocytes. The repression of the HH pathway modulates mTOR signaling and results in impaired oxidative phosphorylation and autophagy. It is conceivable that the synergistic effect of HH and mTOR inhibition could be applied in targeted therapies. Furthermore, the culture of male and female primary hepatocytes demonstrate that sex differences change significantly over time. This should be considered when using primary hepatocytes for initial preclinical studies to analyze potential new drugs. Especially, the alterations in xenobiotic metabolism can have serious effects on pharmacodynamic processes. In addition, the enhancement or reversal of sex-specific expression of some genes in vitro suggests that there must be previously unknown mechanisms that modulate sexual dimorphism independently of the steroids and growth hormones known to date.:Table of Contents 1. Introduction 1.1 Characteristics of the liver 1.2 The hepatic sexual dimorphism 1.3 Interaction of signaling pathways in the liver 1.3.1 The Hedgehog pathway 1.3.2 The mTOR pathway 1.3.3 The role of HH and mTOR in diseases 1.4 Aim of the study 2. Publications 2.1 Cyclopamine and rapamycin synergistically inhibit mTOR signaling in mouse hepatocytes, revealing an interaction of hedgehog and mtor signaling in the liver 2.2 Sex-dependent dynamics of metabolism in primary mouse hepatocytes 33 3. Summary of the thesis 4. References 5. Attachments 5.1 List of abbreviations 5.2 Supplemental Material 5.2.1 Cyclopamine and rapamycin synergistically inhibit mTOR signaling in mouse hepatocytes, revealing an interaction of hedgehog and mtor signaling in the liver 5.2.2 Sex‑dependent dynamics of metabolism in primary mouse hepatocytes 5.3 Description of the author's own contribution 5.3.1 Cyclopamine and rapamycin synergistically inhibit mTOR signaling in mouse hepatocytes, revealing an interaction of hedgehog and mtor signaling in the liver 5.3.2 Sex‑dependent dynamics of metabolism in primary mouse hepatocytes 5.4 Statement of Authorship 5.5 Curriculum vitae 5.6 Publications 5.7 Acknowledgemen