The liver is one of the most sexually dimorphic organs. The hepatic metabolic pathways that are subject to sexual dimorphism include xenobiotic, amino acid and lipid metabolism. Non-alcoholic fatty liver disease and hepatocellular carcinoma are among diseases with sex-dependent prevalence, progression and outcome. Although male and female livers differ in their abilities to metabolize foreign compounds, including drugs, sex-dependent treatment and pharmacological dynamics are rarely applied in all relevant cases. Therefore, it is important to consider hepatic sexual dimorphism when developing new treatment strategies and to understand the underlying mechanisms in model systems. We isolated primary hepatocytes from male and female C57BL6/N mice and examined the sex-dependent transcriptome, proteome and extracellular metabolome parameters in the course of culturing them for 96 h. The sex-specific gene expression of the general xenobiotic pathway altered and the female-specific expression of Cyp2b13 and Cyp2b9 was significantly reduced during culture. Sex-dependent differences of several signaling pathways increased, including genes related to serotonin and melatonin degradation. Furthermore, the ratios of male and female gene expression were inversed for other pathways, such as amino acid degradation, beta-oxidation, androgen signaling and hepatic steatosis. Because the primary hepatocytes were cultivated without the influence of known regulators of sexual dimorphism, these results suggest currently unknown modulatory mechanisms of sexual dimorphism in vitro. The large sex-dependent differences in the regulation and dynamics of drug metabolism observed during cultivation can have an immense influence on the evaluation of pharmacodynamic processes when conducting initial preclinical trials to investigate potential new drugs.

The Hedgehog (Hh) and Wnt/β-Catenin (Wnt) cascades are morphogen pathways whose pronounced influence on adult liver metabolism has been identified in recent years. How both pathways communicate and control liver metabolic functions are largely unknown. Detecting core components of Wnt and Hh signaling and mathematical modeling showed that both pathways in healthy liver act largely complementary to each other in the pericentral (Wnt) and the periportal zone (Hh) and communicate mainly by mutual repression. The Wnt/Hh module inversely controls the spatiotemporal operation of various liver metabolic pathways, as revealed by transcriptome, proteome, and metabolome analyses. Shifting the balance to Wnt (activation) or Hh (inhibition) causes pericentralization and periportalization of liver functions, respectively. Thus, homeostasis of the Wnt/Hh module is essential for maintaining proper liver metabolism and to avoid the development of certain metabolic diseases. With caution due to minor species-specific differences, these conclusions may hold for human liver as well.

In the liver, energy homeostasis is mainly regulated by mechanistic target of rapamycin (mTOR) signalling, which influences relevant metabolic pathways, including lipid metabolism. However, the Hedgehog (Hh) pathway is one of the newly identified drivers of hepatic lipid metabolism. Although the link between mTOR and Hh signalling was previously demonstrated in cancer development and progression, knowledge of their molecular crosstalk in healthy liver is lacking. To close this information gap, we used a transgenic mouse model, which allows hepatocyte-specific deletion of the Hh pathway, and in vitro studies to reveal interactions between Hh and mTOR signalling. The study was conducted in male and female mice to investigate sexual differences in the crosstalk of these signalling pathways. Our results reveal that the conditional Hh knockout reduces mitochondrial adenosine triphosphate (ATP) production in primary hepatocytes from female mice and inhibits autophagy in hepatocytes from both sexes. Furthermore, in vitro studies show a synergistic effect of cyclopamine and rapamycin on the inhibition of mTor signalling and oxidative respiration in primary hepatocytes from male and female C57BL/6N mice. Overall, our results demonstrate that the impairment of Hh signalling influences mTOR signalling and therefore represses oxidative phosphorylation and autophagy.

The Hedgehog signaling pathway regulates many processes during embryogenesis and the homeostasis of adult organs. Recent data suggest that central metabolic processes and signaling cascades in the liver are controlled by the Hedgehog pathway and that changes in hepatic Hedgehog activity also affect peripheral tissues, such as the reproductive organs in females. Here, we show that hepatocyte-specific deletion of the Hedgehog pathway is associated with the dramatic expansion of adipose tissue in mice, the overall phenotype of which does not correspond to the classical outcome of insulin resistance-associated diabetes type 2 obesity. Rather, we show that alterations in the Hedgehog signaling pathway in the liver lead to a metabolic phenotype that is resembling metabolically healthy obesity. Mechanistically, we identified an indirect influence on the hepatic secretion of the fibroblast growth factor 21, which is regulated by a series of signaling cascades that are directly transcriptionally linked to the activity of the Hedgehog transcription factor GLI1. The results of this study impressively show that the metabolic balance of the entire organism is maintained via the activity of morphogenic signaling pathways, such as the Hedgehog cascade. Obviously, several pathways are orchestrated to facilitate liver metabolic status to peripheral organs, such as adipose tissue.

The Hedgehog (Hh) signaling pathway is essential for maintaining homeostasis during embryogenesis and in adult tissues. In the liver, dysregulation of this pathway often leads to liver cancer development. Recent studies also suggest that disturbances in the Hh pathway can affect liver metabolism in healthy livers through interactions with other signaling pathways, such as the Wnt/β-catenin pathway. As a result, the Hh pathway has emerged as a promising target for therapeutic intervention. However, little is known about the effects of Hh modulators on healthy hepatocytes. In our study, we investigated the effects of the Hh agonists SAG (300 nM) and triamcinolone acetonide (40 µM), as well as the antagonists RU-SKI 43 (100 nM), cyclopamine (5 µM), budesonide (25 µM), GANT61 (0.5 µM), and vismodegib (1 µM) on healthy mouse and human primary hepatocytes in vitro. We employed toxicological, transcriptomic, proteomic, and functional assays, including proliferation and Seahorse assays. Our results show that these compounds significantly impact metabolic pathways such as lipid and glucose metabolism at both transcriptional and protein levels. Mechanistically, our data suggest the involvement of both canonical and non-canonical Hedgehog pathways, a phenomenon not previously described in hepatocytes. These findings highlight the diverse effects of these compounds on signaling and key metabolic functions in the liver, which emphasizes the need to investigate the hepatic Hh cascade and its metabolic control in depth. As the compounds regulate different aspects of metabolism, they need to be carefully studied in appropriate model systems for specific therapeutic use.

The Hedgehog (Hh) pathway is a morphogenic pathway that plays an important role not only in tissue differentiation and embryonic development. It also regulates the homeostasis in adult tissues, despite a decreased activity compared to embryonic tissue. The Hh pathway interacts with many other important pathways which regulate metabolism. Studies of Matz-Soja et al. show that one of these pathways is the mechanistic target of rapamycin (mTor). This serine/threonine protein kinase forms two different complexes mTORC1 and mTORC2. Whereas mTORC1 regulates autophagy, cell growth and cell proliferation, mTORC2 is responsible for cell survival and the reorganization of the cytoskeleton. In our studies, we examine the interactions of Hh and mTor signaling and their impact on liver metabolism. Various hepatocyte specific knockout mouse models with activated and inactivated Hh signaling have been developed to examine the crosstalk of Hh and mTor signaling in liver metabolism. The repression of the Hh pathway was achieved by the deletion of Smoothened (Smo), an activating receptor in the Hh signaling cascade. The activation of Hh was carried out through the deletion of the receptor protein Patched 1 (Ptch1), which is a repressing receptor in the Hh signaling cascade. For further studies on the direct impact of the inhibition of the two pathways we incubated C57BL6N cells with Cyclopamine and Rapamycin, respectively. In addition, Seahorse technology was used to reveal changes in glycolytic and mitochondrial metabolism. Furthermore, western Blotting, qPCR and immunohistochemistry were used for additional analyses of primary hepatocytes from all sources. Our experiments show the repression of mTor and the mTORC2 component Rictor in primary male hepatocytes with inactivated Hh pathway. The mTORC1 complex seems not to be affected. Consistently with this, hepatocytes with activated Hh Signaling show an increase in the phosphorylation of mTor and an altered parenchymal distribution of the phosphorylated proteins from pan-lobular to pericentral. However, the mRNA levels of mTORC1 complex molecules such as mTor, mlst8 and Raptor are increased in hepatocytes from mice with repressed Hh signaling in the hepatocytes. In contrast to primary male hepatocytes, primary female hepatocytes from Smo Knockout mice show changes in mitochondrial function such as increased proton leak and decreased ATP production. The latter is consistent with primary male hepatocytes from C57BL6N mice treated with Cyclopamine. These cells show also an increased non-mitochondrial oxygen consumption as well as decreased glycolytic and respiratory activity. Furthermore, Cyclopamine and Rapamycin seem to have a synergistic effect in the repression of mTor signaling on primary male hepatocytes from C57BL6N mice. Our results suggest an interaction between Hh and mTor signaling, which influences the energy metabolism of cultured primary hepatocytes with distinct effects on male and female metabolism.