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.
