Eugenia Marbach-Breitrück1, Madlen Matz-Soja2, Ute Abraham3, Wolfgang Schmidt-Heck4, Susanne Sales5, Christiane Rennert6, Matthias Kern7, Susanne Aleithe8, Luise Spormann9, Carlo Thiel9, Raffaele Gerlini10, Katrin Arnold9, Nora Klöting7, Reinhard Guthke4, Damjana Rozman11, Raffaele Teperino12, Andrej Shevchenko5, Achim Kramer3, Rolf Gebhardt13. 1. Rudolf-Schönheimer-Institute of Biochemistry, Faculty of Medicine, Leipzig University, Leipzig, Germany; Institute of Biochemistry, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Germany. 2. Rudolf-Schönheimer-Institute of Biochemistry, Faculty of Medicine, Leipzig University, Leipzig, Germany. Electronic address: madlen.matz@medizin.uni-leipzig.de. 3. Laboratory of Chronobiology, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Germany. 4. Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute, Jena, Germany. 5. Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany. 6. Rudolf-Schönheimer-Institute of Biochemistry, Faculty of Medicine, Leipzig University, Leipzig, Germany; Department of Hepatobiliary Surgery and Visceral Transplantation, University Hospital, Leipzig University, Leipzig, Germany. 7. IFB Adiposity Diseases, University of Leipzig, Leipzig, Germany. 8. Rudolf-Schönheimer-Institute of Biochemistry, Faculty of Medicine, Leipzig University, Leipzig, Germany; Clinic and Polyclinic of Neurology, Faculty of Medicine, Leipzig University, Germany. 9. Rudolf-Schönheimer-Institute of Biochemistry, Faculty of Medicine, Leipzig University, Leipzig, Germany. 10. Institute of Experimental Genetics (IEG), HDC, Neuherberg, Germany. 11. Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Slovenia. 12. Institute of Experimental Genetics (IEG), HDC, Neuherberg, Germany; DZD, German Center for Diabetes Research, Neuherberg, Germany. 13. Rudolf-Schönheimer-Institute of Biochemistry, Faculty of Medicine, Leipzig University, Leipzig, Germany. Electronic address: rgebhardt@medizin.uni-leipzig.de.
Abstract
BACKGROUND & AIMS: The mammalian circadian clock controls various aspects of liver metabolism and integrates nutritional signals. Recently, we described Hedgehog (Hh) signaling as a novel regulator of liver lipid metabolism. Herein, we investigated crosstalk between hepatic Hh signaling and circadian rhythm. METHODS: Diurnal rhythms of Hh signaling were investigated in liver and hepatocytes from mice with ablation of Smoothened (SAC-KO) and crossbreeds with PER2::LUC reporter mice. By using genome-wide screening, qPCR, immunostaining, ELISA and RNAi experiments in vitro we identified relevant transcriptional regulatory steps. Shotgun lipidomics and metabolic cages were used for analysis of metabolic alterations and behavior. RESULTS: Hh signaling showed diurnal oscillations in liver and hepatocytes in vitro. Correspondingly, the level of Indian Hh, oscillated in serum. Depletion of the clock gene Bmal1 in hepatocytes resulted in significant alterations in the expression of Hh genes. Conversely, SAC-KO mice showed altered expression of clock genes, confirmed by RNAi against Gli1 and Gli3. Genome-wide screening revealed that SAC-KO hepatocytes showed time-dependent alterations in various genes, particularly those associated with lipid metabolism. The clock/hedgehog module further plays a role in rhythmicity of steatosis, and in the response of the liver to a high-fat diet or to differently timed starvation. CONCLUSIONS: For the first time, Hh signaling in hepatocytes was found to be time-of-day dependent and to feed back on the circadian clock. Our findings suggest an integrative role of Hh signaling, mediated mainly by GLI factors, in maintaining homeostasis of hepatic lipid metabolism by balancing the circadian clock. LAY SUMMARY: The results of our investigation show for the first time that the Hh signaling in hepatocytes is time-of-day dependent, leading to differences not only in transcript levels but also in the amount of Hh ligands in peripheral blood. Conversely, Hh signaling is able to feed back to the circadian clock.
BACKGROUND & AIMS: The mammalian circadian clock controls various aspects of liver metabolism and integrates nutritional signals. Recently, we described Hedgehog (Hh) signaling as a novel regulator of liver lipid metabolism. Herein, we investigated crosstalk between hepatic Hh signaling and circadian rhythm. METHODS: Diurnal rhythms of Hh signaling were investigated in liver and hepatocytes from mice with ablation of Smoothened (SAC-KO) and crossbreeds with PER2::LUC reporter mice. By using genome-wide screening, qPCR, immunostaining, ELISA and RNAi experiments in vitro we identified relevant transcriptional regulatory steps. Shotgun lipidomics and metabolic cages were used for analysis of metabolic alterations and behavior. RESULTS: Hh signaling showed diurnal oscillations in liver and hepatocytes in vitro. Correspondingly, the level of Indian Hh, oscillated in serum. Depletion of the clock gene Bmal1 in hepatocytes resulted in significant alterations in the expression of Hh genes. Conversely, SAC-KO mice showed altered expression of clock genes, confirmed by RNAi against Gli1 and Gli3. Genome-wide screening revealed that SAC-KO hepatocytes showed time-dependent alterations in various genes, particularly those associated with lipid metabolism. The clock/hedgehog module further plays a role in rhythmicity of steatosis, and in the response of the liver to a high-fat diet or to differently timed starvation. CONCLUSIONS: For the first time, Hh signaling in hepatocytes was found to be time-of-day dependent and to feed back on the circadian clock. Our findings suggest an integrative role of Hh signaling, mediated mainly by GLI factors, in maintaining homeostasis of hepatic lipid metabolism by balancing the circadian clock. LAY SUMMARY: The results of our investigation show for the first time that the Hh signaling in hepatocytes is time-of-day dependent, leading to differences not only in transcript levels but also in the amount of Hh ligands in peripheral blood. Conversely, Hh signaling is able to feed back to the circadian clock.