Accepted Manuscript Tick-Tock Hedgehog-Mutual crosstalk with liver circadian clock promotes liver steatosis Eugenia Marbach-Breitrück, Madlen Matz-Soja, Ute Abraham, Wolfgang Schmidt-Heck, Susanne Sales, Christiane Rennert, Matthias Kern, Susanne Aleithe, Luise Spormann, Carlo Thiel, Raffaele Gerlini, Katrin Arnold, Nora Klöting, Reinhard Guthke, Damjana Rozman, Raffaele Teperino, Andrej Shevchenko, Achim Kramer, Rolf Gebhardt PII: S0168-8278(19)30065-0 DOI: https://doi.org/10.1016/j.jhep.2019.01.022 Reference: JHEPAT 7247 To appear in: Journal of Hepatology Received Date: 8 June 2018 Revised Date: 20 December 2018 Accepted Date: 16 January 2019 Please cite this article as: Marbach-Breitrück, E., Matz-Soja, M., Abraham, U., Schmidt-Heck, W., Sales, S., Rennert, C., Kern, M., Aleithe, S., Spormann, L., Thiel, C., Gerlini, R., Arnold, K., Klöting, N., Guthke, R., Rozman, D., Teperino, R., Shevchenko, A., Kramer, A., Gebhardt, R., Tick-Tock Hedgehog-Mutual crosstalk with liver circadian clock promotes liver steatosis, Journal of Hepatology (2019), doi: https://doi.org/10.1016/j.jhep. 2019.01.022 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Tick-Tock Hedgehog-Mutual crosstalk with liver circadian clock promotes liver steatosis Eugenia Marbach-Breitrück1,2#, Madlen Matz-Soja1#*, Ute Abraham3, Wolfgang Schmidt-Heck4, Susanne Sales5, Christiane Rennert1,6, Matthias Kern7, Susanne Aleithe1,8, Luise Spormann1, Carlo Thiel1, Raffaele Gerlini9, Katrin Arnold1, Nora Klöting7, Reinhard Guthke4, Damjana Rozman10, Raffaele Teperino9,11, Andrej Shevchenko5, Achim Kramer3, Rolf Gebhardt1* 1 Rudolf-Schönheimer-Institute of Biochemistry, Faculty of Medicine, University of Leipzig, Leipzig, Germany 2Institute of Biochemistry, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health 3Laboratory of Chronobiology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health 4Leibniz Institute for Natural Product Research and Infection Biology – Hans Knoell Institute, Jena, Germany 5Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany 6 Department of Hepatobiliary Surgery and Visceral Transplantation, University Hospital, Leipzig University, Leipzig, Germany 7IFB Adiposity Diseases, University of Leipzig, Leipzig, Germany 8Clinic and Polyclinic of Neurology, Faculty of Medicine, Leipzig University 9Institute of Experimental Genetics (IEG) - HDC Ingolstädter Landstraße 1, 85764 Neuherberg 10Institute of Biochemistry, Faculty of Medicine, University of Ljubljana 11DZD – German Center for Diabetes Research – 85764 Neuherberg #These authors contributed equally as first authors, respectively Correspondence:* To whom correspondence should be addressed. Rolf Gebhardt; Madlen Matz-Soja, Rudolf-Schönheimer-Institute of Biochemistry, Johanissallee 30, 04103 Leipzig, Germany: [email protected]; [email protected] leipzig.de, telephone: +49 341/9722100, +49 341/9722117; fax: +49 341/9722109 1 Key words: Liver; Hepatocytes; Hedgehog; Circadian Rhythm, Steatosis Electronic word count: 6832 words Number of figures and tables: 7 Figures Conflict of interest: There is no conflict of interest. The co-author Susanne Sales has left the field of liver sciences and is not available regarding the COI. Financial support: This work was supported by the Federal Ministry of Education and Research (Germany) within the research network Systems Medicine of the Liver (LiSyM) [grantnumber031L0054 and 031L0037] and the Virtual Liver Network (VLN) [grant number 0315735, 0315736, 0315755, and 0315775], the Deutsche Forschungsgemeinschaft (Bonn, Germany) [grant MA 6610/2-1]. Author Contributions: Eugenia Marbach-Breitrück: acquisition of data; analysis and interpretation of data; drafting of the manuscript; Madlen Matz-Soja: acquisition of data; analysis and interpretation of data; drafting of the manuscript; Ute Abraham: acquisition of bioluminescence data; analysis and interpretation of data; Wolfgang Schmidt-Heck: acquisition of microarray data, statistical analysis; Susanne Sales: acquisition of lipidomic data; Christiane Rennert: acquisition of data; Matthias Kern: acquisition of metabolic cage data; Susanne Aleithe: acquisition of data; Luise Spormann: Western Blot analyses; Carlo Thiel: acquisition of lipidomic data; Raffaele Gerlini: acquisition of data; Katrin Arnold: acquisition of data; Nora Klöting: acquisition of metabolic cage data; Reinhard Guthke: acquisition of microarray data, 2 statistical analysis; Damjana Rozman: providedPER2::LUC reporter mice, critical revision of the manuscript for important intellectual content; Raffaele Teperino: acquisition of data; Andrej Shevchenko: acquisition of lipidomic data; Achim Kramer: interpretation of data; critical revision of the manuscript for important intellectual content; Rolf Gebhardt: study concept and design, interpretation of data; drafting of the manuscript; obtained funding; study supervision; critical revision of the manuscript for important intellectual content. Abbreviations: CD (control diet), CER (ceramides), CHOL (cholesterol), CE (cholesterol esters), DAG (diacylglycerols), HFD (high fat diet), HSCs (hepatic stellate cells), Hh (Hedgehog), PC (phosphatidylcholines), PE (phosphatidylethanolamine), SCN (hypothalamic suprachiasmatic nuclei), SM (sphingomyelins), TAG (triacylglycerides), VLDL (very low density lipoproteins), ZT (zeitgeber time) Abstract 3 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. Here, 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 hepatic lipid metabolism homeostasis by balancing the circadian clock. (Electronic word count: 248) 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 4 in the amount of HH ligands in peripheral blood. Conversely, Hh signaling is able to feed back to the circadian clock. Highlights: Hh signaling shows diurnal oscillations in liver and hepatocytes in vitro and in vivo Hh signaling feeds-back on the liver clock via GLI transcription factors The amplitude of the oscillations of the liver clock is decreased in hepatocytes from Smo- knockout mice Rhythmicity of many metabolic pathways, including hepatic lipid metabolism, is affected by oscillating Hh signaling Diurnal timing of starvation differently affects the clock-hedgehog module Introduction Circadian rhythm plays a substantial role in regulating physiology and behavior. In mammals, the natural light-dark cycle synchronizes the central circadian pacemaker in the SCN, which, in turn, coordinates the rhythms of autonomous clocks in peripheral tissues such as the liver [1]; these tissues adapt their functions to the rhythmic cycles of feeding and activity. Indeed, most metabolic and secretory functions of the liver show pronounced circadian rhythms [2],[3].In particular, liver carbohydrate and lipid metabolism, which are crucial for energy supply of the entire organism, oscillate throughout the day. The importance of this daily control is emphasized by epidemiological and experimental evidence that interruption or perturbation of circadian rhythms increases the risk for various types of liver disease and may even contribute to diabetes, obesity, metabolic syndrome and cancer [4–6]. On the molecular level, circadian oscillations in the liver are regulated by several transcriptional and translational