From the Institute of Neurobiology of the University of Lübeck Director: Prof. Dr. rer. nat. H. Oster “The interplay of central and peripheral circadian clocks in white adipose function and metabolic homeostasis” Dissertation for Fulfillment of Requirements for the Doctoral Degree of the University of Lübeck from the Department of Natural Sciences Submitted by Isa Kolbe from Geesthacht, Germany Lübeck 2017 1 First referee: Prof. Dr. rer. nat. Henrik Oster Second referee: Prof. Dr. rer. nat. Stefan Anemüller Date of oral examination: 13. Oktober 2017 Approved for printing. Lübeck, 24. Oktober 2017 2 Declaration Herewith, I confirm that I have written the present PhD thesis independently and with no other sources and aids than quoted. Lübeck, Mai 2017 Isa Kolbe 3 “Zeit. Es gibt Kalender und Uhren, um sie zu messen, aber das will wenig besagen, denn jeder weiß, dass einem eine einzige Stunde wie eine Ewigkeit vorkommen kann, mitunter kann sie aber auch wie in einem Augenblick vergehen – je nachdem, was man in dieser Stunde erlebt. Denn Zeit ist Leben” Michael Ende 4 Contents Summary ........................................................................................................................................ 8 Zusammenfassung ..................................................................................................................... 10 Abbreviations ............................................................................................................................... 12 1 Introduction ............................................................................................................................... 15 1.1 Biological Rhythms .......................................................................................................... 15 1.2 Properties of circadian clocks ........................................................................................ 16 1.3 Molecular mammalian circadian timing system ........................................................... 18 1.4 The master clock in the suprachiasmatic nucleus (SCN) .......................................... 20 1.4.1 SCN anatomy ............................................................................................................ 23 1.4.2 SCN output ................................................................................................................ 25 1.5 Peripheral clocks .............................................................................................................. 27 1.6 Circadian clock hierarchy ................................................................................................ 27 1.6.1 Peripheral clock synchronization ............................................................................ 28 1.7 Circadian clocks and metabolism .................................................................................. 30 1.7.1 Metabolic phenotypes of circadian clock gene mutant mice .............................. 31 1.7.1.1 Bmal1 .................................................................................................................. 32 1.7.1.2 Clock .................................................................................................................... 32 1.7.1.3 Period .................................................................................................................. 32 1.7.1.4 Cryptochrome ..................................................................................................... 33 1.7.1.5 Nuclear receptors .............................................................................................. 33 1.7.2 SCN lesion and metabolism .................................................................................... 33 1.8 Peripheral clocks as metabolic regulators .................................................................... 34 1.9 Adipose tissue .................................................................................................................. 35 1.9.1 WAT innervation........................................................................................................ 35 1.9.2 Fatty acid metabolism .............................................................................................. 36 1.9.2.1 Lipogenesis ........................................................................................................ 36 1.9.2.2 Lipolysis .............................................................................................................. 37 1.9.4. Endocrine function of adipose tissue .................................................................... 38 1.9.4.1 Leptin ................................................................................................................... 39 1.9.5. The adipose clock .................................................................................................... 40 1.9.5.1 AT specific clock disruption and metabolism ................................................ 41 5 1.9.5.2 Regulation of AT by clock regulated hormones ............................................ 42 1.9.5.3 Glucocorticoids .................................................................................................. 43 1.9.5.4 Insulin .................................................................................................................. 43 1.10 Aims of this work ............................................................................................................ 45 2 Material and Methods ............................................................................................................. 46 2.1 Animal experiments ......................................................................................................... 46 2.1.1 Animal housing and breeding ................................................................................. 46 2.1.3 SCN mutant (SCN-KO) mouse breeding .............................................................. 46 2.1.4 Wheel-running analysis ............................................................................................ 47 2.1.5 Energy expenditure .................................................................................................. 48 2.1.6 Temperature measurement ..................................................................................... 48 2.1.7 Tissue collection and blood collection ................................................................... 49 2.1.9 Restricted feeding ..................................................................................................... 49 2.2 Immunobiological experiments ...................................................................................... 49 2.2.1 Leptin enzyme-linked immunosorbent assay (ELISA) ........................................ 49 2.3 Molecular biological experiments .................................................................................. 50 2.3.1 DNA extraction from mouse ear biopsies ............................................................. 50 2.3.2 Genotyping PCRs ..................................................................................................... 51 2.3.2.1 Bmal1-flox genotyping ...................................................................................... 51 2.3.2.2 Synaptotagmin10-Cre genotyping .................................................................. 51 2.3.3 mRNA isolation and cDNA synthesis..................................................................... 53 2.3.4 Quantitative real-time PCR (qPCR) ....................................................................... 54 2.4 Metabolic experiments .................................................................................................... 55 2.4.1 Glucose, insulin and pyruvate tolerance tests ...................................................... 55 2.5 Microarray hybridizations ................................................................................................ 56 2.5.1 Circadian rhythm analysis ....................................................................................... 57 2.5.2 Gene enrichment analysis ....................................................................................... 57 2.6 Magnetic resonance imaging ......................................................................................... 57 2.7 Statistical analysis ............................................................................................................ 58 3 Results ...................................................................................................................................... 59 3.1 Behavior and metabolic phenotype of SCN-KO mice ................................................ 59 3.2 White adipose tissue microarray analysis .................................................................... 66 6 3.3 Influence of SCN and peripheral clocks on body weight and glucose homeostasis ................................................................................................................................................... 77 4 Discussion ................................................................................................................................ 83 4.1 Metabolic phenotype of SCN-KO mice ......................................................................... 84 4.2 Transcriptional alteration in eWAT in the absence of the master pacemaker SCN86 4.3 Body weight regulation and glucose homeostasis in synchronized and unsynchronized conditions ...................................................................................................