1. Introduction INHIBITION OF THE PENTOSE PHOSPHATE PATHWAY (PPP), BUT NOT OF GLYCOLYSIS, REVERSIBLY ABOLISHES CELLULAR TIMEKEEPING Introduction Circadian rhythms are cell-autonomous phenomena found throughout biology and have been shown to regulate many aspects of health and disease 1. Despite this almost ubiquitous observation of cellular timekeeping, the genes generally proposed to be responsible show little or no homology between kingdoms. Recent work, however, has observed that oxidation-reduction cycles of peroxiredoxin enzymes exhibit a circadian rhythm which is conserved between plants, animals and fungi 2 and it is suggested that this might represent a cellular oscillation in management of Reactive Oxygen Species (ROS) which is common to all organisms with circadian clocks. In this work, we investigate carbohydrate metabolism as a potential source of reducing equivalents which we posit could underlie this rhythm in redox balance. Introduction Priya Crosby 1, Kevin Feeney 1, Sofia Henriques da Costa 2, Christian Frezza 2 and John O’Neill 1 1.MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK 2.MRC Cancer Unit, University of Cambridge, Hutchison/MRC Research Centre, Cambridge Biomedical Campus, Cambridge, UK Metabolic Regulation of Cellular Circadian Timekeeping REFERENCES 1.Reddy, A. B. & O’Neill, J. S. Healthy clocks, healthy body, healthy mind. Trends Cell Biol (2010) 2.Edgar, R. S. et al. Europe Peroxiredoxins are conserved markers of circadian rhythms. Nature (2012). 3.Frezza, C. et al. Haem oxygenase is synthetically lethal with the tumour suppressor fumarate hydratase. Nature (2011). ACKNOWLEDGMENTS Many thanks to the Ned Hoyle and to Michael Hastings for discussions, and to Mario de Bono and Rob Kay for equipment loans Introduction a) Application of hexokinase inhibitor 2- deoxyglucose in ratio with glucose leads to rapid loss of PER2:: LUCIFERASE rhythms which can be recovered with wash off of the drug (±SD, n=4) b) Bypass of glycolysis by replacing glucose with pyruvate as the primary carbon source does not show the same abolition of PER2 rhythmicity (±SD, n=3, normalised) c) Replacing glucose with pyruvate does cause significant period lengthening (p=0.0003) d) Fumarate Hydratase KO human kidney cells3, which lack a competent Krebs cycle, still retain rhythms in clock gene expression (±SD, n=3, normalised), although e) they do so with a shorter period (p=0.0146) Introduction ba INTRODUCTION 3 / CIRCADIAN FLUX THROUGH PRIMARY METABOLISM a) Schematic describing the mammalian cycle of core clock gene expression and the use of luciferase fusion reporters with this system to provide a real-time report of cellular circadian state b) Schematic of primary carbohydrate metabolism c) Catabolism of glucose in early carbohydrate metabolism and its subsequent export into surrounding media varies in a circadian manner, as shown by longitudinal mass spec analysis of outflow media from PER2::LUC mouse fibroblasts in a perfused culture system (representative, n=3, R 2 =0.6276 ) METABOLIC PERTURBATION CAN INFLUENCE THE CELLULAR CLOCK PPP FLUX IS CIRCADIAN AND DIFFERS FROM TOTAL GLYCOLYTIC FLUX a) Flux through the PPP varies in a circadian manner, established by comparison of the ratio of triply:unlabeled glycolytic products produced from 1,2,3-C 13 labeled glucose using longitudinal mass spec analysis of PER2::LUC mouse fibroblasts in perfused cell culture (representative, n=2, R 2 =0.5749) b) As total glycolytic flux increases, PPP flux also increases, but this PPP flux increase is greater than that which can be considered concomitant. Introduction a) Application of PPP inhibitors diphenyleneiodonium (DPI) and dehydroepiandrosterone (DHEA) shows similar loss of per2:luc rhythmicity to that seen with 2-DG and can also be recovered with wash-off (representative, n=4) b) conversely, application of glycolytic inhibitor heptelidic acid (HA) shows no such effect (±SD, n=4) and c) has no significant effect on period d) HA does cause a significant decrease in maximal glycolytic capacity (±SD, n=5) Cellular rhythms are not metabolically compensated Carbohydrate metabolism, and associated production of redox co-factors NADH and NADPH, varies on a circadian basis Manipulation of carbohydrate metabolism can affect cellular timekeeping Inhibition of the pentose phosphate pathway, but not of glycolysis, leads to reversible abolition of circadian ‘clock gene’ rhythmicity Flux through the PPP is circadian and is distinguishable from glycolytic flux This provides a platform from which to further investigate whether the PPP and the related cellular redox state might play a more general role in cellular circadian timekeeping That the systems of the PPP and ROS detoxification might be intrinsically linked with cellular timekeeping appears increasingly likely given the wide evolutionary conservation of these processes CONCLUSION c a b d e Pentose Phosphate Pathway Glycolysis Krebs Cycle NADH NADPH mPer2Luc LuciferasePER2 + Luciferin LIGHT bmal1/clock BMAL1/CLOCK a b c Luciferase ab a d b c