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Transcript
Metabolic Regulation of Cellular Circadian Timekeeping Priya Crosby1, Kevin Feeney1, Sofia Henriques da Costa2, Christian Frezza2 and John O’Neill1 Media Change 500 250 24 48 72 96 120 144 50 0 + Luciferin bmal1/clock Period, hrs 50 * 23 FH1-/-bmal1:luc pFH bmal1:Luc 22 21 Introduction 20 12 24 36 48 60 FH1-/-bmal1:luc pFH bmal1:Luc 72 Time, hrs Luciferase b c 800 Media Metabolite Concentration, au PER2::LUC Bioluminescence, cps Increasing Glycolytic Flux 4000 0.2 3500 Total extracellular pyruvate/lactate PER2::LUC 36 48 12 24 36 48 60 72 84 60 72 84 3250 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, R2=0.6276 ) 96 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 3500 0.6 24 36 48 60 72 3000 84 0.9 0.10 0.8 0.08 0.7 0.06 Increasing PPP Flux 0.04 0.02 12 24 36 48 60 72 0.6 0.5 84 Time • Manipulation of carbohydrate metabolism can affect cellular timekeeping 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) Time, hrs Increased PPP flux 0.12 • Cellular rhythms are not metabolically compensated Introduction 3000 96 0.7 Introduction Total extracellular pyruvate/lactate CONCLUSION Time, hrs 3750 0.1 0 4000 Ratio labelled:unlabelled metabolite 1.0 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-C13 labeled glucose using longitudinal mass spec analysis of PER2::LUC mouse fibroblasts in perfused cell culture (representative, n=2, R2=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. ** Glycolysis 100µM heptelidic acid 0.8 0.14 b 9 Control 4500 Time, hrs Introduction 23.5 5000 • Carbohydrate metabolism, and associated production of redox co-factors NADH and NADPH, varies on a circadian basis 8 7 6 5 4 3 2 1 0 -1 24.0 Increasing Glycolytic Flux 0.9 0.5 12 4250 0.3 0.0 24 84 ** Krebs Cycle NADH 24.5 200 0 0.4 -2 Control 100µM Heptelidic Acid *** NADH 0.5 PER2::LUC Bioluminescence Pentose Phosphate Pathway 72 Maximal Glycolytic Capacity (ECAR), mpH/min -3 600 25.0 -4 G G HA PI EA icle D 2D 2D H D Veh M M 0uM nM m m 40 0uM 10 20 b Luc c NADPH 60 20 Introduction d 25.5 Introduction 400 mPer2 48 10 PER2 a Period, hrs BMAL1/CLOCK 36 INHIBITION OF THE PENTOSE PHOSPHATE PATHWAY (PPP), BUT NOT OF GLYCOLYSIS, REVERSIBLY ABOLISHES CELLULAR TIMEKEEPING LIGHT Luciferase 24 a) Application of hexokinase inhibitor 2deoxyglucose 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) b THROUGH PRIMARY METABOLISM a 12 Ratio labelled:unlabelled metabolite PER2::LUC 3hr smoothed 1.0 Introduction Time, hrs e 0 24 27.8 mM glucose, 50 mM pyruvate, no pyruvate no glucose 100 0 25 23 Time, hrs d a 26 0 168 *** 27 27.8 mM glucose, no pyruvate 50 mM pyruvate, no glucose Introduction Introduction 0 c Period, hrs PER2::LUC bioluminescence, cps Media Change 0 100 Media Metabolite Concentration, au b 10:20 mM glucose:2-DG 20:10 mM glucose:2-DG Percentage PER2::LUC Bioluminescence 30 mM glucose Ratio labelled:unlabelled pyruvate and lactate a 750 PPP FLUX IS CIRCADIAN AND DIFFERS FROM TOTAL GLYCOLYTIC FLUX PER2::LUC Bioluminescence Circadian rhythms are cell-autonomous phenomena found throughout biology and have been shown to regulate many aspects of health and disease1. 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 fungi2 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. 3 / CIRCADIAN FLUX METABOLIC PERTURBATION CAN INFLUENCE THE CELLULAR CLOCK a Percentage bmal1:luc Bioluminescence INTRODUCTION 2. MRC Cancer Unit, University of Cambridge, Hutchison/MRC Research Centre, Cambridge Ratio C13-labelled:unlabelled lactate and pyruvate 1. MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK Biomedical Campus, Cambridge, UK • 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