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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