Download Introduction - MRC Laboratory of Molecular Biology

Insulin resets the circadian clock in “non-metabolic” cells
via induction of clock gene per2
Priya Crosby1 and John S. O’Neill1
1. MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge,
UK
AFFECTS THE CIRCADIAN CLOCK IN ‘NONMETABOLIC’ CELL TYPES
Luciferase
+ Luciferin
bmal1/cloc
k
PER
2
mPer
2
Introduction
1000
Luciferase
Lu
c
24
48
72
96
120
144
600
0
24
48
72
96
Time, hrs
120
144
168
Period, hrs
4
8
12
16
Zeitgeber Time of Insulin Addition
20
24
Phase Shift, hrs
24
36
48
60
72
84
*
*
50
4
3
2
1
0
1
-1
10
100
1000
Insulin Concentration, nM
vehicle
insulin
300
addition
100
0
48
vehicle
+ insulin
150
200
250
800
60
72
84
96
108
6
12
18
Time, hrs
a) Schematic of the mammalian cycle of core clock gene expression and the use of
the PERIOD2:: LUCIFERASE reporter with this system to provide a real-time report
of cellular circadian state b) An acute (3hr) bolus of insulin (600nM) induces a
phase-shift in PER2 expression in perfused immortalised fibroblasts
(representative, n=3) . This acute induction in PER2 and subsequent phase shift is
also observed in c) neuronal cultures (n=8, ±SEM) but d) is not observed in
fibroblast that express luciferase constitutively (NIH 3T3 SV40::LUC, n=3, ±SEM)
insulin addition
control + insulin
rapamycin + vehicle
rapamycin + insulin
UO126 + insulin
600
400
Introduction
200
0
0
12
24
Time, hrs
36
48
mPer2
CONCLUSIO
N
• Induction of PER2 by insulin is sufficient to modulate
key
parameters of the cellular clock
• This effect is dose-dependent and is independent of
prior circadian phase.
12
24
36
48
60
Addition
drug addition
p70S6
K
Schematic highlighting components of the insulin signalling pathway implicated in the
induction of PERIOD2 protein as a result of extracellular insulin application
200
0
PER2
?
vehicle
LY294002
Time, hrs
Time, hrs
c
eIF4E
400
0
rapamyci
n
mTORC
mTORC
11
drug + insulin addition
600
PKB
GLUT4
vesicle
Introduction
200
MEK 1/2
Introduction
a) In the absence of serum and B27,
insulin acts as a major zeitgeber for
cellular time keeping and its application
affects the b) phase (n=4, ±SEM) c)
period (n=7, ±SD) and d) amplitude
(n=4, ±SD) of the cellular circadian
rhythm, as observed using PER2:: LUC
bioluminescence e) The magnitude of
this effect is dose dependent, but is
evident even with insulin
concentrations as low as 1nM (n=6,
±SEM)
4 / EFFECT OF INSULIN IS VIA
PI3K BUT IS GLUCOSE
INDEPENDENT
a
b
400
100
UO126
PIP3
glucose
starvatio
n
Amplitude PER2::LUC Bioluminescence a.u.
6
IRS-1
PKC
0
96
IRS
PI3K
26
vehicle
Introduction
300000
25
+ insulin
5
168
24
Period, hrs
LY29400
2
Introduction
d
Introduction
0
23
200
e
2000
200000
700
22
Time, hrs
Bolus
100000
+ insulin
+ vehicle
Bioluminescence, cps
PER2::LUC Bioluminescence a.u.
Addition
vehicle
-4
0
12
d
800
0
-12
0
Time, hrs
900
4
**
**
-8
Introduction
insulin
receptor
+ insulin
500
0
c
500
+ vehicle
+ insulin
4000
12
PER2::LUC Bioluminescence, cps
BMAL1/CL
OCK
400
2 / INSULIN INDUCES PRODUCTION OF NASCENT
PER2
cc
8
600
PER2::LUC Bioluminescence, cps
b
6000
a
PER2::LUC Bioluminescence, cps
a
PER2::LUC Bioluminescence a.u.
LIGHT
b
media change
PER2::LUC Bioluminescence, cps
The mammalian
circadian clock is an endogenous daily rhythm in
behavioural and cellular activity that is reset by
appropriate physiological and environmental cues.
Prominent among these is feeding time, which can reset
the clock in peripheral tissue. When this occurs out of
phase with other timing cues, such as light, the result is a
disruption of internal timing — associated with an
increased risk of pathologies such as type II diabetes and
obesity1. Previous work has suggested that the metabolic
hormone insulin plays a role in food-resetting circadian
rhythms in liver and adipose tissue2, but has not been
able to explain the basis of this resetting in other “nonmetabolic” tissue types. Here, we show that
administration of insulin at physiologically-relevant
concentrations stimulates acute expression of the
circadian clock gene PER2 in a range of non-metabolic
cell
types.
3 / ACUTE AND LONG TERM INSULIN APPLICATION
1 / INSULIN IMPACTS ON ALL ASPECTS
OF
CELLULAR CIRCADIAN TIMEKEEPING
Phase shift, hrs
INTRODUCTION
a)PER2 induction and subsequent
phase-shift occurs in the absence of
extracellular glucose (representative,
n=3) b) PER2 induction is attenuated
in the presence PI3K inhibitor
LY294002 (n=4, ±SEM) and c) in the
presence of mTOR inhibitor
rapamycin but not in the presence of
MEK1/2 inhibitor UO126 (n=4,
±SEM)
72
• These effects are independent of glucose availability,
but
are reliant on PI3K and its downstream
components.
Our data suggest that insulin alone may be
sufficient to
account for entrainment of mammalian
circadian
rhythms by feeding time
REFERENCES
1. Reddy, A. B. & O’Neill, J. S. Healthy clocks, healthy body, healthy mind. Trends Cell Biol
(2010)
2. Sato et al., The Role of the Endocrine System in Feeding-Induced Tissue-Specific
Circadian Entrainment, Cell Reports (2014)
ACKNOWLEDGMENTS
Many thanks to the O’Neill lab and to Michael Hastings for discussions, and to Mario de Bono
and Rob Kay for equipment loans