V12 Circadian rhythms are coupled to metabolism

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V12 Circadian rhythms are coupled to metabolism
Review The suprachiasmatic nuclei (SCN) of the
hypothalamus are the principal circadian
pacemaker in mammals, They drive the sleepwake
cycle and coordinate subordinate clocks in other
tissues. Current understanding The molecular
clockwork within the SCN is being modeled as a
combination of transcriptional and
posttranslational negative feedback
loops. Protein products of Period and
Cryptochrome genes periodically suppress their
own expression.
ONeill et al. Science, 320, 949 (2008)
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Circadian rhythms are coupled to metabolism
Open question It is unclear how long-term,
high-amplitude oscillations with a daily period
are maintained. In particular, transcriptional
feedback loops are typically less precise than
the oscillation of the circadian clock and
oscillate at a higher frequency than one cycle
per day. Possible explanations given in V11 -
phosphorylation causes delay, - secondary loops
give stabilization.
ONeill et al. Science, 320, 949 (2008)
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Intro Metabolites in E. coli
Each distinct substrate occurs in an average of
2.1 reactions.
Ouzonis, Karp, Genome Res. 10, 568 (2000)
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Intro Metabolism Citrate Cycle (TCA cycle) in
E.coli
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Intro Coupling of gene transcription and
metabolites
Solid arrows indicate direct associations between
genes and proteins (via transcription and
translation), between proteins and proteins (via
direct physical interactions), between proteins
and metabolites (via direct physical interactions
or with proteins acting as enzymatic catalysts),
and the effect of metabolite binding to genes
(via direct interactions). Lines show direct
effects, with arrows standing for activation, and
bars for inhibition. The dashed lines represent
indirect associations between genes that result
from the projection onto 'gene space'. For
example, gene 1 deactivates gene 2 via protein 1
resulting in an indirect interaction between gene
1 and gene 2 (drawn after Brazhnik00).
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Review (V11) circadian rhythms in mammals
Ko Takahashi Hum Mol Genet 15, R271 (2006)
Biological Sequence Analysis
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SS 2009 lecture 11
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Evidence for coupling of circadian clocks with
metabolism

• Recombinant cyanobacterial proteins can sustain
  circadian cycles of autophosphorylation in vitro,
  in the absence of transcription,
• (2) intracellular signaling molecules cyclic
  adenosine diphosphateribose (cADPR) and Ca2 are
  essential regulators of circadian oscillation in
  Arabidopsis and Drosophila.
• This indicates that transcriptional mechanisms
  may not be the sole, or principal, mediator of
  circadian pacemaking.

ONeill et al. Science, 320, 949 (2008)
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Example of a gene regulatory network
ONeill and co-workers now show that the
transcriptional feedback loops of theSCN are
sustained by cytoplasmic cAMP signaling. cAMP
signaling determines their canonical properties
of amplitude, phase, and period. Roles of
cAMP? In molluscs, birds, and the mammalian SCN,
cAMP is implicated in entrainment or maintenance
of clocks, or both, or mediation of clock output.
It has not been considered as part of the core
oscillator. This extends the concept of the
mammalian pacemaker beyond transcriptional
feedback to incorporate its integration with
rhythmic cAMP-mediated cytoplasmic signaling.
ONeill et al. Science, 320, 949 (2008)
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What is cAMP
Cyclic adenosine monophosphate (cAMP) is a second
messenger that is important in many biological
processes. cAMP is derived from ATP and used
for intracellular signal transduction in many
different organisms, conveying the cAMP dependent
pathway. In humans, cyclic AMP works by
activating cAMP-dependent protein kinase. Cyclic
AMP binds to specific locations on the regulatory
units of the protein kinase, and causes
dissociation between the regulatory and catalytic
subunits Thus it activates the catalytic units
and enables them to phosphorylate substrate
proteins.
www.wikipedia.org
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Side functions of cAMP
There are some minor PKA-independent functions of
cAMP, e.g. activation of calcium channels. This
provides a minor pathway by which growth hormone
releasing hormone causes release growth
hormone Picture Epinephrine (adrenaline) binds
its receptor, that associates with an
heterotrimeric G protein. The G protein
associates with adenylyl cyclase that converts
ATP to cAMP, spreading the signal
www.wikipedia.org
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Cyclic cAMP levels in mouse brain
We tracked the molecular oscillations of the SCN
as circadian emission of bioluminescence by
organo-typical slices from transgenic mouse
brain. Rhythmic luciferase activity controlled
by the Per1 promoter (Per1luciferase) revealed
circadian transcription, and a fusion protein of
mPER2 and LUCIFERASE (mPER2LUC) reported
circadian protein synthesis rhythms.
Circadian oscillation of cAMP concentration
(blue) and PER2LUC bioluminescence (red), as
well as cAMP concentration in SCN slices treated
with MDL-12,330A (MDL) or with forskolin plus
IBMX.
ONeill et al. Science, 320, 949 (2008)
Interpretation Under these conditions, the cAMP
content of the SCN was circadian.
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Effect of MDL
Idea can one show that cAMP is the reason for
the oscillations? Realization need to suppress
cAMP-production in the cell. Experiment treat
SCN slices with MDL, a potent, irreversible
inhibitor of adenylyl cyclase (that synthesizes
cAMP) to reduce concentrations of cAMP to basal
levels.
Interpretation MDL rapidly suppressed circadian
CREluciferase activity, presumably through loss
of cAMP-dependent activation of CRE sequences.
This caused a dose-dependent decrease in the
amplitude of cycles of circadian transcription
and protein synthesis observed with
mPer1luciferase and mPER2LUC.
ONeill et al. Science, 320, 949 (2008)
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MDL also affects the synchronization of the clock
Prolonged exposure to mild levels of MDL (1.0 mM)
suppressed and desynchro-nized the
transcriptional cycles of SCN cells.
ONeill et al. Science, 320, 949 (2008)
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Can one block cAMP action?
Time of application of ZD7288
Idea If cAMP sustains the clock, interference
with cAMP effectors should compromise pacemaking.
PlanA treat brain slices with inhibitors of
cAMP-dependent protein kinase. This had no
effect, however, on circadian gene expression in
the SCN. PlanB But cAMP also acts through
hyperpolarizing cyclic nucleotidegated ion (HCN)
channels and through the guanine
nucleotideexchange factors Epac1 and Epac2
(Epac, exchange protein directly activated by
cAMP).
The irreversible HCN channel blocker ZD7288,
which would be expected to hyperpolarize the
neuronal membrane, dose-dependently damped
circadian gene expression in the SCN. This is
consistent with disruption of transcriptional
feedback rhythms by other manipulations that
hyperpolarize clock neurons.
ONeill et al. Science, 320, 949 (2008)
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Can cAMP stimulation be recoved?
Idea Direct activation of the effectors might
compensate, therefore, for inactivation of
Adenylate Cyclase by MDL. Observation A
hydrolysis-resistant Epac agonist transiently
activated oscillations in transcriptional
activity in SCN treated with MDL.
ONeill et al. Science, 320, 949 (2008)
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slowing cAMP synthesis
Idea if cAMP signaling is an integral component
of the SCN pacemaker, altering the rate of cAMP
synthesis should affect circadian period.
Experiment 9-(Tetrahydro-2-furyl)-adenine
(THFA) is a noncompetitive inhibitor of adenylate
cyclase that slows the rate of Gs-stimulated cAMP
synthesis, which attenuates peak concentrations.
Interpretation THFA dose-dependently increased
the period of circadian pacemaking in the SCN,
from 24 to 31 hours, with rapid reversal upon
washout
ONeill et al. Science, 320, 949 (2008)
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Conclusions on cAMP-coupling
Circadian pacemaking in mammals is
sustained. Its canonical properties of
amplitude, phase, and period are determined by a
reciprocal interplay in which transcriptional and
posttranslational feedback loops drive rhythms of
cAMP signaling. Dynamic changes in cAMP
signaling, in turn, regulate transcriptional
cycles. Thus, output from the current cycle
constitutes an input into subsequent cycles.
The interdependence between nuclear and
cytoplasmic oscillator elements we describe for
cAMP also occurs in the case of Ca2 and
cADPR. This highlights an important newly
recognized common logic to circadian pacemaking
in widely divergent taxa.
ONeill et al. Science, 320, 949 (2008)
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Implications?

• These studies raise the question of which
  mechanisms couple oscillations of intracellular
  signaling molecules to the transcriptional
  feedback loops of circadian clocks.
• Of the cAMP effectors studied by ONeill et al.,
  only inhibition of
• the hyperpolarization-activated cyclic
  nucleotidegated ion channel or
• the guanine nucleotideexchange factors Epac 1
  and Epac 2
• suppressed circadian gene expression.

Harrising Nitabach Science, 320, 879 (2008)
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Implications?
Application of an Epac agonist resulted in the
phosphorylation and increased activity of cAMP
response elementbinding (CREB) protein, a
transcription factor. This suggests that
changes in cAMP signaling could feed into the
circadian transcriptional oscillator by
regulating the expression of genes that contain
binding sites for CREB. Such genes include the
circadian clock genes Per1 and Per2.
Harrising Nitabach Science, 320, 879 (2008)
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Effect of cADPR in plants
Dodd et al. determined that cADPR concentration
peaks during the early hours of the day. This
fluctuation was abolished in plants with
defective clock function, indicating that the
circadian clock regulates cADPR concentration.
cADPR is synthesized from nicotinamide adenine
dinucleotide by the enzyme ADP ribosyl cyclase.
Nicotinamide, at 10 to 50 mM concentrations,
inhibited ADP ribosyl cyclase and weakened
circadian Ca2i oscillation in plant cells.
Dodd et al. also found a correlation between
the expression of circadian- and cADPR-regulated
genes. Moreover, decreasing the cellular
concentration of cADPR lengthened the period of
circadian gene expression. The authors suggest
that circadian- regulated cADPR-derived Ca2
signaling may configure part of the feedback loop
that controls the clock (see the figure).
Imaizumi et al. Science, 318, 1730 (2007)
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Example of a gene regulatory network
The results of Dodd et al. raise interesting
questions. The phytohormone abscisic acid,
thought to lengthen the clock period, induces
cADPR production, and cADPR gene expression
overlaps with that of genes controlled by
abscisic acid. Does abscisic acid affect the
clock partly through cADPR derived
signals? Also, assuming that both IP3-and
cADPR-dependent pathways are involved in
generating circadian Ca2i oscillation, do they
interact with each other?
Imaizumi et al. Science, 318, 1730 (2007)
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Example of a gene regulatory network
Dodd et al. found that a pharmacological
inhibitor (U73122 at 1 µM) of IP3 production did
not affect daily Ca2i oscillation. Because
IP3 concentrations were not analyzed, more
research is needed to understand the relative
roles of both cADPR and IP3. In particular,
identification of the plant genes that encode the
enzymes that produce cADPR and the proteins that
control Ca2 release by cADPR and IP3 are
required to analyze the functions of these
signaling molecules in plants.
Imaizumi et al. Science, 318, 1730 (2007)
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Current evidence
Recent finding activity of sirtuin-1 (Sirt1), a
longevity-associated protein belonging to a
family of NAD-activated histone deacetylases
oscillates in a circadian fashion.
Eckel-Mahan Sassone-Corsi, Nat Struct Mol Biol.
16, 462 (2009)
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Outlook1
Whether there are mammalian metabolite
oscillations analogous to those of the yeast
metabolic cycle is still unclear, but it remains
a tantalizing possibility. The fact that
cellular demands are met temporally as a function
of the cells metabolic cycle is likely to be
true for all cells, regardless of the organism.
In the context of mammalian Sirt1 circadian
activity, it seems likely that metabolite
oscillations in the coenzyme NAD must also occur
in a cyclical, circadian manner. If metabolite
fluctuations are organized temporally in a
circadian manner, what might this mean
physiologically?
Eckel-Mahan Sassone-Corsi, Nat Struct Mol Biol.
16, 462 (2009)
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Outlook 2
The central functions of NAD in DNA repair, gene
silencing, the cell cycle and circadian control
indicate that the consequences of its aberrant
regulation could be numerous and physiologically
severe. It is conceivable that food restriction
impinges on circadian rhythms because it disrupts
NAD-NADH cycling, essentially allowing the redox
state of individual cells and tissues to alter
rhythmicity.
Eckel-Mahan Sassone-Corsi, Nat Struct Mol Biol.
16, 462 (2009)
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Outlook 3
The absence of ClockBmal1 dimerization in the
presence of increased levels of oxidized NAD is
one piece of evidence supporting this idea. As
such, it is easy to imagine sophisticated schemes
coordinating SCN-driven rhythms with those of a
phase-shifted periphery for drug administration
and efficacy. Already there are numerous drugs,
perhaps most commonly known within cancer
chemotherapeutic strategies, administered
following a circadian protocol so that the
maximal benefit might be achieved from their use.
Eckel-Mahan Sassone-Corsi, Nat Struct Mol Biol.
16, 462 (2009)
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Additional slides
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Cross-talk
By activating Sirt1, NAD conjoins two feedback
loops necessary for cross-talk between the
circadian clock and metabolite production. The
NAD-salvage pathway is important for regulating
intracellular NAD levels. After the conversion
of nicotinamide (NAM) into nicotinamide
mononucleotide (NMN) by NAM phosphoribosyl
transferase (NAMPT), NMN is further modified into
NAD by the nicotinamide mononucleotide adenylyl
transferases (Nmnat1, 2 and 3).
Whereas NAM inhibits Sirt1 activity,
NAD-activated Sirt1 feeds back into the
NAD-salvage pathway by directly regulating Nampt
gene expression in a ClockBmal1-dependent
manner. By this mechanism, NAD conjoins the two
feedback loops, contributing to the fine tuning
necessary for achieving energy balance.
Eckel-Mahan Sassone-Corsi, Nat Struct Mol Biol.
16, 462 (2009)