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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.
O‘Neill et al.
Science, 320, 949 (2008)
Biological Sequence Analysis
SS 2009
lecture 12
1
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.
O‘Neill et al.
Science, 320, 949 (2008)
Biological Sequence Analysis
SS 2009
lecture 12
2
Intro: Metabolites in E. coli
Each distinct substrate occurs in an average of 2.1 reactions.
Ouzonis, Karp, Genome Res. 10, 568 (2000)
Intro: Metabolism: Citrate Cycle (TCA cycle) in E.coli
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]).
Review (V11): circadian rhythms in mammals
Ko & Takahashi Hum Mol Genet 15, R271 (2006)
Biological Sequence Analysis
SS 2009
lecture 11
6
Evidence for coupling of circadian clocks with metabolism
(1) Recombinant cyanobacterial proteins can sustain circadian cycles of
autophosphorylation in vitro, in the absence of transcription,
(2) intracellular signaling molecules cyclic adenosine diphosphate–ribose
(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.
O‘Neill et al.
Science, 320, 949 (2008)
Example of a gene regulatory network
O’Neill 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.
O‘Neill et al.
Science, 320, 949 (2008)
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 cAMPdependent 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
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
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 (Per1::luciferase)
revealed circadian transcription, and a
fusion protein of mPER2 and
LUCIFERASE (mPER2::LUC) reported
circadian protein synthesis rhythms.
O‘Neill et al.
Science, 320, 949 (2008)
Circadian oscillation of cAMP
concentration (blue) and
PER2::LUC bioluminescence
(red), as well as cAMP
concentration in SCN slices
treated with MDL-12,330A (MDL)
or with forskolin plus IBMX.
Interpretation: Under these conditions, the
cAMP content of the SCN was circadian.
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.
O‘Neill et al.
Science, 320, 949 (2008)
Interpretation: MDL rapidly suppressed
circadian CRE::luciferase activity,
presumably through loss of cAMPdependent activation of CRE sequences.
This caused a dose-dependent decrease in
the amplitude of cycles of circadian
transcription and protein synthesis
observed with mPer1::luciferase and
mPER2::LUC.
MDL also affects the synchronization of the clock
Prolonged exposure to mild
levels of MDL (1.0 mM)
suppressed and desynchronized the transcriptional cycles
of SCN cells.
O‘Neill et al.
Science, 320, 949 (2008)
Can one block cAMP action?
Idea: If cAMP sustains the clock,
interference with cAMP effectors should
compromise pacemaking.
Time of application of ZD7288
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 nucleotide–gated
ion (HCN) channels and through the
guanine nucleotide–exchange factors
Epac1 and Epac2 (Epac, exchange
protein directly activated by cAMP).
O‘Neill et al.
Science, 320, 949 (2008)
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.
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.
O‘Neill et al.
Science, 320, 949 (2008)
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
O‘Neill et al.
Science, 320, 949 (2008)
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.
O‘Neill et al.
Science, 320, 949 (2008)
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 O’Neill et al., only inhibition of
- the hyperpolarization-activated cyclic nucleotide–gated ion channel or
- the guanine nucleotide–exchange factors Epac 1 and Epac 2
suppressed circadian gene expression.
Harrising & Nitabach
Science, 320, 879 (2008)
Implications?
Application of an Epac agonist resulted in the phosphorylation and increased
activity of cAMP response element–binding (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)
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 [Ca2+]i 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)
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 [Ca2+]i oscillation, do they interact with each other?
Imaizumi et al.
Science, 318, 1730 (2007)
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 [Ca2+]i 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)
Current evidence
Eckel-Mahan & Sassone-Corsi,
Nat Struct Mol Biol. 16, 462 (2009)
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.
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 cell’s
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)
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)
Outlook 3
The absence of Clock–Bmal1 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)
Additional slides
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 Clock–Bmal1dependent 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)