MAMMALS ON ICE : MAMMALS ON ICE : HIBERNATIONHIBERNATION
www.carleton.ca/~kbstoreywww.carleton.ca/~kbstorey
Myotis lucifugus, little brown bat
Spermophilus tridecemlineatus,13-lined ground squirrel
Spermophilus richardsonii,Richardson’s ground squirrel
• Seasonal phenomenon
• Pre-hibernation hyperphagia
• Gain up to 40% of body mass
• Need polyunsaturated fats
• Find hibernaculum: dark, near 0°C
• drop in body temperature• reduced heart rate• apnoic breathing• some muscle atrophy • periods of torpor lasting weeks• non-REM sleep• oleamide increases in brain
• suppression of carbohydrate oxidation
• RQ of 0.7 = lipid oxidation
Stewart JM, Boudreau NM, Blakely JA &Storey KB. 2002. J. Thermal Biol. 27, 309-315.
Oleamide in groundsquirrel brain
(ng/g)
Euthermic Hibernating0
200
400
600
800
Month
Body temperature
MR falls to fraction of normal
• Metabolism inhibited causing Tb to fall
• Metabolic rate falls to <5% of normal
• Smaller animals cool down faster
• Q10 values up to 15
• Reversible in arousal
• Torpor bout duration 4 days to 2 weeks
GENESGENES
Transcription
RNAsRNAs
Control byControl bytranscriptional regulationtranscriptional regulation
Control byControl bytranslational regulationtranslational regulationTranslation
PROTEINSPROTEINS(ENZYMES)(ENZYMES)
Control byControl byproteasesproteases INACTIVEINACTIVE
ENZYMEENZYME
NoModification
FUNCTIONALFUNCTIONALENZYMESENZYMES
Covalentmodification
Degradation
Control by post-Control by post-translationaltranslationalmodificationmodification
ACTIVEACTIVEENZYMESENZYMES
Inhibitionand
Activation
Control at level ofControl at level ofenzyme functionenzyme function
METABOLISM IN METABOLISM IN HIBERNATIONHIBERNATION
• mRNA synthesis • Protein synthesis • Ion Pumping • Fuel use (esp. CHO)• O2 consumed
ATP turnover to <5% of normal
PRINCIPLES OF PRINCIPLES OF HIBERNATIONHIBERNATION
1. Metabolic rate reduction
2. Control by protein kinases(SAPKs, 2nd messenger PKs)
3. Selective gene activation
Nucleus
GENESON/OFF
mRNAs
[ i + e Factors]
PROTEINS
Ca+2
KINASES (2nd)
PATHWAYS
SMW
CHO
AA
ATP
?SAPK
ATP
ADP
MITOGENES
FAT
[Trans.F] Na
K
ETC
P PROT
Nucleus
GENESON/OFF
mRNAs
[ i + e Factors]
PROTEINS
Ca+2
KINASES (2nd)
PATHWAYS
SMW
CHO
AA
ATP
?SAPK
ATP
ADP
MITOGENES
FAT
[Trans.F] Na
K
ETC
P PROT
HIBERNATION INDUCED HIBERNATION INDUCED CHANGESCHANGES
• Protein Synthesis slows to 1% • Pumps & Channels closed • Energy Production slows to 5% • Energy Utilization slows to 2%• Few ‘SAP’ kinases activated
• Gene ‘inactivation’ ( mRNA )• Few Genes activated (1-2%)
PROTEIN KINASESPROTEIN KINASES
PROTEIN
nATP nADP
PROTEIN-(P)n
• Covalent modification by phosphorylation
• Families of protein kinases: PKA (cAMP), PKG (cGMP), CaM (Ca2+), PKC (Ca2+, PL,DG)
• SAPKs : daisy chain phosphorylations
• Regulation via interconversion of active vs subactive forms of protein substrates
• p38, ERK (1/2), JNK, AMPK, AKT (mTOR)
PP
PP PP
PP
PiPiProtein
Phosphatase
ATPATP ADPADPProtein Kinase
P & deP enzymesseparate on ion
exchange columns
ReversibleReversiblephosphorylationphosphorylation
control of enzymescontrol of enzymes
PATHWAY CONTROL IN PATHWAY CONTROL IN HIBERNATIONHIBERNATION
• Glycolysis (GP, GS, PFK, PK)• Fat synthesis (ATP-CL, ACC)• CHO fuel use (PDH)• Translation (eIF2α, eEF2)• Ion pumps (NaK, Ca-ATPase)
• the usual suspects, TextBook
Phospho / de-Phospho
1. Novel PhosphoEnzymes: BioInformatics + Phospho-analyses 2. 32P-ATP labeling studies3. Purification / Properties 4. Structure / Function5. Phospho-sites
Post-translational Modifications:Post-translational Modifications: The Next Generation The Next Generation
Novel Phosphorylation ControlCK, GDH, Hexokinase, G6PDH,
LDH, NADP-IDH, α-GPDH, AMPD, GAPDH, FBPase, Antioxidant
enzymes
PTM: Acetylation, Methylation, SUMOylation
HIBERNATION INDUCED HIBERNATION INDUCED CHANGESCHANGES
• Protein Synthesis slows to 1% • Pumps & channels closed • Energy Production slows to 5% • Energy Utilization slows to 2%• Few ‘SAP’ kinases activated
• Gene ‘inactivation’ ( mRNA )• Few Genes activated (1-2%)
TURNING OFF GENES:TURNING OFF GENES:Role of EpigeneticsRole of Epigenetics
Epigenetics:Epigenetics: - Stable changes in gene activity that do not involve changes in DNA sequence
Common mechanisms:Common mechanisms: - DNA methylation - Histone modification / histone variants e.g. acetylation, phosphorylation - Regulatory non-coding RNAs
Transcription Transcription Suppression in Hibernator Suppression in Hibernator
MuscleMuscle
• Phospho-Histone H3 (Ser10) levels reduced
• Acetyl-Histone H3 (Lys23) levels reduced * Both inhibit Transcription ** Both inhibit Transcription *
• Histone Deacetylase activity increased 80%
• HDAC 1 & 4 protein levels increased
• RNA Polymerase II activity Decreased
Regulatory non-coding RNAs Regulatory non-coding RNAs
• Small RNAs ~22 nucleotides in length
• Highly conserved across species
• Bind to 3’ UTR of mRNAs
• Could be 1000, affect 60 % of genes
• Disease involvement
• Act to :
- Block translation of mRNA - Target mRNA for degradation
microRNAmicroRNA
Are miRNAs differentially regulated Are miRNAs differentially regulated in hibernators?in hibernators?
• Yes! Selected miRNAs were regulated in heart, muscle & kidney of hibernating 13-lined ground squirrels
(Morin, Dubuc & Storey, 2008, Biochim Biophys Acta 1779:628-633)
miRNA Fold change Process in higher vertebrates
Mir-1 2.0 Myogenesis
Mir-133a 2.4 Myogenesis
Mir-206 2.6 Myogenesis
Let-7 2.0 Cell cycle
Mir-26 2.4 Hypoxia
Mir-451 2.6 Erythropoiesis
HIBERNATION INDUCED HIBERNATION INDUCED CHANGESCHANGES
• Protein Synthesis slows to 1% • Pumps & channels closed • Energy Production slows to 5% • Energy Utilization slows to 2%• Few ‘SAP’ kinases activated
• Gene ‘inactivation’ ( mRNA )• Few Genes activated (1-2%)
ROLE OF ROLE OF TRANSCRIPTIONTRANSCRIPTION
• Global rate of mRNA synthesis depressed. Method: nuclear run-on
• Are selected genes up-regulated ?
• TO ASSESS GENE UPREGULATION:TO ASSESS GENE UPREGULATION:
What new mRNAs are created - cDNA library, Gene Chip
Sequenced genome(s) as of 2011 Sequenced genome(s) as of 2011
cDNA ArrayscDNA Arrays- Methods- Methods- MaterialsMaterials- SourcesSources- Publications- Publications
GENE CHANGES IN GENE CHANGES IN HIBERNATIONHIBERNATION
• cDNA Library screen - Mitochondrial Genes - AOE - FABP, CPT, etc. - Shock proteins (GRP, HSP) - Transcription factors • DNA Chip ~1-2%
CONTROL REGION OF A CONTROL REGION OF A TYPICAL EUKARYOTIC GENETYPICAL EUKARYOTIC GENE
Epigenetics = OFF) : Epigenetics = OFF) : • microRNAmicroRNA
• phospho-RNA Polymerasephospho-RNA Polymerase• Histones modified Histones modified
• HDAC / HAT changesHDAC / HAT changes
TRANSCRIPTION FACTORS TRANSCRIPTION FACTORS
• ATF (Glucose Regulated Proteins)
• HIF (O2), HSF (Hsp)
• NFkB (IkB-P), Nrf-2 (**), NRF-1
• PPAR, PGC, RXR, chREBP, CREB-P
• STAT, SMAD, p53-P, HNF, AP (1,2)
• Methods: EMSA, CHiP
Nrf2/ARE pathwayNrf2/ARE pathway
Antioxidant proteins(e.g. GSTs, HO1)
ARE
Nucleus
Cytoplasm
Small Maf
Nrf2
Keap1
Actin
Reactive Oxygen Species (ROS)
Activation
Dissociation
Nrf2 P
Nrf2 P
Small Maf Nrf2 P
Nrf-2Nrf-2• Increased Nrf-2 protein & P
• Increased Nrf-2 in the Nucleus
• Increased levels of co-Tf: MafG
• Downstream gene activation:
• GST, HO-1, HO-2, Peroxiredoxin
• Thioredoxin, SOD (Cu/Zn & Mn)
Nrf2/ARE pathwayNrf2/ARE pathway
Antioxidant proteins(e.g. GSTs, HO1)
ARE
Nucleus
Cytoplasm
Small Maf
Nrf2
Keap1
Actin
Reactive Oxygen Species (ROS)
Activation
Dissociation
Nrf2 P
Nrf2 P
Small Maf Nrf2 P
Protein Regulation of Nrf2Protein Regulation of Nrf2
100 kDa
57 kDa
E H
Nrf2
Actin
Nrf2
Euthermicvs
Hibernating
Squirrel Nrf2 57kDa and 100kDa protein expression(hibernating vs.euthermic)
00.5
11.5
22.5
33.5
44.5
B.A.T. Brain Heart Kidney Liver Lung Muscle W.A.T.rati
o:
hib
ern
ati
ng
vs
. eu
the
rmic
57KDa
100KDa
* *
* *
* *
**
*
RatioHib:Euth
Nrf2 distribution between nuclearNrf2 distribution between nuclearand cytoplasmic fractionsand cytoplasmic fractions
E H E H Nucleus Cytoplasm
57 kDa
100 kDa
Nucleus CytoplasmE H E H
57 kDa
100 kDa
Moved to nucleus
Nrf2 (57 KDa and 100 KDa) Protein Expression inMuscle Nuclear and Cytoplasmic Fractions
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Muscle Nuclear Fraction Muscle Cytoplasmic Fraction
Hib
ern
atin
g v
s. E
uth
erm
ic
57KDa
100KDa
**
*
Muscle
Hib
: E
uth
Nrf2 (57 KDa and 100 KDa) Protein Expression in LiverNuclear and Cytoplasmic Fractions
00.20.40.60.8
11.21.41.6
Liver Nuclear Fraction Liver Cytoplasmic Fraction
Hib
ern
atin
g v
s. E
uth
erm
ic
57KDa
100KDa
*
*
Liver
Hib
: E
uth
Nrf2 Timecourse in HeartNrf2 Timecourse in Heart
• Nrf2 protein in early and late hibernation
Up-regulation “cascade”.
Active incold room
(37°C)
Entrance
(T-drop)
Early Hibern.
(~5-7°C)
Late Hibern.
(~5-7°C)
Early Arousal(T-gain)
FullyAroused(37°C)
Nrf2
CuZnSOD
AFAR1
HO-1
PeroxiredoxinsPeroxiredoxins
• Detoxify / reduce hydroperoxides
• Expressed at high levels
• ARE in promoter region of Prdx genes
•Nrf2 activated
0
2
4
6
8
10
12
14
16
18
Brown adipose tissue Heart
scan
ned
in
ten
sit
y o
f h
ibern
ati
ng
versu
s
eu
therm
ic
peroxiredoxin-1
peroxiredoxin-2
peroxiredoxin-3
BAT
Heart
Euth Hib
Prdx1 Prdx3Prdx2Peroxiredoxin protein levels
Euth HibEuth HibR
ati
o b
and inte
nsi
tyH
ib :
Euth
Peroxiredoxin ActivityPeroxiredoxin Activity
• Protein level correlates with increased activity
• Assays in BAT and heart with thioredoxin, thioredoxin reductase and NADPH:
Kim et al., 2005
0
0.5
1
1.5
2
2.5
3
3.5
4
Brown adipose tisuue Heart
Rat
io o
f hib
erna
ting
vers
us e
uthe
rmic
act
ivity
Relative activity: Hibernating: euthermic
A
ctiv
ity
rati
o
Hib
: E
uth
BAT Heart
Nrf ConclusionNrf Conclusion Activation of the Nrf2 pathway:Activation of the Nrf2 pathway:
Activated in early-late torpor, along with downstream gene protein products
Increased PRDX, HO & TRX protein and activity
Result:Result:
Detoxification of ROS, intracellular signaling control
GENE CHIPS TRANSCRIPTION FACTORPROFILING
Data LeadsData Leads
ELISAs in plates
Confirm by EMSA
Confirm byRT-PCR,
Northern blots
TfDownstream genes
ROLE & CONTROL OF SYSTEM
Transgenics
Cell AssayRNAi Knock out Epigenetics
FUNCTIONAL ASSAYS
Protein levels - enzyme assay - antibodies : protein - functional analysis e.g. HIF EPO
Where do we go from Where do we go from here?here?
• Applications of MRD research • Novel phosphorylations• Atrophy, hypertrophy -- autophagy for survival • Turning it all off -- microRNA• Epigenetics & adaptation • Life span extension• Antioxidant Defense• Cell cycle suppression• Unity through evolution
NEW DIRECTIONSNEW DIRECTIONS
Novel phosphorylations Novel phosphorylations
PP
PP PP
PP
PiPiProtein
Phosphatase
ATPATP ADPADPProtein Kinase
HIBERNATIONHIBERNATION• J. STOREY• S. EDDY• D. HITTEL• J. MacDONALD• A. FAHLMAN• P. MORIN• C. HOLDEN• H. MEHRANI• J. NI
• M. HAPSATOU• S. TESSIER• M. WU• S. BROOKS• C. FRANK• J. HALLENBECK• D. THOMAS• A. RUBTSOV• J. STEWART
www.carleton.ca/~kbstoreywww.carleton.ca/~kbstorey
Funded by NSERC CanadaFunded by NSERC Canada