MAMMALS ON ICE : HIBERNATORS

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MAMMALS ON ICE HIBERNATORS
www.carleton.ca/kbstorey
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Model Hibernators
Spermophilus richardsonii, Richardsons ground
squirrel
Spermophilus tridecemlineatus, 13-lined ground
squirrel
Myotis lucifugus, little brown bat
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• Seasonal phenomenon
• Pre-hibernation hyperphagia
• Gain up to 40 of body mass
• Need polyunsaturated fats
• Find hibernaculum dark, near 0C

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What happens?

• 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

MR falls to fraction of normal
Stewart JM, Boudreau NM, Blakely JA Storey KB.
2002. J. Thermal Biol. 27, 309-315.
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• Metabolism inhibited causing Tb to fall
• Metabolic rate falls to lt5 of normal
• Smaller animals cool down faster
• Q10 values up to 15
• Reversible in arousal
• Torpor bout duration 4 days to 2 weeks

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METABOLIC RATE DEPRESSION
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GENES
Control by transcriptional regulation
Transcription
RNAs
Control by translational regulation
Translation
Control by proteases
No Modification
PROTEINS (ENZYMES)
INACTIVE ENZYME
Degradation
Covalent modification
Control by post- translational modification
FUNCTIONAL ENZYMES
Inhibition and Activation
Control at level of enzyme function
ACTIVE ENZYMES
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METABOLISM IN HIBERNATION

• mRNA synthesis
• Protein synthesis
• Ion Pumping
• Fuel use (esp. CHO)
• O2 consumed

ATP turnover to lt5 of normal
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PRINCIPLES OF HIBERNATION

• 1. Metabolic rate reduction
• 2. Control by protein kinases(SAPKs, 2nd
  messenger PKs)
• 3. Selective gene activation

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i e Factors
Nucleus
mRNAs
GENES ON/OFF
CHO
PROTEINS
Trans.F
Na
ATP
K
PATHWAYS
AA
SAPK
P
PROT
?
SMW
FAT
ADP
ATP
KINASES (2nd)
MITO
ETC
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HIBERNATION INDUCED CHANGES

• 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)

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PROTEIN KINASES

• Covalent modification by phosphorylation
• Families of protein kinases PKA (cAMP), PKG
  (cGMP), CaM (Ca2), PKC (Ca2, PL,DG)
• SAPKs daisy chain phosphorylations
• Regulation is via interconversion of active
  vs subactive forms of protein substrates

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Reversiblephosphorylation control of enzymes
P deP enzymesseparate on ionexchange columns
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PATHWAY CONTROL IN HIBERNATION
Phospho / de-Phospho

• Glycolysis (GP, GS, PFK, PK)
• Fat synthesis (ATP-CL, ACC)
• CHO fuel use (PDH)
• Translation (eIF2a, eEF2)
• Ion pumps (NaK-ATPase, Ca-ATPase)

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• Novel PhosphoEnzymes
• BioInformatics Phospho-analyses
• 2. 32P-ATP labeling studies
• 3. Purification / Properties
• 4. Structure / Function
• 5. Phospho-sites

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HIBERNATION INDUCED CHANGES

• 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)

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TURNING OFF GENESRole of Epigenetics
Epigenetics - Stable changes in gene
activity that do not involve changes in DNA
sequence Common mechanisms - DNA
methylation - Histone modification / histone
variants e.g. acetylation,
phosphorylation - Regulatory non-coding RNAs
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Transcription Suppression in Hibernator Muscle

• Phospho-Histone H3 (Ser10) levels reduced
• Acetyl-Histone H3 (Lys23) levels reduced
  Both inhibit Transcription
• Histone Deacetylase activity increased 80
• HDAC 1 4 protein levels increased
• RNA Polymerase II activity Decreased

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Regulatory non-coding RNAs
microRNA the New Frontier

• Small RNAs 22 nucleotides in length
• Highly conserved across species
• Bind to 3 UTR of mRNAs
• Act to
• - Block translation of mRNA - Target
  mRNA for degradation

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Are miRNAs differentially regulated in
hibernators?

• Yes! Selected miRNAs were regulated in heart,
  muscle and kidney of hibernating 13-lined ground
  squirrels (Morin, Dubuc Storey, 2008, Biochim
  Biophys Acta 1779628-633)

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METABOLIC RATE DEPRESSION
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HIBERNATION INDUCED CHANGES

• 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)

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ROLE OF TRANSCRIPTION

• Global rate of mRNA synthesis depressed.
  Method nuclear run-on
• Are selected genes up-regulated ?
• TO ASSESS GENE UPREGULATION
• What new mRNAs are created - cDNA
  library, Gene Chip

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cDNA ARRAY SCREENING
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• cDNA Arrays- Methods
• Materials
• Sources- Publications

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GENE CHANGES IN HIBERNATION
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CONTROL REGION OF A TYPICAL EUKARYOTIC GENE
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Nrf2/ARE pathway
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NRF-2

• Increased NFR-2 protein
• Increased NFR-2 in the Nucleus
• Increased levels of co-Tf MafG
• Downstream gene activation
• GST, HO-1, HO-2, Peroxiredoxin
• Thioredoxin, SOD (Cu/Zn Mn)

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Nrf2/ARE pathway
Reactive Oxygen Species (ROS)
Actin
Keap1
Dissociation
Nrf2
Cytoplasm
Nucleus
Activation
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Protein Regulation of Nrf2
100 kDa 57 kDa
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Nrf2 distribution between nuclearand cytoplasmic
fractions
Moved to nucleus
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Nrf2 Timecourse in Heart

• Nrf2 protein in early and late hibernation
• ? Up-regulation cascade.

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Peroxiredoxins

• Detoxify / reduce hydroperoxides
• Expressed at high levels
• ARE in promoter region of Prdx
  genes
• Nrf2 activated

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Peroxiredoxin Activity

• Protein level correlates with increased activity
• Assays in BAT and heart with thioredoxin,
  thioredoxin reductase and NADPH

Kim et al., 2005
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Conclusions

• Activation of the Nrf2 pathway
• Activated in early-late torpor, along with
  downstream gene protein products
• Increased PRDX, HO TRX protein and activity
  
• Result
• ? Detoxification of H2O2, intracellular
  signaling control

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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

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ENDURANCE EXERCISE
VS
HIBERNATION
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RESCULPTING MUSCLE IN HIBERNATION vs ATROPHY

• Torpor inactivity for up to 8-9 mo
• Brief arousals, no exercise
• Muscles retain full function in spring
• Muscle loss / wasting minimal

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ATROPHY vs HIBERNATION DIFFERENCES

• GRP HSP chaperones
• Glycolysis
• Fat Metabolism genes
• Antioxidant genes
• Proteolysis complex UB
• Serpins Apoptosis inhibitors
• Myosin protein synthesis

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ATROPHY HIBERNATIONSIMILARITIES

• Protein synthesis

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EXERCISE HIBERNATIONSIMILARITIES

• Oxidative stress markers
• NFkB (Tf) , IkB-P
• Nrf-2 (Tf) Antioxidant enzymes
• STAT (Tf)
• HIF (Tf)
• Ferritin, Transferrin receptor
• common to atrophy, hibr. exercise

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EXERCISE HIBERNATION SIMILARITIES -2

• Myosin change Heavy Light chains
• Antioxidant enzymes mRNA protein
• Fat oxidation (CPT1)
• Mitochondrial numbers (NAD6, COX1/IV)
• HO-1, PPARa
• Heat shock proteins
• Apoptosis inhibitors
• Human gene map for performanceMed Sci
  Sports Exercise (2004) 36 1451-1469

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ATROPHY vs HIBERNATION NEW DIRECTIONS

• Hibernation more closely mimics
  EXERCISE !
• UNANSWERED QUESTIONS
• The Tfs Rb-P, ETS, chREBP, AP (1,2)
• Cell cycle control kinases
• Chromatin resculpting (Histones, SIRT,
  HDAC)
• Bcl3, PARP, ELK-P, ERG, CREB(P), MyoD/G
  ID proteins

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HIBERNATION

• J. STOREY
• S. EDDY
• D. HITTEL
• J. MacDONALD
• A. FAHLMAN
• P. MORIN
• C. HOLDEN
• H. MEHRANI

• J. NI
• M. HAPSATOU
• J. HALLENBECK
• D. THOMAS
• A. RUBTSOV
• J. STEWART
• S. BROOKS
• C. FRANK

Funded by NSERC Canada
www.carleton.ca/kbstorey
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TRANSCRIPTION FACTORS

• ATF (Glucose Regulated Proteins)
• HIF (O2), HSF (Hsp)
• NFkB (IkB-P), Nrf-2 (GST), NRF-1
• PPAR, PGC, RXR, chREBP, CREB-P
• STAT, SMAD, p53-P, HNF, AP (1,2)
• Methods EMSA, CHiP

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GENES
Control by transcriptional regulation
Transcription
RNAs
Control by translational regulation
Translation
Control by proteases
No Modification
PROTEINS (ENZYMES)
INACTIVE ENZYME
Degradation
Covalent modification
Control by post- translational modification
FUNCTIONAL ENZYMES
Inhibition and Activation
Control at level of enzyme function
ACTIVE ENZYMES
www.carleton.ca/kbstorey
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(No Transcript)
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HIBERNATION HORMONES

• Prehibernation (summer) Adiposity
• Leptin GH Ghrelin
• Hibernation (fall) BMR
• Leptin

KEY removing the metabolic signal
of leptin during prehibernation
fattening
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HIBERNATING BRAIN

• Decrease metabolic activity overall
• Some areas decrease to 0.04 of active
• Decrease substrate useDecrease biosynthesis
• Exception Suprachiasmatic nucleus (a)
  endogenous circadian oscillator (b) time
  keeper (c) if destroyed hibernation ceases