ANTIOXIDANT DEFENSE: LESSONS FROM MAMMALIAN HIBERNATORS

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ANTIOXIDANT DEFENSE LESSONS FROM MAMMALIAN
HIBERNATORS
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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

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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METABOLISM IN HIBERNATION

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

ATP turnover to lt5 of normal
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PRINCIPLES OF HIBERNATION

• 1. Control by protein kinases (SAPKs)
• 2. Metabolic rate reduction
• 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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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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HIBERNATION INDUCED CHANGES

• Protein Synthesis slows to 1
• Pumps channels closed
• Energy Production slows to 5
• Energy Utilization slows to 2
• Gene inactivation ( mRNA )
• Few SAP kinases activated
• Few Genes activated

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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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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
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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 at low body temperature

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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 peroxiredoxin protein and activity
• Result
• ? Detoxification of H2O2, intracellular
  signaling control

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Heme Oxygenase
NADP CO iron
Heme Oxygenase
NADPH
NADPH O2
Biliverdin Reductase
NADP
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What is protective about HO?
Substrate Free heme - liberated from
heme-proteins under oxidative stress -
highly lipophilic, can intercalate into the lipid
bilayers

• Products
• CO
• - Suppresses apoptosis via activation of p38
  MAPK
• Bilirubin
• - Antioxidant
• Iron
• - Oxidant (sequestered by co-induced ferritin)

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Regulation of HO1

• DURING HIBERNATION
• mRNA increased in brain, kidney, liver and lung
• Protein increased in brain, kidney, liver and
  heart
• HO1 unchanged in brown adipose and muscle

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Thioredoxin System
Redox balancing systems
Imbalance in hibernating ground squirrel intestine
?
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Thioredoxin Protein levels
Trx1 Cytosolic and nuclear
form Trx2 Mitochondrial
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• Protective Role of Trx in Hibernation
• Up-regulation of Trx1 and Trx2 in BAT and liver-
  suggests NRF-2 driven transcription
• - protects tissues from oxidative stress-
  suppresses apoptosis response to oxidative stress
  
• Reduced Trx1 and Trx2 in brain, heart, kidney and
  muscle suggests - these organs less
  susceptible to oxidative stress during
  hibernation/arousal- rely on other antioxidant
  defense (e.g. the GSH system)

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Thioredoxin reductase 2
TrxR2
TrxR2 Mitochondrial form
Elevated TrxR2 protein suggests - higher
activity of the enzyme - a need for higher
rates of Trx reduction in hibernation or
arousal
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ENDURANCE EXERCISE
VS
HIBERNATION
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RESCULPTING MUSCLE IN HIBERNATION vs ATROPHY

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

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ATROPHY vs HIBERNATIONSIMILARITIES

• Protein synthesis
• Oxidative stress markers
• NFkB (Tf) , IkB-P
• Nrf-2 (Tf) Antioxidant enzymes
• STAT (Tf)
• HIF (Tf)

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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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ENDURANCE EXERCISE MOLECULAR CHANGES

• Myosin change HC / LC
• Antioxidant enzymes mRNA protein
• Protein synthesis
• Carbohydrate oxidation (HK, Glut 4) Fat
  oxidation (CPT1)
• Pyruvate dehydrogenase active
• Mitochondrial numbers (NAD6, COX1/IV)
• HO-1, PPARa
• Human gene map for performance Med
  Sci Sports Exercise (2004) 36 1451-1469

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HIBERNATING MUSCLE NOT ANTI-EXERCISE !

• Gene up-regulation
• HO-1, PPAR, mitochondrial genes, CPT-1
• Myosin myosin 5C, 7A, 9B, L6, L4
• ----------------------------------------------
  ----
• Ferritin H L chains (HIF)
• Transferrin receptor 2 (HIF)
• AOE glutathione peroxidase 2,
  glutathione S-transferase A2,
  peroxiredoxin 1, thioredoxin
• Apoptosis inhibitors

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MUSCLE RESCULPTING IN EXERCISE VS HIBERNATION

• SIMILARITIES (Common increases in Tf cassettes)
  
• - Myosin regulation
• - Mitochondrial genes
• - NRF-2 ? Heme oxygenase et al.
• - PPAR
• ----------------------------
• DIFFERENCES (Oppositely directed Tf cassettes)
• - PDH (synthesis of components)
• - CHO oxidation enzymes/transporters
• - Protein synthesis
• ----------------------------
• NEW DIRECTIONS T.B.A.
• - Antioxidant enzymes
• - Ferritin Transferrin receptor

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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 (Histone, 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
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