MAMMALS ON ICE : HIBERNATION

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1 MAMMALS ON ICE : Spermophilus tridecemlineatus, 13-lined ground squirrel Spermophilus richardsonii, Richardson s ground squirrel Myotis lucifugus, little brown bat Seasonal phenomenon re-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 Oleamide in ground squirrel brain (ng/g) Body temperature Month suppression of carbohydrate oxidation RQ of 0.7 = lipid oxidation MR falls to fraction of normal 0 Euthermic Hibernating Stewart JM, Boudreau NM, Blakely JA & Storey KB J. Thermal Biol. 27,

2 Metabolism inhibited causing Tb to fall Metabolic rate falls to <5% of normal Smaller animals cool down faster Q 10 values up to 15 Reversible in arousal Torpor bout duration 4 days to 2 weeks Hibernation METABOLIC RATE DERESSION Estivation Anoxia Freezing Diapause No Modification FUNCTIONAL ENZYMES Inhibition and Activation ACTIVE ENZYMES Transcription Translation GENES Control by transcriptional regulation RNAs ROTEINS (ENZYMES) Covalent modification Control at level of enzyme function Control by translational regulation Control by posttranslational modification Control by proteases Degradation INACTIVE ENZYME METABOLISM IN mrna synthesis rotein synthesis Ion umping Fuel use (esp. CHO) O 2 consumed AT turnover to <5% of normal 2

3 RINCILES OF Nucleus GENES ON/OFF [Trans.F] mrnas CHO [ i + e Factors] ROTEINS Na 1. Metabolic rate reduction 2. Control by protein kinases (SAKs, 2 nd messenger Ks) 3. Selective gene activation? SAK KINASES (2 nd ) ATHWAYS SMW AT AD GENES AT AA ROT MITO Ca +2 FAT K ETC? Nucleus GENES ON/OFF [Trans.F] SAK mrnas CHO ATHWAYS SMW AD [ i + e Factors] ROTEINS AT AA Ca +2 ROT FAT Na K INDUCED CHANGES rotein Synthesis slows to 1% umps & Channels closed Energy roduction slows to 5% Energy Utilization slows to 2% Few SA kinases activated KINASES (2 nd ) AT GENES MITO ETC Gene inactivation ( mrna ) Few Genes activated (1-2%) 3

4 ROTEIN ROTEIN KINASES nat nad ROTEIN-()n Covalent modification by phosphorylation Families of protein kinases: KA (cam), KG (cgm), CaM (Ca 2+ ), KC (Ca 2+, L,DG) SAKs : daisy chain phosphorylations Regulation via interconversion of active vs subactive forms of protein substrates p38, ERK (1/2), JNK, AMK, AKT (mtor) AT i rotein Kinase rotein hosphatase & de enzymes separate on ion exchange columns AD Reversible phosphorylation control of enzymes ATHWAY CONTROL IN hospho / de-hospho Glycolysis (G, GS, FK, K) Fat synthesis (AT-CL, ACC) CHO fuel use (DH) Translation (eif2α, eef2) Ion pumps (NaK, Ca-ATase) the usual suspects, TextBook 1. Novel hosphoenzymes: BioInformatics + hospho-analyses AT labeling studies 3. urification / roperties 4. Structure / Function 5. hospho-sites 4

5 ost-translational Modifications: The Next Generation Novel hosphorylation Control CK, GDH, Hexokinase, G6DH, LDH, NAD-IDH, α-gdh, AMD, GADH, FBase, Antioxidant enzymes TM: Acetylation, Methylation, SUMOylation INDUCED CHANGES rotein Synthesis slows to 1% umps & channels closed Energy roduction slows to 5% Energy Utilization slows to 2% Few SA kinases activated Gene inactivation ( mrna ) Few Genes activated (1-2%) TURNING OFF GENES: Role 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 5

6 Transcription Suppression in Hibernator Muscle hospho-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 olymerase II activity Decreased Regulatory non-coding RNAs microrna 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 Are mirnas differentially regulated 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: ) Cuellar TL, McManus MT. J Endocrinol. 187(3): , mirna Fold change rocess in higher vertebrates Mir Myogenesis Mir-133a 2.4 Myogenesis Mir Myogenesis Let Cell cycle Mir Hypoxia Mir Erythropoiesis 6

7 Turning it all off METABOLIC RATE DERESSION Anoxia Hibernation Estivation Freezing Diapause INDUCED CHANGES rotein Synthesis slows to 1% umps & channels closed Energy roduction slows to 5% Energy Utilization slows to 2% Few SA kinases activated Gene inactivation ( mrna ) Few Genes activated (1-2%) ROLE OF TRANSCRITION Global rate of mrna synthesis depressed. Method: nuclear run-on Are selected genes up-regulated? TO ASSESS GENE UREGULATION: What new mrnas are created - cdna library, Gene Chip Sequenced genome(s) as of

8 cdna ARRAY SCREENING Euthermic Hibernating cdna Arrays - Methods - Materials - Sources - ublications GENE CHANGES IN cdna Library screen - Mitochondrial Genes -AOE - FAB, CT, etc. - Shock proteins (GR, HS) - Transcription factors DNA Chip ~1-2% CONTROL REGION OF A TYICAL EUKARYOTIC GENE Epigenetics = OFF) : microrna phospho-rna olymerase Histones modified HDAC / HAT changes 8

9 TRANSCRITION FACTORS ATF (Glucose Regulated roteins) HIF (O 2 ), HSF (Hsp) NFkB (IkB-), Nrf-2 (), NRF-1 AR, GC, RXR, chreb, CREB- STAT, SMAD, p53-, HNF, A (1,2) Methods: EMSA, CHi Small Maf /ARE pathway Reactive Oxygen Species (ROS) Small Maf Keap1 ARE Dissociation Activation Actin Nucleus Cytoplasm Antioxidant proteins (e.g. GSTs, HO1) Nrf-2 Increased Nrf-2 protein & Increased Nrf-2 in the Nucleus Increased levels of co-tf: MafG Downstream gene activation: GST, HO-1, HO-2, eroxiredoxin Thioredoxin, SOD (Cu/Zn & Mn) Small Maf /ARE pathway Reactive Oxygen Species (ROS) Small Maf Keap1 ARE Dissociation Activation Actin Nucleus Cytoplasm Antioxidant proteins (e.g. GSTs, HO1) 9

10 rotein Regulation of Euthermic vs Hibernating ratio: hibernating vs. euthermic Ratio 2.5 Hib:Euth E H 100 kda 57 kda Actin Squirrel 57kDa and 100kDa protein expression (hibernating vs.euthermic) B.A.T. Brain Heart Kidney Liver Lung Muscle W.A.T. 57KDa 100KDa distribution between nuclear and cytoplasmic fractions Liver Nuclear and Cytoplasmic Fractions Moved to nucleus Hib : Euth Hibernating vs. Euthermic Muscle Hib : Euth Hibernating vs. Euthermic (57 KDa and 100 KDa) rotein Expression in Liver Liver Nuclear Fraction (57 KDa and 100 KDa) rotein Expression in Muscle Nuclear and Cytoplasmic Fractions Muscle Nuclear Fraction Liver Cytoplasmic Fraction Muscle Cytoplasmic Fraction 57KDa 100KDa 57KDa 100KDa 57 kda 100 kda 57 kda 100 kda Nucleus Cytoplasm E H E H Nucleus Cytoplasm E H E H Timecourse in Heart CuZn SOD AFAR1 HO-1 Active in Entrance cold room (37 C) (T-drop) Early Hibern. (~5-7 C) Late Hibern. (~5-7 C) Early Arousal (T-gain) Fully Aroused (37 C) protein in early and late hibernation Up-regulation cascade. eroxiredoxins Detoxify / reduce hydroperoxides Expressed at high levels ARE in promoter region of rdx genes activated BAT Heart scanned intensity of hibernating versus euthermic Ratio band intensity Hib : Euth eroxiredoxin protein levels rdx1 Euth Hib Brown adipose tissue rdx2 Euth Hib Heart rdx3 Euth Hib peroxiredoxin-1 peroxiredoxin-2 peroxiredoxin-3 10

11 eroxiredoxin Activity rotein level correlates with increased activity Assays in BAT and heart with thioredoxin, thioredoxin reductase and NADH: Kim et al., 2005 Ratio of hibernating versus euthermic activity Activity ratio Hib : Euth Relative activity: Hibernating: euthermic Brown adipose tisuue BAT Heart Nrf Conclusion Activation of the pathway: Activated in early-late torpor, along with downstream gene protein products Increased RDX, HO & TRX protein and activity Result: Detoxification of ROS, intracellular signaling control GENE CHIS Confirm by RT-CR, Northern blots Tf Data Leads Downstream genes ROLE & CONTROL OF SYSTEM Transgenics Cell Assay RNAi Knock out Epigenetics FUNCTIONAL ASSAYS TRANSCRITION FACTOR ROFILING ELISAs in plates Confirm by EMSA rotein levels - enzyme assay - antibodies : protein - functional analysis e.g. HIF EO Where do we go from 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 DIRECTIONS 11

12 RIMATE!! GREY MOUSE LEMUR Hibernation and medicine rimates!! Novel phosphorylations Atrophy Hypertrophy AT i rotein Kinase rotein hosphatase AD 12

13 Turning it all off Epigenetics in Adaptation Life span extension Unavoidable metabolic costs 13

14 Unity through Evolution WWCeD J. STOREY S. EDDY D. HITTEL J. MacDONALD A. FAHLMAN. MORIN C. HOLDEN H. MEHRANI J. NI M. HASATOU S. TESSIER M. WU S. BROOKS C. FRANK J. HALLENBECK D. THOMAS A. RUBTSOV J. STEWART Funded by NSERC Canada 14