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Hypertrophic signalling

Hypertrophic signalling

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Hypertrophic signalling

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  1. Hypertrophic signalling • Identify contraction-induced growth signals • Describe the composition and regulation of mTORC1 • Describe the effectors of mTOR • Explain the role of mTOR in muscle hypertrophy • Muscle contraction • Diet • Growth factors

  2. Consequences of contraction • Intracellular calcium increase • ATP (energy) turnover • Muscle: Oxygen depletion, AMP accumulation • Systemic: nutrient mobilization • Membrane permeability • Growth factor release • Peptides: IGF-1, FGF, HGF • Lipids: PGF2a, PGE2 • Systemic hormones • Insulin, GH, adrenaline

  3. Exercise induces mTOR activity • Rats trained to lift 60%BW vest • Phosphorylation by WB • Protein synthesis over 16 h • Rapamycin blocks Akt phosphorylation mTOR phosphorylation Bolster & al., 2003 Kubica & al., 2005

  4. Rapamycin blocks hypertrophy • Synergist ablation • Cyclosporin to block Cn • Rapamycin to block mTOR • CsA muscles hypertrophy • Rap muscles don’t Bodine & al., 2001

  5. Why mTOR? • Powerful, multiplex regulator of protein synthesis and growth • Translation efficiency • Translational regulation/selection • Protein degradation • Activated by diverse growth and function relevant stimuli • Contraction/exercise • Nutrients • Hormones (insulin, IGF, HGH)

  6. Mammalian Target of Rapamycin Pro-growth stimuli mTORC mTOR Contraction p38 Protein synthesis (hypertrophy) Deldicque & al., 2005

  7. Two mTOR Complexes Rapamycin sensitive Rapamycin insensitive mTORC2 Composition mTOR GbL (mLST8) PRR5, mSin1 RICTOR Regulation Growth factors (PI3K/akt) mTORC1 (RICTOR) Targets Cytoskeleton (esp yeast) Proteasome (AktFOXO) Glycogen synthesis (GSK3) PKC • mTORC1 Composition • mTOR • GbL (mLST8) dispensible • PRAS40 • RAPTOR • Regulation • Growth factors (PI3K/akt) • Nutrients (TSC1/2, Rag) • Redox • Targets • Ribosomal biogenesis (p70S6k) • Translation (4EBP1) • Autophagy

  8. Core mTORC1 control • Active complex requires Rheb-GTP • Rheb GTPase • GTPase-Activating Protein (GAP) • Guanine Exchange Factor (GEF) • mTOR autophos S2481 • TSC 1/2 • Tuberous Sclerosis Complex • Major site of GF/energy reg. • GEF unknown/unnecessary • Translationally Controlled Tumor Protein TSC1 Rheb-GDP TSC2 TCTP(?) Rheb-GTP mTOR GbL Substrate RAPTOR

  9. Growth Factors and “Energy” • Phosphatidylinositol 3’ kinase (PI3K) • PIP2PIP3 • PDK1 • Akt • Extracellular-signal Regulated Kinase (ERK) • P38MK2 • AMPK (activates TSC2) • GSK3 (activates TSC2) • Hypoxia • HIFREDD ERK2 MK2 Akt GSK3 TSC1 TSC2 AMPK Rheb-GTP Rheb-GDP REDD

  10. Amino Acids • Branched-chain AA • Leucine, isoleucine, valine • Rag-GTPase • Ragulator AA-sensitive GEF • Translocation to Rheb-richlysosomes Rag-GDP TSC2 Ragulator RagB-GTP RAPTOR Rab7/ lysosome GbL mTOR Rheb-GTP AA-starved mTOR is distributed through the cytoplasm, and becomes localized to lysosomes rapidly on AA feeding Sanack & al., 2008

  11. Growth factors and overload • Insulin • Suppressed at low (<60% VO2max) intensity • Neutral at high (>80% VO2max) • Insulin-like growth factor-1 • Elevated after resistance exercise (up to 2 days) • Powerful growth stimulator • Insulin and IGF-1 Receptors • Insulin receptor substrate 1(IRS1) • PI3KAkt • ERK, p38, PLC IGF-1 expression after synergist ablation (Adams & al 2002)

  12. IGF-1 promotes muscle growth • Infused into muscle (notsystemic) • Activation of Akt, mTOR • p70S6k, 4EBP1 Adams & McCue 1998

  13. Overload seems independent of IGF-1 • Muscle hypertrophy by synergist ablation in IGF-1R knockout • Cardiac hypertrophy by swim-training in p70S6k knockout WT MKR-/- 35 d 7 d 0 d Plantaris mass after synergist ablation Spangenburg & al 2008 Heart weights after 8 weeks swimming (McMullen & al., 2004)

  14. Amino acid feeding • AA feeding alone increases mTOR &PS • Protein feeding with exercise gives much better/faster mTOR activation • No difference in hypertrophy (22 weeks) mTOR phosphorylation post-exercise with or without protein feeding (Hulmi & al 2009)

  15. Metabolic effects • Elevated AMP • AMP Kinase  TSC2 --| mTOR • Permissive? • GSK3 • InsulinAkt--|GSK3 • Oxygen • Hypoxia Inducible Factor REDDTSC2 • ROS directly oxidize cysteines AICAR-induced activation of AMPK blocks AA-induced protein synthesis (Pruznak & al., 2008)

  16. Intermediate summary • Exercise-related stresses tend to block mTOR during exercise and activate mTOR after exercise • Energetic stresses during exercise: Low O2, high AMP • Recovery processes/hormones after exercise • Nutrient mobilization • Insulin • IGF-1 • Acute mTOR signaling correlates with hypertrophy under normal conditions • Not in Insulin/IGF-1 receptor defective models • Not in p70 S6k defective models

  17. Correlation and causation 8000 6000 4000 Placebo Type II fiber area Protein 2000 0 5 10 15 20 Fold phosphorylation of p70S6k Muscle mass gain after 6 weeks HFES correlates with p70S6k phosphorylation at 6 hours. (Baar & Esser 1999) Fiber size after 3 weeks training vs p70S6k phosphorylation. (Hulmi & al 2009)

  18. mTOR effectors • Ribosome assembly • p70S6kRPS6 • 5’-TOP mRNAs (ribosome components) • Translational efficiency • 4EBP--|eIF4E • Cap dependent translation • Transcription factors • Akt/SGK--|FOXO • NFAT3, STAT3 • IRS-1 (negative feedback)

  19. Protein translation • Initiation • eIF4 recognition and melting of 7’mG cap • eIF4E cap-binding subunit • 4EBP competition with eIF4F scaffold • Recruit 40S ribosome • met-tRNA • eIF2 GTP-dependent met-tRNA loader • Recruit 60S ribosome • Start codon

  20. Initiation Transition to elongation Pre-initiation complex Fig 17-9

  21. Protein translation • Elongation • tRNA recruitment • eEF1 GTP-dependent tRNA carrier • GTP hydrolysis with peptide bond formation • Ribosome advance • eEF2 GTP-dependent procession • GTP hydrolysis with advance

  22. eEF1 Cycle Elongation Cycle Elongation eEF2 cycle Fig 17-10

  23. 3’ untranslated region structure • Post-transcriptional control • 2° and 3° structure of mRNA • Analogous to DNA promoter • 5’ Tract of Oligopyrimidines • Ribosomal proteins • eEF1, eEF2 • “Highly structured” 5’ cap • Ribosome scanning • Growth factors, cell cycle control • Internal Ribosome Entry Site (IRES) • Inflammation, angiogenesis Phosphorylated RPS6 favors these Active eIF4 complex favors these

  24. Species differences • Most proteins conserved yeast-human • Regulatory processes differ • S cerevisiae have 2 TORs • Drosophila akt doesn’t directly regulate TSC2 • C Elegans has no TSC1/2; transcriptional repression of RAPTOR via FOXO • S cerevisiae mTOR independent of Rheb

  25. Summary • High force contractions induce multiple signaling modes • Metabolites, growth factors, mechanical • Hypertrophy closely linked with mTOR • GF signaling • Metabolite signaling • mTOR is a powerful control of protein accretion • Makes more ribosomes via p70S6k • General translation efficiency via 4EBP • Reduced degradation via FOXO, NFAT3