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

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

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

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

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)

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

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

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 GLGL Rheb-GTP Rheb-GDP RAPTOR mTOR TSC2 TSC1 TCTP(?) Substrate

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 Rheb-GTPRheb-GDP TSC2 TSC1 Akt ERK2MK2 GSK3 AMPK REDD

Amino Acids Branched-chain AA – Leucine, isoleucine, valine – Rag-GTPase – Ragulator AA-sensitive GEF – Translocation to Rheb-rich lysosomes GLGL RagB-GTP Rag-GDP RAPTOR mTOR TSC2Ragulator Rab7/ lysosome Sanack & al., 2008 Rheb-GTP AA-starved mTOR is distributed through the cytoplasm, and becomes localized to lysosomes rapidly on AA feeding

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)

IGF-1 promotes muscle growth Infused into muscle (not systemic) – Activation of Akt, mTOR – p70S6k, 4EBP1 Adams & McCue 1998

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

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)

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)

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

Correlation and causation Muscle mass gain after 6 weeks HFES correlates with p70S6k phosphorylation at 6 hours. (Baar & Esser 1999) Fold phosphorylation of p70S6k Type II fiber area Placebo Protein Fiber size after 3 weeks training vs p70S6k phosphorylation. (Hulmi & al 2009)

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)

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

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

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

Elongation Elongation Cycle eEF1 Cycle Fig eEF2 cycle

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

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

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