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![Page 1: Hypertrophic signalling Identify contraction-induced growth signals Describe the composition and regulation of mTORC1 Describe the effectors of mTOR Explain.](https://reader030.fdocuments.net/reader030/viewer/2022032709/56649eab5503460f94bb154a/html5/thumbnails/1.jpg)
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
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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
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Exercise induces mTOR activity• Rats trained to lift 60%BW vest• Phosphorylation by WB• Protein synthesis over 16 h• Rapamycin blocks
Akt
ph
osp
ho
ryla
tion
mT
OR
ph
osp
ho
ryla
tion
Bolster & al., 2003Kubica & al., 2005
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Rapamycin blocks hypertrophy• Synergist ablation
– Cyclosporin to block Cn– Rapamycin to block mTOR
• CsA muscles hypertrophy• Rap muscles don’t
Bodine & al., 2001
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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)
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Mammalian Target of Rapamycin
Deldicque & al., 2005
mTORC
Pro-growth stimuli
mTOR
Protein synthesis(hypertrophy)
Contraction p38
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Two mTOR ComplexesRapamycin sensitive
• mTORC1 Composition– mTOR– GL (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– GL (mLST8)– PRR5, mSin1– RICTOR
• Regulation– Growth factors (PI3K/akt)– mTORC1 (RICTOR)
• Targets– Cytoskeleton (esp yeast)– Proteasome (AktFOXO)– Glycogen synthesis (GSK3)– PKC
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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
GL
Rheb-GTP
Rheb-GDP
RAPTOR
mTOR
TSC2
TSC1
TCTP(?)
Substrate
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Growth Factors and “Energy”• Phosphatidylinositol 3’ kinase (PI3K)
– PIP2PIP3– PDK1– Akt
• Extracellular-signal Regulated Kinase (ERK)• P38MK2• AMPK (activates TSC2)• GSK3 (activates TSC2)• Hypoxia
– HIFREDDRheb-GTP Rheb-GDP
TSC2TSC1
AktERK2 MK2
GSK3
AMPK
REDD
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Amino Acids• Branched-chain AA
– Leucine, isoleucine, valine– Rag-GTPase– Ragulator AA-sensitive GEF– Translocation to Rheb-rich
lysosomes GL
RagB-GTP
Rag-GDP
RAPTOR
mTOR
TSC2 Ragulator
Rab7/ lysosome
Sanack & al., 2008
Rheb-GTP
AA-starved mTOR is distributed through the cytoplasm, and becomes localized to lysosomes rapidly on AA feeding
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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)– PI3KAkt– ERK, p38, PLC IGF-1 expression after synergist
ablation (Adams & al 2002)
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IGF-1 promotes muscle growth• Infused into muscle (not
systemic)– Activation of Akt, mTOR– p70S6k, 4EBP1
Adams & McCue 1998
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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
WT MKR-/-
35 d7 d0 d
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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)
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Metabolic effects• Elevated AMP
– AMP Kinase TSC2 --| mTOR– Permissive?
• GSK3– InsulinAkt--|GSK3
• Oxygen– Hypoxia Inducible Factor
REDDTSC2– ROS directly oxidize cysteines
AICAR-induced activation of AMPK blocks AA-induced protein synthesis (Pruznak & al., 2008)
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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
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Correlation and causation
Muscle mass gain after 6 weeks HFES correlates with p70S6k phosphorylation at 6 hours. (Baar & Esser 1999)
2000
4000
6000
8000
0 5 10 15 20
Fold phosphorylation of p70S6k
Typ
e II
fib
er
are
a
PlaceboProtein
Fiber size after 3 weeks training vs p70S6k phosphorylation. (Hulmi & al 2009)
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mTOR effectors• Ribosome assembly
– p70S6kRPS6– 5’-TOP mRNAs (ribosome components)
• Translational efficiency– 4EBP--|eIF4E– Cap dependent translation
• Transcription factors– Akt/SGK--|FOXO– NFAT3, STAT3
• IRS-1 (negative feedback)
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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
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InitiationPre-initiation complex Transition to elongation
Fig 17-9
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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
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Elongation
Elongation CycleeEF1 Cycle
Fig 17-10
eEF2 cycle
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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
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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
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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