hypertrophic signalling
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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. - PowerPoint PPT PresentationTRANSCRIPT
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
ph
osp
ho
ryla
tion
mT
OR
ph
osp
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ryla
tion
Bolster & al., 2003Kubica & 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 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
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
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
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
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)
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
WT MKR-/-
35 d7 d0 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– InsulinAkt--|GSK3
• Oxygen– Hypoxia Inducible Factor
REDDTSC2– 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)
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)
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)
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
InitiationPre-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 CycleeEF1 Cycle
Fig 17-10
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