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

ho

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