Transcript Calcium Signaling

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
Akt phosphorylation
Rats trained to lift 60%BW vest
Phosphorylation by WB
Protein synthesis over 16 h
Rapamycin blocks
mTOR phosphorylation
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Kubica & al., 2005
Bolster & al., 2003
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
Pro-growth stimuli
mTORC
Contraction
mTOR
p38
Protein synthesis
(hypertrophy)
Deldicque & al., 2005
Two mTOR Complexes
Rapamycin sensitive
• mTORC1 Composition
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mTOR
GbL (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
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mTOR
GbL (mLST8)
PRR5, mSin1
RICTOR
• Regulation
– Growth factors (PI3K/akt)
– mTORC1 (RICTOR)
• Targets
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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
TSC1
• TSC 1/2
– Tuberous Sclerosis Complex
– Major site of GF/energy reg.
Rheb-GDP
TSC2
TCTP(?)
Rheb-GTP
GbL
mTOR
RAPTOR
• GEF unknown/unnecessary
– Translationally Controlled Tumor Protein
Substrate
Growth Factors and “Energy”
• Phosphatidylinositol 3’ kinase (PI3K)
– PIP2PIP3
– PDK1
– Akt
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Extracellular-signal Regulated Kinase (ERK)
P38MK2
ERK2
MK2
Akt
AMPK (activates TSC2)
GSK3
TSC1
GSK3 (activates TSC2)
TSC2
AMPK
Hypoxia
Rheb-GTP
Rheb-GDP
– HIFREDD
REDD
Amino Acids
• Branched-chain AA
– Leucine, isoleucine, valine
– Rag-GTPase
– Ragulator AA-sensitive GEF
– Translocation to Rheb-rich
lysosomes
Rag-GDP
TSC2
Ragulator
RagB-GTP
GbL
RAPTOR
mTOR
Rab7/
lysosome
Rheb-GTP
Sanack & al., 2008
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
WT
MKR-/35 d
7d
0d
Plantaris mass after synergist
ablation Spangenburg & al 2008
Heart weights after 8 weeks
swimming (McMullen & al., 2004)
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
Type II fiber area
8000
6000
4000
2000
0
Muscle mass gain after 6 weeks HFES
correlates with p70S6k phosphorylation at 6
hours. (Baar & Esser 1999)
Placebo
Protein
5
10
15
Fold phosphorylation of p70S6k
20
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
Fig 17-9
Transition to elongation
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
eEF1 Cycle
Elongation Cycle
eEF2 cycle
Fig 17-10
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
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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