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Cellular Regulation of Anabolism and Catabolism
Erich RothMedizinische Universität Wien
Klinik für Chirurgie/Forschungslaboratorien
Cellular Regulation of Anabolism and Catabolism
• Amino acid metabolism• Protein synthesis• Proteindegradation• Energy metabolism
• Hormons, Interleukins, growth factors • Signaling molecules • Neuromuscular disorder
Composition of human muscle
g/kg
Dry solid 230
Extracellular water 120
Intracellular water 650
Three ways to atrophy of skeletal muscle
• Protein catabolism mediated by catabolic factors
• Disuse of skeletal muscle• Sarcopenia of old age
One-Year Outcomes in Survivors of the Acute Respiratory Distress SyndromeHerridge MS et al. New England J Med 348 (2003)
• …the patients have persistent functional limitation one year after discharged from the ICU largely as a result of muscle wasting and weakness
• ..the impaired muscle function had an important effect on the long term outcomes on these patients
Disuse: Immobilisation of the knee – effect on thight muscles
Four weeks:
loss of 53 % of muscle strength of knee extension
Quadriceps circumference was decreased for 21 %
Sarcopenia of Old Age• Reduction of muscle mass:
10 % until the age of 60 yr30 to 40 % until the age of 80 yr
up to 30 % between 80 to 90 yr
• Reduction of muscle strength> 60yr3 % loss of grip stength per year: male4 % loss female
Strength, but not muscle mass, is associated with mortality in older adults.
AB. Newman et al. J Gerontology 61A;2006
Men, grip strength, and mortality. Kaplan–Meier survival curves for grip strength groups (<30, 30–<40, 40–<50, 50 kg). Intervals of 10 kg of grip strength were used to approximate men's standard deviation = 8.5 and to distribute the number of events
Men, leg strength, and mortality. Kaplan–Meier survival curves for leg strength groups (<90, 90–<130, 130–<170, 170 Nm). Intervals of 40 Nm of quadriceps strength were used to approximate men's standard deviation = 33.8 and to distribute the number of events
M Visser et al, J Gerontol A Biol Sci Med Sci (2005)
Muscle mass, muscle strength, and muscle fat infiltration as predictors of incident mobility limitations in well-functioning older persons
Muscle fiber specific apoptosis and TNF-alpha signaling in sarcopenia are attenuated by life long calorie restriction
Phillips T et al. FASEB 19 (2005)
Muscle fiber specific apoptosis and TNF-alpha signaling in sarcopenia are attenuated by life long calorie restriction
Phillips T et al. FASEB 19 (2005)
Sarcopenia: An inflammatory process with an increased insulin
resistance?
Regulation of atrophy
Catabolic stimuli in critically ill patients: supposed causes
• Increased catabolic/Inflammatory mediators
• Reduced anabolic stimulators
• Polyneuropathy• Loss of electrical excitability of the
sarcolemma• Neuromuscluar blockade by drugs
Catabolic factors of skeletal muscle catabolism
• Interleukins: TNF-α, IL-1, IL-6, IFN-γ• Myostatin: member of transforming
growth factor family (increased in AIDS)• Glucocorticoids (affecting the IGF-I
pathway)
• Reduced levels of growth hormone• testosteron
Denervation
• Denervated muscle quickly atrophy due to increased proteolysis
• Long-term denervation leads to myofibre death
• Denervation as a result from blocked signals of physical disruption at the neuromuscular junction (synapse)
Nucleus
Proliferation
MAFbxMURF1
PHAS1 = 4E-BP1
eIF-4ETranslation-initiator
IGF-1
IRS-1PI3K
Akt-1 GSK3ßeIF-2BTranslationinitiator
mTOR
P70S6K
Translation-initiator + elongation
Rapamycin + FKBP12
amino acids
RAS
RAF
MEK
ERK
SHIP2PTEN
FOXO
Glucocorticoide
+
Apoptosis(BAD / BAX)
_
+
_
mechanischer Reiz
Integrine/Vinculin/Talin
PI3K
Akt-1
mTOR
NFAT
MGF
Ca+-Calmodulin
Calcineurin
CainMCIP1
Hypertrophy
Signalling
DNA
Protein
RNA
Metabolic action
Transkription
Proteome
Translation
Metabolome
Genome
Gene regulation
Transcriptional profile of a myotube starvation model of atrophy.
E.J.Stevenson et al. J Appl Physiology 98;2005
Altered Marker genes
• Muscle contraction (4)• Structural components (6)• Cytoskeleton (4)
• Proteolytic enzyms (3)• Biosynthesis (3)
• Oxidative stress (1)• Signaling (15)• Growth factors (9)
Protein level
Protein synthesis Protein degradation
Protein synthesis
Stress and protein turnover
Slow wasting condition is found in mild injury, mal-nutrition, cancer, immobilization.
Rapid wasting occurs after severe injury, burns and infection.
One genome - different proteomes
• > 35,000 coding genes in higher organisms• Differently spliced• Posttranslational modified• 100,000 proteins / organism• Selectively expressed in tissues and cells• > 20,000 proteins / cell
Proteomic analysis of rat soleus muscle undergoing hindlimb suspension-induced atrophy
RJ Isfort et al, Proteomics 2 (2002)
Proteomic analysis of altered protein expression in skeletal muscle of rats in a hypermetabolic state
induced by burn sepsis
X Duan et al, Biochem J (2006)
Significantly regulated proteins in EDL muscle following burn-CLP treatment
X Duan et al, Biochem J (2006)
Proteomics
Stimulation of Protein degradation
Occurrence of oxygen radicals
Energy regulation
Protein degradation
SH Lecker et al, J Nutr (1999)
Muscle protein breakdown and the critical role of the ubiquitin-proteasome pathway in normal and
disease states
Abnormal proteins
Short-lived normal proteins
Long-lived normal proteins
Proteins of the Endoplasmic Reticulum
Extracellular proteinsSurface Receptors
Mitochondrial proteins
Ubiquitin-Proteasome
pathway
Lysosomes
Mitochondrialproteases
Disuse
• Calcium overload• Increased calpain activity, increased
degradation of sarcomeric proteins• Stimulation of calpases activities (ROS?)• Degradation of intact actomyosin
complexes • Proteasome system degrade monomeric
contractile proteins
Proteasome mediated proteolysis
• Either by 20S or 26S proteasome• 26S is composed of the 20S core proteasome with a
regulatory 19 S complex connected to each end• 19S complex posses ATPase activity
• 26S pathway – ubiquitin convalently binds to protein substrates
• Unfolding of protein by 19S ATP dependent process
• Degradation of the protein in the 20S core proteasome (oxidized protein degradated without ubiquitination)
..moreover• Ubiquitin-activating enzymes (E1)• Ubiquitin-conjugating enzymes (E2) Ubiquitin
protein ligase enzymes (E3)
• Specific ligases: atrogin 1 muscle ring finger-1 (both upregulated by ROS) out of stimulation of two
genes: MAFbx (muscle RING Finger !) MuRF1 (muscle Atrophy F-box) (upregulated 10 times by IL-1 and Dexamthason)
B Biedermann, Schweiz Med Forum 11 (2002)
Metabolism
Energy metabolismOxygen Radicals
Energy metabolism in sepsis• Decrease in oxygen extraction – increase
in tissue oxygen tension• Reduced levels of ATP and ADP• Increased level of AMP – AMPK activation• Mitochondrial dysfunction through an
impairment in complex I mediated respiration
• 30 % reduction in the ability to utilize oxygen during exercise
Consequences of AMPK activation on metabolism during single bouts of exercise
WW Winder, J Appl Physiol. 2001; 91:1017-28
Decreased antioxidative metabolites in skeletal muscle
• Chemoluminescensce + 100 %• Mn-Superoxiddismutase - 46 %• Catalase - 83 %• Glutathioneperoxidase - 55 %
S.Llesuy et al. Free Rad Biol Med 16 (1994)
Amino acid Metabolism
Roth E, et al.: Clin Nutr 1:25, 1982 Vinnars E, et al.: Ann Surg 182:665, 1975 Askanazi J, et al.: Ann Surg 192:78, 1980 Elwyn DH, et al.: in Walser M & Williamson JR (eds.) Metabolism and Clinical Implications
of Branched Chain Amino Acids and Ketoacids. Elsevier NY 1981, pp 547-552
0
10
20co
ntro
l
Pos
top.
Inju
ry &
Infe
ctio
n
Abd
omin
al S
epsi
s (S
urvi
vors
)
Abd
omin
al S
epsi
s (N
onsu
rviv
ors)
Sta
rvat
ion
(4 d
ays)
mm
ol/l
Muscle Glutamine
AMPK pathway
Cellular redox state
Osmo-signaling
Translation
glutaminyl-tRNA synthetase
ATP↓ AMPK
GSH↓ redox-sensitive kinases
cell volume ↓
Fas ↑
Erk ↓
p38 ↓
mTOR ↓p70s6k
4E-BP1
GLUTAMINEDEPLETION
...
QRS:Gln:ASK1 complex ↓ JNK
apoptosis
catabolic pathways ↑anabolic pathways ↓protein synthesis ↓
TRX:ASK1 ↓ JNK
translation ↓
autophagic-proteolysis ↑
?
delayed Hsp70-induction
NF-kB
AP-1
Anabolic Regulation
Muscle anabolism
• Increased IGF-I, especially of one isoform stimulated especially by strechting exercise
• Growth hormone• Testosterone• IL-12, IL-15• Overexpression of the oncogene „ski“
Growth factors, especially IGF-I, affect neuronal function
• Myelination• Prevention of apoptotic death• Stimulation of axonal sprouting• Repair of damaged axons
Any hope ?Which concepts are reliable?
Electrostimulation
• Fast-to-slow muscle conversion
• Increased respiratory chain activity and efficiency
• Increased amount of cytochrome a, a3, b562, c, c1
STRETCHING
G.Goldspink et al. J.Physiol.1999:516
AM Petersen et al, J Appl Physiol (2005)
The anti-inflammatory effect of exercise
TNF
IL-6
IL-6
AM Petersen et al, J Appl Physiol (2005)
Metabolic effects of IL-6 released by muscle fibers
IL-6
IL-6
IL-6
MGF and the regulation of muscle strength.
Geoff GoldspinkRoyal Free & University College Medical School, London, UK
MGF (autocrine) reading frame shift 49 base insert
*
3 4 5 2
Human IGF–I gene
Mechanical signals including local cell damage
3 4 5
3 4Hormones effect on the liver
Alternative Splicing of the Human IGF-I Gene
IGF – IEa (systemic)
IGF - IEb
643
5
Promoter 2Promoter 1
1 6
6
The effect of treating the muscles of mdx dystrophic mice with MGF in a plasmid vector after only 3 weeks on muscle strength
-10
-5
0
5
10
15
20
25
30
35
40
Perc
enta
ge o
f tre
ated
mus
cle
teta
nic
forc
e co
mpa
red
to th
eir c
ontra
late
ral
untre
ated
mus
cle
(%)
MGF Vector
***
MGF peptide use for generic treatment for damage and repair
1) Rescue and repair of muscle in muscular dystrophy, (ALS) and other diseases.
2) Postural problems arising from muscle weakness.
3) Muscle cachexia in cancer, HIV, COPD, cardiac and renal disease.
4) Age-related muscle loss – sarcopenia.
Nutrition
• Can reduce but not avoid protein catabolism
• Exert anabolic effect
Short-term bed rest impairs amino acid-induced protein anabolism in humans
• 14 day period of strict bed rest or controlled ambulation
• Weight-maintaining diet
• 3h infusion of an amino acid mixture• Determination of whole-body protein kinetics
Bed rest leads to reduced stimulation of protein synthesis by amino acid administration
G Biolo et al, J Physiol (2004)
Latency and duration of stimulation of human muscle protein synthesis during
continuous infusion of amino acidsJ Bohé et al, J Physiol (2001)
Time course of rate of synthesis of mixed muscle proteins
Time course of serum insulin and glucose
J Bohé et al, J Physiol (2001)
Rates of synthesis of mixed muscle proteins and muscle fractions
(myofibrillar, sarcoplasmic and mitochondrial proteins)
Protein synthesis during and post-exercise
• Protein synthesis is decreased during exercise• PS is increased post exercise untill hr 38
• Exercise stimulates protein translation by increasing the group 4 eurkaryotic initiation factor eIF4E
• Post meal exercise composition (CH+ proteins/AAs) influence the availability of eIF4E
Pathophysiology: Impressive for me…
• Prognostic importance of muscle strength and fat-infiltration
• Role of oxygen radicals in protein degradation– antioxidants
• Caloric restriction: reduced degradation process – insulin resistance ??
• Role of IL- 6: inflammatory vs anabolic stimulus?
Conclusion• Pathogenesis of protein catabolism is
multifactorial…..
• Therapeutical interventions:• Exercise – electrostimulation (?)• Appropriate nutrition• Endocrine and mediator-directed strategies• Behavior, emotional interactions – well-being,
anti-stressing
0
200
400
600
800
1000
1200
up to 800 801-2000 2001-3000 3001-4000 4001-5000 5001-6000
area of fibres (square micrometer)
numb
er of
fibres injection
control
25% increase in x sectional area
Muscle Fibre Size After Injection Of MGF Muscle Fibre Size After Injection Of MGF cDNAcDNA
Molecular events after stretching
• Increased myoblast proliferation• Increased COX2 mRNA stimulation of
mTOR and rapamycin-p70 S6 kinase• Satellite cell activation by HGF and NO • Increase of mechano growth factor (MGF)
Coupling of Voltage-sensitive Sodium Channel Activity to Stretch-induced Amino Acid Transport in Skeletal Muscle in Vitro
• Stretching of tissue-cultured skeletal myotobes stimulates amino acid uptake
• Serum factors are not required for stretch-induced AA uptake
• Alterations in the cell‘s voltage-sensitive sodium channel and sodium pump activity
HH Vandenburgh, S Kaufman, J Biol Chem (1982)
Ubiquitin – Proteasom System
Mitch et al., New England Journal of Medicine 1996
Disuse Spinal cord isolation:
• Atrophy of slow and fast extensor muscles• The slow rat soleus atrophied by ~ 50 % within 15 days• Myofibrillar protein content, myosin heavy chain ~ 50 % • Actin maintained at control level
Molecular mechanism: • reduction in ribosomal RNA and protein translational
capacity• Unsufficient RNA substrate for translating key
-sarcomeric proteins compromising the myofibril fraction
DisuseConditions:
Triggers &Signals:
TargetSystems:
Reduced muscle tension(e.g. bedrest, immobilization, denervation,
unloading, spaceflight)
• Akt• mTOR• p70S6kinase• 4E-BP1
• Glucocorticoids• myostatin• NF-kappaB• Reactive oxygen species
Protein synthesisrate
Protein degradationrate
or
RW Jackman, SC Kandarian. Am J Physiol Cell Physiol (2004)
Disuse of Skeletal Muscle
Cell volume and hormone action
TIPS-October 1992 [Vol.13]Current awareness
Muscle tissue changes with aging
• Reduction in muscle cell number, in muscle twitch time and force
• Reduction in sarcoplasmic reticulum• Reduction in calcium pumping capacity• Disorganisation of sarcomere spacing• Centralisation of muscle nuclei along the
muscle fiber
Muscle Cachexia: Current Concepts of Intracellular mechanism and Molecular Regulation
PO Hasselgren, JE Fischer Ann Surg 233 (2001)