Cellular Regulation of
Anabolism and Catabolism
Erich Roth
Medizinische 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 Syndrome
Herridge 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 yr
30 to 40 % until the age of 80 yr
up to 30 % between 80 to 90 yr
• Reduction of muscle strength
> 60yr
3 % loss of grip stength per year: male
4 % loss
female
Strength, but not muscle mass, is
associated with mortality in older adults.
AB. Newman et al. J Gerontology 61A;2006
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
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
Muscle mass, muscle strength, and muscle fat
infiltration as predictors of incident mobility
limitations in well-functioning older persons
M Visser et al, J Gerontol A Biol Sci Med Sci (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)
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)
Signalling
IGF-1
mechanischer Reiz
_
Integrine/Vinculin/Talin
IRS-1
RAS
PI3K
SHIP2
PTEN
PI3K
RAF
Akt-1
Cain
MCIP1 Ca+-Calmodulin
Glucocorticoide
NFAT
Akt-1
+
MEK
mTOR
Calcineurin
Apoptosis
(BAD / BAX)
_
FOXO
mTOR
MGF
Nucleus
+
Rapamycin
+ FKBP12
amino acids
ERK
PHAS1 = 4E-BP1
Hypertrophy
GSK3ß
eIF-2B
Translation
initiator
P70S6K
Translation-initiator
+ elongation
Proliferation
MAFbx
MURF1
eIF-4E
Translation-initiator
DNA
Genome
Transkription
RNA
Translation
Protein
Metabolic
action
Proteome
Metabolome
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
Energy
regulation
Occurrence of
oxygen radicals
Stimulation of
Protein degradation
Protein degradation
Muscle protein breakdown and the critical role of
the ubiquitin-proteasome pathway in normal and
disease states
Abnormal proteins
Short-lived normal proteins
UbiquitinProteasome
pathway
Long-lived normal proteins
Proteins of the
Endoplasmic Reticulum
Lysosomes
Extracellular proteins
Surface Receptors
Mitochondrial proteins
Mitochondrial
proteases
SH Lecker et al, J Nutr (1999)
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 metabolism
Oxygen 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
Starvation (4 days)
Abdominal Sepsis
(Survivors)
Abdominal Sepsis
(Nonsurvivors)
10
Injury & Infection
mmol/l
20
Postop.
control
Muscle Glutamine
0




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 547552
AMPK pathway
ATP↓
GLUTAMINE
DEPLETION
catabolic pathways ↑
AMPK
anabolic pathways ↓
protein synthesis ↓
?
Cellular redox state
GSH↓
TRX:ASK1 ↓
redox-sensitive
kinases
JNK
NF-kB
AP-1
Osmo-signaling
cell volume ↓
Fas ↑
delayed Hsp70-induction
Erk ↓
p38 ↓
Translation
p70s6k
mTOR ↓
4E-BP1
autophagic-proteolysis ↑
translation ↓
...
glutaminyl-tRNA synthetase
QRS:Gln:ASK1 complex ↓
JNK
apoptosis
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
IL-6
TNF
IL-6
The
anti-inflammatory
effect of exercise
AM Petersen et al, J Appl Physiol (2005)
Metabolic effects of IL-6 released by
muscle fibers
IL-6
IL-6
IL-6
AM Petersen et al, J Appl Physiol (2005)
Geoff Goldspink
Royal Free & University College Medical
School, London, UK
MGF and the regulation of
muscle strength.
Alternative Splicing of the Human
IGF-I Gene
3
Promoter 1
2
IGF - IEb
5
4
3
6
Promoter 2
Human IGF–I gene
Mechanical
signals including
local cell damage
6
IGF – IEa (systemic)
5
4
3
Hormones
effect on the
liver
1
4
3
4
MGF (autocrine)
reading frame
shift
5
6
*
49 base insert
The effect of treating the muscles of mdx dystrophic mice with MGF
in a plasmid vector after only 3 weeks on muscle strength
Percentage of treated muscle tetanic
force compared to their contralateral
untreated muscle (%)
40
***
35
30
25
20
15
10
5
0
-5
-10
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 acidinduced 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 acids
J Bohé et al, J Physiol (2001)
Time course of
serum insulin and glucose
Time course of rate of synthesis of
mixed muscle proteins
Rates of synthesis
of mixed muscle proteins and muscle fractions
(myofibrillar, sarcoplasmic and mitochondrial proteins)
J Bohé et al, J Physiol (2001)
Protein synthesis during and postexercise
• 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…..
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•
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•
•
Therapeutical interventions:
Exercise – electrostimulation (?)
Appropriate nutrition
Endocrine and mediator-directed strategies
Behavior, emotional interactions – well-being,
anti-stressing
Muscle Fibre Size After Injection Of MGF
cDNA
1200
1000
25% increase
in x sectional
area
number of fibres
800
injection
control
600
400
200
0
up to 800 801-2000 2001-3000 3001-4000 4001-5000 5001-6000
area of fibres (square micrometer)
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 stretchinduced 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
Disuse of Skeletal Muscle
Disuse
Conditions:
Triggers &
Signals:
Reduced muscle tension
(e.g. bedrest, immobilization, denervation,
unloading, spaceflight)
• Akt
• mTOR
• p70S6kinase
• 4E-BP1
• Glucocorticoids
• myostatin
• NF-kappaB
• Reactive oxygen species
or
Target
Systems:
Protein synthesis
rate
Protein degradation
rate
RW Jackman, SC Kandarian. Am J Physiol Cell Physiol (2004)
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)