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

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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

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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

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up to 800 801-2000 2001-3000 3001-4000 4001-5000 5001-6000

area of fibres (square micrometer)

numb

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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)