86.Sarcopenia Prevention by Growth Hormone

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86.Sarcopenia Prevention by Growth Hormone

 

 

 

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Abbreviations …………………………………………………………………………………………………………….. 8

Figures and Tables ……………………………………………………………………………………………………. 11

INTRODUCTION ………………………………………………………………………………………………….. 13

REVIEW ………………………………………………………………………………………………………………… 17

Chapter 1: What is Sarcopenia? ………………………………………………………………………………. 18

  1. Definitions of sarcopenia …………………………………………………………………………………… 18

1.1. The origins of the word “Sarcopenia” …………………………………………………………… 18

1.2. First definitions based only on muscle mass ………………………………………………….. 18

1.3. Limits of only using muscle mass to define sarcopenia …………………………………… 19

1.4. Consensus definitions of sarcopenia ……………………………………………………………… 20

1.5. Convergences and differences of the various definitions …………………………………. 22

1.5.1. Sarcopenia as a syndrome not a disease ……………………………………………… 22

1.5.2. Not only muscle mass ………………………………………………………………………. 23

1.5.3. Diagnosis and strategy of case finding ……………………………………………….. 24

1.6. Prevalence of Sarcopenia …………………………………………………………………………….. 26

  1. Making a Diagnosis of sarcopenia ………………………………………………………………………. 28

2.1. Muscle mass assessment ……………………………………………………………………………… 28

2.2. Strength assessment ……………………………………………………………………………………. 30

2.3. Physical performance assessment …………………………………………………………………. 32

  1. Muscle characteristic changes during aging leading to sarcopenia ………………………….. 33

3.1. Loss of muscle mass …………………………………………………………………………………… 33

3.2. Loss of muscle strength ………………………………………………………………………………. 35

  1. Chapter 1 abstract …………………………………………………………………………………………….. 37

Chapter 2: Sarcopenia-related cellular and molecular skeletal muscle alterations ……… 38

  1. Cellular and molecular mechanisms controlling proteins synthesis and degradation …. 38

1.1. Protein synthesis ………………………………………………………………………………………… 38

1.1.1. Transcriptional activity of muscle fiber ………………………………………………. 39

1.1.2. Translational activity of muscle fiber …………………………………………………. 39

1.2. Proteolysis systems …………………………………………………………………………………….. 44

1.2.1. Ca2+-dependent pathway: calpains and caspases ………………………………….. 44

1.2.2. Overview of the ubiquitine-proteasome-dependent system ……………………. 45

1.2.3. Overview of Autophagy ……………………………………………………………………. 47

1.2.4. UPS and autophagy regulation…………………………………………………………… 49

1.3. Myostatin: master regulator of muscle mass …………………………………………………. 52

  1. Role of Mitochondria in Cellular Homeostasis …………………………………………………….. 54

2.1. Mitochondrial biogenesis …………………………………………………………………………….. 54

2.1.1. Mitochondrial biogenesis pathway …………………………………………………….. 54

2.1.2. Mitochondrial biogenesis pathway up-streams …………………………………….. 56

2.2. Mitochondria as a source of reactive oxygen species ………………………………………. 58

2.3. The mitochondrial apoptotic machinery ………………………………………………………… 58

2.4. The dynamic nature of mitochondria …………………………………………………………….. 60

  1. Sarcopenia-related skeletal muscle alterations ……………………………………………………… 61

3.1. Protein turnover alterations ………………………………………………………………………….. 62

3.1.1. Sarcopenia-associated protein synthesis impairment…………………………….. 62

3.1.2. Sarcopenia-associated protein degradation impairment ………………………… 66

3.2. Mitochondria dysfunctions and sarcopenia ……………………………………………………. 69

3.2.1. Reduced mitochondrial content and function with age ………………………….. 69

3.2.2. The vicious cycle between oxidative stress and mitochondrial dysfunction in the aged musc. 70

3.2.3. Possible involvement of mitochondria dynamics in sarcopenia ……………… 71

3.2.4. Mitochondria-mediated apoptosis in sarcopenia ………………………………….. 72

3.3. Satellite cells impairment ……………………………………………………………………………. 74

  1. Chapter 2 abstract …………………………………………………………………………………………….. 76 5 Sarcopenia: Mechanisms and Prevention – Role of Exercise and Growth Hormone – Involvement of Oxidative Stress and Glucose-6-phosphate Dehydrogenase – 2014

Chapter 3: The contribution of oxidative stress to sarcopenia …………………………………… 77

  1. Generalities on oxidative stress ………………………………………………………………………….. 77

1.1. Definitions ………………………………………………………………………………………………… 77

1.2. Theories of aging related to oxidative stress …………………………………………………. 78

  1. Oxidative stress in sarcopenic skeletal muscle ……………………………………………………… 79

2.1. Increased RONS production in skeletal muscle is associated with sarcopenia ……. 79

2.1.1. Mitochondria as sources of RONS …………………………………………………….. 80

2.1.2. Free iron accumulation is associated with sarcopenia …………………………… 83

2.1.3. Increased Xanthine oxidase activity as source of RONS……………………….. 84

2.1.4. NADPH Oxidase and Nitric oxide Synthase as sources of RONS ? ……….. 85

2.2. Increased oxidative damage in skeletal muscle is associated with sarcopenia …….. 85

2.2.1. Protein oxidative damage: Protein carbonylation and nitrosylation ………… 86

2.2.2. Lipid oxidative damage: Lipid peroxidation………………………………………… 87

2.2.3. Nucleic acids oxidative damage…………………………………………………………. 87

2.3. Antioxidant defenses, aging and sarcopenia …………………………………………………… 89

2.3.1. Enzymatic antioxidant systems are impaired during aging and sarcopenia 90

2.3.2. Non enzymatic antioxidant systems are impaired during aging and sarcopenia …………….. 92

2.3.3. Repair systems seem to be impaired during aging………………………………… 93

2.4. Mechanistic links between oxidative stress and sarcopenia ……………………………… 93

2.4.1. Link between oxidative stress and impaired satellite cells activity …………. 93

2.4.2. Oxidative stress could disturb protein turn-over …………………………………… 94

2.4.3. Oxidative stress and muscle contractile qualities …………………………………. 96

  1. Chapter 3 abstract …………………………………………………………………………………………….. 97

Chapter 4: Strategies against sarcopenia ………………………………………………………………….. 98

  1. Exercise as the perfect strategy against sarcopenia ……………………………………………….. 98

1.1. Exercise during aging improves protein turnover …………………………………………… 99

1.2. Exercise during aging decreases apoptosis …………………………………………………… 101

1.3. Exercise during aging stimulates satellite cells …………………………………………….. 102

1.4. Exercise during aging improves mitochondrial functions and dynamics ………….. 103

1.5. Exercise during aging would restore a young redox status …………………………….. 104

  1. Alternative strategies to exercise for fighting sarcopenia ……………………………………… 106

2.1. Possible antioxidant strategies to attenuate sarcopenia ………………………………….. 106

2.2. Exercise and antioxidant supplementation at old age …………………………………….. 109

2.3. Hormones replacement-therapies as a possible strategy ………………………………… 111

  1. The Glucose-6-Phosphate Dehydrogenase as potential target to fight sarcopenia ……. 116

3.1. G6PDH biochemistry and regulation in skeletal muscle ………………………………… 116

3.2. G6PDH, NADPH, antioxidant defenses and sarcopenia ………………………………… 118

3.3. G6PDH, apoptosis and sarcopenia ……………………………………………………………… 120

3.4. G6PDH, NADPH, ribose-5-phosphate and sarcopenia ………………………………….. 121

  1. Chapter 4 abstract …………………………………………………………………………………………… 124 Table of

SYNTHESIS AND OBJECTIVES …………………………………………………………………………. 125

PERSONAL CONTRIBUTION …………………………………………………………………………….. 129

Study 1: Growth hormone replacement therapy prevents sarcopenia by a dual mechanism: improvement of protein balance and of antioxidant defenses …………………………………….. 130

Study 2: Glucose-6-phosphate dehydrogenase overexpression improves body composition and physical performance in mice ………………………………………………………………………….. 155

Study 3: Redox status in resting conditions and in response to pro-oxidizing stimuli: impact of glucose-6-phospahe dehydrogenase overexpression ……………………………………………… 176

GENERAL DISCUSSION ……………………………………………………………………………………… 194

CONCLUSION ……………………………………………………………………………………………………… 202

REFERENCES ……………………………………………………………………………………………………….. 205

PUBLICATIONS AND PRIZES ……………………………………………………………………………. 233

ANNEXE ………………………………………………………………………………………………………………. 236

Sarcopenia Prevention by Growth Hormone


Sarcopenia is the progressive generalized loss of skeletal muscle mass, strength, and function which occurs as a consequence of aging. With a growing older population, there has been great interest in developing approaches to counteract the effects of sarcopenia, and thereby reduce the age-related decline and disability. This paper reviews (1) the mechanisms of sarcopenia, (2) the diagnosis of sarcopenia, and (3) the potential interventions for sarcopenia. Multiple factors appear to be involved in the development of sarcopenia including the loss of muscle mass and muscle fibers, increased inflammation, altered hormonal levels, poor nutritional status, and altered renin–angiotensin system. The lack of diagnostic criteria to identify patients with sarcopenia hinders potential management options. To date, pharmacological interventions have shown limited efficacy in counteracting the effects of sarcopenia. Recent evidence has shown benefits with angiotensin-converting enzyme inhibitors; however, further randomized controlled trials are required. Resistance training remains the most effective intervention for sarcopenia; however, older people maybe unable or unwilling to embark on strenuous exercise training programs.


Maintaining muscle function is vital to maintain functional independence. In our growing older population, muscle mass and force reach their peak between the second and fourth decades of life and thereafter show a steady decline with age.1 Sarcopenia is a syndrome characterized by progressive generalized loss of skeletal muscle mass and strength. It is usually accompanied by physical inactivity, decreased mobility, slow gait, and poor physical endurance which are also common features of the frailty syndrome.2 Rockwood et al3 described the concept of frailty as “a multidimensional syndrome which involves loss of reserves (energy, physical activity, cognition, and health) which gives rise to increased vulnerability”. Frailty involves a cumulative decline in multiple physiological systems including a decline in the neuromuscular system which is linked to the development of sarcopenia in later life. The loss of muscle mass during the ageing process is clinically important as it leads to reduced strength and exercise capacity, both of which are required to undertake normal daily living activities. Moreover, loss of muscle mass is a strong predictor of mortality in later life.4


It has been estimated that up to 15% of people older than 65 years and as many as 50% of people older than 80 years have sarcopenia.5 Sarcopenia has a major impact on public health and the cost in the united States alone was estimated at $18.5 billion in 2000.6 With the increasing number of older people worldwide, the cost is ever increasing. There has, therefore, been great interest in developing approaches to counteract the effects of sarcopenia and thereby help in reducing the age-related decline and disability.


Understanding the mechanisms that have been implicated in the development of sarcopenia can help direct sarcopenia treatments. Research is still ongoing but as yet no primary cause of sarcopenia has been identified. Multiple factors appear to be involved in the development of sarcopenia (Figure 1). A reduction in muscle strength is primarily linked to a reduction in overall muscle mass.7 This reduction in muscle mass may occur due to a combination of the loss of muscle fibers as well as muscle fiber atrophy with a preferential atrophy of type 2 fast twitch fibers. Denervation of motor units which is then reinnervated with slow motor units can lead to increased muscle fatigability.8 Although the overall biological mechanism of sarcopenia is not fully understood, observational studies have shown that satellite cells which are involved in muscle regeneration are much lower in older people and, therefore, could play a role in sarcopenia.


Osteopenia and sarcopenia develops rapidly during disuse. The study investigated whether intermittent parathyroid hormone (1-34) (PTH) and growth hormone (GH) administered alone or in combination could prevent or mitigate disuse osteopenia and sarcopenia in rats. Disuse was achieved by injecting 4IU botulinum toxin A (BTX) into the right hindlimb musculature of 12-14-week-old female Wistar rats. Seventy-two rats were divided into six groups: 1. Baseline; 2. Ctrl; 3. BTX; 4. BTX+GH; 5. BTX+PTH; 6.

BTX+PTH+GH. PTH (1-34) (60μg/kg/day) and GH (5mg/kg/day). The animals were sacrificed after 6weeks of treatment. Sarcopenia was established by histomorphometry, while the skeletal properties were determined using DXA, μCT, mechanical testing, and dynamic bone histomorphometry. Disuse resulted in lower muscle mass (-63%, p<0.05), trabecular BV/TV (-28%, p<0.05), Tb.Th (-11%, p<0.05), lower diaphyseal cortical thickness (-10%, p<0.001), and lower bone strength at the distal femoral metaphysis (-27%, p<0.001) compared to Ctrl animals. PTH fully counteracted the immobilization-induced lower BV/TV, Tb.Th, and distal femoral metaphyseal strength. GH increased muscle mass (+17%, p<0.05) compared to BTX, but did not prevent the immobilization-induced loss of bone strength, BV/TV, and cortical trabecular thickness. Combination of PTH and GH increased distal femoral metaphyseal bone strength (+45%, p<0.001), BV/TV (+50%, p<0.05), Tb.Th (+40%, p<0.05), and whole femoral aBMD (+15%, p<0.001) compared to BTX and muscle mass (+21%, p<0.05) compared to BTX+PTH. In conclusion, PTH and GH in combination is more efficient at preventing the disuse-related deterioration of bone strength, density, and micro-architecture than either PTH or GH given as monotherapy. Furthermore, GH, either alone or in combination with PTH, attenuated disuse-induced loss of muscle mass. The combination of PTH and GH resulted in a more effective treatment than PTH and GH as monotherapy.


The aim of our study was to elucidate the role of growth hormone (GH) replacement therapy in three of the main mechanisms involved in sarcopenia: alterations in mitochondrial biogenesis, increase in oxidative stress, and alterations in protein balance. We used young and old Wistar rats that received either placebo or low doses of GH to reach normal insulin-like growth factor-1 values observed in the young group. We found an increase in lean body mass and plasma and hepatic insulin-like growth factor-1 levels in the old animals treated with GH. We also found a lowering of age-associated oxidative damage and an induction of antioxidant enzymes in the skeletal muscle of the treated animals. GH replacement therapy resulted in an increase in the skeletal muscle protein synthesis and mitochondrial biogenesis pathways. This was paralleled by a lowering of inhibitory factors in skeletal muscle regeneration and in protein degradation. GH replacement therapy prevents sarcopenia by acting as a double-edged sword, antioxidant and hypertrophic.
SARCOPENIA is a syndrome characterized by progressive and generalized loss of skeletal muscle mass and strength with a risk of adverse outcomes such as physical disability, poor quality of life, and death (1). This loss of muscle occurs at a rate of 3%–8% per decade after the age of 30 with a higher rate of muscle loss at advanced age (2). Recent estimates show that one-quarter to one-half of men and women aged 65 and older are likely sarcopenic (3). Progressive sarcopenia is ultimately central to the development of frailty, an increased likelihood of falls, and impairment of the ability to perform activities of daily living (1). The logical endpoint of severe sarcopenia is loss of quality of life and ultimately institutionalization (4).


The importance of maintaining muscle mass and physical and metabolic functions in the elderly adults is well recognized. Less appreciated are the diverse roles of muscle throughout life and the importance of muscle in preventing some of the most common and increasingly prevalent clinical conditions, such as obesity and diabetes (4). Skeletal muscle atrophy is a common feature in several chronic diseases and conditions. It reduces treatment options and positive clinical outcomes as well as compromising quality of life and increasing morbidity and mortality (4). Individuals with limited reserves of muscle mass respond poorly to stress (4). In support of the importance of maintaining skeletal muscle mass, strength, and function, a recent study has demonstrated that all-cause, as well as cancer-based, mortality is lowest in men in the highest tertile of strength, an indicator of high muscle mass (5).


If there is a preexisting deficiency of muscle mass before trauma, the acute loss of muscle mass and function may push an individual over a threshold that makes recovery of normal function unlikely to ever occur. For this reason, more than 50% of women older than 65 years who break a hip in a fall never walk again (6).
Several hormones have been suggested to have an impact on muscle mass, strength, and function (7). Among them, growth hormone (GH) has been one of the most studied (7). Levels of GH are usually lower in the elderly subjects, and the amplitude and frequency of pulsatile GH release are significantly reduced (7). Thus, it has been hypothesized that GH would be useful in preventing the age-related loss of muscle mass (8).
In our study, we aimed to elucidate the role of GH replacement therapy in four of the main mechanisms involved in the onset and progression of sarcopenia: alteration in mitochondrial biogenesis, increase in oxidative stress, increase in protein degradation, and lowering in the rate of protein synthesis (9,10).
In this study, we present the existing evidence behind the argument that restoration of GH profile is a good intervention to improve or preserve skeletal muscle mass in old animals.


Materials and Methods
Animals and Treatment
Ten young (aged 1 month) and 20 old (aged 22 months) male Wistar rats, maintained under controlled light and temperature conditions, were used in the study. We chose 22-month-old rats because previous studies have reported that sarcopenia is evident at this age in this species (11). The animals were fed a normal rat chow (A.04; Panlab, Barcelona, Spain) and had free access to tap water. Half of the old animals (n = 10) were treated daily with two subcutaneous doses of GH (2 mg/kg/day diluted in saline; Omnitrope, Sandoz, Spain), one at 10.00 hours and another at 17.00 hours for 8 weeks. Control animals were injected with the same amount of vehicle (saline solution) as GH-treated rats. After 8 weeks of treatment, rats were sacrificed by cervical dislocation followed by decapitation, and troncular blood was collected and processed to measure plasma insulin-like growth factor-1 (IGF-1). Gastrocnemius muscle, liver, and heart were collected and immediately frozen in liquid nitrogen. The study was conducted following recommendations from the institutional animal care and use committee, according to the Guidelines for Ethical Care of Experimental Animals of the European Union. The Committee of ethics in research from the University Complutense of Madrid granted ethical approval.


We have previously shown that young animals do not show any effect when submitted to our GH treatment because they have high endogenous GH levels and also do not show alterations that could get ameliorated (12–14). This is why this experimental group has not been included in the study.


Body Composition Study
All rats were weighted weekly to determine changes in body weight during the study. After the rats were sacrificed, total body fat was determined by the specific gravity index (SGI), which shows the proportion between lean mass and body fat (15). This can be calculated comparing the animal’s carcass weight (animal without head, hair, and viscera) in the air (Wa) and in the water (Ww), using the following formula: SGI = Wa/[(Wa − Ww)] (assuming the specific gravity of water at 21°C to be 1) (15).


IGF-1 Levels
Plasma and hepatic IGF-1 levels were measured as previously described (16) by an specific radioimmunoassay, using reagents kindly provided by the National Hormone and Pituitary Program from the National Institute of Diabetes and Digestive and Kidney Diseases and a secondary antibody obtained in our laboratory.


Determination of Oxidative Damage in Gastrocnemius Muscle
Oxidative modification of total proteins in gastrocnemius muscles was assessed by immunoblot detection of protein carbonyl groups using the “OxyBlot” protein oxidation kit (Millipore, MA) as previously described (17).


Oxidative DNA damage was measured by 8-hydroxy-2′-deoxyguanosine (8-OHdG). A commercially available enzyme-linked immunoassay (Highly Sensitive 8-OHdG Check; Japan Institute for the Control of Aging, Japan) was used to measure oxidized DNA in isolated muscle DNA samples. DNA was extracted from the muscle via the High Pure PCR Template Preparation Kit (Roche, GmbH, Germany) according to the manufacturer’s protocol. DNA was used if it had a minimum 260:280 ratio of 1.8. The assay was performed following the manufacturer’s directions. Briefly, 50 µL of DNA were incubated with the primary antibody, washed, and then incubated in secondary antibody. The chromogen (3,3′,5,5′-tetramethylbenzidine) was added to each well and incubated at room temperature in the dark for 15 minutes. The reaction was terminated, and the samples were read at an absorbance of 450nm. Samples were normalized to the DNA concentration measured via a plate spectrophotometer for nucleic acids (ND-2000; NanoDrop, Wilmington, DE). All analyses were done in triplicate.


Determination of Citrate Synthase and Glucose-6-Phosphate Dehydrogenase Activities in Gastrocnemius

Muscle
Citrate synthase assay was performed in the gastrocnemius muscle following the method of Srere (18). Results were obtained in nmol × mg of protein−1 × minute−1. Values were normalized to those observed in the samples obtained from the young group, which were assigned a value of 100%.


Glucose-6-phosphate dehydrogenase (G6PDH) activity was determined following the method of Waller and coworkers (19). Results have been expressed in nmol × mg of protein−1 × minute−1.


Protein concentrations were determined by Bradford’s method (20) by using bovine serum albumin as standard.


Immunoblot Analysis
Aliquots of muscle lysate (50–120 µg of proteins) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The whole gastrocnemius was used to ensure homogeneity. Proteins were then transferred to nitrocellulose membranes, which were incubated overnight at 4°C with appropriate primary antibodies: anti-myf5 (1:200; Santa Cruz Biotechnology Inc., Santa Cruz, CA); anti-p70S6K (1:1,000; Cell Signaling); anti-phosphorylated p70S6K (1:1,000; Cell Signaling); anti-myostatin (1:1,000; Abcam, UK); anti-catalase (1:5,000; Sigma Aldrich, MO); anti-G6PDH (1:1,000; Abcam); anti-Gpx (1:2,000; Abcam); anti-cytochrome c (1:1,000; Santa Cruz Biotechnology); anti-PGC-1α (1:1,000; Cayman); anti-AKT (1:1,000; Cell Signaling); anti-phosphorylated AKT (1:1,000; Cell Signaling); anti-p38 (1:1,000; Cell Signaling); anti-phosphorylated p38 (1:1,000; Cell Signaling); anti-MuRF-1 (1:200; Santa Cruz Biotechnology Inc.); anti-MAFbx (1:500; Abcam); anti-Nrf1 (1:200; Santa Cruz Biotechnology Inc.); and anti-p21 (1:200; Santa Cruz Biotechnology Inc.). Thereafter, membranes were incubated with a secondary antibody for 1 hour at room temperature. Specific proteins were visualized by using the enhanced chemiluminescence procedure as specified by the manufacturer (Amersham Biosciences, Piscataway, NJ). Autoradiographic signals were assessed by using a scanning densitometer (Bio-Rad, Hercules, CA). Data were represented as arbitrary units of immunostaining. To check for differences in loading and transfer efficiency across membranes, an antibody directed against α-actin (1:1,000; Sigma Aldrich) was used to hybridize with all the membranes previously incubated with the respective antibodies. For the western blotting quantifications, we first normalized all the proteins measured to α-actin. Samples from each group were run on the same gel.

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