L17 Muscle building drugs 1 slide per page.pdf

NEUR3101 Muscle building drugs and performance supplements Dr Vita Birzniece SoMS, Faculty of Medicine & Health Learning objectives To understand the main mode of action of an anabolic drug clenbuterol Describe the mechanism by which a dietary supplement creati...

NEUR3101 Muscle building drugs and performance supplements Dr Vita Birzniece SoMS, Faculty of Medicine & Health Learning objectives To understand the main mode of action of an anabolic drug clenbuterol Describe the mechanism by which a dietary supplement creatine has its mode of action and explain the role of the “creatine phosphate shuttle” To understand the effects of anabolic steroids and growth hormone on muscle mass and function To understand the principles of hormone doping detection Muscle building drugs and performance supplements Muscle building drugs – to improve body composition Doping – to improve fitness  2018 Winter Olympic Games An athlete from Russia Nadezhda Sergeeva, bobsleigh, tested positive for trimetazdine, a medication used for angina sufferers  World Anti-Doping Agency (WADA) prohibited list 1) Anabolic androgenic steroids (testosterone, androsterone, oxandrolone, stanozol) 2) Other anabolic agents (SARMs, tibolone, clenbuterol) 3) Peptide hormones, growth factors and related substances (erythropoietins, LH, growth hormone, ghrelin, FGFs, VEGF) 4) Beta-2 adrenoreceptor agonists (salbutamol, formoterol, clenbuterol) 5) Hormone and metabolic modulators (aromatase inhibitors, antiestrogen fulvestrant, SERMs, myostatin inhibitors, insulin, AMKP activator AICAR) 6) Diuretics and masking agents (furosemide, albumin, dextran) 7) Narcotics and cannabinoids 8) Blood doping 9) Gene doping https://www.wada-ama.org/en/content/what-is-prohibited  2-adrenergic receptor agonist Clenbuterol Clenbuterol induces anabolic action by stimulating 2-adrenergic receptors in the sarcolemma of skeletal muscle. Clenbuterol is a structural analog of naturally occurring catecholamines, such as adrenaline. Clenbuterol increases muscle mass by inhibiting myofibrillar protein breakdown.  Clenbuterol prevents protein degradation Pathway of action of 2-adrenergic agonists  Stimulates G-protein Activates Adenylate cyclase cAMP Activates protein kinase  The protein kinase catalyses protein phosphorylation Clenbuterol  2-adrenergic agonists increase: Muscle mass Aerobic exercise capacity CNS stimulation Blood pressure Oxygen transport Lipolysis, fatty acid oxidation and metabolic rate loss of fat mass  Clenbuterol used in cycles Anabolic doses of clenbuterol: Therapeutic dose 20 µg twice daily. Anabolic – used up to 120-140 µg/day. Dosage schedule: 2 weeks on 2 weeks off The rational behind this is to try and minimize the 2-adrenergic receptor down-regulation which is known to occur with repeated stimulation.  Clinical trials with clenbuterol  The plasma creatine kinase (skeletal muscle specific enzyme) was greatly elevated in subjects. As this is a marker of skeletal muscle damage, muscle biopsies were taken from patients to determine the nature of muscle damage.  The biopsies showed an interesting muscle fibre specific damage pattern with only the slow Type 1 fibres being damaged.  In control subjects who in addition to clenbuterol took the 2-adrenergic antagonist propranolol, only 50% of the anabolic response was inhibited. Questions – is there additional non-beta adrenergic pathway? why only type 1 fibres are damaged? To determine is there any additional mechanism for clenbuterol action, skinned muscle fibre experiments were used A segment of a skeletal muscle fibre that has been skinned and tied to a sensitive force transducer for isometric force recording In this technique the surface membrane of the skeletal muscle fibre is removed and with it all the 2-adrenergic receptors. The t-tubular system, sarcoplasmic reticulum, and contractile proteins all retain normal function. So any effects will be via a none beta-2 pathway. Bakker AJ, Head SI, Wareham AC, Stephenson DG (1998). Effect of clenbuterol on sarcoplasmic reticulum function in single skinned mammalian skeletal muscle fibers. American Journal of Physiology; Vol274:pages C1718-26 Aim – to find out the cause of the different effects of clenbuterol in slow and fast skeletal muscle fibres. Skinned EDL (fast-twitch) and soleus (slow-twitch) muscles were treated with clenbuterol. Results  Clenbuterol acts directly on the SR making calcium to leak out, therefore less Ca2+ can be released from SR during excitation reducing the force the muscle can produce in response to an action potential  The effect is greater in slow-twitch muscle  In the fast-twitch muscle, baseline Ca2+ remains elevated and genes are activated that stimulate muscle hypertrophy Bakker AJ, Head SI, Wareham AC, Stephenson DG (1998). Effect of clenbuterol on sarcoplasmic reticulum function In single skinned mammalian skeletal muscle fibers. American Journal of Physiology; Vol274:pages C1718-26 Sarcoplasmic reticulum  Additional mechanism of clenbuterol Phosphorylation of the ryanodine receptor, Ca2+ release channel on the sarcoplasmic reticulum (SR) calcium efflux from the SR.  Why slow-twitch muscle was damaged by clenbuterol and fast-twitch muscle was not so affected? Fast-twitch muscle: more calsequestrin and Ca2+ uptake pumps  Side effects of clenbuterol  Ventricular cardiac hypertrophy, palpitations, increased blood pressure, sudden heart failure Reason - it also acts through β1-adrenoreceptors  Headaches, tremors, cramps, anxiety, sweating, nausea Solution? Selective β2-adrenoreceptor agonist – Formoterol – only acts on muscle, no cardiac side effects; has no effect on SR Ca2+ leak  Conclusion Clenbuterol  Increases muscle mass by preventing muscle breakdown  Stimulates 2-adrenergic receptors on sarcolemma  Induces SR Ca2+ leak, especially in slow-twitch muscle  Fast-twitch muscle not affected by Ca2+ leak because of more calsequestrin and Ca2+ uptake pumps, so SR is not depleted of Ca2+ and muscle force is maintained.  As baseline Ca2+ is elevated in fast-twitch muscle, it results in muscle hypertrophy.  CREATINE – dietary performance supplement  Creatine phosphate system Creatine kinase Active muscle for muscle contraction The Creatine/Phosphocreatine cycle acts as a temporal buffer of ATP during the first seconds of intense skeletal muscle contraction before glycolysis and mitochondrial mechanisms can respond to the increased demand for ATP. Without this buffering most skeletal muscles would run out of ATP after less than one second of intense exercise (Hultman & Greenhaff, 1999). Changes in Type II skeletal muscle ATP and phosphocreatine during sprinting During sprinting, PCr levels drop to maintain relatively stable ATP levels Physiology of Sport and Exercise, 5th edition, Kenney WL, 2012 Main substrates which supply ATP: phosphocreatine; muscle and liver glycogen; blood glucose and fatty acids. 1 1. Phosphocreatine is used in the first few seconds of high intensity exercise to maintain ATP levels in the time taken for 3 glycolysis to start working. 2. During high-intensity contraction only carbohydrate is used and this is broken down in glycolysis to yield pyruvate and ATP. 3. During low intensity contraction most energy comes from oxidative phosphorylation of carbohydrate and fat, this occurs in the mitochondria. 2 Creatine/phosphocreatine cycle catalysed by creatine kinase Mi-CK, the mitochondrial isoform MM-CK, the M-Line isoform linked to oxidative phosphorylation linked to glycolysis Pi Pi PCr  The Phosphocreatine Shuttle ATP is used by the contractile apparatus during muscle activity and as a result produces ADP. ADP is re-phosphorylated (adding phosphate group from phosphocreatine) to ATP by MM-CK, the muscle isoform of creatine kinase localised to the M-line of the contractile apparatus. Creatine is produced as a result of this conversion and diffuses to the mitochondria. Here ATP is used to phosphorylate creatine to phosphocreatine by Mi-CK. Phosphocreatine then diffuses back to the contractile proteins where it can be used to maintain the levels of ATP. The advantage of this system is that phosphocreatine and creatine are smaller and less negatively charged than ATP or ADP, this means that they diffuse faster and cross the mitochondrial membrane faster. Thus, not a lot of ATP is moving around, ATP is produced where it is needed.  Fibre type specificity of the creatine kinase enzymes Type 1 slow fibres up to 15-35% of the total creatine kinase is in the mitochondrial isoform Mi-CK (sMtCK). Type 2 fast fibres only up to 2-3% of the creatine kinase is in the mitochondrial isoform Mi-CK. Therefore, in fast-twitch muscle most of the ATP is produced quickly by the MM CK right next to the contractile proteins where it is needed.  Why do we take creatine? Clearly the aim of taking creatine is to maintain ATP levels during high intensity exercise. In the diet we get creatine from meat and fish. It is also made endogenously in the liver and kidneys, and pancreas. 95% of creatine is found in skeletal muscles the remaining 5% spread amongst brain, cardiac muscle, retina and sperm.  How do we take creatine? Doses of 20 g per day for a period of five days. This increases cellular creatine levels. The supplementation is continued at a 2-5 g/day. 20 to 30% of the increase is in the form of phosphocreatine. Vegetarians benefit the most from creatine supplementation. Result - increase of 0.7 to 2 kg of lean body mass.  The effect of creatine on muscle function Creatine improves fatigue. In combination with resistance training, creatine use is associated with greater increase in strength, related to the increased ability to train at higher intensities. Creatine stimulates intermittent high-energy activity (increase in sprint power) which favours the recruitment of fast type 2 muscle fibres. This is what we predict from the distribution of the different isoforms of the creatine kinase because in the fast-twitch muscle the energy would largely come from PCr at M-line of contractile apparatus and glycolysis.  Side effects of creatine Gastric distress, bloating and diarrhoea This is prevented by ingesting it with food Dehydration Drink plenty of water Should not be taken if history of kidney disease or diabetes  Growth hormone (GH) and androgens (testosterone) – major anabolic hormones Growth hormone Androgens Anabolic androgenic steroids Male sex hormones – endogenous (Testosterone, Dihydrotestosterone (DHT)) – exogenous (Sustanon, Nandrolone (Deca-Durabolin)) Anabolic: positive nitrogen balance, protein synthesis Androgenic: masculinization Growth hormone Testosterone  Growth hormone (GH) and androgens (testosterone) – major anabolic hormones The increase in GH during exercise 75-90% of VO2max 40-66% of VO2max 25-33% of VO2max The higher exercise intensity, the greater the GH increase Sutton J, Lazarus L; J Appl Physiol 1976;41(4):523-7  Dose-dependent effect of testosterone on muscle mass Change in lean body mass Change in leg press strength 12 100 * * 10 * 80 8 * kg 60 6 * kg 4 40 2 20 0 0 25 50 125 300 600 -20 Testosterone dose (mg/week) 25 50 125 300 600 Testosterone dose (mg/week) Bhasin et al., 2001 Am J Physiol Endocrinol Metab 281:E1172-81  Testosterone vs exercise Exercise Placebo Testosterone Placebo Testosterone Testosterone - 600 mg/week for 10 weeks Exercise - weightlifting sessions 3 times a week High doses of testosterone increase muscle strength, the effect similar to that of exercise Bhasin et al., N Engl J Med 1996; 35:1-7  The effect of androgens on muscle Reduced Central nitrogen loss effects of through androgens hepatic urea (fatigue, cycle training) Lam, Poljak, McLean, Bahl, Ho, Birzniece; Josiak K, et al. J Cachexia Sarcopenia Muscle. 2014 Dec;5(4):287-96 Eur J Endocrinol. 2017;176(4):489-496.  Growth hormone mode of action Insulin-like growth factor Heinemeier et al., 2012 Scand J Med Sci Sports  Benefits of GH on connective tissue Shorter recovery time after soft tissue injury GH increases collagen synthesis GH effect on tendon GH effect on muscle 6 6 Fold change over placebo 5 5 4 4 3 3 2 2 1 1 0 0 IGF-I Collagen I Collagen III Collagen IGF-I Collagen I Collagen III Collagen Myofibrillar mRNA mRNA mRNA FSR mRNA mRNA mRNA FSR FSR Ten healthy men, GH for 14 days; 33.3 μg/kg/d and 50 μg/kg/d for the 1st and 2nd week. FSR – fractional synthesis rate Doessing et al., 2010 J Physiol 588:341-51 Change in anaerobic exercise capacity (sprint) over 8 weeks 12 * 10 8 * 6 % 250 mg/week 2 mg/day 4 2 0 Placebo T GH GH+T testosterone Meinhardt et al., Ann Intern Med. 2010; 152:568-77  Suppressed GH – anaerobic metabolism Stimulated Glucose Fatty acid Glycolysis Glucose Glycogen FA-CoA Lactate Pyruvate -oxidation acetyl-CoA Pyruvate FA-CoA PDHK4 TCA CO2 H+ H2O Mitochondria Respiratory Chain ATP ADP Sjogren et al AJP 2007;293:364-71  The effects of growth hormone  Increases muscle mass (also water retention)  Reduces fat mass by stimulating lipolysis  No major effect on muscle strength  No major effect on aerobic capacity  Stimulates anaerobic exercise capacity  Stimulates collagen formation, tissue repair  Increases tolerance for hard training  Anabolic steroid side effects Gynecomastia Acne Hirsutism Sexual dysfunction Aggression  Anabolic steroid side effects Behavioural and psychiatric side effects: Depression, mania Increased risk of other drug and alcohol abuse Increased risk of suicidal and homicidal death Cardiovascular complications: Reduction in HDL, increase in LDL cholesterol Myocardial hypertrophy, sudden cardiac death Suppression of hypothalamic-pituitary-testicular axis: Reduction in testicular size, sperm count Sexual dysfunction Gynecomastia in men, breast atrophy in women Hirsutism, clitoral enlargement, menstrual irregularity in women Acne, male pattern balding Increase in hemoglobin, hematocrit Liver dysfunction, neoplasms Tendon, ligament and joint injuries Androgen dependence Increase in mortality - 5 times that of controls (Parssinen et al, 2000 Int J Sports Med 21:225-7) Effects /side effects?  Growth hormone side effects Acromegaly: Coarse facial features; enlarged jaw, frontal bossing, enlarged nose, lips, tongue, widely spaced teeth Large hands, feet, arthropathy Carpal tunnel syndrome Cardiomegaly, hepatomegaly, nephromegaly, splenomegaly, enlargement of thyroid gland, prostate, development of polyps Increased risk of diabetes, hypertension, cancer Life expectancy reduced by 10 years (Rajasoorya et al. Clin Endocrinol. 1994;41:95)  Summary Testosterone prevents protein loss by inhibiting hepatic urea cycle, stimulates muscle protein production, stimulates satellite cells, inhibits myostatin, stimulates muscle IGF-1, stimulates myotube formation increases muscle mass improves muscle perfusion, oxygenation, promotes central stimulatory effect, reduces fatigue, increases muscle strength Growth hormone stimulates muscle and tendon collagen production, stimulates muscle IGF-1, increases muscle mass (not all functionally active muscle mass) stimulates anaerobic metabolism increases sprint capacity  Take-home message Growth hormone good for sprinting Side effects!!! Testosterone increases strength  Doping detection In-competition Out-of-competition Athletes passport? Urine tests Blood tests 278,047 samples analysed in 2019, 0.97% positive Anabolic agents 44% of all reported findings Diuretics and other masking agents 16% 2-adrenoreceptor agonists 4% Growth hormone, growth factors 3% https://www.wada-ama.org/sites/default/files/resources/files/2019_anti-doping_testing_figures_en.pdf  Detecting steroid doping 1. Urinary testosterone:epitestosterone (T/E) ratio Testosterone Epitestosterone (inactive epimer of testosterone, Increases with secreted from testes, end ratio exogenous testosterone 1:1 with testosterone) Suppressed by exogenous testosterone Cut off = 4  Detecting steroid doping 2. Carbon Isotope Ratio (mass spectrometry) – 13C isotope more prevalent in animal/plant carbon sources – Measure 13C/12C ratio in testosterone – Diet rich in maize or sugar cane (Africa) will result in higher 13C – Extensive sample preparation required Testosterone Ben Johnson – 1988 Liudmyla Blonska 90y old cyclist; age-based record 2018 Stanozolol 2002, 2008 Stanozolol Epitrenbolone; in-competition testing  GH detection - The hGH Isoform Test 1) Pituitary hGH Recombinant hGH 22 kD hGH 22 kD hGH and monomer various other exclusively isoforms 2) Exogenous GH will suppress pituitary GH secretion Ratio: recombinant (22 kD) hGH / pituitary hGH Window of detection up to 36 hrs - good for out-of-competition testing  GH detection The Biomarker Test The Biomarker Test detects unnatural increases in two GH action markers (IGF-1 and PIIINP) that are produced after the injection of hGH and can detect doping with hGH up to 14 days after use Suggested cut off 3.7 Detection time - up to 2 weeks after stopping GH abuse Smaller increase of GH markers in women Combined testosterone and GH abuse – easier to detect Powrie JK et al., Growth Horm IGF Res 17, 220–226, 2007  GH abuse Terry Newton - 2009 Mike Jacobs - 2011 Marion Jones - 2000 Patrik Sinkewitz - 2011 Andrus Veerpalu - 2011 Jarrell Miler - 2019 GH difficult to detect because of the short half-life, small window of detection, biomarker test not highly specific, “safe” normative values  Quiz What did Harry Potter use? Enlarged jaw – GH? Androgenic steroids and growth hormone Bulky muscle – testosterone? Important questions to answer  Explain the mechanisms for androgen-induced anabolic effect  Explain how growth hormone stimulates anaerobic exercise capacity  Explain two main modes of clenbuterol action  Explain the effect of SR Ca2+ leak and how slow- and fast-twitch muscles cope with that  Explain the role of ryanodine receptors, calsequestrin and Ca2+ uptake pumps  Explain the mechanisms why creatine may stimulate sprint capacity and reduce fatigue  Provide comparison of severity of side effects for misuse of testosterone, growth hormone, clenbuterol and creatine  Explain the principles of GH and testosterone doping detection