Vetenskaplig rapport. Application

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1 Vetenskaplig rapport FO Optimizing effects of sprint exercise by nutritional supply in both males and females Eva Jansson Karolinska institutet, Institutionen för laboratoriemedicin Bakgrund och syfte med projektet The aim of the project was to study the acute response to sprint exercise and especially how nutritional supply (or lack of) can direct this process either towards muscle hypertrohy (muscle protein synthesis (MPS) - mtor pathway) or mitochondrial biogenesis (AMPK pathway) in both males and females. Focus has been upon factors of importance for the switch between the activation of these two pathways. It was hypothesised that: 1. Males compared to females obtain a smaller or no increase in mtor - and larger activation of AMPK signalling and smaller down regulation of the myostatin gene after sprint exercise in fasting condition, which favour an activation of mitochondrial biogenesis, but not MPS and muscle hypertrohy. 2. Oral nutrients (essential amino acids and maltodextrin) ingested during and after sprint exercise generates a switch from AMPK to mtor signalling in skeletal muscle, in parallell with an activation of hvps34 and MPS in both males and females, favouring hypertrohy. 3. Muscle cells, in vitro, exposed to serum obtained after sprint exercise with nutritional supply as compared to fasting condition develop a greater hypertrophy and greater decrease in mrna for myostatin (favouring hypertrophy). Kortfattad teori- och metoddiskussion An appropriate sprint training program, including various componenets such as training of speed, acceleration and strength could be expected to increase % type IIA fibres and muscle cross-sectional area [1-5]. However, the results from these sprint studies are not consistent regarding the hypertrophic response. One explanation may be gender differences in combination with variations in nutrition results in various response to sprint exercise [6-8]. Moreover, a 30-s bout of sprint exercise [9, 10], activates AMPK [11]. Does an activation of AMPK inhibit muscle protein synthesis (MPS)? Effects of nutrition on muscular response to sprint training has not yet been studied. If training of speed and acceleration leads to a decreased MPS, this may counteract the gains by preceeding strength training. Therefore, it is important to optimize the spint training with regard to post exercise MPS by eg nutritional supply. Projektets viktigaste resultat och effekter Study I. Title: Sprint exercise enhances skeletal muscle p70s6k phosphorylation and more so in women than in men. Publication see list AIM: Sprint exercise is characterized by repeated sessions of brief intermittent exercise at a high relative workload. However, little is known about the effect on mtor pathway, an important link in the regulation of muscle protein synthesis. An earlier training study showed a greater increase in muscle fibre cross-sectional area in women than men. Therefore, we tested the hypothesis that the activation of mtor signalling is more pronounced in women than in men. Healthy men (n=9) and women (n=8) performed three bouts of 30-s sprint exercise with 20-min rest in between. METHODS: Multiple blood samples were collected over time, and muscle biopsy specimens were obtained at rest and 140 min after the last sprint. RESULTS: Serum insulin increased by sprint exercise and more so in women than in men [gender (g) time (t)]: P=0.04. In skeletal muscle, phosphorylation of Akt increased by 50% (t, P=0.001) and mtor by 120% (t, P=0.002) independent of gender. The elevation in p70s6k phosphorylation was larger in women (g t, P=0.03) and averaged 230% (P=0.006) as compared to 60% in men (P=0.04). Phosphorylation rps6 increased by 660% over time independent of gender (t, P=0.003). Increase in the phosphorylation of p70s6k was directly related to increase in serum insulin (r=0.68, P=0.004). CONCLUSION: It is concluded that repeated 30-s all-out bouts of sprint exercise separated by 20 min of rest increases Akt/mTOR signalling in Secondly, signalling downstream of mtor was stronger in women than in men after sprint exercise indicated by the increased phosphorylation of p70s6k. Study II. Nutrient ingestion increased mtor signaling, but not hvps34 activity in human skeletal muscle after sprint exercise. Publication see list AIM: Nutrient provision after sprint exercise enhances mammalian target of rapamycin (mtor) signaling. One suggested nutrient sensor is the class III phosphatidylinositol 3-kinase, vacuolar protein sorting 34 (Vps34), not previously studied in human It is hypothesized that oral ingestion of essential amino acids (EAA) and carbohydrates (Carb) increases Vps34 activity and mtor signaling in human skeletal muscle after sprint exercise. METHODS: Nine subjects were performed 3 30-sec all-out sprints with or without ingestion of EAA + Carb or placebo drinks in a randomized order with a month interval. Muscle biopsies were performed at rest and 140 min after last sprint and analyzed for p-mtor, p-p70s6k, p-eef2 and for hvps34 activity and hvps34 protein content. Venous blood samples were collected and analyzed for amino acids, glucose, lactate, and insulin, see also protocol II. RESULTS: During the sprint exercise session, EAA, glucose, and insulin in blood increased significantly more in EAA + Carb than in placebo. P-mTOR and p-p70s6k were significantly increased above rest in EAA + Carb (P = 0.03, P = 0.007) 140 min after last sprint, but not in placebo. Activity and protein expression of hvps34 were not significantly changed from rest in EAA + Carb 140 min after the last sprint. However, hvps34 activity and protein expression tended to increase in placebo (both P = 0.08). CONCLUSION: On the contrary to the hypothesis, no increase in activation of hvps34 was found following sprint exercise in EAA + Carb condition. In spite of this, the results support an activation of mtor during this condition. However, this does not exclude the permissive role of hvps34 in mediating the amino acid-induced activation of mtor and muscle protein synthesis.

2 Study III. Nutrient ingestion, muscle protein synthesis and global gene expression in human skeletal muscle after repeated sprint exercise. Manuscript in progress. AIM: It is hypothesised that nutritional supply (essential amino acids and maltodextrin) ingested during and after sprint exercise generates a switch from AMPK to mtor signalling in skeletal muscle, in parallel with an increase in muscle protein synthesis. METHODS: Twelve subjects performed 3 30-sec all-out sprints with or without ingestion of EAA + Carb or placebo drinks in a randomized order with a month interval. Two post biopsies were taken with 2 hours in between, first biopsy 80 min and second 200 min after last sprint. In vivo protein synthesis rates was measured using a stable isotope technique. The subject received an intravenous injection of a stable isotope of phenylalanine (D5-phenylalanine) to label the precursor pool for protein synthesis. The infusion started directly before the first bout of exercise. The two post biopsies, taken with 2 hours in between were used for measuring the fractional synthesis rate (FSR): The amount of the labelled phenylalanine incorporated into muscle protein over these 2 hours represents the in vivo protein synthesis rates. The amount of labelled phenylalanine in the free amino acid pool and in the muscle protein was determined by mass spectrometry. FSR was calculated by dividing the amount of labelled phenylalanine incorporated in these 2 hours by the amount in the free amino acid (precursor) pool. Muscle signaling was analysed for p-mtor, p-p70s6k, AKT in the post exercise biopsies. Global gene expressions was analysed using affymetrix chip (Human Gene 2.1 ST). Venous blood samples were collected and analysed for amino acids, glucose, lactate, and insulin. PRELIMINARY RESULTS: The experimental parts of the study and all analyses for protein synthesis (FSR), Western blot (pmtor p-p70) and blood data (insulin, glucose, lactate, amino acids) are finished and data evaluation are ongoing and preliminary data were presented in August 2014 on the Scandianavian Physiological Society Congres in Stockholm. The conlusions so far are: Given that oral ingestion of EEA+Carb increases plasma leucine after sprint exercise, muscle protein synthesis increased and is suggested to be activated through activated AA transport (increassed gene expression of SNAT 2) and subsequent Akt/mTOR signaling. Study IV. In vitro studies of muscle cell and hypertrophy after repeated sprint exercise. The study is to be started during spring AIM: For a deeper understanding of the mechanisms behind sprint exercise induced adaptation of skeletal muscle, experiments are performed in vitro by using primary cell cultures of human myoblasts and myotubes. The idea with these experiments is to study the effects of systemic or extra cellular factors including nutritional factors on muscle fibre hypertrophy. Cell experiments will be conducted where the hypothesis is tested that myotubes exposed to sprint-serum, in vitro, in nutrient supply condition develop hypertrophy. The idea with this part of the project is to study the integrated effect of exercise-induced changes in systemic factors with or without nutritional supply on myotube size. Resultatets relevans för idrotten The results in the present study will have direct implications for designing performance-related training programs for athlets at various levels including the elite. The results will also increase the understanding of muscular response to severe metabolic stress, how to optimize positive and reduce negative effects by nutritional supply and how to drive the response into a desired direction depending on the particular demands of physical capacities in various types of sports. For example a sprinter needs both a large muscle mass and fast contracting muscles to develop high power (force x speed). If sprint training (speed and acceleration) leads to a decreased muscle protein synthesis this may counteract the gains obtained by strength training sessions. Therefore, it is important to optimize the sprint training sesssions with regard to post exercise muscle (myofibrillar) protein syntehsis. On the other hand, a growing body of evidence suggests that sprint exercise, except for improving specifically sprint performance, may represent a time-efficient strategy to induce adaptations normally associated with endurance training [1]. However, in this case it may be of importance to potentiate the stimulation of mitochondrial biogenesis instead of switching towards hypertrohy. In the last decade, the popularity of team and court sports, which require the athlete to sprint intermittently over the course of the game, has increased [2]. Populärvetenskaplig sammanfattning av projektresultaten En populärvetenskaplig artikel om vår och andras forskning om sprintträning finns i CIFs tidning Svensk Idrottsforskning 2013 nr 2 Nedan ges en sammanfattning av det som har starkast koppling till det aktuella forskningsprojektet. Sprintträning med och utan intag av näring Många medel- och långdistanslöpare har börjat komplettera sin vanliga träning med sprintträning för att nå bättre tider. Det är även populärt inom lagidrotter och nyligen har forskning visat att sprintträning eller sprintintervallträning också kan ge positiva effekter på hälsan. Det gäller inte bara för friska utan även för personer med och utan riskfaktorer, som till exempel övervikt och nedsatt insulinkänslighet eller kronisk sjukdom. Vid ett 30-sekunders all-out sprintarbete aktiveras både snabba och långsamma muskelfibrer och man uppnår en mycket hög hastighet på nedbrytningen av muskelglykogen och ATP, speciellt i snabba fibrer. Denna snabba energiförlust kan påverka vilka träningseffekter som uppnås och det är därför intressant att studera effekter av näringsintag i samband med sprintträning. Våra studier, utan intag av näring, visar att VEGF, som är en viktig tillväxtfaktor för ökningen av antalet kapillärer, steg trefaldigt, efter tre 30-sekunders sprint med 20 minuters vila mellan varje sprint. Detta indikerar att det finns förutsättningar för nybildning av kapillärer efter sprintarbete. När det gäller anpassning av kapilläriseringen så avgörs dock slutresultatet av en finstämd balans mellan förändringar i stimulerande och hämmande faktorer för kapillärtillväxt. Detsamma gäller för förändringar i muskelmassa som bestäms av balansen mellan proteinsyntes och proteinnedbrytning i muskulaturen. Ett samtidigt påslag på hypertrofisignalering (signaler för aktiverad syntes och hämmad nedbrytning) och hypotrofisignalering (signaler för aktiverad nedbrytning och hämmad syntes) kan leda till utebliven muskeltillväxt. Våra studier av genuttryck visar att många system för både aktivering och hämning av såväl proteinsyntes som proteinnedbrytning är påslagna samtidigt. Det kan vara svårt att hitta den rätta balansen mellan till exempel arbets- och viloperiodens karaktär och längd för att optimera träningseffekterna. Våra studier tyder på en aktivering av hypotrofisignalering efter sprintarbete speciellt för männen genom aktivering av t ex SMAD 3 och utebliven aktivering

3 (fosforylering) av markörer för hypertrofisignalering - mtor och p70. För kvinnorna såg vi däremot en aktivering av dessa markörer för hypertrofisignalering i muskulaturen efter 30-sekunders sprintarbeten. En tidigare fyra veckor lång sprintträningsstudie stödjer dessa fynd. Kvinnorna i studien fick en ökad tvärsnittsyta av snabba muskelfibrer men inte männen. Dessa fynd ledde fram till att vi ville studera effekten av intag av näring, och i första hand aminosyror i samband med sprintträning. De grenade aminosyrorna i blodet, till exempel leucin, sjunker vid upprepade sprintarbeten, vilket kan bero på att de bryts ner. Sänkningen skulle även kunna bero på att aminosyrorna används som byggstenar för en ökad proteinsyntes i muskulaturen. Flera faktorer talar dock emot detta. Männen, som uppvisade den största sänkningen av leucin i blodet, hade ingen eller endast en mycket liten ökning av hypertrofisignaleringsmarkörerna mtor och p70 i muskulaturen, det vill säga liten förutsättning för muskeltillväxt. Snarare skulle det kunna vara så att de sjunkande blodnivåerna av leucin tyder på en brist på byggstenar för proteinsyntes i muskulaturen och därmed att hypertrofisignaleringen uteblir bland männen i studien. Från studier av styrketräning så vet man att intag av aminosyror i samband med ett träningspass ökar förutsättningarna för hypertrofi. Effekten av att intag aminosyror i samband med sprintarbete är mer okänd. De flesta tidigare studier på sprintarbete är utförda utan intag av näring i samband med träningspassen. Vi har nyligen slutfört en studie, där vi undersökt effekten av intag av aminosyror i en dryck före och efter ett sprintträningspass. Vi undersökte effekten på genuttryck, cellsignalering och muskelproteinsyntes och även hur en muskulaturen känner av den ökade nivån av aminosyror i blodet och muskeln, sk aminosyratransportörer och/eller aminosyrasensorer. Våra preliminära resultat tyder på att muskelproteinsyntesen ökar under förutsättning att, drycken leder till en tydlig ökning av nivån av aminosyrorna i blodet. Förutsättningar tycks finnas för att denna effekt på muskelproteinsyntesen medieras via aminosyratransportören SNAT 2, som i sin tur leder till en stimulering av hypertrofisignaleringsproteinerna mtor och p70. Våra studier av en annan aminosyrasensor, Vps34, som studerats av oss första gången i människans skelettmuskulatur, gav inget stöd för att detta protein skulle mediera effekten av den ökade tillgången på aminosyror på hypertrofisignalering och muskelproteinsyntes. Övrigt Är projektet avslutat? Nej Kvar att göra inom forskningsprojektet - Framtida forskning Study II - Manuscript: Sprint exercise and global gene expression in skeletal muscle in males and females Study III - Manuscript: Nutrition, muscle protein synthesis, global gene expression after sprint exercise Study IV - The study will be performed during spring 2015 Results from this project will be presented in a doctoral thesis by Håkan Rundqvist Bilder, tabeller och diagram (bifogas som pdf) Uppladdad fil: Fem nyckelord skelettmuskulatur, kön, nutrition, sprint, proteinsyntes Redovisning av publicering Typ Manuskript Titel, tidskrift, volym Rundqvist HC., Lilja M., Rooyackers O., Murray JT., *Esbjörnsson M. & *Jansson E. Nutrient ingestion increased mtor signaling, but not hvps34 activity in human skeletal muscle after sprint exercise. Physiol Rep Oct;1(5) :e doi: /phy2.76. Epub 2013 Sep 23. Esbjörnsson M., Rundqvist HC., Mascher H., Österlund T., Rooyackers O., Blomstrand E. & Jansson E. Sprint exercise enhances skeletal muscle p70s6k phosphorylation and more so in females than in males. Acta Physiol. 205:411-22, Esbjörnsson M., Rooyackers O., Norman B., Rundqvist HC., Nowak J., Bülow J., Simonsen L. & Jansson E. Gender specific levels of plasma and muscle amino acids before and after repeated bouts of sprint exercise. Scand J Med Sci Sports. 22: , Rundqvist HC., Rooyackers O., Österlund T., Esbjörnsson M., Jansson E. Nutrient ingestion, muscle protein synthesis and global gene expression in human skeletal muscle after sprint exercise. Manuskript Montelius A., Esbjörnsson M., Rundqvist HC., Österlund T. & Jansson E. Sprint exercise and global gene expression in skeletal muscle in males

4 and females. Populärvetenskapliga publikationer Jansson E., Rundqvist H., Norman B., Lilja M., Montelius A., Österlund T., & Esbjörnsson M.. Sprintträning inte bara för sprintlöpare. Svensk idrottsforskning, vol. 2, p , 2013 Redovisning av deltagande med muntlig presentation Typ Titel Plats/namn/datum Oral amino acids, sprint exercise and muscle protein synthesis. Sprint exercise and myoblast proliferation. SPS, Scandinavian Physiological Society, Stockholm, Sweden 22 aug, Rundqvist HC, Österlund T, Montelius A, Moberg M, Rooyackers O, Esbjörnsson M, Jansson E. The 13th Biennial Conferenc Advances in Skeletal Muscle Biology in Health and Disease, Gainesville Florida, March 5-7, Fischer H, Alam S, Rundqvist HC, Montelius A, Norman B, Esbjörnsson M, Jansson E. Lack of alpha-actinin-3 attenuates m- TOR signaling in human skeletal muscle after sprint exercise. The 13th Biennial Conferenc Advances in Skeletal Muscle Biology in Health and Disease, Gainesville Florida, March 5-7, 2014 Norman B., Esbjörnsson M., Östlund T., Rundqvist HC., Jansson E. Altered glycogen metabolism in alphaactinin-3 deficient individuals. Vps34 in human Effects of exercise and oral amino acids. The effect of sprint exercise on expression of myostatin and SMAD7 in Global muscle gene expression after spring exercise. The effect of sprint exercise on expression of myostatin and SMAD7 in Regulation of SMAD7 and myostatin gene expression after sprint exercise in Vps34 activity in human Effects of exercise and oral amino acids. ECSS i Barcelona. Norman B., Esbjörnsson M., Glenmark B., Lindholm M., Jansson E Rundqvist H C., Lilja M., Murray TJ., Rooyackers O., Esbjörnsson M. & Jansson E Österlund T., Rundqvist H C., Esbjörnsson M. & Jansson E Montelius M., Rundqvist HC., Esbjornsson M. & Jansson E. 59th annual meeting "Exercise is Medicine", San F, California, USA may 29-june 2, 2012 Rundqvist H C., Esbjörnsson M., Österlund T. & Jansson E Oct 10-13, Esbjörnsson M., Rundqvist HC., Österlund T., Montelius A. & Eva Jansson. Oct 10-13, Jansson E., Rundqvist HC., Lilja MR., MurrayJT., Rooyackers O. & Esbjörnsson M. Lack of alpha-actinin-3 attenuates m- TOR signaling in human skeletal muscle after sprint exercise. Oct 10-13, Norman B, Esbjörnsson M, Österlund T, Rundqvist HC, Jansson E.

5 Global Muscle Gene Expression After Sprint Exercise. Oct 10-13, Montelius A, Rundqvist HC, Esbjörnsson M, Jansson E.