Do different levels of lactate have an impact on mTORC1-signal and protein synthesis induced by resistance exercise?

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Do different levels of lactate have an

impact on mTORC1-signal and protein

synthesis induced by resistance exercise?

Rasmus Liegnell

The Swedish School of Sport and Health Sciences, GIH

Master Degree Project 30 credits: 43:2018

Master Program in Sport Science 2016-2018

Supervisor: Marcus Moberg

Examiner: Örjan Ekblom

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Abstract

Aim:

To investigate the role of lactate as a potential signaling molecule, for the molecular mechanisms underlying acute resistance exercise adaptation in the muscle cell.

Method:

Four women and four men recruited to perform two trials of unilateral leg extension exercise performed with either an infusion of sodium lactate or isotonic saline (placebo), in a randomized, blinded, crossover fashion. To enable measurement of muscle protein FSR, an oral dose of 2H2O was consumed the day before the trials. Sixteen blood

samples were taken for direct analysis of lactate, glucose, Na+, K+ and pH. Five muscle biopsies were obtained from vastus lateralis during each trial and analyzed for 2H-protein incorporation using mass spectrometry. Furthermore, in sampled muscle tissue, the degree of protein phosphorylation in the mTORC1 signaling pathway was determined by

immunoblotting and levels of lactate were determined spectrophotometrically.

Results:

There were no significant differences in load, repetitions or time under tension between the trials. Blood levels of lactate reached a maximal level of 3.00 ± 0.26 mmol/L post 6th_{set in the placebo trial (P<0.05 vs. Rest). In the lactate trial, a peak of 6.94 ± 0.47 mmol/L}

was reached post 6th _{set with levels significantly higher than placebo up to 45 min recovery}

(P<0.05 vs. Rest and Placebo). In both trials the muscle levels of lactate increased above rest both post exercise and following 90 min of recovery, with significantly higher levels in the lactate trial (P<0.05 vs. Rest and Placebo). The phosphorylation of mTOR at Ser2448, p70S6K1 at Thr389, and p44 at Thr202/Tyr204 increased immediately after resistance exercise and remained elevated throughout the entire recovery period (P<0.05). The phosphorylation of PRAS40 at Ser183, 4E-BP1 both at Ser65 and Thr37-46 decreased immediately after resistance exercise, whereas that of eEF2 at Thr56 decreased following 90 and 180 min of recovery (P<0.05). There were no differences between trials for any intracellular signaling protein on group basis.

Conclusion:

An infusion of a sodium lactate solution during resistance exercise increases whole venous blood and muscle levels of lactate, without altering time under tension, load or repetition lifted during exercise. Resistance exercised induced mTORC1-signaling is not altered by a sodium lactate infusion at the group level in this population but there are underpowered differences between men and women.

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“Our understanding of the role of lactate in energy metabolism has changed so much in recent years that it is appropriate to speak about a shift of paradigm. Furthermore, new evidence continues to accumulate at a rapid pace, partly supporting and partly challenging existing theories. We hope this means that our understanding of this important area of metabolism will continue to improve.” (Åstrand et al., 2003)

1. Introduction

In sport science and particularly in the pursuit of strength and improved performance, one tissue is primary of interest, the skeletal muscle. The amount and properties of contractile unites in the muscle is a key factor for quality of life and performance in sports (Ruiz et al., 2008, Blair, 2009). Depending on age and sex, the skeletal muscle constitutes between 30 to 40% of the body weight (Janssen et al., 2000, Collins and Partridge, 2005) and through its plasticity, constantly adapts to the exposure and requirements in life. The muscles´ ability to develop force correlates with size and contraction rate of the skeletal muscle (Close, 1972). Increased level of loading such as strength training or heavy daily activities results over time in hypertrophy through repeated stimulation of protein synthesis in the muscle cell. With repeated sessions of resistance exercise over time, growth of contractile proteins is the primary outcome in the muscle cell that contributes to hypertrophy (Marcotte et al., 2015, Goodman et al., 2011).

The underlying process of how resistance exercise stimulates the rate of protein synthesis in the muscle cell, ultimately leading to hypertrophy, is still far from understood (Damas et al., 2018) but in general, three potential mechanisms are most often discussed in the literature. Mechanical stimuli of the muscle and especially the total number of activated motor units in muscle fibers is seen as the main factor for hypertrophy (Dankel et al., 2017, Schoenfeld et al., 2016, Mitchell et al., 2012, Burd et al., 2010b). High load with few repetitions rapidly recruits a large number of muscle fibers, whereas lower load and higher number of repetitions can be used for equal fiber activation (Dankel et al., 2017). Total volume seems to be the major factor in mechanical loading also with regards to the acute stimulation of myofibrillar protein synthesis (Burd et al., 2010a). Muscle damage as a result of heavy, and especially eccentric, exercise is a mechanism of debate that is suggested to stimulate the rate of protein synthesis acutely, explained by the process of repairing the fiber (Damas et al., 2018).

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2 The last and most intriguing mechanism is the involvement of metabolic stress, where

mechanical stimuli leads to metabolic changes, both in the extra- and intracellular milieu, that ultimately stimulates the rate of protein synthesis. Further in this master thesis the mechanism of metabolic stress will be of interest.

1.1 Protein synthesis and the mTOR signaling pathway

Resistance exercise has been shown to acutely stimulate the rate of protein synthesis within the muscle cell (Chesley et al., 1992), and primarily synthesis of the myofibrillar protein pool (Wilkinson et al., 2008, Burd et al., 2011, Moore et al., 2009). In molecular biology, protein synthesis refers to the translation from nucleic acids to protein, the process when amino acids via peptide bonds links to form a protein. A complicated and energy demanding process which involves, to mention a few important molecules, mRNA, tRNA, ribosomes and amino acids (Erlandson-Albertsson and Gullberg, 2007, Ma and Blenis, 2009, Thougaard et al., 2015). Several chemical signals are stimulated within the cell when loaded contractions of the skeletal muscle occurs, and since the translation is energy demanding most organisms have evolved mechanism to efficiently shift between states of anabolism and catabolism, and in mammals a major control site for regulating growth is the mechanistic target of rapamycin (mTOR) (Goodman, 2014, Laplante and Sabatini, 2012, Maiese, 2016, Ma and Blenis, 2009, Mahoney et al., 2009).

The mTOR protein is a serine/threonine kinase that integrates various growth signals such as mechanical stress and regulates processes such as hypertrophy. The mTOR kinase, discovered in 1994, is the core of the so called mTOR protein complex (mTORC1), which is a key

complex of a pathway that regulates several biological processes. Even if many signaling pathways are able to influence the rate of protein synthesis, mTORC1 appears to be the major regulator and is thus termed “master regulator of cell growth”. (Goodman, 2014, Maiese, 2016, Mahoney et al., 2009). A strong correlation between muscle growth and the activation of mTORC1 has been established in mice during progressive overload (Baar and Esser, 1999). The importance of mTORC1 signaling in muscle cells after exercise with regard to the

stimulation of protein synthesis and muscle growth has also been shown in humans by pharmacological inhibition of the protein complex (Drummond et al., 2009).

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3 An increase in 70 kDa ribosomal protein S6 kinase beta-1 (p70S6K1) phosphorylation,

a main read out of mTORC1 activity, after a single resistance exercise bout also reveals a close relationship with improvements in fat free mass and strength in trained individuals over time (Terzis et al., 2008).

1.2 Downstream of mTORC1

The translation of nucleic acids to protein includes three main phases, initiation, elongation and termination. When activated, mTORC1 targets different enzymes of importance in the process of translation (fig. 1). Two of the most studied and well characterized targets are the p70S6K1_{and the eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) which}

are involved in the regulation of translation via their influence on both initiation and elongation (Goodman, 2014, Holz et al., 2005).

Fig. 1. Simplified figure of the mTORC1 pathway based on previous work (Mahoney et al., 2009, Goodman, 2014, Bahrami et al., 2014, Maiese, 2016, Moberg, 2016a). Blue figures represent stimulators and yellow inhibitors. When an upstream stimulus such as mechanical stimuli via for example resistance exercise occur, mTORC1 upregulates and launches a link of downstream events. Phosphorylation of p70S6K1_{both upregulate events such as phosphorylation of eIF4B but also inhibit} the inhibition of eEF2. An upregulation of mTORC1 also phosphorylate 4E-BP1 which releases its binding to eIF4E, that’s of importance for cell growth.

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4 Translation initiation refers to the gathering of ribosomal subunits with the initiator tRNA and eukaryotic translation initiation factors (eIFs) (Mahoney et al., 2009). 4E-BP1 has been shown to play a crucial role in the regulation of cap-dependent initiation, and the phosphorylation of 4E-BP1 is a major target of mTORC1, which plays a significant role in the regulation of protein synthesis. When 4E-BP1 gets phosphorylated it releases its binding to eIF4E and thus enabling the interacting between proteins eIF4E and eIF4G, resulting in the forming of an eIF4F complex and recruitment of ribosomal subunits needed for decoding mRNA

(Goodman, 2014, Mahoney et al., 2009). mTORC1 also phosphorylates, and thus activates, p70S6K1 via phosphorylation of the threonine 389 (Thr389) site. Activated p70S6K1 further regulates factors in translation initiation through phosphorylation of eIFs via ribosomal S6 protein or direct trough phosphorylation of eIF4B (Suzuki et al., 2008, Raught et al., 2004). Another target of p70S6K1 _{is the eukaryotic elongation factor 2 (eEF2) kinase which}

phosphorylates eEF2 on the Thr56 _{site, thus releasing its inhibitory action on the elongation}

and accordingly stimulates protein synthesis (Wang et al., 2001, Hizli et al., 2013, Goodman, 2014, Bahrami et al., 2014) In summary both p70S6K1_{and 4E-BP1 is regulated by mTORC1}

through phosphorylation at specific sites and have basically a similar output, regulation of translation initiation and in some part elongation. If these proteins are repeatedly stimulated over time, it ultimately leads to cell growth and ribosomal biogenesis (Goodman, 2014, Bahrami et al., 2014, Mahoney et al., 2009).

1.3 Measuring protein synthesis

After a mechanical stimulus such as resistance exercise, the degree of protein phosphorylation in the mTORC1 pathway can be determined by immunoblotting in sampled muscle tissue. However, to enable a direct measurement of the protein synthesis, a labeled molecule called tracer is needed. The tracer acts as a precursor for protein synthesis, and when enriching the body with that tracer, it will add to the endogenous pool and become incorporated into protein over time (Gasier et al., 2010). Reliable measurements using tracers in form of isotopically labeled amino acids such as [13C]leucine or [13C]-, [15N]-, or [2H]phenylalanine have traditionally been used to measure protein synthesis, but since the labeled amino acids is in need of continuous infusion, free-living activities are difficult to execute during the infusion (Gasier et al., 2010).

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5 Besides of labeled amino acids, deuterium oxide (2H2O) or so called heavy water can be used

as a valid tracer when measuring protein synthesis for days and even weeks in free-living environment (Wilkinson et al., 2015, Wilkinson et al., 2014, MacDonald et al., 2013).

2_H

2O is consumed orally and equilibrates rapidly within the body water pool through the

exchange of hydrogen (H) and deuterium (2H). 2H incorporates into metabolic pools and tissues, such as muscle tissue thru the amino acid pool and various amino acids such as alanine (Wilkinson et al., 2014, Gasier et al., 2010). Alanine undergoes rapid turnover and holds four sites where H can be replaced by 2H, allowing high sensitivity detection.

Furthermore, the 2H-labeling of alanine is about equal to that of 2H2O, it´s the concentration

of incorporated 2H2O in the body water that determines the percentage of 2H-labeled alanine,

not the amount of alanine (Gasier et al., 2010). When the 2_{H-labeled alanine is incorporated}

into the protein the enrichment of 2_{H is measured in sampled muscle tissue (Biolo et al.,}

1992). Muscle protein fractional synthetic rate (FSR) can subsequently be calculated by the change in enrichment between two biopsies divided by the time of incorporation and

precursor pool enrichment (Foster et al., 1993). In summary the mTORC1 signaling pathway controls anabolic actions in the muscle cell, which could be non-quantitatively via

immunoblotting, but to enable direct measures of the elongated protein synthesis a tracer is needed to calculate FSR.

1.4 Upstream of mTORC1

While the molecular events that occur downstream of mTORC1 are rather well described there is insufficient knowledge concerning the upstream mechanisms leading to mTORC1 activation. Factors in addition to mechanical stimuli that affect the signaling of mTORC1 such as intake of amino acids, insulin release, growth hormones and energy deficiency (fig. 1) (Maiese, 2016, Goodman, 2014, Moberg et al., 2014, Laplante and Sabatini, 2012). One suggested mechanism to control muscle growth via resistance exercise is the process of mechanotransduction (Goldmann, 2012), in which the stimulus of muscle contraction is transformed into biochemical signals (Dankel et al., 2017). There are additional suggested mechanisms by which resistance exercise is able to regulate mTORC1 activity. Down regulation of mTORC1 due to energy deficiency depends on the intracellular levels of ATP.

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6 When reduced, AMP-activated protein kinase (AMPK) is activated and acts as a metabolic checkpoint, suppressing the mTORC1-pathway direct via the regulatory associated protein of mTOR (Raptor) (Mihaylova and Shaw, 2011). Furthermore, mechanical stimuli is suggested to increase the expression of different Insulin-Like Growth Factors (IGFs) (Goodman, 2014). In cultured muscle cells the release of IGF-1 has been shown acutely after mechanical stimuli (Perrone et al., 1995), and IGF-1 stimulate protein synthesis as well as inhibit protein

degradation when measured in rat skeletal myotubes (Gulve and Dice, 1989). IGF-1 will phosphorylate lipids on the plasma membrane and activate the phosphatidylinositol 3-kinase (PI3K) (Dardevet et al., 1996) leading to activation of intracellular serine/threonine kinase called Akt (King et al., 2015, Manning and Cantley, 2007). When phosphorylated, Akt can stimulate mTORC1 signaling either directly thru the phosphorylation of the mTORC1

inhibitor Proline-rich Akt substrate of 40 kDa (PRAS40) (Sancak et al., 2007) or via tuberous sclerosis 2 (TSC2) (Manning and Cantley, 2007, Goodman, 2014). The importance of IGF-1 and evidence supporting that IGF-1/PI3K/Akt-dependent activation of mTORC1 signaling is a required response for hypertrophy after resistance exercise in human muscle has albeit later been challenged (Philp et al., 2011), and the relationship is not seen in vivo when targeting p70S6K1 (Mitchell et al., 2013). When PI3K is inhibited in rodents during a bout of eccentric resistance exercise (O'Neil et al., 2009) or mechanical overload (Miyazaki et al., 2011) no difference in mTORC1 activation was noted compared with when PI3K was active, suggested alternative signaling pathways such as intracellular phosphatic acid (PA). PA is sufficient for the mechanical activation of mTOR signaling (Hornberger et al., 2006) and strongly

suggested to play a key role in the mTORC1 signaling response to mechanical stimuli. PA may also explain the PI3K/Akt-independent activation of mTORC1 (Goodman, 2014). In addition to these mechanisms an upstream stimulus that have been suggested to affect the mTORC1 signaling is lactate (Nalbandian and Takeda, 2016, Schoenfeld, 2013, Oishi et al., 2015, Willkomm et al., 2014).

1.5 Lactate

In 1780 a swede by the name Carl Wilhelm Scheele discovered an acid in sour milk and named it “mjölksyra”, in English; lactate based on the Latin word for milk. In the early 19th

century Berzelius discovered that the meat from slaughtered stags contained lactate and his conclusion was that the levels were higher in animals who struggled more before their death.

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7 By the middle of 19th century it was reported that lactate from milk and muscle had different properties, and during early 1900s several studies led to the view of lactate being a residual product, formed in the absence of O2,resulting in acidification of the muscle (Ferguson et al.,

2018, Robergs et al., 2004, Philp et al., 2005).

These early assumptions are based on correlations, since lactate is easy to measure in the blood and an increased concentration correlate with perceived fatigue, leading to the paradigm that lactate is the cause of fatigue, which lack biochemical support (Robergs et al., 2004). Lactate is a part in the metabolic process of glycolysis, the breakdown of carbohydrate to resynthesize ATP. In the process of glycolysis, the product pyruvate may proceed in two directions, via mitochondria and oxidative phosphorylation or through the conversion to lactate (Erlandson-Albertsson and Gullberg, 2007, Lännergren et al., 2012, Haff and Triplett, 2016, Åstrand et al., 2003). When ATP is broken down, H+ _{is released within the muscle cell}

and as long as the energy demand is met by mitochondrial respiration no H+ _accumulation

occurs since the H+_{are oxidized in the electron transport chain called Krebs cycle (Robergs et}

al., 2004, Erlandson-Albertsson and Gullberg, 2007). At higher exercise intensities pyruvate and the coenzyme NAD+ will increase above levels that can´t be handled by the mitochondria and formation of lactate occur from pyruvate when catalyzed by the enzyme lactate

dehydrogenase (LDH) (Haff and Triplett, 2016, Robergs et al., 2004).

From a biochemical point of view the production of lactate is beneficial, the LDH reaction allows continued ATP to be regenerated from glycolysis, the reaction is alkalinizing to the cell and in net consumes 2 H+ (Robergs et al., 2004). Lactate is the end product of glycolysis and after lactate is produced, the molecule can be removed via monocarboxylate transporter (MCT) from the cell and consumed in other tissue such as other muscle cells and organs (Brooks, 2009, Sonveaux et al., 2008, Adeva-Andany et al., 2014, Nalbandian and Takeda, 2016, Ferguson et al., 2018, Gladden, 2004, Philp et al., 2005, Robergs et al., 2004). Lactate is used as energy directly by other tissue for example via muscle cells switching from net lactate production to net lactate consumption during exercise, if more muscle mass is involved (Richter et al., 1988) or indirect through gluconeogenesis in the liver via the Cori cycle (van Hall, 2010, Haff and Triplett, 2016, Ferguson et al., 2018). Lactate also fuel the brain (Ferguson et al., 2018, Bouzat et al., 2014, Pellerin et al., 1998) and heart, especially during exercise (van Hall, 2010).

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8 Lactate infusion during exercise increase oxidation of lactate while glucose oxidation

decreased without contributing to perceived fatigue (Miller et al., 2002a, Miller et al., 2002b), uptake is depending on circulating levels of the molecule (Gertz et al., 1988).

1.6 Lactate as a signaling molecule

The mechanisms by which mTORC1 signaling and protein synthesis is stimulated after resistance exercise are, as noted above, not well known but there are proposed mechanisms such as mechanotransduction involving PA. Resistance exercise with low-load has a similar effect on hypertrophy compared to higher load if lifted to failure (Mitchell et al., 2012), or even more effective in phosphorylation of p70S6K1_{and 4E-BP1 than high-load low volume}

exercise (Burd et al., 2010b). Blood flow restriction during low-load resistance exercise has been shown to induce a stronger p70S6K1 phosphorylationcompared with low-load exercise without blood flow restriction. As a result of the blood flow restriction, lactate concentration where significantly higher compared to the situation without blood flow restriction (Fujita et al., 2007). In contrast, shorter set-rest has shown to blunt the effect on protein synthesis and mTOR signaling pathway, however with focus on lactate the difference in plasma levels between groups where minor. Furthermore, the exercise volume drastically decreased with shorter set-rest and is a potential explanation for reduced phosphorylation of p70S6K1 (McKendry et al., 2016).

Resistance exercise protocols that accumulate a significant amount of metabolites within the muscle indicates a positive impact on hypertrophy and it is speculated if the metabolites themselves are stimulatory factors for the hypertrophic response or if the accumulation merely is a consequence of the mechanical stress (Dankel et al., 2017, Schoenfeld, 2013). Thresholds for muscle activation is decreased by the presents of metabolites (Sahlin et al., 1997, Houtman et al., 2003) and as a result, more of the muscles fibers are activated under metabolic stress. Generating uncertainty whether it is the metabolic stress or increased fiber activation that is of the greatest importance for hypertrophy (Schoenfeld, 2013). In vitro, muscle cells from mice, treated with lactate significantly increased the phosphorylation of p70S6K1 _{and mTOR}

compared with control (Oishi et al., 2015). Muscle cells from rat incubated with lactate increased the expression of mitochondria related proteins via the production of reactive oxygen compounds (ROS) which suggest that the potential signaling properties of lactate might be indirect (Hashimoto et al., 2007).

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9 According to the in vitro data and indications from in vivo studies, it is possible that an

increase in levels of lactate during exercise could have a positive effect on mTORC1

signaling pathway and protein synthesis, either direct or indirect via reduction of the threshold for muscle fiber activation (Schoenfeld, 2013, Nalbandian and Takeda, 2016).

In the last five years three reviews (Nalbandian and Takeda, 2016, Schoenfeld, 2013, Dankel et al., 2017) all highlight the problem of isolating metabolites as individual factors in studies. The reviews raise the need for better controlled studies on trained individuals to convey the knowledge of lactates role as a signal molecule forward.

2.0 Aim

The purpose of this study is to investigate the role of lactate, as a potential signaling molecule, for the mechanisms underlying resistance exercise adaptation in the muscle cell. In creating, as far as possible, two similar situations where the difference is the levels of lactate in the participants bloodstream and muscles during resistance exercise. Knowledge and a broader understanding of this area is of important for all athletes and individuals with goals to improve muscle growth and strength, as well as in a rehabilitation purpose as potentially higher levels of lactate, achieved thru shorter set-rest could be beneficial for hypertrophy when for example a joint or ligament can’t be loaded as usual due to an injury.

2.1 Research questions

Do different levels of lactate during resistance exercise affect the stimulation of the mTORC1 signaling pathway during recovery from exercise?

Do different levels of lactate during resistance exercise affect the stimulation of protein synthesis 0-3 h and 0-24 h after the exercise session?

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3.0 Method

Study design; randomized controlled trial, with single-blinded trials utilizing a crossover design.

3.1 Pre-study infusion testing

Since an infusion of sodium lactate during resistance exercise never has been studied and the sodium lactate solution was custom-made for the purpose of this study, pre-testing of the infusion and protocol occurred. The pre-study testing consisted an infusion of a hypertonic sodium lactate solution (Na+ 1000 mmol/L, lactate 1000 mmol/L at pH 6.2-6.5, prepared by APL, Sweden), different doses and intensity during exercise was tested to set the experimental protocol. The dose of sodium lactate was based on previous studies (Miller et al., 2005,

Bouzat et al., 2014, Buckley et al., 2001, Miller et al., 2002a, Miller et al., 2002b, van Hall et al., 2009, Ryan et al., 1979, Mustafa and Leverve, 2002, Schiffer et al., 2011). To obtain a balance between infusion, clearance and lactate production during the heavy resistance exercise, pre-study testing occurred at a total of seven occasions and performed on voluntary employees at the laboratory, the author and supervisor. Infusion dose started at 30

µmol/kg/min and tested both during rest and resistance exercise. After gradually increasing the dose it was set to 50 µmol/kg/min with three boluses á 10 ml over a 50 – 60-minute period (fig. 2), due to the increase in lactate clearance during resistance exercise. The exercise

protocol that was established did not differ between sodium lactate and saline infusion.

Fig. 2. Overview of the infusion protocol. Boluses infused immediately after finished exercise set and when blood sample had been obtained, post warm up, the 2th _{and 4}th _{set in} both trials with either saline or sodium lactate.

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3.2 Participants

Eight healthy volunteers, four women and four men participated in the study. Inclusion criteria were performance of moderate resistance exercise of the lower limbs one to two times a week for at least one year and being able to lift a one repetition maximum (1RM) with a minimum, 100% of their body weight in kg (BW) for the women and 120% of BW for the men per leg. Participants stated they were free from drugs, performing enhancing supplements and did not use nicotine around the trials.

Table 1. Participant characteristics

3.3 General experimental design

After the participants presented interested in study via e-mail, a first of total five occasions at the Åstrand Laboratory of Work Physiology (table 2.), where booked for preliminary

assessments. During the first occasion participants received written and oral information about the experimental trial (appendix 1), signed informed consent and filled in a health status form and after that, pre-testing (described below) occurred.

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12 If the participant met the criteria for enrollment, the next four occasions where each

performed approximately one week apart. In most cases the participant visited the lab at the same weekday for four weeks. To confirm that the maximal strength test was representative and to minimize the acute effects of neural adaptions to the protocol, two familiarizing exercise sessions where performed before the experimental trials. In order to enable measurement of muscle protein FSR participant consumed an oral dose of 2H2O the day

before the experimental trials. During the trials, participants performed a unilateral leg

extension exercise with either an infusion of sodium lactate or placebo in the form of saline. A total of five muscle biopsies were collected from the exercising leg during each trial. The exercise protocol was set so that the load, number of repetitions and time under tension would be similar between the two experimental trials.

Table 2. Overview of the five occasions at the Åstrand Laboratory of Work Physiology

3.3.1 Preliminary assessments

Leg strength. Maximal knee extensions were tested using a leg extension machine (Star Trac,

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13 The test protocol to determine the 1RM for each leg is based on previous protocols (Haff and Triplett, 2016, Delorme and Watkins, 1948) but slightly modified since 1RM is tested

unilaterally at the same occasion. The protocol was performed as follows; participants was fastened with a belt over the hip and informed to keep hands in the lap throughout the protocol. Light weighted warm up, 10 repetitions with full extension in the knee joint and with the foot and knee straight forward in sagittal plane starting with non-dominant leg. The participant then performs six reps of unilateral leg extensions, with light load 20-30 kg. Between non-dominant and dominant leg, the participant rests two and a half minutes before the next step. Weight is set to a level that the participant can handle for two to three

repetitions. After three sets of warm up the weight is increased successively to failure, with five minutes of rest between each attempt. Maximal leg strength was determined to calculate the load at the designated percentage of 1RM used in the experimental protocol.

Anaerobic capacity. During a five minutes rest following the 1RM assessment, the participant

was informed of the following 30 second all out sprint test, performed on cycle ergometer (SRM, Germany) to estimate anaerobic capacity. Before the sprint test a capillary blood sample via fingertip was taken to measure the resting levels of lactate in venous blood. The sprint test was performed sitting and started with a warm up for five minutes at 100 watts. To initiate the test, the participant began pedaling up to about 80 rpm, followed with a few seconds countdown where the cadence was increased (Bellardini et al., 2009). A sprint of 30 seconds was then performed with maximal effort at a fixed cadence of 115 rpm, with the participants being heavily encouraged by the test leaders during the sprint. The cycle

ergometer software (SRMX training software) samples power output continuously every 0.5 s which enables determination of peak power and mean power during the test. After testing, capillary blood was taken after one minute to analyze the lactate levels after the anaerobic maximal effort.

3.3.2 Familiarizing exercise session

During the familiarizing exercise sessions, participants were accustomed to the exercise protocol by performing the protocol with both legs, one at a time. The protocol started with three sets of warm up, separated by two minutes of rest, were the load was based on 0, 25 and 50% of their individual 1RM.

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14 Thereafter the participants performed six sets of heavy resistance exercise to fatigue, starting at 80% of their 1 RM, gradually reducing the load so that at least eight, but not more than ten repetitions where performed. Time under tension was supervised so that the sessions were as similar as possible. Participants received feedback if they needed to slow down or increase the contraction speed. The instructions given was; explosive contraction to straight angle in the knee joint and controlled eccentric contraction down to full flexion then repeat. The test leaders assessed whether the range of motion was sufficient and if the weight should be altered. Between every heavy set was there a three-minute set-rest. Before the first exercise session leg volume and area were assessed and after the second exercise session a baseline saliva sample collected for controlling basal 2H enrichment in the body. Here participants were also provided with the first 2H2O dose based on body weight.

Leg volume. Leg volume (V) was calculated using the formula V = (L/12π) · (C12_{+ C2}2

+ C32_{) − [(S − 0.4)/2] · L · [(C1 + C2 + C3)/3]. Surface measurements of participants’ thigh}

length, from trochanter major to the lateral femur epicondyle (L), three circumferences, up ¼ of L (C1) mid ½ of L (C2) and low ¾ of L (C3) together with skinfold at C2, anterior on thigh (S). (Tothill and Stewart, 2002, Andersen and Saltin, 1985)

Muscle area (bone free). The muscle area AM presented in table 1 was calculated using the

formula AM = 0,649 · ((C2/ π – S)2 – (0,3 · dE)2). In addition to the factors already mentioned

under leg volume, epicondyle diameter of femur dE is present in the formula. (Knapik et al.,

1996)

3.3.3 Experimental trials

The day before the first experiment participants consumed 3.5 mg/kg body weight of a liquid (Cambridge Isotope Laboratory) containing 70% 2H2O with the aim to enrich the body water

with 0.3-0.4% 2_H

2O for proper measurement of FSR (Gasier et al., 2012). The isotope was

sterile filtered and divided into 50 ml doses that were instructed to be consumed separated by at least one hour between 8 am and 8 pm the day prior to trial. By dividing the dose over a longer period, the risk of dizziness and nausea was reduced, which is a known possible side effect of drinking 2H2O (Gasier et al., 2010, Wilkinson et al., 2015). Participants reported to

the laboratory 07.00 am after having been fasted for at least nine hours, refrained from alcohol, caffeine and nicotine upon the experiments.

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15 Resistance and endurance type of exercise was not allowed during the two days prior to

arrival. The experiment started with the subject taking a supine position followed by the insertion of a peripheral venous catheter (PVC) into a forearm vein in both arms, to enable blood sampling in the right arm and infusion of sodium lactate/saline in left (fig. 3). Blood samples (á 5 ml) were collected into Lithium Heparin-tubes and immediately analyzed for levels of lactate, sodium (Na+), pH, glucose, and potassium (K+) in the blood. Rest of the sample maintained on ice for approx. 60 min before being centrifuged at 3000 g, 10 min in 4°C. The plasma thus obtained was transferred to Eppendorf tubes and placed in liquid nitrogen for storage at -80°C until further analysis. Following 30 min of rest, and the clearance for participation by control of normal blood parameters, a baseline muscle biopsy sample was obtained from the quadriceps m. vastus lateralis in the randomized leg using a Weil-Blakesley conchotome (AB Wisex, Mölndal, Sweden) under local anesthesia (Carbocain 20 mg/ml), using the conchotome for 62 of 80 of the biopsies (Henriksson, 1979). In the residual 18 biopsies the Bergström needle technique was used (Bergstrom, 1975).

Fig. 3. Picture of the set up with infusion in left arm and venous blood sampling in the right. The participants were not allowed to us the device´s handles during exercise to minimize local lactate production.

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16 Muscle tissue samples were immediately blotted free from blood and frozen in liquid nitrogen for further storage at - 80°C until analysis. The participants were subsequently seated in the leg extension machine and infusion started. During the first 20 minutes of infusion the

participants rested, and blood sample was analyzed every 10th minute. After the resting period resistance exercise was performed as described in section 3.3.2, with blood sampling after warm up, the 2nd, 4th and 6th set and analyzed directly after sampling. Immediately after the 6th set the infusion stopped and participant rapidly placed in supine position for second muscle biopsy. For the following 180 minutes the participants rested in a supine position, blood samples and biopsies were repeated according to figure 4.

After the 180-minute post exercise biopsy participants received 34 g of protein, 21 g CHO and 6,5 g of fat via a shake of whey protein (Enervit 100% whey, vanilla flavor) and a protein bar (Enervit 26% protein, coco crunch flavor). Participants were instructed to consume 24 g of whey protein in the late evening, and otherwise live and eat as usual until 10.00 pm when next fasting begun. The participants then reported to the lab the next day 07.00 am for the 24 h biopsy and 16th blood sample. The terminal half-life of 2H2O is seven to ten days and to

receive a similar body water enrichment of 2H2O between trials, participants consumed

another dose of 0.15 ml/kg/day between trials, 24 h before performing the second experiment trial that took place seven to ten days after the first (Speakman and Hambly, 2016, Gasier et al., 2010). During the second trial, type of infusion and exercise leg was changed according to randomization.

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3.4 Visual Analog Scale

In order to monitor perceived pain and discomfort of the subjects during infusion, a visual analog scale (VAS) were used. VAS is a measuring instrument that allows the participant to move a marker over a 100-millimeter-long line where zero stands for no pain and 100 mm for the worst imaginable pain. The values are then collected and presented in a subjective pain experience from 0 to 100 (Williamson and Hoggart, 2005). Participants estimated a central sensation and a local sensation in the leg that was exercising three times during infusion, 15 minutes into infusion, post warm up and after 5th _{set. The purpose of monitoring pain between}

the infusions was to evaluate if lactate contributes to the sensation of pain during exercise.

3.5 Blood analysis

Venous whole blood lactate and glucose: Concentrations of lactate and glucose during the

trials were determined using BIOSEN C_line Clinic/GP+ Glucose/Lactate Measuring System (EKF Diagnostics, Cardiff, UK). Immediately when the blood was sampled, 20 µl was added to a one ml test tube provided with the analytical kit and prefilled with a hemolyzing solution to dilute the sample 1:51. The machine was calibrated every 60 minutes and every test were performed in duplicates and the mean calculated between samples to increase validity.

Venous whole blood Na+, K+, pH: During the infusion of sodium lactate and saline the levels

of electrolytes (Na+ & K+) and the blood pH was supervised in the blood samples using a i-STAT1, handheld blood analyzer (Abbott Point of Care, USA) calibrated before every trial. 95 µl of blood was pipetted into an EG6+ _{cartridge, inserted into the i-STAT1 and analyzed}

within minutes for the monitoring of participant’s safety.

3.6 Muscle tissue analysis

Muscle samples were after the trials lyophilized and carefully dissected clean from blood, lipid droplets and connective tissue under a light microscope (VWR, USA), leaving small bundles of muscle fibers that were mixed to obtain a homogenous sample. After muscle samples were cleaned free from contaminating tissue and mixed, the muscle samples of lyophilized muscle were stored at -80°C and aliquots for analysis weighed on an analytical balance scale (XA105, Dual Range, Mettler Toledo, Switzerland) calibrated before the measuring of muscle samples.

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3.7 Immunoblotting

To enable immunoblotting the muscle sample were homogenized as described in detail by Moberg et al. (2016b), briefly: 3 mg of lyophilized muscle were homogenized in ice-cold buffer utilizing a Bullet blender™. The homogenates were then rotated for 30 min at 4°C and centrifuged at 10,000 g for 10 min. The supernatant was collected and protein concentration in an aliquot of the supernatant was determined using the Pierce™ 660nm protein assay (Thermo Scientific). The remaining supernatant and pellet was then stored at -80°C for further analysis. The muscle homogenates were diluted with 4 x Laemmli sample buffer (Bio-Rad, Richmond, CA) and homogenizing buffer to obtain a final protein concentration of 1.5 µg/µl. The samples were heated at 95°C for 5 minutes to denature the proteins, and subsequently stored at -20°C until loading. From each sample, 22.5 µg of protein was loaded onto 26-well Criterion TGX™ gradient gels (4–20% acrylamide; Bio-Rad) and electrophoresis was performed at 300V for 30 min on ice. The gel was then equilibrated in transfer buffer and proteins were transferred to polyyinylidine fluoride membranes (Bio-Rad) at a current of 300 mA for 3 h at 4°C. The membranes were stained with MemCode™ Reversible Protein Stain Kit (Thermo Scientific) so that equal loading and transfer could be confirmed. The

membranes were then destained, cut into strips containing each target protein and assembled so that the entire sample set were exposed to the same blotting conditions. After the strips were assembled in containers divided by the target protein, they were blocked in Tris-buffered saline (TBS; 20 mM Tris base, 137 mM NaCl, pH 7.6) containing 5% nonfat dry milk for 1 h at room temperature.

Membranes were thereafter incubated overnight with primary antibodies from rabbit, diluted in TBS supplemented with 0.1% Tween 20, containing 2.5% nonfat dry milk (TBS-TM). Next day membranes were washed with TBS-TM and incubated with secondary antibodies from rabbit for 1 h at room temperature, washed with TBS-TM 2 x 1 min, 3 x 10 min and then washed with TBS 4 x 5 min. Target proteins on the membranes were visualized by applying Super Signal West Femto™ Chemiluminescent Substrate (Thermo Scientific), followed by detection on a Molecular Imager ChemiDoc XRS system and quantification of the resulting bands with the contour tool in the Quantity One software (version 4.6.3; Bio-Rad). Following visualization, the membranes were stripped of the phosphospecific antibodies utilizing

Restore Western Blot Stripping Buffer (Thermo Scientific) for 30 min at room temperature, washed and reprobed with primary antibodies to detect the total amount of each protein,

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19 as described above. The levels of all phosphoproteins were normalized to the total level of the corresponding protein. (Moberg et al., 2016b)

3.8 Muscle protein fractional synthetic rate

In order to quantify muscle protein FSR approx. 4 mg of lyophilized muscle from each muscle sample was sent on dry ice to the Clinical Metabolomics Core Facilility at

Rigshospitalet in Copenhagen. The analysis was conducted according to the method described by Wilkinson et al. (2014), but with the difference that a liquid chromatography with the combination of two mass analyzers (LC-MS/MS) was used instead of a gas chromatography – mass spectrometry (GC-MS) for detection of 2_{H-alanine in plasma.}

3.9 Muscle levels of lactate

For determination of muscle levels of lactate approx. 2 mg of lyophilized muscle was extracted in 100 µl of ice-cold trichloroacetic acid using a glass pestle. Samples were subsequently centrifuged for 10 min at 3000g 4°C, the remaining supernatant collected and neutralized using 1M KOH. Levels of lactate were subsequently determined

spectrophotometrically as described by Bergmeyer (1974).

3.10 Ethics

During the trials, pain and discomfort during biopsies and insertion of PVCs can be an

unpleasant experience for the participants. Also, the fact that biopsies will leave sore legs and scars for up to month’s after trials is an ethical concern. Participants can also experience discomfort prior to the infusion. To prepare participants for the trials they received both oral and written information (appendix 1) and had the chance to walk away from the trial at any point. The benefits with participation are to get a broader understanding of exercise

physiology and helping to understand the unknown. All participants were given access to their own physiological test data. At the first occasion, immense importance was given to choosing participants who were interested in the research questions and had a main interested in

exercise physiology, not in the financial compensation (3000 SEK). Ethical approval number: 2017/1139-31/4 was granted 2017-06-21 by the Regional Ethical Review Board in Stockholm and the study conformed to the code of the Helsinki Declaration.

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3.11 Statistics

When parametric statistics is present, the mean values and standard error of mean (SEM) are

presented as mean ± SEM. A two-way ANOVA (time x intervention) repeated-measurements,

applied to compare intracellular signaling changes, muscle levels of lactate, blood markers from exercise data and whole blood lactate data during exercise. In case of a significant main effect or an interaction, Fisher´s post hoc test was performed. For significant differences between situations in the exercise performance data (load, repetitions and time under tension) a two-way dependent t-test was used. For non-parametric data as pain and discomfort values are presented and Friedman´s two-way ANOVA used for statistically significant between trials. Statistical analyses were performed with STATISTICA and in Excel. P < 0.05 was considered statistically significant.

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4.0 Results

4.1 Exercise data

Total number of repetitions in the resistance exercise performance averaged 82 ± 1.8 in the placebo trial and 82 ± 2.0 in the lactate trial, the average time under tension 212 ± 8.0 in the placebo trial and 212 ± 9.9 for the lactate trial. Six of the participants lifted the exact same load in both trials while one male participant lifted 2.5 kg less in one set (placebo, non-dominant leg) and one female participant lifted 2.5 kg less in four sets and 7.5 kg less in the last set (lactate, non-dominant leg). There are no significant differences in load (P=0.11), repetitions (P=0.28) or time under tension (P=0.27) between trials.

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Venous whole blood Na+, K+, pH and glucose: No significant differences in resting values

between the trials. When sodium lactate infusion started the pH increased and continued to stay elevated compared with placebo during the lactate trial (P<0.05), peaking 60 min post exercise before descending toward resting values. In the placebo trial, the pH decreased as an effect of exercise and were lower post exercise compared with rest (P<0.05). Changes in Na+ and K+ followed the same trend during trials, blood levels increased during exercise/infusion (P<0.05). There were no significant differences between trials in Na+. K+ were reduced during the lactate trial compared with the placebo trial (P<0.05) and levels went back to baseline in recovery were in the placebo trial the levels stayed elevated even in the 180 min recovery sample. There were no significant differences in the levels of glucose between trials but an effect of time after 90 min recovery in both trials, with levels lower than baseline. All

participants were within the reference range of physiological normal values during the trials in both sodium lactate and saline infusions. Values presented in graphs bellow is from the blood samples 1 (baseline), 4 (post WU), 7 (post exercise), 10 (30 min recovery), 12 (60 min recovery), 13 (90 min recovery) and 15 (180 min recovery) (fig. 4).

Fig. 5. Venous whole blood concentrations of sodium (A), pH (B), potassium (C) and glucose (D) during the two trials. Values are presented as mean ± SEM for 8 participants. *P< 0.05 vs. rest and #_{P< 0.05 vs. trial.}

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Perceived pain and discomfort: There were no significant differences between the placebo

trial and the lactate trial in neither an experienced centralized, nor a leg pain. One participant experienced the first trial as more centrally painful (placebo), one participant experienced the second trial more centrally painful (placebo). Three participants experienced the first trials as more painful in the leg (two placebo, one lactate). One participant experienced both more centrally and leg pain post 5th set in the second trial (lactate). Remaining estimates is within 10 millimeters between the trials, none of the participants stated after the trials that they could feel in which trial they received sodium lactate or placebo.

Fig. 6. Individual values from all 8 participants presented in the graph with different symbols. VAS Centrally present the perceived pain and discomfort in the participants bodies during the trials and VAS Leg the perceived pain and discomfort in the participants exercising leg.

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4.2 Lactate data

Venous whole blood lactate: Blood levels of lactate did not differ at baseline between the

trials. In the placebo trial did the effect of resistance exercise raised the levels significant to 2.26 ± 0.24 mmol/L post 2nd set and stayed significant elevated up to 45 min recovery with a peak of 3.00 ± 0.26 mmol/L post 6th set (P<0.05). In the lactate trial did the levels increase to 1.34 ± 0.08 at the 10-minute infusion sample as an effect of intervention (P<0.05). Levels remained elevated during the infusion and raised to 1.85 ± 0.14 mmol/L after 20 min of infusion, with a peak of 6.94 ± 0.47 mmol/L post 6th_{and significant elevated levels up to 60}

min recovery after resistance exercise (P<0.05). Different levels between the trials from 10 min infusion to 45 min recovery with 131% higher peak in lactate compared with placebo (P<0.05).

Muscle levels of lactate: There were no differences in muscle levels of lactate at baseline

between trials. In both trials levels increased from baseline to post resistance exercise with a mean of 21.7 ± 2.6 mmol/g dry weight for the placebo trial and 26.1 ± 2.6 mmol/g in the lactate trial and stayed elevated at 90 min recovery (P<0.05). The ANOVA revealed a main effect of trial with significantly higher muscle levels of lactate present in the lactate trial.

Fig. 7. Venous whole blood concentrations of lactate (A) and levels of lactate in lyophilized muscle (B). Values are presented as mean ± SEM for 8 participants. *P< 0.05 vs. rest and #P< 0.05 vs. trial.

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4.3 Intracellular signaling

Downstream events: The phosphorylation of mTOR at Ser2448 and p70S6K1 atThr389 increased immediately after resistance exercise in both trials and remained elevated up to 180 min recovery compared with baseline (P<0.05). Mean of mTOR at Ser2448 increased post 69% in the placebo trial, and in the lactate trial the mean increased post resistance exercise 72% compared with baseline. Mean of p70S6K1 atThr389 increased with a fold change of 5 compared with rest values in both the placebo trial and the lactate trial with elevated levels even at 180 min in both trials (P<0.05). No difference between trials in the phosphorylation of mTOR at Ser2448_{and p70}S6K1 _at_Thr389_.

Fig. 8. Phosphorylation of mTOR at Ser2448_{for 8 participants (A), when divided by 4 women (B) and} 4 men (C). Values in graphs are presented as means ± SEM*P < 0.05 vs. rest. Representative

phosphorylated bands (upper panel) and total protein (lower panel), from one participant is shown above graph B and C. The two sets of bands have been rearranged to fit the order of trials in the graph.

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26 Fig. 9. Phosphorylation of p70 at Thr389_{for 8 participants (A), when divided by 4 women (B) and 4} men (C). Values in graphs are presented as means ± SEM*P < 0.05 vs. rest. Representative

Phosphorylation of 4E-BP1 both at Ser65 and Thr37-46 decreased significantly immediately after resistance exercise and the repressed phosphorylation of 4E-BP1 at Ser65 returned to baseline at 180 min (data not shown) and at 90 min for that of Thr37-46 (P<0.05). In the placebo trial mean of 4E-BP1 at Ser65 was repressed 54% (data not shown) and at Thr37-46 repressed 43% compared with baseline post resistance exercise. In the lactate trial mean of 4E-BP1 at Ser65 was repressed 53% (data not shown) and at Thr37-46 repressed 46% compared with baseline post resistance exercise. Phosphorylation of eEF2 at Thr56 decreased

significantly 90 min post resistance exercise (P<0.05). In the placebo trial, repressed 52% compared with baseline at 90 min recovery and in the lactate trial, repressed 32% at 90 min recovery. The reduced eEF2 phosphorylation was maintained significant even at 24 h compared with baseline (P<0.05) (data not shown).

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27 No significant main effect of intervention in downstream events. The number of participants in the present thesis were underpowered for statistical between sex comparisons, however values and numeric differences for the female and male participants are presented in the figures 8-13.

Fig. 10. Phosphorylation of 4EB-P1 at Thr37-46_{for 8 participants (A), when divided by 4 women (B)} and 4 men (C). Values in graphs are presented as means ± SEM*P < 0.05 vs. rest. Representative phosphorylated bands (upper panel) and total protein (lower panel), from one participant is shown above graph B and C. The two sets of bands have been rearranged to fit the order of trials in the graph.

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28 Fig. 11. Phosphorylation of eEF2 at Thr56_{for 8 participants (A), when divided by 4 women (B) and 4} men (C). Values in graphs are presented as means ± SEM*P < 0.05 vs. rest. Representative

Upstream events: The phosphorylation of p44 at Thr202/Tyr204 and TSC2 at Ser1387 increased statistically immediately after resistance exercise (P<0.05). The degree of phosphorylation of p44 at Thr202/Tyr204 was elevated with a fold change of 4 in the placebo trial and 3 in the lactate trial and stayed up to 90 min recovery compared with baseline. The phosphorylation of TSC2 at Ser1387 increased in the placebo trial immediately after resistance exercise 26% and for the lactate trial 20% compared with baseline, with elevated phosphorylation 180 min in both trials (data not shown) (P<0.05), with no differences between trials at any time point. In both trials the phosphorylation of PRAS40 at Ser183 _{decreased significantly immediately after}

resistance exercise, 31% in the placebo trial and 30% in the lactate trial (P<0.05), returning to baseline levels after 90 min recovery (data not shown).

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29 There were no significant differences in phosphorylation of AMPK after resistance exercise, all 8 participants increased the degree of phosphorylation in the placebo trial. post resistance exercise, and 7 of 8 in the lactate trial, resulting in a P=0.057 for time. No significant main effect of intervention in upstream events. The number of participants in the present thesis were underpowered for statistical between sex comparisons, however values and numeric differences for the female and male participants are presented in the figures 8-13.

Fig. 12. Phosphorylation of p44 at Thr202_/Tyr204_{for 8 participants (A), when divided by 4 women (B)}

and 4 men (C). Values in graphs are presented as means ± SEM*P < 0.05 vs. rest. Representative phosphorylated bands (upper panel) and total protein (lower panel), from one participant is shown above graph B and C. The two sets of bands have been rearranged to fit the order of trials in the graph.

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30 Fig. 13. Phosphorylation of AMPK for 8 participants (A), when divided by 4 women (B) and 4 men (C). Values in graphs are presented as means ± SEM. Representative phosphorylated bands (upper panel) and total protein (lower panel), from one participant is shown above graph B and C. The two sets of bands have been rearranged to fit the order of trials in the graph.

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4.4 Muscle protein fractional synthetic rate

FSR data is presented for three participants, two men and one woman. Participant 1 and 2 performed the placebo trial first followed by the lactate trial while participant 3 performed lactate trial, followed by the placebo trial. The mean value, FSR (% · h-1) for placebo 0.13 ± 0.07 in the 0-3 h calculation and 0.17 ± 0.10 for lactate. In the 0-24 h calculation the mean for placebo were 0.11 ± 0.02 and 0.11 ± 0.01 for lactate. FSR underpowered for statistical

analysis.

Fig. 9. Individual values for 3 participants presented in the graph with different symbols. Mean ± SEM of the individual values representing the forth symbol. FSR calculated from 2_{H-labeled alanine} detected by LC-MS/MS between baseline (0 h) and 180 min recovery (3 h) biopsies.

Fig. 10. Individual values for 3 participants presented in the graph with different symbols. Mean ± SEM of the individual values representing the forth symbol. FSR calculated from 2H-labeled alanine detected by LC-MS/MS between baseline (0 h) and 24 h biopsies.

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5.0 Discussion

To the authors knowledge is this the first time the effects of a sodium lactate infusion during resistance exercise has been studied in human skeletal muscle. Here with the aim to evaluate the effects on regulation of muscle protein turnover. The main findings in this thesis was that different levels of lactate during resistance exercise do not affect the mTORC1 signaling pathway, compared with placebo up to 24 hours in a moderately trained group of men and women. An infusion of sodium lactate increases the levels of venous whole blood lactate during resistance exercise and the lactate levels in muscle up to 90 min post exercise without affecting the performance, perceived pain or discomfort during the trials.

Performance during resistance exercise had a similar outcome with regard to load, numbers of repetitions, and time under tension. There was expected differences between the sexes in load lifted. Six of the participants lifted the exact same load in both trials (3 women, 3 men) while a male participant lifted 2.5 kg less in one set and one female participant lifted 2.5 kg less in four sets and 7.5 kg less in the last set. In both cases was it the participants non-dominant leg which hurled less load and especially in the woman’s case, is it possible that the 1RM-test or the estimated working capacity in her non-dominant leg wasn´t completely representative. One aggravating circumstance in the pre-testing was that the sodium lactate infusion has a 30 days durability date and since the trials were very comprehensive and demanding, participants needed to be booked for their occasions at the laboratory before infusion was ordered. The participant that varied most between trials in load lifted was also the first pre-tested, hence had it gone 38 days between pre-testing and first familiarizing session, which may potentially affect the 1RM. Or it could simply have been the participant's daily fitness.

In the placebo trial, resistance exercise raised the levels of lactate to a mean peak of 3.00 ± 0.26 mmol/L post 6th_{set, and in the lactate trial to a 131% higher level with a peak of 6.94 ±}

0.47 mmol/L post 6th, measured in venous whole blood. When the pre-study infusion testing occurred did the levels of lactate during rest stayed lower than expected. The volunteers did not raise their levels of lactate over 2.00 mmol/L during rest and even throughout the

resistance exercise, the levels of lactate remained low, approx. 3.00 to 4.00 mmol/L, without boluses. This was unexpected since other studies that have investigated the effect of a sodium lactate infusion during rest and cycle ergometer exercise have had higher levels of lactate during rest (Miller et al., 2005, Miller et al., 2002a, Miller et al., 2002b, Ryan et al., 1979).

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33 There are aggravating circumstances when comparing blood data between studies, due to the fact that the infusion is transported into muscle, the levels would be higher in arterial blood, but the resistance exercise will elevate the levels of lactate in venous blood. Since infusion and resistance exercise is combined, the situation that occur physiologically is special. The studies conducted by Miller et al. (2005, 2002a, 2002b) studied men and sampled arterial or arterialized venous blood, also presented plasma data, not whole blood and due to the removal of erythrocytes in plasma compared to whole blood, plasma concentration of lactate would have been higher (Persson et al., 1995). One lactate infusion study compared values from arterial and venous blood in men during handgrip exercise, the mean value for the arterial blood lactate was about 100% higher compared to the venous blood (Buckley et al., 2001). Studies with longer periods of rest during infusion have raised the levels of lactate higher, Miller et al. (2005) had a mean value over 4 mmol/L after a 90 min resting infusion and Bourzat et al. (2014) a mean of 6.1 mmol/L after 3 h of infusion at rest. In these studies, the resting values increased during the infusion, indicating that the system need to be saturated by the lactate infusion during rest. Nevertheless, Bourzat et al. (2014) studied patients in a coma with severe traumatic brain injury, their overall fitness levels are unknown and seeing that the ability to turnover lactate mainly occurs in the bodies fat free mass (van Hall, 2010) it is a possibility that patients in a coma don´t have the fat free mass as the participants of this thesis. Based on the results by Buckley et al. (2001) and due to the results that the infusion is

transported into muscles, the levels would be higher in arterial blood and the sampling of venous blood represent the product of uptake and lactate released from the working muscles. The decision was made to use venous blood because of the benefits, for once, when lactate is analyzed in a sport situation as for example Wingate test, venous whole blood is used and the levels of lactate more comparable than arterial blood. Also due to the risks and difficulties associated with insertion of an artery catheter. Because levels were significantly higher in the muscle samples with a mean of 21.7 ± 2.6 mmol/g dry weight for the placebo trial and 26.1 ± 2.6 mmol/g in the lactate trial, the infusions filled its purpose even if levels of lactate in the blood is difficult to compare with previous work.

Although the system seems to be saturated by higher doses and longer periods of infusion at rest and potentially greater differences in lactate levels during resistance exercise, the infusion is associated with risks, where harmful increases in pH and Na+ is a potential outcome. In the placebo trial did pH decrease during exercise, because of the muscle contractions leading to a release of lactate and H+_{, resistance exercise is acidic to the bloodstream.}

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34 pH did increase during the sodium lactate infusion and stayed elevated during the lactate trial, a larger sodium lactate infusion dose could potentially have resulted in alkalosis post exercise even though infusion stopped. The pH increased during the lactate trial was expected and monitored during both trials. Na+ were also expected to increase, since the sodium lactate solution is hypertonic, but the ANOVA did not find any effects on intervention, only on an increase over time in both trials. Mean peak values for the lactate trial were 143 mmol/L compared with 142 mmol/L for the placebo trial, 145 mmol/L are considered to be hypernatremia. The placebo trial infusion solution, saline is isotonic and will not raise the levels of Na+. Blood levels of circulating Na+ and K+ increased during resistance exercise as a result of the electrical activity in the exercising muscles (Medbo and Sejersted, 1990). The differences in blood levels of K+ between the lactate trial and the placebo trial was seen also by Miller et al. (2005) and since the lactate infusion solution contains Na+_{and the Na}+_/K+

relationship during resistance exercise is complexed, is it likely that the activity of Na+_{- K}+

pumps are affected by the infusion of the hypertonic Na+ _{solution and thus lowers the levels of}

K+ _{circulating in venous whole blood (Tong et al., 1999, Perry et al., 2016). In summary,}

alkalosis appears to be a greater risk than hypernatremia during the sodium lactate infusion.

There were no differences between placebo and lactate in neither an experienced centralized, nor a leg pain. No previous study has reported any pain or discomfort and there are no biochemical indications that the molecule would perceived painful, as presented in the background (Lactate 1.5). Even though a sodium lactate infusion is not the same situation as lactate produced during heavy exercise, it’s an interesting area and since VAS is an easy method to estimate pain during resistance exercise, the results contributes to a deeper understanding of the lactate molecule. The trend was that participants who estimated differences between the trials, estimated more pain their first trial, regardless to the type of infusion. Most likely was it less stressful the second time due to expectations and that the participants were experienced with biopsies, blood samples, infusion etc.

The phosphorylation of mTOR at Ser2448 and p70S6K1 atThr389 increased immediately after resistance exercise in both trials and remained elevated up to 180 min recovery compared with baseline. Burd et al. (2010b) also reported a significant main effect of time in the phosphorylation of mTOR at Ser2448 up to 4 h post resistance exercise and similar to the present results, phosphorylation status returned to baseline after 24 h.

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35 The phosphorylation of p70S6K1 atThr389 is specific for mTORC1 (Pearson et al., 1995) and similar increases induced by resistance exercise up to 180 min recovery has previously been reported (Apro et al., 2015, Moberg, 2016a, Moberg et al., 2014, Moberg et al., 2016b, Burd et al., 2010b). The approximate 5-fold change in phosphorylation of p70S6K1 atThr389 after resistance exercise in the fasted state is comparable with previous results (Apro et al., 2015, Moberg, 2016a) and amino acid ingestion appears to be required for an even larger increase in phosphorylation of p70S6K1 atThr389 (Moberg et al., 2014, Moberg et al., 2016b).

For the second most studied and well characterized target of mTORC1, 4E-BP1 a reduced phosphorylation at Ser65 and Thr37-46 were noticed in both trials immediately after resistance exercise. The phosphorylation status of 4E-BP1is high at rest and a decrease post resistance exercise in fasting participants seen also by Apró et al. (2015) and Moberg (2016a). Food intake and especially amino acids is needed to get a more pronounced phosphorylation of 4E-BP1 (Moberg et al., 2016b) and an expected increase in phosphorylation from the increased mTOR activity (Gingras et al., 1999) is not seen directly after resistance exercise here, nor in the studies mentioned above (Apro et al., 2015, Moberg, 2016a). The reduction is probably influenced by the fact that, 4E-BP1 and p70S6K1 both gets phosphorylated by mTOR, and since 4E-BP1 is highly phosphorylated at rest, the increased phosphorylation of p70S6K1 at Thr389 which occurs immediately after resistance exercise is prioritized and likely prevents the phosphorylation of 4E-BP1 at Ser65 and Thr37-46.

The phosphorylation of eEF2 was the only signaling protein that remained significantly altered from baseline at 24 h post resistance exercise. Previous cell-data have shown that eEF2 is phosphorylated by p70S6K1 and markedly inhibits its activity (Wang et al., 2001). This is however not supported by other studies on human muscle nor by the result in the present thesis (Apro et al., 2015, Moberg, 2016a). eEF2 responded to the resistance exercise, but with a delay compared with p70S6K1 and when resistance exercise has been performed with

additional supplementation of amino acids, the phosphorylation of p70S6K1 atThr389 increased without changes in eEF2 phosphorylation (Moberg et al., 2016b, Moberg et al., 2014). All in all, this suggests that the phosphorylation of eEF2 isn´t depending on p70S6K1 (Mieulet et al., 2007) but rather governed by the muscle contractions itself.

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36 In summary, previous studies showing an increase in p70S6K1 atThr389 phosphorylation post low-load high volume resistance exercise that was comparable with high-load low volume exercise indicated that it could be an endogenous effect mediated by e.g. lactate (Burd 2010b). A potential effect that also Moberg (2016a) reported in a concurrent exercise study on men were the levels of lactate was significantly higher in the concurrent exercise trial compared with only resistance exercise, and the phosphorylation of p70S6K1 atThr389 significantly elevated post exercise in the concurrent trial. In vitro studies were lactate have played a regulatory role in the myogenesis of myoblasts and were different concentration affected the signals of adaptations in muscle cells in response to endurance exercise, have cultured cells with lactate levels of either 10 or 20 mmol/L (Willkomm et al., 2014, Hashimoto et al., 2007). However, the results of this thesis do not support this notion on a group of moderately trained men and women and based on the in vitro studies possibly higher levels of lactate is required to see an effect.

In the results, underpowered differences between sexes is presented in figures 8-13. In this population the effects were in general similar post resistance exercise between the sexes, but for the four women the intracellular response was repressed in the lactate trial during 90 min and 180 min recovery. In the case of the four men the lactate infusion accelerated the response following 90 min and 180 min recovery. Potential sex differences were also noted for AMPK- and mitogen-activated protein kinase, MAPK-signaling. For AMPK this mainly related to differences in time for peak activation, but for p44 at

Thr202/Tyr204 phosphorylation, males exhibited an amplified response during recovery while females exhibited a reduced response in the lactate trial. The thesis was not designed to answer questions about differences between sexes and since the previous lactate infusion exercise studies (Miller et al., 2005, Miller et al., 2002a, Miller et al., 2002b, Buckley et al., 2001, Ryan et al., 1979) involved only males there were no obvious rationale for differences between males and females to take into consideration when designing the method. However, the inclusion of both sexes physiology is a strength to this thesis. The dose of sodium lactate is based on body weight, not based on fat free mass. This could potentially be an explanation in the differences seen between sex since women tend to have lower levels of fat free mass (Nickerson, 2018). Even though adipose tissue contributes, the turnover of lactate mainly occurs in the bodies fat free mass, and if adipose tissue contributes to the uptake of lactate is unknown (van Hall, 2010). This could have resulted in a relative higher level of lactate for the women´s fat free mass to turnover.

Do different levels of lactate have an impact on mTORC1-signal and protein synthesis induced by resistance exercise?