FACULTY OF SCIENCE UNIVERSITY OF COPENHAGEN

PhD thesis by KRISTIAN KIILERICH

PDH REGULATION IN SKELETAL MUSCLE

Academic advisor: Henriette Pilegaard Submitted: 17-12-2010

ACKNOWLEDGEMENTS 

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LIST OF PAPERS 

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ENGLISH ABSTRACT 

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DANSK RESUMÉ 

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STANDING ON THE SHOULDERS OF GIANTS 

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METABOLISM 

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PYRUVATE DEHYDROGENASE COMPLEX 

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Regulation of PDHa activity 

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PDP and PDK 

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Tissue distribution PDP 

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Tissue distribution of PDK 

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Site specificity and PDK 

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Regulation of PDP and PDK activity 

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Regulation of PDP and PDK expression 

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PDH-E1α phosphorylation 

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Fiber type and PDH regulation 

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Activity level and PDH regulation 

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PGC-1α and metabolism 

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PGC-1α and substrate regulation 

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AIMS 

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METHODS 

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Human experiments 

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Mice models 

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Determination of PDHa activity 

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SDS-PAGE and immunoblotting 

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RNA isolation and real time PCR 

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RESULTS AND DISCUSSION 

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Summary of thesis articles 

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Is exercise-induced PDH regulation affected by muscle type? (Study 1) 

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Effect of muscle glycogen on PDH regulation (Study 2) 

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The impact of physical inactivity on exercise-induced PDH regulation (Study 3) 

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Elucidating the importance of PGC-1α in PDH regulation in skeletal muscle at rest and in response to fasting and during recovery from exercise (Study 4)  Integrated Discussion 

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Muscle type 

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Muscle glycogen 

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Plasma FFA 

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Physical inactivity 

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Phosphorylation and PDHa activity 

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New phosporylation sites 

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Methodological Discussion 

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Limitations in PDHa activity determination 

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The mice models 

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CONCLUSIONS AND PERSPECTIVES 

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REFERENCE LIST 

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APPENDIX (STUDY 1­4) 

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ACKNOWLEDGEMENTS I would like to thank Academy of Muscle Biology, Exercise & Health Research, the Lundbeck Foundation and Centre of Inflammation and Metabolism (CIM) for the financial support. Henriette Pilegaard is the one that has motivated as well as made it possible to do this thesis, for that I’m very thankful. Her positive attitude and patience have been qualities that I couldn’t have done without. She has always been willing to share and advise me with her great knowledge within the area of human physiology. Besides I appreciated her fascination for the German soccer team around the mid-70s, and the Swedish DJ “Familjen” and lately Donny Osmond. Last but not least she is singlehandedly the reason for my newest hobby: racing bikes, a hobby I have enjoyed very much. The thesis would not have been possible without help from and collaboration with several people: I would like to thank the Institute of Sport Science at the University of Copenhagen, especially the “fredags hockey” gang and Jesper B Brik who always has had the time to help me in the laboratory. Furthermore I would like to thank Bente Klarlund Pedersen and the rest of the CIM people. From Henriette Pilegaards laboratory I would especially like to thank, Mikkel Gudmundsson, Lotte Leick, Helle Adser, Anne Hviid Jakobsen, Sune Mattsson Johansen, Ninna Iversen, Rasmus Sjørup Biensø, Stine Ringholm Jørgensen, Jesper Olesen, Maja Munk Nielsen, Jakob Grunnet Knudsen, Mads Bønnelycke. Some of you have helped me with academic and laboratory tasks, others have made my lunch breaks funny and interesting, both parts have been cruzential for me. Furthermore I would like to thank the co-authors on the articles of my thesis for the collaboration. I am also grateful to the anonymous reviewer who is responsible for correcting rabbis to rabbit under the “development of antibodies” in one of the articles.

Finally, I would like to thank my family and Julia for always supporting me during the thesis.

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LIST OF PAPERS The present thesis is based on the following review and the four papers listed below. The papers are included in the appendix.

STUDY: 1. Kiilerich K, Birk JB, Damsgaard R, Wojtaszewski JF, Pilegaard H. Regulation of PDH in human arm and leg muscles at rest and during intense exercise. Am J Physiol Endocrinol Metab. 2008 Jan;294(1) 2. Kiilerich K, Gudmundsson M, Birk JB, Lundby C, Taudorf S, Plomgaard P, Saltin B, Pedersen PA, Wojtaszewski JF, Pilegaard H. Low muscle glycogen and elevated plasma free fatty acid modify but do not prevent exercise-induced PDH activation in human skeletal muscle. Diabetes. 2010 Jan;59(1):2632. 3. Kiilerich K, Ringholm S, Biensø RS, Fisher J, Iversen N, Van Hall G, Wojtaszewski JFP, Saltin B, Lundby C, Calbet JAL, Pilegaard H Exercise-induced Pyruvate Dehydrogenase Activation Is Not Affected by Seven Days of Bed Rest. Submitted to Am J Physiol Endocrinol Metab. 4. Kiilerich K, Adser H, Jakobsen AH, Pedersen PA, Hardie DG, Wojtaszewski JF, Pilegaard H. PGC-1α Increases PDH Content But Does Not Change Acute PDH Regulation In Mouse Skeletal Muscle. Am J Physiol Regul Integr Comp Physiol. 2010 Nov;299(5):R1350-9.

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ENGLISH ABSTRACT Pyruvate dehydrogenase (PDH) decarboxylates pyruvate into acetyl-CoA and links glycolysis with the Krebs cycle. Because PDH is the only step where carbohydrate-derived substrate can enter the mitochondria and become completely oxidized, PDH activity can potentially determine if glycogen / glucose is oxidized completely, or whether pyruvate is converted to lactate. Activity of PDH in the active form (PDHa) is overall determined by the degree of PDH-E1α phosphorylation, where PDH-E1α dephosphorylation activates PDH, while PDH-E1α phosphorylation inactivates PDH. The PDH-E1α phosphorylation state is determined by the overall content / activity of the regulatory proteins PDH kinase (PDK), of which there are 4 isoforms, and PDH phosphatase (PDP), of which there are 2 isoforms. The overall aim of the PhD project was to elucidate 4 issues. 1: Role of muscle type in resting and exercise-induced PDH regulation in human skeletal muscle. 2: Effect of muscle glycogen on PDH regulation in human skeletal muscle at rest and during exercise. 3: The impact of physical inactivity on PDH regulation in human skeletal muscle at rest and during exercise. 4: Elucidating the importance of PGC-1α in PDH regulation in mouse skeletal muscle at rest and in response to fasting and during recovery from exercise. The studies indicate that the content of PDH-E1α in human muscle follows the metabolic profile of the muscle, rather than the myosin heavy chain fiber distribution of the muscle. The larger lactate accumulation in arm than leg muscles during exercise in humans may be the result of lower PDH-E1α content and not a muscle type dependent difference in PDH regulation. Both low muscle glycogen and increased plasma FFA are associated with upregulation of PDK4 protein and less exercise-induced increase in PDHa activity in human skeletal muscle. It may be noted that the increased PDK4 protein associated with elevated plasma FFA occurs already 2 hours after different dietary intake. A week of physical inactivity (bed rest), leading to whole body glucose intolerance, does not affect muscle PDH-E1α content, or the exercise-induced PDH regulation. Use of peroxisome proliferator activated receptor γ coactivator 1α (PGC-1α) knockout mice and muscle-specific PGC-1α overexpressing mice suggests that PGC1α increases PDH-E1α content, but does not have clear effects on PDK4 protein content or regulation of PDHa activity in response to fasting and in recovery from exercise in mice. Overall, there is a very close association between the phosphorylation degree of the 3 measured phosphorylation sites and the PDHa activity. This association is very strong at the onset of an

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exercise bout, but becomes weaker during the later part of an exhaustive high-intensity exercise bout, this indicates that additional regulatory mechanisms are involved in this phase of exercise. Mass spectrometry has identified a new phosphorylation site, along with 4 PDH-E1α-acetylation sites, which may have regulatory effects that can explain the current findings. This remains to be determined in future studies.

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DANSK RESUMÉ Pyruvate dehydrogenase (PDH) decarboxylerer pyruvat til acetyl-CoA og forbinder derved glykolysen med Krebs cyklus. Da PDH er det eneste trin, hvor kulhydrat-deriveret substrat kan komme ind i mitokondrierne og blive fuldstændig forbrændt, kan PDH aktiviteten potentielt være bestemmende for om glykogen/glukose forbrændes fuldstændigt, eller om pyruvat omdannes til laktat. Aktiviteten af PDH i den aktive form (PDHa) bestemmes overordnet af fosforyleringsgraden af PDH-E1α, hvor PDH-E1α defosforylering betyder, at PDH er aktiv, mens PDH-E1α fosforylering betyder, at PDH er inaktiv. Endvidere bestemmes fosforyleringen af PDH overordnet af mængden/aktiviteten af de regulatoriske proteiner PDH kinase (PDK), hvoraf der eksisterer 4 isoformer, og PDH phosphatase (PDP), hvor der findes 2 isoformer. Projektets formål var overordnet at belyse 4 problemstillinger. 1: Betydning af muskeltypen for hvile og arbejds-induceret PDH regulering i human skeletmuskulatur. 2: Muskel-glykogens effekt på PDH regulering i human skeletmuskulatur i hvile og under arbejde. 3: Fysisk inaktivitets indvirkning på PDH regulering i human skeletmuskulatur i hvile og under arbejde. 4: Belyse PGC1α’s rolle i PDH regulering i skeletmuskulaturen i hvile samt ved faste og i restitutionsperioden efter arbejder. Studierne viser, at indholdet af PDH-E1α i human muskulatur synes at følge musklens metaboliske profil, frem for myosin heavy chain fordelingen. Tillige synes den større laktatakkumulering i arm- end benmuskulatur under arbejde hos mennesker at kunne være en følge af mindre mængde PDH-E1α, men ikke en forskel i PDH reguleringen i de forskellige muskeltyper. Både lavt muskelglykogen og øget plasma frie fede syre (FFA) er associeret med opregulering af PDK4 protein og mindre arbejds-induceret stigning i PDHa aktiviteten i human skeletmuskulatur. Det kan bemærkes, at den FFA associerede PDK4 stigning indtræffer blot 2 timer efter forskelligt kostindtag. Endvidere vises, at en uges fysisk inaktivitet (sengeleje), der fører til helkrops-glukoseintolerance, ikke ændrer muskulaturens PDH-E1α indhold eller den arbejdsinducerede PDH regulering. Brug af peroxisome proliferator activated receptor γ coactivator 1α (PGC-1α) knockout-mus og muskel-specifikke PGC-1α overekspressionsmus tyder på, at PGC-1α øger PDH-E1α-indholdet, men ikke har klar effekt på PDK4 proteinindholdet eller på regulering af PDHa aktiviteten ved faste og i restitutionsperioden efter arbejde.

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Overordnet er der en meget tæt association mellem PDHa aktivitet og fosforyleringsgrad af PDHE1α på de tre målte fosforyleringssites i starten af et arbejde, mens denne association bliver mindre omkring udmattelse i høj intensitets arbejde. Dette indikerer, at yderligere regulatoriske mekanismer er involveret i denne fase af et arbejde. Dette kunne muligvis omfatte et ved massespektroskopi nyt identificeret fosforyleringssite eller endnu ikke opdagede fosforyleringssites. Men der er tillige fundet 4 PDH-E1α-acetyleringssites, som ligeledes kunne tænkes at have en regulatorisk virkning. Dette kunne være interessant at belyse i fremtidige studier.

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STANDING ON THE SHOULDERS OF GIANTS One of the most important contributors to establishing human physiology as a research area in Copenhagen was Schack August Steenberg Krogh (1874-1949). In 1897 August Krogh began working in the Laboratory of Medical Physiology under Professor Christian Bohr, and in 19081945 he became the head of the newly established department of zoophysiology at the University of Copenhagen. In 1910 he published 7 articles in “Skandinavischen Archiv für Physiologie” with the collective title “The Mechanism of Gas-Exchange“, where he argued that uptake of oxygen and the elimination of carbon dioxide in the lungs took place by diffusion alone. This statement was arguing against Christian Bohr, who believed that exchange of oxygen and carbon dioxide occurred via a nervous controlled secretion. The conclusion was printed in bold, which was not the normal practice of the journal. Along with the description of lung function August Krogh investigated the regulation of respiration and circulation in response to exercise together with Johannes Lindhard. Therefore, a method to measure exercise intensity precisely was needed leading to the construction of Kroghs bicycle ergometer, which made it possible to study human exercise in a more controlled manner. August Krogh was the first to describe the mechanism of capillary regulation in skeletal muscle and it was his work on the capillary system that rewarded him the Nobel Prize in Physiology or Medicine in 1920. The main discovery was the opening of previously closed capillaries in response to exercise (Anne Lykke Poulsen, 2009). Investigation of respiration and the circulatory regulation during exercise was in the following years pursued by August Erik Hohwü-Christensen and Ove Hansen, who elucidated the effect of prolonged exercise and diet on respiratory exchange ratio (RER) (Christensen EH and Hansen O, 1939).

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METABOLISM Metabolism is derived from the Greek word Metabolismos, which means “change” or “transform”. The first study in metabolism was published in 1614 in the book Ars de static medicina (“art of static medicine”, where static means in balance) (Figure 1), by Santorio Santorius from the University of Padua, Italy. For a period of thirty years Sanctorius weighed himself, in a self constructed weighing chair, everything he ate and drank, as well as his urine and feces. He compared the weight of what he had eaten to that of his waste products, the latter being considerably smaller. He produced his theory of insensible perspiration as an attempt to account for this (LINK, 2010e). This study may not have the great scientific significance, but illustrates an early interest and curiosity about the area metabolism.

Figure 1: Cover page, and the self constructed weighing chair.

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What Santorio Santorius called “insensible perspiration” was elucidated by Antoine Lavoisier (26 August 1743 – 8 May 1794), who was a French scientist in the area of chemistry and biology. Lavoisier conducted experiments that showed that respiration was essentially a slow combustion of organic material using inhaled oxygen, and thereby challenged the scientific paradigm at that time, the phlogiston theory, which postulated that materials released a substance called phlogiston when they burned (LINK, 2010a). Lavoisier was the first to use a calorimeter in the attempt to measure heat production, as a result of respiration in a guinea pig. The construction of the calorimeter was very simple but sophisticated. The outer shell was packed with snow to maintain a constant temperature of 0°C around the inner shell that was filled with ice. The guinea pig in the center of the chamber produced heat which melted the ice, and the water was collected and weighed to estimate the metabolism. Lavoisier conclusion on this experiment was that respiratory gas exchange is due to combustion, like that of a candle burning (LINK, 2010a) (Figure 2).

Figure 2: The metabolic chamber and Antoine Lavoisier conducting an experiment.

Biochemistry as a research area were emerging along the 19th century, and especially the work of Friedrich Wöhler proving chemical synthesis of urea proved that the organic compounds and chemical reactions found in cells were in principle not different than any other part of chemistry

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(LINK, 2010c). Along with that, the studies of fermentation of sugar to alcohol in yeast by Louis Pasteur and the discovery of enzymes in the beginning of the 20th century, did along with other studies ultimately established biochemistry as a research area (LINK, 2010b), and in 1906 “Biochemische Zeitschrift” was published for the first time, with Carl Alexander Neuberg (18771956) as first editor, today the journal is known as FEBS (LINK, 2010d). With the development of new techniques, and the awareness of biochemistry as a research area, the biochemical reactions in metabolism were organized into metabolic pathways, including glycolysis, where glucose is converted to pyruvate, beta-oxidation, where fatty acids are degraded to acetyl-CoA and the tricarboxylic acid cycle (TCA cycle), which is responsible for chemical conversion of carbohydrates, fats and proteins into carbon dioxide and water, while transferring energy in the form of transferred energy-rich electrons to NAD+, forming NADH. As the techniques in modern biochemistry became more sophisticated, it became possible to investigate these metabolic pathways not only in whole animals, but also to examine a unique metabolic reaction in cells. The German-born British physician and biochemist Hans Adolf Krebs (25 August 1900 – 22 November 1981), made major effort in elucidating these metabolic pathways, not the least by the identification of the TCA cycle, which earned him the Nobel Prize in 1953 (LINK, 2010f). Important contribution to understanding pathways and interaction between different pathways in human metabolism, has also been produced by Philip John Randle (16 July 1926 - 26 September 2006), who described the Glucose Fatty Acid Cycle (also known as the Randle cycle), first put forward in a paper in The Lancet in 1963 (Randle et al., 1963), based on the demonstration that fatty acids reduce the oxidation of sugar by muscle. Randle describes the cycle: “(1) the relationship between glucose and FFA metabolism is reciprocal and not dependent;

(2) in vivo, oxidation of FFA and ketone bodies released into circulation in diabetes and starvation may inhibit catabolism of glucose in muscle; (3) in vitro, the oxidation of FFA released from muscle triacylglycerol may have similar effects; (4) these effects of FFA and ketone body oxidation are mediated by inhibition of the pyruvate dehydrogenase (PDH) complex, phosphofructo 1kinase (PFK1), and hexokinase (HK); (5) the essential mechanism is an increase in the mitochondrial ratio of [acetyl CoA] / [CoA], which inhibits the PDH complex directly, and which indirectly leads to inhibition of PFK1 by citrate and of HK by glucose-6-phosphate; and (6) the effect of physiological concentrations of insulin to activate glucose transport in heart muscle is

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inhibited by FFA and ketone bodies.” (Randle et al., 1994). Hence, the Glucose Fatty Acid Cycle describes the mechanism behind maintaining a constant plasma glucose concentration, and suggests a mechanism that determines the choice of substrate combusted by skeletal muscle. A key regulatory protein in this “Randle cycle” is the pyruvate dehydrogenase complex, which, as stated by Randle, has a central position in metabolism, and considered an important enzyme in regulating metabolism. This thesis focuses on the regulation of pyruvate dehydrogenase complex (PDC).

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PYRUVATE DEHYDROGENASE COMPLEX In every cell that contains mitochondria, the pyruvate dehydrogenase complex catalyzes the irreversible oxidative decarboxylation of pyruvate to form acetyl-CoA (Figure 3). This reaction links glycolysis and the citric acid cycle and is in skeletal muscle determining the rate of complete carbohydrate oxidation through the TCA cycle. In liver cells, the product of the pyruvate dehydrogenase complex, acetyl-CoA, is also used for fatty acid and cholesterol synthesis, and in adipose tissue it can be used for fatty acid synthesis (Behal et al., 1993). Thus, control of the pyruvate dehydrogenase complex plays an important role in dictating the fuel used by various tissues of the body, and the forthcoming pages will introduce the reader to the structure of the complex, and the regulation of the activity of the complex with focus on skeletal muscle.

Figure 3: Reaction mechanism of the pyruvate dehydrogenase complex. The enzymes of the complex are represented as follows: E1, pyruvate dehydrogenase (decarboxylating); E2, dihydrolipoamide acetyltransferase; E3, dihydrolipoamide dehydrogenase. The broken lines attached to E2 represents the rotating lipoamide arm of the enzyme. The products of the reaction, acetyl-CoA, NADH and CO2, are shown in the boxes. Four enzyme cofactors of the complex, namely thiamine pyrophosphate, coenzyme A, FAD and NAD, are derived from members of the vitamin-B group. Picture and text from (Tyler, 1992)

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Figure 4: Surface shaded representations of three-dimensional reconstructions of S. cerevisiae PDH complex and its subcomplexes viewed along a 3-fold axis of symmetry. A, tE2; B, the tE2·BP-E3 subcomplex has 12 copies of BP-E3 (red) buried deep inside the 12 pentagonal openings of the tE2 scaffold (green); C, structure of the wild-type PDH complex consisting of the tE2 inner core (green) with the BP-E3 components (red) bound on the inside and the tetrameric E1 molecules (yellow) bound on the outside. Binding of E1 to the tE2 core increases the diameter of the structure from 250 to 500 Å; D, cutaway reconstruction of the PDH complex from C showing the disposition of BPE3 and E1 relative to tE2. Text and figure from (Reed, 2001).

The mammalian PDC is a multifunctional enzyme complex, and around 50 years ago the structure and components of the complex were identified from isolated enzymes (KOIKE et al., 1963a). It was demonstrated that the individual enzymes are linked by non-covalent bonds, and that each of these functional units is composed of multiple copies of three enzymes, which act in sequence to catalyze the transformation of pyruvate to acetyl-CoA (Behal et al., 1993,KOIKE et al., 1963b). The three enzymes are in the literature described with several different names, but in this thesis,

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the subunits will be referred to as E1; Pyruvate dehydrogenase (EC 1.2.4.1), E2; dihydrolipoamide acetyltransferase (EC 2.3.1.12) and E3; dihydrolipoamide dehydrogenase (EC 1.8.1.4). PDC is made up of 60 E2, which are constructing an inner core of the PDC (Figure 4), and each E2 subunit has a “superarm” (Patel MS and Roche TE, 1990). PDH-E1, which is attached to the “superarm”, is a heterotetramer (α2β2) responsible for decarboxylation of pyruvate (Behal et al., 1993,Khailova et al., 1982). E3 is responsible for oxidation of the reduced lipoyl group of E2, and the subsequent production of NADH (Reed, 2001). Beside the three enzymes, the complex consists of a structural component, the E3-binding protein (E3BP). Each pyruvate dehydrogenase complex is composed of 216 subunits (60 E2, 60 E1α, 60 E1β, 12 E3BP, and 24 E3), which sum up to about 9.5 MD (Harris et al., 2002). In addition, the regulatory proteins pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP), which will be described in the following, is also associated with the complex.

Regulation of PDHa activity The activity of PDH has proven to be very sensitive and tightly regulated when the body is exposed to metabolic stress, as during fasting and physical exercise. The activity of PDH in the active form (referred to as PDHa) is increased in response to acute exercise and is upregulated already after 1 min of exercise (Howlett et al., 1998). In addition, a dose response relationship exists between exercise intensity and PDHa activation (Howlett et al., 1998). Many studies have by now shown the relationship between exercise and PDHa activity, and the activity of PDHa within the first 2 hours of exercise seems to be relative stable when exercise is perform at moderate intensity (Mourtzakis et al., 2006,Pilegaard et al., 2006,Putman et al., 1993,Ward et al., 1982,Watt et al., 2002), working intensities in these studies has been in the range of 45–55% VO2 max or wattmax. But when the exercise period exceeds 2 hours, the activity of PDHa is decreasing towards the resting value of the PDHa activity (Mourtzakis et al., 2006,Pilegaard et al., 2006,Watt et al., 2004). Whether the change in PDHa activity during prolonged exercise is reflecting the change in substrate utilization during exercise, or if the decrease in PDHa activity is the initially tricking factor changing substrate utilization is unknown. In addition, the PDHa activity is also regulated very tightly, during fasting situation, and by different diets (Behal et al., 1993,Harris et al., 2002,Peters, 2003), and

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downregulation of PDHa in rest in response to a high fat diet, or a period of starvation, have been found quit a few times (Peters et al., 1998). PDHa activity is overall regulated in two manners. The simplest of these is the down regulation of PDHa activity by an inhibitory effect of the reaction end products, acetyl-CoA and NADH, which thus provide a direct negative feedback on PDHa activity, (Harris et al., 2002,Behal et al., 1993). By contrast, an upregulation of PDHa activity by the substrate, pyruvate, has not been demonstrated (Constantin-Teodosiu et al., 2004). The second regulatory mechanism is covalent modification of PDHa, which was discovered more than 40 years ago. Linn and colleagues described how PDH was regulated via phosphorylation / dephosphorylation (Linn et al., 1969) at three specific serine residues, on the α subunit of the PDH-E1α component (Yeaman et al., 1978). PDH-E1α protein has a 29-long amino acid transit peptide, and when these amino acids are included in the sequence, the phosphorylation sites are found at Ser232 (site 3), Ser293 (site 1) and Ser300 (site 2) (Korotchkina and Patel, 2001a,Yeaman et al., 1978). If PDH-E1α is dephosphorylated the enzyme is active, but if one of the three sites are phosphorylated PDH-E1α are inactive (Sugden and Randle, 1978). Pyruvate dehydrogense kinase (PDK) is responsible for phosphorylation and thus inactivation of PDH, whereas pyruvate dehydrogenase phosphatase (PDP) is responsible for dephosphorylation thereby activating PDH (Holness & Sugden, 2003). There is broad consensus that PDHa activity is predominantly regulated through the phosphorylation / dephosphorylation cycle (Harris et al., 2002,Watt et al., 2004). Therefore understanding the biology of PDK and PDP and there interaction with PDH-E1α, will tell a lot about the regulation of PDHa activity.

PDP and PDK Four isoforms of (PDK)1-4, and 2 isoforms of (PDP)1 and 2 have been described (Roche et al., 2001,Patel and Korotchkina, 2001). The distribution of the different isoforms is tissue specific (Bowker-Kinley et al., 1998,Gudi et al., 1995,Huang et al., 2003), and furthermore the different isoforms of kinases and phosphatases have distinct kinetic properties to each phosphorylation site (Korotchkina and Patel, 1995,Patel and Korotchkina, 2001). Finally the activity level of the different PDK’s and PDP’s is also unique, hence it’s different substrates that activates and

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inactivates the different isoforms (Harris et al., 2002). As a consequence the regulation of PDHa activity is very complex and specialized from tissue to tissue, depending on the isoform expression pattern.

Tissue distribution PDP By using northern blotting, a strong mRNA expression of PDP1 was demonstrated in heart and brain cells, and to a lesser extent also in skeletal muscle cells, while no expression of PDP2 was evident in the muscle cell in rats (Huang et al., 2003). In humans, the presence of PDP1 and PDP2 mRNA in skeletal muscle was demonstrated by real-time PCR, but PDP2 was considerably lower expressed than PDP1 (Pilegaard et al., 2006). Therefore, it is believed the PDP1 is the phosphatase responsible for the PDP-mediated regulation of PDH in skeletal muscle (Harris et al., 2002,Holness and Sugden, 2003).

Tissue distribution of PDK PDK1 has been reported to have a limited distribution in the body, and has only been found in the heart (Bowker-Kinley et al., 1998), at mRNA level, in pancreas (Sugden et al., 2001a) at the protein level and to a small extent at protein level in skeletal muscle (Peters et al., 2001a). PDK2 is strongly expressed in all examined tissues on mRNA level (Bowker-Kinley et al., 1998), while PDK3 is expressed in testis, kidney and brain (Bowker-Kinley et al., 1998,Huang et al., 1998) but also at the mRNA level in human skeletal muscle (Pilegaard et al., 2006). PDK4 is expressed in several tissues, including heart, skeletal muscle, liver, kidneys and pancreas (Holness and Sugden, 2003), so PDK4 is expressed in tissues influencing whole body energy metabolism, hence the heart and skeletal muscles use to a large extend glucose for combustion, liver and kidney synthesize glucose, and finally pancreas produces glucose-regulating hormones (insulin, glucagon). There is a broad consensus that PDK2 and PDK4 have the overall responsibility for the PDK-mediated regulation of PDH in human skeletal muscle (Harris et al., 2002,Holness and Sugden, 2003).

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Site specificity and PDK It has been demonstrated that each PDK isoform has site-specific activity level. Thus, for site 1 the PDK activity towards the phosphorylation site is PDK2>PDK4>PDK1>PDK3, for site 2 the hierarchy is PDK3>PDK4>PDK2>PDK1, finally site 3 is only phosphorylated by PDK1, phosphorylation of any of the sites inactivates the PDC complex (Patel and Korotchkina, 2001,Korotchkina and Patel, 2001b). Beside the unique distribution of the kinase isoforms in the tissue, the different sites have unique kinetics properties. Thus, site 1 is phosphorylated 4.5 times faster than site 2 and 16 times faster than site 3. By contrast, no difference in dephosphorylation of the three sites is evident (Korotchkina & Patel, 1995).

Regulation of PDP and PDK activity Substrates for the reaction catalyzed by PDH (pyruvate, NAD+ and CoA), up-regulate the PDHa activity. This upregulation is the result of an inhibition of PDK activity (Sugden et al., 1995). Opposite PDK activity is stimulated by the products of the reaction, NADH and acetyl-CoA, and consequently PDHa activity is reduced (Behal et al., 1993). Again there are differences in isoform properties, and for example pyruvate inhibits PDK2 stronger than PDK4 (Bowker-Kinley et al., 1998). Upregulation of PDK activity via increases in acetyl-CoA/CoA and NADH/NAD+ is thought to occur as a result of changes in the acetylation degree and reduction/oxidation degree of the lipoyl moieties of the E2 subunit (Ravindran et al., 1996). PDP1 activity is upregulated by Ca2+, whereas Ca2+ has no effect on PDP2 (Huang et al., 1998). Muscle activity leads to increased cytoplasmic Ca2+ concentration, and consequently increased mitochondrial Ca2+ concentration. This stimulates the activity of PDP1 and therefore PDHa activity is upregulated (Denton et al., 1996). In addition, in vitro studies have shown a downregulation of PDP activity by an increased NADH/NAD ratio (Pettit et al., 1975), and insulin has been suggested to increase PDP1 and 2 activity (Caruso et al., 2001,Mandarino et al., 1990,Patel and Korotchkina, 2001) via protein kinase C-delta, resulting in an upregulation of PDHa activity (Caruso et al., 2001).

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Regulation of PDP and PDK expression The more long term regulation of PDHa activity is by means of regulation of PDP and PDK expression. PDK2 and PDK4 expression has been shown to be very sensitive to substrate accessibility. Diet interventions as fasting did prove to have a huge impact on PDK4 regulation. Wu et al. (, 1999a) showed that fasting rats for 48 h markedly induced PDK4 but not PDK2 mRNA and protein expression in skeletal muscle compared with rats on normal diets, while the expression of PDK2 and PDK4 mRNA as protein level, increased in the kidneys and liver in response to 48 h fasting (Wu et al., 2000). Similarly, PDK4 transcription and mRNA content have been shown to increase in human skeletal muscle after only 20 hours of fasting (Pilegaard et al., 2003). Fasting has also been demonstrated to down-regulate PDP1 and PDP2 in rat kidney, while in rat cardiac muscle tissue only PDP2 was down-regulated by fasting (Huang et al., 2003). In a human study subjects consumed a high fat / low carbohydrate diet and a marked increase of PDK activity was evident already after 3 days of diet intervention and on the 6’th day the PDHa activity was also decreased compared with the pre condition (Peters et al., 1998). Later the same group published data on the specific PDK isoforms, and it was shown that PDK4 mRNA and protein increased in response to the diet intervention already after the first day, while the intervention had no effect on PDK2 mRNA or protein (Peters et al., 2001b). It have been elucidated that the adaptive increases in PDK4 in response to fasting is mediated by PPARα (Wu et al., 2001,Sugden et al., 2001b), and the regulation of PDK4 via PPARα was investigated and it was found that in the fed state, acute (24 h) activation of PPARα by WY14643 failed to modify PDK4 protein expression in soleus, but modestly enhanced PDK4 protein expression in anterior tibialis (Holness et al., 2002). It has been shown that skeletal muscle up-regulates PDK4 transcription and PDK4 mRNA content in human and rodent skeletal muscle during prolonged exercise and especially during fasting and recovery from exercise (Nordsborg et al., 2010,Pilegaard et al., 2000,Pilegaard et al., 2002,Pilegaard et al., 2005,Mourtzakis et al., 2006). On the contrary, no changes were detected in PDK4 protein in human skeletal muscle during prolonged exercise (Watt et al., 2004). However, 2-3 fold increases have been observed in PDK4 protein in human skeletal muscle after 6 hours recovery (Kiilerich et al., 2008). Because the other PDK’s do not change at the mRNA or protein level in response to acute exercise (Pilegaard et al., 2006), PDK4 appears to be the key PDK

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regulator of PDHa activity during prolonged exercise, unless the activity of the other PDK’s changes. In addition, as PDP1 mRNA has been reported to decrease in human skeletal muscle in recovery from acute exercise (Pilegaard et al., 2006), a concerted regulation of PDP1 and PDK4 could be the ultimate determinant of the PDHa activity in response to acute exercise. Furthermore increased plasma FFA by intralipid infusion increased PDK4 mRNA content at basal level (Pilegaard et al., 2006). The co-existence between increased plasma FFA and increased expression of PDK2 and PDK4 at both mRNA and protein level with a concomitant downregulation of PDP1 protein and reduced PDHa activity in skeletal muscle, further supports that FFA may be a regulatory factor of PDK and PDP expression and thus PDHa activity (Bajotto et al., 2004,Peters et al., 2001a). Finally, it’s been demonstrated in human skeletal muscle that insulin down-regulates the expression of PDK2 and PDK4 mRNA (Majer et al., 1998). What is responsible for the upregulation of PDK4 in response to exercise, high fat diet, fasting and FFA infusion? It is well known that a rise in the mitochondrial ratio of NADH:NAD+, which typically rises with increased fatty acid β-oxidation, induces PDK4 upregulation (Harris et al., 2002). Since all the above mentioned interventions will induce a rise in FFA in the blood plasma, there is reason to believe that it could be one mechanism leading to the increased PDK4. But it does not explain why there is an increased upregulation of PDK4 in the recovery period after exercise, where the FFA level in the plasma has normalized. But an explanation could be that the muscle glycogen level has an impact on PDK4 regulation, because PDK4 transcription and mRNA content was shown to be enhanced in a muscle glycogen depleted leg (Pilegaard et al., 2002). However the manipulation with muscle glycogen in the previous study was also associated with changes in plasma FFA as well as insulin, making it difficult to give an account of the possible influence from this factor as well. In addition no studies have tried to elucidate the combined effect of FFA and muscle glycogen, so the PDK4 regulation in response to muscle glycogen remains speculative.

PDH-E1α phosphorylation Even though it’s more than 40 years ago that the relationship between PDHa activity and phosphorylation was described (Linn et al., 1969), measurements of PDH-E1α phosphorylation in

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skeletal muscle was first published in 2006 (Pilegaard et al., 2006). In this study the phosphorylation of the two phosphorylation sites of regulatory importance in skeletal muscle, Ser293 (site 1) and Ser300 (site 2) was measured (Sugden and Holness, 2003). During a 4 hours intra lipid infusion, the degree of phosphorylation increased significantly compared with the control (saline infusion), but the activity of PDHa was not different between the groups. The infusion was followed by a 3 hour exercise bout, which dramatically decreased the phosphorylation level on both sites, and resulted in a higher PDHa activity. During the exercise, the phosphorylation degree seemed to increase, along with a decrease in PDHa activity. Thus the PDH-E1α phosphorylation pattern followed the PDHa activity during exercise, while the changes in PDH-E1α phosphorylation and PDHa activity in resting conditions appeared disassociated and the authors speculated that other phosphorylation sites might be involved.

Fiber type and PDH regulation Muscle specific regulation of PDH has only been studied to a limited extend, and the conclusions to draw from these studies are not straight forward. In a rat study (Sugden et al., 2000), PDK2 and 4 protein expression was determined in slow-twitch (soleus) and fast-twitch (anterior tibialis) muscle in response to 48h fasting, and both muscle types showed an increase of PDK4 protein in response to fasting, while anterior tibialis also had an increase in PDK2 protein in response to fasting, another study from the same group conformed the upregulation of PDK4 protein in response to 24h fasting, where they found an starvation enhanced PDK4 protein expression in both soleus and anterior tibialis muscles, with the greater response in anterior tibialis (Holness et al., 2002). Furthermore it seemed that the PDK in the different muscle types had a different sensitivity to be regulated by pyruvate. Hence pyruvate is known to inactivate PDK thereby activating PDH, and it seemed that pyruvate had an unequal effect on PDK, because PDH activation was smaller in anterior tibialis than soleus in response to refeeding after 48h fasting (Sugden et al., 2000). The same pattern was found in a high fat diet protocol in rats (Holness et al., 2000). Later a study examined the response of PDK1, 2 and 4 mRNA and protein (all the rat kinases), to a 48h fasting protocol in three different rat skeletal muscles, white gastrocnemius (WG), red gastrocnemius (RG) and soleus. In that study WG was shown to have a smaller amount of PDK1 and PDK4 protein than the two other muscles, both in the fed and fasted stated (Peters et al., 2001a).

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Another rat study analysed the transcription of metabolic genes in white and red gastrocnemius muscle in response to prolonged low intensity exercise, and showing that PDK4 transcription seemed to be regulated in a fiber type specific manner with most marked changes in the white glycolytic muscle (Hildebrandt et al., 2003). So there seem to be reason to believe that PDH can be regulated differently in different muscle types, but the physiological impact has not been investigated clearly, and human data are lacking in this area.

Activity level and PDH regulation There haven’t been published much work on how a long term endurance training period affect PDH regulation. But one study has elucidated the protein response of PDH and PDK’s in response to an 8 weeks endurance training protocol (LeBlanc et al., 2004b), the main findings was that the total activity of PDH total and PDK increased. On the protein level PDH-E1α increased, while the exercise period had no effect on protein levels of PDH-E2 and PDH-E3. Protein level of PDK1, PDK2 and PDK4 was measured, and only PDK2 was affected by the 8 weeks training protocol, with a modest increase. The authors concluded that the muscles increased the maximal capacity for utilize carbohydrate, as the activity of PDH increased, and that the increase in PDK2 reflected greater metabolic sensitivity towards pyruvate, since PDK2 is tightly regulated by pyruvate (Patel and Korotchkina, 2001). Another study measured the PDHa activity in response to acute exercise before and after a 7 weeks endurance exercise period. And they reported a smaller increase in PDHa activity in response to the acute exercise after the training period then before, and they speculated it could be because of lower pyruvate content after the endurance exercise period (LeBlanc et al., 2004a). One big weakness about this study is that the acute exercise was performed on the same absolute workload, meaning the subjects worked on different relative workloads, which could be the reason for the observed smaller increase in PDHa activity in response to exercise, since it follows the relative workload (Howlett et al., 1998). Finally a study with short term interval training for 2 weeks show that PDHa activity was enhanced in response to an acute exercise bout after the training period. In this study the subjects performed the acute exercise on the same relative workload before and after the exercise period, this reflect a greater capacity for carbohydrate metabolism (Burgomaster et al., 2006). Not many studies have looked

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on PDP, but in a rat study with a group of rats exercise for 8 weeks, it was found that the PDP activity increased (a trend) in response to training, and that the protein level of PDP1 but not PDP2 increased in soleus and red gastrocnemius (LeBlanc et al., 2008). Finally a study by Ward et al. (, 1986) elucidated both the role of immobilization and strength training in triceps in a cross-over study. They found that neither of the interventions changed the total PDHa activity, but at rest PDH in the active form was lower in the detraining group compared to the strength training group, finally the PDHa activity in response to an acute exercise bout was lower in the immobilized intervention than the strength training intervention. Thus most of the studies examining activity level and PDH regulation have elucidated PDH regulation in response to an increase in activity level and only one study has investigated the impact of inactivity on PDHa activity without further molecular biology analyses.

PGC-1α and metabolism What could be the mediating factor for all these adaptations, and changes in activity and expression pattern of PDH-E1α, PDK’s and PDP’s described above in response to training, diet and fasting? A lot of attention has been given to the transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α since it in 1998 was discovered and identified as a co-activator of PPARγ in brown adipose tissue (Puigserver et al., 1998). PGC-1α is preferentially expressed in tissues with high oxidative capacity such as heart, skeletal muscle and brown adipose tissue (Puigserver et al., 1998,Puigserver and Spiegelman, 2003). PGC-1α activates a long list of transcription factors that regulate nuclear genes. To mention some, nuclear respiratory factor (NRF) 1 and NRF2 and estrogen-related receptor α (ERRα) regulates genes encoding proteins in mitochondrial biogenesis and ERRα also affects angiogenesis (Arany et al., 2008,Mootha et al., 2003,Mootha et al., 2004,Schreiber et al., 2004,Wu et al., 1999b). Furthermore PGC-1α activates peroxisome proliferator-activated receptor (PPAR)α and PPARδ, which affects fatty acid oxidation (Vega et al., 2000,Wang et al., 2003). Finally PGC-1α binds and co-activates the transcriptional function of NRF1 on the promoter for mitochondrial transcription factor A (mtFTA) (Wu et al.,

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1999b), which is an activator of mitochondrial encoding genes, whereby a direct link between PGC-1α and regulation of mitochondrial biogenesis exist. So it is well established that PGC-1α plays an important role in maintaining the glucose lipid and energy homeostasis, and is important for mitochondrial biogenesis.

PGC-1α and substrate regulation Beside of being important for mitochondrial biogenesis, it is believed that PGC-1α has a regulatory effect on the choice of substrate (Calvo et al., 2008,Wende et al., 2007). In the study by Calvo and colleagues (, 2008), performance tests were performed on muscle specific overexpression PGC-1α mice, and WT littermates. Overall it was found that the overexpressing mice performed better in an endurance exercise test, and very interestingly the mice did not exceed 1 in RER values, even at maximal running capacity. When the RER values were compared between PGC-1α overexpressing mice and WT mice at the same percentage of maximal speed the PGC-1α transgenic mice continued to exhibit lower RER values, thus the RER value was lower in overexpression then in WT mice both at the same absolute and same relative exercise intensity, indicating that the PGC1α transgenic mice utilize more fatty acid during exercise then WT. Therefore there is reason to believe that PGC-1α can affect the choice of substrate combusted by the muscles.

Figure 5: Schematic illustration of the suggested peroxisome proliferator-activated receptor-γ coactivator (PGC)-1αmediated upregulation of pyruvate dehydrogenase kinase (PDK)4 leading to increased fat oxidation in skeletal muscle.

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ERRα oestrogen-related receptor α, PDC pyruvate dehydrogenase complex, TCA tricarboxylic acid. From (Olesen et al., 2010).

The mechanism behind the substrate regulation could be and upregulation of PDK4 by PGC-1α, because it has been shown that overexpression of PGC-1α in C2C12 myotubes decreases glucose oxidation rates, with a concomitant increase in PDK4 expression (Wende et al., 2005), so it was suggested that this decrease in glucose oxidation is achieved via upregulation of PDK4 (Wende et al., 2005,Zhang et al., 2006). Apparently PGC-1α co-activates the transcription factor ERRα which induces PDK4 expression (Huss et al., 2002,Huss et al., 2004,Wende et al., 2005,Zhang et al., 2006), (see figure 5). Several studies have indicated a link between PGC-1α and PDK4 regulation. But no studies has elucidated if it has an effect on the PDHa activity, or if this suggested PGC-1α mediated PDK4 regulation has an impact on PDHa regulation in response to physical stress as fasting and endurance exercise, where PDK4 expression is regulated and potentially exerting an important regulatory role.

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AIMS The overall objective of this PhD thesis was to gain deeper understanding of the regulation of pyruvate dehydrogenase in skeletal muscle.

The key questions to be answered were:

•

Is exercise-induced PDH regulation affected by muscle type.

•

Effect of muscle glycogen on the PDH regulation.

•

The impact of physical inactivity on exercise-induced PDH regulation.

•

Role of PGC-1α in PDH regulation in skeletal muscle at rest and in response to fasting and during recovery from exercise.

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METHODS Below follows a brief description of the experimental models used in the thesis and a detailed presentation of the main laboratory analysis performed; RT-PCR using real time PCR, western blotting and PDHa activity.

Human experiments Several different human experimental setups were used and young healthy male subject participated in the human studies (studt 1,2,3). They are all detailed described in the respective articles. Muscle biopsies were obtained from vastus lateralis in all these studies, and in study 1 biopsies were also sampled from deltoid and triceps. Venflon sampling was performed in all the human studies, and in study 3 cauterization of the femoral artery and vein was also included.

Mice models The generation and phenotype of the whole body PGC-1α KO and the muscle specific PGC-1α overexpressing mice (MCK PGC-1α) mice, used in study 4, have been described in detail elsewhere (Lin et al., 2002,Lin et al., 2004). For the PGC-1α KO strain, littermate PGC-1α KO and WT mice were obtained by crossing of heterozygote parents, while littermate MCK PGC-1α and WT mice were obtained by crossing a MCK PGC-1α and a WT parent. The genotypes of the mice were determined by PCR-based genotyping, as previously described (Leick et al., 2008). Mice were kept on a 11:13 hour light:dark cycle and received standard rodent chow (Altromin no. 1324, Chr. Pedersen, Ringsted, Denmark).

Determination of PDHa activity Determination of PDHa activity is conducted in an assay that consists of three main steps: - Homogenization - Formation of acetyl-CoA

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- Determination of acetyl-CoA content For determination of PDHa activity ≈10 mg wet weight muscle were used. This was homogenized on ice in a homogenization buffer with volume equivalent to 30 × muscle weight (ConstantinTeodosiu et al., 1991) using a motor-driven mini-glass homogenizer (Pilegaard et al., 2006). A given volume of homogenate and pyruvate is added to a preheated mixture containing NAD, Coenzyme A and thiamine pyrophosphate (TPP), whereby the following reaction proceeds: ,

1

From this mixture 200 μl of sample are transferred after 45, 90 and 135 seconds to tubes containing PCA (perchloric acid) that stops the reaction and hence production of acetyl-CoA. For each homogenate, a duplicate is made with pyruvate, and a blank in which water is added instead of pyruvate, i.e. each sample results in 9 tubes. The more PDHa activity in the muscle sample the more acetyl-CoA is produced. The amount of acetyl-CoA is subsequently determined in an assay, based on two enzymatic reactions: 2

2 3

The purpose of reaction (2) is to produce

14

C marked oxaloacetate, because this is not

commercially available. This 14C marked oxaloacetate is added to the samples obtained from reaction 1, after 45, 90 and 135 seconds. This leads to reaction (3), where acetyl-CoA is the limiting factor, because 14C oxaloacetate is produced in excess, and the produced 14C-citrate reflects the amount of acetyl-CoA produced in reaction (1) 1:1. The excess 14C oxaloacetate is converted to 14

C aspartate by the addition of glutamic oxaloacetic transaminase (GOT) and glutamate in excess

(Cederblad et al., 1990). Finally 14C-aspartate is removed by adding Dowex, which has an ion exchange resin that binds aspartate (Constantin-Teodosiu et al., 1991).

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Earlier in the assay N-ethylmaleimid (NEM) was added. This compound binds 14C-citrate, thereby making sure reaction 3 runs at maximal speed, 14C-citrate can be determined in a scintillation counter after adding the liquid scintillation cocktail, Ultima Gold (PerkinElmer). Ultima Gold will capture the β-rays emitted from 14C-citrate and emit photons, which the scintillation counter will detect (TRICARB 2300 TR, Packard). The fourth part of the assay is construction of a standard curve, reflecting the relationship between the amount of acetyl-CoA and counts from the scintillation counter. The difference in acetyl-CoA concentration between sampling points (45, 90 and 135 sec) is then converted into a PDHa activity in mmol acetyl-CoA • min-1 • kg-1 ww. The PDHa was then later normalized to total creatine content, normalizing the samples to the content of muscle tissue.

SDS-PAGE and immunoblotting The immunoblotting technique was used in all studies in order to measure phosphorylation level and content or of examined proteins. First step in this process was to produce lysate. This was done by homogenizing muscle samples followed by centrifugation, and transferring the supernatant to a new tube. Afterwards the protein content in the muscle lysates was determined by the bicinchoninic acid method using bovine serum albumin standards (Pierce Biotechnology Inc, IL, USA). The lysates were diluted in sample buffer containing sodium dodecyl sulphate (SDS). The proteins hereby denaturants and the unfolded proteins were then separated by size (molecular mass) through a polyacrylamide gel electrophoresis. Hereafter the proteins were transferred to a polyvinylidine difloride (PVDF) membrane, and the membrane was blocked in either milk or bovine serum albumin (BSA). Incubation with primary antibody was done 2 hours a room temperature, or overnight at 4°C, followed by one hour incubation with secondary antibody, conjugated with horseradish peroxidise. Immobilon Western (Millipore Corporation, MA) was used as detection system. Bands were visualized using an Eastman Kodak Co. Image Station 2000MM. Bands were quantified using Kodak Molecular Imaging Software v. 4.0.3, and protein content was expressed as arbitrary units relative to control samples loaded on each gel. All antibodies used were validated using recombinant protein, and ensured that the amount of protein loaded was within the linear range for all antibodies used.

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RNA isolation and real time PCR The reverse transcription and real time PCR technique used in study 3 and 4 is described in great detail in following articles (Bustin, 2000,Bustin, 2002,Bustin et al., 2005,Heid et al., 1996,Lundby et al., 2005,Pilegaard et al., 2000). RNA isolation was performed on ~18 mg muscle tissue with the guanidinium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987) with modifications (Pilegaard et al., 2000). The next step involved synthesis of complementary DNA (cDNA) from the extracted RNA using reverse transcription (RT) with unspecific “oligo dT” primers. The oligo dTs hybridize to the poly(A) tail of mRNA (Merkel et al., 1975) thus ribosomal RNA is not copied into cDNA. RT was performed using the Superscript II RNase H-system (Invitrogen), and the sample was diluted in nuclease-free water. Real-time PCR was performed with an ABI 7900 sequence-detection system (Applied Biosystems, Foster City, CA), carried out using the fluorescence resonance energy transfer (FRET) technology, in which Taqman probes labeled with a quencher dye at the 3´end and a reporter dye at the 5´end are used. Primers and TaqMan probes for amplifying gene-specific mRNA fragments were designed using the human/mice database from ensembl.org and Primer Express (Applied Biosystems). All probes were 5´-FAM (reporter) and 3´-TAMRA (quencher) labeled, and primers and probes were obtained from TAG Copenhagen (Copenhagen, Denmark). During PCR amplification the Taqman probe binds to its target cDNA sequence, and the 5'- nuclease activity of the DNA polymerase cleaves the probe and thereby splits the reporter and quencher. When the reporter is no longer quenched, the fluorescence emitted by the reporter is detected by sensors. After 40 temperature cycles, the PCR run was finished and an amplification plot plotting cycle on a linear scale against fluorescence on a logarithmic scale was produced. Cycle threshold (Ct) can thereafter be defined, and reflects the number of cycle during the amplification required to reach a specific fluorescence level. The Ct of each sample thus gives an indirect measure of the mRNA content of the specific target gene where a low Ct indicates a higher initial content of cDNA of the specific target gene. Ct was converted to a relative amount by use of a standard curve constructed from a serial dilution of a pooled RT sample run together with the samples. The standard curve furthermore serves as a control of the amplification, as the slope of the curve gives a measure of the amplification efficiency.

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Figure 6: (1) Intact probes, reporter fluorescence is quenched. (2) Probes and the complementary DNA strand are hybridized. (3) During PCR, the probe is degraded by the Taqman polymerase and the fluorescent reporter released. From (LINK, 2010g)

For normalization of the samples was used the OliGreen method, described in (Lundby et al., 2005). This method is measuring the total amount of single stranded (ss) DNA in the cDNA samples. These values were then used to normalize the mRNA data from the real time PCR analyses.

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RESULTS AND DISCUSSION The questions raised under “Aim of the thesis” were each addressed by a specific study. Below follows a short summary of the main results and conclusions of each article included in the thesis with emphasis on the novel findings provided by the study. Thereafter results obtained in the 4 studies will form the basis for an integrated discussion, and finally a section discussing some of the methods used and their limitations.

Summary of thesis articles

Is exercise-induced PDH regulation affected by muscle type? (Study 1) The main finding of the study was that the PDH-E1α content follows the metabolic profile of the muscle rather than the MHC fiber type distribution. This conclusion was based on the observation that two muscles, vastus lateralis and deltoid, with a similar MHC fiber type distribution had different levels of PDH-E1α content, and these differences followed the differences in CS activity. In addition it seemed that PDH regulation was the same in vastus lateralis and triceps, because the PDHa activity normalized to the total amount of PDH-E1α was identical in the two muscles, implying that muscle type did not seem to influence exercise-induced PDH regulation. Finally it was observed that after an initial increase from rest to 10 min of exercise, the PDHa activity decreased (non-significantly) from 10 min to 30 min of exercise, and this decrease in PDHa activity was not reflected by an increase of PDH-E1α phosphorylation, indicating that additional regulations of PDH exist. In addition, lactate accumulation during exercise was higher in triceps than in vastus lateralis, and this may be due to a lower PDH capacity rather than different PDH regulation in triceps and vastus lateralis.

Effect of muscle glycogen on PDH regulation (Study 2) The main findings of this study were that exercise increases PDHa activity in human skeletal muscle despite enhanced plasma FFA levels or reduced muscle glycogen, but both reduced muscle

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glycogen concentration and elevated plasma FFA levels were associated with reduced exerciseinduced PDH activation. Because there was no difference in the PDHa activity in the control leg and glycogen depleted leg in the high FFA trial, high FFA levels and low muscle glycogen levels do not seem to have additive down regulatory effects on the exercise-induced PDH regulation. Regulation of PDK4 protein expression seems to be a likely mediator of these effects of muscle glycogen and FFA on the PDHa activity. In general there was in this study a very tight relationship between PDK4 protein, PDH-E1α phosphorylation and PDHa activity. In addition, to the best of my knowledge this is also the first study to show a very fast upregulation of PDK4 protein in response to diet intervention performed only 2 hours before the first sampling point.

The impact of physical inactivity on exercise-induced PDH regulation (Study 3) The main findings of this study were that although 7 days of bed rest induced whole body glucose intolerance and reduced glucose uptake by the exercising muscle, exercise-induced PDH regulation in skeletal muscle was unchanged. Thus the inactivity period had no impact on PDH regulation. This indicates that the response to inactivity is not simply the opposite of the response to activity, but it cannot be ruled out that longer periods of physical inactivity will have effects.

Elucidating the importance of PGC-1α in PDH regulation in skeletal muscle at rest and in response to fasting and during recovery from exercise (Study 4) The main findings of this study were that PGC-1α expression regulates the total amount of PDHE1α as well as the phosphorylation state and activity of PDH in skeletal muscle at rest. However, fasting- and exercise-induced PDH regulation in skeletal muscle does not require PGC-1α. In general the relationships between PDH-E1α phosphorylation and PDHa activity were not that obvious as in Study 1, 2 and 3. These KO and TG mice were surly a good model to elucidate if PGC-1α had a significant role of regulating the PDH regulation. But the mice models also showed limitations, because the relation between PDK4 protein and PDH-E1α phosphorylation was less clear.

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Integrated Discussion Muscle type In study 1 muscles with different MHC fiber type were analysed and the regulation of PDH in response to exercise was investigated. First of all the highest content of PDH-E1α was located in vastus lateralis, and deltoid and triceps had the same, but significantly lower level of PDH-E1α than vastus lateralis. Hence it clearly shows that the MHC fiber type distribution does not determine the content of PDH-E1α, but that the PDH-E1α content rather follows the general oxidative capacity of the muscle. PDK4 protein was not measured in this study, and it could be interesting to see if the PDK4 expression pattern is different between the human muscles with different metabolic properties, as there could be reason to believe from animal studies, as explained above (see Fiber type and PDH regulation). The absolute level of PDHa activity does not as such reflect the regulation of each PDH-E1α molecule but is more a physiological measure reflecting the oxidative capacity of the muscle. Therefore when interpreting PDHa activity data in situations where the content of PDH-E1α is different in the analysed muscles, it is very interesting to regulate the PDHa activity data to the total content of PDH-E1α and thereby elucidate if there are differences in the regulation of each PDH-E1α. There did not seem to be any major differences in PDH regulation between vastus lateralis and triceps, because although the site 1 (Ser293) phosphorylation differed, the site 2 (Ser300) phosphorylation and PDHa activity normalized to PDH-E1α content were similar in the two muscles. So overall there seem to be no regulatory differences between PDH in the two muscles. PGC-1α overexpressing mice used in study 4 have previously been reported to have higher expression of oxidative proteins and PGC-1α KO mice to have lower (Leick et al., 2008,Lin et al., 2004). In accordance, in the present study the PGC-1α KO mice had lower, and the PGC-1α overexpressing mice had higher cyt c protein content than there WT littermates. This was associated with a markedly lower content of PDH-E1α in the KO than the WT littermates, and opposite there was a higher content of PDH-E1α in the overexpressing mice than the WT littermates. The higher oxidative capacity of the skeletal muscle of the overexpressing mice was associated with a modest increase in PDK4 protein in comparison with the WT mice (it reached only significance in the “exercise recovery” intervention.). But the PGC-1α KO mice had the same

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PDK4 protein content as the WT. Based on this observation it is concluded that PGC-1α in not required for normal PDK4 protein expression and that PDK4 expression does not follow the metabolic profile of the muscle. In addition, the similar fasting and exercise-induced PDH regulation in the very oxidative muscles of PGC-1α overexpression mice and the glycolytic muscles of the PGC-1α KO mice suggests that the metabolic profile is not of major importance in these responses. In general the overall conclusion from the above mentioned studies is that the acute regulation of PDH is the same in the different muscle types. But variations of the total capacity of PDH-E1α, may have a great impact on muscle physiology.

Muscle glycogen The lower muscle glycogen content in deltoid than triceps and vastus lateralis in Study 1 and the higher muscle glycogen after bed rest than before in Study 3, without differences in the PDHa activity at rest, suggests that a smaller change in resting muscle glycogen is not important for resting PDHa activity. This is supported by the mouse study (Study 4), where the PGC-1α overexpression mice had a higher muscle glycogen level than the WT littermates, while there was no difference in muscle glycogen between the PGC-1α KO and the WT littermates. PDHa activity regulated to the total content of PDH-E1α was similar in the various genotypes, in the FAST intervention, supports that resting muscle glycogen level are not critical for PDHa activity. It was concluded that deltoid was not recruited to the same degree as the two other muscles during the exercise bout in Study 1, and therefore it was not relevant directly to compare the exerciseinduced PDHa activity in deltoid with triceps of vastus lateralis. But the finding that the lower muscle glycogen use in deltoid during exercise was associated with less exercise-induced PDHa activity, and PDH dephosphorylation, does support a potential role of muscle glycogen in PDH regulation, although other factores may also be involved. Study 2 was therefore specifically designed to elucidate the role of muscle glycogen on PDH regulation. The findings that decreased muscle glycogen was associated with increased PDK4 content, increased PDH-E1α phosphorylation, and decreased PDHa activity provided further evidence that muscle glycogen levels do affect exercise-induced PDH regulation in human skeletal muscle. However in Study 3

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(bed rest study) enhanced glycogen content in the muscle after the bed rest period did not change the exercise-induced PDHa activity, suggesting that a certain critical low level of muscle glycogen needs to be reached before a muscle glycogen associated regulation contributes, as previously suggested for gene regulation (Pilegaard et al., 2002). The above mentioned glycogen-dependent regulation of gene expression may take place through glycogen regulatory enzymes such as protein phosphatase 1 (PP1) and glycogen synthase kinase 3 (GSK3), which are bound to the glycogen scaffold, and released when the glycogen content decreases (Pilegaard et al., 2002). This may explain the lack of influence of resting muscle glycogen on PDH regulation at rest, because there probably exist some kind of equilibrium between the normal amount of muscle glycogen and the total content of PP1 and GSK3. That could also explain why there is no difference between the PGC-1α overexpressing and WT mice, because the resting muscle glycogen as such is irrelevant, whereas the net glycogen breakdown which was identical in the two mice groups, is determining the release of PP1 and GSK3. Study 2 clearly suggests that the net breakdown of muscle glycogen had an impact on PDHa activity rather than the initial level. Furthermore it might be speculated that the downregulation of PDHa activity observed from 10 min of exercise to 30 min of exercise in study 1 is a consequence of the substantial muscle glycogen breakdown (>200 mmol/kg dry wt). Because PDK4 protein content was not measured in that study, it can only be speculated if PDK4 expression was changed. However because a previous study has not observed changes in PDK4 protein content during exercise (Watt et al., 2004) this does not seem likely. In addition the lack of change in phosphorylation of PDH during the exercise further support that neither PDK4 expression nor activity was affected, and it further stresses that there is a regulatory mechanism regulating the activity of PDHa in a manner independent of phosphorylation, or on undiscovered phosphorylation sites.

Plasma FFA In study 2 the plasma FFA levels were manipulated by a diet intervention, which showed that elevated plasma FFA concentrations were associated with less exercise-induced PDHa activity. It is suggested that this in part could be due to an enhanced PDK4 expression leading to a higher phosphorylation level and hence the lower PDHa activity observed. These data fit very well with

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what has previously been published on FFA mediated regulation of PDK4 expression in rodent skeletal muscle (Bajotto et al., 2004) as well as human skeletal muscle (Peters et al., 2001b,Pilegaard et al., 2006). Together this supports a role of FFA in regulation of PDK4 and concomitantly PDHa activity in human skeletal muscle. However in Study 3 the plasma level of palmitate was lower after bed rest than before, reflecting general lower plasma FFA levels, and this was not associated with changes in the PDK4 protein content in response to the bed rest period, or changes in PDHa activity at rest or in response to exercise. Because the content of muscle glycogen increased in response to the bed rest period, and the relative work load is expected to be higher after the bed rest period, these multiple factors may have influenced the final PDHa activity and this may explain why the FFA level did not seem to affect the PDHa activity in this study. In addition it could be speculated that PDK4 expression relies more on acute changes in FFA levels, and does not respond to slow adaptive changes in the plasma FFA. However the study by Bajotto et al. (, 2004), on the Otsuka Long-Evans Tokushima Fatty rats (OLETF), showed that an increased FFA level was followed by an enhanced PDK4 protein expression after 8 weeks which as such does not support that PDK4 protein is unaffected by slow adaptive changes. Finally the decreased level of palmitate after bed rest in Study 3 was very modest, reflecting a modest decrease in plasma FFA. Hence it could be speculated, that the plasma FFA levels have to fluctuate with a certain amount before it will influence the PDK4 protein expression.

Physical inactivity Study 3 was designed to elucidate the impact of physical inactivity on exercise-induced PDHa activity, and the data clearly show no difference on PDHa activation in response to the inactivity period. This is unexpected based on previous findings that 5 weeks of immobilization of triceps resulted in less exercise-induced PDHa activation (Ward et al., 1986), however this difference could be due to the duration of inactivity, or the muscle group studied. It could be speculated that a longer period of inactivity might have an effect on PDH, but there is not even a modest difference in PDH phosphorylation level or PDHa activity, before and after bed rest in Study 3. Regarding the mouse model used in Study 4, it has previously been reported that the muscle specific PGC-1α overexpressing mice have a phenotype like trained mice, and the PGC-1α KO mice the opposite. So considering the PGC-1α overexpressing mice adapted as highly physically

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active and PGC-1α KO mice as inactive is appropriate in terms of the cellular adaptations that have taken place in skeletal muscle of these mice. From that point of view it can also be concluded that there is no differences in the acute PDH regulation related to the endurance capacity of the mice. However the capacity for carbohydrate metabolism is clearly different due to the lager amount of PDH-E1α in the PGC-1α overexpressing mice and lower in the PGC-1α KO mice, but it seems that the regulation of PDH at the molecule level is the same, independent of the endurance capacity.

Phosphorylation and PDHa activity Does phosphorylation state reflect PDHa activity? Based on study 1,2 and 3 as well as previous findings (Pilegaard et al., 2006), there definitely exists a strong relationship between the degree of phosphorylation and PDHa activity. In Study 1 there is a strong relationship between phosphorylation and PDHa activity for vastus lateralis r2=0,54 and r2=0,67 for ser293 and ser300, respectively, and for triceps r2=0,23 and r2=0,34, respectively. The reason why the regression for triceps is not that tight could be, that it has a smaller content of PDH-E1α, and at the molecular level PDHa is activated to the highest degree possible reflecting that PDH in triceps was measured in full activation. This means that a further dephosphorylation will not be followed by an increase in PDHa activity. Thus if the workload had been smaller, it might be that the regression would have been better, and the regression from deltoid supports this possibility because deltoid appears to have been recruited to a smaller extend than triceps and the regression for deltoid was r2=0,31 and r2=0,45 for the two sites. Another interesting observation from Study 1 was the downregulation although non-significant of PDHa activity between 10 min of exercise and 30 min of exercise, while the degree of phosphorylation was the same at these time points. So it could be speculated that the degree of PDH phosphorylation and PDHa activity is very tightly regulated at the onset and first part of exercise, while other mechanisms contribute at the later stages closer to exhaustion. With these observations in mind it’s clear that the regression between PDH phosphorylation and PDHa activity would have been greater if only data from rest and 10 min of exercise was included in the regression. However, during prolonged exercise an observed decrease in PDHa activity at 3h of exercise was associated with increased PDH phosphorylation (Pilegaard et al., 2006). This may indicate that the intensity or duration of the exercise plays a role in this type of regulation.

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In the low FFA trial in Study 2 a more marked phosphorylation on ser295 and ser300 was observed in the glycogen depleted leg than in the control leg and this was associated with a smaller upregulation of PDHa activity in response to exercise. The same pattern was observed in the high FFA trial, but the differences did not reach statistical significance. This resulted in a very tight relationship between phosphorylation and PDHa activity in Study 2, and of notice is also that the exercise bout was relatively short (20 min). Hence the regression between PDHa activity and phosphorylation on ser293 and ser300 was r2=0,39 r2=0,49 respectively if all samples were included. But making the regression for CON leg only gave a tighter regression, r2=0,47 r2=0,55. In Study 3, a clear upregulation of PDHa activity with a simultaneous dephosphorylation of ser293, ser295 and ser300, in response to the exercise bout was observed. The upregulation of PDHa activity was exactly the same before as after the bed rest, and that was reflected by exactly the same dephosphorylation pattern, the association between ser293, ser295 and ser300 and PDHa activity was r2=0,47, r2=0,27 r2=0,54 respectively. Hence also in this study is the conclusion that the phosphorylation level reflects the PDHa activity. In this study the exercise bout was 45 min at 60% Watt max (pre bed rest level), and in accordance the activity of PDHa and the degree of dephosphorylation was also smaller than in the 2 previous studies. Based on study 1,2 and 3 it therefore seems to be reasonably to conclude that the degree of exercise-induced dephosphorylation reflects the increase in PDHa activity, and that this relation is strongest at the onset of exercise, while other mechanisms may contribute more close to exhaustion of high intensity exercise. However in Study 4 the relationship between PDH phosphorylation and PDHa activity was very vague, likely indicating that other mechanisms may play a role in mice than in humans.

New phosporylation sites The observation that the tightness of the relationship between PDHa activity and PDH phosphorylation on ser293 and ser300 varies, suggests that that other phosphorylation sites or other post translational modifications may be responsible. In accordance, during my PhD, a new phosphorylation site on PDH-E1α was reported (see figure 7). The new site is located at Ser295, and an antibody against the site was developed with the help from D. Grahame Hardie.

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Figure 7: A screenshot from www.phosida.com.

In general the conclusion about the new phosphorylation site, studied in study 3 and 4 was that it followed the phosphorylation pattern observed for ser293 and Ser300, and therefore it did not seem to explain the previously observed disassociation between PDHa activity and PDH phosphorylation at rest (Pilegaard et al., 2006). Whether ser295 phosphorylation can explain the disassociation observed in study 1 during the last 20 min of exercise remains however to by determined. Interestingly, as evident from the screenshot (Figure 7) a newly identified phosphorylation site on tyrosine301 has recently been detected. There is to my knowledge no published data on this site, but maybe the phosphorylation pattern of that site could add new knowledge to the dissociation of PDH phosphorylation and PDHa activity in skeletal muscle. On figure 7 the grey vertical bar on the left hand side presents sites of posttranslational modifications, determined by mass spectrometry. The sites of Y,T and S represent phosphorylation sites of tyrosine, threonine and serine, respectively. Besides regulation via phosphorylation, it is shown that PDH-E1α has 4 sites of acetylation on the lysine amino acid (K). To my knowledge there have not been published any studies examining the biological importance of the regulation of PDHa activity via acetylation. This is another candidate for explaining the disassociation mentioned earlier.

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Methodological Discussion

Limitations in PDHa activity determination The regulation of PDHa via phosphorylation is included in the assay, because the buffer contains phosphatase and kinase inhibitors. But allosteric regulation of PDHa activity is not detected by this assay, because this kind of regulation relays very much on the chemical environment in which the PDH complex is present, and the mitochondrial membrane is destroyed in the PDH assay used in the current studies. Other studies have used isolated mitochondria for determining the PDH activity (Rasmussen et al., 2003) and the use of such a technique in the protocols used in the present thesis might add to the understanding of PDH regulation.

The mice models In Study 4 an experiment with a group of mice was not presented in the manuscript. The mice performed acute exercise (10 min running at 20° incline at 15,9 meter/min), and were sacrificed immediately after the exercise bout. However this exercise bout failed to show an upregulation of PDHa activity in both WT groups, and therefore the data were not included in the study. This stresses the issue that mice in many terms have a different metabolism compared with humans, and therefore underlines the caution to be taking when trying to transfer findings in mice models to humans.

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CONCLUSIONS AND PERSPECTIVES Overall, a very close association was observed between the PDHa activity and the phosphorylation degrees of the 2 previously described phosphorylation sites ser293 and ser300, as well as the newly investigated ser295. This association was especially strong during the early part of an exercise bout, but became weaker later during high intensity exercise or at fatigue. Study 1 elucidated if the regulation of PDH is different between muscles with different MHC fiber type distribution and metabolic profile. It was shown that the content of PDH-E1α follows the oxidative profile of the muscle rather than the distribution of MHC muscle fibres. Furthermore it was concluded that the larger lactate accumulation in triceps than in vastus lateralis, may be due to lower PDH capacity in tricpes, rather than differences in PDH regulation between the two muscles. Study 2 elucidated the role of muscle glycogen in PDH regulation. There was showed a clear regulation of PDK4 protein in response to both changes in muscle glycogen and plasma FFA concentrations. The response in PDK4 protein was proven to be very fast, as differences were observed only 2h after dietary manipulation. Furthermore a very tight association between the PDK4 protein level, the degree of phosphorylation on PDH-E1α and the PDHa activity was evident. This suggests that the regulation of PDHa activity in response to FFA and muscle glycogen is exerted via regulation of the PDK4 protein content. Study 3 elucidated the effect of a period of physical inactivity on the exercise-induced regulation of PDH. The study showed that the degree of phosphorylation and PDHa activity, in response to exercise, was exactly the same before and after the inactivity period. Study 4 elucidated the role of PGC-1α in PDH regulation. It was shown that PGC-1α increased the content of PDH-E1α, but did not seem to affect the regulation of PDH in response to fasting, or in recovery from exercise. PGC-1α did not seem to have an effect on PDK4 protein expression, indicating that the regulation of PDK4 protein occurs independent of PGC-1α, or that multiple pathways exist enabling a compensation when PGC-1α is knocked out.

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In addition, mice may not to be a good model to study exercise-induced PDHa activation and PDH phosphorylation, because we were unable to induce an upregualtion of PDHa activity in response to an exercise bout. In future studies it would be interesting to determine if the newly discovered phosphorylation site on PDH has a phosphorylation pattern different from the 3 examined phosphorylation sites. Furthermore it would be very interesting to elucidate the importance of acetylation in PDH regulation, and identify the proteins that acetylate and deacetylate the PDH-E1α protein.

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REFERENCE LIST

Anne Lykke Poulsen. Forskning i bevægelse. Museum Tusculanums Forlag, 2009. Arany Z, Foo S Y, Ma Y, Ruas J L, Bommi-Reddy A, Girnun G, Cooper M, Laznik D, Chinsomboon J, Rangwala S M, Baek K H, Rosenzweig A, Spiegelman B M. HIF-independent regulation of VEGF and angiogenesis by the transcriptional coactivator PGC-1alpha. Nature 2008; 451(7181): 1008-1012. Bajotto G, Murakami T, Nagasaki M, Tamura T, Tamura N, Harris R A, Shimomura Y, Sato Y. Downregulation of the skeletal muscle pyruvate dehydrogenase complex in the Otsuka Long-Evans Tokushima Fatty rat both before and after the onset of diabetes mellitus. Life Sci 2004; 75(17): 21172130. Behal R H, Buxton D B, Robertson J G, Olson M S. Regulation of the pyruvate dehydrogenase multienzyme complex. Annu Rev Nutr 1993; 13: 497-520. Bowker-Kinley M M, Davis W I, Wu P, Harris R A, Popov K M. Evidence for existence of tissue-specific regulation of the mammalian pyruvate dehydrogenase complex. Biochem J 1998; 329 ( Pt 1): 191-196. Burgomaster K A, Heigenhauser G J, Gibala M J. Effect of short-term sprint interval training on human skeletal muscle carbohydrate metabolism during exercise and time-trial performance. J Appl Physiol 2006; 100(6): 2041-2047. Bustin S A. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 2000; 25(2): 169-193. Bustin S A. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 2002; 29(1): 23-39. Bustin S A, Benes V, Nolan T, Pfaffl M W. Quantitative real-time RT-PCR--a perspective. J Mol Endocrinol 2005; 34(3): 597-601. Calvo J A, Daniels T G, Wang X, Paul A, Lin J, Spiegelman B M, Stevenson S C, Rangwala S M. Musclespecific expression of PPARgamma coactivator-1alpha improves exercise performance and increases peak oxygen uptake. J Appl Physiol 2008; 104(5): 1304-1312. Caruso M, Maitan M A, Bifulco G, Miele C, Vigliotta G, Oriente F, Formisano P, Beguinot F. Activation and mitochondrial translocation of protein kinase Cdelta are necessary for insulin stimulation of pyruvate dehydrogenase complex activity in muscle and liver cells. J Biol Chem 2001; 276(48): 4508845097. Cederblad G, Carlin J I, Constantin-Teodosiu D, Harper P, Hultman E. Radioisotopic assays of CoASH and carnitine and their acetylated forms in human skeletal muscle. Anal Biochem 1990; 185(2): 274278. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal Biochem 1987; 162(1): 156-159. Christensen EH, Hansen O. Zur Methodik der respiratorischen Quotient-Bestimmungen in Ruhe and bei Arbeit. Skand Arch Physiol 1939; 81: 137-171. Constantin-Teodosiu D, Cederblad G, Hultman E. A sensitive radioisotopic assay of pyruvate dehydrogenase complex in human muscle tissue. Anal Biochem 1991; 198(2): 347-351. Constantin-Teodosiu D, Peirce N S, Fox J, Greenhaff P L. Muscle pyruvate availability can limit the flux, but not activation, of the pyruvate dehydrogenase complex during submaximal exercise in humans. J Physiol 2004; 561(Pt 2): 647-655.

45

Denton R M, McCormack J G, Rutter G A, Burnett P, Edgell N J, Moule S K, Diggle T A. The hormonal regulation of pyruvate dehydrogenase complex. Adv Enzyme Regul 1996; 36: 183-198. Gudi R, Bowker-Kinley M M, Kedishvili N Y, Zhao Y, Popov K M. Diversity of the pyruvate dehydrogenase kinase gene family in humans. J Biol Chem 1995; 270(48): 28989-28994. Harris R A, Bowker-Kinley M M, Huang B, Wu P. Regulation of the activity of the pyruvate dehydrogenase complex. Adv Enzyme Regul 2002; 42: 249-259. Heid C A, Stevens J, Livak K J, Williams P M. Real time quantitative PCR. Genome Res 1996; 6(10): 986994. Hildebrandt A L, Pilegaard H, Neufer P D. Differential transcriptional activation of select metabolic genes in response to variations in exercise intensity and duration. Am J Physiol Endocrinol Metab 2003; 285(5): E1021-E1027. Holness M J, Bulmer K, Gibbons G F, Sugden M C. Up-regulation of pyruvate dehydrogenase kinase isoform 4 (PDK4) protein expression in oxidative skeletal muscle does not require the obligatory participation of peroxisome-proliferator-activated receptor alpha (PPARalpha). Biochem J 2002; 366(Pt 3): 839-846. Holness M J, Kraus A, Harris R A, Sugden M C. Targeted upregulation of pyruvate dehydrogenase kinase (PDK)-4 in slow-twitch skeletal muscle underlies the stable modification of the regulatory characteristics of PDK induced by high-fat feeding. Diabetes 2000; 49(5): 775-781. Holness M J, Sugden M C. Regulation of pyruvate dehydrogenase complex activity by reversible phosphorylation. Biochem Soc Trans 2003; 31(Pt 6): 1143-1151. Howlett R A, Parolin M L, Dyck D J, Hultman E, Jones N L, Heigenhauser G J, Spriet L L. Regulation of skeletal muscle glycogen phosphorylase and PDH at varying exercise power outputs. Am J Physiol 1998; 275(2 Pt 2): R418-R425. Huang B, Gudi R, Wu P, Harris R A, Hamilton J, Popov K M. Isoenzymes of pyruvate dehydrogenase phosphatase. DNA-derived amino acid sequences, expression, and regulation. J Biol Chem 1998; 273(28): 17680-17688. Huang B, Wu P, Popov K M, Harris R A. Starvation and diabetes reduce the amount of pyruvate dehydrogenase phosphatase in rat heart and kidney. Diabetes 2003; 52(6): 1371-1376. Huss J M, Kopp R P, Kelly D P. Peroxisome proliferator-activated receptor coactivator-1alpha (PGC1alpha) coactivates the cardiac-enriched nuclear receptors estrogen-related receptor-alpha and -gamma. Identification of novel leucine-rich interaction motif within PGC-1alpha. J Biol Chem 2002; 277(43): 40265-40274. Huss J M, Torra I P, Staels B, Giguere V, Kelly D P. Estrogen-related receptor alpha directs peroxisome proliferator-activated receptor alpha signaling in the transcriptional control of energy metabolism in cardiac and skeletal muscle. Mol Cell Biol 2004; 24(20): 9079-9091. Khailova L S, Severin S E, Hubner G, Neef H, Schellenberger A. Effect of pyruvate and its analogs on the thiamine pyrophosphate binding in the active center of muscle pyruvate dehydrogenase. FEBS Lett 1982; 139(1): 49-52. Kiilerich K, Birk JB, Saltin B, Pedersen PA, Wojtaszewski JFP, Pilegaard H. Exercise induces increased PDK4 expression in human skeletal muscles independent of a fasting effect. Diabetes 57(suppl. 1):A308. 2008. Ref Type: Abstract

46

KOIKE M, Reed L J, CARROLL W R. alpha-Keto acid dehydrogenation complexes. IV. Resolution and reconstitution of the Escherichia coli pyruvate dehydrogenation complex. J Biol Chem 1963a; 238: 3039. KOIKE M, Reed L J, CARROLL W R. alpha-Keto acid dehydrogenation complexes. IV. Resolution and reconstitution of the Escherichia coli pyruvate dehydrogenation complex. J Biol Chem 1963b; 238: 3039. Korotchkina L G, Patel M S. Mutagenesis studies of the phosphorylation sites of recombinant human pyruvate dehydrogenase. Site-specific regulation. J Biol Chem 1995; 270(24): 14297-14304. Korotchkina L G, Patel M S. Probing the mechanism of inactivation of human pyruvate dehydrogenase by phosphorylation of three sites. J Biol Chem 2001a; 276(8): 5731-5738. Korotchkina L G, Patel M S. Site specificity of four pyruvate dehydrogenase kinase isoenzymes toward the three phosphorylation sites of human pyruvate dehydrogenase. J Biol Chem 2001b; 276(40): 3722337229. LeBlanc P J, Howarth K R, Gibala M J, Heigenhauser G J. Effects of 7 wk of endurance training on human skeletal muscle metabolism during submaximal exercise. J Appl Physiol 2004a; 97(6): 2148-2153. LeBlanc P J, Mulligan M, Antolic A, Macpherson L, Inglis J G, Martin D, Roy B D, Peters S J. Skeletal muscle type comparison of pyruvate dehydrogenase phosphatase activity and isoform expression: effects of obesity and endurance training. Am J Physiol Regul Integr Comp Physiol 2008; 295(4): R1224R1230. LeBlanc P J, Peters S J, Tunstall R J, Cameron-Smith D, Heigenhauser G J. Effects of aerobic training on pyruvate dehydrogenase and pyruvate dehydrogenase kinase in human skeletal muscle. J Physiol 2004b; 557(Pt 2): 559-570. Leick L, Wojtaszewski J F, Johansen S T, Kiilerich K, Comes G, Hellsten Y, Hidalgo J, Pilegaard H. PGC1alpha is not mandatory for exercise- and training-induced adaptive gene responses in mouse skeletal muscle. Am J Physiol Endocrinol Metab 2008; 294(2): E463-E474. Lin J, Wu H, Tarr P T, Zhang C Y, Wu Z, Boss O, Michael L F, Puigserver P, Isotani E, Olson E N, Lowell B B, Bassel-Duby R, Spiegelman B M. Transcriptional co-activator PGC-1 alpha drives the formation of slow-twitch muscle fibres. Nature 2002; 418(6899): 797-801. Lin J, Wu P H, Tarr P T, Lindenberg K S, St-Pierre J, Zhang C Y, Mootha V K, Jager S, Vianna C R, Reznick R M, Cui L, Manieri M, Donovan M X, Wu Z, Cooper M P, Fan M C, Rohas L M, Zavacki A M, Cinti S, Shulman G I, Lowell B B, Krainc D, Spiegelman B M. Defects in adaptive energy metabolism with CNS-linked hyperactivity in PGC-1alpha null mice. Cell 2004; 119(1): 121-135. LINK. http://en.wikipedia.org/wiki/Antoine_Lavoisier. 2010a. Ref Type: Online Source LINK. http://en.wikipedia.org/wiki/Biochemistry. 2010b. Ref Type: Online Source LINK. http://en.wikipedia.org/wiki/Friedrich_W%C3%B6hler. 2010c. Ref Type: Online Source LINK. http://en.wikipedia.org/wiki/Metabolism. 2010d. Ref Type: Online Source LINK. http://en.wikipedia.org/wiki/Sanctorius. 2010e. Ref Type: Online Source

47

LINK. http://nobelprize.org/nobel_prizes/medicine/laureates/1953/krebs.html. 2010f. Ref Type: Online Source LINK. http://upload.wikimedia.org/wikipedia/commons/1/1d/TaqMan_Probes.jpg. 2010g. Ref Type: Online Source Linn T C, Pettit F H, Reed L J. Alpha-keto acid dehydrogenase complexes. X. Regulation of the activity of the pyruvate dehydrogenase complex from beef kidney mitochondria by phosphorylation and dephosphorylation. Proc Natl Acad Sci U S A 1969; 62(1): 234-241. Lundby C, Nordsborg N, Kusuhara K, Kristensen K M, Neufer P D, Pilegaard H. Gene expression in human skeletal muscle: alternative normalization method and effect of repeated biopsies. Eur J Appl Physiol 2005; 95(4): 351-360. Majer M, Popov K M, Harris R A, Bogardus C, Prochazka M. Insulin downregulates pyruvate dehydrogenase kinase (PDK) mRNA: potential mechanism contributing to increased lipid oxidation in insulin-resistant subjects. Mol Genet Metab 1998; 65(2): 181-186. Mandarino L J, Consoli A, Kelley D E, Reilly J J, Nurjhan N. Fasting hyperglycemia normalizes oxidative and nonoxidative pathways of insulin-stimulated glucose metabolism in noninsulin-dependent diabetes mellitus. J Clin Endocrinol Metab 1990; 71(6): 1544-1551. Merkel C G, Kwan S, Lingrel J B. Size of the polyadenylic acid region of newly synthesized globin messenger ribonucleic acid. J Biol Chem 1975; 250(10): 3725-3728. Mootha V K, Handschin C, Arlow D, Xie X, St Pierre J, Sihag S, Yang W, Altshuler D, Puigserver P, Patterson N, Willy P J, Schulman I G, Heyman R A, Lander E S, Spiegelman B M. Erralpha and Gabpa/b specify PGC-1alpha-dependent oxidative phosphorylation gene expression that is altered in diabetic muscle. Proc Natl Acad Sci U S A 2004; 101(17): 6570-6575. Mootha V K, Lindgren C M, Eriksson K F, Subramanian A, Sihag S, Lehar J, Puigserver P, Carlsson E, Ridderstrale M, Laurila E, Houstis N, Daly M J, Patterson N, Mesirov J P, Golub T R, Tamayo P, Spiegelman B, Lander E S, Hirschhorn J N, Altshuler D, Groop L C. PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet 2003; 34(3): 267-273. Mourtzakis M, Saltin B, Graham T, Pilegaard H. Carbohydrate metabolism during prolonged exercise and recovery: interactions between pyruvate dehydrogenase, fatty acids, and amino acids. J Appl Physiol 2006; 100(6): 1822-1830. Nordsborg N B, Lundby C, Leick L, Pilegaard H. Relative workload determines exercise-induced increases in PGC-1alpha mRNA. Med Sci Sports Exerc 2010; 42(8): 1477-1484. Olesen J, Kiilerich K, Pilegaard H. PGC-1alpha-mediated adaptations in skeletal muscle. Pflugers Arch 2010; 460(1): 153-162. Patel MS, Roche TE. Molecular biology and biochemistry of pyruvate dehydrogenase complexes. FASEB J 1990; 14: 3224-3233. Patel M S, Korotchkina L G. Regulation of mammalian pyruvate dehydrogenase complex by phosphorylation: complexity of multiple phosphorylation sites and kinases. Exp Mol Med 2001; 33(4): 191-197. Peters S J. Regulation of PDH activity and isoform expression: diet and exercise. Biochem Soc Trans 2003; 31(Pt 6): 1274-1280.

48

Peters S J, Harris R A, Heigenhauser G J, Spriet L L. Muscle fiber type comparison of PDH kinase activity and isoform expression in fed and fasted rats. Am J Physiol Regul Integr Comp Physiol 2001a; 280(3): R661-R668. Peters S J, Harris R A, Wu P, Pehleman T L, Heigenhauser G J, Spriet L L. Human skeletal muscle PDH kinase activity and isoform expression during a 3-day high-fat/low-carbohydrate diet. Am J Physiol Endocrinol Metab 2001b; 281(6): E1151-E1158. Peters S J, St Amand T A, Howlett R A, Heigenhauser G J, Spriet L L. Human skeletal muscle pyruvate dehydrogenase kinase activity increases after a low-carbohydrate diet. Am J Physiol 1998; 275(6 Pt 1): E980-E986. Pettit F H, Pelley J W, Reed L J. Regulation of pyruvate dehydrogenase kinase and phosphatase by acetylCoA/CoA and NADH/NAD ratios. Biochem Biophys Res Commun 1975; 65(2): 575-582. Pilegaard H, Birk J B, Sacchetti M, Mourtzakis M, Hardie D G, Stewart G, Neufer P D, Saltin B, Van H G, Wojtaszewski J F. PDH-E1alpha dephosphorylation and activation in human skeletal muscle during exercise: effect of intralipid infusion. Diabetes 2006; 55(11): 3020-3027. Pilegaard H, Keller C, Steensberg A, Helge J W, Pedersen B K, Saltin B, Neufer P D. Influence of preexercise muscle glycogen content on exercise-induced transcriptional regulation of metabolic genes. J Physiol 2002; 541(Pt 1): 261-271. Pilegaard H, Ordway G A, Saltin B, Neufer P D. Transcriptional regulation of gene expression in human skeletal muscle during recovery from exercise. Am J Physiol Endocrinol Metab 2000; 279(4): E806E814. Pilegaard H, Osada T, Andersen L T, Helge J W, Saltin B, Neufer P D. Substrate availability and transcriptional regulation of metabolic genes in human skeletal muscle during recovery from exercise. Metabolism 2005; 54(8): 1048-1055. Pilegaard H, Saltin B, Neufer P D. Effect of short-term fasting and refeeding on transcriptional regulation of metabolic genes in human skeletal muscle. Diabetes 2003; 52(3): 657-662. Puigserver P, Spiegelman B M. Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1 alpha): transcriptional coactivator and metabolic regulator. Endocr Rev 2003; 24(1): 78-90. Puigserver P, Wu Z, Park C W, Graves R, Wright M, Spiegelman B M. A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell 1998; 92(6): 829-839. Putman C T, Spriet L L, Hultman E, Lindinger M I, Lands L C, McKelvie R S, Cederblad G, Jones N L, Heigenhauser G J. Pyruvate dehydrogenase activity and acetyl group accumulation during exercise after different diets. Am J Physiol 1993; 265(5 Pt 1): E752-E760. Randle P J, GARLAND P B, HALES C N, NEWSHOLME E A. The glucose fatty-acid cycle. Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet 1963; 1(7285): 785-789. Randle P J, Priestman D A, Mistry S C, Halsall A. Glucose fatty acid interactions and the regulation of glucose disposal. J Cell Biochem 1994; 55 Suppl: 1-11. Rasmussen U F, Krustrup P, Kjaer M, Rasmussen H N. Experimental evidence against the mitochondrial theory of aging. A study of isolated human skeletal muscle mitochondria. Exp Gerontol 2003; 38(8): 877-886. Ravindran S, Radke G A, Guest J R, Roche T E. Lipoyl domain-based mechanism for the integrated feedback control of the pyruvate dehydrogenase complex by enhancement of pyruvate dehydrogenase kinase activity. J Biol Chem 1996; 271(2): 653-662.

49

Reed L J. A trail of research from lipoic acid to alpha-keto acid dehydrogenase complexes. J Biol Chem 2001; 276(42): 38329-38336. Roche T E, Baker J C, Yan X, Hiromasa Y, Gong X, Peng T, Dong J, Turkan A, Kasten S A. Distinct regulatory properties of pyruvate dehydrogenase kinase and phosphatase isoforms. Prog Nucleic Acid Res Mol Biol 2001; 70: 33-75. Schreiber S N, Emter R, Hock M B, Knutti D, Cardenas J, Podvinec M, Oakeley E J, Kralli A. The estrogen-related receptor alpha (ERRalpha) functions in PPARgamma coactivator 1alpha (PGC-1alpha)induced mitochondrial biogenesis. Proc Natl Acad Sci U S A 2004; 101(17): 6472-6477. Sugden M C, Bulmer K, Augustine D, Holness M J. Selective modification of pyruvate dehydrogenase kinase isoform expression in rat pancreatic islets elicited by starvation and activation of peroxisome proliferator-activated receptor-alpha: implications for glucose-stimulated insulin secretion. Diabetes 2001a; 50(12): 2729-2736. Sugden M C, Bulmer K, Gibbons G F, Holness M J. Role of peroxisome proliferator-activated receptoralpha in the mechanism underlying changes in renal pyruvate dehydrogenase kinase isoform 4 protein expression in starvation and after refeeding. Arch Biochem Biophys 2001b; 395(2): 246-252. Sugden M C, Holness M J. Recent advances in mechanisms regulating glucose oxidation at the level of the pyruvate dehydrogenase complex by PDKs. Am J Physiol Endocrinol Metab 2003; 284(5): E855-E862. Sugden M C, Kraus A, Harris R A, Holness M J. Fibre-type specific modification of the activity and regulation of skeletal muscle pyruvate dehydrogenase kinase (PDK) by prolonged starvation and refeeding is associated with targeted regulation of PDK isoenzyme 4 expression. Biochem J 2000; 346 Pt 3: 651-657. Sugden M C, Orfali K A, Holness M J. The pyruvate dehydrogenase complex: nutrient control and the pathogenesis of insulin resistance. J Nutr 1995; 125(6 Suppl): 1746S-1752S. Sugden P H, Randle P J. Regulation of pig heart pyruvate dehydrogenase by phosphorylation. Studies on the subunit and phosphorylation stoicheiometries. Biochem J 1978; 173(2): 659-668. Tyler D. THE MITOCHONDRION IN HEALTH & DISEASE. VCH Publishers, Inc., 1992. Vega R B, Huss J M, Kelly D P. The coactivator PGC-1 cooperates with peroxisome proliferator-activated receptor alpha in transcriptional control of nuclear genes encoding mitochondrial fatty acid oxidation enzymes. Mol Cell Biol 2000; 20(5): 1868-1876. Wang Y X, Lee C H, Tiep S, Yu R T, Ham J, Kang H, Evans R M. Peroxisome-proliferator-activated receptor delta activates fat metabolism to prevent obesity. Cell 2003; 113(2): 159-170. Ward G R, MacDougall J D, Sutton J R, Toews C J, Jones N L. Activation of human muscle pyruvate dehydrogenase with activity and immobilization. Clin Sci (Lond) 1986; 70(2): 207-210. Ward G R, Sutton J R, Jones N L, Toews C J. Activation by exercise of human skeletal muscle pyruvate dehydrogenase in vivo. Clin Sci (Lond) 1982; 63(1): 87-92. Watt M J, Heigenhauser G J, Dyck D J, Spriet L L. Intramuscular triacylglycerol, glycogen and acetyl group metabolism during 4 h of moderate exercise in man. J Physiol 2002; 541(Pt 3): 969-978. Watt M J, Heigenhauser G J, LeBlanc P J, Inglis J G, Spriet L L, Peters S J. Rapid upregulation of pyruvate dehydrogenase kinase activity in human skeletal muscle during prolonged exercise. J Appl Physiol 2004; 97(4): 1261-1267. Wende A R, Huss J M, Schaeffer P J, Giguere V, Kelly D P. PGC-1alpha coactivates PDK4 gene expression via the orphan nuclear receptor ERRalpha: a mechanism for transcriptional control of muscle glucose metabolism. Mol Cell Biol 2005; 25(24): 10684-10694.

50

Wende A R, Schaeffer P J, Parker G J, Zechner C, Han D H, Chen M M, Hancock C R, Lehman J J, Huss J M, McClain D A, Holloszy J O, Kelly D P. A role for the transcriptional coactivator PGC-1alpha in muscle refueling. J Biol Chem 2007; 282(50): 36642-36651. Wu P, Blair P V, Sato J, Jaskiewicz J, Popov K M, Harris R A. Starvation increases the amount of pyruvate dehydrogenase kinase in several mammalian tissues. Arch Biochem Biophys 2000; 381(1): 1-7. Wu P, Inskeep K, Bowker-Kinley M M, Popov K M, Harris R A. Mechanism responsible for inactivation of skeletal muscle pyruvate dehydrogenase complex in starvation and diabetes. Diabetes 1999a; 48(8): 1593-1599. Wu P, Peters J M, Harris R A. Adaptive increase in pyruvate dehydrogenase kinase 4 during starvation is mediated by peroxisome proliferator-activated receptor alpha. Biochem Biophys Res Commun 2001; 287(2): 391-396. Wu Z, Puigserver P, Andersson U, Zhang C, Adelmant G, Mootha V, Troy A, Cinti S, Lowell B, Scarpulla R C, Spiegelman B M. Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell 1999b; 98(1): 115-124. Yeaman S J, Hutcheson E T, Roche T E, Pettit F H, Brown J R, Reed L J, Watson D C, Dixon G H. Sites of phosphorylation on pyruvate dehydrogenase from bovine kidney and heart. Biochemistry 1978; 17(12): 2364-2370. Zhang Y, Ma K, Sadana P, Chowdhury F, Gaillard S, Wang F, McDonnell D P, Unterman T G, Elam M B, Park E A. Estrogen-related receptors stimulate pyruvate dehydrogenase kinase isoform 4 gene expression. J Biol Chem 2006; 281(52): 39897-39906.

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APPENDIX (STUDY 1-4)

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STUDY 1

Am J Physiol Endocrinol Metab 294: E36–E42, 2008. First published October 23, 2007; doi:10.1152/ajpendo.00352.2007.

Regulation of PDH in human arm and leg muscles at rest and during intense exercise Kristian Kiilerich,1,2,3 Jesper B. Birk,1,5 Rasmus Damsgaard,1,4 Jørgen F. P. Wojtaszewski,1,5 and Henriette Pilegaard1,2,3 1

Copenhagen Muscle Research Centre, 2Centre of Inflammation and Metabolism, and 3Department of Molecular Biology, August Krogh Building, University of Copenhagen; 4Rigshospitalet, Copenhagen; and 5Section of Human Physiology, Department of Exercise and Sport Sciences, University of Copenhagen, Copenhagen, Denmark Submitted 6 June 2007; accepted in final form 8 October 2007

ARM AND LEG SKELETAL MUSCLES in humans differ in their metabolic response to exercise. Examples are a larger glucose extraction in arm than in leg muscles and a markedly higher net lactate release at similar relative exercise intensities (1). Moreover, the fatty acid uptake as well as the contribution of lipids for the energy yield are less in the arms (27). Also, the action of insulin appears to be most pronounced in the arm muscles with a larger insulin-stimulated glucose uptake and a further lowering of the free fatty acid (FFA) uptake (19, 27). These differences in metabolism comparing arm and leg muscles can hardly be explained by fiber type, blood flow, or oxygen delivery alone, as these variables are quite similar in the muscles of the upper and lower limbs (1). The interest in

understanding especially the difference in lactate release is emphasized by lactate being a central player in both cellular and whole body metabolism (12). Training status could play a role, but even in the most well-trained endurance athletes using their arms just as much as their legs, a high dependency of carbohydrates and high lactate production are still observed in the arm muscles (8). This indicates that the difference in metabolic response to exercise between arm and leg muscles could be related to the metabolic profile of the involved muscles, regulation of the glycolytic flux, and/or entry of carbohydrate-derived fuel into the mitochondria. Muscle type-related distributions of metabolic enzymes have been demonstrated in human muscles, with higher phosphofructokinase (PFK) activity in the arm muscle triceps than in the leg muscles soleus and vastus lateralis (25) and higher citrate synthase (CS) and 3-hydroxyacyl-CoA dehydrogenase (HAD) activity in soleus and vastus lateralis than in triceps (11, 25). Oxidative muscles are typically characterized by a high proportion of myosin heavy chain (MHC) type I fibers and glycolytic muscles by a high proportion of MHC type II fibers. However, the different adaptability of metabolic enzymes and MHC isoforms (13) indicates that a given MHC muscle fiber type may have a wide range of metabolic capacity (21, 25), and that metabolically related parameters may not necessarily show a tight relationship with MHC composition. The activities of the metabolic enzymes mentioned above are all sensitive to endurance exercise training, with upregulation of CS (7) and HAD (28) activity and downregulation of PFK activity (28), underlining the major impact of physical activity on skeletal muscle metabolic profile (11, 23, 24, 28, 29). It may be that the differences in substrate use between arm and leg muscles solely can be explained by the lower respiratory capacity of arm than leg muscles. This could be the case, even in highly trained cross-country skiers, for whom very intense use of the arm muscles still renders the oxidative enzyme capacity of the arm muscles lower than that of the leg muscles (8). The pyruvate dehydrogenase complex (PDC) regulates the entry of carbohydrate-derived fuel into the mitochondria for oxidation by catalyzing the decarboxylation of pyruvate to acetyl-CoA. Lactate production will be expected to be related to the capacity and ability of the PDC to convert pyruvate to acetyl-CoA. PDC is composed of three catalytic proteins (E1, E2, and E3), a structural protein (E3BP), and two regulatory proteins, pyruvate dehydrogenase kinase (PDK) and pyruvate

Address for reprint requests and other correspondence: K. Kiilerich, August Krogh Bldg., Dept. of Molecular Biology, Universitetsparken 13, 2100 Copenhagen, Denmark (e-mail: [email protected]).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

pyruvate dehydrogenase; pyruvate dehydrogenase activity; pyruvate dehydrogenase phosphorylation; muscle type

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Kiilerich K, Birk JB, Damsgaard R, Wojtaszewski JP, Pilegaard H. Regulation of PDH in human arm and leg muscles at rest and during intense exercise. Am J Physiol Endocrinol Metab 294: E36–E42, 2008. First published October 23, 2007; doi:10.1152/ajpendo.00352.2007.—To test the hypothesis that pyruvate dehydrogenase (PDH) is differentially regulated in specific human muscles, regulation of PDH was examined in triceps, deltoid, and vastus lateralis at rest and during intense exercise. To elicit considerable glycogen use, subjects performed 30 min of exhaustive arm cycling on two occasions and leg cycling exercise on a third day. Muscle biopsies were obtained from deltoid or triceps on the arm exercise days and from vastus lateralis on the leg cycling day. Resting PDH protein content and phosphorylation on PDH-E1␣ sites 1 and 2 were higher (P ⱕ 0.05) in vastus lateralis than in triceps and deltoid as was the activity of oxidative enzymes. Net muscle glycogen utilization was similar in vastus lateralis and triceps (⬇50%) but less in deltoid (likely reflecting less recruitment of deltoid), while muscle lactate accumulation was ⬇55% higher (P ⱕ 0.05) in triceps than vastus lateralis. Exercise induced (P ⱕ 0.05) dephosphorylation of both PDH-E1␣ site 1 and site 2 in all three muscles, but it was more pronounced at PDH-E1␣ site 1 in triceps than in vastus lateralis (P ⱕ 0.05). The increase in activity of the active form of PDH (PDHa) after 10 min of exercise was more marked in vastus lateralis (⬇246%) than in triceps (⬇160%), but when it was related to total PDH-E1␣ protein content, no difference was evident. In conclusion, PDH protein content seems to be related to metabolic enzyme profile, rather than myosin heavy chain composition, and less PDH capacity in triceps is a likely contributing factor to higher lactate accumulation in triceps than in vastus lateralis.

REGULATION OF PDH IN HUMAN ARM AND LEG MUSCLES

dehydrogenase phosphatase (PDP). The E1 subunit, pyruvate dehydrogenase (PDH), is responsible for catalyzing the decarboxylation of pyruvate. Therefore, regulation of this PDH component of the enzyme complex could be important for the mitochondrial choice of substrate at rest and during exercise. Thus, to provide information on mechanisms underlying differences in carbohydrate metabolism in human muscles, the aim of the present study was to test the hypothesis that differences in exercise-induced activation of PDH in part can explain the dissimilarity in carbohydrate dependency between arm and leg skeletal muscles during exercise. This is examined by investigating PDH regulation in human arm and leg muscles characterized by different metabolic and MHC profiles. MATERIALS AND METHODS

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Muscle glycogen, lactate, and enzyme activity. Muscle specimens were freeze-dried and dissected free of blood, fat, and connective tissue under the microscope, and muscle glycogen content was determined as glycosyl units after acid hydrolysis using an automatic spectrophotometer as previously described (16). Muscle lactate concentrations of freeze-dried samples were determined fluorometrically (16). The activity of CS, HAD, lactate dehydrogenase (LDH), and PFK was analyzed spectrophotometrically as previously described (11). Muscle lysate. Muscle pieces were homogenized in an ice-cold buffer (10% glycerol, 20 mM Na-pyrophosphate, 150 mM NaCl, 50 mM HEPES, 1% NP-40, 20 mM ␤-glycerophosphate, 10 mM NaF, 1 mM EDTA, 1 mM EGTA, 2 mM PMSF, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, 2 mM Na3VO4, 3 mM benzamidine, pH 7.5) for 20 s using a homogenizer (PT 3100, Kinematica). Homogenates were rotated end over end for 1 h at 4°C. Lysates were generated by centrifugation (17,500 g) for 20 min at 4°C. Protein content in lysates was measured by the bicinchoninic acid method (Pierce Chemical). SDS-PAGE and Western blotting. The protein expression and phosphorylation of sites 1 and 2 were measured in muscle lysate by SDS-PAGE (Tris 䡠 HCl 10% gel, Bio-Rad) and Western blotting using polyvinylidene difluoride (PVDF) membrane and semi-dry transfer. After the transfer, the PVDF membrane was blocked for 1 h at room temperature [TBS with Tween (TBST) ⫹ 2% skim milk], followed by incubation with primary antibody overnight at 4°C (TBST ⫹ 2% skim milk). The following day, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (Dako) for 1 h at room temperature (TBST ⫹ 2% skim milk). Immobilon Western (Millipore) was used as a detection system. Bands were visualized using an Eastman Kodak Image Station 2000MM. Bands were quantified using Kodak Molecular Imaging Software v.4.0.3, and protein content was expressed in units relative to control samples loaded on each gel. Protein levels of the PDH-E1␣ subunit and phosphorylation of site 1 (Ser293) and site 2 (Ser300) of PDH-E1␣ were determined using antibodies generated in sheep as previously described (22). Activity of the active form of PDH. The activity of the active form of PDH (PDHa) was determined as previously described (9, 10, 26) after homogenizing ⬇10 mg of muscle tissue for 50 s in the modified glycogen synthase kinase-3 buffer given above, using a glass homogenizer (Kontes), and quickly (10 –15 s) freezing the samples in liquid nitrogen. It is normal procedure that PDHa activity is adjusted to total creatine. However, because of previous findings in rodents that glycolytic type II-rich muscles have a higher content of creatine than oxidative type I muscles (14), the PDHa activity was in the present study normalized to total PDH-E1␣ protein content as measured by Western blotting. Statistics. Values presented are means ⫾ SE. Two-way ANOVA for repeated measures was applied to evaluate the effect of muscle type and time. One-way ANOVA for repeated measures was used to test for differences in fiber type distribution and enzyme activity between muscle types. The Student-Newman-Keuls post hoc test was used to locate differences. Differences were considered significant at P ⱕ 0.05, and a tendency is reported when 0.05 ⱕ P ⱕ 0.1. Statistical calculations were performed using SigmaStat v.2.03. RESULTS

Muscle fiber composition. The percent occurrence of type I fibers was similar in vastus lateralis (52%) and deltoid (54%), whereas triceps (32%) contained less type I fibers than vastus and deltoid (P ⱕ 0.05; Table 1). Enzyme activity. The activity of CS was ⬃50% higher in vastus lateralis (31.3 mmol 䡠min⫺1 䡠kg dry wt⫺1) than in both triceps (18.8 mmol 䡠min⫺1 䡠kg dry wt⫺1) and deltoid (17.1

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Subjects. Eight healthy male subjects with normal physical activity level, an average age of 26 yr (range: 23–30 yr), weight 85 kg (range: 71–96 kg), and height 185 cm (range: 179 –192 cm) participated in the study. The maximum power (wattmax) was 121 ⫾ 5 and 303 ⫾ 12 W (average ⫾ SE) for arm and leg, respectively. The subjects were given both written and oral information about the experimental protocol and procedures and were informed about any discomfort that might be associated with the experiment before they gave their written consent. The study was performed according to the Declaration of Helsinki and was approved by the Copenhagen and Frederiksberg Ethics Committee, Denmark. Experimental protocol. Approximately 1 wk before the first trial, the subjects performed preexperimental tests to determine wattmax for arm and leg exercise separately. The wattmax was determined for arm cycling and leg cycling separately using incremental tests where the resistance was increased (arm, 12 W; leg, 30 W) every second minute until exhaustion. Wattmax was estimated from the time to exhaustion and workload. Each subject completed three experimental days, one day with leg cycling using an electronic ergometer (Monark 839E) and two days with arm cycling using an arm crank ergometer (Monark 891E), which was adjusted so that the shoulder of the subject leveled the crank. The day before each experimental trial, the subjects refrained from exercise. On the experimental day, the subjects arrived at the laboratory 2.5 h after consuming a standardized breakfast [77 percent energy (E%) carbohydrate; 11 E% protein; 12 E% fat], regulated for bodyweight and activity level (34). Muscle biopsies were obtained from either deltoid or triceps on the arm exercise days and from vastus lateralis on the day of leg cycling. Three incisions for muscle biopsies were made under local anesthesia (lidocaine and epinephrine). After a resting muscle biopsy was obtained using the percutaneous needle biopsy technique (3) with suction, a blood lactate sample was taken using finger prick and analysis of mixed blood lactate with Lactate Pro LT-1710 (Arkray). Thereafter, the subjects performed 30 min of exercise. The aim of the protocol was to elicit considerable glycogen use, and the exercise protocol therefore aimed at reaching exhaustion after 30 min of exercise. Pilot tests showed that an intensity of 80% wattmax for arm and 70% wattmax for leg exercise was a suitable starting intensity. The exercise intensity was adjusted if needed to ensure exhaustion after 30 min of exercise, based on reports from the subject on exertion level. Whereas the two arm exercise trials increased the heart rate to 155 ⫾ 6 and 156 ⫾ 4 beats/min at the end of exercise, the heart rate reached 174 ⫾ 3 beats/min at 30 min of exercise in the leg cycling trial. Additional blood lactate samples and biopsies were taken after 10 and 30 min of exercise. The biopsies were taken from separate incisions and were rapidly frozen in liquid nitrogen and stored at ⫺80°C until analysis. Muscle fiber types. Fiber analyses were carried out with ATPase histochemistry, as previously described (4, 5). Tema (Scanbeam, Hadsund, Denmark) was used as the image program. Because of a lack of tissue, only samples from four to five subjects were analyzed.

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REGULATION OF PDH IN HUMAN ARM AND LEG MUSCLES

Table 1. Resting values of triceps, vastus lateralis, and deltoid

Muscle fiber, % Type I Type I/IIa Type IIa Type IIx Enzyme activity, mmol 䡠 min⫺1 䡠 kg dry wt⫺1 CS HAD LDH PFK PDH PDH protein content Absolute phosphorylation on site 1 Absolute phosphorylation on site 2 Relative phosphorylation on site 1 Relative phosphorylation on site 2 PDHa activity, mmol 䡠 min⫺1 䡠 kg wet wt⫺1 Absolute Relative

Triceps

Vastus

32⫾4.8 19⫾5.4 35⫾5.6 14⫾8.8

52⫾6.7* 16⫾5.6 20⫾4.3 12⫾5.5

Deltoid

54⫾6.9* 8⫾5.3 29⫾1.6 9⫾7.7

18.8⫾3.2 25.8⫾3.2 924⫾91 411⫾49

31.3⫾2.3*† 39.5⫾2.8*† 752⫾109 375⫾17

17.1⫾2.4 29.9⫾4.8* 1,023⫾132 370⫾13

1.2⫾0.2 0.7⫾0.2 0.6⫾0.2 0.6⫾0.1 0.5⫾0.1

1.8⫾0.1*† 1.5⫾0.2*† 1.3⫾0.2*† 0.9⫾0.1‡ 0.8⫾0.1*

1.2⫾0.1 0.8⫾0.0 0.6⫾0.0 0.7⫾0.1 0.6⫾0.1

0.82⫾0.19 0.62⫾0.18

0.90⫾0.15 0.53⫾0.09

1.0⫾0.16 0.91⫾0.12

mmol䡠min⫺1 䡠kg dry wt⫺1) (P ⱕ 0.05) (Table 1). The HAD activity was ⬇20% higher in deltoid (29.9 mmol䡠min⫺1 䡠kg dry wt⫺1) than in triceps (25.8 mmol 䡠min⫺1 䡠kg dry wt⫺1) (P ⱕ 0.05) and ⬇50 and ⬇30% higher in vastus lateralis (39.5 mmol䡠min⫺1 䡠kg dry wt⫺1) than in triceps and deltoid, respectively (P ⱕ 0.05) (Table 1). There were no differences in total LDH (752–1,023 mmol䡠min⫺1 䡠kg dry wt⫺1) or PFK (370 – 411 mmol䡠min⫺1 䡠 kg dry wt⫺1) activity among the three muscles (Table 1). Muscle glycogen. The glycogen concentration at rest was lower in deltoid (366 mmol/kg dry wt) than in triceps (457 mmol/kg dry wt) and vastus lateralis (454 mmol/kg dry wt) (Table 2). Net glycogen utilization during exercise was similar in vastus (205 mmol/kg dry wt) and triceps (269 mmol/kg dry wt) but lower (P ⱕ 0.05) in deltoid (90 mmol/kg dry wt). The average net glycogen utilization rate from onset of exercise to 10 min of exercise was 11.4 and 8.4 mmol 䡠kg dry wt⫺1 䡠min⫺1 in triceps and vastus lateralis, respectively, and for the last 20 min of exercise was 7.2 mmol䡠kg dry wt⫺1 䡠min⫺1 in both muscles.

Table 2. Muscle glycogen concentration and lactate accumulation in triceps, vastus lateralis, and deltoid

Glycogen, mmol/kg dry wt Rest 10 min of exercise 30 min of exercise Lactate, mmol/kg dry wt Rest 10 min of exercise 30 min of exercise

Triceps

Vastus

Deltoid

457⫾24 349⫾29† 188⫾23†‡

454⫾22 381⫾15† 249⫾28†‡*

366⫾18*§ 304⫾13 276⫾14

18.5⫾4.1 80.2⫾10.2† 90.3⫾10.5†

12.6⫾3.0 50.4⫾6.8†* 60.4⫾12.0†*

17.3⫾3.0 34.8⫾10.5 42.9⫾7.3

Values are means ⫾ SE; n ⫽ 7– 8. During exercise, statistical analysis was performed only on triceps and vastus. †Different from preexercise within same muscle, P ⱕ 0.05. ‡Different from 10 min of exercise within same muscle, P ⱕ 0.05. *Different from triceps, P ⱕ 0.05. §Different from vastus, P ⱕ 0.05. AJP-Endocrinol Metab • VOL

On the basis of these findings, it is concluded that both triceps and vastus lateralis were intensely activated during the full 30 min of exercise, and a direct comparison on PDH regulation during this exercise protocol is only reasonable for these two muscles, assuming that net muscle glycogen use is a reflection of muscle recruitment. Therefore, the exercise data below are only statistically analyzed for vastus lateralis and triceps. Muscle lactate. There was at rest no difference in the lactate concentration in triceps (18.5 mmol/kg dry wt), vastus (12.6 mmol/kg dry wt), and deltoid (17.3 mmol/kg dry wt) (Table 2). Exercise increased muscle lactate concentration after both 10 (⬇315% increase) and 30 min (⬇380% increase) of exercise compared with rest for triceps and vastus (P ⱕ 0.05) (Table 2). The level of muscle lactate was higher in triceps than in vastus lateralis after both 10 and 30 min of exercise (P ⱕ 0.05) (Table 2). PDH-E1␣ protein and phosphorylation state. PDH-E1␣ protein content was higher in vastus lateralis than in triceps and deltoid (P ⱕ 0.05) (Table 1). Phosphorylation of the PDH-E1␣ subunit at sites 1 and 2 was higher in vastus lateralis than in triceps and deltoid at rest (P ⱕ 0.05) (Table 1). Considering the relative phosphorylation (normalized to PDH-E1␣ content) of sites 1 and 2, there was no difference between vastus lateralis and deltoid, but a more marked phosphorylation of the two sites was still evident in vastus relative to triceps (P ⱕ 0.05) (Table 1). Exercise induced a pronounced dephosphorylation of sites 1 and 2 in both triceps and vastus lateralis (P ⱕ 0.05). After 10 and 30 min of exercise, phosphorylation of site 1 was 21 and 14% of the resting level in triceps and 29 and 24% in vastus lateralis, respectively (Fig. 1C). A more pronounced phosphorylation of the PDH-E1␣ subunit on site 1 was present in vastus lateralis compared with triceps during exercise (P ⱕ 0.05) (Fig. 1B). Relative to the level at rest, the phosphorylation level of site 2 was reduced (P ⱕ 0.05) in triceps to 6 and 4% and in vastus lateralis to 19 and 16% after 10 and 30 min of exercise,

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Values are means ⫾ SE. For the enzyme activity and the pyruvate dehydrogenase (PDH) data: n ⫽ 6 – 8. For muscle fiber analysis: triceps and vastus, n ⫽ 4; deltoid, n ⫽ 5. CS, citrate synthase; HAD, 3-hydroxyacyl-CoA dehydrogenase; LDH, lactate dehydrogenase; PFK, phosphofructokinase; PDHa, active form of PDH. *Different from triceps, P ⱕ 0.05. †Different from deltoid, P ⱕ 0.05. ‡Different from triceps with use of paired t-test, P ⱕ 0.05.

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respectively (Fig. 1D). No difference was apparent between the muscles in site 2 phosphorylation during exercise (Fig. 1D). When converting the absolute phosphorylation to the relative phosphorylation, no difference was evident between triceps and vastus lateralis during exercise. PDHa activity. There was no difference in the PDHa activity among the three muscles at rest (0.82–1.0 mmol䡠min⫺1 䡠kg wet wt⫺1) (Table 1). In triceps, an ⬇160% increase in PDHa activity was evident after 10 min of exercise compared with the activity at rest (Fig. 1A) (P ⱕ 0.05), with no significant difference between rest and 30 min of exercise (Fig. 1A). In vastus lateralis, the PDHa activity increased ⬇246 and ⬇170% after 10 and 30 min of exercise, respectively, relative to rest (Fig. 1A) (P ⱕ 0.05). After 10 min of exercise, the absolute PDHa activity was ⬇45% higher in vastus lateralis than in triceps (P ⱕ 0.05) (Fig. 1A), but after 30 min of exercise, there was no longer a difference between the two muscles (Fig. 1A). Normalizing the PDHa activity to PDH-E1␣ protein content resulted, however, in similar PDHa activity in vastus lateralis and triceps. AJP-Endocrinol Metab • VOL

The relationship between phosphorylation on sites 1 and 2 and the PDHa activity shows that a high PDHa activity is associated with an extremely low phosphorylation on both site 1 and site 2 (Fig. 2, A and B). Within the lower level of PDHa activity, there was, however, a large range in phosphorylation and, therefore, not the same strong relation between activity and phosphorylation. Analyzing the data for phosphorylation and activity using a monoexponential model gives, for triceps, r2 ⫽ 0.23 and r2 ⫽ 0.34 for sites 1 and 2 and, for vastus, r2 ⫽ 0.54 and r2 ⫽ 0.67 for sites 1 and 2, respectively. DISCUSSION

The main findings of the present study are that the PDH content follows the metabolic profile of the muscle rather than the MHC fiber type distribution, and that a smaller exercise-induced increase in PDHa activity in triceps than in vastus lateralis can be explained by a lower content of PDH in triceps rather than differences in activation of each PDH molecule. In addition, the lower PDHa activity in triceps

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Fig. 1. Effect of 30 min of arm [80% maximum power (wattmax)] or leg exercise (70% wattmax) in triceps, vastus lateralis, and deltoid. A: absolute PDHa activity (where PDHa is the active form of pyruvate dehydrogenase). B: detection by Western blot phosphorylation (phos). C and D: absolute phosphorylation of PDH-E1␣ site 1 (C) and PDH-E1␣ site 2 (D). Muscle samples were taken before exercise (pre) and after 10 (10’) and 30 min (30’) of exercise. Values are means ⫾ SE. For triceps, n ⫽ 6 – 8. For vastus lateralis and deltoid, n ⫽ 7– 8. Due to less recruitment of deltoid, statistical comparison during exercise is done only for triceps and vastus lateralis. *Different from triceps, P ⱕ 0.05. †Different from preexercise within same muscle, P ⱕ 0.05. §Tendency to differ from triceps, P ⫽ 0.085.

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during exercise was associated with higher lactate accumulation in this muscle. Because endurance training has been shown to increase oxidative capacity (2) as well as PDH-E1␣ protein content (15), it would be expected that the most endurance trained muscle had the largest amount of PDH-E1␣ protein. In accordance, PDH-E1␣ protein content and CS and HAD activity were higher in vastus lateralis than in the two arm muscles, and the linear correlation between PDH-E1␣ protein and CS (r2 ⫽ 0.70) and HAD (r2 ⫽ 0.49) activity further underlines that the PDH capacity follows the oxidative potential of the muscle. In contrast, the PDH-E1␣ protein content did not follow the MHC fiber type composition, as vastus lateralis and deltoid had similar MHC fiber type distribution, but vastus lateralis had higher PDH-E1␣ protein content than deltoid. A similar indication for divergence between metabolic characteristics and MHC fiber type has previously been reported for gene expression profile and MHC fiber type distribution in human skeletal muscle (25). The present findings of lower PDHa activity and higher lactate accumulation in triceps than in vastus lateralis are in line with the possibility that limited PDHa activity can be at least a contributing factor for the larger lactate accumulation and release from arm than leg muscles during exercise (1). Moreover, the observation that the PDHa activity normalized to AJP-Endocrinol Metab • VOL

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Fig. 2. Relationship between PDHa activity and PDH-E1␣ site 1 phosphorylation (A) and PDH-E1␣ site 2 phosphorylation (B) in deltoid (squares), triceps (triangles), and vastus lateralis (circles). Exponential regression: site 1, r2 ⫽ 0.31, 0.23, and 0.54; site 2, r2 ⫽ 0.45, 0.34, and 0.67.

the PDH-E1␣ protein content was the same in triceps and vastus lateralis at each of the three time points indicates that each PDH molecule is activated to the same degree in triceps and vastus lateralis, meaning that the higher absolute PDHa activity and likely flux through PDH in vastus lateralis was due to greater PDH capacity in vastus lateralis than in triceps rather than differences at the molecule level. Thus it is suggested that, because of a lower capacity of PDH, the flux through the PDH complex is lower in triceps, resulting in the conversion of pyruvate to lactate. However, it may be noted that triceps showed a more marked dephosphorylation of site 1 than vastus lateralis during exercise, indicating that there may indeed be some differences in regulation of the phosphorylation state of PDH in triceps and vastus lateralis. The present finding of more pronounced phosphorylation of sites 1 and 2 in vastus lateralis than in triceps at rest, as an absolute value as well as when normalized to PDH protein content, would be expected to cause a lower PDHa activity in vastus lateralis than in triceps. The fact that no differences in the PDHa activity were observed among the three muscles at rest indicates that the PDHa activity is not regulated only by these two phosphorylation sites, which is supported by previous findings (22) where intralipid infusion resulted in an increased phosphorylation of PDH without an effect on the PDHa activity. Because regulation of PDHa activity at site 3 usually is considered negligible in human skeletal muscle (20), these findings may indicate that other covalent regulations of PDH exist. While a discrepancy seems to exist between PDHa activity and the degree of phosphorylation at rest, a stronger correlation exists at higher PDHa activities, but still the decline in PDHa activity from 10 to 30 min of exercise was not associated with a stronger phosphorylation of sites 1 and 2. Thus the covalent regulation of PDHa activity in skeletal muscle during exercise also seems to be more complex and, perhaps, is not only explained by phosphorylation of sites 1 and 2. The exercise-induced activation of PDHa activity corresponds well with previous human studies on vastus lateralis (18, 22, 26, 31, 32). In these studies, the intensity has in general been moderate [45–55% maximal oxygen consumption ˙ O2 max) or wattmax], and only one study examined PDHa (V activity during intense exercise (6), where subjects performed ˙ O2 max. graded 10-min sessions of exercise at 60 and 90% V Previous studies (32, 33) have reported a PDHa activity of ⬇2.8 mmol䡠min⫺1 䡠kg wet wt⫺1 after 10 min of exercise at ˙ O2 max, corresponding to a 3-fold increase compared 55% V with rest, and an ⬇3.5-fold increase in PDHa activity was ˙ O2 max (30). This is found after 5 min of exercise at 55% V rather similar to the level obtained in the present study, where the PDHa activity in vastus lateralis was ⬇3.1 mmol䡠min⫺1 䡠kg ˙ max, correwet wt⫺1 after 10 min of exercise at 70 – 80% W sponding to an ⬇3.5-fold increase relative to rest. Thus the PDHa activity and the relative increase in PDHa activity do not seem to be higher in the present study with more intense exercise than in protocols with low and intermediate exercise intensities. This observation can probably be explained by an upper limit of total PDH activity, which has been reported to be ⬇3.6 mmol 䡠min⫺1 䡠kg wet wt⫺1 (17). This number is, however, sensitive to aerobic exercise training, as 8 wk of aerobic exercise was found to increase total PDH activity from ⬇3.8 to ⬇4.9 mmol 䡠min⫺1 䡠kg wet wt⫺1 (15). It may therefore be

REGULATION OF PDH IN HUMAN ARM AND LEG MUSCLES

ACKNOWLEDGMENTS We thank the subjects for participating in the study. We acknowledge the excellent technical assistance of Carsten Nielsen and Ditte Kjærsgaard Klein and the excellent guidance of Ylva Hellsten for fiber type analysis. We thank D. Grahame Hardie, Dundee, University, Dundee, Scotland, UK, for the kind donation of valuable tools for this study. GRANTS The study was supported by grants from The Ministry of Culture Committee on Sports Research and Danish Medical Research Council, the Danish Diabetes Association, the Novo Nordisk Foundation, the Copenhagen Muscle Research Centre, and an Integrated Project from the European Union (contract LSHM-CT-2004-005272). J. F. P. Wojtaszewski was supported by a Hallas Møller Fellowship from the Novo Nordisk Foundation. The Centre of Inflammation and Metabolism is supported by the Danish National Research Foundation (grant no. 02-512-555); The Copenhagen Muscle Research Centre is supported by grants from The University of Copenhagen and Rigshospitalet, Copenhagen, Denmark. AJP-Endocrinol Metab • VOL

REFERENCES 1. Ahlborg G, Jensen-Urstad M. Metabolism in exercising arm vs. leg muscle. Clin Physiol 11: 459 – 468, 1991. 2. Baldwin KM, Klinkerfuss GH, Terjung RL, Mole PA, Holloszy JO. Respiratory capacity of white, red, and intermediate muscle: adaptative response to exercise. Am J Physiol 222: 373–378, 1972. 3. Bergstro¨m J. Muscle electrolytes in man determined by neutron activation analysis on needle biopsy speciments. A study on normal subjects, kidney patients, and paitients with chronic diarrhea. Scand J Clin Lab Invest 68: 1–110, 1962. 4. Brooke MH, Kaiser KK. Muscle fiber types: how many and what kind? Arch Neurol 23: 369 –379, 1970. 5. Brooke MH, Kaiser KK. Three “myosin adenosine triphosphatase” systems: the nature of their pH lability and sulfhydryl dependence. J Histochem Cytochem 18: 670 – 672, 1970. 6. Burgomaster KA, Heigenhauser GJ, Gibala MJ. Effect of short-term sprint interval training on human skeletal muscle carbohydrate metabolism during exercise and time-trial performance. J Appl Physiol 100: 2041–2047, 2006. 7. Bylund AC, Bjuro T, Cederblad G, Holm J, Lundholm K, Sjostroom M, Angquist KA, Schersten T. Physical training in man. Skeletal muscle metabolism in relation to muscle morphology and running ability. Eur J Appl Physiol Occup Physiol 36: 151–169, 1977. 8. Calbet JA, Holmberg HC, Rosdahl H, van Hall G, Jensen-Urstad M, Saltin B. Why do arms extract less oxygen than legs during exercise? Am J Physiol Regul Integr Comp Physiol 289: R1448 –R1458, 2005. 9. Cederblad G, Carlin JI, Constantin-Teodosiu D, Harper P, Hultman E. Radioisotopic assays of CoASH and carnitine and their acetylated forms in human skeletal muscle. Anal Biochem 185: 274 –278, 1990. 10. Constantin-Teodosiu D, Cederblad G, Hultman E. A sensitive radioisotopic assay of pyruvate dehydrogenase complex in human muscle tissue. Anal Biochem 198: 347–351, 1991. 11. Essen-Gustavsson B, Henriksson J. Enzyme levels in pools of microdissected human muscle fibres of identified type. Adaptive response to exercise. Acta Physiol Scand 120: 505–515, 1984. 12. Gladden LB. Lactate metabolism: a new paradigm for the third millennium. J Physiol 558: 5–30, 2004. 13. Henriksson J, Reitman JS. Time course of changes in human skeletal muscle succinate dehydrogenase and cytochrome oxidase activities and maximal oxygen uptake with physical activity and inactivity. Acta Physiol Scand 99: 91–97, 1977. 14. Kushmerick MJ, Moerland TS, Wiseman RW. Mammalian skeletal muscle fibers distinguished by contents of phosphocreatine, ATP, and Pi. Proc Natl Acad Sci USA 89: 7521–7525, 1992. 15. LeBlanc PJ, Peters SJ, Tunstall RJ, Cameron-Smith D, Heigenhauser GJ. Effects of aerobic training on pyruvate dehydrogenase and pyruvate dehydrogenase kinase in human skeletal muscle. J Physiol 557: 559 –570, 2004. 16. Lowry OH, Passonneau JV. A Flexible System of Enzymatic Analysis. New York: Academic, 1972. 17. Mandarino LJ, Wright KS, Verity LS, Nichols J, Bell JM, Kolterman OG, Beck-Nielsen H. Effects of insulin infusion on human skeletal muscle pyruvate dehydrogenase, phosphofructokinase, and glycogen synthase. Evidence for their role in oxidative and nonoxidative glucose metabolism. J Clin Invest 80: 655– 663, 1987. 18. Mourtzakis M, Saltin B, Graham T, Pilegaard H. Carbohydrate metabolism during prolonged exercise and recovery: interactions between pyruvate dehydrogenase, fatty acids, and amino acids. J Appl Physiol 100: 1822–1830, 2006. 19. Olsen DB, Sacchetti M, Dela F, Ploug T, Saltin B. Glucose clearance is higher in arm than leg muscle in type 2 diabetes. J Physiol 565: 555–562, 2005. 20. Patel MS, Korotchkina LG. Regulation of mammalian pyruvate dehydrogenase complex by phosphorylation: complexity of multiple phosphorylation sites and kinases. Exp Mol Med 33: 191–197, 2001. 21. Pettit FH, Pelley JW, Reed LJ. Regulation of pyruvate dehydrogenase kinase and phosphatase by acetyl-CoA/CoA and NADH/NAD ratios. Biochem Biophys Res Commun 65: 575–582, 1975. 22. Pilegaard H, Birk JB, Sacchetti M, Mourtzakis M, Hardie DG, Stewart G, Neufer PD, Saltin B, van Hall G, Wojtaszewski JF. PDH-E1alpha dephosphorylation and activation in human skeletal muscle during exercise: effect of intralipid infusion. Diabetes 55: 3020 –3027, 2006.

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speculated that the normally physically active subjects in the present study were close to the maximal PDHa activity after 10 min of exercise. Furthermore, no study has reported PDHa activities reaching total PDH activity during exercise. Studies with low-intensity exercise suggest that a high PDHa activity found after 10 min of exercise (32) is sustained during the initial 2 h of exercise, followed by a decline toward resting level as exercise duration proceeds beyond 2 h (18, 22). In the present study with high-intensity exercise, the PDHa activity was highest after 10 min of exercise, and for triceps, the PDHa activity returned to resting level after 30 min of exercise. Thus it seems that the exercise-induced upregulation of PDHa activity is rather independent of exercise intensity, while the intensity is important for the timing of the downregulation of PDHa activity in both types of muscles investigated. The reduction in PDHa activity may be related to decreasing glycogen levels in the exercising muscle, and, as high-intensity exercise consumes a great amount of muscle glycogen, this may contribute to the early downregulation of PDHa activity observed in both vastus lateralis and triceps at 30 min of exercise in the present study. As explained, the deltoid muscle was not included in the comparison of exercise-induced PDH regulation between muscles because of less marked net glycogen usage in deltoid and thus, most likely, less recruitment of deltoid. Interestingly, however, the smaller net glycogen utilization in deltoid was associated with less marked changes in PDHa activity and PDH-E1␣ phosphorylation in response to arm cycling, indicating that local factors rather than systemic factors are critical in regulating PDH during this type of exercise. Whether the level of muscle glycogen itself could be a determining factor is presently not known. In conclusion, the present findings show that, in humans, PDH protein content follows the metabolic profile of the muscle rather than MHC fiber type distribution. While PDH protein content and exercise-induced PDH activation are higher in vastus lateralis than in triceps, the PDH molecule seems to be regulated similarly in the two muscles. This suggests that a lower PDH capacity in triceps, rather than less PDH activation of the PDH molecule, is likely a contributing factor to differential carbohydrate metabolism in triceps vs. vastus lateralis.

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23. Pilegaard H, Ordway GA, Saltin B, Neufer PD. Transcriptional regulation of gene expression in human skeletal muscle during recovery from exercise. Am J Physiol Endocrinol Metab 279: E806 –E814, 2000. 24. Pilegaard H, Saltin B, Neufer PD. Effect of short-term fasting and refeeding on transcriptional regulation of metabolic genes in human skeletal muscle. Diabetes 52: 657– 662, 2003. 25. Plomgaard P, Penkowa M, Leick L, Pedersen BK, Saltin B, Pilegaard H. The mRNA expression profile of metabolic genes relative to MHC isoform pattern in human skeletal muscles. J Appl Physiol 101: 817– 825, 2006. 26. Putman CT, Spriet LL, Hultman E, Lindinger MI, Lands LC, McKelvie RS, Cederblad G, Jones NL, Heigenhauser GJ. Pyruvate dehydrogenase activity and acetyl group accumulation during exercise after different diets. Am J Physiol Endocrinol Metab 265: E752–E760, 1993. 27. Sacchetti M, Olsen DB, Saltin B, van Hall G. Heterogeneity in limb fatty acid kinetics in type 2 diabetes. Diabetologia 48: 938 –945, 2005. 28. Saltin B, Gollnick PD. Skeletal muscle adaptability: significance for metabolism and performance. In: Handbook of Physiology. Skeletal Muscle. Bethesda, MD: Am. Physiol. Soc. 1983, sect. 10, chapt. 19, p. 555– 631.

29. Sjodin B, Thorstensson A, Frith K, Karlsson J. Effect of physical training on LDH activity and LDH isozyme pattern in human skeletal muscle. Acta Physiol Scand 97: 150 –157, 1976. 30. Stellingwerff T, Watt MJ, Heigenhauser GJ, Spriet LL. Effects of reduced free fatty acid availability on skeletal muscle PDH activation during aerobic exercise. Am J Physiol Endocrinol Metab 284: E589 – E596, 2003. 31. Ward GR, Sutton JR, Jones NL, Toews CJ. Activation by exercise of human skeletal muscle pyruvate dehydrogenase in vivo. Clin Sci (Lond) 63: 87–92, 1982. 32. Watt MJ, Heigenhauser GJ, Dyck DJ, Spriet LL. Intramuscular triacylglycerol, glycogen and acetyl group metabolism during 4 h of moderate exercise in man. J Physiol 541: 969 –978, 2002. 33. Watt MJ, Heigenhauser GJ, LeBlanc PJ, Inglis JG, Spriet LL, Peters SJ. Rapid upregulation of pyruvate dehydrogenase kinase activity in human skeletal muscle during prolonged exercise. J Appl Physiol 97: 1261–1267, 2004. 34. World Health Organization. Energy and Protein Requirements. Geneva: WHO, 1985.

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STUDY 2

ORIGINAL ARTICLE

Low Muscle Glycogen and Elevated Plasma Free Fatty Acid Modify but Do Not Prevent Exercise-Induced PDH Activation in Human Skeletal Muscle Kristian Kiilerich,1,2,3 Mikkel Gudmundsson,1,2,3 Jesper B. Birk,1,4 Carsten Lundby,1,5 Sarah Taudorf,1,2,6 Peter Plomgaard,1,2,6 Bengt Saltin,1,5 Per A. Pedersen,3 Jorgen F.P. Wojtaszewski,1,4 and Henriette Pilegaard1,2,3

OBJECTIVE—To test the hypothesis that free fatty acid (FFA) and muscle glycogen modify exercise-induced regulation of PDH (pyruvate dehydrogenase) in human skeletal muscle through regulation of PDK4 expression. RESEARCH DESIGN AND METHODS—On two occasions, healthy male subjects lowered (by exercise) muscle glycogen in one leg (LOW) relative to the contra-lateral leg (CON) the day before the experimental day. On the experimental days, plasma FFA was ensured normal or remained elevated by consuming breakfast rich (low FFA) or poor (high FFA) in carbohydrate, 2 h before performing 20 min of two-legged knee extensor exercise. Vastus lateralis biopsies were obtained before and after exercise. RESULTS—PDK4 protein content was ⬃2.2- and ⬃1.5-fold higher in LOW than CON leg in high FFA and low FFA, respectively, and the PDK4 protein content in the CON leg was approximately twofold higher in high FFA than in low FFA. In all conditions, exercise increased PDHa (PDH in the active form) activity, resulting in similar levels in LOW leg in both trials and CON leg in high FFA, but higher level in CON leg in low FFA. PDHa activity was closely associated with the PDH-E1␣ phosphorylation level. CONCLUSIONS—Muscle glycogen and plasma FFA attenuate exercise-induced PDH regulation in human skeletal muscle in a nonadditive manner. This might be through regulation of PDK4 expression. The activation of PDH by exercise independent of changes in muscle glycogen or plasma FFA suggests that exercise overrules FFA-mediated inhibition of PDH (i.e., carbohydrate oxidation), and this may thus be one mechanism behind the health-promoting effects of exercise. Diabetes 59:26–32, 2010

From the 1Copenhagen Muscle Research Centre, University of Copenhagen, Copenhagen, Denmark; the 2Centre of Inflammation and Metabolism, University of Copenhagen, Copenhagen, Denmark; the 3Department of Biology, University of Copenhagen, Copenhagen, Denmark; the 4Section of Human Physiology, Department of Exercise and Sport Sciences, University of Copenhagen, Copenhagen, Denmark; 5Rigshospitalet, Section 7652, Copenhagen, Denmark; and 6Rigshospitalet, Section 7641, Copenhagen, Denmark. Corresponding author: Kristian Kiilerich, [email protected]. Received 15 July 2009 and accepted 2 October 2009. Published ahead of print at http://diabetes.diabetesjournals.org on 15 October 2009. DOI: 10.2337/db09-1032. © 2010 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by -nc-nd/3.0/ for details. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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I

nsulin resistance has been suggested to be associated with dysregulation of the pyruvate dehydrogenase complex in skeletal muscle, but the underlying mechanism remains unclear (1–3). However, it has been suggested that elevated plasma free fatty acid (FFA) concentrations is the initial triggering event leading to downregulation of PDH (pyruvate dehydrogenase) activity and thus potentially contributing to insulin resistance (1). The pyruvate dehydrogenase complex occupies a central role in carbohydrate metabolism, catalyzing the first irreversible step in mitochondrial glucose metabolism, and hence determines the fate of carbohydrates in skeletal muscle metabolism (4). Regulation of PDH activity in human skeletal muscle is believed mainly to be mediated through changes in the phosphorylation state of site one (Ser293) and two (Ser300) on the PDH-E1␣ subunit, where dephosphorylation activates (5). The known regulatory kinases and phosphatases include four isoforms of PDH kinase (PDK1– 4) and two PDH phosphatases (PDP1–2) (6 – 8). Of these, PDK2, PDK4, and PDP1 are thought to be the important isoforms in skeletal muscle (4,8,9). Skeletal muscle PDH activity is affected by fasting, high-fat diet, and exercise (4,10 –13). Factors responsible for this regulation have been suggested to include changes in plasma FFA concentration (1,12,14), muscle glycogen content (15–17), plasma insulin levels (14,18 –20), and intracellular Ca2⫹ concentration (21,22). Thus, regulation of PDH seems to be under both local and systemic control (17). Insulin activates PDH at least in part through downregulation of PDK4 expression as shown at the protein level in rat skeletal muscle (14). Also based on findings in rat skeletal muscle, plasma FFA is also believed to reduce PDH activity through a peroxisome proliferator–activated receptor (PPAR)-␣–mediated upregulation of PDK4 protein (14). Similarly, manipulation of the muscle glycogen content in humans has indicated that lowering of muscle glycogen upregulates PDK4 at the transcriptional and mRNA level (15,16). As previously suggested, such a glycogen-dependent regulation of gene expression may take place through glycogen regulatory enzymes such as protein phosphatase 1 (PP1) and glycogen synthase kinase 3 (GSK3), which are bound to the glycogen scaffold, but released when the glycogen content decreases (15). However, in these studies, plasma FFA and muscle glycogen were manipulated simultaneously, making it impossible to discriminate between the role of muscle glycogen and FFA. Therefore, the aim of the present study was to test the diabetes.diabetesjournals.org

K. KIILERICH AND ASSOCIATES

Blood sampling

Muscle biopsies from both legs

75% Wattmax -15h

-2h

0

One leg glycogen depletion

Breakfast • high fat = high FFA trial • low fat = low FFA trial

Pre

65% Wattmax 10 min

20 min Post ex

FIG. 1. A schematic overview of the experimental setup. Each subject completed the experiment on two separate days, with the only difference being the breakfast consumed. Therefore, each subject completed both the high FFA and low FFA trial. Exercise (ex) was performed as two-legged knee extensor exercise.

hypothesis that both low muscle glycogen and elevated FFA modify exercise-induced PDH regulation in human skeletal muscle independent of each other, potentially through regulation of PDK4 expression. RESEARCH DESIGN AND METHODS Eight healthy normally physical active male subjects with an average age of 26.5 years (range 22–31), weight 80.6 kg (60.4 –99.8), and stature of 184.6 cm (175–193) participated in the study. The average peak oxygen uptake of the subjects was 51.8 ml O2 䡠 min⫺1 䡠 kg⫺1 (47.9 –55). The subjects were given both written and oral information about the experimental protocol and procedures and were informed about any discomfort that might be associated with the experiment before they gave their written consent. The study was performed according to the Declaration of Helsinki and was approved by the Copenhagen and Frederiksberg Ethics Committee, Denmark (H-C-2007-0085). Experimental protocol. Approximately 2 weeks before the first trial, peak oxygen uptake and Wattmax of the subjects were determined by an incremental bicycle test. Furthermore, Wattmax during two-legged knee extensor exercise was determined by an incremental test, with a starting resistance of 48 – 60 W and increasing the load by 12 W every 2 min. The maximal resistance that could be sustained for 2 min was set as Wattmax. Each subject completed two experimental trials, which consisted of identical exercise protocols, but differed in the dietary protocol. The subjects were instructed to eat food rich in carbohydrates 5 days before a glycogen depletion protocol. Before reporting at the laboratory on the day of glycogen depletion, the subjects consumed a prepackaged standardized meal regulated to body weight and activity level (23), with 77% energy (%E) carbohydrate, 10E% protein, and 13%E fat. The day before each experimental trial, the subjects arrived at the laboratory between 4:00 and 6:00 P.M. To reduce muscle glycogen in one leg, the subjects performed a one-legged cycling exercise protocol, consisting of 20 min continuous cycling (10 min 65% Wattmax and 10 min 55% Wattmax) followed by intermittent one-legged cycling as previously described (15). The depletion leg was randomly selected. To lower glycogen stores in the liver, and thus to minimize glycogen resynthesis, the subjects furthermore performed 30 min of arm cycling. After the glycogen depletion, they were given a dinner low in carbohydrates (1%E carbohydrate, 26%E protein, and 73%E fat) to prevent muscle glycogen resynthesis. On the experimental day (Fig. 1), the subjects arrived at the laboratory in the morning 2 h after intake of a prepacked breakfast either high in fat (high FFA) (3%E carbohydrate, 18%E protein, and 79%E fat) or high in carbohydrate (low FFA) (74%E carbohydrate, 12E% protein, and 14%E fat). This breakfast was the only difference between the trials and aimed at obtaining similar insulin levels, whereas FFA levels were different in the two trials. The two trials were separated by at least 10 days and were performed in random order. A venous catheter was inserted in either v. cephalica or v. mediana cubiti, and a resting blood sample was taken. Furthermore, two incisions were made in the middle part of vastus lateralis of each leg under local anesthesia (lidocaine), and a resting biopsy was obtained from the glycogen depleted leg (LOW) and the nonexercised leg (CON) using the diabetes.diabetesjournals.org

percutaneous needle biopsy technique (24), with suction. Thereafter, the subjects performed a two-legged knee extensor exercise bout at 75% Wattmax for 10 min followed by 10 min at 65% Wattmax. Immediately at the end of the 20-min exercise period, a muscle biopsy was obtained simultaneously from each leg through the prior made new incisions. Additional blood samples were taken after 10 and 20 min of exercise. The work that each leg performed was evaluated using strain gauge. The LOW and CON leg did an equal amount of work in both trials. Blood parameters. Plasma FFA was measured with a Wako FA kit (Wako Chemical, Neuss, Germany) and an automatic spectrophotometer (Cobas FARA 2; Roche Diagnostic, Basel, Switzerland). Plasma insulin was measured with an insulin enzyme-linked immunosorbent assay (ELISA) kit (DakoCytomation, Glostrup, Denmark). Muscle glycogen. Muscle specimens were freeze-dried and dissected free of blood, fat, and connective tissue under the microscope, and muscle glycogen content was determined as glycosyl units after acid hydrolysis (25) using an automatic spectrophotometer (Cobas FARA 2, Roche Diagnostic, Switzerland). Muscle lysate. Muscles pieces were homogenized in an ice-cold buffer (10% glycerol, 20 mmol/l Na-pyrophosphate, 150 mmol/l NaCl, 50 mmol/l HEPES, 1% NP-40, 20 mmol/l ␤-glycerophosphate, 10 mmol/l NaF, 1 mmol/l EDTA, 1 mmol/l EGTA, 2 mmol/l phenylmethylsulfonyl fluoride, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, 2 mmol/l Na3VO4, 3 mmol/l benzamidine, pH 7.5) for 20 s using a polytron (PT 1200; Kinematica AG, Switzerland). Homogenates were rotated end over end for 1 h at 4°C. Lysates were generated by centrifugation (17,500g) for 20 min at 4°C. Protein content in lysates was measured by the bicinchoninic acid method (Pierce, Rockford, IL). SDS-PAGE and Western blotting. PDH-E1␣ and PDK4 protein expression and phosphorylation of PDH-E1␣ site 1 and 2 were measured in muscle samples by SDS-PAGE (Tris-HCl 10% gel, Bio-Rad, Denmark) and Western blotting using PVDF membrane and semi-dry transfer. After the transfer, the PVDF membrane was blocked overnight at 4°C (Tris-buffered saline with Tween [TBST] ⫹ 2% skim milk). The following day, the membrane was incubated with primary antibody (in TBST ⫹ 2% skim milk) for 2 h at room temperature and thereafter washed in TBST and incubated with horseradish peroxidase– conjugated secondary antibody (Dako, Denmark) for 1 h at room temperature (TBST ⫹ 2% skim milk). Immobilon Western (Millipore, Billerica, MA) was used as a detection system. Bands were visualized using an Eastman Kodak Image Station 2000MM. Bands were quantified using Kodak Molecular Imaging Software version 4.0.3, and protein content was expressed in units relative to control samples loaded on each gel. Protein levels of the PDH-E1␣ subunit and phosphorylation of site 1 and 2 of PDH-E1␣ were determined using antibodies generated in sheep as previously described (12) and PDK4 protein by in-house–made antibodies generated in rabbit (26). PDHa activity. The activity of PDHa (PDH in the active form) was determined as previously described (27–29) after homogenizing ⬃10 mg muscle tissue for 50 s in a glass homogenizer (Kontes) and quickly (10 –15 s) freezing the samples in liquid nitrogen. The PDHa activity was adjusted to total creatine in each muscle sample. Statistical analysis. Values presented are means ⫾ SE. Two-way ANOVA for repeated measures was applied to evaluate the effect of exercise and trial (low DIABETES, VOL. 59, JANUARY 2010

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GLYCOGEN AND FFA IN PDH REGULATION

TABLE 1 FFA and insulin before exercise, 10 min into exercise, and immediately after 20 min of two-legged knee extensor exercise

714 ⫾ 142* 497 ⫾ 61*† 564 ⫾ 70*

193 ⫾ 30 188 ⫾ 24 223 ⫾ 29

24 ⫾ 3 13 ⫾ 1 35 ⫾ 11

39 ⫾ 9 19 ⫾ 3 25 ⫾ 5

Data are means ⫾ SE. The experimental protocol was performed on two occasions with either fat-rich (high FFA) or carbohydrate-rich (low FFA) breakfast 2 h prior to the first blood samples. *Significant difference between the trials, P ⱕ 0.05. †Significantly different from pre-exercise, P ⱕ 0.05. FFA vs. high FFA) as well as the effect of exercise and leg (CON versus LOW). The Student-Newman-Keuls post hoc test was used to locate differences. Differences were considered significant at P ⱕ 0.05. Statistical calculations were performed using SigmaStat Version 2.03.

RESULTS

Plasma FFA and insulin. The plasma FFA concentration was in the high FFA trial ⬃3.7-fold higher (P ⱕ 0.05) at rest and ⬃2.5-fold higher (P ⱕ 0.05) during exercise than in the low FFA trial. In the high FFA trial, the plasma FFA concentration was at 10 min of exercise reduced (P ⱕ 0.05) relative to pre-exercise. There was no difference in plasma insulin levels between the trials or over time (Table 1). Muscle glycogen. Within each trial, muscle glycogen concentration was in the LOW leg ⬃46 and ⬃37% of the level in the CON leg before and after exercise, respectively (P ⱕ 0.05). Exercise lowered (P ⱕ 0.05) muscle glycogen in both legs in both trials (Fig. 2). Muscle lactate. The muscle lactate concentration was similar in CON and LOW leg before exercise in both trials (Table 2). Muscle lactate concentration was 2.5-fold higher (P ⱕ 0.05) after exercise than before exercise in the low FFA trial. Muscle lactate concentration was after exercise in low FFA trial 2.4-fold higher (P ⱕ 0.05) in CON leg than in LOW leg. In addition the muscle lactate concentration after exercise in CON leg was 1.1 fold higher (P ⱕ 0.05) in high FFA trial then low FFA trial. Muscle glucose-6-phosphate. The muscle glucose-6phosphate concentration was similar in LOW and CON leg before exercise in both trials. No changes were observed over time in muscle glucose-6-phosphate concentration in the LOW leg in either trial, whereas the glucose-6-phosphate concentration in the CON leg was increased (P ⱕ 0.05) ⬃2.5-fold after exercise compared with before exercise in both trials. The concentration of muscle glucose-6phosohate was ⬃2.6-fold higher (P ⱕ 0.05) in the CON leg than LOW leg after exercise in the low FFA trial (Table 2). Muscle glucose. The muscle glucose concentration was similar in the LOW and CON leg before exercise in both trials. No changes were observed over time in muscle glucose concentration in the LOW leg in either trial, whereas an approximately threefold increase (P ⱕ 0.05) was observed after exercise in the CON leg relative to before exercise in both trials. The muscle glucose concentration was ⬃2.6-fold higher (P ⱕ 0.05) in the CON leg than in the LOW leg in both trials (Table 2). 28

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500

†

-1

Low FFA

Muscle glycogen mmol · kg dry wt

Plasma FFA (␮mol/l) Pre-exercise 10 min exercise Post-exercise Plasma insulin (pmol/l) Pre-exercise 10 min exercise Post-exercise

High FFA

CON LOW

600

400

† 300

* * † †

200

*

* 100

0 Pre

Post Ex

high FFA

Pre

Post Ex

low FFA

FIG. 2. Content of muscle glycogen in vastus lateralis before and after 20 min of two-legged knee extensor exercise (Ex). At the initiation of this exercise, muscle glycogen was reduced in one leg (LOW leg) (䡺) by one-legged exercise the day before (14 h prior) and high-fat diet overnight, whereas the other leg (CON leg) (f) had normal glycogen levels. The experimental protocol was performed on two occasions with either fat-rich (high FFA) or carbohydrate-rich (low FFA) breakfast 2 h before the first biopsies. Values are means ⴞ SE. *Significantly different from CON at given time point and within given trial, P < 0.05. †Significantly different from Pre within given leg and trial.

PDHa activity. Before exercise, PDHa activity was similar in the two legs in both trials. Exercise increased (P ⱕ 0.05) the PDHa activity in both trials and legs. In the high FFA trial, the increase in PDHa activity with exercise was similar in the CON leg (3.8-fold) and LOW leg (3.7-fold; Fig. 4A). In the low FFA trial, exercise increased (P ⱕ 0.05) PDHa activity 5.7-fold in the CON leg and 5.1-fold in the LOW leg, resulting in higher (P ⱕ 0.05) PDHa activity level in the CON than LOW leg after exercise. TABLE 2 Muscle lactate, glucose-6-phosphate, and muscle glucose concentrations (in mmol/kg dry wt) in vastus lateralis muscle before and immediately after 20 min of two-legged knee extensor exercise High FFA CON LOW Muscle lactate Pre-exercise Post-exercise Glucose-6phosphate Pre-exercise Post-exercise Muscle glucose Pre-exercise Post-exercise

Low FFA CON LOW

15 ⫾ 2 35 ⫾ 10‡

13 ⫾ 2 24 ⫾ 5

16 ⫾ 2 40 ⫾ 10†

13 ⫾ 2 17 ⫾ 2*

1.0 ⫾ 0.2 2.3 ⫾ 0.2†

0.8 ⫾ 0.2 1.1 ⫾ 0.2

0.8 ⫾ 0.1 2.3 ⫾ 0.5†

0.7 ⫾ 0.2 0.9 ⫾ 0.2*

1.8 ⫾ 0.3 5.3 ⫾ 1.2†

1.8 ⫾ 0.3 2.1 ⫾ 0.3*

1.7 ⫾ 0.3 5.1 ⫾ 1.0†

1.8 ⫾ 0.3 1.9 ⫾ 0.4*

Data are means ⫾ SE. The LOW leg had reduced muscle glycogen due to one-legged exercise the day before (14 h prior) and high-fat diet overnight, whereas the prior nonexercised CON leg had normal glycogen levels. The experimental protocol was performed on two occasions with either fat-rich (high FFA) or carbohydrate-rich (low FFA) breakfast 2 h before the first biopsies. *Significant difference between CON and LOW, P ⱕ 0.05. †Significantly different from pre-exercise, P ⱕ 0.05. ‡Significant difference between trials, P ⱕ0.05. diabetes.diabetesjournals.org

K. KIILERICH AND ASSOCIATES

FIG. 3. Representative Western blots for PDK4 protein and for the phosphorylation of PDH-P1 and PDH-P2 shown for the samples of one subject. Ex, exercise.

B

6

PDHa activity -1 -1 (mmol · min · kg wet wt)

4

†

*

† 3

2

*

*

‡

†

0.6

*

† 0.4

0.2

0.0

0 Pre

Post Ex

high FFA

Pre

Post Ex

Pre

low FFA

D

1.6 1.4

*

1.2 1.0 0.8 0.6

†

†

*

*

†

0.4

Post Ex

high FFA

†

0.2

PDH-E1α site 2 phosphorylation AU

PDH-E1α α site 1 phosphorylation AU

*

0.8

‡ †

1

C

1.0

†

5

PDK4 protein AU

A

Furthermore, the PDHa activity in the CON leg after exercise was 1.3-fold higher (P ⱕ 0.05) in the low FFA trial than in the high FFA trial. PDK4 protein. The PDK4 protein content was higher (P ⱕ 0.05) in the LOW leg than in the CON leg before and after exercise in both trials (⬃1.5-fold higher [P ⱕ 0.05] level in the high FFA trial and ⬃2.2-fold higher [P ⱕ 0.05] level in the low FFA trial). PDK4 protein content in the CON leg was 19% lower (P ⱕ 0.05) after exercise than before in the high FFA trial, and PDK4 protein content in the LOW leg was 21% lower (P ⱕ 0.05) after exercise than before in the low FFA trial (Fig. 3 and Fig. 4B). Before exercise, PDK4 protein content in the CON leg was approximately twofold higher (P ⱕ 0.05) in the high FFA trial than in the low FFA trial.

Pre

Post Ex

low FFA

1.6

*

1.4 1.2 1.0

† 0.8

‡ †

0.6

†

*

0.4

† 0.2 0.0

0.0 Pre

Post Ex

high FFA

Pre

Post Ex

low FFA

Pre

Post Ex

high FFA

Pre

Post Ex

low FFA

FIG. 4. A: Activity of PDH in the active form (PDHa activity). B: PDK4 protein expression. C: PDH-E1␣ site 1 phosphorylation. D: PDH-E1␣ site 2 phosphorylation in both vastus lateralis muscles before and immediately after 20 min of two-legged knee extensor exercise. At the initiation of this exercise, muscle glycogen was reduced in one leg (LOW leg) (䡺) by one-legged exercise the day before (14 h prior) and high-fat diet overnight, whereas prior non-exercised leg (CON leg) (f) had normal glycogen levels. The experimental protocol was performed on two occasions with either fat-rich (high FFA) or carbohydrate-rich (low FFA) breakfast 2 h before the first biopsies. Values are means ⴞ SE. *Significantly different from CON at given time point and within given trial, P < 0.05. †Significantly different from Pre within given leg and trial, P < 0.05. ‡Significantly different from low FFA trial, P < 0.05. diabetes.diabetesjournals.org

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GLYCOGEN AND FFA IN PDH REGULATION

PDH-E1␣ protein and phosphorylation. The PDH-E1␣ protein content was the same in the two legs throughout each protocol. The phosphorylation data are presented as relative phosphorylation (normalized to the PDH-E1␣ content). Exercise induced (P ⱕ 0.05) a dephosphorylation of PDH-P1 (to 20 – 45% of prelevel) and PDH-P2 (34 – 65% of prelevel) in both trials and legs (Fig. 3 and Fig. 4C and D). In the low FFA trial, phosphorylation on PDH-P1 and PDH-P2 was greater (P ⱕ 0.05) in the LOW leg than in the CON leg at all time points, with ⬃1.4-fold before exercise and ⬃2.8-fold after exercise, respectively. No trial effect was apparent for PDH-P1 phosphorylation in the CON leg. After exercise, phosphorylation on PDH-P2 was in the CON leg ⬃2.9-fold higher (P ⱕ 0.05) in the high FFA trial than in the low FFA trial. DISCUSSION

The main findings of the present study are that exercise increases PDHa activity in human skeletal muscle despite enhanced plasma FFA levels, but both reduced muscle glycogen concentration and elevated plasma FFA levels are associated with reduced exercise-induced PDH activation. In addition, the results support that the observed relationship between these metabolic parameters and regulation of PDH may be mediated through effects on PDK4 expression before exercise. The present finding that exercise increased the PDHa activity at least threefold independent of differences in muscle glycogen concentration, and despite enhanced plasma FFA levels, demonstrates that mechanisms other than muscle glycogen and plasma FFA dominate exercise-induced PDH regulation in human skeletal muscle. Increases in mitochondrial calcium levels are likely important, since mitochondrial calcium concentration increases during exercise (4,21) and calcium has been shown to activate PDP1 leading to dephosphorylation and activation of PDH (30). The impact of exercise on PDH regulation in skeletal muscle unrelated to the metabolic status of the cell and body may reflect that exercise can overcome potential inhibition of carbohydrate utilization present in resting skeletal muscle when circulating FFA levels are increased (2). Thus, although there was no effect of elevated FFA on PDHa activity at rest in the present study, maybe due to the overnight high-fat diet in both trials, elevated FFA levels have been shown to induce insulin resistance (31,32), and individuals with enhanced circulating FFA levels like type 2 diabetic subjects may experience FFA-mediated insulin resistance at rest (33), potentially in part because of FFA-induced downregulation of PDH (1). Therefore, the observed upregulation of PDHa activity by exercise despite elevated plasma FFA in the present study in accordance with previous findings (26,34,35) supports that a beneficial effect of physical activity may include that FFAs do not prevent exerciseinduced PDH activation. However, the observation that the highest exerciseinduced PDHa activity and largest PDH dephosphorylation were present when the muscle glycogen level was only moderately reduced and when FFA remained close to baseline levels (193 ␮mol/l) indicates that metabolic factors do adjust the exercise-induced activation of PDH. In addition, the smaller increase in PDHa activity and the smaller decline in PDH dephosphorylation in response to exercise when muscle glycogen was reduced or plasma 30

DIABETES, VOL. 59, JANUARY 2010

FFA concentration was elevated initially suggest that each of these factors may modify exercise-induced PDH regulation. Such an effect of FFA is in accordance with previous studies, indicating that FFA downregulates PDHa activity or increases PDH phosphorylation in rat (1) and human (12) skeletal muscle at rest. A relation between muscle glycogen levels and PDHa activity and PDH phosphorylation is in line with our previous finding that further lowering of muscle glycogen during high-intensity exercise was associated with reduced PDHa activity (17). Of notice is also that the same degree of repression of PDHa activity and PDH dephosphorylation changes was evident when muscle glycogen was lowered to 268 mmol/kg dry wt or plasma FFA was elevated to 714 ␮mol/l, and when both these changes were present simultaneously, no further effect was found. Thus, the impact on PDH regulation associated with low muscle glycogen and elevated plasma FFA was not additive, which may suggest that a similar underlying mechanism could be involved in exerting this effect on PDHa activity. The possibility that changes in PDK4 protein expression may have mediated the observed association between muscle glycogen and PDH regulation as well as between plasma FFA and PDH regulation is supported by the observation that the highest PDH activation and largest dephosphorylation occurred in the leg and trial with lowest PDK4 protein expression. The downregulation of PDK4 protein content when plasma FFA was reduced by a carbohydrate-rich breakfast in the present study supports previous studies showing that an elevated FFA level is associated with increased PDK4 expression (1,36). Although plasma insulin was at basal level and similar in the two trials when the pre-exercise biopsy was obtained, a transient increase in plasma insulin in response to the meal has without doubt occurred and cannot be excluded to have had a contributing influence on the observed PDK4 expression, as previous studies have demonstrated that insulin can regulate PDK4 expression (3,14). However, PDK4 mRNA downregulation in human skeletal muscle is typically not observed until after 3 h of insulin infusion (unpublished data, from our laboratory), and although posttranscriptional regulation cannot be ruled out, these findings do suggest that insulin changes elicited by the meal unlikely have been important in the quick reduction in PDK4 protein content observed in the present study. The current finding that PDK4 protein content was higher in the muscle with low glycogen than the control muscle both before and after exercise and in both trials indicates that reduced muscle glycogen levels could be an initiating signal to increase PDK4 expression. These findings are in accordance with previous human studies showing a similar association between lowered muscle glycogen and regulation of PDK4 mRNA expression, but changes in muscle glycogen was in these previous studies accompanied by changes in plasma insulin and/or plasma FFA (15,16). In the present study, however, the carbohydrate-rich breakfast meal in one trial ensured lowered plasma FFA levels and normalized plasma insulin, leaving only muscle glycogen different. Of notice is that the low muscle glycogen leg had exercised intensively ⬃14 h before, and thus it cannot be ruled out that other exerciseassociated signals have initiated the induction of PDK4 expression in this muscle. In addition, we have recently shown that PDK4 protein content is increased 6 h after a prolonged exercise session, and although this change was associated with reduced muscle glycogen, a decline in diabetes.diabetesjournals.org

K. KIILERICH AND ASSOCIATES

muscle glycogen was not required to obtain an increase in PDK4 protein expression after prolonged exercise in that study (26). On the other hand, lowering of plasma FFA by the carbohydrate-rich breakfast in the present study was associated with a 50% reduction in PDK4 protein content in the control leg but not in the low glycogen leg, supporting that reduced muscle glycogen may indeed have induced PDK4 protein expression. Thus, taken together, the association between changes in muscle glycogen and PDK4 protein as well as between plasma FFA levels and PDK4 protein content supports that PDK4 expression may be regulated by each of these metabolic parameters. Moreover, the association between PDK4 protein content and both PDH phosphorylation and PDHa activity also supports that such PDK4 expression changes may have a functional role in regulating substrate utilization in human skeletal muscle to match availability. The lowering of PDK4 protein content just 2 h after the carbohydrate-rich meal, and the reduction in response to 20 min of exercise, clearly shows that regulation of PDK4 protein content is fast. To our knowledge, no previous studies have reported such quick regulation of PDK4 protein content in skeletal muscle, although changes have been reported in PDK4 protein after 48 h of fasting in rats (14) and 1 day of high-fat diet in humans (37), and we recently have shown an upregulation of PDK4 protein 6 h after a single exercise session (26). Glycolytic flux has been suggested to be one factor regulating PDH and hence the flux through the pyruvate dehydrogenase complex (29,35,38). Based on measurements of muscle glucose and muscle glucose-6-phosphate concentrations, the current findings may suggest that glycolytic flux did not entirely determine the PDH activity during exercise. Thus, the clear differences in exerciseinduced muscle glucose-6-phosphate and muscle glucose responses in the normal and the low muscle glycogen muscles in the high FFA trial were not associated with differences in PDH regulation, and despite similar muscle glucose-6-phosphate and muscle glucose levels in the normal glycogen leg in the two trials, PDHa activity was higher in the low FFA trial than in the high FFA trial. Such interpretation is supported by the observation that lower glycogen utilization in the low glycogen muscle than in the normal glycogen muscle occurred without influence on PDH regulation in the high FFA trial. But at the same time, the present data are also consistent with the previous indications (29,36,37) that PDH activation plays a role in determining the balance between glycolytic/glycogenolytic flux and oxidation. Hence, accumulation of glycolytic intermediates may depend on how well glycolytic flux and PDH activity match, and the lack of accumulation of intermediates in the low leg in both trials may reflect a balanced glycolytic flux and PDH activity, whereas glycolytic flux may have exceeded PDH activity in the CON leg, leading to accumulation of glucose-6-phosphate. In conclusion, muscle glycogen and plasma FFAs modify exercise-induced PDH regulation in human skeletal muscle in a nonadditive manner, which might be through glycogen and FFA-mediated regulation of PDK4 expression. However, of notice is that marked exercise-induced activation of PDH was still present when plasma FFA was elevated, which suggests that beneficial effects of physical activity include that exercise overrules FFA-mediated inhibition of carbohydrate oxidation. diabetes.diabetesjournals.org

ACKNOWLEDGMENTS

This study was supported by grants from the Danish Medical Research Council and the Novo Nordisk Foundation, Denmark. The Centre of Inflammation and Metabolism is supported by the Danish National Research Foundation (grant 02-512-555). No other potential conflicts of interest relevant to this article were reported. The authors thank the subjects for participating in the study and D. Grahame Hardie, Dundee University, Dundee, Scotland, U.K., for the kind donation of valuable tools for this study. REFERENCES 1. Bajotto G, Murakami T, Nagasaki M, Tamura T, Tamura N, Harris RA, Shimomura Y, Sato Y. Downregulation of the skeletal muscle pyruvate dehydrogenase complex in the Otsuka Long-Evans Tokushima Fatty rat both before and after the onset of diabetes mellitus. Life Sci 2004;75:2117– 2130 2. Kelley DE, Mandarino LJ. Hyperglycemia normalizes insulin-stimulated skeletal muscle glucose oxidation and storage in noninsulin-dependent diabetes mellitus. J Clin Invest 1990;86:1999 –2007 3. Majer M, Popov KM, Harris RA, Bogardus C, Prochazka M. Insulin downregulates pyruvate dehydrogenase kinase (PDK) mRNA: potential mechanism contributing to increased lipid oxidation in insulin-resistant subjects. Mol Genet Metab 1998;65:181–186 4. Harris RA, Bowker-Kinley MM, Huang B, Wu P. Regulation of the activity of the pyruvate dehydrogenase complex. Adv Enzyme Regul 2002;42:249 – 259 5. Linn TC, Pettit FH, Reed LJ. Alpha-keto acid dehydrogenase complexes. X. Regulation of the activity of the pyruvate dehydrogenase complex from beef kidney mitochondria by phosphorylation and dephosphorylation. Proc Natl Acad Sci U S A 1969;62:234 –241 6. Bowker-Kinley MM, Davis WI, Wu P, Harris RA, Popov KM. Evidence for existence of tissue-specific regulation of the mammalian pyruvate dehydrogenase complex. Biochem J 1998;329:191–196 7. Gudi R, Bowker-Kinley MM, Kedishvili NY, Zhao Y, Popov KM. Diversity of the pyruvate dehydrogenase kinase gene family in humans. J Biol Chem 1995;270:28989 –28994 8. Huang B, Wu P, Popov KM, Harris RA. Starvation and diabetes reduce the amount of pyruvate dehydrogenase phosphatase in rat heart and kidney. Diabetes 2003;52:1371–1376 9. Holness MJ, Sugden MC. Regulation of pyruvate dehydrogenase complex activity by reversible phosphorylation. Biochem Soc Trans 2003;31:1143– 1151 10. Peters SJ, Harris RA, Heigenhauser GJ, Spriet LL. Muscle fiber type comparison of PDH kinase activity and isoform expression in fed and fasted rats. Am J Physiol Regul Integr Comp Physiol 2001;280:R661–R668 11. Pilegaard H, Saltin B, Neufer PD. Effect of short-term fasting and refeeding on transcriptional regulation of metabolic genes in human skeletal muscle. Diabetes 2003;52:657– 662 12. Pilegaard H, Birk JB, Sacchetti M, Mourtzakis M, Hardie DG, Stewart G, Neufer PD, Saltin B, van Hall G, Wojtaszewski JF. PDH-E1alpha dephosphorylation and activation in human skeletal muscle during exercise: effect of intralipid infusion. Diabetes 2006;55:3020 –3027 13. Ward GR, Sutton JR, Jones NL, Toews CJ. Activation by exercise of human skeletal muscle pyruvate dehydrogenase in vivo. Clin Sci (Lond) 1982;63: 87–92 14. Wu P, Inskeep K, Bowker-Kinley MM, Popov KM, Harris RA. Mechanism responsible for inactivation of skeletal muscle pyruvate dehydrogenase complex in starvation and diabetes. Diabetes 1999;48:1593–1599 15. Pilegaard H, Keller C, Steensberg A, Helge JW, Pedersen BK, Saltin B, Neufer PD. Influence of pre-exercise muscle glycogen content on exerciseinduced transcriptional regulation of metabolic genes. J Physiol 2002;541: 261–271 16. Pilegaard H, Osada T, Andersen LT, Helge JW, Saltin B, Neufer PD. Substrate availability and transcriptional regulation of metabolic genes in human skeletal muscle during recovery from exercise. Metabolism 2005; 54:1048 –1055 17. Kiilerich K, Birk JB, Damsgaard R, Wojtaszewski JF, Pilegaard H. Regulation of PDH in human arm and leg muscles at rest and during intense exercise. Am J Physiol Endocrinol Metab 2008;294:E36 –E42 18. Caruso M, Maitan MA, Bifulco G, Miele C, Vigliotta G, Oriente F, Formisano P, Beguinot F. Activation and mitochondrial translocation of DIABETES, VOL. 59, JANUARY 2010

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protein kinase Cdelta are necessary for insulin stimulation of pyruvate dehydrogenase complex activity in muscle and liver cells. J Biol Chem 2001;276:45088 – 45097 19. Mandarino LJ, Consoli A, Kelley DE, Reilly JJ, Nurjhan N. Fasting hyperglycemia normalizes oxidative and nonoxidative pathways of insulinstimulated glucose metabolism in noninsulin-dependent diabetes mellitus. J Clin Endocrinol Metab 1990;71:1544 –1551 20. Patel MS, Korotchkina LG. Regulation of mammalian pyruvate dehydrogenase complex by phosphorylation: complexity of multiple phosphorylation sites and kinases. Exp Mol Med 2001;33:191–197 21. Denton RM, McCormack JG, Rutter GA, Burnett P, Edgell NJ, Moule SK, Diggle TA. The hormonal regulation of pyruvate dehydrogenase complex. Adv Enzyme Regul 1996;36:183–198 22. Huang B, Gudi R, Wu P, Harris RA, Hamilton J, Popov KM. Isoenzymes of pyruvate dehydrogenase phosphatase: DNA-derived amino acid sequences, expression, and regulation. J Biol Chem 1998;273:17680 –17688 23. World Health Organization. Energy and Protein Requirements. Geneva, World Health Organization, 1985 24. Bergstro¨m J. Muscle electrolytes in man determined by neutron activation analysis on needle biopsy specimens: a study on normal subjects, kidney patients, and patients with chronic diarrhea. Scandinavian Journal of Clinical and Laboratory Investigation 1962;68:1–110 25. Lowry OH, Passonneau JV. A Flexible System of Enzymatic Analysis. New York, Academic Press, 1972 26. Kiilerich K, Birk JB, Saltin B, Bune L, Pedersen PA, Wojtaszewski JFP, Pilegaard H. Exercise induces increased PDK4 expression in human skeletal muscles independent of a fasting effect (Abstract). Diabetes 2008;57(Suppl. 1):A308 27. Cederblad G, Carlin JI, Constantin-Teodosiu D, Harper P, Hultman E. Radioisotopic assays of CoASH and carnitine and their acetylated forms in human skeletal muscle. Anal Biochem 1990;185:274 –278 28. Constantin-Teodosiu D, Cederblad G, Hultman E. A sensitive radioisotopic assay of pyruvate dehydrogenase complex in human muscle tissue. Anal Biochem 1991;198:347–351

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29. Putman CT, Spriet LL, Hultman E, Lindinger MI, Lands LC, McKelvie RS, Cederblad G, Jones NL, Heigenhauser GJ. Pyruvate dehydrogenase activity and acetyl group accumulation during exercise after different diets. Am J Physiol 1993;265:E752–E760 30. Denton RM. Regulation of mitochondrial dehydrogenases by calcium ions. Biochim Biophys Acta 2009;1787;1309 –1316 31. Boden G, Chen X, Ruiz J, White JV, Rossetti L. Mechanisms of fatty acid-induced inhibition of glucose uptake. J Clin Invest 1994;93:2438 –2446 32. Hevener AL, Reichart D, Janez A, Olefsky J. Thiazolidinedione treatment prevents free fatty acid-induced insulin resistance in male Wistar rats. Diabetes 2001;50:2316 –2322 33. Boden G. Effects of free fatty acids (FFA) on glucose metabolism: significance for insulin resistance and type 2 diabetes. Exp Clin Endocrinol Diabetes 2003;111:121–124 34. Bradley NS, Heigenhauser GJ, Roy BD, Staples EM, Inglis JG, LeBlanc PJ, Peters SJ. The acute effects of differential dietary fatty acids on human skeletal muscle pyruvate dehydrogenase activity. J Appl Physiol 2008;104: 1–9 35. St Amand TA, Spriet LL, Jones NL, Heigenhauser GJ. Pyruvate overrides inhibition of PDH during exercise after a low-carbohydrate diet. Am J Physiol Endocrinol Metab 2000;279:E275–E283 36. Schummer CM, Werner U, Tennagels N, Schmoll D, Haschke G, Juretschke HP, Patel MS, Gerl M, Kramer W, Herling AW. Dysregulated pyruvate dehydrogenase complex in Zucker diabetic fatty rats. Am J Physiol Endocrinol Metab 2008;294:E88 –E96 37. Peters SJ, Harris RA, Wu P, Pehleman TL, Heigenhauser GJ, Spriet LL. Human skeletal muscle PDH kinase activity and isoform expression during a 3-day high-fat/low-carbohydrate diet. Am J Physiol Endocrinol Metab 2001;281:E1151–E1158 38. Howlett RA, Parolin ML, Dyck DJ, Hultman E, Jones NL, Heigenhauser GJ, Spriet LL. Regulation of skeletal muscle glycogen phosphorylase and PDH at varying exercise power outputs. Am J Physiol 1998;275: R418 –R425

diabetes.diabetesjournals.org

STUDY 3

Exercise-induced Pyruvate Dehydrogenase Activation Is Not Affected by Seven Days of Bed Rest Kristian Kiilerich1,2,3, Stine Ringholm1,2,3, Rasmus S Biensø1,2,3, James Fisher4*, Ninna Iversen1,2,3, Gerrit Van Hall5, Jorgen, F.P. Wojtaszewski2,6, Bengt Saltin2,7, Carsten Lundby2,7, Jose AL Calbet8*, Henriette Pilegaard1,2,3 1

Centre of Inflammation and Metabolism. 2Copenhagen Muscle Research Centre. 3Department of

Biology, August Krogh Building, University of Copenhagen, Denmark. 4School of Sport and Exercise

Sciences,

University

of

Birmingham.

5

Metabolic

Mass-Spectrometry

Facility,

Rigshospitalet and Department of Biomedical Sciences, Faculty of Health Sciences, University of Copenhagen, Denmark. 6Section of Human Physiology, Department of Exercise and Sport Sciences, University of Copenhagen, Denmark. 7Rigshospitalet, Section 7652, Copenhagen, Denmark. 8

Department of Physical Education, University of Las Palmas de Gran Canaria.

*On leave at Copenhagen Muscle Research Centre, Rigshospitalet, Denmark, during the experiment. Running title: Physical inactivity and exercise induced PDH activation Correspondence; Kristian Kiilerich August Krogh Building, Department of Biology Universitetsparken 13, 2100 Copenhagen, Denmark. Phone: (+45) 35321688 E-mail: [email protected]

1

Abstract Objective: Test the hypothesis that physical inactivity impairs the exercise-induced modulation of pyruvate dehydrogenase (PDH). Research Design and Methods: 6 healthy normally physically active male subjects completed 7 days of bed rest. Before and immediately after the bed rest, subjects completed an OGTT and a onelegged knee extensor exercise bout (45min at 60% Wmax) with muscle biopsies obtained from vastus lateralis before, immediately after exercise, and at 3h of recovery. Blood samples were taken from the femoral vein and artery before and after 40min of exercise. Results: Glucose intake elicited a lager insulin response after bed rest then before, indicating glucose intolerance. Muscle glycogen content tended to be higher after bed rest than before, but muscle glycogen breakdown in response to exercise was similar before and after bed rest. There were no differences in lactate release/uptake across the exercising muscle before and after bed rest, but glucose uptake after 40min of exercise was larger before bed rest than after. PDH protein content did not change in response to bed rest or in response to the exercise intervention. Exercise increased the activity of PDH in the active form (PDHa) and induced dephosphorylation of PDHE1α on Ser293, ser295 and Ser300, with no difference before and after bed rest. Conclusions: The present findings show that although 7 days of bed rest induced whole body glucose intolerance, exercise-induced PDH regulation in skeletal muscle was not changed. This suggests that exercise-induced PDH regulation in skeletal muscle is maintained in glucose intolerant (e.g. insulin resistant) individuals.

2

Introduction It is well established that skeletal muscle is a highly plastic tissue capable of adapting quickly to both use and disuse (1). Such skeletal muscle adaptations affect muscle substrate choice and utilization (2) and can have a major impact on whole body metabolism and hence the health of the individual (3). Insulin-mediated glucose uptake by resting skeletal muscle is influenced by both increases and reductions in physical activity (4-6). Physical activity level also influences substrate utilization during exercise with increased fat oxidation after a period of exercise training (7;8). Such changes in metabolism render the muscle more metabolically efficient and consequently enhance muscle endurance (8). But the impact of reductions in normal physical activity level on skeletal muscle substrate utilization during exercise has been less investigated, although this is the major problem in a western lifestyle. It is possible that a reduction in physical activity level induces an opposite change in skeletal muscle substrate utilization to that observed with training. However, as physical inactivity has been shown to induce insulin resistance and hence reduce glucose uptake at a given insulin concentration at rest (9), it is also possible that carbohydrate use during exercise is compromised when the physical activity level is reduced. This remains to be clarified. The mechanism underlying modifications in substrate utilization during exercise following a change in physical activity level is unresolved. This may involve adjustments in the expression/activity of membrane transporters and metabolic enzymes (10;11), as well as capillarization (12), but changes in the acute regulation of substrate choice in skeletal muscle during exercise may indeed also contribute to the observed changes in substrate oxidation. When examining the choice of substrate, the regulation of the pyruvate dehydrogenase complex (PDC) is of special interest. PDC is responsible for catalyzing the decarboxylation of pyruvate to acetyl-CoA, and represents the only entry of carbohydrate-derived substrate into the mitochondria for oxidation, and thus determines which metabolic pathway the carbohydrate undergoes (13). Therefore, regulation of PDC is believed to be important for the mitochondrial choice of substrate both at rest and during exercise. The PDH component of the complex catalyzes the decarboxylation of pyruvate to form acetyl-CoA, and the activity of PDH determines the overall activity of the complex. PDH activity is regulated by a phosphorylation/dephosphorylation cycle catalyzed by PDH kinases (PDK) and PDH phosphatases (PDP) (14). The activity of PDH in the active form (PDHa) increases in human skeletal muscle during exercise as first demonstrated by Ward et al (15) and an associated dephosphorylation of PDH-E1α has been shown more recently (16-18). The exercise-induced PDHa

3

activation increases with increasing power output (19), and the up-regulation of PDHa activity in the initial part of an exercise bout occurs concomitant with increased carbohydrate use (17;20). A reduction in PDHa activity towards the resting level is observed in the later part of both prolonged low intensity exercise (18;21;22) and high-intensity exercise (16) potentially reflecting reduced carbohydrate oxidation. In accordance with a potential role of PDH in the observed changes in fat utilization in skeletal muscle with training (8), the exercise-induced increase in PDHa activity has been shown to be lower after a period of endurance exercise training than before, when exercising at the same absolute intensity (23). Such a reduced PDHa activity during exercise may indicate that less carbohydrate is oxidized in the trained muscle during exercise at a given absolute intensity, although a study examining dog muscle reported that PDHa activity does not necessarily reflect the level of carbohydrate oxidation during steady state contractions (24). The impact of physical inactivity on exercise-induced PDH regulation is however unknown. The response may be opposite of the training effect (23), but as insulin resistance has been shown to be associated with a reduced insulin stimulated increase in PDH activity in skeletal muscle (25) and physical inactivity to induce insulin resistance, exercise-induced PDH regulation may also be impaired. Therefore the aim of the present study was to investigate the impact of physical inactivity on exercise induced PDH regulation. Normally physically active male subjects underwent 7 days of bed rest. An acute exercise trial with muscle biopsies obtained before and after exercise and with leg a-v differences was performed before and after the bed rest period.

4

Research Design and Methods Subjects Six healthy normally physically active male subjects with an average age, weight, height, body mass index and maximal oxygen consumption of 28.7±5.3 (range: 22-36) yr, 82.2±12.3 (range: 66-102) kg, 183.1±7.6 (range: 173-195) cm, 24.4±2.2 (range: 20-27) kg·m-2 and 4.1±1.0 (range: 2.8-5.4) L·min-1 (means ±SD) respectively completed seven days of bed rest. The subjects were given both written and oral information about the experimental protocol and procedures, and were informed about any discomfort that might be associated with the experiment before they gave their written consent. The study was performed according to the Declaration of Helsinki and was approved by the Copenhagen and Frederiksberg Ethics Committee, Denmark (H-A-2008-024). Bed rest Seven days of bed rest were used as a physical inactivity intervention. The subjects were placed in hospital beds with manual head and leg elevation adjustments. During the seven days bed rest period, the subjects were allowed to sit up for 5 hours a day and they were transported to the restroom, the TV lounge and outside in a wheelchair. Oral glucose tolerance test An oral glucose tolerance test (OGTT) was performed between six and ten days before the onset of bed rest and six days into the bed rest. Each subject consumed 1g of glucose per kg body mass, with each gram of glucose dissolved in 6.67 ml water. Blood was sampled 30, 60 and 120 min after glucose intake, and the samples were subsequently analyzed for plasma insulin and glucose (Department of Clinical Biochemistry, Rigshospitalet, Denmark). Pre-testing Each of the 6 subjects performed a one-legged knee extensor exercise performance test to determine the workload to be used during the experiments. The workload was gradually increased every 2 minutes and the highest load, which could be sustained for 2 min, was set as the maximal load (Wattmax) (26).

5

Experimental protocol After consuming a standardized breakfast with energy content adjusted for body weight (30 kJ·kg-1), the subject reported to the laboratory between 07.00-09.00 a.m. Under local anesthesia (2% lidocain) a catheter was placed in one femoral vein and artery, with the Seldinger technique. Using a modified ergometer bicycle, a one-legged knee extensor exercise bout (45 min at 60% Wmax) was performed. Muscle biopsies were obtained from vastus lateralis before (Pre) and immediately after exercise (Post) and 3 hours into recovery (3h Rec) (Figure 1), using the percutaneous needle biopsy technique (27) with suction. All muscle biopsies were taken through separate incisions, and quickly frozen in liquid nitrogen (