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FACULTY OF SCIENCE UNIVERSITY OF COPENHAGEN PhD thesis Rasmus Sjørup Biensø Regulation of PDH, GS and insulin signalling in skeletal muscle; effect of physical activity level and inflammation Academic advisor: Henriette Pilegaard Submitted: 31/01/14

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Page 1: Regulation of PDH, GS and insulin signalling in skeletal ...20Sj%F8rup%20Biens%F8.pdf · training on glucose-mediated regulation of PDH and GS in skeletal muscle in elderly men, 3)

F A C U L T Y O F S C I E N C E U N I V E R S I T Y O F C O P E N H A G E N

 

PhD thesis Rasmus Sjørup Biensø

Regulation of PDH, GS and insulin signalling in skeletal muscle; effect of physical activity level and inflammation

Academic advisor: Henriette Pilegaard

Submitted: 31/01/14

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Contents  

Acknowledgements  .........................................................................................................................................................  5  Summary  .............................................................................................................................................................................  6  Resume (Danish summary)  ...........................................................................................................................................  8  List of manuscripts  ........................................................................................................................................................  10  INTRODUCTION  ........................................................................................................................................................  13  

Metabolic flexibility during rest and exercise  ................................................................................................  13  Insulin resistance  ......................................................................................................................................................  16  Insulin signaling  ........................................................................................................................................................  17  

Protein kinase B/ Akt  .........................................................................................................................................  18  Exercise-induced Akt regulation  ....................................................................................................................  18  

Glucose uptake and glucose metabolism  ..........................................................................................................  19  TBC1D4  ..................................................................................................................................................................  19  Insulin-induced TBC1D4 regulation  .............................................................................................................  19  Exercise-induced TBC1D4 regulation  ..........................................................................................................  20  GLUT4  ....................................................................................................................................................................  20  Exercise-induced GLUT4 regulation  ............................................................................................................  21  HKII  .........................................................................................................................................................................  21  Exercise-induced HKII regulation  .................................................................................................................  21  Fate of Glucose-6-phosphate  ...........................................................................................................................  21  

Glycogen  synthase  ..................................................................................................................................................  22  GS  regulation  .......................................................................................................................................................  22  Insulin-­‐mediated  GS  regulation  ...................................................................................................................  22  Exercise-induced GS regulation  .....................................................................................................................  23  

Pyruvate  dehydrogenase  .....................................................................................................................................  24  PDH structure  .......................................................................................................................................................  24  PDH regulation  .....................................................................................................................................................  25  Diabetes and PDK4  .............................................................................................................................................  26  Fasting and HFD-induced PDH regulation  .................................................................................................  27  Exercise-induced PDH regulation  ..................................................................................................................  28  PDH and inflammation  ......................................................................................................................................  29  

Physical activity  ........................................................................................................................................................  30  Physical activity level – physiological adjustments  .................................................................................  30  Physical  activity  level  –  and  molecular  adaptations  ...........................................................................  32  

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Akt  ............................................................................................................................................................................  32  TBC1D4  ..................................................................................................................................................................  32  GLUT4  ....................................................................................................................................................................  32  HKII  .........................................................................................................................................................................  32  GS  .............................................................................................................................................................................  32  PDH  ..........................................................................................................................................................................  33  Oxidative proteins  ...............................................................................................................................................  33  

AIMS  .................................................................................................................................................................................  35  METHODS  ......................................................................................................................................................................  36  

Human subjects  .........................................................................................................................................................  36  Mice  ..............................................................................................................................................................................  36  Experimental  protocols  .......................................................................................................................................  36  Bed  rest  ..................................................................................................................................................................  36  LPS  ............................................................................................................................................................................  36  Catheterization  ...................................................................................................................................................  37  Hyperinsulinemic  euglycemic  clamp  ........................................................................................................  37  Interleukin-­‐6  injection  ....................................................................................................................................  37  

In  vivo  tests  ...............................................................................................................................................................  38  Oral  glucose  tolerance  test  ............................................................................................................................  38  Exercise  performance  ......................................................................................................................................  38  VO2max  test  .............................................................................................................................................................  38  

Laboratory  analyses  ..............................................................................................................................................  39  Muscle  biopsies  ..................................................................................................................................................  39  Freeze-­‐drying  ......................................................................................................................................................  39  Muscle  glycogen  .................................................................................................................................................  39  Muscle  creatine  ...................................................................................................................................................  40  Muscle  G-­‐6-­‐P  and  lactate  ................................................................................................................................  40  mRNA  isolation  and  PCR  ................................................................................................................................  41  SDS-­‐page  and  immunoblotting  ....................................................................................................................  41  Gels  ...........................................................................................................................................................................  42  GS  activity  ..............................................................................................................................................................  43  PDHa  activity  .......................................................................................................................................................  43  Enzyme  activity  ..................................................................................................................................................  45  

Statistics  .......................................................................................................................................................................  45  INTEGRATED DISCUSSION  ................................................................................................................................  47  

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Physical  activity  level  ............................................................................................................................................  47  Effects  of  physical  activity  level  on  key  proteins  in  insulin  signaling  and  glucose  uptake  in  skeletal  muscle  ....................................................................................................................................................  47  Effects  of  physical  activity  level  on  GS  regulation  in  skeletal  muscle  .........................................  50  Effects  of  physical  activity  level  on  PDH  regulation  in  skeletal  muscle  .....................................  53  

Regulation  of  PDH  during  and  after  exercise  .............................................................................................  56  IL-­‐6  ...........................................................................................................................................................................  56  Inflammation  and  PDH  regulation  during  exercise  ............................................................................  57  

Conclusion  .......................................................................................................................................................................  60  Perspective  .......................................................................................................................................................................  62  Reference  List  ...............................................................................................................................................................  63   Study I including co-authorship statement Study II including co-authorship statement Study III including co-authorship statement Study IV including co-authorship statement

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Acknowledgements

The present thesis is based on experimental work conducted at the section for Molecular and

Integrative Physiology, Department of Biology, University of Copenhagen in the period 2011-2013.

The funding was provided by the Lundbeck Foundation, Department of Biology, University of

Copenhagen and Danish Medical Research Council for the PhD scholarship. Additional founding

was obtained from Lundbeck, Danish Medical Research Council, KIF, NOVO and CIM.

First and foremost I would like to thank my supervisor Henriette Pilegaard for the excellent

guidance and support throughout my time as PhD student in her research group, for that I am very

thankful and it would not have been possible without you.

During my time as PhD student I have had the pleasure working with numerous great colleagues in

HP lab, at the Centre of Inflammation and Metabolism (CIM) and colleagues on the 2th floor at

AKB thanks for the scientific discussions and for making science even more fun.

From the HP lab I would like to thank Jesper Olesen, Stine Ringholm Jørgesen, Jakob Grunnet

Knudsen, Maja Munk Nielsen, Anders Gudiksen, Nina Brandt, Caroline Maag Khristensen, Signe

Larsson, Simon Meinertz, Line van Hauen, Mikkel Sillesen Matzen, Jens Frey Halling, Ella

Joensen, Lærke Bernholdt and former members of the HP group Lotte Leick, Kristian Kiilerich.

Furthermore I would like to thank the co-authors for their collaboration and the subjects for their

participation in the studies.

Finally, I owe my girlfriend Karina a great thank for invaluable support and for showing patients

and indulgence especially during the last month of thesis writing

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Summary The aims of the present thesis were to investigate 1) The impact of physical inactivity on insulin-

stimulated Akt, TBC1D4 and GS regulation in human skeletal muscle, 2) The impact of exercise

training on glucose-mediated regulation of PDH and GS in skeletal muscle in elderly men, 3) The

impact of inflammation on resting and exercise-induced PDH regulation in human skeletal muscle

and 4) The effect of IL-6 on PDH regulation in mouse skeletal muscle.

Study I demonstrated that bed rest–induced insulin resistance was associated with reduced insulin-

stimulated GS activity and Akt signaling as well as decreased protein level of HKII and GLUT4 in

skeletal muscle. Iαn addition, the ability of acute exercise to increase insulin-stimulated glucose

extraction was maintained after 7 days of bed rest. However, acute exercise after bed rest did not

fully normalize the ability of skeletal muscle to extract glucose to the level seen when exercise was

performed before bed rest.

Study II demonstrated that exercise training-improved glucose regulation in elderly healthy subjects

was associated with increased HKII, GLUT4, Akt2, PDK2, GS and PDH-E1α protein content.

Moreover, exercise training resulted in an enhanced response of TBC1D4 and GS and a reduced

PDHa activity in response to glucose intake relative to before training.

Study III demonstrated that LPS-induced inflammation with elevated plasma TNFα concentration

did not affect resting or exercise-induced AMPK and PDH regulation in human skeletal muscle.

Study IV demonstrated that an IL-6 injection reduced PDHa activity in skeletal muscle from fed

mice and increased the PDHa activity in skeletal muscle from fasted mice without any change in

phosphorylation level. An IL-6 injection increased AMPK and ACC phosphorylation in mouse

skeletal muscle only in the fasted state. In addition, the effects of IL-6 on PDH were rather modest

relative to the changes observed with fasting.

In conclusion, 1) decreased glucose transport/phosphorylation and decreased non-oxidative glucose

metabolism seemed to contribute to the decreased skeletal muscle glucose extraction with physical

inactivity in humans, and physical inactivity did not affect the ability of exercise to enhance insulin-

mediated skeletal muscle glucose extraction. 2) Exercise training-improved glucose handling in

aged human skeletal muscle was associated with increased content of key proteins in glucose

metabolism and acute molecular changes towards improved glucose uptake and storage. 3) Short-

term inflammation did not seem to influence resting or exercise-induced fat and carbohydrate

utilization in human skeletal muscle. 4) IL-6 may regulate the PDHa activity in mouse skeletal

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muscle, but the effect seems to depend on the energy state of the muscle, and AMPK may be

involved in the fasted state.

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Resume (Danish summary)

Formålet med denne PhD afhandling var at undersøge 1) effekt af fysisk inaktivitet på den insulin

stimulerede regulering af Akt, TBC1D4 og GS i human skeletmuskulatur, 2) effekt af træning på

glukose-induceret PDH og GS regulering i skeletmuskulaturen hos ældre mænd, 3) effekt af

inflammation på hvile og arbejds-induceret PDH regulering i human skeletmuskulatur og 4) effekt

af en enkelt IL-6 injektion på PDH regulering i skeletmuskulatur hos mus.

Studie 1 viste, at sengeleje-induceret insulinresistent var associeret med reduceret insulin-stimuleret

GS-aktivitet og Akt-signalering, samt reduceret HKII og GLUT4 proteinindhold i

skeletmuskulaturen. Desuden var evnen til at øge den insulin-stimulerede GS aktivitet efter akut

arbejde bibeholdt efter 7 dages sengeleje. Efter 7 dages sengeleje var et enkelt akut arbejdet dog

ikke nok til at normalisere muskulaturens glukoseekstraktion til det samme niveau, som før

sengeleje.

Studie II viste, at trænings-induceret forbedring af glukosereguleringen hos ældre, raske mænd var

associeret med øget proteinindhold af GLUT4, HKII, Akt2, PDK2, GS og PDHH-E1α i

skeletmuskulaturen. Derudover øgede træning det glukose-medierede respons på TBC1D4 i forhold

til før træning, og glukoseindtag øgede GS-aktiviteten samt reducerede PDHa aktiviteten i

skeletmuskulaturen kun i den trænede tilstand.

Studie III viste, at LPS-medieret inflammation, med forhøjet plasmakoncentration af TNFα ikke

ændrede hvile eller arbejds-induceret AMPK og PDH regulering i human skeletmuskulatur.

Studie IV viste, at en IL-6 injektion reducerede PDHa aktiviteten i skeletmuskulaturen hos mus og

øgede PDHa aktiviteten i fastede mus dog uden at ændre på fosforyleringsniveauet. En IL-6

injektion øgede kun AMPK og ACC fosforylerigen i fastede mus. Endvidere var de IL-6-medierede

effekter på PDH kun moderate i forhold til de ændringer, der sås ved faste.

Samlet kan det konkluderes, at 1) reduceret glukosetransport/fosforylering og reduceret non-

oxidativ glukosemetabolisme kan være årsagen til den reducerede glukoseekstraktion i

skeletmuskulaturen, som forekommer ved fysisk inaktivitet. Fysisk inaktivitet synes ikke at ændre

effekten af et forudgående arbejde på skeletmuskulaturens insulinfølsomhed. 2) den forbedrede

glukosehåndtering efter træning hos ældre mænd var associeret med øget proteinindhold af centrale

proteiner i glukosemetabolismen og akutte molekylære ændringer mod forbedret optag og lagring af

glukose. 3) Akut inflammation ændrede tilsyneladende ikke hvile eller arbejds-induceret

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substratvalg i human skeletmuskulatur. 4) IL-6 synes at regulere PDH i skeletmuskulaturen, men

energitilstanden synes at influere på denne effekt, og AMPK kan muligvis være involveret ved lav

energitilstand i muskulaturen.

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List of manuscripts

The present thesis is based on the following manuscripts referred to through this thesis as study I-IV

Study

I: Biensø RS, Ringholm S, Kiilerich K, Aachmann-Andersen NJ, Krogh-Madsen

R, Guerra B, Plomgaard P, van Hall G, Treebak JT, Saltin B, Lundby C, Calbet

JA,Pilegaard H, Wojtaszewski JF. GLUT4 and glycogen synthase are key players in

bed rest-induced insulin resistance. Diabetes. 2012 May;61(5):10909.

II: Biensø RS, Olesen J, Gliemann L, Smith JF, Matzen MS, Wojtaszewski JFP,

Hellsten Y, Pilegaard H. Effects of exercise training on regulation of skeletal muscle

glucose metabolism in skeletal muscle of elderly men. Submitted Journal of

Gerontology: Medical Scinces 2014

III: Biensø RS, Olesen J, van Hauen L, Meinertz S, Halling JF, Gliemann L,

Plomgaard P, Pilegaard H.. Exercise-induced AMPK and pyruvate dehydrogenase

regulation is maintained during LPS-induced inflammation. Submitted Pflugers

Archive, European Journal of Physiology 2014

IV: Biensø RS, Knudsen JG, Brandt N, Pedersen PA, Pilegaard H. Effects of IL-6 on

pyruvate dehydrogenase regulation in mouse skeletal muscle. Pflugers Arch. 2013

Nov 14.

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Work was contributed to the following manuscript not included in the present thesis

Knudsen JG, Murholm M, Carey AL, Biensø RS, Basse AL, Allen TL, Hidalgo J, Kingwell

BA, Febbraio MA, Hansen JB, Pilegaard H. Role of IL-6 in Exercise Training- and Cold-

Induced UCP1 Expression in Subcutaneous White Adipose Tissue. PLoS One. 2014 Jan

8;9(1):e84910

Guerra B, Ponce-González JG, Morales-Alamo D, Guadalupe-Grau A, Kiilerich K, Fuentes

T, Ringholm S, Biensø RS, Santana A, Lundby C, Pilegaard H, Calbet JA. Leptin signaling in

skeletal muscle after bed rest in healthy humans. European Journal of Applied physiology. 2014

Feb;114(2):345-57.

Nellemann B, Vendelbo MH, Nielsen TS, Bak AM, Høgild M, Pedersen SB, Biensø RS,

Pilegaard H, Møller N, Jessen N, Jørgensen JO. Growth hormone-induced insulin resistance in

human subjects involves reduced pyruvate dehydrogenase activity. Acta Physiologia (Oxf). 2013

Oct 21.

Kenneth Allen Dyar, Stefano Ciciliot, Lauren Emily Wright, Rasmus Sjørup

Biensø, Guidantonio Malagoli Tagliazucchi,Vishal Rajesh Patel, Mattia Forcato, Marcia

Ivonne Peña Paz,Anders Gudiksen, Francesca Solagna, Mattia Albiero, Irene Moretti, Kristin

Lynn Eckel-Mahan, Pierre Baldi, Paolo Sassone-Corsi, Rosario Rizzuto, Silvio

Bicciato, Henriette Pilegaard, Bert Blaauw, Stefano Schiaffino. Muscle insulin sensitivity and

glucose metabolism are controlled by the intrinsic muscle clock Molecular Metabolism. 01/2013

Gliemann L, Schmidt JF, Olesen J, Biensø RS, Peronard SL, Grandjean SU, Mortensen SP,

Nyberg M, Bangsbo J, Pilegaard H, Hellsten Y. Resveratrol blunts the positive effects of exercise

training on cardiovascular health in aged men. Journal of Physiology. 2013 Oct 15;591(Pt 20):5047-

59.

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Bach E, Nielsen RR, Vendelbo MH, Møller AB, Jessen N, Buhl M, K-Hafstrøm T, Holm L,

Pedersen SB, Pilegaard H, Biensø RS, Jørgensen JO, Møller N.Direct effects of TNF-α on local

fuel metabolism and cytokine levels in the placebo-controlled, bilaterally infused human leg:

increased insulin sensitivity, increased net protein breakdown, and increased IL-6 release. Diabetes.

2013 Dec;62(12):4023-9.

Buhl M, Bosnjak E, Vendelbo MH, Gjedsted J, Nielsen RR, K-Hafstrøm T, Vestergaard ET,

Jessen N, Tønnesen E, Møller AB, Pedersen SB, Pilegaard H, Biensø RS, Jørgensen JO,

Møller N. J Direct effects of locally administered lipopolysaccharide on glucose, lipid, and protein

metabolism in the placebo-controlled, bilaterally infused human leg.

Clinical Endocrinol Metababolism. 2013 May;98(5):2090-9.

Haase TN, Ringholm S, Leick L, Biensø RS, Kiilerich K, Johansen S, Nielsen MM,

Wojtaszewski JF, Hidalgo J, Pedersen PA, Pilegaard H Role of PGC-1α in exercise and fasting-

induced adaptations in mouse liver. American Journal of Physiology, Regulatory Integrative

Comparative Physiology. 2011 Nov;301(5):R1501-9.

Ringholm S, Biensø RS, Kiilerich K, Guadalupe-Grau A, Aachmann-Andersen NJ, Saltin B,

Plomgaard P, Lundby C, Wojtaszewski JF, Calbet JA, Pilegaard H. Bed rest reduces metabolic

protein content and abolishes exercise-induced mRNA responses in human skeletal muscle.

American Journal Physiology, Endocrinol Metabolism. 2011 Oct;301(4):E649-58

Kiilerich K, Ringholm S, Biensø RS, Fisher JP, Iversen N, van Hall G, Wojtaszewski JF,

Saltin B, Lundby C, Calbet JA, Pilegaard H. Exercise-induced pyruvate dehydrogenase

activation is not affected by 7 days of bed rest. Journal of Applied Physiology (1985). 2011

Sep;111(3):751-7.

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INTRODUCTION

The development of lifestyle related metabolic diseases including insulin resistance and type 2

diabetes (T2D) are increasing all over the world. It was estimated that 200 million people were

glucose intolerant (Zimmet et al. 2001) and 171 million people had T2D in 2000 and a prognosis

estimates that this number will increase to 366 million people in the year 2030 (Wild et al. 2004).

The development of glucose intolerance, insulin resistance and T2D is strongly correlated with

aging and the number of elderly people with T2D is predicted to be doubled in the year 2030

(Glumer et al. 2003;Wild et al. 2004). Together this underlines the importance of understanding the

etiology of lifestyle related metabolic diseases in order to be able to cope with the future challenges

and hopefully prevent these predictions.

Metabolic flexibility during rest and exercise Skeletal muscle is one of the largest organs in the body and skeletal muscle metabolism has

therefore a major impact on whole body metabolism. Skeletal muscle has normally an incredible

ability to adjust the substrate utilization according to the demands. This includes the changes in

substrate utilization from rest to exercise, where the energy turn-over increases and there is a need

for a marked increase in substrate utilization, as well as the ability to shift between fat and

carbohydrates, when substrate availability changes after a meal or during prolonged exercise. This

ability of skeletal muscle to adjust according to demands can be termed metabolic flexibility

(Corpeleijn et al. 2009;Kelley et al. 1999;Kelley, Mandarino 2000;Storlien et al. 2004). Previous

studies have most often used “metabolic flexibility” to describe the ability to switch between fat and

carbohydrate oxidation (Corpeleijn et al. 2009;Kelley et al. 1999;Kelley, Mandarino 2000;Meex et

al. 2010;Storlien et al. 2004), but also the regulation of fat oxidation (Corpeleijn et al. 2009;Kelley,

Mandarino 2000) and the ability to increase glucose utilization in response to enhanced availability

and increased workload (Crewe et al. 2013;Prior et al. 2013). Metabolic flexibility was first

introduced by Kelley and Mandarino in 2000 (Kelley, Mandarino 2000), where they described the

metabolic inflexibility of oxidative fuel selection in skeletal muscle of insulin resistant, obese

subjects and in T2D subjects emphasizing that a key aspect of skeletal muscle metabolic fitness is

the capacity to switch between fuels. They illustrated (Figure 1, left) that insulin resistant, obese

subjects and T2D patients had a diminished or blunted ability to increase respiratory quotient (RQ)

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during insulin stimulation due to a reduced insulin-mediated suppression of fat oxidation and they

suggested this as part of the observed insulin resistance (Kelley, Mandarino 2000). This point has

Fig 1. Illustration of metabolic flexibility in obese subjects and T2D patients as the ability to increase the respiratory

quotient (RQ) during a hyperinsulinemic euglycemic clamp (left) (Kelley et al. 2000), and metabolic flexibility and

inflexibility after a meal (right) (Corpeleijn et al. 2008).

also schematically been illustrated by the response of lipid and carbohydrate oxidation after a meal

in a metabolic flexible and inflexible subject (Figure 1, right).

Exercise is the most potent physiological enhancer of whole body and muscle metabolism with an

intensity dependent increase in energy expenditure of the working muscle (van Loon et al. 2001).

While carbohydrate utilization dominates the initial phase of exercise (Figure 2, left), a switch

towards fat oxidation takes place as the exercise is prolonged. This was presented already 75 years

ago by Christensen and Hansen showing a decline in RQ during prolonged exercise reflecting

increased fat oxidation (E Christensen, O Hansen 1939;E.Christensen, O.Hansen 1939) (Figure 2,

right).

Fig 2. The respiratory quotient (R.Q) during 30 min (left) and up to 240 min (right) of exercise. The figure to the right

shows the RQ during exercise when on a carbohydrate (K-h) diet, protein rich (N) diet and fat (F) diet (Christensen et

al. 1939).

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This shift in substrate choice takes place as carbohydrate availability decreases and enables muscle

contractions to continue although potentially at a lower exercise intensity. The mechanisms behind

this shift is not fully understood, but the interplay between fat and carbohydrate utilization is

thought to be a contributing factor (Garland et al. 1962;Randle et al. 1963;Randle et al.

1978;Randle 1986;Randle et al. 1988) as originally described in the glucose fatty-acid cycle for

resting conditions proposed by Randle et al. The original idea was that muscle derives energy

through a tight regulation between glucose and fat metabolism (Randle et al. 1963), where fatty

acids released from muscle or adipose tissue impose restrictions on muscle glucose metabolism, and

where glucose uptake imposes restrictions on fatty acid release from triglycerides (Randle et al.

1963). This idea was later modified with the discovery that lipolysis is independent of glucose

metabolism and regulated by hormones, and the observation that FFA oxidation can inhibit glucose

oxidation (Randle et al. 1988).

Recently, a less marked increase in the respiratory exchange ratio (RER) during exercise was

demonstrated in obese, older glucose intolerant subjects than in normal glucose tolerant subjects

reflecting metabolic inflexibility during submaximal exercise (Prior et al. 2013) (Figure 3). This

suggests a link between insulin-mediated and exercise-induced metabolic inflexibility in glucose

intolerant subjects.

Figure 3. Respiratory exchange ratio (RER) during exercise at 50% and 60% of VO2max in normal glucose tolerant

subjects (NGT) and subjects with impaired glucose tolerance (IGT) (Prior et al. 2013).

Taken together, disturbances in the interaction between fat and carbohydrate may lead to inflexible

skeletal muscle fat and carbohydrate utilization, which may influence skeletal muscle and whole

body metabolism both during exercise and at rest, where it may result in insulin resistance.

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Insulin resistance Insulin regulates a large number of cellular processes in many tissues including adipose tissue, liver

and skeletal muscle. In adipose tissue insulin stimulates glucose uptake and triglyceride synthesis

and inhibits the release of FFA (Kiens et al. 1989;NOVAK et al. 1965), while insulin inhibits the

release of glucose from the liver by inhibiting hepatic gluconeogenesis and glycogenolysis and

stimulating glycogen storage (DeFronzo et al. 1981;Donkin et al. 1997). In skeletal muscle, insulin

stimulates glucose uptake, storage and oxidation and inhibits glycogenolysis (Bogardus et al.

1984;Cohen 1979;DANFORTH 1965;DeFronzo et al. 1979;DeFronzo et al. 1981), and prior

exercise has been shown to enhance the insulin-mediated effects on glucose uptake and glycogen

storage in skeletal muscle (Wojtaszewski et al. 2000;Bergstrom, Hultman 1966). Insulin resistance

is defined as reduced insulin-mediated cellular responses (Hojlund, Beck-Nielsen 2006;Mandarino

et al. 1987;Mandarino et al. 1996;Perseghin et al. 1996;Schinner et al. 2005) and T2D is most often

characterized by the combined existence of peripheral insulin resistance and β-cell dysfunction

(Donath et al. 2009;Kahn et al. 2006;Stumvoll et al. 2005). The general view is that insulin

resistance is the first step in the development of T2D and that T2D only develops if the beta-cells

are unable to compensate for the reduced insulin sensitivity (Kahn et al. 2006;Stumvoll et al. 2005).

Hence, while a normal glucose tolerant individual will compensate for reduced insulin sensitivity

with increased β-cell insulin release, a person with impaired glucose tolerance will respond with a

reduced insulin secretion and T2D patients will be unable to compensate for reduced insulin

sensitivity due to β-cell dysfunction. A schematic illustration (Figure 4) of this scenario originally

presented by Stumvoll et al (Stumvoll et al. 2005) places insulin resistance as the first step in the

development of T2D and underlines the utmost importance in understanding how to prevent and

potentially reverse the insulin resistant state before β-cell dysfunction sets in.

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Fig 4 Schematic illustration of the hyperbolic relation between insulin release and insulin sensitivity in a normal

glucose tolerant individual (Normal), while this relationship is abolished in people with impaired glucose tolerance

(IGT) and type 2 diabetes (T2DM) due to an inability to compensate sufficiently for reduced insulin sensitivity.

(adapted from Stumvoll 2005 by Kahn, hull 2006)

Insulin resistance may occur in several insulin-sensitive tissues (Schinner et al. 2005;Tsatsoulis et

al. 2013). Although the insulin regulated processes in adipose tissue and liver may be impaired and

contribute to insulin resistance (DeFronzo et al. 1985;Smith et al. 1999;Tsatsoulis et al. 2013),

skeletal muscle is at least quantitatively by far the most important insulin sensitive tissue (Baron et

al. 1988). Hence, glucose uptake by skeletal muscle constitutes 75-95% of insulin-stimulated

glucose uptake (Baron et al. 1988). Therefore even small changes in skeletal muscle glucose uptake

and glucose metabolism can have a major impact on whole body metabolism. This includes skeletal

muscle insulin resistance characterized by for example reduced insulin-stimulated glucose uptake

and glycogen storage, compared with normal insulin sensitive muscles (DeFronzo et al.

1985;Himsworth 2011;Mandarino et al. 1987). An understanding of insulin signaling in skeletal

muscle in both insulin sensitive and insulin resistant states is therefore important.

Insulin signaling The plasma glucose concentration is regulated to be kept around 5 mM in the post absorptive phase

and during fasting (Hommes et al. 1991). But after a meal, the blood glucose concentration will

increase and glucose will activate secretion of insulin from the pancreas. The increased insulin level

will stimulate signaling pathways in skeletal muscle leading to increased glucose uptake, storage

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and oxidation. The first step in the insulin signaling pathway is the activation and phosphorylation

of the insulin receptor (IR) at the plasma membrane. This is followed by the docking and

phosphorylation of the Insulin receptor substrate (IRS)1(Yenush, White 1997). Phosphatidylinositol

3-kinase p85 regulatory subunit binds to IRS and activates the p110 catalytic subunit, that becomes

active and phosphorylates phosphatidylinositol (4,5)-biphosphate (PIP2) to phosphatidylinositol

(3,4,5)-triphosphate (PIP3) (Summers, Birnbaum 1997). The increase in PIP3 will phosphorylate and

activate the phosphatidylinositol dependent kinase, which then phosphorylates protein kinase B

(Akt).

Protein kinase B/ Akt

Akt exists as 2 isoforms, Akt1 and Akt2, and it has been suggested that Akt2 plays a larger role in

insulin-stimulated glucose uptake than Akt1 (Vind et al. 2012;Whiteman et al. 2002). Akt is

activated by phosphorylation on two phosphorylation sites termed Akt Thr308 and Akt Ser473

((Alessi et al. 1996;Summers et al. 1999) and concomitantly phosphorylate downstream targets

including TBC1D4 and glycogen synthase kinase (GSK)-3. As TBC1D4 is a key factor in GLUT4

translocation and GSK3 involved in regulating glycogen synthase (GS), Akt is seen as a central

factor in insulin-mediated intracellular signaling towards glucose uptake and glycogen storage

(Figure 4 and 5). While there is no clear line regarding the effect of aging on Akt protein content in

skeletal muscle (Consitt et al. 2013;Leger et al. 2008;Wilkes et al. 2009), lower insulin-stimulated

Akt Ser473 phosphorylation and Akt activity has been observed with aging (Leger et al. 2008;Wilkes

et al. 2009), although Akt phosphorylation has also been reported to be similar in young and elderly

subjects (Consitt et al. 2013). The reduced Akt phosphorylation may be compared with the lower

insulin-stimulated Akt Thr308and Akt Ser473 phosphorylation observed in insulin resistant subjects

(Hojlund, Beck-Nielsen 2006;Hojlund et al. 2009;Karlsson et al. 2005;Meyer et al. 2002;Vind et al.

2011;Vind et al. 2012) indicating that an impaired insulin-stimulated Akt response in aged skeletal

muscle may have similarities with observations in insulin resistant subjects.

Exercise-induced Akt regulation

An increase in Akt Thr308 and Akt Ser473 phosphorylation has been observed in rodent skeletal

muscle immediately after exercise relative to resting skeletal muscle (Howlett et al. 2006;Sakamoto

et al. 2004). However, Akt phosphorylation is similar in a rested and a prior exercised muscle in

both humans and rodents 3-4 h after exercise (Wojtaszewski et al. 1997;Wojtaszewski et al.

2000;Wojtaszewski et al. 2002b;Arias et al. 2007;Hansen et al. 1998).

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Glucose uptake and glucose metabolism

TBC1D4

The TBC1D4 protein has been shown to be a direct target of Akt, hence the name Akt substrate of

160 kD (zeiger 04). TBC1D4 contains a Rab GTPase activating protein, which has a reduced

activity when phosphorylated leading to an increase in Rab-GTP and the exocytosis of the GLUT4

vesicles to the plasma membrane (Bruss et al. 2005;Cartee, Wojtaszewski 2007;Karlsson et al.

2005;Zeigerer et al. 2004) (Figure 5).

Fig 5 Insulin stimulated and contraction stimulated phosphorylation of TBC1D4 and the following exocytosis of

GLUT4 vesicles (CArtee and wojtaszewski 2007).

Insulin-induced TBC1D4 regulation

The insulin-stimulated GLUT4 translocation is dependent on the RabGAP on TBC1D4 observed in

mice skeletal muscle (Zeigerer et al. 2004;Karlsson et al. 2005). TBC1D4 has been shown to be

phosphorylated on 9 different phosphorylation sites (REF). Furthermore it has been observed in

humans that insulin stimulation further increases the phosphorylation on several specific

phosphorylation sites (Consitt et al. 2013;Treebak et al. 2009;Treebak et al. 2010;Treebak et al.

2014;Vind et al. 2011). The insulin-stimulated phosphorylation of specific sites on TBC1D4 in

skeletal muscle is reduced in type 2 diabetic patients compared with healthy subjects (Consitt et al.

2013;Hojlund et al. 2008;Vind et al. 2011) and this effect has been suggested to contribute to the

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reduced glucose uptake observed in insulin resistant skeletal muscle (Hojlund et al. 2008;Vind et al.

2011). In addition, insulin-stimulated TBC1D4 phosphorylation has been shown to be reduced in

skeletal muscle of elderly subjects (Consitt et al. 2013)

Exercise-induced TBC1D4 regulation

TDC1D4 was first shown to be phosphorylated by insulin, and shortly after it was observed that

insulin and exercise together increased the overall phosphorylation of TBC1D4 in mouse (Bruss et

al. 2005) and human skeletal muscle (O'Gorman et al. 2006;Treebak et al. 2009).

A single 1 hour exercise bout or longer has been reported to increase TBC1D4 phosphorylation in a

site-specific manner (Howlett et al. 2008;Treebak et al. 2009;Treebak et al. 2014) and with an

elevated phosphorylation level 3 hours into recovery (Howlett et al. 2008;Treebak et al. 2009). The

phosphorylation level in recovery from exercise was further increased with insulin stimulation

(Treebak et al. 2009;Vind et al. 2011), but this seemed to be exercise intensity dependent. In one

study the subjects exercised 60 min at 60% of VO2max (Howlett et al. 2008) and in another study 60

min at 80% of peak work-load and 5 min with 100% peak work load. The more intense workload in

the Treebak et al study resulted in the detection of increased site-specific phosphorylation on

TBC1D4 (Treebak et al. 2009). In addition, the enhanced phosphorylation of TBC1D4 by

combining exercise and insulin could explain some of the increased glucose uptake in the prior

exercised muscle (Treebak et al. 2009).

GLUT4 Because glucose transport across the sarcolemma in part is determined by both the protein content

of GLUT4 and by the translocation of GLUT4 to the membrane (Goodyear, Kahn 1998;Mueckler

1990), improvements in either of these can enhance glucose uptake. In accordance, the insulin

stimulated glucose uptake correlates with the GLUT4 protein content in the muscle (Christ-Roberts

et al. 2004;Dela et al. 1993;Dela et al. 1994b;Frosig et al. 2007).

Aging has often been associated with decreased GLUT4 protein in human (Houmard et al. 1995)

and mouse skeletal muscle (Kern et al. 1992), although no change in GLUT4 protein content has

also been reported with age (Cox et al. 1999). As with aging there is some controversy about

whether or not the GLUT4 protein content in skeletal muscle is changed with insulin resistance and

T2D, because some studies have reported no change (Kahn et al. 1992;Pedersen et al. 1990;Dela et

al. 1994b) and some a decrease in GLUT4 protein content in skeletal muscle with aging and T2D

(Kampmann et al. 2011).

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Exercise-induced GLUT4 regulation

Even a single exercise bout has been observed to increase the GLUT4 protein content in rat (Ren et

al. 1994) and human skeletal muscle. The contraction-induced translocation of GLUT4 to the

plasma membrane occurring already during exercise is in part mediated by AMPK as shown in

AMPK KO mice (Kramer et al. 2006). Studies suggest that GLUT4 re-locates back to the

cytoplasmic vesicles within 2h after the end of exercise (Goodyear et al. 1990). This indicates that

the increased insulin-stimulated glucose uptake several hours after an exercise session is not due to

an initially elevated number of GLUT4 in the plasma membrane.

HKII When glucose has entered the muscle cell through GLUT4, it is phosphorylated by hexokinase

(HK)II, into glucose-6-phosphate (G-6-P), which is unable to leave the muscle cell. The

phosphorylation of glucose by HKII has been suggested as a limiting step in glucose uptake and

studies in genetically manipulated mice support this possibility (Fueger et al. 2004a;Fueger et al.

2004b;Wasserman et al. 2011;Wasserman, Ayala 2005). In addition, insulin resistant subjects and

T2D patients have reduced HKII activity (Vestergaard et al. 1995) and reduced insulin-stimulated

muscle G-6-P compared with healthy subjects (Cline et al. 1999;Rothman et al. 1995). Furthermore

as aging can be associated with reduced insulin sensitivity (Consitt et al. 2013;Cox et al. 1999;Dela

et al. 1996) and HKII activity also is reduced with age, HKII may be suggested to contribute to the

reduced glucose uptake with aging (Consitt et al. 2013;Cox et al. 1999;Dela et al. 1996).

Exercise-induced HKII regulation

Even a single exercise bout has been shown to increase HKII activity as well as HKII transcription

and mRNA content in human skeletal muscle in recovery from exercise (O'Doherty et al.

1996;Pilegaard et al. 2000). In addition, the exercise-induced HKII mRNA response has been

reported to be similar in T2D patients and control subjects, while the HKII activity has been

reported to be lower in T2D patients (Cusi et al. 2001;Vestergaard et al. 1995) suggesting a less

efficient phosphorylation of glucose in T2D patients than in healthy controls.

Fate of Glucose-6-phosphate

The G-6-P in the muscle cell will either be incorporated into muscle glycogen or through glycolysis

be converted to pyruvate, which either will be converted to acetyl CoA in the mitochondria or

lactate in the cytosol (Randle et al. 1978;Randle 1986;Randle et al. 1988;Randle et al. 1963). GS

catalyzes the incorporation of G-6-P into glycogen in the cytosol (Leloir, CARDINI 1955;Leloir,

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CARDINI 1957), while the pyruvate dehydrogenase (PDH) complex (PDC) catalyzes the oxidative

decarboxylation of pyruvate to acetyl CoA in the mitochondria (Randle et al. 1978;Randle 1986).

Hence GS and PDC are key enzymes in non-oxidative and oxidative skeletal muscle glucose

metabolism, respectively.

Glycogen  synthase  

GS  regulation  Glycogen is a branched polymer and GS incorporates glucose from uridine diphosphate glucose

(UDP-glc) into glycogen (Leloir, CARDINI 1957). GS is a member of the A-type

glycosyltransferase family that catalyzes the formation of the glycosidic linkages of glycogen using

UDP-glucose as the donor (Roach 2002;Roach et al. 2012). GS is regulated by phosphorylation on

at least 9 serine residues (Roach et al. 1977;Roach 1990;Roach 2002;Roach et al. 2012;Roach,

Larner 1977;Skurat et al. 1994;Skurat, Roach 1995;Skurat, Roach 1996) and allosterically by G-6-P

(Leloir, CARDINI 1957;Roach et al. 1976;Roach, Larner 1976;Roach et al. 1977) (Figure 6). When

GS is phosphorylated it becomes inactivated, however the allosteric regulation by G-6-P overrules

the covalent modification (Leloir, CARDINI 1957;Roach et al. 1976;Roach, Larner 1976;Roach et

al. 1977). The main regulatory sites on GS are 2+2a, 3a and 3b (Skurat et al. 1994;Skurat, Roach

1995).

Insulin-­‐mediated  GS  regulation  Insulin is a very potent activator of GS activity both through the increase in cellular G-6-P and

through reduced GSK-3 activity by Akt-mediated phosphorylation, leading to dephosphorylation of

sites 3a, 3b, 3c and 4 (Roach et al. 1977;Skurat, Roach 1995;Skurat et al. 2000) (Figure 6). GS is

dephosphorylated by protein phosphorylase (PP)1 (Cohen 1989), the activity of which is regulated

by its subunit GM. When GM is bound to PP1, the PP1-GM complex is active and dephosphorylates

GS (Cohen 1989;Nielsen, Wojtaszewski 2004;Roach 2002). Insulin resistant subjects and T2D

patients have reduced GS activity and a reduced insulin-mediated effect on GS regulation (Christ-

Roberts et al. 2004;Hojlund, Beck-Nielsen 2006;Hojlund et al. 2008;Hojlund et al. 2009;Perseghin

et al. 1996;Vind et al. 2011;Cline et al. 1999). One of the molecular changes observed in skeletal

muscle of insulin resistant subjects is a defect in insulin-mediated dephosphorylation of GS site

2+2a (Hojlund et al. 2003;Hojlund et al. 2009;Glintborg et al. 2008;Vind et al. 2011) resulting in a

hyper-phosphorylation of GS site 2+2a (Hojlund et al. 2003;Vind et al. 2011), although not

observed in all studies (Hojlund et al. 2006).

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Fig 6. The regulation of glycogen synthase (GS) by insulin (left) and exercise (right). Insulin stimulated the Akt/PKB-

GSK-3 pathway (modified from Roach 2002 and Nielsen et al 2004)

In addition the insulin-stimulated GS activity in elderly subjects has been reported to be reduced

despite a reduced phosphorylation level on GS site 2 and site 3a compared with young subjects

(Poulsen et al. 2005). Furthermore, the GS protein content has also been shown to be reduced in

aged subjects (Poulsen et al. 2005). Indicating a discrepancy between GS activity and

phosphorylation level in elderly subjects

Exercise-induced GS regulation

During exercise glycogen is a key substrate for supplying the muscle with energy and restoring

glycogen after use has high priority for the muscle (E Christensen, O Hansen 1939;Green 1991). GS

activity has been shown to increase in human skeletal muscle after a single bout of exercise in part

due to dephosphorylation of GS site 2+2a, 3a and 3a+3b (Jensen et al. 2012;Prats et al. 2009),

while several studies have reported that GS activity is not increased during exercise, but

immediately after exercise (Chasiotis et al. 1983;Katz, Raz 1995;Kida et al. 1989). As

phosphorylation on site 2+2a is regulated by AMPK (Huang, Krebs 1977;Jorgensen et al.

2004;Wojtaszewski et al. 2002a), adrenaline (Hiraga, Cohen 1986) and the glycogen content in the

muscle (DANFORTH 1965;Nielsen, Wojtaszewski 2004;Wojtaszewski et al. 2002a) (Figure 6,7),

these factors are thought to mediate the observed changes at 2+2a in response to exercise. GS

remains dephosphorylated and GS activity elevated for several hours after a single bout of exercise

(Prats et al. 2005;Wojtaszewski et al. 1997;Wojtaszewski et al. 2000;Wojtaszewski et al. 2003).

This increased GS activity after exercise is related to lower glycogen content in skeletal muscle as

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both observed in rats (DANFORTH 1965) and humans (Nielsen et al. 2001;Wojtaszewski et al.

2001).

Figure 7. Relationship between GS activity and glycogen content in skeletal muscle in fed and 16h fasted mice as well

as after treatment with epinephrine (DANFORTH 1965).

The elevated GS activity in recovery from exercise can be further increased with insulin stimulation

(Christ-Roberts, Mandarino 2004;Frosig et al. 2007;Prats et al. 2009;Wojtaszewski et al.

1997;Wojtaszewski et al. 2000;Wojtaszewski et al. 2003), which is associated with a further

dephosphorylation of GS site 2+2a and 3a (Prats et al. 2009) This elevated GS activity is thought to

be a key factor in the effect of prior exercise on insulin-stimulated glucose uptake (Wojtaszewski et

al. 1997;Wojtaszewski et al. 2000;Wojtaszewski et al. 2003).

Pyruvate  dehydrogenase  The PDC is the only entry for carbohydrate-derived substrate into the mitochondria for oxidation

and is therefore central in the switching between carbohydrates and fat oxidation. PDC ensures

carbohydrate oxidation when carbohydrates are abundant and inhibition of carbohydrate oxidation

when carbohydrates are scarce (Peters et al. 1998;Putman et al. 1993;Randle et al. 1978;Randle et

al. 1988;St Amand et al. 2000).

PDH structure

PDC is a multi-enzyme complex composed of several copies of the heterotetramer (α2β2) pyruvate

dehydrogenase (PDH) (E1), dihydrolipoamide acetyltransferase (E2), dihydrolipoamide

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dehydrogenase (E3) and dihydrolipoamide dehydrogenase-binding protein (E3BP) (Holness,

Sugden 2003;Patel, Korotchkina 2001;Sugden, Holness 1994).

PDH regulation

The activity of PDH in the active form (PDHa) is tightly regulated when energy demand and

substrate availability changes during exercise and when availability of glucose changes during

glucose intake, fasting and high fat diet (HFD). Although the activity of PDC to some extend is

regulated by product inhibition and stimulated by the substrates (Garland et al. 1963;Kanzaki

1969;Kelley et al. 1993), the main regulation is thought to be by phosphorylation of at least 4 serine

residues in PDH-E1α located at 232 (site 3), 293 (site 1), 300 (site 2) and 295 (site 4) (Gnad et al.

2011;Kiilerich et al. 2010a;Sugden et al. 1978;Linn et al. 1969). More recently, PDH has also been

reported to be regulated by acetylation, but with the effects exerted on the phosphorylation level of

PDH-E1α (Gnad et al. 2011;Jing et al. 2013).

The phosphorylation level of these sites is regulated by 4 isoforms of PDH kinase (PDK) (Jing et al.

2013;Korotchkina et al. 1995;Korotchkina, Patel 2001;Patel, Korotchkina 2001), which

phosphorylate and inactivate the complex, and 2 isoforms of PDH phosphatase (PDP) (Patel,

Korotchkina 2006;Roche et al. 2001) (Fig 7), which dephosphorylate and activate the complex

(Figure 8). The distribution of the 4 PDK’s is tissue specific. While PDK1 is primarily expressed in

heart and pancreas islets (Bowker-Kinley et al. 1998;Sugden, Holness 2003), PDK2 is expressed in

most tissues (Bowker-Kinley et al. 1998), and PDK3 is expressed in testis, kidney and brain

(Bowker-Kinley et al. 1998;Huang et al. 1998). PDK4 is primarily expressed in heart, skeletal

muscle, liver, kidney and pancreatic islets (Bowker-Kinley et al. 1998;Holness et al. 2000). PDP1 is

primarily expressed in heart, brain and skeletal muscle (Huang et al. 1998;Huang et al. 2003), while

PDP2 is expressed in liver, adipose tissue and kidney (Huang et al. 1998;Huang et al. 2003).

Furthermore both PDP isoforms have been detected at the mRNA level in human skeletal muscle by

using real time PCR (Pilegaard et al. 2006). The phosphorylation level of PDH-E1α will therefore

at any given time depend on the balance between the activities of the PDK’s and PDP’s, In addition,

the different PDK isoforms have different affinities for the various phosphorylation sites on the

PDH-E1α subunit (Korotchkina, Patel 2001), which can further play a role in the regulation of the

phosphorylation state.

The activities of the PDK’s and PDP’s can be modified both by changes in expression,

phosphorylation and/or allosteric regulation of the proteins (Patel, Korotchkina 2006). The activity

of the PDK’s is regulated by the acetylation and reduction state of PDH-E2 with PDK activated by

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an increase in mitochondrial acetyl CoA/CoA and NADH/NAD+ concentration ratios, which may

occur during high rates of β-oxidation as well as PDH activity (Cate, Roche 1978;Randle et al.

1988;Roche et al. 1989). Furthermore, PDK activity is reduced by pyruvate (Cooper et al.

1974;Cooper et al. 1975;Priestman et al. 1996). PDP1 is stimulated by Ca2+ (Huang et al. 1998) and

PDP2 activity has previously been reported to be regulated by insulin in skeletal muscle (Caruso et

al. 2001) through phosphorylation-induced translocation to the mitochondria (Caruso et al. 2001)

(Figure 8).

Fig 8. Illustration of the regulation of the pyruvate dehydrogenase complex (PDC) by PDP and PDK, and the interaction

between the different factors involved in PDK and PDP regulation (Holness et al. 2003).

Diabetes and PDK4

An increased protein content of PDK4 has been observed in a rat model for diabetes and the authors

suggest that the higher PDK4 protein content was induced by elevated FFA plasma levels (Bajotto

et al. 2004). In a human study, PDH activity during a hyperinsulinemic euglycemic clamp was

reduced in T2D patients relative to healthy subjects (Mandarino et al. 1990;Mandarino et al. 1996).

The lower PDH activity indicates a reduced glucose oxidation and this could be a possible

mechanism behind insulin resistance. This is in line with the observed reduced glucose oxidation

and increased lipid oxidation in skeletal muscle during insulin stimulation in diabetic rats (Wu et al.

1998) and in T2D patients (Kelley, Mandarino 2000). In addition is has been reported that T2D

patients have elevated PDK4 mRNA content in skeletal muscle compared with glucose tolerant

subjects (Kulkarni et al. 2012).

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Fasting and HFD-induced PDH regulation

Fasting has been shown to increase PDK4 transcription and mRNA content in human skeletal

muscle (Pilegaard et al. 2003a) and PDK4 mRNA and protein content in rat (Wu et al. 1998;Wu et

al. 2000) and mouse (Kiilerich et al. 2010a) skeletal muscle with no change in skeletal muscle

PDK2 protein content (Wu et al. 1998;Wu et al. 2000). Similarly skeletal muscle PDK activity has

been reported to increase in human and rat skeletal muscle with fasting (Fuller, Randle 1984;Peters

et al. 1998;Sale, Randle 1980). In accordance with the observed PDK regulation, the two PDP

isoforms have been shown to be down regulated in rat skeletal muscle at both the mRNA and

protein level by 48 hours of fasting (Huang et al. 2003). Hence, the hyper-phosphorylation of PDH-

E1α during fasting shown in mouse skeletal muscle (Kiilerich et al. 2010a) seems to be due to a

combination of increased PDK4 (Kiilerich et al. 2010a;Wu et al. 1998;Wu et al. 2000) and

decreased PDP protein content. Furthermore, in agreement with an the increased PDH-E1α

phosphorylation, skeletal muscle PDHa activity has been shown to be reduced in mice after 24

hours of fasting (Kiilerich et al. 2010a) suggesting that inhibition of PDHa activity contributes to

reducing carbohydrate oxidation and increasing fat oxidation in skeletal muscle during fasting.

Another model used to investigate the regulation of PDH during changes in substrate availability

and use is high fat diet (HFD). Intake of saturated fat has been shown to increase PDK activity in

rat oxidative skeletal muscle (Peters et al. 2001a) and a human study has shown that decreased RER

after 3 days on low carbohydrate diet/rich in saturated fat was associated with increased PDK

activity and reduced PDHa activity in skeletal muscle (Peters et al. 2001b). This suggests that PDK-

mediated inhibition of PDH contributes to an increased fat oxidation during HFD (Jansson, Kaijser

1982;Peters et al. 1998;Peters et al. 2001b;Putman et al. 1993). Furthermore, intake of a

carbohydrate rich breakfast meal after an overnight HFD has been shown to reduce PDK4 protein

content in human skeletal muscle relative to a high fat rich breakfast after an o/n HFD

demonstrating very rapid regulation of PDK4 protein in humans by changes in carbohydrate

availability (Kiilerich et al. 2010b). A tendency for an increase in PDK4 mRNA content in human

skeletal muscle after only 4h of intralipid infusion (Pilegaard et al. 2006) supports that fatty acids

play a role in this regulation of PDH during fasting and HFD.

Only limited information exists on PDK4 regulation with aging, but there are indications that PDK4

at least in rat muscle is increased with aged (Bajotto et al. 2004).

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Exercise-induced PDH regulation

The first study to show an exercise-induced increase in PDHa activity in human skeletal muscle

used cycling exercise with 1 min exercise and 3 min rest until exhaustion (Ward et al. 1982). Later

it has been demonstrated that PDHa activity increases in human skeletal muscle already after just 1

min (Howlett et al. 1998) and that the PDHa activity is increased in an exercise intensity dependent

manner (Howlett et al. 1998). A proposed contributing mechanism for the increase in PDHa activity

during exercise is a combination of increased accumulation of Ca+ and pyruvate (Constantin-

Teodosiu et al. 2004;Huang et al. 2003) with concomitant effects on the activity of PDP1 and

PDK’s. In accordance, a single exercise bout has been shown to result in marked dephosphorylation

of PDH-E1α in human skeletal muscle (Kiilerich et al. 2008;Pilegaard et al. 2006;Kiilerich et al.

2010b;Kiilerich et al. 2011) supporting a key role of phosphorylation status in PDHa activity also

during exercise. The impact of plasma FFA concentration on exercise-induced PDH regulation has

been examined by modulating the FFA concentration through HFD, nicotinic acid and intralipid

infusion (Kiilerich et al. 2010b;Pilegaard et al. 2006;Stellingwerff et al. 2003). Reduction in plasma

free fatty acid (FFA) concentration by ingestion of the antilipolytic dryg nicotinic acid resulted in a

higher level of PDHa activity and higher RER value in response to exercise (Stellingwerff et al.

2003). In accordance, enhanced plasma FFA concentration by o/n HFD has been shown to reduce

the exercise-induced increase in PDHa activity and reduced the decrease in PDH phosphorylation at

Ser300 (Kiilerich et al. 2010b).

Further studies have shown that the PDHa activity in human skeletal muscle remains elevated at a

rather constant level up for to about 2 hours of low intensity exercise followed by a decrease

towards the resting level as exercise proceeds above 2 hours (Mourtzakis et al. 2006;Pilegaard et al.

2006;Watt et al. 2004). Although a single exercise bout has been shown to increase PDK4

transcription and mRNA content in human skeletal muscle in response to exercise (Pilegaard et al.

2002;Pilegaard et al. 2003a), the increase is first pronounced some hours into recovery (Pilegaard et

al. 2000;Pilegaard et al. 2002). In accordance, the decrease in PDHa activity during prolonged

exercise, as the duration exceeds 2 hours of exercise, does not seem to be explained by an increase

in PDK4 protein content, but may be due to an increase in PDK activity (Watt et al. 2004). Opposite

of expected, intralipid infusion resulting in elevated plasma FFA level, maintained the level of

PDHa activity and of PDH dephosphorylation after 3 hours of two-legged knee extensor exercise

relative to a decrease observed at 3 hours of exercise in the control trial. (Pilegaard et al. 2006).

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This suggests that elevated circulating FFA are not sufficient to obtain the down-regulation of

PDHa activity late during prolonged exercise and that additional factors also play a role.

Previous findings in gene modified mice showing that AMPK KO mice exhibited a more marked

exercise-induced increase in PDHa activity and PDH dephosphorylation than wildtype mice

indicated that AMPK may exert an inhibitory effect on exercise-induced PDH regulation. The

concomitant observation that in vitro muscle incubation with AICAR had no effect on PDH

phosphorylation did however not support such a link between AMKP and PDH (Klein et al. 2007).

On the other hand, the observations in AMPK KO mice (Klein et al. 2007) combined with the

observations that plasma interleukin (IL)-6 increased during prolonged exercise (Keller et al.

2001;Steensberg et al. 2002), IL-6 infusion increased fat oxidation in human skeletal muscle (van et

al. 2003;Wolsk et al. 2010) and IL-6 injections increased AMPK phosphorylation in rat skeletal

muscle (Kelly et al. 2009;Ruderman et al. 2006) may suggest that an IL-6-AMPK-PDH mediated

regulation is involved in regulating the shift towards fat oxidation in skeletal muscle during

prolonged exercise. However, the potential role of IL-6 in regulating skeletal muscle PDH

regulation is unresolved.

PDH and inflammation

One of the factors suggested to play a role in lifestyle related metabolic diseases is low-grade

inflammation characterized by 2-3 fold elevated level of circulating cytokines (Dandona et al.

2004). One of these cytokines is the pro-inflammatory cytokine, tumor necrosis factor (TNF)α,

which has been observed to be elevated in the plasma of obese individuals and T2D patients as well

as in elderly subjects (Bruunsgaard et al. 2001;Bruunsgaard, Pedersen 2003;Hotamisligil

1999;Plomgaard et al. 2005;Stephens et al. 1997). Furthermore TNFα has been reported to induce

insulin resistance in cell culture as well as in mouse and human skeletal muscle (Hotamisligil

1999;Hotamisligil 1999;Plomgaard et al. 2005;Stephens et al. 1997). Hence, 2 hours of TNFα

infusion has been shown to reduce glucose uptake during a hyperinsulinemic euglycemic clamp in

humans (Plomgaard et al. 2005) suggesting that elevated circulating TNFα plays a role in the

development of insulin resistance. Although very little information exists on this, results from a few

studies do indicate an inhibitory effect of inflammation on PDHa activity. Hence, incubation of

cardio myocytes with TNF-α for 24 hours has been shown to reduce the PDH activity (Zell et al.

1997). Furthermore, 24 hour of LPS infusion increased PDK4 mRNA and protein content, reduced

PDP1 mRNA content and reduced the PDHa activity in rat skeletal muscle (Crossland et al. 2008).

Similarly, sepsis in rats induced by E. coli has been shown to decrease PDH activity (vary 1999,

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vary martin 1993) and increase PDK activity in isolated mitochondria from skeletal muscle (Vary,

Martin 1993;Vary et al. 1998;Vary, Hazen 1999). However the impact of inflammation on PDH

regulation in human skeletal muscle at rest and during exercise is unknown.

Physical activity Several factors contribute to the development of metabolic inflexibility including insulin resistance

in skeletal muscle including genetics, microbiota and lifestyle related factors like obesity and

physical inactivity (Fletcher et al. 2002;Meng et al. 2013;Morine et al. 2010). The major impact of a

physical active lifestyle in reducing the risk of developing lifestyle related diseases and in

improving health in general underlines, however, the special importance of physical activity in this

(Pedersen, Saltin 2006).

The effects of endurance exercise training in preventing lifestyle related metabolic diseases involve

numerous types of adaptations in several tissues of the body including skeletal muscle. These

adaptations are initiated in response to each single exercise bout and are thought to arise from

cumulative effects of each exercise bout (Pilegaard et al. 2000). However, the metabolic challenges

during exercise with enhanced fat and carbohydrate utilization in skeletal muscle may also provide

beneficial effects through the increased removal of fatty acids and/or glucose from the blood. In

addition, the molecular changes in skeletal muscle induced by endurance exercise training improve

both metabolic capacity and flexibility (Meex et al. 2010), which together will improve metabolic

regulation and exercise endurance performance. Hence, an understanding of the impact of physical

activity in the prevention of lifestyle related metabolic diseases requires both an examination of

metabolic regulation during exercise as well as the adaptations to exercise training.

Physical activity level – physiological adjustments

It is well established that the physical activity level has a major impact on both cardiovascular and

metabolic parameters with effects on maximal oxygen uptake, exercise endurance and metabolic

flexibility. Previous studies have used both decreased and increased level of physical activity to

examine the impact of physical activity on skeletal muscle metabolism.

Several studies have reported that exercise training improved insulin sensitivity and/or insulin

responsiveness in skeletal muscle (Dela et al. 1994a;Mikines et al. 1988;Consitt et al. 2013;Frosig

et al. 2007), which is related to the improved capacity for glucose handling. Similarly, many studies

have demonstrated increased exercise endurance using for example an ergometer bike or one-

legged knee extensor exercise (Meex et al. 2010;Perry et al. 2008;Yeo et al. 2008;Pilegaard et al.

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2003b). Lifelong exercise trained subjects have also been shown to have markedly higher exercise

endurance during cycling than age-matched untrained subjects (Iversen et al. 2011). Furthermore

exercise training has been reported to improve glucose regulation both using hyperinsulinemic

euglycemic clamp and an oral glucose tolerance test (REF; REF). This indicates that elderly

subjects have maintained the ability to adapt to exercise training (Consitt et al. 2013;Cox et al.

1999;Iversen et al. 2011). However, the underlying mechanisms behind the improved glucose

regulation with exercise training in elderly subjects are not fully resolved.

The National Space Agency (NASA) was among the first to investigate the effect of physical

inactivity on the human body through the investigations of the effects of weightlessness during

space travel (Carlson 1967;Lipman et al. 1970;Lipman et al. 1972;Lutwak L, Whedon GD

1959;Mikines et al. 1989;Mikines et al. 1991;Saltin et al. 1968). In addition, one of the first studies

investigating the impact of physical inactivity on cardiovascular functions showed that only 20 days

of bed rest elevated the heart rate during submaximal exercise due to reduced stroke volume and

reduced maximal oxygen uptake due to reduced maximal stroke volume and cardiac output (Saltin

et al. 1968).

Mikines et al (Mikines et al. 1989;Mikines et al. 1991) investigated the effect of 7 days of bed rest

on both whole body and skeletal muscle insulin sensitivity using the hyper-insulinemic euglycemic

clamp technique. They observed that insulin-stimulated glucose uptake in the legs was reduced

following 7 days in bed, clearly demonstrating a decreased skeletal muscle insulin sensitivity.

Furthermore, using a glucose clamp, the same study also showed that the glucose-stimulated insulin

secretion was increased after bed rest (Mikines et al. 1989). Even as little as 3 days in bed have later

been shown to increase heart rate during a submaximal exercise bout, decrease glucose tolerance

and increase the plasma insulin level during an oral glucose tolerance test (Smorawinski et al.

2000).

Detraining can be used as another model of physical activity level to study insulin sensitivity. Dela

et al. observed a decrease in insulin sensitivity after only 6 days of detraining, underlining the

impact of the physical activity level on skeletal muscle insulin sensitivity (Dela et al. 1992).

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Physical  activity  level  –  and  molecular  adaptations  

Akt

Several studies have demonstrated that exercise training increases both Akt 1 and Akt 2 protein

content in human skeletal muscle (Consitt et al. 2013;Vind et al. 2011;Yfanti et al. 2011). However,

no studies have reported the impact of physical inactivity on insulin-mediated Akt regulation.

TBC1D4

Exercise training has been reported to increase TBC1D4 protein content in young subjects (Frosig

et al. 2007), but not in aged subjects (Consitt et al. 2013;Vind et al. 2011). Furthermore, exercise

training has been shown to reverse the impaired insulin-stimulated TBC1D4 phosphorylation

observed in T2D patients (Vind et al. 2011) and elderly subjects (Consitt et al. 2013) underlining

the impact of exercise training on key factors in insulin-mediated cellular responses. However, no

studies have examined the effect of physical inactivity on insulin-mediated TBC1D4 regulation.

GLUT4

Skeletal muscle GLUT4 protein content has been reported to be reduced following 10 days of

detraining (McCoy et al. 1994) and in accordance exercise training has in many studies been shown

to increase the GLUT4 protein content in skeletal muscle of both young and aged subjects (Consitt

et al. 2013;Cox et al. 1999;Dela et al. 1994c;Frosig et al. 2007). However, no studies have

investigated the effect of physical inactivity on GLUT4 protein content in skeletal muscle.

(Kraniou et al. 2006)

HKII

Although hexokinase is considered a glycolytic enzyme, numerous studies have shown that HKII

activity (Bernadr R, Peter J. 1969) and HKII protein content can increase in mouse skeletal muscle

and skeletal muscle of young subjects with endurance exercise training (Frosig et al. 2007;Leick et

al. 2008). However, no studies have investigated the effect of physical inactivity on HKII protein

content in skeletal muscle or whether exercise training can also enhance HKII protein in elderly

subjects.

GS

Exercise training increases total GS activity in human skeletal muscle both in healthy subjects

(Frosig et al. 2007;Vind et al. 2011) and in T2D patients (Perseghin et al. 1996;Vind et al. 2011). In

line, skeletal muscle GS protein content has been reported to increase following 10 weeks of

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endurance exercise training (Vind et al. 2011), but not to change with 3 weeks of one-legged knee

extensor exercise training (Frosig et al. 2007). The improved insulin-stimulated glucose uptake

observed after an exercise training period may thus in part be due to an increased capacity for GS

synthesis if the exercise training period is of sufficient duration (Christ-Roberts et al. 2004;Frosig et

al. 2007;Vind et al. 2011). The GS activity has also been observed in T2D patients indicating that

improved glucose handling in T2D patients with exercise training include increased GS activity

(Perseghin et al. 1996;Vind et al. 2011). However the impact of physical inactivity on prior

exercise enhancement of insulin-stimulated GS regulation and the impact of exercise training on

skeletal muscle GS regulation in elderly subjects are unresolved.

PDH

The protein content of PDH-E1α in skeletal muscle is increased in human skeletal muscle with

exercise training (Burgomaster et al. 2008;LeBlanc et al. 2004b;Sjogaard et al. 2013) and the

oxidative capacity has been shown to correlate with the protein content of PDH-E1α (LeBlanc et al.

2004a;Love et al. 2011). In line with the increased PDH-E1α protein content, PDK2 protein has

also been shown to increase with exercise training, while PDK4 protein has been reported to remain

unchanged with exercise training (LeBlanc et al. 2004b). The exercise-induced PDHa activity has

been shown to depend on the relative exercise intensity (Howlett et al. 1998). In accordance, PDHa

activity during exercise at the same absolute workload was demonstrated to be lower after an

exercise training period than before (LeBlanc et al. 2004a). However, 7 days in bed have been

reported not to affect the exercise-induced PDH regulation in human skeletal muscle (Kiilerich et

al. 2011) indicating that PDH regulation is affected differently by increased and increased physical

activity.

Oxidative proteins

Numerous previous studies have demonstrated that endurance exercise training increases skeletal

muscle oxidative capacity through enhanced activity and/or protein content of oxidative proteins

and increased capillarization (Henriksson, Reitman 1977;Iversen et al. 2011;Klausen et al.

1981;LeBlanc et al. 2004b;Leick et al. 2008). In accordance, RER during submaximal exercise has

repeatedly been shown to be lower after endurance exercise training than before (Henriksson,

Reitman 1976;Kiens et al. 1993) reflecting a change in substrate utilization towards increased fat

oxidation (Kiens 2006). Furthermore, 6 weeks of exercise training followed by detraining

demonstrated that the activity of oxidative enzymes decreased abruptly when the exercise training

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34    

was terminated (Henriksson, Reitman 1977). In line several studies have later reported reduced

citrate synthase (CS) activity after a period with physical inactivity (Coyle et al. 1985;Jansson et al.

1988;Richter et al. 1989), while 7 days of bed rest only resulted in a visual decrease in skeletal

muscle CS and 3-hydroxyacyl-CoA-dehydrogenase (HAD) activity. Such reduced capacity for

substrate oxidation may reduce the ability of skeletal muscle to remove both glucose and FFA from

the blood (Mikines et al. 1991).

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AIMS The overall aim of this PhD thesis was to investigate 1) the impact of physical activity level on

glucose/insulin mediated regulation of PDH, GS and insulin signaling in skeletal muscle and 2) the

role of IL-6 and inflammation in exercise-induced PDH regulation in skeletal muscle.

The following questions will be addressed

1) The impact of physical inactivity on insulin-stimulated Akt, TBC1D4 and GS regulation in

human skeletal muscle.

2) The impact of exercise training on gluce-mediated regulation of PDH and GS in skeletal

muscle in elderly men.

3) The impact of inflammation on resting and exercise-induced regulation in human skeletal

muscle.

4) The effect of IL-6 on PDH regulation in mouse skeletal muscle

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METHODS In the following section a description of the methods used in this thesis will be described.

Human subjects Human subjects participated in study I, II, and III. In study I, 12 normally physically active young

male subjects with an average age 26±3 years. In study II, 13 elderly male subjects with an average

aged of 65±1 years and in study III 9 untrained subjects age with an average age of 20.8±1 years

participated. Ethical approvals study I, H-C-2007-0085, study II H-2-2011-079 and study III, H-1-

2012-108. All the subjects were informed of the risk and discomfort associated with the

participation in all studies and the experimental protocols were explained in details. The subjects all

provided there oral and written consent to participate. All studies were conducted in accordance

with the guidelines of the Declaration of Helsinki.

Mice In study IV, 8 weeks old female C57/B6 mice (Taconic, Lille Skensved, Denmark) were used. The

mice had free access to a normal chow diet (Altromin 1324, Brogaarden, Lynge, Denmark) and

water ad libitum. The mice had a 12:12 light-dark cycle. The mice were housed together until the

day before the experimental day, where they were housed individually. The ethical approval in

study IV, 2009, 561 1607.

Experimental  protocols  

Bed  rest    The protocol used in study I, were a 7 days bed rest with performance tests and hyperinsulinemic

euglycemic clamp before and after the. The subjects performed a one-legged knee extensor

endurance exercise test to exhaustion and a VO2max test before and after the 7 days in bed. The

subjects were allowed and encouraged to sit in a wheel chair for up to 5 hours a day, to avoid

cardiovascular complication. The subjects were transported in wheelchairs to the bathroom.

Rigshospitalets kitchen delivered all meals to the subjects during the bed rest period, where the

subjects were allowed to eat ad libitum.

LPS  Lipopolysaccharide (LPS) endotoxin is a protein located in the cell wall of gram negative bacteria

and is the compound that our immune system reacts to during a bacterial infection (Elin et al. 1981).

The LPS can create acute inflammation in subjects when injected with a dose of LPS. The LPS will

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indorse the macrophages to produce TNFα and IL-6 (Andreasen et al. 2008;Frost et al. 2002). In

study III untrained and trained subjects were injected with 0.3 ng/kg LPS based on previous studies

(Andreasen et al. 2008;Andreasen et al. 2011) and pilot trials on 3 subjects. The LPS injection was

given 2 hours before a 10 min one-legged knee extensor exercise bout at 60% of Wattpeak. Biopsies

were obtained before and after the exercise bout.

Catheterization    In study I and III, the subjects had catheters placed in the femoral vein (18 Ga. X 8”, Arrow,

Athlone, Ireland) and femoral artery (20 Ga. X 5”, Arrow, Athlone, Ireland). In study I catheters

were placed in the femoral vein of both legs and one femoral artery, in the rested and exercise leg

respectively. The femoral vein and artery was found with a combination of palpation and ultra

sound. The area where the catheter was inserted was shaved free of hair and disinfected with

ethanol. When the area is clean it was covered with a sterile cloth. The vein was localized with ultra

sound punctuated with a needle and the catheter was inserted via a guide wire. A similar procedure

was performed with the artery. The catheters was flushed with sterile saline and taped to the skin to

minimize irritation during the exercise bout.

Hyperinsulinemic  euglycemic  clamp    There are several ways to assess insulin sensitivity. One method is with the hyper insulinemic

euglycemic clamp technic first described by deFronzo in 1979 (DeFronzo et al. 1979). Insulin (Acta

rapid, Novo nordisk, Denmark) was infused into an arm vein in the subject at a given infusion rate

while the blood glucose concentration was kept at approximately 5mM by a continuous infusion of

glucose. The amount of glucose infused to keep glucose 5mM was a reflection of whole body

insulin sensitivity (DeFronzo et al. 1979). The clamp technique was used in study I with an insulin

infusion rate of 50 mU min-1· m-2. This results in plasma concentration between 400-500 pmol/l that

was within the normal physiological range. The blood glucose concentration was measured during

the clamp to make sure that the blood glucose does not drop to a critical low level. The plasma

glucose was measured on a Radiometer ABL 725 series every 5-10 min and the glucose infusion

rate was adjusted accordingly. The muscle tissue will during a hyper insulinemic clamp be affected

by elevated plasma insulin, but the plasma glucose concentration around 5mM.

Interleukin-­‐6  injection    In study IV, mice were injected with 3 ng/g recombinant mouse (rm) IL-6 or phosphate buffer

saline (PBS) in the fed or fasted state (16-18h). The quadriceps muscles and trunk blood were

obtained 30 min or 60 min after the injection.

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In  vivo  tests  

Oral  glucose  tolerance  test  Another and more simple way to examine potential changed in glucose metabolism was to use an

oral glucose tolerance test (OGTT) that was first described by Boudouin 1908 with a 100 gr.

glucose load. In study II the subjects consumed a glucose drink containing 1 g glucose per kg body

weight. Blood samples were obtained at 15, 30, 45, 60, 90 and 120 min and plasma glucose, insulin

and C-peptide concentrations were measured. This will give an indication of the glucose tolerance

of the subjects. The muscle tissue during a OGTT potentially be affected by the transient elevation

in both plasma glucose and insulin concentrations.

Exercise  performance  Performance tests were used in study I, II and III to determine fitness and exercise endurance of the

subjects. A one-legged knee extensor exercise was used in study 1, 3 and 4. The one-legged knee

extensor exercise was first described by Andersen el al (Andersen et al. 1985), where they used a

modified Krogh bicycle ergometer. For the studies described in this thesis the test was performed

on a modified Monach Ergometer bicycle ergometer, but with almost the same setup as in 1985.

The bike was mounted with a rod and attached to one side of the crankset, making it possible for the

subject to kick either with the right or left leg. The subject was instructed to continue until

exhaustion, that was when it was no longer possible to keep up a frequency of 60 per min. The

subjects kicked with a frequency of 60 kicks/min. The test begins with a 5 min warm up followed

by the test a graded incremental test with an increase in resistance (6W) every second minute. The

endurance test was started with different load with either untrained (18W) or trained (24W).

VO2max  test  The subjects maximal oxygen uptake in study I, II and III using a graded bicycle ergometer test.

The test is performed on an electric Monach Ergometer bike (Monark 839E, Sweden), a modified

version of the original first described by August Krogh in 1911-1913 (Krogh 1913). Oxygen uptake

was determined using an online system (COSMED Quark b2). The test was started with a 5 min

warm up, followed by an incremental increase with 30 W every minute. The subjects respiratory

exchange ratio should be ∼1.10-1.15 at exhaustion and the test should optimally last between 5-10

min (Astrand, Rodahl 1977).

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Laboratory  analyses  

Muscle  biopsies  Muscle biopsies were obtained from the vastus lateralis muscle in study I, II and III. Before the

muscle biopsy was obtained, the area on the thigh was wiped with chlorhexidine and the skin area

was anesthetized (lidocain) and an insertion was made in the skin. Muscle biopsies were obtained

using the percutaneous needle biopsy technique (Bergstrom 1962) modified with suction (Evans et

al. 1982) from individual insertions in the skin. The biopsies were quickly dissected free from

connective tissue and visual blood and frozen in liquid nitrogen within 25 sec and stored at -80°C.

Freeze-­‐drying    In study I, II and III the muscle samples were freeze-dried to make it possible to separate blood and

connective tissue from the muscle tissue. Muscle pieces to measure PDHa activity and mRNA were

cut off while 80-100 mg of the remaining samples were freeze-dried. The samples were placed in -

25°C and 0.1 bar pressure for at least 48 h that allows the frozen water in the muscle samples to

evaporate. After 48 h, the samples were moved to room temperature for 1 h ad 0.1 bar to be ready

for dissection. Before starting the dissection, a water calibration was made to make it possible to

calculate the exact dry weight of the samples. One sample was weighted at 20 s, 30 s, 40 s, 50 s, 60

s, 90 s and 120 s after the release of the vacuum. The muscle samples were dissected free from

blood and connective tissue and weighted out to western blotting analyses, glycogen content,

enzyme activity and perchloric acid (PCA) extract. Due to the very low water content within the

sample <10% the protein phosphorylation and enzyme activity is maintained in the freeze-dried

muscle samples (Essen et al. 1975;Schantz, Henriksson 1987).

Muscle  glycogen  The muscle glycogen content was determined in ∼0.8 mg dry weight or 225 µg protein in

homogenate samples in study I, III, and IV and in ∼20 mg wet weight in study II as previously

described (Passonneau, Lauderdale 1974) was boiled for 2 hours in 1 M HCl, which hydrolyses the

glycogen into glycosyl units. The samples were quick spun and the solution free of tissue is

transferred to a new tube. The samples were then loaded to a white mikrotiter plate (Whatman,

Frisenette, Ebeltoft, Denamark) and a reaction mix is added. The mix contains Trisbuffer, H2O,

ATP, MgCl, DTT and NADP+. This assay is based on the auto fluorescence properties of NADPH.

By adding the enzyme G-6-PDH, any G-6-P and NADP+ are converted into 6- phosphogluconic

acid and NADPH that was measured spectophotometrically.

Glycogen --->boiling for 2hours ----> Glucose

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G-6-P + NADP+ ---G-6-PDH ---> 6PG + NADPH + H+

Glucose + ATP ---> HK ---> G-6-P + ADP

The glycogen concentration was based calculated on a standard curve made from standards loaded

together with the samples on the plate.

Muscle  creatine  The creatine content was determined in the homogenate used for determining PDHa activity. The

creatine content was use to normalized the PDHa activity, to the total muscle content in each

sample. The creatine was extracted with 0.6 M PCA for 1 hour at room temperature. The reaction

was neutralized with KHCO3 and the samples were centrifuges at 10.000 g for 3 min and

supernatant was transferred to new tubes. The creatine content was determined by using the auto

fluorescence of NADPH in the reaction described in fig xx. The samples and a standard with a

known concentration of creatine is loaded to a white microtiter plate a mix containing: Hepes,

MgCl2, phosphoenol pyruvate, NADH, pentaphosphate pentasodium, ATP, pyruvate kinase (PK)

and lactate dehydrogenase (LDH) is added to each well on the plate and incubated for 20 min. The

plate was then analyzed in a Flouroscan (Thermo Sientific, Rockford, IL, USA) and creatine kinase

(CK) was added to start the reaction described below.

Creatine + ATP ----CK----> P-Creatine + ADP

ADP + P-Pyruvate ----PK----> ATP + Pyruvate

Pyruvate + NADH + H ---LDH---> Lactate + ADP

Muscle  G-­‐6-­‐P  and  lactate  For measuring lactate and G-6-P in muscle 0.8 mg of freeze dried muscle was hydrolyzed with 0.6

M PCA to extract the metabolites (Bergmeyer H.U. 1965). The samples were kept on ice and the

PCA was added the samples were then placed 30 min at 5C°. The sample was transferred to new

tubes and neutralized with KHCO3.

The G-6-P content in the muscle samples was determined in the PCA extract. In a similar way

described above in the glycogen and creatine assay. The samples was loaded on a white microtiter

plate and a mix containing Trisbuffer, H2O, ATP MgCl, DTT and NADP was added and analyses in

a flouroscan G-6-PDH enzyme was added to start the reaction described below. The samples was

incubated for 30 min and analyzed again.

G-6-P + NADP+ ---G-6-PDH ---> 6PG + NADPH + H+

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Glucose + ATP ---> HK ---> G-6-P + ADP

The muscle content of lactate was determined in the PCA extract from the muscle samples. The

samples were loaded on a white microtiter plate and a mix containing Glycylglycine, H2O, NAD+

and Glutamic acid. The plate was analyzed in a floroscan and a enzyme mix containing LDH and

glutamic-pyruvate transaminase was added to start the reaction described below and incubated for

60 min. The plate was then analyzed in a flouroscan and the lactate content was calculated.

L-Lactat + NAD+ ---LDH---> Pyruvat + NADH + H+

Pyruvat + Lglutamat ---GPT---> Lalanine + α-Oxoglutarate

mRNA  isolation  and  PCR  Total RNA was isolated from ∼25 mg muscle with the guanidinium thiocyanate (GT)-phenol-

chloroform extraction method as previously described (Chomczynski, Sacchi 1987) and modified

by Pilegaard et al (Pilegaard et al. 2000). The muscle samples were weighted of in a sterile tube in a

-20°C freezer. The samples were homogenized in an ice-cold GT and β-mercaptoethanol (BME)

solution using a TissueLyserII (Quiagen, Germany) and placed on ice. The GT and BME denature

proteins including RNases. The RNA was extracted by adding phenol, chloroform:isoamyl (49:1)

and NaOAc and the samples were shaken vigorously and placed on ice for 15 min followed by

centrifugation at 12.000 g for 20 min at 4°C. This results in separation into an upper aqueous phase

and a lower organic phase. The organic phase contains proteins while the aqueous phase contains

the RNA. The aqueous phase was carefully transferred to a new tube. Ice-cold isopropanol (-20°C)

was added and the samples vortexed vigorously followed by incubation at -20°C for at least 15 min.

The samples were centrifuged at 12.000 g for 10 min at 4°C resulting in an RNA pellet. The

supernatant was poured off and 75% EtOH in DEPC H2O was added followed by centrifugation at

12.000 g for 5 min at 4°C. The wash step was repeated followed by vacuum drying of the tubes and

resuspension of the pellet in 1 µl/mg DEPC H2O containing 0.1 mM EDTA. It is important to work

with sterile tubes and tips and to keep the samples on ice at all times to keep RNase inactive.

SDS-­‐page  and  immunoblotting    The method was described and presented in 1979 (Towbin et al. 1979) and later modified (Burnette

1981). The method used in our laboratory is further modified from the original description. The

analyses were made with either homogenate or lysate made from the homogenized samples. The

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42    

muscle samples were homogenized in an ice cold buffer containing 50 mmol/l HEPES, 150 mmol/l

NaCl, 20 mmol/l Na4P2O7, 20 mmol/l β-glycerophosphate, 10 mmol/l Na3VO4, 2 mmol/l EDTA,

1%, NP-40, 10%, glycerol, 2 mmol/l phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 20 µg/ml

aprotinin and 3 mmol/l benzamidine using a TissuLyserII (Quiagen, Germany) for 2 min. Freeze-

dried samples from the human studies (study I, II and III) were diluted 1:80, while wet weight

samples were diluted 1:20 in the mouse study (Study IV). Afterwards the samples were rotated end

over end for 1 hour. A part of the homogenate is transferred to new tubes, while the remaining of

the samples were centrifuged at 17500 g at 4°C for 20 min to obtain lysate (the supernatant), that

were transferred to new tubes. The homogenates and lysates are stored at -80°C

The protein concentration in the homogenates and lysates was determined in microtiter plates using

the bicinchoninic acid method (Pierce Chemical). This method uses that the reaction between Cu2+

and protein under alkali conditions reduced Cu2+ to Cu+ that produces a color shift, detectable at a

wavelength at 550 nm. This value is converted to a protein concentration based on albumin

standards (Thermo Sientific, Rockford, IL, USA) loaded on the plate.

To prepare the samples for SDS-PAGE the homogenate and lysate samples were mixed with

sample buffer, containing Tris Base, Dithiothreitol (DTT), sodium dodecyl sulfate (SDS), glycerol

and bromphenol blue. The samples were heated to 96 °C for 3 min, that denatures the proteins and

SDS coats the proteins with negative charges, according to the size of the protein approximately

one SDS molecule for every two amino acid residues.

Gels  The hand casted or pre-made gels (24 well, Bio-Rad, Hercules, CA, USA) were placed into a gel

electrophoresis cell and running buffer solution containing Tris-base, glycine, SDS amd H2O is

added. The samples were loaded onto the gel, together with a protein standard and a protein ladder

in each side of the gel. The protein standards were used to normalize between gels and to evaluate

both the run across each gel and the quality of the blotting process. The ladder helps to identify the

relevant part of the gel for visualizing a specific protein of interest. An electric current was applied

to the electrophoresis cell, 150 V and 40 mA per gel in the cell. The SDS coated proteins is

negatively charged and will know be pulled through the gel according to charge and therefore the

size of the protein. When the samples were running through the gel, a color front was created by the

sample buffer, helping to estimate when the samples have proceeded sufficiently, through the gel.

The proteins in the gel were transferred to a polyvinylidene fluoride (PVDF) membrane

(Immobilon-P membrane, Millipore, Billerica, MA, USA) by using the semi dry blotting. The gel

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43    

piece of interest was cut out of the gel and placed in a sandwich consisting of 3x2 pieces of filter

paper with the gel in between. The sandwich was stacked on a blotting apparatus (Transblot DF

semi-dry Electrophoretic transfer cell, Bio-Rad, Hercules, CA, USA) and an electric field was

applied at 20 V and 0.8 mA per cm2 to horizontally drawn out the proteins and captured them in the

membrane.

After the protein transfer it is important to block the membrane in either bovine serum albumin

(BSA), skin milk solution or fish gelatin to ensure that the PVDF membrane was blocked with

protein. This improves specific binding of the primary antibody to the protein of interest. The

membrane was then incubated overnight in primary antibodies diluted in BSA.

The following day the membrane was washed in tris buffered saline (TBS) with tween 20 (TBST)

and species specific secondary antibodies (DAKO, Glostrup, Denmark) against the primary

antibody was added. Horse radish peroxidase is attached to the secondary antibody and addition of

ECL (Luminata, Millipore, Billerica, MA, USA) reagent containing luminol reagts a creates a

photon which is detected by a digital photo analyzing system (Image Quant LAS4000, Munich,

Germany and Carestream Health, Rochester, NY, USA). The proteins will in this way be visualized

as band on the PVDF membrane. The bands can then be analyzed and quantified using computer

software (Image Quant TL, Munich, Germany and Carestream MI SE, Rochester, NY, USA). The

intensity of the bands on each gel was divided with the average of the loaded standard on the same

gel, making it possible to compare the intensity of samples loaded on different gels.

GS  activity  The GS activity was determined in muscle homogenate as previously described (Thomas et al.

1968) but modified to microtiter plates. A total of 125µg protein was used from each sample and

diluted to a concentration of 1 µg/µl in a new tube. The GS activity was calculated as UDP-glucose

incorporated into glycogen per minute per gram tissue in the presence of low concentration of G-6-

P (0.02 mM), a physiological concentration (0.17 mM) of G-6-P and a high concentration (8 mM)

of G-6-P to get the total activity. These values were then used to calculate the percentage of GS G-

6-P independent GS activity (0.02mM / 8 mM) (GS I-form) and the GS fractional velocity (GS

%FV) (0.17mM/ 8 mM).

PDHa  activity    The activity of PDH in the active form (PDHa) is determined based on the rate of acetyl-CoA

formation and the following determination of acetyl-CoA using a radioisotopic assay.

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44    

The protocol was modified from the methods described in two original manuscripts (Cederblad et

al. 1990;Constantin-Teodosiu et al. 1991). Approx. ∼10 mg of frozen muscle sample was

homogenized in 225 µl ice-cold homogenizing buffer containing Sucrose, KCl, MgCl2, EGTA, Tris

HCl, NaF, DCA and Triton X-100 in a glass tube on ice for 50 sec using a motor-driven

homogenizer and a glass pluger (Kontest, Vineland, NJ, USA). This process will gently tear apart

the tissue and the without disrupting the PDC in the mitochondria. The homogenate is transferred to

a tube containing the rest of the homogenizing buffer up to a total volume given by 30 x weight of

the sample and the sample is quickly frozen in liquid nitrogen.

The homogenate was transferred to an assay buffer solution containing Tris-base, EDTA and

MgCl2 and kept at 37°C. To start the reaction, pyruvate is added, and after 45 s, 90 s and 135 s

sample was transferred to a new tube containing PCA. Duplicates are made and a reaction with

water added instead of pyruvate is also performed to serve as “black”. The total of 9 samples was

neutralized after 5 min incubation by addition of potassium bicarbonate. The samples were

centrifuged at 10000 g for 3 min. The supernatant contains the acetyl-CoA of which the content was

determined as previously described (Cederblad et al. 1990) the following 2 enzymatic reactions. A) 14C-aspartate+ 2-oxogluterat ⇔14C-Oxaloacetate + L-glutamate catalyzed by aspartate

aminotransferase and B) 14C-Oxaloacetat + acetyl CoA ⇔ 14C-citrate + CoASH catalyzed by citrate

synthase. Reaction A was necessary because 14-C- oxaloacetate is not commercially available. After

incubation the unreacted 14C- oxaloacetate is transaminated back to 14C-aspartate in a reaction

catalyzed by glutamic oxaloacetic transaminase (GOT) followed by separation of 14C-aspartate

from the formed 14C-citrate using DOWEX, which is a cation exchange resin (Figure 9).

Standards with different concentration of acetyl-CoA were running along with the samples and

were used to create a standard curve. 14C-citrate was measured in the scintillation counter. The

count for each of the samples was related to the standard curve and an average of the duplicates was

calculated. After taking dilutions during the assay into account, the PDHa activity was given in

mmol·min-1·kg-1.

The total creatine content in each sample is extracted using the PCA method (Bergmeyer H.U.

1965). The method is described under creatine content.

Each sample was normalized to the total creatine content in the sample, to adjust for the presence of

non-muscle tissue in the samples (St Amand et al. 2000).

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45    

Fig 9 the reactions in the PDHa assay drawn by Prof. Henriette Pilegaard (Cederblad et al. 1990;Constantin-Teodosiu et

al. 1991)

Enzyme  activity  The enzyme activity of 3-hydroxyacyl-CoA and citrate synthase in study I were measured in muscle

homogenate as previously described (Lowry, Passonneau 1972).

Statistics In all manuscripts the values were presented as mean±SE. Two way ANOVA tests were used to

evaluate the effects in study I a Two-way ANOVA with repeated measures was applied to evaluate

the effect of exercise and insulin before as well as after bed rest. Also, a two-way ANOVA with

repeated measures was used to evaluate the effect of bed rest and insulin within the rested leg as

well as within the exercised leg. The data were log transformed if normality or equal variance tests

failed. If significant main effects were found, the Student-Newman-Keuls post hoc test was used to

locate differences. Differences were considered significant at P <0.05. A tendency is reported for

0.05 # P , 0.1. Statistical calculations were performed using SigmaStat version 3.1.

In study II a student paired t-test was used to test the effect of exercise training on fasting plasma

insulin and glucose as well as protein content. Two-way ANOVA with repeated measures was used

to test the effect of exercise training and glucose intake on plasma glucose, insulin and c-peptide as

well as skeletal muscle protein and protein phosphorylation levels. When a main effect was

observed, a Student-Newman-Keuls post hoc test was used to locate differences between groups if

the data set was log transformed if the data did not pass equal variance test. The data were

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46    

considered significant at P<0.05 and a tendency is reported when 0.05≤P<0.1. Statistical

calculations were performed in SigmaPlot 11.0.

In study III A Two Way ANOVA with repeated measures was used to evaluate the effect of LPS

and exercise. The data were log transformed if normality or equal variance test failed. If a

significant main effect was detected, the student Newman-Keuls test was used to locate differences.

Differences are considered significant at p<0.05 and a tendency is reported for 0.05≤p<0.1.

Statistical calculations were performed using SigmaPlot 11.0

In study IV a two-way ANOVA tests were used to evaluate the effect of fasting and IL-6 injection

by testing within each time point and within each condition. The data were log transformed if equal

variance test failed. If a main effect was observed, the Student–Newman–Keuls post hoc test was

used to locate differences. In addition, a Student’s t test was used to evaluate the effect of IL-6

within a given time point and condition. Differences were considered significant when P <0.05 and

a tendency is reported for 0.05≤P<0.1. The statistical tests were performed using SigmaPlot 11.0.

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47    

INTEGRATED DISCUSSION In the following the data from the 4 manuscripts of the present thesis will be discussed. Additional

data from the 4 studies not included in the manuscripts and unpublished data from a mouse

HFD/exercise training study, a single LPS injection in mice, a single AICAR injection in mice and

a study examining the impact of glucose intake in recovery from exercise in elite athletes will also

be integrated.

Physical  activity  level  

Effects  of  physical  activity  level  on  key  proteins  in  insulin  signaling  and  glucose  uptake  in  skeletal  muscle  The findings in Study I that 7 days of bed rest reduced whole body and skeletal muscle insulin

sensitivity is in accordance with previous bed rest studies (Mikines et al. 1989;Mikines et al. 1991).

Similarly, in Study I prior exercise enhanced the insulin-induced increase in the arterio-venous

glucose difference as previously demonstrated (Wojtaszewski et al. 1997;Wojtaszewski et al. 2000).

Together this provides the setting for studying the underlying mechanisms behind physical

inactivity induced insulin resistance. Study I shows also for the first time that bed rest did not

abolish the ability of exercise to increase insulin sensitivity of skeletal muscle 3h after exercise,

although the glucose uptake was at a lower level than before bed rest. Furthermore, the

observations that exercise training in elderly men resulted in lower plasma insulin concentration

during an OGGT in the trained state than the untrained together with similar plasma glucose

responses indicate that exercise training induced an increase in insulin sensitivity in aging skeletal

muscle. This is in agreement with previous studies using either an OGTT (Cox et al. 1999) or a

hyperinsulinemic euglycemic clamp (Dela et al. 1996) and provides the background for exploring

the underlining mechanisms behind exercise training-induced improvements in glucose handling in

elderly subjects.

The decrease in HKII and GLUT4 protein content in skeletal muscle of young subjects with bed

rest in Study I is in accordance with many previous studies showing an increase with exercise

training (Christ-Roberts et al. 2004;Cox et al. 1999;Dela et al. 1994c;Frosig et al. 2007). In

addition, the increase in GLUT4 and the tendency for an increase in HKII protein with exercise

training in the elderly subjects in Study II show that this ability was maintained in the elderly

subjects as was also observed for oxidative proteins in the same study (Olesen et al., unpublished

data). Together these findings support that the protein level of both HKII and GLUT4 depends on

the physical activity level in both young and aged individuals. Because HKII and GLUT4 are

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48    

central in skeletal muscle glucose uptake, these changes have likely contributed to the observed

changes in glucose regulation in Study I and II.

In study I, 7 days of bed rest resulted in lower protein content of Akt1 and Akt2 in skeletal muscle

to about 50-70% of the level before bed rest and reduced the Akt Thr308 and Ser473 phosphorylation

level even after normalization to Akt protein in the resting leg before insulin and in both legs after

insulin infusion. However, the insulin-induced increase in Akt Thr308 and Ser473 phosphorylation

during a hyperinsulinemic euglycemic clamp was unaffected by bed rest. The reduced Akt1 and

Akt2 protein content in Study I is in contrast to the lack of change in Akt protein in a similar bed

rest study in subjects with low birth weight (Mortensen et al. 2013), while the reduced Akt

phosphorylation level with bed rest in Study I is in accordance with the reduced Akt Ser473

phosphorylation in the previous bed rest study (Mortensen et al. 2013). The reduced Akt1 and Akt2

protein levels in Study I are however in line with the observed increase in total Akt protein in

previous exercise training studies in young subjects (Frosig et al. 2007;Vind et al. 2011) and does

suggest that the physical activity level can influence Akt protein and phosphorylation level.

Similarly, the observed almost 2 fold increase in Akt2 protein and the more marked increase in

glucose-stimulated absolute Akt Ser473 phosphorylation in the elderly subjects after exercise

training in Study II are in accordance with the findings in a previous study examining elderly aged

subjects (Vind et al. 2011). These observations support that the Akt protein level is sensitive to the

physical activity level and demonstrates that aging muscle maintains the ability to adapt in insulin

signaling components like Akt to exercise training as for HKII and GLUT4. Of notice is that the

glucose intake-induced increase in Akt phosphorylation in Study II was similar before and after

exercise training, when Akt phosphorylation was normalized to the Akt2 protein level. This

suggests that changes in Akt protein content underlines the changes observed in Akt

phosphorylation with exercise training in the elderly subjects, while physical inactivity in young

subjects influences not only protein content, but also additional mechanisms. Although this

mechanism may be lost with aging it may suggest that the changes induced with physical inactivity

and exercise training are not direct opposites, when it comes to Akt regulation.

Previous studies have reported that the protein content of Akt1 and Akt2 in skeletal muscle was

unaffected in skeletal muscle of T2D patients (Vind et al. 2011), although total Akt protein content

has been found to be reduced in another T2D human study (Christ-Roberts et al. 2004). In addition,

as Akt is central in insulin signaling (Hojlund, Beck-Nielsen 2006;Hojlund et al. 2009;Karlsson et

al. 2005;Meyer et al. 2002;Vind et al. 2012) the observed changes in Akt in T2D patients have been

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49    

suggested to contribute to insulin resistance (Hojlund, Beck-Nielsen 2006;Hojlund et al.

2009;Karlsson et al. 2005;Meyer et al. 2002;Vind et al. 2012). Therefore, the observed changes in

Akt protein and Akt phosphorylation level in Study I and II may in part explain the reduced insulin

sensitivity with bed rest observed in Study I and the seemingly increased insulin sensitivity with

exercise training in Study II.

In addition, the observations that bed rest in Study I lowered both Akt1 and Akt2 protein content in

skeletal muscle, while exercise training only increased Akt2 protein content in the elderly subjects

in Study II may suggest that Akt1 is sensitive to physical inactivity but not exercise training or that

the exercise training-induced regulation of Akt1 is age-dependent. While the previous exercise

training studies in young subjects did not discriminate between the isoforms (Christ-Roberts et al.

2004;Frosig et al. 2007), a comparison of the Akt1 and Akt2 protein levels in skeletal muscle of the

untrained subjects in Study III and well trained young subjects, shows that both Akt1 and Akt 2

protein are higher in exercise trained than untrained young subjects (Figure 10).

Akt1 Akt2

Akt

pro

tein

(AU

)

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4Untrained Trained #

#

Fig 10 Akt1 and Akt2 protein content in untrained and trained subjects. The untrained subjects are from Study III.

#:significantly different from untrained, P<0.05

Although it is possible that an exercise training-induced increase in Akt1 protein requires more than

8 weeks of regular physical activity, the present findings do bring up the possibility that the ability

of exercise training to increase Akt1 protein in skeletal muscle is lost with age. As results from

Akt1 overexpression in mice (Lai et al. 2004) have suggested that especially Akt1 plays a role in

translational regulation and hence potentially hypertrophy, an age-related impairment in Akt1

regulation may be speculated to affect the ability to increase muscle mass rather than to improve

glucose regulation with exercise training at increasing age.

The observations that 7 days of bed rest in Study I did not affect either TBC1D4 protein level or the

insulin-induced TBC1D4 phosphorylation at Ser588, Thr642 or Ser751 are surprising based on the

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50    

reduced Akt phosphorylation level. However, a similar lack of change in both total TBC1D4

protein and insulin-stimulated TBC1D4 phosphorylation has been observed in normal birth weight

subjects in a previous 10 day bed rest study (Mortensen et al. 2013). Moreover, in accordance with

the unchanged TBC1D4 protein with physical inactivity in Study I, exercise training in Study II did

not affect TBC1D4 protein in skeletal muscle of the elderly subjects. On the other hand, previous

studies have reported that TBC1D4 protein tended to increase with exercise training in young

subjects (Frosig et al. 2007) and increased in aged subjects (Vind et al. 2012), while another study

reported no change (Consitt et al. 2013). This may indicate that physical inactivity and exercise

training do not necessarily impose opposite effects on TBC1D4 protein in young subjects and that

the ability to increase TBC1D4 protein with exercise training depends on the exact exercise training

protocol. Moreover, the previous finding that exercise training enhanced the insulin-stimulated

TBC1D4 phosphorylation in skeletal muscle of young subjects (Consitt et al. 2013;Frosig et al.

2007) suggests that insulin-stimulated TBC1D4 phosphorylation is not inversely affected by

physical inactivity and exercise training in young subjects as observed for Akt1, Akt2, GLUT4 and

HKII. Furthermore, the observation that glucose intake-induced TBC1D4 phosphorylation was

enhanced after the exercise training period in the elderly subjects in Study II is in accordance with a

previous finding in elderly subjects (Consitt et al. 2013), and suggests that the ability to enhance

insulin-stimulated GLUT4 translocation with exercise training is maintained in aged skeletal

muscle.

The similar regulation of TBC1D4 before and after bed rest in Study I together with the similar

insulin-stimulated increase in the arterio-venous glucose difference before and after bed rest

supports that TBC1D4 is important in glucose uptake. It has been suggested that TBC1D4 in part

can explain the ability of prior exercise to enhance insulin-stimulated skeletal muscle glucose

uptake. However, the observations in Study I, that the insulin-stimulated regulation of TBC1D4

phosphorylation was similar in the rested and prior exercised leg, although the arterio-venous

glucose difference was higher in the prior exercised leg than the rested leg, do not support an

important role of TBC1D4 in this regulation.

Effects  of  physical  activity  level  on  GS  regulation  in  skeletal  muscle  In study I, bed rest did not affect GS protein content or total GS activity in skeletal muscle of young

healthy subjects, although the latter seemed lower than before bed rest. These observations are in

accordance with previous findings showing that exercise training in young subjects did not change

GS protein content in skeletal muscle (Frosig et al. 2007). On the other hand, GS protein content

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51    

and total GS activity increased in skeletal muscle with exercise training in the elderly men in study

II, which is in line with previous observations in aged subjects (Vind et al. 2011). The different

effects of exercise training in young (Frosig et al. 2007) and elderly subjects (Study II) with an

increase in both GS protein and GS activity only in the elderly subjects may suggest that an initial

lower level in the elderly subjects than the young has resulted in a more marked effect of exercise

training in the aged subjects. However, the previous findings that GS protein level and total GS

activity in skeletal muscle were similar in T2D patients and healthy controls (Vind et al. 2011) may

suggest that the different observations rather are due to the exercise training protocols. Hence, the

high-intensity bicycling and cross fit performed by the elderly subjects in Study II may have

provided a stronger stimulus for GS regulation than the one-legged knee extensor exercise used in

the previous study (Frosig et al. 2007).

While bed rest in Study I did not influence the insulin-stimulated dephosphorylation of GS site 3a,

and insulin did increase GS activity also after bed rest, the GS activity tended to be lower and the

insulin-induced dephosphorylation of GS site 2+2a was blunted in the rested leg after 7 days in bed

in Study I. This may in part resemble the impaired insulin-stimulated GS activation and GS site

2+2a dephosphorylation reported in skeletal muscle of insulin resistant subjects and T2D patients

(Glintborg et al. 2008;Hojlund et al. 2003;Hojlund et al. 2006;Hojlund et al. 2008;Vind et al. 2011).

Moreover, the elderly subjects in Study II had before exercise training no change in GS activity or

GS site 2+2a phosphorylation after glucose intake, but GS activity increased and GS site 2+2a

phosphorylation decreased in response to glucose intake after the exercise training period. Taken

together, it may therefore be speculated that 7 days in bed resulted in an impaired insulin-induced

regulation of GS activity due to dysregulation of GS site 2+2a and that this modification is similar

to the changes observed in skeletal muscle of T2D patients (Vind et al. 2011) and potentially also

elderly untrained subjects. In addition, as exercise training in young subjects has been shown not to

change insulin-induced GS activity and/or GS site 2+2a phosphorylation in skeletal muscle, the

changes observed in the elderly subjects in Study II may be explained by an initial impairment in

GS regulation in the elderly subjects in Study II and in previous studies (DeFronzo 1981;Dela et al.

1996;Poulsen et al. 2005).

In study I, a single bout of exercise resulted in higher GS activity in the prior exercised leg before

insulin and GS activity appeared higher in the prior exercised leg than the rested after insulin-

stimulation. This may suggest that regulation of GS at least in part contributed to the enhanced

insulin-stimulated arterio-venous glucose difference in the prior exercised leg relative to the rested

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52    

leg as previously suggested (Wojtaszewski et al. 1997;Wojtaszewski et al. 2000). Furthermore, the

observations that insulin decreased GS site 3a phosphorylation only in the prior exercised leg and

the 2+2a phosphorylation level was lower in the prior exercised leg than in the rested leg before and

after insulin support that modifications of sites 3a and 2+2a likely have played a role in the higher

GS activity in the prior exercised leg than the rested leg in Study I. This is however different from a

previous study showing that prior exercise resulted in a further dephosphorylation of GS site 2+2a

(Prats et al. 2009). In addition, Study I showed for the first time that 7 days in bed did not abolish

the ability of prior exercise to enhance insulin-stimulated skeletal muscle glucose removal, although

the arterio-venous glucose difference was at a lower level after bed rest than before. This ability

may in part be explained by the maintained effect of prior exercise on basal GS activity, although

GS activity only tended to be higher in the prior exercised leg than the rested leg after bed rest.

Furthermore, the exercise bout seemed to prevent the bed rest-induced GS site 2+2

hyperphosphorylation, but the insulin-mediated regulation of GS site 2+2a was not restored.

Together these findings support that GS site 2+2a regulation is important in the observed GS

dysregulation in Study I and II.

As muscle glycogen is one of the factors thought to regulate GS site 2+2a phosphorylation (Nielsen,

Wojtaszewski 2004), the observed changes in muscle glycogen may have played a role in the

modifications in GS site 2+2a phosphorylation in Study I and II. Hence, the lower muscle glycogen

in the prior exercise leg both before and after bed rest in Study I likely contributed to the higher GS

activity in that muscle as previously suggested (Wojtaszewski et al. 1997;Wojtaszewski et al.

2000;Wojtaszewski et al. 2002b). Similarly, the elevated muscle glycogen levels in both legs after

bed rest relative to before in Study I and the elevated muscle glycogen after exercise training

relative to before in Study II may have contributed to the observed elevated GS site 2+2a

phosphorylation, although other factors most likely also have been in play. The link between

muscle glycogen levels and GS site 2+2a phosphorylation during recovery from exercise was

further investigated in an additional study, where elite athletes exercised for 4h at 73% of HRmax

and received either glucose or water for the initial 4h of recovery (Figure 10). During the 24h of

recovery the energy intake was similar in the two groups. Muscle glycogen was markedly reduced

after exercise in both groups, and while glucose intake resulted in an increase in muscle glycogen at

4h of recovery, no change was observed with H2O intake. However, muscle glycogen was similar at

24 h of recovery. These changes in muscle glycogen were associated with lower GS site 2+2a

phosphorylation in the H2O group than the glucose group at 4h and seemingly less than half in the

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53    

H2O group than in glucose group at 24h of recovery. As reduced GS site 2+2a phosphorylation

reflects increased GS activity (Jensen et al. 2012;Prats et al. 2009), a higher GS activity may be

assumed in the H2O group than in the glucose group at 4h of recovery, which is in accordance with

the expected effect of reduced muscle glycogen on GS activity (Nielsen et al. 2001;Wojtaszewski et

al. 1997;Wojtaszewski et al. 2000). However, these findings may also suggest that suppression of

muscle glycogen resynthesis by H2O intake after exercise results in elevated GS activity even 24h

after exercise although muscle glycogen has been restored (Figure 11 left, 11 right).

Fig 11 Muscle glycogen (Left) and GS site 2+2a phosphorylation (Right) in elite athletes before (Pre) and after 4h of

exercise (Post) as well as at 4h of recovery with either H2O (black) or glucose (grey) intake from Post to 4h (4h) and at

24h of recovery (24h). GS phosphorylation is normalized to GS protein content and given in arbitrary units (AU).

Values are mean±SE. *: significantly different from Pre within given group, P<0.05; #: significantly different from

H2O group at given time point, P<0.05; §: significantly different from post exercise, P<0.05 and £: significantly

different from 4h after exercise, P<0.05 and † significantly different from H2O group located with t-test. Parentheses

indicate tendency(Bogardus et al. 1984;Frosig et al. 2007;Perseghin et al. 1996).

Effects  of  physical  activity  level  on  PDH  regulation  in  skeletal  muscle  Previous published results from the same experiment as Study I showed that 7 days of bed rest did

not affect resting PDH-E1α protein, PDH phosphorylation levels or exercise-induced PDH

regulation in human skeletal muscle (Kiilerich et al. 2011). This is in contrast to previous studies

demonstrating that PDH-E1α protein content increases with exercise training in skeletal muscle of

young subjects (LeBlanc et al. 2004a) and unpublished results showing that the PDH-E1α protein

content and the PDH phosphorylation level are higher in well trained young subjects than in the

untrained young subjects from Study III (FIGUR 12). The different sensitivity of PDH-E1α protein

Tri11 Glycogen concentration

Pre Post 4h 24h

Gly

coge

n (m

mol

/kg

dw)

0

200

400

600

800

H2O Glucose

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Pre Post 4h 24h

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54    

to decreased and increased physical activity indicates that basal PDH-E1α maintenance does not

require regular muscle contractions, while increased muscle activity will elicit an increased capacity

for glucose oxidation in accordance with an increased demand from the working muscle.

Fig 12 PDH-E1α, PDHSer295 phosphorylation/PDH-E1α, PDK2 and PDK4 protein content in untrained and trained

subjects additional data from study II, #:significantly different from untrained, P<0.05

The observation that PDH-E1α protein content and PDH phosphorylation increased with exercise

training in the elderly subjects in Study II as observed in young subjects (LeBlanc et al. 2004a)

shows that aged skeletal muscle maintains the ability to increase the capacity for glucose oxidation

with exercise training. The similar increase in PDH-E1α and PDK2, but no change in PDK4 protein

content, in study II suggests that PDK2 may have mediated the increased phosphorylation of PDH

in the trained state in Study II. This is in accordance with unpublished results showing higher PDK2

protein in well trained young subjects than the untrained young subjects in Study III (Figure 11).

The observations that PDK4 protein did not follow the increase in PDH-E1α protein with exercise

training in Study II and the similar level of PDK4 protein in untrained and trained young subjects

(unpublished results; Figure 11) may suggest that a trained muscle independent of age possesses

less capacity for PDK4-mediated PDH inactivation per PDH-E1α molecule. Unpublished results

from a mouse study demonstrated a similar parallel increase in PDK2 and PDH-E1α in skeletal

muscle with exercise training of mice on HFD, but also a parallel increase in PDK4 protein

matching the increase in PDH-E1α. On the other hand, while HFD did not affect PDK2 and PDH-

E1α protein, PDK4 protein content increased providing a likely mechanism for a reduced PDHa

activity as observed in untrained HFD mice (also relative to PDH-E1α protein) reflecting an

enhanced fatty acid oxidation during the HFD. Although the increase in PDK4 protein with exercise

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55    

training in the mouse study (Figure 13) may be species specific, it seems likely that it is due to the

exercise training being combined with HFD intake. Hence, taken together these findings support

previous studies (LeBlanc et al. 2004b;LeBlanc et al. 2004a) and show that endurance exercise

training increases PDH-E1α protein content in skeletal muscle and that physical activity level

regulates PDK2 protein, while PDK4 protein mainly is regulated by substrate availability (Peters et

al. 1998;Peters et al. 2001b).

Fig 13 Protein content of PDH-E1α, PDK2 and PDK4 (left) and PDK4/PDH-E1α (right) after 16 weeks on chow, high

fat diet (HFD) or HFD and exercise training (Ex). Values are mean±SE. #:significantly different from chow, P<0.05; *:

significantly different from HDF, P<0.05.

In accordance with previous studies (Peters et al. 1998;Peters et al. 2001b;Peters et al. 2001a). The

above mentioned HFD mouse study demonstrated that 4 month of HFD resulted in decreased

resting PDHa activity in mouse skeletal muscle (unpublished data). In addition, 4 month of HFD

combined with exercise training in mice increased resting PDHa activity relative to sedentary HDF

mice. This may suggest that the exercise trained muscle maintained the ability to oxidize

carbohydrates to the same extend as chow fed mice. Still, no difference was observed in the RER

during submaximal treadmill exercise. However, it may be speculated that the effect of the

enhanced resting PDHa activity, which was matched by a similar increase in PDH-E1α protein, first

becomes evident during more intense exercise, where carbohydrate oxidation is dominant and

required (unpublished data). This remains to be determined.

Previous unpublished results from our group has shown that PDH phosphorylation and PDHa

activity were unaffected during the 3h hyperinsulinemic euglycemic clamp in the same experiment

as used in Study I (Kiilerich, Biensø, Ringholm, Wojtaszewski, Calbet, Pilegaard) both before and

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56    

after bed rest. However, while insulin was elevated, the plasma glucose concentration was kept

constant and relatively low (5 mM) in Study I, and the use of an OGGT in Study II aimed at

providing a more physiological condition with a transient increase in plasma glucose and plasma

insulin. The findings in Study II that glucose intake did not affect PDHa activity in skeletal muscle

of the elderly subjects in the untrained state and even reduced PDHa activity after the exercise

training period were therefore unexpected. Because a previous study has reported increased PDHa

activity in human skeletal muscle with glucose intake (Pehleman et al. 2005), the lack of change in

Study II may indicate that age or training status has played a role. However, the observation that

glucose intake reduced the PDHa activity in the elderly subjects only after the exercise training

period may suggest that exercise training prevented an otherwise impaired regulation in the elderly

untrained subjects in Study II. In addition, the observed combined regulation of PDH and GS after

the exercise training period may suggest that glucose oxidation is inhibited to prioritize glycogen

storage after the glucose intake, although this suggestion remains to be examined.

Regulation  of  PDH  during  and  after  exercise  

IL-­‐6    The previous finding that PDHa activity after an initial increase decreases towards resting level as

the exercise duration proceeds above 2h has led to the suggestion that PDH is involved in regulating

the switch from carbohydrate to fat oxidation during prolonged exercise, although PDH does not

seem to be the only factor (Mourtzakis et al. 2006;Pilegaard et al. 2006;Watt et al. 2004). Study IV

aimed at elucidating the potential role of IL-6 in this regulation. A single injection of recombinant

IL-6 was given to fed and fasted mice to mimic the transient IL-6 increase in response to exercise

during different metabolic states. The injection increased the plasma IL-6 9.5 fold, which is rather

similar to what has previously been reported in mice running on a treadmill (Nedachi et al. 2008).

The observation that IL-6 reduced PDHa activity in the fed state was in line with the hypothesis that

IL-6 reduced glucose oxidation in skeletal muscle through an inhibition of PDH. The finding, that

IL-6 did not affect AMPK and ACC phosphorylation in the fed state, indicated however that this

IL-6-mediated effect on PDH was not through AMPK, which is not in agreement with a previous

study in rats reporting that IL-6 increased the AMPK phosphorylation in skeletal muscle (Kelly et

al. 2009). However, the observation that AMPK and ACC phosphorylation was increased in the

fasted mice 1h after the injection together with an IL-6-induced increase in PDHa activity may

suggest that an IL-6-AMPK-PDH pathway does exist in fasting conditions although the effect is

opposite of expected based on a previous study in AMPK KO mice (Klein et al. 2007). The idea

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57    

that PDH should be a direct target for AMPK was also investigated in a pilot study. Fed mice were

injected with the AMPK activator AICAR. AMPK phosphorylation increased but no change was

observed in PDHa activity or PDH phosphorylation (data not shown). Furthermore, IL-6 was also

injected at a higher dose resulting in 10 fold higher IL-6 plasma concentration than in Study IV

(data not shown), but this did not affect PDHa activity or PDH phosphorylation in skeletal muscle.

Taken together, it seems that IL-6 can regulate PDH in mouse skeletal muscle, but that the level of

IL-6 and the metabolic condition of the muscle influence this regulation. In addition, whether such

an IL-6-mediated regulation of PDH takes place during prolonged exercise remains to be

determined.

 Inflammation  and  PDH  regulation  during  exercise  Several studies have found that insulin resistant subjects have an impaired ability to switch to

glucose oxidation during an hyperinsulinemic euglycemic clamp (Kelley, Mandarino

2000;Mandarino et al. 1996). The aim of Study III was to investigate whether inflammation is

associated with a similar metabolic inflexibility in exercise-induced PDH and AMPK regulation in

human skeletal muscle reflecting the ability to enhance substrate oxidation. The observation in

Study III that PDH and AMPK regulation in human skeletal muscle was not changed at rest or

during exercise after LPS injection resulting in a 17 fold increase in plasma TNFα indicates that low

grade inflammation does not affect substrate oxidation at rest and in the initial phase of exercise.

These findings are in line with the observation that local infusion of TNFα or LPS did not change

PDH phosphorylation in human skeletal muscle (Bach et al. 2013;Buhl et al. 2013), but different

from studies in rats and another study in humans reporting that PDH or AMPK regulation was

changed during sepsis or LPS infusion (Andreasen et al. 2008;Andreasen et al. 2011;Crossland et

al. 2008;Vary, Martin 1993;Vary et al. 1998;Vary, Hazen 1999). Additional observations in a pilot

study that PDH Ser300 phosphorylation increased in mouse skeletal muscle 2h after a single LPS

injection compared with saline (unpublished results; FIGURE 14) do support the latter findings in

rats and humans. Although species differences may explain the different effects of LPS on PDH

regulation in Study III and the previous studies (Crossland et al. 2008;Vary, Martin 1993;Vary et al.

1998;Vary, Hazen 1999), it is also possible that the level of cytokines like TNFα is important for

the effects on PDH. Hence, the pilot study in mice resulted in 23 fold increase in plasma TNFα,

while plasma TNFα increased 17 fold in Study III. But this remains to be clarified.

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58    

Saline LPS

PD

H S

er30

0 pho

spho

ryla

tion

(AU

)

0

1

2

3

4

5

6

7

(*)

Fig 14 PDH Ser300 phosphorylation 2h after saline or LPS injection. Values are mean±SE. (*): tendency to be

significantly different from saline, 0.05≤p<0.1.

As mentioned previous published results (Kiilerich et al. 2011) from the same experiment as Study

I have shown that the exercise-induced regulation of PDHa activity and PDH phosphorylation was

similar before and after bed rest, even though Study I shows that muscle insulin sensitivity was

reduced. However, the exercise-induced AMPK phosphorylation was abolished after the bed rest

(Ringholm et al. 2011). In accordance with the observations in Study III, this effect did not seem to

be caused by inflammation, because systemic inflammation was not detected after bed rest (Olesen

et al, unpublished data). Taken together, these observations suggest that the exercise-induced

regulation of PDH is rather robust, although previous studies have observed effects of intralipid and

high fat diet (Kiilerich et al. 2011;Pilegaard et al. 2006). In addition, the maintained exercise-

induced PDH and AMPK regulation in Study III together with previous studies reporting that

inflammation induces insulin resistance (Bach et al. 2013;Buhl et al. 2013;Plomgaard et al. 2005)

suggests that insulin sensitivity and exercise-induced substrate regulation are affected differently

during inflammation, although it cannot be ruled out that more prolonged inflammation also will

affect exercise-induced metabolic flexibility.

PDH regulation during recovery from exercise

PDH has also been suggested to play a role in substrate regulation in recovery from exercise. To

investigate this further, PDH regulation was examined in the elite athletes described above after

prolonged exercise with either glucose or only water during the initial 4h of recovery (Figure 15).

PDH phosphorylation was at 4h lower in the glucose group than the water group indicating a higher

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59    

glucose oxidation when glucose was available. However, the PDH phosphorylation level was still

higher, reflecting reduced PDHa activity, 4h after glucose intake than before exercise, which

suggests that the muscle prioritizes glycogen resynthesis rather than glucose oxidation in recovery

from prolonged endurance exercise, when glucose is available during recovery, as also suggested

above after glucose intake without acute exercise in Study II

Fig 15. Phosphorylation of PDH Ser300 (site p2) in elite athletes before (Pre) and after 4h of exercise at 73% HRmax

(Post), 4h after exercise with H2O (black) or glucose intake (grey) fra Post to 4h (4h) and at 24h of recovery (24h).

Values are mean±SE. *: significantly different from Pre within given group, P<0.05; #: significantly different from H2O

group at given time point, P<0.05; §: significantly different from post exercise, P<0.05. Parentheses indicate a

tendency.

PDH site p2 phosphorylation

Pre Post 4h 24h

PDH

site

p2

phos

phor

ylat

ion

/ PD

H to

tal

0.0

0.5

1.0

1.5

2.0

2.5

H2O Glucose

*

*

**

*(*)

(#)(ß)

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60    

Conclusion • Study I demonstrated that bed rest–induced insulin resistance was associated with reduced insulin-

stimulated GS activity and Akt signaling as well as decreased protein level of HKII and GLUT4 in

skeletal muscle. This suggests that decreased glucose transport/phosphorylation and decreased non-

oxidative glucose metabolism contributed to the decreased skeletal muscle glucose extraction with

physical inactivity in humans. In addition, the ability of acute exercise to increase insulin-stimulated

glucose extraction was maintained after 7 days of bed rest. However, acute exercise after bed rest

did not fully normalize the ability of skeletal muscle to extract glucose to the level seen, when

exercise was performed before bed rest.

• Study II demonstrated that exercise training-improved glucose regulation in elderly healthy

subjects was associated with increased HKII, GLUT4, Akt2, PDK2, GS and PDH-E1α protein

content. Moreover, exercise training resulted in an enhanced glucose-stimulated TBC1D4 response

relative to before training and GS activity increased while PDHa activity decreased after glucose

intake only in the trained state. This shows that aged human skeletal muscle can adapt to exercise

training. In addition, these results suggest that increased content of key proteins in glucose

metabolism and acute molecular changes towards improved glucose uptake and storage contributed

to the observed exercise training-improved glucose handling in aged human skeletal muscle.

• Study III demonstrated that LPS-induced inflammation with elevated plasma TNFα concentration

did not affect resting or exercise-induced AMPK and PDH regulation in human skeletal muscle.

This suggests that short-term inflammation did not influence resting or exercise-induced fat and

carbohydrate utilization in human skeletal muscle.

• Study IV demonstrated that IL-6 reduced PDHa activity in skeletal muscle form fed mice, and

increased the PDHa activity in skeletal muscle from fasted mice without any change in

phosphorylation level. IL-6 injection increased AMPK and ACC phosphorylation in mouse skeletal

muscle only in the fasted state. This suggests that IL-6 regulates PDHa activity in mouse skeletal

muscle, but that the effect depends on the energy state of the muscle. In addition, AMPK may be

involved in the fasted state.

Taken together the present findings suggest that modifications in key factors in skeletal muscle

glucose uptake and storage contribute to the altered insulin-stimulated glucose handling with

changes in physical activity level. Furthermore, the maintained exercise-induced metabolic

regulation in skeletal muscle during acute inflammation suggests that exercise-induced metabolic

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61    

flexibility is unaffected during low grade inflammation. In addition, IL-6 may regulate substrate

utilization in skeletal muscle through effects on PDH.

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62    

Perspective

Several questions have already been answered with this thesis, but there were several interesting

findings that could be further pursued.

In study II the unchanged PDHa activity with glucose stimulation before the training intervention in

the elderly subjects was unexpected and the decreased PDHa activity after the 8 weeks of training

was also unexpected. Because a previous study observed increased PDHa activity with glucose

stimulation (Pehlemand), it may be speculated that aging caused this. Hence, it would be interesting

to conduct the same experiment in untrained and trained young subjects. In line with this a similar

experiment would be interesting to perform in T2D patients.

Most of the previous studies investigating the effect of insulin on PDH regulation have used a

hyperinsulinemic euglycemic clamp and observed no effect of insulin. It may be speculated that the

lack of an elevation in plasma glucose concentration is the reason for the absent PDH regulation.

Therefore it would be interesting to perform a hyperglycemic clamp in both healthy subjects and

insulin resistant subjects.

The current study did not observe any effects of acute inflammation on PDH and AMPK regulation.

However, it may be due to the relatively short period of inflammation. Therefore, it would be

interesting to investigate the effect of more prolonged inflammation on PDH regulation in skeletal

muscle both in humans and rodents, with both healthy and diseased individuals.

The present findings indicated that IL-6 may regulate PDH in skeletal muscle. However this was

based on injection of recombinant IL-6. Therefore it would be interesting to further examine the

potential role of IL-6 in PDH regulation during prolonged exercise for example by using IL-6

knockout mice.

Three phosphorylation sites in PDH-E1α have been analyzed in the current PhD studies. However,

a fourth site is known (Ser232). This site was previously thought not to play a role in skeletal muscle,

but recent studies do indicate that this may not be a correct conclusion. Therefore, it would be

interesting to supplement the present analyses with phosphorylation status of this site, Ser232.

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63    

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Zeigerer,A.,  McBrayer,M.K.,  and  McGraw,T.E.  2004.  Insulin  stimulation  of  GLUT4  exocytosis,  but  not  its  inhibition  of  endocytosis,  is  dependent  on  RabGAP  AS160.  Mol.  Biol.  Cell  15:4406-­‐4415.  

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Study I

Biensø RS, Ringholm S, Kiilerich K, Aachmann-Andersen NJ, Krogh-Madsen R, Guerra

B, Plomgaard P, van Hall G, Treebak JT, Saltin B, Lundby C, Calbet JA,Pilegaard H, Wojtaszewski

JF. GLUT4 and glycogen synthase are key players in bed rest-induced insulin resistance. Diabetes. 2012

May;61(5):10909.

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GLUT4 and Glycogen Synthase Are Key Players in BedRest–Induced Insulin ResistanceRasmus S. Biensø,1,2,3 Stine Ringholm,1,2,3 Kristian Kiilerich,1,2,3 Niels-Jacob Aachmann-Andersen,1,4

Rikke Krogh-Madsen,1,2,5 Borja Guerra,6 Peter Plomgaard,1,2,5 Gerrit van Hall,1,4 Jonas T. Treebak,1,7

Bengt Saltin,1,4 Carsten Lundby,1,4 Jose A.L. Calbet,6 Henriette Pilegaard,1,2,3

and Jørgen F.P. Wojtaszewski1,7

To elucidate the molecular mechanisms behind physical inactivity–induced insulin resistance in skeletal muscle, 12 young, healthymale subjects completed 7 days of bed rest with vastus lateralismuscle biopsies obtained before and after. In six of the subjects,muscle biopsies were taken from both legs before and after a 3-hhyperinsulinemic euglycemic clamp performed 3 h after a 45-min,one-legged exercise. Blood samples were obtained from one fem-oral artery and both femoral veins before and during the clamp.Glucose infusion rate and leg glucose extraction during the clampwere lower after than before bed rest. This bed rest–inducedinsulin resistance occurred together with reduced muscleGLUT4, hexokinase II, protein kinase B/Akt1, and Akt2 proteinlevel, and a tendency for reduced 3-hydroxyacyl-CoA dehydro-genase activity. The ability of insulin to phosphorylate Akt andactivate glycogen synthase (GS) was reduced with normal GSsite 3 but abnormal GS site 2+2a phosphorylation after bed rest.Exercise enhanced insulin-stimulated leg glucose extractionboth before and after bed rest, which was accompanied by higherGS activity in the prior-exercised leg than the rested leg. The pres-ent !ndings demonstrate that physical inactivity–induced insulinresistance in muscle is associated with lower content/activity ofkey proteins in glucose transport/phosphorylation and storage.Diabetes 61:1090–1099, 2012

Lifestyle-related diseases like type 2 diabetes arerapidly increasing worldwide, and there is strongevidence that physical inactivity contributes tothis development (1). The prediabetic and di-

abetic states are characterized by peripheral insulin re-sistance (2), and due to the large mass of skeletal muscle,the insulin action in this tissue has major in"uence onwhole-body insulin sensitivity, especially in lean adults (3).Insulin sensitivity in skeletal muscle is to a large extent adynamic parameter that can be enhanced by exercisetraining and decreased by physical inactivity (4–7). Thus,research attempting to combat diseases related to insulin

resistance has focused on mechanisms by which the sen-sitivity of the glucose uptake to insulin is regulated.

Type 2 diabetes patients with peripheral insulin re-sistance have impaired GLUT4 translocation in skeletalmuscle (8). Moreover, muscle insulin–resistant individualsand type 2 diabetes patients have impaired insulin-stimulatedAkt Thr308 and Ser473 phosphorylation (9–11), lower insulin-stimulated Akt substrate of 160 kDa (AS160/TBC1D4)phosphorylation on multiple sites (7,9–11), and impairedinsulin-stimulated glycogen synthase (GS) activity (9). Theimpaired GS activity in type 2 diabetes patients is associ-ated with hyperphosphorylation of GS site 2+2a duringinsulin stimulation, whereas GS site 3a phosphorylationappears to be unaffected (9,12), indicating that GS phos-phorylation downstream of Akt is maintained despite lowerAkt phosphorylation. Exercise training has been shownto enhance the capacity for glucose transport and glyco-gen storage in skeletal muscle by increasing GLUT4,hexokinase II (HKII), and TBC1D4 protein content as wellas GS activity in both healthy and type 2 diabetic patients,and these adaptations may contribute to the training-inducedimprovements in insulin sensitivity (13–15). In addition, theimproved insulin sensitivity observed after exercise traininghas been reported to be associated with improved, and infact normalized, insulin-induced phosphorylation of TBC1D4in type 2 diabetes (7). In line with the changes observed withtraining, physical inactivity has been demonstrated to induceinsulin resistance (5,16). However, to the best of ourknowledge, no study has reported the effects of physicalinactivity on key proteins in glucose metabolism in humanskeletal muscle.

Muscle insulin sensitivity has been shown to be in-creased in the period after a single bout of exercise (17).Thus, enhanced insulin-mediated glucose clearance is ob-served for up to 48 h after a single exercise bout (17–19). Ithas been shown in rat skeletal muscle that the increasedinsulin sensitivity to glucose uptake is associated with anincreased GLUT4 translocation to the plasma membrane (20).Moreover, multiple studies have documented that insulin-mediated activation of the most proximal insulin signalingelements (e.g., insulin receptor, insulin receptor substrate,phosphatidylinositol-3 kinase, and Akt) is not enhancedafter a single bout of exercise (21–23). Interestingly, recentstudies have revealed that basal and insulin-induced TBC1D4phosphorylation are increased in the period after a singlebout of exercise (24,25), although unchanged TBC1D4phosphorylation has also been reported, but in a study notdetecting increased glucose uptake after exercise (26).Studies have also reported an increased GS activity 3–4 hafter exercise (27), and thus a combination of increasedphosphorylation of TBC1D4 (regulating glucose transport)

From the 1Copenhagen Muscle Research Centre, University of Copenhagen,Copenhagen, Denmark; the 2Centre of In"ammation and Metabolism, Uni-versity of Copenhagen, Copenhagen, Denmark; the 3Department of Biology,University of Copenhagen, Copenhagen, Denmark; the 4Rigshospitalet, Sec-tion 7652, Copenhagen, Denmark; the 5Rigshospitalet, Section 7641, Copen-hagen, Denmark; the 6Department of Physical Education, University of LasPalmas de Gran Canaria, Las Palmas de Gran Canaria, Spain; and the7Molecular Physiology Group, Department of Exercise and Sport Sciences,University of Copenhagen, Copenhagen, Denmark.

Corresponding author: Rasmus S. Biensø, [email protected] 27 June 2011 and accepted 16 January 2012.DOI: 10.2337/db11-0884B.G. and J.A.L.C. were visiting CopenhagenMuscle Research Centre, Rigshospitalet

7652, during this study.! 2012 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 pro!t,and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

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ORIGINAL ARTICLE

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and increased GS activity (regulating glycogen synthesis)may explain the increased sensitivity of insulin to stimu-late glucose uptake 3–4 h after a single bout of exercise.

Knowing the underlying molecular mechanisms behindphysical inactivity–induced insulin resistance and the im-pact of physical inactivity on exercise-induced insulinsensitivity may provide the basis for developing pharma-ceutical agents and interventions that can counteract thedevelopment of insulin resistance in skeletal muscle dur-ing bed rest after surgery/illness as well as induced bya physically inactive lifestyle. The aim of the current studywas to test the hypotheses that 1) physical inactivity–induced insulin resistance in human skeletal muscle is dueto dysregulation of both GS and TBC1D4 and 2) exercise-induced enhancement of insulin sensitivity is lost withphysical inactivity.

RESEARCH DESIGN AND METHODSSubjects. Twelve young, healthy male subjects participated in the study(Table 1). The subjects were physically active, as they all walked or bicycledfor .1 h/day. The subjects were given both oral and written information aboutthe experimental protocol and procedures as well as the discomfort they mightexperience during the experiment. After having received this information, thesubjects gave their written consent. The study was performed according to theDeclaration of Helsinki and was approved by the Copenhagen and Freder-iksberg Ethics Committee in Denmark (H-C-2007-0085).Bed rest. The subjects were placed in bed for 7 days and were given in-formation about the importance of not moving their legs. All transport ofsubjects took place in wheelchairs and subjects were told to sit down duringshowers. The subjects were encouraged to sit in the wheelchair for up to 5 h perday to avoid vascular complications. The daily energy intake during the bed restcame from meals containing 10–20% protein, 50–60% carbohydrates, and25–35% fat. The meals were served ad libitum.Physical performance and body composition. Twelve subjects were in-cluded in the bed rest part (Table 1). Six of these subjects were chosen randomlyto follow a study protocol involving one-legged exercise and a hyperinsulinemiceuglycemic clamp. The characteristics of these subjects are given in brackets inTable 1.

Approximately 2 weeks before the bed rest, maximal oxygen uptake (VO2max)was determined by an incremental test on a cycle ergometer (Monarch Ergo-medic) for all 12 subjects. Body composition and body weight were determinedby dual-emission X-ray absorptiometry scanning (GE Healthcare). The VO2max

test and the dual-emission X-ray absorptiometry scan were repeated at the endof the bed rest period. In addition, the six subjects participating in the exerciseand clamp protocol performed an incremental one-legged knee extensor exer-cise test to exhaustion on a modi!ed cycle ergometer bike (Monarch Ergo-medic) to determine Wattmax as previously described (28).Experimental protocol. The day before the experimental day (both beforeand after the bed rest period), the subjects consumed a prepackaged, stan-dardized meal and evening snack regulated for body weight (14.3 kcal/kg and2.9 kcal/kg, respectively). On the experimental day, the subjects arrived to thelaboratory in the morning. Catheters were placed in the femoral vein of each legand in one femoral artery. Three and a half hours after consuming a stan-dardized breakfast (2.9 kcal/kg), the subjects completed 45 min of one-leggedknee extensor exercise at 60% of Wattmax. This relatively low intensity waschosen to ensure that the subjects would be able to complete the exercise

bout after the bed rest. Three hours into recovery, a 3-h euglycemic hyper-insulinemic clamp with a constant rate of insulin infusion (50 mU/[min $ m2])(Actrapid; Novo Nordisk, Bagsvaerd, Denmark) was performed. Arterial bloodsamples were obtained every 5–10 min for immediate determination of plasmaglucose concentration (Radiometer ABL 725 series Acid-Base Analyser).Euglycemia was maintained by subsequent adjustment of the glucose infusionrate (GIR). The duration of 3 h was chosen to approach clamp steady-stateconditions at the sampling time points.

Muscle biopsies were obtained from the vastus lateralis of both the pre-viously exercised (Ex leg) and the rested leg (Rest leg) both before (B) and after(I) the clamp using the needle biopsy technique (29) with suction. Incisions forbiopsies were made under local anesthesia (lidocaine; AstraZeneca, Södertälje,Sweden) and individual incisions were used for each biopsy. The biopsies werequickly frozen in liquid nitrogen (within 10–20 s) after removing visual bloodand connective tissue.

Before as well as at 2, 2.5, and 3 h of the insulin clamp, blood samples weredrawn from the three femoral catheters and placed in EDTA-treated tubes.Plasma was collected by centrifugation and stored at 280°C.Plasma analyses. The femoral arterial and venous plasma glucose con-centrations were measured using an acid-base analyzer (Radiometer ABL 725series Acid-Base Analyser), and arterial insulin concentration was measuredby ELISA (K6219; DAKO, Glostrup, Denmark). Glucose extraction across theleg was calculated as the difference between the glucose concentration in thearterial and venous plasma samples. The plasma palmitate concentration wasdetermined by gas chromatography (30)

Muscle samples were freeze dried for .48 h. Visible blood, fat, and con-nective tissue were removed under a microscope.

Glycogen was measured as glycosyl units after acid hydrolysis using anautomatic spectrophotometer as previously described (31,32).Enzyme activities. The activity of 3-hydroxyacyl-CoA dehydrogenase (HAD)and citrate synthase (CS) was analyzed spectrophotometrically as previouslydescribed (32). The GS activity was determined in muscle homogenates aspreviously described (22,33).SDS-PAGE and Western blotting. Muscle homogenate, muscle lysate, andprotein concentration determination were prepared as previously described(25,34).

The protein or phosphorylation of b-actin, HKII, GLUT4, Akt, GS kinase 3b(GSK-3b), and TBC1D4 in muscle lysates and GS protein and protein phos-phorylation in muscle homogenates were measured by SDS-PAGE and West-ern blotting (25,34). Primary polyclonal antibodies against b-actin (4967; CellSignaling Technology, Beverly, MA), HKII (2867; Cell Signaling Technology),GLUT4 (ABR-PAI1065; Thermo Scienti!c, Golden, CO), Akt1 and Akt2 (2967and 3063, respectively; Cell Signaling Technology), GSK-3a and -3b (9338 and9315, respectively; Cell Signaling Technology), and TBC1D4 protein (ABCAM,Cambridge, U.K.) as well as against protein phosphorylation of Akt Thr308 (05-669;Upstate Biotechnology, Lake Placid, NY) and Ser473 (05-802; Millipore, Bedford,MA), GSK-3a Ser21 and GSK-3b Ser9 (9331 and 9323, respectively; CellSignaling Technology), TBC1D4 Ser588 and Thr642 (3028-P2 and 3028-P1,respectively; Symansis, Auckland, N.Z.), and TBC1D4 Ser751 (provided byGraham Hardie, Dundee University, Dundee, U.K.) were used. Primary poly-clonal antibodies against GS protein (provided by Oluf Pedersen, University ofCopenhagen) and phosphospeci!c antibodies against 2+2a, 1b (provided byGraham Hardie), and 3a (3891; Cell Signaling Technology) were also used.Membranes probed with phosphospeci!c antibodies were stripped to allowfor determination of the level of the corresponding protein as previouslydescribed (25).Statistics. Values presented are means6 SE. Two-way ANOVA with repeatedmeasures was applied to evaluate the effect of exercise and insulin before aswell as after bed rest. Also, a two-way ANOVA with repeated measures was

TABLE 1Subject characteristics before and after bed rest

Subject characteristics Before bed rest After bed rest

Age (years) 26 6 3 (29 6 5) —Height (cm) 181 6 2 (183 6 7) —Weight (kg) 75.2 6 3.2 (81.6 6 4.9) 75.1 6 3.3 (81.4 6 5.0)VO2max (L/min) 3.9 6 0.2 (4.1 6 1.0) 3.7 6 0.2 (3.9 6 0.4)†Leg muscle mass (kg) 20.6 6 1.0 (21.7 6 1.9) 20.0 6 0.9 (21.0 6 1.7)†Percentage of whole body fat (kg) 18.2 6 2.1 (21.7 6 3.3) 18.5 6 2.1 (21.7 6 3.1)

Age, height, weight, VO2max, leg muscle mass, and percentage of whole body fat before and after 7 days of bed rest. Values are means6 SE; n = 12.Values given in parentheses represent the six subjects randomly chosen for the invasive exercise and clamp study. †Signi!cantly different frombefore bed rest, P , 0.05.

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used to evaluate the effect of bed rest and insulin within the rested leg as wellas within the exercised leg. The data were log transformed if normality orequal variance tests failed. If signi!cant main effects were found, theStudent-Newman-Keuls post hoc test was used to locate differences.Differences were considered signi!cant at P , 0.05. A tendency is reportedfor 0.05 # P , 0.1. Statistical calculations were performed using SigmaStatversion 3.1.

RESULTS

Physical characteristics and performance. Total bodyweight and BMI were unaffected by 7 days of bed rest(Table 1). Leg muscle mass was reduced 3 6 0.7% (P ,0.05), whereas whole body fat did not change signi!cantlywith bed rest (Table 1). Seven days of bed rest reducedwhole body VO2max 5 6 1.4% (P , 0.05) (Table 1).Protein content and enzyme activity. Skeletal muscleHAD activity tended to decrease 8 6 4.7% (0.05 # P , 0.1)by bed rest, whereas CS activity did not change signi!-cantly (Table 2). The protein content of HKII (66 6 15%),GLUT4 (74 6 14%), Akt1 (56 6 6%), and Akt2 (68 6 7%)was reduced after bed rest relative to before (P , 0.05),whereas the protein level of TBC1D4 and GS and GS totalactivity were unchanged with bed rest (Fig. 1 and Table 2).Plasma insulin. The average plasma insulin concentrationbefore exercise, immediately after, and 3 h into recoverywas not signi!cantly different before and after bed rest,with an average of 32 6 15 mmol/L before and 74 6 20mmol/L after bed rest. However, during the last hour of theclamp, plasma insulin concentration tended to be higherafter (452 6 66 pmol/L) than before (341 6 30 pmol/L) bedrest (0.05 # P , 0.1) (Table 3).Plasma palmitate. The plasma palmitate concentrationwas unaffected by bed rest both before and after the in-sulin clamp. Insulin reduced the plasma palmitate con-centration both before and after bed rest (P , 0.05).Plasma glucose and GIR. The arterial plasma glucoseconcentration during the clamp was similar, 4.9 6 0.1mmol/L before and after bed rest. The GIR did not reacha steady state even after 180 min (Fig. 2A). At all timepoints after 50 min, GIR was higher before than afterbed rest (P , 0.05). During the last hour of the clamp,

GIR after bed rest was 75 6 12% of the level before bedrest (P , 0.05) (Fig. 2A).Glucose extraction. Glucose extraction was evaluatedduring the last hour as the arterio-venous concentration dif-ference across the leg. It has previously been reported thatglucose extraction increased only in the exercising leg duringthe one-legged exercise (35). Three hours after exercise,glucose extraction across the leg was similar in the exercisedand rested leg, re"ecting that the acute exercise effect onglucose extraction was fully diminished at 3 h of recoveryfrom exercise (corresponding to pre-insulin) (Fig. 2B). Thiswas seen both before and after bed rest. Insulin infusion in-creased glucose extraction both in the prior-exercised andthe rested leg, both before and after bed rest (Fig. 2B). Duringthe last hour of the clamp before bed rest, glucose extrac-tion in the rested leg was 11-fold (P , 0.05), and the prior-exercised leg sixfold (P , 0.05), higher after insulin than atbasal. After bed rest, glucose extraction was six- and seven-fold (P , 0.05) higher after insulin than at basal in the restedand prior-exercised leg, respectively. The insulin-stimulatedglucose extraction in the rested leg after bed rest was78 6 10% (P , 0.05) of the level before bed rest. Also,insulin-stimulated glucose extraction before bed rest was1.6 6 0.2-fold (P , 0.05), and after bed rest 1.9 6 0.2-fold(P, 0.05), higher in the prior-exercised than in the restedleg (Fig. 2B).Muscle glycogen. Three hours after exercise, muscleglycogen content was ;20% (P , 0.05) lower in the prior-exercised than the rested leg. This was observed both

TABLE 2Muscle protein content and activity before and after bed rest

Protein Before bed rest After bed rest

GLUT4 protein 1 6 0.18 0.74 6 0.14†HKII protein 1 6 0.14 0.66 6 0.15†Akt1 protein 1 6 0.13 0.56 6 0.06†Akt2 protein 1 6 0.13 0.68 6 0.07†TBC1D4 protein 1 6 0.22 0.79 6 0.21GS protein 1 6 0.15 0.82 6 0.21GS total activity(nmol $ min21 $ mg21) 30.7 6 2.3 30.8 6 1.5

HAD activity(mmol $ min21 $ kg21) 23.5 6 1.2 21.4 6 1.4(†)

CS activity(mmol $ min21 $ kg21) 44.9 6 3.8 41.9 6 4.2

GLUT4 protein, HKII protein, Akt2 protein, TBC1D4 protein, and GSprotein content, as well as GS total activity (nmol $ min21 $ mg21),HAD activity (mmol $ min21 $ kg21 dry weight), and CS activity(mmol $ min21 $ kg21 dry weight) in vastus lateralis before and after7 days of bed rest. Target protein content is normalized to b-actinprotein content. Values are means6 SE; n = 12. †Signi!cantly differentfrom before bed rest, P , 0.05. (†)Tendency, 0.05 # P , 0.1.

FIG. 1. Representative blots showing GLUT4, HKII, Akt1, Akt2, TBC1D4,and GS protein before and after 7 days of bed rest in rested (Rest) andprior-exercised (Ex) leg for the same subject. Although the averageAkt2 protein content of all subjects is reduced with bed rest (Table 2),it may be noted that the abundance of Akt2 is not reduced with bed restin the subject used for the representative blots. kD, kilodaltons.

BED REST–INDUCED INSULIN RESISTANCE

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before and after bed rest. There was no change in themuscle glycogen level in response to insulin infusion. Bedrest per se tended to enhance the muscle glycogen content;20% (0.05 # P , 0.1) in the rested leg (Fig. 2C).Muscle signalingAkt phosphorylation. Akt Thr308 phosphorylation wassimilar in the rested and exercised leg at 3 h of recoveryboth before and after bed rest. Insulin infusion increasedAkt Thr308 phosphorylation similarly four- to !vefold in bothlegs before and after bed rest. No effect of prior exercise wasobserved on insulin-induced Akt phosphorylation. However,bed rest decreased Akt Thr308 phosphorylation 20–30% (P ,0.05) after the clamp (Fig. 3A and C). This decrease wasobserved in both the prior-exercised and rested leg. A similarphosphorylation pattern was observed for Akt Ser473 (datanot shown). Thus, the insulin resistance induced by bedrest was associated with decreased signaling through Akt,even after accounting for decreased Akt2 protein expres-sion (Table 2).GSK-3 phosphorylation. The phosphorylation pattern ofAkt target sites (GSK-3a Ser21 and GSK-3b Ser9) did not re-"ect the 20–30% lower Akt phosphorylation after bed rest.Phosphorylation of GSK-3b Ser9 was however 10–20%(P , 0.05) lower in the prior-exercised than the restedleg at 3 h recovery both before and after bed rest, but theability of insulin to induce phosphorylation was apparentlynot signi!cantly changed by either bed rest or prior exer-cise (Fig. 3B and C). Effects of insulin on GSK-3a Ser21

phosphorylation were too small to be detected with theantibody applied (data not shown).TBC1D4 phosphorylation. Being a direct target of AktTBC1D4 phosphorylation was evaluated by the use of phos-phospeci!c antibodies toward three phosphorylationsites (Ser588, Thr642, and Ser751). The general phosphory-lation pattern revealed by these antibodies was similar(Fig. 4). Thus, both before and after bed rest, TBC1D4 phos-phorylation 3 h after exercise was similar in the rested andprior-exercised leg. Insulin stimulation increased the TBC1D4phosphorylation 1.5- to 3-fold (P , 0.05) in both legsbefore and after bed rest, and there was no change in theinsulin-stimulated TBC1D4 phosphorylation with bed rest(Fig. 4).Glycogen synthase activity and phosphorylationGS activity. The GS activity was evaluated both as %I-form(not shown) and percent fractional velocity (%FV). Thetwo measures of GS activity revealed the same pattern ofregulation. GS %FV activity was enhanced (P , 0.05) inthe prior-exercised leg relative to the rested leg 3 h afterexercise both before (P , 0.05) and after bed rest (0.05 #P , 0.1). Insulin increased GS activity in both legs beforeand after bed rest and with a similar magnitude (P , 0.05).

However, in both legs, the level of GS activity tended to belower after bed rest than before (0.05 # P , 0.1) (Fig. 5A).GS phosphorylation. GS phosphorylation at site 2+2awas, in general, elevated after bed rest relative to before(P , 0.05). Site 2+2a phosphorylation was 30% (P , 0.05)lower in the prior-exercised leg than in the rested leg 3 hafter exercise only before bed rest (Fig. 5B and C). Insulinstimulation decreased site 2+2a phosphorylation by 70%(P , 0.05) before but not after bed rest.

GS site 3a phosphorylation was reduced 40% (P , 0.05)by insulin in the rested leg after bed rest and was reduced45% in the prior-exercised leg (P , 0.05) before andtended to be reduced 35% (0.05 # P , 0.1) after bed restin response to insulin. Bed rest resulted in an apparent(P , 0.05) 1.2-fold higher GS site 3a phosphorylation inboth legs 3 h after exercise (Fig. 5B and D). GS phos-phorylation at site 1b was not different between legs andwas not affected by insulin, exercise, or bed rest (data notshown).

DISCUSSION

The main !ndings of the current study are that bed rest–induced insulin resistance is associated with reducedinsulin-stimulated muscular GS activity and Akt signalingas well as decreased protein level of HKII and GLUT4.Thus, this study may indicate that decreased glucose ex-traction with bed rest occurs as a consequence of bothdecreased glucose transport/phosphorylation as well asdecreased nonoxidative glucose metabolism in skeletalmuscle. In addition, the ability of acute exercise to increaseinsulin-stimulated glucose extraction is well maintainedeven after 7 days of bed rest. However, acute exerciseafter bed rest does not fully normalize the ability of skel-etal muscle to extract glucose to the level seen when ex-ercise is performed before bed rest.

The observation that 7 days of bed rest resulted in lowerinsulin-stimulated glucose extraction across the leg is inaccordance with previous bed rest studies (5,16). Un-fortunately, we are not able to evaluate whether this relatesto changes in blood "ow. However, based on previousobservations, insulin-stimulated blood "ow is expected todecrease with bed rest (5,36). Thus, it seems unlikely thatthe decreased glucose extraction observed after bed restis a compensation for an increased blood "ow. Thus, weare con!dent that the decreased leg glucose extractionobserved re"ects decreased leg glucose uptake after bedrest.

The current study provides some indications for the mo-lecular mechanisms behind the observed bed rest–inducedinsulin resistance. Although it is assumed that the observed

TABLE 3Plasma concentrations before and after bed rest

Before bed rest After bed restPlasma parameter B I B I

Glucose (mmol/L) 5.3 6 0.03 4.9 6 0.1* 5.3 6 0.08 5.0 6 0.07*Insulin (pmol/L) 32 6 15 341 6 30* 74 6 20 452 6 66*(†)Palmitate (mmol/L) 183 6 11.1 20 6 1.3* 166 6 9.8 20 6 3.3*

Glucose (mmol/L), insulin (pmol/L), and palmitate (mmol/L) concentration in arterial blood plasma before and after 7 days of bed rest.Samples were obtained before (B) and after (I) a 3-h hyperinsulinemic euglycemic clamp performed 3 h after a one-legged exercise bout.Values are means 6 SE; n = 6. *Signi!cantly different from B, P , 0.05. (†)Tends to be signi!cantly different from before bed rest,0.05 # P , 0.1.

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molecular changes are due to the physical inactivity initself, a contribution from a potential positive energy bal-ance cannot be ruled out (37). But the !nding that the bodyweight was maintained by the ad libitum food intake dur-ing the bed rest period indicates that the subjects were inenergy balance. Central to the observed bed rest–inducedinsulin resistance is the !nding that both basal and insulin-stimulated GS activity was lower after bed rest than be-fore. This impairment is likely one explanatory factor forthe decreased leg glucose extraction. The observed lowerGLUT4 protein content after bed rest is similar to the pre-viously reported decrease in GLUT4 protein after a periodof physical inactivity in trained humans (38) and rats (39).This suggests a similar effect of bed rest and detraining onthe regulation of GLUT4 expression. Although our datasuggest that physical inactivity–induced insulin resistance isassociated with reduced GLUT4 abundance, it is notablethat other studies have found no relationship betweenGLUT4 protein content and insulin action (40). Further-more, the lower HKII and GLUT4 protein levels after bedrest likely decrease glucose transport and phosphorylationin skeletal muscle. These modi!cations are likely addi-tional explanatory factors. Although we can only speculateas to the mechanism for the latter observations, it is temptingto suggest that the absence of muscle use during bed restleads to lack of stimulation of transcriptional/translationalprocesses normally seen in contracting skeletal muscle(41,42).

The current study does not provide insight as to whethertranslocation of GLUT4 to the plasma membrane is alsocompromised in bed rest–induced insulin resistance asseen in, for example, insulin-resistant muscle (40). But the20–30% decreased protein and phosphorylation level ofAkt after bed rest, similar to what has recently been ob-served with reduced levels of physical activity (43), maysuggest such a mechanism because impaired Akt phos-phorylation is seen in skeletal muscle of type 2 diabetessubjects. A possible upstream regulator responsible for theobserved decreased Akt phosphorylation could be phospha-tidylinositol-3 kinase, but due to lack of suf!cient tissue, thiscannot be elucidated further in the current study. However,the impaired phosphorylation on both of the key regulatorysites for Akt activity did not translate to two endogenoussubstrates of Akt. Thus, in contrast to insulin resistance intype 2 diabetes subjects (7), basal and insulin-induced phos-phorylation of TBC1D4 was fully normal on multiple pro-posed Akt target sites after bed rest. In addition, the presentobservations do not indicate that reduced TBC1D4 phos-phorylation is responsible for muscle insulin resistance afterbed rest. However, other mechanisms involved in the GLUT4recruitment to the plasma membrane, dependent on Akt,may be impaired (e.g., cytoskeleton rearrangements) (44).

Interestingly, the impaired activity and activation of GSafter bed rest might be linked to impaired covalent regu-lation of the enzyme. Thus, whereas GS site 3a dephos-phorylation was not compromised after bed rest, theability of insulin to induce dephosphorylation on site 2+2awas lost. Furthermore, the general level of site 2+2aphosphorylation was increased and potentially contribut-ing to the suppressed GS activity after bed rest. Such apattern of dysregulation of site 2+2a rather than site 3a is

FIG. 2. A: GIR during 3 h of hyperinsulinemic euglycemic clamp initiated3 h after 45 min of one-legged knee extensor exercise before and after 7days’ bed rest. B: Arterio-venous (a-v) glucose difference across rested(Rest leg) and prior-exercised (Ex leg) vastus lateralis during 3 h ofhyperinsulinemic euglycemic clamp performed 3 h after 45 min of one-legged knee extensor exercise before and after bed rest. Samples wereobtained before (B) as well as during (I) the clamp, presented here bythe accumulated data during the last hour of the clamp (2–3 h). C:Muscle glycogen concentration (mmol/kg dry weight [dw]) in rested andprior-exercised leg before (B) and after (I) 3 h of hyperinsulinemic eugly-cemic clamp performed 3 h after one-legged knee extensor exercise before

and after bed rest. Values are means 6 SE; n = 6. *Signi!cantly dif-ferent from B, P < 0.05. †Signi!cantly different from before bed rest,P < 0.05. #Signi!cantly different from Rest leg at given time point,P < 0.05. (†)Tendency, 0.05 £ P < 0.1.

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FIG. 3. Akt Thr308 phosphorylation (A), GSK-3b Ser9 phosphorylation (B), and representative blots (C) in rested (Rest leg) and prior-exercised(Ex leg) vastus lateralis before (B) and after (I) 3-h hyperinsulinemic euglycemic clamp performed 3 h after one-legged knee extensor exercisebefore and after 7 days of bed rest. AU, arbitrary units; kD, kilodaltons. Values are means 6 SE; n = 6. *Signi!cantly different from B, P < 0.05.†Signi!cantly different from before bed rest, P < 0.05. #Signi!cantly different from Rest leg at given time point, P < 0.05. (#)Tendency, 0.05 £P < 0.1.

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in agreement with observations in muscle insulin resistancerelated to obesity and type 2 diabetes (9,12). Site 2+2a isapparently not a direct target of either GSK-3 or Akt. Yet it isuncertain whether the impaired Akt signaling indirectlyrelates to the lack of site 2+2a dephosphorylation seen afterbed rest. Site 2+2a is directly targeted by multiple kinases,but so far we have not been able to !nd indications thatthese kinases are dysregulated with bed rest. Thus, the fullynonresponsive site 1b phosphorylation indicates that neithercalcium calmodulin–dependent kinase nor cAMP-dependentprotein kinase is regulated under these conditions (45).Also, AMP-activated protein kinase activity/phosphorylationis not regulated by either insulin or bed rest (data notshown). In addition, both GS site 3 and site 2+2a aredephosphorylated by the multisubstrate phosphatase (PP1)(46), and a dysregulation of PP1 activity would be expectedto affect phosphorylation at both sites, and not only site2+2a as seen in the current study.

Previous studies have suggested that GS site 2+2a phos-phorylation is affected by muscle glycogen levels, and thatGS site 2+2a phosphorylation is a possible link between theinverse relationship between muscle glycogen and GS ac-tivity (47,48). Thus, although mechanistically unresolved,the lower GS activity and higher site 2+2a phosphorylation

after bed rest might be related to the higher muscle glyco-gen level after bed rest. Of notice is, however, that the dif-ferences in muscle glycogen concentration eliciting changesin GS activity in previous studies (47–50) are larger than the20% difference seen in the current study. This makes it lesslikely that the enhanced glycogen level is the only reasonfor the differences observed in the current study. The!ndings that resting muscle glycogen was elevated despiteimpaired insulin-induced glucose extraction and reducedGS activity after bed rest may at !rst seem contradictory.However, the impact of the dysregulated GS activation onthe glycogen level likely !rst becomes evident when a sig-ni!cant amount of glycogen has to be synthesized (e.g., inthe period after glycogen-depleting exercise).

The !nding that insulin-stimulated GS activity in theprior-exercised leg was higher than in the rested leg bothbefore and after bed rest is in line with previous studies(14,22) and indicates that activation of GS may play a rolein the exercise-induced enhanced glucose uptake severalhours after exercise both before and after the inactivityperiod. The similar insulin-mediated regulation of GS site 3aand Akt phosphorylation in the rested and prior-exercisedleg both before and after bed rest is also in accordancewith earlier studies (22,48) and suggests that changes in

FIG. 4. TBC1D4 Ser588 phosphorylation (A), TBC1D4 Ser751 phosphorylation (B), TBC1D4 Thr642 phosphorylation (C), and representative blots(D) in rested (Rest leg) and prior-exercised (Ex leg) vastus lateralis before (B) and after (I) 3-h hyperinsulinemic euglycemic clamp performed 3 hafter one-legged knee extensor exercise before and after 7 days of bed rest. AU, arbitrary units; kD, kilodaltons. Values are means 6 SE; n = 6.*Signi!cantly different from B, P < 0.05.

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GS site 3a and Akt phosphorylation do not contribute tothe observed exercise-induced effects on GS activity. Previous!ndings show that reduced muscle glycogen is associatedwith dephosphorylation of GS site 2+2a (34), and thus themore marked dephosphorylation of GS site 2+2a could bea link between the reduced muscle glycogen and the in-creased GS activity in the prior-exercised leg. Previous studiesshowing that prior exercise increased TBC1D4 phosphoryla-tion both before and after insulin stimulation (25) suggesta role of TBC1D4 in the enhanced glucose clearance ina prior-exercised muscle. However, the current study does notcon!rm these observations, although it cannot be excludedthat a more intense or more prolonged bout of exercisewould elicit such changes. Thus, the analyses performed inthis study do not bring further insights to the mechanismsbehind exercise-induced enhanced insulin sensitivity but doprovide the important observation that the mechanisms stillseem fully functional in bed rest–induced insulin-resistantmuscle.

We are well aware of the limitation in the study by havinginvestigated the effect of acute exercise in only six subjects,leaving us in some cases with near-signi!cant trends. Stillwe are able to draw some conclusions to potential mecha-nisms involved in bed rest–induced insulin resistance inskeletal muscle. Thus bed rest–induced insulin resistance inskeletal muscle relates to decreased expression and impaired

activation of central players regulating glucose uptake andstorage in skeletal muscle, together with a possible im-paired insulin-stimulated blood "ow. In addition, the similarenhancement of glucose removal by prior exercise beforeand after bed rest demonstrates that the ability of exerciseto increase skeletal muscle insulin sensitivity is maintainedafter 7 days of physical inactivity.

ACKNOWLEDGMENTS

This study was supported by grants from the LundbeckFoundation, Denmark; The Danish Medical Research Council,Denmark; and the Novo Nordisk Foundation, Denmark. TheCentre of In"ammation and Metabolism (CIM) is supportedby a grant from the Danish National Research Foundation(02-512-55). The Copenhagen Muscle Research Centre issupported by a grant from the Capital Region of Denmark.CIM and the Molecular Physiology Group (Department ofExercise and Sport Sciences) are part of UNIK: Food,Fitness & Pharma for Health and Disease, supported bythe Danish Ministry of Science, Technology, and Innova-tion. The experimental part was funded by a grant to B.S.,while laboratory analyses were funded by grants to H.P.and J.F.P.W.

No potential con"icts of interest relevant to this articlewere reported.

FIG. 5. Glycogen synthase (GS) activity (%FV) (A), representative blots (B), GS site 3a phosphorylation (C), and GS site 2+2a phosphorylation(D) in rested (Rest leg) and prior-exercised (Ex leg) vastus lateralis before (B) and after (I) 3-h hyperinsulinemic euglycemic clamp performed 3 hafter one-legged knee extensor exercise before and after 7 days of bed rest. AU, arbitrary units; kD, kilodaltons. Values are means 6 SE; n = 6.*Signi!cantly different from B, P< 0.05. †Signi!cantly different from before bed rest, P< 0.05. #Signi!cantly different from Rest leg at given timepoint, P < 0.05. Symbols within parentheses indicate tendency, 0.05 £ P < 0.1.

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R.S.B. designed the study, took part in conducting thehuman experiments, performed the laboratory analyses,wrote the manuscript, and commented on the manuscript.S.R. designed the study, took part in conducting the humanexperiments, performed the laboratory analyses, and com-mented on the manuscript. K.K., C.L., and P.P. designed thestudy, took part in conducting the human experiments, andcommented on the manuscript. N.-J.A.-A., R.K.-M., B.G., G.v.H.,and J.A.L.C. took part in conducting the human experi-ments and commented on the manuscript. J.T.T. providedvaluable guidance for speci!c laboratory analyses andcommented on the manuscript. B.S. designed the studyand commented on the manuscript. H.P. and J.F.P.W. de-signed the study, took part in conducting the human ex-periments, assisted with writing the manuscript, andcommented on the manuscript. J.F.P.W. is the guarantor ofthis work and, as such, had full access to all the data in thestudy and takes responsibility for the integrity of the dataand the accuracy of the data analysis.

Parts of this study were presented in poster form at the70th Scienti!c Sessions of the American Diabetes Associ-ation, Orlando, Florida, 25–29 June 2010.

The authors would like to thank the subjects for theirextraordinary effort during the study. In addition, theauthors have sincere appreciation for the whole Copenha-gen 2008 Bed Rest Team for a fantastic collaboration. Theauthors are grateful to Oluf Pedersen (University of Copen-hagen), Graham Hardie, and Carol Mackintosh (DundeeUniversity) for donating antibodies essential for this study.

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Regulation of PDH, GS and insulin signalling in skeletal muscle; effect of physical activity level and inflammation

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GLUT4 and glycogen synthase are key players in bed rest-induced insulin resistance

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Study II

Biensø RS, Olesen J, Gliemann L, Smith JF, Matzen MS, Wojtaszewski JFP, Hellsten Y,

Pilegaard H. Effects of exercise training on regulation of skeletal muscle glucose metabolism in

skeletal muscle of elderly men. Submitted Journal of Gerontology: Medical Scinces 2014

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1

Effects of exercise training on regulation of skeletal muscle glucose

metabolism in skeletal muscle of elderly men

1Rasmus Sjørup Biensø, 1Jesper Olesen, 2Lasse Gliemann, 2Jacob Friis Smith, 1Mikkel Sillesen Matzen, 2Jørgen F. P. Wojtaszewski, 2Ylva Hellsten, 1Henriette Pilegaard

1Centre of Inflammation and Metabolism, The August Krogh Centre, Molecular Integrative Physiology Department of Biology, University of Copenhagen

2The August Krogh Centre, Department of Nutrition, Exercise and Sports, University of Copenhagen

Short title: Skeletal muscle glucose regulation in elderly

Key word: Physical activity, aging, pyruvate dehydrogenase, glycogen synthase, insulin signaling

Corresponding author: Henriette Pilegaard

Universitetsparken 13

2100 Copenhagen Ø,

Email: [email protected]

Telephone:+4535321687

Fax:+4535321567

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Abstract

BACKGROUND: The aim was to investigate the molecular mechanisms behind exercise training-

induced improvements in glucose regulation in aged subjects, METHODS: Twelve elderly males

completed 8 weeks of exercise training. Before and after the training period, the subjects were given

an oral glucose tolerance test (OGTT) and a muscle biopsy was obtained from the vastus lateralis

before and 45 min into the OGTT. Blood samples were collected before and up to 120 min after

glucose intake. RESULTS: exercise training increased Hexokinase II, GLUT4, Akt2, glycogen

synthase (GS), pyruvate dehydrogenase (PDH)-E1 and PDK2 protein and glycogen content in

skeletal muscle. Furthermore, in response to glucose GS activity was increased and the

dephosphorylation on GS site 2+2a and 3a was enhanced after the training intervention. The

glucose-mediated insulin stimulation of TBC1D4 Thr642 phosphorylation was increased. The PDHa

activity was reduced following glucose intake and without changes in phosphorylation level on

PDH-E1. CONCLUSIONS: This suggests that exercise training improves glucose regulation in

elderly subjects by enhancing the capacity for glucose uptake and improving glycogen storage

rather than oxidation.

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Introduction

Aging is associated with impaired glucose metabolism as demonstrated both by reduced glucose

tolerance during an oral glucose tolerance test [1] and reduced glucose disposal during a

hyperinsulinemic euglycemic clamp [2;3]. Such changes may pre-dispose to diseases like Type 2

diabetes [4] and prevention of age-related dysfunctional glucose metabolism through physical

activity is therefore important. Regular physical activity has been shown to increase glucose

tolerance [1;5] and insulin sensitivity in elderly subjects [3] to a similar extent as in young subjects

[6;7]. Aging and exercise training associated changes in glucose tolerance and insulin sensitivity

involve several tissues, but changes in skeletal muscle metabolism have a particularly large impact

on whole body metabolism [8].

Insulin-stimulated glucose uptake in skeletal muscle involves glucose transporter (GLUT)4

translocation to the plasma membrane regulated through phosphorylation and activation of Akt and

concomitant phosphorylation and inactivation of Akt substrate of 160 kD (AS160/TBC1D4) [9]. G-

6-P will in a resting muscle cell either be incorporated into glycogen or be oxidized within the

mitochondria. The rate limiting enzyme in glycogen formation is glycogen synthase (GS), while the

pyruvate dehydrogenase (PDH) complex converts pyruvate to acetyl CoA in an irreversible step,

which represents the only entry of carbohydrate-derived substrates into the mitochondria for

oxidation [10]. GS is stimulated allosterically by G-6-P [11] and inactivated by phosphorylation

[12;13] Insulin is known to increase the activity of GS through insulin-stimulated activation of Akt

and concomitant phosphorylation and inactivation of GSK3[12;13]. Activity of PDH in the active

form (PDHa) is primarily determined by the phosphorylation level of the PDH-E1α subunit

regulated by PDH kinases (PDK), which phosphorylate and inactivate PDH, and PDH phosphatases

(PDP), which dephosphorylate and activate PDH [14;15]. PDHa activity has previously been shown

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to increase in human skeletal muscle in response to oral glucose intake [16], and to either increase

[17] or remain unchanged [18] during a hyperinsulinemic euglycemic clamp in humans. In

accordance with an insulin-mediated regulation of PDHa activity, insulin has been reported to

increase PDP activity [19] and to reduce PDK4 protein in rat skeletal muscle [20].

Previous studies have observed either reduced or unchanged GLUT4 [21;22] protein content in

human skeletal muscle with increasing age. Moreover, the recent finding that aging was associated

with impaired insulin-induced TBC1D4 phosphorylation in skeletal muscle [2] suggests that

reduced insulin-mediated GLUT4 translocation may contribute to insulin resistance in aged

subjects. Furthermore, skeletal muscle HKII activity has been shown to be lower in elderly than in

young subjects [23] indicating that a reduced ability to maintain the transmembrane glucose

gradient also may affect skeletal muscle glucose uptake in aged subjects. Knowledge about

potential age-related effects on GS and PDH regulation in skeletal muscle is scarce. However total

GS activity has been reported to be reduced with age [24]. Moreover insulin-stimulated GS

activation has been demonstrated to be impaired in Type 2 diabetic (T2D) patients [22;25].

Similarly, an insulin-induced increase in PDHa activity has been reported to be abolished in Type 2

diabetes patients [17]. In line with this observation, T2D has been shown to be associated with

impaired insulin-mediated down-regulation of PDK4 in skeletal muscle suggesting that PDK4-

induced inhibition of PDH and hence inhibition of glucose oxidation may contribute to insulin

resistance [26]. Together, this indicates that both oxidative and non-oxidative glucose removal are

reduced in skeletal muscle of T2D patients. This may suggest that GS and PDH dysregulation also

contributes to age-associated glucose intolerance and insulin resistance.

Exercise training has previously been shown to increase skeletal muscle protein content of GLUT4,

HKII, Akt, TBC1D4, GS, PDK2 and PDH-E1α in young subjects [7;22;27] and similarly for

GLUT4 and Akt2, but not TBC1D4 in elderly subjects [2;28]. Furthermore, exercise training has

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been demonstrated to enhance insulin-mediated TBC1D4 and GS regulation in young subjects as

well as to reverse the age-associated impairment of insulin-stimulated TBC1D4 phosphorylation

[2;22].

Therefore, the purpose of the present study was to test the hypotheses that exercise training-induced

improvements in whole body glucose metabolism in elderly men are associated with increased

content of key factors involved in GS and PDH regulation in skeletal muscle, and enhanced acute

regulation of GS and PDH in skeletal muscle upon glucose intake.

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Methods

Results from the current experiment have previously been published [29]

Subjects. Twelve physically inactive but healthy male subjects 60-72 years of age with a average

body mass index 26.0±0.5 participated in the study. Subject characteristics have previously been

published [29] including fasting glucose (5.3±0.1 mmol/l) and fasting blood insulin concentration

(49.3±12 pmol/l) [29].

Exercise training. The subjects exercise trained for 8 weeks, 4 days a week as previously described

[29]. The training was a combination of high intensity cycling exercise (Spinning) 2 times per

week, strength and mobility training (crossfit) once per week and a 5 km walk once a week.

Experimental setup. Before and after the intervention period, the subjects were challenged with an

oral glucose tolerance test (OGTT; 1g·kg-1 body mass). A muscle biopsy was obtained from vastus

lateralis using the Bergström needle biopsy method with suction and blood samples were obtained

before and up to 2h after (see supplementary for details),

Plasma and muscle analyses: see supplementary

Statistics: see supplementary

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Results

Performance

Exercise training increased (p<0.05) VO2max as previously published [29].

Blood analyses

Fasting glucose and insulin

Fasting plasma insulin and plasma glucose concentrations were not different before and after the 8

weeks of exercise training as in part previously reported [29].

OGTT

Plasma glucose: The plasma glucose response during the OGTT was similar in the untrained and

trained state increasing (p<0.05) in both conditions to approximately 8 mM 30 min after glucose

intake and returning to the basal level (5 mM) at 120 min after intake. There was no difference in

the plasma glucose concentration between the untrained and the trained state (Fig 1A).

Plasma Insulin: The plasma insulin concentration increased (p<0.05) ~10 fold 60 min after the

glucose intake and remained elevated (p<0.05) 120 min after intake relative to before both in the

untrained and trained state. Moreover, the plasma insulin concentration was at 15 min after glucose

intake higher (p<0.05) and at 90 and 120 min lower (p<0.05) in the trained than in the untrained

state (Fig 1B). The insulin AUC was 15% lower (p<0.05) after exercise training than before

exercise training. There was no difference in the HOMA-IR index before and after exercise training.

Plasma C-peptide: The plasma C-peptide concentration increased (p<0.05) similarly in the

untrained and trained state to 5 fold at 60 min after glucose intake and remained in both conditions

elevated (p<0.05) at 120 min relative to before intake. Moreover, as for the plasma insulin

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concentration, the C-peptide concentration was at 90 and 120 min after glucose intake lower

(p<0.05) in the trained than in the untrained state (Fig 1C). The C-peptide AUC was 15% lower

(p<0.05) after exercise training than before.

Muscle analyses

Muscle glycogen

The muscle glycogen concentration increased (p<0.05) from 496±29 mmol·kg-1dw before to

686±43 mmol·kg-1dw after exercise training.

Protein content

Skeletal muscle protein content of GLUT4, Akt2, GS, PDK2 and PDH-E1 (all p<0.05) and HKII

(0.05≤p<0.1) increased 1.2 to 1.8 fold with exercise training. There was no difference in Akt1,

TBC1D4, GSK-3 or PDK4 protein content before and after exercise training (Fig 2).

Intracellular signaling

Akt phosphorylation: Forty five min after glucose intake, the absolute Akt Thr308 phosphorylation

increased (p<0.05) 2.8 and 3.7 fold in the untrained and trained skeletal muscle, respectively, with

no difference between training status (Fig 3A). Insulin-stimulated Akt phosphorylation occurs

primarily if not exclusive on Akt2 in human skeletal muscle [36], The absolute phosphorylation on

Akt Ser473 increased (p<0.05) 2.1 and 3 fold in the untrained and trained state, respectively,

reaching a 2 fold higher (p<0.05) level in the trained than untrained state (Fig 3B)

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TBC1D4 phosphorylation: The absolute TBC1D4 Thr642 phosphorylation in skeletal muscle

increased (p<0.05) 1.8 fold in the untrained state and 2.5 fold in the trained state reaching a higher

(p<0.05) level in the trained than the untrained condition (Fig 3C).

GSK3β phosphorylation: Absolute GSK3β Ser9 phosphorylation in skeletal muscle increased

(p<0.05) similarly 1.3-1.4 fold 45 min after glucose intake before and after the exercise training

period with no difference between the two conditions (Fig 4A).

Glycogen synthase regulation

GS site 2+2a: Glucose intake had no effect on the absolute GS site 2+2a phosphorylation in skeletal

muscle before exercise training, but after exercise training the absolute GS site 2+2a

phosphorylation decreased (p<0.05) 20% in response to glucose intake. Furthermore, the absolute

GS site 2+2a phosphorylation was in the trained state 1.4 fold higher (p<0.05) in the basal state and

tended to be 1.2 fold higher (0.05≤p<0.1) 45 min after glucose intake than in the untrained state

(Fig 4B).

GS site 3a: The absolute GS site 3a phosphorylation in skeletal muscle tended to decrease

(0.05≤p<0.1) 25% in response to glucose intake before exercise training and decreased (p<0.05)

40% with glucose intake after exercise training. The absolute GS site 3a phosphorylation was 1.3-

1.7 fold higher (p<0.05) in the trained state than in the untrained state (Fig 4C).

GS activity: The GS activity (I-form) in skeletal muscle did not change with glucose intake before

exercise training, but increased (p<0.05) 1.3 fold 45 min after glucose intake in the exercise trained

state. There was no difference in GS activity between the untrained and trained state (Fig 4D).

PDH regulation

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PDH phosphorylation: There was no effect of glucose intake on the absolute or the normalized PDH

site 1 and site 2 phosphorylation in skeletal muscle. Exercise training increased (p<0.05) the

absolute phosphorylation level of both sites with 1.3-1-8 fold, but this effect was removed by

normalization to PDH-E1α protein, except for site 2 phosphorylation in the basal state (Fig 5A,Fig

5B; Supplementary Fig 3A; Fig 3B).

PDHa activity: There was no effect of glucose intake on skeletal muscle PDHa activity before

exercise training, but glucose intake decreased (p<0.05) the PDHa activity 30% after the exercise

training period (Fig 5C).

PDK4 protein:

PDK4 protein content overall (untrained and trained state together) tended to be lower (0.05≤p<0.1)

after glucose intake (Fig 6B).

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Discussion

The main findings of the present study are that exercise training-improved glucose regulation in

elderly healthy subjects was associated with increased HKII, GLUT4, Akt2, PDK2, GS and PDH-

E1 protein content. Moreover, exercise training resulted in an enhanced response of TBC1D4 and

GS and a reduced PDHa activity in response to glucose intake relative to before training.

The present observation that the plasma glucose response to an oral glucose intake was similar

before and after the exercise training period shows that glucose tolerance was unaffected by the

exercise training intervention. However, there was a strong indication of improved insulin

sensitivity after training in the present study as the insulin and C-peptide plasma concentrations as

well as AUC for insulin and C-peptide were lower during the OGTT after training than before. Such

an exercise training effect is in accordance with the observations in previous studies using

hyperinsulinemic euglycemic clamp in elderly subjects demonstrating enhanced whole body insulin

action [1;2] as well as improved insulin sensitivity in skeletal muscle after exercise training [2;6].

The finding in the current study that glucose intake induced a more marked absolute TBC1D4

phosphorylation after exercise training than before is in accordance with a recent study

demonstrating that aged subjects increased the insulin-stimulated TBC1D4 phosphorylation during

a hyperinsulinemic euglycemic clamp [2]. This indicates that exercise training enhances skeletal

muscle GLUT4 translocation upon glucose intake in elderly subjects as previously reported in

young subjects [2]. Although the effect of exercise training on TBC1D4 phosphorylation

normalized to TBC1D4 protein did not reach statistical significance in the present study, the lack of

change in TBC1D4 protein content with exercise training both in the present and the previous study

[2;22] suggests that the observed training effect on TBC1D4 phosphorylation upon glucose intake is

due to acute regulation rather than a change in TBC1D4 protein content. Although Akt Thr308

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phosphorylation after glucose intake was similar in the untrained and trained state, the present

observation that Akt Ser473 phosphorylation level after glucose intake was higher in the trained

muscle than the untrained is in line with the higher TBC1D4 phosphorylation after the training

period. This indicates that Akt mediate the exercise training effect on TBC1D4 phosphorylation in

the present study, which is different from previous findings [2] reporting that insulin-stimulated Akt

Ser473 phosphorylation could not explain the observed exercise training-induced changes in

TBC1D4 phosphorylation. This difference between the two studies may be related to the use of

hyperinsulinemic euglycemic clamp [2] and an oral glucose intake. In addition, the observed higher

plasma insulin concentration in the trained state than in the untrained 15 min after glucose intake

may have contributed to the enhanced Akt Ser473 response in the present study. The observed

increase in GLUT4 and HKII protein content with exercise training in the current study is in line

with previous studies showing that exercise training increases skeletal muscle GLUT4 protein

content in aged subjects [1;28]. Together these observations indicate that exercise training-induced

increased capacity for glucose transport and phosphorylation also contributes to improved

regulation of glucose uptake in elderly subjects.

The observation that glucose intake did not affect GS activity in the elderly subjects in the untrained

state is in contrast with previous observations in young subjects [16] suggesting an age-associated

impairment in GS regulation [24]. However, the finding that glucose intake induced an increase in

GS activity in the elderly subjects after the exercise training period in the present study may

indicate that exercise training restored the ability of the aged muscle to activate GS in response to

glucose intake.

The observation that glucose intake in the untrained state did not change GS site 2+2a

phosphorylation is in contrast to the previously observed GS site 2+2 dephosphorylation in skeletal

muscle of middle aged [25] and young subjects [24;25;31], but is in line with the unchanged GS

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activity in the present study. Furthermore, the observed dephosphorylation of GS site 2+2a in

response to glucose intake after the exercise training period is in accordance with the observed

increase in GS activity upon glucose intake in the exercise trained state. The present observation

that glucose uptake-stimulated phosphorylation of GSK-3β was similar before and after the exercise

training period suggests that a difference in GSK-3 is not a likely explanation for the changes in GS

regulation with exercise training. However, previous studies have indicated that GS site 2+2a

hyperphosphorylation may contribute to impairment of insulin-induced GS activation in T2D

patients [25] and in young subjects after 1 week of bed rest [31]. This suggests that the exercise

training-induced change in glucose intake-stimulated site 2+2a phosphorylation may explain the

associated changes in GS activation in the present study. The increased muscle glycogen

concentration with exercise training is a possible reason for the elevated level of GS 2+2a

phosphorylation after exercise training, because GS site 2+2a is regulated by the glycogen level

[37]. But the lack of change in muscle glycogen after glucose intake does not support that muscle

glycogen influenced the acute changes in GS site 2+2a phosphorylation in the present study.

The finding that PDHa activity was unaffected by the glucose intake in the untrained state is not in

accordance with the previously reported increase in PDHa activity in young subjects during an

OGTT [16]. It is possible that the elderly subjects in the present study had an impaired PDH

regulation in response to glucose intake in the untrained state, but this needs to be further

investigated. The finding that glucose intake even reduced skeletal muscle PDHa activity in the

trained state was unexpected and has not previously been reported. However, it may suggest that

downregulation of PDHa activity combined with an increased GS activity after glucose intake in the

trained state serves to reduce carbohydrate oxidation ensuring that carbohydrate enters glycogenesis

rather than oxidation.

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The observation that the exercise training-induced elevation in basal PDH-E1α phosphorylation was

associated with a similar increase in PDH-E1α protein content indicates that the same fraction of

PDH-E1α molecules was phosphorylated in the trained and the untrained state. Furthermore, the

concomitant exercise training-induced increase in PDK2 protein suggests that the enhanced PDH-

E1α phosphorylation was executed by PDK2. In addition the observed overall tendency for

reduction in PDK4 protein in response to glucose intake may be expected to influence the

phosphorylation status of PDH-E1α and thus affect PDHa activity. However the lack of change in

PDH-E1α phosphorylation with glucose intake in the trained state shows that the regulation of

PDHa activity was independent of the phosphorylation status of site 1 and 2 on PDH-E1α. As PDH-

E1α has been shown to be phosphorylated on at least 2 further sites [38] as well as being regulated

by acetylation [38], additional post-translational modifications may explain the observed regulation

of PDHa activity in the trained state.

In conclusion, the present findings suggest that exercise training-improved glucose regulation in

elderly subjects is associated with enhanced potential for GLUT4 translocation, glucose

phosphorylation and intracellular glucose removal to glycogen synthesis rather than oxidation.

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Funding

This work was supported by grants from the Ministry of Culture. The Centre of Inflammation and

Metabolism (CIM) is supported by a grant from the Danish National Research Foundation

(DNRF55). The Centre for Physical Activity Research (CFAS) is supported by a grant from

Trygfonden. CIM is part of the UNIK Project: Food, Fitness & Pharma for Health and Disease,

supported by the Danish Ministry of Science, Technology.

Acknowledgement

We would like to thank Ninna Iversen, Marie Henriksen, Line Nielsen, Simon Granjean and

Sebastian Peronard for technical assistance. The authors are grateful to Oluf Pedersen (University of

Copenhagen), Graham Hardie and Carol Mackintosh (Dundee University) for donating antibodies

essential for this study.

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31 Bienso RS, Ringholm S, Kiilerich K, Aachmann-Andersen NJ, Krogh-Madsen R, Guerra B, Plomgaard P, van HG, Treebak JT, Saltin B, Lundby C, Calbet JA, Pilegaard H, Wojtaszewski JF: GLUT4 and glycogen synthase are key players in bed rest-induced insulin resistance. Diabetes 2012;61:1090-1099.

32 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.

33 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.

34 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.

35 Højlund K, Birk JB, Klein DK, Levin K, Rose AJ, Hansen BF, Nielsen JN, Beck-Nielsen H, Wojtaszewski JF: Dysregulation of glycogen synthase. J Clin Endocrinol Metab 2009;94:4547-4556.

36 Vind BF, Birk JB, Vienberg SG, Andersen B, Beck-Nielsen H, Wojtaszewski JF, Hojlund K: Hyperglycaemia normalises insulin action on glucose metabolism but not the impaired activation of AKT and glycogen synthase in the skeletal muscle of patients with type 2 diabetes. Diabetologia 2012;55:1435-1445.

37 Friedrichsen M, Birk JB, Richter EA, Ribel-Madsen R, Pehmoller C, Hansen BF, Beck-Nielsen H, Hirshman MF, Goodyear LJ, Vaag A, Poulsen P, Wojtaszewski JF: Akt2 influences glycogen synthase activity in human skeletal muscle through regulation of NH(2)-terminal (sites 2 + 2a) phosphorylation. Am J Physiol Endocrinol Metab 2013;304:E631-E639.

38 Gnad F, Gunawardena J, Mann M: PHOSIDA 2011: the posttranslational modification database. Nucleic Acids Res 2011;39:D253-D260.

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19

Fig. 1. Plasma glucose (A), plasma insulin (B) and plasma C-peptide (C) during a 120 min oral

glucose tolerance test in untrained (UT) (●) and exercise trained (T) (○) aged male subjects,. *:

Significantly different from before glucose intake OGTT, p<0.05. #: Significantly different from

UT, p<0.05. Symbols within parentheses indicate tendency for statistical significance, 0.05≤p<0.1.

Fig. 2. Skeletal muscle protein content of hexokinase (HK)II, glucose transporter (GLUT)4, protein

kinase B(Akt)1, Akt2, TBC1D4, glycogen synthase (GS), pyruvate dehydrogenase (PDH)-E1 and

glycogen synthase kinase (GSK)3 in untrained (UT) and exercise trained (T). # Significantly

different from UT, p<0.05. Symbols within parentheses indicate tendency for statistical

significance, 0.05≤p<0.1.

Fig. 3. Skeletal muscle Akt Thr308 phosphorylation (A), Akt Ser473 (B) and TBC1D4 Thr642

phosphorylation (C) and glycogen synthase kinase (GSK)-3 Ser9 phosphorylation (D) before (Pre)

and 45 min into and oral glucose tolerance test (Post) in skeletal muscle of untrained (Untrained)

and exercise trained (Trained) aged male subjects. *: Significantly different from Pre p<0.05. #:

Significantly different from UT, p<0.05.

Fig. 4. Skeletal muscle Glycogen synthase (GS) site 2+2a phosphorylation (A), GS site 3a

phosphorylation (B) and GS activity (I-form) (C) before (Pre) and 45 min into and oral glucose

tolerance test (Post) in untrained (Untrained) and exercise trained (Trained) aged male subjects. *:

Significantly different from Pre p<0.05, #: Significantly different from UT, p<0.05. Symbols within

parentheses indicate tendency for statistical significance, 0.05≤p<0.1.

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20

Fig. 5. Skeletal muscle pyruvate dehydrogenase (PDH) site 1 phosphorylation (A), PDH site 2

phosphorylation (B) and PDH activity in the active form (C) before (Pre) and 45 min into and oral

glucose tolerance test (Post) in skeletal muscle of untrained (Untrained) and exercise trained

(Trained) aged male subjects. *: Significantly different from Pre p<0.05, #: Significantly different

from UT, p<0.05. Symbols within parentheses indicate tendency for statistical significance,

0.05≤p<0.1.

Fig. 6. Skeletal muscle pyruvate dehydrogenase kinase (PDK)2 (A) and PDK4 (B) before (Pre) and

45 min into and oral glucose tolerance test (Post) in untrained (Untrained) and exercise trained

(Trained) aged male subjects. *: Significantly different from Pre p<0.05, #: Significantly different

from UT, p<0.05. Symbols within parentheses indicate tendency for statistical significance,

0.05≤p<0.1.

Fig.7 Representative blots before (B) and 45 min into and oral glucose tolerance test (G) in skeletal

muscle of untrained (UT) and exercise trained (T) subjects.

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Time (min)0 20 40 60 80 100 120

Pla

sma

gluc

ose

(mm

ol/l)

0

2

4

6

8

10

UT T

(#)*

*

*

*

*

**

*

Time (min)0 20 40 60 80 100 120

Pla

sma

insu

lin (p

mol

/l)

0

200

400

600

800

1000

#

#

#

*

*

*

*

*

* *

*

*

**

*

Time (min)0 20 40 60 80 100 120

Plas

ma

C-p

eptid

e (p

mol

/l)

0

500

1000

1500

2000

2500

3000

3500

4000#

#

*

**

**

*

*

**

* **

Fig.1

A)

B)

C)

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Pro

tein

con

tent

(AU

.)

0.0

0.5

1.0

1.5

2.0

2.5 UTT

(#)

#

#

##

HKII GLUT4 Akt1 Akt2 TBC1D4 GS PDH-E1 GSK-3

Fig. 2

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Untrained Trained

Akt S

er47

3 pho

spho

ryla

tion

(AU

)

0.0

0.5

1.0

1.5

2.0

2.5

*

*#

Untrained Trained

Akt T

hr30

8 pho

spho

ryla

tion

(AU

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8 Pre Post

*

*

Fig. 3

A)

Untrained Trained

TBC

1D4

Thr64

2 pho

spor

ylat

ion

(AU

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

*

*#

C)

C) Untrained Trained

GSK

3- S

er9 p

hosp

hory

latio

n (A

U)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

**

B)

D)

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Untrained Trained

GS

site

3a

phos

phor

ylat

ion

(AU

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

*

*#

#

Untrained Trained

GS

activ

ity (

I-for

m)

0

5

10

15

20

25

30

*

Untrained Trained

GS

site

2+2

a ph

osph

oryl

atio

n (A

U)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4 Pre Post

*

#(#)

Fig. 4

A)

B)

C)

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Untrained Trained

PDH

site

1 p

hosp

hory

latio

n (A

U)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4Pre Post

(#) #

Untrained Trained

PDH

site

2 p

hosp

hory

latio

n (A

U)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

##

Fig.5

A)

B)

Untrained Trained

PDH

a ac

tivity

/ to

tal C

r (m

mol

·kg-1

·min

-1)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

*

C)

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Untrained Trained

PDK2

pro

tein

(AU

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2 Pre Post

# #

Fig.6

A)

Untrained Trained

PD

K4

prot

ein

(AU

)

0.0

0.2

0.4

0.6

0.8

1.0 (*)B)

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GS site 3a

TBC1D4 Thr642

GAPDH

HKII

PDH site 2

GLUT4

PDK4

PDH-E1

GSK-3 Ser9

GSK-3

GS site 2+2a AKT2

Akt Thr308

PDH site 1 GS total

Akt1

TBC1D4

UT UT T T B G B G

UT UT T T B G B G 50 kD

37 kD

150kD 100 kD

50 kD 37 kD

75 kD 50 kD

75 kD 50 kD

250 kD 150 kD

100 kD 75 kD

50 kD 37 kD

50 kD 37 kD

75 kD 60 kD

50 kD 37 kD

250 kD 150 kD

100 kD 75 kD

100 kD 75 kD

50 kD 37 kD

50 kD 37 kD

50 kD 37 kD

75 kD 60 kD

Akt Ser473

50 kD 37 kD

PDK2

Fig.7

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Supplemenray methods

Ethics: The subjects were informed of the risk and discomfort associated with the experimental

protocol and provided oral and written consent to participate. The study was approved by the Ethics

Committee of Copenhagen and Frederiksberg communities (H-2-2011-079) and was conducted in

accordance with the guidelines of the Declaration of Helsinki.

Experimental setup. The subject arrived to the laboratory after 10-12h of fasting and 24h after the

last exercise session. A venflon was inserted into an antecubital vein in the forearm and a blood

sample was taken. The glucose beverage was ingested within 4 min and additional blood samples

were obtained 15 min, 30 min, 45 min, 60 min, 90 min and 120 min after the glucose intake and a

second biopsy 45 min after the glucose intake. The incisions for the biopsy needles were made

under local anesthesia (lidocain; AstraZeneca, Södertälje, Sweden) and new incisions were made

for each biopsy. Superficial blood was quickly removed from the biopsies, which were frozen in

liquid nitrogen within 20 seconds and stored at -80°C.

Blood parameters: The blood plasma was analyzed for glucose, insulin and C-peptide

concentrations at Department of Clinical Biochemistry at Rigshospitalet, Copenhagen, Denmark.

Muscle analyses

Muscle biopsies. Each muscle biopsy was cut in smaller pieces for determination of PDHa activity

and the rest was freeze-dried for at least 48h. The freeze-dried samples were dissected free of blood,

fat and connective tissue under the microscope.

Muscle glycogen. The muscle glycogen concentration was determined as previously described [30]

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SDS-PAGE and western blotting. The freeze-dried muscle tissue was homogenized using a Tissue

LyserII (Qiagen, Hilden, Germany) and muscle lysate was prepared as previously described [31].

SDS-PAGE and western blotting were performed as previously described [31] using hand casted

gels. Protein and phosphorylation levels were determined in muscle lysate using antibodies towards

HKII, GSK-3 Ser9, GSK-3, Akt Ser473, Akt1 and Akt2 (#2867, #9323, #9315, #9271, #2967,

#3063 respectively; Cell Signaling Technology, Berverly, MA, USA), GLUT4 (#998-101; ABR,

Golden, CO, USA), PDK2 (#ST1643, Millipore, Billerica, MA, USA), Akt Thr308 (#05-669,

Upstate Biotechnology, Lake Placid, NY, USA), TBC1D4 (#AB24469; Abcam, Cambridge, MA,

USA), TBC1D4 Thr642 (#3028 p1; Symansis, New Zealand) as well as PDH site1 (Ser293), PDH site

2 (Ser300) phosphorylation, PDH-E1 and PDK4 (provided by Graham Hardie, Dundee University,

Dundee, U.K) or in muscle homogenate using antibodies towards GS 2+2a, GS 3a (provided by

Graham Hardie) and GS (provided by Oluf Pedersen, University of Copenhagen, Denmark). The

bands on the membranes were visualized with ECL reagent (Millipore, Billerica, MA, USA) in a

digital image system (GE healthcare, München, DE).

PDHa activity. PDHa activity in each sample was determined in muscle homogenate as previously

described [32-34].

GS activity. GS activity in each sample was determined in muscle homogenate as previously

described [35]

Statistics. Values are presented as mean ± SE unless otherwise stated. A student paired t-test was

used to test the effect of exercise training on fasting plasma insulin and glucose as well as protein

content. Two-way ANOVA with repeated measures was used to test the effect of exercise training

and glucose intake on plasma glucose, insulin and c-peptide as well as skeletal muscle protein and

protein phosphorylation levels. When a main effect was observed, a Student-Newman-Keuls post

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hoc test was used to locate differences between groups if the data set was log transformed if the data

did not pass equal variance test. The data were considered significant at P<0.05 and a tendency is

reported when 0.05≤P<0.1. Statistical calculations were performed in SigmaPlot 11.0.

Supplementary Results

Plasma glucose

There was no difference in plasma glucose when comparing the area under the curve (AUC) for

glucose before and after the training intervention.

Intracellular signaling

GSK3β phosphorylation: Normalized GSK3β Ser9 phosphorylation in skeletal muscle increased

(p<0.05) similarly 1.3-1.4 fold 45 min after glucose intake before and after the exercise training

period with no difference between the two conditions (Fig 1A).

Akt phosphorylation: Akt Thr308 phosphorylation normalized to Akt2 protein content increased

(p<0.05) 2.5 fold after glucose intake both before and after exercise training with no difference

between the conditions (supplementary Fig 1B).

Akt Ser473 phosphorylation normalized to Akt2 protein increased (p<0.05) similarly 2.2 fold and 3.5

fold in the untrained and trained state, respectively, with no difference between training status

(supplementary Fig 1C).

TBC1D4 phosphorylation: TBC1D4 Thr642 phosphorylation normalized to TBC1D4 protein content

increased (p<0.05) similarly ~2.5 fold in response to glucose intake in the untrained and trained

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state with no difference in TBC1D4 phosphorylation level before and after exercise training

(supplementary Fig 1D.

Glycogen synthase regulation

GS site 2+2a: GS site 2+2a phosphorylation normalized to GS protein increased (p<0.05) ~1.8 fold

with glucose intake before exercise training, but was unchanged in response to glucose intake after

the exercise training period (Supplementary Fig 2A).

GS site 3a: GS site 3a phosphorylation normalized to GS protein showed a similar pattern as the

absolute phosphorylation level both in response to glucose intake and exercise training

(Supplementary Fig 2B).´

PDH regulation

PDH phosphorylation: There was no effect of glucose intake on the normalized PDH site 1 and site

2 phosphorylation in skeletal muscle. Exercise training had no effect on PDH phosphorylation when

normalization to PDH-E1α protein, except for site 2 phosphorylation in the basal state

(Supplementary Fig 3A, 3B).

Supplementary figure legends

Supplementary figure 1. Skeletal muscle protein kinase B (PKB/Akt) Thr308 normalized to Akt2

protein (A) Akt Thr473 normalized to Akt2 protein (B) glycogen synthase kinase (GSK)-3 Ser9

phosphorylation normalized to GSK3 protein (C) and TBC1D4 Thr642 phosphorylation normalized

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to TBC1D4 protein (D) before (Pre) and 45 min into and oral glucose tolerance test (Post) in

untrained (Untrained) and exercise trained (Trained) aged male subjects. *: Significantly different,

from Pre p<0.05, #: Significantly different from UT, p<0.05. Symbols within parentheses indicate

tendency for statistical significance, 0.05≤p<0.1.

Supplementary figure. 2. Skeletal muscle glycogen synthase (GS) site 2+2a phosphorylation

normalized to GS protein (A) and GS site 3a phosphorylation normalized to GS protein (B) before

(Pre) and 45 min into and oral glucose tolerance test (Post) in skeletal muscle of untrained

(Untrained) and exercise trained (Trained) aged male subjects. *: Significantly different, from Pre

p<0.05, #: Significantly different from UT, p<0.05. Symbols within parentheses indicate tendency

for statistical significance, 0.05≤p<0.1.

Supplementary figure.3. Skeletal muscle pyruvate dehydrogenase (PDH) site 1 phosphorylation

normalized to PDH-1Eα protein (A) and PDH site 2 phosphorylation normalized to PDH-1Eα

protein (B) before (Pre) and 45 min into and oral glucose tolerance test (Post) in untrained

(Untrained) and exercise trained (Trained) aged male subjects. #: Significantly different from UT,

p<0.05.

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Untrained Trained

Akt

Ser

473 p

hosp

hory

latio

n / A

kt2

prot

ein

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

**

Untrained Trained

TBC

1D4

Thr64

2 pho

spho

ryla

tion

/ TBC

1D4

prot

ein(

AU)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

*

*

Untrained TrainedGSK

3 S

er9 p

hosp

hory

latio

n / G

SK3

pro

tein

(AU

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0 Pre Post

*

*

A)

C)

Supplementary fig. 1

Untrained TrainedAKT

Thr30

8 pho

spho

ryla

tion

/ Akt

2 pr

otei

n (A

U)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

*

(#)

*

B)

D)

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Untrained TrainedGS

site

2+2

a ph

osph

oryl

atio

n / G

S pr

otei

n (A

U)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6 Pre Post

(#)

*

Untrained TrainedGS

site

3a

phos

phor

ylat

ion

/ GS

prot

ein

(AU

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

(*)

*

#

#

A)

C)

B)

Supplementary fig. 2

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Supplementary fig. 3

Untrained Trained

PDH

site

1 p

hosp

hory

latio

n/ P

DH

-E1a

pro

tein

(AU

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6 Pre Post

A)

Untrained Trained

PDH

site

2 p

hosp

hory

latio

n / P

DH

-E1

pro

tein

(AU

)

0.0

0.5

1.0

1.5

2.0

2.5

#

B)

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THE FACULTY OF SCIENCE,U N I V E R S I T Y O F C O P E N H A G E N

ffiThe phD School of scIENCE -ffi

-"6d#'Lra+r-

3A. Co-authorship statement

All papers/manuscripts with multiple authors enclosed as annexes to a PhD thesis synopsis

should contain a co-author statement, stating the PhD student's contribution to the paper.

2.Title of PhD thesis

Regulation of PDH, GS and insulin signalling in skeletal muscle; effect of physical activity level and inflammation

3.This co-authorship declaration applies to the following paper

Effects of exercise training on regulation of skeletal muscle glucose metabolism in skeletal muscle of elderly men

The extent of the PhD student's contribution to the article is assessed on the following scale

A. has contributed to the work (0-33%)

B. has made a substantial contribution (34-66%)

C. did the majority of the work independently (67-100%).

w

1. General information

PhD student

NameRasmus SjOrup BiensoCiv.reg.n o. (lf not applicable, then birth dote)[email protected] of Biology

Please mark

PhD Schemex 5+3 Scheme 4+4 Scheme Industr ial PhD

Principal supervisor

NameHenriette Pi legaardE-mai lHPi [email protected]

7/tRevised 29 Jonuary 2073

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THE FACULTYU N I V E R S I T Y O F

OF SCIENCE,C O P E N H A G E N

The PhD School of SCIENCE

4. Declaration on the individual elements Extent (A, B, C)

l. Formulation in the concept phase of the basic scientific problem on the basis of

theoretical questions which require clarification, including a summary of the general

questions which it is assumed will be answerable via analyses or concrete

experiments/investigations.

C

2. Planning of experiments/analyses and formulation of investigative methodology in

such a way that the questions asked under (1) can reasonably be expected to be

answered, including choice of method and independent methodological development.

C

3. Involvement in the analysis or the concrete experiments/investigation.C

4. Presentation, interpretation and discussion of the results obtained in article form. C

5. Material in the paper from another degree / thesis :Articles/work published in connectionwith another degredthesis must notform part of the PhD thesis.Data collected and preliminary work carried out as part of another degree/thesis may be part of the PhD thesis if

further research, analysis andwriting are carried out as part of the PhD study.

Does the paper contain data material, which has also formed part of a previous degree / thesis(e.g. your masters degree)

Please indicate which desree / thesis:

Yes: E

No: E

Percentage of the paper that

Percentage of the paper that

is from the PhD degree work

is from the other degree / thesis

%

%

Etl

Please indicate which specific part(-s) of the paper that has been produced as part of the PhD study:

6.Signatures of co-authors :

i+li- lt,1

Jacob Friis Smith

2/3Revised 29 Januory 20L3

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THE FACULTYU N I V E R S I T Y O F

OF SCIENCE,C O P E N H A G E N

The PhD School of SCIENCE

n/a- - flo/lMikkel Sillesen Matzen kahlmJargen Frank Pind Wojtaszewski t{1ulYlvaHellsten Ir

U\fKWHenriette Pilegaard rhlou^*)

T.Signature

By signing the document, the PhD student hereby declares that the above information is correct.

03o2 2o /V(dd.mm.yy14t)

when completed, the form shoutd be sent to the relevant department's phD secretary who on your behalf wiltforward it to the PhD School ([email protected]).

To find your departments phD secretary, please see:

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Study III

Biensø RS, Olesen J, van Hauen L, Meinertz S, Halling JF, Gliemann L, Plomgaard P,

Pilegaard H.. Exercise-induced AMPK and pyruvate dehydrogenase regulation is maintained

during LPS-induced inflammation. Submitted Pflugers Archive, European Journal of Physiology

2014

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Exercise-induced AMPK and pyruvate dehydrogenase regulation is

maintained during LPS-induced inflammation

1Rasmus Sjørup Biensø, 1Jesper Olesen, 1Line van Hauen, 1Simon Meinertz, 1Jens Frey Halling,

2Lasse Gliemann, 3Peter Plomgaard, 1Henriette Pilegaard

1Centre of Inflammation and Metabolism, CFAS and August Krogh Centre, Department of Biology,

University of Copenhagen, Denmark

2Department of Nutrition Sport and Exercise, University of Copenhagen, Denmark

3Centre of Inflammation and Metabolism and The Centre for Physical Activity Research (CFAS),

Department of Clinical Biochemistry, Rigshospitalet, Denmark

Keywords: LPS, TNFα, skeletal muscle, substrate oxidation, human, AMPK, exercise

Corresponding author

Henriette Pilegaard

Universitesparken 13, 4sal

2100 Copenhagen Ø

Mail: [email protected]

Phone no. : +45 35321687

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ABSTRACT

The aim of the present study was to examine the effect of LPS-induced inflammation on AMPK and

PDH regulation in human skeletal muscle at rest and during exercise. Nine young healthy

physically inactive male subjects completed two trials. In an LPS trial, the subjects received a single

lipopolysaccharide (LPS) injection (0.3ng/kg body weight) and blood samples and vastus lateralis

muscle biopsies were obtained before and 2h after the LPS injection and immediately after a 10 min

one-legged knee extensor exercise bout performed approximately 2½ hour after the LPS injection.

The exercise bout with muscle samples obtained before and immediately after was repeated in a

control trial without LPS injection. The plasma tumor necrosis factor (TNF)α concentration

increased 17 fold 2h after LPS relative to before. Muscle lactate and muscle glycogen were

unchanged from before to 2h after LPS and exercise increased muscle lactate and decreased muscle

glycogen in the control (P<0.05) and the LPS (0.05≤P<0.1) trial with no differences between the

trials. AMP-activated kinase (AMPK), acetyl-CoA carboxylase (ACC) and pyruvate dehydrogenase

(PDH) phosphorylation as well as PDHa activity were unaffected 2h after LPS relative to before.

Exercise decreased (P<0.05) PDH and increased (P<0.05) AMPK and ACC phosphorylation as well

as increased (P<0.05) PDHa activity similarly in the LPS and control trial. In conclusion, LPS-

induced inflammation does not affect resting or exercise-induced AMPK and PDH regulation in

human skeletal muscle. This suggests that metabolic flexibility during exercise is maintained during

short-term low grade inflammation in humans.

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INTRODUCTION

Skeletal muscle has an extraordinary ability to regulate substrate choice and utilization according to

availability [13;32]. The exercise-induced enhancement of glucose and fat oxidation in skeletal

muscle ensures ATP production for muscle contractions and the interaction between fatty acids and

glucose regulates the relative fatty acid and glucose oxidation contributes to efficient substrate

utilization [32]. Regulation of substrate choice and substrate utilization may however be influenced

by metabolic changes and contribute to metabolic dysfunction. For example chronically elevated

plasma free fatty acid (FFA) levels will inhibit glucose oxidation and elevated plasma glucose may

inhibit fat oxidation in skeletal muscle [19;23;27;32]. Similarly, metabolically related diseases are

often associated with low grade inflammation characterized by chronically elevated levels of

circulating cytokines [25] like the pro-inflammatory cytokine tumor necrosis factor (TNF).

Previous studies have linked TNFα to insulin resistance in rat and human skeletal muscle [17;30],

as well as indicated TNFα-mediated effects on substrate utilization [35;40;47].

The pyruvate dehydrogenase (PDH) complex has a key position in the regulation of substrate choice

as it catalyzes the decarboxylation of pyruvate to acetyl CoA, which represents the entry of

carbohydrate-derived substrate into the mitochondria for oxidation [33]. In accordance, exercise has

been shown to induce a rapid increase in the activity of PDH in the active form (PDHa) in human

skeletal muscle [18] concomitant with increased glucose oxidation [31]. Furthermore, elevated

plasma FFA has been shown to reduce the exercise-induced increase in PDHa activity in human

skeletal muscle [20] supporting that PDH contributes to regulating substrate utilization during

exercise as part of the interaction between fatty acids and carbohydrates [32;33]. The PDHa activity

is thought mainly to be regulated by phosphorylation of the PDH-E1 subunit [22;28] determined

by the activity of PDH kinases (PDK), which phosphorylate and inactivate the enzyme and PDH

phosphatases (PDP), which dephosphorylate and activate PDH. Previous studies have shown that

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the PDK4 protein content is up-regulated in rat and/or human skeletal muscle by fasting and high-

fat diet [26;46] and this regulation has been suggested to contribute to the associated changes in

PDHa activity [16]. Although the PDK4 protein content has been shown to be unaffected during

even prolonged exercise, the PDK4 protein content has been demonstrated to be rapidly regulated in

human skeletal muscle by carbohydrate availability [20] indicating a potential role of acute changes

in PDK4 protein content in PDH regulation.

Several previous studies suggest that inflammation influences PDH regulation. Repeated E-coli

injections in rats have been shown to reduce the concentration of active PDH complex (PDC) and

increase PDK activity [39;40]. Moreover the observations that treatment with TNFα reduced PDH

activity in rat cardiomyocytes [47] and human immune cells [36] as well as the finding that

treatment with TNF binding protein alleviated the effects of inflammation on PDH activity [40]

indicate that TNFα influences PDH regulation. These findings are further supported by a recent

study showing that LPS infusion for 24 hours decreased PDHa activity and increased PDK4 protein

content in rat skeletal muscle [12]. On the other hand, local infusion of LPS or TNF in one leg did

not change resting PDH-E1 phosphorylation in human skeletal muscle [6;9], which may suggest

that the impact of inflammation on PDH regulation at rest depends on species, dose or duration of

the treatment.

The intracellular energy sensor, AMP-activated protein kinase (AMPK), is also a key factor in the

regulation of substrate utilization. AMPK activity is increased both by AMP mediated allosteric

regulation and by phosphorylation [44] leading to stimulation of several downstream processes

aiming at increasing ATP production. In accordance, exercise increases AMPK phosphorylation

and activity in skeletal muscle leading to an enhancement of fat oxidation in skeletal muscle

through AMPK-mediated phosphorylation and inactivation of acetyl-CoA carboxylase (ACC) [43].

As ACC catalyzes the production of malonyl CoA, an inactivation of ACC leads to reduced

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production of malonyl-CoA with concomitantly less inhibition of carnitine palmitoyltransferase I

and an increased fatty acid oxidation [15].

AMPK has been suggested to have anti-inflammatory effects [14], and AMPK phosphorylation has

been reported to increase in human skeletal muscle after an LPS injection, although a concomitant

increase in ACC phosphorylation was not observed [3]. This may suggests that inflammation

modify AMPK-mediated intracellular signaling in resting skeletal muscle.

Although previous studies indicate that inflammation affects PDH and AMPK regulation, it is yet

unresolved how inflammation influences exercise-induced AMPK and PDH regulation in human

skeletal muscle. Therefore the aim of the present study was to investigate the effects of LPS-

induced inflammation on AMPK and PDH regulation in human skeletal muscle at rest and in

response to an acute exercise bout.

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METHODS

Subjects

Nine physically inactive young healthy male subjects in the range from 20-26 years of age with an

average body mass index of 25.6±1.3 (mean±SE) participated in the study. The subjects were

physically active physical less than 1h per week. Each subject underwent a health examination by a

medical doctor. The subjects could participate in the study if the VO2max was less than 45 ml·min-

1·kg-1 and was approved by the medical doctor. The subjects were given both written and oral

information about the study and the subjects gave their written consent to participate in the study.

The study performed according to the Declaration of Helsinki and approved by the Copenhagen and

Frederiksberg Ethical committee in Denmark (H-1-2012-108).

Pre-testing

The VO2max was measured for each subject using an incremental ergometer bicycle test (Monarch

Ergomedic 839E) for use as inclusion criteria. In addition, Wattmax was determined during an

incremental one-legged knee extensor exercise test on a modified ergometer bicycle as previously

described [29]. The Wattmax was used to determine the resistance during the experimental trials.

Experimental protocol

The subjects completed a LPS trial and a control trial separated by a least 7 days. For both trials, the

subjects were instructed to eat a carbohydrate rich meal 1h before arriving to the laboratory.

LPS trial. After arriving to the laboratory, a catheter was placed in the femoral artery and in the

femoral vein of one leg and a venflon was placed in an antecubital vein in the forearm. Blood

samples were obtained from the femoral catheters and a muscle biopsy was obtained from the

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vastus lateralis muscle using the needle biopsy method [8] with suction. Insertions for biopsies were

made under local anesthesia (Lidocaine, AstraZeneca, Södertälje, Sweden). A LPS solution (100

ng/ml) was either freshly prepared from a LPS stock (The Clinical Center, Critical Care Medicine

Department, Bethesda, MD, USA) or used within a week from the preparation with storage at -

20°C. An intravenous injection of 0.3 ng/kg LPS was given through an arm venflon approximately

2.5 hours after the subjects had eaten breakfast. Additional blood samples were obtained 30 min, 60

min, 90 min and 120 min after LPS injection and additional muscle biopsies at 1 hour and 2 hour

after LPS was given. The 1 hour biopsy, all venous blood samples and arterial blood samples

obtained at 30, 60 and 90 min after the LPS injection are not used in the present study except the

venous sample before LPS for comparison of the arterial and venous TNFα level. After the 2h blood

and tissue sampling, the subjects were transferred to a chair connected to a one-legged knee

extensor ergometer bicycle with the back of the chair lying down. The right foot of the subject was

tied to a rod connected to the crank set of the modified ergometer bicycle. The subjects first

performed 1 min passive knee extensions to warm up the leg followed by 5 min exercise at 50% of

Wattmax and 5 min exercise at 60% of Wattmax. Additional blood samples were obtained 8 min into

the exercise and an additional muscle biopsy was obtained from the vastus lateralis muscle of the

working leg immediately as the exercise was terminated after 10 min of exercise.

Individual insertions were made for each biopsy. Visual fat, connective tissue and blood were

removed from the biopsies, which were quickly frozen in liquid nitrogen. The muscle biopsies were

stored at -80°C. The blood was collected in EDTA containing tubes, which were centrifuged and

plasma was collected and stored at -80°C.

Ear temperature, mean arterial blood pressure (MAP) and heart rate were recorded every 15 min

until 3h after the LPS injection. MAP and heart rate monitoring continued until at least 4 hours after

the LPS injection to insure that normal blood pressure regulation was re-established.

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Control trial. After arriving to the laboratory, a venflon was inserted in a vein in the forearm and a

blood sample was obtained through the venflon. In addition, a muscle biopsy was obtained from the

vastus lateralis muscle as described above. The subjects were placed in the chair connected to the

one-legged knee extensor ergometer bicycle with the back of the chair lying down. The right leg of

the subject was tied to the modified ergometer bicycle and 1 min of passive knee extensions were

performed followed by 5 min exercise at 50% Wattmax and 5 min exercise at 60% Wattmax as in the

LPS trial. An additional blood sample was drawn after 8 min of exercise and an additional Vastus

lateralis muscle biopsy was obtained from the working leg immediately after 10 min of exercise.

Blood and muscle biopsies were handled as described for the LPS trial above.

Plasma analyzes

Plasma glucose and insulin. The blood was analyzed immediately for blood glucose (ABL,

Radiometer 725 series Acid-Base Analyzer, Denmark) and insulin was measured at The

Department of Clinical Biochemistry at Rigshospitalet, Copenhagen, Denmark.

Plasma TNF. The plasma TNF was determined using a MSD multi-spot 96 wells plate with pre-

coated antibodies (MesoScaleDiscovery, Gaithersburg, ML, USA). The plates were measured on

MSD Sector Image 2400 plate reader. The data were analyzed using the Discovery Workbench 3.0

(MSD). The results were converted to a concentration by use of a standard curve constructed from a

serial dilution of recombinant TNF run alongside on each plate.

Muscle analyses

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Freeze-drying. Muscle biopsies were freezed-dried for at least 48 hours and the samples were

dissected free from visual blood and connective tissue under the microscope. The muscle tissue was

weighted out for the different analyses and stored at -80°C.

Muscle glycogen. The muscle glycogen concentration was determined on freeze-dried muscle tissue

as glycosyl units after acid hydrolysis as previously described [24]

Muscle lactate. PCA extract was made on freeze-dried muscle tissue as previously described [7].

Muscle lactate was measured using the auto-fluorescence ability of NADH as previously described

[7].

SDS-PAGE and Western blotting. The freeze-dried muscle was homogenized using a Tissue LyserII

(Qiagen, Hilden, Germany) and the homogenized muscle samples were made into lysates as

previously described [38]. The protein concentration was determined with the bicinchoninic acid

method (Pierce, ThermoScientific, Rockford, IL, USA) using BSA as a standard.

The lysate samples were loaded on hand casted gels (7.5%-10%). After the gel electrophoresis, the

proteins were blotted from the gel to a PVDF membrane (Millipore, Bedford, USA), blocked in 3%

fish gelatin solution and incubated with antibodies. The protein content and phosphorylation level

were determined using antibodies towards AMPK Thr172 phosphorylation (2535; Cell Signaling

Technology, Berverly, MA, USA), ACC Ser79 phosphorylation (07-303; Millipore, Bedford, USA),

PDH Ser293 (site1), PDH Ser300 (site 2), PDH Ser295 (site 4) phosphorylation, PDH-E1 protein,

AMPKα2 protein and PDK4 protein (provided by Graham Hardie, Dundee University, Dundee,

U.K). ACC protein content was detected using streptavidin-HRP (Dako, Glostrup, Denmark). The

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bands on the membranes were visualized with ECL reagent (Millipore, Billerica, MA, USA) in a

digital image system (GE healthcare, München, DE).

PDHa activity. The PDHa activity was determined as previously described [10;11;31]). In brief,

muscle homogenate was prepared on ice from wet weight muscle tissue using a glass homogenizer

(Kontes, Vineland, NJ, USA). Pyruvate was converted to acetyl CoA followed by determination of

the acetyl CoA content by use of a radioactive assay. The PDHa activity was determined as the rate

of conversion of pyruvate to acetyl CoA and normalized to the total creatine content in each sample

as previously described [34].

Statistics

Values presented are means±SE. A paired t-test was used to test the effect of LPS at rest. A Two

Way ANOVA with repeated measures was used to evaluate the effect of LPS and exercise. The data

were log transformed if normality or equal variance test failed. If a significant main effect was

detected, the student Newman-Keuls test was used to locate differences. Differences are considered

significant at p<0.05 and a tendency is reported for 0.05≤p0.1. Statistical calculations were

performed using SigmaPlot 11.0

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RESULTS

Physical parameters

The mean arterial pressure was stable around 85-90 mmHg and the ear temperature was unchanged

around 37-37.6°C. The heart rate increased (P<0.05) from 63±3 before LPS to 83±4 beats/min 3h

after the LPS injection (Table 1).

Plasma parameters

Plasma glucose and insulin

The arterial plasma glucose concentration decreased (P<0.05) 2h after LPS relative to before the

LPS injection. Exercise did not change the plasma glucose concentration in the LPS trial (Table 1).

The arterial plasma insulin concentration decreased (P<0.05) 2h after LPS injection relative to

before LPS. Exercise did not affect the plasma insulin level in the LPS trial (Table 1).

Plasma TNFα

Plasma TNFα was determined as a measure of the LPS induced inflammation. The plasma TNF

concentration before LPS injection was similar in the femoral artery and femoral vein suggesting

that arterial and venous levels can be compared at least in a non-inflammatory state. The arterial

plasma TNF concentration increased (P<0.05) ~17 fold 2h after LPS injection relative to before

LPS. Exercise did not change the plasma TNF concentration in either trial. The arterial plasma

TNF concentration was 13-15 fold higher (P<0.05) in the LPS trial than the venous plasma TNF

in the control trial before and at the end of exercise (Table 2).

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Muscle analyses

Muscle glycogen

The muscle glycogen concentration was unaffected from before LPS injection to 2h after LPS.

Exercise reduced (p<0.05) muscle glycogen 27% in the control trial and tended to reduce

(0.05<P<0.1) muscle glycogen 6% in the LPS trial. There was no significant difference in the

muscle glycogen concentration between the two trials neither before nor after exercise (Table 3).

Muscle lactate

Muscle lactate did not change from before the LPS injection to 2h after LPS. Exercise increased

(P<0.05) muscle lactate 6.8 fold in the control trial and tended to increase (0.05<P<0.1) muscle

lactate 4.9 fold in the LPS trial with no difference between the two trials neither before exercise nor

after exercise (Table 3).

AMPK and ACC phosphorylation

There was no difference in AMPK Thr172 and ACC Ser79 phosphorylation in skeletal muscle before

and 2 hours after LPS injection (Figure 1A and 1C).

The exercise bout increased (P<0.05) skeletal muscle AMPK phosphorylation ~3 fold and ACC

phosphorylation 6-7 fold in the control and LPS trial with no difference in the responses between

the trials. The AMPK and ACC phosphorylation levels were similar in the two trials both before

and after exercise (Figure 1B and 1D).

PDH-E1 phosphorylation

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PDH-E1 phosphorylation at Ser293, Ser300 and Ser295 was unchanged 2 hours after LPS injection

relative to before (Figure 2A, 2C and 2E).

Exercise decreased (P<0.05) the PDH-E1 phosphorylation at site Ser293 40-50%, site300 60-70%

and site Ser295 30-40 % in the control and LPS trial with no difference in the responses between the

trials. The PDH-E1 phosphorylation level was for each of the three sites similar in the control and

LPS trial both before and after exercise (Figure 2B, 2D,2F).

PDHa activity

The PDHa activity was unchanged 2 hours after LPS injection relative to before LPS. Exercise

increased (P<0.05) the PDHa activity ~1.8 fold in the control trial and ~2.2 fold in the LPS trial

with no difference in the response between the trials. The PDHa activity was similar in the two

trials both before and after exercise (Figure 3).

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DISCUSSION

The main findings of the present study are that LPS-induced inflammation with elevated plasma

TNFα concentration does not affect the exercise-induced AMPK and PDH regulation in human

skeletal muscle. In addition, short-term inflammation does not affect AMPK and PDH regulation at

rest.

The present human study used a single LPS injection to induce a controlled inflammation in young

volunteers as a model for low grade inflammation as frequently used [4;5;36;37]. The present

observation that the LPS injection increased the plasma TNFα concentration 17 fold to 15 ng/l 2

hours after the LPS injection is in accordance with several previous studies [4;5;37] and shows that

the anticipated inflammatory state was obtained.

The observation that skeletal muscle PDHa activity and PDH phosphorylation did not change 2h

after LPS injection in the current study is different from previous observations in rats [12;39;40].

Hence 24h of LPS infusion in rats has been shown to reduce the PDHa activity in skeletal muscle

[12] and sepsis induced by repeated treatments with E-coli was associated with reduced

concentration of active PDC in skeletal muscle [41]. The additional finding that treatment with

TNF binding protein prevented the E-coli-induced reduction in PDH activity suggested that TNF

is important in the LPS-induced effects on PDH during sepsis in rats [40]. Although the different

observations may be due to species or model differences, it seems possible that the dose and the

duration of treatment could be important. While a single injection of 0.3 ng·kg-1 was used in the

present study, effects on PDH was observed in rats with 24h of LPS infusion resulting in an 8.9 fold

increase in plasma TNFα [2;12]. In addition, while the present study did not observe any change in

muscle lactate 2h after the LPS injection, a previous rat study using a high dose of LPS infusion

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(150µg·kg-1·h-1) reported that muscle lactate increased after 2 hours of LPS treatment [2] indicating

that the inflammation inhibited the PDH.

Previous studies have also indicated a link between TNFα and AMPK, although different effects

have been reported. The unchanged AMPK phosphorylation 2h after the LPS injection in the

present human study is hence not in accordance with a previous study showing that treatment of

muscle cells with TNFα decreased the AMPK activity [35]. The current finding is in line with

previous observations in humans, although AMPK phosphorylation increased in human skeletal

muscle 4h after a single LPS injection [3]. This may suggest that the different observations are due

to model differences and the present findings do not oppose the possibility that LPS-induced

inflammation in humans increases AMPK phosphorylation in skeletal muscle.

The present study is (to our knowledge) the first to examine the impact of LPS-induced

inflammation on exercise-induced metabolic regulation in humans. The exercise-induced decrease

in PDH phosphorylation and increase in AMPK and ACC phosphorylation as well as in PDHa

activity in the control trial are as expected based on many similar previous studies [20;21;28;45].

The present finding that the exercise-induced regulation of PDH phosphorylation and PDHa activity

was similar in the LPS trial, where TNFα was elevated, as in the control trial is however not as

hypothesized. As PDH activation increases carbohydrate oxidation [31;42] and AMPK-mediated

ACC inactivation increases fat oxidation [15;43], these observations suggest that human skeletal

muscle maintains the ability to increase carbohydrate and fat oxidation in response to exercise

despite the presence of short-term systemic inflammation. This conclusion is supported by the

similar increase in the muscle lactate concentration in the two trials in the present study.

The lack of effect of inflammation on exercise-induced PDH and AMPK regulation suggests that

exercise-induced metabolic flexibility is maintained during inflammation, which is in contrast to

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previous studies showing that elevated systemic TNFα levels [30] and a single LPS injection [1]

induce whole body insulin resistance in humans. These findings may suggest that insulin-signaling

and exercise-induced metabolic regulation are affected differently by inflammation. However of

notice is that the lack of effect on the exercise-induced responses was observed 2½ hours after LPS

injection, while the LPS-induced lowering of insulin sensitivity was demonstrated 420 min after

LPS was injected. On the other hand, the previously reported TNFα-induced insulin resistance was

observed within 2h of TNFα infusion with similar plasma levels of TNFα [30] as in the present

study, while the previous LPS study resulted in more than 50 fold higher plasma TNFα

concentration [1] than in the present and previous TNFα study [30]. Although the TNFα infusion

did result in a constant elevation in plasma TNFα, while the present study induced a gradual

increase in plasma TNF, these considerations do support a different impact of TNFα on insulin-

mediated glucose uptake than on exercise-induced AMPK and PDH regulation in human skeletal

muscle.

As the purpose of the present study was to examine the potential effect of inflammation with

elevated plasma TNFα, the exercise bout was placed at the time point where the plasma TNFα

concentration was expected to peak. In accordance, the observation that plasma TNFα was at a

similar level (15 fold elevated) and not further increased at the sampling time point during exercise

(approximately 2½ h after LPS injection) relative to 2h after LPS is in line with previous studies

showing that plasma TNF peaks at approximately 2h after a single LPS injection [1;3;5;37].

However, it is certainly possible that more long-term inflammation with more sustained elevation in

plasma TNF will affect the exercise-induced AMPK and PDH regulation in skeletal muscle, but

this remains to be determined.

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As previous studies examining the impact of LPS-induced inflammation and TNFα on PDH

regulation have observed that a down-regulation of the PDHa activity was associated with increased

PDK activity [39;40] and PDK4 protein content [12], it may be that inflammation-induced effects

on PDH regulation require changes in PDK4 expression. The present observation that the lack of

effect of inflammation on PDH regulation was associated with unchanged PDK4 protein content in

skeletal muscle does support this possibility that changes in PDK4 protein underlies part of the

effect of inflammation on PDH.

In conclusion, a single LPS injection resulting in 17 fold increase in plasma TNFα concentration did

not change AMPK and PDH phosphorylation and/or activity in human skeletal muscle at rest and

did not affect exercise-induced AMPK and PDH regulation in human skeletal muscle. This suggests

that the ability of skeletal muscle to increase glucose and fat oxidation during exercise is maintained

during short-term inflammation with elevated plasma TNFα levels. Hence, short-term low grade

inflammation does not seem to elicit metabolic inflexibility during exercise in humans as has been

reported for insulin resistance.

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Acknowledgement

We would very much like to thank to subjects for the participation in the study. In addition, we are

very grateful to Professor AF. Suffredini, National Institutes of Health, Bethesta, USA, for

providing the LPS for the present study. The study was supported by The Danish Council for

Independent Research, Danish Medical Research Council and The Danish Ministry of Culture for

Sports Research. The Centre of Inflammation and Metabolism (CIM) is supported by a grant from

the Danish National Research Foundation (DNRF55). The Centre for Physical Activity Research

(CFAS) is supported by a grant from Trygfonden. CIM is part of the UNIK Project: Food, Fitness &

Pharma for Health and Disease, supported by the Danish Ministry of Science, Technology, and

Innovation. CIM is a member of DD2 - the Danish Centre for Strategic Research in Type 2 Diabetes

(the Danish Council for Strategic Research, grant no. 09-067009 and 09-075724). The Copenhagen

Muscle Research Centre (CMRC) is supported by a grant from the Capital Region of Denmark.

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28. Pilegaard H, Birk JB, Sacchetti M, Mourtzakis M, Hardie DG, Stewart G, Neufer PD, Saltin B, van HG, Wojtaszewski JF (2006) PDH-E1alpha dephosphorylation and activation in human skeletal muscle during exercise: effect of intralipid infusion. Diabetes 55:3020-3027

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36. Sutendra G, Dromparis P, Bonnet S, Haromy A, McMurtry MS, Bleackley RC, Michelakis ED (2011) Pyruvate dehydrogenase inhibition by the inflammatory cytokine TNFalpha contributes to the pathogenesis of pulmonary arterial hypertension. J Mol Med (Berl) 89:771-783

37. Taudorf S, Krabbe KS, Berg RM, Pedersen BK, Moller K (2007) Human models of low-grade inflammation: bolus versus continuous infusion of endotoxin. Clin Vaccine Immunol 14:250-255

38. Treebak JT, Frosig C, Pehmoller C, Chen S, Maarbjerg SJ, Brandt N, Mackintosh C, Zierath JR, Hardie DG, Kiens B, Richter EA, Pilegaard H, Wojtaszewski JF (2009) Potential role of TBC1D4 in enhanced post-exercise insulin action in human skeletal muscle. Diabetologia 52:891-900

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40. Vary TC, Hazen SA, Maish G, Cooney RN (1998) TNF binding protein prevents hyperlactatemia and inactivation of PDH complex in skeletal muscle during sepsis. J Surg Res 80:44-51

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42. Watt MJ, Heigenhauser GJ, LeBlanc PJ, Inglis JG, Spriet LL, Peters SJ (2004) Rapid upregulation of pyruvate dehydrogenase kinase activity in human skeletal muscle during prolonged exercise. J Appl Physiol 97:1261-1267

43. Winder WW, Hardie DG (1996) Inactivation of acetyl-CoA carboxylase and activation of AMP-activated protein kinase in muscle during exercise. Am J Physiol 270:E299-E304

44. Winder WW, Hardie DG (1999) AMP-activated protein kinase, a metabolic master switch: possible roles in type 2 diabetes. Am J Physiol 277:E1-10

45. Wojtaszewski JF, MacDonald C, Nielsen JN, Hellsten Y, Hardie DG, Kemp BE, Kiens B, Richter EA (2003) Regulation of 5'AMP-activated protein kinase activity and substrate utilization in exercising human skeletal muscle. Am J Physiol Endocrinol Metab 284:E813-E822

46. Wu P, Sato J, Zhao Y, Jaskiewicz J, Popov KM, Harris RA (1998) Starvation and diabetes increase the amount of pyruvate dehydrogenase kinase isoenzyme 4 in rat heart. Biochem J 329 ( Pt 1):197-201

47. Zell R, Geck P, Werdan K, Boekstegers P (1997) TNF-alpha and IL-1 alpha inhibit both pyruvate dehydrogenase activity and mitochondrial function in cardiomyocytes: evidence for primary impairment of mitochondrial function. Mol Cell Biochem 177:61-67

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Table 1: Physiological and plasma parameters in the LPS trial

Pre 2h LPS/Pre exercise 3h LPS

Temperature (°C) 37.4±0.2 37.3±0.1 37.6±0.2

MAP (mmHg) 87.8±2.9 88.0±3.4 87.9±3.6

Heart rate (BPM) 63±3 68±4 86±4*

Arterial plasma glucose (mmol/l) 6.4±0.6 5.0±0.1 * 4.9±0.2

Arterial plasma insulin (pmol/l) 135.8±18.2 65.3±10.8* 69.3±8.9

Table 2: Plasma TNFα concentrations

Pre LPS 2h LPS/Pre Exercise Exercise

TNF (ng/l) Control - 0.97±0.1 1.0±0.1

LPS 0.90±0.1 15.5±1.6 *# 13.6±1.3*#

Table 3: Muscle glycogen and lactate concentrations

Pre LPS 2h LPS/Pre Exercise Post Exercise

Glycogen (mmol·kg-1 dw) Control - 365±38 267±44*

LPS 329±32 391±49 367±35(*)

Lactate (mmol·kg-1 dw) Control - 9.7±1.9 66.4±15.6*

LPS 10.9±1.3 10.8±1.4 53.2±13.3(*)

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Table and figure legends

Table 1

Ear temperature (˚C), mean arterial pressure (MAP; mmHg) and heart rate (beats/min) before (Pre

LPS) and 2h (2h LPS) and 3h (3h LPS) after a single LPS injection as well as arterial glucose

(mmol/liter) and arterial insulin (pmol/liter) at Pre LPS, 2h LPS corresponding to before exercise

(Pre Exercise) and at 8 min of exercise (Exercise). The values are mean±SE. *:significantly

different from Pre LPS, P<0.05.

Table 2

Arterial (LPS trial) and venous (control trial) plasma tumor necrosis factor (TNF)α (ng/liter)

concentration before a single LPS injection (Pre LPS) and 2h after the LPS injection corresponding

to before exercise (2h LPS/Pre Exercise) and 8 min into an one-legged knee extensor exercise bout

(Exercise). The values are mean±SE. *: significantly different from Pre LPS, P<0.05; #:

significantly different from control at the given time point, P<0.05.

Table 3

Glycogen (mmol·kg-1 dry weight) and lactate (mmol·kg-1 dry weight) concentration in the vastus

lateralis muscle before a single injection of LPS (Pre LPS), 2h after the LPS injection corresponding

to before exercise (2h LPS/Pre Exercise) and immediately after 10 min of one-legged knee extensor

exercise (Post exercise). The values are mean±SE. *: significantly different from Pre LPS, P<0.05;

(*): tends to be significantly different from Pre LPS, 0.05≤P<0.1

Figure 1

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AMPK Thr172 phosphorylation normalized to AMPKα2 protein content A) before (Pre LPS) and 2

h after LPS (2h LPS) and B) before (Pre Exercise) and immediately after 10 min of one-legged knee

extensor exercise (Post Exercise). ACC Ser 79 phosphorylation normalized to ACC2 protein

content C) Pre LPS and 2h LPS as well as D) Pre Exercise and Post Exercise. The results are

presented as arbitrary Units (AU). Values are mean±SE. *: significantly different from Pre

Exercise, P<0.05.

Figure 2

PDH Ser293 phosphorylation normalized to PDH-E1α protein content A) before (Pre LPS) and2 h

after LPS (2h LPS) and B) before (Pre Exercise) and immediately after 10 min of one-legged knee

extensor exercise (Post Exercise). PDH Ser300 phosphorylation normalized to PDH-E1α protein

content C) Pre LPS and 2h LPS as well as D) Pre Exercise and Post Exercise. PDH site Ser295

phosphorylation normalized to PDH-E1α protein content E) Pre LPS and 2h LPS as well as F) Pre

Exercise and Post Exercise. The results are presented as arbitrary Units (AU). Values are mean±SE.

*: significantly different from Pre Exercise, P<0.05.

Figure 3

PDHa activity (mmol ·min-1·kg-1) A) before (Pre LPS) and2 h after LPS (2h LPS) and B) before

(Pre Exercise) and immediately after 10 min of one-legged knee extensor exercise (Post Exercise).

The PDHa activity is normalized to total creatine in the samples. Values are mean±SE. *:

significantly different from Pre Exercise, P<0.05.

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Pre LPS 2 h LPS

AMPK

Thr

172 p

hosp

hory

latio

n / A

MPK

2

prot

ein

(AU

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Pre Exercise Post Exercise

AMPK

Thr

172 p

hosp

hory

latio

n / A

MPK

2

prot

ein

(AU

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0 Control LPS *

*

Pre LPS 2 h LPS

ACC

Ser

79 p

hosp

hory

latio

n / A

CC

pro

tein

(AU

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Pre Exercise Post Exercise

ACC

Ser

79 p

hosp

hory

latio

n / A

CC

pro

tein

(AU

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0 * *

A) B)

C) D)

AMPK Thr172

ACC ser227

ACC protein

Pre Post Pre Post

LPS Control

LPS Pre 2h

LPS Pre 2h Pre Post Pre Post

LPS Control

AMPK 2

ACC ser227

ACC protein

AMPK Thr172

AMPK 2

Rested Exercise

75 kD 50 kD

75 kD 50 kD

75 kD 50 kD

75 kD 50 kD

250 kD 150 kD

250 kD 150 kD

250 kD 150 kD

250 kD 150 kD

Figure 1

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Pre LPS 2 h LPS

PDH

Ser

295 p

hosp

hory

latio

n / P

DH

-E1

pro

tein

(AU

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Pre Exercise Post Exercise

PDH

Ser

295 p

hosp

hory

latio

n / P

DH

-E1

pro

tein

(AU

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

* *

E) F) LPS

Pre 2h

PDH Ser295

PDH- E1

PDH Ser295

PDH- E1

50 kD 37 kD

50 kD 37 kD

Pre Post Pre Post

LPS Control

50 kD 37 kD

50 kD 37 kD

Pre LPS 2 h LPS

PDH

Ser

300 p

hosp

hory

latio

n / P

DH

-E1

pro

tein

(AU

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Pre Exercise Post Exercise

PDH

Ser

300 p

hosp

hory

latio

n / P

DH

-E1

pro

tein

(AU

)0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

* *

50 kD 37 kD

50 kD 37 kD

PDH Ser300

PDH- E1

Pre Post Pre Post

LPS Control

50 kD 37 kD

50 kD 37 kD

LPS Pre 2h

PDH Ser300

PDH- E1

C)

Pre LPS 2 h LPSPD

H S

er29

3 pho

spho

ryla

tion

/ PD

H-E

1 p

rote

in (A

U)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Pre Exercise Post Exercise

PDH

Ser

293 p

hosp

hory

latio

n / P

DH

-E1

pro

tein

(AU

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2 Control LPS

* *

A) B)

LPS Pre 2h

PDH Ser293

PDH- E1

50 kD 37 kD

50 kD 37 kD

LPS Control

Pre Post Pre Post

50 kD 37 kD

50 kD 37 kD

PDH Ser293

PDH- E1

Rested Exercise Figure 2

D)

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Figure 3 Rested

Pre LPS 2 h LPS

PD

H a

ctiv

ity (m

mol

·kg-1

·min

-1) /

tota

l Cr

0,0

0,5

1,0

1,5

2,0

2,5

Exercise

Pre Exercise Post Exercise

PD

H a

ctiv

ity (m

mol

·kg-1

·min

-1) /

tota

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0,0

0,5

1,0

1,5

2,0

2,5

Control LPS

*

*A) B)

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THE FACULTY OF SCIENCE,U N I V E R S I T Y O F C O P E N H A G E N

The PhD School of SCIENCE ffi

3A. Co-authorship statement

All papers/manuscripts with multiple authors enclosed as annexes to a PhD thesis synopsis

should contain a co-author statement, stating the PhD student's contribution to the paper.

I;Tifle,, Of,,PhD the$ij

Regulation of PDH, GS and insulin signalling in skeletal muscle; effect of physical activity level and inflammation

Exercise-induced AMPK and pyruvate dehydrogenase regulation is maintained during LPS-induced

inflammation.

The extent of the PhD student's contribution to the article is assessed on the followine scale

A. has contributed to the work (0-33%)

B. has made a substantial contribution (34-66%)

C. did the majority of the work independently (67-100%).

ffi

1. General inforrnation

PhD student

NameRasmus Sjorup BiensoCiv.reg.n o. (lf not applicable, then birth date)[email protected] of Biology

Please markPhD Scheme

X 5+3 Scheme 4+4 Scheme Industrial PhD

Principal supervisor

NameHenriette PilegaardE-mai lHPi [email protected]

t/3Revised 29 Jonuary 2073

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THE FACULTY OF SCIENCE,U N I V E R S I T Y O F C O P E N H A G E N

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t. po..ulation in the concept phase of the basic scientific problem on the basis of

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experiments/inve stigations.

C

Z. ftunnltrg of experiments/analyses and formulation of investigative methodology in

such a way that the questions asked under (l) can reasonably be expected to be

answered, including choice of method and independent methodological development.C

3. Involvement in the analysis or the concrete experiments/investigation.C

4. P.esentation, interpretation and discussion of the results obtained in article form. c

5. Material in the paper from another degree I thesis :

Articles/work pubt*nia n connection with another degree/thesis must no1lform part of the PhD thesis-

Data collectei and preliminary work carried aut as pirt of another degree/thesis may be part of the PhD thesis if

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Does the paper contain data material, which has also formed part of a previous degree / thesis

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please indicate which specific part(-s) of the paper that has been produced as part of the PhD study:

6.Signatures of co-authors :

Date Name sisnat? ,.,r

'Lc'lt.- lLl

Jesper Olesen 7, ///1,Line Van Hauen / d- ,,;-"/'.

t 8/t - /LrSimon Meinertz -r-r,%

7Z li - lt{Jens Frey Halling trNI{dL\}.,Lasse Gliemann

*,* 6t0,4,Aru/./l

2/3Revised 29 January 2013

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THE FACULTYU N I V E R S I T Y O F

OF SCIENCE,C O P E N H A G E N

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Study IV

Biensø RS, Knudsen JG, Brandt N, Pedersen PA, Pilegaard H. Effects of IL-6 on pyruvate

dehydrogenase regulation in mouse skeletal muscle. Pflugers Arch. 2013 Nov 14.

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SIGNALING AND CELL PHYSIOLOGY

Effects of IL-6 on pyruvate dehydrogenase regulationin mouse skeletal muscle

Rasmus S. Biensø & Jakob G. Knudsen & Nina Brandt &Per A. Pedersen & Henriette Pilegaard

Received: 26 June 2013 /Revised: 28 October 2013 /Accepted: 29 October 2013# The Author(s) 2013. This article is published with open access at Springerlink.com

Abstract Skeletal muscle regulates substrate choice accord-ing to demand and availability and pyruvate dehydrogenase(PDH) is central in this regulation. Circulating interleukin(IL)-6 increases during exercise and IL-6 has been suggestedto increase whole body fat oxidation. Furthermore, IL-6 hasbeen reported to increase AMP-activated protein kinase(AMPK) phosphorylation and AMPK suggested to regulatePDHa activity. Together, this suggests that IL-6 may be in-volved in regulating PDH. The aim of this study was toinvestigate the effect of a single injection of IL-6 on PDHregulation in skeletal muscle in fed and fasted mice. Fed and16–18 h fasted mice were injected with either 3 ng·g−1 re-combinant mouse IL-6 or PBS as control. Fasting markedlyreduced plasma glucose, muscle glycogen, muscle PDHaactivity, as well as increased PDK4 mRNA and protein con-tent in skeletal muscle. IL-6 injection did not affect plasmaglucose or muscle glycogen, but increased AMPK and ACCphosphorylation and tended to decrease p38 protein content inskeletal muscle in fasted mice. In addition IL-6 injectionreduced PDHa activity in fed mice and increased PDHa

activity in fasted mice without significant changes in PDH-E1α phosphorylation or PDP1 and PDK4 mRNA and proteincontent. The present findings suggest that IL-6 contributes toregulating the PDHa activity and hence carbohydrate oxida-tion, but the metabolic state of the muscle seems to determinethe outcome of this regulation. In addition, AMPK and p38may contribute to the IL-6-mediated PDH regulation in thefasted state.

Keywords IL-6 . Pyruvate dehydrogenase activity . AMPK .

p38 . Skeletal muscle

Introduction

Skeletal muscle has an exceptional ability to adjust the sub-strate choice during fasting and exercise according to thesubstrate availability [9, 24]. During prolonged exercise, skel-etal muscle first oxidizes blood glucose and stored glycogen,but an increasing fraction of the energy is derived from fatoxidation as the exercise proceeds [37, 38]. This ensures thatexercise can continue, although at a lower exercise intensity.Similarly, fasting is associated with a switch in substrateoxidation from carbohydrates to fat markedly increasing sur-vival without food intake.

The pyruvate dehydrogenase complex (PDC) is central inthe regulation of substrate choice in skeletal muscle. PDCconverts pyruvate into acetyl CoA thereby linking glycolysiswith the citric acid cycle and represents the only entry forcarbohydrate-derived substrate into the mitochondria for oxi-dation. PDC is composed of several copies of three catalyticenzymes, which includes the pyruvate dehydrogenase (PDH)-E1α. Fasting has been shown to downregulate the activity ofPDH in the active form (PDHa) [43]. In addition, PDHaactivity increases with exercise [37], but declines towardsresting level as the exercise duration exceeds 2 h [25, 39].

R. S. Biensø : J. G. Knudsen :N. Brandt :H. PilegaardCentre of Inflammation and Metabolism and The August KroghCentre, University of Copenhagen, Copenhagen, Denmark

R. S. Biensø : J. G. Knudsen :N. Brandt : P. A. Pedersen :H. PilegaardDepartment of Biology, University of Copenhagen, Copenhagen,Denmark

H. Pilegaard (*)Universitesparken 13, 2100 Copenhagen Ø, Denmarke-mail: [email protected]

Present Address:N. BrandtExercise and Metabolic Disease Research Laboratory, TranslationalSciences Section, School of Nursing, University of California, LosAngeles, CA, USA

Pflugers Arch - Eur J PhysiolDOI 10.1007/s00424-013-1399-5

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Although PDHa activity can be allosterically regulated [2],phosphorylation/dephosphorylation of the α subunit of PDH-E1 is thought to be the mainmechanism regulating the activityof PDH. Phosphorylation and concomitant inactivation ofPDH is catalyzed by pyruvate dehydrogenase kinases(PDK), and dephosphorylation and concomitant activationof PDH is catalyzed by pyruvate dehydrogenase phosphatases(PDP). The decrease in PDHa activity during fasting seems tobe due to increased PDK4 protein and decreased PDP1 proteincontent in the muscle [43], while the reduction duringprolonged exercise does not appear to be caused by increasedPDK4 protein content but may be due to increased PDKactivity as previously reported [39]. The underlying mecha-nisms initiating the regulation of PDH during prolonged ex-ercise remains unresolved.

Interleukin (IL)-6 is a myokine released from contractingmuscle during exercise and suggested to exert autocrine,paracrine and endocrine effects [23]. The plasma IL-6 con-centration has been reported to increase in both humans andmice during prolonged exercise [20, 21]. In humans, theincrease in plasma IL-6 concentration during exercise is de-tectable after about 2 hour with moderate exercise intensityand increases further with exercise duration [12, 34], while anincrease in plasma IL-6 has been detected already after 30 minof intense exercise in mice [20]. Recombinant IL-6 has beenshown to increase free fatty acid release from myotubes [1]and increase fat oxidation in human skeletal muscle in vivo[35, 42]. In addition, IL-6 has been reported to increase AMP-activated protein kinase (AMPK) phosphorylation in rat skel-etal muscle [13], and rodent studies suggest that AMPK mayregulate PDH [17, 32]. Furthermore, IL-6 has been shown toincrease phosphorylation of the stress mitogen activated pro-tein (MAP) kinase p38 in incubated human muscle [6] andMAPK p38 has been demonstrated to be involved in theregulation of IL-6 transcription [36]. Furthermore, previousfindings suggest an association between increased p38 phos-phorylation and decreased PDHa activity in cardiomyocytes[29]. Together this suggests that IL-6 could be involved inregulating the substrate choice of muscle either by directlyregulating PDH or potentially via AMPK and p38. Therefore,the aim of the present studywas to examine the isolated effectsof a single injection of recombinant mouse (rm) IL-6 on PDHregulation in mouse skeletal muscle. As IL-6 normally iselevated during exercise in the presence of severe metabolicchallenge, the effect of IL-6 injection was determined in boththe fed and fasted state.

Methods

Mice Female C57BL/6 mice (Taconic, Lille Skensved, Den-mark) 8 weeks of age were used in the study. Female mice wereused because a previous study showed no indications of

gender-specific regulation of PDH [14] and because regularoestrous cycle has been reported to be absent in the majority offemale mice housed in large groups [41]. The mice had adlibitum access to water and normal chow diet (Altromin 1324,Brogaarden, Lynge, Denmark) and had a 12:12 light–darkcycle. The experiments were approved by the Danish AnimalExperimental expectorate (license no. 2009, 561 1607) andcomplied by the European Convention for the protection ofvertebrate animals used for experiments and other scientificpurpose (Council of Europe no. 123. Strasbourg, France 1985).

Experimental setup The mice were housed individually 24 hbefore initiation of the experiment and divided into two groupseither fed or fasted for 16–18 h. The mice were given anintraperitoneal injection of either phosphate-buffered saline(PBS) or rm IL-6 (3 ng·g−1) dissolved in PBS. The mice wereeuthanized by cervical dislocation 30 or 60 min after the injec-tion. Quadriceps muscles were removed and frozen in liquidnitrogen. Trunk blood was collected in EDTA containing tubesand plasma was collected after centrifugation at 2,600×g ,15 min, 4 °C. Muscle and plasma samples were stored at −80 °C.

Plasma glucose and plasma IL-6 Plasma glucose concentra-tion was analyzed fluorometrically [18]. The plasma IL-6concentration was determined using Meso Scale Discovery(Rockville, MD, USA) as described by the manufacturer.

Muscle glycogen Muscle samples were hydrolyzed in 1 MHCl and the muscle glycogen concentration was determinedfluorometrically as glycosyl units as previously described [22].

RNA isolation and reverse transcription Quadriceps musclesfrom the mice were crushed in liquid nitrogen to ensurehomogeneity of each sample. RNA was isolated using aguanidinium thiocyanate phenol-choloroform method as pre-viously described [4, 26] except that the samples were ho-mogenized using a Tissue LyserII (Qiagen, Hilden, Germany).Reverse transcription was performed using the Superscript IIRNase H- system (Invitrogen, Carlsbad, CA, USA) as previ-ously described [26].

Real time PCR mRNA content was determined by use of the5′ fluorogenic nuclease assay with TaqMan probes (ABIPRISM 7900 Sequence Detection System; AppliedBiosystems, Foster City, CA, USA) as previously described[19]. Primer and probe sequences are given in Table 1. Cyclethreshold values reflecting the content of a specific mRNAwere converted to an arbitrary amount by use of a standardcurve, constructed from a serial dilution of a representativesample run together with the unknown samples. For eachsample the amount of target cDNAwas normalized to the totalsingle stranded (ss)DNA content in the sample determined byOliGreen as previously described [19].

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SDS-PAGE and western blotting Crushed quadriceps muscleswere homogenized using a Tissue LyserII (Quagen, Hilden,Germany) and muscle lysate was prepared as previously de-scribed [25]. The protein content in each sample was determinedusing the bicinchoninic acidmethod (Pierce, Rockford, IL, USA).

Protein content and protein phosphorylation were deter-mined in muscle lysates using pre casted gels (Biorad, Hercu-les, CA, USA) or hand-casted gels and SDS-PAGE and West-ern blotting as previously described [16, 25] with primarypolyclonal antibodies against Signal Transducer and Activatorof Transcription (STAT)3 (9139; Cell Signaling Technology,Berverly, MA, USA), p38 (9212; Cell Signaling Technology),acetyl CoA carboxylase (ACC; P0397; DAKO, Glostrup, Den-mark), AMPKα2, PDP1, PDK4, and PDH-E1α (provided byProfessor Graham Hardie, Dundee University, Dundee, UK)and primary phospho-specific antibodies against STAT3 Tyr705

and AMPK Thr172 (9138 and 2535, respectively, Cell Signal-ing Technology), ACC Ser79 (07-303; Millipore, Bedford,USA), PDH-E1α site 1 (Ser293), site 2 (Ser300), and site 4(Ser295; provided by Professor Graham Hardie). Secondaryantibodies used were all species-specific horseradish peroxi-dase conjugated immunoglobulin (DakoCytomation, Glostrup,Denmark). The bands were visualized with ECL reagent(Millipore, Billerica, MA, USA) in a photo image system andquantified (Carestream Health, Rochester, NY, USA). ThePDP1 protein band was verified with recombinant protein.GAPDH protein was determined (2118, Cell Signaling Tech-nology) and showed no changes between groups.

PDHa activity PDHa activity was determined in muscle ho-mogenates as previously described [3, 5, 25, 31]. PDHaactivity in each sample was adjusted to the total creatinecontent in the homogenate as previously described [33].

Statistics Values are presented as means±SE. Two-wayANOVA tests were used to evaluate the effect of fasting andIL-6 injection by testing within each time point and within eachcondition. The data were log transformed if equal variance testfailed. If a main effect was observed, the Student–Newman–Keuls post hoc test was used to locate differences. In addition, aStudent’s t test was used to evaluate the effect of IL-6 within agiven time point and condition. Differences were consideredsignificant whenP <0.05 and a tendency is reported for 0.05≤P<0.1. The statistical tests were performed using SigmaPlot 11.0.

Results

Plasma IL-6

The plasma IL-6 concentration was measured to examine thetime course of plasma IL-6 after an IL-6 injection. The IL-6T

able1

Prim

erandTaqm

anprobes

Forw

ardprim

ers

Reverse

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anprobes

IL-6

5′GCTTA

ATTA

CACATGTTCTCTGGGAAA3′

5′CAAGTGCATCATCGTTGTTCATA

C3′

5′ATCAGAATTGCCATTGCACAACTCTTTTCTCAT′3

PDK4

5′GGAAGTA

TCGACCCAAACTGTGA′3

5′GGTCGCAGAGCATCTTTGC3′

5′CACTCAAAGGCATCTTGGACTA

CTGCTA

CCA3′

PDP1

5′CGGGCACTGCTA

CCTA

TCCTT′3

5′ACAATTTGGACGCCTCCTTA

CT3′

5′AGTGGCACAAGCACCCCAATGATTA

CTTC3′

Sequencesof

forw

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ersas

wellasTaqm

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used

forreal-tim

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R

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interleukin6,PDK4pyruvatedehydrogenasekinase

4,PDP1pyruvatedehydrogenasephosphatase1

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injection increased (P <0.05) the plasma IL-6 concentration9.5-fold in fedmice and 40-fold in fastedmice 30min after theinjection relative to the PBS injected mice. After 60 min, theplasma IL-6 concentration was still elevated (P <0.05) 5-foldin fed mice and 12-fold in fasted mice relative to the PBS-injected mice (Fig. 1a).

In the IL-6-injected mice, the plasma IL-6 level was 2.4-fold higher (P <0.05) in fasted than fed mice at 30 min, whichmay be due to differences in blood volume or differences inuptake or removal of IL-6 from the blood. In the PBS-injectedmice, fasting reduced (P <0.05) the plasma IL-6 concentrationto 60 % compared with the fed mice (Fig. 1a). However, ofnotice is that such effect was not present at 30 min suggestingthat additional factors have contributed to the effect observedat 60 min (Fig. 1a).

STAT3 phosphorylation

STAT3 phosphorylation was determined in skeletal muscle toconfirm that IL-6 injections induced IL-6-mediated intracel-lular responses.

IL-6 injection increased (P <0.05) STAT3 Tyr705 phos-phorylation in skeletal muscle 2-fold in fed mice and 2.3-foldin fasted mice relative to PBS at 30 min after injection. At60 min, the injection of IL-6 increased (P <0.05) the STATTyr705 phosphorylation in skeletal muscle 2.2-fold in fed and2-fold in fasted mice relative to PBS-injected mice (Fig. 1b).

In the IL-6-injected mice, STAT3 Tyr705 phosphorylationwas at 30 min 1.8-fold higher (P <0.05) in the fasted than thefed mice (Fig. 1b).

Plasma glucose

The blood glucose concentration wasmeasured to describe themetabolic status of the mice during fasting and after IL-6injection. The IL-6 injection did not change the plasma

glucose concentration relative to PBS, neither in the fed northe fasted mice (Fig. 2a).

Plasma glucose concentration was at approximately 8 mMin fed mice independent of injection and decreased (P <0.05)to approximately 5 mM in the fasted mice both in the PBS andthe IL-6-injected mice (Fig. 2a).

Muscle glycogen

Muscle glycogen concentration was measured to describe themetabolic status of the mice during fasting and after IL-6injection. There was a main effect (P <0.05) of IL-6 injectionon muscle glycogen content at 30 min, but no changes wereobserved at 60 min (Fig. 2b).

Fasting reduced (P <0.05) the muscle glycogen contentwith 50–60 % compared with the fed mice independent ofinjection (Fig. 2b).

Signaling

AMPK, ACC and p38 phosphorylation were determined toexamine the effects of IL-6 on intracellular signaling in skel-etal muscle.

AMPK

There were no effects of IL-6 on AMPK Thr172 phosphoryla-tion in skeletal muscle of the fed mice. In the fasted mice, theIL-6 injection increased (P <0.05) AMPK Thr172 phosphory-lation 1.3-fold relative to PBS at 60 min (Fig. 3a).

In addition, at 30 min fasting tended to increase (0.05≤P <0.1) AMPK Thr172 phosphorylation 1.2-fold in the PBS-injected mice and in the IL-6-injected mice fasting increased(P <0.05) AMPK phosphorylation 1.2-fold relative to fed(Fig. 3a).

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Fig. 1 Plasma concentration ofIL-6 and STAT3 phosphorylation.Plasma IL-6 concentration(nanograms per milliliter) (a) andSTAT3 phosphorylation (b) in fed(FED) and fasted (FASTED) mice30 and 60 min after a singleinjection of either PBS or rmIL-6.Values are means±SE; n=8. *P <0.05, significantly different fromPBS within given condition andtime point; #P<0.05,significantly different from FEDwithin given treatment and timepoint

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ACC

The IL-6 injection did not change ACC Ser79 phosphorylationin skeletal muscle of the fed mice. In the fasted mice, the IL-6injection increased (P <0.05) the ACC phosphorylation 1.4-fold in the fastedmice relative to the PBSmice at 60min whenusing a t test (Fig. 3b).

In line with the AMPK results, 30 min after injection, ACCSer79 phosphorylation was 1.5-fold higher (P <0.05) in fastedthan in fed mice injected with PBS and tended to be 1.4-foldhigher (0.05≤P <0.1) in fasted than in fed mice injected withIL-6 (Fig. 3b).

p38

The IL-6 injection did not change the phosphorylation level ofp38 when normalized to p38 protein content in skeletal mus-cle of fed or fasted mice (Fig. 3c). However, the IL-6 injectiontended to decrease (0.05≤P <0.1) the p38 protein content with70–85 % compared with PBS injection both at 30 and 60 min(Fig. 3c, d).

In addition, p38 protein content was 1.5- to 1.8-fold higher(P <0.05) in the fasted than the fed mice both at 30 and 60min(Fig. 3d).

IL-6 mRNA

IL-6 injection did not change the mRNA content of IL-6 inskeletal muscle of the fed and fasted mice (Fig. 4).

Furthermore, fasting increased (P <0.05) the IL-6 mRNAlevel 1.8- to 2.0-fold at 30 and 60 min but only in the IL-6-injected mice (Fig. 4).

PDH regulation

The mRNA and protein levels of PDK4 and PDP1 weredetermined together with the PDHa activity and PDH

phosphorylation to examine the effects of fasting and IL-6injection on PDH regulation.

PDK4 and PDP1 mRNA

IL-6 injection did not change the PDK4 mRNA content inskeletal muscle of the fed and fasted mice (Fig. 5a), but thePDP1 mRNA content tended to be higher (0.05≤P <0.1)60 min after IL-6 injection than PBS (Fig. 5c).

Fasting increased (P <0.05) the PDK4mRNA content 5- to6-fold Fig. 5a) and decreased (P <0.05) the PDP1 mRNAcontent with 60 % (Fig. 5c).

PDK4 and PDP1 protein

The PDK4 protein content in skeletal muscle increased (P <0.05) 1.6-fold in the fasted mice relative to the fed with noeffect of IL-6 injection (Fig. 5b). The PDP1 protein contentdid not change with IL-6 injection or with fasting (Fig. 5d).

PDHa activity

The PDHa activity in skeletal muscle tended overall to belower (0.05≤P <0.1) in the IL-6-injected fed mice than thePBS injected fed mice. In addition, the PDHa activity was inthe fed mice at 30 min 30 % lower (P <0.05) after IL-6injection than after PBS when using a t test. IL-6 injectionin the fasted mice increased (P <0.05) PDHa activity 2-fold at30 min and 1.8-fold at 60 min.

Furthermore, fasting overall reduced (P <0.05) the PDHaactivity in skeletal muscle to 15–30 % of the level in fed mice(Fig. 6a).

PDH-E1α phosphorylation

Overall, the effects on PDH-E1α phosphorylation were sim-ilar for the investigated PDH-E1α phosphorylation sites. IL-6

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Fig. 2 Plasma glucose concentration and muscle glycogen. Plasma glu-cose concentration (millimolar) (a) and muscle glycogen (millimoles perkilogram) (b), in skeletal muscle of fed (FED) and fasted (FASTED) mice30 and 60 min after a single injection of either PBS or rmIL-6. Values are

means±SE; n =8. *P <0.05, significantly different from PBS withingiven condition and time point; #P <0.05, significantly different fromFED within given treatment and time point. Line indicates an overalleffect

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injection did not change PDH-E1α phosphorylation in the fedor fasted mice (Fig. 6b–d).

Fasted mice had 1.7- to 2.1-fold higher (P <0.05) PDH-E1α phosphorylation than fed mice independent of injection(Fig. 6b–d).

Discussion

The main finding of the present study is that IL-6 injectionsregulated PDHa activity in mouse skeletal muscle but differ-ently in fed and fasted conditions. In addition, an IL-6 injec-tion increased AMPK and ACC phosphorylation in mouseskeletal muscle only in the fasted state. This indicates that

AMPK did not mediate the IL-6-induced reduction in PDHaactivity in the fed state but could potentially be involved in theincrease in the fasted state. Furthermore, IL-6 reduced p38protein content in skeletal muscle in the fasted state, making itpossible that p38 could play a role in the observed IL-6-induced change in PDHa activity in the fasted state.

Skeletal muscle regulates substrate choice according toavailability and is therefore capable of coping with majormetabolic changes during exercise and fasting [2, 38]. Theprevious findings that IL-6 infusion in humans enhances fatoxidation in skeletal muscle [35] and that plasma IL-6 isincreased during prolonged exercise [21] suggest that IL-6could be involved in regulating substrate choice during exer-cise. The observation that the increase in plasma IL-6 after the

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FED FASTED FED FASTEDPBS IL-6 PBS IL-6 PBS IL-6 PBS IL-6

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Fig. 3 Phosphorylation of AMPK, ACC, and p38. AMPK Thr172 phos-phorylation (a), ACC Ser79 phosphorylation (b), p38 Ser15 phosphory-lation (c), and p38 protein content (d) in skeletal muscle of fed (FED)and fasted (FASTED) mice 30 and 60 min after a single injection of eitherPBS or rmIL-6. Values are means±SE; n =8. *P<0.05, significantly

different from PBS within given condition and time point; #P<0.05,significantly different from FED within given treatment and time point.Symbols within parentheses indicate a statistical tendency, 0.05≤P<0.1.Line indicates an overall effect

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IL-6 injection in the present study was similar to the increasereported after a single exercise bout in mice [20], shows thatthe obtained IL-6 levels are at a physiologically relevant level.Furthermore, the increase in phosphorylation of the IL-6signaling marker STAT3 in skeletal muscle in the currentstudy demonstrates that IL-6 elicited an intracellular responsein skeletal muscle at the investigated time points. Together thissuggests that the present experimental setting can be used as amodel to investigate the isolated effects of IL-6 on PDHregulation in skeletal muscle during exercise. In addition, thepronounced fasting-induced decrease in muscle glycogen andplasma glucose concentration shows that the fasted mice wereseverely metabolically challenged. This provides a model toinvestigate the potential impact of the metabolic state for IL-6-mediated effects on PDH regulation in skeletal muscle.

The observation that IL-6 downregulated PDHa activity inskeletal muscle in the fed state is in accordance with the

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Fig. 5 mRNA and protein content of PDK4 and PDP1. PDK4 mRNA (a),PDK4 protein content (b), PDP1 mRNA (c), and PDP1 protein content (d)in skeletal muscle of fed (FED) and fasted (FASTED) mice 30 and 60 minafter a single injection of either PBS or rmIL-6. Values are means±SE; n=8.

*P<0.05, significantly different from PBS within given condition and timepoint; #P<0.05, significantly different fromFEDwithin given treatment andtime point. Symbols within parentheses indicate a statistical tendency, 0.05≤P<0.1. Line indicates an overall effect

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Fig. 4 IL-6mRNA content. IL-6 mRNA in skeletal muscle of fed (FED)and fasted (FASTED) mice 30 and 60 min after a single injection of eitherPBS or rmIL-6. Values are means±SE; n =8. #P<0.05, significantlydifferent from FED within given treatment and time point

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previously shown IL-6-induced fat oxidation [35, 42] andsuggests that IL-6 may contribute to regulating substratechoice during exercise towards increased skeletal muscle fatoxidation in part by regulating PDH. The previous observa-tion that an IL-6 injection increased AMPK phosphorylationin rat skeletal muscle suggests that AMPK may be mediatingIL-6-induced regulation [13]. Furthermore, AMPKα2 knock-out (KO) mice have been reported to exhibit an enhancedexercised-induced increase in PDHa activity [17] and theAMPK activator AICAR has been shown to increase PDK4mRNA in skeletal muscle [11]. Together this suggests that IL-6 mediates effects on PDH via AMPK. However, the presentobservation that AMPK and ACC phosphorylations wereunaffected by IL-6 injections in the fed state does not supportthat AMPK was directly involved in the observed IL-6-induced reduction in PDHa activity in the fed state.

The present findings that IL-6 elicited an increase in skel-etal muscle PDHa activity in the fasted state, despite the IL-6-induced downregulation in the fed state, indicate that themetabolic status influences the impact of IL-6 on PDH regu-lation. As IL-6 injections in the fasted state were associatedwith increased AMPK and ACC phosphorylation at 60 min, itis possible that AMPK was involved in the IL-6-mediatedPDH regulation at this time point. However, this potentialeffect of AMPK on PDH is opposite of the apparent AMPK-mediated suppression of the exercise-induced PDHa activa-tion, as suggested from the more marked exercise-inducedincrease in PDHa activity in AMPKα2 KO than wild-type(WT) mice [17]. However, the AMPKα2 KO mice in theprevious study had lower muscle glycogen and plasma glu-cose after exercise than WT [17]. Therefore, the more markedPDH activation in AMPKα2 KO mice may not be due to the

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PDH-E1

Fig. 6 PDHa activity and phosphorylation of PDH. PDHa activity (mil-limoles per minute per kilogram) (a), PDH Ser293 phosphorylation (b),PDH Ser300 phosphorylation (c), and PDH Ser295 phosphorylation (d) inskeletal muscle of fed (FED) and fasted (FASTED) mice 30 and 60 minafter a single injection of either PBS or rmIL-6. Values are means±SE;

n =8. *P <0.05, significantly different from PBS within given conditionand time point; #P<0.05, significantly different from FED within giventreatment and time point; ¤0.05≤P<0.1, tendency for a difference be-tween IL-6 and PBS. Symbols within parentheses indicate a statisticaltendency 0.05≤P<0.1. Line indicates an overall effect

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lack of AMPK, but rather that the AMPKα2 KO mice weremore metabolically challenged than WT as also suggested inthe previous study [17, 25]. Furthermore, as low muscleglycogen has been suggested to enhance exercise-inducedIL-6 expression and release from skeletal muscle [12] themore marked PDHa activation in the AMPKα2 KO mice inthe previous study [17] may have been due to increased IL-6levels rather than lack of AMPK. This possibility is supportedby the previous finding that AICAR incubation elicited ahigher IL-6 release from AMPK kinase dead mouse musclethan WT muscle [7] and is also in line with the present IL-6-mediated increase in PDHa activity in the fasted state. Inaddition, the present findings that IL-6 injection tended toreduce p38 protein content in the fasted state may suggest thatp38 potentially have contributed to the observed IL-6-inducedincrease in PDHa activity, because increased p38 phosphory-lation has been associated with decreased PDHa activity incardiomyocytes [29]. Of notice, the IL-6-induced downregu-lation of p38 protein only 30 and 60 min after injection issurprisingly fast and indicates that IL-6 either inhibits p38synthesis or increases degradation.While a previous study hasshown that IL-6 regulates p38 phosphorylation in skeletalmuscle [40], no previous studies have to our knowledgereported IL-6-mediated regulation of p38 protein. However,it may be speculated that IL-6 mediates the regulation of p38mRNA targeting miRNA’s leading to reduced translation ofp38, but this remains to be determined.

As PDHa activity is known to be regulated by changes inNADH/NAD+, ATP/ADP, and acetyl CoA/acetyl [9, 24, 30],it may be speculated that IL-6 has mediated effects via chang-es in one of these ratios. Indeed a previous study has shownthat IL-6 incubation of rat EDL decreased the ATP concentra-tion and elevated the AMP concentration suggesting that IL-6-induced changes in nucleotides could play a role in the ob-served IL-6-mediated regulation of PDHa activity in the pres-ent study. An IL-6-induced decrease in ATP and increase inAMP concentrations would be expected to be associated withan enhanced PDHa activity, which is in accordance with thechanges observed in the fasted state in the present study.However, changes in ATP, ADP and AMP as well asNADH/NAD+ and acetyl CoA/acetyl are thought to exerteffects on PDHa activity through changes in PDH-E1α phos-phorylation [10, 24]. Because IL-6 injections did not affectPDH-E1α phosphorylation significantly neither in the fed northe fasted state in the present study, changes in these param-eters do not appear to be a likely mechanism for the observedIL-6-induced effects on PDH regulation.

It should be noted that IL-6 induced an increase in PDHaactivity when PDHa activity was at a very low level due to thefasting conditions. This means that the PDHa activity after IL-6 injections in the fasted state still was much lower than thelevel of PDHa activity after IL-6 induced downregulation ofPDH in the fed state. The mechanism behind such a switch is

unknown and additional studies are needed to answer this.The observations that PDHa activity, PDH-E1α phosphoryla-tion, PDK4 and PDP1 mRNA, as well as PDK4 protein weremarkedly changed by fasting in the present study are inaccordance with previous studies [27, 28]. This shows thatdifferences could be detected at the mRNA, protein phosphor-ylation, and activity level. Furthermore, the finding thatfasting elicited 90% reduction in PDHa activity in PBS, whileIL-6 injection only resulted in a 30 % reduction demonstratesthat the effect of 18 h of fasting is much more marked than asingle IL-6 injection. This may indicate that IL-6 only con-tributes to the regulation of PDH for example by sensitizingPDHa to the metabolic needs of the cell. However, it may alsobe worth noting that the magnitude of reduction in PDHaactivity in response to IL-6 injection is quite similar to thereduction previously reported in the exercise-induced increasein PDHa activity in human skeletal muscle, when muscleglycogen had been lowered prior to exercise [15]. This makesit possible that an enhanced IL-6 release contributed to thesmaller exercise-induced increase in PDHa activity whenmuscle glycogen was reduced in the previous study [15].Moreover, the similar magnitude of change in the presentstudy and the previous human study indicates that the ob-served change in PDHa activity with IL-6 injections is similarto changes observed in a physiological setting in humans [15].

PDH-E1α phosphorylation is known to be the dominantregulatory mechanism determining PDHa activity. The un-changed PDH-E1α phosphorylation with IL-6 injection inthe present study indicates that the observed changes in PDHaactivity was not due to changes in phosphorylation level. Suchdiscrepancy between PDHa activity and PDH-E1α phosphor-ylation has previously been reported in resting human andmouse skeletal muscle [14, 25], although the previous studiesobserved changes in PDH-1Eα phosphorylation withoutchanges in PDHa activity. As effects of PDK and PDP ex-pression and/or activity would be expected to influence thephosphorylation level of PDH-E1α, these PDH regulatoryproteins do not seem to be in play in the IL-6-mediated effects.This suggestion is in line with the unaffected PDK4 and PDP1protein level with a single IL-6 injection. Furthermore, theunchanged plasma glucose concentration and unchangedmuscle glycogen in the fed state upon IL-6 injection (althoughan overall difference was observed for muscle glycogen)indicate that IL-6 did not change the carbohydrate availabilityin the fed state. Thus, it seems unlikely that changes in themetabolic state have contributed to the observed downregula-tion of PDHa activity. It may therefore be speculated that IL-6has elicited alternative mechanisms like changes in acetylationstate, because several acetylation sites have been identified onPDH [8].

Based on the previous findings that reduced muscle glyco-gen has been shown to enhance IL-6 transcription in humanskeletal muscle [12] it may be expected that fasting would

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elevate muscle IL-6mRNA levels. However, the finding that afasting-induced elevation in IL-6 mRNA only was observedin IL-6 injected mice suggests that low glycogen is not suffi-cient to increase IL-6 mRNA, and that IL-6 exerts a positivefeedback on the expression of IL-6 when muscle glycogenlevels are low. In addition, the IL-6 induced intracellularsignaling observed both in the fed and fasted state while IL-6 mRNA only increased significantly in the fasted state in thepresent study, may suggest that an IL-6-mediated effect on IL-6 expression requires a factor which is only available whenmuscle glycogen is low. As p38 has been implicated in regu-lating IL-6 expression [36] the observed fasting-induced in-crease in p38 protein content may have contributed to theelevated IL-6 mRNA. However, the observed reduction inp38 protein content with IL-6 injection does not support thispossibility. Hence, the mechanism behind the fasting-inducedincrease in skeletal muscle IL-6 mRNA only when IL-6 wasinjected remains to be determined.

In conclusion, a single IL-6 injection reduces PDHa activityin mouse skeletal muscle in the fed state and increases PDHaactivity in the fasted state. This suggests that IL-6-mediatedPDH regulation contributes to regulating substrate choice, butthat the metabolic state determines the outcome. IL-6 appearsto regulate PDHa activity without clear changes in PDH-E1αphosphorylation and AMPK and p38 may be involved in theIL-6-mediated PDH regulation in the fasted state.

Acknowledgments This study was funded by the Danish MedicalResearch Council for Independent Research and the Novo nordisk Foun-dation. We would also like to thank Prof. Graham Hardy (DundeeUniversity, Dundee, U.K) for providing the PDH phospho specific,PDK4 and PDP1 antibodies. Centre of Inflammation and Metabolism,(CIM) is supported by a grant from the Danish National ResearchFoundation (# 02-512-55).

Open Access This article is distributed under the terms of the CreativeCommons Attribution License which permits any use, distribution, andreproduction in any medium, provided the original author(s) and thesource are credited.

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