A schematic illustration of cellular events in the energy metabolism of human skeletal muscles. Free fatty acids (FFA) are taken up from the blood stream to be transported into the cell (fatty acid binding proteins, FABP; membrane = m / cytosolic = c) and into the mitochondria (carnitine palmitoyl transferase, CPT) providing Acetyl-CoA or reestified and stored as triacylglycerol (TAG). The FFA come either from those bound to albumin in the blood stream or from hydrolysis of TAG catalysed by lipoprotein lipase (LPL). A critical regulation is the AMP activation of protein kinase (AMPK) which brings about a reduction in malonyl-CoA via a phosphorylation of Acetyl-CoA carboxylase (ACC) opening for FA to be bound to CPT, which is a prerequisite for long chain FA to be transported into the mitochondria. Illustrated is also how the pyruvate dehydrogenase kinase (PDK) gene is activated by muscle contraction which in turn may contribute to the deactivation of pyruvate dehydrogenase (PDH) inhibiting decarboxylation of pyruvate and the formation of Acetyl-CoA. This process is enhanced when muscle glycogen stores are low (energy crisis). The IL-6 gene is also activated by muscle contractions and the protein is produced in prolonged exercise and released to the blood stream for possible action in the liver, enhancing liver glucose production. As for the PDK gene this activation is faster and more pronounced when muscle glycogen stores are low. (Pilegaard H et al., Am. J. Physiol. 279: E806-E814, 2000.; Steensberg A et al. Rapid rapport, J. Physiol. (London) 529: 237-243, 2000; Wojtaszewski J.F.P. et al. Rapid rapport, J Physiol 528, 221-226, 2000)

The Copenhagen Muscle Research Centre
Rigshospitalet - University of Copenhagen
Founded by the Danish National Research Foundation

Annual Report 2000

CMRC Research Highlights 2000

August Krogh raised the question of what determines the relative role of lipids and carbohy-drates to be used for combustion in skeletal muscle. This most critical problem has been dis-cussed ever since, and now substantial new insight has been provided by recent CMRC re-search. Substrate availability, the transport into the muscle cell and further into the mito-chondria are possible limiting and regulatory steps, as is the metabolism of fatty acids and glucose. Major efforts by CMRC researchers during the last couple of years have pinpointed not only where some of these regulations occur, but also brought about the possible underly-ing mechanisms. The transport capacity for fatty acids (FA) in skeletal muscle of humans is low, but can be enlarged by muscle usage and the proper diet. It is questionable, however, whether cytosolic concentrations of FA is ever limiting, rather our present findings indicate that the entrance into the mitochondria and the rate by which FA are metabolised in the b-oxidation are the critical steps. Moreover, exercise-induced activation of AMP protein kinase (AMPK) has now also been demonstrated to occur in human muscle, which indicates a cen-tral regulatory role for AMPK as highlighted in an editorial in J. Physiol. written in connection with a CMRC article on the subject. The AMPK is proposed via phosphorylation of Acetyl CoA carboxylase to reduce the malonyl CoA and thereby give access for FA binding to CPT-1 and transport into the mitochondria (see figure on front page).

Traditionally, metabolic regulations during exercise are thought to be mediated by changes either in cytosolic Ca2+, or in the energy and redox state of the muscle. However, CMRC re-searchers have clearly demonstrated that muscle contraction can activate genes, silent at rest to be transcribed and a protein to be made during the exercise and that these proteins may play a regulatory role for the metabolic response.

One example is PDK4, the activity of which when increased will downregulate the PDHa, re-ducing the flux of pyruvate into the mitochondria. During exercise lasting one hour or more, the PDK4 gene is transcribed and significant mRNA levels are apparent within 2-3 hours of exercise. Low muscle glycogen stores further enhance the expression of this gene during exercise, suggesting that it is an important mechanism to reduce mitochondrial pyruvate utilisation(see figure on front page).

Another example is the IL-6 gene. At the mRNA level, barely any IL-6 gene is present in the resting muscle, but with exercise a very marked elevation in transcription is observed, and mRNA and IL-6 proteins are expressed. As is the case for PDK4, this process is further ac-celerated with low muscle glycogen. IL-6 is known to stimulate the glucose output from hepa-tocytes, which means that the muscle derived IL-6 from contracting skeletal muscles may signal the need for an elevated liver glucose output to maintain the blood glucose level as more glucose is taken up by the contracting skeletal muscles when muscle glycogen stores are diminished; a scheme proposed in an editorial in the Journal of Physiology, accompany-ing our article on the subject (see figure on front page).

A schematic illustration of the Na+/K+ pumps in human skeletal muscle. The new observation is that the pumps are not only localised in the cell wall of the muscle fibre but also present adjacent to it, being recruited to maintain the Na+/K+ homeostasis in the muscle when strained (Juel C et al. Am J Physiol 278, R400-R406, 2000).

In another area related to transport of ions, a crucial finding has been made. It relates to Na+/K+ exchange in skeletal muscle and the localisation of the Na+/K+ pumps. The traditional view has been that the number of pumps is permanently located as an integrative part of the sarcolemma. However, CMRC researchers have revealed that the number of pumps in the membrane may vary and that it can acutely become increased with muscle contractions when the fluxes for both Na+ and K+ are elevated (see figure on inside cover). The finding is very controversial, but well substantiated. It brings a new dimension to the understanding of how K+ homeostasis in blood and muscle is maintained when threatened by an imbalance, e.g. during intense muscular work. The Na+/K+ pumps present in the sarcolemma cannot only be activated, but more pumps can be recruited from pools adjacent to the plasma mem-brane.

Other Research Activities

During the year 2000 CMRC research has in all principle aspect followed the research plan. It is of note that our research has "gone more molecular" in regard to activation of certain genes and signalling in skeletal muscle. The primary reason for this is that genes encoding for proteins related to energy metabolism of muscle are not only being activated quickly in exercise, but the proteins are produced as well, having an impact on metabolic regulation.

In regard to publications, two CMRC researchers defended their doctoral theses and we had four Ph.D. defences of which two took place at the University of North Texas at Forth Worth. The number of original articles (44) is somewhat lower than in previous years, but their im-pact was higher as judged by the journals in which they appeared. We published 11 articles in our "preferred" journal, the Journal of Physiology (London) that has an impact factor of ~4.4. Three of the articles were highlighted by the journal in an editorial-perspective. In addi-tion, we published 6 articles in journals with an impact factor of ~10.0. Of the remaining origi-nal articles, 11 were published in the American Journal of Physiology and another 3 in jour-nals of similar impact. We published 19 book chapters or reviews, of which one was pub-lished in Physiological Reviews. In the area of "knowledge dissemination" we had 16 publica-tions and in fact, CMRC researchers had one article in Scientific American. At the national level, Ugeskrift for Læger had a special issue on skeletal muscle and health in which the dominant fraction of articles were written by CMRC researchers.

All in all, CMRC presented itself quite strongly in 2000.

Collaboration with senior researchers outside CMRC,
resulting in publications in 2000.

In Denmark

· Jens Bülow, Klinisk Fysiologisk Afd, Bispebjerg Hospital.
· Ulla Feldt Rasmussen, Rigshospitalet
· Kari Mikines, Urologisk afd., KAS Gentofte
· Allan Vaag, Steno Diabetes Center.
· Niels Ørtenblad, University of Southern Denmark, Odense.

Internationally

· Hua Ai, Institute of Sports Medicine, Beijing Medical University, China.
· James Fluckey, Donald W, Reynolds Center on Aging, Univ. of Arkansas, USA.
· Laurie Goodyear, Harvard Medical School, Boston, USA
· Terry E. Graham, Dept. of Human Biology and Nutrition, University of Guelph, Canada
· Simon Green, Dept. of Physiology, Technological University of Brisbane, Australia
· Mark Hargreaves, Dept. of Physiology, Deakin University, Australia
· Cecilia Holm, Dep of Cell and Molecular Biology, University of Lund, Sweden.
· Ingibjörg Jonsdottir, Dept. of Physiol. and Pharmacol., Univ. of Gothenburg, Sweden.
· Satu Koskinen, Dept. of Health Sciences, University of Jyväskylä, Finland.
· Jozef Langfort, Dep. Appl. Physiology, The Polish Academy of Science, Poland.
· P. Darrell Neufer, John B. Pierce Lab. Foundation and Yale Univ., New Haven, USA.
· Timo Takala, Dept. of Health Sciences, University of Jyväskylä, Finland.
· Gale D. Thomas, Univ. of Texas Southwestern Medical School, Dallas, USA.

Exchange of researchers

For visiting researchers, please see enclosure 1, list of CMRC personnel 2000.

CMRC Researchers abroad ( > one month)

Jens R. Daugaard,
· post doctoral fellow, University of Barcelona, Spain, March - September 2000.

Bente Kiens and Erik A. Richter,
· visiting professors at School of Health Sciences, Deakin University, Melbourne, Aus-tralia, October 2000 - January 2001.

Henriette Pilegaard,
· post doctoral fellow at the John B. Pierce Laboratory Foundation, Yale University, New Haven, USA, March - May and September - December 2000.

Adam Steensberg,
· pre-Ph.D. student, Dept. of Physiology, University of Melbourne, Australia, Novem-ber-December 2000.

Conferences, etc.

Arranged by CMRC

One-day symposium entitled
· "Unsolved issues in cardiovascular regulation during exercise; how close are we to an answer?". Held at Carlsberg Academy, 15/11-2000 in honour of professor Jere H. Mitchell, UT Southwestern Medical Center, Dallas who at the same time be-came honorary doctor of medicine at the University of Copenhagen.

Conference entitled
· "Mechanisms to secure tissue oxygenation in humans; adaptive and evolution-ary aspects". Held at the Royal Danish Academy of Sciences and Letters, 25-29 No-vember 2000. Approximately 65 participants of whom 20 were international authori-ties on altitude physiology/medicine, invited to discuss the findings from the CMRC high altitude expedition 1998 which were presented by CMRC researchers. The meeting was highly successful and gave the proper base for finalising the scientific articles to be published. Moreover, some of the invited researchers participated in 3 follow-up symposia of which one was arranged by CMRC and held at Rigshospitalet. In addition to the funding from the Danish National Research Foundation, the confer-ence was supported by Carlsbergfondet and Radiometer Medical A/S.

Attended by CMRC researchers

Jens R. Daugaard,
· invited speaker, Acta Physiologica Scandinavica Symposium, Stockholm, May 2000.

Flemming Dela,
· invited speaker. 11th International Conference on Biochemistry of Exercise, Little Rock, Arkansas, USA, May 2000.

Henrik Galbo,
· invited speaker. 11th International Conference on Biochemistry of Exercise, Little Rock, Arkansas, USA, May 2000.

José Gonzáles-Alonso,
· invited speaker. Human Physiology Symposium, The Physiological Society, Aber-deen, UK, September 2000.

Bente Kiens and Erik A. Richter,
· invited speakers. Australian Physiological Society, Melbourne, October 2000.

Bente K. Pedersen,
· invited speaker. The London Marathon Conference, London, UK, April 2000.
· invited speaker. Int. Congress on Obesity. Antwerpen, May 2000.
· invited speaker. British Society of Nutrition, London, December 2000.

Bengt Saltin.
· invited speaker. Two lectures at the London Marathon Conference, London, UK, April 2000.
· invited speaker. Int. Congress on Obesity. Title: Gene regulation in obesity. Antwer-pen, May 2000.
· memorial lecture for professor Ethan Nadel, Yale University. ACSM Congress, Indi-anapolis, June 2000.
· invited speaker. 20th Int. OUEH Symposium. Title: The elderly worker. Kitakyushu, Japan, October 2000.

Examiner/opponent at doctoral dissertations

Bente K. Pedersen,
· Norges Idrettshøjskole, Oslo, December 2000.

Thorkil Ploug,
· University of Southern Denmark, December 2000.

Bengt Saltin,
· Norges Idrettshøjskole, Oslo, February 2000.
· University of Umeå, Medical Faculty, November 2000.

Committee work, etc.

Flemming Dela,
· member of the organising committee of the 18th Annual Meeting of the Anglo-Danish Dutch Diabetes Group.

Michael Kjær,
· member of a committee on physical exercise and health under the General Director-ate for Health.
· member of a committee on interaction between nutrition and physical activity for health under the General Directorate for Nutrition.
· member of a working group examining physical activity in the elderly under the Minis-try of Social Affairs.
Bente K. Pedersen,
· member of a committee on physical exercise and health under the General Director-ate for Health.
· member of the National Nutritional Council.

Bengt Saltin,
· member of a 7-person panel evaluating basic biological and physiological research in Norway, 10 days in total, March and September 2000, resulting in a report, published by the Research Council of Norway: "Research in Biology and relevant areas of Bio-chemistry in Norwegian Universities, Colleges and Research Institutes".
· member of a committee on physical exercise and health under the General Director-ate for Health.
· member of a committee on interaction between nutrition and physical activity for health under the General Directorate for Nutrition.
· chairman, The Baltic Summer School Board.
· chairman, Anti Doping Danmark.
· member of the World Anti Doping Agency's (WADA) Health, Medical, and Research Committee.

Research education

Ph.D. courses

Michael Kjær,
· organiser and lecturer, Ph.D. course, University of Copenhagen, November 2000. Title: Forskningsmetoder indenfor arbejdsfysiologi og idrætsmedicin.

Thorkil Ploug,
· lecturer, Ph.D. course, Danmarks Farmaceutiske Højskole, October 2000. Title: Bio-logical membranes: drug targets and absorption barriers.

Erik A. Richter,
· lecturer, Ph.D. course, University of Southern Denmark, June 2000. Title: Regulation of glucose utilization during exercise.

Bengt Saltin,
· lecturer, Ph.D. course, University of Umeå, Sweden, November 2000. Title: Rehabili-tation medicine, focus on muscle and the elderly.

Jørgen Wojtaszewski,
· lecturer, Ph.D. course, University of Southern Denmark, June 2000. Title: "Effects of exercise on insulin resistant muscle".

Other research education

The Baltic Summer School 2000.
Theme: "Current trends in immunology and signal transduction", Kiel 18-29 September 2000. Arranged jointly by the universities in Copenhagen (CMRC), Lund, and Kiel. 60 students from a large number of countries in the world participated in the theoretical course in Kiel. Of the 20 students who continued with lab courses, 5 worked in Denmark and 3 at CMRC.

Jesper L. Andersen,
· teacher and lecturer by special invitation, University of Thessaloniki, Greece, 20-30 January 2000.

Gerrit v. Hall, José AL Calbet, Mikael Sander, Bengt Saltin,
· teachers and lecturers by special invitation, University of La Paz Medical Faculty and University of Cochabamba Medical Faculty, Bolivia, 1 week in February 2000.

Bente K. Pedersen,
· teacher and lecturer by special invitation, Summer School on Exercise and Immune Function, Rome, 1 week, September, 2000.

Bengt Saltin,
· teacher and lecturer by special invitation, staff course on basic metabolism, University of Seoul, Korea, 3-7 May 2000.

Collaboration with Industry

Several CMRC researchers have contacts with Industry, primarily with Novo Nordisk, but also with other companies such as Arla Foods and its daughter companies. With the former, the research is on fundamental issues or clinical evaluation of new products. At the moment, there are no intentions within CMRC to seek for intellectual property rights or patents. The goal of the work with the dairy company is to produce products (protein hydrolysates) which if patented include CMRC rights.

CMRC "external" funding in Dkr.
(defined as funding primarily meant to be used in 2000)

Research councils, etc.
· Danish Research Academy: 100.000
· European Commission: 360.000
· Kulturministeriets Udvalg for idrætsforskning: 724.000
· Nordisk Forskningskomité: 95.000
· Statens Naturvidenskabelige Forskningsråd: 300.000
· Statens Sundhedsvidenskabelige Forskningsråd: 1.447.000

Major private foundations
· Carlsbergfondet: 50.000
· The Diabetes Association: 263.000
· The Danish Heart Association: 350.000
· Novo Nordisk Foundation: 422.000
· Velux Foundation: total 5.000.000, CMRC: 3.000.000

Other private foundations
· The Gatorade Sport Science Institute: 240.000
· Frk. P.A. Brandts Legat: 46.000
· Radiometer: 50.000
· Team Danmark: 460.000
· Anonymous Foundation v/Dir. E. K. Hansen: 460.000

"Ved nedenstående underskrift bekræftes det, at nærværende beretning og det for år 2000 tidligere indsendte regnskab til tilhørende noter og oversigter indeholder alle oplysninger, som vedrører årets aktiviteter i Center for Muskelforskning under Danmarks Grundforskningsfond"

København, den 2. august 2001

Bengt Saltin
Centerleder

Rigshospitalet, section 7652, Blegdamsvej 9
DK-2100 Copenhagen Ø, Denmark

Phone: +45 3545 7621
Fax: +45 3545 7634
E-mail: E-mail: cmrc@rh.dk