This document summarizes research on analyzing metabolic fluxes in human muscle using phosphorus NMR spectroscopy. It discusses: 1. Why studying cellular metabolism in muscle is important, as it can provide insights into whole-body health and lead to medical advances. 2. How phosphorus NMR spectroscopy combined with force measurements can quantify metabolic fluxes non-invasively in vivo by monitoring changes in phosphocreatine and pH levels. 3. Findings that both elevated metabolite levels and a muscle activation factor like calcium are needed to initiate and sustain high glycolysis rates in muscle.

1. An analysis of metabolic fluxes in contracting human muscle Gregory J. Crowther Dept. of Physiology & Biophysics University of Washington (Seattle) ATP

2. Energy metabolism in muscle H + & Lactate Glucose ATP ATP supply ATP demand (contractile cost) (oxidative phosphorylation) (glycolysis)

3. Questions Why study cellular metabolism in muscle ? How can metabolic fluxes be quantified using phosphorus NMR spectroscopy? What turns glycolysis on and off? How is muscle metabolism affected by type 1 diabetes mellitus? Intro: Methods: Results:

4. Definitions NMR nuclear magnetic resonance PCr phosphocreatine (an ATP buffer) HP hexose phosphates (substrates of glycolysis) “ metabolites” P i , ADP, AMP (products of ATP hydrolysis)

5. Introduction: Why study cellular metabolism in muscle ?

6. Why study cellular metabolism ? C ellular metabolism . . . • is central to the existence of all cells. • has important whole-organ and whole-body consequences. • can be harnessed to spawn advances in medicine and industry. GAPDH glycogen GP PFK PGK PK lactate PGI PGM ALD PGM ENO LDH

7. Why study cellular metabolism ? X C ellular metabolism . . . • is central to the existence of all cells. • has important whole-organ and whole-body consequences. • can be harnessed to spawn advances in medicine and industry. GAPDH glycogen GP PFK PGK PK lactate PGI PGM ALD PGM ENO LDH

8. Why study cellular metabolism ? X C ellular metabolism . . . • is central to the existence of all cells. • has important whole-organ and whole-body consequences. • can be harnessed to spawn advances in medicine and industry. GAPDH glycogen GP PFK PGK PK lactate PGI PGM ALD PGM ENO LDH GAPDH PFK PGK PK ethanol PGI HK ALD PGM ENO PDC glucose ADH

9. Why study cellular metabolism ? We don’t know . . . • What are the pathway flux rates in vivo ? • How are these rates up- and downregulated? GAPDH glycogen GP PFK PGK PK lactate PGI PGM ALD PGM ENO LDH

10. Why study cellular metabolism in muscle ? Advantages of muscle . . . • huge range of fluxes • ~40% of human body mass • critical to physical performance and well-being • can study muscles specialized for different tasks McArdle et al., Essentials of exercise physiology , 1994

11. Methods: How can metabolic fluxes be quantified using phosphorus NMR spectroscopy?

12. The basic idea Force [PCr] Time

13. Simultaneous collection of NMR and force data • in vivo • noninvasive NMR coil force transducer

14. NMR reveals metabolic changes • good time resolution

15. [ATP] remains constant during exercise and recovery ischemia exercise [PCr] Time [ATP] [P i ]

16. PCr keeps [ATP] constant Therefore changes in [PCr] reflect changes in ATP production/consumption. ADP + PCr +  H + Cr + ATP ATP ADP + P i +  H + PCr +  H + Cr + P i

17. Quantifying contractile cost ATP consumption at the start of ischemic exercise is not “contaminated” by glycolytic or oxidative ATP production.

18. Quantifying oxidative phosphorylation rate constant k PCr = 1/tau rapid recovery --> high k PCr --> high oxidative capacity

19. Quantifying glycolysis Recall: Under ischemic conditions, glycolytic H + production = (  pH)*(  ) + (  )*(  PCr)  is the muscle buffer capacity ( Conley et al., Am J Physiol 273 : C306, 1997 ) PCr +  H + Cr + P i

20. Results: What turns glycolysis on and off?

21. Potential controllers of glycolysis Metabolites P i , ADP, and AMP are substrates and allosteric activators of glycolytic enzymes Calcium glycolysis  stimulation frequency (Conley et al., Am J Physiol 273 : C306, 1997) Hexose phosphates substrates for glycolysis + + + + + GAPDH glycogen GP PFK PGK PK lactate PGI PGM ALD PGM ENO LDH

22. Turning off glycolysis after exercise calcium important Post-exercise time Glycolytic rate calcium unimportant

23. Evidence of post-exercise glycolysis • pH falls due to continued lactic acid production • [PCr] rises due to continued glycolytic ATP production

24. Agreement of two estimates of post-exercise glycolysis

25. Time course of post-exercise glycolysis The cessation of glycolysis must reflect the decline of a muscle activation-related factor such as calcium. calcium unimportant calcium important

26. Turning on glycolysis at the start of exercise What mechanism is responsible for this delayed onset?

27. Testing the importance of metabolites 2 bouts of exercise Will glycolysis begin sooner in Bout 2 (when metabolites are high) than in Bout 1 (when metabolites are low)?

28. Glycolysis begins earlier in Bout 2 than in Bout 1

29. The onset of glycolysis coincides with elevated metabolites aerobic rest onset of flux [P i ] 3.4-3.8 16.2-20.6 [ADP] 0.013-0.014 0.080-0.121 [AMP] 2x10 -5 -3x10 -5 0.0008-0.0012 (All concentrations in mM.)

30. Summary of glycolysis data To initiate and sustain high rates of glycolysis, both elevated metabolite levels and a muscle activation-related factor such as calcium are needed.

31. Results (continued): How is muscle metabolism affected by type 1 diabetes mellitus?

32. Little is known about muscle metabolism in people with type 1 diabetes Rat models suggest that diabetes-induced changes may be completely reversed by insulin treatment (Ianuzzo et al., J Appl Physiol 52 : 1471, 1982; Noble & Ianuzzo, Am J Physiol 249 : E360, 1985). We asked whether careful treatment of type 1 diabetes with insulin restores human muscle metabolism to normal.

33. Subjects 10 men with type 1 diabetes and 10 male age- and activity-matched control subjects All diabetic subjects used insulin injections to keep blood glucose levels under good clinical control : • glycosylated hemoglobin (Hb A1c ) levels  7% • no glucose in the urine

34. No significant difference in force

35. Glycolysis occurs “early and often” in diabetic subjects Earlier onset Higher peak rate

36. Oxidative recovery rate is slower in diabetic subjects

37. Contractile cost: difference not significant

38. Summary of diabetes data Elimination of symptoms of type 1 diabetes does not normalize muscle properties. The observed abnormalities in glycolytic and oxidative fluxes suggest that diabetes causes a shift in the metabolic profile of the muscle.

39. Future work

40. Acknowledgments Advisers Kevin Conley Marty Kushmerick UW Departments Physiology & Biophysics Radiology Funding National Institutes of Health National Science Foundation

41. Acknowledgments (continued) Coworkers Cathy Amara Iris Asllani Outi Hyyti Melissa Lambeth Donghoon Lee Dave Marcinek Ken Marro Daryl Monear Brad Moon Eric Shankland Rudy Stuppard Nina V Ø llestad Coauthors Mike Carey Rod Gronka Sharon Jubrias Will Kemper Jerry Milstein

42. This talk, like glycolysis after exercise, is over.