A mechanism is only as important as its functional impact in the whole organism.

1. The foundation of our understanding of metabolic physiology is built on discoveries in fundamental, but isolated model systems. Results from genes to organelles and cells may belie the physiognome. A mechanism is only as important as its functional impact in the whole organism.

2. Well-Controlled Animal Models Bridge Cell Biology to the Physiology of Exercise

3. The Study of Glucoregulation

4. Provocative or Sensitizing Tests Physical Exercise Hormone and Metabolic Challenges (e.g. hyperinsulinemic, euglycemic glucose clamps) Etcetera

5. Four Grams of Glucose

6. Maintaining 4 Grams of Glucose in the BloodSedentary, Postabsorptive Brain Fat Glucose ~4 grams Liver Blood Liver Muscle

7. Maintaining 4 Grams of Glucose in the BloodFeeding Suppression (Insulin) Brain Fat Glucose ~4 grams Liver Blood Liver GI Tract Stimulus (Insulin) Muscle

8. Maintaining 4 Grams of Glucose in the BloodExercise Stimulus Brain Fat Glucose ~4 grams Liver Blood Liver Muscle Stimulus

9. Why don’t we get hypoglycemic when we exercise? If the liver does not release more glucose during exercise . . . Hypoglycemia rapidly ensues Exercise 6 Glucose Utilization mg･kg-1･min-1 0 6 Hepatic Glucose Production mg･kg-1･min-1 0 100 Arterial Plasma Glucose mg·dl-1 0 -30 0 60 Time (minutes)

10. Five Guiding Principles to Study of Metabolism in vivo Glucose metabolism is all about flux control. Glucose flux control is distributed amongst distinct systems that require an in vivo model to be fully understood. Glucose fluxes are most sensitively regulated and therefore best studied in theconscious state. Novel animal models can be used to bridge basic and clinical research. Provocative tests are often necessary to precipitate phenotypes and reveal functional limitations.

11. Endocrine and Sympathetic Nerve Response to Exercise Exercise 16 120 Glucagon Arterial Insulin 80 Arterial 12 Glucagon pg·ml-1 µU·ml-1 40 8 Insulin 0 0 300 Norepinephrine Arterial Catecholamines 200 pg·ml-1 100 Epinephrine 0 -60 -30 0 30 60 90 120 150 Time (min)

12. Investigator sees… Liver sees… head and upper extremities pancreas liver gut heart and lungs A Minimal Overview of the Circulation arterial trunk and lower extremities portal vein venous

13. Chronically-Catheterized Conscious Dog Model

14. Investigator sees… Liver sees… arterial trunk and Basal Exercise head and upper extremities pancreas lower Basal liver extremities Exercise gut 300 300 portal vein Portal Vein Hepatic Vein venous 200 200 Arterial Plasma Glucagon (pg·ml-1) Plasma Epinephrine (pg·ml-1) Portal Vein 100 100 Artery Hepatic Vein heart and lungs 0 0 -50 0 50 100 150 -50 0 50 100 150 Time (min) Time (min)

15. Protocols: Role of Glucagon 150 -120 min 0 -40 Basal Equilibration Moderate Treadmill Exercise Somatostatin + [3-3H]glucose + [U-14C]alanine Exercise-Simulated Intraportal Insulin Basal Intraportal Insulin Saline Variable Glucose Protocol A Basal Intraportal Glucagon Protocol B Exercise-Simulated Intraportal Glucagon Basal Intraportal Glucagon

16. Exercise as a model to study glucagon action Exercise 150 Simulated Glucagon 100 Arterial Glucagon pg/ml 50 Basal Glucagon 0 15 Basal Glucagon Arterial Insulin 10 µU/ml 5 Simulated Glucagon 0 -60 -30 0 30 60 90 120 150 Time (min)

17. Exercise-induced Increment in Glucagon Stimulates Hepatic Glucose Production Exercise 120 Simulated Glucagon Arterial Plasma Glucose mg·dl-1 80 Basal Glucagon 40 0 Simulated Glucagon 10 Hepatic Glucose Production mg·kg-1·min-1 8 6 4 Basal Glucagon 2 0 -40 0 30 60 90 120 150

18. Exercise-induced Increment in Glucagon Stimulates Gluconeogenesis from Alanine 400 300 200 100 0 400 300 200 100 0 Exercise Simulated Glucagon Gluconeogenesis from Alanine (% Basal) Basal Glucagon Simulated Glucagon Intrahepatic Gluconeogenic Efficiency from Alanine (% Basal) Basal Glucagon -60 -30 0 30 60 90 120 150 Time (min)

19. Comparison of the Effects of Similar Increases in Glucagon at Rest and during Exercise 6 5 Increase in Endogenous Glucose Production (mg·kg-1·min-1) 4 3 2 1 0 Rest Exercise

20. Why is Glucagon so Effective during Exercise? Brain Autonomic Nerve Activity Adrenal Working Muscle Epi Intestine ? Adipose Glycerol NEFA Pancreas Amino Acids IL6 Lactate Amino Acids RBP4 Glucose 4 grams Substrates GNG Glucagon Signals Insulin Gly Liver

21. Why is Glucagon so Effective during Exercise? Brain Autonomic Nerve Activity Adrenal Working Muscle Body is in a ‘Gluconeogenic Mode’ Epi Intestine ? Adipose Glycerol NEFA Pancreas Amino Acids IL6 Lactate Amino Acids RBP4 Glucose 4 grams Substrates GNG Glucagon Signals Insulin Gly Liver

22. Why is Glucagon so Effective during Exercise? Brain Autonomic Nerve Activity Adrenal Working Muscle Body is in a ‘Gluconeogenic Mode’ Epi Intestine ? Adipose Glycerol NEFA Pancreas Amino Acids IL6 Lactate Amino Acids RBP4 Glucose 4 grams Substrates GNG Glucagon Signals Insulin Gly Effects are Potentiated by the Fall in Insulin Liver

23. Why is Glucagon so Effective during Exercise? Brain Autonomic Nerve Activity Adrenal Working Muscle Body is in a ‘Gluconeogenic Mode’ Epi Intestine ? Adipose Glycerol NEFA Pancreas Amino Acids IL6 Lactate Amino Acids  Glucose Uptake Prevents Hyperglycemia RBP4 Glucose 4 grams Substrates GNG Glucagon Signals Insulin Gly Effects are Potentiated by the Fall in Insulin Liver

24. Protocol: Study of Splanchnic Amino Acid Metabolism during Exercise -120 min -30 0 150 Equilibration Basal Treadmill Exercise 15 13 [5- N]Glutamine + [1- C]Leucine

25. The Exercise-induced Glucagon Response is Essential to the Increment in Hepatic Glutamine Extraction Simulated Glucagon Basal Glucagon 0.60 * Hepatic * Fractional 0.40 * Glutamine † Extraction 0.20 † 0.00 Basal 25-50 75-100 125-150 Basal 25-50 75-100 125-150 Exercise Duration Exercise Duration (min) (min)

26. The Exercise-induced Glucagon Response Drives Urea Formation in the Liver Simulated Glucagon Basal Glucagon 20 * Net Hepatic * 15 * Urea Output 10 -1 -1 (mol· kg ･min ) 5 0 Basal 25-50 75-100 125-150 Basal 25-50 75-100 125-150 Exercise Duration (min) Exercise Duration (min)

27. The Exercise-induced Glucagon Response is Required for the Accelerated transfer of Glutamine Amide Nitrogen to Urea in the Liver 3.0 Formation of Urea from 2.0 Glutamine Amide Nitrogen during Exercise 1.0 -1 -1 (mol· kg ･min ) 0.0 Basal Simulated Glucagon Glucagon

28. Energy State and the Liver

29. Energy State in the Liver is Controlled by Glucagon

30. Studies using the Phloridzin-Euglycemic Clamp further Illustrate the Role of Glucagon in Liver Energy Balance *

31. Blood is Regulated like a HomeostatLiver is the Battery (rechargeable)

32. Substrates and Signals Implicated in Control of Glucose Fluxes to Working Muscle during Exercise Brain Sensors Carotid Sinus Liver/Portal Vein Working Muscle Autonomic Nerve Activity Feedback Chemical Mechanical Feedforward Adrenal Epi Intestine IL6 Adipose Working Muscle Glycerol NEFA Pancreas Amino Acids IL6 Lactate Amino Acids RBP4 Glucose 4 grams Glucagon GNG Substrates Insulin Signals Gly Liver

33. What about the Famous Catecholamine Response to Exercise? Epinephrine plays little to no role in control of glucose production during exercise.Moates et al Am J Physiol 255: E428-E436, 1988. Hepatic nerves are not necessary for the exercise-induced rise in glucose production.Wasserman et al Am J Physiol 259: E195-E203, 1990. Liver specific blockade of both - and -adrenergic receptors do not attenuate the increase in glucose production during exercise.Coker et al Am J Physiol 273: E831-E838, 1997. Coker et al Am J Physiol 278: 444-451, 2000.

34. Catecholamines • Essential, in association with the fall in insulin, for extrahepatic substrate mobilization during exercise. • Muscle glycogenolysis • Adipose tissue lipolysis

35. NEFA Flux is Accelerated during Moderate Exercise by Increased Lipolysis and Decreased Re-esterification ATP TG FFA G3P Glucose FFA FFA TG N E TG Glycerol T G Glycerol G l y c e r o l

36. NEFA Flux is Accelerated during Moderate Exercise by Increased Lipolysis and Decreased Re-esterification ATP TG FFA G3P Glucose FFA FFA TG N E TG Glycerol T G Glycerol G l y c e r o l

37. Four Grams of GlucoseControlling Rate of Removal Extracellular Intracellular glucose 6-phosphate glucose • hexokinase # • hexokinase compartmentation • spatial barriers • blood flow • capillary recruitment • spatial barriers Membrane • transporter # • transporter activity

38. Strategy Selectively remove sites of resistance to MGU in conscious mice by using transgenic mice or pharmacological methods.

39. Ohm’s Law Applied to Glucose Influx Current (I) V1 V2 V3 V4 Resistor3 Resistor1 Resistor2 V1 = I · Resistor1 V2 = I · Resistor2 V3 = I · Resistor3 Glucose Influx (Ig) Ga Ge Gi 0 RExtracell RTransport RPhosp Gextracell= Ig · Rextracell Gtransport= Ig · Rtransport Gphos = Ig ·RPhosp

40. Ohm’s Law to Determine Sites of Resistance to Muscle Glucose Uptake Ga Ge Gi 0 GLUT4Tg HKTg GLUT4Tg HKTg Glucose Influx WT Transgenics

41. Artery Chronically Catheterized, Conscious Unstressed Mouse Sample [3-3H]Glc Blood Insulin Glucose [2-14C]DG Vein From: Glucose Clamping the Conscious Mouse by Vanderbilt MMPC 2005 ptf 2002/jea 2005

42. Metabolic Control Analysis of MGU Control Coefficient( C ) = lnRg/ln[E] Sum of Control Coefficients in a Defined Pathway is 1 i.e. Cd + Ct + Cp = 1

43. Control Coefficients for MGU by Mouse Muscle Comprised of Type II Fibers Rest Insulin (~80 µU/ml)

44. Exercise Protocol -90 0 5 30 min Sedentary or Exercise Acclimation Excise Tissues [2-3H]DG Bolus

45. Sedentary and ExercisingMice WT GLUT4Tg HKTg HKTg + GLUT4Tg Exercise Sedentary 250 200 Blood Glucose (mg·dl-1) * * * 150 * * * * * 100 * * 50 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 Time (min) Time (min) Fueger et al. Am J Physiol; 286: E77-84, 2004

46. Sedentary and ExercisingMice † † † † † † † † HKTG HKTg + GLUT4Tg GLUT4Tg Gastrocnemius 40 Sedentary 20 Exercise 0 SVL 20 Muscle Glucose Uptake (mol·100g-1·min-1) 10 0 Soleus 100 50 0 WT Fueger et al. Am J Physiol; 286: E77-84, 2004

47. Control Coefficients for MGU by Mouse Muscle Comprised of Type II Fibers Rest Insulin (~80 µU/ml) Exercise

48. Distributed Control of Muscle Glucose Uptake Transport is clearly the primary barrier to muscle glucose uptake in the fasted, sedentary state. Transport is so effectively regulated by exercise and insulin that the membrane is no longer the primary barrier to muscle glucose uptake. The resistance to insulin-stimulated muscle glucose uptake with high fat feeding is due, in large part, to defects in the delivery of glucose to the muscle. The vast majority of the literature on the regulation of glucose uptake is comprised of studies in isolated muscle tissue or cells that are blind to fundamental control mechanisms involved in muscle glucose uptake.

49. Four Grams of Glucose glucose 6-phosphate gluconeogenic precursors glycogen Membrane Intracellular Extracellular glucose 6-phosphate glucose Liver Extracellular Membrane Intracellular The distributed control of blood glucose allows for more precise control of glucose homeostasis, multiple mechanisms of glucose flux control, and multiple targets to correct dysregulation of metabolism such as is seen in diabetes Carefully conducted studies in the whole animal are necessary to ascribe function to putative controllers of glucose homeostasis.