1. Regulation of Muscle Glucose Uptake David Wasserman Light Hall Rm. 823

2. Regulation and Assessment of Muscle Glucose Uptake Processes and Reactions Involved Methods of Measurement Physiological Regulation by Exercise and Insulin Metabolic Control Analysis

3. To Understand Glucose Metabolism In Vivo, One must Understand Regulation by Muscle Muscle is ~90% of insulin sensitive tissue Muscle metabolism can increase ~10x in response to exercise Muscle is a major site of insulin resistance in diabetes

4. blood flow capillary recruitment spatial barriers Extracellular Membrane hexokinase # hexokinase compartmentation spatial barriers Intracellular glucose 6-phosphate glucose transporter # transporter activity Muscle Glucose Uptake in Three Easy Steps

5. InsideOutside Glc 6-P Glycogen Synthesis Glycolysis Glc (-) HK II Glucose is not “officially” taken up by muscle until it is phosphorylated. Glucose phosphorylation traps carbons in the muscle and primes it for metabolism. GLUT4 GLUT1

6. Feedback Inhibition of HK II by Glc 6-P distributes Control of Muscle Glucose Uptake to Glycogen Synthesis/Breakdown and Glycolytic Pathways d[Glc 6-P]/dt = Flux HK + Flux Phos - Flux GS - Flux Gly

7. Regulation and Assessment of Muscle Glucose Uptake Processes and Reactions Involved Methods of Measurement Physiological Regulation by Exercise and Insulin Metabolic Control Analysis

8. Tools for Studying Muscle Glucose Uptake in vivo Why in vivo? Why conscious?

9. Methods to Assess Muscle Glucose Uptake in vivo Arteriovenous differences Isotope dilution ([3- 3 H]glucose) [ 3 H]2-deoxyglucose Positron emission tomography ([ 18 F]deoxyglucose)

10. Arteriovenous Differences Multiple measurements over time Invasive, but applicable to human subjects Requires accurate blood flow measure Not applicable to small animals Sensitive analytical methods are needed to measure small arteriovenous difference In Out

11. Isotope dilution ([3- 3 H]glucose) Minimal invasiveness Applicable to human subjects Applicable with 6,6[ 2 H]glucose also Whole body measure; not muscle specific

12. [ 3 H]2-deoxyglucose Sensitive Tissue specific 2-deoxyglucose metabolism may differ from that for glucose Usually single endpoint measure Generally not applied to people. 2DG 2DG-P IntracellularWaterExtracellularWater

13. Positron emission tomography ([ 18 F]deoxyglucose) Minimal invasiveness Applicable to human subjects 2-deoxyglucose metabolism may differ from that for glucose Single point derived from curve fitting/compartmental analysis Requires expensive equipment ROI must be stationary (i.e. cannot study during exercise)

14. Regulation and Assessment of Muscle Glucose Uptake Processes and Reactions Involved Methods of Measurement Physiological Regulation by Exercise and Insulin Metabolic Control Analysis

15. You can’t Study Muscle Glucose Metabolism in vivo without a Sensitizing Test Reason: In the fasted, non-exercising state muscle glucose metabolism is too low. Sensitizing Tests: Insulin clamp, Exercise

16. HK II 3 1 2 GLUT4 3 2 1 G ptf 2002 FastedInsulin or Exercise G G G G G G G G G G G P G P G G G GG G G GG G G G G G G G G G G P G P G P G

17. Basal Insulin Contractions Insulin + Contractions 0 4 8 12 0 1 2 3 4 5 Soleus Cell Surface GLUT4 Content pmol/(g wet wt) µmol/(mlhr) From Lund et al. PNAS 92: 5817, 1995 Relationship of Muscle Cell Surface GLUT4 to Uptake of a Glucose Analog Soleus 3-O-methylglucose Uptake

18. Insulin signals the translocation of GLUT4 to the cell membrane by a series of protein phosphorylation rxns

19. GLUT4 Translocation Glucose Insulin- stimulated Muscle I IRS-1 P85P110 PI3K P P PDK1 PIP2 PIP3 AKT mTORGSK3 PDE3b inhibit lipolysis stimulate gly syn stimulate protein syn P P

20. Exercise and Insulin act through Different Mechanisms

21. GLUT4 Translocation Glucose AMPKK AMPK Working Muscle    NOS aPKC ERK? p38 FAT Acetyl-CoA Carboylase (ACC)  Fat Metabolism P [AMP] CaMKK CaMKII [Ca ++ ] P

22. Contraction- and Insulin-stimulated Muscle Glucose Uptake use Separate Cell Signaling Pathways Contraction does not phosphorylate proteins involved in early insulin signaling steps. Wortmannin eliminates insulin- but not contraction- stimulated increases in glucose transport. Insulin and contraction recruit GLUT4 from different pools. Effects of insulin and exercise on glucose transport are additive. Insulin resistant states are not always ‘contraction resistant’.

23. Glucose 6-Phosphate Glycogen Synthesis Glycolysis Insulin Stimulation Glucose 6-Phosphate Glycogen Synthesis Glycolysis Exercise

24. Somatostatin (SRIF) infusion creates an insulin-free environment Basal Exercise SRIF plus Insulin SRIF minus Insulin Control Time (min) 1030 6090 10 ± 1 8 ± 1 7 ± 27 ± 1 6 ± 1 <2 13 ± 110 ± 2 9 ± 19 ± 2 7 ± 1

25. Exercise stimulates glucose utilization independent of insulin action in vivo 0 2 4 6 8 10 -300306090 Time (min) Glucose Disappearance (mg·kg -1 ·min -1 ) + Insulin - Insulin Exercise

26. What is the stimulus/stimuli that signal the working muscle to take up more glucose? Shift in muscle Ca ++ stores Increase in muscle adenine nucleotide levels Increase in muscle blood flow

27. Insulin-stimulated glucose utilization is increased during exercise 0 6 12 18 10 100 1000 Insulin (µU/ml) Glucose Disappearance (mg·kg -1 ·min -1 ) Rest 1 Exercise

28. Proposed mechanisms by which acute exercise enhances insulin sensitivity Increased muscle blood flow Increased capillary surface area Direct effect on working muscles Indirect effect mediated by insulin-induced suppression of FFA levels

29. Regulation and Assessment of Muscle Glucose Uptake Processes and Reactions Involved Methods of Measurement Physiological Regulation by Exercise and Insulin Metabolic Control Analysis

30. Muscle Glucose Uptake (MGU) Requires Three Steps HK II GLUT4 Intracellular Blood Extracellular 3 2 1 Delivery Phosphorylation Transport G G G G P G P ptf 2002 G G G G GG G G G G G G G

31. An Index of MGU (R g ) in vivo using 2-Deoxy- [ 3 H]glucose 10 2 10 3 10 4 051015202530 Time (min) Plasma [2- 3 H]DG (dpm·  l -1 ) Bolus [2- 3 H]DGP tissue (t) AUC [2- 3 H] DG plasma Glucose plasma R g = ptf 2002

32. Ohm’s Law V1V1 V2V2 V3V3 V4V4 Resistor 1 Resistor 3 Resistor 2 Current (I) I·Resistor 1 = V 1 -V 2 I·Resistor 2 = V 2 -V 3 I·Resistor 3 = V 3 -V 4

33. Excise Tissues Acclimation Insulin (0 or 4 mU·kg -1 ·min -1 ) Euglycemic Clamp [2-3H]DG Bolus -15030 min0-905 Hyperinsulinemic Euglycemic Clamp in the Mouse Mice are 5 h fasted at the time of the clamp

34. Ohm’s Law GaGa GeGe GiGi 0 Glucose Influx WT GLUT4 Tg HK Tg GLUT4 Tg HK Tg Transgenics

35. Saline-infused and Insulin-clamped Mice Saline Insulin-clamped 0 50 100 Soleus 0 10 20 Gastrocnemius WT * * GLUT4 Tg * * † HK Tg * * ‡ ‡ HK Tg + GLUT4 Tg * * ‡ ‡ † Muscle Glucose Uptake (  mol·100g -1 ·min -1 )

36. Metabolic Control Analysis of MGU Control Coefficient E =  lnR g /  ln[E] Sum of Control Coefficients in a Defined Pathway is 1 i.e. C d + C t + C p = 1

37. Control Coefficients for MGU by Mouse Muscle Comprised of Type II Fibers DeliveryTransportPhosphorylation 0.10.90.0 0.50.10.4 Rest Insulin (~80 µU/ml)

38. HK II 3 1 2 GLUT4 3 2 1 G ptf 2002 FastedInsulin G G G G G G G G G G G P G P G G G GG G G GG G G G G G G G G G G P G P G P G

39. Functional Barriers to MGU during Exercise ExtracellularMembraneIntracellular glucose 6-phosphate glucose

40. Exercise Protocol Acclimation Sedentary or Exercise -60 30 min 50 [2- 3 H]DG Bolus Excise Tissues

41. Sedentary and Exercising Mice 0 20 40 0 10 20 * Muscle Glucose Uptake (  mol·100g -1 ·min -1 ) Sedentary Exercise SVL Gastrocnemius 0 50 100 Soleus * † † * GLUT4 Tg * WT * † * HK Tg + GLUT4 Tg HK TG † *

42. Control Coefficients for MGU by Mouse Muscle Comprised of Type II Fibers DeliveryTransportPhosphorylation 0.10.90.0 0.50.10.4 Rest Insulin (~80 µU/ml)

43. Control Coefficients for MGU by Mouse Muscle Comprised of Type II Fibers DeliveryTransportPhosphorylation 0.10.90.0 0.50.10.4 0.20.0 0.8 Rest Insulin (~80 µU/ml) Exercise

44. HK II 3 1 2 GLUT4 3 2 1 G ptf 2002 SedentaryExercise G G G G G G G G G G G P G P G G G GG G G GG G G G G G G G G G G P G P G P G

45. Functional Barriers to MGU in Dietary Insulin Resistance ExtracellularMembraneIntracellular glucose 6-phosphate glucose

46. Descriptive Characteristics B = p < 0.001 vs. HKII TG chow fed mice A = p < 0.001 vs. WT chow fed mice 20 ± 3Fasting Insulin ( mU·mL -1 ) 166 ± 6Fasting Glucose ( mg·dL -1 ) 26 ± 1Body Weight ( g ) 27n Chow WT 52 ± 13 A 196 ± 5 A 36 ± 2 A 17 High Fat 37 ± 16 B 167 ± 7 35 ± 1 B 17 High Fat 21 ± 2 167 ± 7 26 ± 1 22 Chow HKII TG

47. Saline-infused and Insulin-clamped Mice Muscle Glucose Uptake µmol·100g -1 ·min -1 Insulin -clamped Saline-infused Chow Fed 0 5 10 15 * † * 0 5 10 15 * † * WTHK Tg SVL Gastroc High Fat Fed * ‡ * * * ‡ WTHK Tg Fueger et al. Diabetes. 53:306-14, 2004..

48. Increasing conductance of the muscle vasculature will prevent insulin resistance in high fat fed mice. Hypothesis

49. Ohm’s Law and Insulin Resistance GaGa GeGe GiGi 0 Glucose Influx WT PDE5(-) Sildenafil was used to inhibit PDE5 activity and increase vascular conductance

50. Blood vessel relaxation PDE5 Inhibition and Vascular Conductance Nitric Oxide PKG cGMP 5’GMP Natriuretic Peptides & Guanylins sGC pGC PDE5 Inhibitor Vascular Smooth Muscle Cell

51. Index of Glucose Uptake during an Insulin Clamp in High Fat Fed Mice Muscle Glucose Uptake µmol·100 g tissue -1 ·min -1 0 50 60 70 80 90 Soleus 10 GastrocnemiusSVL Vehicle Sildenafil/L-arginine * * *

52. 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.

53. Summary ExtracellularMembraneIntracellular glucose 6-phosphate glucose Control of Muscle Glucose Uptake is Distributed between Glucose Delivery to Muscle, Glucose Transport into Muscle, and Glucose Phosphorylation into Muscle. Treatment of Insulin Resistance may Involve any one or More of the Three Steps