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Regulation of Muscle Glucose Uptake

Regulation of Muscle Glucose Uptake. David Wasserman Light Hall Rm. 823. Regulation and Assessment of Muscle Glucose Uptake. Processes and Reactions Involved Methods of Measurement Physiological Regulation by Exercise and Insulin Metabolic Control Analysis.

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Regulation of Muscle Glucose Uptake

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

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

  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 = FluxHK + FluxPhos - FluxGS - FluxGly

  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 ([3-3H]glucose) • [3H]2-deoxyglucose • Positron emission tomography ([18F]deoxyglucose)

  10. Arteriovenous Differences In • 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 Out

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

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

  13. Positron emission tomography ([18F]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. G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G P P P P P Fasted Insulin or Exercise 1 1 2 2 GLUT4 3 HK II 3 HK II ptf 2002

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

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

  19. I PIP2 PIP3 P P P P PDK1 AKT PDE3b mTOR GSK3 stimulate protein syn stimulate gly syn inhibit lipolysis Glucose Insulin-stimulated Muscle PI3K IRS-1 P85 P110 GLUT4 Translocation

  20. Exercise and Insulin act through Different Mechanisms

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

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

  24. Exercise Basal Time (min) 10 30 60 90 SRIF plus 10 ± 1 8 ± 1 7 ± 2 7 ± 1 6 ± 1 Insulin SRIF minus <2 <2 <2 <2 <2 Insulin Control 13 ± 1 10 ± 2 9 ± 1 9 ± 2 7 ± 1 Somatostatin (SRIF) infusion creates an insulin-free environment

  25. Exercise stimulates glucose utilization independent of insulin action in vivo Exercise 10 + Insulin - Insulin 8 Glucose Disappearance 6 4 (mg·kg-1·min-1) 2 0 -30 0 30 60 90 Time (min)

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

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

  31. An Index of MGU (Rg) in vivo using 2-Deoxy-[3H]glucose [2-3H]DGPtissue (t) Rg = • Glucoseplasma AUC [2-3H] DGplasma Bolus 104 Plasma [2-3H]DG (dpm·l-1) 103 102 0 5 10 15 20 25 30 Time (min) ptf 2002

  32. Current (I) V1 V2 V3 V4 Resistor3 Resistor1 Resistor2 Ohm’s Law I·Resistor1 = V1-V2 I·Resistor2 = V2-V3 I·Resistor3 = V3-V4

  33. Hyperinsulinemic Euglycemic Clamp in the Mouse -150 -90 0 5 30 min Insulin (0 or 4 mU·kg-1·min-1) Acclimation Euglycemic Clamp Excise Tissues [2-3H]DG Bolus Mice are 5 h fasted at the time of the clamp

  34. Ga Ge Gi 0 GLUT4Tg HKTg GLUT4Tg HKTg Ohm’s Law Glucose Influx WT Transgenics

  35. ‡ * ‡ * * * ‡ * ‡ * * * † † WT HKTg GLUT4Tg HKTg + GLUT4Tg Saline-infused and Insulin-clamped Mice 100 Soleus 50 Muscle Glucose Uptake (mol·100g-1·min-1) 0 Saline Insulin-clamped 20 Gastrocnemius 10 0

  36. Metabolic Control Analysis of MGU • Control CoefficientE = lnRg/ln[E] • Sum of Control Coefficients in a Defined Pathway is 1 i.e. Cd + Ct + Cp = 1

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

  38. G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G P P P P P Fasted Insulin 1 1 2 2 GLUT4 3 HK II 3 HK II ptf 2002

  39. Functional Barriers to MGU during Exercise glucose 6-phosphate glucose Extracellular Membrane Intracellular

  40. Exercise Protocol -60 0 5 30 min Sedentary or Exercise Acclimation Excise Tissues [2-3H]DG Bolus

  41. † * † * * † † * † * * † † * † * HKTG HKTg + GLUT4Tg GLUT4Tg Sedentary and ExercisingMice Gastrocnemius 40 * 20 0 Sedentary SVL 20 Muscle Glucose Uptake (mol·100g-1·min-1) Exercise * 10 0 Soleus 100 * 50 0 WT

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

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

  44. G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G P P P P P Sedentary Exercise 1 1 2 2 GLUT4 3 HK II 3 HK II ptf 2002

  45. Functional Barriers to MGU in Dietary Insulin Resistance glucose 6-phosphate glucose Extracellular Membrane Intracellular

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

  47. Saline-infused and Insulin-clamped Mice High Fat Fed * ‡ * * ‡ * WT HKTg Chow Fed † 15 * Gastroc 10 * 5 0 Muscle Glucose Uptake µmol·100g-1·min-1 15 SVL † * 10 Saline-infused * Insulin -clamped 5 0 WT HKTg 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. Ga Ge Gi 0 Ohm’s Law and Insulin Resistance Glucose Influx WT PDE5(-) Sildenafil was used to inhibit PDE5 activity and increase vascular conductance

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

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