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

Regulation of Muscle lucose Uptake David Wasserman Light Hall Rm. 823

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

To Understand lucose 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

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

Outside Inside (-) lycogen Synthesis lc LUT4 LUT1 lc HK II lc 6-P lycolysis lucose is not officially taken up by muscle until it is phosphorylated. lucose phosphorylation traps carbons in the muscle and primes it for metabolism.

Feedback Inhibition of HK II by lc 6-P distributes Control of Muscle lucose Uptake to lycogen Synthesis/Breakdown and lycolytic Pathways d[lc 6-P]/dt = Flux HK + Flux Phos - Flux S - Flux ly

Tools for Studying Muscle lucose Uptake in vivo Why in vivo? Why conscious?

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

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

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

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

Positron emission tomography Minimal invasiveness ([ 18 F]deoxyglucose) 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)

You can t Study Muscle lucose 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

Fasted Insulin or Exercise 1 1 LUT4 2 2 HK II 3 HK II 3 P P P P P ptf 2002

Relationship of Muscle Cell Surface LUT4 to Uptake of a lucose Analog 12 Soleus 3-O-methylglucose Uptake µmol/(ml hr) 8 4 0 5 Soleus Cell Surface LUT4 Content pmol/(g wet wt) 4 3 2 1 0 Basal Insulin Contractions Insulin + Contractions From Lund et al. PNAS 92: 5817, 1995

Insulin signals the translocation of LUT4 to the cell membrane by a series of protein phosphorylation rxns

lucose I Insulinstimulated Muscle P PIP2 PIP3 PI3K IRS-1 P85 P110 P P PDK1 P AKT LUT4 Translocation mtor stimulate protein syn SK3 stimulate gly syn

Exercise and Insulin act through Different Mechanisms

lucose Working Muscle AMPKK AMPK P [AMP] CaMKII P CaMKK LUT4 Translocation NOS apkc p38 ERK? FAT Acetyl-CoA Carboylase (ACC) [Ca ++ ] Fat Metabolism

Contraction- and Insulin-stimulated Muscle lucose Uptake use Separate Cell Signaling Pathways Contraction does not phosphorylate proteins involved in early insulin signaling steps. Wortmannin eliminates insulin- but not contractionstimulated increases in glucose transport. Insulin and contraction recruit LUT4 from different pools. Effects of insulin and exercise on glucose transport are additive. Insulin resistant states are not always contraction resistant.

Insulin Stimulation lycogen Synthesis lucose 6-Phosphate lycolysis Exercise lycogen Synthesis lucose 6-Phosphate lycolysis

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

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

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

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

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

Muscle lucose Uptake (MU) Requires Three Steps Blood Extracellular Intracellular LUT4 HK II 1 Delivery 2 3 Transport Phosphorylation P P ptf 2002

Ohm s Law Current (I) V 1 V 2 V 3 V 4 Resistor 1 Resistor 2 Resistor 3 I Resistor 1 = V 1 -V 2 I Resistor 2 = V 2 -V 3 I Resistor 3 = V 3 -V 4

Mouse Model Catheterization of jugular vein and carotid artery Experiment performed ~5-7 days postoperative Sampling is performed 5 h after food is removed [2-3 H]deoxyglucose infused to measure an index of muscle glucose influx

Hyperinsulinemic Euglycemic Clamp -150-90 0 5 30 min Acclimation Insulin (0 or 4 mu kg -1 min -1 ) Euglycemic Clamp Mice are 5 h fasted at the time of the clamp [2-3H]D Bolus Excise Tissues

Ohm s Law lucose Influx a e i 0 WT Transgenics LUT4 Tg HK Tg LUT4 Tg HK Tg

Saline-infused and Insulin-clamped Mice Muscle lucose Uptake ( mol 100g -1 min -1 ) 100 50 0 10 Soleus * * 20 astrocnemius * * * * Saline Insulin-clamped * * 0 WT LUT4 Tg HK Tg HK Tg + LUT4 Tg

Metabolic Control Analysis of MU 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

Control Coefficients for MU by Mouse Muscle Comprised of Type II Fibers Delivery Transport Phosphorylation Rest Insulin (~80 µu/ml) 0.1 0.9 0.0 0.5 0.1 0.4

Fasted Insulin 1 1 LUT4 2 2 HK II 3 HK II 3 P P P P P ptf 2002

Functional Barriers to MU during Exercise Extracellular Membrane Intracellular glucose glucose 6-phosphate

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

Sedentary and Exercising Mice 40 20 astrocnemius * * * * Muscle lucose Uptake ( mol 100g -1 min -1 ) 0 20 10 SVL * Sedentary Exercise * * * 0 100 Soleus * * 50 * 0 WT LUT4 Tg HK T HK Tg + LUT4 Tg

Control Coefficients for MU by Mouse Muscle Comprised of Type II Fibers Delivery Transport Phosphorylation Rest Insulin (~80 µu/ml) 0.1 0.9 0.0 0.5 0.1 0.4

Control Coefficients for MU by Mouse Muscle Comprised of Type II Fibers Delivery Transport Phosphorylation Rest Insulin (~80 µu/ml) Exercise 0.1 0.9 0.0 0.5 0.1 0.4 0.2 0.0 0.8

Sedentary Exercise 1 1 LUT4 2 2 HK II 3 HK II 3 P P P P P ptf 2002

Functional Barriers to MU in Dietary Insulin Resistance Extracellular Membrane Intracellular glucose glucose 6-phosphate

Saline-infused and Insulin-clamped Mice Chow Fed High Fat Fed 15 10 5 astroc * * * * Muscle lucose Uptake µmol 100g-1 min-1 0 Saline-infused Insulin -clamped 15 10 5 SVL * * * * 0 WT HK Tg WT HK Tg Fueger et al. Diabetes. 53:306-14, 2004..

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

Ohm s Law and Insulin Resistance lucose Influx a e i 0 WT PDE5(-) Sildenafil was used to inhibit PDE5 activity and increase vascular conductance

PDE5 Inhibition and Vascular Conductance Natriuretic Peptides & uanylins pc Nitric Oxide sc PDE5 cmp PK Blood vessel relaxation 5 MP Vascular Smooth Muscle Cell PDE5 Inhibitor

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

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

astrocnemius Akt/PKB after an Insulin Clamp in High Fat Fed Mice Sildenafil + L-Arginine Vehicle Anti-Akt/PKB Anti-phospho- Akt/PKB Anti-APDH 200 150 Anti-Akt/PKB 100 50 Anti-phospho- Akt/PKB 0 125 100 75 50 25 0 Sildenafil/L-arginine Control

An Index of MU (R g ) in vivo using 2-Deoxy- [ 3 H]glucose R g = [2-3 H]DP tissue (t) AUC [2-3 H] D plasma Bolus 10 4 lucose plasma Plasma [2-3 H]D (dpm l -1 ) 10 3 10 2 0 5 10 15 20 25 30 Time (min) ptf 2002

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