regulation of muscle glucose uptake

Regulation of Muscle Glucose Uptake

David WassermanDavid WassermanLight Hall Rm. 823Light 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

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

• 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

InsideOutside

Glc 6-P

Glycogen Synthesis

Glycolysis

GlcGlc

(-)

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.

GLUT4GLUT1

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

Tools for Studying Muscle Glucose Uptake in vivo

• Why in vivo?

• Why conscious?

Methods to Assess Muscle Glucose Uptake in vivo

• Arteriovenous differences

• Isotope dilution ([3-3H]glucose)

• [3H]2-deoxyglucose

• Positron emission tomography ([18F]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

In

Out

Isotope dilution ([3-3H]glucose)

• Minimal invasiveness

• Applicable to human subjects

• Applicable with 6,6[2H]glucose also

• Whole body measure; not muscle specific

[3H]2-deoxyglucose

• Sensitive• Tissue specific• 2-deoxyglucose

metabolism may differ from that for glucose

• Usually single endpoint measure

• Generally not applied to people.

2DG2DG 2DG-P

IntracellularIntracellularWaterWater

ExtracellularExtracellularWaterWater

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)

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

HK II 3

1

2

HK II

GLUT4

3

2

1

G

ptf 2002

Fasted Insulin or Exercise

G GG

G G

G GG

GGG

PG PG

GG G G

G G

G G

GG G G

G

G GG G

G

PGPG PG

Basal Insulin ContractionsInsulin + Contractions

0

4

8

12

0

1

2

3

4

5

Soleus Cell Surface

GLUT4 Content

pmol/(g wet wt)

µmol/(ml•hr)

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

Insulin signals the translocation of GLUT4 to the cell membrane

by a series of protein phosphorylation rxns

GLUT4 Translocation

Glucose Insulin-stimulated Muscle

I

IRS-1 P85 P110

PI3K

P

P PDK1

PIP2 PIP3

AKT

mTOR GSK3 PDE3binhibit

lipolysisstimulate gly syn

stimulate protein syn

P

P

Exercise and Insulin act through Different Mechanisms

GLUT4 Translocation

Glucose

AMPKK

AMPK

Working Muscle

NOS aPKCERK?p38

FATAcetyl-CoA

Carboylase (ACC)

Fat Metabolism

P

[AMP]

CaMKK

CaMKII

[Ca++]

P

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’.

Glucose 6-Phosphate

Glycogen Synthesis

Glycolysis

Insulin Stimulation

Glucose 6-Phosphate

Glycogen Synthesis

Glycolysis

Exercise

Somatostatin (SRIF) infusion creates an insulin-free environment

Basal Exercise

SRIF plusInsulinSRIF minusInsulinControl

Time (min) 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

02468

10

-30 0 30 60 90Time (min)

Glucose Disappearance

(mg·kg-1·min-1)

+ Insulin- Insulin

Exercise

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

0

6

12

18

10 100 1000

Insulin (µU/ml)

Glucose Disappearance (mg·kg-1·min-1)

Rest

1

Exercise

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 Glucose Uptake (MGU) Requires Three Steps

HK II

GLUT4Intracellular

Blood

Extracellular

3

2

1 Delivery

Phosphorylation

Transport

G GG

GP

GP

ptf 2002

G

GGG GG GG G G

G

GG

An Index of MGU (Rg) in vivo using 2-Deoxy-[3H]glucose

102

103

104

0 5 10 15 20 25 30Time (min)

Plasma [2-3H]DG (dpm·l-1)

Bolus

[2-3H]DGPtissue (t)

AUC [2-3H] DGplasma• Glucoseplasma

Rg =

ptf 2002

Ohm’s Law

V1 V2 V3 V4

Resistor1 Resistor3Resistor2

Current (I)

I·Resistor1 = V1-V2 I·Resistor2 = V2-V3 I·Resistor3 = V3-V4

ExciseTissues

Acclimation Insulin (0 or 4 mU·kg-1·min-1)Euglycemic Clamp

[2-3H]DGBolus

-150 30 min0-90 5

Hyperinsulinemic Euglycemic Clamp in the Mouse

Mice are 5 h fasted at the time of the clamp

Ohm’s Law

Ga Ge Gi 0

Glucose Influx

WT

GLUT4Tg

HKTg

GLUT4Tg

HKTg

Transgenics

Saline-infused and Insulin-clamped Mice

SalineInsulin-clamped

0

50

100Soleus

0

10

20 Gastrocnemius

WT

*

*

GLUT4Tg

*

*

†

HKTg

*

* ‡

‡

HKTg + GLUT4Tg

*

* ‡

‡

†

Muscle Glucose Uptake

(mol·100g-1·min-1)

Metabolic Control Analysis of MGU

• Control CoefficientE = lnRg/ln[E]

• Sum of Control Coefficients in a Defined Pathway is 1i.e. Cd + Ct + Cp = 1

Control Coefficients for MGU by Mouse Muscle Comprised of Type II Fibers

Delivery Transport Phosphorylation

0.1 0.9 0.0

0.5 0.1 0.4

Rest

Insulin(~80 µU/ml)

HK II 3

1

2

HK II

GLUT4

3

2

1

G

ptf 2002

Fasted Insulin

G GG

G G

G GG

GGG

PG PG

GG G G

G G

G G

GG G G

G

G GG G

G

PGPG PG

Functional Barriers to MGU during Exercise

Extracellular Membrane Intracellular

glucose 6-phosphate

glucose

Exercise Protocol

Acclimation Sedentary or Exercise

-60 30 min50

[2-3H]DG Bolus

Excise Tissues

Sedentary and Exercising Mice

0

20

40

0

10

20

*


(mol·100g-1·min-1)

SedentaryExercise

SVL

Gastrocnemius

0

50

100Soleus

*

†

†

*

GLUT4Tg

*

WT

*† *

† *

† *

HKTg

+ GLUT4Tg

HKTG

† *

† *

† *



0.1 0.9 0.0

0.5 0.1 0.4

Rest

Insulin(~80 µU/ml)



0.1 0.9 0.0

0.5 0.1 0.4

0.2 0.0 0.8

Rest

Insulin(~80 µU/ml)

Exercise

HK II 3

1

2

HK II

GLUT4

3

2

1

G

ptf 2002

Sedentary Exercise

G GG

G G

G GG

GGG

PG PG

GG G G

GG

G G

GG G G

G

G GG G

G

PGPG PG

Functional Barriers to MGU in Dietary Insulin Resistance


glucose 6-phosphate

glucose

Descriptive Characteristics

B = p < 0.001 vs. HKIITG chow fed miceA = 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 ± 13A

196 ± 5A

36 ± 2A

17

High Fat

37 ± 16B

167 ± 7

35 ± 1B

17

High Fat

21 ± 2

167 ± 7

26 ± 1

22

Chow

HKIITG

Saline-infused and Insulin-clamped Mice


µmol·100g-1·min-1

Insulin -clamped

Saline-infused

Chow Fed

0

5

10

15

*

†*

0

5

10

15

*

†*

WT HKTg

SVL

Gastroc

High Fat Fed

*‡*

** ‡

WT HKTg

Fueger et al. Diabetes. 53:306-14, 2004..

Increasing conductance of the muscle vasculature will prevent insulin resistancein high fat fed mice.

Hypothesis

Ohm’s Law and Insulin Resistance

Ga Ge Gi 0

Glucose Influx

WT

PDE5(-)

Sildenafil was used to inhibit PDE5 activity and increase vascular conductance

Blood vessel relaxation

PDE5 Inhibition and Vascular Conductance

Nitric Oxide

PKG

cGMP

5’GMP

Natriuretic Peptides & Guanylins

sGC

pGC

PDE5

PDE5 Inhibitor

Vascular Smooth Muscle Cell

Index of Glucose Uptake during an Insulin Clamp in High Fat Fed Mice


µmol·100 g tissue-1·min-1

0

50

60

70

80

90

Soleus

10

Gastrocnemius SVL

VehicleSildenafil/L-arginine

*

**

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.

Summary


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

regulation of muscle glucose uptake

Documents

exercise muscle

fluxgly regulation

insulin clamp

insulin act

human subjectsapplicable

cell membrane

diabetesmembrane transporter

sensitizing tests