Regulation and Assessment 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

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-phosphateglucose

• 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






Insulin


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

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

Intracellular

WaterExtracellular

Water

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)






Insulin


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

GG

G

GGG

PG P

G

GG G G

G G

G G

GG

G G

G

GG

G GG

PG

PG

PG

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/(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

stimulate

gly syn

stimulate

protein syn

P

P

Exercise and Insulin act through

Different Mechanisms

GLUT4

Translocation

Glucose

AMPKK

AMPK

Working

Muscle

NOS aPKC

ERK?p38

FAT

Acetyl-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 plusInsulin

SRIF minusInsulin

Control

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

0

2

4

6

8

10

-30 0 30 60 90

Time (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






Insulin


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

G

GG GG G

G G GG

GG

Ohm‟s Law

V1 V2 V3 V4

Resistor1 Resistor3Resistor2

Current (I)

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

Mouse Model

• Catheterization of jugular vein and carotid artery

• Experiment performed ~5-7 days postoperative

• Sampling is performed 5 h after food is removed

• [2-3H]deoxyglucose infused to measure an index of muscle glucose influx

Excise

Tissues

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

Euglycemic Clamp

[2-3H]DG

Bolus

-150 30 min0-90 5

Hyperinsulinemic Euglycemic Clamp

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

Saline

Insulin-clamped

0

50

100

Soleus

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 1

i.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)




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

GG

G

GGG

PG P

G

GG G G

G G

G G

GG

G G

G

GG

G GG

PG

PG

PG

Functional Barriers to

MGU during Exercise

Extracellular Membrane Intracellular

glucose

6-phosphateglucose

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

*

Muscle Glucose

Uptake

( mol·100g-1·min-1)

Sedentary

Exercise

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

GG

G

GGG

PG P

G

GG G G

GG

G G

GG

G G

G

GG

G GG

PG

PG

PG

Functional Barriers to

MGU in Dietary Insulin Resistance


glucose

6-phosphateglucose

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 resistance

in 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

Muscle Glucose

Uptake

µmol·100 g tissue-1·min-1

0

50

60

70

80

90

Soleus

10

Gastrocnemius SVL

Vehicle

Sildenafil/L-arginine

*

**

Summary


glucose

6-phosphateglucose

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

Gastrocnemius Akt/PKB after an Insulin Clamp

in High Fat Fed Mice

Anti-Akt/PKB

Anti-phospho-

Akt/PKB

Anti-GAPDH

Sildenafil + L-Arginine

Vehicle

0

25

50

75

100

125

Sildenafil/L-arginine Control

Anti-phospho-

Akt/PKB

0

50

100

150

200

Anti-Akt/PKB

An Index of MGU (Rg) in vivo using 2-Deoxy-

[3H]glucose

102

103

104

0 5 10 15 20 25 30

Time (min)

Plasma

[2-3H]DG

(dpm· l-1)

Bolus

[2-3H]DGPtissue (t)

AUC [2-3H] DGplasma• Glucoseplasma

Rg =

ptf 2002

Descriptive Characteristics

B = p < 0.001 vs. HKIITG 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 ± 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

Documents

Regulation and Assessment of Muscle Glucose Uptake