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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

Regulation of Muscle Glucose Uptake

David Wasserman

Light Hall Rm. 823

regulation and assessment of muscle glucose uptake
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
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
slide4
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

slide5
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.

d glc 6 p dt flux hk flux phos flux gs flux gly
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

regulation and assessment of muscle glucose uptake1
Regulation and Assessment of Muscle Glucose Uptake
  • Processes and Reactions Involved
  • Methods of Measurement
  • Physiological Regulation by Exercise and Insulin
  • Metabolic Control Analysis
methods to assess muscle glucose uptake in vivo
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
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

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

positron emission tomography 18 f deoxyglucose
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)
regulation and assessment of muscle glucose uptake2
Regulation and Assessment of Muscle Glucose Uptake
  • Processes and Reactions Involved
  • Methods of Measurement
  • Physiological Regulation by Exercise and Insulin
  • Metabolic Control Analysis
you can t study muscle glucose metabolism in vivo without a sensitizing test
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

slide16
G

G

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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

relationship of muscle cell surface glut4 to uptake of a glucose analog
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

slide18

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

slide19
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

slide21
[AMP]

P

P

NOS

aPKC

ERK?

[Ca++]

p38

FAT

Acetyl-CoA Carboylase (ACC)

 Fat Metabolism

Glucose

Working Muscle

AMPKK

CaMKII

AMPK

CaMKK

GLUT4 Translocation

contraction and insulin stimulated muscle glucose uptake use separate cell signaling pathways
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’.
slide23
Glycogen Synthesis

Glucose 6-Phosphate

Glycolysis

Exercise

Glycogen Synthesis

Glucose 6-Phosphate

Glycolysis

Insulin Stimulation

somatostatin srif infusion creates an insulin free environment
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
exercise stimulates glucose utilization independent of insulin action in vivo
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)

what is the stimulus stimuli that signal the working muscle to take up more glucose
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
ExerciseInsulin-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)

proposed mechanisms by which acute exercise enhances insulin sensitivity
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
regulation and assessment of muscle glucose uptake3
Regulation and Assessment of Muscle Glucose Uptake
  • Processes and Reactions Involved
  • Methods of Measurement
  • Physiological Regulation by Exercise and Insulin
  • Metabolic Control Analysis
muscle glucose uptake mgu requires three steps
G

G

G

G

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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

an index of mgu r g in vivo using 2 deoxy 3 h glucose
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

slide32
Current (I)

V1

V2

V3

V4

Resistor3

Resistor1

Resistor2

Ohm’s Law

I·Resistor1 = V1-V2

I·Resistor2 = V2-V3

I·Resistor3 = V3-V4

hyperinsulinemic euglycemic clamp in the mouse
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

slide34
Ga

Ge

Gi

0

GLUT4Tg

HKTg

GLUT4Tg

HKTg

Ohm’s Law

Glucose Influx

WT

Transgenics

saline infused and insulin clamped mice

*

*

*

*

*

*

*

*

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

metabolic control analysis of mgu
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
Control Coefficients for MGU by Mouse Muscle Comprised of Type II Fibers

Rest

Insulin

(~80 µU/ml)

slide38
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G

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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

functional barriers to mgu during exercise
Functional Barriers to MGU during Exercise

glucose

6-phosphate

glucose

Extracellular

Membrane

Intracellular

exercise protocol
Exercise Protocol

-60

0

5

30 min

Sedentary or Exercise

Acclimation

Excise

Tissues

[2-3H]DG

Bolus

sedentary and exercising mice
† *

† *

*

† *

† *

*

† *

† *

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

control coefficients for mgu by mouse muscle comprised of type ii fibers1
Control Coefficients for MGU by Mouse Muscle Comprised of Type II Fibers

Rest

Insulin

(~80 µU/ml)

control coefficients for mgu by mouse muscle comprised of type ii fibers2
Control Coefficients for MGU by Mouse Muscle Comprised of Type II Fibers

Rest

Insulin

(~80 µU/ml)

Exercise

slide44
G

G

G

G

G

G

G

G

G

G

G

G

G

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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

functional barriers to mgu in dietary insulin resistance
Functional Barriers to MGU in Dietary Insulin Resistance

glucose

6-phosphate

glucose

Extracellular

Membrane

Intracellular

descriptive characteristics
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

saline infused and insulin clamped mice1
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..

hypothesis
Increasing conductance of the muscle vasculature will prevent insulin resistance

in high fat fed mice.

Hypothesis
slide49
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

slide50
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

index of glucose uptake during an insulin clamp in high fat fed mice
Index of Glucose Uptake during an Insulin Clamp in High Fat Fed Mice

90

*

80

Vehicle

70

Sildenafil/L-arginine

60

Muscle Glucose

Uptake

µmol·100 g tissue-1·min-1

50

*

*

10

0

Soleus

Gastrocnemius

SVL

distributed control of muscle glucose uptake
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
Summary

glucose

6-phosphate

glucose

Extracellular

Membrane

Intracellular

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

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