regulation of muscle glucose uptake
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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
Regulation and Assessment of Muscle Glucose Uptake
• Processes and Reactions Involved• Methods of Measurement• Physiological Regulation by Exercise and
Insulin• Metabolic Control Analysis
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)
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
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
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
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
*
Muscle Glucose Uptake
(mol·100g-1·min-1)
SedentaryExercise
SVL
Gastrocnemius
0
50
100Soleus
*
†
†
*
GLUT4Tg
*
WT
*† *
† *
† *
HKTg
+ GLUT4Tg
HKTG
† *
† *
† *
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)
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
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
Extracellular Membrane Intracellular
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
Muscle Glucose Uptake
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
Muscle Glucose Uptake
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
Extracellular Membrane Intracellular
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
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