View
791
Download
1
Category
Tags:
Preview:
DESCRIPTION
Greg Crowther's dissertation defense slides.
Citation preview
1
An analysis of metabolic fluxesin contracting human muscle
Gregory J. Crowther
Dept. of Physiology & Biophysics
University of Washington (Seattle)
ATP
2
Energy metabolism in muscle
H+ & Lactate
Glucose
ATP
ATP supplyATP demand
(contractile cost)
(oxidative phosphorylation)
(glycolysis)
3
Questions
Why study cellular metabolism in muscle?
How can metabolic fluxes be quantified using phosphorus NMR spectroscopy?
What turns glycolysis on and off?
How is muscle metabolism affected by type 1 diabetes mellitus?
Intro:
Methods:
Results:
4
Definitions
NMR nuclear magnetic resonance
PCr phosphocreatine (an ATP buffer)
HP hexose phosphates (substrates of glycolysis)
“metabolites” Pi, ADP, AMP (products of ATP hydrolysis)
5
Introduction:
Why study cellular metabolism in muscle?
6
GAPDH
glycogen
GP
PFK
PGK
PK
lactate
PGI
PGM
ALD
PGM
ENO
LDH
Why study cellular metabolism?
Cellular metabolism . . .
• is central to the existence of all cells.
• has important whole-organ and whole-body consequences.
• can be harnessed to spawn advances in medicine and industry.
7
X
GAPDH
glycogen
GP
PFK
PGK
PK
lactate
PGI
PGM
ALD
PGM
ENO
LDH
Why study cellular metabolism?
Cellular metabolism . . .
• is central to the existence of all cells.
• has important whole-organ and whole-body consequences.
• can be harnessed to spawn advances in medicine and industry.
8
X
GAPDH
glycogen
GP
PFK
PGK
PK
lactate
PGI
PGM
ALD
PGM
ENO
LDH
Why study cellular metabolism?
GAPDH
PFK
PGK
PK
ethanol
PGI
HK
ALD
PGM
ENO
PDC
glucose
ADH
Cellular metabolism . . .
• is central to the existence of all cells.
• has important whole-organ and whole-body consequences.
• can be harnessed to spawn advances in medicine and industry.
9
Why study cellular metabolism?
We don’t know . . .
• What are the pathway flux rates in vivo?
• How are these rates up- and downregulated?
GAPDH
glycogen
GP
PFK
PGK
PK
lactate
PGI
PGM
ALD
PGM
ENO
LDH
10
Why study cellular metabolism in muscle?
Advantages of muscle . . .
• huge range of fluxes
• ~40% of human body mass
• critical to physical performance and well-being
• can study muscles specialized for different tasks McArdle et al., Essentials of
exercise physiology, 1994
11
Methods:
How can metabolic fluxes be quantified using phosphorus NMR spectroscopy?
12
The basic idea
Force
[PCr]
Time
13
Simultaneous collection of NMR and force data
NMR coil
force transducer
• in vivo
• noninvasive
14
NMR reveals metabolic changes
10
20
30[PCr](mM)
250 300 350 400 450
Time (s)
• good time resolution
Am
plitu
de
-10-50510
Chemical shift (ppm)
HP
Pi PCr
ATPpH
15
[ATP] remains constant during exercise and recovery
[PCr]
Time
[ATP]
[Pi]
ischemia
exercise
16
PCr keeps [ATP] constant
ADP + PCr + H+ Cr + ATP
ATP ADP + Pi + H+
PCr + H+ Cr + Pi
Therefore changes in [PCr] reflect changes in ATP production/consumption.
17
Quantifying contractile cost
ATP consumption at the start of ischemic exercise is not “contaminated” by glycolytic or oxidative ATP production.
10
15
20
25
30
35
[PCr](mM)
0 50 100
Time (s)
y = -0.244x + 32.508
18
Quantifying oxidative phosphorylation
15
25
35
[PCr] (mM)
0 50 100 150 200
Recovery time (s)
tau
rate constant kPCr = 1/tau
rapid recovery --> high kPCr --> high oxidative capacity
19
Quantifying glycolysis
Recall:
Under ischemic conditions,
glycolytic H+ production = (pH)*() + ()*(PCr)
is the muscle buffer capacity
(Conley et al., Am J Physiol 273: C306, 1997)
PCr + H+ Cr + Pi
20
Results:
What turns glycolysis on and off?
21
Potential controllers of glycolysis
Metabolites
Pi, ADP, and AMP are substrates and allosteric activators of glycolytic enzymes
Calcium
glycolysis stimulation frequency (Conley et al., Am J Physiol 273: C306, 1997)
Hexose phosphates
substrates for glycolysis
+
+
++
+
GAPDH
glycogen
GP
PFK
PGK
PK
lactate
PGI
PGM
ALD
PGM
ENO
LDH
22
Turning off glycolysis after exercise
0
1
2
3
4
[HP](mM)
0 20 40 60
ischemic exercise
ischemic rest
Post-exercise time (s)
0
5
10
15
20
25
30
[Pi]
(mM)
calcium important
Post-exercise time
Gly
coly
tic r
ate
calcium unimportant
23
Evidence of post-exercise glycolysis
• pH falls due to continued lactic acid production
• [PCr] rises due to continued glycolytic ATP production
0
1
2
PCr(mM)
0 15 30 45 60
Post-exercise time (s)
-0.15
-0.1
-0.05
0
pH
0 15 30 45 60
24
Agreement of two estimatesof post-exercise glycolysis
0
1
2
3
4
Glycolytic H+ production (mM)
0 15 30 45 60
Post-exercise time (s)
"pH method""PCr method"
25
Time course of post-exercise glycolysis
The cessation of glycolysis must reflect the decline of a muscle activation-related factor such as calcium.
0
0.05
0.1
0.15
Glycolytic rate
(mM H+/s)
0 20 40 60
Post-exercise time (s)
ischemic exercise
ischemic rest
calcium unimportant
calcium important
26
Turning on glycolysis at the start of exercise
What mechanism is responsible for this delayed onset?
0123456
lactate (mM)
6.95
7
7.05
7.1
7.15
pH
Time
27
Testing the importance of metabolites
2 bouts of exercise
Will glycolysis begin sooner in Bout 2 (when metabolites are high) than in Bout 1 (when metabolites are low)?
10
20
[P i](mM)
0
75
150
[ADP](M)
0 50 100
Time (s)
10
20
30
[PCr](mM)
ischemic exercise
ischemic rest
Bout 1 Bout 2
28
Glycolysis begins earlier in Bout 2 than in Bout 1
0
2
4
6
8
Gly
coly
tic
H+
pro
duct
ion (
mM
)
5 15 25 35 5 15 25 35
Time (s)
5 15 25 35
Bout 1(B1 = 12 s) (B1 = 24 s)
* * *
Bout 2Bout 2
29
The onset of glycolysis coincides with elevated metabolites
aerobic rest onset of flux
[Pi] 3.4-3.8 16.2-20.6
[ADP] 0.013-0.014 0.080-0.121
[AMP] 2x10-5-3x10-5 0.0008-0.0012
(All concentrations in mM.)
30
Summary of glycolysis data
To initiate and sustain high rates of glycolysis, both elevated metabolite levels and a muscle activation-related factor such as calcium are needed.
31
Results (continued):
How is muscle metabolism affected by type 1 diabetes mellitus?
32
Little is known about muscle metabolism in people with type 1 diabetes
Rat models suggest that diabetes-induced changes may be completely reversed by insulin treatment (Ianuzzo et al., J Appl Physiol 52: 1471, 1982; Noble & Ianuzzo, Am J Physiol 249: E360, 1985).
We asked whether careful treatment of type 1 diabetes with insulin restores human muscle metabolism to normal.
33
Subjects
10 men with type 1 diabetes and 10 male age- and activity-matched control subjects
All diabetic subjects used insulin injections to keep blood glucose levels under good clinical control:
• glycosylated hemoglobin (HbA1c) levels 7%
• no glucose in the urine
34
No significant difference in force
10
30
50
70% of max
force
0 50 100
Time (s)
CONTROL
DIABETIC
6.5
6.7
6.9
7.1
pH
0 50 100
Time (s)
10
30
50
70% of max
force
CONTROL
DIABETIC
*
35
Glycolysis occurs “early and often” in diabetic subjects
Earlier onset Higher peak rate
0
1
2
3
4
5
Gly
coly
tic
H+
pro
duct
ion (
mM
)
0 5 10 15 20 25 30
Time (s)
CONTROL DIABETIC
*
*
0
0.1
0.2
0.3
0.4
0.5
Peak
gly
coly
tic
rate
(mM
H+
/s)
CONTROL DIABETIC
*
36
Oxidative recovery rateis slower in diabetic subjects
10
15
20
25
30
[PC
r] (
mM
)
0 25 50 75
Recovery time (s)
CONTROL
DIABETIC
0
0.005
0.01
0.015
0.02
0.025
0.03
kPC
r (1
/s)
CONTROL DIABETIC
*
37
Contractile cost: difference not significant
0
0.25
0.5
0.75
1
1.25
Contractile cost(mM PCr/s)
CONTROL DIABETIC
P = 0.12
38
Summary of diabetes data
Elimination of symptoms of type 1 diabetes does not normalize muscle properties.
The observed abnormalities in glycolytic and oxidative fluxes suggest that diabetes causes a shift in the metabolic profile of the muscle.
39
Future work
40
Acknowledgments
Advisers
Kevin Conley
Marty Kushmerick
UW Departments
Physiology & Biophysics
Radiology
Funding
National Institutes of Health
National Science Foundation
41
Acknowledgments(continued)
Coworkers
Cathy Amara
Iris Asllani
Outi Hyyti
Melissa Lambeth
Donghoon Lee
Dave Marcinek
Ken Marro
Daryl Monear
Brad Moon
Eric Shankland
Rudy Stuppard
Nina VØllestad
Coauthors
Mike Carey
Rod Gronka
Sharon Jubrias
Will Kemper
Jerry Milstein
42
This talk, like glycolysis after exercise, is over.
Recommended