Transcript Metabolic Fates of Muscle Pyruvate Under Different

Hyperoxia decreases muscle
glycogenolysis, pyruvate and lactate
production and efflux during
steady-state exercise.
Trent Stellingwerff1, Melanie Hollidge2, Paul J. LeBlanc3,
George J.F. Heigenhauser2 and Lawrence L. Spriet1
1Department
of Human Health and Nutritional Sciences, University of Guelph, Guelph,
Canada; 2Department of Medicine, McMaster University, Hamilton, Canada; 3Department of
Physical Education and Kinesiology, Brock University, St. Catharines, Canada.
Am J Physiol Endocrinol Metab 290 (6): E1180-E1190, 2006
Effects of hyperoxia during steady-state exercise
• Numerous studies have reported decreases in blood lactate
and decreases in RER during hyperoxia, suggesting that
muscle metabolism is altered during aerobic exercise by
decreasing reliance on CHO and promoting fat oxidation.
(Welch RG, review, 1987)
• However, exercise measurements of whole-body O2 uptake
during hyperoxia are technically difficult and can lead to
overestimates of VO2 and artificially low RER. (Welch RG, review, 1987)
• Total paucity of muscle measurements involving steady-state
exercise (>5min) and hyperoxia:
- Graham et al. showed decrease muscle lactate and no change
in glycogen utilization over 40 min of cycling exercise
(Graham et al. J Appl Physiol 63 (4): 1457-1462, 1987).
Major findings of previous hyperoxia study
Stellingwerff et al. J Appl Physiol 98: 250-256, 2005
• Conversely, we found that hyperoxic breathing reduced the
breakdown of glycogen over 15 min of steady-date cycling.
• There was no effect of hyperoxia on carbohydrate oxidation
(as estimated from PDH activation) as compared to normoxia.
• Few studies have examined effects of hyperoxia using arterial
and venous (a-v) blood sampling. But, primary findings are that
hyperoxia has no effect on lactate efflux, suggesting lactate
production is decreased (Knight et al. J Appl Physiol 81: 246-251, 1996;
Mourtzakis et al. J Appl Physiol 97: 1796-1802, 2004; Pedersen et al. Acta Physiol Scand 166: 309-318, 2004).
• The mechanism(s) responsible for the decrease in muscle
glycogenolysis and blood lactate during hyperoxia are still not
clear.
Purpose
Through the use of a-v line and blood flow methodology,
coupled with muscle biopsy sampling, this study had
2 primary aims:
I. To determine if the decreased muscle and blood lactate that is
normally found with hyperoxic vs.normoxic breathing is due to:
1) a decreased glycogenolysis leading to decreased
muscle pyruvate and lactate production
AND/OR
2) decreased pyruvate and lactate efflux.
II. To elucidate the mechanisms behind the decreased
glycogenolysis and decreased blood lactate.
We accomplished this by measuring glycogenolysis
and the 5 major fates of pyruvate:
Glycogen
Glucose
G-6-P
Pyruvate
1
Pyruvate
5
Pyruvate
PDH
2
Efflux
Aceytl-CoA
4
Lactate
3
Lactate
Efflux
During 40 min of cycling at 70% VO2 peak while subjects
breathed either 21 or 60% O2.
Hypothesis
We hypothesized that hyperoxia would:
I. Confirm our previous findings of a decreased muscle
glycogenolysis, and no change in pyruvate oxidation via PDH.
II. Which would result in decreased muscle pyruvate and
lactate production,
III. Leading to decreased muscle pyruvate and lactate
release.
IV. Mechanisms would be mediated through attenuated
accumulations of ADPf and AMPf and epinephrine during
exercise.
Exercise and Sampling Protocol
RESTING
-20min
BLD#2
-10min
Age:
Weight:
VO2peak:
22.3  1.2 yr,
76.1  4.3 kg, and
52.8  3.0 ml · kg-1 · min-1
EXERCISE
20min of resting breathing at
either 21% or 60% inspired O2
BLD#1
7 active males
0min
BIO#1
40min of cycling at 70% VO2max with continued
21% or 60% inspired O2
BLD#3
5min
BLD#4
BLD#5
10min
20min
BIO#2
BIO#3
BLD#6
30min
Muscle Biopsy Sample
a-v difference blood draws with blood flow measurements (avg. of 3 per time point)
BLD#7
40min
BIO#4
No change in leg O2 delivery (CaO2 x leg blood flow)
~7% increase in CaO2 with hyperoxia (21.1 ± 0.9 vs. 19.6 ± 0.9 ml/dl)
10
-1
Venous Blood Flow (L · min )
*
*
8
*
*
*
*
*
*
~8% dec.
6
4
21% O2
60% O2
2
0
-20
-10
0
10
Time (min)
20
30
40
Decreased muscle glycogenolysis with hyperoxia
†
-1
Glycogen (umoles glycosyl units · g dry wt)
400
300
21% O2
60% O2
200
100
0
Net glycogen breakdown
No change in PDH activation between trials
4.0
-1
PDHa (mmol ·kg w.w. · min )
3.5
-1
3.0
2.5
2.0
1.5
21% O2
60% O2
1.0
0.5
0.0
0
10
20
Time (min)
30
40
Decreased pyruvate production during hyperoxia
30
†
†
Lactate Accum.
Lactate Efflux
PDHa flux
Pyruvate Efflux
Rate of pyruvate production
-1
-1
(mmoles · min · leg )
†
†
750
†
†
20
600
15
450
10
300
†
5
150
†
†
0
0
21% 60%
0-10 min
21% 60%
21% 60%
21% 60%
21% 60%
10-20 min
20-40 min
0-40 min
0-40 min
Total pyruvate production over 40 min
(mmole)
25
900
Pyruvate Efflux
870.1
PDHa Flux
4.6%
750
734.8
Lactate Efflux
Lactate Accum.
4.0%
600
Note: Pyruvate Accum.
(negligible)
69.4%
450
82.4%
300
†
150
21.8%
4.2%
10.0%
3.6%
21% 60%
0
Total pyruvate production over 40 min
(mmoles)
Decreased total pyruvate production during hyperoxia
†
900
Decreased rate of lactate production during hyperoxia
4
21% O2
60% O2
-1
0
Efflux
-2
-4
†
-6
14
21%
12
-8
Lactate Accumulation
Lactate Efflux
-10
†
60%
21%
-12
6
21%
60%
60%
-14
10
0-10 min
8
21%
60%
0
10
20
10-20 min
30
20-40 min
Time (min)
4
40
0-40 min
Rate of lactate production
-1
-1
(mmoles · min · leg )
Lactate Flux (mmol · min )
2
Decreased arterial lactate during hyperoxia
7
Exercise
Arterial lactate (mM)
*†
*†
6
*†
*†
*†
5
21% O2‡
60% O2
4
*
*
*
*
*
3
2
1
0
-20
-10
0
10
Time (min)
20
30
40
High Energy Phosphates
• Increased oxidative phosphorylation potential
during hyperoxia
- PCr utilization
- ADPf accumulation
- AMPf accumulation
Suggests decreased energy supply by
substrate level phosphorylation
Decreased epi concentrations during hyperoxia
6
Exercise
*†
Arterial epinephrine (nM)
5
*†
*†
21% O2
4
*
60% O2
3
*
*
2
*
*
*
*
1
0
-20
-10
0
10
Time (min)
20
30
40
Overview of skeletal muscle regulation during hyperoxia
Hyperoxia resulted in
“tighter” metabolic
control between
glycogenolysis
and CHO oxid. (PDHa)Similar to that found following
even short-term
endurance training
hyperoxia vs. normoxia
(mmol/min/leg)
Glycogen
16%
=
Glucose
5
Pyruvate
27%
Efflux
G-6-P
15%
1
Pyruvate
Pyruvate
Production
PDH
2
=
Aceytl-CoA
4
Lactate
Efflux
3
62%
Lactate
Production
56%
Epi
Lactate
+
blood
Glycogen
Glycogenolysis
G-6-P
PM
cytosol
+
FFA-FABP
NC Pyruvate
OM
IM
G-1-P
Lactate
ATP
ADP
Cr
PCr
ATP
ADP
CPT-I
CAT
NC PDH
CPT-1I
ATP ADP
b-oxidation
matrix
fatty
acyl-CoA
acetyl-CoA
+
?
? NADH
TCA Cycle
Oxidative ATP Provision
delivery)
O2 (O
(PiO )
2
2
NC
ADP + Pi
ACKNOWLEDGEMENTS
LABMATES at Guelph
Advisor: Lawrence Spriet
Veronic Bezaire
Rebecca Tunstall
Kerry Mullen
Katie Junkin
Jason Talanian
Jane Rutherford
Graham Holloway
Clinton Bruce
Chris Perry
Brianne Thrush
Angela Smith
ACKNOWLEDGEMENTS
LABMATES in Maastricht
Advisor: Luc van Loon
Milou Beelen, Hanneke Boon, Richard Jonkers, Rene Koopman,
Ralph Manders, Bart Pennings, Joan Senden,
Kristof Vanschoonbeek, Lex Verdijk, Antoine Zornec
Hyperoxia Study II: Overall Study Outline
100% O2 from wall
BIOPSIES
Mixer
(Delivers 21 or 60% O2
to the subject)
21% O2
OR
60% O2
Subject on Bike
Arterial Sampling
500 L
Tissot
(Mixed O2
Storage Vesicle)
BLOOD
Venous Sampling
Inspired O2
Blood Flow Measurments
(avg of 3, via thermodilution)
Regulation of Oxidative Phosphorylation
3 ADP + 3 Pi + NADH + ½ O2 + H+ → 3 ATP + NAD+ + H2O
ATP
FFA-FABP
cytosol
OM
IM
Pyruvate
Cr
PCr
ATP
ADP
CPT-I
PDH
CAT
CPT-1I
b-oxidation
fatty
acyl-CoA
+
NADH
(DF Wilson)
Lactate
ADP
TCA cycle
ATP ADP
matrix
acetyl-CoA
Oxidative ATP Provision
O2
ADP + Pi
Regulation of Substrate Phosphorylation
During exercise situations with increasing intensity, when ATP production
from oxidative phosphorylation cannot match the rate of ATP hydrolysis,
the shortfall in oxidative energy supply is made up by substrate phosphorylation
1. PCr utilization in the creatine kinase reaction:
PCr + ADP + H+
ATP + Cr
2. and the metabolism of glycogen with lactate formation:
Glycogen + 3 ADP + 3 Pi → 3 ATP + 2 lactate + 2 H+
FFA-ALB
blood
cytosol
PM
Glucose
HK
LIPASE
TG
Lactate
Glucose
FFA-FABP
G-6-P
PFK
Glycogen
PHOS
G-1-P
ATP
NAD
fatty
acyl-CoA
ATP
NADH NAD
NADH
Pyruvate
OM
IM
matrix
Lactate
ADP
Cr
PCr
ATP
ADP
LDH
CPT-I
CAT
PDH
CPT-1I
b-oxidation
fatty
acyl-CoA NAD
NAD
ATP ADP
NADH
H+
acetyl-CoA
NAD
NADH
CO2
TCA
cycle
NADH
E
T
C
H+
H20
H+
O2