Hyperoxia decreases muscle Hyperoxia decreases muscle glycogenolysis, pyruvate and lactate glycogenolysis, pyruvate and lactate

production and efflux during production and efflux during steady-state exercise.steady-state exercise.

Trent StellingwerffTrent Stellingwerff11, Melanie Hollidge, Melanie Hollidge22, Paul J. LeBlanc, Paul J. LeBlanc33, , George J.F. HeigenhauserGeorge J.F. Heigenhauser22 and Lawrence L. Spriet and Lawrence L. Spriet11

11Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Canada; Canada; 22Department of Medicine, McMaster University, Hamilton, Canada; Department of Medicine, McMaster University, Hamilton, Canada; 33Department of Department of

Physical Education and Kinesiology, Brock University, St. Catharines, Canada. Physical Education and Kinesiology, Brock University, St. Catharines, Canada.

Am J Physiol Endocrinol MetabAm J Physiol Endocrinol Metab 290 (6): E1180-E1190, 2006 290 (6): E1180-E1190, 2006

Effects of hyperoxia during steady-state exerciseEffects of hyperoxia during steady-state exercise

• Numerous studies have reported decreases in blood lactate Numerous studies have reported decreases in blood lactate and decreases in RER during hyperoxia, suggesting that and decreases in RER during hyperoxia, suggesting that muscle metabolism is altered during aerobic exercise by muscle metabolism is altered during aerobic exercise by decreasing reliance on CHO and promoting fat oxidation. decreasing reliance on CHO and promoting fat oxidation. (Welch RG, review, 1987)(Welch RG, review, 1987)

• • However, exercise measurements of whole-body OHowever, exercise measurements of whole-body O22 uptake uptake

during hyperoxia are technically difficult and can lead to during hyperoxia are technically difficult and can lead to overestimates of VOoverestimates of VO22 and artificially low RER. and artificially low RER. (Welch RG, review, 1987)(Welch RG, review, 1987)

•• Total paucity of muscle measurements involving steady-state Total paucity of muscle measurements involving steady-state exercise (>5min) and hyperoxia:exercise (>5min) and hyperoxia:

- Graham - Graham et al.et al. showed decrease muscle lactate and no change showed decrease muscle lactate and no change in glycogen utilization over 40 min of cycling exercisein glycogen utilization over 40 min of cycling exercise (Graham et al. (Graham et al. J Appl PhysiolJ Appl Physiol 63 (4): 1457-1462, 1987). 63 (4): 1457-1462, 1987).

Major findings of previous hyperoxia study Major findings of previous hyperoxia study Stellingwerff et al. J Appl Physiol 98: 250-256, 2005

• Conversely, we found that Conversely, we found that hyperoxic breathing reduced the hyperoxic breathing reduced the breakdown of glycogen over 15 min of steady-date cyclingbreakdown of glycogen over 15 min of steady-date cycling..

• • There was no effect of hyperoxia on carbohydrate oxidation There was no effect of hyperoxia on carbohydrate oxidation (as estimated from PDH activation) as compared to normoxia.(as estimated from PDH activation) as compared to normoxia.

•• The mechanism(s) responsible for the decrease in muscle The mechanism(s) responsible for the decrease in muscle glycogenolysis and blood lactate during hyperoxia are still not glycogenolysis and blood lactate during hyperoxia are still not clear.clear.

• • Few studies have examined effects of hyperoxia using arterial Few studies have examined effects of hyperoxia using arterial and venous (a-v) blood sampling. But, primary findings are tand venous (a-v) blood sampling. But, primary findings are that hat hyperoxia has no effect on lactate efflux, suggesting lactate hyperoxia has no effect on lactate efflux, suggesting lactate production is decreased production is decreased (Knight (Knight et al. J Appl Physiolet al. J Appl Physiol 81: 246-251, 1996; 81: 246-251, 1996;Mourtzakis Mourtzakis et al.et al. J Appl PhysiolJ Appl Physiol 97: 1796-1802, 2004; Pedersen 97: 1796-1802, 2004; Pedersen et al. Acta Physiol Scandet al. Acta Physiol Scand 166: 309-318, 166: 309-318, 2004).2004).

PurposePurpose

I. To determine if the decreased muscle and blood lactate that isI. To determine if the decreased muscle and blood lactate that is normally found with hyperoxic vs.normoxic breathing is due to:normally found with hyperoxic vs.normoxic breathing is due to:

1) a decreased glycogenolysis leading to decreased 1) a decreased glycogenolysis leading to decreased muscle pyruvate and lactate production muscle pyruvate and lactate production

AND/ORAND/OR

2) decreased pyruvate and lactate efflux.2) decreased pyruvate and lactate efflux.

II. To elucidate the mechanisms behind the decreasedII. To elucidate the mechanisms behind the decreased glycogenolysis and decreased blood lactate. glycogenolysis and decreased blood lactate.

Through the use of a-v line and blood flow methodology, Through the use of a-v line and blood flow methodology, coupled with muscle biopsy sampling, this study had coupled with muscle biopsy sampling, this study had 2 primary aims:2 primary aims:

We accomplished this by measuring glycogenolysis and the 5 major fates of pyruvate:

PyruvatePyruvate11

PyruvatePyruvate

LactateLactate33

G-6-PG-6-P

GlycogenGlycogen

Aceytl-CoAAceytl-CoA

PyruvatePyruvate55

LactateLactate44

GlucoseGlucose

Efflux

Efflux PDH 2

During 40 min of cycling at 70% VO2 peak while subjects breathed either 21 or 60% O2.

HypothesisHypothesis

I. Confirm our previous findings of a decreased muscle I. Confirm our previous findings of a decreased muscle glycogenolysis, and no change in pyruvate oxidation via PDH.glycogenolysis, and no change in pyruvate oxidation via PDH.

We hypothesized that hyperoxia would:We hypothesized that hyperoxia would:

II. Which would result in decreased muscle pyruvate and II. Which would result in decreased muscle pyruvate and lactate production,lactate production,

III. Leading to decreased muscle pyruvate and lactate III. Leading to decreased muscle pyruvate and lactate release.release.

IV.IV. Mechanisms would be mediated through attenuated Mechanisms would be mediated through attenuated accumulations of ADPaccumulations of ADPff and AMP and AMPf f and epinephrine during and epinephrine during

exercise.exercise.

-20min-20min -10min 0min 5min 10min 20min 30min 40min -10min 0min 5min 10min 20min 30min 40min

BIO#1 BIO#2 BIO#3 BIO#4BIO#1 BIO#2 BIO#3 BIO#4

BLD#1 BLD#2 BLD#3 BLD#4 BLD#5 BLD#6 BLD#7BLD#1 BLD#2 BLD#3 BLD#4 BLD#5 BLD#6 BLD#7

20min of resting breathing at 40min of cycling at 70% VO20min of resting breathing at 40min of cycling at 70% VO 22max with continued max with continued

either 21% or 60% inspired Oeither 21% or 60% inspired O22 21% or 60% inspired O 21% or 60% inspired O 22

RESTINGRESTING EXERCISE EXERCISE

Exercise and Sampling ProtocolExercise and Sampling Protocol

Muscle Biopsy SampleMuscle Biopsy Samplea-v difference blood draws with blood flow measurements (avg. of 3 per time point)a-v difference blood draws with blood flow measurements (avg. of 3 per time point)

7 active males7 active malesAge: 22.3 Age: 22.3 1.2 yr, 1.2 yr, Weight: 76.1 Weight: 76.1 4.3 kg, and 4.3 kg, andVOVO2peak2peak: 52.8 : 52.8 3.0 ml · kg-1 · min-1 3.0 ml · kg-1 · min-1

No change in leg ONo change in leg O22 delivery (CaO delivery (CaO22 x leg blood flow) x leg blood flow)

~7% increase in CaO~7% increase in CaO22 with hyperoxia (21.1 with hyperoxia (21.1 ± 0.9 vs. 19.6 ± 0.9 vs. 19.6 ± 0.9 ml/dl)± 0.9 ml/dl)

~8% dec.~8% dec.

Net glycogen breakdown

Gly

coge

n (

umol

es g

lyco

syl u

nits

· g

-1 d

ry w

t)

0

100

200

300

400

21% O2

60% O2

†

Decreased muscle glycogenolysis with hyperoxia Decreased muscle glycogenolysis with hyperoxia

No change in PDH activation between trials No change in PDH activation between trials

Time (min)

0 10 20 30 40

PD

Ha

(mm

ol ·

kg w

.w.-1

· m

in-1

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

21% O2

60% O2

Decreased pyruvate production during hyperoxiaDecreased pyruvate production during hyperoxia

†

† †

†

†

†

900

750

600

450

300

150

0

0-10 min 10-20 min 20-40 min 0-40 min 0-40 min

Ra

te o

f p

yru

vate

pro

duc

tion

(m

mol

es ·

min

-1 ·

leg-1

)

0

5

10

15

20

25

30 Lactate Accum.Lactate EffluxPDHa fluxPyruvate Efflux

21% 60% 21% 60% 21% 60% 21% 60% 21% 60%

Tota

l pyru

vate

pro

ductio

n o

ver 40

min

(mm

ole)

†

†

†

Decreased total pyruvate production during hyperoxiaDecreased total pyruvate production during hyperoxia

Lactate Accum.Lactate Accum.

Lactate EffluxLactate Efflux

PDHa FluxPDHa Flux

Pyruvate EffluxPyruvate Efflux

Note: Pyruvate Accum.Note: Pyruvate Accum. (negligible) (negligible)

Tot

al p

yruv

ate

prod

uctio

n ov

er 4

0 m

inT

otal

pyr

uvat

e pr

oduc

tion

over

40

min

(mm

oles

)(m

mol

es)

21% 60%21% 60%

00

150150

300300

450450

600600

750750

900900††

††

870.1870.1

734.8734.8

4.6%4.6%

69.4%69.4%

21.8%21.8%

4.2%4.2%

4.0%4.0%

82.4%82.4%

10.0%10.0%3.6%3.6%

Decreased rate of lactate production during hyperoxia Decreased rate of lactate production during hyperoxia

Decreased arterial lactate during hyperoxiaDecreased arterial lactate during hyperoxia

High Energy PhosphatesHigh Energy Phosphates

• Increased oxidative phosphorylation potentialIncreased oxidative phosphorylation potential

during hyperoxia during hyperoxia

- PCr utilization- PCr utilization- ADPADPf f accumulationaccumulation- AMPAMPf f accumulationaccumulation

Suggests decreased energy supply by Suggests decreased energy supply by substrate level phosphorylationsubstrate level phosphorylation

Decreased epi concentrations during hyperoxiaDecreased epi concentrations during hyperoxia

PyruvatePyruvate11

PyruvatePyruvate

LactateLactate33

G-6-PG-6-P

GlycogenGlycogen

Aceytl-CoAAceytl-CoA

PyruvatePyruvate55

LactateLactate44

GlucoseGlucose

Efflux

EffluxPDH

2

Overview of skeletal muscle regulation during hyperoxiaOverview of skeletal muscle regulation during hyperoxia

==

==

hyperoxia vs. normoxiahyperoxia vs. normoxia(mmol/min/leg)(mmol/min/leg)

16%16%

15%15%

56%56%

27%27%

62%62%

Hyperoxia resulted in Hyperoxia resulted in ““tighter” metabolic tighter” metabolic control between control between glycogenolysisglycogenolysisand CHO oxid. (PDHa)- and CHO oxid. (PDHa)- Similar to that found followingSimilar to that found followingeven short-term even short-term endurance training endurance training

Production

Production

FFA-FABP

Pyruvate Lactate

CAT

CPT-I

CPT-1I

PDH

fattyacyl-CoA

acetyl-CoA-oxidation

OMIM

matrixmatrix

Oxidative ATP ProvisionOxidative ATP Provision

NADH O2 ADP + Pi

GlycogenGlycogen

G-6-PG-6-P G-1-PG-1-P

GlycogenolysisGlycogenolysis

++

(O(O22 delivery) delivery)

(PiO(PiO22))

NC

+

??

TCA Cycle

??

NC

NC

bloodblood

cytosolcytosol

PMPM

EpiEpi

++

Lactate

ATPATP ADPADP

PCrPCrCrCr

ATPATP ADPADP

ATPATP ADPADP

ACKNOWLEDGEMENTSACKNOWLEDGEMENTS

LABMATES at GuelphLABMATES at Guelph

Advisor: Lawrence SprietAdvisor: Lawrence Spriet

Veronic BezaireVeronic BezaireRebecca TunstallRebecca Tunstall

Kerry MullenKerry MullenKatie JunkinKatie Junkin

Jason TalanianJason TalanianJane RutherfordJane Rutherford

Graham HollowayGraham HollowayClinton BruceClinton BruceChris PerryChris Perry

Brianne ThrushBrianne ThrushAngela SmithAngela Smith

ACKNOWLEDGEMENTSACKNOWLEDGEMENTS

LABMATES in MaastrichtLABMATES in Maastricht Advisor: Luc van LoonAdvisor: Luc van Loon

Milou Beelen, Hanneke Boon, Richard Jonkers, Rene Koopman,Milou Beelen, Hanneke Boon, Richard Jonkers, Rene Koopman,Ralph Manders, Bart Pennings, Joan Senden, Ralph Manders, Bart Pennings, Joan Senden, Kristof Vanschoonbeek, Lex Verdijk, Antoine ZornecKristof Vanschoonbeek, Lex Verdijk, Antoine Zornec

Hyperoxia Study II: Overall Study OutlineHyperoxia Study II: Overall Study Outline

Mixer(Delivers 21 or 60% O2

to the subject)

100% O100% O22 from wall from wall

21% O21% O22 OR OR 60% O60% O22

500 LTissot

(Mixed O2

Storage Vesicle)Inspired OInspired O22

BIOPSIESBIOPSIES

BLOODBLOOD

Subject on BikeSubject on Bike

Blood Flow MeasurmentsBlood Flow Measurments(avg of 3, via thermodilution)(avg of 3, via thermodilution)

Arterial SamplingArterial Sampling

Venous SamplingVenous Sampling

cytosol

FFA-FABP

Pyruvate Lactate

CAT

CPT-I

CPT-1I

PDH

fattyacyl-CoA

acetyl-CoA-oxidation

OMIM

matrix

ATP ADP

PCrCr

ATP ADP

ATP ADP

Oxidative ATP ProvisionOxidative ATP Provision

NADH O2 ADP + Pi

+

Regulation of Oxidative PhosphorylationRegulation of Oxidative Phosphorylation

3 ADP + 3 Pi + NADH + ½ O3 ADP + 3 Pi + NADH + ½ O22 + H + H++ → 3 ATP + NAD → 3 ATP + NAD++ + H + H22OO

TCA cycle

(DF Wilson)(DF Wilson)

Regulation of Substrate PhosphorylationRegulation of Substrate Phosphorylation

During exercise situations with increasing intensity, when ATP production During exercise situations with increasing intensity, when ATP production from oxidative phosphorylation cannot match the rate of ATP hydrolysis, from oxidative phosphorylation cannot match the rate of ATP hydrolysis, the shortfall in oxidative energy supply is made up by substrate phosphorylationthe shortfall in oxidative energy supply is made up by substrate phosphorylation

2. and the metabolism of glycogen with lactate formation:2. and the metabolism of glycogen with lactate formation:

Glycogen + 3 ADP + 3 Pi → 3 ATP + 2 lactate + 2 HGlycogen + 3 ADP + 3 Pi → 3 ATP + 2 lactate + 2 H++

1. PCr utilization in the creatine kinase reaction:1. PCr utilization in the creatine kinase reaction:

PCr + ADP + HPCr + ADP + H++ ATP + Cr ATP + Cr

GlucoseFFA-ALB

Glycogen

G-6-P G-1-P

bloodcytosol

FFA-FABPTG

Pyruvate Lactate

GlucosePHOSHK

fattyacyl-CoA

CAT

CPT-I

CPT-1I

PDH

fattyacyl-CoA

acetyl-CoA-oxidation

TCAcycleCO2

NAD

NADH

NAD

NADH

NADH

NADH

NAD

NADETC

O2H20

H+ H+

H+

ATP ADP

PCrCr

ATP ADP

NAD

NADH

PFK

LDH

ATP ADP

PM

OMIM

matrix

LIPASE

ATP

Lactate