Metabolic aspects of human exercise performance at ......Maximal pulmonary ventilation (from Pugh et al., 1964) VO 2 OP LQ-1) 0 1 2 3 4 V E S Q-1) 0 50 100 150 200 REST. 0 m 5800 m

Metabolic aspects of human exercise

performance at altitude. A holistic approach.

Paolo CerretelliIstituto di Bioimmagini e Fisiologia Molecolare

CNR – Segrate (Mi)

Rovereto, November 12, 2015

The effects of Hypoxia on physical performance

have been assessed as functions of:

a) Exposure duration < 10 days : acute and subacute

1-3 months : subchronic and chronic

> 12 months : partial adaptation

From birth : full adaptation

b) Metabolic level Resting

Submaximal workload

Maximal workload (VO2max)

Supramaximal workload (>VO2max up to “peak”)

at the integrative, at the cellular and at the molecular level

..

A) The integrative level

A1. Maximal and submaximal

aerobic performance

Maximal pulmonary ventilation

(from Pugh et al., 1964)

VO2 (l·min

-1)

0 1 2 3 4

VE

BT

PS (

l·m

in-1

)

0

50

100

150

200

REST

. 0 m

5800 m

(380 torr)

7440 m

(300 torr)

.

T

Maximal Heart Rate

0 1 2 3 4 5 6 7 8110

120

130

140

150

160

170

180

190

200

210

Untrained lowlanders

Trained lowlanders

Skyrunners

Tibetans 2nd

Elite climbers

A. D. P.

B. C.

Altitude (km)

HR

max (

b·m

in-1

)

Acute Hypoxia

Hb concentration as a function of altitude

0 1 2 3 4 5 6 7 810

12

14

16

18

20

22Lowlanders Skyrunners Tibetans 2ndElite climbers

Altitude (km)

Hb

(g%

)

0

20

40

60

80

100

0 5050 7600

Lowlanders

Skyrunners

Sherpas

Tib.2nd

% Hb oxygen saturation at exhaustion %

Hb

O2

Altitude (m)

Cardiac output in élite climbers

rest max exercise

0

5

10

15

20

25

30

350m

5050m

Q (

l·m

in-1

)

.

.

Altitude (km)

0 1 2 3 4 5 6 7 8 9

VO

2m

ax

( %

s.l

.)

0

20

40

60

80

100

Acute hypoxia

Chronic hypoxia

VO2max as a function of altitude.

(From Cerretelli and Hoppeler, Handbook of Physiology, APS, 1996)

(from Cerretelli, J.Appl.Physiol., 1976)

Effects of rapid reoxygenation on VO2max of

acclimatized Caucasians at Mt Everest base

camp (5450 m)

.

120

100

80

60760 600 400 760 600 400 760 600 400

PB (torr)

Chronic

hypoxia

Air Air

O2

O2

% h

r ma

x

(%Q

ma

x)

%V

O2m

ax

.

%V

O2m

ax

.

.*

VO2max as a function of altitude:

training and ethnic variability

.

92

83

74

60

53

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6

Sea level

VO

2m

ax (

% s

.l.)

Tibetans 2nd

Sherpas

Skyrunners

Caucasians 11days

Caucasians 31days

.

0 2 4 6 8 1010

20

30

40

50

60

70

80

90

100

Altitude (km)

DVO2max at 5050 m. as a function of initial

maximal aerobic power

.

Normoxic VO2max (ml·kg-1·min-1)

20 30 40 50 60 70 80

DV

O2m

ax

(%

)

-50

-40

-30

-20

-10

0

Untrained lowlandersTrained lowlanders

Tibetans

Sherpas

.

.

(From Marconi et al., J.Physiol. (London), 2004)

(from Marconi et al., 2006)

Evolution of VO2max during prolonged

altitude exposure

.

VO2 max in élite himalayan climbers

30

40

50

60

70

80

90

10 20 30 40 50 60

Age(yr)

VO

2m

x (

ml/

kg

.min

)

Elite

marathon

runners

Untrained

population

Professional alpine

guides

.

.

(from Cerretelli, J.Appl.Physiol., 1976)

Effects of rapid reoxygenation on VO2max of

acclimatized Caucasians at Mt Everest base

camp (5450 m)

.

120

100

80

60760 600 400 760 600 400 760 600 400

PB (torr)

Chronic

hypoxia

Air Air

O2

O2

% h

r ma

x

(%Q

ma

x)

%V

O2m

ax

.

%V

O2m

ax

.

.*

walking 6 km h-1

0 5 10 15 20

20

25

30

35Tibetan migrants (1300 m)Nepali (1300 m)Tibetans (4300 m)

.

Italians (122 m)

slope (%)

net

VO

2 (

ml

kg

-1 m

in-1

)

Walking economy

(from Marconi et al., in preparation)

*

(from Cerretelli, High Altitude Medicine & Biology., 2009)

Conclusions A 1

VO2max decreases as a parabolic function of altitude.The rate of decrease is surprisingly very similar in acute and chronic hypoxia. Peripheral factors, possibly at the muscle level, appear to play a major role in chronic conditions.

Altitude natives are characterized by higher VO2max than acclimatized lowlanders at any given altitude. However, altitude exposure for over two years tends to reduce the gap.

There is a large scatter among various groups in the drop of VO2max as a function of altitude.

Within any given ethnic group, individuals with greater maximum aerobic power undergo at 5050 m. a larger drop of VO2max.

Elite himalayan climbers are not characterized by particularly high VO2max absolute levels.

Sudden reoxygenation does not allow to resume initial normoxic VO2max.

Walking economy is greater in altitude natives thanks to higherefficiency of oxidative phosphorylation

.

.

.

.

.

.

*

*

A 2. Maximal anaerobic performance

Extreme Altitude Survival Test 1 and 2

(1994-1997)

Mt Everest advanced base camp

(6400 m)

Altitude (km)

0 1 2 3 4 5 6 7 8 9

[La] b

pea

k (

mM

)

0

3

6

9

12

15

HA Tibetan refugees

Operation Everest II

Caucasian lowlanders

Altitude natives

EAST 1997

Caucasian lowlanders

(personal observation, 1994)

Sherpas

(personal observation, 1994)

2nd generation Tibetans

acute hypoxia

La[max] as a function of altitude

Arterial lactate concentration and vastus lateralis

lactate content: denial of the “lactate paradox”

from Van Hall et al., J Physiol (London), 2009

LDH activity in muscle in acute and

chronic hypoxia

from Van Hall et al., J Physiol (London), 2009

Conclusion A 2

Is there a “lactate paradox”?

The data of the preceding figure are the main basis of the so-called “lactate

paradox”, i.e. the apparent decrease of the subject’s ” maximal glycolytic

capacity” in acclimatized lowlanders and altitude natives.

The above definition has been recently challenged since it is based

on blood lactate data. In fact, muscle lactate determinations do not evidence

impairment of anaerobic glycolysis in altitude adapted individuals:

whence the recent contention by Van Hall et al.(2009) that the lactateparadox does not exist. The discrepancy between muscle and blood lactatelevels at exhaustion could be the consequence of an impaired function of thelactate transporters in the sarcolemma .

*

B) The cellular and subcellular

level

0

10

20

30

40

50

60

70

I IIA IIB

Fibre types

Fib

res (

%)

Controls

Nepali

Tibetans

Bolivians

Fiber types distribution

Morphometry and Enzymes in muscle after the 1986

Swiss Mt. Everest expedition (n=7subjects)

VARIABLE % change

Muscle mass -11

Fiber diameter -15

(central) -55

-26

-18

Mitochondrial volume density (total)

(sub-sarcolemmal)

HK -8

PFK +6

LDH 0

CS

(citric acid cycle)-23

MDH -20

CYTOX

(respiratory chain)-23

HADH

(beta-oxidation of fatty acids)-27

HBDH

(utilization of ketone bodies)-27

0

2

4

6

8

10

C UM AM EC Sh Bo Ma Ne Ti

%

from Cerretelli, Textbook of Exercise Physiology, SEU, Roma 2001

Mitochondrial volume density in various

altitude and sea level populations

Conclusion B

Muscle fiber types distribution is the same in altitude natives and in lowlanders and is independent of ethnicity.

Oxidative enzymes activity is reduced in acclimatized subjects.

Mitochondrial volume density is low in altitude natives, independent of their ethnic background. In Caucasians, it undergoes reduction in the course of acclimatization.

C) The molecular level

C1) The role of the Hypoxia Inducible

Factor ( HIF-1)

The interpretation of most functional responses of metazoan organisms to

decreased oxygen partial pressure is supported and implemented by the

discovery of a number of adaptive mechanisms for oxygen sensing and

signal transduction promoted by a protein, the Hypoxia Inducible Factor

(HIF-1). HIF-1, a dimer a and β, is expressed in all cell types and has

been identified in all species suggesting that its appearance represented an

adaptation essential to metazoan evolution. HIF-1 is a transcription

factor regulating the expression of hundreds of genes in response to

changes in oxygen availability. The HIF-1α subunit of the dimer is

continuously synthesized and is eliminated by proteasomal

degradation under well oxygenated conditions

HIF-1 : a Master Regulator of oxygen

homeostasis

Regulates erythropoiesis (EPO) and vascularization(VEGF).

Activates transcription of genes encoding glucosetransporters and glycolytic enzymes.

Activates transcription of the PDK 1 gene shuntingpyruvate away from mitochondria.

Represses mitochondrial biogenesis and respiration thuspreventing increased levels of reactive oxygen speciesand consequent cell dysfunction.

Increases mitochondrial autophagy

Coordinates a switch in the composition of cytochrome coxidase (COX) increasing the efficiency of the latterunder hypoxic conditions.

Oxygen sensing, gene expression, and

adaptive responses to hypoxia

From Semenza, 2011

well oxygenated hypoxia

Regulation of glucose metabolism in response

to changes in cellular oxygen levels

From Semenza, 2011

V.L. enzyme profiles after progressive increase

of altitude exposure

(18 days) (66 days)

Hypoxia and reactive oxygen species (ROS) prevent proteasomal degradation of HIF-1α, resulting in increased levels of HIF-1 (see h).

The latter regulates transcription of genes enhancing a number of metabolic adaptations: a) a switch from COX4-1 to COX4-2 subunit,

thereby increasing the efficiency of oxidative phosphorylation (the latter may depend also on the complex interaction among myoglobin,

nitric oxide, and COX, see i);

b) inactivation of pyruvate dehydrogenase (PDH), induced by PDK1 (a gene expressing PDH kinase);

c) inhibition of mitochondrial biogenesis;

d) increased mitochondrial autophagy;

e) activated transcription of genes encoding glucose transporter GLUT 1;

f) activated transcription of genes encoding plasma membrane lactate transporter 4 (MCT4);

g) increased activity of lactate dehydrogenase (LDH).

IMM and OMM refer to the inner and outer mitochondrial membrane, respectively; FIH 1 is a factor inhibiting HIF-1; BNIP3 is a cell

death-related gene; Bcl2 and Beclin 1 are proteins involved in the regulation of macroautophagy; C-MYC is a transcription factor

promoting mitochondrial biogenesis; MXI-1 is a gene competing with C-MYC; PCG-1β is a transcription factor involved in

mitochondrial biogenesis; LON gene encodes a protease required for the degradation of the subunit COX4-1; Fo and F1 ATPase are the

rotary motors driving ATP synthase; NO is nitric oxide; CoQ is Coenzyme Q10, an electron carrier in the mitochondrial respiratory

chain. (For more details, see Semenza , 2007; Zhang et al, 2007 and 2008).

N.B. Role of

IGF1/AKT/mTOR

FoxO signaling

Ubiquitin/proteasome

Regulation of glucose metabolism in response

to changes in cellular oxygen levels

From Semenza, 2011

The regulation of energy metabolism in hypoxia (modified from Semenza, 2009)

PDK1

PDH

*

5

ROS

*BNIP3

Beclin1

Mitochondrial

Autophagy

COX4-2

COX4-1

*

PM

OMM

GLUT1

*Lactateext

Glucoseext* MCT4

Mitochondrial

Biogenesis

PDK-1

PDH

*

5

ROS

*BNIP3

Beclin1

Mitochondrial

Autophagy

COX4-2

COX4-1

*

PM

OMM

GLUT1

*Lactateext

Glucoseext* MCT4

Mitochondrial

Biogenesis

PGC-1*

PGC- α/β

C-MYC

Schematic representation of proteomic results of anaerobic (alactacid and

glycolytic) metabolisms in vastus lateralis muscle.

(from Levett et al., Proteomics 2015)

CKM, creatine kinase

PYGM, glycogen phosphorylase

PGM1, phosphoglucomutase

ALDOA, bisphosphate aldolase A

TPI1, triosephosphate isomerase

GAPDH, glyceraldehyde-3-phosp dehyd

PGK1, phosphoglycerate kinase 1

ENO3, beta-enolase

PKM2, pyruvate kinase

LDHA, lactate dehydrogenase A

Fructose-6-phosphate

Glycogen

Glucose-6-phosphateGlucose-1-phosphate

ALDOA

GAPDH

TPI1

PGK1

PGAM2

ENO3

PKM2

LDHA

PGM1

PYGM

Fructose-1,6-bisphosphate

1,3-Diphosphoglycerate

3-Phosphoglycerate

2-Phosphoglycerate

Phosphoenolpyruvate

PyruvateLactate

Glyceraldehyde-3-

phosphate

Dihydroxyacetone

phosphate

Anaerobic glycolytic metabolism

creatine

CKM

NAD

NADH + H

NADHNAD

ADP ATP

ATPADP

+

+

+

Anaerobic alactacid metabolism

phosphocreatine

A

% of spot volume variation > 30

% of spot volume variation > 20 < 30

% of spot volume variation < 20

Legend

EBC

BEBC

BEBCAEBC

AEBC BEBC

AEBC

AEBC

AEBC

AEBC

AEBC

AEBC

AEBC

BEBC

BEBC

BEBC

BEBC

LDHB

Group A: Base Camp laboratory staff

n = 5, two females, three males)

sojourning at EBC for the duration of the

expedition

Group B: climbers

n = 6, males

who ascended higher on Mount Everest

Schematic representation of proteomic results of aerobic metabolisms in vastus

lateralis muscle.

(from Levett et al., Proteomics 2015)

Pyruvate

Acetyl-CoA

Pyruvate

dehydrogenase

complex

Citrate

a-Ketoglutaratedehydrogenasecomplex

OGDH

IDH2

DLDSDHA

Isocitrate

Succinate

Fumarate

Malate

Succinyl-CoA

a-Ketoglutarate

Oxaloacetate

TCA

CYCLE

Mito

chon

drial m

atrix

NAD

NADH + H+

NADH

dehydrogenase

Succinate

dehydrogenase

Cytochrome c oxidase

Cytochrome

c reductase

ATP synthase

Complex I

Complex II

Complex III

Complex IV

Complex V

UQ

Cyt c1

Cyt c

Cyt b

Cytos

ol

UQ

ACADVL

ECI1

ACADS

Acyl-CoA (short e medium chain)

Acyl-CoA (long chain)

2-Enoyl-CoA (long chain)

2-Enoyl-CoA

Fatty acidβ-OXIDATION

SDHA

UQCRC1

FAD

FADH2

NAD

NADH + HNAD

NADH + H

NAD

NADH + H

FAD

FADH2

NAD

NADH + H

+

+

+

+

+

+

+

+ DLD

Malate

Oxaloacetate

Malate

Oxaloacetate

NAD

NADH + H

+

NADH + H

+

+

NAD+

MDH1 A

NADP+

NADPH

+

EBC

AEBC

AEBC BEBC

BEBC

BEBC

AEBC

AEBC

BEBC

BEBC

BEBC

BEBC

BEBC

BEBC

MDH1, cytosolic malate dehyd

DLD, dihydrolipoyl dehyd

ACADVL, very long-chain acyl-CoA

ACADS, short-chain acyl-CoA dehy

ECI1, 3,2-transenoyl-CoA isomerase

IDH2, isocitrate dehydrogenase 2

OGDH, 2-oxoglutarate) dehyd

SDHA, succinate dehydrogenase

UQCRC1, cytochrome b-c1 complex

Group A: Base Camp laboratory staff

n = 5, two females, three males)

sojourning at EBC for the duration of the

expedition

Group B: climbers

n = 6, males

who ascended higher on Mount Everest

Schematic representation of α-ketoglutarate metabolic pathway

PDH2, prolyl hydroxylase 2

FASN, fatty acid synthase

IDH1, isocitrate dehydrogenase 1

GLSN, glutamine synthetase

GSS, glutathione synthetase

↑, increase

↓, decrease

=, absence of variation

(from Levett et al.,

Proteomics 2015)

Group A: Base Camp laboratory staff

n = 5, two females, three males)

sojourning at EBC for the duration of the expedition

ASL, group A sea level

AEBC, group A Everest Base Camp

Group B: climbers

n = 6, males

who ascended higher on Mount Everest

BSL, group B sea level

BEBC, group B Everest Base Camp

Acknowledgments

Dr. Mauro Marzorati & Dr. Claudio Marconi

contributed a great deal of work on Himalayan

natives and acclimatized Caucasians in the

Pyramid laboratory at Lobuche (m.5050), Nepal.

Prof. Cecilia Gelfi and Dott.ssa Manuela Moriggi

developed muscle proteomics in humans and

applied it to high altitude studies.

The proteomic contribution in the

study of man at altitude

The definition of proteome

The proteome is defined by all proteins expressedby the genome in a given space (the cell) at a giventime

Why the proteome?

The proteome is the protein complement of a genome representing its end product.

The proteome is in a highly dynamic state of synthesis and degradation also as a consequence of environmental changes.

The proteome does include also post -translational modifications.

Kda

104

60

40

25

15

4 5 6 7

10

LDH

G3P2

PGM2

Mb

GST-P1

ECHM

NUGM

pH

C2) High altitude Sherpas vs. lowlanders:

“differential proteomics”

0

1

2

3

4

5

GST P1-1 ECH GAPHD

a.u. N Tib I Tib 2

0

0,5

1

1,5

2

2,5

LDH PGA NUGM Mb

a.u.

* * * ** * *

* *

* *

* *

**

**

(Gelfi et al., FASEB J., 2004)

Results