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
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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
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