Altitude acclimatization and energy metabolic adaptations in skeletal muscle during exercise H. J. GREEN, J. R. SUTTON, E. E. WOLFEL, J. T. REEVES, G. E. BUTTERFIELD, AND G. A. BROOKS Department of Kinesiolugy, University of Waterloo, Waterloo, Ontario NZL 3Gl, Canada; Department of Biological Sciences, Cumberland College of Health Sciences, Lidcombe, New South Wales, Australia; Cardiovascular Pulmonary Research Laboratory and Division of Cardiology, University of Colorado, Medical Sciences Center, Denver, Colorudo 80262; Palo Alto Veterans Administration Medical Center and Stanford University, Palo Alto 94304; and University of California, Berkeley, California 94720

GREEN,H.J.,J. G. E. BUTTERFIELD,

R. SUTTON, E.E. WOLFEL, J. T. REEVES, AND G. A. BROOKS. Altitude a~clhatiza-

tion and energy metabolic adaptations

in skeletul muscle during

exercise.J. Appl. Physiol. 73(6): 2701-2708, 1992.-To determine whether the working muscleis able to sustain ATP homeostasisduring a hypoxic insult and the mechanismsassociated with energy metabolic adaptations during the acclimatization process,seven male subjects [23 t 2 (SE) yr, 72.2 of 1.6 kg] were given a prolonged exercise challenge (45 min) at sea level (SL), within 4 h after ascent to an altitude of 4,300 m (acute hypoxia, AH), and after 3 wk of sustainedresidenceat 4,300m (chronic hypoxia, CH). The prolonged cycle test conducted at the sameabsoluteintensity and representing51 t 1% of SL maximal aerobic power (HO,,,,) and between 64 t 2 (AH) and 66 t 1% (CH) at altitude was performed without a reduction in ATP concentration in the working vastus lateralis regardless of condition. Compared with rest, exercise performed during AH resulted in a greater increase (P < 0.05) in musclelactate concentration (5.11t 0.68 to 22.3t 6.1 mmoVkg dry wt) than exercise performed either at SL (5.88 t 0.85 to 11.5 -t 3.1) or CH (5.99 t 0.88to 12.4 ~fr2.1). These differences in lactate concentration have been shownto reflect differences in arterial lactate concentration and glycolysis (Brooks et al. J. Appl. PhysioE.71: 333-341,1991).The reduction in glycolysis at least between AH and CH appears to be accompaniedby a tighter metabolic control. During CH, free ADP waslower and the ATP-to-free ADP ratio wasincreased(P < 0.05) compared with AH. With acclimatization, no change was found in the maximal activity of succinate dehydrogenasemeasuredat SL and CH: 63.9 t 6.1 vs. 68.6 t 4.9 mmol . kg protein-’ min-‘, respectively. Similarly, acclimatization failed to alter fiber area, the number of capillaries per fiber, and the capillary-tofiber area ratio. These results suggestthat the reduction in glycolytic flux that occurs with acclimatization cannot be explained by changesin the muscle oxidative potential, muscle fiber size, or musclefiber capillarization. l

metabolism;glycolysis

DESPITE NUMEROUS STUDIES examiningthephysiological alterations occurring during exercise after acute exposure to hypoxia and the adaptive effects of sustained residence at altitude, several issues remain unresolved. One such issue is the question of the effects of altitude on energy metabolic behavior and substrate utilization dur-

ing exercise and how alterations in energy metabolic behavior are influenced by adaptations at the level of the working muscle. During an acute hypoxic insult, performance of submaximal exercise at a given absolute power output results in an exaggeration in blood and muscle lactate concentration in comparison to sea level (for review, see Ref. 14). With acclimatization, the increase in blood lactate concentration is blunted (39). Recently, it has been demonstrated using blood flow measurements across the working limbs in conjunction with arterial and venous determinations of lactate concentration that the altered lactate response is at least partly due to variations in lactate release (2). The alterations in lactate release occur during exercise at altitude without any compromise in oxidative phosphorylation as evaluated by the constancy in 0, consumption (VO,) (2). Both the cause and significance of the reduction in lactate accumulation during exercise at altitude or during hypobaric hypoxia after acclimatization, a phenomenon that has been referred to as the lactate paradox (19), are in dispute. If, as indicated; the alteration in lactate release from the working muscle occurs as a result of variations in glycolytic flux (5), the mechanism involved could be due to alterations in metabolic control at some rate-limiting site such as phosphofructokinase (PFK) or to mass action effects (7). With regard to the former mechanism, glycolysis at the level of PFK could be altered in response to a change in phosphorylation state. As an example, during exercise soon after ascent to altitude, alterations in the levels of the effecters of PFK activity might result, such as ATP, ADP, AMP, and Pi (3O), secondary to a declining intracellular PO,. With acclimatization, adaptations may be involved in restoring phosphorylation state toward sea level values, providing for a reduction in glycolysis (18). Evidence that the phosphorylation state may be involved in explaining the lactate paradox, at least in high-altitude natives, has recently been published (20, 28). Alternatively, the timedependent response in glycolysis with altitude exposure may be simply explained by a tightening of metabolic control, with pyruvate production better matched to utilization by mitochondrial respiration. Alterations in gly2701

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2702

ALTITUDE

ACCLIMATIZATION

colysis by mass action, independent of phosphorylation state, could occur at any one of a number of sites (7). Whether lowlanders who display the lactate paradox within a few weeks after exposure to altitude utilize the same metabolic strategy as native highlanders is unknown. Adaptations at the level of the skeletal muscle remain as a potentially viable mechanism to explain the lactate paradox, particularly in view of the evidence linking adaptations in muscle capillary and muscle oxidative potential to the lower lactate response observed during submaximal exercise after training (l&21). Although the limited evidence available would suggest that increases in mitochondrial capacity are not involved (17, 22, 23), decreases in muscle fiber cross-sectional area and an increase in capillary-to-fiber area ratios do occur (17, 22) and could be implicated in the altered metabolic response. Our objective here and in other recent studies (5,6,38) was to examine the mechanisms associated with the altered lactate release observed with exercise both soon after arrival at altitude and after a period of residence. We have hypothesized that the reduced lactate release previously noted (3) after acclimatization is associated with an reduction in glycolysis, secondary to an increased phosphorylation potential in the working muscle. Additionally, we have proposed that the altered metabolic response during exercise after acclimatization is accompanied by adaptations in capillarity but not in mitochondrial potential. The information collected for this study was part of a much larger study investigating altitude acclimatization on a wide range of phenomena. METHODS

Experimental design. The basic experimental design consisted of the measurement of central and peripheral circulatory phenomena as well as the muscle metabolic responses to a submaximal exercise challenge at sea level (SL), after a cu t e exposure to 4,300 m (acute hypoxia, AH), and after 3 wk of full-time residence at 4,300 m (chronic hypoxia, CH). Before each submaximal work session, the subjects underwent a progressive maximal cycle test for evaluation of 0, flux (maximal aerobic power, To2 max). The data regarding central and peripheral circulatory processes involved in 0, transport, including cardiac output, muscle blood flow, hematology, and blood volume, have been reported elsewhere (38). Subjects. The seven male volunteers were sea level residents with no recent history of altitude exposure and, with one exception, no history of regular participation in physical exercise. The one subject who had been active on a regular basis had terminated his training -21 mo before the beginning of the SL measurements. Selected characteristics of the subjects included an age of 23 & 2 (SE) yr and an average weight of 72.0 t 1.6 kg. As required, the study was approved by the appropriate Human Subjects Committees at the University of Colorado Health Center, the Palo Alto Veterans Administration Hospital, and the University of California, Berkeley. Subjects were accepted into the study only after undergoing a medical examination and providing their written

AND

EXERCISE

METABOLISM

consent- Control of the dietary pattern of the subjects both before and during the altitude exposure was accomplished by having an enforced food and formula diet provided in amounts necessary to maintain an isocaloric state. At altitude, the physical activity level was matched to sea level in an attempt to avoid the complicating effects of deconditioning. The SL I$ maxof the subjects, as determined during a progressive cycle test to exhaustion, amounted to 3.55 t 0.09 Ifmin. Repeated measures of VO 2 maxwith use of the same exercise protocol at different times during acclimatization were 2.69 t 0.09 (day 5) and 2.72 t 0.8 l/min (day 18). The drop in vo2max during exposure to altitude amounted to 25-27% of the SL value (38). Prolonged exercise challenge. The same absolute power output was used for each test session and involved cpntinuous cycle exercise for 45 min. As expected, the VO, in liters per minute during the steady state exercise was similar *during the three test sessions (38). As a percentage Of v”2 max?the power output represented 51 t 1% at sea level and 64 t 2 and 66 t 1% for the two exercise sessions at altitude. Only in the case of acute exposure were two subjects not able to complete a planned 45-min period of exercise. For the two subjects in this category, exercise was terminated at 30 min. Muscle sampling. Before the beginning of exercise, the subjects were prepared for measurements of blood flow and arterial and venous composition of blood gases and metabolites (femoral arterial and venous catheterization) and for hemodynamic measurements and muscle biopsies. During this time, the vastus lateralis muscle was also prepared for the extraction of samples by the percutaneous needle biopsy technique. For each exercise challenge, biopsies were extracted from two different locations: one location was used before exercise and one location was used immediately after the exercise for each of the experimental conditions. Two samples were extracted before the exercise and one after the exercise. Throughout the course of the experiment, biopsies were equally distributed between the right and left vastus lateralis. In the case of preexercise, the first biopsy was rapidly extracted from the muscle and rapidly plunged into liquid nitrogen. This biopsy was stored at low temperature (-70 to -8OOC) and subsequently used for analyses of tissue metabolites. The second biopsy obtained from the same incision was used for the determination of histochemical properties and enzyme activities. This tissue was also stored at low temperature before analysis. After 45 min of exercise, a single biopsy was rapidly obtained, rapidly frozen, and used for analysis of tissue metabolite concentrations. Analytic procedures. Muscle tissue samples were analyzed for adenine nucleotides (ATP, ADP, and AMP) and inosine monophosphate (IMP) concentrations with use of high-performance liquid chromatography according to procedures previously published from our laboratory (15). The standards were obtained from Sigma Chemical and dissolved in water redistilled in potassium permanganate. Muscle phosphocreatine (PCr), phosphate (PJ, creatine (Cr), glycogen, and selected glycolytic intermediates were performed using fluorometric procedures (15, 27). All analyses were performed on

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ALTITUDE

ACCLIMATIZATION

AND

EXERCISE

METABOLISM

2703

freeze-dried tissue. To minimize the effect of various nase (AdK) reactions. For ADP,, the calculation involved amounts of blood and connective tissue in each sample, use of the measured concentrations of ATP, PCr, and Cr all metabolite concentrations, except glucose, pyruvate, in conjunction with H+ and free Mg” (26). Muscle pH and lactate samples, were corrected to total Cr, based on was determined by the regression formula established by the average total Cr content established for each subject. Sahlin et al. (33) for dynamic work with use of the meaBecause the uncertainty regarding fluid shifts associated sured values for lactate and pyruvate. Free Mg2+ was aswith altitude exposure, the concentrations of glucose, sumed to be 1.0 mM wet wt (26). Calculation of AMP, lactate, and pyruvate in muscle were not corrected for involved using the measured calculation of ATP and the the extracellular contamination. For a given subject, all calculated level of ADP, The equilibrium constants used metabolite measurements were performed during the for the CPK and AdK reactions (KcpK and K,,,) at 38OC same analytic session. were 1.66 X 10’ M-l and 1.05, respectively. Resting free Pi Tissue histochemical properties examined included was assumed to be 10.8 mmol/kg dry wt, and exercise free fiber type composition, fiber area, and capillarity and Pi was estimated as follows: resting concentration of fiber succinate dehydrogenase (SDH) activity. Fiber spe- PCr - exercise concentration + resting concentration of cific types (I, IIa, IIb) were identified using the procePi* This calculation is based on the approach used by dures of Brooke and Kaiser (4) as modified by Doriguzzi Dudley et al. (II), which assumes a stoichiometry beet al. (9). Fiber area was determined on SDH-stained tween increases in Pi and decreases in PCr during consections with use of planimetry on subsamples of specific tractile activity (10). fiber types. Where possible 220 fibers of each type were Cytosolic NAD+/NADH was determined using the measured. Capillaries were visualized using the periodic equilibrium reaction involving lactate dehydrogenase. acid-Schiff procedure (31). Capillarization was expressed An equilibrium constant of 1.1 X lo-l1 M-l was used (37). as the number of capillaries in contact with each fiber Statisticul analysis. A two-way analysis of variance type and subtype (cap/fiber), the number of capillaries with repeated measures was used to examine the metaper unit fiber area (cap/fiber area), and the number of bolic data for statistical significance. The two experimencapillaries in a field of defined area (mean cap density) tal factors included time (pre- vs. postexercise) and conwithout regard to type or number of fibers. Fiber type dition (SL, AH, CH). In the case of the histochemical and SDH activity was assessed using microphotometry (32). enzymatic data where only a single value was obtained This involved measuring the optical density (OD) for each condition, one-way analysis of variance procechanges in the core of the fiber at a wavelength of 546 nm dures were employed. Where significance was found, by the use of a Zeiss Zonax microscope photometer Newman-Keuls post hoc procedures were applied to de(MPMO,). The OD measured within the fiber core was termine differences between specific means. All comparidetermined relative to the OD at the mounting medium, sons were based on a confidence level 295% (P < 0.05). glass slide, and cover slip just outside the section being measured. Corrections were also made for “nothing dehy- RESULTS drogenase” activity, which represented the nonspecific reduction of the reaction indicator, nitroblue tetrazoHigh-energy phosphates and metabolites. The prolium. All measurements were end-point measurements, longed exercise protocol utilized in this study failed to obtained after the reaction had been terminated. All elicit changes in ATP concentration regardless of these procedures have been described in detail elsewhere whether the exercise was performed at sea level after (16, 17). acute exposure to altitude or after a period of acclimatiMeasurements of the maximal activity of a number of zation (Table I). Similarly, both total ADP and AMP enzymes representative of different energy metabolic were unaffected by the exercise in any of the conditions pathways and segments were also made on homogenates examined. Exercise did elicit an increase in IMP concenof mixed fiber type, These measurements performed on tration; however, the magnitude of the increase was not the preexercise muscle sample obtained during each con- differentiated by environmental condition (Table 1). As dition included phosphorylase, PFK, hexokinase (HK), expected, decreases in PCr and increases in Cr occurred 3-hydroxyacyl-CoA dehydrogenase, and SDH. The actividuring exercise. A condition effect was also found for ties of these enzymes were measured fluorometrically at PCr and Cr (Table 1). The condition effect, which was room temperature by use of the linear portion of the reac- found only between acute hypoxia and chronic hypoxia, tion time curve. Muscle homogenization (1:lOO dilution) resulted in higher PCr values and lower Cr values during was performed at 0-4OC in 0.17 M phosphate buffer at exercise after acclimatization. A condition effect was also pH 7.4, which contained 0.02% bovine serum albumin found for ATP. In t$he case of ATP, the average concenand 5 mM 2-mercaptoethanol. Specific details of the en- trations (rest plus exercise) observed during chronic hypzymatic procedures appear in an earlier publication (17). oxia were higher than those observed either at sea level Maximal enzyme activities are expressed on the basis of or acute hypoxia. No interaction effects were found for muscle protein content. Protein was measured by the any of the variables examined. Lowry technique with use of the modification of SchacVarious indexes used to characterize phosphorylation terle and Pollack (36) applicable to small tissue samples. potential are provided in Table 2. As expected, exercise Data analysis. Free ADP (ADP,) and free AMP (AMP,) resulted in increases in ADP,, AMP,, and calculated Pi concentrations were calculated as has been done previand a reduction in ATP/ADP,. When the various condi3usly (10) on the basis of the near-equilibrium properties tions were compared, differences were found only for 3f the creatine phosphokinase (CPK) and adenylate ki- ADP, and ATP/ADP, where the calculated concentraDownloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on September 21, 2018. Copyright © 1992 American Physiological Society. All rights reserved.

2704

ALTITUDE

ACCLIMATIZATION

1. Alterations in high-energy phosphate compounds and metubolites in vastus luteralis muscle after prolonged exercise at sea level, acute hypuxiu, and chronic hypoxia

TABLE

Acute Hypoxia

Chronic Hypoxia

21.3t0.82 19.6t0.88

22.3t0.47 21.4t0.83

24.11rO.75 24.5~0.83

2.6520.20 3,OO_tO.12

2.99-t-0.10 3.33t0.35

2.97t0.14 3.18tO.28

0.16t0.01 0.1420.02

0.16t0.02 0.20t0.06

0.15kO.02 0.14-tO.02

24.lkO.84 22.8k0.82

25e4t0.46 25.OkO.72

27.2kO.82 27.92zO.89

0.122t0.03 0.157t0.07

0.113t0.04 0.676-t-0.39

0.061t0.07 0.379to. 13

67.5t3.4 44.1t4.6

63.3t3.4 35.lk4.6

67.0t3.8 52.424.6

40.922*8 64.3k5.8

45.1t2.8 70.8t6,6

41.4t2.4 56.5k4.7

Sea L&V64

ATP Pre Post ADP Pre Post AMP Pre Post TAN Pre Post IMP Pre Post PCr Pre Post Cr Pre Post

AND EXERCISE

METABOLISM

3. Alterutiuns in glucose and selected glyculytic intermediates in vastus lateralis muscle ulith prolonged exercise at sea level, acute hypoxia, and chrunic hypoxia TABLE

Glucose Pre Post G-I-P Pre Post F-6-P Pre Post DHAP Pre Post Pyruvate Pre Post

Sea Level

Acute Hypoxia

Chronic Hypoxia

2.85t0.29 3,21+0.74

3.15&0.41 3.89-t0.65

3.49t0.67 4.48+0.70

0.042+0.01 0.060+0.01

0.064+0.02 0.082t0.04

0.031_+0.01 0.042+0.01

0,06lt0.02 0.045+0.02

0.060t0.02 0.061t0.02

0.040+0.01 0.071t0.02

0.399Iko.27 0.199+0.02

0.152+0.04 0.213t0.03

0.166t0.03 0.217t0.03

0.18lt0.08 0.182t0.08

0.184+_0.09 0.361+0.17

0.115+0.03 0.166t0.04

Values are means t SE in mmol/kg dry wt; n = 7. G-l-P, glucose l-phosphate; F-6-P, fructose 6-phosphate; DHAP, dihydroxyacetone phosphate. No significant effects of exercise or condition were observed.

-

Values are means t SE in mmoI/kg dry wt. Pre, preexercise; Post, postexercise. For ATP, total adenine nucleotides (TAN), phosphocreatine (PCr), and creatine (Cr), a main effect for condition was found (P < 0.05). For ATP and TAN, chronic hypoxia > sea level and acute hypoxia; for PCr, chronic hypoxia > acute hypoxia; for Cr, acute hypoxia > chronic hypoxia. For IMP, PCr, and Cr, a main effect for time was found (P < 0.05). No interaction effects occurred.

2. Alterations in energy state in vastus lateralis muscle with prolonged exercise at sea level, acute hypoxia, and chronic hypoxia

TABLE

ADP,, pmol/kg dry wt Pre Post AMPf, pmol/kg dry wt Pre Post Pi, mmol/kg dry wt Pre Post ATP/A13Pf Pre Post

Sea Level

Acute Hypoxia

86.2t8.6 188t25

106t-8.8 226-t32

98.6t7.7 172t21

0*35t_O*O6 2.02t0.59

0.50NL09 2.3120.58

0.40tO.07 1.3lt0.36

10.8*0 33.8t-6.2

lO.BtO 37.8t6.7

10.820 29.5k3.8

260t24 119tl9

218,tl9 109&22

255+24 160t23

Chronic Hypoxia

Values are means t, SE; n = 7. ADPf and AMP,, free ADP and free AMP. For all variables, a main effect for time was found (P < 0.05). For ADPf and ATP/ADPf, a main effect for condition occurred (P < 0.05). For ADPE, chronic hypoxia < acute hypoxia; for ATPIADP,, chronic hypoxia > acute hypoxia. No interaction effects were present.

tions (rest plus exercise) during acute hypoxia were greater for ADP, and lower for ATP/ADP, than at chronic hypoxia. In no case did condition affect the magnitude of the exercise response. Glycugen, glucose, and gycolytic intermediates. The changes in glycogen and selected glycolytic intermediates during exercise under the various experimental conditions are presented in Table 3 and Figs. 1 and 2. In

SEA LEVEL

ACUTE HYPOXIA

CHRONIC HYPOXIA

FIG. 1. Muscle glycogen concentration in vastus lateralis muscle during submaximal exercise at sea level, on acute exposure to altitude (acute hypoxia), and after acclimatization (chronic hypoxia). Open columns, preexercise, hatched columns, postexercise. Values are means & SE; n = 7. A main effect for both condition and time was found (P < 0.05) as well as a condition X time interaction (P < 0.05). * Significantly different from sea level (P < 0.05). “f Significantly different from acute hypoxia (P < 0.05). $ Significantly different from preexercise (P < 0.05).

general, exercise was accompanied by decreases in muscle glycogen (Fig. 1) and increases in lactate concentration (Fig. 2). In the case of glycogen, lower resting concentrations were found after acute exposure to altitude than at sea level or after chronic altitude exposure. In addition, acclimatization influenced the postexercise concentration. The glycogen concentration was lower after exercise during acute hypoxia than during chronic hypoxia. In the case of lactate, the resting concentration was not affected by altitude exposure. However, exercise performed under acute altitude exposure resulted in higher muscle lactate concentrations than when performed at sea level or after acclimatization. With regard to the other glycolytic intermediates examined, glucose, glucose l-phosphate, fructose 6-phosphate (F-6-P), dihy-

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ALTITUDE

ACCLIMATIZATION

30

5. Effects of altitude acclimatization on maximal activities of enzymes involved in energy metabolism in uastus lateralis muscle

2 15 E w I0 2 L 5 5

Sea Level

Acute Hypoxia

63.9t6.1

61.4k5.9

68.6t4.9

20.2t2.4 11.2t0.44

23.2t2.0 10.9t0.88

22.1t1.2 13.0+0.82*‘/-

43.lt2.1

41.4t2.0

40.2+1.3

133t7.4 1.82t0.11

lOlt7.5* 1.71t0.11

1x&4.4* 2.06&0.13

Chronic Hypoxia

l

ACUTE

SEA LEVEL

CHRONIC

l

HYPOXIA HYPOXIA 2. Muscle lactate concentration in vastus lateralis muscle during submaximal exercise at sea level, on acute exposure to altitude (acute hypoxia), and after acclimatization (chronic hypoxia). Open columns, preexercise; hatched columns, postexercise. Values are means LL SE; n = 6. A main effect for both condition and time was found (P < 0.05) as well as a condition X time interaction (P c 0.05). * Significantly different from sea level (P < 0.05). t Significantly different from acute hypoxia (P < 0.05). # Sighificantly different from preexercise (P < 0.05). TABLE 4. Alterations in NADVNADH, lactute-topyruuate ratio, and pH in vastus lateralis muscle with prolonged exercise at sea level, acute hypoxia, and chronic hypoxia Sea Level

Post

SDH, mmol . kg protein-‘. min-’ HAD, mmol . kg protein-’ s min-’ HK, mmol kg protein-‘. mine1 Phosph, mmol kg protein-‘. min-’ PFK, mmol kg protein-‘. min-’ Protein, mg/g l

FIG.

Lactate/pyruvate Pre Post NAD+/NADH Pre Post PH Pre

2705

TABLE

3’ 25 d T’ 20 2

0

AND EXERCISE METABOLISM

Acute Hypoxia

Chronic Hypoxia

54.1-t41 101+3u

59.7k19 121t44

199t97 95t42

167t79

151+50

114276

133t39

7.03ztQ.004 7~01-1-0.012

7.04tO.004 6.97*0.024*$

60&16 64.Ok 19

7.03tO.004 7.01+0.008~

Values are means t SE; n = 5. A main effect of time was found for all variables (P < 0.05). A condition and an interaction effect were found only for pH (P < 0.05). * Significantly different from sea level (P < 0.05). t Significantly different from acute hypoxia (P < 0.05).$ Significantly different from Pre (P < 0.05).

droxyacetone phosphate (DHAP), and pyruvate, neither exercise nor altitude resulted in significant changes (Table 3). Exercise resulted in increases in the lactate-to-pyruvate ratio and decreases in pH and NADVNADH (Table 4). However, only in the case of pH was a condition effect found. Although no differences were found in preexercise pH between the different conditions, postexercise pH was affected by the environmental state in which the exercise was performed. The pH after exercise in acute hypoxia was lower than the pH after exercise at either sea level or chronic hypoxia. Muximal enzyme activities and histochemical properties, The maximal activity of several enzymes used to

represent the potential of various metabolic pathways and segments used in energy metabolism indicated that altitude had an effect in altering only HK and PFK (Table 5), In the case of HK, a 16% increase over sea level values was observed after acclimatization, whereas in the

Values are means f SE; n = 7, SDH, succinate dehydrogenase; HAD, 3-hydroxyacyl-CoA dehydrogenase; HK, hexokinase; Phosph, phosphorylase; PFK, phosphofructokinase. * Significantly different from sea level (P -C0.05). t Significantly different from acute hypoxia (P < 0.05).

TABLE 6. Oxidative and glycolytic potential in specific fiber types of vastus lateralis muscle at sea level, acute hypoxia, and chronic hypoxia Fiber TYPe

I IIa IIb

Sea Level

Acute Hypoxia

Chronic Hypoxia

0.289t0.04

0.295t0.03

0.295t0*02

0.227+0.03

0.206tO.02

0.219t0.04

0.213tO.03

0.208t0.02 0.207kO.02

Values are means & SE measured as SDH activity in optical density units; n = 5. A main effect (P < 0.05) was found between fiber types for SDH (type I > type IIa = type IIb).

case of PFK, a 24% decrease occurred after acute exposure to altitude. Although there was some evidence of recovery in PFK activity with acclimatization, the value remained - 14% below the maximal activity measured at sea level. No effect of hypoxia was observed on the maxima1 activities of SDH, 3-hydroxyacyl-CoA dehydrogenase, and phosphorylase. Protein yields were not different between the different conditions. Enzyme activity, specifically SDH, was also measured in single muscle cells of different types by use of microphotometry (Table 6). Using SDH as a measure of oxidative potential, it was found that neither acute nor chronic hypoxia affected the maximal activity in type I, IIa, or IIb fibers. Other histochemical properties examined included fiber type distribution, fiber size, and capillarization (Table 7). As expected, fiber type distribution was not affected by the experimental conditions. Similarly, the areas of the specific fiber types, although demonstrating a trend toward lower values wit.h acute hypoxia, were not significantly altered. Capillarization was represented by the number of capillaries per fiber, the number of capillaries per unit area of specific fiber types, and the number of capillaries in a unit area of the tissue section. Of these measures of capillarization, only the number of capillaries per fiber was altered. In the case of the number of capillaries per fiber, the affect of acute hypoxia was to reduce the number compared with both sea level and chronic hypoxia. The alteration in number of capil-

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27U6

ALTITUDE

ACCLIMATIZATION

7. Fiber type distribution, area, capillary rtumber, and capillary-to-fiber area ratio in vastus lateralis muscle at sea level, acute hypoxia, and chronic hypoxia

TABLE

Type, 70 I IIa

IIb Area, pm2 X 103 I

IIa IIb Cap/fiber I IIa IIb Cap/fiber area, loo3 pm2 I

IIa IIb Mean cap density, cap. lo2 pm

Sea Level

Acute Hypoxia

Chronic Hypoxia

54.9t7.9 35.6t5.6 9.5t2.6

54.6t7.4 36.3t6.0 9.642.8

43.Ckb7.9 43.7k6.3 13.5t1.7

6.28-10.39 7.05tl.l 4.99zt0.58

5.25kO.71 6.00t1.3 4.21~~0.33

6.23t0.43 6.98kU.90 5&3-t-0.95

5.5920.58 5.84tl.O 3.76&O. 14

4.87_tU.58 4.66~0.74 3.39-1-0.29

6.06kO.47 5.89kO.72 5.OlkO.69

0.899-t-0.09 0.830tU.06 0.788t0.10

0.941t0.06 0.805t0.05 0.826kO.11

0.978+0.05 0.851,tO.O5 0.910+,0.06

0.347t0.03

0.352t0.02

0.361+0.02

Values are means + SE; n = 5. A fiber type main effect (P < 0.05) was found for composition (type IIa = type I > type IIb) and cap/fiber (type Ha = type I > type I&). A condition main effect (P < 0.05) was found only for cap/fiber (sea level = chronic hypoxia > acute hypoxia). No interaction

effects

were

found.

laries per fiber was not reflected in an alteration ber of capillaries per fiber area.

in num-

A major finding of this study was that, at least for the exercise challenge employed, compensatory mechanisms are able to defend ATP homeostasis in the working vastus lateralis muscle during acute exposure to 4,300 m. With acclimatization to altitude, working ATP concentrations are maintained despite a reduction in muscle lactate concentration. These changes occur at the same absolute work intensity in the absence of any difference in VO, between conditions (38). Furthermore, these metabolic adaptations appear not to depend on changes in muscle mitochondrial capacity, fiber size, or capillarization. The observation that Vop and, therefore, oxidative phosphorylation is protected after ascent and acclimatization to 4,300 m during moderate-intensity exercise has been previously documented, and the central circulatory adjustments involved in maintaining adequate 0, supply to the working muscles have been described (2,3). Using isotopic tracers, Brooks et al. (5) were able to establish that the changes in lactate release reflect primarily changes in blood lactate appearance rate, Collectively, these findings indicate that exercise performed soon after ascent to altitude compared with sea level results in an exaggeration in the blood and muscle lactate concentration and that the elevations in concentration are due to an elevation in glycolysis. During exercise after acclimatization, glycolytic flux is reduced and the reduction results in a more appropriate matching to tricarboxylic acid cycle turnover. At least for submaximal exercise, it thus appears that the lactate paradox, characterized by a

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reduced blood lactate accumul tion after acclimatization (19), can be accounted for by reduction in glycolysis. Changes in glycolytic flux rate could conceivably occur as a result of a change in the availability of substrate, namely, glucose 6-phosphate, by a direct alteration at some nonequilibrium site in the glycolytic pathway itself, such as between F-6-P and fructose 1,6-biphosphate (F1,6-P), OFby both mechanisms. Flux through the F-6-P/ F-1,6-P site is controlled by the activity of PFK, generally acknowledged to be the major rate-limiting enzyme in glycolysis (30). To examine whether the activity of PF&ould have been affected during exercise under the various experimental conditions, we have examined some of the known modulators of PFK activity, namely ATP, ADP, AMP,, and Pi (7,30). Prolonged exercise regardless of condition resulted in elevations of ADP, AMP, and Pi* Although decreases in ATP concentration with-exercise would have been expected (29) given the increases in IMP that occurred with exercise ?no significant changes were found. We attribute this to a lack of sensitivity in ou r analytic procedures and the inability to detect changes given the relatively small percent changes that would be expected in ATP concentration. In the absent eofan altitude effect on changes in other effecters of PFK (7), the finding of elevations in ADP,, AMP,, and free Pi, although offering a tentative explanation for the relatively greater increase in glycolysis observed with exercise in general, may also account for the differences observed between the different experimental conditions. A clear trend was evident with ADP,, AMP,, and ATP/ADP, changing in a direction during exercise with acute hypoxia that is consistent with a greater activation of PFK. In the case of ADPf and the ATP/ATP, greater elevations were found (P < 0.05) during acute hypoxia than during chronic hypoxia, and in the case of AMP, the increase approached significance (P < 0.10). The fact that F-6-P did not increase with exercise regardless of condition would suggest that the activity of PFK wa .s sufficient to handle the substrate regardless of condition. The increased formation of F-6P that would be expected during exercise with acute hypoxia to support the increased glycolytic flux rate (30) appears to be closely coordinated with elevations in PFK activity. Another possibility to explain the alteration in glycolysis noted during exercise in acute hypoxia and after acclimatization is simply a result of a mismatching of pyruvate formation by glycolysis and pyruvate disposal by oxidative phosphorylation. This effect, referred to as a mass action effect, occurs when flux is accelerated at some specific site in glycolysis such as at the level of glycogenolysis (7). As a consequence of the relative overstimulation of glycolysis, lactate formation is accelerated, because pyruvate formation exceeds that which can be taken up by the mitochondria. Although exercise resulted in increases in lactate-to-pyruvate ratio and decreases in the cytosolic NAD+/NADH that would be expetted (34), the basic response pattern observed at sea level was not modified during exercise performed at altitude, regardless of the time of residence. Alterations in glycogenolysis in the working muscle at altitude could be facilitated by the epinephrine background, which has

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been noted to drop substantially with residence at altitude (5). Epinephrine, probably operating through alterations in phosphorylase activity, has been shown to alter muscle glycogenolysis during exercise (25). The decrease in muscle and blood lactate concentration with exercise at a given absolute irO, noted with acclimatization (3, 5) has been reported after training (21). Two explanations have been provided to explain the lower lactate response. Donovan and Brooks (8), using [2-3H]- and [U=14C]lactate to measure lactate turnover, have concluded that the depression in lactate is not due to a reduction in glycolysis but rather an increased clearance of the lactate. In contrast, the most popular view is that the decrease in lactate concentration is, in fact, due to a depression in glycolysis (12, 21). The depression in glycolysis has been postulated to be secondary to a training-induced increase in mitochondrial density and oxidative potential (12, 21). However, recent findings using abbreviated training periods would suggest that increases in oxidative potential as well as other adaptations in support of aerobic-based metabolism are not necessary for the blunting of the lactate response (13,16). It is also evident that increases in oxidative potential and/ or muscle fiber capillarization are not necessary for the reduction in lactate release and glycolysis that occurred in this study during exercise after acclimatization. With 3 wk of acclimatization, no change was found in the maximal activity of SDH, the enzyme used to characterize oxidative potential in homogenates of mixed-fiber type. This finding was also supported using microphotometric determinations, where SDH activity was found not to change with either acute or chronic hypoxia regardless of fiber type. With more sustained exposure to altitude, however, reductions in muscle oxidative potential apparently occur (17, 23). The other adaptive indexes examined would also appear incapable of explaining the lower glycolytic involvement noted after acclimatization. Changes in 3-hydroxyacyl-CoA dehydrogenase, a measure of ,&oxidative potential, as well as total phosphorylase, a measure of glycogenolytic potential, did not occur, as has been noted earlier (23, 40). Reductions in the maximal activity of PFK, a measure of glycolytic potential, did occur, the reduction being observed both after acute ascent and after acclimatization. At present, the mechanisms underlying the reduction in PFK, particularly after acute ascent, are unknown. The fact that PFK was depressed both on ascent to altitude and after acclimatization in the face of a pronounced alteration in glycolysis would suggest that the altered PFK activity is without consequence. In addition, increases in HK activity did occur. The increase in HK activity may have been in response to the increase in blood glucose dependency that results both during rest and exercise with acclimatization (6). Increased glucose flux through HK would provide a calorie-efficient fuel (kcal/l 0,) for the tissue. This has been previously postulated as a beneficial adaptation to hypoxic environments (19), and some experimental support for the hypothesis has recently been provided (20). We have also examined a number of other potential peripheral adaptations to acclimatization such as fiber area and fiber cat$llarization. For all three fiber tunes- no

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reduction in size was observed with acclimatization. Reductions in fiber size have been noted in humans exposed to more severe and sustained chronic hypoxic environments (17, 22). Acclimatization also failed to elicit alterations in the various indexes used to characterize fiber capillarization, namely, the number of capillaries per fiber and the number of capillaries per unit area. Although increases in number of capillaries per fiber have been noted to occur with hypoxia (I, 24), this is not a consistent finding (17). Indeed, the index most often affected by acclimatization is the number of capillaries per unit fiber area, where observed increases are primarily mediated by fiber area reductions (17, 22). The contractile history is also known to have a important effect on muscle size and capillarization (35), and this may also complicate the interpretation of the changes that occur. In this study, careful attempts were made to control the activity pattern of the subjects during acclimatization, and activity was kept at a very low level. A surprising finding was the reduction in the number of capillaries per fiber noted with acute exposure to altitude. At present, we are at a loss to explain the finding. The potential for sampling error must be acknowledged. In summary, it would appear that regulatory mechanisms are able to defend muscle ATP homeostasis during prolonged whole body exercise of moderate intensity after acute exposure to an O,-deprived environment, at least to an altitude of 4,300 m. Because exercise VO, is preserved under such conditions, it is possible that the metabolic adjustments, including an increase in glycolysis, serve to protect oxidative phosphorylation as the primary mechanism of maintaining ATP concentration in the working muscle. With acclimatization, ATP homeostasis during exercise can be maintained in the face of a substantial reduction in lactate production. This metabolic adjustment results in a tighter coupling between oxidative phosphorylation and glycolytic flux. It is concluded that the reductions in glycolytic rate with acclimatization to moderate altitude are not dependent on peripheral adaptations such as increases in oxidative potential or capillarization or on adaptations to energetic efficiency, as indicated by the unchanged 0, flux rates during submaximal exercise. The capable assistance of M. Ball-Burnett, N. Kowalchuk, and P. Kosteniuk is gratefully acknowledged. This study was supported by the Natural Science and Engineering Research Council (Canada) and National Heart, Lung, and Blood Institute Grant HL-14985. Address for reprint requests: H. J. Green, Dept. of Kinesiology, University of Waterloo, Waterloo, Ontario NZL 3G1, Canada. Received 10 February 1992; accepted in final form 6 July 1992. REFERENCES

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