INTRODUCTION

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A KEY CHALLENGE IMPOSED ON the various physiological systems during exercise, and particularly during exercise involving large muscle groups, is preservation of energy balance in the contracting muscle while at the same time optimizing the contribution of oxidative phosphorylation to ATP resynthesis (19). This challenge is made particularly difficult during exercise in hypoxic environments where the availability of O₂ is compromised. Evidence that compensatory adjustments can be invoked, at least during submaximal exercise at moderate levels of hypobaric hypoxia (equivalent to 4,300 m), is provided by the whole body, steady-state levels of O₂ consumption (O₂), which appear unchanged from those measured under normoxic conditions at similar power output (2, 30). These compensatory adjustments appear to involve a complex interplay between both central and peripheral processes.

The major central responses to exercise in acute hypoxia compared with normoxia appear to involve additional increases in ventilation, cardiac output, and blood flow, all designed to protect O₂ delivery to the contracting muscle (27). Peripheral adjustments in the contracting muscle cell also appear to occur. Although ATP levels remain reasonably well preserved, further reductions in phosphocreatine (PCr) and further increases in lactate have been observed (14). The exaggerated increase in the metabolites [inorganic phosphate (P_i), ADP] arising from the high-energy phosphate reactions are thought to have both direct and indirect effects in exploiting available O₂ to maintain oxidative phosphorylation in hypoxia. On the one hand, one or more of these metabolites act directly to stimulate mitochondrial respiration, whereas on the other hand, the metabolites provide for increased glycolytic flux. The increased glycolytic flux provides reducing equivalents (H⁺) to the mitochondria, increasing mitochondrial redox potential, which provides for a compensation to hypoxia (7). Even though these compensatory adjustments appear successful in maintaining energy balance and the level of oxidative phosphorylation, there is a price to pay. Under these conditions, glycogen depletion rates are accelerated and lactate (and H⁺) accumulate in the muscle (14), potentially challenging substrate availability and management of by-products.

From a peripheral perspective, meaningful adaptations to chronic hypoxia would appear to involve improving the balance between ATP utilization and ATP synthesis and in the process maintaining a higher phosphorylation potential. This appears to be what occurs. A 21-day period of acclimatization appears to result in an improvement in phosphorylation potential (and lower accumulation of by-products) during moderate exercise in conjunction with a lower glycolytic flux as indicated both by lower muscle lactate levels (14) and lactate release (3). Unclear at present is what mechanisms are responsible for these changes.

Adaptations in central processes do not appear to be involved. Even though arterial O₂ content is dramatically improved during chronic altitude exposure as a result of increases in both arterial hemoglobin (Hb) concentration and arterial Hb saturation (2, 30), arterial O₂ delivery is not improved because of reductions in leg blood flow (2, 30). Under these conditions, O₂ is maintained by a widened arteriovenous (a-v) O₂ difference across the contracting muscle. The widened a-v O₂ difference would suggest that acclimatization-induced changes occur in the muscle cell. One hypothesis, based on evidence obtained from high-altitude adapted natives, is that a metabolic reorganization occurs, resulting in an increased aerobic metabolic control and a decreased glycolytic potential (17, 18). However, analysis of skeletal muscle from subjects acclimatized to 4,300 m have revealed no differences in the maximal activities of enzymes representative of these pathways measured at the beginning of and following chronic residence (14, 31). At present, it is unclear what changes occur in the short-term acclimatized muscle to explain the altered metabolic response.

Differences also may exist between acclimation- and acclimatization-based studies and the environment employed to assess the effects of chronic hypoxia. In previous mountaineering studies, a reduction in the maximal activities of several mitochondrial enzymes has been observed in the absence of changes in a range of glycolytic enzymes (21). These findings suggest that it is possible that an altitude expedition, given the multiple stressors involved, may produce a substantially different response than observed during contrived acclimation experiments, attempting to examine the singular effects of chronic hypoxia (30). Moreover, it is not at all clear whether the metabolic adaptations observed during hypoxia following short-term altitude residence (14) also occur when exercise is performed in normoxia. Indeed, it is possible, given the potential complications of the added energy costs associated with hyperpnea and the work of breathing, that the acclimatization effects may be even more pronounced. In the only study published to date, a higher ATP demand-supply coupling was observed in long-term residents during exercise at low altitude compared with sea-level residents (25). However, as indicated, long-term residents may also display a reorganization in muscle energy metabolic pathways (17, 18).

In this study, our objectives were to investigate the muscle metabolic adaptations that occur during exercise at sea level in response to a mountaineering expedition and in relation to the changes that occur in mitochondrial potential. We have hypothesized that the expedition would result in an improved energy balance in contracting muscle as indicated by a higher phosphorylation potential. Moreover, we also have postulated that the metabolic adaptations would occur in the absence of changes in mitochondrial potential as indicated by the maximal activities of representative enzymes of the citric acid cycle.

	METHODS

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The exercise tests from which measurements of muscle metabolic behavior were made were performed under standard conditions at the University of Waterloo (altitude 317 m) ~1 wk prior to and 3 to 4 days following the return to base camp after a mountaineering expedition. The expedition was to the summit of Mount Denali (6,194 m), which is located in Alaska. On day 1, base camp was established at 2,100 m following arrival at Talkeetna, Alaska. Eighteen days were needed to attain the summit. Two days later the mountaineers arrived at the initial base camp and returned to Talkeetna. From Talkeetna, the subjects were transported to the University of Waterloo. All testing and sampling protocols were conducted at the University of Waterloo, following approval by the Office of Human Research and Animal Care and after obtaining written consent by each participant. Because the expedition itself was self-initiated, without involvement of the University of Waterloo or any of the scientists, it was not appropriate to obtain consent for that aspect of the study.

Subjects. The five male subjects were mountaineers who had volunteered for the expedition. The mean age was 28 ± 2 (SE) yr, weight 76.9 ± 4.3 kg, and height 173.6 ± 3.6 cm. All subjects were regularly active, particularly during the months preceding the expedition. None of the expedition members was involved in any hypoxic training preceding the expedition. In addition, none of the participants had been on a mountaineering expedition for at least 6 mo prior to Mount Denali. After the expedition the mountaineers were generally inactive prior to arrival at the University of Waterloo with most of the time spent in traveling. The peak O₂ obtained during a progressive cycle test to fatigue was 52.3 ± 2.1 ml · kg¹ · min¹. This value was not changed by the expedition.

Exercise protocol. The submaximal cycling protocol consisted of cycling for two consecutive 20-min periods at two different power outputs, designed to elicit ~60 and 75% of the prealtitude peak O₂. This protocol, using the same absolute power output, was also performed following the expedition. In this study, we have used a two-stage protocol to increase O₂ demand and ATP flux in the contracting muscle. Our rationale was that this type of protocol would be more effective in unmasking any metabolic adaptations that might exist. Gas exchange (O₂, CO₂ uptake, and expired ventilation) was measured using an open-circuit system that has been commonly employed by our group (22). Gas collection was performed at rest and during a 4- to 5-min period prior to the completion of each stage of the exercise. The gas analyzers (Beckman OM-11 and LB-2) and the pneumotachograph (Hewlett Packard 4730A) were calibrated on each testing day using common, precisely determined reference gases (gas analyzers) and a 3-liter syringe (pneumotachograph). The exercise was performed on an electrically braked cycle ergometer (Quinton 870) that was calibrated on each testing day. All details of the procedures and the effects of the mountaineering expedition on the gas exchange responses, as well as related measures, obtained during progressive and prolonged exercise, appear elsewhere (12). In this study, the O₂ data are reported because it is important to the interpretation of the muscle metabolic adaptations that occurred.

Analytic procedures. For analysis of the metabolic response to exercise, both fluorometric (15) and ion-pair reversed-phase high-performance liquid chromatography (HPLC) (23) techniques were employed on freeze-dried tissue. The measurement of glycogen, selected glycolytic intermediates, high-energy phosphates, and the metabolites creatine (Cr) and P_i were measured fluorometrically following extraction by perchloric acid and neutralization by K₂HPO₄. The concentrations of inosine monophosphate (IMP) and the adenine nucleotides (ATP, ADP, and AMP) were measured on the extracts using HPLC procedures. All values for a given individual were expressed relative to average total Cr content for that individual. This procedure adjusts for blood and connective tissue contamination. Neither training nor exercise altered total Cr content. Muscle glucose and lactate were not corrected for extracellular water content because of the uncertainty of water content in this space. These procedures were essentially as performed previously by our laboratory (10, 14).

Statistical procedures. The effects of acclimatization (Pre vs Post) on resting adenine nucleotides and IMP were analyzed using a one-way ANOVA procedure. When both acclimatization state and exercise served as the independent variables, a two-way ANOVA was employed for repeated measures. When significant differences were found, Newman-Keuls procedures were used to locate differences between specific means. Significance was set at the 0.05 level.

	RESULTS

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Respiratory gases. The two-stage, submaximal cycle exercise resulted in progressive increases in O₂ regardless of the acclimatization state (Table 1). After the mountaineering expedition, O₂ was reduced at both power outputs. No differences in resting O₂ were observed.

View this table: [in this window] [in a new window]   Table 1.   Effects of acclimatization on O2 during rest and exercise

Muscle metabolism. Acclimatization was without effect in altering the resting muscle total adenine nucleotide content or its constituents ATP, ADP, and AMP (Table 2). Similarly, no effect of acclimatization was observed in PCr, P_i, and Cr measured in resting muscle.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Effects of acclimatization and exercise on changes in phosphocreatine (PCr), creatine (Cr), and inorganic phosphate (Pi). Values are means ± SE, n = 5 male subjects. Pre, preacclimatization; Post, postacclimatization. For PCr, Cr, and Pi, main effects (P < 0.05) of both exercise and acclimatization were found. For exercise, PCr 0 > 3 = 20 > 40 min; for Cr and Pi, 0 < 3 = 20 < 40 min. For acclimatization, Post > Pre for all variables.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Effects of exercise and acclimatization on the total adenine nucleotides (TAN). Values are means ± SE, n = 5 male subjects. ADP, adenosine diphosphate; AMP, adenosine monophosphate; ATP, adenosine triphosphate. No main effects for either exercise or acclimatization were found.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Effects of exercise and acclimatization on inosine monophosphate (IMP). Values are means ± SE, n = 5 male subjects. Main effects (P < 0.05) of both exercise and acclimatization were found. For exercise, 0 = 3 = 20 < 40 min; for acclimatization, Pre > Post.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effects of exercise and acclimatization on muscle glycogen. Values are means ± SE, n = 5 male subjects. Main effects (P < 0.05) of both exercise and acclimatization were found. For exercise, 0 > 3 = 20 > 40 min; for acclimatization, Pre < Post.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Effects of exercise and acclimatization on muscle pyruvate, lactate, and the lactate/pyruvate ratio. Values are means ± SE, n = 5 male subjects. For lactate, main effects (P < 0.05) were found for both exercise and acclimatization. For exercise, 0 < 3 = 20 < 40 min. For acclimatization, Pre > Post. For the lactate/pyruvate, a main effect (P < 0.05) was found for exercise. For exercise, 0 < 3 = 20 < 40 min.

View larger version (10K): [in this window] [in a new window]   Fig. 6.   Effects of acclimatization on the maximal activities of citrate synthase (CS) and lactate dehydrogenase (LDH) in vastus lateralis. Values are means ± SE, n = 6. *Significantly different from Pre (P < 0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Based on the results that we have observed, it can be concluded, as hypothesized, that the expedition to Mount Denali results in a tighter coupling between the processes involved in ATP utilization and the processes involved in ATP synthesis. Moreover, the metabolic adaptations that were observed occurred in the absence of changes in mitochondrial potential. Evidence that a tighter coupling has occurred is indicated by the lower muscle IMP (used as a more sensitive indicator of changes in ATP) and higher PCr levels (observed during exercise following the expedition). Moreover, as in the earlier study (14), lower muscle lactates also were found, suggesting a possible reduction in glycolytic flux rate with acclimatization.

The similarity in findings between this study and an earlier study (14) is noteworthy given the different test conditions and altitude exposures. In the earlier study, acclimatization was examined following 3-wk residence at 4,300 m. The subjects were studied under carefully controlled dietary and exercise conditions. In addition, the acclimatization effects were examined during exercise following both acute and chronic exposure to altitude and compared with the preacclimatized response, measured during exercise under normoxic conditions. In the present study, a mountaineering expedition involving a 21-day sojourn to 6,194 m (Mount Denali) was used as the hypoxic stimulus. Moreover, acclimatization responses were examined only under normoxic conditions. Given the similarity in responses between the two studies, at least qualitatively, it would appear that several potentially confounding influences associated with a mountaineering expedition such as diet, exercise, and cold exposure do not substantially modify the basic acclimatization responses that occur in contracting muscle. Interestingly, the metabolic responses to acclimatization are also similar to that reported for high-altitude natives tested at sea level and compared with unacclimatized controls (25).

Our results also indicate that the acclimatization effects on muscle metabolism persist for 3-4 days following return to sea level. Other acclimatization effects also appear to persist during this time frame. Blood Hb concentration ([Hb]) levels, considered as a hallmark of the response to chronic hypoxia (2, 30), remained significantly elevated (15.0 ± 0.49 vs. 15.8 ± 0.41 g/100 ml) (12). These results are consistent with a previous report (8), which has shown that significant elevations in blood [Hb] persist for at least 1 wk following return to sea level. Changes in several other properties such as the muscle Na⁺-K⁺-ATPase content (11) and the sarcoplasmic reticulum Ca²⁺-ATPase activity (13) also indicate the persistence of acclimatization effects.

Our study appears to be the first to report metabolic adaptations in the contracting muscle while exercising at sea level following short-term acclimatization to altitude. However, in this study, unlike an earlier study (14), we have found that these adaptations were accompanied by an increase in net mechanical efficiency as indicated by the lower exercise O₂. A higher mechanical efficiency also has been reported for high-altitude residents while exercising at sea level (20). It is clear from the present results, the limitations of the small subject number not withstanding, that the acclimatization effects on muscle metabolism that are designed to defend against chronic hypoxia were also expressed during exercise in normoxia. The importance of increasing the work efficiency as part of the adaptative strategy in defending against O₂ limitations and restoring a tighter coupling between ATP utilization and ATP synthesis has been proposed previously (17). This study appears to be the first to demonstrate that such a strategy can be induced through a relatively short period of acclimatization.

The metabolic adaptation, which is perceived to be central to the other metabolic changes that were observed with chronic hypoxia, is the improved matching that occurs between the rates of synthesis and utilization of ATP (1). The improved matching results in a higher PCr content and consequently an expected lower accumulation of the free adenine nucleotides ADP and AMP and a lower P_i, given the equilibrium nature of the Cr phosphokinase and adenylate kinase reactions (1, 7). What is unclear are the mechanisms responsible for this acclimatization effect. Several possibilities exist. One possibility is that chronic hypoxia results in an improved ATP synthesis, mediated by an increase in either the rate of PCr hydrolysis, glycolysis, or oxidative phosphorylation. All of these possibilities appear remote since resting PCr and the maximal activities of Cr phosphokinase were unaltered (11), muscle lactate concentrations were reduced, and whole body O₂ kinetics measured during the non-steady state and O₂ measured during the steady state were both depressed following acclimatization (Ref. 12 and M. J. MacDonald, H. J. Green, H. L. Naylor, C. Otto, and R. L. Hughson, unpublished observation). At least at sea level, whole body O₂ accurately reflects the O₂ used by the contracting muscles (9).

The reduction in O₂ was unexpected given the results of previous experiments that have not found any changes in either whole body O₂ immediately after chronic exposure to hypoxia (2) or 1 wk following return to sea level(8) or in leg O₂ (2) when the acclimatization effects were examined during normoxia only. Our results indicate that the depression in whole body O₂, induced by hypoxia, is also expressed during exercise in normoxia and persists for at least 3 to 4 days following return to sea level. It is possible, based on the work of Grassi et al. (8), that the adaptive effect is lost by 1 wk of deacclimatization. Interestingly, a greater work efficiency has been reported previously by Hochachka et al. (20) on high-altitude natives examined under normoxic conditions and compared with sea-level control subjects. In the case of the high-altitude natives, the increase in cycling efficiency persists for a longer period during residence at sea level than 3 to 4 days.

The lower O₂ that we have observed is also consistent with an increase in work efficiency, particularly since resting O₂ remained unaffected by the altitude expedition (12). The increase in efficiency indicates that a given amount of work may be performed at lower ATP synthesis rates and specifically lower rates of oxidative phosphorylation. In related papers, we have found that the cation ATPases may be implicated in this response since downregulation uses found for both the sarcolemmal and t-tubule Na⁺-K⁺-ATPase and the sarcoplasmic reticulum Ca²⁺-ATPase (11, 13). A downregulation could result in a lower ATPase activity, lower ATP utilization, and reduced cycling rates (20).

We also have observed that muscle lactate content was reduced following the expedition, an event that was first observed at 3 min of exercise, similar to the improved high-energy phosphate state. Lower muscle lactates have been previously reported during exercise at altitude following 3 wk acclimatization (14). Lower muscle lactates could be explained by a decrease in glycolytic flux rates or increased removal either from the muscle or by processes within the muscle. Measurements of a-v differences across the contracting muscle in conjunction with blood flow determinations have confirmed that acclimatization results in a decrease in lactate release regardless of whether the exercise is performed in hypoxia or normoxia (3). In addition, measurements using stable isotopes have supported decreased lactate production from the contracting muscle with acclimatization (6).

The possibility that decreases in glycolytic flux may have occurred with acclimatization, contributing, at least partly, to the lower muscle lactate observed with exercise, is supported by the metabolite profile. The more preserved PCr levels observed following the expedition resulted in a lower P_i and would be expected to result in a lower free ADP and AMP (1, 7). These metabolites have been implicated in the activation of both phosphorylase and phosphofructokinase (7), the enzymes controlling glycogenolysis and glycolysis, respectively (26). Reductions in glycogenolysis would be expected to result in a higher muscle glycogen level during exercise following acclimatization. Although this is what we observed, the higher level could not be attributed only to a decreased depletion rate. Increases in glucose oxidation, known to occur with acclimatization at least when tested under hypoxic conditions (5), also could have decreased the utilization of muscle glycogen stores.

The higher glycogen levels that we observed following acclimatization appear to be due to the higher resting concentration and not due to a differential depletion during the exercise itself. Because higher resting glycogen levels are not normally reported with short-term acclimatization (14), the increases were probably mediated following return to sea level.

It is noteworthy that the decreases in muscle lactate levels with acclimatization was observed early in the exercise. During this period, the lactate/pyruvate ratio was not reduced, suggesting that the cytosolic redox potential was not alleviated by acclimatization. The cytosolic redox potential has been used as an indication of cellular hypoxia (7). Conceivably, this could have occurred because of a downregulation in ATP utilization.

It has been postulated previously that the lower muscle lactate observed with chronic hypoxia results from a reorganization in the metabolic pathways, characterized by an increased pyruvate kinase to LDH ratio (17). It is highly unlikely that this occurred in the present study because LDH was elevated. Unfortunately, pyruvate kinase was not measured. Moreover, an upregulation in oxidative potential relative to the glycolytic potential also has been proposed as a meaningful adaptation (17, 28). In this study, as in previous studies, oxidative potential, as measured by the maximal activity of the marker enzyme CS or MDH (31), appeared unchanged. Because no change was observed in total phosphorylase, which exists in constant proportion with enzymes of the glycolytic pathway (11), acclimatization-induced reductions would not be expected. Unfortunately, technical problems and limitations in tissue availability prevented measurement of phosphofructokinase, recognized as the rate-limiting enzyme in glycolysis. It should be emphasized as well that the enzymes selected to characterize oxidative phosphorylation may not be rate limiting.

In summary, evidence has been provided that indicates that a mountaineering expedition elicits the same basic response in exercise muscle metabolism as observed during residence for a similar period of time at 4,300 m. Moreover, it also appears that the metabolic adaptations can be manifested at sea level, at least within the first 3 to 4 days after the expedition. However, it is suggested that the acclimatization effects on muscle metabolism, at least for mountaineering, occur as a result of an increase in work efficiency and not of reorganization of the metabolic pathways within the muscle.