High-altitude acclimation increases the triacylglycerol/fatty acid cycle at rest and during exercise

Grant B. McClelland1,3, Peter W. Hochachka1, Shannon P. Reidy2, and Jean-Michel Weber2

1 Department of Zoology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, 2 Department of Biology, University of Ottawa, Ottawa, Ontario, Canada K1N 6N5; and 3 Department of Integrative Biology, University of California, Berkeley, Berkeley, California 94720


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

High-altitude acclimation alters lipid metabolism during exercise, but it is unknown whether this involves changes in rates of lipolysis or reesterification, which form the triacylglycerol/fatty acid (TAG/FA) cycle. We combined indirect calorimetry with [2-3H]glycerol and [1-14C]palmitate infusions to simultaneously measure total lipid oxidation, lipolysis, and rate of appearance (Ra) of nonesterified fatty acids (NEFA) in high-altitude-acclimated (HA) rats exercising at 60% maximal O2 uptake (VO2 max). During exercise, relative total lipid oxidation (%VO2) equaled sea-level control (SL) values; however, acclimation greatly stimulated lipolysis (+75%) but had no effect on Ra NEFA. As a result, TAG/FA cycling increased (+119%), due solely to an increase in recycling (+144%) within adipocytes. There was no change in either group in these variables with the transition from rest to exercise. We conclude that, in HA, 1) acclimation is a potent stimulator of lipolysis; 2) rats do not modify TAG/FA cycling with the transition to exercise; and 3) in normoxia, HA and SL derive the same fraction of their total energy from lipids and carbohydrates.

rats; exercise intensity; lipid metabolism; lipolysis; nonesterified fatty acids; reesterification; carbohydrates; glycerol; oxidative fuel; maximal oxygen uptake


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE EFFECTS OF ENVIRONMENTAL HYPOXIA on metabolic fuel selection during exercise have attracted much attention, because their study provides fundamental insights into the complex relationship between oxygen and substrate supply to living cells. We have recently shown (14, 15) that the relative contribution of lipids and carbohydrates to energy metabolism is not affected by acclimation to hypoxia, even though favoring carbohydrates would offer significant savings in oxygen. Animals running at the same percentage of their aerobic capacity (%VO2 max) oxidize the same proportions of lipids and carbohydrates when exercising at their respective acclimation PO2. Therefore, high-altitude acclimation does not modify this seemingly robust relationship between exercise intensity (defined as %VO2 max) and the relative importance of the different oxidative fuels (3, 21). However, acclimation may have several other important effects on fuel selection that remain to be addressed. For example, it is not known whether the above conclusion still holds when both experimental groups, acclimated and nonacclimated, exercise under normoxic conditions. Furthermore, acclimation causes intriguing changes in lipid metabolism, even though relative fat oxidation appears to remain constant. In particular, plasma fatty acid (FA) concentration can be elevated through acclimation but not as a result of increased release into the circulation (15), and the mechanism behind this effect is not understood. It is conceivable that acclimation could modify the rate of lipolysis [FA mobilization from triacylglycerol (TAG)] or have an impact on FA reesterification, the only possible fate other than oxidation. Lipolysis always exceeds oxidative needs; therefore, a fraction of all the FAs released must be reesterified. Simultaneous flux through lipolysis and reesterification form the TAG/FA cycle, a substrate cycle known to play an important role in adjusting FA supply during exercise (28). Substrate cycles consist of two opposing reactions, require energy input, but result in no net generation of product. The role of such cycles in the regulation of fuel utilization has never been investigated in altitude-acclimated individuals. At sea level, the TAG/FA cycle allows lipid metabolism to adjust to changes in metabolic rate when the demand for FAs is rapidly modified. The percentage of all FAs released being reesterified drops from ~70% at rest to ~25% with the onset of moderate exercise, thereby increasing availability for oxidation (28). Conversely, percent reesterification increases to >90% after the end of prolonged exercise to prevent the overshoot in plasma FA concentration from reaching toxic levels (13, 28). It is not clear whether acclimation will influence TAG/FA cycling or whether this cycle will play a different role than in individuals exercising at sea level in normoxia. Decreasing cycling would save energy, and ultimately oxygen, but increased cycling may be the preferred response if, for instance, high-altitude acclimation triggers a decreased demand for circulatory FA with no change or an increase in lipolysis. Finally, two pathways are available for TAG/FA cycling: intracellular cycling (where the FAs remain in the cell in which they were released through lipolysis) and extracellular cycling (where reesterification occurs only after FA transit through the circulation). Interestingly, both pathways can be modified independently. In humans, for instance, burn trauma mainly affects intracellular cycling (27), whereas cold stress preferentially targets extracellular cycling (25). It is not clear which pathway(s) will be influenced by hypoxia acclimation if any.

Here, we use indirect calorimetry simultaneously with continuous tracer infusions in high-altitude-acclimated (HA) and sea-level control (SL) rats at rest and exercising at 60% VO2 max to address the following questions. 1) Does exercise intensity (defined as %VO2 max) also determine the relative importance of lipids and carbohydrates when SL and HA individuals are both exercised under normoxic conditions? 2) Does high-altitude acclimation have an effect on lipolytic rate in resting or running individuals? 3) Is TAG/FA cycling affected by acclimation, and, if so, what pathway(s) is/are implicated at rest and during exercise?


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. All procedures used in this study were approved by the animal ethics committees of the Universities of British Columbia and Ottawa. Twenty-four female Wistar rats (Charles River, Canada, St Hyacinthe, QC, Canada) were randomly assigned to two groups, one kept under normoxic, sea-level conditions (SL) and the other under hypobaric hypoxia equivalent to a high altitude of 4,300 m (HA). Each group had free access to food (Rodent Lab Diet, PMI Nutrition International, St. Louis, MO: protein, 28%; fat, 12%; carbohydrates (CHO), 60%), water, and a water supplement (Transgel, Charles River, St Hyacinthe, QC, Canada). The HA rats were acclimated to hypoxia by progressive decrease in pressure over a 10-day period, as described previously (14), by use of an on-line vacuum system (Animal Care and Veterinary Services, University of Ottawa). Once the final pressure of 450 mmHg was reached, it was not allowed to vary by more than ±10%, and the animals were acclimated to these conditions for 8 wk before experiments.

Exercise protocols. Aerobic capacity (VO2 max) was estimated from previous measurements on untrained rats. Because VO2 max is not significantly different between untrained SL and HA rats [unlike male Wistars, (1)] exercising in normoxia (SL = 82 ± 2, HA = 80 ± 3 ml · kg-1 · min-1, FIO2 = 0.2094; G. B. McClelland and P. W. Hochachka, unpublished data), both groups were run at the same average speed of 12.33 ± 0.13 m/min. Oxygen consumption (VO2) was monitored continuously, starting at a speed of 12.5 m/min. If VO2 failed to stabilize after 30 min of exercise, the speed was decreased by 0.5 m/min steps until a plateau was reached. Running speed was never decreased below 11 m/min.

Glycerol and nonesterified FA kinetics. When acclimation was completed, the animals had jugular and carotid artery catheters surgically implanted under halothane anesthesia, as described before (14). They were given buprenorphine (0.01 mg/kg) before and for 48 h after surgery. All rats were closely monitored and allowed to recover for 2-4 days after surgery. HA rats spent the first 24 h of recovery in normoxia before returning to the hypobaric chambers until the exercise trials 24-48 h later. On the experimental day, extensions were added to the catheters, and the animals were placed on a Plexiglas-enclosed treadmill (Columbus Instruments, Columbus, OH). Both catheters were fed out of this respirometer, and the jugular catheter was connected to a calibrated syringe pump (Harvard Apparatus, South Natick, MA) for tracer infusion. A resting infusion of [2-3H]glycerol (0.5-1.0 Ci/mmol) and [1-14C]palmitate (50-60 mCi/mmol) was performed at 0.6 ml/h for 60 min in the resting state. The infusion rate was then increased to 1.0 ml/h for 60 min of exercise and 2 min of postexercise recovery. The infusate was prepared in sterile physiological saline containing 1.1 µM delipidated albumin. Resting infusion rates of [2-3H]glycerol were 1,408,593 ± 18,523 and 1,200,164 ± 177,694 dpm · kg-1 · min-1 for SL and HA, respectively. [1-14C]palmitate infusion rates were 1,599,987 ± 20,713 and 1,673,479 ± 58,613 dpm · kg-1 · min-1. Infusion rates were increased for exercise and recovery (+67%) to minimize changes in specific activity (12). Arterial blood samples (250-300 µl each) were drawn after 50 min of resting infusion, after 15, 30, 45, and 60 min of exercise, and 2 min after the end of exercise. The samples were centrifuged immediately, and the plasma was stored in glass tubes at -20°C. Throughout the experiments, simultaneous measurements of VO2 and carbon dioxide production (VCO2) were made using an EcoOxymax system (Columbus Instruments), as detailed elsewhere (6). Gas exchange data were collected every 30 s for the final 15 min of the resting infusion, during exercise, and in recovery. At the end of each experiment, the animals were allowed to recover for >= 2 days (HA kept under hypobaric hypoxia), before the gas exchange measurements were repeated but in the absence of isotope infusion.

Sample analysis. Plasma samples were prepared as published previously (2). Briefly, 100-150 µl of plasma were placed in 25 ml of chloroform-methanol (Folch 2:1 vol/vol), shaken, filtered (Whatman no. 1), and rinsed with Folch (5 ml, 2:1 vol/vol). Distilled water (7.5 ml, or 0.25 × final volume) was added to each tube, and the layers were separated by centrifugation (833 g for 10 min). Chloroform (10 ml) was added to the aqueous phase, and methanol-water (7.5 ml; 40:30 vol/vol) was added to the organic phase. Phases were separated after centrifugation and dried on a rotating evaporator (Büchi RE 121 Rotovapor; 65-70°C; 130-150 rpm). Ten milliliters of 99% ethanol were added to the aqueous phase to aid evaporation. The aqueous phase was redissolved in ethanol-water (1:1 vol/vol), and total 3H activity was counted in the equivalent of 5 µl of plasma. To determine percent total 3H activity in glycerol, the equivalent of 15 µl of plasma was spotted on a silica gel plate (60 F254, Merck) for thin-layer chromatography. The plate was developed with Folch (40:24 vol/vol), and sections (1 × 1 cm) were scraped into separate vials before ethanol-water (3 ml; 1:1 vol/vol) and 8 ml of ACS II scintillation fluid (Packard) were added for counting. Glycerol concentration ([glycerol]) was determined spectrophotometrically (26). The organic phase was redissolved in hexane-isopropanol (3:2 vol/vol), and total 14C activity was determined. The equivalent of 100 µl of plasma was methylated to measure the concentrations of individual nonesterified FA ([NEFA]) by gas chromatography (2, 13).

Calculations. Rates of lipid and CHO oxidation were calculated from VO2 and VCO2 with the equations of Frayn (7), after correcting for nitrogen excretion by use of an average value of 0.1042 mg/min from 24-h-fasted rats (17). The rate of appearance (Ra) of palmitate was calculated with the steady-state equation of Steele (23). Ra NEFA was calculated as Ra palmitate divided by the fractional contribution of palmitate to total NEFA. Ra glycerol was calculated using the steady-state (rest and exercise) or the non-steady-state equations (recovery) of Steele (23), assuming a volume of distribution of 150 ml/kg (5). Total, intra- and extracellular TAG/FA cycling were calculated as in Wolfe et al. (28). Results were analyzed using t-test and one- or two-way analyses of variance (ANOVA). When tests for normality failed, a Mann-Whitney ranked-sum test or a Kruskal-Wallis ANOVA was used. Pairwise multiple comparisons were made with the Student-Newman-Keuls or Dunnett's methods. All percentages were arcsine square root transformed. Bonferroni-adjusted levels of significance were used to take into account the number of tests performed. All the values presented are means ± SE.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Respirometry. VO2, VCO2, and respiratory exchange ratios (RER) of SL and HA rats running in normoxia at 60% VO2 max appear in Fig. 1. Values are averages of all exercise trials, pooled from experiments with and without tracer infusions. Exercise VO2, VCO2, and RER values were not significantly different between experiments when tracer infusion was or was not performed, for both HA and SL rats (P > 0.05). There were no significant differences in VO2, VCO2, or RER between the two groups of rats at any individual time point before, during, and after exercise (P > 0.05; Fig. 1). At the beginning of exercise, mean RER values were 0.858 ± 0.012 and 0.853 ± 0.011, whereas at the end of exercise they were 0.797 ± 0.011 and 0.821 ± 0.006 in SL and HA, respectively (Fig. 1). Likewise, the rates of CHO oxidation were not significantly different between SL and HA (P > 0.05). CHO oxidation went from 757 ± 61 and 728 ± 56 µM O2 · kg-1 · min-1 at 15 min of exercise to 432 ± 87 and 531 ± 36 at the end of exercise in SL and HA, respectively (Fig. 2A). The percent contribution of CHO to VO2 was also not different between SL and HA (Fig. 2B; P > 0.05). At the beginning of exercise, CHO oxidation contributed to 46 ± 3 and 45 ± 3% of VO2 in SL and HA, respectively, and it decreased to 36 ± 2% (HA) and 28 ± 4% (SL) by the end of exercise. At the beginning of exercise, absolute rates of lipid oxidation were not different between the two groups (796 ± 60 vs. 704 ± 34 µM O2 · kg-1 · min-1 for SL and HA, respectively; P > 0.05). These rates increased during activity to reach 938 ± 27 (SL) and 827 ± 38 µM O2 · kg-1 · min-1 (HA) by the end of exercise (Fig. 3A). Expressed as relative contributions to total metabolic rate, they were 48 ± 3 and 46 ± 3% of VO2 (SL vs. HA) initially but increased to 63 ± 4 and 56 ± 2% (SL vs. HA) by the end of exercise (Fig. 3B).


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Fig. 1.   Oxygen consumption (VO2; A), carbon dioxide production (VCO2; B), and respiratory exchange ratio [(RER) = VO2/VCO2; C] in high-altitude-acclimated (HA, ) and sea-level control (SL, ) rats during exercise at 60% of their estimated maximal O2 uptake (VO2 max); n = 13 for SL rats, and n = 7 for HA rats. REC, recovery. Values presented are means ± SE.



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Fig. 2.   Total carbohydrate (CHO) oxidation (µmol O2 · kg-1 · min-1; A) and expressed relative to total VO2 (%; B) in rats running at 60% VO2 max. Symbols are the same as in Fig. 1.



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Fig. 3.   Total lipid oxidation (µmol O2 · kg-1 · min-1; A) and expressed relative to total VO2 (%; B) in rats running at 60% of their estimated VO2 max. Symbols are the same as in Fig. 1.

Glycerol and NEFA kinetics. Table 1 shows average resting and exercise values for NEFA and glycerol metabolism. Although resting [NEFA] (P = 0.34) and [glycerol] (P = 0.07) were not different, during exercise, HA animals had a lower mean NEFA concentration (P = 0.002) and a higher mean [glycerol] (P < 0.001) than the SL controls. Mean Ra NEFA was the same in the two groups (47 ± 3 vs. 48 ± 3 µM · kg-1 · min-1, P = 0.096), but lipolytic rate was significantly higher in HA than in SL (Ra glycerol of 56 ± 5 vs. 32 ± 2 µM · kg-1 · min-1; P < 0.001).

                              
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Table 1.   Average resting and exercise values for NEFA and glycerol metabolism

Temporal changes in glycerol concentration, specific activity, and Ra are shown in Fig. 4. Palmitate concentration ([palmitate]) and Ra palmitate were not different between SL and HA (Fig. 5, A and C; P > 0.05). On the other hand, [NEFA] was lower in HA than in SL after 45 and 60 min of exercise (Fig. 6A; P = 0.012) and during recovery (P = 0.02). Ra NEFA was not significantly different between the two groups at any time (Fig. 6C; P > 0.05).


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Fig. 4.   Plasma concentration (mM; A), specific activity (dpm/mM; B), and rate of appearance (Ra) of glycerol (µmol · kg-1 · min-1; C) before (REST), during, and after (REC, recovery) 60 min of exercise at 60% of estimated VO2 max under normoxia. Rats were SL (, n = 7) or HA (, n = 8). *Significantly different from SL (P < 0.05).



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Fig. 5.   Plasma concentration (µmol/l; A), specific activity (dpm/mM; B), and Ra palmitate (µmol · kg-1 · min-1; C) before (REST), during, and after (REC) 60 min of exercise at 60% of estimated VO2 max under normoxia. Rats were SL (n = 7) or HA (n = 8). Symbols are the same as in Fig. 4.



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Fig. 6.   Plasma concentration (µmol/l; A) and Ra of total nonesterified fatty acids (Ra NEFA; µmol · kg-1 · min-1; B) before (REST), during, and after (REC) 60 min of exercise at 60% of estimated VO2 max under normoxia. Rats were SL (n = 7) or HA (n = 8). Symbols are the same as in Fig. 4. *Significantly different from SL (P < 0.05).

TAG/FA cycling. In the resting state, mean total TAG/FA cycling was increased in HA but not to a point significantly different from SL (P = 0.23), whereas the percentage of FA recycled was the same in the two groups (67 ± 12 and 65 ± 6% for HA and SL, respectively; P > 0.05). During exercise, total cycling in HA (138 ± 15 vs. 63 ± 6 in SL; P < 0.001) and percent recycling (74 ± 1 vs. 67 ± 3% in SL; P < 0.001) were both higher (Fig. 7). These differences were entirely due to an acclimation-induced increase in intracellular TAG/FA cycling (from 50 ± 8 to 122 ± 15 µM · kg-1 · min-1 in SL vs. HA; P = 0.001), because extracellular cycling was not different between the two groups (P > 0.05). Intra- and extracellular cycling over time are shown in Fig. 8. None of the parameters related to TAG/FA cycling (i.e., absolute total cycling, %recycled, intra-, and extracellular cycling) showed a significant change between rest and exercise in either group (Table 1; P > 0.05).


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Fig. 7.   Rate of total fatty acids (FA) recycled (µmol · kg-1 · min-1; A) and percentage of FA recycled back into triacylglycerol (TAG; B) before (REST), during, and after (REC) 60 min of exercise at 60% of estimated VO2 max under normoxia. Rats were SL (n = 5) or HA (n = 8). Symbols are the same as in Fig. 4.



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Fig. 8.   Rate of intracellular FA recycling (µmol · kg-1 · min-1; A) within adipocytes and rate of extracellular FA recycling (B) before (REST), during, and after (REC) 60 min of exercise at 60% of estimated VO2 max under normoxia. Rats were SL (n = 5) or HA (n = 8). Symbols arethe same as in Fig. 4.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study supports current models of fuel selection showing that mammals exercising at the same percent VO2 max derive the same fraction of their total energy from lipids and carbohydrates, (3, 21). Animals whose aerobic capacity is decreased in hypoxia, even after prolonged exposure (HA), follow the same general pattern of fuel oxidation as SL controls (14, 15), even when HA rats are running under normoxic conditions (Figs. 2 and 3). We also show that acclimation to hypoxia strongly stimulates lipolysis (+75%) and the TAG/FA substrate cycle (+119%), whereas Ra NEFA remains unaffected. The increase in absolute (µmol FA reesterified · kg-1 · min-1) and relative cycling (%total FA released that are reesterified back to TAG) is entirely due to a sharp rise in intracellular cycling (+144%), because the extracellular component of the cycle is insensitive to altitude acclimation. However, the TAG/FA cycle is not involved in regulating FA availability in running rats, because its flux does not change significantly at the beginning of exercise or in early recovery, as it does in humans (28).

Oxidative fuel selection. During exercise, rates of lipid oxidation were lower (although not significantly different) after acclimation, but they were decreased in proportion to metabolic rate. This was reflected in the relative contributions of lipids and carbohydrates that were the same for SL and HA (Figs. 2 and 3). Therefore, the principle that relative exercise intensity (%VO2 max) determines the mixture of fuels oxidized appears to be very robust, because it has now been observed under many circumstances where aerobic capacity is modified experimentally. The same relationship between fuel selection and %VO2 max appears before (SL controls) as well as after acclimation to hypoxia (HA), and this conclusion holds true when HA animals run in normoxia (Figs. 2 and 3) or in hypoxia (14, 15). Moreover, it applies to all mammalian species measured to date, spanning a surprisingly wide range of body sizes and aerobic capacities (rats, goats, dogs, and humans; see Ref. 21). So far, the only clear exceptions are for individuals fed a very high fat diet over a long period of time who oxidize a significantly higher percentage of lipids than controls fed a normal diet (9, 21) and for women (8). Changes in fuel selection with increasing work intensity may reflect the progressive recruitment of different populations of muscle fibers. Therefore, future work examining correlations between fiber composition, fiber type recruitment, and fuel selection may provide important new insights on this issue. The disproportionately high reliance on lipids shown after feeding a high-fat diet may be explained by changes in muscle composition, fiber recruitment, or both.

Lipolysis and NEFA kinetics. This study is the first to investigate the effects of high-altitude acclimation on glycerol kinetics. It shows that plasma [glycerol] and lipolytic rate (Ra glycerol) are both significantly increased by HA acclimation (Table 1; Fig. 4), whereas plasma [NEFA] and Ra NEFA are not (Table 1; Figs. 5 and 6). Reasons for this response are unclear, because the demand for FA by working muscles is not higher in HA than in SL during exercise. Catecholamines, norepinephrine in particular, are well-known stimulators of lipolysis (22), and it is possible that their plasma concentration is increased after prolonged exposure to hypoxia. No such change was observed in humans after relatively acute exposure to hypoxia of <= 5 days (18), but individuals acclimated at 4,300 m for a 3-wk period had higher plasma norepinephrine levels than normoxic controls during running (20). Therefore, the higher lipolytic rate observed here in the HA group may have been caused by elevated plasma norepinephrine levels compared with SL. Unlike most other mammals measured to date (26, 28), rats did not show any significant change in lipolytic rate (Ra glycerol) or Ra NEFA between rest, exercise, and recovery (Figs. 4-6). Interestingly, the rate of total lipid oxidation increases in both groups with the onset of exercise, suggesting an increase in 1) extraction rates of circulating NEFA at the muscle, 2) the percentage of NEFA taken up by the muscle that was oxidized, or 3) the contribution of muscle TAG to overall energy expenditure. This latter scenario seems the most likely, on the basis of the low Ra NEFA and higher postexercise muscle TAG depletions seen in trained HA rats running under hypoxic conditions (15). However, we cannot exclude the possibility that an increase in Ra glycerol takes place in this species, for example at lower exercise intensity or when work is prolonged for more than 60 min.

TAG/FA cycling. Our results show that the TAG/FA substrate cycle is very active in rats at rest and during moderate exercise (Figs. 7 and 8). Most of the reesterification takes place directly within the cells where the FA are originally released by lipolysis (77-89% of total cycling is intracellular; Table 1), similar to recent data on fasted rats at rest [57% intracellular (10)]. The extracellular reesterification pathway is a minor component of the TAG/FA cycle (only 11-23% of total reesterification takes place after FA transit through the circulation), and absolute flux through this pathway does not change with acclimation to hypoxia (Table 1). The most dramatic effect of high-altitude acclimation on lipid metabolism is a 119% increase in intracellular TAG/FA cycling (Table 1; Figs. 7 and 8), and this response is directly linked to the stimulation of lipolysis (Fig. 4), because Ra NEFA remains unchanged (Figs. 5 and 6). This may not be unique to rats, since a human acclimation study found that resting [glycerol] was higher, whereas [NEFA] did not change, suggesting an increase in recycling as well (29).

Regulation of TAG/FA cycling. Regulation of the TAG/FA cycle is poorly understood but is probably complex, involving many factors. Some of the potential regulators include hormones, substrate concentrations, adipose tissue blood flow, and the interactions among them. None of these factors has been studied in relation to substrate cycling after altitude acclimation. At SL, esterification of FA to TAG in adipose tissue is stimulated by insulin and the production of glycerol 3-phosphate via glycolysis, but insulin can also inhibit hormone-sensitive lipase. Hyperglycemia is thought to increase reesterification by insulin action and to a greater extent than it decreases lipolysis, so that TAG/FA cycling increases (16). Insulin is increased during acclimation (22), but it is not known whether [glucose] was higher in HA rats in the experiments presented here. However, [glucose] did not change during exercise before or after acclimation in trained rats (14). Interestingly, in isolated perfused adipose tissue [lactate] enhances reesterification without affecting glycerol release (4). Low [lactate] seen in most acclimation studies and equivalent [lactate] in SL and HA rats (14) point to other mechanisms as potential regulators of TAG/FA cycling. As mentioned before, lipolysis is stimulated via a beta -adrenergic mechanism normally turned on with exercise by epinephrine or sympathetic stimulation. Catecholamines also affect adipose tissue blood flow (11), via an alpha -adrenergic mechanism, and may be limiting to the release of FA into the circulation by decreasing the availability of albumin binding sites. As well, high plasma [NEFA] are thought to feed back on adipose tissue and increase the rate of TAG/FA cycling, lipolysis being unaffected. [NEFA] is the same or lower in HA than in SL (Fig. 6), suggesting that this is not the mechanism at work here. Leptin also plays an important role in the control of TAG homeostasis in adipocytes and nonadipocytes (19, 24). Very recent work shows that leptin administration stimulates TAG/FA cycling in rabbits (S. P. Reidy and J.-M. Weber, unpublished observations). Therefore, it is conceivable that the activation of TAG/FA cycling observed after acclimation to hypoxia is mediated by an increase in circulating leptin concentration. The question of how hormones, or other factors in general, are affected by altitude acclimation and the influence of exercise intensity remains to be fully investigated.

Cost of TAG/FA cycling. Substrate cycles require energy input and the cost of the TAG/FA cycle has been estimated at 144 kcal/mol of triacylglycerol recycled. In men, the cost is fairly low at 1.2% of total energy expenditure at rest, but this value drops during exercise to 0.5% due to the increase in metabolism and decrease in percent FA recycled (28). Even though the cost is low, decreasing this cycle may be beneficial at altitude to save energy under hypoxic conditions. As we have shown, acclimation increases the rate of the TAG/FA cycling with no change in resting VO2 (Fig. 1; Table 1). Therefore, the cost of this cycle was higher in HA at 7.4% of total energy expenditure compared with 2.9% at SL. During exercise, FA recycling remains higher in HA rats, but metabolic rate increases in both groups. This results in the cost's dropping to 4.1 and 1.8% of metabolic rate in HA and SL, respectively. The differences between SL and HA rats and the difference between rats and humans remain to be explained. Moreover, increased recycling in humans is accompanied by an increase in metabolic rate. This was not seen here in these rats; in fact, there was a decrease in the average metabolic rate during exercise despite an over twofold increase in the TAG/FA cycle. HA rats must presumably decrease the rate of other, yet-unknown, cellular processes to avoid hypermetabolism.

Conclusions and implications. This is the first study to measure the TAG/FA cycle after high-altitude acclimation and the first to measure this cycle and the rate of lipolysis in running rats. High-altitude acclimation alone greatly stimulates lipolysis. The FA released must be reesterified back to TAG within the adipocyte, because Ra NEFA is unaffected by acclimation. This high rate of cycling may be necessary to protect against dangerously high intracellular FA concentrations. However, the TAG/FA cycle is not used by rats to increase the availability of FA at the onset of exercise when the demand for fatty acids increases. The regulation of this response is currently unknown and warrants further investigation. Finally, the general model that relative exercise intensity (%VO2 max) controls substrate oxidation rates is supported by this study on HA rats running under normoxia. Evidence for this model as a generalized condition found in all mammals under all conditions is growing. Indeed, it may be the exceptions to this rule that offer the most fertile paths for future research.


    ACKNOWLEDGEMENTS

G. McClelland was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) postgraduate scholarship and a University of British Columbia Graduate Fellowship. This study was funded through NSERC operating grants to J.-M. Weber and P. W. Hochachka.


    FOOTNOTES

Address for reprint requests and other correspondence: G. McClelland, Dept. of Integrative Biology, Univ. of California, Berkeley, 3060 Valley Life Sciences Bldg. #3140, Berkeley, CA, 94720-3140 (E-mail: grantm{at}socrates.berkeley.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 27 November 2000; accepted in final form 9 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abdelmalki, A, Fimbel S, Mayet-Sornay MH, Sempore B, and Favier R. Aerobic capacity and skeletal muscle properties of normoxic and hypoxic rats in response to training. Pflügers Arch 431: 671-679, 1996[ISI][Medline].

2.   Bernard, SF, Reidy SP, Zwingelstein G, and Weber JM. Glycerol and fatty acid kinetics in rainbow trout: effects of endurance swimming. J Exp Biol 202: 279-288, 1999[Abstract/Free Full Text].

3.   Brooks, GA, and Mercier J. Balance of carbohydrate and lipid utilization during exerice: the "crossover" concept. J Appl Physiol 76: 2253-2261, 1994[Abstract/Free Full Text].

4.   Bülow, J. Lipid mobilization and utilization. In: Principles of Exercise Biochemistry, edited by Poortmans JR. Basel, Switzerland: Karger, 1988, p. 140-163.

5.   Coppack, SW, Persson M, Judd RL, and Miles JM. Glycerol and nonesterified fatty acid metabolism in human muscle and adipose tissue in vivo. Am J Physiol Endocrinol Metab 276: E233-E240, 1999[Abstract/Free Full Text].

6.   Fournier, RA, and Weber JM. Locomotory energetics and metabolic fuel reserves of the Virginia opossum. J Exp Biol 197: 1-16, 1994[Abstract/Free Full Text].

7.   Frayn, KN. Calculation of substrate oxidation rates in vivo from gaseous exchange. J Appl Physiol 55: 628-634, 1983[Abstract/Free Full Text].

8.   Horton, TJ, Pagliassotti MJ, Hobbs K, and Hill JO. Fuel metabolism in men and women during and after long-duration exercise. J Appl Physiol 85: 1823-1832, 1998[Abstract/Free Full Text].

9.   Jansson, E, and Kaijer L. Effect of diet on the utilization of blood-borne and intramuscular substrates during exercise in man. Acta Physiol Scand 115: 19-30, 1982[ISI][Medline].

10.   Kalderon, B, Mayorek N, Berry E, Zevit N, and Bar-Tana J. Fatty acid cycling in the fasted rat. Am J Physiol Endocrinol Metab 279: E221-E227, 2000[Abstract/Free Full Text].

11.   Larsen, T, Myhre K, Vik-Mo H, and Mjos OD. Adipose tissue perfusion and fatty acid release in exercising rats. Acta Physiol Scand 113: 111-116, 1981[ISI][Medline].

12.   Levy, JC, Brown G, Matthews DR, and Turner RC. Hepatic glucose output in humans measured with labeled glucose to reduce negative errors. Am J Physiol Endocrinol Metab 257: E531-E540, 1989[Abstract/Free Full Text].

13.   McClelland, G, Zwingelstein G, Taylor CR, and Weber JM. Increased capacity for circulatory fatty acid transport in a highly aerobic mammal. Am J Physiol Regulatory Integrative Comp Physiol 266: R1280-R1286, 1994[Abstract/Free Full Text].

14.   McClelland, GB, Hochachka PW, and Weber JM. Carbohydrate utilization during exercise after high-altitude acclimation: a new perspective. Proc Natl Acad Sci USA 95: 10288-10293, 1998[Abstract/Free Full Text].

15.   McClelland, GB, Hochachka PW, and Weber JM. Effect of high-altitude acclimation on NEFA turnover and lipid utilization during exercise in rats. Am J Physiol Endocrinol Metab 277: E1095-E1102, 1999[Abstract/Free Full Text].

16.   Miyoshi, H, Shulman GI, Peters EJ, Elahi D, and Wolfe RR. Hormonal control of substrate cycling in humans. J Clin Invest 81: 1545-1555, 1988[ISI][Medline].

17.   Parrilla, R. Flux of metabolic fuels during starvation in the rat. Pflügers Arch 374: 3-7, 1978[ISI][Medline].

18.   Raff, H. Endocrine adaptation to hypoxia. In: Handbook of Physiology, edited by Fregly MJ, and Blatteis CM. New York: Oxford U, 1996, p. 1259-1275.

19.   Reidy, SP, and Weber JM. Leptin: an essential regulator of lipid metabolism. Comp Biochem Physiol A Physiol 125: 285-297, 2000[ISI].

20.   Roberts, AC, Reeves JT, Butterfield GE, Mazzeo RS, Sutton JR, Wolfel EE, and Brooks GA. Altitude and beta -blockade augment glucose utilization during submaximal exercise. J Appl Physiol 80: 605-615, 1996[Abstract/Free Full Text].

21.   Roberts, TJ, Weber JM, Hoppeler H, Weibel ER, and Taylor CR. Design of oxygen and substrate pathways. II. Defining the upper limits of carbohydrate and fat oxidation. J Exp Biol 199: 1651-1658, 1996[Abstract/Free Full Text].

22.   Sawhney, RC, Malhotra AS, and Singh T. Glucoregulatory hormones in man at high altitude. Eur J Appl Physiol 62: 286-291, 1991.

23.   Steele, R. Influences of glucose loading and of injected insulin on hepatic glucose output. Ann NY Acad Sci 82: 420-430, 1959[ISI].

24.   Unger, RH, Zhou YT, and Orci L. Regulation of fatty acid homeostasis in cells: novel role of leptin. Proc Natl Acad Sci USA 96: 2327-2332, 1999[Abstract/Free Full Text].

25.   Vallerand, AL, Zamecnik J, Jones PJH, and Jacobs I. Cold stress increases lipolysis, FFA Ra and TG/FFA cycling in humans. Aviat Space Environ Med 70: 42-50, 1999[ISI][Medline].

26.   Weber, JM, Roberts TJ, and Taylor CR. Mismatch between lipid mobilization and oxidation: glycerol kinetics in running African goats. Am J Physiol Regulatory Integrative Comp Physiol 264: R797-R803, 1993[Abstract/Free Full Text].

27.   Wolfe, RR, Herndon DN, Jahoor F, Miyoshi H, and Wolfe M. Effect of severe burn injury on substrate cycling by glucose and fatty acids. N Engl J Med 317: 403-408, 1987[Abstract].

28.   Wolfe, RR, Klein S, Carraro F, and Weber JM. Role of triglyceride-fatty acid cycle in controlling fat metabolism in humans during and after exercise. Am J Physiol Endocrinol Metab 258: E382-E389, 1990[Abstract/Free Full Text].

29.   Young, PM, Sutton JR, Green HJ, Reeves JT, Rock PB, Houston CS, and Cymerman A. Operation Everest II: metabolic and hormonal responses to incremental exercise to exhaustion. J Appl Physiol 73: 2574-2579, 1992[Abstract/Free Full Text].


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