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Am J Physiol Regul Integr Comp Physiol 291: R1100-R1110, 2006. First published May 11, 2006; doi:10.1152/ajpregu.00858.2005
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ENVIRONMENTAL, EXERCISE AND RESPIRATORY PHYSIOLOGY

Manipulation of dietary carbohydrates after prolonged effort modifies muscle sarcoplasmic reticulum responses in exercising males

T. A. Duhamel, J. G. Perco, and H. J. Green

Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada

Submitted 7 December 2005 ; accepted in final form 10 May 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 GRANTS
 REFERENCES
 
The hypothesis tested was that disturbances in the sarcoplasmic reticulum (SR) Ca2+-cycling responses to exercise would associate with muscle glycogen reserves. Ten untrained males [peak O2 consumption (VO2 peak) = 3.41 ± 0.20 (SE) l/min] performed a standardized cycle test (~70% VO2 peak) on two occasions, namely, following 4 days of a high (Hi CHO)- and 4 days of a low (Lo CHO)-carbohydrate diet. Both Hi CHO and Lo CHO were preceded by a session of prolonged exercise designed to deplete muscle glycogen. SR Ca2+ cycling in crude homogenates prepared from vastus lateralis samples indicated higher (P < 0.05) Ca2+ uptake (µM·g protein–1·min–1) in Hi CHO compared with Lo CHO at 30 min (2.93 ± 0.10 vs. 2.23 ± 0.12) and at 67 min (2.77 ± 0.16 vs. 2.10 ± 0.12) of exercise, the point of fatigue in Lo CHO. Similar effects (P < 0.05) were noted between conditions for maximal Ca2+-ATPase (µM·g protein–1·min–1) at 30 min (142 ± 8.5 vs. 107 ± 5.0) and at 67 min (130 ± 4.5 vs. 101 ± 4.7). Both phase 1 and phase 2 Ca2+ release were 23 and 37% higher (P < 0.05) at 30 min of exercise and 15 and 34% higher (P < 0.05), at 67 min during Hi CHO compared with Lo CHO, respectively. No differences between conditions were observed at rest for any of these SR properties. Total muscle glycogen (mmol glucosyl units/kg dry wt) was higher (P < 0.05) in Hi CHO compared with Lo CHO at rest (+36%), 30 min (+53%), and at 67 min (+44%) of cycling. These results indicate that exercise-induced reductions in SR Ca2+-cycling properties occur earlier in exercise during low glycogen states compared with high glycogen states.

calcium ion regulation; diet

PROLONGED MODERATE TO HEAVY exercise results in a disturbance in muscle sarcoplasmic reticulum (SR) Ca2+ cycling when assessed "in vitro" (3, 47). The disturbance in Ca2+ cycling consists of reductions in Ca2+ release (8–10) and in reductions in Ca2+ uptake (3, 8, 9, 50). Structural modifications to the Ca2+ release channel (CRC) or ryanodine receptor (11) and to the Ca2+-ATPase enzyme (27) appear to be, at least in part, responsible for the depression in Ca2+ release and Ca2+ uptake, respectively. Interestingly, at least in humans, prolonged exercise does not result in membrane damage as assessed by the ratio of the maximal Ca2+-ATPase activity (Vmax), measured in the presence and absence of the ionophore A-12387 (9). Similarly, no changes appear to occur in the binding affinity of the enzyme for Ca2+ as measured by the Hill coefficient (nH) and the Ca2+ needed to elicit 50% of Vmax (Ca50) (9). The efficiency of Ca2+-transport also appears to be protected during prolonged exercise since the ratio of Ca2+ uptake to Ca2+-ATPase activity does not change significantly (9). Collectively, these measurements suggest that, at least for Ca2+-uptake, the problem underlying the exercise-induced reduction is restricted to the Ca2+-ATPase activity and, specifically, to Vmax.

Prolonged moderate to heavy cycle exercise in humans also results in a progressive reduction in glycogen content in the contracting vastus lateralis muscle (20). The association between the changes in muscle glycogen and SR function has led to the speculation of a mechanistic link between the two. The speculation is supported by recent evidence associating the depletion of muscle glycogen with reduced Ca2+ transport properties in rat (24), mouse (4), and toad (45) skeletal muscle.

Two theories have been proposed to account for the association between glycogen and SR function. Both theories are based on the existence of a SR-glycogenolytic complex containing glycogen phosphorylase, glycogen debranching enzyme, many of the enzymes involved in the glycolytic pathway, and creatine phosphokinase (CPK), which is located in close proximity to the SR (52). One theory is that glycogen depletion may acutely regulate SR Ca2+ transport function as a result of disturbances in energy homeostasis in the region of the SR Ca2+-ATPase (6). Specifically, studies have been published demonstrating the role of high-energy phosphate transfer (35) and glycolysis (53) in supporting high phosphorylation potentials, which are needed for optimal Ca2+-ATPase function.

The second theory to account for the link between glycogen and SR Ca2+-handling properties is based on the structural alterations induced by direct effects of substrate loss (24) or by interrupting second messenger signaling pathways (25). According to this theory, it is the structural alterations that accompany the dissociation of the SR and glycogen-glycolytic complex that promote the dysregulation in SR behavior.

A limitation in the studies published to date (24) that have directly measured SR Ca2+-handling properties in vitro has been the failure to directly manipulate muscle glycogen reserves. In this study, we have used a session of prolonged exercise to substantially deplete glycogen reserves in the active muscles (14, 20) and employed either a low carbohydrate (CHO) diet to maintain low muscle glycogen concentrations or a diet high in CHO to induce a supercompensation (2). These protocols result in substantially different resting glycogen levels, which provide the opportunity to investigate the relationship of substrate reserve during prolonged fatiguing exercise. Interestingly, this appears to be the first study to have directly employed this strategy to examine the effects of glycogen on SR Ca2+ cycling.

The purpose of this study was to investigate the relationship between muscle glycogen concentration and SR Ca2+ regulation during prolonged moderate-intensity cycling exercise. We have hypothesized that, at similar exercise durations, the reductions in Vmax, Ca2+ uptake, and Ca2+ release would be attenuated in the high CHO vs. the low CHO dietary conditions. The changes in these properties would occur in the absence of changes in Ca2+ sensitivity, Ca2+ uptake efficiency, and membrane permeability for Ca2+.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 APPENDIX
 GRANTS
 REFERENCES
 
Subjects

Ten healthy, male volunteers who were recreationally active but not exercising on a regular basis (i.e., <1 time/wk) were recruited for the study. The physical characteristics of the subjects included age, 19.9 ± 0.5 (SE) yr; height, 183 ± 2.5 cm; and weight, 87.1 ± 2.4 kg. Maximal aerobic power (VO2 peak), as determined during a progressive cycle test to fatigue, was 3.41 ± 0.20 l/min and 39.2 ± 1.8 ml·kg–1·min–1. As required, the study was approved by the Office of Human Ethics and Animal Care, and all volunteers were made fully aware of all procedures before written consent was obtained.

Experimental Design

Two dietary conditions were investigated, namely high CHO (Hi CHO) and low CHO (Lo CHO). The diets, implemented for 4 days immediately after an initial session of prolonged fatiguing exercise designed to substantially deplete glycogen reserves in the contracting muscles, were introduced to manipulate muscle glycogen by varying the CHO intake over a 4-day period. After the 4-day period, a standardized prolonged cycling task to fatigue was performed. The experimental design required all participants to complete the Lo CHO trial before the Hi CHO trial. This was necessary so that tissue samples could be obtained during Hi CHO at a time matched to fatigue during Lo CHO. A minimum of 28 days separated each condition for each participant. Although this design invites the possibility of an order effect, we do not feel that this occurred. We have shown that, for all of the metabolic and SR measurements reported, normal values occur within days after prolonged exercise (Green, unpublished observations).

The Hb (g/100 g) was 16.4 ± 0.4 and 16.5 ± 0.4 for the Lo CHO and Hi CHO groups, respectively. Comparable values for Hct (%) were 46.0 ± 0.6 and 45.9 ± 0.9 for the two groups. No significant differences were observed between groups for either of the hematological properties.

Dietary Manipulation

To maximize the differences between diets for total CHO intake relative to fat-free mass (FFM) for each volunteer, we employed a protocol similar to that described by Tarnopolsky et al. (46). This protocol involved altering the average daily total caloric intake and also the relative contribution of total daily calories from CHO, fat, and protein sources. Generally, participants ingested a higher number of total calories and a higher percentage of total calories from CHO sources during Hi CHO compared with Lo CHO. For the Lo CHO condition, the dietary manipulation provided 1.4 ± 0.1 g CHO·kg FFM–1·day–1 for the 4-day period after the glycogen depletion protocol, whereas, during the Hi CHO condition, the volunteers ingested 9.8 ± 0.6 g CHO·kg FFM–1·day–1 during the 4-day period after the glycogen depletion protocol. To ensure that minimal brain glucose requirements were met, absolute CHO intake was always greater than 85 g CHO/day for all volunteers during Lo CHO. Participants were asked to limit their physical activity to low levels of exertion (i.e., walking) and were asked to refrain from ingestion of caffeine and alcohol for the 4-day period before testing.

Test Session Protocol

The prolonged exercise test sessions were conducted at approximately the same time of day for each participant. Approximately 3–4 h before reporting to the laboratory, participants ingested a meal replacement supplement (Ensure, 250 kcal; 53.7% CHO, 31.5% fat, 14.8% protein; Ross Laboratories, Montreal, Canada).

Participants reported to the laboratory ~60 min before the beginning of the prolonged cycling task in each condition. During this period, preparations were made for tissue sampling from the vastus lateralis using the needle biopsy technique under local anesthesia [2% xylocaine with epinephrine (Epi); see Ref. 1]. In Lo CHO, three sites were prepared (randomized between legs) for sampling at rest, at 30 min of exercise, and at the time of fatigue. During the Hi CHO condition, four sites were prepared for tissue sampling at rest, at 30 min of exercise, at time matching fatigue in Lo CHO and at fatigue during Hi CHO. Two separate samples were extracted at each time point to secure sufficient tissue (~100 mg) to be able to assess muscle metabolic and SR Ca2+-handling properties. The prolonged exercise task was performed at ~70% VO2 peak. Exercise was continued until volitional fatigue, defined as the point when the participant could not maintain at least 50 revolutions/min, with verbal encouragement. Blood and respiratory gas samples were collected before (0 min) and throughout the prolonged exercise task at regular intervals.

Analytical Techniques

Respiratory measurements. VO2 peak was measured during progressive cycle exercise to fatigue using procedures previously described in our laboratory (17). For the standardized cycling task, respiratory gas samples were collected for 4- to 5-min periods before exercise and at regular intervals throughout the exercise. Gas exchange [VO2, CO2 production (VCO2)] was measured using an open-circuit system that has been regularly employed by our group (22). The respiratory exchange ratio (RER) was defined as the ratio of VCO2 to VO2. All exercise tests were performed on an electrically braked cycle ergometer (Quinton 870) that was calibrated each testing day. Environmental conditions in the laboratory during test days averaged ~21°C and ~50% relative humidity.

Blood metabolites. Blood samples were obtained from a 20-gauge catheter (Angiocath) inserted in a dorsal hand vein for determination of Hb, hematocrit (Hct), glucose, lactate (Lac), and the catecholamines Epi and norepinephrine (NE). Hct was measured in triplicate using standard techniques and corrected for trapped plasma (0.96) and whole body Hct differences (0.91). Hb was also measured in triplicate using standard cyanomethemoglobin methods. Blood for glucose and Lac determinations was deproteinized by using ice-cold perchloric acid, centrifuged to remove the pelleted proteins, and neutralized with ice-cold KHCO3. Samples were stored at –80°C and subsequently analyzed by fluorometric techniques (26). Plasma Epi and NE concentrations were assessed using HPLC techniques with electrochemical detection as previously described (16).

Muscle metabolism. For analysis of the muscle metabolic response to exercise, only tissue samples that had been rapidly extracted from the vastus lateralis, plunged into liquid N2, and stored at –80°C were used. The measurement of glycogen, high-energy phosphates [ATP, phosphocreatine (PCr)], and the metabolites inorganic phosphate (Pi), creatine (Cr), and Lac were measured on freeze-dried tissue using fluorometric techniques after extraction by perchloric acid and neutralization by K2HPO4 (19). Total glycogen content (glucosyl units) was measured after hydrolysis with hydrochloric acid, whereas the assessment of the subfractions proglycogen and macroglycogen was based on the acid solubility characteristics as detailed by Marchand et al. (29). The concentration of inosine 5'-monophosphate (IMP) was 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 exercise nor experimental treatment (Hi CHO vs. Lo CHO) altered total Cr content.

SR. To characterize the effects of exercise and dietary manipulation on SR Ca2+-cycling responses, we have measured a variety of properties. These properties, all measured on whole muscle homogenates, included the kinetic properties of the Ca2+-ATPase, namely Vmax, the free Ca2+ concentration ([Ca2+]f) needed to obtain Ca50 and the slope of the relationship between Ca2+-ATPase activity, (using 20–80% of Vmax) and [Ca2+]f (nH). The ionophore ratio calculated by measuring Vmax, with and without the Ca2+ ionophore A-23187, provided a measure of changes in membrane permeability to Ca2+. Potential changes in isoform content of the Ca2+-ATPase were determined by comparing sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 1 and SERCA 2a between experimental conditions. For functional assessment of the SR, both Ca2+ uptake and Ca2+ release (phase 1 and phase 2) were analyzed. The apparent coupling ratio, defined as the ratio of Ca2+ uptake to Vmax, was used to provide an indication of the efficiency of Ca2+ transport in the SR. It should be noted that, in this study, coupling ratios were measured only at submaximal levels of [Ca2+]f.

Homogenates for measurement of SR properties were prepared from muscle samples (40–60 µg) diluted 11:1 (vt/wol) in ice-cold homogenizing buffer containing (in mmol/l): 250 sucrose, 5 HEPES, 0.2 phenylmethylsulfonyl fluoride, and 0.2% sodium azide (NaN3). The samples were hand homogenized with a Duall glass-on-glass hand homogenizer (Duall 20; Kontes). The homogenate was separated into multiple aliquots, quick-frozen in liquid N2, and stored at –80°C pending measurement of specific SR properties. Specific descriptions for the measurement of each of these properties appear in the APPENDIX.

All SR measurements were performed in duplicate, whereas the measurement of protein using the procedure of Lowry as modified by Schacterle and Pollock (39) was done in triplicate. As with the blood and muscle metabolite measurements, all samples for a given individual and a given property were measured during the same analytical session.

To examine for changes in Ca2+-ATPase content, the isoforms SERCA 1 and SERCA 2a were measured in homogenates from Lo CHO and Hi CHO conditions using SDS-PAGE and Western blotting. Immunoblotting was performed using the primary monoclonal antibodies specific for human SERCA 1 (MA3–912; Affinity Bioreagents) and SERCA 2a (MA3–919; Affinity Bioreagents). Each homogenate sample (10 µg) was loaded in each lane for SDS-PAGE with all samples analyzed in duplicate. For Western immunoblots, each sample was compared with the same standard that was comprised of a reference tissue sample from the vastus lateralis collected earlier and stored at –80°C. All samples for a given individual were run in duplicate on separate gels with a standard on the same day. The values were initially expressed as a percentage of the standard and then as a percent of Hi CHO. All procedures have been described in a recent publication from our laboratory (8).

Statistics. A one-way ANOVA was used to compare differences between the different sampling times within each condition. A two-way ANOVA was used to discriminate between differences resulting from dietary conditions and sampling time for matched samples (i.e., rest, 15 min, and 30 min of exercise and at a time corresponding to fatigue in LO CHO during both conditions). Where significant differences were found, Newman-Keul's post hoc procedures were used to compare specific means. Significance was accepted at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 APPENDIX
 GRANTS
 REFERENCES
 
Diet Analyses

As planned, the Hi CHO diet resulted in a higher average daily calorie intake, a higher percent CHO and lower percent fat and percent protein (Table 1). Both Lo CHO and Hi CHO were also different from the normal individual diet practiced before the experiment (habitual diet). The habitual diet was intermediate between the other two diets both in terms of average daily calorie intake and the percent calories derived from each of the three food categories.


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  		Table 1. Dietary analyses both before the experimental period and during the low-and high-carbohydrate manipulations

 
Endurance Time

Cycle time to fatigue was significantly longer after the Hi CHO diet compared with the Lo CHO diet. For the Lo CHO condition, cycle time was 66.7 ± 5.9 min compared with 103 ± 9.3 min for Hi CHO, an increase of ~54%.

Respiratory Gas Exchange

Although increases were observed from rest to exercise in VO2 and VCO2, no drifts beyond 15 min of exercise were found for either of these respiratory measurements (Fig. 1). The dietary manipulation was effective in altering the RER, both at rest and during exercise. At all matching time periods, RER was higher with the Hi CHO diet compared with Lo CHO diet. Time-dependent differences were also observed for RER. For Lo CHO, RER increased at 15 min of exercise compared with rest, remained stable during the next 15 min of exercise, and then declined at fatigue. For Hi CHO, the RER was also highest at 15 min of exercise and then declined progressively until fatigue.


Figure 1
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  		Fig. 1. Effects of dietary alterations after glycogen depletion exercise on respiratory gas exchange during prolonged cycling in males. A: O2 consumption (VO2); B: CO2 production (VCO2); C: respiratory exchange ratio (RER). Values are means ± SE (n = 10 subjects). Lo CHO, low-carbohydrate diet; Hi CHO, high-carbohydrate diet. The exercise times, 67 and 103 min, represent the average times to fatigue for the Lo CHO and Hi CHO conditions, respectively. Main effects (P < 0.05) were observed for time for VO2 and VCO2. For all variables, 0 < all time points. P < 0.05, significantly different from Lo CHO (#), significantly different from 0 min (*), significantly different from 15 min ({dagger}), significantly different from 30 min ({ddagger}), and significantly different from 67 min ({lozenge}).

 
Blood Metabolites and Hormones

For Lo CHO, reductions in blood glucose were observed at both 30 min and at 67 min, the point of fatigue in Lo CHO (Fig. 2). For Hi CHO, a reduction in blood glucose was observed only at 103 min of exercise, also the point of fatigue for this condition. A comparison between conditions indicated higher levels of blood glucose for Hi CHO at 30 min and at 67 min of exercise. Changes in blood Lac were only observed for exercise with no differences detected between conditions (Fig. 2). For blood Lac, increases were observed from rest throughout the exercise time points that were examined. This was a main effect that was independent of condition.


Figure 2
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  		Fig. 2. Blood glucose (A) and lactate (B) responses to prolonged cycling after glycogen-depleting exercise and dietary manipulation in males. Values are means ± SE (n = 10 subjects). The exercise times, 67 and 103 min, represent the average times to fatigue for the Lo CHO and Hi CHO conditions, respectively. P < 0.05, significantly different from 0 min (*), significantly different from 15 min ({dagger}), significantly different from 67 min ({lozenge}), and significantly different from Lo CHO (#). For blood lactate, a main effect (P < 0.05) of exercise was observed. For exercise, 0 < 15 = 30 = 67 min.

 
Both plasma Epi and NE increased with exercise, with the pattern dependent on the experimental condition (Fig. 3). During Lo CHO, increases in Epi were observed during the first 15 min of exercise followed by further increases at fatigue. In the case of Hi CHO, initial increases in Epi were also observed at 15 min followed by further increases at 67 and 103 min of exercise. Differences between conditions for Epi were only observed at 67 min of exercise where the concentration was higher in Lo CHO. For NE, increases were also observed in both conditions by 15 min of exercise. For Lo CHO, the increase in NE was progressive until the end of exercise. In the case of Hi CHO, further increases were more delayed, not observed until 67 min of exercise. This was followed by additional elevations as the exercise progressed to fatigue. At all exercise time points except 15 min, a lower NE was observed in Hi CHO compared with Lo CHO.


Figure 3
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  		Fig. 3. Plasma epinephrine (A) and norepinephrine (B) responses to prolonged cycling after glycogen-depleting exercise and dietary manipulation in males. Values are means ± SE (n = 10 subjects). The exercise times, 67 and 103 min, represent the average times to fatigue for the Lo CHO and Hi CHO conditions, respectively. P < 0.05, significantly different from 0 min (*), significantly different from 15 min ({dagger}), significantly different from 30 min ({ddagger}), significantly different from 67 min ({lozenge}), and significantly different from Lo CHO (#).

 
Muscle Substrate and Metabolites

Before and throughout the exercise, total muscle glycogen reserves were persistently higher in Hi CHO compared with Lo CHO (Fig. 4). For Lo CHO, glycogen concentrations were reduced at both 30 and 67 min of exercise. Progressive reductions in total glycogen were also observed throughout exercise in Hi CHO. Proglycogen responses to exercise and dietary manipulation followed the same pattern as total glycogen. Macroglycogen, which represented the smallest subfraction, was reduced during exercise in Hi CHO in a pattern similar to total glycogen and proglycogen. In Lo CHO, however, reductions were only observed at 30 min of exercise.


Figure 4
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  		Fig. 4. Effects of glycogen-depleting exercise and dietary manipulation on total muscle glycogen concentration and subfractions during prolonged cycling in males. A: total glycogen; B: proglycogen; C: macroglycogen. Values are means ± SE (n = 10 subjects). The exercise times, 67 and 103 min, represent the average times to fatigue for the Lo CHO and Hi CHO conditions, respectively. P < 0.05, significantly different from 0 min (*), significantly different from 30 min ({dagger}), significantly different from 67 min ({ddagger}), and significantly different from Lo CHO (#).

 
The concentration of ATP was not altered either by exercise or by condition (Table 2). For PCr, regardless of condition, exercise induced a reduction in concentration that was fully manifested at 30 min, the initial sampling point. No further changes were observed beyond this time. Both Cr and Pi displayed similar but opposite effects to PCr, namely an increase at 30 min of exercise with no further changes at fatigue (Lo CHO). However, at fatigue in Hi CHO, a reduction in Pi content was observed. No differences between conditions were observed for PCr or the metabolites Cr and Pi. The increase in muscle Lac with exercise was different between experimental conditions. In the case of Lo CHO, the increase observed at 30 min of exercise persisted until fatigue. For Hi CHO, the increase observed at 30 min was followed by progressive decreases at both 67 and 103 min of exercise. At 30 min and at 67 min of exercise, Lac was higher in Hi CHO compared with Lo CHO. For IMP, only an effect of exercise was observed. Exercise resulted in elevations of IMP regardless of condition.


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  		Table 2. Effects of glycogen-depleting exercise plus dietary manipulation on muscle high-energy phosphates and metabolites during prolonged exercise in males

 
SR Properties

Of the three properties used to characterize the kinetic characteristics of the Ca2+-ATPase, namely Vmax, nH, and Ca50, only Vmax was changed with exercise and dietary condition (Figs. 5 and 6). In the case of Lo CHO, a 32% reduction was observed in Vmax by 30 min of exercise. No further changes were observed beyond this time point. For Hi CHO, initial reductions were not detected until fatigue, namely 103 min. At fatigue, the reduction amounted to 27% compared with preexercise. Compared with Lo CHO, the Vmax was higher in Hi CHO at both 30 and 67 min.


Figure 5
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  		Fig. 5. Kinetic properties of the sarcoplasmic reticulum Ca2+-ATPase during prolonged cycling after a session of glycogen-depleting exercise and dietary manipulation. Values are means ± SE (n = 10 subjects). Vmax, maximal Ca2+-ATPase activity (A); Ca50, Ca2+ concentration at one-half Vmax (B). The Hill slope (C) was determined from Hill plots employing the relationship between Ca2+ concentration and Ca2+-ATPase activity using a section of the curve corresponding to 20–80% Vmax. P < 0.05, significantly different from 0 min (*), significantly different from 30 min ({dagger}), significantly different from 67 min ({ddagger}), and significantly different from Lo CHO (#). The exercise times, 67 and 103 min, represent the average times to fatigue for the Lo CHO and Hi CHO conditions, respectively.

 

Figure 6
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  		Fig. 6. Results of a typical volunteer illustrating the relationship between Ca2+-ATPase activity and Ca2+ concentration at matched cycling time points after glycogen-depleting exercise and either Lo CHO or Hi CHO dietary manipulation. The matched time point was at Lo CHO-fatigue, which represented an average 66.7 ± 5.9 min. See METHODS for a description of the kinetic properties that were obtained from the curves.

 
The changes in Vmax were also accompanied by changes in Ca2+ uptake during exercise but not at rest (Fig. 7). For Lo CHO, Ca2+ uptake was depressed by 32% at 30 min of exercise and by 36% at fatigue. In contrast, initial reductions in Ca2+ uptake in Hi CHO did not occur until 103 min of exercise. At fatigue in this condition, the reduction in Ca2+ uptake amounted to 24%. At 30 and 67 min of exercise, Ca2+ uptake was 31 and 32% higher in Hi CHO compared with Lo CHO.


Figure 7
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  		Fig. 7. Sarcoplasmic reticulum Ca2+ uptake responses during prolonged cycling after glycogen-depleting exercise and dietary manipulation. Values are means ± SE (n = 10 subjects). The exercise times, 67 and 103 min, represent the average times to fatigue for the Lo CHO and Hi CHO conditions, respectively. P < 0.05, significantly different from 0 min (*), significantly different from 30 min ({dagger}), significantly different from 67 min ({ddagger}), and significantly different from Lo CHO (#).

 
The apparent coupling ratio, an indirect measure of the energetic efficiency of Ca2+ transport, was not affected by either exercise or condition (Table 3). Similarly, the ionophore ratio, which is an indirect index of membrane damage, remained unchanged with exercise after the dietary manipulations (Table 3).


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  		Table 3. Coupling and ionophore ratios during prolonged cycling after glycogen-depleting exercise and dietary manipulation

 
Ca2+ release kinetics were assessed as two distinct phases as described by Tupling and Green (49). Phases 1 and 2 were both affected by exercise and the dietary condition (Fig. 8). For Lo CHO, the reduction in phase 1 of Ca2+ release was fully manifested by 30 min of exercise. In contrast, for Hi CHO, initial reductions in phase 1 Ca2+ release were not observed until 103 min of exercise. At 30 and 67 min of exercise, phase 1 Ca2+-release was higher in Hi CHO compared with Lo CHO. Differences were also observed between conditions for phase 2 Ca2+ release. At 30 and at 67 min of exercise, phase 2 Ca2+-release was higher in Hi CHO than Lo CHO. This occurred because of the more rapid reduction in Ca2+ release in Lo CHO compared with Hi CHO during exercise.


Figure 8
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  		Fig. 8. Effects of glycogen-depleting exercise plus dietary alterations on sarcoplasmic reticulum Ca2+ release during prolonged cycling. Values are means ± SE (n = 10 subjects). A: phase 1 Ca2+ release; B: phase 2 Ca2+ release. The exercise times, 67 and 103 min, represent the average times to fatigue for the Lo CHO and Hi CHO conditions, respectively. P < 0.05, significantly different from 0 min (*), significantly different from 30 min ({dagger}), significantly different from 67 min ({ddagger}), and significantly different from Lo CHO (#).

 
The Ca2+-ATPase isoform content, both SERCA 1 and SERCA 2a, was not different between conditions. When compared with the Hi CHO condition, which was standardized to 100%, SERCA 1 and SERCA 2a for Lo CHO were 104 ± 9.3% and 101 ± 9.8%, respectively.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
APPENDIX
 GRANTS
 REFERENCES
 
As hypothesized, we have found that manipulation of muscle glycogen levels by prior exercise and diet was associated with modification of the SR Ca2+-cycling responses to prolonged cycling. Specifically, our data indicate that exercise-induced reductions in SR Ca2+ uptake and Ca2+ release properties occur earlier in exercise during low glycogen states compared with high glycogen states. Furthermore, our data indicate that the exercise-induced reductions in SR Ca2+ uptake were mediated by reductions in SR Ca2+-ATPase activity, since reductions in this property followed a similar time line as that observed for Ca2+ uptake during both Lo CHO and Hi CHO. Because no differences were found between conditions at rest for any of the SR properties examined, it appears that an exercise-related stimulus contributed to the reductions in SR Ca2+-handling properties observed during exercise. Our data also indicate that neither exercise nor muscle glycogen content directly affects the sensitivity of the Ca2+-ATPase, as demonstrated by the lack of change in Ca50 and nH.

This study appears to be the first to investigate the relationship between muscle glycogen content and SR function in human skeletal muscle by directly manipulating muscle glycogen levels through a variation of a previously published exercise plus dietary protocol known to prolong exercise time to exhaustion (46). With our protocol, resting total muscle glycogen was 57% higher with the Hi CHO compared with Lo CHO. At comparable exercise time points, total glycogen levels in the Hi CHO remained between 112% (30 min) to 79% (fatigue-Lo CHO) elevated over Lo CHO. At fatigue, both Lo CHO and Hi CHO, no differences existed between conditions in glycogen concentration in the vastus lateralis. As in previous studies (15), initial total glycogen levels were associated with a large increase in endurance time (54%). Our study is the first to demonstrate that endogenous total glycogen levels directly impact SR Ca2+-handling properties (i.e., Ca2+ uptake and Ca2+ release) during prolonged exercise.

Although our observations do not allow for the identification of a specific mechanism to account for the suggested regulation of SR function by cellular glycogen concentration, these results do add to the growing body of evidence that suggests that cellular glycogen levels directly influence SR Ca2+ regulation. One prominent theory involves a direct link between the glycogen-glycolytic complex itself and SR regulation. Glycogen has been associated with the SR via a complex involving phosphorylase, glycogen synthase, glycogen debranching enzyme, and enzymes involved in the glycolytic pathway in addition to CPK (51, 52). Recently, a regulatory subunit (a muscle protein belonging to a family of glycogen and protein phosphatase 1 catalytic binding subunits) has been shown to bind glycogen and the glycogenolytic complex to the SR (37). The current wisdom is that, with reductions in cellular glycogen, the glycogenolytic complex is dislodged, resulting in structural modifications to the SR and changes in Ca2+-cycling behavior (24). Cuenda et al. (6) have reported that the dislocation of inactive glycogen phosphorylase from the SR-glycogenolytic complex, as a consequence of reductions in glycogen, results in structural alterations to the SR Ca2+-ATPase as mediated by changes in the region of the adenine nucleotide binding site of the enzyme.

It is possible that the progressive loss of glycogen observed during exercise in the current study also affected SR Ca2+-ATPase by a similar mechanism. Numerous studies, particularly in humans (3, 8, 9, 50), have reported reductions in Vmax during prolonged exercise. Moreover, with this type of exercise, the effect appears to be specific to Vmax since no changes were observed in the nH and Ca50, which are both measures of the Ca2+-binding affinity of the enzyme (8, 9). Previous studies have also demonstrated that the reduction in Vmax during sustained repetitive activity is associated with changes in the region of the adenine nucleotide binding site measured with the competitive inhibitor fluorescein isothiocyanate (27, 31). The changes in Vmax that we have observed in this study are consistent with a similar structural alteration. If such is the case, our finding suggests that the higher glycogen content observed during exercise with Hi CHO compared with Lo CHO protected the structural integrity of the enzyme in the region of the nucleotide binding site. Given the large differences in cellular glycogen that existed between conditions at rest, it might be expected that resting SR Ca2+-handling properties would be different. However, our data indicate that an exercise-related stimulus was required to trigger the observed difference in SR Ca2+-handling properties during exercise. It is possible that the depletion of glycogen during the first 30 min of exercise during Lo CHO caused glycogen levels to fall below a critical level of glycogen required to maintain SR function, thereby adversely affecting SR Ca2+-handling properties. Given that muscle glycogen content was elevated during the Hi CHO condition, it is possible that this critical limit of glycogen was not passed until the late stages of exercise (i.e., >67 min) during this condition, which may explain why the exercise-induced reductions in SR properties were observed much later during Hi CHO compared with Lo CHO.

The apparent coupling ratio, which we have defined as the ratio of the Ca2+ uptake measured at the highest [Ca2+]f, namely 2,000 nM, to Vmax was unaffected by exercise or by condition. This suggests that the efficiency of Ca2+ transport was unchanged with manipulation of total muscle glycogen either at rest or during exercise. One factor that can influence the energetic costs of inducing a net reduction of [Ca2+]f by transport in the SR is passive leak of Ca2+ from the CRC, the Ca2+-ATPase, or via the membrane (7, 30). Although we found no change in the ionophore ratio, calculated as the ratio of Ca2+-ATPase measured with and without the Ca2+-ionophore A-23187, a crude measure of membrane intactness, no measurements were made of leak from the CRC and the Ca2+-ATPase.

Our study also appears to be the first to report an effect of glycogen content on Ca2+ release in human skeletal muscle. We have found that, as with Ca2+ uptake, both phases 1 and 2 of Ca2+ release were depressed much earlier in exercise during Lo CHO compared with Hi CHO. Numerous studies have also documented exercise-induced reductions in Ca2+ release during prolonged exercise (10), apparently also as a result of structural modifications to the CRC (11). Assuming a similar effect of exercise in this study, it would appear that the cellular glycogen levels may also affect the functional characteristics of the CRC during exercise. At present, it is not clear how this would occur.

Also of potential significance in the interpretation of the prior exercise dietary regimes on SR Ca2+-cycling behavior during exercise are the substrate and energetic considerations. Conceivably, changes in substrate preference and/or phosphorylation potential could modify the effects of exercise on SR function. In this study, the level of VO2 during exercise was unchanged between conditions, indicating the same strain on the processes involved in oxidative phosphorylation. However, the Lo CHO condition, compared with the Hi CHO condition, resulted in a lower RER and, as a consequence, a higher calculated fat oxidation during exercise. It is possible that the difference in fat and/or CHO oxidation between conditions could, in isolation, selectively affect the SR responses to exercise. Few studies in the literature appear to have examined this possibility.

It is also possible that cellular glycogen status could have affected phosphorylation potential and/or metabolite accumulation in working muscle. To examine this possibility, we have measured the concentration of the high energy phosphates ATP and PCr and selected metabolites Pi, Cr, and Lac. We have also measured IMP, which is regarded as a more sensitive indicator of changes in ATP concentration (21). Although we found the expected changes in these compounds, given the intensity of exercise (32), only in the case of Lac could we find differences between Lo CHO and Hi CHO. As reported previously (44), Lac was higher during exercise in Hi CHO compared with Lo CHO, a difference that probably reflects a higher rate of glycogenolysis/glycolysis given the high concentration of the substrate glycogen (5). The elevation in Lac observed during exercise with Hi CHO is relatively modest. There is little evidence to suggest that Lac at this concentration could result in alterations in Ca2+-cycling function when assessed in vitro. Care must be exercised in the interpretation of these results since our measurements reflect global cellular measurements. As a consequence, it is possible that regional differences could have occurred, as an example, in the vicinity of the SR, where it has been shown that Ca2+ cycling is very much dependent on maintaining high ATP-to-ADP ratios and low accumulation of metabolites (23).

Also to be considered is the possible effect of the experimental manipulation on the behavior of the Ca2+-ATPase and the CRC as a result of second messenger regulation. In this study, we have confirmed the reports of others (13), namely that a Hi CHO compared with a Lo CHO diet after exercise results in a pronounced blunting of the time-dependent increase with exercise in both plasma Epi and NE concentration. Increases in Epi are known to result in a cAMP-mediated increase in the phosphorylation of phospholamban (28), a regulatory protein associated with the SERCA 2a isoform. Because the vastus lateralis in humans is composed of a mixture of fiber types (38), the homogenate would contain both SERCA 1 and SERCA 2a isoforms, and consequently some degree of phosphorylation of phospholamban would be expected. It is possible that a different level of phosphorylation of phospholamban occurred during exercise between the two conditions in this study given the large difference in Epi concentrations. However, the functional consequences are probably of minimal significance since we have reported that only Vmax was altered with exercise and condition, whereas the effects of phosphorylation of phospholamban appear to increase the affinity of the Ca2+ pump for Ca2+. Direct phosphorylation of the CRC has also been reported to increase the open state of the CRC (28, 34). Compared with the Hi CHO condition, the higher Epi levels in Lo CHO could potentially result in an increased open state of the CRC and increased Ca2+ release in vivo. It should be emphasized that it is not at all clear if these events possibly present "in vivo" are, in fact, measured in vitro.

There is emerging evidence that the glycogen subfractions, namely proglycogen and macroglycogen, may have special significance in cellular function (41). For this reason, we measured these subfractions in conjunction with total glycogen to determine if the change in a particular subfraction during exercise more closely paralleled the changes in SR Ca2+-cycling behavior. As expected, proglycogen represented the most predominant subfraction (29). In general, proglycogen utilization paralleled the time-dependent changes during exercise observed for total glycogen. However, macroglycogen utilization was attenuated after 30 min of exercise during Lo CHO compared with Hi CHO, which can be attributed to the lower availability of this glycogen subfraction during Lo CHO compared with Hi CHO (42). Although our data do not clearly indicate a role for glycogen subfractions in the regulation of SR function, the possibility still exists. It should be acknowledged that our measurements provide no indication as to whether there were regional differences in the deposition and utilization of the subfractions. Previous research by Fridén et al. (12) has shown that aerobic exercise promotes a more emphasized intermyofibrillar utilization, possibly in the region of the SR.

It should be emphasized that, since the vastus lateralis in humans is composed of a mixture of fiber types (38), the changes in SR function that we have observed with exercise could be restricted to a specific fiber type population. Conceivably, this could occur because of differences in recruitment between the different fiber types resulting in different glycogen reserves or because of fiber type differences in regulation of the Ca2+-ATPase and CRC. Previous studies employing prolonged exercise of comparable intensity have documented a progressive depletion in glycogen in both type 1 and type IIA fibers (18), which would be expected to represent in excess of 90% of the fiber population of the vastus lateralis (38). It is possible, however, that different rates of glycogen utilization in specific fiber type pools could result in a variable effect of glycogen on SR responses during exercise. The existence of different SERCA isoforms between the type I and type IIA fibers in combination with different regulatory control of the catalytic activity of the enzyme (47) could also result in different responses between the different fiber types. Unfortunately, the SR measurements that we performed are not possible at the single fiber level.

In summary, our results indicate that a preliminary session of exercise followed by 4 days of a diet rich in CHO (Hi CHO) or poor in CHO (Lo CHO) resulted in pronounced differences in resting muscle glycogen. The differences in muscle glycogen were also accompanied by differences in the SR Ca2+-cycling responses in working muscle. For both Ca2+ uptake and Ca2+ release (phases 1 and 2), exercise-induced reductions in SR function occurred earlier during low glycogen states compared with high glycogen states. The reduction in Ca2+ uptake appeared to be because of reductions in maximal Ca2+-ATPase activity, since exercise did not effect on Ca2+-binding affinity. We have also shown that the more pronounced disturbance in SR Ca2+-cycling behavior observed in muscles with low glycogen concentrations is also accompanied by a reduced time to fatigue, which occurred in the absence of changes in phosphorylation potential.

Perspectives

Our study appears to be the first to manipulate muscle glycogen reserves and to study the modifications that occur in SR function. Although our results are consistent for a role for this substrate in SR Ca2+ responses, other factors may also be involved. Conceivably, the exercise and dietary protocols that we have used could result in other changes in addition to muscle glycogen. By design, the dietary manipulation resulted in large differences between conditions in both the daily percentage and total calories derived from fat and CHO. Consequently, it is possible that the effects on SR Ca2+ cycling that we have observed between experimental treatments during exercise may not be specific to one substrate. It is also possible that the session of exercise used to reduce muscle glycogen may have affected SR function in a diet-specific manner. If such was the case, the effects would only apply to the exercise state since no differences were observed between conditions at rest on the SR properties assessed. The impact of our study must be put in context. Our results clearly demonstrate a relationship between glycogen level and SR function in muscles. As such, this study should serve as a catalyst to additional research design to systematically isolate the potential role of other factors that might be involved. In exercising humans, this task is not without difficulty.


   APPENDIX
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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GRANTS
 REFERENCES
 
The Ca2+-ATPase was measured at 37°C with the coupled enzyme system pyruvate kinase (PK)/lactate dehydrogenase (LDH) as previously described for whole homogenates (43, 48) using the following reaction buffer (in mmol/l): 200 KCI, 20 HEPES, 15 MgCl2, 1 EGTA, 10 NaN3, 5 ATP, and 10 phosphoenolpyruvate (PEP). The pH of the buffer was adjusted to 7.0 at 37°C. Just before starting the reaction, 18 U/ml LDH, 18 U/ml PK, 0.3 mmol/l NADH, 25 µl of homogenate, and 1 µmol/l Ca2+ ionophore A-23187 (Sigma C-7522) were added to a cuvette (containing 1 ml reaction buffer), and the assay was performed at 37°C and 340 nm (Shimadzu UV 160). The reaction was initiated by adding 1 µl of 100 mmol/l CaCl2. Vmax and Ca2+ dependency of Ca2+-ATPase was obtained by successive additions of 0.5 µl of 100 mmol/l CaCl2. The Ca2+-independent (basal) ATPase activity was measured in the presence of 40 µmol/l cyclopiazonic acid. The measurement of [Ca2+]f was assessed separately using dual-wavelength spectrofluorometry and the Ca2+ fluorescent dye indo 1. Calculation of the kinetic properties was as previously detailed (40).

Ca2+ uptake in the homogenate was also measured using the ratiometric method as originally described by O'Brien (33) and subsequently modified by our laboratory (40) based on dual-emission spectrofluorometry and indo 1. Muscle homogenate (40 µl) was added to a 2-ml reaction mixture containing (in mmol/l): 200 KCl, 20 HEPES, 10 NaN3, 10 PEP, 5 oxalate, 0.005 N,N,N',-N',-tetrakis(2-pyridyl-methyl)ethylenediamine 1.5 µmol/l indo 1, 18 U/ml LDH, and 18 U/ml PK. In addition, 2.5 µl of CaCl2 (10 mmol/l) were added to produce a consistent starting [Ca2+]f of ~3.5 µmol/l. To initiate the reaction, 5 mmol/l ATP were added. The maximal rate of Ca2+ uptake, obtained by differentiating the linear fit curve, was determined at [Ca2+]f corresponding to 500, 1,000, 1,500 ,and 2,000 nmol/l using a single assay. In our hands, the limited sensitivity of the indo 1 procedure prevented reliable measurements of Ca2+ uptake above 2,000 nmol/l. This procedure also uses oxalate to bind the Ca2+ taken up by the SR to avoid backinhibition of the enzyme (43).

Ca2+ release was measured in a single assay immediately after Ca2+ uptake according to the methods of Ruell et al. (36) with modifications from our laboratory (49). After active loading of the SR and plateauing of [Ca2+]f, 20 mmol/l of the Ca2+-releasing agent 4-chloro-m-cresol was added. This procedure produces a biphasic Ca2+ release, consisting of a rapid (phase 1) and a slow (phase 2) phase that can be quantified separately (49). These measurements have been performed with oxalate, which was used in the reaction mixture to assess Ca2+ uptake. A potential limitation of the assay is that the rate of dissociation of Ca2+ from oxalate could affect the kinetics of Ca2+ release. This is an unavoidable limitation of the assay. Loading Ca2+ in the lumen of the SR without Ca2+ takes 40–60 min in human muscle homogenates, a period during which the stability of the preparation becomes problematic (Green, unpublished observation).


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 GRANTS
REFERENCES
 
The financial support provided by the Gatorade Sports Science Institute (The Quaker Oats Company) for the research is gratefully acknowledged.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. J. Green, Dept. of Kinesiology, Univ. of Waterloo, Waterloo, Ontario, Canada N2L 3G1 (e-mail: green{at}healthy.uwaterloo.ca)

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.


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