Effect of induced metabolic acidosis on human skeletal muscle metabolism during exercise

M. G. Hollidge-Horvat, M. L. Parolin, D. Wong, N. L. Jones, and G. J. F. Heigenhauser

Department of Medicine, McMaster University Medical Centre, Hamilton, Ontario, Canada L8N 3Z5


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The roles of pyruvate dehydrogenase (PDH), glycogen phosphorylase (Phos), and their regulators in lactate (Lac-) metabolism were examined during incremental exercise after ingestion of 0.3 g/kg of either NH4Cl [metabolic acidosis (ACID)] or CaCO3 [control (CON)]. Subjects were studied at rest, at rest postingestion, and during continuous steady-state cycling at three stages (15 min each): 30, 60, and 75% of maximal oxygen uptake. Radial artery and femoral venous blood samples, leg blood flow, and biopsies of the vastus lateralis were obtained during each power output. ACID resulted in significantly lower intramuscular concentration of [Lac-] (ACID 40.8 vs. CON 56.9 mmol/kg dry wt), arterial whole blood [Lac-] (ACID 4.7 vs. CON 6.5 mmol/l), and leg Lac- efflux (ACID 3.05 vs. CON 6.98 mmol · l-1 · min-1). The reduced intramuscular [Lac-] resulted from decreases in pyruvate production due to inhibition of glycogenolysis, at the level of Phos a, and phosphofructokinase, together with an increase in the amount of pyruvate oxidized relative to the total produced. The reduction in Phos a activity was mediated through decreases in transformation, decreases in free inorganic phosphate concentration, and decreases in the posttransformational allosteric regulator free AMP. Reduced PDH activity occurred with ACID and may have resulted from alterations in the concentrations of acetyl-CoA, free ADP, pyruvate, NADH, and H+, leading to greater relative activity of the kinase. The results demonstrate that imposed metabolic acidosis in skeletal muscle results in decreased Lac- production due to inhibition of glycogenolysis at the level of Phos and increased pyruvate oxidation at PDH.

glycogen phosphorylase; pyruvate dehydrogenase; carbohydrate; fat; lactate metabolism


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

SYSTEMIC ACIDOSIS in humans decreases lactate concentration ([Lac-]) in the blood during exercise (22, 40, 44, 48). Data extrapolated from animal studies have suggested that this results from changes in the rates of glycogenolysis, glycolysis, and Lac- efflux, but the mechanisms have not been elucidated (30, 47, 63). The reduction in [Lac-] has been attributed primarily to the influence of pH on glycolysis at the level of phosphofructokinase (PFK) (35, 37, 67), but the in vivo effects of acidosis on the regulatory enzymes glycogen phosphorylase (Phos) and pyruvate dehydrogenase (PDH) have not been established. Phos and PDH occupy key, flux-generating control points for glycogenolysis and oxidative phosphorylation in the tricarboxylic acid cycle (TCA), respectively, and therefore ultimately influence lactate production. Lactate accumulation results from conversion of nonoxidized pyruvate to lactate by lactate dehydrogenase (LDH) and as such will be influenced by both pyruvate production from glycogen and pyruvate oxidation by PDH (14, 41).

In an effort to clarify the nature of the effects of acidosis on lactate production, we chose an oral dose of ammonium chloride (NH4Cl) previously shown to induce a sufficient metabolic acidosis to influence plasma [Lac-] during exercise (40, 66). Continuous, dynamic steady-state exercise at low, moderate, and high intensity was chosen to follow the metabolic effects and compare fuel utilization against previously described carbohydrate and fat contributions at these intensities (55, 56). This is the first in vivo study in humans to examine the key regulatory enzymes and their controllers during continuous dynamic steady-state exercise under acidotic conditions.

The aim of the present study was, first, to determine the effect of metabolic acidosis on the key regulatory enzymes Phos and PDH and their respective controllers; second, to measure the effect of metabolic acidosis on glycolytic intermediates and muscle pyruvate production and oxidation; third, to measure muscle lactate accumulation, production, and efflux; and finally, to determine if acidosis has any effects on glucose uptake and free fatty acid (FFA) utilization during exercise.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects

Eight healthy male volunteers participated in the study [age, 27 ± 1.9 (SE) yr; height, 185 ± 3.4 cm; and weight, 82.7 ± 3.7 kg]. Written consent was obtained from each subject after explanation of the purposes and associated risks of the study protocol. The study was approved by the Ethics Committees of both McMaster University and McMaster University Medical Centre.

Preexperimental Protocol

All subjects completed an initial incremental maximal exercise test on a cycle ergometer to determine maximal oxygen uptake (VO2 max) and maximal work capacity with a metabolic measurement system (Quinton Q-Plex 2, Quinton Instruments, Seattle, WA). Mean VO2 max for the group was 3.6 ± 0.3 l/min. None of the subjects was well trained, but all participated in some form of regular activity. Each subject was instructed to refrain from caffeine, alcohol, and exercise 24 h before each trial, and studies were carried out at the same time of day.

Experimental Protocol

Each subject participated in two experimental trials separated by 2-3 wk and were randomized to receive capsules with either 0.3 g/kg of NH4Cl (ACID) or 0.3 g/kg of CaCO3 (CON). On the morning of each trial, the subjects reported to the laboratory after consumption of a standard light meal consisting primarily of carbohydrates. The exercise portion of the protocol consisted of three levels of continuous, steady-state exercise on a cycle ergometer at 30, 60, and 75% of VO2 max, each maintained for 15 min, which began after insertion of arterial and femoral venous catheters and ingestion of the required capsules (Fig. 1).


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Fig. 1.   Experimental protocol. Total of 2 trials were completed per subject. Either 0.3 g/kg CaCO3 (CON) or 0.3 g/kg NH4Cl (ACID) was ingested over time indicated, each constituting 1 trial. Arterial and femoral venous blood samples were taken at times indicated, as well as leg blood flow. Exercise bouts refer to 3 continuous exercise power outputs of 15 min each: 30, 60, and 75% indicate cycling intensity of 30, 60, and 75% maximal oxygen uptake (VO2 max), respectively. Muscle biopsies were taken at times indicated. Respiratory measurements were taken immediately after blood sample and blood flow.

A radial artery was catheterized with a Teflon catheter (20 gauge, 3.2 cm, Baxter, Irvine, CA) percutaneously after the area was anesthetized with 0.5 ml of 2% lidocaine without epinephrine (5). A femoral vein was catheterized percutaneously for insertion of the thermodilution catheter (model no. 93-135-6F, Baxter) with the Seldinger technique (5) after administration of 3-4 ml of lidocaine without epinephrine. Both the arterial and femoral venous catheters were maintained patent with sterile, nonheparinized, isotonic saline solution. Arterial and femoral venous blood samples were simultaneously taken at rest, postingestion, and during each of the three exercise bouts at 7 and 11 min. Single leg blood flow measurements were collected after blood sampling at the same time points. Single leg blood flow was determined with the thermodilution technique as described by Andersen and Saltin (1): 10 ml of nonheparinized isotonic saline were injected and leg blood flow was calculated by a portable cardiac output monitor (Spacelab, Redmond, WA). At least three measurements were recorded at each sampling point and then averaged.

A total of five percutaneous needle biopsies of the vastus lateralis were taken, one at rest, one at rest postingestion, and three during exercise at the end of each power output. The resting biopsies were obtained with the subject lying on a bed. The resting and exercise biopsies were obtained on opposite legs and then reversed for the second trial. Biopsy sites were prepared by making an incision through the deep fascia under local anesthetic (2% lidocaine without epinephrine) as described by Bergström (4). Respiratory measurements of ventilation (VE), rate of oxygen consumption (VO2), rate of CO2 production (VCO2) and respiratory exchange ratio (RER) were measured at 5 and 11 min of each exercise stage.

Muscle Analysis

Muscle samples were immediately frozen in liquid N2. A small piece (10-35 mg) was chipped from each biopsy (under liquid N2) for determination of the fraction of PDH in the active a form (PDHa) as previously described (15, 56). The remainder of the sample was freeze-dried, dissected free of blood and connective tissue, and powdered. One aliquot was analyzed for Phos activity according to the methods of Young et al. (69). Briefly, a 3- to 4-mg sample of muscle was homogenized at -20°C in 0.2 ml of 100 mM of Tris · HCl (pH 7.5) containing glycerol, potassium fluoride, and EDTA. Homogenates were then diluted with 0.8 ml of the same buffer without glycerol and homogenized further at 0°C. Total (a + b) Phos activity (measured in the presence of 3 mM AMP) and Phos in the active a form (Phos a; measured in the absence of added AMP) were measured at 30°C with a spectrophotometer. Maximum velocity (Vmax) was derived from the equation described by Lineweaver and Burk (45), 1/V = (Km/Vmax)(1/S) + (1/Vmax), where V is the initial reaction rate expressed in mmol · kg-1 · min dry wt-1, S is the inorganic phosphate (Pi) concentration in mmol/l, and Km is a Michaelis-Menten constant of 26.2 mmol/l. The mole fraction of Phos a is presented as a percentage and calculated from Vmax a/Vmax(a + b) × 100. Phos a measurements were made only on exercise samples for two reasons. First, resting samples must be kept at room temperature for ~30 s before freezing for accuracy, which would have required two additional biopsies (59). Ethically this was not acceptable for the provision of one measurement. Second, the changes at rest and postingestion were not a main focus of this study. A second aliquot was used to determine muscle glycogen fluorometrically with the enzymatic end-point method described by Bergmeyer (3). A third aliquot of dry muscle was extracted in 0.5 M PCA and 1 mM EDTA, neutralized to pH 7.0 with 2.2 M KHCO3, and analyzed for acetyl-CoA, free CoASH, total CoA, acetylcarnitine, free carnitine, and total carnitine according to the methods of Cederblad et al. (8). A fourth aliquot was used to determine ATP, pyruvate, lactate, phosphocreatine (PCr), creatine, glucose, glucose 6-phosphate (G-6-P), glucose 1-phosphate (G-1-P), fructose 6-phosphate (F-6-P), and glycerol 3-phosphate (G-3-P) concentrations using the methods described by Bergmeyer (3) and adapted for fluorometry. All muscle metabolites were normalized to the highest total creatine content for a given individual (<OVL>×</OVL>TCr = 133.4 ± 11.4 mmol/kg dry wt) to correct for nonmuscle contamination. Free contents of ADP and AMP (ADPf and AMPf, respectively) were calculated as described by Dudley et al. (20), with the reactants and equilibrium constants of the near-equilibrium reactions catalyzed by creatine kinase and adenylate kinase. ADPf was estimated with the measured ATP, PCr, and creatine contents and an estimated H+ concentration ([H+]) {calculated indirectly from muscle [Lac-] and [pyruvate] with the regression equation of Sahlin et al. (61)}. From this information, the concentration of AMPf was determined assuming a Keq of 1.05 for the adenylate kinase reaction. Free Pi content was calculated from the sum of the estimated resting free Pi concentration of 10.8 mmol/kg dry wt (20) and the Delta PCr, Delta G-6-P, Delta F-6-P, and Delta G-3-P between rest and each time point during exercise. For the purposes of ADPf, AMPf, and free Pi calculations, no differences were observed between the rest and postingestion values and therefore the mean of the two values was taken as the resting value.

Blood Sampling and Analysis

Arterial and femoral venous blood samples (~10 ml) were collected into heparinized plastic syringes and placed on ice. One portion (1-2 ml) of each blood sample was analyzed for blood gas determination (AVL 995 automatic blood gas analyzer), O2 and CO2 content (Cameron Instrument, Port Arkansas, TX), and hemoglobin (OSM3 Hemoximeter, Radiometer, Copenhagen, Denmark). A second portion of each sample was deproteinized with 6% perchloric acid and stored at -20°C until analysis for glucose, lactate, and glycerol according to the methods of Bergmeyer (3) adapted for fluorometry. The third portion of blood was immediately centrifuged at 15,900 g for 2 min, and the plasma supernatant was frozen and later analyzed for FFA (Wako, NEFA C test kit, Wako Chemical, Montreal, Canada). Hematocrit was determined on blood samples with a heparinized microcapillary tube centrifuged for 5 min at 15,000 g.

Leg Uptake and Release of Metabolites, O2, CO2, and Respiratory Quotient

Uptake and release of metabolites (glucose, glycerol, and lactate) were calculated from their whole blood measurements in arterial and femoral venous blood and leg blood flow according to the Fick equation. Because there were differences in the hematocrit over time within a condition and between matched arterial and femoral venous samples, venous samples were corrected for fluid shifts. Fluid shifts for the whole blood measurements were corrected with the differences in hemoglobin (Hb) to calculate a percent change in blood volume (%Delta BV) from the equation (assuming no change in intravascular hemoglobin) (29)
%&Dgr;BV = [(Hb<SUB>arterial</SUB>/Hb<SUB>venous</SUB>) − 1] × 100
This value was then multiplied by the measured venous value to yield a corrected value, which was used in determining flux for that metabolite. Plasma FFA venous values were also corrected with changes in plasma protein concentration to correct for changes in plasma water (29). The leg O2 uptake and CO2 production were calculated from their respective arterial and femoral venous content differences and blood flow.

Subjects exercised in steady state, and no significant differences occurred in blood flows or metabolite concentrations between the 7- and the 11-min sampling points at each power output; therefore, the two values were averaged to obtain one value for each exercise level. Reported values are for the single leg only.

Calculations

Flux through Phos was calculated from the differences in glycogen utilization divided by time. PDHa flux was estimated from the PDHa as measured in wet tissue and converted to dry tissue by use of the wet-to-dry ratio. Pyruvate production was calculated from the sum of the rates of glycogen breakdown and glucose uptake minus the sum of the rates of accumulation of muscle glucose, G-6-P, and F-6-P. Lactate production was calculated from the sum of the rates of muscle lactate accumulation and lactate release. Pyruvate oxidation was calculated as pyruvate production minus lactate production. All values are reported in millimoles per kilograms per minute dry weight and are for single leg only. All values were calculated in three carbon units and assume a wet muscle mass of 5 kg.

Intramuscular pH

Intramuscular pH (pHi) was calculated from the [Lac-] and [pyruvate] according to the methods of Harris and Hultman (28). However, Hultman et al. (34) found that calculated pHi is 0.2 pH units more than measured pHi with ingestion of the same dose of NH4Cl that we used. Therefore, our estimated values from the [Lac-] and [pyruvate] were calculated and then reduced by 0.2 pH units.

Statistical Analysis

Data were analyzed using two-way ANOVA with repeated measures (time × treatment) except were otherwise stated. When a significant F ratio was found, the Newman-Keuls post hoc test was used to compare means. Data are presented as means ± SE. Significance was accepted at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Muscle Metabolism

Phos. Phos a decreased as exercise intensity increased in both conditions. At 75% VO2 max, Phos a was significantly lower during ACID (ACID 21.6 ± 4.5 vs. CON 29.8 ± 5.9 mmol · kg-1 · min dry wt-1; Fig. 2).


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Fig. 2.   Phosphorylase a (Phos a) in mole fraction % during cycling at various power outputs. + Significantly different from CON at matched time points. There was a significant main effect of time with each condition. Values are means ± SE.

Glycogen. Resting and postingestion muscle glycogen levels were not different between conditions (Table 1). Muscle glycogen content decreased with increasing power output but to a significantly greater degree with CON (Table 1). During the complete exercise study, total muscle glycogen utilization was 327 ± 22 mmol/kg dry wt during CON compared with 229 ± 34 mmol/kg dry wt with ACID. This corresponded to a 30% sparing of glycogen during ACID. Muscle glycogen utilization at each power output was similar between conditions at 30% VO2 max but was significantly lower at both 60 (75 ± 20 vs. 113 ± 7 mmol/kg dry wt) and 75% VO2 max (103 ± 16 vs. 157 ± 18 mmol/kg dry wt) during ACID compared with CON, respectively (Fig. 3).

                              
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Table 1.   Muscle glycogen and glycolytic intermediate contents in vastus lateralis at rest, postingestion, and during cycle ergometry at 30, 60, and 75% VO2 max after either CON or ACID



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Fig. 3.   Glycogen utilization for each exercise intensity. Data are means ± SE. + Significantly different from CON at matched time points. dw, Dry wt.

Glucose, G-6-P, F-6-P, G-1-P, and G-3-P. Intramuscular accumulation of glucose increased with each power output level comparably between conditions (Table 1). Intramuscular [G-6-P] and [F-6-P] increased with exercise and were significantly higher with ACID at all three power outputs (Table 1). Muscle [G-1-P] and [G-3-P] were similar between conditions, increasing with each power output (Table 1).

Lactate and pyruvate. Muscle [Lac-] increased with increasing power output but was significantly lower with ACID at both 60 (ACID 20.5 ± 4.2 vs. CON 34.6 ± 7.0 mmol/kg dry wt) and 75% VO2 max (ACID 40.8 ± 7.4 vs. CON 56.9 ± 8.6 mmol/kg dry wt; Fig. 4). Similarly, muscle [pyruvate] was significantly lower with ACID during 75% VO2 max (ACID 0.44 ± 0.05 vs. CON 0.69 ± 0.09 mmol/kg dry wt) compared with CON (Fig. 4).


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Fig. 4.   Muscle lactate (A) and pyruvate (B) concentrations with ACID and CON at rest and each exercise intensity. + Significantly different from CON at matched time points. Values are means ± SE. There was a significant main effect of time for both lactate and pyruvate, specifically at 60 and 75% VO2 max.

PDHa. Resting (ACID 0.79 ± 0.13 vs. CON 0.68 ± 0.19 mmol · kg-1 · min wet wt-1) and postingestion (ACID 0.79 ± 0.12 vs. CON 0.78 ± 0.24 mmol · kg-1 · min wet wt-1) PDHa levels were not different between conditions (Fig. 5). PDHa levels increased progressively with cycling but were significantly lower at 30 (2.35 ± 0.34 vs. 2.94 ± 0.33), 60 (3.29 ± 0.27 vs. 3.96 ± 0.34), and 75% VO2 max (3.91 ± 0.15 vs. 4.77 ± 0.10) during ACID compared with CON, respectively (Fig. 5).


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Fig. 5.   Muscle pyruvate dehydrogenase activity (PDHa) with ACID and CON at rest and each exercise intensity. + Significantly different from CON at matched time points. Data are means ± SE. ww, Wet wt. There was a significant main effect of time for each condition.

CoA, carnitine, and acetylated forms. Total muscle CoA was not different between conditions at rest or during exercise (Table 2). Acetyl-CoA concentration ([acetyl-CoA]) increased with power output with ACID, these increases with exercise being significantly higher compared with matched time points during CON (Table 2). In addition, the resting postingestion [acetyl-CoA] was significantly higher with acidosis (Table 2). Free CoASH declined equally between conditions with exercise intensity (Table 2). Acetylcarnitine followed a similar pattern to acetyl-CoA and did not differ between conditions (Table 2). Muscle total carnitine content increased significantly from rest to 75% VO2 max to the same degree in each condition, whereas free carnitine decreased in a reciprocal manner with increasing power output. There were no differences between conditions for either total or free carnitine contents (Table 2).

                              
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Table 2.   Muscle acetyl group content in vastus lateralis at rest, postingestion, and during cycle ergometry at 30, 60, and 75% VO2 max after either CON or ACID

ATP, ADPf, AMPf, free Pi, and PCr. Muscle [ATP] was unaltered by exercise or as a result of acidosis. Muscle ADPf and AMPf concentrations increased with each level, but both were significantly lower with ACID during 75% VO2max (Table 3). Free [Pi] increased with power output equally between conditions. PCr concentrations decreased with increasing power output similarly between conditions (Table 3).

                              
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Table 3.   Muscle high energy phosphate content in vastus lateralis at rest, postingestion, and during cycle ergometry at 30, 60, and 75% VO2 max after either CON or ACID

Pyruvate production and oxidation and lactate production and oxidation. Pyruvate production increased with power output but was significantly lower at each power output during ACID. Pyruvate oxidation increased as power output increased but was significantly lower with ACID. Relative pyruvate oxidation expressed as the percentage of pyruvate produced that was oxidized was similar between conditions at 30% but was significantly higher at both 60 and 75% VO2 max during ACID (Table 4). Lactate production was also significantly lower with ACID at all exercise time points (Table 4).

                              
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Table 4.   Muscle pyruvate production, oxidation, and lactate production at 30, 60, and 75% VO2 max after either CON or ACID

Blood Metabolites, Blood Flow, and Exchange Across the Leg

Blood pH, PCO2, and HCO-3. Arterial pH, PCO2, HCO-3, venous pH, and HCO-3 (Fig. 6; Table 5) were all significantly lower with ACID compared with CON at rest postingestion and each of the three power outputs.


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Fig. 6.   Arterial blood pH (A), PCO2 (B), and HCO-3 (C) during ACID and CON at rest and each exercise intensity. + Significantly different from CON at matched time points. Values are means ± SE.


                              
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Table 5.   Arterial concentration of blood-borne substrates during rest, postingestion, and cycle ergometry at 30, 60, and 75% VO2 max after either CON or ACID

Blood lactate and flux. Arterial [Lac-] increased progressively with power output but was lower at both 60 and 75% VO2 max with ACID (Table 5). Net release of Lac- across the leg increased with power output but was significantly lower at both 60 and 75% VO2 max with ACID (Fig. 7).


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Fig. 7.   Glucose uptake (A) and lactate release (B) across leg during ACID and CON at rest and for each power output. + Significantly different from CON at matched time points. There was a significant main effect of time for glucose uptake and lactate release. Values are means ± SE.

Blood FFA and glycerol. Arterial plasma [FFA] declined progressively with power output and was significantly lower during ACID compared with CON at postingestion, 30 and 60% VO2 max (Table 5). FFA uptake occurred across the leg during CON and increased progressively with each power output. However, during ACID at 30 and 75% VO2 max a net release occurred, whereas at 60% VO2 max a net uptake occurred, which was significantly lower than CON (Fig. 8). Arterial [glycerol] increased with each power output, but this increase was significantly lower with ACID (Table 5). During CON, glycerol release occurred across the leg at 30% VO2 max, whereas at 60 and 75% VO2max an uptake across the leg was observed. However, during ACID a net release across the leg occurred at all three power outputs (Fig. 8).


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Fig. 8.   Glycerol flux (A) and free fatty acid (FFA) flux (B) across leg during ACID and CON at rest and each power output. + Significantly different from CON at matched time point. There was a significant main effect of time with each condition. Values are means ± SE.

Leg blood flow and leg respiratory quotient. Leg blood flow increased progressively from rest to 60% VO2 max to the same extent between conditions. However, at 75% VO2 max leg blood flow was significantly lower with ACID (Table 6). Leg O2 uptake was not different between conditions; however, leg CO2 production was significantly higher during all three power outputs with ACID (Table 6). Similarly, leg respiratory quotient was significantly higher during all three levels and postingestion with ACID compared with CON (Table 6).

                              
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Table 6.   Leg blood flow, RQ, CO2 production, and O2 uptake at rest, postingestion, and during cycle ergometry at 30, 60, and 75% VO2 max after either CON or ACID

Blood glucose and flux. Arterial [glucose] (Table 5) and leg glucose uptake (Fig. 7) were similar at all power outputs between conditions.

Respiratory Gas Exchange Variables

Whole body VO2 increased similarly between conditions with each power output (Table 7). Whole body VCO2 was significantly lower during 60 (2.47 ± 0.12 vs. 2.57 ± 0.14) and 75% (3.29 ± 0.15 vs. 3.40 ± 0.17) VO2 max in ACID compared with CON, respectively. The RER was also significantly lower during ACID at 75% VO2 max (Table 7). VE was significantly higher during ACID compared with CON at each power output (Table 7).

                              
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Table 7.   Respiratory variables during cycle ergometry at 30, 60, and 75% VO2 max after either CON or ACID


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study examined the effects of acidosis on the metabolic responses in exercising muscle during continuous dynamic exercise for three power outputs: 30, 60, and 75% VO2 max. The main effects of induced acidosis during exercise included: a reduction in intramuscular lactate accumulation due to reduced lactate production; reduced lactate efflux from the exercising leg; and a greater reduction in Phos a than PDHa.

Lactate production within the muscle is dependent on the balance between the rates of pyruvate production and oxidation. Intramuscular lactate is formed from pyruvate by the action of the near-equilibrium enzyme LDH (50). Pyruvate is the end product of glycolysis from glycogen degradation and glucose uptake. Pyruvate is oxidized via the TCA cycle and the electron transport chain after being converted to acetyl-CoA by PDHa. Lactate production was reduced by 46% during the second power output and by 48% in the third with ACID (Table 4). Not only was less Lac- produced at both of these intensities, but the amount of pyruvate oxidized relative to that produced was significantly higher with ACID, resulting in a 14% increase during the second power output and 12% with the third (Table 4). These changes resulted from the effect of acidosis on the rate-limiting enzymes Phos, PFK, and PDH. The main control points for glycogenolysis and glycolysis involve Phos and PFK, respectively, whereas entry into the oxidative pathways is controlled by PDH.

Phos

Phos is the flux-generating enzyme responsible for glycogen degradation within skeletal muscle and is subject to both covalent and allosteric regulation. Phos a is considered the active form, active in the absence of AMPf, whereas Phos b, the less active form, requires AMPf (10). Covalent Phos b to a transformation is mediated by phosphorylase kinase a, which is activated by either an increase in cytosolic Ca2+ concentration or epinephrine (60). Posttransformation allosteric regulation of Phos b by the activators AMP, IMP, and Pi and the inhibitors ATP and G-6-P is also important (14).

ACID had a transformational effect, significantly reducing Phos a at the highest power output, resulting in a 34% reduction in muscle glycogen utilization. The reduction in Phos a transformation presumably resulted from the direct inhibition of phosphorylase kinase b by [H+], which has been previously demonstrated in intensely exercising muscle when the pHi is ~6.6, the same value estimated in the present study with ACID (9). In addition, the reduced availability of the substrate (HPO2-4) for Phos may have played a role (42), as the proportion of free Pi in the HPO2-4 form falls by 30% as the pH falls from 7 to 6.5 (10).

AMP and IMP have been shown to stimulate Phos a (2, 60) and Phos b (2, 10) through posttransformational regulation. AMP acts on Phos a by reducing the Km for Pi from 26.2 mM to 11.8 mM in the presence of 0.01 mM AMP (2, 60). In the present study, AMP accumulation was blunted with ACID, falling well below the 0.01 mM required for activation. In support of the close relationship between Phos activity, glycogenolytic flux, and AMPf concentration, other studies utilizing caffeine ingestion (12), increased FFA availability (21, 51), and short-term training (11) have demonstrated glycogen sparing during exercise, associated with blunted AMPf accumulation. The reduction in AMPf concentration combined with the reduced availability of free Pi would prevent or reduce allosteric activation of Phos a and b. IMP activates Phos b at a Km of 1.2 mM (2). IMP was not measured in the present study but has been shown previously to increase with exercise and acidosis (10, 19), which may have activated Phos b, thereby contributing to the maintenance of glycogenolytic flux.

G-6-P inhibits Phos b through end-product inhibition, at concentrations of 0.3 mM and above (14). [G-6-P] values with ACID are above this value and therefore would inhibit Phos at the higher power outputs. The [G-6-P] was highest at the end of the second power output and then decreased by the end of the third power output. The elevated values at the end of the second power output may have inhibited Phos b, thereby further reducing glycogenolytic flux and accounting for the lower [G-6-P] seen at the end of the third power output. This is confirmed by the simultaneous glycogen sparing that occurred. Previous investigation has alluded to this reduced glycolytic flux from inhibition before G-1-P with acidosis (66).

PFK

PFK is a nonequilibrium enzyme that converts F-6-P to fructose 1, 6-bisphosphate with consumption of ATP (50), with the relative activity being reflected by changes in [F-6-P] and [G-6-P] with which it is in equilibrium. Increases in [H+] have been shown to affect the kinetic and structural organization of PFK, which ultimately results in increased affinity of the ATP-binding site and reduced affinity of the F-6-P binding site (6, 18, 67). The magnitude of pH inhibition can be partially overcome by increases in the [F-6-P] only to a pH of 6.5, a value achieved with intense exercise (37). Previous human exhaustive exercise protocols have seen similar changes reflecting reductions in PFK activity with reduced pHi (39, 64). This increase in [F-6-P], although necessary to maintain partial enzyme activity, results in a reciprocal rise in the [G-6-P], which inhibits Phos and therefore reduces substrate supply. The results of the present study support this explanation. ACID resulted in a lower pHi, higher [F-6-P], and higher [G-6-P] compared with CON (Table 1). This, together with the pattern of reduced [G-6-P] after the second power output with ACID, reflects both a progressive increase in PFK inhibition and the reciprocal inhibition of Phos flux and substrate supply that results when [F-6-P] increases to maintain flux through PFK. This reduced substrate supply and glycogenotlytic flux is further evidenced by the glycogen sparing that occurred during the third power output.

The combined results demonstrate reduced glycogenolytic and glycolytic flux due to acidotic inhibition of both Phos and PFK, resulting in the observed decrease in pyruvate production (Table 4).

PDH

PDH is a mitochondrial enzyme that catalyzes the decarboxylation of glycolytically derived pyruvate to acetyl-CoA for entry into the TCA cycle and therefore reflects the rate of entry of carbohydrate into the oxidative pathways. PDH transformation between the active PDHa and inactive PDHb forms is regulated by the balance between PDH kinase (PDHK; deactivating) and PDH phosphate (PDHP; activating; Refs. 58, 68). The relative phosphatase-kinase activity is controlled by the mitochondrial acetyl-CoA-to-CoASH, ATP-to-ADP, and NADH-to-NAD+ ratios and the allosteric regulators Ca2+, pyruvate, and H+ (56, 58, 68).

Acetyl-CoA-to-CoASH ratio. ACID significantly elevated [acetyl-CoA] at rest and throughout exercise, probably reflecting an increased utilization of intramuscular triacylglyerides (TAG) (Tables 2 and 5). It appears in the present study that intramuscular TAG was used rather than adipose tissue TAG, as evidenced by reduced arterial FFA and glycerol concentrations and marked efflux from the exercising leg with increasing power output. Previous studies have demonstrated that acidosis impairs adipose tissue lipolysis by inhibiting the cAMP-dependent activation of the hormone sensitive lipase (HSL) (23, 36, 40). Activation of the HSL is the rate-limiting and flux-generating step of TAG mobilization from adipose tissue. However, the rate of plasma FFA release is a poor estimate of lipolysis because the endothelial lining of the capillaries contains lipoprotein lipases; glycerol release provides a better estimate of lipolysis because adipose tissue lacks glycerol kinase (16). Precise regulation of intramuscular lipolysis is not known but is thought to involve a HSL that is activated by a cAMP cascade and is sensitive to pH (25, 52). Intramuscular lipolysis has been shown to occur during intense exercise in isolated muscle preparations (62, 63) when the [H+] is increased. Also, in exercising humans, Jones et al. (38) studied FFA turnover and found simultaneous increases in plasma [glycerol] with decreases in FFA turnover, indicating intramuscular TAG utilization when FFA release from adipose tissue is significantly reduced. All of the above studies support our present findings of increased intramuscular TAG hydrolysis. Interestingly, the acetyl-CoA-to-CoASH ratio did not differ between conditions. Because acetyl-CoA stimulates PDHK whereas CoASH stimulates PDHP, with the net PDHa resulting from both effects, it is possible that the increased [acetyl-CoA] has the dominating effect on PDHa acting through increased kinase activity (68).

ATP-to-ADP ratio. This ratio affects PDHK only because ATP is the substrate for the reaction and therefore competes with its product ADP, which inhibits catalytic activity (68). The [ATP] did not change with exercise nor was it different between conditions. However, the [ADPf] was significantly lower with ACID during 75% VO2 max (Table 3). The ATP-to-ADP ratio declined with power output but to a lesser extent with ACID, which could have resulted in greater PDHK activity and therefore contributed to the lower PDHa observed with ACID.

NADH-to-NAD+ ratio. The NADH-to-NAD+ ratio was not measured in the present study. Previous studies utilizing indirect techniques have shown that the redox state and NADH-to-NAD+ ratios decline with high intensity exercise, indicating a more oxidized state (13, 27, 65). Based on these studies, the NADH-to-NAD+ ratio should be declining, leading to an increase in PDHa. However, in the present study, PDHa was lower with ACID. It seems likely that changes in the NADH-to-NAD+ ratio may have played only a minor role, compared with the effects of the other regulators, including Ca2+, and pyruvate as previously suggested during high-intensity exercise in humans (56). In addition, the increased utilization of intramuscular TAG with ACID may have sufficiently increased the NADH concentration, resulting in greater activation of the kinase and subsequently a lower PDHa (55).

Pyruvate. [Pyruvate] increases with exercise were lower with ACID, and although [pyruvate] increased up to 60% VO2 max, it then declined by the end of the third power output (Fig. 4). Pyruvate acts directly on the kinase to inhibit activity and thus maintain PDHa activity. In the present study, with ACID, [pyruvate] was well below the Ki (0.5-2.0 mM) for PDHK and could have contributed to the reduced PDHa (46). The importance of [pyruvate] on PDHa has been demonstrated in human diet-manipulation studies (54, 57). Total pyruvate production was lower with ACID compared with CON and resulted from decreases in glycogenolysis and glycolysis. The total amount of pyruvate oxidized was also lower with ACID but exactly matched the ~4 mmol · kg-1 · min dry wt-1 reduction in PDHa compared with CON (Table 4; Fig. 5). Despite lower PDHa, the proportion of pyruvate oxidized was higher with ACID during both 60 and 75% VO2 max compared with CON (Table 4). The reduction in intramuscular [pyruvate] with ACID from the second to third power output likely resulted from a marked inhibition in glycogenolysis and glycolysis with decreased pyruvate production due to the severe acidosis and an increase in pyruvate oxidation as PDHa increased with power output. These combined results suggest a better match between glycogenolytic flux and flux through PDHa with subsequently lower pyruvate accumulation as also shown in previous studies (32, 56). Thus acidosis results in relatively lower PDH inhibition (18%; Fig. 5) than Phos inhibition (27%; Fig. 2) at 75% VO2 max.

Hydrogen ions and calcium. Increases in [Ca2+] have been shown to increase PDHa transformation by activating PDHP and inhibiting PDHK. Mitochondrial estimates of [Ca+2] have been confirmed to be within the physiological range of calcium to cause half-maximal activation (0.2-0.3 µM; Ref. 17). [Ca2+] should have been similar between conditions because subjects exercised at the same intensities in both trials.

Hydrogen ion has been shown to activate PDHa in acidotic perfused rat hearts (53) and has been attributed to the differences in pH optimum of PDHK and PDHP. PDHK has a pH optimum of 7.0-7.2, with increased inhibition as pH falls, whereas PDHP has a pH optimum of 6.7-7.1 (33). Based on these data, our original hypothesis was that, like the heart, metabolic acidosis would increase PDHa. However, the results of the present study indicate that PDHa is lower during ACID with each of the three power outputs. This can be explained by the influence of acetyl-CoA, ADP, pyruvate, and possibly NADH that cumulatively resulted in greater activation of PDHK, leading to reduced PDHa transformation. [H+] may have had an effect but was overridden or masked by the other covalent modifiers.

Lactate Metabolism

Intramuscular lactate accumulation reflects the balance between the rates of lactate production and efflux from the muscle (24, 41). The results of previous studies employing both metabolic acidosis (31, 40, 48, 63, 66) and respiratory acidosis (22, 26, 63) were similar to the present data in showing elevated [F-6-P] (inferring PFK inhibition), glycogen sparing, and reductions in both intramuscular and plasma [Lac-] compared with control conditions. Respiratory acidosis has less of an overall effect on these parameters compared with metabolic acidosis, possibly due to the enhanced catecholamine release accompanying hypercapnia (22). However, none of these studies was able to elucidate the mechanisms responsible for these universally observed changes accompanying acidosis.

The mechanisms responsible for lactate production by muscle have been controversial; conventionally, lactate production has been attributed to O2 limitation at the mitochondria (24, 43). However, in the present study, the differences in lactate accumulation between conditions clearly did not result from an O2 limitation at the mitochondria because leg O2 uptake was similar, despite a reduction in blood flow with ACID during the highest power output (Table 6). Oxygen delivery was maintained with ACID due to a pH-mediated rightward shift of the oxyhemoglobin dissociation curve, augmenting oxygen availability to the working muscle (35).

Lactate Transport

Intramuscular lactate accumulation is also a function of the rate of efflux from the muscle (41). ACID significantly reduced both arterial whole blood [Lac-] and efflux from the leg during 60 and 75% VO2 max. Blood [Lac-] represents the balance of lactate entry from muscle and uptake by inactive tissue (7). The present results during ACID may be explained by both an impairment of the lactate transport out of the exercising leg and enhanced uptake by nonexercising tissue.

Lactate transport across the sarcolemma occurs via a monocarboxylate lactate-proton cotransport protein and as such is the rate-limiting step in lactate efflux. Diffusion of lactate in the undissociated form of lactic acid in the direction of the transmembrane [Lac-] and [H+] occurs, but it accounts for only 20% of lactate efflux, although higher contributions are seen with higher lactate and H+ concentrations. The monocarboxylate carrier accounts for 70-90% of lactate transport across the physiological range of [Lac-] (41, 49). Kinetic studies of the transporter with sarcolemmal vesicles have shown it to have a high affinity for L-lactate, to be sensitive to changes in [Lac-] and [H+] gradients, and to cotransport lactate and H+ in a 1:1 ratio (41). In the present study with ACID, there was a decrease in the intramuscular [Lac-] and a decrease in the femoral venous [Lac-], suggesting that the lower lactate efflux resulted from the reduced [Lac-] gradient. However, the magnitude of the reduced lactate efflux was greater than the reduction in intramuscular [Lac-] (56 vs. 28% respectively), suggesting a role for the transporter. The altered [H+] gradient with ACID may have affected H+ binding and off-loading and therefore transport (41). In addition, the elevated [H+] within the muscle is expected to increase the proportion of lactate in the associated form and thereby reduce the availability of Lac- to the transporter. ACID also induced a reduction in extracellular [HCO-3], which may have contributed to the reduced lactate efflux from the muscle. Investigators examining lactate efflux under varied external [HCO-3] in isolated muscle preparations have found reduced lactate efflux with low external [HCO-3] (30, 47, 63). In addition, studies comparing metabolic and respiratory acidosis in exercising humans (22, 31, 40, 44) consistently demonstrate greater reductions in the appearance of plasma lactate when acidosis is accompanied by reductions in [HCO-3]. During ACID in the present study, extracellular [HCO-3] was significantly reduced in both arterial and femoral venous blood at rest postingestion and with each of the three power outputs, which is in agreement with the above evidence (Table 5). Additionally, enhanced lactate uptake by inactive tissue may have contributed to the lower arterial [Lac-] with ACID. Uptake by inactive tissue has been shown to occur during exercise (24). Normally, with exercise, increases in blood [H+] and blood [Lac-] create an inwardly directed [H+] and [Lac-] gradient, which would facilitate uptake. Acidosis enhances the [H+] gradient and therefore may enhance uptake compared with control conditions.

In conclusion, imposition of a metabolic acidosis results in a complex series of metabolic effects during exercise, reflecting changes in the activity of key regulatory enzymes and fuel utilization. The main findings of the study demonstrate that the reduced intramuscular [Lac-] seen with acidosis results from a combined reduction in pyruvate production and an enhanced percentage of pyruvate oxidized, due to a better match between the rate of glycogenolysis and the rate of flux through PDHa. The reduced pyruvate production results from decreased glycogenolytic flux from a direct acidotic depression of Phos a transformation and reduced availability of substrate due to greater prevalence of H2PO-4. Glycogenolytic flux via Phos b was also reduced because of the acidotic reduction in the allosteric modulators ADPf and AMPf. Acidotic inhibition of PFK resulted in elevations in [F-6-P] and, consequently, [G-6-P], which also contributed to the reduced glycogenolytic flux via end-product inhibition. The combined results of reduced glycogenolytic and glycolytic flux resulted in glycogen sparing and reduced pyruvate production, particularly at 75% VO2 max when pHi was the lowest. The reduction in carbohydrate utilization was accompanied by increased intramuscular TAG utilization and occurred because of the acidotic inhibition of adipose tissue lipolysis. This increased use of FFA was associated with elevations in [acetyl-CoA]. The increased [acetyl-CoA], decreased [ADP], and decreased [pyruvate] collectively contributed to the reduced PDHa transformation.

The decreased blood [Lac-] seen with acidosis may reflect inhibition of the lactate transporter by an altered [H+] gradient or the reduced extracellular [HCO-3], rather than changes in the [Lac-] gradient. Inactive muscle may have contributed significantly to the reduced arterial [Lac-], but, the contribution was not assessed in the present study. The present data demonstrate that the reduced intramuscular lactate accumulation during acidosis results from both decreased production and enhanced oxidation through modulation of the key regulatory enzymes Phos, PFK, and PDHa.


    ACKNOWLEDGEMENTS

We gratefully acknowledge the following individuals for excellent technical assistance: G. Obminski, Dr. M. Ganagaragah, T.M. Bragg, and J. Otis.


    FOOTNOTES

This work was supported by grants from the Medical Research Council of Canada (MRC). M. G. Hollidge-Horvat was supported by an MRC Studentship, M. L. Parolin was supported by an National Sciences and Engineering Research Council studentship, and G. J. F. Heigenhauser is a Career Investigator of the Heart and Stroke Foundation of Ontario (no. I-2576).

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: G. J. F. Heigenhauser, Dept. of Medicine, McMaster Univ. Medical Centre, 1200 Main St. West, Hamilton, Ontario, Canada L8N 3Z5 (E-mail: heigeng{at}fhs.csu.McMaster.ca).

Received 24 February 1999; accepted in final form 3 June 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Endocrinol Metab 277(4):E647-E658
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