Department of Medicine, McMaster University Medical Centre, Hamilton, Ontario, Canada L8N 3Z5
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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METHODS |
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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 (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
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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 (O2), rate of
CO2 production
(
CO2) 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 atBlood 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 atLeg 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 (%
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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 [LacStatistical 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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Muscle Metabolism
Phos. Phos a decreased as exercise intensity increased in both conditions. At 75%
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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%
O2 max but was
significantly lower at both 60 (75 ± 20 vs. 113 ± 7 mmol/kg dry
wt) and 75%
O2 max
(103 ± 16 vs. 157 ± 18 mmol/kg dry wt) during ACID compared
with CON, respectively (Fig. 3).
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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%
O2 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%
O2 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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PDHa. Resting
(ACID 0.79 ± 0.13 vs. CON 0.68 ± 0.19 mmol · kg1 · 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%
O2 max (3.91 ± 0.15 vs. 4.77 ± 0.10) during ACID compared with CON,
respectively (Fig. 5).
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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%
O2 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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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%
O2max (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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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% O2 max
during ACID (Table 4). Lactate production was also significantly lower with ACID at all exercise time points (Table 4).
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Blood Metabolites, Blood Flow, and Exchange Across the Leg
Blood pH, PCO2, and HCO
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Blood lactate and flux. Arterial
[Lac] increased
progressively with power output but was lower at both 60 and 75%
O2 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%
O2 max with ACID (Fig.
7).
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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%
O2 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%
O2 max a net
release occurred, whereas at 60%
O2 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%
O2 max, whereas at 60 and 75%
O2max 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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Leg blood flow and leg respiratory
quotient. Leg blood flow increased progressively from
rest to 60% O2 max to
the same extent between conditions. However, at 75%
O2 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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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
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DISCUSSION |
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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%
O2 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 (HPO24)
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% O2 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%
O2 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%
O2 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%
O2 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 [LacThe 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 [LacLactate 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%
O2 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.
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ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge the following individuals for excellent technical assistance: G. Obminski, Dr. M. Ganagaragah, T.M. Bragg, and J. Otis.
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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.
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