Vol. 273, Issue 4, R1211-R1218, October 1997
Carbohydrate utilization in rat soleus muscle is influenced by
carbonic anhydrase III activity
Claude H.
Côté,
Guylaine
Perreault, and
Jérôme
Frenette
Hormonal Bioregulation Research Unit, Laval University
Hospital Research Center, Ste-Foy, Quebec, Canada G1V 4G2
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ABSTRACT |
Inhibition of carbonic anhydrase III (CA
III; EC 4.2.1.1) activity in type I muscle can influence resistance to
fatigue and glycogen utilization. Our aim was to determine if CA III
inhibition could influence muscle pH and glycolytic rate. Muscle pH,
hexosemonophosphates (HMP), glycolytic intermediates, ATP, and creatine
phosphate (CP) were measured at rest and during a fatigue protocol in
rat soleus muscles in vitro with or without CA inhibitors (CAI). In
resting muscles, CAI resulted in a significant drop in pH (7.11 vs.
7.06, P < 0.05) and in a two- to
threefold increase in HMP content compared with control muscles.
Measurements of HMP and glycolytic intermediates during the fatigue
protocol suggested, however, that the glycolytic flux was not
influenced. Globally, muscles incubated with CAI showed larger
perturbations of their CP and ATP content than control muscles. The
accumulation of HMP induced by the CAI was found to be totally
dependent on the combined presence of external glucose and contractile
activity, suggesting that inhibiting CA III may augment the
responsitivity of the contraction-induced glucose uptake process.
muscle fatigue; sulfonamide; glycolysis; hexosemonophosphate; glucose transport; contraction
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INTRODUCTION |
CARBONIC ANHYDRASE III (CA III; EC 4.2.1.1) is
predominantly found in skeletal muscle, liver, and adipose tissue (28). In muscle, CA III is especially abundant in the cytosol of type I
slow-twitch fibers where it may count for as much as 15% of the
cytosolic protein mass (11, 12). CA III activity is ~5-10 times
lower in type IIa fibers, whereas it is basically undetectable in type
IIb fibers (12). The membrane-bound CA IV isoform is also present in
sarcoplasmic reticulum (SR) (4, 11) and sarcolemmal muscle preparations
(16). Finally, ambiguity still exists regarding the presence of the
mitochondrial isoform CA V in rodent mammalian muscle.
Since CA III's discovery, it has been postulated that its unique
function in skeletal muscle was to facilitate the diffusion of
CO2 (2, 15, 17, 31). However, a
strong case can be presented that suggests that this may not be its
only cellular function. First, CA III has a very low hydratase activity
compared with the other cytosolic isoforms CA I and CA II, the activity of CA III being ~1% of that for CA II. Second, there is a virtual absence of correlation between the oxidative capacity of a given muscle
fiber type and its CA III activity and content (12), whereas a highly
significant negative correlation exists between the level of CA III
activity and those of various glycolytic enzymes (1).
Third, when rats are made hypo- or hyperthyroid, several changes occur
at the energy metabolism level that are coupled with rapid increase and
decrease, respectively, in CA III expression. However, even during the
transition, a tight coupling persists between the activity of key
glycolytic enzymes and CA III activity, whereas no tight relationship
can be observed with marker enzymes of the oxidative metabolism (30).
In other words, muscles with low glycolytic potentials appear to have a
strong need for CA III.
We have previously demonstrated that soleus (Sol) muscle incubated with
CA inhibitors (CAIs) can show an increased resistance to fatigue
compared with control (Ctr) muscles and that this effect is
attributable to the loss of cytosolic CA III activity (9, 13). An
analysis of the kinetics of the effect on fatigue showed that the
largest difference between Ctr and methazolamide (Meth) muscles was
observed in the early minutes of the fatigue protocol, a period when
creatine phosphate (CP) hydrolysis and glycolysis are the predominant
energy metabolic pathways (13). Geers and Gros (15), using in vitro
muscles, also found that CAI could influence muscle contractility and
CP concentrations. We subsequently proposed, on the basis of studies
that examined glucose and glycogen metabolism under conditions of CA
inhibition, that CA III may have a direct or indirect downregulatory
effect on the glycogenolytic and/or glycolytic rate (9).
Indeed, it was found that Sol muscles deprived of their CA III activity
were using their glycogen at a higher rate than the Ctr muscles when
measured over a 45-min in vitro fatigue protocol. According to this
hypothesis, the inhibition of the CA III activity should allow a
greater rate of carbohydrate utilization, which can translate into a
lower fatigability, depending on the exercise duration. Because the
handling of CO2 by CA III may be
involved in maintaining the cytosolic pH and because the key enzymes of
glycolysis and glycogenolysis are both sensitive to pH fluctuations, it
is therefore possible that inhibition of the CA III activity may
influence one or both of these metabolic pathways. The objective of
this study was, therefore, to determine if inhibition of CA III
activity can influence 1) muscle pH
at rest and during sustained muscle activity,
2) the accumulation of various
glycolytic and glycogenolytic metabolites, and
3) the glycolytic rate.
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MATERIALS AND METHODS |
Measurement of contractile properties.
For all experiments presented, isometric contractile properties were
measured before initiation of the fatigue protocols as described
previously (13). Female Wistar rats weighing 140-160 g were
anesthetized with pentobarbital sodium (50 mg/kg ip), and whole Sol
muscles were carefully dissected. Rats were killed by means of
pneumothorax performed under anesthesia. Contractile properties were
measured in vitro in a buffered physiological solution (Krebs-Ringer
bicarbonate) supplemented with curare (20 mg/ml) and glucose (2 mg/ml),
unless otherwise specified, and maintained at 25°C. One tendon was
attached to a rigid support at the bottom of the bath, and the other
end was connected to an isometric force transducer (Grass FT-03)
through a stainless steel hook. Muscles were stimulated with 25-V
square pulses of 0.2 ms duration delivered through platinum field
electrodes. The bathing solution of the experimental group contained a
CAI in the form of 1 mM Meth. This concentration represents the highest amount of this compound that can be solubilized in the solution used
for these experiments. In a small number of experiments, 0.1 mM
ethoxyzolamide (Ethox) was also used as a CAI, and although it
theoretically inhibits roughly 80% of the Sol muscle CA III activity
compared with 100% inhibition for Meth, no difference was found
between the two CAIs. Therefore, all metabolic data were pooled. These
CAIs were selected mostly for their characteristics of high
permeability through the cellular membrane (18). Previous studies in
our laboratory indicate that the time course for the effect of Meth on
fatigability is ~10 min (13).
For all protocols, an initial period of 30 min was allowed for muscles
to equilibrate with the incubation medium (with or without CAI). During
this period, muscles were adjusted to their optimum length, defined as
the length at which maximal isometric twitch tension was produced. This
process usually required 10-12 twitch contractions. At the end of
the equilibration period, one single twitch contraction was elicited,
and the following measurements were obtained: maximum twitch tension,
contraction time (time-to-peak tension), and one-half relaxation time
(RT1/2). After measurement of
the twitch parameters, muscles were stimulated for 1 s at frequencies of 35, 50, 80, 100, and 120 Hz to determine maximum tetanic tension (Po). Each tetanic contraction
was separated by a 60-s resting period. Ten minutes after the last
tetanic contraction, muscles were submitted to a fatigue protocol that
consisted of eliciting one 0.5-s tetanic contraction at 10 Hz every 5 s
for 30 min. This stimulation frequency produces a partially fused
tetanic contraction, with tension reaching ~50% of
Po at the beginning of the test. This is the same test used in previous experiments (9, 13); it was
designed to induce a significant amount of tension loss over a
relatively long period to allow recruitment of the aerobic metabolism
and therefore increased CO2
production.
The first series of experiments, including the determination of muscle
pH and measurement of glucose 6-phosphate
(G-6-P), fructose 6-phosphate
(F-6-P), fructose 1,6-phosphate
(F-1,6-P), glyceraldehyde
3-phosphate (G-3-P), pyruvate (Pyr),
lactate (Lact), CP, and ATP was performed with Sol muscles incubated in
the presence of exogenous glucose in the bathing solution as described
previously. Other series of experiments were performed thereafter. In
one of these, G-6-P concentration was
also measured in Sol muscles submitted to the same experimental
protocol described previously (with or without CAI) but with an
incubation medium containing no glucose and one containing 25 µM
cytochalasin B (Cyto) to abolish glucose uptake by the muscle. Finally,
to verify the specificity of the inhibitor, the extensor digitorum
longus (EDL) muscle, a type II muscle known to be devoid of CA III
activity (26), was submitted to the same experimental protocol used for
Sol muscle in the presence or in absence of CAI, and
G-6-P concentrations were determined.
Determination of muscle pH.
The homogenate method was used to investigate the influence of CA III
inhibition on muscle pH variation. All muscle samples obtained in situ
at rest and at various times during the in vitro fatigue protocol (0, 1, 3, 5, 15, and 30 min) were frozen with metal tongs precooled in
liquid nitrogen ~3 s after interruption of the fatigue test. The
muscle samples were weighed frozen, dissected free of tendons and other
nonmuscular elements, and then homogenized at 0°C in 5 vol of 145 mM KCl, 10 mM NaCl, and 5 mM iodoacetic acid. The pH of the homogenate
was measured at 38°C according to the method described by Sahlin
(27).
Measurement of glycolytic intermediates.
Muscle samples were frozen at rest and at various times during the
fatigue protocol as described previously. Samples were homogenized at
0°C in 5 vol (wt/vol) of 0.6 M perchloric acid and centrifuged at
5,000 g for 10 min. The supernatant
was recovered and neutralized with 2 M
KHCO3. Measurements of
G-6-P,
F-6-P, F-1,6-P,
G-3-P, Pyr, Lact, CP, and ATP
concentrations were obtained fluorometrically or spectrophotometrically
as described by Lowry and Passonneau (21) and Bergmeyer (3).
Statistical analysis.
All results for metabolite concentrations are expressed in millimoles
per gram wet muscle, unless specified otherwise, and are presented as
means ± SE. Data for muscle metabolite concentrations and muscle pH
were analyzed by a two-way analysis of variance for repeated measures
followed by Fisher's protected least significant differences test when
a significant F ratio was obtained.
Data on muscle contractile properties were analyzed by a one-way
analysis of variance. In all cases, the level of significance was set
at P < 0.05.
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RESULTS |
Data for isometric contractile properties of Sol muscles from Ctr and
experimental groups are shown in Table 1.
All measurements were unaffected by CAI except for
RT1/2, which was significantly prolonged, an effect previously shown to be related to the inhibition of the SR-CA IV isoform (15). As shown in Fig.
1, when incubated in presence of CAI,
muscles showed an increased resistance to fatigue compared with Ctr
muscles. The difference in tension production could be observed as soon
as 1 min after initiation of the fatigue protocol.
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Fig. 1.
Effects of inhibition of carbonic anhydrase III (CA III) activity with
carbonic anhydrase inhibitors (CAIs) on fatigue. Rat soleus (Sol)
muscles were incubated in the presence of CAI (0.1 mM ethoxyzolamide or
1 mM methazolamide; data pooled) or without inhibitor [control
(Ctr)] for ~45 min before a 30-min stimulation protocol.
Stimulation consisted of 1 500-ms train at 10 Hz every 5 s for 30 min
(n = 30-65 muscles in each
group). A significant difference was found for all time points between
1 and 30 min (analysis of variance, P < 0.05).
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Mean pH value for muscles incubated with CAI at time
0 of the fatigue protocol was significantly different
from the value obtained with Ctr muscles (Table
2). Fluctuations in muscle pH during
contraction, although quite small, were more pronounced in the Ctr
group than in the CAI muscles. Indeed, significant decreases were
observed in Ctr after 5, 15, and 30 min of stimulation compared with
the value at time 0, whereas the
difference was almost significant already at 3 min
(P = 0.06). In the CAI group, no
acidification took place during the first 3 min of the fatigue protocol, a period when CP utilization is important, and significant changes appeared only after 15 min.
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Table 2.
Values for pH obtained during fatigue protocol for soleus muscles
incubated with or without carbonic anhydrase inhibitor
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Values for metabolite concentrations of in vivo muscles at rest and for
muscles submitted to the in vitro fatigue protocol when glucose was
present in the bathing solution are presented in Tables
3 and 4. Very significant
differences between both groups in terms of hexosemonophosphate (HMP)
content are already present before the start of the fatigue
protocol. Although it is clear that the dissection, preparation, and
incubation procedure did not affect Ctr muscles, as shown by the fact
that values for HMP at rest in vivo and at time
0 of the in vitro protocol are similar, large increases
in G-6-P and
F-6-P levels are found in the muscles
incubated with CAI. On the contrary, values for all glycolytic
intermediates located at steps after the phosphofructokinase (PFK)
reaction did not show such an increase.
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Table 3.
Concentrations of various hexosemonophosphates at rest and during
fatigue protocol in soleus muscles from control and CAI groups
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Table 4.
Concentrations of various 3-C glycolytic intermediates at rest and
during fatigue protocol in soleus muscles from control and CAI
groups
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Variations in HMP content during the stimulation protocol were also
quantitatively and qualitatively different between both groups (Table
3). Significant increases in the levels of
G-6-P during the fatigue protocol only
occurred in the Ctr group, with values for
G-6-P content in the CAI group even
showing a significant decrease after 1 min compared with the value at
time 0.
G-6-P content was approximately two-
to fourfold greater in the CAI group than in the Ctr group for all time
points selected. Levels of F-6-P in
Ctr muscle did increase gradually during the fatigue test in parallel
with the values for G-6-P. However,
F-6-P levels in the CAI muscles had a
tendency to show changes in a direction opposite to the one for
G-6-P, with a significant accumulation already present after 1 min of stimulation compared with the value at
time 0. Concentrations of
F-6-P became similar in both groups at
20 min. At all time points selected, no significant difference was
found for F-1,6-P between Ctr and CAI.
The ratio
F-6-P/F-1,6-P, which can be used to determine if an accumulation of metabolite occurred at a critical point of the glycolytic flux, was three- to
fivefold higher in the CAI group for all time points (Table 3). Muscle
content of G-3-P, Pyr, and Lact during
the fatigue protocol is shown in Table 4. Only Lact levels increased
very significantly over time during the fatigue test, but, as is the case for G-3-P and Pyr, no difference
could be found between groups. Overall, one is left with the conclusion
that the glycolytic flux was similar in both Ctr and muscles deprived
of CA activity.
To investigate the origin of this accumulation of HMPs in the muscles
incubated with CAI, G-6-P measurements
were made in both Ctr and CAI Sol muscles incubated in a bathing
solution with (+) and without (
) glucose; also, for each
condition (+ or glucose),
G-6-P levels were obtained on muscles
submitted to the usual contractile activity preceding the fatigue test
and in muscles that were installed under passive tension in the bath
but not submitted to any contractile activity at all for the 45-min
period. Because hexokinase (HK) (type II, EC 2.7.1.1) cannot provide G-6-P to the muscle cell in the
absence of external glucose, glycogenolysis becomes the only possible
pathway to produce G-6-P. In the
absence of external glucose, CAIs do not have an effect on
G-6-P accumulation, regardless of
whether muscles undergo contractile activity during the 45-min
incubation (Fig. 2). Interestingly, the
accumulation of G-6-P previously
described in the presence of external glucose cannot be demonstrated if
muscles are not submitted to contractile activity during the
incubation. To further confirm these data, experiments were conducted
with muscles incubated in the presence of external glucose and
undergoing contractile activities but with the bathing solution
containing Cyto (25 µM), an inhibitor of glucose uptake. Under such
conditions, CAI could not induce any increase in
G-6-P content compared with Ctr
muscles (Fig. 2). When similar protocols were carried out with the EDL
muscle, which is basically devoid of CA III activity, no influence of CAI on HMP content was observed. Values of 0.143 ± 0.050 and 0.130 ± 0.030 were obtained for Ctr and CAI groups, respectively,
suggesting that the accumulation of HMPs observed with the Sol muscles
is a specific effect related to the inhibition of CA III.
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Fig. 2.
Effect of inhibition of CA III activity on accumulation of
glucose-6-phosphate (G-6-P) in
muscles incubated with or without exogenous glucose and submitted or
not to contractile activity before measurement. Rat Sol muscles were
incubated without CA inhibitor (Ctr) or with CAI as described in Fig.
1. In one group, the bathing solution did not contain glucose
(Glc), whereas in the other group (+Glc)
D-glucose was present. Muscles
with and without glucose were subdivided into groups submitted (+Con)
or not submitted (Con) to contractile activity (twitch and
tetanic contractions; see MATERIALS AND
METHODS) before muscle sampling. In one group,
cytochalasin B (Cyto, 25 µM) was added to the bathing medium
containing glucose. Muscles were sampled at time
0 of the fatigue protocol. Values are means ± SE
(n = 4-8 determinations in each
group). A significant accumulation of
G-6-P in presence of CAI was only
noticed when glucose and contractile activity were present.
* Significantly different from respective Ctr value,
P < 0.05.
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Results obtained for ATP and CP contents in Ctr and CAI groups just
before and during the fatigue test are shown in Figs. 3 and 4. For
both ATP and CP levels, values at time
0 did not differ from those obtained on Sol muscles
from resting (in vivo) anesthetized rats (3.03 and 13.3 µmol/g,
respectively). Values for ATP levels in Ctr muscles did show a small
but significant increase at 5 min but did not differ significantly from
the value at time 0 thereafter during
the protocol, while significant drops in ATP content did occur at 10 and 30 min in the CAI group. Muscle CP levels decreased significantly
in both groups by roughly 50% after the 30-min period. The absolute
value for CP content in the CAI group was significantly lower than the
Ctr value only at time 5 min in the
protocol. This kinetic of CP disappearance may appear slow compared
with many other contractile protocols and has to be related to the fact
that 1) the contractile protocol used was of a fairly moderate intensity with a high rest-to-work ratio
and 2) type I muscle fibers have a
much lower rate of CP hydrolysis than the mixed or fast-twitch muscles
used in most published experiments. Using mean values for CP content,
one can estimate that the rate of CP utilization in the first 5 min of the protocol was higher in muscles with the presence of CAI than in the
Ctr muscles (Fig. 5).
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Fig. 3.
ATP concentrations in Sol muscles sampled during the in vitro fatigue
protocol. Values at times 0, 1, 5, 10,
and 30 min are from Sol muscles
incubated in vitro with CAI for 45 min before the fatigue protocol or
without CAI (Ctr). Values are expressed as a percentage of the value at
time 0. All muscles were incubated in
a solution containing glucose (n = 5-8 determinations for each group at all time points). Fatigue
protocol was as described in Fig. 1, with time
0 as the start of the protocol. Values are means ± SE. There are no significant differences between both groups at all
time points. * Significantly different from the value at
time 0 (P < 0.05)
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Fig. 4.
Creatine phosphate (P) concentrations in Sol muscles sampled during the
in vitro fatigue protocol. Rat Sol muscles incubated in vitro with CAI
for 45 min before the fatigue protocol or without CAI (Ctr) were freeze
clamped at times 0, 1, 5, 10, and
30 min. All muscles were incubated in
a solution containing glucose (n = 5-8 determinations for each group at all time points). Fatigue
protocol was as described in Fig. 1, with time
0 as the start of the protocol. Values are means ± SE. * Significantly different from respective control value
(P < 0.05)
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Fig. 5.
Rate of creatine phosphate (PCr) disappearance in Sol muscles sampled
at various times during the in vitro fatigue protocol. Rates were
calculated with the use of absolute values shown in Fig. 4. For the 4 different intervals selected, values are from Sol muscles incubated in
vitro with CAI (Meth) for 45 min before the fatigue protocol or without
CAI (Ctr). All muscles were incubated in a solution containing glucose
(n = 5-8 determinations for each
group at all time points). Fatigue protocol was as described in Fig. 1,
with time 0 as the start of the
protocol.
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DISCUSSION |
Muscle contractile properties.
The data presented for contractile properties are in agreement with
previously published reports using rat Sol muscle (7, 8). The results
obtained for Po and other
isometric contractile properties in this study confirmed the integrity
of the isolated muscles used. Aside from
RT1/2, which is significantly
prolonged in the presence of CAI, the inhibitors used do not interfere
with the cellular reactions involved in the contractile process. Such a
prolongation of RT1/2 in rat Sol
muscles incubated in vitro in the presence of CAI has been confirmed by
others (15), and thorough investigations have revealed that the
prolongation is linked to inhibition of the SR-CA IV isoform,
which can influence calcium release and reuptake by the SR.
Possible mechanisms underlying this effect have been discussed in
detail in previous papers (9, 10, 13).
Muscle pH.
The pH values obtained for skeletal muscle with the homogenate
technique are, in general, significantly higher, by roughly 0.1 unit,
than the values obtained with pH microelectrodes (23), and this
difference is likely related to the presence of noncytosolic fluids.
Although absolute values must be looked at lightly, this technique may
be useful in the comparison of relative changes of two different groups
submitted to a similar protocol. The homogenate pH reported here, 7.11 ± 0.01, is similar to the value of 7.09 ± 0.06 reported by
Troup et al. (31) and the value of 7.16 reported by Geers and Gros
(15), who used the 5,5-dimethyl-2,4-oxazolidinedione method. The lower muscle pH in the Meth group before the
beginning of the fatigue protocol (7.11 vs. 7.06) is consistent with
the results presented by Geers and Gros (15), who observed a 0.1-unit drop in pH in Sol muscle incubated for 6 h (compared with ~45 min in
the present study) with a CAI. They attributed this effect to the
suppression of facilitated CO2
diffusion and to a reduction in
CO2 diffusivity. Under these
conditions, CO2 level should
increase inside the muscle cell and the pH should drop because of the
low uncatalyzed hydration activity that occurs naturally. No
significant decrease in pH was noticed during the first 5 min of the
fatigue protocol in the CAI group, whereas a significant decrease of pH occurred in the Ctr group during this same period. CP hydrolysis and
glycolysis are two energy metabolic pathways used by the muscle during
this early period. Hydrolysis of CP is a proton-consuming reaction,
whereas glycolysis leads to H+
production. The fact that no visible decrease in pH occurred in muscles
deprived of CA activity during the early portion of the fatigue test is
consistent with the higher rate of CP utilization observed in this
group compared with Ctr muscle and suggests that CA III can have a
significant impact on proton accumulation. Globally, even though the pH
data must be interpreted with care, we nevertheless believe that
1) the fact that the pH value
measured in both groups of muscles was identical for the last 25 min of
the stimulation protocol, at a time when oxidative metabolism is
active, does not support the idea that CA III is there only to
facilitate the diffusion of CO2
and 2) it is very unlikely that the
increase in resistance to fatigue induced by CAI is the direct result
of changes in cytosolic pH.
CA III and carbohydrate metabolism.
The values reported for resting content of HMPs compare well with the
data reported from other laboratories (29). Surprisingly, differences
in HMP concentrations were already present before initiation of the
fatigue test. Because muscles underwent 10-12 twitch contractions
followed by 5-6 600-ms tetanic contractions during this ~45-min
period, it is obvious that the metabolic rate does not have to be very
high to see the metabolic influence of CAI. Three main metabolic
components control the concentration of HMPs: two generating pathways
and one consuming chain of reactions. G-6-P can be generated through
glycogenolysis and through phosphorylation of exogenous glucose by HK,
whereas degradation of HMPs is totally dependent on the glycolytic
flux, which is considered to be mostly under the influence of PFK and
possibly pyruvate dehydrogenase (PDH, EC 1.2.4.1) also. At a first
look, data obtained for HMPs and glycolytic intermediates located past
the PFK step clearly suggest that 1)
HMP production was overriding utilization in the absence of CA III
activity during the period preceding the stimulation protocol and
2) there was no obvious elevation of
the glycolytic rate in the CAI group compared with Ctr muscle. Thus it
seems clear that, in the minutes before the initiation of the fatigue test, at a time when energy demand was low, PFK was obviously blocking
the breakdown of HMP, the concentration of which kept increasing
despite the well-known negative feedbacks that should have been exerted
on HK and glycogenolysis. PKF is an allosteric enzyme that could be
influenced by CA III activity whose modulation at rest and during
exercise is quite complex (20, 24). Whether the small decrease in pH
measured before the fatigue test could have induced PFK blockade and
the subsequent HMP accumulation deserves further examination with more
accurate methods.
The most significant finding of the present series of experiments is
that the accumulation of HMP observed is dependent on both the presence
of external glucose and contractile activity. Although the amount of
contractile activity performed during the equilibration period was not
enough to increase the G-6-P level in
Ctr muscle, G-6-P content was
increased by 100% in muscles incubated with CAI. If one assumes the
noninvolvement of HK activity in this scheme, this suggests that
inhibiting the CA activity of the Sol muscle does in some way enhance
the responsitivity of the contraction-induced glucose uptake process.
However, a mechanistic explanation for this influence would clearly be
speculative at this point in the investigation. Skeletal muscle is a
very important tissue for glucose disposal and contains two
facilitative glucose transporters, GLUT-4 and GLUT-1. GLUT-4 is
specially abundant in oxidative muscles like the Sol muscle (25) and is
regulated by both insulin and contraction through two different
intracellular processing schemes possibly involving the contribution of
two different pools of GLUT-4 transporters (22) where
phosphorylation-dephosphorylation reactions are numerous. Although the
possible influence of CAI on insulin-induced glucose uptake needs to be
investigated to get a more global picture of the impact of CA activity
on muscle glucose uptake at rest and during exercise, the fact remains
that this first demonstration of the influence of CA activity on the uptake of exogenous energy substrate strengthens the notion that CA III
is possibly a key modulator of energy metabolism.
The influence of CAI on carbohydrate metabolism during the fatigue test
seems to be, however, quite different from what was noticed during the
pretest period. Once the stimulation protocol was initiated, there was
a gradual increase in HMP concentrations in the Ctr Sol muscles, with
the first significant difference observed after 5 min of stimulation
for G-6-P; after 20 min of stimulation, the ratio
F-6-P/F-1,6-P
in Ctr muscles was twice as high as the value at time
0, indicating that PFK is somehow limiting the flux. On
the contrary, HMPs did not increase over time during the fatigue test
in the CAI group. If one assumes that the flux through PFK is the same
in both groups during the fatigue test, as the values for 3-C compounds
would suggest, it is then implied that HMP production in the CAI group
is smaller than in the Ctr muscles. However, this does not agree with
the previous demonstration of an increased glycogen consumption during the same fatigue test with CAI. Thus a strict logical approach would
suggest that, during the fatigue protocol, a time when the cellular
environment is obviously very different from the situation at rest, CAI
can probably impact on some key downstream reactions, like the one
catalyzed by PDH, making increases in 3-C compounds invisible. Such an hypothesis would agree with the work of
Geers et al. (14), who found that CAI could increase oxygen consumption of rat Sol muscle incubated in vitro during very long periods by
~25%. PDH is a multienzyme complex that bridges glycolysis and the
tricarboxylic acid cycle and plays a pivotal role in the regulation of
carbohydrate metabolism as a whole. Its modulation is quite complex and
involves, among other things, a phosphorylation-dephosphorylation cascade.
Globally, the evidence is quite convincing that
1) the energy demand was higher in
the group deprived of CA activity than in the Ctr group, as suggested
by data for CP, ATP, and HMP and 2)
in the presence of CAI, the Sol muscle has an increased reliance on
carbohydrate for ATP production during contractile activity. The
overall impression is that inhibition of CA leads to some loss of
homeostatic control as it specifically relates to energy metabolism at
large. The higher rate of CP disappearance noted during the first 5 min
could be one of the metabolic mechanisms that allows muscles with CAI
to sustain higher tension production during the early part of the
fatigue test. However, there seems to be a price to pay for this
consistently higher level of muscle performance recorded during the
30-min test as, for one, ATP level decreased to unexpectedly low levels
after 30 min of contractile activity in the presence of CAI. On the
basis of this observation, one would predict that the positive
influence of CAI on fatigue should diminish gradually thereafter,
assuming that the difference in ATP levels between Ctr and CAI muscle
keeps increasing.
More specifically, two key observations deserve more in-depth analysis.
First, it is clear that the action of CAI on HMP is restricted only to
muscles undergoing some contractile activity; second, evidence shows
that the influence of CAI (and consequently of CA III) is not the same
during rest and sustained contractile activity. Such a contrast in
enzymatic behavior could possibly be related to the fact that CA III is
the only member of the CA family of isozymes to possess both a
hydratase and a phosphatase activity. The possible physiological or
biological significance of the phosphatase activity of CA III has
remained elusive until quite recently (19). Indeed, recent results
suggest that CA III, under conditions leading to increased oxidative
stress (like exercise) and, consequently, to CA III glutathiolation (5, 6), could possibly act as a protein phosphatase with tyrosine, serine,
and threonine as preferred substrates. This opens a very exciting and
interesting avenue for the biology of CA III in skeletal muscle because
both carbohydrate and lipid metabolism in this tissue contain cellular
cascades that involve protein phosphatases. It is therefore quite
possible that the influence of CA III under resting and contracting
conditions may be very different, because CA III could act as a
hydratase and/or phosphatase, respectively, under such
conditions.
In conclusion, we have shown that inhibition of the CA activity of the
Sol muscle can slightly influence muscle pH. The metabolic correlates
obtained during these same experiments suggest that the increased
resistance to fatigue induced by the CA inhibitor may be partially
related to an accumulation of glycolytic substrates that occurred
during the period of incubation before the fatigue protocol. Inhibition
of CA activity in an oxidative type I muscle is undoubtedly linked to a
loss of regulatory metabolic processes. More specific experiments will
have to be conducted to identify more precisely the underlying
regulatory mechanism(s) through which inhibition of CA III can
influence muscle metabolism.
Perspectives
The present results can be added to the data obtained previously for
glycogen utilization that show that it is increased when CA activity is
abolished. Globally, it is becoming evident that an active CA III in a
type I muscle contributes to the sparing of the glycogen store and
could therefore be part of the complex and still-debated glucose free
fatty acid (FFA) cycle. Glycogen sparing is amazingly highly efficient
in type I muscle fibers, where the highest CA III contents and
activities are found. The present data that show that CA III may
influence glucose uptake during contractile activity are provocative
and very interesting. Whether CA III has a direct effect on
carbohydrate utilization or an indirect action linked to the
preferential use of FFA as oxidative substrate also remains to be
investigated. Type I fibers do possess an important pool of
triglycerides, and it has been estimated that ~50% of the fat
oxidized during exercise of moderate intensity comes from this pool.
Because skeletal muscle hormone-sensitive lipase is sensitive to pH
fluctuations and is activated through a cascade of phosphatase
reactions, CA III could also potentially participate in the modulation
of FFA metabolism. The fact that CA III is found in very high
concentrations in the three specific tissues controlling energy
metabolism (fat cells, skeletal muscle, and liver) makes it a good
candidate for a potential role as a metabolic modulator.
| |
ACKNOWLEDGEMENTS |
We thank Lucie Turcotte for typing the manuscript and N. Jomphe for
technical assistance. Methazolamide was generously provided by Cyanamid
Canada.
| |
FOOTNOTES |
This work was supported by grants from Natural Sciences and Engineering
Research Council of Canada and the Fonds de la Recherche en Santé
du Québec. J. Frenette was the recipient of a scholarship from
the Fonds Concerté d'Aide à la Recherche.
Address for reprint requests: C. Côté, Laval Univ. Hospital
Center, Research Center, Rm. 9500, 2705 Blvd. Laurier, Ste-Foy,
Québec, Canada G1V 4G2.
Received 5 September 1996; accepted in final form 22 May 1997.
| |
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