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INTRODUCTION |
Skeletal muscle is a heterogeneous tissue, which responds
differentially to a large variety of stimuli (1, 2). Modifications of
basal contractile activity, induced by denervation or chronic low
frequency stimulation, promote an adaptive response. These adaptations
are reflected in muscle phenotype; i.e. there are variations
in muscle type fiber composition, characterized by different
biochemical and physiological properties.
One consequence of denervation or chronic low frequency stimulation in
rabbit fast twitch skeletal muscle is the phenotypic transition from
fast twitch to slow twitch fiber type (3-6). These changes involve
variations in enzyme activities and, in some case, isozyme expression.
Fast twitch fibers are characterized by the predominance of anaerobic
glycolysis, while in slow twitch fibers aerobic glycolysis is the main
pathway by which energy demands are supplied during contraction. Thus,
glucose metabolism is involved in this process.
The rate-limiting enzyme of glycolysis is 6-phosphofructo-1-kinase
(PFK-1),1 which is regulated
by a large number of modulators. The bisphosphorylated sugar fructose
2,6-bisphosphate (Fru-2,6-P2) has been identified as a
potent positive effector of this enzyme in several mammalian tissues
(7). Moreover, modifications of Fru-2,6-P2 by factors such
as hormonal or contraction pattern have been related with variations of
the glycolytic rate (8-11).
The intramuscular concentration of Fru-2,6-P2 is controlled
by the bifunctional enzyme
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2),
which is responsible for the synthesis and degradation of this
metabolite. This enzyme has several isoforms, the relative abundance of
which depends on the metabolic properties of the tissue. These isozymes
differ in size, kinetics, and immunologic properties as well as in
their response to phosphorylation by protein kinases. Skeletal muscle
expresses two of these isoforms. It mainly contains muscle isozyme
(M-type), and a small amount of the liver isozyme (L-type) (12-16).
Both isoforms are encoded by the same gene, as a result of alternative
splicing, whose expression is under hormonal control (17).
Moreover, contractile activity has a regulatory effect on some proteins
related with glucose metabolism in fast twitch skeletal muscle. The
absence of contractions induces an increase in GLUT-1 (18) and HK-II
(19, 20), while an increase in contractile activity induces a
rise in GLUT-4 (21) and HK-II (22). These studies highlight the
importance of muscle contraction in the regulation of protein
expression related with glucose metabolism.
Here we examine the influence of contractile activity on
Fru-2,6-P2 metabolism and its regulation through
PFK-2/FBPase-2. We measured the changes in this enzyme induced by
denervation or by chronic low frequency stimulation at 10 Hz in fast
twitch rabbit skeletal muscle.
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EXPERIMENTAL PROCEDURES |
Chemicals--
[
-32P]ATP (5000 Ci/mmol),
[
-32P]dCTP (3000 Ci/mmol), and Hybond N filters were
purchased from Amersham Pharmacia Biotech. The random priming DNA
labeling kit was from Roche Molecular Biochemicals. Enzymes and
biochemical reagents were from either Roche Molecular Biochemicals or
Sigma. All other reagents were of analytical grade.
Treatment of Animals--
Adult White New Zealand rabbits
weighing between 3.0 and 3.5 kg were fed ad libitum and
housed in animal quarters with a 12-h light, 12-h dark cycle. For
denervation studies, animals were anesthetized by intramuscular
injection of 20 mg/kg ketamine (Ketolar, Parke-Davis) and a 2-ml
solution of 0.5% azepromazine (Calmoneosan, Smithkline Beecham), the
peroneal nerve was unilaterally severed, and 10-15 mm of the distal
stump was removed. Finally, the proximal part of the nerve was ligated
in the Biceps femoris muscle to avoid reinnervation. The contralateral
control leg was sham-operated.
For stimulation studies, under the same anesthesia protocol as in
denervated animals, 1 week before the onset of stimulation period,
electrodes were implanted laterally to the peroneal nerve as described
elsewhere (23). This allowed the continuous stimulation at 10-Hz trains
(trains of pulses of 18 mA for 0.15 ms) for 24 h/day.
After various periods of denervation or stimulation (1, 2, 4, 7, or 14 days), right (control) and left (denervated or stimulated) tibialis
anterior (TA) muscles were carefully exposed. Both muscles were removed
simultaneously by two researchers and quickly frozen with aluminum
clamps precooled in liquid N2. The frozen samples were
stored at 
80 °C until analysis.
Assay of Metabolites--
Samples of frozen muscle were powdered
in a stainless steel percussion mortar precooled in liquid
N2 and used for the measurement of metabolites.
Glycogen was extracted from about 10 mg of muscle by alkaline
treatment. The hydrolysis and measurement of glucose produced were
carried out using the anthrone method (24). ATP, glucose 6-phosphate
(Glc-6-P) and fructose 6-phosphate (Fru-6-P) were extracted by acid
treatment from about 20 mg in ice-cold 0.5 M perchloric
acid. After neutralization and centrifugation, aliquots of the extracts
were used. Measurements were carried out using enzymatic methods with
fluorimetric techniques (25).
For measuring fructose 2,6-bisphosphate (Fru-2,6-P2), about
30 mg of frozen muscle powder was homogenized in 10 volumes of 50 mM NaOH and kept at 90 °C for 10 min. The extract was
neutralized with 250 mM sodium acetate (pH 4.0), and the
soluble material was used for determination of Fru-2,6-P2
(26).
Partial Purification of
6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase--
Muscle
samples were homogenized in 5 volumes of 20 mM TES, 100 mM KCl, 1 mM dithiothreitol, 5 mM
EDTA, 5 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride, and 2.5 ml/liter leupeptin, pH 7.5. The crude extracts were
fractionated with 6% (w/v) polyethylene glycol 6000 and centrifuged at
26,000 × g for 30 min. The resulting supernatants were
fractionated again with 15% (w/v), polyethylene glycol 6000 and
centrifuged in the same conditions described before. Finally, the
pellets were resuspended in a half volume of initial tissue weight with homogenizing medium and used to measure PFK-2/FBPase-2 activity (27).
Enzyme Assays--
PFK-2 activity was measured as described by
El-Maghrabi et al. (28). Partially purified (6-21% PEG) TA
muscle PFK-2 activity was assayed at 30 °C in 0.1 ml of 20 mM TES, 10 mM KCl, 50 mM MgCl2, 25 mM KH2PO4,
0.5 mM EDTA, 0.5 mM EGTA, 1.2 mM
phenylmethylsulfonyl fluoride, 2.5 mg/liter leupeptin, 25 mM ATP, 25 mM Fru-6-P under optimal conditions
and 0.5 mM ATP, 0.1 mM Fru-6-P under suboptimal conditions.
The reaction was stopped by adding 5 µl of 2.5 N NaOH,
and samples were held at 90 °C for 10 min, after which time samples were used for assay of Fru-2,6-P2.
FBPase-2 activity was measured by the production of
[32P]Pi from
[2-32P]Fru-2,6-P2, which was synthesized as
described by El-Maghrabi et al. (28). The reaction was
carried out at 30 °C in 50 mM Hepes buffer (pH 7.5)
containing 50 mM KCl, 5 mM
KH2PO4, 2 mM EDTA, 1 mM
dithiothreitol, 2 mM MgCl2, 5 mM
[2-32P]Fru-2,6-P2 (200,000 cpm/assay). The
product of the reaction, fructose 6-phosphate, was removed by an
enzymatic system consisting of 0.1 mM NADP+, 9 units/ml phosphoglucoisomerase, and 4 units/ml glucose 6-phosphate dehydrogenase. The reaction was stopped by the addition of 1 volume of
0.1 M NaOH. Blanks did not exceed 0.2% of the applied radioactivity.
Treatment of PFK-2 with the Catalytic Subunit of Cyclic
AMP-dependent Protein Kinase--
Partial purified PFK-2
from control, denervated, and stimulated TA muscle were incubated at
30 °C for 20 min with the catalytic subunit of cyclic
AMP-dependent protein kinase (1 milliunit/ml) in a final
volume of 0.1 ml containing 100 mM Hepes (pH 7.1), 1 mM dithiothreitol, 0.1% bovine serum albumin. The reaction
was started by the addition of 1 mM ATP-Mg2+
and stopped by the addition of 5 mM EDTA. Samples were
removed and assayed for PFK-2 activity (28, 29).
Western Blot Analysis of PFK-2/FBPase-2--
Control,
denervated, and stimulated muscle PFK-2/FBPase-2 were partially
purified by PEG fractionation followed by SDS-polyacrylamide gel
electrophoresis in a 10% acrylamide gel. Proteins were then transferred to a nitrocellulose membrane (Immobilon-P, polyvinylidene difluoride, Millipore Corp.). For immunodetection, two antibodies were
used: polyclonal antiserum MCL-2 (30) against L-type and M-type
PFK-2/FBPase-2 isozymes and polyclonal antiserum CL against L-type PFK-2/FBPase-2 isozyme (31). Both antibodies were a
generous gift from Dr. Hue. MCL-2 and CL IgG were produced in rabbits. Primary antibody was preincubated for 1 h with a secondary
antibody (peroxidase-conjugated anti-rabbit). To prevent an excess of
secondary antibody, a third IgG, against a maize protein that is not
present in mammalian tissue, was added and incubated for 1 h.
Later, this mixture was used for incubating the membrane (2 h) and for
detecting PFK-2/FBPase-2 by enhanced chemiluminescense, (ECL, Amersham
Pharmacia Biotech). A control test of rabbit blood ruled out any
possible disturbance attributable to saturation of the secondary with
the tertiary antibody.
RNA Isolation and Northern Blot Analysis--
Total RNA from
muscle was extracted using the acid guanidinium
isothiocyanate/phenol/chloroform method as described by Chomczynski and
Sacchi (32). All samples had a 260/280 absorbance ratio above 1.7.
After quantification, total RNA (15-30 µg) was denatured at 65 °C
in the presence of formamide, formaldehyde, and ethidium bromide (33)
to allow the visualization of RNA. RNA was separated on a 1.2%
agarose/formaldehyde gel and blotted on Hybond N filters. The RNA in
gels and filters was visualized with ethidium bromide and photographed
by UV transillumination to ensure the integrity of RNA, to check the
loading of equivalent amounts of total RNA, and to confirm proper
transfer. RNA was transferred in 10× standard saline citrate (SSC;
0.15 M NaCl and 0.015 M sodium citrate, pH 7.4). After transfer, RNA was fixed by irradiating the membrane with UV
light for 5 min.
Blots were initially prehybridized for 6 h at 42 °C in 50%
(v/v) formamide, 2× PSE (1× PSE: 0.01 M Pipes, 0.4 M NaCl, 1 mM EDTA at pH 6.4), 0.5% (w/v) SDS,
5× Denhart's solution (1× Denhart's solution: 0.2 g/liter Ficoll,
0.2 g/liter polyvinyl pyrrolidone, and 0.2 g/liter bovine serum
albumin) and 0.5 mg/ml of heat-denatured herring sperm DNA.
Hybridization was allowed to proceed overnight at 42 °C in the same
medium. The cDNA probe was labeled with [32P]dCTP by
random oligonucleotide priming, and it was included at 2 × 106 cpm/ml.
Filters were washed twice in 2× SSC at room temperature for 5 min and
twice in 0.1× SSC plus 0.1% SDS at 50 °C for 30 min.
The abundance of specific PFK-2/FBPase-2 mRNA was quantified by
scanning densitometry of autoradiograms as described above, and data
were expressed as a percentage of control values.
Other Methods--
Protein was determined with bovine serum
albumin as a standard (34). Statistical significance of differences was
assessed by nonparametric Kruskal-Wallis test. Results were considered significant when p was <0.05.
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RESULTS |
Effects of Muscle Denervation or Stimulation on Muscle
Fructose-2,6-bisphospate Level--
Fru-2,6-P2 levels were
determined at different times after TA muscles were denervated or
stimulated. Both treatments increased Fru-2,6-P2 (Fig.
1).
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Fig. 1.
Time course of changes in
Fru-2,6-P2 concentration. Fru-2,6-P2
levels were measured from neutralized alkaline extract under conditions
described under "Experimental Procedures." Values of control
( ), denervated ( ), and stimulated muscle ( ) are shown. Each
point shows the mean ± S.E. for 4-6 samples. *, significant
difference (p < 0.05) between levels of control and
denervated or stimulated muscles.
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In denervated muscle, a significant increase occurred 2 days after
nerve section, and it persisted throughout the 14 days of the
experiment. At the end of this period Fru-2,6-P2
concentration was approximately 3-4-fold higher than the control value.
Chronic low frequency stimulation also increased
Fru-2,6-P2. By day 1, there was a significant increase,
which persisted at day 4. The peak at day 4 was approximately 4-5-fold
higher than control. As stimulation continued, the concentration
declined to control values at day 30 (results not shown).
Fru-2,6-P2 values in control remained constant throughout
the experiment.
Because levels of Fru-2,6-P2 in skeletal muscle are
controlled by substrate availability (12), its precursors (Glc-6-P, Fru-6-P, ATP) were studied. Denervation or stimulation had different effects on the intracellular concentration of these metabolites (Table
I).
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Table I
Effects of denervation or chronic low frequency on metabolite levels of
rabbit fast twitch skeletal muscle
Glc-6-P, Fru-6-P, and ATP were measured from neutralized perchloric
acid extracts as described under "Experimental Procedures." Values
are means ± S.E., n = 3-6.
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In denervated muscle, Glc-6-P and Fru-6-P decreased by more than 50%
at day 4, and their concentration remained significantly lower than
that of control muscle. ATP showed a significant decrease at day 4, which persisted for 14 days.
In stimulated muscle, although Glc-6-P was already lower on the first
day, Fru-6-P followed the same pattern as in denervated muscle.
However, in stimulated muscle these metabolites returned to control
values at day 14. ATP was constant throughout this period.
Effects of Denervation or Stimulation on PFK-2 and
FBPase-2 Activities--
In order to explain the increases
observed in Fru-2,6-P2 after denervation or stimulation of
muscle, we measured the enzyme activity for the synthesis and
degradation of this bisphosphorylated metabolite, using partial
purified (6-21% PEG) TA muscle homogenate.
Denervation and stimulation increased PFK-2 activity (Fig.
2A). In denervated muscle,
kinase activity increased significantly at day 1, reaching the highest
value at day 2. PFK-2 activity then decreased rapidly to control
values. The pattern of change in stimulated muscle was similar to the
changes in denervated muscle. Kinase activity increased significantly
at day 1, reached a maximum at day 4, and declined thereafter to
control values. The response of FBPase-2 activity depended on the
muscle treatment (Fig. 2B). In denervated muscle, there
was a significant decrease at day 1, which was maintained
for the rest of the study. In contrast, in stimulated muscle
FPBase-2 increased at day 1, reached a maximum at day 4, and
recovered basal level at day 7.
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Fig. 2.
Time course of PFK-2/FBPase-2
activities. PFK-2/FBPase-2 activities were measured from
extract treated with PEG 6-21% under conditions described under
"Experimental Procedures." A, the time course of PFK-2
activity; B, the time course of FBPase-2 activity;
C, the ratio PFK-2/FBPase-2 from denervated (),
stimulated (), or control muscle (). Each point shows the
mean ± S.E. for 4-6 samples. *, significant difference
(p < 0.05) between zero point and values after the
onset of stimulation.
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The effect on the kinase/phosphatase activity ratio also depended on
muscle treatment (Fig. 2C). After denervation, the ratio increased significantly at day 1. By day 2, the ratio (2.36 ± 0.30) was approximately 10-fold higher than control, and it declined thereafter to control values. In contrast, stimulation did not modify
the ratio (0.17-0.34 ± 0.05).
Effects of sn-Glycerol 3-Phosphate and Treatment of Catalytic
Subunit of Cyclic AMP-dependent Protein Kinase on PFK-2
Activity--
Because the PFK-2/FBPase-2 ratio in denervated muscle
was similar to that previously described for liver (35), we proposed a
kinetic study. As described previously (12, 35), inhibition by
sn-glycerol 3-phosphate and cyclic AMP-dependent
protein kinase phosphorylation can be used to differentiate M-type
isozyme from L-type. To assess whether the L-type isozyme was present
in denervated muscle, we measured the kinase activity in the presence
of sn-glycerol 3-phosphate. Moreover, we carried out a
treatment with the catalytic subunit of cyclic
AMP-dependent protein kinase.
PFK-2 activity from denervated muscle was inhibited by a low
concentration of sn-glycerol 3-phosphate. In contrast, PFK-2 activity from stimulated muscle was unaltered at any of the
concentrations tested. As expected, activity from control muscle was
not modified (Fig. 3).
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Fig. 3.
Effects of sn-glycerol
3-phosphate on PFK-2 activity from control, denervated, and stimulated
rabbit tibialis anterior muscle. Partially purified PFK-2 from
control (), 2-day denervated (), and 2-day stimulated ()
muscles were assayed in suboptimal conditions in the presence of 0.1 mM fructose 6-phosphate and 0.5 mM
ATP-Mg2+ and with the indicated concentrations of
sn-glycerol 3-phosphate at pH 7.1. Each point shows the
mean ± S.E. for 4-6 samples. *, significant difference
(p < 0.05) between levels of control and denervated or
stimulated muscles.
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When extracts were incubated in the presence of the catalytic subunit
of cyclic AMP-dependent protein kinase, PFK-2 activity from
denervated muscles decreased by more than 50%, whereas the PFK-2
activity from control and stimulated muscle did not show any change
(Table II). Simultaneously, the extracts
were also incubated in the presence of Mg2+ to activate
phosphatases. This treatment had no effect on PFK-2 activity,
indicating that the enzyme was not phosphorylated at the start of
incubation.
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Table II
Effects of treatment of PFK-2 with the catalytic subunit of cAMP
protein kinase
Partially purified PFK-2 from control, 2-day denervated and 2-day
stimulated muscles were incubated with 1 milliunit of catalytic subunit
of cyclic AMP-dependent protein kinase in a final volume of
0.1 ml containing 20 mM Hepes buffer, pH 7.1, 0.1% bovine
serum albumin, 5 mM MgCl2, 1 mM
ATP-Mg2+, and 1 mM dithiothreitol at 30 °C for
20 min. Samples (40 µl) were taken to measure PFK-2 activity as
described under "Experimental Procedures." The results (mean ± S.E.) of three separate experiments are shown.
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Effects of Denervation or Stimulation on PFK-2/FPBase-2
Transcript--
In order to assess the effects of contractile muscular
activity on the expression of PFK-2/FPBase-2, we determined the
mRNA levels.
RNA was obtained from control, denervated, and stimulated TA muscle on
the second day of treatment. The abundance of PFK-2/FPBase-2 mRNA
was determined by Northern blot analysis (Fig.
4).
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Fig. 4.
Expression of PFK-2/FBPase-2 mRNA on
control, denervated, and stimulated rabbit tibialis anterior
muscle. Total RNA was purified from 2-day denervated
(D), 2-day stimulated (S), and contralateral
tibialis anterior muscles (C). RNA was measured
spectrophotometrically, and the integrity and relative amounts of RNA
in each sample used were checked by ethidium bromide staining on the
same gel (bottom of A). 20 µg of total RNA from
the different groups was applied on gels. PFK-2/FBPase mRNA was
detected after hybridization with rat PFK-2/FBPase cDNA probe, as
described under "Experimental Procedures." A representative
autoradiogram is shown in A (top). Autoradiograms
were subjected to scanning densitometry. The results (mean ± S.E.)of three separate experiments are shown and expressed as a
percentage of control value (B). *, significant difference
(p < 0.05) between levels of control and denervated or
stimulated muscles.
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Denervated muscle showed a 25% reduction in PFK-2/FPBase-2 mRNA
content, and a further reduction was noted at day 7 (results not
shown). In contrast, stimulation for 2 days led to a sharp increase
(50%) in the specific mRNA levels.
Effects of Denervation or Stimulation on the Quantity of
PFK-2/FPBase-2 Protein--
In order to determine the relative amounts
of PFK-2/FBPase-2 protein, we used polyclonal antiserum MCL-2 (30),
which recognizes L- and M-type isoforms. The signal intensity from
denervated muscle (Fig. 5A,
lane D) was 10% of control muscle
(lane C). In contrast, the signal from stimulated
muscle (Fig. 5A, lane S) increased 2.5-fold.
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Fig. 5.
Western blot analysis of PFK-2/FBPase-2
in control, denervated, and stimulated rabbit tibialis anterior
muscle. Samples of control, denervated, and 2-day stimulated
muscles were partially purified by polyethylene glycol fractionation,
as described under "Experimental Procedures." SDS-polyacrylamide
gel electrophoresis (40 µg of protein/lane) was then
analyzed by Western blot using polyclonal antiserum MCL-2 (30) against
L-type and M-type PFK-2/FBPase-2 isozymes (A) and polyclonal
antiserum CL against L-type PFK-2/FBPase-2 isozyme specifically (31). A
liver sample was used as a positive control of L-type (lane
L) (B). C, control muscle;
lane D, 2-day denervated muscle; lane
S, 2-day-stimulated muscle.
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To determine the L-type isoform, we used the polyclonal antiserum CL
raised against the L-type isozyme (31). The signal from denervated
muscle (lane D) was twice as strong as that
signal from control muscle (lane C) (Fig.
5B).
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DISCUSSION |
We examined the influence of contractile activity on
Fru-2,6-P2 concentration and the influence of
PFK-2/FBPase-2 activity on fast twitch skeletal muscle. To this end we
chose two situations: (i) denervated fast twitch skeletal muscle, which
is characterized by the absence of contractions, and (ii) chronic low
frequency stimulated fast twitch skeletal muscle, which is
characterized by increased contractile activity.
Our results show that both conditions induced an increase in
Fru-2,6-P2 and a modification of the L- and M-type
PFK-2/FBPase-2 isozyme expression. Moreover, we found these changes may
depend on contractile activity. Denervation provoked an increase in
L-type isozyme, while chronic low frequency stimulation induced an
increase in M-type isozyme.
It has been found that Fru-2,6-P2 concentration is
dependent on substrate availability (7); however, the lack of
variations in Fru-6-P and ATP in our experiments suggested that
Glc-6-P, Fru-6-P, and ATP were not responsible for the increase in
Fru-2,6-P2. Because Fru-2,6-P2 concentration is
also dependent on synthesis/degradation equilibrium, we focused our
attention on the study of PFK-2/FBPase-2 activities.
In denervated muscle, the increased PFK-2 activity and the concomitant
reduction in FBPase-2 could thus explain the increase in
Fru-2,6-P2. The PFK-2/FBPase-2 ratio increased after
denervation from 0.3 to 2.4. This change toward values characteristic
of liver-type isozyme was interpreted as resulting from increased
L-type isozyme synthesis. Since there is evidence that L-type could be
differentiated from M-type by the inhibitory effect of
sn-glycerol-3-phosphate and by the lower kinase activity
after phosphorylation by cyclic AMP-dependent protein
kinase (35), we proposed using these characteristics to examine the
possible change from M-type to L-type. Our findings are consistent with
an increase in L-type isozyme. This possibility was supported by an
increased signal in Western blot analysis, with specific
L-PFK-2/FBPase-2 antibody. In conclusion, denervation elicited a change
from M-type to L-type in fast twitch skeletal muscle.
In stimulated muscle, the increase in Fru-2,6-P2 was
coincident with increases in kinase activity, which confirms previous results (11). Moreover, stimulation induces increases in
FBPase-2. The PFK-2/FBPase-2 ratio therefore remained
constant throughout the stimulation period. This ratio was similar to
values described elsewhere in skeletal muscle from rat (14). The lack
of variation in this ratio indicated that the isozyme type was not
modified. This was confirmed by the absence of
sn-glycerol-3-phosphate inhibition (Fig. 3) and the lack of
effect of the catalytic subunit of cyclic AMP-dependent
protein kinase on kinase activities (Table II). Since isozyme
expression did not appear to have changed, the increase in kinase and
phosphatase activity was attributed to a rise in M-type PFK-2/FBPase-2
protein. This assumption was confirmed by the stronger signal in
denervated than that in control muscle by Western blot analysis with
the specific M-type PFK-2/FBPase-2 antibody, and this increase was
coincident with a rise in PFK-2/FBPase-2 mRNA. In conclusion,
stimulation increased M-type in fast twitch rabbit skeletal muscle.
The variation in contractile activity induced by denervation (3) or
stimulation (6) has been described as causing transformation of fast
twitch fibers into slow twitch fibers. In concordance with our results,
these variations are manifested as modifications of enzyme activities
and/or its isozyme distribution. In denervated fast twitch muscle,
phosphoglycerate mutase isozyme pattern changes from MM-type toward MB
and BB forms (36). Moreover, there is an increase in GLUT-1 (18, 37)
and HK-II (19, 20). In the same way, muscle hyperactivity induced by
stimulation causes increases in GLUT-4 (21) and HK-II (22), while
lactate dehydrogenase isozyme changes its isozyme distribution toward
H-type (38). The literature shows that contractile activity modifies
protein expression (for a review, see Ref. 6). Furthermore, in
transgenic mice the existence of nerve- and
activity-dependent elements in the proximal promoter region
of the human aldolase A gene has recently been identified, suggesting
that multiple transcription programs are involved in fiber specificity
(39).
The relationship between the transcriptional mechanism involved in
denervation and/or chronic low frequency stimulation and the expression
of L- and M-type PFK-2/FBPase isozyme in skeletal muscle remains to be elucidated.
In summary, in this paper we present evidence showing that
PFK-2/FBPase-2 isozyme expression is modulated by contractile activity. The absence of muscular activity induced by denervation increases the
expression of L-type PFK-2/FBPase-2, while the muscular hyperactivity induced by chronic low frequency stimulation increases the expression of M-type PFK-2/FBPase-2. Moreover, both treatments cause a huge rise
in Fru-2,6-P2 concentration.
,
TOP
ABSTRACT
INTRODUCTION
Recipient of research fellowships (Ayudas para la
Incorporación de Doctores y Tecnólogos) from the Ministerio
de Educación y Cultura, Spain. To whom correspondence should be
addressed: Dept. Ciencias Fisiologicas I, Facultad de Medicina,
Universidad de Barcelona, C/Casanova 143, E-08036 Barcelona, Spain.
Tel.: 34-93-402-19-19; Fax: 34-93-403-58-82; E-mail:
jcadefau@medicina.ub.es.
