Download The effect of endurance-training on the maximum activities of

Bioscience Reports 3, 831-835 (1983)
Printed in Great Britain
The e f f e c t
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of e n d u r a n c e - t r a i n i n g on the maximum
a c t i v i t i e s of h e x o k i n a s e , 6 - p h o s p h o l r u c t o k i n a s e ,
c i t r a t e s y n t h a s e , and o x o g l u t a r a t e d e h y d r o g e n a s e
in red and w h i t e m u s c l e s of the rat
Paramjeet K. SOAR% C. T. Mervyn DAVIES% Peter H. FENTEM*,
and Eric A. NEWSHOLME**
*Department of Physiology and Pharmacology,
Medical School, Queen's Medical Centre,
University of Nottingham, Nottingham, U.K.; and
**Department of Biochemistry, University of Oxford,
South Parks Road, Oxford OXl 3QU, U.K.
(Received 18 July 1983]
Adult female rats were subjected to an eleven-week
endurance-training programme, and, for the first time,
the maximum activities of enzymes that can indicate
the quantitative capacities of both anaerobic glycolysis
a n d the Krebs c y c l e in muscle (viz. 6-phosphof r u c t o k i n a s e and o x o g l u t a r a t e dehydrogenase respectively)
w e r e m e a s u r e d in h e a r t plus white and
f a s t - o x i d a t i v e skeletal muscle.
No changes were
observed in heart muscle.
In fast-oxidative skeletal
muscle~ activities of hexokinase, citrate synthase, and
oxoglutarate dehydrogenase were increased by 51~ 267
and 33% r e s p e c t i v e l y but there was no e f f e c t on
6-phosphofructokinase.
These results demonstrate that
in red m u s c l e t h e r e is no e f f e c t of this training
programme on the anaerobic capacity but that of the
aerobic system is increased by one third.
In white
skeletal muscle, only the activity of citrate synthase
was increased, which indicates that this activity may
not provide even qualitative information about changes
in capacity of the Krebs cycle.
The m a x i m u m activities of hexokinase (EC 2.7.1.1), 6-phosphof r u c t o k i n a s e (EC 2 . 7 . 1 . 1 I ) , and o x o g l u t a r a t e dehydrogenase (EC
1.2.~.2) in muscle have been shown to provide quantitative indices of
the maximum capacities of glucose utilization, glycogen degradation to
l a c t i c acid, and the Krebs cycle~ rexpectively (see Crabtree &
Newsholme, 1972; Newsholme et al.~ 1980).
Indeed, of the enzymes
that comprise the Krebs cycle and electron transport that have been
investigated, only oxoglutarate dehydrogenase activity has been shown
to correlate with the maximum flux through the cycle (Alp et al.,
1976; Read et a]., 1977; Newshotme~ 1980; Cooney et a l , i9gt;
Newsholme & Paul~ 1983). Although it has been well established that
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The Biochemical Society
SOAR
832
ET AL.
endurance-training of rats and man increases the capacities of the
o x i d a t i v e p r o c e s s e s in m u s c l e , including t he K rebs c y c l e and
e l e c t r o n - t r a n s f e r chain (for review, see Gollnick & Saltin~ 1992), t here
are no reports of the e f f e c t of training on oxoglutarate dehydrogenase
activity; most studies have used c i t r a t e synthase or succinate dehydrogenase activities which do not correl at e with the cycle flux (see
above reference).
H e n c e conclusions concerning the quantitative
e f f e c t s of endurance-training on the maximum capacity of the Krebs
cycle cannot be made with any certainty.
Consequently~ the e f f e c t s
of endurance-training of rats on the maximum activities of hexokinase,
6-phosphofructokinase, oxoglutarate dehydrogenase, and an additional
Krebs-cycte enzym% c i t r a t e synthase (EC #.1.3.7), were investigated.
Th e e f f e c t s of t r a i ni ng on three different types of muscle were
i n v e s t i g a t e d : t h e superficial portion of the vastus lateralis~ which
contains predominantly fast-twitch glycolytic fibres (Type IIB); the
d e e p p o r t i o n of the g a s t r o c n e m i u s , which contains predominantly
f a s t - t w i t c h oxidative fibre (Type IIA) (see Pet er et a l , 1972), and
cardiac muscle.
Materials
and Methods
Chemicals and enzymes
All c h e m i c a l s and enzymes were obtained from the Boehringer
C o r p . ( L o n d o n ) Ltd.~ Lewes~ Sussex, e x c e p t for the following:
5~5'-dithiobis- (2-nitrobenzoic acid) and ethyleneglycol-bis-B-amino-ethyt
e t h e r ) N N ' - t e t r a a c e t i c acid were obtained from Sigma Chemical Co.,
Poole, Dorset; Tes ( 2- ( [ 2-hydroxyl- 1,1 -his- ( hyd roxym ethyl ) ethyl ] am ino ) e t h a n e s u l p h o n i c acid) was obtained from Calbiochem-Behring Corp.,
Bishops Stortford~ Herts.
Animals
Female Wistar rats (initial age l l weeks) obtained from the 3oint
Animal Breeding Unit9 School of Agricultur% Sutton Bonnington~ were
housed in groups of five in a t e m p e r a t u r e - c o n t r o l l e d room (20~
with
a 12-h dark-light cycle and were provided with free access to food
and water. The animals were introduced to the motor-driven treadmill
for 3 successive days for 5-10 min daily at 10 m/min. Those animals
that responded readily to the treadmill were assigned to the exercising
group. The sedentary group were limited to cage activity only but in
every other way were t r e a t e d identically to the exercising group. The
training procedure employed was the one-step programme as described
by Booth (1977): the animals were run on a treadmill for 10 min per
day at 27 m per min for 5 days per week for 2 weeks; this was
followed by running for 100 rain per day at 27 m per rain for 7 days
per week for 9 weeks.
Each animal was exercised at the same time
each day and sacrificed 24 h following the last exercise period.
Preparation of homogenates
An imals w e r e killed by cervical dislocation, and muscles
rapidly dissected and~ for all assays except 6-phosphofructokinas%
immediately homogenized.
For 6-phosphofructokinase assay,
pieces of muscle were frozen by dropping into liquid nitrogen and
were
were
small
were
ENDURANCE-TRAINING
AND ENZYME
ACTIVITIES
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stored at -70~
for 2 months.
Preliminary studies showed that this
t r e a t m e n t did not a f f e c t the maximum activity of this enzyme.
For
assay of hexokinase, muscle was homogenized in 10 vol. of ext ract i on
medium consisting of 50 mM triethanolamine/HCl, i mm EDTA, 2 mM
MgCI2, and 30 mM mercaptoethanol at pH 7.5.
For 2-oxoglutarate
dehydrogenase and c i t r a t e synthase the muscle was homogenized in 5
vol. of ex t r a c t i on medium which consisted of 250 mM mannitol, 5 mM
Tes (Na salt), and 1 mM EGTA at pH 7.4. For 6-phosphofructokinase
the ex tr act i on medium consisted of 50 mM Tris/HCl, 5 mM MgCl2~ l
mM EDTA, and 20 mM mercaptoethanol at pH g.2.
Assay of enzyme activity
H e x o k i n a s e and 6-phosphofructokinase were assayed as described
previously (Zammit & Newsholme, 1976; Opie & Newsholm% I967).
For cardiac muscle, the crude extract was used for a c t i v i t y assays,
but for skeletal muscle the extract was centrifuged at 1000 g for 2
min p r i o r to use.
A c t i v i t i e s of 2-oxoglutarate dehydrogenase and
citrate synthase were measured as described by Cooney et al. (19g 1).
All activities were measured on a Gilford recording spectrophotometer
(Model 240) at 25~
Expression of results
All activities are presented as lamol/min per g of fresh muscle.
Preliminary experiments established that provided glycogen stores in
the muscle are normal there is no effect of training on the protein
content.
R e s u l t s and D i s c u s s i o n
The l l - w e e k endurance-training programme used in this study did
not cause any change in the activities of the enzymes of glycolysis or
the Krebs cycle in cardiac muscle (Table 1). This suggests that the
maximum capacities of both anaerobic and aerobic processes in cardiac
muscle are unaffected by the intensity of training resulting from the
regime employed in this study. Similarly, this type of training had no
effect on the a c t i v i t y of 6-phosphofructokinase in fast-oxidative or
white skeletal muscle, suggesting t h a t the maximum capacity of the
a n a e r o b i c process was unaffected.
In the fast-oxidative skeletal
muscle, the maximum activities of hexokinase, citrate synthase, and
2-oxoglutarate dehydrogenase were increased b y 51%, 26%, and 33%
respectively. These results indicate an improved capacity for glucose
utilization and aerobic metabolism via the the Krebs cycle. Moreover,
these f i n d i n g s suggest that the magnitude of change of the two
processes in response to training is similar. A more intensive training
programme is known to increase hexokinase a c t i v i t y in red muscle by
170% (Baldwin et al., 1973) and on the basis of the present findings
i t is suggested that the increase in oxoglutarate dehydrogenase a c t i v i t y
and hence the aerobic c a p a c i t y would be of a similar order of
magnitude.
The only e f f e c t of endurance-training on white muscle was an
increase in citrate synthase a c t i v i t y of about 2g%.
Since there was
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ET AL.
Table I. Effect of exercise-training on activities of
hexokinase, 6-phosphofructokinase, citrate synthase,
and oxoglutarate dehydrogenase in three muscles of the rat
Rats were endurance-trained and enzyme activities were measured as
described in Materials and Methods.
Activities are presented as
means
• S.E.M.
for five s e p a r a t e
animals
in each group.
Statistical significance of the difference in activities between
control and exercise-trained rats (Student's t-test) is indicated
by *(P < 0.025); **(P < 0.01); ***(P < 0.005).
Condition
of animal
(Hmol/min
Heart
Enzyme activities
per g fresh wt. at 25~
Gastrocnemius
Vastus
lateralis
Hexokinase
Sedentary control
Exercise-trained
6.3 + 0.18
5.4 + 0.15
0.86 + 0.05
1.3 • 0.06*
0.51 + 0.03
0.60 + 0.03
6-Phosphofructokinase
Sedentary control
Exercise-trained
13.1 + 1.0
11.5 + 0.4
43.6
40.1
-+ 1.7
+ 2.3
48.4
51.8
+ 1.6
+ 3.2
Citrate synthase
Sedentary control
Exercise-trained
91.2 + 7.2
85.4 + 4.3
11.5
14.5
-+ 0.22
+ 0.59**
2.9
3.7
+ 0.09
+- 0.02**
Oxoglutarate dehydrogenase
Sedentary control
Exercise trained
7.5 + 0.34
8.3 + 0.59
1.2
1.6
+ 0.06
+ 0.08***
0.53 + 0.06
0.42 + 0.02
no change in oxoglutarate dehydrogenase activity, it is unlikely that
this increase in citrate synthase a c t i v i t y is indicative of an increase in
the maximum capacity of the Krebs cycle in this muscle. This finding
raises doubts as to whether changes in citrate synthase can provide
even q u a l i t a t i v e i n f o r m a t i o n about e f f e c t s of training or other
conditions on the maximum capacity of the Krebs cycle.
The early
stages of the Krebs cycle can be used to provide 2-oxoglutarate not
for further oxidation in the cycle but for the formation of glutamine
(Newsholme, 1976).
It is suggested that the increase in a c t i v i t y of
c i t r a t e synthase increases the m a x i m u m c a p a c i t y for glutamine
formation in white muscle of trained rats. An increased capacity for
glutamine production may be necessary to satisfy increased requirements for glutamine utilization by kidney; increased rates of production of acids such as fatty acid and lactic acid during each training
session may result in a higher rate of proton excretion by the kidney
in trained animals and hence an increased demand for glutamine by
the kidney (for review see Newsholme & Leech, 1993).
ENDURANCE-TRAINING
AND
ENZYME
ACTIVITIES
References
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