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_ LIBRARY
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This is to certify that the
thesis entitled
Exercise-Induced Biochemical Alterations
in DifferenT.Types of Skeletal Muscle

presented by
Robert Charles Hickson
has been accepted towards fulfillment

of the requirements for

Ph.D. dpmppin Physical Education

 

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ABSTRACT

EXERCISE-INDUCED BIOCHEMICAL ALTERATIONS
IN DIFFERENT TYPES OF SKELETAL MUSCLE

BY

Robert Charles Hickson

The purpose of this study was to determine the effects of
aerobic and anaerobic programs of endurance running on selected
enzymatic activities in the left plantaris, the left soleus, and the
"white" area of the left vastus lateralis muscles of the adult male
albino rat. The training regimens were the Controlled Running Wheel
programs previously reported from this laboratory. Biochemical
determinations of the muscle homogenates were made on the levels of
activity of phosphoglucomutase, an enzyme of glycogenolysis; phosphoglu-
coisomerase, an enzyme of glycolysis, lactate dehydrogenase, an enzyme
concerned with anaerobic metabolism of glycolytically-formed pyruvate;
and fumarase, a mitochondrial enzyme of the tricarboxylic acid cycle.
Enzyme activity ratios also were investigated in each of the fiber
types.

Eighty-four normal, male, albino rats of the Sprague-Dawley
strain were randomly assigned to three treatment groups. The treatment

groups were a sedentary-control group (CON); a high intensity,

Robert Charles Hickson

short-duration running group (SHT); and a low—intensity, long-duration
running group (LON). The animals were provided food and water
ad'libitum.

Initiation of treatments for all animals began at 84 days of
age. Performance criteria were used as the basis of animal selection
for subsequent investigation. Animals were sacrificed 72 hours after
their last training period. Biochemical analyses were performed before
the initiation of treatments (O-wk) and after eight (8-wk) and sixteen
(lG-wk) weeks of exercise. The final sample consisted of 36 animals
with 4 animals in each treatment—duration subgroup.

The results did not reflect a differentiation of metabolic
activity between the SHT and LON groups. This observation was
supported by the large number of significant overall training (TRAIN)
effects found at 16 wks and by the fact that there were no significant
LON vs SHT contrasts at that time.

Decreases in glycogenolytic capacity as measured by phos—
phoglucomutase and increases in tricarboxylic acid cycle activity as
measured by fumarase, in all muscles of the TRAIN group at 16 wks,
suggest an exercise-induced inverse relationship between levels of
these enzymes. This result is opposite to that expected with
maturation. In addition, decreases in enzyme ratios of variable
metabolic organization in all muscles, along with a decrease in the
lactate dehydrogenase activity of the "white" vastus lateralis muscle,
imply an increased dependence upon oxidative metabolism by the TRAIN

group.

Robert Charles Hickson

Alterations in some of the constant proportion enzyme ratios
occurred as a result of either maturation or training. The variable
enzyme ratio data support the validity of histochemical fiber-typing

methods.

EXERCISE-INDUCED BIOCHEMICAL ALTERATIONS

IN DIFFERENT TYPES OF SKELETAL MUSCLE

BY

Robert Charles Hickson

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Health, Physical Education, and Recreation

1974

DEDICATION

TO

William W. Heusner

ii

ACKNOWLEDGMENTS

Sincere thanks to Dr. William W. Heusner and Dr. Wayne D.

Van Huss for making my four years at Michigan State an unsurpassed
experience.

Special appreciation is extended to Dr. John E. Wilson for his
enthusiasm, scholarly input, and direction toward the completion of
this study; and for the use of his laboratory.

Gratitude is extended to Dr. Loran Bieber for his meaningful
assistance on the doctoral committee.

The following people have contributed many ways throughout this
study: JoAnn LaFay, David Anderson, Phil Felgner, Roland and Sharon
Roy, Kwok-Wai Ho, Ken Stevens, Nancy Williams, Marjorie Middel, Laura
Zydbel, Robin Krause, Fayann Lippincott, Bob Holmes, Bob Wells, Albert
Chov, Barbara Binns, and my mother and father-in-law Kenneth and
-Dorothy Jones.

I owe a debt of thanks for reaching this goal to my mother,
father, and Aunt Doris, and even Sister Mary Collette, and Father Bob
Dahlke S. J.

A most important person whom I have not forgotten is my new
bride Susie Jones (Hickson). She has been understanding, patient, and

instrumental in assisting me toward the completion of this dissertation.

iii

TABLE OF

LIST OF TABLES . . . . . . .

LIST OF FIGURES O O O O O O 0

Chapter

I.

II.

III.

IV.

THE PROBLEM. . . . . .

CONTENTS

Page

0 O O O O O O O O O Viii

Statement of the Problem. . . . . . . . . . . 3
Rationale. . . . . . . . . . . . . . . . 4
Significance of the Problem. . . . . . . . . . 5
Limitations of the Study. . . . . . . . . . . 5
REVIEW OF RELATED LITERATURE . . . . . . . . . . 7
Overview of Skeletal Muscle Enzyme Activities. . . . 7
Glycogenolytic, Glycolytic, Gluconeogenic Enzymes
and Related Muscle Constituents. . . . . . . . 15
Mitochondrial Enzymes and Related Muscle Constituents . 21
METHODS AND MATERIALS . . . . . . . . . . . . 29
Sampling Procedures . . . . . . . . . . . . 29
Research Design. . . . . . . . . . . . . . 30
Treatment Groups . . . . . . . . . . . . . 31
Duration Groups. . . . . . . . . . . . . . 33
Acute and Chronic Groups. . . . . . . . . . . 33
Experimental Procedures . . . . . . . . . . . 35
Animal Care . . . . . . . . . . . . . . . 35
Sacrifice Procedures . . . . . . . . . . . . 36
Biochemical Procedures . . . . . . . . . . . 37
Statistical Procedures . . . . . . . . . . . 41
RESULTS AND DISCUSSION . . . . . . . . . . . . 43
Training Results . . . . . . . . . . . . . 43
Muscle Weight Results. . . . . . . . . . . . 45
Enzyme Activity Results . . . . . . . . . . . 51
Enzyme Activity Ratios Results. . . . . . . . . 70
‘Discussion . . . . . . . . . . . . . . . 88

iv

Chapter
V. SUMMARY, CONCLUSIONS, AND
S‘mary I I C O O 0
Conclusions . . . .
Recommendations . . .
REFERENCES. . . . . . . .

APPENDIX

A. Training Programs . . .

RECOMMENDATIONS

Page
95
95
96
97

99

108

LIST OF TABLES

Table Page
1. Experimental design with final cell frequencies . . . . 31

2. Controlled running wheel exercise programs for SHT and
LON groups at the thirty-seventh day of training . . . 34

3. Two-way multivariate analysis of variance for plantaris
and soleus muscle weights demonstrating the effects of
treatment and duration of treatment . . . . . . . 48

4. One-way multivariate analysis of variance for muscle
weights, demonstrating the effects of treatment
duration 0 O I O O O I O O O I O O O O O 49

5. Summary of duration effects for muscle weights with
significant mean differences. . . . . . . . . . 50

6. Two-way multivariate analysis of variance for enzyme
activities in three voluntary muscle types demonstrating
the effects of treatment and duration of treatment . . 53

7. One-way multivariate analysis of variance for enzyme
activities in three voluntary muscle fiber types
demonstrating the treatment effects . . . . . . . 55

8. Summary of treatment effects for enzyme activities with
significant mean differences. . . . . . . . . . 58

9. One-way multivariate analysis of variance for enzyme
activities in three voluntary muscle fiber types
demonstrating the effects of treatment duration . . . 63

10. Summary of duration effects for enzyme activities with
significant mean differences. . . . . . . . . . 66

ll. Two-way multivariate analysis of variance for enzyme
activity ratios in three voluntary muscle types
demonstrating the effects of treatment and duration
of treatment . . . . . . . . . . . . . . . 73

vi

Table Page

12. One-way multivariate analysis of variance for enzyme
activity ratios in three voluntary muscle fiber types
demonstrating the treatment effects . . . . . . . 74

13. Summary of treatment effects for enzyme activity ratios
with significant mean differences. . . . . . . . 77

14. One-way multivariate analysis of variance for enzyme
activity ratios in three voluntary muscle fiber types

demonstrating the effects of treatment duration . . . 81

15. Summary of duration effects for enzyme activity ratios
with significant mean differences. . . . . . . . 85

vii

LIST OF FIGURES

Figure A Page

1. Mean daily per cent shock free time (PSF) and per cent
expected meters (PEM) for CRW SHORT . . . . . . . 44

2. Mean daily per cent shock free time (PSF) and per cent
expected meters (PEM) for CRW LONG . . . . . . . 46

3. Plantaris and soleus muscle weights across all
treatments and durations of treatments . . . . . . 47

4. Enzyme activities in the plantaris, soleus, and "white"
vastus lateralis muscles across all treatments and
durations of treatments . . . . . . . . . . . 52

5. Enzyme activity ratios representing variable metabolic
organization in the plantaris, soleus, and "white"
vastus lateralis muscles across all treatments and
durations of treatments . . . . . . . . . . . 71

6. Enzyme activity ratios representing constant metabolic
organization in the plantaris, soleus, and "white"
vastus lateralis muscles across all treatments and
durations of treatments . . . . . . . . . . . 72

viii

CHAPTER I

THE PROBLEM

Histochemical observations of mammalian skeletal muscle have
demonstrated a heterogeneous distribution of metabolic characteristics.
Attempts have been made to classify skeletal muscle fibers according to
cellular characteristics (93, 77). These investigations have produced
a taxonomy of skeletal muscle, predicated primarily on the identifi-
cation of three fiber types. The definitions of the metabolic charac-
teristics of red, white, or intermediate fibers, however, have not
been entirely consistent with each other (9, 14, 15, 21, 26, 27, 28,
42, 74).

Physiological studies, based on contractile properties of
muscle, coupled with histochemical and biochemical determinations have
provided evidence for an innovative reclassification of fibers to fast-
twitch red, fast-twitch white, and slow-twitch red (10). Additional
biochemical research on skeletal muscle fiber types warranted a further
modification in nomenclature to fast-twitch oxidative-glycolytic (FOG),
fast-twitch glycolytic (PG), and slow—twitch oxidative (50) respectively
(82).

Fiber populations of five mammals were categorized histo-

chemically according to their percentages of FOG, PG, and SO fibers (1).

Whole muscles showed little fiber-type homogeneity, although certain
muscles were able to meet the criteria of being predominantly composed
of one fiber population.

Some evidence has been presented to indicate a coordination of
metabolism at the molecular level. Determinations of glycolytic
enzyme activity in skeletal muscle have indicated that the five enzymes
of the "phosphotriose glycerate phosphate group" are represented in
constant proportions and in equimolar concentrations in all types of
skeletal muscle (99, 83, 70). However, a recent paper by Dalrymple
(19) has challenged this concept. Constant proportions of groups of
mitochondrial enzymes also were established (84); but unlike the
succession of glycolytic enzymes from triose phosphate isomerase to
enolase, no sequential metabolic arrangement of constant-proportion
mitochondrial enzymes was found.

A comparison of enzyme-activity ratios in various muscle types
has exposed both a metabolic constancy and a metabolic differentiation
(13, 85). Arduous endurance training can induce constant-proportion
increases in the activity of the citric acid cycle, the electron
transport system, and related enzymes and constituents in skeletal
muscle (54, 56, 57, 25). The level of aerobic stress appears to be the
primary stimulus in evoking appropriate metabolic responses. Most of
the studies involving moderate aerobic exercise programs have not
shown any resultant modifications of enzymatic activity.

Experimentation with anaerobic training programs is rare at
this time. Staudte, Exner, and Pette (92) subjected rats to a high-
intensity, short-duration running program and discovered that the

soleus, a slowbtwitch oxidative muscle, responded with a shift toward

glycolytic metabolism as judged by enzyme levels more than did the
rectus femoris muscle which contains almost no slow-oxidative
characteristics. It is worthwhile to postulate that the normal
glycolytic capacity of the rectus femoris may be sufficient to cope
with this degree of stress. If this is the case, adaptation of the
rectus femoris might be observed only following more strenous workloads
generating a high level of oxygen debt.

In light of these findings, this study was undertaken to
determine the effects of specifically designed aerobic and anerobic
endurance interval training programs on enzymatic activities which are
representative of appropriate pathways of energy-supplying metabolism
in skeletal muscle. Specific exercise regimens should represent a form
of metabolic specialization. This raised the question of how the
constant-proportion as well as the differentiated enzymatic activity
ratios of skeletal muscle metabolism would respond to these different
stresses. The responses of specific fiber types to the training

programs also were investigated.

Statement of the Problem

The purpose of this study was to determine the effects of
aerobic and anaerobic programs of endurance running on selected
enzymatic activities in the left plantaris, the left soleus, and the
"white" area of the left vastus lateralis muscles of the adult male
albino rat. The training regimens were the Controlled Running Wheel
programs previously reported from this laboratory (101). Various
durations of the exercise programs were incorporated in the study.

Biochemical determinations were made on the levels of activity of

phosphoglucomutase, an enzyme of glycogenolysis; phosphoglucoisomerase,
an enzyme of glycolysis; lactate dehydrogenase, an enzyme concerned
with anaerobic metabolism of glycolytically-formed pyruvate, and
furmarase, a mitochondrial enzyme of the tricarboxylic acid cycle.
Enzyme activity ratios also were investigated in each of the fiber

types.

Rationale

The muscles designated for this study were selected to
represent fast-twitch oxidative-glycolytic, fast-twitch glycolytic,
and slowbtwitch oxidative fiber types. Another criteria for selection
was anatomical location. It was assumed that any resultant changes due
to the exercise programs would be best reflected by muscles "actively"
affected by the training regimens. Since "whole" muscle fiber homo-
geneity appears to be the exception, the muscles were chosen with
emphasis toward a prevalent fiber-type population. In the rat, the
plantaris was reported to contain 53% FOG, 41% PG, and 6% SO fibers;
the soleus, 16% FOG, 0% PG, and 84% SO; and the vastus lateralis, 56%
FOG, 42% PG, and 2% SO (1). Only the "white" area of the vastus
lateralis which has predominantly FG fiber characteristics was used
for this investigation.

It was hypothesized that the response to the increased energy
requirements imposed by the training programs would be specific.
Therefore, the enzymes were chosen to represent enzyme activity states
in four separate biochemical pathways. Since the different exercise
regimens could be expected to result in some degree of metabolic

specialization, an adaptation of both constant and variable enzymatic

activity ratios was expected. A differential response by fiber types
to the training regimens also was anticipated.

Exercise-induced biochemical alterations probably are dependent
upon the duration of the training program. Thus, zerobweek, eight-week,
and sixteen-week periods of training were incorporated. Furthermore,
the responses to training may be reflected differently immediately
after an exercise bout and following a recovery period. To test this
hypothesis, animals were sacrificed fifteen minutes and seventy-two

hours after their last exercise bout within each duration group.

Significance of the Problem

Determinations of the biochemical effects of various training
programs should contribute to an understanding of the functional nature
of the aerobic-anaerobic continuum. The study of exercise-related
alterations by muscle fiber types may provide further insight into the
mechanisms of metabolic adaptation. A potential overall contribution
of this study is that the information obtained may provide a basis for
the appropriate prescription of exercise to improve human health and

work performance.

Limitations of the Study

 

1. Although specific muscle fiber populations were employed, the
results may reflect an integrated response of all fiber types.

2. The results of animal studies cannot be applied directly to
humans.

3. In vitro enzymatic assays may not be representative of in vivo

cellular activities.

The exercise programs were designed to represent specific
aerobic and anaerobic interval training routines. However, at
this time it is impossible to design and conduct a program of
exercise for animals which is either purely aerobic or
anaerobic in nature.

No control over the shock stimulus to run was included in the
study.

The small sample size may have limited the power of the
statistical analyses.

Enzymatic activities were expressed as Units/gm of muscle. It
appears from the literature that biochemical differences in
activity levels are not masked when expressed in this manner,

but that may not be the case.

CHAPTER II

REVIEW OF RELATED LITERATURE

In an attempt to facilitate a discussion of the interrelation-
ships between various biochemical parameters, especially enzymatic
activities, and exercise in skeletal muscle, this chapter has been
organized into three sub-divisions. First, an overview is presented
of enzymatic studies that are uniquely connected with skeletal muscle
fiber types. Second, there is a discussion of the effects of exercise
on glycogenolytic, glycolytic, gluconeogenic and related constituents.
Finally, the focus is on the influence of exercise on mitochondrial
enzymes and related constituents.

Overview of Skeletal Muscle
Enzyme Activities

 

An early investigation of skeletal muscle was performed on the
African migratory locust (99). Three types of voluntary-muscles were
compared with respect to structure, function and enzymatic activity.

A flight muscle used for enduring work performance exhibited high
levels of aerobic metabolism, maximum mitochondria, and a moderate but
specific endoplasmic reticulum. A hind leg muscle used for jumping had
anaerobic metabolic patterns with minimum mitochondria, a maximum

number of myofibrils, and a moderate endoplasmic reticulum. The

flexor tibiae exhibited contractile tonus, a specific arrangement of
pairs of mitochondria in the vicinity of the Z discs and an extended
endoplasmic reticulum. All three muscles had a constant relation
between cytochrome c content and the volume fraction of mitochondria
estimated by electron microscopic pictures. Lactate dehydrogenase
activity was very low in the flight muscle and highest in the jumping
muscle. Qualitative differences in locust muscles were previously
reported by Delebruck, Zebe, and Bucher (22). The relationships
between the five enzymes of the glycolytic pathway from triose-3-
phosphate to phosphoenolpyruvate were relatively constant in the three
types of muscle.

Subsequent examination of the same "phosphotriose-
glyceratephosphate group" of enzymes again showed nearly constant
ratios in various muscle types from phylogenetically different
animals which suggested a coordination at the molecular level (83).

It was suggested that since this quintet forms the only major
unbranched segment of the glycolytic chain, the limited variability of
the ratios of their activities might be explained by the close
functional connection of these enzymes. Phosphoglucomutase and
several other enzymes exhibited similar constant proportions with this
quintet of enzymes. Supportive evidence for the constant-proportion
group of the Embden-Meyerhof pathway was presented by Mier and Cotton
(70). A significant finding which may have escaped notice by Vogell
and coworkers (99) is that the actual pattern represents equimolar
amounts of these enzymes. They concluded that the cell necessarily
produces molecules of each of these five enzymes in precisely equal

numbers and that synthesis is controlled by a single operator gene.

Moreover, one single molecule of mRNA is synthesized from the DNA
strand per operon rather than a separate mRNA molecule for each
structural gene.

The constant-proportion concept for the glycolytic enzymes was
studied in developing red (trapezius) and white (longissimus) muscles
of pigs from the fetal stage to 24 weeks (19). Adult levels of enzymes
were evident two weeks postnatal. Enzyme activities were two- to
three-fold greater in the longissimus than in the trapezius muscles.
Enzyme activity ratios based on glyceraldehyde-3-phosphate dehydrogenase
were not constant in fetal and day-one samples but were constant during
later stages of growth. At 105 days gestation, glyceraldehyde-3-
phosphate dehydrogenase and enolase activities were different from
triose phosphate isomerase, phosphoglycerate kinase, and phosphogly-
cerate mutase enzyme patterns. From this evidence, the single operon
hypothesis of Mier and Cotton was questioned.

A constancy of the respiratory chain molar ratio of cytochrome
a to cytochrome c has been found in different types of muscle (84),
and the DPN content in various types of mitochondria has been shown to
be constant when expressed as a ratio of molar concentrations to
cytochrome c (61). When expressed relative to cytochrome c turnover
and not activity per gm tissue, malate dehydrogenase and glutamate-
oxaloacetate transaminase were constant, succinic dehydrogenase and
pyruvate oxidase varied slightly and TPN-specific isocitrate dehydro-
genase, glutamate dehydrogenase, and glycerol-l-P oxidase varied in
the range of two orders of magnitude in different mitochondria.

The constant-proportion enzymes in the mitochondria were not

sequential as was observed for the five glycolytic enzymes. The

10

importance of auxiliary enzymes of the tricarboxylic acid cycle was
emphasized in the constant ratio of glutamate-oxaloacetate transaminase
to cytochrome c.

Hexokinase, an enzyme linked with glucose phosphorylation, was
found to be highest in red muscle (semitendinosus) of the rabbit and
to vary inversely with phosphorylase which is maximally active in
white muscle (adductor magnus) (16). Hexokinase activities correlate
approximately with citric acid cycle capacities as determined by
succinate oxidase. Observations from the literature (8, 15) suggest
that muscles with an active hexokinase may preferentially accumulate
glycogen when excess glucose is present. It also was concluded that
phosphorylase plays a minor role in muscles with predominantly
oxidative metabolism and that there is a significant increase in
phosphorylase activity levels with muscle dissuse.

Crabtree and Newsholme (18) measured the maximal activities of
hexokinase, phosphorylase, and phosphofructokinase in extracts from a
variety of muscles. In all vertebrate muscles examined, the activity
of phosphofructokinase was very similar to that of phosphorylase which
was highest in white muscle. These findings are consistent with the
concept that glycogenolytic and glycoltyic control are mediated by
phosphorylase and phosphofructokinase respectively (103). In addition,
the work supported and extended the conclusion of Burleigh and
Schimke (16) that utilization of glycogen is more important and
utilization of glucose is less important in white than in red muscles.
In vertebrates the activity of hexokinase is greater in red (rabbit
semitendinosus and rat heart) than in white muscles. This observation

suggests that glucose is a more important energy source for contraction

11

in red than in white muscle. However, phosphorylase and phospho-
fructokinase levels are higher than hexokinase levels in all red and
most white mammalian muscles. An examination of human skeletal muscle
disclosed lower levels of lactate dehydrogenase and total phosphorylase
in the soleus than in either the gastrocnemius or vastus lateralis (41).
No intermuscular differences were found to exist for levels of activity
of hexokinase, succinate dehydrogenase and creatine phosphokinase.
Biopsy samples from the soleus contained approximately 80% slow-twitch
(ST) fibers. Fibers from the gastrocnemius and vastus lateralis
averaged 57% ST.

In the guinea pig, the LDH activity level of a fast-twitch
white muscle (white vastus lateralis) was found to be higher than in
either fast-twitch red (red vastus lateralis) or slowbtwitch red
(soleus) muscles (81). The LDH activity in the red vastus lateralis
was significantly higher than in the soleus. The LDHS (muscle-type)
was shown to be predominant in both fast-twitch white and fast-twitch

red fibers, whereas LDH (heart-type) was predominant in the slow-

1
twitch red fibers.

In a classical study Bass et a1. (13) attempted to uncover the
systems of energy-supplying metabolism which are subject to differ-
entiation in muscle. This question was examined by comparing enzyme
activity levels and ratios of these activities in various muscle types
of higher animals. The ratios were intended to reflect quantitative

relations of metabolic systems at the level of enzymatic organization.

Nearly constant values were found for the following ratios:

l2

Enzymes Pathways Represented

Phosphorylase Glycogenolysis
Triosephosphate Dehydrogenase Glycolysis

Glycerophosphate Dehydrogenase Mitochondrial Glycerophosphate Oxidation
Triosephosphate Dehydrogenase Glycolysis

Triospehosphate Dehydrogenase Glycolysis

 

 

 

 

Lactate Dehydrogenase Lactate Fermentation
Hexokinase Glucose Phosphorylation
Citrate Synthetase Citric Acid Cycle

3-Hydroxyl-CoA Dehydrogenase Fatty Acid Oxidation

Citrate Synthetase Citric Acid Cycle

The lack of variability in these ratios was assumed to indicate constant
organization of enzyme activity patterns. It was postulated, there—
fore, that the relationships between these metabolic systems are not
subject to differentiation.

Metabolic variability was observed in the following ratios:

Enz es Pathways Represented

Triosephosphate Dehydrogenase Glycolysis
3-Hydroxyl-CoA Dehydrogenase Fatty Acid Oxidation

 

 

Triosephosphate Dehydrogenase Glycolysis

 

 

 

 

 

 

 

 

Citrate Synthetase Citric Acid Cycle
Lactate Dehydrogenase Lactate Fermentation
Citrate Synthetase Citric Acid Cycle
Phosphorylase Glycogenolysis
Hexokinase Glucose Phosphorylation
Hexosediphosphatase Gluconeogenesis
Hexokinase Glucose Phosphorylation

It was concluded that these variable enzymatic activity ratios are
discriminative and can be used to classify distinct metabolic types

of muscle.

13

Fast-twitch white muscles exhibit high levels of glycogenolytic,
glycolytic, lactate fermentation, mitochondrial glycerophosphate
oxidation, and gluconeogenic enzyme activities. Low activities of
glucose phosphorylation, citric acid cycle, and fatty acid oxidation
are found in white muscle. An opposite pattern of these enzyme
activities is found in red (slow-twitch) muscle. Hexokinase activity
parallels that of citrate synthetase in both muscle types. However,
mitochondrial glycerophosphate dehydrogenase and hexosediphosphatase
activities are high in white muscle indicating the importance of
gluconeogenesis starting from glycerophosphate or triosephosphate in
this type of fiber. Higher levels of fructose 1,6-diphosphatase have
been previously reported in white muscle than in red muscle, and a
plausible relationship between gluconeogenesis activity and glycero-
phosphate content is implied (62, 75).

Ratios of these enzyme activities might help to clarify the
pattern of metabolic differentiation not only in red and white muscles
but in mixed-fiber populations of heterogeneous muscles.

Pette (85) further illustrated metabolic differentiation in
two muscles with similar functional characteristics. The energy
expenditures of the flight muscles of the Apis Mellifera (honey bee)
and the Bombyx Mori (silk moth) are both based on aerobic metabolism.
However, the predominant substrates for catabolism are carbohydrate
and fat respectively.

It was concluded that further clarification of the differences
and similarities between the energy metabolism of red and white muscle
is needed. The constancy of some ratios suggests a fixed pattern of

the metabolic systems at the level of molecular organization. However,

14

specific metabolic specialization may alter the constant-proportion
enzyme activity groups. These variations can be regarded as the
expression of metabolic differentiation. Differentiation then could

be interpreted as an optimal adaptation of the energy supply to the
requirements of muscle function, and different metabolic patterns could
be associated with different functional activities. The optimal way

to investigate differences in metabolic type is at the level of
enzymatic organization.

Staudte and Pette (91) studied fifty-one muscle specimens from
a variety of animals of different species and phyla. Linear corre-
lations of enzyme activities were observed between glycogen phosphory-
lase and triosephosphate dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase
and citrate synthetase, and hexokinase and citrate synthetase. In
muscles containing creatine kinase, a linear correlation existed between
the activity of that enzyme and the activity of triosephosphate
dehydrogenase. Although these results reinforce the general validity
of the concept that constant proportions do exist between certain
metabolic enzymes in muscle, deviations were found which indicate the
possibility of independent regulation of the constant-proportion
enzymes.

Administration of thyroid hormones has been shown to induce
changes in energy metabolism in red (soleus), white (rectus femoris),
and heart muscles of rats (63). Hexokinase activity increased in
white muscle, while both hexokinase and glycerophosphate dehydrogenase
activities increased in red muscle. In heart muscle, only glycero-
phosphate dehydrogenase activity increased. The ratio of hexokinase/

citrate synthetase increased in white and red muscle only; whereas,

l5

the ratio of glycerophosphate dehydrogenase/triosephosphate dehydro-
genase increased only in red and heart muscle. These results indeed
suggest that various metabolic factors can affect the constant-
proportion enzymatic pattern.

Glycogenolytic, Glycolytic, Gluconeogenic Enzymes
and Related Muscle Constituents

 

 

Introductory investigations using biopsy samples of the vastus
lateralis muscle showed phosphorylase a activity to increase with
prolonged training (96). The experimental subjects were healthy adult
males who were trained to increase maximum V02 for sixteen weeks on a
bicycle ergometer. After exercise to fatigue, both the trained
subjects and a group of sedentary controls had decreased activities of
phosphorylase a. Neither training nor exercise to fatigue affected
phosphorylase b activity.

Endurance training on a treadmill for six months produced no
changes in the activities of phosphorylase, lactate dehydrogenase, or
mitochondrial a-glycerophosphate dehydrogenase in either the semi-
membranosus or vastus lateralis of the lesser bushbaby (Galago sene-
galensis) (29). Forced swimming in rats for 6 hours did not produce
any changes in phosphorylase (43). Intermittent isometric activity
has induced elevated phosphorylase activities in the rectus femoris
muscles of female and male rats (33, 34).

Peter, Jeffress, and Lamb (80) demonstrated increases in
activity levels of hexokinase in red and white quadriceps of adult
guinea pigs who had been trained every other day for 21 days on a
treadmill driven at 1.9 km/hr. The results were significant when

expressed as Units/gm muscle and Units/mg protein. Lamb et a1. (64)

16

also observed increases in levels of hexokinase activity in the same

muscles of guinea pigs both immediately and 48 hours after exercise.

The results of both studies revealed hexokinase activity to be higher
in red than in white skeletal muscle.

A similar adaptation to endurance exercise in rats was
reported by Holloszy (57). The levels of activity of hexokinase
increased two-fold in exercised gastrocnemius muscle. In contrast, the
levels of activity of phosphorylase, phosphofructokinase, pyruvate
kinase, and lactate dehydrogenase were unaffected by the exercise
program. Baldwin et a1. (6) also has shown that rats who were
endurance trained by treadmill running had increased levels of
hexokinase: 170% in the red portion of quadriceps muscle, 50% in the
soleus, and 30% in the white portion of the quadriceps as well as a
15% decrease of lactate dehydrogenase in this area. In the red muscle
decreases of approximately 20% occurred in the activity levels of
phosphorylase, phosphofructokinase, glyceraldehyde-3-phosphate dehydro-
genase, pyruvate kinase, lactate dehydrogenase and cytoplasmic
a-glycerophosphate dehydrogenase of the trained group. A 50% increase
in cytoplasmic a-glycerophosphate dehydrogenase activity and 18-35%
increases in the levels of phosphorylase, phosphofructokinase,
glyceraldehyde-3-phosphate dehydrogenase and pryuvate kinase was
evident in the soleus muscle of the exercised animals. The increases
of hexokinase and phosphorylase activities in soleus muscle were
inconsistent with previous results (18).

Gollnick et a1. (40) subjected six healthy male subjects to a
five-month program of endurance training on a bicycle ergometer. The

level of activity of phosphofructokinase (PFK) increased 117% in the

l7

biopsied vastus lateralis muscle. Histochemical analysis showed
percentages of slowbtwitch and fast-twitch fibers to remain unchanged
after training when identified by myosin ATPase. Oxidative potential
(DPNH-Diaphorase) and glycolytic capacity (a-glycerophosphate dehydro-
genase) increased in both fiber types. Slow-twitch fibers were larger
after training than before training. The relative area the slow—twitch
fibers occupied in the muscle was higher after training. The soleus
fiber type in the rat is similar to the slow-twitch human type. These
findings suggest increased glycolytic capacity of slow-twitch fibers
after training. Increased levels of activities of PER (83%) also have
been shown in boys 11-13 years old after an endurance training program
(31).

Staudte, Exner, and Pette (92) imposed a high-intensity,
short-duration (80 m/min, 30° incline, 45 sec) training program on
female rats four times daily for three weeks. Increases in hexokinase
and citrate synthetase activities occurred with training in the rectus
femoris and soleus muscles. The adaptability of the soleus muscle was
again more pronounced. It acquired a shorter contraction time and
increases in creatine kinase and glycolytic triosephosphate dehydro-
genase activities. The authors noted that physiological and bio-
chemical changes in muscle may not correlate with higher performance.
However, different responses of fast- and slow-twitch muscles can be
representative of specific adaptations.

Aldolase activity in rat skeletal muscle has been shown to
increase after prolonged treadmill running (49). However, in another
study, training plus sustained running raised aldolase activity in

red muscle only of rats on a low vitamin C diet (23). Hearn (51)

18

reported no differences in aldolase levels of the gastrocnemius muscle
after a five-week swimming program and detraining.

Lactate dehydrogenase (LDH) activity was found to increase in
the vastus lateralis muscles of four male subjects after prolonged
severe exercise (59). These findings are similar to those of Dieter,
Altland, and Highman (24) who showed increased activity levels of LDH
in red and white muscles of cold-acclimated rats that were exercised
for up to nine hours. LDH levels of unacclimated rats increased up
to five hours and then dropped off. However, the levels of activity
of LDH were decreased in the gastrocnemius muscles of rats trained by
swimming (37). Acute exercise did not alter these levels. Molé et a1.
(73) observed no significant shift in LDH isozyme patterns in the
gastrocnemius muscles of endurance-trained rats. Mitochondrial and
cytoplasmic glutamate-pyruvate transaminase activities (GPT) increased
in the same muscle with training. This result led to the conclusion
that muscle which has adapted to endurance exercise has an increased
capacity to generate alanine from pyruvate and citric acid cycle
intermediates from glutamate.

Elevated glycogen synthetase activities in both the I (inde—
pendent or active) and D (dependent or inactive) forms were elicited in
human vastus lateralis muscle following a five-month aerobic training
program (95). After exercise to fatigue, both I and D values were
lower in all groups with the sedentary group demonstrating the greatest
decline in synthetase I values. Synthetase I and D values were
markedly elevated after exhaustive exercise of 3-5 min duration.
Jeffress, Peter, and Lamb (58) also demonstrated increased I and I + D

forms of glycogen synthetase in skeletal muscle of guinea pigs after

19

training. Total synthetase activity was higher in the red area than
in the white area of the vastus lateralis muscle. Sustained swimming
for six hours raised glycogen synthetase I and D activities in hindleg
muscle of rats (17). This effect was still evident after six hours of
rest. Glycogen content and glycogen synthetase I activity (% of total)
has been shown to vary inversely in skeletal muscle (20). This concept
was investigated by Roch-Norlund (89) who found that glycogen
depletion by exercise increased I activity in normal subjects and in
diabetics to a lesser extent.

Various intracellular ATPase's are responsible for forming
ATP from appropriate precursors as immediate sources of energy. Study
of these enzymes when related to exercise has yielded a diversity of
results. Low-intensity endurance swim training produced an increase
in myosin ATPase activity of the soleus muscle, but not the extensor
digitorum longus muscle, in young rats (94). Histochemical analysis
showed an 11.8% increase in the number of muscle fibers with high
actomyosin ATPase activity in the soleus muscles of the trained
animals. These effects were not apparent if the exercise was initiated
in adult animals. Bagby, Sembrowich, and Gollnick (3) determined the
effects of endurance training (28.4 m/min, 60 min/day, five days/week)
and sprint training (80.4 m/min, 30-sec alternated exercise and rest
periods, 18 bouts) for 11 weeks on myosin ATPase activity and fiber
composition of rat gastrocnemius muscle. Neither exercise program
altered the levels of myosin ATPase. It was calculated that the small
percentage (8%) of slowhtwitch fibers in the gastrocnemius could
account for only minor changes in myosin ATPase activity were they all

to change from slow-twitch to fast-twitch fibers. These results agree

20

with those of previous studies (87) in which swimming 30 min/day for
six weeks produced no changes in calcium-activated myosin ATPase.
Neither myosin ATPase nor actomysin ATPase in vastus lateralis and
vastus intermedius muscles of the lesser bushbaby were affected by
endurance training (29). Contrasting results of increased myosin
ATPase activity were obtained by Marikova (68) and Wilkerson and
Evonuk (102) in response to moderate and exhaustive swimming programs
respectively.

The response to exercise of creatine phosphokinase (CPK), an
enzyme involved in the release of stored ATP and an immediate source
of energy, has not been consistent. Kendrick-Jones and Perry (60)
demonstrated increased levels of CPK activity in rat hind-leg muscle
as a result of prolonged treadmill exercise. Their activity values
were related to the wet weight of the total protein-nitrogen content
after removal of all enzyme from the tissue. Dieter (23) also found
increased CPK activity per mg protein in homogenates of red (soleus)
and white (anterior portion of biceps femoris) muscles of guinea pigs.
The animals were maintained on low levels of vitamin C and were
exercised for 10 hours following six weeks of treadmill training.
Rawlinson and Gould (87) did not find any change in CPK activity in
skeletal muscle of rats swim-trained 30 min/day for six 5-day weeks.

Oscai and Holloszy (76) studied the effects of endurance
training in rats on enzymes involved in the regeneration of ATP. Both
mitochondrial ATPase activity and cytochrome C content, which served
as a marker for the respiratory chain, increased two-fold in the
gastrocnemius muscles of the exercised animals. These effects were

viewed as a coordinated, quantitatively related increase in the

21

components of mitochondrial cristae as an adaptation to exercise. The
levels of activity of mitochondrial creatine phosphokinase and
adenylate kinase did not increase with training, and were reduced when
expressed per mg of mitochondrial protein. These results provided
evidence for a change in mitochondrial composition. Cytoplasmic
creatine phosphokinase.and adenylate kinase were unchanged. These
findings lend credence to the hypothesis that endurance exercise can
increase the ability of the muscle to regenerate ATP aerobically while
not altering the ability of muscle to form ATP anerobically.

Mitochondrial Enzymes and Related

Muscle Constituents

Moderate exercise regimens do not produce changes in mito-
chondrial enzyme levels. Hearn and Waino (48) subjected rats to 30
minutes of daily swimming for five to eight weeks and found no increase
in succinic dehydrogenase (SDH) activity in gastrocnemius muscle.

Under similar training procedures, Gould and Rawlinson (43) and

Hearn (51) found no differences in malic dehydrogenase and cytochrome
oxidase activities respectively. Succinate cytochrome c reductase
activity was elevated in the latter study. Neither cold acclimation
nor treadmill running caused any change in SDH activity in the
gastrocnemius muscles of white mice (46). These results were expressed
as activity per mg of protein.

Strenuous aerobic endurance training has been shown to markedly
influence mitochondrial capacity. Holloszy (54) subjected rats to
treadmill running for 12 weeks with the workload gradually being
increased to 120 minutes at 31 m/min on a 8° incline with twelve

30-sec. intervals at 42 m/min interspersed every 10 minutes. The

22.

total protein content of the mitochondrial fraction of the gastrocnemius
muscle increased approximately 60% and the capacity of the mitochondrial
fraction to oxidize pyruvate expressed as pl 02 hr/g, doubled in the

trained animals. SDH, DPNH dehydrogenase, DPNH cytochrome c

reductase, succinate oxidase, and cytochrome oxidase, when expressed
per gm of muscle, increased approximately two-fold in hindlimb muscles
in response to training. The change in DPNH dehydrogenase activity
was not significant when expressed as activity per mg of mitochondrial
protein. Cytochrome c content also doubled which suggested that the
rise in respiratory enzyme activity was due to an increase in enzyme
protein. The increase in electron transport capacity was associated
with a concommitant rise in capacity to produce ATP. SDH activity
values after mild exercise were not changed. This data reinforces

the hypothesis that mild or moderate exercise is not sufficient to
increase respiratory enzyme activity in rats. By comparison, it has
been shown that SDH activity (Vo2 ml/gm/min) in human vastus lateralis
muscle is increased by six weeks of activity such as occasional
football, bicycling 15-30 minutes daily, or long distance walks on
Sunday (98). Bicycle ergometer training for five months (40) and six
weeks (31) induced increased levels of SDH activity in the vastus
lateralis muscles of adults by 95% and in boys 11-13 years old by 30%
respectively.

The adaptive increase in respiratory enzymes due to strenuous
exercise led Holloszy et al. (56) to investigate exercise-related
effects on the citric acid cycle and related enzymes. Cytochrome
content and SDH activity, which served as respiratory chain.markers,

increased two-fold in the gastrocnemius muscles of trained rats. The

23

activities of citrate synthetase and DPN-specific isocitrate dehydro-
genase also doubled. These results suggested a quantitative coordi-
nation of the respiratory chain enzymes with some citric acid cycle
enzymes. The levels of activity of a-ketoglutarate dehydrogenase and
mitochondrial malate dehydrogenase increased approximately 50% while
glutamate dehydrogenase increased only 35% providing further evidence
of an exercise adaptation of muscle mitochondria. These findings
showed that all citric acid cycle enzymes do not undergo parallel
changes in conjunction with respiratory constituents in response to
exercise.

Barnard and Peter (11) also have investigated the effects of
exercise on cytochromes. Concentrations of cytochromes a and c were
not increased until the sixth week of training in the gastrocnemius
muscles of guinea pigs. The cytochrome c changes were not has high
as those reported by Holloszy (54); however, the experimental animals
were different. The coefficients of correlation were 0.70 between
cytochrome c concentration and performance of isolated muscle
(electrical stimulation), 0.37 between cytochrome c concentration and
running time to exhaustion, and 0.26 between running time to exhaustion
and performance of isolated muscle. Cytochrome c concentration
increased more in young guinea pigs that were trained (87%) than in
adults that were trained (68%). The results indicated that improvement
in performance capacity of the whole animal, as measured by running
time to exhaustion, is largely independent of cytochrome concentration
or performance capacity of the isolated muscle. The importance of
relating biochemical adaptations in skeletal muscle to the performance

of the muscle itself and not to the whole animal was emphasized.

24

The oxidative capacity of six trained adult non-human primates
(Galago senegalensis) was shown to increase after six months of
endurance running (29). Cytochrome a and c in the semimembranosus and
vastus lateralis muscles and SDH in the vastus lateralis muscle were
elevated as a result of training. The tibialis anterior muscle showed
a decrease in the number of fast-twitch glycolytic fibers which
paralleled an increase in fast-twitch oxidative-glycolytic fibers.
The training program did not elicit any changes in soleus and plantaris
fiber-type populations, but fast-twitch oxidative-glycolytic fibers
were significantly larger in the plantaris and soleus muscles of the
trained Galagos than in a group of untrained control animals. These
observations demonstrated that muscles vary in response to treadmill
exercise depending on their anatomical location and action as well as
fiber-type composition.

Dohm et a1. (25) studied the effects of training, exercise,
and diet on citric acid cycle activity in skeletal muscle of the rat.
High carbohydrate and high fat diets did not produce any alteration of
citric acid cycle enzyme activity. However, training did increase the
activities of all citric acid cycle enzymes and cytochrome oxidase in
gastrocnemius muscle. The increased activities of citrate synthetase,
isocitrate dehydrogenase, succinate dehydrogenase and malate dehydro-
genase with training were parallel to those reported by Holloszy and co-
workers (56). This was to be expected since the training programs were
similar. The magnitude of increase of cytochrome oxidase in Dohm's study
was not as marked as previously reported (54). Relative mitochondrial
yield and succinate oxidation by isolated mitochondria also increase in

quadriceps muscle with training. The enzyme activities, relative

25

mitochondrial yield and mitochondrial succinate oxidation were the
same in exhausted untrained rats and rested untrained animals.
Exhausted trained rats had significantly lower values for isocitrate
dehydrogenase, succinate dehydrogenase, cytochrome oxidase, relative
mitochondrial yield and succinate oxidation by mitochondria than did
rested trained rats. The results were the same whether activity was
expressed per gm of tissue or per milligram of protein.

Baldwin et al. (4) studied which specific fiber type or types
contribute to the increase in oxidative capacity following a program
of strenuous endurance running. The soleus muscle and the arbitrarily
divided superfical white and deep-red portion of the quadriceps were
selected as being representative of intermediate, white and red muscle
of the rat. The activities of cytochrome oxidase and citrate synthetase
and the concentration of cytochrome c increased approximately two-fold
in all three muscle types. This data was not in agreement with that
from histochemical studies which suggested that the exercise-induced
increase in oxidative capacity of muscles is due to the conversion of
white into red fibers (9, 26, 35). Since the respiratory capacity
response to exercise was approximately two-fold in all three muscles,
the proportionate contribution from.each fiber population remained
unchanged.

Holloszy and Oscai (55) studied the influence of endurance
exercise on a—glycerophosphate dehydrogenase activity. Although
cytochrome oxidase levels and cytochrome c concentration doubled in
the gastrocnemius muscles of trained rats, neither mitochondrial nor
cytoplasmic a-glycerophosphate dehydrogenase activity was affected by

the training program. The mitochondrial protein fraction of the

26

gastrocnemius muscle increased 60% in the exercised group. Thus,
expressing cytochrome oxidase activity per milligram of mitochondrial
protein eliminated any differences due to exercise. In addition,
mitochondrial a-glycerophosphate dehydrogenase activity, when expressed
per milligram of mitochondrial protein, was significantly lower in the
trained group than in the sedentary group. These findings support the
position that the adaptive response of skeletal muscle to exercise
involves a change in mitochondrial cristae rather than simply an
increase in size or number of mitochondria. The fact that glycerophos-
phate shuttle activity was not increased and may even have been
decreased, led to the hypothesis of lower glycolytic activity and/or a
shift in carbon source for the citric acid cycle from carbohydrate to
fat. Edgerton et a1. (29) also reported no training differences in
d-glycerophosphate dehydrogenase activity in vastus lateralis and
semimembranosus muscles of the lesser bushbaby.

Molé, Oscai, and Holloszy (72) have provided enzymatic evidence
of a shift toward fatty acid oxidation as an adaptive response of
skeletal muscle to endurance exercise. The levels of activity of
carnitine palmityltransferase, palmityl CoA dehydrOgenase, and
mitochondrial ATP-dependent palmityl CoA synthetase doubled in
gastrocnemius and quadriceps muscles of trained rats. Mitochondrial
protein increased 60%. In contrast, lipoprotein lipase activity was
unaffected by endurance training in the quadriceps muscle groups of
rats (2). In rat muscle stimulated to contract repetitively in situ,
the level of activity of carnitine palmityltransferase was greater in
the slow—twitch soleus than in the fast-twitch tibialis anterior (5).

Both muscles had similar rates of decline of work during prolonged

27

stimulation, although the soleus maintained greater relative and
absolute work capacities. It was suggested that the primary energy
source for the sustained work was the capacity of the red and inter-
mediate fibers in the muscles to oxidize fatty acids. Fatty acid
oxidation has been shown to increase significantly after a program of
endurance training (71, 72, 105). Triglyceride concentrations have
been shown to decrease more in red muscle than in white or intermediate
muscle after exhaustive exercise (88).

Mitochondrial protein, as reported by Barnard, Edgerton, and
Peter (9), did not increase in guinea pig gastrocnemius and plantaris
muscles after nine weeks of training. Training for eighteen weeks did
result in a significant increase in mitochondrial yield. Electron
microscopic studies have provided conflicting results. Gollnick et a1.
(38) observed that the fine structure of human vastus lateralis muscle
after exhaustive exercise was not different from that of rested
muscle, although the electron micrographs of fatigued muscles displayed
an almost complete absence of glycogen particles. In a subsequent
study, Gollnick and King (39) found greater numbers of mitochondria in
the gastrocnemius muscles of trained rats. These mitochondria also
appeared to be larger and to have more densely packed cristae than did
those of the sedentary animals. In animals that were sacrificed
immediately after exhaustive running, the mitochondria showed swelling,
cristae degeneration and tissue edema. Exhaustive swimming did not
produce any of these effects. Terjung et a1. (97) also ran trained
rats to exhaustion. No evidence was found of leakage of soluble

enzymes from the mitochondria of gastrocnemius muscle. Electron

28

microscopic inspection revealed the mitochondria to be well preserved
in the exhausted animals with no evidence of disruption or impairment

of function.

CHAPTER III

METHODS AND MATERIALS

In classical exercise physiology, gross measurements of total-
body oxygen debt and oxygen uptake have been used to reflect metabolic
responses to physical activity. Exhaustive or nearly exhaustive
training programs lead to greater tolerance of oxygen debt which
presumably reflects a greater capacity for generation of metabolic
energy via anaerobic (e.g., glycolytic) metabolism. Such programs are
described as being of the "anaerobic endurance" type and are character-
ized by maximal workloads maintained for relatively short periods of
time. In contrast, training of an "aerobic endurance" nature is
thought to be dependent chiefly upon oxidative muscle metabolism and
tends to increase total-body oxygen uptake capacity. Moderate or light
workloads maintained for relatively long periods of time are typical of
aerobic endurance exercise programs. The current study was designed to
investigate cellular level alterations in selected working muscles

following various durations of aerobic and anaerobic endurance training.

Sampling Procedures
Eighty-four normal, male, albino rats of the Sprague-Dawley

strain were used for this study. The animals were received in two

29

30

shipments of 72 and 12 animals respectively. All of the animals were
randomly assigned to treatment groups and were given a period of ten
days to adjust to laboratory conditions before the treatments were
begun. Shipment-one animals were trained for eight and sixteen weeks
duration, while those in shipment-two served as zero-week controls.

Initiation of treatments for all animals began at 84 days of age.

Research Design

 

The study was organized as a 3x3x2 factorial design. The first
factor, Treatment, consisted of three groups: (Short) an exercise
group which was subjected to an anaerobic endurance training program,
(Long) an exercise group which was subjected to an aerobic endurance
training program, and (Control) a sedentary-control group. The second
factor, Duration, consisted of three groups: (O-wk) a group that was
trained for zero weeks, (8-wk) a group that was trained for eight
weeks, and (16-wk) a group that was trained for sixteen weeks. The
third factor, Sacrifice Time After Exercise, consisted of two groups:
(Acute) those animals sacrificed fifteen minutes after their last
exercise bout, and (Chronic) those animals sacrificed seventy-two
hours after their last exercise bout.

For the trained animals, percent of expected meters run (PEM)
served as the major performance criterion for selection of the final
sample (101). In order to achieve a cell size of n=4, animals with
the highest training performance over each duration period were
selected. A representation of the experimental design with final cell

frequencies can be seen in Table l.

31

Table l.--Experimental design with final cell frequencies.

 

 

 

 

 

 

 

 

O-weeks 8-weeks 16-weeks
Treatment Sacrifice Time Sacrifice Time Sacrifice Time
Acute Chronic Acute Chronic Acute Chronic
Control 4 4 4 4 4 4
Short 4 4 4 4 4 4
Long 4 4 4 4 4 4

 

Treatment Grogpg

The exercise treatments were performed on a Controlled Running
Wheel (CRW) developed at the Human Energy Research Laboratory, Michigan
State University. The CRW apparatus can be_described as, ". . . a
unique animal-powered wheel which is capable of inducing small
laboratory animals to participate in highly specific programs of
controlled, reproducible exercise" (101). The animals learn to run by
avoidance-response operant conditioning. A low-intensity controlled
shock current provides motivation for the animals to run.

Following body weight recording at the start of each treatment
period, each animal was placed in an individual running wheel. A light
above the running wheel signaled the start of each work interval. If
the animal responded to the light by running at or faster than a
preset speed, the light was extinguished and shock was avoided. The
initial time during which the light was on is termed the "acceleration
period." If the animal was not running at the predetermined speed by
the end of the acceleration period, the light was turned off and a

current was applied to the grid which serves as the running surface.

32

If the animal attained the prescribed speed while being shocked, the
shock was immediately discontinued. If, when running, the animal
slowed down below the prescribed speed, the light and shock sequence
was repeated. A typical running program consisted of alternate work
and rest periods. During the work periods, the wheel was free to turn;
whereas, during the rest periods, the wheel was braked automatically to
prevent spontaneous activity. A specified number of alternated work
and rest periods (repetitions) constituted one bout of exercise. A
single training period would include several such bouts separated by_a
relatively long time between bouts.

The three training groups in the study were as follows:

Short (SHT)

 

This group was subjected to a highrintensity, short-duration
endurance program which was progressive in nature. That is, the
intensity of the program was gradually increased until on the thirty-
seventh day of training, and thereafter, the animals were expected to
complete eight bouts of exercise with 2.5 minutes of inactivity between
bouts. Each bout consisted of six repetitions of 10 seconds of work
alternated with 40 seconds of rest. During the work intervals, these
animals were required to run at the relatively fast speed of 99 m/min.

A description of the complete training program is given in Appendix A.

Long (LON)

This group was subjected to a low-intensity, long-duration
endurance program which was also progressive in nature. By the thirty-
seventh day of training, and thereafter, the animals were expected to

complete four bouts of exercise with 2.5 minutes of inactivity between

33

bouts. Each bout consisted of one repetition of 12.5 minutes of
continuous work. This group ran at a speed of 36 m/min during the
work intervals. A description of the complete training program is
given in Appendix A. Table 2 illustrates the controlled running wheel
regimens for both exercise groups which was imposed on the thirty-

seventh and all ensuing days of training.

Control (CON)

 

These animals did not receive any type of forced exercise and
were maintained in individual, sedentary cages during the adjustment

and treatment periods.

Duration Groups

 

Three durations of the treatments were studied. Eight animals
from each treatment group were killed at zero, eight, and sixteen
weeks. These groups are referred to as 0—wk, 8-wk, and l6-wk animals
respectively. The animals were 84, 140, and 196 days of age at

sacrifice.

Acute and Chronic Groups

 

Four animals from each duration group were sacrificed at
fifteen minutes (Acute) and at seventy-two hours (Chronic) after their
last exercise bout. Acute and chronic measures were incorporated into
the experimental design to distinguish immediate and transitory
responses to exercise from gradual metabolic adaptations to training.
In the current investigation only the data from the chronically

exercised animals are reported.

34

 

 

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35

Experimental Procedures

All exercise treatments were conducted five days per week
between 12:00 noon and 9:00 p.m. Body weights of the exercised animals
were recorded before and after each exercise period. The intensity of
the exercise was gradually increased up to eight weeks and then held
constant through sixteen weeks.

The performance data for each trained animal were recorded
daily. Total meters run (TMR) and total expected meters (TEM) were
used to calculate percent to expected meters (PEM): PEM - 100
(TMR/TEM). PEM values were used to evaluate performance and to select
animals for inclusion in the final sample. Total work time (TWT) and
cumulative duration of shock (CDS) were used to calculate percent
shock-free time (PSF): PSF = 100 - 100 (CDS/TWT). PSF values served

as an additional criterion to evaluate performance in some cases.

Animal Care

 

All of the animals were housed in standard, individual,
sedentary cages (24 cm x 18 cm x 18 cm) throughout the entire investi-
gation. Since rats normally are more active at night than during
daylight hours, the light sequence in the animal quarters was auto-
matically set to reverse the rat's active period by having the lights
off between 1:00 p.m. and 1:00 a.m.

A relatively constant environment was maintained for the
animals by daily handling as well as by temperature, barometric
pressure and humidity control. The cages were cleaned regularly. All
animals had access to food (Wayne Laboratory Blox) and water 29_

libitum.

36

Sacrifice Procedures

Four sacrifices of twelve animals each were conducted either
fifteen minutes or seventy-two hours after eight and sixteen weeks of
treatments. An additional sacrifice of twelve 0-wk animals was
conducted following the lO-day adjustment period. Originally, each
O-wk animal was assigned randomly to one of the three training groups
as an Acute animal (see table 1). Since no training had occurred at
this time, it was decided that these same 12 animals could be used as
O-wk Chronic subjects. Therefore, to provide a balanced statistical
design, each 0-wk animal also was assigned randomly to a second but
different training group as a Chronic animal.

On the sacrifice day, final body weights were recorded and
each animal was anesthetized by an intraperitoneal injection
(4 mg/lOOg body weight) of a 6.48% Halatal solution (sodium pentobar-
bital). After several tissues had been removed by other investigators,
the left hindlimb was skinned and the superior posterior crural muscles
were exposed by reflecting the overlying tissues. The left plantaris
and soleus muscles were removed separately. The quadriceps group then
was exposed and a portion from the white area of the vastus lateralis
muscle was removed. Each muscle was weighed, held in forceps and quick
frozen in 2-methylbutane (isopentane). The isopentane had been
previously cooled to a viscous fluid (-l40 to -l85°C) by liquid
nitrogen. The frozen muscles were placed in aluminum 35-mm film
containers and stored at -20°C until the biochemical analyses could be

run.

37

Biochemical Procedures

The glycogenolytic enzymes, phosphorylase and phosphoglucomutase,
are responsible for the conversion of glycogen to glucose-G-phosphate.
Furthermore, the activity of phosphoglucomutase has been shown to be
proportional to the activities of the ”phosphotriose glycerate
phosphate group" of glycolytic enzymes in various types of muscles (83).
Phosphoglucomutase was selected to represent glycogenolytic activity in
this investigation.

Glucose-6-phosphate is the common intermediate produced by
glycogenolysis and phosphorylation of blood glucose and may be
metabolized further via either the glycolytic or the hexose monophosphate
shunt (HMS) pathways. However, metabolism via the HMS is low in
skeletal muscle (47). Thus, the relative levels of phosphoglucoiso-
merase, presumed to reflect glycolytic capacity, and phosphogluco-
mutase were used to estimate the relative contributions of blood
glucose and glycogen as sources of glucose-6-phosphate for glycolytic
metabolism.

It generally is accepted that mammalian lactate dehydrogenase
exists as five tetrameric isozymes. One isozyme, LDHS, is predominate
in muscle and catalyzes the anaerobic conversion of pyruvate to
lactate. It also appears that this enzyme is regulatory in function
(36). In this study, total lactate dehydrogenase was chosen to
represent anaerobic glycolysis.

Fumarase and citrate synthetase, enzymes of the tricarboxylic
acid cycle, can serve as markers for the mitochondrial matrix (67).
Wilson (104) has shown that fumarase and citrate synthetase exist in

an approximately constant ratio in all types of rat brain mitochondria.

38

Unpublished observations by Wilson have disclosed rather invariant
cytochrome/fumarase ratios in mitochondria from a variety of mammalian
tissues. Furthermore, in pilot work for this investigation, relatively
constant cytochrome oxidase/fumarase ratios were found in different
types of rat skeletal muscle. Fumarase was used in this study to
represent tricarboxylic acid cycle activity.

All biochemicals required for the assays were obtained from
the Sigma Chemical Co. except for glucose 1,6-diphosphate which was

obtained from the Boehringer-Mannheim Corp.

Preparation of Homogenates

 

Muscle homogenates were prepared in 10-15 volumes of 0.1 M
potassium phosphate (pH 7.0). The muscles were chopped into a fine
mince with scissors and homogenized in a Servall Omnimixer at maximum
power for 15 seconds. This procedure was followed by 15 passes in a
glass-Teflon homogenizer. The samples were maintained in an ice bath
during the preparation and kept at -20°C until assayed for enzyme

activity.

Enzyme Assays

Phosphoglucomutase, phosphoglucoisomerase, and lactate
dehydrogenase assays were performed in a Turner Spectrophotometer.
Fumarase activity was determined in a Coleman-Hitachi 124 Spectrophoto-
meter. The sample compartment was thermostated at 25°C for all
assays. Enzyme activities were determined by analyzing the muscles of
each animal at the same time as those of the other animals in its
duration group. All enzyme activities were recorded on a Sargent

Recorder and have been expressed as.Units/g fresh wt. of muscle. An

39

enzyme unit is the amount of enzyme catalyzing the formation of one u
mole of product per minute.
Phosphoglucomutase (PGM) catalyzes the following reaction of

glycogenolysis:
Glucose l-phosphate I Glucose 6—phosphate

Phosphoglucomutase (a-D-glucose—l,6 bisphosphate:a-D-glucose-l-phosphate

phosphotransferase EC 2.7.5.1) was assayed by the method of Shonk and

Boxer (90)- The enzyme activity was determined by measuring the

reduction of NADP+ at 340 nm. The assay medium contained: 55 mM

aspes (pH 7.6), 5.55 mM EDTA, 15.55 mM Mgc12, 0.34 mM mum»+ 1.0 u

glucose-6-phosphate dehydrogenase, 3.2 mM glucose-l-phosphate, and

5.0 uM glucose-1,6-diphosphate. Samples of homogenate (10-25 ul) plus

water were added to the reaction medium for a total volume of 1 m1.
Phosphoglucoisomerase (PGI) catalyzes the following reaction of

glycolysis:
a-D-Glucose 6-phosphate I a-D-fructose 6-phosphate

Phosphoglucoisomerase (D-glucose-6-phosphate ketolisomerase EC
5.3.1.9) was assayed by the method of Shonk and Boxer (90). The
enzyme activity was determined by measuring the reduction of NADP+ at
340 nm. The assay medium contained: 50 mM HEPES (pH 7.6), 5 mM EDTA,
10 mM MgC12, 0.34 mM NADP+, 1.0 U glucose-6-phosphate dehydrogenase,
and 3.0 mM fructose-G-phosphate. Samples of homogenate (1-5 ul) plus
water were added to the reaction medium for a total volume of 1 ml.
Lactate dehydrogenase (LDH) catalyzes the following anaerobic

reaction of glycolytically formed pyruvate:

40
+ + +
Pyruvate + NADH+H + Lactate + NAD

Lactate dehydrogenase (L-Lactate:NAD+oxidoreductase EC 1.1.1.27) was
assayed by a method adapted from Shonk and Boxer (90). The enzyme
activity was determined by measuring the oxidation of NADH at 340 nm.
The assay medium contained: 0.03 M potassium phosphate (pH 7.4),
1.0 mM sodium pyruvate (pH 7), and 0.17 mM NADH. Samples of homogenate
(1-5 ul) plus water were added to the reaction medium for a total
volume of 1 ml.

Fumarase (FUM) catalyzes the following reaction of the

tricarboxylic acid cycle:

Fumarate + H20 2 L-malate

Fumarase (L-Malate hydro-lyase EC 4.2.1.2) was assayed by the method
of Racker (86). The enzyme activity was determined by measuring the
formation of fumarate from malate at 240 nm. The assay medium
contained: 56 mM potassium phosphate (pH 7.4) and 56 mM Lemalate.
Samples of homogenate (10-50 ul) plus water were added to the reaction
medium for a total volume of 1 ml. No further increase in fumarase
activity was observed after treatment of the homogenates with 0.5%
(v/v) Triton x-100. Thus, complete disruption of the mitochondria

apparently had occurred during homogenization.

Enzyme Activity Ratios

 

The following three enzyme activity ratios, reflecting constant
metabolic organization, were calculated for each of the three

voluntary muscles:

41

 

 

 

 

 

Phosphoglucomutase . Glycogenolysis
Phosphoglucoisomerase representing Glycolysis
Phosphoglucoisomerase . Glycolysis .
Lactate Dehydrogenase representing Lactate Fermentation
Phosphoglucomutase representing glycogenolysis

 

Lactate Dehydrogenase Lactate Fermentation

Three enzyme activity ratios, reflecting variable metabolic
organization, also were calculated for each of the three voluntary

muscles:

Lactate Dehydrogenase Lactate Fermentation

 

 

 

 

 

 

Fumarase representing Tricarboxylic Acid Cycle
Phosphoglucoisomerase Glycolysis

Fumarase representing Tricarboxylic Acid Cycle
Phozphoglucomutase . Glycogenolysis

Fumarase representing Tricarboxylic Acid Cycle

Statistical Procedures

The experimental design satisfied the assumptions for multi-
variate analysis of variance (MANOVA) procedures. The data were
analyzed using the Finn's Multivarance routine as modified and adapted
for the Control Data Computer 6500 by V. M. Scheifley and W. H. Schmidt.
Treatment and duration of treatment constituted the category variables.

Separate overall MANOVA analyses were performed for the
following seven sets of dependent variables: plantaris and soleus
muscle weights, enzyme activities for each of the three muscles, and
enzyme activity ratios for each of the three muscles. Significant
interaction effects were followed by appropriate one-way multivariate
analyses of variance across all treatments within each duration and

across all durations within each treatment. Significant main-effect

42

contrasts, with a concomitant nonsignificant interaction effect, also
justified use of the one-way fixed-effects MANOVA across levels of
that independent variable within each level of the other category
variable.

An F-ratio for the multivariate test of the equality of mean
vectors was used to determine a significant multivariate effect.
Univariate F's were used to obtain an estimate of the relative contri-
bution of each variable involved in the analysis. Step-down F's will
not be reported since no 2_priori knowledge of the importance of the
dependent variables was available and thus no logical ordering the
variables could be accomplished prior to the analyses. An experiment-
wide alpha level of .05 was used for all multivariate procedures.
Significant contrasts were reported at the .10 level in order to avoid
overlooking potentially meaningful differences which may have been

hidden by the sample size.

CHAPTER IV

RESULTS AND DISCUSSION

This chapter is divided into five main sections. The training
results from the controlled running wheel programs are given first.
Data on muscle weights, enzyme activities, and enzyme activity ratios
follow in that order. An overall discussion of the more important

findings is presented last.

Training Results

For both training groups, a criterion of 75 was set for the
percent of expected meters (PEM). This value represents the minimum
acceptable performance level of the animals on the CRW programs. The
training results for the SHT group are shown in Fig. 1. The data
indicate that these animals maintained a PEM of approximately 80 during
the most vigorous stages of training when running velocities of 90 and
99 m/min were required. A search of the literature has revealed no
other programs for the training of small animals which have incorporated
such high running velocities. Percent shock—free time (PSF) values
were approximately 85 to 90 during the more strenuous portions of the
training program. These results show that the animals generally

responded to the conditioned light stimuli rather than to the

43

44

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unconditioned shock stimuli. It is evident that the SHT running
program is a satisfactory interval training regimen of high intensity
endurance exercise.

Training data for the LON program are presented in Fig. 2. PEM
values were approximately 85 for the last fourteen weeks of training.
A running velocity of 36 m/min was required throughout this period. The
PSF values were in the range of 85 to 95 during the last eleven weeks
of training. These results again provide evidence that the animals
responded well to the training regimen. The running speed used in the
LON program is similar to that of 31 m/min employed by Holloszy (54).
However, Holloszy ran animals continuously for periods of up to two
hours. By contrast, the LON program was designed to simulate long-
duration low-intensity interval training and is characterized by a
maximum work time of fifty minutes split up into a small number of
bouts of running with a recovery interval between bouts. The

physiological changes induced by these two programs need not coincide.

Muscle Weight Results

 

Plantaris and soleus muscle weights are shown across all
treatments and durations of treatments in Fig. 3. The vertical bars
represent standard errors of the means. Overall MANOVA results for
treatment and duration of treatment are presented in Table 3. Neither
treatment nor the treatment x duration interaction produced a signifi-
cant multivariate effect. An overall significant effect was found for
duration of treatment. Subsequent one-way MANOVA results for duration
within each of the treatment groups are shown in Table 4. A summary of

these effects is is included in Table 5.

46

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Plantaris

Muscle

Fig. 3 Plantaris and soleus muscle weights across all treatments

and durations of treatments.

48

Table 3.--Two-way multivariate analysis of variance for plantaris and
soleus muscle weights demonstrating the effects of treatment
and duration of treatment.

 

 

 

Helmert Multivariate
EffeCt Contrast
F-Ratio P Less Than

Treatment TRAINa-CON .35 .710

SHT-LON . .52 .602
Duration 0 vs 8,16 92.91 .001**

8 vs 16 2.33 .117
Interaction . . 1.27 .279

 

aTRAIN group contains pooled muscle weights of SHT and LON
groups.

*Significant at p < .10.

**Significant at p < .05.

49

Table 4.--One-way multivariate analysis of variance for muscle weights,
demonstrating the effects of treatment duration.

 

Multivariate Results

 

Univariate Results

 

 

 

Contrast Treatment Muscle
("ks) Gr°up F-Ratio P Less Than F P Less Than
0 vs 8 CON 10.38 .006** Plantaris 21.83 .001**
Soleus .02 .894
SHT 4.66 .046** Plantaris 10.32 .011**
Soleus .94 .357
LON 10.83 .005** Plantaris 16.72 .003**
Soleus .47 .509
0 vs 16 CON 13.20 .003** Plantaris 22.33 .001**
Soleus 2.45 .152
SHT 28.13 .001** Plantaris 47.80 .001**
Soleus 25.78 .001**
LON 45.25 .001** Plantaris 91.67 .001**
Soleus 16.47 .003**
8 vs 16 CON 1.30 .325 Plantaris 2.84 .127
Soleus .44 .523
SHT 1.35 .313 Plantaris .46 .517
Soleus 2.88 .124
LON .98 .417 Plantaris 1.55 .244
Soleus 2.05 .186
*Significant at p < .10.
**Significant at p < .05.

50

Table 5.--Summary of duration effects for muscle weights with
significant mean differences.

 

 

 

 

Duration Treatment Multivariate Results Mean Differencesa
Contrast Groups
(wks) F-Ratio P Less Than Plantaris Soleus
0 vs 8 CON 10.38 .006** +45
SHT 4.66 .046** +35
LON 10.83 .005** +35
0 vs 16 CON 13.20 .003** +30
SHT 28.13 .001** +40 +55
LON 42.25 .001** +40 +45
8 vs 16 CON 1.30 .325
SHT 1.35 .313
LON .98 .417

 

aSignificant mean differences for muscle weights are tabulated
as percent increase over time. Percentages are rounded to nearest
multiple of five. Effects which are attributed to training appear in
boldface. Maturation-related effects are shown in italics. Effects
which may be due to unexplained or measurement artifacts are given in
small type.

*Significant at p < .10.

**Significant at p < .05.

51

Significant multivariate effects between 0 wks and 8 wks were
evident in all treatment groups. Both plantaris and soleus weights
were greater after 8 wks. However, examination of the univariate F
statistics revealed that the plantaris weight was the source of the
multivariate effect in all three groups.

Statistically significant multivariate effects also were
observed in all training groups between 0 wks and 16 wks. Greater
weights were found in both muscles after 16 wks. The plantaris weight
again was the source of the significance in the CON group; however, in
the SHT and LON groups both the plantaris and soleus weights were
responsible for the "significant effect." These results might be
interpreted as evidence that the training programs produced increases
in soleus weight between 0 and 16 wks beyond those normally expected
to occur with maturation. However, the large random variability
between groups at 0 wks (see Fig. 3) makes this conclusion unwarranted.

No multivariate effects were observed in the muscle weights

between 8 wks and 16 wks.

Enzyme Activity Results
Mean enzyme activity responses in the three voluntary muscle
fiber types, across all treatments and duration of treatments, appear
graphically in Fig. 4. The vertical bars represent standard errors of
the means. Overall MANOVA results for treatment and duration of
treatment are shown in Table 6. The multivariate test for the
treatment x duration interaction was significant in all muscle types.

Separate treatment and duration effects also were significant for each

52

 

 

 

 

 

 

 

I 0.
Venus Le'srolis

0
“units"

 

Fig. 4 Enzyme activities in the plantaris, soleus. and "while" vastus lateralis

mules across all trsolmmrs ma Motion: of lrealmsnts.

53

Table 6.--Two-way multivariate analysis of variance for enzyme

activities in three voluntary muscle types demonstrating the
effects of treatment and duration of treatment.

 

Multivariate Results

 

 

Muscle Effect :::::::t
F—Ratio P Less Than
Plantaris Treatment TRAINa-CON 5.84 .003**
SHT-LON 1.13 .365
Duration 0 vs 8,16 7.00 .004**
8 vs 16 19.95 .001**
Treatment . . 2.11 .017**
Duration
Soleus Treatment TRAINa-CON 7.22 .001**
SHT-LON . 22 .922
Duration 0 vs 8,16 6.24 .001**
8 vs 16 27.37 .001**
Treatment . . 2.14 .015**
Duration
"White" Treatment TRAINa-CON 10 . 17 . 001**
Vastus SHT-LON 1.38 .269
Lateralis
Duration 0 vs 8,16 7.34 .001**
8 vs 16 14.42 .001**
Treatment . . 2.37 .007**
Duration

 

a
TRAIN represents pooled enzyme activities of SHT and LON

group.

*Significant at p < .10.

**Significant at p < .05.

54

muscle; however, these main effects are statistically dependent upon

the treatment x duration interactions.

Treatment Effects
One-way MANOVA results for enzyme activities, comparing
treatment effects at 8 wks and 16 wks, are presented in Table 7. A
summary of all treatment contrasts, including identification of enzymes

which account for the significant effects, is shown in Table 8.

SHT-CON

There were no significant SHT training effects, when the enzyme
levels were tested simultaneously, other than marginal significance in
the soleus muscle at 8 wks. Decreased lactate dehydrogenase activity
in the SHT group was the source of that effect. Nonsignificant but
specific metabolic activity patterns were apparent. The SHT group had
levels of lactate dehydrogenase activity which were slightly lower than
those of the CON group in all fiber types at 8 and 16 wks with the
exception of the plantaris at 16 wks. The high-intensity trained
animals, as compared to the controls, displayed decreased activity
levels of phosphoglucomutase in the plantaris and "white" vastus
lateralis muscles at 16 wks. Glycolytic activity, as measured by
phosphoglucoisomerase levels, tended to be greater in the soleus of the
SHT group but lower in the plantaris and vastus muscles of that group
at 8 and 16 wks. Fumarase activities of the SHT group generally were
higher than those of the CON group in all muscles for both durations of

training.

555

Table 7.--0ne-way multivariate analysis of variance for enzyme activities in three voluntary
muscle fiber types demonstrating the treatment effects.

 

 

 

 

Multivariate Results Univariate Results
Muscle Contrast Duration Enzyme
(wks) F-Ratio P Less Than F P Less Than

Plantaris SHT-CON 8 .88 .530 FUM .92 .362
- LDH .57 .470
PGI 1.01 .341
PGM .29 .605
16 .75 .595 FUM .09 .767
LDH .43 .531
PGI .11 .753
PGM .18 .686
LON-CON 8 1.42 .334 FUM .48 .505
LDH .30 .597
PGI 2.95 .120
PGM .03 .862

16 9.20 .010** FUM 22.93 .001**
LDH 1.43 .262
PGI .45 .521

PGM 5.02 .052*
LON-SHT a 1.40 .338 mm .23 .640
LDH .14 .713
PGI 2.99 .118
PGM .31 .593
16 2.05 .207 FUM 4.54 .062
LDH 1.35 .274
PGI .00 .959
PGM .58 .468
mama-con a . 39 . 522 run 1. 17 . 307
LDH .73 .417
PGI .97 .351
PGM .01 .913

16 7.89 .014“4 FUM 18.49 .002**
LDH .51 .495
PGI .55 .477

PGM 4.62 .060‘

aTRAIN represents pooled enzyme activities of SHT and LON groups.
*Significant at p < .10.

"Significant at p < .05.

556

 

 

 

 

Multivariate Results Univariate Results
Muscle Contrast Duration Enzyme
(wks) F-Ratio P Less Than F P Less Than
Soleus SHT—CON 8 3.57 .081* FUM .68 .432
LDH 9.16 .014**
PGI .22 .652
PGM .14 .715
16 2.13 .195 FUM 3.21 .107
LDH .17 .690
PGI 1.01 .342
PGM 1.09 .323
LON-CON 8 3.13 .103 FUM , .08 .789
LDH 2.13 .178
PGI 6.36 .033
PGM .01 .925
16 13.92 .004** FUM 12.01 .007**
LDH 4.35 .066*
PGI 2.07 .184
PGM 7.47 .023**
LON-SHT 8 3.22 .098* FUM .72 .417
LDH . 3.57 .091*
PGI 2.77 .130
PGM .14 .717
16 .68 .629 FUM .03 .859
LDH 1.96 .195
PGI .02 .884
PGM .21 .655
TRAINa-CON e 3.49 .oe4t run .03 .867
LDH 7.72 .0224.
PGI 3.80 .083*
PGM .01 .919
16 15.36 .003** FUM 15.18 .004**
LDH 2.56 .144
PGI 3.06 .115
PGM 8.35 .018**

a
TRAIN represents pooled enzyme activities of SHT and LON groups.

*Significant at p < .10.

**Significant at p < .05.

57

Table 7.--Continued.

 

 

 

 

M .
Muscle Contrast Duration ultivariate Results Enzyme Univariate Results
("*8) F-Ratio P Less Than F P Less Than
”White" SHT-CON 8 1.22 .392 FUM 1.28 .287
Vastus LDH 2.16 .176
Lateralis PGI 2.06 .185
PGM . 33 . 578
16 .72 .607 FUM .01 .941
LDH .81 .392
PGI .56 .474
PGM 1.33 .278
LON-CON 8 1.54 .302 FUM .73 .416
LDH 8.35 .018
PGI .14 .721
PGM 4.12 .073
16 17.24 .002** FUM 9.48 .013**
LDH 10.81 .010“
PGI 3.11 .112
PGM 9.91 .012‘"
LON-SHT 8 .95 .498 FUM 1.98 .193
LDH .03 .867
PGI 1.12 .318
PGM 2.30 .164
16 1.92 .227 FUM 2.17 .175
LDH .75 .410
PGI .06 .820
PGM .33 .580
" FUM .03 .867
-CON 3 1.82 .244
TRAIN LDH 10.48 .010
PGI 1.07 .327
PGM 2.16 .176
16 16.05 .002** FUM 7.31 .024**
LDH 10.87 .009**
PGI 3.61 .090*
PGM 10.91 .009**

 

aTRAIN represents pooled enzyme activities of SHT and LON groups.
*Significant at p < .10.

MSignificant at p < .05.

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59

LON -CON
Examination of the generalized multivariate F-ratios indicated

significant LON training effects at 16 wks in all three muscles.

Plantaris.--The enzyme activities of fumarase and phosphogluco-
mutase contributed most to the significant multivariate effect in the
plantaris. The activity level of fumarase was increased and that of
phosphoglucomutase was decreased after 16 wks of LON training. These
results must be viewed with some reservation. The moderate random
variabilities between treatment groups at 0 wks would support the
existence of real fumarase and phosphoglucomutase differences at 16 wks
(see Fig. 4). On the other hand, the relatively large variabilities
that were observed between durations within the CON group must be
considered. The question is whether the patterns of the CON-group
fumarase and phosphoglucomutase means across time were due to systematic
or random factors.

Data on the levels of these two enzymes in similar animals
sacrificed at the same time for a companion study suggest that small
but systematic measurement errors depressed all of the plantaris
fumarase and phosphoglucomutase values obtained in this investigation
at 8 wks. If that were the case, it would appear that maturation
tended to decrease fumarase and increase phosphoglucomutase activities
over time. Under this hypothesis, there is no reason to question the
existence of significant LON training effects in those two enzymes at
16 wks.

There is no evidence to support the alternative hypothesis

that the variabilities in the CON group fumarase and phosphoglucomutase

60

means across time are reflective of purely random factors, but that
possibility cannot be ignored. If only random factors were operating,
the assumption of meaningful LON training effects on fumarase and

phosphoglucomutase at 16 wks is not justified.

Soleus.--Fumarase, lactate dehydrogenase and phosphoglucomutase
enzyme activities were responsible for the LON training effect at
16 wks in the soleus. Fumarase activity increased while the activities
of lactate dehydrogenase and phosphoglucomutase decreased as a result

of long-endurance running.

"White" Vastus Lateralis.--The LON training effect in the
"white" vastus lateralis was produced by alterations in fumarase,
lactate dehydrogenase, and phosphoglucomutase at 16 wks. Fumarase
activity almost doubled in the LON group; however, the magnitude of
this increase should be viewed in light of the rather large random
variability observed between groups at 0 wks. The activities of
lactate dehydrogenase and phosphoglucomutase were lower in this group

than in the CON group.

LON-SHT

The only significant difference between enzyme levels in LON
groups was observed in the soleus at 8 wks. The activity of lactate
dehydrogenase was slightly lower in the SHT group than in the LON group
at that time. The nonsignificant but consistent results across
muscles in each of the four enzymes might suggest a metabolic

divergence between the training groups at 16 wks.

61

TRAIN-CON

This contrast was established to determine if an overall
interval training response, obtained by combining enzyme activities for
the SHT and LON groups, would be reflected by some general metabolic
adaptations. It would appear that this analysis was justified. That
is, although the results of the LON-SHT contrast suggest differential
training effects, the final enzyme levels in both training groups were
consistently either above or below those of the CON group in all
muscles.

Several important overall training effects were identified.
Significant multivariate F-values were obtained for the soleus at

8 wks and for all three skeletal muscles at 16 wks.

Plantaris.--Fumarase and phosphoglucomutase activities were
identified as reflecting a general training effect in the plantaris at
16 wks. The direction and magnitude of the enzyme changes were similar
to those found in the LON-CON contrast, and the interpretation of these
results is subject to the limitation mentioned in the discussion of

that contrast.

Soleus.--At 8 wks, there was a significant overall training
effect of decreased lactate dehydrogenase activity in the soleus. The
apparent general increase in soleus phosphoglucoisomerase activity at
that time must be regarded cautiously due to the large variability
found in the CON group means across durations.

The activities of fumarase and phosphoglucomutase accounted

for the multivariate overall training effect in the soleus at 16 wks.

62

Changes in these enzymes were similar to those observed in the LON-CON

contrast.

"White" Vastus Lateralis.--The significant overall training
effect in the "white" vastus muscle was mediated by alterations in all
of the enzymes. Fumarase, lactate dehydrogenase, and phosphoglucomutase
activities responded in the same direction as they did in the LON—CON
contrast. In addition, the activity of phosphoglucoisomerase decreased

with training.

Duration Effects
One-way multivariate analysis of variance results for enzyme
activities, comparing duration effects within each of the treatment
groups, are presented in Table 9. A summary of all duration contrasts,
including identification of enzymes which account for significant

effects, is shown in Table 10.

0 vs 8 wks

 

The one-way multivariate F-ratios indicate the existence of
significant 0 vs 8-wk effects for all treatment groups in the
plantaris, for the CON and SHT groups in the soleus, and for all groups

in the "white" vastus lateralis.

Plantaris.--The activity of fumarase in the plantaris was
significantly affected in all treatment groups with lower levels being
observed at 8 wks than at 0 wks. As discussed earlier, these changes
probably were due to a systematic maturation effect which operated
across all groups. The activity of phosphoglucomutase was lower in

the CON group after 8 wks. This observation also appears to be

63

Table 9.-~One-way multivariate analysis of variance for enzyme activities in three voluntary
muscle fiber types demonstrating the effects of treatment duration.

 

 

 

 

Muscle Contrast Treatment Multivariate Results Enzyme Univariate Results
("k“) Gr°up P-Ratio 9 Less Than P 9 Less Than
Plantaris 0 vs 8 CON 5.89 .028 FUM 30.96 .001**
LDH 1.63 .234
PGI 2.69 .135
PGM 3.41 .098*
SHT 8.36 .013'* FUM 10.65 .010**
LDH 5.27 .047“'
PGI .42 .532
PGM .86 .378
LON 11.72 .005*' FUM 36.64 .001**
LDH 1.14 .314
PGI 4.36 .066*
PGM 1.05 .333
0 vs 16 CON 2.84 .123 FUM 7.70 .022
LDH 5.34 .046
PGI 1.33 .279
PGM 1.77 .216
SHT 2.02 .211 FUM .86 .377
LDH 3.52 .094
PGI .01 .915
PGM .423 .532
LON 2.09 .201 FUM 7.11 .026
LDH 2.13 .178
PGI .56 .472
PGM .48 .508
8 vs 16 CON 3.15 .102 FUM 11.78 .008
LDH 5.10 .050
PGI 3.99 .077
PGM 5.13 .050
SHT 8.44 .012** FUM 5.58 .043**
LDH 8.56 .017**
PGI .38 .552
PGM .23 .644
LON 12.55 .005“ FUM 43.24 .001**
LDH 2.73 .133
PGI 2.05 .186
PGM .29 .602

*Significant at p < .10.

'*Significant at p < .05.

64

 

 

 

 

Multivari t
Muscle Contrast Treatment a e Results Enzyme Univariate Results
("k3, Group P-Ratio P Less Than P P Less Than
Soleus 0 vs 8 CON 7.60 .016** FUM 2.51 .147
LDH 4.23 .070*
PGI 23.61 .001**
PGM 1.36 .273
SHT 18.35 .022** FUM 6.13 .035**
LDH 10.43 .010**
PGI 71.71 .001**
PGM 1.87 .205
LON 3.10 .105 FUM .01 .912
LDH 2.47 .151
PGI 12.85 .006
PGM .76 .405
0 vs 16 CON 12.25 .005** FUM 1.79 .214
LDH 10.46 .010**
PGI .61 .453
PGM .75 .410
SHT 10.82 .007** FUM 1.26 .291
LDH 10.80 .010**
PGI 16.09 .003**
PGM 7.51 .023**
LON 5.24 .037** FUM 4.46 .064*
LDH .47 .511
PGI 13.50 .005“r
a PGM 14.31 .004“
8 vs 16 CON 8.55 .012** FUM 4.17 .072*
LDH 11.54 .008'*
PGI 21.16 .001**
PGM .34 .577
SHT 27.16 .001** FUM 7.32 .024**
LDH 19.71 .002**
PGI 87.22 .001**
PGM .03 .857
LON 4.89 .043** FUM 1.33 .278
LDH 2.90 .123
PGI 24.42 .001**
PGM 1.29 .286

*Significant at p < .10.

**Significant at p < .05.

(55

Table 9.--Continued.

 

 

 

 

Multivari t R
Muscle Contrast Treatment a e esults Enzyme Univariate Results
("k3, Gr°up P-Ratio P Less Than P P Less Than
"white" 0 vs 8 CON 3.34 .091* FUM 2.07 .184
Vastus LDH 5.34 .046**
Lateralis PGI 1.29 .286
PGM 5.77 .040**
SHT 30.09 .001** FUM .79 .396
LDH 28.45 .091.1.
PGI .28 .609
PDM 21.32 .001**
LON 8.37 .013** FUM 4.94 .053*
LDH 13.01 .006“
PGI .85 .380
PGM 21.52 .001**
0 vs 16 con 16.59 .002** FUM .34 .575
LDH 10.82 .009**
PGI 23.84 .001**
PGM 6.94 .027**
SHT 1.44 .329 FUM .01 .930
LDH .18 .679
PGI 2.95 .120
PGM .04 .856
LON 2.22 .183 FUM 3.07 .114
w" .20 .665
P61 3.10 .112
PGM .04 .839
8 vs 16 CON 6.68 .021** FUM .91 .365
LDH 13.28 .005**
PGI 1.98 .193
PGM 11.53 .008“
SHT 18.79 .002*‘ FUM .67 .435
LDH 19.41 .002**
PGI 1.74 .220
PGM 15.25 .004**
LON 4.44 .052* FUM 7.85 .021**
LDH 8.41 .018**
PGI .01 .938
PGM 16.99 .003**

 

*Significant at p < .10.

**Significant at p < .05.

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67

maturation related. The level of activity of lactate dehydrogenase was
lower in the SHT group, but phosphoglucoisomerase activity was somewhat
higher in the LON group after 8 wks. These changes most likely are

indicative of real training effects.

Soleus.--A lower level of phosphoglucoisomerase at 8 wks was
primarily responsible for the significant 0 vs 8-wk duration effect in
the soleus muscle of the CON group. The significant multivariate
effect in the SET group was the result of decreased activities of
fumarase, lactate dehydrogenase, and phosphoglucoisomerase at 8 wks.
The phosphoglucoisomerase results found in the CON and SHT groups are
suspect. A maturation effect does not seem likely since the 0 to 8-wk
trend seen in the CON group was reversed by 16 wks. No claim can be
made for a training effect in the SET group since the decrease in the
SHT group was less than that in the CON group. At present, the
depressed phosphoglucoisomerase values obtained at 8 wks should be
considered to be unexplained artifacts. The fumarase level decrease
in the SHT group also should be discounted due to the large random

variability observed between groups at 0 wks.

"White" Vastus Lateralis.--The multivariate 0 vs 8-wks
duration effects in the three treatment groups were primarily due to
lower lactate dehydrogenase and phosphoglucomutase activities in the
"white” vastus muscle after 8 wks. In addition, the fumarase level
of the LON group was decreased at this time. The lactate dehydrogenase
decreases in the two training groups were of considerably greater
magnitude than that in the CON group and probably represent training

effects. The change in phosphoglucomutase level in the LON group also

68

seems to be exercise related, but no claim can be made for that in the
SET group. The fumarase results should be ignored because of the large

random variability observed between groups at 0 wks.

0 vs 16 wks
Significant 0 vs 16 wks duration effects were evident for all
treatment groups in the soleus muscle and for the control group in the

”white” vastus muscle.

Soleus.--Lactate dehydrogenase activity in the soleus was
increased between 0 and 16 wks in the CON group. The overall SHT-group
effect was attributable to increased lactate dehydrogenase and
phosphoglucoisomerase levels and to a decreased phosphoglucomutase
level after 16 wks. The enzymes alterations observed in the LON group
after 16 wks were increased fumarase and phosphoglucoisomerase
activities and decreased phosphoglucomutase activity. All of these
alterations in lactate dehydrogenase and phosphoglucoisomerase
activities probably were maturation dependent, whereas those seen in
fumarase and phosphoglucomutase levels appear to have been produced by

the training regimens.

"White" Vastus Lateralis.--The significant response in the

 

"white" vastus lateralis of the CON group at 16 wks can be attributed
to maturative increases in levels of activity of lactate dehydrogenase,
phosphoglucoisomerase, and phosphoglucomutase. It should be noted
that the two training programs apparently delayed these age-related

changes.

69

8 vs 16 wks
Significant multivariate contrasts between 8 and 16 wks were

observed for the SHT and LON groups in the plantaris and for all three

treatment groups in the other two muscles.

Plantaris.--The univariate F results indicate that fumarase
activity in the plantaris was higher in both the SHT and LON groups
after 16 wks than after 8 wks. However, the apparent change in the
SET group could have been an artifact resulting from systematic factors
operating at 8 wks. The increase in the LON group may have been
inflated by these same factors but probably reflects a true training
effect. The SHT group had a training-related increase in lactate

dehydrogenase activity during this period of time.

Soleus.--The enzyme activities that were altered in the soleus
muscles of the CON and SHT groups between 8 and 16 wks of training were
those of fumarase, lactate dehydrogenase, and phosphoglucoisomerase.
Activity levels of lactate dehydrogenase increased greatly and levels
of phosphoglucoisomerase almost doubled in both groups between 8 and
16 wks. In contrast, fumarase activity decreased in the CON group and
increased in the SHT group. For the LON group, an increase in
phosphoglucoisomerase activity was the only source of the multivariate
effect. Of all of these changes, the only one which definitely can be
attributed to training is the increased fumarase activity in the SHT

group.

“White" Vastus Lateralis.--Increases in lactate dehydrogenase

and phosphoglucomutase activities were accountable for the multivariate

7O

effect in the ”white“ vastus lateralis muscles of all three treatment
groups between 8 and 16 wks. All of these changes appear to be due to
maturation. Fumarase levels were nearly doubled as a result of the LON

program during the second half of the training period.

Enzyme Activitngatio Results

The term "variable metabolic organization," when used to
describe an enzyme activity ratio, refers to a ratio that has essentially
different ranges of values in various types of skeletal muscles.
”Constant metabolic organization" indicates an enzyme activity ratio
that has overlapping ranges of values that do not differentiate
skeletal-muscle types.

Mean enzyme activity ratios representing variable metabolic
organization and constant metabolic organization in the three voluntary
muscle-fiber types, across all treatments and durations of treatments,
are found in Figs. 5 and 6. The vertical bars represent standard
errors of the means. Overall MANOVA results for treatment and duration
of treatment appear in Table 11. The multivariate test for the
treatment x duration interaction was significant in the soleus muscle
with borderline significance in the plantaris muscle. Separate

treatment and duration effects also were significant for each muscle.

Treatment Effects
One-way MANOVA results for enzyme activity ratios, comparing
treatment effects at 8 and 16 wks, are presented in Table 12. A
summary of all treatment contrasts, including identification of enzyme

ratios which account for the significant effects, is shown in Table 13.

 

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end auctions of treatments.

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end durations of treatments.

vastus Ioterolus moles across all treatments

73

Table ll.--Two-way multivariate analysis of variance for enzyme
activity ratios in three voluntary muscle types demonstrating
the effects of treatment and duration of treatment.

 

Multivariate Results

 

 

Helmert
Muscle Effect Contrast F-Ratio P Less Than
Plantaris Treatment TRAINa-CON 2.71 .040**
SHT-LON 1.12 .381
Duration 0 vs 8,16 4.26 .005**
8 vs 16 5.72 .001**
Treatment x . . 1.50 .095*
Duration
Soleus Treatment TRAINa-CON 5. 97 . 001**
Duration 0 vs 8,16 10.39 .001**
8 vs 16 16.08 .001**
Treatment x - . 3.29 .001**
Duration
"White" Treatment TRAINa-CON 7 . 23 . 001**
Vastus SHT-LON 1.06 .417
Lateralis
Duration 0 vs 8,16 4.65 .004**
8 vs 16 6.40 .001**
Treatment x . . 1.17 .299
Duration

 

aTRAIN represents pooled enzyme activities of SHT and LON
group.

*Significant at p < .10.

**Significant at p < .05.

'74

Table 12.--One-way multivariate analysis of variance for enzyme activity ratios in three
voluntary muscle fiber types demonstrating the treatment effects.

 

 

 

 

Muscle Contrast Duration Multivariate Results Enzyme Univariate Results
(WKS) E-Ratio P Less Than Ratios F P Less Than
Plantaris SHT-CON 8 1.14 .469 LDH/FUM .79 .397
PGI/FUM 4.69 .059
PGM/FUM .00 .982
PGI/LDH .40 .544
EON/LDH 2.06 .185
PGM/PGI 2.94 .120
16 .78 .626 LDH/FUM .05 .834
PGI/FUM .96 .352
PGM/FUM 1.42 .263
PGI/LDH 2.66 .137
PGM/LDH .69 .426
PGM/PGI .01 .929
LON-CON 8 .72 .660 LDH/FUM 1.37 .271
PGI/FUM 1.52 .249
PGM/FUM 1.02 .339
PGI/LDH 7.25 .025
PGM/LDH .01 .934
PGM/PGI 2.69 .136
16 5.51 .060* LDH/FUM 21.50 .001**
PGI/FUM 11.22 .009"
PGM/FUM 32.98 .001**
PGI/LDH .24 .634
PGM/LDH 1.47 .256
PGM/PGI 1.58 .240
LON-SHT 8 1.02 .518 LDH/FUM .03 .859
PGI/FUM 6.21 .034
PGM/FUM .28 .612
PGI/LDH 3.58 .091
PGM/LDH 1.44 .261
PGM/PGI 5.32 .047
16 .92 .562 LDH/FUM 4.54 .062
PGI/FUM .68 .431
PGM/FUM 3.38 .099
PGI/LDH 2.76 .131
PGM/LDH .01 .911
PGM/PGI .50 .497
mama-CON 8 . 84 . 594 LDH/FUM 2 . 1 3 . 179
PGI/FUM .00 .988
PGM/FUM .75 .410
PGI/LDH 4.06 .075
PGM/LDH .62 .450
PGM/PGI .32 .588
16 5.38 .063* LDH/FUM 17.00 .003**
PGI/FUM 11.51 .008**
PGM/FUM 31.03 .001“
PGI/LDH .15 .707
PGM/LDH 2.15 .176
PGM/PGI 1.09 .324

aTRAIN represents pooled enzyme activity ratios of SHT and LON group.
'Significant at p < .10.

*‘Significant at p < .05.

Table 12.--Continued.

'75

 

Multivariate Results

 

Univariate Results

 

 

Muscle Contrast Duration Enzyme
("k”) P-Ratio P Less Than Rati°° P P Less Than
Soleus SHT-CON 8 .64 .705 LDH/FUM 1.10 .321
PGI/FUM .00 .971
PGM/FUM .02 .890
PGI/LDH .54 .480
PGM/LDH .58 .466
PGM/PGI .08 .787
16 2.91 .160 LDH/FUM 5.92 .038
PGI/FUM .82 .388
PGM/FUM 10.69 .010
PGI/LDH .15 .708
PGM/LDH 1.19 .304
PGM/PGI 3.21 .107
LON-CON 8 5.30 .064* LDH/FUM 1.73 .221
PGI/FUM 4.69 .059*
PGM/FUM .02 .897
PGI/LDH 8.45 .017**
PGM/LDH .41 .538
PGM/PGI 1.48 .255
16 10.85 .019** LDH/FUM 49.74 .001**
PGI/FUM 4.89 .054*
PGM/FUM 44.64 .001**
PGI/LDH 9.38 .014**
PGM/LDH .09 .774
PGM/PGI 11.00 .009**
LON—SHT 8 1.97 .267 LDH/FUM .06 .807
PGI/FUM 1.10 .321
PGM/FUM .00 .956
PGI/LDH .67 .436
PGM/LDH .12 .742
PGM/PGI .14 .722
16 .54 .760 LDH/FUM 2.01 .190
PGI/FUM .10 .756
PGM/FUM .26 .623
PGI/LDH 1.43 .262
PGM/LDH 1.19 .303
PGM/PGI .01 .918
mama-con 8 3 . 97 . 102 LDH/FUM 2. 77 . 131
PGI/FUM 3.59 .091
PGM/FUM .03 .856
PGI/LDH 8.33 .018
PGM/LDH .87 .374
PGM/PGI 1.42 .263
16 13.22 .013** LDH/FUM 53.64 .001**
PGI/FUM 5.61 .o42**
PGM/FUM 55.07 .001*9
PGI/LDH 8.10 .019*'
PGM/LDH .08 .779
PGM/PGI 14.21 .005**

aTRAIN represents pooled enzyme activity ratios of SHT and LON group.

*Significant at p < .10.

HSignificant at p < .05.

76

Table 12.--Continued.

 

 

 

 

Muscle Contrast Duration Multivariate Results Enzyme Univariate Results
("k“) F-Ratio P Less Than Rat1°° F P Less Than

”White” SHT-CON 8 .62 .717 LDH/FUM 2.69 .136
Vastus PGI/FUM 2.67 .137
Lateralis PGM/FUM 1.04 .335
PGI/LDH .38 .551
PGM/LDH 4.79 .057
PGM/PGI 3.59 .091
16 .57 .742 LDH/FUM 2.90 .123
PGI/FUM 1.88 .204
PGM/FUM 4.74 .058
PGI/LDH .01 .941
PGM/LDH .12 .737
PGM/PGI .13 .727
LON-CON 8 1.75 .307 LDH/FUM .03 .857
PGI/FUM .33 .580
PGM/FUM .04 .840
PGI/LDH 2.26 .167
PGM/LDH .05 .829
PGM/PGI 1.22 .299

16 8.91 .027** LDH/FUM 29.86 .001“

PGI/FUM 20.76 .001**

PGM/FUM 49.28 .001**
PGI/LDH .81 .393
PGM/LDH .26 .622
PGM/PGI 3.09 .113
LON-SHT 8 .90 .570 LDH/FUM 1.76 .217
PGI/FUM 2.89 .123
PGM/FUM .61 .456
PGI/LDH 1.66 .230
PGM/LDH 4.02 .076
PGM/PGI 4.80 .056
16 1.32 .410 LDH/FUM 1.58 .240
PGI/FUM 1.19 .304
PGM/FUM 2.64 .139
PGI/LDH .27 .619
PGM/LDH .00 .966
PGM/PGI .32 .584
TRAINa-CON 8 1.47 .371 LDH/FUM .96 .353
PGI/FUM .10 .757
PGM/FUM .48 .507
PGI/LDH .98 .348
PGM/LDH .81 .391
PGM/PGI .00 .994

16 8.15 .o31** LDH/FUM 31.17 .001**

PGI/FUM 21.45 .001“

PGM/FUM 51.38 .001“
PGI/LDH .55 .478
PGM/LDH .38 .553
PGM/PGI 2.90 .123

 

aTRAIN represents pooled enzyme activity ratios of SHT and LON group.
*Significant at p < .10.

“Significant at p < .05.

77

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78

SHT-CON

No statistically significant SHT training effects were observed
when all of the enzyme ratios were tested simultaneously. However,
subjectively a distinct metabolic pattern appeared to exist in the
three variable activity ratios. These ratios generally were lower in
the SHT group than in the CON group, at.both 8 wks and 16 wks in all
three fiber types. No identifable pattern was found for the ratios of

constant metabolic organization.

LON -CON

Inspection of the one-way multivariate F-ratios revealed
significant multivariate training effects in the soleus and "white"
vastus lateralis muscles at 16 wks. Marginal effects were observed in

the plantaris at 16 wks and the soleus at 8 wks.

Plantaris.--In the plantaris muscle, enzyme activity ratios of
variable metabolic organization were uniquely affected by the LON
program. These ratios were approximately one-third to one-half lower

in the LON group than in the CON group at 16 wks.

Soleus.--The LON program induced pronounced changes in the
phosphoglucoisomerase/fumarase and phosphoglucoisomerase/lactate
dehydrogenase ratios in the soleus after 8 wks of training. Both ratios
appeared to be elevated in the LON group; however, these results should
be overlooked due to the pattern of variability found in the CON group
means across durations.

At 16 wks, the multivariate effect was at least partially

attributable to training-related decreases in all three variable-ratio

79

enzymes. Activity ratios of constant metabolic organization behaved
differently. Phosphoglucoisomerase/lactate dehydrogenase was increased
by the LON program. Phosphoglucomutase/phosphoglucoisomerase was

lower in the LON group than in the CON group, but this finding is
suspect due to the variability observed in the 0-wk data as well as in

the CON group values across durations.

"White" Vastus Lateralis.-—The significant multivariate LON
training effect at 16 wks in the "white" vastus lateralis was caused by
decreases of approximately one-half in the variable enzyme ratios.
These changes must be viewed with caution due to the random variability
seen at 0 wks. However, that variability is the direct result of the
single relatively low fumarase value obtained in the CON group at 0 wks
(see Fig. 4). No such anomalities were observable in the 16-wk enzyme
data, and thus it appears that these decreases in the variable enzyme

ratios at 16 wks probably represent actual training effects.

LON-SHT

Neither 8 wks nor 16 wks of training produced any significant
differences in enzyme activity ratios between the LON and SHT groups.
Certain ratios appeared subjectively to be specifically altered by each
training regimen in all three muscles. The phosphoglucomutase/
phosphoglucoisomerase ratios were higher in the SHT group than in the
LON group at both 8 and 16 wks. In contrast, the phosphoglucoisomerase/
lactate dehydrogenase ratios were higher in the LON group at both of

these durations.

80

TRAIN-CON
An overall interval training response was significant in the
soleus and "white" vastus lateralis muscles at 16 wks. Marginal

significance was observed in the plantaris at this duration.

Plantaris.--The overall training effect at 16 wks in the
plantaris was modulated entirely by changes in enzyme activity ratios
which are representative of variable metabolic organization. The
adaptation was similar to that found in the LON—CON contrast for the

plantaris.

Soleus.--Significant training changes at 16 wks in the soleus
appear to be regulated by both activity ratios of variable and constant
metabolic organization. The pattern of response was similar to that
observed in the LON-CON contrast for this muscle, and the interpretation
of these results is subject to the limitation mentioned in the dis-

cussion of that contrast.

"White" Vastus Lateralis.--The multivariate overall training
effect at 16 wks in the "white" vastus lateralis was predominantly
related to changes in the variable enzyme ratios. As in the plantaris
and soleus, the shifts in these ratios were similar to those produced

by the LON program alone.

Duration Effects
One-way MANOVA results for enzyme activity ratios, illustrating
duration effects within each of the treatment groups, are presented

in Table 14. A summary of all duration contrasts, including

8].

Table l4.--One~way multivariate analysis of variance for enzyme activity ratios in three
voluntary muscle fiber types demonstrating the effects of treatment duration.

 

 

 

 

Muscle Contrast Treatment Multivariate Results Enzyme Univariate Results
(wks) Group P-Ratio P Less Than Ratios P P Less Than
Plantaris 0 vs 8 CON 1.27 .426 LDH/FUM 5.20 .049
PGI/FUM 4.81 .056
PGM/FUM .29 .602
PGI/LDH .08 - .785
PGM/LON 1.62 .236
PGM/PGI .88 .375
SHT 2.74 .175 LDH/FUM 1.72 .222
PGI/FUM 10.02 .012
PGM/FUM 1.63 .233
PGI/LDH 2.30 .164
PGM/LDH ..04 .838
PGM/PGI .22 .651
LON 7.57 .035" LDH/FUM 22.48 .001**
PGI/FUM 48.20 .001'*
PGM/FUM 3.22 .106
PGI/LDH 14.51 .004“
PGM/LDH .51 .494
PGM/PGI 6.21 .034"
0 vs 16 CON 2.43 .205 LDH/FUM 9.27 .014
PGI/FUM 5.22 .048
PGM/FUM 6.51 .031
PGI/LDH 2.02 .189
PGM/LDH .00 .993
PGM/PGI .62 .452
SHT .57 .741 LDH/FUM 2.05 .186
PGI/FUM .62 .453
PGM/FUM .01 .913
PGI/LDH 3.00 .117
PGM/LDH 2.43 .154
PGM/PGI .26 .624
LON 1.33 .410 LDH/FUM .27 .616
PGI/FUM .26 .620
PGM/FUM 2.75 .132
PGI/LDH .07 .792
PGM/LDH 2.26 .167
PGM/PGI 2.33 .161
8 vs 16 CON .26 .930 LDH/run .20 .662
PGI/FUM .57 .469
PGM/FUM ‘ .65 .441
PGI/LDH .22 .651
PGM/LDH 1.20 .302
PGM/PGI 1.44 .260
SHT 2.57 .190 LDH/FUM .18 .684
PGI/FUM 5.52 .043
PGM/FUM 1.35 .275
PGI/LDH 4.75 .057
PGM/LDH .92 .362
PGM/PGI .02 .882
LON 5.09 .069‘ LDH/FUM 19.06 .OO2**
PGI/FUM 39.30 .001‘*
PGM/FUM 5.68 .041“
PGI/LDH 11.80 .008**
PGM/LDH .02 .896
PGM/PGI 1.95 .197

'Significant at p < .10.

‘*Significant at p < .05.

82

Table l4.--Continued.

 

Multivariate Results

 

Univariate Results

 

 

Muscle Contrast Treatment Enzyme
("k“) cm“? F-Ratio P Less Than “a“ P P Less Than
Soleus 0 vs 8 CON 6.85 .042** LDH/FUM 19.51 .002“
PGI/FUM 36.18 .001**
PGM/FUM 8.64 .017**
PGI/LDH 26.57 .001**
PGM/LDH .12 .741
PGM/PGI 4.10 .074'
SHT 5.27 .065* LDH/FUM 1.75 .219
PGI/FUM 17.86 .002**
PGM/FUM 1.39 .269
PGI/LDH 8.85 .016*'
PGM/LDH 2.45 .152
PGM/PGI 30.27 .001"
LON 1.12 .479 LDH/FUM .77 .403
PGI/FUM 9.51 .013
PGM/FUM .29 .606
PGI/LDH 4.66 .059
PGM/LDH .18 .684
PGM/PGI 3.43 .097
0 vs 16 CON 10.11 0.21“ LDH/FUM 34.84 .001**
PGI/FUM 5.13 .050**
PGM/FUM .12 .739
PGI/LDH 2.61 .141
PGM/LDH 8.47 .017“
PGM/PGI .19 .675
SHT 4.31 .090* LDH/FUM 8.15 .019**
PGI/FUM 3.68 .087*
PGM/FUM 8.57 .017"
PGI/LDH .01 .918
PGM/LDH 14.69 .004**
PGM/PGI 10.16 .011**
LON 6.66 .044“ LDH/FUM 2.22 .171
PGI/FUM .44 .526
PGM/FUM 14.46 .004“
PGI/LDH 6.75 .029"
PGM/LDH 10.47 .010**
PGM/PGI 13.19 .006“
8 vs 16 CON 7.75 .034'* LDH/FUM 45.92 .001“
PGI/FUM 40.22 .001"
PGM/FUM 7.39 .024'*
PGI/LDH 13.37 .005“
PGM/LDH 1.35 .276
PGM/PGI 3.89 .080.
SET 6.30 0.48'* LDH/FUM 6.62 .030**
PGI/FUM 21.34 .001"
PGM/FUM 6.17 .035"
PGI/LDH 6.37 .033"
PGM/LDH 10.71 .010H
PGM/PGI 40.43 .001**
LON 3.05 .150 LDH/FUM .00 .988
PGI/FUM 9.00 .015
PGM/FUM 2.07 .184
PGI/LDH 10.05 .011
PGM/LDH 3.93 .079
PGM/PGI 11.69 .008

'Significant at p < .10.

1..significant at p < .05.

83

 

 

 

 

Muscle Contrast Treatment Multivariate Results Enzyme Univariate Results
("k') Group r-Ratio P Less Than Ratios P P Less Than

“White" 0 vs 8 CON 2.23 .228 LDH/FUM 5.11 .050
Vastus PGI/FUM .22 .654
Lateralis PGM/FUM 6.49 .031
PGI/LDH 7.12 .026
PGM/LDH 1.20 .302
PGM/PGI 16.28 .003

SHT 3.63 .092* LDH/FUM 10.95 .009**
PGI/FUM .03 .877
PGM/FUM 2.84 .127

PGI/LDH 10.19 .011**
PGM/LDH .00 1.000
PGM/PGI 2.08 .183
LON 3.97 .102 LDH/FUM .85 .381
PGI/FUM 5.95 .037
PGM/FUM .37 .557
PGI/LDH 14.35 .004
PGM/LDH .48 .508
PGM/PGI 28.17 .001
0 vs 16 CON 1.42 .384 LDH/FUM .86 .378
PGI/FUM 2.08 .183
PGM/FUM .77 .402
PGI/LDH .89 .370
PGM/LDH .08 .788
PGM/PGI 1.37 .272
SHT 3.01 .126 LDH/FUM .17 .691
PGI/FUM 1.73 .221
PGM/FUM .00 .948
PGI/LDH 3.19 .108
PGM/LDH .00 1.000
PGM/PGI 2.28 .165
LON 1.29 .421 LDH/FUM 1.65 .231
PGI/FUM .22 .650
PGM/FUM 1.68 .227
PGI/LDH 1.81 .211
PGM/LDH .16 .701
PGM/PGI 3.29 .103
8 vs 16 CON 2.71 .177 LDH/FUM 5.86 .039
PGI/FUM 1.26 .290
PGM/FUM 7.00 .027
PGI/LDH 3.38 .099
PGM/LDH 1.18 .305
PGM/PGI 8.46 .017
SET 1.77 .273 LDH/FUM 7.08 .026
PGI/FUM .27 .615
PGM/FUM 2.03 .188
PGI/LDH 3.50 .094
PGM/LDH .00 1.000
PGM/PGI .24 .633
LON 2.91 .160 LDH/FUM 2.08 .183
PGI/FUM 5.51 .044
PGM/FUM 1.38 .270
PGI/LDH 6.80 .028
PGM/LDH .63 .446
PGM/PGI 13.61 .005

 

*Significant at p < .10.

4'significant at p < .05.

84

identification of enzyme ratios which account for the significant

effects, is shown in Table 15.

0 vs 8 wks

A check of the multivariate F-ratios for 0 vs 8 wks revealed
significant multivariate effects for the LON group in the plantaris and
for the CON group in the soleus. Marginal significance was found for

the SHT group in both the soleus and "white" vastus lateralis muscles.

Plantaris.--Increases in the enzyme activity ratios of lactate
dehydrogenase/fumarase, phosphoglucoisomerase/fumarase, and_
phosphoglucoisomerase/lactate dehydrogenase as well as a decrease in
phosphoglucomutase/phosphoglucoisomerase all contributed to the overall
significant effect of the LON training regimen at 8 wks. Only the
phosphoglucoisomerase/lactate dehydrogenase increase appears to be

definitely attributable to training.

Soleus.--All of the variable enzyme ratios were decreased in the
soleus muscles of the CON group at 8 wks.~ In the constant-ratio organi-
zational scheme, phosphoglucoisomerase/lactate dehydrogenase was
decreased and phosphoglucomutase/phosphoglucoisomerase was increased in
the soleus muscles of the CON group at 8 wks. The changes in the soleus
associated with the SHT group at 8 wks were decreased phospho-
glucoisomerase/fumarase, decreased phosphoglucoisomerase/lactate
dehydrogenase, and increased phosphoglucomutase/phosphoglucoisomerase.
No importance can be attached to these alterations in the soleus. The
lactate dehydrogenase/fumarase and phosphoglucomutase/ fumarase results

must be discounted due to the large random variabilities observed

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'86

between groups at 0 wks. Possible measurement artifacts in phosphoglu-

coisomerase at 8 wks cast doubt on the changes in the other three ratios.

"White" Vastus Lateralis.--At 8 wks, the SHT group had a
decreased lactate dehydrogenase/fumarase ratio and an increased
phosphoglucoisomerase/lactate dehydrogenase ratio. Only the latter
effect can be ascribed to training. It shOuld be noted that both this
increase in the SHT group and the parallel nonsignificant increase in
the LON group were caused by significant training-related decreases in
lactate dehydrogenase levels between 0 and 8 wks. The seemingly
similar change in the CON group, on the other hand, was the result of

a maturative increase in phosphoglucoisomerase activity.

0 vs 16 wks

 

Significant multivariate F-values for 0 vs 16 wks were obtained
only in the soleus muscle. All three of the treatment groups exhibited
overall duration effects.

The variable enzyme ratios of lactate dehydrogenase/fumarase
and phosphoglucoisomerase/fumarase were increased in the soleus
muscles of the CON group at 16 wks. However, the constant enzyme
ratios of phosphoglucomutase/lactate dehydrogenase was decreased at
this time. These changes appear to be related to age.

All of the variable enzyme ratios in the soleus were altered
significantly in the SHT group between 0 and 16 wks. The lactate
dehydrogenase/fumarase and phosphoglucoisomerase/fumarase ratios were
increased while the phosphoglucomutase/fumarase ratio was decreased.
The constant proportion ratios of phosphoglucomutase/lactate dehydro-

genase and phosphoglucomutase/phosphoglucoisomerase both were decreased

87

approximately one-third at the termination of the study. Only the
phosphoglucomutase/phosphoglucoisomerase decrease appears to be a
training effect.

The LON group had a decreased phosphoglucomutase/fumarase ratio
in the soleus at 16 wks. The three constant-organization enzyme ratios
also were affected in the LON group. The phosphoglucoisomerase/lactate
dehydrogenase ratio was increased, whereas the changes of the other
two ratios were similar to those found in the SHT group. Again, the
only change which can be attributed to training is that which occurred

in the phosphoglucomutase/phosphoglucoisomerase ratio.

8 vs 16 wks

The multivariate F-values for 8 vs 16 wks confirmed the enzyme-
ratio changes in the plantaris muscles of the LON group to be
'marginally significant.‘ The CON and SHT groups experienced signifi-
cant enzyme-ratio changes in the soleus muscle during the latter half

of the treatment period.

P1antaris.--The three enzyme ratios of variable metabolic
organization and the phosphoglucoisomerase/lactate dehydrogenase ratio
all decreased between 8 and 16 wks as a result of the LON training

program.

Soleus.--Between 8 and 16 wks, all three of the enzyme ratios
of variable metabolic organization significantly increased in the
soleus muscles of both the CON and SHT groups except for the
phosphoglucomutase/fumarase ratio in the SHT group which decreased.

Alterations in enzyme ratios representing constant metabolic

88

organization varied considerably. The CON and SHT groups each displayed
an increase in the phosphoglucoisomerase/lactate dehydrogenase ratio

and a decrease in the phosphoglucomutase/phosphoglucoisomerase ratio
between 8 and 16 wks of training. In addition, the phosphoglucomutase/
lactate dehydrogenase ratio decreased in the SHT group. None of these

8 to 16-wk ratio changes can be ascribed definitely to training.

Discussion

 

The results of this investigation suggest that over time the
LON and SHT training programs tend to produce similar metabolic
adaptations in skeletal muscle. This observation is supported by the
large number of significant overall training (TRAIN) effects found at
16 wks and by the fact that there were no significant LON vs SHT
contrasts at that time (Tables 8 and 13). It also can be seen that at
16 wks the LON and SHT groups exhibited parallel responses across all
three muscles in all four enzymes (Fig. 4). The patterns of change
found in the enzyme ratios are nearly as conclusive (Figs. 5 and 6).
The mean value of the CON group separated those of the LON and SHT
groups at 16 wks in only two instances: phosphoglucoisomerase/lactate
dehydrogenase in the plantaris and phosphoglucomutase/lactate dehydro-
genase in the soleus (Fig. 6).

Unpublished data from a companion study tend to both confirm
and refute the theory that there may be no lasting differential effects
of the LON and SHT training regimens. The mean numbers of split fibers
in the soleus muscle, although quite different after 4 wks of training,
were similar in LON and SHT groups of animals at 8 and 12 wks. The

functional significance of fiber splitting as an adaptation to

89

training has yet to be clarified, but there is little doubt that at
least transient metabolic adjustments must take place during the
splitting phenomenon. In the same study, phosphorylase activity was
demonstrated histochemically in ten preselected areas of the plantar
flexor muscles. After 4 wks of training, histochemical photometry
revealed differences between the LON and SHT groups in two areas of the
gastrocnemius and in one area each of the plantaris and soleus muscles.
By 8 wks, the number of muscle areas in which there were LON vs SHT
differences had decreased to two. A difference in phosphorylase
activity was discernible in only one mixed-fiber area of the gastro-
cnemius at 12 wks. It is clear that these histochemical phosphorylase
observations are in general agreement with the biochemical phosphogluco-
mutase results obtained in the present investigation. In contrast to
the evidence which suggests that no differences or only temporary
differences exist between the effects of the LON and SHT programs,
other histochemical data from the companion study support the concept
of at least some continuing differential training responses. For
example, Sudan Black 8 was used to demonstrate total lipid content.
Higher values were obtained for the LON group than for the SHT group
in all ten muscle areas after 8 wks of training. Several of these
differences in lipid content were magnified at 12 wks. No 16-wk
histochemical results are available as yet.

Regardless of whether or not there are permanent differences
in the effects of the LON and SHT programs, the parameters selected
for study in this investigation did not reflect a differentiation of

metabolic activities between the two groups. Therefore, this

9O

discussion will be based chiefly upon the results obtained in the CON
and combined TRAIN groups.

Since no training had occurred at 0 wks, the means of the three
treatment groups all reflect control values, and the patterns of the
group means at that time are representative of random group variability.
Furthermore, for those parameters in which a smooth maturation effect
is not apparent in the CON group across time (Figs. 4, S, and 6),
alterations in the CON group means at 8 and 16 wks may be suspected of
being due to either random factors or measurement artifacts. These
multiple control values have been used subjectively to discriminate
between those statistically significant effects that may be due to
maturation or artifacts and those that actually appear to be caused by
training.

The pattern of responses of the TRAIN group in all muscles at
16 wks suggests a decrease in glycogenolytic capacity as measured by
phosphoglucomutase levels. In addition, increases in fumarase levels
and decreases in enzyme ratios of variable metabolic organization in
all muscles, as well as a decrease in the lactate dehydrogenase level
of the "white" vastus lateralis muscle, imply increased dependence upon
oxidative metabolism by the TRAIN group.

Burleigh and Schimke (16) have observed an inverse relationship
between phosphorylase and hexokinase activities in mammalian skeletal
muscles. A similar relationship has been found between phosphogluco-
mutase and mitochondrial hexokinase levels in rat liver, diaphragm,
kidney, heart, brain, testis, spleen, lung, small intestine, and
pancreas (Wilson, unpublished report). Thus, in this investigation the

decreased phosphoglucomutase levels in the TRAIN group might reflect

91

an increased dependence upon blood glucose as a source of carbon for
glycolysis. This hypothesis is plausible since the decreases in
phosphoglucomutase levels in the plantaris and soleus muscles of the
TRAIN group were not accompanied by decreases in the activity of
phosphoglucoisomerase. However, it should be noted that the decreases
in the variable enzyme ratios, particularly those in
phosphoglucoisomerase/fumarase, indicate that other sources of fuel
besides carbohydrates may have been at least partially responsible
for the increases in tricarboxylic acid cycle activity as measured by
fumarase. There is strong evidence from other studies that endurance
training can elicit a shift toward increased fatty acid oxidation with
subsequent participation of triglycerides as well as carbohydrates for
energy supply (71, 72, 105, 88). Unfortunately, the current data do
not provide a means of detecting alterations in beta oxidation.

Various investigators have concluded that glucose is more
important than glycogen as an energy source for contraction in red
muscle and, conversely, that glycogen is more important in white muscle
(16, 18). The decreased phosphoglucomutase results that occurred with
endurance training seem to indicate that the relative importance of
glucose utilization can be increased in all muscle fiber types. It
should be noted that the percentage decrease in phosphoglucomutase
activity was approximately the same in the three muscles of the TRAIN
group.

The decreased dependence on glycogenolysis in all muscle types
which was found in the present study is in partial disagreement with
the results of Baldwin et al. (6) who reported that endurance exercise

did not induce phosphorylase changes in white quadriceps, whole

92

quadriceps, and soleus muscles of the rat. Only the red quadriceps, a
portion of muscle with a fiber population similar to that of the
plantaris, displayed decreased levels of phosphorylase in the exercised
group. Baldwin and his coworkers also reported that decreased lactate
dehydrogenase levels occurred in red, white, and whole quadriceps
muscles as a result of training. In the current study, lower lactate
dehydrogenase activity was found only in the "white" vastus lateralis
of the TRAIN group at 16 wks.

There have been several other studies of the effects of
exercise on glycogenolytic capacity. All of these have yielded
results which conflict with the data obtained in this investigation.
No changes in phosphorylase levels were found when the exercise
regimen used was of a endurance nature (29, 43, 57). Isometric strength
exercise even produced increases in phosphorylase activity (33, 34).

Bolloszy et a1. (56) observed a 50 percent increase in
mitochondrial malate dehydrogenase activity in rat gastrocnemius muscle
after twelve weeks of long-duration endurance running. As described by
Ariano, Armstrong, and Edgerton (l), the medial and lateral aspects of
the gastrocnemius muscle have fiber-type characteristics that resemble
those of the plantaris. The reaction which fumarase catalyzes in the
tricarboxylic acid cycle is just prior to that which is catalyzed by
malate dehydrogenase. Thus, the 45 percent increase in fumarase
activity that occurred in the plantaris muscle of the LON group at
16 wks is almost an identical manifestation of training as that
previously reported.

Endurance exercise has been shown to cause a two-fold increase

in the activity of citrate synthetase, an enzyme of the tricarboxylic

93

acid cycle, and similar increases in cytochrome oxidase activity and

cytochrome c content in three types of rat muscle (4). These results

were interpreted as being incompatible with the histochemically based

hypothesis that an exercise-related increase in the oxidative capacity

of muscle may be due to a mutation of white to red fibers (9, 26, 35).

In this investigation, increases in fumarase levels of different

magnitudes were observed in the three muscles of the TRAIN group at T
16 wks. Furthermore, the fact that statistically significant decreases
in lactate dehydrogenase activity were found only in the soleus at

8 wks and in the "white" vastus lateralis at 16 wks suggests that
unequal alterations in anaerobic metabolism occurred in the three
muscles. It would appear from these results that the possibility of
fiber-type conversions in skeletal muscle cannot be discounted as yet.

For a given carbohydrate molecule, anaerobic metabolism
releases much less chemical energy than does aerobic metabolism. The
observed increases at 16 wks in fumarase activity appear to indicate
that the efficiency of energy production was increased in all three
muscles of the TRAIN group. The increased availability of energy
acquired through the tricarboxylic acid cycle might help to explain the
results of other studies in which there were no exercise-related
changes in creatine phosphokinase, adenylate kinase, and ATPase--
enzymes involved in the regeneration of ATP (50, 76, 87).

Staudte and Pette (91) suggest that deviations in the constant
proportion enzymes are possible. Kubista, Kubi§tova, and Pette (63)
administered thyroid hormone to male rats and found markedly altered
constant enzyme ratios in the soleus muscle but not in the rectus

femoris muscle. The results of the present study reinforce the

94

general concept of constant proportion enzyme groups (Fig. 6). However,
some of the constant enzyme ratios do appear to be altered either by
maturation or training (Tables 13 and 15).

The 0 vs l6-wks contrast for enzyme activities in the "white"
vastus lateralis muscle suggests that there were significant maturational
increases in lactate dehydrogenase, phosphoglucoisomerase, and phos-
phoglucomutase levels (Table 10). From inspection of the l6-wk TRAIN
vs CON contrast for this muscle, it seems that the overall training
effect was to delay the age-related increases in these enzymes.

Studies by Bass et a1. (13) and Pette (85) have shown that the
variable enzyme activity ratios can be used to classify distinct types
of muscle. Examination of these ratios in Fig. 5 illustrates their
usefulness. The highest ratios are seen in the "white" vastus
lateralis, a fast-twitch glycolytic muscle; and the lowest ratios are
seen in the soleus, a slow-twitch oxidative muscle. Intermediate
values are seen in the plantaris, a fast-twitch oxidative-glycolytic
muscle. The variable enzyme ratio data support the validity of the

histochemical fiber typing methods (1, 82).

CHAPTER V

SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS

Summary

The purpose of this study was to determine the effects of
aerobic and anaerobic programs of endurance running on selected
enzymatic activities in the left plantaris, the left soleus, and the
"white" area of the left vastus lateralis muscles of the adult male
albino rat. The training regimens were the Controlled Running Wheel
programs previously reported from this laboratory (101). Biochemical
determinations of the muscle homogenates were made on the levels of
activity of phosphoglucomutase, an enzyme of glycogenolysis: phos-
phoglucoisomerase, an enzyme of glycolysis: lactate dehydrogenase, an
enzyme concerned with anaerobic metabolism of glycolytically-formed
pyruvate; and fumarase, a mitochondrial enzyme of the tricarboxylic
acid cycle. Enzyme activity ratios also were investigated in each of
the fiber types.

Animals were randomly assigned to CON, SHT, and LON treatment
groups. Initiation of treatments for all animals began at 84 days of
age. Performance criteria were used as the basis of animal selection
for subsequent investigation. Animals were sacrificed 72 hours after

their last training period. Biochemical analyses were performed

95

 

96

before the initiation of treatments and after eight and sixteen weeks
of exercise. The final sample size consisted of 36 animals with four
animals in each treatment-duration subgroup.

The results suggested that, over time, the two training
programs tend to produce similar metabolic adaptations in skeletal
muscle. This observation is supported by the large number of signifi-
cant overall training (TRAIN) effects found at 16 wks and by the fact
that there were no significant LON vs SHT contrasts at that time.

An inverse relationship of decreased glycogenolytic capacity,
as measured by phosphoglucomutase, and increased tricarboxylic acid
cycle activity, as measured by fumarase, was evident in all muscles of
the TRAIN group at 16 wks. In addition, decreases in enzyme ratios of
variable metabolic organization in all muscles, along with a decrease
in the lactate dehydrogenase activity of the white "vastus" lateralis
muscle, imply an increased dependence upon oxidative metabolism by the
TRAIN group.

Some of the constant proportion enzyme ratios were altered
either by maturation or training. The variable enzyme ratio data

support the validity of histochemical fiber typing methods.

Conclusions

 

The results of this study have led to the following conclusions:
1. The parameters selected for study did not reflect a differ-
entiation of metabolic activities between the SHT and LON
groups.
2. ‘The pattern of responses of the TRAIN group in all muscles at

16 wks suggests a decrease in glycogenolytic capacity as

97

measured by phosphoglucomutase levels. In addition, increases
in fumarase levels and decreases in enzyme ratios of variable
metabolic organization in all muscles, as well as a decrease
in the lactate dehydrogenase level of the "white" vastus
lateralis muscle, imply increased dependence upon oxidative
metabolism by the TRAIN group.

The decreased phosphoglucomutase results that occurred with
endurance training seem to indicate that the relative importance
of glucose utilization can be increased in all muscle fiber
types.

The results of the present study reinforce the general concept
of constant proportion enzyme groups. However, some of the
constant enzyme ratios do appear to be altered either by
maturation or training.

The variable enzyme ratio data support the validity of histo-

chemical fiber typing methods.

Recommendations
Studies of the biochemical responses to specific exercise
regimens of acetylocholinesterase and related enzymes at the
motor end plate are needed.
Biochemical, histochemical, and contractile investigations of
the effects of exercise on the different types of motor units
are needed.
Studies of the hormonal influences on exercise performance and

metabolism are necessary.

98

High-intensity exercise regimens for animals should be
developed to facilitate study of the adaptations to anaerobic
training.

Power-type events such as high-jumping and weight-lifting are
needed for animals so that resultant adaptations can be
compared with those resulting from activities across the
endurance continuum.

Both biochemical and histochemical analyses are needed for
complete muscle evaluations.

The effects of specific exercise regimens should be studied
through muscle biopsys in humans. These studies should
include various age groups as well as both sexes.

In any follow-up of this investigation, mitochondrial hexo-
kinase activity should be included as a dependent variable.
The incorporation of some parameter of fatty acid oxidation

also would be beneficial.

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APPENDIX A

TRAINING PROGRAMS

APPENDIX A
TRAINING PROGRAMS
Standard Eight-Week, Short-Duration, High-Intensity

Endurance Training Program for Postpubertal and
Adult Male Rats in Controlled-Running Wheels

 

 

Total
Acc- Time Time Total
eler- Work Repeti- Bet- of Total Wbrk
Day Day ation Time Rest tions No. ween Run Prog. Exp. Time
of of Time (min: Time par of Bouts Shock Speed (min: Meters (sec)
Wk. Wk. Tr. (sec) sec) (sec) Bout Bouts (min) (ma) (m/min) sec) TEM TNT
0 4-T -2 3.0 40:00 10 l 1 5.0 0.0 27 40:00 ---. ---
S-F -1 3.0 40:00 10 1 l 5.0 0.0 27 40:00 --- -—-
1 1=M 1 3.0 00:10 10 40 3 5.0 1.2 27 49:30 540 1200
2-T 2 3.0 00:10 10 40 3 5.0 1.2 27 49:30 540 1200
3-W 3 3.0 00:10 10 40 3 5.0 1.2 27 49:30 540 1200
4-T 4 2.5 00:10 10 40 3 5.0 1.2 36 49:30 720 1200
5-F 5 2.0 00:10 10 40 3 5.0 1.2 36 49:30 720 1200
2 l-M 6 1.5 00:10 10 28 4 5.0 1.2 45 51:40 840 1120
2-T 7 1.5 00:10 15 27 4 5.0 1.2 54 59:00 972 1080
3-W 8 1.5 00:10 15 27 4 5.0 1.2 54 59:00 972 1080
4-T 9 1.5 00:10 15 27 4 5.0 1.2 54 59:00 972 1080
5-F 10 1.5 00:10 15 27 4 5.0 1.2 54 59:00 972 1080
3 1-M 11 1.5 00:10 15 27 4 5.0 1.2 54 59:00 972 1080
2-T 12 1.5 00:10 20 23 4 5.0 1.2 63 59:40 966 920
3-W 13 1.5 00:10 20 23 4 5.0 1.2 63 59:40 966 920
4-T 14 1.5 00:10 20 23 4 5.0 1.2 63 59:40 966 920
S-F 15 1.5 00:10 20 23 4 5.0 1.2 63 59:40 966 920
4 l-M 16 1.5 00:10 20 23 4 5.0 1.2 63 59:40 966 920
2-T 17 1.5 00:10 25 20 4 5.0 1.0 72 60:00 960 800
3dW 18 1.5 00:10 25 20 4 5.0 1.0 72 60:00 960 800
4-T 19 1.5 00:10 25 20 4 5.0 1.0 72 60:00 960 800.
S-F 20 1.5 00:10 25 20 4 5.0 1.0 72 60:00 960 800
5 l-M 21 1.5 00:10 25 20 4 5.0 1.0 72 60:00 960 800
2-T 22 1.5 00:10 30 16 4 5.0 1.0 81 55:40 864 640
3-W 23 1.5 00:10 30 16 4 5.0 1.0 81 55:40 864 640
4-T 24 1.5 00:10 30 16 4 5.0 1.0 81 55:40 864 640
5=F 25 1.5 00:10 30 16 4 5.0 1.0 81 55:40 864 640
6 l-M 26 1.5 00:10 30 16 4 5.0 1.0 81 55:40 864 640
2-T 27 2.0 00:10 35 10 5 5.0 1.0 90 54:35 750 500
3-W 28 2.0 00:10 35 10 5 5.0 1.0 90 54:35 750 500
4-T 29 2.0 00:10 35 10 5 5.0 1.0 90 54:35 750 500
58? 30 2.0 00:10 35 10 5 5.0 1.0 90 54:35 750 500
7 1-M 31 2.0 00:10 35 10 5 5.0 1.0 90 54:35 750 500
2-T 32 2.0 00:10 35 7 8 2.5 1.0 90 54:50 840 560
3dW 33 2.0 00:10 35 7 8 2.5 1.0 90 54:50 840 560
4-T 34 2.0 00:10 35 7 8 2.5 1.0 90 54:50 840 560
S-F 35 2.0 00:10 35 7 8 2.5 1.0 90 54:50 840 560
8 l-M 36 2.0 00:10 35 7 8 2.5 1.0 90 54:50 840 560
2-T 37 2.0 00:10 40 6 8 2.5 1.0 99 52.10 792 480
3-W 38 2.0 00:10 40 6 8 2.5 1.0 99 52:10 792 480
4-T 39 2.0 00:10 40 6 8 2.5 1.0 99 52:10 792 480
5-F 40 2.0 00:10 40 6 8 2.5 1.0 99 52:10 792 480

 

This standard program was designed using male rats of the Sprague-Dawley strain. All
animals were between 70 and 170 days-of-age at the beginning of the program. The duration and
intensity of the program were established so that 75 per cent of all such animals should have PSF
and PER scores of 75 or higher during the final two weeks. Alterations in the work time, number
of bouts, or time between bouts can be used to affect changes in these values. Other strains or
ages of animals could be expected to respond differently to the program.

All animals should be exposed to a minimum of one week of voluntary running in a wheel
prior to the start of the program. Failure to provide this adjustment period will impose a double
learning situation on the animals and will seriously impair the effectiveness of the training
programs.

108

1(19

Standard Eight-Week, Long-Duration, Low-Intensity
Endurance Training Program for Postpubertal and
Adult Male Rats in Controlled-Running Wheels

 

 

Total
Acc- Time Time Total
eler- Work Repeti- Bet- of Total Work
Day Day ation Time Rest tions No. ween Run Prog. Exp. Time
of of Time (min: Time per of Bouts Shock Speed (min: Meters (sec)
Wk. Wk. Tr. (sec) sec) (sec) Bout Bouts (min) (ma) (m/min) sec) TEM TWT
0 4-T -2 3.0 40:00 10 l 1 5.0 0.0 27 40:00 --- ---
Sn? -1 3.0 40:00 10 1 1 5.0 0.0 27 40:00 --- -—-
l l-M 1 3.0 00:10 10 40 3 5.0 1.2 27 49:30 540: 1200
2=T 2 3.0 00:10 10 40 3 5.0 1.2 27 49:30 540 1200
3=W 3 3.0 00:10 10 40 3 5.0 1.2 27 49:30 540 1200
4=T 4 2.5 00:20 10 30 2 5.0 1.2 27 34:40 540 1200
5a? 5 2.5 00:30 15 20 2 5.0 1.2 27 34:30 540 1200
2 1=M 6 2.0 00:40 20 15 2 5.0 1.2 36 34:20 720 1200
2=T 7 2.0 00:50 25 12 2 5.0 1.2 36 34:10 720 1200
3iW 8 1.5 01:00 30 10 2 5.0 1.2 36 34:00 720 1200
4=T 9 1.5 02:30 60 4 2 5.0 1.2 36 31:00 720 1200
S-F 10 1.0 02:30 60 4 2 5.0 1.2 36 31:00 720 1200
3 1-M 11 1.0 02:30 60 4 2 5.0 1.2 36 31:00 720 1200
2=T 12 1.0 05:00 0 1 5 2.5 1.2 36 35:00 900 1500
3-W 13 1.0 05:00 0 1 5 2.5 1.2 36 35:00 900 1500
4=T 14 1.0 05:00 0 l 5 2.5 1.2 36 35:00 900 1500
5=F 15 1.0 05:00 0 1 5 2.5 1.2 36 35:00 900 1500
4 lnM 16 1.0 05:00 0 1 5 2.5 1.2 36 35:00' 900 1500
2=T 17 1.0 07:30 0 l 4 2.5 1.0 36 37:30 1080 1800
3=W 18 1.0 07:30 0 1 4 2.5 1.0 36 37:30 1080 1800
4-T 19 1.0 07:30 0 l 4 2.5 1.0 36 37:30 1080 1800
5=F 20 1.0 07:30 0 l 4 2.5 1.0 36 37:30 1080 1800
5 1=M 21 1.0 07:30 0 1 4 2.5 1.0 36 37:30 1080 1800
2=T 22 1.0 07:30 O 1 5 2.5 1.0 36 47:30 1350 2250
3=W 23 1.0 07:30 0 1 S 2.5 1.0 36 47:30 1350 2250
4-T 24 1.0 07:30 0 l 5 2.5 1.0 36 47:30 1350 2250
S-F 25 1.0 07:30 0 1 5 2.5 1.0 36 47:30 1350 2250
6 1=M 26 1.0 07:30 0 l 5 2.5 1.0 36 47:30 1350 2250
2-T 27 1.0 10:00 0 l 4 2.5 1.0 36 47:30 1440 2400
3=W 28 1.0 10:00 0 l 4 2.5 1.0 36 47:30 1440 2400
4-T 29 1.0 10:00 0 1 4 2.5 1.0 36 47:30 1440 2400
5=F 30 1.0 10:00 0 l 4 2.5 1.0 36 47:30 1440 2400
7 1-M 31 1.0 10:00 0 1 4 2.5 1.0 36 47:30 1440 2400
2-T 32 1.0 10:00 0 1 5 2.5 1.0 36 60:00 1800 3000
3=W 33 1.0 10:00 0 1 5 2.5 1.0 36 60:00 1800 3000
4=T 34 1.0 10:00 0 l 5 2.5 1.0 36 60:00 1800 3000
5-F 35 1.0 10:00 0 1 5 2.5 1.0 36 60:00 1800 3000
8 1-M 36 1.0 10:00 0 1 5 2.5 1.0 36 60:00 1800 3000
2=T 37 1.0 12:30 0 1 4 2.5 1.0 36 57:30 1800 3000
3=W 38 1.0 12:30 0 1 4 2.5 1.0 36 57:30 1800 3000
4=T 39 1.0 12:30 0 1 4 2.5 1.0 36 57:30 1800 3000
SaF 40 1.0 12:30 0 l 4 2.5 1.0 36 57:30 1800 3000

 

This standard program was designed using male rats of the Sprague-Dawley strain. All
animals were between 70 and 170 days-of-age at the beginning of the program. The duration and
intensity of the program were established so that 75 per cent of all such animals should have PSF
and PER scores of 75 or higher during the final two weeks. Alterations in the rest time,
repetitions per bout, number of bouts, or time between bouts can be used to affect changes in
these values. Other strains or ages of animals could be expected to respond differently to the
program.

All animals should be exposed to a minimum of one week of voluntary running in a wheel
prior to the start of the program. Failure to provide this adjustment period will impose a

double learning situation on the animals and will seriously impair the effectiveness of the
training program.

   

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