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Metabolic Research Centre, University of Wollongong, NSW, Australia,
*
Copenhagen Muscle Research Centre, August Krogh Institute, University of Copenhagen, Denmark, and
Department of Biological Sciences, University of Wollongong, NSW, Australia
2To whom correspondence should be addressed at Copenhagen Muscle Research Centre, Dept. Human Physiology, Universitetsparken 13, DK-2100 Copenhagen Ø, Denmark. E-mail: Jhelge{at}aki.ku.dk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • training • fatty acid • rat • diet • phospholipid • fiber types • insulin action
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
The control and regulation of membrane phospholipid composition are not
clearly understood; however, dietary fatty acid profile significantly
influences the incorporation of fatty acids into the membrane in both
rats (Ayre and Hulbert 1996
, Pan and Storlien 1993
) and humans (McMurchie et al. 1996
). In
rats these changes in muscle membrane fatty acid composition occur
within days, and remodeling approaches a plateau within 3 to 4 wk (Pan
and Storlien, unpublished observations). The membrane fatty acid
composition is a dynamic system, which constantly adapts to changes in
its precursor, the endogenous and exogenous fatty acid supply.
Therefore factors which affect the drainage of the precursor pool could
influence membrane phospholipid fatty acid composition. Evidence
suggests that fatty acids are selectively recruited from fat depots
(Raclot and Groscolas 1993
, Raclot et al. 1997
) and oxidized preferentially in both rats (Leyton et al. 1987
) and humans (Jones et al. 1985
).
Consistent with this, the lipolytic stimulus of vigorous exercise also
appears to selectively mobilize unsaturated fatty acids (Mougios et al. 1995
).
During low to moderate intensity, exercise induced fat oxidation
contributes significantly to energy turnover, and it is possible that a
recurring preferential recruitment and oxidation of fatty acids could
modify the fatty acid composition of adipose tissue and fat stored
within the muscle, and subsequently affect muscle membrane phospholipid
fatty acid composition. In humans, Thomas and colleagues
(1977)
found a small but significant decrease in the content of
palmitate and a borderline significant increase in the sum of C18–20
fatty acids in vastus lateralis muscle, when endurance-trained and
sedentary males were compared. More recent work by Andersson and
colleagues (1998)
demonstrated that 6 wk of low-intensity
exercise training resulted in significant changes in muscle
phospholipid fatty acid composition with a significant increase in
oleic acid and a decrease in arachidonic acid. Presently only limited
information is available showing how changes in dietary fatty acid
supply interact with regular exercise and whether this affects muscle
phospholipid fatty acid composition.
The aim of this study was to investigate the effects of, and interactions between, exercise training and dietary fatty acid profile on skeletal muscle phospholipid fatty acid composition in rats.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The University of Wollongong Animal Experimentation Ethics Committee approved the experiment. Rats (n = 62) were randomly allocated to one of three dietary groups and consumed either a saturated high-fat diet (n = 22, SAT)3 , a monounsaturated high-fat diet (n = 20, MONO) or a high carbohydrate diet (n = 20, CHO). Each dietary group was randomly divided into two subgroups, of which one was trained and the other remained sedentary. After 4 wk, rats were anesthetized and muscle tissue excised.
Animal care.
Male Wistar rats (initial wt. 240–270 g) were acquired from the Animal
Resource Center (Perth, Australia) and housed three per cage at 21
± 2°C (12:12-h light/dark cycle, 1800–0600 h) for 1 wk before
random allocation to three dietary groups. Each group was fed one of
three diets for 4 wk. The diets were a high-fat diet rich in
saturated fatty acids [65% of total energy (E%) fat, 20 E% protein,
15 E% carbohydrate], a high-fat diet rich in MONO fatty acids (65
E% fat, 20 E% protein, 15 E% CHO) and a high CHO (10 E% fat, 20 E%
protein, 70 E% CHO) (Table 1
).The fatty acid compositions of each of the three diets are listed in
Table 2.
Rats were given free access to food and water. During the experimental
period, body weight was recorded weekly.
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Each dietary group was randomly divided into two subgroups, one of which was trained for 4 wk; the other group remained sedentary. Rats were trained in the hours before the dark cycle 6 d/wk on a custom-built nine-lane motorized treadmill equipped with a rear electrical shock grid. Training was initiated at 15 min, 20 m/min, 0 degrees incline and in the course of 2 wk speed, incline and duration were progressively increased such that the rats were running for 60 min at 28 m/min on a 5° incline at the end of the second week. Over the third week the incline was gradually increased to 10° and the final load, 60 min at 28 m/min on a 10° incline, was maintained until the experiment was terminated. To avoid possible effects of routine handling, sedentary rats were habituated to the treadmill 3 d/wk for 10 min at 0° incline and at the same speed as the trained rats. After the completion of the training program, a total of seven animals had to be discarded from the trained groups (four SAT, two MONO, one CHO), since more than 5% of total running time was lost due to injured feet or toe nails.
Tissue sampling procedures.
At the conclusion of the experiment, all rats were food-deprived for 5 h during the morning and were then anesthetized by intraperitoneal injection of pentobarbitol (60 mg/100 g body wt of Nembutal, Abbott, Sydney, Australia) during the early afternoon to avoid diurnal effects. Red quadriceps, white quadriceps and soleus muscles were removed, freeze clamped and quickly stored in liquid nitrogen and subsequently the heart was stopped. All tissue was stored at -80°C until analyzed.
Biochemical analysis.
All red quadriceps muscle was analyzed, and this analysis was
complemented by a random selection of four white quadriceps and four
soleus from each of the six groups. Extraction and derivatization of
the fatty acid components of muscle phospholipids were described in
full detail elsewhere (Pan and Storlien 1993
). In brief,
muscle tissue was homogenized in 2:1 (vol/vol) chloroform/methanol and
total lipid extracts prepared according to Folch et al. (1957)
. Phospholipids were isolated from less polar lipids by
solid-phase extraction on Sepp-Pak silica cartridges (Waters,
Milford, MA). The phospholipids were then transmethylated and the
methyl fatty acids separated, identified and quantified by gas
chromatography. The content of individual fatty acids in the
phospholipids extracted from the muscles was expressed as a percentage
of the total fatty acids that were identified. Individual fatty acids
that made up less than 1% of the total in all groups are not shown in
the tables but are included in the statistical analyses. Two fatty acid
indices were derived from these results; the average degree of
unsaturation, the Unsaturation Index (UI, was calculated as the average
number of double bonds per fatty acid residue multiplied by 100) and
the total percentage of long-chain polyunsaturated fatty acids
(PUFA) with 
20 carbon units (
C20–22 PUFA). Furthermore, the
activity of one of the enzymes involved in the fatty acid biosynthesis,
5-desaturase, was estimated according to the product-precursor
ratio between arachidonic acid [20:4(n-6)] and eicosatrienoic acid
[20:3(n-6)].
Statistics.
Results are expressed as means ± SEM. Statistical analyses were performed using the SAS system release 6.10 (SAS Institute Inc. Cary, NC). Three-way analysis of variance (ANOVA) with diet (CHO, MONO, SAT), exercise (training-sedentary) and muscle (red quadriceps, white quadriceps and soleus) as factors was applied to the data set. Due to the number of samples analyzed, this is an unbalanced design and is thus susceptible to interpretational problems. To circumvent this problem, two-way ANOVA using diet and exercise as factors was applied to the data set for each muscle. In this way the data were evaluated in a balanced design. Wherever two-way ANOVA revealed significant effects, a Student-Newman-Keuls test was used to discern differences between groups. Statistical significance of differences was set at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The rats were not kept in metabolic cages, and thus diet intake was not measured. There may have been somewhat more energy consumed in rats fed fat- compared to carbohydrate-rich diet. But since body weight increased similarly in the groups, this would indicate that energy intake and thus nutrient intake was approximately similar. A difference in diet intake would also lead to a difference in vitamin and mineral intake; however, since the rats should be more than safely above minimum requirements, this should not be an issue. Furthermore, the key issue of the study is the activity effect, which is within the dietary groups.
There was a significant effect of regular exercise on all muscles
analyzed for linoleic acid [18:2(n-6)], arachidonic acid
[20:4(n-6)], docosahexaenoic acid [22:6(n-3)], the sum of
long-chain polyunsaturates (20–22 PUFA), the unsaturation index
and the 5-desaturase activity (Table 3
).The relative difference in two representative fatty acids and two
composite measures of fatty acid composition by regular exercise
training are shown in Figure 1.
Since different numbers of samples were analyzed between muscle groups,
two-way ANOVA was also performed on each of the three muscles to
complement the statistical analysis. The individual membrane
phospholipid fatty acid composition of the three muscles analyzed, red
quadriceps, white quadriceps and soleus, are tabulated in Tables 4
, 5
and 6,
respectively. In each of the three muscles analyzed, regular exercise
induced a significantly higher relative content of linoleic
[18:2(n-6)], a lower content of arachidonic [20:4(n-6)] (in white
quadriceps it tended to be lower P = 0.07) and a lower
amount of polyunsaturated long-chain fatty acids (Tables 4
, 5
and 6)
. The UI was significantly lower in the trained animals for red and
white quadriceps, but not for soleus (Tables 4
and 5)
. In red
quadriceps muscle regular exercise also induced a significantly lower
relative content of palmitic acid (16:0), a higher content of
stearic acid (18:0) and a lower activity of the 5-desaturase
(Table 4)
. These changes were not present in white quadriceps or
soleus, possibly due to the smaller number of muscles analyzed in each
group (n = 4), as opposed to the red quadriceps
(n = 8–10).
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20–22 PUFA), the ratio
of (n-6) to (n-3) fatty acids [(n-6)/(n-3)] and the 5-desaturase
activity were all higher in white quadriceps than in red quadriceps and
soleus (P < 0.05). There were also significant
differences between red quadriceps and soleus; however most of them
were smaller in magnitude than when either muscle was compared to white
quadriceps.
The dietary fatty acid composition significantly affected the
phospholipid fatty acid composition in the muscles (Tables 4
, 5
and 6)
.
The sum of saturated fatty acids was significantly higher in rats fed
the SAT compared to the MONO and CHO diet for each of the three muscles
(Tables 4
, 5
and 6)
. As expected from the fatty acid profile of the
MONO diet (Table 2)
, the individual contents of oleic acid
[18:1(n-9)] and vaccenic acid [18:1(n-7)] were significantly higher
in rats fed the MONO than the SAT diet in each of the three muscles
(Tables 4
, 5
and 6)
. The CHO and MONO diets both contained canola oil
as the only fat source, and thus by design the dietary fatty acid
profiles were similar, if not identical (Table 2)
. However since the
absolute fat intake was dramatically different (42 vs 400 g/kg diet in
rats fed the CHO and fat diets, respectively), the role of the
endogenous fatty acid synthesis complicates the interpretation of a
pure diet effect. It is noteworthy that the content of palmitic acid
(16:0), one of the major endogenously synthesized fatty acids, was
significantly higher in rats fed the CHO than in those fed the
fat-rich diets in all three muscles (Tables 4
, 5
and 6)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In skeletal muscle, regular exercise induces a multitude of adaptations
that enhances the oxidative capacity of the muscle cell and its ability
to maintain homeostasis during sustained stress (Saltin and Gollnick 1983
). Homeostasis is linked to the transport and
maintenance of ion and metabolite concentrations within the cell, both
of which are influenced by membrane function, which subsequently is
influenced by membrane phospholipid fatty acid composition. In this
study, regular exercise-induced changes in the muscle membrane
phospholipid fatty acid profile and should thus be considered as a
modulator of muscle phospholipid fatty acid composition.
Two studies in humans addressed a similar issue to this present study.
A recent report by Andersson and colleagues (1998)
demonstrated that 6 wk of low-intensity exercise training resulted
in a larger decrease in arachidonic acid [20:4(n-6)] and a larger
increase in oleic acid [18:1(n-9)] when compared to the changes in a
sedentary group consuming the same diet. This change matched the
training-induced decrease in the membrane content of arachidonic
acid observed in the present study; however, training increased oleic
acid membrane content in the human training study compared to an
increase in linoleic acid [18:2(n-6)] in the present study.
The results of Andersson et al. (1998)
and the present
study can be contrasted to data from Thomas and colleagues
(1977)
. In a cross-sectional design of trained vs.
untrained individuals with no dietary control, a small but significant
decrease in the content of palmitate and a borderline significant
increase in the sum of C18–20 in vastus lateralis muscle were apparent
in trained compared to sedentary males. Dietary fatty acid composition
is a potent moderator of membrane fatty acid composition (Pan and Storlien 1993
). Possibly in the Thomas study the trained
individuals were eating a lower saturated/higher unsaturated fat diet
(Thomas et al. 1977
). This suggests the interesting
possibility that individuals who exercise regularly may compensate for
the increased utilization of unsaturated fatty acids by modulating
dietary fatty acid profile. They may do this merely as an associated
lifestyle change or it may reflect a homeostatic regulatory mechanism.
This latter possibility could be tested in experimental animals by
allowing access to diets with differing fatty acid unsaturation
concomitant with a training program.
One other study in experimental animals has looked at voluntary
"wheel running" exercise and muscle membrane lipid composition.
Kriketos and co-workers (1995)
found no changes in
phospholipid fatty acid composition in extensor digitorum longus muscle
and only a significant decline in docosahexaenoic acid [22:6(n-3)] in
soleus muscle in trained vs. untrained female rats. The differences
between that study and the current one may reflect differences in the
mode of exercise, with wheel exercise being done in short,
high-intensity bursts which may utilize glucose almost entirely for
fuel.
This study does not provide direct evidence to clarify the mechanism
explaining an independent effect of exercise on muscle phospholipid
fatty acid profile. However, as noted previously, numerous studies have
now shown that lipolytic stimuli, including exercise, preferentially
mobilize unsaturated fatty acids from fat depots (Raclot and Groscolas 1993
); in addition, these unsaturated fatty acids are
more likely to be oxidized for energy in both rats (Leyton et al. 1987
) and humans (Jones et al. 1985
).
Arguably, some correlate of physical activity rather than the activity
itself could be responsible for the changes in phospholipid fatty acid
composition. Gudbjarnason (1989)
showed that catecholamine stress can
alter the fatty acid composition of cardiac phospholipids. However in
that study catecholamine stress caused an increase in arachidonic acid
[20:4(n-6)] and docosahexaenoic acid [22:6(n-3)] content, and a
decrease in linoleic acid [18:2(n-6)] content of cardiac
phospholipids. This is exactly the opposite of the changes we observed
in skeletal muscle following regular exercise training in the current
study, and thus it appears unlikely that the changes we observed are
due to catecholamine stress during the training regime.
Another possible mechanism underlying the changes in fatty acid
composition after training is an increased lipid peroxidation caused by
the increased exposure to reactive oxygen species produced during
regular exercise (Dillard et al. 1978
). Evidence from
red blood cells indicates that phospholipids containing PUFA are more
susceptible to lipid peroxidation (Chiu et al. 1989
),
and one study found that exercise affected the fatty acid profile in
rat muscle mitochondria (Dohm et al. 1975
). However more
studies are needed to determine to what extent exposure to different
modes, duration and intensities of exercise will lead to significant
lipid peroxidation.
An interesting aspect of this study is the observed difference between
muscle types in phospholipid fatty acid composition. The white
quadriceps (mainly fast twitch glycolytic fibers) had a significantly
higher degree of unsaturation, indicated by a higher UI, a higher
content of C20–22 PUFA, and a higher 5-desaturase activity than
either soleus (mainly slow oxidative fibers) or red quadriceps (a
mixture of slow oxidative and oxidative glycolytic fibers) muscle. From
the discussion above, it is tempting to speculate that the higher
degree of unsaturation in the white quadriceps relates to the lower
turnover of fat, and thus oxidation of unsaturated fatty acids, that is
inherent with the fast twitch glycolytic fibers. However, we could also
speculate that the increased unsaturation seen in the muscle with
predominantly fast twitch fibers may relate to the necessity for rapid
ion flux. It has been shown that the intrinsic activity of the
sodium-potassium pump is increased in a more unsaturated lipid
environment (Dr. Paul Else, personal communication).
In the literature it is well-documented that insulin sensitivity is
related to muscle phospholipid fatty acid composition (Borkman et al. 1993
), and it was demonstrated that a reduction in
C20–22 PUFA (C20–22, PUFA) in muscle membranes is linked to an
impaired insulin action (Pan et al. 1995
). In the
present study the decreased unsaturation and content of C20–22 fatty
acids in the membrane indicate that training might attenuate the
exercise-induced improvement in insulin action through an effect on
the skeletal muscle membrane lipid profile. Further studies must
investigate the role of exercise intensity in this effect on membrane
fatty acid composition and possibly insulin sensitivity. In any case,
attention to the dietary lipid profile, emphasizing intake of PUFA
during a training program, may be important to achieve the full
potential for individuals with predisposition to, or already existing,
obesity and diabetes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: CHO, carbohydrate rich diet;
E%, percent of total energy; MONO, fat-rich diet containing
predominantly monounsaturated fatty acids; PUFA, polyunsaturated
long-chain fatty acids; SAT, high-fat diet containing
predominantly saturated fatty acids; UI, Unsaturation Index.
Manuscript received January 22, 1999. Initial review completed March 11, 1999. Revision accepted June 16, 1999.
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McMurchie E. J., Margetts B. M., Beilin L. J., Croft K. D., Vandongen R., Armstrong B. Dietary-induced changes in the fatty acid composition of human cheek cell phospholipids: correlation with changes in the dietary polyunsaturated/saturated fat ratio. Am. J. Clin. Nutr. 1996;39:975-980
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(P < 0.01) CHO vs. SAT; $ (P < 0.01) SAT vs. (CHO and
MONO).