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Effect of aspartate, asparagine, and

carnitine supplementation in the diet
on metabolism of skeletal muscle
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Dulcineia Abdalla
Physiology & Behavior

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 Physiology& Behavior,Vol. 57, No. 2, pp. 367-371, 1995
Pergamon Copyright© 1995ElsevierScienceLtd
Printedin the USA.All rightsreserved
0031-9384/95$9.50 + O0
0031-9384(94)00243-6

Effect of Aspartate, Asparagine, and Carnitine

Supplementation in the Diet on Metabolism of
Skeletal Muscle During a Moderate Exercise
A. H. L A N C H A , JR.,*' M. B. RECCO,* D. S. P. A B D A L L A t A N D R. CURI:~

*Physical Education School, Biodynamic Department, tPharmaceutical Sciences Faculty, and ¢Institute of
Biomedical Sciences, Physiology and Biophysics Department, Sro Paulo Universi~, Brasil

Received 12 January 1994

LANCHA, JR., A. H., M. B. RECCO, D. S. P. ABDALLA AND R. CURl. Effect of aspartate, asparagine, and carnitine
supplementation in the diet on metabolism of skeletal muscle during a moderate exercise. PHYSIOL BEHAV 57(2) 367-371,
1995.--The present study examined the effect of diet supplementationof oxaloacetate precursors (aspartate and asparagine) and
carnitine on muscle metabolism and exercise endurance. The results suggest that the diet supplementationincreased the capacity
of the muscle to utilize FFA and spare glycogen. Time to exhaustion was about 40% longer in the experimentalgroup compared
to the control, which received commercial diet only. These findings suggest that oxaloacetate may be important to determine the
time to exhaustion during a prolonged and moderate exercise.

Exercise endurance Oxaloacetateprecursors Muscleglycogen

Glycemia Bloodlactate and plasma free fatty acids

DURING moderate physical effort (aerobic exercise), type I
fibres are constantly used. This type of muscle fibre shows a
very high content of mitochondria compared to types IIa and
IIb, and presents a high activity of the Krebs cycle. The bio-
chemical characteristics of red muscle allow the utilization of
free fatty acids as the major fuel during a prolonged exercise
(19,21). Surprisingly, however, the time of exhaustion, under
these conditions, is directly related to the muscle glycogen
content at rest (2,12). In fact, the total amount of glycogen in
the muscles of the whole body is enough to provide energy
for 71 min of exercise only (17). As a consequence, it is un-
likely that the energy required by marathon runners, for ex-
ample, should be provided solely by glycogen. Therefore, the
relationship between muscle glycogen content and exercise
endurance remains unclear.
Several studies have shown significant activity of pyruvate
carboxylase in the muscle (22). This enzyme forms oxaloacetate
from pyruvate and is stimulated by acetyl coenzyme A (acetyl-
coA) and ATP in the liver and isolated pancreatic islets (7). The
condensation of oxaloacetate and acetyl-coA to citrate by citrate
synthase initiates the Krebs cycle. This is a key reaction for ox-
idation of acetyl-coA either from pyruvate through pyruvate de-
hydrogenase or oxidation of fatty acids. Therefore, the generation
of oxaloacetate represents a limiting step in the control of oxi-
dation of fatty acids. Studies of Rowan et al. (21) and those of
our laboratory (13) have found that muscle pyruvate carboxylase
activity does increase during exercise. These findings suggest
that pyruvate carboxylase may play a key role to control the
Krebs cycle activity during prolonged exercise. These data led
us to speculate whether glycogen improves endurance to exercise
by provision of oxaloacetate.
Recently, Miller et al. (14) demonstrated that sedentary rats
have low plasma levels of asparagine, aspartate, and carnitine
compared to exercise-trained rats. In the same study, it was found
that plasma levels of asparagine, aspartate, and carnitine mark-
edly decrease after exhaustion to exercise, compared to the rest
condition, in exercised-trained rats. These findings suggest that
these amino acids may be used to provide oxaloacetate in the
skeletal muscles (15). In fact, it has been shown that aspartate
amino transferase activity increases with exercise (18).
The present study was carried out to futher examine the hy-
pothesis that oxaloacetate might play a key role for the estab-
lishment of exhaustion in rats submitted to prolonged exercise,
as was also suggested in our previous report (13). For this pur-
pose, rats fed a commercial diet received a supplementation of
carnitine, asparagine, and aspartate in the drinking water. Car-
nitine was also added to the diet because this metabolite is a
limiting factor for the transport and oxidation of fatty acids (23),
so the maximal capacity of fatty acids oxidation under all con-
ditions was ensured. Aspartate and asparagine are precursors of
oxaloacetate (17). The experiments aimed to increase the amount
of oxaloacetate available and to improve the biochemical capac-

Requests for reprints should be addressed to Antonio Herbert Lancha, Jr., Physical Education School, S~o Paulo University, Av. Prof. Mello de
Moraes 65, 05508-900-CidadeUniversitSria,S~o Paulo, Brazil.

367
368
ity of the muscle for oxidation of fatty acids through the Krebs
cycle.
METHOD
Animals
Male albino Wistar rats weighing 250 g were obtained from
the Department of Physiology and Biophysics, Institute of Bio-
medical Sciences, USP, S~o Paulo. The rats were housed five to
a cage, kept at 23°C, light/dark cycle of 12/12 h, and fed a diet
consisting of approximately 52% carbohydrate, 21% protein, and
4% fat (Nuvilab CR 1, Nuvital Nutrients ltda. Estrada Ribeira
3001, Km 3, Curitiba, Brasil).
Chemicals and Enzymes
All chemicals for buffer preparation were obtained from Re-
agen, S~o Paulo. Lactate dehydrogenase, NAD, DTNB (5,5-di-
thio-bis-2-nitrobenzoicacid), Triton X-100, pyruvate and acetyl-
coA were obtained from Sigma Chemical (St. Louis, MO).
Analysis of Plasma Amino Acids
Blood was collected in heparinized tubes and a pool of plasma
from six rats per group was prepared. Plasma was treated with
sulfossalicylic acid (50 mg per ml), homogenized, and centri-
fuged at 2500 rpm. The supernatant was separated, filtered (0.22
/~m), and injected in a Beckman amino acid autoanalyser (7200
model).
Diet Supplementation
The control group (C) received the commercial chow only,
whereas the supplemented diet group (S) received aspartate (45
mg. kg- ~"day- ~), asparagine (45 mg. kg- ~.day- 1), and carnitine
(90 mg'kg-~'day -~) in the drinking water. Both groups were
submitted to the following conditions: a) no activity (sedentary
rats), b) spontaneous physical activity in a rotating wheel cage,
and c) swimming training for 5 weeks. Swimming-trained rats
were then studied: a) just before the habitual daily exercise to
investigate the training effect, b) after 1 h of exercise; and c) after
exhaustion due to exercise. Body weight gain and daily food
intake were also determined in all groups.
Sedentary Condition
The rats received the two types of diets during 5 weeks and
were maintained in a cage (17 cm high, 33 cm large, and 44 cm
lenght) all the time.
Spontaneous Physical Activity
The rats were kept individually in a rotating wheel cage (24)
during 5 weeks. This condition allows the rats to develop a highly
aerobic exercise.
Swimming Training
To familiarize the rats with the swimming system (4), the rats
exercised for 5 days with a growing load that finally reached 5%
of body weight, allowing an aerobic physical effort of about 6 0 -
70% (data obtained from preliminary experiments). Afterwards,
the two groups (C and S) were trained 1 h daily, 5 days a week,
for 5 weeks. Rats trained to swimming were then killed at rest,
after 1 h of exercise and after exhaustion had been reached.
Swimming was always performed at a water temperature of 32°C
between 1100 and 1300 h. The 5% body weight load was the one
used during all experiments.
LANCHA, JR. ET AL.
Determination of the Time of Exhaustion
The rats were assumed to be exhausted by swimming because
they lost the capacity to keep the nose off the water. At this stage,
the time to exhaustion was recorded and the rats were immedi-
ately killed.
Experimental Procedure
The animals were always killed by decapitation between 1100
and 1300 h. Whole blood was collected for the measurements of
glucose, free fatty acids (FFA), and lactate. The liver, gastroc-
nemius (white portion), and soleus muscles were removed for
determination of glycogen. Citrate synthase activity was mea-
sured as an indication of the efficiency of the schedule of exercise
training imposed.
Blood Measurements
Whole blood glucose was determined by the method of Du-
bowiski (8), lactate according Engle and Jones (9), and free fatty
acids (FFA) by the method of Falholt et al. (10).
Glycogen Content in Muscles and Liver
Glycogen content in soleus and gastrocnemius muscles and
the liver was determined by the method of Hassid and Abrahams
(11).
Citrate Synthase Assay
Soleus and gastrocnemius muscles were dissected, freeze-
clamped, and stored in liquid nitrogen prior to enzyme assays.
Preliminary studies showed that this treatment did not affect the
maximum activity of the enzyme. The tissues were homogenized
in a Polytron (PCU-2 at position 3/4). The extraction medium for
citrate synthase (EC.4.1.3.7) contained 50 mM Tris-HC1 and 1
mM EDTA; the final pH was 7.4.
Citrate synthase activity was measured as previously de-
scribed (5,6). For all enzyme assays, 0.05% Triton X-100 was
added to the system to complete the extraction of the enzyme.
The final volume of the assay mixtures in all cases was 1.0 ml.
Citrate synthase was assayed by following the rate of change in
A-412. All spectrophotometric measurements were performed in
a Gilford recorder spectrophotometer (Model Response) at 25°C.
For the enzyme studied, preliminary experiments established
that extraction and assay procedures produced maximum enzyme
activities, as described by Crabtree and Newsholme (3). The ac-
tivities of citrate synthase are expressed as mol of substrate uti-
lized per minute per gram of fresh weight.
Statistical Analysis
Statistical differences between controls and diet-supple-
mented rats were assessed by the Student's t-test as indicated in
the tables. Significance was related to a value of p < 0.05.
RESULTS AND DISCUSSION
Body weight gain and daily food intake were not different
among the groups (data not shown). These findings indicate that
the diet supplementation and the exercise training imposed did
not affect food intake and body weight gain of the rats.
The efficiency of amino acids supplementation in the diet was
evaluated by the analysis of the amino acids concentration in
plasma of all groups. Supplementation of aspartate, asparagine,
and carnitine in the diet caused an increase of about 30% of the
concentration of aspartate in plasma in either the sedentary or
O X A L O A C E T A T E A N D EXERCISE RESISTANCE 369

trained rats (data not shown). These findings indicate that the the performance in a second session of exercise remains to be
supplementation, used effectively, increased the availability of investigated.
the amino acids for muscles.

Citrate Synthase Glycogen Content

Compared to controls, citrate synthase activity in soleus mus-
cle of swimming-trained rats presented an increment of 2.4-fold
(data not shown). These findings indicate that swimming asso-
ciated with a load of 5% body weight attached to the tail increases
the oxidative capacity of trained muscle (1).
Blood Measurements
As shown in Table 1, S rats presented increased blood lactate
(33%) and FFA levels (99%) and decreased glycemia (12%)
compared to controls under sedentary conditions. These differ-
ences were fully abolished by spontaneous physical activity, ex-
ercise training, and swimming for 1 h, except for lactate of swim-
ruing-trained rats. At the time of exhaustion, plasma FFA levels
were higher (65%) in the S group. Because the uptake of FFA
by muscles and liver basically depends on plasma concentration
(16), it remains unknown whether high plasma FFA levels in S
rats resulted from an increased lipolysis in adipose tissue and/or
low FFA utilization by the muscle and liver.
Under sedentary condition, the increased provision of acetyl-
coA in the S group possibly inhibited pyruvate dehydrogenase
activity and so the conversion of pyruvate to lactate was en-
hanced. As a consequence, blood glucose levels decreased and
plasma lactate levels increased. When physical activity was im-
posed, however, the capacity of the muscle to utilize acetyl-coA
was augmented, as indicated by the increase of citrate synthase
activity, which converts acetyl-coA and oxaloacetate to citrate,
and the differences observed were reverted in rats submitted to
spontaneous activity, The reason for the maintenance of high
plasma lactate levels in rats trained to swimming remains un-
known. The S group, nevertheless, reached exhaustion, present-
ing high levels of plasma FFA. The importance of this fact for
The diet supplementation of asparagine, aspartate, and car-
nitine markedly increased glycogen content in gastrocnemius
(22%) and soleus (2.7-fold) muscles of sedentary rats (Table 2).
Spontaneous physical activity reverted the effect of the diet in
the soleus glycogen and did not change it in the gastrocnemius.
Muscle glycogen content was not different between the two
groups in rats submitted to swimming training, swimming for 1
h, and after exhaustion. Therefore, although the diet supplemen-
tation enhanced glycogen content in the muscles, glycogen
breakdown was very similar between groups during exercise.
Liver glycogen content was not different between S and C groups
in all conditions studied.
The accumulation of glycogen in the skeletal muscles of S
rats under sedentary conditions is possibly due to the g l u c o s e -
FFA cycle (20). Increased rates of FFA utilization by the muscle
would elevate the production of ATP and citrate through the
Krebs cycle. These products of fatty acid oxidation (ATP and
citrate) would inhibit phosphofrutokinase (PFK) activity (16,17),
increasing the amount of glucose-6 phosphate available for gly-
cogen synthesis.
Time of Exhaustion
The S group swam for a longer period of time than control
rats before exhaustion. The values obtained in were (in minutes):
423.8 _+ 27.7 and 297.8 _ 42.02 (mean _+ SE of 24 rats) for S
and C rats, respectively. There was a 42% increase for the period
of time to exhaustion in the diet-supplemented group. These re-
sults show that the addition of asparagine, aspartate, and carnitine
to the diet of the rats is able to improve endurance to aerobic
exercise.

TABLE 1
BLOOD MEASUREMENTSOF THE CONTROL (C) AND THE DIET-SUPPLEMENTED(S)
GROUPS UNDER VARIOUS CONDITIONS

Glycemia FFA Lactate

Conditions Groups (mg/100 ml) (mEq/l) (mmol/l)

Sedentary
C 134.7 + 17. 0.158 + 0.059 3.28 + 0.79
S 120.1 + 7.8* 0.315 + 0.142" 4.39 + 1.37"
Spontaneous activity
C 147.0 + 25.6 0.211 + 0.163 3.37 + 0.74
S 143.8 + 20.6 0.382 + 0.247 3.38 + 0.70
Swimming training
C 107.7 + 8.9 0.161 + 0.103 3.05 + 0.79
S 105.8 + 6.2 0.207 + 0.196 4.02 + 1.06t
One-hour swimming
C 159.9 + 30.0 0.798 + 0.388 3.83 + 0.90
S 152.4 + 36.9 0.821 + 0.471 4.15 + 1.15
Exhaustion swimming
C 46.1 + 27.8 0.379 + 0.173 7.07 + 2.73
S 48.0 + 37.9 0.626 + 0.381" 5.53 + 3.36

*t The two groups were submitted to the following conditions: sedentary, spontaneous
physical activity, swimming training, swimming for 1 h, and exhaustion to swimming. For
details see the Method section. The values are presented as means + SD of 10 rats.
*p < 0.01, tP < 0.05.
370 LANCHA, JR. ET AL.

TABLE 2
GLYCOGEN CONTENT OF THE CONTROL (C) AND THE DIET-SUPPLEMENTED(S)
GROUPS IN LIVER AND GASTROCNEMIUS (WHITE PORTION) AND
SOLEUS MUSCLE UNDER VARIOUS CONDITIONS

Liver Gastrocnemius Soleus

Conditions Groups (mg/100 mg) (mg/100 mg) ~mg/100 mg)

Sedentary
C 3.62 + 1.88 0.45 +0.11 0.53 +0.11
S 2.74 + 0.72 0.55 + 0.08* 1.43 + 0.30#
Spontaneous activity
C 3.50 + 1.79 0.17 + 0.04 0.51 + 0.39
S 3.17 + 1.39 0.46 + 0.23t 0.53 + 0.39
Swimming training
C 4.70 + 1.99 0.37 + 0.18 1.50 + 0.52
S 4.29 + 0.51 0.40 + 0.26 1.34 + 0.36
One-hour swimming
C 2.77 + 1.07 0.70 + 0.36 1.32 + 0.49
S 3.26 + 1.06 0.76 + 0.38 1.59 + 0.85
Exhaustion swimming
C 0.27 + 0.11 0.23 + 0.15 0.70 + 0.20
S 0.43 + 0.21 0.20 + 0.09 0.57 + 0.08

The two groups were submitted to the following conditions: sedentary, spontaneous
physical activity, swimming training, swimming for 1 hour and exhaustion to swimming.
For details see materials and methods. The values are presented as mean + and standard
deviation of 10 rats.
*t Indicate statistical differences for comparison between S and C rats in all conditions:
* p < 0.05, t P < 0.01.

Concluding Remarks
The diet supplementation of asparagine, aspartate, and car-
nitine enhanced the capacity o f the muscle to utilize FFA, sparing
glycogen, as indicated by the results o f glycemia, plasma FFA,
and lactate levels and muscle glycogen content. Because muscle
glycogen is strongly related to endurance exercise (2,12), the
glycogen finding in itself suggests that the S rats were able to
resist to a physical effort for a longer period of time compared
to controls. The authors believe this could be an important sup-
port to the hypothesis for the role o f glycogen as a source o f
oxaloacetate for oxidation of FFA during a prolonged exercise.
On this basis, depletion o f glycogen would enable the muscle to
utilize acetyl-coA from FFA and so lead to exhaustion.
Further experiments must be performed to clarify several
points of this study. However, the findings presented here cer-
tainly contribute to the understanding of the peripheral metabolic
control for the establishment of exercise exhaustion.
ACKNOWLEDGEMENTS
The authors are grateful to Dr. E. A. Newsholme for the constant
encouragement; to Dr. E. Kokubun for the discussion of this study; and
to S. Gabay for revision of the manuscript. This research was supported
by FAPESP and CNPq.

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