Download Effect of carnitine supplementation on mitochondrial

Indian Journal of Experimental Biology
Vol. 48, May 2010, pp. 503-510
Effect of carnitine supplementation on mitochondrial enzymes in liver and
skeletal muscle of rat after dietary lipid manipulation and physical activity
Jyothsna Karanth & K Jeevaratnam1*
Biochemistry and Nutrition Discipline, Defence Food Research Laboratory, Mysore 570 011, India
Received 25 September 2009; revised 14 December 2009
Effect of carnitine supplementation in enhancing fat utilization was investigated by looking into its effects on
mitochondrial respiratory enzymes activity in liver and muscle as well as on membrane fatty acid profile in rats fed with
hydrogenated fat (HF) and MUFA-rich peanut oil (PO) with or without exercise. Male Wistar rats were fed HF-diet
(4 groups, 8 rats in each group) or PO-diet (4 groups, 8 rats in each group), with or without carnitine for 24 weeks. One
group for each diet acted as sedentary control while the other groups were allowed swimming for 1 hr a day, 6 days/week,
for 24 weeks. The PO diet as well as exercise increased the activities of mitochondrial enzymes, NADH dehydrogenase,
NADH oxidase, cytochrome C reductase, cytochrome oxidase, while carnitine supplementation further augmented the
oxidative capacity of both liver and muscle significantly by enhancing the activity of carnitine palmitoyl transferase and the
respiratory chain enzymes. These effects can be attributed to the enhanced unsaturated fatty acids in phospholipids of
mitochondria and may be due to increased fluidity of the membrane in these rats. Results of this study show a significant
health promoting effects of carnitine supplementation which could be further augmented by regular exercise.
Keywords: Carnitine supplementation, Dietary fat, Liver, Membrane phospholipids composition, Mitochondrial enzymes,
Physical activity, Skeletal muscle
Dietary fat and exercise are known determinants of
good health. Many a studies have shown that the
different type of dietary fat influenced the fatty acid
composition of plasma and cellular membranes1,2
including mitochondrial membrane and their
function3,4,5. Fatty acid compositional changes due to
dietary fat (monounsaturated or polyunsaturated fatty
acids) were associated with extensive changes in
mitochondrial enzyme activities3. Carnitine palmitoyl
transferase (CPT) activity is also influenced by
dietary fat4. It has been reported that in addition to
dietary fat (virgin olive oil or sunflower oil); physical
exercise also altered the fatty acid profile of
mitochondrial membrane5 and increased the activity
of cytochrome oxidase in liver and skeletal muscle6.
However, there have been contradicting reports about
the influence of physical training on hepatic
mitochondrial function, i.e., either a decrease in
oxidative capacity7 or no change in the oxidative
capacity or an increased cytochrome oxidase activity6.
________________
*
Correspondent author
1
Present address : Department of Biochemistry and Molecular
Biology, Pondicherry University, Kalapet,
Puducherry 605 014, India
Telephone: 0413-2654421 (O); 0413-2256813 (R)
E-mail: [email protected]
A higher level of CPT activity was observed in muscle
of endurance athletes8. Supplementation of
L-Carnitine, an obligatory cofactor for mitochondrial
fatty acid oxidation, has been shown to increase
pyruvate dehydrogenase complex, NADH–cytochrome
C reductase, succinate–cytochrome C reductase and
cytochrome oxidase in muscle of endurance athletes9,
while the CPT activity remained unchanged8.
L-carnitine supplementation could enhance fat
utilization in rats fed either hydrogenated fat (HF) or
MUFA-rich peanut oil (PO) diet with or without
exercise10. Dietary fat type and regular exercise are
known to modulate fatty acid profile of mitochondrial
membranes and associated with extensive changes in
the activity of mitochondrial enzymes. Carnitine
supplementation has been hypothesized to improve
exercise performance in humans by increasing
respiratory chain enzymes activity. Previously, we
have reported that L-carnitine supplementation could
enhance fat utilization in rats fed either hydrogenated
fat (HF) or MUFA-rich peanut oil (PO) diet with or
without exercise10. Hence, the present study was
undertaken to assess the influence of the type of dietary
fat and regular exercise of moderate intensity
(swimming for 1 hr) for 6 months duration on the liver
mitochondrial membrane lipid profile and the activities
504
INDIAN J EXP BIOL, MAY 2010
of enzymes such as NADH dehydrogenase, NADH
oxidase, cytochrome C reductase, cytochrome oxidase
and carnitine palmitoyl transferase in liver and muscle
mitochondria, and also to assess the effect of dietary
supplementation of carnitine on these parameters. The
dietary fats chosen were hydrogenated fat and peanut
oil. The former is used in the preparation of food for
Indian armed forces as it gives long shelf life, while
the peanut oil is predominantly used in southern
part of India.
Materials and Methods
The Meals-Ready to-Eat (MRE) ration developed
at Defence Food Research Laboratory, Mysore for
Indian armed forces was used as diet for the study.
Each ration pack consisted of sooji halwa, vegetable
pulav, chapati, potato peas curry, soft bar, tealeaves,
skimmed milk powder and sugar. The ration was
prepared either with hydrogenated vegetable fat,
vanaspati (HF) as source of SFA or with refined
peanut oil (PO) as a source of MUFA-rich fat.
Carbohydrates, fat and proteins contributed 58, 27 and
15 % respectively, of the total energy in this diet. The
fatty acid profile of the Indian vanaspati (Dalda) and
peanut oil analyzed by gas chromatography has been
reported earlier11. Chemicals used were of AR grade
obtained from either E.Merck or SRL (India). The
fatty acid methyl esters (FAME) standards were
procured from Sigma (USA). L-Carnitine as LCarnitine tartarate was kindly gifted by Lonza, Basel
(Switzerland). L- Carnitine was given at the level of
0.5 % on dry weight basis of diet.
Experimental protocol⎯Male Wistar rats (64),
bred in animal house of Defence Food Research
Laboratory, Mysore, and weighing 140 ± 10 g were
used. The Institutional Animal Ethics Committee had
approved this animal experimentation. Rats were
housed individually in stainless steel cage with a 12hr L/D cycle, relative humidity and temperature under
control. After being on the MRE ration diet for 2
weeks rats were grouped randomly into 8 groups of 8
rats in each group. Groupings and treatment protocol
are summarized in Table 1. Rats were given preweighed diet and water ad libitum. Left out diet in
each cage was collected over a week to quantify the
food consumption of rats weekly for 6 months of
experimental period. The composition of the diet has
been reported earlier10.
Tissue preparation and analytical methods⎯At the
end of the experimental period, the animals were
allowed to rest for about 20 hr after the exercise;
fasting blood was drawn into heparinised tube and later
killed by cervical dislocation between 9-10 am. Liver
and muscle from left hind limb were excised, rinsed in
ice-cold physiological saline and kept on ice until
processed. Blood was centrifuged to obtain plasma,
while the sediment after removing the buffy coat was
washed twice with isotonic phosphate buffer, pH 7.5 to
obtain pure erythrocytes. The plasma, a part of liver
and muscle were immediately frozen and stored at 20oC until the analysis of free carnitine according to the
method of Wieland et al12.
A part of erythrocytes was used to determine the
osmotic fragility according to Kaplay13. Results were
expressed as % haemolysis in 0.4% NaCl, taking the
haemolysis in 0.1% NaCl as 100%. The remaining
erythrocytes were used for the preparation of ghosts
according to the procedure of Dodge et al14. The
erythrocytes were lysed with 20 milliosmolar (mOsm)
phosphate buffer, pH 7.5 and centrifuged at 20000 g
for 40 min. The pellet (ghosts) was washed thrice
subsequent to haemolysis. The ghosts were then
incubated in 320 mOsm (isotonic) phosphate buffered
saline solution, pH7.5 at 37oC for 1 hr during which the
membrane resealed15,16. The membrane protein was
estimated by Lowry et al17.
Pyrene was used as the fluorescent probe to monitor
the fluidity of erythrocyte membrane because of its
lateral diffusing capability in the hydrophobic region of
the membranes18. Incorporation of pyrene into the
ghost membrane was accomplished by adding a
concentrated solution of pyrene in alcohol to the
aqueous suspension of membranes (0.2 mg of
erythrocyte membrane protein) and incubating at 25oC
with gentle stirring for 30 min. Steady state
fluorescence measurements were performed at 90o to
the exciting beam, 342 nm using Elico SL 174
spectrofluorometer. Dimer: monomer (D/M) ratios
were estimated by comparing the fluorescence intensity
Table 1⎯Treatment groups
Group
1
2
3
4
5
6
7
8
Treatment
HFS – fed with hydrogenated fat diet and sedentary
POS – fed with peanut oil (MUFA-rich) diet and
sedentary
HFE – Exercising HF
POE – Exercising PO
HFCRS – Sedentary HF supplemented with carnitine
POCRS – Sedentary PO supplemented with carnitine
HFCRE – Exercising HF supplemented with carnitine
POCRE – Exercising PO supplemented with carnitine
KARANTH & JEEVARATNAM: CARNITINE, DIETARY LIPID, EXERCISE & MITOCHONDRIAL ENZYMES
at 480 nm to that at 386 nm, using 342 nm as the
excitation wavelength in the erythrocyte ghosts
incorporated with various concentrations (10-30 µM)
of pyrene16.
Liver was homogenized in ice-cold buffered 0.25 M
sucrose containing 1 mM ethylene diamine tetra acetic
acid (EDTA) and used for preparation of mitochondria
as described elsewhere19. Muscle mitochondria was
prepared from finely minced muscle tissue and gently
homogenized with a manual glass homogenizer in icecold Tris buffer cocktail as described by Jackman and
Willis20. Protein was determined by the modified biuret
method21.
The frozen and thawed mitochondria from liver
and muscle were used for various mitochondrial
enzyme
assays.
Standard
spectrophotometric
procedures were used for the assay of NADH oxidase,
NADH
dehydrogenase,
NADH
cytochrome
C reductase and cytochrome oxidase19. The carnitine
palmitoyl transferase (CPT) was assayed according to
the method of Bieber et al22.
Lipid peroxidation in these mitochondria was also
assessed based on malondialdehyde (MDA) formation
and assayed by the thiobarbituric acid method after
washing mitochondrial preparation with phosphate
buffer to remove sucrose in the samples23.
The remaining hepatic mitochondria were used for
the extraction of fat and analysis of fatty acid profile
of phospholipids. The lipids were extracted according
to Folch procedure24 using chloroform and methanol
at 2:1 containing 0.01 % butylated hydroxy toluene
(BHT) as antioxidant.
Thin layer chromatographic separation of
phospholipids was carried out on alkaline silica gel.
The plates (20 cm and 0.25 mm layer thickness) were
prepared with alkaline silica gel G (E. Merck) using
0.01 M Na2CO3 solution and were activated for 1 hr at
110oC before use. The plates were first developed
with hexane/methanol/acetone (80:20:1). After air
drying for 30 min, they were subsequently developed
at right angle with chloroform/methanol/glacial acetic
acid/water (50:45:3.5:0.15). Iodine vapors were used
for detection and identification of the various
fractions by comparing with Rf of authentic standards.
The fractions corresponding to phosphotidylcholine,
phosphotidylethanolamine and cardiolipin were
scrapped from the TLC plate and eluted with
chloroform/methanol (2:1 containing 0.01 % BHT).
The individual fractions were esterified in methanolic
BF3. The fatty acid methyl esters (FAME) were then
505
analysed by gas liquid chromatography in a Chemito
8610 model GC. The column used was Supelco BPX
70. Column temperature was set at 110 to 220o C, 6oC
/min, N2 was used as carrier gas using flame
ionization detector. The fatty acids (FAME’s) were
identified by comparison with authentic standard
mixtures. Peak areas of individual fractions were
calculated by an integrator and expressed as relative
percents of total fatty acids extracted.
Statistical analysis⎯The results represent mean ±
standard error of values from 8 animals. A two-way
ANOVA was performed to evaluate the effect of
dietary fat and physical activity on each variable.
Significant (P<0.05) interaction terms were evaluated
by Duncan’s test.
Results
There was no appreciable difference in body
weight or weight gain between the groups fed on HF
and PO, sedentary as well as exercising groups. Food
consumption was not significantly different across the
various groups though there was a mild increase in
exercising rats (data not shown).
Effect on plasma and tissue free carnitine
level⎯The plasma free carnitine was higher in POfed rats as compared to HF-fed ones, while tissue
level was not altered (Table 2). Regular exercise
decreased the free carnitine level in plasma, liver and
muscle from all the groups of non-carnitine
supplemented rats. There was a significant increase in
free carnitine level in all carnitine supplemented ones
including exercising rats as compared to
corresponding non-supplemented groups.
Effects on liver and muscle mitochondrial CPT
activity⎯Dietary fat had no influence on the hepatic
Table 2⎯Effect of dietary fat, carnitine and exercise on free
carnitine in plasma, muscle and liver
Groups*
HFS
POS
HFE
POE
HFCRS
POCRS
HFCRE
POCRE
Plasma
Liver (nmol/g) Muscle(nmol/g)
(µmol/L)
[Values are mean + SE of 8 rats]
118 ± 6a
178 ± 14a
33.2 ± 1.7a
b
b
138 ± 3
211 ± 18b
37.9 ± 1.6
30.0 ± 1.8a
105 ± 3a
146 ± 18c
a
a
117 ± 4
187 ± 9a
32.9 ± 1.2
c
c
53.6 ± 3.1
180 ± 3
328 ± 18d
194 ± 9c
337 ± 12d
64.3 ± 2.0d
c
d
51.1 ± 2.0
163 ± 6
350 ± 13d
195 ± 7c
365 ± 20d
59.2 ± 1.8d
Values in the same column that do not share the same superscript
letters are significantly different at P<0.05.
*
Groups as in Table 1
INDIAN J EXP BIOL, MAY 2010
506
and muscle mitochondrial CPT activity except for an
increased in PO fed rats, while regular exercise
increased this enzyme activity in both the tissues
(Table 3). The carnitine supplementation further
increased CPT activity in both the tissues, more so in
HFCRE and POCRE rats.
Effect on activities of liver and muscle
mitochondrial enzymes⎯Activities of hepatic
mitochondrial enzymes, NADH dehydrogenase,
NADH oxidase, cytochrome C reductase, cytochrome
oxidase were significantly higher in PO fed group.
Regular exercise further increased these activities.
Activities of these enzymes were significantly higher
in carnitine fed rats from both the dietary groups as
compared to non-carnitine supplemented ones more
so in PO fed exercising rats (Table 3). Dietary fat did
not alter the activities of muscle mitochondrial
enzymes while regular exercise was found to enhance
the activity of these enzymes in both the dietary
conditions. Carnitine fed rats showed significantly
higher activities of these enzymes as compared to
non-carnitine supplemented ones more so in HFCRE
and POCRE groups (Table 3).
Effect on the fatty acid composition of hepatic
mitochondrial membrane⎯Fatty acid composition of
PC was greatly influenced by the dietary fatty acids
(Table 4). Rats fed on HF diet showed higher palmitic
acid and lower stearic acid levels as compared to PO
fed ones, while carnitine supplementation resulted in
decreased palmitic acid in all the groups and a
differential response for stearic acid, increase in HF
and a decrease in PO fed. Linoleic and arachidonic
acids increased in all dietary groups supplemented
with carnitine. Regular exercise found to increase
unsaturated fatty acids such as linoleic and
arachidonic in all the groups. The effect of dietary
fatty acids on the fatty acid composition of PE has
been given in Table 4. A lower palmitic acid and
higher unsaturated fatty acids such as oleic, linoleic
and arachidonic acid were observed in PO fed rats as
compared to HF, while exercise augmented this
response. The magnitude of above response was
greater in all the carnitine supplemented rats. The
cardiolipin fraction from hepatic mitochondria
showed higher oleic acid in HF fed rats, while higher
levels of linoleic acid were observed in PO fed ones.
Regular exercise induced a mild increase in the
unsaturated fatty acids in both the dietary groups,
while carnitine supplementation led to further
increase in unsaturated fatty acids along with a
significant increase in arachidonic acid in both the
dietary groups (Table 4).
Effect on the liver and muscle mitochondrial lipid
peroxidation⎯The level of MDA was higher in
Table 3⎯Effects of dietary fat, carnitine and exercise on activity of liver and muscle mitochondrial enzymes
[Values are mean ± SE of 8 rats]
Parameter/
Groups*
Liver
HFS
POS
HFE
POE
HFCRS
POCRS
HFCRE
POCRE
Muscle
HFS
POS
HFE
POE
HFCRS
POCRS
HFCRE
POCRE
CPT
(nmole/mg/min)
NADH dehydrogense
(nmole/mg/min)
NADH oxidase
(nmole/mg/min)
Cytochrome C reductase Cytochrome oxidase
(nmole/mg/min)
(nmole/mg/min)
5.3 ± 0.22a
6.1 ± 0.34a
5.7 ± 0.13b
7.0 ± 0.55c
5.3 ± 0.22a
6.1 ± 0.34a
5.7 ± 0.13b
7.0 ± 0.55c
1583 ± 44a
2032 ± 117b
1637 ± 53a
2823 ± 118c
3313 ± 133d
3427 ± 127e
3268 ± 151d
3430 ± 474f
268 ± 1.3a
499 ± 2.9b
392 ± 4.9c
521 ± 3.2d
419 ± 2.2e
565 ± 6.9f
432 ± 2.0g
689 ± 3.9h
65 ± 4.5a
75 ± 1.7b
65 ± 4.4a
128 ± 13c
181 ± 28d
199 ± 16e
200 ± 6f
220 ± 18g
782 ± 7a
989 ± 7b
1100 ± 12c
1167 ± 6d
2153 ± 7e
2274 ± 18f
3146 ± 11g
3637 ± 25h
0.5 ± 0.05a
0.6 ± 0.02a
0.8 ± 0.02b
0.9 ± 0.09c
0.6 ± 0.03d
0.7 ± 0.05e
1.0 ± 0.05f
1.2 ± 0.08f
431 ± 23a
446 ± 24a
613 ± 39b
782 ± 25c
438 ± 29a
473 ± 26a
966 ± 39d
1094 ± 63e
90 ± 6a
73 ± 3b
108 ± 5c
110 ± 12c
224 ± 18d
244 ± 12e
319 ± 27f
339 ± 12f
29 ± 1.7a
30 ± 1.3a
31 ± 1.5a
46 ± 2.7b
23 ± 2.4a
27 ± 1.2a
33 ± 2.5ab
64 ± 5.0c
521 ± 30a
560 ± 32a
580 ± 47ab
678 ± 45b
605 ± 29ab
659 ± 54ab
652 ± 5ab
813 ± 44c
Values in the same column that do not share the same superscript letters are significantly different at P<0.05
*
Groups as in Table 1
KARANTH & JEEVARATNAM: CARNITINE, DIETARY LIPID, EXERCISE & MITOCHONDRIAL ENZYMES
507
Table 4⎯Fatty acid composition (% of total fatty acids) of hepatic mitochondrial membrane phosphotidyl choline,
phosphotidyl ethanolamine and cardiolipin
Groups*
HFS
POS
HFE
POE
HFCRS
POCRS
HFCRE
POCRE
16:0
46.7
33.8
35.8
31.9
39.9
30.5
29.1
21.1
Phosphotidyl choline
18:0
18:1
18:2
21.7
4.7
4.4
25.8
10.4
5.4
28.9
4.4
5.9
27.8
7.9
7.9
25.6
5.6
6.3
19.9
4.6
7.9
30.9
5.3
8.3
24.3
6.3
11.6
20:4
4.0
4.7
5.4
6.6
17.0
20.3
19.4
24.4
16:0
31.6
23.9
29.9
23.5
29.9
19.6
19.1
15.5
Phosphotidyl ethanolamine
18:0
18:1 18:2
25.3
3.5
4.9
25.9
4.4
5.5
26.5
6.4
5.6
25.9
6.6
6.6
15.8
3.9
6.9
23.8
10.0 11.4
28.9
4.1
8.9
24.9
15.1 14.5
20:4
5.3
5.4
6.8
7.0
16.7
17.9
17.9
18.9
16:0
5.8
2.6
4.8
2.6
5.7
2.2
4.7
2.1
Cardiolipin
18:0 18:1 18:2
3.5
44.5 12.1
2.3
22.4 51.7
3.9
48.9 15.2
3.6
30.0 54.2
4.6
47.5 13.3
2.7
14.1 52.5
4.9
50.2 17.4
3.4
16.8 53.6
20:4
2.9
7.5
4.2
7.8
21.5
23.5
25.5
26.6
*
Groups as in Table 1
mitochondria of liver and muscle from PO-fed rats as
compared to HF fed ones. Regular exercise resulted in
an increase in MDA formation in liver and muscle
mitochondria, more so in PO-fed rats (Table 5).
Carnitine supplementation increased MDA level in
hepatic mitochondria from both the dietary groups
and exercise resulted in a further increase in MDA,
while muscle mitochondria showed lower MDA
levels except for POCRE rats.
Effect on the fluidity and fragility of
erythrocytes⎯The dietary fat and exercise did not
have appreciable influence on the fluidity of
erythrocyte
membrane,
while
carnitine
supplementation increased the fluidity significantly as
compared to non-supplemented groups (Fig. 1).
Fragility of erythrocytes was not affected by the
dietary fat, while regular exercise decreased it
significantly (Table 5). Carnitine supplementation
also significantly decreased the fragility of
erythrocytes as compared to respective non-carnitine
fed groups.
Discussion
The present study deals with the effect of dietary
fat, carnitine supplementation and exercise on the
activities of membrane bound enzymes in
mitochondria from liver and muscle of the rats.
Regular moderate exercise were shown to ameliorate
deleterious effects of hydrogenated fat, while
carnitine supplementation further enhanced fat
utilization, even in sedentary HF-fed rats10. The
results of the present study showed an increased
activity of oxidative enzymes in hepatic mitochondria
from PO-fed rats, while regular exercise enhanced the
activities of these enzymes in both liver and
muscle from both the dietary groups. Carnitine
supplementation led to a significant increase in
activities of these enzymes in both the tissues, more
so in PO-fed exercising rats.
The most widely established function of carnitine
is its obligate role in the transport of long chain fatty
acid in to the mitochondria, where they are
metabolized through β-oxidation and the Kreb’s
cycle. The regular physical exercise reduced free
carnitine content especially in muscle due to increased
flux of fatty acid by carnitine (transport of fatty acid
through inner membrane) for β-oxidation in muscle
mitochondria. Earlier observation that regular
physical exercise increased free fatty acids in skeletal
muscle10, resulting in an increased CPT activity. The
intake of PO-diet resulted in higher activity of CPT in
liver and skeletal muscle as compared to HF-fed ones.
The fatty acid composition of these diets might have
played a role in the elevation of CPT in accordance
with earlier reports4,25. Considering the effect of
carnitine on CPT and other enzymes as described
below in muscle confirm increased fatty acid
oxidation with carnitine supplementation. Moreover,
carnitine supplementation increased the free carnitine
level in plasma and muscle in sedentary as well as
exercising rats leading to enhanced fatty acid
oxidation26. The increased fatty acid oxidation as
indicated by high FFA level and low triglyceride10 in
liver can also be attributed to the increased free
carnitine level in carnitine supplemented rats in the
present study. This finding is in agreement with the
earlier observation where conditions known to
stimulate fatty acid oxidation such as starvation and
nephrosis were associated with higher carnitine
level27, however, the nature of this association
is not clear.
The PO-diet containing about 13% linoleic acid has
also resulted in higher activities of all the respiratory
enzymes as compared to HF-fed rats as reported
508
INDIAN J EXP BIOL, MAY 2010
Table 5⎯Effects of dietary lipid, carnitine supplementation and exercise on liver and muscle mitochondrial lipid peroxidation and
osmotic fragility of erythrocytes.
[Values are mean ± SE of 8 rats]
Groups*
Lipid peroxidation (nmole/g)
Liver
HFS
POS
HFE
POE
HFCRS
POCRS
HFCRE
POCRE
Osmotic fragility (% hemolysis)
of erythrocytes
Muscle
a
0.22 ± 0.011
0.28 ± 0.018ab
0.24 ± 0.010a
0.31± 0.039b
0.30 ± 0.106b
0.42 ± 0.036c
0.34 ± 0.036b
0.47 ± 0.029c
0.27 ± 0.010ad
0.31 ± 0.010ab
0.33 ± 0.013b
0.47 ± 0.037c
0.24 ± 0.013d
0.28 ± 0.006ad
0.26 ± 0.007ad
0.43 ± 0.008c
86 ± 3.4a
79 ± 3.8a
59 ± 6.6b
57 ± 5.1b
66 ± 3.4b
61 ± 2.8b
51 ± 3.9b
41 ± 5.4c
Values in the same column that do not share the same superscript letters are significantly different at P<0.05.
Groups as in Table 1
*
Fig. 1⎯Effect of dietary fat, carnitine supplementation and
exercise on the fluidity of erythrocyte membrane through
dimer:monomer (D:M) ratio of pyrene incorporated erythrocyte
ghosts. [Values are mean ± SE of 6 experiments. Values having
different alphabets (m.n.o.p.q and r) comparing various treatments
are significantly different among various groups at P<0.05.]
previously in rats fed on linoleic acid compared to
beef tallow diet28. Regular physical exercise resulted in a
significantly increased activity of all respiratory chain
enzymes in liver as well as muscle in accordance with
the earlier reports7,29. Exercise has also shown to
enhance mitochondria electron transport chain activity in
older
human
skeletal
muscle30.
Carnitine
supplementation with these diets has further enhanced
the respiratory enzymes activities along with profound
increase in free carnitine levels in plasma, liver and
muscle. The effect of carnitine supplementation was
even greater in exercising rats compared to other
groups. Previously, exercise training and carnitine
supplementation in diet were shown to increase carnitine
levels, triglyceride utilization and endurance in rats31.
Surprisingly the activities of respiratory enzymes were
higher in sedentary rats fed on carnitine supplemented
diet. Recently, L-carnitine supplementation and physical
exercise were shown to restore age-associated decline in
mitochondrial functions in the rat32. The mechanism
whereby L-carnitine induces this shift in respiratory
chain enzymes pattern is not known yet.
In present study, the effects of dietary fat and
exercise on hepatic mitochondrial fatty acid profiles
were in agreement with those found by Quiles et al5,
but significant increase in 20:4 level was the most
interesting results obtained with respect to the effect of
carnitine supplementation (Table 4). The enhanced
CPT and oxidative enzymes activity may be attributed
to the higher unsaturated fatty acid incorporation into
the membrane5 resulting in an increased fluidity. As
such we have observed an increased fluidity of the
erythrocyte ghost membrane and increased lipid
peroxidation in mitochondria further supports the
above conclusions. However, the lipid peroxidation
was reduced in muscle mitochondria of carnitine fed
rats. The earlier study on carnitine as an antioxidant has
shown that carnitine protects muscle from oxidative
stress due to dietary lipids or exercise33.
In summary, the PO diet, exercise and carnitine
supplementation increases the activities of
mitochondrial enzymes, probably due to the changes
in mitochondrial membrane fatty acid composition
and fluidity. Carnitine supplementation increases the
oxidative capacity of both liver and muscle in
sedentary and exercising rats by enhancing the
activity of respiratory enzymes and also influences
lipid profile of liver mitochondria.
KARANTH & JEEVARATNAM: CARNITINE, DIETARY LIPID, EXERCISE & MITOCHONDRIAL ENZYMES
Acknowledgement
Financial support by Defence Research and
Development Organisation in the form of Senior
Research Fellowship to one of the authors, JK is
acknowledged.
References
1 Periago J L, Suarez M D & Pita M L, Effect of dietary olive
oil, corn oil and medium chain triglyceride on the lipid
composition of rat red blood cell membranes, J Nutr, 120
(1990) 986.
2 Suarez A, Ramrez M C D, Faus M J & Gil A, Dietary longchain polyunsaturated fatty acids influence tissue fatty acid
composition in rats at weaning, J Nutr, 126 (1996) 887.
3 Barzantti V, Battino M, Baracca M, Cavazzoni M, Cocchi M,
Noble M, Maranesi M, Turchetto E & Lenaz G, The effect of
dietary lipid changes on the fatty acid composition and
function of liver, heart and brain mitochondria in the rat at
different ages, Brit J Nutr, 71 (1994) 193.
4 Power G W, Yaqoob P, Harvey D J, Newsholme E A &
Calder P C, The effect of dietary lipid manipulation on
hepatic mitochondrial phospholipid fatty acid composition
and carnitine palmitoyl transeferase I activity, Biochem Mol
Biol Internl, 34 (1994) 671.
5 Quiles J L, Huertas J R, Manas M, Battino M & Mataix J,
Physical exercise affects the lipid profile of mitochondrial
membranes in rats fed with virgin olive oil or sunflower oil,
Brit J Nutr, 81 (1999) 21.
6 Quiles J L, Huertas J R, Mañas M, Ochoa J J, Battino M &
Mataix J, Dietary fat type and regular exercise affect
mitochondrial composition and function depending on
specific tissue in the rat, J Bioenerg Biomembr, 33 (2001)
127.
7 Terblanche S E, Gohil K, Packer L, Henderson S & Brooks
G A, The effects of endurance training and exhaustive
exercise on mitochondrial enzymes in tissues of the rat
(Rattus norvegicus), Comp Biochem Physiol, 128A (2001)
889.
8 Arenas J, Huertas R, Campos Y, Diaz A T, Villalon J M &
Vilas E, Effects of L-carnitine on the pyruvate
dehydrogenase complex and carnitine palmitoyl transferase
activities in muscle of endurance athletes, FEBS Lett, 341
(1994) 91.
9 Huertas R, Campos Y, Diaz E, Esteban J, Vechietti L,
Montanari G, D’Iddio S, Corsi M & Arenas J, Respiratory
chain enzymes in muscle of endurance athletes: Effect of Lcarnitine, Biochem Biophys Res Commun, 188 (1992) 102.
10 Jyothsna Karanth & Jeevaratnam K, Effect of dietary lipid,
carnitine and exercise on lipid profile in rat blood, liver and
muscle, Indian J Exp Biol, 47 (2009) 748.
11 Jyothsna Karanth, Kumar R & Jeevaratnam K, Response of
antioxidant system against dietary fat and exercise on rat
tissues, Indian J Physiol Pharmacol, 48 (2004) 446.
12 Wieland O, Duefall T & Paetzke-Brummer I, Free and
esterified carntine, Colorimetric method, in: Meth Enz Anal.
Vol III (Metabolites, 3) edited by H U Bergemeyer, J
Bergemeyer & M Grab (VCH Publishers) 1985, 481.
13 Kaplay S S, Lack of association between human erythrocyte
membrane acetylcholinesterase and osmotic fragility,
Biochem Med, 19 (1978) 299.
509
14 Dodge J T, Mitchell C & Hanahan D J, The preparation and
characteristics of hemoglobin- free ghosts of human
erythrocytes, Arch Biochem Biophys, 100 (1963) 119.
15 Chang T, Sharpless P B, Devenport D A, Radunsky M B &
Yu H, Osmotic deformation of red blood cell ghosts induced
by carbohydrates, Biochem Biophys Acta, 731 (1983) 346.
16 Jeevaratnam K & Vaidyanathan C S, Acute toxicity of
methyl isocyanate in rabbit: In vitro and in vivo effects on
rabbit erythrocyte membrane, Arch Environ Contam Toxicol,
22 (1992) 300.
17 Lowry O H, Rosebrough N J, Farr A L & Randal R J, Protein
measurement with the folin phenol reagent, J Biol Chem,
193 (1951) 265.
18 Kurup C K R, Aithal H N & Ramasarma T, Increase in
hepatic mitochondria on administration of ethyl-p-chlorophenoxyisobutyrate to the rat, Biochem J, 116 (1970) 773.
19 Jeevaratnam K, Vidya S & Vaidyanathan C S, In vitro and in
vivo effect of methyl isocyanate on rat liver mitochondrial
respiration, Toxicol Appl Pharmacol, 117 (1992) 172.
20 Jackman M R & Willis W T, Characteristics of mitochondria
isolated from type I and type IIb skeletal muscle, Am J
Physiol, 270 (1996) C673.
21 Gornall A G, Bardwill G J & David M M, Determination of
serum proteins by means of biuret reaction, J Biol Chem,
117 (1949) 751.
22 Bieber L L, Abraham T & Helmrath T, A rapid
spectrophotometric assay for carnitine palmitoyl transferase,
Anal Biochem, 50 (1972) 509.
23 Jeevaratnam K, Vidya S & Vaidyanathan C S, Role of lipid
peroxidation in impairment of mitochondrial function at
complex I by methyl isocyanate treatment of rats, Biochem
Mol Biol Internl, 30 (1993) 411.
24 Folch J, Lees M & Stanley G H S, A simple method for the
isolation and purification of total lipids from animal tissues, J
Biol Chem, 226 (1957) 497.
25 Power G W & Newsholme E A, Dietary fatty acids influence
the activity and metabolic control of mitochondrial carnitine
palmitoyl transferase I in rat heart and skeletal muscle, J nutr,
127 (1997) 2142.
26 Bacurau R F P, Navarro F, Bassil R A, Meneguello M O,
Santos R V T, Almeida A L R & Rosa F B P C, Does
exercise training interfere with the effects of L- carnitine
supplementation? Nutr Res, 19 (2003) 337.
27 Shurbaji A, Berglund L, Berge R K, Cederblad G & Humble
E, On the interrelationship between hepatic carnitine, fatty
acid oxidation and triglyceride biosynthesis in nephrosis,
Lipids, 32 (1999) 547.
28 Abuirmeileh N M & Elson C E, The influence of linoleic
acid intake on membrane-bound respiratory activities, Lipids,
15 (1980) 918.
29 Davies K J A, Packer A L & Brooks G A, Biochemical
adaptation of mitochondria muscle and whole–animal
respiration to endurance training, Arch biochem Biophys,
209 (1981) 539.
30 Menshikova E V, Ritov V B, Fairfull L, Ferrell R E, Kelly D
E & Goodpaster B H, Effects of exercise on mitochondrial
content and function in aging human skeletal muscle, J
Gerontol, 61A (2006) 534.
31 Kim E. Park H & Cha Y S, Exercise training and
supplementation with carnitine and antioxidants increases
carnitine stores, triglyceride utilization, and endurance in
exercising rats, J Nutr Sci Vitaminol, 50 (2004) 335.
510
INDIAN J EXP BIOL, MAY 2010
32 Bernard A, Rigault C, Mazue F, Le Borgne F & Demarquoy
J, L-Carnitine supplementation and physical exercise restore
age-associated decline in some mitochondrial functions in
the rat, J Gerontol 63A (2008) 1027.
33 Jyothsna Karanth & Jeevaratnam K, Oxidative stress and
antioxidant status in rat blood, liver and muscle: effect of
dietary lipid, carnitine and exercise, Int J Vitam Nutr Res,
75 (2005) 333.