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Fatty acid metabolism in dystrophic muscle in vitro
Life Sciences Vol . 8, Part II, pp . 21-26, 1969 . Printed in Great Britain.
Pergamon Press
FATTY ACID METABOLISM IN DYSTROPHIC MUSCLE IN VITRO C. H . LTn, A . J . Hudson and K . P . Strickland Department of Biochemistry, University of Western Ontario London, Canada
(Received 9 September 1968 ; in final form 25 October 1968) Recent biochemical studies of mitochondria in dystrophic muscle have revealed metabolic defects that are undoubtedly a major factor in the myopathic process .
A type of skeletal muscle disease has been described in which the
mitochondria showed a loosely coupled state of oxidative phosphorylation (1,2) and Coleman et al. (3) have described a type of myopathy with mitochondrial enzyme hyperactivity .
In the latter case, there was an abnormal accumulation
of fat that the authors postulated was due to an impaired capacity of mitochondria to oxidize fat .
An abnormal
increase of lipid in the skeletal
muscle in some types of muscular dystrophy is well known and it is probable that mitochondria have an important role in its accumulation .
Since excess
lipid is present in the skeletal muscle of dystrophic mice, a study was made of
fatty acid oxidation in muscle homogenate and of pyruvic and fatty acid
oxidation in muscle mitochondria to determine whether a defect exists .
The
amount of lipid is also dependent upon the rate at which it is formed and therefore fatty acid synthesis was also investigated . :Saterials and Methods Dystrophic mice (Strain 129) and their littermate controls
(ages 60-98
days) of both sexes were decapitated and the hind leg muscles were rapidly removed, weighed and suspended in ice-cold 0 .25M sucrose containing 1 mM EDTA . The muscle was cut into small pieces and homogenized gently in a loose-fitting all glass Potter-Elvejehm homogenizer for 60 seconds .
Homogenization was
carried out another two minutes in a tightly fitting homogenizer .
21
The
Vol. 8, No . 2
DYSTROPHIC MUSCLE
22
homogenate was centrifuged at 600 F for 10 minutes at 40C.
The supernatant
fraction was carefully pipetted and used for incubation . For isolation of mitochondria the medium
(0 .251 sucrose, 10 m', l Tris
buffer, p1l 7 .2 and 0 .5 mM EDTA) of Bode and Klingenberg (4) was used .
The
muscle was subpended in nine volumes of the above medium containing 1 .0 mg
per
30 ml of Nagarse proteinase (Enzyme Development Corp ., ICew York, N.Y .) . Homogenization was carried out as described above. 350 g
After centrifugation at
the supernatant was carefully removed and centrifuged at 9000 g for 10
minutes .
The mitochondrial pellet was washed once (recentrifugation at 11,000
g for 8 minutes), resuspended in isolation medium and used directly for incubation . Albumin-bound palmitate-l-C14 was prepared as described by Nilstein and Driscole (5) with the molar ratio of albumin to palmitate at 0 .14 (6) .
:1ito-
chondrial protein was determined by the method of Lowry et al ,(7) using bovine serum albumin as standard
(standardized against purified ammonium sulfate) .
With muscle supernatant the incubation mixture contained 20 m:1 sodium phosphate buffer, pl° 7 .4, 1 .6
itil
All', 1 .5 m:l D,L-carnitine 1101, 12 .5 y l
Coenzyme A, 6 .8
at
palmitate-1-C14
(8 .9 x 104 disintegrations per minute) and 0 .7 ml of muscle
cytochrome c,
7 .5 dl MgCl 2 , 50 m:l KCl, 50 ull albumin-bound
supernatant in a final volume of 2 .0 ml .
The mitochondrial fraction was
incubated under similar conditions except
that a Kreb's cycle tntermediate
(C 4 dicarboxylic acids,
0 .123 m'1) and 200 - 280 ug mitochondrial protein were
added.i n a final volume of 1 .0 ml .
For pyruvate-3-C 14 oxidation the same
medium was used but D,L-carnitine was omitted and ADP (1 .6 m>1)
replaced All' .
The pyruvate-3-C 14 concentration was 1 .0 m:l (approx . 60,000 disintegrations per minute) . With each system the reaction mixture was placed in the main chamber of a Warburg flask (pyruvate-3-C 14 in side arm) .
C140
2
released was trapped by
0 .2 ml 10% KOH on folded filter paper in the center well .
After pre-incubation
Vol. 8, No . 2
DYSTROPHIC MUSCLE
23
at 37 0C for 5 minutes (and pyruvate tipped in) the system was closed and shaking was continued for 15 minutes for mitochondria and 30 minutes for muscle The reaction was terminated by tipping 0 .2 ml 62 .5 . citric acid
supernatant .
from the side arm and shaken for 10 minutes .
The filter paper together with
the washings from the center well were transferred to
Conway vessels .
CO 2 ,
released from the KOH by acidification with 6N 11 250 4 , was trapped in hydroxide of Hyamine (Packard Instrument Corp .) in the center compartment .
The Hyamine
solution was diluted to 5 .0 ml with methanol and 1 .0 ml was diluted with 10 ml scintillation solution
(1 .0 g PPO and 0 .1 g POPOP. per liter of toluene) for
radioactivity measurement .
A Model 6725 Nuclear Chicago Liquid Scintillation
System was used for counting with correction for quenching made by the channels ratio method . Fatty acid synthesis in muscle was studied using the method of Allman and Cibson
(8) .
In this procedure, muscle homogenate from non-starved animals was
prepared in four volumes of ice-cold - 0.111 KPO4 buffer, pll 7 .5 and centrifuged in a No . 40 Dead 1.00 minutes .
(Spinto Model L ultracentrifuge) at 35,000 rpm (71,000 g)
The supernatant was used directly as the enzyme source .
method of incubation was the same as described by Allman and Cibson figure 4 of
for
The
(8) in
their article with the exception that citrate replaced isocitrate .
Following incubation, the amount of acetate-2-C 14 incorporated into the combined non-saponified and saponified fatty acid fraction was determined by the method of Fritz and 11su
(9) . Results
The mitochondrial yield (mg protein per gram wet weight muscle) was 1 .98 ± 0 .2799 (S .D .) muscle .
for dystrophic muscle and 3 .36 ± 0 .345
(S .D .)
for normal
The difference in these results is highly significant (P - 0 .001)
and in agreement with the observation of Scrivastava (1968) . Experiments with the supernatant fraction of muscle homogenate showed
24
Vol . 8, No . 2
DYSTROPHIC MUSCLE
TABLE I The Oxidation of Pyruvate-3-C 14 and Palmitate-1-C 14 by Mitochondria from Muscle of Dystrophic `lice (D) and their Littermate Controls (C) C4 Dicarboxylic Acid Added
Number of Pairs
Oxidation Pair Comparison (mumoles C1402119 protein t S .E .) D/C x 100
A. Pvruvate-3-C 14 Malate Succinate B . Palmitate-1-C Malate Succinate
9
212 .2 t 28 .2
358 .9 ± 23 .10
61 .7 t 7 .23 a
7
194 .0 t 21 .20
332 .1 ± 11 .83
60 .46 ± 8 .44b
8
65 .11 ± 9 .48
94 .18 t 2 .47
69 .0 ± 10 .54c
12
50 .81 t 4 .21
86 .42 t 5 .19
58 .7 ± 3 .81 d
14
S .E . - Standard Lrror Probabilities (P) derived from Student's "_ t" Test : cP = 0 .02, dP . 0 .001
aP - 0 .001, bf - 0 .003,
TABLE II The Synthesis of Fatty Acid from Acetate-2-C 14 by high Speed Supernatant from Muscle Fomogenate of Dystrophic Mice (D) and their Littermate Controls (C) Number of Pairs
15
Acetate-2-C 14 Incorporation (m.umoles/mg protein t S .E .)
Pair Comparison D/C x 100
3 .02 t 0 .187
145 .94 t 12 .72a
4 .21 *_ 0 .383
S .E . = Standard Lrror Probability (P) derived from Student's "t" Test :
aY - 0 .005
that the oxidation of palmitate-1-C14 was markedly reduced in the tissue of dystrophic mice as compared with the littermate control .
In pair comparison
studies on 7 pairs the average oxidation by dystrophic muscle was only 21 per cent of the control value (P - 0 .001) .
The results on palmitate and pyruvate
DYSTROPHIC MUSCLE
Vol . 8, No. 2
25
oxidation by muscle mitochondria of normal and dystrophic mice are shown in Table 1 .
A Kreb's cycle intermediate is required and,
as shown, a decrease in
both palmitate and pyruvate oxidation in the muscle mitochondria of dystrophic mice was observed using either malate or succinate . A study of fatty acid synthesis revealed a highly significant increase in the incorporation of acetate-2-C 14 into the fatty acids of dystrophic muscle as compared with the littermate control (Table II) .
The results indicate that
fatty acid synthesis is increased in dystrophic muscle . Discussion The evidence obtained from the present study indicates that two types of defect may contribute to an accumulation of fat in the skeletal muscle of dystrophic mice .
There is both an impaired capacity of mitochondria to
oxidize pyruvic and fatty acid and an increased synthesis of fatty acid in the dystrophic muscle .
IAiether the impairment of palmitate and pyruvate oxidation
is due to common or separate defects has not been determined but there may be a common cause in which enzyme interiction is impaired by a fundamental structural defect in the mitochondrion .
Lvidence of this is the marked
reduction of mitochondrial protein in dystrophic muscle that reflects a loss of mitochondria .
Because of the reduced oxidative activity per unit weight of
mitochondrial protein it is probable that some mitochondria are functioning poorly . The increase in fatty acid synthesis is probably secondary to the disorder in mitochondria and indicates either an enhanced production of fatty acid synthetase or modification in the activity of this enzyme . Acknowledgements The skillful technical assistance of Miss Karen Ponath is gratefully acknowledged . of Canada .
This work was supported by the Muscular Dystrophic Association
DYSTROPHIC MUSCLE
28
Vol . 8, No. 2
References
I*p .
1.
L . ERNSTER and R . LUFT,
2.
G . K . VAN WIJNGAARDEN, J . BETIII .L1, A . I? . F . H . PIEIJER, W . CIL HULSMANN and C . A. FELTKAMP, brain 90, 577 (1967) .
3.
R . F . COMIAN, A . W. NIENHUIS, W . J . BROWN, T . L . MUNSAT and C . N . PEARSON, J . A . M. A. 1~99 , 624 (1967) .
4.
C . BODE and M . KLINGENBFRC, Biochem. Z . 341, 271 (1965) .
5.
S . 14 . MILSTEIN and L . H . DRISCOLE, J . Biol . Chem . 234,
6.
I . B . FRITZ,
7.
0 . H . LOWRY, N . J . ROSEBROUGH, A . L. FARR and R. J . RANDAL, J . Biol . Chem . 193, 265 (1951) .
8.
D . M . ALLION and D . M . GIBSON, J . Lipid Res . 6,
9.
I . B . FRITZ and M. f . IISU, J . Biol . Chem . 242, 865 (1967) .
10 .
Science 129,
U . SCRIVASTAVA,
Cell Res . 32, 26
(1963) .
19 (1959) .
334 (1959) .
Can. J . Biochem. 46 4 6 , 35 (1968) .
51 (1965) .
Pergamon Press
FATTY ACID METABOLISM IN DYSTROPHIC MUSCLE IN VITRO C. H . LTn, A . J . Hudson and K . P . Strickland Department of Biochemistry, University of Western Ontario London, Canada
(Received 9 September 1968 ; in final form 25 October 1968) Recent biochemical studies of mitochondria in dystrophic muscle have revealed metabolic defects that are undoubtedly a major factor in the myopathic process .
A type of skeletal muscle disease has been described in which the
mitochondria showed a loosely coupled state of oxidative phosphorylation (1,2) and Coleman et al. (3) have described a type of myopathy with mitochondrial enzyme hyperactivity .
In the latter case, there was an abnormal accumulation
of fat that the authors postulated was due to an impaired capacity of mitochondria to oxidize fat .
An abnormal
increase of lipid in the skeletal
muscle in some types of muscular dystrophy is well known and it is probable that mitochondria have an important role in its accumulation .
Since excess
lipid is present in the skeletal muscle of dystrophic mice, a study was made of
fatty acid oxidation in muscle homogenate and of pyruvic and fatty acid
oxidation in muscle mitochondria to determine whether a defect exists .
The
amount of lipid is also dependent upon the rate at which it is formed and therefore fatty acid synthesis was also investigated . :Saterials and Methods Dystrophic mice (Strain 129) and their littermate controls
(ages 60-98
days) of both sexes were decapitated and the hind leg muscles were rapidly removed, weighed and suspended in ice-cold 0 .25M sucrose containing 1 mM EDTA . The muscle was cut into small pieces and homogenized gently in a loose-fitting all glass Potter-Elvejehm homogenizer for 60 seconds .
Homogenization was
carried out another two minutes in a tightly fitting homogenizer .
21
The
Vol. 8, No . 2
DYSTROPHIC MUSCLE
22
homogenate was centrifuged at 600 F for 10 minutes at 40C.
The supernatant
fraction was carefully pipetted and used for incubation . For isolation of mitochondria the medium
(0 .251 sucrose, 10 m', l Tris
buffer, p1l 7 .2 and 0 .5 mM EDTA) of Bode and Klingenberg (4) was used .
The
muscle was subpended in nine volumes of the above medium containing 1 .0 mg
per
30 ml of Nagarse proteinase (Enzyme Development Corp ., ICew York, N.Y .) . Homogenization was carried out as described above. 350 g
After centrifugation at
the supernatant was carefully removed and centrifuged at 9000 g for 10
minutes .
The mitochondrial pellet was washed once (recentrifugation at 11,000
g for 8 minutes), resuspended in isolation medium and used directly for incubation . Albumin-bound palmitate-l-C14 was prepared as described by Nilstein and Driscole (5) with the molar ratio of albumin to palmitate at 0 .14 (6) .
:1ito-
chondrial protein was determined by the method of Lowry et al ,(7) using bovine serum albumin as standard
(standardized against purified ammonium sulfate) .
With muscle supernatant the incubation mixture contained 20 m:1 sodium phosphate buffer, pl° 7 .4, 1 .6
itil
All', 1 .5 m:l D,L-carnitine 1101, 12 .5 y l
Coenzyme A, 6 .8
at
palmitate-1-C14
(8 .9 x 104 disintegrations per minute) and 0 .7 ml of muscle
cytochrome c,
7 .5 dl MgCl 2 , 50 m:l KCl, 50 ull albumin-bound
supernatant in a final volume of 2 .0 ml .
The mitochondrial fraction was
incubated under similar conditions except
that a Kreb's cycle tntermediate
(C 4 dicarboxylic acids,
0 .123 m'1) and 200 - 280 ug mitochondrial protein were
added.i n a final volume of 1 .0 ml .
For pyruvate-3-C 14 oxidation the same
medium was used but D,L-carnitine was omitted and ADP (1 .6 m>1)
replaced All' .
The pyruvate-3-C 14 concentration was 1 .0 m:l (approx . 60,000 disintegrations per minute) . With each system the reaction mixture was placed in the main chamber of a Warburg flask (pyruvate-3-C 14 in side arm) .
C140
2
released was trapped by
0 .2 ml 10% KOH on folded filter paper in the center well .
After pre-incubation
Vol. 8, No . 2
DYSTROPHIC MUSCLE
23
at 37 0C for 5 minutes (and pyruvate tipped in) the system was closed and shaking was continued for 15 minutes for mitochondria and 30 minutes for muscle The reaction was terminated by tipping 0 .2 ml 62 .5 . citric acid
supernatant .
from the side arm and shaken for 10 minutes .
The filter paper together with
the washings from the center well were transferred to
Conway vessels .
CO 2 ,
released from the KOH by acidification with 6N 11 250 4 , was trapped in hydroxide of Hyamine (Packard Instrument Corp .) in the center compartment .
The Hyamine
solution was diluted to 5 .0 ml with methanol and 1 .0 ml was diluted with 10 ml scintillation solution
(1 .0 g PPO and 0 .1 g POPOP. per liter of toluene) for
radioactivity measurement .
A Model 6725 Nuclear Chicago Liquid Scintillation
System was used for counting with correction for quenching made by the channels ratio method . Fatty acid synthesis in muscle was studied using the method of Allman and Cibson
(8) .
In this procedure, muscle homogenate from non-starved animals was
prepared in four volumes of ice-cold - 0.111 KPO4 buffer, pll 7 .5 and centrifuged in a No . 40 Dead 1.00 minutes .
(Spinto Model L ultracentrifuge) at 35,000 rpm (71,000 g)
The supernatant was used directly as the enzyme source .
method of incubation was the same as described by Allman and Cibson figure 4 of
for
The
(8) in
their article with the exception that citrate replaced isocitrate .
Following incubation, the amount of acetate-2-C 14 incorporated into the combined non-saponified and saponified fatty acid fraction was determined by the method of Fritz and 11su
(9) . Results
The mitochondrial yield (mg protein per gram wet weight muscle) was 1 .98 ± 0 .2799 (S .D .) muscle .
for dystrophic muscle and 3 .36 ± 0 .345
(S .D .)
for normal
The difference in these results is highly significant (P - 0 .001)
and in agreement with the observation of Scrivastava (1968) . Experiments with the supernatant fraction of muscle homogenate showed
24
Vol . 8, No . 2
DYSTROPHIC MUSCLE
TABLE I The Oxidation of Pyruvate-3-C 14 and Palmitate-1-C 14 by Mitochondria from Muscle of Dystrophic `lice (D) and their Littermate Controls (C) C4 Dicarboxylic Acid Added
Number of Pairs
Oxidation Pair Comparison (mumoles C1402119 protein t S .E .) D/C x 100
A. Pvruvate-3-C 14 Malate Succinate B . Palmitate-1-C Malate Succinate
9
212 .2 t 28 .2
358 .9 ± 23 .10
61 .7 t 7 .23 a
7
194 .0 t 21 .20
332 .1 ± 11 .83
60 .46 ± 8 .44b
8
65 .11 ± 9 .48
94 .18 t 2 .47
69 .0 ± 10 .54c
12
50 .81 t 4 .21
86 .42 t 5 .19
58 .7 ± 3 .81 d
14
S .E . - Standard Lrror Probabilities (P) derived from Student's "_ t" Test : cP = 0 .02, dP . 0 .001
aP - 0 .001, bf - 0 .003,
TABLE II The Synthesis of Fatty Acid from Acetate-2-C 14 by high Speed Supernatant from Muscle Fomogenate of Dystrophic Mice (D) and their Littermate Controls (C) Number of Pairs
15
Acetate-2-C 14 Incorporation (m.umoles/mg protein t S .E .)
Pair Comparison D/C x 100
3 .02 t 0 .187
145 .94 t 12 .72a
4 .21 *_ 0 .383
S .E . = Standard Lrror Probability (P) derived from Student's "t" Test :
aY - 0 .005
that the oxidation of palmitate-1-C14 was markedly reduced in the tissue of dystrophic mice as compared with the littermate control .
In pair comparison
studies on 7 pairs the average oxidation by dystrophic muscle was only 21 per cent of the control value (P - 0 .001) .
The results on palmitate and pyruvate
DYSTROPHIC MUSCLE
Vol . 8, No. 2
25
oxidation by muscle mitochondria of normal and dystrophic mice are shown in Table 1 .
A Kreb's cycle intermediate is required and,
as shown, a decrease in
both palmitate and pyruvate oxidation in the muscle mitochondria of dystrophic mice was observed using either malate or succinate . A study of fatty acid synthesis revealed a highly significant increase in the incorporation of acetate-2-C 14 into the fatty acids of dystrophic muscle as compared with the littermate control (Table II) .
The results indicate that
fatty acid synthesis is increased in dystrophic muscle . Discussion The evidence obtained from the present study indicates that two types of defect may contribute to an accumulation of fat in the skeletal muscle of dystrophic mice .
There is both an impaired capacity of mitochondria to
oxidize pyruvic and fatty acid and an increased synthesis of fatty acid in the dystrophic muscle .
IAiether the impairment of palmitate and pyruvate oxidation
is due to common or separate defects has not been determined but there may be a common cause in which enzyme interiction is impaired by a fundamental structural defect in the mitochondrion .
Lvidence of this is the marked
reduction of mitochondrial protein in dystrophic muscle that reflects a loss of mitochondria .
Because of the reduced oxidative activity per unit weight of
mitochondrial protein it is probable that some mitochondria are functioning poorly . The increase in fatty acid synthesis is probably secondary to the disorder in mitochondria and indicates either an enhanced production of fatty acid synthetase or modification in the activity of this enzyme . Acknowledgements The skillful technical assistance of Miss Karen Ponath is gratefully acknowledged . of Canada .
This work was supported by the Muscular Dystrophic Association
DYSTROPHIC MUSCLE
28
Vol . 8, No. 2
References
I*p .
1.
L . ERNSTER and R . LUFT,
2.
G . K . VAN WIJNGAARDEN, J . BETIII .L1, A . I? . F . H . PIEIJER, W . CIL HULSMANN and C . A. FELTKAMP, brain 90, 577 (1967) .
3.
R . F . COMIAN, A . W. NIENHUIS, W . J . BROWN, T . L . MUNSAT and C . N . PEARSON, J . A . M. A. 1~99 , 624 (1967) .
4.
C . BODE and M . KLINGENBFRC, Biochem. Z . 341, 271 (1965) .
5.
S . 14 . MILSTEIN and L . H . DRISCOLE, J . Biol . Chem . 234,
6.
I . B . FRITZ,
7.
0 . H . LOWRY, N . J . ROSEBROUGH, A . L. FARR and R. J . RANDAL, J . Biol . Chem . 193, 265 (1951) .
8.
D . M . ALLION and D . M . GIBSON, J . Lipid Res . 6,
9.
I . B . FRITZ and M. f . IISU, J . Biol . Chem . 242, 865 (1967) .
10 .
Science 129,
U . SCRIVASTAVA,
Cell Res . 32, 26
(1963) .
19 (1959) .
334 (1959) .
Can. J . Biochem. 46 4 6 , 35 (1968) .
51 (1965) .