NAOKO HARADA Shiztioka Women's College, 3-2-3, Nunohashi, llamamatsu, Shizuoka-ken, Japan ABSTRACT It has been shown previously by the author that the structure of rat liver mitochondria is changed by a low casein diet. In the present experiments, using the free fatty acid producing substances Ca2* and thyroxine, and the free fatty acid sodium oleate, the influence of a 4% casein diet on the in vitro swelling process of purified rat liver mito chondria was investigated by following the decrease in optical density at 520 nm after addition of one of the swelling agents. Swelling due to hypotonicity was also investigated. Rats were fed the test diets for 80 to 100 days. Mitochondria were purified by washing three times; after each wash the isotonic suspension was centrifuged at 3,000 X g for 6 minutes. The results show that the in vitro swelling of mitochondria induced by Ca2* or thyroxine was inhibited greatly in the early period of swelling in rats fed a low casein diet. By comparison to this, there was not a notable effect of a low casein diet on the rate of swelling induced by sodium oleate. But, in the case of sodium oleate, the values of optical density were always higher in rats fed a low casein diet, and the differences between two groups were always significant. Swelling due to hypotonicity was not affected at all by a low casein diet. J. Nutr. 106: 1637-1642, 1976. INDEXING KEY WORDS protein deficiency •mitochondria • swelling •calcium ion •thyroxine •sodium oleate •hypotonicity In a previous report by the author (1), it was shown that the Qo2 and respiratory control index of liver mitochondria from rats fed a low casein diet were higher than those from control rats. However, on examining electron micrographs of liver mitochondria isolated from rats fed a low casein diet washed only once, some of the mitochondria had assumed a cresecent or doughnut shape. In essential fatty acid deficiency, liver mitochondria are easily induced to swell (2, 3), and respiratory activity is impaired (4, 5). Moreover, many cresecent-shapcd mitochondria were observed by the electron microscope (4). Since mitochondria from rats fed a low casein diet had a higher respiratory activ-
ity, while those from fatty acid deficient rats and rats subjected to other nutritional deficiency had impaired respiratory activity (4-8), the type of morphological changes induced by a low casein diet must be unique. In the present experiments, in vitro swelling characteristics of mitochondria were investigated because the swelling phenomena have been frequently used to study structural alterations of mitochondria in relation to biochemical properties. Mitochondria are induced to swell under various conditions (9-16). Not only are they induced to swell by various swelling Receivedfor publicationMarch2, 1976.
16.37
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Characteristic Inhibition of in vitro Swelling of Purified Rat Liver Mitochondria Induced by Ca2+ or Thyroxine by Feeding Rats a Low Casein Diet
1638
XAOKO HARADA
used. They were housed in individual cages in a temperature-controlled room maintained at about 22°. The composition of diets is shown in table 1. As in a previous report (1), the low casein diet contained 4% casein, and the control diet contained 25% casein; but, the amount of vitamins added were dif ferent. In the previous report (1), the vitamin mixture was added at one third of the level used by Harper (19), but in the present experiments, the amount used by Harper was added. Plan of experiments. The experiments were carried out in two phases as experi ments 1 and 2. In both experiments, the rats were di vided into two groups, one group was fed a low casein diet, and the other a control diet. Both groups were fed the test diets ad libitum for 80 to 100 days. Then, the rats were killed, the liver mitochondria were isolated, and in experiment 1, swell ing of mitochondria induced by Ca2*, thyroxine and hypotonicity was measured, and in experiment 2, swelling induced by sodium oleate was measured. Preparation of mitochondria. The swell ing of mitochondria is affected greatly by co-existing substances and organelles other than mitochondria in the preparation. Con tamination of lysosomes is especially un desirable because free fatty acid produc tion would be accelerated by lysosomal enzymes. Therefore, in the present experi ments, to minimize the contamination, mitochondria were isolated by low speed TABLE 1 centrifugation 3,000 X g. The purity of Composition of diet1 the mitochondrial fraction was investi gated, and contamination by other cell organelles was found to be considerably lowered by such procedure than by one using centrifugation at 10,000 X g (20). Two kinds of media were used for the preparation of mitochondria. Preparation Casein«-Starch2Corn medium 1 contained 0.21 M mannitol, 0.07 oilMineral M sucrose, 0.1 mM EDTA, 0.01 M Tris B3Vitaminmixture buffer (pH 7.4) and 0.2% bovine serum mixture*Choline albumin. Preparation medium 2 contained chlorideLowcaseindiet485.6550.250.15Highcaseindiet2564.6550.250.15 onlv 0.21 M mannitol, 0.07 M sucrose and 1Diets were supplemented further with 6,000 IU 0.01 M Tris buffer (pH 7.4). retinol, 600 IU ergocalciferol and 100 mg DL-«Rats were killed; the livers were re tocopherol per kg diet. 2Commercial name moved and homogenized with preparation "Matsunorin" obtained from Matsutani Kagakukogyo Company, Japan. 3According to Harper medium 1. After removal of nuclei and (19). cell debris by low speed centrifugation,
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agents, but also by physical conditions such as hypotonicity (13, 14). The bio chemical mechanisms by which various agents have swelling action upon mito chondria have been investigated. Two biochemical mechanisms are known; swell ing of mitochondria is induced by accel eration of production of free fatty acids (12, 14) and by lipid peroxide formation (15, 16). Most swelling agents have been shown to cause swelling of mitochondria by accelerating production of free fatty acids (14). Recently, it was shown that there is a parallelism between mitochondrial swelling and phospholipase A activ ity, and free fatty acids would be pro duced as a result of phospholipase A action (17, 18). In the present experiments, Ca2* and thyroxine were used as representative swelling agents which accelerate produc tion of free fatty acids. Ca2t is also known to stimulate phospholipase A activity (17). The influence of a low casein diet upon the swelling of mitochondria induced by these agents was investigated. Free fatty acids themselves are also known to be swelling agents (12-14, 17); therefore, sodium oleate was also used. To compare the swelling due to these agents with the purely physical process, the influence of a low casein diet upon the hypotonie swell ing process was investigated. Animals and diets. Male rats of the Wistar strain, weighing about 170 g, were METHODS
LOW CASEIN DIET AND SWELLING OF MITOCHONDRIA
TABLE 3 The effect of low casein diet on liver mitochondrial swelling induced by Ca?+ (experiment 1) '
Incubation timemin0 CaseinO.D,
5 10 20 304%
CaseinO.D.
520 nm/mg 520 nm/mg protein1.736±0.054l protein1.699 ±0.090 1.301±0.1033 1.640±0.095 1.125 ±0.077» 1.451 ±0.125 0.889 ±0.0673 1.202±0.074 0.745±0.0513 1.083±0.06725%
1The value was expressed as optical density at 520 nm divided by protein content in 3 ml of re action mixture. 2Mean±so; five rats per group. 3Significantly different from the mean for the re spective 4% casein group. P < 0.01.
termined by the method of Lowry et al. (21). The significance of the difference be tween two groups of values was deter mined by the Student's t test (22).
RESULTS In table 2 are shown the final body weight, liver weight and number of rats per group. Ca-^-induced swelling. Mean values of optical density at 520 nm per fixed amount of protein at various time intervals during incubation with swelling medium contain ing Ca-+ were calculated for two groups (table 3). Mean values were significantly higher using mitochondria from rats fed a low casein diet than those from control rats. As can be seen from the value at zero time and 5 minutes, the slope of the curve for the first 5 minutes was steep TABLE 2 using mitochondria from rats fed a con Final body weight, liver weight and number trol diet, but it was very gentle using of rats per group those from rats fed a low casein diet. Thyroxine-indttced swelling. Mean values Dietgroup4% Rats/ body liver of optical density at 520 nm per fixed group weightNo. weightg5.8±0.3 amount of protein at various time intervals during incubation with swelling medium gExperiment containing thyroxine were calculated for 15 two groups (table 4). As in the case of 209±61 Ca2%mean values were significantly higher Casein Casein4% 25% 349±13Experiment 5 10.5 ±0.76.4±0.1using mitochondria from rats fed a low casein diet than those from control rats. 27 Casein 25% CaseinFinal 7 ' Mean dbso.
221 ±14 355± 7Final 11.7±0.8
1 Guaranteed reagent, Nakarai Kyoto, Japan. "Extra pure reagent, Nakarai Kyoto, Japan.
Chemicals Ltd., Chemicals Ltd.,
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mitochondria were sedimentad by centrifugation at 3,000 X g for 6 minutes. This mitochondrial fraction was washed once with preparation medium 1, and after that, was further purified by washing two more times with preparation medium 2. The final two washes were required be cause contamination with bovine serum albumin and EDTA could inhibit the swelling of mitochondria. After each wash, mitochondria were sedimented by centrifugation at 3,000 X g for 6 minutes. Assay of mitochondrial swelling jyrocess. Tests of mitochondrial swelling were car ried out using the method of Lehninger ( 13 ). Mitochondria from 4 g of the liver were suspended in 2 ml of preparation medium 2, and the suspension was kept in ice. Aliquots of this suspension were added to 3.0 ml of swelling medium containing 0.125 M KC1, 0.02 M Tris buffer (pH 7.4) and one of the swelling agents to produce an initial absorbancy of between 0.4 and 0.6 at 520 nm. The final concentrations of swelling agents in swelling media were as follows: Cado, 1 mM, DL-thyroxine,1 10 /¿M,or sodium oleate,2 30 /XM.In the case of hypotonie swelling, mitochondrial sus pension was added to the medium con taining only 0.02 M Tris buffer (pH 7.4). Incubations were carried out at 25°,and the decrease of optical density at 520 nm was followed for 20 or 30 minutes, and only in the case of sodium oleate, it was followed until the curves reached a plateau. In all the experiments, protein was dc-
1639
1G40
NAOKO HARADA TABLE 4
TABLE 6
The effect of low casein diet on liver milochondrial swelling induced by thyroxine (experiment 1) '
The effect of low casein diet on liver mitochondrial swelling induced by hypotonicity (experiment 1) '
CaseinO.D.
Incubation timemin010 CaseinO.D.
520 nm/mg 620 nm/mg protein1.678±0.0482 protein1.678±0.097
510 20 304%
1.272±0.030 1.180±0.035 1.119±0.041 1.091±0.05125%
1.134±0.0723 1.027±O.OG23 0.874±0.0(>03 0.799±0.0033
1The value was expressed as optical density at 520 nm divided by protein content in 3 ml of reaction mixture. 2Mean±SD;five rats per group. 'Sig nificantly different from the mean of the respective 4% casein group, P < 0.01.
Sodium oleate-induced swelling. Mean values of optical density at 520 nm per fixed amount of protein at various time in tervals during incubation with swelling medium containing sodium oleate were calculated for two groups (table 5). The mean values were always significantly higher using mitochondria from rats fed a low casein diet than those from control rats. There was a significant difference at zero time between the two groups. Since some time was required, after addition of mitochondrial suspension for mixing the reaction mixture, before recording the zero time value this difference is assumed to be due to the rapid effect of sodium oleate.
CaseinO.D.
520 nm/mg 620 nm/mg protein0.729±0.0312 protein0.699±0.012 204%
0.579±0.035 0.551±0.013 0.545±0.02325% 0.535±0.012
1The value was expressed as optical density at 520 nm divided by protein content in 3 ml of re action mixture. 2Mean±sD,five rats per group.. There were no significant differences between groups due to dietary protein level.
Hypotonie swelling. Mean values of optical density at 520 nm per fixed amount of protein at various time intervals during incubation in hypotonie medium were cal culated for the two groups (table 6). The mean values were only slightly higher using mitochondria from rats fed a low casein diet than those from control rats, and the differences were not significant.
DISCUSSION In the present experiments when rats were fed a low casein diet, the decrease of optical density at 520 nm was inhibited during the early period of swelling when Ca2t or thyroxine were used as swelling agents. On the other hand, when sodium oleate was used as a swelling agent, the zero time value was already different be tween the two groups, and thereafter, TABLE 5 values of two groups were always signifi The effect of low casein diet on liver mitochondrial swelling induced by sodium oleate (experiment A)1'1 cantly different. In the case of Ca2* or thyroxine, free Incubation fatty acids are produced during incubation CaseinO.D. timemin0 CaseinO.D. of mitochondria probably by the action of mitochondrial phospholipase A (17). Free 6eO nm/mg 500 nm/mg fatty acids have a powerful detergent ac protein1.715±0.058'protein1.532±0.0664 tion, especially at high concentration. Con 0.797±0.11f>« sidering these facts, the following two 0.992±0.112 1020 0.446±0.0684 mechanisms can be considered for the re 0.779±0.087 at plateau4% 0.574±0.02425% 0.362±0.026< sults of the present experiments. 1. As can be seen from table 5, the same 1The value was expressed as optical density at amount of free fatty acids always cause a 520 nm divided by protein content in 3 ml of re action mixture. *Elapsed time for the analysis greater swelling in the mitochondria from after adding mitochondria! suspension for mixing rats fed a 25% casein diet than those from the reaction mixture was about 30 seconds. 3Mean±so; seven rats per group. «Significantly rats fed a 4% casein diet. This would different from the mean of the respective 4% casein probably be caused by the fact that mito chondria from rats fed a 25% casein diet group, P < 0.01.
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Incubation timemin0 CaseinO.D.
LOW CASEIN DIET AND SWELLING OF MITOCHONDRIA
purely physical process was not affected by feeding rats a low casein diet. However, in a previous report by the author (1), electron micrographs revealed expansion of mitochondrial volume in puri fied liver mitochondria from rats fed a low casein diet, but not in those from control rats. The following reasons for the dis crepancy between this observation and the results of the present experiments are proposed: 1. In the previous report (1), when electron micrographs of enlarged mito chondria were taken, the composition of the 4% casein diet fed to rats was differ ent from that of the present experiments. The vitamin mixture was added at one third of the level used by Harper (19), rather than at the level used by Harper. Also, vitamin E was not added in the diet used in the previous report, but in the present experiments, 100 mg vitamin E/kg of diet was added. Therefore, the combi nation of the deficiency of protein and vitamins together might result in an ac celeration of the swelling of the mito chondria. Biomcmbranes would be fragile due to a deficiency of protein and vitamin E. Murata and Ikehata (23) reported that hemolysis was accelerated in rats fed a low vitamin E, low protein diet. 2. Mitochondria from rats fed a low casein diet might be less resistant to me chanical treatments, such as rcsuspending and centrifuging many times. In the pre vious report (1), when the electron micro graphs of enlarged mitochondria was taken, mitochondria were washed several times. 3. In the previous report (1), EDTA was always present in preparation me dium, but in the present experiments, EDTA was omitted from preparation medium 2. Presence of EDTA mignt have led to different results. The particularly, EDTA is known as a powerful inhibitor of phospholipase A (17). To ascertain whether the effects ob served have any relationship with the fat content of the liver, liver fat content was determined for the two groups. The values were, expressed as percentage of dry weight, for the 4% casein diet group, 20.49 ±8.04,3 and for the 25% casein diet » Mean ±SD five rats.
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are always more susceptible to the de tergent action of sodium oleate than those from rats fed a 4% casein diet. Especially in the case of sodium oleate, there was al ready a significant difference at zero time. The zero time value of the 4% casein diet group incubated with sodium oleate was not different from the Cajt and thyroxine group, but the value of the control group with sodium oleate was lower than the other group. This would mean that mito chondria from rats fed a control diet were affected more by the rapid detergent action of sodium oleate. This different be havior of mitochondria from two groups to the same amount of sodium oleate might be the reason for the inhibitory effect of a low casein diet on swelling of mitochon dria induced by Ca-+ or thyroxine. 2. In the case of Ca-* or thyroxine, in the early period of incubation, production of free fatty acids from mitochondria might be inhibited in rats fed a low casein diet, probably by inhibition of mitochondrial phospholipase A. It was suggested bv Waite et al. (17) that the role of phos pholipase A in swelling of mitochondria is to catalyze the hydrolysis of phospholipid at a key locale. It was also shown that hydrolysis of phospholipids was ac celerated very much by the presence of Ca-* during the first 5 minutes incubation of mitochondria. Thus, the swelling of mitochondria induced by Ca2f or thyrox ine during the first 5 minutes might be caused by hydrolysis of phospholipids at a key locale, thus resulting in changes in the nature of mitochondrial membrane. There fore, inhibition of swelling of mitochondria in ratsor fed a 4% during casein diet induced by Ca'-'* thyroxine the early period of incubation might owe to inhibition of phospholipase A activity. Further investigations should distinguish which of these two mechanisms is respon sible for the inhibitory effect of a low casein diet on swelling of mitochondria induced by Ca2t or thyroxine, or whether a combination of these two mechanisms is responsible. Compared to these cases using various swelling agents, hypotonie swelling of mi tochondria was not affected at all by feed ing rats a low casein diet. This means that swelling of mitochondria induced by a
11)41
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NAOKO HARADA
LITERATURE CITED 1. Harada, N. (1967) Morphological and res piratory changes in rat liver mitochondria resulting from a low casein diet. J. Nutr. 93, 263-272. 2. Hayashida, T. & Portman, O. W. (1960) Swelling of liver mitochondria from rats fed diets deficient in essential fatty acids. Proc. Soc. Exp. Biol. Med. 103, 656-659. 3. Johnson, R. M. (1963) Swelling studies on liver mitochondria from essential fatty acid deficient rats. Exp. Cell Res. 32, 118129. 4. Levin, E., Johnson, R. M. & Albert, S. (1957) Mitochondrial changes associated with essential fatty acid deficiency in rats. J. Biol. Chem. 228, 15-21. 5. Ito, T. & Johnson, R. M. (1964) Effects of a nutritional deficiency of unsaturated fats on rat liver mitochondria. I. Respiratory con trol and adenosine triphosphate-inorganic orthophosphate exchange activity. J. Biol. Chem. 239, 3201-3208. 6. Hall, J. C., Sordahl, L. A. & Stefko, P. L. ( 1960 ) The effect of insulin on oxidative phosphorylation in normal and diabetic mito chondria. J. Biol. Chem. 235, 1536-1539. 7. Burch, H. B., Hunter, F. E. Jr., Combs, A. N. & Schutz, B. A. (1960) Oxidative enzymes and phosphorylation in hepatic mitochondria from riboflavin-deficient rats. J. Biol. Chem. 235, 1540-1544. 8. Koppel, C. L. & Tandler, B. (1975) Riboflavin and mouse hepatic cell structure and function. Mitochondrial oxidative metabolism 'Unpublished
data.
in severe deficiency states. J. Nutr. 105, 562570. 9. Hunter, F. E. Jr. & Ford, L. (1955) Inactivation of oxidative and phosphorylative systems in mitochondria by preincubation with phosphate and other ions. J. Biol. Chem. 216, 357-369. 10. Tapley, D. F. (1956) The effect of thyrox ine and other substances on the swelling of isolated rat liver mitochondria. J. Biol. Chem. 222, 325-339. 11. Lehninger, A. L. & Schneider, M. (1959) Mitochondrial swelling induced by glutathion. J. Biochem. Biophys. Cytol. 5, 109-116. 12. Lehninger, A. L. & Remmert, L. F. (1959) An endogenous uncoupling and swelling agent in liver mitochondria and its enzymic formation. J. Biol. Chem. 234, 2459-2464. 13. Lehninger, A. L. (1959) Reversal of vari ous types of mitochondria! swelling by adeno sine triphosphate. J. Biol. Chem. 234, 24652471. 14. Wojtczak, L. & Lehninger, A. L. (1961) Formation and disappearance of an en dogenous uncoupling factor during swelling and contraction of mitochondria. Biochim. Biophys. Acta 51, 442-456. 15. Hoffsten, P. E., Hunter, F. E. Jr., Gebicki, J. M. & Weinstein,under J. (1962) of "lipid peroxide" conditions Formation which lead to swelling and lysis of rat liver mitochondria. Biochem. Biophys. Res. Commun. 7, 276280. 16. Hunter, F. E. Jr., Gebicki, J. M., Hoffsten, P. E., Weinstein, J. & Scott, A. (1963) Swelling and lysis of rat liver mitochondria induced by ferrous ions. J. Biol. Chem. 238, 828-835. 17. Waite, M., Deenen, L. L. M. Van, Ruigrok, T. J. C. & Elbers, P. F. (1969) Relation of mitochondrial phospholipase A activity to mitochondrial swelling. J. Lipid Res. 10, 599-608. 18. Waite, M. & Golde, L. M. Van (1988) Di etary induced alterations in swelling charac teristics and endogenous phospholipase Az activity of rat liver mitochondria. Lipids 3, 449-452. 19. Harper, A. E. (1959) Amino acid balance and imbalance. I. Dietary level of protein and amino acid imbalance. J. Nutr. 68, 405418. 20. Harada, N. & Sato, T. (1975) Some ad vantages of using low speed centrifugation for the purification of rat liver mitochondria. Agr. Biol. Chem. 39, 899-900. 21. Lowry, O. H., Rosebrough, N. J., Fan-, A. L. & Randall, R. J. (1951) Protein measure ment with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. 22. Snedecor, G. W. (1948) Statistical meth ods. Iowa State Univ. Press, Ames, Iowa, Chapters 3 and 4. 23. Murata, K. & Ikehata, H. (1965) Proteinvitamin E relation and the hemolysis test. Agr. Biol. Chem. 29, 809-812.
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group, 10.54 ±0.83. The results show tliat there was significantly more fat in the liver of rats fed the low casein diet. But, as can be seen from the so, the liver fat content of rats fed a low casein diet was more variable than that of control rats. On the contrary, the fluctuation in the swell ing process induced by Ca2+ and thyroxine was not different between the two groups as can be seen from the SD (tables 3 and 4). Moreover, in another experiment, when 1% orotic acid was added to the 25% casein diet, severe fatty liver was induced, but swelling of isolated mitochondria from such rats using Ca-* as the swelling agent was not inhibited, but rather, a little activated.4 From these facts, it would be reasonable to suppose that increased fat in livers of rats fed the 4% casein diet would not be of importance for the results of the present experiments.
ity, while those from fatty acid deficient rats and rats subjected to other nutritional deficiency had impaired respiratory activity (4-8), the type of morphological changes induced by a low casein diet must be unique. In the present experiments, in vitro swelling characteristics of mitochondria were investigated because the swelling phenomena have been frequently used to study structural alterations of mitochondria in relation to biochemical properties. Mitochondria are induced to swell under various conditions (9-16). Not only are they induced to swell by various swelling Receivedfor publicationMarch2, 1976.
16.37
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Characteristic Inhibition of in vitro Swelling of Purified Rat Liver Mitochondria Induced by Ca2+ or Thyroxine by Feeding Rats a Low Casein Diet
1638
XAOKO HARADA
used. They were housed in individual cages in a temperature-controlled room maintained at about 22°. The composition of diets is shown in table 1. As in a previous report (1), the low casein diet contained 4% casein, and the control diet contained 25% casein; but, the amount of vitamins added were dif ferent. In the previous report (1), the vitamin mixture was added at one third of the level used by Harper (19), but in the present experiments, the amount used by Harper was added. Plan of experiments. The experiments were carried out in two phases as experi ments 1 and 2. In both experiments, the rats were di vided into two groups, one group was fed a low casein diet, and the other a control diet. Both groups were fed the test diets ad libitum for 80 to 100 days. Then, the rats were killed, the liver mitochondria were isolated, and in experiment 1, swell ing of mitochondria induced by Ca2*, thyroxine and hypotonicity was measured, and in experiment 2, swelling induced by sodium oleate was measured. Preparation of mitochondria. The swell ing of mitochondria is affected greatly by co-existing substances and organelles other than mitochondria in the preparation. Con tamination of lysosomes is especially un desirable because free fatty acid produc tion would be accelerated by lysosomal enzymes. Therefore, in the present experi ments, to minimize the contamination, mitochondria were isolated by low speed TABLE 1 centrifugation 3,000 X g. The purity of Composition of diet1 the mitochondrial fraction was investi gated, and contamination by other cell organelles was found to be considerably lowered by such procedure than by one using centrifugation at 10,000 X g (20). Two kinds of media were used for the preparation of mitochondria. Preparation Casein«-Starch2Corn medium 1 contained 0.21 M mannitol, 0.07 oilMineral M sucrose, 0.1 mM EDTA, 0.01 M Tris B3Vitaminmixture buffer (pH 7.4) and 0.2% bovine serum mixture*Choline albumin. Preparation medium 2 contained chlorideLowcaseindiet485.6550.250.15Highcaseindiet2564.6550.250.15 onlv 0.21 M mannitol, 0.07 M sucrose and 1Diets were supplemented further with 6,000 IU 0.01 M Tris buffer (pH 7.4). retinol, 600 IU ergocalciferol and 100 mg DL-«Rats were killed; the livers were re tocopherol per kg diet. 2Commercial name moved and homogenized with preparation "Matsunorin" obtained from Matsutani Kagakukogyo Company, Japan. 3According to Harper medium 1. After removal of nuclei and (19). cell debris by low speed centrifugation,
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agents, but also by physical conditions such as hypotonicity (13, 14). The bio chemical mechanisms by which various agents have swelling action upon mito chondria have been investigated. Two biochemical mechanisms are known; swell ing of mitochondria is induced by accel eration of production of free fatty acids (12, 14) and by lipid peroxide formation (15, 16). Most swelling agents have been shown to cause swelling of mitochondria by accelerating production of free fatty acids (14). Recently, it was shown that there is a parallelism between mitochondrial swelling and phospholipase A activ ity, and free fatty acids would be pro duced as a result of phospholipase A action (17, 18). In the present experiments, Ca2* and thyroxine were used as representative swelling agents which accelerate produc tion of free fatty acids. Ca2t is also known to stimulate phospholipase A activity (17). The influence of a low casein diet upon the swelling of mitochondria induced by these agents was investigated. Free fatty acids themselves are also known to be swelling agents (12-14, 17); therefore, sodium oleate was also used. To compare the swelling due to these agents with the purely physical process, the influence of a low casein diet upon the hypotonie swell ing process was investigated. Animals and diets. Male rats of the Wistar strain, weighing about 170 g, were METHODS
LOW CASEIN DIET AND SWELLING OF MITOCHONDRIA
TABLE 3 The effect of low casein diet on liver mitochondrial swelling induced by Ca?+ (experiment 1) '
Incubation timemin0 CaseinO.D,
5 10 20 304%
CaseinO.D.
520 nm/mg 520 nm/mg protein1.736±0.054l protein1.699 ±0.090 1.301±0.1033 1.640±0.095 1.125 ±0.077» 1.451 ±0.125 0.889 ±0.0673 1.202±0.074 0.745±0.0513 1.083±0.06725%
1The value was expressed as optical density at 520 nm divided by protein content in 3 ml of re action mixture. 2Mean±so; five rats per group. 3Significantly different from the mean for the re spective 4% casein group. P < 0.01.
termined by the method of Lowry et al. (21). The significance of the difference be tween two groups of values was deter mined by the Student's t test (22).
RESULTS In table 2 are shown the final body weight, liver weight and number of rats per group. Ca-^-induced swelling. Mean values of optical density at 520 nm per fixed amount of protein at various time intervals during incubation with swelling medium contain ing Ca-+ were calculated for two groups (table 3). Mean values were significantly higher using mitochondria from rats fed a low casein diet than those from control rats. As can be seen from the value at zero time and 5 minutes, the slope of the curve for the first 5 minutes was steep TABLE 2 using mitochondria from rats fed a con Final body weight, liver weight and number trol diet, but it was very gentle using of rats per group those from rats fed a low casein diet. Thyroxine-indttced swelling. Mean values Dietgroup4% Rats/ body liver of optical density at 520 nm per fixed group weightNo. weightg5.8±0.3 amount of protein at various time intervals during incubation with swelling medium gExperiment containing thyroxine were calculated for 15 two groups (table 4). As in the case of 209±61 Ca2%mean values were significantly higher Casein Casein4% 25% 349±13Experiment 5 10.5 ±0.76.4±0.1using mitochondria from rats fed a low casein diet than those from control rats. 27 Casein 25% CaseinFinal 7 ' Mean dbso.
221 ±14 355± 7Final 11.7±0.8
1 Guaranteed reagent, Nakarai Kyoto, Japan. "Extra pure reagent, Nakarai Kyoto, Japan.
Chemicals Ltd., Chemicals Ltd.,
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mitochondria were sedimentad by centrifugation at 3,000 X g for 6 minutes. This mitochondrial fraction was washed once with preparation medium 1, and after that, was further purified by washing two more times with preparation medium 2. The final two washes were required be cause contamination with bovine serum albumin and EDTA could inhibit the swelling of mitochondria. After each wash, mitochondria were sedimented by centrifugation at 3,000 X g for 6 minutes. Assay of mitochondrial swelling jyrocess. Tests of mitochondrial swelling were car ried out using the method of Lehninger ( 13 ). Mitochondria from 4 g of the liver were suspended in 2 ml of preparation medium 2, and the suspension was kept in ice. Aliquots of this suspension were added to 3.0 ml of swelling medium containing 0.125 M KC1, 0.02 M Tris buffer (pH 7.4) and one of the swelling agents to produce an initial absorbancy of between 0.4 and 0.6 at 520 nm. The final concentrations of swelling agents in swelling media were as follows: Cado, 1 mM, DL-thyroxine,1 10 /¿M,or sodium oleate,2 30 /XM.In the case of hypotonie swelling, mitochondrial sus pension was added to the medium con taining only 0.02 M Tris buffer (pH 7.4). Incubations were carried out at 25°,and the decrease of optical density at 520 nm was followed for 20 or 30 minutes, and only in the case of sodium oleate, it was followed until the curves reached a plateau. In all the experiments, protein was dc-
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TABLE 6
The effect of low casein diet on liver milochondrial swelling induced by thyroxine (experiment 1) '
The effect of low casein diet on liver mitochondrial swelling induced by hypotonicity (experiment 1) '
CaseinO.D.
Incubation timemin010 CaseinO.D.
520 nm/mg 620 nm/mg protein1.678±0.0482 protein1.678±0.097
510 20 304%
1.272±0.030 1.180±0.035 1.119±0.041 1.091±0.05125%
1.134±0.0723 1.027±O.OG23 0.874±0.0(>03 0.799±0.0033
1The value was expressed as optical density at 520 nm divided by protein content in 3 ml of reaction mixture. 2Mean±SD;five rats per group. 'Sig nificantly different from the mean of the respective 4% casein group, P < 0.01.
Sodium oleate-induced swelling. Mean values of optical density at 520 nm per fixed amount of protein at various time in tervals during incubation with swelling medium containing sodium oleate were calculated for two groups (table 5). The mean values were always significantly higher using mitochondria from rats fed a low casein diet than those from control rats. There was a significant difference at zero time between the two groups. Since some time was required, after addition of mitochondrial suspension for mixing the reaction mixture, before recording the zero time value this difference is assumed to be due to the rapid effect of sodium oleate.
CaseinO.D.
520 nm/mg 620 nm/mg protein0.729±0.0312 protein0.699±0.012 204%
0.579±0.035 0.551±0.013 0.545±0.02325% 0.535±0.012
1The value was expressed as optical density at 520 nm divided by protein content in 3 ml of re action mixture. 2Mean±sD,five rats per group.. There were no significant differences between groups due to dietary protein level.
Hypotonie swelling. Mean values of optical density at 520 nm per fixed amount of protein at various time intervals during incubation in hypotonie medium were cal culated for the two groups (table 6). The mean values were only slightly higher using mitochondria from rats fed a low casein diet than those from control rats, and the differences were not significant.
DISCUSSION In the present experiments when rats were fed a low casein diet, the decrease of optical density at 520 nm was inhibited during the early period of swelling when Ca2t or thyroxine were used as swelling agents. On the other hand, when sodium oleate was used as a swelling agent, the zero time value was already different be tween the two groups, and thereafter, TABLE 5 values of two groups were always signifi The effect of low casein diet on liver mitochondrial swelling induced by sodium oleate (experiment A)1'1 cantly different. In the case of Ca2* or thyroxine, free Incubation fatty acids are produced during incubation CaseinO.D. timemin0 CaseinO.D. of mitochondria probably by the action of mitochondrial phospholipase A (17). Free 6eO nm/mg 500 nm/mg fatty acids have a powerful detergent ac protein1.715±0.058'protein1.532±0.0664 tion, especially at high concentration. Con 0.797±0.11f>« sidering these facts, the following two 0.992±0.112 1020 0.446±0.0684 mechanisms can be considered for the re 0.779±0.087 at plateau4% 0.574±0.02425% 0.362±0.026< sults of the present experiments. 1. As can be seen from table 5, the same 1The value was expressed as optical density at amount of free fatty acids always cause a 520 nm divided by protein content in 3 ml of re action mixture. *Elapsed time for the analysis greater swelling in the mitochondria from after adding mitochondria! suspension for mixing rats fed a 25% casein diet than those from the reaction mixture was about 30 seconds. 3Mean±so; seven rats per group. «Significantly rats fed a 4% casein diet. This would different from the mean of the respective 4% casein probably be caused by the fact that mito chondria from rats fed a 25% casein diet group, P < 0.01.
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Incubation timemin0 CaseinO.D.
LOW CASEIN DIET AND SWELLING OF MITOCHONDRIA
purely physical process was not affected by feeding rats a low casein diet. However, in a previous report by the author (1), electron micrographs revealed expansion of mitochondrial volume in puri fied liver mitochondria from rats fed a low casein diet, but not in those from control rats. The following reasons for the dis crepancy between this observation and the results of the present experiments are proposed: 1. In the previous report (1), when electron micrographs of enlarged mito chondria were taken, the composition of the 4% casein diet fed to rats was differ ent from that of the present experiments. The vitamin mixture was added at one third of the level used by Harper (19), rather than at the level used by Harper. Also, vitamin E was not added in the diet used in the previous report, but in the present experiments, 100 mg vitamin E/kg of diet was added. Therefore, the combi nation of the deficiency of protein and vitamins together might result in an ac celeration of the swelling of the mito chondria. Biomcmbranes would be fragile due to a deficiency of protein and vitamin E. Murata and Ikehata (23) reported that hemolysis was accelerated in rats fed a low vitamin E, low protein diet. 2. Mitochondria from rats fed a low casein diet might be less resistant to me chanical treatments, such as rcsuspending and centrifuging many times. In the pre vious report (1), when the electron micro graphs of enlarged mitochondria was taken, mitochondria were washed several times. 3. In the previous report (1), EDTA was always present in preparation me dium, but in the present experiments, EDTA was omitted from preparation medium 2. Presence of EDTA mignt have led to different results. The particularly, EDTA is known as a powerful inhibitor of phospholipase A (17). To ascertain whether the effects ob served have any relationship with the fat content of the liver, liver fat content was determined for the two groups. The values were, expressed as percentage of dry weight, for the 4% casein diet group, 20.49 ±8.04,3 and for the 25% casein diet » Mean ±SD five rats.
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are always more susceptible to the de tergent action of sodium oleate than those from rats fed a 4% casein diet. Especially in the case of sodium oleate, there was al ready a significant difference at zero time. The zero time value of the 4% casein diet group incubated with sodium oleate was not different from the Cajt and thyroxine group, but the value of the control group with sodium oleate was lower than the other group. This would mean that mito chondria from rats fed a control diet were affected more by the rapid detergent action of sodium oleate. This different be havior of mitochondria from two groups to the same amount of sodium oleate might be the reason for the inhibitory effect of a low casein diet on swelling of mitochon dria induced by Ca-+ or thyroxine. 2. In the case of Ca-* or thyroxine, in the early period of incubation, production of free fatty acids from mitochondria might be inhibited in rats fed a low casein diet, probably by inhibition of mitochondrial phospholipase A. It was suggested bv Waite et al. (17) that the role of phos pholipase A in swelling of mitochondria is to catalyze the hydrolysis of phospholipid at a key locale. It was also shown that hydrolysis of phospholipids was ac celerated very much by the presence of Ca-* during the first 5 minutes incubation of mitochondria. Thus, the swelling of mitochondria induced by Ca2f or thyrox ine during the first 5 minutes might be caused by hydrolysis of phospholipids at a key locale, thus resulting in changes in the nature of mitochondrial membrane. There fore, inhibition of swelling of mitochondria in ratsor fed a 4% during casein diet induced by Ca'-'* thyroxine the early period of incubation might owe to inhibition of phospholipase A activity. Further investigations should distinguish which of these two mechanisms is respon sible for the inhibitory effect of a low casein diet on swelling of mitochondria induced by Ca2t or thyroxine, or whether a combination of these two mechanisms is responsible. Compared to these cases using various swelling agents, hypotonie swelling of mi tochondria was not affected at all by feed ing rats a low casein diet. This means that swelling of mitochondria induced by a
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LITERATURE CITED 1. Harada, N. (1967) Morphological and res piratory changes in rat liver mitochondria resulting from a low casein diet. J. Nutr. 93, 263-272. 2. Hayashida, T. & Portman, O. W. (1960) Swelling of liver mitochondria from rats fed diets deficient in essential fatty acids. Proc. Soc. Exp. Biol. Med. 103, 656-659. 3. Johnson, R. M. (1963) Swelling studies on liver mitochondria from essential fatty acid deficient rats. Exp. Cell Res. 32, 118129. 4. Levin, E., Johnson, R. M. & Albert, S. (1957) Mitochondrial changes associated with essential fatty acid deficiency in rats. J. Biol. Chem. 228, 15-21. 5. Ito, T. & Johnson, R. M. (1964) Effects of a nutritional deficiency of unsaturated fats on rat liver mitochondria. I. Respiratory con trol and adenosine triphosphate-inorganic orthophosphate exchange activity. J. Biol. Chem. 239, 3201-3208. 6. Hall, J. C., Sordahl, L. A. & Stefko, P. L. ( 1960 ) The effect of insulin on oxidative phosphorylation in normal and diabetic mito chondria. J. Biol. Chem. 235, 1536-1539. 7. Burch, H. B., Hunter, F. E. Jr., Combs, A. N. & Schutz, B. A. (1960) Oxidative enzymes and phosphorylation in hepatic mitochondria from riboflavin-deficient rats. J. Biol. Chem. 235, 1540-1544. 8. Koppel, C. L. & Tandler, B. (1975) Riboflavin and mouse hepatic cell structure and function. Mitochondrial oxidative metabolism 'Unpublished
data.
in severe deficiency states. J. Nutr. 105, 562570. 9. Hunter, F. E. Jr. & Ford, L. (1955) Inactivation of oxidative and phosphorylative systems in mitochondria by preincubation with phosphate and other ions. J. Biol. Chem. 216, 357-369. 10. Tapley, D. F. (1956) The effect of thyrox ine and other substances on the swelling of isolated rat liver mitochondria. J. Biol. Chem. 222, 325-339. 11. Lehninger, A. L. & Schneider, M. (1959) Mitochondrial swelling induced by glutathion. J. Biochem. Biophys. Cytol. 5, 109-116. 12. Lehninger, A. L. & Remmert, L. F. (1959) An endogenous uncoupling and swelling agent in liver mitochondria and its enzymic formation. J. Biol. Chem. 234, 2459-2464. 13. Lehninger, A. L. (1959) Reversal of vari ous types of mitochondria! swelling by adeno sine triphosphate. J. Biol. Chem. 234, 24652471. 14. Wojtczak, L. & Lehninger, A. L. (1961) Formation and disappearance of an en dogenous uncoupling factor during swelling and contraction of mitochondria. Biochim. Biophys. Acta 51, 442-456. 15. Hoffsten, P. E., Hunter, F. E. Jr., Gebicki, J. M. & Weinstein,under J. (1962) of "lipid peroxide" conditions Formation which lead to swelling and lysis of rat liver mitochondria. Biochem. Biophys. Res. Commun. 7, 276280. 16. Hunter, F. E. Jr., Gebicki, J. M., Hoffsten, P. E., Weinstein, J. & Scott, A. (1963) Swelling and lysis of rat liver mitochondria induced by ferrous ions. J. Biol. Chem. 238, 828-835. 17. Waite, M., Deenen, L. L. M. Van, Ruigrok, T. J. C. & Elbers, P. F. (1969) Relation of mitochondrial phospholipase A activity to mitochondrial swelling. J. Lipid Res. 10, 599-608. 18. Waite, M. & Golde, L. M. Van (1988) Di etary induced alterations in swelling charac teristics and endogenous phospholipase Az activity of rat liver mitochondria. Lipids 3, 449-452. 19. Harper, A. E. (1959) Amino acid balance and imbalance. I. Dietary level of protein and amino acid imbalance. J. Nutr. 68, 405418. 20. Harada, N. & Sato, T. (1975) Some ad vantages of using low speed centrifugation for the purification of rat liver mitochondria. Agr. Biol. Chem. 39, 899-900. 21. Lowry, O. H., Rosebrough, N. J., Fan-, A. L. & Randall, R. J. (1951) Protein measure ment with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. 22. Snedecor, G. W. (1948) Statistical meth ods. Iowa State Univ. Press, Ames, Iowa, Chapters 3 and 4. 23. Murata, K. & Ikehata, H. (1965) Proteinvitamin E relation and the hemolysis test. Agr. Biol. Chem. 29, 809-812.
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group, 10.54 ±0.83. The results show tliat there was significantly more fat in the liver of rats fed the low casein diet. But, as can be seen from the so, the liver fat content of rats fed a low casein diet was more variable than that of control rats. On the contrary, the fluctuation in the swell ing process induced by Ca2+ and thyroxine was not different between the two groups as can be seen from the SD (tables 3 and 4). Moreover, in another experiment, when 1% orotic acid was added to the 25% casein diet, severe fatty liver was induced, but swelling of isolated mitochondria from such rats using Ca-* as the swelling agent was not inhibited, but rather, a little activated.4 From these facts, it would be reasonable to suppose that increased fat in livers of rats fed the 4% casein diet would not be of importance for the results of the present experiments.
