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Myofibrillar protein degradation and muscle proteinases in normal and diabetic rats
BIOCHEMICAL
MEDICINE
21,
33-39
(1979)
Myofibrillar Protein Degradation and Muscle Proteinases in Normal and Diabetic Rats B. DAHLMANN,~.
SCHROETER, L. HERBERTZ, AND H. REINAUER
Diabetes-Forschungsinstirut, Biochemical Department, Auj’m Hennekamp 65, 4000 Diisseldorf, Federal Republic of Germany Received
September
26, 1978
In diabetes mellitus, a known protein-wasting metabolic condition, the outflow of amino acids from muscle is enhanced, which may result from reduced protein synthesis, increased protein breakdown, or both (1, 2). Recent experiments (3) gave strong evidence that insulin controls muscle mass by regulating protein synthesis and protein breakdown. In accordance with this assumption we have previously shown, that in diabetic rats the muscle alkaline proteolytic activity was increased, whereas the activities of acidic proteinases did not change (4). These experiments of course did not give evidence whether the alkaline proteinases degrade myofilbrillar proteins to which they are partly associated. On the other hand, the breakdown of myofibrillar protein can be estimated by the renal excretion of 3-methylhistidine (5-9). Therefore, in the present experiments we measured muscle proteolytic activities, as well as the urinary excretion of 3-methylhistidine, in rats after induction of diabetes and subsequent insulin treatment. METHODS
Male rats of wistar HaN strain with initial body weight ranging from 180-200 g were obtained from Winkelmann, Paderborn, GFR. After fasting for 24 hr, 145 rats were divided into four groups. Group A. Each of 50 animals was injected intravenously with 100 mg of streptozotocin/kg body wt. Streptozotocin (Upjohn Co., Kalamazoo, Mich.) was dissolved in cold 0.1 M phosphate-citrate buffer, pH 4.5. Group B. Twenty-five animals were injected each with 100 mg streptozotocin/kg body wt. Beginning at the ninth day after the induction 33 OOM-2944/79/010033-07$U2.00/0 Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
34
DAHLMANN
ET AI
of diabetes the animals were treated with 3 IU of insulin/l00 g body wt daily (Insulin from Novo Industrie GmbH Pharmaceutics, Mainz, GFR). Insulin was applied in each case at 4 P.M. Group C. Ten animals were injected each with 100 mg of streptozotocinlkg body wt. Beginning at the second day after the induction of diabetes, the animals were treated with 5 IU of insulin/100 g body wt by daily subcutaneous injections. Group D. Fifty-five rats were used as untreated (single injection of phosphate-citrate buffer, pH 4.5) controls. Diabetic state was defined by increased blood glucose levels, ketonuria, polyphagia, and decreased levels of blood insulin. Rats were killed by a blow on the head, and hind leg skeletal muscles were quickly removed and freed of fat and connective tissue. The preparation of the muscle homogenate and measurement of the alkaline (at pH 9) proteolytic activity by hydrolysis of [‘4Clhemoglobin was performed as described elsewhere (4). Overall aminopeptidase activity was measured by the hydrolysis of leucine-4-nitroanilide (2.5 mM) dissolved in 0.05 M Tris-HCI buffer, pH 8.5, containing 1 M KC1 and 5% (v/v) dimethyl formamide. This substrate solution (0.5 ml) and 0.2 ml of the muscle homogenate (5 mg protein/ml) were mixed and incubated at 37°C for 60 min. The reaction was stopped by the addition of 0.3 ml of 8% (w/v) trichloroacetic acid. After centrifugation (5 min. 1l,OOOg), the concentration of the enzymatically liberated nitroaniline was measured at 405 nm (10). Linear relationships between the amount of muscle tissue homogenate (up to 10 mg protein/ml) and hydrolysis of [14C]hemog10bin and leucine-4-nitroanilide, respectively, under the conditions used, were established in preliminary experiments. Protein concentration was measured with the biuret method with LAB-TROL (Dade, Miami, Fla.,) as a standard. Urine samples (24 hr) of the rats from groups A, C, and D were collected in metabolic cages. For 3-methylhistidine analysis, an aliquot of the urine sample was hydrolyzed for 18 hr at 110°C in 6 N HCI, evaporated, and chromatographed on a column (0.6 x 25 cm) of Durrum DC-4A. Elution of the amino acids was accomplished according to a modified program of Biotronik using an automatic amino acid analyzer BT 6000 E equipped with a fluorescence detector BT 6630 (Biotronik, Miinchen, GFR). RESULTS The overall activity of muscle alkaline proteinases did not change in the group of control rats (group D) during the experimental period, whereas 4 days after onset of diabetes (group A) the specific activity was significantly elevated and increased further to about 250% when compared with the controls (Fig. 1). The same significant rise was observed in the
MUSCLE
PROTEOLYSIS
0
2
4
6
IN DIABETIC
6 IO days
RATS
35
12 14 16 16
FIG. 1. Muscle alkaline proteolytic activity of normal (0, group D) and diabetic (A, group A) rats. STR, streptozotocin. Means 2 SEM are indicated. The increase of activity was significant (Student’s t test, P < 0.05) 4 days after onset of diabetes.
overall activity of skeletal muscle aminopeptidases in diabetic rats (Fig. 2). Treatment of diabetic rats with insulin for 8 days (group B) reduced the muscle aminopeptidase activity to control level (Fig. 3). In a similar way, the activity of muscle alkaline proteinases significantly declined after 8 days of treatment of diabetic rats with 3 IU of insulin000 g body wt (Fig. 3). In rats treated with insulin daily beginning 2 days after the induction of diabetes (group C), muscle alkaline proteolytic activity and aminopeptidase activity were also normalized within 1 week (Fig. 3). Corresponding to the low activity of the muscle proteinases in the group of control rats (group D), their daily excretion of 3-methylhistidine did not exceed 0.7 Fmole/24 hr during the experimental period, whereas the 3-methylhistidine excretion of rats 2 days after induction of diabetes rose to 1.9 pmole/24 hr (Fig. 4). After 9 and 15 days of diabetes, the excretion rate was about five-fold higher than in normal rats. This drastic increase of the 3-methylhistidine excretion was repressed by treatment of the diabetic animals with insulin (group C). Normalization of the 3-methylhistidine excretion was attained after 5 days of insulin treatment of the diabetic rats.
36
DAHLMANN
ET AL AL..
4 1 .$I 3
0 2
4
6
6
10
12 14
, 16 16
days FIG. 2. Muscle aminopeptidase activity of normal (0, group D) and diabetic (A, group A) rats. STR, streptozotocin. Means ? SEM are indicated. The increase of activity was significant (Student’s t test, P < 0.05) 2 days after onset of diabetes.
5 UJ insulin
3 UJ. insulin
c
1
4 ““4 \ ‘--y-1;I z!L‘\ IL- ------ --+I -------____A -0“‘c., \ '
.
L
1
I
I
1
1
I
I
1
I
0
2
4
6
6
10
12
14
16
18
days
FIGURE
3
MUSCLE
PROTEOLYSIS
IN DIABETIC
RATS
37
DISCUSSION
In previous reports (4,11), it was shown that the muscle alkaline proteolytic activity in rats, which is partly associated with the myofibrillar fraction, increases in the diabetic state and decreases after insulin treatment of the diabetic rats. In the present experiments, the overall activities of muscle aminopeptidases was measured at the same time and concomitant change of aminopeptidase activity with the alkaline proteinases could be shown. These data suggest that the nonlysosomal, alkaline proteolytic system shows a highly adaptive change of its activity. The insulin-dependent alteration of enzyme pattern does not prove necessarily that there is some causal correlation between these findings and muscle protein degradation. Therefore, the excretion of 3-methylhistidine into urine of diabetic and control rats was measured, because the excretion of this amino acid derivative reflects the amount of degraded actomyosin in skeletal muscle (5-9). In fact we could show that the elevation of muscle proteolytic activities is accompanied by a four- to five-fold increased excretion rate of 3-methylhistidine. Furthermore, since insulin treatment of the diabetic rats resulted in normalization of both the proteolytic activities and the urinary excretion of 3-methylhistidine, it is very likely that the alkaline proteinases and aminopeptidases of the skeletal muscle are involved in the degradation process of actomyosin. This is supported by a report about the ability of muscle serine proteinase to degrade actin and myosin in vitro (12). The enzyme, whose activity is increased in dystrophic muscle, has optimum activity in the alkaline pH range (13,14). On the other side it is reported that acidic proteinases are responsible for autolysis in genetically dystrophic (15,16) and denervated skeletal muscle (17,18), and it has been shown that cathepsin B and D (19) and other unidentified acidic proteinases from muscle (20,21) are able to degrade myosin and actin in vitro. Since protein degradation in normal and dystrophic muscles is lowered in vitro by the bacterial proteinase inhibitors, pepstatin (22) and leupeptin (23), it may be possible that cathepsin B and D play a role in muscle protein turnover. On the other hand, leupeptin also inhibits the alkaline proteinase which was recently isolated from rat skeletal muscle in our laboratory (24). Further both FIG. 3. Muscle alkaline proteolytic activity and aminopeptidase activity in diabetic rats treated with insulin. Alkaline proteolytic activity (A) and aminopeptidase activity (A) in diabetic rats treated with 3 IU insulin/lOOgbody wffday (group B). After ldays treatment with insulin, no significant difference (Student’s t test) was observed for the activities as compared to the activities in the control rats (group D). Alkaline proteolytic activity (0) and aminopep tidase activity (W) in diabetic rats treated with 5 IU insulin400 g body wt/day (group C). Means * SEM are indicated. The activities after 7 days treatment with insulin were not significantly different (Student’s t test) from the activities measured in the control group (group D).
38
DAHLMANN
ET AI
streptozotocin insulin I I
-
4i \\
i \
\
\
\ I
D>++&j I
o
L
2
4
6
1 6 days
I 10
1 12
1 14
I 16
I 16
FIG. 4. Urinary excretion of 3-methylhistidine in normal (0, group D), diabetic (A, group A) and insulin (5 III)-treated diabetic rats (m, group C). Means + SEM are indicated. The increase of 3-methylhistidine excretion of diabetic rats was significant (Student’s t test, P < 0.01) as compared to the control rats. After 5 days of insulin treatment of diabetic rats. their 3-methylhistidine excretion was no more different (Student’s t test) from the values measured
in control
rats.
cathepsins are located in lysosomes, but lysosomes do not engulf myofibril or actin and myosin filaments (25,26). Consequently, if in vivo lysosomal enzymes participate in the degradation of myofibrillar proteins, the initiating step for actomyosin degradation has to be an extralysosomal disruption of the myofibrils by nonlysosomal proteinases. As the muscle alkaline proteinases and also the aminopeptidases show adaptive behavior to the insulin level and run parallel to the 3-methylhistidine excretion in diabetic rats, we suggest that this degradative system is responsible for the breakdown of skeletal muscle myofibrils in diabetic rats. SUMMARY
In diabetic rats, the overall activities of alkaline proteinases and aminopeptidases in skeletal muscle are increased two- to three-fold. The elevation of proteolytic activities may be responsible for the massive breakdown of myofibrillar proteins indicated by a five-fold increase of 3-methylhistidine excretion into urine. Insulin treatment of the diabetic rats results in the normalization of both muscle proteolytic activities as
MUSCLE
PROTEOLYSIS
IN DIABETIC
39
RATS
well as the 3-methylhistidine excretion. Our data suggest that in diabetic rats the enhanced muscle protein catabolism is effected by the increased activities of alkaline proteinases and aminopeptidases and that insulin is a regulatory hormone of protein catabolism. ACKNOWLEDGMENTS This work was supported by the Ministerium fur Wissenschaft und Forschung des Landes Nordrhein-Westfalen, Dusseldorf, and the Bundesministerium fur Jugend, Familie und Gesundheit, Bonn, G.F.R. We thank Mr. L. Bohne for excellent technical assistance.
REFERENCES 1. Cahill, G. F. Jr., Aoki, T. T., and Marliss, E. B., in “Handbook of Physiology, Section 7: Endocrinology,” Vol. I, p. 563. Williams & Wilkens. Baltimore, 1972. 2. Felig, P., Metabolism 22, 179 (1973). 3. Fulks, R. M., Li, I. B., and Goldberg, A. L., J. Biol. Chem. 250, 2% (1975). 4. Rothig, H. J., Stiller, N., Dahlmann, B., and Reinauer, H., Horm. Metabol. Res. 10, 101 (1978). 5. Asatoor, A. M., and Armstrong, M. D., Biochem. Biophys. Res. Commun. 26, 168 (1%7). 6. Johnson, P., Harris, C. I., and Perry, S. V., Biochem. J. 105, 361 (1%7). 7. Haverberg, L. N., Omstedt, P. T., Munro, H. N.. and Young, V. R., B&hem. Biophys. Acta
67 (1975).
405,
8. Young, V. R., Haverberg, L. N., Bilmazes, C., and Munroe, H. N.,
Metabolism
22,
1429 (1973). 9.
Haverberg, L. N., Deckelbaum, L., Bilmazes. C., Munroe. H. N., and Young, V. R.,
IO. II. 12. 13. 14.
Wachsmuth, E. D., Fritze, I., and Ptleiderer, G., Biochemistry 5, 169 (1966). Mayer, M., Amin. R., and Shafrir, E., Arch. Biochem. Biophys. 161, 20 (1974). Yasogawa, N., Sanada, Y., and Katunuma, N.. J. Biochem. 83, 1355 (1978). Sanada, Y., Yasogawa, N., and Katunuma, N.. J. Biochem. 83, 27 (1978). Katunuma, N., Yasogawa, N., Kito. K.. Sanada, Y., Kawai, H., and Miyoshi, K., J.
Biochem.
Biochem.
J. 152, 503 (1975).
83,
625
(1978).
15. Tappel, A. L., Zalkin, H.. Caldwell, K. A., Desai, I. D., and Shibko, S. ,Arch. Biochem. Biophys. 96, 340 (l%2). 16. Pennington, R. J., Biochem. J. 88, 64 (1963). 17. Pluskal, M. G.. and Pennington, R. J., Biochem. Sot. Trans. 1, 1307 (1973). 18. McLaughlin, J., and Bosmann, H. B., Int. J. Biochem. 7, 125 (1976). 19. Schwartz, W. N., and Bird, J. W. C., Biochem. J. 167, 811 (1977). 20. Arakawa, N.. Fujiki. S., Inagaki, C., and Fujimaki, M., &r. B;ol. Chem. 40, 1265 (1976). 21. Okitani, A., Matsukura, U., Otsuka. Y.. Watanabe, M.. and Fujimaki, M., Agr. Biol. Chem.
22.
23. 24. 25. 26.
41, 1821 (1977).
Iodice, A. A., Life Sci. 19, 1351 (1976). Libby, P., and Goldberg, A. L., Science 199, 534 (1978). Dahlmann, B., and Reinauer, H., Biochem. J. 171, 803 (1978). Schiaftino, S., and Hanzilokova, V., J. U/trastruct. Res. 39, 1 Lockshin, R. A., and Beaulaton. J., J. Ultrastruct. Res. 46, 63
(1972). (1974).
MEDICINE
21,
33-39
(1979)
Myofibrillar Protein Degradation and Muscle Proteinases in Normal and Diabetic Rats B. DAHLMANN,~.
SCHROETER, L. HERBERTZ, AND H. REINAUER
Diabetes-Forschungsinstirut, Biochemical Department, Auj’m Hennekamp 65, 4000 Diisseldorf, Federal Republic of Germany Received
September
26, 1978
In diabetes mellitus, a known protein-wasting metabolic condition, the outflow of amino acids from muscle is enhanced, which may result from reduced protein synthesis, increased protein breakdown, or both (1, 2). Recent experiments (3) gave strong evidence that insulin controls muscle mass by regulating protein synthesis and protein breakdown. In accordance with this assumption we have previously shown, that in diabetic rats the muscle alkaline proteolytic activity was increased, whereas the activities of acidic proteinases did not change (4). These experiments of course did not give evidence whether the alkaline proteinases degrade myofilbrillar proteins to which they are partly associated. On the other hand, the breakdown of myofibrillar protein can be estimated by the renal excretion of 3-methylhistidine (5-9). Therefore, in the present experiments we measured muscle proteolytic activities, as well as the urinary excretion of 3-methylhistidine, in rats after induction of diabetes and subsequent insulin treatment. METHODS
Male rats of wistar HaN strain with initial body weight ranging from 180-200 g were obtained from Winkelmann, Paderborn, GFR. After fasting for 24 hr, 145 rats were divided into four groups. Group A. Each of 50 animals was injected intravenously with 100 mg of streptozotocin/kg body wt. Streptozotocin (Upjohn Co., Kalamazoo, Mich.) was dissolved in cold 0.1 M phosphate-citrate buffer, pH 4.5. Group B. Twenty-five animals were injected each with 100 mg streptozotocin/kg body wt. Beginning at the ninth day after the induction 33 OOM-2944/79/010033-07$U2.00/0 Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
34
DAHLMANN
ET AI
of diabetes the animals were treated with 3 IU of insulin/l00 g body wt daily (Insulin from Novo Industrie GmbH Pharmaceutics, Mainz, GFR). Insulin was applied in each case at 4 P.M. Group C. Ten animals were injected each with 100 mg of streptozotocinlkg body wt. Beginning at the second day after the induction of diabetes, the animals were treated with 5 IU of insulin/100 g body wt by daily subcutaneous injections. Group D. Fifty-five rats were used as untreated (single injection of phosphate-citrate buffer, pH 4.5) controls. Diabetic state was defined by increased blood glucose levels, ketonuria, polyphagia, and decreased levels of blood insulin. Rats were killed by a blow on the head, and hind leg skeletal muscles were quickly removed and freed of fat and connective tissue. The preparation of the muscle homogenate and measurement of the alkaline (at pH 9) proteolytic activity by hydrolysis of [‘4Clhemoglobin was performed as described elsewhere (4). Overall aminopeptidase activity was measured by the hydrolysis of leucine-4-nitroanilide (2.5 mM) dissolved in 0.05 M Tris-HCI buffer, pH 8.5, containing 1 M KC1 and 5% (v/v) dimethyl formamide. This substrate solution (0.5 ml) and 0.2 ml of the muscle homogenate (5 mg protein/ml) were mixed and incubated at 37°C for 60 min. The reaction was stopped by the addition of 0.3 ml of 8% (w/v) trichloroacetic acid. After centrifugation (5 min. 1l,OOOg), the concentration of the enzymatically liberated nitroaniline was measured at 405 nm (10). Linear relationships between the amount of muscle tissue homogenate (up to 10 mg protein/ml) and hydrolysis of [14C]hemog10bin and leucine-4-nitroanilide, respectively, under the conditions used, were established in preliminary experiments. Protein concentration was measured with the biuret method with LAB-TROL (Dade, Miami, Fla.,) as a standard. Urine samples (24 hr) of the rats from groups A, C, and D were collected in metabolic cages. For 3-methylhistidine analysis, an aliquot of the urine sample was hydrolyzed for 18 hr at 110°C in 6 N HCI, evaporated, and chromatographed on a column (0.6 x 25 cm) of Durrum DC-4A. Elution of the amino acids was accomplished according to a modified program of Biotronik using an automatic amino acid analyzer BT 6000 E equipped with a fluorescence detector BT 6630 (Biotronik, Miinchen, GFR). RESULTS The overall activity of muscle alkaline proteinases did not change in the group of control rats (group D) during the experimental period, whereas 4 days after onset of diabetes (group A) the specific activity was significantly elevated and increased further to about 250% when compared with the controls (Fig. 1). The same significant rise was observed in the
MUSCLE
PROTEOLYSIS
0
2
4
6
IN DIABETIC
6 IO days
RATS
35
12 14 16 16
FIG. 1. Muscle alkaline proteolytic activity of normal (0, group D) and diabetic (A, group A) rats. STR, streptozotocin. Means 2 SEM are indicated. The increase of activity was significant (Student’s t test, P < 0.05) 4 days after onset of diabetes.
overall activity of skeletal muscle aminopeptidases in diabetic rats (Fig. 2). Treatment of diabetic rats with insulin for 8 days (group B) reduced the muscle aminopeptidase activity to control level (Fig. 3). In a similar way, the activity of muscle alkaline proteinases significantly declined after 8 days of treatment of diabetic rats with 3 IU of insulin000 g body wt (Fig. 3). In rats treated with insulin daily beginning 2 days after the induction of diabetes (group C), muscle alkaline proteolytic activity and aminopeptidase activity were also normalized within 1 week (Fig. 3). Corresponding to the low activity of the muscle proteinases in the group of control rats (group D), their daily excretion of 3-methylhistidine did not exceed 0.7 Fmole/24 hr during the experimental period, whereas the 3-methylhistidine excretion of rats 2 days after induction of diabetes rose to 1.9 pmole/24 hr (Fig. 4). After 9 and 15 days of diabetes, the excretion rate was about five-fold higher than in normal rats. This drastic increase of the 3-methylhistidine excretion was repressed by treatment of the diabetic animals with insulin (group C). Normalization of the 3-methylhistidine excretion was attained after 5 days of insulin treatment of the diabetic rats.
36
DAHLMANN
ET AL AL..
4 1 .$I 3
0 2
4
6
6
10
12 14
, 16 16
days FIG. 2. Muscle aminopeptidase activity of normal (0, group D) and diabetic (A, group A) rats. STR, streptozotocin. Means ? SEM are indicated. The increase of activity was significant (Student’s t test, P < 0.05) 2 days after onset of diabetes.
5 UJ insulin
3 UJ. insulin
c
1
4 ““4 \ ‘--y-1;I z!L‘\ IL- ------ --+I -------____A -0“‘c., \ '
.
L
1
I
I
1
1
I
I
1
I
0
2
4
6
6
10
12
14
16
18
days
FIGURE
3
MUSCLE
PROTEOLYSIS
IN DIABETIC
RATS
37
DISCUSSION
In previous reports (4,11), it was shown that the muscle alkaline proteolytic activity in rats, which is partly associated with the myofibrillar fraction, increases in the diabetic state and decreases after insulin treatment of the diabetic rats. In the present experiments, the overall activities of muscle aminopeptidases was measured at the same time and concomitant change of aminopeptidase activity with the alkaline proteinases could be shown. These data suggest that the nonlysosomal, alkaline proteolytic system shows a highly adaptive change of its activity. The insulin-dependent alteration of enzyme pattern does not prove necessarily that there is some causal correlation between these findings and muscle protein degradation. Therefore, the excretion of 3-methylhistidine into urine of diabetic and control rats was measured, because the excretion of this amino acid derivative reflects the amount of degraded actomyosin in skeletal muscle (5-9). In fact we could show that the elevation of muscle proteolytic activities is accompanied by a four- to five-fold increased excretion rate of 3-methylhistidine. Furthermore, since insulin treatment of the diabetic rats resulted in normalization of both the proteolytic activities and the urinary excretion of 3-methylhistidine, it is very likely that the alkaline proteinases and aminopeptidases of the skeletal muscle are involved in the degradation process of actomyosin. This is supported by a report about the ability of muscle serine proteinase to degrade actin and myosin in vitro (12). The enzyme, whose activity is increased in dystrophic muscle, has optimum activity in the alkaline pH range (13,14). On the other side it is reported that acidic proteinases are responsible for autolysis in genetically dystrophic (15,16) and denervated skeletal muscle (17,18), and it has been shown that cathepsin B and D (19) and other unidentified acidic proteinases from muscle (20,21) are able to degrade myosin and actin in vitro. Since protein degradation in normal and dystrophic muscles is lowered in vitro by the bacterial proteinase inhibitors, pepstatin (22) and leupeptin (23), it may be possible that cathepsin B and D play a role in muscle protein turnover. On the other hand, leupeptin also inhibits the alkaline proteinase which was recently isolated from rat skeletal muscle in our laboratory (24). Further both FIG. 3. Muscle alkaline proteolytic activity and aminopeptidase activity in diabetic rats treated with insulin. Alkaline proteolytic activity (A) and aminopeptidase activity (A) in diabetic rats treated with 3 IU insulin/lOOgbody wffday (group B). After ldays treatment with insulin, no significant difference (Student’s t test) was observed for the activities as compared to the activities in the control rats (group D). Alkaline proteolytic activity (0) and aminopep tidase activity (W) in diabetic rats treated with 5 IU insulin400 g body wt/day (group C). Means * SEM are indicated. The activities after 7 days treatment with insulin were not significantly different (Student’s t test) from the activities measured in the control group (group D).
38
DAHLMANN
ET AI
streptozotocin insulin I I
-
4i \\
i \
\
\
\ I
D>++&j I
o
L
2
4
6
1 6 days
I 10
1 12
1 14
I 16
I 16
FIG. 4. Urinary excretion of 3-methylhistidine in normal (0, group D), diabetic (A, group A) and insulin (5 III)-treated diabetic rats (m, group C). Means + SEM are indicated. The increase of 3-methylhistidine excretion of diabetic rats was significant (Student’s t test, P < 0.01) as compared to the control rats. After 5 days of insulin treatment of diabetic rats. their 3-methylhistidine excretion was no more different (Student’s t test) from the values measured
in control
rats.
cathepsins are located in lysosomes, but lysosomes do not engulf myofibril or actin and myosin filaments (25,26). Consequently, if in vivo lysosomal enzymes participate in the degradation of myofibrillar proteins, the initiating step for actomyosin degradation has to be an extralysosomal disruption of the myofibrils by nonlysosomal proteinases. As the muscle alkaline proteinases and also the aminopeptidases show adaptive behavior to the insulin level and run parallel to the 3-methylhistidine excretion in diabetic rats, we suggest that this degradative system is responsible for the breakdown of skeletal muscle myofibrils in diabetic rats. SUMMARY
In diabetic rats, the overall activities of alkaline proteinases and aminopeptidases in skeletal muscle are increased two- to three-fold. The elevation of proteolytic activities may be responsible for the massive breakdown of myofibrillar proteins indicated by a five-fold increase of 3-methylhistidine excretion into urine. Insulin treatment of the diabetic rats results in the normalization of both muscle proteolytic activities as
MUSCLE
PROTEOLYSIS
IN DIABETIC
39
RATS
well as the 3-methylhistidine excretion. Our data suggest that in diabetic rats the enhanced muscle protein catabolism is effected by the increased activities of alkaline proteinases and aminopeptidases and that insulin is a regulatory hormone of protein catabolism. ACKNOWLEDGMENTS This work was supported by the Ministerium fur Wissenschaft und Forschung des Landes Nordrhein-Westfalen, Dusseldorf, and the Bundesministerium fur Jugend, Familie und Gesundheit, Bonn, G.F.R. We thank Mr. L. Bohne for excellent technical assistance.
REFERENCES 1. Cahill, G. F. Jr., Aoki, T. T., and Marliss, E. B., in “Handbook of Physiology, Section 7: Endocrinology,” Vol. I, p. 563. Williams & Wilkens. Baltimore, 1972. 2. Felig, P., Metabolism 22, 179 (1973). 3. Fulks, R. M., Li, I. B., and Goldberg, A. L., J. Biol. Chem. 250, 2% (1975). 4. Rothig, H. J., Stiller, N., Dahlmann, B., and Reinauer, H., Horm. Metabol. Res. 10, 101 (1978). 5. Asatoor, A. M., and Armstrong, M. D., Biochem. Biophys. Res. Commun. 26, 168 (1%7). 6. Johnson, P., Harris, C. I., and Perry, S. V., Biochem. J. 105, 361 (1%7). 7. Haverberg, L. N., Omstedt, P. T., Munro, H. N.. and Young, V. R., B&hem. Biophys. Acta
67 (1975).
405,
8. Young, V. R., Haverberg, L. N., Bilmazes, C., and Munroe, H. N.,
Metabolism
22,
1429 (1973). 9.
Haverberg, L. N., Deckelbaum, L., Bilmazes. C., Munroe. H. N., and Young, V. R.,
IO. II. 12. 13. 14.
Wachsmuth, E. D., Fritze, I., and Ptleiderer, G., Biochemistry 5, 169 (1966). Mayer, M., Amin. R., and Shafrir, E., Arch. Biochem. Biophys. 161, 20 (1974). Yasogawa, N., Sanada, Y., and Katunuma, N.. J. Biochem. 83, 1355 (1978). Sanada, Y., Yasogawa, N., and Katunuma, N.. J. Biochem. 83, 27 (1978). Katunuma, N., Yasogawa, N., Kito. K.. Sanada, Y., Kawai, H., and Miyoshi, K., J.
Biochem.
Biochem.
J. 152, 503 (1975).
83,
625
(1978).
15. Tappel, A. L., Zalkin, H.. Caldwell, K. A., Desai, I. D., and Shibko, S. ,Arch. Biochem. Biophys. 96, 340 (l%2). 16. Pennington, R. J., Biochem. J. 88, 64 (1963). 17. Pluskal, M. G.. and Pennington, R. J., Biochem. Sot. Trans. 1, 1307 (1973). 18. McLaughlin, J., and Bosmann, H. B., Int. J. Biochem. 7, 125 (1976). 19. Schwartz, W. N., and Bird, J. W. C., Biochem. J. 167, 811 (1977). 20. Arakawa, N.. Fujiki. S., Inagaki, C., and Fujimaki, M., &r. B;ol. Chem. 40, 1265 (1976). 21. Okitani, A., Matsukura, U., Otsuka. Y.. Watanabe, M.. and Fujimaki, M., Agr. Biol. Chem.
22.
23. 24. 25. 26.
41, 1821 (1977).
Iodice, A. A., Life Sci. 19, 1351 (1976). Libby, P., and Goldberg, A. L., Science 199, 534 (1978). Dahlmann, B., and Reinauer, H., Biochem. J. 171, 803 (1978). Schiaftino, S., and Hanzilokova, V., J. U/trastruct. Res. 39, 1 Lockshin, R. A., and Beaulaton. J., J. Ultrastruct. Res. 46, 63
(1972). (1974).