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Metabolic adaptation in muscle and brown adipose tissues to insulin deficiency in streptozotocin treated rats
Comp. Biochem Physiol., Vol. 66B, pp. 391 to 395
0305-0491,'80,'0701-0391S02.00/0
© Pergamon Press Lid 1980. Printed in Great Britain
METABOLIC ADAPTATION IN MUSCLE A N D BROWN ADIPOSE TISSUES TO INSULIN DEFICIENCY IN STREPTOZOTOCIN TREATED RATS R. RAURAMAA,M. HARRI,E. PUHAKAINEN,P. KUUSELA and O. H,~NNINEN Department of Physiology, University of Kuopio, P.O. Box 138, SF-70101 Kuopio 10, Finland (Received 12 November 1979)
Abstract--l. Glycogen stores in skeletal muscle were increased in diabetic rats compared to controls 2 months after streptozotocin treatment. 2. The increase in muscle glycogen stores seemed to occur faster in soleus muscle compared to gastrocnemius muscle. 3. Insulin deficiency caused by streptozotocin administration had only minimal effects on the enzyme activities either in skeletal muscle or in brown adipose tissue. INTRODUCTION
MATERIALS AND METHODS
Skeletal muscle is one of the main target tissues of insulin (Clausen, 1975; Olefsky et al., 1976). This hormone is required among other functions for glucose uptake (e.g. Berger et al., 1975). One could therefore expect lowered glycogen stores in muscle in insulin deficiency. This has indeed been demonstrated, by Roch-Norlund (1972) in human diabetics, both in juvenile and maturity-onset types. G o o d m a n et al. (1974) and K a m m & Strope (1974) have observed lowered muscle glycogen content also in diabetic rats. On the other hand, Armstrong & Ianuzzo (1977) found no differences in the amount of muscle glycogen between control and diabetic rats. The level of tissue glycogen depends not only on the penetration and deposition of glucose in cells but also on the ability of cells to consume glycosyl units. Glycolytic enzymes have been reported to be lower in skeletal muscles in experimental (Murphy & Anderson, 1974; Ianuzzo & Armstrong, 1976) and in human (Vondra et al., 1977) diabetes. Also mitochondrial enzyme activities are depressed in experimental (Ianuzzo et al., 1974) and in human (Vondra et al., 1977) diabetes. These would tend to save glycogen stores. Similar effects have free fatty acids and ketone bodies, the availability of which increases when the antilipolytic action of insulin (Desai & Hollenberg, 1975) diminishes in diabetes. The present series of experiments was started to clarify the metabolic reserves for exercise in experimental diabetes in rats. For this purpose the glycogen levels and the activities of phosphofructokinase, citrate synthase and malate dehydrogenase were followed in two muscles representing different functional types. Samples were taken from the medial head of gastrocnemius, which is composed of all three types of muscle fibres, and from soleus containing only slow and intermediate fibres (e.g. Stein & Padykula, 1962). For comparison the enzyme levels in brown adipose tissue were followed, since this forms its own type of tissue with high consumption of energy substrates. It is also sensitive to several exogenous and endogenous stimuli (e.g. Harri & Valtola, 1975; Harri & Narvola, 1979).
Male albino rats (Wistar/af/Han/Mol (Kuo 67)) weighing 210-225 g at the beginning of the study were used. Rats were housed in net floor cages at 20 _+ 2'C with a daily 14:10 light-dark cycle. They were fed rat food pellets (Hankkija Co., Finland) ad libitum, and tap water was always available. Diabetes was produced by an intravenous (femoral vein) injection of streptozotocin (kindly donated by Dr E. W. Dulin, Upjohn Co., Kalamazoo, MI, U.S.A.) (75 mg/kg) to pentobarbital (30--50mg/kg, intraperitoneally) anaesthetized rats. Streptozotocin was dissolved in 0.9°0 NaCI-O.1 M citrate buffer, pH 4.5 always immediately before injection. Control rats received one injection of buffer only. Urinary glucose and possible ketonuria was measured 3 days after streptozotocin treatment with the aid of reagent sticks (Keto-Diastix, Ames Co., Great Britain). One week, one month and two months after the streptozotocin injection, rats were anaesthetized again, and, in addition to blood, the following tissue samples were taken: interscapular brown adipose tissue, soleus and the medial head of gastrocnemius muscles. Visible connective tissue and blood clots were removed. Tissue samples were frozen with liquid nitrogen and kept at -80°C until analysed. Blood samples for glucose determinations (by glucose oxidase-peroxidase method, Glox Novum, Kabi Ab, Sweden) were drawn by cardiac puncture and placed in tubes containing ftuoride-oxalate. For the determination of glycogen content, the muscle samples were homogenized in 1 M HC1 and heated at 100°C for 2 hr. The glycogen content was then analysed as glycosyl units according to the method of Hultman (1967). For enzyme assays the tissue samples were weighed and homogenized in a Potter Elvehjem glass homogenizer in Tris-HCl buffer (0. l M, pH 7.6) to a 2'7o homogenate (w/v), and centrifuged for 10rain at 1000g at 4°C to remove unbroken cells and particulate debris. The supernatants were used for determining the enzyme activities. The protein contents were estimated by the phenol method of Lowry et al. (195l). The activity of phosphofructokinase (PFK) (EC 2.7.1.11) was determined according to BostriSm el al. (1974). Citrate synthase (CS) (EC 4.1.3.7) activity was analysed according to Srere (1969) and malate dehydrogenase (MDH) (EC 1.1.1.37) activity by the method of Englard & Siegel (1969). The measurements were made at 37c'C with a Cary 118 double-beam spectrophotometer. Enzyme activities were calculated as micromoles of substrate utilized per min and g of fresh tissue weight and per mg of protein. 391
392
R. RAURAMAAet al.
Table 1. Final body weights and blood glucose levels in control and in diabetic rats l week, I month and 2 months after streptozotocin treatment Body w e i g h t (g)
Blood g l u c o s e (n~nol/1)
Group
Controls
Diabetics
Controls
Diabetics
1 Week
24828.6
218t3.7"
6.2~3.1
16.2t2.08"*
(s]
1 Month 2 Months
(s)
291t8.1 (9]
262~8.8
337t5.9
278t8.4
(4)
19.9-].61
6.0tO. ?~
(7)
(9)
U)
6.4:0,27
(1])
(9)
(4)
21.iti.02"**
(8)
(n)
Means, _+SEM have been given, number of rats in parentheses. Asterisks indicate significant difference between controls and diabetics as follows: *P < 0.05, **P < 0.01, ***P < 0.001 and NS not significant.
RESULTS
Streptozotocin treated rats gained weight more slowly than did the controls. The difference was observable already 1 week after the streptozotocin injection. The success in producing diabetes was evident from the high blood glucose values (Table 1). Diabetic rats had also a marked glucosuria but not ketonuria. Glycogen was determined in soleus and gastrocnemius muscles 1 and 2 months after the induction of diabetes. After 1 week neither muscle showed any changes in glycogen levels (not shown in Fig. 1). After I month the diabetic rats had higher glycogen stores in soleus muscle compared to the controls. No difference was observed in glycogen content of gastrocnemius muscle until 2 months had elapsed from the streptozotocin administration. Then the diabetic rats had significantly higher glycogen levels than their controls (Fig. 1). Table 2 shows that the activity of phosphofructokinase was much lower in soleus than in gastrocnem i u s muscle. On the other hand no difference in the phosphofructokinase activities could be found between control and experimental rats within the fol-
low-up period of 2 months. Table 2 shows also citrate synthase and malate dehydrogenase activities in the muscles studied. The citrate synthase activity was alike in both groups. Also the behaviour of malate dehydrogenase activity was comparable to that of citrate synthase. There was, however, a small decline of activity in the soleus of diabetic rats. There were no differences either between the control and experimental group, when the enzyme activities were calculated on protein basis instead of tissue fresh weight. In brown adipose tissue the activities of both citrate synthase and malate dehydrogenase (Fig. 2) displayed similar pattern. In the acute phase of insulin deficiency, there was a tendency for increased activities. However, 1 month after the streptozotocin treatment, the trend was reversed, i.e. the diabetic rats had now slightly lower enzyme activities. After 2 months of insulin deficiency the activity of citrate synthase and malate dehydrogenase were at the control level.
DISCUSSION
Control rats had lower glycogen content in soleus than in gastrocnemius muscle in accordance to the
SOLEUS
GASTROCNEMFUS O0 N.S.
7,0 ~"
OtI~O
ttOtD
5.o
3.0 ~ E
2.0
i
..A
55
//A
"/
,#~
;
'
." . /z
~.o
<5 one
month
two
months
one
month
two
months
Fig. 1. Glycogen levels in soleus and gastrocnemius muscle one and two months after streptozotocin treatment (hatched columns). Open columns give control data. Means and SEM and the number of rats in parentheses have been given. Asterisks indicate significant difference between controls and diabetics as follows: *P < 0.05, **P < 0.01, ***P < 0.001 and NS not significant.
Metabolic reserves in diabetic rat muscle
393
Table 2. The activities of phosphofructokinase, citrate synthase and malate dehydrogenase in different muscles of control and diabetics 2 months after streptozotocin treatment
Enzyme/ Muscle
Controls
Diabetics
0.73~.071
0.642.065
Phosphofructokinase: Soleus
(8) Gastrocnemius
N.S.
(9)
5.31~.307
6.06~.236
(9)
N.S.
(11)
Citrate synthase: Soleus
31.6!2.07
30.0~1.28
(8) Gastrocnemius
N.S.
(11)
28.3~5.40
18.9!2.90
N.S.
(9) Malate dehydro£enase: Soleus
275~14.4 (8)
240~7.7 (]1)
Gastrocnemius
182130.4
151217.3
(7)
N.S.
(11)
For explanations, see Table 1. results published by Armstrong & Ianuzzo (1977). Furthermore, in controls the glycogen content of both muscles agreed their values, whereas slightly higher levels in rat soleus have been obtained by Moorthy & Gould (1969). Thus, unlike in human skeletal muscle 500
one week
(e.g. Ess6n, 1977h rats have different glycogen contents in muscles depending perhaps on the fibre composition. In acute experimental diabetes Goodman et al. (1974) have demonstrated lowered glycogen level in one month
two months
400
@
oo
300
200
// // //
100
// //
(4) /~///
(9)
"/
CS
MDH
cs
MDH
(8) CS
MDH
Fig. 2. Citrate synthase (CS) and malate dehydrogenase (MDH) activities (U/g) in interscapular brown adipose tissue of controls (open columns) and of diabetics (hatched columns) 1 week, 1 month and 2 months after streptozotocin treatment. For other explanations see Fig. 1.
394
R. RAURAMAAet al.
gastrocnemius muscle of ketotic rats 3 days after the streptozotocin (125mg/kg) treatment. The present findings indicate that the chronic diabetic state leads in rats to an elevation in muscle glycogen stores. This has not previously been reported. Most authors have followed metabolic changes in muscle only for short time periods. Chen et al. (1977) have, however, published results indicating a tendency to an increase in the glycogen content of gastrocnemius muscle 6 weeks after streptozotocin (60 mg/kg) injection. The present data suggests that glycogen accumulation occurs faster and is relatively much greater in soleus than in gastrocnemius muscle. There are several possible mechanisms, which could explain the elevation in muscle glycogen in chronic experimental diabetes. Due to hyperglycemia more glucose is available for filling of the glycogen stores. Thus, one reason could be simply a greater diffusion gradient across the cell membranes. In the cells glucose in rapidly metabolized. This also occurs in depancreatized dogs, as reported already over 40 years ago by Soskin & Levine (1937). Moorthy & Gould (1969) have shown in vitro that the activity of glycogen synthase can be increased about 20-fold with glucose concentration increasing from 10 to 20 mM, if small amounts of insulin (0.1 U/ml) are present. Since our rats showed no ketonuria, and the administered dose was only 75 mg/kg of streptozotocin, they probably had still some endogenous insulin secretion left. Rakieten et al. (1963) showed that streptozotocin in a dose of 50 mg/kg does not lead to ketonuria. Increases in muscle glycogen of our streptozotocin treated rats cannot be due to dehydration secondary to hyperglycemia, since the protein concentrations in tissue samples were the same both in control and experimental rats. The present results show that the enzyme levels remained practically unchanged in both muscles at least up to 2 months after streptozotocin administration. The enzyme levels present in tissue would allow the consumption of glycosyl units and elevated glycogen stores cannot be due to depressed keyenzyme amounts of glycolysis and of Kreb's cycle. For some reason, our results do not confirm the findings of lowered enzyme activities in skeletal muscles of streptozotocin diabetic rats (Ianuzzo et al., 1974; Ianuzzo & Armstrong, 1976). Still one reasonably good explanation for elevated muscle glycogen levels could be the increased availability of free fatty acids for energy production due to diminished antilipolytic action of insulin (Schnatz et al., 1972). Recently, we have also found that the lipoprotein lipase activity in muscle tissue is significantly higher in diabetic rats compared to controls (Rauramaa et al., 1980). Furthermore, since in the resting state skeletal muscles use mostly free fatty acids (e.g. Felig & Wahren, 1975) the glycogen stores in muscles tend to remain unused. Besides increasing the muscle glycogen stores increased fatty acid oxidation also leads to inhibition of phosphofructokinase activity (Rennie et al., 1976). The role of insulin in the metabolism of brown adipose tissue is the regulation of glucose uptake and its conversion to lipids (Shackney & Joel, 1966). In this study the effect of insulin deficiency was evaluated in respect to oxidative metabolism. Like in muscle tissue
the differences in enzyme activities were surprisingly small also in brown adipose tissue between controls and diabetic rats. In conclusion, present study suggests that one manifestation of deranged carbohydrate metabolism in rats suffering from chronic insulin deficiency is an increase in muscle glycogen stores. It is also apparent that changes of enzyme activities in glycolysis and Kreb's cycle are minimal both in muscle and in brown adipose tissue of diabetic rats. These findings suggest that streptozotocin diabetic rats should have in principle comparable metabolic ability to the control animals to perform muscular work. SUMMARY
The effect of insulin deficiency for 1 week, 1 month and 2 months on skeletal muscle glycogen stores as well as on some enzyme activities in skeletal muscle and brown adipose tissue was studied in adult streptozotocin treated rats. After 1 month, the diabetic rats had higher glycogen reserves in soleus muscle than controls. No further change was found 1 month later. In gastrocnemius muscle no difference was found between control and diabetic rats until 2 months after streptozotocin treatment. Then the diabetic rats had also significantly higher glycogen stores. The effect of 2 months' insulin deficiency on the activities ofphosphofructokinase, citrate synthase and malate dehydrogenase was evaluated in soleus ano gastrocnemius muscles. As comparison these two Kreb's cycle enzymes were determined in brown adipose tissue. With the exception of a small and reversible decline in malate dehydrogenase activity in brown adipose tissue 1 month after the induction of diabetes, there were practically no differences in the enzyme activities between control and diabetic groups in either tissues. In conclusion, skeletal muscles of diabetic rats contain more glycogen than those of control rats. The effect of insulin deficiency on enzyme activities in muscle and brown adipose tissue was surprisingly small. REFERENCES
ARMSTRONG R. B. & IANUZZO C. D. (1977) Exerciseinduced muscle glycogen depletion and repletion in diabetic rats. Life Sci. 20, 301-308. BERGER M., HAGG S. & RUDERMANN. B. (1975) Glucose metabolism in perfused skeletal muscle. Interaction of insulin and exercise on glucose uptake. Biochem. J. 146, 23l 238. BOSTR()MS., FAHLENM., HJALMARSON/~. & JOHANSSONR. (1974) Activities of rat muscle enzymes after acute exercise. Acta physiol, scand. 90, 544-554. CHEN V., IANUZZOC. D. &WILLIAMS C. (1977) Exerciseinduced changes in muscle glycogen concentration in streptozotocin-diabetic rats. J. Physiol. 59P. CLAUSENT. (1975) Metabolic effects on muscular tissue. In Insulin, part 2, pp. 32%370. (Edited by HASSELBLATT A. V. 8z BRUCKHAUSENF.), Springer, Berlin. DESA1K. & HOLLENBERGC. H. (1975) Regulation, by insulin, of lipoprotein lipase and phosphodiesterase activities in rat adipose tissue. Israel J. Med. Sci. 11, 540--550. EARL D. C. N. & KORNER A. (1965j The isolation and properties of cardiac ribosomes and polysomes. Biochem. J. 94, 721 726. ENGLARD S. & SIEGEL L. (1969) Mitochondrial L-malatc dehydrogenase on beef heart. In Methods in Euzymology
Metabolic reserves in diabetic rat muscle
395
(Edited by LOWENSTEINJ. M.) Vol. 13, pp. 99-106. Aca- OLEFSKY J., BACON V. C. 8¢~ BAUR S. (1976) Insulin receptors of skeletal muscle: Specific insulin binding sites and demic Press, New York. demonstration of decreased numbers of sites in obese ESSEN B. (1977) Intramuscular substrate utilization during rats. Metabolism 25, 179-19 I. prolonged exercise. In The Marathon: Physiological, medical, epidemiological and psychological studies RAKIETEN N., RAKIETEN M. L. & NADKARNI M. V. (1963) Studies on the diabetogenic action of streptozotocin (Edited by MILVY P.). Ann. N.Y. Acad. Sci. 301, 30-44. (NSC-37917). Cancer Chemother. Rep. 29, 91 98. FELIG P. 8~, WAHRENJ. (1975) Fuel homeostasis in exercise. RAURAMAAR., KUUSELAP. & HIETANEN E. (1980) Adipose. N. Engl. J. Med. 293, 1078 1084. muscle and lung tissue lipoprotein lipase activities of GOODMAN M. N., BERGER M. & RUDERMAN N. B. (1974) young streptozotocin treated rats. Horm. Metah. Res. In Glucose metabolism in rat skeletal muscle at rest. Effect press. of starvation, ketone bodies and free fatty acids. Diabetes RENNIE M., WINDER W. M. & HOLLDSZY J. O. (1976) A 23, 881-888. sparing effect of increased free fatty acids on muscle glyHARRI M. N. E. & VALTOLAJ. (1975) Comparison of the cogen content in exercising rats. Biochem. J. 156, effects of physical exercise, cold acclimation and 647 655. repeated injections of isoprenaline on rat muscle ROCH-NORLUND A. E. (1972) Muscle glycogen and glycoenzymes. Acta physiol, scand. 95, 391 399. gen synthase in diabetic man. Scand. J. din. Lab. Ira?est. HARRI M. N. E. & NARVOLA I. (1979) Physical training 29, (suppl. 125). under the influence of beta blockade in rats: effect on SCHNATZ J. D., FORMANIAK J. M. & CHLOUVERAKIS C. adrenergic responses. Eur. J. Appl. Physiol. 41, 199-210. (1972) The origin of hypertriglyceridemia in streptozotoHULTMAN E. (1967) Muscle glycogen in man determined in cin diabetic rats. Diabetologia 8, 125-130. needle biopsy specimens. Method and normal values. SHACKNEY E. & JOEL C. D. (1966) Stimulation of glucose Scand. J. clin. Lab. Invest. 19, 209-217. metabolism in brown adipose tissue by addition of insuIANUZZO C. D., LESSERM. & BATTISTA F. (1974) Metabolic lin in vitro. J. biol. Chem. 241, 4004-4010. adaptations in skeletal muscle of streptozotocin diabetic SOSKIN S. & LEVINER. (1937) A relation between the blood rats. Biochem. biophys. Res. Commun. 58, 107-111. sugar level and the rate of sugar utilization affecting the IANUZZO C. D. & ARMSTRONGR. B. (1976) Phosphofructokinase and succinate dehydrogenase activities of northeories of diabetes. Am. J. Physiol. 120, 761-770. mal and diabetic rat skeletal muscle. Horm. Metab. Res. SRERE P. A. (1969) Citrate synthase. In Methods in Enzymo8, 244-245. logy, (Edited by LOWENSTEIN J. M.), Vol. 13, pp. 3 19, KAMM D. E. & STROPE G. L. (1974) Effect of acid-base Academic Press, New York. status on tissue glycogen in normal and diabetic rats. STEIN J. M. & PADYKULAH. A. (1962) Histochemical clasAm. J. Physiol. 226, 371-376. sification of individual skeletal muscle fibers of the rat. LOWRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALL Am. J. Anat. 110, 103-115. R. J. (1951) Protein measurement with the Folin phenol VONDRA K., RATH R. & BASS A. (1975) Activity of some reagent. J. biol. Chem. 193, 265-275. enzymes of energy metabolism in striated muscle of obese subjects with respect to body composition. Horm. MOORTHY K. A. & GOULD M. K. (1969) Synthesis of glycoMetab. Res. 7, 47%480. gen from glucose and lactate in isolated rat soleus musVONDRA K., RATH R., BASSA., SLABOCHOV./~Z., TEISINGER cle. Archs Biochem. Biophys. 130, 399-407. J. & VITEK V. (1977) Enzyme activities in quadriceps MURPHY E. D. & ANDERSSONJ. W. (1974) Tissue glycolytic femoris muscle of obese diabetic male patients. Diahetoand gluconeogenic enzyme activities in mildly and Iogia 13, 527 529. moderately diabetic rats: Influence of tolbutamide administration. Endocrinology 94, 27-34.
0305-0491,'80,'0701-0391S02.00/0
© Pergamon Press Lid 1980. Printed in Great Britain
METABOLIC ADAPTATION IN MUSCLE A N D BROWN ADIPOSE TISSUES TO INSULIN DEFICIENCY IN STREPTOZOTOCIN TREATED RATS R. RAURAMAA,M. HARRI,E. PUHAKAINEN,P. KUUSELA and O. H,~NNINEN Department of Physiology, University of Kuopio, P.O. Box 138, SF-70101 Kuopio 10, Finland (Received 12 November 1979)
Abstract--l. Glycogen stores in skeletal muscle were increased in diabetic rats compared to controls 2 months after streptozotocin treatment. 2. The increase in muscle glycogen stores seemed to occur faster in soleus muscle compared to gastrocnemius muscle. 3. Insulin deficiency caused by streptozotocin administration had only minimal effects on the enzyme activities either in skeletal muscle or in brown adipose tissue. INTRODUCTION
MATERIALS AND METHODS
Skeletal muscle is one of the main target tissues of insulin (Clausen, 1975; Olefsky et al., 1976). This hormone is required among other functions for glucose uptake (e.g. Berger et al., 1975). One could therefore expect lowered glycogen stores in muscle in insulin deficiency. This has indeed been demonstrated, by Roch-Norlund (1972) in human diabetics, both in juvenile and maturity-onset types. G o o d m a n et al. (1974) and K a m m & Strope (1974) have observed lowered muscle glycogen content also in diabetic rats. On the other hand, Armstrong & Ianuzzo (1977) found no differences in the amount of muscle glycogen between control and diabetic rats. The level of tissue glycogen depends not only on the penetration and deposition of glucose in cells but also on the ability of cells to consume glycosyl units. Glycolytic enzymes have been reported to be lower in skeletal muscles in experimental (Murphy & Anderson, 1974; Ianuzzo & Armstrong, 1976) and in human (Vondra et al., 1977) diabetes. Also mitochondrial enzyme activities are depressed in experimental (Ianuzzo et al., 1974) and in human (Vondra et al., 1977) diabetes. These would tend to save glycogen stores. Similar effects have free fatty acids and ketone bodies, the availability of which increases when the antilipolytic action of insulin (Desai & Hollenberg, 1975) diminishes in diabetes. The present series of experiments was started to clarify the metabolic reserves for exercise in experimental diabetes in rats. For this purpose the glycogen levels and the activities of phosphofructokinase, citrate synthase and malate dehydrogenase were followed in two muscles representing different functional types. Samples were taken from the medial head of gastrocnemius, which is composed of all three types of muscle fibres, and from soleus containing only slow and intermediate fibres (e.g. Stein & Padykula, 1962). For comparison the enzyme levels in brown adipose tissue were followed, since this forms its own type of tissue with high consumption of energy substrates. It is also sensitive to several exogenous and endogenous stimuli (e.g. Harri & Valtola, 1975; Harri & Narvola, 1979).
Male albino rats (Wistar/af/Han/Mol (Kuo 67)) weighing 210-225 g at the beginning of the study were used. Rats were housed in net floor cages at 20 _+ 2'C with a daily 14:10 light-dark cycle. They were fed rat food pellets (Hankkija Co., Finland) ad libitum, and tap water was always available. Diabetes was produced by an intravenous (femoral vein) injection of streptozotocin (kindly donated by Dr E. W. Dulin, Upjohn Co., Kalamazoo, MI, U.S.A.) (75 mg/kg) to pentobarbital (30--50mg/kg, intraperitoneally) anaesthetized rats. Streptozotocin was dissolved in 0.9°0 NaCI-O.1 M citrate buffer, pH 4.5 always immediately before injection. Control rats received one injection of buffer only. Urinary glucose and possible ketonuria was measured 3 days after streptozotocin treatment with the aid of reagent sticks (Keto-Diastix, Ames Co., Great Britain). One week, one month and two months after the streptozotocin injection, rats were anaesthetized again, and, in addition to blood, the following tissue samples were taken: interscapular brown adipose tissue, soleus and the medial head of gastrocnemius muscles. Visible connective tissue and blood clots were removed. Tissue samples were frozen with liquid nitrogen and kept at -80°C until analysed. Blood samples for glucose determinations (by glucose oxidase-peroxidase method, Glox Novum, Kabi Ab, Sweden) were drawn by cardiac puncture and placed in tubes containing ftuoride-oxalate. For the determination of glycogen content, the muscle samples were homogenized in 1 M HC1 and heated at 100°C for 2 hr. The glycogen content was then analysed as glycosyl units according to the method of Hultman (1967). For enzyme assays the tissue samples were weighed and homogenized in a Potter Elvehjem glass homogenizer in Tris-HCl buffer (0. l M, pH 7.6) to a 2'7o homogenate (w/v), and centrifuged for 10rain at 1000g at 4°C to remove unbroken cells and particulate debris. The supernatants were used for determining the enzyme activities. The protein contents were estimated by the phenol method of Lowry et al. (195l). The activity of phosphofructokinase (PFK) (EC 2.7.1.11) was determined according to BostriSm el al. (1974). Citrate synthase (CS) (EC 4.1.3.7) activity was analysed according to Srere (1969) and malate dehydrogenase (MDH) (EC 1.1.1.37) activity by the method of Englard & Siegel (1969). The measurements were made at 37c'C with a Cary 118 double-beam spectrophotometer. Enzyme activities were calculated as micromoles of substrate utilized per min and g of fresh tissue weight and per mg of protein. 391
392
R. RAURAMAAet al.
Table 1. Final body weights and blood glucose levels in control and in diabetic rats l week, I month and 2 months after streptozotocin treatment Body w e i g h t (g)
Blood g l u c o s e (n~nol/1)
Group
Controls
Diabetics
Controls
Diabetics
1 Week
24828.6
218t3.7"
6.2~3.1
16.2t2.08"*
(s]
1 Month 2 Months
(s)
291t8.1 (9]
262~8.8
337t5.9
278t8.4
(4)
19.9-].61
6.0tO. ?~
(7)
(9)
U)
6.4:0,27
(1])
(9)
(4)
21.iti.02"**
(8)
(n)
Means, _+SEM have been given, number of rats in parentheses. Asterisks indicate significant difference between controls and diabetics as follows: *P < 0.05, **P < 0.01, ***P < 0.001 and NS not significant.
RESULTS
Streptozotocin treated rats gained weight more slowly than did the controls. The difference was observable already 1 week after the streptozotocin injection. The success in producing diabetes was evident from the high blood glucose values (Table 1). Diabetic rats had also a marked glucosuria but not ketonuria. Glycogen was determined in soleus and gastrocnemius muscles 1 and 2 months after the induction of diabetes. After 1 week neither muscle showed any changes in glycogen levels (not shown in Fig. 1). After I month the diabetic rats had higher glycogen stores in soleus muscle compared to the controls. No difference was observed in glycogen content of gastrocnemius muscle until 2 months had elapsed from the streptozotocin administration. Then the diabetic rats had significantly higher glycogen levels than their controls (Fig. 1). Table 2 shows that the activity of phosphofructokinase was much lower in soleus than in gastrocnem i u s muscle. On the other hand no difference in the phosphofructokinase activities could be found between control and experimental rats within the fol-
low-up period of 2 months. Table 2 shows also citrate synthase and malate dehydrogenase activities in the muscles studied. The citrate synthase activity was alike in both groups. Also the behaviour of malate dehydrogenase activity was comparable to that of citrate synthase. There was, however, a small decline of activity in the soleus of diabetic rats. There were no differences either between the control and experimental group, when the enzyme activities were calculated on protein basis instead of tissue fresh weight. In brown adipose tissue the activities of both citrate synthase and malate dehydrogenase (Fig. 2) displayed similar pattern. In the acute phase of insulin deficiency, there was a tendency for increased activities. However, 1 month after the streptozotocin treatment, the trend was reversed, i.e. the diabetic rats had now slightly lower enzyme activities. After 2 months of insulin deficiency the activity of citrate synthase and malate dehydrogenase were at the control level.
DISCUSSION
Control rats had lower glycogen content in soleus than in gastrocnemius muscle in accordance to the
SOLEUS
GASTROCNEMFUS O0 N.S.
7,0 ~"
OtI~O
ttOtD
5.o
3.0 ~ E
2.0
i
..A
55
//A
"/
,#~
;
'
." . /z
~.o
<5 one
month
two
months
one
month
two
months
Fig. 1. Glycogen levels in soleus and gastrocnemius muscle one and two months after streptozotocin treatment (hatched columns). Open columns give control data. Means and SEM and the number of rats in parentheses have been given. Asterisks indicate significant difference between controls and diabetics as follows: *P < 0.05, **P < 0.01, ***P < 0.001 and NS not significant.
Metabolic reserves in diabetic rat muscle
393
Table 2. The activities of phosphofructokinase, citrate synthase and malate dehydrogenase in different muscles of control and diabetics 2 months after streptozotocin treatment
Enzyme/ Muscle
Controls
Diabetics
0.73~.071
0.642.065
Phosphofructokinase: Soleus
(8) Gastrocnemius
N.S.
(9)
5.31~.307
6.06~.236
(9)
N.S.
(11)
Citrate synthase: Soleus
31.6!2.07
30.0~1.28
(8) Gastrocnemius
N.S.
(11)
28.3~5.40
18.9!2.90
N.S.
(9) Malate dehydro£enase: Soleus
275~14.4 (8)
240~7.7 (]1)
Gastrocnemius
182130.4
151217.3
(7)
N.S.
(11)
For explanations, see Table 1. results published by Armstrong & Ianuzzo (1977). Furthermore, in controls the glycogen content of both muscles agreed their values, whereas slightly higher levels in rat soleus have been obtained by Moorthy & Gould (1969). Thus, unlike in human skeletal muscle 500
one week
(e.g. Ess6n, 1977h rats have different glycogen contents in muscles depending perhaps on the fibre composition. In acute experimental diabetes Goodman et al. (1974) have demonstrated lowered glycogen level in one month
two months
400
@
oo
300
200
// // //
100
// //
(4) /~///
(9)
"/
CS
MDH
cs
MDH
(8) CS
MDH
Fig. 2. Citrate synthase (CS) and malate dehydrogenase (MDH) activities (U/g) in interscapular brown adipose tissue of controls (open columns) and of diabetics (hatched columns) 1 week, 1 month and 2 months after streptozotocin treatment. For other explanations see Fig. 1.
394
R. RAURAMAAet al.
gastrocnemius muscle of ketotic rats 3 days after the streptozotocin (125mg/kg) treatment. The present findings indicate that the chronic diabetic state leads in rats to an elevation in muscle glycogen stores. This has not previously been reported. Most authors have followed metabolic changes in muscle only for short time periods. Chen et al. (1977) have, however, published results indicating a tendency to an increase in the glycogen content of gastrocnemius muscle 6 weeks after streptozotocin (60 mg/kg) injection. The present data suggests that glycogen accumulation occurs faster and is relatively much greater in soleus than in gastrocnemius muscle. There are several possible mechanisms, which could explain the elevation in muscle glycogen in chronic experimental diabetes. Due to hyperglycemia more glucose is available for filling of the glycogen stores. Thus, one reason could be simply a greater diffusion gradient across the cell membranes. In the cells glucose in rapidly metabolized. This also occurs in depancreatized dogs, as reported already over 40 years ago by Soskin & Levine (1937). Moorthy & Gould (1969) have shown in vitro that the activity of glycogen synthase can be increased about 20-fold with glucose concentration increasing from 10 to 20 mM, if small amounts of insulin (0.1 U/ml) are present. Since our rats showed no ketonuria, and the administered dose was only 75 mg/kg of streptozotocin, they probably had still some endogenous insulin secretion left. Rakieten et al. (1963) showed that streptozotocin in a dose of 50 mg/kg does not lead to ketonuria. Increases in muscle glycogen of our streptozotocin treated rats cannot be due to dehydration secondary to hyperglycemia, since the protein concentrations in tissue samples were the same both in control and experimental rats. The present results show that the enzyme levels remained practically unchanged in both muscles at least up to 2 months after streptozotocin administration. The enzyme levels present in tissue would allow the consumption of glycosyl units and elevated glycogen stores cannot be due to depressed keyenzyme amounts of glycolysis and of Kreb's cycle. For some reason, our results do not confirm the findings of lowered enzyme activities in skeletal muscles of streptozotocin diabetic rats (Ianuzzo et al., 1974; Ianuzzo & Armstrong, 1976). Still one reasonably good explanation for elevated muscle glycogen levels could be the increased availability of free fatty acids for energy production due to diminished antilipolytic action of insulin (Schnatz et al., 1972). Recently, we have also found that the lipoprotein lipase activity in muscle tissue is significantly higher in diabetic rats compared to controls (Rauramaa et al., 1980). Furthermore, since in the resting state skeletal muscles use mostly free fatty acids (e.g. Felig & Wahren, 1975) the glycogen stores in muscles tend to remain unused. Besides increasing the muscle glycogen stores increased fatty acid oxidation also leads to inhibition of phosphofructokinase activity (Rennie et al., 1976). The role of insulin in the metabolism of brown adipose tissue is the regulation of glucose uptake and its conversion to lipids (Shackney & Joel, 1966). In this study the effect of insulin deficiency was evaluated in respect to oxidative metabolism. Like in muscle tissue
the differences in enzyme activities were surprisingly small also in brown adipose tissue between controls and diabetic rats. In conclusion, present study suggests that one manifestation of deranged carbohydrate metabolism in rats suffering from chronic insulin deficiency is an increase in muscle glycogen stores. It is also apparent that changes of enzyme activities in glycolysis and Kreb's cycle are minimal both in muscle and in brown adipose tissue of diabetic rats. These findings suggest that streptozotocin diabetic rats should have in principle comparable metabolic ability to the control animals to perform muscular work. SUMMARY
The effect of insulin deficiency for 1 week, 1 month and 2 months on skeletal muscle glycogen stores as well as on some enzyme activities in skeletal muscle and brown adipose tissue was studied in adult streptozotocin treated rats. After 1 month, the diabetic rats had higher glycogen reserves in soleus muscle than controls. No further change was found 1 month later. In gastrocnemius muscle no difference was found between control and diabetic rats until 2 months after streptozotocin treatment. Then the diabetic rats had also significantly higher glycogen stores. The effect of 2 months' insulin deficiency on the activities ofphosphofructokinase, citrate synthase and malate dehydrogenase was evaluated in soleus ano gastrocnemius muscles. As comparison these two Kreb's cycle enzymes were determined in brown adipose tissue. With the exception of a small and reversible decline in malate dehydrogenase activity in brown adipose tissue 1 month after the induction of diabetes, there were practically no differences in the enzyme activities between control and diabetic groups in either tissues. In conclusion, skeletal muscles of diabetic rats contain more glycogen than those of control rats. The effect of insulin deficiency on enzyme activities in muscle and brown adipose tissue was surprisingly small. REFERENCES
ARMSTRONG R. B. & IANUZZO C. D. (1977) Exerciseinduced muscle glycogen depletion and repletion in diabetic rats. Life Sci. 20, 301-308. BERGER M., HAGG S. & RUDERMANN. B. (1975) Glucose metabolism in perfused skeletal muscle. Interaction of insulin and exercise on glucose uptake. Biochem. J. 146, 23l 238. BOSTR()MS., FAHLENM., HJALMARSON/~. & JOHANSSONR. (1974) Activities of rat muscle enzymes after acute exercise. Acta physiol, scand. 90, 544-554. CHEN V., IANUZZOC. D. &WILLIAMS C. (1977) Exerciseinduced changes in muscle glycogen concentration in streptozotocin-diabetic rats. J. Physiol. 59P. CLAUSENT. (1975) Metabolic effects on muscular tissue. In Insulin, part 2, pp. 32%370. (Edited by HASSELBLATT A. V. 8z BRUCKHAUSENF.), Springer, Berlin. DESA1K. & HOLLENBERGC. H. (1975) Regulation, by insulin, of lipoprotein lipase and phosphodiesterase activities in rat adipose tissue. Israel J. Med. Sci. 11, 540--550. EARL D. C. N. & KORNER A. (1965j The isolation and properties of cardiac ribosomes and polysomes. Biochem. J. 94, 721 726. ENGLARD S. & SIEGEL L. (1969) Mitochondrial L-malatc dehydrogenase on beef heart. In Methods in Euzymology
Metabolic reserves in diabetic rat muscle
395
(Edited by LOWENSTEINJ. M.) Vol. 13, pp. 99-106. Aca- OLEFSKY J., BACON V. C. 8¢~ BAUR S. (1976) Insulin receptors of skeletal muscle: Specific insulin binding sites and demic Press, New York. demonstration of decreased numbers of sites in obese ESSEN B. (1977) Intramuscular substrate utilization during rats. Metabolism 25, 179-19 I. prolonged exercise. In The Marathon: Physiological, medical, epidemiological and psychological studies RAKIETEN N., RAKIETEN M. L. & NADKARNI M. V. (1963) Studies on the diabetogenic action of streptozotocin (Edited by MILVY P.). Ann. N.Y. Acad. Sci. 301, 30-44. (NSC-37917). Cancer Chemother. Rep. 29, 91 98. FELIG P. 8~, WAHRENJ. (1975) Fuel homeostasis in exercise. RAURAMAAR., KUUSELAP. & HIETANEN E. (1980) Adipose. N. Engl. J. Med. 293, 1078 1084. muscle and lung tissue lipoprotein lipase activities of GOODMAN M. N., BERGER M. & RUDERMAN N. B. (1974) young streptozotocin treated rats. Horm. Metah. Res. In Glucose metabolism in rat skeletal muscle at rest. Effect press. of starvation, ketone bodies and free fatty acids. Diabetes RENNIE M., WINDER W. M. & HOLLDSZY J. O. (1976) A 23, 881-888. sparing effect of increased free fatty acids on muscle glyHARRI M. N. E. & VALTOLAJ. (1975) Comparison of the cogen content in exercising rats. Biochem. J. 156, effects of physical exercise, cold acclimation and 647 655. repeated injections of isoprenaline on rat muscle ROCH-NORLUND A. E. (1972) Muscle glycogen and glycoenzymes. Acta physiol, scand. 95, 391 399. gen synthase in diabetic man. Scand. J. din. Lab. Ira?est. HARRI M. N. E. & NARVOLA I. (1979) Physical training 29, (suppl. 125). under the influence of beta blockade in rats: effect on SCHNATZ J. D., FORMANIAK J. M. & CHLOUVERAKIS C. adrenergic responses. Eur. J. Appl. Physiol. 41, 199-210. (1972) The origin of hypertriglyceridemia in streptozotoHULTMAN E. (1967) Muscle glycogen in man determined in cin diabetic rats. Diabetologia 8, 125-130. needle biopsy specimens. Method and normal values. SHACKNEY E. & JOEL C. D. (1966) Stimulation of glucose Scand. J. clin. Lab. Invest. 19, 209-217. metabolism in brown adipose tissue by addition of insuIANUZZO C. D., LESSERM. & BATTISTA F. (1974) Metabolic lin in vitro. J. biol. Chem. 241, 4004-4010. adaptations in skeletal muscle of streptozotocin diabetic SOSKIN S. & LEVINER. (1937) A relation between the blood rats. Biochem. biophys. Res. Commun. 58, 107-111. sugar level and the rate of sugar utilization affecting the IANUZZO C. D. & ARMSTRONGR. B. (1976) Phosphofructokinase and succinate dehydrogenase activities of northeories of diabetes. Am. J. Physiol. 120, 761-770. mal and diabetic rat skeletal muscle. Horm. Metab. Res. SRERE P. A. (1969) Citrate synthase. In Methods in Enzymo8, 244-245. logy, (Edited by LOWENSTEIN J. M.), Vol. 13, pp. 3 19, KAMM D. E. & STROPE G. L. (1974) Effect of acid-base Academic Press, New York. status on tissue glycogen in normal and diabetic rats. STEIN J. M. & PADYKULAH. A. (1962) Histochemical clasAm. J. Physiol. 226, 371-376. sification of individual skeletal muscle fibers of the rat. LOWRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALL Am. J. Anat. 110, 103-115. R. J. (1951) Protein measurement with the Folin phenol VONDRA K., RATH R. & BASS A. (1975) Activity of some reagent. J. biol. Chem. 193, 265-275. enzymes of energy metabolism in striated muscle of obese subjects with respect to body composition. Horm. MOORTHY K. A. & GOULD M. K. (1969) Synthesis of glycoMetab. Res. 7, 47%480. gen from glucose and lactate in isolated rat soleus musVONDRA K., RATH R., BASSA., SLABOCHOV./~Z., TEISINGER cle. Archs Biochem. Biophys. 130, 399-407. J. & VITEK V. (1977) Enzyme activities in quadriceps MURPHY E. D. & ANDERSSONJ. W. (1974) Tissue glycolytic femoris muscle of obese diabetic male patients. Diahetoand gluconeogenic enzyme activities in mildly and Iogia 13, 527 529. moderately diabetic rats: Influence of tolbutamide administration. Endocrinology 94, 27-34.