Training Adaptations in Skeletal Muscle of Juvenile Diabetics D. L COSTILL, P. CLEARY, W. J. FINK, C. FOSTER, J. L. IVY, AND F. WITZMANN


Skeletal muscles from 12 male, juvenile-onset diabetics (JD) and 13 nondiabetics (ND) were studied to determine the effects of endurance training on mitochondrial enzyme activities, lipoprotein lipase (LPL) activity, and the oxidation of lipids (l4C-palmityl CoA) in vitro. Ten weeks of endurance running (30 min/day, 5 days/wk) resulted in 11.0 and 12.9% gains in aerobic capacity for the JD and ND groups (P > 0.05), respectively. Both groups showed significant (P < 0.05) increases in muscle LPL, carnitine palmityl transferase, succinate dehydrogenase, and hexokinase activities with training. Though the pretraining capacities for 14C-palmityl CoA oxidation were similar for both ND and JD groups, the diabetics showed a 4 1 % greater improvement in the measurement of muscle lipid oxidation after training than did the ND group. The principal finding of this research was that skeletal muscle of juvenile diabetics who are in moderate insulin balance shows adaptations to endurance training that are similar to those of nondiabetic men. DIABETES 28:818-822, September 1979.


here is evidence that the insulin-dependent diabetic individual has an abnormal protein turnover in heart and skeletal muscle, resulting in altered enzyme activities and abnormal carbohydrate (CHO) and fat metabolism.2>28-33 Since skeletal muscle represents a large mass of metabolically active tissue, a reduction in muscle enzymes could have a profound influence on lipid metabolism and subsequent plasma clearance of triglycerides. Previous research with nondiabetic rodents and human beings has shown that chronic exercise results in significant increases in the activities of enzymes that affect CHO, fat, and protein metabolism in the trained musculature.1'18 Unfortunately,

From the Human Performance Laboratory, Ball State University, Muncie, Indiana 47306. Received for publication 5 April 1979 and in revised form 31 May 1979.


there is no information available to describe the traininginduced alterations in lipid uptake and utilization in the muscle of human diabetics. Thus, the purpose of this research was to study the effects of endurance training on mitochondrial enzyme activities, lipoprotein lipase activities, and the oxidation of lipids (14C-palmityl CoA) in vitro in skeletal muscle of juvenile diabetics (JD). METHODS AND MATERIALS

Subjects. The subjects were 12 male, juvenile-onset diabetics (JD) and 13 nondiabetic men (ND). The latter group (ND) was found to have normal blood glucose and insulin responses to a 75 gm oral glucose tolerance test. The age of these subjects averaged (±SE) 21.1 yr (±1.4) and 23.3 yr (±1.1) for the JD and ND groups, respectively: Other relevant subject data, obtained at the beginning of the investigation, are given in Table 1. The subjects in the JD group had been diabetic for from 3 mo to 11 yr, and they generally took insulin (Lente) in an abdominal site once per day (avg. = 48 U/day; range = 20-58 U/day) in the early morning. For the most part, the exercise was performed at 4 to 6 h after the insulin injection. Glucosuria was checked and recorded by diabetic subjects two to three times each day. The subjects generally reported fewer episodes of glucosuria during the training period, but these were strictly subjective observations, since we had no untrained observations for comparison. All subjects were given a thorough physical examination and were fully informed of the risks and stresses associated with this research before giving their written consent to participate. None of the subjects exhibited any symptoms of neuropathy. No patient received any medicine except insulin or had any history of angina pectoris, cardiac arrhythmias, or pulmonary disease. Auscultation of heart and lungs and ECG in six precordial leads were normal in all subjects. No patient had signs or symptoms of other endocrine disease. None of the subjects had participated in any form of physical training for at least 3 mo preceding this investigation.



Testing. Before and after the training program, each subject's maximal oxygen uptake (Vo2 max) was measured during treadmill running. During the test the treadmill speed was kept constant at 161 m/min, with the grade increased by 2% every 2 min, until exhaustion. Respiratory exchange was measured with a semiautomated system34 and heart rates were monitored electrocardiographically. Three minutes after the Vo2 max test, venous blood was sampled and analyzed for lactic acid determination.23 After an overnight fast, and, before the JD subjects took insulin, venous blood samples were obtained from each of the participants and were analyzed for serum glucose, triglyceride, glycerol, free fatty acids (FFA), and cholesterol. The procedures for these measurements are described elsewhere.11 Immediately after the blood was drawn, four biopsy specimens were obtained from the gastrocnemius muscle. One tissue sample was mounted in OCT, frozen in isopentane cooled to the temperature of liquid nitrogen, and later sectioned in a cryostat (-20 °C) for histochemical measurements of muscle fiber composition.30 The other biopsy specimens, with the exception of that used for 14 C-palmityl CoA oxidation, were individually weighed, frozen, and stored in liquid nitrogen. These pieces of tissue were subsequently analyzed for succinate dehydrogenase (SDH), malate dehydrogenase (MDH), hexokinase (HK), carnitine palmityltransferase (CPT), and lipoprotein lipase (LPL). The muscle sample used to determine the oxidation of 14C-palmityl CoA in vitro was weighed and homogenized immediately for assay. The activities of muscle SDH,12 MDH,11 HK,3 and LPL29 were determined by previously published methods.23 CPT activity was assayed by the method of Bieber et al. 4 In this measurement, a muscle specimen, weighing roughly 10 mg, was homogenized in a 1-to-40 dilution with 250 mM sucrose/0.2 mM EDTA. An aliquot of this homogenate was incubated in a tris-buffered cocktail (2.5 ml) containing 1.65 mM EDTA, 0.03 mM palmityl-CoA, 0.44 mM carnitine, and 0.121 mM DTNB. The reaction was measured spectrophotometrically at 412 nm, 25 °C. The rate of palmityl-CoA oxidation in vitro was determined by modification of the Fritz and Yue method.13 The sample (15 to 25 mg) was placed in 1.5 ml of cold (4°C) 175 mM KCI/2.5 mM EDTA (300 mosmol/kg) and homogenized using a Teflon tissue homogenizer. Duplicate 500-/xl aliquots of this homogenate were placed in each of two incubation flasks. The flasks contained 2.5 ml of an incubation medium13 and optimal amounts of 14Cpalmityl CoA and palmityl-CoA in final concentrations of 0.1 AIM (40,000 dpm) and 6.6 fxM, respectively. The samples were subsequently incubated for 30 min in a shaker bath set at 37 °C and 40 oscillations per minute. The reaction was stopped with the addition of 1.0 ml of IN perchloric acid. A strip of filter paper, soaked in 0.5 ml of NCS (a quaternary ammonium base), in a shell vial was used to trap the 14CO2 produced. After allowing the NCS to absorb all the CO2 in the vial, the shell vial, filter paper, and NCS were placed in 15 ml of toluene scintillation fluid and their 14C activity was measured. Training. After the preliminary tests, the JD and ND subjects initiated a 10-wk running program, exercising 5 days per week, 30 min per day. The intensity of each training


TABLE 1 Mean (±SE) information concerning subjects before training Ht (cm)

Wt (kg)

% Fat

% ST


178.9 (2.5)

67.3 (2.8)

10.6 (0.9)

52.0 (3.6)


181.0 (2.8)

71.5 (3.1)

15.2 (1.0)

56.6 (2.4)


Abbreviations: % Fat = skinfold estimate of body fat,31 and % ST = percentage slow-twitch fibers in gastrocnemius muscle.

session was determined by the subjects. In general, the running speed was gradually increased throughout the 10-wk training period. Based on the preliminary treadmill test and the subjects' running speed, we estimated that the training required the men to use 60 to 70% of their aerobic capacities. Each exercise session was supervised by laboratory personnel, and information regarding training and insulin treatment was recorded regularly. No attempts were made to control the subjects' diets. Statistical treatment. Mean differences between pre- and post-training values were tested for significance (P < 0.05) with a Mest for paired observations. Differences between the means for the JD and ND groups were computed with a Mest for independent groups. RESULTS

Both groups of subjects showed similar gains in Vo2 max— 11.0% for the JD and 12.9% for the ND groups. In both cases, the increases in Vo2 max were significant (P < 0.05). These and other pertinent data from the maximal treadmill tests are presented in Table 2. Mean body weights and the percentage of body weight composed of fat remained unchanged with training. Likewise, the fractional composition of slow-twitch fibers in the gastrocnemius muscle was unaffected by the endurance training program. Consequent to training and the frequency of insulin reactions, the JD men reduced their insulin dose by an average of 23%. Most of this adjustment in insulin treatment occurred in the first 2 to 3 wk of training. Thereafter, the amount of insulin administered remained relatively constant. Table 2 also contains the mean fasting serum values for glucose, triglyceride, cholesterol, glycerol, and free fatty acids. Despite the reduction in insulin administration, the JDs' fasting blood glucose after training was significantly lower (P < 0.05) than during the pretraining tests. These subjects also showed significant (P < 0.05) mean reductions in serum triglyceride and cholesterol as a result of the training program. None of the serum values for the ND group was altered by the training. As anticipated, serum glucose values were significantly higher for the JD than for the ND men. With the exception of triglyceride, this was the only significant difference between the mean serum values for the two groups. After training, serum triglyceride decreased significantly (P < 0.05) for the JD subjects (Table 2). Pretraining muscle enzyme activities were similar for both groups. The single exception was carnitine palmityltransferase (CPT) activity, which averaged 29% higher in the muscle of the JD men than the ND group. These and other mean enzyme activities are presented in Table 3. Endurance



TABLE 2 Mean (±SE) data obtained from the juvenile diabetic and nondiabetic groups before (pre) and after (post) training Nondiabetics



+ 114%






49.2 (2.8)

54.6* (2.4)

48.5 (2.6)

202 (1) 10.4 (0.9)

197* (3) 11.1 (0.9)

199 (2) 11.7 (0.5)

54.8* (1.6) 192*

13.2 (1.8)

11.0* (2.0)

Triglyceride (mM)

0.826 (0.099)

Cholesterol (mM)


*o X

Treadmill Test Vo2 max (ml/kg-min"1) HRmax (beats/min)


c c •a o



12.6 (0.8)

a o

5.1t (0.1)

5.1t (0.1)

o >-

0.678* (0.096)

1.537t (0.275)

1.270t (0.140)


4.56 (0.64)

4.09 (0.24)

4.44 (0.26)

4.33 (0.25)

0. i

Glycerol (mM)

0.074 (0.016)

0.100 (0.044)

0.050 (0.004)

0.061 (0.009)

Free fatty acids (mM)

0.304 (0.081)

0.297 (0.104)

0.263 (0.034)

0.243 (0.042)

Lactic acid:}: (mM) Fasting blood Glucose (mM)


* Denotes significant difference between pre- and posttraining means. t Denotes significant difference between means for JD and ND groups. $ Blood was sampled 3 min afterthe maximal oxygen uptake test.

training produced a marked increase in mean muscle LPL, CPT, SDH, and HK activities for the diabetics and nondiabetics. These changes in muscle enzyme activities were similar for both groups. Although muscle MDH showed increases with training, only the ND means were significantly (P < 0.05) changed. Figure 1 illustrates the mean rates for 14C-palmityl CoA oxidation. Though the pretraining rates of 14Cpalmityl CoA oxidation were similar for both the ND and JD groups, the diabetic group showed a significantly (P < 0.05) greater capacity for this lipid oxidation in vitro after training than did the nondiabetics. The post-training mean values for 14C-palmityl CoA oxidation was 41 % higher in the diabetic than in the nondiabetic subjects. TABLE 3 Means (±SE) for muscle lipoprotein lipase (LPL), carnitine palmityltransferase (CPT), succinate dehydrogenase (SDH), malate dehydrogenase (MDH), and hexokinase (HK) activities measured before (pre) and after (post) training in the diabetic (JD) and nondiabetic (ND) men. Variable g/min) LPL CPT SDH MDH HK


Diabetic Pre 0.88 0.89t 6.9 53.3 2.2

(0.16) (0.05) (0.6) (5.3) (0.2)



1.27* (0.28) 1.01'(0.04) 8.6* (1.6) 56.8 (5.7) 2.7* (0.2)

0.87(0.21) 0.68 (0.04) 7.4 (0.8) 51.4 (3.1) 2.2 (0.2)

Post 1.11* 0.91* 10.0* 61.4* 2.7*

(0.33) (0.04) (1.1) (2.4) (0.2)

* Denotes significant change (P < 0.05) from the pretraining mean, t Denotes significant difference (P < 0.05) between means before training for the diabetic and nondiabetic subjects.





FIGURE 1. Mean rates of "C-palmityl CoA oxidation for diabetic and nondiabetic subjects before (pre) and after (post) training. Black vertical bars denote SE of means, while asterisks indicate significant difference between pre- and posttraining means.


The principal finding of this research was that juvenile diabetics who are in moderate insulin balance show adaptations to endurance training that are not different from those of the nondiabetic. Specifically, the capacity to consume oxygen during exhaustive exercise (Vo2 max, ml/kg-min" 1 ) was similar in the JD and ND subjects both before and after training. This latter point clearly demonstrates that juvenile diabetics adapt to endurance exercise as well as do nondiabetics and confirms earlier findings by Larsson et al. 21 Previous studies, however, have reported lower Vo2 max values for untrained juvenile diabetics than for an age-matched group of untrained nondiabetics.20>21>32 The present data suggest that such differences in diabetic and control subjects might be attributed to differences in pretraining activity levels rather than to limitations induced by diabetes per se. Endurance training is known to enhance the muscle's capacity for oxidative metabolism.1-18 This fact has been attributed to both quantitative and qualitative changes in muscle mitochondria.18 Krebs cycle enzyme activities, measured before and after training, are frequently used to reflect changes in the muscle's oxidative potential. The values reported here for SDH are similar to those previously cited for untrained and moderately trained men.12 Muscle MDH activity, on the other hand, is an enzyme less specific to mitochondrial activity, which may explain its smaller change with endurance training. Drug-induced diabetic animals have lower than normal muscle hexokinase (HK) activities.9 The present data, however, demonstrate that insulin-treated diabetics have normal



HK activities (Table 3). One might speculate that insulin deprivation, as seen in the diabetic animals, probably lowers HK concentration as a result of the reduced glucose entry into the muscle fiber. Thus, one might speculate that insulin-treated diabetics have normal mitochondrial and glucose-transport enzyme activities as a result of near normal substrate flux within the muscle. Despite the fact that untreated diabetics generally show reduced LPL activity,5 the LPL activity of the JD was the same as that of the ND. Pretraining muscle CPT activity was significantly higher in the JD. This was not surprising, since it has been shown that diabetics have elevated liver CPT activity.24-27 Norum27 found that diabetics also have a greater capacity to oxidize FFA. However, in the present study, the capacity of the muscle to oxidize FFA, as measured by the oxidation of 14C-palmityl CoA in vitro, did not differ between groups before training. Therefore, it would appearthat the JD muscle was able to hydrolyze triglycerides, transport FFA into the mitochondria, and oxidize FFA normally before training. Studies have demonstrated that endurance training also enhances the capacity for lipid metabolism.25'26 Lower respiratory-exchange ratios have been observed among trained as compared with untrained subjects at the same relative exercise intensity. This supports the concept of an elevated extraction and oxidation of FFA during exercise after training.14 The LPL and CPT responses to training were similar for both the JD and ND groups. The 14Cpalmityl CoA oxidation capacity of the JD muscle increased noticeably more than that of the ND muscle, but it was still within the range found for trained men.10 Therefore, the changes in lipid metabolism caused by training appear to be normal among juvenile diabetics. It should be noted that the changes in CPT activity and in oxidation of palmityl CoA in vitro with training did not parallel each other. Since CPT is not rate limiting to the entry of palmityl CoA into the mitochondria,35 there is no reason to expect a constant relationship with the 14CoA evolution in our muscle homogenate. It is acknowledged that the 14C-palmityl CoA oxidation is probably less than maximal, but this is also true of the enzymes measured here. We are assuming that these pre- and posttraining values are relative to changes associated with the oxidative capacity of muscle. Serum triglyceride (TG) concentrations were also normal in the two groups and showed a decrease with training. Since the JD subjects were moderately well controlled on insulin before training, one would not expect them to be hypertriglyceridemic. As stated earlier, endurance training results in a slower rate of glycogenolysis in working muscle and a shift in the carbon source for the citric acid cycle.16-17 It is conceivable, therefore, that endurance training played a role in lowering the fasting serum TG by facilitating the uptake and utilization of FFA. With endurance training, muscle glycogen storage is enhanced15-19 and there is a diminished insulin response to a given carbohydrate oral feeding.6"8-22 These changes along with an increased hexokinase activity could account, in part, for the decreased blood glucose concentration and daily insulin requirement seen in the JD after training.


Thus, we can conclude that the skeletal muscle of insulintreated diabetics is capable of making the same adaptive changes to endurance training as nondiabetics. ACKNOWLEDGMENTS

This research was supported by grants from the National Institutes of Health (HL 20408-02) and the American Diabetes Association, Indiana Affiliate. REFERENCES 1 Baldwin, K. M., Klinkerfuss, G. H., Terjung, R. L, Mole, P. A., and Holloszy, J. 0.: Respiratory capacity of white, red and intermediate muscle: adaptive response to exercise. Am. J. Physiol. 222:373-78, 1972. 2 Berger, M., Hagg, S. A., and Ruderman, N. B.: Effects of starvation and diabetes on muscle glucose and alanine metabolism. Diabetes 22 (Suppl. 1):292, 1973. 3 Bergmeyer, H. U., editor: Enzymes as biochemical reagents. In Methods of Enzymatic Analysis, Vol. 1. New York, Academic Press, 1974, pp. 425-522. 4 Bieber, L. L, Abraham, T., and Helmrath, T.: A rapid spectrophotometric assay for camitine palmityltransferase, Anal. Biochem. 50: 509-18, 1972. 5 Biemar, E. L: Insulin and hypertriglyceridemias. Isr. J. Med. Sci. 8:303-07, 1972. "Bjorntorp, P., Berchtold, P., Grinky, G., Lindholm, B., Sanne, H., Tibblin, G., and Wilhelmsen, L: Effects of physical training on glucose tolerance, plasma insulin and lipids, and on body composition in men after myocardial infarction. Acta Med. Scand. 792:439-43, 1972. 7 Bjorntorp, P., de Jonnge, K., Sjostrom, L et al.: The effect of physical training on insulin production in obesity. Metab. Clin. Exp. 79: 631-38, 1970. " Bjorntorp, P., Fahl6n, M., Grimby, G. et al.: Carbohydrate and lipid metabolism in middle-aged, physically well-trained men. Metab. Clin. Exp. 27:1037-44, 1972. "Colowick, S. P., Ceri, G. T., and Slein, M. W.: The effect of adrenal cortex and anterior pituitary extracts and insulin on the hexokinase reaction. J. Biol. Chem. 768:583-96, 1947. 10 Costill, D. L, Fink, W. J., Getchell, L H., Ivy, J. L, and Witzmann, F. A.: Lipid metabolism in skeletal muscle of endurance trained males and females. J. Appl. Physiol. In press, 1979. 11 Costill, D. L, Coyle, E. F., Fink, W. J., Lesmes, G. R., and Witzmann, F. A.: Adaptations in skeletal muscle following strength training. J. Appl. Physiol.: Respirat-Environ. Exercise Physiol. 46:96-99, 1979. 12 Costill, D. L, Daniels, J., Evans, W., Fink, W., Krahenbuhl, G., and Saltin, B.: Skeletal muscle enzymes and fiber composition in male and female track athletes. J. Appl. Physiol. 40:149-54, 1976. '•''Fritz, I. B., and Yue, K. T.: Long-chain camitine acyltransferase and the role of acylcarnitine derivatives in the catalytic increase of fatty acid oxidation induced by camitine. J. Lipid Res. 4:279-88, 1963. 14 Gollnick, P. D.: Free fatty acid turnover and the availability of substrates as a limiting factor in prolonged exercise. In The Marathon: Physiological, Medical, Epidemiological, and Psychological Studies. Milvy, P., Ed. New York, New York Academy of Science, 1977, p. 66. 15 Grollman, S.: A study of oxygen debt in the albino rat. J. Exp. Zool. 728:511-23, 1955. l(i Hermansen, L., Hultman, E., and Saltin, B.: Muscle glycogen during prolonged severe exercise. Acta Physiol. Scand. 77:129-39, 1967. 17 Holloszy, J. O. et al.: Biochemical adaptations to endurance exercise in skeletal muscle. In Muscle Metabolism During Exercise. Pernow, P., and Saltin, B., Eds. New York, Plenum, 1971, pp. 51-61. '" Holloszy, J. O., Oscai, L B., Donn, I. J., and Mole, P. A.: Mitochondrial citric acid cycle and related enzymes: adaptive response to exercise. Biochem. Biophys. Res. Commun. 40:1368-73, 1970. 19 Lamb, D. R., Peter, J. B., Jeffrees, R. R., and Wallace, H. A.: Glycogen, hexokinase, and glycogen synthetase adaptations to exercise. Am. J. Physiol. 27 7:1628-32, 1969. 20 Larsson, Y., Persson, B., Sterky, G., and Thorin, C : Functional adaptation to rigorous training and exercise in diabetic and non-diabetic adolescents. J. Appl. Physiol. 79:629-35, 1964. 21 Larsson, Y., Sterky, G., Ekengren, K., and Moller, T.: Physical fitness and the influence of training in diabetic adolescent girls. Diabetes 7 7:109-13, 1962. 22 Lohmann, D., Liebold, F., Heilmann, W., Senger, H., and Pohl, A.: Diminished insulin response in highly trained athletes. Metab. Clin. Exp. 27:521-24, 1978. 23 Lowry, O. H., and Passonneau, J. V.: A Flexible System of Enzymatic Analysis. New York, Academic Press, 1972. 24 Mehlmane, M. A. et al.: Metabolism turnover time, half life, and body pool of camitine " C in normal alloxan diabetic and insulin treated rats. Life Sci. 8:465, 1969.


TRAINING ADAPTATIONS IN SKELETAL MUSCLE C£ JUVENILE DIABETICS 25 Mole, P. A., and Holloszy, J. 0.: Exercise induced increase in the capacity of skeletal muscle to oxidize palmitate. Proc. Soc. Exp. Biol. Med. 734:789-92, 1970. 26 Mole, P. A., Oscai, L P., and Holloszy, J. 0.: Adaptations of muscle to exercise. Increase in the levels of palmityl CoA synthetase, carnitine palmityltransferase and palmityl CoA dehydrogenase, and in the capacity to oxidize fatty acids. J. Clin. Invest. 50:2323-30, 1971. 27 Norum, K. Ft.: Activation of palmityl CoA: carnitine palmityltransferase in livers from fasted, fat-fed, or diabetic rats. Biochem. Biophys. Acta 98:654, 1965. 2H Nakajima, K., and Ishikawa, E.: Hormonal and dietary control of enzymes in the rat. J. Biochem. (Tokyo) 68:829-34, 1971. 29 Nilsson-Ehle, N.J., and Shotz, M.: A stable, radioactive substrate emulsion for assay of lipoprotein lipase. J. Lipid Res. 7 7:536-41, 1976.


30 Padykula, H. A., and Herman, E.: The specificity of the histochemical method for adenosine triphosphatase. J. Histochem. Cytochem. 3:170-95, 1955. 31 Sloan, A. W.: Estimation of body fat in young men. J. Appl. Physiol. 23:311-15, 1967. 32 Sterky, G.: Physical work capacity in diabetic school children. Acta Pediatr. 52:1-7, 1963. 33 Weber, G.: Integrative action of insulin at the molecular level. Isr. J. Med. Sci. 8:325-40, 1972. 34 Wilmore, J. H., and Costill, D. L: Semiautomated systems approach to the assessment of oxygen uptake during exercise. J. Appl. Physiol. 36:618-20, 1974. 35 Witzmann, F. A.: Changes in muscle lipid metabolism with endurance training in man. Thesis, Ball State University, Muncie, IN, 1978.


Training adaptations in skeletal muscle of juvenile diabetics.

Training Adaptations in Skeletal Muscle of Juvenile Diabetics D. L COSTILL, P. CLEARY, W. J. FINK, C. FOSTER, J. L. IVY, AND F. WITZMANN SUMMARY Ske...
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