Effect of Acute Exercise on Insulin Binding to Erythrocytes in Type II Diabetes

Transcription

1 Endocrinol. Japon. 1982, 29 (5), Effect of Acute Exercise on Insulin Binding to Erythrocytes in Type II Diabetes MAKOTO IWASAKI, MASASHI KOBAYASHI, SEIJI OHGAKU, HIROSHI MAEGAWA AND YUKIO SHIGETA The Third Department of Medicine, Shiga University of Medical Science. Seta, Ohtsu, Shiga Abstract We studied the effect of acute short-term exercise on insulin binding to erythrocytes in patients with non-insulin dependent diabetes mellitus and normal controls. In the normal controls, insulin binding was increased immediately after the exercise and returned to the basal level at 30 min after the exercise. This increase was due to an affinity change without any change in the insulin receptor number. In contrast, insulin binding tended to decrease in the diabetics after the exercise, and this change was due to a decreased affinity without any change in the number of insulin receptors. These results indicate that the control mechanism of insulin receptor affinity in response to exercise is different in non-insulin dependent diabetics and normals. Many investigators reported that the affinity of insulin receptors can change in variable conditions such as acromegaly (Muggeo et al., 1978), fasting states (Olefsky, 1976; Kolterman et al., 1979), streptozotocininduced diabetics (Kobayashi and Olefsky, 1979), the condition under chronic hypergravitation (Kobayashi et al., 1980) and L- Dopa administration (Iwasaki et al., 1982). Physical exercise has been shown to cause an increase in insulin binding. Pedersen et al. reported that insulin binding to both erythrocytes and monocytes was increased after exercise in patients with insulindependent diabetes mellitus (Pedersen et al., 1980). However, there has been no insulin Received May 6, This study was supported in part by Grant No from the Ministry of Education, Science and Culture and a Research Grant for Intractable Diseased from the Ministry of Health and Welfare of Japan. binding study during exercise in patients with non-insulin dependent diabetes mellitus (NIDDM). In the present study we studied the effect of exercise on insulin binding to erythrocytes in patients with non-insulin dependent diabetes mellitus in order to examine whether insulin binding to erythrocytes in type II diabetics may react differently with exercise. Subjects Materials and Methods Erythrocytes from seven male non-insulin dependent diabetics without diabetic complications were examined for insulin binding before and after exercise. Their age, body weight and % of ideal body weight were 39 }8 years, 65 }12 kg and %, respectively (Mean }SD). Three of them were treated with oral hypoglycemic agents and the rest with diet thereby only. Eight healthy non-obese male volunteers were

2 562 IWASAKI et al. Endocrinol. Japon. October 1982 studied as controls and their age, weight and % of ideal body weight were 28 }4 years, 62 }7 kg and 103 }10%, respectively. The control subjects had no history of illness known to affect glucose metabolism and group was engaged in regular physical exercise before the study. After a twelve hour overnight fast, the subjects were asked to jog for 30 min at a speed of 6 km/h on a treadmill (model 18-54, Quinton Instrument Co., Seattle., Washinton, USA). The exercise was of moderate intensity, raising the heart rate to beats/min. The intensity of the exercise was approximately 30 to 40% of their VO2 max. Twenty ml heparinized blood samples were obtained from the antecubital vein before, five and 30 minutes after the end of the exercise. Cell Preparation and Insulin Binding Studies Materials Purified single peak pork insulin was a gift from Shimizu Pharmaceutical Co.. The purity of this insulin was equal to that of monocomponent insulin judged by the electrophoretic method and biological potency was 26 U/mg. Na125 I was purchased from New England Nuclear, Di-n-butyl phthalate from Nakarai Chemicals, Bovine Serum Albumin (BSA) (Fraction V) from Armour Chemical Co. and Lymphoprep(R) (Ficoll-Hypaque gradient solution) from Daiichi Kagaku Yakuhin. throcytes were incubated with 125I-insulin (0.2 ng/ml) and unlabeled insulin at various concentrations ranging from 0 to 100 ng/ml in a total volume of 0.5 ml in a plastic tube at 15 Ž, shaken by hand every 60 min. Incubation was terminated by removing 100ƒÊl aliquots from the cell suspension and immediately centrifuging the cells in plastic microcentrifuge tubes containing 100ƒÊl of dibutyl phthalate and the 200ƒÊl of cold buffer. The cells formed a pellet under a layer of dibutyl phthalate and the radioactivity was determined. Nonspecific binding was assessed by incubating cells with 100ƒÊg/ml of unlabeled insulin, and the binding data were obtained by subtracting the nonspecific binding. Analytical procedures Plasma glucose was measured by the glucose oxidation method. Plasma insulin, growth hormone and glucagon were determined by a double antibody radioimmunoassay. Plasma ketone bodies were determined colorimetrically by the modification of Salway's method (Yamashita et al., 1981), which is based on a reaction of acetoacetate with diazonium salt (paranitrophenol-diazonium fluoroborate). Plasma free fatty acid was measured by the method of Itaya (Itaya and Ui, 1965). Statistical analyses were done by the paired Student's t-test. Results Fig. 1 shows the changes in plasma glucose, insulin, glucagon, growth hormone, free fatty acid and ketone body concent- during the study. In normal sub- rations jects there were no significant changes in the concentrations of these hormones and Binding studies metabolites during the exercise except for Methods of preparation of erythrocytes and insulin binding assay have been described elsewhere growth hormone which increased immediately after the exercise. In the diabetics (Kobayashi et al., 1980). In brief, 3-5 ml of blood was drawn from the subjects with a heparinized there were no significant changes in the syringe. The blood was centrifuged at 2000 rpm for levels of the hormones and metabolites 10 min at 4 Ž to separate plasma for the determination of insulin, glucose, other hormones and metabolites. Buffy coat on a pellet of erythrocytes was The insulin binding to erythrocytes in during the exercise. removed with a pasteur pipette. Erythrocytes were normal subjects was significantly increased isolated by Ficoll-Hypaque gradient method. Erythrocytes were then washed twice with buffer con- within five minutes after exercise at a sisting of HEPES (N-2-Hydroxyethylpiperazine-N Œ-2- tracer insulin concentration (5.80 }0.60 vs ethane-sulfonic acid) 50 mm and 1% of BSA, ph }0.54% per 2.4 ~106 erythrocytes/mm3, Cells were resuspended in 3 volumes of the buffer, P<0.05; Mean }SEM), then reduced to almost the initial level 30 min after the and this suspension was used for the receptor assay. Cells were counted with an Industrial D model Coulter counter with an aperture of 100ƒÊM. Ery- exercise (5.63 }0.49%) (Fig. 2a). The

3 Vol.29, No.5 EFFECT OF ACUTE EXERCISE ON INSULIN BINDING 563 Fig. 2b. Scatchard analysis of the data of Fig. 2a. Fig. 1. Changes in plasma hormones and metabolites of normal subjects are shown on the left and those of diabetics on the right. The values are Mean }SEM. (*: P<0.05). Fig. 2c. Average affinity plot of the data of Fig. 2a. Fig. 2a. Change in insulin binding to erythrocytes from normal subjects before and after exercise. Before ( - ), immediately after ( ~- ~) and 30 min after ( - E- ) exercise (Mean }SEM). (*: P<0.05, Before vs. immediately after exercise). Scatchard analysis of the binding data (Fig. 2b) shows that this increased binding is due to an increased affinity without a change in the number of insulin redeptors. Average affinity plot clearly shows increased affinity at lower levels of insulin receptor occupancy immediately after the exercise (Fig. 2c). In contrast, the insulin binding to erythrocytes tended to decrease at 5 min after exercise and demonstrated a significant decrease 30 min after excercise in diabetics

4 564 IWASAKI et al. Endocrinol. Japon. October 1982 (Fig. 3a). The Scatchard analysis shows that this decreased binding is due to a decreased affinity without a change in the number of insulin receptors (Fig. 3b). The average affinity plot shows that there are no significant differences among affinities of the three insulin binding curves in the non-insulin dependent diabetics (Fig. 3c). Discussion Fig. 3a. Change in insulin binding to erythrocytes from diabetics before and after exercise. Before ( œ- œ), immediately after ( ~--- ~) and 30 min after ( - E- ) exercise (Mean }SEM). (*:P< 0.05, Before vs. 30 min after exercise). Fig. 3b. Scatchard analysis of the data in Fig. 3a. Fig. 3c. Average affinity plot of the data in Fig. 3a. It is well known that the affinity and the number of insulin receptors are affected by variable physiological factors (Roth et al., 1976; Olefsky, 1976; Gavin et al., 1974). A marked increase in affinity has been reported in several conditions such as fasting states (Bar et al., 1976; Olefsky and Kobayashi, 1978; Kolterman et al., 1979), acromegaly (Muggeo et al., 1978; Muggeo et al., 1979), L-Dopa administration (Iwasaki et al., 1982), streptozotocin-induced diabetes (Kobayashi and Olefsky, 1979) and exercise (Pedersen et al., 1980). Pedersen reported that acute exercise caused an increase in insulin binding to erythrocytes and monocytes in insulin-dependent diabetics. In the present study insulin binding in non-insulin dependent diabetics was decreased after the exercise, whereas insulin binding was increased in normal subjects. We do not know the reason why insulin receptors showed opposite responses in the two groups. Koivisto reported that the rise in insulin binding to monocytes was less in the obese than the normal subjects during exercise (Koivisto et al., 1980). The decreased sensitivity to insulin is well documented in obesity and non-insulin dependent diabetes mellitus (Olefsky, 1976). These altered responses of insulin receptors to exercise, fasting and etc. may be related to the decrease in insulin sensitivity in NIDDM. Although, in diabetic patients, the mean values for insulin bindings at 30 min after

5 Vol.29, No.5 EFFECT OF ACUTE EXERCISE ON INSULIN BINDING 565 exercise further decreased compared to the values immediately after the exercise, individual diabetic subjects behave differently, i. e. three patients out of seven showed further decreased binding, whereas four out of seven showed increased binding at 30 min after exercise compared to those immediately after exercise. Among the various hormones measured in normal subjects, growth hormone was significantly increased during exercise. In diabetics, growth hormone had a tendency to increase during exercise, but this rise was not statistically significant. Recently we have shown that an increased growth hormone concentration may be related to a change in receptor affinity (Iwasaki et al., 1982). Thus, the increase in growth hormone concentration during exercise may increase insulin receptor affinity in normals. However the increase in the growth hormone concentration does not seem to influence the affinity change in insulin receptors in non-insulin dependent diabetic subjects. We have measured various metabolites that may affect the affinity of insulin receptors, such as ketone bodies and fatty acid, but no substance was identified as the cause of the affinity change. There were no significant changes in the plasma concentration of several hormones and metabolites except for growth hormone, which could be responsible for the affinity change in normal subjects. The control mechanism of insulin receptor affinity could be regulated by several factors which still remain unknown. In conclusion we showed that in noninsulin dependent diabetics the affinity change in insulin receptors during exercise was different from that in normal subjects. Acknowledgment This work was supported in part Grant No from the Ministry of Education, Science and Culture and a Research Grant for Intractable Disease from the Ministry of Health and Welfare of Japan. References Bar, R. S., Gorden, P., Roth, J., Kahn, C. R., De- Meyts, P. (1976). Fluctuations in the affinity and concentration of insulin receptors on circulating monocytes of obese patients: effect of starvation, refeeding and dieting. J. Clin. Invest. 58, Freychet, P., Roth, J., Neville, D. M., Jr. (1971). Monoiodoinsulin: demonstration of its biological activity and binding to fat cells and liver membrane. Biochem. Biophys. Res. Commun. 43, Gavin, J. R., Roth, J., Neville, D. M., Jr., De- Meyts, P., Buell, D. W. (1974). Insulin-dependent regulation of insulin receptor concentrations: a direct demonstration in cell culture. Proc. Natl. Acad. Sci. U.S.A. 71, Insel, J. R., Kolterman, O. G., Saekow, M., Olefsky, J. M. (1980). Short-term regulation of insulin receptor affinity in man. Diabetes 29, Itaya, K., Ui, M. (1965). Colorimetric determination of free fatty acid in biological fluids. J. Lip. Res. 6, Iwasaki, M., Kobayashi, M., Ohgaku, S., Maegawa, H., Shigeta, Y. (1982). Effect of L-Dopa administration on insulin binding : possible role of growth hormone in regulation of insulin receptor affinity. Horm. Metab. Res. 14, Kobayashi, M., Olefsky, J. M. (1979). Effect of streptozotocin-induced diabetes on insulin binding, glucose transport and intracellular glucose metabolism in-isolated rat adipocytes. Diabetes 28, Kobayashi, M., Ohgaku, S., Iwasaki, M., Harano, Y., Maegawa, H. Shigeta, Y. (1980a) Evaluation of the method of insulin binding in human erythrocytes. Endocrinol. Japon. 27, Kobayashi, M., Mondon, C. E., Oyama, J. (1980b). Insulin binding and glucose uptake of adipocytes in rats adapted to hypergravitational force. Am. J. Physiol. 238, E Kolterman, O. G., Saekow, M., Olefsky, J. M. (1979). The effects of acute and chronic starvation on insulin binding to isolated human adipocytes. J. Clin. Endocrinol. Metab. 48, Koivisto, V. A., Soman, V. R., Felig, P. (1980). Effects of acute exercise on insulin binding to monocytes in obesity. Metabolism 29, Muggeo, M., Bar, R. S., Roth, J., Kahn, C. R. (1978). Two abnormalities in insulin binding to its receptor in the insulin resistance of acromegaly. Clin. Res. 26, 310A. Muggeo, M., Bar, R. S., Roth, J., Kahn, C. R., Gorden, P. (1979). The insulin resistance in acromegaly: evidence for two alterations in the insulin receptor on circulating monocytes. J. Clin. Endocrinol. Metab. 48, Olefsky, J. M. (1976a). The insulin receptor: its

6 566 Endocrinol. Japon. October 1982 role in insulin resistance in obesity and diabetes. Diabetes 25, Olefsky, J. M. (1976b). Effect of fasting on insulin binding, glucose transport, and glucose oxidation in isolated rat adipocytes. J. Clin. Invest. 58, Olefsky, J. M., Kobayashi, M. (1978). Mechanism of the fasting-induced increase in insulin binding to rat adipocytes. J. Clin. Invest. 61, Pederson, O., Beck-Nielsen, H., Heding, L. (1980). Increased insulin receptors after acute exercise in patients with insulin-dependent diabetes mellitus. N. Engl. J. Med. 302, Roth, J., Kahn, C. R., Lesniak, M. A., Gorden, P., DeMeyts, P., Megyesi, K., Neville, D. M. Jr., Gain, J. R., III, Soil, A. H., Freychet, P., Goldfine, I. D., Bar, R. S., Archer, J. A. (1976). Receptors for insulin, NSILA-S and growth hormone: applications to disease states in man. Recent Prog. Horm. Res. 31, Yamashita, Y., Harano, Y., Hidaka, H., Kosugi, K., Yasumoto, K., Shigeta, Y. (1981). Simplified method for the differential determination of blood ketone bodies. Jpn. J. Clin. Pathol. 29, (in Japanese).