Abnormal feeding behavior and insulin replacement in STZ-induced diabetic rats

Physiology& Behavior, Vol. 47, pp. 731-734. ©Pergamon Press plc, 1990. Printed in the U.S.A.

0031-9384/90 $3.00 + .00

Abnormal Feeding Behavior and Insulin Replacement in STZ-Induced Diabetic Rats YOHNOSUKE SHIMOMURA,I MASAKI TAKAHASHI, HIROYUKI SHIMIZU, NORIYUKI SATO, YUTAKA UEHARA, MAYUMI NEGISHI, TOSHIHIKO INUKAI, ISAO KOBAYASHI AND SETSUO KOBAYASHI

Division of Endocrinology, Department of Medicine, the First Department of Internal Medicine Gunma University of Medicine, Maebashi, Japan R e c e i v e d 29 A u g u s t 1989

SHIMOMURA, Y., M. TAKAHASHI, H. SHIMIZU, N. SATO, Y. UEHARA, M. NEGISHI, T. INUKAI, I. KOBAYASHI AND S. KOBAYASHI. Abnormal feeding behavior and insulin replacement in STZ-induced diabetic rats. PHYSIOL BEHAV 47(4) 731-734, 1990. --The present studies were undertaken to investigate whether or not decreased ambulatory activity, including abnormal feeding behavior in diabetic rats, will be simultaneously normalized by insulin administration. To do this, we used the Gunma University-type automatic apparatus for continuous and direct measurement of ambulation and drinking. In this study, 3 U NPH insulin were administered at 1800, just before the dark phase, and 2 U were administered at 0600, just before the light phase. With these insulin doses, we found that 5 weeks were needed to normalize ambulatory activity, 4 weeks were necessary for food intake, 6 weeks for drinking and 2 weeks for body weight. Since ambulatory activity is reported to be related to changes in dopamine turnover, further studies are in progress to determine whether or not dopamine turnover is normalized when there is no difference in ambulatory activity due to insulin replacement. Ambulation

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RECENTLY Marshall et al. (7) reported that amphetamineinduced wheel running and stereotyped behavior, which depend on the function of central dopaminergic neurons (3), were decreased in rats with alloxan-induced diabetes in a relatively short time, 90 minutes. In addition, they concluded that these phenomena appear to be the results of the diabetic state, since amphetamine-induced stereotyped behavior could be reinstated in alloxan-treated rats by administration of protamine zinc insulin for 10 days (7). We recently found, in striatum dopaminergic neurons of STZ-induced diabetic rats, metabolic abnormalities associated with decreased ambulatory activity (13). However, little is known about whether or not this decreased ambulatory activity, as well as drinking and feeding behavior, is normalized by insulin administration. To do this, we used the Gunma University-type automatic apparatus, described previously (5, 16, 17), for continuous and direct measurement of ambulation and drinking.

Insulin replacement

tank and a drinking spout (SE TV-25, Ohara and Co.). When the rats drank, water fell into the cartridge drop by drop, and the number of drops was counted electrically. Ambulation and drinking were simultaneously measured in 10 cages of this type. The activity counts were recorded every hour and automatically printed out. Experiments were performed on adult male Wistar rats (Imai animal center, Saitama, Japan), weighing 200-250 g at the start of the experiment. They were kept in a controlled environment (23---2°C) on a 12-hr light (0600-1800 hr), 12-hr dark (18000600 hr) cycle. Rats were habituated to the cage for several weeks and given ground Purina rat chow (Ralston-Purina, St. Louis, MO) before measurements were started. We observed ambulatory activity, including feeding behavior, for 7 days in the control rats (Cont) ( n = 15), which were injected with the vehicle (acidified saline) solution. Test rats were made diabetic by injection of STZ (Sigma Chemical Co., St. Louis, MO; 60 mg/kg IP), which was dissolved in saline acidified to pH 4.5 with citrate (1). Twentyfour hours after STZ administration, diabetes was verified by glucosuria and blood glucose level (>200 mg/dl, n = 10). We observed ambulatory activity, including feeding behavior, in diabetic rats for 4 weeks. Rats were then injected subcutaneously with NPH insulin at various doses. Thereafter, we have studied whether or not the abnormal ambulatory activity, and abnormal feeding behavior in the diabetic rats were normalized by insulin administration.

METHOD The automatic apparatus for measurement of ambulation and drinking was assembled by a medical instrument maker (Ohara and Co. Ltd., Tokyo, Japan). A tilting floor grid (40 cm long x 25 cm wide x 25 cm high) that recorded movement in only one direction was used to measure ambulatory activity. A cartridge that made a water drop of 0.05 ml was inserted between a water

~Requests for reprints should be addressed to Yohnosuke Shimomura, MD, Division of Endocrinology, Department of Medicine, Gunma University Hospital, 3-39 Showa-Machi, Maebashi, 371, Japan.

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Methods for frequent sampling of blood, such as cutting the tail or puncturing the heart, allow few samples to be taken. Moreover, a serious disadvantage of these methods is the induction of hormonal imbalance because the animal is subjected to stress. Therefore, we modified the technique of Steffen's method (14) to collect blood samples in unrestrained and unanesthetized rats by inserting a cannula at the entrance of the venae cavae into the right atrium. Blood glucose level was measured by the glucose oxidase method. For statistical study, analysis of variance (one-way ANOVA with repeated measures) was employed and Newman-Keuls test was used for individual comparison when appropriate, and p < 0 . 0 5 was the criterion for statistical significance.

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In the control rats, the mean ambulatory activity was 371.8 ---54.3 for 24 hours. After administration of STZ, ambulatory activity decreased further each day. After 4 weeks of STZ administration, mean ambulatory activity of 134.8±9.5 for 24 hours was observed (p<0.01 compared to control). After insulin administration, ambulatory activity increased. There was no significant decrease compared to control after 5 weeks of insulin administration (Fig. 2).

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In the first study, we administered various doses of insulin to normalize blood glucose level for 14 days. As shown in Fig. 1, mean blood glucose level in the control group was 133.9±4.3 mg/dl at 0600 and 131.6±3.4 mg/dl at 1800. Thus, there was no diurnal difference between the dark and light phases. When 4 U insulin were administered at 1800, just before the dark phase, marked hyperglycemia was apparent at the end of the light phase (449.0 ± 37.4 mg/dl at 1500,447.3 ± 49.1 mg/dl at 1800). Therefore, insulin was administered twice a day. When 3 U insulin were administered at 1800 and 1 U at 0600, hyperglycemia was observed at the end of the dark and light phases (387.8±39.5 mg/dl at 0600, 420.5 ±27.9 mg/dl at 1500). After the treatment with 4 U at 1800, just before the dark phase, and 2 U at 0600, just before the dark phase, hyperglycemia was evident at 1500 (271.1 --46.0 mg/dl) and at 1800 (177.6±42.5 mg/dl). The best diurnal rhythm of blood glucose level, in our doses, was obtained when 3 U insulin were administered at 1800 and 2 U at 0600 (158.0± 27.2 mg/dl at 0300, 130.0±16.1 mg/dl at 1500) (Fig. 1). Therefore, we administered 3 U of insulin at 1800 and 2 U at 0600 for 8 weeks to normalize abnormal feeding behavior in diabetic rats.

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FIG. 1. Diurnal rhythm in blood glucose level after treatment with insulin. Mean blood glucose level in control rats was 133.9 _+4.3 mg/dl at 0600 and 131.6---3.4 mg/dl at 1800. When 4 U insulin were administered at 1800, marked hyperglycemia was observed at the end of the light phase. When 3 U insulin were administered at 1800 and 1 U at 0600, hyperglycemia was evident at the end of the dark and light phases. The best diurnal rhythm in blood glucose was obtained when 3 U were administered at 1800 and 2 U at 0600.

which was 1.7 times that of control. After treatment with insulin, food intake decreased daily. Increase compared to controls was significantly the same 4 weeks after insulin administration (Fig. 4). Thereafter, there was no difference compared to controls throughout the observation time.

Changes in Drinking Behavior In the control rats, a mean drinking volume was 45.9 - 1.5 ml. After administration of STZ, drinking volume increased continu-

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Mean body weight was 358.4---7.5 g in the control rats. After administration of STZ, body weight decreased continuously. Four weeks later, decrease in body weight was about 60 g compared to control rats (Fig. 3). Two weeks after treatment with insulin, body weight increased significantly compared to controls, and thereafter, body weight increased normally (Fig. 3).

Changes in Food Intake In the control rats, mean food intake was 33.2---0.7 g. We observed the changes in food intake for 4 weeks after STZ administration and for 8 weeks after insulin replacement. After administration of STZ, food intake increased continuously. After 4 weeks of STZ administration, food intake was 58.8---3.7 g

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FIG. 2. Changes in ambulatory activity. After administration of STZ, ambulatory activity decreased day by day. Ambulatory activity continued to increase during insulin replacement. There was no significant decrease compared to control after 5 weeks of insulin administration. *p<0.05; **p<0.01, compared to control.

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ously. After 4 weeks of STZ, a mean drinking volume was 345.0 - 35.9 ml, which was 7.5 times that of control. After insulin treatment, drinking volume continued to decrease. There was no significant increase greater than that of the control rats after 6 weeks of insulin administration (Fig. 5). DISCUSSION

In these studies, we found that decreased ambulatory activity, increased drinking volume, increased food intake and decreased body weight in the diabetic rats could be normalized by insulin administration. In the first experiment, 3 U of insulin at 1800 (just before the dark phase), and 2 U at 0600 (just before the light phase) were determined to be the best dosages to normalize high levels of blood glucose in the diabetic rats.

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FIG. 5. Changes in drinking volume. In the diabetic state, drinking volume increased continuously. Four weeks after STZ, mean drinking volume was 345.0 +-35.9 ml, which was 7.5 times control. There was no significant increase over control rats after 6 weeks of insulin administration. *p<0.05; **p<0.01; ***p<0.001, compared to control.

FIG. 3. Changes in body weight. Mean body weight was 358.4---7.5 g in the control rats. After administration of STZ, body weight decreased continuously. Two weeks after treatment with insulin, a significant increase over control was observed.

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FIG. 4. Changes in food intake. Food intake increased continuously after administration of STZ. After four weeks of STZ administration, food intake was 58.8+-3.7 g which was 1.7 times control. No significant increase compared to control was observed after 4 weeks of insulin administration. **p<0.0l; ***p<0.001, compared to control.

Various doses of insulin have been reported in previous papers to normalize abnormal feeding behavior in diabetic rats. Lozovsky et al. (6) reported that treatment with two subcutaneous 2 U/kg injections of protamine zinc insulin (PZI) on days 1 and 2, and 8 U/kg thereafter for 12 days, normalized increased DA receptor sensitivity in diabetic rats. Marshall et al. (8) reported that alloxan-treated rats receiving PZI 1-2 U in the light phase and 1 U in the dark phase for 5 days became nearly as anorexic to amphetamine as normal rats. In addition, Trulson et al. (18) suggested that 5 U/kg PZI every 12 hours for 10 days resulted in restoration of dopamine metabolism to the normal control level. In another experiment, Marshall et al. (7) reported that treatment with 2 U PZI on the first day increased to 5 U by the fourth day, and 2 U twice a day on the 4th to 10th days, during the light phase, and 1 U during the light phase dramatically and significantly affected stereotyped behavior in diabetic rats. Moreover, Chu et al. (2) reported that 5 U/kg PZI administered every 12 hours for 7 days significantly increased motor activity of diabetic rats. Although various doses of insulin have been reported to affect feeding behavior (2, 6, 7, 8, 18), to our knowledge no papers show whether diurnal rhythm in blood glucose level was normalized when blood samples were collected (6-8, 18). We think that further studies are needed to determine relations between diurnal rhythm in blood glucose and insulin dose. In our case, 3 U of insulin were administered at 1800 (just before the dark phase), and 2 U at 0600 (just before the light phase) normalized abnormal feeding behavior in diabetic rats. These insulin doses brought almost normalization of ambulatory activity in 5 weeks, food intake in 4 weeks, drinking in 6 weeks, body weight in 2 weeks. On the other hand, Marshall et al. (7, 8, 12) recently reported that amphetamine-induced stereotypy and locomotor activity, which depended on the function of central dopaminergic neurons, were decreased in alloxan-induced diabetic rats. It is reported that locomotor activity induced by amphetamine depended more on brain DA levels than on noradrenaline levels (10, 12, 15, 19, 20). Evidence is also accumulating that associates ambulatory activity and stereotyped behavior with dopaminergic nerve activity of the nucleus accumbens and striatum, respectively (4,9). Sailer et al. (1 l) reported that striatal DA metabolite levels were reduced 3 and 6 weeks after alloxan administration. Therefore, hyperglycemia would result in decreased rates of DA release and turnover. We recently found metabolic abnormalities associated with

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decreased ambulatory activity (13) in the striatum dopaminergic neurons of STZ-induced diabetic rats. In addition, through hourly observations, we found significant decrease in dopamine turnover (DOPAC/DA) compared to that in control rats in the dark phase when ambulatory activity was lower (2000) (control rats: 0.112 -+ 0.001, diabetic rats: 0.102-+0.003, p < 0 . 0 5 ) , but found no difference in dopamine turnover in the light phase when there was no

difference in ambulatory activity (0800) (Cont: 0.061---0.003, DM: 0.055-+0.006, ns). These results strongly suggest that changes in dopamine turnover are important in the regulation of ambulatory activity. Further studies are in progress to determine whether or not striatal DA metabolism in diabetic rats will normalize when insulin induces no difference in ambulatory activity.

REFERENCES 1. Arison, R. N.; Ciaccio, E. I.; Glitzer, M. S.; Cassaro, J. A.; Pruss, M. P. Light and electron microscopy of lesions in rats rendered diabetic with streptozotocin. Diabetes 16:51-56; 1967. 2. Chu, P. C.; Lin, M. T.; Shian, L. R.; Leu, S. Y. Alterations in physiologic functions and in brain monoamine content in streptozotocin-diabetic rats. Diabetes 35:481-485; 1986. 3. Hoffman, D. C.; Beninger, R. J. The DI dopamine receptor antagonist SCH 23390 reduces locomotor activity and rearing in rats. Pharmacol. Biochem. Behav. 22:341-342; 1985. 4. Kelly, P. H.; Seviour, P. W.; Iversen, S. D. Amphetamine and apomorphine responses in the rat following 6-OHDA lesions of the nucleus accumbens septi and corpus striatum. Brain Res. 94:507-522; 1975. 5. Kuribara, H.; Hayashi, T.; Alam, M. R.; Tadokoro, S.; Miura, T. Automatic measurement of drinking in rats. Effects of hypophysectomy. Pharmacol. Biochem. Behav. 9:697-702; 1978. 6. Lozovsky, D.; Sailer, C. F.; Kopin, I. Dopamine receptor binding is increased in diabetic rats. Science 214:1031-1033; 1981. 7. Marshall, J. F. Further analysis of the resistence of the diabetic rat to d-amphetamine. Pharmacol. Biochem. Behav. 8:281-286; 1978. 8. Marshall, J. F.; Friedman, M. I.; Heffner, T. G. Reduced anorexic and locomotor-stimulant action of D-amphetamine in alloxan-diabetic rats. Brain Res. 111:428-432; 1976. 9. Pijnenburg, A. J. J.; Van Rossum, J. M. Stimulation of locomotor activity following injection of dopamine into the nucleus accumbens. J. Pharm. Pharmacol. 25:1003-1005; 1973. 10. Randrup, A. J.; Munkvad, I. Role of catecholamine in the amphetamine excitation response. Nature 211:540; 1966. 11. Saller, C. F. Dopaminergic activity is reduced in diabetic rats. Neurosci. Lett. 49:301-306; 1984. 12. Scheel-Kruger, J.; Randrup, A. Stereotyped hyperactive behavior

13.

14. 15.

16.

17. 18. 19.

20.

produced by dopamine in the absence of noradrenaline. Life Sci. 6:1389; 1967. Shimomura, Y.; Shimizu, H.; Takahashi, M.; Sato, N.; Uehara, Y.; Suwa, K.; Kobayashi, I.; Tadokoro, S.; Kobayashi, S. Changes in ambulatory activity and dopamine turnover in streptozotocin-induced diabetic rats. Endocrinology 123:2621-2625; 1988. Steffens, A. B. A method for frequent sampling of blood and continuous infusion of fluids in the rat without disturbing the animal. Pharmacol. Behav. 4:833-836; 1969. Stock, J. M.; Reck, R. H. Antagonism of D-amphetamine by alpha-methyl-para-tyrosine. Behavioral evidence for the participation of catecholamine stores and synthesis in the amphetamine stimulant response. Neuropharmacology 9:249-263; 1970. Tadokoro, S.; Kuribara, H.; Shirasaka, K.; Fujimoto, K.; Alam, M. R. Automatic measurement of diurnal rhythms of naive behaviors in rats and its pharmaco-toxico applications. Adv. Biosci. 41:77-84; 1982. Tadokoro, S.; Kuribara, H.; Shirasaka, K.; Alam, M. R.; Fujimoto, K. Fully automatic measurement of behavioral rhythms in rats and its applications. Jpn. J. Neuropharmacol. 3:785-803; 1981. Trulson, M. E.; Himmel, C. D. Decreased brain dopamine synthesis rate and increased [3H] spiroperidol binding in streptozotocin-diabetic rats. Pharmacol. Biochem. Behav. 40:1456-1459; 1983. Thornburg, J. E.; Moore, K. E. The relative importance of dopaminergic and noradrenergic neuronal systems for the stimulation of locomotor activity induced by amphetamine and other drugs. Neuropharmacology 12:853-866; 1973. Weissman, A.; Koe, B. K.; Tenen, S. Anti-amphetamine effects following inhibition of tyrosine hydroxylase. J. Pharmacol. Exp. Ther. 151:339-352; 1966.