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Reduced anorexic and locomotor-stimulant action of D-amphetamine in alloxan-diabetic rats
428
Brain Rese,rch. 111 (1976) 42~ 432 ~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The NctherkN;d,.
Reduced anorexic and locomotor-stimulant action of D-amphetamine in alloxan-diabetic rats
JOHN F. MARSHALL, MARK 1. FRIEDMAN* and THOMAS G. HEFFNER Psychobiology Program, Departments of Psychology and Psychiatry, Department of Psychology ( M.I.F.) and Department of Biology (T.G.H.), University of Pittsburgh, Pittsburgh, Pa. 15260 (U.S.A.)
(Accepted April 13th, 1976)
During an investigation of the rat with diabetes mellitus induced by atloxan injections, we have observed that such animals are resistant to the anorexic action of amphetamine. Moreover, diminished responsiveness to amphetamine is not limited to feeding-related behaviors, since diabetic rats show little of the normal increase in motor activity following amphetamine injection. These findings suggest to us that alloxan diabetes might induce an alteration in central structures responsible for amphetamine's behavioral actions. The present communication documents these behavioral changes and suggests that they cannot be explained by a reduction in the accumulation of amphetamine in the plasma or brains of diabetic rats. Several possible mechanisms for this altered drug response are discussed. Experiment 1 compared the anorexic action of amphetamine in diabetic and control rats. Twenty female albino Sprague-Dawley rats (200-250 g; Zivic-Miller, Pittsburgh) were housed individually in wire-mesh cages with Purina lab chow and tap water available ad libitum, except as noted. Lights in the colony room were on between 0800 and 2000 h. Ten rats were made diabetic by an injection of alloxan monohydrate (200 mg/kg, dissolved in 0.9 ~ saline, s.c., Sigma) under light ether anesthesia. The remaining l 0 rats were etherized but not injected. During the first week after alloxan, the presence of diabetes was confirmed by glyeosuria (Tes-Tape, Lilly), marked polydipsia and failure to gain weight 3,8. Tests for amphetamine anorexia began 12-14 days after the alloxan injection, and continued thereafter at 4 day intervals. All rats were deprived o f food (but not of water) 24 h before each test. On each test day, the rats received an i.p. injection of 0.9 ~ NaCI or of D-amphetamine sulfate (Sigma ; 0.25, 0.50, or 1.00 mg/kg, as the salt) 15 min before food was introduced into the home cages. Food intake ( ! 0.1 g, corrected for spillage) was measured duringthe following 45 min. When injected with saline, both alloxan-treated and control rats ate substantial amounts of food (4.4-5.2 g) following 24 h deprivation. Amphetamine produced a * Present address: Psychology Department, University of Massachusetts, Amehrst, Mass., U.S.A.
429 b o Controls etics
[3 Controls • Insulin-Treated
Diabetics
2 ~3 2
o~o~5 o;o ,;o
Amphetomine (mg/kg)
Isotonic Amphetamine Saline (0.50mg/kg)
Fig. 1. The left frame (a) depicts the dose-response relationship for amphetamine anorexia in control and alloxan-diabetic rats. Points indicate mean intakes in 45 rain tests; brackets show one S.E.M. The right frame (b) depicts the restoration of anorexia to amphetamine (0.5 mg/kg) in alloxan-treated rats given protamine zinc insulin. Bars indicate means: brackets show one S.E.M.
profound dose-dependent decrease in the food intake of control rats. However, these doses of amphetamine caused only a minor reduction in feeding in alloxan-diabetic animals (Fig. la). In a second experiment, we examined whether the resistance to amphetamine's anorexic action seen in alloxan-treated animals is attributable to the lack of insulin. The preceding experiment was repeated using 14 female rats, except that the 8 diabetic animals were given insulin replacement therapy starting 2 days after the alloxan injection. The diabetic rats received 1-2 I.U. of protamine zinc insulin (Lilly) s.c. during the light part of their cycle and 1 I.U. during the dark. This schedule of insulin reversed the excessive glycosuria, reestablished weight gain and prevented the polydipsia 3. Five days after the initiation of insulin injections, testing for amphetamine anorexia began as described in the previous experiment. Food intake was measured following the i.p. injection of isotonic saline or 0.5 mg/kg D-amphetamine sulfate, with 4 days separating the tests. Alloxan-treated rats receiving insulin showed an anorexia to amphetamine nearly as great as that seen in normal animals (Fig. lb). The suppression of eating produced by 0.5 mg/kg o-amphetami/ae in insulin-treated diabetics (58 ~o) is similar to that seen in control animals of this (78 ~o) and the previous ( 6 8 ~ ) experiment, but differs markedly from the minimal suppression (16 ~ ) seen in the diabetic animals of experiment 1. These findings suggest that the resistance to amphetamine's anorexic action is due to the absence of insulin and cannot be attributed to a non-pancreatic action of alloxan. Experiment 3 was performed to test whether the resistance to amphetamine's action observed in alloxan-diabetic rats extended to behaviors other than feeding. Here amphetamine's capacity to stimulate locomotor activity was studied in 16 female rats. Eight were made diabetic with alloxan, while 8 served as normal controls. Seven
430 days later, testing for locomotor activity was begun. The rats were placed in ;t ;viremesh cage (36 cm 41 cm 18 cm high) containing a single photobeam mounted 4.8 cm off the floor and remained there for a 30 min adaptation period before drug treatment. On the first tesl day, the rats were then given isotonic saline (2 ml/kg) and the number of photobeam interruptions was recorded during the subsequent 90 rain. On the following day the test was repeated, except that the rats received 1.0 mg/kg D-amphetamine sulfate following the adaptation period. Control rats showed the expected hyperactivity after receiving amphetamine (Table 1). Although the diabetic rats were also more active after amphetamine than after saline, amphetamine-induced hyperactivity in such animals was much tess than that seen in controls (F 7.77: P 0.01). Direct observation of several rats during the test confirmed that whereas normal rats given amphetamine were continually exploring the cage, the diabetic rats appeared much less activated. In no case were interfering behaviors, such as stereotyped sniffing or gnawing, observed in either group. The fourth experiment examined the possibility that the diminished responsiveness of diabetic animals to amphetamine was due to decreased accumulation of the drug in blood plasma or brain. Forty female rats were housed and tested as in experiment I. Eighteen rats received alloxan, while the remaining 22 rats served as controls. Two weeks later all rats were deprived of food for 24 h. D-Jail]Amphetamine sulfate (7.76 Ci/mmole, New England Nuclear) was added to solutionscontainingeither 0.5 or 1.0 mg D-amphetamine sulfate per ml of 0.9 NaCI to give a final specific activity of 20/zCi/mg for both solutions. Groups of 4-7 rats were given 1 ml/kg i.p. of one of the above solutions and were killed 15 or 60 min later (corresponding to the start and end of the test period for amphetamine's effects on feeding). The animals killed 60 rain after [3H]amphetamine were given access to food, as in experiment l. Aliquots of whole brain homogenates and of plasma (blood collected from the neck) were later assayed for [:~H]amphetamine according to the method of Glowinski et al.;. The results of this assay (Table 11) indicate that diabetic rats show minor decreases (10-30 ~i;) in the accumulation of [all]amphetamine in plasma and brain, as compared with controls. However, it appears that the slight decrease in accumulation of amTABLE I Effect o[alloxan diabetes on locomotor activity [b/lowing amphetamine
Mean _L S.E.M. photobeam interruptions during 90 min after isotonic saline or o-amphetamine sulfate (1.0 mg/kg). Subjects per group are given in parentheses, t values indicate difference~ compared with respective control groups. Group
Control (8) Diabetic (8)
Treatment Isotonic saline
Amphetamine
79 i 13 56 i 10"
607 -3_92 274 L 60**
* t ~: 2.39, P -: 0.05. ** t = 6.06, P ,:- 0.001.
¢'¢5
"¢1"
Accumulation o l D - 3H,'amphetamine in plasma aml brain o['normal and alloxan diabetic rats
TABLE II
Plasma
1 8 8 0 2 5- 1440 1 6 1 0 2 5- ll01
Brain
9434 5- 148 7468 5- 306
Plasma
65938 ± 1607 59557 5- 2535
Brain
6068 5- 497 4053 ± 147
Plasma
49312 5- 4071 40754 5- 2608
Brain
60 mitt
Brain
2412 ± 162 1732 5- 118
15 min
1.0 mg/kg
Plasma
22420 ± 2355 20334 5- 249
60 rain
3552 5- 402 2743 5- 38
15 rain
0.5 mg/kg
Mean 5- S.E.M. disint./min/ml (for plasma) and disint./min/g (for brain)at 15 and 60 rain after D-[3H]amphetamine sulfate. 4-7 animals were used per group. Rats given 0.5 mg/kg amphetamine received 10 uCi/kg D-['~H]amphetamine; those given 1.0 mg/kg received 20 /¢Ci/kg D-[aH]amphetamine. Group
Controls Diabetics
432 phetamine seen in diabetics cannot explain the attenuated behavioral effects of this drug. The diabetic rats receiving 1 mg/kg o f amphetamine eat far more than controls receiving half this dose even though higher levels o f [ZH]amphetamine in both plasma and brain are achieved after 1 mg/kg in diabetics than after 0.5 mg/kg in controls. Of course, these data do not eliminate the possibility that in diabetic rats less amphetaamine reaches the specific sites responsible for its behavioral effects. The present experiments demonstrate: (1) that rats made diabetic by the injection o f alloxan are resistant to two different behavioral effects o f amphetamine, (2) that this resistance appears dependent upon the absence of insulin and (3) that the altered responsiveness does not seem due to reduced drug levels in plasma or brain. Because o f the considerable literature supporting the involvement of central catecholamine(CA)-containing systems in both amphetamine's anorexic ~.~,7 and locomotor-stimulant actions '~,7, it is possible that the diabetic state interferes with amphetamine's action on these pathways. This might be achieved, for example, by a reduction in amphetamine-induced CA release or hyposensitivity of receptors to released CA. Regardless o f the mechanism, the present results clearly indicate a reduced responsiveness to amphetamine in alloxan-diabetic rats and suggest the possibility of an altered central nervous response to drugs in human diabetes metlitus.
1 Ahlskog, J. E. and Hoebel, B. G., Overeating and obesity from damage to noradrenergic system in the brain, Science, 182 (1973) 166-168. 2 Fibiger, H. C., Zis, A. P. and McGeer, E. G., Feeding and drinking deficits after 6-hydroxydopamine administration in the rat : similarities to the lateral hypothalamic syndrome, Brain Research, 55 (1973) 135-148. 3 Friedman, M. I., Effects of alloxan diabetes on hypothalamic hyperphagia and obesity, Amer. J. PhysioL, 222 (1972) 174-178. 4 Glowinski, J., Axelrod, J. and lversen, L. L., Regional studies of catecholamines in the rat brain. IV. Effects of drugs on the disposition and metabolism of HS-norepinephrine and HS-dopamine, J. Pkarmacol. exp. Ther., 153 (1966) 30-41. 5 lversen, S. D., 6-Hydroxydopamine: a chemical lesion technique for studying the role of amine neurotransmitters in behaviour. In F. O. Schmitt and F. G. Worden (Eds.), The Neurosciences Third Stud), Program, M.I.T. Press, Cambridge, Mass., 1974, pp. 705-711. 6 Kumaresan, P. and Turner, C. W., Effect of alloxan on feed consumption and replacement therapy with graded levels of insulin in rats, Proc. Soc. exp. Biol. (N. Y.), 122 (1966) 526-527. 7 Weissman, A., Koe, B. K. and Tenen, S., Antiamphetamine effects following inhibition of tyrosine hydroxylase, J. Pharmacol. exp. Ther., 151 (1966) 339-352.
Brain Rese,rch. 111 (1976) 42~ 432 ~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The NctherkN;d,.
Reduced anorexic and locomotor-stimulant action of D-amphetamine in alloxan-diabetic rats
JOHN F. MARSHALL, MARK 1. FRIEDMAN* and THOMAS G. HEFFNER Psychobiology Program, Departments of Psychology and Psychiatry, Department of Psychology ( M.I.F.) and Department of Biology (T.G.H.), University of Pittsburgh, Pittsburgh, Pa. 15260 (U.S.A.)
(Accepted April 13th, 1976)
During an investigation of the rat with diabetes mellitus induced by atloxan injections, we have observed that such animals are resistant to the anorexic action of amphetamine. Moreover, diminished responsiveness to amphetamine is not limited to feeding-related behaviors, since diabetic rats show little of the normal increase in motor activity following amphetamine injection. These findings suggest to us that alloxan diabetes might induce an alteration in central structures responsible for amphetamine's behavioral actions. The present communication documents these behavioral changes and suggests that they cannot be explained by a reduction in the accumulation of amphetamine in the plasma or brains of diabetic rats. Several possible mechanisms for this altered drug response are discussed. Experiment 1 compared the anorexic action of amphetamine in diabetic and control rats. Twenty female albino Sprague-Dawley rats (200-250 g; Zivic-Miller, Pittsburgh) were housed individually in wire-mesh cages with Purina lab chow and tap water available ad libitum, except as noted. Lights in the colony room were on between 0800 and 2000 h. Ten rats were made diabetic by an injection of alloxan monohydrate (200 mg/kg, dissolved in 0.9 ~ saline, s.c., Sigma) under light ether anesthesia. The remaining l 0 rats were etherized but not injected. During the first week after alloxan, the presence of diabetes was confirmed by glyeosuria (Tes-Tape, Lilly), marked polydipsia and failure to gain weight 3,8. Tests for amphetamine anorexia began 12-14 days after the alloxan injection, and continued thereafter at 4 day intervals. All rats were deprived o f food (but not of water) 24 h before each test. On each test day, the rats received an i.p. injection of 0.9 ~ NaCI or of D-amphetamine sulfate (Sigma ; 0.25, 0.50, or 1.00 mg/kg, as the salt) 15 min before food was introduced into the home cages. Food intake ( ! 0.1 g, corrected for spillage) was measured duringthe following 45 min. When injected with saline, both alloxan-treated and control rats ate substantial amounts of food (4.4-5.2 g) following 24 h deprivation. Amphetamine produced a * Present address: Psychology Department, University of Massachusetts, Amehrst, Mass., U.S.A.
429 b o Controls etics
[3 Controls • Insulin-Treated
Diabetics
2 ~3 2
o~o~5 o;o ,;o
Amphetomine (mg/kg)
Isotonic Amphetamine Saline (0.50mg/kg)
Fig. 1. The left frame (a) depicts the dose-response relationship for amphetamine anorexia in control and alloxan-diabetic rats. Points indicate mean intakes in 45 rain tests; brackets show one S.E.M. The right frame (b) depicts the restoration of anorexia to amphetamine (0.5 mg/kg) in alloxan-treated rats given protamine zinc insulin. Bars indicate means: brackets show one S.E.M.
profound dose-dependent decrease in the food intake of control rats. However, these doses of amphetamine caused only a minor reduction in feeding in alloxan-diabetic animals (Fig. la). In a second experiment, we examined whether the resistance to amphetamine's anorexic action seen in alloxan-treated animals is attributable to the lack of insulin. The preceding experiment was repeated using 14 female rats, except that the 8 diabetic animals were given insulin replacement therapy starting 2 days after the alloxan injection. The diabetic rats received 1-2 I.U. of protamine zinc insulin (Lilly) s.c. during the light part of their cycle and 1 I.U. during the dark. This schedule of insulin reversed the excessive glycosuria, reestablished weight gain and prevented the polydipsia 3. Five days after the initiation of insulin injections, testing for amphetamine anorexia began as described in the previous experiment. Food intake was measured following the i.p. injection of isotonic saline or 0.5 mg/kg D-amphetamine sulfate, with 4 days separating the tests. Alloxan-treated rats receiving insulin showed an anorexia to amphetamine nearly as great as that seen in normal animals (Fig. lb). The suppression of eating produced by 0.5 mg/kg o-amphetami/ae in insulin-treated diabetics (58 ~o) is similar to that seen in control animals of this (78 ~o) and the previous ( 6 8 ~ ) experiment, but differs markedly from the minimal suppression (16 ~ ) seen in the diabetic animals of experiment 1. These findings suggest that the resistance to amphetamine's anorexic action is due to the absence of insulin and cannot be attributed to a non-pancreatic action of alloxan. Experiment 3 was performed to test whether the resistance to amphetamine's action observed in alloxan-diabetic rats extended to behaviors other than feeding. Here amphetamine's capacity to stimulate locomotor activity was studied in 16 female rats. Eight were made diabetic with alloxan, while 8 served as normal controls. Seven
430 days later, testing for locomotor activity was begun. The rats were placed in ;t ;viremesh cage (36 cm 41 cm 18 cm high) containing a single photobeam mounted 4.8 cm off the floor and remained there for a 30 min adaptation period before drug treatment. On the first tesl day, the rats were then given isotonic saline (2 ml/kg) and the number of photobeam interruptions was recorded during the subsequent 90 rain. On the following day the test was repeated, except that the rats received 1.0 mg/kg D-amphetamine sulfate following the adaptation period. Control rats showed the expected hyperactivity after receiving amphetamine (Table 1). Although the diabetic rats were also more active after amphetamine than after saline, amphetamine-induced hyperactivity in such animals was much tess than that seen in controls (F 7.77: P 0.01). Direct observation of several rats during the test confirmed that whereas normal rats given amphetamine were continually exploring the cage, the diabetic rats appeared much less activated. In no case were interfering behaviors, such as stereotyped sniffing or gnawing, observed in either group. The fourth experiment examined the possibility that the diminished responsiveness of diabetic animals to amphetamine was due to decreased accumulation of the drug in blood plasma or brain. Forty female rats were housed and tested as in experiment I. Eighteen rats received alloxan, while the remaining 22 rats served as controls. Two weeks later all rats were deprived of food for 24 h. D-Jail]Amphetamine sulfate (7.76 Ci/mmole, New England Nuclear) was added to solutionscontainingeither 0.5 or 1.0 mg D-amphetamine sulfate per ml of 0.9 NaCI to give a final specific activity of 20/zCi/mg for both solutions. Groups of 4-7 rats were given 1 ml/kg i.p. of one of the above solutions and were killed 15 or 60 min later (corresponding to the start and end of the test period for amphetamine's effects on feeding). The animals killed 60 rain after [3H]amphetamine were given access to food, as in experiment l. Aliquots of whole brain homogenates and of plasma (blood collected from the neck) were later assayed for [:~H]amphetamine according to the method of Glowinski et al.;. The results of this assay (Table 11) indicate that diabetic rats show minor decreases (10-30 ~i;) in the accumulation of [all]amphetamine in plasma and brain, as compared with controls. However, it appears that the slight decrease in accumulation of amTABLE I Effect o[alloxan diabetes on locomotor activity [b/lowing amphetamine
Mean _L S.E.M. photobeam interruptions during 90 min after isotonic saline or o-amphetamine sulfate (1.0 mg/kg). Subjects per group are given in parentheses, t values indicate difference~ compared with respective control groups. Group
Control (8) Diabetic (8)
Treatment Isotonic saline
Amphetamine
79 i 13 56 i 10"
607 -3_92 274 L 60**
* t ~: 2.39, P -: 0.05. ** t = 6.06, P ,:- 0.001.
¢'¢5
"¢1"
Accumulation o l D - 3H,'amphetamine in plasma aml brain o['normal and alloxan diabetic rats
TABLE II
Plasma
1 8 8 0 2 5- 1440 1 6 1 0 2 5- ll01
Brain
9434 5- 148 7468 5- 306
Plasma
65938 ± 1607 59557 5- 2535
Brain
6068 5- 497 4053 ± 147
Plasma
49312 5- 4071 40754 5- 2608
Brain
60 mitt
Brain
2412 ± 162 1732 5- 118
15 min
1.0 mg/kg
Plasma
22420 ± 2355 20334 5- 249
60 rain
3552 5- 402 2743 5- 38
15 rain
0.5 mg/kg
Mean 5- S.E.M. disint./min/ml (for plasma) and disint./min/g (for brain)at 15 and 60 rain after D-[3H]amphetamine sulfate. 4-7 animals were used per group. Rats given 0.5 mg/kg amphetamine received 10 uCi/kg D-['~H]amphetamine; those given 1.0 mg/kg received 20 /¢Ci/kg D-[aH]amphetamine. Group
Controls Diabetics
432 phetamine seen in diabetics cannot explain the attenuated behavioral effects of this drug. The diabetic rats receiving 1 mg/kg o f amphetamine eat far more than controls receiving half this dose even though higher levels o f [ZH]amphetamine in both plasma and brain are achieved after 1 mg/kg in diabetics than after 0.5 mg/kg in controls. Of course, these data do not eliminate the possibility that in diabetic rats less amphetaamine reaches the specific sites responsible for its behavioral effects. The present experiments demonstrate: (1) that rats made diabetic by the injection o f alloxan are resistant to two different behavioral effects o f amphetamine, (2) that this resistance appears dependent upon the absence of insulin and (3) that the altered responsiveness does not seem due to reduced drug levels in plasma or brain. Because o f the considerable literature supporting the involvement of central catecholamine(CA)-containing systems in both amphetamine's anorexic ~.~,7 and locomotor-stimulant actions '~,7, it is possible that the diabetic state interferes with amphetamine's action on these pathways. This might be achieved, for example, by a reduction in amphetamine-induced CA release or hyposensitivity of receptors to released CA. Regardless o f the mechanism, the present results clearly indicate a reduced responsiveness to amphetamine in alloxan-diabetic rats and suggest the possibility of an altered central nervous response to drugs in human diabetes metlitus.
1 Ahlskog, J. E. and Hoebel, B. G., Overeating and obesity from damage to noradrenergic system in the brain, Science, 182 (1973) 166-168. 2 Fibiger, H. C., Zis, A. P. and McGeer, E. G., Feeding and drinking deficits after 6-hydroxydopamine administration in the rat : similarities to the lateral hypothalamic syndrome, Brain Research, 55 (1973) 135-148. 3 Friedman, M. I., Effects of alloxan diabetes on hypothalamic hyperphagia and obesity, Amer. J. PhysioL, 222 (1972) 174-178. 4 Glowinski, J., Axelrod, J. and lversen, L. L., Regional studies of catecholamines in the rat brain. IV. Effects of drugs on the disposition and metabolism of HS-norepinephrine and HS-dopamine, J. Pkarmacol. exp. Ther., 153 (1966) 30-41. 5 lversen, S. D., 6-Hydroxydopamine: a chemical lesion technique for studying the role of amine neurotransmitters in behaviour. In F. O. Schmitt and F. G. Worden (Eds.), The Neurosciences Third Stud), Program, M.I.T. Press, Cambridge, Mass., 1974, pp. 705-711. 6 Kumaresan, P. and Turner, C. W., Effect of alloxan on feed consumption and replacement therapy with graded levels of insulin in rats, Proc. Soc. exp. Biol. (N. Y.), 122 (1966) 526-527. 7 Weissman, A., Koe, B. K. and Tenen, S., Antiamphetamine effects following inhibition of tyrosine hydroxylase, J. Pharmacol. exp. Ther., 151 (1966) 339-352.