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Life Sciences. Vol. 62. No. 1. pp. PL 14. 1998 Copyright 0 1998 Elsevier Science Inc. printed in the USA. All rights r-ed C?24-3205,98 $19.00 + 00
PII soo24-3205(97)01041-2 PHARMACOLOGY LETTER.9 Accelemted Communication
MODULATION BY SERUM GLUCOSE LEVELS ON MORPHINE-INDUCED ANTINOCICEPTIVE EFFECT IN C57BL/KSJ-DBIDB MICE Junzo Kamei, Midori Sodeyama, Masahiro Ohsawa, Mari Kimura* and Shun-ichi Tanaka* Department of Pathophysiology & Therapeutics, Faculty of Pharmaceutical Sciences, Hoshi University, Tokyo 142, Japan. *Third Department of Internal Medicine, Yokohama City University, School of Medicine, Yokohama, 236, Japan. (SubmittedAugust 12, 1997; accepted September 15, 1997; received in final form October 9, 1997)
Abstract: The role of serum glucose levels on the sensitivity to the antinociceptive effect of morphine in streptozotocin-induced diabetic mice and C57BLKsJ dbldb mice were examined. The sensitivity to the antinociceptive effect of morphine was significantly reduced in streptozotocin-induced diabetic mice as compared with age-matched nondiabetic mice. Pretreatment with insulin (3 U/kg, s.c.) significantly reduced the serum glucose levels of streptozotocin-induced diabetic mice as compared with those of untreated diabetic mice. However, post-drug (morphine) tail-flick latency was not affected by pretreatment with insulin. The antinociceptive effect of morphine was also significantly reduced in C57BL/KsJ-db/db mice as compared with age-matched control mice. When CS-045 was administered to C57BLKsJ-dbldb mice, the serum glucose levels were significantly reduced. There was no significant difference in the antinociceptive effect of morphine between CS-O45-treated C57BL/KsJdb/db mice and C57BL/KsJdb/++ mice. Adoptive transfer of supematant of the spleen cell homogenate from C57BL/KsJ-dbldb mice to naive ICR mice had no significant effect on the recipients’ antinociceptive sensitivities to S.C. morphine. These findings support the our previous suggestion that some factor(s) derived from spleen mononuclear cells is the prime factor involving the insulin-insensitive mechanisms for the reduction of p-opioid agonistinduced antinociception during the severe stages of diabetes. 0 1998EIS~G~ Science hc. Key Words: morphine, CX’BL/KsJ-db/db mice., antinociception, serum glucose level, diabetic mice
Many researchers have demonstrated that animals with diabetes are significantly less sensitive to the antinociceptive effect of p-opioid agonists, such as morphine and DAMGO ([D-Ala’ N-MePhe4 Gly’-011 enkephalin) (e.g., 1, 2). The antinociceptive potency of DAMGO, based on the tail-flick test, was significantly decreased in diabetic mice, which had been rendered diabetic by an injection of streptozotocin 1 and 2 weeks previously, as compared to its effect in age-matched controls (3). Sensitivity to the antinociceptive potency of DAMGO returned to normal when insulin was administered to streptozotocin-induced l-week diabetic mice (3). However, pretreatment with insulin had no significant effect on the sensitivity to the antinociceptive potency of DAMGO in J. Kami, Ph.D., Department of Pathophysiology & Therapeutics, Correspondence: Faculty of Pharmaceutical Sciences, Hoshi University, 4-41, Ebara 2-chome, Shinagawaku, Tokyo 142, Japan. FAX: 81-3-5498-5029
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2-week diabetic mice (3). Based on these results, we proposed that the serum glucose levels may possibly be responsible for the altered potency of p-opioid agonists only during the incipient stages of diabetes in streptozotocin-induced diabetic mice, a model animal of insulin-dependent diabetic mellitus (type I diabetes) (3). On the other hand, we recently found that splenectomy in diabetic mice reversed the sensitivity to CL-opioid receptor-mediated pharmacological actions to the level seen in non-diabetic mice (4-6). Furthermore,we also demonstrated that the supernatant of spleen cell homogenate from diabetic mice to naive mice significantly reduced the u-opioid receptor mediated functions in these mice, without changing serum glucose levels (45,7). These results led us to propose the hypothesis that some factor(s) derived fromspleen cells may influence to the opioid receptor-mediated functions in diabetic mice (1,4,5,7). It has been also reported that the antinociceptive potency of morphine was decreased in spontaneous diabetic (C57BL/KsJ_db/db) mice, a model animal of noninsulin-dependent diabetic mellitus (type II diabetes) (2). However, the role of hyperglycemia in reduction of morphine antinociception in spontaneous diabetic mice has not been established. In the present study, therefore, to examine the role of serum glucose levels on the sensitivity to the antinociceptive effect of morphine in C57BL/KsJ diabetic mice, the effect of (k)-5-[4-(6-hydroxy-2, 5, 7, 8-tetramethylchroman-2yhnethoxy)benzyl]-2,4_thiazolldinedione ((X-045), a new oral antidiabetic agent (8), on the antinociceptive effect of morphine was examined. CS-045 is a new oral antidiabetic agent that was effective in insulin-resistant diabetic animal models, including C57BLKsJ diabetic mice. CS-045 was not effective in the streptozotocin treated mouse, an insulindeficient diabetic animal model (8). Furthermore, the role of factor(s) derived from spleen cells on the antinociceptive effect of morphine in C57BLKsJ diabetic mice was also examined. Materials and Methods Animals
Male C57BL/KsJ diabetic (dbidb) mice and C57BL/KsJ non-diabetic (db/++) mice were generously supplied by Dr. Fujiwara (Institute of Experimental Animals, Tokyo University, School of Medicine, Tokyo, Japan) and were maintained in Yokohama City University, School of Medicine. C57BL/KsJ-db/db mice were used for experiments at 20 24 weeks of age. Male ICR mice weighing about 20 g at the beginning of the experiments were purchased from Tokyo Animal Laboratory Inc., Tokyo, Japan. They had free access to food and water in an animal room which was maintained at 24 + 1 “C with a 12-hr light/dark cycle. The studies were carried out in accordance with the Declaration of Helsinki and/or with the guide for the care and use of laboratory animals as adopted by the committee on care and use of laboratory animals of Hoshi University which is accredited by the Ministry of Education, Science, Sports and Culture. Induction of diabetes bv streptozotocin
Male ICR mice were rendered diabetic i.v.) prepared in 0.1 N citrate buffer at pH with the vehicle alone. The experiments streptozotocin or vehicle. Serum glucose method (9). Preoaration
ofsupernatant
ofspleen
by an injection of streptozotocin (200 rug/kg, 4.5. Age-matched control mice were injected were conducted 2 weeks after injection of level was estimated utilizing the o-toluidine
cell homogenate
Supematant of spleen cell homogenate was prepared by a method previously mice were removed and described (7). In brief, spleens of C57BLKsJ-db/db homogenized in 2 vol. of ice-cold saline. Ihe homogenate was centrifuged at 100,000 x g for 1 hr at 4 “C. The supematant of the spleen cell homogenate from each C57BL/KsJ-
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db/db mouse (-350 ~1) was injected into each recipient ICR mouse via the tail vein. Control naive ICR mice received injections of supematant of the spleen cell homogenate from C57BL/KsJ-db/++ mice. Ihe experiments were conducted 2 weeks after adoptive transfer of supernatant of the spleen cell homogenate. Assay of the extent of antinociception
Antinociceptive response was evaluated by the tail-flick test. The intensity of a heat lamp was adjusted to provide a pre-drug latency time in the tail-flick response of 2-4 sec. A cut-off latency of 15 set was used to prevent injury to the tail. Animals not responding within 15 set were removed and assigned a score of 15 sec. ‘Ihe percent maximum possible effect (%MPE) was calculated for each animal according to the formula: %MPE = 100 x (post-drug latency -pre-drug latency) / (15 - pre-drug latency). Data are expressed as mean + S.E. The Newman-Keuls test was used for statistical analysis. The drugs used were morphine (Sankyo Co., Tokyo, Japan), porcine insulin (Biomedical Technologies Inc., Stoughton, MA, USA) and streptozotocin (Sigma Chemical Co., St. Louis, MO, USA). (=)-5-[4-(6-hydroxy-2,5,7,8-tetramethylchroman-2yhnethoxy)benzyl]-2,4_thiazolldinedione (CS-045) was generously supplied by Sankyo Co. Morphine was dissolved in saline. The assessment of antinociceptive effects was conducted 30, 60, 90 and 120 min after S.C. administration of morphine (2.5 mg/kg). Insulin (3 U/kg, s.c.) was administered to mice 30 min before morphine treatment. C57BL/KsJdb/db and C57BL/KsJdb/++ mice given powdered MM-l chow alone or MM-l chow with 0.2 % CS-045 added for 3 days. Results As shown in Table 1, serumglucose levels in mice which had been rendered diabetic by streptozotocin were significantly elevated as compared to those in age-matched nondiabetic mice. Furthermore, serum glucose levels in C57BL/KsJ-db/db mice were significantly elevated as compared to those in age-matched C57BWKsJ-db/++ mice. TABLE 1 Effect of insulin and CS-045 on the serum glucose levels in diabetic mice. Groups of animals StreDtozotocin_induced untreated non-diabetic mice insulin-treated non-diabetic mice untreated diabetic mice insulin-treated diabetic mice SDontaneous diabetic mice untreated C57BL/KsJ-db/++ mice CS-045-treated C57BL/KsJ-db/++ mice untreated C57BL/KsJ-dbldb mice CS-045-treated C57BL/KsJ-db/db mice
serum glucose levels (mg/dl) 214.1 + 6.9 79.6 + 20.0* 772.9 + 12.5 280.1 + 31.5* 166.3 132.7 480.0 270.7
f * f 2
5.5 8.3 10.9 27.5*
* donetes significant difference from respective untreated group, P
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Fig. 1. A) Effect of insulin on the antinociceptive effect of morphine in streptozotocin-induced diabetic mice, and B) effect of CS-045 on the antinociceptive effect of morphine in C57BL/KsJ-db/db mice. 0: vehicletreated non-diabetic mice, a: insulin-treated non-diabetic mice, n: vehicletreated diabetic mice, A: insulin-treated diabetic mice, 0: vehicle-treated C57BL/KsJ-db/++ mice, +: CS-045-treated C57BL/KsJ-dbltt mice, 3: vehicle-treated C57BL/KsJdb/db mice, w: CS-045-treated C57BL/KsJdb/db mice. The antinociceptive effects were measured 30,60,90 and 120 min after morphine (2.5 n-&kg, s.c.) injection. Each point represents the mean with S.E. for 9 to 10 mice in each group. *P
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mg/dl, n=4). Adoptive transfer of supematant of the spleen cell homogenate from C57BUKsJdbldb mice to naive ICR mice had no significant effect on the recipients’ antinociceptive sensitivities to S.C. morphine (2.5 mg/kg) when assessed 2 weeks after transfer of supematant of the spleen cell homogenate from C57BLKsJ-dbldb mice (tteatrrxmt with supematant of the spleen cell homogenate from C57BL/KsJdb/++ mice, 91.6 -c 6.4 %MPE, n=4; treatment with supematant of the spleen cell homogenate from C57BL/KsJ-db/db mice 97.1 f 3.2 %MPE, n=5). Discussion This study shows that when insulin was administered to diabetic mice to lower their serum glucose levels, their sensitivity to the antinociceptive effect of morphine was still significantly lower than in non-diabetic mice. This result is support our previous suggestion that hyperglycemia might not be responsible for the altered potency of popioid agonists at the severe stages of diabetes (3). On the contrary, this study also shows that when CS-045 was administered to C57BLKsJdbldb mice to normalize serum glucose levels, their sensitivity to the antinociceptive effect of morphine returned to control values. However, CS-045, by itself, had no effect on the antinociceptive effect in C57BL/KsJdb/++ mice. These results suggest that higher serum glucose levels are the prime factor affecting the altered antinociceptive effect of morphine in C57BL/KsJ-dbldb mice, a spontaneous diabetic mice. However, the mechanism of this differential modulation of the antinociceptive effect of morphine by serum glucose levels in streptozotocin-induced diabetic mice and C57BL/KsJdb/db mice remains to be clarified. We previously demonstrated that when insulin was administered to streptozotocin-induced diabetic mice which had been rendered diabetic l-week previously to lower their serum glucose levels, their sensitivity to the antinociceptive effect of DAMGO returned to control values (3). However, when insulin was administered to 2-week diabetic mice to normalize serum glucose levels, sensitivity to the analgesic potency of DAMGO was still significantly lower than in control mice (3). Based on these results, we proposed that that higher serum glucose levels are the prime factor affecting the altered effect of CL-opioid agonists during the incipient stages of diabetes (3). However, hyperglycemia might not be responsible for the altered effect of p-opioid agonists at the severe stages (namely, consequence of hyperglycemia and/or hypoinsluinemia) of diabetes (3). We previously reported that diabetic mice are hyperresponsive to &opioid receptor-mediated functions in consequence of the selective down-regulation of p-opioid receptor mediated functions (10,ll). In this regard, we recently found that the spontaneous locomotor activity in streptozotocin-induced diabetic mice was significantly increased 2 weeks after streptozotocin treatment (12,13). Since, the enhanced spontaneous locomotor activity in 2-weeks diabetic mice was significantly reduced by pretreatment with naltrindole, a selective &opioid receptor antagonist, we suggested that this enhanced activity in diabetic mice may be due to the compensatory up-regulation of &opioid receptormediated functions (13). Furthermore, when insulin was administered to 2-weeks diabetic mice to lower their serum glucose levels, their spontaneous locomotor activity was still significantly greater than that in insulin-treated age-matched non-diabetic mice (5). However, the enhanced spontaneous locomotor activity in diabetic mice was restored to the level seen in age-matched non-diabetic mice by insulin-replacement therapy (5). Ihe serum glucose levels in streptozotocin-induced diabetic mice was about 1.6-fold higher than that in C57BL/KsJ-db/db mice. In contrast to the hyperglycemic and hypoinsulinemic produced by streptozotocin-induced diabetic mice, spontaneous diabetic mice are hyperglycemic and hyperinsulinemic (14). It seems likely, therefore, further hyperglycemic and/or hypoinsulinemic may be responsible for the difference of the modulation by serum glucose levels to the antinociceptive effect of morphine
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between streptozotocin-induced diabetic mice and C57BL/KsJ-dbidb mice. On the hand, CS-045 decreased the plasma glucose and insulin levels in C57BL/KsJ-db/db (8). It seems likely, therefore, not only hyperglycemic but also hyperinsulinemic nx responsible for the reduction of the antinociceptive effect of p-opioid receptor agon C57BL/KsJ-db/db mice. However, there is no information available regarding the I of insulin on the antinociceptive effects of p.-opioid receptor agonists. Further studic necessary before this possibility can be established with greater certainty. We previously reported that some factor(s) derived from spleen mononuclear may play an important role in the reduction of p-opioid receptor-met antinociception in streptozotocin-induced diabetic mice (1,4,5,7). However, in the PI study, we observed that adoptive transfer of supematant of the spleen cell hornog from C57BL/KsJ-db/db mice to naive ICR mice had no significant effect on the reciI antinociceptive sensitivity to morphine. These findings support the our pre suggestion that some factor(s) derived from spleen mononuclear cells is the prime involving the insulin-insensitive mechanisms for the reduction of p-opioid ag induced antinociception during the severe stages of diabetes (35). Although the mechanism is unclear, it is possible that some factor(s) derived from spleen monon cells may be produced as the results of consequence further hyperglycemia ; hypoinsulinemia conditions. References 1. J. KAMEI, Y. OHHASHI, T. Aoki, N. KAWASHIMA and Y. KASUYA Brain Res 199-203 (1992). 2. G.S. SIMON and W.L. DEWEY, J. Pharmacol. Exp. Ther. 218 318-323 (1981). 3. J. KAMEI, N. KAWASHIMA and Y. KASUYA, Life Sci. 52 53-60 (1992). 4. J. KAMEI and A. SAITOH, Neurosci. Lett. 210 54-60 (1996). 5. J. KAMEI and A. SAITOH, Life Sci. 60 1699-1708 (1997). 6. J. KAMEI, N. KAWASHIMA and Y. KASUYA, Brain Res. 576 139-142 (1992). 7. J. KAMEI, Y. IWAMOTO, M. MISAWA, H. NAGASE and Y. KASUYA, Neuropharmacology 33 1553-1558 (1994). 8. T. FUJIWARA, M. WADA, K. FUKUDA, M. FUKAMI, S. YOSHIOKA, T. YOSH and H. HORIKOSHI, Metabolism 40 1213-1218 (1991). 9. K.M. DUBOWSKI, Clin. Chem. 8 215-235 (1962). 10. J. KAMEI, Y. IWAMOTO, M. MISAWA, H. NAGASE and Y. KASUYA, Life Sci PL121-PL126 (1994). 11. J. KAMEI, A. SAITOH, M. OHSAWA, T. SUZUKI, M. MISAWA, H. NAGASE at KASUYA, Eur. J. Pharmacol. 276 131-135 (1995). 12. J. KAMEI, M. OHSAWA, A. SAITOH, Y. IWAMOTO, T. SUZUKI, M. MISAWA NAGASE and Y. KASUYA, J. Pharmacol. Exp. Ther. 274 700-706 (1995). 13. J. KAMEI, A. SAITOH, Y. IWAMOTO, M. FUNADA, T. SUZUKI, M. MISAWA, NAGASE and Y. KASUYA, Neurosci. Lett. 178 69-72 (1994). 14. D.L. COLEMAN and K.P. HUMMEL, Diabetologia 3 238-248 (1967).
PII soo24-3205(97)01041-2 PHARMACOLOGY LETTER.9 Accelemted Communication
MODULATION BY SERUM GLUCOSE LEVELS ON MORPHINE-INDUCED ANTINOCICEPTIVE EFFECT IN C57BL/KSJ-DBIDB MICE Junzo Kamei, Midori Sodeyama, Masahiro Ohsawa, Mari Kimura* and Shun-ichi Tanaka* Department of Pathophysiology & Therapeutics, Faculty of Pharmaceutical Sciences, Hoshi University, Tokyo 142, Japan. *Third Department of Internal Medicine, Yokohama City University, School of Medicine, Yokohama, 236, Japan. (SubmittedAugust 12, 1997; accepted September 15, 1997; received in final form October 9, 1997)
Abstract: The role of serum glucose levels on the sensitivity to the antinociceptive effect of morphine in streptozotocin-induced diabetic mice and C57BLKsJ dbldb mice were examined. The sensitivity to the antinociceptive effect of morphine was significantly reduced in streptozotocin-induced diabetic mice as compared with age-matched nondiabetic mice. Pretreatment with insulin (3 U/kg, s.c.) significantly reduced the serum glucose levels of streptozotocin-induced diabetic mice as compared with those of untreated diabetic mice. However, post-drug (morphine) tail-flick latency was not affected by pretreatment with insulin. The antinociceptive effect of morphine was also significantly reduced in C57BL/KsJ-db/db mice as compared with age-matched control mice. When CS-045 was administered to C57BLKsJ-dbldb mice, the serum glucose levels were significantly reduced. There was no significant difference in the antinociceptive effect of morphine between CS-O45-treated C57BL/KsJdb/db mice and C57BL/KsJdb/++ mice. Adoptive transfer of supematant of the spleen cell homogenate from C57BL/KsJ-dbldb mice to naive ICR mice had no significant effect on the recipients’ antinociceptive sensitivities to S.C. morphine. These findings support the our previous suggestion that some factor(s) derived from spleen mononuclear cells is the prime factor involving the insulin-insensitive mechanisms for the reduction of p-opioid agonistinduced antinociception during the severe stages of diabetes. 0 1998EIS~G~ Science hc. Key Words: morphine, CX’BL/KsJ-db/db mice., antinociception, serum glucose level, diabetic mice
Many researchers have demonstrated that animals with diabetes are significantly less sensitive to the antinociceptive effect of p-opioid agonists, such as morphine and DAMGO ([D-Ala’ N-MePhe4 Gly’-011 enkephalin) (e.g., 1, 2). The antinociceptive potency of DAMGO, based on the tail-flick test, was significantly decreased in diabetic mice, which had been rendered diabetic by an injection of streptozotocin 1 and 2 weeks previously, as compared to its effect in age-matched controls (3). Sensitivity to the antinociceptive potency of DAMGO returned to normal when insulin was administered to streptozotocin-induced l-week diabetic mice (3). However, pretreatment with insulin had no significant effect on the sensitivity to the antinociceptive potency of DAMGO in J. Kami, Ph.D., Department of Pathophysiology & Therapeutics, Correspondence: Faculty of Pharmaceutical Sciences, Hoshi University, 4-41, Ebara 2-chome, Shinagawaku, Tokyo 142, Japan. FAX: 81-3-5498-5029
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2-week diabetic mice (3). Based on these results, we proposed that the serum glucose levels may possibly be responsible for the altered potency of p-opioid agonists only during the incipient stages of diabetes in streptozotocin-induced diabetic mice, a model animal of insulin-dependent diabetic mellitus (type I diabetes) (3). On the other hand, we recently found that splenectomy in diabetic mice reversed the sensitivity to CL-opioid receptor-mediated pharmacological actions to the level seen in non-diabetic mice (4-6). Furthermore,we also demonstrated that the supernatant of spleen cell homogenate from diabetic mice to naive mice significantly reduced the u-opioid receptor mediated functions in these mice, without changing serum glucose levels (45,7). These results led us to propose the hypothesis that some factor(s) derived fromspleen cells may influence to the opioid receptor-mediated functions in diabetic mice (1,4,5,7). It has been also reported that the antinociceptive potency of morphine was decreased in spontaneous diabetic (C57BL/KsJ_db/db) mice, a model animal of noninsulin-dependent diabetic mellitus (type II diabetes) (2). However, the role of hyperglycemia in reduction of morphine antinociception in spontaneous diabetic mice has not been established. In the present study, therefore, to examine the role of serum glucose levels on the sensitivity to the antinociceptive effect of morphine in C57BL/KsJ diabetic mice, the effect of (k)-5-[4-(6-hydroxy-2, 5, 7, 8-tetramethylchroman-2yhnethoxy)benzyl]-2,4_thiazolldinedione ((X-045), a new oral antidiabetic agent (8), on the antinociceptive effect of morphine was examined. CS-045 is a new oral antidiabetic agent that was effective in insulin-resistant diabetic animal models, including C57BLKsJ diabetic mice. CS-045 was not effective in the streptozotocin treated mouse, an insulindeficient diabetic animal model (8). Furthermore, the role of factor(s) derived from spleen cells on the antinociceptive effect of morphine in C57BLKsJ diabetic mice was also examined. Materials and Methods Animals
Male C57BL/KsJ diabetic (dbidb) mice and C57BL/KsJ non-diabetic (db/++) mice were generously supplied by Dr. Fujiwara (Institute of Experimental Animals, Tokyo University, School of Medicine, Tokyo, Japan) and were maintained in Yokohama City University, School of Medicine. C57BL/KsJ-db/db mice were used for experiments at 20 24 weeks of age. Male ICR mice weighing about 20 g at the beginning of the experiments were purchased from Tokyo Animal Laboratory Inc., Tokyo, Japan. They had free access to food and water in an animal room which was maintained at 24 + 1 “C with a 12-hr light/dark cycle. The studies were carried out in accordance with the Declaration of Helsinki and/or with the guide for the care and use of laboratory animals as adopted by the committee on care and use of laboratory animals of Hoshi University which is accredited by the Ministry of Education, Science, Sports and Culture. Induction of diabetes bv streptozotocin
Male ICR mice were rendered diabetic i.v.) prepared in 0.1 N citrate buffer at pH with the vehicle alone. The experiments streptozotocin or vehicle. Serum glucose method (9). Preoaration
ofsupernatant
ofspleen
by an injection of streptozotocin (200 rug/kg, 4.5. Age-matched control mice were injected were conducted 2 weeks after injection of level was estimated utilizing the o-toluidine
cell homogenate
Supematant of spleen cell homogenate was prepared by a method previously mice were removed and described (7). In brief, spleens of C57BLKsJ-db/db homogenized in 2 vol. of ice-cold saline. Ihe homogenate was centrifuged at 100,000 x g for 1 hr at 4 “C. The supematant of the spleen cell homogenate from each C57BL/KsJ-
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db/db mouse (-350 ~1) was injected into each recipient ICR mouse via the tail vein. Control naive ICR mice received injections of supematant of the spleen cell homogenate from C57BL/KsJ-db/++ mice. Ihe experiments were conducted 2 weeks after adoptive transfer of supernatant of the spleen cell homogenate. Assay of the extent of antinociception
Antinociceptive response was evaluated by the tail-flick test. The intensity of a heat lamp was adjusted to provide a pre-drug latency time in the tail-flick response of 2-4 sec. A cut-off latency of 15 set was used to prevent injury to the tail. Animals not responding within 15 set were removed and assigned a score of 15 sec. ‘Ihe percent maximum possible effect (%MPE) was calculated for each animal according to the formula: %MPE = 100 x (post-drug latency -pre-drug latency) / (15 - pre-drug latency). Data are expressed as mean + S.E. The Newman-Keuls test was used for statistical analysis. The drugs used were morphine (Sankyo Co., Tokyo, Japan), porcine insulin (Biomedical Technologies Inc., Stoughton, MA, USA) and streptozotocin (Sigma Chemical Co., St. Louis, MO, USA). (=)-5-[4-(6-hydroxy-2,5,7,8-tetramethylchroman-2yhnethoxy)benzyl]-2,4_thiazolldinedione (CS-045) was generously supplied by Sankyo Co. Morphine was dissolved in saline. The assessment of antinociceptive effects was conducted 30, 60, 90 and 120 min after S.C. administration of morphine (2.5 mg/kg). Insulin (3 U/kg, s.c.) was administered to mice 30 min before morphine treatment. C57BL/KsJdb/db and C57BL/KsJdb/++ mice given powdered MM-l chow alone or MM-l chow with 0.2 % CS-045 added for 3 days. Results As shown in Table 1, serumglucose levels in mice which had been rendered diabetic by streptozotocin were significantly elevated as compared to those in age-matched nondiabetic mice. Furthermore, serum glucose levels in C57BL/KsJ-db/db mice were significantly elevated as compared to those in age-matched C57BWKsJ-db/++ mice. TABLE 1 Effect of insulin and CS-045 on the serum glucose levels in diabetic mice. Groups of animals StreDtozotocin_induced untreated non-diabetic mice insulin-treated non-diabetic mice untreated diabetic mice insulin-treated diabetic mice SDontaneous diabetic mice untreated C57BL/KsJ-db/++ mice CS-045-treated C57BL/KsJ-db/++ mice untreated C57BL/KsJ-dbldb mice CS-045-treated C57BL/KsJ-db/db mice
serum glucose levels (mg/dl) 214.1 + 6.9 79.6 + 20.0* 772.9 + 12.5 280.1 + 31.5* 166.3 132.7 480.0 270.7
f * f 2
5.5 8.3 10.9 27.5*
* donetes significant difference from respective untreated group, P
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Fig. 1. A) Effect of insulin on the antinociceptive effect of morphine in streptozotocin-induced diabetic mice, and B) effect of CS-045 on the antinociceptive effect of morphine in C57BL/KsJ-db/db mice. 0: vehicletreated non-diabetic mice, a: insulin-treated non-diabetic mice, n: vehicletreated diabetic mice, A: insulin-treated diabetic mice, 0: vehicle-treated C57BL/KsJ-db/++ mice, +: CS-045-treated C57BL/KsJ-dbltt mice, 3: vehicle-treated C57BL/KsJdb/db mice, w: CS-045-treated C57BL/KsJdb/db mice. The antinociceptive effects were measured 30,60,90 and 120 min after morphine (2.5 n-&kg, s.c.) injection. Each point represents the mean with S.E. for 9 to 10 mice in each group. *P
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mg/dl, n=4). Adoptive transfer of supematant of the spleen cell homogenate from C57BUKsJdbldb mice to naive ICR mice had no significant effect on the recipients’ antinociceptive sensitivities to S.C. morphine (2.5 mg/kg) when assessed 2 weeks after transfer of supematant of the spleen cell homogenate from C57BLKsJ-dbldb mice (tteatrrxmt with supematant of the spleen cell homogenate from C57BL/KsJdb/++ mice, 91.6 -c 6.4 %MPE, n=4; treatment with supematant of the spleen cell homogenate from C57BL/KsJ-db/db mice 97.1 f 3.2 %MPE, n=5). Discussion This study shows that when insulin was administered to diabetic mice to lower their serum glucose levels, their sensitivity to the antinociceptive effect of morphine was still significantly lower than in non-diabetic mice. This result is support our previous suggestion that hyperglycemia might not be responsible for the altered potency of popioid agonists at the severe stages of diabetes (3). On the contrary, this study also shows that when CS-045 was administered to C57BLKsJdbldb mice to normalize serum glucose levels, their sensitivity to the antinociceptive effect of morphine returned to control values. However, CS-045, by itself, had no effect on the antinociceptive effect in C57BL/KsJdb/++ mice. These results suggest that higher serum glucose levels are the prime factor affecting the altered antinociceptive effect of morphine in C57BL/KsJ-dbldb mice, a spontaneous diabetic mice. However, the mechanism of this differential modulation of the antinociceptive effect of morphine by serum glucose levels in streptozotocin-induced diabetic mice and C57BL/KsJdb/db mice remains to be clarified. We previously demonstrated that when insulin was administered to streptozotocin-induced diabetic mice which had been rendered diabetic l-week previously to lower their serum glucose levels, their sensitivity to the antinociceptive effect of DAMGO returned to control values (3). However, when insulin was administered to 2-week diabetic mice to normalize serum glucose levels, sensitivity to the analgesic potency of DAMGO was still significantly lower than in control mice (3). Based on these results, we proposed that that higher serum glucose levels are the prime factor affecting the altered effect of CL-opioid agonists during the incipient stages of diabetes (3). However, hyperglycemia might not be responsible for the altered effect of p-opioid agonists at the severe stages (namely, consequence of hyperglycemia and/or hypoinsluinemia) of diabetes (3). We previously reported that diabetic mice are hyperresponsive to &opioid receptor-mediated functions in consequence of the selective down-regulation of p-opioid receptor mediated functions (10,ll). In this regard, we recently found that the spontaneous locomotor activity in streptozotocin-induced diabetic mice was significantly increased 2 weeks after streptozotocin treatment (12,13). Since, the enhanced spontaneous locomotor activity in 2-weeks diabetic mice was significantly reduced by pretreatment with naltrindole, a selective &opioid receptor antagonist, we suggested that this enhanced activity in diabetic mice may be due to the compensatory up-regulation of &opioid receptormediated functions (13). Furthermore, when insulin was administered to 2-weeks diabetic mice to lower their serum glucose levels, their spontaneous locomotor activity was still significantly greater than that in insulin-treated age-matched non-diabetic mice (5). However, the enhanced spontaneous locomotor activity in diabetic mice was restored to the level seen in age-matched non-diabetic mice by insulin-replacement therapy (5). Ihe serum glucose levels in streptozotocin-induced diabetic mice was about 1.6-fold higher than that in C57BL/KsJ-db/db mice. In contrast to the hyperglycemic and hypoinsulinemic produced by streptozotocin-induced diabetic mice, spontaneous diabetic mice are hyperglycemic and hyperinsulinemic (14). It seems likely, therefore, further hyperglycemic and/or hypoinsulinemic may be responsible for the difference of the modulation by serum glucose levels to the antinociceptive effect of morphine
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between streptozotocin-induced diabetic mice and C57BL/KsJ-dbidb mice. On the hand, CS-045 decreased the plasma glucose and insulin levels in C57BL/KsJ-db/db (8). It seems likely, therefore, not only hyperglycemic but also hyperinsulinemic nx responsible for the reduction of the antinociceptive effect of p-opioid receptor agon C57BL/KsJ-db/db mice. However, there is no information available regarding the I of insulin on the antinociceptive effects of p.-opioid receptor agonists. Further studic necessary before this possibility can be established with greater certainty. We previously reported that some factor(s) derived from spleen mononuclear may play an important role in the reduction of p-opioid receptor-met antinociception in streptozotocin-induced diabetic mice (1,4,5,7). However, in the PI study, we observed that adoptive transfer of supematant of the spleen cell hornog from C57BL/KsJ-db/db mice to naive ICR mice had no significant effect on the reciI antinociceptive sensitivity to morphine. These findings support the our pre suggestion that some factor(s) derived from spleen mononuclear cells is the prime involving the insulin-insensitive mechanisms for the reduction of p-opioid ag induced antinociception during the severe stages of diabetes (35). Although the mechanism is unclear, it is possible that some factor(s) derived from spleen monon cells may be produced as the results of consequence further hyperglycemia ; hypoinsulinemia conditions. References 1. J. KAMEI, Y. OHHASHI, T. Aoki, N. KAWASHIMA and Y. KASUYA Brain Res 199-203 (1992). 2. G.S. SIMON and W.L. DEWEY, J. Pharmacol. Exp. Ther. 218 318-323 (1981). 3. J. KAMEI, N. KAWASHIMA and Y. KASUYA, Life Sci. 52 53-60 (1992). 4. J. KAMEI and A. SAITOH, Neurosci. Lett. 210 54-60 (1996). 5. J. KAMEI and A. SAITOH, Life Sci. 60 1699-1708 (1997). 6. J. KAMEI, N. KAWASHIMA and Y. KASUYA, Brain Res. 576 139-142 (1992). 7. J. KAMEI, Y. IWAMOTO, M. MISAWA, H. NAGASE and Y. KASUYA, Neuropharmacology 33 1553-1558 (1994). 8. T. FUJIWARA, M. WADA, K. FUKUDA, M. FUKAMI, S. YOSHIOKA, T. YOSH and H. HORIKOSHI, Metabolism 40 1213-1218 (1991). 9. K.M. DUBOWSKI, Clin. Chem. 8 215-235 (1962). 10. J. KAMEI, Y. IWAMOTO, M. MISAWA, H. NAGASE and Y. KASUYA, Life Sci PL121-PL126 (1994). 11. J. KAMEI, A. SAITOH, M. OHSAWA, T. SUZUKI, M. MISAWA, H. NAGASE at KASUYA, Eur. J. Pharmacol. 276 131-135 (1995). 12. J. KAMEI, M. OHSAWA, A. SAITOH, Y. IWAMOTO, T. SUZUKI, M. MISAWA NAGASE and Y. KASUYA, J. Pharmacol. Exp. Ther. 274 700-706 (1995). 13. J. KAMEI, A. SAITOH, Y. IWAMOTO, M. FUNADA, T. SUZUKI, M. MISAWA, NAGASE and Y. KASUYA, Neurosci. Lett. 178 69-72 (1994). 14. D.L. COLEMAN and K.P. HUMMEL, Diabetologia 3 238-248 (1967).