J Pharmacol Sci 93, 74 – 79 (2003) Journal of Pharmacological Sciences ©2003 The Japanese Pharmacological Society Full Paper The Tricyclic Increases Plasma Glucose Levels of Mice

Yumi Sugimoto*, Kiyo Inoue, and Jun Yamada Department of Pharmacology, Kobe Pharmaceutical University, Motoyamakita-machi, Higashinada-ku, Kobe 658-8558 Japan

Received May 12, 2003; Accepted July 3, 2003

Abstract. Effects of the clomipramine on plasma glucose levels in mice were studied. Clomipramine at doses ranging 5 – 20 mg/kg elicited significant hyperglycemia in mice. Hyperglycemia elicited by clomipramine was not reduced by pretreatment with the 5- hydroxytryptamine (5-HT) depleter p-chlorophenylalanine. The 5-HT1/2/5/7- antagonist and the 5-HT2A/2B/- LY 53857 enhanced clomipramine- induced hyperglycemia, while the 5-HT1A/1B-receptor antagonist ()- and the 5-HT3/4- receptor antagonist did not affect it. The 5-HT2B/2C-receptor antagonist SB 206553 facilitated hyperglycemia induced by clomipramine, although the 5-HT2A-receptor antagonist was without effect. Clomipramine-induced hyperglycemia was reduced by prior adrenalectomy. These results suggest that clomipramine induces hyperglycemia in mice by blocking the 5-HT2B and/or 5-HT2C receptors, which results in facilitation of release.

Keywords: clomipramine, 5-hydroxytryptamine (5-HT), hyperglycemia, 5-HT2B/2C receptor, antidepressant

Introduction regulation. The activation of the central 5-HT1A, 5-HT2A, and 5-HT2B/2C receptor elevates plasma glucose levels of A number of observations suggest that enhancement rats (9 – 12). It is well known that stress elevates blood of neurotransmitters such as noradrenaline and 5- glucose levels. Since depressive patients are considered hydroxytryptamine (5-HT) leads to improvement of sensitive to stress, glucose homeostasis may be affected depression (1). Clomipramine is a tricyclic antidepres- more easily than normal subjects. Therefore, it is impor- sant like and is widely used in the therapy tant to clarify the effects of on blood for depression (1, 2). It is recognized that clomipramine glucose regulation. We previously reported that anti- is also available for treatment of obsessive compulsive depressants, selective reuptake inhibitors disorder (OCD) (2 – 4). In mice, clomipramine reduces (SSRIs) including , , and zimeli- immobility in the forced swimming test, which is the dine, cause hyperglycemia in mice, although mecha- behavioral model for antidepressants (5, 6). It also nisms for hyperglycemia elicited by these SSRIs may inhibits the OCD animal model, marble burying not be identical (13, 14). Nevertheless, clomipramine is behavior in mice (7). Antidepressants inhibit reuptake widely used for the therapy of depression. It powerfully of noradrenaline and/or 5-HT at nerve terminals and inhibits 5-HT reuptake similar to SSRIs, but it is not elevate these amine levels at the synaptic cleft (1, 8). yet clear whether clomipramine affects plasma glucose Clomipramine can inhibit reuptake of both noradre- levels. In this study, therefore, we investigated the naline and 5-HT, although clomipramine inhibits 5-HT effects of clomipramine on plasma glucose levels of reuptake more strongly than it inhibits noradrenaline mice. reuptake (1, 8). It has been reported that 5-HT is involved in glucose

*Corresponding author. FAX: +81-78-441-7574 E-mail: [email protected]

74 Clomipramine-Induced Hyperglycemia 75

Materials and Methods 5mg/kg elicited significant hyperglycemia in mice. The hyperglycemia lasted for at least 60 min at 10 and Animals 20 mg/kg of clomipramine. Male ddY mice weighing 28 – 32 g were obtained from SLC Japan, Inc. (Hamamatsu). Mice were given free access to food and water and they were housed under a controlled 12-h/12-h light-dark cycle (light from 7:00 a.m. to 7:00 p.m.), with room temperature at 23 M 1LC and humidity at 55 M 5%. The experimental procedure was approved by the Kobe Pharmaceutical University Animal Care and Use Committee.

Drug treatment Clomipramine HCl, p-chlorophenylalanine methyl- ester HCl (PCPA), methysergide hydrogen maleate, LY 53857 maleate, SB 206553 HCl, ketanserin tartrate, tropisetron HCl, and ()-propranolol HCl were obtained from Sigma (St. Louis, MO, USA). Methysergide, LY 53857, ketanserin, and ()-propranolol were dis- solved in saline. Tropisetron was dissolved in a few drops of 0.1 N HCl and diluted with saline. All were injected i.p. at a volume of 0.1 ml/10 g. PCPA was injected i.p. 72, 48, and 24 h before injection of clomipramine. 5-HT-receptor antagonists were given 30 min before the injection of clomipramine. Fig. 1. Effects of clomipramine on the plasma glucose of mice. Determination of plasma glucose levels Results are shown as the mean M S.E.M. (N  5 – 8). Clomipramine Mice were decapitated and blood was collected in was given i.p. **P0.01. tubes containing NaF. Plasma glucose was measured following the method described in our previous study (12).

Adrenalectomy Adrenalectomy was performed under anesthesia with Na at 50 mg/kg. NaCl (0.5%) was given to adrenalectomized mice to maintain mineral balance. Experiments were carried out one week after the opera- tion. After the experiments, adrenalectomized mice were dissected to confirm that the adrenal gland was completely removed.

Statistics Dose-related effects on plasma glucose levels were evaluated by one-way analysis of variance (ANOVA) followed by Dunnett’s test. Other results were analyzed by two-way ANOVA followed by Tukey’s test.

Results Fig. 2. Effects of PCPA on clomipramine-induced hyperglycemia Effects of clomipramine on plasma glucose levels of in mice. Results are shown as the mean M S.E.M. (N  7 – 9). Clomi- mice pramine at 20 mg/kg was given i.p. PCPA at 400 mg/kg was injected i.p. 72, 48, and 24 h before injection of clomipramine. Effects of clomipramine on plasma glucose levels are Plasma glucose levels were determined 30 min after the injection shown in Fig. 1. Clomipramine above the dosage of of clomipramine. ***P0.001 vs saline of respective group. 76 Y Sugimoto et al

Effects of PCPA on hyperglycemia induced by clomi- nists on clomipramine (20 mg/kg)-induced hyper- pramine glycemia in mice. The 5-HT1A/1B-receptor antagonist Figure 2 shows the effects of the 5-HT depleter PCPA ()-propranolol and the 5-HT3/4-receptor antagonist on hyperglycemia induced by clomipramine. PCPA did tropisetron did not affect hyperglycemia elicited by not affect clomipramine-induced hyperglycemia. clomipramine. Hyperglycemia induced by clomipramine was enhanced by pretreatment with the 5-HT1/2/5/7- Effects of ()-propranolol, tropisetron methysergide, receptor antagonist methysergide and the 5-HT2A/2B/2C- and LY 53857 on clomipramine-induced hyperglycemia receptor antagonist LY 53857. Figure 3 shows the effects of 5-HT-receptor antago-

Fig. 3. Effects of methysergide, tropisetron, ()-propranolol, and LY 53857 on clomipramine-induced hyperglycemia in mice. Results are shown as the mean M S.E.M. (N  5 – 8). Clomipramine was given i.p. at 20 mg/kg. 5-HT-receptor antagonists were injected i.p. 30 min before clomipramine. Plasma glucose levels were determined 30 min after the injection of clomipramine. ***P0.001 vs respective saline-pretreated group. ##P0.01, ###P0.001 vs saline + clomipramine-treated group. Clomipramine-Induced Hyperglycemia 77

Fig. 4. Effects of ketanserin and SB 206553 on clomipramine-induced hyper- glycemia in mice. Results are shown as the mean M S.E.M. (N  5 – 8). Clomipramine was given i.p. at 20 mg/kg. Ketanserin and SB 206553 were injected i.p. 30 min before clomipramine. Plasma glucose levels were determined 30 min after the injection of clomipramine. ***P0.001 vs respective saline-pretreated group. ###P0.001 vs saline + clomipramine-treated group.

clomipramine-induced hyperglycemia, although the 5- HT2A-receptor antagonist ketanserin was without effect.

Effects of clomipramine on blood glucose levels in adrenalectomized mice Figure 5 demonstrates blood glucose levels in adrena- lectomized mice. As shown in the results, clomipramine- induced hyperglycemia was strongly inhibited by prior adrenalectomy.

Discussion

Many antidepressants improve depression by modify- ing neurotransmission of biogenic amines such as nor- adrenaline, 5-HT, or (1, 8). Although 5-HT participates in glucose regulation (9 – 11), effects of antidepressants on glucose homeostasis are not fully established. The present results indicate that the tricyclic Fig. 5. Effects of clomipramine in adrenalectomized mice. Results antidepressant clomipramine above the dosage of 5 mg are shown as the mean M S.E.M. (N  6 – 7). Clomipramine at 20 mg /kg significantly induced hyperglycemia in mice. Pre- /kg was given i.p. Plasma glucose levels were determined 30 min after the injection of clomipramine. ***P0.001 vs saline of vious studies have shown that clomipramine reduces respective group. ###P0.001 vs clomipramine-treated mice of the immobility in the forced swimming test or inhibits sham group. marble burying behavior (5 – 7). The doses at which clomipramine inhibited behaviors in these models are higher than those showing hyperglycemia in this study. Effects of ketanserin and SB 206553 on clomipramine- It was reported that another tricyclic antidepressant induced hyperglycemia imipramine induces hyperglycemia in fasted rabbits and The effects of ketanserin and SB 206553 on clomi- it antagonizes insulin-induced hypoglycemia (15). We pramine-induced hyperglycemia are shown in Fig. 4. previously reported that SSRIs fluoxetine or fluvox- The 5-HT2B/2C-receptor antagonist SB 206553 amplified amine also elevated plasma glucose levels in non-fasted 78 Y Sugimoto et al mice (14). Our present results with clomipramine further hyperglycemia induced by clomipramine is closely asso- shows that the antidepressants may affect glucose ciated with direct effects of clomipramine on the 5-HT2 homeostasis. receptor. In previous studies, it was recognized that clomi- The 5-HT2 receptor is subdivided into the 5-HT2A, pramine strongly blocks reuptake of 5-HT. The 5-HT 5-HT2B, and 5-HT2C receptors (17). As shown in the uptake IC50 value of clomipramine was 1.5 nM, which results, the 5-HT2A-receptor antagonist ketanserin did was lower than that of the SSRI, fluvoxamine (3.8 nM) not affect clomipramine-induced hyperglycemia. The (8). This suggests that clomipramine-induced hyper- 5-HT2B/2C-receptor antagonist SB 206553 apparently glycemia may be related to inhibition of 5-HT reuptake. enhanced hyperglycemia induced by clomipramine. Thus, we examined the effects of the 5-HT depleter These results suggest that the selective blockade of PCPA on hyperglycemia elicited by clomipramine. The the 5-HT2B/2C receptor increases clomipramine-induced dose of PCPA used in this study reduced brain 5-HT hyperglycemia in mice. It is not known whether clomi- levels by 74% and inhibited fluvoxamine-induced pramine has affinity with the 5-HT2B receptor. However, hyeprglycemia in mice (14). However, PCPA did not a previous report demonstrated that tricyclic anti- affect clomipramine-induced hyperglycemia. Therefore, depressants including imipramine or clomipramine have clomipramine-induced hyperglycemia is not related to an appropriate affinity for the 5-HT2C-receptor subtype its inhibition of 5-HT reuptake. We previously demon- (16). Clomipramie may act as a 5-HT2C-receptor antago- strated that SSRIs, fluoxetine, and fluvoxamine could nist, since it inhibited 5-HT-stimulated phosphoinositide induce hyperglycemia in mice, although these hyper- hydrolysis in the choroid plexus, which is an index of a glycemic effects were not equal (14). The 5-HT depleter 5-HT2C-receptor-mediated response (16). Therefore, it is PCPA reduces hyperglycemia induced by fluvoxamine, suggested that clomipramine may induce hyperglycemia while hyperglycemic effects of fluoxetine are resistant in mice as a result of blockade of the 5-HT2C receptor. to 5-HT depletion (14). These findings suggest that It is well known that adrenaline released from the clomipramine-induced hyperglycemia may be similar to adrenal gland has an important role in regulating blood that induced by fluoxetine. glucose levels and that it elevates plasma glucose levels Clomipramine has an affinity for 5-HT2 receptors and by inhibition of insulin release, glycolysis, or inhibition the direct interaction with this receptor may be related to of glucose uptake in tissues. Therefore, we studied pharmacological effects of clomipramine (16). Thus, to effects of adrenalectomy on clomipramine-hyper- clarify the involvement of 5-HT-receptor subtypes, we glycemia. As shown in the results, adrenalectomy examined effects of several 5-HT-receptor antagonists significantly inhibited clomipramine-induced hyper- including antagonists of 5-HT2 receptors on clomi- glycemia. Therefore, adrenaline release from the adrenal pramine-induced hyperglycemia. Clomipramine-elicited gland may be involved in hyperglycemic effects of hyperglycemia was enhanced by the 5-HT1/2/5/7-receptor clomipramine. As discussed above, the inhibition of antagonist methysergide and the non-selective 5-HT2- the 5-HT2C receptor may be related to clomipramine- receptor antagonist LY 53857. However, the 5-HT1A/1B- induced hyperglycemia. Recent reports demonstrated receptor antagonist ()-propranolol and the 5-HT3/4- that the 5-HT2C receptor tonically inhibits noradrenaline receptor antagonist tropisetron did not affect hyper- release from the noradrenergic neurons in the brain and glycemia induced by clomipramine. These results that the blockade of the 5-HT2C receptor facilitates suggest that clomipramine-induced hyperglycemia is noradrenaline release determined by microdialysis (18). related to the 5-HT2 receptor but not to the 5-HT1A/1B These results indicate that the inhibition of the central and 5-HT3/4 receptors. The present results have shown 5-HT2C receptor by clomipramine may activate the that blockade of the 5-HT2 receptor results in facilitation sympatho-adrenal system, leading to the facilitation of of hyperglycemia induced by clomipramine. The in- adrenaline release. It is not yet clear whether clomi- volvement of 5-HT2 receptors in the pharmacological pramine directly stimulates adrenal medulla and adrena- effects of clomipramine has been reported. We pre- line release and further studies are required. viously demonstrated that the 5-HT2-receptor antagonist In summary, the present results suggest that clomi- LY 53857, at the same dose as used in this study, pramine induced an apparent hyperglycemia in mice. enhanced the anti-immobility effects of clomipramine Clomipramine-induced hyperglycemia is not associated (6). It indicates that the 5-HT2 receptor is involved in with its inhibition of 5-HT reuptake. It is also indicated the pharmacological effects of clomipramine. Thus, that clomipramine-induced hyperglycemia is related to the enhancing effects of LY 53857 on clomipramine- its inhibition of the 5-HT2B/2C receptor, probably the 5- induced hyperglycemia are consistent with results HT2C receptor, which is mediated by adrenaline release. obtained with the forced swimming test. Therefore, Clomipramine-Induced Hyperglycemia 79

Acknowledgment 9 Chaouloff F, Jeanrenaud B. 5-HT1A and alpha-2 receptors mediate the hyperglycemic and hypoinsulinemic This work was supported in part by a Grant-in-Aid for effects of 8-hydroxy-2-(di-n-propylamino)tetralin in the con- scious rat. J Pharmacol Exp Ther. 1987;243:1159–1166. Scientific Research (C) from the Ministry of Education, 10 Chaouloff F, Gunn SH, Young JB. Central 5-hydroxy- Culture, Sports, Science and Technology of Japan. tryptamine2 receptors are involved in the adrenal catecholamine- releasing and hyperglycemic effects of the 5-hydroxytryp- References tamine indirect d- in the conscious rat. J Pharm Exp Ther. 1992;260:1008–1016. 1 Briley M, Moret C. Neurobiological mechanisms involved in 11 Sugimoto Y, Yamada J, Yoshikawa T, Horisaka K. Effects of antidepressant therapies. J Clin Neuropharmacol. 1993;16:24– the 5-HT2C/2B receptor agonist 1-(3-chlorophenyl) on 35. the plasma glucose levels of rats. Eur J Pharmacol. 1996; 2 McTavish D, Benfield P. Clomipramine. An overview of its 307:75–80. pharmacological properties and a review of its therapeutic use in 12 Sugimoto Y, Yamada J, Kimura I, Watanabe Y, Horisaka K. obsessive compulsive disorder and panic disorder. Drugs. The effects of the serotonin1A receptor agonist on 1990;39;136–153. the blood glucose and pancreatic hormones in rats. Jpn J 3 Lopez-Ibor JJ Jr, Saiz J, Cottraux J, et al. Double-blind Pharmacol. 1992;60:145–148. comparison of fluoxetine versus clomipramine in the treatment 13 Sugimoto Y, Inoue K, Yamada J. Involvement of serotonin in of obsessive compulsive disorder. Eur Neuropsychopharmacol. -induced hyperglycemia in mice. Biol Pharm Bull. 1996;6:111–118. 1999;22:1240–1241. 4 Rouillon F. A double-blind comparison of fluvoxamine and 14 Yamada J, Sugimoto Y, Inoue K. Selective serotonin reuptake clomipramine in OCD. Eur Neuropsychopharmacol. 1998;8: inhibitors fluoxetine and fluvoxamine induce hyperglycemia by S260–S261. different mechanisms. Eur J Pharmacol. 1999;382:211–215. 5 Porsolt RD, Bertin A, Jalfre M. Behavioural despair in mice: 15 Gupta B, Awasthi A, Jaju BP. Effect of acute & chronic a primary screening test for antidepressants. Arch Int Pharma- imipramine treatment on glucose homeostasis. Indian J Med codyn. 1977;229:327–336. Res. 1992;96:65–71. 6 Yamada J, Sugimoto Y. Differential effects of the 5-HT2 recep- 16 Pälvimaki E P, Roth BL, Majasuo H, et al. Interactions of tor antagonist on the anti-immobility effects noradrenaline and selective serotonin reuptake inhibitors with the serotonin 5-HT2C serotonin reuptake inhibitors in the forced swimming test. receptor. Psychopharmacology (Berl). 1996;126:234–240. Brain Res. 2002;958:161–168. 17 Baxter G, Kennett G A, Blaney F, Blackburn T. 5-HT2 receptor 7 Njung’e K, Handley SL. Effects of 5-HT uptake inhibitors, subtypes: a family re-united? Trends Pharmacol Sci. 1995;16: and antagonists on the burying of harmless objects by 105–110. mice:a putative test for anxiolytic agents. Br J Pharmacol. 18 Millan M J, Dekeyne A, Gobert A. Serotonin (5-HT)2C receptors 1991;104:105–112. tonically inhibit dopamine (DA) and noradrenaline (NA), but 8 Hyttel J. Pharmacological characterization of the selective not 5-HT, release in the frontal cortex, in vivo. Neuro- serotonin reuptake inhibitors(SSRI’s). Int J Pharmacol. 1994;9: pharmacology. 1998;37:953–955. 19–26.