MATERIALS AND METHODS

Animals: Study was done in the laboratories of Department of Pharmacology, Karadeniz Technical University, Faculty of Medicine. BALB/c mice (20-40 g) of either sex were used in the experiments. The animals were housed in cages with free access to food and water and were not starved before the experiments. The cages were placed in a quiet and temperature-humidity controlled

room (22±2°C and 60±5%, respectively) in which a 12:12 h light-dark cycle was maintained. Experiments were conducted between 9:00 and 17:00 h to minimize diurnal variation. The experimental protocol was approved by the Institutional Animal Ethics Committee of Karadeniz Technical University, Medical School (2004/10). Animals were allowed a 1-week acclimatization period before being used in experiments.

Drugs: Paroxetine hydrochloride (Novartis, Istanbul, Turkey), verapamil, serotonin, methysergide, ketanserin and glucose (Sigma) were dissolved in distilled water and diazoxide (Sigma) was dissolved in 0.1 M NaOH. All drugs were injected intraperitoneally in a dose volume of no more than 10 mL kg^-1 of body weight.

Experimental protocols: Blood glucose levels were measured before the drug application procedure in all groups. Blood was collected by puncture of the distal end of the mouse tail and blood drops were placed on the test zone of the strip for immediate measurement of glycemia using a glucometer device (MediSense-Optium, Abbott, UK). Animals with a pre-treatment blood glucose level between 60-160 mg dL^-1 (normoglycemic) were divided into 2 main groups. While in first group the effect of paroxetine on the blood glucose levels in 3 different doses (5, 10, 20 mg kg^-1) and the possible mechanism were investigated, in the second group, an intraperitoneal glucose tolerance test (IGTT) was applied and the effect of paroxetine (5, 10, 20 mg kg^-1) and serotonin (20 mg kg^-1) on this test was investigated.

The experimental groups and the time intervals of blood glucose level measurements were shown in Table 1. The doses of the drugs were decided with literature review.

Table 1:	Experimental groups: group 1 (normoglycemic mice), group 2 (intraperitoneal glucose tolerance test in normoglycemic mice) (In each subgroup number of animals were 8)

Intraperitoneal glucose tolerance test (IGTT): Normoglycemic mice (group 2) were fasted overnight (12 h). A bolus of glucose (1 g kg^-1 b.wt.) was administered by i.p. injection (Gletsu et al., 2005). Blood samples were taken before i.p. glucose injection and 5, 10, 15, 20, 30, 35, 45, 75 and 120 min after the injection as shown in Fig. 1. Thereafter, the mice were allowed free access to food and water.

Statistical analysis: The data were expressed as the Mean±SEM. Statistical analyses were carried out using the one way ANOVA test followed by the post hoc Bonferroni test when needed (Mohajeri et al., 2009; Chaudhari et al., 2010).

Fig. 1:	Intraperitoneal glucose tolerance test (IGTT)

The level of significance was set at p<0.05.

RESULTS

Paroxetine decreased blood glucose levels at almost all doses used and at all times tested in the first group of animals when compared with the pre-treatment blood glucose values and also when compared each corresponding value with saline group (Fig. 2a, p<0.006) and in the intraperitoneal glucose tolerance test (Fig. 2b, p<0.006).

While diazoxide (an ATP-dependent potassium channel opener, 25 mg kg^-1) increased blood glucose levels significantly at 60, 90 and 120. min when given alone (Fig. 3a), paroxetine at the 10 mg kg^-1 dose given after diazoxide, reversed this increase and caused a decrease in blood glucose levels (Fig. 3b).

The hypoglycemic effect of paroxetine at the 10 mg kg^-1 dose was not inhibited by application of the voltage-sensitive calcium channel antagonist verapamil (25 mg kg^-1) (Fig. 4).

Fig. 2:	The effect of paroxetine on blood glucose level in normoglycemic mice and in intraperitoneal glucose tolerance test (B). Results are expressed as Mean±SEM. *p<0.05, compared with the mean of pre-treatment blood glucose values, a: p<0.05, compared with saline group. S: Saline, PX: Paroxetine

Fig. 3:	The effect of diazoxide (a) on blood glucose level and the effect of paroxetine (PX) on the increase in blood glucose level induced by diazoxide when applied after diazoxide (b). Results are expressed as Means±SEM. *p<0.05, compared with the mean of pre- treatment blood glucose values

Similar to paroxetine, serotonin 20 mg kg^-1 also decreased blood glucose levels significantly in normoglycemic mice (Fig. 5a, p<0.005). In the intraperitoneal glucose tolerance test applied to animals in the second group, the increase in blood glucose levels due to glucose administration was completely prevented both by paroxetine at the 10 mg kg^-1 dose and serotonin 20 mg kg^-1 dose. Paroxetine and serotonin also decreased blood glucose levels to below pre-treatment blood glucose values when compared to both pre-treatment blood glucose levels and blood glucose levels in the saline group (Fig. 5b).

Both methysergide (a 5-HT_1/2B/2C receptor antagonist, 1 mg kg^-1) and ketanserin (a 5-HT_2A receptor antagonist, 1 mg kg^-1) inhibited the decreasing effect of paroxetine on blood glucose levels (Fig. 6a). Similar to paroxetine, the hypoglycemic effect of serotonin was also inhibited by these two antagonists (Fig. 6b).

Fig. 4:	The effect of paroxetine (PX) on blood glucose level in the presence of verapamil. Results are expressed as Means±SEM. *p<0.05, compared with the mean of pre- treatment blood glucose values

While inhibition of voltage-sensitive calcium channels via verapamil did not prevent hypoglycemia induced by paroxetine (Fig. 4), ketanserin used in combination with verapamil reversed this hypoglycemia (Fig. 7).

DISCUSSION

In our study, paroxetine caused a decrease in blood glucose levels in normoglycemic mice. There have been no previous investigations evaluating the direct effect of paroxetine on blood glucose in humans or animals. Some indirect results have been obtained from previous studies which suggested increased insulin sensitivity in humans (Weber-Hamann et al., 2006), hyperglycemia observed in a woman with panhypopituitarism (Petty, 1996) and increased glucose metabolism in the brain in depressed patients (Kennedy et al., 2001) and hypoglycemia in mice (Kesim et al., 2006).

The main mechanism involved in decreasing blood glucose levels is the secretion of insulin from pancreatic beta cells. Glucose is the main stimulant triggering this secretion (Davis, 2006; Yang and Gills, 2004). A method of testing the secretion of insulin induced by glucose in clinical practice is the glucose tolerance test. While paroxetine caused a decrease in blood glucose levels, it also altered the pattern of the glucose tolerance test by preventing the expected increase due to glucose. In additional, paroxetine decreased blood glucose levels to below pre-treatment values. The decrease in glucose level in spite of the overload of glucose may be related to a possible increase in insulin secretion caused by paroxetine.

Fig. 5:	The effect of serotonin (5-HT) on blood glucose level in normoglycemic mice and the effect of 5-HT and paroxetine (PX) in intraperitoneal glucose tolerance test (B). Results are expressed as Mean±SEM. a: p<0.05, compared with the mean of pre- treatment blood glucose values; b: p<0.05, compared with saline group; c: p<0.05, compared with 5-HT group. S: Saline, PX: Paroxetine, 5-HT: Serotonin

The coupling factor for the secretion of insulin from beta cells is known to be intracellular calcium. An increase in intracellular calcium is provided both by calcium influx via. voltage-sensitive calcium channels and release from intracellular stores (Davis, 2006; Yang and Gills, 2004; Wasada, 2002; Straub and Sharp, 2002). The mediators of this release from intracellular stores are inositol triphosphate (IP₃) and cAMP. The contribution of ATP which is the product of glucose metabolism has also been noted. Closure of ATP-dependent potassium channels causes a depolarization which triggers the opening of voltage-sensitive calcium channels.

While the ATP-dependent potassium channel opener, diazoxide, caused an increase in blood glucose level, possibly by preventing insulin secretion due to inhibition of membrane depolarization, paroxetine alone at the 10 mg kg^-1 dose given after diazoxide reversed this increase and caused a decrease in blood glucose to a level lower than pre-treatment measurements. Each drug showed its own but opposite effect on blood glucose. In the combination group of paroxetine with diazoxide, diazoxide did not prevent the hypoglycemic effect of paroxetine. If the hypoglycemic effect of paroxetine is related to the potassium channels, this can be explained by the reclosure of ATP-dependent potassium channels by paroxetine which were previously opened by diazoxide. In the literature, there are no studies showing the direct effect of paroxetine on ATP-dependent potassium channels in the pancreas. However, there are two studies reporting the inhibitory effect of paroxetine on TREK channels (a type of potassium channel expressed throughout the central nervous system) and G-protein activated potassium channels (Kobayashi et al., 2006; Thummler et al., 2007). Hence if the effect of paroxetine does not depend on any interaction with ATP-dependent potassium channels, then this effect may be due to another mechanism activated by paroxetine.

Fig. 6:	The effect of serotonin (5-HT) (A) and paroxetine (PX) (B) on blood glucose level in the presence of methysergide (M) and ketanserin (K). Results are expressed as Mean±SEM. *p<0.05, compared with the mean of pre-treatment blood glucose values, a: p<0.05, compared with 5-HT group; b: p<0.05, compared with paroxetine group

Fig. 7:	The effect of paroxetine (PX) on blood glucose level in the presence of verapamil plus ketanserin in normoglycemic mice. Results are expressed as Mean±SEM

The opening of voltage-sensitive calcium channels situated in the membrane also caused an increase in intracellular calcium level. The calcium channel blocker, verapamil, has been previously reported to cause hyperglycemia following a decrease in insulin secretion in vitro (Semple et al., 1983). Paroxetine was shown to be a calcium channel blocking agent in some smooth muscles (Yaris et al., 2003). The hypoglycemic effect of paroxetine in our study is thought to be attributed to its calcium channel blocking effect. However, verapamil, a specific blocker of these channels did not show any effect on blood glucose level in our study and paroxetine decreased blood glucose level in the presence of verapamil. We can conclude that paroxetine shows its hypoglycemic effect via. different mechanisms rather than calcium channels. Another finding supporting this suggestion was the ineffectiveness of verapamil on the hypoglycemia induced by paroxetine administered after verapamil.

Paroxetine, as an inhibitor of 5-HT re-uptake may be expected to strengthen serotonergic action in the central nervous system (Bourin et al., 2001). The results of other in vitro studies suggest that this action may be valid in some peripheral systems. Thus, the strengthening of serotonergic action may be observed in the periphery and possibly in the pancreas. The similar effects of 5-HT and paroxetine seen in our study, such as decreasing blood glucose levels, support this idea. In addition, the inhibitory actions of the 5-HT antagonists were similar both for 5-HT and paroxetine. It was previously shown that 5-HT is present in the nerves innervating the pancreas in many mammalian species (Adeghate et al., 1999). The identification of serotoninergic nerves in the periaciner regions of the pancreas indicates that serotonin may play a role in the regulation of acinar secretion and stimulates the endocrine secretagogue cells. It was also shown that serotonin elicited a concentration-dependent increase in insulin secretion and thereafter, a decrease in blood glucose levels (Adeghate et al., 1999; Sugimoto et al., 2003; Yamada et al., 1999; Sugimoto et al., 1990). In our study, correlated with literature, the decreasing effect of serotonin on blood glucose level was also thought to be related to the increase in insulin secretion stimulated by serotonin. 5-HT_1A and 5-HT_2A receptors are thought to be involved in glucose regulation (Sugimoto et al., 2005). The decreasing effect of 5-HT on blood glucose levels in our study is possibly related to this secretory action. 5-HT_2A receptors which are thought to involved in the secretory action of 5-HT, are coupled to the enzyme phospholipase C situated in the membrane. The activation of this enzyme is known to cause the production of IP₃ and release of calcium from intracellular stores. Although it was not proved that paroxetine caused a decrease in blood glucose levels which was directly related to an increase in insulin secretion, it can be speculated that the effect of paroxetine on blood glucose level might have been secondary to an increase in insulin secretion via serotonergic mechanisms and/or receptors. Further studies such as determining insulin level, or incubating pancreatic beta cells with paroxetine will help to clarify the decreasing effect of paroxetine on blood glucose level.

In conclusion, paroxetine causes a decrease in blood glucose level probably by an increase in insulin secretion and this increase is provided possibly by intracellular mechanisms mediated by serotonergic receptors and postreceptor events, rather than an influx of calcium from the extracellular medium.

ACKNOWLEDGMENT

This study was supported by Karadeniz Technical University Research Fund (Grant No. 2004.114.003.11). The authors wish to thank Founder of Karadeniz Technical University for their special contribution. Part of this study is presented as poster presentation in 4th Panhellenic Congress of Pharmacology, 19-20 May 2006, Greece and published in the issue of Review of Clinical Pharmacology and Pharmacokinetics devoted to this congress and also presented an oral presentation and poster presentation in 19th National Congress of Pharmacology, Turkish Pharmacological Society. 24-27 September 2007, Turkey. The manuscript is edited professionally by International Science Editing service for English language.