Leptin is an adipokine whose blood level depends on fat stores in the body. This hormone is transported from the blood to the brain through a specific transport system in the blood-brain barrier (1). Leptin receptor was detected in brain structures involved in food intake control (2, 3). Therefore, leptin may readily affect the function of neuronal circuits involved in food intake control and modify their response to other neurotransmitters (4). It seems that the interaction between leptin, a factor involved in the long-term appetite control, and other peptidergic systems involved in the short-term appetite regulation is crucial for the normal feeding behaviour. Leptin was found to enhance the suppressory effect of some brain-gut peptides, such as cholecystokinin (5), GLP-1, and its agonist, exendin-4, on food consumption in the rat (6, 7). Little is known, however, about the mechanism of these interactions. Possibly, both hormones induce the same intracellular signal transduction pathways. A similar effect has been described recently for cholecystokinin and epidermal growth factor (8).

However, the active form of leptin receptor (Ob-Rb) was found both in gut endocrine cells and neurons producing GLP-1. This suggests that leptin may affect the secretion of GLP-1 that, in turn, might mediate the effects of leptin on feeding centres. In the central nervous system, leptin receptor was identified in GLP-1-containing neurons in the nucleus of the solitary tract (NTS) in animals (9, 10) and leptin was found to activate GLP-1-containing neurons in this nucleus (11). On the other hand, food deprivation that is associated with the decreased plasma leptin concentration resulted in the reduction of GLP-1 content in the hypothalamus (12), an event that may be attributed to altered proglucagon gene expression in the NTS (13) and that may be reversed by leptin injection (12). NTS neurons project to the paraventricular and dorsomedial nucleus in the hypothalamus (14). Hence, GLP-1 released from leptin-stimulated neurons in the NTS might modify the activity of hypothalamic neurons involved in the appetite control. Indeed, GLP-1 injected into the dorsomedial (15) or paraventricular (16) nuclei inhibited food intake. Taken together, due to possible leptin - GLP-1 interaction in the brain, the current food intake controlled by GLP-1 would depend on energetic stores of the body reflected by changes in leptin levels.

Interaction between leptin and GLP-1 may be also initiated outside the central nervous system. GLP-1 is synthesized and released from endocrine L cells localized in the distal intestine soon after food intake. Similarly, leptin is produced in the stomach and released after food consumption (17). Leptin was found to stimulate GLP-1 release from L cells (18). A very short half-life of GLP-1 in plasma strongly suggests that this peptide is a paracrine signal that produces its effects locally, in the gut (19). The GLP-1 receptor was found in vagal afferent terminals in the gut thus suggesting that this peptide may modify the activity of these terminals (20). Consistently, GLP-1 was shown to stimulate the electrical activity of afferent neurons that innervate the digestive tract (21). These neurons convey information from the gut to the NTS and then to higher feeding centres in the hypothalamus. Hence, it seems that GLP-1 released peripherally might mediate the effect of leptin and augment the leptin-dependent satiety signals.

Therefore, using exendin (9 - 39), a GLP-1 antagonist, to block GLP-1 receptor, a possible role of this peptide for leptin-dependent effects on food consumption was examined in the rat.


MATERIALS AND METHODS
Animals

The experiments were carried out on male Wistar rats with the initial body weight 280 - 350 g. The animals were maintained in individual cages throughout the experiment. They were kept in a 12:12 h light-dark cycle with lights on at 06.00, at 22°C. Rat standard chow and tap water were given ad libitum throughout the study. All the rats had been handled daily several days before the beginning of the experiment to minimize stress that was shown to alter significantly the feeding response to peptides (22). The experiments were approved by the Local Ethical Committee at the Medical University of Lodz.

Drugs

Exendin (9 - 39) and leptin (116 - 130) amide were purchased from BACHEM, Poznan, Poland. Just before the experiment the appropriate solutions of exendin (9 - 39), leptin, or both peptides dissolved in saline were prepared. All drugs were injected 0.5 - 1 hr before lights off according to the schedule described below.

Surgical procedure and cannula placement verification

Randomly selected rats were anaesthetised with pentobarbital (30 mg/kg bw.) and a stainless steel cannula was implanted into the lateral brain ventricle as described previously (23). After the surgery, the rats were housed individually and handled every day to adapt them to the experimental procedure. During a 5-day recovery all rats regained their pre-surgical body weight and were used in the first series of experiments (see below). At the end of this experiment 5 µl of 5 % Evans blue solution was injected through the cannula to each animal under pentobarbital anaesthesia. Rats were decapitated 10 min later, the brain was isolated, frozen and cut into slices to asses whether the dye was evenly dispersed within brain ventricles.

Experimental design

In the first series of experiments, the effect of central GLP-1 receptor blockade on the leptin-induced effect on food intake was investigated. During the experiment, each rat received two subsequent intracerebroventricular injections of drugs or 0.9% saline. The second injection was made10 min after the first one. Hence, the animals were given either exendin (9 - 39) (10 µg/5 µl) + 5 µl saline, leptin (5 µg/5 µl) + 5 µl saline, the same dose of exendin (9 - 39) + leptin or saline + saline.

In the second series, the effect of peripheral GLP-1 receptor blockade on the leptin-induced effect on food intake was investigated. To this aim, rats were injected twice intraperitoneally with the same combinations of drugs: exendin (9 - 39) (50 µg/ 0,2 ml per rat), leptin (100 µg/ 0,2 ml per rat) and saline.

In both series 24-hour food and water intake as well as body weight were measured every day 2 days before and 2 days after the injection. Every day each rat was provided with the pre-weighed amount of food (50g) and a calibrated bottle containing water (250 ml). A small diameter (3-4 mm) of the attachment fastened to the bottle did not allow the fluid to flow out when the rat stopped drinking. The next day the food remaining in a cage was carefully collected and weighed again. The difference between the initial and final weight of food indicated the amount consumed within 24 h. Similarly, we measured the volume of consumed water.

Statistical analysis

The results are expressed as means ± SEM of 6 - 11 animals. The effects of drug treatment on food and water intake as well as body weight over the time course of the experiment were estimated using two-way ANOVA (drug treatment x time) for repeated measures on one factor (time) followed by the post-hoc least significant difference (LSD) test (Statistica, StatSoft, Krakow, Poland) with p < 0.05 considered to be significant.


RESULTS
Basal values have been expressed as a mean of measures performed within 2 subsequent 24-hour periods at the beginning of the experiment. There were no significant differences between all groups in both series as to basal food and water intake as well as body weight.

In the first series, when drugs were injected intracerebroventricularly, ANOVA revealed a significant effect of drug (P < 0.02), time (P < 0.001) and drug x time interaction (P < 0.001) on food consumption. Leptin reduced significantly (P < 0.001) food intake in rats injected previously with exendin (9 - 39) (Fig. 1A). However, the suppressory effect of exendin (9 - 39) + leptin was less pronounced than that seen in animals treated with leptin only (P < 0.05).

ANOVA showed a significant effect of time (P < 0.001) and time and drug interaction (P < 0.001) on body weight. Leptin injected to rats previously treated with exendin (9 - 39) significantly (P < 0.01) reduced body weight 24 h after the injection (Fig. 1B) and this effect did not differ significantly from that produced by leptin alone.

According to ANOVA, there was a significant effect of drug (P < 0.05), time (P < 0.001) and drug x time (P < 0.02) on water intake. Leptin (P < 0.001) and exendin (9 - 39) (P < 0.05) injected separately diminished markedly water consumption 24 h after the injection as compared to the respective initial values in each group (Fig. 1C). There were no marked differences in water intake between rats treated with leptin, exendin (9 - 39) or both 24 h after the injection.

Fig. 1. Food intake (panel A), body weight (panel B), and water intake (panel C) in exendin (9 - 39)- (Ex 9 - 39) and leptin- (Lep) treated rats 24 and 48 h after intracerebroventricular injection of each drug (mean ± SEM). All comparisons were made with respect to the basal value in each group.* P < 0.05; ** P < 0.01; *** P < 0.001.

In the second series, where both drugs were injected intraperitoneally, ANOVA indicated a significant effect of time (P < 0.02) and drug x time interaction (P < 0.01) on food consumption. Previous injection of exendin (9 - 39) completely abolished the suppressory effect of leptin on food intake (Fig. 2A).

ANOVA revealed a significant effect of time only on body weight (P < 0.001). Except for rats treated with leptin alone, the animals of all remaining groups significantly gained body weight 24 h after the injection (Fig. 2B).

ANOVA showed a significant effect of time only (P < 0.05) on water intake. None of the drugs administered in either combination affected significantly water intake after the injection (Fig. 2C).

Fig. 2. Food intake (panel A), body weight (panel B), and water intake (panel C) in exendin (9-39)- (Ex 9-39) and leptin- (Lep) treated rats 24 and 48 h after intraperitoneal injection of each drug (mean ± SEM). All comparisons were made with respect to the basal value in each group.* P < 0.05; ** P < 0.01; *** P < 0.001.

DISCUSSION
Although the role for leptin in the regulation of appetite has been established, the mechanisms by which leptin controls feeding behaviour is not clear. In recent years a growing body of evidence has accumulated that leptin somehow interacts with other hormones involved in food intake control. Some of them appear to mediate leptin effects on feeding centres. For instance, the pharmacological blockade of corticotropin-releasing hormone (24), interleukin-1 (25), neurotensin (26), or melanocortin (27) receptor in the brain was proved to attenuate the response of feeding centres to leptin. Since GLP-1 is produced both in the brain and in the digestive system and the distribution of leptin receptor indicates that this hormone may interact with GLP-1 at both sites, we examined whether GLP-1 is involved in the mediation of leptin effects on feeding behaviour.

We have found that the GLP-1 antagonist injected intraperitoneally prior to leptin completely abolished its suppressory effect on food intake and body weight gain. This indicates that the active peripheral GLP-1 receptor is necessary for the mediation of the leptin effect on food consumption. The physiological mechanism of this interaction may occur at two levels. Firstly, leptin might stimulate GLP-1 release from endocrine L cells in the intestine (18). GLP-1, in turn, would enhance the activity of the satiety centre. Consequently, the blockade of GLP-1 receptor prevented leptin effects on these centres as shown in this study. Secondly, the activation of both GLP-1 and leptin receptors could be necessary to additively stimulate peripheral neural pathways activating satiety centres. Since leptin (28) and GLP-1 (29) receptor were found in nodose ganglion cells in the rat, it seems that both peptides may act in concert to stimulate vagal afferent terminals. Intact vagal transmission was shown to be necessary for the effect of both GLP-1 (30) and leptin (31) on food intake thus suggesting that the interaction between these two hormones may take place at the level of vagal sensory inputs. It was consistently shown that leptin and GLP-1 injected intraperitoneally act additively to reduce food consumption in the rat (6). Alternatively, exendin (9-39) injected intraperitoneally could, to some extend, cross the blood-brain barrier and block the central GLP-1 receptor preventing the interaction between leptin and GLP-1 in the brain.

As expected, intracerebroventricular leptin markedly reduced food intake. This effect was similar to that reported by others (32, 33). Pre-treatment with the GLP-1 receptor antagonist, however, significantly attenuated the inhibitory effect of leptin on food consumption although a significant suppressory response to this hormone could be still seen. This finding is consistent with that reported earlier by Goldstone et al. (9). In contrast to that study, however, we have found that GLP-1 receptor blockade did not prevent from the leptin-dependent body weight reduction. Unlike Goldstone et al. we injected the GLP-1 antagonist prior to leptin and this methodological difference might account for some discrepancy in our results. Altogether, it seems that central GLP-1 mediates in part the effects of leptin on feeding centres. Possibly, the effect of central leptin on feeding behaviour is mediated not only by GLP-1 but also by other neuropeptidergic systems (24 - 27) that remain active in exendin (9 - 39)-treated rats. This could explain the partial effect of the GLP-1 receptor blockade on the inhibitory effect of leptin on feeding centres obtained in the present study.

Interestingly, only central but not peripheral leptin produced a significant effect on water intake. This finding is consistent with results obtained by Patel and Ebenezel (31) who found no effect of leptin administered intraperitoneally on water intake and Zorilla et al. (34) who reported the reduced water consumption after the intracerebroventricular leptin injection in the rat. Possibly, the direct effect produced by the intracerebroventricularly infused peptide is more efficient than the indirect activation of the thirst centre by peripheral leptin.

Since leptin receptor was found in brain areas involved in food but not water intake control (2, 35), it seems that the reduction in water consumption was secondary to the decrease of appetite. This is supported by the finding that both food and water consumption tended to decrease 24 h and then to increase 48 h after injection. On the other hand, exendin (9 - 39) was shown to reduce selectively water intake without any considerable effect on food consumption. This is surprising given the previously reported suppressive effect of GLP-1 on thirst (23, 36, 37). Possibly, GLP-1 might affect thirst through two independent mechanisms. The first one would be closely related to the GLP-1-produced decrease in food and, in turn, water consumption. Accordingly, GLP-1-induced suppression of feeding resulted in the respective reduction of water intake. The second mechanism could be related to the direct impact of GLP-1 on structures involved in thirst control. It seems that GLP-1 might stimulate these areas, an event seen in some hypothalamic nuclei involved in the maintenance of water balance of the body (38). Apparently, the GLP-1-ergic transmission is necessary for the control of water intake and the disruption of this signalling results in the diminished thirst. In contrast to feeding behaviour, no considerable effect of GLP-1 receptor blockade on leptin-induced inhibition of water consumption was seen.

In conclusion, it is suggested that GLP-1 receptor blockade due to either peripheral or central injection of a GLP-1 antagonist attenuates the effect of leptin on feeding centres. Thus, intact GLP-1 signalling is necessary to mediate the effect of exogenous leptin on food but not water intake in the rat. Moreover, our findings produce further evidence for close interactions between long- and short-term factors regulating the activity of feeding centres.

Acknowledgements: This work was supported by the Medical University of Lodz, grant No. 502-16-509.


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