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Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements

Vagal afferent nerve fibres are involved in the transmission to the central nervous system of information relating to food intake and immune reactions. Leptin is involved in the control of food intake and has specific receptors in afferent vagal neurones. To investigate the role of these receptors, we studied the effects of leptin on single vagal afferent activities from intestinal mechanoreceptors in anaesthetized cats. The activity of 35 intestinal vagal mechanoreceptors was recorded by means of glass microelectrodes implanted in the nodose ganglion. Leptin (10 μg), administered into the artery irrigating the upper part of the intestine, induced activation in 17 units (P < 0.001), inhibition in 8 units (P < 0.05), and had no effect on 10 units. The excitatory effects of leptin were blocked by the endogenous interleukine-1β receptor antagonist, (Il-1ra, 250 μg, i.a.). Cholecystokinin (CCK, 10 μg, i.a.) induced an activatory response only in the two types of units which were responsive to leptin alone. When leptin was administered after CCK, its excitatory effects were enhanced and its inhibitory effects were blocked, whereas it had no effect on the units which were not affected by leptin alone. The interactions between leptin and CCK are specific ones, since other substances (phenylbiguanide, a serotoninergic agonist; substance P) known to activate the mechanoreceptors did not modify the effects of leptin. These results indicate that leptin appears to play a role in the control of immune responses and food intake via intestinal vagal afferent nerve fibres and that there is a functional link between leptin and Il-1β.

Leptin is a 16 kDa protein produced by the ob gene, which mainly controls food intake and energy expenditure by acting on the hypothalamus (Zhang et al. 1994; Pelleymounter et al. 1995). However, leptin-specific receptors have been found to exist in several peripheral tissues (Dal Fara et al. 2000), especially in the sympathetic prevertebral ganglionic neurones in rats and mice (Miller et al. 1999), in the intestinal intramural plexuses in guinea- pigs (Liu et al. 1999) and in afferent and efferent vagal neurones in rats (Buyse et al. 2001). These findings suggest that leptin may also play an extra-hypothalamic role. Although leptin is synthesized mainly by adipose tissues in proportion to the body fat mass in numerous species (Campfield et al. 1995), other cell types, namely stomach epithelial cells, seem to be able to produce this substance in both rats and humans (Bado et al. 1998; Sobhani et al. 2000). In addition, many data published in the literature have suggested that leptin may not only be involved in the control of food intake and body weight, but also contribute to modifying immune responses (Bik et al. 2001). Leptin receptors have been described in macrophages and granulocytes in the lamina propria of the rat jejunum (Lostao et al. 1998). In addition, it has been reported that under pathological conditions (such as septicaemia, some carcinomas or chronic inflammatory diseases of the digestive tract), the plasmatic leptin level is no longer correlated with the body fat (Janik et al. 1997; Barbier et al. 1998; Torpy et al. 1998).

Although the long-term effects of leptin on body weight control have been thoroughly investigated (Pelleymounter et al. 1995), very little is known so far as to how circulating leptin may quickly activate the central nervous system by stimulating visceral afferent neurones. Previous studies (Wang et al. 1997, 2000; Shiraishi et al. 1999; Yuan et al. 1999) have focused on the activation of gastric and hepatic vagal afferent nerve fibres by leptin.

Gastrointestinal mechanoreceptors are known to be sensitive to numerous substances, including locally released neurotransmitters and hormones such as cholecystokinin (CCK), substance P (SP) and serotonin (5HT) (Grovum & Leek, 1982; Davison, 1986; Mei & Lucchini, 1992; Blackshaw & Grundy, 1993). Despite their simple structure (free endings) visceral sensory endings correspond to various functional receptors due to their complex biochemical membrane equipment (ionic channels and receptors). Some vagal afferent nerve fibres, probably those endowed with chemosensitive mechanoreceptors, are involved in the transmission to the central nervous system of information relating to food intake (Schwartz, 2000) and the immune system (Bret-Dibat et al. 1995). The effects of interleukine-1β (Il-1β) on gastric and hepatic afferent nerve fibres have been extensively studied (Niijima, 1996; Bucinskaite et al. 1997; Ek et al. 1998). Since the existence of some strong functional links (such as intestino-gastric reflexes controlling gastric emptying and intestinal motility) has been clearly established (Gonella et al. 1987), it is proposed in the present study to investigate the effects of leptin on the activity of intestinal vagal chemosensitive mechanoreceptors. At the same time, the interactions between leptin, CCK and Il-1β are also studied with a view to determining how these agents contribute to the control of food intake and immune responses.

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements

The experimental procedures described here were carried out in keeping with the European guidelines for the care and use of laboratory animals (Council Directive 86/6009/EEC).

The experimental methods used were practically the same as those described by Mei (1978) and Abysique et al. (1999).

Thirty-five adult European cats of either sex weighing 2.8 to 5.0 kg (mean, 3.3 kg) were used. Animals had fasted (water ad libitum) for 24 h before undergoing surgery.

Surgical preparation

Anaesthesia.

Anaesthesia was induced by a gaseous mixture (2 % halothane-98 % air; Bélamont, Neuilly-sur-Seine, France), the trachea was cannulated and a catheter was inserted into a radial vein, through which the main anaesthetic (chloralose, 80 mg kg−1, i.v., Sigma, Saint Quentin-Fallavier, France) was administered intravenously. To prevent the occurrence of any movement artefacts due to electrical stimulation of the nerves, the animals were neuromuscularly blocked by intravenously administering Flaxedil (gallamine triethiodide, 4 mg kg−1, i.v., Sigma) and ventilated with a respiratory pump. Pupil dilation, palpebral reflex, heart rate and blood pressure were monitored throughout the experiments in order to determine whether further anaesthesia and curarization were necessary.

Fixation of animals and body temperature.

Cats were fixed in a Horsley-Clarke apparatus in the supine position, and their body temperature was maintained at 37 ± 1 °C by means of a heating blanket.

Administration of pharmacological substances.

Pharmacological drugs were administered locally through a catheter introduced into the right femoral artery so that its tip was located at the level of the coeliac artery that irrigates the upper part of the small intestine. Its position was checked post mortem by injection of 1 % Methylene Blue (Sigma) into the catheter.

Isolated small intestine loop.

A segment of small intestine was isolated between two cannulas. The first of these, which was connected to a vertically adjustable reservoir filled with physiological solution, was inserted into the proximal jejunum, so that various perfusion pressures could be obtained. The second cannula, which was used for emptying the intestinal loop, was placed 20 cm lower down, and was connected to a pressure transducer (Telco, 94250 Gentilly-Seine, France).

Dissection of the vagus nerve and nodose ganglion.

The right cervical vagus nerve was dissected along 1 cm in the neck and was placed on stimulating electrodes composed of two platinum wires placed in a Plexiglass gutter. The electrodes were connected to a stimulator (Grass S88). The right nodose ganglion was dissected out in the pharyngeal area, 4–5 cm above the stimulating electrode. It was then placed on a special metallic plate, designed to prevent the occurrence of any artefactual respiratory and circulatory movements.

Lethal procedures.

At the end of each experiment, the cats were killed with an overdose of barbiturates (sodium thiopentone, 70 mg kg−1, i.v., Sanofi, Libourne, France).

Stimulation techniques

Stimulation of the vagus nerve.

Rectangular stimulation shocks were delivered by means of a Grass stimulator in series connected to an isolation unit. With the electrical shocks used (1 ms in duration, and 15–30 V in amplitude), it was possible to monitor all the responses by the recording electrode. This set-up recorded the conduction velocity (approximately 1 m s−1) of the unmyelinated vagal fibres.

Mechanical stimulation.

The intestinal loop was isolated and subjected to mechanical distensions performed by vertically moving the reservoir containing the physiological solution. With our experimental protocol, it is possible to make pressure adjustments ranging between 0 and 100 cmH2O. Distensions of 20 cmH2O were applied to test whether the receptors were mechanosensitive. With these stimuli, it was possible to selectively activate intestinal mechanoreceptors and thus to analyse their electrical activity by recording their activity at nodose ganglion level. In addition, moderate digital compression was applied to locate the mechanoreceptors more exactly.

Chemical stimulation.

When interoceptors were identified as mechanoreceptors based on their response to mechanical stimulation, intra-arterial chemical stimuli were applied. All the substances were diluted in 1 ml of physiological saline and administered within 5 s. Each injection was followed by a flush with 1 ml of saline solution. To prevent the occurrence of any tachyphylaxic disorders, the time elapsing between successive injections was set at 60 min. When testing whether CCK, SP and phenylbiguanide (PBG) potentiated the effects of leptin, however, this interval was reduced to 20 min, since this was the time at which the maximum enhancing effects were observed with several other drugs (M. Bouvier, unpublished observations).

Drugs

The following drugs were used: murine leptin lyophilized from a sterile filtered solution in phosphate-buffered saline (PBS) (AMGEN Thousand Oaks, USA), recombinant human interleukine-1β receptor antagonist (Il-1ra, AMGEN), cholecystokinin (CCK; Sigma, Saint Quentin-Fallavier, France), substance P (SP; Sigma), phenylbiguanide (PBG; RBI, Sigma) and atropine sulphate salt (Sigma).

The doses of CCK, SP and PBG used to activate vagal afferent nerve fibres were 10 μg (8.75, 7.42, and 56.43 nmol, respectively). These doses were similar to those previously used by Mei et al. (1996) to stimulate intestinal vagal mechanoreceptors. These authors also recorded the activities of the receptors from nodose cell bodies. The dose of Il-1ra (250 μg i.e. 14.71 nmol) was calculated based on the study by Luheshi et al. (1999). The weight of the animals was not taken into account because the drugs were injected locally into the intestine.

Electrophysiological techniques

The activity of vagal afferent units was recorded in the nodose ganglion via an extracellular glass microelectrode filled with 3 m KCl solution. It has been reported that recordings obtained by means of glass electrodes filled with KCl (3 m) are similar to those obtained with glass electrodes filled with NaCl (2 m; Mei, 1968), or with metal electrodes (S. Gaigé, unpublished observations), which clearly indicates that using KCl (3 m) does not affect the discharge pattern of activity of vagal afferent neurones under our experimental conditions. The electrode was set in the gastro-intestinal area of the nodose ganglion, which is located in its caudal part (Mei, 1970, Zhuo et al. 1997) using a micromanipulator (Mei, 1983). Electrodes were prepared using a glass vertical stretching apparatus: their diameters ranged from 1 to 3 μm and their resistances from 2 to 5 MΩ. The discharge patterns of the vagal afferent units were displayed on an oscilloscope (TEKTRONIX, type R564B, Beaverton, OR, USA) and a thermosensitive paper recorder (Astromed, type DASH IV, 78190 Trappes, France). The activity of these units was also recorded on magnetic tapes (DTR biologic, 38640 Claix, France) for further data processing. With the amplitude discrimination (WPI, model 121, New Haven, CT, USA) and shape recognition (MacLab, New Haven, CT, USA) procedures used, it was possible to select the electrical activity of a single afferent fibre and thus to work under unitary recording conditions. Data were processed by a computer in the form of frequency histograms showing the number of action potentials per second (AP s−1). To ensure that the full effects were displayed, the recordings started 2 min before the injections were administered and lasted for 17 min.

Statistical analysis

When the activity of several mechanoreceptors was recorded in the same animal, only the first unit recorded was included in the statistical analysis. For each injection, we calculated the mean discharge frequency (AP s−1) of afferent neurones for 40 s before the administration of the drug and the mean discharge frequency for the 40 s period starting at the peak effect. The peak effect was the optimum (maximum or minimum) instantaneous discharge frequency, depending on the type of unit and the drug injected. For each injection, we calculated the percentage of variation of the mean discharge frequencies. Results quoted are the mean percentages ±s.e.m. The values in parentheses are the mean discharge frequency before the injection ±s.e.m. vs. the mean discharge frequency after the injection ±s.e.m. Student's paired t test was used to assess the significance of the results. Differences were taken to be significant at P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements

Thirty-five vagal neurones of the small intestine responding to mechanical stimulation were studied. All these neurones had unmyelinated fibres with conduction velocities ranging between 0.8 and 1.2 m s−1. Their basal discharge frequencies ranged from 1.31 to 2.23 AP s−1 (mean value: 1.75 ± 0.31 AP s−1). Leptin was injected intra-arterially (i.a.) in the following doses: 0.1, 1 and 10 μg (6.25, 62.50 and 625.00 pmol).

Effects of leptin on the activity of intestinal mechanoreceptors

Leptin was administered to 35 mechanoreceptors and produced the following effects: 17 mechanoreceptors were activated (Fig. 1B), 8 were inhibited and 10 showed no effect. The activated neurones will be referred to here as type 1 units and the inhibited neurones, as type 2 units.

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Figure 1. Effects of leptin (10 μg, i.a.) on the activity of type 1 intestinal vagal mechanoreceptor before and after administration of CCK (10 μg, i.a.)

A (control), the basal activity consisted of two bursts of spikes. B, leptin administration (arrow) induced a strong activation of the receptor. C, leptin administration (arrow) 20 min after CCK induced practically continuous discharges of the receptor, indicating that the effects of leptin were enhanced by previous injection of CCK. B, 10 s after A; C, 80 min after B.

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The responses of seven type 1 units were investigated at three increasing doses of leptin: 0.1, 1 and 10 μg. At all these doses, leptin significantly increased (P < 0.01) the mean discharge frequency (18.63 ± 8.34, 50.25 ± 8.89 and 93.78 ± 25.02 %, respectively) (2.51 ± 0.47 vs. 2.85 ± 0.51, 2.40 ± 0.32 vs. 3.49 ± 0.45 and 1.76 ± 0.32 vs. 3.17 ± 0.52 AP s−1, respectively). These effects had the following latencies: 9.4 ± 2.1, 8.4 ± 1.4 and 7.9 ± 1.5 s, respectively, and the following durations: 219.6 ± 42.4, 300.4 ± 73.9 and 325.7 ± 98.4 s, respectively. The dose-response curve indicates that a dose of 10 μg can be routinely employed to stimulate intestinal chemosensitive mechanoreceptors (Fig. 2).

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Figure 2. Dose-response curve of the effects of leptin (0.1, 1 and 10 μg, i.a.) on the activity of type 1 intestinal vagal mechanoreceptors before and after CCK (10 μg, i.a.)

At each dose the excitatory effects of leptin were enhanced after CCK administration. **P < 0.01 (n= 7).

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In the 17 type 1 units, 10 μg leptin significantly increased (P < 0.001) the mean discharge frequency (Fig. 3A) by 91.96 ± 23.83 % (1.64 ± 0.27 vs. 2.93 ± 0.51 AP s−1), with a latency of 6.8 ± 0.7 s and a duration of 234.2 ± 43.7 s (Fig. 4B).

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Figure 3. Statistical analysis of the effects of leptin (Lep), SP, CCK and PBG on the activity of intestinal mechanoreceptors

Leptin, CCK, SP and PBG were intra-arterially administered at a dose of 10 μg. A, type 1 units; B, type 2 units. In A and B, columns C give the mean value of the control responses as 100 %. The effects of various drugs are expressed as mean values compared with the control value. The vertical bars at the top of the columns give the s.e.m.. In type 1 units, all the drugs induced excitatory effects, whereas in type 2 units, only CCK induced excitatory effects. In type 2 units, SP and PBG had no effect, while leptin induced inhibitory effects. *P< 0.05; **P< 0.01; ***P< 0.001; ns: not significant. n (the number of units) is indicated in parentheses above the histograms.

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Figure 4. Histograms comparing the effects of arterially administered leptin alone (10 μg), and those of leptin after CCK (10 μg, i.a.) on the activity of a type 1 intestinal vagal mechanoreceptor

A, control; B, leptin administration (arrow); C, leptin administration (arrow) 20 min after CCK. The effects of leptin were enhanced after CCK. Top traces in insets in A, B and C are the same reference waveform as that recorded at the beginning of A. AP s−1: action potential per second.

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In the eight type 2 mechanoreceptors, 10 μg leptin significantly decreased (P < 0.05) the mean discharge frequency (Fig. 3B) by 16.07 ± 4.85% (1.84 ± 0.39 vs. 1.62 ± 0.41 AP s−1), with a latency of 9.0 ± 0.9 s and a duration of 133.4 ± 10.0 s.

In 10 mechanoreceptors, 10 μg leptin did not affect the mean discharge frequency.

It is worth noting that there was no significant difference between the spontaneous responses recorded from type 1 units (1.64 ± 0.27 AP s−1) and those from type 2 units (1.84 ± 0.39 AP s−1).

The effects of the vehicle were investigated in the case of each unit and no significant changes in spontaneous activity were recorded after intra-arterial administration. In seven mechanoreceptors, leptin (10 μg, i.a.) administered after injecting atropine (250 μg), a muscarinic receptor blocking agent, had no effect on either the excitatory responses of the type 1 units (n= 4) or the inhibitory responses of the type 2 units (n= 3). Sectioning of the vagus nerve, performed caudally to the nodose ganglion, abolished the spontaneous activity of all three types of units (type 1, n= 5; type 2, n= 4; and leptin insensitive, n= 3) and no effects of leptin on the chemosensitive mechanoreceptors were observed (n= 9). At all the doses tested, leptin injection does not modify the intraluminal intestinal pressure and affected neither the arterial pressure (107.61 ± 9.21 vs. 110.81 ± 11.03 mmHg) nor cardiac frequency (74.23 ± 0.67 vs. 72.84 ± 0.98 beats min−1; n= 35).

In five cats, we recorded the activity of several mechanoreceptors. In one of these five cats, one type 1 unit was activated by leptin and one type 2 unit was inhibited by leptin. In the other four cats, all three types of units (type 1, type 2 and leptin-insensitive) were identified.

Effects of intestinal distension on the three types of mechanoreceptors depending on the effects of leptin

The effects of a 20 cmH2O distension of the intestine on the three types of units defined in terms of their response to leptin were analysed. They each responded differently to mechanical stimulation. In the 17 type 1 units, distension significantly (P < 0.001) increased the mean discharge frequency by 118.80 ± 7.81 % (1.72 ± 0.06 vs. 3.71 ± 0.07 AP s−1). In the eight type 2 units, distension significantly (P < 0.0001) increased the mean discharge frequency by 242.30 ± 27.66 % (1.76 ± 0.12 vs. 5.81 ± 0.24 AP s−1). In the 10 leptin-insensitive units, distension significantly (P < 0.0001) increased the mean discharge frequency by 260.49 ± 13.86 % (1.76 ± 0.09 vs. 6.29 ± 0.29 AP s−1). There was no significant difference between the responses to intestinal distension observed in the type 2 units and the leptin-insensitive units, whereas these responses were significantly (P < 0.001) different from those of the type 1 units.

Effects of CCK, SP and PBG on the activity of intestinal mechanoreceptors

Effects of CCK.

CCK (10 μg) increased the mean discharge frequency in all the units tested (n= 18), whether they were type 1 (n= 10) or type 2 units (n= 8). The mean discharge frequency of the units that did not respond to leptin was not affected by the injection (n= 10). In the type 1 units, a significant increase (P < 0.001) in the mean discharge frequency (Fig. 3A) was observed, amounting to 803.64 ± 203.66 % (1.75 ± 0.28 vs. 15.82 ± 2.41 AP s−1), with a latency of 8.2 ± 0.9 s and a duration of 514.4 ± 96.2 s. The significant increase (P < 0.01) in the mean discharge frequency of 265.45 ± 99.31 % (2.16 ± 0.51 vs. 7.74 ± 0.87 AP s−1) observed with the type 2 units after CCK (Fig. 3B) had a latency of 8.1 ± 1.0 s and a duration of 210.5 ± 52.8 s. A significant difference (P < 0.01) was found to exist between the activatory effects of CCK on type 1 and type 2 units.

Effects of SP.

SP at a dose of 10 μg had no effect on the type 2 units (n= 4; Fig. 3B); whereas in the case of the type 1 units SP significantly increased (P < 0.05) the mean discharge frequency by 140.43 ± 47.95 % (1.47 ± 0.28 vs. 3.63 ± 0.54 AP s−1) with a latency of 9.6 ± 2.3 s and a duration of 93.2 ± 9.8 s (n= 5; Fig. 3A).

Effects of PBG.

PBG at a dose of 10 μg had no effect on the type 2 units (n= 3; Fig. 3B), but in the case of the type 1 units it significantly increased (P < 0.05) the mean discharge frequency by 818.03 ± 202.41 % (1.24 ± 0.62 vs. 11.25 ± 0.81 AP s−1) with a latency of 13.3 ± 2.8 s and a duration of 45.3 ± 5.7 s (n= 4; Fig. 3A).

Effects of leptin on the activity of intestinal mechanoreceptors before and after administration of CCK, SP and PBG

Effects of leptin before and after CCK.

In type 1 units, leptin was administered at doses of 0.1, 1 and 10 μg, 20 min after CCK injection and in all the cases studied, CCK was found to significantly enhance (P < 0.01) the effects of leptin described above (Fig. 1 and Fig. 2). In all seven units tested at these three doses (0.1, 1 and 10 μg), a significant increase (P < 0.01) of 97.03 ± 23.25 % (2.02 ± 0.30 vs. 3.94 ± 0.67 AP s−1), 188.11 ± 47.00 % (1.72 ± 0.24 vs. 4.80 ± 0.75 AP s−1) and 320.56 ± 115.46 % (2.15 ± 0.36 vs. 8.93 ± 1.17 AP s−1), respectively, was observed in the mean discharge frequency (Fig. 5A). On the other hand, the latencies (12.3 ± 1.8, 12.0 ± 1.9 and 8.0 ± 0.9 s, respectively) and durations (161.4 ± 21.7, 156.9 ± 24.9 and 235.3 ± 50.0 s, respectively) were not significantly different from those obtained with leptin before CCK administration. Moreover, the latencies of the effects of leptin did not differ significantly from those of the effects of CCK (Fig. 4C).

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Figure 5. Statistical analysis of the effects of leptin (Lep, 10 μg, i.a.) on the intestinal vagal mechanoreceptors of types 1 and 2 before and after CCK, SP and PBG

CCK, SP and PBG were intra-arterially administered at a dose of 10 μg. In each group of traces: Aa (n= 10), Ba (n= 5) and Ca(n= 4) showed the effects on type 1 units. Ab(n= 8), Bb (n= 4) and Cb (n= 3) showed the effects on type 2 units. Aa and b, effects of CCK (arrow). Ba and b, effects of SP (arrow). Ca and b, effects of PBG (arrow). For each group of traces, column C gives the mean value of the control response as 100%. Note that the effects of leptin were enhanced after CCK but remained unchanged after SP or PBG. *P< 0.05; **P< 0.01; ns, not significant.

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The injection of vehicle, 20 min after CCK had no effect (n= 4).

In the type 2 units, CCK injection 20 min before leptin (10 μg), significantly (P < 0.01) decreased the inhibitory effects of leptin described above (n= 8; Fig. 5A).

Two injections of 10 μg leptin performed at an interval of 20 min both induced a similar increase in the discharge frequencies of the type 1 units (n= 4), and a similar decrease in the discharge frequencies of the type 2 units (n= 4).

Effects of leptin before and after SP.

In the five type 1 units tested with leptin and SP, injecting leptin alone (10 μg) significantly increased (P < 0.01) the mean discharge frequency of 96.07 ± 24.53 % (1.53 ± 0.68 vs. 2.98 ± 0.71 AP s−1) with a latency of 10.6 ± 1.5 s and a duration of 150.8 ± 23.4 s (Fig. 5B). Leptin administered to these five units 20 min after SP injection (10 μg) also induced a significant (P < 0.01) increase of 109.86 ± 28.43 % (1.51 ± 0.56 vs. 3.08 ± 0.80 AP s−1) in the mean discharge frequency with a latency of 13.6 ± 2.9 s and a duration of 184.8 ± 49.6 s (Fig. 5B). Four type 2 units were tested with leptin and SP. Leptin injected alone (10 μg) significantly inhibited (P < 0.05) the mean discharge frequency by 14.38 ± 2.23 % (1.73 ± 0.21 vs. 1.49 ± 0.32 AP s−1) with a latency of 9.3 ± 0.7 s and a duration of 118.7 ± 12.9 s (Fig. 5B). Leptin administered 20 min after SP injection also significantly inhibited (P < 0.05) the mean discharge frequency by 15.72 ± 3.74 % (1.88 ± 0.41 vs. 1.58 ± 0.37 AP s−1) with a latency of 9.3 ± 0.8 s and a duration of 120.3 ± 21.4 s. The excitatory and inhibitory effects of leptin observed before SP did not differ significantly from those observed after SP (Fig. 5B).

Effects of leptin before and after PBG.

In the four type 1 units tested with leptin and PBG, injecting leptin alone (10 μg) significantly increased (P < 0.01) the mean discharge frequency by 92.35 ± 31.02 % (1.62 ± 0.33 vs. 2.99 ± 0.21 AP s−1) with a latency of 13.3 ± 2.8 s and a duration of 159.5 ± 31.1 s (Fig. 5C). Leptin administered at the same dose 20 min after PBG (10 μg) induced a significant increase (P < 0.01) in the mean discharge frequency of 89.13 ± 14.51 % (1.48 ± 0.71 vs. 2.96 ± 0.55 AP s−1) with a latency of 10.5 ± 1.3 s and a duration of 186.5 ± 34.6 s (Fig. 5C). Three type 2 units were tested with leptin and PBG. Leptin injected alone (10 μg) significantly inhibited (P < 0.05) the mean discharge frequency by 17.08 ± 4.91 % (1.81 ± 0.36 vs. 1.49 ± 0.44 AP s−1) with a latency of 8.8 ± 0.2 s and a duration of 154.3 ± 21.5 s (Fig. 5C). Leptin administered at the same dose 20 min after PBG injection also significantly inhibited (P < 0.05) the mean discharge frequency by 16.04 ± 3.92 % (1.83 ± 0.21 vs. 1.53 ± 0.46 AP s−1) with a latency of 8.9 ± 0.5 s and a duration of 132.8 ± 30.1 s (Fig. 5C). Previously administering PBG did not significantly affect the responses to leptin injection of the units of either type.

Effects of leptin on the activity of intestinal mechanoreceptors before and after injection of Il-1ra.

In the 10 type 1 units tested with Il-1ra, 10 μg leptin injection alone significantly increased (P < 0.01) the mean discharge frequency by 147.54 ± 43.66 % (1.61 ± 0.23 vs. 4.06 ± 0.41 AP s−1), with a latency of 8.30 ± 1.11 s and a duration of 273.8 ± 72.3 s (Fig. 6B and Fig. 7B). Injecting leptin at the same dose 5 min after Il-1ra (250 μg) had no further significant effects on the mean discharge frequency (Fig. 6C and Fig. 7C). The excitatory effects of leptin were thus significantly (P < 0.01) inhibited by Il-1ra (Fig. 8).

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Figure 6. Effects of leptin (10 μg, i.a.) on the activity of a type 1 intestinal mechanoreceptor before and after administration of Il-1ra (250 μg, i.a.)

A (control), the receptor weakly discharged (action potential of large amplitude). B, leptin administration (arrow) strongly activated the receptor. C, 5 min after Il-1ra, administration of leptin (arrow) induced no effect. B, 10 s after A; C, 65 min after B.

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Figure 7. Histograms comparing the effects of arterially administered leptin alone (10 μg), with those of leptin after Il-1ra (250 μg, i.a.) on the activity of a type 1 intestinal vagal mechanoreceptor

A, control. B, leptin administration (arrow). C, leptin administration (arrow) 5 min after Il-1ra. The excitatory effect of leptin (B) was blocked by Il-1ra (C). Top traces in insets in A, B and C are the same reference waveform as that recorded at the beginning of A.

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Figure 8. Statistical analysis of the effects of leptin (Lep, 10 μg, i.a.) before and after administration of Il-1ra (250 μg, i.a.) on the type 1 intestinal vagal mechanoreceptors

Column C gives the mean value of the control response as 100 %. Note that leptin administered after Il-1ra had no effect. **P < 0.01; ns: not significant (n= 10).

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Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements

All the mechanoreceptors investigated in the present study were in series muscle mechanoreceptors, as confirmed by the fact that they responded to a moderate distension of the intestine (Grundy, 1992). They were probably tension receptors located in the intraganglionic laminar ending (Phillips & Powley, 2000).

Leptin and food intake

The present results show that leptin can affect the spontaneous discharge patterns of numerous (71%) vagal chemosensitive mechanoreceptors in the intestine in two ways: by significantly increasing the discharge frequencies of 68 % of the vagal afferent nerve fibres (type 1 units) and significantly decreasing those of the remaining 32 % (type 2 units). These results are consistent with the in vitro data published in rats, showing that the effects of leptin on the vagal afferent nerve fibres innervating the stomach are also of two kinds (Wang et al. 1997). Based on the effects of leptin, three different types of intestinal vagal mechanoreceptors were identified (type 1, type 2 and leptin-insensitive units). Units of all these types were present in the same animal. The three types of neurones involved cannot be differentiated in terms of the basal discharge frequencies, which were all identical. However, the differences between their responses to mechanical stimulation as well as to CCK administration indicate that three different populations of mechanoreceptors were involved. Both type 2 neurones and leptin-insensitive neurones are strongly sensitive to distension, but the former are CCK sensitive as well. Also strongly CCK sensitive are type 1 neurones which, however, are less distension sensitive than the two other categories. The presence of two populations of mechano- and chemosensitive neurones was confirmed by the fact that SP and PBG activate type 1 but not type 2 units. Leptin and CCK insensitive units might be purely mechanoreceptors, whereas both type 1 and type 2 units are mechanosensitive receptors, which belong to two different populations having different functions.

Although CCK always induced an increase in the discharge frequency of both type 1 and type 2 units, these effects differed in their intensity and their duration between these two populations of units. This finding supports the existence of two different populations. However, the latency of the effects of CCK was similar with both types of units, which indicates that CCK affects vagal sensory neurones via a similar mechanism (namely, a direct activation via CCK receptors).

The fact that all the mechanoreceptors in our study were connected to type C fibres indicates that all the neurones studied have small cell bodies (about 25 μm in diameter) (Harper & Lawson, 1985) and they cannot therefore be differentiated in terms of their morphology.

The two types of afferent nerve fibres cannot be differentiated in terms of their basal discharge frequencies, which are identical. However, since the type 1 and type 2 units responded in significantly different ways to CCK, they seem to belong to two different populations. This assumption was confirmed by the fact that only the type 1 units were activated by both SP and PBG. Previously published data have indicated that the percentage of vagal chemosensitive mechanoreceptors responding to one or several chemicals varies (Mei et al. 1996).

The short latency of the effects of leptin strongly suggests that this substance acts directly on the sensory endings. It is worth noting that this latency is similar to that recorded in the case of CCK, with which a direct effect may also occur (Mei, 1978; Mei et al. 1996).

The methods employed here were identical to those previously described by other authors for studying the discharge frequencies of vagal afferent units (Mei et al. 1996). The effects observed here were not due to arterial distension associated with drug injection (1 ml within 5 s), since the discharge frequencies were not affected when vehicle alone was injected. Although some substances are able to activate the cell bodies of vagal afferent neurones directly (Dun et al. 1991), the effects of leptin observed here were definitely of peripheral origin. As a matter of fact, these effects were completely abolished upon sectioning the ipsilateral vagus nerve caudally to the ganglion. The peripheral action of leptin has been confirmed by the lack of any systemic effects of the drug on cardiac frequency and arterial pressure (Haynes, 2000). In addition, the fact that leptin injection resulted in no changes in the intestinal intraluminal pressure indicates that these effects are probably not due to motor activation. Moreover, atropine, which is a muscarinic receptor blocking agent generally used to inhibit motor activity, failed to modify the effects of leptin. Our results are in keeping with those obtained by Mei et al. (1996), showing that most of the vagal mechanoreceptors in the intestine (71 %) are chemosensitive. But the fact that some of them (29 %) are not affected by the substances tested indicates that these mechanoreceptors either are not sensitive to endogenous substances, or are not chemosensitive at all. All the mechanoreceptors responding to leptin are spontaneously active. Identical data have been published on the stomach vagal afferent neurones (Wang et al. 1997). We have observed that leptin injection never results in activation of previously silent units, indicating that the mechanoreceptors activated by leptin are only low-threshold muscle receptors (Grundy, 1992). Moreover the disappearance of spontaneous activity after sectioning of vagus nerve caudally to nodose ganglion confirms the peripheral origin of the activity recorded in our experiments.

These results showing that CCK can activate intestinal vagal mechanoceptors are in agreement with previously published data (Blackshaw & Grundy, 1990; Mei & Lucchini, 1992). As established above, the present effects are peripheral sensory effects, since the activation of afferent neurones always occurred prior to the motor effects, as described by Mei et al. (1996). These authors have also reported that the sensory effects of CCK persist under atropine. Although the activatory responses of type 1 units to CCK are significantly larger in amplitude and duration to those of type 2 units, the similarity between their latencies strongly suggests that a similar mechanism of activation is involved. In 15 experiments, leptin was administered both before CCK (first injection) and 20 min after CCK (second injection). The effects induced by the second injection differed significantly from those observed after the first one. In type 1 units, the effects of leptin are enhanced by CCK, whereas in type 2 units, they are inhibited by CCK. This is not a tachyphylaxic phenomenon, because two injections of leptin given 20 min apart have identical effects on the vagal mechanoreceptors. The enhancement of leptin by CCK was not due to motor activation, which had recovered its basal level before the leptin injection was performed. Concerning the mechanism whereby CCK enhanced the effects of leptin, this enhancement cannot have been due to the effect of CCK on the basal activity, because leptin was injected 20 min after CCK, the effects of which last approximately 8 min. The discharge frequency of the mechanoreceptors was therefore similar before injection of leptin alone and before injection of leptin 20 min after CCK. Indeed, although gastric leptin has been found to be rapidly released from the fundic mucosa in response to feeding, data in the literature indicate that, in rats in vivo, gastric leptin is released into the blood compartment 15 min after perfusion of the stomach with CCK (Bado et al. 1998). This time is consistent with the effects we have observed. These effects might result from an increase in the concentration of circulating leptin. This finding supports the idea that plasma leptin may affect the vagal intestinal afferents, but we cannot rule out the possibility that gastric leptin secreted within the fundic mucosa may diffuse into the intestinal mucosa in the vicinity of vagal afferent nerve endings (Wang et al. 2000). Changes in the effects of leptin on vagal mechanoreceptors occurring in response to CCK have also been described in the stomach (Wang et al. 1997), but these changes involve different modes of interaction. CCK does not affect the leptin-induced activation of the vagal mechanoreceptors in the stomach, but those which are not initially affected by leptin become leptin sensitive after CCK. This difference does not seem to be due simply to differences in the doses injected or the times elapsing between CCK and leptin injections. It probably reflects the fact that the patterns of innervation differ between these organs. SP and PBG, which are routinely employed for activating sensory visceral receptors (Paintal, 1973; Mei et al. 1996), activate type 1 units but do not alter the activatory effects of leptin, which indicates that leptin/CCK interactions are actually specific ones. Previous authors have described the relationships between leptin and CCK, and also, in the case of some effects, the need for the simultaneous occurrence of the two substances (see Wang et al. 2000; Lewin & Bado, 2001). In mice, intraperitoneal injections of these two substances induce an early satiety signal. But when delivered separately, the same doses have no effect (Barrachina et al. 1997). In mice, the effects of leptin on the loss of body weight are also enhanced by CCK (Matson et al. 2000). Upon studying the expression of c-fos protein in the hypothalamus in the same species, Wang et al. (1998) observed similar enhancing effects. These authors reported that simultaneous injections of CCK and leptin increased the number of neurones activated in the paraventricular nucleus by about 50 % in comparison with CCK alone.

As postulated by Wang et al. (1997, 1998), and Buyse et al. (2001), post-prandially released CCK and circulating leptin might have combined effects on vagal afferent fibres and thus generate a satiety signal to the central nervous system. Depending on its plasmatic level, leptin may therefore modulate the combination of messages arising from the stomach and intestine and thus optimize the satiety signal.

Leptin and inflammation

Our results show that injecting Il-1ra, an endogenous peptide which is a specific Il-1β receptor antagonist, inhibits the excitatory response to leptin. This is consistent with data obtained on rats by Luheshi et al. (1999), showing that the effects of leptin on food intake and body temperature involve Il-1β. As a matter of fact, intracerebroventricular injection of Il-1ra inhibits the cessation of food intake and the increase in the body temperature induced by leptin. In addition, in mice deprived of Il-1β, no decrease in food intake occurred after leptin administration (Faggioni et al. 1998). Moreover some studies on rodents with genetic abnormalities as regards leptin production or leptin receptor synthesis have shown that a deficit occurs in the level of the animals proinflammatory cytokine expression (Loffreda et al. 1998). On the other hand, numerous data in the literature point to the existence of a correlation between the release of interleukin-1β and the plasmatic leptin levels. In humans as well as in animals, most of the studies available so far have shown that any inflammation, whatever its origin, induces a release of Il-1β that results in an increase in the plasmatic leptin level (Barbier et al. 1998; Faggioni et al. 1998; Arnarlich et al. 1999; Francis et al. 1999). Lastly, Lostao et al. (1998) have described the presence of leptin receptors in the plasma membrane of immune cells located in the lamina propria of the small intestine in rats.

Our results also confirm that leptin is involved, via intestinal vagal afferent fibres, in the mechanisms responsible for inflammation, and probably for a process with which it is generally associated, namely anorexia (Sarraf et al. 1997; Gualillo et al. 2000).

One of our noteworthy results involves the effects of leptin, which were found to depend strongly on Il-1β receptors. The possible existence of a functional link between leptin and Il-1β should therefore be taken into account when dealing not only with the control of immune responses but also with the control of food intake.

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Acknowledgements

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements

The authors would like to thank AMGEN (Thousand Oaks, USA) for generous gifts of leptin and Il-1ra. We thank Drs R. M. Bluthé and R. Dantzer (Bordeaux, France) for their support in this study. We also wish to express our thanks to Drs N. Mei and S. Lucchini for kindly participating in the experiments and for their valuable critical reading of the manuscript. We appreciated the helpful assistance of Mrs F. Farnarier in the preparation of this manuscript.