INTRODUCTION

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GLUCAGON-LIKE PEPTIDE-1-(7-36) amide (GLP-1) is a posttranslational product of the proglucagon molecule that is secreted by distal intestinal L cells in response to food entering the gut, particularly carbohydrate-rich foods (8, 13, 22). GLP-1 is a member of the incretin family (5, 9, 14, 27), a group of peptides that facilitate the release of insulin from pancreatic B cells in response to glucose ingestion (4). GLP-1 is also localized within brain and may act in brain to modulate feeding (21, 22, 29, 32). Turton et al. (32) noted decreased feeding following third cerebroventricular (i3vt) infusion of GLP-1 at doses of 10 and 100 µg in fasted rats, and Navarro et al. (21) noted decreased feeding and drinking following lateral ventricular infusion of GLP-1 at doses of 1 and 2 µg in fasted rats. Reduced feeding has also been noted following fourth ventricular administration of GLP-1 (20). Peripheral administration of GLP-1 does not alter feeding (32), suggesting that the feeding-inhibitory action of GLP-1 is restricted to action within brain.

Increasing evidence suggests that the hypothalamic paraventricular nucleus (PVN) participates in the feeding-inhibitory action of exogenous GLP-1. The PVN contains a high density of GLP-1-immunoreactive nerve fibers and terminals (15, 16), exhibits GLP-1 binding (11, 32), and contains GLP-1 receptor mRNA (26). Moreover, i3vt infusion of GLP-1 results in the expression of c-fos within the PVN (32, 33). PVN infusions of GLP-1 at doses of 100 and 200 ng dose dependently suppress the consumption of a palatable liquid diet by ~25 and 50%, respectively, in non-food-deprived rats (19). Intra-PVN GLP-1 suppresses feeding at dose levels substantially lower than those required via the i3vt route, suggesting that ventricular administration of GLP-1 may act via the PVN to suppress food intake in the rat.

Although PVN infusion of GLP-1 reduces food intake, it is unclear as to whether this effect reflects "satiety" or is a consequence of potential malaise-inducing effects of GLP-1. Not only does i3vt infusion of GLP-1 induce c-fos expression within the PVN and central amygdala (32, 33), but selective brain stem nuclei also express c-fos after i3vt GLP-1, including the area postrema, nucleus of the solitary tract, and lateral parabrachial nucleus (33). This pattern of c-fos activity after intracerebroventricular infusion of GLP-1 is similar to the pattern obtained after peripheral administration of the satiety hormone cholecystokinin (CCK; 6, 17) and the emetic agent lithium chloride (LiCl; 28). The patterns of c-fos expression following central GLP-1 and peripheral CCK or LiCl suggests that GLP-1 reduces feeding by causing illness. i3vt infusion of 3 and 10 µg GLP-1 induces a conditioned taste aversion (CTA), as indexed by decreased latency to reject saccharin in an intraoral paradigm in rats (30, 34), and 10 µg of i3vt GLP-1 decreases saccharin preference in a standard two-bottle CTA preference procedure in rats (30). On the other hand, another study reported that lateral ventricular infusion of 1 µg GLP-1, a dose that suppresses feeding by ~50%, does not induce CTA (29).

The present study sought to determine whether the feeding-inhibitory action of PVN GLP-1 (19) is associated with illness as indexed by a CTA procedure. In the first experiment, rats were exposed to a novel taste, saccharin, and then received either PVN infusion of GLP-1 (0, 100, or 200 ng) immediately afterward or a PVN infusion of 200 ng GLP-1 18 h later. CTA was assessed using a two-bottle preference procedure (10, 24). Using the same group of rats, the second experiment examined the impact of PVN infusion of GLP-1 (0, 100, or 200 ng) on locomotor behavior for 1 h, and the third experiment examined the impact of PVN infusion of GLP-1 (0, 100, or 200 ng) on feeding and drinking for 30 min in non-food-deprived rats. The induction of a CTA would suggest that PVN GLP-1 might reduce feeding by producing malaise, whereas a disruption of locomotion would suggest that PVN GLP-1 might reduce feeding by altering nonfeeding behaviors (e.g., via sedation or hyperactivity). The notion that the feeding-inhibitory action of PVN GLP-1 represents satiety would be weakened by the demonstration that GLP-1 either induces CTA or interferes with motor behavior.

	MATERIALS AND METHODS

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Animals. The animals were 59 adult male Sprague-Dawley albino rats (Harlan Industries, Houston, TX) weighing 250-300 g at the beginning of the study. The animals were housed individually in standard plastic rodent cages and were allowed a 1-wk adaptation period before the onset of behavioral testing to acclimate them to daily handling and colony maintenance procedures. The animal holding room was maintained at 23 ± 1.0°C, with a 12:12-h light-dark lighting schedule. The rats received continuous access to rodent pellet-chow (Teklad) throughout the experiment except as specified in the testing procedure.

Drugs. The GLP-1 solutions (100 and 200 ng) were prepared by dissolving GLP-1 (Pfizer) into Ringer solution (composed of 120.0 mM NaCl, 2.0 mM CaCl₂, and 2.0 mM NaHCO₃). A norepinephrine (NE) solution (25 nmol) was prepared by dissolving (±)-arterenol hydrochloride (Sigma) into Ringer solution. All solutions were calculated as the weight of the base molecule in 0.5 µl of Ringer solution.

Surgical procedure. Before surgery, each rat was injected intraperitoneally with 0.4 mg/kg atropine sulfate (to minimize bronchial secretions) and then anesthetized using separate injections (5 min apart) of ketamine (Ketaset: 60 mg/kg ip) and pentobarbital sodium (20 mg/kg ip). With the upper incisor bar of the stereotaxic instrument positioned at 3.0 mm below the interaural line, the tip of a stainless steel 22-gauge guide cannula was positioned 1.6 mm caudal to bregma, 0.2 mm lateral to the midline, and 7.2 mm below the surface of the skull. The shaft of each unilateral guide cannula was affixed to the skull with stainless steel screws and a pedestal of dental acrylic. After surgery, each rat was injected intramuscularly with penicillin (300,000 units each). A 7-day recovery period followed surgery, during which the rats were weighed daily and had continuous access to water and food pellets in the home cage.

Taste aversion training. Before surgery, the rats were trained to consume water from metal sipping tubes attached to Wahmann 100-ml graduated drinking bottles during 30-min trials on 5 consecutive baseline days. The sipper tubes were inserted through holes in the wire mesh above each cage. Fluid intakes were directly measured from the bottles to the nearest 0.5 ml and recorded for each rat. All fluid intake trials were conducted in the home cages beginning 2 h before dark onset. Food was not available during the fluid intake trials. A 23.5-h water deprivation schedule was implemented during the first five baseline trials to motivate drinking. After surgery, the rats underwent an additional eight consecutive baseline drinking trials, a training day, and four extinction trials, during which a less rigorous 7-h water deprivation schedule was implemented.

On the training day, the rats were randomly assigned to four separate GLP-1 treatment groups (0, 100, or 200 ng or 200 ng noncontingent) based on quantitatively similar water intakes recorded during the last three baseline drinking trials. Each rat was presented with a 0.1% saccharin solution [1.0 g saccharin (Sigma)/1.0 liter distilled water] instead of water during a 30-min training session. Immediately after the 30-min access to saccharin, the bottles were removed and each rat received a single PVN infusion of either 0, 100, or 200 ng GLP-1, except for the 200 ng GLP-1 noncontingent treatment group. The microinjector needle was positioned within each unilateral guide cannula so as to extend 0.5 mm beyond the tip of the cannulas. A volume of 0.5 µl was injected over a 10-s period, and the injector was left in place for an additional 50 s to allow diffusion of the drug solution into the injection site. The noncontingent group received a PVN infusion of 200 ng GLP-1 18 h after the training session to control for nondrug conditioning factors following exposure to the novel taste. After the training day, the rats underwent 4 consecutive extinction days, during which a two-bottle preference procedure consisting of 0.1% saccharin in one Wahmann tube and distilled water in another Wahmann tube was given during a 30-min period. Bottle position was alternated daily to control for position effects. Access to food and tap water was given between every extinction session.

Measures of locomotion. After the CTA testing, the rats were assigned to one of three GLP-1 treatment conditions (0, 100, or 200 ng). The distribution of rats into groups for the locomotion testing was counterbalanced so that equal numbers of rats were taken from each of the CTA treatments (0, 100, and 200 ng GLP-1 and 200 ng GLP-1 noncontingent). On the test day, each rat received a single PVN infusion of either 0, 100, or 200 ng GLP-1 as specified in the CTA experiment and then was placed into an activity chamber. Locomotion testing began 5 min after PVN infusion of GLP-1. All activity measures were collected for each rat on a single test day.

An automated Digiscan-16 system was used to monitor activity. The system included four optical beam activity monitors (model RXYZCM-16; Omnitech Electronics, Columbus, OH) composed of 16 vertical and 16 horizontal infrared sensors. Each monitor surrounded an acrylic activity monitor cage (40 × 40 × 30.5 cm), which was completely enclosed by Plexiglas. Ventilation was provided by 0.5-cm airholes in the top panel. The monitors and cages were located in a sound-proofed room with a 40-dB white-noise generator operating continuously. A multiplexor-analyzer (model DCM-4, Omnitech Electronics) in an adjacent room monitored beam breaks from the optical beam activity monitors and tracked the simultaneous interruption of beams. The multiplexor-analyzer updated the animal's position in the acrylic cage every 10 ms using a 100% real-time conversion system. Computerized integration of the data obtained from the monitor recorded general activity using total distance (in cm) and other locomotor behaviors as the dependent measures. A selector switch on the multiplexor analyzer was set to print updated totals for each test cage at successive 5-min intervals for 1 h during the light phase. Room lights were on during the PVN infusions of GLP-1 but were off during locomotor testing.

Measures of feeding and drinking. After locomotor testing, each rat underwent a series of eight baseline ingestion trials. Beginning each day 2 h before dark onset, each rat was weighed, handled, and transferred to a clean home cage outfitted with a wire grid floor. A cardboard pad positioned under the grid floor was used to collect food spillage. Approximately 14 g of the pellet diet was placed on the grid floor of each cage, and each cage was provided with a water bottle. After 30-min access, the remaining food and spillage were removed from the cage and food intake was recorded to the nearest 0.1 g (corrected for spillage). Water intakes were recorded to the nearest 0.1 ml. The rats were then allowed free access to food and water until the feeding trial on the next day.

Before drug testing, the rats were assigned to one of three GLP-1 treatment conditions (0, 100, or 200 ng). The distribution of rats into groups for the ingestion test was counterbalanced so that equal numbers of rats were taken from each of the locomotion treatments (0, 100, and 200 ng GLP-1). On the test day, each rat received a single PVN infusion of either 0, 100, or 200 ng GLP-1 as described in the CTA experiment. Five minutes later, each rat was given access to food and water and intakes were recorded as in the baseline ingestion trials. The GLP-1 test day was followed by two baseline feeding trials in which no injections were given to minimize drug-carryover effects. On the final ingestion test day, a single PVN infusion of 25 nmol NE was administered to each rat to determine the probable locus of the guide cannula. The procedure for the PVN infusion of NE was identical to the GLP-1 infusion procedure. Prior studies have indicated that this dose of NE reliably increases feeding when infused into the PVN (25, 36). The NE response was used to verify that the PVN infusion site remained patent throughout the experimental procedures.

Histological analyses. At the conclusion of the experiment, each rat was overdosed with pentobarbital sodium (60 mg/kg ip) and perfused through the heart with 0.9% saline followed by 10% Formalin. Further fixation in 10% Formalin proceeded for at least 72 h before each brain was sectioned. Alternate 80-µm frozen sections were photographically enlarged (×7) and compared with the atlas plates from Paxinos and Watson (23) to verify cannula placements. PVN and non-PVN groups were formed based on the histological analyses.

Data analyses. For the CTA data, separate one-way analyses of variance (ANOVAs; SigmaStat) using the between-group factor of GLP-1 treatment (0, 100, or 200 ng or 200 ng noncontingent) were computed to compare baseline water intakes averaged across the last three baseline trials as well as saccharin intakes on the training day. A two-way ANOVA (SuperAnova) with a between-group factor of GLP-1 treatment (0, 100, or 200 ng or 200 ng noncontingent) and a within-group factor of extinction day (1- 4) was used to compare saccharin suppression ratio [saccharin intake/(saccharin intake  water intake)] calculated for each rat on extinction days 1- 4. The locomotion data were analyzed by a two-way ANOVA (SuperAnova) with a between-group factor of GLP-1 treatment (0, 100, or 200 ng) and a within-group factor of time (12 separate 5-min intervals, 5-60 min). The dependent variables were measures of locomotor activity, including total distance (cm), stereotypy time (s), and rest time (s) recorded for each rat. For the feeding and drinking tests, separate one-way ANOVAs (SigmaStat) using the between-group factor of GLP-1 treatment (0, 100, or 200 ng) were computed to compare baseline food and water intakes averaged across the last five baseline trials for PVN and non-PVN groups. Separate one-way ANOVAs (SigmaStat) using the between-group factor of GLP-1 treatment (0, 100, or 200 ng) were computed to compare food and water intakes on the GLP-1 test day for PVN and non-PVN groups. Each ANOVA was supplemented with a post hoc Student-Newman-Keuls test when appropriate. Differences are noted as statistically significant for probability values less than 0.05 for all analyses.

	RESULTS

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Histology and NE-feeding response. To be assigned to a PVN group, each rat had to fulfill an anatomic criterion based on cannula placement and a behavioral criterion based on the feeding response following PVN infusion of 25 nmol NE. Rats that exhibited cannula placements within the medial parvicellular and anterior parvicellular aspects of the PVN following blind histological inspection fulfilled the anatomic criterion. Fifty-two rats were determined to exhibit the proper anterior-posterior, medial-lateral, and dorsal-ventral coordinate placements within the PVN. Seven rats had cannula placements that were ventral and/or lateral to the PVN, and their data were not analyzed. Among rats that sustained accurate cannula placements within the PVN, only rats that exhibited a 0.5-g or greater increase in food intake after PVN infusion of 25 nmol NE relative to their mean baseline food intake (averaged across the last 5 baseline feeding trials for each rat) were assigned to the PVN group. Thirty-nine rats with proper PVN cannula placements met the feeding criterion and were therefore assigned to the PVN group. In the CTA experiment, data from five rats were discarded because the rats were not observed to drink the saccharin solution on the GLP-1 training day. Twenty rats did not fulfill the anatomic or behavioral criteria and were assigned to a non-PVN control (Con) group for comparison to the feeding and drinking responses exhibited by the PVN group.

PVN GLP-1 and CTA. Average group water intakes (mean ± SE) on the final 3 baseline drinking days were 1.9 ± 0.33, 2.3 ± 0.54, 1.8 ± 0.69, and 2.0 ± 0.46 ml for the 0-, 100-, and 200-ng and 200-ng noncontingent treatments, respectively. A one-way ANOVA using the between-group factor of GLP-1 treatment revealed that baseline water intakes were not significantly different [F(3,33) = 0.14, P = 0.93]. Average group saccharin intakes (mean ± SE) on the training day were 2.5 ± 0.46, 4.0 ± 0.75, 2.5 ± 0.99, and 1.8 ± 0.67 ml for the 0-, 100-, and 200-ng and 200-ng noncontingent treatments, respectively. A one-way ANOVA using the between-group factor of GLP-1 treatment revealed that saccharin intakes were not significantly different [F(3,33) = 1.29, P = 0.30]. Figure 1 depicts percent saccharin suppression ratios for each of the four GLP-1 treatment groups (0, 100, or 200 ng or 200 ng noncontingent) for the 4 extinction days. Rats receiving a PVN infusion of 0 ng GLP-1 [vehicle (Veh)] exhibited a mean saccharin suppression ratio of ~0.80 across the 4 extinction days. PVN infusion of GLP-1 (100 or 200 ng or 200 ng noncontingent) did not markedly alter mean saccharin suppression ratios. A two-way ANOVA using saccharin suppression ratio revealed no significant effect of GLP-1 treatment [F(3,30) = 1.26, P = 0.31] and no significant interaction between GLP-1 treatment and extinction day [F(9,90) = 1.00, P = 0.45].

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Mean saccharin suppression ratios (%) for each group of rats on extinction days 1- 4 after hypothalamic paraventricular nucleus (PVN) infusion of glucagon-like peptide-1-(736) amide (GLP-1) [0 (Veh), 100, or 200 ng or 200 ng noncontingent (NC)] on the training day. Vertical lines extending above and below each symbol represent SE.

PVN GLP-1 and Locomotion. Figure 2 depicts mean group cumulative total distances (cm; Fig. 2A), cumulative stereotypy times (s; Fig. 2B), and cumulative rest times (s; Fig. 2C) over a 60-min period after PVN infusion of the doses of GLP-1 (0, 100, or 200 ng). Rats receiving a PVN infusion of Veh exhibited an average cumulative total distance of 3,452 cm, a cumulative stereotypy time of 413 s, and a cumulative rest time of 3,264 s. PVN infusion of GLP-1 (100 or 200 ng) did not significantly alter cumulative total distance [F(2,35) = 0.04, P = 0.97], cumulative stereotypy time [F(2,35) = 0.72, P = 0.50], or cumulative rest time [F(2,35) = 0.26, P = 0.77]. Moreover, although there was a significant effect of time (data not presented) for each measure, there were no significant GLP-1 dose × time interactions for cumulative total distance [F(22,385) = 0.22, P = 1.00], cumulative stereotypy time [F(22,385) = 0.56, P = 0.95], or cumulative rest time [F(22,385) = 0.52, P = 0.97].

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Mean group cumulative distances (cm; top), stereotypy times (s; middle), and rest times (s; bottom) after PVN infusion of either 0, 100, or 200 ng GLP-1. Vertical lines extending above and below each symbol represent SE.

PVN GLP-1 and ingestive behavior. Average group food intakes (mean ± SE) on the final 5 baseline feeding days were 2.7 ± 0.19, 2.8 ± 0.19, and 2.4 ± 0.17 g for the 0-, 100-, and 200-ng GLP-1 treatments, respectively. A one-way ANOVA using the between-group factor of GLP-1 treatment revealed that baseline food intakes were not significantly different [F(2,38) = 1.44, P = 0.25]. Average group water intakes (mean ± SE) on the final 5 baseline drinking days were 3.6 ± 0.30, 3.3 ± 0.30, and 3.1 ± 0.27 ml for the 0-, 100-, and 200-ng GLP-1 treatments, respectively. A one-way ANOVA using the between-group factor of GLP-1 treatment revealed that baseline water intakes were not significantly different [F(2,38) = 0.82, P = 0.48].

Figure 3 depicts mean group food intakes (Fig. 3A) and water intakes (Fig. 3B) after PVN infusion of the doses of GLP-1 (0, 100, or 200 ng). Rats receiving a PVN infusion of Veh consumed 2.5 g of food during the 30-min ingestion test. PVN infusion of 100 or 200 ng GLP-1 resulted in decreases of food intake of 40 and 60%, respectively. For food intake, a one-way ANOVA revealed a significant effect of GLP-1 treatment [F(2,38) = 8.33, P < 0.001]. A post hoc Student-Newman-Keuls test revealed decreased food intake after PVN infusion of either 100 or 200 ng GLP-1, relative to PVN infusion of Veh. However, food intake was not significantly different between the 100- and 200-ng GLP-1 treatments. Rats receiving a PVN infusion of Veh consumed 2.6 ml of water during the 30-min ingestion test. PVN infusion of 100 and 200 ng GLP-1 resulted in decreases of water intake of 65 and 54%, respectively. For water intake, a one-way ANOVA revealed a significant effect of GLP-1 treatment [F(2,38) = 8.87, P < 0.0008]. A post hoc Student-Newman-Keuls test revealed decreased water intake following PVN infusion of either 100 or 200 ng GLP-1, relative to PVN infusion of Veh. However, water intake was not significantly different between the 100- and 200-ng GLP-1 treatments.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Mean group food intakes (A) and water intakes (B) after PVN infusion of either 0, 100, or 200 ng GLP-1. Vertical lines extending above each bar represent SE. Numbers in parentheses indicate no. of animals. * Significant drug-treatment differences (P < 0.05).

Feeding and drinking data were analyzed for rats exhibiting cannula placements ventral and/or lateral to the PVN (group anatomic Con). For the anatomic Con group, average group food intakes (mean ± SE) on the final 5 baseline feeding days were 2.5 ± 0.26, 2.3 ± 0.20, and 2.3 ± 0.28 g for the 0-, 100-, and 200-ng GLP-1 treatments, respectively. A one-way ANOVA using the between-group factor of GLP-1 treatment revealed that baseline food intakes were not significantly different [F(2,19) = 0.19, P = 0.83]. For the anatomic Con group, average group water intakes (mean ± SE) on the final 5 baseline drinking days were 3.9 ± 0.35, 3.0 ± 0.42, and 3.2 ± 0.44 ml for the 0-, 100-, and 200-ng GLP-1 treatments, respectively. A one-way ANOVA using the between-group factor of GLP-1 treatment revealed that baseline water intakes were not significantly different [F(2,19) = 0.97, P = 0.40].

Figure 4 depicts mean group food intakes (Fig. 4A) and water intakes (Fig. 4B) following infusion of the doses of GLP-1 (0, 100, or 200 ng) for the anatomic Con group. Anatomic Con rats receiving an infusion of Veh consumed 1.6 g of food during the 30-min ingestion test. Anatomic Con rats receiving an infusion of GLP-1 (100 or 200 ng) exhibited food intakes similar to food intake following Veh treatment. For food intake, a one-way ANOVA revealed no significant effect of GLP-1 treatment in anatomic Con rats [F(2,19) = 0.15, P = 0.86]. Anatomic Con rats that received an infusion of Veh consumed 1.9 ml of water during the 30-min ingestion test. Anatomic Con rats receiving an infusion of GLP-1 (100 or 200 ng) exhibited water intakes similar to water intake following Veh treatment. For water intake, a one-way ANOVA revealed no significant effect of GLP-1 treatment in anatomic Con rats [F(2,19) = 1.94, P = 0.17].

View larger version (10K): [in this window] [in a new window]   Fig. 4.   Mean group food intakes (A) and water intakes (B) after infusion of either 0, 100, or 200 ng GLP-1 into brain sites ventral and/or lateral to the PVN (group anatomic control). Vertical lines extending above each bar represent SE. Numbers in parentheses indicate no. of animals.

	DISCUSSION

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Intraventricular administration of GLP-1 reduces feeding in rats (20, 21, 29, 30, 32), an effect that may reflect the induction of malaise. The malaise view is supported by reports in which intracerebroventricular GLP-1 infusion induces c-fos expression within the area postrema, nucleus of the solitary tract, and lateral parabrachial nucleus (33), a pattern that has been characterized as similar to that induced by the prototypical CTA-inducing agent LiCl (34). The demonstration that intracerebroventricular infusion of GLP-1 can induce CTA at doses that reduce food intake (30; but see Ref. 29 for an exception) suggests that malaise may contribute in part to the anorexic effect of intracerebroventricular GLP-1.

In an earlier experiment (19), we suggested that ventricular administration of GLP-1 could act within the PVN to suppress feeding. This notion was supported by localization of GLP-1 within the PVN (11, 16, 26), by activation of c-fos within the PVN (32, 33), and by our finding that PVN infusion of GLP-1 reduces consumption of a palatable liquid diet in rats (19). Indirect support came from the observation that lower dose levels were required to induce anorexia for intra-PVN administration of GLP-1 relative to intracerebroventricular administration of GLP-1 (19, 21, 32). The intent of the present experiment was to evaluate whether PVN infusion of GLP-1 induces CTA at doses that suppress food intake. Our present findings indicate that intra-PVN administration of 100 or 200 ng GLP-1 significantly reduces feeding without the concomitant induction of significant CTA. These results were obtained using one protocol for assessment of CTA. It is possible that these findings are specific for this CTA paradigm.

Alternative strategies can be brought to bear on the issue of whether GLP-1 induces malaise and in turn reduces feeding. No study to date has examined the effects of intra-PVN administration of GLP-1 on malaise as assessed by the intraoral taste paradigm (30) or the plasma oxytocin paradigm (35). Another approach would be to evaluate whether GLP-1 induces the behavioral satiety sequence in the sham-feeding paradigm or in freely feeding rats (1, 12). Intracerebroventricular infusion of GLP-1 is sufficient to inhibit sham-feeding in rats (3). Moreover, intra-PVN administration of GLP-1 at 100 and 200 ng reduces feeding without disrupting the behavioral satiety sequence (18). These findings suggest that GLP-1 may serve as a satiety signal in the absence of gastric and postgastric feedback. A final approach would be to evaluate whether pharmacological inactivation of the GLP-1 receptor increases feeding. Increased feeding would not be expected were GLP-1 to suppress feeding via the induction of malaise. The malaise view is not supported by the observation of increased feeding following intracerebroventricular infusion of the GLP-1 receptor antagonist exendin-(939) at light onset in fasted rats (32). Furthermore, the finding that PVN infusion of exendin-(939) stimulates feeding (31) provides strong support for the notion that this peptide does not reduce feeding via the induction of malaise.

The specificity of the behavioral effects of GLP-1 was examined in the present study. Measures of locomotion were recorded after intra-PVN administration of GLP-1 to assess whether GLP-1 disrupts locomotion, an effect that in turn could reduce feeding. The present experiments demonstrated that PVN infusion of GLP-1 (100 and 200 ng), at doses that suppressed feeding, did not significantly alter locomotion.

The present experiments also extend our earlier findings that PVN infusion of GLP-1 (100 and 200 ng) significantly reduces feeding (19) to include an inhibitory action of this peptide on drinking. The antidipsogenic action of GLP-1 may be a prandial effect (i.e., reduced feeding leads to reduced food-associated drinking) or may reflect an independent action of this peptide on neurons that control water intake. A prandial interpretation is consistent with the demonstration that intracerebroventricular administration of the GLP-1 antagonist exendin-(939) significantly increased food intake but did not increase water intake (21). In support of the latter possibility are studies that document innervation of drinking-related brain sites by GLP-1-positive neurons and studies suggesting that intracerebroventricular administration of GLP-1 reduces water intake in rats made thirsty by water (but not food) deprivation or by intracerebroventricular administration of angiotensin (29).

Perspectives

The PVN, however, may comprise only a portion of a central network of GLP-1 neurons. The present studies suggest that GLP-1 neurons within the PVN may play a role in the modulation of feeding. Whether that role is exclusive to the PVN awaits experiments in which 1) ablation of the PVN is shown to obviate the anorexic action of intracerebroventricular GLP-1 and 2) in which intra-PVN administration of a GLP-1 receptor antagonist minimizes the anorexic action of intracerebroventricular GLP-1. The functional significance of GLP-1 neurons in cortex and brain stem remains unknown. Fourth ventricular infusions of GLP-1 reduce feeding (20), an effect presumably mediated by brain stem GLP-1 receptors within structures such as the nucleus of the solitary tract and area postrema (11). The contribution of these sites to GLP-1 anorexia awaits studies in which GLP-1 is infused into discrete brain stem sites before measures of feeding, drinking, and CTA.