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APPETITE, OBESITY, DIGESTION, AND METABOLISM

Attenuation of lipopolysaccharide anorexia by antagonism of caudal brain stem but not forebrain GLP-1-R

Graduate Groups of Psychology and Neuroscience, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Submitted 15 March 2004 ; accepted in final form 22 June 2004

	ABSTRACT

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The central glucagon-like peptide-1 (GLP-1) system has been implicated in the control of feeding behavior. Here we explore GLP-1 mediation of the anorexic response to administration of systemic LPS and address the relative importance of caudal brain stem and forebrain GLP-1 receptor (GLP-1-R) for the mediation of the response. Fourth-intracerebroventricular delivery of the GLP-1-R antagonist exendin-(9–39) (10 µg) did not itself affect food intake in the 24 h after injection but significantly attenuated the otherwise robust (60%) reduction in food intake obtained after LPS (100 µg/kg) treatment. This result highlights a role for caudal brain stem GLP-1-R in the mediation of LPS anorexia but does not rule out the possibility that forebrain receptors also contribute to the response. Forebrain contribution was addressed by delivery of the GLP-1-R antagonist to the third ventricle with the caudal flow of cerebrospinal fluid blocked by occlusion of the cerebral aqueduct. Exendin-(9–39) delivery thus limited to forebrain did not attenuate the anorexic response to LPS. These data suggest that LPS anorexia is mediated, in part, by release of the native peptide acting on GLP-1-R within the caudal brain stem.

feeding behavior; cachexia; neural systems; nucleus of the solitary tract; glucagon-like peptide-1 receptor

A number of findings taken together suggest glucagon-like peptide-1 (GLP-1) as another potentially important link in the downstream mediation of LPS anorexia. Central GLP-1 treatment itself induces a dose-related inhibition of feeding (14, 20, 23). In addition, stimulation of endogenous GLP-1 activity has been implicated in the mediation of the anorexic effects of LiCl and oxytocin (14, 16, 19). In each case, doses that reduce food intake increase c-Fos expression in GLP-1 neurons, and importantly, the anorexic effects are attenuated or eliminated with central injection of a GLP-1-receptor (GLP-1-R) antagonist (14, 16, 19). c-Fos expression in GLP-1 neurons is also enhanced by systemic LPS treatment (15), although pharmacological evidence supporting a critical contribution of GLP-1 to LPS anorexia has not yet been provided. Here, the GLP-1-mediation hypothesis is explored by intracerebroventricular delivery of exendin-(9–39), an antagonist of the GLP-1-R, alone and in combination with systemic LPS injection.

GLP-1 neurons are situated in the medulla, primarily in the caudal nucleus of the solitary tract (8, 11). They project both within the caudal brain stem and to forebrain substrates. Hypothalamic receptors have received the most attention with respect to feeding actions of GLP-1-R stimulation (e.g., Ref. 10) although anorexic responses have been observed after caudal brain stem as well as forebrain delivery of the peptide (6, 19, 20). A GLP-1 contribution to LPS anorexia, then, could be mediated through either or both forebrain or brain stem receptor targets. In experiment 1, the GLP-1-R antagonist exendin-(9–39) is delivered to the fourth ventricle alone or before LPS treatment. An attenuation of LPS anorexia would be consistent with the GLP-1-R mediation hypothesis and indicate that the peptide's contribution is transmitted, at least in part, via receptor populations in the caudal brain stem. The possible contribution of specifically forebrain GLP-1-R is evaluated in experiment 2, where the antagonist is delivered through third-intracerebroventricular cannula in rats with the caudal flow of cerebrospinal fluid (CSF) blocked by occlusion of the cerebral aqueduct (17).

	MATERIALS AND METHODS

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Subjects

Male Sprague-Dawley rats (Charles River) weighing 350–450 kg at the time of surgery were housed individually in hanging stainless cages under a 12:12-h light-dark cycle. Pelleted food (Purina 5001) and water were available ad libitum.

Surgery

Rats were anesthetized with ketamine (90 mg/kg), xylazine (2.7 mg/kg), and acepromazine (0.64 mg/kg) delivered intramuscularly. Rats in experiment 1 received a fourth-intracerebroventricular guide cannula (Plastics One, 22-G) with its tip positioned 2.0 mm above the fourth cerebral ventricle (coordinates: on the midline, 2.5 mm anterior to the occipital suture, and 4.5 mm ventral to the dura). Rats in experiment 2 received two cannulas: one as a guide for third-intracerebroventricular injections with its tip positioned 2.0 mm above the ventricle (coordinates: on the midline, 2.0 mm posterior to bregma and 7.5 mm ventral to dura) and the other (Plastics One, 19-G) for placement of the aqueduct plug (coordinates: 7.0 mm posterior to bregma, 1.0 mm lateral to the midline with an 11° angle toward midline, and 5.0 mm ventral to dura). Cannulas were cemented in place with dental acrylic and jeweler's screws attached to the skull and closed with an obturator.

Procedure

Cannula verification. At least 7 days after surgery, intracerebroventricular cannula placement was assessed by measurement of the sympathoadrenal mediated glycemic response to 5-thio-D-glucose [210 µg in 2 µl of artificial cerebral spinal fluid (aCSF)] (3). The criterion for subject inclusion in the experiments was at least a doubling of plasma glucose level 1 h after injection.

Habituation training. On several occasions rats were handled before lights out, and on one occasion they received intracerebroventricular and intraperitoneal vehicle injections to adapt them to these procedures.

Experiment 1: Effect of Fourth-Intracerebroventricular Exendin-(9–39) on LPS Anorexia

Rats received fourth-intracerebroventricular injections 30 min before lights out, followed immediately by intraperitoneal injection. Three conditions were run across separate test days with at least 3 days between tests. All rats (n = 16) were tested under two control conditions: intracerebroventricular vehicle (2 µl aCSF) + intraperitoneal vehicle [isotonic saline (1.0 ml/kg)], and exendin-(9–39) (10 µg; California Peptide Research, Napa, CA) + intraperitoneal vehicle. These two conditions were presented in counterbalanced order across subjects. For one group of rats (n = 8), the last condition was a fourth-intracerebroventricular injection of exendin-(9–39), followed immediately by an intraperitoneal injection of LPS [Sigma-Aldrich, St. Louis, MO; 100 µg/kg body wt in saline vehicle (1.0 ml/kg)]. The last condition for a second group (n = 8) was a fourth-intracerebroventricular vehicle injection followed by LPS. Groups were matched for body weight. Tolerance to the ingestive effects of LPS (7) precluded a within-subjects comparison of these two injection conditions. The LPS dose selected is commonly tested (e.g., Refs. 7, 15), which, in our experience, yields 60% suppression of 24-h intake. For each condition, cumulative food intake was measured 2, 4, 6, 8, and 24 h after injection.

Experiment 2: LPS Anorexia with Third-Intracerebroventricular Exendin-(9–39) and Aqueduct Occlusion

The conditions tested were identical to those of experiment 1, except for the intracerebroventricular placement and a variation in testing order. All rats (n = 19) received intracerebroventricular vehicle + intraperitoneal vehicle and intracerebroventricular exendin-(9–39) + intraperitoneal vehicle conditions, with condition order counterbalanced across rats. On the third test day the aqueduct was occluded by injection of silicon grease (5 µl). Separate groups of rats received intracerebroventricular vehicle + intraperitoneal LPS (n = 10) or intracerebroventricular exendin-(9–39) + intraperitoneal LPS (n = 9). Groups were matched for body weight. After the 24-h intake measure, the effectiveness of the aqueduct plug was verified by the absence of glycemic response to third-intracerebroventricular injection of 5-thio-D-glucose. Three subjects failed to meet this criterion and were excluded from the analysis.

Data Analysis

For each experiment, separate two-way ANOVAs (time x condition) were run for two-condition pairs as follows. The effect of exendin-(9–39) itself on cumulative intake was evaluated by within-subjects comparison of vehicle + vehicle and exendin + vehicle conditions. LPS anorexia was evaluated by within-subjects comparison of vehicle + vehicle and vehicle + LPS conditions. The ability of the antagonist to attenuate the effect of LPS was evaluated by between-subject comparison of vehicle + LPS and exendin + LPS conditions. In addition, within-subjects differences between vehicle + vehicle and exendin + LPS conditions were also evaluated.

	RESULTS

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Experiment 1

Figure 1 shows the overall effects of treatment condition on cumulative intake during the 24 h after injections. The intake suppression with LPS relative to the vehicle condition was pronounced overall [F(1,7) = 19.02, P < 0.004] and grew in magnitude over time [interaction: F(4,28) = 7.93, P < 0.0003], amounting to 45% of the baseline value 24 h after treatment. Comparison between the LPS and the LPS + exendin conditions revealed a significant antagonism of the anorexic response [F(1,14) = 5.09, P < 0.05], which was most pronounced at the later time points [interaction: F(4,56) = 5.28, P < 0.002]. The reversal of LPS anorexia, however, was not complete; overall, the difference in cumulative intakes between vehicle and LPS + exendin condition was not significant [F(1,7) = 3.93, not significant (NS)], but the difference between conditions was evident at the later time points [interaction: F(4,28) = 7.96, P < 0.0003]. Importantly, the antagonist itself had no effect on cumulative intake [condition: F(1,15) = 0.0, NS; interaction: F(4,60) = 0.81, NS].

View larger version (18K): [in this window] [in a new window]   Fig. 1. Cumulative intake values for the 4 conditions of experiment 1. Rats received an intraperitoneal [saline (Sal)] and a fourth-intracerebroventricular injection [artificial cerebrospinal fluid (aCSF) or exendin-(9–39)] in counterbalanced order before 1 of 2 LPS conditions [fourth-intracerebroventricular saline or exendin-(3–39) and intraperitoneal LPS].

The results of experiment 2 (Fig. 2) show that third-intracerebroventricular administration of exendin-(9–39), in contrast with the first experiment, failed to attenuate LPS anorexia [vehicle + LPS vs. exendin + LPS: F(1,14) = 0.31, NS]. In every other respect (degree of LPS anorexia, lack of effect of antagonist given alone), the results were comparable to those of experiment 1.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Cumulative intake values for the 4 conditions of experiment 2. Rats received an intraperitoneal (saline) and a third-intracerebroventricular injection [aCSF or exendin-(9–39)] in counterbalanced order before the occlusion of the cerebral aqueduct. After occlusion, rats received 1 of the 2 LPS conditions [third-intracerebroventricular saline or exendin-(3–39) and intraperitoneal LPS].

	DISCUSSION

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The present study highlights, for the ingestive response to LPS, the significance of GLP-1 projections intrinsic to the caudal brain stem and discounts a role for GLP-1 projections to receptor populations in the forebrain. The role of the brain stem is indicated by the effectiveness of the fourth-intracerebroventricular route of antagonist administration. Antagonist delivered and limited to forebrain, by contrast, yielded no attenuation of LPS anorexia. With unrestricted flow of CSF, we would predict that third-intracerebroventricular antagonist administration would indeed attenuate the anorexic response but infer on the basis of the present results that the reversal would reflect action on GLP-1-R in the caudal brain stem. It is important to note, first, that the negative results concerning basal forebrain contributions were based on third-intracerebroventricular delivery of antagonist, which may not have effectively accessed tissue at appreciable distances from the ventricle. Thus, although the present results minimize roles for the paraventricular and arcuate hypothalamic nuclei and other sites proximal to the ventricle, further work in which antagonist is delivered to yet other sites, such as the dorsomedial hypothalamus and central nucleus of the amygdala, should be pursued.

Our results indicating a minimal contribution of forebrain GLP-1-Rs should not be readily applied to discussions of GLP-1 contributions to feeding control more generally or to the GLP-1-R mediation of the anorexic effects of other treatments. GLP-1-Rs are expressed in hypothalamic neurons of demonstrated importance for energy homeostasis. Direct action of GLP-1 on POMC neurons in arcuate nucleus and on oxytocin neurons (24) and CRH neurons in paraventricular hypothalamus (18), for example, may modulate neuroendocrine, autonomic, and/or feeding responses influenced by activity at these sites. We had noted that intake suppression was observed when GLP-1 itself was delivered at low doses to the hypothalamic paraventricular nucleus (10). Interestingly, a recent study focusing on GLP-1 mediation of LiCl anorexia yielded conclusions precisely opposing those clearly drawn from our analysis of intake suppression after LPS treatment. It had already been shown (14) that GLP-1-R antagonist delivered to the lateral ventricle attenuates the intake suppressive effects of systemic LiCl administration. Given the caudal flow of CSF, those results did not address brain stem vs. forebrain receptor mediation. Billes and colleagues (1), however, went further to show that fourth-intracerebroventricular antagonist administration did not influence the anorexic response, indicating a forebrain GLP-1-R mediation of LiCl anorexia. It appears the GLP-1 system plays multiple roles in the control of feeding behavior. A better understanding of the system will require identification of the relevant circuits in both forebrain and caudal brain stem and an appreciation of their relative contributions under different physiological conditions.

	GRANTS

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The work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-21397 and DK-42284.

	FOOTNOTES

 

Address for reprint requests and other correspondence: H. J. Grill, Graduate Groups of Psychology and Neuroscience, Univ. of Pennsylvania, 3720 Walnut St., Philadelphia, PA 19104 (E-mail: grill{at}psych.upenn.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 287/5/R1190    most recent 00163.2004v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (16) Citing Articles via Google Scholar Google Scholar Articles by Grill, H. J. Articles by Kaplan, J. M. Search for Related Content PubMed PubMed Citation Articles by Grill, H. J. Articles by Kaplan, J. M.