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Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom
Address all correspondence and requests for reprints to: Dr. C. B. Lawrence, Faculty of Life Sciences, The University of Manchester, Michael Smith Building, Manchester M13 9PT, United Kingdom. E-mail: Catherine.lawrence{at}manchester.ac.uk.
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and IL-1β protein in macrophages and/or microglia in selected brain areas, including the meninges, and periventricular brain regions. Intracerebroventricular injection of GALP in rats stimulated food intake over 1 h but decreased feeding and body weight at 24 h and caused a rise in core body temperature over 8 h. Coinfusion of the IL-1 receptor antagonist had no effect on the GALP-induced orexigenic response but significantly reduced the longer-term actions of GALP observed at 24 h and its effect on body temperature. Furthermore, the actions of GALP on feeding, body weight, and body temperature were significantly reduced in IL-1/β-, IL-1β-, or IL-1 type I receptor (IL-1RI)-deficient mice. These data suggest that GALP induces expression of IL-1 in the brain, and its anorexic and febrile actions are mediated by this cytokine acting via IL-1 type I receptor. |
Introduction |
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Recent data suggest that GALP promotes feeding in the rat via activation of known orexigenic neurons that contain orexin and neuropeptide Y (NPY) (11, 12), but the mediators of the anorexic effect are unknown. Central administration of GALP in rats or mice induces a specific pattern of Fos activation in the brain (10, 13, 14), with robust c-Fos protein expression observed in the ependymal cells of the ventricles and periventricular regions, a response not seen with galanin. The cells that are activated in the periventricular hypothalamic region are astrocytes, rather than neurons or microglia (13). Activated astrocytes are involved in inflammatory responses to infection and injury in the brain, and release key mediators of inflammation, such as cytokines (15). Analogous to actions of GALP, infection and inflammation are associated with negative energy balance, characterized by weight loss, reduced food intake, and fever (rise in core body temperature). The prostaglandin-dependent rise in temperature and anorexia induced by GALP are similar in intensity and pattern to those produced by the cytokine IL-1 (16). IL-1 is a classical mediator of inflammation that is found in two forms, IL-1 and IL-1β, and acts via the IL-1 type I receptor (IL-1RI), although recent data also suggest that IL-1 may also act through a novel receptor (17, 18). The similarities between the cytokine-mediated response to infection and the actions of GALP on energy balance raise the possibility that GALP may be inducing anorexia, body weight loss, and fever nonphysiologically.
The overall aim of the present study, therefore, was to test the hypothesis that IL-1 mediates the actions of GALP on energy balance in the rodent. First, the effect of GALP on IL-1 and IL-1β expression in the brain of the rat was investigated. After this, the effect of inhibiting endogenous IL-1 was assessed in the rat by coadministering GALP with the IL-1 receptor antagonist (IL-1RA). Finally, the IL-1 ligand and receptor responsible for the actions of GALP on energy balance were assessed by testing the effects of GALP in transgenic mice deficient in IL-1/β, IL-1β, or IL-1RI.
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Materials and Methods |
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/β- and IL-1β-deficient male mice (IL-1/β–/– and IL-1β–/–) were kindly provided by Dr. Yoichiro Iwakura, and had been backcrossed for eight generations on a C57BL/6 background. IL-1RI-deficient male mice (IL-1RI–/–) were obtained from Charles River, and had previously undergone nine backcrosses on a C57BL/6 background by Professor Martin Nicklin (University of Sheffield, Sheffield, UK). Toll-like receptor 4-deficient mice (TLR4–/–) were also on a C57BL/6 background and were provided by Dr. Richard Grencis (University of Manchester, Manchester, UK). IL-1/β–/–, IL-1β–/–, IL-1RI–/–, and TLR4–/– mice and wild-type control (C57BL/6) mice were kept and bred at the Faculty of Life Sciences animal facility (University of Manchester) and used at a weight range of 26–32 g. All animals were housed at a constant ambient temperature of 21 ± 2 C on a 12-h light, 12-h dark cycle (lights on 0800 h). Rodent chow (Beekay International, Hull, UK) and tap water were provided ad libitum. All procedures conformed to the requirements of the United Kingdom Animals (Scientific Procedures) Act, 1986.
Materials
Rat GALP (Bachem, Merseyside, UK) and rat galanin (Bachem) were used at an equimolar concentration of 1.5 nmol (in 1 µl). Human IL-1RA (R&D Systems, Abingdon, UK) was administered in three separate injections at a concentration of 200 µg/injection (in 2 µl). Lipopolysaccharide (LPS; 0127:B8; Sigma-Aldrich Co. Ltd., Poole, UK) was administered peripherally at 100 µg/kg or 100 ng icv. All compounds were dissolved in sterile saline and stored at –20 C until required.
Intracerebroventricular cannulation and injections
Rats or mice were anesthetized with isoflurane (1.5–3% in O2) and stereotaxically implanted with guide cannula into the lateral cerebral ventricle. Injection coordinates relative to bregma for rats were posterior 0.8 mm, lateral 1.5 mm, and ventral 4 mm, according to the atlas of Paxinos and Watson (19), and for mice were posterior 0.2 mm, lateral 1 mm, and ventral 2.5 mm, according to the atlas of Franklin and Watson (20). In experiments involving measurements of core body temperature, radiotransmitters (TA10TA-F40 for rats and TA10TA-F20 for mice; Data Sciences, Minneapolis, MN) were implanted abdominally into the peritoneum at the same time as cannulation. After surgery, mice were housed individually and allowed to recover for at least 7 d. Rats were also left to recover for the same period and housed individually 24–48 h before experimental injections. Animals were assigned randomly to treatment groups, and each animal was used only once. All icv injections were performed in conscious unrestrained rats or lightly restrained mice commencing 2 h after lights on (1000 h) and were given in a total volume of 1–3 µl for rats or 1 µl for mice. After injections, animals were given a preweighed amount of food that was reweighed at several time points later.
ELISA
Rat plasma IL-6 concentrations were analyzed by ELISA (Duoset; R&D Systems), according to the manufacturer’s instructions.
Experiment 1: effect of GALP on IL-1 or IL-1β expression
Rats (n = 3 per group) were given a single icv injection of rat GALP (1.5 nmol in 1 µl), rat galanin (1.5 nmol in 1 µl), or vehicle (1 µl sterile saline). After injections, animals were given a preweighed amount of food that was reweighed 2 h later. Animals were then culled at 2, 4, 8, or 24 h after injection with an overdose of sodium pentobarbitone (90 mg/kg ip; Sagatal; Rhône-Mérieux, Harlow, UK) and transcardially perfused with heparinized saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB). Plasma was obtained from anti-coagulated (sodium citrate) blood samples taken by cardiac puncture, immediately before perfusions.
After transcardial perfusion, brains were removed, postfixed in the same fixative overnight, immersed in 30% sucrose until the tissue sank, and then frozen. Coronal brain sections were cut at 30 µm on a freezing sledge microtome and stored in cryoprotectant at –20 C until required.
Immunohistochemistry for IL-1 or IL-1β was performed on free-floating sections at room temperature, unless otherwise stated. To remove endogenous peroxidase activity, sections were treated with 1.5% H2O2 (in 20% methanol, 0.2% Triton X-100 in 0.1 M PB). After preincubation in 2% normal horse serum (in 0.1% saponin) or 10% normal rabbit serum (in 0.3% Triton) for 1 h for IL-1 or IL-1β, respectively, the sections were incubated at 4 C in either goat antirat IL-1 (1 µg/ml in 2% normal horse serum/0.1% saponin; R&D systems) or sheep antirat IL-1β antibody diluted 1:1000 (in 2% normal rabbit serum/0.3% Triton; National Institute for Biological Standards and Control, Potters Bar, UK). After 24 h, sections were rinsed in PB/0.1% saponin (for IL-1), or PB/0.3% Triton (for IL-1β) and incubated in the appropriate secondary biotinylated antibody (antigoat IgG 1:500 in 2% normal horse serum/0.1% saponin for IL-1, or antisheep IgG 1:500 in 2% normal rabbit serum/0.3% Triton for IL-1β; Vector Laboratories Ltd., Peterborough, UK) for 2 h. All sections were then washed in 0.1 M PB, immersed in avidin-biotin-peroxidase complex (ABC; Vector) for 30 min, rinsed in 0.1 M PB, and color developed using a 0.05% diaminobenzidine solution (in 0.01% H2O2). Sections were mounted onto gelatin-coated slides and dried, and coverslips were applied before viewing under a light microscope.
In a separate experiment, rats (n = 2–3 per group) were given an icv injection of LPS (100 ng in 1 µl) or vehicle (1 µl saline). Food intake and body weight were assessed 24 h later, or animals were culled at 2, 8, or 24 h, and immunohistochemistry for IL-1 or IL-1β was performed as above.
Experiment 2: phenotype of IL-1- and IL-1β-expressing cells
For dual-fluorescence immunohistochemistry, rat brain sections (taken from experiment 1) were treated with 2% normal horse serum (for IL-1) or 10% normal donkey serum (for IL-1β) before incubation for 24 h at 4 C in the goat antirat IL-1 antibody (1 µg/ml; in 2% normal horse serum/0.1% saponin) or the sheep antirat IL-1β antibody (1:1000; in 2% normal rabbit serum/0.3% Triton), along with either a mouse monoclonal antibody against glial fibrillary acidic protein (1:64,000; Sigma-Aldrich) to detect astrocytes, rabbit anti-Iba1 (1:5000; Wako Pure Chemicals Ltd., Osaka, Japan) for microglia and macrophages, or mouse monoclonal anti-NeuN (1:1000; Chemicon International, Millipore, Watford, UK) for neurons. After washes in 0.1 M PB/0.1% saponin (for IL-1) or 0.1 M PB/0.3% Triton (for IL-1β), sections were incubated in an antigoat Alexa 594 (for IL-1; Molecular Probes; Invitrogen, Paisley, UK) or antisheep Alexa 594 secondary antibody (for IL-1β; Molecular Probes), along with the appropriate Alexa 488 IgG antibody (antirabbit or antimouse; Molecular Probes). After additional washes (in 0.1 M PB), sections were mounted and coverslipped with ProLong Gold mounting medium (Invitrogen) before examination under an Olympus wide-field fluorescence microscope.
Experiment 3: effect of IL-1RA on GALP-induced changes in food intake, body weight, and core body temperature in rats
Rats (n = 8–13 per group) were given an icv injection of either vehicle (1 µl saline) or GALP (1.5 nmol in 1 µl). IL-1RA (200 µg in 2 µl) or vehicle (2 µl saline) was administered icv at –1, 0, and 1 h relative to GALP injections. Food consumption was measured at 1 and 24 h and body weight at 24 h after injections. Core body temperature was monitored continuously for 24 h by remote radiotelemetry.
Experiments 4–6: effect of GALP on food intake, body weight, and core body temperature in IL-1/β–/–, IL-1β–/–, and IL-1RI–/– mice
In separate experiments, IL-1/β–/–, IL-1β–/–, or IL-1RI–/– mice and their respective wild-type C57BL/6 controls (n = 5/group) were injected icv with either vehicle (1 µl saline) or GALP (1.5 nmol in 1 µl). Because GALP does not stimulate food intake at 1 h after injection in mice (7, 10), food consumption was measured only at 24 h. Body weight was also assessed 24 h after injections, and core body temperature was monitored continuously for 24 h by remote radiotelemetry.
Experiments 7 and 8: effect of GALP or LPS on food intake, body weight, and core body temperature in TLR4–/– mice
The effects of GALP on the IL-1 system are similar to those seen after injection of LPS, the bacterial endotoxin that mediates its actions via the receptor TLR4. Thus, to test whether the actions of GALP on energy homeostasis were due to endotoxin sample contamination, its effects were tested in TLR4–/– mice. The icv injections of GALP (1.5 nmol) or vehicle (1 µl saline) were administered to TLR4–/– or wild-type mice (n = 4–5 per group). To confirm the lack of responsiveness of TLR4–/– to LPS, a separate experiment was performed whereby TLR4–/– or wild-type mice were given an ip injection of LPS (100 µg/kg) or vehicle (5 ml/kg saline; n = 4–5 per group). For both experiments, food intake and body weight were assessed at 24 h, and core body temperature was monitored continuously for 24 h by remote radiotelemetry.
Statistical analyses
All data are presented as mean ± SEM. Body temperatures are plotted as the mean change from the point of injection (time zero) over 10 h. The integrated temperature response between 0 and 10 h for rats [area under the curve (AUC)] was calculated for each animal by the trapezoidal method. Average AUC values were then determined for each treatment group. For mice, AUC was calculated between 2 and 10 h to avoid the conflicting hypothermia that was observed between 0 and 2 h after GALP treatment. For both rats and mice, only data up to 10 h was analyzed, because GALP-induced changes in temperature are no longer apparent after this time (6, 10). In experiments involving LPS, the dose administered did not cause hypothermia in control wild-type mice but an increase in core body temperature that lasted approximately 8 h. Thus, in experiments using LPS, core body temperature was analyzed by calculating AUC from 0–8 h.
Statistical comparisons for food intake, body weight, and AUC were performed using a one-way ANOVA followed by a Tukey’s multiple comparisons test for the experiment using IL-1RA in rats, or for experiments employing transgenic mice, a parametric two-way ANOVA followed by Scheffé multiple comparisons test was used. For data involving transgenic mice, statistical significances are plotted on figures for GALP treatment vs. their respective controls only. Statistical significance was taken when P < 0.05.
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and IL-1β expression
No cells expressing IL-1 or IL-1β were found in the brain after icv injection of vehicle (Fig. 1
, A, E, I, and M) or galanin (1.5 nmol; data not shown) at any time point studied or in any area of the brain. However, IL-1 and IL-1β immunoreactive cells were observed in the brain after icv injection of GALP (Fig. 1). At 2, 4 (data not shown), and 8 h after GALP treatment, IL-1- and IL-1β-positive cells were found within the ependymal cells lining the ventricles and in the brain parenchyma surrounding the ventricles (the lateral, dorsal third, third, and fourth ventricles). The staining in the parenchyma appeared to be almost limited exclusively to the areas surrounding the ventricles and was strongest in the peri-third hypothalamic region (Fig. 1, F, G, N, and O). IL-1β-positive cells were also seen within the meninges (Fig. 1, J and K) and the choroid plexus (data not shown). The cells expressing IL-1β in the meninges were predominantly in the ventral region of the brain. However, in contrast to IL-1β, only a few cells expressing IL-1 were identified in the meninges (Fig. 1C).
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-positive cells in the parenchyma and ependymal lining appeared to be maximal at 2 h compared with 8 h. In contrast, IL-1β-expressing cells in the parenchyma and ependymal cells increased from 2–8 h after GALP injection, although the number of cells expressing IL-1β in the meninges appeared to be maximal at 2 h. There were no apparent cells immunoreactive for either IL-1 or IL-1β at 24 h after injection of GALP in any brain region apart from a few scattered cells in the ependymal lining of the ventricles (Fig. 1
, H and P).
Morphologically, the IL-1β-positive cells in the meninges and the choroid plexus were spherical and are likely to represent macrophages. In contrast, IL-1- and IL-1β-expressing cells within the brain parenchyma and the ependymal lining had a distinct morphology that resembled microglia.
IL-1- and IL-1β-expressing cells were also observed in the brains of mice treated with 1.5 nmol GALP (data not shown). The effect of GALP on IL-1 and IL-1β protein was studied only at 2 h after injection in mice but was found to be expressed in an identical pattern to that observed in rats at the equivalent time point. In particular, strong expression was seen in the ependymal cells of the ventricles and the periventricular regions.
To assess whether icv injection of GALP caused a general inflammatory response in the periphery, plasma IL-6 concentration was analyzed as a marker of systemic inflammation in the rat. There was no change in plasma IL-6 concentration at all time points (2, 4, 8, and 24 h) analyzed, and in all samples, IL-6 concentrations were below the limit of detection (data not shown).
To compare the effect of GALP on IL-1 expression in the brain, a separate group of animals were injected icv with LPS (100 ng), a known stimulator of IL-1 expression. LPS caused a significant reduction in food intake (vehicle, 27.8 ± 0.7 g; LPS, 17.9 ± 3.5 g; P < 0.05) and body weight (vehicle, 10 ± 1 g; LPS, –1 ± 3 g; P < 0.01) at 24 h in rats. This anorexic effect of LPS was similar in magnitude to that observed after injection of GALP into rats (see experiment 3 and Refs. 6 and 13). This dose of LPS (100 ng) was then used to assess the effect of LPS on expression of IL-1 in the same brain regions that were positive for IL-1 after icv injection of GALP. The icv injection of LPS caused an induction of IL-1β in the meninges at 2 h, which peaked at 8 h (Fig. 1, R and S) and was almost absent at 24 h (Fig. 1T). In general, there were more cells expressing IL-1β at 8 h after injection of LPS compared with GALP (Fig. 1K). However, in contrast to GALP, LPS did not induce a robust expression of IL-1β in the parenchyma of the brain surrounding the ventricles at any time point studied (Fig. 1, V–X), and only the occasional cell was detected for IL-1β immunoreactivity, albeit at a much lower intensity (Fig. 1W), although a few IL-1β-positive cells were detected in the ependymal cells lining the ventricles. No cells expressing IL-1β were observed in the brain after icv injection of vehicle at any time point. The icv injection of LPS also induced the expression of IL-1 in the same areas as IL-1β was found. However, the effect of LPS on IL-1 was minimal, and only a few weakly stained IL-1-positive cells were observed (data not shown).
Experiment 2: phenotype of IL-1- and IL-1β-expressing cells
To determine the phenotype of the GALP-induced IL-1- and IL-1β-expressing cells in the rat, double-fluorescence immunohistochemistry was performed to colocalize IL-1 or IL-1β with astrocytes, microglia, or neurons. Figure 2 illustrates that the IL-1β-positive cells in the parenchyma surrounding the ventricles were microglia (Fig. 2F) and not astrocytes (Fig. 2I) or neurons (Fig. 2L). The IL-1β-expressing cells in the meninges (Fig. 2C) and choroid plexus (not shown) were also positive for Iba1, a marker for microglia and macrophages, and are therefore likely to be macrophages due to their spherical morphology.
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-expressing cells in the parenchyma were microglia and not astrocytes or neurons (data not shown). Because there were only a few cells expressing IL-1 in the meninges, the phenotype of these cells could not be determined.
Microglia and macrophages, astrocytes, and neurons were detected in the brains of vehicle-injected rats, but they did not express IL-1 or IL-1β (data not shown).
Experiment 3: effect of IL-1RA on actions of GALP in the rat
In rats, central administration of IL-1RA alone had no effect on food intake at 1 and 24 h or body weight at 24 h compared with vehicle-treated animals. The icv injection of GALP stimulated food intake at 1 h but suppressed feeding at 24 h after treatment (1 h: vehicle, 0.2 ± 0.1 g vs. GALP, 4.9 ± 0.9 g, P < 0.01; 24 h: vehicle, 21.3 ± 0.1 g vs. GALP, 14.1 ± 1.4 g, P < 0.05; Fig. 3A). IL-1RA had no affect on GALP-induced feeding at 1 h but significantly inhibited the reduction in feeding at 24 h in response to GALP, and the values for GALP- plus IL-1RA-treated rats were no different from vehicle-injected rats (1 h: vehicle, 0.2 ± 0.1 g vs. GALP+IL-1RA, 5.9 ± 1.1 g, P < 0.001; 24 h: vehicle, 21.3 ± 0.1 g vs. GALP+IL-1RA, 19.8 ± 2.1 g; Fig. 3A). The reduction in the change in body weight 24 h after central administration of GALP was partially reversed by IL-1RA treatment (vehicle, 3 ± 3 g vs. GALP, –11 ± 2 g vs. GALP+IL-1RA, –2 ± 3 g; P < 0.001, GALP vs. vehicle; Fig. 3B), and there was no significant difference between vehicle- and GALP- plus IL-1RA-treated rats.
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, C and D). Although IL-1RA administration alone had no effect on core body temperature compared with vehicle-treated rats, it significantly reduced the rise in core body temperature in rats treated with GALP (Fig. 3, C and D). IL-1RA did not completely inhibit the GALP-induced rise in body temperature, because an immediate rise in temperature was observed in GALP- plus IL-1RA-treated rats, which was temporally identical to rats injected with GALP alone. However, IL-1RA appeared to affect the maintenance of the temperature response to GALP. The duration of the febrile response in animals treated with IL-1RA plus GALP was shorter than in animals treated with GALP alone, and body temperature of rats injected with GALP plus IL-1RA was similar to vehicle-treated animals by 6 h after injections.
Experiment 4: effect of GALP on food intake, body weight, and core body temperature in IL-1/β–/– mice
The icv injection of GALP in wild-type mice had no effect on food intake but caused a significant reduction in the change in body weight at 24 h after injection. However, 24 h after icv injection of GALP in IL-1/β–/– mice, there was no significant difference in food intake and body weight compared with vehicle-treated IL-1/β mice (Fig. 4, A and B).
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/β–/– mice, a drop in core body temperature was observed that reached a nadir at 30 min (Table 1 and Fig. 4, C and D). The extent of hypothermia after GALP injection was not significantly different between wild-type and IL-1/β–/– mice. In wild-type mice, this period of hypothermia was followed by an immediate rise in core body temperature that lasted until approximately 10 h after injection (Table 2 and Fig. 4C). However, there was no significant difference in the change in core body temperature after central injection of GALP in IL-1/β–/– mice compared with vehicle-treated IL-1/β–/– mice 2–10 h after injection (Fig. 4D).
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, A and B). However, there was a nonsignificant trend in the extent of body weight change being less in the IL-1β–/– compared with wild-type mice (difference in change in body weight compared with respective vehicle-treated mice: wild-type, –1.32 ± 0.32 g vs. IL-1β–/–, –1.01 ± 0.22 g; P > 0.05).
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, C and D, and Table 1). The hypothermic effect of GALP in IL-1β–/– mice was more prolonged than in wild-type mice, and significant differences were observed up to 90 min after injection of GALP in IL-1β–/– mice compared with vehicle-treated mice (change in temperature at 60 min: vehicle, 0.7 ± 0.3 g vs. GALP, –4.2 ± 1.5 g, P < 0.05; 90 min: vehicle, 0.3 ± 0.3 g vs. GALP; –3.7 ± 2.0 g, P < 0.05). However, this greater effect of GALP on hypothermia in IL-1β–/– mice was likely to be due to one animal that had an extreme and prolonged response. In wild-type mice, the transient period of hypothermia was followed by a prolonged rise in core body temperature that was significantly different from vehicle-treated wild-type mice over 2–10 h (Table 2). However, after the hypothermic phase in GALP-treated IL-1β–/– mice, body temperature was the same as in IL-1β–/– mice injected with vehicle (Table 1). Furthermore, there was no significant difference in body temperature over 2–10 h in GALP- vs. vehicle-treated IL-1β–/–, even after the animal with the prolonged hypothermic response had been omitted (data not shown).
Experiment 6: effect of GALP on food intake, body weight, and core body temperature in IL-1RI–/– mice
It has been suggested that in addition to the known IL-1 receptor, IL-1RI, IL-1 may also signal via an unidentified novel receptor (17, 18). Thus, to identify whether IL-1RI mediates the actions of IL-1, the effect of GALP was tested in IL-1RI–/– mice. The icv injection of 1.5 nmol GALP in wild-type mice led to a significant reduction in food intake and change in body weight at 24 h. However, GALP administration had no significant effect on food intake and body weight in IL-1RI–/– mice at 24 h compared with vehicle-treated IL-1RI–/– mice (Fig. 6, A and B).
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and Fig. 6, C and D). The extent of hypothermia was not significantly different in both wild-type and IL-1RI–/– mice after GALP injection. In wild-type mice, this period of hypothermia was followed by a significant rise in core body temperature that lasted until approximately 8 h after injection, an effect that was not observed in IL-1RI–/– mice (Fig. 6, C and D, and Table 2).
Experiments 7 and 8: effect of GALP or LPS on food intake, body weight, and core body temperature in TLR4–/– mice
The icv injection of GALP had no effect on food intake at 24 h in wild-type or TLR4–/– mice. However, GALP administration caused a significant reduction in the change in body weight in both wild-type and TL4R–/– mice. In addition, GALP caused an increase in core body temperature in wild-type and TLR4–/– mice, and temperature was significantly elevated from 2–10 h (Table 3).
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). Core body temperature was elevated after peripheral injection of LPS in wild-type mice, but there was no significant difference in temperature over 8 h in TLR4–/– mice treated with LPS compared with vehicle-injected TLR4–/– mice. |
Discussion |
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The anorexic and febrile responses observed after GALP injection in rats and mice are similar to how rodents respond to an immune challenge and in particular to injection of the proinflammatory cytokine IL-1. Peripheral or central injection of IL-1 or IL-1β in rodents reduces food intake and body weight and causes fever (16, 23, 24, 25), effects that are similar in magnitude and profile to GALP. These similarities between IL-1 and GALP led us to test our main hypothesis that IL-1 mediates the effects of GALP on energy balance, and questions the physiological relevance of the effects of GALP on energy balance. The present study demonstrates that central administration of GALP (but not galanin) in rodents induced IL-1 and IL-1β expression in the brain, and the effects of GALP on feeding, body weight, and core body temperature are mediated by IL-1 acting via IL-1RI. These data suggest that GALP may induce an inflammatory response in the brain. However, these effects of GALP on inflammation are likely to be localized to the brain, because GALP did not affect plasma IL-6 levels (a marker of peripheral infection).
In rats, inhibition of endogenous IL-1, using the antagonist IL-1RA, significantly reduced the GALP-induced reduction in food intake and body weight at 24 h but had no effect on the acute orexigenic action. The latter result is not surprising because studies in the rat have demonstrated that the acute stimulatory effect of GALP on feeding is mediated by the orexigenic neuropeptides orexin and NPY (11, 12). IL-1RA also partially reduced the rise in core body temperature observed in rats after GALP, with a predominant effect on the maintenance of the febrile response. The failure of IL-1RA to completely reverse the GALP-induced rise in body temperature is not unusual because several studies have demonstrated the inability of central administration of IL-1RA to fully inhibit the febrile response to LPS (26, 27). Thus, to avoid any possible uncertainties about the dose of IL-1RA, time of administration, and/or its short half-life, we studied the effects of GALP in mice that were defective in the IL-1 system. Our data show that the acute hypothermia induced by GALP is not mediated by IL-1 or IL-1β. However, the GALP-induced hyperthermia is due to the action of IL-1β on the IL-1RI, because mice deficient in IL-1β or IL-1RI were resistant to these effects of GALP temperature. Because IL-1β–/– mice also fail to show GALP-induced hyperthermia, the contribution of IL-1 in the febrile effects of GALP cannot be fully disregarded but is unlikely, because IL-1β is the predominant ligand involved in the febrile response in rodents (25, 28). It is also difficult to conclude whether IL-1 or IL-1β is involved in the anorexic effects of GALP, because this neuropeptide did not significantly reduce food intake in wild-type mice in experiments involving IL-1/β- and IL-1β-deficient mice. However, one or both of these IL-1 ligands mediate the effects of GALP on food intake through IL-1RI, because IL-1RI–/– mice did not respond to the anorexic actions of GALP, which is in contrast to the wild-type control mice. In addition, because both IL-1/β–/– and IL-1RI–/– mice were resistant to the actions of GALP on body weight, and IL-1β–/– mice still lost body weight in response to GALP (albeit less than in wild-type mice), IL-1 must be playing a role in the effect of GALP on body weight. Because in our hands, IL-1 is equally or more potent at reducing food intake and body weight compared with IL-1β in mice (unpublished observation), it can be hypothesized that the effects of GALP on food intake and body weight are through the actions of both IL-1 and IL-1β, but the febrile response is mediated by IL-1β. However, to fully understand the contribution of IL-1 in the actions of GALP, studies would need to be performed in IL-1-deficient mice.
Central administration of GALP (but not galanin) in rats stimulated the expression of IL-1 and IL-1β in the parenchyma surrounding the ventricles, predominantly in the region surrounding the third ventricle of the hypothalamus, the ependymal cells of the ventricles. GALP also stimulated expression of IL-1β in the meninges and choroid plexus, whereas only a few cells expressing IL-1 were found in these regions. The IL-1- and IL-1β-positive cells found in the parenchyma were microglia, whereas the cells expressing IL-1β in the meninges and choroid plexus were macrophages. The time course of expression of IL-1 vs. IL-1β appeared to be slightly different, with maximal expression of IL-1 being earlier compared with IL-1β. The relevance of the temporal difference between IL-1 and IL-1β expression and the lack of IL-1 expression in the meninges is unclear. However, to verify these qualitative findings, the expression of the two IL-1 ligands needs to be assessed quantitatively (e.g. by ELISA). The effect of central administration of LPS on IL-1 expression in the rat was also assessed. Although IL-1β-positive cells were found within the meninges and the ependymal cells of the ventricles after icv LPS, there were very few cells expressing IL-1β found within the parenchyma surrounding the ventricles. The lack of IL-1β-positive cells in the parenchyma is in contrast to the effect seen here with GALP and suggests that this neuropeptide is more potent at inducing IL-1β in the brain after central administration compared with the inflammatory stimulus LPS.
The mechanism by which GALP leads to this production of IL-1 in the brain also remains to be determined. Galanin and GALP are reported to act via the three known galanin receptors GALR1–3. In vitro studies show that galanin exhibits equal affinity for all three receptors, whereas GALP has higher affinity for GALR2 and GALR3 (1, 29). However, it is likely that GALP is initiating IL-1 expression via a novel receptor because galanin did not induce expression of this cytokine in the brain. These findings are in agreement with data from other groups, suggesting that GALP acts via a novel receptor. For example, GALP produces several different physiological responses compared with galanin (6, 13, 30), and the anorexic actions of GALP are still present in mice deficient in GALR1 or GALR2 (8).
Central administration of GALP induces a specific pattern of Fos activation in the brain compared with galanin (13, 14, 31). In particular, GALP activates cells within the periventricular hypothalamic area (13), the region where microglial IL-1 expression is observed here. It is unlikely that GALP directly activates microglia to express IL-1, because the cells that express Fos in the periventricular hypothalamic area are astrocytes and not microglia or neurons (13). The actions of GALP on astrocytes may be direct because galanin receptors are expressed on astrocytes in culture (32); however, this effect may also be due to a novel GALP receptor because galanin does not induce periventricular hypothalamic Fos expression (13). The relevance of the GALP-induced astrocytic activation is unclear and remains to be tested, but these cells have been shown to release prostaglandins that may be involved in the febrile response to GALP. It is also possible that IL-1 production within microglia is mediated via key inflammatory mediators released by astrocytes.
Peripheral infections activate the immune system leading to cytokine production, which results in changes in brain-mediated responses including sickness behavior, fever, and anorexia. The effects of GALP in the present study on feeding, body weight, body temperature, and IL-1 expression are analogous to those observed during infection and inflammation. The endotoxin LPS is a component of gram-negative bacteria cell walls and is a potent inducer of the inflammatory response. Like GALP, peripheral or central administration of LPS to rodents causes a reduction in food intake and body weight and a rise in core body temperature (33). LPS stimulates the expression of IL-1 in the brain (34, 35, 36), and its effects on feeding, body weight, and temperature are mediated by this cytokine (27, 37, 38), actions similar to GALP in the present study. GALP mRNA in the arcuate nucleus and pituitary are increased in response to LPS administration in rats (39, 40). It is possible, therefore, that LPS may mediate its affects on IL-1, and hence fever and anorexia, via actions of GALP. To test this hypothesis, the effects of LPS in a GALP-deficient mouse, or in the presence of a GALP-specific antagonist, needs to be determined. However, because the actions of GALP are similar to those of LPS, it could be argued that the preparations of GALP used in the present study were contaminated with endotoxin. To test this, we injected GALP into mice deficient in the receptor that is responsible for the actions of LPS, TLR4 (41). In contrast to LPS, which had no effect in TLR4–/– mice, GALP caused a reduction in body weight and a rise in body temperature in these mice. These data confirm that the GALP samples do not contain endotoxin or that GALP is a ligand for TLR4.
Taken together, the findings in the present study question the relevance of GALP in the physiological regulation of food intake. It is possible that endogenous GALP has an acute physiological effect in rats to increase feeding via the actions of orexin and/or NPY (11, 12). However, in contrast, the anorexia, body weight loss, and fever induced by central administration of GALP may represent a non-GALR-mediated nonspecific sickness response that is dependent on IL-1. Caution is therefore needed in the interpretation of studies involving the central administration of GALP.
In summary, we have demonstrated that the anorexic and febrile effects of GALP in the rodent are mediated by IL-1 in the brain. The relevance of the localized IL-1 production in response to central administration of GALP and how this relates to energy balance remain to be determined. However, because GALP neurons are located in the arcuate nucleus, they are ideally positioned to receive and respond to peripheral signals, and it is therefore interesting to speculate that GALP may be an intermediary in how peripheral infections lead to anorexic and febrile effects.
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Disclosure Summary: The authors have nothing to disclose.
First Published Online July 10, 2008
Abbreviations: AUC, Area under the curve; GALP, galanin-like peptide; icv, intracerebroventricular; IL-1RA, IL-1 receptor antagonist; IL-1RI, IL-1 type I receptor; LPS, lipopolysaccharide; NPY, neuropeptide Y; PB, phosphate buffer; TLR4, Toll-like receptor 4.
Received February 22, 2008.
Accepted for publication June 27, 2008.
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and interleukin-1β. Eur Cytokine Netw 9:279–288[Medline] involves different mechanisms. Biochem Biophys Res Commun 162:591–596[CrossRef][Medline], β, and receptor antagonist in rat brain. Ann NY Acad Sci 840:128–138[CrossRef][Medline]
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) in core body temperature (C and D) in rats. GALP (1.5 nmol in 1 µl) or vehicle (1 µl saline) was administered icv in rats. Three separate icv injections of 200 µg (in 2 µl) of IL-1RA or vehicle (2 µl saline) were given at 1 h before, at the time of, and 1 h after injection of GALP (or vehicle). Food intake was measured at 1 and 24 h and body weight at 24 h after injections. Body temperature was monitored continuously by remote radiotelemetry for 24 h, but for clarity, data are shown only for 0–10 h. Analysis of the change in core body temperature over 0–10 h after injections is illustrated in D as the AUC (C·h). Data are mean ± SEM for n = 8–13 rats per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. vehicle-treated animals and ##, P < 0.01; ###, P < 0.001 vs. IL-1RA-treated animals.
