Agouti-Related Peptide, Neuropeptide Y and Somatostatin-Producing Neurons Are Targets for Ghrelin Actions in the Rat Hypothalamus
Abstract;, http://www.100md.com
Ghrelin, the endogenous ligand of the GH secretagogue receptor, acts at central level to elicit GH release and regulate food intake. To elucidate the neural circuit that exerts its effects, we measured the expression of hypothalamic neuropeptides involved in weight regulation and GH secretion after ghrelin administration. Adult male rats, fed or fasted for 72 h, were treated centrally (intracerebroventicularly) with a single dose of ghrelin (5 µg). After 2, 4, and 6 or 8 h, agouti-related peptide, melanin-concentrating hormone, neuropeptide Y, prepro-orexin, GHRH, and somatostatin mRNA levels were measured by in situ hybridization. We found that ghrelin increased agouti-related peptide and neuropeptide Y expression in the arcuate nucleus of the hypothalamus of fed and fasted rats. In contrast, no change was demonstrated in the mRNA levels of the other neuropeptides studied at any time evaluated. Finally, we examined the effect of ghrelin on GHRH and somatostatin mRNA levels in GH-deficient (dwarf) rats. Our results show that ghrelin increases somatostatin mRNA levels in the hypothalamus of these rats. This study furthers our understanding of the molecular basis and mechanisms involved in the effect of ghrelin on food intake and GH secretion.
Introductionan22+w3, 百拇医药
GH SECRETAGOGUES (GHSs) are artificial compounds that release GH in all species tested to date. Until 1999, these molecules mimicked an unknown endogenous factor that activates the GHS receptor (GHS-R) (1). The earlier cloning of GHS-R suggested that an endogenous ligand for this receptor might exist (2). Indeed, after intensive research by different groups, the isolation of an endogenous ligand of the GHS-R, ghrelin (3), was recently reported. The purified ligand was found to be a peptide of 28 amino acids, in which the serine 3 residue was n-octanoylated. More recently, a second endogenous ligand for the GHS-R, des-Gln14-ghrelin, whose biological activity and sequence are identical to ghrelin except for one glutamine in position 14, has been purified and characterized (4). These peptides have been shown to exert a very potent and specific GH-releasing activity in vitro and in vivo as well as to increase the transcription rate of the Pit gene (5, 6). Taking into account that ghrelin is secreted prevalently from the stomach and circulates in normal subjects at considerable plasma concentrations, it has been postulated that this molecule is secreted from the stomach, circulates in the bloodstream, and stimulates GH synthesis and secretion by the somatotrophs (3). Moreover, ghrelin has emerged as a regulatory signal involved in energy homeostasis (7), reproduction (8), and gastrointestinal (9, 10) and cardiovascular (11, 12, 13), function among others.
Recent data have led to the recognition that ghrelin plays an important role in energy homeostasis, and the evidence is as follows. Ghrelin administration induces a positive energy balance in rodents by decreasing fat utilization without significantly changing energy expenditure or locomotor activity (14, 15). The effect of ghrelin appears to be exerted at the central level, and its chronic administration is associated with metabolic changes that lead to an efficient metabolic state, resulting in increased body weight and fat mass (14, 15). In keeping with this, an inverse relationship has been reported between plasma ghrelin levels and body mass index in humans (16).#[&+h30, 百拇医药
Recent findings have demonstrated the interaction between ghrelin and orexigenic systems in the rat hypothalamus. Therefore, GHS-R mRNA is expressed in neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons in the arcuate nucleus of the hypothalamus (ARC) (17, 18, 19), and the central administration of ghrelin increases the mRNA content of NPY and AgRP in the ARC (15, 20, 21). On the other hand, the lack of expression of the GHS-R in the lateral hypothalamus (LHA) (17), where a large number of orexigenic neurons are located (22, 23), suggested that this hypothalamic nucleus was unlikely to be a target of the effects of ghrelin on food intake. However, some of the biological effects of ghrelin might be mediated by as yet unknown receptors, different from the only one cloned until now (24, 25). In keeping with this possibility, it was found that ghrelin administration leads to increased c-Fos expression in neurons of the LHA (26). Taking into consideration that the main central roles of ghrelin appear to be those related to the regulation of body weight and GH secretion, we decided to study the hypothalamic expression of neuropeptides implicated in both processes. Specifically, the functional linkage of ghrelin and body weight homeostasis was carried out by measuring hypothalamic AgRP, melanin-concentrating hormone (MCH), NPY, and prepro-orexin (prepro-OX) mRNA levels. Furthermore, we examined the effect of ghrelin on AgRP and NPY in the adaptation to fasting, because their responses are frequently dependent on the nutritional status of the animals (27, 28).
Finally, we assessed the effect of ghrelin on GHRH and somatostatin (SST) mRNA levels. These two neuropeptides are the major regulators of GH secretion (29). Furthermore, in view of the fact that GH feeds back to regulate GHRH and SST expression at the hypothalamic level, we also studied the effects of ghrelin in an experimental model of GH deficiency, namely the dwarf Lewis rat (30, 31, 32). This model displays an autosomal recessive mutation due to a selective failure in GHRH signaling in the somatotrope cell, which results in almost undetectable plasma GH levels (30, 31). Therefore, these rats provide a unique opportunity to study the regulation of GHRH and SST in the practical absence of endogenous GH.@2, 百拇医药
Materials and Methods@2, 百拇医药
Experimental animals@2, 百拇医药
We used four different rats models: 1) adult male Sprague Dawley rats (250–300 g, 9–11 wk old; Animalario General University of Santiago de Compostela, Santiago de Compostela, Spain), 2) dwarf (HsdOla:dw-4) Lewis rats (150–175 g, 10–12 wk old; Harlan Ibérica, Barcelona, Spain), 3) Lewis rats age-matched (250–300 g, 10–12 wk old) with dwarf Lewis rats (Charles River, Lyon, France), and 4) Lewis rats weight-matched (150–175 g, 6–8 wk old) with dwarf Lewis rats. The animals were allowed free access to standard laboratory pellets of rat chow and tap water. The protocols were approved by the ethics committee of the University of Santiago de Compostela, and experiments were performed in accordance to the rules of laboratory animal care and international law on animal experimen-tation.
Implantation of intracerebroventricular (icv) cannulas and ghrelin treatment-\7w, 百拇医药
Chronic icv cannulas were implanted under ketamine-xylazine anesthesia (50 mg/kg, ip), as described previously (33, 34) and were demonstrated to be located in the lateral ventricle by methylene blue staining. The animals were caged individually and used for the experiments 1 wk later. During this postoperative recovery period the rats became accustomed to the handling procedure under nonstressful conditions. After this time one group of rats continued to have food available ad libitum (fed rats), and the other group was deprived of food for 72 h (fast 72 h). To confirm the efficiency of the food deprivation, plasma leptin levels were measured in the described experimental groups of animals, using a commercial kit (Linco Research, Inc., St. Charles, MO) (35). Food deprivation for 72 h significantly decreased plasma leptin levels (fed rats, 5.1 ± 0.7 ng/ml; fast 72 h rats, 0.1 ± 0.04 ng/ml; P < 0.001). Thereafter, rats received either a single administration of ghrelin (Bachem, Bubendorf, Switzerland; 5 µg/rat dissolved in 5 µl distilled water saturated with argon) or vehicle (control rats). Two, 4, 6, or 8 h after the treatment, vehicle- and ghrelin-treated animals were quickly decapitated in an independent room, and their brains were removed rapidly. For in situ hybridization, the whole brains were maintained at -80 C until processed. All treatments started in the lights on phase (0900 h). Six to eight animals were used in each experimental group.
In situ hybridization\o&3r/&, 百拇医药
Coronal hypothalamic sections (16 µm) were cut on a cryostat and immediately stored at -80 C until hybridization. For AgRP, MCH, NPY, prepro-OX, GHRH, and SST mRNA detection we employed the specific antisense oligodeoxynucleotides (Table 1). These probes were 3'-end labeled with [{alpha} -35S]deoxy-ATP using terminal deoxynucleotidyl transferase. The specificity of the probes was confirmed by incubating the sections with an excess of the unlabeled probes (Fig. 1). In situ hybridizations were performed as previously reported (34, 36). The frozen sections were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at room temperature for 30 min. They were then dehydrated using 70%, 80%, 90%, 95%, and absolute ethanol (5 min each). The hybridization was carried out overnight at 37 C in a moist chamber. Hybridization solution contained 5 x 105 (AgRP, prepro-OX, SST, and GHRH) or 1 x 106 cpm (MCH and NPY) per slide of the labeled probe, 4x standard saline citrate (SSC), 50% deionized formamide, 1x Denhardt’s solution, 10% dextran sulfate, and 10 µg/ml sheared, single-stranded salmon sperm DNA. Afterward, the hybridization sections were sequentially washed in 1x SSC at room temperature, four times in 1x SSC at 42 C (30 min/wash), and once in 1x SSC at room temperature (1 h), and then rinsed in water and ethanol. Finally, the sections were air-dried and exposed to Hyperfilm ß-Max (Amersham International, Little Chalfont, UK) at room temperature for 4–6 d for AgRP, MCH, NPY, SST, and prepro-OX and for 14 d for GHRH. After the films were developed, sections were dipped in Ilford K5 autoradiographic emulsion (Ilford, UK) and exposed for 2 wk (AgRP, MCH, NPY, SST, and prepro-OX) or 6 wk (GHRH) at 4 C. The slides were then developed in Kodak D-19 developer (Eastman Kodak Co., Rochester, NY), fixed (Kodak fixer), and counterstained with methylene blue.
fig.ommitteedigm, http://www.100md.com
Table 1. Antisense oligonucleotides for rat AgRP, rat GHRH, rat MCH, rat NPY, rat prepro-OX, and rat SSTigm, http://www.100md.com
fig.ommitteedigm, http://www.100md.com
Figure 1. Left panels: A, Brightfield photomicrographs of cells in the PeN that were incubated with a 35S-labeled antisense oligonucleotide SST probe (magnification, x400). B, Brightfield photomicrographs of cells in the ARC that were incubated with a 35S-labeled antisense oligonucleotide GHRH probe (magnification, x400). C, Brightfield photomicrographs of cells in the ARC that were incubated with a 35S-labeled antisense oligonucleotide AgRP probe (magnification, x200). D, Cells in the ARC that were incubated with a 35S-labeled antisense oligonucleotide NPY probe (magnification, x200). E, Cells in the LHA that were incubated with a 35S-labeled antisense oligonucleotide MCH probe (magnification, x200). F, Cells in the LHA that were incubated with a 35S-labeled antisense oligonucleotide prepro-OX probe (magnification, x200). Right panels, Autoradiographic images (magnification, x1) of representative brain coronal sections, incubated with a 35S-labeled antisense oligonucleotide probe in the absence (control) or presence of crescent amounts of nonlabeled (x100, x200, and x500) oligonucleotide probes. A, SST; B, GHRH; C, AgRP; D, NPY; E, MCH; F, prepro-OX. 3V, Third ventricle.
To compare anatomically similar regions, the slides were matched according to the rat brain atlas of Paxinos and Watson (37). The slides from control and treated animals at each treatment time were always exposed to the same autoradiographic film. All sections were scanned, and the specific hybridization signal was quantified by densitometry using a digital imaging system (Molecular Analyst, Bio-Rad Laboratories, Inc., Richmond, CA) (34, 38). The OD of the hybridization signal was determined and subsequently corrected by the OD of its adjacent background value. For this reason a rectangle, with the same dimensions in each case, was drawn enclosing the hybridization signal over each nucleus and over adjacent brain areas of each section (background). We used 16–20 sections for each animal (4–5 slides, 4 sections/slide). The mean of these 16–20 values was used as the densitometry value for each animal. The coefficients of intraindividual variation were 5.8% for AgRP, 6.3% for MCH, 6.9% for NPY, 5.4% for prepro-OX, 6.6% for GHRH, and 6.0% for SST.
Statistical analysis and data presentationhk, http://www.100md.com
In experiments with wild-type normal rats, the data were analyzed using a nonparametric Mann-Whitney test. We compared control with treated animals. To analyze the time dependence effect of the treatment, we performed an ordinary ANOVA followed by a post hoc Bonferroni test for the treated groups at the different time points. For each test, significance was set at P < 0.05.hk, http://www.100md.com
In experiments with dwarf Lewis rats treated with ghrelin, the data were analyzed using ANOVA. Therefore, we compared Lewis rats (age- and weight-matched) treated with vehicle, dwarf rats treated with vehicle, and dwarf rats treated with ghrelin. The data (mean ± SEM) were expressed as the percentage of change in relation to Lewis rats age-matched and treated with vehicle (100%).hk, http://www.100md.com
Because of the presence of circadian changes in the hypothalamic mRNA content of the neuropeptides evaluated in this study (39, 40, 41, 42, 43, 44), the results were normalized by making comparisons with control animals killed the same day and at the same hour. The coefficients of interindividual variation were 20.7% for AgRP, 12.7% for MCH, 20.9% for NPY, 10.9% for prepro-OX, 13.1% (wild-type rats) and 14.4% (dwarf rats) for GHRH, and 10.8% (wild-type rats) and 12.7% (dwarf rats) for SST.
In each case the data (mean ± SEM) were expressed as the percentage of change in relation to control values (vehicle-treated animals = 100%). All statistical analyses and graphical representations presented in this work have been performed using 1 data point/animal, obtained from the mean of the 16–20 sections for each animal.|u8cz8e, 百拇医药
Results|u8cz8e, 百拇医药
In agreement with previous reports (36, 45), our in situ hybridization histochemistry showed SST mRNA-positive cells distributed widely throughout the hypothalamus. The highest density of positive cells was located in the periventricular nucleus of the hypothalamus (PeN) (Fig. 1A). In situ hybridization with an oligoprobe for GHRH mRNA demonstrated labeling in numerous cells in the ventrolateral part of the ARC (36, 45) (Fig. 1B).|u8cz8e, 百拇医药
In agreement with previous reports (46) our in situ hybridization histochemistry showed AgRP (Fig. 1C) and NPY (Fig. 1D) mRNA-positive cells located in the ARC. In situ hybridization with oligoprobes for MCH (Fig. 1E) and prepro-OX (Fig. 1F) mRNA demonstrated labeling of numerous cells in the LHA, as previously reported (46, 47). In all cases, incubation of the sections with an excess (100-, 200-, and 500-fold) of the unlabeled probes displaced the positive signal (Fig. 1, right panels).
Treatment with ghrelin increases AgRP and NPY mRNA levels in the ARC of fed ratsl, http://www.100md.com
Using in situ hybridization analysis, a significant increase in AgRP (Fig. 2A) and NPY (Fig. 2B mRNA levels was observed in the ARC after treatment with ghrelin. This increase was transient, being evident 2 and 4 h after treatment in the case of AgRP and 2 h after treatment in the case of NPY. On the other hand, we did not find any change in the mRNA content of MCH, prepro-OX (in the LHA), GHRH (in the ARC), and SST (in the PeN) at any time point (2, 4, and 6 h) as assessed by in situ hybridization (Fig. 3).l, http://www.100md.com
fig.ommitteedl, http://www.100md.com
Figure 2. A, Upper panel, Autoradiographic images (magnification, x5) of representative brain coronal sections, incubated with a 35S-labeled antisense oligonucleotide AgRP probe, in the vehicle- and ghrelin-treated groups for 2 h. Areas delineated in both sections are shown at higher magnification in the bottom (magnification, x60). Lower panel, AgRP mRNA levels in the ARC of the described experimental groups. *, P < 0.05 vs. vehicle treated for 2 and 4 h, respectively. 3V, Third ventricle. B, Upper panel, Autoradiographic images (magnification, x5) of representative brain coronal sections, incubated with a 35S-labeled antisense olignucleotide NPY probe, in the vehicle-treated group and the ghrelin-treated group for 2 h. Areas delineated in both sections are shown at higher magnification in the bottom (magnification, x60). 3V, Third venticle. Lower panel, NPY mRNA levels in the ARC of the described experimental groups. *, P < 0.05 vs. vehicle treated for 2 h.
fig.ommitteed|-j, 百拇医药
Figure 3. Effect of ghrelin on mRNA levels of MCH in the LHA (A), prepro-OX in the LHA (B), GHRH in the ARC (C), and SST in the PeN (D).|-j, 百拇医药
Treatment with ghrelin increases NPY and AgRP mRNA levels in the ARC of fasted rats|-j, 百拇医药
Using in situ hybridization analysis, a significant increase in AgRP (Fig. 4A) and NPY (Fig. 4B) mRNA levels was observed in the ARC after 72 h of food deprivation. In fasted rats treated with ghrelin, we observed a further increase in AgRP and NPY mRNA content in the ARC 4 h after the treatment compared with fasted vehicle-treated rats (Fig. 4).|-j, 百拇医药
fig.ommitteed|-j, 百拇医药
Figure 4. A, Upper panel, Autoradiographic images (magnification, x1.6) of representative brain coronal sections, incubated with a 35S-labeled antisense oligonucleotide AgRP probe, in the vehicle-treated group for 4 h, the 72-h fasted group treated with vehicle for 4 h, and the 72-h fasted group treated with ghrelin for 4 h. Areas delineated in the sections are shown at higher magnification in the bottom (magnification, x15). 3V, Third ventricle. Lower panel, AgRP mRNA levels in the ARC of the described experimental groups. *, P < 0.05 vs. vehicle-treated for 2, 4, and 8 h, respectively; ***, P < 0.001 vs. vehicle-treated for 4 h; ##, P < 0.01, 72-h fasted treated with vehicle for 4 h vs. 72-h fasted treated with ghrelin for 4 h. B, Upper panel, Autoradiographic images of representative brain coronal sections, incubated with a 35S-labeled antisense oligonucleotide NPY probe, in the vehicle-treated group for 4 h, the 72-h fasted group treated with vehicle for 4 h, and the 72-h fasted group treated with ghrelin for 4 h. Areas delineated in the sections are shown at higher magnification in the bottom. 3V, Third ventricle. Lower panel, NPY mRNA levels in the ARC of the described experimental groups. *, P < 0.05 vs. vehicle treated for 2 h and 4 h, respectively; **, P < 0.01 vs. vehicle treated for 8 h; ***, P < 0.001 vs. vehicle for 4 h; #, P < 0.05, 72-h fasted treated with vehicle for 4 h vs. 72-h fasted treated with ghrelin for 4 h.
Ghrelin increases SST mRNA levels in the PeN of dwarf Lewis rats5, 百拇医药
Using in situ hybridization analysis, we detected a significant decrease in SST mRNA content in the PeN of dwarf Lewis rats compared with vehicle-treated, wild-type (age- and weight-matched) Lewis rats (Fig. 5A. Treatment with ghrelin increased SST mRNA levels in the PeN of dwarf Lewis rats 6 h after treatment, reaching levels observed in vehicle-treated (age- and weight-matched) Lewis rats (Fig. 5A). The GHRH mRNA content in the ARC was increased in dwarf Lewis rats compared with vehicle-treated wild-type (age- and weight-matched) Lewis rats. Ghrelin did not change GHRH mRNA levels in dwarf Lewis rats 6 h after treatment (Fig. 5B).5, 百拇医药
fig.ommitteed5, 百拇医药
Figure 5. A, Upper panel, Autoradiographic images (magnification, x1) of representative brain coronal sections, incubated with a 35S-labeled antisense oligonucleotide SST probe, in age-matched Lewis rats treated with vehicle for 6 h, weight-matched Lewis rats treated with vehicle for 6 h, dwarf Lewis rats treated with vehicle for 6 h, and dwarf Lewis rats treated with ghrelin for 6 h. Areas delineated in the sections are shown at higher magnification in the bottom (magnification, x10). 3V, Third ventricle. Lower panel, SST mRNA levels in the PeN of the described experimental groups. ***, P < 0.001 vs. dwarf Lewis vehicle treated for 6 h; ###, P < 0.001 vs. age-matched Lewis rats treated with vehicle for 6 h; !!!, P < 0.001 vs. weight-matched Lewis rats treated with vehicle for 6 h. B, Upper panel, Autoradiographic images (x1) of representative brain coronal sections, incubated with a 35S-labeled antisense oligonucleotide GHRH probe, in age-matched Lewis rats treated with vehicle for 6 h, weight-matched Lewis rats treated with vehicle for 6 h, dwarf Lewis rats treated with vehicle for 6 h, and dwarf Lewis rats treated with ghrelin for 6 h. Areas delineated in the sections are shown at higher magnification in the bottom (x6). 3V, Third ventricle. Lower panel, GHRH mRNA levels in the ARC of the described experimental groups. ###, P < 0.001 vs. age-matched Lewis rats treated with vehicle for 6 h; !!!, P < 0.001 vs. weight-matched Lewis rats treated with vehicle for 6 h.(Luisa M. Seoane Miguel López Sulay Tovar Felipe F. Casanueva Rosa Señarís and Carlos Diégu)
Ghrelin, the endogenous ligand of the GH secretagogue receptor, acts at central level to elicit GH release and regulate food intake. To elucidate the neural circuit that exerts its effects, we measured the expression of hypothalamic neuropeptides involved in weight regulation and GH secretion after ghrelin administration. Adult male rats, fed or fasted for 72 h, were treated centrally (intracerebroventicularly) with a single dose of ghrelin (5 µg). After 2, 4, and 6 or 8 h, agouti-related peptide, melanin-concentrating hormone, neuropeptide Y, prepro-orexin, GHRH, and somatostatin mRNA levels were measured by in situ hybridization. We found that ghrelin increased agouti-related peptide and neuropeptide Y expression in the arcuate nucleus of the hypothalamus of fed and fasted rats. In contrast, no change was demonstrated in the mRNA levels of the other neuropeptides studied at any time evaluated. Finally, we examined the effect of ghrelin on GHRH and somatostatin mRNA levels in GH-deficient (dwarf) rats. Our results show that ghrelin increases somatostatin mRNA levels in the hypothalamus of these rats. This study furthers our understanding of the molecular basis and mechanisms involved in the effect of ghrelin on food intake and GH secretion.
Introductionan22+w3, 百拇医药
GH SECRETAGOGUES (GHSs) are artificial compounds that release GH in all species tested to date. Until 1999, these molecules mimicked an unknown endogenous factor that activates the GHS receptor (GHS-R) (1). The earlier cloning of GHS-R suggested that an endogenous ligand for this receptor might exist (2). Indeed, after intensive research by different groups, the isolation of an endogenous ligand of the GHS-R, ghrelin (3), was recently reported. The purified ligand was found to be a peptide of 28 amino acids, in which the serine 3 residue was n-octanoylated. More recently, a second endogenous ligand for the GHS-R, des-Gln14-ghrelin, whose biological activity and sequence are identical to ghrelin except for one glutamine in position 14, has been purified and characterized (4). These peptides have been shown to exert a very potent and specific GH-releasing activity in vitro and in vivo as well as to increase the transcription rate of the Pit gene (5, 6). Taking into account that ghrelin is secreted prevalently from the stomach and circulates in normal subjects at considerable plasma concentrations, it has been postulated that this molecule is secreted from the stomach, circulates in the bloodstream, and stimulates GH synthesis and secretion by the somatotrophs (3). Moreover, ghrelin has emerged as a regulatory signal involved in energy homeostasis (7), reproduction (8), and gastrointestinal (9, 10) and cardiovascular (11, 12, 13), function among others.
Recent data have led to the recognition that ghrelin plays an important role in energy homeostasis, and the evidence is as follows. Ghrelin administration induces a positive energy balance in rodents by decreasing fat utilization without significantly changing energy expenditure or locomotor activity (14, 15). The effect of ghrelin appears to be exerted at the central level, and its chronic administration is associated with metabolic changes that lead to an efficient metabolic state, resulting in increased body weight and fat mass (14, 15). In keeping with this, an inverse relationship has been reported between plasma ghrelin levels and body mass index in humans (16).#[&+h30, 百拇医药
Recent findings have demonstrated the interaction between ghrelin and orexigenic systems in the rat hypothalamus. Therefore, GHS-R mRNA is expressed in neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons in the arcuate nucleus of the hypothalamus (ARC) (17, 18, 19), and the central administration of ghrelin increases the mRNA content of NPY and AgRP in the ARC (15, 20, 21). On the other hand, the lack of expression of the GHS-R in the lateral hypothalamus (LHA) (17), where a large number of orexigenic neurons are located (22, 23), suggested that this hypothalamic nucleus was unlikely to be a target of the effects of ghrelin on food intake. However, some of the biological effects of ghrelin might be mediated by as yet unknown receptors, different from the only one cloned until now (24, 25). In keeping with this possibility, it was found that ghrelin administration leads to increased c-Fos expression in neurons of the LHA (26). Taking into consideration that the main central roles of ghrelin appear to be those related to the regulation of body weight and GH secretion, we decided to study the hypothalamic expression of neuropeptides implicated in both processes. Specifically, the functional linkage of ghrelin and body weight homeostasis was carried out by measuring hypothalamic AgRP, melanin-concentrating hormone (MCH), NPY, and prepro-orexin (prepro-OX) mRNA levels. Furthermore, we examined the effect of ghrelin on AgRP and NPY in the adaptation to fasting, because their responses are frequently dependent on the nutritional status of the animals (27, 28).
Finally, we assessed the effect of ghrelin on GHRH and somatostatin (SST) mRNA levels. These two neuropeptides are the major regulators of GH secretion (29). Furthermore, in view of the fact that GH feeds back to regulate GHRH and SST expression at the hypothalamic level, we also studied the effects of ghrelin in an experimental model of GH deficiency, namely the dwarf Lewis rat (30, 31, 32). This model displays an autosomal recessive mutation due to a selective failure in GHRH signaling in the somatotrope cell, which results in almost undetectable plasma GH levels (30, 31). Therefore, these rats provide a unique opportunity to study the regulation of GHRH and SST in the practical absence of endogenous GH.@2, 百拇医药
Materials and Methods@2, 百拇医药
Experimental animals@2, 百拇医药
We used four different rats models: 1) adult male Sprague Dawley rats (250–300 g, 9–11 wk old; Animalario General University of Santiago de Compostela, Santiago de Compostela, Spain), 2) dwarf (HsdOla:dw-4) Lewis rats (150–175 g, 10–12 wk old; Harlan Ibérica, Barcelona, Spain), 3) Lewis rats age-matched (250–300 g, 10–12 wk old) with dwarf Lewis rats (Charles River, Lyon, France), and 4) Lewis rats weight-matched (150–175 g, 6–8 wk old) with dwarf Lewis rats. The animals were allowed free access to standard laboratory pellets of rat chow and tap water. The protocols were approved by the ethics committee of the University of Santiago de Compostela, and experiments were performed in accordance to the rules of laboratory animal care and international law on animal experimen-tation.
Implantation of intracerebroventricular (icv) cannulas and ghrelin treatment-\7w, 百拇医药
Chronic icv cannulas were implanted under ketamine-xylazine anesthesia (50 mg/kg, ip), as described previously (33, 34) and were demonstrated to be located in the lateral ventricle by methylene blue staining. The animals were caged individually and used for the experiments 1 wk later. During this postoperative recovery period the rats became accustomed to the handling procedure under nonstressful conditions. After this time one group of rats continued to have food available ad libitum (fed rats), and the other group was deprived of food for 72 h (fast 72 h). To confirm the efficiency of the food deprivation, plasma leptin levels were measured in the described experimental groups of animals, using a commercial kit (Linco Research, Inc., St. Charles, MO) (35). Food deprivation for 72 h significantly decreased plasma leptin levels (fed rats, 5.1 ± 0.7 ng/ml; fast 72 h rats, 0.1 ± 0.04 ng/ml; P < 0.001). Thereafter, rats received either a single administration of ghrelin (Bachem, Bubendorf, Switzerland; 5 µg/rat dissolved in 5 µl distilled water saturated with argon) or vehicle (control rats). Two, 4, 6, or 8 h after the treatment, vehicle- and ghrelin-treated animals were quickly decapitated in an independent room, and their brains were removed rapidly. For in situ hybridization, the whole brains were maintained at -80 C until processed. All treatments started in the lights on phase (0900 h). Six to eight animals were used in each experimental group.
In situ hybridization\o&3r/&, 百拇医药
Coronal hypothalamic sections (16 µm) were cut on a cryostat and immediately stored at -80 C until hybridization. For AgRP, MCH, NPY, prepro-OX, GHRH, and SST mRNA detection we employed the specific antisense oligodeoxynucleotides (Table 1). These probes were 3'-end labeled with [{alpha} -35S]deoxy-ATP using terminal deoxynucleotidyl transferase. The specificity of the probes was confirmed by incubating the sections with an excess of the unlabeled probes (Fig. 1). In situ hybridizations were performed as previously reported (34, 36). The frozen sections were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at room temperature for 30 min. They were then dehydrated using 70%, 80%, 90%, 95%, and absolute ethanol (5 min each). The hybridization was carried out overnight at 37 C in a moist chamber. Hybridization solution contained 5 x 105 (AgRP, prepro-OX, SST, and GHRH) or 1 x 106 cpm (MCH and NPY) per slide of the labeled probe, 4x standard saline citrate (SSC), 50% deionized formamide, 1x Denhardt’s solution, 10% dextran sulfate, and 10 µg/ml sheared, single-stranded salmon sperm DNA. Afterward, the hybridization sections were sequentially washed in 1x SSC at room temperature, four times in 1x SSC at 42 C (30 min/wash), and once in 1x SSC at room temperature (1 h), and then rinsed in water and ethanol. Finally, the sections were air-dried and exposed to Hyperfilm ß-Max (Amersham International, Little Chalfont, UK) at room temperature for 4–6 d for AgRP, MCH, NPY, SST, and prepro-OX and for 14 d for GHRH. After the films were developed, sections were dipped in Ilford K5 autoradiographic emulsion (Ilford, UK) and exposed for 2 wk (AgRP, MCH, NPY, SST, and prepro-OX) or 6 wk (GHRH) at 4 C. The slides were then developed in Kodak D-19 developer (Eastman Kodak Co., Rochester, NY), fixed (Kodak fixer), and counterstained with methylene blue.
fig.ommitteedigm, http://www.100md.com
Table 1. Antisense oligonucleotides for rat AgRP, rat GHRH, rat MCH, rat NPY, rat prepro-OX, and rat SSTigm, http://www.100md.com
fig.ommitteedigm, http://www.100md.com
Figure 1. Left panels: A, Brightfield photomicrographs of cells in the PeN that were incubated with a 35S-labeled antisense oligonucleotide SST probe (magnification, x400). B, Brightfield photomicrographs of cells in the ARC that were incubated with a 35S-labeled antisense oligonucleotide GHRH probe (magnification, x400). C, Brightfield photomicrographs of cells in the ARC that were incubated with a 35S-labeled antisense oligonucleotide AgRP probe (magnification, x200). D, Cells in the ARC that were incubated with a 35S-labeled antisense oligonucleotide NPY probe (magnification, x200). E, Cells in the LHA that were incubated with a 35S-labeled antisense oligonucleotide MCH probe (magnification, x200). F, Cells in the LHA that were incubated with a 35S-labeled antisense oligonucleotide prepro-OX probe (magnification, x200). Right panels, Autoradiographic images (magnification, x1) of representative brain coronal sections, incubated with a 35S-labeled antisense oligonucleotide probe in the absence (control) or presence of crescent amounts of nonlabeled (x100, x200, and x500) oligonucleotide probes. A, SST; B, GHRH; C, AgRP; D, NPY; E, MCH; F, prepro-OX. 3V, Third ventricle.
To compare anatomically similar regions, the slides were matched according to the rat brain atlas of Paxinos and Watson (37). The slides from control and treated animals at each treatment time were always exposed to the same autoradiographic film. All sections were scanned, and the specific hybridization signal was quantified by densitometry using a digital imaging system (Molecular Analyst, Bio-Rad Laboratories, Inc., Richmond, CA) (34, 38). The OD of the hybridization signal was determined and subsequently corrected by the OD of its adjacent background value. For this reason a rectangle, with the same dimensions in each case, was drawn enclosing the hybridization signal over each nucleus and over adjacent brain areas of each section (background). We used 16–20 sections for each animal (4–5 slides, 4 sections/slide). The mean of these 16–20 values was used as the densitometry value for each animal. The coefficients of intraindividual variation were 5.8% for AgRP, 6.3% for MCH, 6.9% for NPY, 5.4% for prepro-OX, 6.6% for GHRH, and 6.0% for SST.
Statistical analysis and data presentationhk, http://www.100md.com
In experiments with wild-type normal rats, the data were analyzed using a nonparametric Mann-Whitney test. We compared control with treated animals. To analyze the time dependence effect of the treatment, we performed an ordinary ANOVA followed by a post hoc Bonferroni test for the treated groups at the different time points. For each test, significance was set at P < 0.05.hk, http://www.100md.com
In experiments with dwarf Lewis rats treated with ghrelin, the data were analyzed using ANOVA. Therefore, we compared Lewis rats (age- and weight-matched) treated with vehicle, dwarf rats treated with vehicle, and dwarf rats treated with ghrelin. The data (mean ± SEM) were expressed as the percentage of change in relation to Lewis rats age-matched and treated with vehicle (100%).hk, http://www.100md.com
Because of the presence of circadian changes in the hypothalamic mRNA content of the neuropeptides evaluated in this study (39, 40, 41, 42, 43, 44), the results were normalized by making comparisons with control animals killed the same day and at the same hour. The coefficients of interindividual variation were 20.7% for AgRP, 12.7% for MCH, 20.9% for NPY, 10.9% for prepro-OX, 13.1% (wild-type rats) and 14.4% (dwarf rats) for GHRH, and 10.8% (wild-type rats) and 12.7% (dwarf rats) for SST.
In each case the data (mean ± SEM) were expressed as the percentage of change in relation to control values (vehicle-treated animals = 100%). All statistical analyses and graphical representations presented in this work have been performed using 1 data point/animal, obtained from the mean of the 16–20 sections for each animal.|u8cz8e, 百拇医药
Results|u8cz8e, 百拇医药
In agreement with previous reports (36, 45), our in situ hybridization histochemistry showed SST mRNA-positive cells distributed widely throughout the hypothalamus. The highest density of positive cells was located in the periventricular nucleus of the hypothalamus (PeN) (Fig. 1A). In situ hybridization with an oligoprobe for GHRH mRNA demonstrated labeling in numerous cells in the ventrolateral part of the ARC (36, 45) (Fig. 1B).|u8cz8e, 百拇医药
In agreement with previous reports (46) our in situ hybridization histochemistry showed AgRP (Fig. 1C) and NPY (Fig. 1D) mRNA-positive cells located in the ARC. In situ hybridization with oligoprobes for MCH (Fig. 1E) and prepro-OX (Fig. 1F) mRNA demonstrated labeling of numerous cells in the LHA, as previously reported (46, 47). In all cases, incubation of the sections with an excess (100-, 200-, and 500-fold) of the unlabeled probes displaced the positive signal (Fig. 1, right panels).
Treatment with ghrelin increases AgRP and NPY mRNA levels in the ARC of fed ratsl, http://www.100md.com
Using in situ hybridization analysis, a significant increase in AgRP (Fig. 2A) and NPY (Fig. 2B mRNA levels was observed in the ARC after treatment with ghrelin. This increase was transient, being evident 2 and 4 h after treatment in the case of AgRP and 2 h after treatment in the case of NPY. On the other hand, we did not find any change in the mRNA content of MCH, prepro-OX (in the LHA), GHRH (in the ARC), and SST (in the PeN) at any time point (2, 4, and 6 h) as assessed by in situ hybridization (Fig. 3).l, http://www.100md.com
fig.ommitteedl, http://www.100md.com
Figure 2. A, Upper panel, Autoradiographic images (magnification, x5) of representative brain coronal sections, incubated with a 35S-labeled antisense oligonucleotide AgRP probe, in the vehicle- and ghrelin-treated groups for 2 h. Areas delineated in both sections are shown at higher magnification in the bottom (magnification, x60). Lower panel, AgRP mRNA levels in the ARC of the described experimental groups. *, P < 0.05 vs. vehicle treated for 2 and 4 h, respectively. 3V, Third ventricle. B, Upper panel, Autoradiographic images (magnification, x5) of representative brain coronal sections, incubated with a 35S-labeled antisense olignucleotide NPY probe, in the vehicle-treated group and the ghrelin-treated group for 2 h. Areas delineated in both sections are shown at higher magnification in the bottom (magnification, x60). 3V, Third venticle. Lower panel, NPY mRNA levels in the ARC of the described experimental groups. *, P < 0.05 vs. vehicle treated for 2 h.
fig.ommitteed|-j, 百拇医药
Figure 3. Effect of ghrelin on mRNA levels of MCH in the LHA (A), prepro-OX in the LHA (B), GHRH in the ARC (C), and SST in the PeN (D).|-j, 百拇医药
Treatment with ghrelin increases NPY and AgRP mRNA levels in the ARC of fasted rats|-j, 百拇医药
Using in situ hybridization analysis, a significant increase in AgRP (Fig. 4A) and NPY (Fig. 4B) mRNA levels was observed in the ARC after 72 h of food deprivation. In fasted rats treated with ghrelin, we observed a further increase in AgRP and NPY mRNA content in the ARC 4 h after the treatment compared with fasted vehicle-treated rats (Fig. 4).|-j, 百拇医药
fig.ommitteed|-j, 百拇医药
Figure 4. A, Upper panel, Autoradiographic images (magnification, x1.6) of representative brain coronal sections, incubated with a 35S-labeled antisense oligonucleotide AgRP probe, in the vehicle-treated group for 4 h, the 72-h fasted group treated with vehicle for 4 h, and the 72-h fasted group treated with ghrelin for 4 h. Areas delineated in the sections are shown at higher magnification in the bottom (magnification, x15). 3V, Third ventricle. Lower panel, AgRP mRNA levels in the ARC of the described experimental groups. *, P < 0.05 vs. vehicle-treated for 2, 4, and 8 h, respectively; ***, P < 0.001 vs. vehicle-treated for 4 h; ##, P < 0.01, 72-h fasted treated with vehicle for 4 h vs. 72-h fasted treated with ghrelin for 4 h. B, Upper panel, Autoradiographic images of representative brain coronal sections, incubated with a 35S-labeled antisense oligonucleotide NPY probe, in the vehicle-treated group for 4 h, the 72-h fasted group treated with vehicle for 4 h, and the 72-h fasted group treated with ghrelin for 4 h. Areas delineated in the sections are shown at higher magnification in the bottom. 3V, Third ventricle. Lower panel, NPY mRNA levels in the ARC of the described experimental groups. *, P < 0.05 vs. vehicle treated for 2 h and 4 h, respectively; **, P < 0.01 vs. vehicle treated for 8 h; ***, P < 0.001 vs. vehicle for 4 h; #, P < 0.05, 72-h fasted treated with vehicle for 4 h vs. 72-h fasted treated with ghrelin for 4 h.
Ghrelin increases SST mRNA levels in the PeN of dwarf Lewis rats5, 百拇医药
Using in situ hybridization analysis, we detected a significant decrease in SST mRNA content in the PeN of dwarf Lewis rats compared with vehicle-treated, wild-type (age- and weight-matched) Lewis rats (Fig. 5A. Treatment with ghrelin increased SST mRNA levels in the PeN of dwarf Lewis rats 6 h after treatment, reaching levels observed in vehicle-treated (age- and weight-matched) Lewis rats (Fig. 5A). The GHRH mRNA content in the ARC was increased in dwarf Lewis rats compared with vehicle-treated wild-type (age- and weight-matched) Lewis rats. Ghrelin did not change GHRH mRNA levels in dwarf Lewis rats 6 h after treatment (Fig. 5B).5, 百拇医药
fig.ommitteed5, 百拇医药
Figure 5. A, Upper panel, Autoradiographic images (magnification, x1) of representative brain coronal sections, incubated with a 35S-labeled antisense oligonucleotide SST probe, in age-matched Lewis rats treated with vehicle for 6 h, weight-matched Lewis rats treated with vehicle for 6 h, dwarf Lewis rats treated with vehicle for 6 h, and dwarf Lewis rats treated with ghrelin for 6 h. Areas delineated in the sections are shown at higher magnification in the bottom (magnification, x10). 3V, Third ventricle. Lower panel, SST mRNA levels in the PeN of the described experimental groups. ***, P < 0.001 vs. dwarf Lewis vehicle treated for 6 h; ###, P < 0.001 vs. age-matched Lewis rats treated with vehicle for 6 h; !!!, P < 0.001 vs. weight-matched Lewis rats treated with vehicle for 6 h. B, Upper panel, Autoradiographic images (x1) of representative brain coronal sections, incubated with a 35S-labeled antisense oligonucleotide GHRH probe, in age-matched Lewis rats treated with vehicle for 6 h, weight-matched Lewis rats treated with vehicle for 6 h, dwarf Lewis rats treated with vehicle for 6 h, and dwarf Lewis rats treated with ghrelin for 6 h. Areas delineated in the sections are shown at higher magnification in the bottom (x6). 3V, Third ventricle. Lower panel, GHRH mRNA levels in the ARC of the described experimental groups. ###, P < 0.001 vs. age-matched Lewis rats treated with vehicle for 6 h; !!!, P < 0.001 vs. weight-matched Lewis rats treated with vehicle for 6 h.(Luisa M. Seoane Miguel López Sulay Tovar Felipe F. Casanueva Rosa Señarís and Carlos Diégu)