Reduction in Adiposity Affects the Extent of Afferent Projections to Growth Hormone-Releasing Hormone and Somatostatin Neurons and the Degre
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《内分泌学杂志》
Prince Henry’s Institute of Medical Research (J.I., I.J.C.), Clayton, Victoria 3168, Australia
AgResearch Invermay (T.R.M.), Mosgiel, New Zealand
Institut National de la Sante et de la Recherche Medicale Unité 378 (P.C.), Institut Francois Magendie, F-33077 Cedex, Bordeaux, France
Abstract
Various neuropeptides and neurotransmitters affect GH secretion by acting on GHRH and somatostatin (SRIF) cells. GH secretion is also affected by alteration in adiposity, which could be via modulation of GHRH and SRIF cells. We quantified colocalization of neuropeptides in GHRH and SRIF cells and afferent projections to these cells in lean (food restricted) and normally fed sheep (n = 4/group). The number of GHRH-immunoreactive (IR) cells in the arcuate nucleus was higher in lean animals, but the number of SRIF-IR cells in the periventricular nucleus was similar in the two groups. A subpopulation of GHRH-IR cells colocalized neuropeptide Y in lean animals, but this was not seen in normally fed animals. GHRH/galanin (GAL) colocalization was higher in lean animals with no difference in numbers of GHRH/tyrosine hydroxylase or GHRH/GAL-like peptide cells. SRIF/enkephalin colocalization was lower in lean animals. The percentage of GHRH neurons receiving SRIF input was similar in lean and normally fed animals, but more GHRH cells received input from enkephalin afferents in normally fed animals. The percentage of SRIF cells receiving GHRH, neuropeptide Y, GAL, and orexin afferents was higher in lean animals. These findings provide an anatomical evidence of central mechanism(s) by which appetite-regulating peptides and dopamine could regulate GH secretion. Increased input to SRIF cells in lean animals may be inhibitory and permissive of increased GH. The appearance of NPY in GHRH cells of lean animals may be a mechanism for regulation of increasing GH secretion with reduced adiposity.
Introduction
GH PLAYS AN plays an important role in the maintenance of body growth and lean body mass (1, 2). Secretion of GH from pituitary somatotropes is stimulated by GHRH and inhibited by somatostatin (SRIF) (1, 2). The GHRH neurones from the arcuate nucleus (ARC) and the hypophysiotropic SRIF neurons from the periventricular nucleus (PeV) of the hypothalamus project to the external zone of the median eminence (ME) and secrete peptides into the hypophysial portal circulation to act upon the pituitary somatotropes (1, 2, 3, 4, 5).
Reduction in body weight causes an increase in the secretion of GH in sheep (4, 6, 7) and humans (8, 9, 10) Nutritional status or the level of adiposity also influences the expression of genes encoding neuropeptide Y (NPY), agouti-related protein, enkephalin (ENK), melanin-concentrating hormone (MCH), cocaine- and amphetamine-regulated transcript, GHRH, and SRIF in the ovine hypothalamus (7, 11, 12) and in rodents (for review, see Ref.13). In sheep, hypothalamic expression of genes for proopiomelanocortin, dynorphin (DYN), and orexin (ORX) do not change with altered levels of adiposity in sheep hypothalamus (7, 14).
It has also been shown that GHRH and SRIF levels, measured by push-pull perfusion of the ME, change with body condition in rats (15). Thus, changes in GH secretion could be effected by action of peripheral factors (such as leptin) on the appetite-regulating peptide (ARP) producing cells in the hypothalamus that, in turn, affect GHRH and SRIF secretion into hypophyseal portal blood. Alternatively, peripheral factors could act directly on GHRH and/or SRIF cells. The signaling form of the leptin receptor is found in GHRH and SRIF cells (16, 17), as well as the hypothalamic cells that produce the ARP mentioned above (16, 18). Ghrelin is another hormone that acts on GHRH and SRIF cells (19, 20) and stimulates GH secretion and food intake through central mechanisms (21, 22, 23). In particular, the ghrelin receptor is found in GHRH cells (24).
The regulation of GH secretion is very complex and appears to involve a variety of neurotransmitters and neuropeptides, including ARP (for review, see Refs.1 and 2). Nevertheless, there is limited knowledge of the extent of colocalization of different peptides in GHRH and SRIF cells, and little is known of the afferent input to these cells from ARP-producing cells in the hypothalamus. Galanin (GAL) (25), NPY (26), and tyrosine hydroxylase (TH) (27) are produced by subpopulations of GHRH neurons in the rat ARC, and NPY is colocalized in GHRH-producing cells of the infundibular nucleus of the human brain (28). To our knowledge, no similar data exist for other important laboratory species, such as sheep. Furthermore, no information is available regarding the effects of alteration in the adiposity on the degree of colocalization and/or afferent input to GHRH and SRIF cells in any species. Therefore, the aim of the present study was to determine the extent of colocalization of neuropeptides within GHRH and SRIF cells and the extent of afferent input (projections) to these cells from the ARP systems of the hypothalamus in animals of different body weight. To this end, we used sheep as an experimental model because GH physiology in the sheep resembles that of the human (4, 6, 7, 8, 9, 10).
Materials and Methods
Ethics
All procedures and tissue collection were carried out with the prior approval of the Animal Experimentation Ethics Committees of Monash Medical Centre and the Victorian Institute of Animal Science.
Animals
Adult Corriedale ewes were ovariectomized (OVX) at least 1 month before use to prevent the cyclic and seasonal alterations in the secretion and action of gonadal steroids that could create differences between animals, especially those of different body condition. A protocol for the dietary manipulation of sheep and the endocrine and metabolic consequences has been described previously (7, 12). Briefly, adult OVX ewes with a mean (±SEM) body weight of 50 ± 1 kg were randomly divided into two groups (n = 4) and were either fed an ad libitum diet of pasture hay (normally fed) or a restricted diet of approximately 500 g grass hay per day per sheep (lean). The lean animals also received straw to add bulk to their diet. Both groups were maintained under normal external environmental conditions and were weighed monthly. Once the body weight of the lean animals fell to less than 40 kg, body weight was held at this level for 3 months by slightly altering the food provided at the discretion of the animal caregiver. At the time of tissue collection, normally fed animals weighed 60.2 ± 0.8 kg, and lean animals weighed 39 ± 1 kg (P < 0.001). Such dietary restriction results in alterations in increased GH mRNA levels in the pituitary gland (4), increased peripheral plasma levels of GH (7, 12), reduced hypophyseal portal levels of SRIF (4), increased mRNA expression for GHRH, and reduced expression of mRNA for SRIF (12) and altered expression of genes for ARP in the hypothalamus (7, 12).
Tissue collection and processing
Single jugular blood samples were collected at 1000 h before perfusion for the measurement of GH and leptin levels by RIA. The animals were not fasted before necropsy, which was carried out between 1000 and 1200 h. The sheep were administered 25,000 IU heparin (iv) 5 min before being killed by a lethal dose of sodium pentobarbital (Lethabarb, May & Baker Pty Ltd., Melbourne, Australia). The heads were flushed via the carotid arteries with 2 liters heparinized (12,500 IU/liter) 0.9% saline followed by 1 liter Zamboni’s fixative in 0.1 M phosphate buffer (PB; pH 7.4) and 0.5 liter of Zamboni’s fixative containing 20% sucrose. The preoptic area/hypothalamic blocks were dissected from the brains and sunk in 30% sucrose in 0.1 M PB for 4–5 d at 4 C. The tissue blocks were frozen on powdered dry ice and stored at –20 C until used. Coronal sections of 40 μm were cut on a cryostat and stored in cryoprotectant solution until used for immunohistochemistry (17, 18).
Double-labeling immunohistochemistry
The details of the antisera used for double-labeling immunohistochemistry are given in Table 1. To determine the extent of colocalization and/or input to GHRH- and SRIF-immunoreactive (IR) cells from a range of hypothalamic neuropeptides, four anatomically matched sections at intervals of 480 μm through the rostro-caudal limits of ARC and PeV region of each lean and normally fed animal were processed using free-floating double-labeling immunohistochemistry (29, 30). Briefly, the sections were washed in 0.05 M PBS and treated with 1% sodium borohydride (Sigma-Aldrich, St. Louis, MO) in 0.1 M PB for 20 min and washed. After blocking (5% normal goat serum and 0.3% Triton X-100 in 0.1 M PB) for 30 min, sections were incubated with either rabbit polyclonal anti-GHRH or anti-SRIF antibody for 48 h at 4 C. After washing, sections were then incubated with goat antirabbit Alexa 546 (red, 1:500; Molecular Probes, Eugene, OR) in 0.1 M PB for 1 h and washed. Sections were blocked in normal donkey or goat serum for 30 min. Subsequently, sections were incubated with a relevant primary antibody for immunostaining of the second neuropeptide/neurotransmitter of interest for 48 h at 4 C, and bound antibodies were detected using goat antimouse Alexa 488 (green, 1:500; Molecular Probes). In some cases, binding of primary antibody was visualized using goat antirabbit, horse antimouse, or goat antiguinea pig biotinylated secondary antibody (1:250; Vector Laboratories, Burlingame, CA) and avidin-fluorescein isothiocyanate (FITC) conjugate (1:500; Pierce, Rockford, IL) as appropriate. Sections were washed in 0.1 M PB and wet-mounted with antifade medium (Dako Corp., Carpenteria, CA) onto gel-coated slides. To determine the codistribution of DYN with GHRH cells, consecutive sections were processed using single-labeling procedures described above because both of the primary antibodies were raised in rabbits. Sections were then counterstained with 0.3% Sudan Black to block nonspecific autoimmunofluorescence.
Immunohistochemical control procedures
In cases where these antibodies had not been previously used in sheep, specificity of immunostaining was validated by preabsorption with the relevant peptide and negative controls (omission of the primary antibody) as previously described (17, 18, 29, 30). A negative control was always included in each procedure. All immunostaining was abolished after control procedures (data not shown).
Microscopic analysis
Microscopy.
Tissue sections processed for double-labeling immunofluorescence were analyzed using an Olympus BMX 50 microscope (Olympus Corp. Ltd., Tokyo, Japan) equipped with mercury light and Texas red and FITC band filter system. Double labeling was visualized by switching between the filters, single- and double-labeled cells were counted unilaterally in each section for each animal, and the total was recorded for each animal for each group. Data are presented as mean (±SEM) percentage of double-labeled cells.
Confocal microscopy.
The use of confocal microscopy to identify putative afferent input to neurons in the brain has been successfully used in our laboratory (29, 31, 32, 33) as well as others (34, 35). This allows examination of a larger number of cells than is feasible with electron microscopy. Semiquantitative estimates of close contacts or putative inputs to GHRH- and SRIF-IR cells from systems producing various peptides and neurotransmitters were made using Fluoview (FV 300) Laser Scanning Confocal Microscope System (Olympus Corp. Ltd.) with a 60x (1.4 numerical aperture) objective. FITC and Texas red were excited simultaneously with the 488- and 568-nm lines of an argon ion laser system. To determine the relative percentage of close contacts between IR varicosities and GHRH-IR and SRIF-IR, the cells were examined unilaterally in anatomically matched sections from each animal (n = 4/group). Cells were randomly selected and sampled using a 60x objective (1.4 numerical aperture) in an area of 0.35 mm2 determined by a grid in the eye piece (29, 31). The grid was moved to five different fields in the ARC and PeV regions, and GHRH-IR and SRIF-IR cells were counted. Optical sections of 1-μm thickness were captured, and two separate stacks of images were generated, one for each fluorophore. The optical sections were then merged to give a composite image. In addition, pairs of 1-μm optical images in the same plane for each fluorophore were merged individually and examined using Adobe PhotoShop computer software (version 5.2; Adobe Systems, Mountain View, CA) to determine whether putative close contacts were real. Yellow pixilation (yellow labeling after colocalization of red and green) or close apposition of green and red fluorescence, which was consistent in three to five individually merged images, were considered to be putative synaptic contacts between axon terminals and perikarya. The data were analyzed by ANOVA and presented as mean (±SEM) percentage of GHRH-IR and SRIF-IR cells receiving putative input. Confocal images demonstrating double-labeling and close contacts were prepared as photomicrographs using Adobe PhotoShop 5.2 software. In all cases, only brightness and contrast were adjusted. All data were collected and analyzed by a single investigator.
RIA
Plasma GH concentrations were measured in one assay (sensitivity, 0.5 ng/ml) according to the method of Thomas et al. (36) with NIDDK-oGH-1–4 as standard and NIDDK-anti-oGH-2 antiserum. Plasma leptin levels were measured in one assay (sensitivity, 100 pg/ml) by the method of Blache et al. (37).
Results
Distribution of GHRH- and SRIF-IR cells
Food restriction significantly reduced mean body weight (P < 0.001), omental fat weight (P < 0.0001), and mean plasma leptin levels (P < 0.03) and significantly (P < 0.05) increased mean plasma GH levels (Table 2). There was a greater (P < 0.01) mean (±SEM) number of GHRH-IR cells in the ARC of lean animals than in normally fed animals (Table 1 and Fig. 1, A and B). The mean number of hypophysiotropic SRIF cells in the PeV did not differ significantly in lean and normally fed animals (Table 2 and Fig. 1, C and D). These findings concur with the earlier in situ hybridization studies in the sheep (12).
Colocalization in GHRH and SRIF cells
The distribution of cells containing NPY, ORX, MCH, TH, GAL, and long-form leptin receptor (Ob-Rb) was similar to that which we have reported previously for the sheep hypothalamus (17, 18). GAL-like peptide (GALP)-IR cell bodies were present in the mediolateral division of the ARC (see Fig. 3H).
Double-labeling procedures revealed that virtually 100% of GHRH cells expressed Ob-Rb (Fig. 2C, arrows). We have reported the colocalization of Ob-Rb to SRIF cells (17) and ARP producing cells previously (18). Subpopulations of GHRH-IR cells were also immunostained for NPY, GAL, GALP, and TH. The extent of peptidergic colocalization within GHRH-IR cells is shown in Table 3, and examples are shown in Fig. 3. Colocalization between NPY and GHRH-IR cells was observed in lean animals only (17.25 ± 1.10%). The mean (±SEM) percent colocalization of GHRH-IR with GAL-IR was higher (P < 0.05) in lean animals than in normally fed animals. Only a few DYN-IR cells were seen in the lateral division of the ARC, and processing of consecutive sections showed that these did not overlap with the distribution of GHRH-IR cells (data not shown).
TH-, ENK-, and DYN-IR cells were found in the PeV, but no cells were found to be IR for NPY, GAL, or GALP in this region. Despite overlapping distribution, DYN-IR cells were distinct from SRIF-IR cells (data not shown). A subpopulation of SRIF-IR cells also immunostained for ENK (Fig. 4, A–C) and TH (Fig. 4, D–F). The estimate of colocalization between ENK and SRIF was lower (P < 0.01) in lean animals than in normally fed animals. A subpopulation of SRIF-IR cells also immunostained for TH in the PeV, but the extent of this colocalization was similar in lean and normally fed animals (Table 3).
Input to GHRH-IR cells
ENK-, SRIF-, and ORX-IR fibers were present in all divisions of the ARC. The extent of close contacts (putative input) to GHRH cells from these various afferents is presented in Table 4, and examples are shown in Fig. 5. Confocal microscopic analysis revealed that SRIF- and ENK-IR fibers/varicosities were in close contact to GHRH-IR cells (Fig. 5), but ORX-IR fibers did not come into close contact with GHRH-IR cells. The mean (±SEM) percentage of GHRH-IR cells receiving input from ENK-IR was significantly (P < 0.05) lower in the lean than in the normally fed animals, but the percent age of GHRH-IR cells receiving input from SRIF-IR fibers did not differ between the two groups (Table 4). No input to GHRH-IR cells was seen from fibers/varicosities that were IR for NPY, GAL, GALP, ORX, and MCH.
Input to SRIF cells in the PeV
The extent of afferents to SRIF cells is seen in Table 5, and examples are shown in Fig. 6. Fibers and varicosities that immunostained for GHRH, NPY, GAL, and ORX were extensively distributed throughout the PeV, but there was only sparse distribution of fibers/varicosities that were IR for MCH in this region. Confocal analysis of the SRIF-IR cells indicated close apposition to varicosities that were IR for GHRH, NPY, GAL, and ORX (Fig. 6). The extent of these close appositions was significantly higher (P < 0.01) for GHRH and NPY and higher (P < 0.05) for GAL and ORX in the lean animals than in the normally fed animals (Table 5).
Discussion
This study provides, for the first time, anatomical evidence on the potential role of various ARP in the central regulation of GHHR and SRIF cells, which is relevant to the control of GH secretion from the anterior pituitary somatotropes. Previously, only minimal information was available regarding peptides that colocalize to GHRH and SRIF neurons and in regard to the neuronal input to these neuroendocrine cells. The present work details the extent of colocalization and afferent input from ARP-producing cells to the GHRH and SRIF cells in the ovine brain. These data thus provide an anatomical substrate for further studies to define neural control of GHRH and SRIF secretion and GH secretion, especially the role of ARP.
In sheep and humans, reduction in the body weight and/or fasting is associated with increased plasma levels of GH (4, 6, 7, 8, 9, 10, 11, 12). In accordance with this, the number of GHRH-IR cells was higher in lean animals than in normally fed animals, suggesting increased synthesis of GHRH; this may lead to greater secretion of GHRH in lean animals, but this has not been demonstrated. Indeed, our earlier work showed no difference in hypophysial portal levels (4), but this could be due to difficulties in measurement of GHRH in plasma due to high levels of nonbiologically active GHRH immunoreactive protein fragments secreted from ectopic organs (38) and possible binding proteins. The present findings are consistent with earlier results obtained by in situ hybridization (12). The number of SRIF-IR cells did not change with reduction in body weight, although we previously reported reduced mRNA levels for SRIF in the PeV of lean OVX ewes (12), and SRIF levels in hypophyseal portal blood are reduced with lowered body weight (4). It is possible, therefore, that reduced gene expression and reduced secretion occurs, but there is no change in the number of SRIF cells. On the other hand, our earlier report indicated that reduction in SRIF mRNA levels was confined to the rostral region of the PeV, and the present work reports on the entire extent of this region.
Colocalization of neuropeptides/transmitters and Ob-Rb in GHRH cells
Reduction in body weight of sheep by food restriction is associated with reduction in the amount of omental adipose tissue and a reduction in circulating leptin levels. This could have a direct effect on the SRIF and GHRH cells. Ob-Rb has been reported in both of these cell types, but we confirmed that this was the case in the ovine GHRH cells. Although colocalization of leptin receptor and GHRH has been reported in the rat (16), the extent of this was previously unknown, and the earlier study did not distinguish between the long and short forms of the receptor. We found that virtually 100% of these cells express Ob-Rb, indicating that changes in the function of these cells with altered body weight could be due to direct effects of leptin. Ob-Rb mRNA expression also changes with alterations in the body weight, being up-regulated with food restriction and reduced body weight (39, 40). Because more GHRH cells are seen by immunohistochemistry in lean animals (present study), and there is increased gene expression for GHRH (12), the increase in activity of these cells may be accompanied by increased level of Ob-Rb expression in GHRH cells, although this remains to be determined. We recently showed that the level of expression of Ob-Rb increased in NPY-producing cells of the ARC in lean animals (40), suggesting that function of the receptors could increase. A similar increase in function could occur in GHRH cells, although detailed cellular analysis would be required to ascertain whether this is the case. Because reduction in body weight also leads to changes in levels of circulating metabolites such as free fatty acids (7), it is possible that these changes may affect the GH axis.
Subpopulations of GHRH-IR cells also immunostained for NPY, GAL, GALP, and TH. Our finding that NPY is produced in a subset of GHRH cells in the ARC of sheep concurs with data obtained from rat (26) and human (28) brains. We showed cellular colocalization of NPY and GHRH in 17% of GHRH cells in the ARC of lean sheep, but this was not seen in normally fed animals. There is substantial up-regulation of NPY expression in the lean condition (7, 40), and the present work shows that at least part of this may occur in GHRH cells. Thus, a subpopulation of GHRH cells appears to be activated by reduction in body weight, accompanied by increased expression of NPY in the same cells. NPY stimulates GH secretion in the sheep (41, 42), bovine (43), and human (44), so the up-regulation of the GH axis that occurs with reduced body weight could be due to mechanisms, within GHRH cells, that involve NPY. On the other hand, NPY reduces GH secretion in the rat (45, 46, 47), but reduction in body weight causes increased GH secretion in this species (41).
Intracerebroventricular infusion of leptin causes a small increase in GH secretion in sheep (48), but the effect is similar in lean and normally fed animals (48). Leptin, however, reduces NPY expression in animals of normal body weight (49) and has no effect on NPY expression in lean animals (Clarke, I. J., unpublished data), so the effect is unlikely to be mediated through NPY. It seems more likely that the effect of leptin is directly on the SRIF and GHRH cells (vide supra) or by neuronal afferents that are not NPY cells.
TH immunostaining identified dopaminergic neurons, and previous reports indicated between 32 (27) and 90% (50) colocalization of TH and GHRH in cells of the rat mediobasal hypothalamus. We found that approximately one quarter of GHRH cells also immunostained for TH, which was unaffected by alteration in body weight. Because dopamine (DA) is not secreted into the hypophysial portal blood of sheep (51), it is unlikely that hypothalamic DA and GHRH have a synergistic effect on GH release from pituitary somatotropes as suggested by Serri et al. (52) for the rat. Autocrine regulation of GHRH synthesis by DA remains a possibility, at least in a subset of the cells. GAL colocalizes with GHRH in the rat hypothalamus (25, 53), and exogenous administration of GAL stimulates GH secretion in rats (54, 55), sheep (56), and humans (57, 58). GAL has also been reported to stimulate GHRH and SRIF release from the rat (59) and bovine (60) ME. A small subpopulation of GHRH-IR cells immunostained for GAL, with slightly, but significantly, increased numbers of costaining cells in the lean animals. Although Barker-Gibb et al. (6) reported an increase in GAL immunoreactivity in the ovine ME of lean OVX ewes, GAL mRNA expression was not altered significantly (7). Colocalization of GAL within a subpopulation of GHRH-IR cells supports the notion that the former has a role in the modulation of GHRH secretion (56). Because only 2% of GHRH-IR cells had GALP-like immunoreactivity, a significant role for this peptide in autocrine function seems unlikely.
Afferent input to GHRH cells
Confocal microscopy provides a good estimation of synaptic interaction and close contacts between afferents and cell bodies. Although this does not provide definitive identification of synapses, identification is being widely used to assess the synaptic interactions (29, 31, 32, 33, 34, 35) because it allows the screening of a much larger number of cells. Our confocal microscope analysis strongly suggests that GHRH-IR cells receive input from SRIF and ENK systems. As in the rat (61, 62), SRIF provides input to the one third of the GHRH neurons in the sheep. GHRH mRNA expression and GH levels are elevated, whereas SRIF mRNA expression is reduced in the lean condition (12), so this might be brought about by reciprocal regulation of GHRH and SRIF cells.
SRIF has an inhibitory influence on GHRH cells (1, 2), but the extent of SRIF input to GHRH cells (cells receiving input) does not differ with alteration in body weight, even though there are increased numbers of GHRH-IR cells in lean animals. The precise origin of SRIF projections to GHRH cells is not known but could be either the PeV or the ventromedial hypothalamic nucleus, in which many SRIF neurons are found (5, 14). At least some of the SRIF cells of the ventromedial nucleus project to the ARC nucleus as revealed by retrograde labeling studies (Qi, Y. and I. J. Clarke, unpublished data). Expression of SRIF mRNA in both regions is reduced with lowered body weight (12).
ENK are opioid peptides (Leu-ENK and met-ENK), derived from the preproenkephalin gene. These peptides bind predominantly to the -subtype of opioid receptors and stimulate GH secretion rodents (63) and humans (64). We found evidence of reduced ENK input to GHRH-IR cells in lean animals, suggesting some plasticity of input regulated by body condition. ENK-producing neurons are localized in the PeV, paraventricular hypothalamic nucleus, and ventromedial hypothalamic nucleus, and ENK gene expression is differentially altered in all of these regions with alteration in the body weight in the sheep (7). However, the exact source of ENK input to the GHRH cells is not clear because ARC nucleus receives afferents from PeV, paraventricular nucleus, and ventromedial hypothalamus region (Qi, Y. and I. J. Clarke, unpublished data). The extent of leptin receptor and ghrelin receptor in ENK neurons has been not reported for any species, so it is not known whether these factors are important in the regulation of this system. ENK input to a subpopulation of GHRH cells provides an evidence of a mechanism by which opioids could regulate GHRH and GH secretion, but the paradoxical reduction in lean animals of afferents from a system that stimulates GH secretion requires further investigation.
Colocalization of neuropeptides/neurotransmitters and projections to SRIF cells
Although SRIF-producing neurons are found in several areas of the hypothalamus (5), those that project to the external zone of the ME to secrete the peptide into hypophyseal portal blood are confined to the PeV (1, 2, 3). A subset (around 11%) of SRIF cells also immunostained for ENK, and we report the novel finding that the extent of this colocalization is reduced in lean animals. The means by which ENK stimulates GH secretion could be autocrine, within SRIF cells, to reduce expression of the gene for SRIF. Previous studies in our laboratory have shown that ENK expression is increased and SRIF expression is reduced in the PeV with reduction in the body weight of OVX ewes (7, 12), suggestive of an association between these peptides. A small subset of SRIF-IR cells (3%) was also immunopositive for TH, which is another novel finding, but the extent of colocalization did not change with altered body weight.
Afferent input to SRIF cells
Our data strongly suggest that subpopulations (up to one quarter) of SRIF neurons in the PeV of the ewe brain receive afferent input from GHRH, NPY, GAL, and ORX systems. For each of these systems, putative input to SRIF neurons was higher in lean animals than in normally fed animals. There is increased expression of GHRH, NPY, and GAL in the ARC of lean animals (6, 7, 12), and this could explain the increase in input to SRIF-producing cells in the PeV. Such a direct link, specifically relating to these systems remains to be demonstrated by detailed tracing studies, but previously demonstrated bidirectional connections between ARC and PeV in rat (61, 62) and sheep (65) provide evidence of reciprocal communication between these two regions. Increased expression of GHRH and increased GHRH input to SRIF cells in lean animals may be a means of inhibiting SRIF synthesis and release into the portal circulation, thus allowing increased GH secretion from the pituitary.
NPY negatively regulates GH secretion and is thought to negatively regulate SRIF cells in the rat (66). On the other hand, NPY stimulates GH secretion in the sheep and other species (vide supra). Increased input to SRIF cells located in the PeV from NPY afferents in lean animals could negatively regulate the SRIF cells, consistent with a stimulatory effect on GH secretion. In lean animals, we found increased colocalization of GAL and GHRH in the ARC and increased afferent input to PeV-located SRIF cells. The latter could be related to the former, but tracing studies are required to confirm this. Because centrally administered GAL stimulates GH secretion in sheep (56), GAL may be inhibitory to SRIF, and increased input to SRIF cells in the PeV of lean animals would be consistent with increased GH secretion in this condition.
ORX-producing cells are localized in the lateral hypothalamus, the perifornical area, and the dorsomedial nucleus of the hypothalamus, and these cells send widespread projections to various regions of the brain in the sheep (31). ORX functions to regulate food intake and neuroendocrine cells as well as in arousal (for review, see Ref.67). We previously reported that ORX-IR fibers were distributed widely in the preoptic area and the PeV region, with an evidence of direct synaptic input to GnRH cells (31), and we now report evidence of direct input to the SRIF cells of the PeV. This provides an anatomical substrate to support recent in vitro and in vivo studies that demonstrate a role for ORX in the regulation of GH, at least in the rat (68, 69, 70). To support a role for ORX in the regulation of SRIF cells, the ORX type-1 receptor is found on SRIF cells of the PeV region of the rat brain (71). In the sheep, however, Sartin et al. (72) found that intracerebroventricular (0.03, 0.3, or 3 μg/kg body weight), or iv infusion (3 μg/kg body weight) of porcine ORX-B did not affect GH secretion in the sheep, and nothing is known about ORX action on SRIF secretion.
Ghrelin is one peripheral factor that regulates GH secretion, with a possible role in the regulation of food intake (21, 22). Because exogenous administration of ghrelin induces fos expression in the ORX cells of the lateral hypothalamic area in rodents (19), and our earlier tracing studies indicated a pathway from the lateral hypothalamic area to the PeV/preoptic area of the ovine brain (31), it is possible that indirect regulation of ORX cells by ghrelin is a means of regulation of SRIF cells. ORX cells also express Ob-Rb, so regulation of these cells by leptin (16, 18) is another means by which SRIF function could be indirectly affected by altered body weight (that alters the level of leptin in plasma and alters Ob-Rb levels).
The findings of this study are summarized in Fig. 7. Subpopulations of GHRH-producing neurons in the ARC also produce NPY, TH, GAL, and GALP, and there is evidence of afferent input from neuronal systems that produce ENK and SRIF. Subpopulations of SRIF-producing neurons in the PeV also produce ENK and TH and appear to receive input from neuronal systems that produce GHRH, NPY, GAL, and ORX. These findings provide an anatomical evidence of potential role of ARP in the central regulation of GHRH and SRIF secretion and subsequent changes in the GH secretion from the anterior pituitary with alteration in the adiposity.
Acknowledgments
We thank Mr. Bruce Doughton, Ms. Karen Briscoe, and Ms. Lynda Morrish for animal care and tissue collection and Sue Panckridge for assisting in preparing the photomicrographs. We also thank Dr. D. Blache (University of Western Australia, Perth, Western Australia) for leptin assays and NIH for RIA reagents for GH assays. Dr. Iqbal was supported by Prince Henry’s Institute of Medical Research.
Footnotes
Abbreviations: ARC, Arcuate nucleus; ARP, appetite-regulating peptide; DA, dopamine; DYN, dynorphin; ENK, enkephalin; FITC, fluorescein isothiocyanate; GAL, galanin; GALP, GAL-like peptide; IR, immunoreactive; MCH, melanin-concentrating hormone; ME, median eminence; NPY, neuropeptide Y; Ob-Rb, long-form leptin receptor; ORX, orexin; OVX, ovariectomized; PB, phosphate buffer; PeV, anteroventral periventricular nucleus; SRIF, somatostatin-releasing inhibiting factor; TH, tyrosine hydroxylase.
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AgResearch Invermay (T.R.M.), Mosgiel, New Zealand
Institut National de la Sante et de la Recherche Medicale Unité 378 (P.C.), Institut Francois Magendie, F-33077 Cedex, Bordeaux, France
Abstract
Various neuropeptides and neurotransmitters affect GH secretion by acting on GHRH and somatostatin (SRIF) cells. GH secretion is also affected by alteration in adiposity, which could be via modulation of GHRH and SRIF cells. We quantified colocalization of neuropeptides in GHRH and SRIF cells and afferent projections to these cells in lean (food restricted) and normally fed sheep (n = 4/group). The number of GHRH-immunoreactive (IR) cells in the arcuate nucleus was higher in lean animals, but the number of SRIF-IR cells in the periventricular nucleus was similar in the two groups. A subpopulation of GHRH-IR cells colocalized neuropeptide Y in lean animals, but this was not seen in normally fed animals. GHRH/galanin (GAL) colocalization was higher in lean animals with no difference in numbers of GHRH/tyrosine hydroxylase or GHRH/GAL-like peptide cells. SRIF/enkephalin colocalization was lower in lean animals. The percentage of GHRH neurons receiving SRIF input was similar in lean and normally fed animals, but more GHRH cells received input from enkephalin afferents in normally fed animals. The percentage of SRIF cells receiving GHRH, neuropeptide Y, GAL, and orexin afferents was higher in lean animals. These findings provide an anatomical evidence of central mechanism(s) by which appetite-regulating peptides and dopamine could regulate GH secretion. Increased input to SRIF cells in lean animals may be inhibitory and permissive of increased GH. The appearance of NPY in GHRH cells of lean animals may be a mechanism for regulation of increasing GH secretion with reduced adiposity.
Introduction
GH PLAYS AN plays an important role in the maintenance of body growth and lean body mass (1, 2). Secretion of GH from pituitary somatotropes is stimulated by GHRH and inhibited by somatostatin (SRIF) (1, 2). The GHRH neurones from the arcuate nucleus (ARC) and the hypophysiotropic SRIF neurons from the periventricular nucleus (PeV) of the hypothalamus project to the external zone of the median eminence (ME) and secrete peptides into the hypophysial portal circulation to act upon the pituitary somatotropes (1, 2, 3, 4, 5).
Reduction in body weight causes an increase in the secretion of GH in sheep (4, 6, 7) and humans (8, 9, 10) Nutritional status or the level of adiposity also influences the expression of genes encoding neuropeptide Y (NPY), agouti-related protein, enkephalin (ENK), melanin-concentrating hormone (MCH), cocaine- and amphetamine-regulated transcript, GHRH, and SRIF in the ovine hypothalamus (7, 11, 12) and in rodents (for review, see Ref.13). In sheep, hypothalamic expression of genes for proopiomelanocortin, dynorphin (DYN), and orexin (ORX) do not change with altered levels of adiposity in sheep hypothalamus (7, 14).
It has also been shown that GHRH and SRIF levels, measured by push-pull perfusion of the ME, change with body condition in rats (15). Thus, changes in GH secretion could be effected by action of peripheral factors (such as leptin) on the appetite-regulating peptide (ARP) producing cells in the hypothalamus that, in turn, affect GHRH and SRIF secretion into hypophyseal portal blood. Alternatively, peripheral factors could act directly on GHRH and/or SRIF cells. The signaling form of the leptin receptor is found in GHRH and SRIF cells (16, 17), as well as the hypothalamic cells that produce the ARP mentioned above (16, 18). Ghrelin is another hormone that acts on GHRH and SRIF cells (19, 20) and stimulates GH secretion and food intake through central mechanisms (21, 22, 23). In particular, the ghrelin receptor is found in GHRH cells (24).
The regulation of GH secretion is very complex and appears to involve a variety of neurotransmitters and neuropeptides, including ARP (for review, see Refs.1 and 2). Nevertheless, there is limited knowledge of the extent of colocalization of different peptides in GHRH and SRIF cells, and little is known of the afferent input to these cells from ARP-producing cells in the hypothalamus. Galanin (GAL) (25), NPY (26), and tyrosine hydroxylase (TH) (27) are produced by subpopulations of GHRH neurons in the rat ARC, and NPY is colocalized in GHRH-producing cells of the infundibular nucleus of the human brain (28). To our knowledge, no similar data exist for other important laboratory species, such as sheep. Furthermore, no information is available regarding the effects of alteration in the adiposity on the degree of colocalization and/or afferent input to GHRH and SRIF cells in any species. Therefore, the aim of the present study was to determine the extent of colocalization of neuropeptides within GHRH and SRIF cells and the extent of afferent input (projections) to these cells from the ARP systems of the hypothalamus in animals of different body weight. To this end, we used sheep as an experimental model because GH physiology in the sheep resembles that of the human (4, 6, 7, 8, 9, 10).
Materials and Methods
Ethics
All procedures and tissue collection were carried out with the prior approval of the Animal Experimentation Ethics Committees of Monash Medical Centre and the Victorian Institute of Animal Science.
Animals
Adult Corriedale ewes were ovariectomized (OVX) at least 1 month before use to prevent the cyclic and seasonal alterations in the secretion and action of gonadal steroids that could create differences between animals, especially those of different body condition. A protocol for the dietary manipulation of sheep and the endocrine and metabolic consequences has been described previously (7, 12). Briefly, adult OVX ewes with a mean (±SEM) body weight of 50 ± 1 kg were randomly divided into two groups (n = 4) and were either fed an ad libitum diet of pasture hay (normally fed) or a restricted diet of approximately 500 g grass hay per day per sheep (lean). The lean animals also received straw to add bulk to their diet. Both groups were maintained under normal external environmental conditions and were weighed monthly. Once the body weight of the lean animals fell to less than 40 kg, body weight was held at this level for 3 months by slightly altering the food provided at the discretion of the animal caregiver. At the time of tissue collection, normally fed animals weighed 60.2 ± 0.8 kg, and lean animals weighed 39 ± 1 kg (P < 0.001). Such dietary restriction results in alterations in increased GH mRNA levels in the pituitary gland (4), increased peripheral plasma levels of GH (7, 12), reduced hypophyseal portal levels of SRIF (4), increased mRNA expression for GHRH, and reduced expression of mRNA for SRIF (12) and altered expression of genes for ARP in the hypothalamus (7, 12).
Tissue collection and processing
Single jugular blood samples were collected at 1000 h before perfusion for the measurement of GH and leptin levels by RIA. The animals were not fasted before necropsy, which was carried out between 1000 and 1200 h. The sheep were administered 25,000 IU heparin (iv) 5 min before being killed by a lethal dose of sodium pentobarbital (Lethabarb, May & Baker Pty Ltd., Melbourne, Australia). The heads were flushed via the carotid arteries with 2 liters heparinized (12,500 IU/liter) 0.9% saline followed by 1 liter Zamboni’s fixative in 0.1 M phosphate buffer (PB; pH 7.4) and 0.5 liter of Zamboni’s fixative containing 20% sucrose. The preoptic area/hypothalamic blocks were dissected from the brains and sunk in 30% sucrose in 0.1 M PB for 4–5 d at 4 C. The tissue blocks were frozen on powdered dry ice and stored at –20 C until used. Coronal sections of 40 μm were cut on a cryostat and stored in cryoprotectant solution until used for immunohistochemistry (17, 18).
Double-labeling immunohistochemistry
The details of the antisera used for double-labeling immunohistochemistry are given in Table 1. To determine the extent of colocalization and/or input to GHRH- and SRIF-immunoreactive (IR) cells from a range of hypothalamic neuropeptides, four anatomically matched sections at intervals of 480 μm through the rostro-caudal limits of ARC and PeV region of each lean and normally fed animal were processed using free-floating double-labeling immunohistochemistry (29, 30). Briefly, the sections were washed in 0.05 M PBS and treated with 1% sodium borohydride (Sigma-Aldrich, St. Louis, MO) in 0.1 M PB for 20 min and washed. After blocking (5% normal goat serum and 0.3% Triton X-100 in 0.1 M PB) for 30 min, sections were incubated with either rabbit polyclonal anti-GHRH or anti-SRIF antibody for 48 h at 4 C. After washing, sections were then incubated with goat antirabbit Alexa 546 (red, 1:500; Molecular Probes, Eugene, OR) in 0.1 M PB for 1 h and washed. Sections were blocked in normal donkey or goat serum for 30 min. Subsequently, sections were incubated with a relevant primary antibody for immunostaining of the second neuropeptide/neurotransmitter of interest for 48 h at 4 C, and bound antibodies were detected using goat antimouse Alexa 488 (green, 1:500; Molecular Probes). In some cases, binding of primary antibody was visualized using goat antirabbit, horse antimouse, or goat antiguinea pig biotinylated secondary antibody (1:250; Vector Laboratories, Burlingame, CA) and avidin-fluorescein isothiocyanate (FITC) conjugate (1:500; Pierce, Rockford, IL) as appropriate. Sections were washed in 0.1 M PB and wet-mounted with antifade medium (Dako Corp., Carpenteria, CA) onto gel-coated slides. To determine the codistribution of DYN with GHRH cells, consecutive sections were processed using single-labeling procedures described above because both of the primary antibodies were raised in rabbits. Sections were then counterstained with 0.3% Sudan Black to block nonspecific autoimmunofluorescence.
Immunohistochemical control procedures
In cases where these antibodies had not been previously used in sheep, specificity of immunostaining was validated by preabsorption with the relevant peptide and negative controls (omission of the primary antibody) as previously described (17, 18, 29, 30). A negative control was always included in each procedure. All immunostaining was abolished after control procedures (data not shown).
Microscopic analysis
Microscopy.
Tissue sections processed for double-labeling immunofluorescence were analyzed using an Olympus BMX 50 microscope (Olympus Corp. Ltd., Tokyo, Japan) equipped with mercury light and Texas red and FITC band filter system. Double labeling was visualized by switching between the filters, single- and double-labeled cells were counted unilaterally in each section for each animal, and the total was recorded for each animal for each group. Data are presented as mean (±SEM) percentage of double-labeled cells.
Confocal microscopy.
The use of confocal microscopy to identify putative afferent input to neurons in the brain has been successfully used in our laboratory (29, 31, 32, 33) as well as others (34, 35). This allows examination of a larger number of cells than is feasible with electron microscopy. Semiquantitative estimates of close contacts or putative inputs to GHRH- and SRIF-IR cells from systems producing various peptides and neurotransmitters were made using Fluoview (FV 300) Laser Scanning Confocal Microscope System (Olympus Corp. Ltd.) with a 60x (1.4 numerical aperture) objective. FITC and Texas red were excited simultaneously with the 488- and 568-nm lines of an argon ion laser system. To determine the relative percentage of close contacts between IR varicosities and GHRH-IR and SRIF-IR, the cells were examined unilaterally in anatomically matched sections from each animal (n = 4/group). Cells were randomly selected and sampled using a 60x objective (1.4 numerical aperture) in an area of 0.35 mm2 determined by a grid in the eye piece (29, 31). The grid was moved to five different fields in the ARC and PeV regions, and GHRH-IR and SRIF-IR cells were counted. Optical sections of 1-μm thickness were captured, and two separate stacks of images were generated, one for each fluorophore. The optical sections were then merged to give a composite image. In addition, pairs of 1-μm optical images in the same plane for each fluorophore were merged individually and examined using Adobe PhotoShop computer software (version 5.2; Adobe Systems, Mountain View, CA) to determine whether putative close contacts were real. Yellow pixilation (yellow labeling after colocalization of red and green) or close apposition of green and red fluorescence, which was consistent in three to five individually merged images, were considered to be putative synaptic contacts between axon terminals and perikarya. The data were analyzed by ANOVA and presented as mean (±SEM) percentage of GHRH-IR and SRIF-IR cells receiving putative input. Confocal images demonstrating double-labeling and close contacts were prepared as photomicrographs using Adobe PhotoShop 5.2 software. In all cases, only brightness and contrast were adjusted. All data were collected and analyzed by a single investigator.
RIA
Plasma GH concentrations were measured in one assay (sensitivity, 0.5 ng/ml) according to the method of Thomas et al. (36) with NIDDK-oGH-1–4 as standard and NIDDK-anti-oGH-2 antiserum. Plasma leptin levels were measured in one assay (sensitivity, 100 pg/ml) by the method of Blache et al. (37).
Results
Distribution of GHRH- and SRIF-IR cells
Food restriction significantly reduced mean body weight (P < 0.001), omental fat weight (P < 0.0001), and mean plasma leptin levels (P < 0.03) and significantly (P < 0.05) increased mean plasma GH levels (Table 2). There was a greater (P < 0.01) mean (±SEM) number of GHRH-IR cells in the ARC of lean animals than in normally fed animals (Table 1 and Fig. 1, A and B). The mean number of hypophysiotropic SRIF cells in the PeV did not differ significantly in lean and normally fed animals (Table 2 and Fig. 1, C and D). These findings concur with the earlier in situ hybridization studies in the sheep (12).
Colocalization in GHRH and SRIF cells
The distribution of cells containing NPY, ORX, MCH, TH, GAL, and long-form leptin receptor (Ob-Rb) was similar to that which we have reported previously for the sheep hypothalamus (17, 18). GAL-like peptide (GALP)-IR cell bodies were present in the mediolateral division of the ARC (see Fig. 3H).
Double-labeling procedures revealed that virtually 100% of GHRH cells expressed Ob-Rb (Fig. 2C, arrows). We have reported the colocalization of Ob-Rb to SRIF cells (17) and ARP producing cells previously (18). Subpopulations of GHRH-IR cells were also immunostained for NPY, GAL, GALP, and TH. The extent of peptidergic colocalization within GHRH-IR cells is shown in Table 3, and examples are shown in Fig. 3. Colocalization between NPY and GHRH-IR cells was observed in lean animals only (17.25 ± 1.10%). The mean (±SEM) percent colocalization of GHRH-IR with GAL-IR was higher (P < 0.05) in lean animals than in normally fed animals. Only a few DYN-IR cells were seen in the lateral division of the ARC, and processing of consecutive sections showed that these did not overlap with the distribution of GHRH-IR cells (data not shown).
TH-, ENK-, and DYN-IR cells were found in the PeV, but no cells were found to be IR for NPY, GAL, or GALP in this region. Despite overlapping distribution, DYN-IR cells were distinct from SRIF-IR cells (data not shown). A subpopulation of SRIF-IR cells also immunostained for ENK (Fig. 4, A–C) and TH (Fig. 4, D–F). The estimate of colocalization between ENK and SRIF was lower (P < 0.01) in lean animals than in normally fed animals. A subpopulation of SRIF-IR cells also immunostained for TH in the PeV, but the extent of this colocalization was similar in lean and normally fed animals (Table 3).
Input to GHRH-IR cells
ENK-, SRIF-, and ORX-IR fibers were present in all divisions of the ARC. The extent of close contacts (putative input) to GHRH cells from these various afferents is presented in Table 4, and examples are shown in Fig. 5. Confocal microscopic analysis revealed that SRIF- and ENK-IR fibers/varicosities were in close contact to GHRH-IR cells (Fig. 5), but ORX-IR fibers did not come into close contact with GHRH-IR cells. The mean (±SEM) percentage of GHRH-IR cells receiving input from ENK-IR was significantly (P < 0.05) lower in the lean than in the normally fed animals, but the percent age of GHRH-IR cells receiving input from SRIF-IR fibers did not differ between the two groups (Table 4). No input to GHRH-IR cells was seen from fibers/varicosities that were IR for NPY, GAL, GALP, ORX, and MCH.
Input to SRIF cells in the PeV
The extent of afferents to SRIF cells is seen in Table 5, and examples are shown in Fig. 6. Fibers and varicosities that immunostained for GHRH, NPY, GAL, and ORX were extensively distributed throughout the PeV, but there was only sparse distribution of fibers/varicosities that were IR for MCH in this region. Confocal analysis of the SRIF-IR cells indicated close apposition to varicosities that were IR for GHRH, NPY, GAL, and ORX (Fig. 6). The extent of these close appositions was significantly higher (P < 0.01) for GHRH and NPY and higher (P < 0.05) for GAL and ORX in the lean animals than in the normally fed animals (Table 5).
Discussion
This study provides, for the first time, anatomical evidence on the potential role of various ARP in the central regulation of GHHR and SRIF cells, which is relevant to the control of GH secretion from the anterior pituitary somatotropes. Previously, only minimal information was available regarding peptides that colocalize to GHRH and SRIF neurons and in regard to the neuronal input to these neuroendocrine cells. The present work details the extent of colocalization and afferent input from ARP-producing cells to the GHRH and SRIF cells in the ovine brain. These data thus provide an anatomical substrate for further studies to define neural control of GHRH and SRIF secretion and GH secretion, especially the role of ARP.
In sheep and humans, reduction in the body weight and/or fasting is associated with increased plasma levels of GH (4, 6, 7, 8, 9, 10, 11, 12). In accordance with this, the number of GHRH-IR cells was higher in lean animals than in normally fed animals, suggesting increased synthesis of GHRH; this may lead to greater secretion of GHRH in lean animals, but this has not been demonstrated. Indeed, our earlier work showed no difference in hypophysial portal levels (4), but this could be due to difficulties in measurement of GHRH in plasma due to high levels of nonbiologically active GHRH immunoreactive protein fragments secreted from ectopic organs (38) and possible binding proteins. The present findings are consistent with earlier results obtained by in situ hybridization (12). The number of SRIF-IR cells did not change with reduction in body weight, although we previously reported reduced mRNA levels for SRIF in the PeV of lean OVX ewes (12), and SRIF levels in hypophyseal portal blood are reduced with lowered body weight (4). It is possible, therefore, that reduced gene expression and reduced secretion occurs, but there is no change in the number of SRIF cells. On the other hand, our earlier report indicated that reduction in SRIF mRNA levels was confined to the rostral region of the PeV, and the present work reports on the entire extent of this region.
Colocalization of neuropeptides/transmitters and Ob-Rb in GHRH cells
Reduction in body weight of sheep by food restriction is associated with reduction in the amount of omental adipose tissue and a reduction in circulating leptin levels. This could have a direct effect on the SRIF and GHRH cells. Ob-Rb has been reported in both of these cell types, but we confirmed that this was the case in the ovine GHRH cells. Although colocalization of leptin receptor and GHRH has been reported in the rat (16), the extent of this was previously unknown, and the earlier study did not distinguish between the long and short forms of the receptor. We found that virtually 100% of these cells express Ob-Rb, indicating that changes in the function of these cells with altered body weight could be due to direct effects of leptin. Ob-Rb mRNA expression also changes with alterations in the body weight, being up-regulated with food restriction and reduced body weight (39, 40). Because more GHRH cells are seen by immunohistochemistry in lean animals (present study), and there is increased gene expression for GHRH (12), the increase in activity of these cells may be accompanied by increased level of Ob-Rb expression in GHRH cells, although this remains to be determined. We recently showed that the level of expression of Ob-Rb increased in NPY-producing cells of the ARC in lean animals (40), suggesting that function of the receptors could increase. A similar increase in function could occur in GHRH cells, although detailed cellular analysis would be required to ascertain whether this is the case. Because reduction in body weight also leads to changes in levels of circulating metabolites such as free fatty acids (7), it is possible that these changes may affect the GH axis.
Subpopulations of GHRH-IR cells also immunostained for NPY, GAL, GALP, and TH. Our finding that NPY is produced in a subset of GHRH cells in the ARC of sheep concurs with data obtained from rat (26) and human (28) brains. We showed cellular colocalization of NPY and GHRH in 17% of GHRH cells in the ARC of lean sheep, but this was not seen in normally fed animals. There is substantial up-regulation of NPY expression in the lean condition (7, 40), and the present work shows that at least part of this may occur in GHRH cells. Thus, a subpopulation of GHRH cells appears to be activated by reduction in body weight, accompanied by increased expression of NPY in the same cells. NPY stimulates GH secretion in the sheep (41, 42), bovine (43), and human (44), so the up-regulation of the GH axis that occurs with reduced body weight could be due to mechanisms, within GHRH cells, that involve NPY. On the other hand, NPY reduces GH secretion in the rat (45, 46, 47), but reduction in body weight causes increased GH secretion in this species (41).
Intracerebroventricular infusion of leptin causes a small increase in GH secretion in sheep (48), but the effect is similar in lean and normally fed animals (48). Leptin, however, reduces NPY expression in animals of normal body weight (49) and has no effect on NPY expression in lean animals (Clarke, I. J., unpublished data), so the effect is unlikely to be mediated through NPY. It seems more likely that the effect of leptin is directly on the SRIF and GHRH cells (vide supra) or by neuronal afferents that are not NPY cells.
TH immunostaining identified dopaminergic neurons, and previous reports indicated between 32 (27) and 90% (50) colocalization of TH and GHRH in cells of the rat mediobasal hypothalamus. We found that approximately one quarter of GHRH cells also immunostained for TH, which was unaffected by alteration in body weight. Because dopamine (DA) is not secreted into the hypophysial portal blood of sheep (51), it is unlikely that hypothalamic DA and GHRH have a synergistic effect on GH release from pituitary somatotropes as suggested by Serri et al. (52) for the rat. Autocrine regulation of GHRH synthesis by DA remains a possibility, at least in a subset of the cells. GAL colocalizes with GHRH in the rat hypothalamus (25, 53), and exogenous administration of GAL stimulates GH secretion in rats (54, 55), sheep (56), and humans (57, 58). GAL has also been reported to stimulate GHRH and SRIF release from the rat (59) and bovine (60) ME. A small subpopulation of GHRH-IR cells immunostained for GAL, with slightly, but significantly, increased numbers of costaining cells in the lean animals. Although Barker-Gibb et al. (6) reported an increase in GAL immunoreactivity in the ovine ME of lean OVX ewes, GAL mRNA expression was not altered significantly (7). Colocalization of GAL within a subpopulation of GHRH-IR cells supports the notion that the former has a role in the modulation of GHRH secretion (56). Because only 2% of GHRH-IR cells had GALP-like immunoreactivity, a significant role for this peptide in autocrine function seems unlikely.
Afferent input to GHRH cells
Confocal microscopy provides a good estimation of synaptic interaction and close contacts between afferents and cell bodies. Although this does not provide definitive identification of synapses, identification is being widely used to assess the synaptic interactions (29, 31, 32, 33, 34, 35) because it allows the screening of a much larger number of cells. Our confocal microscope analysis strongly suggests that GHRH-IR cells receive input from SRIF and ENK systems. As in the rat (61, 62), SRIF provides input to the one third of the GHRH neurons in the sheep. GHRH mRNA expression and GH levels are elevated, whereas SRIF mRNA expression is reduced in the lean condition (12), so this might be brought about by reciprocal regulation of GHRH and SRIF cells.
SRIF has an inhibitory influence on GHRH cells (1, 2), but the extent of SRIF input to GHRH cells (cells receiving input) does not differ with alteration in body weight, even though there are increased numbers of GHRH-IR cells in lean animals. The precise origin of SRIF projections to GHRH cells is not known but could be either the PeV or the ventromedial hypothalamic nucleus, in which many SRIF neurons are found (5, 14). At least some of the SRIF cells of the ventromedial nucleus project to the ARC nucleus as revealed by retrograde labeling studies (Qi, Y. and I. J. Clarke, unpublished data). Expression of SRIF mRNA in both regions is reduced with lowered body weight (12).
ENK are opioid peptides (Leu-ENK and met-ENK), derived from the preproenkephalin gene. These peptides bind predominantly to the -subtype of opioid receptors and stimulate GH secretion rodents (63) and humans (64). We found evidence of reduced ENK input to GHRH-IR cells in lean animals, suggesting some plasticity of input regulated by body condition. ENK-producing neurons are localized in the PeV, paraventricular hypothalamic nucleus, and ventromedial hypothalamic nucleus, and ENK gene expression is differentially altered in all of these regions with alteration in the body weight in the sheep (7). However, the exact source of ENK input to the GHRH cells is not clear because ARC nucleus receives afferents from PeV, paraventricular nucleus, and ventromedial hypothalamus region (Qi, Y. and I. J. Clarke, unpublished data). The extent of leptin receptor and ghrelin receptor in ENK neurons has been not reported for any species, so it is not known whether these factors are important in the regulation of this system. ENK input to a subpopulation of GHRH cells provides an evidence of a mechanism by which opioids could regulate GHRH and GH secretion, but the paradoxical reduction in lean animals of afferents from a system that stimulates GH secretion requires further investigation.
Colocalization of neuropeptides/neurotransmitters and projections to SRIF cells
Although SRIF-producing neurons are found in several areas of the hypothalamus (5), those that project to the external zone of the ME to secrete the peptide into hypophyseal portal blood are confined to the PeV (1, 2, 3). A subset (around 11%) of SRIF cells also immunostained for ENK, and we report the novel finding that the extent of this colocalization is reduced in lean animals. The means by which ENK stimulates GH secretion could be autocrine, within SRIF cells, to reduce expression of the gene for SRIF. Previous studies in our laboratory have shown that ENK expression is increased and SRIF expression is reduced in the PeV with reduction in the body weight of OVX ewes (7, 12), suggestive of an association between these peptides. A small subset of SRIF-IR cells (3%) was also immunopositive for TH, which is another novel finding, but the extent of colocalization did not change with altered body weight.
Afferent input to SRIF cells
Our data strongly suggest that subpopulations (up to one quarter) of SRIF neurons in the PeV of the ewe brain receive afferent input from GHRH, NPY, GAL, and ORX systems. For each of these systems, putative input to SRIF neurons was higher in lean animals than in normally fed animals. There is increased expression of GHRH, NPY, and GAL in the ARC of lean animals (6, 7, 12), and this could explain the increase in input to SRIF-producing cells in the PeV. Such a direct link, specifically relating to these systems remains to be demonstrated by detailed tracing studies, but previously demonstrated bidirectional connections between ARC and PeV in rat (61, 62) and sheep (65) provide evidence of reciprocal communication between these two regions. Increased expression of GHRH and increased GHRH input to SRIF cells in lean animals may be a means of inhibiting SRIF synthesis and release into the portal circulation, thus allowing increased GH secretion from the pituitary.
NPY negatively regulates GH secretion and is thought to negatively regulate SRIF cells in the rat (66). On the other hand, NPY stimulates GH secretion in the sheep and other species (vide supra). Increased input to SRIF cells located in the PeV from NPY afferents in lean animals could negatively regulate the SRIF cells, consistent with a stimulatory effect on GH secretion. In lean animals, we found increased colocalization of GAL and GHRH in the ARC and increased afferent input to PeV-located SRIF cells. The latter could be related to the former, but tracing studies are required to confirm this. Because centrally administered GAL stimulates GH secretion in sheep (56), GAL may be inhibitory to SRIF, and increased input to SRIF cells in the PeV of lean animals would be consistent with increased GH secretion in this condition.
ORX-producing cells are localized in the lateral hypothalamus, the perifornical area, and the dorsomedial nucleus of the hypothalamus, and these cells send widespread projections to various regions of the brain in the sheep (31). ORX functions to regulate food intake and neuroendocrine cells as well as in arousal (for review, see Ref.67). We previously reported that ORX-IR fibers were distributed widely in the preoptic area and the PeV region, with an evidence of direct synaptic input to GnRH cells (31), and we now report evidence of direct input to the SRIF cells of the PeV. This provides an anatomical substrate to support recent in vitro and in vivo studies that demonstrate a role for ORX in the regulation of GH, at least in the rat (68, 69, 70). To support a role for ORX in the regulation of SRIF cells, the ORX type-1 receptor is found on SRIF cells of the PeV region of the rat brain (71). In the sheep, however, Sartin et al. (72) found that intracerebroventricular (0.03, 0.3, or 3 μg/kg body weight), or iv infusion (3 μg/kg body weight) of porcine ORX-B did not affect GH secretion in the sheep, and nothing is known about ORX action on SRIF secretion.
Ghrelin is one peripheral factor that regulates GH secretion, with a possible role in the regulation of food intake (21, 22). Because exogenous administration of ghrelin induces fos expression in the ORX cells of the lateral hypothalamic area in rodents (19), and our earlier tracing studies indicated a pathway from the lateral hypothalamic area to the PeV/preoptic area of the ovine brain (31), it is possible that indirect regulation of ORX cells by ghrelin is a means of regulation of SRIF cells. ORX cells also express Ob-Rb, so regulation of these cells by leptin (16, 18) is another means by which SRIF function could be indirectly affected by altered body weight (that alters the level of leptin in plasma and alters Ob-Rb levels).
The findings of this study are summarized in Fig. 7. Subpopulations of GHRH-producing neurons in the ARC also produce NPY, TH, GAL, and GALP, and there is evidence of afferent input from neuronal systems that produce ENK and SRIF. Subpopulations of SRIF-producing neurons in the PeV also produce ENK and TH and appear to receive input from neuronal systems that produce GHRH, NPY, GAL, and ORX. These findings provide an anatomical evidence of potential role of ARP in the central regulation of GHRH and SRIF secretion and subsequent changes in the GH secretion from the anterior pituitary with alteration in the adiposity.
Acknowledgments
We thank Mr. Bruce Doughton, Ms. Karen Briscoe, and Ms. Lynda Morrish for animal care and tissue collection and Sue Panckridge for assisting in preparing the photomicrographs. We also thank Dr. D. Blache (University of Western Australia, Perth, Western Australia) for leptin assays and NIH for RIA reagents for GH assays. Dr. Iqbal was supported by Prince Henry’s Institute of Medical Research.
Footnotes
Abbreviations: ARC, Arcuate nucleus; ARP, appetite-regulating peptide; DA, dopamine; DYN, dynorphin; ENK, enkephalin; FITC, fluorescein isothiocyanate; GAL, galanin; GALP, GAL-like peptide; IR, immunoreactive; MCH, melanin-concentrating hormone; ME, median eminence; NPY, neuropeptide Y; Ob-Rb, long-form leptin receptor; ORX, orexin; OVX, ovariectomized; PB, phosphate buffer; PeV, anteroventral periventricular nucleus; SRIF, somatostatin-releasing inhibiting factor; TH, tyrosine hydroxylase.
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