| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Cardiff School of Biosciences (N.M.T., J.S.D., T.W.), Cardiff University, Cardiff CF10 3US, United Kingdom; Department of Medical Nutrition (A.M.), Karolinska Institutet, Huddinge University Hospital, Novum S-14186, Sweden; and Department of Neuroendocrinology (P.A.H.), Division of Neuroscience and Psychological Medicine, Imperial College London, Hammersmith Hospital, London W12 0NN, United Kingdom
Address all correspondence and requests for reprints to: Dr. Timothy Wells, School of Biosciences, Cardiff University, P.O. Box 911, Museum Avenue, Cardiff CF10 3US, United Kingdom. E-mail: wellst{at}cardiff.ac.uk.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
It has been known for several decades that starvation influences the secretion of GH. In humans, short-term (24 h) starvation is associated with augmented GH secretion (7), whereas in rats, more prolonged periods of food deprivation (up to 163 h) result in progressive suppression of the amplitude of GH secretory episodes (8, 9). It is likely that the short-term effects of starvation on GH secretion are mediated by ghrelin because continuous exposure to the ghrelin mimetic, GH-releasing peptide-6 (GHRP-6), enhances GH secretion in both rats (10) and humans (11). However, more prolonged (1 wk) exposure to GHRP-6 produces only a transient acceleration in growth, which is associated with a desensitization of the hypothalamo-pituitary-GH axis to further GH secretagogue treatment (12, 13, 14). We reasoned from our previous studies that because this desensitization was accompanied by an elevation in periventricular somatostatin (SRIF) mRNA expression (14), prolonged exposure to ghrelin may also mediate the long-term effect of starvation on GH secretion viz. suppression of GH pulsatility. The effects of prolonged GHS-R activation on the hypothalamo-pituitary GH axis have been investigated in the studies described below.
In this series of experiments, we employed two contrasting paradigms of GHS-R activation: continuous infusion, to mimic gastric ghrelin production in response to fasting; and patterned infusion, more likely to reflect the fluctuations in circulating ghrelin evoked by intermittent feeding (15) or the activity of putative ghrelinergic neurons (1, 16, 17). To determine the effectiveness of these treatment regimens in accelerating skeletal growth in the context of GH-deficient dwarfism, these studies were performed in the transgenic growth-retarded (Tgr) rat (18, 19). In this model of moderate GH deficiency, the hypothalamo-pituitary-GH axis is suppressed by the expression of human (h)GH in the arcuate GH-releasing factor (GRF) neurons (18). However, unlike other rat models of GH-deficient dwarfism, skeletal growth can be accelerated in Tgr rats by GHS treatment (14, 19). We used this model to characterize the pattern-dependent effects of ghrelin and GHRP-6 on the acceleration of skeletal growth and the spontaneous secretion of GH. In addition, we examined the hypothalamic mechanisms that may underlie this ghrelin-induced pattern-dependent desensitization. A preliminary report of part of this work has previously been communicated (20).
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Experiment 1: effect of patterned iv infusion of rat ghrelin on growth in Tgr rats
Male Tgr rats (14 wk old; weighing 180–206 g) were placed in metabolic cages for 4 d before the implantation of single-bore iv cannulae into the right jugular vein under halothane anesthesia. Animals were permitted at least 48 h of recovery, during which time body weight and food intake were monitored daily. After this period, rats received an iv infusion of either vehicle [sterile saline containing BSA (1 mg/ml) and heparin (10 U/ml)], given intermittently [300 µl (2 min) pulses every 3 h] or vehicle containing rat ghrelin given either continuously (80 µg/d at 100 µl/h) or in 10-µg (300 µl) pulses every 3 h for 7 d. Body weight and food intake were monitored daily throughout the 7-d infusion period, and one animal showing erratic food intake/weight loss was excluded from further analysis. At the end of the infusion protocol, the rats were reanesthetized and killed by cervical dislocation.
Experiment 2: effect of patterned iv infusion of GHRP-6 on GH pulsatility
Male Tgr rats (14 wk old; weighing 180–205 g) were prepared as described above with double-bore iv cannulae and permitted at least 48 h recovery. After this period, rats received an iv infusion of either vehicle (as above), given intermittently (as above), or vehicle containing GHRP-6, given either continuously (80 µg/d) or in 10-µg (300 µl) pulses every 3 h for 7 d. In addition, all animals received a maintenance infusion of vehicle (50 µl pulse every hour) through the second arm of the cannula for 6 d. At the beginning of the seventh day of infusion, the maintenance infusion was succeeded by a 12-h period of automated blood sampling, during which 150-µl samples of 1:5 whole blood were collected every 10 min from each rat. At the end of this period, these samples were vortexed, centrifuged, and 100 µl 1:5 plasma subsamples were separated and stored at -20 C for subsequent determination of plasma rat GH (rGH) concentration. Body weight and food intake were monitored daily throughout the 7-d infusion period. At the end of the infusion/sampling protocol, the rats were killed by a bolus iv injection of Expiral (sodium pentabarbitone; Sanofi Animal Health, Watford, Herts, UK).
Experiment 3: hepatic expression of P450 2C11 and P450 2C12 expression in Tgr rats
We have previously used the hepatic expression profiles of P450 2C11 and 2C12 mRNAs as surrogate markers of the spontaneous GH secretory pattern in dw/dw rats (21, 22). Because these parameters have not previously been determined in Tgr rats, groups of 15-wk-old male and female WT and Tgr littermates were weighed, killed, and liver samples taken post mortem into 1x SET buffer [20 mM Tris HCl (pH 7.5), containing 1% sodium dodecyl sulphate and 10 mM EDTA].
To determine the effect of patterned ghrelin infusion on these hepatic markers of rGH secretion, livers were removed postmortem from male Tgr rats treated in experiment 1. After excision, livers were immersed in 1x SET buffer and snap frozen on dry ice before subsequent determination of hepatic IGF-1, P450 2C11, and 2C12 mRNA levels.
Experiment 4: effect of patterned iv infusion of GHRP-6 on sensitivity to GRF
Male Tgr rats (14 wk old; weighing 183–241 g) were prepared as above with double-bore iv cannulae and permitted at least 48 h of recovery. After this period, rats received an iv infusion of either vehicle or vehicle containing GHRP-6, given either continuously or in pulses (as in experiment 2), for 7 d. In addition, all animals received a maintenance infusion of vehicle (50 µl pulse every hour) through the second arm of the cannula for 6 d. At the beginning of the seventh day of infusion, a 100-µl blood sample was withdrawn from the indwelling vascular cannulae before and at 5, 15, and 30 min after a bolus iv injection of 1 µg GRF [rat GRF(1–29)NH2 in 100 µl vehicle]. These injections were timed to coincide with or replace the pulse of GHRP-6 timed to be received from the automated infusion system. These blood samples were centrifuged, and 2x 25 µl plasma samples were separated and stored at -20 C for subsequent determination of plasma rGH concentration. Body weight and food intake were monitored daily throughout the 7-d infusion period. At the end of the infusion/sampling protocol, the rats were killed by a bolus iv injection of Expiral (sodium pentabarbitone).
Experiment 5: effect of patterned iv infusion of rat ghrelin on hypothalamic gene expression in Tgr rats
To examine the hypothalamic mechanisms underlying the pattern-dependent effects of ghrelin on the hypothalamo-pituitary GH axis, brains were removed postmortem from the male Tgr rats treated in experiment 1. After excision, brains were snap frozen in isopentane at -20 C for subsequent determination of hypothalamic GHS-R, GRF, and SRIF mRNA expression.
Peptides used
Rat ghrelin was generously supplied by Pharmacia (Stockholm, Sweden). GHRP-6 and GRF [rat GRF(1–29)NH2] were generously donated by Novo Nordisk A/S (Bagsværd, Denmark).
Tissue analyses
Plasma rGH concentrations were determined by RIA, with the results expressed in terms of the reference preparation RP-2, using the reagents generously supplied by the National Institute of Diabetes and Digestive and Kidney Diseases (intraassay variation 1.2%; sensitivity 0.25ng/ml).
Effects on skeletal growth were determined by measurement of tibial epiphysial plate width (23). Dissected tibia were fixed in 10% buffered formal saline for 2 d and decalcified in 10% EDTA (in 0.3 M NaOH) for 2–3 wk. After embedding in paraffin wax, 8 µm longitudinal/anterior-posterior sections were taken and stained using Masson’s trichrome (24). Epiphysial plate widths were measured using an ocular graticule under light microscopy, with a mean of nine readings taken for each bone.
Measurement of hypothalamic gene expression
Coronal brain sections (12 µm) cut through the periventricular and arcuate nuclei of the hypothalamus were taken at -16 C, thaw mounted onto gelatin and chrome alum-coated slides, and stored at -70 C for subsequent in situ hybridization. 35S-UTP (NEN Life Science Products, Stevenage, Hertfordshire, UK) was used to synthesize radiolabeled antisense probes using an SP6/T7 transcription kit (Roche Molecular Biochemicals, Lewes, UK) and purified on Sephadex G50 columns (Pharmacia, Uppsala, Sweden). The cDNA constructs used as templates were as follows: recombinant GRF, a 500-bp fragment corresponding to exon 5; GHS-R, the full-length type 1a kindly donated by Dr. A. Howard (Merck Research Laboratories, Rahway, NJ). No detectable signals were obtained from corresponding sense RNA probes used as negative controls. SRIF mRNA was detected using a 48-base oligonucleotide as previously described (25). Frozen sections were thawed at room temperature, fixed in 4% paraformaldehyde, acetylated, dehydrated through graded ethanol solutions, and then delipidated in chloroform before hybridization, as previously described (25). Sections were hybridized overnight at 45 C. The following day, posthybridization washes were performed including a ribonuclease digestion. Slides were then air dried and apposed to autoradiographic film (BioMax MR, Kodak, Rochester, NY) for up to 1 wk. X-ray images were analyzed densitometrically using NIH Image software (http://rsb.info.nih. gov/nih-image/). For each rat, two to four anatomically matched sections (identified by counterstaining with cresyl violet) were analyzed for each probe, with data generated in the form of integrated OD. Because of the arbitrary nature of these OD units, the relative expression of the different mRNA species cannot be determined.
Hepatic P450 2C11, 2C12, and IGF-1 mRNA expression
Liver 2C11, 2C12, and IGF-1 mRNAs were determined by ribonuclease protection/solution hybridization assays using specific [35S]UTP-labeled mRNA probes, as previously described (26, 27, 28). The concentration of total nuclei acids (tNAs) in each sample was quantified using a fluorometric assay (29). Quantitation was achieved by comparison with a standard curve obtained from hybridizations to liver tNA calibrated to known amounts of mRNA synthesized in vitro. Samples were analyzed in triplicate and the results expressed as attomoles of mRNA per microgram DNA. The interassay variations were assessed using internal tNA standards.
Statistical analysis
The secretory profiles of rGH were subjected to PULSAR analysis with the cut-off parameters set to give a false-positive error rate of 5% [G(1) = 3.98; G(2) = 2.40; G(3) = 1.68; G(4) = 1.24; G(5) = 0.93] (30). Because Tgr males display a 3- to 3.5-hourly GH pulse frequency (19) and the pulse frequency was not significantly affected by ghrelin infusion (Table 1
), pulse amplitude was determined by calculating the mean peak height of the four highest peaks in each 12-h profile.
|
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
) and epiphyseal plate widths (Fig. 1B). During the first 2 d of treatment, growth acceleration was identical following either pattern of ghrelin infusion [growth rate: 4.0 ± 1.0 g/d (c-ghrelin: P < 0.01; 4.9 ± 0.4 g/d (p-ghrelin: P < 0.001) vs. -0.1 ± 0.6 g/d (vehicle)]. After the second day, the rate of body weight gain decelerated in those animals receiving continuous ghrelin infusion [growth rate (d 2–7): 0.9 ± 0.5 g/d, P < 0.01 vs. d 0–2 rate]. However, at the end of the infusion period, this group had a significantly higher weight gain than that observed in the vehicle-treated group (P < 0.001). In contrast, the Tgr rats receiving the pulsed infusion of ghrelin continued to gain weight at a faster rate than the vehicle-treated animals [growth rate (d 2–7): 2.6 ± 0.2 g/d; P < 0.01 vs. -0.2 ± 0.3 g/d (vehicle treated)].
|
). This effect on skeletal growth was not observed after continuous ghrelin treatment. Infusion of rat ghrelin stimulated a small increase in pituitary weight, which was only statistically significant after pulsatile ghrelin infusion. Average daily food intake was unaffected by either pattern of ghrelin infusion (data not shown).
Experiment 2: effect of patterned iv infusion of GHRP-6 on GH pulsatility
Intravenous infusion of GHRP-6 produced the expected pattern-dependent acceleration in body weight gain and skeletal growth (tibial epiphyseal plate width; data not shown) as previously reported (14, 19) and as seen above for rat ghrelin (Fig. 1, A and B). Automated serial blood sampling on the last day of infusion revealed that vehicle-infused Tgr rats continued to display a pattern of rGH secretion characteristic of male rats, with a pulse amplitude similar to that previously reported in the Tgr model (18, 19) (Fig. 2 and Table 1). After 6 d of continuous GHRP-6 infusion, the amplitude of the four highest rGH secretory episodes in each profile was significantly suppressed (Fig. 2 and Table 1), but the total number of peaks and peak length were not significantly affected (Table 1). Mean interpeak interval was more than doubled and the area under the curve almost halved (Table 1), but neither of these parameters were statistically significant. One animal treated with continuous GHRP-6 produced one large episode of GH secretion (peak amplitude 120 ng/ml) in a 12-h profile that was otherwise similar to the other profiles in this treatment group. This animal was excluded from PULSAR analysis. In contrast, pulsatile infusion of GHRP-6 augmented the amplitude of the four highest rGH secretory episodes (Fig. 2 and Table 1) and produced synchronization of these secretory events. In addition, this treatment significantly elevated the total rGH secretory output (area under the curve), although the total number of peaks, peak length, and interpeak interval were not significantly affected.
|
). Thus, Tgr rats exhibited the normal sexually dimorphic hepatic expression of these transcripts, P450 2C11 mRNA expression being high in male and low in female Tgr rats (P < 0.001), and P450 2C12 mRNA expression being low in male and high in female Tgr rats (P < 0.001). In contrast, hepatic IGF-1 mRNA expression was significantly reduced in Tgr rats of both sexes (P < 0.01) and paralleled the changes previously reported in circulating IGF-1 (19).
|
), we used these markers to investigate the effect of ghrelin infusion on GH pulsatility using livers obtained at the end of experiment 1. We were surprised to observe that the expression of these transcripts was unaltered by either pattern of ghrelin infusion (Fig. 3). This confirmed that baseline secretion of rGH was not elevated by continuous infusion and also suggested that residual GH release continued to be secreted in a pulsatile pattern characteristic of male rats. Hepatic IGF-1 mRNA expression was unaltered by either pattern of ghrelin infusion (Fig. 3).
|
). Despite the large variation in the peak plasma levels achieved, this response to GRF treatment was not diminished by either pattern of GHRP-6 infusion (Fig. 4). The degree of variation in responsiveness to GRF may reflect the possible coincidence of the GRF bolus with the refractory periods related to the underlying endogenous GH episodes.
|
). Continuous ghrelin infusion elevated GHS-R and SRIF mRNA expression in the arcuate and periventricular nuclei respectively (Fig. 5, A and C). Although showing a similar pattern, arcuate GRF mRNA expression was not statistically significant (Fig. 5B). Although intermittent ghrelin infusion also elevated arcuate GHS-R mRNA expression (Fig. 5A), neither arcuate GRF nor periventricular SRIF mRNA expression was significantly elevated by this treatment (Fig. 5, B and C). Neither pattern of ghrelin infusion had any significant effect on SRIF mRNA expression in the arcuate nuclei (Fig. 5D).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In this study, intermittent infusion of ghrelin elicited an acceleration in weight gain, which was sustained over the 7-d infusion period. This growth response is similar to that seen with the ghrelin mimetic, GHRP-6, in both normal (12) and Tgr (14) rats and is likely to be due to the skeletal (Fig. 1B) and anabolic effects of augmented GH release because this treatment does not produce any observable change in fat deposition (33). Sustained GH responses and elevated circulating IGF-1 have previously been recorded following daily administration of the GHS L-692,585 in beagles (34) and MK-677 in man (35). Although acute ghrelin treatment promotes feeding behavior (3, 4), a chronic elevation in cumulative food intake is seen more readily following central infusion (2, 3) than peripheral administration (2). Thus, the absence of any observable orexigenic action of ghrelin in this study was not unexpected and has previously been reported in Tgr rats after GHRP-6 treatment (14).
The amplitude of GH pulses in male rats is largely determined by the secretion of GRF from the terminals of arcuate neurons (36). The elevation in amplitude of the GH episodes following intermittent GHS-R activation (Fig. 2 and Table 1), coupled with our previous observation that pulsatile infusion of GHRP-6 increases arcuate GRF mRNA expression (14), suggests that GRF release is augmented by this treatment. In addition, the synchronization of the GH profiles following intermittent GHS-R activation (Fig. 2) implies a predominantly central action, triggering arcuate GRF secretion. However, we cannot exclude the possibility that in the context of reduced GRF mRNA expression in the Tgr model (18), the functional antagonism of SRIF following GHS-R activation may make a significant contribution to the enhanced GH secretion.
Periodic fluctuations in circulating ghrelin may promote GRF-induced GH secretion and contribute to the tone of the hypothalamo-pituitary-GH axis. However, this contribution appears to be minimal in normally fed animals because ghrelin-knockout mice do not display a growth-retarded phenotype (37). It should also be noted that the concentrations of circulating ghrelin achieved in this and other studies (1, 38) are likely to be supraphysiological when compared with the preprandial elevation in circulating ghrelin reported in humans (15) and the levels reported in fasted and fully fed rats (2).
In contrast to the sustained growth acceleration produced by intermittent ghrelin treatment, continuous infusion produced only a transient increase in body weight gain, without any observable change in skeletal growth. Initially, continuous ghrelin infusion stimulates weight gain at the same rate as that seen with intermittent treatment. This initial growth response may be related to an enhancement of the pulsatility of GH secretion reported to occur with short-term continuous GHS-R activation (10, 11, 39). This initial rate of growth is not sustained. A similar fatigue in the growth response to continuous GHRP-6 has previously been reported in male Tgr rats (14) and also occurs in normal female rats (12, 13). A number of possible mechanisms may contribute to this phenomenon.
First, it is well documented that continuous GHS-R activation results in desensitization of the GRF-GH axis to further GHS treatment (10, 11, 40, 41). Although the waning systemic IGF-1 levels remain elevated in humans following prolonged daily administration of the long-acting GHS, MK-677 (35, 42, 43), the effect of chronic continuous iv infusion is equivocal. Previous iv infusion studies have reported that circulating IGF-1 levels are elevated in normal male rats with a high dose of hexarelin (44), decreased in normal female rats with a low dose of GHRP-6 (13), or unaltered in male Tgr rats with a low dose of GHRP-6 (14). Although circulating IGF-1 levels were not measured in the current study, the absence of a change in the low expression of hepatic IGF-1 mRNA in male Tgr rats with continuous ghrelin infusion (Fig. 3) corroborates our previous study (14). In Tgr rats, the desensitization of the GRF-GH axis to a bolus injection of GHRP-6 occurs after only 3 d of continuous GHRP-6 infusion, i.e. at the same time as the deceleration in growth rate (14). Without the additional drive to the GRF-GH axis by ghrelin, continuously infused Tgr rats gain weight at the same rate as vehicle-infused controls.
However, it is evident that ghrelin is also adipogenic (2), an action that is observed most clearly in the context of GH deficiency (45). Thus, the residual weight gain seen in Tgr rats given continuous ghrelin is likely to be due to the adipogenic effect of this treatment (33). Because GH is lipolytic, it is possible that in the context of continuous ghrelin exposure, the hypothalamo-pituitary-GH axis may even be suppressed.
To explore this possibility, we measured GH profiles in Tgr rats given either continuous or intermittent GHS treatment. This revealed that continuous GHS-R activation suppressed the amplitude of the spontaneous episodes of GH secretion. This result differs from an earlier study in which GH secretion was unaltered in normal male rats following a continuous iv infusion of a 30-fold higher dose of the GHS hexarelin (44). Because the GH peaks measured in the current study were close to the detection limit of the assay, we used the hepatic expression of the cytochrome P450 2C11 and 2C12 mRNA as surrogate markers of the residual GH secretion following ghrelin infusion. The absence of any significant alteration in CYP2C11 and 2C12 mRNA expression suggests that the residual GH secretion continued to be released in a frequency characteristic of male rats without any elevation in baseline secretion (21). These results concurred with the measured GH profiles, which were reminiscent of circulating GH levels in the profoundly GH-deficient dw/dw rat (21, 46). Thus, in addition to the desensitization of the GRF-GH axis, the suppression of spontaneous GH secretory episodes represents a further mechanism contributing to the waning growth response to continuous GHS-R activation. Several hypothalamic mechanisms may contribute to this suppression of the GRF-GH axis.
The most direct mechanism of desensitization would result from down-regulation of GHS-R expression. Although it is not always possible to extrapolate from transcript to protein levels, our data do not support this hypothesis. Arcuate GHS-R mRNA expression was almost doubled following continuous ghrelin treatment (Fig. 5A), which is consistent with our previous observations that continuous infusion of GHRP-6 up-regulates GHS-R mRNA expression in both Tgr (14) and normal rats (47). Thus, down-regulation of GHS-R expression does not appear to underlie the desensitization of the hypothalamo-pituitary-GH axis to continuous GHS-R activation. However, the level of GHS-R protein is extremely low and below the level of detection for the purposes of quantification. Therefore, we cannot rule out the possibility that reduced GHS-R mRNA translation could result in a reduction in GHS-R protein levels and a reciprocal elevation in mRNA expression.
As already indicated, the amplitude of the episodes of GH secretion is largely determined by the quantity of GRF released (36). This, coupled with the fact that GHS-induced GH secretion in vivo is mediated predominantly by the arcuate GRF neurons (48, 49), suggests that a reduction in GRF secretion may be responsible for the desensitization of the GRF-GH axis. It should be noted that the production of GRF mRNA is not reduced by continuous ghrelin treatment but is, if anything increased, as previously reported for GHRP-6 (14). Similarly, in conjunction with numerous previous reports (10, 11, 40, 41), our data indicate that, despite the observed suppression of GH secretion, pituitary somatotrophs remain sensitive to GRF exposure (Fig. 4). This combination of facts suggests that either the activity of the GRF neurons is suppressed or the stimulus-secretion process is uncoupled by continuous ghrelin exposure. The amount of GRF secreted could be assessed by analysis of GH responses to SRIF withdrawal (50), but to combine this procedure with patterned ghrelin infusions would be technically demanding and outside the scope of the current study. Evidence from experiments measuring the induction of Fos expression (the protein product of the immediate early gene c-fos), indicates that continuous central infusion of GHRP-6 in normal male rats suppresses the GHS-induced activation of arcuate neurons (51). But this method does not permit analysis of either baseline or spontaneous neuronal activity.
Although no discernible GHS-R expression has been detected within the periventricular nuclei (52), a significant up-regulation of periventricular SRIF mRNA expression was found to occur following continuous ghrelin exposure (Fig. 5B). This finding is consistent with previous studies using GHRP-6 in both normal (51) and Tgr (14) rats and was not accompanied by parallel changes in arcuate SRIF mRNA expression. Should this increase in periventricular SRIF mRNA expression be associated with elevated peptide secretion, this could represent a potent mechanism to suppress GH secretion. The fact that somatotrophs in rats continuously infused with GHRP-6 remain sensitive to GRF treatment and that GHS-induced Fos responses in arcuate neurons are suppressed by continuous GHS-R activation (51) suggests that any increase in SRIF secretion primarily inhibits the activity of the GRF neurons. This is possible because GRF neurons express binding sites for SRIF (53) and somatostatinergic neurons project from the periventricular to the arcuate nuclei (54). It has also been demonstrated that the inhibition of GHS-induced Fos responses in GRF neurons by octreotide is reduced in SRIF receptor subtype 2 knockout mice (55).
A fourth possible mechanism contributing to the suppression of the GRF-GH axis in this study relates to the etiology of growth retardation in the Tgr rat. In this model, the hypothalamo-pituitary-GH axis is suppressed by the expression of hGH in the arcuate GRF neurons under the control of the GRF promoter (18). Elevating arcuate hGH expression would result in a further suppression of GRF expression, inhibition of the activity of GRF neurons (55) and an increase in periventricular SRIF expression. This combination would lead to a reduction in GRF secretion. Although we cannot exclude this possibility, we think the contribution of transgene-derived hGH to the observed response is minimal because continuous GHS-R activation produces a similar biphasic growth-promoting effect in normal female rats (12, 13) and suppresses the Fos responses of GRF neurons in normal male rats (51).
The results we report here are strikingly similar to those previously reported for prolonged periods of starvation in rats. Although short-term fasting is associated with an increased sensitivity to GRF (56) and augmented GH release (7) in humans, the opposite effect on GH secretion is seen with prolonged starvation in rats (8, 9). In this species a progressive reduction in the amplitude of GH secretory episodes occurs with food deprivation, which is reversed by refeeding (9). This suppression occurs without any change in the peak GH response to exogenous GRF treatment (57) and is accompanied by a 3- to 4-fold increase in both circulating SRIF-like immunoreactivity (57) and intrahypophysial SRIF (58).
It is clear that both short-term starvation (2) and prolonged food deprivation (59) increase plasma ghrelin concentration in rats. The data we present here suggest that the effects of prolonged starvation on the hypothalamo-pituitary-GH axis may be mediated by long-term exposure to elevated circulating ghrelin. Thus, in the short term, starvation-induced ghrelin release may promote GH secretion, thereby accessing an alternative energy supply via the lipolytic action of GH. In the longer term, however, ghrelin may reverse this effect.
In conclusion, these results demonstrate that the growth-promoting action of ghrelin and the GHSs is pattern dependent. The combination of patterned iv infusions with the monitoring of GH pulsatility reveals that although intermittent GHS-R activation augments GH release, prolonged continuous exposure to ghrelin suppresses the amplitude of the spontaneous episodes of GH secretion. In addition, these results provide evidence that this suppression in GH secretion may be mediated by an elevation in SRIF production and that elevated circulating ghrelin may be responsible for the effects of starvation on GH secretion.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Abbreviations: GHRP-6, GH-releasing peptide-6; GHS-R, GH secretagogue receptor; GRF, GH-releasing factor; h, human; rGH, rat GH; SRIF, somatostatin; Tgr, transgenic growth retarded; tNA, total nuclei acid; WT, wild-type.
Received April 11, 2003.
Accepted for publication July 17, 2003.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  L. Pacifico, C. Anania, J. F Osborn, E. Ferrara, E. Schiavo, M. Bonamico, and C. Chiesa Long-term effects of Helicobacter pylori eradication on circulating ghrelin and leptin concentrations and body composition in prepubertal children Eur. J. Endocrinol., March 1, 2008; 158(3): 323 - 332. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Stevanovic, V. Milosevic, D. Nesic, V. Ajdzanovic, V. Starcevic, and W. B. Severs A BRIEF COMMUNICATION: Central Effects of Ghrelin on Serum Growth Hormone and Morphology of Pituitary Somatotropes in Rats. Experimental Biology and Medicine, November 1, 2006; 231(10): 1610 - 1615. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. C. Martini, R. Fernandez-Fernandez, S. Tovar, V. M. Navarro, E. Vigo, M. J. Vazquez, J. S. Davies, N. M. Thompson, E. Aguilar, L. Pinilla, et al. Comparative Analysis of the Effects of Ghrelin and Unacylated Ghrelin on Luteinizing Hormone Secretion in Male Rats Endocrinology, May 1, 2006; 147(5): 2374 - 2382. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Wei, X. Qi, J. Reed, J. Ceci, H.-Q. Wang, G. Wang, E. W. Englander, and G. H. Greeley Jr. Effect of chronic hyperghrelinemia on ingestive action of ghrelin Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2006; 290(3): R803 - R808. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Ghizzoni, G. Mastorakos, A. Vottero, M. Ziveri, I. Ilias, and S. Bernasconi Spontaneous Growth Hormone (GH) Secretion Is Not Directly Affected by Ghrelin in Either Short Normal Prepubertal Children or Children with GH Neurosecretory Dysfunction J. Clin. Endocrinol. Metab., November 1, 2004; 89(11): 5488 - 5495. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. Davies, D. A. Carter, and T. Wells Photic Stimulation Inhibits Growth Hormone Secretion in Rats: A Hypothalamic Mechanism for Transient Entrainment Endocrinology, June 1, 2004; 145(6): 2950 - 2958. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. P. Goldstone, E. L. Thomas, A. E. Brynes, G. Castroman, R. Edwards, M. A. Ghatei, G. Frost, A. J. Holland, A. B. Grossman, M. Korbonits, et al. Elevated Fasting Plasma Ghrelin in Prader-Willi Syndrome Adults Is Not Solely Explained by Their Reduced Visceral Adiposity and Insulin Resistance J. Clin. Endocrinol. Metab., April 1, 2004; 89(4): 1718 - 1726. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. M. Thompson, D. A. S. Gill, R. Davies, N. Loveridge, P. A. Houston, I. C. A. F. Robinson, and T. Wells Ghrelin and Des-Octanoyl Ghrelin Promote Adipogenesis Directly in Vivo by a Mechanism Independent of the Type 1a Growth Hormone Secretagogue Receptor Endocrinology, January 1, 2004; 145(1): 234 - 242. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||
, P < 0.05; 
