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Section of Endocrinology and Metabolism, Department of Medicine, University of Illinois at Chicago (J.K., T.G.U., L.A.F, R.D.K.); and Department of Medicine, Chicago Veterans Administration Health Care System, West Side Division (T.G.U.), Chicago, Illinois 60612
Address all correspondence and requests for reprints to: R. D. Kineman, Section of Endocrinology and Metabolism, Department of Medicine, University of Illinois at Chicago, 1819 West Polk, M/C 640, Chicago, Illinois 60612. E-mail: kineman{at}uic.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The spontaneous dwarf rat (SDR) provides a unique opportunity to study the regulation of GHRH and GHRH-R expression in the complete absence of the negative feedback effects of endogenous GH. This dwarf model displays a selective absence of GH, as a consequence of a point mutation within the GH gene, which creates a premature, in-phase stop codon (8). SDR pituitary GH mRNA levels are less than 3% of normal controls, and there is no immunodetectable GH within the pituitary or the systemic circulation (8). Unlike other animal models of GH-deficiency, PRL and TSH are relatively normal (9, 10). SDRs are 75% smaller than their normal counterparts, and the pituitary is proportionately reduced in size. Within the SDR pituitary, 50% of the cells are not immunoreactive for any pituitary hormone (11). However, these cells do contain a highly developed organelle system that is characteristic of GH-producing cells (8, 11). The proportion of immunonegative cells is similar to that which normally contains GH (12), suggesting that these cells are somatotropes that lack GH secretory granules. Although GH synthesis is absent, the SDR GHRH-R signaling system seems to be intact, because GHRH treatment in vitro results in an increase in GH mRNA that is proportional to the response observed in normal pituitary cell cultures (8). Therefore, in the present study, we have used the SDR model to determine whether modulation of somatotrope sensitivity by regulation of GHRH-R synthesis is a component of GH negative feedback regulation, by examining the effects of GH/insulin-like growth factor-I (GH/IGF-I) replacement on SDR GHRH-R mRNA levels.
In addition, we have used this animal model to further explore GH-dependent changes in hypothalamic neuropeptide expression that could ultimately lead to the suppression of GHRH synthesis. We know that only a few GHRH-producing neurons within the arcuate nucleus express the GH receptor (GH-R) (13). However, many neuropeptide Y (NPY) neurons located within the arcuate nucleus express GH-R mRNA (14, 15) and respond to systemic administration of GH by increasing c-fos expression (16, 17). These same neurons have been shown to interact with hypophysiotropic SS-positive neurons located within the periventricular nucleus (18, 19). This anatomical relationship among GHRH, NPY, and SS suggests that GH mediates the reduction of GHRH synthesis by sequential activation of NPY and SS neurons. To test this possibility, studies were conducted to examine the effects of GH/IGF-I replacement on the relative levels of GHRH, SS, and NPY mRNA in SDR rats.
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Materials and Methods |
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RNA isolation
Total pituitary and hypothalamic RNA was extracted according to
the Tri Reagent protocol (Molecular Research Center, Cincinnati, OH),
with the exception that the aqueous phase was further purified by
extraction with phenol:chloroform:isoamyl alcohol (25:24:1; pH 5.2;
Fisher Scientific, Pittsburgh, PA) to improve the efficiency of the RT
reaction (21). RNA was then precipitated with isopropanol; and the
pellet was washed with 70% ethanol, air dried, and resuspended in TE
buffer (10 mM Tris-HCL, pH 7.6, 1 mM EDTA). The
concentration and purity of RNA were determined by spectrophotometric
analysis at OD 260/280 nm. Total RNA recovered was 36.9 ± 5 µg
from normal pituitaries, 7.1 ± 1.4 µg from SDR pituitaries,
44.2 ± 1.7 µg from normal hypothalami, and 48 ± 1.8 µg
from SDR hypothalami.
RT-PCR
Pituitary GHRH-R. GHRH-R mRNA levels were assessed using
quantitative RT-PCR, as previously described (21). To correct for
RT-PCR efficiency, a synthetic RNA, homologous to the endogenous rat
GHRH-R mRNA with an internal 235-bp fragment excised (RPS-1), was added
to each sample before RT, and the generated complementary DNAs (cDNAs)
were amplified using a primer set specific for the rat GHRH-R cDNA
sequence. To correct for variations in total RNA,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was amplified
from a separate aliquot of the RT reaction. PCR products were
transferred to nylon membranes and hybridized with specific
radiolabeled probes. A phosphorimager was used to visualize the
amplified products, and image analysis was used to evaluate band
intensity. The relative abundance of GHRH-R mRNA was calculated by the
equation: GHRH-R x 1/RPS-1 x 1/GAPDH.
Pituitary transcription factor-1 (Pit-1). Pit-1 mRNA levels were quantified by RT-PCR using a set of primers (sense, 5'-AACGTGATGTCCACAGCGACAG-3' and antisense, 5'-TCGGCAGATGGTTGTTTGACTG-3') specific to the rat Pit-1 cDNA sequence (22). PCR conditions were identical to those for GHRH-R/RPS-1 (21), with the exception that the annealing temperature was 65 C. The PCR reaction was run for 28 cycles, which was within the linear portion of the amplification curve. The product was gel electrophoresed, transferred to a nylon membrane, and hybridized with a radiolabeled probe generated by PCR. A single band corresponding to the predicted size (390 bp) was amplified. Pit-1 mRNA values were normalized by GAPDH values to correct for variations in starting mRNA concentrations.
Northern blot analysis of hypothalamic GHRH, SS, and NPY mRNA
Forty-percent of the total RNA isolated from a single
hypothalamus (
18 µg) was electrophorectically separated on a 0.4
M formaldehyde-1% agarose gel, transferred to a nylon
membrane, and hybridized (24 h at 42C) sequentially with
32P-labeled cDNA probes specific for rat GHRH (23), SS
(24), and NPY (25), as previously described (26). Membranes were
exposed to the phophorscreen for 1–3 days, and the intensity of the
hybridization signals were determined by image analysis.
GHRH-induced intracellular cAMP generation
Anterior pituitaries from SDRs and control rats were
enzymatically and mechanically dissociated into single cells, as
previously described (21, 27). Cells were then washed and resuspended
in 
-MEM (Gibco BRL, Grand Island, NY) supplemented with 0.1% BSA
and antibiotics. Approximately 1.5 x 106 and 3.5
x 106 cells were recovered from each SDR and normal
pituitary, respectively. Cell viability after dissociation was
consistently greater than 95%, as assessed by the exclusion of trypan
blue. Cells were plated at a density of 50,000 cells/well in 1 ml
-MEM supplemented with 10% horse serum and placed in an humidified
atmosphere containing 95% air-5% CO2. After a 3-day
culture period, wells were rinsed with serum-free medium and
preincubated for 1 h. Medium was then removed and replaced with 1
ml of fresh medium to which was added rat GHRH (Bachem, Torrence, CA)
to achieve a final concentration of 0.001–10 nM. Cultures
were incubated for 15 min, and cells were extracted with 0.1
M HCl in 95% ethanol for assay of cAMP (28).
Systemic administration of GH and IGF-I
SDRs were anesthetized using ketamine/xylazine and osmotic
minipumps (model 1003D, Alzet Co., Palo Alto, CA), containing rat GH
(10 µg/µl; NIDDK National Hormone and Pituitary Program) or vehicle
(saline) were implanted sc (five animals/treatment group). In a
separate experiment, SDRs (five animals/group) were implanted with
osmotic pumps containing recombinant human IGF-I (5 µg/µl;
Genentech, Inc., South San Francisco, CA) or vehicle (saline). The
pumps released hormone solutions at a rate of 1 µl/h. Seventy-two
hours after pump placement (1000–1200 h), rats were killed; and
hypothalami and pituitaries were rapidly removed and frozen for GHRH-R,
Pit-1, GHRH, SS, and NPY mRNA determinations. Serum IGF-I levels were
assessed by double-antibody RIA, as described (29), after removal of
the binding proteins by Sep-Pak C18 reverse-phase
cartridges (Millipore, Milford, MA). Synthetic recombinant human IGF-I,
used as a standard, was provided by Genentech, whereas IGF-I antiserum
(UBK 487) was provided by the National Hormone and Pituitary Program,
NIH.
Statistical analysis
All results are expressed as mean ± SEM.
Comparisons between groups were made by Student’s t test.
P < 0.05 was considered significant. All comparisons
were limited to samples electrophoresed on the same gel.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. SDR hypothalami
contained 39% more GHRH mRNA, compared with age-matched normal
controls (P < 0.01). In contrast, SS and NPY mRNA
levels in SDRs were reduced by 43% and 30% of normal values,
respectively (P < 0.01) .
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. Pituitary GHRH-R mRNA levels in SDRs
were 56% greater than those of normal controls (P <
0.05), consistent with the increase in hypothalamic GHRH mRNA, whereas
Pit-1 mRNA did not differ between animal groups. These differences in
GHRH-R mRNA levels can be attributed to changes in the level of GHRH-R
mRNA in individual somatotropes, because the relative abundance of
somatotrope-like cells is comparable (50%) in SDRs and normal rats
(8, 11, 12). To determine whether the elevated levels of GHRH-R mRNA in
the SDRs resulted in an increase in GHRH-R function, SDR and normal
pituitary cell cultures were challenged with GHRH (0.001–10
nM for 15 min), and intracellular cAMP concentrations were
determined (Fig. 3). SDR pituitary cells
were hyperresponsive to 1 and 10 nM GHRH, which induced a
rise in intracellular cAMP concentrations 50% greater than that
observed in control cultures (P < 0.05 and
P < 0.01, respectively).
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. GH treatment resulted in an 11%
increase in body weight from pretreatment values and a 600% increase
in circulating IGF-I concentrations, compared with vehicle-treated
controls. IGF-I replacement had a less dramatic (but significant)
effect on body weight gain (1% of pretreatment values;
P < 0.05), although circulating IGF-I concentrations
reached values comparable with that found after GH treatment (550% of
vehicle-treated controls; P < 0.01).
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. SDRs treated with GH
had 60% less hypothalamic GHRH mRNA than vehicle-treated controls
(P < 0.01). The reduction in GHRH mRNA levels was
accompanied by a reciprocal increase in SS and NPY mRNA levels (31%,
and 27% of vehicle treated controls, respectively; P
< 0.05). In contrast, IGF-I treatment did not alter hypothalamic GHRH,
SS, or NPY mRNA levels (Fig. 5).
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), in
parallel with its effects on hypothalamic GHRH mRNA. IGF-I infusion
also resulted in a 30% reduction in pituitary GHRH-R mRNA levels
(P < 0.05). Neither GH nor IGF-I significantly altered
pituitary Pit-1 mRNA levels (data not shown).
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Discussion |
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These results raise the question: what mediates GH-induced suppression of GHRH-R synthesis? It is unlikely that GH itself exerts a direct inhibitory role, because we (21) and others (31) have shown that in vitro treatment with GH does not alter somatotrope sensitivity to a subsequent GHRH challenge. A more likely candidate for a direct modulator of GHRH-R synthesis is its own endogenous ligand, GHRH. Several lines of evidence predict that GHRH stimulation should augment GHRH-R gene expression. Passive immunization of neonatal rats (birth to day 10) with GHRH antiserum resulted in a 70% decrease in pituitary GHRH-R mRNA levels, compared with normal serum-treated controls (1), and a comparable reduction in in vitro GHRH responsiveness (32). A reduction in GHRH-R mRNA levels was also observed after injection of GH, which was used to circumvent the fall in endogenous GH observed after GHRH antiserum treatment (1). However, the combination of GHRH antiserum and GH did not result in a greater suppression of GHRH-R mRNA levels than that observed with each hormone alone, suggesting a common mechanism of action, which likely involves modulation by GHRH. Such a mechanism is supported by the observation that treatment of neonatal rats with GH reduces hypothalamic GHRH expression and decreases pituitary responsiveness to GHRH (33), whereas coadministration of GHRH and GH restores pituitary sensitivity to the ligand. These data, combined with the positive correlation between GHRH and GHRH-R mRNA levels observed in the present study, suggest that GHRH is required to maintain its own receptor synthesis and function. A direct effect of GHRH on GHRH-R expression has been recently reported in vitro, where treatment of rat pituitary cell cultures for 12 h with GHRH resulted in a 6-fold increase in GHRH-R mRNA levels (3). In that study, GHRH-R mRNA levels were also increased by in vitro exposure to dibutyryl cAMP, suggesting that GHRH activates the cAMP second-messenger system to augment GHRH-R synthesis. This hypothesis is compatible with the demonstration of cAMP response elements within the 5'-flanking region of the human (34) and rat (35) GHRH-R genes.
The positive correlation between the GHRH and GHRH-R observed in the present study at first seems contradictory with our previous report, where treatment of primary rat pituitary cell cultures with GHRH for 4 h resulted in a 50% reduction in GHRH-R mRNA levels, in vitro (21). However, it is well documented in other G-protein-coupled receptor systems that the modulation of receptor synthesis and sensitivity (positive or negative) is dependent on concentration, frequency, and duration of ligand stimulation. For example, pulsatile delivery and low concentrations of GnRH prime gonadotropes, resulting in an increase in GnRH-R mRNA levels (36), whereas continuous exposure to high agonist concentrations down-regulate GnRH-R synthesis (37). Similar biphasic effects of ligand stimulation have been observed for the TSH (38, 39) and ß-adrenergic (40) receptors. When the current in vivo report is compared with our previous in vitro observations, it is clear that the pattern and effective concentration of GHRH interacting with the somatotrope population is drastically different. In the SDR model, endogenous GHRH is likely delivered to the pituitary in a pulsatile manner against a background of intermittent SS tone, which would act to temper GHRH action. In vitro, GHRH is introduced, in the absence of any inhibitory factors, resulting in unopposed ligand stimulation. Thus, the somatotrope can sense the relative pattern of GHRH input and respond accordingly by up-regulating or downregulating GHRH-R synthesis, thereby providing an additional layer of control that ultimately leads to maintenance of GH synthesis and release in a well-defined range.
Pit-1 is required for the expression of GH and the GHRH-R, as demonstrated by the lack of GH and GHRH-R mRNA in the Pit-1-defective Snell dwarf (dw/dw) mouse (41). This link between Pit-1 and the GHRH-R is substantiated by the observations that sequences corresponding to the Pit-1 response element are present within the GHRH-R gene promoter region and that Pit-1 can activate expression of a GHRH-R promoter-driven reporter gene in a heterologous cell system (41). Although Pit-1 is clearly necessary for GHRH-R expression, GHRH has also been shown to increase Pit-1 expression in vitro (42). The effect of GHRH on Pit-1 mRNA levels is attributed to activation of the cAMP second-messenger system, because the nonspecific activation of adenylate cyclase activity by forskolin can mimic the stimulatory action of GHRH (42). In the present study, we sought to determine whether the increase in GHRH-R mRNA levels was accompanied by an increase in Pit-1 gene expression. Despite the elevated levels of GHRH and GHRH-R mRNAs in SDR pituitaries, there was no difference in Pit-1 mRNA levels. Likewise, there was no effect of GH or IGF-I replacement on the level of Pit-1 mRNA in the SDR pituitary, suggesting that there is sufficient GHRH input in the SDR model (in the absence or presence of GH) to maintain Pit-1 expression.
The relative level of NPY expression, in the SDR hypothalamus, corresponded to that observed for SS. In the absence of GH, SS and NPY mRNA levels were low, compared with normal controls, whereas GH replacement resulted in a concomitant increase in both SS and NPY mRNA levels. This observation is consistent with the hypothesis that GH-induced inhibition of GHRH mRNA is dependent on the activation of hypothalamic NPY neurons (15). GH-R expression has been demonstrated in several areas of the hypothalamus, including the arcuate nucleus (6, 13, 43). Though few GHRH-producing neurons within the arcuate nucleus express GH-R mRNA, NPY-containing neurons (found in close proximity to GHRH-producing neurons) do express the GH-R (14, 15). The same neurons respond to GH by increasing both NPY (14) and c-fos expression (16, 17). Hypophysectomy results in the selective reduction in both GH-R and NPY mRNA within the arcuate nucleus (14, 44) and a rise in GHRH mRNA (4). Despite the relationship between NPY and GHRH, there is no direct evidence that NPY neurons form synapses with GHRH neurons. However, NPY axons do project to the periventricular nucleus (19), where SS-containing hypophysiotropic neurons are located, and NPY/SS synaptic connections have been identified within this region (18). SS neurons have been shown to synapse with GHRH-containing dendrites (45, 46), and SS receptors have been colocalized to GHRH-immunopositive neurons within the arcuate nucleus (47, 48). These anatomical associations, coupled with the reciprocal relationship between GHRH and NPY/SS mRNA levels observed in the SDR model, strongly suggest that GH mediates the reduction of GHRH mRNA by the sequential activation of NPY and SS neurons. However, a direct effect of NPY on SS or GHRH gene expression remains to be demonstrated.
In contrast to the effect of GH replacement on hypothalamic gene expression, IGF-I treatment did not alter GHRH, SS, or NPY mRNA levels. These observations parallel those in the dw/dw rat, where systemic administration of GH, but not IGF-I, suppressed elevated levels of GHRH mRNA (29). However, in the same study, intracerebroventricular administration of IGF-I clearly decreased GHRH mRNA. IGF-I and IGF-I receptor mRNA are found throughout the brain, including the hypothalamus (49, 50), and GH treatment can increase central expression of IGF-I (51, 52). Therefore, it has been postulated that the IGF-I critical for hypothalamic negative feedback regulation is of central origin. Although systemic IGF-I was ineffective in modulating hypothalamic function, IGF-I infusion did result in a 30% decrease in pituitary GHRH-R mRNA levels. IGF-I has a profound inhibitory effect on basal and stimulated GH release from pituitary cell cultures (53, 54) by directly suppressing GH gene expression (54, 55, 56) and has also been shown to reduce the stimulatory effect of dibutyryl cAMP and forskolin on GH release and synthesis (57). Because GHRH is believed to modulate GHRH-R synthesis by activation of the cAMP second-messenger signaling pathway, elevation of circulating IGF-I levels in SDRs could interfere with intracellular signaling events and blunt somatotrope responsiveness to endogenous GHRH, leading to a reduction in GHRH-R mRNA levels. Therefore, the suppression of GHRH-R mRNA levels, observed after systemic administration of GH, could be attributed, in part, to a direct pituitary effect of circulating IGF-I.
In summary, the results of the present study demonstrate that the mechanism of GH autofeedback regulation is a multilevel process that includes the reduction in pituitary GHRH-R synthesis and sensitivity, as well as a reduction in hypothalamic GHRH expression. We have also demonstrated that GH-dependent reduction of hypothalamic GHRH expression is accompanied by a concomitant increase in SS and NPY mRNA, suggesting that activation of SS and/or NPY neurons act as intermediates in GH feedback inhibition of GHRH gene expression. Therefore, we can conclude from these data that GH acts to suppress its own synthesis and release by decreasing hypothalamic stimulatory tone, increasing hypothalamic inhibitory tone and reducing the responsiveness of the pituitary somatotrope to stimulatory hypothalamic input.
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Acknowledgments |
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Footnotes |
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2 Visiting Scientist from the Department of Medicine, Nippon Medical
School, Sendagi 1–1-5, Bunkyo-ku, Tokyo 113, Japan. Recipient of the
Japan Private School Promotion Foundation Award for Overseas
Training.
Received January 13, 1998.
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