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Endocrinology Vol. 146, No. 3 1506-1513
Copyright © 2005 by The Endocrine Society

Partial Reversibility of Growth Hormone (GH) Deficiency in the GH-Releasing Hormone (GHRH) Knockout Mouse by Postnatal Treatment with a GHRH Analog

Maria Alba, Andrew V. Schally and Roberto Salvatori

Division of Endocrinology and Ilyssa Center for Molecular and Cellular Endocrinology, The Johns Hopkins University School of Medicine (M.A., R.S.), Baltimore, Maryland 21287; and Endocrine and Polypeptide Cancer Institute, Veterans Affairs Medical Center and Tulane University School of Medicine (A.V.S.), New Orleans, Louisiana 70112

Address all correspondence and requests for reprints to: Dr. Roberto Salvatori, Division of Endocrinology, The Johns Hopkins University School of Medicine, 1830 East Monument Street #333, Baltimore, Maryland 21287. E-mail: salvator{at}jhmi.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The proliferation of pituitary somatotroph cells and the synthesis and secretion of GH require the hypothalamic peptide GH-releasing hormone (GHRH). Accordingly, we have shown that mice with targeted disruption [knockout (KO)] of the GHRH gene (GHRHKO) have isolated GH deficiency (GHD) and anterior pituitary hypoplasia. The weight of GRHRKO mice is about 60% that of normal mice by 12 wk of age. The phenotype is strikingly similar to that observed in the mouse with mutated GHRH receptor (little). It is not known whether exposure to endogenous GHRH during intrauterine growth is necessary for postnatal GH secretion, and whether GHD due to congenital lack of GHRH activity would be reversible by treatment with GHRH during the postnatal period. To answer this question, we treated GHRHKO mice with a long-acting superactive GHRH analog (JI-38) at two ages: from wk 2–6 (2 µg, twice a day) and from wk 12–16 (4 µg, twice a day). Normal littermates served as controls. At both ages JI-38 caused growth acceleration, increase in size of the pituitary gland, increase in pituitary GH mRNA and GH protein levels and serum GH, and significant increase in liver IGF-I mRNA, although none of these parameters was fully normalized. Our findings demonstrate that GHD and pituitary hypoplasia in GHRHKO mice may be partially reversed by long-term treatment with a GHRH analog, and that somatotroph cells maintain responsiveness to GHRH even if this factor is absent during intrauterine development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE SECRETION OF GH by somatotroph cells of the pituitary gland is under control of the hypothalamic GH-releasing hormone (GHRH) (1). Accordingly, mutations in the GHRH receptor (GHRH-R) cause isolated GH deficiency (IGHD) in both humans and mice (2, 3, 4, 5). The little mouse (gene symbol lit) is a well known mouse strain with autosomal recessive IGHD caused by a missense mutation in the extracellular domain of the GHRH-R that impairs the ability of the receptor to bind to GHRH (6). The pituitary of the little mouse is hypoplastic due to a deficiency of somatotroph cells that, although present in the ventrolateral portion of the gland, do not colonize the caudomedial area in the absence of GHRH stimulation (5). Similarly, humans with mutated GHRH-R genes have radiological evidence of pituitary hypoplasia (7, 8). We recently demonstrated that targeted ablation of the GHRH gene causes IGHD and pituitary hypoplasia in mice (9).

It is unclear whether GHD and somatotroph hypoplasia caused by the lack of GHRH during the development of the pituitary gland could be reversed during postnatal life. This question cannot be answered in the little model, because the lack of a functional GHRH-R cannot be bypassed. In adult humans, the presence of GHRH-secreting tumors causes somatotroph cell proliferation, proving that somatotroph cells maintain the ability to proliferate during postnatal life (10). Therefore, we hypothesized that even in the absence of intrauterine GHRH, somatotroph cells could proliferate and start producing GH when stimulated by GHRH.

Because GHRH has a very short half-life (11), we used an agonistic analog of GHRH (JI-38) that has been shown to have a much higher potency than the native peptide (12). In rats whose GHRH-producing neurons had been destroyed after birth with monosodium glutamate, this molecule (at an approximate dose of 66 µg/kg·d) has been shown to restore normal growth by stimulating GH synthesis and IGF-I production (13). In the present study we used a higher dose (~660 µg/kg·d at the initiation of treatment) to ensure maximal efficacy. We show that parenteral administration of JI-38 is able to partially reverse GHD in both young and adult mice with congenital lack of GHRH.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
GHRHKO mice were generated by mating +/– females and –/– males. Animals were ear-tagged, and tails were clipped at 10 d of age. Genotype was determined by Southern blot analysis (with 6-h transfer), as previously described (9).

To evaluate the effects of the GHRH analog JI-38 at different ages, we injected eight GHRHKO mice (4 males, 4 females) with 2 µg JI-38, sc, twice daily from age 2–6 wk (young group) or with placebo. A second group of adult GHRHKO mice (four males and four females) was treated with 4 µg JI-38, sc, twice a day from 12–16 wk of age (adult group) or with placebo. Dosage of JI-38 was doubled in adult groups in the attempt of adjusting to body weight (approximately twice the weight of young animals). Normal (+/+) sex- and age-matched mice served as controls. The sc route of administration was chosen based on previous reports that in rats JI-38 had a higher potency after sc than after iv administration, possibly due to resistance of the analog to enzymatic degradation in sc tissues (12).

The young mice were weaned at 3 wk of age and housed based on sex and treatment. All mice experienced controlled environment with 14-h light, 10-h dark cycles at 21 C and 23% humidity and were fed standard mouse/rat food (Prolab RMH2500, PMI Nutrition International, Brentwood, MO) and water ad libitum.

Animals were weighed once a week. Body weights were recorded to the nearest 0.1 g using a daily calibrated electronic balance (Scout Pro Balance, Ohaus Corp., Pine Brook, NJ). At the end of the treatment period, animals were euthanized with an overdose of halothane (Sigma-Aldrich Corp., St. Louis, MO), and blood was obtained by cardiac puncture. Body length was determined as nose to anus measurement using an electronic digital caliper (Control Co., Friendswood, TX).

All procedures were approved by The Johns Hopkins Institutional animal care committee.

GH serum measurement
Thirty minutes before death, all animals were injected sc with JI-38. Blood was collected by cardiac puncture (immediately after euthanasia) in the three young groups (placebo-treated, JI-38-treated, and control animals). Serum was frozen, and GH was assayed by Anilytics (Gaithersburg, MD) using a mouse-specific RIA.

GH mRNA measurement
For each experiment, pituitary glands of each group were harvested and pooled, and total RNA was isolated using TRIzol reagent (Invitrogen Life Technologies, Inc., Carlsbad, CA) according to the manufacturer’s recommendations. RNA was quantified using spectrophotometric analysis at OD 260/280 nm (DU 640 spectrophotometer, Beckman Coulter, Fullerton CA).

Pituitary GH mRNA content was quantified by Northern analysis of 3 µg total RNA, using a 671-bp 32P-labeled mouse cDNA probe, obtained by PCR amplification of GH sequences from mouse pituitary cDNA (sense, 5'-TCCTGACCGTCAGCCTGCTCT-3'; antisense, 5'-GAGGCACAGGAGAGTGCAGCA-3'). The intensity of the band corresponding to GH mRNA was quantified by phosphorimager (Molecular Imager FX, Bio-Rad Laboratories, Inc., Hercules, CA), and results were normalized by comparison with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression, measured by hybridization with 32P-labeled mouse GAPDH cDNA probe after stripping the blots. Sex-mixed (male and female) pooled pituitaries were used as normal control.

Pituitary GH protein measurement
Pooled pituitary extracts used for RNA extraction were also used for protein extraction according to the recommendations of TRIzol’s manufacturer. The protein concentration was determined using the bicinchoninic acid method (Micro BCA Protein Assay Kit, Pierce Chemical Co., Rockford, IL). Two mcirograms of proteins from each sample were boiled in 2x sample buffer, resolved on 15% SDS-PAGE, and electrotransferred onto a polyvinylidene difluoride membrane (Immobilon-P, Millipore Corp., Bedford, MA). After 1-h blocking in Tris-buffered saline, 0.02% Tween 20, and 5% milk, membranes were incubated for 2 h at room temperature with rabbit antimouse GH antibody (National Hormone and Peptide Program, Harbor-University of California-Los Angeles Medical Center, Torrance, CA) at a 1:80,000 dilution. After washing, membranes were incubated 1 h with goat antirabbit immunoglobulin G horseradish peroxidase-conjugated (1:3,000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The membranes were washed, and proteins were detected by enhanced chemiluminescence (Amersham Biosciences, Chicago, IL). Band size was determined by comparison with a Full Range Rainbow protein weight marker (Amersham Biosciences).

Serum protein and liver mRNA of IGF-I
Serum IGF-I levels were measured by Anilytics, Inc., using rat IGF-I RIA (DSL-2900, Diagnostic Systems Laboratories, Webster, TX) after acid-ethanol extraction, following the manufacturer’s recommendations. Liver RNA was extracted using TRIzol reagent. Measurement of mRNA for IGF-I was performed by Northern blotting (15 µg RNA/lane) using 32P-labeled mouse IGF-I cDNA probe (donated by Dr. D. LeRoith, NIH, Bethesda, MD). The intensity of the bands was analyzed by phosphorimager, and the results were normalized to GAPDH cDNA after stripping the blot.

Statistical analysis
Results are expressed as the mean ± SD. Statistical analysis of the data was performed by ANOVA using the SPSS statistical package (SPSS, Inc., Chicago, IL), with post hoc analysis by Bonferroni’s method. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Body weight
Young animals.
At the end of 4 wk of treatment, body weights were significantly higher in JI-38-injected animals than in placebo-treated mice (Fig. 1Go). Although treated male mice weighed less than the control group, the weight of treated females was not significant different from that of the control group (14.0 ± 1.4 vs. 17.2 ± 2.2 g; Fig. 1Go).



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  		FIG. 1. Weight curves (in grams) of the young group (2–6 wk old). Bars represent SDs. *, P < 0.05. At the end of treatment with JI-38, the treated group reached a significantly higher weight than the placebo group. The weight of the treated female group was not significantly different from that of the control +/+ mice.

 
Adult animals.
JI-38 injected animals did not achieve a significantly higher body weight compared with the sex- and age-matched, placebo-treated group (males, 16.2 ± 0.4 vs. 13.6 ± 1.1 g; females, 15.0 ± 1.6 vs. 12.0 ± 1.3 g), and they weighed significantly less than control animals (Fig. 2Go).



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  		FIG. 2. Weight curves (in grams) of the adult group (12–16 wk old). Bars represent SDs. *, P < 0.05. No significant difference was seen between JI-38-treated and placebo groups at the end of treatment.

 
Body length
Young animals.
The nose to anus length of the JI-38-injected animals was significantly greater than that of the placebo treated group in both males groups, but was less than that in the control groups (Fig. 3AGo).



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  		FIG. 3. Length measurement (nose to anus, in centimeters) after 4 wk of treatment with JI-38 in the young group (A) and the adult group (B). Bars represent SDs. *, P < 0.05. All JI-38-treated groups had significantly increased body length compared with placebo-treated animals.

 
Adult animals.
Body length after therapy with JI-38 was significantly greater than that after placebo treatment, but less than that in controls (Fig. 3BGo).

Serum GH
Serum GH was measured only in the young groups, on serum collected at the time of death, 30 min after the last JI-38 injection (also administered to placebo-treated animals). Results are shown in Fig. 4Go. As expected, GH levels (in nanograms per milliliter) were significantly lower in placebo-treated GHRHKO female animals than in normal female controls. This difference, although marked, did not reach statistical significance in male animals (1.92 ± 1.56 vs. 9.74 ± 3.14). In both sexes, serum GH levels were higher in JI-38-treated animals compared with placebo-treated animals. Surprisingly, the levels reached in JI-38-treated animals were significantly higher than those in wild-type controls.



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  		FIG. 4. Serum GH levels (in nanograms per milliliter) in the young group 30 min after JI-38 injection. *, P < 0.05; **, P < 0.01.

 
Total pituitary RNA
Young animals.
The average RNA content per pituitary of JI-38-treated animals showed a 3-fold increase compared with that in placebo-treated males and females, but not to the level in normal mice. Nevertheless, the average total pituitary RNA level of the treated groups was only 30% that of the sex-matched control groups (Fig. 5AGo).



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  		FIG. 5. Total pituitary RNA content (in micrograms per pituitary) in the young group (A) and the adult group (B). Each columnis the result of extraction from four pituitary glands pooled at the time of RNA extraction.

 
Adult group.
Similar to the young group, the average yield of total RNA from pituitaries from adult JI-38-treated females and males was higher than that in placebo animals, but lower than that in controls (Fig. 5BGo).

Pituitary GH mRNA
Young animals.
The GH mRNA content was 3.5 times higher in JI-38-treated females and 5 times higher in JI-38-treated males compared with the placebo group (Fig. 6AGo) and reached 82% and 86% of the GH mRNA levels in the control animals, indicating that most of the increase in the total pituitary mRNA content was related to GH expression.



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  		FIG. 6. Pituitary GH mRNA measurement in the young group (A) and the adult group (B), measured by Northern blot and quantified by phosphorimager. For the control group, RNA from two +/+ females and two +/+ males was used. The results are expressed in arbitrary units and are normalized to GAPDH mRNA after stripping the membrane.

 
Adult animals.
As in the case of young animals, GH mRNA was markedly increased in JI-38-treated animals, reaching 92% in females and 107% in males of that in the age-mixed control group (Fig. 6BGo).

Pituitary GH protein content
The results of a Western blot performed on pituitary extracts are shown in Fig. 7Go. This is a representative blot of three different Western blot experiments performed on the same pituitary extracts. No GH protein was detected in the testis (as expected). No detectable signal was observed using 2 µg protein extract in placebo-treated GHRHKO animals, whereas GH signal was detected in JI-38-treated animals and (at higher intensity) in normal controls.



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  		FIG. 7. Western blot analysis of protein extracts from testis (T; negative control) and pooled pituitary glands of normal controls (Con), placebo-treated (Pl), and JI-38-treated (JI-38) animals from the young groups. F, Females; M, males. The expected size of mouse GH is 22 kDa.

 
Liver IGF-I mRNA and serum IGF-I
Young animals.
As shown in Fig. 8AGo, male animals treated with JI-38 had a significantly greater expression of liver IGF-I mRNA compared with the placebo group, although this expression was still significantly less than that in the control group. The difference between treated females and placebo was not statistically significant.



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  		FIG. 8. Liver IGF-I mRNA in the young group (A) and the adult group (B), measured by Northern blot and quantified by phosphorimager. The results are expressed in arbitrary units and are normalized to GAPDH mRNA. Bars on columnsrepresent SDs. *, P < 0.05. No significant increase was detected between JI-38-treated young females and the placebo group.

 
Surprisingly, no difference in serum IGF-I was found between JI-38-treated and placebo-treated animals of either sex (Fig. 9AGo), and in both groups, the levels were significantly lower than controls (males, 342.5 ± 40.2 vs. 301.5 ± 32.8 vs. 579.9 ± 140.8; P < 0.05; females, 299.9 ± 11.7 vs. 280.4 ± 19.3 vs. 620.3 ± 156.0 ng/ml; P < 0.05).



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  		FIG. 9. Serum IGF-I (in nanograms per milliliter) in the young group (A) and the adult group (B) after 4-wk treatment with JI-38 or placebo. Bars represent SDs. *, P < 0.05. At no point did treated animals have higher serum IGF-I levels compared with placebo groups.

 
Adult animals.
mRNA for liver IGF-I in JI-38-treated adults was significantly greater than that in the placebo-treated animals, both males and females, and significantly less than that in wild-type controls (Fig. 8BGo). As observed in the younger animals, this finding did not correlate with plasma IGF-I levels, which remained almost within the same range as in the placebo group, both of which were significantly lower than the control values (males, 332.9 ± 4.3 vs. 400.3 ± 90.5 vs. 492.5 ± 57.9 ng/ml; P < 0.05; females, 382.3 ± 122.0 vs. 365.4 ± 78.6 vs. 655.6 ± 84.5 ng/ml; P < 0.05; Fig. 9BGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
GHRH is a neuropeptide secreted by the hypothalamus that acts on the pituitary, inducing proliferation of somatotroph cells and controlling the synthesis and release of GH (1). The lack of GHRH stimulus causes GHD and pituitary hypoplasia, as described in the naturally occurring, GH-deficient little mouse (5). Because in the little animal the GHRH-R is nonfunctional, it is impossible to determine whether GHD may be reversed by GHRH.

We recently created a mouse in which targeted ablation of the GHRH gene causes GHD and pituitary hypoplasia (9). Because the production of GHRH is abolished in the GHRHKO mouse at the embryonic stage and the function of the GHRH-R is probably preserved, this mouse offers a modifiable model of GHRH deficiency. The aim of this study was to investigate whether postnatal treatment with a GHRH analog could reverse GHD in the GHRHKO mouse, and if similar results could be achieved at two different stages of life. To this end, we used a long-acting analog (JI-38) of human GHRH-(1–29)-NH2.

In our study, groups of 2-wk-old GHRHKO mice (young animals) and 12-wk-old GHRHKO mice (adult animals) were treated twice a day for 4 wk by sc injection of GHRH analog. At the end of the treatment periods, the level of total pituitary RNA was 3–10 times higher in the JI-38-treated than in placebo animals, but remained lower than that in the control group. Because we could not measure the actual size of the pituitaries and examine their histology, we could not determine whether JI-38 caused mainly somatototroph cell proliferation or hypertrophy. In interpreting the total pituitary RNA data, it should be taken into consideration that GHRHKO mice, despite JI-38-induced growth, remained smaller than +/+ mice. Therefore, it is conceivable that if the total RNA could be adjusted for head or brain size, this difference could be narrowed. Indeed, when we measured the content of mRNA for pituitary GH and adjusted it for GAPDH mRNA levels, we found a dramatic increase in GH expression in the treated group, ranging from 82–107% compared with the sex- and age-matched controls, whereas nontreated animals had levels of pituitary GH mRNA that were between 17–24% of normal.

Consistent with the finding of an increased content of GH mRNA, Western blot analysis showed that GH protein content was increased in JI-38-treated animals compared with placebo-treated animals, but was still lower than that in normal wild-type controls. Accordingly, serum GH levels (obtained 30 min after JI-38 injection in all groups) were significantly increased in JI-38 animals compared with placebo-treated mice, confirming that JI-38 increased GH production in GHRHKO animals. Surprisingly, serum GH levels in JI-38-treated animals were even higher than those in normal controls. This result does not match the mRNA and protein contents. One possible explanation of the higher GH release in JI-38-treated GHRHKO animals than in normal controls is that prolonged and intermittent JI-38 treatment may have caused an up-regulation of the GHRH-R (14). The acute exposure to JI-38 in animals with up-regulation of the receptor may have a more rapid and preferential effect on the release of preformed GH.

Treatment with JI-38 was able to induce a significant acceleration of longitudinal growth in young animals and, interestingly, an even more significant increase in body length in the older group. Growth is possible in adult mice, because the mouse growth plate does not fuse long after puberty. Surprisingly, these length findings were not accompanied by an increase in serum IGF-I levels, which remained similar to those in the placebo groups in both young and adult mice. Serum IGF-I is mostly an expression of liver IGF-I production (15). It is possible that the initial days or weeks of treatment are needed to reverse somatotroph cells hypoplasia, and that the increase in GH secretion occurred in the later part of the treatment period, not allowing an increase in liver IGF-I expression robust enough to be detected by serum measurements. However, serum IGF-I is only a marker of GH action. Bone growth is a complex event in which a number of hormonal factors are involved (16). Although the IGF-I KO mouse has a dramatic growth failure due to a severe reduction in serum IGF-I (17), the liver IGF-I-deficient mouse displays relatively normal growth despite a 75% reduction in serum IGF-I compared with the controls (18, 19), indicating that the autocrine/paracrine growth actions of GH do not require liver IGF-I. Accordingly, GH itself has direct effects on the growth plate, by acting directly on its receptor (20), by stimulating local IGF-I production (21), or, as recently suggested, by promoting local IGF-II production (22). Because treatment with JI-38 was able to partially reverse the pituitary hypoplasia and almost completely restore the expression of GH, we hypothesize that the significant increase in body length observed in the treated groups may be due to this local action of GH.

Interestingly, when we measured the expression of liver IGF-I mRNA, we found significantly higher levels in treated animals compared with the placebo group, except in young females (P = 0.813). This finding is in contrast with the serum IGF-I levels and cannot be easily explained. We speculate that serum IGF-I is a less sensitive index of liver IGF-I production than direct measurement of liver IGF-I mRNA.

The fact that JI-38 was able to partially reverse GHD is clearly shown by the observation that in young animals the final weight and length reached in the JI-38-treated group were significantly greater than those in the placebo group. The young females treated with JI-38 gained weight to the point that no significant differences were found between them and the control group. However, in adult animals, despite an increase in body length, there was no significant weight difference between JI-38-treated and placebo groups. Although we have not performed any study to determine body composition in GHRHKO animals, they appear to be more obese compared with +/+ mice. We believe that the reason for the lack of weight difference may be the GHD status itself, which is associated with decreased lean body mass and central obesity in both men (23, 24) and rodents (25, 26, 27). The weight gain associated with JI-38-induced longitudinal growth may have been lessened by the lipolytic activity cause by the reversal of GHD. Additional studies are being designed to test this hypothesis.

In conclusion, we report that GHD and pituitary hypoplasia can be partially reversed by treatment with a GHRH analog in GHRHKO mice, showing that the lack of GHRH during development does not permanently impair somatotroph cell responsiveness to this factor. Because the treatment was performed for only a relatively short period of time, it cannot be excluded that a longer treatment, different regimens, or an earlier initiation may improve the final outcome and fully normalize growth.


   Footnotes
 
This work was supported by National Institutes of Health Grants R03-HD-042465-01 and R03-HD-046641-01. M.A. was supported in part by a fellowship from Pfizer, Inc.

First Published Online November 24, 2004

Abbreviations: GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; GHD, GH deficiency; GHRH, GH-releasing hormone; GHRH-R, GH-releasing hormone receptor; IGHD, isolated GH deficiency; KO, knockout.

Received August 10, 2004.

Accepted for publication November 19, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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