GHRH excess and blockade in X-LAG syndrome

Abstract

X-linked acrogigantism (X-LAG) syndrome is a newly described form of inheritable pituitary gigantism that begins in early childhood and is usually associated with markedly elevated GH and prolactin secretion by mixed pituitary adenomas/hyperplasia. Microduplications on chromosome Xq26.3 including the GPR101 gene cause X-LAG syndrome. In individual cases random GHRH levels have been elevated. We performed a series of hormonal profiles in a young female sporadic X-LAG syndrome patient and subsequently undertook in vitro studies of primary pituitary tumor culture following neurosurgical resection. The patient demonstrated consistently elevated circulating GHRH levels throughout preoperative testing, which was accompanied by marked GH and prolactin hypersecretion; GH demonstrated a paradoxical increase following TRH administration. In vitro, the pituitary cells showed baseline GH and prolactin release that was further stimulated by GHRH administration. Co-incubation with GHRH and the GHRH receptor antagonist, acetyl-(d-Arg(2))-GHRH (1-29) amide, blocked the GHRH-induced GH stimulation; the GHRH receptor antagonist alone significantly reduced GH release. Pasireotide, but not octreotide, inhibited GH secretion. A ghrelin receptor agonist and an inverse agonist led to modest, statistically significant increases and decreases in GH secretion, respectively. GHRH hypersecretion can accompany the pituitary abnormalities seen in X-LAG syndrome. These data suggest that the pathology of X-LAG syndrome may include hypothalamic dysregulation of GHRH secretion, which is in keeping with localization of GPR101 in the hypothalamus. Therapeutic blockade of GHRH secretion could represent a way to target the marked hormonal hypersecretion and overgrowth that characterizes X-LAG syndrome.

Keywords: GHRH; GPR101; gigantism; growth hormone; pituitary.

Figure 1.

Histological and immunohistochemical appearance of the pituitary lesion Histological sections were stained with hematoxylin and eosin (A, B, C), and with silver to highlight the reticulinic fibres (D, E) or immunostained with the CAM 5.2 monoclonal mouse antibody against cytokeratins 8 and 18 (F), with polyclonal rabbit antibody against growth hormone (G) or against prolactin (H), or with the MIB-1 mouse monoclonal antibody against Ki67 (I). Hematoxylin and eosin-stained sections (A-C) show the abundance of acidophilic somatotroph cells. Argyrophilic staining show that reticulinic fibres are still present in most parts of the lesion (D), indicating its hyperplastic nature, but tend to disappear in areas (E), suggesting development of an adenoma within the hyperplastic tissue. Pituitary cells contain cytokeratin filaments, most often diffusely distributed in the cytoplasm but concentrated in a perinuclear dot in a few cells (F). Many cells contain growth hormone (G) or prolactin (H). About 5% of pituitary cells are proliferating as indicated by Ki67 immunostaining (I). Bars correspond to 400 μm in A, to 100 μm in B, D, E, G, H, I, to 50 μm in F and to 25 μm in C.

Figure 2.

Dynamic and pulsatility testing. Elevated growth hormone (GH), prolactin (PRL) and GHRH levels were seen throughout an extended testing period of 180 minutes (Panel A). GH levels are seen to peak at 150 min after an earlier GHRH rise between 105min and 135 min. A TRH test induced an immediate and marked increase in GH levels and GHRH remained largely unchanged (Panel B). GnRH administration led to suppression of GH levels at 30 mins, whereas GHRH levels remained unaltered and prolactin rose mildly (Panel C). GH and PRL are measured in ng/mL; GHRH is measured in pg/mL.

Figure 2.

Dynamic and pulsatility testing. Elevated growth hormone (GH), prolactin (PRL) and GHRH levels were seen throughout an extended testing period of 180 minutes (Panel A). GH levels are seen to peak at 150 min after an earlier GHRH rise between 105min and 135 min. A TRH test induced an immediate and marked increase in GH levels and GHRH remained largely unchanged (Panel B). GnRH administration led to suppression of GH levels at 30 mins, whereas GHRH levels remained unaltered and prolactin rose mildly (Panel C). GH and PRL are measured in ng/mL; GHRH is measured in pg/mL.

Figure 2.

Dynamic and pulsatility testing. Elevated growth hormone (GH), prolactin (PRL) and GHRH levels were seen throughout an extended testing period of 180 minutes (Panel A). GH levels are seen to peak at 150 min after an earlier GHRH rise between 105min and 135 min. A TRH test induced an immediate and marked increase in GH levels and GHRH remained largely unchanged (Panel B). GnRH administration led to suppression of GH levels at 30 mins, whereas GHRH levels remained unaltered and prolactin rose mildly (Panel C). GH and PRL are measured in ng/mL; GHRH is measured in pg/mL.

Figure 3.

The in vitro dose-effect on GH (A, C) and PRL (B, D) secretion in somatotroph cells after overnight incubation with (A,B) GHRH, GnRH1–5, (C,D) octreotide (OCT) cabergoline (CAB) or pasireotide (PAS). The results are expressed as the mean percentage of PRL or GH change compared to the values of control wells (ctrl). * : p<0.05.

Figure 4.

The in vitro dose-effect on GH (A) and PRL (B) secretion in somatotroph cells after overnight incubation with GHSR agonist (MK 0677) or GHSR-inverse agonist (MSP). The results are expressed as the mean percentage of PRL or GH change compared to the values of control wells (ctrl). * : p<0.05.

Figure 5.

The in vitro dose-effect on GH (A) and PRL (B) secretion in somatotroph cells after overnight incubation with GHRH-R antagonist, with or without GHRH at 10⁻¹⁰ M. The results are expressed as the mean percentage of PRL or GH change compared to the values of control wells (ctrl). * : p<0.05.