Alain Enjalbert

A Pituitary Cell-Restricted T Box Factor, Tpit, Activates POMC Transcription in Cooperation with Pitx Homeoproteins

The pituitary gland has provided unique insight into molecular mechanisms and regulatory factors controlling both differentiation and gene transcription. We identified Tpit, a novel T box factor only present in the two pituitary POMC-expressing lineages, the corticotrophs and melanotrophs, and apparently in no other tissue, including hypothalamic POMC neurons. In pituitary cells, Tpit activation of POMC gene transcription requires cooperation with Pitx1, the two factors binding to contiguous sites within the same regulatory element. In gain-of-function experiments, Tpit induces POMC expression in undifferentiated pituitary cells, indicating that it can initiate differentiation into POMC-expressing lineages. TPIT gene mutations were found in patients with isolated deficiency of pituitary POMC-derived ACTH, in support of an essential role of Tpit for differentiation of the pituitary POMC lineage.

Imprinting of the Gsα gene GNAS1 in the pathogenesis of acromegaly

Approximately 40% of growth hormone-secreting pituitary adenomas have somatic mutations in the GNAS1 gene (the so-called gsp oncogene). These mutations at codon 201 or codon 227 constitutively activate the alpha subunit of the adenylate cyclase-stimulating G protein G(s). GNAS1 is subject to a complex pattern of genomic imprinting, its various promoters directing the production of maternally, paternally, and biallelically derived gene products. Transcripts encoding G(s)alpha are biallelically derived in most human tissues. Despite this, we show here that in 21 out of 22 gsp-positive somatotroph adenomas, the mutation had occurred on the maternal allele. To investigate the reason for this allelic bias, we also analyzed GNAS1 imprinting in the normal adult pituitary and found that G(s)alpha is monoallelically expressed from the maternal allele in this tissue. We further show that this monoallelic expression of G(s)alpha is frequently relaxed in somatotroph tumors, both in those that have gsp mutations and in those that do not. These findings imply a possible role for loss of G(s)alpha imprinting during pituitary somatotroph tumorigenesis and also suggest that G(s)alpha imprinting is regulated separately from that of the other GNAS1 products, NESP55 and XLalphas, imprinting of which is retained in these tumors.

Decreased Expression of the Two D2 Dopamine Receptor Isoforms in Bromocriptine-Resistant Prolactinomas

Bromocriptine or other dopamine agonists are usually effective for the treatment of prolactin-secreting adenomas. Five to 18% of prolactinomas, however, do not respond to such therapy. We have shown previously that such resistance to bromocriptine correlates with reduced binding to the D2 receptor subtype of dopamine, the major PRL inhibiting factor. In the present work, we demonstrated that reduced binding actually corresponds to decreased expression of the gene coding for the D2 receptor in the pituitary from bromocriptine-resistant patients, as shown by 4-fold lower levels of the corresponding mRNAs compared to those coding for actin. The existence of two D2 receptor isoforms, D2S and D2L generated by alternative splicing, has been described in several tissues, including the pituitary. Both are negatively coupled to adenylyl cyclase and inhibit prolactin secretion, but, in addition, the shortest one (D2S) is more efficiently coupled to phospholipase C. Consequently, we also investigated whether expression of a particular D2 receptor isoform was preferentially affected in resistant adenomas. The proportion of messengers corresponding to the short receptor isoform (D2S) was lower in resistant compared to responsive adenomas: D2S/D2L = 0.74 +/- 0.08 and 1.00 +/- 0.07, respectively. In parallel, much lower levels of D2 receptor mRNAs were found in growth hormone-secreting adenomas, with a D2S/D2L ratio comparable to those of both normal human pituitary and bromocriptine-sensitive prolactinomas (1.05 +/- 0.11). Thus, resistance to bromocriptine therapy seems to involve defects in D2 dopamine receptor expression and possibly in posttranscriptional splicing.

Endogenous opiates block dopamine inhibition of prolactin secretion in vitro.

OPIATES stimulate prolactin secretion in vivo; plasma prolactin levels are increased by morphine administration in normal1 and steroid-primed male rats2, as well as in immature3 and oestrogen-treated female rats4. Met-enkephalin and β-endorphin were also stimulatory1–6 as were enkephalin analogues3,7,8. Naloxone or naltrexone, specific opiate antagonists, reduce basal prolactin levels when given alone1,3,9, and block, at least partially, the effect of opiate agonists1–4,7,8. None of these drugs is active in vitro when tested alone on anterior pituitaries3 or dispersed pituitary cells2,4. However, as we have previously shown in a preliminary experiment, the inhibitory effect of dopamine on prolactin secretion in vitro was antagonised by the addition of morphine to the incubation medium10. In the present work, we have attempted to clarify further the mode of interaction of opiates with prolactin secretion. We confirmed that morphine, Met-enkephalin and β-endorphin do not affect the spontaneous release of prolactin in vitro, but found that they suppress the inhibitory effect of dopamine on prolactin release. We have also characterised the specificity and the kinetics of this interaction.

Stimulation of in vitro Prolactin Release by Vasoactive Intestinal Peptide

VIP stimulated prolactin secretion from incubated rat hemipituitaries. Under the same conditions, the secretion of GH, LH, FSH was not affected. The stimulation of prolactin was dose-dependent, with an apparent affinity of VIP of 10.9 +/- 3.1 nM and a maximal stimulation of 57.7 +/- 4.2%. Secretin, a structurally related peptide, was also active at higher concentrations whereas another partial analogue, glucagon, was ineffective. The effect of VIP was not blocked by alpha-flupentixol, a potent dopaminergic antagonist, at concentrations which antagonized the dopamine inhibition of prolactin secretion. Stimulation by VIP and TRH was additive. Neither Met-enkephalin nor naloxone interfered with the response to VIP. It thus seems that specific VIP receptors are present on pituitary prolactin cells. VIP, present in the mediobasal hypothalamus and detected in the hypothalamo-hypophyseal portal blood therefore is a good candidate as a physiological PRF.

18F-FDG Avidity of Pheochromocytomas and Paragangliomas: A New Molecular Imaging Signature?

Our objective was to evaluate 18F-FDG PET uptake in patients with nonmetastatic and metastatic chromaffin-derived tumors. Methods: Twenty-eight consecutive unrelated patients with chromaffin tumors, including 9 patients with genetically determined disease, were studied. A combination of preoperative imaging work-up, surgical findings, and pathologic analyses was used to classify the patients into 2 groups: those with nonmetastatic disease (presumed benign, n = 18) and those with metastatic tumors (n = 10). 18F-FDG PET was performed in all cases. Visual and quantitative analyses were individually graded for each tumor. Somatic mutations of the succinate dehydrogenase subunits B and D and Von-Hippel Lindau genes were also evaluated in 6 benign sporadic tumor samples. Results: All but 2 patients showed significantly increased 18F-FDG uptake on visual analysis. The maximum standardized uptake value (SUVmax) ranged from 1.9 to 42 (mean ± SD, 8.2 ± 9.7; median, 4.6) in nonmetastatic tumors and 2.3 to 29.3 (mean ± SD, 9.7 ± 8.4; median, 7.4) in metastatic tumors. No statistical difference was observed between the groups (P = 0.44), but succinate dehydrogenase–related tumors were notable in being the most 18F-FDG–avid tumors (SUVmax, 42, 29.3, 21, 17, and 5.3). Succinate dehydrogenase and Von-Hippel Lindau–related tumors had a significantly higher SUVmax than did neurofibromatosis type 1 and multiple endocrine neoplasia type 2A syndrome–related tumors (P = 0.02). 18F-FDG PET was superior to 131I-metaiodobenzylguanidine in all metastatic patients but one. By contrast, 18F-FDG PET underestimated the extent of the disease, compared with 6-18F-fluorodopa PET, in 5 patients with metastatic pheochromocytoma. However, succinate dehydrogenase mutations (germline and somatic) and functional dedifferentiation do not adequately explain 18F-FDG uptake since most tumors were highly avid for 18F-FDG. Conclusion: 18F-FDG PET positivity is almost a constant feature of pheochromocytomas and paragangliomas. It may be considered a molecular signature of such tumors, although which aspect of the plethora of molecular changes associated with dedifferentiation, germline genetic defects, or the adaptive response to hypoxia is responsible for this characteristic requires further elucidation.

Genetic analysis in young patients with sporadic pituitary macroadenomas: besides AIP don't forget MEN1 genetic analysis

CONTEXT Germline mutations in the aryl hydrocarbon receptor interacting protein gene (AIP) have been identified in young patients (age ≤30 years old) with sporadic pituitary macroadenomas. Otherwise, there are few data concerning the prevalence of multiple endocrine neoplasia type 1 (MEN1) mutations in such a population. OBJECTIVE We assessed the prevalence of both AIP and MEN1 genetic abnormalities (mutations and large gene deletions) in young patients (age ≤30 years old) diagnosed with sporadic and isolated macroadenoma, without hypercalcemia and/or MEN1-associated lesions. DESIGN The entire coding sequences of AIP and MEN1 were screened for mutations. In cases of negative sequencing screening, multiplex ligation-dependent probe amplification was performed for the detection of large genetic deletions. PATIENTS AND SETTINGS One hundred and seventy-four patients from endocrinology departments of 15 French University Hospital Centers were eligible for this study. RESULTS Twenty-one out of 174 (12%) patients had AIP (n=15, 8.6%) or MEN1 (n=6, 3.4%) mutations. In pediatric patients (age ≤18 years old), AIP/MEN1 mutation frequency reached nearly 22% (n=10/46). AIPmut and MEN1mut were identified in 8/79 (10.1%) and 1/79 (1.2%) somatotropinoma patients respectively; they each accounted for 4/74 (5.4%) prolactinoma (PRL) patients with mutations. Half of those patients (n=3/6) with gigantism displayed mutations in AIP. Interestingly, 4/12 (33%) patients with non-secreting adenomas bore either AIP or MEN1 mutations, whereas none of the eight corticotroph adenomas or the single thyrotropinoma case had mutations. No large gene deletions were observed in sequencing-negative patients. CONCLUSION Mutations in MEN1 can be of significance in young patients with sporadic isolated pituitary macroadenomas, particularly PRL, and together with AIP, we suggest genetic analysis of MEN1 in such a population.

Differential Modulation of D1 and D2 Dopamine‐Sensitive Adenylate Cyclases by 17β‐Estradiol in Cultured Striatal Neurons and Anterior Pituitary Cells

Abstract: Primary cultures of anterior pituitary cells from female rats and of mouse embryonic striatal neurons were used to study the effects of 17β‐estradiol on D1‐ and D2‐dopamine (DA)‐sensitive adenylate cyclase. 17β‐Estradiol pretreatment (10−9M, 72 h) suppressed the D2‐DA‐induced inhibition of adenylate cyclase activity in anterior pituitary cells. The steroid (10−9M, 24 h) also blocked the D2‐DA‐evoked response in striatal neurons whereas it enhanced by twofold the D1‐DA‐induced stimulation of the enzyme activity in these neurons. All these effects of the steroid were dose dependent and specific, as neither 17α‐estradiol, dexamethasone, nor progesterone used at the same concentration (10−9M) was effective. Furthermore, the modulation of DA‐sensitive adenylate cyclases by the steroid required long‐term exposure of living cells to 17β‐estradiol since neither 17β‐estradiol pretreatment for 4 h nor its addition to broken cells directly into the adenylate cyclase assay induced any alteration in the DA‐sensitive adenylate cyclase activity. These results are in agreement with a genomic effect of the steroid. Using both anterior pituitary cells and striatal neurons in culture, 17β‐estradiol affected neither the total number of DA (D1 and D2) receptors nor the estimated number of adenylate cyclase catalytic units. Therefore, it is suggested that the steroid modifies the coupling process by a mechanism that still has to be elucidated. These results demonstrate an effect of 17β‐estradiol on DA target cells in both systems. The steroid‐induced changes in DA receptivity could account, at least in part, for the modulation of hormonal (in the anterior pituitary) and behavioral (in the striatum) responses mediated by DA observed under various physiological or pathological conditions.

Evidence for a Direct Action of Neuropeptide Y in the Rat Pituitary Gland

Neuropeptide Y (NPY) has recently been localized in the rat hypothalamus. In order to evaluate the putative effects of NPY on pituitary function, its action was examined on anterior pituitary cells in culture. Also, an immunocytochemical method was used with the aim of localizing endogenous NPY-like material at the cellular and subcellular levels of the pituitary gland. In vitro studies using dispersed anterior pituitary cells indicated that NPY (10(-6) to 10(-9)M) increased the secretion of luteinizing hormone, growth hormone and prolactin, whereas beta-lipotropin hormone and thyrotropin secretions were not affected. The presence of endogenous NPY was demonstrated in gonadotrophs, somatotrophs, corticotrophs and some lactotrophs, but not in thyrotrophs. In immunoreactive cells, NPY-like material was detected in the cytoplasmic matrix, in the secretory granules and in the nucleus distributed primarily in the euchromatin, in the vicinity of the heterochromatin. NPY-like immunoreactivity was also observed at the plasma membrane but only scarcely. These biochemical and immunocytochemical results indicate that NPY may play a direct regulatory role in adenohypophyseal secretion.

Autoradiographic localization of a non-reducible somatostatin analog (125I-CGP 23996) binding sites in the rat brain: Comparison with membrane binding

The regional distribution of somatostatin binding sites in the rat brain was determined by quantitative autoradiography, using 125I-CGP 23996, a non-reducible somatostatin analog. In preliminary experiments, kinetic properties of 125I-CGP 23996 binding to rat brain membranes and slide mounted frozen brain sections were compared and found similar. In addition, distribution of 125I-CGP 23996 and 125I-N-Tyr-SRIF14 binding sites on membrane prepared from 10 different rat brain structures were closely correlated (r = 0.91, 2 p less than 0.01), indicating that the non-reducible analog recognizes the same binding site as the Tyr-extended native peptide. Highest levels of 125I-CGP 23996 binding sites were found in anterior temporal, frontal and cingular cortex as well as hippocampus. Moderate levels were found in the remaining part of the limbic system including amygdala, olfactory tubercles and bed nucleus of the stria terminalis. In the brain stem, nuclei involved in the auditory system such as the ventral cochlear nucleus and the superior olive nucleus, contained high levels of 125I-CGP 23996 binding sites. The distribution of 125I-CGP 23996 binding sites roughly correlated with that of the endogenous peptide in most structures, except in the mediobasal hypothalamus.