Dominique Grouselle

Loss of constitutive activity of the growth hormone secretagogue receptor in familial short stature

The growth hormone (GH) secretagogue receptor (GHSR) was cloned as the target of a family of synthetic molecules endowed with GH release properties. As shown recently through in vitro means, this receptor displays a constitutive activity whose clinical relevance is unknown. Although pharmacological studies have demonstrated that its endogenous ligand--ghrelin--stimulates, through the GHSR, GH secretion and appetite, the physiological importance of the GHSR-dependent pathways remains an open question that gives rise to much controversy. We report the identification of a GHSR missense mutation that segregates with short stature within 2 unrelated families. This mutation, which results in decreased cell-surface expression of the receptor, selectively impairs the constitutive activity of the GHSR, while preserving its ability to respond to ghrelin. This first description, to our knowledge, of a functionally significant GHSR mutation, which unveils the critical importance of the GHSR-associated constitutive activity, discloses an unusual pathogenic mechanism of growth failure in humans.

Loss of somatostatin-like immunoreactivity in the frontal cortex of Alzheimer patients carrying the apolipoprotein epsilon 4 allele

We measured somatostatin-like immunoreactivity, using a radioimmunoassay which does not cross react with cortistatin-like immunoreactivity, in postmortem frontal cortex (Brodmann area 9) from 32 patients, of different apolipoprotein E genotypes, and presenting with different degrees of cognitive impairment. Eleven subjects and eight patients presented with no (controls) or limited memory impairments (Borderline), respectively. Six patients with clinical criteria for possible Alzheimers disease also presented with clinical or brain imaging of cerebrovascular disease (mixed dementia) and seven patients were classified as Alzheimers disease (AD). In the 6 months preceeding their deaths, all subjects had been evaluated by Folsteins Mini Mental State examination (MMS). Sixty nine percent of patients with MMS >20 did not carry the epsilon 4 allele while 66% of patients with MMS <10 did. Somatostatin concentrations (ng/mg wet weight) were significantly lower in the patients carrying the epsilon 4 allele (E2/3: 0.71 +/- 0.05, n = 19 vs. E4: 0.42 +/- 0.06, n = 13; mean +/- SEM, P < 0.001). These results, which are reminiscent of those obtained on cholinergic markers, suggest that apolipoprotein E4 is involved in the somatostatinergic dysfunction early after the onset in AD.

Ghrelin and Obestatin Circadian Levels Differentiate Bingeing-Purging from Restrictive Anorexia Nervosa

CONTEXT Anorexia nervosa (AN) patients present with restrictive food behavior (AN-R). Some of them develop episodes of bulimia (AN-BP) without any clear pathophysiological explanation to date. Their clinical differentiation is important but not easily performed. Orexigenic/anorexigenic peptides measurements could provide some clues for that matter. OBJECTIVE The objective of the study was to determine whether the circadian profile of total and acylated ghrelin, obestatin, and peptide YY (PYY) levels is different in AN-R subjects when compared with AN-BP patients. DESIGN AND SETTINGS This was a cross-sectional study in an endocrinological unit. PATIENTS AND CONTROL SUBJECTS Four groups of age-matched young women: 22 AN-R, 10 AN-BP, 16 normal-weight bulimia nervosa (BN), and nine controls. MAIN OUTCOME MEASURES Twelve-point circadian profiles of plasma total and acylated ghrelin, obestatin, and PYY were measured. RESULTS Total and acylated ghrelin and obestatin circadian levels were increased in AN-R when compared with controls but decreased in both AN-BP and BN groups (P < 0.001). PYY was decreased in all groups with eating disorders. Acylated to total ghrelin ratio was decreased in AN-BP and BN (P < 0.001), whereas obestatin to acylated ghrelin and PYY to acylated ghrelin ratios were increased in both groups with bingeing-purging behavior (P < 0.0001). CONCLUSIONS Patients with AN-associated bingeing-purging behavior present a very different profile of appetite regulatory peptides when compared with the pure restrictive type. The assessment of ghrelin (and eventually obestatin) could be of particular interest for differential diagnosis. Very low ghrelin levels and increased anorexigenic to orexigenic peptide ratios suggest either a lack of adaptation to a starvation state or a higher facility to cope with undernutrition.

Genetic deletion of somatostatin receptor 1 alters somatostatinergic transmission in the mouse retina

In the mammalian retina, sparse amacrine cells contain somatostatin-14 (SRIF) which acts at multiple levels of neuronal circuitry through distinct SRIF receptors (sst(1-5)). Among them, the sst1 receptor has been localised to SRIF-containing amacrine cells in the rat and rabbit retina. Little is known about sst1 receptor localisation and function in the mouse retina. We have addressed this question in the retina of mice with deletion of sst1 receptors (sst1 KO mice). In the retina of wild type (WT) mice, sst1 receptors are localised to SRIF-containing amacrine cells, whereas in the retina of sst1 KO mice, sst1 receptors are absent. sst1 receptor loss causes a significant increase in retinal levels of SRIF, whereas it does not affect SRIF messenger RNA indicating that sst1 receptors play a role in limiting retinal SRIF at the post-transcriptional level. As another consequence of sst1 receptor loss, levels of expression of sst2 receptors are significantly higher than in control retinas. Together, these findings provide the first demonstration of prominent compensatory regulation in the mouse retina as a consequence of a distinct SRIF receptor deletion. The fact that in the absence of the sst1 receptor, retinal SRIF increases in concomitance with an increase in sst2 receptors suggests that SRIF may regulate sst2 receptor expression and that this regulatory process is controlled upstream by the sst1 receptor. This finding can be important in the design of drugs affecting SRIF function, not only in the retina, but also elsewhere in the brain.

Plasma and hypothalamic peptide-hormone levels regulating somatotroph function and energy balance in fed and fasted states: a comparative study in four strains of rats.

Both growth hormone (GH)/insulin growth factor (IGF)‐1 axis and energy balance have been implicated in longevity independently. The aim of the present study was to characterize the effect of a 72‐h fasting period at 3 months of age in four different rat strains: (i) Wistar and (ii) Fischer 344 rats, which develop obesity with age, and (iii) Brown Norway and (iv) Lou C rats, which do not. Wistar rats ate more, were significantly bigger, and presented with higher plasma leptin and lower ghrelin levels and hypothalamic growth hormone‐releasing hormone (GHRH) content than rats from the three other strains. Plasma insulin and IGF‐1 levels were lower in Brown Norway and Lou C rats, and somatostatin content was lower in Brown Norway rats only. Glycaemia was lower in Lou C rats that displayed a lower relative food intake compared to Fischer and Wistar rats. Brown Norway rats showed a greater caloric efficiency than the three other strains. Concerning major hypothalamic neuropeptides implicated in feeding, similar amounts were detected in the four strains for neuropeptide Y, agouti‐related peptide, galanin, melanin‐concentrating hormone, α‐melanocortin‐stimulating hormone (α‐MSH) and corticotropin‐releasing hormone. Orexin A appeared to be slightly elevated in Fischer rats and cocaine amphetamine‐regulated transcript (CART)55−102 diminished in Brown Norway. At the mRNA level, orexin A, GHSR1, α‐MSH and CART expression were higher in Wistar and Lou C rats. Principal component analysis confirmed the presence of two main factors in the ad libitum rat population; the first being associated with growth‐related parameters and the second being associated with food intake regulation. Hypothalamic GHRH and somatostatin content were positively correlated with feeding‐related neuropeptides such as α‐MSH for GHRH, and orexin A and CART for both peptides. Plasma ghrelin levels were negatively correlated with leptin and IGF‐1 levels. Finally, a 72‐h fasting period affected minimally body weight, plasma IGF‐1 and leptin levels in Lou C rats compared to the three other strains, and plasma insulin levels were less affected in Brown Norway rats. In conclusion, Wistar shorter life span is consistent with its already fatter phenotype at 3 months of age. In terms of IGF‐1, glycaemia and leptin responses to fasting, the Lou strain, which presents with a low food intake/body weight and caloric efficiency, is the least affected. The link between food intake regulation, GH axis and ageing is further demonstrated by principal component analysis, where GHRH and somatostatin were found to be strongly associated with energy homeostasis parameters.

Identification of Endocrine Cell Populations Expressing the AT1B Subtype of Angiotensin II Receptors in the Anterior Pituitary.

Angiotensin II (Ang II) participates in the regulation of anterior pituitary hormone secretion by acting either directly on the anterior pituitary or indirectly on the hypothalamus. When applied directly on pituitary cells, Ang II increases both ACTH and PRL secretion and has also been reported to affect GH secretion. Three distinct subtypes of Ang II receptors (AT1A, AT1B, and AT2) have been identified; they are unequally distributed and differently regulated in various tissues. We have previously demonstrated that only AT1A receptors are present in the hypothalamus while anterior pituitary cells express predominantly the AT1B subtype. Using in situ hybridization in combination with immunohistochemistry, the aim of the present study was to identify the phenotype of the endocrine cell expressing AT1B receptor messenger RNA (mRNA) in the anterior pituitary of adult male Sprague-Dawley rats. Expression of AT1B receptor mRNA was present in 33.9 +/- 1.0% of anterior pituitary cells. AT1B mRNA is predominantly expressed by lactotropes (78.2 +/- 2.1% of AT1B mRNA-expressing cells) and to a lower degree by corticotropes (18.3 +/- 2.1%) and is not detectable in somatotropes, mammosomatotropes, gonadotropes, or thyrotropes. These results indicate that in adult male rats, Ang II, which has been shown to be synthesized in gonadotropes, can directly stimulate PRL and ACTH release from lactotropes and corticotropes through activation of AT1B receptors. As only 53.8 +/- 2.7% of lactotropes and 23.6 +/- 2.8% of corticotropes expressed AT1B mRNA, our findings suggest a functional heterogeneity of both cell types regarding their sensitivity to Ang II.

Ghrelin-Derived Peptides: A Link between Appetite/Reward, GH Axis, and Psychiatric Disorders?

Psychiatric disorders are often associated with metabolic and hormonal alterations, including obesity, diabetes, metabolic syndrome as well as modifications in several biological rhythms including appetite, stress, sleep–wake cycles, and secretion of their corresponding endocrine regulators. Among the gastrointestinal hormones that regulate appetite and adapt the metabolism in response to nutritional, hedonic, and emotional dysfunctions, at the interface between endocrine, metabolic, and psychiatric disorders, ghrelin plays a unique role as the only one increasing appetite. The secretion of ghrelin is altered in several psychiatric disorders (anorexia, schizophrenia) as well as in metabolic disorders (obesity) and in animal models in response to emotional triggers (psychological stress …) but the relationship between these modifications and the physiopathology of psychiatric disorders remains unclear. Recently, a large literature showed that this key metabolic/endocrine regulator is involved in stress and reward-oriented behaviors and regulates anxiety and mood. In addition, preproghrelin is a complex prohormone but the roles of the other ghrelin-derived peptides, thought to act as functional ghrelin antagonists, are largely unknown. Altered ghrelin secretion and/or signaling in psychiatric diseases are thought to participate in altered appetite, hedonic response and reward. Whether this can contribute to the mechanism responsible for the development of the disease or can help to minimize some symptoms associated with these psychiatric disorders is discussed in the present review. We will thus describe (1) the biological actions of ghrelin and ghrelin-derived peptides on food and drugs reward, anxiety and depression, and the physiological consequences of ghrelin invalidation on these parameters, (2) how ghrelin and ghrelin-derived peptides are regulated in animal models of psychiatric diseases and in human psychiatric disorders in relation with the GH axis.

The proprotein convertase PC2 is involved in the maturation of prosomatostatin to somatostatin-14 but not in the somatostatin deficit in Alzheimer's disease.

A somatostatin deficit occurs in the cerebral cortex of Alzheimers disease patients without a major loss in somatostatin-containing neurons. This deficit could be related to a reduction in the rate of proteolytic processing of peptide precursors. Since the two proprotein convertases (PC)1 and PC2 are responsible for the processing of neuropeptide precursors directed to the regulated secretory pathway, we examined whether they are involved first in the proteolytic processing of prosomatostatin in mouse and human brain and secondly in somatostatin defect associated with Alzheimers disease. By size exclusion chromatography, the cleavage of prosomatostatin to somatostatin-14 is almost totally abolished in the cortex of PC2 null mice, while the proportions of prosomatostatin and somatostatin-28 are increased. By immunohistochemistry, PC1 and PC2 were localized in many neuronal elements in human frontal and temporal cortex. The convertases levels were quantified by Western blot, as well as the protein 7B2 which is required for the production of active PC2. No significant change in PC1 levels was observed in Alzheimers disease. In contrast, a marked decrease in the ratio of the PC2 precursor to the total enzymatic pool was observed in the frontal cortex of Alzheimer patients. This decrease coincides with an increase in the binding protein 7B2. However, the content and enzymatic activity of the PC2 mature form were similar in Alzheimer patients and controls. Therefore, the cortical somatostatin defect is not due to convertase alteration occuring during Alzheimers disease. Further studies will be needed to assess the mechanisms involved in somatostatin deficiency in Alzheimers disease.

Altered morphology of rod bipolar cell axonal terminals in the retinas of mice carrying genetic deletion of somatostatin subtype receptor 1 or 2

Somatostatin (SRIF), similar to other neuropeptides, is likely to influence the morpho‐functional characteristics of neurons. We studied possible morphological alterations of mouse retinal neurons following genetic deletion of SRIF subtype receptor 1 [sst1 knockout (KO)] or 2 (sst2 KO). In sst1 KO retinas, axonal terminals of rod bipolar cells (RBCs), identified with protein kinase C immunoreactivity, were 25% larger than in controls. In contrast, in sst2 KO retinas, RBC axonal terminals were significantly smaller (−14%). No major ultrastructural differences were observed between control and KO RBCs. In sst2 KO retinas, SRIF levels decreased by about 35%, while both sst1 receptor mRNA and protein increased by about 170% and 100%, respectively. This compares to previous results reporting an increase of both retinal SRIF and sst2 receptors following sst1 receptor deletion. Together, these findings suggest that, on the one hand, sst1 receptor deletion induces over‐expression of sst2 receptors, and vice versa; on the other hand, that an imbalance in sst1 and sst2 receptor expression and/or changes in the levels of retinal SRIF induced by sst1 or sst2 receptor deletion are responsible for the morphological changes in RBC axonal terminals. Similar alterations of RBC terminals were observed in KO retinas at 2 weeks of age (eye opening). In addition, reverse transcription‐polymerase chain reaction analysis of the expression of sst2 and sst1 receptors in developing sst1 and sst2 KO retinas, respectively, demonstrated that these receptors are up‐regulated at or near eye opening. These findings suggest that the integrity of the somatostatinergic system during development is necessary for proper RBC maturation.

An Enzyme Immunoassay for Rat Growth Hormone: Validation and Application to the Determination of Plasma Levels and in Vitro Release

A competitive enzyme immunoassay for rat growth hormone (rGH) has been developed using polyclonal anti-rGH antibodies and an acetylcholinesterase (EC 3.1.1.7.) enzymatic tracer coupled covalently with rGH. The assay was performed in 96-well microtiter plates coated with rabbit polyclonal anti-goat immunoglobulin antibodies. Molecular sieve filtration and Western blot analysis revealed a single immunoreactive peak for rat plasma or pituitary extracts. Cross-reactivity with other rat pituitary hormones or human GH was less than 1%. Assay of samples in a concentration range of 0.7 to 69 ng/ml by enzyme immunoassay and radioimmunoassay were well correlated (r = 0.87 and 0.85 respectively for plasma and culture medium samples). Intra- and inter-assay variations in plasma were 4 (n = 24) and 14% (n = 9) respectively. Minimal detectable amounts of rGH were 0.6 ng/ml. A two-site immunometric assay also developed with the same antibodies allowed a detection threshold of 0.25 ng/ml.