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Among the factors playing a crucial role in the regulation of energy metabolism, gastro-intestinal peptides are essential signals to maintain energy homeostasis as they relay to the central nervous system the informations about the nutritional status of the body. Among these factors, preproghrelin is a unique prohormone as it encodes ghrelin, a powerful GH secretagogue and the only orexigenic signal from the gastrointestinal tract and obestatin, a proposed functional ghrelin antagonist. These preproghrelin-derived peptides may contribute to balance energy intake, metabolism and body composition by regulating the activity of the GH/IGF-1 axis and appetite. Whereas the contribution of ghrelin has been well characterized, the role of the more recently identified obestatin, in this regulatory process is still controversial. In this chapter, we describe the contribution of these different preproghrelin-derived peptides and their receptors in the regulation of GH secretion and feeding. Data obtained from pharmacological approaches, mutant models and evaluation of the hormones in animal and human models are discussed., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A somatostatin deficit occurs in the cerebral cortex of Alzheimer's 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 Alzheimer's 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 Alzheimer's 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 Alzheimer's disease. Further studies will be needed to assess the mechanisms involved in somatostatin deficiency in Alzheimer's disease.

External clues for neuron development include extracellular matrix (ECM) molecules. To explore ECM influence on the early development of peptide phenotype in the CNS, we have compared pro-TRH levels in primary cultures of rat hypothalamic cells plated either on poly-lysine (PL) (control) or on PL plus one of various ECM molecules at 10 microgram/ml. Fetal day 17 cells plated at a density of 1250/mm(2) were grown in a serum free medium made of Neurobasal medium supplemented with B27 (GIBCO). Cultures, consisting mainly of neurons, were analyzed at DIV 2. ECM proteins induced morphological effects in agreement with previously published studies. The amount of pro-TRH per dish, quantified by Western blotting, was increased to 275% for laminin, 191% for fibronectin and 173% for tenascin-C (control=100%); there was no effect of vitronectin. Laminin or fibronectin did not change pro-TRH mRNA or TRH levels but enhanced levels of the pro-protein convertase PC1 suggesting that the ECM molecules did regulate the translational status of pro-TRH. In conclusion, we have shown that some ECM proteins increased pro-TRH level in vitro; this may contribute to the enhancement of pro-TRH levels observed early in vivo in the hypothalamus.

Immunoreactive (IR) proTRH forms were characterized in human hypothalamic tissue with two antisera raised against a hepta- and a decapeptide containing the TRH progenitor sequence (-Gln-His-Pro-Gly-). A similar study was performed in human normal and adenomatous anterior pituitaries, tissues in which TRH synthesis has been previously suggested. IR-proTRH was found in all the samples ranging from 42-775 fmol/mg proteins. Size exclusion chromatography identified a major 25-35 kDa form and a minor 4-8 kDa form. The existence of the major form was confirmed by immunoblotting. The results suggest that both human hypothalamic and normal or adenomatous anterior pituitary tissues synthesize similar IR-proTRH forms.

Enzyme immunoassays (EIAs) for Thyroliberin (TRH) and TRH-elongated peptides were developed. Three haptens less than E-H-P-NH2 (TRH). Less than E-H-P-OH (TRH-OH), and S-K-R-Q-H-P-G-K-R-F (P10) were conjugated by the use of different heterobifunctional cross-linking agents either to sun-flower globulin as carrier or to acetylcholinesterase as tracer. For a same hapten, the same chemical group in the peptide was used to prepare the immunogen and the enzyme conjugate. These EIAs were performed with a second antibody solid phase technique using acetylcholinesterase as label. They permitted the measurement of TRH and TRH-elongated peptides with a sensitivity threshold of 10 fmol/well for TRH and 2 fmol/well for P10. TRH EIA only detected authentic TRH whereas TRH-OH EIA detected TRH and TRH peptides elongated on C terminal part. Anti-P10 serum was specific of TRH peptides elongated both on C and N terminal parts and no cross reactivity was observed with TRH. Using these assays, TRH and TRH-elongated peptides were determined in crude or chromatographed mouse and rat hypothalamus tissular extracts. Several TRH extended forms were identified by P10 EIA, whereas TRH-OH EIA permitted detection of both TRH and TRH-elongated peptides in chromatographed extracts. Authentic TRH was measured by TRH EIA both in crude and chromatographed hypothalamic extracts. These assays can permit the study of the processing and maturation of TRH.

Two antisera (Anti-P7 and Anti-P10) were raised against (-Gln-His-Pro-Gly-) elongated peptides: P7 Gln-His-Pro-Gly-Lys-Arg-Phe) and P10 (Ser-Lys-Arg-Gln-His-Pro-Gly-Lys-Arg-Phe). They recognized TRH extended peptides but not TRH. A RIA against P7 and a highly sensitive enzyme immunoassay against P10 were used to identify two major high mol wt forms of 25-35 K and 6-8 K in chromatography fractions of adult and fetal mouse as well as adult rat hypothalami. The existence of the largest form was confirmed by immunoblotting with Anti-P7. During mouse hypothalamus development in vivo and in vitro, the ratio of TRH content vs. P10-associated immunoreactivity increased several times. This suggests that these Pro-TRH peptides are precursors of TRH biosynthesis and indicate an acceleration of TRH processing during development. Double immunostaining with A-TRH and A-P7 of hypothalamic cells taken on the 16th fetal day and cultured for 6, 12, and 18 days in vitro (DIV) revealed three populations of neurons: 1) a very minor population (approximately 2%) of small round cells positive with A-TRH only; 2) a major population of neurons positive with both A-TRH and A-P7. 3) multipolar neurons positive with A-P7 only (up to approximately 45% after 18 DIV). The respective distribution of TRH and P7 along neurites also varied with time in culture. Whatever perikarya staining, TRH was restricted to short neurites and growth cones before synapse formation and, during synapse development, to varicosities and terminal boutons. However even at the latest stage examined some varicosities and terminal boutons were positive with A-P7 only. These results suggest a preferential processing of pro-TRH at a post-Golgi step during axonal transport to growth cones and synaptic boutons.