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D. S. C. Jones and A. P. F. Flint

ABSTRACT

Concentrations of oxytocin in corpora lutea from pregnant and non-pregnant sheep rose during luteinization to peak between days 5 and 9 after oestrus before falling to low levels on days 12 and 14. Concentrations of the 608-base mRNA encoding the oxytocin-neurophysin prohormone, measured by Northern blotting using a cRNA probe, were highest before day 5, and fell to low levels by day 9. The decline in oxytocin during the second half of the oestrous cycle and in pregnancy could therefore be accounted for by reduced luteal concentrations of oxytocin-neurophysin mRNA. The rate of decline in prohormone mRNA concentration between days 3 and 16 followed first order kinetics, suggesting that gene expression ceased early in the cycle. The lag between peak oxytocin-neurophysin mRNA and peak oxytocin concentrations may represent the time taken for post-translational processing of the prohormone.

J. Endocr. (1988) 117, 409–414

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S Wei, Y Feng, FY Che, H Pan, N Mzhavia, LA Devi, AA McKinzie, N Levin, WG Richards and LD Fricker

ProSAAS is a neuroendocrine peptide precursor that potently inhibits prohormone convertase 1 in vitro. To explore the function of proSAAS and its derived peptides, transgenic mice were created which express proSAAS using the beta-actin promoter. The body weight of transgenic mice was normal until approximately 10-12 weeks, and then increased 30-50% over wild-type littermates. Adult transgenic mice had a fat mass approximately twice that of wild-type mice, and fasting blood glucose levels were slightly elevated. In the pituitary, the levels of several fully processed peptides in transgenic mice were not reduced compared with wild-type mice, indicating that the proSAAS transgene did not affect prohormone convertase 1 activity in this tissue. Because the inhibitory potency of proSAAS-derived peptides towards prohormone convertase 1 is much greater in the absence of carboxypeptidase E activity, the proSAAS transgene was also expressed in carboxypeptidase E-deficient Cpe (fat/fat) mice. Although the transgenic mice were born in the expected frequency, 21 of 22 proSAAS transgenic Cpe (fat/fat) mice died between 11 and 26 weeks of age, presumably due to greatly elevated blood glucose. The levels of several pituitary peptides were significantly reduced in the proSAAS transgenic Cpe (fat/fat) mice relative to non-transgenic Cpe (fat/fat) mice, suggesting that the transgene inhibited prohormone convertase 1 in these mice. Taken together, these results are consistent with a role for proSAAS-derived peptides as neuropeptides that influence body weight independently of their function as inhibitors of prohormone convertase 1.

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L Friis-Hansen, KA Lacourse, LC Samuelson and JJ Holst

The maturation of many peptide hormones is attenuated in carboxypeptidase E (CPE)-deficient fat/fat mice, leading to a slowly developing, adult-onset obesity with mild diabetes. To determine the contribution of the hormones generated from the proglucagon precursor to this phenotype, we studied the tissue-specific processing of glucagon and glucagon-like peptide-1 (GLP-1) in these mice. In all tissues examined there was a great reduction in mature amidated GLP-1. Furthermore, a lack of CPE attenuates prohormone convertase processing of proglucagon in both the pancreas and the intestine. These findings suggest that defects in proglucagon processing together with other endocrine malfunctions could contribute to the diabetic and obesity phenotype in fat/fat mice.

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Willis K Samson, Gina L C Yosten, Jaw-Kang Chang, Alastair V Ferguson and Meghan M White

Introduction We have previously reported that obestatin, a 23 amino acid peptide derived from the same prohormone as ghrelin ( Zhang et al . 2005 ), when injected into the lateral cerebroventricle inhibits water drinking in response to dehydration

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PC Guest, SM Abdel-Halim, DJ Gross, A Clark, V Poitout, R Amaria, CG Ostenson and JC Hutton

The biosynthesis and processing of proinsulin was investigated in the diabetic Goto-Kakizaki (GK) rat. Immunofluorescence microscopy comparing GK and Wistar control rat pancreata revealed marked changes in the distribution of alpha-cells and pronounced beta-cell heterogeneity in the expression patterns of insulin, prohormone convertases PC1, PC2, carboxypeptidase E (CPE) and the PC-binding proteins 7B2 and ProSAAS. Western blot analyses of isolated islets revealed little difference in PC1 and CPE expression but PC2 immunoreactivity was markedly lower in the GK islets. The processing of the PC2-dependent substrate chromogranin A was reduced as evidenced by the appearance of intermediates. No differences were seen in the biosynthesis and post-translational modification of PC1, PC2 or CPE following incubation of islets in 16.7 mM glucose, but incubation in 3.3 mM glucose resulted in decreased PC2 biosynthesis in the GK islets. The rates of biosynthesis, processing and secretion of newly synthesized (pro)insulin were comparable. Circulating insulin immunoreactivity in both Wistar and GK rats was predominantly insulin 1 and 2 in the expected ratios with no (pro)insulin evident. Thus, the marked changes in islet morphology and PC2 expression did not impact the rate or extent of proinsulin processing either in vitro or in vivo in this experimental model.

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LE Pritchard, AV Turnbull and A White

Bioactive peptides derived from the prohormone, pro-opiomelanocortin (POMC), are generated in neurons of the hypothalamus and act as endogenous ligands for the melanocortin-4 receptor (MC4R), a key molecule underlying appetite control and energy homeostasis. It is therefore important to understand many aspects of POMC gene regulation in the brain, as pharmacological manipulation of POMC expression/processing could be a potential strategy to combat obesity. Most studies that have analysed POMC gene expression in the hypothalamus have focused on gene transcription experiments. Ultimately, however, factors that regulate post-translational processing and secretion of peptides will have most bearing on melanocortin signalling. This article focuses on (a) current evidence that POMC is involved in obesity, (b) how POMC transcription is regulated in the hypothalamus, (c) the mechanism by which proteolytic processing of POMC is controlled in the hypothalamus and what peptides are produced and (d) which POMC-derived peptides are the most potent ligands at the melanocortin receptor in vitro and in vivo. It seems that post-translational cleavage of POMC in the hypothalamus may be regulated with respect to energy requirement. We predict that further research into hypothalamic POMC processing, and the proteolytic enzymes involved, may yield important new clues on how flux through the MC4R pathway is regulated.

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CJ Pemberton, TG Yandle, CJ Charles, MT Rademaker, GD Aitken and EA Espiner

Whereas numerous studies have examined the cardiac tissue content and secretion of atrial natriuretic peptide (ANP), the response of brain natriuretic peptide (BNP) in states of experimental cardiac overload is less well documented. Our recent partial cloning of the ovine BNP gene has enabled us to study changes in cardiac tissue concentration, together with tissue and circulating molecular forms of ANP and BNP, in response to cardiac overload induced by rapid ventricular pacing (n = 7) and aortic coarctation (n = 6). In normal sheep, although highest levels of BNP were found in atrial tissue (15-fold those of the ventricle), the BNP/ANP concentration ratio in the ventricles was 10- to 20-fold higher than the ratio calculated for atrial tissue. Compared with normal sheep, significant depletion of both ANP and BNP concentrations within the left ventricle occurred after rapid ventricular pacing. Size exclusion and reverse phase HPLC analysis of atrial and ventricular tissue extracts from normal and overloaded sheep showed a single peak of high molecular weight BNP consistent with the proBNP hormone. In contrast, immunoreactive BNP extracted from plasma drawn from the coronary sinus was all low molecular weight material. Further analysis of plasma BNP using ion exchange HPLC disclosed at least 3 distinct immunoreactive peaks consistent with ovine BNP forms 26-29 amino acid residues in length. These findings show that BNP is stored as the prohormone in sheep cardiac tissues and that complete processing to mature forms occurs at the time of secretion. The capacity to process the prohormone at secretion is not impaired by chronic heart failure.

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JC Whitley, C Moore, AS Giraud and A Shulkes

High concentrations of a peptide related to gastrin-releasing peptide (GRP) are produced in the utero-placental unit of the human and sheep and secreted into the general circulation. This suggests an endocrine role in addition to its role as a neurotransmitter/neuromodulator. The GRP is larger than the previously described form GRP(1-27) but it is not known whether the larger form is the product of a related GRP-like gene or differences in post-translational processing. We have therefore cloned the gene for the sheep homologue of the GRP gene and determined its distribution. Only a single GRP gene was found in the sheep. This had a similar organisation to the human GRP gene with three exons and two introns. The larger form of GRP in the pregnant endometrium therefore appears to be the result of an alteration in processing of the GRP prohormone. The expression of GRP mRNA in the pregnant uterus was extraordinarily high comprising one-third of all mRNA synthesised by the pregnant endometrium. As the endometrial GRP mRNA arises solely from the glandular epithelium, the localised synthesis of GRP mRNA would be far higher. GRP mRNA was expressed in a wide variety of fetal tissues (fundus, colon, jejunum, ileum, duodenum, kidney, adrenal, lung, heart and pancreas) with a corresponding presence of GRP immunoreactivity. The expression of GRP in the fetal lung was biphasic with peaks at mid-term and near parturition but none in the adult supporting the concept of a specific developmental role of GRP in the lung.

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R. Suzuki, A. Takahashi and Y. Takei

ABSTRACT

The presence of a C-type natriuretic peptide (CNP) has been reported in the brain of mammals, birds, amphibians and teleost fishes, mostly as a 22-residue peptide (CNP-22). In the present study, we attempted to isolate natriuretic peptides from an elasmobranch, Triakis scyllia, using a chick rectum-relaxant assay, and different molecular forms of CNP were found in the brain and heart. Only CNP-22 was recovered from the brain, as is the case in other vertebrates. A large amount of prohormone (proCNP or CNP-115) and small amounts of its C-terminal peptides (CNP-38 and CNP-39) were isolated from the atrium and ventricle, however. No CNP-22 was recovered from the heart. Natriuretic peptides other than CNP were not isolated from Triakis heart and brain by the rectumrelaxant assay. The 22 residues at the C-terminal region of proCNP, i.e. CNP-22, were well conserved when Triakis and mammals were compared, although the sequence homology of the N-terminal segment of proCNP was very low. Not only was CNP-22 identical but the N-terminal segments of proCNP were also quite similar when Triakis and another elasmobranch, Scyliorhinus canicula, were compared. These data suggest that, in elasmobranchs, CNP is a primary hormone in the natriuretic peptide family, and also that CNP is processed differently in the brain and heart.

Journal of Endocrinology (1992) 135, 317–323

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L. Shukovski, J. K. Findlay and A. I. Smith

ABSTRACT

Acid extracts of bovine preovulatory granulosa cells and corpora lutea (CL) were subjected to high-performance liquid chromatography (HPLC) and found to contain two peaks of immunoreactive (ir) oxytocin (OT), one corresponding to authentic OT and the second eluting 8 min later. The second peak was more abundant than authentic irOT in preovulatory follicles and in the early CL, but became less abundant as the CL matured (mid luteal) and was close to the limit of detection in the late CL. This peak could be detected only by an OT antiserum which recognized both the biologically active form of OT, as well as the post-translational processing intermediate Gly10-extended oxytocin. A second more specific OT antiserum (OT-933) did not recognize the second peak as strongly. Further analysis of the second peak revealed a complex of OT bound to its neurophysin (NP-I) which could be dissociated under denaturing conditions. Furthermore, we were able to create this complex in vitro by combining the two materials together under acid conditions, similar to the pH predicted in secretory granules, but not under neutral conditions. Measuring irNP-I by radioimmunoassay showed a single peak with a similar retention time to the OT/NP-I complex, confirming the identity of the unknown peak. Incubation of CL slices in culture showed a time-related release of both OT and NP-I, with OT having a greater rate of release in the mid luteal CL.

These data suggest the presence of an OT/NP-I complex in the bovine preovulatory granulosa cells and CL, as well as the unbound peptide presumably within the secretory granules. The ratio of OT/NP-I complex and free peptide changes with ageing of the the CL, perhaps indicating regulated differences in the post-translational processing of the prohormone.

Journal of Endocrinology (1991) 128, 305–314