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Rebecca McGirr, Leonardo Guizzetti and Savita Dhanvantari

L cells, and by the primary and secondary structures of proglucagon. The prohormone processing enzyme carboxypeptidase E (CPE) is expressed in both alpha and L cells, while PC1/3 is found in L cells and PC2 in alpha cells. Several lines of evidence

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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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EH Leiter

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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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P J Fowke and S C Hodgkinson

Abstract

Insulin-like growth factor binding protein-3 (IGFBP-3) is known to modulate the actions of insulin-like growth factors (IGF)-I and -II at the level of the cell. Proposed mechanisms include association of IGFBP-3 with cell surface proteoglycan, with cell surface binding proteins, proteolysis and/or internalization of IGFBP-3. In previous studies we have characterized a protein of 40 kDa in extracts of ovine pancreas and muscle which binds IGFBP-3 on ligand blot analyses. This paper reports the identity of the pancreatic species as procarboxypeptidase A (peptidyl-l-amino acid hydrolase, E.C. 3.4.17.1; proCPA). Identity was established by amino terminal sequence analysis, binding studies with pure bovine carboxypeptidase A (CPA) and observations that the binding activity was present in pancreatic secretions consistent with the role of proCPA as a secretory zymogen. The binding activity was inhibited by unlabelled IGFBP-3 at high doses (10 μg/ml) and reduced but not abolished by preincubation of 125I-IGFBP-3 with excess IGF-I. Digestion of 125I-IGFBP-3 with mature CPA produced a 26 kDa product. Modification of IGFBP-3 by CPA or binding to proCPA may provide a mechanism for modulation of IGFBP activity and hence IGF action.

Journal of Endocrinology (1996) 150, 51–56

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E. REID

Different batches of crude bovine pituitary extract were compared with respect to the 'D/G ratio', i.e. the ratio of diabetogenic activity in cats to growth-promoting activity in rats. The ratio was low for some extracts which had been incubated as in the experiments of Marks & Young [1940], but wide variations were found with 'normal' (non-incubated) extracts, for some of which the ratio was notably high.

Purified preparations of the diabetogenic factor isolated from crude extracts appeared to consist predominantly of growth hormone (GH), as shown particularly by examination in the ultracentrifuge (see Addendum).

The possibility that 'purified GH' might, in common with crude extracts, vary in D/G ratio, received little support from tests performed on semi-purified GH fractions isolated from crude extracts, or on purified GH preparations subjected to mildly destructive treatments such as incubation with carboxypeptidase, which did not impair either diabetogenic or growth-promoting activity. Tests with the GH preparation (pig) of Raben & Westermeyer [1952] did not confirm their claim that it lacks diabetogenic activity.

The growth-promoting activity of GH is reduced by ACTH or posterior-pituitary extract, and enhanced by thyrotrophin. The diabetogenic activity of GH is known to be enhanced by ACTH under certain conditions. Variations among crude extracts in D/G ratio may thus be attributable to variations in the content of certain hormones other than GH.

Data are presented for changes in body-weight in cats treated with crude pituitary extracts or purified GH.

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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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C. L. Coulter, I. R. Young, C. A. Browne and I. C. McMillen

ABSTRACT

We have investigated the possible role of the fetal pituitary and ACTH in the control of the synthesis and post-translational processing of the enkephalin precursor, proenkephalin A (proEnk A), in the fetal sheep adrenal gland in late gestation.

Fetal hypophysectomy (n = 8) or sham operations (n = 4) were performed between 109 and 118 days of gestation. At 138–139 days, either ACTH(1–24) (10·5 μg/0·24 ml saline per h, n = 4) was infused intravenously for 72 h into hypophysectomized fetal sheep or 0·9% (w/v) NaCl alone (0·24 ml/h, n = 4) was infused for 72 h into hypophysectomized fetal sheep and sham-operated animals. At the end of the infusion the pregnant ewe was killed and left or right adrenal glands (n = 12) were collected from the fetal sheep that were intact and given saline (Intact + sal; n = 4), hypophysectomized and given saline (Hx + sal; n = 4) and hypophysectomized and given ACTH (Hx + ACTH; n = 4). Each adrenal was homogenized in acid (acetic acid (1 mol/l)/HCl (20 mmol/l)/2-mercaptoethanol (0·2%)). After centrifugation, the supernatant was loaded onto a Sephadex G-75 column (2·0 × 50 cm), eluted at 80 ml/24 h and fractions were collected (5 ml, n = 42). An aliquot of each fraction (2 ml) was dried down prior to enzymatic digestion (trypsin/carboxypeptidase B) and oxidation with H2O2, and assay for methionine-O-enkephalin (immunoreactive Met-O-Enk).

The total adrenal content of immunoreactive Met-O-Enk was significantly greater in the Hx + ACTH group (326·2 ±66·7 (s.e.m.)ng/adrenal) when compared with either the Intact + sal group (152·7 ±44·0 ng/adrenal) or the Hx + sal group (112·1 ±20·8 ng/adrenal). In the adrenal glands from all fetuses immunoreactive Met-O-Enk was found in four molecular weight ranges: < 12 kDa, 12–7 kDa, 7–3 kDa and < 3 kDa. There was no significant difference between the Hx + sal and Hx + ACTH groups in the proportion of immunoreactive Met-O-Enk present in each of the molecular weight ranges in the adrenals and therefore the data from these groups were combined for further statistical analysis. The proportion of immunoreactive Met-O-Enk in the > 12 kDa range was significantly less in the Intact + sal group (5·5 ±2·3%) when compared with the hypophysectomized sheep with or without ACTH replacement (18·7 ± 4·5%).

These data demonstrate that fetal hypophysectomy alters the molecular weight profile of Enk-containing peptides in the adrenal of the fetal sheep and whilst ACTH replacement in the hypophysectomized fetus does not alter the post-translational processing of the Enk-containing peptides, it stimulates an increase in the total amount of immunoreactive Met-O-Enk in the fetal adrenal in late gestation.

Journal of Endocrinology (1992) 134, 369–375

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Rohit Singhal, Kartik Shankar, Thomas M Badger and Martin J Ronis

endogenous estrogen, 17β-estradiol (E 2 ). Mature ovariectomized female Sprague–Dawley rats were fed with a diet containing SPI and followed by E 2 infusion in half of the rats. The effects of feeding SPI in the presence or absence of E 2 were compared with

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Robson A S Santos, Anderson J Ferreira, Thiago Verano-Braga and Michael Bader

-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1–9 . Circulation Research 87 E1 – E9 . ( doi:10.1161/01.RES.87.5.e1 ) Donoghue M Wakimoto H Maguire CT Acton S Hales P Stagliano N Fairchild-Huntress V Xu J Lorenz JN Kadambi V