Identification of the Alzheimer's disease amyloid precursor protein (APP) and its homologue APLP2 as essential modulators of glucose and insulin homeostasis and growth.

The amyloid precursor protein (APP), the source of the neurotoxic amyloid beta (A beta) peptide involved in Alzheimer's disease (AD), belongs to a conserved family of related proteins. In mammals, the APP family contains amyloid precursor-like protein 1 (APLP1) and amyloid precursor-like protein 2 (APLP2). Whilst a number of activities have been attributed to the APP family, an overall function has not been definitively established. While ablating either the APP or APLP2 gene in mice produces minimal phenotypic change, the combined knockout of these genes in mice causes postnatal mortality. Postnatal survival therefore requires a shared but unknown function of APP and APLP2. To investigate the biochemical basis for the postnatal lethality, plasma was analysed from double knockout mice (APP-/- APLP2-/-) 2 days before birth, at gestational day E17, and from mice at 12-16 h after birth. The postnatal double knockouts had 66% lower plasma glucose levels than their wild-type controls and 50% lower than their single knockout counterparts. Interestingly, the postnatal double knockouts displayed hyperinsulinaemia, as shown by inappropriate plasma insulin levels, given their degree of hypoglycaemia. The single knockout mice also showed hyperinsulinaemia and had 31% lower plasma glucose than the wild-types. While the double knockouts did not survive more than 24 h after birth, the single knockouts reached adulthood and their hypoglycaemia continued. Therefore, APP and APLP2 expression modulates plasma insulin and glucose concentrations. Plasma calcium, magnesium and phosphate were also significantly reduced in the double knockouts compared to the wild-types, and they showed distinctive growth restriction, suggesting the involvement of a metabolic impairment. These results link the expression of the APP and APLP2 genes with glucose homeostasis and growth and therefore identify a novel function for the APP family.

Breeding stock-specific variation in peptidylglycine alpha-amidating monooxygenase messenger ribonucleic acid splicing in rat pituitary.

Peptidylglycine alpha-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the carboxyl-terminal amidation of glycine-extended peptides in a two-step reaction involving a monooxygenase and a lyase. Several forms of PAM messenger RNA result from alternative splicing of the single copy PAM gene. The presence of alternately spliced exon A between the two enzymatic domains allows endoproteolytic cleavage to occur in selected tissues, generating soluble monooxygenase and membrane lyase from integral membrane PAM. While using an exon A antiserum, we made the unexpected observation that Charles River Sprague Dawley rats expressed forms of PAM containing exon A in their pituitaries, whereas Harlan Sprague Dawley rats did not. Forms of PAM containing exon A were expressed in the atrium and hypothalamus of both types of Sprague Dawley rat, although in different proportions. PAM transmembrane domain splicing also differed between rat breeders, and full-length PAM-1 was not prevalent in the anterior pituitary of either type of rat. Despite striking differences in PAM splicing, no differences in levels of monooxygenase or lyase activity were observed in tissue or serum samples. The splicing patterns of other alternatively spliced genes, pituitary adenylate cyclase-activating polypeptide receptor type 1 and cardiac troponin T, did not vary with rat breeder. Strain-specific variations in the splicing of transcripts such as PAM must be taken into account in analyzing the resultant proteins, and knowledge of these differences should identify variations with functional significance.

Induction of integral membrane PAM expression in AtT-20 cells alters the storage and trafficking of POMC and PC1.

Peptidylglycine alpha-amidating monooxygenase (PAM) is an essential enzyme that catalyzes the COOH-terminal amidation of many neuroendocrine peptides. The bifunctional PAM protein contains an NH2-terminal monooxygenase (PHM) domain followed by a lyase (PAL) domain and a transmembrane domain. The cytosolic tail of PAM interacts with proteins that can affect cytoskeletal organization. A reverse tetracycline-regulated inducible expression system was used to construct an AtT-20 corticotrope cell line capable of inducible PAM-1 expression. Upon induction, cells displayed a time- and dose-dependent increase in enzyme activity, PAM mRNA, and protein. Induction of increased PAM-1 expression produced graded changes in PAM-1 metabolism. Increased expression of PAM-1 also caused decreased immunofluorescent staining for ACTH, a product of proopiomelanocortin (POMC), and prohormone convertase 1 (PC1) in granules at the tips of processes. Expression of PAM-1 resulted in decreased ACTH and PHM secretion in response to secretagogue stimulation, and decreased cleavage of PC1, POMC, and PAM. Increased expression of a soluble form of PAM did not alter POMC and PC1 localization and metabolism. Using the inducible cell line model, we show that expression of integral membrane PAM alters the organization of the actin cytoskeleton. Altered cytoskeletal organization may then influence the trafficking and cleavage of lumenal proteins and eliminate the ability of AtT-20 cells to secrete ACTH in response to a secretagogue.

Glycine-extended gastrin acts as an autocrine growth factor in a nontransformed colon cell line.

BACKGROUND & AIMS: The hypothesis that progastrin-derived peptides act as autocrine growth factors for colorectal carcinomas has generated considerable interest. However, the influence of autocrine gastrins on nontumorigenic colonic cells has not been investigated. This study tested the above hypothesis in the nontumorigenic, conditionally immortalized mouse colon cell line YAMC. METHODS: The effects of expression of antisense or sense gastrin messenger RNA, treatment with antibodies against progastrin-derived peptides, or treatment with gastrin receptor antagonists on YAMC cell proliferation were measured. RESULTS: YAMC clones expressing antisense gastrin messenger RNA had reduced levels of immunoreactive progastrin-derived peptides and a reduced rate of proliferation, relative to vector only-transfected cells. Glycine-extended gastrin17, but not amidated gastrin17, reversed the antisense-induced inhibition of proliferation and stimulated the proliferation of sense- or vector only-transfected cells. YAMC cells bound 125I-glycine-extended gastrin17 (Kd, 0.36 nmol/L, 1810 sites/cell), but not 125I-amidated gastrin17, and binding was unaffected by gastrin receptor antagonists including benzotript. Proliferation of all YAMC clones was partially inhibited either by an antibody selective for glycine-extended gastrin or by preincubation with benzotript, and the inhibitory effects were additive. CONCLUSIONS: YAMC cells use nonamidated progastrin-derived peptides as autocrine growth factors, partly through binding to an extracellular receptor selective for glycine-extended gastrin, and partly through an intracellular mechanism.

Gastrin processing and secretion in patients with end-stage renal failure.

Gastrin circulates at higher than normal concentrations in patients with end-stage renal failure (ESRF). However, it remains unclear which forms of gastrin are elevated and whether there is also an alteration in the secretory profile after stimulation. In the present study all processed and partially processed forms of circulating gastrin were measured in plasma before and after meal stimulation in ESRF patients and control subjects. Since Helicobacter pylori (HP) infection affects gastrin secretion, HP status was determined. Fasting gastrin-amide (36 +/- 8 pmol/L), gastrin-Gly (55 +/- 16 pmol/L), and total gastrin (218 +/- 32 pmol/L) measured in ESRF/HP-patients were all significantly greater than those in the control group (10 +/- 1, 15 +/- 3, and 17 +/- 2 pmol/L, respectively; P < 0.01). Plasma gastrin-amide (126 +/- 67 pmol/L) and total gastrin (397 +/- 164 pmol/L) were highest in the ESRF/HP+ patients. The proportion of nonamidated gastrin products was 4-fold higher in ESRF patients than in control subjects, suggesting structure-specific changes in gastrin secretion and metabolism, and this was confirmed by chromatography. The meal-stimulated increments in control/HP- and ESRF/HP-groups were similar. However, the ESRF/HP+ group had a markedly potentiated gastrin response. Fasting plasma somatostatin, an inhibitor of gastrin secretion, was also measured and was significantly lower in the ESRF patients than that in the control group. These studies show that the hypergastrinemia associated with renal failure has been underestimated. This is because only amidated products were measured. The potentiated gastrin meal response in ESRF attributed previously to changes in gastrin metabolism are in part explained by the effect of HP infection. The observed diminished somatostatin response suggests that the increase in circulating gastrin in ESRF is the result of loss of inhibition of secretion as well as decreased metabolism. As both amidated and nonamidated gastrin are now considered to have trophic and secretory effects, these findings may explain the gastrointestinal tract hypertrophy often associated with ESRF.

Ontogeny of gastrin and cholecystokinin in the colon and duodenum of sheep.

The different roles of gastrin and cholecystokinin in the fetus compared to the adult may be reflected in different distribution patterns. Re-expression of these fetal patterns is often seen in tumours of the adult. Using region-specific antisera and chromatography, we have determined the ontogeny of amidated gastrin (G-amide), glycine extended gastrin (G-gly), and cholecystokinin (CCK) in various segments of the colon and compared it to the developmental profile in the duodenum. Fetal sheep aged 80-90, 115-125 and 135-144 days (term is 145 days), 7-14 day lamb, and adult sheep were examined. In the colon, higher concentrations of G-amide (2.8 +/- 0.2 pmol/g) and CCK (11.7 +/- 1.6 pmol/g) were measured in the fetus while G-gly (0.7 +/- 0.1 pmol/g) was higher in the adult compared to other age groups. The calculated G-gly/G-amide ratio was 0.4 in the fetus and 1.4 in the adult while the CCK/G-amide ratios were 5 in the fetus and 13 in the adult. The duodenum of the lamb rather than the fetus contained the highest concentrations of G-amide, G-gly and CCK (40.3 +/- 9.7, 2.0 +/- 0.4, 109.0 +/- 14.3 pmol/g, respectively) and at concentrations exceeding that in the colon. The results demonstrate two major developmentally regulated features. Firstly as the colon matures, there is a gradual switch between the expression of the gastrin and CCK genes and secondly, the processing to G-amide is attenuated. These findings suggest that non-amidated gastrin should be examined for a potential role as a growth factor in colorectal carcinogenesis.

Expression, processing, and secretion of gastrin in patients with colorectal carcinoma.

BACKGROUND & AIMS: The relationship between gastrin and the development of colorectal carcinoma (CRC) remains controversial. Problems with previous studies include failure to measure all forms of gastrin, lack of comparison between stored and secreted gastrin, and not controlling for Helicobacter pylori infection (a known cause of hypergastrinemia). The aim of this study was to quantify progastrin and progastrin-derived peptides in the resected tumor and plasma of patients with CRC and in the antrum and plasma of normal subjects. METHODS: Four region-specific gastrin antisera were used to measure progastrin, glycine-extended gastrin, amidated gastrin, and total gastrin. RESULTS: Progastrin, amidated gastrin, total gastrin, and glycine-extended gastrin were detected in 100%, 69%, 56%, and 44% of tumors, respectively (n = 32). When allowing for H. pylori infection, circulating amidated gastrin levels were not significantly elevated in patients with CRC. However, compared with control H. pylori-positive and H. pylori-negative subjects, fasting plasma total gastrin levels were increased in H. pylori-positive (5.2-fold) and H. pylori-negative (2.3-fold) patients with CRC. CONCLUSIONS: Gastrin or its processing intermediates are present in a high proportion of CRCs. Nonamidated gastrin levels are elevated in the circulation of patients with CRC regardless of H. pylori status. We conclude that gastrin should continue to be assessed as a circulating or autocrine growth factor in the development of CRC.

Pharmacokinetics and organ specific metabolism of glycine-extended and amidated gastrin in sheep.

Glycine (Gly)-extended gastrin has been described as the inactive precursor form of the biologically active amidated gastrin. The ratio of Gly-extended to amidated gastrin is higher in the circulation than in tissue, suggesting either differential secretion and/or metabolism. Although the distribution of the precursor form is similar in tissue and circulation to its amidated product, the significance of measurable levels of precursor peptide in the circulation is unknown. In this study, we have examined the pharmacokinetic properties and organ-specific metabolism of both the Gly-extended and the amidated forms of gastrin-17 (G-17-Gly and G-17-amide) in the conscious sheep. The metabolic clearance rate, half disappearance time, and production rates were similar for both G-17-Gly and G-17-amide. G-17-Gly was extracted across the head, kidney, and lung but not across the gut and liver. Similarly, G-17-amide was extracted across the head, gut, lung, and kidney but not across the liver. G-17-Gly had no biological activity as evidenced by its failure to stimulate somatostatin secretion nor was there any measurable conversion to amidated gastrin in the circulation. We conclude that the presence of G-17-Gly in the circulation is not the result of a slower clearance and that circulating G-17-Gly is not a precursor for circulating gastrin-amide. The results of this study provide important baseline data for understanding the dynamics of the precursor product relationship between G-Gly and G-amide.