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The completion of the sequencing of the human genome and the concurrent, rapid development of high-throughput proteomic methods have resulted in an increasing need for automated approaches to archive proteomic data in a repository that enables the exchange of data among researchers and also accurate integration with genomic data. PeptideAtlas (http://www.peptideatlas.org/) addresses these needs by identifying peptides by tandem mass spectrometry (MS/MS), statistically validating those identifications and then mapping identified sequences to the genomes of eukaryotic organisms. A meaningful comparison of data across different experiments generated by different groups using different types of instruments is enabled by the implementation of a uniform analytic process. This uniform statistical validation ensures a consistent and high-quality set of peptide and protein identifications. The raw data from many diverse proteomic experiments are made available in the associated PeptideAtlas repository in several formats. Here we present a summary of our process and details about the Human, Drosophila and Yeast PeptideAtlas builds.

The present study explores the potential utility of peripheral versus central administration of glucagon-like peptide-1 (GLP-1) receptor agonists in the regulation of feeding behavior in Wistar and Zucker obese rats. Acute central (intracerebroventricular [i.c.v.]) and peripheral (subcutaneous [s.c.]) administration of both GLP-1 (7-36) amide and exendin-4 resulted in a reduction in food intake for at least 4 hours, exendin-4 being much more potent than GLP-1 (7-36) amide, especially after peripheral administration. Both Zucker obese rats (fa/fa) and their lean littermates (Fa/-) responded to acute central and peripheral administration of exendin-4. Moreover, in situ hybridization revealed specific labeling for the mRNA for GLP-1 receptors in several brain areas of both the obese and lean rats. The presence of this receptor was also detected by affinity cross-linking assays. Long-term s.c. administration of exendin-4 (1 single injection per day, 1 hour prior to the onset of the dark phase of the cycle) decreased daily food intake and practically blocked weight gain in obese rats. In contrast to previous studies, these findings show that peripheral (s.c.) administration of both GLP-1 receptor agonists also induces satiety and weight loss in rats, and suggest the potential usefulness of exendin-4 as a therapeutic tool for the treatment of diabetes and/or obesity.

Peripheral administration of large doses of lithium chloride (LiCl) to rats causes a spectrum of effects that are consistent with visceral illness. LiCl reduces food intake, decreases salt ingestion after sodium depletion, induces pica, and produces robust conditioned taste aversions. Because some of the effects of peripheral LiCl are mimicked by centrally administered glucagon-like peptide-1 (7-36) amide (GLP-1), we hypothesized that this peptide is involved in the neural pathways by which LiCl causes visceral illness. To test this hypothesis, we pretreated rats with a selective and potent GLP-1 receptor antagonist given directly into the third ventricle via an indwelling cannula before administration of peripheral LiCl. The GLP-1 receptor antagonist completely blocked the effect of LiCl to reduce food intake, induce pica, and produce a conditioned taste aversion. The same dose of GLP-1 receptor antagonist did not reverse the LiCl-induced reduction in NaCl intake. The data indicate a role for GLP-1 receptors in the CNS pathway that mediates some of the effects of visceral illness.

This study was designed to determine the contribution of the central nervous system (CNS) to the effects of glucagon-like peptide-1-(7-36) amide (tGLP-1) on arterial blood pressure and heart rate in rats. Accordingly, intracerebroventricular administration of the peptide produced an increase in cardiovascular parameters, which was blocked by previous administration of exendin-(9-39) through the same route, but not when it was intravenously injected. Intravenous administration of tGLP-1 produced a significant increase in arterial blood pressure and heart rate, which was blocked by the previous intracerebroventricular or intravenous administration of exendin-(9-39). Bilateral vagotomy blocked the stimulating effect of intracerebroventricular tGLP-1 administration on arterial blood pressure and heart rate. Also, bilateral vagotomy prevented the blocking effect of intracerebroventricular but not of intravenous exendin-(9-39) on cardiovascular parameters after intravenous administration of tGLP-1. These findings suggest that the action of tGLP-1 on cardiovascular parameters is under a dual control generated in the CNS and in peripheral structures and that the neural information emerging in the brain is transmitted to the periphery through the vagus nerve.

Central infusion of glucagon-like peptide-1-(7-36) amide (GLP-1) and intraperitoneal (i.p.) injection of lithium chloride (LiCl) produce similar patterns of c-Fos induction in the rat brain. These similarities led us to assess the hypothesis that neuronal activity caused by i.p. injection of LiCl involves activation of central GLP-1 pathways. We therefore determined if third-ventricular (i3vt) infusion of a GLP-1 receptor antagonist would block LiCl-induced c-Fos expression in the brainstem. Relative to rats pretreated with i3vt infusion of vehicle, pretreatment with the potent GLP-1 receptor antagonist, des His1 Glu9 exendin-4 (10.0 microgram), significantly attenuated LiCl-induced (76 mg/kg; i.p.) c-Fos expression in several brainstem regions, including the area postrema, the nucleus of the solitary tract, and the lateral parabrachial nucleus. While central infusion of des His1 Glu9 exendin-4 also blocked GLP-1-induced (10.0 microgram) anorexia and c-Fos expression, the antagonist produced no independent effects on food intake or c-Fos expression. These results suggest that LiCl-induced c-Fos expression in the rat brainstem is mediated, at least in part, by GLP-1 receptor signaling., (Copyright 1998 Elsevier Science B.V.)

GLP-1-(7-36)-amide and exendin-4-(1-39) are glucagon-like peptide-1 (GLP-1) receptor agonists, whereas exendin-(9-39) is the only known antagonist. To analyze the transition from agonist to antagonist and to identify the amino acid residues involved in ligand activation of the GLP-1 receptor, we used exendin analogs with successive N-terminal truncations. Chinese hamster ovary cells stably transfected with the rat GLP-1 receptor were assayed for changes in intracellular cAMP caused by the test peptides in the absence or presence of half-maximal stimulatory doses of GLP-1. N-terminal truncation of a single amino acid reduced the agonist activity of the exendin peptide, whereas N-terminal truncation of 3-7 amino acids produced antagonists that were 4-10-fold more potent than exendin-(9-39). N-terminal truncation of GLP-1 by 2 amino acids resulted in weak agonist activity, but an 8-amino acid N-terminal truncation inactivated the peptide. Binding studies performed using 125I-labeled GLP-1 confirmed that all bioactive peptides specifically displaced tracer with high potency. In a set of exendin/GLP-1 chimeric peptides, substitution of GLP-1 sequences into exendin-(3-39) produced loss of antagonist activity with conversion to a weak agonist. The results show that receptor binding and activation occur in separate domains of exendin, but they are more closely coupled in GLP-1.

This study was designed to determine the interactions of peptide exendin-(9-39) with the effect of glucagon-like peptide-1-(7-36) (GLP-1 (7-36)) amide and of exendin-4 on arterial blood pressure and heart rate in the rat. Both GLP-1 (7-36) amide and exendin-4 produced a dose-dependent increase in systolic, diastolic and mean arterial blood pressure, as well as in heart rate, although the effect of exendin-4 was more prolonged. These data indicate a longer functional half-life in vivo for exendin-4 as compared to GLP-1 (7-36) amide, which may have therapeutical applications. The antagonist effect of exendin-(9-39) on these cardiovascular parameters was also tested with 3000 ng of exendin-(9-39) intravenously administered 5 min before i.v. injection of 10 ng of either GLP-1 (7-36) amide or exendin-4. Under these experimental conditions the effect of the latter two peptides on arterial blood pressure and heart rate was blocked. By contrast, single administration of exendin-(9-39) did not modify cardiovascular parameters. These findings indicate that exendin-4 is an agonist and that exendin-(9-39) is an antagonist of the action of GLP-1 (7-36) amide on arterial blood pressure and heart rate. Therefore, the action of GLP-1 (7-36) amide on these parameters seems to be mediated through its own receptors.

This study was designed to determine the possible role of brain glucagon-like peptide-1 (GLP-1) receptors in feeding behavior. In situ hybridization showed colocalization of the mRNAs for GLP-1 receptors, glucokinase, and GLUT-2 in the third ventricle wall and adjacent arcuate nucleus, median eminence, and supraoptic nucleus. These brain areas are considered to contain glucose-sensitive neurons mediating feeding behavior. Because GLP-1 receptors, GLUT-2, and glucokinase are proteins involved in the multistep process of glucose sensing in pancreatic beta cells, the colocalization of specific GLP-1 receptors and glucose sensing-related proteins in hypothalamic neurons supports a role of this peptide in the hypothalamic regulation of macronutrient and water intake. This hypothesis was confirmed by analyzing the effects of both systemic and central administration of GLP-1 receptor ligands. Acute or subchronic intraperitoneal administration of GLP-1 (7-36) amide did not modify food and water intake, although a dose-dependent loss of body weight gain was observed 24 h after acute administration of the higher dose of the peptide. By contrast, the intracerebroventricular (i.c.v.) administration of GLP-1 (7-36) amide produced a biphasic effect on food intake characterized by an increase in the amount of food intake after acute i.c.v. delivery of 100 ng of the peptide. There was a marked reduction of food ingestion with the 1,000 and 2,000 ng doses of the peptide, which also produced a significant decrease of water intake. These effects seemed to be specific because i.c.v. administration of GLP-1 (1-37), a peptide with lower biological activity than GLP-1 (7-36) amide, did not change feeding behavior in food-deprived animals. Exendin-4, when given by i.c.v. administration in a broad range of doses (0.2, 1, 5, 25, 100, and 500 ng), proved to be a potent agonist of GLP-1 (7-36) amide. It decreased, in a dose-dependent manner, both food and water intake, starting at the dose of 25 ng per injection. Pretreatment with an i.c.v. dose of a GLP-1 receptor antagonist [exendin (9-39); 2,500 ng] reversed the inhibitory effects of GLP-1 (7-36) amide (1,000 ng dose) and exendin-4 (25 ng dose) on food and water ingestion. These findings suggest that GLP-1 (7-36) amide may modulate both food and drink intake in the rat through a central mechanism.

Glucagon-like peptide 1 (GLP-1) is an insulinotropic hormone released after nutrient ingestion which is known to augment insulin secretion, inhibit glucagon release, and promote insulin-independent glucose disposition. To determine the overall effect of GLP-1 on glucose disposition after a meal we studied a group of healthy, conscious baboons before and after intragastric glucose administration during infusions of saline, and two treatments to eliminate the action of GLP-1: (a) exendin-[9-39] (Ex-9), a peptide receptor antagonist of GLP-1; or (b) an anti-GLP-1 mAb. Fasting concentrations of glucose were higher during infusion of Ex-9 than during saline (4.44 +/- 0.05 vs. 4.16 +/- 0.05 mM, P < 0.01), coincident with an elevation in the levels of circulating glucagon (96 +/- 10 vs. 59 +/- 3 ng/liter, P < 0.02). The postprandial glycemic excursions during administration of Ex-9 and mAb were greater than during the control studies (Ex-9 13.7 +/- 2.0 vs. saline 10.0 +/- 0.8 mM, P = 0.07; and mAb 13.6 +/- 1.2 vs. saline 10.6 +/- 0.9 mM, P = 0.044). The increments in insulin levels throughout the absorption of the glucose meal were not different for the experimental and control conditions, but the insulin response in the first 30 min after the glucose meal was diminished significantly during treatment with Ex-9 (Ex-9 761 +/- 139 vs. saline 1,089 +/- 166 pM, P = 0.044) and was delayed in three of the four animals given the neutralizing antibody (mAb 946 +/- 262 vs. saline 1,146 +/- 340 pM). Thus, elimination of the action of GLP-1 impaired the disposition of an intragastric glucose meal and this was at least partly attributable to diminished early insulin release. In addition to these postprandial effects, the concurrent elevation in fasting glucose and glucagon during GLP-1 antagonism suggests that GLP-1 may have a tonic inhibitory effect on glucagon output. These findings demonstrate the important role of GLP-1 in the assimilation of glucose absorbed from the gut.

Rab3 proteins are low molecular weight guanine nucleotide-binding proteins that belong to the Ras superfamily and are believed to play a role in the final steps of exocytosis. To examine potential interactions of these proteins with signaling pathways that mediate pepsinogen secretion from gastric chief cells, we synthesized peptides corresponding to the effector domain of Rab3. In the absence of added calcium [calcium concentration ([Ca2+]) < 1 nM], a maximal concentration (15 microM) of the Rab3 effector domain peptide or Rab3AL peptide, containing alanine and leucine substitutions, stimulated the release of 62 and 66%, respectively, of total pepsinogen from streptolysin O-permeabilized chief cells. A Rab2AL peptide, corresponding to the Rab2 effector domain, and modified (scrambled and truncated) Rab3AL peptides did not alter secretion from permeabilized cells. An additive secretory response was observed when 5 microM Rab3AL peptide was combined with increasing calcium ([Ca2+] < 1 nM to 3 microM). In contrast, adding up to 3 mM adenosine 3',5'-cyclic monophosphate (cAMP) had no effect on Rab3AL peptide-induced secretion, and Rab3AL peptide did not alter endogenous cAMP production. The addition of a nonhydrolyzable GTP analogue [0.01 to 100 microM guanosine 5'-O-(3-thiotriphosphate)] potentiated the secretory response to Rab3AL peptide. This potentiated response indicates that other GTP-binding proteins are involved in calcium-independent secretion. Preincubation of cells with streptolysin O (10-30 min), to allow egress of cytosolic constituents, enhanced the response to Rab3AL peptide, suggesting that the target(s) for this peptide is (are) anchored to chief cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Exendin-4 is a novel peptide from Heloderma suspectum venom which is 53% homologous with glucagon-like peptide-1 GLP-1-(7-36)NH2, a stimulant of cAMP-dependent H+ production in rat parietal cells. It was the aim of the present study to determine whether this effect of GLP-1-(7-36)NH2 is shared by exendin-4, and whether the responses to either peptide are blocked by exendin-(9-39)NH2, a competitive specific exendin receptor antagonist. In enriched rat parietal cells H+ production was measured indirectly by [14C]aminopyrine accumulation. cAMP production was determined by radioimmunoassay. [125I]GLP-1-(7-36)NH2 was prepared using chloramine T followed by high pressure liquid chromatography (HPLC) purification. Exendin-4 (10(-12) - 10(-8) M) stimulated [14C]aminopyrine accumulation in a concentration-dependent manner (EC50 = 7.6 x 10(-11) M). At the maximally effective concentration (10(-9) M) exendin-4 was as effective as GLP-1-(7-36)NH2 reaching 70-80% of the response to 10(-4) M histamine. Likewise, exendin-4 (10(-11) - 10(-7) M) stimulated parietal cell cAMP production up to 2.8-fold. Maximal stimulation by exendin-4 of [14C]aminopyrine accumulation was not affected by ranitidine (10(-4) M), but was concentration-dependently reduced by exendin-(9-39)NH2 (10(-11) - 10(-7) M). At the maximal concentration, exendin-(9-39)NH2 completely abolished the responses to 10(-9) M exendin-4 and to 10(-9) M GLP-1-(7-36)NH2 while not altering stimulation by 10(-4) M histamine. Binding of [125I]GLP-1-(7-36)NH2 to enriched parietal cells was displaced by exendin-4 (Ki = 4.6 x 10(-10) M) as well as by exendin-(9-39)NH2 (Ki = 4.0 x 10(-9) M).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of exendin-4, a peptide of the secretin-glucagon family with high homology of amino acid sequence with glucagon-like peptide-1 (GLP-1), on gastric hormone release was investigated in the isolated perfused rat stomach. Exendin-4 dose dependently stimulated somatostatin release up to 9-fold at a concentration of 10(-7) M whereas gastrin release was inversely inhibited by up to 63%. These effects could partially be reduced by concomitant perfusion of truncated exendin-4, exendin(9-39)amide. Similarly, stimulation of somatostatin secretion and inhibition of gastrin release induced by GLP-1(7-36)amide was partially reversed by exendin-4 (9-39)amide. These data are consistent with the assumption that exendin-4 and truncated GLP-1amide exert their effects on gastric D and G cell by interaction with the same receptor.

Exendin-4 purified from Heloderma suspectum venom shows structural relationship to the important incretin hormone glucagon-like peptide 1-(7-36)-amide (GLP-1). We demonstrate that exendin-4 and truncated exendin-(9-39)-amide specifically interact with the GLP-1 receptor on insulinoma-derived cells and on lung membranes. Exendin-4 displaced 125I-GLP-1, and unlabeled GLP-1 displaced 125I-exendin-4 from the binding site at rat insulinoma-derived RINm5F cells. Exendin-4 had, like GLP-1, a pronounced effect on intracellular cAMP generation, which was reduced by exendin-(9-39)-amide. When combined, GLP-1 and exendin-4 showed additive action on cAMP. They each competed with the radio-labeled version of the other peptide in cross-linking experiments. The apparent molecular mass of the respective ligand-binding protein complex was 63,000 Da. Exendin-(9-39)-amide abolished the cross-linking of both peptides. Exendin-4, like GLP-1, stimulated dose dependently the glucose-induced insulin secretion in isolated rat islets, and, in mouse insulinoma beta TC-1 cells, both peptides stimulated the proinsulin gene expression at the level of transcription. Exendin-(9-39)-amide reduced these effects. In conclusion, exendin-4 is an agonist and exendin-(9-39)-amide is a specific GLP-1 receptor antagonist.

To find mammalian analogues of exendin-4, a peptide from Helodermatidae venoms that interacts with newly discovered exendin receptors on dispersed acini from guinea pig pancreas, we examined the actions of recent additions to the vasoactive intestinal peptide/secretin/glucagon family of regulatory peptides. In every respect tested, the truncated form of glucagon-like peptide-1, GLP-1(7-36)NH2, mimicked the actions of exendin-4. Like exendin-4, GLP-1(7-36)NH2 caused an increase in acinar cAMP without stimulating amylase release. GLP-1(7-36)NH2-induced increases in cAMP were inhibited progressively by increasing concentrations of the specific exendin-receptor antagonist, exendin(9-39)NH2. In dispersed acini from guinea pig and rat pancreas, concentrations of GLP-1(7-36)NH2 that stimulated increases in cAMP caused potentiation of cholecystokinin-induced amylase release. Binding of 125I-[Y39]exendin-4 or 125I-GLP-1(7-36)NH2 to dispersed acini from guinea pig pancreas was inhibited by adding increasing concentrations of unlabeled exendin-4 or GLP-1(7-36)NH2. We conclude that the mammalian peptide GLP-1(7-36)NH2 interacts with exendin receptors on dispersed acini from guinea pig pancreas. Exendin(9-39)NH2, a competitive antagonist of the actions of GLP-1(7-36)NH2 in pancreatic acini, may be a useful tool for examining the physiological actions of this peptide.

A radioimmunoassay was developed to detect the cholecystokinin (CCK)-associated nonapeptide (CAP-9) that forms the COOH terminus of pig preproCCK. This peptide (Ser-Ala-Glu-Glu-Tyr-Glu-Tyr-Thr-Ser) is presumably produced at the time that the tyrosine-sulfated octapeptide CCK8(s) is cleaved from preproCCK. Radioimmunoassay of a dried methanol extract of pig brain revealed no detectable CAP-9 immunoreactivity, whereas acid desulfation of the dried methanol extract prior to radioimmunoassay resulted in easily measurable concentrations of CAP-9 immunoreactivity. Two peptides, CAP-9 and des-Ser9-CAP-9, were purified from a methanol extract of 8 kg of commercially obtained whole pig brains. Amino acid analysis showed that each peptide has both tyrosines sulfated. Thus, the likely sequence of CCK post-translational processing events is sulfation of the three tyrosines in the COOH terminus of preproCCK followed by peptide cleavage and appearance of CCK8(s) and CAP-9(s,s).

Fractionation on Sephadex G50 gel of methanol extracts of rat intestine revealed two molecular forms of cholecystokinin (CCK) of about equal immunopotency: one form has an elution volume between CCK33 and CCK12; the other elutes in the salt region as does authentic CCK8. Purification and sequencing have demonstrated that the smaller molecular form is CCK8 with a sequence identical to the pork and sheep CCK8's that had previously been sequenced. Purification and sequencing of the larger molecular form reveals that it is a 22 amino acid C-terminal CCK fragment identical with pig CCK22 except that glycine instead of serine is present at the nineteenth residue from the C-terminus. This sequence is consistent with that predicted by cloned cDNA encoding preprocholecystokinin from a rat medullary thyroid carcinoma. CCK22 has not previously been reported to be a prominent molecular form in either pig or dog intestines.