Wirkung und Wirkungsweise von Insulin-like Growth-factor-I auf das proliferative Wachstum neuroendokriner Tumorzellen am Beispiel der humanen Karzinoidzelllinie BON

Karzinoide sind neuroendokrine Tumoren des gastroenteropankreatischen Systems. Bis heute gibt es mit Ausnahme eines chirurgischen Eingriffes keine adäquate kurativeTherapiemöglichkeit. Die Expression des IGF-I-Rezeptors und von IGF-I konnte bei dieser Tumorart bereits nachgewiesen werden. Diese beiden Teile des IGF-Systems sind von besonderer Bedeutung für das Entstehen und das Erhalten verschiedener Tumorarten und gelten heute als sehr interessanter Ansatzpunkt für die Erforschung neuer Therapiestrategien. Ziel dieser Arbeit war es, am Beispiel der humanen Karzinoid-Zellinie BON, das proliferative Wachstum dieser Tumorart unter dem Einfluß von IGF-I zu untersuchen und mögliche, für die Signalvermittlung verantwortliche Transduktionswege zu entschlüsseln. In einem ersten Schritt konnte durch Rezeptor-Bindungsstudien gezeigt werden, dass BON-Zellen einen funktionstüchtigen IGF-I-Rezeptor tragen. Im Radioimmunoassay wurde zudem die IGF-I-Synthese quantitativ nachgewiesen. Die Stimulation der Zellen mit IGF-I bewirkt eine deutliche und signifikante Steigerung des proliferativen Zellwachstums. In Versuchen mit den Hemmstoffen PD 98059 als spezifischer Inhibitor der MAP-Kinasen ERK1 und 2 sowie mit LY 294002 als selektiven PI-3Kinase-Inhibitor konnte darüber hinaus gezeigt werden, dass die Signalkaskaden dieser beiden Kinasefamilien für die Signaltransduktion nach Bindung von IGF-I an seinen Rezeptor von wesentlicher Bedeutung sind. Aus diesen Hemmungsversuchen ergeben sich zudem Hinweise für eine mögliche Verknüpfung beider Transduktionswege. Im MAP-Kinase-Assay wurden die Einflüsse auf die MAP-Kinase visualisiert. Abschließend wurde durch Transfektionsstudien mit einem IGF-I-Promotor die Bedeutungen beider Signaltransduktionswege auch auf die IGF-I-Produktion der BON-Zellen nachgewiesen. Zusammen mit dem beschriebenen quantitativen Nachweis von IGF-I ergeben sich damit deutliche Hinweise für einen auto- beziehungsweise parakrinen Wirkungsmechanismus dieses Peptides bei BON-Zellen. Das IGF-I-System stellt damit einen interessanten Ansatz für die Entwicklung neuer therapeutischer Strategien zur Behandlung neuroendokriner Tumoren des gastroenteropankreatischen Systems dar. Um das IGF-I-vermittelte Wachstum also zu unterbinden erscheinen mehrere Ansatzpunkte möglich: (1.) Auf der Ebene des Rezeptors durch Antagonisierung des IGF-I-Rezeptors, (2.) auf Cytoplasmaebene durch Unterbrechung der Signaltransduktionswege beziehungsweise Antagonisierung des autokrin sezernierten IGF-I oder (3.) auf Genebene durch Störung der IGF-I-Expression selbst

Cosecretion of amylin and insulin from isolated rat pancreas

AbstractAmylin, a 37 amino acid C-terminal amidated peptide is an integral part of secretory granules of pancreatic β-cells. Utilizing a specific radioimmunoassay system we demonstrate in the present study a cosecretion of amylin and insulin from the isolated rat pancreas. The secretion pattern of both peptides during glucose or glucose plus arginine stimulation is identical. The molar ratio of amylin amounts to 10% of that of insulin. The biological significance of amylin is still unknown, but a paracrine/endocrine role in glucose homeostasis is speculated

Analysis of airway secretions in a model of sulfur dioxide induced chronic obstructive pulmonary disease (COPD)

Hypersecretion and chronic phlegm are major symptoms of chronic obstructive pulmonary disease (COPD) but animal models of COPD with a defined functional hypersecretion have not been established so far. To identify an animal model of combined morphological signs of airway inflammation and functional hypersecretion, rats were continuously exposed to different levels of sulfur dioxide (SO2, 5 ppm, 10 ppm, 20 ppm, 40 ppm, 80 ppm) for 3 (short-term) or 20–25 (long-term) days. Histology revealed a dose-dependent increase in edema formation and inflammatory cell infiltration in short-term-exposed animals. The submucosal edema was replaced by fibrosis after long-term-exposure. The basal secretory activity was only significantly increased in the 20 ppm group. Also, stimulated secretion was significantly increased only after exposure to 20 ppm. BrdU-assays and AgNOR-analysis demonstrated cellular metaplasia and glandular hypertrophy rather than hyperplasia as the underlying morphological correlate of the hypersecretion. In summary, SO2-exposure can lead to characteristic airway remodeling and changes in mucus secretion in rats. As only long-term exposure to 20 ppm leads to a combination of hypersecretion and airway inflammation, only this mode of exposure should be used to mimic human COPD. Concentrations less or higher than 20 ppm or short term exposure do not induce the respiratory symptom of hypersecretion. The present model may be used to characterize the effects of new compounds on mucus secretion in the background of experimental COPD

Impaired Hyperglycemia-Induced Delay in Gastric Emptying in Patients With Type 1 Diabetes Deficient for Islet Amyloid Polypeptide

OBJECTIVE—Slowing of gastric emptying by hyperglycemia, a physiological response to minimize postprandial hyperglycemia, may be impaired in patients with type 1 diabetes. The causes and consequences on glucose homeostasis are unknown

Priming effect of glucagon-like peptide-1 (7-36) amide, glucose-dependent insulinotropic polypeptide and cholecystokinin-8 at the isolated perfused rat pancreas

The priming effect of glucagon-like peptide-1 (7-36) amide (GLP-1 (7-36) amide), glucose-dependent insulin-releasing polypeptide (GIP) and cholecystokinin-8 (CCK-8) on glucose-induced insulin secretion from rat pancreas was investigated. The isolated pancreas was perfused in vitro with Krebs-Ringer bicarbonate buffer containing 2.8 mmol/l glucose. After 10 min this medium was supplemented with GLP-1 (7-36) amide, GIP or CCK-8 (10, 100, 1000 pmol/l) for 10 min. After an additional 10 min period with 2.8 mmol/l glucose alone, insulin secretion was stimulated with buffer containing 10 mmol/l glucose for 44 min. In control experiments the typical biphasic insulin response to 10 mmol/l glucose occurred. Pretreatment of the pancreas with GIP augmented insulin secretion: 10 pmol/l GIP enhanced only the first phase of the secretory response to 10 mmol/l glucose; 100 and 1000 pmol/l GIP stimulated both phases of hormone secretion. After exposure to CCK-8, enhanced insulin release during the first (at 10 and 1000 pmol/l CCK-8) and the second phase (at 1000 pmol/l) was observed. Priming with 100 pmol/l GLP-1 (7-36) amide significantly amplified the first and 1000 pmol/l GLP-1 (7-36) amide both secretion periods. 10 pmol/l GLP-1 (7-36) amide had no significant effect. All three peptide hormones influenced the first, quickly arising secretory response more than the second phase. Priming with forskolin (30 mM) enhanced the secretory response to 10 mM glucose plus 0.5 nM GLP-1 (7-36) amide 4-fold. With a glucose-responsive B-cell line (HIT cells), we investigated the hypothesis that the priming effect of GLP-1 (7-36) amide is mediated by the adenylate cyclase system. Priming with either IBMX (0.1 mM) or forskolin (2.5 [mu]M) enhanced the insulin release after a consecutive glucose stimulation (5 mM). This effect was pronounced when GLP-1 (7-36) amide (100 pM) was added during glucose stimulation. Priming capacities of intestinal peptide hormones may be involved in the regulation of postprandial insulin release. The incretin action of these hormones can probably, at least in part, be explained by these effects. The priming effect of GLP-1 (7-36) amide is most likely mediated by the adenylate cyclase system.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29450/1/0000532.pd

Second extracellular loop of human glucagon-like peptide-1 receptor (GLP-1R) differentially regulates orthosteric but not allosteric agonist binding and function

The glucagon-like peptide-1 receptor (GLP-1R) is a prototypical family B G protein-coupled receptor that exhibits physiologically important pleiotropic coupling and ligand-dependent signal bias. In our accompanying article (Koole, C., Wootten, D., Simms, J., Miller, L. J., Christopoulos, A., and Sexton, P. M. (2012) J. Biol. Chem. 287, 3642-3658), we demonstrate, through alanine-scanning mutagenesis, a key role for extracellular loop (ECL) 2 of the receptor in propagating activation transition mediated by GLP-1 peptides that occurs in a peptide- and pathway-dependent manner for cAMP formation, intracellular (Ca 2+ i) mobilization, and phosphorylation of extracellular signal-regulated kinases 1 and 2 (pERK1/2). In this study, we examine the effect of ECL2 mutations on the binding and signaling of the peptide mimetics, exendin-4 and oxyntomodulin, as well as small molecule allosteric agonist 6,7-dichloro-2-methylsulfonyl-3-tert-butylaminoquinoxaline (compound 2). Lys-288, Cys-296, Trp-297, and Asn-300 were globally important for peptide signaling and also had critical roles in governing signal bias of the receptor. Peptide-specific effects on relative efficacy and signal bias were most commonly observed for residues 301-305, although R299A mutation also caused significantly different effects for individual peptides. Met-303 was more important for exendin-4 and oxyntomodulin action than those of GLP-1 peptides. Globally, ECL2 mutation was more detrimental to exendin-4-mediated Ca 2+ i release than GLP-1(7-36)-NH 2, providing additional evidence for subtle differences in receptor activation by these two peptides. Unlike peptide activation of the GLP-1R, ECL2 mutations had only limited impact on compound 2 mediated cAMP and pERK responses, consistent with this ligand having a distinct mechanism for receptor activation. These data suggest a critical role of ECL2 of the GLP-1R in the activation transition of the receptor by peptide agonists

Postnatal Development of Numbers and Mean Sizes of Pancreatic Islets and Beta-Cells in Healthy Mice and GIPRdn Transgenic Diabetic Mice

The aim of this study was to examine postnatal islet and beta-cell expansion in healthy female control mice and its disturbances in diabetic GIPRdn transgenic mice, which exhibit an early reduction of beta-cell mass. Pancreata of female control and GIPRdn transgenic mice, aged 10, 45, 90 and 180 days were examined, using state-of-the-art quantitative-stereological methods. Total islet and beta-cell volumes, as well as their absolute numbers increased significantly until 90 days in control mice, and remained stable thereafter. The mean islet volumes of controls also increased slightly but significantly between 10 and 45 days of age, and then remained stable until 180 days. The total volume of isolated beta-cells, an indicator of islet neogenesis, and the number of proliferating (BrdU-positive) islet cells were highest in 10-day-old controls and declined significantly between 10 and 45 days. In GIPRdn transgenic mice, the numbers of islets and beta-cells were significantly reduced from 10 days of age onwards vs. controls, and no postnatal expansion of total islet and beta-cell volumes occurred due to a reduction in islet neogenesis whereas early islet-cell proliferation and apoptosis were unchanged as compared to control mice. Insulin secretion in response to pharmacological doses of GIP was preserved in GIPRdn transgenic mice, and serum insulin to pancreatic insulin content in response to GLP-1 and arginine was significantly higher in GIPRdn transgenic mice vs. controls. We could show that the increase in islet number is mainly responsible for expansion of islet and beta-cell mass in healthy control mice. GIPRdn transgenic mice show a disturbed expansion of the endocrine pancreas, due to perturbed islet neogenesis