Cooper, Garth J.S.Bernard, ChoongStojkovic, Mirjana2007-06-282007-06-281999Thesis (PhD--Biological Sciences)--University of Auckland, 1999http://hdl.handle.net/2292/577Full text is available to authenticated members of The University of Auckland only.It has previously been shown that the amylin gene is strongly expressed in pancreatic islet β-cells (1). In this thesis, a clonal insulinoma βTC-6 F7 cell line (2) was used as a model to study insulin and amylin co-secretion. Polyclonal antibodies were raised against rat amylin and bovine insulin in rabbits and guinea pigs, respectively. Rabbit D # 56 antiserum (Ka = 4.61/nM) was used at a final dilution of 1:40,000, and guinea pig I # 1 antiserum (Ka = 81 I/nM) at a final dilution of 1: 100,000 antiserum, in the development of amylin and insulin radioimmunoassays (RIA), respectively. The amylin and insulin RIAs have provided high degrees of sensitivity, with minimum detectable concentrations of 4.6 ± 1.5 pM and 19.6 ± 2.7 pM, (n = l0), respectively. The rat / mouse amylin assay employed synthetic rat amylin as standards from concentrations of 2.5 to 340 pM. The rat insulin assay employed human insulin as standards from 17.5 to 2,480 pM. Intra- and inter-assay coefficients of variation corresponding to both insulin and amylin RIAs were less then l0% and 15%, respectively. The mean values of intracellular amylin and insulin in βTC-6 F7 cells were 27.1 ± 1.7 ng/100 μg cellular protein and 1,840 ± 59 ng/100 μg, respectively. Experimental results showed that the mean value of the non-stimulated molar amylin / insulin ratio was 0.023 ± 0.04, which was close to that reported for normal islets (3,4,5). The ratio was slightly, but not significantly changed when the βTC-6F7 cells were stimulated with increasing concentrations of D(+)-glucose and L-arginine (ANOVA, p> 0.05). Both peptides responded differently to glucose and L-arginine. Glucose potentiated more insulin and amylin production than did L-arginine (ANOVA. p < 0.05). The glucose concentration-response curves for both peptides were shifted to the left when compared to that reported for normal islet β-cells (6,7). Measured EC50 values were: 0.24 ± 4.2 mM for glucose stimulation of insulin secretion; and 0.36 ± 4.2 mM for amylin secretion. The L-arginine concentration-response curves derived for both peptides resembled those previously reported (8). Measured EC50 values were: 5.7 ± 8.2 mM L-arginine for insulin secretion, and 4.9 ± 10.4 mM L-arginine for amylin secretion. Maximum insulin was secreted at l0 mM of glucose, while amylin secretion reached its maximum at 15 mM glucose. At l5 mM of L-arginine both peptides reached maximum secretion. 4.1 ± 0.7% of total cell insulin and 2.9 ± 0.5% of total cell amylin were released in 1h at37°C and 0 mM of glucose, while at 10 mM glucose, 8.4 ± 0.5% of total cell insulin but only 4.9 ± 0.5% of total cell amylin were released. When the cells were stimulated with l0 mM glucose, increasing concentrations of somatostatin-14(SST-14) and the rat somatostatin receptor-5 and -3 (rSSTR5 / 3) selective ligand, BIM23268 (9), resulted in the proportional decrease of insulin and amylin release (ANOVA, p<0.05). Both insulin and amylin responses obtained for SST-14 were found to be slightly but not significantly different, when compared to BIM23268 (ANOVA, p < 0.05). Both peptides were released in a non-parallel manner, both for increasing concentrations of SST-14. and BIM23268 (multiple regression, p < 0.05 and p > 0.05, respectively). Increasing concentrations of SST-14 consistently suppressed amylin more effectively than insulin, whereas BIM23268 suppressed insulin more effectively (Newman-Keuls post hoc comparison test, p < 0.05). Molecular and pharmacological evidence from this thesis have underscored the expression of multiple SSTR subtypes in βTC- F7 cells. Experimental results from the reverse transcriptase-polymerase chain reaction (RT-PCR) studies confirmed the presence of messenger ribonucleic acid (mRNA) corresponding to both SSTR3 and 5 in these cells. SST-14,BIM23268, and the SSTR3-selective ligand, BIM23056 (9), displaced the binding of [125I-Tyr11]SST-14 in βTC-6 F7 cells. SST-14 was more potent in displacing labelled ligand [125I-Tyr11]SST-14 than was BIM23268, while BIM23056 had the lowest potency at 50% binding. The pattern of endogenously expressed SSTR subtypes on βTC-6 F7 cells detected in this thesis, suggests that both mSSTR3 and 5 could play roles in the suppression of amylin and insulin secretion.enRestricted Item. Available to authenticated members of The University of Auckland.Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated.https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htmThesisCopyright: The authorQ112849960