EFFECTIVENESS OF NATEGLINIDE ON IN WTRO 75 NATEGLINIDE REPAGLINIDE \\" II GLYBURIDE GLIMEPIRIDE FIGURE 1 Chemical structure of test antidiabetic drugs. experimental condition. Tubes were incubated at 37C with intermittent hand shaking for 1hour. At the end of incubation, islet media (500 1/ tube) were transferred to 96 deep-well plates and stored at -20C for subsequent insulin analysis. Insulin Scintillation Proximity Assay (SPA) The incubation media were diluted by factors ranging from lx to 20x depending on the concen-trations of glucose/drugs. The diluted super-natant was assayed for insulin content with SPA. [15] The assay employed commercially available products including a guinea pig antirat insulin specific antibody (Linco Research Inc) and scin-tillation proximity Type I reagent coupled to protein A (Amersham Life Science), and was performed as a single step assay. All samples were assayed in duplicate. The preparation of 96 well sample plates was made by sequentially adding standard/test samples, anti-insulin serum, [125I]-insulin tracer, and SPA reagent, and the final volume equaled 175 1/ well. The plates were incubated and vortexed on a titer plate shaker for approximately 16 hours at room temperature. The plates were then placed into a Wallac Microbeta 1450 Liquid Scintillation Counter to be read under a normalization protocol. The output was in counts per minute (CPM). Data Analysis The sample insulin concentration was calculat- ed by utilizing a template set up in Excel spreadsheet that possesses statistical analysis function. The calculated concentration was adjusted eventually to reflect the degree of dilution. The intra- and inter-assay coeffi’cients of variation were generally between 5% and 8%. To allow comparison of the hypoglycemic drug- induced insulin secretion from overnight SU- treated and untreated islets, data were expressed as percent of the appropriate con-trois. That is to say, insulin secretion stimulat-ed by secreta- gogues in SU-treated and untreated groups was normalized, respectively, with the amount of insulin without secreta-gogues in SU-treated and untreated groups. This transformation cor- rected for the increase of control values elicited by overnight treat-ment of SUs. All values were expressed as the mean _SEM. Statistical signifi- cance was determined with t-test (single-tailed). P < 0.05 was considered significantly different. RESULTS Drug-induced Insulin Secretion from Islets with Overnight Treatment of 100nM GLY It was assumed that prolonged treatment with SUs would impair islet responsiveness to physiological and pharmacological stimuli. The effectiveness of four hypoglycemic agents, NAT, GLY, GLI, and REP, to stimulate insulin release from islets cultured overnight (18 hours) in the presence of 100nM GLY was evaluated and was compared to that from overnight cultured islets without GLY to determine the magnitude of desensitization induced by GLY. In the acute experiments following overnight incubation, the concentrations of test drugs were so chosen that they were comparably effective in stimulating insulin release at glucose concentration of 8mM, i.e., NAT at 5M, GLY at 100nM, GLI at 100nM, and REP at 50nM. These hypoglycemic agents INTERNATIONAL JOURNAL OF EXPERIMENTAL DIABETES RESEARCH