74 S. HU et al. ]B-cell refractoriness after prolonged exposure to SUs remain at present unclear. Nateglinide (NAT), a novel D-phenylalanine derivative, shares the mechanism of action with SUs to act on KATP channels in pancreatic ]B-cells. This drug, compared to other marketed KATP channel-blocking hypoglycemic agents, has demonstrated unique characteristics including a rapid onset, short duration of action, sensitivity to ambient glucose, and resistance to metabolic inhi- bition, suggesting some aspects of the signaling pathway(s) mediating NAT’s action are novel and distinct from those mediating the effects of SUs and REP. This study was designed to evaluate the ability of NAT and the com-parators, glyburide (GLY), glimepiride (GLI), and repaglinide (REP) to stimulate insulin secretion from isolated rat islets undergoing prolonged pre-exposure to SUs, GLY or tolbutamide (TOL). Using static incuba- tion method, normal rat islets were incubated overnight (---18hours) in the absence and pre- sence of therapeutically relevant concentrations of GLY or TOL before their acute responsiveness to NAT, GLY, REP, and GLI was determined. Our results showed that lasting treatment of GLY or TOL markedly attenuated the islets’ acute response to GLY, GLI and REP, while NAT retained ability to stimulate insulin secretion from SU-desensitized islets. These findings are sugges- tive of common signaling pathways for the action of SUs and REP. On the other hand, the effective- ness of NAT in SU-desensitized islets could imply the involvement of an action site and/or a recep- tor/ effector pathway different from those of SUs and REP. The unique "desensitization-resistant" properties of NAT may explain some of the phar- macological differences between those of NAT and those of SUs and REP. [12,13] MATERIALS AND METHODS Islet Isolation Pancreas were dissected from normal fed male Sprague Dawley rats (250-275g), which were INTERNATIONAL JOURNAL OF EXPERIMENTAL DIABETES RESEARCH euthanized with Na pentobarbital i.p. at 120mg/kg. Islets of Langerhans were isolated by librase digestion (0.5mg/ml, Boehringer Mannheim, Germany) followed by a Ficoll gradient centrifugation. [14] Static Incubation of Islets Desensitized to SUs Freshly isolated islets were handpicked under a stereomicroscope by gentle suction through a large firepolished pipette (--4001xm diameter) into 60 15 mm Petri dishes (Corning) contain- ing 25ml of DMEM (Dulbecco’s Modified Eagle Medium, Gibco BRL) supplemented with 10% fetal calf serum, 5mM glucose (G5- DMEM) and 1% BSA (BSA was present in all incubation media throughout the exper- iments). Islets were preincubated in a humidified atmosphere of 95% 02 and 5% CO2 at 37C for I hour. The islets, after 1 hour preincubation, were selected in batches of two and placed into borosilicate glass tubes (1275mm) contain- ing 500txl G8-DMEM in the presence or absence of 100nM or 10txM GLY or 100txM TOL, and incubated over night (--18 hours) in a humidified atmosphere of 95% 02/5% CO2 at 37C. Acute experiments were performed the following morning to determine the ability of islets to respond to NAT and other insulinotropic agents. 500txl 2x treatment of the concentrations of test hypoglycemic drugs was added to the tubes (to form 1 ml final volume) containing either GLY/TOL-treated or GLY/TOL-untreated islets. The concentrations of test hypoglycemic drugs were so chosen that they were comparably effective in control, i.e., 5txM NAT, 100nM GLY, 100nM GLI and 50nM REP. All test drugs were first dissolved in DMSO to form a stock solution of 10 or 30mM and stored at 4C. The stock solutions were further diluted with incubation media to the desired concentrations before each experiment. Eight independent samples were run for each