76 S. HU et al. stimulated insulin secretion during 1 hour static incubation from islets cultured overnight without 100nM GLY by a respective 270 44%, 292 42%, 301 26% and 430 +_ 35%. In parallel experiments on islets cultured overnight with 100nM GLY, the stimulation factors were, respectively, 354 + 19%, 302 + 13%, 340 + 12%, and 352 + 7%. These data are illustrated in Figure 2, in which the stimulation factors of all test agents were not statistically different between islet groups without and with pretreatment of 100nM GLY. Thus, prolonged treatment with glyburide at 100nM did not appear to render the islets insensitive to subsequent application of insulin secretagogues. Drug-induced Insulin Secretion from Islets with Overnight Treatment of 10M GLY When the concentration of GLY for overnight treatment was raised by 100-fold to 10zM, the pattern of the acute response of islets to insulin secretagogues has changed and the results are shown in Figure 3. The ability of NAT to stimulate insulin secretion did not significantly differ between the GLY untreated control (126 20%) and the GLY pretreated groups (155_ 14%). Conversely, the acute stimulation factors by GLY, REP and GLI in the groups without or []- 100 nM GLY overnight 100 nM GLY overnight 500 400 300 200 100 _T_ Control uM NAT 100 nM GLY 50 nM REP 100 nM GLI FIGURE 2 Insulin release during I hour static incubation in response to acute challenges by antidiabetic agents from rat islets following overnight treatment without (empty bars) and with (filled bars) 100nM GLY at G8. Data are expressed as % of appropriate controls GLY, n 16) *p < 0.05 when compare between data with and without pretreatment. INTERNATIONAL JOURNAL OF EXPERIMENTAL DIABETES RESEARCH 300 [] 10 uM GLY overnight 10 uM GLY overnight Control uM NAT 100 nM GLY 50 nM REP 100 nM GLI FIGURE 3 Insulin release during hour static incubation in response to acute challenge by antidiabetic agents from rat islets following overnight treatment without (empty bars) and with (filled bars) 10M GLY at G8. Data were pooled from 16 independent samples/condition and expressed as % of appropriate controls (+GLY). *p < 0.05 when compare between data with and without pretreatment. Basal insulin secretion without and with GLY pretreatment was, respectively, 265 25 and 413 62 U/islet/lh. with 10 M GLY pretreatment were, respectively, 173 + 21% or 112 + 15% (NS) with GLY, 191 18% or 112 + 11% (p < 0.05) with REP, 251 33% or 138 + 17% (p<0.05) with GLI. Thus, the ability of GLY, REP and GLI to stimulate insulin secretion was attenuated following lasting treatment of islets with 10 pM GLY compared to that without pretreatment. The data demonstrat- ed that 10M GLY induced islet desensitization leading to hypo-responsiveness of islets to the subsequent application of GLY, REP and GLI. NAT was the only agent that was able to maintain its insulinotropic efficacy intact in desensitized islets. Drug-induced Insulin Secretion from Islets with Overnight Treatment of 100mM TOL To further confirm SU induction of islet desensitization and the effectiveness of NAT, tolbutamide (TOL), a classic first generation SU, was also used as a desensitizing agent for the assessment of the ability of four hypoglycemic drugs on secretory response of islets. The stimulation factors of the test drugs during 1 hour static incubation following overnight