Humans with type-2 diabetes (TTDM) have hyperglycemia (11 mM) and impaired glucose mediated insulin secretion, characterized by impaired first phase insulin release and pulsatile insulin release. Culture of islets from non-diabetic humans in very high glucose concentrations (20-30 mM) for 96 h causes impaired first phase insulin release (FPIR). We sought to determine (1) if human islets cultured at glucose of 11 mM (comparable to TTDM) recapitulates impaired insulin secretion in TTDM, specifically impaired FPIR and insulin pulse mass with an increased proinsulin/insulin (PI/I) secretion ratio, and (2) if these changes can be attenuated by addition of diazoxide to islets cultured at glucose 11 mM. Islets cultured at glucose 11 mM for 96 h had 75% depleted insulin stores (P < 0.05), decreased FPIR and insulin pulse mass (P < 0.05) and a 3-fold increase in the ratio of PI/I islet content and secretion ratio (P < 0.05). Addition of diazoxide to islets cultured at 11 mM glucose decreased insulin secretion during static incubation leading to relative preservation of insulin stores and enhanced insulin secretion during subsequent perifusion; FPIR increased by 162%, (P < 0.05) and insulin pulse mass by 150%, (P < 0.05) vs. no diazoxide. The mean islet PI/I content and islet PI/I secretion ratio were also decreased by 70%, (P < 0.05) by prior addition of diazoxide to islets during culture at glucose of 11 mM. FPIR and insulin pulse mass were related to islet insulin stores (P < 0.001 for FPIR and P < 0.001 for pulse amplitude). In conclusion, the pattern of defects of insulin secretion in present in TTDM (impaired FPIR and pulsatile insulin secretion, increased PI/I ratio) can be recapitulated in human islets cultured at a glucose of 11 mM for 96 h. These defects can be at least partially offset by concurrent inhibition of insulin secretion by diazoxide which also preserves insulin stores. Defective insulin secretion in TTDM may be at least in part due to depletion of available insulin stores secondary to chronic increased demand (insulin resistance and hyperglycemia) in the setting of a decreased cell mass.