In health, most insulin is secreted in pulses. Type 2 diabetes mellitus (TTDM) is characterized by impaired pulsatile insulin secretion with a defect in insulin pulse mass. It has been suggested that this defect is partly due to chronic overstimulation of beta-cells imposed by insulin resistance and hyperglycemia, which results in depletion of pancreatic insulin stores. It has been reported that in TTDM overnight inhibition of insulin secretion (induction of beta-cell rest) leads to quantitative normalization of pulsatile insulin secretion upon subsequent stimulation. Recently, decreased orderliness of insulin secretion has been recognized as another attribute of impaired insulin secretion in TTDM. In the current studies we sought to address at the level of the isolated islet whether chronic elevated glucose concentrations induce both defects involved in impaired insulin secretion in TTDM: deficiency and decreased orderliness of insulin secretion. We use the concept of beta-cell rest, induced by a novel beta-cell selective K(ATP)-channel opener (KCO), NN414 (6-chloro-3-(1-methylcyclopropyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide), to test whether preservation of insulin stores leads to normalization of both processes in response to glucose stimulation. Human islets were isolated from three cadaveric organ donors and studied in perifusion experiments and static incubation. Acute activation of K(ATP)-channels suppressed insulin secretion from perifused human islets by approximately 90% (P < 0.0001). This KCO also inhibited glucagon secretion in a dose-dependent manner (P = 0.01). Static incubation at 11 and 16 vs. 4 mM glucose for 96 h decreased islet insulin stores by approximately 80% and 85% (P < 0.0001, respectively). In subsequent perifusion experiments, total insulin secretion ( approximately 30%; P < 0.01) from these islets and insulin pulse mass ( approximately 40%; P < 0.05) were both decreased (11 vs. 4 mM). The inhibition of insulin secretion during static incubation with KCO reduced the loss of islet insulin stores in a dose-dependent manner (P < 0.0001) and resulted in increased total insulin secretion (2.6-fold; P < 0.01) and insulin pulse mass (2.5-fold; P < 0.05) during subsequent perifusion. The orderliness of insulin secretion was significantly reduced after chronic incubation of human islets at 11 mM glucose (P = 0.04), but induction of beta-cell rest at 11 mM failed to normalize the regularity of insulin secretion during subsequent perifusion. We conclude that physiological increased glucose concentrations (11 mM), which are frequently observed in diabetes, lead to a loss of islet insulin stores and defective pulsatile insulin secretion as well as reduced orderliness of insulin secretion. Induction of beta-cell rest by selective activation of beta-cell K(ATP)-channels preserves insulin stores and pulsatile insulin secretion without restoring the orderliness of insulin secretion. Therefore, the concept of beta-cell rest may provide a strategy to protect beta-cells from chronic overstimulation and to improve islet function. Impaired glucose-regulated insulin secretion in TTDM may, however, partially involve mechanisms that are distinct from insulin stores and insulin secretion rates.