Insulin exocytosis in Goto-Kakizaki rat β-cells subjected to long-term glinide or sulfonylurea treatment

Sulfonylurea and glinide drugs display different effects on insulin granule motion in single β-cells in vitro. We therefore investigated the different effects that these drugs manifest towards insulin release in an in vivo long-term treatment model. Diabetic GK (Goto-Kakizaki) rats were treated with nateglinide, glibenclamide or insulin for 6 weeks. Insulin granule motion in single β-cells and the expression of SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) proteins were then analysed. Perifusion studies showed that decreased first-phase insulin release was partially recovered when GK rats were treated with nateglinide or insulin for 6 weeks, whereas no first-phase release occurred with glibenclamide treatment. In accord with the perifusion results, TIRF (total internal reflection fluorescence) imaging of insulin exocytosis showed restoration of the decreased number of docked insulin granules and the fusion events from them during first-phase release for nateglinide or insulin, but not glibenclamide, treatment; electron microscopy results confirmed the TIRF microscopy data. Relative to vehicle-treated GK β-cells, an increased number of SNARE clusters were evident in nateglinide- or insulin-treated cells; a lesser increase was observed in glibenclamide-treated cells. Immunostaining for insulin showed that nateglinide treatment better preserved pancreatic islet morphology than did glibenclamide treatment. However, direct exposure of GK β-cells to these drugs could not restore the decreased first-phase insulin release nor the reduced numbers of docked insulin granules. We conclude that treatment of GK rats with nateglinide and glibenclamide varies in long-term effects on β-cell functions; nateglinide treatment appears overall to be more beneficial.

Diazoxide Attenuates Glucose-Induced Defects in First-Phase Insulin Release and Pulsatile Insulin Secretion in Human Islets

Humans with type-2 diabetes mellitus (TTDM) have hyperglycemia (∼11 mm) and impaired glucose-mediated insulin secretion characterized by impaired first-phase insulin release (FPIR) and pulsatile insulin release. Culture of islets from nondiabetic humans in very high glucose concentrations (∼20–30 mm) for 96 h causes impaired FPIR. We sought to determine 1) whether human islets cultured at a glucose concentration of approximately 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) whether these changes can be attenuated by addition of diazoxide to islets cultured with 11 mm glucose. Islets cultured with 11 mm glucose for 96 h had 75% depleted insulin stores (P < 0.05), decreased FPIR and insulin pulse mass (P < 0.05), and an approximately 3-fold increase in the ratio of PI/I islet content and in secretion ratio (P < 0.05). Addition of diazoxide to islets cultured with 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 approximately 70% (P < 0.05) by prior addition of diazoxide to islets during culture with 11 mm glucose. 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 present in TTDM (impaired FPIR and pulsatile insulin secretion, increased PI/I ratio) can be recapitulated in human islets cultured with 11 mm glucose 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 β-cell mass.