Effects of short-term culturing on islet phosphoinositide and insulin secretory responses to glucose and carbachol.

Abstract

The ability of glucose and carbachol, alone or in combination, to stimulate islet cell phosphoinositide (PI) hydrolysis and insulin secretory responses in freshly isolated or in 20-24 h cultured rat islets was assessed. In freshly isolated, 3H-inositol-prelabeled islets, 20 mM glucose alone or 1 mM carbachol alone stimulated significant increments in 3H-inositol efflux and inositol phosphate (IP) accumulation. When stimulated with both agonists, a dramatic and synergistic effect on IP accumulation was noted. Carbachol (1 mM) alone had no sustained stimulatory effect on insulin secretion. Glucose (20 mM) alone induced a biphasic insulin secretory response. When compared to prestimulatory secretory rates of 18 +/- 4 pg/islet/min, peak first and second phase responses now averaged 422 +/- 61 and 1016 +/- 156 pg/islet/min, respectively. In contrast to freshly studied islets, culturing islets for 20-24 h in CMRL-1066 medium attenuated all measured responses. The increases in 3H-inositol efflux rates in response to glucose, carbachol, or their combination were significantly less than those observed with fresh islets. The IP responses were also attenuated. Second phase insulin secretory responses to 20 mM glucose alone 68 +/- 9 pg/islet/min) or the combination of 20 mM glucose plus 1 mM carbachol (358 +/- 85 pg/islet/min) were also significantly decreased when compared with fresh islets. We conclude from these studies that the process of culturing islets for one day in CMRL-1066 significantly decreases islet cell PI hydrolysis and insulin secretory responsiveness. These observations may help to explain the discordant conclusions reached concerning the involvement of PI hydrolysis and protein kinase C activation in the regulation of insulin release from freshly isolated versus cultured islets.