EFFECTS OF SHORT-TERM CULTURING ON ISLET PHOSPHOINOSITIDE AND INSULINSECRETORY RESPONSES TO GLUCOSE AND CARBACHOL

The ability of glucose and carbachol, alone or in combination, to stim ulate islet cell phosphoinositide (PI) hydrolysis and insulin secretor y responses in freshly isolated or in 20-24 h cultured rat islets was assessed. In freshly isolated, H-3-inositol-prelabeled islets, 20 mM g lucose alone or 1 mM carbachol alone stimulated significant increments in H-3-inositol efflux and inositol phosphate (IP) accumulation. When stimulated with both agonists, a dramatic and synergistic effect on I P accumulation was noted. Carbachol (1 mM) alone had no sustained stim ulatory effect on insulin secretion. Glucose (20 mM) alone induced a b iphasic insulin secretory response. When compared to prestimulatory se cretory rates of 18+/-4 pg/islet/min, peak first and second phase resp onses of freshly isolated islets to 20 mM glucose averaged 126+/-24 an d 520+/-82 pg/islet/min, respectively. In the presence of both glucose (20 mM) and carbachol (1 mM), peak first and second phase responses n ow averaged 422+/-61 and 1016+/-156 pg/islet/min, respectively. In con trast to freshly studied islets, culturing islets for 20-24 h in CMRL- 1066 medium attenuated all measured responses. The increases in H-3-in ositol efflux rates in response to glucose, carbachol, or their combin ation were significantly less than those observed with fresh islets. T he IP responses were also attenuated. Second phase insulin secretory r esponses to 20 mM glucose alone (68+/-9 pg/islet/min) or the combinati on of 20 mM glucose plus 1 mM carbachol (358+/-85 pg/islet/min) were a lso significantly decreased when compared with fresh islets. We conclu de from these studies that the process of culturing islets for one day in CMRL-1066 significantly decreases islet cell PI hydrolysis and ins ulin secretory responsiveness. These observations may help to explain the discordant conclusions reached concerning the involvement of PI hy drolysis and protein kinase C activation in the regulation of insulin release from freshly isolated versus cultured islets.