Alterations of insulin secretion from mouse islets treated with sulphonylureas: perturbations of Ca2+ regulation prevail over changes in insulin content

Abstract

1. To determine how pretreatment with sulphonylureas alters the beta cell function, mouse islets were cultured (18 - 20 h) without (controls) or with (test) 0.01 microM glibenclamide. Acute responses to glucose were then determined in the absence of glibenclamide. 2. Test islets were insensitive to drugs (sulphonylureas and diazoxide) acting on K+-ATP channels, and their [Ca2+]i was already elevated in the absence of stimulation. 3. Insulin secretion was increased in the absence of glucose, and mainly stimulated between 0 - 10 instead of 7 - 20 mM glucose in controls. The maximum response was halved, but this difference disappeared after correction for the 45% decrease in the islet insulin content. 4. The first phase of glucose-induced insulin secretion was abrogated because of a paradoxical decrease of the high basal [Ca2+]i in beta cells. The second phase was preserved but occurred with little rise of [Ca2+]i. These abnormalities did not result from alterations of glucose metabolism (NADPH fluorescence). 5. In islets cultured with 50 microM tolbutamide, glucose induced biphasic increases in [Ca2+]i and insulin secretion. The decrease in the secretory response was matched by the decrease in insulin content (45%) except at maximal glucose concentrations. Islets pretreated with tolbutamide, however, behaved like those cultured with glibenclamide if tolbutamide was also present during the acute functional tests. 6. In conclusion, treatment with a low glibenclamide concentration causes long-lasting blockade of K+-ATP channels and rise of [Ca2+]i in beta cells. Glucose-induced insulin secretion occurs at lower concentrations, is delayed and is largely mediated by a modulation of Ca2+ action on exocytosis. It is suggested that glucose regulation of insulin secretion mainly depends on a K+-ATP channel-independent pathway during in vivo sulphonylurea treatment.

Figure 1

Influence of glibenclamide pretreatment of mouse islets on the concentration-dependence of glucose-induced insulin secretion. The islets were cultured for 18–20 h in a control medium (Ctrl) or in a medium containing 0.01 μM glibenclamide (Glib). After preincubation without glibenclamide (Ctrl) or with glibenclamide (Glib), batches of three islets were incubated in the presence of different glucose concentrations without glibenclamide. Results are presented as absolute values (A) or as a percentage of the islet insulin content (B), which is shown in the inset. Values are means±s.e.mean for 25 batches of islets from five separate experiments.

Figure 2

Influence of glibenclamide pretreatment of mouse islets on the changes in cytoplasmic [Ca²⁺]_i produced by tolbutamide and diazoxide. The islets were cultured for 18–20 h in a control medium (Ctrl) or in a medium containing 0.01 μM glibenclamide (Glib). After preincubation without or with glibenclamide, the islets were then perifused with a medium that did not contain glibenclamide. Tolbutamide (A) and diazoxide (B) were added to a medium containing 10 and 15 mM glucose respectively. The traces correspond to means±s.e.mean for 11–12 islets from three separate experiments.

Figure 3

Influence of glibenclamide pretreatment of mouse islets on the kinetics of glucose-induced insulin secretion. The islets were cultured for 18–20 h in a control medium (Ctrl) or in a medium containing 0.01 μM glibenclamide (Glib). After preincubation without or with glibenclamide, batches of 25 islets were then perifused with a medium that did not contain glibenclamide. The glucose concentration was increased from 3 to 15 mM as indicated. Results are presented as absolute rates of secretion (A) or as a percentage of the islet insulin content (B), which is shown in the inset. Values are means±s.e.mean for five separate experiments.

Figure 4

Influence of glibenclamide pretreatment of mouse islets on the changes in cytoplasmic [Ca²⁺]_i (A) and NAD(P)H fluorescence (B) produced by glucose. The islets were cultured for 18–20 h in a control medium or in a medium containing 0.01 μM glibenclamide. After preincubation without or with glibenclamide, the islets were then perifused with a medium that did not contain glibenclamide. The glucose concentration was increased from 3 to 15 mM as indicated. The traces correspond to means±s.e.mean for 18–19 islets from four separate experiments. In (B) the changes in NAD(P)H fluorescence are expressed as a percentage of the signal recorded in the presence of 3 mM glucose. This basal fluorescence was similar in the two groups of islets.

Figure 5

Influence of tolbutamide pretreatment of mouse islets on the concentration-dependence of glucose-induced insulin secretion. The islets were cultured for 18–20 h in a control medium (Ctrl) or in a medium containing 50 μM tolbutamide (Tolb). After preincubation without or with tolbutamide, batches of three islets were then incubated in the presence of different glucose concentrations without tolbutamide. Results are presented as absolute values (A) or as a percentage of the islet insulin content (B), which is shown in the inset. Values are means±s.e.mean for 20 batches of islets from four different cultures.

Figure 6

Influence of tolbutamide pretreatment of mouse islets on the kinetics of glucose-induced insulin secretion. The islets were cultured for 18–20 h in a control medium (Ctrl) or in a medium containing 50 μM tolbutamide (Tolb). After preincubation without or with tolbutamide, batches of 25 islets were then perifused with a medium that did not contain tolbutamide except for one series of tolbutamide-pretreated islets (C and D) which were perifused with 250 μM tolbutamide (•). The glucose concentration was increased from 3 to 15 mM as indicated. Results are presented as absolute rates of secretion (A and C) or as a percentage of the islet insulin content (B and D), which is shown in the insets. Values are means±s.e.mean for five separate experiments.

Figure 7

Influence of tolbutamide pretreatment of mouse islets on the changes in cytoplasmic [Ca²⁺]_i produced by glucose. The islets were cultured for 18–20 h in a control medium (Ctrl) or in a medium containing 50 μM tolbutamide (Tolb). After preincubation without or with tolbutamide, the islets were then perifused with a medium which contained either no tolbutamide (A) or 250 μM tolbutamide (B), and in which the concentration of glucose was increased from 3 to 15 mM as indicated. The traces correspond to means±s.e.mean for 11–12 islets from three separate experiments.