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Abstract
Background
Glucocorticoids play important roles in the regulation of glucose homeostasis. It is well known that glucocorticoids reduce hepatic and peripheral tissue sensitivity to insulin, but the roles of glucocorticoids on insulin secretion and synthesis in pancreatic beta cells are still unclear. We have investigated the direct effects of glucocorticoids on insulin secretion and synthesis in rat insulinoma (INS-1) cells.
Methods
Insulin content and 11.2 mM glucose-stimulated insulin secretion (GSIS) were measured in INS-1 cells after culture with or without 1 µM dexamethasone (DEX). Preproinsulin mRNA levels were analyzed by real-time RT-PCR and normalized to the internal control. Effect of RU486 on DEX-induced inhibition of GSIS and preproinsulin mRNA synthesis was evaluated.
Results
Insulin content of INS-1 cells cultured in RPMI containing 11.2 mM glucose in the presence of DEX was not different from that of control cells. After 1-h preincubation in 2.8 mM glucose, basal insulin secretion from cells treated with DEX did not differ from that of controls, but GSIS was significantly reduced in the cells treated with DEX in comparison to control cells. The expression of preproinsulin mRNA relative to beta-actin mRNA was also lower in the cells treated with DEX. Glucocorticoid receptor antagonist improved DEX-induced inhibition of GSIS and preproinsulin mRNA synthesis.
Figures and Tables
Fig. 1
Effect of dexamethasone (DEX) on intracellular insulin content in INS-1 cells. Insulin content was measured from the lysates of INS-1 cells cultured in RPMI containing 11.1 mM glucose in the absence (white bar) or presence (black bar) of DEX for 1 day and 2 days. Insulin amount was normalized by total protein amount in the cell lysates. Values are means ± SEM.
Fig. 2
Effect of DEX on glucose-stimulated insulin secretion (GSIS) in INS-1 cells. GSIS was evaluated from INS-1 cells in the absence (white bar) or presence (black bar) of DEX for 1 day (A) and 2 days (B). The amount of secreted insulin was normalized by total protein amount in the cell lysates. In the cells cultured with DEX, basal insulin secretion (2.8 mM) did not differ from that of controls, but GSIS (11.2 mM) was significantly decreased in comparison to control cells.
*p < 0.05 versus cells in 2.8 mM glucose.
†p < 0.05 versus cells with vehicle in 11.2 mM glucose.
Fig. 3
Effect of RU486 on DEX-induced inhibition of insulin secretion in INS-1 cells. The cells were cultured with or withiout RU486 in the presence or absence of DEX for 2 days and glucose-stimulated insulin secretion was measured by rat insulin radioimmunoassay. The amount of secreted insulin was normalized by total protein amount in the cell lysates. Values are means ± SEM.
*p < 0.05 versus cells with DEX in 11.2 mM glucose.
Fig. 4
Effect of DEX and RU486 on preproinsulin mRNA in INS-1 cells. After the cells were cultured with or withiout RU486 in the absence (white bar) or presence (black bar) of DEX for 2 days, preincubated in 2.8 mM KRBB-HEPES for 2 hours, and then cultured in KRBB-HEPES containing 2.8 mM and 11.2 mM glucose for 3 hours. The expression levels of preproinsulin mRNA and beta-actin were analyzed by real-time RT-PCR. The preproinsulin mRNA expression was normalized to beta-actin mRNA expression in the corresponding sample. Values are means ± SEM.
*p < 0.05 versus cells with vehicle in 2.8 mM glucose.
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