N-acetylcysteine protects pancreatic islet against glucocorticoid toxicity

Abstract

Objectives: Reactive oxygen species (ROS) are involved in many physiological and pathological processes. In the present study, we analysed whether the synthetic glucocorticoid dexamethasone induces oxidative stress in cultured pancreatic islets and whether the effects of dexamethasone on insulin secretion, gene expression, and viability can be counteracted by concomitant incubation with N-acetylcysteine (NAC).

Methods: ROS production was measured by dichlorofluorescein (DCFH-DA) assay, insulin secretion by radioimmunoassay, intracellular calcium dynamics by fura-2-based fluorescence, gene expression by real-time polymerase chain reaction analyses and cell viability by the MTS assay.

Results: Dexamethasone (Dexa) increased ROS production and decreased glucose-stimulated insulin secretion after 72 hours incubation. Intracellular ROS levels were decreased and the insulin secretion capacity was recovered by concomitant treatment with Dexa+NAC. The total insulin content and intracellular Ca2+ levels were not modulated in either Dexa or Dexa+NAC groups. There was a decrease in the NAD(P)H production, used as an indicator of viability, after dexamethasone treatment. Concomitant incubation with NAC returned viability to control levels. Dexa also decreased synaptotagmin VII (SYT VII) gene expression. In contrast, the Dexa+NAC group demonstrated an increased expression of SYT VII compared to controls. Surprisingly, treatment with NAC decreased the gene expression of the antioxidant enzyme copper zinc superoxide dismutase soluble.

Discussion: Our results indicate that dexamethasone increases ROS production, decreases viability, and impairs insulin secretion in pancreatic rat islets. These effects can be counteracted by NAC, which not only decreases ROS levels but also modulates the expression of genes involved in the secretory pathway and those coding for antioxidant enzymes.

Figure 1.

Effects of Dexa and NAC treatment on ROS generation. Total ROS production was measured by DCFH-DA oxidation. Islets were treated with 1 µmol/l of dexamethasone (Dexa) for 72 hours in the presence or absence of 1 mmol/l of NAC (Dexa + NAC) and assayed in the presence of 25 mmol/l glucose. N = 5–8. *P < 0.05, compared to control; ANOVA followed by Bonferroni.

Figure 2.

(A) Effects of Dexa and NAC treatment on insulin secretion. Islets were treated with 1 µmol/l of dexamethasone (Dexa) for 72 hours in the presence or absence of 1 mmol/l of NAC (Dexa + NAC). After the culture period, groups of five islets were pre-incubated in Krebs-bicarbonate buffer containing 5.6 mmol/l of glucose and then incubated with 2.8 mmol/l of glucose or 25 mmol/l glucose. After 1 hour of incubation, supernatants were collected and insulin was measured by RIA. N = 20–30. *P < 0.05; ANOVA followed by Bonferroni. (B) Effect of Dexa and NAC on total insulin content in cultured rat islets. After 72 hours of culture, groups of five islets were homogenized and the total insulin content was measured by RIA (N = 18).

Figure 3.

Effects of Dexa and NAC treatment on pancreatic islet cell viability. Islets were treated with 1 µmol/l of dexamethasone for 72 hours in the presence or absence of 1 mmol/l of NAC. NAD(P)H production, as an indicator of viability, was accessed using the MTS method. N = 5. *P < 0.05; ANOVA followed by Bonferroni.

Figure 4.

Glucose-induced [Ca⁺²]i changes in pancreatic islets after 72 hours of dexamethasone and Dexa plus NAC treatment. Bars represent mean values of at least five independent experiments.

Figure 5.

Effects of Dexa and NAC treatment on the gene expressions of (A) calcium channel, voltage-dependent, CaB2, (B) SYT VII, (C) syntaxin 1, (D) VAMP-2, (E) Cu/ZnSOD, (F) MnSOD, (G) catalase, and (H) GPx. After reverse transcription, real-time analyses were performed using Gapdh as an internal control. The experiments were performed after 72 hours of culture. N= 6–8 independent experiments. *P < 0.05, ANOVA followed by Bonferroni.