N-acetylcysteine protects pancreatic islet against glucocorticoid toxicity

Figures & data

Table 1. Sequences of the primers used for the real-time RT–PCR gene expression quantification

Gene	GenBank acess	5′-Primer sequence-3′
Beta-actin	NM_031144	Fw: AGAGGGAAATCGTGCGTGAC
		Rv: CGATAGTGATGACCTGACCGT
Catalase	NM_012520	Fw: GATGAAGCAGTGGAAGGAGC
		Rv: TGCCATCTCGTCGGTGAAA
CaV.beta2	NM_053851	Fw: TGCCCCTGAAGAGAGAAA
		Rv: CTGAGGATGAACAAGAGGAG
CuZnSOD	NM_017050	Fw: TGAAGAGAGGCATGTTGGAGA
		Rv: TCATCTTGTTTCTCGTGGACC
Gapdh	NM_017008	Fw: TCACCACCATGGAGAAGGC
		Rv: GCTAAGCAGTTGGTGGTGCA
Glutathione peroxidase	NM_030826	Fw: CCCTCAAGTATGTCCGACCC
		Rv: GCAGGAAGGTAAAGAGCGGG
MnSOD	NM_017051	Fw: CCTCCCTGACCTGCCTTACGACTA
		Rv: TTCAGATTGTTCACGTAGGTCGCG
Synaptotagmin 7	NM_021659	Fw: AGACCAAGGTGAAACGGAAGA
		Rv: CTGAGCTTGTCTTTGTCCATG
Syntaxin 1	NM_053788	Fw: ATGAAGGACCGAACCCAGGAGGC
		Rv: TCTATCCCAAAGATGCCCCCGA
VAMP-2	NM_012663	Fw: GCATCTCTCCTACCCTTTCA
		Rv: TTTAGGGGTCTGAGGGTACA

Fw, forward (sense) primer; Rv, reverse (antisense) primer. All amplicons were in the size ranging from 95 to 170 bp. Gapdh was used as housekeeping gene.

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.