NADPH oxidase 2-derived reactive oxygen species mediate FFAs-induced dysfunction and apoptosis of β-cells via JNK, p38 MAPK and p53 pathways

Abstract

Dysfunction of β-cell is one of major characteristics in the pathogenesis of type 2 diabetes. The combination of obesity and type 2 diabetes, characterized as 'diabesity', is associated with elevated plasma free fatty acids (FFAs). Oxidative stress has been implicated in the pathogenesis of FFA-induced β-cell dysfunction. However, molecular mechanisms linking between reactive oxygen species (ROS) and FFA-induced β-cell dysfunction and apoptosis are less clear. In the present study, we test the hypothesis that NOX2-derived ROS may play a critical role in dysfunction and apoptosis of β-cells induced by FFA. Our results show that palmitate and oleate (0.5 mmol/L, 48 h) induced JNK activation and AKT inhibition which resulted in decreased phosphorylation of FOXO1 following nuclear localization and the nucleocytoplasmic translocation of PDX-1, leading to the reducing of insulin and ultimately dysfunction of pancreatic NIT-1 cells. We also found that palmitate and oleate stimulated apoptosis of NIT-1 cells through p38MAPK, p53 and NF-κB pathway. More interestingly, our data suggest that suppression of NOX2 may restore FFA-induced dysfunction and apoptosis of NIT-1 cells. Our findings provide a new insight of the NOX2 as a potential new therapeutic target for preservation of β-cell mass and function.

Figure 1. FFA-induced increased ROS generation in NIT-1 cells.

ROS levels were dose- and time-dependently increased by exposure of NIT-1 cells to palmitate and oleate (A, B). The effects of different inhibitors of ROS-generating systems: DPI (NOX, 2.5 µmol/L), L-NAME (nitric oxide synthases, 50 µmol/L), Rotenone (mitochondrial respiratory chain, 1 µmol/L) and Oxypurinol (xanthine oxidase, 50 µmol/L) on palmitate- and oleate-induced ROS generation were analyzed (C). NIT-1 cells were transiently transfected with siRNA-NOX2 for 48 h followed by palmitate and oleate (0.5 mmol/L) treatment for 48 h, and ROS generation was measured (D). Data are present as mean ± S.E.M., n = 3 independent experiments. *p<0.05 and **p<0.01 by ANOVA test (Palmitate/Oleate v.s. control). †p<0.05 and ††p<0.01 by ANOVA test (siNOX2+Palmitate/Oleate v.s. Palmitate/Oleate).

Figure 2. FFA- induced dysfunction and apoptosis of NIT-1 cells.

Palmitate and oleate (0.5 mmol/L, 48 h) decreased glucose-stimulated insulin secretion (GSIS, 20 mmol/L D-glucose in KRBH) (A), reduced insulin mRNA level shown by real-time PCR (B), induced apoptosis assessed by double-staining with Annexin V and PI (C) and increased lipid accumulation detected by Oil Red O Staining in NIT-1 cells (D). Data are present as mean ± S.E.M., n = 3 independent experiments. *p<0.05 and **p<0.01 by ANOVA test (Palmitate/Oleate v.s. control).

Figure 3. The signal transduction pathways involved in FFA-induced dysfunction of NIT-1 cells.

Palmitate and oleate (0.5 mmol/L, 48 h) enhanced the phosphorylation of PTEN and JNK, and reduced the phosphorylation of Akt (A). FOXO1 content was impaired in cytoplasm but elevated in nucleus, while level of PDX-1 was increased in cytoplasm but decreased in nucleus (B). Data are present as mean ± S.E.M., n = 3 independent experiments. *p<0.05 and **p<0.01 by ANOVA test (Palmitate/Oleate v.s. control).

Figure 4. Molecular mechanisms involved in apoptosis of β-cells induced by FFA.

Palmitate and oleate (0.5 mmol/L, 48 h) stimulated the phosphorylation of p53 and p38MAPK (A). Palmitate and oleate induced degradation of I-κB, phosphorylation of I-κB in ³²Ser (B), translocation of Bax into mitochondrial (B), release and translocation of cytochrome C (B, C). Data are present as mean ± S.E.M., n = 3 independent experiments. *p<0.05 and **p<0.01 by ANOVA test (Palmitate/Oleate v.s. control).

Figure 5. Suppression of NOX2 restores FFA-induced dysfunction and apoptosis of NIT-cells.

NIT-1 cells were transiently transfected with siRNA-NOX2 for 48 h followed by palmitate and oleate (0.5 mmol/L) treatment for 48 h. The release of insulin (A), expression of insulin and activation of PTEN-JNK pathway (B), p38MAPK and p53 pathways (C) were measured. Data are present as mean ± S.E.M., n = 3 independent experiments. *p<0.05 and **p<0.01 by ANOVA test (Palmitate/Oleate v.s. control). †p<0.05 and ††p<0.01 by ANOVA test (siNOX2+Palmitate/Oleate v.s. Palmitate/Oleate).