Antioxidant effect of angiotensin (1‑7) in the protection of pancreatic β cell function

Abstract

It is well known that the local renin-angiotensin system (RAS) is activated in the diabetic state, which results in an increase in the level of oxidative stress injury to pancreatic β cells. The angiotensin‑converting enzyme 2 (ACE2)/angiotensin (1‑7) [Ang (1‑7)]/Mas axis is a negative regulator of the classical renin‑angiotensin system. In order to investigate the antioxidant effect of Ang (1‑7) on pancreatic β cells, INS‑1 cells were cultured and oxidative stress was induced by treatment with H2O2. Glucose‑stimulated insulin secretion (GSIS), the generation of reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and glucose-stimulated calcium (GSCa) responses in β cells were determined following treatment with Ang (1-7). It was observed that H2O2 significantly impaired the insulin secreting function, increased the production of ROS, and also decreased the levels of GSCa and MMP. Pre‑treatment with Ang (1‑7) alleviated these effects and treatment with A779 [antagonist of Ang (1‑7)] prevented the effects of Ang (1-7). Based on these findings, it was concluded that Ang (1‑7) can protect pancreatic β cells from oxidative injury and such protection can be blocked by its antagonist A779.

Figure 1

GSIS of INS-1 cells was tested at 3.3 and 16.7 mM glucose. At the higher glucose concentration, the insulin release index of the INS-1 cells was 2.9, demonstrating healthy INS-1 cell function and glucose sensitivity. ^*P<0.05. GSIS, glucose-stimulated insulin secretion; OD, optical density.

Figure 2

Effects of Ang (1-7) (10⁻⁸ mol/l) and A779 (10⁻⁶ mol/l) on the GSIS from INS-1 cells in the presence of H₂O₂. Insulin secretion was markedly reduced (51.8%) by H₂O₂ treatment which was partly reversed by Ang (1-7) (34.1%). ^*P<0.05. Data are presented as the mean ± standard error of the mean (n=3). Ang (1-7), angiotensin (1-7); GSIS, glucose-stimulated insulin secretion; OD, optical density.

Figure 3

Effect of Ang (1-7) and A779 on the generation of ROS stimulated by H₂O₂. (A) Effects of Ang (1-7) (10⁻⁸ mol/l) and A779 (10⁻⁶ mol/l) on the glucose (16.7 mmol/l)-stimulated insulin release from INS-1 cells in the presence of H₂O₂. ROS levels were elevated markedly (46.8%) following H₂O₂ treatment which was partly reversed by Ang (1-7) (27.1%). This effect was blocked by A779. (B) The effect of Ang (1-7) and A779 on the generation of ROS stimulated by H₂O₂ was tested by flow cytometry. ^*P<0.05; ^***P<0.001. Data are presented as the mean ± standard error of the mean (n=3). Ang (1-7), angiotensin (1-7); ROS, reactive oxygen species; DHE, dihydroethidium.

Figure 4

Ang (1-7) restores glucose-stimulated calcium in the presence of H₂O₂. (A) H₂O₂ clearly decreased the calcium fluorescence intensity when compared with the control groups. Pre-treatment with 10⁻⁸ mol/l Ang (1-7) for 2 h prior to adding H₂O₂ upregulates calcium fluorescence significantly, and treatment with A779 (10⁻⁸ mol/l for 2 h) can selectively inhibit this effect. Ang (1-7) restored the amplitude of calcium (first phase of insulin secretion) in the presence of H₂O₂. (B) Graph of calcium fluorescence intensity. Pre-treatment with 10⁻⁸ mol/l Ang (1-7) for 2 h prior to adding H₂O₂ upregulates calcium fluorescence by 25% (P<0.05), and A779 (10⁻⁸ mol/l for 2 h) can selectively inhibit this effect (P<0.05). ^*P<0.05 and ^***P<0.001. Data are presented as the mean ± standard error of the mean (n=3). Ang (1-7), angiotensin (1-7).

Figure 5

Ang (1-7) restored the MMP in the presence of H₂O₂. (A) Flow cytometry using JC-1 in INS-1 cells. (B) JC-1 staining in INS-1 cells. MMP was evaluated by laser confocal microscopy. Ang (1-7) decreased the green fluorescence (column 1) and increased the red fluorescence (column 2) when compared with the Con + H₂O₂ groups. Ang (1-7) restores MMP in the presence of H₂O₂. The staining was quantified and presented as a graph. Ang (1-7) treatment reversed the decreases in the MMP induced by H₂O₂ (15 min at 250 µM H₂O₂), and this effect was blocked by A779. ^*P<0.05 and ^***P<0.001. Data are presented as means ± standard error of the mean (n=3). MMP, mitochondrial membrane potential; Con, control.