The Effect And Mechanism Study Of Sodium Arsenite On Pancreatic Beta-Cells Function

IntroductionArsenic is a naturally occurring element that is ubiquitously present in the environment in both organic and inorganic forms. Human exposure to the generally more toxic inorganic arsenic (iAs) occurs in environmental or occupational settings, as well as through medicinal arsenical use. The main source of human environmental exposure is through consumption of water containing elevated levels of arsenic, primarily from natural contamination. It is estimated that 200 million people are being under the threat of high arsenic in drinking water in the world. Chronic exposure to high levels of iAs is associated with a wide range of human ailments including cancer, arteriosclerosis, hypertension and T2D.Type 2 diabetes (T2D) has become a serious public health problem throughout the world. It has been estimated that approximately 150 million people worldwide had T2D in the year 2000, with the prediction that this number could double by 2025. Many factors, including genetic elements and lifestyle are involved in the incidence of diabetes. However, a link between environmental exposures and diabetes has also been established but has received little attention by the medical community.Although the evidence for a causal association between iAs exposure and T2D is not unequivocally established, epidemiologic studies carried out in Taiwan, Bangladesh, Sweden, and Mexico have shown a strong diabetogenic effect of arsenic in humans. More recently, Meliker et al. report a modest but significant association between iAs exposure and T2D in residents of Michigan with average iAs level in drinking water of 0.011 ppm. In addition, a cross-sectional study carried out in 788 adults reveals a strong positive association between low-level arsenic exposure and the prevalence of T2D in the USA. These new epidemiological studies provided additional support for the importance of arsenic exposure in the development of T2D.A key driver in the pathogenesis of T2D is the impairment of pancreaticβ-cell function, with the hallmark ofβ-cell function being glucose-stimulated insulin secretion (GSIS). According to the currently accepted hypothesis, the control of GSIS in beta-cells depends largely on glucose metabolism in which glycolytic and oxidative phosphorylation triggers a sequence of signaling events, including increased ATP production and ATP/ADP ratio, leading to insulin secretion. Emerging evidence, including our own suggests that, in addition to ATP and ATP/ADP ratio, reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), derived from glucose metabolism, serve as one of the metabolic signals for GSIS. Thus, endogenous antioxidant enzymes that can be robustly induced in response to oxidative stress have the potential to blunt such a glucose-triggered ROS signal and inhibit GSIS.Accumulating data, including our previous studies suggest that arsenic exposure is associated with increased oxidative stress. A key cellular component that defends cells against oxidative damage is NF-E2-related factor 2 (Nrf2):a transcription factor that regulates both constitutive and inducible expression of many antioxidant/detoxification enzymes. However, this same Nrf2-driven induction of endogenous antioxidant enzymes, meant to maintain intracellular redox homeostasis and limit oxidative damage, may also have the potential, as a side effect, to diminish ROS that function as intracellular signals.So based on this study on pancreatic (3-cell line INS-1(832/13), firstly, try to study the effect onβ-cell GSIS function, cell viability, insulin content and expression. Secondly, determine the oxidative stress level and endogenous antioxidant level of low level arsenite treated cells, and try to figure out the role of Nrf2 inβ-cell GSIS function regulation. Thirdly, arsenite effect on the consensus GSIS pathway also determined.Materials and Methods1. Cell culture, arsenite treatment and plasmid transfection. INS-1(832/13) cells were cultured in RPMI medium 1640 supplemented with 10% fetal bovine serum (FBS),10 mM glucose,25 mM HEPES,2 mM L-glutamine,50μMβ-mercaptoethanol, 100 U of penicillin/ml, and 100μg of streptomycin/ml. Cultures were maintained at 37℃in a humidified 5% CO2 atmosphere. INS-1 (832/13) cells were treated for 96 h by sodium arsenite (0,0.05,0.1,0.25, 0.5uM), and the medium was changed every 48 h. The Nrf2 plasmid was transfected by Lipofectamine 2000 reagent.2. Measurement of insulin secretion. Levels of secreted insulin were normalized to DNA content. Insulin measurements were determined using RIA kit with rat insulin as the standard.3. Antioxidant Response Element reporter assay. INS-1 (832/13) cells were transducted by Lentivirus including ARE-Luciferase reporter, when the cells were grown to-90% confluency and sub-cultured in medium containing 0.35μg/ml of puromycin. The luciferase activity was measured by Dual-Luciferase Reporter Assay System according to the manufacturer's protocol. The luciferase activity was normalized to cell viability which was determined using a Non-Radioactive Cell-Proliferation Assay Kit.4. Intracellular peroxide determination. The cells were labeled by the probe CM-H2DCFDA, and intracellular peroxide levels were measured by flow cytometry.5. Measurement of intracellular glutathione (GSH). Cells were sonicated in cold PBS immediately after collection followed by centrifugation at 12,000g for 5 min. The resulting supernatants were used for measurement of GSSG and total glutathione. Levels of total glutathione (GSH+GSSG) and GSSG in cells were measured immediately after collection using BIOXYTECH GSH/GSSG-412 kit (OxisResearch, Portland, OR) according to the manufacturer's protocols.6. Measurement of H2O2-scavenging activity. The H2O2 remaining in the cells supernatants was measured using Amplex Red Hydrogen Peroxide Assay Kit. The difference in H2O2 concentrations between lysate-treated and a PBS control represents the H2O2-scavenging activity contributed by cells. Protein concentrations were determined by Bio-Rad protein assay using BSA as a standard.7. Quantitative real-time RT-PCR analysis. Total RNA was isolated with TRIzoland then subjected to cleanup using RNase-Free DNase Set and RNeasy Mini kit. The primers Real-time fluorescence detection was carried out using an ABI PRISM 7900 Sequence Detector.8. Western blot analysis. Isolation of cell fractions and Western blotting was performed for the protein dertermination. Antibodies for Nrf2 (sc-13032; 1:500), glucokinase (GCK, sc-7908; 1:1000), glucose transporter 2 (Glut2, sc-9117; 1:1000), potassium inwardly rectifying channel, subfamily J, member 11 (KCNJ11, also termed KIR6.2, sc-11226; 1:500), and sulfonylurea receptor 1 (SUR1, sc-25683; 1:1000) were from Santa Cruz Biotechnology. Antibodies for Lamin A (L1293; 1:2500) andβ-actin (A1978; 1:2000) were purchased from Sigma.9. Measurement of ATP. Cells were washed three times with ice-cold Kreb's buffer with the same concentrations of glucose as treatments and lysed in ATP releasing buffer followed by centrifugation at 12,000g for 5 min. The resulting supernatants were used immediately for measurement of ATP. ATP levels were measured using an ATP Bioluminescent Assay Kit.10. Measurements of mitochondrial mass. Mitochondrial mass was determined by flow cytometry and confocal microscope using the fluorescent probe MitoTracker green. The final concentration of the probe used was 75 nM and the pre-loading time was 30 min. In the flow cytometry measurements, dead cells and clumps were eliminated based upon Forward Scatter vs. Side Scatter measurement, and untreated cells provided a source of comparison.11. Oxygen consumption rate (OCR). OCR was measured by the XF24 Extracellular Flux Analyzer. The basal OCR and OCR response were determined 3-5 min in 3 mM or 20 mM glucose Kreb's buffer.Results1. Arsenite exposure effects on the GSIS function in pancreatic beta-cells.When exposure of INS-1(832/13) cells to arsenite at high level arsenite (≥1μM), the cells viability were decreased and some cells were dead. Exposure of the cells to arsenite at non-cytotoxic concentrations (≤0.5μM) for 96 hrs resulted in a dose-dependent reduction in insulin secretion in response to glucose stimulation, whereas the KC1, caused a significant increase of insulin release in the cells treated with 0.5μM arsenite. In contrast to the decreased GSIS, elevated basal insulin release (in presence of 3 mM glucose) was observed in the arsenite-treated cells. This result is likely due to the increase in gene expression and protein levels of insulin in the cells.2. Arsenite effects on the Nrf2-mediated antioxidant response and the role of Nrf2 in pancreatic beta-cells GSIS regulation.The accumulation of Nrf2 in nuclear fractions, activation of ARE reporter and significant induction of Nrf2-target genes indicate an activation of Nrf2-mediated adaptive response in the arsenite-exposed cells. The intracellular GSH and intracellular H2O2-scavenging activity were dose-dependently increased by arsenite exposure. The basal (in the presence of 3 mM glucose) intracellular peroxide level was significantly increased by arsenite exposure, but the glucose stimulated peroxide rate was decreased dose-dependantly, which may have contributed to the decreased GSIS function.Consistent with this notion, INS-1(832/13) cells challenged with another Nrf2 activator sulforaphane or overexpressed Nrf2 exhibit a modest, but significant decrease in GSIS.3. Arsenite exposure effects on the consensus GSIS pathway.In the current study, the gene and protein expressions of major glucose transporter Glut2, metabolism enzyme Gck, SUR1 and KIR6.2 showed no significant decrease in the arsenite-exposed cells. ATP production is the primary regulator of KATP, thus the ATP levels under low-and high-glucose conditions were determined in the arsenite-exposed cells. However, no decrease in glucose-stimulated ATP production was observed in the cells. In contrast to the significant reduction in GSIS, arsenite exposure did not decrease, but dose-dependently enhanced mitochondrial mass. The results in this section suggest the impaired GSIS of INS-1(832/13) cell caused by chronic arsenite exposure is not associated with the consensus GSIS pathway.Conclusion1. Exposure of INS-1(832/13) cells to arsenite at non-cytotoxic concentrations for 96 hrs resulted in a dose-dependent reduction in insulin secretion in response to glucose stimulation. In contrast to the decreased GSIS, elevated basal insulin release was observed in the arsenite-treated cells.2. Aresnite exprosure activate Nrf2-mediated antioxidant response, and the induction of endogenous antioxidants in the presence of oxidative stress may blunt this signal resulting in reduced GSIS. Consistent with this notion, INS-1(832/13) cells challenged with another Nrf2 activator sulforaphane or overexpressed Nrf2 exhibit a modest, but significant decrease in GSIS. This is the evidence of the regulation role of Nrf2 in pancreatic beta-cells GSIS function.3. The gene and protein expressions of Glut2, Gck, SUR1 and KIR6.2 showed no significant decrease in the arsenite-exposed cells. However, no decrease in glucose-stimulated ATP production and mitochondrial mass were observed in the cells. The results in this section suggest the impaired GSIS of INS-1(832/13) cell caused by prolonged arsenite exposure is not associated with the consensus GSIS pathway.