亚砷酸钠对胰岛β细胞功能的影响及其机制研究
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摘要
前言
砷是一种自然环境中广泛存在的类金属元素,以有机胂和无机砷两种形式存在,而无机砷的毒性较有机胂强。人类可以通过环境、职业暴露或者使用含砷药物而接触到无机砷(iAs),而环境砷暴露主要通过饮用自然界中被砷污染的水源。全世界约有2亿人正处于慢性饮水型高砷暴露的威胁中,我国大陆地区饮水型砷中毒病区中的高砷暴露人群已接近300万,随着调查的深入,慢性饮水型高砷暴露人群的数目正在不断扩大。慢性饮水型砷暴露不仅能够引起砷性皮肤损伤,砷性皮肤癌及其它内脏器官肿瘤,还与心血管疾病、高血压和糖尿病等常见疾病的发生关系密切。
2型糖尿病(T2D)已经成为一个全球性的公共卫生问题。截至到2000年全球有大约1.5亿人患有2型糖尿病,预计到2025年将有3亿人会罹患2型糖尿病。遗传和不良生活方式等因素都与糖尿病的发病有关。而环境危险因素暴露与糖尿病发生也存在一定程度的联系,目前还未得到医学界的广泛关注。
环境砷暴露和2型糖尿病的因果关系还不明确,但对台湾、孟加拉、瑞典和墨西哥等砷暴露人群的流行病学调查研究显示环境砷暴露与糖尿病的发生关系密切。最近,Meliker等对美国密歇根州饮用水中无机砷为0.011 ppm的居民进行的研究中发现低砷暴露和2型糖尿病发生存在显著相关性。另外一项对美国788位成年人的横断面调查研究显示低砷暴露与2型糖尿病的流行存在明显的正相关。这些近期的流行病学研究也都说明砷污染是暴露人群的2型糖尿病发生的重要因素之一。
2型糖尿病的发生是胰岛β细胞功能障碍和胰岛素抵抗共同作用的结果。胰岛β细胞具有对葡萄糖刺激反应释放胰岛素的功能,即葡萄糖刺激的胰岛素分泌(GSIS)功能,而胰岛细胞GSIS功能的降低是2型糖尿病发生的原因之一。根据目前的研究,β细胞的GSIS过程受细胞内葡萄糖代谢的直接影响,即从最初的葡萄糖摄入和磷酸化,胞质和线粒体内的糖代谢,及随后产生的信号,最终导致胰岛素分泌。目前认为葡萄糖糖酵解和氧化磷酸化过程引起的ATP/ADP比值的变化是β细胞信号转导中的关键信号,但葡萄糖代谢和胰岛素分泌偶联的确切信号目前还不十分清楚。前期研究发现活性氧(ROS),如糖代谢过程中产生的过氧化氢(H2O2),可以作为信号参与GSIS过程。因此,氧化应激诱导的内源性抗氧化酶的升高很可能拮抗葡萄糖引起的ROS信号,从而抑制GSIS功能。
以往的研究表明,砷暴露能够诱导机体和细胞处于氧化应激状态,同时活化核转录因子-E2类相关因子2(Nrf2),而Nrf2作为细胞内抵御氧化损伤的关键蛋白调节许多结构型和诱导型抗氧化酶和解毒酶的表达。Nrf2调节多种内源性抗氧化酶,如：血红素氧化酶1 (Hmox1), NAD (P) H:醌氧化还原酶1 (NQO1),过氧化氢酶(CAT),γ-谷氨酰半胱氨酸连接酶催化亚基(Gclc)和调节亚基(Gclm)和硫氧还蛋白1 (Srxn1),维持细胞内氧化还原状态,保护细胞免受氧化损伤,但是这种内源性抗氧化酶的升高还存在一种潜在的副作用,就是降低细胞内ROS作为第二信使的功能。
本研究拟通过对胰岛β细胞系INS-1(832／13)的细胞试验研究,首先探讨砷暴露对β细胞GSIS功能、细胞活力、胰岛素含量及胰岛素基因表达的影响。其次,通过测定低剂量亚砷酸钠暴露引起的氧化应激水平和细胞的内源性抗氧化水平,研究核转录因子Nrf2在调节胰岛β细胞GSIS功能中的作用。最后,研究同样条件下的砷暴露对胰岛β细胞经典GSIS过程中各环节的影响,进一步探讨砷暴露对胰岛β细胞GSIS功能影响的机制。
材料与方法
1、细胞培养,砷处理和质粒转染：
INS-1(832／13)细胞常规培养于RPMI 1640培养液中,培养液含10%胎牛血清(FBS),10mM葡萄糖,25mMHEPES,50μMβ-巯基乙醇,100 U／ml青霉素,100μg／ml链霉素,37℃,5%的二氧化碳培养箱中。
细胞染毒：INS-1(832／13)细胞亚砷酸钠(0,0.05,0.1,0.25,0.5 gM)染毒96 h,每48 h重新换含砷培养液继续培养。Nrf2质粒转染应用Lipofectamine 2000试剂完成。
2、胰岛素测定：采用放射免疫测定试剂盒法测定,应用大鼠胰岛素作为标准标定样品浓度,结果用细胞的DNA含量校正。
3、抗氧化反应元件报告基因活性检测：INS-1(832／13)细胞转导含ARE报告基因的病毒颗粒后,应用0.35μg/ml puromycin的选择培养基筛选细胞为稳定细胞系。荧光素酶的活性测定采用双荧光素酶报告基因检测系统,按照试剂盒说明书操作。荧光素酶活性用细胞活力校正。细胞活力测定采用非放射性细胞增殖检测试剂盒测定。
4、细胞内过氧化氢的测定：细胞内过氧化氢应用CM-H2DCFDA探针标记,应用流式细胞仪测定荧光强度。
5、细胞内谷胱甘肽浓度的测定：砷处理后的细胞用预冷的PBS冲洗三次后收集细胞,超声破碎细胞,12000 g,4℃离心5 min,上清用于氧化型和总谷胱甘肽的测定。总谷胱甘肽浓度和氧化型谷胱甘肽的测定按照BIOXYTECH GSH/GSSG-412试剂盒说明书进行。
6、过氧化氢清除能力的测定：砷处理后细胞的上清液中剩余的过氧化氢浓度测定应用Amplex Red过氧化氢检测试剂盒测定,细胞上清液中的过氧化氢浓度之差反映细胞的过氧化氢清除能力。上清液中蛋白质浓度由Bio-Rad蛋白检测试剂盒标定,牛血清白蛋白作为标准。
7、实时定量RT-PCR分析基因表达：TRIzol法抽提细胞总RNA,经无RNA酶的DNA酶处理和RNeasy Mini试剂盒纯化。实时荧光检测使用ABI PRISM 7900 Sequence Detector检测。
8、Western Blot免疫印迹检测蛋白表达：细胞总蛋白和细胞核组分的分离和Western Blot用于检测Nrf2、GCK, Glut2、KIR6.2、SUR1、LaminA、β-actin蛋白表达水平。
9、细胞内ATP含量的测定：砷处理结束的细胞用预冷的Kreb's缓冲液冲洗,加入ATP释放缓冲液,超声裂解,12000 g,4℃离心5 min,上清液立即用于测定。ATP水平的测定使用ATP生物发光检测试剂盒,结果由蛋白浓度校正。
10、细胞内线粒体含量测定：细胞内线粒体应用绿色荧光探针MitoTracker Green标记,采用流式细胞仪和共聚焦显微镜检测。探针的应用浓度为75 nM,预染时间为30 min。在流式细胞仪测定过程中,死细胞和细胞团通过前向散射与侧散射被淘汰,未经处理的细胞作为对照。
11、细胞耗氧率(OCR)的测定：OCR测定应用XF24 Extracellular Flux分析仪,基础耗氧率和葡萄刺激的OCR变化连续3-5 min测定缓冲液中氧分压,计算细胞耗氧率。
结果
1、亚砷酸钠对胰岛p细胞GSIS功能的影响
INS-1(832／13)细胞砷暴露96 h后,高剂量(>0.5μM)砷处理导致细胞活力下降,当砷浓度高于1μM时观察到明显的细胞毒性。INS-1(832／13)细胞暴露在非毒性剂量(<0.5μM)的亚砷酸钠,葡萄糖刺激的胰岛素分泌减少,并且这种减少与砷暴露浓度存在剂量效应关系。相对于GSIS功能降低,砷处理却使细胞基础胰岛素分泌(3 mM葡萄糖)增加。慢性低砷暴露升高细胞胰岛素基因表达和细胞内胰岛素含量。
2、亚砷酸钠对胰岛p细胞Nrf2介导的抗氧化反应及Nrf2对GSIS功能的影响
低剂量亚砷酸钠暴露使细胞核蛋白Nrf2水平升高、抗氧化反应元件(ARE)-荧光素酶活性升高、Nrf2的下游靶基因的表达升高、细胞内谷胱甘肽浓度升高。低砷暴露使细胞内过氧化氢的清除能力升高,而且与砷暴露浓度存在剂量依赖性。亚砷酸钠使细胞内基础的过氧化物水平升高,却明显抑制葡萄糖刺激的细胞内过氧化物生成百分率,这与砷暴露导致的胰岛细胞的GSIS功能降低相关。
Nrf2的激活剂SFN或Nrf2质粒的转染影响胰岛p细胞GSIS功能,验证了Nrf2介导的抗氧化反应在调节GSIS功能中的作用。
3、亚砷酸钠对胰岛β细胞经典GSIS途径的影响
亚砷酸钠对经典GSIS通路中Glut2、GCK, Kcnj11、SUR1等关键基因和蛋白的表达未见影响；测定低砷暴露细胞在低糖和高糖条件下的ATP水平,没有观察到砷暴露细胞的葡萄糖刺激ATP生成的变化；与GSIS功能显著降低相比,砷暴露却升高细胞内线粒体含量。对经典GSIS途径中各环节的研究表明砷暴露所引起的INS-1(832／13)细胞GSIS功能降低不是由于砷影响GSIS过程的经典途径。
结论
1、在未发现明显细胞毒性的低剂量砷暴露96 h刺激基础胰岛素分泌的同时,降低高糖刺激的胰岛素分泌,降低β细胞对葡萄糖的反应性。
2、亚砷酸钠暴露激活Nrf2介导的抗氧化反应,这种升高的抗氧化反应拮抗作为信号的ROS,从而影响细胞GSIS功能。Nrf2激活剂SFN处理或过表达Nrf2抑制β细胞的GSIS功能,进一步验证了砷引起的Nrf2活化在调节胰岛β细胞GSIS功能中的作用。
3、亚砷酸钠对经典GSIS通路中Glut2、GCK、Kcnj11、Surl的基因和蛋白表达没有影响。未观察到细胞的葡萄糖刺激ATP生成的变化,提示低剂量亚砷酸钠引起的胰岛β细胞GSIS功能抑制不是因为砷对经典GSIS途径的影响。
Introduction
Arsenic 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 Methods
1. 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.
Results
1. 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.
Conclusion
1. 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.
砷是一种自然环境中广泛存在的类金属元素,以有机胂和无机砷两种形式存在,而无机砷的毒性较有机胂强。人类可以通过环境、职业暴露或者使用含砷药物而接触到无机砷(iAs),而环境砷暴露主要通过饮用自然界中被砷污染的水源。全世界约有2亿人正处于慢性饮水型高砷暴露的威胁中,我国大陆地区饮水型砷中毒病区中的高砷暴露人群已接近300万,随着调查的深入,慢性饮水型高砷暴露人群的数目正在不断扩大。慢性饮水型砷暴露不仅能够引起砷性皮肤损伤,砷性皮肤癌及其它内脏器官肿瘤,还与心血管疾病、高血压和糖尿病等常见疾病的发生关系密切。
2型糖尿病(T2D)已经成为一个全球性的公共卫生问题。截至到2000年全球有大约1.5亿人患有2型糖尿病,预计到2025年将有3亿人会罹患2型糖尿病。遗传和不良生活方式等因素都与糖尿病的发病有关。而环境危险因素暴露与糖尿病发生也存在一定程度的联系,目前还未得到医学界的广泛关注。
环境砷暴露和2型糖尿病的因果关系还不明确,但对台湾、孟加拉、瑞典和墨西哥等砷暴露人群的流行病学调查研究显示环境砷暴露与糖尿病的发生关系密切。最近,Meliker等对美国密歇根州饮用水中无机砷为0.011 ppm的居民进行的研究中发现低砷暴露和2型糖尿病发生存在显著相关性。另外一项对美国788位成年人的横断面调查研究显示低砷暴露与2型糖尿病的流行存在明显的正相关。这些近期的流行病学研究也都说明砷污染是暴露人群的2型糖尿病发生的重要因素之一。
2型糖尿病的发生是胰岛β细胞功能障碍和胰岛素抵抗共同作用的结果。胰岛β细胞具有对葡萄糖刺激反应释放胰岛素的功能,即葡萄糖刺激的胰岛素分泌(GSIS)功能,而胰岛细胞GSIS功能的降低是2型糖尿病发生的原因之一。根据目前的研究,β细胞的GSIS过程受细胞内葡萄糖代谢的直接影响,即从最初的葡萄糖摄入和磷酸化,胞质和线粒体内的糖代谢,及随后产生的信号,最终导致胰岛素分泌。目前认为葡萄糖糖酵解和氧化磷酸化过程引起的ATP/ADP比值的变化是β细胞信号转导中的关键信号,但葡萄糖代谢和胰岛素分泌偶联的确切信号目前还不十分清楚。前期研究发现活性氧(ROS),如糖代谢过程中产生的过氧化氢(H2O2),可以作为信号参与GSIS过程。因此,氧化应激诱导的内源性抗氧化酶的升高很可能拮抗葡萄糖引起的ROS信号,从而抑制GSIS功能。
以往的研究表明,砷暴露能够诱导机体和细胞处于氧化应激状态,同时活化核转录因子-E2类相关因子2(Nrf2),而Nrf2作为细胞内抵御氧化损伤的关键蛋白调节许多结构型和诱导型抗氧化酶和解毒酶的表达。Nrf2调节多种内源性抗氧化酶,如：血红素氧化酶1 (Hmox1), NAD (P) H:醌氧化还原酶1 (NQO1),过氧化氢酶(CAT),γ-谷氨酰半胱氨酸连接酶催化亚基(Gclc)和调节亚基(Gclm)和硫氧还蛋白1 (Srxn1),维持细胞内氧化还原状态,保护细胞免受氧化损伤,但是这种内源性抗氧化酶的升高还存在一种潜在的副作用,就是降低细胞内ROS作为第二信使的功能。
本研究拟通过对胰岛β细胞系INS-1(832／13)的细胞试验研究,首先探讨砷暴露对β细胞GSIS功能、细胞活力、胰岛素含量及胰岛素基因表达的影响。其次,通过测定低剂量亚砷酸钠暴露引起的氧化应激水平和细胞的内源性抗氧化水平,研究核转录因子Nrf2在调节胰岛β细胞GSIS功能中的作用。最后,研究同样条件下的砷暴露对胰岛β细胞经典GSIS过程中各环节的影响,进一步探讨砷暴露对胰岛β细胞GSIS功能影响的机制。
材料与方法
1、细胞培养,砷处理和质粒转染：
INS-1(832／13)细胞常规培养于RPMI 1640培养液中,培养液含10%胎牛血清(FBS),10mM葡萄糖,25mMHEPES,50μMβ-巯基乙醇,100 U／ml青霉素,100μg／ml链霉素,37℃,5%的二氧化碳培养箱中。
细胞染毒：INS-1(832／13)细胞亚砷酸钠(0,0.05,0.1,0.25,0.5 gM)染毒96 h,每48 h重新换含砷培养液继续培养。Nrf2质粒转染应用Lipofectamine 2000试剂完成。
2、胰岛素测定：采用放射免疫测定试剂盒法测定,应用大鼠胰岛素作为标准标定样品浓度,结果用细胞的DNA含量校正。
3、抗氧化反应元件报告基因活性检测：INS-1(832／13)细胞转导含ARE报告基因的病毒颗粒后,应用0.35μg/ml puromycin的选择培养基筛选细胞为稳定细胞系。荧光素酶的活性测定采用双荧光素酶报告基因检测系统,按照试剂盒说明书操作。荧光素酶活性用细胞活力校正。细胞活力测定采用非放射性细胞增殖检测试剂盒测定。
4、细胞内过氧化氢的测定：细胞内过氧化氢应用CM-H2DCFDA探针标记,应用流式细胞仪测定荧光强度。
5、细胞内谷胱甘肽浓度的测定：砷处理后的细胞用预冷的PBS冲洗三次后收集细胞,超声破碎细胞,12000 g,4℃离心5 min,上清用于氧化型和总谷胱甘肽的测定。总谷胱甘肽浓度和氧化型谷胱甘肽的测定按照BIOXYTECH GSH/GSSG-412试剂盒说明书进行。
6、过氧化氢清除能力的测定：砷处理后细胞的上清液中剩余的过氧化氢浓度测定应用Amplex Red过氧化氢检测试剂盒测定,细胞上清液中的过氧化氢浓度之差反映细胞的过氧化氢清除能力。上清液中蛋白质浓度由Bio-Rad蛋白检测试剂盒标定,牛血清白蛋白作为标准。
7、实时定量RT-PCR分析基因表达：TRIzol法抽提细胞总RNA,经无RNA酶的DNA酶处理和RNeasy Mini试剂盒纯化。实时荧光检测使用ABI PRISM 7900 Sequence Detector检测。
8、Western Blot免疫印迹检测蛋白表达：细胞总蛋白和细胞核组分的分离和Western Blot用于检测Nrf2、GCK, Glut2、KIR6.2、SUR1、LaminA、β-actin蛋白表达水平。
9、细胞内ATP含量的测定：砷处理结束的细胞用预冷的Kreb's缓冲液冲洗,加入ATP释放缓冲液,超声裂解,12000 g,4℃离心5 min,上清液立即用于测定。ATP水平的测定使用ATP生物发光检测试剂盒,结果由蛋白浓度校正。
10、细胞内线粒体含量测定：细胞内线粒体应用绿色荧光探针MitoTracker Green标记,采用流式细胞仪和共聚焦显微镜检测。探针的应用浓度为75 nM,预染时间为30 min。在流式细胞仪测定过程中,死细胞和细胞团通过前向散射与侧散射被淘汰,未经处理的细胞作为对照。
11、细胞耗氧率(OCR)的测定：OCR测定应用XF24 Extracellular Flux分析仪,基础耗氧率和葡萄刺激的OCR变化连续3-5 min测定缓冲液中氧分压,计算细胞耗氧率。
结果
1、亚砷酸钠对胰岛p细胞GSIS功能的影响
INS-1(832／13)细胞砷暴露96 h后,高剂量(>0.5μM)砷处理导致细胞活力下降,当砷浓度高于1μM时观察到明显的细胞毒性。INS-1(832／13)细胞暴露在非毒性剂量(<0.5μM)的亚砷酸钠,葡萄糖刺激的胰岛素分泌减少,并且这种减少与砷暴露浓度存在剂量效应关系。相对于GSIS功能降低,砷处理却使细胞基础胰岛素分泌(3 mM葡萄糖)增加。慢性低砷暴露升高细胞胰岛素基因表达和细胞内胰岛素含量。
2、亚砷酸钠对胰岛p细胞Nrf2介导的抗氧化反应及Nrf2对GSIS功能的影响
低剂量亚砷酸钠暴露使细胞核蛋白Nrf2水平升高、抗氧化反应元件(ARE)-荧光素酶活性升高、Nrf2的下游靶基因的表达升高、细胞内谷胱甘肽浓度升高。低砷暴露使细胞内过氧化氢的清除能力升高,而且与砷暴露浓度存在剂量依赖性。亚砷酸钠使细胞内基础的过氧化物水平升高,却明显抑制葡萄糖刺激的细胞内过氧化物生成百分率,这与砷暴露导致的胰岛细胞的GSIS功能降低相关。
Nrf2的激活剂SFN或Nrf2质粒的转染影响胰岛p细胞GSIS功能,验证了Nrf2介导的抗氧化反应在调节GSIS功能中的作用。
3、亚砷酸钠对胰岛β细胞经典GSIS途径的影响
亚砷酸钠对经典GSIS通路中Glut2、GCK, Kcnj11、SUR1等关键基因和蛋白的表达未见影响；测定低砷暴露细胞在低糖和高糖条件下的ATP水平,没有观察到砷暴露细胞的葡萄糖刺激ATP生成的变化；与GSIS功能显著降低相比,砷暴露却升高细胞内线粒体含量。对经典GSIS途径中各环节的研究表明砷暴露所引起的INS-1(832／13)细胞GSIS功能降低不是由于砷影响GSIS过程的经典途径。
结论
1、在未发现明显细胞毒性的低剂量砷暴露96 h刺激基础胰岛素分泌的同时,降低高糖刺激的胰岛素分泌,降低β细胞对葡萄糖的反应性。
2、亚砷酸钠暴露激活Nrf2介导的抗氧化反应,这种升高的抗氧化反应拮抗作为信号的ROS,从而影响细胞GSIS功能。Nrf2激活剂SFN处理或过表达Nrf2抑制β细胞的GSIS功能,进一步验证了砷引起的Nrf2活化在调节胰岛β细胞GSIS功能中的作用。
3、亚砷酸钠对经典GSIS通路中Glut2、GCK、Kcnj11、Surl的基因和蛋白表达没有影响。未观察到细胞的葡萄糖刺激ATP生成的变化,提示低剂量亚砷酸钠引起的胰岛β细胞GSIS功能抑制不是因为砷对经典GSIS途径的影响。
Introduction
Arsenic 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 Methods
1. 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.
Results
1. 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.
Conclusion
1. 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.
引文
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10 Arsenic and prostate cancer. Harv Mens Health Watch,2009.14(1):p.5,7.
11 Baastrup, R., et al., Arsenic in drinking-water and risk for cancer in Denmark. Environ Health Perspect,2008.116(2):p.231-7.
12 Chen, C.J., et al., Cancer potential in liver, lung, bladder and kidney due to ingested inorganic arsenic in drinking water. Br J Cancer,1992.66(5):p.888-92.
13 Liao, CM., et al., Risk assessment of arsenic-induced internal cancer at long-term low dose exposure. J Hazard Mater,2009.165(1-3):p.652-63.
14 Chiou, J.M., et al., Arsenic ingestion and increased microvascular disease risk:observations from the south-western arseniasis-endemic area in Taiwan, Int J Epidemiol,2005.34(4):p. 936-43.
15 Engel, R.R., et al., Vascular effects of chronic arsenic exposure:a review. Epidemiol Rev,1994. 16(2):p.184-209.
16 Huang, Y.K., et al., Arsenic methylation capability and hypertension risk in subjects living in arseniasis-hyperendemic areas in southwestern Taiwan. Toxicol Appl Pharmacol,2007.218(2): p.135-42.
17 Rahman, M., Arsenic and hypertension in Bangladesh. Bull World Health Organ,2002.80(2): p.173.
18 Rahman, M., et al., Hypertension and arsenic exposure in Bangladesh. Hypertension,1999. 33(1):p.74-8.
19 Meliker, J.R., et al., Arsenic in drinking water and cerebrovascular disease, diabetes mellitus, and kidney disease in Michigan:a standardized mortality ratio analysis. Environ Health,2007. 6:p.4.
20 Chen, C.J., et al., Arsenic and diabetes and hypertension in human populations:a review. Toxicol Appl Pharmacol,2007.222(3):p.298-304.
21 Kile, M.L. and D.C. Christiani, Environmental arsenic exposure and diabetes. Jama,2008. 300(7):p.845-6.
22 Zimmet, P., K.G Alberti, and J. Shaw, Global and societal implications of the diabetes epidemic. Nature,2001.414(6865):p.782-7.
23 Navas-Acien, A., et al., Arsenic exposure and prevalence of type 2 diabetes in US adults. Jama, 2008.300(7):p.814-22.
24 Tseng, C.H., The potential biological mechanisms of arsenic-induced diabetes mellitus. Toxicol Appl Pharmacol,2004.197(2):p.67-83.
25 Kajimoto, Y. and H. Kaneto, Role of oxidative stress in pancreatic beta-cell dysfunction. Ann N YAcad Sci,2004.1011:p.168-76.
26 Navas-Acien, A., et al., Arsenic exposure and type 2 diabetes:a systematic review of the experimental and epidemiological evidence. Environ Health Perspect,2006.114(5):p.641-8.
27 Chiu, H.F., et al., Does arsenic exposure increase the risk for diabetes mellitus? J Occup Environ Med,2006.48(1):p.63-7.
28 Nabi, A.H., M.M. Rahman, and L.N. Islam, Evaluation of Biochemical Changes in Chronic Arsenic Poisoning among Bangladeshi Patients. Int J Environ Res Public Health,2005.2(3-4): p.385-93.
29 Rahman, M., G Wingren, and O. Axelson, Diabetes mellitus among Swedish art glass workers--an effect of arsenic exposure? Scand J Work Environ Health,1996.22(2):p.146-9.
30 Coronado-Gonzalez, J.A., et al., Inorganic arsenic exposure and type 2 diabetes mellitus in Mexico. Environ Res,2007.104(3):p.383-9.
31 Izquierdo-Vega, J.A., et al., Diabetogenic effects and pancreatic oxidative damage in rats subchronically exposed to arsenite. Toxicol Lett,2006.160(2):p.135-42.
32 Paul, D.S., et al., Molecular mechanisms of the diabetogenic effects of arsenic:inhibition of insulin signaling by arsenite and methylarsonous Acid. Environ Health Perspect,2007.115(5): p.734-42.
33 Diaz-Villasenor, A., et al., Sodium arsenite impairs insulin secretion and transcription in pancreatic beta-cells. Toxicol Appl Pharmacol,2006.214(1):p.30-4.
34 Fridlyand, L.E. and L.H. Philipson, Does the glucose-dependent insulin secretion mechanism itself cause oxidative stress in pancreatic beta-cells? Diabetes,2004.53(8):p.1942-8.
35 Pi, J., et al., Reactive oxygen species as a signal in glucose-stimulated insulin secretion. Diabetes,2007.56(7):p.1783-91.
36 Woods, C.G, et al., Dose-dependent transitions in Nrf2-mediated adaptive response and related stress responses to hypochlorous acid in mouse macrophages. Toxicol Appl Pharmacol,2009. 238(1):p.27-36.
37 Newgard, C.B., et al., Stimulus/secretion coupling factors in glucose-stimulated insulin secretion:insights gained from a multidisciplinary approach. Diabetes,2002.51 Suppl 3:p. S389-93.
38 Li, X., et al., Arsenic methylation capacity and its correlation with skin lesions induced by contaminated drinking water consumption in residents of chronic arsenicosis area. Environ Toxicol,2009.
39 Majumdar, K.K., et al., Systemic manifestations in chronic arsenic toxicity in absence of skin lesions in West Bengal. Indian J Med Res,2009.129(1):p.75-82.
40 Paul, P.C., et al., Skin cancers in chronic arsenic toxicity-a study of predictive value of some proliferative markers. Indian J Pathol Microbiol,2004.47(2):p.206-9.
41 Rahman, M., et al., Arsenic exposure and age and sex-specific risk for skin lesions:a population-based case-referent study in Bangladesh. Environ Health Perspect,2006.114(12):p. 1847-52.
42 Ferreccio, C., et al., Lung cancer and arsenic exposure in drinking water:a case-control study in northern Chile. Cad Saude Publica,1998.14 Suppl 3:p.193-8.
43 Hopenhayn-Rich, C., et al., Bladder cancer mortality associated with arsenic in drinking water in Argentina. Epidemiology,1996.7(2):p.117-24.
44 Kwok, R.K., A review and rationale for studying the cardiovascular effects of drinking water arsenic in women of reproductive age. Toxicol Appl Pharmacol,2007.222(3):p.344-50.
45 Pi, J., et al., Vascular dysfunction in patients with chronic arsenosis can be reversed by reduction of arsenic exposure. Environ Health Perspect,2005.113(3):p.339-41.
46 Hsueh, Y.M., et al., Genetic polymorphisms of oxidative and antioxidant enzymes and arsenic-related hypertension. J Toxicol Environ Health A,2005.68(17-18):p.1471-84.
47 Datta, D.V., et al., Chronic oral arsenic intoxication as a possible aetiological factor in idiopathic portal hypertension (non-cirrhotic portal fibrosis) in India. Gut,1979.20(5):p. 378-84.
48 Navas-Acien, A., et al., Rejoinder:Arsenic exposure and prevalence of type 2 diabetes: updated findings from the National Health Nutrition and Examination Survey,2003-2006. Epidemiology,2009.20(6):p.816-20; discussion e1-2.
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39 Izquierdo-Vega, J.A., et al., Diabetogenic effects and pancreatic oxidative damage in rats subchronically exposed to arsenite. Toxicol Lett,2006.160(2):p.135-42.
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51 Diaz-Villasenor, A., et al., Sodium arsenite impairs insulin secretion and transcription in pancreatic beta-cells. Toxicol Appl Pharmacol,2006.214(1):p.30-4.
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58 Bazuine, M., et al., Arsenite stimulated glucose transport in 3T3-L1 adipocytes involves both Glut4 translocation and p38 MAPK activity. Eur J Biochem,2003.270(19):p.3891-903.
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2 Abernathy, C.O., et al, Arsenic:health effects, mechanisms of actions, and research issues. Environ Health Perspect,1999.107(7):p.593-7.
3 Gebel, T.W., Arsenic and drinking water contamination. Science,1999.283(5407):p.1458-9.
4 金银龙,梁超轲,何公理,曹静祥,中国地方性砷中毒分布调查(总报告).卫生研究,2003.32(6)：p.519-40.
5 Ahsan, H., et al., Arsenic exposure from drinking water and risk of premalignant skin lesions in Bangladesh:baseline results from the Health Effects of Arsenic Longitudinal Study. Am J Epidemiol,2006.163(12):p.1138-48.
6 Ahsan, H., et al., Arsenic metabolism, genetic susceptibility, and risk of premalignant skin lesions in Bangladesh. Cancer Epidemiol Biomarkers Prev,2007.16(6):p.1270-8.
7 Brown, K.G, et al., A dose-response analysis of skin cancer from inorganic arsenic in drinking water. Risk Anal,1989.9(4):p.519-28.
8 Chen, Y.C., et al., Arsenic methylation and skin cancer risk in southwestern Taiwan. J Occup Environ Med,2003.45(3):p.241-8.
9 Col, M., et al., Arsenic-related Bowen's disease, palmar keratosis, and skin cancer. Environ Health Perspect,1999.107(8):p.687-9.
10 Arsenic and prostate cancer. Harv Mens Health Watch,2009.14(1):p.5,7.
11 Baastrup, R., et al., Arsenic in drinking-water and risk for cancer in Denmark. Environ Health Perspect,2008.116(2):p.231-7.
12 Chen, C.J., et al., Cancer potential in liver, lung, bladder and kidney due to ingested inorganic arsenic in drinking water. Br J Cancer,1992.66(5):p.888-92.
13 Liao, CM., et al., Risk assessment of arsenic-induced internal cancer at long-term low dose exposure. J Hazard Mater,2009.165(1-3):p.652-63.
14 Chiou, J.M., et al., Arsenic ingestion and increased microvascular disease risk:observations from the south-western arseniasis-endemic area in Taiwan, Int J Epidemiol,2005.34(4):p. 936-43.
15 Engel, R.R., et al., Vascular effects of chronic arsenic exposure:a review. Epidemiol Rev,1994. 16(2):p.184-209.
16 Huang, Y.K., et al., Arsenic methylation capability and hypertension risk in subjects living in arseniasis-hyperendemic areas in southwestern Taiwan. Toxicol Appl Pharmacol,2007.218(2): p.135-42.
17 Rahman, M., Arsenic and hypertension in Bangladesh. Bull World Health Organ,2002.80(2): p.173.
18 Rahman, M., et al., Hypertension and arsenic exposure in Bangladesh. Hypertension,1999. 33(1):p.74-8.
19 Meliker, J.R., et al., Arsenic in drinking water and cerebrovascular disease, diabetes mellitus, and kidney disease in Michigan:a standardized mortality ratio analysis. Environ Health,2007. 6:p.4.
20 Chen, C.J., et al., Arsenic and diabetes and hypertension in human populations:a review. Toxicol Appl Pharmacol,2007.222(3):p.298-304.
21 Kile, M.L. and D.C. Christiani, Environmental arsenic exposure and diabetes. Jama,2008. 300(7):p.845-6.
22 Zimmet, P., K.G Alberti, and J. Shaw, Global and societal implications of the diabetes epidemic. Nature,2001.414(6865):p.782-7.
23 Navas-Acien, A., et al., Arsenic exposure and prevalence of type 2 diabetes in US adults. Jama, 2008.300(7):p.814-22.
24 Tseng, C.H., The potential biological mechanisms of arsenic-induced diabetes mellitus. Toxicol Appl Pharmacol,2004.197(2):p.67-83.
25 Kajimoto, Y. and H. Kaneto, Role of oxidative stress in pancreatic beta-cell dysfunction. Ann N YAcad Sci,2004.1011:p.168-76.
26 Navas-Acien, A., et al., Arsenic exposure and type 2 diabetes:a systematic review of the experimental and epidemiological evidence. Environ Health Perspect,2006.114(5):p.641-8.
27 Chiu, H.F., et al., Does arsenic exposure increase the risk for diabetes mellitus? J Occup Environ Med,2006.48(1):p.63-7.
28 Nabi, A.H., M.M. Rahman, and L.N. Islam, Evaluation of Biochemical Changes in Chronic Arsenic Poisoning among Bangladeshi Patients. Int J Environ Res Public Health,2005.2(3-4): p.385-93.
29 Rahman, M., G Wingren, and O. Axelson, Diabetes mellitus among Swedish art glass workers--an effect of arsenic exposure? Scand J Work Environ Health,1996.22(2):p.146-9.
30 Coronado-Gonzalez, J.A., et al., Inorganic arsenic exposure and type 2 diabetes mellitus in Mexico. Environ Res,2007.104(3):p.383-9.
31 Izquierdo-Vega, J.A., et al., Diabetogenic effects and pancreatic oxidative damage in rats subchronically exposed to arsenite. Toxicol Lett,2006.160(2):p.135-42.
32 Paul, D.S., et al., Molecular mechanisms of the diabetogenic effects of arsenic:inhibition of insulin signaling by arsenite and methylarsonous Acid. Environ Health Perspect,2007.115(5): p.734-42.
33 Diaz-Villasenor, A., et al., Sodium arsenite impairs insulin secretion and transcription in pancreatic beta-cells. Toxicol Appl Pharmacol,2006.214(1):p.30-4.
34 Fridlyand, L.E. and L.H. Philipson, Does the glucose-dependent insulin secretion mechanism itself cause oxidative stress in pancreatic beta-cells? Diabetes,2004.53(8):p.1942-8.
35 Pi, J., et al., Reactive oxygen species as a signal in glucose-stimulated insulin secretion. Diabetes,2007.56(7):p.1783-91.
36 Woods, C.G, et al., Dose-dependent transitions in Nrf2-mediated adaptive response and related stress responses to hypochlorous acid in mouse macrophages. Toxicol Appl Pharmacol,2009. 238(1):p.27-36.
37 Newgard, C.B., et al., Stimulus/secretion coupling factors in glucose-stimulated insulin secretion:insights gained from a multidisciplinary approach. Diabetes,2002.51 Suppl 3:p. S389-93.
38 Li, X., et al., Arsenic methylation capacity and its correlation with skin lesions induced by contaminated drinking water consumption in residents of chronic arsenicosis area. Environ Toxicol,2009.
39 Majumdar, K.K., et al., Systemic manifestations in chronic arsenic toxicity in absence of skin lesions in West Bengal. Indian J Med Res,2009.129(1):p.75-82.
40 Paul, P.C., et al., Skin cancers in chronic arsenic toxicity-a study of predictive value of some proliferative markers. Indian J Pathol Microbiol,2004.47(2):p.206-9.
41 Rahman, M., et al., Arsenic exposure and age and sex-specific risk for skin lesions:a population-based case-referent study in Bangladesh. Environ Health Perspect,2006.114(12):p. 1847-52.
42 Ferreccio, C., et al., Lung cancer and arsenic exposure in drinking water:a case-control study in northern Chile. Cad Saude Publica,1998.14 Suppl 3:p.193-8.
43 Hopenhayn-Rich, C., et al., Bladder cancer mortality associated with arsenic in drinking water in Argentina. Epidemiology,1996.7(2):p.117-24.
44 Kwok, R.K., A review and rationale for studying the cardiovascular effects of drinking water arsenic in women of reproductive age. Toxicol Appl Pharmacol,2007.222(3):p.344-50.
45 Pi, J., et al., Vascular dysfunction in patients with chronic arsenosis can be reversed by reduction of arsenic exposure. Environ Health Perspect,2005.113(3):p.339-41.
46 Hsueh, Y.M., et al., Genetic polymorphisms of oxidative and antioxidant enzymes and arsenic-related hypertension. J Toxicol Environ Health A,2005.68(17-18):p.1471-84.
47 Datta, D.V., et al., Chronic oral arsenic intoxication as a possible aetiological factor in idiopathic portal hypertension (non-cirrhotic portal fibrosis) in India. Gut,1979.20(5):p. 378-84.
48 Navas-Acien, A., et al., Rejoinder:Arsenic exposure and prevalence of type 2 diabetes: updated findings from the National Health Nutrition and Examination Survey,2003-2006. Epidemiology,2009.20(6):p.816-20; discussion e1-2.
49 Hohmeier, H.E., et al., Isolation of INS-1-derived cell lines with robust ATP-sensitive K+ channel-dependent and-independent glucose-stimulated insulin secretion. Diabetes,2000. 49(3):p.424-30.
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