Arsenic, Nrf2 and Autophagy Dysfunction in Type II Diabetes

项目摘要 慢性砷暴露是一个全球性的公共卫生问题， 患某些类型癌症的风险，以及包括II型糖尿病在内的代谢性疾病。 砷相关的II型糖尿病最近在美国人群中被证实， 墨西哥、加拿大、台湾和孟加拉国。我们以前曾报道过，低，环境 相关剂量的砷在后期阻断自噬。自噬是细胞降解的关键 自噬途径和自噬功能障碍被认为是致病变化的组成部分， 发生在脂肪细胞、β-胰岛细胞、肝细胞、骨骼肌和肾系膜细胞中， 糖尿病进展。我们还发现，自噬途径的失调导致了 关键的抗氧化转录因子核因子红细胞衍生的-2-样的延长激活 2（Nrf2）。Nrf 2通常在胞质溶胶中被Kelch样ECH相关蛋白结合和降解， 蛋白1（Keap 1）在Keap 1的氧化修饰后在蛋白质水平上上调 （Keap 1-C151依赖性，规范）或通过在细胞分裂期间将Keap 1隔离到自噬体中， 自噬功能障碍（p62依赖性，非典型）。虽然通过Nrf 2激活来控制 经典机制是保护性的，Nrf 2的长期非经典激活导致细胞 功能障碍和组织损伤，指示Nrf 2的“黑暗面”。因此，我们假设， 由于砷介导的自噬通量的阻断， 是砷在促进II型糖尿病中必不可少的。这一假设得到了 以下证据：1）慢性砷暴露降低葡萄糖摄取和胰岛素信号转导， 3 T3-L1脂肪细胞，2）Keap 11 KD/Lepob/ob小鼠，一种持续性糖尿病的遗传小鼠模型 Nrf 2激活，显示受损的脂肪形成，以及降低胰岛素敏感性和葡萄糖 与Lepob/ob对照相比的耐受性，和3）Atg 7的β细胞特异性敲低，Atg 7是一种关键的自噬， 起始蛋白，导致p62和多聚泛素化蛋白水平增加，伴随β- 细胞损失和胰岛素产生减少。 我们已经产生了大量的数据，表明砷阻断了自噬， 自噬体-溶酶体融合步骤。三个核心SNARE蛋白Stx 17、SNAP 29和VAMP 8 介导融合，SNAP 29介导Stx 17与外部自噬体之间的相互作用。 在溶酶体膜上存在VAMP 8。我们相信基因切除任何这些 三种融合蛋白将阻碍自噬体-溶酶体融合，导致Nrf 2 激活，这将模拟砷在促进II型糖尿病中的作用。所以我们提出 收件人： 目的1：确定砷阻断自噬体的分子机制。 溶酶体融合，导致Nrf 2活化延长。 目的2：确定自噬功能障碍是否导致Nrf 2激活延长 在肌肉、肾脏、胰腺、肝脏和脂肪细胞中诱导代谢重编程。 目的3：确定自噬功能障碍和延长的Nrf 2激活表型是否 砷在II型糖尿病肾病中的作用 在SNAP 29 f/f、Nrf 2-/-和SNAP 29 f/f/Nrf 2-/-小鼠中的糖尿病模型中。 影响：对自噬功能障碍的详细和彻底的了解，以及长期的 在砷诱导的代谢性疾病中，Nrf 2的激活将被证明是非常有价值的， 预防和治疗策略，以及生物标志物的鉴定， 处于危险中的人群。

Project Summary Chronic arsenic exposure is a worldwide public health concern correlated with an increased risk of developing certain types of cancer, as well as metabolic diseases including type II diabetes. Arsenic-linked type II diabetes has recently been shown among populations in the United States, Mexico, Canada, Taiwan, and Bangladesh. We have previously reported that low, environmentally relevant doses of arsenic block autophagy at a later stage. Autophagy is a key cellular degradation pathway and autophagic dysfunction is thought to be an integral part of pathogenic changes that occur in adipocytes, β-islet cells, hepatocytes, skeletal muscle, and kidney mesangial cells during diabetes progression. We have also shown that dysregulation of the autophagy pathway results in prolonged activation of the key antioxidant transcription factor nuclear factor erytheroid-derived-2-like 2 (Nrf2). Nrf2, which is normally bound and degraded in the cytosol by Kelch-like ECH-associated protein 1 (Keap1), is upregulated at the protein level following oxidative modification of Keap1 (Keap1-C151 dependent, canonical) or by sequestration of Keap1 into autophagosomes during autophagy dysfunction (p62-dependent, non-canonical). While controlled Nrf2 activation through the canonical mechanism is protective, prolonged non-canonical activation of Nrf2 causes cellular dysfunction and tissue damage, indicative of a “dark side” to Nrf2. Therefore, we hypothesize that the prolonged activation of Nrf2, resulting from arsenic-mediated blockage of autophagy flux, is essential for arsenic in promoting type II diabetes. This hypothesis is supported by the following evidence: 1) chronic arsenic exposure decreases glucose uptake and insulin signaling in 3T3-L1 adipocytes, 2) Keap11 KD/Lepob/ob mice, a genetic mouse model for diabetes with persistent Nrf2 activation, display impaired adipogenesis, as well as decreased insulin sensitivity and glucose tolerance compared to Lepob/ob controls, and 3) β-cell specific knockdown of Atg7, a key autophagy initiation protein, results in increased levels of p62 and poly-ubiquitinated proteins, accompanied by β- cell loss and decreased insulin production. We have generated a large amount of data indicating that arsenic blocks autophagy at the autophagosome-lysosome fusion step. Three core SNARE proteins Stx17, SNAP29, and VAMP8 mediate fusion, with SNAP29 mediating the interaction between Stx17 on the outer autophagosomal membrane and VAMP8 on the lysosomal membrane. We believe that genetic ablation of any of these three fusion proteins will hinder autophagosome-lysosome fusion and result in prolonged Nrf2 activation, which will mimic the effect of arsenic in promoting type II diabetes. Therefore, we propose to: Aim 1: Determine the molecular mechanism by which arsenic blocks autophagosome- lysosome fusion, leading to prolonged Nrf2 activation. Aim 2: Determine if prolonged Nrf2 activation resulting from autophagic dysfunction induces metabolic reprogramming in muscle, kidney, pancreas, liver and fat cells. Aim 3: Determine if autophagy dysfunction and prolonged Nrf2 activation phenocopy arsenic in exacerbating insulin resistance, obesity, and diabetic nephropathy using type II diabetes models in SNAP29f/f, Nrf2-/-, and SNAP29f/f/Nrf2-/- mice. Impact: A detailed and thorough understanding of autophagy dysfunction and the prolonged activation of Nrf2 in arsenic-induced metabolic disease will prove extremely valuable in the generation of preventive and therapeutic strategies, as well as in the identification of biomarkers, for the populations at risk.