长爪沙鼠骨骼肌Kv2.1通道氧化修饰在老年胰岛素抵抗中作用及机制研究

The incidence of insulin resistance in the elderly increased significantly, studies have shown that oxidative stress is the important reason for it, but the exact mechanism has not been clearly elucidated till now. We and other studies have shown the similar metabolic characteristics between human and gerbil, therefore, we speculate that the incidence of insulin resistance is also increased in the elderly gerbil. Voltage-gated potassium channel 2.1 subtype (Kv2.1) is the target of ROS. Some studies have shown that the oxidative modification of the channel induces cell apoptosis, and influences the normal distribution of insulin receptors in the cell membrane, therefore we speculate that in the important tissue of insulin utilization, namely skeletal muscle, Kv2.1 channel oxidation may mediate the process of insulin resistance in the elderly through the above process. First, in this study we will exam the insulin sensitivity of different aged gerbil to determine the characteristics of insulin resistance in elderly gerbil; second, to get the insulin resistance animal model by establishment the criteria of insulin resistance in elderly gerbil, then analyze the association between the oxidation of Kv2.1 channel in the skeletal muscle and insulin resistance, so as to assess the action of Kv2.1 channel oxidation in the process of elderly insulin resistance; third, we will analyze the specific role of Kv2.1 channel oxidation in the pathological process of different aged insulin resistance; last, we will investigated the molecular mechanism of Kv2.1 channel oxidation in the process of insulin resistance by the technique of patch-clamp and gene mutation. This study will provide the basis for the establishment of the evaluation criteria for insulin resistance in the elderly gerbil and its animal model, and also for the elucidation of the mechanism for insulin resistance in elderly.

老年人群胰岛素抵抗明显增加，现认为氧化应激是其重要原因，但机制尚未阐明。本课题组和其他研究显示长爪沙鼠血糖代谢表型与人相似，推测老年长爪沙鼠胰岛素抵抗亦升高。研究表明Kv2.1通道是活性氧作用的靶分子，该通道氧化修饰可诱导细胞凋亡，并影响胰岛素受体在细胞膜上正常分布，为此推测在胰岛素利用的重要组织骨骼肌中，Kv2.1通道氧化修饰可能通过上述环节介导老年长爪沙鼠胰岛素抵抗过程。本研究通过检测不同年龄沙鼠胰岛素敏感性，确立老年沙鼠胰岛素抵抗特征；通过建立老年长爪沙鼠胰岛素抵抗评价标准，获得胰岛素抵抗模型动物，分析骨骼肌Kv2.1通道氧化修饰与胰岛素抵抗的关联，评价其在老年沙鼠胰岛素抵抗中作用；并比较该通道氧化修饰在不同年龄胰岛素抵抗过程中的作用特点；用膜片钳和基因突变等方法，探讨Kv2.1通道氧化修饰作用的分子机制。本研究为建立老年长爪沙鼠胰岛素抵抗评价标准和模型动物及阐明其发生机制提供依据。

目前，我国人口老龄化程度日益加重，由此引发的T2DM、心脑血管疾病等给社会来沉重负担，而上述疾病都与胰岛素抵抗密切相关。研究表明年龄增大是胰岛素抵抗的重要危险因素，开展老龄人口胰岛素抵抗机制研究，将有助于揭示老年疾病发生机制。但受道德伦理限制，机制研究中的常用的干预手段不能在人类研究中开展。长爪沙鼠是研究糖尿病较为理想的动物模型，本课题组前期在长爪沙鼠糖尿病高发群体研究中发现血糖随年龄升高。但迄今为止，国内外尚无老龄长爪沙鼠胰岛素抵抗指标的正常值范围，亦未见长爪沙鼠与人类血糖重要代谢调控过程的比较。.我们首先在不同月龄沙鼠中观察其胰岛素抵抗及代谢表型，发现上述指标随月龄增加呈恶化趋势；我们用Hoffman和百分位数方法，确定了成年组和老年组沙鼠胰岛素敏感指数（QUICKI）和空腹血糖正常值的参考区间。分别为成年组：0.318-0.420和3.1-5.2mmol/L；老年组：0.286-0.389和4.6-6.5mmol/L。然后，我们发现HOMA-IRI与骨骼肌质量指数显著相关，应用近年建立的骨骼肌组织胰岛素抵抗指标（Mus-IRI），我们进一步发现骨骼肌组织胰岛素抵抗与沙鼠月龄、氧化应激及血糖和血脂水平呈正相关，提示骨骼肌组织可能参与了老年沙鼠胰岛素抵抗过程，并提示氧化应激可能参与该调控过程。.我们在机制研究中发现，C2C12骨骼肌细胞转染Kv2.1通道后，葡萄糖摄取能力提高，而细胞氧化应激后，膜胰岛素受体分布下降，葡糖糖摄取降低；细胞经补充胆固醇后，胰岛素受体分布增加，糖摄取能力升高，说明Kv2.1通道氧化应激可能参与了年龄相关的胰岛素抵抗过程。 .此外，我们也在人群中发现随年龄增长，骨骼肌质量下降，而骨骼肌组织及整体水平胰岛素抵抗升高，代谢异常发生率增加。在人群中编码Kv2.1通道的KCNB1基因多态与骨骼肌组织胰岛素抵抗相关。.总之，本研究建立了老年沙鼠胰岛素抵抗的参考区间，为构建老年胰岛素抵抗长爪沙鼠模型提供了依据，同时也表明长爪沙鼠与人类有着相似的血糖代谢表型及调控机制，为应用该动物开展人类胰岛素抵抗机制研究提供了充分证据。