TLR4调控TMEM24介导代谢炎症性β细胞功能静默的机制研究

Nutrient-energy stress induced metainflammation was the important mechanism of diabetes, and was lack of specific targets for intervention. Our previous studies have demonstrated that glucolipotoxicity induced dysfunction in β cell through metainflammation by activating TLR4 signal way directly, but the underlying mechanismthe remained unresolved. New studies have shown that TMEM24 was a key lipid transport protein and it could regulate the impulsive secretion of insulin, and our preliminary experiments showed that induction of TMEM24 overexpression could attenuate TLR4 induced β cell dysfunction.Therefore, we presumed that the activating TLR4 induced by glucolipotoxicity may obstruct the secretion of insulin vesicles and lead β cell dysfunction by regulating TMEM24. To confirm our hypothesis, we will conduct the research from molecular, cell, tissue and animal holistic levels. We will establish the models of obese mice induced by high-fat diet, diabetic mice and glucolipotoxicity β-cell; induce TMEM24/TLR4 over-expression or conditional knockout TMEM24 /TLR4 gene; filter mice with different gene phenotypes by hybridization; and regulate the activity of proteins using agonists or inhibitors to investigated whether TLR4 can induce β cell dysfunction by regulating TMEM24 protein. This study will show the mechanism of sustained damage in β cell induced by metainflammation from the new viewpoint of TMEM24, and provide the intervention target for metainflammation and a novel therapeutic way for type 2 diabetes.

营养过剩压力诱导的代谢性炎症是糖尿病的重要致病机制，目前尚无靶向干预措施。我们前期发现糖脂毒性可直接激活β细胞TLR4信号通路从而诱导代谢性炎症反应，导致β细胞功能静默，但机制有待进一步明确。研究显示TMEM24是调节胰岛素脉冲性分泌的关键脂质转运蛋白，我们预实验发现诱导TMEM24过表达可削弱TLR4导致的β细胞功能障碍。为此，我们提出假说：糖脂毒性激活的TLR4可能通过调控TMEM24功能影响胰岛素囊泡分泌，导致β细胞功能静默的发生。为了验证这一假说，我们将通过构建饮食诱导的肥胖小鼠，糖尿病小鼠及糖脂毒性β细胞模型，采用过表达或组织特异性敲除TMEM24或TLR4基因，杂交获得不同基因型小鼠，并运用激动剂或抑制剂调控蛋白活性等手段，从分子、细胞、组织以及动物整体水平等多层面探讨TLR4是否通过调控TMEM24蛋白导致β细胞功能静默及机制，为靶向干预代谢性炎症相关药物的研发提供潜在靶点。

营养过剩压力诱导的代谢性炎症是糖尿病的重要致病机制，目前尚无靶向干预措施。本项以脂毒性或炎症性β细胞模型以及肥胖SD大鼠、TLR4KO大鼠模型作为研究对象，研究糖脂毒性通过TLR4调控TMEM24诱导β细胞功能损伤的机制，并探索GPR40激动剂/ Exendin-4 /二甲双胍对β细胞糖脂毒性的保护作用。结果显示，炎症因子IL6和IL11参与了糖毒性诱导的胰岛纤维化及胰岛功能障碍；生信分析提示 NOD 样受体 (NLR) 信号通路与糖尿病发病的密切相关。进一步证实TLR4KO可改善肥胖大鼠胰岛β细胞功能；体外沉默TLR4可以抑制棕榈酸（PA）诱导的 β 细胞凋亡、胰岛素分泌障碍；PA对胰岛β细胞TMEM24表达的抑制作用具有浓度和时间依赖性；调控TMEM24表达水平可影响PA对β细胞的损害作用；TLR4介导的β细胞脂毒性损害与抑制MEM24-pPKC-PIP2-IP3通路有关；过表达TMEM24可拮抗TLR4介导的β细胞脂毒性损害；TLR4和TMEM24共定位于细胞质，两者存在相互作用。GPR40激动剂（TAK875）可通过拮抗TLR4-NF-κB活化减缓脂毒性β细胞凋亡，改善胰岛素分泌功能；同样，我们也发现 Exendin-4 /二甲双胍与GPR40激动剂具有类似的作用。总之，TLR4是介导β细胞糖脂毒性损害的枢纽信号，其作用与抑制TMEM24-pPKC-PIP2-IP3通路有关。GPR40激动剂/Exendin-4/二甲双胍可通过抑制TLR4-NF-κB通路拮抗代谢性炎症诱导的β细胞损伤。我们的研究揭示了代谢性炎症在胰岛功能障碍中的关键作用，TMEM24是改善胰岛功能的潜在药物靶点。我们的研究揭示了GPR40激动剂TAK875，GLP-1，二甲双胍拮抗糖脂毒性β细胞损伤的新机制，也为TLR4作为减重以及干预β细胞代谢性损伤的治疗靶点提供了实验证据。

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