TLR4参与高脂诱导的胰岛β细胞功能损伤的生物学机制研究

Obesity is a risk factor of type 2 diabetes .Pancreatic β cells play a key role during obesity. Obesity is associated with low levels of the inflammatory process. But the molecular mechanism of β cell dysfunction during obesity is still unknow. The Toll-like receptor4 (TLR4), one of the pattern recognition receptors, has an important role in inflammation and immunity, and its expression has been reported in most tissues including islet β cells. Because it can be activated by lipopolysaccharide and saturated fatty acids, which are inducers of obesity, TLR4 may be a candidate for participation in the cross-talk between inflammatory and metabolic signals. The current study was undertaken to determine whether TLR4 mediates islet dysfunction in obesity induced by high-fat(HF) feeding. Whether TLR4 signaling contributes to the link between nutrient excess, inflammation and islet β cell dysfunction in vivo is an important unanswered question. We also investigate the effects of myeloid infiltrating cells in islet dysfunction during obesity. Whether myeloid infiltrating cells accelerates the islet dysfunction through activating the inflammation signaling is still unknown. Here, to investigate the expression of TLR4 in islet during obesity, mice will be randomly divided into HF and ND groups. To explore the interaction between toll-like receptor 4 with islet β cell function during diet induced obesity, weight、insulin sensitivity、triglyceride(TG)、total cholesterol (TC) and fasting serum insulin will be measured; Glucose tolerance test and insulin releasing test will be performed; the level of TLR4 in the islet will be determined with realtime PCR or immunohistochemical. Moreover, we plan to observe the change of TNFa 、IL-6 and MCP-1 mRNA in primary islet after high-fat diet in TLR4-Widetype(C57BL/SNJ) and TLR4-knockout (C57BL/ScNJ) mice. Obesity leads to elevated plasma free fatty acids (FFAs). Acute FFA exposure stimulates insulin secretion, but prolonged FFA exposure decreases glucose-stimulated insulin secretion(GSIS). To determine whether TLR4 signaling mediates deleterious effects of FFAs in β cells, we incubated mouse β cell lines MIN6 cells in the presence of palmitate and investigated the effects of prolonged exposure to palmitate on GSIS in MIN6 β cells. The mRNA levels of cytokine genes including IL-6, 、TNFa and MCP-1 in MIN6 cells after palmiate exposure will be measured. In conclusion, the aim of the present study is to discover the molecular mechanism of TOLL-like Receptor 4 involved in the β cell Dysfunction during Ddiet induced Obesity and the effect of TLR4 signaling on myeloid cells for islet dysfuncuion in diet induced obesity. This study will provide a new idea and a new strategy for the protection of β cell function during diet induced obesity.

肥胖是2型糖尿病的高危因素，在肥胖-糖尿病前期-糖尿病这一变化进展中，胰岛β细胞功能受损起着关键的作用。大量研究发现，肥胖常伴随着低水平的炎症过程,但肥胖状态下胰岛β细胞功能受损的具体机制不明。本课题组的前期研究发现，持续高脂喂养导致肥胖小鼠胰岛功能明显受损，且随着高脂喂养时间的延长，胰岛β细胞中TLR4的表达水平显著升高。相反，TLR4敲除小鼠却能抵抗高脂诱导的肥胖，改善胰岛β细胞功能。因此，我们提出TLR4参与调控高脂饮食诱导的胰岛β细胞功能损伤的假设。本研究拟利用TLR4敲除小鼠，交叉骨髓移植技术，从动物水平、原代胰岛和细胞水平验证该假设。拟明确高脂损伤β细胞的机制是否同β细胞本身的TLR4活化密切相关，并初步排除骨髓浸润细胞进入胰岛，激活炎症信号通路而造成的β细胞损伤。本项目为探讨肥胖损伤胰岛β细胞功能的机制开辟了新思路和新策略。

肥胖是2型糖尿病的重要诱发因素，然而持续高脂饮食诱导的肥胖损伤胰岛beta细胞的确切机制仍不是非常清楚。我们发现高脂诱导的肥胖小鼠和转基因db/db,ob/ob小鼠胰岛内TLR4表达水平均显著上调。而面对同样的高脂持续刺激24周，TLR4-/- HF组小鼠体重，肝脏含量，附睾脂肪垫含量，以及血糖，胰岛素水平均低于WT HF组。糖耐量试验，胰岛素耐量试验以及胰岛素释放试验提示，WT HF组小鼠胰岛功能受损，TLR4-/- HF组小鼠胰岛功能较WT HF组明显改善。各组胰岛beta细胞电镜，WT HF组beta细胞胰岛素分泌颗粒数量增多，TLR4-/- HF组beta细胞超微结构接近WT ND组。分离原代胰岛检测葡萄糖刺激的胰岛素分泌，TLR4-/- HF组小鼠胰岛分泌功能好于WT HF组, TLR4-/- HF组24周，胰岛内IL-6mRNA，TNFa mRNA，MCP-1 mRNA水平显著低于WT HF组。WT HF组胰岛出现巨噬细胞浸润，而TLR4-/-HF组未见明显巨噬细胞浸润。提示，高脂饮食诱导的肥胖胰岛功能受损可能同上调胰岛beta细胞表面的TLR4的表达水平，并且招募巨噬细胞进入胰岛，损伤胰岛功能相关。本研究探讨了肥胖胰岛β细胞功能受损的发病机理，TLR4是介导高脂诱导的胰岛β细胞功能损伤的关键蛋白，该研究将为治疗以及开发新的治疗肥胖药物提供理论依据和试验铺垫。