游离脂肪酸受体GPR40对胰腺β细胞胰岛素合成分泌的调控及配体不依赖受体内吞机制的研究

发布时间：2018-06-13 18:01

本文选题：胰岛素 + 蛋白激酶C　；参考：《浙江大学》2014年博士论文

【摘要】：游离脂肪酸作为人体重要的营养物质,同时又是重要的信使物质,通过G蛋白偶联受体(GPCRs)起调节代谢等生理作用。最近研究表明,长链脂肪酸亚油酸是胰岛p细胞高表达的GPR40受体的内源性配体,可以通过GPR40呈葡萄糖依赖性的促进胰岛素合成和分泌。此外GPR40还能辅助DPP-IV抑制剂促进胰岛素的分泌。本文为了解析GPR40增强胰岛素分泌的机制,采用cAMP响应元件(CRE)荧光素酶报告系统检测到在细胞水平上游离脂肪酸和forskolin共孵育会显著提高荧光素酶活性,其小分子激动剂GW9508也有同样效果,并能被特异拮抗剂GW1100抑制。此外,PDE的普遍抑制剂IBMX和两种异构体抑制剂MMPX,Rolipram证实了PDE1能协助亚油酸介导的荧光素酶活性提高,而非PDE4。进一步的Gs小肽拮抗实验证实亚油酸和forskolin协同介导的荧光素酶活性的提高并不通过Gs蛋白,而Gq的特异性抑制剂UBO-QIC能显著地抑制这种GPR40介导的CREB活性增强。PKC抑制剂和cAMP ELISA检测验证了Gq/PKC/CREB通路而非cAMP的激活来协助cAMP/PKA通路发挥增效作用,并且PKC通过磷酸化CREB的Ser133,121位点来激活转录,8-CPT-cAMP也证实了cAMP是该通路中重要的一环。用艾塞那肽取代forskolin发现,亚油酸同样有协同艾塞那肽增强荧光素强度的作用,并且通过Gq/PKC/CREB信号通路来协同GLP-1R/Gs/PKA通路。将细胞水平的实验在SD大鼠上重复后,发现亚油酸同样协同forskolin或exentin-4来促进胰岛素的分泌,相比于单独作用得到显著提高。用PKC抑制剂Go6983和Gq抑制剂UBO-QIC处理后能显著抑制胰岛素的分泌,在敲除Gq亚基的小鼠上也同样抑制了亚油酸和forskolin或exendin-4激起的胰岛素分泌。因此亚油酸能协同forskolin和exentin-4促进胰岛素的分泌,随后我们发现利拉鲁肽侧链带有棕榈酸修饰,而棕榈酸是GPR40的内源性配体,利拉鲁肽和艾塞那肽对于激活GLP-1R具有相同的活性,将侧链用做亚油酸修饰发现能激活GPR40,并也能激活GLP-1R。在同时转染GPR40和GLP-1R的细胞株中,利拉鲁肽活性提高了20%。我们的研究为有效地治疗糖尿病提供了新的思路。在稳定表达GPR40-EGFP的HEK293细胞中,GPR40具有配体依赖型内吞和配体不依赖型内吞两种方式。不仅在HEK293细胞中,在β-TC-6,GPR40内源性细胞中过表达GPR40也发现有配体不依赖型内吞的存在。用FITC-anti-Flag抗体标记GPR40受体的实验表明在荧光显微镜下观察到的GPR40大量聚集在胞质内不是由于受体高表达所致,而是由于配体不依赖型内吞造成的。Arrestin-3和GRK2敲除实验发现配体依赖型内吞依赖于GRK2和arrestin-3,而配体不依赖内吞则不依赖GRKs和arrestins。此外,GPR40配体依赖型内吞能与Rab4/Rab5共定位,而配体不依赖型内吞及再循环则不经过Rab4通路,而能同Rab5共定位。由于脂肪酸家族具有高度同源性,因此该发现能运用到其他脂肪酸家族成员。并且受体经历不同的内吞途径,如果能找到与配体不依赖型内吞相关的结合蛋白,则可以用来筛选阻断自激活内吞行为的化合物。
[Abstract]:Free fatty acids are important nutrients and important messenger substances, which regulate metabolism through the G protein coupling receptor (GPCRs). Recent studies have shown that long chain fatty acid linoleic acid is an endogenous ligand for the high expression of GPR40 receptor in pancreatic islet P cells. It can promote the pancreas through GPR40 to promote the pancreas. In addition, GPR40 also assists the DPP-IV inhibitor to promote insulin secretion. In order to analyze the mechanism of GPR40 enhancement of insulin secretion, the cAMP response element (CRE) luciferase reporter system has detected a significant increase in luciferase activity at the upper level of cell level and forskolin, and its small molecules The agonist GW9508 also has the same effect and can be suppressed by the specific antagonist GW1100. In addition, the universal inhibitor IBMX of PDE and the two isomer inhibitor MMPX, Rolipram confirmed that PDE1 can help the linoleic acid mediated luciferase activity increase, but not PDE4. further Gs small peptide antagonistic experiments confirm the synergistic fluorescence mediated by linoleic acid and forskolin The enhancement of the activity of the enzyme does not pass through the Gs protein, and the specific inhibitor UBO-QIC of Gq can significantly inhibit the GPR40 mediated CREB activity and enhance the.PKC inhibitor and cAMP ELISA detection to verify the Gq/PKC/CREB pathway rather than cAMP activation to assist the cAMP/PKA pathway to exert synergism, and PKC through the phosphorylation of the locus. Activation of transcription, 8-CPT-cAMP also confirmed that cAMP is an important link in the pathway. Using erisannin instead of forskolin, it is also found that linoleic acid also synergistically enhances fluorescein intensity with the synergistic effect of the Gq/PKC/CREB signaling pathway to the GLP-1R/Gs/PKA pathway. The cell level experiment was repeated on SD rats and the oil Suboil was found. The acid also synergies with forskolin or exentin-4 to promote insulin secretion, which is significantly increased compared to the individual effect. The secretion of insulin can be inhibited significantly with the PKC inhibitor Go6983 and the Gq inhibitor UBO-QIC. The insulin secretion induced by linoleic acid and forskolin or exendin-4 is also inhibited in the mice that knock off the Gq subunit. This linoleic acid can promote the secretion of insulin with forskolin and exentin-4. Then we found that the Liraru peptide side chain is modified with palmitic acid, and palmitic acid is an endogenous ligand for GPR40. The Liraru peptide and erenin have the same activity for activating GLP-1R. The side chain is used as linoleic acid modification to activate GPR40 and also activate GL. P-1R. in the cells transfected with GPR40 and GLP-1R simultaneously, the activity of the L La r peptide increased by 20%.. Our research provides a new way of thinking for the effective treatment of diabetes.
In HEK293 cells that stably express GPR40-EGFP, GPR40 has two ways of ligand dependent endocytosis and ligand endocytosis. Not only in HEK293 cells, the presence of GPR40 in beta -TC-6 and GPR40 endogenous cells is also found in the presence of ligands without dependent endocytosis. The experiment of labeling GPR40 receptors with FITC-anti-Flag antibodies indicates that the fluorescence is in the presence of fluorescence. The massive aggregation of GPR40 observed under the microscope is not due to the high expression of the receptor, but the.Arrestin-3 and GRK2 knockout experiments caused by the ligand undependent endocytosis found that the ligand dependent endocytosis depends on the GRK2 and arrestin-3, while the ligand does not depend on the endocytosis in addition to the GRKs and arrestins., and the GPR40 ligand dependent type. TAC can co locate with Rab4/Rab5, while ligand independent endocytosis and recycling can co locate with Rab5 without Rab4 pathway.
Because the fatty acid family is highly homologous, the discovery can be applied to other fatty acid family members. And the receptors experience different endocytic pathways, and if they can find binding proteins associated with the ligand without endocytosis, they can be used to screen the compounds that block self activated endocytosis.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R965

【共引文献】