Objective To elucidate GPR40/FFA1 and its downstream signaling pathways in regulating insulin secretion. Methods GPR40/FFA1 expression was detected by immunofluorescence imaging. We employed linoleic acid (LA), a free...Objective To elucidate GPR40/FFA1 and its downstream signaling pathways in regulating insulin secretion. Methods GPR40/FFA1 expression was detected by immunofluorescence imaging. We employed linoleic acid (LA), a free fatty acid that has a high affinity to the rat GPR40, and examined its effect on cytosolic free calcium concentration ([Ca2+]i) in primary rat β-cells by Fluo-3 intensity under confocal microscopy recording. Downregulation of GPR40/FFA1 expression by antisense oligonucleotides was performed in pancreatic β-cells, and insulin secretion was assessed by enzyme-linked immunosorbent assay. Results LA acutely stimulated insulin secretion from primary cultured rat pancreatic islets. LA induced significant increase of [Ca2+]i in the presence of 5.6 mmol/L and 11.1 mmol/L glucose, which was reflected by increased Fluo-3 intensity under confocal microscopy recording. LA-stimulated increase in [Ca2+]i and insulin secretion were blocked by inhibition of GPR40/FFA1 expression in β-cells after GPR40/FFA1-specific antisense treatment. In addition, the inhibition of phospholipase C (PLC) activity by U73122, PLC inhibitor, also markedly inhibited the LA-induced [Ca2+]i increase. Conclusion LA activates GPR40/FFA1 and PLC to stimulate Ca2+ release, resulting in an increase in [Ca2+]i and insulin secretion in rat islet β-cells.展开更多
背景与目的:磷脂酶C-γ1(phospholipase C gamma1,PLC-γ1)是跨膜信号转导中关键和重要的一个信号中介,是细胞增殖与细胞凋亡调控的一个重要分子,最近研究发现它在大肠癌等许多肿瘤组织中呈过表达状态,与肿瘤的发生、发展有密切关系。...背景与目的:磷脂酶C-γ1(phospholipase C gamma1,PLC-γ1)是跨膜信号转导中关键和重要的一个信号中介,是细胞增殖与细胞凋亡调控的一个重要分子,最近研究发现它在大肠癌等许多肿瘤组织中呈过表达状态,与肿瘤的发生、发展有密切关系。本研究主要探讨阻断PLC-γ1信号通路后对大肠癌LoVo细胞增殖、凋亡的影响,及其上述影响的信号机制。方法:以人大肠癌LoVo细胞作为研究模型,利用PLC-γ1特异的化学阻断剂U73122处理以阻断LoVo细胞中PLC-γ1信号通路,通过绘制细胞生长曲线、PI单染的流式细胞仪检测细胞周期而评估对其细胞增殖的影响,通过细胞形态观察及DNA片段琼脂糖凝胶电泳评估是否启动细胞凋亡,同时检测阻断PLC-γ1信号通路后HSP70、Caspase-3表达水平的变化来探讨可能的信号机制。结果:阻断PLC-γ1信号通路明显减缓大肠癌LoVo细胞的生长,其细胞增殖抑制率随药物作用时间和浓度的增加逐渐增高,10μmol/LU73122作用24、48h后其抑制效果可分别达到35%和45%,使LoVo细胞G1期细胞比例增加,而S期细胞比例降低,延缓细胞从G1期向S期的过渡,即抑制细胞周期的进行,阻断PLC-γ1信号通路后LoVo细胞未能出现凋亡特征性的形态学改变,DNA琼脂糖凝胶电泳未能检测到凋亡特征的梯状带的出现,不能引起Caspase-3的激活,同时PLC-γ1信号通路的阻断可上调HSP70的表达水平,HSP70的分子伴侣作用可能是其抑制大肠癌细胞周期进行的机制。结论:阻断磷脂酶C-γ1信号通路能够抑制大肠癌LoVo细胞的过度增殖、抑制其细胞周期的进行,其机制可能是通过上调热休克蛋白70的表达水平而实现,但不能启动LoVo细胞凋亡,磷脂酶C-γ1不是调控LoVo细胞凋亡的关键信号分子。展开更多
基金Supported by Grant from Department of Education of Liaoning Province (2008810)
文摘Objective To elucidate GPR40/FFA1 and its downstream signaling pathways in regulating insulin secretion. Methods GPR40/FFA1 expression was detected by immunofluorescence imaging. We employed linoleic acid (LA), a free fatty acid that has a high affinity to the rat GPR40, and examined its effect on cytosolic free calcium concentration ([Ca2+]i) in primary rat β-cells by Fluo-3 intensity under confocal microscopy recording. Downregulation of GPR40/FFA1 expression by antisense oligonucleotides was performed in pancreatic β-cells, and insulin secretion was assessed by enzyme-linked immunosorbent assay. Results LA acutely stimulated insulin secretion from primary cultured rat pancreatic islets. LA induced significant increase of [Ca2+]i in the presence of 5.6 mmol/L and 11.1 mmol/L glucose, which was reflected by increased Fluo-3 intensity under confocal microscopy recording. LA-stimulated increase in [Ca2+]i and insulin secretion were blocked by inhibition of GPR40/FFA1 expression in β-cells after GPR40/FFA1-specific antisense treatment. In addition, the inhibition of phospholipase C (PLC) activity by U73122, PLC inhibitor, also markedly inhibited the LA-induced [Ca2+]i increase. Conclusion LA activates GPR40/FFA1 and PLC to stimulate Ca2+ release, resulting in an increase in [Ca2+]i and insulin secretion in rat islet β-cells.
文摘背景与目的:磷脂酶C-γ1(phospholipase C gamma1,PLC-γ1)是跨膜信号转导中关键和重要的一个信号中介,是细胞增殖与细胞凋亡调控的一个重要分子,最近研究发现它在大肠癌等许多肿瘤组织中呈过表达状态,与肿瘤的发生、发展有密切关系。本研究主要探讨阻断PLC-γ1信号通路后对大肠癌LoVo细胞增殖、凋亡的影响,及其上述影响的信号机制。方法:以人大肠癌LoVo细胞作为研究模型,利用PLC-γ1特异的化学阻断剂U73122处理以阻断LoVo细胞中PLC-γ1信号通路,通过绘制细胞生长曲线、PI单染的流式细胞仪检测细胞周期而评估对其细胞增殖的影响,通过细胞形态观察及DNA片段琼脂糖凝胶电泳评估是否启动细胞凋亡,同时检测阻断PLC-γ1信号通路后HSP70、Caspase-3表达水平的变化来探讨可能的信号机制。结果:阻断PLC-γ1信号通路明显减缓大肠癌LoVo细胞的生长,其细胞增殖抑制率随药物作用时间和浓度的增加逐渐增高,10μmol/LU73122作用24、48h后其抑制效果可分别达到35%和45%,使LoVo细胞G1期细胞比例增加,而S期细胞比例降低,延缓细胞从G1期向S期的过渡,即抑制细胞周期的进行,阻断PLC-γ1信号通路后LoVo细胞未能出现凋亡特征性的形态学改变,DNA琼脂糖凝胶电泳未能检测到凋亡特征的梯状带的出现,不能引起Caspase-3的激活,同时PLC-γ1信号通路的阻断可上调HSP70的表达水平,HSP70的分子伴侣作用可能是其抑制大肠癌细胞周期进行的机制。结论:阻断磷脂酶C-γ1信号通路能够抑制大肠癌LoVo细胞的过度增殖、抑制其细胞周期的进行,其机制可能是通过上调热休克蛋白70的表达水平而实现,但不能启动LoVo细胞凋亡,磷脂酶C-γ1不是调控LoVo细胞凋亡的关键信号分子。
基金Supported by Guangdong Natural Science Foundation(No.S2012010008170)Specialized Research Fund for the Doctoral Program of Higher Education(No.20120001120096)National Basic Research Program of China(973 Program,No.2014CB910200)~~
