G蛋白偶联受体40在游离脂肪酸影响小鼠胰岛NIT-1细胞增殖中的作用

目的 探讨G蛋白偶联受体40在游离脂肪酸影响小鼠胰岛NIT-1细胞增殖中的作用.方法 细胞分为空白对照组、棕榈酸组、硬脂酸组、油酸组、亚油酸组、棕榈酸+油酸组,各组采用游离脂肪酸孵育12、48 h,比较各组干预对NIT-1细胞增殖的影响,细胞增殖采用CCK-8法、5'-溴-2脱氧尿苷(Brdu)-ELISA法检测.转染G蛋白偶联受体40 siRNA以抑制G蛋白偶联受体40在NIT-1细胞表达,细胞分为空转组、对照siRNA转染组、G蛋白偶联受体40 siRNA转染组,分别观察不同游离脂肪酸干预12、48 h后细胞增殖情况.应用Western blot观察游离脂肪酸孵育及抑制G蛋白偶联受体40表达对Egr-1表达的影响.采用方差分析进行统计学分析.结果 各组游离脂肪酸孵育12 h均可促进NIT-1细胞生长.棕榈酸及硬脂酸孵育48 h后细胞增殖较对照组明显下降,油酸及亚油酸孵育48 h后仍促进细胞增殖.棕榈酸和油酸共孵育48 h后,细胞增殖较棕榈酸、硬脂酸单独孵育明显增加.抑制G蛋白偶联受体40表达后,分别予棕榈酸、硬脂酸孵育NIT-1细胞12、48 h,G蛋白偶联受体40 siRNA转染组细胞吸光度值较空转组无明显差异;予油酸、亚油酸孵育NIT-1细胞12、48 h,G蛋白偶联受体40 siRNA转染组细胞吸光度值与空转组相比明显减少.G蛋白偶联受体40 siRNA转染组细胞子棕榈酸/油酸共孵育12、48 h后,吸光度值较空转组下降(CCK-8法:q值分别为7.834、8.236,均P<0.05;Brdu-ELISA法:q值分别为7.981、5.376,均P<0.05).Western blot显示棕榈酸孵育对Egr-1蛋白表达水平无明显影响,油酸孵育后可见Egr-1表达明显上调,抑制G蛋白偶联受体40表达后,油酸刺激Egr-1水平较空转组明显降低.结论 饱和脂肪酸对胰岛β细胞的双向调节作用不依赖G蛋白偶联受体40;而不饱和脂肪酸对胰岛B细胞的增殖促进作用及对饱和脂肪酸长期负性作用的抑制至少部分通过G蛋白偶联受体40介导,这一作用伴有Egr-1表达上调.上述结果提示G蛋白偶联受体40可能参与调节胰岛B细胞代偿.

G-protein-coupled receptor 40 mediates the effects of free fatty acid on proliferation in mouse islet beta-cell line NIT-1

Objective To investigate whether G-pretein-couple receptor 40 (GPR40) mediates the effects of free fatty acid ( FFA) on proliferation of mouse insulin NIT-1 cells. Methods The expression of GPR40 in NIT-1 cells was inhibited by siRNA, and the effects of 12 or 48 h incubation with saturated fatty acid (SFA) and unsaturated fatty acid (UFA)on proliferation of NIT-1 cell transfected with GPR40 siRNA were observed. Cell proliferation was detected by cell counting kit-8 and Brdu-ELISA methods. The expression of Egr-1 in NIT-1 cells transfected with GPR40 siRNA or treated with FFAs was examined by Western blot. Analysis of variance was used for data analysis. Results A 12-h exposure to different groups of FFA and 48-h exposure to oleate or linoleic acid stimulated the proliferation of NIT-1 cells, whereas 48-h exposure to palmitate or sterate inhibited the proliferation of NIT-1 cells. After co-incubation with palmitate and oleate for 48 h, the proliferation of NIT-1 cells was significantly increased when compared with that in palmitate or sterate treated cells. The mock-, control siRNA-, and GPR40 siRNA-transfected cells were supplemented with saturated fatty acids (palmitate or sterate), no significant difference in the absorbance values was indicated between the GPR40 siRNA tranfected cells and mock. Incubated with oleate or linoleic acid for 12 and 48 h, the absorbance values of GPR40 siRNA tranfected cells were less than that of the mock. After co-incubation with palmitate and oleate for 12 and 48 h, the absorbance values of GPR40 siRNA tranfected cells were less than that of mock (CCK-8: q was 7.834 and 8.236,P<0.05;Brdu-ELISA:q was 7.981 and 5.376, P<0.05). Western blot showed that Egr-1 was markedly activated by oleate, which was significantly inhibited by transfection with GPR40 siRNA. Conclusion The bilateral effects of SFA may be GPR40 independent UFA promotes islet beta-cell proliferation, and counteracts the negative effects of SFA, which is mediated at least in part through GPR40; the process is accompanied by increased expression of Egr-1. Our data suggest that GPR40 might be implicated in the control of beta-cell compensation and GPR40 probably provide a link between obesity and type 2 diabetes mellitus.