探讨胰岛素-mTOR通路对小鼠白色前脂肪细胞成脂分化的影响
发布时间：2019-04-04 12:14
【摘要】：研究表明,胰岛素对动物脂肪细胞增殖、分化和糖脂代谢均发挥重要作用,其对前脂肪细胞成脂分化的调控作用是通过影响mTORC1的活化以及GLUT-4的转运效率等实现的。然而目前胰岛素发挥调控前脂肪细胞分化的主要途径尚未明确。基于前期实验结果,我们猜测mTOR信号通路是胰岛素发挥调控前脂肪细胞成脂分化的主要途径。为了验证该假设,我们体外诱导了Lepr~(db/db)肥胖与WT非肥胖小鼠白色皮下原代脂肪干细胞(sASCs)成脂分化,将其分为4组:单纯成脂诱导组、成脂诱导加mTORC1抑制剂(A-769662)组、成脂诱导加GLUT-4抑制剂(细辛脂素)组以及成脂诱导加mTORC1及GLUT-4双抑制组。使用qPCR以及Western blot技术分别检测mTOR、GLUT-4和PPARγ2的表达变化特征。实验结果总结如下。1.mTORC1抑制组(1)Leprdb/db小鼠sASCs成脂抑制率为(50.32±3.17)%;m TOR基因表达量无显著变化,而其蛋白磷酸化水平下降了(69.13±4.26)%;GLUT-4基因表达量和蛋白水平均无显著变化;PPARγ2基因和蛋白水平分别下降了(74.62±4.48)%和(61.52±2.28)%。(2)WT小鼠sASCs成脂抑制率为(60.17±3.45)%;mTOR基因表达量无显著变化,而其蛋白磷酸化水平下降了(66.22±4.66)%;GLUT-4基因表达量和蛋白水平均无显著变化;PPARγ2基因和蛋白水平分别下降(68.73±4.87)%和(71.68±2.12)%。2.GLUT-4抑制组(1)Leprdb/db小鼠sASCs成脂抑制率为(30.44±2.21)%;mTOR基因表达量且其蛋白磷酸化水平均无显著变化;GLUT-4基因表达量上调了(98.26±5.56)%,而其蛋白水平则下降了(70.41±3.57)%;PPARγ2基因和蛋白水平分别下降了(42.44±4.10)%和(46.24±2.99)%。(2)WT小鼠s ASCs成脂抑制率为(35.43±2.72)%;mTOR基因表达量及其蛋白磷酸化水平均无显著变化;GLUT-4基因表达量上调了(88.14±4.43)%,而其蛋白水平则下降了(64.57±3.23)%;PPARγ2基因和蛋白水平分别下降了(48.62±4.17)%和(49.35±3.54)%。3.mTORC1及GLUT-4双抑制组(1)Leprdb/db小鼠sASCs成脂抑制率为(70.41±3.17)%;m TOR基因表达量无显著变化,但其蛋白磷酸化水平则下降了(72.06±2.28)%;GLUT-4基因表达量上调了(102.31±4.46)%,但其蛋白水平则下降了(71.43±3.68)%;PPARγ2基因及蛋白水平分别下降了(78.53±5.06)%和(84.48±2.30)%。(2)WT小鼠s ASCs成脂抑制率为(70.75±3.17)%;mTOR基因表达量无显著变化,但其蛋白磷酸化水平则下降了(63.46±4.33)%;GLUT-4基因表达量上调了(87.02±5.54)%,但其蛋白水平下降了(62.83±4.16)%,PPARγ2基因及蛋白水平分别下降了(73.31±4.25)%和(76.56±3.65)%。综合以上分别抑制胰岛素信号通路不同关键分子的实验结果,我们证明胰岛素信号可能主要通过影响m TOR信号通路发挥对小鼠前脂肪细胞成脂分化的调控作用,且胰岛素-mTOR信号通路对于WT小鼠sASCs成脂分化的主导作用更为明显。
[Abstract]:The results show that insulin plays an important role in the proliferation, differentiation and glucose-lipid metabolism of adipocytes. The regulation of insulin on adipocyte adipogenesis is achieved by affecting the activation of mTORC1 and the transport efficiency of GLUT-4. However, the main pathway of insulin regulation of preadipocyte differentiation is not clear. Based on the previous results, we hypothesized that mTOR signaling pathway is the main pathway of insulin regulation of adipocyte adipogenesis. In order to test this hypothesis, we induced adipogenic differentiation of Lepr~ (db/db) and WT non-obese white subcutaneous adipose stem cells (sASCs) in vitro, and divided them into four groups: adipogenic induction group; Lipogenic induction plus mTORC1 inhibitor group, lipogenic induction plus GLUT-4 inhibitor group and lipogenic induction plus mTORC1 and GLUT-4 double inhibition group. The expression characteristics of mTOR,GLUT-4 and PPAR 纬 2 were detected by qPCR and Western blot, respectively. The results were summarized as follows: 1. The inhibition rate of sASCs lipid formation in Leprdb/db mice was (50.32 卤3.17)% in mTORC1 inhibition group, while the expression of mTOR gene did not change significantly, but the protein phosphorylation level decreased by (69.13 卤4.26)%. There was no significant change in the expression and protein level of GLUT-4 gene, but the level of PPAR 纬 2 gene and protein decreased by (74.62 卤4.48)% and (61.52 卤2.28)%, respectively. (2) the inhibition rate of sASCs lipid formation in WT mice was (60.17 卤3.45)%. The protein phosphorylation level of mTOR gene decreased by (66.22 卤4.66)%, and there was no significant change in GLUT-4 gene expression and protein level. The levels of PPAR 纬 2 gene and protein decreased by (68.73 卤4.87)% and (71.68 卤2.12)%, respectively, and the inhibition rate of sASCs lipid formation in Leprdb/db mice was (30.44 卤2.21)% in the 2.GLUT-4 inhibition group (1). The expression of mTOR gene was up-regulated by (98.26 卤5.56)%, while the protein level of GLUT-4 gene decreased by (70.41 卤3.57)%. The levels of PPAR 纬 2 gene and protein decreased by (42.44 卤4.10)% and (46.24 卤2.99)%, respectively. (2) the inhibition rate of s ASCs lipid formation in WT mice was (35.43 卤2.72)%, and the expression of mTOR gene and its protein phosphorylation level had no significant change. The expression of GLUT-4 gene was up-regulated by (88.14 卤4.43)%, while its protein level decreased by (64.57 卤3.23)%. The levels of PPAR 纬 2 gene and protein decreased by (48.62 卤4.17)% and (49.35 卤3.54)%, respectively. (1) the inhibition rate of sASCs lipid formation in Leprdb/db mice was (70.41 卤3.17)% in both groups of mTORC1 and GLUT-4. The protein phosphorylation level of m-TOR gene was decreased by (72.06 卤2.28)%, and the expression of GLUT-4 gene was up-regulated by (102.31 卤4.46)%, but its protein level decreased by (71.43 卤3.68)%. The levels of PPAR 纬 2 gene and protein decreased by (78.53 卤5.06)% and (84.48 卤2.30)%, respectively. (2) the inhibition rate of ASCs lipid formation in WT mice was (70.75 卤3.17)%. There was no significant change in the expression of mTOR gene, but the protein phosphorylation level decreased by (63.46 卤4.33)%. The expression of GLUT-4 gene was up-regulated by (87.02 卤5.54)%, but its protein level decreased by (62.83 卤4.16)%, and the level of PPAR 纬 2 gene and protein decreased by (73.31 卤4.25)% and (76.56 卤3.65)%, respectively. Based on the above experimental results of inhibiting different key molecules of insulin signaling pathway, we have demonstrated that insulin signal may play a regulatory role in adipocyte adipogenesis by affecting m-TOR signaling pathway, and it is suggested that insulin signaling may play an important role in regulating adipocyte differentiation. Moreover, insulin-mTOR signaling pathway plays a more important role in adipogenic differentiation of sASCs in WT mice.
【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R589.2
本文编号：2453798
[Abstract]:The results show that insulin plays an important role in the proliferation, differentiation and glucose-lipid metabolism of adipocytes. The regulation of insulin on adipocyte adipogenesis is achieved by affecting the activation of mTORC1 and the transport efficiency of GLUT-4. However, the main pathway of insulin regulation of preadipocyte differentiation is not clear. Based on the previous results, we hypothesized that mTOR signaling pathway is the main pathway of insulin regulation of adipocyte adipogenesis. In order to test this hypothesis, we induced adipogenic differentiation of Lepr~ (db/db) and WT non-obese white subcutaneous adipose stem cells (sASCs) in vitro, and divided them into four groups: adipogenic induction group; Lipogenic induction plus mTORC1 inhibitor group, lipogenic induction plus GLUT-4 inhibitor group and lipogenic induction plus mTORC1 and GLUT-4 double inhibition group. The expression characteristics of mTOR,GLUT-4 and PPAR 纬 2 were detected by qPCR and Western blot, respectively. The results were summarized as follows: 1. The inhibition rate of sASCs lipid formation in Leprdb/db mice was (50.32 卤3.17)% in mTORC1 inhibition group, while the expression of mTOR gene did not change significantly, but the protein phosphorylation level decreased by (69.13 卤4.26)%. There was no significant change in the expression and protein level of GLUT-4 gene, but the level of PPAR 纬 2 gene and protein decreased by (74.62 卤4.48)% and (61.52 卤2.28)%, respectively. (2) the inhibition rate of sASCs lipid formation in WT mice was (60.17 卤3.45)%. The protein phosphorylation level of mTOR gene decreased by (66.22 卤4.66)%, and there was no significant change in GLUT-4 gene expression and protein level. The levels of PPAR 纬 2 gene and protein decreased by (68.73 卤4.87)% and (71.68 卤2.12)%, respectively, and the inhibition rate of sASCs lipid formation in Leprdb/db mice was (30.44 卤2.21)% in the 2.GLUT-4 inhibition group (1). The expression of mTOR gene was up-regulated by (98.26 卤5.56)%, while the protein level of GLUT-4 gene decreased by (70.41 卤3.57)%. The levels of PPAR 纬 2 gene and protein decreased by (42.44 卤4.10)% and (46.24 卤2.99)%, respectively. (2) the inhibition rate of s ASCs lipid formation in WT mice was (35.43 卤2.72)%, and the expression of mTOR gene and its protein phosphorylation level had no significant change. The expression of GLUT-4 gene was up-regulated by (88.14 卤4.43)%, while its protein level decreased by (64.57 卤3.23)%. The levels of PPAR 纬 2 gene and protein decreased by (48.62 卤4.17)% and (49.35 卤3.54)%, respectively. (1) the inhibition rate of sASCs lipid formation in Leprdb/db mice was (70.41 卤3.17)% in both groups of mTORC1 and GLUT-4. The protein phosphorylation level of m-TOR gene was decreased by (72.06 卤2.28)%, and the expression of GLUT-4 gene was up-regulated by (102.31 卤4.46)%, but its protein level decreased by (71.43 卤3.68)%. The levels of PPAR 纬 2 gene and protein decreased by (78.53 卤5.06)% and (84.48 卤2.30)%, respectively. (2) the inhibition rate of ASCs lipid formation in WT mice was (70.75 卤3.17)%. There was no significant change in the expression of mTOR gene, but the protein phosphorylation level decreased by (63.46 卤4.33)%. The expression of GLUT-4 gene was up-regulated by (87.02 卤5.54)%, but its protein level decreased by (62.83 卤4.16)%, and the level of PPAR 纬 2 gene and protein decreased by (73.31 卤4.25)% and (76.56 卤3.65)%, respectively. Based on the above experimental results of inhibiting different key molecules of insulin signaling pathway, we have demonstrated that insulin signal may play a regulatory role in adipocyte adipogenesis by affecting m-TOR signaling pathway, and it is suggested that insulin signaling may play an important role in regulating adipocyte differentiation. Moreover, insulin-mTOR signaling pathway plays a more important role in adipogenic differentiation of sASCs in WT mice.
【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R589.2
【参考文献】
相关期刊论文 前2条
1 唐海双;王清松;焦炳华;杨生生;;GLUT4在胰岛素调控葡萄糖转运中作用[J];生命的化学;2014年02期
2 Eun-Hee Kim;Chan Yeong Heo;;Current applications of adipose-derived stem cells and their future perspectives[J];World Journal of Stem Cells;2014年01期
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