探討胰島素-mTOR通路對小鼠白色前脂肪細胞成脂分化的影響
發(fā)布時間：2019-04-04 12:14
【摘要】：研究表明,胰島素對動物脂肪細胞增殖、分化和糖脂代謝均發(fā)揮重要作用,其對前脂肪細胞成脂分化的調控作用是通過影響mTORC1的活化以及GLUT-4的轉運效率等實現(xiàn)的。然而目前胰島素發(fā)揮調控前脂肪細胞分化的主要途徑尚未明確�；谇捌趯嶒灲Y果,我們猜測mTOR信號通路是胰島素發(fā)揮調控前脂肪細胞成脂分化的主要途徑。為了驗證該假設,我們體外誘導了Lepr~(db/db)肥胖與WT非肥胖小鼠白色皮下原代脂肪干細胞(sASCs)成脂分化,將其分為4組:單純成脂誘導組、成脂誘導加mTORC1抑制劑(A-769662)組、成脂誘導加GLUT-4抑制劑(細辛脂素)組以及成脂誘導加mTORC1及GLUT-4雙抑制組。使用qPCR以及Western blot技術分別檢測mTOR、GLUT-4和PPARγ2的表達變化特征。實驗結果總結如下。1.mTORC1抑制組(1)Leprdb/db小鼠sASCs成脂抑制率為(50.32±3.17)%;m TOR基因表達量無顯著變化,而其蛋白磷酸化水平下降了(69.13±4.26)%;GLUT-4基因表達量和蛋白水平均無顯著變化;PPARγ2基因和蛋白水平分別下降了(74.62±4.48)%和(61.52±2.28)%。(2)WT小鼠sASCs成脂抑制率為(60.17±3.45)%;mTOR基因表達量無顯著變化,而其蛋白磷酸化水平下降了(66.22±4.66)%;GLUT-4基因表達量和蛋白水平均無顯著變化;PPARγ2基因和蛋白水平分別下降(68.73±4.87)%和(71.68±2.12)%。2.GLUT-4抑制組(1)Leprdb/db小鼠sASCs成脂抑制率為(30.44±2.21)%;mTOR基因表達量且其蛋白磷酸化水平均無顯著變化;GLUT-4基因表達量上調了(98.26±5.56)%,而其蛋白水平則下降了(70.41±3.57)%;PPARγ2基因和蛋白水平分別下降了(42.44±4.10)%和(46.24±2.99)%。(2)WT小鼠s ASCs成脂抑制率為(35.43±2.72)%;mTOR基因表達量及其蛋白磷酸化水平均無顯著變化;GLUT-4基因表達量上調了(88.14±4.43)%,而其蛋白水平則下降了(64.57±3.23)%;PPARγ2基因和蛋白水平分別下降了(48.62±4.17)%和(49.35±3.54)%。3.mTORC1及GLUT-4雙抑制組(1)Leprdb/db小鼠sASCs成脂抑制率為(70.41±3.17)%;m TOR基因表達量無顯著變化,但其蛋白磷酸化水平則下降了(72.06±2.28)%;GLUT-4基因表達量上調了(102.31±4.46)%,但其蛋白水平則下降了(71.43±3.68)%;PPARγ2基因及蛋白水平分別下降了(78.53±5.06)%和(84.48±2.30)%。(2)WT小鼠s ASCs成脂抑制率為(70.75±3.17)%;mTOR基因表達量無顯著變化,但其蛋白磷酸化水平則下降了(63.46±4.33)%;GLUT-4基因表達量上調了(87.02±5.54)%,但其蛋白水平下降了(62.83±4.16)%,PPARγ2基因及蛋白水平分別下降了(73.31±4.25)%和(76.56±3.65)%。綜合以上分別抑制胰島素信號通路不同關鍵分子的實驗結果,我們證明胰島素信號可能主要通過影響m TOR信號通路發(fā)揮對小鼠前脂肪細胞成脂分化的調控作用,且胰島素-mTOR信號通路對于WT小鼠sASCs成脂分化的主導作用更為明顯。
[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 緯 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 鹵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 鹵4.26)%. There was no significant change in the expression and protein level of GLUT-4 gene, but the level of PPAR 緯 2 gene and protein decreased by (74.62 鹵4.48)% and (61.52 鹵2.28)%, respectively. (2) the inhibition rate of sASCs lipid formation in WT mice was (60.17 鹵3.45)%. The protein phosphorylation level of mTOR gene decreased by (66.22 鹵4.66)%, and there was no significant change in GLUT-4 gene expression and protein level. The levels of PPAR 緯 2 gene and protein decreased by (68.73 鹵4.87)% and (71.68 鹵2.12)%, respectively, and the inhibition rate of sASCs lipid formation in Leprdb/db mice was (30.44 鹵2.21)% in the 2.GLUT-4 inhibition group (1). The expression of mTOR gene was up-regulated by (98.26 鹵5.56)%, while the protein level of GLUT-4 gene decreased by (70.41 鹵3.57)%. The levels of PPAR 緯 2 gene and protein decreased by (42.44 鹵4.10)% and (46.24 鹵2.99)%, respectively. (2) the inhibition rate of s ASCs lipid formation in WT mice was (35.43 鹵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 鹵4.43)%, while its protein level decreased by (64.57 鹵3.23)%. The levels of PPAR 緯 2 gene and protein decreased by (48.62 鹵4.17)% and (49.35 鹵3.54)%, respectively. (1) the inhibition rate of sASCs lipid formation in Leprdb/db mice was (70.41 鹵3.17)% in both groups of mTORC1 and GLUT-4. The protein phosphorylation level of m-TOR gene was decreased by (72.06 鹵2.28)%, and the expression of GLUT-4 gene was up-regulated by (102.31 鹵4.46)%, but its protein level decreased by (71.43 鹵3.68)%. The levels of PPAR 緯 2 gene and protein decreased by (78.53 鹵5.06)% and (84.48 鹵2.30)%, respectively. (2) the inhibition rate of ASCs lipid formation in WT mice was (70.75 鹵3.17)%. There was no significant change in the expression of mTOR gene, but the protein phosphorylation level decreased by (63.46 鹵4.33)%. The expression of GLUT-4 gene was up-regulated by (87.02 鹵5.54)%, but its protein level decreased by (62.83 鹵4.16)%, and the level of PPAR 緯 2 gene and protein decreased by (73.31 鹵4.25)% and (76.56 鹵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.
【學位授予單位】：山東師范大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R589.2
本文編號：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 緯 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 鹵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 鹵4.26)%. There was no significant change in the expression and protein level of GLUT-4 gene, but the level of PPAR 緯 2 gene and protein decreased by (74.62 鹵4.48)% and (61.52 鹵2.28)%, respectively. (2) the inhibition rate of sASCs lipid formation in WT mice was (60.17 鹵3.45)%. The protein phosphorylation level of mTOR gene decreased by (66.22 鹵4.66)%, and there was no significant change in GLUT-4 gene expression and protein level. The levels of PPAR 緯 2 gene and protein decreased by (68.73 鹵4.87)% and (71.68 鹵2.12)%, respectively, and the inhibition rate of sASCs lipid formation in Leprdb/db mice was (30.44 鹵2.21)% in the 2.GLUT-4 inhibition group (1). The expression of mTOR gene was up-regulated by (98.26 鹵5.56)%, while the protein level of GLUT-4 gene decreased by (70.41 鹵3.57)%. The levels of PPAR 緯 2 gene and protein decreased by (42.44 鹵4.10)% and (46.24 鹵2.99)%, respectively. (2) the inhibition rate of s ASCs lipid formation in WT mice was (35.43 鹵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 鹵4.43)%, while its protein level decreased by (64.57 鹵3.23)%. The levels of PPAR 緯 2 gene and protein decreased by (48.62 鹵4.17)% and (49.35 鹵3.54)%, respectively. (1) the inhibition rate of sASCs lipid formation in Leprdb/db mice was (70.41 鹵3.17)% in both groups of mTORC1 and GLUT-4. The protein phosphorylation level of m-TOR gene was decreased by (72.06 鹵2.28)%, and the expression of GLUT-4 gene was up-regulated by (102.31 鹵4.46)%, but its protein level decreased by (71.43 鹵3.68)%. The levels of PPAR 緯 2 gene and protein decreased by (78.53 鹵5.06)% and (84.48 鹵2.30)%, respectively. (2) the inhibition rate of ASCs lipid formation in WT mice was (70.75 鹵3.17)%. There was no significant change in the expression of mTOR gene, but the protein phosphorylation level decreased by (63.46 鹵4.33)%. The expression of GLUT-4 gene was up-regulated by (87.02 鹵5.54)%, but its protein level decreased by (62.83 鹵4.16)%, and the level of PPAR 緯 2 gene and protein decreased by (73.31 鹵4.25)% and (76.56 鹵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.
【學位授予單位】：山東師范大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R589.2
【參考文獻】
相關期刊論文 前2條
1 唐海雙;王清松;焦炳華;楊生生;;GLUT4在胰島素調控葡萄糖轉運中作用[J];生命的化學;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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