探討胰島素-mTOR通路對(duì)小鼠白色前脂肪細(xì)胞成脂分化的影響
發(fā)布時(shí)間：2019-04-04 12:14
【摘要】：研究表明,胰島素對(duì)動(dòng)物脂肪細(xì)胞增殖、分化和糖脂代謝均發(fā)揮重要作用,其對(duì)前脂肪細(xì)胞成脂分化的調(diào)控作用是通過影響mTORC1的活化以及GLUT-4的轉(zhuǎn)運(yùn)效率等實(shí)現(xiàn)的。然而目前胰島素發(fā)揮調(diào)控前脂肪細(xì)胞分化的主要途徑尚未明確�；谇捌趯�(shí)驗(yàn)結(jié)果,我們猜測(cè)mTOR信號(hào)通路是胰島素發(fā)揮調(diào)控前脂肪細(xì)胞成脂分化的主要途徑。為了驗(yàn)證該假設(shè),我們體外誘導(dǎo)了Lepr~(db/db)肥胖與WT非肥胖小鼠白色皮下原代脂肪干細(xì)胞(sASCs)成脂分化,將其分為4組:單純成脂誘導(dǎo)組、成脂誘導(dǎo)加mTORC1抑制劑(A-769662)組、成脂誘導(dǎo)加GLUT-4抑制劑(細(xì)辛脂素)組以及成脂誘導(dǎo)加mTORC1及GLUT-4雙抑制組。使用qPCR以及Western blot技術(shù)分別檢測(cè)mTOR、GLUT-4和PPARγ2的表達(dá)變化特征。實(shí)驗(yàn)結(jié)果總結(jié)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(dá)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(dá)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(dá)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(dá)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(dá)量且其蛋白磷酸化水平均無顯著變化;GLUT-4基因表達(dá)量上調(diào)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(dá)量及其蛋白磷酸化水平均無顯著變化;GLUT-4基因表達(dá)量上調(diào)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(dá)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(dá)量上調(diào)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(dá)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(dá)量上調(diào)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(hào)通路不同關(guān)鍵分子的實(shí)驗(yàn)結(jié)果,我們證明胰島素信號(hào)可能主要通過影響m TOR信號(hào)通路發(fā)揮對(duì)小鼠前脂肪細(xì)胞成脂分化的調(diào)控作用,且胰島素-mTOR信號(hào)通路對(duì)于WT小鼠sASCs成脂分化的主導(dǎo)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(xué)位授予單位】：山東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R589.2
本文編號(hào)：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(xué)位授予單位】：山東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R589.2
【參考文獻(xiàn)】
相關(guān)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(diào)控葡萄糖轉(zhuǎn)運(yùn)中作用[J];生命的化學(xué);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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