Translocation and Expression of GLUT-4 in Bone Marrow Mesenchymal Stem Cells Transfected with Akt Gene of Rat Ex Vivo_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

SHAO Lei ¹ , ZHOU Shuchun ¹ , XU Guiping ² ,  KONG Hongliang ²

1.Department of Cardiology, The Second Affiliated Hospital, Zhengzhou University, Zhengzhou 450014, China;;
2.Department of Cardiology, Liaoning Provincial People’s Hospital, Shenyang 110016, China;

Corresponding author：

KONG Hongliang, Email: khl339@sina.com

Keywords：

Rat; Bone marrow mesenchymal stem cells; Akt gene transfection; Hypoxia; 2-［3H］-deoxy-D-glucose intake; Glucose transporters-4

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Objective To elucidate whether glucose transporters-4 （GLUT-4） takes part in glucose uptake of mesenchymal stem cells （MSCs） and whether Akt gene improves translocation and expression of GLUT-4 in MSCs under hypoxic environment ex vivo.
Methods MSCs, transfected by Akt gene and no, were cultured with normoxia (5% CO2) or hypoxia (94%N2, 1%O2 and 5% CO2) at 37 ℃ for 8 h. Glucose uptake was assayed by using radiation isotope 2-［3H］-deoxy-Dglucose (3H-G) and the expression of GLUT-4 protein and mRNA was assayed by immunocytochemistry, Western blot and RT-PCR, respectively.
Results ①3 H-G intake of MSCs was significantly increased in hypoxiatransfection group than that in hypoxia-non-transfection 〔（1.39±0.13） fold, P＜0.05〕, but which was lower than that in normoxia-non-transfection group, P＜0.05. ②GLUT-4 was expressed by MSCs under any conditions. Compared with normoxia-non-transfection group, hypoxia decreased the expressions of GLUT-4 mRNA and protein significantly (P＜0.05). ③Compared with hypoxianontransfection group, the expression of GLUT-4 〔mRNA（1.756±0.152） fold, total protein in cell （1.653±0.312） fold, protein in plasma membrane （2.041±0.258） fold〕 was increased in hypoxia-transfection group significantly (P＜0.05), but which was lower than that in normoxianontransfection group (P＜0.05). ④There was significantly positive relation between 3H-G intake and GLUT-4 protein expression in plasma membrane (r=0.415, P=0.001).
Conclusion GLUT-4 may take part in glucose uptake of MSCs, and the capability of Akt gene to improve MSCs anti-hypoxia may be finished by its role in increasing the expression and translocation of GLUT-4.

Citation： SHAO Lei ,ZHOU Shuchun,XU Guiping,KONG Hongliang. Translocation and Expression of GLUT-4 in Bone Marrow Mesenchymal Stem Cells Transfected with Akt Gene of Rat Ex Vivo. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2010, 17(1): 52-56. doi: Copy

1.	Ernens I, Goodfellow SJ, Innes F, et al. Hypoxic stress suppresses RNA polymerase Ⅲ recruitment and tRNA gene transcription in cardiomyocytes[Ｊ]. Nucleic Acids Res, 2006; 34(1): 286２94.
2.	Ullah MS, Davies AJ, Halestrap AP. The plasma membrane lactate transporter MCT4, but not MCT1, is upregulated by hypoxia through a HIF1αdependent mechanism [Ｊ]. J Biol Chem, 2006; 281(14): 90309037.
3.	Wollert KC, Drexler H. Clinical applications of stem cells for the heart [Ｊ]. Circ Res, 2005; 96(2): 151163.
4.	Kong HL, Liu NN, Huo X, et al. Cell multiplication, apoptosis and pAkt protein expression of bone mesenchymal stem cells of rat under hypoxia environment [Ｊ]. JNMU, 2007; 21(4): 233239.
5.	孔宏亮, 刘宁宁, 霍鑫，等. 缺氧对大鼠骨髓间充质干细胞增殖、凋亡及葡萄糖摄取和Akt表达的影响 [Ｊ].中国现代医学杂志， 2008; 18（10）： 13741382.
6.	孔宏亮, 刘宁宁, 张海，等. 缺氧环境下大鼠骨髓间充质干细胞的凋亡及Fas、FasL的表达 [Ｊ]. 中国组织化学与细胞化学杂志, 2007; 16(5): 538543.
7.	齐国先，孔宏亮，霍鑫，等. 缺氧环境下大鼠骨髓间充质干细胞凋亡相关蛋白和mRNA的表达 [Ｊ]. 中国组织化学与细胞化学杂志, 2008; 17(2): 138143.
8.	孔宏亮, 张利群, 曹善峰，等. Akt基因转染对骨髓间充质干细胞缺氧时凋亡和增殖的影响 [Ｊ]. 中国组织化学与细胞化学杂志, 2008; 17(3): 225231.
9.	霍鑫，孔宏亮，胡新华，等. pEGFPC1/Akt体外转染骨髓间充质干细胞后对血管内皮生长因子表达的影响 [Ｊ]. 中华实验外科杂志, 2008; 25(2)： 149151.
10.	霍鑫, 冯金炜, 刘方峰, 等. pEGFPC1/Akt体外转染骨髓间充质干细胞对后肢缺血大鼠血管生成的影响 [Ｊ]. 中国普外基础与临床杂志, 2008; 15(6): 402407.
11.	霍鑫, 孔宏亮, 刘方峰, 等. 无血清条件对诱导转染pEGFPC1/Akt的骨髓间充质干细胞凋亡的影响 [Ｊ]. 中国普外基础与临床杂志, 2008; 15(7): 479483.
12.	霍鑫，唐海燕，孔宏亮，等. SCF对体外转染pEGFPC1/Akt的骨髓间充质干细胞中ckit、Akt及VEGF表达的影响 [Ｊ]. 中国普外基础与临床杂志, 2008; 15(2): 116121.
13.	Hemmings BA. Akt signaling: linking membrane events to life and death decisions [Ｊ]. Science, 1997; 275(5300): 628630.
14.	Downward J. Mechanisms and consequences of activation of protein kinase B/Akt [Ｊ]. Curr Opin Cell Biol, 1998; 10(2): 262267.
15.	Foster LJ, Li D, Randhawa VK, et al. Insulin accelerates interendosomal GLUT4 traffic via phosphatidylinositol 3kinase and protein kinase B [Ｊ]. J Biol Chem, 2001; 276(47): 4421244221.
16.	Sweeney G, Garg RR, Ceddia RB, et al. Intracellular delivery of phosphatidylinositol (3,4,5)trisphosphate causes incorporation of glucose transporter 4 into the plasma membrane of muscle and fat cells without increasing glucose uptake [Ｊ]. J Biol Chem, 2004; 279(31): 3223332242.
17.	Maffucci T, Brancaccio A, Piccolo E, et al. Insulin induces phosphatidylinositol3phosphate formation through TC10 activation [Ｊ]. EMBO J, 2003; 22(16): 41784189.
18.	JeBailey L, Rudich A, Huang X, et al. Skeletal muscle cells and adipocytes differ in their reliance on TC10 and Rac for insulininduced actin remodeling [Ｊ]. Mol Endocrinol, 2004; 18(2): 359372.
19.	Kupriyanova TA, Kandror KV. Akt2 binds to Glut4containing vesicles and phosphorylates their component proteins in response to insulin [Ｊ]. J Biol Chem, 1999; 274(3): 14581464.
20.	Bae SS, Cho H, Mu J, et al. Isoformspecific regulation of insulindependent glucose uptake by Akt/protein kinase B [Ｊ]. J Biol Chem, 2003; 278(49): 4953049536.
21.	Chen X, AlHasani H, Olausson T, et al. Activity, phosphorylation state and subcellular distribution of GLUT4targeted Akt2 in rat adipose cells [Ｊ]. J Cell Sci, 2003; 116(Pt 17): 35113518.
22.	Kane S, Sano H, Liu SC, et al. A method to identify serine kinase substrates. Akt phosphorylates a novel adipocyte protein with a Rab GTPaseactivating protein (GAP) domain [Ｊ]. J Biol Chem, 2002; 277(25): 2211522118.
23.	Bae SS, Cho H, Mu J, et al. Isoformspecific regulation of insulindependent glucose uptake by Akt/protein kinase B [Ｊ]. J Biol Chem, 2003; 278(49): 4953049536.
24.	Welsh GI, Hers I, Berwick DC, et al. Role of protein kinase B in insulinregulated glucose uptake [Ｊ]. Biochem Soc Trans, 2005; 33(Pt 2): 346349.
25.	Berwick DC, Dell GC, Welsh GI, et al. Protein kinase B phosphorylation of PIKfyve regulates the trafficking of GLUT4 vesicles[Ｊ]. J Cell Sci, 2004; 117(Pt 25): 59855993.

1. Ernens I, Goodfellow SJ, Innes F, et al. Hypoxic stress suppresses RNA polymerase Ⅲ recruitment and tRNA gene transcription in cardiomyocytes[Ｊ]. Nucleic Acids Res, 2006; 34(1): 286２94.
2. Ullah MS, Davies AJ, Halestrap AP. The plasma membrane lactate transporter MCT4, but not MCT1, is upregulated by hypoxia through a HIF1αdependent mechanism [Ｊ]. J Biol Chem, 2006; 281(14): 90309037.
3. Wollert KC, Drexler H. Clinical applications of stem cells for the heart [Ｊ]. Circ Res, 2005; 96(2): 151163.
4. Kong HL, Liu NN, Huo X, et al. Cell multiplication, apoptosis and pAkt protein expression of bone mesenchymal stem cells of rat under hypoxia environment [Ｊ]. JNMU, 2007; 21(4): 233239.
5. 孔宏亮, 刘宁宁, 霍鑫，等. 缺氧对大鼠骨髓间充质干细胞增殖、凋亡及葡萄糖摄取和Akt表达的影响 [Ｊ].中国现代医学杂志， 2008; 18（10）： 13741382.
6. 孔宏亮, 刘宁宁, 张海，等. 缺氧环境下大鼠骨髓间充质干细胞的凋亡及Fas、FasL的表达 [Ｊ]. 中国组织化学与细胞化学杂志, 2007; 16(5): 538543.
7. 齐国先，孔宏亮，霍鑫，等. 缺氧环境下大鼠骨髓间充质干细胞凋亡相关蛋白和mRNA的表达 [Ｊ]. 中国组织化学与细胞化学杂志, 2008; 17(2): 138143.
8. 孔宏亮, 张利群, 曹善峰，等. Akt基因转染对骨髓间充质干细胞缺氧时凋亡和增殖的影响 [Ｊ]. 中国组织化学与细胞化学杂志, 2008; 17(3): 225231.
9. 霍鑫，孔宏亮，胡新华，等. pEGFPC1/Akt体外转染骨髓间充质干细胞后对血管内皮生长因子表达的影响 [Ｊ]. 中华实验外科杂志, 2008; 25(2)： 149151.
10. 霍鑫, 冯金炜, 刘方峰, 等. pEGFPC1/Akt体外转染骨髓间充质干细胞对后肢缺血大鼠血管生成的影响 [Ｊ]. 中国普外基础与临床杂志, 2008; 15(6): 402407.
11. 霍鑫, 孔宏亮, 刘方峰, 等. 无血清条件对诱导转染pEGFPC1/Akt的骨髓间充质干细胞凋亡的影响 [Ｊ]. 中国普外基础与临床杂志, 2008; 15(7): 479483.
12. 霍鑫，唐海燕，孔宏亮，等. SCF对体外转染pEGFPC1/Akt的骨髓间充质干细胞中ckit、Akt及VEGF表达的影响 [Ｊ]. 中国普外基础与临床杂志, 2008; 15(2): 116121.
13. Hemmings BA. Akt signaling: linking membrane events to life and death decisions [Ｊ]. Science, 1997; 275(5300): 628630.
14. Downward J. Mechanisms and consequences of activation of protein kinase B/Akt [Ｊ]. Curr Opin Cell Biol, 1998; 10(2): 262267.
15. Foster LJ, Li D, Randhawa VK, et al. Insulin accelerates interendosomal GLUT4 traffic via phosphatidylinositol 3kinase and protein kinase B [Ｊ]. J Biol Chem, 2001; 276(47): 4421244221.
16. Sweeney G, Garg RR, Ceddia RB, et al. Intracellular delivery of phosphatidylinositol (3,4,5)trisphosphate causes incorporation of glucose transporter 4 into the plasma membrane of muscle and fat cells without increasing glucose uptake [Ｊ]. J Biol Chem, 2004; 279(31): 3223332242.
17. Maffucci T, Brancaccio A, Piccolo E, et al. Insulin induces phosphatidylinositol3phosphate formation through TC10 activation [Ｊ]. EMBO J, 2003; 22(16): 41784189.
18. JeBailey L, Rudich A, Huang X, et al. Skeletal muscle cells and adipocytes differ in their reliance on TC10 and Rac for insulininduced actin remodeling [Ｊ]. Mol Endocrinol, 2004; 18(2): 359372.
19. Kupriyanova TA, Kandror KV. Akt2 binds to Glut4containing vesicles and phosphorylates their component proteins in response to insulin [Ｊ]. J Biol Chem, 1999; 274(3): 14581464.
20. Bae SS, Cho H, Mu J, et al. Isoformspecific regulation of insulindependent glucose uptake by Akt/protein kinase B [Ｊ]. J Biol Chem, 2003; 278(49): 4953049536.
21. Chen X, AlHasani H, Olausson T, et al. Activity, phosphorylation state and subcellular distribution of GLUT4targeted Akt2 in rat adipose cells [Ｊ]. J Cell Sci, 2003; 116(Pt 17): 35113518.
22. Kane S, Sano H, Liu SC, et al. A method to identify serine kinase substrates. Akt phosphorylates a novel adipocyte protein with a Rab GTPaseactivating protein (GAP) domain [Ｊ]. J Biol Chem, 2002; 277(25): 2211522118.
23. Bae SS, Cho H, Mu J, et al. Isoformspecific regulation of insulindependent glucose uptake by Akt/protein kinase B [Ｊ]. J Biol Chem, 2003; 278(49): 4953049536.
24. Welsh GI, Hers I, Berwick DC, et al. Role of protein kinase B in insulinregulated glucose uptake [Ｊ]. Biochem Soc Trans, 2005; 33(Pt 2): 346349.
25. Berwick DC, Dell GC, Welsh GI, et al. Protein kinase B phosphorylation of PIKfyve regulates the trafficking of GLUT4 vesicles[Ｊ]. J Cell Sci, 2004; 117(Pt 25): 59855993.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Translocation and Expression of GLUT-4 in Bone Marrow Mesenchymal Stem Cells Transfected with Akt Gene of Rat Ex Vivo

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