Characterization of bovine glucose transporter 1 kinetics and substrate specificities in Xenopus oocytes
Characterization of bovine glucose transporter 1 kinetics and substrate specificities in Xenopus oocytes
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DOI：
10.3168/jds.2011-4430
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发表时间：
2012-03-01
影响因子：
3.5
通讯作者：
Zhao, F. -Q.
中科院分区：
文献类型：
--
作者：
Bentley, P. A.;Shao, Y.;Zhao, F. -Q.
葡萄糖是乳糖合成的重要底物，也是牛奶生产中的重要能量来源。因此，乳腺中的葡萄糖摄取在乳汁合成中起着关键作用。促进性葡萄糖转运体(GLUT)介导乳腺的葡萄糖摄取。葡萄糖转运蛋白1(GLUT1)是牛乳腺中表达的主要促糖转运蛋白，定位于乳腺上皮细胞的基底膜。因此，葡萄糖转运蛋白1被认为在哺乳期葡萄糖摄取中起主要作用。本研究的目的是利用非洲爪哇卵母细胞模型来确定牛GLUT1的转运动力学特性和底物专一性。牛GLUT1(BGLUT1)通过显微注射体外转录的cRNA在非洲爪哇卵母细胞中表达，并定位于质膜，导致葡萄糖摄取增加。这种bGLUT1介导的葡萄糖摄取被特定的促进性葡萄糖运输抑制剂、细胞松弛素B和根霉素所显著抑制。利用放射性标记的2-脱氧-D-葡萄糖和Michaelis-Menten动力学原理，在零反式条件下对牛和人GLUT1进行了动力学分析。牛GLUT1对2-脱氧-D-葡萄糖的米氏常数(K-m)为9.8+/-3.0 mM，与人GLUT1的11.7+/-3.7 mM相近。BGLUT1转运受甘露糖和半乳糖的抑制，但不受果糖的抑制，提示bGLUT1可能也能转运甘露糖和半乳糖。我们的数据为bGLUT1在乳汁合成中跨乳腺上皮细胞吸收葡萄糖的运输特性提供了功能洞察力。
Glucose is an essential substrate for lactose synthesis and an important energy source in milk production. Glucose uptake in the mammary gland, therefore, plays a critical role in milk synthesis. Facilitative glucose transporters (GLUT) mediate glucose uptake in the mammary gland. Glucose transporter 1 (GLUT1) is the major facilitative glucose transporter expressed in the bovine mammary gland and has been shown to localize to the basolateral membrane of mammary epithelial cells. Glucose transporter 1 is, therefore, thought to play a major role in glucose uptake during lactation. The objective of this study was to determine the transport kinetic properties and substrate specificity of bovine GLUT1 using the Xenopus oocyte model. Bovine GLUT1 (bGLUT1) was expressed in Xenopus oocytes by microinjection of in vitro transcribed cRNA and was found to be localized to the plasma membrane, which resulted in increased glucose uptake. This bGLUT1-mediated glucose uptake was dramatically inhibited by specific facilitative glucose transport inhibitors, cytochalasin B, and phloretin. Kinetic analysis of bovine and human GLUT1 was conducted under zero-trans conditions using radio-labeled 2-deoxy-D-glucose and the principles of Michaelis-Menten kinetics. Bovine GLUT1 exhibited a Michaelis constant (K-m) of 9.8 +/- 3.0 mM for 2-deoxy-D-glucose, similar to 11.7 +/- 3.7 mM for human GLUT1. Transport by bGLUT1 was inhibited by mannose and galactose, but not fructose, indicating that bGLUT1 may also be able to transport mannose and galactose. Our data provides functional insight into the transport properties of bGLUT1 in taking up glucose across mammary epithelial cells for milk synthesis.