人关节软骨细胞中表达的易化型葡萄糖转运蛋白的分子特征及部分cDNA克隆；胰岛素样生长因子-I对2-脱氧葡萄糖摄取的刺激作用以及葡萄糖剥夺对基质金属蛋白酶-2分泌的上调作用
Molecular characterization and partial cDNA cloning of facilitative glucose transporters expressed in human articular chondrocytes; stimulation of 2-deoxyglucose uptake by IGF-I and elevated MMP-2 secretion by glucose deprivation.
作者信息
Richardson S, Neama G, Phillips T, Bell S, Carter S D, Moley K H, Moley J F, Vannucci S J, Mobasheri A
机构信息
Connective Tissue Research Group, Department of Veterinary Preclinical Sciences, Faculty of Veterinary Science, University of Liverpool, UK.
出版信息
Osteoarthritis Cartilage. 2003 Feb;11(2):92-101. doi: 10.1053/joca.2002.0858.
OBJECTIVE
Recent evidence suggests that human chondrocytes express several facilitative glucose transporter (GLUT) isoforms and also that 2-deoxyglucose transport is accelerated by cytokine stimulation. The aim of the present investigation was to determine if human articular chondrocytes express any of the recently identified members of the GLUT/SLC2A gene family and to examine the effects of endocrine factors, such as insulin and IGF-I on the capacity of human chondrocytes for transporting 2-deoxyglucose.
DESIGN/METHODS: PCR, cloning and immunohistochemistry were employed to study the expression of GLUT/SLC2A transporters in normal human articular cartilage. The uptake of 2-deoxyglucose was examined in monolayer cultured immortalized human chondrocytes following stimulation with TNF-alpha, insulin and IGF-I. Levels of MMP-2 were assessed by gelatin zymography following glucose deprivation of alginate cultures.
RESULTS
Using PCR we detected transcripts for eight glucose transporter isoforms (GLUTs 1, 3, 6, 8, 9, 10, 11 and 12) and for a fructose transporter (GLUT5) in human articular cartilage. Expression of GLUT1, GLUT3 and GLUT9 proteins in normal human articular cartilage was confirmed by immunohistochemistry. The uptake of 2-deoxyglucose was dependent on time and temperature, inhibited by cytochalasin B and phloretin, and significantly accelerated in chondrocyte cultures stimulated with IGF-I. However, 2-deoxyglucose uptake was unaffected by short and long-term insulin treatment, which ruled out a functional role for insulin-sensitive GLUT4-mediated glucose transport. Furthermore, secretion of MMP-2 was increased in alginate cultures deprived of glucose.
CONCLUSIONS
The data supports a critical role for glucose transport and metabolism in the synthesis and degradation of cartilage.
目的
近期证据表明，人类软骨细胞表达多种易化葡萄糖转运体（GLUT）亚型，并且细胞因子刺激可加速2-脱氧葡萄糖的转运。本研究的目的是确定人类关节软骨细胞是否表达GLUT/SLC2A基因家族最近鉴定出的任何成员，并研究胰岛素和IGF-I等内分泌因子对人类软骨细胞转运2-脱氧葡萄糖能力的影响。
设计/方法：采用PCR、克隆和免疫组织化学方法研究GLUT/SLC2A转运体在正常人关节软骨中的表达。在用TNF-α、胰岛素和IGF-I刺激后，检测单层培养的永生化人类软骨细胞对2-脱氧葡萄糖的摄取。在用藻酸盐培养物进行葡萄糖剥夺后，通过明胶酶谱法评估MMP-2的水平。
结果
通过PCR，我们在人类关节软骨中检测到8种葡萄糖转运体亚型（GLUT1、3、6、8、9、10、11和12）以及一种果糖转运体（GLUT5）的转录本。免疫组织化学证实了GLUT1、GLUT3和GLUT9蛋白在正常人关节软骨中的表达。2-脱氧葡萄糖的摄取依赖于时间和温度，受细胞松弛素B和根皮素抑制，并且在IGF-I刺激的软骨细胞培养物中显著加速。然而，短期和长期胰岛素处理对2-脱氧葡萄糖摄取没有影响，这排除了胰岛素敏感的GLUT4介导的葡萄糖转运的功能作用。此外，在缺乏葡萄糖的藻酸盐培养物中，MMP-2的分泌增加。
结论
数据支持葡萄糖转运和代谢在软骨合成与降解中的关键作用。
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