Evidence for high-cavacity bidirectional glucose transport across the endoplasmic reticulum membrane by genetically encoded fluorescence resonance energy transfer nanosensors
Evidence for high-cavacity bidirectional glucose transport across the endoplasmic reticulum membrane by genetically encoded fluorescence resonance energy transfer nanosensors
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DOI：
10.1128/mcb.25.24.11102-11112.2005
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发表时间：
2005-12-01
影响因子：
5.3
通讯作者：
Frommer, WB
中科院分区：
文献类型：
--
作者：
Fehr, M;Takanaga, H;Frommer, WB
肝细胞释放葡萄糖对维持血糖水平很重要。葡萄糖-6-磷酸磷酸酶，催化糖异生的最终代谢步骤，面对内质网(ER)腔。因此，内质网中产生的葡萄糖要么在释放到循环之前从内质网输出到胞浆中，要么直接通过囊泡途径输出。为了测量葡萄糖的内质网转运，基于荧光共振能量转移的纳米传感器被定位于HepG2细胞的胞浆或内质网管腔。在5 mM葡萄糖的灌流过程中，胞浆水平维持在接近外供的80%，表明质膜转运超过了葡萄糖磷酸化的速率。内质网内的葡萄糖水平和动力学与胞浆水平几乎没有区别，这表明葡萄糖在内质网膜上的快速双向转运。一个包含快速双向电流变传输的动力学模型与观察到的动力学很好地吻合。质膜和内质膜葡萄糖转运对细胞松弛素B的敏感性不同，对半乳糖的吸收和释放表现出不同的相对动力学，表明两种膜上的催化活性不同。内质网膜上存在一个高容量的葡萄糖转运系统，这与肝细胞葡萄糖通过胞浆通过一组尚未确定的蛋白质输出的假设是一致的。
Glucose release from hepatocytes is important for maintenance of blood glucose levels. Glucose-6-phosphate phosphatase, catalyzing the final metabolic step of gluconeogenesis, faces the endoplasmic reticulum (ER) lumen. Thus, glucose produced in the ER has to be either exported from the ER into the cytosol before release into circulation or exported directly by a vesicular pathway. To measure ER transport of glucose, fluorescence resonance energy transfer-based nanosensors were targeted to the cytosol or the ER lumen of HepG2 cells. During perfusion with 5 mM glucose, cytosolic levels were maintained at similar to 80% of the external supply, indicating that plasma membrane transport exceeded the rate of glucose phosphorylation. Glucose levels and kinetics inside the ER were indistinguishable from cytosolic levels, suggesting rapid bidirectional glucose transport across the ER membrane. A dynamic model incorporating rapid bidirectional ER transport yields a very good fit with the observed kinetics. Plasma membrane and ER membrane glucose transport differed regarding sensitivity to cytochalasin B and showed different relative kinetics for galactose uptake and release, suggesting catalysis by distinct activities at the two membranes. The presence of a high-capacity glucose transport system on the ER membrane is consistent with the hypothesis that glucose export from hepatocytes occurs via the cytosol by a yet-to-be-identified set of proteins.