Ahmed N, Kansara M, Berridge M V
Malaghan Institute of Medical Research, Wellington School of Medicine, P.O. Box 7060, Wellington South, New Zealand.
Biochem J. 1997 Oct 15;327 ( Pt 2)(Pt 2):369-75. doi: 10.1042/bj3270369.
Activation of the respiratory burst imposes acute metabolic demands on phagocytic cells. These are met by mobilizing internal energy stores and by increasing the utilization of exogenous energy, including glucose in the circulation. To determine whether the increased glucose uptake that is known to be associated with the respiratory burst involves the regulation of glucose transporter molecules, the intrinsic transport properties of glucose transporters on the macrophage cell line RAW 264.7 were determined after activation with PMA, N-formyl-methionine-leucine-phenylalanine (fMLP) and the cytokines granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3). Treatment with PMA resulted in a 2-fold increase in respiratory burst activity within 10 min; this was associated with a 30-50% increase in 2-deoxyglucose uptake and a 4-fold increase in transporter affinity for glucose. Similarly, fMLP, GM-CSF and IL-3 treatments stimulated 2-deoxyglucose uptake that was associated with a 3-4-fold increase in transporter affinity for glucose. To determine whether the changes observed in 2-deoxyglucose uptake in response to PMA, fMLP and growth factors were influenced by phosphorylation of the sugar, 3-O-methylglucose, which is not phosphorylated, was used. Increased 3-O-methylglucose uptake and increased transporter affinity for glucose were also observed after PMA, fMLP and GM-CSF treatments. Whereas both fMLP and GM-CSF stimulated superoxide production, IL-3 failed to activate respiratory burst activity. The protein kinase inhibitors genistein and staurosporine inhibited the increase in 2-deoxyglucose uptake observed with fMLP and GM-CSF, and partly reversed the affinity increase towards that of untreated control cells. In contrast, the phosphatidylinositol 3-kinase inhibitor wortmannin had little effect on 2-deoxyglucose uptake in response to these activators. Western blotting with subtype-specific antisera showed that Glut-3 was the predominant transporter on RAW 264.7 cells. These studies demonstrate that acute regulation of glucose transporters occurs in response to activators that promote respiratory burst activity, and show that this regulation involves both tyrosine kinases and protein kinase C activity.
呼吸爆发的激活对吞噬细胞提出了急性代谢需求。这些需求通过动员内部能量储备以及增加对外源能量（包括循环中的葡萄糖）的利用来满足。为了确定已知与呼吸爆发相关的葡萄糖摄取增加是否涉及葡萄糖转运蛋白分子的调节，在用佛波酯（PMA）、N-甲酰甲硫氨酰亮氨酰苯丙氨酸（fMLP）以及细胞因子粒细胞/巨噬细胞集落刺激因子（GM-CSF）和白细胞介素3（IL-3）激活巨噬细胞系RAW 264.7后，测定了葡萄糖转运蛋白的内在转运特性。用PMA处理在10分钟内使呼吸爆发活性增加了2倍；这与2-脱氧葡萄糖摄取增加30 - 50%以及转运蛋白对葡萄糖的亲和力增加4倍相关。同样，fMLP、GM-CSF和IL-3处理刺激了2-脱氧葡萄糖摄取，这与转运蛋白对葡萄糖的亲和力增加3 - 4倍相关。为了确定在对PMA、fMLP和生长因子的反应中观察到的2-脱氧葡萄糖摄取变化是否受糖的磷酸化影响，使用了不被磷酸化的3-O-甲基葡萄糖。在PMA、fMLP和GM-CSF处理后也观察到3-O-甲基葡萄糖摄取增加以及转运蛋白对葡萄糖的亲和力增加。虽然fMLP和GM-CSF都刺激了超氧化物的产生，但IL-3未能激活呼吸爆发活性。蛋白激酶抑制剂染料木黄酮和星形孢菌素抑制了fMLP和GM-CSF诱导的2-脱氧葡萄糖摄取增加，并部分将亲和力增加逆转至未处理对照细胞的水平。相反，磷脂酰肌醇3-激酶抑制剂渥曼青霉素对这些激活剂诱导的2-脱氧葡萄糖摄取几乎没有影响。用亚型特异性抗血清进行的蛋白质印迹分析表明，Glut-3是RAW 264.7细胞上的主要转运蛋白。这些研究表明，葡萄糖转运蛋白的急性调节发生在对促进呼吸爆发活性的激活剂的反应中，并表明这种调节涉及酪氨酸激酶和蛋白激酶C的活性。
