Phenobarbital 對胎盤葡萄糖轉運蛋白GLUT1及GLUT3影響之研究

葡萄糖轉運蛋白在分佈在身體的各種組織，負責將受質運送入細胞內產生ATP以供應活動所須的能量，主要表現在胎盤組織的葡萄糖轉運蛋白是葡萄糖轉運蛋白型一及型三，負責將母體中的葡萄糖運送至胎兒體內，以提供胎兒生長發育所需。GLUT1及GLUT3這兩種在膜上所發現的葡萄糖轉運蛋白具有類似的動力學特性，而且是鈉不依賴性的，而對D型的葡萄糖運送的選擇性大於L型。為了研究葡萄糖轉運蛋白的轉錄量是否會受到核內受體的調控，實驗中使用各種的核內受體的活化者來處理BeWo細胞株，BeWo細胞株是一種源於人類的絨毛膜癌細胞，可用在模擬胎盤的in vitro的模式中。現階段實驗的結果中，首先要確定在BeWo細胞中有那些轉運蛋白的基因會被表現，以反轉錄-聚合酶連鎖反應可發現BeWo細胞中表現的轉運蛋白有運送肉鹼(carnitine)的OCTN2及葡萄糖轉運蛋白型一及型三。之後以各種核內受體的活化者來處理BeWo細胞，包括PXR的活化者rifampicin，FXR的活化者CDCA，及CAR的活化者phenobarbital。處理之後，以反轉錄-聚合酶連鎖反應結果發現以0.1 mM的phenobarbital處理後GLUT1及融合膜蛋白syncytin的mRNA轉錄量有下降的情形，而以rifampicin及CDCA處理後則無顯著的變化。為了進一步了解phenobarbital對GLUT1及GLUT3的影響，我們利用核內受體CAR的直接活化者CITCO及AMP-protein kinase的活化者AICAR，來探討BeWo細胞中的葡萄糖轉運蛋白型一及型三的調控模式，結果發現CITCO和濃度0.1 mM的phenobarbital對於GLUT1的轉錄量情況是相似的，因此推論在低濃度下的phenobarbital可能是透過CAR來調控GLUT1；而GLUT3則沒明顯變化。然而在濃度為0.5 mM及1 mM的情況下，GLUT1的轉錄量反而會增加，而此情況和AICAR處理之後GLUT1及GLUT3的轉錄表現情況相似，因此可知道在高濃度的phenobarbital會活化AMPK進而調節GLUT1的表現。除此之外，在以氚標示的2去氧D葡萄糖來觀察BeWo細胞對葡萄糖的攝取量時其動力學參數為Km= 0.55±0.01mM，Vm= 28.37±1.93nmol/10min.mg /protein，並有一個不飽和參數κ=1.46±0.08mL/10min.mg /protein，在加入0.5 mM及1 mM濃度的phenobarbital之後24小時，得到Km值均有增加，不飽和常數k值減小，但是其Vmax則變化不大(Km= 1.04±0.03 mM，Vm= 30.35±1.57 nmol/10min.mg /protein，κ=0.89±0.04mL/ 10 min.mg /protein)，而以1 mM濃度的phenobarbital處理24小時之後，其動力學參數Km為1.05±0.22 mM；Vm為34.52±4.49 nmol/10min.mg/protein；不飽合常數k為1.38±0.13 mL/10min.mg /protein。以1 mM濃度的phenobarbital處理72小時之後，其動力學參數Km為1.08±0.23 mM；Vm為24.49±4.16 nmol/10min.mg/protein；不飽合常數k為0.83±0.07 mL/10min.mg /protein。因此可以推論在加入較高濃度的phenobarbital經過24小時及72小時處理之後，會降低葡萄糖轉運蛋白對葡萄糖的親和能力，若是以0.5 mM或是1 mM的phenobarbital處理24小時後，其在生理狀態下的葡糖糖濃度對葡萄糖轉運蛋白的運送能力則影響不大，然而在1 mM的phenobarbital處理72小時之後會降低葡萄糖轉運蛋白的運送能力。

關鍵字

胎盤；葡萄糖轉運蛋白；子宮內胎兒生長遲緩

並列摘要

The sodium-independent facilitated glucose transporters, GLUT1 and GLUT3, are expressed in placental tissues and are responsible for glucose transfer from maternal to the fetus. Deficiency in glucose levels may cause detrimental effects on fetal growth and development. It was reported that phenobarbital can be related to occurrence of intrauterine growth retardation (IUGR). Although phenobarbital is known to be an indirect activator of CAR, an orphan nuclear receptor, the interaction between phenobarbital and placental glucose transporters is far from clear. To investigate the impacts of phenobarbital on the expression and functions of placental GLUT1 and GLUT3, compounds including Phenobarbital (an indirect CAR activator), CITCO (a direct CAR activator) and AICAR (an AMPK activator) were used to treat BeWo cells, a cell line derived from human choriocarcinoma and has therefore been used as an in vitro placenta model. In the RT-PCR study, the results showed that, at a concentration of 0.1 mM, phenobarbital down-regulated m-RNA levels of both syncytin and GLUT1, but not GLUT3. However, at concentrations of 0.5 mM and 1 mM, phenobarbital increased m-RNA levels of GLUT1 but decreased that of GLUT3. The treatment of CITCO caused similar effects comparable to that of phenobarbital at a concentration of 0.1 mM. On the other hand, AICAR showed similar effects comparable to that of phenobarbital at concentrations of 0.5 mM and 1 mM. In the cellular uptake study, the results showed that the Km and Vm values of 3H-2-deoxy-D-glucose in un-treated BeWo cells are 0.55±0.01 mM and 28.37±1.93 nmol/10min-mg/ protein, respectively. There is also with a non-saturable constant k=1.46±0.08mL/10min.mg /protein .After the treatment (24 hours) of 0.5 mM phenobarbital, the Km and Vm values of 3H-2-deoxy-D-glucose are 1.04±0.03 mM and 30.35±1.57 nmol/10min-mg/ protein, respectively. Non-saturable constant k is 0.89±0.04mL/ 10 min.mg /protein. After the treatment (24 hours and 72 hours) of 1 mM phenobarbital, the Km and Vm values of 3H-2-deoxy-D-glucose are 1.05±0.22mM and 34.52±4.49 nmol/10min-mg/ protein (24 hours), 1.08±0.23 mM and 24.49±4.16 nmol/10min.mg/protein (72 hours) respectively. Non-saturable constant k is 1.38±0.13 mL/ 10 min.mg /protein(24 hours) and 0.83±0.07 mL/10min.mg /protein(72 hours). In conclusion, the impacts of phenobarbital on GLUT1 and GLUT3 are concentration-dependent, in which phenobarbital activates CAR at low concentration and AMPK at high concentration, respectively. Given that plasma levels of phenobarbital are about 0.1 mM, this study indicate that phenobarbital may decrease placental glucose transfer by activating CAR. After the treatment of 0.5 mM and 1 mM phenobarbital, Vmax is unchanged but the value of Km increase 24 hours later.After 1 mM phenobarbital treatment, Vmax is decreased than control. These results suggest that the affinity of glucose transporters decrease after phenobarbital treatment. In higher concentration of phenobarbital treatment, the transport ability of glucose transporters is no significantly changed under physiological condition after 24 hours. However, higher concentration of phenobarbital (1 mM) treat 72 hours, the transport ability of glucose transporters is somehow decreased.

並列關鍵字

placenta ； glucose transporter ； GLUT1 ； GLUT3 ； intrauterine growth retadation(IUGR) ； phenobarbital

參考文獻

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Phenobarbital 對胎盤葡萄糖轉運蛋白GLUT1及GLUT3影響之研究

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