Biotin uptake by human intestinal and liver epithelial cells: role of the SMVT system
Biotin uptake by human intestinal and liver epithelial cells: role of the SMVT system
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DOI：
10.1152/ajpgi.00059.2003
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发表时间：
2003-07-01
影响因子：
4.5
通讯作者：
Said, HM
中科院分区：
文献类型：
--
作者：
Balamurugan, K;Ortiz, A;Said, HM
已经确定人肠和肝上皮细胞通过Na+依赖性载体介导的机制转运生物素。钠依赖性多种维生素转运蛋白（SMVT）是一种生物素转运蛋白，在两种细胞类型中均有表达。然而，SMVT对这些细胞总载体介导的生理（纳摩尔）浓度生物素摄取的相对贡献尚不清楚。解决这个问题是很重要的，特别是鉴于最近确定的第二个人的高亲和力生物素摄取机制，在纳摩尔范围内运作。因此，我们采用了生理学方法来表征纳摩尔浓度范围内人源性肠Caco-2和HepG 2细胞对生物素的摄取。我们还采用了一种分子生物学方法，用特异性小干扰RNA（siRNA）选择性沉默这些细胞的内源性SMVT，然后检查载体介导的生物素摄取。结果表明，在Caco-2和HepG 2细胞中，生物素摄取的初始速率作为浓度的函数在0.1至50 nM范围内呈线性。此外，我们发现向孵育培养基中加入100 nM未标记生物素、脱硫生物素或泛酸对2.6 nM [H-3]生物素的摄取没有影响。用SMVT特异性siRNA预处理Caco-2和HepG 2细胞显著降低了SMVT mRNA和蛋白水平。此外，siRNA预处理严重抑制Caco-2和HepG 2细胞对载体介导的[H-3]生物素（2.6 nM）的摄取（P 0.01）。这些结果表明，最近描述的人高亲和力生物素摄取系统在肠和肝上皮细胞中没有功能。此外，结果提供了强有力的证据，SMVT是主要的（如果不是唯一的）生物素摄取系统，在这些细胞中运作。
It has been well established that human intestinal and liver epithelial cells transport biotin via an Na+-dependent carrier-mediated mechanism. The sodium-dependent multivitamin transport (SMVT), a biotin transporter, is expressed in both cell types. However, the relative contribution of SMVT toward total carrier-mediated uptake of physiological (nanomolar) concentrations of biotin by these cells is not clear. Addressing this issue is important, especially in light of the recent identification of a second human high-affinity biotin uptake mechanism that operates at the nanomolar range. Hence, we employed a physiological approach of characterizing biotin uptake by human-derived intestinal Caco-2 and HepG2 cells at the nanomolar concentration range. We also employed a molecular biology approach of selectively silencing the endogenous SMVT of these cells with specific small interfering RNAs (siRNAs), then examining carrier-mediated biotin uptake. The results showed that in both Caco-2 and HepG2 cells, the initial rate of biotin uptake as a function of concentration over the range of 0.1 to 50 nM to be linear. Furthermore, we found that the addition of 100 nM unlabeled biotin, desthiobiotin, or pantothenic acid to the incubation medium had no effect on the uptake of 2.6 nM [H-3]biotin. Pretreatment of Caco-2 and HepG2 cells with SMVT specific siRNAs substantially reduced SMVT mRNA and protein levels. In addition, carrier-mediated [H-3]biotin (2.6 nM) uptake by Caco-2 and HepG2 cells was severely (P 0.01) inhibited by the siRNAs pretreatment. These results demonstrate that the recently described human high-affinity biotin uptake system is not functional in intestinal and liver epithelial cells. In addition, the results provide strong evidence that SMVT is the major (if not the only) biotin uptake system that operates in these cells.