Ikaga Reina, Namekata Iyuki, Kotiadis Vassilios N, Ogawa Haruko, Duchen Michael R, Tanaka Hikaru, Iida-Tanaka Naoko
Department of Advanced Biosciences, Graduate School of Humanities and Sciences, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan.
Department of Food Science, Otsuma Women's University, Chiyoda-ku, Tokyo 102-8357, Japan.
Biochem Biophys Rep. 2015 Sep 18;4:187-195. doi: 10.1016/j.bbrep.2015.09.009. eCollection 2015 Dec.
Aquaporin-8 (AQP8), a member of the aquaporin water channel family, is expressed in various tissue and cells, including liver, testis, and pancreas. AQP8 appears to have functions on the plasma membrane and/or on the mitochondrial inner membrane. Mitochondrial AQP8 with permeability for water, HO and NH has been expected to have important role in various cells, but its information is limited to a few tissues and cells including liver and kidney. In the present study, we found that AQP8 was expressed in the mitochondria in mouse adipose tissues and 3T3-L1 preadipocytes, and investigated its role by suppressing its gene expression.
AQP8-knocked down (shAQP8) cells were established using a vector expressing short hairpin RNA. Cellular localization of AQP8 was examined by western blotting and immunocytochemistry. Mitochondrial function was assessed by measuring mitochondrial membrane potential, oxygen consumption and ATP level measurements.
In 3T3-L1 cells, AQP8 was expressed in the mitochondria. In shAQP8 cells, mRNA and protein levels of AQP8 were decreased by about 75%. The shAQP8 showed reduced activities of complex IV and ATP synthase; it is probable that the impaired mitochondrial water handling in shAQP8 caused suppression of the electron transport and ADP phosphorylation through inhibition of the two steps which yield water. The reduced activities of the last two steps of oxidative phosphorylation in shAQP8 cause low routine and maximum capacity of respiration and mitochondrial hyperpolarization.
Mitochondrial AQP8 contributes to mitochondrial respiratory function probably through maintenance of water homeostasis.
The AQP8-knocked down cells we established provides a model system for the studies on the relationships between water homeostasis and mitochondrial function.
水通道蛋白8（AQP8）是水通道蛋白家族的成员之一，在包括肝脏、睾丸和胰腺在内的多种组织和细胞中表达。AQP8似乎在质膜和/或线粒体内膜上发挥作用。具有水、过氧化氢和氨通透性的线粒体AQP8有望在各种细胞中发挥重要作用，但其信息仅限于包括肝脏和肾脏在内的少数组织和细胞。在本研究中，我们发现AQP8在小鼠脂肪组织和3T3-L1前脂肪细胞的线粒体中表达，并通过抑制其基因表达来研究其作用。
使用表达短发夹RNA的载体建立AQP8敲低（shAQP8）细胞。通过蛋白质印迹和免疫细胞化学检测AQP8的细胞定位。通过测量线粒体膜电位、耗氧量和ATP水平来评估线粒体功能。
在3T3-L1细胞中，AQP8在线粒体中表达。在shAQP8细胞中，AQP8的mRNA和蛋白质水平降低了约75%。shAQP8显示复合物IV和ATP合酶的活性降低；很可能shAQP8中受损的线粒体水处理通过抑制产生水的两个步骤导致电子传递和ADP磷酸化受到抑制。shAQP8中氧化磷酸化最后两步的活性降低导致呼吸的常规和最大能力降低以及线粒体超极化。
线粒体AQP8可能通过维持水平衡来促进线粒体呼吸功能。
我们建立的AQP8敲低细胞为研究水平衡与线粒体功能之间的关系提供了一个模型系统。
