The new approach to the diabetogenic mechanism and the new therapy : The disturbance of insulin secretion by oxidized lipoproteins and its relief

我们研究了低密度脂蛋白(LDL)是否影响胰岛β细胞的胰岛素分泌。我们推测修饰后的低密度脂蛋白通过脂蛋白受体摄取低密度脂蛋白后对胰岛β细胞功能有影响，因此我们首先检查了胰岛β细胞中是否存在脂蛋白受体。用抗大鼠胰岛素抗体、抗低密度脂蛋白受体(LDL-R)抗体和抗清道夫受体抗体(CD36和SRB1)对胰岛β细胞进行免疫染色。抗胰岛素抗体阳性细胞同时进行抗低密度脂蛋白受体抗体、抗CD36抗体和抗SRB1抗体染色。这些结果表明，β细胞不仅有普遍表达的低密度脂蛋白受体，还伴随有清道夫受体。为了研究各受体在β细胞中的表达情况，我们制备了大鼠肝、肾、胰岛和Hit-T15细胞的总rna，这些细胞系来源于Hamst…。更多的呃胰岛β细胞。大鼠肝脏分别表达低密度脂蛋白受体、SRB1和CD36的mRNA。大鼠肾脏既有低密度脂蛋白受体又有SRB1受体的mRNA，但没有CD36的mRNA，这在任何地方都已经报道过。在大鼠胰岛和HIT-T15细胞中，低密度脂蛋白受体、SRB1和CD36的mRNA表达水平与肝脏相同。因此，推测脂蛋白可能通过受体被摄取，并影响胰岛素的分泌。为了检测HIT-T15细胞对脂蛋白的摄取，用DiI标记的天然低密度脂蛋白(NLDL)、氧化型低密度脂蛋白(OxLDL)和乙酰化低密度脂蛋白(AcLDL)培养HIT-T15细胞。荧光显微镜下可见HIT-T15细胞清晰可见，提示每种低密度脂蛋白均已内化到细胞内。这些结果表明，每种低密度脂蛋白都是通过每种受体摄取的。接下来，我们检测了用每种低密度脂蛋白培养的HIT-T15细胞的胰岛素分泌情况。将氧化型低密度脂蛋白(25或50μg/ml)加入HIT-T15细胞培养24或48小时。葡萄糖浓度分别为1、3、5 mg/ml时，oxLDL对胰岛素分泌和细胞内胰岛素含量的影响呈剂量和时间依赖性。加入oxLDL后，胰岛素基因的表达显著降低。在nLDL和添加AcLDL的培养中未发现这些结果。HIT-T15细胞胰岛素代谢的这些改变是由oxLDL引起的，而不是AcLDL引起的。结论oxLDL作用于胰岛β细胞，干扰胰岛素基因表达，降低细胞内胰岛素含量和胰岛素分泌。这些已发表在《生物化学生物物理学A-摩尔细胞生物学L》(1687：173-180,2005)上。较少

We have studied whether low-density lipoprotein(LDL) affected on the insulin secretion of pancreatic beta cells. We speculated that the modified LDLs affected on beta cell function after the uptake of LDL through lipoprotein receptor, so at first we checked that the lipoprotein receptors were found in the pancreatic beta cells. The immunostaining of the beta cells was performed with anti-rat insulin antibody, anti-LDL receptor(LDL-R) antibody, and anti-scavenger receptor antibodies (CD36 and SRB1). The positively stained cells with anti-insulin antibody were also stained with anti-LDL-R antibody, anti-CD36 antibody and anti-SRB1 antibody. These results indicated that the beta cells had not only LDL receptor, which was ubiquitously expressed, but also scavenger receptors concomitantly. In order to study how mRNA of each receptor was expressed in beta cells, we prepared the total RNA from rat liver, kidney, islets of pancreas and Hit-T15 cells, which were the cell line derived from hamst … More er pancreatic beta cells. The rat liver expressed each mRNA of LDL-R, SRB1, and CD36. The rat kidney had both receptors mRNA of LDL-R and SRB1, but not CD36 mRNA, which had already been reported in anywhere. In the rat islets and Hit-T15 cells each mRNA of LDL-R, SRB1 and CD36 was expressed as same as liver. Therefore it was suggested that lipoproteins would be up taken through the receptors and affect on insulin secretion. In order to check the lipoprotein uptake, Hit-T15 cells were cultured with DiI-labeled native LDL (nLDL), oxidized LDL (oxLDL), and acetylated LDL (AcLDL). Hit-T15 cells were clearly marked under the fluorescence microscopy, which suggested that every LDLs were internalized into the cells. These results suggested that each LDL was uptaken through each receptor. Next we checked the insulin secretion in Hit-T15 cells cultured with each LDL. OxLDL (25 or 50 μg/ml) were added into the culture of Hit-T15 cells for 24 or 48 hours. The glucose concentrations of the medium were set at 1,3,and 5mg/ml. The insulin secretion and the intracellular insulin contents in the culture with oxLDL were disturbed in the dose and time dependent manner. The expression of insulin gene was significantly decreased in the oxLDL addition. These results were not found in nLDL and AcLDL added culture. These changes of the insulin metabolism in Hit-T15 cells were caused by oxLDL and not by AcLDL. As conclusions oxLDL affected on the beta cells disturbing insulin gene espression and decreasing the intracellular insulin contents and the insulin seceretion. These have been published in Biochim Biophys A - Mol Cell Biol L(1687:173-180,2005). Less