Gstraunthaler G, Gersdorf E, Fischer W M, Joannidis M, Pfaller W
Institutes of Physiology and Medical Biology and Genetics, University of Innsbruck, Austria.
Ren Physiol Biochem. 1990 May-Jun;13(3):137-53. doi: 10.1159/000173360.
The established renal epithelial cell line LLC-PK1 retained in tissue culture several differentiated properties of renal proximal tubular cells. By adapting LLC-PK1 cells to glucose-free culture conditions, we recently succeeded in isolating a gluconeogenic strain of LLC-PK1 cells capable of growing in the absence of hexoses. In contrast to the parental wild type, the isolated strain expressed fructose-1,6-bisphosphatase activity and was, therefore, designated LLC-PK1-FBPase+. Besides the differences in glucose metabolism, the isolated gluconeogenic substrain differs form the parental wild type with respect to morphological appearance and the expression of apical membrane marker enzymes. LLC-PK1-FBPase+ cells display a drastic accumulation of autophagic vacuoles, disappearance of apical membrane alkaline phosphatase activity, and increased gamma-glutamyltranspeptidase activity. In order to find out whether or not a low alkaline phosphatase activity in combination with the enhanced formation of autophagic vacuoles is related to a change in apical membrane surface, we utilized a combined light and electron microscopic morphometric procedure to determine the absolute amount of organelle volumes and membrane surface areas. This stereologic approach shows that LLC-PK1-FBPase+ cells display a tenfold increase in the volume of autophagic vacuoles and the lysosomal compartment. Analysis of lysosomal enzyme activities, however, revealed no changes as compared to wild-type cells. The apical membrane surface of gluconeogenic cells was found to be increased by 80%. Karyotype analysis revealed that LLC-PK1 wild-type cells were diploid, whereas FBPase+ cells exhibited polyploidy with a high percentage of tetraploid nuclei. Culturing LLC-PK1-FBPase+ cells in the presence of 5 mM glucose does not abolish the morphological and biochemical changes described, indicating the stability of the FBPase+ strain.
已建立的肾上皮细胞系LLC-PK1在组织培养中保留了肾近端小管细胞的几种分化特性。通过使LLC-PK1细胞适应无葡萄糖培养条件，我们最近成功分离出一种能在无己糖条件下生长的糖异生型LLC-PK1细胞株。与亲代野生型相比，分离出的细胞株表达果糖-1,6-二磷酸酶活性，因此被命名为LLC-PK1-FBPase+。除了葡萄糖代谢的差异外，分离出的糖异生亚株在形态外观和顶膜标记酶的表达方面也与亲代野生型不同。LLC-PK1-FBPase+细胞表现出自噬泡的大量积累、顶膜碱性磷酸酶活性的消失以及γ-谷氨酰转肽酶活性的增加。为了确定低碱性磷酸酶活性与自噬泡形成增加是否与顶膜表面积的变化有关，我们采用了光镜和电镜相结合的形态计量学方法来确定细胞器体积和膜表面积的绝对量。这种体视学方法表明，LLC-PK1-FBPase+细胞的自噬泡和溶酶体区室体积增加了10倍。然而，溶酶体酶活性分析显示与野生型细胞相比没有变化。发现糖异生细胞的顶膜表面积增加了80%。核型分析显示LLC-PK1野生型细胞是二倍体，而FBPase+细胞表现为多倍体，四倍体核的比例很高。在5 mM葡萄糖存在的情况下培养LLC-PKl-FBPase+细胞并不能消除上述形态和生化变化，这表明FBPase+细胞株具有稳定性。
