Glycolate and glyoxylate metabolism in HepG2 cells
Glycolate and glyoxylate metabolism in HepG2 cells
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DOI：
10.1152/ajpcell.00238.2004
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发表时间：
2004-11-01
影响因子：
5.5
通讯作者：
Holmes, RP
中科院分区：
文献类型：
--
作者：
Baker, PRS;Cramer, SD;Holmes, RP
尽管人类肝细胞草酸合成在原发性高草酸尿症等疾病中具有临床意义，但其合成还没有很好的定义。为了进一步确定这些步骤，研究了草酸前体乙醇酸和乙二醇酸在HepG2细胞中向草酸的代谢以及可能涉及的途径。这些细胞被发现含有草酸、乙醛和乙醇酸作为细胞内的代谢物，并将草酸和乙醇酸排泄到培养液中。乙醇酸比乙醛更有效地被细胞吸收，但乙醛更有效地转化为草酸。只有当细胞暴露在高浓度时，外源乙醇酸才能形成草酸。与人类肝细胞不同，HepG2细胞中的过氧化物体不参与乙醇酸代谢。与纯化的乳酸脱氢酶孵育表明，该酶负责在HepG2细胞中将乙醇酸代谢为草酸。只有当细胞膜被Triton X-100渗透时，才能观察到从1(4C)标记的乙醇酸中形成C-14标记的甘氨酸。这些结果表明，在这些细胞中，过氧化物酶体对乙醇酸的通透性受到限制。线粒体具有丙氨酸乙醛转氨酶(AGT)2和乙氧基酸还原酶(AGT)两种活性，分别能将乙醛转化为甘氨酸和乙醇酸。逆转录聚合酶链式反应(RT-PCR)证实AGT2基因在HepG2细胞中有表达。这些结果表明，HepG2细胞将有助于阐明人肝细胞中与草酸合成相关的非过氧化体代谢。
Oxalate synthesis in human hepatocytes is not well defined despite the clinical significance of its overproduction in diseases such as the primary hyperoxalurias. To further define these steps, the metabolism to oxalate of the oxalate precursors glycolate and glyoxylate and the possible pathways involved were examined in HepG2 cells. These cells were found to contain oxalate, glyoxylate, and glycolate as intracellular metabolites and to excrete oxalate and glycolate into the medium. Glycolate was taken up more effectively by cells than glyoxylate, but glyoxylate was more efficiently converted to oxalate. Oxalate was formed from exogenous glycolate only when cells were exposed to high concentrations. Peroxisomes in HepG2 cells, in contrast to those in human hepatocytes, were not involved in glycolate metabolism. Incubations with purified lactate dehydrogenase suggested that this enzyme was responsible for the metabolism of glycolate to oxalate in HepG2 cells. The formation of C-14-labeled glycine from 1(4C)-labeled glycolate was observed only when cell membranes were permeabilized with Triton X-100. These results imply that peroxisome permeability to glycolate is restricted in these cells. Mitochondria, which produce glyoxylate from hydroxyproline metabolism, contained both alanine: glyoxylate aminotransferase (AGT)2 and glyoxylate reductase activities, which can convert glyoxylate to glycine and glycolate, respectively. Expression of AGT2 mRNA in HepG2 cells was confirmed by RT-PCR. These results indicate that HepG2 cells will be useful in clarifying the nonperoxisomal metabolism associated with oxalate synthesis in human hepatocytes.