Overexpression of carbamoyl-phosphate synthase 1 significantly improves ureagenesis of human liver HepaRG cells only when cultured under shaking conditions
Overexpression of carbamoyl-phosphate synthase 1 significantly improves ureagenesis of human liver HepaRG cells only when cultured under shaking conditions
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DOI：
10.1016/j.mito.2019.02.005
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发表时间：
2019-07-01
期刊：
影响因子：
4.4
通讯作者：
Hoekstra, Ruurdtje
中科院分区：
文献类型：
--
作者：
Adam, Aziza A. A.;van der Mark, Vincent A.;Hoekstra, Ruurdtje
高氨血症是终末期肝衰竭患者发生肝性脑病的重要原因。因此，降低高氨血症是生物人工肝支持（BAL）的必要条件。人肝HepaRG细胞的氨清除主要通过可逆固定为氨基酸发生，而不可逆转化为尿素是有限的。与人类肝脏相比，三种尿素循环（UC）酶氨甲酰磷酸氨甲酰酶1（CPS 1）、鸟氨酸转氨甲酰酶（OTC）和氨甲酰酶1的表达和活性较低。为了提高HepaRG细胞作为BAL生物组分的能力，在两种培养条件下测定了其UC的速率限制因子：静态和动态培养基流（DMF），通过振荡实现。HepaRG细胞越来越多地将递增的精氨酸剂量转化为尿素，表明精氨酸酶活性不限制尿素生成。也不是OTC活性，作为一个稳定的HepaRG线过表达OTC表现出90和15.7倍的OTC转录和活性水平上调，没有改善尿素生成。然而，一个稳定的HepaRG线过表达CPS1表现出增加线粒体应激和减少肝分化，而不促进CPS1转录水平或尿素生成在静态培养条件下，然而，它表现出4.3倍增加尿素生成下DMF。这与CPS1转录和活性水平增加> 2倍、线粒体丰度增加和肝分化有关。出乎意料的是，其他几个UC基因的转录水平增加了6.8倍。我们的结论是，在HepaRG细胞中，通过CPS1过表达可以改善尿素生成，但是，仅与DMF培养相结合，这表明低CPS1水平和静态培养（可能是由于线粒体不足）都限制了UC。
Hyperammonemia is an important contributing factor to hepatic encephalopathy in end-stage liver failure patients. Therefore reducing hyperammonemia is a requisite of bioartificial liver support (BAL).Ammonia elimination by human liver HepaRG cells occurs predominantly through reversible fixation into amino acids, whereas the irreversible conversion into urea is limited. Compared to human liver, the expression and activity of the three urea cycle (UC) enzymes carbamoyl-phosphate synthasel (CPS1), omithine transcarbamoylase (OTC) and arginasel, are low. To improve HepaRG cells as BAL biocomponent, its rate limiting factor of the UC was determined under two culture conditions: static and dynamic medium flow (DMF) achieved by shaking. HepaRG cells increasingly converted escalating arginine doses into urea, indicating that arginase activity is not limiting ureagenesis. Neither was OTC activity, as a stable HepaRG line overexpressing OTC exhibited a 90- and 15.7-fold upregulation of OTC transcript and activity levels, without improvement in ureagenesis. However, a stable HepaRG line overexpressing CPS1 showed increased mitochondrial stress and reduced hepatic differentiation without promotion of the CPS1 transcript level or ureagenesis under static culturing conditions, yet, it exhibited a 4.3-fold increased ureagenesis under DMF. This was associated with increased CPS1 transcript and activity levels amounting to > 2-fold, increased mitochondrial abundance and hepatic differentiation. Unexpectedly, the transcript levels of several other UC genes increased up to 6.8-fold. We conclude that ureagenesis can be improved in HepaRG cells by CPS1 overexpression, however, only in combination with DMF-culturing, suggesting that both the low CPS1 level and static-culturing, possibly due to insufficient mitochondria, are limiting UC.