Jain, Aakriti; (2019) Differential ammonium detoxification capacity influences mitochondrial anaplerotic pathways. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The tricarboxylic acid (TCA) cycle is a major pathway in central carbon metabolism that supports energy production and anabolic processes required for cancer cell survival and proliferation. To maintain flux through the TCA cycle, individual anaplerotic reactions need to coordinate with cataplerotic reactions and other anaplerotic pathways. This work uses in vitro reconstitution of mitochondrial metabolism to explore the mechanisms through which glutamine- and glucose-derived anaplerotic pathways are coordinated. In cell-free mitochondria, increased ammonium levels, as a consequence of glutamine deamidation, increased reductive amination through glutamate dehydrogenase (GDH) and flux through transaminases for anaplerosis. Ammonium-mediated changes to glutamine-derived anaplerosis were correlated with increased pyruvate-derived anaplerosis evidenced by increased flux through pyruvate carboxylase (PC). Addition of exogenous substrates to invoke flux through the urea cycle, as an alternative pathway to detoxify ammonium, rescued the effects of ammonium on GDH and PC. The studies in cell-free mitochondria revealed that acute changes in intramitochondrial ammonium levels coordinated glutamine- and glucose-derived anaplerosis without gene expression changes. To determine if chronic changes in gene expression could induce ammonium-mediated coordination of anaplerosis, enzymes that may affect intramitochondrial ammonium levels in whole cells were modulated. Overexpression of the urea cycle enzyme, carbamoyl-phosphate synthetase 1 (CPS1) was associated with decreased flux through PC and increased sensitivity to oxidative stress. Analysis of mRNA expression data from human cancers showed that CPS1 mRNA expression was down-regulated in hepatocellular carcinoma (HCC). A mouse model of HCC, which exhibited the same changes in gene expression on the protein level, was used to show that attenuation of CPS1 expression was associated with increased reductive amination through GDH and increased PC-derived anaplerosis. Altogether, this study provides evidence suggesting that down-regulation of the urea cycle and ammonium-mediated coordination of anaplerosis may promote survival and proliferation of HCC cells.

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