Abstract
SUMMARYCitrate lies at a critical node of metabolism, linking tricarboxylic acid metabolism and lipogenesis via acetyl-coenzyme A. Recent studies have observed that deficiency of the sodium-dependent citrate transporter (NaCT), encoded by SLC13A5, dysregulates hepatic metabolism and drives pediatric epilepsy. To examine how NaCT contributes to citrate metabolism in cells relevant to the pathophysiology of these diseases, we apply 13C isotope tracing to SLC13A5-deficient hepatocellular carcinoma (HCC) cells and primary rat cortical neurons. Exogenous citrate appreciably contributes to intermediary metabolism only under hypoxic conditions. In the absence of glutamine, citrate supplementation increases de novo lipogenesis and growth of HCC cells. Knockout of SLC13A5 in Huh7 cells compromises citrate uptake and catabolism. Citrate supplementation rescues Huh7 cell viability in response to glutamine deprivation or Zn2+ treatment, and NaCT deficiency mitigates these effects. Collectively, these findings demonstrate that NaCT-mediated citrate uptake is metabolically important under nutrient-limited conditions and may facilitate resistance to metal toxicity.
Highlights
Citrate serves as a critical substrate for biosynthesis, acetylation, and the regeneration of NAD(P)H
Extracellular citrate uptake is tissue specific Recent findings have highlighted the importance of circulating tricarboxylic acid (TCA) intermediates as metabolic substrates or regulators of tissue function (Mills et al, 2018)
Citrate is not present in typical culture media such as DMEM, RPMI, or OptiMEM, we found that complete medium including 10% fetal bovine serum (FBS) contained 16 ± 5 mM citrate, significantly lower than that observed in human plasma (Figure 1B)
Summary
Citrate serves as a critical substrate for biosynthesis, acetylation, and the regeneration of NAD(P)H. Citrate is exported to the cytosol via the mitochondrial citrate transporter (SLC25A1/CTP) and metabolized by ATP-citrate lyase (ACLY) to generate acetyl-coenzyme A (acetyl-CoA) for downstream metabolic processes, including lipid biosynthesis and acetylation (Wakil and Abu-Elheiga, 2009). Mitochondrial production of citrate is the primary source for most cells, plasma concentrations are relatively high (Costello and Franklin, 2016). Dysregulation of plasma citrate homeostasis has pathophysiological consequences including impaired blood clotting and bone disorders (Costello and Franklin, 2016; Zuckerman and Assimos, 2009). The functional importance of exogenous citrate transport by cells has garnered increasing interest (Bhutia et al, 2017; Costello and Franklin, 1991, 2016)
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