Carbon sources and pathways for citrate secreted by human prostate cancer cells determined by NMR tracing and metabolic modeling

Abstract

Significance The human prostate accumulates high luminal citrate levels to serve sperm viability. There is only indirect qualitative evidence about metabolic pathways and carbon sources maintaining these levels. Human citrate-secreting prostate cancer cells were supplied with 13 C-labeled substrates, and NMR spectra of extracellular fluid were recorded. We report absolute citrate production rates of prostate cells and direct evidence that glucose is the main carbon source for secreted citrate. Pyruvate carboxylase provides sufficient anaplerotic carbons to support citrate secretion. Glutamine carbons exchange with carbons for secreted citrate but are likely not involved in its net synthesis. Moreover, we developed metabolic models employing the 13 C distribution in extracellular citrate as input to assess intracellular pathways followed by carbons toward citrate.

CPT1a regulates the delivery of extracellular fatty acids for cardiolipin turnover in prostate cancer cells

“…1a). Similar studies using 13 Cglucose and -glutamine in cultured cells have shown that 3 to 6 hours of label exposure is sufficient for isotopic steady state 25,26 . We separately noted that doubling of media volume to 2 mL was necessary for a 12 to 24 hour experiment, in order to maintain media substrates above 50% (Extended Data Fig.…”

“…In contrast, extracellular [U- 13 C]-palmitate contributed less than 15% of carbons to TCA cycle metabolites (Fig, 1f and Extended Data Fig. 1c-d).…”

“…(F) Fractional contribution of [U- 13 C]-glucose to pyruvate (red), [U- 13 C]-glutamine to intracellular glutamine (blue), [U- 13 C]-FA 16:0 to palmitoyl-carnitine (orange) after 6 hours of labelling. [U- 13 We achieved sufficient labelling of the respective intracellular TCA cycle precursor pools after 6 hours (pyruvate >65%, glutamine >85%, and palmitoyl-carnitine >60%; Fig. 1f) and confirmed the uptake of extracellular labelled substrates.…”

“…1f) and confirmed the uptake of extracellular labelled substrates. The fractional contribution of [U- 13 C]-glucose and [U- 13 C]-glutamine, calculated as the weighted sum by the number of labelled carbons 27 , combined ranged from 60 to 80% for all measured TCA cycle metabolites (Fig. 1f).…”

Activity-Based Self-Enriched SERS Sensor for Blood Metabolite Monitoring

“…Magnetic resonance spectroscopy and mass spectrometry are currently popular techniques for metabolic phenotyping in biological and medical settings. − Despite their advantages, these techniques are not suitable for sensing metabolites at the point of care due to heavy reliance on high-cost advanced equipment and professional technicians. In this regard, surface-enhanced Raman spectroscopy (SERS) has emerged as a promising optical technique that allows for sensitive detection of metabolites by enhancing the Raman signals of molecules, mainly via excitation of the localized surface plasmon resonance (LSPR) of metal nanostructures. − Especially, over 100 medically relevant metabolites can be catalyzed by specific enzymes to generate stoichiometric H 2 O 2 , − a reactive compound that can be quantified by optical probes operating through activity-based sensing, such as through H 2 O 2 -mediated boronate oxidation. − For example, we and other groups have developed boronate-based SERS nanoprobes enabling detection of H 2 O 2 and many different metabolites in body fluids, living cells, and tissues. − Nevertheless, these SERS assays are not compatible with practical large-scale metabolic assays because synthesis of nanoprobes is labor-intensive, and more importantly, can suffer from batch-to-batch variability.…”