The Quantitative Relationship Between Isotopic And Net Contributions Of Glucose And Lactate To The TCA Cycle Of Cancer Cells

The fuel sources of cancer cells remain an important research topic in tumor metabolism field.Glucose is one of the main carbon sources and energy sources in the canonical opinion.However,recent studies demonstrated that lactate is also a fuel for cancer cells and its carbon contribution to TCA cycle is larger than that of glucose.Therefore,the recent studies sparkled a debate about the major carbon source for the TCA cycle.Noticeably,the key data to assess lactate as a preferred fuel to glucose in the recent studies are all the labeling percentages of TCA cycle intermediates labeled with 13C-lactate and 13C-glucose.Although the experiments demonstrated unambiguously a significant larger labeling percentage of TCA cycle intermediates labeled with 13C-lactate than that labeled with 13C-glucose,it cannot be inferred that lactate contributes more carbon than glucose to TCA cycle for isotopic labeling does not equal to net contribution.Consequently,the conclusions which based merely on isotopic labeling in the recent studies may be untenable.Therefore,to analyze which one contribute more to TCA cycle between glucose and lactate,not only isotopic labeling but also the net consumption data should be determined.We did these measurements using 13C-labeled glucose and lactate in cultured cells.The results showed that lactate’s labeling efficiency to TCA cycle intermediates is higher than that of glucose in most conditions,however,the net contribution to TCA cycle of lactate is 0.Furthermore,according to the mathematical models we established,we deduced a mathematical equation:CG-CG’=CL’-CL.Here CG and CL refer the contributions to TCA cycles of glucose and lactate measured by net consumption,while CG’ and CL’ refer the same meaning but measured by isotopic labeling.The equation clearly pointing out that the isotopic labeling data overestimates the net contribution of lactate to TCA cycle meanwhile underestimates the net contribution of glucose to TCA cycle in a same amount.This is because glucose and lactate must be converted to pyruvate before entering into TCA cycle,and the conversion between pyruvate and lactate are fully reversible,isotopic exchange could occur between glucose-derived pyruvate and lactate:the conversion of glucose-derived pyruvate to lactate counteracts the consumption of exogenous lactate,however,the counteracted part cannot be reflected by isotopic labeling of the TCA cycle intermediates.Therefore,after excluding the interference of isotopic exchange at pyruvate and lactate,the major carbon contribution(acetyl-CoA)to TCA cycle should be glucose rather than lactate.Theoretically,the principle is also applicable to cancer cells in vivo.Although it had been known for a Iong time that isotopic exchange could deviate isotopic labeling data from net flux data,a lot of research work overlooked this point.Consequently,many conclusions based on merely isotopic labeling are in fact misleading.There are some corrections about the impropriate interpretations of isotopic labeling results,however,they are all qualitative.Our work analyzes the problem in a quantitative way for the first time,and we deduce a simple mathematical equation which reflect isotopic exchange.The equation could also be applied in other metabolic reactions which involve isotopic exchange.Therefore,it has great significance for the understanding of isotopic labeling and the proper application of isotopic labeling.