A relative metabolic flux analysis model of glucose anaplerosis

Abstract

Glucose provides substrate for the predominant anaplerotic pathway which involves the activity of pyruvate carboxylase (PC). PC-mediated anaplerosis has been extensively studied as a metabolic regulator in glycolytic cells during tumorigenesis and metastasis. Herein, inaccuracies in established methods to measure relative intracellular flux through PC are highlighted and a compartmentalized condensed metabolic network (CCMN) is used to resolve the total malate pool into relative contributions from PC and other sources by metabolic flux analysis (MFA) with [U–¹³C₆]glucose tracing. Performance of the CCMN method is evaluated in breast cancer cell lines that are exposed to small molecules targeting metabolism. Across conditions and cell lines, the CCMN approach yields results nearest to an accepted gold-standard methodology, using [3–¹³C]glucose, or even exposes the gold standard's limitations. The CCMN method does not require a separate experiment with a much more costly and generally less informative metabolic tracer, such as [3–¹³C]glucose, and in some cases, may outperform its application.