Figure 4

Metformin reduces glucose metabolism through the citric acid cycle. (A) Schematic depicting glucose carbon flow into glycolysis and the citric acid cycle (CAC). The usage of tracer metabolites such as [U-¹³C]glucose where all carbons (¹²C, white) are replaced by ¹³C (black circles) allows for the measurement of CAC activity by gas chromatography/mass spectrometry (GC/MS) analysis and isotopomer enrichments. (B-F) MCF7 and MCF10A cells were treated with ddH₂O (control) or metformin (0.5 mM or 5.0 mM) for 24 hours. Cells were then incubated with [U-¹³C]glucose (m + 6) for 1 hour. (B) Intracellular lactate to pyruvate ratio induced by metformin treatment, displayed as fold change from untreated conditions. (C) Enrichment of citrate (m + 2) and (m + 4), (D), isocitrate (m + 2) (E) and alpha-ketoglutarate (m + 2) upon incubation with [U-¹³C]glucose and quantified as mass isotopomer distributions. (F) CAC intermediates reorganization upon metformin treatment. The sum of the ion intensities for all the isotopomers of each individual CAC intermediate was normalized to the sum of the ion intensities for all the isotopomers of all combined CAC intermediates. For B-E, data are presented as mean ± SEM of a representative experiment performed in triplicate of three independent experiments for control and 0.5 mM metformin treatments, and two independent experiments for 5.0 mM metformin treatment. *P <0.05, Student’s t-test. For F, data are presented as mean of a representative experiment performed in triplicate of three independent experiments for control and 0.5 mM metformin treatments, and two independent experiments for 5.0 mM metformin treatment. CAC: citric acid cycle.