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. 2020 Jan 22;23(2):100858. doi: 10.1016/j.isci.2020.100858

Figure 4.

Figure 4

Exposure to Excess Glucose Enhances Mitochondrial Pyruvate Metabolism

(A–F) INS-1 cells were kept in physiological glucose or pre-exposed to excess glucose for 20–24 h, then transferred to complete serum-free Seahorse base-media containing 3 mM glucose for 2 h, after which OCR was measured. (A) Diagram depicting the media composition and glucose concentrations throughout the different stages of the experimental timeline for (B)–(F). (B) OCR trace along a mitochondrial stress test at 3 mM glucose with INS-1 cells that were either pre-exposed to excess glucose for 20 h (dark gray squares, triangles, and exes) or kept in physiological glucose (light gray circles, diamonds, and stars). Vehicle (circles and squares), etomoxir (stars and exes), or UK5099 (diamonds and triangles) was injected into the media in between measurements, as indicated by the arrow. Arrows also indicate when oligomycin (O), FCCP (f), and antimycin A (AA) were introduced into the cell media. Data shown are the average and standard error of four to six replicates. (C and D) Calculated OCR corresponding to ATP-coupled respiration (C) or maximal respiratory capacity (D) from the experiment shown in (B). Data shown are the average and standard error from four to six replicates, and is representative of at least three separate experiments. (E) Calculated OCR corresponding to the ATP-coupled respiration of INS-1 cells at 3 mM glucose, which was acutely treated at the time of OCR measurements with various sub-optimum concentrations of UK5099, as indicated. (F) Calculated OCR corresponding to the ATP-coupled respiration of INS-1 cells at 3 mM glucose measured with sodium pyruvate (hatched bars) or without (solid bars) in the media. Cells that had been pre-exposed to excess glucose are shown in dark gray bars, whereas cells that were kept in physiological glucose are shown in light gray bars. (E and F) Data shown are the average and standard error of three separate experiments, with four to six replicates each. # indicate no statistical significance, * indicate 0.05 < p < 0.02, ** indicate 0.02 < p < 0.01, and *** indicate 0.01 < p < 0.003, as determined by Student's t tests.