Fig 1.
Pathways of H+ production and ATP production and consumption during measurement of glycolytic capacity.
Relevant pathways under basal conditions (a), and during the conventional (b) or improved (c) assay of glycolytic capacity. Thicker arrows denote higher flux, dotted arrows denote zero flux. Double bars perpendicular to pathway arrows indicate sites of inhibition by the indicated compounds (cycloheximide was added only where indicated in later figures). Other additions are indicated with labelled, open block arrows. Only the protons associated with lactate- and HCO3-, which contribute to extracellular acidification, are specified; the ionization states of pyruvate and adenine nucleotides are not shown.
Fig 2.
Increased glycolytic rate following inhibition of the F1FO-ATP synthase in C2C12 myoblasts.
Raw traces of (a) oxygen consumption rate (OCR) and (b) extracellular acidification rate (ECAR) after sequential additions of 10 mM glucose, 2 μg/mL oligomycin, and 100 mM 2-deoxyglucose. One representative experiment is shown. c: Respiratory (open column sections) and glycolytic (blue column sections) proton production rates (PPR) of the experiment exemplified in a and b calculated using Eq 1. Coloured wedges indicate glycolysis under basal conditions (blue) and apparent glycolytic capacity (green), with the difference between these defined as apparent glycolytic reserve. Data are means ± SEM of n = 6 independent biological replicates.
Fig 3.
Assay of glycolytic capacity by inhibition of electron transport in C2C12 myoblasts.
Raw traces of (a) oxygen consumption rate (OCR) and (b) extracellular acidification rate (ECAR) after sequential addition using ports A-C of 10 mM glucose, followed by vehicle and then either 2 μg/mL oligomycin (black), or 1 μM rotenone plus 1 μM myxothiazol (red). One representative experiment is shown. c: Respiratory (open column sections) and glycolytic (blue column sections) proton production rates (PPR) of the experiment exemplified in a and b calculated using Eq 1. Data are means ± SEM of n = 4 independent biological replicates. *p ≤ 0.05. Statistical analysis was of glycolytic proton production rates only (blue column sections). w, well; A, B, C, addition ports. These data are replotted after Fig 4, where another addition (in port D) is also shown.
Fig 4.
Effects of activating additional ATP consumers on glycolytic rate in C2C12 myoblasts.
a: Respiratory (open column sections) and glycolytic (blue column sections) proton production rates after sequential additions as shown of 10 mM glucose, 1 μM rotenone plus 1 μM myxothiazol, 200 μM monensin, 1 μM FCCP, and 1 mM ouabain, calculated using Eq 1. Data are means ± SEM of n = 4 independent biological replicates. Statistical analysis was of glycolytic proton production rates only (blue column sections). w, well; A, B, C, addition ports. b: Lactate accumulation predicted by glycolytic PPR (left) and measured, in the proposed assay for maximum glycolytic capacity. Data are means ± SEM of n = 3 independent biological replicates. n.s.: not significant; **p ≤ 0.01; ***p ≤ 0.005.
Fig 5.
Effects of attenuating ATP demand using cycloheximide on assay of glycolytic capacity in C2C12 myoblasts.
Raw traces of oxygen consumption rate (OCR) (a, c) and extracellular acidification rate (ECAR) (b, d) after sequential addition using ports A-D of 10 mM glucose, followed by vehicle, and then 2 μg/mL oligomycin (black), or 1 μM rotenone plus 1 μM myxothiazol (red), and then vehicle (black) or 200 μM monensin plus 1 μM FCCP (red). One representative experiment is shown. e: Respiratory (open column sections) and glycolytic (blue column sections) proton production rates (PPR) of the experiment exemplified in a-d calculated using Eq 1. Data are means ± SEM of n = 4 independent biological replicates. n.s.: not significant; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.005. Statistical analysis was of glycolytic proton production rates only (blue column sections). w, well; A, B, C, D, addition ports. A representative raw data file is appended here (S1 Table) with description (S1 Text).
Fig 6.
Effects of attenuating ATP demand using cycloheximide on assay of glycolytic capacity in HEK293 fibroblasts.
Raw traces of oxygen consumption rate (OCR) (a, c) and extracellular acidification rate (ECAR) (b, d) after sequential addition using ports A-D of 10 mM glucose, followed by vehicle or 10 μM cycloheximide (CHX), and then 2 μg/mL oligomycin (black), or 1 μM rotenone plus 1 μM myxothiazol (red), and then vehicle (black) or 200 μM monensin plus 1 μM FCCP (red). One representative experiment is shown. e: Respiratory (open column sections) and glycolytic (blue column sections) proton production rates (PPR) of the experiment exemplified in a-d calculated using Eq 1. Data are means ± SEM of n = 4 independent biological replicates. n.s.: not significant; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.005. Statistical analysis was of glycolytic proton production rates only (blue column sections). w, well; A, B, C, D, addition ports.
Fig 7.
The optimized assay of maximum glycolytic capacity.
Raw traces of (a) oxygen consumption rate (OCR) and (b) extracellular acidification rate (ECAR) in HEK293 fibroblasts after sequential additions of 10 mM glucose, 1 μM rotenone plus 1 μM myxothiazol, and 200 μM myxothiazol plus 1μM FCCP. c: Respiratory (open column sections) and glycolytic (blue column sections) proton production rates (PPR) of the experiment exemplified in A and B calculated using Eq 1. Shaded wedges indicate glycolysis under basal conditions (lightest), ATP demand-limited glycolytic rate (medium), and maximum glycolytic capacity (darkest), with the difference between the basal rate and the maximum glycolytic capacity defined as the glycolytic reserve; Data are means ± SEM of n = 4 independent biological replicates.