Quantifying intracellular rates of glycolytic and oxidative ATP production and consumption using extracellular flux measurements

Abstract

Partitioning of ATP generation between glycolysis and oxidative phosphorylation is central to cellular bioenergetics but cumbersome to measure. We describe here how rates of ATP generation by each pathway can be calculated from simultaneous measurements of extracellular acidification and oxygen consumption. We update theoretical maximum ATP yields by mitochondria and cells catabolizing different substrates. Mitochondrial P/O ratios (mol of ATP generated per mol of [O] consumed) are 2.73 for oxidation of pyruvate plus malate and 1.64 for oxidation of succinate. Complete oxidation of glucose by cells yields up to 33.45 ATP/glucose with a maximum P/O of 2.79. We introduce novel indices to quantify bioenergetic phenotypes. The glycolytic index reports the proportion of ATP production from glycolysis and identifies cells as primarily glycolytic (glycolytic index > 50%) or primarily oxidative. The Warburg effect is a chronic increase in glycolytic index, quantified by the Warburg index. Additional indices quantify the acute flexibility of ATP supply. The Crabtree index and Pasteur index quantify the responses of oxidative and glycolytic ATP production to alterations in glycolysis and oxidative reactions, respectively; the supply flexibility index quantifies overall flexibility of ATP supply; and the bioenergetic capacity quantifies the maximum rate of total ATP production. We illustrate the determination of these indices using C2C12 myoblasts. Measurement of ATP use revealed no significant preference for glycolytic or oxidative ATP by specific ATP consumers. Overall, we demonstrate how extracellular fluxes quantitatively reflect intracellular ATP turnover and cellular bioenergetics. We provide a simple spreadsheet to calculate glycolytic and oxidative ATP production rates from raw extracellular acidification and respiration data.

Keywords: ATP; ECAR; OCR; bioenergetics; energy metabolism; glycolysis; metabolic index; mitochondria; oxidative phosphorylation.

Figure 1.

Maximum extramitochondrial ATP yields and P/O ratios for the catabolism of conventional substrates by isolated mammalian mitochondria and physiological substrates by mammalian cells and calculation of the rates of ATP production from glycolysis, tricarboxylic acid cycle, β-oxidation, and oxidative phosphorylation using oxygen consumption rate. A, maximum extramitochondrial ATP yields and P/O ratios. The maximum net yield of glycolytic ATP/mol of glucose or glycogen converted to pyruvate (and then to lactate or oxidized through the TCA cycle), ATP_glyc, is given in column g, after correction for ATP used to activate glucose or glycogen (calculated using columns b and c and columns e and f). Column o gives ATP_ox, the maximum oxidative yield of ATP/mol of substrate oxidized by pyruvate dehydrogenase, TCA cycle, β-oxidation, and electron transport chain, including substrate-linked phosphorylation in the TCA cycle and NADH equivalents imported from glycolytic reactions, and corrected for ATP used to activate substrates other than glucose (calculated using columns b and c and columns h and n). Values are given with bars (i.e. 1.63 = 1.636363 …) to emphasize that they are not integers or approximations but exact values arising from the arithmetic of small integers as shown (values for glycogen incorporate an assumption about branching, so they are less precise and are therefore rounded to two decimal places). Column p gives the maximum total yield of ATP per mol substrate (sum of columns g and o). Column q expresses this maximum yield of ATP per mol of oxygen atoms [O] consumed (i.e. the maximum P/O ratio for the reactions in column b). The values in each column are calculated row-by-row as follows. During oxidation of succinate to malate by isolated mitochondria, succinate enters on the dicarboxylate carrier in exchange for malate and is oxidized to fumarate by succinate dehydrogenase, reducing Q to QH₂. QH₂ is oxidized by the electron transport chain, reducing 1 [O] to H₂O and driving 6 H⁺ from the matrix to the intermembrane space. The fumarate is hydrated to malate by fumarase and exits in exchange for incoming succinate. The 6 H⁺ re-enter the matrix through the ATP synthesis machinery, which translocates 8 H⁺ through the ATP synthase and 3 H⁺ through the phosphate and adenine nucleotide carriers for every 3 ATP generated, giving an H⁺/ATP ratio of 11:3 (see “Results”). In this way, the oxidation of succinate causes phosphorylation of 6 H⁺/O × 3/11 ATP/H⁺ = 1.63 ATP molecules/[O] reduced to water. This is the maximum P/O ratio for oxidation of succinate by mitochondria. During oxidation of glycerol 3-phosphate to dihydroxyacetone phosphate by isolated mitochondria, mitochondrial glycerol 3-phosphate dehydrogenase reduces Q to QH₂, which is oxidized as above, with the same P/O_max of 1.63. During oxidation of pyruvate plus malate by isolated mitochondria, pyruvate enters mitochondria on the pyruvate carrier (electroneutrally with a proton) and is oxidized to acetyl-CoA and CO₂ by pyruvate dehydrogenase. Malate enters in exchange for citrate and is oxidized to oxaloacetate by malate dehydrogenase. The two dehydrogenation reactions form a total of 2 NADH. Citrate synthase uses acetyl-CoA and oxaloacetate to form citrate, which exits the mitochondria with a proton (balancing the proton imported with pyruvate) on the tricarboxylate carrier in exchange for incoming malate. The 2 NADH are oxidized, driving pumping of 20 H⁺ and generating 20 × 3/11 = 5.45 ATP with a P/O_max of 2.72. During oxidation of malate plus glutamate by isolated mitochondria, malate enters electroneutrally on the dicarboxylate carrier in exchange for 2-oxoglutarate, and glutamate enters on the glutamate-aspartate carrier electrogenically with a proton (which is therefore unavailable for ATP synthesis; column l) in exchange for aspartate. Malate dehydrogenase produces oxaloacetate plus 1 NADH, which is oxidized, driving pumping of 10 H⁺, and then aspartate aminotransferase uses oxaloacetate and glutamate to generate 2-oxoglutarate and aspartate, which exit in exchange for incoming malate and glutamate. Overall, 9 H⁺ are translocated, driving synthesis of 9 × 3/11 = 2.45 ATP/[O]. During glycolysis of glucose to lactate by cells, 1 ATP is consumed at hexokinase, and 1 ATP is consumed at phosphofructokinase to yield 2 trioses, each of which generates 1 ATP at phosphoglycerate kinase and 1 ATP at pyruvate kinase, for a net yield of 2 ATP/glucose. The 2 NADH generated at glyceraldehyde phosphate dehydrogenase are reoxidized during reduction of pyruvate to lactate by lactate dehydrogenase, and 2 lactates are exported from the cell accompanied by the 2 H⁺ generated by the conversion of 1 uncharged glucose to 2 anionic lactate⁻ molecules. Overall, glycolysis to lactate produces 2 ATP/glucose (1 ATP/lactate). During glycolysis of glucose to pyruvate by cells followed by oxidation of pyruvate to bicarbonate by pyruvate dehydrogenase and the TCA cycle, 2 ATP are formed by glycolytic reactions per glucose. In addition, 2 ATP are formed in the mitochondrial matrix during oxidative metabolism by the substrate-linked reaction at succinyl-CoA synthetase. Each of these ATPs is exported to the cytosol, using 1 H⁺ in the process, giving a net cytosolic ATP yield from succinyl-CoA synthetase of 2 × (1 − 3/11) = 1.45 ATP/glucose. NADH generated at glyceraldehyde phosphate dehydrogenase enters the mitochondria on the malate-aspartate shuttle, driven by re-entry of 2 of the 20 subsequently translocated H⁺ (column l), or on the glycerol 3-phosphate shuttle (which allows the reducing equivalents to enter the electron transport chain without passing through complex I, so pumping 12 H⁺, 8 fewer than normal for matrix NADH; column l). The 2 pyruvates from glycolysis are fully oxidized by pyruvate dehydrogenase and the TCA cycle, generating 8 NADH and 2 QH₂ (driving pumping of 92 H⁺). The sum of 110 (or 104 if the glycerol 3-phosphate shuttle is used) translocated H⁺ yields a maximum of 110 × 3/11 = 30 (or 104 × 3/11 = 28.36 ATP, which, together with the substrate-linked ATP production, gives a net oxidative yield of 31.45 (or 28.81) ATP/glucose. The overall ATP yield is the sum of the glycolytic and oxidative yields: 33.45 (or 31.81) ATP/glucose or a P/O_max of 2.78 (or 2.651). During catabolism of glycogen, the yields are the same as those for catabolism of glucose, except that less ATP is needed for the initial activation reactions. About 90% of the linkages in glycogen are α-1,4, which are split by the addition of phosphate, yielding glucose 1-phosphate and bypassing the consumption of ATP at hexokinase. The remainder are α-1,6, which are hydrolyzed to yield glucose, which requires activation at hexokinase. On average, activation therefore requires ∼0.1 ATP at hexokinase and 1 ATP at phosphofructokinase, increasing the ATP yield of glycogen catabolism by ∼0.9 ATP/glucose compared with catabolism of glucose itself, giving the yields of ATP/glucose unit and P/O_max ratios shown. Complete oxidation of pyruvate by cells bypasses glycolysis and generates 1 ATP/pyruvate at succinyl-CoA synthetase in the matrix (0.72 ATP/pyruvate after export of ATP). Proton pumping yields 46 × 3/11 = 12.54 ATP/pyruvate, for a sum of 13.27 ATP/pyruvate or a P/O_max of 2.654. During complete oxidation of palmitate by cells, palmitate activation to palmitoyl-CoA generates AMP from ATP, effectively using 2 ATP/palmitate. Palmitoyl-CoA enters the matrix electroneutrally as palmitoyl carnitine on the carnitine transporter, and then β-oxidation to 8 acetyl-CoA yields 7 NADH and 7 QH₂, and oxidation of 8 acetyl-CoA in the TCA cycle yields 24 NADH, 8 QH₂, and 8 matrix ATP. The maximum overall yield is 112.90 ATP/palmitate and a P/O_max of 2.45. Oxidation of other fatty acids gives slightly different yields and P/O_max values; the monounsaturated oleate, whose oxidation generates 1 fewer QH₂ and 6 fewer H⁺ translocated than the corresponding saturated fatty acid, is calculated out as an example. HK, hexokinase; PFK, phosphofructokinase; PK, pyruvate kinase; PGK, phosphoglycerate kinase; ACS, acyl- CoA synthase; SCS, succinyl-CoA synthetase. Purple highlights isolated mitochondria; gray highlights cells (and the darker band highlights complete oxidation of glucose by cells). B, calculation of yields of ATP per oxygen consumed. Non-repeating values are rounded to two decimal places for assumed estimates and to three decimal places for real numbers. Columns s–u divide the overall P/O ratio in A (column q) into components dependent on different subsets of the total mitochondrial oxygen consumption, to enable calculations of glycolytic and oxidative ATP yields from experimental oxygen consumption data (glycolytic ATP yields from glycolysis to lactate are calculated from extracellular acidification, but glycolytic ATP yields from glycolysis to pyruvate (subsequently oxidized) are calculated from oxygen consumption). Column s gives the glycolytic ATP yield from conversion of glucose or glycogen to pyruvate subsequently oxidized to bicarbonate (which depends on total mitochondrial oxygen consumption). Column t gives the P/O ratio for substrate-linked reactions in the TCA cycle and β-oxidation (which depends on total mitochondrial oxygen consumption), and column u gives the P/O ratio for oxidation of NADH and QH₂ derived from pyruvate dehydrogenase, TCA cycle, and β-oxidation (which depends on coupled mitochondrial oxygen consumption). Column v gives the overall sum of these partial P/O ratios, which are the same as column q in A. β-ox, β-oxidation; oxphos, oxidative phosphorylation. C, the total rate of ATP production, J_{ATP production}, is the sum of measurable extracellular rates (PPR and OCR) multiplied by the appropriate ATP/lactate ratio from A or P/O ratio from B.

Figure 2.

Substrate catabolism by glycolysis and oxidation and related extracellular measurements. A, steady-state relationship between rates of glycolytic and oxidative ATP production and rate of ATP consumption, connected by ATP (or other linked variables, such as ATP/ADP or phosphorylation potential) as an intermediate metabolite. B, pathways of glucose and glycogen catabolism giving rise to measurable extracellular rates of acid production (PPR) and oxygen consumption (OCR). The black outline represents the mitochondrial inner membrane enclosing the mitochondrial matrix. Blue section, reactions linked directly to PPR_glyc. Exogenous glucose that enters the cell (or endogenous glycogen) is converted to pyruvate, yielding net ATP. Pyruvate can be converted to lactate, consuming glycolytic NADH and causing extracellular acidification (PPR_glyc). Red section, reactions linked directly to PPR_resp and OCR. Reducing equivalents from glycolysis are delivered into the matrix by one of two shuttles (C). Pyruvate also enters the matrix and is fully oxidized to CO₂ by pyruvate dehydrogenase and the tricarboxylic acid cycle, yielding further reducing equivalents. Electrons (e⁻) are fed into the respiratory complexes of the electron transport chain, driving oxygen consumption. OCR is shown divided into parts (red arrows), where OCR_leak is OCR due to proton leak reactions not coupled to oxidative phosphorylation (OCR_leak = OCR_oli − OCR_r/m) and OCR_coupled is OCR coupled to oxidative phosphorylation. C, detail of shuttles for mitochondrial import of reducing equivalents shown in B. Left, malate-aspartate shuttle; right, glycerol 3-phosphate shuttle. GA3P, glyceraldehyde 3-phosphate; OAA, oxaloacetate; OG, 2-oxoglutarate; G3P, glycerol 3-phosphate; DHAP, dihydroxyacetone phosphate. NADH + H⁺ cycles in the steady state, causing no net change in H⁺, and is shown as NADH₂ to reduce confusion over sources of PPR.

Figure 3.

Raw extracellular flux data and its conversion into rates of ATP production (J_ATP). A and B, raw traces of extracellular acidification and oxygen consumption by C2C12 myoblasts. A basal measurement was recorded in the absence of exogenous substrate, followed by the addition of 10 mm glucose, vehicle control (DMSO; maximum concentration <0.05% (v/v)), 2 μg/ml oligomycin (oli), and 1 μm rotenone with 1 μm myxothiazol (rot/myx). Points within gray regions were assumed to be at or near steady state and were part of the data set used to calculate values shown in sequential columns in C and D. Points are means ± S.E. (error bars) of n = 4 independent experiments. The final protein content of each well was typically 10–15 μg. C, J_{ATP production} for each time point marked in gray in A and B, divided into J_ATPglyc and J_ATPox. Aggregate data from multiple experiments, including those in A and B, gave rise to basal n = 24, glucose n = 36, and glucose + oligomycin n = 19. D, data in C further divided into the component reactions of J_ATPglyc and J_ATPox. J_ATPglyc is divided into ATP production during glycolytic production of pyruvate that is either converted to lactate (J_{ATPglyc to lac}) or imported into the matrix and oxidized (J_{ATPglyc to bicarbonate}).J_ATPox is divided into ATP production from oxidation of glycolytic NADH (J_ATPox-glyc), from oxidation of reducing equivalents generated within the mitochondria (J_ATPox-oxphos) and from succinyl-CoA synthetase (J_ATPox-SCS).

Figure 4.

Visualization of bioenergetic phenotypes. A single raw data set from C2C12 myoblasts is presented in different ways in different panels. A, column plot of raw ECAR and OCR values plotted on the same y axis, with no exogenous substrate (basal) and in the presence of different combinations of glucose (g), oligomycin (o), FCCP (F), rotenone plus myxothiazol (r/m), and monensin (mon) as shown. B, data from A plotted as points with (ECAR, OCR) as (x, y) values. C, data from A converted to J_ATPglyc and J_ATPox. D, data from C presented as stacked columns summing to J_{ATP production}. E, data from C plotted as points with (J_ATPglyc, J_ATPox) as (x, y) values. Constructed lines through each point show slopes denoting J_ATP proportionality (slope = 1 denotes equal J_ATP by source) and of −1 (denoting the same J_ATP as the point) through each of the data points. Aggregate data from multiple experiments are presented, giving n values for each condition as follows: basal = 24, glucose = 36, glucose + oligomycin = 19, glucose + oligomycin + FCCP = 12, glucose + rotenone/myxothiazol = 8, glucose + rotenone/myxothiazol + monensin = 8. Values are means ± S.E. (error bars) of n independent experiments.

Figure 5.

Metabolic indices. In all panels, the solid red line connects points with different values of J_{ATP production} but the same value of J_ATPglyc/J_{ATP production} as the point with added glucose (other lines through the origin indicate different values of J_ATPglyc/J_{ATP production}), and lines of slope = −1 connect combinations of J_ATPglyc and J_ATPox that sum to the same value of J_{ATP production} (iso-J_ATP). A, GI = (100 × J_ATPglyc/J_{ATP production}). Vertical and horizontal dotted lines, values of the (J_ATPglyc, J_ATPox) coordinate point for C2C12 cells with glucose (GI = 44%). The thick line of slope = 1 connects points with GI = 50% and denotes the threshold for the a primarily glycolytic cell, defined as points with GI > 50% lying within the blue shaded area. B, CI = GI_{condition 2} − GI_{condition 1} caused by a change in glycolysis, indicated by the curved arrow showing the effect of added glucose on J_ATPox. C, PI = GI_{condition 1} − GI_{condition 2} caused by a change in oxidative reactions, indicated by the curved arrows showing the effects of “removal” of oligomycin (light blue) and “removal” of rotenone plus myxothiazol (dark blue) on J_ATPglyc. D, bioenergetic capacity and bioenergetic scope. Solid black lines denote the empirical (vertical) maximum for J_ATPglyc and the theoretical (horizontal) maximum for J_ATPox. The dotted black line indicates GI_mc = GI_{max capacity} = 57%, passing though the theoretical bioenergetic maximum J_ATP of 109 pmol of ATP/min/μg of protein. The bioenergetic capacity is indicated, after scaling to correct for the change in GI needed to achieve maximum J_{ATP production}. The shaded box indicates the bioenergetic scope: all possible points of J_{ATP production}, bounded by the maximum capacities of each supply flux. Thick black arrows indicate ATP supply flexibility; thick red arrows indicate ATP demand flexibility (see “Discussion”). E, SFI = 100 × θ°/90°, showing the range over which ATP demand can be met by shifting J_{ATP production} between J_ATPglyc and J_ATPox. The thick arrows indicate SFI in the presence of glucose, and the thin arrow associated with θ₁ indicates how SFI would increase at lower ATP turnover. The dashed arrow associated with θ₂ indicates how SFI would decrease at higher ATP turnover.

Figure 6.

Rates of consumption of ATP by different pathways in C2C12 myoblasts. A, steady-state rates of glycolytic ATP production (J_ATPglyc) and oxidative ATP production (J_ATPox) sum to the total rate of ATP production (J_{ATP production}) and hence to the total rate of ATP consumption by multiple ATP consumers (J_{ATP consumers}). ATP production and consumption are connected by ATP (or other linked variables, such as ATP/ADP or phosphorylation potential) as the common intermediate. B, absolute J_ATPglyc and J_ATPox supplying individual ATP consumers, calculated from the decrease in ECAR and OCR caused by the addition of different inhibitors of specific ATP consumption pathways (colored bars) or unassigned (by difference from the total J_{ATP consumption} in the absence of inhibitors). The third bar shows the set of values for J_ATPox stacked above the set of values for J_ATPglyc to display total assigned and unassigned rates of ATP consumption. C, data from B scaled to the uninhibited totals for J_ATPglyc, J_ATPox, and J_{ATP consumption} as 100%. No significant differences were found by unpaired t test between percentage contribution of J_ATPglyc and percentage contribution of J_ATPox for any single consumer or for “unassigned” percentage contribution of J_ATP. The third bar shows the individual values for J_ATPox stacked above the corresponding individual values for J_ATPglyc to emphasize the contributions of each ATP consumption pathway to J_{ATP consumption}. Compounds used to define individual pathways of ATP consumption were as follows: 10 μm cycloheximide (protein synthesis), 25 μm MG132 (26S proteasome activity), 1 mm ouabain (plasma membrane Na⁺ cycling), 1 μm nocodazole (tubulin dynamics), 0.5 μm thapsigargin (intracellular Ca²⁺ cycling), and 0.25 μm latrunculin A (actin dynamics). Values are means ± S.E. (error bars) of n = 6–8 independent experiments/compound.