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

Open AccessPublished:April 28, 2017DOI:https://doi.org/10.1074/jbc.M116.774471
      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.

      Introduction

      Cells require energy to run the reactions that maintain their viability, growth, and proper function. The dominant currency of chemical energy in cells is ATP, which is produced mostly by two pathways: glycolysis in the cytosol and oxidative phosphorylation in the mitochondria. The rates of these pathways are cumbersome to measure directly. They can be followed using the fluxes of isotopic tracers, such as 13C and 31P, between metabolic pools (e.g. Refs.
      • Thompson J.
      • Torchia D.A.
      Use of 31P nuclear magnetic resonance spectroscopy and 14C flurography in studies of glycolysis and regulation of pyruvate kinase in Streptococcus lactis.
      and
      • Metallo C.M.
      • Walther J.L.
      • Stephanopoulos G.
      Evaluation of 13C isotopic tracers for metabolic flux analysis in mammalian cells.
      ). However, they can also be estimated relatively easily and quickly from the rates of linked reactions: extracellular acidification resulting from glycolytic conversion of uncharged glucose to 2 lactate plus 2 H+ and oxygen consumption to oxidize pyruvate and other substrates and to support oxidative phosphorylation.
      The rate of glycolysis in cell culture has been measured in a variety of ways, including using the rates of production of lactate and protons. Our focus here is on measurement of extracellular proton production, which can quantitatively report the rate of glycolysis to lactate and can be measured using pH electrodes or by fluorescent indicators simultaneously with oxygen consumption in commercial instruments (
      • Wu M.
      • Neilson A.
      • Swift A.L.
      • Moran R.
      • Tamagnine J.
      • Parslow D.
      • Armistead S.
      • Lemire K.
      • Orrell J.
      • Teich J.
      • Chomicz S.
      • Ferrick D.A.
      Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells.
      ,
      • Mookerjee S.A.
      • Brand M.D.
      Measurement and analysis of extracellular acid production to determine glycolytic rate.
      ,
      • Mookerjee S.A.
      • Goncalves R.L.S.
      • Gerencser A.A.
      • Nicholls D.G.
      • Brand M.D.
      The contributions of respiration and glycolysis to extracellular acid production.
      ).
      Historically, the rate of oxygen consumption has been measured using Clark oxygen electrodes. These can consume significant amounts of oxygen and therefore work best in well-mixed, bulk aqueous solutions, limiting amenable experimental systems to suspended material, including cells and isolated mitochondria. More recent methods use fluorescent sensors, which have high sensitivity and bind negligible amounts of oxygen and can therefore be used to measure oxygen consumption rates in small volumes of aqueous media above adherent mitochondria and cell cultures. Modern commercial fluorescence-based instruments for the measurement of oxygen consumption rates (
      • Wu M.
      • Neilson A.
      • Swift A.L.
      • Moran R.
      • Tamagnine J.
      • Parslow D.
      • Armistead S.
      • Lemire K.
      • Orrell J.
      • Teich J.
      • Chomicz S.
      • Ferrick D.A.
      Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells.
      ) incorporate sophisticated corrections for oxygen diffusion (
      • Gerencser A.A.
      • Neilson A.
      • Choi S.W.
      • Edman U.
      • Yadava N.
      • Oh R.J.
      • Ferrick D.A.
      • Nicholls D.G.
      • Brand M.D.
      Quantitative microplate-based respirometry with correction for oxygen diffusion.
      ) and have been described extensively in both practical (
      • Mookerjee S.A.
      • Brand M.D.
      Measurement and analysis of extracellular acid production to determine glycolytic rate.
      ,
      • Nicholls D.G.
      • Darley-Usmar V.M.
      • Wu M.
      • Jensen P.B.
      • Rogers G.W.
      • Ferrick D.A.
      Bioenergetic profile experiment using C2C12 myoblast cells.
      ) and theoretical contexts (
      • Brand M.D.
      • Nicholls D.G.
      Assessing mitochondrial dysfunction in cells.
      ).
      Combining the extracellular measurement of rates of acidification and oxygen consumption into one assay provides a powerful way to assess the total energy metabolism of a cell (i.e. the total rate of ATP cycling through production by both glycolysis and oxidative reactions and consumption by the pathways that impose an ATP demand). A description of the bioenergetic phenotype of a cell is the highly desired outcome of many investigations and can be an important reporter of cellular status and behavior. However, despite the potential utility of simultaneous measurement of glycolysis (by extracellular acidification) and oxidative phosphorylation (by oxygen consumption) to quantify ATP turnover, most current analyses directly compare extracellular acidification rate (ECAR,
      The abbreviations used are: ECAR
      extracellular acidification rate
      FCCP
      carbonyl cyanide p-trifluoromethoxyphenylhydrazone
      P/O ratio
      mol of ATP made/mol of oxygen atoms ([O]) consumed
      H+/O ratio
      mol of protons translocated/mol of oxygen atoms ([O]) consumed
      H+/ATP ratio
      mol of protons translocated/mol of ATP consumed or generated
      Q
      ubiquinone
      QH2
      ubiquinol
      OCR
      oxygen (O2) consumption rate
      OCRtot
      total OCR
      OCRr/m
      OCR in the presence of rotenone plus myxothiazol
      OCRoli
      OCR in the presence of oligomycin
      OCRmito
      OCRtot − OCRr/m
      OCRcoupled
      OCRtot − OCRoli
      PPR
      proton production rate
      PPRglyc
      PPR linked to lactate production
      PPRtot
      total PPR
      PPRresp
      proton production rate linked to HCO3 production
      ATPglyc
      net amount of ATP produced by glycolysis to pyruvate (pyruvate subsequently reduced to lactate or oxidized to bicarbonate)
      ATPox
      net amount of ATP produced by oxidative reactions (oxidative phosphorylation and tricarboxylic acid cycle, etc.)
      JATPglyc
      net rate of ATP production by glycolysis to pyruvate (pyruvate subsequently reduced to lactate or oxidized to bicarbonate)
      JATPglyc to lac
      net rate of ATP production by glycolysis to pyruvate reduced to lactate
      JATPglyc to bicarbonate
      net rate of ATP production by glycolysis to pyruvate oxidized to bicarbonate
      JATPox
      net rate of ATP production by oxidative reactions (oxidative phosphorylation and tricarboxylic acid cycle, etc.)
      JATP production
      JATPglyc + JATPox
      GI
      glycolytic index (100 × JATPglyc/JATP production)
      WI
      Warburg index (GI − GIo for a chronic change from baseline GIo)
      CI
      Crabtree index (GIcondition 2 − GIcondition 1 following a change in glycolysis)
      PI
      Pasteur index (GIcondition 1 − GIcondition 2 following a change in oxidative reactions)
      SFI
      supply flexibility index (100 × θ°/90°)
      mpH unit
      milli-pH unit
      TCA
      tricarboxylic acid
      BP
      buffering power (ΔpH/nmol H+)
      SCS
      succinyl-CoA synthetase.
      in mpH units/min) to oxygen consumption rate (OCR, in pmol O2/min). This direct comparison can be very misleading, for five main reasons.
      First, the rate of change of pH depends on the buffering power (BP) of the medium (BP = change in pH/nmol of H+). A given rate of glycolytic proton production will cause high rates of pH change in a lightly buffered medium but low rates of pH change in a well-buffered medium. Correction for the buffering power of the medium and conversion of ECAR to total proton production rate (PPRtot = ECAR/BP) (
      • Mookerjee S.A.
      • Goncalves R.L.S.
      • Gerencser A.A.
      • Nicholls D.G.
      • Brand M.D.
      The contributions of respiration and glycolysis to extracellular acid production.
      ) is essential for quantitative interpretation of raw data.
      Second, during catabolism of glucose, there are two main pathways that contribute to extracellular acidification: conversion of glucose to lactate plus H+ (PPRglyc) and conversion of glucose to bicarbonate plus H+ (PPRresp). Acidification associated with bicarbonate production can be small, intermediate, or dominant, or all of these within one experiment, so it must be assessed and subtracted before the proton production rate can be equated to the lactate production rate (PPRglyc = PPRtot − PPRresp) (
      • Mookerjee S.A.
      • Goncalves R.L.S.
      • Gerencser A.A.
      • Nicholls D.G.
      • Brand M.D.
      The contributions of respiration and glycolysis to extracellular acid production.
      ).
      Third, pyruvate produced by glycolysis has two different fates: reduction to lactate or oxidation to bicarbonate. For correct assessment of the rate of glycolysis to pyruvate, with either subsequent fate of the pyruvate, the rates of these two components must be converted to the same units (e.g. glucose consumed/min) and summed.
      Fourth, even when the rates of glycolysis and oxidative metabolism are correctly assessed, the two pathways produce very different amounts of ATP per glucose. To characterize a cell as “very glycolytic,” the majority of its ATP should come from glycolysis rather than oxidative reactions. The rates of the two pathways should therefore be converted into the same units (ATP production) before they are compared. As shown in Fig. 1, glucose catabolism produces 2 ATP/glucose from glycolysis but up to 31. 45¯ ATP/glucose from oxidative reactions during the complete oxidation of glucose to bicarbonate. This gearing is easily accounted for by comparing the rates of the two pathways in the same units (i.e. ATP produced per time).
      Figure thumbnail gr1
      Figure 1Maximum 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), ATPglyc, 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 ATPox, 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/Omax 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/Omax 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/Omax 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/Omax 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/Omax 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/Omax of 2. 45¯. Oxidation of other fatty acids gives slightly different yields and P/Omax 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, JATP 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.
      Fifth, because of the leak of protons across the mitochondrial inner membrane, not all oxygen consumption is coupled to ATP synthesis, so it is coupled oxygen consumption, not total oxygen consumption, that should be considered.
      Once these factors are considered, the rationale for calculating JATPglyc and JATPox is clear. The method we introduce here builds on our previous deconvolution of glycolytic and respiratory sources of acidification (
      • Mookerjee S.A.
      • Brand M.D.
      Measurement and analysis of extracellular acid production to determine glycolytic rate.
      ,
      • Mookerjee S.A.
      • Goncalves R.L.S.
      • Gerencser A.A.
      • Nicholls D.G.
      • Brand M.D.
      The contributions of respiration and glycolysis to extracellular acid production.
      ) and extends this analysis to the calculation of rates of intracellular ATP production by glycolysis and oxidative reactions from extracellular measurements of rates of acidification and oxygen consumption. Once the rates of ATP production are known, we show how they can be used to quantify and interpret classical qualitative indicators of cellular energy metabolism, the Warburg, Crabtree, and Pasteur effects, and to quantify the flexibility of substrate use and the bioenergetic capacity of cells. We use this method to characterize the bioenergetic phenotype of C2C12 myoblasts under different conditions and to assess whether different ATP-consuming reactions draw preferentially on glycolytic or oxidative ATP production.

      Results

      Updated consensus view of maximum ATP yields by mitochondria and cells catabolizing different substrates

      The yield of ATP from oxidative phosphorylation per oxygen atom ([O]) consumed, the P/O ratio, has been investigated for 70 years using isolated mitochondria. When the mechanism was thought to be analogous to substrate-level phosphorylation, the P/O ratio was assumed to be a small integer, with a maximum value of 3 for oxidation of NAD-linked substrates. Once a chemiosmotic mechanism was accepted, it became clear that the maximum P/O ratio for reducing equivalents entering the electron transport chain is the number of protons pumped out of the matrix (H+/O) divided by the number needed to make each ATP as protons re-enter the matrix (H+/ATP). H+/O and H+/ATP are themselves composed of simple combinations of a few small integers resulting from the underlying molecular mechanisms, so the maximum P/O ratio is generally not an integer. Brand (
      • Brand M.D.
      The efficiency and plasticity of mitochondrial energy transduction.
      ) summarized the then-consensus values of H+/O, H+/ATP, and P/O for different substrates, based on empirical measurements refined by theoretical mechanistic models. The H+/O ratio for oxidation of simple NAD-linked substrates by mitochondria was (and remains) 10. The H+/ATP ratio at the F1FO-ATP synthase is the number of protons driven through the c-ring of the FO subunit during a complete rotation of the F1 subunit, divided by the 3 ATP synthesized per rotation. Based on the reported 10 c-subunits/c-ring in yeast FO, the value for H+/ATP was therefore 10 H+ translocated/rotation, plus 3 H+ used to translocate the 3 ATP made or consumed, giving an H+/ATP ratio of 13:3. Combining the H+/O and H+/ATP values gave 10 × 3/13 for the maximum P/O ratio for oxidation of simple NAD-linked substrates by mitochondria, close to 2.308.
      In Fig. 1A, we update the H+/ATP and P/O values in Ref.
      • Brand M.D.
      The efficiency and plasticity of mitochondrial energy transduction.
      , based on subsequent refinement of the structural model of FO. There are now thought to be eight c-subunits in the c-ring of the mammalian F1FO-ATP synthase, not 10 (
      • Watt I.N.
      • Montgomery M.G.
      • Runswick M.J.
      • Leslie A.G.
      • Walker J.E.
      Bioenergetic cost of making an adenosine triphosphate molecule in animal mitochondria.
      ). Assuming 8 H+ translocated per rotation, H+/ATP changes from 13:3 to 11:3, altering the theoretical P/O ratios and potential yields of ATP. For example, for oxidation of pyruvate plus malate by isolated mitochondria, the revised maximum P/O ratio is 10 × 3/11, close to 2.727 (Fig. 1A) and substantially larger than the previous value of 2.308 (
      • Brand M.D.
      The efficiency and plasticity of mitochondrial energy transduction.
      ).
      Fig. 2 shows the pathways involved when glucose (or glycogen) is used as the substrate for cellular ATP production, and Fig. 1A provides the associated accounting. During cellular glycolysis to lactate, the net yield is 2 ATP/glucose (or 2.9 ATP/glucose unit in glycogen, assuming 90% α-1,4 glycosidic bonds). During complete oxidation of glucose, glycolysis yields 2 ATP/glucose, and oxidative phosphorylation plus the tricarboxylic acid cycle yields up to 31. 45¯ ATP/glucose; the maximum total yield is 33. 45¯ ATP/glucose and the maximum overall P/O ratio is 2. 78¯. During complete oxidation of glycogen, these values are 2.9, 31.45, 34.35, and 2.86, respectively. The full set of updated values for the oxidation of a variety of substrates by mitochondria and cells and the assumptions behind the calculations are detailed in Fig. 1A.
      Figure thumbnail gr2
      Figure 2Substrate 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 PPRglyc. 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 (PPRglyc). Red section, reactions linked directly to PPRresp 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 OCRleak is OCR due to proton leak reactions not coupled to oxidative phosphorylation (OCRleak = OCRoli − OCRr/m) and OCRcoupled 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.

      Definition of amounts and rates of glycolytic and oxidative ATP production

      The vast majority of ATP made in cells comes either from glycolysis (ATPglyc) or from oxidative reactions (ATPox); the corresponding rates (denoted by J) are JATPglyc and JATPox. If we apply the principles of modular kinetic analysis (
      • Brand M.D.
      Top down metabolic control analysis.
      ), the complex network of metabolic reaction rates and concentrations of metabolic intermediates involved in ATP turnover collapses to a simple model, represented in Fig. 2A, in which JATPglyc and JATPox are represented by arrows that point to a common intermediate (ATP, ATP/ADP, phosphorylation potential, etc.), describing the two major pathways by which ATP is generated in cells. A third arrow (JATP consumption) points away from this intermediate and represents all of the pathways that consume ATP.
      The relevant reactions of ATP production in the steady state are made explicit in Fig. 2B. We define ATPglyc as the net amount of ATP made by substrate-level phosphorylation during glycolytic conversion of glycogen or glucose (or other sugars) to pyruvate. The pyruvate has two possible reaction fates (Fig. 2B); therefore, the total JATPglyc is the sum of these two rates. Pyruvate can be reduced to lactate using the NADH produced at glyceraldehyde 3-phosphate dehydrogenase, driving no further ATP synthesis; the amount of ATP produced during glucose catabolism by this route (ATPglyc to lac) can be calculated from total lactate production, and the portion of JATPglyc by this route can be calculated from the rate of lactate production. Lactate production can be measured directly or calculated from the associated extracellular acidification (PPRglyc; see below). Alternatively, the pyruvate can be oxidized by the mitochondria, in which case the reducing equivalents on glycolytic NADH are also oxidized by the mitochondria, and the further ATP that is generated from this NADH is counted as part of ATPox. This portion of JATPglyc (JATPglyc to bicarbonate) can be calculated from the associated rate of oxygen consumption, assuming full oxidation of the pyruvate to bicarbonate (see below and Figure 1, Figure 2). Note that this definition of ATPglyc includes all of the ATP made during glycolysis to pyruvate, not just the ATP that is made with lactate as the final product. This definition of ATPglyc is rational because it describes overall flux through glycolysis. However, it contrasts with a different common definition of glycolytic ATP production, which is strictly that of glycolysis to lactate. It is important to be clear about which definition is being used in the literature in any particular context.
      We define ATPox as the net amount of ATP made during oxidative metabolism, both oxidative phosphorylation (using reducing equivalents from both glycolysis and pyruvate oxidized to bicarbonate) and substrate-level phosphorylation at succinyl-CoA synthetase as carbon flows around the tricarboxylic acid cycle. The reducing equivalents carried on cytosolic NADH derived from glycolysis can enter the mitochondrial matrix by two different routes, the malate-aspartate shuttle and the glycerol 3-phosphate shuttle (Fig. 2, B and C); the two routes give slightly different ATP yields. The rate of oxidative ATP production (JATPox) can be calculated from the rate of oxygen consumption for complete oxidation of a defined substrate (Figure 1, Figure 2).
      The total rate of ATP production JATP production = JATPglyc + JATPox. In the steady state, JATP production is equal to the total rate of ATP consumption, JATP consumption.

      The model system; C2C12 cells consuming glycogen or glucose by defined pathways

      Indirect estimation of rates of ATP production from extracellular acid and oxygen fluxes as described below is a very powerful approach to understanding cellular bioenergetics, providing important information from simple measurements. In support of this view, the reaction stoichiometries for the complete oxidation of different substrates are unambiguous (Fig. 1), the contribution of glycolytic lactate production to overall extracellular acidification can be readily calculated from measured extracellular changes in pH and oxygen concentration (
      • Mookerjee S.A.
      • Goncalves R.L.S.
      • Gerencser A.A.
      • Nicholls D.G.
      • Brand M.D.
      The contributions of respiration and glycolysis to extracellular acid production.
      ), and the maximum P/O values predicted by our current understanding of ATP synthesis (Fig. 1) are constant (although subject to possible future change with further refinement of the mechanistic models of the proton pumps).
      Although this approach is unambiguous for defined substrates, physiologically relevant mixes of different substrates lead to greater calculation uncertainty. In addition, when cells are growing, flux through anabolic pathways (such as the pentose phosphate pathway) may be quantitatively important. Under these conditions, the relationships between the extracellular measurements and the ATP production by the two major pathways are poorly defined unless the carbon pathways and fluxes are known or assumed. This is because the respiratory quotient (CO2 produced/O2 consumed), which is used to define PPRglyc, depends on the substrate and the extent to which it is fully oxidized (although the respiratory quotients for complete oxidation of conventional substrates range only from about 0.7 to 1.2 (
      • Mookerjee S.A.
      • Goncalves R.L.S.
      • Gerencser A.A.
      • Nicholls D.G.
      • Brand M.D.
      The contributions of respiration and glycolysis to extracellular acid production.
      ), so assuming a value of 1.0 is usually adequate for semi-quantitative estimation). Similarly, the overall maximum P/O ratio is the weighted mean of the P/O values of all the oxidative pathways. For the complete oxidation pathways described in Figure 1, Figure 2, the range of maximum P/O values is small (the value for glycogen is only 17% greater than the value for palmitate), and the assumption of an average value would have only small effects on the calculations. However, other more unusual, partial, or complicated pathways have more extreme respiratory quotients and P/O ratios.
      Fortunately, it is possible to avoid these complications when demonstrating the underlying concepts, by designing experiments that minimize the number of parallel metabolic routes. To keep things relatively straightforward, in the present paper, we consider only the simplest case of glucose or glycogen being converted to lactate or fully oxidized to bicarbonate. ATP yields during the metabolism of other substrates or mixtures of substrates could be analyzed in the same way, but these cases require knowledge or assumptions about the relative fluxes through each metabolic pathway, complicating the analysis.
      This approach to predicting cellular ATP production is accurate to the extent that the assumptions behind the calculations fit the experimental system. The initial model that we present here assumes that cells given no exogenous substrate metabolize only endogenous glycogen, and when external glucose is provided, this glucose is the sole substrate. In either case, we assume that glucose units are only converted to lactate or fully oxidized to bicarbonate, that reducing equivalents on extramitochondrial NADH enter the matrix primarily (90%) through the malate-aspartate shuttle, and that there are no anabolic reactions or cell growth.
      We have chosen a cell model (C2C12 myoblasts) and experimental design that fit these assumptions within the error of our measurements. C2C12 myoblasts were used in previous work that was foundational to the data we present here (
      • Mookerjee S.A.
      • Brand M.D.
      Measurement and analysis of extracellular acid production to determine glycolytic rate.
      ,
      • Mookerjee S.A.
      • Goncalves R.L.S.
      • Gerencser A.A.
      • Nicholls D.G.
      • Brand M.D.
      The contributions of respiration and glycolysis to extracellular acid production.
      ,
      • Mookerjee S.A.
      • Nicholls D.G.
      • Brand M.D.
      Determining maximum glycolytic capacity using extracellular flux measurements.
      ), and in other work using the Seahorse XF extracellular flux analyzer (
      • Nicholls D.G.
      • Darley-Usmar V.M.
      • Wu M.
      • Jensen P.B.
      • Rogers G.W.
      • Ferrick D.A.
      Bioenergetic profile experiment using C2C12 myoblast cells.
      ). Glucose uptake by C2C12 myoblasts is largely insulin-independent, occurring primarily through the GLUT1 transporter (
      • Mangnall D.
      • Bruce C.
      • Fraser R.
      Insulin-stimulated glucose uptake in C2C12 myoblasts.
      ,
      • Nedachi T.
      • Kanzaki M.
      Regulation of glucose transporters by insulin and extracellular glucose in C2C12 myotubes.
      ). Under our standard experimental conditions, all of the extracellular acidification and oxygen consumption can be quantitatively assigned to glycolysis or oxidative reactions (
      • Mookerjee S.A.
      • Goncalves R.L.S.
      • Gerencser A.A.
      • Nicholls D.G.
      • Brand M.D.
      The contributions of respiration and glycolysis to extracellular acid production.
      ). Cells were assayed in a minimal salts medium following a short period of starvation, conditions very likely to severely slow or stop cell growth during the measurements. Of course, the conclusions reached below are only applicable to this non-growing state; a more sophisticated analysis would be required for growing cells.

      Calculation of ATP production rates (JATPglyc and JATPox) from extracellular acidification rate and oxygen consumption rate

      As diagrammed in Fig. 1C, JATP production is the sum of JATPglycand JATPox. JATPglyc is itself the sum of two parts. The first is ATP produced by glycolysis to pyruvate that is subsequently converted to lactate. This is calculated from extracellular acidification rate as PPRglyc × ATP/lactate. Because 2 lactates are produced per glucose or glucose residue, ATP/lactate is half the value for ATP/glucose given in Fig. 1A, column g (i.e. 1.0 for glucose, 1.45 for glycogen). The second is ATP produced by glycolysis to pyruvate that is subsequently converted to bicarbonate. This is calculated from the oxygen consumption rate by multiplying the mitochondrial OCR by the P/O ratio attributable to glycolytic production of pyruvate that is then fully oxidized (Fig. 1B, column s), with a conversion factor of 2 to account for the switch from oxygen atoms ([O]) in P/O to molecules (O2) in OCR. Thus, JATPglyc to bicarbonate = OCRmito × 2P/Oglyc. Total JATPglyc is therefore PPRglyc × ATP/lactate + OCRmito × 2P/Oglyc (Fig. 1C). As a side note, glycolysis to pyruvate precedes both lactate production and oxidation to bicarbonate; for this reason, it is incorrect to use any glycolytic inhibitor that acts before lactate dehydrogenase, such as 2-deoxyglucose, to attempt to distinguish between glycolysis and respiration; both processes will be prevented by such inhibitors when the main substrate is glycogen or glucose. The approach outlined here avoids the need to inhibit glycolysis.
      JATPox is calculated from the mitochondrial oxygen consumption rate, the portion of the total oxygen consumption rate that is sensitive to the mitochondrial electron transport inhibitors rotenone and myxothiazol (OCRmito = OCRtot − OCRr/m). OCRmito can be further divided into the phosphorylating or coupled rate, which is sensitive to the ATP synthase inhibitor oligomycin (OCRcoupled = OCRtot − OCRoli), and the non-coupled or leak rate, which is the mitochondrial OCR in the presence of oligomycin (OCRleak = OCRoli − OCRr/m). Comparing OCR before and after oligomycin addition causes a slight underestimate of OCRcoupled. The underestimate (<10%) results from the oligomycin-dependent hyperpolarization of the mitochondrial inner membrane, which provokes a slight increase in OCRoli, and can be corrected for (
      • Affourtit C.
      • Brand M.D.
      Measuring mitochondrial bioenergetics in INS-1E insulinoma cells.
      ) as is done here and in supplemental Table 1. JATPox is then the sum of two parts. First, the ATP production rate attributable to oxidative phosphorylation is equal to the coupled OCR multiplied by the P/O ratio of oxidative phosphorylation; OCRcoupled × 2P/Ooxphos (Fig. 1B, column u). This portion of JATPox is driven by reducing equivalents generated during both glycolysis and the oxidation reactions of pyruvate dehydrogenase plus the tricarboxylic acid cycle. Second, substrate-level phosphorylation at succinyl-CoA synthetase during activity of the tricarboxylic acid cycle is driven by OCRmito, not just OCRcoupled. This portion of JATPox is determined by multiplying the mitochondrial oxygen consumption rate by the P/O ratio attributable to tricarboxylic acid cycle flux: OCRmito × 2P/OTCA (Fig. 1B, column t). Therefore, the total rate of oxidative ATP production JATPox = OCRcoupled × 2P/Ooxphos + OCRmito × 2P/OTCA.

      Experimental determination of rates of ATP production by glycolysis and oxidative metabolism

      Here, we apply the calculations above to determine the rates of ATP production by glycolysis, JATPglyc, and oxidative metabolism, JATPox, using C2C12 myoblasts in adherent cell culture.
      Fig. 3 shows raw extracellular flux data and the overall values and individual components of JATPglyc and JATPox during a partial cell respiratory control assay (
      • Brand M.D.
      • Nicholls D.G.
      Assessing mitochondrial dysfunction in cells.
      ). Fig. 3A shows raw ECAR traces, and Fig. 3B shows raw OCR traces during the course of a single set of experiments. No substrate was present under basal conditions, which were followed by sequential additions at the points indicated of glucose, dimethyl sulfoxide vehicle (DMSO, added as a control for other data sets), oligomycin, and finally rotenone plus myxothiazol. The shaded regions define each condition after the system approached steady state, but before other additions. Under the basal condition, ECAR was low and OCR was high because the cells produced ATP primarily by oxidative phosphorylation from endogenous substrates, assumed to be glycogen (see below). After the addition of glucose, ECAR increased, reflecting a shift to glycolysis using the new substrate, and OCR decreased, reflecting the shift away from oxidative phosphorylation (the Crabtree effect). The subsequent addition of oligomycin inhibited the mitochondrial ATP synthase, so ECAR increased, reflecting a greater requirement for glycolytic ATP, and OCR decreased, reflecting the inhibition of oxidative phosphorylation. The remaining mitochondrial OCR represents electron transport driving the cycle of proton pumping and proton leak across the mitochondrial inner membrane. The addition of rotenone plus myxothiazol fully inhibited mitochondrial electron transport, so it had little further effect on ECAR but decreased OCR to the non-mitochondrial rate, which was subtracted from all other OCR values below.
      Figure thumbnail gr3
      Figure 3Raw extracellular flux data and its conversion into rates of ATP production (JATP). 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, JATP production for each time point marked in gray in A and B, divided into JATPglyc and JATPox. 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 JATPglyc and JATPox. JATPglyc is divided into ATP production during glycolytic production of pyruvate that is either converted to lactate (JATPglyc to lac) or imported into the matrix and oxidized (JATPglyc to bicarbonate).JATPox is divided into ATP production from oxidation of glycolytic NADH (JATPox-glyc), from oxidation of reducing equivalents generated within the mitochondria (JATPox-oxphos) and from succinyl-CoA synthetase (JATPox-SCS).
      JATPglyc and JATPox were calculated as described above (and in the worked example below) from a larger data set that included the data in Fig. 3, A and B (see “Experimental procedures”). Fig. 3C shows the results. We assumed that, under basal conditions, full oxidation of endogenous glycogen drove ATP production. There was no glycolysis to lactate; the calculations showed that the low basal rate of extracellular acidification in Fig. 3A was caused entirely by production of bicarbonate, not lactate. Notably, if cells were oxidizing glutamine and fats (derived, for example, from autophagy) instead of glycogen (respiratory quotient 0.6 as compared with 1), the assumption of full oxidation of glycogen would have led to negative PPRglyc values. PPRglyc was close to zero, validating our assumption. Most of the ATP was produced by oxidative reactions (oxidative phosphorylation plus substrate-linked phosphorylation in the tricarboxylic acid cycle), with only a small contribution from glycolysis (conversion of glycogen to pyruvate that was subsequently converted to bicarbonate, not lactate). The total rate of ATP production was 41.7 pmol of ATP/min/μg of cellular protein.
      Upon the addition of glucose to provide exogenous substrate, the cells began to run glycolysis to lactate, leading to a 32% increase in the total ATP production rate to 55.2 pmol of ATP/min/μg of cellular protein. This is equivalent to about 0.2 fmol of ATP/s/cell. At the same time, oxidative ATP production decreased by 19.6% (from 38.3 to 30.8 pmol/min/μg of protein). Presumably, the extra ATP production by glycolysis increased the cellular phosphorylation potential, partially suppressing oxidative ATP production by the Crabtree effect and recruiting extra ATP demand to give new, higher, steady-state rates of ATP production and demand. The increase in lactate production increased the extracellular acidification rate substantially, but this effect was partially masked by a small decrease in bicarbonate production, which had been high in the basal state. The resulting change in ECAR of about 2.8-fold shown in Fig. 3A (0.94 with glucose/0.34 basal) reveals the weakness of using ECAR as a direct measure of glycolysis, because it would greatly underestimate the true change of about 7.3-fold in the glycolytic ATP production rate (Fig. 3C).
      Upon the addition of oligomycin to prevent oxidative phosphorylation, the ATP demand was supplied almost entirely by glycolysis to lactate, with a tiny fraction from substrate-level phosphorylation at succinyl-CoA synthetase (SCS) accompanying the residual tricarboxylic acid cycle flux needed to drive proton leak (Fig. 3C). The increase in glycolysis caused a further increase in ECAR, which was again partially masked by the decrease in bicarbonate production (ECAR increased 1.6-fold compared with glucose (Fig. 3A), but JATPglyc increased 2.1-fold (Fig. 3C). Note that this normal cellular ATP demand is insufficient to drive the glycolytic rate to its maximum capacity, which is revealed only when the demand is greatly increased (see below and see Ref.
      • Mookerjee S.A.
      • Nicholls D.G.
      • Brand M.D.
      Determining maximum glycolytic capacity using extracellular flux measurements.
      ). These calculations illustrate the deeper insights into cellular bioenergetics that can be gained by the simple conversion of ECAR and OCR data into ATP production rates.
      Fig. 3D shows the contributions of each component of glycolytic and oxidative ATP production to the total rates of ATP production shown in Fig. 3C. The relative contribution toJATPglyc of ATP production by glycolysis to lactate varied from zero in the absence of added glucose (the calculated contribution was slightly negative, within the noise of the measurement) to 100% in the presence of glucose plus oligomycin, illustrating the dynamic switching between the different fates of glycolytic pyruvate as substrate supply and ATP demand were manipulated. In contrast, the relative contributions to JATPox of the oxidation of glycolytic and TCA-derived reducing equivalents and substrate-level phosphorylation by SCS did not change during complete oxidation of glycogen or glucose, except when the ATP synthase was inhibited by oligomycin. This follows from the fixed stoichiometric relationship between these reactions for a given substrate (Fig. 1B).
      In the supplemental material, we present these calculations as a simple Excel spreadsheet in which experimental parameters and raw ECAR and OCR data for non-growing cells metabolizing glucose may be entered on the left, and calculated values of JATPglyc and JATPox appear on the right.
      As a worked example, this spreadsheet calculates the results underlying Fig. 3, C and D; in each well, glucose, oligomycin, and rotenone plus myxothiazol were added stepwise, and ECAR and OCR at each step were measured. The C2C12 cells were assayed at 37 °C in KRPH medium at pH 7.4 with a buffering power of 0.045 mpH/pmol of H+/7 μl (measured as in Ref.
      • Mookerjee S.A.
      • Goncalves R.L.S.
      • Gerencser A.A.
      • Nicholls D.G.
      • Brand M.D.
      The contributions of respiration and glycolysis to extracellular acid production.
      ). At pH 7.4, the combined hydration/dissociation constant of CO2 to HCO3 + H+ is 6.093 (
      • Mookerjee S.A.
      • Goncalves R.L.S.
      • Gerencser A.A.
      • Nicholls D.G.
      • Brand M.D.
      The contributions of respiration and glycolysis to extracellular acid production.
      ). These values are entered in cells G5–G9 under “Constants & Conditions” in the spreadsheet and are used in the subsequent calculations.
      The “Data” portion of the spreadsheet is for input of raw values of protein, ECAR, and OCR data for individual wells and can be extended down as required. The worked example shows data from experiments of the type shown in Fig. 3, A and B, for a hypothetical well with the mean ECAR, OCR, and protein content of the data sets underlying Fig. 3C. The protein content of the well was 15 μg of protein (entered into cells D17–D19). Under basal conditions (medium only, with no exogenous substrate), the raw OCR was 182 pmol of O2/min (entered into cell E17). In the presence of added glucose, OCR dropped to 158 pmol of O2/min (cell E18), then to 62 pmol O2/min with oligomycin (cell E19), and finally to 42 pmol of O2/min with rotenone plus myxothiazol. To keep things simple, we assume that the rate of respiration with oligomycin (cells F17–F19) was unaffected by the addition of glucose (this could be checked and corrected if important conclusions depended on it) and that non-mitochondrial respiration with rotenone plus myxothiazol (cells G17–G19) was a constant. The raw ECAR values were 5.2 mpH/min at basal level (cell H17), 19.7 mpH/min after glucose addition (cell H18), and 34.2 mpH/min after oligomycin addition (cell H19).
      The “Calculations” portion of the spreadsheet applies normalizations and corrections and calculates JATPglyc, JATPox, and JATPtot from the constants and data entered. Mitochondrial respiration per μg of protein is determined in column J,
      (OCRtotOCRr/m)/μg of protein=OCRmito
      (Eq. 1)


      where (182 − 42)/15 = 9.3 pmol of O2/min/μg of protein.
      The respiration rate/μg of protein coupled to ATP production is determined in column K using a correction for oligomycin-induced hyperpolarization of the mitochondrial membrane of 0.908, taken from Ref.
      • Affourtit C.
      • Brand M.D.
      Measuring mitochondrial bioenergetics in INS-1E insulinoma cells.
      ,
      ((OCRtotOCRoli)×hyperpolarization correction factor)/μg of protein=OCRcoupled
      (Eq. 2)


      where ((182 − 62) × 0.908)/15 = 7.3 pmol of O2/min/μg of protein. Total PPR/μg of protein is determined in column L,
      (ECARtot/buffering power)/μg of protein=PPRtot
      (Eq. 3)


      where (5.2/0.045)/15 = 7.7 pmol of H+/min/μg of protein. The respiratory portion of PPRtot is determined using Equation 3 from Ref.
      • Mookerjee S.A.
      • Goncalves R.L.S.
      • Gerencser A.A.
      • Nicholls D.G.
      • Brand M.D.
      The contributions of respiration and glycolysis to extracellular acid production.
      and assuming a maximum H+/O2 value of 1 (see “The model system: C2C12 cells consuming glycogen or glucose by defined pathways”) in column M,
      OCRmito×max*H+/O2×(10pHpK1)/(1+(10pHpK1)
      (Eq. 4)


      where 9.3 × 1 × (10(7.4 − 6.093))/(1 + 10(7.4 − 6.093)) = 8.9 pmol H+/min/μg of protein. The glycolytic portion of PPR is determined in column N,
      PPRtotPPRresp=PPRglyc
      (Eq. 5)


      where 7.7 − 8.9 = −1.2 pmol of H+/min/μg of protein. Using the OCRcoupled, OCRmito, PPRresp, and PPRglyc values obtained above, rates of glycolytic and oxidative ATP production are then calculated as shown using the equations described in the text and presented in Fig. 1C. Basal JATPglyc is calculated using a P/Oglyc of 0.242 for glycogen (see Fig. 1B) in column O,
      (PPRglyc×ATP/lactate)+(OCRmito×2P/Oglyc)=JATPglyc
      (Eq. 6)


      where (−1.2 × 1) + (9.3 × 2 × 0.242) = 3.3 pmol of ATP/min/μg of protein. Basal JATPox is calculated using a P/Ooxphos of 2.486 and P/OTCA of 0.121 in column P,
      (OCRcoupled×2P/Ooxphos)+(OCRmito×2P/OTCA)=JATPox
      (Eq. 7)


      where (7.3 × 2 × 2.486) + (9.3 × 2 × 0.121) = 38.4 pmol of ATP/min/μg of protein, assuming that in the basal state, endogenous glycogen is the primary fuel source for ATP production and that glycolytic NADH transport into the mitochondrial matrix is driven 90% by the malate-aspartate shuttle and 10% by the glycerol 3-phosphate shuttle, for an estimated P/Ooxphos ratio of (0.9 × 2.5) + (0.1 × 2.364) = 2.486.
      In sum, the calculations for the basal condition are 1) OCRmito = (182 − 42)/15 = 9.3 pmol of O2/min/μg of protein; 2) OCRcoupled = ((182 − 62) × 0.908)/15 = 7.3 pmol of O2/min/μg of protein; 3) PPRtot = 5.2/0.045/15 = 7.7 pmol of H+/min/μg of protein; 4) PPRresp = 9.3 × 1 × (10(7.4 − 6.093))/(1 + 10(7.4 − 6.093)) = 8.9 pmol of H+/min/μg of protein; 5) PPRglyc = 7.7 − 8.9 = −1.2 pmol of H+/min/μg of protein; 6) JATPglyc = (−1.2 × 1) + (9.3 × 2 × 0.242) = 3.3 pmol of ATP/min/μg protein; 7) JATPox = (7.3 × 2 × 2.486) + (9.3 × 2 × 0.121) = 38.4 pmol of ATP/min/μg of protein.
      These same calculations are applied to the measurements in the presence of glucose and glucose plus oligomycin, in both cases with the appropriate P/Oglyc value of 0.167 for glucose (see Fig. 1).
      For glucose, 1) OCRmito = (158 − 42)/15 = 7.7 pmol of O2/min/μg of protein; 2) OCRcoupled = ((158 − 62) × 0.908)/15 = 5.8 pmol of O2/min/μg of protein; 3) PPRtot = 19.7/0.045/15 = 29.2 pmol of H+/min/μg; 4) PPRresp = 7.7 × 1 × (10(7.4 − 6.093))/(1 + 10(7.4 − 6.093)) = 7.4 pmol of H+/min/μg of protein; 5) PPRglyc = 29.2 - 7.4 = 21.8 pmol of H+/min/μg of protein; 6) JATPglyc = (21.8 × 1) + (7.7 × 2 × 0.167) = 24.4 pmol of ATP/min/μg of protein; 7) JATPox = (5.8 × 2 × 2.486) + (7.7 × 2 × 0.121) = 30.8 pmol of ATP/min/μg of protein.
      For oligomycin, 1) OCRmito = (62 − 42)/15 = 1.3 pmol of O2/min/μg of protein; 2) OCRcoupled = ((62 − 62) × 0.908)/15 = 0 pmol of O2/min/μg of protein; 3) PPRtot = 34.2/0.045/15 = 50.7 pmol of H+/min/μg of protein; 4) PPRresp = 1.3 × 1 × (10(7.4 − 6.093))/(1 + 10(7.4 − 6.093)) = 1.3 pmol of H+/min/μg of protein; 5) PPRglyc = 50.7 − 1.3 = 49.4 pmol of H+/min/μg of protein; 6) JATPglyc = (49.4 × 1) + (1.3 × 2 × 0.167) = 49.8 pmol of ATP/min/μg of protein; 7) JATPox = (0 × 2 × 2.486) + (1.3 × 2 × 0.121) = 0.3 pmol of ATP/min/μg of protein.
      For each condition, the total JATP production is calculated as JATPglyc + JATPox (column Q).

      Characterization of the cellular bioenergetic phenotype

      Describing cellular bioenergetics in terms of the total rate of ATP production (JATP production), divided into glycolytic (JATPglyc) and oxidative sources (JATPox) (Figs. 2A and 3C) allows quantitative and comprehensive bioenergetic analysis that is not possible with raw data. To illustrate this, a single extended data set is represented in several different ways in Figure 4, Figure 5. The raw data consisted of ECAR and OCR values for C2C12 myoblasts compiled from Fig. 3 and different portions of assays for cell respiratory control (exemplified in Refs.
      • Mookerjee S.A.
      • Goncalves R.L.S.
      • Gerencser A.A.
      • Nicholls D.G.
      • Brand M.D.
      The contributions of respiration and glycolysis to extracellular acid production.
      and
      • Brand M.D.
      • Nicholls D.G.
      Assessing mitochondrial dysfunction in cells.
      ) and glycolytic capacity (exemplified in Ref.
      • Mookerjee S.A.
      • Nicholls D.G.
      • Brand M.D.
      Determining maximum glycolytic capacity using extracellular flux measurements.
      ). For each assay, we took basal measurements with no exogenous substrate, followed by measurements after the addition of 10 mm glucose. For the cell respiratory control assay, measurements continued after sequential additions of 2 μg/ml oligomycin and 1 μm FCCP (and of rotenone plus myxothiazol; not shown here). For the glycolytic capacity assay, measurements after glucose addition continued after the sequential additions of 1 μm rotenone plus 1 μm myxothiazol and then 20 μm monensin.
      Figure thumbnail gr4
      Figure 4Visualization 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 JATPglyc and JATPox. D, data from C presented as stacked columns summing to JATP production. E, data from C plotted as points with (JATPglyc, JATPox) as (x, y) values. Constructed lines through each point show slopes denoting JATP proportionality (slope = 1 denotes equal JATP by source) and of −1 (denoting the same JATP 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 thumbnail gr5
      Figure 5Metabolic indices. In all panels, the solid red line connects points with different values of JATP production but the same value of JATPglyc/JATP production as the point with added glucose (other lines through the origin indicate different values of JATPglyc/JATP production), and lines of slope = −1 connect combinations of JATPglyc and JATPox that sum to the same value of JATP production (iso-JATP). A, GI = (100 × JATPglyc/JATP production). Vertical and horizontal dotted lines, values of the (JATPglyc, JATPox) 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 = GIcondition 2 − GIcondition 1 caused by a change in glycolysis, indicated by the curved arrow showing the effect of added glucose on JATPox. C, PI = GIcondition 1 − GIcondition 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 JATPglyc. D, bioenergetic capacity and bioenergetic scope. Solid black lines denote the empirical (vertical) maximum for JATPglyc and the theoretical (horizontal) maximum for JATPox. The dotted black line indicates GImc = GImax capacity = 57%, passing though the theoretical bioenergetic maximum JATP 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 JATP production. The shaded box indicates the bioenergetic scope: all possible points of JATP 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 JATP production between JATPglyc and JATPox. 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.
      Fig. 4A shows the data set described above in a conventional column plot, typically used for comparing the raw rates of acidification or respiration under different conditions. Clarifying the relationship of the overall bioenergetics between two conditions or samples is the implied goal of the scatter plots in Fig. 4B, where pairs of ECAR and OCR values are used as (x, y) coordinates. Comparison of two such points, representing two different conditions, cell types, cell treatments, etc., can give a crude idea of how the ATP production phenotype differs between the two samples. However, it is impossible to determine from Fig. 4B which source of ATP production predominates, and in general, no clear information can be obtained because of the arbitrary scaling between ECAR and OCR, the entangled relationships of ECAR and OCR to the rates of glycolysis and oxidative phosphorylation, and the unequal proportionality of each rate to ATP production, discussed above.
      In contrast, converting ECAR and OCR measurements to rates of ATP synthesis as in Fig. 3C eliminates the above problems and allows direct comparison of the rates of glycolytic and oxidative ATP production. Fig. 4C shows the calculated values of JATPglyc and JATPox side-by-side, allowing comparison of their relative contributions and how each changes with conditions. This is much more meaningful than the equivalent raw data plot in Fig. 4A. Fig. 4D shows JATPglyc and JATPox as stacked columns, allowing comparison of the summed JATP production between conditions (expanding the data set shown in Fig. 3C), which is impossible to do with the raw ECAR and OCR values and difficult to do with the side-by-side representations of JATPglyc and JATPox.
      Fig. 4E shows scatter plots of JATPglyc and JATPox, which allow bioenergetic phenotypes to be characterized by the absolute positions of the points and allow the relationships between the different conditions to be characterized by the relative positions of the points. The representation of JATP production as a single (JATPglyc, JATPox) point in Fig. 4E allows us to identify whether a cell is primarily glycolytic or primarily oxidative in a way that the ECAR/OCR point in Fig. 4B cannot. In Fig. 4E, C2C12 myoblasts in the basal condition were primarily oxidative, but after the addition of glucose, they produced ATP at roughly similar rates by each pathway; this important conclusion cannot be deduced from Fig. 4B. In the presence of inhibitors of oxidative phosphorylation, they were almost entirely glycolytic, as expected. The most dramatic difference between B and E of Fig. 4 is the position of the point with glucose plus oligomycin plus FCCP. This point had high uncoupled leak respiration, appearing at high OCR in Fig. 4B, but low JATPox because of the inhibition of the ATP synthase by oligomycin and the uncoupling effect of FCCP, so it appears near theJATPglyc axis on Fig. 4E, which is much more appropriate when considering bioenergetics. The slight inhibitory effect of FCCP on JATPglyc observed previously (
      • Mookerjee S.A.
      • Nicholls D.G.
      • Brand M.D.
      Determining maximum glycolytic capacity using extracellular flux measurements.
      ) is also obvious in Fig. 4, D and E, because JATPglyc and JATP production decreased upon the addition of FCCP. In the presence of glucose plus oligomycin, the cells were almost entirely glycolytic, but this is less apparent from Fig. 4B, in which the non-coupled respiration driving proton leak was not subtracted, leading to the incorrect impression that the cells were still partially oxidative. In addition, by eliminating the large contribution of bicarbonate production to the extracellular acidification rate when respiration rates were high, Fig. 4E clearly shows the large increase in glycolytic rate upon the addition of mitochondrial inhibitors, which is much less obvious in the change in total ECAR in Fig. 4B (compare also the glycolytic contribution in Fig. 3C with the ECAR changes in Fig. 3A).

      The bioenergetic space plot

      Once ECAR and OCR data are disentangled and converted to the same units (JATPglyc and JATPox), the geometric relationships revealed in the “bioenergetic space plot” (Fig. 4E) allow the following powerful observations.
      First, we define a line through the origin with a slope of 1, connecting all points at which JATPglyc and JATPox are identical (ATP production is 50% glycolytic). Points below this line represent cells that derive >50% of their ATP by glycolysis, and points above the line represent cells that derive more than 50% of their ATP by oxidative reactions. It is easy to see that C2C12 cells under basal conditions or with added glucose were more oxidative than glycolytic (both points lie above the 50% line), whereas in the presence of mitochondrial poisons, they were primarily glycolytic (the points lie below the 50% line).
      Second, any line connecting all points with the same total rate of ATP production, JATP production, has a slope of −1. The value of JATP production is indicated by either of the axis intersections of this line. If cells are flexible in their sources of ATP supply, they can readily move along such an “iso-JATP” line when conditions change, maintaining the same rate of ATP supply by varying the proportions derived from glycolysis and oxidative phosphorylation (this underlies the idea of indices quantifying the Crabtree and Pasteur effects and the flexibility of ATP supply; see below). When oligomycin was added to C2C12 cells using glucose, they responded flexibly by increasing the glycolytic rate to almost completely compensate for the inhibition of oxidative phosphorylation; the point with oligomycin lies near the iso-JATP line for glucose.
      Third, if cellular ATP turnover increases due to increases in either supply or demand, then cells move to parallel iso-JATP lines further from the origin (this underlies the idea of indexing the bioenergetic capacity of cells and, more generally, the idea of cellular bioenergetic scope; see below). It is easy to see that when glucose was added to the C2C12 cells under basal conditions, the rate of ATP production (and, since the system was in steady state, also the rate of ATP consumption) increased substantially. Similarly, the addition of monensin (to drive increased ATP demand by the Na+/K+-ATPase) increased the rate of ATP production; the point with glucose plus rotenone plus myxothiazol plus monensin lies on a higher iso-JATP line than the other points. Conversely, if ATP demand decreases or if cells are inflexible to changes in conditions and cannot compensate for a decreased ATP supply through one route by increasing another, then they would move to parallel iso-JATP lines closer to the origin, as seen with the addition of FCCP to the condition with glucose plus oligomycin, which directly compromises glycolytic rate, probably by acidifying the cytosol (
      • Mookerjee S.A.
      • Nicholls D.G.
      • Brand M.D.
      Determining maximum glycolytic capacity using extracellular flux measurements.
      ).

      Quantifying cellular bioenergetic phenotypes

      The glycolytic index and the Warburg effect

      The glycolytic index describes the degree to which a cell uses glycolysis to meet its total ATP demand. Cells with chronically increased GI values exhibit a Warburg effect. This definition is related to the underlying history and current use of the term “Warburg effect,” requiring some historical explanation. In some yeast grown under certain conditions, the presence of oxygen suppresses glycolysis to ethanol and favors oxidative phosphorylation, an observation now known as the Pasteur effect (
      • Stickland L.
      The Pasteur effect in normal yeast and its inhibition by various agents.
      ). Because the oxygen-independent reactions of glycolysis in mammalian cells terminate in lactate rather than ethanol, the analogous expectation is that cells exposed to oxygen (e.g. in culture) should produce little lactate. The Warburg effect describes the observation by Warburg that cancers, both tumor tissue slices and Ehrlich ascites cells, produce surprising amounts of lactate relative to non-cancerous cells (
      • Warburg O.
      On the origin of cancer cells.
      ), inconsistent with the Pasteur effect. The cause of the Warburg effect was originally proposed to be the accumulation of irreversible mitochondrial damage, but such damage was subsequently shown not to be required for significant lactate production. Still, the Warburg effect continues to underlie the view that pathological processes such as cancer subvert “normal” bioenergetic regulation to favor glycolysis (and disengage respiration), even under aerobic conditions, and that, by extension, high rates of glycolysis maintain, may report, and may be targeted to attenuate the pathologic severity of cancer. This reasoning has been extended to the hypothesis that the source of bioenergetic supply is significant as a physiological signal or regulatory mechanism under both normal and pathological conditions (e.g. see Refs.
      • Mouradian M.
      • Kikawa K.D.
      • Dranka B.P.
      • Komas S.M.
      • Kalyanaraman B.
      • Pardini R.S.
      Docosahexaenoic acid attenuates breast cancer cell metabolism and the Warburg phenotype by targeting bioenergetic function.
      and
      • Epstein T.
      • Xu L.
      • Gillies R.J.
      • Gatenby R.A.
      Separation of metabolic supply and demand: aerobic glycolysis as a normal physiological response to fluctuating energetic demands in the membrane.
      ), an inversion of the observation that transcriptional regulation of metabolic enzymes is frequently altered in cancer (
      • Hanahan D.
      • Weinberg R.A.
      Hallmarks of cancer: the next generation.
      ). If these ideas are true, the presence of a Warburg effect in a cell should be enormously informative. However, despite an early proposal by Warburg to quantify his observations (using the Meyerhof quotient), there is currently no systematic approach for defining, quantifying, or analyzing the Warburg effect in terms of ATP turnover. In this section, we propose such an approach.
      For the glycolytic index, the total ATP production rate (JATP production = JATPglyc + JATPox) must be considered. We define the glycolytic index GI = (100 × JATPglyc/JATP production). A cell whose ATP comes entirely from glycolysis to lactate therefore has a glycolytic index of 100%; a cell whose ATP comes entirely from oxidative reactions would theoretically have a GI = 0%, and a cell whose ATP comes equally from ATPglyc and ATPox has GI = 50%. A cell is primarily glycolytic when the majority of its ATP comes from glycolysis; otherwise, it is primarily oxidative.
      Fig. 5A demonstrates this characterization in the bioenergetic space plot. When provided with external glucose in aerobic culture, C2C12 myoblasts had JATPglyc = 24.4 ± 1.8 and JATPox = 30.8 ± 2.4, for a total of 55.2 ± 3.0 pmol of ATP/min/μg of protein (mean ± S.E., n = 36). Their glycolytic index was 24.4/55.2 = 44.2 ± 6.7%, insufficient (although not by much) to allow them to be described as primarily glycolytic. The threshold for this determination is shown as a line through the origin with slope = 1; points below this line have GI > 50% (blue shading in Fig. 5A) and describe primarily glycolytic cells; points above the line represent primarily oxidative cells.
      The glycolytic index normalizes JATPglyc to JATP production, accounting for any differences in total ATP production between samples and yielding a value that can be compared with another even when the total ATP production rate is different. In addition, the normalization clarifies when the glycolytic index is unchanged between two points of comparison, which would fall on a single line through the origin even if their absolute rates of glycolysis (and therefore lactate production) were very different (all other positions on the red line in Fig. 5A).
      Using the glycolytic index, the Warburg effect can be described as a chronically increased GI relative to some baseline, caused by a change in the enzymic machinery of the cell. By extension, a Warburg index (WI) can be defined as the difference between these points: WI = GI − GIo, where GIo refers to the baseline. Importantly, Warburg’s initial observation (and conception of the eponymous effect) was based on indirect measurement of lactate production (reviewed in Ref.
      • Koppenol W.H.
      • Bounds P.L.
      • Dang C.V.
      Otto Warburg's contributions to current concepts of cancer metabolism.
      ), suggesting that rapid glycolysis was required mostly for regenerating NAD+ (with glucose catabolism ending with lactate) and not for generating biosynthetic intermediates (which would not yield lactate).

      The Crabtree and Pasteur indices

      Unlike the Warburg effect, which describes chronic bioenergetic alteration relative to a baseline, the Crabtree and Pasteur effects reflect the acute shift of ATP supply between glycolysis and oxidative phosphorylation in response to rapid external changes, typically before and after the addition of a substrate. As with the Warburg index, the names Crabtree index and Pasteur index are deliberate reflections of the Crabtree effect and Pasteur effect, respectively, as explained below.
      The Pasteur effect initially described slow yeast growth despite rapid glucose utilization under fermentative conditions, which shifted upon the addition of oxygen to slow glucose utilization and rapid yeast growth (
      • Barnett J.A.
      • Entian K.D.
      A history of research on yeasts 9: regulation of sugar metabolism.
      ). Although this was perhaps due to anaerobic suppression of fatty acid and sterol synthesis and not a preference for a bioenergetically productive reaction over a less productive one, it was later recognized that O2 does directly suppress glycolysis in yeast and mammalian samples (
      • Barnett J.A.
      • Entian K.D.
      A history of research on yeasts 9: regulation of sugar metabolism.
      ). The Pasteur effect (as proposed by Warburg, describing a link between the pathways of fermentation and respiration) is currently understood in these latter terms, as the suppression of glycolysis by mitochondrial respiratory activity (or, in reverse, glycolytic activation by inhibition of mitochondrial respiration). The Pasteur index could, therefore, be equally described as the “glycolysis suppression index.” In terms of ATP supply, as we propose here, the Pasteur effect is simply the suppression of glycolytic ATP generation by ATP supplied by oxidative phosphorylation. The Crabtree effect is the inverse of the Pasteur effect, in which the introduction of a fermentable carbon source (e.g. glucose) suppresses respiration and oxidative phosphorylation as the glycolytic rate increases (
      • Gerencser A.A.
      • Neilson A.
      • Choi S.W.
      • Edman U.
      • Yadava N.
      • Oh R.J.
      • Ferrick D.A.
      • Nicholls D.G.
      • Brand M.D.
      Quantitative microplate-based respirometry with correction for oxygen diffusion.
      ,
      • Crabtree H.
      Observations on the carbohydrate metabolism of tumours.
      ,
      • Ibsen K.H.
      The Crabtree effect: a review.
      ). The Crabtree index could, therefore, be equally described as the “respiration suppression index.” In terms of ATP supply, the Crabtree effect is simply the suppression of oxidative phosphorylation by ATP supplied by glycolysis. Thus, the Crabtree effect is the change in JATPox when JATPglyc is altered, and the Pasteur effect is the change in JATPglyc when JATPox is altered (Fig. 2A). From these definitions, we draw the Crabtree and Pasteur indices as described below.
      Fig. 5B demonstrates how the Crabtree effect (the change in JATPox when JATPglyc is altered) can be represented in the bioenergetic space plot. In addition to the glucose data point (24.4, 30.8) shown in Fig. 5A, Fig. 5B shows the point for C2C12 myoblasts under basal conditions with no exogenous substrate (3.4 ± 0.5, 38.3 ± 2.3). The glycolytic index of the cells without glucose was 3.4/(38.3 + 3.4) = 8.2%, whereas GI with glucose = 44.2%. This information can be used to quantify the Crabtree effect as a Crabtree index.
      The Crabtree effect can be observed as a decrease in the raw oxygen consumption rate after the addition of glucose in the subset of data shown in Fig. 3B. In the full data set underlying Fig. 3C, the mean decrease in raw oxygen consumption rate was 17%. This decrease is slightly greater for JATPox in Figs. 3C, 4C, and 5B, calculated as the x-value difference of 38.3 − 30.8 = 7.5 pmol ATP/min/μg of protein between the points before and after the glucose addition, a decrease in JATPox of 19.6%. It is tempting to conclude that the Crabtree index (the change in JATPox upon the addition of glucose) should have a value of 19.6%. However, to ensure that the Crabtree index is not confounded by any associated changes in total ATP production rate, JATPox should be scaled to JATP production. Therefore, the Crabtree index is best calculated as the change in the percentage of ATP production that is oxidative, not the absolute change. Under basal conditions without glucose (condition 1), oxidative ATP production was 38.3/(38.3 + 3.4) = 91.8%, and with glucose (condition 2), it was 30.8/(30.8 + 24.4) = 55.8%. The Crabtree index in these cells was therefore 91.8 − 55.8 = 36.0%. Because these values are scaled to JATP production, any change in JATPox describes an equal and opposite change in JATPglyc. Mathematically, this calculation of the Crabtree index is then the same as the value of the glycolytic index in condition 2 minus the value of the glycolytic index in condition 1 (44.2 − 8.2 = 36.0%) (Fig. 5B), so the Crabtree index is conveniently defined as CI = GIcondition 2 − GIcondition 1 caused by a change in glycolysis.
      This Crabtree index of 36% illustrates that more than one-third of JATP production shifted from JATPox to JATPglyc, reflecting a strong depression of oxidative ATP production by added glucose in these C2C12 cells. It reflects the underlying bioenergetics much more accurately than the simple change in oxygen consumption rate (17%) for three reasons. First, calculating the value of the Crabtree index through JATP excludes non-mitochondrial respiration, which is an irrelevant confounder. Second, it can account for all ATP synthesized by the oxidative route, regardless of substrate and P/O ratios, in a way that OCR itself cannot. Third, it is unaffected by any changes in ATP turnover. In Fig. 5B, the addition of glucose increasedJATP production from 41.7 to 55.2 units, partially obscuring the Crabtree effect; the Crabtree index corrects for such changes. In the other extreme, a hypothetical Crabtree index of 0% would mean that even if the total ATP production rate and therefore respiration rate were to decrease upon the addition of glucose, there would be no change in the proportion of ATP derived from oxidative reactions and no Crabtree effect but simply a proportional depression of ATP turnover following the black GI = 8% line in Fig. 5B.
      Theoretically, a negative Crabtree effect could occur if glycolytic activity were forced to slow (e.g. by adding an inhibitor to fully prevent glucose transport in the condition with glucose). We did not carry out such an experiment, but if it were to return all of the rates to basal, the Crabtree index would have been −36% (8–44% GI units); partial glycolytic inhibition would lead to smaller absolute values of CI.
      Fig. 5C demonstrates how the Pasteur effect (the alteration in JATPglyc when JATPox is altered) can be represented in the bioenergetic space plot. In addition to the point for C2C12 myoblasts with glucose present (24.4, 30.8) shown in Fig. 5A, Fig. 5C shows the point with glucose plus oligomycin (50.0, 0.5), where GI = 99% (there is still a small JATPox from substrate-linked phosphorylation in the tricarboxylic acid cycle even when oxidative phosphorylation is fully inhibited by oligomycin), and the point with glucose plus rotenone plus myxothiazol (47.7, 0; GI = 100%). This information can be used to quantify the Pasteur effect as a Pasteur index.
      Using the same logic as for the Crabtree index, the Pasteur index can be defined as PI = GIcondition 1 − GIcondition 2 caused by a change in oxidative reactions. Although the Pasteur effect classically describes a decrease in JATPglyc when oxygen is added to an anaerobic culture (in other words, when JATPox is increased), it can be generalized to include a negative Pasteur effect when JATPox under aerobic conditions is inhibited. In this spirit, the (negative) Pasteur effect can be qualitatively observed as an increase in the raw ECAR trace after the addition of oligomycin (Fig. 3A); it is qualitative because of the confounding effects of decreased respiratory acidification on raw ECAR values. Starting from the point with glucose (GI = 44%) in Fig. 5C, the addition of oligomycin moved the cells to GI = 99%, giving PI a value of 44 − 99 = −55%. Similarly, the addition of rotenone plus myxothiazol moved the cells to GI = 100%, giving PI a value of 44 − 100 = −56%. If instead we choose to consider the inhibited conditions to be the initial ones, with the experimental manipulation being the “removal” of the inhibitors, then these values change sign (the “removal” of oligomycin activates oxidative phosphorylation and gives a Pasteur index of 55%).

      Indexing bioenergetic activity; bioenergetic capacity, bioenergetic scope, and the supply flexibility index

      Important additional pieces of information that can be determined using this approach are the bioenergetic capacity of cells (an extension of the more limited idea of “spare respiratory capacity” (
      • Brand M.D.
      • Nicholls D.G.
      Assessing mitochondrial dysfunction in cells.
      ,
      • Nicholls D.G.
      Spare respiratory capacity, oxidative stress and excitotoxicity.
      )) and the bioenergetic scope within which the cells operate, including their flexibility to respond to changes in ATP demand or to change the source of their ATP supply.
      By measuring glycolytic capacity, an upper limit can be set for JATPglyc. Glycolytic capacity is reached when ATP demand in the absence of oxidative ATP synthesis just exceeds JATPglyc and can be achieved experimentally in these C2C12 cells by adding rotenone plus myxothiazol to fully inhibit respiration and adding monensin to stimulate ATP-requiring Na+ cycling across the plasma membrane (
      • Mookerjee S.A.
      • Nicholls D.G.
      • Brand M.D.
      Determining maximum glycolytic capacity using extracellular flux measurements.
      ). The upper limit to JATPglyc measured in this way was 62.5 pmol of ATP/min/μg of protein (Fig. 4, C–E).
      Similarly, the maximum capacity of oxidative reactions to produce ATP is reached when ATP demand in the absence of glycolytic ATP synthesis just exceeds JATPox. The upper limit for JATPox is hard to achieve experimentally, because FCCP and monensin uncouple oxidative phosphorylation, and no other convenient reactions that demand high rates of ATP generation and can be turned on pharmacologically have been identified. However, as a surrogate, we can measure the maximum respiration rate in the presence of a mitochondrial uncoupler, such as FCCP, assuming that if the mitochondria were still coupled they could use this respiration to drive ATP synthesis, and treat the resulting calculated value of JATPox as a hypothetical maximum. This assumption will fail in cells, such as brown adipocytes, in which the maximum rate of ATP synthesis is substantially less than the maximum rate of electron transport (
      • Nicholls D.G.
      Brown adipose tissue mitochondria.
      ), but in most cells, including the C2C12 myoblasts considered here, it will probably cause little if any overestimation of the upper limit to JATPox. From the mitochondrial respiration rate in the presence of glucose + oligomycin + FCCP shown in Fig. 4A, the hypothetical maximum value of JATPox was 46.5 pmol of ATP/min/μg of protein. However, because the mitochondria were uncoupled, the actual value of JATPox (from substrate-linked phosphorylation in the tricarboxylic acid cycle) was 1.3 pmol of ATP/min/μg of protein, as shown in Fig. 4C.
      These maximum values of JATPglyc and JATPox define the bioenergetic capacity of the cells. As shown in Fig. 5D, the maximum individual capacities of JATPglyc and JATPox in the bioenergetic space plot intersect at (62.5, 46.5) for a theoretical maximum bioenergetic capacity of 62.5 + 46.5 = 109.0 pmol of ATP/min/μg of protein. At this maximum point, the glycolytic index (GImax capacity) would be 62.5/109 = 57.3%, making C2C12 myoblasts primarily glycolytic when running at their maximum ATP production rate. Compared with the actual value of JATP production in the presence of glucose (55.2), the bioenergetic capacity was 109/55.2 = 197% of the rate with glucose (Fig. 5D). This bioenergetic capacity of 197% of the rate with glucose (alternatively, a reserve capacity of 109.0 − 55.2 = 53.8 pmol of ATP/min/μg of protein) reveals that the C2C12 cells under our experimental assay conditions with added glucose were operating comfortably within their capacity to generate ATP and were well set up to respond to any acute increases in ATP demand by increasing either glycolytic or oxidative ATP production, or both.
      The maximum values of JATPglyc and JATPox define the full extent of the bioenergetic space that a cell can access without changing its metabolic machinery by alterations in, for example, enzyme concentration. This “bioenergetic scope” is defined by the shaded area in Fig. 5, D and E. Because it is rectilinear, the area and shape of this shaded area is fully defined by the value of the glycolytic index at maximum capacity, GImax capacity.
      The bioenergetic scope of different cells or of the same cells treated in different ways can be compared quantitatively. For example, it is possible to say that cell X has 3.7 times the bioenergetic scope of cell Y if the area bounded by JATPglyc and JATPox is 3.7 times greater for cell X than for cell Y, regardless of the dimensions of either area. By comparing values of GImax capacity, it is possible to describe how different values of the maximum glycolytic and oxidative ATP production capacities allow this difference in metabolic scope.
      The Crabtree and Pasteur indices quantify the response of one supply arm in Fig. 2A when the other supply arm is altered, after scaling to remove any confounding effects of changes in their sum (JATP production). A special case of this is the extent to which a cell shows flexibility to make compensatory changes in one supply pathway when the other is changed, without any such effect on JATP production. A fully flexible cell could alter either supply arm to provide 0–100% of total supply (and so change its glycolytic index) without affecting JATP production, whereas a less flexible cell would reach a limit beyond which it could not maintain JATP production with only a single supply arm operating. The extent to which a cell can meet a given ATP demand through either respiration or glycolysis (ATP supply flexibility) can be readily appreciated in a bioenergetic space plot. Perturbations to either JATPglyc or JATPox that do not alter total ATP demand would cause sliding of the initial point along its iso-JATP line of slope −1, because the decrease in one rate was matched by an equal increase in the other, denoted by the thick black arrows in Fig. 5D. If a perturbation caused a shift that would exceed the maximum capacity of the summoned rate as shown, JATP production would necessarily decrease. The flexibility of substrate use can be captured quantitatively in the supply flexibility index, which has a value of 100% where ATP demand can be fully satisfied by any combination of JATPglyc and JATPox, lower values as the excursions along lines of slope −1 hit maximum capacities, and a value of 0% at the maximum ATP production capacity.
      The supply flexibility index (SFI) can be defined as 100 × θ°/90°, where θ is the angle (in degrees) defined by the lines along which a supply arm reaches its maximum capacity for the given value of JATP production, as shown in Fig. 5E. For C2C12 cells with glucose, SFI was 100 × 79°/90° = 87%. This value shows that the cells had high flexibility to switch between ATP production pathways (87% of the maximum possible). The limitation was caused by the iso-JATP line leaving the area of bioenergetic scope if the cells were forced to rely solely on oxidative ATP production, although they could stay within this area if forced to rely solely on glycolytic ATP production (thick black arrows in Fig. 5E). At the slightly lower ATP demand under basal conditions (JATP production = 41.7, thin arrows in Fig. 5E), SFI would rise to 100% as the line stayed within both limits of the bioenergetic scope. At the higher hypothetical ATP demand under glucose, oligomycin, and FCCP exposure (dotted arrows in Fig. 5E), SFI would drop further and be limited by both JATPox and JATPglyc, with a value of 28%. At the maximum ATP demand when the cells were at bioenergetic capacity, SFI would decrease to 0%.

      Profiling ATP consumers

      To date, the major consumers of ATP generated by oxidative phosphorylation in a cell have been profiled by inferring their rates from measurements of the decrease in cellular oxygen consumption rate when individual ATP-consuming pathways are inhibited pharmacologically (
      • Felber S.M.
      • Brand M.D.
      Concanavalin A causes an increase in sodium permeability and intracellular sodium content of pig lymphocytes.
      ,
      • Brand M.D.
      • Felber S.M.
      The intracellular calcium antagonist TMB-8 [8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate] inhibits mitochondrial ATP production in rat thymocytes.
      ,
      • Buttgereit F.
      • Brand M.D.
      A hierarchy of ATP-consuming processes in mammalian cells.
      ,
      • Buttgereit F.
      • Brand M.D.
      • Müller M.
      Con A induced changes in energy metabolism of rat thymocytes.
      ,
      • Birket M.J.
      • Orr A.L.
      • Gerencser A.A.
      • Madden D.T.
      • Vitelli C.
      • Swistowski A.
      • Brand M.D.
      • Zeng X.
      A reduction in ATP demand and mitochondrial activity with neural differentiation of human embryonic stem cells.
      ). However, both glycolysis and oxidative reactions generate cellular ATP that can be used by ATP consumption pathways (Fig. 6A). We have extended the previous approach (
      • Felber S.M.
      • Brand M.D.
      Concanavalin A causes an increase in sodium permeability and intracellular sodium content of pig lymphocytes.
      ,
      • Brand M.D.
      • Felber S.M.
      The intracellular calcium antagonist TMB-8 [8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate] inhibits mitochondrial ATP production in rat thymocytes.
      ,
      • Buttgereit F.
      • Brand M.D.
      A hierarchy of ATP-consuming processes in mammalian cells.
      ,
      • Buttgereit F.
      • Brand M.D.
      • Müller M.
      Con A induced changes in energy metabolism of rat thymocytes.
      ,
      • Birket M.J.
      • Orr A.L.
      • Gerencser A.A.
      • Madden D.T.
      • Vitelli C.
      • Swistowski A.
      • Brand M.D.
      • Zeng X.
      A reduction in ATP demand and mitochondrial activity with neural differentiation of human embryonic stem cells.
      ) by combining it with the analysis described above, generating a complete ATP consumption profile for these C2C12 myoblasts in the presence of glucose, with both ATPglyc and ATPox accounted for. Fig. 6B shows the extent of inhibition of JATPglyc and JATPox by inhibitors of specific ATP-consuming pathways, allowing about 43% of glycolytic, 81% of oxidative, and 62% of total ATP production to be associated with specific cellular ATP-consuming processes, particularly protein synthesis and actin dynamics.
      Figure thumbnail gr6
      Figure 6Rates of consumption of ATP by different pathways in C2C12 myoblasts. A, steady-state rates of glycolytic ATP production (JATPglyc) and oxidative ATP production (JATPox) sum to the total rate of ATP production (JATP production) and hence to the total rate of ATP consumption by multiple ATP consumers (JATP 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 JATPglyc and JATPox 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 JATP consumption in the absence of inhibitors). The third bar shows the set of values for JATPox stacked above the set of values for JATPglyc to display total assigned and unassigned rates of ATP consumption. C, data from B scaled to the uninhibited totals for JATPglyc, JATPox, and JATP consumption as 100%. No significant differences were found by unpaired t test between percentage contribution of JATPglyc and percentage contribution of JATPox for any single consumer or for “unassigned” percentage contribution of JATP. The third bar shows the individual values for JATPox stacked above the corresponding individual values for JATPglyc to emphasize the contributions of each ATP consumption pathway to JATP 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.
      Several reports have proposed preferential consumption in a cell of locally synthesized ATP (primarily glycolytic) over the bulk ATP pool (both oxidative and glycolytic) (
      • Epstein T.
      • Xu L.
      • Gillies R.J.
      • Gatenby R.A.
      Separation of metabolic supply and demand: aerobic glycolysis as a normal physiological response to fluctuating energetic demands in the membrane.
      ,
      • Tourmente M.
      • Villar-Moya P.
      • Rial E.
      • Roldan E.R.
      Differences in ATP generation via glycolysis and oxidative phosphorylation and relationships with sperm motility in mouse species.
      ,
      • James A.D.
      • Patel W.
      • Butt Z.
      • Adiamah M.
      • Dakhel R.
      • Latif A.
      • Uggenti C.
      • Swanton E.
      • Imamura H.
      • Siriwardena A.K.
      • Bruce J.I.
      The plasma membrane calcium pump in pancreatic cancer cells exhibiting the Warburg effect relies on glycolytic ATP.
      ). The idea that a spatial preference exists where ATP is consumed near its site of production extends back to earlier work examining consumption of ATP made by phosphoglycerate kinase by the Na+/K+ pump in erythrocytes (
      • Mercer R.W.
      • Dunham P.B.
      Membrane-bound ATP fuels the Na/K pump.
      ) and vascular smooth muscle (
      • Paul R.J.
      • Bauer M.
      • Pease W.
      Vascular smooth muscle: aerobic glycolysis linked to sodium and potassium transport processes.
      ). Fig. 6C shows the same data as Fig. 6B normalized to 100% to make it easier to see whether any major ATP consumer has a marked preference for one source of ATP in preference to the other. Within the relatively large errors caused by measuring small percentage changes in ECAR and OCR, no such preference was apparent.

      Discussion

      The updated theoretical maximum yields of ATP shown in Fig. 1A provide a summary of the current consensus ATP/glucose and P/O ratios by mitochondria and cells catabolizing different substrates. These values are of wide significance for biochemistry, physiology, and ecology and should replace the older estimates still found in some reviews and textbooks.
      We describe a method for converting extracellular measurements of proton production and oxygen consumption rates (Fig. 3, A and B) into estimated rates of ATP production from glycolysis and oxidative phosphorylation using these values (Fig. 3C). This method improves upon previous approaches in several ways. It accounts for the entire ATP turnover in the cell, not just the part linked to respiration, and it resolves technical issues related to interpreting the extracellular measurements that allow their use in a fully quantitative, rather than qualitative or semiquantitative, way. To keep things simple, the calculations here are restricted to the simple pathways of glucose and glycogen metabolism outlined in Fig. 2. However, if the rates and pathways of metabolism of other substrates or substrate mixes are known or can be reasonably assumed, in principle, the method is fully extendable to more complicated situations, such as cells growing in culture medium.
      The definition of glycolytic ATP used here is biochemically sound and is the most correct way to quantify the rate of glycolytic ATP production. However, there is often great interest in knowing the rates of cellular ATP production from the anaerobic reactions of glycolysis terminating in lactate production. This provides information about how much ATP a cell can generate under anaerobic conditions and also how much lactate-linked ATP production may occur under aerobic conditions. As shown in Fig. 3, in C2C12 myocytes, pyruvate derived from exogenous glucose primarily terminates with lactate; a much smaller proportion (with much greater ATP-generating yield) is oxidized. Although it is sometimes helpful to know the rate of ATP production associated with lactate production, and this is readily calculated using our analysis, a much richer picture is obtained by the more complete analysis described here.
      Once the rates of ATP production are measured, we show how a bioenergetic space plot (Fig. 4E) can reveal many aspects of the rates and flexibility of cellular bioenergetics. In particular, we have proposed indices that describe different bioenergetic phenomena using the same glycolytic index units (Fig. 5A). These units offer a unified and quantitative way to describe the bioenergetic status and flexibility of a cell, including its acute supply flexibility (The Crabtree and Pasteur indices; Fig. 5, B and C) and its capacities of total JATP production (bioenergetic capacity; Fig. 5D) and of supply flexibility (supply flexibility index; Fig. 5E). The proposed analysis method and tools extend or complement several existing models for conceptualizing and quantifying bioenergetic states, including cell respiratory control (
      • Brand M.D.
      • Nicholls D.G.
      Assessing mitochondrial dysfunction in cells.
      ), spare respiratory capacity (
      • Yadava N.
      • Nicholls D.G.
      Spare respiratory capacity rather than oxidative stress regulates glutamate excitotoxicity following partial respiratory inhibition of mitochondrial complex I with rotenone.
      ), the direct analysis of ECAR/OCR ratios and (ECAR, OCR) two-dimensional plots (

      Technical brief: Identifying metabolic phenotype switches in cancer cells using the Seahorse XF Analyzer in a hypoxic environment. Agilent Technologies. www.agilent.com/cs/library/technicaloverviews/public/Tech-Brief-Hypoxia.pdf.

      ), the use of OCR and ECAR values to estimate ATP production (
      • Correia-Melo C.
      • Marques F.D.
      • Anderson R.
      • Hewitt G.
      • Hewitt R.
      • Cole J.
      • Carroll B.M.
      • Miwa S.
      • Birch J.
      • Merz A.
      • Rushton M.D.
      • Charles M.
      • Jurk D.
      • Tait S.W.
      • Czapiewski R.
      • et al.
      Mitochondria are required for pro-ageing features of the senescent phenotype.
      ), the bioenergetic health index (
      • Chacko B.K.
      • Zhi D.
      • Darley-Usmar V.M.
      • Mitchell T.
      The bioenergetic health index is a sensitive measure of oxidative stress in human monocytes.
      ), and the mitochondrial oncobioenergetic index (
      • Vayalil P.K.
      • Landar A.
      Mitochondrial oncobioenergetic index: a potential biomarker to predict progression form indolent to agressive prostate cancer.
      ). Other “glycolytic index” models use different measured variables, including positron emission tomography measurements of glucose and O2 consumption (
      • Vaishnavi S.N.
      • Vlassenko A.G.
      • Rundle M.M.
      • Snyder A.Z.
      • Mintun M.A.
      • Raichle M.E.
      Regional aerobic glycolysis in the human brain.
      ) and transcript levels of lactate dehydrogenase variants (
      • Arora R.
      • Schmitt D.
      • Karanam B.
      • Tan M.
      • Yates C.
      • Dean-Colomb W.
      Inhibition of the Warburg effect with a natural compound reveals a novel measurement for determining the metastatic potential of breast cancers.
      ).
      The implications of using bioenergetic space to visualize “bioenergetic scope,” or the possible ways that a cell can respond to changing bioenergetic conditions, are interesting to consider. Within the area in Fig. 5 (D and E) that defines the bioenergetic scope, the coordinate (JATPglyc, JATPox) can move in bioenergetic space to maintain different steady states of ATP turnover. Increases in ATP consumption would cause this point to move above a line with a slope of −1 through the initial condition (e.g. the point with added glucose), and decreases in ATP consumption would cause it to move below this line. If JATPglyc and JATPox responded equally to changes in ATP consumption, the glycolytic index would not change, and the point would move directly away from or toward the origin along the line defining the original value of GI denoted by the thick red arrows in Fig. 5D (ATP demand flexibility). At high ATP consumption rates, the line of constant GI might hit a capacity limit as shown, in which case ATP supply could still increase, but only by altering the value of GI until the point reached the limit of bioenergetic capacity at GImax capacity. If JATPglyc and JATPox responded unequally to changes in ATP consumption even at the initial condition, the glycolytic index would change immediately, and the point would move along some more complicated trajectory in bioenergetic space. Similar considerations apply when alterations in ATP consumption are driven by increases or decreases in ATP production.
      Our finding of no apparent preferential use of JATPglyc or JATPox by any of the major ATP consumers under the conditions assayed (Fig. 6) agrees with some (
      • Mercer R.W.
      • Dunham P.B.
      Membrane-bound ATP fuels the Na/K pump.
      ), but not all (
      • Beutler E.
      • Guinto E.
      • Kuhl W.
      • Matsumoto F.
      Existence of only a single pool of adenine triphosphate in human erythrocytes.
      ) work on the possibility that preferential glycolytic ATP consumption occurs in membrane-localized “pools.” It is possible that the rates of ATP consumption that we have assigned to specific consumers are overestimates, if the compounds used dampen ATP turnover more nonspecifically (although we attempted to minimize this possibility by assaying over a short time) or if the effects of two inhibitors overlap significantly. Nevertheless, the value of this approach to measuring rates of ATP consumption in cells, which could be increased with more refined measurements, is clear.
      Overall, our analysis shows that detailed descriptions and conceptual analyses of ATP production and consumption by different processes within cells can be derived from simple, non-invasive, non-terminal extracellular measurements of acidification and oxygen consumption using commercially available instruments.

      Experimental procedures

      Reagents

      Chemicals were from Sigma. Cell culture reagents and consumables were from Corning. Seahorse XF consumables were from Agilent. The bicinchoninic (BCA) protein assay was from Thermo.

      Cells

      Mouse C2C12 myoblasts (
      • Blau H.M.
      • Chiu C.P.
      • Webster C.
      Cytoplasmic activation of human nuclear genes in human heterocaryons.
      ) (ATCC) were cultured in Dulbecco’s modified Eagle’s medium containing 25 mm glucose and 2 mm glutamine, with added 10% (v/v) fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin. 24 h before the assay, cells were plated in 100 μl of culture medium at 25,000–30,000 cells/well in a 24-well polystyrene Seahorse V7-PS Flux plate with no additional coating. 25 min before the assay, cells were washed three times with and then incubated in 500 μl of Krebs-Ringer phosphate HEPES (KRPH) medium (2 mm HEPES, 136 mm NaCl, 2 mm NaH2PO4, 3.7 mm KCl, 1 mm MgCl2, 1.5 mm CaCl2, 0.1% (w/v) fatty-acid-free bovine serum albumin, pH 7.4 at 37 °C).

      Seahorse XF assays

      At the start of the assay, medium was replaced with 500 μl of fresh KRPH. Cell respiratory control (
      • Brand M.D.
      • Nicholls D.G.
      Assessing mitochondrial dysfunction in cells.
      ) and associated extracellular acidification were assayed in a Seahorse XF-24 extracellular flux analyzer by the addition via ports A–D of 10 mm glucose, 2 μg/ml oligomycin, 1 μm FCCP, and 1 μm rotenone with 1 μm myxothiazol. For assaying glycolytic capacity, the additions (ports A–C) were 10 mm glucose, 1 μm rotenone with 1 μm myxothiazol, and 20 μm monensin. For isolating ATP consumers, the additions (ports A–D) were 10 mm glucose, vehicle or inhibitor (see Fig. 6), 2 μg/ml oligomycin, and 1 μm rotenone with 1 μm myxothiazol. All inhibitors and ionophores were prepared in DMSO or water. Two or three measurement cycles of 1-min mix, 1-min wait, and 3-min measure were carried out during each phase of the experiment. Each measurement cycle is represented by a time point in the raw trace data in the figures. Following the assay, Seahorse plate wells were washed three times with 250 μl of bovine serum albumin-free KRPH. 25 μl of radioimmune precipitation assay lysis medium (150 mm NaCl, 50 mm Tris, 1 mm EGTA, 1 mm EDTA, 1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.1% (v/v) SDS, pH 7.4, at 22 °C) was added. Plates were incubated on ice for 30 min and agitated on a plate shaker at 1200 rpm for 5 min. The protein concentration in each well (typically 10–15 μg/well) was measured by a BCA assay according to the manufacturer’s instructions for a 96-well format assay (Thermo). Rates of oxygen consumption and extracellular acidification were expressed relative to the protein content of the appropriate well.

      Analysis of XF measurements

      The raw values of ECAR and OCR were subdivided into component rates (
      • Mookerjee S.A.
      • Goncalves R.L.S.
      • Gerencser A.A.
      • Nicholls D.G.
      • Brand M.D.
      The contributions of respiration and glycolysis to extracellular acid production.
      ). Briefly, for ECAR, the total rate of change of pH was first converted to total proton production rate (PPRtot) and then divided into proton production rates originating from respiratory bicarbonate production (PPRresp) (using OCR data) and glycolytic lactate production (PPRglyc). For OCR, mitochondrial oxygen consumption rate (OCRmito) was defined as total oxygen consumption rate (OCRtot) minus the oxygen consumption rate (OCRr/m) in the presence of the respiratory chain poisons rotenone and myxothiazol (OCRmito = OCRtot − OCRr/m), and the phosphorylating or coupled rate was defined as the total oxygen consumption rate minus the oligomycin-insensitive oxygen consumption rate (OCRoli), with a small additional correction by 9.2% to compensate for changes in mitochondrial protonmotive force upon the addition of oligomycin (
      • Mookerjee S.A.
      • Goncalves R.L.S.
      • Gerencser A.A.
      • Nicholls D.G.
      • Brand M.D.
      The contributions of respiration and glycolysis to extracellular acid production.
      ) (see supplemental Table 1). Thus, OCRcoupled = 0.908 × (OCRtot − OCRoli).
      The measured buffering power of KRPH medium (typically used with cells) is the same when determined using a glass pH electrode or the pH sensor in a Seahorse assay cartridge (
      • Mookerjee S.A.
      • Brand M.D.
      Measurement and analysis of extracellular acid production to determine glycolytic rate.
      ). However, despite our earlier assertion, we found that the Seahorse pH sensor detected only about 77% of the change measured by a glass pH electrode in MAS-1 medium (
      • Rogers G.W.
      • Brand M.D.
      • Petrosyan S.
      • Ashok D.
      • Elorza A.A.
      • Ferrick D.A.
      • Murphy A.N.
      High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria.
      ) (typical for assaying isolated mitochondria). Therefore, the buffering power of MAS-1 and possibly other media of high osmotic and/or low ionic strength should be measured using the same instrument as the experiment.

      Author contributions

      S. A. M. and M. D. B. conceived the project; S. A. M. carried out all experiments. S. A. M. and M. D. B. wrote the manuscript. A. A. G. and D. G. N. contributed to the development and direction of the project and reviewed the manuscript.

      Acknowledgments

      We thank Samantha J. Antonio for technical assistance in data collection.

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