IAPP toxicity activates HIF1α/PFKFB3 signaling delaying ß-cell loss at the expense of ß-cell function.

The islet in type 2 diabetes (T2D) is characterized by amyloid deposits derived from islet amyloid polypeptide (IAPP), a protein co-expressed with insulin by ß-cells. In common with amyloidogenic proteins implicated in neurodegeneration, human IAPP (hIAPP) forms membrane permeant toxic oligomers implicated in misfolded protein stress. Here, we establish that hIAPP misfolded protein stress activates HIF1α/PFKFB3 signaling, this increases glycolysis disengaged from oxidative phosphorylation with mitochondrial fragmentation and perinuclear clustering, considered a protective posture against increased cytosolic Ca2+ characteristic of toxic oligomer stress. In contrast to tissues with the capacity to regenerate, ß-cells in adult humans are minimally replicative, and therefore fail to execute the second pro-regenerative phase of the HIF1α/PFKFB3 injury pathway. Instead, ß-cells in T2D remain trapped in the pro-survival first phase of the HIF1α injury repair response with metabolism and the mitochondrial network adapted to slow the rate of cell attrition at the expense of ß-cell function.

Functional characterization of the oxidative capacity of mitochondria and glycolytic assessment in benthic aquatic organisms.

The metabolism of benthic aquatic invertebrates, populating transitional water ecosystems, is influenced by both physiological and environmental factors, thus involving an adjustment of physiological processes which has a metabolic cost. In order to discover changes in metabolic pathways in response to specific factors, it's firstly necessary characterizing the principal cellular metabolic activities of the small benthic aquatic organisms. We approach here the bioenergetic state issue of two benthic organisms, i.e. Lekanesphaera monodi and Gammarus insensibilis, evidencing that no apparent and statistically significative differences between them in aerobic as well in glycolytic capacities are detected, except for COX activity.

Regional assessment of energy-producing metabolic activity in the endothelium of donor corneas.

PURPOSE: We characterized mitochondrial respiration and glycolysis activity of human corneal endothelium, and compared metabolic activity between central and peripheral regions. METHODS: Endothelial keratoplasty-suitable corneas were obtained from donors aged 50 to 75 years. The endothelium-Descemet membrane complex (EDM) was isolated, and 3-mm punches were obtained from central and peripheral regions. Endothelium-Descemet membrane punches were assayed for mitochondrial respiration (oxygen consumption) and glycolysis (extracellular acidification) using an extracellular flux analyzer. Enzymatic (citrate synthase, glucose hexokinase) and mitochondrial density (MitoTracker) assays also were performed. RESULTS: Ten corneas were analyzed per assay. Metabolic activity for mitochondrial respiration and glycolysis showed expected changes to assay compounds (P < 0.01, all pairwise comparisons). Basal mitochondrial respiration and glycolysis activity did not differ between regions (P > 0.99). Similarly, central versus peripheral activity after assay compound treatment showed no significant differences (P > 0.99, all time points). The intracorneal coefficient of variation for basal readings between two and four peripheral punches was 18.5% of the mean. Although peripheral samples displayed greater enzymatic activity than central samples (P < 0.05), similar to extracellular flux results, mitochondrial density did not differ between regions (P = 0.78). CONCLUSIONS: Extracellular flux analysis of oxygen and pH is a valid technique for characterizing metabolic activity of human corneal endothelium. This technique demonstrates high reproducibility, allows quantification of metabolic parameters using small quantities of live cells, and permits estimation of overall metabolic output. Neither oxygen consumption nor extracellular acidification differed between central and peripheral regions of transplant suitable corneas in this series. Our results show that endothelial cell health can be quantified biochemically in transplant suitable corneas.

Physiological responses and energy expenditure during competitive fencing.

Fencing is an Olympic sport in which athletes fight one against one using bladed weapons. Contests consist of three 3-min bouts, with rest intervals of 1 min between them. No studies investigating oxygen uptake and energetic demand during fencing competitions exist, thus energetic expenditure and demand in this sport remain speculative. The aim of this study was to understand the physiological capacities underlying fencing performance. Aerobic energy expenditure and the recruitment of lactic anaerobic metabolism were determined in 15 athletes (2 females and 13 males) during a simulation of fencing by using a portable gas analyzer (MedGraphics VO2000), which was able to provide data on oxygen uptake, carbon dioxide production and heart rate. Blood lactate was assessed by means of a portable lactate analyzer. Average group energetic expenditure during the simulation was (mean ± SD) 10.24 ± 0.65 kcal·min(-1), corresponding to 8.6 ± 0.54 METs. Oxygen uptakeand heart rate were always below the level of anaerobic threshold previously assessed during the preliminary incremental test, while blood lactate reached its maximum value of 6.9 ± 2.1 mmol·L(-1) during the final recovery minute between rounds. Present data suggest that physical demand in fencing is moderate for skilled fencers and that both aerobic energy metabolism and anaerobic lactic energy sources are moderately recruited. This should be considered by coaches when preparing training programs for athletes.

Characterization of fructose-1,6-bisphosphate aldolase during anoxia in the tolerant turtle, Trachemys scripta elegans: an assessment of enzyme activity, expression and structure.

One of the most adaptive facultative anaerobes among vertebrates is the freshwater turtle, Trachemys scripta elegans. Upon a decrease in oxygen supply and oxidative phosphorylation, these turtles are able to reduce their metabolic rate and recruit anaerobic glycolysis to meet newly established ATP demands. Within the glycolytic pathway, aldolase enzymes cleave fructose-1,6-bisphosphate to triose phosphates facilitating an increase in anaerobic production of ATP. Importantly, this enzyme exists primarily as tissue-specific homotetramers of aldolase A, B or C located in skeletal muscle, liver and brain tissue, respectively. The present study characterizes aldolase activity and structure in the liver tissue of a turtle whose survival greatly depends on increased glycolytic output during anoxia. Immunoblot and mass spectrometry analysis verified the presence of both aldolase A and B in turtle liver tissue, and results from co-immunoprecipitation experiments suggested that in the turtle aldolase proteins may exist as an uncommon heterotetramer. Expression levels of aldolase A protein increased significantly in liver tissue to 1.59±0.11-fold after 20 h anoxia, when compared to normoxic control values (P<0.05). A similar increase was seen for aldolase B expression. The overall kinetic properties of aldolase, when using fructose-1,6-bisphosphate as substrate, were similar to that of a previously studied aldolase A and aldolase B heterotetramer, with a Km of 240 and 180 nM (for normoxic and anoxic turtle liver, respectively). Ligand docking of fructose-1,6-bisphosphate to the active site of aldolase A and B demonstrated minor differences in both protein:ligand interactions compared to rabbit models. It is likely that the turtle is unique in its ability to regulate a heterotetramer of aldolase A and B, with a higher overall enzymatic activity, to achieve greater rates of glycolytic output and support anoxia survival.

The effect of time-under-tension and weight lifting cadence on aerobic, anaerobic, and recovery energy expenditures: 3 submaximal sets.

We examined the aerobic and anaerobic energy expenditures of weight lifting (bench press); submaximal work was kept constant among protocols. Ten male subjects (age, 23.2 ± 3.1 years; height, 177.3 ± 5.3 cm; weight, 82.1 ± 11.5 kg) were randomly assigned to 3 lifting sessions of 3 sets of 5 repetitions at 70% 1 repetition maximum (1RM) using 3 lifting cadences: 1.5 s down and 1.5 s up (15 s per set), 4 s down and 1 s up (25 s per set), and 1 s down and 4 s up (25 s per set). No differences were found among the aerobic exercise energy expenditures for each lifting cadence. However, anaerobic energy expenditure was significantly different among protocols: 1.5 down-1.5 up, 16.5 ± 8.1 kJ; 4 down-1 up, 21.6 ± 8.1 kJ; and 1 down-4 up, 26.7 ± 7.2 kJ (p = 0.001). Excess postexercise oxygen consumption (EPOC; after each set) was lower for 1.5 down-1.5 up, 38.6 ± 17.8 kJ; versus 4 down-1 up, 50.2 ± 23.5 kJ; and 1 down-4 up, 50.0 ± 22.6 kJ (p = 0.002). Total energy expenditure also was significantly less for 1.5 up-1.5 down, 60.2 ± 23.8 kJ; versus 4 down-1 up, 80.0 ± 27.7 kJ; and 1 down-4 up, 84.2 ± 28.3 kJ (p = 0.001). Differences in EPOC and total energy expenditure with submaximal lifting were based not on the amount of work performed or with a particular eccentric-concentric cadence, but on the time to completion of the weight lifting exercise - time-under-tension; longer submaximal lifting times had greater energy expenditure.

Comparative bioenergetic assessment of transformed cells using a cell energy budget platform.

The aberrant expression and functional activity of proteins involved in ATP production pathways may cause a crisis in energy generation for cells and compromise their survival under stressful conditions such as excitation, starvation, pharmacological treatment or disease states. Under resting conditions such defects are often compensated for, and therefore masked by, alternative pathways which have significant spare capacity. Here we present a multiplexed 'cell energy budget' platform which facilitates metabolic assessment and cross-comparison of different cells and the identification of genes directly or indirectly involved in ATP production. Long-decay emitting O(2) and pH sensitive probes and time-resolved fluorometry are used to measure changes in cellular O(2) consumption, glycolytic and total extracellular acidification (ECA), along with the measurement of total ATP and protein content in multiple samples. To assess the extent of spare capacity in the main energy pathways, the cells are also analysed following double-treatment with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone and oligomycin. The four-parametric platform operating in a high throughput format has been validated with two panels of transformed cells: mouse embryonic fibroblasts (MEFs) lacking the Krebs cycle enzyme fumarate hydratase (Fh1) and HeLa cells with reduced expression of pyrimidine nucleotide carrier 1. In both cases, a marked reduction in both respiration and spare respiratory capacity was observed, accompanied by a compensatory activation of glycolysis and consequent maintenance of total ATP levels. At the same time, in Fh1-deficient MEFs the contribution of non-glycolytic pathways to the ECA did not change.

Proteomic assessment of the acute phase of dystrophin deficiency in mdx mice.

Duchenne muscular dystrophy (DMD) is caused by the absence of a functional dystrophin protein and is modeled by the mdx mouse. The mdx mouse suffers an early necrotic bout in the hind limb muscles lasting from approximately 4 to 7 weeks. The purpose of this investigation was to determine the extent to which dystrophin deficiency changed the proteome very early in the disease process. In order to accomplish this, proteins from gastrocnemius from 6-week-old C57 (n = 6) and mdx (n = 6) mice were labeled with fluorescent dye and subjected to two-dimensional differential in-gel electrophoresis (2D-DIGE). Resulting differentially expressed spots were excised and protein identity determined via MALDI-TOF followed by database searching using MASCOT. Proteins of the immediate energy system and glycolysis were generally down-regulated in mdx mice compared to C57 mice. Conversely, expression of proteins involved in the Kreb's cycle and electron transport chain were increased in dystrophin-deficient muscle compared to control. Expression of cytoskeletal components, including tubulins, vimentin, and collagen, were increased in mdx mice compared to C57 mice. Importantly, these changes are occurring at only 6 weeks of age and are caused by acute dystrophin deficiency rather than more chronic injury. These data may provide insight regarding early pathologic changes occurring in dystrophin-deficient skeletal muscle.

Energy expenditure characteristics of weight lifting: 2 sets to fatigue.

We investigated the work performed and energy expenditure characteristics within and among 2 sets of the bench press at 70%, 80%, and 90% of 1 repetition maximum (1RM). For both sets fatigue was the end point. We asked: do multiple sets affect subsequent work output along with aerobic, anaerobic, and excess postexercise oxygen consumption (EPOC) contributions? Ten males participated. Work was significantly less for the 2nd set within the 70% and 80% protocols, but not the 90% protocol. Anaerobic (glycolytic) energy expenditure was less for the 2nd set within all protocols. However, within all protocols, the work / energy expenditure ratio was not different between sets. Overall work was significantly different among protocols, becoming less as the weight lifted was increased: 70%, 637.1 ± 122.4 J; 80%, 512.4 ± 93.4 J; 90%, 324.7 ± 92.6 J (p < 0.001). EPOC was not different among protocols after the 1st set, 2nd set, or combined overall. Moreover, the overall EPOC did not correlate with overall work performed (r = 0.31, p = 0.11). EPOC overall did correlate with aerobic (r = 0.68, p < 0.001) and anaerobic (r = 0.65, p < 0.001) energy expenditures. In terms of a work / energy expenditure ratio, the least amount of completed work at 90% 1RM required greater energy expenditure as compared with 70% and 80% because of an EPOC that is similar for all. As more work is completed (i.e., lower weight, more repetitions), aerobic and anaerobic exercise energy expenditures appear to increase accordingly, yet absolute EPOC remains essentially unchanged, contributing less to the overall energy expenditure.

Pyruvate kinase type M2: a key regulator of the metabolic budget system in tumor cells.

Cell proliferation only proceeds when metabolism is capable of providing a budget of metabolic intermediates that is adequate to ensure both energy regeneration and the synthesis of cell building blocks in sufficient amounts. In tumor cells, the glycolytic pyruvate kinase isoenzyme M2 (PKM2, M2-PK) determines whether glucose is converted to lactate for regeneration of energy (active tetrameric form, Warburg effect) or used for the synthesis of cell building blocks (nearly inactive dimeric form). This review discusses the regulation mechanisms of pyruvate kinase M2 expression by different transcription factors as well as the regulation of pyruvate kinase M2 activity by direct interaction with certain oncoproteins, tyrosine and serine phosphorylation, binding of phosphotyrosine peptides, association with other glycolytic and non glycolytic enzymes, the promyelocytic leukemia tumor suppressor protein, as well as metabolic intermediates. An intervention in the regulation mechanisms of the expression, activity and tetramer to dimer ratio of pyruvate kinase M2 has severe consequences for metabolism as well as proliferation and tumorigenic capacity of the cells which makes this enzyme a promising target for potential therapeutic approaches. The quantification of the dimeric form of pyruvate kinase M2 (Tumor M2-PK) in plasma and stool allows early detection of tumors and therapy control. Several different mechanisms may induce a translocation of pyruvate kinase M2 into the nucleus. The role of pyruvate kinase M2 in the nucleus is complex as witnessed by evidence of its effect both as pro-proliferative as well as pro-apoptotic stimuli.

Sustained oscillations for density dependent Markov processes.

Simulations of models of epidemics, biochemical systems, and other bio-systems show that when deterministic models yield damped oscillations, stochastic counterparts show sustained oscillations at an amplitude well above the expected noise level. A characterization of damped oscillations in terms of the local linear structure of the associated dynamics is well known, but in general there remains the problem of identifying the stochastic process which is observed in stochastic simulations. Here we show that in a general limiting sense the stochastic path describes a circular motion modulated by a slowly varying Ornstein-Uhlenbeck process. Numerical examples are shown for the Volterra predator-prey model, Sel'kov's model for glycolysis, and a damped linear oscillator.

KSR2 is an essential regulator of AMP kinase, energy expenditure, and insulin sensitivity.

Kinase suppressors of Ras 1 and 2 (KSR1 and KSR2) function as molecular scaffolds to potently regulate the MAP kinases ERK1/2 and affect multiple cell fates. Here we show that KSR2 interacts with and modulates the activity of AMPK. KSR2 regulates AMPK-dependent glucose uptake and fatty acid oxidation in mouse embryonic fibroblasts and glycolysis in a neuronal cell line. Disruption of KSR2 in vivo impairs AMPK-regulated processes affecting fatty acid oxidation and thermogenesis to cause obesity. Despite their increased adiposity, ksr2(-/-) mice are hypophagic and hyperactive but expend less energy than wild-type mice. In addition, hyperinsulinemic-euglycemic clamp studies reveal that ksr2(-/-) mice are profoundly insulin resistant. The expression of genes mediating oxidative phosphorylation is also downregulated in the adipose tissue of ksr2(-/-) mice. These data demonstrate that ksr2(-/-) mice are highly efficient in conserving energy, revealing a novel role for KSR2 in AMPK-mediated regulation of energy metabolism.

Parallel activation of mitochondrial oxidative metabolism with increased cardiac energy expenditure is not dependent on fatty acid oxidation in pigs.

Steady state concentrations of ATP and ADP in vivo are similar at low and high cardiac workloads; however, the mechanisms that regulate the activation of substrate metabolism and oxidative phosphorylation that supports this stability are poorly understood. We tested the hypotheses that (1) there is parallel activation of mitochondrial and cytosolic dehydrogenases in the transition from low to high workload, which increases NADH/NAD+ ratio in both compartments, and (2) this response does not require an increase in fatty acid oxidation (FAO). Anaesthetized pigs were subjected to either sham treatment, or an abrupt increase in cardiac workload for 5 min with dobutamine infusion and aortic constriction. Myocardial oxygen consumption and FAO were increased 3- and 2-fold, respectively, but ATP and ADP concentrations did not change. NADH-generating pathways were rapidly activated in both the cytosol and mitochondria, as seen in a 40% depletion in glycogen stores, a 3.6-fold activation of pyruvate dehydrogenase, and a 50% increase in tissue NADH/NAD+. Simulations from a multicompartmental computational model of cardiac energy metabolism predicted that parallel activation of glycolysis and mitochondrial metabolism results in an increase in the NADH/NAD+ ratio in both cytosol and mitochondria. FAO was blocked by 75% in a third group of pigs, and a similar increase in and the NAHD/NAD+ ratio was observed. In conclusion, in the transition to a high cardiac workload there is rapid parallel activation of substrate oxidation that results in an increase in the NADH/NAD+ ratio.

Bovine sperm capacitation: assessment of phosphodiesterase activity and intracellular alkalinization on capacitation-associated protein tyrosine phosphorylation.

Mammalian sperm capacitation is the obligatory maturational process leading to the development of the fertilization-competent state. Heparin is known to be a unique species-specific inducer of bovine sperm capacitation in vitro and glucose a unique inhibitor of this induction. Heparin-induced capacitation of bovine sperm has been shown to correlate with protein kinase A (PKA)-dependent protein tyrosine phosphorylation driven by an increase in intracellular cAMP. This study examines the possible roles of cyclic nucleotide phosphodiesterase (PDE) activity and intracellular alkalinization on bovine sperm capacitation and the protein tyrosine phosphorylation associated with it. Measurement of whole cell PDE kinetics during capacitation reveals neither a substantial change with heparin nor one with glucose: PDE activity is effectively constitutive in maintaining intracellular cAMP levels during capacitation. In contrast to a transient increase in intracellular pH, a sustained increase in medium pH by switching from 5% CO(2)/95% air incubation to 1% CO(2)/99% air incubation over 4 hr in the absence of heparin resulted in an increase in protein tyrosine phosphorylation and in the extent of induced acrosome reaction comparable to that observed following heparin-induced capacitation in 5% CO(2). These results suggest that increased bicarbonate-dependent adenylyl cyclase activity, driven by alkalinization, increases intracellular cAMP and so increases PKA activity mediating protein tyrosine phosphorylation. Quantitative analysis of the lactic acid production rate by bovine sperm glycolysis accounts fully for intracellular acidification sufficient to offset heparin-induced alkalinization, thus inhibiting capacitation. The mechanism by which heparin uniquely induces intracellular alkalinization in bovine sperm leading to capacitation remains obscure, inviting future investigation.

A hierarchical mixture of Markov models for finding biologically active metabolic paths using gene expression and protein classes.

With the recent development of experimental high-throughput techniques, the type and volume of accumulating biological data have extremely increased these few years. Mining from different types of data might lead us to find new biological insights. We present a new methodology for systematically combining three different datasets to find biologically active metabolic paths/patterns. This method consists of two steps: First it synthesizes metabolic paths from a given set of chemical reactions, which are already known and whose enzymes are co-expressed, in an efficient manner. It then represents the obtained metabolic paths in a more comprehensible way through estimating parameters of a probabilistic model by using these synthesized paths. This model is built upon an assumption that an entire set of chemical reactions corresponds to a Markov state transition diagram. Furthermore, this model is a hierarchical latent variable model, containing a set of protein classes as a latent variable, for clustering input paths in terms of existing knowledge of protein classes. We tested the performance of our method using a main pathway of glycolysis, and found that our method achieved higher predictive performance for the issue of classifying gene expressions than those obtained by other unsupervised methods. We further analyzed the estimated parameters of our probabilistic models, and found that biologically active paths were clustered into only two or three patterns for each expression experiment type, and each pattern suggested some new long-range relations in the glycolysis pathway.

Pre-conditioning activates adenosine utilization in a cost-effective way during myocardial ischaemia.

During pre-conditioning the interstitial concentration of adenosine, in contrast to lactate, presents a die-away curve-pattern for every successive episode of ischaemia. This die-away pattern might not necessarily be attributed to diminished adenosine production. The present study was undertaken to investigate whether pre-conditioning alters the metabolic turnover of adenosine as observed by the lactate production during ischaemia. Interstitial levels of metabolites in pre-conditioned (n=21) and non-preconditioned (n=21) porcine hearts were monitored with microdialysis probes inserted in both ischaemic and non-ischaemic tissue in an open chest heart model. Three subgroups perturbated with either plain microdialysis buffer (control), buffer containing adenosine (375 microM), or buffer containing deoxyadenosine (375 microM) were studied. All animals were subjected to 90 min of equilibrium microdialysis before 40 min of regional myocardial ischaemia and 120 min of reperfusion. Pre-conditioning consisted of four repetitive episodes of 10 min of ischaemia and 20 min of reperfusion. Significantly higher levels of inosine and lactate were found in the ischaemic tissue of the pre-conditioned subgroup receiving adenosine (P < 0.05) compared with the other two subgroups receiving deoxyadenosine and plain buffer, respectively. This difference was only valid for pre-conditioned ischaemic myocardium, and hence equal amounts of inosine and lactate were produced in the non-preconditioned ischaemic myocardium regardless of the presence of adenosine or deoxyadenosine. In the non-ischaemic myocardium baseline levels of metabolites were measured in all subgroups. Pre-conditioning favoured degradation of exogenous adenosine to inosine successively ending up in enhanced lactate production. This was probably because of the involvement of the hexose monophosphate pathway in the pre-conditioned ischaemic myocardium. This route may therefore be supplementary in energy metabolism as a metabolic flow can be started by adenosine ending up in lactate without initial adenosine 5'-triphosphate (ATP) investment. Utilization of adenosine in this way may also explain the successive die-away pattern of adenosine seen in consecutive pre-conditioning cycles.

Energy cost and cardiorespiratory demands of nunchaku exercise.

BACKGROUND: The purpose was to examine the energy cost and cardiorespiratory demands of nunchaku exercise, one of the martial arts performed with an instrument. METHODS: Nine male martial art practitioners (age 26.4 (+/-SD) 3.4 years, VO2max 54.1+/-9.0 ml x kg(-1)x min(-1)) completed three nunchaku exercise trials, lasting 20 sec, 1 min and 5 min. Cardiorespiratory response was followed by the Cosmed K4 portable respiratory analysis system. MEASURES: Oxygen consumption (VO2) was determined during pre-exercise rest, the exercise period and the recovery phase (10-15 min), and energy release from lactate (La) production was calculated assuming that an increase of 1 mmol x l(-1) corresponds to aVO2 of 3 ml O2 (di Prampero 1981). RESULTS: The overall energy cost of nunchaku exercise attained 76+/-16 kJ (229+/-48 kJ x min(-1)), 98+/-23 kJ and 271+/-72 kJ (54+/-14 kJ x min(-1)) for the 20 sec, 1 min and 5 min nunchaku exercise, respectively. From the point of view of the energy sources (alactic, lactic and oxidative), 20-sec performance was essentially "anaerobic alactic" (69:15:16%), 1 min exercise "alactic-oxidative" (49:16:35%) and 5-min performance an "oxidative" exercise workload (17% alactic: 6% lactic: 77% oxidative, respectively). The intensity of the exercise, on the average, corresponded to 43, 49 and 56% of VO2max and 69, 72 and 76% of HRmax, for the 20 sec, 1 min and 5 min exercises, respectively. CONCLUSIONS: Nunchaku exercise elicits high alactic and oxidative energy sources, but its demand for anaerobic glycolytic pathway seem to be relatively low, regardless the duration of the exercise. The energy cost (EC) for 20-sec to 5-min lasting exercise may be described by power function EC=10.84.t(-0.522) (n=24, r2=0.95), where EC is in kJ x min(-1) x kg(-1) and t in seconds.

Re-interpreting anaerobic metabolism: an argument for the application of both anaerobic glycolysis and excess post-exercise oxygen comsumption (EPOC) as independent sources of energy expenditure.

Due to current technical difficulties and changing cellular conditions, the measurement of anaerobic and recovery energy expenditure remains elusive. During rest and low-intensity steady-state exercise, indirect calorimetric measurements successfully represent energy expenditure. The same steady-state O2 uptake methods are often used to describe the O2 deficit and excess post-oxygen consumption (EPOC): 1 l O2 = 5 kcal = 20.9 kJ. However, an O2 deficit plus exercise O2 uptake measurement ignores energy expenditure during recovery, and an exercise O2 uptake plus EPOC measurement misrepresents anaerobic energy expenditure. An alternative solution has not yet been proposed. Anaerobic glycolysis and mitochondrial respiration are construed here as a symbiotic union of metabolic pathways, each contributing independently to energy expenditure and heat production. Care must be taken when using O2 uptake alone to quantify energy expenditure because various high-intensity exercise models reveal that O2 uptake can lag behind estimated energy demands or exceed them. The independent bioenergetics behind anaerobic glycolysis and mitochondrial respiration can acknowledge these discrepancies. Anaerobic glycolysis is an additive component to an exercise O2 uptake measurement. Moreover, it is the assumptions behind steady-state O2 uptake that do not permit proper interpretation of energy expenditure during EPOC; 1 l O2 not = 20.9 kJ. Using both the O2 deficit and a modified EPOC for interpretation, rather than one or the other, leads to a better method of quantifying energy expenditure for higher intensity exercise and recovery.