Calculation of ATP production rates using the Seahorse XF Analyzer.

Oxidative phosphorylation and glycolysis are the dominant ATP-generating pathways in mammalian metabolism. The balance between these two pathways is often shifted to execute cell-specific functions in response to stimuli that promote activation, proliferation, or differentiation. However, measurement of these metabolic switches has remained mostly qualitative, making it difficult to discriminate between healthy, physiological changes in energy transduction or compensatory responses due to metabolic dysfunction. We therefore present a broadly applicable method to calculate ATP production rates from oxidative phosphorylation and glycolysis using Seahorse XF Analyzer data and empirical conversion factors. We quantify the bioenergetic changes observed during macrophage polarization as well as cancer cell adaptation to in vitro culture conditions. Additionally, we detect substantive changes in ATP utilization upon neuronal depolarization and T cell receptor activation that are not evident from steady-state ATP measurements. This method generates a single readout that allows the direct comparison of ATP produced from oxidative phosphorylation and glycolysis in live cells. Additionally, the manuscript provides a framework for tailoring the calculations to specific cell systems or experimental conditions.

Mitochondrial Activity and Skeletal Muscle Insulin Resistance in Kidney Disease.

Insulin resistance is a key feature of the metabolic syndrome, a cluster of medical disorders that together increase the chance of developing type 2 diabetes and cardiovascular disease. In turn, type 2 diabetes may cause complications such as diabetic kidney disease (DKD). Obesity is a major risk factor for developing systemic insulin resistance, and skeletal muscle is the first tissue in susceptible individuals to lose its insulin responsiveness. Interestingly, lean individuals are not immune to insulin resistance either. Non-obese, non-diabetic subjects with chronic kidney disease (CKD), for example, exhibit insulin resistance at the very onset of CKD, even before clinical symptoms of renal failure are clear. This uraemic insulin resistance contributes to the muscle weakness and muscle wasting that many CKD patients face, especially during the later stages of the disease. Bioenergetic failure has been associated with the loss of skeletal muscle insulin sensitivity in obesity and uraemia, as well as in the development of kidney disease and its sarcopenic complications. In this mini review, we evaluate how mitochondrial activity of different renal cell types changes during DKD progression, and discuss the controversial role of oxidative stress and mitochondrial reactive oxygen species in DKD. We also compare the involvement of skeletal muscle mitochondria in uraemic and obesity-related muscle insulin resistance.

Control of pancreatic ß-cell bioenergetics.

The canonical model of glucose-stimulated insulin secretion (GSIS) by pancreatic ß-cells predicts a glucose-induced rise in the cytosolic ATP/ADP ratio. Such bioenergetic sensitivity to metabolic fuel is unusual as it implies that ATP flux is governed, to a significant extent, by ATP supply, while it is predominantly demand-driven in other cell types. Metabolic control is generally shared between different processes, but potential control of ATP consumption over ß-cell bioenergetics has been largely ignored to date. The present paper offers a brief overview of experimental evidence that demonstrates ATP flux control by glucose-fuelled oxidative phosphorylation. Based on old and new data, it is argued that ATP supply does not hold exclusive control over ATP flux, but shares it with ATP demand, and that the distribution of control is flexible. Quantification of the bioenergetic control distribution will be important from basic and clinical perspectives, but precise measurement of the cytosolic ATP/ADP ratio is complicated by adenine nucleotide compartmentalisation. Metabolic control analysis of ß-cell bioenergetics will likely clarify the mechanisms by which glucose and fatty acids amplify and potentiate GSIS, respectively. Moreover, such analysis may offer hints as to how ATP flux control shifts from ATP supply to ATP demand during the development of type 2 diabetes, and why prolonged sulfonylurea treatment causes ß-cell deterioration.

Mitochondrial involvement in skeletal muscle insulin resistance: A case of imbalanced bioenergetics.

Skeletal muscle insulin resistance in obesity associates with mitochondrial dysfunction, but the causality of this association is controversial. This review evaluates mitochondrial models of nutrient-induced muscle insulin resistance. It transpires that all models predict that insulin resistance arises as a result of imbalanced cellular bioenergetics. The nature and precise origin of the proposed insulin-numbing molecules differ between models but all species only accumulate when metabolic fuel supply outweighs energy demand. This observation suggests that mitochondrial deficiency in muscle insulin resistance is not merely owing to intrinsic functional defects, but could instead be an adaptation to nutrient-induced changes in energy expenditure. Such adaptive effects are likely because muscle ATP supply is fully driven by energy demand. This market-economic control of myocellular bioenergetics offers a mechanism by which insulin-signalling deficiency can cause apparent mitochondrial dysfunction, as insulin resistance lowers skeletal muscle anabolism and thus dampens ATP demand and, consequently, oxidative ATP synthesis.

Palmitate-induced changes in energy demand cause reallocation of ATP supply in rat and human skeletal muscle cells.

Mitochondrial dysfunction has been associated with obesity-related muscle insulin resistance, but the causality of this association is controversial. The notion that mitochondrial oxidative capacity may be insufficient to deal appropriately with excessive nutrient loads is for example disputed. Effective mitochondrial capacity is indirectly, but largely determined by ATP-consuming processes because skeletal muscle energy metabolism is mostly controlled by ATP demand. Probing the bioenergetics of rat and human myoblasts in real time we show here that the saturated fatty acid palmitate lowers the rate and coupling efficiency of oxidative phosphorylation under conditions it causes insulin resistance. Stearate affects the bioenergetic parameters similarly, whereas oleate and linoleate tend to decrease the rate but not the efficiency of ATP synthesis. Importantly, we reveal that palmitate influences how oxidative ATP supply is used to fuel ATP-consuming processes. Direct measurement of newly made protein demonstrates that palmitate lowers the rate of de novo protein synthesis by more than 30%. The anticipated decrease of energy demand linked to protein synthesis is confirmed by attenuated cycloheximide-sensitivity of mitochondrial respiratory activity used to make ATP. This indirect measure of ATP turnover indicates that palmitate lowers ATP supply reserved for protein synthesis by at least 40%. This decrease is also provoked by stearate, oleate and linoleate, albeit to a lesser extent. Moreover, palmitate lowers ATP supply for sodium pump activity by 60-70% and, in human cells, decreases ATP supply for DNA/RNA synthesis by almost three-quarters. These novel fatty acid effects on energy expenditure inform the 'mitochondrial insufficiency' debate.

Palmitate-induced impairment of glucose-stimulated insulin secretion precedes mitochondrial dysfunction in mouse pancreatic islets.

It has been well established that excessive levels of glucose and palmitate lower glucose-stimulated insulin secretion (GSIS) by pancreatic ß-cells. This ß-cell 'glucolipotoxicity' is possibly mediated by mitochondrial dysfunction, but involvement of bioenergetic failure in the pathological mechanism is the subject of ongoing debate. We show in the present study that increased palmitate levels impair GSIS before altering mitochondrial function. We demonstrate that GSIS defects arise from increased insulin release under basal conditions in addition to decreased insulin secretion under glucose-stimulatory conditions. Real-time respiratory analysis of intact mouse pancreatic islets reveals that mitochondrial ATP synthesis is not involved in the mechanism by which basal insulin is elevated. Equally, mitochondrial lipid oxidation and production of reactive oxygen species (ROS) do not contribute to increased basal insulin secretion. Palmitate does not affect KCl-induced insulin release at a basal or stimulatory glucose level, but elevated basal insulin release is attenuated by palmitoleate and associates with increased intracellular calcium. These findings deepen our understanding of ß-cell glucolipotoxicity and reveal that palmitate-induced GSIS impairment is disconnected from mitochondrial dysfunction, a notion that is important when targeting ß-cells for the treatment of diabetes and when assessing islet function in human transplants.

Insulin acutely improves mitochondrial function of rat and human skeletal muscle by increasing coupling efficiency of oxidative phosphorylation.

Insulin is essential for the regulation of fuel metabolism and triggers the uptake of glucose by skeletal muscle. The imported glucose is either stored or broken down, as insulin stimulates glycogenesis and ATP synthesis. The mechanism by which ATP production is increased is incompletely understood at present and, generally, relatively little functional information is available on the effect of insulin on mitochondrial function. In this paper we have exploited extracellular flux technology to investigate insulin effects on the bioenergetics of rat (L6) and human skeletal muscle myoblasts and myotubes. We demonstrate that a 20-min insulin exposure significantly increases (i) the cell respiratory control ratio, (ii) the coupling efficiency of oxidative phosphorylation, and (iii) the glucose sensitivity of anaerobic glycolysis. The improvement of mitochondrial function is explained by an insulin-induced immediate decrease of mitochondrial proton leak. Palmitate exposure annuls the beneficial mitochondrial effects of insulin. Our data improve the mechanistic understanding of insulin-stimulated ATP synthesis, and reveal a hitherto undisclosed insulin sensitivity of cellular bioenergetics that suggests a novel way of detecting insulin responsiveness of cells.

Novel insights into pancreatic ß-cell glucolipotoxicity from real-time functional analysis of mitochondrial energy metabolism in INS-1E insulinoma cells.

High circulating glucose and non-esterified (free) fatty acid levels can cause pancreatic ß-cell failure. The molecular mechanisms of this ß-cell glucolipotoxicity are yet to be established conclusively. In the present paper we report on the involvement of mitochondrial dysfunction in fatty-acid-induced ß-cell failure. We have used state-of-the-art extracellular flux technology to functionally probe mitochondrial energy metabolism in intact INS-1E insulinoma cells in real-time. We show that 24-h palmitate exposure at high glucose attenuates the glucose-sensitivity of mitochondrial respiration and lowers coupling efficiency of glucose-stimulated oxidative phosphorylation. These mitochondrial defects coincide with an increased level of ROS (reactive oxygen species), impaired GSIS (glucose-stimulated insulin secretion) and decreased cell viability. Palmitate lowers absolute glucose-stimulated respiration coupled to ATP synthesis, but does not affect mitochondrial proton leak. Palmitate is not toxic when administered at low glucose unless fatty acid ß-oxidation is inhibited. Palmitoleate, on the other hand, does not affect mitochondrial respiration, ROS levels, GSIS or cell viability. Although palmitoleate protects against the palmitate-induced ROS increase and cell viability loss, it does not protect against respiratory and insulin secretory defects. We conclude that mitochondrial dysfunction contributes to fatty-acid-induced GSIS impairment, and that glucolipotoxic cell viability and GSIS phenotypes are mechanistically distinct.

Mitochondrial involvement in sarcopenia.

Sarcopenia lowers the quality-of-life for millions of people across the world, as accelerated loss of skeletal muscle mass and function contributes to both age- and disease-related frailty. Physical activity remains the only proven therapy for sarcopenia to date, but alternatives are much sought after to manage this progressive muscle disorder in individuals who are unable to exercise. Mitochondria have been widely implicated in the etiology of sarcopenia and are increasingly suggested as attractive therapeutic targets to help restore the perturbed balance between protein synthesis and breakdown that underpins skeletal muscle atrophy. Reviewing current literature, we note that mitochondrial bioenergetic changes in sarcopenia are generally interpreted as intrinsic dysfunction that renders muscle cells incapable of making sufficient ATP to fuel protein synthesis. Based on the reported mitochondrial effects of therapeutic interventions, however, we argue that the observed bioenergetic changes may instead reflect an adaptation to pathologically decreased energy expenditure in sarcopenic muscle. Discrimination between these mechanistic possibilities will be crucial for improving the management of sarcopenia.

Nitrite lowers the oxygen cost of ATP supply in cultured skeletal muscle cells by stimulating the rate of glycolytic ATP synthesis.

Dietary nitrate lowers the oxygen cost of human exercise. This effect has been suggested to result from stimulation of coupling efficiency of skeletal muscle oxidative phosphorylation by reduced nitrate derivatives. In this paper, we report the acute effects of sodium nitrite on the bioenergetic behaviour of cultured rat (L6) myocytes. At odds with improved efficiency of mitochondrial ATP synthesis, extracellular flux analysis reveals that a ½-hour exposure to NaNO2 (0.1-5 µM) does not affect mitochondrial coupling efficiency in static myoblasts or in spontaneously contracting myotubes. Unexpectedly, NaNO2 stimulates the rate of glycolytic ATP production in both myoblasts and myotubes. Increased ATP supply through glycolysis does not emerge at the expense of oxidative phosphorylation, which means that NaNO2 acutely increases the rate of overall myocellular ATP synthesis, significantly so in myoblasts and tending towards significance in contractile myotubes. Notably, NaNO2 exposure shifts myocytes to a more glycolytic bioenergetic phenotype. Mitochondrial oxygen consumption does not decrease after NaNO2 exposure, and non-mitochondrial respiration tends to drop. When total ATP synthesis rates are expressed in relation to total cellular oxygen consumption rates, it thus transpires that NaNO2 lowers the oxygen cost of ATP supply in cultured L6 myocytes.

Acute bioenergetic insulin sensitivity of skeletal muscle cells: ATP-demand-provoked glycolysis contributes to stimulation of ATP supply.

Skeletal muscle takes up glucose in an insulin-sensitive manner and is thus important for the maintenance of blood glucose homeostasis. Insulin resistance during development of type 2 diabetes is associated with decreased ATP synthesis, but the causality of this association is controversial. In this paper, we report real-time oxygen uptake and medium acidification data that we use to quantify acute insulin effects on intracellular ATP supply and ATP demand in rat and human skeletal muscle cells. We demonstrate that insulin increases overall cellular ATP supply by stimulating the rate of glycolytic ATP synthesis. Stimulation is immediate and achieved directly by increased glycolytic capacity, and indirectly by elevated ATP demand from protein synthesis. Raised glycolytic capacity does not result from augmented glucose uptake. Notably, insulin-sensitive glucose uptake is increased synergistically by nitrite. While nitrite has a similar stimulatory effect on glycolytic ATP supply as insulin, it does not amplify insulin stimulation. These data highlight the multifarious nature of acute bioenergetic insulin sensitivity of skeletal muscle cells, and are thus important for the interpretation of changes in energy metabolism that are seen in insulin-resistant muscle.

Mutagenesis of the Sauromatum guttatum alternative oxidase reveals features important for oxygen binding and catalysis.

The alternative oxidase (AOX) is a non-protonmotive ubiquinol oxidase that is found in mitochondria of all higher plants studied to date. To investigate the role of highly conserved amino acid residues in catalysis we have expressed site-directed mutants of Cys-172, Thr-179, Trp-206, Tyr-253, and Tyr-299 in AOX in the yeast Schizosaccharomyces pombe. Assessment of AOX activity in isolated yeast mitochondria reveals that mutagenesis of Trp-206 to phenylalanine or tyrosine abolishes activity, in contrast to that observed with either Tyr-253 or 299 both mutants of which retained activity. None of the mutants exhibited sensitivity to Q-like inhibitors that differed significantly from the wild type AOX. Interestingly, however, mutagenesis of Thr-179 or Cys-172 (a residue implicated in AOX regulation by alpha-keto acids) to alanine not only resulted in a decrease of maximum AOX activity but also caused a significant increase in the enzyme's affinity for oxygen (4- and 2-fold, respectively). These results provide important new insights in the mechanism of AOX catalysis and regulation by pyruvate.

Respiratory Parameters for the Classification of Dysfunctional Insulin Secretion by Pancreatic Islets.

The development of obesity and type 2 diabetes (T2D) has been associated with impaired mitochondrial function. In pancreatic beta (ß) cells, mitochondrial energy metabolism plays a central role in triggering and controlling glucose-stimulated insulin secretion (GSIS). Here, we have explored whether mitochondrial bioenergetic parameters assessed with Seahorse extracellular flux technology can quantitatively predict insulin secretion. We metabolically stressed male C57BL/6 mice by high-fat feeding (HFD) and measured the glucose sensitivity of islet respiration and insulin secretion. The diet-induced obese (DIO) mice developed hyperinsulinemia, but no pathological secretory differences were apparent between isolated DIO and chow islets. Real-time extracellular flux analysis, however, revealed a lower respiratory sensitivity to glucose in DIO islets. Correlation of insulin secretion with respiratory parameters uncovers compromised insulin secretion in DIO islets by oxidative power. Normalization to increased insulin contents during DIO improves the quantitative relation between GSIS and respiration, allowing to classify dysfunctional properties of pancreatic insulin secretion, and thereby serving as valuable biomarker for pancreatic islet glucose responsiveness and health.

On the role of uncoupling protein-2 in pancreatic beta cells.

Pancreatic beta cells secrete insulin when blood glucose levels are high. Dysfunction of this glucose-stimulated insulin secretion (GSIS) is partly responsible for the manifestation of type 2 diabetes, a metabolic disorder that is rapidly becoming a global pandemic. Mitochondria play a central role in GSIS by coupling glucose oxidation to production of ATP, a signal that triggers a series of events that ultimately leads to insulin release. Beta cells express a mitochondrial uncoupling protein, UCP2, which is rather surprising as activity of such a protein is anticipated to lower the efficiency of oxidative phosphorylation, and hence to impair GSIS. The mounting evidence demonstrating that insulin secretion is indeed blunted by UCP2 agrees with this prediction, and has provoked the idea that UCP2 activity contributes to beta cell pathogenesis and development of type 2 diabetes. Although this notion may be correct, the evolved function of UCP2 remains unclear. With this paper we aim to provide a brief account of the present state of affairs in this field, suggest a physiological role for UCP2, and highlight some of our own recent results.

Dynamic regulation of uncoupling protein 2 content in INS-1E insulinoma cells.

Uncoupling protein 2 (UCP2) regulates glucose-stimulated insulin secretion in pancreatic beta-cells. UCP2 content, measured by calibrated immunoblot in INS-1E insulinoma cells (a pancreatic beta-cell model) grown in RPMI medium, and INS-1E mitochondria, was 2.0 ng/million cells (7.9 ng/mg mitochondrial protein). UCP2 content was lower in cells incubated without glutamine and higher in cells incubated with 20 mM glucose, and varied from 1.0-4.4 ng/million cells (2.7-14.5 ng/mg mitochondrial protein). This dynamic response to nutrients was achieved by varied expression rates against a background of a very short UCP2 protein half-life of about 1 h.

Compelling EPR evidence that the alternative oxidase is a diiron carboxylate protein.

The alternative oxidase is a respiratory chain protein found in plants, fungi and some parasites that still remains physically uncharacterised. In this report we present EPR evidence from parallel mode experiments which reveal signals at approximately g=16 in both purified alternative oxidase protein (g=16.9), isolated mitochondrial membranes (g=16.1), and in trypanosomal AOX expressed in Escherichia coli membranes (g=16.4). Such signals are indicative of a dicarboxylate diiron centre at the active site of the enzyme. To our knowledge these data represent the first EPR signals from AOX present in its native environment.

Uncoupling protein-2 contributes significantly to high mitochondrial proton leak in INS-1E insulinoma cells and attenuates glucose-stimulated insulin secretion.

Proton leak exerts stronger control over ATP/ADP in mitochondria from clonal pancreatic beta-cells (INS-1E) than in those from rat skeletal muscle, due to the higher proton conductance of INS-1E mitochondria [Affourtit and Brand (2006) Biochem. J. 393, 151-159]. In the present study, we demonstrate that high proton leak manifests itself at the cellular level too: the leak rate (measured as myxothiazol-sensitive, oligomycin-resistant respiration) was nearly four times higher in INS-1E cells than in myoblasts. This relatively high leak activity was decreased more than 30% upon knock-down of UCP2 (uncoupling protein-2) by RNAi (RNA interference). The high contribution of UCP2 to leak suggests that proton conductance through UCP2 accounts for approx. 20% of INS-1E respiration. UCP2 knock-down enhanced GSIS (glucose-stimulated insulin secretion), consistent with a role for UCP2 in beta-cell physiology. We propose that the high mitochondrial proton leak in beta-cells is a mechanism which amplifies the effect of physiological UCP2 regulators on cytoplasmic ATP/ADP and hence on insulin secretion.

Energization-dependent endogenous activation of proton conductance in skeletal muscle mitochondria.

Leak of protons into the mitochondrial matrix during substrate oxidation partially uncouples electron transport from phosphorylation of ADP, but the functions and source of basal and inducible proton leak in vivo remain controversial. In the present study we describe an endogenous activation of proton conductance in mitochondria isolated from rat and mouse skeletal muscle following addition of respiratory substrate. This endogenous activation increased with time, required a high membrane potential and was diminished by high concentrations of serum albumin. Inhibition of this endogenous activation by GDP [classically considered specific for UCPs (uncoupling proteins)], carboxyatractylate and bongkrekate (considered specific for the adenine nucleotide translocase) was examined in skeletal muscle mitochondria from wild-type and Ucp3-knockout mice. Proton conductance through endogenously activated UCP3 was calculated as the difference in leak between mitochondria from wild-type and Ucp3-knockout mice, and was found to be inhibited by carboxyatractylate and bongkrekate, but not GDP. Proton conductance in mitochondria from Ucp3-knockout mice was strongly inhibited by carboxyatractylate, bongkrekate and partially by GDP. We conclude the following: (i) at high protonmotive force, an endogenously generated activator stimulates proton conductance catalysed partly by UCP3 and partly by the adenine nucleotide translocase; (ii) GDP is not a specific inhibitor of UCP3, but also inhibits proton translocation by the adenine nucleotide translocase; and (iii) the inhibition of UCP3 by carboxyatractylate and bongkrekate is likely to be indirect, acting through the adenine nucleotide translocase.

Identification of a mitochondrial alcohol dehydrogenase in Schizosaccharomyces pombe: new insights into energy metabolism.

In the present study we have shown that mitochondria isolated from Schizosaccharomyces pombe exhibit antimycin A-sensitive oxygen uptake activity that is exclusively dependent on ethanol and is inhibited by trifluoroethanol, a potent inhibitor of ADH (alcohol dehydrogenase). Ethanol-dependent respiratory activity has, to our knowledge, not been reported in S. pombe mitochondria to date, which is surprising as it has been concluded previously that only one ADH gene, encoding a cytosolic enzyme, occurs in this yeast. Spectrophotometric enzyme assays reveal that ADH activity in isolated mitochondria is increased approximately 16-fold by Triton X-100, which demonstrates that the enzyme is located in the matrix. Using genetic knockouts, we show conclusively that the novel mitochondrial ADH is encoded by adh4 and, as such, is unrelated to ADH isoenzymes found in mitochondria of other yeasts. By performing a modular-kinetic analysis of mitochondrial electron transfer, we furthermore show how ethanol-dependent respiratory activity (which involves oxidation of matrix-located NADH) compares with that observed when succinate or externally added NADH are used as substrates. This analysis reveals distinct kinetic differences between substrates which fully explain the lack of respiratory control generally observed during ethanol oxidation in yeast mitochondria.

Measurement of Proton Leak in Isolated Mitochondria.

Oxidative phosphorylation is an important energy-conserving mechanism coupling mitochondrial electron transfer to ATP synthesis. Coupling between respiration and phosphorylation is not fully efficient due to proton leaks. In this chapter, we present a method to measure proton leak activity in isolated mitochondria. The relative strength of a modular kinetic approach to probe oxidative phosphorylation is emphasized.