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Context/Objective Impaired adipose tissue (AT) function might induce recent-onset type 2 diabetes (T2D). Understanding AT energy metabolism could yield novel targets for the treatment of T2D. Design/Patients Male patients with recently-diagnosed T2D and healthy male controls (CON) of similar abdominal subcutaneous AT (SAT)-thickness, fat mass, and age (n = 14 each), underwent hyperinsulinemic-euglycemic clamps with [6,6-2H2]glucose and indirect calorimetry. We assessed mitochondrial efficiency (coupling: state 3/4o; proton leak: state 4o/u) via high-resolution respirometry in superficial (SSAT) and deep (DSAT) SAT-biopsies, hepatocellular lipids (HCL) and fat mass by proton-magnetic-resonance-spectroscopy and -imaging. Results T2D patients (known diabetes duration: 2.5 [0.1; 5.0] years) had 43%, 44%, and 63% lower muscle insulin sensitivity (IS), metabolic flexibility (P Conclusions Impaired mitochondrial function, insulin resistance, and DSAT expansion are AT abnormalities in recent-onset T2D that might promote whole-body insulin resistance and increased substrate flux to the liver.

A Western diet (WD), high in sugars and saturated fats, impairs learning and memory function and contributes to weight gain. Mitochondria in the brain provide energy for neurocognitive function and may play a role in body weight regulation. We sought to determine whether a WD alters behavior and metabolic outcomes in male and female rodents through impacting hippocampal and hypothalamic mitochondrial bioenergetics. Results revealed a sexually dimorphic macronutrient preference, where males on the WD consumed a greater percentage of calories from fat/protein and females consumed a greater percentage of calories from a sugar-sweetened beverage. Both males and females on a WD gained body fat and showed impaired glucose tolerance when compared to same-sex controls. Males on a WD demonstrated impaired hippocampal functioning and an elevated tendency toward a high membrane potential in hippocampal mitochondria. Comprehensive bioenergetics analysis of WD effects in the hypothalamus revealed a tissue-specific adaption, where males on the WD oxidized more fat, and females oxidized more fat and carbohydrates at peak energy demand compared to same-sex controls. These results suggest that adult male rats show a susceptibility toward hippocampal dysfunction on a WD, and that hypothalamic mitochondrial bioenergetics are altered by WD in a sex-specific manner.

In general, metabolic flexibility refers to an organism’s capacity to adapt to metabolic changes due to differing energy demands. The aim of this work is to summarize and discuss recent findings regarding variables that modulate energy regulation in two different pathways of mitochondrial fatty metabolism: β-oxidation and fatty acid biosynthesis. We focus specifically on two diseases: very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) and malonyl-CoA synthetase deficiency (acyl-CoA synthetase family member 3 (ACSF3)) deficiency, which are both characterized by alterations in metabolic flexibility. On the one hand, in a mouse model of VLCAD-deficient (VLCAD−/−) mice, the white skeletal muscle undergoes metabolic and morphologic transdifferentiation towards glycolytic muscle fiber types via the up-regulation of mitochondrial fatty acid biosynthesis (mtFAS). On the other hand, in ACSF3-deficient patients, fibroblasts show impaired mitochondrial respiration, reduced lipoylation, and reduced glycolytic flux, which are compensated for by an increased β-oxidation rate and the use of anaplerotic amino acids to address the energy needs. Here, we discuss a possible co-regulation by mtFAS and β-oxidation in the maintenance of energy homeostasis.

Aim This study sought to provide a statistically robust reference for measures of mitochondrial function from standardized high‐resolution respirometry with permeabilized human skeletal muscle (ex vivo), compare analogous values obtained via indirect calorimetry, arterial‐venous O2 differences and 31P magnetic resonance spectroscopy (in vivo) and attempt to resolve differences across complementary methodologies as necessary. Methods Data derived from 831 study participants across research published throughout March 2009 to November 2019 were amassed to examine the biological relevance of ex vivo assessments under standard conditions, ie physiological temperatures of 37°C and respiratory chamber oxygen concentrations of ~250 to 500 μmol/L. Results Standard ex vivo‐derived measures are lower (Z ≥ 3.01, P ≤ .0258) en masse than corresponding in vivo‐derived values. Correcting respiratory values to account for mitochondrial temperatures 10°C higher than skeletal muscle temperatures at maximal exercise (~50°C): (i) transforms data to resemble (Z ≤ 0.8, P > .9999) analogous yet context‐specific in vivo measures, eg data collected during maximal 1‐leg knee extension exercise; and (ii) supports the position that maximal skeletal muscle respiratory rates exceed (Z ≥ 13.2, P

Biomarker discovery and implementation are at the forefront of the precision medicine movement. Modern advances in the field of metabolomics afford the opportunity to readily identify new metabolite biomarkers across a wide array of disciplines. Many of the metabolites are derived from or directly reflective of mitochondrial metabolism. L-carnitine and acylcarnitines are established mitochondrial biomarkers used to screen neonates for a series of genetic disorders affecting fatty acid oxidation, known as the inborn errors of metabolism. However, L-carnitine and acylcarnitines are not routinely measured beyond this screening, despite the growing evidence that shows their clinical utility outside of these disorders. Measurements of the carnitine pool have been used to identify the disease and prognosticate mortality among disorders such as diabetes, sepsis, cancer, and heart failure, as well as identify subjects experiencing adverse drug reactions from various medications like valproic acid, clofazimine, zidovudine, cisplatin, propofol, and cyclosporine. The aim of this review is to collect and interpret the literature evidence supporting the clinical biomarker application of L-carnitine and acylcarnitines. Further study of these metabolites could ultimately provide mechanistic insights that guide therapeutic decisions and elucidate new pharmacologic targets.

Most anionic metabolites including respiratory substrates, glycolytic adenosine triphosphate (ATP), and small cations that enter mitochondria, and mitochondrial ATP moving to the cytosol, cross the outer mitochondrial membrane (OMM) through voltage dependent anion channels (VDAC). The closed states of VDAC block the passage of anionic metabolites, and increase the flux of small cations, including calcium. Consequently, physiological or pharmacological regulation of VDAC opening, by conditioning the magnitude of both anion and cation fluxes, is a major contributor to mitochondrial metabolism. Tumor cells display a pro-proliferative Warburg phenotype characterized by enhanced aerobic glycolysis in the presence of partial suppression of mitochondrial metabolism. The heterogeneous and flexible metabolic traits of most human tumors render cells able to adapt to the constantly changing energetic and biosynthetic demands by switching between predominantly glycolytic or oxidative phenotypes. Here, we describe the biological consequences of changes in the conformational state of VDAC for cancer metabolism, the mechanisms by which VDAC-openers promote cancer cell death, and the advantages of VDAC opening as a valuable pharmacological target. Particular emphasis is given to the endogenous regulation of VDAC by free tubulin and the effects of VDAC-tubulin antagonists in cancer cells. Because of its function and location, VDAC operates as a switch to turn-off mitochondrial metabolism (closed state) and increase aerobic glycolysis (pro-Warburg), or to turn-on mitochondrial metabolism (open state) and decrease glycolysis (anti-Warburg). A better understanding of the role of VDAC regulation in tumor progression is relevant both for cancer biology and for developing novel cancer chemotherapies., Instituto de Estudios Inmunológicos y Fisiopatológicos

Cancer cells frequently lack nutrients like glucose, due to insufficient vascular networks. Mitochondrial phosphoenolpyruvate carboxykinase, PCK2, has recently been found to mediate partial gluconeogenesis and hence anabolic metabolism in glucose starved cancer cells. Here we show that PCK2 acts as a regulator of mitochondrial respiration and maintains the redox balance in nutrient-deprived human lung cancer cells. PCK2 silencing increased the abundance and interconversion of tricarboxylic acid (TCA) cycle intermediates, augmented mitochondrial respiration and enhanced glutathione oxidation under glucose and serum starvation, in a PCK2 re-expression reversible manner. Moreover, enhancing the TCA cycle by PCK2 inhibition severely reduced colony formation of lung cancer cells under starvation. As a conclusion, PCK2 contributes to maintaining a reduced glutathione pool in starved cancer cells besides mediating the biosynthesis of gluconeogenic/glycolytic intermediates. The study sheds light on adaptive responses in cancer cells to nutrient deprivation and shows that PCK2 confers protection against respiration-induced oxidative stress., Free Radical Biology and Medicine, 176, ISSN:0891-5849, ISSN:1873-4596

International audience; The liver is a major organ that coordinates the metabolic flexibility of the whole body, which is characterized by the ability to adapt dynamically in response to fluctuations in energy needs and supplies. In this context, hepatocyte mitochondria are key partners in fine-tuning metabolic flexibility. Here we review the metabolic and signalling pathways carried by mitochondria in the liver, the major pathways that regulate mitochondrial function and how they function in health and metabolic disorders associated to obesity, i.e. insulin resistance, non-alcoholic steatosis and steatohepatitis and hepatocellular carcinoma. Finally, strategies targeting mitochondria to counteract liver disorders are discussed.

Osteoarthritis (OA) is the most frequent joint disease, however, the etiopathogenesis is still unclear. Chondrocytes rely primarily on glycolysis to meet cellular energy demand, but studies implicate impaired mitochondrial function in OA pathogenesis. The relationship between mitochondrial dysfunction and OA has been established. The aim of the study was to examine the differences in glucose and Fatty Acids (FA) metabolism, especially with regards to metabolic flexibility, in cybrids from healthy (N) or OA donors. Glucose and FA metabolism were studied using D-[14C(U)]glucose and [1-14C]oleic acid, respectively. There were no differences in glucose metabolism among the cybrids. Osteoarthritis cybrids had lower acid-soluble metabolites, reflecting incomplete FA &beta, oxidation but higher incorporation of oleic acid into triacylglycerol. Co-incubation with glucose and oleic acid showed that N but not OA cybrids increased their glucose metabolism. When treating with the mitochondrial inhibitor etomoxir, N cybrids still maintained higher glucose oxidation. Furthermore, OA cybrids had higher oxidative stress response. Combined, this indicated that N cybrids had higher metabolic flexibility than OA cybrids. Healthy donors maintained the glycolytic phenotype, whereas OA donors showed a preference towards oleic acid metabolism. Interestingly, the results indicated that cybrids from OA patients had mitochondrial impairments and reduced metabolic flexibility compared to N cybrids.

Energy balance, mitochondrial dysfunction, obesity, and insulin resistance are disrupted by metabolic inflexibility while therapeutic interventions are associated with improved glucose/lipid metabolism in skeletal muscle. Conjugated linoleic acid mixture (CLA) exhibited anti-obesity and anti-diabetic effects, however, the modulatory ability of its isomers (cis9, trans11, C9, trans10, cis12, C10) on the metabolic flexibility in skeletal muscle remains to be demonstrated. Metabolic inflexibility was induced in rat by four weeks of feeding with a high-fat diet (HFD). At the end of this period, the beneficial effects of C9 or C10 on body lipid content, energy expenditure, pro-inflammatory cytokines, glucose metabolism, and mitochondrial efficiency were examined. Moreover, oxidative stress markers, fatty acids, palmitoyletanolamide (PEA), and oleyletanolamide (OEA) contents along with peroxisome proliferator-activated receptors-alpha (PPAR&alpha, ), AKT, and adenosine monophosphate-activated protein kinase (AMPK) expression were evaluated in skeletal muscle to investigate the underlying biochemical mechanisms. The presented results indicate that C9 intake reduced mitochondrial efficiency and oxidative stress and increased PEA and OEA levels more efficiently than C10 while the anti-inflammatory activity of C10, and its regulatory efficacy on glucose homeostasis are associated with modulation of the PPAR&alpha, /AMPK/pAKT signaling pathway. Our results support the idea that the dissimilar efficacy of C9 and C10 against the HFD-induced metabolic inflexibility may be consequential to their ability to activate different molecular pathways.

Peroxisome proliferator-activated receptor (PPAR)-α activation controls hepatic lipid homeostasis, stimulating fatty acid oxidation, and adapting the metabolic response to lipid overload and storage. Here, we investigate the effect of palmitoylethanolamide (PEA), an endogenous PPAR-α ligand, in counteracting hepatic metabolic inflexibility and mitochondrial dysfunction induced by high-fat diet (HFD) in mice. Long-term PEA administration (30mg/kg/die per os) in HFD mice limited hepatic lipid accumulation, increased energy expenditure, and markedly reduced insulin resistance. In isolated liver mitochondria, we have demonstrated PEA capability to modulate mitochondrial oxidative capacity and energy efficiency, leading to the reduction of intracellular lipid accumulation and oxidative stress. Moreover, we have evaluated the effect of PEA on mitochondrial bioenergetics of palmitate-challenged HepG2 cells, using Seahorse analyzer. In vitro data showed that PEA recovered mitochondrial dysfunction and reduced lipid accumulation in insulin-resistant HepG2 cells, increasing fatty acid oxidation. Mechanistic studies showed that PEA effect on lipid metabolism was limited by AMP-activated protein kinase (AMPK) inhibition, providing evidence for a pivotal role of AMPK in PEA-induced adaptive metabolic setting. All these findings identify PEA as a modulator of hepatic lipid and glucose homeostasis, limiting metabolic inflexibility induced by nutrient overload.

Background Metabolic flexibility is the ability of an organism to switch between substrates for energy metabolism, in response to the changing nutritional state and needs of the organism. On the cellular level, metabolic flexibility revolves around the tricarboxylic acid cycle by switching acetyl coenzyme A production from glucose to fatty acids and vice versa. In this study, we modelled cellular metabolic flexibility by constructing a logical model connecting glycolysis, fatty acid oxidation, fatty acid synthesis and the tricarboxylic acid cycle, and then using network analysis to study the behaviours of the model. Results We observed that the substrate switching usually occurs through the inhibition of pyruvate dehydrogenase complex (PDC) by pyruvate dehydrogenase kinases (PDK), which moves the metabolism from glycolysis to fatty acid oxidation. Furthermore, we were able to verify four different regulatory models of PDK to contain known biological observations, leading to the biological plausibility of all four models across different cells and conditions. Conclusion These results suggest that the cellular metabolic flexibility depends upon the PDC-PDK regulatory interaction as a key regulatory switch for changing metabolic substrates.

Previously published work has demonstrated that overexpression of the insulin receptor isoform A (IR-A) might play a role in cancer progression and metastasis. The IR has a predominant metabolic role in physiology, but the potential role of IR-A in cancer metabolic reprogramming is unknown. We aimed to characterize the metabolic impact of IR-A and its ligand insulin like growth factor 2 (IGF2) in human breast cancer (BC) cells. To establish autocrine IGF2 action, we generated human BC cells MCF7 overexpressing the human IGF2, while we focused on the metabolic effect of IR-A by stably infecting IGF1R-ablated MCF7 (MCF7IGF1R-ve) cells with a human IR-A cDNA. We then evaluated the expression of key metabolism related molecules and measured real-time extracellular acidification rates and oxygen consumption rates using the Seahorse technology. MCF7/IGF2 cells showed increased proliferation and invasion associated with aerobic glycolysis and mitochondrial biogenesis and activity. In MCF7IGF1R-ve/IR-A cells insulin and IGF2 stimulated similar metabolic changes and were equipotent in eliciting proliferative responses, while IGF2 more potently induced invasion. The combined treatment with the glycolysis inhibitor 2-deoxyglucose (2DG) and the mitochondrial inhibitor metformin blocked cell invasion and colony formation with additive effects. Overall, these results indicate that IGF2 and IR-A overexpression may contribute to BC metabolic reprogramming.

Fatty acid oxidation is a central fueling pathway for mitochondrial ATP production. Regulation occurs through multiple nutrient- and energy-sensitive molecular mechanisms. We explored if upregulated mRNA expression of the mitochondrial enzyme pyruvate dehydrogenase kinase 4 (PDK4) may be used as a surrogate marker of increased mitochondrial fatty acid oxidation, by indicating an overall shift from glucose to fatty acids as the preferred oxidation fuel. The association between fatty acid oxidation and PDK4 expression was studied in different contexts of metabolic adaption. In rats treated with the modified fatty acid tetradecylthioacetic acid (TTA), Pdk4 was upregulated simultaneously with fatty acid oxidation genes in liver and heart, whereas muscle and white adipose tissue remained unaffected. In MDA-MB-231 cells, fatty acid oxidation increased nearly threefold upon peroxisome proliferator-activated receptor α (PPARα, PPARA) overexpression, and four-fold upon TTA-treatment. PDK4 expression was highly increased under these conditions. Further, overexpression of PDK4 caused increased fatty acid oxidation in these cells. Pharmacological activators of PPARα and AMPK had minor effects, while the mTOR inhibitor rapamycin potentiated the effect of TTA. There were minor changes in mitochondrial respiration, glycolytic function, and mitochondrial biogenesis under conditions of increased fatty acid oxidation. TTA was found to act as a mild uncoupler, which is likely to contribute to the metabolic effects. Repeated experiments with HeLa cells supported these findings. In summary, PDK4 upregulation implies an overarching metabolic shift towards increased utilization of fatty acids as energy fuel, and thus constitutes a sensitive marker of enhanced fatty acid oxidation. publishedVersion

The heart requires a continuous supply of energy but has little capacity for energy storage and thus relies on exogenous metabolic sources. We previously showed that cardiac MED13 modulates systemic energy homeostasis in mice. Here, we sought to define the extra-cardiac tissue(s) that respond to cardiac MED13 signaling. We show that cardiac overexpression of MED13 in transgenic (MED13cTg) mice confers a lean phenotype that is associated with increased lipid uptake, beta-oxidation and mitochondrial content in white adipose tissue (WAT) and liver. Cardiac expression of MED13 decreases metabolic gene expression in the heart but enhances them in WAT. Although exhibiting increased energy expenditure in the fed state, MED13cTg mice metabolically adapt to fasting. Furthermore, MED13cTg hearts oxidize fuel that is readily available, rendering them more efficient in the fed state. Parabiosis experiments in which circulations of wild-type and MED13cTg mice are joined, reveal that circulating factor(s) in MED13cTg mice promote enhanced metabolism and leanness. These findings demonstrate that MED13 acts within the heart to promote systemic energy expenditure in extra-cardiac energy depots and point to an unexplored metabolic communication system between the heart and other tissues. See also: M Nakamura & J Sadoshima (December 2014)

Summary The transcription factor EB (TFEB) is an essential component of lysosomal biogenesis and autophagy for the adaptive response to food deprivation. To address the physiological function of TFEB in skeletal muscle, we have used muscle-specific gain- and loss-of-function approaches. Here, we show that TFEB controls metabolic flexibility in muscle during exercise and that this action is independent of peroxisome proliferator-activated receptor-γ coactivator1α (PGC1α). Indeed, TFEB translocates into the myonuclei during physical activity and regulates glucose uptake and glycogen content by controlling expression of glucose transporters, glycolytic enzymes, and pathways related to glucose homeostasis. In addition, TFEB induces the expression of genes involved in mitochondrial biogenesis, fatty acid oxidation, and oxidative phosphorylation. This coordinated action optimizes mitochondrial substrate utilization, thus enhancing ATP production and exercise capacity. These findings identify TFEB as a critical mediator of the beneficial effects of exercise on metabolism., Graphical Abstract, Highlights • TFEB regulates mitochondrial biogenesis and function in muscle • Glucose homeostasis in skeletal muscle requires TFEB • The effects of TFEB on muscle metabolism are independent from PGC1α • TFEB coordinates metabolic flexibility during physical exercise, Mansueto et al. show that TFEB acts as a central coordinator of skeletal muscle insulin sensitivity, glucose homeostasis, lipid oxidation, and mitochondrial function in the adaptive metabolic response to physical exercise in a PGC1α-independent manner.

Obesity in humans is often associated with metabolic inflexibility, but the underlying molecular mechanisms remain incompletely understood. The aim of the present study was to investigate how adaptation to prolonged fasting affects energy/nutrient-sensing pathways and metabolic gene expression in skeletal muscle from lean and obese individuals. Twelve lean and 14 nondiabetic obese subjects were fasted for 48 h. Whole body glucose/lipid oxidation rates were determined by indirect calorimetry, and blood and skeletal muscle biopsies were collected and analyzed. In response to fasting, body weight loss was similar in both groups, but the decrease in plasma insulin and leptin and the concomitant increase in growth hormone were significantly attenuated in obese subjects. The fasting-induced shift from glucose toward lipid oxidation was also severely blunted. At the molecular level, the expression of insulin receptor-β (IRβ) was lower in skeletal muscle from obese subjects at baseline, whereas the fasting-induced reductions in insulin signaling were similar in both groups. The protein expression of mitochondrial respiratory chain components, although not modified by fasting, was significantly reduced in obese subjects. Some minor differences in metabolic gene expression were observed at baseline and in response to fasting. Surprisingly, fasting reduced AMPK activity in lean but not in obese subjects, whereas the expression of AMPK subunits was not affected. We conclude that whole body metabolic inflexibility in response to prolonged fasting in obese humans is associated with lower skeletal muscle IRβ and mitochondrial respiratory chain content as well as a blunted decline of AMPK activity.

Corpeleijn E, Hessvik NP, Bakke SS, Levin K, Blaak EE, Thoresen GH, Gaster M, Rustan AC. Oxidation of intramyocellular lipids is dependent on mitochondrial function and the availability of extracellular fatty acids. Am J Physiol Endocrinol Metab 299: E14-E22, 2010. First published May 4, 2010; doi:10.1152/ajpendo.00187.2010.-Obesity and insulin resistance are related to both enlarged intramyocellular triacylglycerol stores and accumulation of lipid intermediates. We investigated how lipid overflow can change the oxidation of intramyocellular lipids (ICLOX) and intramyocellular lipid storage (ICL). These experiments were extended by comparing these processes in primary cultured myotubes established from healthy lean and obese type 2 diabetic (T2D) individuals, two extremes in a range of metabolic phenotypes. ICLs were prelabeled for 2 days with 100 mu M [C-14] oleic acid (OA). ICLOX was studied using a (CO2)-C-14 trapping system and measured under various conditions of extracellular OA (5 or 100 mu M) and glucose (0.1 or 5.0 mM) and the absence or presence of mitochondrial uncoupling [carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP)]. First, increased extracellular OA availability (5 vs. 100 mu M) reduced ICLOX by 37%. No differences in total lipolysis were observed between low and high OA availability. Uncoupling with FCCP restored ICLOX to basal levels during high OA availability. Mitochondrial mass was positively related to ICLOX, but only in myotubes from lean individuals. In all, a lower mitochondrial mass and lower ICLOX were related to a higher cell-associated OA accumulation. Second, myotubes established from obese T2D individuals showed reduced ICLOX. ICLOX remained lower during uncoupling (P

Inflammation is a central feature of various metabolic diseases including obesity and type-II diabetes. For this study, we hypothesized postprandial metabolism following an acute, high fat (HF) meal to be impaired in mice pre-injected with an inflammatory agonist. To this end, C57BL/6J mice were injected with saline or lipopolysaccharide (LPS, 1μg/kgbw) following an overnight fast and gavaged 2hr post-injection with water or a HF meal in liquid form (5kcal; 21.4%SF, 40.8%UF, 27.1%CHO, 10.7%PRO). Blood and muscle samples taken 3hr post-gavage underwent ex vivo analysis. Overall, results demonstrated a metabolic response to a HF meal that was blocked in the presence of LPS. Metabolic flexibility, though unchanged following the HF meal alone, was reduced following the HF meal in the presence of LPS. Additionally, state-4 uncoupled mitochondrial respiration, which was increased following the HF meal, was also reduced following the HF meal in the presence of LPS. Similar near-significant trends were also observed with total palmitate oxidation. Although no independent response to a HF meal or LPS exposure was observed, a unique interaction between treatments significantly diminished ADP dependent, state-3 and maximal respiration. These effects do not appear to be dependent on the production of reactive oxygen species (ROS) since neither the HF meal nor LPS exposure resulted in increased production of ROS. In conclusion, these results demonstrate that acute activation of inflammatory pathways results in alterations in metabolic response to a HF meal in skeletal muscle from mice, although the mechanism underlying these effects is not yet understood. Master of Science

Nitrate and ammonium constitute primary inorganic nitrogen sources that can be incorporated into carbon skeletons in photosynthetic eukaryotes. In Chlamydomonas, previous studies and the present one showed that the mitochondrial AOX is up-regulated in nitrate-grown cells in comparison with ammonium-grown cells. In this work, we have performed a comparative proteomic analysis of the soluble mitochondrial proteome of Chlamydomonas cells growth either on nitrate or ammonium. Our results highlight important proteomics modifications mostly related to primary metabolism in cells grown on nitrate. We could note an up-regulation of some TCA cycle enzymes and a down-regulation of cytochrome c1 together with an up-regulation of l-arginine and purine catabolism enzymes and of ROS scavenging systems. Hence, in nitrate-grown cells, AOX may play a dual role: (1) lowering the ubiquinone pool reduction level and (2) permitting the export of mitochondrial reducing power under the form of malate for nitrate and nitrite reduction. This role of AOX in the mitochondrial plasticity makes logical the localization of Aox1 in a nitrate assimilation gene cluster.