Complex I activity in hypoxia: implications for oncometabolism.

Certain cancer cells within solid tumors experience hypoxia, rendering them incapable of oxidative phosphorylation (OXPHOS). Despite this oxygen deficiency, these cells exhibit biochemical pathway activity that relies on NAD+. This mini-review scrutinizes the persistent, residual Complex I activity that oxidizes NADH in the absence of oxygen as the electron acceptor. The resulting NAD+ assumes a pivotal role in fueling the α-ketoglutarate dehydrogenase complex, a critical component in the oxidative decarboxylation branch of glutaminolysis - a hallmark oncometabolic pathway. The proposition is that through glutamine catabolism, high-energy phosphate intermediates are produced via substrate-level phosphorylation in the mitochondrial matrix substantiated by succinyl-CoA ligase, partially compensating for an OXPHOS deficiency. These insights provide a rationale for exploring Complex I inhibitors in cancer treatment, even when OXPHOS functionality is already compromised.

Viability of HepG2 and MCF-7 cells is not correlated with mitochondrial bioenergetics.

Alterations in metabolism are a hallmark of cancer. It is unclear if oxidative phosphorylation (OXPHOS) is necessary for tumour cell survival. In this study, we investigated the effects of severe hypoxia, site-specific inhibition of respiratory chain (RC) components, and uncouplers on necrotic and apoptotic markers in 2D-cultured HepG2 and MCF-7 tumour cells. Comparable respiratory complex activities were observed in both cell lines. However, HepG2 cells exhibited significantly higher oxygen consumption rates (OCR) and respiratory capacity than MCF-7 cells. Significant non-mitochondrial OCR was observed in MCF-7 cells, which was insensitive to acute combined inhibition of complexes I and III. Pre-treatment of either cell line with RC inhibitors for 24-72 h resulted in the complete abolition of respective complex activities and OCRs. This was accompanied by a time-dependent decrease in citrate synthase activity, suggesting mitophagy. High-content automated microscopy recordings revealed that the viability of HepG2 cells was mostly unaffected by any pharmacological treatment or severe hypoxia. In contrast, the viability of MCF-7 cells was strongly affected by inhibition of complex IV (CIV) or complex V (CV), severe hypoxia, and uncoupling. However, it was only moderately affected by inhibition of complexes I, II, and III. Cell death in MCF-7 cells induced by inhibition of complexes II, III, and IV was partially abrogated by aspartate. These findings indicate that OXPHOS activity and viability are not correlated in these cell lines, suggesting that the connection between OXPHOS and cancer cell survival is dependent on the specific cell type and conditions.

Metabolic management of microenvironment acidity in glioblastoma.

Glioblastoma (GBM), similar to most cancers, is dependent on fermentation metabolism for the synthesis of biomass and energy (ATP) regardless of the cellular or genetic heterogeneity seen within the tumor. The transition from respiration to fermentation arises from the documented defects in the number, the structure, and the function of mitochondria and mitochondrial-associated membranes in GBM tissue. Glucose and glutamine are the major fermentable fuels that drive GBM growth. The major waste products of GBM cell fermentation (lactic acid, glutamic acid, and succinic acid) will acidify the microenvironment and are largely responsible for drug resistance, enhanced invasion, immunosuppression, and metastasis. Besides surgical debulking, therapies used for GBM management (radiation, chemotherapy, and steroids) enhance microenvironment acidification and, although often providing a time-limited disease control, will thus favor tumor recurrence and complications. The simultaneous restriction of glucose and glutamine, while elevating non-fermentable, anti-inflammatory ketone bodies, can help restore the pH balance of the microenvironment while, at the same time, providing a non-toxic therapeutic strategy for killing most of the neoplastic cells.

Proline Oxidation Supports Mitochondrial ATP Production When Complex I Is Inhibited.

The oxidation of proline to pyrroline-5-carboxylate (P5C) leads to the transfer of electrons to ubiquinone in mitochondria that express proline dehydrogenase (ProDH). This electron transfer supports Complexes CIII and CIV, thus generating the protonmotive force. Further catabolism of P5C forms glutamate, which fuels the citric acid cycle that yields the reducing equivalents that sustain oxidative phosphorylation. However, P5C and glutamate catabolism depend on CI activity due to NAD+ requirements. NextGen-O2k (Oroboros Instruments) was used to measure proline oxidation in isolated mitochondria of various mouse tissues. Simultaneous measurements of oxygen consumption, membrane potential, NADH, and the ubiquinone redox state were correlated to ProDH activity and F1FO-ATPase directionality. Proline catabolism generated a sufficiently high membrane potential that was able to maintain the F1FO-ATPase operation in the forward mode. This was observed in CI-inhibited mouse liver and kidney mitochondria that exhibited high levels of proline oxidation and ProDH activity. This action was not observed under anoxia or when either CIII or CIV were inhibited. The duroquinone fueling of CIII and CIV partially reproduced the effects of proline. Excess glutamate, however, could not reproduce the proline effect, suggesting that processes upstream of the glutamate conversion from proline were involved. The ProDH inhibitors tetrahydro-2-furoic acid and, to a lesser extent, S-5-oxo-2-tetrahydrofurancarboxylic acid abolished all proline effects. The data show that ProDH-directed proline catabolism could generate sufficient CIII and CIV proton pumping, thus supporting ATP production by the F1FO-ATPase even under CI inhibition.

Can the Mitochondrial Metabolic Theory Explain Better the Origin and Management of Cancer than Can the Somatic Mutation Theory?

A theory that can best explain the facts of a phenomenon is more likely to advance knowledge than a theory that is less able to explain the facts. Cancer is generally considered a genetic disease based on the somatic mutation theory (SMT) where mutations in proto-oncogenes and tumor suppressor genes cause dysregulated cell growth. Evidence is reviewed showing that the mitochondrial metabolic theory (MMT) can better account for the hallmarks of cancer than can the SMT. Proliferating cancer cells cannot survive or grow without carbons and nitrogen for the synthesis of metabolites and ATP (Adenosine Triphosphate). Glucose carbons are essential for metabolite synthesis through the glycolysis and pentose phosphate pathways while glutamine nitrogen and carbons are essential for the synthesis of nitrogen-containing metabolites and ATP through the glutaminolysis pathway. Glutamine-dependent mitochondrial substrate level phosphorylation becomes essential for ATP synthesis in cancer cells that over-express the glycolytic pyruvate kinase M2 isoform (PKM2), that have deficient OxPhos, and that can grow in either hypoxia (0.1% oxygen) or in cyanide. The simultaneous targeting of glucose and glutamine, while elevating levels of non-fermentable ketone bodies, offers a simple and parsimonious therapeutic strategy for managing most cancers.

Bioenergetic consequences of FoF1-ATP synthase/ATPase deficiency in two life cycle stages of Trypanosoma brucei.

Mitochondrial ATP synthase is a reversible nanomotor synthesizing or hydrolyzing ATP depending on the potential across the membrane in which it is embedded. In the unicellular parasite Trypanosoma brucei, the direction of the complex depends on the life cycle stage of this digenetic parasite: in the midgut of the tsetse fly vector (procyclic form), the FoF1-ATP synthase generates ATP by oxidative phosphorylation, whereas in the mammalian bloodstream form, this complex hydrolyzes ATP and maintains mitochondrial membrane potential (ΔΨm). The trypanosome FoF1-ATP synthase contains numerous lineage-specific subunits whose roles remain unknown. Here, we seek to elucidate the function of the lineage-specific protein Tb1, the largest membrane-bound subunit. In procyclic form cells, Tb1 silencing resulted in a decrease of FoF1-ATP synthase monomers and dimers, rerouting of mitochondrial electron transfer to the alternative oxidase, reduced growth rate and cellular ATP levels, and elevated ΔΨm and total cellular reactive oxygen species levels. In bloodstream form parasites, RNAi silencing of Tb1 by ∼90% resulted in decreased FoF1-ATPase monomers and dimers, but it had no apparent effect on growth. The same findings were obtained by silencing of the oligomycin sensitivity-conferring protein, a conserved subunit in T. brucei FoF1-ATP synthase. However, as expected, nearly complete Tb1 or oligomycin sensitivity-conferring protein suppression was lethal because of the inability to sustain ΔΨm. The diminishment of FoF1-ATPase complexes was further accompanied by a decreased ADP/ATP ratio and reduced oxygen consumption via the alternative oxidase. Our data illuminate the often diametrically opposed bioenergetic consequences of FoF1-ATP synthase loss in insect versus mammalian forms of the parasite.

From Glucose to Lactate and Transiting Intermediates Through Mitochondria, Bypassing Pyruvate Kinase: Considerations for Cells Exhibiting Dimeric PKM2 or Otherwise Inhibited Kinase Activity.

A metabolic hallmark of many cancers is the increase in glucose consumption coupled to excessive lactate production. Mindful that L-lactate originates only from pyruvate, the question arises as to how can this be sustained in those tissues where pyruvate kinase activity is reduced due to dimerization of PKM2 isoform or inhibited by oxidative/nitrosative stress, posttranslational modifications or mutations, all widely reported findings in the very same cells. Hereby 17 pathways connecting glucose to lactate bypassing pyruvate kinase are reviewed, some of which transit through the mitochondrial matrix. An additional 69 converging pathways leading to pyruvate and lactate, but not commencing from glucose, are also examined. The minor production of pyruvate and lactate by glutaminolysis is scrutinized separately. The present review aims to highlight the ways through which L-lactate can still be produced from pyruvate using carbon atoms originating from glucose or other substrates in cells with kinetically impaired pyruvate kinase and underscore the importance of mitochondria in cancer metabolism irrespective of oxidative phosphorylation.

On the Origin of ATP Synthesis in Cancer.

ATP is required for mammalian cells to remain viable and to perform genetically programmed functions. Maintenance of the ΔG'ATP hydrolysis of -56 kJ/mole is the endpoint of both genetic and metabolic processes required for life. Various anomalies in mitochondrial structure and function prevent maximal ATP synthesis through OxPhos in cancer cells. Little ATP synthesis would occur through glycolysis in cancer cells that express the dimeric form of pyruvate kinase M2. Mitochondrial substrate level phosphorylation (mSLP) in the glutamine-driven glutaminolysis pathway, substantiated by the succinate-CoA ligase reaction in the TCA cycle, can partially compensate for reduced ATP synthesis through both OxPhos and glycolysis. A protracted insufficiency of OxPhos coupled with elevated glycolysis and an auxiliary, fully operational mSLP, would cause a cell to enter its default state of unbridled proliferation with consequent dedifferentiation and apoptotic resistance, i.e., cancer. The simultaneous restriction of glucose and glutamine offers a therapeutic strategy for managing cancer.

Consideration of Ketogenic Metabolic Therapy as a Complementary or Alternative Approach for Managing Breast Cancer.

Breast cancer remains as a significant cause of morbidity and mortality in women. Ultrastructural and biochemical evidence from breast biopsy tissue and cancer cells shows mitochondrial abnormalities that are incompatible with energy production through oxidative phosphorylation (OxPhos). Consequently, breast cancer, like most cancers, will become more reliant on substrate level phosphorylation (fermentation) than on oxidative phosphorylation (OxPhos) for growth consistent with the mitochondrial metabolic theory of cancer. Glucose and glutamine are the prime fermentable fuels that underlie therapy resistance and drive breast cancer growth through substrate level phosphorylation (SLP) in both the cytoplasm (Warburg effect) and the mitochondria (Q-effect), respectively. Emerging evidence indicates that ketogenic metabolic therapy (KMT) can reduce glucose availability to tumor cells while simultaneously elevating ketone bodies, a non-fermentable metabolic fuel. It is suggested that KMT would be most effective when used together with glutamine targeting. Information is reviewed for suggesting how KMT could reduce systemic inflammation and target tumor cells without causing damage to normal cells. Implementation of KMT in the clinic could improve progression free and overall survival for patients with breast cancer.

Glutaminases as a Novel Target for SDHB-Associated Pheochromocytomas/Paragangliomas.

Pheochromocytoma/paragangliomas (Pheo/PGL) are rare endocrine cancers with strong genetic background. Mutations in the SDHB subunit of succinate dehydrogenase (SDH) predispose patients to malignant disease with limited therapeutic options and poor prognosis. Using a host of cellular and molecular biology techniques in 2D and 3D cell culture formats we show that SDH inhibition had cell line specific biological and biochemical consequences. Based on our studies performed on PC12 (rat chromaffin cell line), Hela (human cervix epithelial cell line), and H295R (human adrenocortical cell line) cells, we demonstrated that chromaffin cells were not affected negatively by the inhibition of SDH either by siRNA directed against SDHB or treatment with SDH inhibitors (itaconate and atpenin A5). Cell viability and intracellular metabolite measurements pointed to the cell line specific consequences of SDH impairment and to the importance of glutamate metabolism in chromaffin cells. A significant increase in glutaminase-1 (GLS-1) expression after SDH impairment was observed in PC12 cells. GLS-1 inhibitor BPTES was capable of significantly decreasing proliferation of SDH impaired PC12 cells. Glutaminase-1 and SDHB expressions were tested in 35 Pheo/PGL tumor tissues. Expression of GLS1 was higher in the SDHB low expressed group compared to SDHB high expressed tumors. Our data suggest that the SDH-associated malignant potential of Pheo/PGL is strongly dependent on GLS-1 expression and glutaminases may be novel targets for therapy.

Exclusive neuronal detection of KGDHC-specific subunits in the adult human brain cortex despite pancellular protein lysine succinylation.

The ketoglutarate dehydrogenase complex (KGDHC) consists of three different subunits encoded by OGDH (or OGDHL), DLST, and DLD, combined in different stoichiometries. DLD subunit is shared between KGDHC and pyruvate dehydrogenase complex, branched-chain alpha-keto acid dehydrogenase complex, and the glycine cleavage system. Despite KGDHC's implication in neurodegenerative diseases, cell-specific localization of its subunits in the adult human brain has never been investigated. Here, we show that immunoreactivity of all known isoforms of OGDHL, OGDH, and DLST was detected exclusively in neurons of surgical human cortical tissue samples identified by their morphology and visualized by double labeling with fluorescent Nissl, while being absent from glia expressing GFAP, Aldhl1, myelin basic protein, Olig2, or IBA1. In contrast, DLD immunoreactivity was evident in both neurons and glia. Specificity of anti-KGDHC subunits antisera was verified by a decrease in staining of siRNA-treated human cancer cell lines directed against the respective coding gene products; furthermore, immunoreactivity of KGDHC subunits in human fibroblasts co-localized > 99% with mitotracker orange, while western blotting of 63 post-mortem brain samples and purified recombinant proteins afforded further assurance regarding antisera monospecificity. KGDHC subunit immunoreactivity correlated with data from the Human Protein Atlas as well as RNA-Seq data from the Allen Brain Atlas corresponding to genes coding for KGDHC components. Protein lysine succinylation, however, was immunohistochemically evident in all cortical cells; this was unexpected, because this posttranslational modification requires succinyl-CoA, the product of KGDHC. In view of the fact that glia of the human brain cortex lack succinate-CoA ligase, an enzyme producing succinyl-CoA when operating in reverse, protein lysine succinylation in these cells must exclusively rely on propionate and/or ketone body metabolism or some other yet to be discovered pathway encompassing succinyl-CoA.

Therapeutic benefit of combining calorie-restricted ketogenic diet and glutamine targeting in late-stage experimental glioblastoma.

Glioblastoma (GBM) is an aggressive primary human brain tumour that has resisted effective therapy for decades. Although glucose and glutamine are the major fuels that drive GBM growth and invasion, few studies have targeted these fuels for therapeutic management. The glutamine antagonist, 6-diazo-5-oxo-L-norleucine (DON), was administered together with a calorically restricted ketogenic diet (KD-R) to treat late-stage orthotopic growth in two syngeneic GBM mouse models: VM-M3 and CT-2A. DON targets glutaminolysis, while the KD-R reduces glucose and, simultaneously, elevates neuroprotective and non-fermentable ketone bodies. The diet/drug therapeutic strategy killed tumour cells while reversing disease symptoms, and improving overall mouse survival. The therapeutic strategy also reduces edema, hemorrhage, and inflammation. Moreover, the KD-R diet facilitated DON delivery to the brain and allowed a lower dosage to achieve therapeutic effect. The findings support the importance of glucose and glutamine in driving GBM growth and provide a therapeutic strategy for non-toxic metabolic management.

The RCR and ATP/O Indices Can Give Contradictory Messages about Mitochondrial Efficiency.

Mitochondrial efficiency is typically taken to represent an animal's capacity to convert its resources into ATP. However, the term mitochondrial efficiency, as currently used in the literature, can be calculated as either the respiratory control ratio, RCR (ratio of mitochondrial respiration supporting ATP synthesis to that required to offset the proton leak) or as the amount of ATP generated per unit of oxygen consumed, ATP/O ratio. The question of how flexibility in mitochondrial energy properties (i.e., in rates of respiration to support ATP synthesis and offset proton leak, and in the rate of ATP synthesis) affects these indices of mitochondrial efficiency has tended to be overlooked. Furthermore, little is known of whether the RCR and ATP/O ratio vary in parallel, either among individuals or in response to environmental conditions. Using data from brown trout Salmo trutta we show that experimental conditions affect mitochondrial efficiency, but the apparent direction of change depends on the index chosen: a reduction in food availability was associated with an increased RCR (i.e., increased efficiency) but a decreased ATP/O ratio (decreased efficiency) in liver mitochondria. Moreover, there was a negative correlation across individuals held in identical conditions between their RCR and their ATP/O ratio. These results show that the choice of index of mitochondrial efficiency can produce different, even opposing, conclusions about the capacity of the mitochondria to produce ATP. Neither ratio is necessarily a complete measure of efficiency of ATP production in the living animal (RCR because it contains no assessment of ATP production, and ATP/O because it contains no assessment of respiration to offset the proton leak). Consequently, we suggest that a measure of mitochondrial efficiency obtained nearer to conditions where respiration simultaneously offsets the proton leak and produce ATP would be sensitive to changes in both proton leakage and ATP production, and is thus likely to be more representative of the state of the mitochondria in vivo.

Functional cyclophilin D moderates platelet adhesion, but enhances the lytic resistance of fibrin.

In the course of thrombosis, platelets are exposed to a variety of activating stimuli classified as 'strong' (e.g. thrombin and collagen) or 'mild' (e.g. ADP). In response, activated platelets adhere to injured vasculature, aggregate, and stabilise the three-dimensional fibrin scaffold of the expanding thrombus. Since 'strong' stimuli also induce opening of the mitochondrial permeability transition pore (MPTP) in platelets, the MPTP-enhancer Cyclophilin D (CypD) has been suggested as a critical pharmacological target to influence thrombosis. However, it is poorly understood what role CypD plays in the platelet response to 'mild' stimuli which act independently of MPTP. Furthermore, it is unknown how CypD influences platelet-driven clot stabilisation against enzymatic breakdown (fibrinolysis). Here we show that treatment of human platelets with Cyclosporine A (a cyclophilin-inhibitor) boosts ADP-induced adhesion and aggregation, while genetic ablation of CypD in murine platelets enhances adhesion but not aggregation. We also report that platelets lacking CypD preserve their integrity in a fibrin environment, and lose their ability to render clots resistant against fibrinolysis. Our results indicate that CypD has opposing haemostatic roles depending on the stimulus and stage of platelet activation, warranting a careful design of any antithrombotic strategy targeting CypD.

Mycoplasma infection and hypoxia initiate succinate accumulation and release in the VM-M3 cancer cells.

Succinate is known to act as an inflammatory signal in classically activated macrophages through stabilization of HIF-1α leading to IL-1ß production. Relevant to this, hypoxia is known to drive succinate accumulation and release into the extracellular milieu. The metabolic alterations associated with succinate release during inflammation and under hypoxia are poorly understood. Data are presented showing that Mycoplasma arginini infection of VM-M3 cancer cells enhances the Warburg effect associated with succinate production in mitochondria and eventual release into the extracellular milieu. We investigated how succinate production and release was related to the changes of other soluble metabolites, including itaconate and 2-HG. Furthermore, we found that hypoxia alone could induce succinate release from the VM-M3 cells and that this could occur in the absence of glucose-driven lactate production. Our results elucidate metabolic pathways responsible for succinate accumulation and release in cancer cells, thus identifying potential targets involved in both inflammation and hypoxia. This article is part of a Special Issue entitled 20th European Bioenergetics Conference, edited by László Zimányi and László Tretter.

Mitochondrial Substrate-Level Phosphorylation as Energy Source for Glioblastoma: Review and Hypothesis.

Glioblastoma multiforme (GBM) is the most common and malignant of the primary adult brain cancers. Ultrastructural and biochemical evidence shows that GBM cells exhibit mitochondrial abnormalities incompatible with energy production through oxidative phosphorylation (OxPhos). Under such conditions, the mitochondrial F0-F1 ATP synthase operates in reverse at the expense of ATP hydrolysis to maintain a moderate membrane potential. Moreover, expression of the dimeric M2 isoform of pyruvate kinase in GBM results in diminished ATP output, precluding a significant ATP production from glycolysis. If ATP synthesis through both glycolysis and OxPhos was impeded, then where would GBM cells obtain high-energy phosphates for growth and invasion? Literature is reviewed suggesting that the succinate-CoA ligase reaction in the tricarboxylic acid cycle can substantiate sufficient ATP through mitochondrial substrate-level phosphorylation (mSLP) to maintain GBM growth when OxPhos is impaired. Production of high-energy phosphates would be supported by glutaminolysis-a hallmark of GBM metabolism-through the sequential conversion of glutamine → glutamate → alpha-ketoglutarate → succinyl CoA → succinate. Equally important, provision of ATP through mSLP would maintain the adenine nucleotide translocase in forward mode, thus preventing the reverse-operating F0-F1 ATP synthase from depleting cytosolic ATP reserves. Because glucose and glutamine are the primary fuels driving the rapid growth of GBM and most tumors for that matter, simultaneous restriction of these two substrates or inhibition of mSLP should diminish cancer viability, growth, and invasion.

Divalent cation chelators citrate and EDTA unmask an intrinsic uncoupling pathway in isolated mitochondria.

We demonstrate a suppression of ROS production and uncoupling of mitochondria by exogenous citrate in Mg2+ free medium. Exogenous citrate suppressed H2O2 emission and depolarized mitochondria. The depolarization was paralleled by the stimulation of respiration of mitochondria. The uncoupling action of citrate was independent of the presence of sodium, potassium, or chlorine ions, and it was not mediated by the changes in permeability of the inner mitochondrial membrane to solutes. The citrate transporter was not involved in the citrate effect. Inhibitory analysis data indicated that several well described mitochondria carriers and channels (ATPase, IMAC, ADP/ATP translocase, mPTP, mKATP) were not involved in citrate's effect. Exogenous MgCl2 strongly inhibited citrate-induced depolarization. The uncoupling effect of citrate was demonstrated in rat brain, mouse brain, mouse liver, and human melanoma cells mitochondria. We interpreted the data as an evidence to the existence of a hitherto undescribed putative inner mitochondrial membrane channel that is regulated by extramitochondrial Mg2+ or other divalent cations.

Simultaneous measurement of mitochondrial respiration and ATP production in tissue homogenates and calculation of effective P/O ratios.

The use of tissue homogenate has greatly aided the study of the functioning of mitochondria. However, the amount of ATP produced per oxygen molecule consumed, that is, the effective P/O ratio, has never been measured directly in tissue homogenate. Here we combine and refine existing methods previously used in permeabilized cells and isolated mitochondria to simultaneously measure mitochondrial ATP production (JATP) and oxygen consumption (JO2) in tissue homogenate. A major improvement over existing methods is in the control of ATPases that otherwise interfere with the ATP assay: our modified technique facilitates simultaneous measurement of the rates of "uncorrected" ATP synthesis and of ATP hydrolysis, thus minimizing the amount of tissue and time needed. Finally, we develop a novel method of calculating effective P/O ratios which corrects measurements of JATP and JO2 for rates of nonmitochondrial ATP hydrolysis and respiration, respectively. Measurements of JATP and JO2 in liver homogenates from brown trout (Salmo trutta) were highly reproducible, although activity declined once homogenates were 2 h old. We compared mitochondrial properties from fed and food-deprived animals to demonstrate that the method can detect mitochondrial flexibility in P/O ratios in response to nutritional state. This method simplifies studies examining the mitochondrial bioenergetics of tissue homogenates, obviating the need for differential centrifugation or chemical permeabilization and avoiding the use of nonmitochondrial ATPase inhibitors. We conclude that our approach for characterizing effective P/O ratio opens up new possibilities in the study of mitochondrial function in very small samples, where the use of other methods is limited.

The total and mitochondrial lipidome of Artemia franciscana encysted embryos.

Encysted embryos (cysts) of the crustacean Artemia franciscana exhibit enormous tolerance to adverse conditions encompassing high doses of radiation, years of anoxia, desiccation and extreme salinity. So far, several mechanisms have been proposed to contribute to this extremophilia, however, none were sought in the lipid profile of the cysts. Here in, we used high resolution shotgun lipidomics suited for detailed quantitation and analysis of lipids in uncharacterized biological membranes and samples and assembled the total, mitochondrial and mitoplastic lipidome of Artemia franciscana cysts. Overall, we identified and quantitated 1098 lipid species dispersed among 22 different classes and subclasses. Regarding the mitochondrial lipidome, most lipid classes exhibited little differences from those reported in other animals, however, Artemia mitochondria harboured much less phosphatidylethanolamine, plasmenylethanolamines and ceramides than mitochondria of other species, some of which by two orders of magnitude. Alternatively, Artemia mitochondria exhibited much higher levels of phosphatidylglycerols and phosphatidylserines. The identification and quantitation of the total and mitochondrial lipidome of the cysts may help in the elucidation of actionable extremophilia-affording proteins, such as the 'late embryogenesis abundant' proteins, which are known to interact with lipid membranes.