A Ca2+-Dependent Mechanism Boosting Glycolysis and OXPHOS by Activating Aralar-Malate-Aspartate Shuttle, upon Neuronal Stimulation.

Calcium is an important second messenger regulating a bioenergetic response to the workloads triggered by neuronal activation. In embryonic mouse cortical neurons using glucose as only fuel, activation by NMDA elicits a strong workload (ATP demand)-dependent on Na+ and Ca2+ entry, and stimulates glucose uptake, glycolysis, pyruvate and lactate production, and oxidative phosphorylation (OXPHOS) in a Ca2+-dependent way. We find that Ca2+ upregulation of glycolysis, pyruvate levels, and respiration, but not glucose uptake, all depend on Aralar/AGC1/Slc25a12, the mitochondrial aspartate-glutamate carrier, component of the malate-aspartate shuttle (MAS). MAS activation increases glycolysis, pyruvate production, and respiration, a process inhibited in the presence of BAPTA-AM, suggesting that the Ca2+ binding motifs in Aralar may be involved in the activation. Mitochondrial calcium uniporter (MCU) silencing had no effect, indicating that none of these processes required MCU-dependent mitochondrial Ca2+ uptake. The neuronal respiratory response to carbachol was also dependent on Aralar, but not on MCU. We find that mouse cortical neurons are endowed with a constitutive ER-to-mitochondria Ca2+ flow maintaining basal cell bioenergetics in which ryanodine receptors, RyR2, rather than InsP3R, are responsible for Ca2+ release, and in which MCU does not participate. The results reveal that, in neurons using glucose, MCU does not participate in OXPHOS regulation under basal or stimulated conditions, while Aralar-MAS appears as the major Ca2+-dependent pathway tuning simultaneously glycolysis and OXPHOS to neuronal activation.SIGNIFICANCE STATEMENT Neuronal activation increases cell workload to restore ion gradients altered by activation. Ca2+ is involved in matching increased workload with ATP production, but the mechanisms are still unknown. We find that glycolysis, pyruvate production, and neuronal respiration are stimulated on neuronal activation in a Ca2+-dependent way, independently of effects of Ca2+ as workload inducer. Mitochondrial calcium uniporter (MCU) does not play a relevant role in Ca2+ stimulated pyruvate production and oxygen consumption as both are unchanged in MCU silenced neurons. However, Ca2+ stimulation is blunt in the absence of Aralar, a Ca2+-binding mitochondrial carrier component of Malate-Aspartate Shuttle (MAS). The results suggest that Ca2+-regulated Aralar-MAS activation upregulates glycolysis and pyruvate production, which fuels mitochondrial respiration, through regulation of cytosolic NAD+/NADH ratio.

Bioenergetic changes in response to sperm capacitation and two-way metabolic compensation in a new murine model.

The acquisition of fertilizing ability by mammalian spermatozoa, known as "capacitation," includes processes that depend on particular metabolic pathways. This has led to the hypothesis that ATP demands might differ between capacitated and non-capacitated cells. Mouse sperm can produce ATP via OXPHOS and aerobic glycolysis, an advantageous characteristic considering that these cells have to function in the complex and variable environment of the female reproductive tract. Nonetheless, despite evidence showing that both metabolic pathways play a role in events associated with mouse sperm capacitation, there is contradictory evidence regarding changes promoted by capacitation in this species. In addition, the vast majority of studies regarding murine sperm metabolism use Mus musculus laboratory strains as model, thus neglecting the wide diversity of sperm traits of other species of Mus. Focus on closely related species with distinct evolutionary histories, which may be the result of different selective pressures, could shed light on diversity of metabolic processes. Here, we analyzed variations in sperm bioenergetics associated with capacitation in spermatozoa of the steppe mouse, Mus spicilegus, a species with high sperm performance. Furthermore, we compared sperm metabolic traits of this species with similar traits previously characterized in M. musculus. We found that the metabolism of M. spicilegus sperm responded to capacitation in a manner similar to that of M. musculus sperm. However, M. spicilegus sperm showed distinct metabolic features, including the ability to perform cross-pathway metabolic compensation in response to either respiratory or glycolytic inhibition, thus revealing a delicate fine-tuning of its metabolic capacities.

Role of UCP2 in the Energy Metabolism of the Cancer Cell Line A549.

The uncoupling protein UCP2 is a mitochondrial carrier for which transport activity remains controversial. The physiological contexts in which UCP2 is expressed have led to the assumption that, like UCP1, it uncouples oxidative phosphorylation and thereby reduces the generation of reactive oxygen species. Other reports have involved UCP2 in the Warburg effect, and results showing that UCP2 catalyzes the export of matrix C4 metabolites to facilitate glutamine utilization suggest that the carrier could be involved in the metabolic adaptations required for cell proliferation. We have examined the role of UCP2 in the energy metabolism of the lung adenocarcinoma cell line A549 and show that UCP2 silencing decreased the basal rate of respiration, although this inhibition was not compensated by an increase in glycolysis. Silencing did not lead to either changes in proton leakage, as determined by the rate of respiration in the absence of ATP synthesis, or changes in the rate of formation of reactive oxygen species. The decrease in energy metabolism did not alter the cellular energy charge. The decreased cell proliferation observed in UCP2-silenced cells would explain the reduced cellular ATP demand. We conclude that UCP2 does not operate as an uncoupling protein, whereas our results are consistent with its activity as a C4-metabolite carrier involved in the metabolic adaptations of proliferating cells.

Reproductive senescence impairs the energy metabolism of human luteinized granulosa cells.

RESEARCH QUESTION: Female age is the single greatest factor influencing reproductive performance and granulosa cells are considered as potential biomarkers of oocyte quality. Is there an age effect on the energy metabolism of human mural granulosa cells? DESIGN: Observational prospective cohort and experimental study including 127 women who had undergone IVF cycles. Women were allocated to two groups: a group of infertile patients aged over 38 years and a control group comprising oocyte donors aged less than 35 years. Individuals with pathologies that could impair fertility were excluded from both groups. Following oocyte retrieval, cumulus and granulosa cells were isolated and their bioenergetic properties (oxidative phosphorylation parameters, rate of aerobic glycolysis and adenine nucleotide concentrations) were analysed and compared. RESULTS: Human mural luteinized granulosa and cumulus cells present high rates of aerobic glycolysis that cannot be increased further when mitochondrial ATP synthesis is inhibited. Addition of follicular fluid to the experimental media is necessary to reach the full respiratory capacity of the cells. Granulosa cells from aged women present lower mitochondrial respiration (12.8 ± 1.6 versus 11.2 ± 1.6 pmol O2/min/mg; P = 0.046), although mitochondrial mass is not decreased, and lower aerobic glycolysis, than those from young donors (12.9 ± 1.3 versus 10.9 ± 0.5 mpH/min/mg; P = 0.009). The concurrent decrease in the two energy supply pathways leads to a decrease in the cellular energy charge (0.87 ± 0.01 versus 0.83 ± 0.02; P < 0.001). CONCLUSIONS: Human mural luteinized granulosa cells exhibit a reduction in their energy metabolism as women age that is likely to influence female reproductive potential.

c-Met Signaling Is Essential for Mouse Adult Liver Progenitor Cells Expansion After Transforming Growth Factor-ß-Induced Epithelial-Mesenchymal Transition and Regulates Cell Phenotypic Switch.

Adult hepatic progenitor cells (HPCs)/oval cells are bipotential progenitors that participate in liver repair responses upon chronic injury. Recent findings highlight HPCs plasticity and importance of the HPCs niche signals to determine their fate during the regenerative process, favoring either fibrogenesis or damage resolution. Transforming growth factor-ß (TGF-ß) and hepatocyte growth factor (HGF) are among the key signals involved in liver regeneration and as component of HPCs niche regulates HPCs biology. Here, we characterize the TGF-ß-triggered epithelial-mesenchymal transition (EMT) response in oval cells, its effects on cell fate in vivo, and the regulatory effect of the HGF/c-Met signaling. Our data show that chronic treatment with TGF-ß triggers a partial EMT in oval cells based on coexpression of epithelial and mesenchymal markers. The phenotypic and functional profiling indicates that TGF-ß-induced EMT is not associated with stemness but rather represents a step forward along hepatic lineage. This phenotypic transition confers advantageous traits to HPCs including survival, migratory/invasive and metabolic benefit, overall enhancing the regenerative potential of oval cells upon transplantation into a carbon tetrachloride-damaged liver. We further uncover a key contribution of the HGF/c-Met pathway to modulate the TGF-ß-mediated EMT response. It allows oval cells expansion after EMT by controlling oxidative stress and apoptosis, likely via Twist regulation, and it counterbalances EMT by maintaining epithelial properties. Our work provides evidence that a coordinated and balanced action of TGF-ß and HGF are critical for achievement of the optimal regenerative potential of HPCs, opening new therapeutic perspectives. Stem Cells 2019;37:1108-1118.

The landscape of mitochondrial small non-coding RNAs in the PGCs of male mice, spermatogonia, gametes and in zygotes.

BACKGROUND: Mitochondria are organelles that fulfill a fundamental role in cell bioenergetics, as well as in other processes like cell signaling and death. Small non-coding RNAs (sncRNA) are now being considered as pivotal post-transcriptional regulators, widening the landscape of their diversity and functions. In mammalian cells, small RNAs encoded by the mitochondrial genome, mitosRNAs were discovered recently, although their biological role remains uncertain. RESULTS: Here, using specific bioinformatics analyses, we have defined the diversity of mitosRNAs present in early differentiated germ cells of male mice (PGCs and spermatogonia), and in the gametes of both sexes and in zygotes. We found strong transcription of mitosRNAs relative to the size of the mtDNA, and classifying these mitosRNAs into different functional sncRNA groups highlighted the predominance of Piwi-interacting RNAs (piRNAs) relative to the other types of mitosRNAs. Mito-piRNAs were more abundant in oocytes and zygotes, where mitochondria fulfill key roles in fecundation process. Functional analysis of some particular mito-piRNAs (mito-piR-7,456,245), also expressed in 3T3-L1 cells, was assessed after exposure to RNA antagonists. CONCLUSIONS: As far as we are aware, this is the first integrated analysis of sncRNAs encoded by mtDNA in germ cells and zygotes. The data obtained suggesting that mitosRNAs fulfill key roles in gamete differentiation and fertilization.

Leishmania infantum modulates host macrophage mitochondrial metabolism by hijacking the SIRT1-AMPK axis.

Metabolic manipulation of host cells by intracellular pathogens is currently recognized to play an important role in the pathology of infection. Nevertheless, little information is available regarding mitochondrial energy metabolism in Leishmania infected macrophages. Here, we demonstrate that during L. infantum infection, macrophages switch from an early glycolytic metabolism to an oxidative phosphorylation, and this metabolic deviation requires SIRT1 and LKB1/AMPK. SIRT1 or LBK1 deficient macrophages infected with L. infantum failed to activate AMPK and up-regulate its targets such as Slc2a4 and Ppargc1a, which are essential for parasite growth. As a result, impairment of metabolic switch caused by SIRT1 or AMPK deficiency reduces parasite load in vitro and in vivo. Overall, our work demonstrates the importance of SIRT1 and AMPK energetic sensors for parasite intracellular survival and proliferation, highlighting the modulation of these proteins as potential therapeutic targets for the treatment of leishmaniasis.

Uncoupling Protein 2 (UCP2) Function in the Brain as Revealed by the Cerebral Metabolism of (1-13C)-Glucose.

The mitochondrial aspartate/glutamate transporter Aralar/AGC1/Slc25a12 is critically involved in brain aspartate synthesis, and AGC1 deficiency results in a drastic fall of brain aspartate levels in humans and mice. It has recently been described that the uncoupling protein UCP2 transports four carbon metabolites including aspartate. Since UCP2 is expressed in several brain cell types and AGC1 is mainly neuronal, we set to test whether UCP2 could be a mitochondrial aspartate carrier in the brain glial compartment. The study of the cerebral metabolism of (1-13C)-glucose in vivo in wild type and UCP2-knockout mice showed no differences in C3 or C2 labeling of aspartate, suggesting that UCP2 does not function as a mitochondrial aspartate carrier in brain. However, surprisingly, a clear decrease (of about 30-35 %) in the fractional enrichment of glutamate, glutamine and GABA was observed in the brains of UCP2-KO mice which was not associated with differences in either glucose or lactate enrichments. The results suggest that the dilution in the labeling of glutamate and its downstream metabolites could originate from the uptake of an unlabeled substrate that could not leave the matrix via UCP2 becoming trapped in the matrix. Understanding the nature of the unlabeled substrate and its precursor(s) as alternative substrates to glucose is of interest in the context of neurological diseases associated with UCP2.

Reshaping of Human Macrophage Polarization through Modulation of Glucose Catabolic Pathways.

Macrophages integrate information from the tissue microenvironment and adjust their effector functions according to the prevalent extracellular stimuli. Therefore, macrophages can acquire a variety of activation (polarization) states, and this functional plasticity allows the adequate initiation, regulation, and resolution of inflammatory responses. Modulation of the glucose metabolism contributes to the macrophage adaptation to the surrounding cytokine milieu, as exemplified by the distinct glucose catabolism of macrophages exposed to LPS/IFN-γ or IL-4. To dissect the acquisition of macrophage effector functions in the absence of activating cytokines, we assessed the bioenergetic profile of macrophages generated in the presence of GM-CSF (GM-MØ) or M-CSF (M-MØ), which do not release pro- or anti-inflammatory cytokines unless subjected to additional activating stimuli. Compared to M-MØ, GM-MØ displayed higher oxygen consumption rate and aerobic glycolysis (extracellular acidification rate [ECAR]), as well as higher expression of genes encoding glycolytic enzymes. However, M-MØ exhibited a significantly higher oxygen consumption rate/ECAR ratio. Surprisingly, whereas aerobic glycolysis positively regulated IL1B, TNF, and INHBA mRNA expression in both macrophage subtypes, mitochondrial respiration negatively affected IL6, IL1B, TNF, and CXCL10 mRNA expression in M-MØ. The physiological significance of these results became evident under low oxygen tensions, as hypoxia enhanced ECAR in M-MØ via HIF-1α and HIF-2α, increased expression of glycolytic enzymes and GM-MØ-specific genes, and diminished expression of M-MØ-associated genes. Therefore, our data indicate that GM-MØ and M-MØ display distinct bioenergetic profiles, and that hypoxia triggers a transcriptomic switch in macrophages by promoting a HIF-1α/HIF-2α-dependent increase in ECAR.

Differences in ATP Generation Via Glycolysis and Oxidative Phosphorylation and Relationships with Sperm Motility in Mouse Species.

Mouse sperm produce enough ATP to sustain motility by anaerobic glycolysis and respiration. However, previous studies indicated that an active glycolytic pathway is required to achieve normal sperm function and identified glycolysis as the main source of ATP to fuel the motility of mouse sperm. All the available evidence has been gathered from the studies performed using the laboratory mouse. However, comparative studies of closely related mouse species have revealed a wide range of variation in sperm motility and ATP production and that the laboratory mouse has comparatively low values in these traits. In this study, we compared the relative reliance on the usage of glycolysis or oxidative phosphorylation as ATP sources for sperm motility between mouse species that exhibit significantly different sperm performance parameters. We found that the sperm of species with higher oxygen consumption/lactate excretion rate ratios were able to produce higher amounts of ATP, achieving higher swimming velocities. Additionally, we show that the species with higher respiration/glycolysis ratios have a higher degree of dependence upon active oxidative phosphorylation. Moreover, we characterize for the first time two mouse species in which sperm depend on functional oxidative phosphorylation to achieve normal performance. Finally, we discuss that sexual selection could promote adaptations in sperm energetic metabolism tending to increase the usage of a more efficient pathway for the generation of ATP (and faster sperm).

Laminar shear stress regulates mitochondrial dynamics, bioenergetics responses and PRX3 activation in endothelial cells.

Endothelial cells in the vascular system are constantly subjected to the frictional force of shear stress due to the pulsatile nature of blood flow. Although several proteins form part of the shear stress mechano-sensing pathway, the identification of mechano-transducing pathways is largely unknown. Given the increasing evidence for a signaling function of mitochondria in endothelial cells, the aim of this study was to investigate their role as mechano-sensor organelles during laminar shear stress (LSS). We demonstrated that LSS activates intracellular signaling pathways that modulate not only mitochondrial dynamics but also mitochondrial function. At early time points of LSS, the fission-related protein Drp1 was recruited from the cytosol to mitochondria and activated mitochondrial fission. LSS-dependent increase in intracellular Ca(2+) concentration was indispensable for mitochondrial fission. As alterations in mitochondrial dynamics have been related to changes in bioenergetics profiles, we studied mitochondrial function after LSS. We found that LSS decreased respiration rate, increased mitochondrial membrane potential and promoted the mitochondrial generation of ROS with the subsequent oxidation and activation of the antioxidant enzyme PRX3. Our data support a novel and active role for mitochondria in endothelial cells as active players, able to transduce the mechanical force of shear stress in the vascular endothelium into a biological response.

Performance of Rodent Spermatozoa Over Time Is Enhanced by Increased ATP Concentrations: The Role of Sperm Competition.

Sperm viability, acrosome integrity, motility, and swimming velocity are determinants of male fertility and exhibit an extreme degree of variation among closely related species. Many of these sperm parameters are associated with sperm ATP content, which has led to predictions of trade-offs between ATP content and sperm motility and velocity. Selective pressures imposed by sperm competition have been proposed as evolutionary causes of this pattern of diversity in sperm traits. Here, we examine variation in sperm viability, acrosome integrity, motility, swimming velocity, and ATP content over time, among 18 species of closely related muroid rodents, to address the following questions: (a) Do sperm from closely related species vary in ATP content after a period of incubation? (b) Are these differences in ATP levels related to differences in other sperm traits? (c) Are differences in ATP content and sperm performance over time explained by the levels of sperm competition in these species? Our results revealed a high degree of interspecific variability in changes in sperm ATP content, acrosome integrity, sperm motility and swimming velocity over time. Additionally, species with high sperm competition levels were able to maintain higher levels of sperm motility and faster sperm swimming velocity when they were incubated under conditions that support sperm survival. Furthermore, we show that the maintenance of such levels of sperm performance is correlated with the ability of sperm to sustain high concentrations of intracellular ATP over time. Thus, sperm competition may have an important role maximizing sperm metabolism and performance and, ultimately, the fertilizing capacity of spermatozoa.

Glucagon regulation of oxidative phosphorylation requires an increase in matrix adenine nucleotide content through Ca2+ activation of the mitochondrial ATP-Mg/Pi carrier SCaMC-3.

It has been known for a long time that mitochondria isolated from hepatocytes treated with glucagon or Ca(2+)-mobilizing agents such as phenylephrine show an increase in their adenine nucleotide (AdN) content, respiratory activity, and calcium retention capacity (CRC). Here, we have studied the role of SCaMC-3/slc25a23, the mitochondrial ATP-Mg/Pi carrier present in adult mouse liver, in the control of mitochondrial AdN levels and respiration in response to Ca(2+) signals as a candidate target of glucagon actions. With the use of SCaMC-3 knock-out (KO) mice, we have found that the carrier is responsible for the accumulation of AdNs in liver mitochondria in a strictly Ca(2+)-dependent way with an S0.5 for Ca(2+) activation of 3.3 ± 0.9 µm. Accumulation of matrix AdNs allows a SCaMC-3-dependent increase in CRC. In addition, SCaMC-3-dependent accumulation of AdNs is required to acquire a fully active state 3 respiration in AdN-depleted liver mitochondria, although further accumulation of AdNs is not followed by increases in respiration. Moreover, glucagon addition to isolated hepatocytes increases oligomycin-sensitive oxygen consumption and maximal respiratory rates in cells derived from wild type, but not SCaMC-3-KO mice and glucagon administration in vivo results in an increase in AdN content, state 3 respiration and CRC in liver mitochondria in wild type but not in SCaMC-3-KO mice. These results show that SCaMC-3 is required for the increase in oxidative phosphorylation observed in liver mitochondria in response to glucagon and Ca(2+)-mobilizing agents, possibly by allowing a Ca(2+)-dependent accumulation of mitochondrial AdNs and matrix Ca(2+), events permissive for other glucagon actions.

Regulation of death induction and chemosensitizing action of 3-bromopyruvate in myeloid leukemia cells: energy depletion, oxidative stress, and protein kinase activity modulation.

3-Bromopyruvate (3-BrP) is an alkylating, energy-depleting drug that is of interest in antitumor therapies, although the mechanisms underlying its cytotoxicity are ill-defined. We show here that 3-BrP causes concentration-dependent cell death of HL60 and other human myeloid leukemia cells, inducing both apoptosis and necrosis at 20-30 µM and a pure necrotic response at 60 µM. Low concentrations of 3-BrP (10-20 µM) brought about a rapid inhibition of glycolysis, which at higher concentrations was followed by the inhibition of mitochondrial respiration. The combination of these effects causes concentration-dependent ATP depletion, although this cannot explain the lethality at intermediate 3-BrP concentrations (20-30 µM). The oxidative stress caused by exposure to 3-BrP was evident as a moderate overproduction of reactive oxygen species and a concentration-dependent depletion of glutathione, which was an important determinant of 3-BrP toxicity. In addition, 3-BrP caused glutathione-dependent stimulation of p38 mitogen-activated protein kinase (MAPK), mitogen-induced extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK), and protein kinase B (Akt)/mammalian target of rapamycin/p70S6K phosphorylation or activation, as well as rapid LKB-1/AMP kinase (AMPK) activation, which was later followed by Akt-mediated inactivation. Experiments with pharmacological inhibitors revealed that p38 MAPK activation enhances 3-BrP toxicity, which is conversely restrained by ERK and Akt activity. Finally, 3-BrP was seen to cooperate with antitumor agents like arsenic trioxide and curcumin in causing cell death, a response apparently mediated by both the generation of oxidative stress induced by 3-BrP and the attenuation of Akt and ERK activation by curcumin. In summary, 3-BrP cytotoxicity is the result of several combined regulatory mechanisms that might represent important targets to improve therapeutic efficacy.

Fatty acids revert the inhibition of respiration caused by the antidiabetic drug metformin to facilitate their mitochondrial ß-oxidation.

While metformin has been widely used to treat type 2 diabetes for the last fifty years, its mode of action remains unclear. Hence, we investigated the short-term alterations in energy metabolism caused by metformin administration in 3T3-L1 adipocytes. We found that metformin inhibited mitochondrial respiration, although ATP levels remained constant as the decrease in mitochondrial production was compensated by an increase in glycolysis. While AMP/ATP ratios were unaffected by metformin, phosphorylation of AMPK and its downstream target acetyl-CoA carboxylase augmented. The inhibition of respiration provoked a rapid and sustained increase in superoxide levels, despite the increase in UCP2 and superoxide dismutase activity. The inhibition of respiration was rapidly reversed by fatty acids and thus respiration was lower in treated cells in the presence of pyruvate and glucose while rates were identical to control cells when palmitate was the substrate. We conclude that metformin reversibly inhibits mitochondrial respiration, it rapidly activates AMPK without altering the energy charge, and it inhibits fatty acid synthesis. Mitochondrial ß-oxidation is facilitated by reversing the inhibition of complex I and, presumably, by releasing the inhibition of carnitine palmitoyltransferase. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

A critical tyrosine residue determines the uncoupling protein-like activity of the yeast mitochondrial oxaloacetate carrier.

The mitochondrial Oac (oxaloacetate carrier) found in some fungi and plants catalyses the uptake of oxaloacetate, malonate and sulfate. Despite their sequence similarity, transport specificity varies considerably between Oacs. Indeed, whereas ScOac (Saccharomyces cerevisiae Oac) is a specific anion-proton symporter, the YlOac (Yarrowia lipolytica Oac) has the added ability to transport protons, behaving as a UCP (uncoupling protein). Significantly, we identified two amino acid changes at the matrix gate of YlOac and ScOac, tyrosine to phenylalanine and methionine to leucine. We studied the role of these amino acids by expressing both wild-type and specifically mutated Oacs in an Oac-null S. cerevisiae strain. No phenotype could be associated with the methionine to leucine substitution, whereas UCP-like activity was dependent on the presence of the tyrosine residue normally expressed in the YlOac, i.e. Tyr-ScOac mediated proton transport, whereas Phe-YlOac lost its protonophoric activity. These findings indicate that the UCP-like activity of YlOac is determined by the tyrosine residue at position 146.

Capacitation promotes a shift in energy metabolism in murine sperm.

In mammals, sperm acquire fertilization ability after a series of physiological and biochemical changes, collectively known as capacitation, that occur inside the female reproductive tract. In addition to other requirements, sperm bioenergetic metabolism has been identified as a fundamental component in the acquisition of capacitation. Mammalian sperm produce ATP through two main metabolic processes, oxidative phosphorylation (OXPHOS) and aerobic glycolysis that are localized to two different flagellar compartments, the midpiece, and the principal piece, respectively. In mouse sperm, the occurrence of many events associated with capacitation relies on the activity of these two energy-producing pathways, leading to the hypothesis that some of these events may impose changes in sperm energetic demands. In the present study, we used extracellular flux analysis to evaluate changes in glycolytic and respiratory parameters of murine sperm that occur as a consequence of capacitation. Furthermore, we examined whether these variations affect sperm ATP sustainability. Our results show that capacitation promotes a shift in the usage ratio of the two main metabolic pathways, from oxidative to glycolytic. However, this metabolic rewiring does not seem to affect the rate at which the sperm consume ATP. We conclude that the probable function of the metabolic switch is to increase the ATP supply in the distal flagellar regions, thus sustaining the energetic demands that arise from capacitation.

Identification of the mitochondrial carrier that provides Yarrowia lipolytica with a fatty acid-induced and nucleotide-sensitive uncoupling protein-like activity.

Uncoupling proteins (UCPs) are mitochondrial carriers distributed throughout the eukaryotic kingdoms. While genes coding for UCPs have been identified in plants and animals, evidences for the presence of UCPs in fungi and protozoa are only functional. Here, it is reported that in the yeast Yarrowia lipolytica there is a fatty acid-promoted and GDP-sensitive uncoupling activity indicating the presence of a UCP. The uncoupling activity is higher in the stationary phase than in the mid-log growth phase. The in silico search on the Y. lipolytica genome led to the selection of two genes with the highest homology to the UCP family, XM_503525 and XM_500457. By phylogenetic analysis, XP_503525 was predicted to be an oxaloacetate carrier while XP_500457 would be a dicarboxylate carrier. Each of these two genes was cloned and heterologously expressed in Saccharomyces cerevisiae and the resulting phenotype was analyzed. The transport activity of the two gene products confirmed the phylogenetic predictions. In addition, only mitochondria isolated from yeasts expressing XP_503525 showed bioenergetic properties characteristic of a UCP: the proton conductance was increased by linoleic acid and inhibited by GDP. It is concluded that the XM_503525 gene from Y. lipolytica encodes for an oxaloacetate carrier although, remarkably, it also displays an uncoupling activity stimulated by fatty acids and inhibited by nucleotides.

Lipotoxicity, fatty acid uncoupling and mitochondrial carrier function.

Diseases like obesity, diabetes or generalized lipodystrophy cause a chronic elevation of circulating fatty acids that can become cytotoxic, a condition known as lipotoxicity. Fatty acids cause oxidative stress and alterations in mitochondrial structure and function. The uncoupling of the oxidative phosphorylation is one of the most recognized deleterious fatty acid effects and several metabolite transporters are known to mediate in their action. The fatty acid interaction with the carriers leads to membrane depolarization and/or the conversion of the carrier into a pore. The result is the opening of the permeability transition pore and the initiation of apoptosis. Unlike the other members of the mitochondrial carrier superfamily, the eutherian uncoupling protein UCP1 has evolved to achieve its heat-generating capacity in the physiological context provided by the brown adipocyte and therefore it is activated by the low fatty acid concentrations generated by the noradrenaline-stimulated lipolysis.

Spin-state-dependent electrical conductivity in single-walled carbon nanotubes encapsulating spin-crossover molecules.

Spin crossover (SCO) molecules are promising nanoscale magnetic switches due to their ability to modify their spin state under several stimuli. However, SCO systems face several bottlenecks when downscaling into nanoscale spintronic devices: their instability at the nanoscale, their insulating character and the lack of control when positioning nanocrystals in nanodevices. Here we show the encapsulation of robust Fe-based SCO molecules within the 1D cavities of single-walled carbon nanotubes (SWCNT). We find that the SCO mechanism endures encapsulation and positioning of individual heterostructures in nanoscale transistors. The SCO switch in the guest molecules triggers a large conductance bistability through the host SWCNT. Moreover, the SCO transition shifts to higher temperatures and displays hysteresis cycles, and thus memory effect, not present in crystalline samples. Our results demonstrate how encapsulation in SWCNTs provides the backbone for the readout and positioning of SCO molecules into nanodevices, and can also help to tune their magnetic properties at the nanoscale.