Age-associated alterations of brain mitochondria energetics.

The study aimed to explore the role of age-associated elevated cytosolic Ca2+ in changes of brain mitochondria energetic processes. Two groups of rats, young adults (4 months) and advanced old (24 months), were evaluated for potential alterations of mitochondrial parameters, the oxidative phosphorylation (OxPhos), membrane potential, calcium retention capacity, activity of glutamate/aspartate carrier (aralar), and ROS formation. We demonstrated that the brain mitochondria of older animals have a lower resistance to Ca2+ stress with resulting consequences. The suppressed complex I OxPhos and decreased membrane potential were accompanied by reduction of the Ca2+ threshold required for induction of mPTP. The Ca2+ binding sites of mitochondrial aralar mediated a lower activity of old brain mitochondria. The altered interaction between aralar and mPTP may underlie mitochondrial dysregulation leading to energetic depression during aging. At the advanced stages of aging, the declined metabolism is accompanied by the diminished oxidative background.

Malate-aspartate shuttle promotes l-lactate oxidation in mitochondria.

Metabolism in cancer cells is rewired to generate sufficient energy equivalents and anabolic precursors to support high proliferative activity. Within the context of these competing drives aerobic glycolysis is inefficient for the cancer cellular energy economy. Therefore, many cancer types, including colon cancer, reprogram mitochondria-dependent processes to fulfill their elevated energy demands. Elevated glycolysis underlying the Warburg effect is an established signature of cancer metabolism. However, there are a growing number of studies that show that mitochondria remain highly oxidative under glycolytic conditions. We hypothesized that activities of glycolysis and oxidative phosphorylation are coordinated to maintain redox compartmentalization. We investigated the role of mitochondria-associated malate-aspartate and lactate shuttles in colon cancer cells as potential regulators that couple aerobic glycolysis and oxidative phosphorylation. We demonstrated that the malate-aspartate shuttle exerts control over NAD+ /NADH homeostasis to maintain activity of mitochondrial lactate dehydrogenase and to enable aerobic oxidation of glycolytic l-lactate in mitochondria. The elevated glycolysis in cancer cells is proposed to be one of the mechanisms acquired to accelerate oxidative phosphorylation.

Determination of mitochondrial metabolic phenotype through investigation of the intrinsic inhibition of succinate dehydrogenase.

Many diseases are accompanied by systemic or organ metabolic abnormalities. Therefore, investigation of the roles of mitochondrial dysfunction in the pathogenesis of major diseases requires a methodology that reflects the characteristics of mitochondrial metabolism particular for the organ of origin. We provide evidence that for brain and heart mitochondria the intrinsic inhibition of succinate dehydrogenase (SDH) is a key mechanism for attenuation of mitochondrial respiration and energy production in response to the organ's energy needs. This mechanism also serves to minimize the production of reactive oxygen species when the organ is at rest. Changes in the organ's workloads are accompanied by changes in metabolites that are used by mitochondria as substrates and for modification of energy production at the SDH level. Measurement of the respiratory activity of mitochondria with various substrates and substrate mixtures and use of bovine serum albumin as an SDH inhibitor will be useful for evaluation of metabolic phenotype at the mitochondrial level.

Artefactual formation of pyruvate from in-source conversion of lactate.

RATIONALE: Lactate and pyruvate are high abundance products of glucose metabolism. Analysis of both molecules as part of metabolomics studies in cellular metabolism and physiology have been aided by advances in liquid chromatography-mass spectrometry (LC-MS). METHODS: We used ion pairing-chromatography and negative ion mode ESI on an QExactive HF to perform stable isotope assisted metabolomics profiling of lactate and pyruvate metabolism. RESULTS: Using an LC-MS method for polar metabolite analysis we discovered an artefactual formation of pyruvate from in-source fragmentation of lactate. Surprisingly, this in-source fragmentation has not been previously described, thus we report this identification to warn other investigators. This artefact was detected by baseline chromatographic resolution of lactate and pyruvate by LC with confirmation of this artefact by stable isotope labeling of lactate and pyruvate. CONCLUSIONS: These findings have immediate implications for metabolomics studies by LC-MS and direct infusion MS, especially in negative ion mode, whereby users should resolve lactate from pyruvate or robustly quantify the potential formation of pyruvate from higher abundance lactate in their assays.

Respirometric reserve capacity of cumulus cell mitochondria correlates with oocyte maturity.

PURPOSE: Oocyte competence is critical in success of assisted reproduction. Metabolic signaling between oocyte and cumulus cells within the cumulus-oocyte complex procure oocyte development. This study evaluated the relationship between respirometric activity of cumulus cells and maturity of corresponding oocytes. METHODS: In prospective cohort study, 20 women of age 28-42 undergoing IVF procedure were involved. To evaluate oocyte maturity, the cumulus cells from individual oocytes were assessed flow cytometrically by double labeling of cells with mitochondria specific dyes. The respirometric stress analysis using ATPase inhibitor oligomycin was applied to assess mitochondria metabolic abnormalities. RESULTS: The cumulus cells from each of 327 oocytes were analyzed. The respirometric index of cumulus cells (O'R) strongly correlates with maternal ovarian reserve, showing to be higher in patients with higher AMH (p < 0.0017). Cumulus cells from immature oocytes had severe mitochondria deficiency, i.e., low O'R, than those from mature oocytes (p < 0.02). No significant difference in respirometric capacity was found between cumulus cells associated with good vs poor-quality embryos. CONCLUSIONS: The oocyte maturity is potentially related to the mitochondria activity of cumulus cells.

The control of brain mitochondrial energization by cytosolic calcium: the mitochondrial gas pedal.

This review focuses on problems of the intracellular regulation of mitochondrial function in the brain via the (i) supply of mitochondria with ADP by means of ADP shuttles and channels and (ii) the Ca(2+) control of mitochondrial substrate supply. The permeability of the mitochondrial outer membrane for adenine nucleotides is low. Therefore rate dependent concentration gradients exist between the mitochondrial intermembrane space and the cytosol. The existence of dynamic ADP gradients is an important precondition for the functioning of ADP shuttles, for example CrP-shuttle. Cr at mM concentrations instead of ADP diffuses from the cytosol through the porin pores into the intermembrane space. The CrP-shuttle isoenzymes work in different directions which requires different metabolite concentrations mainly caused by dynamic ADP compartmentation. The ADP shuttle mechanisms alone cannot explain the load dependent changes in mitochondrial energization, and a complete model of mitochondrial regulation have to account the Ca(2+) -dependent substrate supply too. According to the old paradigmatic view, Ca(2+) (cyt) taken up by the mitochondrial Ca(2+) uniporter activates dehydrogenases within the matrix. However, recently it was found that Ca(2+) (cyt) at low nM concentrations exclusively activates the state 3 respiration via aralar, the mitochondrial glutamate/aspartate carrier. At higher Ca(2+) (cyt) (> 500 nM), brain mitochondria take up Ca(2+) for activation of substrate oxidation rates. Since brain mitochondrial pyruvate oxidation is only slightly influenced by Ca(2+) (cyt) , it was proposed that the cytosolic formation of pyruvate from its precursors is tightly controlled by the Ca(2+) dependent malate/aspartate shuttle. At low (50-100 nM) Ca(2+) (cyt) the pyruvate formation is suppressed, providing a substrate limitation control in neurons. This so called "gas pedal" mechanism explains why the energy metabolism of neurons in the nucleus suprachiasmaticus could be down-regulated at night but activated at day as a basis for the circadian changes in Ca(2+) (cyt) . It also could explain the energetic disadvantages caused by altered Ca(2+) (cyt) at mitochondrial diseases and neurodegeneration.

Physiological validation of cell health upon probing with carbon nanotube endoscope and its benefit for single-cell interrogation.

New-generation nanoscale devices for single-cell study are intensively being developed. As has been shown, nanodevices are minimally invasive because of their order-of-magnitude smaller size in comparison to conventional glass pipettes. However, in most studies the evaluation of the nanodevice impact on cell health has not extended to their effects on cell metabolic integrity. In this work we evaluated the degree to which the insertion of a carbon-based nanotube endoscope into a cell induces mechanical and biochemical stress, and affects cellular key metabolic systems. The effects of insertion of the nanotube endoscope on cell morphological and physiological modulations were monitored and compared to those of glass micropipettes. We report that nanotube endoscope insertion does not significantly modulate the plasma membrane and actin network. The cell metabolic mechanisms such as energy production and inositol 1,4,5-trisphosphate-dependent calcium signaling remain preserved for prolonged endoscope presence within a cell. FROM THE CLINICAL EDITOR: In this basic science study, the effects of insertion of carbon nanotube endoscope on cell morphological and physiological modulations were monitored and compared to those of glass micropipettes. Nanotube endoscope insertion is truly minimally invasive: it does not significantly modulate the plasma membrane and actin network; the energy production and inositol 1,4,5-trisphosphate-dependent calcium signaling also remain preserved during prolonged endoscope presence within a cell.

Curcumin and Carnosic Acid Cooperate to Inhibit Proliferation and Alter Mitochondrial Function of Metastatic Prostate Cancer Cells.

Anticancer activities of plant polyphenols have been demonstrated in various models of neoplasia. However, evidence obtained in numerous in vitro studies indicates that proliferation arrest and/or killing of cancer cells require quite high micromolar concentrations of polyphenols that are difficult to reach in vivo and can also be (geno)toxic to at least some types of normal cells. The ability of certain polyphenols to synergize with one another at low concentrations can be used as a promising strategy to effectively treat human malignancies. We have recently reported that curcumin and carnosic acid applied at non-cytotoxic concentrations synergistically cooperate to induce massive apoptosis in acute myeloid leukemia cells, but not in normal hematopoietic and non-hematopoietic cells, via sustained cytosolic calcium overload. Here, we show that the two polyphenols can also synergistically suppress the growth of DU145 and PC-3 metastatic prostate cancer cell cultures. However, instead of cell killing, the combined treatment induced a marked inhibition of cell proliferation associated with G0/G1 cell cycle arrest. This was preceded by transient elevation of cytosolic calcium levels and prolonged dissipation of the mitochondrial membrane potential, without generating oxidative stress, and was associated with defective oxidative phosphorylation encompassing mitochondrial dysfunction. The above effects were concomitant with a significant downregulation of mRNA and protein expression of the oncogenic kinase SGK1, the mitochondria-hosted mTOR component. In addition, a moderate decrease in SGK1 phosphorylation at Ser422 was observed in polyphenol-treated cells. The mTOR inhibitor rapamycin produced a similar reduction in SGK1 mRNA and protein levels as well as phosphorylation. Collectively, our findings suggest that the combination of curcumin and carnosic acid at potentially bioavailable concentrations may effectively target different types of cancer cells by distinct modes of action. This and similar combinations merit further exploration as an anticancer modality.

Mitochondria membrane transformations in colon and prostate cancer and their biological implications.

Mitochondria have emerged as important determinants in cancer progression and malignancy. However, the role of mitochondrial membranes in cancer onset and progression has not been thoroughly investigated. This study compares the structural and functional properties of mitochondrial membranes in prostate and colon cancer cells in comparison to normal mitochondria, and possible therapeutic implications of these membrane changes. Specifically, isolation of cell mitochondria and preparation of inverted sub-mitochondrial particles (SMPs) illuminated significant cancer-induced modulations of membrane lipid compositions, fluidity, and activity of cytochrome c oxidase, one of the key mitochondrial enzymes. The experimental data further show that cancer-associated membrane transformations may account for mitochondria targeting by betulinic acid and resveratrol, known anti-cancer molecules. Overall, this study probes the relationship between cancer and mitochondrial membrane transformations, underlying a potential therapeutic significance for mitochondrial membrane targeting in cancer.

Surface-enhanced Raman spectroscopy as a tool for detecting Ca2+ mobilizing second messengers in cell extracts.

Understanding of calcium signaling pathways in cells is essential for elucidating the mechanisms of both normal cell function and cancer development. Calcium messengers play the crucial role for intracellular Ca(2+) release. We propose a new approach to detecting the calcium second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) in cell extracts using surface-enhanced Raman spectroscopy (SERS). Currently available radioreceptor binding and enzymatic assays require extensive sample preparation and take more than 12 h. With a SERS sensor, NAADP can be detected in less than 1 min without any special sample preparation. To the best of our knowledge, this is the first demonstration of using SERS for calcium signaling applications.

Small diameter carbon nanopipettes.

Nanoscale multifunctional carbon probes facilitate cellular studies due to their small size, which makes it possible to interrogate organelles within living cells in a minimally invasive fashion. However, connecting nanotubes to macroscopic devices and constructing an integrated system for the purpose of fluid and electrical signal transfer is challenging, as is often the case with nanoscale components. We describe a non-catalytic chemical vapor deposition based method for batch fabrication of integrated multifunctional carbon nanopipettes (CNPs) with tip diameters much smaller (10-30 nm) than previously reported (200 nm and above) and approaching those observed for multiwalled carbon nanotubes. This eliminates the need for complicated attachment/assembly of nanotubes into nanofluidic devices. Variable tip geometries and structures were obtained by controlled deposition of carbon inside and outside quartz pipettes. We have shown that the capillary length and gas flow rate have a marked effect on the carbon deposition. This gives us a flexible protocol, useful for growing carbon layers of different thicknesses at selective locations on a glass pipette to yield a large variety of cellular probes in bulk quantities. The CNPs possess an open channel for fluid transfer with the carbon deposited inside at 875 degrees C behaving like an amorphous semiconductor. Vacuum annealing of the CNP tips at temperatures up to 2000 degrees C yields graphitic carbon structures with an increase in conductivity of two orders of magnitude. Penetration of the integrated carbon nanoprobes into cells was shown to produce minimal Ca(2+) signals, fast recovery of basal Ca(2+) levels and no adverse activation of the cellular metabolism during interrogation times as long as 0.5-1 h.

Mitochondrial responses to organelle-specific drug delivering nanoparticles composed of polypeptide and peptide complexes.

Aims: The mechanistic study of the drug carrier-target interactions of mitochondria-unique nanoparticles composed of polypeptide-peptide complexes (mPoP-NPs). Materials & methods: The isolated organelles were employed to address the direct effects of mPoP-NPs on dynamic structure and functional wellbeing of mitochondria. Mitochondria morphology, respiration, membrane potential, reactive oxygen species generation, were examined by confocal microscopy, flow cytometry and oxygraphy. Lonidamine-encapsulated formulation was assessed to evaluate the drug delivery capacity of the naive nanoparticles. Results: The mPoP-NPs do not alter mitochondria structure and performance upon docking to organelles, while successfully delivering drug that causes organelle dysfunction. Conclusion: The study gives insight into interactions of mPoP-NPs with mitochondria and provides substantial support for consideration of designed nanoparticles as biocompatible and efficient mitochondria-targeted platforms.

Membrane processes and biophysical characterization of living cells decorated with chromatic polydiacetylene vesicles.

The structural complexity of the cell membrane makes analysis of membrane processes in living cells, as compared to model membrane systems, highly challenging. Living cells decorated with surface-attached colorimetric/fluorescent polydiacetylene patches might constitute an effective platform for analysis and visualization of membrane processes in situ. This work examines the biological and chemical consequences of plasma membrane labeling of promyelocytic leukemia cells with polydiacetylene. We show that the extent of fusion between incubated lipid/diacetylene vesicles and the plasma membrane is closely dependent upon the lipid composition of both vesicles and cell membrane. In particular, we find that cholesterol presence increased bilayer fusion between the chromatic vesicles and the plasma membrane, suggesting that membrane organization plays a significant role in the fusion process. Spectroscopic data and physiological assays show that decorating the cell membrane with the lipid/diacetylene patches reduces the overall lateral diffusion within the membrane bilayer, however polydiacetylene labeling does not adversely affect important cellular metabolic pathways. Overall, the experimental data indicate that the viability and physiological integrity of the surface-engineered cells are retained, making possible utilization of the platform for studying membrane processes in living cells. We demonstrate the use of the polydiacetylene-labeled cells for visualizing and discriminating among different membrane interaction mechanisms of pharmaceutical compounds.

Screening membrane interactions of pesticides by cells decorated with chromatic polymer nanopatches.

Elucidating the factors contributing to the cell toxicity of pesticides and other environmentally sensitive small molecules is critical for evaluation of their health impacts and for understanding the biological processes that they affect. Disruption and permeation of the plasma membrane, which constitutes the critical interface between the cell and its environment, are recognized initiators of cytotoxicity. We present a new approach for predicting pesticide cytotoxicity through rapid screening of membrane interactions of pesticides using a recently developed live-cell chromatic sensor. The sensing platform comprises living mammalian cells labeled with polydiacetylene (PDA), a chromatic polymer that undergoes intense fluorescence transformations induced by structural perturbations of the membrane bilayer. Within a short time after the addition of membrane-interacting tested compounds to the labeled cells, the PDA patches emit high fluorescence, which can be monitored by conventional spectroscopy and microscopy apparatuses. The chromatic technology facilitates rapid evaluation of membrane activity of pesticide compounds and is capable of distinguishing between toxic effects associated with membrane interactions vs intracellular mechanisms.

Cardiolipin mediates curcumin interactions with mitochondrial membranes.

Curcumin, the main molecular ingredient of the turmeric spice, has been reported to exhibit therapeutic properties for varied diseases and pathological conditions. While curcumin appears to trigger multiple signaling pathways, the precise mechanisms accounting for its therapeutic activity have not been deciphered. Here we show that curcumin exhibits significant interactions with cardiolipin (CL), a lipid exclusively residing in the mitochondrial membrane. Specifically, we found that curcumin affected the structures and dynamics of CL-containing biomimetic and biological mitochondrial membranes. Application of several biophysical techniques reveals the CL-promoted association and internalization of curcumin into lipid bilayers. In parallel, curcumin association with CL containing bilayers increased their fluidity and reduced lipid ordering. These findings suggest that membrane modifications mediated by CL interactions may play a role in the therapeutic functions of curcumin, and that the inner mitochondrial membrane in general might constitute a potential drug target.

Rapamycin increases oxidative metabolism and enhances metabolic flexibility in human cardiac fibroblasts.

Inhibition of mTOR signaling using rapamycin has been shown to increase lifespan and healthspan in multiple model organisms; however, the precise mechanisms for the beneficial effects of rapamycin remain uncertain. We have previously reported that rapamycin delays senescence in human cells and that enhanced mitochondrial biogenesis and protection from mitochondrial stress is one component of the benefit provided by rapamycin treatment. Here, using two models of senescence, replicative senescence and senescence induced by the presence of the Hutchinson-Gilford progeria lamin A mutation, we report that senescence is accompanied by elevated glycolysis and increased oxidative phosphorylation, which are both reduced by rapamycin. Measurements of mitochondrial function indicate that direct mitochondria targets of rapamycin are succinate dehydrogenase and matrix alanine aminotransferase. Elevated activity of these enzymes could be part of complex mechanisms that enable mitochondria to resume their optimal oxidative phosphorylation and resist senescence. This interpretation is supported by the fact that rapamycin-treated cultures do not undergo a premature senescence in response to the replacement of glucose with galactose in the culture medium, which forces a greater reliance on oxidative phosphorylation. Additionally, long-term treatment with rapamycin increases expression of the mitochondrial carrier protein UCP2, which facilitates the movement of metabolic intermediates across the mitochondrial membrane. The results suggest that rapamycin impacts mitochondrial function both through direct interaction with the mitochondria and through altered gene expression of mitochondrial carrier proteins.

Perfusion double-channel micropipette probes for oxygen flux mapping with single-cell resolution.

Measuring cellular respiration with single-cell spatial resolution is a significant challenge, even with modern tools and techniques. Here, a double-channel micropipette is proposed and investigated as a probe to achieve this goal by sampling fluid near the point of interest. A finite element model (FEM) of this perfusion probe is validated by comparing simulation results with experimental results of hydrodynamically confined fluorescent molecule diffusion. The FEM is then used to investigate the dependence of the oxygen concentration variation and the measurement signal on system parameters, including the pipette's shape, perfusion velocity, position of the oxygen sensors within the pipette, and proximity of the pipette to the substrate. The work demonstrates that the use of perfusion double-barrel micropipette probes enables the detection of oxygen consumption signals with micrometer spatial resolution, while amplifying the signal, as compared to sensors without the perfusion system. In certain flow velocity ranges (depending on pipette geometry and configuration), the perfusion flow increases oxygen concentration gradients formed due to cellular oxygen consumption. An optimal perfusion velocity for respiratory measurements on single cells can be determined for different system parameters (e.g., proximity of the pipette to the substrate). The optimum perfusion velocities calculated in this paper range from 1.9 to 12.5 µm/s. Finally, the FEM model is used to show that the spatial resolution of the probe may be varied by adjusting the pipette tip diameter, which may allow oxygen consumption mapping of cells within tissue, as well as individual cells at subcellular resolution.

Danazol alters mitochondria metabolism of fibrocystic breast Mcf10A cells.

Fibrocystic Breast Disease (FBD) or Fibrocystic change (FC) affects about 60% of women at some time during their life. Although usually benign, it is often associated with pain and tenderness (mastalgia). The synthetic steroid danazol has been shown to be effective in reducing the pain associated with FBD, but the cellular and molecular mechanisms for its action have not been elucidated. We investigated the hypothesis that danazol acts by affecting energy metabolism. Effects of danazol on Mcf10A cells homeostasis, including mechanisms of oxidative phosphorylation, cytosolic calcium signaling and oxidative stress, were assessed by high-resolution respirometry and flow cytometry. In addition to fast physiological responses the associated genomic modulations were evaluated by Affimetrix microarray analysis. The alterations of mitochondria membrane potential and respiratory activity, downregulation of energy metabolism transcripts result in suppression of energy homeostasis and arrest of Mcf10A cells growth. The data obtained in this study impacts the recognition of direct control of mitochondria by cellular mechanisms associated with altered energy metabolism genes governing the breast tissue susceptibility and response to medication by danazol.

Mitochondria-Mediated Anticancer Effects of Non-Thermal Atmospheric Plasma.

Non-thermal atmospheric pressure plasma has attracted great interest due to its multiple potential biomedical applications with cancer treatment being among the most urgent. To realize the clinical potential of non-thermal plasma, the exact cellular and molecular mechanisms of plasma effects must be understood. This work aimed at studying the prostate cancer specific mechanisms of non-thermal plasma effects on energy metabolism as a central regulator of cell homeostasis and proliferation. It was found that cancer cells with higher metabolic rate initially are more resistant to plasma treated phosphate-buffered saline (PBS) since the respiratory and calcium sensitive signaling systems were not responsive to plasma exposure. However, dramatic decline of cancer oxidative phosphorylation developed over time resulted in significant progression of cell lethality. The normal prostate cells with low metabolic activity immediately responded to plasma treated PBS by suppression of respiratory functions and sustained elevation of cytosolic calcium. However, over time the normal cells start recovering their mitochondria functions, proliferate and restore the cell population. We found that the non-thermal plasma induced increase in intracellular ROS is of primarily non-mitochondrial origin. The discriminate non-thermal plasma effects hold a promise for clinical cancer intervention.