Aerobic Glycolysis: A DeOxymoron of (Neuro)Biology.

The term 'aerobic glycolysis' has been in use ever since Warburg conducted his research on cancer cells' proliferation and discovered that cells use glycolysis to produce adenosine triphosphate (ATP) rather than the more efficient oxidative phosphorylation (oxphos) pathway, despite an abundance of oxygen. When measurements of glucose and oxygen utilization by activated neural tissue indicated that glucose was consumed without an accompanied oxygen consumption, the investigators who performed those measurements also termed their discovery 'aerobic glycolysis'. Red blood cells do not contain mitochondria and, therefore, produce their energy needs via glycolysis alone. Other processes within the central nervous system (CNS) and additional organs and tissues (heart, muscle, and so on), such as ion pumps, are also known to utilize glycolysis only for the production of ATP necessary to support their function. Unfortunately, the phenomenon of 'aerobic glycolysis' is an enigma wherever it is encountered, thus several hypotheses have been produced in attempts to explain it; that is, whether it occurs in cancer cells, in activated neural tissue, or during postprandial or exercise metabolism. Here, it is argued that, where the phenomenon in neural tissue is concerned, the prefix 'aerobic' in the term 'aerobic glycolysis' should be removed. Data collected over the past three decades indicate that L-lactate, the end product of the glycolytic pathway, plays an essential role in brain energy metabolism, justifying the elimination of the prefix 'aerobic'. Similar justification is probably appropriate for other tissues as well.

Kv7.4 channels regulate potassium permeability in neuronal mitochondria.

Mitochondrial K+ permeability regulates neuronal apoptosis, energy metabolism, autophagy, and protection against ischemia-reperfusion injury. Kv7.4 channels have been recently shown to regulate K+ permeability in cardiac mitochondria and exert cardioprotective effects. Here, the possible expression and functional role of Kv7.4 channels in regulating membrane potential, radical oxygen species (ROS) production, and Ca2+ uptake in neuronal mitochondria was investigated in both clonal (F11 cells) and native brain neurons. In coupled mitochondria isolated from F11 cells, K+-dependent changes of mitochondrial membrane potential (ΔΨ) were unaffected by the selective mitoBKCa channel blocker iberiotoxin and only partially inhibited by the mitoKATP blockers glyburide or ATP. Interestingly, K+-dependent ΔΨ decrease was significantly reduced by the Kv7 blocker XE991 and enhanced by the Kv7 activator retigabine. Among Kv7s, western blot experiments showed the expression of only Kv7.4 subunits in F11 mitochondrial fractions; immunocytochemistry experiments showed a strong overlap between the Kv7.4 fluorescent signal and that of the mitochondrial marker Mitotracker. Silencing of Kv7.4 expression significantly suppressed retigabine-dependent decrease in ΔΨ in intact F11 cells. Expression of Kv7.4 subunits was also detected by western blot in isolated mitochondria from total mouse brain and by immunofluorescence in mouse primary cortical neurons. Pharmacological experiments revealed a relevant functional role for Kv7.4 channels in regulating membrane potential and Ca2+ uptake in isolated neuronal mitochondria, as well as ΔΨ and ROS production in intact cortical neurons. In conclusion, these findings provide the first experimental evidence for the expression of Kv7.4 channels and their contribution in regulating K+ permeability of neuronal mitochondria.

Mitochondrial Transport in Glycolysis and Gluconeogenesis: Achievements and Perspectives.

Some metabolic pathways involve two different cell components, for instance, cytosol and mitochondria, with metabolites traffic occurring from cytosol to mitochondria and vice versa, as seen in both glycolysis and gluconeogenesis. However, the knowledge on the role of mitochondrial transport within these two glucose metabolic pathways remains poorly understood, due to controversial information available in published literature. In what follows, we discuss achievements, knowledge gaps, and perspectives on the role of mitochondrial transport in glycolysis and gluconeogenesis. We firstly describe the experimental approaches for quick and easy investigation of mitochondrial transport, with respect to cell metabolic diversity. In addition, we depict the mitochondrial shuttles by which NADH formed in glycolysis is oxidized, the mitochondrial transport of phosphoenolpyruvate in the light of the occurrence of the mitochondrial pyruvate kinase, and the mitochondrial transport and metabolism of L-lactate due to the L-lactate translocators and to the mitochondrial L-lactate dehydrogenase located in the inner mitochondrial compartment.

Nonalcoholic Fatty Liver Disease (NAFLD). Mitochondria as Players and Targets of Therapies?

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and represents the hepatic expression of several metabolic abnormalities of high epidemiologic relevance. Fat accumulation in the hepatocytes results in cellular fragility and risk of progression toward necroinflammation, i.e., nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and eventually hepatocellular carcinoma. Several pathways contribute to fat accumulation and damage in the liver and can also involve the mitochondria, whose functional integrity is essential to maintain liver bioenergetics. In NAFLD/NASH, both structural and functional mitochondrial abnormalities occur and can involve mitochondrial electron transport chain, decreased mitochondrial ß-oxidation of free fatty acids, excessive generation of reactive oxygen species, and lipid peroxidation. NASH is a major target of therapy, but there is no established single or combined treatment so far. Notably, translational and clinical studies point to mitochondria as future therapeutic targets in NAFLD since the prevention of mitochondrial damage could improve liver bioenergetics.

The occurrence of l-lactate dehydrogenase in the inner mitochondrial compartment of pig liver.

Although pig represents a model species in biomedical research including studies dealing with liver patho-physiology, some aspects of liver metabolism need to be addressed. In particular, whether and how pig mitochondria can metabolize l-lactate remains to be established. We show here that pig liver mitochondria (PLM) possess their own l-lactate dehydrogenase (mL-LDH). This was shown both via immunological analysis and by assaying photometrically the L-LDH reaction in solubilised PLM. The mL-LDH reaction shows hyperbolic dependence on the substrate concentration, it is inhibited by oxamate and proves to differ from the cytosolic activity (cL-LDH), as revealed by the difference found in both pH profiles and temperature dependence of m- and cL-LDH. Titration experiments with digitonin show that mL-LDH is restricted in mitochondrial inner compartment. In agreement with the above findings, three genes in Sus scrofa genome encoded for L-LDH subunits which are predicted to have mitochondrial localization, as investigated by Target P 1.1 and PredSL analysis.

In boar sperm capacitation L-lactate and succinate, but not pyruvate and citrate, contribute to the mitochondrial membrane potential increase as monitored via safranine O fluorescence.

Having ascertained using JC-1 as a probe that, in distinction with the controls, during capacitation boar sperm maintains high mitochondrial membrane potential (ΔΨ), to gain some insight into the role of mitochondria in capacitation, we monitored ΔΨ generation due to externally added metabolites either in hypotonically-treated spermatozoa (HTS) or in intact cells by using safranine O as a probe. During capacitation, the addition to HTS of L-lactate and succinate but not those of pyruvate, citrate and ascorbate + TMPD resulted in increase of ΔΨ generation. Accordingly, the addition of L-lactate and succinate, but not that of citrate, to intact sperm resulted in ΔΨ generation increased in capacitation.

Absorption of monochromatic and narrow band radiation in the visible and near IR by both mitochondrial and non-mitochondrial photoacceptors results in photobiomodulation.

In addition to the major functions performed by in the cell, mitochondria play a major role in cell-light interaction. Accordingly it is generally accepted that mitochondria are crucial in cell photobiomodulation; however a variety of biomolecules themselves proved to be targets of light irradiation. We describe whether and how mitochondria can interact with monochromatic and narrow band radiation in the red and near IR optical regions with dissection of both structural and functional effects likely leading to photobiostimulation. Moreover we also report that a variety of biomolecules localized in mitochondria and/or in other cell compartments including cytochrome c oxidase, some proteins, nucleic acids and adenine nucleotides are light sensitive with major modifications in their biochemistry. All together the reported investigations show that the elucidation of the mechanism of the light interaction with biological targets still remains to be completed, this needing further research, however the light sensitivity of a variety of molecules strongly suggests that photobiomodulation could be used in both in photomedicine and in biotechnology.

Yeast growth in raffinose results in resistance to acetic-acid induced programmed cell death mostly due to the activation of the mitochondrial retrograde pathway.

In order to investigate whether and how a modification of mitochondrial metabolism can affect yeast sensitivity to programmed cell death (PCD) induced by acetic acid (AA-PCD), yeast cells were grown on raffinose, as a sole carbon source, which, differently from glucose, favours mitochondrial respiration. We found that, differently from glucose-grown cells, raffinose-grown cells were mostly resistant to AA-PCD and that this was due to the activation of mitochondrial retrograde (RTG) response, which increased with time, as revealed by the up-regulation of the peroxisomal isoform of citrate synthase and isocitrate dehydrogenase isoform 1, RTG pathway target genes. Accordingly, the deletion of RTG2 and RTG3, a positive regulator and a transcription factor of the RTG pathway, resulted in AA-PCD, as shown by TUNEL assay. Neither deletion in raffinose-grown cells of HAP4, encoding the positive regulatory subunit of the Hap2,3,4,5 complex nor constitutive activation of the RTG pathway in glucose-grown cells due to deletion of MKS1, a negative regulator of RTG pathway, had effect on yeast AA-PCD. The RTG pathway was found to be activated in yeast cells containing mitochondria, in which membrane potential was measured, capable to consume oxygen in a manner stimulated by the uncoupler CCCP and inhibited by the respiratory chain inhibitor antimycin A. AA-PCD resistance in raffinose-grown cells occurs with a decrease in both ROS production and cytochrome c release as compared to glucose-grown cells en route to AA-PCD.

Prostate cancer cells metabolize d-lactate inside mitochondria via a D-lactate dehydrogenase which is more active and highly expressed than in normal cells.

Although D-lactate metabolism has been shown to occur in a variety of mitochondria, the metabolic fate of D-lactate in cancer cells has never been investigated, as it is believed to be exported to the extracellular phase. We show that mitochondria from both cancer (PC-3) and normal (PNT1A) prostate cells can metabolize D-lactate in an energy competent manner. This is due to the mitochondrial D-lactate dehydrogenase, a membrane flavoprotein, the activity and protein level of which are higher in PC-3 than in PNT1A cells, as detected by both kinetic and immunological analysis. D-Lactate can enter prostate mitochondria and cause the export of newly synthesized malate in a carrier-mediated manner, with the rate of malate efflux from mitochondria twofold higher in cancer.

The N-acetylcysteine-insensitive acetic acid-induced yeast programmed cell death occurs without macroautophagy.

Programmed cell death can occur through two separate pathways caused by treatment of Saccharomyces cerevisiae with acetic acid (AA-PCD), which differ from one another essentially with respect to their sensitivity to N-acetylcysteine (NAC) and to the role played by cytochrome c and metacaspase YCA1. Moreover, yeast can also undergo macroautophagy which occurs in NAC-insensitive manner. In order to gain some insight into the relationship between AA-PCD and macroautophagy use was made of WT and knock-out cells lacking YCA1 and/or cytochrome c. We show that i. macroautophagy is modulated by YCA1 and by cytochrome c in a negative and positive manner, respectively, ii. the NAC-insensitive AA-PCD and macroautophagy differ from one another and iii. NAC-insensitive AA-PCD pathway takes place essentially without macroautophagy, even if the shift of extracellular pH to acidic values required for AA-PCD to occur leads itself to increased or decreased macroautophagy in YCA1 or cytochrome c-lacking cells.

The role of mitochondria in yeast programmed cell death.

Mammalian apoptosis and yeast programmed cell death (PCD) share a variety of features including reactive oxygen species production, protease activity and a major role played by mitochondria. In view of this, and of the distinctive characteristics differentiating yeast and multicellular organism PCD, the mitochondrial contribution to cell death in the genetically tractable yeast Saccharomyces cerevisiae has been intensively investigated. In this mini-review we report whether and how yeast mitochondrial function and proteins belonging to oxidative phosphorylation, protein trafficking into and out of mitochondria, and mitochondrial dynamics, play a role in PCD. Since in PCD many processes take place over time, emphasis will be placed on an experimental model based on acetic acid-induced PCD (AA-PCD) which has the unique feature of having been investigated as a function of time. As will be described there are at least two AA-PCD pathways each with a multifaceted role played by mitochondrial components, in particular by cytochrome c.

l-Lactate metabolism in HEP G2 cell mitochondria due to the l-lactate dehydrogenase determines the occurrence of the lactate/pyruvate shuttle and the appearance of oxaloacetate, malate and citrate outside mitochondria.

As part of an ongoing study of l-lactate metabolism both in normal and in cancer cells, we investigated whether and how l-lactate metabolism occurs in mitochondria of human hepatocellular carcinoma (Hep G2) cells. We found that Hep G2 cell mitochondria (Hep G2-M) possess an l-lactate dehydrogenase (ml-LDH) restricted to the inner mitochondrial compartments as shown by immunological analysis, confocal microscopy and by assaying ml-LDH activity in solubilized mitochondria. Cytosolic and mitochondrial l-LDHs were found to differ from one another in their saturation kinetics. Having shown that l-lactate itself can enter Hep G2 cells, we found that Hep G2-M swell in ammonium l-lactate, but not in ammonium pyruvate solutions, in a manner inhibited by mersalyl, this showing the occurrence of a carrier-mediated l-lactate transport in these mitochondria. Occurrence of the l-lactate/pyruvate shuttle and the appearance outside mitochondria of oxaloacetate, malate and citrate arising from l-lactate uptake and metabolism together with the low oxygen consumption and membrane potential generation are in favor of an anaplerotic role for l-LAC in Hep G2-M.

Achievements and perspectives in yeast acetic acid-induced programmed cell death pathways.

The use of non-mammalian model organisms, including yeast Saccharomyces cerevisiae, can provide new insights into eukaryotic PCD (programmed cell death) pathways. In the present paper, we report recent achievements in the elucidation of the events leading to PCD that occur as a response to yeast treatment with AA (acetic acid). In particular, ROS (reactive oxygen species) generation, cyt c (cytochrome c) release and mitochondrial function and proteolytic activity will be dealt with as they vary along the AA-PCD time course by using both wild-type and mutant yeast cells. Two AA-PCD pathways are described sharing common features, but distinct from one another with respect to the role of ROS and mitochondria, the former in which YCA1 acts upstream of cyt c release and caspase-like activation in a ROS-dependent manner and the latter in which cyt c release does not occur, but caspase-like activity increases, in a ROS-independent manner.

Cytochrome c Trp65Ser substitution results in inhibition of acetic acid-induced programmed cell death in Saccharomyces cerevisiae.

To gain further insight into the role of cytochrome c (cyt c) in yeast programmed cell death induced by acetic acid (AA-PCD), comparison was made between wild type and two mutant cells, one lacking cyt c and the other (W65Scyc1) expressing a mutant iso-1-cyt c in a form unable to reduce cyt c oxidase, with respect to occurrence of AA-PCD, cyt c release, ROS production and caspase-like activity. We show that in W65Scyc1 cells: i. no release of mutant cyt c occurs with inhibition of W65Scyc1 cell AA-PCD shown to be independent on impairment of electron flow, ii. there is a decrease in ROS production and an increase in caspase-like activity. We conclude that cyt c release does not depend on cyt c function as an electron carrier and that when still associated to the mitochondrial membrane, cyt c in its reduced form has a role in AA-PCD, by regulating ROS production and caspase-like activity.

The rate of ATP export in the extramitochondrial phase via the adenine nucleotide translocator changes in aging in mitochondria isolated from heart left ventricle of either normotensive or spontaneously hypertensive rats.

To find out whether and how deficit of cellular energy supply from mitochondria to cytosol occurs in aging and hypertension, we used mitochondria isolated from 5 to 72 week-old heart left ventricle of either normotensive (WKY) or spontaneous hypertensive (SH) rats as a model system. Measurements were made of the rate of ATP appearance outside mitochondria, due to externally added ADP, as an increase of NADPH absorbance which occurs when ATP is produced in the presence of glucose, hexokinase and glucose-6-phosphate dehydrogenase. Such a rate proved to mirror the function of the adenine nucleotide translocator (ANT) rather than other processes linked to the both oxidative and substrate level phosphorylation. The changes in both Ki for atractyloside and Km for ADP suggest the occurrence of modification of the ANT conformation during aging in which the ANT Vmax was found to decrease in normotensive but to increase under spontaneously hypertension in 24 week-old rats with a subsequent decrease in both cases. ANT function, as investigated in the ADP physiological range (20-60µM), is expected to decrease in normotensive, but to increase in hypertensive rats up to 48 weeks. Later a decrease in the ATP rate of export outside mitochondria should occur in both cases.

L-lactate metabolism can occur in normal and cancer prostate cells via the novel mitochondrial L-lactate dehydrogenase.

Both normal (PTN1A) and cancer (PC3) prostate cells produce high levels of L-lactate because of a low energy supply via the citric cycle and oxidative phosphorylation. Since some mammalian mitochondria possess a mitochondrial L-lactate dehydrogenase (mLDH), we investigated whether prostate cells can take up L-lactate and metabolize it in the mitochondria. We report here that externally added L-lactate can enter both normal and cancer cells and mitochondria, as shown by both the oxygen consumption and by the increase in fluorescence of NAD(P)H which occur as a result of L-lactate addition. In both cell types L-lactate enters mitochondria in a carrier-mediated manner, as shown by the inhibition of swelling measurements due to the non-penetrant thiol reagent mersalyl. An L-lactate dehydrogenase exists in mitochondria of both cell types located in the inner compartment, as shown by kinetic investigation and by immunological analysis. The mLDHs proved to differ from the cytosolic enzymes (which themselves differ from one another) as functionally investigated with respect to kinetic features and pH profile. Normal and cancer cells were found to differ from one another with respect to mLDH protein level and activity, being the enzyme more highly expressed and of higher activity in PC3 cells. Moreover, the kinetic features and pH profiles of the PC3 mLDH also differ from those of the PNT1A enzyme, this suggesting the occurrence of separate isoenzymes.

Knock-out of metacaspase and/or cytochrome c results in the activation of a ROS-independent acetic acid-induced programmed cell death pathway in yeast.

To gain further insight into yeast acetic acid-induced programmed cell death (AA-PCD) we analyzed the effects of the antioxidant N-acetyl-L-cysteine (NAC) on cell viability, hydrogen peroxide (H(2)O(2)) production, DNA fragmentation, cytochrome c (cyt c) release and caspase-like activation in wild type (wt) and metacaspase and/or cyt c-lacking cells. We found that NAC prevents AA-PCD in wt cells, by scavenging H(2)O(2) and by inhibiting both cyt c release and caspase-like activation. This shows the occurrence of a reactive oxygen species (ROS)-dependent AA-PCD. Contrarily no NAC dependent change in AA-PCD of mutant cells was detectable, showing that a ROS-independent AA-PCD can also occur.

L-lactate generates hydrogen peroxide in purified rat liver mitochondria due to the putative L-lactate oxidase localized in the intermembrane space.

In order to ascertain whether and how mitochondria can produce hydrogen peroxide (H(2)O(2)) as a result of L-lactate addition, we monitored H(2)O(2) generation in rat liver mitochondria and in submitochondrial fractions free of peroxisomal and cytosolic contamination. We found that H(2)O(2) is produced independently on the respiratory chain with 1:1 stoichiometry with pyruvate, due to a putative flavine-dependent L-lactate oxidase restricted to the intermembrane space. The L-lactate oxidase reaction shows a hyperbolic dependence on L-lactate concentration and is inhibited by NAD(+) in a competitive manner, being the enzyme different from the L-lactate dehydrogenase isoenzymes as shown by their pH profiles.