Concept: Citric acid cycle
Cysteine S-nitrosylation is a posttranslational modification by which nitric oxide regulates protein function and signaling. Studies of individual proteins have elucidated specific functional roles for S-nitrosylation, but knowledge of the extent of endogenous S-nitrosylation, the sites that are nitrosylated, and the regulatory consequences of S-nitrosylation remains limited. We used mass spectrometry-based methodologies to identify 1011 S-nitrosocysteine residues in 647 proteins in various mouse tissues. We uncovered selective S-nitrosylation of enzymes participating in glycolysis, gluconeogenesis, tricarboxylic acid cycle, and oxidative phosphorylation, indicating that this posttranslational modification may regulate metabolism and mitochondrial bioenergetics. S-nitrosylation of the liver enzyme VLCAD [very long chain acyl-coenzyme A (CoA) dehydrogenase] at Cys(238), which was absent in mice lacking endothelial nitric oxide synthase, improved its catalytic efficiency. These data implicate protein S-nitrosylation in the regulation of β-oxidation of fatty acids in mitochondria.
BACKGROUND: Increased glycolysis is a hallmark of cancer metabolism, yet relatively little is known about this phenotype at premalignant stages of progression. Periodic ischemia occurs in the premalignant condition Barrett’s esophagus (BE) due to tissue damage from chronic acid-bile reflux and may select for early adaptations to hypoxia, including upregulation of glycolysis. METHODOLOGYPRINCIPAL FINDINGS: We compared rates of glycolysis and oxidative phosphorylation in four cell lines derived from patients with BE (CP-A, CP-B, CP-C and CP-D) in response to metabolic inhibitors and changes in glucose concentration. We report that cell lines derived from patients with more advanced genetically unstable BE have up to two-fold higher glycolysis compared to a cell line derived from a patient with early genetically stable BE; however, all cell lines preserve active mitochondria. In response to the glycolytic inhibitor 2-deoxyglucose, the most glycolytic cell lines (CP-C and CP-D) had the greatest suppression of extra-cellular acidification, but were able to compensate with upregulation of oxidative phosphorylation. In addition, these cell lines showed the lowest compensatory increases in glycolysis in response to mitochondrial uncoupling by 2,4-dinitrophenol. Finally, these cell lines also upregulated their oxidative phosphorylation in response to glucose via the Crabtree effect, and demonstrate a greater range of modulation of oxygen consumption. CONCLUSIONSSIGNIFICANCE: Our findings suggest that cells from premalignant Barrett’s esophagus tissue may adapt to an ever-changing selective microenvironment through changes in energy metabolic pathways typically associated with cancer cells.
Glucose stimulated insulin secretion (GSIS) from pancreatic β-cells is triggered by metabolism of the sugar to increase ATP/ADP ratio that blocks the KATP channel leading to membrane depolarization and insulin exocytosis. Other metabolic pathways believed to augment insulin secretion have yet to be fully elucidated. To study metabolic changes during GSIS, liquid chromatography with mass spectrometry was used to determine levels of 87 metabolites temporally following a change in glucose from 3 mM to 10 mM glucose and in response to increasing concentrations of glucose in the INS-1 832/13 β-cell line. 13C-glucose was used to probe flux in specific metabolic pathways. Results include a rapid increase in ATP/ADP, anaplerotic tricarboxylic acid cycle flux, and increases in the malonyl CoA pathway, support prevailing theories of GSIS. Novel findings include that aspartate used for anaplerosis does not derive from the glucose fuel added to stimulate insulin secretion, glucose flux into glycerol-3-phosphate, and esterification of long chain CoAs resulting in rapid consumption of long chain CoAs and de novo generation of phosphatidic acid and diacylglycerol. Further, novel metabolites with potential roles in GSIS such as 5-aminoimidazole-4-carboxamide ribotide (ZMP), GDP-mannose, and farnesyl pyrophosphate were found to be rapidly altered following glucose exposure.
Hypersaline environments pose major challenges to their microbial residents. Microorganisms have to cope with increased osmotic pressure and low water activity and therefore require specific adaptation mechanisms. Although mechanisms have already been thoroughly investigated in the green alga Dunaliella salina and some halophilic yeasts, strategies for osmoadaptation in other protistan groups (especially heterotrophs) are neither as well known nor as deeply investigated as for their prokaryotic counterpart. This is not only due to the recent awareness of the high protistan diversity and ecological relevance in hypersaline systems, but also due to methodological shortcomings. We provide the first experimental study on haloadaptation in heterotrophic microeukaryotes, using the halophilic ciliate Schmidingerothrix salinarum as a model organism. We established three approaches to investigate fundamental adaptation strategies known from prokaryotes. First, hydrogen-1 nuclear magnetic resonance (1H-NMR) spectroscopy was used for the detection, identification, and quantification of intracellular compatible solutes. Second, ion-imaging with cation-specific fluorescent dyes was employed to analyze changes in the relative ion concentrations in intact cells. Third, the effect of salt concentrations on the catalytic performance of S. salinarum malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICDH) was determined. 1H-NMR spectroscopy identified glycine betaine (GB) and ectoine (Ect) as the main compatible solutes in S. salinarum. Moreover, a significant positive correlation of intracellular GB and Ect concentrations and external salinity was observed. The addition of exogenous GB, Ect, and choline (Ch) stimulated the cell growth notably, indicating that S. salinarum accumulates the solutes from the external medium. Addition of external 13C2-Ch resulted in conversion to 13C2-GB, indicating biosynthesis of GB from Ch. An increase of external salinity up to 21% did not result in an increase in cytoplasmic sodium concentration in S. salinarum. This, together with the decrease in the catalytic activities of MDH and ICDH at high salt concentration, demonstrates that S. salinarum employs the salt-out strategy for haloadaptation.
The development of metabolic approaches towards understanding the origins of life, which have focused mainly on the citric acid (TCA) cycle, have languished-primarily due to a lack of experimentally demonstrable and sustainable cycle(s) of reactions. We show here the existence of a protometabolic analog of the TCA involving two linked cycles, which convert glyoxylate into CO2 and produce aspartic acid in the presence of ammonia. The reactions proceed from either pyruvate, oxaloacetate or malonate in the presence of glyoxylate as the carbon source and hydrogen peroxide as the oxidant under neutral aqueous conditions and at mild temperatures. The reaction pathway demonstrates turnover under controlled conditions. These results indicate that simpler versions of metabolic cycles could have emerged under potential prebiotic conditions, laying the foundation for the appearance of more sophisticated metabolic pathways once control by (polymeric) catalysts became available.
Here, we developed a new synthetic lethal strategy for further optimizing the eradication of cancer stem cells (CSCs). Briefly, we show that chronic treatment with the FDA-approved antibiotic Doxycycline effectively reduces cellular respiration, by targeting mitochondrial protein translation. The expression of four mitochondrial DNA encoded proteins (MT-ND3, MT-CO2, MT-ATP6 and MT-ATP8) is suppressed, by up to 35-fold. This high selection pressure metabolically synchronizes the surviving cancer cell sub-population towards a predominantly glycolytic phenotype, resulting in metabolic inflexibility. We directly validated this Doxycycline-induced glycolytic phenotype, by using metabolic flux analysis and label-free unbiased proteomics.Next, we identified two natural products (Vitamin C and Berberine) and six clinically-approved drugs, for metabolically targeting the Doxycycline-resistant CSC population (Atovaquone, Irinotecan, Sorafenib, Niclosamide, Chloroquine, and Stiripentol). This new combination strategy allows for the more efficacious eradication of CSCs with Doxycycline, and provides a simple pragmatic solution to the possible development of Doxycycline-resistance in cancer cells. In summary, we propose the combined use of i) Doxycycline (Hit-1: targeting mitochondria) and ii) Vitamin C (Hit-2: targeting glycolysis), which represents a new synthetic-lethal metabolic strategy for eradicating CSCs.This type of metabolic Achilles' heel will allow us and others to more effectively “starve” the CSC population.
Chronic fatigue syndrome (CFS) is a highly debilitating disease of unknown aetiology. Abnormalities in bioenergetic function have been cited as one possible cause for CFS. Preliminary studies were performed to investigate cellular bioenergetic abnormalities in CFS patients. A series of assays were conducted using peripheral blood mononuclear cells (PBMCs) from CFS patients and healthy controls. These experiments investigated cellular patterns in oxidative phosphorylation (OXPHOS) and glycolysis. Results showed consistently lower measures of OXPHOS parameters in PBMCs taken from CFS patients compared with healthy controls. Seven key parameters of OXPHOS were calculated: basal respiration, ATP production, proton leak, maximal respiration, reserve capacity, non-mitochondrial respiration, and coupling efficiency. While many of the parameters differed between the CFS and control cohorts, maximal respiration was determined to be the key parameter in mitochondrial function to differ between CFS and control PBMCs due to the consistency of its impairment in CFS patients found throughout the study (p≤0.003). The lower maximal respiration in CFS PBMCs suggests that when the cells experience physiological stress they are less able to elevate their respiration rate to compensate for the increase in stress and are unable to fulfil cellular energy demands. The metabolic differences discovered highlight the inability of CFS patient PBMCs to fulfil cellular energetic demands both under basal conditions and when mitochondria are stressed during periods of high metabolic demand.
High-energy-density, green, safe batteries are highly desirable for meeting the rapidly growing needs of portable electronics. The incomplete oxidation of sugars mediated by one or a few enzymes in enzymatic fuel cells suffers from low energy densities and slow reaction rates. Here we show that nearly 24 electrons per glucose unit of maltodextrin can be produced through a synthetic catabolic pathway that comprises 13 enzymes in an air-breathing enzymatic fuel cell. This enzymatic fuel cell is based on non-immobilized enzymes that exhibit a maximum power output of 0.8 mW cm(-2) and a maximum current density of 6 mA cm(-2), which are far higher than the values for systems based on immobilized enzymes. Enzymatic fuel cells containing a 15% (wt/v) maltodextrin solution have an energy-storage density of 596 Ah kg(-1), which is one order of magnitude higher than that of lithium-ion batteries. Sugar-powered biobatteries could serve as next-generation green power sources, particularly for portable electronics.
Body-wide changes in bioenergetics, i.e., energy metabolism, occur in normal aging and disturbed bioenergetics may be an important contributing mechanism underlying late-onset Alzheimer’s disease (LOAD). We investigated the bioenergetic profiles of fibroblasts from LOAD patients and healthy controls, as a function of age and disease. LOAD cells exhibited an impaired mitochondrial metabolic potential and an abnormal redox potential, associated with reduced nicotinamide adenine dinucleotide metabolism and altered citric acid cycle activity, but not with disease-specific changes in mitochondrial mass, production of reactive oxygen species, transmembrane instability, or DNA deletions. LOAD fibroblasts demonstrated a shift in energy production to glycolysis, despite an inability to increase glucose uptake in response to IGF-1. The increase of glycolysis and the abnormal mitochondrial metabolic potential in LOAD appeared to be inherent, as they were disease- and not age-specific. Our findings support the hypothesis that impairment in multiple interacting components of bioenergetic metabolism may be a key mechanism contributing to the risk and pathophysiology of LOAD.
The study of combined effects of pesticides represents a challenge for toxicology. In the case of the new growing generation of genetically modified (GM) plants with stacked traits, glyphosate-based herbicides (like Roundup) residues are present in the Roundup-tolerant edible plants (especially corns) and mixed with modified Bt insecticidal toxins that are produced by the GM plants themselves. The potential side effects of these combined pesticides on human cells are investigated in this work. Here we have tested for the very first time Cry1Ab and Cry1Ac Bt toxins (10 ppb to 100 ppm) on the human embryonic kidney cell line 293, as well as their combined actions with Roundup, within 24 h, on three biomarkers of cell death: measurements of mitochondrial succinate dehydrogenase, adenylate kinase release by membrane alterations and caspase 3/7 inductions. Cry1Ab caused cell death from 100 ppm. For Cry1Ac, under such conditions, no effects were detected. The Roundup tested alone from 1 to 20 000 ppm is necrotic and apoptotic from 50 ppm, far below agricultural dilutions (50% lethal concentration 57.5 ppm). The only measured significant combined effect was that Cry1Ab and Cry1Ac reduced caspases 3/7 activations induced by Roundup; this could delay the activation of apoptosis. There was the same tendency for the other markers. In these results, we argue that modified Bt toxins are not inert on nontarget human cells, and that they can present combined side-effects with other residues of pesticides specific to GM plants. Copyright © 2012 John Wiley & Sons, Ltd.