Regulation of vascular smooth muscle cell bioenergetic function by protein glutathiolation

AbstractProtein thiolation by glutathione is a reversible and regulated post-translational modification that is increased in response to oxidants and nitric oxide. Because many mitochondrial enzymes contain critical thiol residues, it has been hypothesized that thiolation reactions regulate cell metabolism and survival. However, it has been difficult to differentiate the biological effects due to protein thiolation from other oxidative protein modifications. In this study, we used diamide to titrate protein glutathiolation and examined its impact on glycolysis, mitochondrial function, and cell death in rat aortic smooth muscle cells. Treatment of cells with diamide increased protein glutathiolation in a concentration-dependent manner and had comparably little effect on protein–protein disulfide formation. Diamide increased mitochondrial proton leak and decreased ATP-linked mitochondrial oxygen consumption and cellular bioenergetic reserve capacity. Concentrations of diamide above 200 μM promoted acute bioenergetic failure and caused cell death, whereas lower concentrations of diamide led to a prolonged increase in glycolytic flux and were not associated with loss of cell viability. Depletion of glutathione using buthionine sulfoximine had no effect on basal protein thiolation or cellular bioenergetics but decreased diamide-induced protein glutathiolation and sensitized the cells to bioenergetic dysfunction and death. The effects of diamide on cell metabolism and viability were fully reversible upon addition of dithiothreitol. These data suggest that protein thiolation modulates key metabolic processes in both the mitochondria and cytosol

Mapping the human platelet lipidome reveals cytosolic phospholipase A2 as a regulator of mitochondrial bioenergetics during activation

Human platelets acutely increase mitochondrial energy generation following stimulation. Herein, a lipidomic circuit was uncovered whereby the substrates for this are exclusively provided by cPLA2, including multiple fatty acids and oxidized species that support energy generation via β-oxidation. This indicates that acute lipid membrane remodeling is required to support energetic demands during platelet activation. Phospholipase activity is linked to energy metabolism, revealing cPLA2 as a central regulator of both lipidomics and energy flux. Using a lipidomic approach (LipidArrays), we also estimated the total number of lipids in resting, thrombin-activated, and aspirinized platelets. Significant diversity between genetically unrelated individuals and a wealth of species was revealed. Resting platelets demonstrated ∼5,600 unique species, with only ∼50% being putatively identified. Thrombin elevated ∼900 lipids >2-fold with 86% newly appearing and 45% inhibited by aspirin supplementation, indicating COX-1 is required for major activation-dependent lipidomic fluxes. Many lipids were structurally identified. With ∼50% of the lipids being absent from databases, a major opportunity for mining lipids relevant to human health and disease is presente

Mitochondrial Genetic Background Modulates Bioenergetics and Susceptibility to Acute Cardiac Volume Overload

Dysfunctional bioenergetics has emerged as a key feature in many chronic pathologies such as diabetes and cardiovascular disease. This has led to the mitochondrial paradigm in which it has been proposed that mtDNA sequence variation contributes to disease susceptibility. In the present study we show a novel animal model of mtDNA polymorphisms, the MNX (mitochondrial–nuclear exchange) mouse, in which the mtDNA from the C3H/HeN mouse has been inserted on to the C57/BL6 nuclear background and vice versa to test this concept. Our data show a major contribution of the C57/BL6 mtDNA to the susceptibility to the pathological stress of cardiac volume overload which is independent of the nuclear background. Mitochondria harbouring the C57/BL6J mtDNA generate more ROS (reactive oxygen species) and have a higher mitochondrial membrane potential relative to those with C3H/HeN mtDNA, independent of nuclear background. We propose this is the primary mechanism associated with increased bioenergetic dysfunction in response to volume overload. In summary, these studies support the ‘mitochondrial paradigm’ for the development of disease susceptibility, and show that the mtDNA modulates cellular bioenergetics, mitochondrial ROS generation and susceptibility to cardiac stress

A mitochondria-targeted mass spectrometry probe to detect glyoxals: implications for diabetes

The glycation of protein and nucleic acids that occurs as a consequence of hyperglycaemia disrupts cell function and contributes to many pathologies, including those associated with diabetes and aging. Intracellular glycation occurs following the generation of the reactive 1,2-dicarbonyls methylglyoxal and glyoxal and disruption to mitochondrial function is associated with hyperglycemia. However, the contribution of these reactive dicarbonyls to mitochondrial damage in pathology is unclear due to uncertainties about their levels within mitochondria in cells and in vivo. To address this we have developed a mitochondria-targeted reagent (MitoG) designed to assess the levels of mitochondrial dicarbonyls within cells. MitoG comprises a lipophilic triphenylphosphonium cationic function, which directs the molecules to mitochondria within cells and an o-phenylenediamine moiety that reacts with dicarbonyls to give distinctive and stable products. The extent of accumulation of these diagnostic heterocyclic products can be readily and sensitively quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), enabling changes to be determined. Using the MitoG-based analysis we assessed the formation of methylglyoxal and glyoxal in response to hyperglycaemia in cells in culture and in the Akita mouse model of diabetes in vivo. These findings indicated that the levels of methylglyoxal and glyoxal within mitochondria increase during hyperglycaemia in both cells and in vivo, suggesting that they can contribute to the pathological mitochondrial dysfunction that occurs in diabetes and aging

Lipid Metabolites Enhance Secretion Acting on SNARE Microdomains and Altering the Extent and Kinetics of Single Release Events in Bovine Adrenal Chromaffin Cells

Lipid molecules such as arachidonic acid (AA) and sphingolipid metabolites have been implicated in modulation of neuronal and endocrine secretion. Here we compare the effects of these lipids on secretion from cultured bovine chromaffin cells. First, we demonstrate that exogenous sphingosine and AA interact with the secretory apparatus as confirmed by FRET experiments. Examination of plasma membrane SNARE microdomains and chromaffin granule dynamics using total internal reflection fluorescent microscopy (TIRFM) suggests that sphingosine production promotes granule tethering while arachidonic acid promotes full docking. Our analysis of single granule release kinetics by amperometry demonstrated that both sphingomyelinase and AA treatments enhanced drastically the amount of catecholamines released per individual event by either altering the onset phase of or by prolonging the off phase of single granule catecholamine release kinetics. Together these results demonstrate that the kinetics and extent of the exocytotic fusion pore formation can be modulated by specific signalling lipids through related functional mechanisms

High fat diet induces dysregulation of hepatic oxygen gradients and mitochondrial function in vivo

NAFLD (non-alcoholic fatty liver disease), associated with obesity and the cardiometabolic syndrome, is an important medical problem affecting up to 20% of western populations. Evidence indicates that mitochondrial dysfunction plays a critical role in NAFLD initiation and progression to the more serious condition of NASH (non-alcoholic steatohepatitis). Herein we hypothesize that mitochondrial defects induced by exposure to a HFD (high fat diet) contribute to a hypoxic state in liver and this is associated with increased protein modification by RNS (reactive nitrogen species). To test this concept, C57BL/6 mice were pair-fed a control diet and HFD containing 35% and 71% total calories (1 cal≈4.184 J) from fat respectively, for 8 or 16 weeks and liver hypoxia, mitochondrial bioenergetics, NO (nitric oxide)-dependent control of respiration, and 3-NT (3-nitrotyrosine), a marker of protein modification by RNS, were examined. Feeding a HFD for 16 weeks induced NASH-like pathology accompanied by elevated triacylglycerols, increased CYP2E1 (cytochrome P450 2E1) and iNOS (inducible nitric oxide synthase) protein, and significantly enhanced hypoxia in the pericentral region of the liver. Mitochondria from the HFD group showed increased sensitivity to NO-dependent inhibition of respiration compared with controls. In addition, accumulation of 3-NT paralleled the hypoxia gradient in vivo and 3-NT levels were increased in mitochondrial proteins. Liver mitochondria from mice fed the HFD for 16 weeks exhibited depressed state 3 respiration, uncoupled respiration, cytochrome c oxidase activity, and mitochondrial membrane potential. These findings indicate that chronic exposure to a HFD negatively affects the bioenergetics of liver mitochondria and this probably contributes to hypoxic stress and deleterious NO-dependent modification of mitochondrial proteins

Cell signalling by reactive lipid species: new concepts and molecular mechanisms

The process of lipid peroxidation is widespread in biology and is mediated through both enzymatic and non-enzymatic pathways. A significant proportion of the oxidized lipid products are electrophilic in nature, the RLS (reactive lipid species), and react with cellular nucleophiles such as the amino acids cysteine, lysine and histidine. Cell signalling by electrophiles appears to be limited to the modification of cysteine residues in proteins, whereas non-specific toxic effects involve modification of other nucleophiles. RLS have been found to participate in several physiological pathways including resolution of inflammation, cell death and induction of cellular antioxidants through the modification of specific signalling proteins. The covalent modification of proteins endows some unique features to this signalling mechanism which we have termed the ‘covalent advantage’. For example, covalent modification of signalling proteins allows for the accumulation of a signal over time. The activation of cell signalling pathways by electrophiles is hierarchical and depends on a complex interaction of factors such as the intrinsic chemical reactivity of the electrophile, the intracellular domain to which it is exposed and steric factors. This introduces the concept of electrophilic signalling domains in which the production of the lipid electrophile is in close proximity to the thiol-containing signalling protein. In addition, we propose that the role of glutathione and associated enzymes is to insulate the signalling domain from uncontrolled electrophilic stress. The persistence of the signal is in turn regulated by the proteasomal pathway which may itself be subject to redox regulation by RLS. Cell death mediated by RLS is associated with bioenergetic dysfunction, and the damaged proteins are probably removed by the lysosome-autophagy pathway

α-Tocopherol mediated peroxidation in the copper (II) and met myoglobininduced oxidation of human low density lipoprotein: The influence of lipid hydroperoxides

AbstractThe principal antioxidant in human LDL, α-tocopherol, is converted to the α-tocopheroxyl radical after reaction with peroxyl radicals or Cu2+, and, if it does not terminate with peroxyl radicals, could initiate lipid peroxidation; a phenomenon called ‘tocopherol mediated peroxidation’. Only in the presence of Cu2+ and low levels of lipid hydroperoxides was an α-tocopherol dependent decrease in the resistance of LDL to oxidation detected. This suggests that tocopherol mediated peroxidation will probably not contribute significantly as a pro-oxidant process in those individuals most at risk of developing atherosclerosis through an oxidative mechanism