NADH‐dependent dehydrogenase activity estimation by flow cytometric analysis of 3‐(4,5‐dimethylthiazolyl‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) reduction

Huet, Olivier; Petit, J.; Ratinaud, Marie‐Hélène; Julien, Raymond

doi:10.1002/cyto.990130513

Cited by 56 publications

(33 citation statements)

References 27 publications

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“…Cell viability was assessed by the MTT assay that is based on the ability of viable cells to have sufficient mitochondrial dehydrogenase activity to reduce MTT (19,34). Considering the linear relationship between cell number and absorbance (40), palmitate (100 M for 24 h) in-duced a significant (P Ͻ 0.01) reduction in absorbance from control and produced a significant (P Ͻ 0.01) loss of cell viability (Fig.…”

Section: Resultsmentioning

confidence: 99%

Palmitate-induced cardiac apoptosis is mediated through CPT-1 but not influenced by glucose and insulin

Kong

Rabkin

2002

American Journal of Physiology-Heart and Circulatory Physiology

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Kong, Jennifer Y., and Simon W. Rabkin. Palmitateinduced cardiac apoptosis is mediated through CPT-1 but not influenced by glucose and insulin. Am J Physiol Heart Circ Physiol 282: H717-H725, 2002; 10.1152/ajpheart.00257. 2001.-To test the hypothesis that regulation of palmitate metabolism, through carnitine palmitoyl transferase-1 (CPT-1) or through alterations of glycolysis, was involved in the pathway of palmitate-mediated cell death, cardiomyocytes were cultured from 7-day-old chick embryos. Palmitateinduced cell death, assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, was enhanced by carnitine, a cofactor needed for palmitate transport into mitochondria via CPT-1. Carnitine co-incubation with palmitate significantly (P Ͻ 0.01) increased the amount of apoptotic cells, assessed by propidium iodine staining and fluorescent-activated cell sorting analysis compared with treatment with either palmitate or carnitine alone. The CPT-1 inhibitor oxfenicine significantly (P Ͻ 0.05) blocked the cell death induced by the combination of palmitate and carnitine. The short-chain saturated fatty acid capric acid (100 M), which is not likely transported by CPT-1, did not significantly affect cell viability, whereas the C18 saturated fatty acid stearic (100 M) significantly (P Ͻ 0.01) reduced cell viability and to a similar extent as palmitate. In contrast, there was no significant alteration of palmitate-induced cell death by cotreatment with 100 nM insulin ϩ 2 g/l glucose or 1 mM lactate, which promote ATP generation by glycolysis rather than fatty acid oxidation. Fumonisin did not alter palmitate-induced cell death or apoptosis, suggesting that the effect of palmitate was not operative through increased ceramide synthesis. These results suggest that oxidation of palmitate through CPT-1 is involved in the production of apoptosis in cardiomyocytes.carnitine; fatty acid metabolism; oxfenicine; fumonisin; palmitate; capric acid FATTY ACIDS ARE A PRINCIPAL source of energy for the heart, and the metabolism of fatty acids is of fundamental importance for cardiac function (for reviews, see Refs. 23,24,48). Briefly, fatty acid metabolism involves fatty acids binding to carrier binding proteins that are transported across the sarcolemmal membrane, and once inside the cell are metabolized to long chain acyl-CoA by acyl-CoA synthetase (23,24,48). The acyl moieties are transferred to the mitochondria by a series of enzymes involving carnitine palmitoyl transferase-1 and -2 (CPT-1 and -2) as well as carnitine-acylcarnitine translocase. In the mitochondria, long-chain acyl-CoA is subjected to ␤-oxidation-producing acetyl CoA, a fatty acyl moiety less two carbons, and the by-products, reduced flavin adenine dinucleotide and reduced nicotinamide adenine dinucleotide. Fatty acid oxidation also occurs in peroxisomes (23,24,48) but the amount is small (9).

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Section: Resultsmentioning

confidence: 99%

Palmitate-induced cardiac apoptosis is mediated through CPT-1 but not influenced by glucose and insulin

Kong

Rabkin

2002

American Journal of Physiology-Heart and Circulatory Physiology

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“…Inhibition of mitochondrial protein synthesis decreased the mitochondrial capacity to synthesize ATP (Huet et al, 1992), and abolished cardiolipin asymmetry. Interactions between newly synthesized polypeptides and cardiolipin seem to contribute partly to the asymmetry.…”

Section: Discussionmentioning

confidence: 97%

Direct analysis and significance of cardiolipin transverse distribution in mitochondrial inner membranes

Petit¹,

Huet²,

Gallet³

et al. 1994

European Journal of Biochemistry

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The distribution of cardiolipin across the inner mitochondrial membrane was directly determined by using the ability of the fluorescent dye 1 O-N-nonyl-3,6-bis(dimethylamino)acridine (10-N-nonyl acridine orange) to form dimers when it interacts with the diacidic phospholipid. Two independent methods were employed : (a) a spectrophotometric measurement of 10-N-nonyl acridine orange binding to isolated rat liver mitochondria, mitoplasts and inside-out submitochondrial particles, and (b) a flow-cytometric analysis of specific red fluorescence, emitted when two dye molecules are bound to one membrane cardiolipin ; the stoichiometry of 10-N-nonyl acridine orange binding to phosphatidylserine and phosphatidylinositol, 1 mol dye/mol phospholipid, prevented dye dimerisation and subsequent red-fluorescence appearance. 57% total cardiolipin was present in the outer leaflets of inner membranes of isolated organelles, a distribution confirmed by saturation measurements for mitoplasts and inside-out submitochondrial particles. The same asymmetry was directly observed in situ with mitochondrial membranes of quiescent L1210 cells, and with mitochondrial membranes of respiring yeasts. Nevertheless, alterations in ATP synthesis and inhibition of mitochondrial protein synthesis revealed that cardiolipin distribution was apparently tightly correlated with mitochondrial membrane assembly and activity.

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“…Mitochondrial Enzymology and Immunoblotting-Mitochondria used for ROS assays were frozen at Ϫ70°C and used for assessment of complex II activity and complex II subunit levels. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay allows a rapid assessment of complex II activity on multiple heart preparations (45). The presence of the specific inhibitor malonate enabled complex II activity to be distinguished from those of the other mitochondrial dehydrogenases.…”

Section: Methodsmentioning

confidence: 99%

Mitochondrial Reactive Oxygen Species in Mice Lacking Superoxide Dismutase 2

Morten¹,

Ackrell

Melov

2006

Journal of Biological Chemistry

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. resulting from complex II normalization had no effect on steady state levels due to the rapid conversion to H 2 O 2 , a process presumably aided by the presence of the EUK189, an SOD mimetic.Age-related diseases, including cancer, type 2 diabetes, and Alzheimer and Parkinson diseases, show a clear correlation with mitochondrial dysfunction and reactive oxygen species (ROS) 3 (5-12). Tissues with high energy expenditure such as brain, heart, muscle, and pancreas have significant ATP requirements. The vast majority of such ATP is derived from mitochondrial oxidative phosphorylation, a series of sequential electron transfers along the respiratory chain resulting in the reduction of O 2 to water concomitant with the production of ATP. During the course of normal respiration under state IV conditions, as much as 0.4 -4% (13-15) of all oxygen consumed can be converted into the free radical superoxide, which in turn can be converted to other ROS, although physiological levels may be dependent on age, tissue, and cell type. The majority of mitochondrial superoxide is generated from two respiratory chain complexes, complex I and complex III (16), the former producing superoxide on the matrix side of the mitochondrial membrane and the latter liberating superoxide both to the matrix side and into the intermembrane space (17)(18)(19)(20). The relative importance of the two sites in mitochondrial superoxide production is currently an area of debate (19,21). When electrons are passed from succinate to NAD via energy-dependent reverse electron flow, complex I produces ROS at relatively high rates (14,15,17,(21)(22)(23). During forward electron flow both complexes produce ROS at very low rates unless respiratory chain inhibitors such as the complex I inhibitor rotenone or the complex III inhibitor antimycin A are present (20,24). The potential consequences of this endogenously produced ROS upon mammalian physiology became clearer with the creation of superoxide dismutase null mice.

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NADH‐dependent dehydrogenase activity estimation by flow cytometric analysis of 3‐(4,5‐dimethylthiazolyl‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) reduction

Cited by 56 publications

References 27 publications

Palmitate-induced cardiac apoptosis is mediated through CPT-1 but not influenced by glucose and insulin

Palmitate-induced cardiac apoptosis is mediated through CPT-1 but not influenced by glucose and insulin

Direct analysis and significance of cardiolipin transverse distribution in mitochondrial inner membranes

Mitochondrial Reactive Oxygen Species in Mice Lacking Superoxide Dismutase 2

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