Effect of Nicotinamide Adenine Dinucleotide on the Oxidation-Reduction Potentials of Lipoamide Dehydrogenase from Pig Heart

Maeda-Yorita, Kazuko; Aki, Kenji

doi:10.1093/oxfordjournals.jbchem.a134886

Cited by 26 publications

(26 citation statements)

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“…Therefore, the redox Redox-associated Flavin Autofluorescence Changes-We examined the 458:488 nm intensity ratio as a means of isolating the LipDH response from ETF. LipDH has a red-shifted excitation spectrum in comparison with free flavins and ETF (31). Previous work has used this red shift to partially isolate LipDH fluorescence using 488-nm excitation (17)(18)(19)(20).…”

Section: Resultsmentioning

confidence: 99%

Quantitative NAD(P)H/Flavoprotein Autofluorescence Imaging Reveals Metabolic Mechanisms of Pancreatic Islet Pyruvate Response

Rocheleau

Head

Piston

2004

Journal of Biological Chemistry

182

158

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Glucose-stimulated insulin secretion is a multistep process dependent on ␤-cell metabolic flux. Our previous studies on intact pancreatic islets used two-photon NAD(P)H imaging as a quantitative measure of the combined redox signal from NADH and NADPH (referred to as NAD(P)H). These studies showed that pyruvate, a non-secretagogue, enters ␤-cells and causes a transient rise in NAD(P)H. To further characterize the metabolic fate of pyruvate, we have now developed one-photon flavoprotein microscopy as a simultaneous assay of lipoamide dehydrogenase (LipDH) autofluorescence. This flavoprotein is in direct equilibrium with mitochondrial NADH. Hence, a comparison of LipDH and NAD(P)H autofluorescence provides a method to distinguish the production of NADH, NADPH, or both. Using this method, the glucose dose response is consistent with an increase in both NADH and NADPH. In contrast, the transient rise in NAD(P)H observed with pyruvate stimulation is not accompanied by a significant change in LipDH, which indicates that pyruvate raises cellular NADPH without raising NADH. In comparison, methyl pyruvate stimulated a robust NADH and NADPH response. These data provide new evidence that exogenous pyruvate does not induce a significant rise in mitochondrial NADH. This inability likely results in its failure to produce the ATP necessary for stimulated secretion of insulin. Overall, these data are consistent with either a restricted pyruvate dehydrogenasedependent metabolism or a buffering of the NADH response by other metabolic mechanisms.

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Section: Resultsmentioning

confidence: 99%

Quantitative NAD(P)H/Flavoprotein Autofluorescence Imaging Reveals Metabolic Mechanisms of Pancreatic Islet Pyruvate Response

Rocheleau

Head

Piston

2004

Journal of Biological Chemistry

182

158

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“…3b). The increase in the protein-bound FAD lifetime in high-grade precancers could indicate decreased NADϩ levels in high-grade precancers compared with normal (13).…”

Section: Discussionmentioning

confidence: 99%

“…NADH has a short and long lifetime component, respectively, depending on whether it is in a free or protein-bound state (10), and FAD has a short and long lifetime component, respectively, depending on whether it is in a protein-bound or free state (11). The shorter fluorescence lifetimes of both protein-bound FAD and free NADH are due to dynamic quenching by the adenine moiety (12,13). The fluorescence lifetime of protein-bound NADH depends on the enzyme to which it is bound (14), and this suggests that changes in NADH binding with cancer development can be probed by the lifetime of this fluorophore.…”

mentioning

confidence: 99%

“…The fluorescence lifetime of protein-bound NADH depends on the enzyme to which it is bound (14), and this suggests that changes in NADH binding with cancer development can be probed by the lifetime of this fluorophore. The fluorescence lifetime of protein-bound FAD decreases in the presence of NADϩ because of increased intramolecular dynamic quenching in the presence of NADϩ (13). This suggests that the lifetime of protein-bound FAD is sensitive to changes in NADϩ levels.…”

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confidence: 99%

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In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia

Skala

Riching

Gendron‐Fitzpatrick

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

917

901

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Metabolic imaging of the relative amounts of reduced NADH and FAD and the microenvironment of these metabolic electron carriers can be used to noninvasively monitor changes in metabolism, which is one of the hallmarks of carcinogenesis. This study combines cellular redox ratio, NADH and FAD lifetime, and subcellular morphology imaging in three dimensions to identify intrinsic sources of metabolic and structural contrast in vivo at the earliest stages of cancer development. There was a significant (P < 0.05) increase in the nuclear to cytoplasmic ratio (NCR) with depth within the epithelium in normal tissues; however, there was no significant change in NCR with depth in precancerous tissues. The redox ratio significantly decreased in the less differentiated basal epithelial cells compared with the more mature cells in the superficial layer of the normal stratified squamous epithelium, indicating an increase in metabolic activity in cells with increased NCR. However, the redox ratio was not significantly different between the superficial and basal cells in precancerous tissues. A significant decrease was observed in the contribution and lifetime of protein-bound NADH (averaged over the entire epithelium) in both low-and high-grade epithelial precancers compared with normal epithelial tissues. In addition, a significant increase in the protein-bound FAD lifetime and a decrease in the contribution of protein-bound FAD are observed in high-grade precancers only. Increased intracellular variability in the redox ratio, NADH, and FAD fluorescence lifetimes were observed in precancerous cells compared with normal cells.T he electron transport chain is the most efficient means of energy production in cells. The electron transport chain produces energy in the form of ATP by transferring electrons to molecular oxygen. The metabolic coenzymes FAD and NADH are the primary electron acceptor and donor, respectively, in oxidative phosphorylation. Neoplastic cells have an increased metabolic demand relative to normal cells because of rapid cell division (1), and neoplastic metabolism is associated with changes in the relative concentrations of NADH and FAD (2-4). Many enzymes bind to NADH and FAD in the metabolic pathway (5), and, as favored metabolic pathways shift with cancer progression, the distribution of NADH and FAD binding sites also change (6). Thus, metabolic imaging of the relative amount of NADH and FAD, and their microenvironment (such as binding sites and/or the presence of local quenchers) can shed light on metabolic changes associated with carcinogenesis.The metabolic coenzymes NADH and FAD are autofluorescent and can be monitored nondestructively and without exogenous labels, using optical techniques. The most common optical method for metabolic imaging is the ''redox ratio,'' which is the ratio of the fluorescence intensity of FAD and NADH (7). This optical redox ratio provides relative changes in the oxidationreduction state in the cell. The redox ratio is sensitive to changes in the cellular metabolic rate and...

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“…The spectrum, which voproteins are completely reduced. It was not possible to visualize the possible further autofluorescence was obtained by addition of substrate (octanoate) and rotenone, is typical for the oxidized flavin moiety of quenching effect of dithionite (due to the large overlap of emission spectra of a-lipoamide dehydrogenase and a-lipoamide dehydrogenase (solid line) with the emission maximum at 525 nm (19). This flavoprotein is as the exclusively dithionite-reducible flavin fraction in Fig.…”

Section: Methodsmentioning

confidence: 98%

Flow Cytometric Detection of Mitochondrial Dysfunction in Subpopulations of Human Mononuclear Cells

Kunz

Luley

Winkler

et al. 1997

Analytical Biochemistry

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Effect of Nicotinamide Adenine Dinucleotide on the Oxidation-Reduction Potentials of Lipoamide Dehydrogenase from Pig Heart

Cited by 26 publications

References 0 publications

Quantitative NAD(P)H/Flavoprotein Autofluorescence Imaging Reveals Metabolic Mechanisms of Pancreatic Islet Pyruvate Response

Quantitative NAD(P)H/Flavoprotein Autofluorescence Imaging Reveals Metabolic Mechanisms of Pancreatic Islet Pyruvate Response

In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia

Flow Cytometric Detection of Mitochondrial Dysfunction in Subpopulations of Human Mononuclear Cells

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