Genetically encoded biosensors for evaluating NAD+/NADH ratio in cytosolic and mitochondrial compartments

1. Because it has been shown that a majority of alcoholics are subclinically scorbutic, the metabolism of ethanol was studied in subclinically-scorbutic guinea-pigs. 2. Hepatic alcohol dehydrogenase activity was raised maximally by ethanol within 2 days. 3. In twenty-three subclinically-scorbutic guinea-pigs fed ethanol for 2 weeks, the alcohol dehydrogenase activity (±SD) was 11·5 ± 1·2 units/g of liver protein compared with 8·6 ± 0·6 units/g of liver protein in twenty-three healthy animals fed ethanol. 4. The NAD+/NADH ratio in subclinically-scorbutic guinea-pigs and healthy guinea-pigs fed ethanol, shows that there is more NAD+ available for oxidation of alcohol in subclinically-scorbutic guinea-pigs. These results may explain the increased tolerance of alcoholics to alcohol.

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Cesium Treatment Depresses Glycolysis Pathway in HeLa Cell

Cellular Physiology and Biochemistry ◽

10.33594/000000399 ◽

2021 ◽

Vol 55 (4) ◽

pp. 477-488

Keyword(s):

Hela Cell ◽

Nicotinamide Adenine Dinucleotide ◽

Aerobic Glycolysis ◽

Biological Effects ◽

Glycolytic Enzyme ◽

Nicotinamide Adenine ◽

Reduced Form ◽

Dependent Manner ◽

Nadh Ratio ◽

Glycolysis Pathway

Background/Aims: Cesium (Cs) is an alkali metal element that is of no essential use for humans; it has no known beneficial function that is verified by clinical research. When used as an alternative cancer therapy, it even causes toxicity in high doses. Thus, before using Cs as treatment in clinical settings, it is important to clearly determine its biological effects on cells. However, Cs was found to suppress the proliferation of human cervical cancer cells in a dose-dependent manner, and it was assumed that Cs inhibits the glycolysis pathway. In this study, we clearly determined the step of the glycolysis pathway that is affected by Cs. Methods: The glycolytic enzyme expressions, activities, and metabolite concentrations in HeLa cells were measured by PCR, western blotting, and enzymatic methods, after treating the cells with Cs for 3 days. Results: Cs treatment decreased transcriptional and expression levels of hexokinase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase (PK), and lactate dehydrogenase and the activity of PK. Analysis of glycolysis pathway metabolites revealed that Cs treatment reduces lactate level and increases the level of nicotinamide adenine dinucleotide (oxidized form, NAD+); however, it did not affect the levels of pyruvate and nicotinamide adenine dinucleotide (reduced form, NADH). Increase of the [NAD+]/[NADH] ratio and decrease of the [lactate]/[pyruvate] ratio indicate that Cs treatment inhibits the aerobic glycolysis pathway. Conclusion: Cs treatment inhibits PK activity and increases the [NAD+]/[NADH] ratio. Hence, Cs has been determined to inhibit glycolysis, especially the aerobic glycolysis pathway. These results suggest that suppression of HeLa cell proliferation following Cs treatment was caused by inhibition of aerobic glycolysis by Cs.

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Effect of maternal canine starvation on fetal and neonatal liver metabolism

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1981.240.2.e88 ◽

1981 ◽

Vol 240 (2) ◽

pp. E88-E94

Author(s):

E. L. Miettinen

Keyword(s):

Fatty Acid ◽

Free Fatty Acid ◽

Glucose Concentration ◽

Acid Oxidation ◽

Hepatic Glycogen ◽

Uridine Diphosphate ◽

Nadh Ratio ◽

Free Fatty Acid Oxidation ◽

Diphosphate Glucose ◽

Neonatal Liver

Heptic glycolytic and gluconeogenic intermediates from fasted newborns of five control and five 3-day starved canine mothers (MCS) were studied at 0, 3, 6, 9, and 24 h of age. MCS did not affect fetal hepatic glycogen concentration; however, a significant increase in uridine diphosphate glucose (UDPG) (0.186 vs. 0.106 mumol/g), fructose 6-phosphate (0.084 vs. 0.034), pyruvate (0.321 vs. 0.126), and citrate (0.190 vs. 0.140) concentrations occurred. At 3 h, the intrahepatic glucose concentration among the MCS newborns declined (3.09 vs. 6.34) and remained lower than the controls for up to 9 h. UDPG concentration, however, remained elevated throughout the 24 h. In addition intrahepatic pyruvate was significantly elevated in the MCS group. Elevated phosphoenolpyruvate concentrations were observed between 3 and 6 h. Malate levels were lower than controls between 6 and 9 h and alpha-ketoglutarate was significantly higher between 6 and 24 h. Calculated cytoplasmic NAD/NADH ratio was elevated throughout the 24 h. Hepatic triglycerides were higher than controls up to 9 h. A decline in hepatic triglycerides was observed between 9 and 24 h. The results suggest increased glycolysis and suppressed gluconeogenesis in the MCS puppies, probably because of increased triglyceride synthesis and decreased free fatty acid oxidation resulting in a lack of cytoplasmic NADH.

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PhoY2 of Mycobacteria Is Required for Metabolic Homeostasis and Stress Response

Journal of Bacteriology ◽

10.1128/jb.01556-12 ◽

2012 ◽

Vol 195 (2) ◽

pp. 243-252 ◽

Cited By ~ 14

Author(s):

Chuan Wang ◽

Yi Mao ◽

Jia Yu ◽

Lin Zhu ◽

Ming Li ◽

...

Keyword(s):

Inorganic Phosphate ◽

Stress Conditions ◽

Nutrient Deprivation ◽

Pho Regulon ◽

Metabolic Homeostasis ◽

Content Type ◽

Redox States ◽

Nadh Ratio ◽

Increased Sensitivity

ABSTRACTThe ability of pathogenic mycobacteria to adapt to diverse environments is essential for their success as pathogens. Here we describe a transposon-inactivatedphoY2mutant ofMycobacterium marinum. PhoY2 of mycobacteria is a functional homologue of PhoU inEscherichia coliand an important component of the Pho regulon. We found that PhoY2 is required for maintaining intracellular inorganic phosphate (Pi) homeostasis and balanced energy and redox states. Disruption ofphoY2resulted in elevated levels of intracellular poly-Piand ATP and an elevated NAD+/NADH ratio, and the mutant strain exhibited increased sensitivity to environmental stress conditions, including nutrient deprivation as well as SDS and antibiotic treatments. Taken together, our results suggest that PhoY2 is required for maintaining metabolic homeostasis and adaptation to stress conditions, which may provide an explanation for the suggested role of PhoY2 in drug tolerance.

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Efficient production of androstenedione by repeated batch fermentation in waste cooking oil media through regulating NAD+/NADH ratio and strengthening cell vitality of Mycobacterium neoaurum

Bioresource Technology ◽

10.1016/j.biortech.2019.01.144 ◽

2019 ◽

Vol 279 ◽

pp. 209-217 ◽

Cited By ~ 11

Author(s):

Xiuling Zhou ◽

Yang Zhang ◽

Yanbing Shen ◽

Xiao Zhang ◽

Shuangping Xu ◽

...

Keyword(s):

Batch Fermentation ◽

Waste Cooking Oil ◽

Efficient Production ◽

Cooking Oil ◽

Mycobacterium Neoaurum ◽

Cell Vitality ◽

Repeated Batch Fermentation ◽

Nadh Ratio ◽

Repeated Batch

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Fructose 1,6-Bisphosphatase 2 Plays a Crucial Role in the Induction and Maintenance of Long-Term Potentiation

Cells ◽

10.3390/cells9061375 ◽

2020 ◽

Vol 9 (6) ◽

pp. 1375 ◽

Cited By ~ 2

Author(s):

Przemysław Duda ◽

Tomasz Wójtowicz ◽

Jakub Janczara ◽

Daniel Krowarsch ◽

Aleksandra Czyrek ◽

...

Keyword(s):

Molecular Basis ◽

Late Phase ◽

Long Term Potentiation ◽

Memory Formation ◽

Lactate Shuttle ◽

Term Potentiation ◽

Fructose 1,6 Bisphosphatase ◽

Nadh Ratio ◽

Simultaneous Inhibition

Long-term potentiation (LTP) is a molecular basis of memory formation. Here, we demonstrate that LTP critically depends on fructose 1,6-bisphosphatase 2 (Fbp2)—a glyconeogenic enzyme and moonlighting protein protecting mitochondria against stress. We show that LTP induction regulates Fbp2 association with neuronal mitochondria and Camk2 and that the Fbp2–Camk2 interaction correlates with Camk2 autophosphorylation. Silencing of Fbp2 expression or simultaneous inhibition and tetramerization of the enzyme with a synthetic effector mimicking the action of physiological inhibitors (NAD+ and AMP) abolishes Camk2 autoactivation and blocks formation of the early phase of LTP and expression of the late phase LTP markers. Astrocyte-derived lactate reduces NAD+/NADH ratio in neurons and thus diminishes the pool of tetrameric and increases the fraction of dimeric Fbp2. We therefore hypothesize that this NAD+-level-dependent increase of the Fbp2 dimer/tetramer ratio might be a crucial mechanism in which astrocyte–neuron lactate shuttle stimulates LTP formation.

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Multiphoton fluorescence lifetime imaging microscopy reveals free-to-bound NADH ratio changes associated with metabolic inhibition

Journal of Biomedical Optics ◽

10.1117/1.jbo.19.8.086016 ◽

2014 ◽

Vol 19 (8) ◽

pp. 086016 ◽

Cited By ~ 32

Author(s):

Krystyna Drozdowicz-Tomsia ◽

Ayad G. Anwer ◽

Michael A. Cahill ◽

Kaiser N. Madlum ◽

Amel M. Maki ◽

...

Keyword(s):

Fluorescence Lifetime ◽

Metabolic Inhibition ◽

Fluorescence Lifetime Imaging Microscopy ◽

Fluorescence Lifetime Imaging ◽

Lifetime Imaging ◽

Nadh Ratio

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Regulation of carbohydrate metabolism in lymphoid tissue. Quantitative aspects of [U-14C]glucose oxidation by rat spleen slices

Biochemical Journal ◽

10.1042/bj1480583 ◽

1975 ◽

Vol 148 (3) ◽

pp. 583-594 ◽

Cited By ~ 7

Author(s):

D Suter ◽

M J Weidemann

Keyword(s):

Lymphoid Tissue ◽

Glucose Utilization ◽

Normal Values ◽

Lactate Production ◽

Pentose Phosphate ◽

Glycolytic Metabolism ◽

Anaerobic Conditions ◽

Maximum Value ◽

Nadh Ratio ◽

Regulation Of Carbohydrate Metabolism

When washed spleen slices from fed rats are incubated with 3 mm-[U-14C]glucose, the rate of glucose utilization (46.2 mumol/h per g dry wt.) is sufficient to account, theoretically, for 80% of the O2 consumption. Measurement of net lactate production, however, and the fate of the radioactive carbon, indicates that the contribution of glucose to the respiratory fuel of the tissue is only 25-30% whereas 60-70% of the glucose utilized is converted into lactate. At saturating glucose concentrations (above 5 mm) its contribution to the respiratory fuel of the slice is increased to a maximum value of 34-39%. Only 2% of the glucose utilized is metabolized via the oxidative steps of the pentose phosphate pathway. Starvation for 72 h marginally increases both the rate of glucose utilization (by 21%) and its net contribution to the respiratory fuel (by 29%). Insulin, glucagon, adrenaline and adenosine 3′:5′-cyclic monophosphate have no significant effect on either the rate of glucose utilization or on the pattern of radioactive isotope distribution. The uptake of glucose is increased by only 20%, whereas the production of lactate doubles when slices are incubated under anaerobic conditions. In assessing the suitability of spleen slices for metabolic studies, the only serious major perturbation, compared with the freeze-clamped organ, is an elevated mitochondrial [NAD+]/[NADH] ratio (connected with increased endogenous NH3 production) that is partially restored to normal values on incubation with glucose. Equal proportions of erythrocytes and leucocytes are found in the washed spleen slice. Metabolic contributions of the constituent cell populations in the washed slice are calculated and it is concluded that lymphocytes account for the major part of the glycolytic metabolism (80-90%), whereas the contribution of erythrocytes is insignificant.

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