Ca²⁺ signals of astrocytes are modulated by the NAD⁺/NADH redox state

Astrocytes are important glial cells in the brain providing metabolic support to neurons as well as contributing to brain signaling. These different functional levels have to be highly coordinated to allow for proper cell and brain function. In this study, we show that in astrocytes the NAD(+) /NADH redox state modulates dopamine-induced Ca(2+) signals thereby connecting metabolism and Ca(2+) signaling. Application of dopamine induced a dose-dependent increase in Ca(2+) signal frequency in these cells, which was dependent on D(1) -receptor signaling, glycolytic activity, an increase in cytosolic NADH and inositol 1,4,5-triphosphate receptor operated intracellular Ca(2+) stores. Application of dopamine at a low concentration (1 μM) did not induce an increase in Ca(2+) signal frequency by itself. However, simultaneously increasing cytosolic NADH content either by direct application of NADH or by application of lactate resulted in a pronounced increase in Ca(2+) signal frequency. This increase could be blocked by co-application of pyruvate, suggesting that indeed the NAD(+) /NADH redox state is regulating Ca(2+) signals. We conclude that at the NAD(+) /NADH redox state metabolic and signaling information is integrated in astrocytes, thereby most likely contributing to precisely coordinate these different tasks of astrocytes.     

NADH/NAD redox state of cytoplasmic glycolytic compartments in vascular smooth muscle

The cytoplasmic NADH/NAD redox potential affects energy metabolism and contractile reactivity of vascular smooth muscle. NADH/NAD redox state in the cytosol is predominately determined by glycolysis, which in smooth muscle is separated into two functionally independent cytoplasmic compartments, one of which fuels the activity of Na(+)-K(+)-ATPase. We examined the effect of varying the glycolytic compartments on cystosolic NADH/NAD redox state. Inhibition of Na(+)-K(+)-ATPase by 10 microM ouabain resulted in decreased glycolysis and lactate production. Despite this, intracellular concentrations of the glycolytic metabolite redox couples of lactate/pyruvate and glycerol-3-phosphate/dihydroxyacetone phosphate (thus NADH/NAD) and the cytoplasmic redox state were unchanged. The constant concentration of the metabolite redox couples and redox potential was attributed to 1) decreased efflux of lactate and pyruvate due to decreased activity of monocarboxylate B-H(+) transporter secondary to decreased availability of H(+) for cotransport and 2) increased uptake of lactate (and perhaps pyruvate) from the extracellular space, probably mediated by the monocarboxylate-H(+) transporter, which was specifically linked to reduced activity of Na(+)-K(+)-ATPase. We concluded that redox potentials of the two glycolytic compartments of the cytosol maintain equilibrium and that the cytoplasmic NADH/NAD redox potential remains constant in the steady state despite varying glycolytic flux in the cytosolic compartment for Na(+)-K(+)-ATPase.     

The redox switch/redox coupling hypothesis

We provide an integrative interpretation of neuroglial metabolic coupling including the presence of subcellular compartmentation of pyruvate and monocarboxylate recycling through the plasma membrane of both neurons and glial cells. The subcellular compartmentation of pyruvate allows neurons and astrocytes to select between glucose and lactate as alternative substrates, depending on their relative extracellular concentration and the operation of a redox switch. This mechanism is based on the inhibition of glycolysis at the level of glyceraldehyde 3-phosphate dehydrogenase by NAD(+) limitation, under sufficiently reduced cytosolic NAD(+)/NADH redox conditions. Lactate and pyruvate recycling through the plasma membrane allows the return to the extracellular medium of cytosolic monocarboxylates enabling their transcellular, reversible, exchange between neurons and astrocytes. Together, intracellular pyruvate compartmentation and monocarboxylate recycling result in an effective transcellular coupling between the cytosolic NAD(+)/NADH redox states of both neurons and glial cells. Following glutamatergic neurotransmission, increased glutamate uptake by the astrocytes is proposed to augment glycolysis and tricarboxylic acid cycle activity, balancing to a reduced cytosolic NAD(+)/NADH in the glia. Reducing equivalents are transferred then to the neuron resulting in a reduced neuronal NAD(+)/NADH redox state. This may eventually switch off neuronal glycolysis, favoring the oxidation of extracellular lactate in the lactate dehydrogenase (LDH) equilibrium and in the neuronal tricarboxylic acid cycles. Finally, pyruvate derived from neuronal lactate oxidation, may return to the extracellular space and to the astrocyte, restoring the basal redox state and beginning a new loop of the lactate/pyruvate transcellular coupling cycle. Transcellular redox coupling operates through the plasma membrane transporters of monocarboxylates, similarly to the intracellular redox shuttles coupling the cytosolic and mitochondrial redox states through the transporters of the inner mitochondrial membrane. Finally, transcellular redox coupling mechanisms may couple glycolytic and oxidative zones in other heterogeneous tissues including muscle and tumors.     

Preferential utilization of NADPH as the endogenous electron donor for NAD(P)H:quinone oxidoreductase 1 (NQO1) in intact pulmonary arterial endothelial cells

The goal was to determine whether endogenous cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1) preferentially uses NADPH or NADH in intact pulmonary arterial endothelial cells in culture. The approach was to manipulate the redox status of the NADH/NAD(+) and NADPH/NADP(+) redox pairs in the cytosolic compartment using treatment conditions targeting glycolysis and the pentose phosphate pathway alone or with lactate, and to evaluate the impact on the intact cell NQO1 activity. Cells were treated with 2-deoxyglucose, iodoacetate, or epiandrosterone in the absence or presence of lactate, NQO1 activity was measured in intact cells using duroquinone as the electron acceptor, and pyridine nucleotide redox status was measured in total cell KOH extracts by high-performance liquid chromatography. 2-Deoxyglucose decreased NADH/NAD(+) and NADPH/NADP(+) ratios by 59 and 50%, respectively, and intact cell NQO1 activity by 74%; lactate restored NADH/NAD(+), but not NADPH/NADP(+) or NQO1 activity. Iodoacetate decreased NADH/NAD(+) but had no detectable effect on NADPH/NADP(+) or NQO1 activity. Epiandrosterone decreased NQO1 activity by 67%, and although epiandrosterone alone did not alter the NADPH/NADP(+) or NADH/NAD(+) ratio, when the NQO1 electron acceptor duroquinone was also present, NADPH/NADP(+) decreased by 84% with no impact on NADH/NAD(+). Duroquinone alone also decreased NADPH/NADP(+) but not NADH/NAD(+). The results suggest that NQO1 activity is more tightly coupled to the redox status of the NADPH/NADP(+) than NADH/NAD(+) redox pair, and that NADPH is the endogenous NQO1 electron donor. Parallel studies of pulmonary endothelial transplasma membrane electron transport (TPMET), another redox process that draws reducing equivalents from the cytosol, confirmed previous observations of a correlation with the NADH/NAD(+) ratio.     

Role of NADH/NAD+ transport activity and glycogen store on skeletal muscle energy metabolism during exercise: in silico studies

Skeletal muscle can maintain ATP concentration constant during the transition from rest to exercise, whereas metabolic reaction rates may increase substantially. Among the key regulatory factors of skeletal muscle energy metabolism during exercise, the dynamics of cytosolic and mitochondrial NADH and NAD+ have not been characterized. To quantify these regulatory factors, we have developed a physiologically based computational model of skeletal muscle energy metabolism. This model integrates transport and reaction fluxes in distinct capillary, cytosolic, and mitochondrial domains and investigates the roles of mitochondrial NADH/NAD+ transport (shuttling) activity and muscle glycogen concentration (stores) during moderate intensity exercise (60% maximal O2 consumption). The underlying hypothesis is that the cytosolic redox state (NADH/NAD+) is much more sensitive to a metabolic disturbance in contracting skeletal muscle than the mitochondrial redox state. This hypothesis was tested by simulating the dynamic metabolic responses of skeletal muscle to exercise while altering the transport rate of reducing equivalents (NADH and NAD+) between cytosol and mitochondria and muscle glycogen stores. Simulations with optimal parameter estimates showed good agreement with the available experimental data from muscle biopsies in human subjects. Compared with these simulations, a 20% increase (or approximately 20% decrease) in mitochondrial NADH/NAD+ shuttling activity led to an approximately 70% decrease (or approximately 3-fold increase) in cytosolic redox state and an approximately 35% decrease (or approximately 25% increase) in muscle lactate level. Doubling (or halving) muscle glycogen concentration resulted in an approximately 50% increase (or approximately 35% decrease) in cytosolic redox state and an approximately 30% increase (or approximately 25% decrease) in muscle lactate concentration. In both cases, changes in mitochondrial redox state were minimal. In conclusion, the model simulations of exercise response are consistent with the hypothesis that mitochondrial NADH/NAD+ shuttling activity and muscle glycogen stores affect primarily the cytosolic redox state. Furthermore, muscle lactate production is regulated primarily by the cytosolic redox state.     

Niacin protects the isolated heart from ischemia-reperfusion injury

Nicotinic acid (niacin) has been shown to decrease myocyte injury. Because interventions that lower the cytosolic NADH/NAD(+) ratio improve glycolysis and limit infarct size, we hypothesized that 1) niacin, as a precursor of NAD(+), would lower the NADH/NAD(+) ratio, increase glycolysis, and limit ischemic injury and 2) these cardioprotective benefits of niacin would be limited in conditions that block lactate removal. Isolated rat hearts were perfused without (Ctl) or with 1 microM niacin (Nia) and subjected to 30 min of low-flow ischemia (10% of baseline flow, LF) and reperfusion. To examine the effects of limiting lactate efflux, experiments were performed with 1) Ctl and Nia groups subjected to zero-flow ischemia and 2) the Nia group treated with the lactate-H(+) cotransport inhibitor alpha-cyano-4-hydroxycinnamate under LF conditions. Measured variables included ATP, pH, cardiac function, tissue lactate-to-pyruvate ratio (reflecting NADH/NAD(+)), lactate efflux rate, and creatine kinase release. The lactate-to-pyruvate ratio was reduced by more than twofold in Nia-LF hearts during baseline and ischemic conditions (P < 0.001 and P < 0.01, respectively), with concurrent lower creatine kinase release than Ctl hearts (P < 0.05). Nia-LF hearts had significantly greater lactate release during ischemia (P < 0.05 vs. Ctl hearts) as well as higher functional recovery and a relative preservation of high-energy phosphates. Inhibiting lactate efflux with alpha-cyano-4-hydroxycinnamate and blocking lactate washout with zero flow negated some of the beneficial effects of niacin. During LF, niacin lowered the cytosolic redox state and increased lactate efflux, consistent with redox regulation of glycolysis. Niacin significantly improved functional and metabolic parameters under these conditions, providing additional rationale for use of niacin as a therapeutic agent in patients with ischemic heart disease.     

Non-invasive In-cell Determination of Free Cytosolic [NAD+]/[NADH] Ratios Using Hyperpolarized Glucose Show Large Variations in Metabolic Phenotypes

Accumulating evidence suggest that the pyridine nucleotide NAD has far wider biological functions than its classical role in energy metabolism. NAD is used by hundreds of enzymes that catalyze substrate oxidation and, as such, it plays a key role in various biological processes such as aging, cell death, and oxidative stress. It has been suggested that changes in the ratio of free cytosolic [NAD⁺]/[NADH] reflects metabolic alterations leading to, or correlating with, pathological states. We have designed an isotopically labeled metabolic bioprobe of free cytosolic [NAD⁺]/[NADH] by combining a magnetic enhancement technique (hyperpolarization) with cellular glycolytic activity. The bioprobe reports free cytosolic [NAD⁺]/[NADH] ratios based on dynamically measured in-cell [pyruvate]/[lactate] ratios. We demonstrate its utility in breast and prostate cancer cells. The free cytosolic [NAD⁺]/[NADH] ratio determined in prostate cancer cells was 4 times higher than in breast cancer cells. This higher ratio reflects a distinct metabolic phenotype of prostate cancer cells consistent with previously reported alterations in the energy metabolism of these cells. As a reporter on free cytosolic [NAD⁺]/[NADH] ratio, the bioprobe will enable better understanding of the origin of diverse pathological states of the cell as well as monitor cellular consequences of diseases and/or treatments.     

The redox state of free nicotinamide-adenine dinucleotide in the cytoplasm and mitochondria of rat liver

1. The concentrations of the oxidized and reduced substrates of the lactate-, beta-hydroxybutyrate- and glutamate-dehydrogenase systems were measured in rat livers freeze-clamped as soon as possible after death. The substrates of these dehydrogenases are likely to be in equilibrium with free NAD(+) and NADH, and the ratio of the free dinucleotides can be calculated from the measured concentrations of the substrates and the equilibrium constants (Holzer, Schultz & Lynen, 1956; Bücher & Klingenberg, 1958). The lactate-dehydrogenase system reflects the [NAD(+)]/[NADH] ratio in the cytoplasm, the beta-hydroxybutyrate dehydrogenase that in the mitochondrial cristae and the glutamate dehydrogenase that in the mitochondrial matrix. 2. The equilibrium constants of lactate dehydrogenase (EC 1.1.1.27), beta-hydroxybutyrate dehydrogenase (EC 1.1.1.30) and malate dehydrogenase (EC 1.1.1.37) were redetermined for near-physiological conditions (38 degrees ; I0.25). 3. The mean [NAD(+)]/[NADH] ratio of rat-liver cytoplasm was calculated as 725 (pH7.0) in well-fed rats, 528 in starved rats and 208 in alloxan-diabetic rats. 4. The [NAD(+)]/[NADH] ratio for the mitochondrial matrix and cristae gave virtually identical values in the same metabolic state. This indicates that beta-hydroxybutyrate dehydrogenase and glutamate dehydrogenase share a common pool of dinucleotide. 5. The mean [NAD(+)]/[NADH] ratio within the liver mitochondria of well-fed rats was about 8. It fell to about 5 in starvation and rose to about 10 in alloxan-diabetes. 6. The [NAD(+)]/[NADH] ratios of cytoplasm and mitochondria are thus greatly different and do not necessarily move in parallel when the metabolic state of the liver changes. 7. The ratios found for the free dinucleotides differ greatly from those recorded for the total dinucleotides because much more NADH than NAD(+) is protein-bound. 8. The bearing of these findings on various problems, including the following, is discussed: the number of NAD(+)-NADH pools in liver cells; the applicability of the method to tissues other than liver; the transhydrogenase activity of glutamate dehydrogenase; the physiological significance of the difference of the redox states of mitochondria and cytoplasm; aspects of the regulation of the redox state of cell compartments; the steady-state concentration of mitochondrial oxaloacetate; the relations between the redox state of cell compartments and ketosis.     

The effect of increasing NADH availability on the redistribution of metabolic fluxes in Escherichia coli chemostat cultures

It is generally known that cofactors play a major role in the production of different fermentation products. This paper is part of a systematic study that investigates the potential of cofactor manipulations as a new tool for metabolic engineering. The NADH/NAD+ cofactor pair plays a major role in microbial catabolism, in which a carbon source, such as glucose, is oxidized using NAD+ and producing reducing equivalents in the form of NADH. It is crucially important for continued cell growth that NADH be oxidized to NAD+ and a redox balance be achieved. Under aerobic growth, oxygen is used as the final electron acceptor. While under anaerobic growth, and in the absence of an alternate oxidizing agent, the regeneration of NAD+ is achieved through fermentation by using NADH to reduce metabolic intermediates. Therefore, an increase in the availability of NADH is expected to have an effect on the metabolic distribution. We have previously investigated a genetic means of increasing the availability of intracellular NADH in vivo by regenerating NADH through the heterologous expression of an NAD(+)-dependent formate dehydrogenase and have demonstrated that this manipulation provoked a significant change in the final metabolite concentration pattern both anaerobically and aerobically (Berríos-Rivera et al., 2002, Metabolic engineering of Escherichia coli: increase of NADH availability by overexpressing an NAD(+)-dependent formate dehydrogenase, Metabolic Eng. 4, 217-229). The current work explores further the effect of substituting the native cofactor-independent formate dehydrogenase (FDH) by an NAD(+)-dependent FDH from Candida boidinii on the NAD(H/+) levels, NADH/NAD+ ratio, metabolic fluxes and carbon-mole yields in Escherichia coli under anaerobic chemostat conditions. Overexpression of the NAD(+)-dependent FDH provoked a significant redistribution of both metabolic fluxes and carbon-mole yields. Under anaerobic chemostat conditions, NADH availability increased from 2 to 3 mol NADH/mol glucose consumed and the production of more reduced metabolites was favored, as evidenced by a dramatic increase in the ethanol to acetate ratio and a decrease in the flux to lactate. It was also found that the NADH/NAD+ ratio should not be used as a sole indicator of the oxidation state of the cell. Instead, the metabolic distribution, like the Et/Ac ratio, should also be considered because the turnover of NADH can be fast in an effort to achieve a redox balance.     

Ethanol-induced redox imbalance in rat kidneys

This study reports the effects of long-term ethanol consumption on kidney redox status, in terms of enzymatic mechanisms involved in regulating the cytosolic [NADH]/[NAD(+) ] balance. Wistar rats were treated with ethanol (2 g/kg body weight/24 h) via intragastric intubation for 10 and 30 weeks, respectively. Ethanol administration induced an enhancement of alcohol dehydrogenase activities and affected the capacity of the kidney to prevent NADH accumulation in the cytosol. After 10 weeks, the excess of NADH was balanced by increased activities of malate dehydrogenase and aspartate transaminase. In the event of a longer period of ethanol intake, the kidney was not able to balance the NADH excess, even though an increase in malate dehydrogenase, lactate dehydrogenase, aspartate transaminase, and alanine transaminase activities was noted. The electrophoretic analysis of alcohol dehydrogenase, lactate dehydrogenase, and malate dehydrogenase isoforms revealed differences between control and ethanol-treated animals. The results suggest that rat kidneys have a multicomponent metabolic response to the same daily dose of ethanol that functions to maintain the redox status and which varies with the length of the administration period.     

Biochemical issues in estimation of cytosolic free NAD/NADH ratio

Cytosolic free NAD/NADH ratio is fundamentally important in maintaining cellular redox homeostasis but current techniques cannot distinguish between protein-bound and free NAD/NADH. Williamson et al reported a method to estimate this ratio by cytosolic lactate/pyruvate (L/P) based on the principle of chemical equilibrium. Numerous studies used L/P ratio to estimate the cytosolic free NAD/NADH ratio by assuming that the conversion in cells was at near-equilibrium but not verifying how near it was. In addition, it seems accepted that cytosolic free NAD/NADH ratio was a dependent variable responding to the change of L/P ratio. In this study, we show (1) that the change of lactate/glucose (percentage of glucose that converts to lactate by cells) and L/P ratio could measure the status of conversion between pyruvate + NADH and lactate + NAD that tends to or gets away from equilibrium; (2) that cytosolic free NAD/NADH could be accurately estimated by L/P only when the conversion is at or very close to equilibrium otherwise a calculation error by one order of magnitude could be introduced; (3) that cytosolic free NAD/NADH is stable and L/P is highly labile, that the highly labile L/P is crucial to maintain the homeostasis of NAD/NADH; (4) that cytosolic free NAD/NADH is dependent on oxygen levels. Our study resolved the key issues regarding accurate estimation of cytosolic free NAD/NADH ratio and the relationship between NAD/NADH and L/P.     

Sir2 regulates skeletal muscle differentiation as a potential sensor of the redox state

Sir2 is a NAD(+)-dependent histone deacetylase that controls gene silencing, cell cycle, DNA damage repair, and life span. Prompted by the observation that the [NAD(+)]/[NADH] ratio is subjected to dynamic fluctuations in skeletal muscle, we have tested whether Sir2 regulates muscle gene expression and differentiation. Sir2 forms a complex with the acetyltransferase PCAF and MyoD and, when overexpressed, retards muscle differentiation. Conversely, cells with decreased Sir2 differentiate prematurely. To inhibit myogenesis, Sir2 requires its NAD(+)-dependent deacetylase activity. The [NAD(+)]/[NADH] ratio decreases as muscle cells differentiate, while an increased [NAD(+)]/[NADH] ratio inhibits muscle gene expression. Cells with reduced Sir2 levels are less sensitive to the inhibition imposed by an elevated [NAD(+)]/[NADH] ratio. These results indicate that Sir2 regulates muscle gene expression and differentiation by possibly functioning as a redox sensor. In response to exercise, food intake, and starvation, Sir2 may sense modifications of the redox state and promptly modulate gene expression.     

Soluble adenylyl cyclase regulates the cytosolic NADH/NAD+ redox state and the bioenergetic switch between glycolysis and oxidative phosphorylation

The evolutionarily conserved soluble adenylyl cyclase (sAC, ADCY10) mediates cAMP signaling exclusively in intracellular compartments. Because sAC activity is sensitive to local concentrations of ATP, bicarbonate, and free Ca²⁺, sAC is potentially an important metabolic sensor. Nonetheless, little is known about how sAC regulates energy metabolism in intact cells. In this study, we demonstrated that both pharmacological and genetic suppression of sAC resulted in increased lactate secretion and decreased pyruvate secretion in multiple cell lines and primary cultures of mouse hepatocytes and cholangiocytes. The increased extracellular lactate-to-pyruvate ratio upon sAC suppression reflected an increased cytosolic free [NADH]/[NAD⁺] ratio, which was corroborated by using the NADH/NAD⁺ redox biosensor Peredox-mCherry. Mechanistic studies in permeabilized HepG2 cells showed that sAC inhibition specifically suppressed complex I of the mitochondrial respiratory chain. A survey of cAMP effectors revealed that only selective inhibition of exchange protein activated by cAMP 1 (Epac1), but not protein kinase A (PKA) or Epac2, suppressed complex I-dependent respiration and significantly increased the cytosolic NADH/NAD⁺ redox state. Analysis of the ATP production rate and the adenylate energy charge showed that inhibiting sAC reciprocally affects ATP production by glycolysis and oxidative phosphorylation while maintaining cellular energy homeostasis. In conclusion, our study shows that, via the regulation of complex I-dependent mitochondrial respiration, sAC-Epac1 signaling regulates the cytosolic NADH/NAD⁺ redox state, and coordinates oxidative phosphorylation and glycolysis to maintain cellular energy homeostasis. As such, sAC is effectively a bioenergetic switch between aerobic glycolysis and oxidative phosphorylation at the post-translational level.     

Effects of hypoxia on relationships between cytosolic and mitochondrial NAD(P)H redox and superoxide generation in coronary arterial smooth muscle

Since controversy exists on how hypoxia influences vascular reactive oxygen species (ROS) generation, and our previous work provided evidence that it relaxes endothelium-denuded bovine coronary arteries (BCA) in a ROS-independent manner by promoting cytosolic NADPH oxidation, we examined how hypoxia alters relationships between cytosolic and mitochondrial NAD(P)H redox and superoxide generation in BCA. Methods were developed to image and interpret the effects of hypoxia on NAD(P)H redox based on its autofluorescence in the cytosolic, mitochondrial, and nuclear regions of smooth muscle cells isolated from BCA. Aspects of anaerobic glycolysis and cytosolic NADH redox in BCA were assessed from measurements of lactate and pyruvate. Imaging changes in mitosox and dehydroethidium fluorescence were used to detect changes in mitochondrial and cytosolic-nuclear superoxide, respectively. Hypoxia appeared to increase mitochondrial and decrease cytosolic-nuclear superoxide under conditions associated with increased cytosolic NADH (lactate/pyruvate), mitochondrial NAD(P)H, and hyperpolarization of mitochondria detected by tetramethylrhodamine methyl-ester perchlorate fluorescence. Rotenone appeared to increase mitochondrial NAD(P)H and superoxide, suggesting hypoxia could increase superoxide generation by complex I. However, hypoxia decreased mitochondrial superoxide in the presence of contraction to 30 mM KCl, associated with decreased mitochondrial NAD(P)H. Thus, while hypoxia augments NAD(P)H redox associated with increased mitochondrial superoxide, contraction with KCl reverses these effects of hypoxia on mitochondrial superoxide, suggesting mitochondrial ROS increases do not mediate hypoxic relaxation in BCA. Since hypoxia lowers pyruvate, and pyruvate inhibits hypoxia-elicited relaxation and NADPH oxidation in BCA, mitochondrial control of pyruvate metabolism associated with cytosolic NADPH redox regulation could contribute to sensing hypoxia.     

Investigation of the NADH/NAD+ ratio in Ralstonia eutropha using the fluorescence reporter protein Peredox

Ralstonia eutropha is a hydrogen-oxidizing (“Knallgas”) bacterium that can easily switch between heterotrophic and autotrophic metabolism to thrive in aerobic and anaerobic environments. Its versatile metabolism makes R. eutropha an attractive host for biotechnological applications, including H₂-driven production of biodegradable polymers and hydrocarbons. H₂ oxidation by R. eutropha takes place in the presence of O₂ and is mediated by four hydrogenases, which represent ideal model systems for both biohydrogen production and H₂ utilization. The so-called soluble hydrogenase (SH) couples reversibly H₂ oxidation with the reduction of NAD⁺ to NADH and has already been applied successfully in vitro and in vivo for cofactor regeneration. Thus, the interaction of the SH with the cellular NADH/NAD⁺ pool is of major interest. In this work, we applied the fluorescent biosensor Peredox to measure the [NADH]:[NAD⁺] ratio in R. eutropha cells under different metabolic conditions. The results suggest that the sensor operates close to saturation level, indicating a rather high [NADH]:[NAD⁺] ratio in aerobically grown R. eutropha cells. Furthermore, we demonstrate that multicomponent analysis of spectrally-resolved fluorescence lifetime data of the Peredox sensor response to different [NADH]:[NAD⁺] ratios represents a novel and sensitive tool to determine the redox state of cells.     

Metabolic concomitants in pure, pancreatic beta cells during glucose-stimulated insulin secretion

The role of the redox potential in insulin secretion by beta cells stimulated with high glucose was investigated using an in vitro pancreas perfusion system. To assess glycolytic flux the sum of fructose-1,6-P2 + triose-P was determined in pure beta cells microdissected from lyophilized sections of the isolated perfused pancreas quick frozen during the early insulin secretory response. L-Glycerol 3-phosphate and dihydroxyacetone phosphate were measured as indicators of the free cytosolic [NAD+]/[NADH] ratio and NADH and NADPH were also measured. Fructose-1,6-P2 + triose-P was increased in beta cells simultaneously with the onset of insulin secretion indicating an increase in glucose metabolism had occurred. The ratio of [dihydroxyacetone phosphate]/[L-glycerol 3-phosphate] increased simultaneously with the onset of insulin secretion. NADH content increased only after initiation of insulin secretion and NADPH levels remained unchanged during the early secretory response to high glucose. These data contradict the hypothesis that insulin secretion is triggered by a more reduced cytosolic redox state and instead indicate that insulin secretion is initiated by other metabolic coupling factor(s) generated in beta cells stimulated by high glucose.     

Methylene blue protection against hypoxic injury in primary cultures of rat hepatocyte monolayers

Mitochondrial respiration is inhibited in cells exposed to hypoxia, and the oxidation of NADH to NAD(+) is blocked. As a result, oxidation reactions requiring NAD(+) are blocked, disrupting cellular metabolism. We studied the influence of methylene blue, which oxidizes NADH, on hypoxic damage to primary cultures of rat hepatocyte monolayers. During hypoxic treatment of hepatocytes, aspartate aminotransferase leaked out of the cells into the culture medium. However, addition of methylene blue to the medium repressed the hypoxic leakage of the enzyme. The exposure of hepatocytes to hypoxia decreased the acetoacetate/beta-hydroxybutyrate ratio which reflects the redox state of the cell. The level of the acetoacetate/beta-hydroxybutyrate ratio in hypoxic cells was increased by the addition of methylene blue. These results suggest that methylene blue protects against hypoxic injury due to its oxidation of NADH.     

An increase in the redox state during reperfusion contributes to the cardioprotective effect of GIK solution

This study aimed at determining whether glucose-insulin-potassium (GIK) solutions modify the NADH/NAD(+) ratio during postischemic reperfusion and whether their cardioprotective effect can be attributed to this change in part through reduction of the mitochondrial reactive oxygen species (ROS) production. The hearts of 72 rats were perfused with a buffer containing glucose (5.5 mM) and hexanoate (0.5 mM). They were maintained in normoxia for 30 min and then subjected to low-flow ischemia (0.5% of the preischemic coronary flow for 20 min) followed by reperfusion (45 min). From the beginning of ischemia, the perfusate was subjected to various changes: enrichment with GIK solution, enrichment with lactate (2 mM), enrichment with pyruvate (2 mM), enrichment with pyruvate (2 mM) plus ethanol (2 mM), or no change for the control group. Left ventricular developed pressure, heart rate, coronary flow, and oxygen consumption were monitored throughout. The lactate/pyruvate ratio of the coronary effluent, known to reflect the cytosolic NADH/NAD(+) ratio and the fructose-6-phosphate/dihydroxyacetone-phosphate (F6P/DHAP) ratio of the reperfused myocardium, were evaluated. Mitochondrial ROS production was also estimated. The GIK solution improved the recovery of mechanical function during reperfusion. This was associated with an enhanced cytosolic NADH/NAD(+) ratio and reduced mitochondrial ROS production. The cardioprotection was also observed when the hearts were perfused with fluids known to increase the cytosolic NADH/NAD(+) ratio (lactate, pyruvate plus ethanol) compared with the other fluids (control and pyruvate groups). The hearts with a high mechanical recovery also displayed a low F6P/DHAP ratio, suggesting that an accelerated glycolysis rate may be responsible for increased cytosolic NADH production. In conclusion, the cardioprotection induced by GIK solutions could occur through an increase in the cytosolic NADH/NAD(+) ratio, leading to a decrease in mitochondrial ROS production.     

Hepatic redox state and gluconeogenesis from lactate in vivo in the rat

1. To examine the role of the hepatic redox state on the rate of gluconeogenesis the effects of sodium crotonate injection (6mmol/kg body wt.) on rat liver metabolite concentrations and gluconeogenesis from lactate were studied in vivo. 2. Crotonate caused a marked oxidation of cytoplasmic and mitochondrial redox couples; decreases were observed in the ratios of [lactate]/[pyruvate], [glycerol 3-phosphate]/[dihydroxyacetone phosphate], [hydroxybutyrate]/[acetoacetate] and measured [NAD(+)]/[NADH]. 3. Increases occurred in the liver concentrations of all gluconeogenic intermediates from pyruvate through to glucose 6-phosphate, but there was no change in lactate concentration. 4. To determine whether gluconeogenesis from lactate was altered by the more-oxidized hepatic redox state l-[2-(14)C]lactic acid was infused into the inferior vena cava (50mumol/min per kg body wt.) and the incorporation of radioactivity into blood glucose was measured. 5. Administration of crotonate transiently decreased the rate of lactate incorporation into glucose but within a few minutes the rate of incorporation returned to that of the controls. 6. The results indicate that in these experiments alteration of the NAD(+)-NADH systems of cytoplasm and mitochondria to a more-oxidized state did not change the rate of gluconeogenesis.     

Online in vivo monitoring of cytosolic NAD redox dynamics in Ustilago maydis

Maintenance of metabolic redox homeostasis is essential to all life and is a key factor in many biotechnological processes. Changes in the redox state of NAD affect metabolic fluxes, mediate regulation and signal transduction, and thus determine growth and productivity. Here we establish an in vivo monitoring system for the dynamics of the cytosolic NADH/NAD⁺ ratio in the basidiomycete Ustilago maydis using the ratiometric fluorescent sensor protein Peredox-mCherry. Metabolic redox dynamics were determined in the cytosol of living cells with high time resolution under biotechnologically relevant conditions, i.e. with high cell density and high aeration. Analytical boundary conditions for reliable analysis were determined, and perturbations in C-, N- or O- availability had marked impact on the cytosolic NADH/NAD⁺ ratio. NAD redox dynamics could be manipulated in lines inducibly expressing a water-forming NADH oxidase as a synthetic reductant sink. The establishment of Peredox-mCherry in U. maydis and the analysis of NAD redox dynamics provides a versatile methodology for the in vivo investigation of cellular metabolism, and contributes fundamental knowledge for rational design and optimization of biocatalysts.