NAD Redox Potential Research Articles

NMNAT2 supports vesicular glycolysis via NAD homeostasis to fuel fast axonal transport

BackgroundBioenergetic maladaptations and axonopathy are often found in the early stages of neurodegeneration. Nicotinamide adenine dinucleotide (NAD), an essential cofactor for energy metabolism, is mainly synthesized by Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) in CNS neurons. NMNAT2 mRNA levels are reduced in the brains of Alzheimer’s, Parkinson’s, and Huntington’s disease. Here we addressed whether NMNAT2 is required for axonal health of cortical glutamatergic neurons, whose long-projecting axons are often vulnerable in neurodegenerative conditions. We also tested if NMNAT2 maintains axonal health by ensuring axonal ATP levels for axonal transport, critical for axonal function.MethodsWe generated mouse and cultured neuron models to determine the impact of NMNAT2 loss from cortical glutamatergic neurons on axonal transport, energetic metabolism, and morphological integrity. In addition, we determined if exogenous NAD supplementation or inhibiting a NAD hydrolase, sterile alpha and TIR motif-containing protein 1 (SARM1), prevented axonal deficits caused by NMNAT2 loss. This study used a combination of techniques, including genetics, molecular biology, immunohistochemistry, biochemistry, fluorescent time-lapse imaging, live imaging with optical sensors, and anti-sense oligos.ResultsWe provide in vivo evidence that NMNAT2 in glutamatergic neurons is required for axonal survival. Using in vivo and in vitro studies, we demonstrate that NMNAT2 maintains the NAD-redox potential to provide “on-board” ATP via glycolysis to vesicular cargos in distal axons. Exogenous NAD+ supplementation to NMNAT2 KO neurons restores glycolysis and resumes fast axonal transport. Finally, we demonstrate both in vitro and in vivo that reducing the activity of SARM1, an NAD degradation enzyme, can reduce axonal transport deficits and suppress axon degeneration in NMNAT2 KO neurons.ConclusionNMNAT2 ensures axonal health by maintaining NAD redox potential in distal axons to ensure efficient vesicular glycolysis required for fast axonal transport.

Nicotinamide Improves Anemia and Decreases Reactive Oxygen Species in Sickle Cell Disease Mice

The polymerization of deoxygenated HbS molecules in the red blood cells (RBCs) of patients with sickle cell disease (SCD) causes RBC destruction resulting in chronic pain, debilitating acute pain crises, strokes, multi-organ damage and a reduced life span. Therapeutic options remain limited. Bone marrow transplantation can be curative but is not an option for the majority of patients. Gene therapy interventions that also offer the promise of a cure are under investigation but are not likely to be available to the vast majority of patients in the near future. Hydroxyurea, the first drug approved for treatment of SCD, increases levels of Fetal Hemoglobin (HbF) that inhibit polymerization of HbS molecules but is not effective in all patients while a more powerful HbF-inducing drug, the DNA methyltransferase inhibitor decitabine has yet to be approved. L-glutamine, another approved therapeutic option, increases NAD redox potential and decreases reactive oxygen species (ROS) in the sickle RBCs to reduce symptoms. In our laboratory we have observed that increased ROS is associated with the retention of mitochondria in the SCD RBCs and have hypothesized that the abnormal presence of mitochondria in these cells is a major source of ROS (Jagadeeswaran et al Exp Hematol 50:46-52, 2017). In this investigation we have tested the hypothesis that chronic oral supplementation with nicotinamide, a direct precursor of NAD synthesis, would improve NAD redox potential, decrease mitochondrial retention and ROS in SCD RBCs, and reduce anemia in the SCD mouse model. The effect of nicotinamide was tested in SCD mice whose drinking water was supplemented for three months with 1% nicotinamide. The percentage of RBCs retaining mitochondria and the levels of ROS were determined by flow cytometric assays using the mitochondrial-specific dye TMRM and the ROS probe CM-H2DCFDA, respectively. In SCD mice receiving nicotinamide the fraction of RBCs retaining mitochondria was reduced 22.1% (p<0.05) and the level of ROS in RBCs was reduced 41% (p<0.01) compared to control SCD mice. The reticulocyte percentage was reduced 28% in nicotinamide-treated SCD mice compared to control SCD mice (p<0.01). The total RBC count was 30% higher (p<0.05) in nicotinamide-treated mice (6.61±0.76 X 10 6/μl) compared to control SCD mice (5.08±0.70 X 10 6/μl). Similar differences in hematocrit and total hemoglobin were also observed but failed to reach statistical significance. Total NAD levels were not significantly different in SCD mice receiving nicotinamide compared to control SCD mice (p<0.05), but the NADH/NAD total ratio was increased 2 fold (p<0.05). These results show that oral administration of high doses of nicotinamide decreases mitochondrial retention and ROS in SCD RBCs and improves NAD redox potential and anemia in SCD mice. These effects strongly suggest that additional studies be performed to investigate nicotinamide as a therapeutic option in SCD. [Display omitted] DisclosuresSaunthararajah: EpiDestiny: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

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.

L-Glutamine Therapy Improves Minute Ventilation of Sickle Cell Anemia Patients.

We have demonstrated previously that oral L-glutamine administration can improve NAD redox potential in sickle red blood cells (RBC) and decrease the endothelial adhesion rate of sickle RBC. Additionally, preliminary results showed that L-glutamine therapy improved the exercise endurance of sickle cell anemia patients. Following up on this study, minute ventilation and work rate were evaluated. After proper consent, 6 homozygous sickle cell anemia volunteers participated in this two-part study. The first part was incremental work rate testing where the work rate was increased incrementally. The second part was the constant work rate test, in which 80% of the maximum work rate of the incremental test was utilized. Patients were observed for changes in ventilation. These tests were conducted at baseline and after 8 to 12 weeks of therapy with oral L-glutamine at 30 grams a day. There were 6 patients who completed the incremental work rate test and 4 patients who completed the constant work rate test, respectively, at baseline and after treatment. Two of the patients withdrew prior to the completion of the follow-up testing. The results were as follows: On the incremental test, there was an upward trend in peak work rate with an average of 3.5 ± 8.7 watts/min (88.7 ± 25.6 vs. 91.5 ± 21.7; ρ> 0.05). Minute ventilation (VE) showed an average improvement of −4.8 ± 3.9 liters/min (61.2 ± 9.8 vs. 56.4 ± 9.9; ρ value of 0.05). The improved ventilation may be secondary to increased pulmonary blood flow inferring an increased cardiovascular oxygen delivery. On the constant work rate test, there was significant increase in exercise duration with an average of 1.4 ± 0.8 minutes (5.9 ± 1.6 vs. 7.3 ± 1.1; ρ< 0.05). The minute ventilation (VE) showed an average decrease of −11.0 ± 11.6 liters/min (66.5 ± 24.9 vs. 55.5 ± 14.5; ρ> 0.05). This could be indicative of improved oxygen delivery to the exercising musculature despite the absence of a preparatory exercise training program. The data confirm our preliminary report suggesting that L-glutamine therapy improves the exercise endurance of sickle cell anemia patients.TestMean + SDρ valuePrePostPaired Δ(two-tailed t-test)Incremental Work RatePeak Work Rate (watts/min)88.7 ± 25.691.5 ± 21.73.5 ± 8.70.37VE (liters/minute)61.2 ± 9.856.4 ± 9.9−c4.8 ± 3.90.05Constant Work Rate−Duration (minutes)5.9 ± 1.67.3 ± 1.11.4 ± 0.80.04−VE (liters/minute)66.5 ± 24.955.5 ± 14.5−11.0 ± 11.60.15

L-Glutamine Therapy Increases Exercise Endurance of Sickle Cell Anemia Patients.

We have demonstrated previously that oral L-glutamine administration can improve NAD redox potential in sickle red blood cells (RBC) and decrease the endothelial adhesion rate of sickle RBC. Clinically, there was subjective reporting of increased energy level. To confirm the reporting of increased energy level objectively, a study incorporating exercise tolerance testing was designed. After proper consent, 7 homozygous sickle cell anemia volunteers participated in the study, which was composed of two parts. The first is incremental work rate test where the work rate increased incrementally. The second part is the constant work rate test. In this test the 80% maximum work rate for the incremental test is utilized and the patients are tested for duration of exercise. These tests were conducted at baseline and after 8 to 12 weeks of therapy with oral L-glutamine at 30 grams a day. Two of the patients withdrew prior to completion of the follow up testing. There were 6 patients who completed the incremental work rate test and 4 patients who completed constant work rate test, respectively, at baseline and after treatment. Two of the 6 patients who completed the incremental work rate test at baseline declined to have the constant work rate test, resulting in 4 patients completing the constant work rate test. The results were as following: The work rates were 88 watts at baseline and 91.5 watts after treatment, with p = 0.18, for the incremental work protocol and 5.87 minutes at baseline and 7.27 minutes after treatment, with p = 0.02, for constant work protocol. For the incremental work rate, the data show a trend for increase with L-glutamine treatment. For the constant work protocol, there was a significant improvement in the work rate after treatment with L-glutamine. The data are consistent with the subjective reporting from a previous study suggesting that L-glutamine therapy improved the exercise endurance of sickle cell anemia patientsTestIncrementalConstantMean valuePeak work rate SD±Duration SD±Pre-88 watts 25.58±5.87 minutes 1.65±Post-91.5 watts 21.69±7.27 minutes 1.14±SignificanceP valueOne-tail test0.180.02

Desferrioxamine (DFO) conjugated with starch decreases NAD redox potential of intact red blood cells (RBC): evidence for DFO as an extracellular inducer of oxidant stress in RBC.

Desferrioxamine (DFO) is an important iron-chelating agent. It has also been thought of as an agent with anti-oxidant potential as it chelates ferric iron in various parts of the body. However, there is evidence suggesting that it may paradoxically affect red blood cells (RBCs) by inducing intracellular oxidant stress. Recently we observed that incubation of RBCs with DFO decreases NAD redox potential in normal RBC. To further understand the mechanism of DFO's interaction with RBC, we conducted a study to determine the effect of extracellular DFO upon RBC's redox status. We examined NAD redox potential in intact RBC (N = 7) incubated with DFO conjugated to starch. RBCs were incubated with 4 mM DFO for 3(1/2) hr and with 6 mM DFO for 2 and 3(1/2) hr. Significant decreases in NAD redox potential were observed after the incubations. With 4 mM DFO at the 3 (1/2) hr time point the mean decrease was 12.37% +/- 9.96% (P < 0.0085). With 6 mM DFO, the mean decreases were 18.54% +/- 9.79% (P < 0.0013) and 19.16% +/- 8.78% (P < 0.0006) for the 2 and 3 (1/2) hr incubations, respectively. DFO by itself is very poorly permeable to RBC. Conjugation with starch further ensured impermeability of DFO. The data presented here confirm the oxidant effect of DFO on RBC. The data also demonstrate that the effect of DFO on RBC's NAD redox potential originates extracellularly.

Dependence of energy metabolism on the density of cells in culture.

Metabolism in cultured cells strongly depends on cell density, thus cell density may be an important factor in biotechnological maintenance of cell cultures. Therefore the energy metabolism of three related cell types with different proliferation activity has been characterised: density controlled primary or secondary fibroblasts [pmf], immortalized, but still density controlled 3T3 cells, and SV40-transformed 3T3 lacking growth control. The investigations revealed the decrease of oxygen consumption, net lactate production, ATP-content, NAD-content and NAD-redox potential, and F-actin content with increasing culture density in pmfs, less distinct in 3T3 cells and in SV40-3T3 cells. The main decrease of these factors is related to cell-cell contacts rather than to proliferation which ceases at least two division cycles after most cells contacted a neighbouring cell. SV40-3T3 cells also at preconfluent densities exhibit relatively low metabolic activity as revealed by the above mentioned factors. Supply with metabolites for catalytic processes seems to be the rate limiting factor as deduced from a decreasing NADH/total NAD ratio. SV40-3T3 cells lack a contact mediated reduction of energy metabolism which is in accord with the missing contact inhibition of motility and proliferation in SV40-3T3 cells. Because of a possible association of glycolytic enzymes with actin, F-actin content has been determined. No correlation, however, was revealed by the F-actin-/lactate ratio. This may be due to the fact that about 50% of the lactate released into the culture medium originated to from glutaminolysis rather than glycolysis. Only in SV40-3T3 cells were respiration and lactate production insensitive to glutamine deprivation and in these cells both these parameters did not change significantly, therefore they did not allow testing as to whether f-actin content and glycolytic activity are correlated. Fractionated release of adeninnucleotides revealed that energy charge in the cytosolic fraction is the only factor which strictly correlates with the cessation of proliferation. Thus energy charge of the cytosol seems to be the only factor of these studied here, which can be used as an indicator of a culture's commitment for further proliferation. To compare energy metabolic parameters among different cell types one always has to take into account the density dependence of these factors.

Modulation of three types of potassium selective channels by NAD and other pyridine nucleotides in human pancreatic beta-cells. NAD and K+ channels in human beta-cells.

ATP-regulated potassium (K+ATP) channels play a major role in control of the cell membrane potential in pancreatic β-cells, and therefore, nutrient-induced insulin secretion (for recent reviews see 1–4). It has been well documented that glucose-induced closure of these channels is involved in the initial depolarisation of the membrane(5), and widely accepted that this may occur through an increase in the intracellular ATP:ADP ratio which is responsible for coupling metabolic events to ionic events at the plasma membrane(1,2,4). However, other intracellular modulators of K+ATP channels could also be involved in the regulation of the complex cycles of electrical activity exhibited by the cells in response to glucose stimulation. Changes in the redox potential of the nicotine adenine dinucleotides NAD(P)/NAD(P)H have for many years been thought to be important intracellular signals that could mediate the effects of nutrient secretagogues on insulin secretion(6–9). In more recent studies it has been demonstrated using rodent β-cells stimulated with glucose that increases in the concentration of NAD(P)H occur prior to an increase in [Ca2+]i(10, 11), and also that all four of the pyridine nucleotides will modulate the gating of ATP-sensitive K+ channels in the RINm5F insulin-secreting cell line(12). Using patch clamp techniques we have now examined the effects of NAD and other pyridine nucleotides on the gating of three types of K+ channel in human pancreatic β-cells; the K+ATP channel, the Ca2+ and voltage gated channel, and a novel NAD-activated K+ channel.

Type‐2 astrocytes have much greater susceptibility to heat stress than type‐1 astrocytes

The present investigations were undertaken to examine the susceptibility of type-2 astrocytes to elevated temperature. Type-2 astrocytes are much more easily injured by temperature elevation than type-1 astrocytes. This may be related to cellular redox potential. Type-1 astrocytes have a greater cytosolic NAD redox potential (i.e., higher NADH:NAD levels) than type-2 astrocytes as evidenced by a 9-fold higher ratio of lactate to pyruvate released into the medium by type-1 astrocytes than type-2 astrocytes. Heat stress causes the induction of hsp-72 in both type-2 and type-1 astrocytes; however, hsp-72 protein expression is retained for a longer period of time by the type-2 astrocyte. A possible basis for the greater sensitivity of type-2 astrocytes to stress may be a poorer ability to scavenge free radicals. This differential sensitivity of one neural cell type relative to another to elevated temperature may be of significance in understanding the effects of hyperthermia on the developing brain.

Ca 2+-fatty acid interaction in the control of hepatic gluconeogenesis

Calcium depletion induced by perfusing livers with calcium-free buffer did not alter the rates of basal glucose production from pyruvate or from increasing concentrations of lactate. However, calcium deficiency selectively prevented the fatty acid-induced stimulation of gluconeogenesis from lactate. This effect is not related to the higher NAD redox potential consistently observed in Ca 2+-deficient livers. On the other hand, octanoate was capable of inducing dose-dependent changes in the [pyruvate] 0.5 in calcium-depleted livers perfused with lactate, ruling out that low cellular calcium content could perturb the mitochondrial transport of pyruvate. The observation that the effect of calcium deficiency can be overcome by supraphysiological concentrations of pyruvate supports the proposal that stimulation of the maximal capacity of the gluconeogenic pathway by fatty acid relies largely on the tricarboxylic acid cycle activity, restricted in calcium deficiency conditions.

Control of hepatic gluconeogenesis: Role of fatty acid oxidation

Octanoate has been found to activate the gluconeogenic pathway in perfused isolated rat liver. Whether a net increase in the production of glucose is observed is a function of the relative concentrations of the glucose precursor and the fatty acid. The kinetics of octanoate interaction with the gluconeogenic pathway are influenced by the rate changes induced by decreases in pyruvate concentration as a result of the increased NAD redox potential produced by the oxidation of fatty acid. Taking this into account, two distinct effects of octanoate were identified. The first is an increase in the V max even at the lowest (25 μ m) concentration of the fatty acid tested. The second is a progressive decrease in [pyruvate] 0.5 as a function of octanoate concentration. The latter occurs at low (<0.1 m m), presumably physiological, pyruvate concentrations, when its mitochondrial transport is limiting, indicating that this process must have been activated. The former is observable even at high (>0.5 m m), supraphysiological, concentrations of pyruvate, when its mitochondrial transport is not limiting, indicating that a distal step, presumably pyruvate carboxylation, is activated. The action of octanoate in increasing gluconeogenesis has been found not to be related to a decreased flux through pyruvate dehydrogenase, neither to changes in the NAD redox potential nor to its ability to increase energy production. Actually, the oxygen uptake induced by octanoate was largely accounted for by the production of ketone body and the latter process was found to be independent of variations in energy demand.

Characterization of the alpha-adrenergic stimulation of hepatic respiration.

The alpha-adrenergic agonist phenylephrine induces a biphasic stimulation of respiration in perfused isolated rat liver. The first phase, of rapid onset and short duration, is paralleled by increased glycogenolysis, glycolysis, and NAD redox potential. The second phase lasts for as long as the alpha-agonist is present and is accompanied by increased gluconeogenic flux. Only the second phase of sustained increased respiration is clearly dependent on extracellular Ca2+. In contrast, normal respiratory responses were obtained under Ca2+-loading conditions or in the presence of the Ca2+ ionophore A23187, indicating that the alpha-adrenergic action on respiration is not simply mediated by its ability to increase the cytosolic Ca2+ concentration. No stimulation of gluconeogenesis is observed in the absence of a sustained increase of respiration. However, it is not energy support that leads to the stimulation of glucose production. The adrenergic response is influenced by the nutritional status of the animal and the availability of oxidizable fuels. In livers from starved animals, the alpha-adrenergic respiratory response is abolished when long chain fatty acid oxidation is prevented by the addition of tetradecylglycidate. In the presence of pyruvate the respiratory response is partially restored. It is concluded that increased beta-oxidation is not mandatory for the alpha-adrenergic stimulation of respiration; however, maximal respiratory responses are obtained only when fatty acid oxidation is allowed to proceed. The latter finding appears to be the result of a limited flux through the tricarboxylic acid cycle when long chain fatty acid oxidation is impeded, secondary to a limiting acetyl CoA supply.

Regulation of cyclic photophosphorylation in Rhodospirillum rubrum by the redox state of nicotinamide-adenine dinucleotide

We have investigated the effect of the redox state of added NAD on the rates of anaerobic cyclic photophosphorylation which are supported by membrane vesicles isolated from Rhodospirillum rubrum. As the redox potential of NAD was lowered, the activity decreased according to a typical potentiometric titration. The Nernst plot showed an apparent midpoint potential ( E′ o) of −350 mV and had a slope which corresponded to a two-electron transition. Besides, an almost identical potentiometric relationship was found to exist between the extent of light-elicited ATP formation in anaerobic suspensions of intact R. rubrum cells and the redox potential of intracellular NAD. These results suggest that physiological photophosphorylation in R. rubrum requires the oxidized form of a membrane-bound constituent ( E′ o = −350 mV) whose redox state is controlled by the redox state of cytoplasmic NAD.

The development of gluconeogenesis in rat liver. Controlling factors in the newborn.

1. Measurements in livers of rats delivered by Caesarian section show a rapid change in the relative proportion of adenine nucleotides. By 20min the ATP/ADP ratio had increased from 1.76 to 8.7 and the value of the relationship [ATP][AMP]/[ADP](2) increased from 1.0 to 4.4. These changes are dependent on the availability of oxygen to the animal. 2. The free [NAD(+)]/[NADH] ratio in the liver cytosol increases from 180 after delivery to reach a maximum of 1010 at 2h, before falling to 540 in the 24h-old animal. 3. The mitochondrial NAD redox potential also shows a sharp increase towards a more oxidized state in livers of delivered rats. 4. These results probably indicate that the foetal liver is hypoxic, with oxygenation occurring in the first hour after delivery. 5. Measurements in livers of naturally born rats 2min after birth also suggest that this tissue is hypoxic with an ATP/ADP ratio of 1.83 and a free [NAD(+)]/[NADH] ratio of 117. 6. Concentrations of intermediates in the gluconeogenic pathway have been determined in livers of foetal, 1h-old and 1-day-old rats. These experiments imply a facilitation of lactate dehydrogenase and glucose 6-phosphatase activities by 1h after birth, and a stimulation of phosphoenolpyruvate carboxykinase and glucose 6-phosphatase steps by 1 day after birth. 7. The appearance of gluconeogenesis in livers of newborn rats seems therefore to involve an oxygenation stage followed by an increase in phosphoenolpyruvate carboxykinase activity.