Effect Of NAD Research Articles

Evaluation of NAD+ precursors for improved metabolism and productivity of antibody-producing CHO cell.

In the previous study, the culture medium was treated with nicotinamide adenine dinucleotide (NAD+) under the hypothesis that NAD+ regeneration is a major factor causing excessive lactate accumulation in Chinese hamster ovary (CHO) cells. The NAD+ treatment improved metabolism by not only reducing the Warburg effect but also enhancing oxidative phosphorylation, leading to enhanced antibody production. Building on this, four NAD+ precursors - nicotinamide mononucleotide (NMN), nicotinic acid (NA), nicotinamide riboside (NR), and nicotinamide (NAM) - were tested to elevate intracellular NAD+ levels more economically. First, the ability of CHO cells to utilize both the salvage and Preiss-Handler pathways for NAD+ biosynthesis was verified, and then the effect of NAD+ precursors on CHO cell cultures was evaluated. These precursors increased intracellular NAD+ levels by up to 70.6% compared to the non-treated group. Culture analysis confirmed that all the precursors induced metabolic changes and that NMN, NA, and NR improved productivity akin to NAD+ treatment, with comparable integral viable cell density. Despite the positive effects such as the increase in the specific productivity and changes in cellular glucose metabolism, none of the precursors surpassed direct NAD+ treatment in antibody titer, presumably due to the reduction in nucleoside availability, as evidenced by the decrease in ATP levels in the NAD+ precursor-treated groups. These results underscore the complexity of cellular metabolism as well as the necessity for further investigation to optimize NAD+ precursor treatment strategies, potentially with the supplementation of nucleoside precursors. Our findings suggest a feasible approach for improving CHO cell culture performances by using NAD+ precursors as medium and feed components for the biopharmaceutical production.

NAD+ affects differentially expressed genes-MBOAT2-SLC25A21-SOX6 in experimental autoimmune encephalomyelitis model

Background Nicotinamide adenine dinucleotide (NAD+) plays a key role in neuroinflammation and neurodegeneration and provides anti-inflammatory and neuroprotective effects in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). Aim In this study, we aimed to investigate whether NAD+ affects differentially expressed genes (DEGs) in splenocytes of EAE mice to reveal candidate genes for the pathogenesis of MS. Methods The EAE model was used to perform an intervention on NAD+ to investigate its potential as a protective agent in inflammation and demyelination. Transcriptome analysis of nerve tissue was carried out to gain better insights into NAD+ function. Effects of NAD+ on DEGs in the splenocytes of EAE mice were investigated to determine its anti-inflammatory effect. Results NAD+ in EAE mice showed the clinical score was significantly improved (EAE 3.190 ± 0.473 vs. NAD+ 2.049 ± 0.715). DEGs (MBOAT2, SLC25A21, and SOX6) between the EAE and the EAE + NAD+ groups showed that SOX6 was significantly improved after NAD+ treatment compared with the EAE group, and other indicators were improved but did not reach statistical significance. NAD+ exhibited clinical scores in EAE mice, and key inflammation was ameliorated in EAE mice spleen after NAD+ intervention, while transcriptome analysis between EAE and EAE + NAD+ groups showed several DEGs in the underlying mechanism. Conclusion NAD+ on DEGs attenuates disease severity in EAE. Transcriptome analysis on nerve tissue reveals several protein targets in the underlying mechanisms. However, NAD+ does not significantly improve DEGs in the splenocytes of the EAE model.

NAD+ Precursors and Intestinal Inflammation: Therapeutic Insights Involving Gut Microbiota.

The oxidized form of nicotinamide adenine dinucleotide (NAD+) is a critical metabolite for living cells. NAD+ may act either as a cofactor for many cellular reactions as well as a coenzyme for different NAD+-consuming enzymes involved in the physiological homeostasis of different organs and systems. In mammals, NAD+ is synthesized from either tryptophan or other vitamin B3 intermediates that act as NAD+ precursors. Recent research suggests that NAD+ precursors play a crucial role in maintaining the integrity of the gut barrier. Indeed, its deficiency has been associated with enhanced gut inflammation and leakage, and dysbiosis. Conversely, NAD+-increasing therapies may confer protection against intestinal inflammation in experimental conditions and human patients, with accumulating evidence indicating that such favorable effects could be, at least in part, mediated by concomitant changes in the composition of intestinal microbiota. However, the mechanisms by which NAD+-based treatments affect the microbiota are still poorly understood. In this context, we have focused specifically on the impact of NAD+ deficiency on intestinal inflammation and dysbiosis in animal and human models. We have further explored the relationship between NAD+ and improved host intestinal metabolism and immunity and the composition of microbiota in vivo. Overall, this comprehensive review aims to provide a new perspective on the effect of NAD+-increasing strategies on host intestinal physiology.

NAD+ supplementation improves mAb productivity in CHO cells via a glucose metabolic shift.

Aerobic glycolysis and its by-product lactate accumulation are usually associated with adverse culture phenotypes such as poor cell viability and productivity. Due to the lack of knowledge on underlying mechanisms and accompanying biological processes, the regulation of aerobic glycolysis has been an ongoing challenge in culture process development for therapeutic protein productivity. Nicotinamide adenine dinucleotide (NAD+ ), a coenzyme and co-substrate in energy metabolism, promotes the conversion of inefficient glycolysis into an efficient oxidative phosphorylation (OXPHOS) pathway. However, the effect of NAD+ on Chinese hamster ovary (CHO) cells for biopharmaceutical production has not been reported yet. In this work, we aimed to elucidate the influence of NAD+ on cell culture performance by examining metabolic shifts and mAb productivity. The supplementation of NAD+ increased the intracellular concentration of NAD+ and promoted SIRT3 expression. Antibody titer and the specific productivity in the growth phase were improved by up to 1.82- and 1.88-fold, respectively, with marginal restrictions on cell growth. NAD+ significantly reduced the accumulation of reactive oxygen species (ROS) and the lactate yield from glucose, determined by lactate accumulation versus glucose consumption (YLAC/GLC ). In contrast, OXPHOS capacity and amino acid consumption rate increased substantially. Collectively, these results suggest that NAD+ contributes to improving therapeutic protein productivity in bioprocessing via inducing an energy metabolic shift.

Investigating ethanol production using the Zymomonas mobilis crude extract

Cell-free systems have become valuable investigating tools for metabolic engineering research due to their easy access to metabolism without the interference of the membrane. Therefore, we applied Zymomonas mobilis cell-free system to investigate whether ethanol production is controlled by the genes of the metabolic pathway or is limited by cofactors. Initially, different glucose concentrations were added to the extract to determine the crude extract's capability to produce ethanol. Then, we investigated the genes of the metabolic pathway to find the limiting step in the ethanol production pathway. Next, to identify the bottleneck gene, a systemic approach was applied based on the integration of gene expression data on a cell-free metabolic model. ZMO1696 was determined as the bottleneck gene and an activator for its enzyme was added to the extract to experimentally assess its effect on ethanol production. Then the effect of NAD+ addition at the high concentration of glucose (1 M) was evaluated, which indicates no improvement in efficiency. Finally, the imbalance ratio of ADP/ATP was found as the controlling factor by measuring ATP levels in the extract. Furthermore, sodium gluconate as a carbon source was utilized to investigate the expansion of substrate consumption by the extract. 100% of the maximum theoretical yield was obtained at 0.01 M of sodium gluconate while it cannot be consumed by Z. mobilis. This research demonstrated the challenges and advantages of using Z. mobilis crude extract for overproduction.

NAD+ attenuates cardiac injury after myocardial infarction in diabetic mice through regulating alternative splicing of VEGF in macrophages

Diabetic mellitus (DM) complicated with myocardial infarction (MI) is a serious clinical issue that remained poorly comprehended. The aim of the present study was to investigate the role of NAD+ in attenuating cardiac damage following MI in diabetic mice. The cardiac dysfunction in DM mice with MI was more severe compared with the non-diabetic mice and NAD+ administration could significantly improve the cardiac function in both non-diabetic and diabetic mice after MI for both 7 days and 28 days. Moreover, application of NAD+ could markedly reduce the cardiac injury area of DM complicated MI mice. Notably, the level of NAD+ was robustly decreased in the cardiac tissue of MI mice, which was further reduced in the DM complicated mice and NAD+ administration could significantly restore the NAD+ level. Furthermore, NAD+ was verified to facilitate the angiogenesis in the MI area of both diabetic mice and non-diabetic mice by microfil perfusion assay and immunofluorescence. Additionally, we demonstrated that NAD+ promoted cardiac angiogenesis after myocardial infarction in diabetic mice by promoting the M2 polarization of macrophages. At the molecular level, NAD+ promoted the secretion of VEGF in macrophages and therefore facilitating migration and tube formation of endothelial cells. Mechanistically, NAD+ was found to promote the generation of pro-angionesis VEGF165 and inhibit the generation of anti-angionesis VEGF165b via regulating the alternative splicing factors of VEGF (SRSF1 and SRSF6) in macrophages. The effects of NAD+ were readily reversible on deficiency of it. Collectively, our data showed that NAD+ could attenuate myocardial injury via regulating the alternative splicing of VEGF and promoting angiogenesis in diabetic mice after myocardial infarction. NAD+ administration may therefore be considered a potential new approach for the treatment of diabetic patients with myocardial infarction.

Nicotinamide Adenine Dinucleotide in the Development and Treatment of Cardiac Remodeling and Aging.

Recently, the beneficial effects of nicotinamide adenine dinucleotide (NAD+) as an antiaging and antioxidant molecule have become a focus of research. However, the mechanisms by which NAD+ supplementation affects the associated metabolites under physiological conditions remain unclear. Specifically, although NAD+ is involved in several processes that are dysregulated in cardiovascular diseases, some effects of NAD+ precursors and NAD+ on cardiac diseases have started to gain recognition only recently. To discuss the influence of NAD+ supplementation on adverse cardiac remodeling and aging. Supplementation with NAD+ precursors or nicotinamide riboside, which enhances or supplements the NAD+metabolome, might have a protective effect on the heart. NAD+ can alleviate chronic heart failure via mitochondrial oxidation-reduction (redox) state mechanism. Furthermore, NAD+ replenishment can improve the life span of mice. NAD+ exerts considerable antiaging and antioxidant effects with promising therapeutic effects. However, its effect on humans and its use as a dietary supplement need to be studied further.

Effects of Combined Treatment with Compounds Affecting Mitochondria on Physiological Parameters in Old Monkey.

Correction of mitochondrial function is a promising direction for modulating aging processes. The effect of NAD+ (in the form of its precursor nicotinamide mononucleotide, NMN) and antioxidant MitoQ specifically affecting mitochondria was studied on male rhesus monkeys (Macaca mulatta). The first group of animals initially received only NMN, the second group received only MitoQ, and then both groups received both drugs simultaneously. The above treatment did not lead to changes in the body weight and body temperature, hemostasis, and ECG. The use of MitoQ, but not NMN improved some hematological and biochemical parameters, shifting them to the values of young animals. Changes in these parameters depended on animal age, so the use of MitoQ can only be effective until a certain age. Combined use of the two drugs had no clear advantages over MitoQ alone.

Targeting NAD metabolism regulates extracellular adenosine levels to improve the cytotoxicity of CD8+ effector T cells in the tumor microenvironment of gastric cancer

PurposeNicotinamide adenine dinucleotide (NAD+) is closely related to the pathogenesis of tumors. However, the effect of NAD+ metabolism of gastric cancer (GC) cells on immune cells remains unexplained. We targeted nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme in the NAD+ synthesis salvage pathway, to observe its effect in the immune microenvironment.MethodsNAMPT of GC cell lines was inhibited by using the small molecule inhibitor (FK866) and short hairpin RNA (shRNA). CCK-8 test and flow cytometry were performed to detect cell viability and apoptosis. Immunofluorescence was used to observe changes in mitochondrial membrane potential (MMP).The transfected GC cells (AGS) and patient-derived organoids (PDOs) were cocultured with activated PBMCs, followed by flow cytometric analysis (FCA) for cytokines and inhibitory marker. The level of NAD and ATP of GC cells (AGS & MKN45) was tested combined with NMN and CD39 inhibitor.ResultsTargeting NAD+ by FK866 obviously reduced MMP, which ultimately inhibited proliferation and increased the apoptosis of GC cells. NAMPT silencing reduced intracellular NAD and ATP，further decreased extracellular adenosine. Meawhile, the cytokines of CD8+T cells were significantly increased after cocultured with transfected AGS, and the expression of PD-1 was distinctly decreased. NMN reversed the effect of shNAMPT and enhanced the immunosuppression. Consistent results were obtained by coculturing PBMCs with PDOs.ConclusionRestraining the function of NAMPT resulted in the functional improvement of effector CD8+ T cells by decreasing extracellular adenosine levels and inducing apoptosis of GC cells simultaneously. Therefore, this study demonstrates that NAMPT can be an effective target for gastric cancer immunotherapy.

Nicotinamide Mononucleotide Administration Triggers Macrophages Reprogramming and Alleviates Inflammation During Sepsis Induced by Experimental Peritonitis.

Peritonitis and subsequent sepsis lead to high morbidity and mortality in response to uncontrolled systemic inflammation primarily mediated by macrophages. Nicotinamide adenine dinucleotide (NAD+) is an important regulator of oxidative stress and immunoinflammatory responses. However, the effects of NAD+ replenishment during inflammatory activation are still poorly defined. Hence, we investigated whether the administration of β-nicotinamide mononucleotide (β-NMN), a natural biosynthetic precursor of NAD+, could modulate the macrophage phenotype and thereby ameliorate the dysregulated inflammatory response during sepsis. For this purpose, C57BL6 mice were subjected to the cecal ligation and puncture (CLP) model to provoke sepsis or were injected with thioglycolate to induce sterile peritonitis with recruitment and differentiation of macrophages into the inflamed peritoneal cavity. β-NMN was administered for 4 days after CLP and for 3 days post thioglycolate treatment where peritoneal macrophages were subsequently analyzed. In the CLP model, administration of β-NMN decreased bacterial load in blood and reduced clinical signs of distress and mortality during sepsis. These results were supported by transcriptomic analysis of hearts and lungs 24 h post CLP-induction, which revealed that β-NMN downregulated genes controlling the immuno-inflammatory response and upregulated genes involved in bioenergetic metabolism, mitochondria, and autophagy. In the thioglycolate model, a significant increase in the proportion of CD206 macrophages, marker of anti-inflammatory M2 phenotype, was detected on peritoneal exudate macrophages from β-NMN-administered mice. Transcriptomic signature of these macrophages after bacterial stimulation confirmed that β-NMN administration limited the pro-inflammatory M1 phenotype and induced the expression of specific markers of M2 type macrophages. Furthermore, our data show that β-NMN treatment significantly impacts NAD + metabolism. This shift in the macrophage phenotype and metabolism was accompanied by a reduction in phagolysosome acidification and secretion of inflammatory mediators in macrophages from β-NMN-treated mice suggesting a reduced pro-inflammatory activation. In conclusion, administration of β-NMN prevented clinical deterioration and improved survival during sepsis. These effects relied on shifts in the metabolism of organs that face up an increased energy requirement caused by bacterial infection and in innate immunity response, including reprogramming of macrophages from a highly inflammatory phenotype to an anti-inflammatory/pro-resolving profile.

Effects of NAD+ precursor supplementation on glucose and lipid metabolism in humans: a meta-analysis

BackgroundThis meta-analysis was performed to investigate the effects of nicotinamide adenine dinucleotide (NAD+) precursor supplementation on glucose and lipid metabolism in human body.MethodsPubMed, Embase, CENTRAL, Web of Science, Scopus databases were searched to collect clinical studies related to the supplement of NAD+ precursor from inception to February 2021. Then the retrieved documents were screened, the content of the documents that met the requirements was extracted. Meta-analysis and quality evaluation was performed detection were performed using RevMan5.4 software. Stata16 software was used to detect publication bias, Egger and Begg methods were mainly used. The main research terms of NAD+ precursors were Nicotinamide Riboside (NR), Nicotinamide Mononucleotide (NMN), Nicotinic Acid (NA), Nicotinamide (NAM). The changes in the levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and fasting blood glucose were mainly concerned.ResultsA total of 40 articles were included in the meta-analysis, with a sample of 14,750 cases, including 7406 cases in the drug group and 7344 cases in the control group. The results of meta-analysis showed that: NAD+ precursor can significantly reduce TG level (SMD = − 0.35, 95% CI (− 0.52, − 0.18), P < 0.0001), and TC (SMD = − 0.33, 95% CI (− 0.51, − 0.14), P = 0.0005), and LDL (SMD = − 0.38, 95% CI (− 0.50, − 0.27), P < 0.00001), increase HDL level (SMD = 0.66, 95% CI (0.56, 0.76), P < 0.00001), and plasma glucose level in the patients (SMD = 0.27, 95% CI (0.12, 0.42), P = 0.0004). Subgroup analysis showed that supplementation of NA had the most significant effect on the levels of TG, TC, LDL, HDL and plasma glucose.ConclusionsIn this study, a meta-analysis based on currently published clinical trials with NAD+ precursors showed that supplementation with NAD+ precursors improved TG, TC, LDL, and HDL levels in humans, but resulted in hyperglycemia, compared with placebo or no treatment. Among them, NA has the most significant effect on improving lipid metabolism. In addition, although NR and NAM supplementation had no significant effect on improving human lipid metabolism, the role of NR and NAM could not be directly denied due to the few relevant studies at present. Based on subgroup analysis, we found that the supplement of NAD+ precursors seems to have little effect on healthy people, but it has a significant beneficial effect on patients with cardiovascular disease and dyslipidemia. Due to the limitation of the number and quality of included studies, the above conclusions need to be verified by more high-quality studies.

Effects of NAD and related precursors against aging and Alzheimer’s disease

Effects of NAD and related precursors against aging and Alzheimer’s disease

Effect of NAD+ boosting on kidney ischemia-reperfusion injury.

Acute kidney injury (AKI) is associated with a very high mortality and an increased risk for progression to chronic kidney disease (CKD). Ischemia-reperfusion injury (IRI) is a model for AKI, which results in tubular damage, dysfunction of the mitochondria and autophagy, and in decreased cellular nicotinamide adenine dinucleotide (NAD+) with progressing fibrosis resulting in CKD. NAD+ is a co-enzyme for several proteins, including the NAD+ dependent sirtuins. NAD+ augmentation, e.g. by use of its precursor nicotinamide riboside (NR), improves mitochondrial homeostasis and organismal metabolism in many species. In the present investigation the effects of prophylactic administration of NR on IRI-induced AKI were studied in the rat. Bilateral IRI reduced kidney tissue NAD+, caused tubular damage, reduced α-Klotho (klotho), and altered autophagy flux. AKI initiated progression to CKD, as shown by induced profibrotic Periostin (postn) and Inhibin subunit beta-A, (activin A / Inhba), both 24 hours and 14 days after surgery. NR restored tissue NAD+ to that of the sham group, increased autophagy (reduced p62) and sirtuin1 (Sirt1) but did not ameliorate renal tubular damage and profibrotic genes in the 24 hours and 14 days IRI models. AKI induced NAD+ depletion and impaired autophagy, while augmentation of NAD+ by NR restored tissue NAD+ and increased autophagy, possibly serving as a protective response. However, prophylactic administration of NR did not ameliorate tubular damage of the IRI rats nor rescued the initiation of fibrosis in the long-term AKI to CKD model, which is a pivotal event in CKD pathogenesis.

Characterization of Sodium‐Dependent Pi Transport Inhibition by NAD

Hyperphosphatemia during end-stage renal disease causes many adverse outcomes. To prevent them, several Pi intestinal absorption inhibitors are necessary for targeting either the paracellular (diffusion) or the transcellular (transport) absorption pathway. Nicotinamide adenine dinucleotide (NADH/NAD+) is a classical inhibitor of intestinal and renal Pi transport and is a circadian modulator of phosphatemia. The mechanism of NAD transporter inhibition remains unclear, although a competitive inhibition mechanism has been suggested. In this work, we sought to determine if this mechanism has a direct effect on Pi transporters. The five known rat Na/Pi cotransporters (NaPi2a, NaPi2b, NaPi2c, PiT1, and PiT2) were expressed in Xenopus laevis oocytes. Uptake of 32P-Pi was performed for three days post-injection of 5 ng of the corresponding cRNAs per oocyte and after 30 minutes of preincubation with 0.5 mM NAD, NADH, or NAM. None of the compounds affected the Pi uptake of any of the expressed Pi transporters in oocytes. Pi transport in the brush border membrane vesicles (BBMV) of either rat kidney cortex or jejunum epithelium was also assayed. As expected, it was significantly inhibited by NADH and NAD when BBMV were preincubated for 30 min at room or ice-cold temperature. NAM had no effect, and the effect was specific for Na-dependent Pi uptake (D-glucose uptake was not affected). Dose-response analyses on jejunum or kidney BBMV revealed IC50 values in the micromolar range for 50 µM of Pi uptake. Several Michaelis-Menten kinetics were performed in the presence of different concentrations of NAD, and a Lineweaver-Burk plot suggested changes in affinity (i.e., competitive inhibition), which contrasts with the lack of an effect on the transporters expressed in oocytes. The competitive model was confirmed by non-linear regression of a global fit with shared parameters, providing a Ki value of 538 µM NAD. When the effect of NAD was assayed in Opossum Kidney cells, a proximal tubule cell line model expressing a regulated NaPi2a, no evidence of Pi transport inhibition was observed. To check whether NAD-mediated ribosylation was involved in Pi transport inhibition, a classical ribosylation assay was performed with 32P-NAD, using jejunum and renal BBMV. No evidence of ribosylation was found for NaPi2b or NaPi2a, even after one month of exposure to an X-ray film. This was confirmed using several ribosylation inhibitors (meta-iodobenzylguanidine, novobiocin, vitamin K1, vitamin K3, or 3-methoxybenzamide), which failed to prevent NAD inhibition of Pi transport in BBMV. In conclusion, despite the kinetic findings in BBMV, NAD inhibition of Na/Pi-cotransporters is, most likely, not mediated through a direct interaction with these transporters. Instead, inhibition seems to be mediated through an indirect mechanism acting on components that are absent in Xenopus oocytes and in OK cells, which ends in the modification of Pi transport affinity (Km) and occurs at ice-cold temperature. Nevertheless, the unlikely inhibition of a novel, unknown Pi transporter cannot be completely discarded.

Abstract 503: Nicotinamide Riboside Modulates Late Sodium Current and Cardiac Repolarization

Background: Boosting NAD + content within the heart has emerged as a cardioprotective strategy against heart failure-related remodeling, ischemic injury, and arrhythmias. Our group recently demonstrated that the bioavailable NAD + precursor Nicotinamide Riboside (NR) drives Sirtuin 1-mediated deacetylation of the main cardiac sodium channel Na v 1.5 to increase peak sodium current (I Na ), a major determinant of cardiac excitability and action potential conduction. However, the effects of NAD + precursors and NR on cardiac repolarization are relatively unexplored. Objective: To evaluate the effects of NR on late Na + current and cardiac repolarization. Methods: To study the in vitro effects of NR, whole-cell patch clamp was utilized to measure I Na and late sodium current (I NaL ) in myocytes isolated from 1-3 day old neonatal rats (NRCMs). To study the in vivo effects of NR, 4-5 month-old C57BL/6 mice were placed on either a control diet or a diet supplemented with NR (1%). EKGs were performed at baseline, 6 weeks, and 12 weeks post-diet. Results: NR increased peak I Na (500 uM: +60.1 ± 26%, 5 mM: +74.7 ± 39%) with a non-significant change in absolute I NaL (500 uM: -12.8 ± 9%, 5 mM: -32.4 ± 20%) , leading to decreased I NaL when normalized to peak current (500 uM: -45.2 ± 7.8%, 5 mM: -51.6 ± 9.8%, Figure A,B). Additionally, 12-week dietary NR supplementation shortened QT c in NR-treated mice (-9.1 ± 4.6%) but not in control mice (+1.7 ± 1.3%, Figure C). Conclusion: NAD + supplementation with NR is potentially anti-arrhythmic by decreasing I NaL and shortening QT c . Potential mechanisms include mitigating the effects of oxidative stress and/or modulation of PKA and PKC phosphorylation of Na v 1.5.

Abstract 867: NAD + Repletion Reverses HFpEF by Attenuating Myocardial Metabolic Dysfunction

Background: Heart failure with preserved ejection fraction (HFpEF) is a highly prevalent clinical condition associated with significant morbidity, mortality and health care expenses. Yet, no effective treatment has been identified. We recently demonstrated that concomitant metabolic and hypertensive stress in mice elicited by a combination of high fat diet (HFD) and constitutive nitric oxide synthase inhibition by N [w] -nitro-l-arginine methyl ester (L-NAME) faithfully recapitulates the numerous and myriad features of human HFpEF (paper in press at Nature ). Methods: Wild type C57Bl6 mice were fed with HFD and L-NAME via drinking water for 5-8 weeks. Myocardial mitochondrial morphology was assessed by electron microscopy. Mitochondrial function was assessed by measuring oxygen consumption rates using an oxygen electrode. Protein expression and modification were assessed by Western blotting and immunoprecipitation. Results: Using this novel mouse model, we discovered significant impairment of mitochondrial fatty acid oxidation (FAO) associated with hyperacetylation of key FAO enzymes in HFpEF myocardium. Mechanistically, downregulation of Sirtuin3, the major mitochondrial deacetylase, and deficiency of its co-substrate nicotinamide adenine dinucleotide (NAD + ), culminated in mitochondrial protein hyperacetylation. Strikingly, supplementation with nicotinamide riboside (NR), a NAD + precursor, led to dramatic improvement of mitochondrial function, and importantly, amelioration of the HFpEF phenotype. Conclusion: In summary, we have unveiled that protein hyperacetylation-mediated mitochondrial dysfunction is a crucial mechanism of HFpEF pathogenesis. This is, to our knowledge, the first study identifying a specific signature of metabolic remodeling in HFpEF heart. We also demonstrated the therapeutic effect of NAD + repletion in a preclinical HFpEF model. In next steps, confirming this benefit in a clinical trial is warranted.

Regulation of flowering in guava (Psidium guajava L.) cv. Lucknow-49

Plant growth hormones play a vital role in guava production and are used in the induction of flowering, help in fruit set, fruit thinning, fruit elongation, premature fruit drop prevention, fruiting buds development and inhibiting ripening process. These can also influence the size, appearance and quality parameters of fruits. By using plant hormones, an experiment was carried out in the Experimental Farm and Laboratory, Department of Horticulture, College of Agriculture, Assam Agricultural University, Jorhat during 2016–18 to regulate the flowering of guava in the rainy season and to increase more production and productivity of guava fruit in the winter season. A total of six treatments with four replications and two seasons (rainy and winter) were laid out in a randomized block design. The effect in foliar application of different concentrations of plant growth hormones of T1-Naphthalene acetic acid (NAA at 100 ppm), T2-Naphthalene acetamide (NAD at 40 ppm), T3-Naphthalene acetamide (NAD at 60 ppm), T4-Urea (2%), T5-Urea (5%) and T6-Urea (10%) at flowering stage in the month of April, 2017 helped in deblossoming of flowers, enhanced fruit size, weight, number of flowers and fruits, reduced fruit drop (76.43% in rainy season to 38.26%), decreased the days required for maturity (118 days) and increased the yield (20.73 kg/plant) in the winter season. The effect of NAD @ 60 ppm gave the best result in fulfilling all the physical parameters of the guava fruit in the winter season.

Extracellular NAD+ Suppresses Adrenergic Effects in the Atrial Myocardium of Rats during the Early Postnatal Ontogeny.

The effects of sympathetic cotransmitter NAD+ (10 μM) on bioelectric activity of the heart under conditions of adrenergic stimulation were studied on isolated spontaneously contracting preparations (without stimulation) of the right atrium from 2-7-day-old rats. Action potentials were recorded in the working myocardium using standard microelectrode technique. Perfusion of the right atrium with norepinephrine solution (1 μM) altered the configuration and significantly lengthened the action potentials. NAD + against the background of norepinephrine stimulation significantly decreased the duration of action potentials, in particular, at 25% repolarization. The effect of purine compounds NAD + , ATP, and adenosine on bioelectrical activity of the heart of newborn rats was studied under basal conditions (without norepinephrine stimulation). The effect of NAD + against the background of adrenergic stimulation was more pronounced than under basal conditions and was probably determined by suppression of ICaL, which can be the main mechanism of NAD + action on rat heart.

SIRT2 and Akt mediate NAD+-induced and NADH-induced increases in the intracellular ATP levels of BV2 microglia under basal conditions.

NAD replenishment can restore ATP levels and rescue premature aging in Cockayne syndrome mice. However, there has been no mechanistic study regarding the effects of NAD and NADH on intracellular ATP levels under basal conditions. In our current study, we used BV2 microglia to test our hypothesis that NAD and NADH can increase intracellular ATP levels under basal conditions. We found that both NAD and NADH significantly increased the intracellular ATP levels of BV2 microglia, which were attenuated by SIRT2 siRNA, the SIRT2 inhibitor AGK2, and the phosphatidylinositol 3-kinase/Akt inhibitor LY294002. Our study has also suggested that SIRT2 mediates the NAD-induced and NADH-induced increase in Akt phosphorylation in BV2 microglia. Collectively, our study has suggested that SIRT2 mediates both NAD-induced and NADH-induced increases in the intracellular ATP levels of BV2 microglia by modulating Akt phosphorylation.

Abstract 2675: Metabolic addiction in IDH1 mutant cancers

Abstract Introduction: Recent efforts have revealed IDH1 mutation not only results in marked accumulation of 2-hydroxyglutarate (2-HG), it generates genomic chromatin alterations, altered HIF activity and reprogrammed metabolic flux. We characterized the metabolic perturbations caused by heterozygous mutant IDH1 by comparing endogenous IDH1 mutant glioma cells treated with direct inhibitors of mutant IDH1 (IDH1i) to control cells without treatment, with the overall aim of identifying novel metabolic therapeutic targets. Experimental Procedures: We tested the effect of IDH1i on the in vitro and in vivo phenotype of a panel of patient-derived endogenous IDH1 mutant solid cancer cells (including gliomas, melanoma, and sarcomas). We then assessed the effect of IDH1i on the metabolome of endogenous IDH1 mutant glioma cells using liquid chromatography-mass spectrometry. We further investigated specifically perturbed metabolic pathways using lentiviral knockdown and overexpression systems. Results: We found that IDH1i exposure and the resulting 2-HG depletion had a mixed effect in 12 endogenous mutant IDH1 lines studied both in vitro or in vivo, including an orthotopic glioma xenograft model. For 10 of 12 lines, IDH1i treatment had no demonstrable effect on proliferation or survival, while 2 lines displayed growth inhibition after IDH1i treatment. However, no significant changes were detected in the genomic DNA methylation profiles or the global levels of histone tail marks including H3K4me3, H3K9me2, H3K9me3 and H3K27me3 in IDH1 mutant glioma lines, even after inhibition of mutant IDH1 for 12 months in vitro. Broader metabolite profiling revealed that inhibition of mutant IDH1 significantly altered levels of the canonical metabolite NAD+. We tested the effect of NAD+ depletion using NAD+ biosynthesis inhibitors, and found that endogenously mutant IDH1 cells were highly dependent on NAD+ for survival, whereas proliferation and survival of IDH1/2 wild-type cancer cells were unaffected by NAD+ depletion. Using an inducible mutant IDH1 expression system we discovered that mutant IDH1 reduced intracellular NAD+ levels, rendering mutant IDH1 cells susceptible to further NAD+ depletion. Lack of sufficient NAD+ induced metabolic crisis with reduced ATP levels in IDH1 mutant cells, triggering the intracellular energy sensor AMPK and autophagy. In vivo, NAD+ depletion significantly extended the survival of mice bearing IDH1 mutant xenograft tumors, including intracerebral gliomas. Conclusions: Although most IDH1 mutant cell lines derived from solid cancers were resistant to direct IDH1 inhibition, a subset of IDH1 mutant cancers were found to be sensitive. Mutant IDH1 reprograms metabolism and renders cancer cells highly dependent on NAD+ for survival. This metabolic addiction presents a potential opportunity for metabolically targeted therapeutic development. Citation Format: Andrew S. Chi, Kensuke Tateishi, Wakimoto Hiroaki, Tracy T. Batchelor, Anthony J. Iafrate, Daniel P. Cahill. Metabolic addiction in IDH1 mutant cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2675.