Potential Therapeutic Interventions Targeting NAD+ Metabolism for ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting both upper and lower motor neurons. While there have been many potential factors implicated for ALS development, such as oxidative stress and mitochondrial dysfunction, no exact mechanism has been determined at this time. Nicotinamide adenine dinucleotide (NAD+) is one of the most abundant metabolites in mammalian cells and is crucial for a broad range of cellular functions from DNA repair to energy homeostasis. NAD+ can be synthesized from three different intracellular pathways, but it is the NAD+ salvage pathway that generates the largest proportion of NAD+. Impaired NAD+ homeostasis has been connected to aging and neurodegenerative disease-related dysfunctions. In ALS mice, NAD+ homeostasis is potentially disrupted prior to the appearance of physical symptoms and is significantly reduced in the nervous system at the end stage. Treatments targeting NAD+ metabolism, either by administering NAD+ precursor metabolites or small molecules that alter NAD+-dependent enzyme activity, have shown strong beneficial effects in ALS disease models. Here, we review the therapeutic interventions targeting NAD+ metabolism for ALS and their effects on the most prominent pathological aspects of ALS in animal and cell models.

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Aging and age-related ailments have emerged as critical challenges and great burdens within the global contemporary society. Addressing these concerns is an imperative task, with the aims of postponing the aging process and finding effective treatments for age-related degenerative diseases. Recent investigations have highlighted the significant roles of nicotinamide adenine dinucleotide (NAD+) in the realm of anti-aging. It has been empirically evidenced that supplementation with nicotinamide mononucleotide (NMN) can elevate NAD+ levels in the body, thereby ameliorating certain age-related degenerative diseases. The principal anti-aging mechanisms of NMN essentially lie in its impact on cellular energy metabolism, inhibition of cell apoptosis, modulation of immune function, and preservation of genomic stability, which collectively contribute to the deferral of the aging process. This paper critically reviews and evaluates existing research on the anti-aging mechanisms of NMN, elucidates the inherent limitations of current research, and proposes novel avenues for anti-aging investigations.

Coenzymes in a pre-enzymatic metabolism.

Experiments now support theoretical suggestions that coenzymes mediated key metabolic reactions before the emergence of enzymes. Three coenzymes believed essential to the core metabolism of the last universal common ancestor to extant life (pyridoxal phosphate, adenosine diphosphate, and nicotinamide adenine dinucleotide) were recently found to be active in their corresponding metabolic reactions in the absence of enzymes. These findings suggest an earlier contribution of coenzymes to abiogenesis, ultimately yielding insights into the prebiotic origins of metabolism.

The Rate of NAD+ Breakdown Is Maintained Constant against Deletion or Overexpression of NAD+-Degrading Enzymes in Mammalian Cells.

Cellular NAD+ is continuously degraded and synthesized under resting conditions. In mammals, NAD+ synthesis is primarily initiated from nicotinamide (Nam) by Nam phosphoribosyltransferase, whereas poly(ADP-ribose) polymerase 1 (PARP1) and 2 (PARP2), sirtuin1 (SIRT1), CD38, and sterile alpha and TIR motif containing 1 (SARM1) are involved in NAD+ breakdown. Using flux analysis with 2H-labeled Nam, we found that when mammalian cells were cultured in the absence of Nam, cellular NAD+ levels were maintained and NAD+ breakdown was completely suppressed. In the presence of Nam, the rate of NAD+ breakdown (RB) did not significantly change upon PARP1, PARP2, SIRT1, or SARM1 deletion, whereas stable expression of CD38 did not increase RB. However, RB in PARP1-deleted cells was much higher compared with that in wild-type cells, in which PARP1 activity was blocked with a selective inhibitor. In contrast, RB in CD38-overexpressing cells in the presence of a specific CD38 inhibitor was much lower compared with that in control cells. The results indicate that PARP1 deletion upregulates the activity of other NADases, whereas CD38 expression downregulates the activity of endogenous NADases, including PARP1 and PARP2. The rate of cellular NAD+ breakdown and the resulting NAD+ concentration may be maintained at a constant level, despite changes in the NAD+-degrading enzyme expression, through the compensatory regulation of NADase activity.

Purification and characterization of a Rieske oxygenase and its NADH-regenerating partner proteins.

The Rieske non-heme iron oxygenases (Rieske oxygenases) comprise a class of metalloenzymes that are involved in the biosynthesis of complex natural products and the biodegradation of aromatic pollutants. Despite this desirable catalytic repertoire, industrial implementation of Rieske oxygenases has been hindered by the multicomponent nature of these enzymes and their requirement for expensive reducing equivalents in the form of a reduced nicotinamide adenine dinucleotide cosubstrate (NAD(P)H). Fortunately, however, some Rieske oxygenases co-occur with accessory proteins, that through a downstream reaction, recycle the needed NAD(P)H for catalysis. As these pathways and accessory proteins are attractive for bioremediation applications and enzyme engineering campaigns, herein, we describe methods for assembling Rieske oxygenase pathways in vitro. Further, using the TsaMBCD pathway as a model system, in this chapter, we provide enzymatic, spectroscopic, and crystallographic methods that can be adapted to explore both Rieske oxygenases and their co-occurring accessory proteins.

A rapid and reliable targeted LC-MS/MS method for quantitative analysis of the Tryptophan-NAD metabolic network disturbances in tissues and blood of sleep deprivation mice.

BACKGROUND: TRY-NAD metabolic network includes TRY (tryptophan), 5-HT (5-hydroxytryptamine), KYN (kynurenine), and NAD (nicotinamide adenine dinucleotide) pathway, which plays a significant role in neurological diseases and ageing. It is important to monitor these metabolites for studying the pathological anatomy of disease and treatment of responses evaluation. Although previous studies have reported quantitative methods for several metabolites in the network, the bottlenecks of simultaneously quantifying the whole metabolic network are their similar structures, diverse physico-chemical properties, and instability. Standardized protocols for the whole metabolic network are still missing, which hinders the in-depth study of TRY-NAD metabolic network in laboratory research and clinical screening. RESULTS: We developed a LC-MS/MS method for quantifying 28 metabolites in the TRY-NAD network simultaneously. Optimization was done for the mass spectral parameters, chromatographic conditions and sample pretreatment process. The developed method was fully validated in terms of standard curves, sensitivity, carryover, recovery, matrix effect, accuracy, precision, and stability. The pretreatment of 30 samples only takes 90 min, and the LC-MS/MS running time of one sample is only 13 min. With this method, we bring to light the chaos of global TRY-NAD metabolic network in sleep deprivation mice for the first time, including serum, clotted blood cells, hippocampus, cerebral cortex, and liver. NAD pathway levels in brain and blood decreased, whereas the opposite happened in the liver. The 5-HT pathway decreased and the concentration of KYN increased significantly in the brain. The concentration of many metabolites in KYN pathway (NAD+ de novo synthesis pathway) increased in the liver. SIGNIFICANCE: This method is the first time to determine the metabolites of KYN, 5-HT and NAD pathway at the same time, and it is found that TRY-NAD metabolic network will be disordered after sleep deprivation. This work clarifies the importance of the pH of the extraction solution, the time and temperature control in pretreatment in standardized protocols building, and overcoming the problems of inconsistent sample pretreatment, separation, matrix effect interference and potential metabolite degradation. This method exhibits great prospects in providing more information on metabolic disturbances caused by sleep deprivation as well as neurological diseases and ageing.

The compartment-specific manipulation of the NAD+/NADH ratio affects the metabolome and the function of glioblastoma.

Glioblastoma multiforme (GBM) is the most aggressive type of cancer in the brain and has an inferior prognosis because of the lack of suitable medicine, largely due to its tremendous invasion. GBM has selfish metabolic pathways to promote migration, invasion, and proliferation compared to normal cells. Among various metabolic pathways, NAD (nicotinamide adenine dinucleotide) is essential in generating ATP and is used as a resource for cancer cells. LbNOX (Lactobacillus brevis NADH oxidase) is an enzyme that can directly manipulate the NAD+/NADH ratio. In this study, we found that an increased NAD+/NADH ratio by LbNOX or mitoLbNOX reduced intracellular glutamate and calcium responses and reduced invasion capacity in GBM. However, the invasion was not affected in GBM by rotenone, an ETC (Electron Transport Chain) complex I inhibitor, or nicotinamide riboside, a NAD+ precursor, suggesting that the crucial factor is the NAD+/NADH ratio rather than the absolute quantity of ATP or NAD+ for the invasion of GBM. To develop a more accurate and effective GBM treatment, our findings highlight the importance of developing a new medicine that targets the regulation of the NAD+/NADH ratio, given the current lack of effective treatment options for this brain cancer.

Oxygen alters redox cofactor dynamics and induces metabolic shifts in Saccharomyces cerevisiae during alcoholic fermentation.

Environmental conditions significantly impact the metabolism of Saccharomyces cerevisiae, a Crabtree-positive yeast that maintains a fermentative metabolism in high-sugar environments even in the presence of oxygen. Although the introduction of oxygen has been reported to induce alterations in yeast metabolism, knowledge of the mechanisms behind these metabolic adaptations in relation to redox cofactor metabolism and their implications in the context of wine fermentation remains limited. This study aimed to compare the intracellular redox cofactor levels, the cofactor ratios, and primary metabolite production in S. cerevisiae under aerobic and anaerobic conditions in synthetic grape juice. The molecular mechanisms underlying these metabolic differences were explored using a transcriptomic approach. Aerobic conditions resulted in an enhanced fermentation rate and biomass yield. Total NADP(H) levels were threefold higher during aerobiosis, while a decline in the total levels of NAD(H) was observed. However, there were stark differences in the ratio of NAD+/NADH between the treatments. Despite few changes in the differential expression of genes involved in redox cofactor metabolism, anaerobiosis resulted in an increased expression of genes involved in lipid biosynthesis pathways, while the presence of oxygen increased the expression of genes associated with thiamine, methionine, and sulfur metabolism. The production of fermentation by-products was linked with differences in the redox metabolism in each treatment. This study provides valuable insights that may help steer the production of metabolites of industrial interest during alcoholic fermentation (including winemaking) by using oxygen as a lever of redox metabolism.

Nicotinamide Mononucleotide: Research Process in Cardiovascular Diseases.

Nicotinamide adenine dinucleotide (NAD+) is an essential metabolite that plays a crucial role in diverse biological processes, including energy metabolism, gene expression, DNA repair, and mitochondrial function. An aberrant NAD+ level mediates the development of cardiovascular dysfunction and diseases. Both in vivo and in vitro studies have demonstrated that nicotinamide mononucleotide (NMN), as a NAD+ precursor, alleviates the development of cardiovascular diseases such as heart failure, atherosclerosis, and myocardial ischemia/reperfusion injury. Importantly, NMN has suggested pharmacological activities mostly through its involvement in NAD+ biosynthesis. Several clinical studies have been conducted to investigate the efficacy and safety of NMN supplementation, indicating its potential role in cardiovascular protection without significant adverse effects. In this review, we systematically summarize the impact of NMN as a nutraceutical and potential therapeutic drug on cardiovascular diseases and emphasize the correlation between NMN supplementation and cardiovascular protection.

Dihydrolipoamide dehydrogenase (DLD) is a novel molecular target of bortezomib.

Proteasome inhibitors (PIs), such as bortezomib and calfizomib, were backbone agents in the treatment of multiple myeloma (MM). In this study, we investigated bortezomib interactors in MM cells and identified dihydrolipoamide dehydrogenase (DLD) as a molecular target of bortezomib. DLD catalyzes the oxidation of dihydrolipoamide to form lipoamide, a reaction that also generates NADH. Our data showed that bortezomib bound to DLD and inhibited DLD's enzymatic function in MM cells. DLD knocked down MM cells (DLD-KD) had decreased levels of NADH. Reduced NADH suppressed assembly of proteasome complex in cells. As a result, DLD-KD MM cells had decreased basal-level proteasome activity and were more sensitive to bortezomib. Since PIs were used in many anti-MM regimens in clinics, we found that high expression of DLD correlated with inferior prognosis of MM. Considering the regulatory role of DLD in proteasome assembly, we evaluated DLD targeting therapy in MM cells. DLD inhibitor CPI-613 showed a synergistic anti-MM effect with bortezomib in vitro and in vivo. Overall, our findings elucidated DLD as an alternative molecular target of bortezomib in MM. DLD-targeting might increase MM sensitivity to PIs.

Alcohol- and Low-Iron Induced Changes in Antioxidant and Energy Metabolism Associated with Protein Lys Acetylation.

Understanding the role of iron in ethanol-derived hepatic stress could help elucidate the efficacy of dietary or clinical interventions designed to minimize liver damage from chronic alcohol consumption. We hypothesized that normal levels of iron are involved in ethanol-derived liver damage and reduced dietary iron intake would lower the damage caused by ethanol. We used a pair-fed mouse model utilizing basal Lieber-DeCarli liquid diets for 22 weeks to test this hypothesis. In our mouse model, chronic ethanol exposure led to mild hepatic stress possibly characteristic of early-stage alcoholic liver disease, seen as increases in liver-to-body weight ratios. Dietary iron restriction caused a slight decrease in non-heme iron and ferritin (FeRL) expression while it increased transferrin receptor 1 (TfR1) expression without changing ferroportin 1 (FPN1) expression. It also elevated protein lysine acetylation to a more significant level than in ethanol-fed mice under normal dietary iron conditions. Interestingly, iron restriction led to an additional reduction in nicotinamide adenine dinucleotide (NAD+) and NADH levels. Consistent with this observation, the major mitochondrial NAD+-dependent deacetylase, NAD-dependent deacetylase sirtuin-3 (SIRT3), expression was significantly reduced causing increased protein lysine acetylation in ethanol-fed mice at normal and low-iron conditions. In addition, the detection of superoxide dismutase 1 and 2 levels (SOD1 and SOD2) and oxidative phosphorylation (OXPHOS) complex activities allowed us to evaluate the changes in antioxidant and energy metabolism regulated by ethanol consumption at normal and low-iron conditions. We observed that the ethanol-fed mice had mild liver damage associated with reduced energy and antioxidant metabolism. On the other hand, iron restriction may exacerbate certain activities of ethanol further, such as increased protein lysine acetylation and reduced antioxidant metabolism. This metabolic change may prove a barrier to the effectiveness of dietary reduction of iron intake as a preventative measure in chronic alcohol consumption.

Phillyrin and its metabolites exert antipyretic effects by targeting the NAD+ binding domain of GAPDH, MDH2 and IDH2.

BACKGROUND: Fever is one of the main pathophysiological reactions that occurs during the acute phase of various diseases. Excessive body temperature can lead to various adverse consequences such as brain tissue damage and abnormal immune responses. Phillyrin (Phr) is the main active ingredient in Forsythia suspensa (Thunb.) Vahl (Lian Qiao) and has antipyretic effects; however, its antipyretic mechanism of action remains unclear. PURPOSE: This study aimed to explore the antipyretic mechanisms of Phr and provide a new treatment plan for fever. METHODS: The antipyretic effects of Phr were evaluated using a mouse model of pneumonia fever. The main metabolites of Phr involved in its antipyretic function were identified using a mitochondrial temperature-sensitive probe. Further synthesis of the main metabolite, phillygenin (Phg), an alkynylated probe, was performed, and chemical proteomics was used to capture and analyze its direct target for antipyretic effects. The mechanism of action of Phg and its antipyretic targets was explored using metabolomics and various molecular biology methods. RESULTS: Phr showed significant antipyretic and anti-inflammatory effects in a mouse model of lipopolysaccharide-induced fever. Phg reversibly targeted the nicotinamide adenine dinucleotide (NAD+) binding domain of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), malate dehydrogenase 2 (MDH2), and isocitrate dehydrogenase 2 (IDH2) to inhibit their enzymatic activity. In-depth analysis of cellular metabolomics and mitochondrial stress testing indicated that inhibition of GAPDH, MDH2, and IDH2 enzyme activity by Phg led to a decrease in cellular energy supply and heat production regulated by glycolysis, tricarboxylic acid cycle, and oxidative phosphorylation signaling pathways. Phg specifically targeted macrophages and inhibited LPS-induced macrophage activation by downregulating GAPDH enzyme activity, thereby exerting anti-inflammatory effects. In vivo experiments also confirmed that the antipyretic effect of Phr in LPS-induced fever model mice was related to its main metabolites, Phg and Phg-sulfonate (Phg-S), which directly targeted the NAD+ binding domain of GAPDH, IDH2, and MDH2, inhibiting the activity of these enzymes, thereby reducing energy supply and regulating febrile-related inflammatory factors. CONCLUSION: This study reported for the first time that the antipyretic effect of Phr is produced by targeting GAPDH, IDH2, and MDH2 to regulate energy supply and febrile-related inflammatory factors through its main metabolites Phg and Phg-S. This study not only provides potential drugs for fever treatment but also provides new ideas for improving clinical fever treatment plans.

NAD metabolism and heart failure: Mechanisms and therapeutic potentials.

Nicotinamide adenine dinucleotide provides the critical redox pair, NAD+ and NADH, for cellular energy metabolism. In addition, NAD+ is the precursor for de novo NADP+ synthesis as well as the co-substrates for CD38, poly(ADP-ribose) polymerase and sirtuins, thus, playing a central role in the regulation of oxidative stress and cell signaling. Declines of the NAD+ level and altered NAD+/NADH redox states have been observed in cardiometabolic diseases of various etiologies. NAD based therapies have emerged as a promising strategy to treat cardiovascular disease. Strategies that reduce NAD+ consumption or promote NAD+ production have repleted intracellular NAD+ or normalized NAD+/NADH redox in preclinical studies. These interventions have shown cardioprotective effects in multiple models suggesting a great promise of the NAD+ elevating therapy. Mechanisms for the benefit of boosting NAD+ level, however, remain incompletely understood. Moreover, despite the robust pre-clinical studies there are still challenges to translate the therapy to clinic. Here, we review the most up to date literature on mechanisms underlying the NAD+ elevating interventions and discuss the progress of human studies. We also aim to provide a better understanding of how NAD metabolism is changed in failing hearts with a particular emphasis on types of strategies employed and methods to target these pathways. Finally, we conclude with a comprehensive assessment of the challenges in developing NAD-based therapies for heart diseases, and to provide a perspective on the future of the targeting strategies.

Niacin supplementation in a child with novel MTTN variant m.5670A>G causing early onset mitochondrial myopathy and NAD+ deficiency.

Myopathy is a common manifestation in mitochondrial disorders, but the pathomechanisms are still insufficiently studied in children. Here, we report a severe, progressive mitochondrial myopathy in a four-year-old child, who died at eight years. He developed progressive loss of muscle strength with nocturnal hypoventilation and dilated cardiomyopathy. Skeletal muscle showed ragged red fibers and severe combined respiratory chain deficiency. Mitochondrial DNA sequencing revealed a novel m.5670A>G mutation in mitochondrial tRNAAsn (MTTN) with 88 % heteroplasmy in muscle. The proband also had systemic NAD+ deficiency but rescuing this with the NAD+ precursor niacin did not stop disease progression. Targeted metabolomics revealed an overall shift of metabolism towards controls after niacin supplementation, with normalized tryptophan metabolites and lipid-metabolic markers, but most amino acids did not respond to niacin therapy. To conclude, we report a new MTTN mutation, secondary NAD+ deficiency in childhood-onset mitochondrial myopathy with metabolic but meager clinical response to niacin supplementation.

Engineering of a hydroxysteroid dehydrogenase with simultaneous enhancement in activity and thermostability for efficient biosynthesis of ursodeoxycholic acid.

Hydroxysteroid dehydrogenases (HSDHs) catalyze the oxidation/reduction of hydroxyl/keto groups of steroids with high regio- or stereoselectivity, playing an essential role in producing optically pure chemicals. In this work, a novel approach was developed to simultaneously improve the stability and activity of 7ß-hydroxysteroid dehydrogenase (7ß-HSDH) by combining B-factor analysis and computer-aided prediction. Several advantageous mutants were identified, and the most promising variant, S51Y/P202Y, exhibited 2.3-fold improvements in catalytic activity, 3.3-fold in half-life at 40°C, and 4.7-fold in catalytic efficiency (kcat/Km), respectively. Structural modeling analysis showed that the shortened reversible oxidation reaction catalytic distance and the strengthened residue interactions compared to the wild type were attributed to the improved stability and activity of the obtained mutants. To synthesize ursodeoxycholic acid cost-effectively by mutant S51Y/P202Y, a NAD-kinase was employed to facilitate the substitution of nicotinamide adenine dinucleotide phosphate (NADP+) with nicotinamide adenine dinucleotide (NAD+) in the whole-cell catalysis system. The substrate 7-ketolithocholic acid (100 mM) was converted completely in 0.5 h, achieving a space-time yield of 1,887.3 g L-1 d-1. This work provided a general target-oriented strategy for obtaining stable and highly active dehydrogenase for efficient biosynthesis. IMPORTANCE: Hydroxysteroid dehydrogenases have emerged as indispensable tools in the synthesis of steroids, bile acids, and other steroid derivatives for the pharmaceutical and chemical industries. In this study, a novel approach was developed to simultaneously improve the stability and activity of a hydroxysteroid dehydrogenase by combining B-factor analysis and computer-aided prediction. This semi-rational method was demonstrated to be highly effective for enzyme engineering. In addition, NAD kinase was introduced to convert NAD+ to NADP+ for effective coenzyme regeneration in the whole-cell multienzyme-catalyzed system. This strategy reduces the significant economic costs associated with externally supplemented cofactors in NADP-dependent biosynthetic pathways.

Mitochondrial complex I promotes kidney cancer metastasis.

Most kidney cancers are metabolically dysfunctional1-4, but how this dysfunction affects cancer progression in humans is unknown. We infused 13C-labelled nutrients in over 80 patients with kidney cancer during surgical tumour resection. Labelling from [U-13C]glucose varies across subtypes, indicating that the kidney environment alone cannot account for all tumour metabolic reprogramming. Compared with the adjacent kidney, clear cell renal cell carcinomas (ccRCCs) display suppressed labelling of tricarboxylic acid (TCA) cycle intermediates in vivo and in ex vivo organotypic cultures, indicating that suppressed labelling is tissue intrinsic. [1,2-13C]acetate and [U-13C]glutamine infusions in patients, coupled with measurements of respiration in isolated human kidney and tumour mitochondria, reveal lower electron transport chain activity in ccRCCs that contributes to decreased oxidative and enhanced reductive TCA cycle labelling. However, ccRCC metastases unexpectedly have enhanced TCA cycle labelling compared with that of primary ccRCCs, indicating a divergent metabolic program during metastasis in patients. In mice, stimulating respiration or NADH recycling in kidney cancer cells is sufficient to promote metastasis, whereas inhibiting electron transport chain complex I decreases metastasis. These findings in humans and mice indicate that metabolic properties and liabilities evolve during kidney cancer progression, and that mitochondrial function is limiting for metastasis but not growth at the original site.

Disruption of glucose homeostasis by bacterial infection orchestrates host innate immunity through NAD+/NADH balance.

Metabolic reprogramming is crucial for activating innate immunity in macrophages, and the accumulation of immunometabolites is essential for effective defense against infection. The NAD+/NADH (ratio of nicotinamide adenine dinucleotide and its reduced counterpart) redox couple serves as a critical node that integrates metabolic pathways and signaling events, but how this metabolite couple engages macrophage activation remains unclear. Here, we show that the NAD+/NADH ratio serves as a molecular signal that regulates proinflammatory responses and type I interferon (IFN) responses divergently. Salmonella Typhimurium infection leads to a decreased NAD+/NADH ratio by inducing the accumulation of NADH. Further investigation shows that an increased NAD+/NADH ratio correlates with attenuated proinflammatory responses and enhanced type I IFN responses. Conversely, a decreased NAD+/NADH ratio is linked to intensified proinflammatory responses and restrained type I IFN responses. These results show that the NAD+/NADH ratio is an essential cell-intrinsic factor that orchestrates innate immunity, which enhances our understanding of how metabolites fine-tune innate immunity.

Immune infiltration and prognosis in gastric cancer: role of NAD+ metabolism-related markers.

Background: This study endeavored to develop a nicotinamide adenine dinucleotide (NAD+) metabolism-related biomarkers in gastric cancer (GC), which could provide a theoretical foundation for prognosis and therapy of GC patients. Methods: In this study, differentially expressed genes (DEGs1) between GC and paraneoplastic tissues were overlapped with NAD+ metabolism-related genes (NMRGs) to identify differentially expressed NMRGs (DE-NMRGs). Then, GC patients were divided into high and low score groups by gene set variation analysis (GSVA) algorithm for differential expression analysis to obtain DEGs2, which was overlapped with DEGs1 for identification of intersection genes. These genes were further analyzed using univariate Cox and least absolute shrinkage and selection operator (LASSO) regression analyses to obtain prognostic genes for constructing a risk model. Enrichment and immune infiltration analyses further investigated investigate the different risk groups, and qRT-PCR validated the prognostic genes. Results: Initially, we identified DE-NMRGs involved in NAD biosynthesis, with seven (DNAJB13, CST2, THPO, CIDEA, ONECUT1, UPK1B and SNCG) showing prognostic significance in GC. Subsequent, a prognostic model was constructed in which the risk score, derived from the expression profiles of these genes, along with gender, emerged as robust independent predictors of patient outcomes in GC. Enrichment analysis linked high-risk patients to synaptic membrane pathways and low-risk to the CMG complex pathway. Tumor immune infiltration analysis revealed correlations between risk scores and immune cell abundance, suggesting a relationship between NAD+ metabolism and immune response in GC. The prognostic significance of our identified genes was validated by qRT-PCR, which confirmed their upregulated expression in GC tissue samples. Conclusion: In this study, seven NAD+ metabolism-related markers were established, which is of great significance for the development of prognostic molecular biomarkers and clinical prognosis prediction for gastric cancer patients.

NAD+ deficiency primes defense metabolism via 1O2-escalated jasmonate biosynthesis in plants.

Nicotinamide adenine dinucleotide (NAD+) is a redox cofactor and signal central to cell metabolisms. Disrupting NAD homeostasis in plant alters growth and stress resistance, yet the underlying mechanisms remain largely unknown. Here, by combining genetics with multi-omics, we discover that NAD+ deficiency in qs-2 caused by mutation in NAD+ biosynthesis gene-Quinolinate Synthase retards growth but induces biosynthesis of defense compounds, notably aliphatic glucosinolates that confer insect resistance. The elevated defense in qs-2 is resulted from activated jasmonate biosynthesis, critically hydroperoxidation of α-linolenic acid by the 13-lipoxygenase (namely LOX2), which is escalated via the burst of chloroplastic ROS-singlet oxygen (1O2). The NAD+ deficiency-mediated JA induction and defense priming sequence in plants is recapitulated upon insect infestation, suggesting such defense mechanism operates in plant stress response. Hence, NAD homeostasis is a pivotal metabolic checkpoint that may be manipulated to navigate plant growth and defense metabolism for stress acclimation.

Extracellular NAD+ response to post-hepatectomy liver failure: bridging preclinical and clinical findings.

Liver fibrosis progressing to cirrhosis is a major risk factor for liver cancer, impacting surgical treatment and survival. Our study focuses on the role of extracellular nicotinamide adenine dinucleotide (eNAD+) in liver fibrosis, analyzing liver disease patients undergoing surgery. Additionally, we explore NAD+'s therapeutic potential in a mouse model of extended liver resection and in vitro using 3D hepatocyte spheroids. eNAD+ correlated with aspartate transaminase (AST) and bilirubin after liver resection (AST: r = 0.2828, p = 0.0087; Bilirubin: r = 0.2584, p = 0.0176). Concordantly, post-hepatectomy liver failure (PHLF) was associated with higher eNAD+ peaks (n = 10; p = 0.0063). Post-operative eNAD+ levels decreased significantly (p < 0.05), but in advanced stages of liver fibrosis or cirrhosis, this decline not only diminished but actually showed a trend towards an increase. The expression of NAD+ biosynthesis rate-limiting enzymes, nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide mononucleotide adenylyltransferase 3 (NMNAT3), were upregulated significantly in the liver tissue of patients with higher liver fibrosis stages (p < 0.0001). Finally, the administration of NAD+ in a 3D hepatocyte spheroid model rescued hepatocytes from TNFalpha-induced cell death and improved viability (p < 0.0001). In a mouse model of extended liver resection, NAD+ treatment significantly improved survival (p = 0.0158) and liver regeneration (p = 0.0186). Our findings reveal that eNAD+ was upregulated in PHLF, and rate-limiting enzymes of NAD+ biosynthesis demonstrated higher expressions under liver fibrosis. Further, eNAD+ administration improved survival after extended liver resection in mice and enhanced hepatocyte viability in vitro. These insights may offer a potential target for future therapies.