NAD Metabolism Research Articles

Mitochondrial and peroxisomal NAD+ uptake are important for improved photosynthesis and seed yield under elevated CO2 concentrations.

As sessile organisms, plants must adapt their physiology and developmental processes to cope with challenging environmental circumstances, such as the ongoing elevation in atmospheric carbon dioxide (CO2 ) levels. Nicotinamide adenine dinucleotide (NAD+ ) is a cornerstone of plant metabolism and plays an essential role in redox homeostasis. Given that plants impaired in NAD metabolism and transport often display growth defects, low seed production and disturbed stomatal development/movement, we hypothesized that subcellular NAD distribution could be a candidate for plants to exploit the effects of CO2 fertilization. We report that an efficient subcellular NAD+ distribution is required for the fecundity-promoting effects of elevated CO2 levels. Plants with reduced expression of either mitochondrial (NDT1 or NDT2) or peroxisomal (PXN) NAD+ transporter genes grown under elevated CO2 exhibited reduced total leaf area compared with the wild-type while PXN mutants also displayed reduced leaf number. NDT2 and PXN lines grown under elevated CO2 conditions displayed reduced rosette dry weight and lower photosynthetic rates coupled with reduced stomatal conductance. Interestingly, high CO2 doubled seed production and seed weight in the wild-type, whereas the mutants were less responsive to increases in CO2 levels during reproduction, producing far fewer seeds than the wild-type under both CO2 conditions. These data highlight the importance of mitochondrial and peroxisomal NAD+ uptake mediated by distinct NAD transporter proteins to modulate photosynthesis and seed production under high CO2 levels.

Abstract 6254: Identification of nicotinic acid phosphoribosyltransferase inhibitors with anticancer properties by in silico drug design

Abstract Cancer cells rely heavily on sustained NAD levels to support their survival and proliferation. Thus, depleting NAD levels in tumor cells through interference with their NAD-biosynthetic machinery has been proposed as a promising anticancer strategy. Several pathways contribute to NAD production in mammals, such as the de novo and the Preiss-Handler (PH) pathways, which generate NAD from tryptophan and from nicotinic acid, respectively. In addition, the NAD salvage pathway, which utilizes nicotinamide as a substrate, is also a major NAD-producing route and its rate-limiting enzyme, nicotinamide phosphoribosyltransferase (NAMPT), is commonly overexpressed in a multitude of human cancers. Over the past two decades, several potent NAMPT inhibitors have been developed, such as FK866. However, their clinical efficacy has proven limited, suggesting that the other NAD-producing routes are frequently also active in cancer cells and are responsible for causing resistance to NAMPT inhibitors. Recently, we have shown nicotinic acid phosphoribosyl transferase (NAPRT), the key enzyme in the PH pathway, to play an important role in NAD metabolism in cancer cells. We demonstrated that NAPRT is amplified and overexpressed in several types of solid tumors and that its expression is a critical determinant of cancer cell susceptibility to NAMPT inhibitors. Although several NAPRT inhibitors have been reported, including 2-hydroxy nicotinic acid (2-HNA), their low potency and/or poor solubility limit their clinical applicability. To identify new NAPRT inhibitors with enhanced pharmacological profiles, we performed a high-throughput molecular docking screen, taking advantage of the availability of NAPRT crystal structure. The first 500 hits were visually inspected to prioritize compounds that reproduced, at least in part, the putative binding mode of known substrates and inhibitors and complied with simple physicochemical rules such as Lipinski’s rule of five. In addition, several 2-HNA analogs were selected. Altogether, this led to a final list of 50 compounds that were tested in vitro. We show that four compounds from this list were able to sensitize NAPRT-overexpressing ovarian cancer cells to FK866, resulting in a more significant reduction in intracellular NAD levels, as well as in a much more pronounced cytotoxicity as compared to FK866 alone. The inhibitory constant (Ki) of the new NAPRT inhibitors was found to be in the micromolar range. In conclusion, in silico molecular docking screens are a promising approach to identify new NAPRT inhibitors with anticancer effects. Citation Format: Moustafa Ghanem, Amr Khalifa, Irene Caffa, Aimable Nahimana, Michel Duchosal, Alberto Del Rio, Santina Bruzzone, Francesco Piancente, Alessio Nencioni. Identification of nicotinic acid phosphoribosyltransferase inhibitors with anticancer properties by in silico drug design [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6254.

P-282 Impact of death-associated protein-3 (DAP3) and DAP3 binding cell death enhancer 1 (DELE1) on drug sensitivity in colorectal cancer cells

Death association protein-3 (DAP3) is an important growth/death regulator of cells, has been found to have important clinical value in various cancer. DELE1 (DAP3 Binding Cell Death Enhancer 1) has been reported to be a partner of DAP3 and plays a key part in mitochondrial metabolism and mitochondria stress signalling (1). DELE1 also appears to regulate the apoptotic signalling in cells including cancer cells. However, the role played by the two molecules markedly vary depending on cell and tumour types with only a very limited tumour types have been investigated. High expressions of DAP3 and DELE1 have been found to strongly correlated with poor overall survival, disease free survival and distant metastasis free survival in colorectal cancer (2). The present study was to assess the impact of DAP3 and DELE1 on the drug sensitivity of colorectal cancers in vitro. Human colorectal cancer cell lines, RKO and HT115 were used. RKO and HT115 cells were transfected with the lentivirus particles that contained an anti-DAP3 shRNA to create the DAP3 knockdown cell model, designated as RKODAP3kd and HT115DAP3kd, respectively. A DELE1 siRNA was also applied to these cells in order to silence the expression of DELE1 (the new submodels designated as RKODELE1kd and HT115DELE1kd). The DAP3/DELE1 Dual knockdown cell models were established by using the anti-DAP3 shRNA and DELE1 siRNA combination transfection and are referred here as RKODAP3kd/DELE1kd and HT115DAP3kd/DELE1kd. The knockdown of the respective gene transcripts was confirmed by quantitative PCR analysis and respective proteins by Western blot method. Cytotoxicity tests were performed on these sub-models using 5-fluorouracil (5-FU), Docetaxel (DTX) and Methotrexate (MTX) with different concentrations. Additionally, the mitochondrial response following genetic knockdowns were also evaluated. Knockdown DAP3 and knockdown of DELE1 sensitised the RKO cells to 5-FU, MTX and DTX with a IC50 at for control cells at respectively at 14.2mM, 1.6mM, 57.1nM and for RKODAP3kd at 10.4mM, 1.2mM and 51.0nM. Although knocking down DELE1 had less impact than the DAP3 knockdown, DAP3/DELE1 double knockdown however had more profound effects compared with single knockdowns and with wild type controls (IC50 for 5-FU, MTX and DTX in RKODAP3kd/DELE1kd cells being respectively at 7.0mM, 0.6mM and 1.5nM). In the metabolic analyses including metabolism of glucose, lactate, NAD(P)H and nitrogen dioxide (NO2), we did not detect a significant change following DAP3 knockdown, DELE1 knockdown or the double knockdown, arguing an mitochondria pathway independent regulation of the drug sensitivity by the two molecules. DAP3 and DELE1, important growth/death regulators and prognostic factors of in colorectal cancer, have a role in the chemosensitivity in colorectal cancer cells. The findings suggest that molecules are potential targets in therapeutic intervention in colorectal cancer.

Regulation of NAD Metabolism in Diastolic Dysfunction Induced by Metabolic Stress

Patients of heart failure with preserved ejection fraction (HFpEF) have diastolic dysfunction. Intervention targeting mechanisms of diastolic dysfunction is lacking, leading to a growing population of patients with HFpEF. Diabetes and obesity are key risk factors of HFpEF. NAD depletion is associated with heart failure, diabetes and obesity, and is due to NAD redox imbalance, suppressed synthesis and/or activated consumption pathways. Here, we used mouse models manipulating NAD redox balance and hydrolysis to determine their roles in metabolic cardiomyopathy. Cardiac Ndufs4‐KO mice (cKO) have lowered cardiac NAD/NADH ratio, normal baseline function, geometry and energetics. Chronic diabetic stress induced by streptozotocin (STZ) or diet‐induced obesity (DIO) in wild type (WT) mice promoted diastolic dysfunction, which was further exacerbated in cKO mice with latent cardiac NAD redox imbalance. Elevated oxidative stress by SOD2 acetylation and increased TnI‐S150 phosphorylation by energetic deficiency in diabetic cKO hearts led to exacerbated diastolic dysfunction. Elevation of cardiac NAD levels normalized NAD/NADH ratio, ameliorated these pathogenic mechanisms and diastolic dysfunction in metabolically stressed mice. These results supported the causal role of NAD redox imbalance in metabolic cardiomyopathy.Next, we tested whether deficiency of SARM1, an intracellular NAD hydrolase, will ameliorate diastolic dysfunction induced by metabolic stresses. WT and total SARM1‐KO mice were challenged with diabetic stress or DIO. These metabolic stresses led to a decline in diastolic function, which was improved in SARM1‐KO mice. DIO also promoted cardiac hypertrophy that was ameliorated by SARM1 deficiency. To determine that the improvement of diastolic function by SARM1 deficiency was not due to a reversal of systemic metabolic state, a comprehensive plasma metabolomic analyses was performed. Hyperglycemia was similar in diabetic WT and SARM1‐KO plasma. Metabolomic analyses showed significant changes in 58 out of 298 aqueous metabolites and in 26 out of 85 lipid species measured, when comparing non‐diabetic WT to diabetic WT plasma. When comparing the 84 metabolites with diabetic changes from diabetic WT and diabetic SARM1‐KO plasma, only one of the lipid metabolites (PC 16:0/16:0) showed a mild further decrease in SARM1‐KO plasma. The metabolomic data suggest that WT and SARM1‐KO hearts experienced similar metabolic stress, and that the improvement of diastolic dysfunction by SARM1 deficiency was due to the reversal of cardiac pathogenic mechanisms.To determine how SARM1 deficiency affected cardiac NAD metabolism, transcript levels of genes in NAD metabolic pathways were assessed. Expressions of Bst1 and Cd38 NAD hydrolases did not change in SARM1‐KO hearts, suggesting that SARM1 deficiency did not induce compensatory changes in expression of the other NAD hydrolases. SARM1‐KO hearts showed lower expressions in several other NAD consuming genes (e.g. Sirtuins) and NAD synthesis genes (e.g. Nmnats). Cardiac fibrosis, pro‐fibrotic genes (e.g. ctgf) and Nppb expression were induced in diabetic WT hearts and were suppressed by SARM1 deficiency. In summary, our data support the critical roles of NAD redox imbalance and SARM1 in diastolic dysfunction induced by metabolic stress.

SARM1 NAD Hydrolase Deficiency Normalizes Fibrosis and Ameliorates Cardiac Dysfunction in Diabetic Hearts

Decline in cellular NAD levels emerges as a hallmark of diseases such as diabetes and heart failure. Strategies to activate NAD synthesis pathways and replenish NAD levels have shown promises to treat heart disease. However, how NAD consumption mechanisms lead to NAD depletion and disease pathogeneses are less understood. SARM1 is an intracellular NAD hydrolase, and its role in heart disease has never been investigated. We therefore hypothesized that SARM1 deficiency may protect hearts against diabetic cardiomyopathy. Male C57BL/6 wild‐type (WT) and SARM1‐KO mice were challenged with 16‐week diabetic stress initiated by streptozotocin injections (STZ). Longitudinal cardiac function was measured by echocardiography after 8 or 16 weeks of diabetes, and tissue and plasma samples were harvested for biochemical and molecular analyses. Chronic diabetic stress lowered cardiac NAD levels and led to progressive decline in systolic function of WT mice. Diabetic SARM1‐KO mice showed improved systolic function and increased cardiac NAD levels, compared to diabetic WT mice. Progressive decline in diastolic function induced by chronic diabetes was also improved by SARM1 deficiency. Heart weights and hyperglycemia were similar in diabetic WT or diabetic SARM1‐KO mice. Metabolomic analyses of plasma showed significant changes in 58 out of ~300 aqueous metabolites assessed when comparing non‐diabetic WT to diabetic WT plasma, including increased levels of glucose and branched‐chain amino acids. However, the 58 metabolites showed no changes in levels when comparing diabetic WT to diabetic SARM1‐KO plasma. Similarly, we found that 26 out of 85 lipid species showed significant changes when comparing non‐diabetic WT to diabetic WT plasma, and only one of these 26 lipid metabolites showed a significant change between diabetic WT and diabetic SARM1‐KO plasma. The data suggest that cardioprotection in SARM1‐KO mice was likely due to the reversal of cardiac pathogenic mechanisms but not improved systemic metabolic state.To determine how SARM1 deficiency affects cardiac NAD metabolism, expression of genes in NAD metabolic pathways were assessed. Levels of Bst1 and Cd38 did not change in SARM1‐KO hearts, suggesting that SARM1 deficiency did not induce compensatory changes in expression of the other NAD hydrolases. SARM1‐KO hearts showed lower expressions in some of the NAD consuming genes (e.g. Sirtuins) and NAD synthesis genes (e.g. Nmnats). Cardiac dysfunction marker, Nppb expression, was induced in diabetic WT hearts, and was reversed by SARM1 deficiency. Cardiac fibrosis and pro‐fibrotic genes (e.g. ctgf) were induced in diabetic WT hearts and were suppressed by SARM1 deficiency. Using the total SARM1 KO mice, our data support that SARM1 deficiency normalizes decline in NAD levels and pro‐fibrotic pathways to ameliorate diabetic cardiomyopathy. Further experiments are warranted to dissect the cell‐type specific roles of SARM1 in diabetic hearts.

Integrating Single Cell RNAseq and Computational Modeling to Infer Hepatocyte Metabolic Functionalities during Liver Regeneration

Hepatocytes make up over 80% of liver mass and carry out all essential liver functions such as protein synthesis and detoxification, among others. Recent studies have shown distinct metabolic functions of proliferating and non‐proliferating hepatocytes as a compensatory mechanism during liver regeneration. However, this mechanism has not been studied in the case of alcoholic liver disease (ALD), which is known to disrupt the regenerative capacity and alter the metabolic processes of the liver. We seek to determine if alcohol consumption alters the balance in metabolic functionality between proliferating and non‐proliferating hepatocyte populations during liver regeneration. In the present study, rats were pair‐fed either a chronic ethanol diet or a carbohydrate control diet per Lieber DeCarli calorie‐matched liquid diet protocol. We performed 70% partial hepatectomy (PHx) and liver samples were collected at the baseline and at 24 hours post PHx. Single hepatocytes were isolated by laser capture microdissection (LCM) where the subset of proliferating hepatocytes were identified by Ki67 staining. Single‐cell RNA‐sequencing (scRNA‐seq) was employed for the purpose of identifying transcriptional differences across hepatocyte subtypes (i.e. PHx, diet, proliferation). Principal component analysis (PCA) of all samples showed distinct separation of cells into clusters based on liver regeneration (baseline and PHx samples), but not on diet (control and ethanol) and cellular proliferation (Ki67‐positive vs. negative staining). PCA of baseline samples showed separation of proliferating and non‐proliferating hepatocytes in the ethanol group. We characterized metabolic differences between hepatocyte subtypes using a computational model of genome‐scale metabolic reaction activities. We found that the metabolic compartmentalization separating proliferating from non‐proliferating hepatocytes in the carbohydrate group is absent in the ethanol group. Several functional differentiating factors arose favoring reactive oxygen species detoxification and steroid metabolism in proliferating hepatocytes from ethanol‐adapted animals at baseline, which were absent post PHx. Furthermore, proliferating cells from the carbohydrate group showed higher reaction activity in CoA synthesis, NAD metabolism and inositol phosphate metabolic pathways as compared to their non‐proliferating counterparts, both at baseline and following PHx. Our results also indicate that these pathways are also active in the non‐proliferating hepatocytes following PHx in the ethanol group. Taken together, our combinatorial transcriptomic analysis and computational modeling showed distinct metabolic pathways differentiating proliferating from non‐proliferating hepatocytes at baseline, which is absent in hepatocytes following PHx in the ethanol group. These results point towards an alternative mechanism of metabolic compensation in ethanol‐fed animals such that similar metabolic pathways are active in both proliferating and non‐proliferating hepatocytes. This lack of metabolic compartmentalization may hinder the regenerative response to PHx in the ethanol‐fed animals. Targeting the metabolic division of labor may be a potential intervention to promote liver regenerative capacity in alcoholic liver disease.

Differences in Extracellular NAD+ and NMN Metabolism on the Surface of Vascular Endothelial Cells.

Simple SummaryNicotinamide adenine dinucleotide (NAD+) is a multifunctional metabolite involved in many key cellular processes. Outside the cell, NAD+ or its metabolites are important signaling molecules, related especially to calcium homeostasis, which controls the functioning of the heart. The cleavage of NAD+ or its precursor, nicotinamide mononucleotide (NMN), produces derivatives entering the cell to rebuild the intracellular NAD+ pool, which is important for cells with high energy turnover. Abnormalities in NAD+ and NMN metabolism can lead to cell aging and the development of cardiovascular diseases. In this study, we demonstrated that the extracellular metabolism of NAD+ and NMN is vastly different in the vascular endothelium obtained from different species and locations. This may have implications for strategies to modulate the NAD+ system and may cause difficulties for comparing the results of different reports.The disruption of the metabolism of extracellular NAD+ and NMN may affect related signaling cascades and pathologies, such as cardiovascular or respiratory system diseases. We aimed to study NAD+ and NMN hydrolysis on surface endothelial cells of diverse origins and with genetically modified nucleotide catabolism pathways. We tested lung endothelial cells isolated from C57BL/6 J wild-type (WT) and C57BL/6 J CD73 knockout (CD73 KO) mice, the transfected porcine iliac artery endothelial cell line (PIEC) with the human E5NT gene for CD73 (PIEC CD73), and a mock-transfected control (PIEC MOCK), as well as HMEC-1 and H5V cells. Substrate conversion into the product was followed by high-performance liquid chromatography (HPLC). We showed profound differences in extracellular NAD+ and NMN metabolism related to the vessel origin, species diversity, and type of culture. We also confirmed the involvement of CD38 and CD73 in NAD+ and NMN cleavage.

Interventions in Nicotinamide Adenine Dinucleotide Metabolism, the Intestinal Microbiota and Microcin Peptide Antimicrobials.

A proper NADH/NAD + balance allows for the flow of metabolic and catabolic activities determining cellular growth. In Escherichia coli, more than 80 NAD + dependent enzymes are involved in all major metabolic pathways, including the post-transcriptional build-up of thiazole and oxazole rings from small linear peptides, which is a critical step for the antibiotic activity of some microcins. In recent years, NAD metabolism boosting drugs have been explored, mostly precursors of NAD + synthesis in human cells, with beneficial effects on the aging process and in preventing oncological and neurological diseases. These compounds also enhance NAD + metabolism in the human microbiota, which contributes to these beneficial effects. On the other hand, inhibition of NAD + metabolism has been proposed as a therapeutic approach to reduce the growth and propagation of tumor cells and mitigating inflammatory bowel diseases; in this case, the activity of the microbiota might mitigate therapeutic efficacy. Antibiotics, which reduce the effect of microbiota, should synergize with NAD + metabolism inhibitors, but these drugs might increase the proportion of antibiotic persistent populations. Conversely, antibiotics might have a stronger killing effect on bacteria with active NAD + production and reduce the cooperation of NAD + producing bacteria with tumoral cells. The use of NADH/NAD + modulators should take into consideration the use of antibiotics and the population structure of the microbiota.

Kynurenine importation by SLC7A11 propagates anti-ferroptotic signaling

IDO1 oxidizes tryptophan (TRP) to generate kynurenine (KYN), the substrate for 1-carbon and NAD metabolism, and is implicated in pro-cancer pathophysiology and infection biology. However, the mechanistic relationships between IDO1 in amino acid depletion versus product generation have remained a longstanding mystery. We found an unrecognized link between IDO1 and cell survival mediated by KYN that serves as the source for molecules that inhibit ferroptotic cell death. We show that this effect requires KYN export from IDO1-expressing cells, which is then available for non-IDO1-expressing cells via SLC7A11, the central transporter involved in ferroptosis suppression. Whether inside the "producer" IDO1+ cell or the "receiver" cell, KYN is converted into downstream metabolites, suppressing ferroptosis by ROS scavenging and activating an NRF2-dependent, AHR-independent cell-protective pathway, including SLC7A11, propagating anti-ferroptotic signaling. IDO1, therefore, controls a multi-pronged protection pathway from ferroptotic cell death, underscoring the need to re-evaluate the use of IDO1 inhibitors in cancer treatment.

Dynamic Control Balancing Cell Proliferation and Inflammation is Crucial for an Effective Immune Response to Malaria.

Malaria has a complex pathology with varying manifestations and symptoms, effects on host tissues, and different degrees of severity and ultimate outcome, depending on the causative Plasmodium pathogen and host species. Previously, we compared the peripheral blood transcriptomes of two macaque species (Macaca mulatta and Macaca fascicularis) in response to acute primary infection by Plasmodium knowlesi. Although these two species are very closely related, the infection in M. mulatta is fatal, unless aggressively treated, whereas M. fascicularis develops a chronic, but tolerable infection in the blood. As a reason for this stark difference, our analysis suggests delayed pathogen detection in M. mulatta followed by extended inflammation that eventually overwhelms this monkey’s immune response. By contrast, the natural host M. fascicularis detects the pathogen earlier and controls the inflammation. Additionally, M. fascicularis limits cell proliferation pathways during the log phase of infection, presumably in an attempt to control inflammation. Subsequent cell proliferation suggests a cell-mediated adaptive immune response. Here, we focus on molecular mechanisms underlying the key differences in the host and parasite responses and their coordination. SICAvar Type 1 surface antigens are highly correlated with pattern recognition receptor signaling and important inflammatory genes for both hosts. Analysis of pathogen detection pathways reveals a similar signaling mechanism, but with important differences in the glutamate G-protein coupled receptor (GPCR) signaling pathway. Furthermore, differences in inflammasome assembly processes suggests an important role of S100 proteins in balancing inflammation and cell proliferation. Both differences point to the importance of Ca2+ homeostasis in inflammation. Additionally, the kynurenine-to-tryptophan ratio, a known inflammatory biomarker, emphasizes higher inflammation in M. mulatta during log phase. Transcriptomics-aided metabolic modeling provides a functional method for evaluating these changes and understanding downstream changes in NAD metabolism and aryl hydrocarbon receptor (AhR) signaling, with enhanced NAD metabolism in M. fascicularis and stronger AhR signaling in M. mulatta. AhR signaling controls important immune genes like IL6, IFNγ and IDO1. However, direct changes due to AhR signaling could not be established due to complicated regulatory feedback mechanisms associated with the AhR repressor (AhRR). A complete understanding of the exact dynamics of the immune response is difficult to achieve. Nonetheless, our comparative analysis provides clear suggestions of processes that underlie an effective immune response. Thus, our study identifies multiple points of intervention that are apparently responsible for a balanced and effective immune response and thereby paves the way toward future immune strategies for treating malaria.

Metabolic Disease, NAD Metabolism, Nicotinamide Riboside, and the Gut Microbiome: Connecting the Dots from the Gut to Physiology.

ABSTRACTThe effort to use nutrients as interventions to treat human disease has been important to medicine. A current example in this vein pertains to NAD+ boosters, such as nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN), which are in many clinical trials in a variety of disease conditions. Independent laboratories have shown that ingested NR (or NMN) has mitigating effects on metabolic syndrome in mice. V. V. Lozada-Fernández, O. deLeon, S. L. Kellogg, F. L. Saravia, et al. (mSystems 7:e00230-21, 2022, https://doi.org/10.1128/mSystems.00230-21) show that NR shifts gut microbiome contents and that the transplantation of an NR-conditioned microbiome by fecal transfer reproduces some effects of NR in mice on a high-fat diet. The involvement of the gut microbiome as a factor in NR effects is linked to changes to the gut microbiome and its activity to transform NR and downstream catabolites. This commentary draws attention to these findings and focuses on some puzzling aspects of NAD+ boosters, exploring the still murky interactions between NAD+ metabolism, energy homeostasis, and the gut microbiome.

Mesenchymal stromal cells attenuate alveolar type 2 cells senescence through regulating NAMPT-mediated NAD metabolism

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic and progressive deadly fibrotic lung disease with high prevalence and mortality worldwide. The therapeutic potential of mesenchymal stem cells (MSCs) in pulmonary fibrosis may be attributed to the strong paracrine, anti-inflammatory, anti-apoptosis and immunoregulatory effects. However, the mechanisms underlying the therapeutic effects of MSCs in IPF, especially in terms of alveolar type 2 (AT2) cells senescence, are not well understood. The purpose of this study was to evaluate the role of MSCs in NAD metabolism and senescence of AT2 cells in vitro and in vivo.MethodsMSCs were isolated from human bone marrow. The protective effects of MSCs injection in pulmonary fibrosis were assessed via bleomycin mouse models. The senescence of AT2 cells co-cultured with MSCs was evaluated by SA-β-galactosidase assay, immunofluorescence staining and Western blotting. NAD+ level and NAMPT expression in AT2 cells affected by MSCs were determined in vitro and in vivo. FK866 and NAMPT shRNA vectors were used to determine the role of NAMPT in MSCs inhibiting AT2 cells senescence.ResultsWe proved that MSCs attenuate bleomycin-induced pulmonary fibrosis in mice. Senescence of AT2 cells was alleviated in MSCs-treated pulmonary fibrosis mice and when co-cultured with MSCs in vitro. Mechanistic studies showed that NAD+ and NAMPT levels were rescued in AT2 cells co-cultured with MSCs and MSCs could suppress AT2 cells senescence mainly via suppressing lysosome-mediated NAMPT degradation.ConclusionsMSCs attenuate AT2 cells senescence by upregulating NAMPT expression and NAD+ levels, thus exerting protective effects in pulmonary fibrosis.

Age-Dependent Decline of NAD+-Universal Truth or Confounded Consensus?

Nicotinamide adenine dinucleotide (NAD+) is an essential molecule involved in various metabolic reactions, acting as an electron donor in the electron transport chain and as a co-factor for NAD+-dependent enzymes. In the early 2000s, reports that NAD+ declines with aging introduced the notion that NAD+ metabolism is globally and progressively impaired with age. Since then, NAD+ became an attractive target for potential pharmacological therapies aiming to increase NAD+ levels to promote vitality and protect against age-related diseases. This review summarizes and discusses a collection of studies that report the levels of NAD+ with aging in different species (i.e., yeast, C. elegans, rat, mouse, monkey, and human), to determine whether the notion that overall NAD+ levels decrease with aging stands true. We find that, despite systematic claims of overall changes in NAD+ levels with aging, the evidence to support such claims is very limited and often restricted to a single tissue or cell type. This is particularly true in humans, where the development of NAD+ levels during aging is still poorly characterized. There is a need for much larger, preferably longitudinal, studies to assess how NAD+ levels develop with aging in various tissues. This will strengthen our conclusions on NAD metabolism during aging and should provide a foundation for better pharmacological targeting of relevant tissues.

NAPRT Expression Regulation Mechanisms: Novel Functions Predicted by a Bioinformatics Approach

The nicotinate phosphoribosyltransferase (NAPRT) gene has gained relevance in the research of cancer therapeutic strategies due to its main role as a NAD biosynthetic enzyme. NAD metabolism is an attractive target for the development of anti-cancer therapies, given the high energy requirements of proliferating cancer cells and NAD-dependent signaling. A few studies have shown that NAPRT expression varies in different cancer types, making it imperative to assess NAPRT expression and functionality status prior to the application of therapeutic strategies targeting NAD. In addition, the recent finding of NAPRT extracellular form (eNAPRT) suggested the involvement of NAPRT in inflammation and signaling. However, the mechanisms regulating NAPRT gene expression have never been thoroughly addressed. In this study, we searched for NAPRT gene expression regulatory mechanisms in transcription factors (TFs), RNA binding proteins (RBPs) and microRNA (miRNAs) databases. We identified several potential regulators of NAPRT transcription activation, downregulation and alternative splicing and performed GO and expression analyses. The results of the functional analysis of TFs, RBPs and miRNAs suggest new, unexpected functions for the NAPRT gene in cell differentiation, development and neuronal biology.

Evolution of Natural Lifespan Variation and Molecular Strategies of Extended Lifespan

To understand the genetic basis and the selective forces acting on longevity, it is useful to employ ecologically diverse individuals of the same species, widely different in lifespan. This way, we may capture the experiment of Nature that modifies the genotype arriving at different lifespans. Here, we analyzed 76 ecologically diverse wild yeast isolates and discovered wide diversity of lifespan. We sequenced the genomes of these organisms and analyzed how their replicative lifespan is shaped by nutrients and transcriptional and metabolite patterns. By identifying genes, proteins and metabolites that correlate with longevity across these isolates, we found that long-lived strains elevate intermediary metabolites, differentially regulate genes involved in NAD metabolism and adjust control of epigenetic landscape through conserved, rare histone modifier. Our data further offer insights into the evolution and mechanisms by which caloric restriction regulates lifespan by modulating the availability of nutrients without decreasing fitness.

The 2021 FASEB science research conference on NAD metabolism and signaling

Aging | doi:10.18632/aging.203766. Vera Gorbunova, Marcus Buschbeck, Xiaolu A. Cambronne, Karthikeyani Chellappa, Daniela Corda, Juan Du, Marc Freichel, Jonathan Gigas, Alexander E. Green, Feng Gu, Iva Guberovic, Aravinthkumar Jayabalan, Imrankhan Khansahib, Sarmistha Mukherjee, Andrei Seluanov, Matthew A. Simon, Lars J. Sverkeli, Nora Kory, Daniel C. Levine, Ivan Matic, Andrey Nikiforov, Johannes G.M. Rack, Shin-Ichiro Imai, David A. Sinclair, Debra Toiber, Yongjuan Zhao, Raul Mostoslavsky, Lee Kraus, Andreas H. Guse

Evolution of a histone variant involved in compartmental regulation of NAD metabolism.

NAD metabolism is essential for all forms of life. Compartmental regulation of NAD+ consumption, especially between the nucleus and the mitochondria, is required for energy homeostasis. However, how compartmental regulation evolved remains unclear. In the present study, we investigated the evolution of the macrodomain-containing histone variant macroH2A1.1, an integral chromatin component that limits nuclear NAD+ consumption by inhibiting poly(ADP-ribose) polymerase 1 in vertebrate cells. We found that macroH2A originated in premetazoan protists. The crystal structure of the macroH2A macrodomain from the protist Capsaspora owczarzaki allowed us to identify highly conserved principles of ligand binding and pinpoint key residue substitutions, selected for during the evolution of the vertebrate stem lineage. Metabolic characterization of the Capsaspora lifecycle suggested that the metabolic function of macroH2A was associated with nonproliferative stages. Taken together, we provide insight into the evolution of a chromatin element involved in compartmental NAD regulation, relevant for understanding its metabolism and potential therapeutic applications.

NNMT is induced dynamically during beige adipogenesis in adipose tissues depot-specific manner.

Nicotinamide N-methyltransferase (NNMT) is a novel regulator, shown recently to regulate adipose tissue energy expenditure partly through changing NAD + content, which is essential for mitochondrial. We determine whether NNMT plays important role in energy metabolism during the beige adipogenesis in vivo and in vitro. Male C57BL/6 mice at 8weeks old were exposed to 4 ℃ for 1, 2, 3, 4, and 5days, respectively. Interscapular brown adipose tissue (iBAT), inguinal subcutaneous WAT (sWAT), and epididymal WAT (eWAT) were harvested for gene and protein expression analysis and the correlation analysis. In addition, cultured primary mice brown adipocyte (BA) and white adipocyte (WA) treated with or without β3-adrenoceptor agonist (CL316, 243) were also harvested for these analyses. A combination of NNMT and its related genetic (Nmnat1, Nampt, Cyp2e1, Nrk1, Cd38) and proteic analyses and also the NAD + levels demonstrated the dynamical and depot-specific remodeling of NAD metabolism in different adipose tissues in response to cold exposure. While upon CL316, 243 treatment, gene expression of Nnmt, Nampt, Cyp2e1, and Nrk1 was all significantly decreased in WA but not in BA. The increased NAD + amount in BA and WA during the beige adipogenesis was observed. Besides, it is demonstrated that the expression of NNMT both in sWAT and WA showed significant negative correlation with browning markers UCP-1 and PGC-1α at protein levels. Above all, NNMT was induced in WAT during the 'cold remodeling' phase and correlated negatively with the process of browning in sWAT and WA, indicating the specific role of NNMT in the regulation of energy homeostasis during the process of beige adipogenesis.

Disruptive NADSYN1 Variants Implicated in Congenital Vertebral Malformations.

Genetic perturbations in nicotinamide adenine dinucleotide de novo (NAD) synthesis pathway predispose individuals to congenital birth defects. The NADSYN1 encodes the final enzyme in the de novo NAD synthesis pathway and, therefore, plays an important role in NAD metabolism and organ embryogenesis. Biallelic mutations in the NADSYN1 gene have been reported to be causative of congenital organ defects known as VCRL syndrome (Vertebral-Cardiac-Renal-Limb syndrome). Here, we analyzed the genetic variants in NADSYN1 in an exome-sequenced cohort consisting of patients with congenital vertebral malformations (CVMs). A total number of eight variants in NADSYN1, including two truncating variants and six missense variants, were identified in nine unrelated patients. All enrolled patients presented multiple organ defects, with the involvement of either the heart, kidney, limbs, or liver, as well as intraspinal deformities. An in vitro assay using COS-7 cells demonstrated either significantly reduced protein levels or disrupted enzymatic activity of the identified variants. Our findings demonstrated that functional variants in NADSYN1 were involved in the complex genetic etiology of CVMs and provided further evidence for the causative NADSYN1 variants in congenital NAD Deficiency Disorder.

Dietary Cocoa Flavanols Enhance Mitochondrial Function in Skeletal Muscle and Modify Whole-Body Metabolism in Healthy Mice.

Mitochondrial dysfunction is widely reported in various diseases and contributes to their pathogenesis. We assessed the effect of cocoa flavanols supplementation on mitochondrial function and whole metabolism, and we explored whether the mitochondrial deacetylase sirtuin-3 (Sirt3) is involved or not. We explored the effects of 15 days of CF supplementation in wild type and Sirt3-/- mice. Whole-body metabolism was assessed by indirect calorimetry, and an oral glucose tolerance test was performed to assess glucose metabolism. Mitochondrial respiratory function was assessed in permeabilised fibres and the pyridine nucleotides content (NAD+ and NADH) were quantified. In the wild type, CF supplementation significantly modified whole-body metabolism by promoting carbohydrate use and improved glucose tolerance. CF supplementation induced a significant increase of mitochondrial mass, while significant qualitative adaptation occurred to maintain H2O2 production and cellular oxidative stress. CF supplementation induced a significant increase in NAD+ and NADH content. All the effects mentioned above were blunted in Sirt3-/- mice. Collectively, CF supplementation boosted the NAD metabolism that stimulates sirtuins metabolism and improved mitochondrial function, which likely contributed to the observed whole-body metabolism adaptation, with a greater ability to use carbohydrates, at least partially through Sirt3.