A Molecular Perspective and Role of NAD+ in Ovarian Aging.

The decline in female fecundity is linked to advancing chronological age. The ovarian reserve diminishes in quantity and quality as women age, impacting reproductive efficiency and the aging process in the rest of the body. NAD+ is an essential coenzyme in cellular energy production, metabolism, cell signaling, and survival. It is involved in aging and is linked to various age-related conditions. Hallmarks associated with aging, diseases, and metabolic dysfunctions can significantly affect fertility by disturbing the delicate relationship between energy metabolism and female reproduction. Enzymes such as sirtuins, PARPs, and CD38 play essential roles in NAD+ biology, which actively consume NAD+ in their enzymatic activities. In recent years, NAD+ has gained much attention for its role in aging and age-related diseases like cancer, Alzheimer's, cardiovascular diseases, and neurodegenerative disorders, highlighting its involvement in various pathophysiological processes. However, its impact on female reproduction is not well understood. This review aims to bridge this knowledge gap by comprehensively exploring the complex interplay between NAD+ biology and female reproductive aging and providing valuable information that could help develop plans to improve women's reproductive health and prevent fertility issues.

Excess glucose alone depress young mesenchymal stromal/stem cell osteogenesis and mitochondria activity within hours/days via NAD+/SIRT1 axis.

BACKGROUND: The impact of global overconsumption of simple sugars on bone health, which peaks in adolescence/early adulthood and correlates with osteoporosis (OP) and fracture risk decades, is unclear. Mesenchymal stromal/stem cells (MSCs) are the progenitors of osteoblasts/bone-forming cells, and known to decrease their osteogenic differentiation capacity with age. Alarmingly, while there is correlative evidence that adolescents consuming greatest amounts of simple sugars have the lowest bone mass, there is no mechanistic understanding on the causality of this correlation. METHODS: Bioinformatics analyses for energetics pathways involved during MSC differentiation using human cell information was performed. In vitro dissection of normal versus high glucose (HG) conditions on osteo-/adipo-lineage commitment and mitochondrial function was assessed using multi-sources of non-senescent human and murine MSCs; for in vivo validation, young mice was fed normal or HG-added water with subsequent analyses of bone marrow CD45- MSCs. RESULTS: Bioinformatics analyses revealed mitochondrial and glucose-related metabolic pathways as integral to MSC osteo-/adipo-lineage commitment. Functionally, in vitro HG alone without differentiation induction decreased both MSC mitochondrial activity and osteogenesis while enhancing adipogenesis by 8 h' time due to depletion of nicotinamide adenine dinucleotide (NAD+), a vital mitochondrial co-enzyme and co-factor to Sirtuin (SIRT) 1, a longevity gene also involved in osteogenesis. In vivo, HG intake in young mice depleted MSC NAD+, with oral NAD+ precursor supplementation rapidly reversing both mitochondrial decline and osteo-/adipo-commitment in a SIRT1-dependent fashion within 1 ~ 5 days. CONCLUSIONS: We found a surprisingly rapid impact of excessive glucose, a single dietary factor, on MSC SIRT1 function and osteogenesis in youthful settings, and the crucial role of NAD+-a single molecule-on both MSC mitochondrial function and lineage commitment. These findings have strong implications on future global OP and disability risks in light of current worldwide overconsumption of simple sugars.

Cysteine oxidation as a regulatory mechanism of Arabidopsis plastidial NAD-dependent malate dehydrogenase.

Malate dehydrogenases (MDHs) catalyze a reversible NAD(P)-dependent-oxidoreductase reaction that plays an important role in central metabolism and redox homeostasis of plant cells. Recent studies suggest a moonlighting function of plastidial NAD-dependent MDH (plNAD-MDH; EC 1.1.1.37) in plastid biogenesis, independent of its enzyme activity. In this study, redox effects on activity and conformation of recombinant plNAD-MDH from Arabidopsis thaliana were investigated. We show that reduced plNAD-MDH is active while it is inhibited upon oxidation. Interestingly, the presence of its cofactors NAD+ and NADH could prevent oxidative inhibition of plNAD-MDH. In addition, a conformational change upon oxidation could be observed via non-reducing SDS-PAGE. Both effects, its inhibition and conformational change, were reversible by re-reduction. Further investigation of single cysteine substitutions and mass spectrometry revealed that oxidation of plNAD-MDH leads to oxidation of all four cysteine residues. However, cysteine oxidation of C129 leads to inhibition of plNAD-MDH activity and oxidation of C147 induces its conformational change. In contrast, oxidation of C190 and C333 does not affect plNAD-MDH activity or structure. Our results demonstrate that plNAD-MDH activity can be reversibly inhibited, but not inactivated, by cysteine oxidation and might be co-regulated by the availability of its cofactors in vivo.

NAD+ precursors promote the restoration of spermatogenesis in busulfan-treated mice through inhibiting Sirt2-regulated ferroptosis.

Rationale: In recent years, nicotinamide adenine dinucleotide (NAD+) precursors (Npre) have been widely employed to ameliorate female reproductive problems in both humans and animal models. However, whether and how Npre plays a role in the male reproductive disorder has not been fully clarified. Methods: In the present study, a busulfan-induced non-obstructive azoospermic mouse model was used, and Npre was administered for five weeks following the drug injection, with the objective of reinstating spermatogenesis and fertility. Initially, we assessed the NAD+ level, germ cell types, semen parameters and sperm fertilization capability. Subsequently, testis tissues were examined through RNA sequencing analysis, ELISA, H&E, immunofluorescence, quantitative real-time PCR, and Western blotting techniques. Results: The results indicated that Npre restored normal level of NAD+ in blood and significantly alleviated the deleterious effects of busulfan (BU) on spermatogenesis, thereby partially reestablishing fertilization capacity. Transcriptome analysis, along with recovery of testicular Fe2+, GSH, NADPH, and MDA levels, impaired by BU, and the fact that Fer-1, an inhibitor of ferroptosis, restored spermatogenesis and semen parameters close to CTRL values, supported such possibility. Interestingly, the reduction in SIRT2 protein level by the specific inhibitor AGK2 attenuated the beneficial effects of Npre on spermatogenesis and ferroptosis by affecting PGC-1α and ACLY protein levels, thus suggesting how these compounds might confer spermatogenesis protection. Conclusion: Collectively, these findings indicate that NAD+ protects spermatogenesis against ferroptosis, probably through SIRT2 dependent mechanisms. This underscores the considerable potential of Npre supplementation as a feasible strategy for preserving or restoring spermatogenesis in specific conditions of male infertility and as adjuvant therapy to preserve male fertility in cancer patients receiving sterilizing treatments.

The SARM1 TIR domain produces glycocyclic ADPR molecules as minor products.

Sterile alpha and TIR motif-containing 1 (SARM1) is a protein involved in programmed death of injured axons. Following axon injury or a drug-induced insult, the TIR domain of SARM1 degrades the essential molecule nicotinamide adenine dinucleotide (NAD+), leading to a form of axonal death called Wallerian degeneration. Degradation of NAD+ by SARM1 is essential for the Wallerian degeneration process, but accumulating evidence suggest that other activities of SARM1, beyond the mere degradation of NAD+, may be necessary for programmed axonal death. In this study we show that the TIR domains of both human and fruit fly SARM1 produce 1''-2' and 1''-3' glycocyclic ADP-ribose (gcADPR) molecules as minor products. As previously reported, we observed that SARM1 TIR domains mostly convert NAD+ to ADPR (for human SARM1) or cADPR (in the case of SARM1 from Drosophila melanogaster). However, we now show that human and Drosophila SARM1 additionally convert ~0.1-0.5% of NAD+ into gcADPR molecules. We find that SARM1 TIR domains produce gcADPR molecules both when purified in vitro and when expressed in bacterial cells. Given that gcADPR is a second messenger involved in programmed cell death in bacteria and likely in plants, we propose that gcADPR may play a role in SARM1-induced programmed axonal death in animals.

Targeting NAD+ Metabolism: Preclinical Insights into Potential Cancer Therapy Strategies.

NAD+ is one of the most important metabolites for cellular activities, and its biosynthesis mainly occurs through the salvage pathway using the nicotinamide phosphoribosyl transferase (NAMPT) enzyme. The main nicotinamide adenine dinucleotide (NAD) consumers, poly-ADP-ribose-polymerases and sirtuins enzymes, are heavily involved in DNA repair and chromatin remodeling. Since cancer cells shift their energy production pathway, NAD levels are significantly affected. NAD's roles in cell survival led to the use of NAD depletion in cancer therapies. NAMPT inhibition (alone or in combination with other cancer therapies, including endocrine therapy and chemotherapy) results in decreased cell viability and tumor burden for many cancer types. Many NAMPT inhibitors (NAMPTi) tested before were discontinued due to toxicity; however, a novel NAMPTi, KPT-9274, is a promising, low-toxicity option currently in clinical trials.

Water-soluble 4-(dimethylaminomethyl)heliomycin exerts greater antitumor effects than parental heliomycin by targeting the tNOX-SIRT1 axis and apoptosis in oral cancer cells.

The antibiotic heliomycin (resistomycin), which is generated from Streptomyces resistomycificus, has multiple activities, including anticancer effects. Heliomycin was first described in the 1960s, but its clinical applications have been hindered by extremely low solubility. A series of 4-aminomethyl derivatives of heliomycin were synthesized to increase water solubility; studies showed that they had anti-proliferative effects, but the drug targets remained unknown. In this study, we conducted cellular thermal shift assays (CETSA) and molecular docking simulations to identify and validate that heliomycin and its water-soluble derivative, 4-(dimethylaminomethyl)heliomycin (designated compound 4-dmH) engaged and targeted with sirtuin-1 (SIRT1) in p53-functional SAS and p53-mutated HSC-3 oral cancer cells. We further addressed the cellular outcome of SIRT1 inhibition by these compounds and found that, in addition to SIRT1, the water-soluble 4-dmH preferentially targeted a tumor-associated NADH oxidase (tNOX, ENOX2). The direct binding of 4-dmH to tNOX decreased the oxidation of NADH to NAD+ which diminished NAD+-dependent SIRT1 deacetylase activity, ultimately inducing apoptosis and significant cytotoxicity in both cell types, as opposed to the parental heliomycin-induced autophagy. We also observed that tNOX and SIRT1 were both upregulated in tumor tissues of oral cancer patients compared to adjacent normal tissues, suggesting their clinical relevance. Finally, the better therapeutic efficacy of 4-dmH was confirmed in tumor-bearing mice, which showed greater tNOX and SIRT1 downregulation and tumor volume reduction when treated with 4-dmH compared to heliomycin. Taken together, our in vitro and in vivo findings suggest that the multifaceted properties of water-soluble 4-dmH enable it to offer superior antitumor value compared to parental heliomycin, and indicated that it functions through targeting the tNOX-NAD+-SIRT1 axis to induce apoptosis in oral cancer cells.

NAD-Driven Sirtuin Activation by Cordyceps sinensis Extract: Exploring the Adaptogenic Potential to Promote Skin Longevity.

In recent years, there has been increasing interest in utilizing Traditional Chinese Medicine principles and natural bioactive compounds to combat age-related ailments and enhance longevity. A Cordyceps sinensis mycelium hydroethanolic extract (CsEx), which was standardized in cordycepin and adenosine using UHPLC-DAD, was investigated for its adaptogenic properties using in vitro assays and a double-blind, placebo-controlled clinical trial involving 40 subjects. The CsEx demonstrated activity at a concentration of 0.0006%, significantly increasing sirtuin expression (SirT1: +33%, SirT3: +10%, SirT6: +72%, vs. CTR, p < 0.05) and NAD+ synthesis in HaCat cells (+20% vs. CTR, p < 0.001). Moreover, the CsEx boosted ATP production by 68% in skin cells, correlating with higher skin energy values (+52.0% at D28, p < 0.01) in the clinical trial. Additionally, CsEx notably reduced cytosolic reactive oxygen species (ROS) by 30% in HaCaT cells (p < 0.05) and enhanced collagen production both in vitro (+69% vs. CTR, p < 0.01) and in vivo (+10% vs. D0, p < 0.01), confirmed by ultrasound examination. Furthermore, CsEx's stimulation of fibroblasts, coupled with its antioxidant and energizing properties, led to a significant reduction in wrinkles by 28.0% (D28, p < 0.001). This study underscores Cordyceps sinensis hydroethanolic extract's potential in regulating skin cell energy metabolism and positively influencing the mechanisms associated with skin longevity control.

HIF1α-dependent uncoupling of glycolysis suppresses tumor cell proliferation.

Hypoxia-inducible factor-1α (HIF1α) attenuates mitochondrial activity while promoting glycolysis. However, lower glycolysis is compromised in human clear cell renal cell carcinomas, in which HIF1α acts as a tumor suppressor by inhibiting cell-autonomous proliferation. Here, we find that, unexpectedly, HIF1α suppresses lower glycolysis after the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) step, leading to reduced lactate secretion in different tumor cell types when cells encounter a limited pyruvate supply such as that typically found in the tumor microenvironment in vivo. This is because HIF1α-dependent attenuation of mitochondrial oxygen consumption increases the NADH/NAD+ ratio that suppresses the activity of the NADH-sensitive GAPDH glycolytic enzyme. This is manifested when pyruvate supply is limited, since pyruvate acts as an electron acceptor that prevents the increment of the NADH/NAD+ ratio. Furthermore, this anti-glycolytic function provides a molecular basis to explain how HIF1α can suppress tumor cell proliferation by increasing the NADH/NAD+ ratio.

Zebrafish nampt-a mutants are viable despite perturbed primitive hematopoiesis.

BACKGROUND: Nicotinamide phosphoribosyltransferase (Nampt) is required for recycling NAD+ in numerous cellular contexts. Morpholino-based knockdown of zebrafish nampt-a has been shown to cause abnormal development and defective hematopoiesis concomitant with decreased NAD+ levels. However, surprisingly, nampt-a mutant zebrafish were recently found to be viable, suggesting a discrepancy between the phenotypes in knockdown and knockout conditions. Here, we address this discrepancy by directly comparing loss-of-function approaches that result in identical defective transcripts in morphants and mutants. RESULTS: Using CRISPR/Cas9-mediated mutagenesis, we generated nampt-a mutant lines that carry the same mis-spliced mRNA as nampt-a morphants. Despite reduced NAD+ levels and perturbed expression of specific blood markers, nampt-a mutants did not display obvious developmental defects and were found to be viable. In contrast, injection of nampt-a morpholinos into wild-type or mutant nampt-a embryos caused aberrant phenotypes. Moreover, nampt-a morpholinos caused additional reduction of blood-related markers in nampt-a mutants, suggesting that the defects observed in nampt-a morphants can be partially attributed to off-target effects of the morpholinos. CONCLUSIONS: Our findings show that zebrafish nampt-a mutants are viable despite reduced NAD+ levels and a perturbed hematopoietic gene expression program, indicating strong robustness of primitive hematopoiesis during early embryogenesis.

Channeling Nicotinamide Phosphoribosyltransferase (NAMPT) to Address Life and Death.

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step in NAD+ biosynthesis via salvage of NAM formed from catabolism of NAD+ by proteins with NADase activity (e.g., PARPs, SIRTs, CD38). Depletion of NAD+ in aging, neurodegeneration, and metabolic disorders is addressed by NAD+ supplementation. Conversely, NAMPT inhibitors have been developed for cancer therapy: many discovered by phenotypic screening for cancer cell death have low nanomolar potency in cellular models. No NAMPT inhibitor is yet FDA-approved. The ability of inhibitors to act as NAMPT substrates may be associated with efficacy and toxicity. Some 3-pyridyl inhibitors become 4-pyridyl activators or "NAD+ boosters". NAMPT positive allosteric modulators (N-PAMs) and boosters may increase enzyme activity by relieving substrate/product inhibition. Binding to a "rear channel" extending from the NAMPT active site is key for inhibitors, boosters, and N-PAMs. A deeper understanding may fulfill the potential of NAMPT ligands to regulate cellular life and death.

The NADase CD38 is a central regulator in gouty inflammation and a novel druggable therapeutic target.

OBJECTIVES: Cellular NAD+ declines in inflammatory states associated with increased activity of the leukocyte-expressed NADase CD38. In this study, we tested the potential role of therapeutically targeting CD38 and NAD+ in gout. METHODS: We studied cultured mouse wild type and CD38 knockout (KO) murine bone marrow derived macrophages (BMDMs) stimulated by monosodium urate (MSU) crystals and used the air pouch gouty inflammation model. RESULTS: MSU crystals induced CD38 in BMDMs in vitro, associated with NAD+ depletion, and IL-1ß and CXCL1 release, effects reversed by pharmacologic CD38 inhibitors (apigenin, 78c). Mouse air pouch inflammatory responses to MSU crystals were blunted by CD38 KO and apigenin. Pharmacologic CD38 inhibition suppressed MSU crystal-induced NLRP3 inflammasome activation and increased anti-inflammatory SIRT3-SOD2 activity in macrophages. BMDM RNA-seq analysis of differentially expressed genes (DEGs) revealed CD38 to control multiple MSU crystal-modulated inflammation pathways. Top DEGs included the circadian rhythm modulator GRP176, and the metalloreductase STEAP4 that mediates iron homeostasis, and promotes oxidative stress and NF-κB activation when it is overexpressed. CONCLUSIONS: CD38 and NAD+ depletion are druggable targets controlling the MSU crystal- induced inflammation program. Targeting CD38 and NAD+ are potentially novel selective molecular approaches to limit gouty arthritis.

Enhanced glycolysis in the myometrium with ectopic endometrium of patients with adenomyosis: a preliminary study.

OBJECTIVES: The objective of this study was to investigate the glycolytic activity of adenomyosis, which is characterized by malignant biological behaviors including abnormal cell proliferation, migration, invasion, cell regulation, and epithelial-mesenchymal transition. METHODS: From January 2021 to August 2022, a total of 15 patients who underwent total hysterectomy for adenomyosis and 14 patients who had non-endometrial diseases, specifically with cervical squamous intraepithelial neoplasia and uterine myoma, were included in this study. Myometrium with ectopic endometrium from patients with adenomyosis while normal myometrium from patients in the control group were collected. All samples were confirmed by a histopathological examination. The samples were analyzed by liquid chromatography-mass spectrometry (LC-MS), real-time quantitative PCR, NAD+/NADH assay kit as well as the glucose and lactate assay kits. RESULTS: Endometrial stroma and glands could be observed within the myometrium of patients in the adenomyosis group. We found that the mRNA expressions of HK1, PFKFB3, glyceraldehyde-3-phospate dehydrogenase (GAPDH), PKM2, and PDHA as well as the protein expressions of PFKFB3 were elevated in ectopic endometrial tissues of the adenomyosis group as compared to normal myometrium of the control group. The level of fructose 1,6-diphosphate was increased while NAD + and NAD+/NADH ratio were decreased compared with the control group. Besides, increased glucose consumption and lactate production were observed in myometrium with ectopic endometrium. CONCLUSIONS: We concluded that altered glycolytic phenotype of the myometrium with ectopic endometrium in women with adenomyosis may contribute the development of adenomyosis.

A Transfer Hydrogenation Approach to Activity-Based Sensing of Formate in Living Cells.

Formate is a major reactive carbon species in one-carbon metabolism, where it serves as an endogenous precursor for amino acid and nucleic acid biosynthesis and a cellular source of NAD(P)H. On the other hand, aberrant elevations in cellular formate are connected to progression of serious diseases, including cancer and Alzheimer's disease. Traditional methods for formate detection in biological environments often rely on sample destruction or extensive processing, resulting in a loss of spatiotemporal information. To help address these limitations, here we present the design, synthesis, and biological evaluation of a first-generation activity-based sensing system for live-cell formate imaging that relies on iridium-mediated transfer hydrogenation chemistry. Formate facilitates an aldehyde-to-alcohol conversion on various fluorophore scaffolds to enable fluorescence detection of this one-carbon unit, including through a two-color ratiometric response with internal calibration. The resulting two-component probe system can detect changes in formate levels in living cells with a high selectivity over potentially competing biological analytes. Moreover, this activity-based sensing system can visualize changes in endogenous formate fluxes through alterations of one-carbon pathways in cell-based models of human colon cancer, presaging the potential utility of this chemical approach to probe the continuum between one-carbon metabolism and signaling in cancer and other diseases.

The use of NADH anisotropy to investigate mitochondrial cristae alignment.

Life may be expressed as the flow of electrons, protons, and other ions, resulting in large potential difference. It is also highly photo-sensitive, as a large proportion of the redox capable molecules it relies on are chromophoric. It is thus suggestive that a key organelle in eukaryotes, the mitochondrion, constantly adapt their morphology as part of the homeostatic process. Studying unstained in vivo nano-scale structure in live cells is technically very challenging. One option is to study a central electron carrier in metabolism, reduced nicotinamide adenine dinucleotide (NADH), which is fluorescent and mostly located within mitochondria. Using one and two-photon absorption (340-360 nm and 730 nm, respectively), fluorescence lifetime imaging and anisotropy spectroscopy of NADH in solution and in live cells, we show that mitochondria do indeed appear to be aligned and exhibit high anisotropy (asymmetric directionality). Aqueous solution of NADH showed an anisotropy of ~ 0.20 compared to fluorescein or coumarin of < 0.1 and 0.04 in water respectively and as expected for small organic molecules. The anisotropy of NADH also increased further to 0.30 in the presence of proteins and 0.42 in glycerol (restricted environment) following two-photon excitation, suggesting more ordered structures. Two-photon NADH fluorescence imaging of Michigan Cancer Foundation-7 (MCF7) also showed strong anisotropy of 0.25 to 0.45. NADH has a quantum yield of fluorescence of 2% compared to more than 40% for photoionisation (electron generation), when exposed to light at 360 nm and below. The consequence of such highly ordered and directional NADH patterns with respect to electron ejection upon ultra-violet (UV) excitation could be very informative-especially in relation to ascertaining the extent of quantum effects in biology, including electron and photonic cascade, communication and modulation of effects such as spin and tunnelling.

Substrate-induced condensation activates plant TIR domain proteins.

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain mediate recognition of strain-specific pathogen effectors, typically via their C-terminal ligand-sensing domains1. Effector binding enables TIR-encoded enzymatic activities that are required for TIR-NLR (TNL)-mediated immunity2,3. Many truncated TNL proteins lack effector-sensing domains but retain similar enzymatic and immune activities4,5. The mechanism underlying the activation of these TIR domain proteins remain unclear. Here we show that binding of the TIR substrates NAD+ and ATP induces phase separation of TIR domain proteins in vitro. A similar condensation occurs with a TIR domain protein expressed via its native promoter in response to pathogen inoculation in planta. The formation of TIR condensates is mediated by conserved self-association interfaces and a predicted intrinsically disordered loop region of TIRs. Mutations that disrupt TIR condensates impair the cell death activity of TIR domain proteins. Our data reveal phase separation as a mechanism for the activation of TIR domain proteins and provide insight into substrate-induced autonomous activation of TIR signalling to confer plant immunity.

A NAD(P)H oxidase mimic for catalytic tumor therapy via a deacetylase SIRT7-mediated AKT/GSK3ß pathway.

Nicotinamide adenine dinucleotide (NADH) and its phosphorylated form, NADPH, are essential cofactors that play critical roles in cell functions, influencing antioxidation, reductive biosynthesis, and cellular pathways involved in tumor cell apoptosis and tumorigenesis. However, the use of nanomaterials to consume NAD(P)H and thus bring an impact on signaling pathways in cancer treatment remains understudied. In this study, we employed a salt template method to synthesize a carbon-coated-cobalt composite (C@Co) nanozyme, which exhibited excellent NAD(P)H oxidase (NOX)-like activity and mimicked the reaction mechanism of natural NOX. The C@Co nanozyme efficiently consumed NAD(P)H within cancer cells, leading to increased production of reactive oxygen species (ROS) and a reduction in mitochondrial membrane potential. Meanwhile, the generation of the biologically active cofactor NAD(P)+ promoted the expression of the deacetylase SIRT7, which in turn inhibited the serine/threonine kinase AKT signaling pathway, ultimately promoting apoptosis. This work sheds light on the influence of nanozymes with NOX-like activity on cellular signaling pathways in tumor therapy and demonstrates their promising antitumor effects in a tumor xenograft mouse model. These findings contribute to a better understanding of NAD(P)H manipulation in cancer treatment and suggest the potential of nanozymes as a therapeutic strategy for cancer therapy.

Lactate enhances NMNAT1 lactylation to sustain nuclear NAD+ salvage pathway and promote survival of pancreatic adenocarcinoma cells under glucose-deprived conditions.

The aim of this study was to investigate the underlying molecular mechanism behind the promotion of cell survival under conditions of glucose deprivation by l-lactate. To accomplish this, we performed tissue microarray and immunohistochemistry staining to analyze the correlation between the abundance of pan-Lysine lactylation and prognosis. In vivo evaluations of tumor growth were conducted using the KPC and nude mice xenograft tumor model. For mechanistic studies, multi-omics analysis, RNA interference, and site-directed mutagenesis techniques were utilized. Our findings robustly confirmed that l-lactate promotes cell survival under glucose deprivation conditions, primarily by relying on GLS1-mediated glutaminolysis to support mitochondrial respiration. Mechanistically, we discovered that l-lactate enhances the NMNAT1-mediated NAD+ salvage pathway while concurrently inactivating p-38 MAPK signaling and suppressing DDIT3 transcription. Notably, Pan-Kla abundance was significantly upregulated in patients with Pancreatic adenocarcinoma (PAAD) and associated with poor prognosis. We identified the 128th Lysine residue of NMNAT1 as a critical site for lactylation and revealed EP300 as a key lactyltransferase responsible for catalyzing lactylation. Importantly, we elucidated that lactylation of NMNAT1 enhances its nuclear localization and maintains enzymatic activity, thereby supporting the nuclear NAD+ salvage pathway and facilitating cancer growth. Finally, we demonstrated that the NMNAT1-dependent NAD+ salvage pathway promotes cell survival under glucose deprivation conditions and is reliant on the activity of Sirt1. Collectively, our study has unraveled a novel molecular mechanism by which l-lactate promotes cell survival under glucose deprivation conditions, presenting a promising strategy for targeting lactate and NAD+ metabolism in the treatment of PAAD.

Nicotinamide phosphoribosyltransferase modulates PD-L1 in bladder cancer and enhances immunotherapeutic sensitivity.

Bladder cancer (BLCA) is one of the most prevalent malignancies worldwide with a high mortality rate and poor response to immunotherapy in patients expressing lower programmed death ligand 1 (PD-L1) levels. Nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme responsible for the biosynthesis of nicotinamide adenine dinucleotide (NAD+) from nicotinamide was reported to be overexpressed in various cancers; however, the role of NAMPT in BLCA is obscure. Immunohistochemistry of tissue microarrays, a real-time polymerase chain reaction, Western blotting, proliferation assay, NAD+ quantification, transwell-migration assay, and colony-formation assay were performed to measure NAMPT and PD-L1 expression levels in patients and the effect of NAMPT inhibition on T24 cells. Our study revealed that NAMPT expression was upregulated in BLCA patients with different grades and associated with poor T-cell infiltration. Notably, FK866-mediated NAMPT inhibition decreased cell viability by depleting NAD+, and reducing the migration ability and colony-formation ability of T24 cells. Interestingly, NAMPT negatively regulated PD-L1 under an interferon (IFN)-γ-mediated microenvironment. However, exogenous NAMPT activator has no effect on PD-L1. NAD+ supplementation also only increased PD-L1 in the absence of IFN-γ. Conclusively, NAMPT is crucial for BLCA tumorigenic properties, and it regulates expression of the PD-L1 immune checkpoint protein. NAMPT could be considered a target for modulating sensitivity to immunotherapy.

Kynurenine 3-monooxygenase limits de novo NAD+ synthesis through dietary tryptophan in renal proximal tubule epithelial cell models.

Nicotinamide adenine dinucleotide (NAD+) is a pivotal coenzyme, essential for cellular reactions, metabolism, and mitochondrial function. Depletion of kidney NAD+ levels and reduced de novo NAD+ synthesis through the tryptophan-kynurenine pathway are linked to acute kidney injury (AKI), whereas augmenting NAD+ shows promise in reducing AKI. We investigated de novo NAD+ biosynthesis using in vitro, ex vivo, and in vivo models to understand its role in AKI. Two-dimensional (2-D) cultures of human primary renal proximal tubule epithelial cells (RPTECs) and HK-2 cells showed limited de novo NAD+ synthesis, likely due to low pathway enzyme gene expression. Using three-dimensional (3-D) spheroid culture model improved the expression of tubular-specific markers and enzymes involved in de novo NAD+ synthesis. However, de novo NAD+ synthesis remained elusive in the 3-D spheroid culture, regardless of injury conditions. Further investigation revealed that 3-D cultured cells could not metabolize tryptophan (Trp) beyond kynurenine (KYN). Intriguingly, supplementation of 3-hydroxyanthranilic acid into RPTEC spheroids was readily incorporated into NAD+. In a human precision-cut kidney slice (PCKS) ex vivo model, de novo NAD+ synthesis was limited due to substantially downregulated kynurenine 3-monooxygenase (KMO), which is responsible for KYN to 3-hydroxykynurenine conversion. KMO overexpression in RPTEC 3-D spheroids successfully reinstated de novo NAD+ synthesis from Trp. In addition, in vivo study demonstrated that de novo NAD+ synthesis is intact in the kidney of the healthy adult mice. Our findings highlight disrupted tryptophan-kynurenine NAD+ synthesis in in vitro cellular models and an ex vivo kidney model, primarily attributed to KMO downregulation.NEW & NOTEWORTHY Nicotinamide adenine dinucleotide (NAD+) is essential in regulating mitochondrial function. Reduced NAD+ synthesis through the de novo pathway is associated with acute kidney injury (AKI). Our study reveals a disruption in de novo NAD+ synthesis in proximal tubular models, but not in vivo, attributed to downregulation of enzyme kynurenine 3-monooxygenase (KMO). These findings highlight a crucial role of KMO in governing de novo NAD+ biosynthesis within the kidney, shedding light on potential AKI interventions.