NAD+ supplementation improves mitochondrial functions and normalizes glaucomatous trabecular meshwork features.

Glaucoma is characterized by pathological elevation of intraocular pressure (IOP) due to dysfunctional trabecular meshwork (TM), which is the primary cause of irreversible vision loss. There are currently no effective treatment strategies for glaucoma. Mitochondrial function plays a crucial role in regulating IOP within the TM. In this study, primary TM cells treated with dexamethasone were used to simulate glaucomatous changes, showing abnormal cellular cytoskeleton, increased expression of extracellular matrix, and disrupted mitochondrial fusion and fission dynamics. Furthermore, glaucomatous TM cell line GTM3 exhibited impaired mitochondrial membrane potential and phagocytic function, accompanied by decreased oxidative respiratory levels as compared to normal TM cells iHTM. Mechanistically, lower NAD + levels in GTM3, possibly associated with increased expression of key enzymes CD38 and PARP1 related to NAD + consumption, were observed. Supplementation of NAD + restored mitochondrial function and cellular viability in GTM3 cells. Therefore, we propose that the aberrant mitochondrial function in glaucomatous TM cells may be attributed to increased NAD + consumption dependent on CD38 and PARP1, and NAD + supplementation could effectively ameliorate mitochondrial function and improve TM function, providing a novel alternative approach for glaucoma treatment.

Supplementation with NAD+ and its precursors: A rescue of female reproductive diseases.

Nicotinamide adenine dinucleotide (NAD+) is an essential coenzyme involved in many pathophysiological processes. Supplementation with NAD+ and its precursors has been demonstrated as an emerging therapeutic strategy for the diseases. NAD+ also plays an important role in the reproductive system. Here, we summarize the function of NAD+ in various reproductive diseases and review the application of NAD+ and its precursors in the preservation of reproductive capacity and the prevention of embryonic malformations. It is shown that NAD+ shows good promise as a therapeutic approach for saving reproductive capacity.

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 Effectiveness of Four Nicotinamide Adenine Dinucleotide (NAD+) Precursors in Alleviating the High-Glucose-Induced Damage to Hepatocytes in Megalobrama amblycephala: Evidence in NAD+ Homeostasis, Sirt1/3 Activation, Redox Defense, Inflammatory Response, Apoptosis, and Glucose Metabolism.

The administration of NAD+ precursors is a potential approach to protect against liver damage and metabolic dysfunction. However, the effectiveness of different NAD+ precursors in alleviating metabolic disorders is still poorly elucidated. The current study was performed to compare the effectiveness of four different NAD+ precursors, including nicotinic acid (NA), niacinamide (NAM), nicotinamide riboside (NR), and nicotinamide mononucleotide (NMN) in alleviating high-glucose-induced injury to hepatocytes in a fish model, Megalobrama amblycephala. An in vitro high-glucose model was successfully established to mimic hyperglycemia-induced damage to the liver, which was evidenced by the reduced cell viability, the increased transaminase activity, and the depletion of cellular NAD+ concentration. The NAD+ precursors all improved cell viability, with the maximal effect observed in NR, which also had the most potent NAD+ boosting capacity and a significant Sirt1/3 activation effect. Meanwhile, NR presented distinct and superior effects in terms of anti-oxidative stress, inflammation inhibition, and anti-apoptosis compared with NA, NAM, and NMN. Furthermore, NR could effectively benefit glucose metabolism by activating glucose transportation, glycolysis, glycogen synthesis and the pentose phosphate pathway, as well as inhibiting gluconeogenesis. Moreover, an oral gavage test confirmed that NR presented the most potent effect in increasing hepatic NAD+ content and the NAD+/NADH ratio among four NAD+ precursors. Together, the present study results demonstrated that NR is most effective in attenuating the high-glucose-induced injury to hepatocytes in fish compared to other NAD+ precursors.

The therapeutic perspective of NAD+ precursors in age-related diseases.

Nicotinamide adenine dinucleotide (NAD+) is the fundamental molecule that performs numerous biological reactions and is crucial for maintaining cellular homeostasis. Studies have found that NAD+ decreases with age in certain tissues, and age-related NAD+ depletion affects physiological functions and contributes to various aging-related diseases. Supplementation of NAD+ precursor significantly elevates NAD+ levels in murine tissues, effectively mitigates metabolic syndrome, enhances cardiovascular health, protects against neurodegeneration, and boosts muscular strength. Despite the versatile therapeutic functions of NAD+ in animal studies, the efficacy of NAD+ precursors in clinical studies have been limited compared with that in the pre-clinical study. Clinical studies have demonstrated that NAD+ precursor treatment efficiently increases NAD+ levels in various tissues, though their clinical proficiency is insufficient to ameliorate the diseases. However, the latest studies regarding NAD+ precursors and their metabolism highlight the significant role of gut microbiota. The studies found that orally administered NAD+ intermediates interact with the gut microbiome. These findings provide compelling evidence for future trials to further explore the involvement of gut microbiota in NAD+ metabolism. Also, the reduced form of NAD+ precursor shows their potential to raise NAD+, though preclinical studies have yet to discover their efficacy. This review sheds light on NAD+ therapeutic efficiency in preclinical and clinical studies and the effect of the gut microbiota on NAD+ metabolism.

Nicotinamide Adenine Dinucleotide in Aging Biology: Potential Applications and Many Unknowns.

Recent research has unveiled an expansive role of NAD+ in cellular energy generation, redox reactions, and as a substrate or cosubstrate in signaling pathways that regulate health span and aging. This review provides a critical appraisal of the clinical pharmacology and the preclinical and clinical evidence for therapeutic effects of NAD+ precursors for age-related conditions, with a particular focus on cardiometabolic disorders, and discusses gaps in current knowledge. NAD+ levels decrease throughout life; age-related decline in NAD+ bioavailability has been postulated to be a contributor to many age-related diseases. Raising NAD+ levels in model organisms by administration of NAD+ precursors improves glucose and lipid metabolism; attenuates diet-induced weight gain, diabetes, diabetic kidney disease, and hepatic steatosis; reduces endothelial dysfunction; protects heart from ischemic injury; improves left ventricular function in models of heart failure; attenuates cerebrovascular and neurodegenerative disorders; and increases health span. Early human studies show that NAD+ levels can be raised safely in blood and some tissues by oral NAD+ precursors and suggest benefit in preventing nonmelanotic skin cancer, modestly reducing blood pressure and improving lipid profile in older adults with obesity or overweight; preventing kidney injury in at-risk patients; and suppressing inflammation in Parkinson disease and SARS-CoV-2 infection. Clinical pharmacology, metabolism, and therapeutic mechanisms of NAD+ precursors remain incompletely understood. We suggest that these early findings provide the rationale for adequately powered randomized trials to evaluate the efficacy of NAD+ augmentation as a therapeutic strategy to prevent and treat metabolic disorders and age-related conditions.

NAD+ Precursors Nicotinamide Mononucleotide (NMN) and Nicotinamide Riboside (NR): Potential Dietary Contribution to Health.

PURPOSE OF REVIEW: NAD+ is a vital molecule that takes part as a redox cofactor in several metabolic reactions besides being used as a substrate in important cellular signaling in regulation pathways for energetic, genotoxic, and infectious stress. In stress conditions, NAD+ biosynthesis and levels decrease as well as the activity of consuming enzymes rises. Dietary precursors can promote NAD+ biosynthesis and increase intracellular levels, being a potential strategy for reversing physiological decline and preventing diseases. In this review, we will show the biochemistry and metabolism of NAD+ precursors NR (nicotinamide riboside) and NMN (nicotinamide mononucleotide), the latest findings on their beneficial physiological effects, their interplay with gut microbiota, and the future perspectives for research in nutrition and food science fields. RECENT FINDINGS: NMN and NR demonstrated protect against diabetes, Alzheimer disease, endothelial dysfunction, and inflammation. They also reverse gut dysbiosis and promote beneficial effects at intestinal and extraintestinal levels. NR and NMN have been found in vegetables, meat, and milk, and microorganisms in fermented beverages can also produce them. NMN and NR can be obtained through the diet either in their free form or as metabolites derivate from the digestion of NAD+. The prospection of NR and NMN to find potential food sources and their dietary contribution in increasing NAD+ levels are still an unexplored field of research. Moreover, it could enable the development of new functional foods and processing strategies to maintain and enhance their physiological benefits, besides the studies of new raw materials for extraction and biotechnological development.

The acute effect of different NAD+ precursors included in the combined metabolic activators.

NAD+ and glutathione precursors are currently used as metabolic modulators for improving the metabolic conditions associated with various human diseases, including non-alcoholic fatty liver disease, neurodegenerative diseases, mitochondrial myopathy, and age-induced diabetes. Here, we performed a one-day double blinded, placebo-controlled human clinical study to assess the safety and acute effects of six different Combined Metabolic Activators (CMAs) with 1 g of different NAD+ precursors based on global metabolomics analysis. Our integrative analysis showed that the NAD+ salvage pathway is the main source for boosting the NAD+ levels with the administration of CMAs without NAD+ precursors. We observed that incorporation of nicotinamide (Nam) in the CMAs can boost the NAD+ products, followed by niacin (NA), nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN), but not flush free niacin (FFN). In addition, the NA administration led to a flushing reaction, accompanied by decreased phospholipids and increased bilirubin and bilirubin derivatives, which could be potentially risky. In conclusion, this study provided a plasma metabolomic landscape of different CMA formulations, and proposed that CMAs with Nam, NMN as well as NR can be administered for boosting NAD+ levels to improve altered metabolic conditions.

The Role of NAD+, SIRTs Interactions in Stimulating and Counteracting Carcinogenesis.

The World Health Organization has identified oncological diseases as one of the most serious health concerns of the current century. Current research on oncogenesis is focused on the molecular mechanisms of energy-biochemical reprogramming in cancer cell metabolism, including processes contributing to the Warburg effect and the pro-oncogenic and anti-oncogenic roles of sirtuins (SIRTs) and poly-(ADP-ribose) polymerases (PARPs). However, a clear understanding of the interaction between NAD+, SIRTs in cancer development, as well as their effects on carcinogenesis, has not been established, and literature data vary greatly. This work aims to provide a summary and structure of the available information on NAD+, SIRTs interactions in both stimulating and countering carcinogenesis, and to discuss potential approaches for pharmacological modulation of these interactions to achieve an anticancer effect.

The Central Role of the NAD+ Molecule in the Development of Aging and the Prevention of Chronic Age-Related Diseases: Strategies for NAD+ Modulation.

The molecule NAD+ is a coenzyme for enzymes catalyzing cellular redox reactions in several metabolic pathways, encompassing glycolysis, TCA cycle, and oxidative phosphorylation, and is a substrate for NAD+-dependent enzymes. In addition to a hydride and electron transfer in redox reactions, NAD+ is a substrate for sirtuins and poly(adenosine diphosphate-ribose) polymerases and even moderate decreases in its cellular concentrations modify signaling of NAD+-consuming enzymes. Age-related reduction in cellular NAD+ concentrations results in metabolic and aging-associated disorders, while the consequences of increased NAD+ production or decreased degradation seem beneficial. This article reviews the NAD+ molecule in the development of aging and the prevention of chronic age-related diseases and discusses the strategies of NAD+ modulation for healthy aging and longevity.

Combination of natural polyphenols with a precursor of NAD+ and a TLR2/6 ligand lipopeptide protects mice against lethal γ radiation.

INTRODUCTION: Effective agents that could confer long-term protection against ionizing radiation in vivo would have applications in medicine, biotechnology, and in air and space travel. However, at present, drugs that can effectively protect against lethal ionizing radiations are still an unmet need. OBJECTIVE: To investigate if combinations of natural polyphenols, known for their antioxidant potential, could protect against ionizing radiations. METHODS: Plant-derived polyphenols were screened for their potential ability to confer radioprotection to mice given a lethal whole-body γ radiation (137Cs) dose expected to kill 50% of the animals in 30 days. Telomere and centromere staining, Q-FISH and comet assays were used to investigate chromosomal aberration, micronuclei formation and DNA breaks. Molecular oxidations were investigated by enzyme immunoassays and UPLC-MS/MS. RT-PCR, western blotting and siRNA-induced gene silencing were used to study signaling mechanisms and molecular interactions. RESULTS: The combination of pterostilbene (PT) and silibinin (SIL) was the most effective against γ-irradiation, resulting in 100% of the mice surviving at 30 days and 20% survival at one year. Treatment post γ-irradiation with two potential radiomitigators nicotinamide riboside (NR, a vitamin B3 derivative), and/or fibroblast-stimulating lipoprotein 1 (FSL1, a toll-like receptor 2/6 agonist), did not extend survival. However, the combination of PT, SIL, NR and FSL1 achieved a 90% survival one year post γ-irradiation. The mechanism involves induction of the Nrf2-dependent cellular antioxidant defense, reduction of NF-kB signaling, upregulation of the PGC-1α/sirtuins 1 and 3 axis, PARP1-dependent DNA repair, and stimulation of hematopoietic cell recovery. The pathway linking Nrf2, sirtuin 3 and SOD2 is key to radioprotection. Importantly, this combination did not interfere with X-ray mediated killing of different tumor cells in vivo. CONCLUSION: The combination of the radioprotectors PT and SIL with the radiomitigators NR and FSL1 confer effective, long-term protection against γ radiation in vivo. This strategy is potentially capable of protecting mammals against ionizing radiations.

Current Uncertainties and Future Challenges Regarding NAD+ Boosting Strategies.

Precursors of nicotinamide adenine dinucleotide (NAD+), modulators of enzymes of the NAD+ biosynthesis pathways and inhibitors of NAD+ consuming enzymes, are the main boosters of NAD+. Increasing public awareness and interest in anti-ageing strategies and health-promoting lifestyles have grown the interest in the use of NAD+ boosters as dietary supplements, both in scientific circles and among the general population. Here, we discuss the current trends in NAD+ precursor usage as well as the uncertainties in dosage, timing, safety, and side effects. There are many unknowns regarding pharmacokinetics and pharmacodynamics, particularly bioavailability, metabolism, and tissue specificity of NAD+ boosters. Given the lack of long-term safety studies, there is a need for more clinical trials to determine the proper dose of NAD+ boosters and treatment duration for aging prevention and as disease therapy. Further research will also need to address the long-term consequences of increased NAD+ and the best approaches and combinations to increase NAD+ levels. The answers to the above questions will contribute to the more efficient and safer use of NAD+ boosters.

Insights into the NAD+ biosynthesis pathways involved during meiotic maturation and spindle formation in porcine oocytes.

Treatments that elevate NAD+ levels have been found to improve oocyte quality in mice, cattle, and pigs, suggesting that NAD+ is vital during oocyte maturation. This study aimed to examine the influence of different NAD+ biosynthetic pathways on oocyte quality by inhibiting key enzymes. Porcine oocytes from small antral follicles were matured for 44 h in a defined maturation system supplemented with 2-hydroxynicotinic acid [2-HNA, nicotinic acid phosphoribosyltransferase (NAPRT) inhibitor], FK866 [nicotinamide phosphoribosyltransferase (NAMPT) inhibitor], or gallotannin [nicotinamide mononucleotide adenylyltransferase (NMNAT) inhibitor] and their respective NAD+ pathway modulators (nicotinic acid, nicotinamide, and nicotinamide mononucleotide, respectively). Cumulus expansion was assessed after 22 h of maturation. At 44 h, maturation rates were determined and mature oocytes were fixed and stained to assess spindle formation. Each enzyme inhibitor reduced oocyte maturation rate and adversely affected spindle formation, indicating that NAD+ is required for meiotic spindle assembly. Furthermore, NAMPT and NMNAT inhibition reduced cumulus expansion, whereas NAPRT inhibition affected chromosomal segregation. Treating oocytes with gallotannin and nicotinamide mononucleotide together showed improvements in spindle width, while treating oocytes with 2-HNA and nicotinic acid combined showed an improvement in both spindle length and width. These results indicate that the salvage pathway plays a vital role in promoting oocyte meiotic progression, while the Preiss-Handler pathway is essential for spindle assembly.

NAD supplementation improves mitochondrial performance of cardiolipin mutants.

Cardiolipin (CL) deficiency causes mitochondrial dysfunction and aberrant metabolism that are associated in humans with the severe disease Barth syndrome (BTHS). Several metabolic abnormalities are observed in BTHS patients and model systems, including decreased oxidative phosphorylation, reduced tricarboxylic acid (TCA) cycle flux, and accumulated lactate and D-ß-hydroxybutyrate, which strongly suggests that nicotinamide adenine dinucleotide (NAD) redox metabolism may be altered in CL-deficient cells. In this study, we identified abnormal NAD+ metabolism in multiple BTHS model systems and demonstrate that supplementation of NAD+ precursors such as nicotinamide mononucleotide (NMN) improves mitochondrial function. Improved mitochondrial function in the Drosophila model was associated with restored exercise endurance, which suggests a potential therapeutic benefit of NAD+ precursor supplementation in the management of BTHS patients.

NAD+ Metabolism and Interventions in Premature Renal Aging and Chronic Kidney Disease.

Premature aging causes morphological and functional changes in the kidney, leading to chronic kidney disease (CKD). CKD is a global public health issue with far-reaching consequences, including cardio-vascular complications, increased frailty, shortened lifespan and a heightened risk of kidney failure. Dialysis or transplantation are lifesaving therapies, but they can also be debilitating. Currently, no cure is available for CKD, despite ongoing efforts to identify clinical biomarkers of premature renal aging and molecular pathways of disease progression. Kidney proximal tubular epithelial cells (PTECs) have high energy demand, and disruption of their energy homeostasis has been linked to the progression of kidney disease. Consequently, metabolic reprogramming of PTECs is gaining interest as a therapeutic tool. Preclinical and clinical evidence is emerging that NAD+ homeostasis, crucial for PTECs' oxidative metabolism, is impaired in CKD, and administration of dietary NAD+ precursors could have a prophylactic role against age-related kidney disease. This review describes the biology of NAD+ in the kidney, including its precursors and cellular roles, and discusses the importance of NAD+ homeostasis for renal health. Furthermore, we provide a comprehensive summary of preclinical and clinical studies aimed at increasing NAD+ levels in premature renal aging and CKD.

NAD+ in Alzheimer's Disease: Molecular Mechanisms and Systematic Therapeutic Evidence Obtained in vivo.

Mitochondria in neurons generate adenosine triphosphate (ATP) to provide the necessary energy required for constant activity. Nicotinamide adenine dinucleotide (NAD+) is a vital intermediate metabolite involved in cellular bioenergetics, ATP production, mitochondrial homeostasis, and adaptive stress responses. Exploration of the biological functions of NAD+ has been gaining momentum, providing many crucial insights into the pathophysiology of age-associated functional decline and diseases, such as Alzheimer's disease (AD). Here, we systematically review the key roles of NAD+ precursors and related metabolites in AD models and show how NAD+ affects the pathological hallmarks of AD and the potential mechanisms of action. Advances in understanding the molecular roles of NAD+-based neuronal resilience will result in novel approaches for the treatment of AD and set the stage for determining whether the results of exciting preclinical trials can be translated into the clinic to improve AD patients' phenotypes.

NAD+-Precursor Supplementation With L-Tryptophan, Nicotinic Acid, and Nicotinamide Does Not Affect Mitochondrial Function or Skeletal Muscle Function in Physically Compromised Older Adults.

BACKGROUND: Boosting NAD+ via supplementation with niacin equivalents has been proposed as a potential modality capable of promoting healthy aging and negating age-dependent declines of skeletal muscle mass and function. OBJECTIVES: We investigated the efficacy of NAD+-precursor supplementation (tryptophan, nicotinic acid, and nicotinamide) on skeletal muscle mitochondrial function in physically compromised older adults. METHODS: A randomized, double-blind, controlled trial was conducted in 14 (female/male: 4/10) community-dwelling, older adults with impaired physical function [age, 72.9 ± 4.0 years; BMI, 25.2 ± 2.3 kg/m2]. Participants were supplemented with 207.5 mg niacin equivalents/day [intervention (INT)] and a control product (CON) that did not contain niacin equivalents, each for 32 days. The primary outcomes tested were mitochondrial oxidative capacity and exercise efficiency, analyzed by means of paired Student's t-tests. Secondary outcomes, such as NAD+ concentrations, were analyzed accordingly. RESULTS: Following supplementation, skeletal muscle NAD+ concentrations [7.5 ± 1.9 compared with 7.9 ± 1.6 AU, respectively] in INT compared with CON conditions were not significantly different compared to the control condition, whereas skeletal muscle methyl-nicotinamide levels were significantly higher under NAD+-precursor supplementation [INT, 0.098 ± 0.063 compared with CON, 0.025 ± 0.014; P = 0.001], suggesting an increased NAD+ metabolism. Conversely, neither ADP-stimulated [INT, 82.1 ± 19.0 compared with CON, 84.0 ± 19.2; P = 0.716] nor maximally uncoupled mitochondrial respiration [INT, 103.4 ± 30.7 compared with CON, 108.7 ± 33.4; P = 0.495] improved under NAD+-precursor supplementation, nor did net exercise efficiency during the submaximal cycling test [INT, 20.2 ± 2.77 compared with CON, 20.8 ± 2.88; P = 0.342]. CONCLUSIONS: Our findings are consistent with previous findings on NAD+ efficacy in humans, and we show in community-dwelling, older adults with impaired physical function that NAD+-precursor supplementation through L-tryptophan, nicotinic acid, and nicotinamide does not improve mitochondrial or skeletal muscle function. This study was registered at clinicaltrials.gov as NCT03310034.

Mono(ADP-ribosyl)ation Enzymes and NAD+ Metabolism: A Focus on Diseases and Therapeutic Perspectives.

Mono(ADP-ribose) transferases and mono(ADP-ribosyl)ating sirtuins use NAD+ to perform the mono(ADP-ribosyl)ation, a simple form of post-translational modification of proteins and, in some cases, of nucleic acids. The availability of NAD+ is a limiting step and an essential requisite for NAD+ consuming enzymes. The synthesis and degradation of NAD+, as well as the transport of its key intermediates among cell compartments, play a vital role in the maintenance of optimal NAD+ levels, which are essential for the regulation of NAD+-utilizing enzymes. In this review, we provide an overview of the current knowledge of NAD+ metabolism, highlighting the functional liaison with mono(ADP-ribosyl)ating enzymes, such as the well-known ARTD10 (also named PARP10), SIRT6, and SIRT7. To this aim, we discuss the link of these enzymes with NAD+ metabolism and chronic diseases, such as cancer, degenerative disorders and aging.

Illuminating NAD+ Metabolism in Live Cells and In Vivo Using a Genetically Encoded Fluorescent Sensor.

Understanding of NAD+ metabolism provides many critical insights into health and diseases, yet highly sensitive and specific detection of NAD+ metabolism in live cells and in vivo remains difficult. Here, we present ratiometric, highly responsive genetically encoded fluorescent indicators, FiNad, for monitoring NAD+ dynamics in living cells and animals. FiNad sensors cover physiologically relevant NAD+ concentrations and sensitively respond to increases and decreases in NAD+. Utilizing FiNad, we performed a head-to-head comparison study of common NAD+ precursors in various organisms and mapped their biochemical roles in enhancing NAD+ levels. Moreover, we showed that increased NAD+ synthesis controls morphofunctional changes of activated macrophages, and directly imaged NAD+ declines during aging in situ. The broad utility of the FiNad sensors will expand our mechanistic understanding of numerous NAD+-associated physiological and pathological processes and facilitate screening for drug or gene candidates that affect uptake, efflux, and metabolism of this important cofactor.

NAD+ precursors protect corneal endothelial cells from UVB-induced apoptosis.

Excessive exposure of the eye to ultraviolet B light (UVB) leads to corneal edema and opacification because of the apoptosis of the corneal endothelium. Our previous study found that nicotinamide (NIC), the precursor of nicotinamide adenine dinucleotide (NAD), could inhibit the endothelial-mesenchymal transition and accelerate healing the wound to the corneal endothelium in the rabbit. Here we hypothesize that NIC may possess the capacity to protect the cornea from UVB-induced endothelial apoptosis. Therefore, a mouse model and a cultured cell model were used to examine the effect of NAD+ precursors, including NIC, nicotinamide mononucleotide (NMN), and NAD, on the UVB-induced apoptosis of corneal endothelial cells (CECs). The results showed that UVB irradiation caused apparent corneal edema and cell apoptosis in mice, accompanied by reduced levels of NAD+ and its key biosynthesis enzyme, nicotinamide phosphoribosyltransferase (NAMPT), in the corneal endothelium. However, the subconjunctival injection of NIC, NMN, or NAD+ effectively prevented UVB-induced tissue damage and endothelial cell apoptosis in the mouse cornea. Moreover, pretreatment using NIC, NMN, and NAD+ increased the survival rate and inhibited the apoptosis of cultured human CECs irradiated by UVB. Mechanistically, pretreatment using nicotinamide (NIC) recovered the AKT activation level and decreased the BAX/BCL-2 ratio. In addition, the capacity of NIC to protect CECs was fully reversed in the presence of the AKT inhibitor LY294002. Therefore, we conclude that NAD+ precursors can effectively prevent the apoptosis of the corneal endothelium through reactivating AKT signaling; this represents a potential therapeutic approach for preventing UVB-induced corneal damage.