Inhibition Of NAD Research Articles

Nampt/SIRT2/LDHA pathway-mediated lactate production regulates follicular dysplasia in polycystic ovary syndrome

Decreased nicotinamide adenine dinucleotide (NAD+) content has been shown to contribute to metabolic dysfunction during aging, including polycystic ovary syndrome (PCOS). However, the effect of NAD+ on ovulatory dysfunction in PCOS by regulating glycolysis has not been reported. Based on the observations of granulosa cells (GCs) transcriptome data from the Gene Expression Omnibus (GEO) database, the signal pathways including glycolysis and nicotinate-nicotinamide metabolism were significantly enriched, and most genes of the above pathway like LDHA and SIRT2 were down-regulated in PCOS patients. Therefore, the PCOS rat model was established by combining letrozole with a high-fat diet (HFD), we demonstrate that in vivo supplementation of nicotinamide mononucleotide (NMN) significantly improves the ovulatory dysfunction by facilitating the follicular development, promoting luteal formation, as well the fertility in PCOS rats. Furthermore, target energy metabolomics and transcriptome results showed that NMN supplementation ameliorates the lactate production by activating glycolytic process in the ovary. In vitro, when NAD+ synthesis and SIRT2 expression were inhibited, lactate content in KGN cells was decreased and LDHA expression was significantly inhibited. We confirmed that FK866 can enhance the acetylation of LDHA on 293T cells by Co-immunoprecipitation (Co-IP) assay. We also observed that inhibition of NAD+ synthesis can reduce the activity and increase the apoptosis of KGN cells. Overall, these benefits of NMN were elucidated and the Nampt/SIRT2/LDHA pathway mediated lactate production in granulosa cells played an important role in the improvement of follicular development disorders in PCOS. This study will provide experimental evidence for the clinical application of NMN in the treatment of PCOS in the future.

NMN partially rescues cuproptosis by upregulating sirt2 to increase intracellular NADPH

Cuproptosis is characterized by lipoylated protein aggregation and loss of iron–sulfur (Fe–S) proteins, which are crucial for a wide range of important cellular functions, including DNA replication and damage repair. Sirt2 and sirt4 are lipoamidases that remove the lipoyl moiety from lipoylated proteins using nicotinamide adenine dinucleotide (NAD+) as a cofactor. However, to date, it is not clear whether nicotinamide mononucleotide (NMN), a precursor of NAD+, affects cellular sensitivity to cuproptosis. Therefore, in the current study, cuproptosis was induced by the copper (Cu) ionophore elesclomol (Es) in HeLa cells. It was also found that Es/Cu treatment increased cellular DNA damage level. On the other hand, NMN treatment partially rescued cuproptosis in a dose-dependent manner, as well as reduced cellular DNA damage level. In addition, NMN upregulated the expression of Fe-S protein POLD1, without affecting the aggregation of lipoylated proteins. Mechanistic study revealed that NMN increased the expression of sirt2 and cellular reduced nicotinamide adenine dinucleotide phosphate (NADPH) level. Overexpression of sirt2 and sirt4 did not change the aggregation of lipoylated proteins, however, sirt2, but not sirt4, increased cellular NADPH levels and partially rescued cuproptosis. Inhibition of NAD+ kinase (NADK), which is responsible for generating NADPH, abolished the rescuing function of NMN and sirt2 for Es/Cu induced cell death. Taken together, our results suggested that DNA damage is a characteristic feature of cuproptosis. NMN can partially rescue cuproptosis by upregulating sirt2, increase intracellular NADPH content and maintain the level of Fe-S proteins, independent of the lipoamidase activity of sirt2.

Abstract B007: NAD+ metabolism drives PARPi resistance in ovarian cancer cells

Abstract NAD+ is a coenzyme for redox reactions and a cofactor for enzymes such as PARPs and Sirtuins. The nuclear enzyme PARP1 is a major consumer of NAD+, which is cleaved during the catalytic activation of PARP1 in response to DNA damage. PARP1 inhibitors compete with NAD+ for the catalytic site of PARP1 and reduce the catalytic activity of PARP1 and impair DNA damage repair. Recently PARP inhibitors (PARPi) have shown efficacy in the treatment of ovarian cancers and most patients with defects in homologous repair of DNA damage are now on PARPi maintenance. But, heterogeneous responses to PARPi is limiting their clinical efficacy. To identify the mechanisms of PARPi resistance, we generated a panel of ovarian cancer cells with acquired resistance to niraparib (PARPiR), an FDA approved PARP inhibitor. We demonstrated that the PARPiR cells are also resistant to other FDA approved PARP inhibitors rucaparib and olaparib. This data suggested that a common mechanism of resistance underlies poor response to the FDA approved PARP inhibitors. Since PARP1 activity is tightly controlled by the cellular NAD+ levels, we next investigated the role of NAD+ biosynthesis in PARPi resistance. Metabolomic profiling of the parental and PARPiR cells demonstrated that PARPiR cells have elevated levels of NAD+ metabolism. Indeed, treating the PARPiR cells with inhibitors of NAD+ synthesis restored the sensitivity to PARP inhibitors. Moreover, culturing the previously sensitive parental ovarian cancer cells in NAD+ precursors decreased their sensitivity to PARP inhibitors. These data collectively demonstrated that high levels of NAD+ metabolism causes PARP inhibitor resistance in ovarian cancer cells. Complementary to the metabolomic analysis, results from phenotypic assays in PARPiR cells revealed that high levels of NAD+ maintain cellular quiescence in PARPiR cells. In summary, our results demonstrated that high levels of NAD+ biosynthesis causes PARPi resistance by maintaining cellular quiescence. There is an urgent need to identify the mechanisms of resistance to PARP inhibitors to increase their clinical efficacy. Our study identified that maintenance of quiescence is a fundamental mechanism of PARPi resistance. We also demonstrated that inhibiting NAD+ biosynthesis reverses PARP inhibitor resistance. Citation Format: Subin Myong, Hardik Shah, Benedikt Nagele, Reshma Rajesh, Sridevi Challa. NAD+ metabolism drives PARPi resistance in ovarian cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B007.

Inhibitors of NAD+ Production in Cancer Treatment: State of the Art and Perspectives.

The addiction of tumors to elevated nicotinamide adenine dinucleotide (NAD+) levels is a hallmark of cancer metabolism. Obstructing NAD+ biosynthesis in tumors is a new and promising antineoplastic strategy. Inhibitors developed against nicotinamide phosphoribosyltransferase (NAMPT), the main enzyme in NAD+ production from nicotinamide, elicited robust anticancer activity in preclinical models but not in patients, implying that other NAD+-biosynthetic pathways are also active in tumors and provide sufficient NAD+ amounts despite NAMPT obstruction. Recent studies show that NAD+ biosynthesis through the so-called "Preiss-Handler (PH) pathway", which utilizes nicotinate as a precursor, actively operates in many tumors and accounts for tumor resistance to NAMPT inhibitors. The PH pathway consists of three sequential enzymatic steps that are catalyzed by nicotinate phosphoribosyltransferase (NAPRT), nicotinamide mononucleotide adenylyltransferases (NMNATs), and NAD+ synthetase (NADSYN1). Here, we focus on these enzymes as emerging targets in cancer drug discovery, summarizing their reported inhibitors and describing their current or potential exploitation as anticancer agents. Finally, we also focus on additional NAD+-producing enzymes acting in alternative NAD+-producing routes that could also be relevant in tumors and thus become viable targets for drug discovery.

THU547 Role Of NAD+ Synthesis Pathway In PARP Inhibitor Resistance

Abstract Disclosure: S. Challa: None. ADP-ribosylation is catalyzed by the poly(ADP-ribose) polymerase (PARP) family of enzymes consisting of 17 members that have distinct structural domains, activities, subcellular localizations, and functions. The catalytic activities of PARP enzymes are intimately tied to the synthesis of NAD+, which is consumed during ADPRylation reactions and, thus, must be regenerated. The intracellular salvage pathway utilizing nicotinamide (NAM) is the primary source of NAD+ in the cell. NAM is converted to nicotinamide mononucleotide (NMN) by NAMPT. Nicotinamide mononucleotide adenylyl transferases (NMNATs) catalyze the final step in the NAD+ salvage pathway, combining NMN and ATP to make NAD+. Three different NMNATs, each with a distinct subcellular localization, control the subcellular levels of NAD+ in each compartment: NMNAT-1 in the nucleus, NMNAT-2 is in the outer membrane of golgi, and NMNAT-3 in the mitochondria. We recently demonstrated that such compartment-specific NAD+ synthesis regulates the levels of ADP-ribosylation. Elevated levels of NMNAT-2, the cytosolic NAD+ synthase, reduces nuclear NAD+ levels and PARP1 activity in adipocytes and ovarian cancer cells. In the current study, we aim to delineate the role of compartmentalized synthesis of NAD+ in PARP inhibitor resistance in ovarian cancers. To identify these mechanisms, we generated ovarian cancer cells with acquired resistance to Niraparib, an FDA approved PARP inhibitor (NirR). We found that NirR cells have elevated levels of NAD+ biosynthesis. Moreover, treatment of parental cells with NAD+ precursors reduced the sensitivity to Niraparib while inhibition of NAD+ metabolism in NirR cells increased the sensitivity to Niraparib. Interestingly, the resistant cells have comparable levels of PARP1 activation to the parental cells. These data collectively suggests that NAD+ metabolism confers resistance to Niraparib without altering PARP1 activity. In line with this, NirR cells are sensitive to the effects of depletion of the cytosolic NAD+ biosynthesis, suggesting high NAD+ biosynthesis in NirR cells supports processes regulated by the cytosolic NAD+ which may drive the resistance phenotype. There is an urgent need to identify the mechanisms of resistance to PARP inhibitors to increase their clinical efficacy. This study identifies the NAD+ biosynthesis pathway as a key mechanism of PARP inhibitor resistance. Presentation: Thursday, June 15, 2023

Inhibition of NAD+-Dependent Metabolic Processes Induces Cellular Necrosis and Tumor Regression in Rhabdomyosarcoma Models.

Deregulated metabolism in cancer cells represents a vulnerability that may be therapeutically exploited to benefit patients. One such target is nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD+ salvage pathway. NAMPT is necessary for efficient NAD+ production and may be exploited in cells with increased metabolic demands. We have identified NAMPT as a dependency in rhabdomyosarcoma (RMS), a malignancy for which novel therapies are critically needed. Here we describe the effect of NAMPT inhibition on RMS proliferation and metabolism in vitro and in vivo. Assays of proliferation and cell death were used to determine the effects of pharmacologic NAMPT inhibition in a panel of ten molecularly diverse RMS cell lines. Mechanism of the clinical NAMPTi OT-82 was determined using measures of NAD+ and downstream NAD+-dependent functions, including energy metabolism. We used orthotopic xenograft models to examine tolerability, efficacy, and drug mechanism in vivo. Across all ten RMS cell lines, OT-82 depleted NAD+ and inhibited cell growth at concentrations ≤1 nmol/L. Significant impairment of glycolysis was a universal finding, with some cell lines also exhibiting diminished oxidative phosphorylation. Most cell lines experienced profound depletion of ATP with subsequent irreversible necrotic cell death. Importantly, loss of NAD and glycolytic activity were confirmed in orthotopic in vivo models, which exhibited complete tumor regressions with OT-82 treatment delivered on the clinical schedule. RMS is highly vulnerable to NAMPT inhibition. These findings underscore the need for further clinical study of this class of agents for this malignancy.

Promotion of NAD+ recycling by the hypoxia-induced shift in the lactate dehydrogenase isozyme profile reduces the senescence of human bone marrow-derived endothelial progenitor cells

Expansion of bone marrow-derived endothelial progenitor cells (EPCs) in vitro to obtain required cell numbers for therapeutic applications faces the challenge of growing cell senescence under the traditional normoxic culture condition. We previously found that 1% O2 hypoxic culture condition is favorable for reducing senescence of EPCs, but the mechanisms underlying the favorability are still unclear. Here, we found that, compared with normoxia, hypoxia induced a shift in lactate dehydrogenase (LDH) isozyme profile, which manifested as decreased LDH2 and LDH1 and increased LDH5, LDH4 and total LDHs. Moreover, under hypoxia, EPCs presented higher LDH activity, which could promote the conversion of pyruvate to lactate, as well as a higher level of NAD+, Bcl2 interacting protein 3 (BNIP3) expression and mitophagy. Additionally, under hypoxia, knock-down of the LDHA subunit increased the LDH2 and LDH1 levels and knock-down of the LDHB subunit increased the LDH5 level, while the simultaneous knock-down of LDHA and LDHB reduced total LDHs and NAD+ level. Inhibition of NAD+ recycling reduced BNIP3 expression and mitophagy and promoted cell senescence. Taken together, these data demonstrated that 1% O2 hypoxia induces a shift in the LDH isozyme profile, promotes NAD+ recycling, increases BNIP3 expression and mitophagy, and reduces EPC senescence. Our findings contribute to a better understanding of the connection between hypoxic culture conditions and the senescence of bone marrow-derived EPCs and provide a novel strategy to improve in vitro expansion of EPCs.

Seed pretreatment with melatonin confers cadmium tolerance to chickpea seedlings through cellular redox homeostasis and antioxidant gene expression improvement.

Several phytoremediation strategies have been undertaken to alleviate cadmium (Cd)-mediated injury to crop yield resulting from agricultural land pollution. In the present study, the potentially beneficial effect of melatonin (Me) was appraised. Therefore, chickpea (Cicer arietinum L.) seeds were imbibed for 12 H in distilled water or Me (10µM) solution. Then, the seeds germinated in the presence or the absence of 200µM CdCl2 for 6days. Seedlings obtained from Me-pretreated seeds exhibited enhanced growth traits, reflected by fresh biomass and length increase. This beneficial effect was associated with a decreased Cd accumulation in seedling tissues (by 46 and 89% in roots and shoots, respectively). Besides, Me efficiently protected the cell membrane integrity of Cd-subjected seedlings. This protective effect was manifested by the decreased lipoxygenase activity and the subsequently reduced accumulation of 4-hydroxy-2-nonenal. Melatonin counteracted the Cd-mediated stimulation of the pro-oxidant NADPH-oxidase (90 and 45% decrease compared to non-pretreated Cd-stressed roots and shoots, respectively) and NADH-oxidase activities (almost 40% decrease compared to non-pretreated roots and shoots), preventing, thus, hydrogen peroxide overaccumulation (50 and 35% lesser than non-pretreated roots and shoots, respectively). Furthermore, Me enhanced the cellular content of pyridine nicotinamide reduced forms [NAD(P)H] and their redox state. This effect was associated with the Me-mediated stimulation of the glucose-6-phosphate dehydrogenase (G6PDH) and malate dehydrogenase activities, concomitantly with the inhibition of NAD(P)H-consuming activities. These effects were accompanied by the up-regulation of G6PDH gene expression (45% increase in roots) and the down-regulation of the respiratory burst oxidase homolog protein F (RBOHF) gene expression (53% decrease in roots and shoots). Likewise, Me induced an increased activity and gene transcription of the Asada-Halliwell cycle, namely ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase, concomitantly with a reduction of the glutathione peroxidase activity. This modulating effect led to the restoration of the redox homeostasis of the ascorbate and the glutathione pools. Overall, current results attest that seed pretreatment with Me is effective in Cd stress relief and can be a beneficial crop-protective approach.

Impact of the Stimulation and Inhibition of NAD<sup>+</sup> Biosynthesis on the Maintenance of Pluripotency in Mouse Embryonic Stem Cells

Nicotinamide adenine dinucleotide (NAD+) plays a key role in cellular metabolism and signaling. In recent years, evidence has accumulated that NAD+-dependent processes are involved in the regulation of pluripotency and differentiation of mammalian embryonic stem cells. The major means to maintain NAD+ levels in mammalian cells is through its biosynthesis from various forms of vitamin B3. In this study, we examined how stimulation and inhibition of NAD+ biosynthesis affect the maintenance of the pluripotency of mouse embryonic stem cells E14 Tg2a (E14 cells). The pluripotency status of E14 cells was assessed by immunocytochemical and immunoblotting analysis using antibodies to the pluripotency factor Oct4, as well as by staining for alkaline phosphatase. Using NMR spectroscopy, we have found that the concentration of NAD+ in pluripotent E14 cells cultured in the presence of LIF is about 4 nmol/mg, and it remains unchanged after induction of differentiation with retinoic acid. We have also demonstrated that pharmacological stimulation of NAD+ biosynthesis by nicotinamide riboside increases the level of intracellular NAD+ by 20%, but it does not affect the maintenance of pluripotency in E14 cells. Moreover, under conditions of critical depletion of NAD+ pool by Nampt inhibition with FK866 E14 cells maintained pluripotency, though the expression level of Oct4 was decreased.

A Versatile Continuous Fluorometric Enzymatic Assay for Targeting Nicotinate Phosphoribosyltransferase.

The maintenance of a proper NAD+ pool is essential for cell survival, and tumor cells are particularly sensitive to changes in coenzyme levels. In this view, the inhibition of NAD+ biosynthesis is considered a promising therapeutic approach. Current research is mostly focused on targeting the enzymes nicotinamide phosphoribosyltransferase (NAMPT) and nicotinate phosphoribosyltransferase (NAPRT), which regulate NAD+ biosynthesis from nicotinamide and nicotinic acid, respectively. In several types of cancer cells, both enzymes are relevant for NAD+ biosynthesis, with NAPRT being responsible for cell resistance to NAMPT inhibition. While potent NAMPT inhibitors have been developed, only a few weak NAPRT inhibitors have been identified so far, essentially due to the lack of an easy and fast screening assay. Here we present a continuous coupled fluorometric assay whereby the product of the NAPRT-catalyzed reaction is enzymatically converted to NADH, and NADH formation is measured fluorometrically. The assay can be adapted to screen compounds that interfere with NADH excitation and emission wavelengths by coupling NADH formation to the cycling reduction of resazurin to resorufin, which is monitored at longer wavelengths. The assay system was validated by confirming the inhibitory effect of some NA-related compounds on purified human recombinant NAPRT. In particular, 2-hydroxynicotinic acid, 2-amminonicotinic acid, 2-fluoronicotinic acid, pyrazine-2-carboxylic acid, and salicylic acid were confirmed as NAPRT inhibitors, with Ki ranging from 149 to 348 µM. Both 2-hydroxynicotinic acid and pyrazine-2-carboxylic acid were found to sensitize OVCAR-5 cells to the NAMPT inhibitor FK866 by decreasing viability and intracellular NAD+ levels.

Apigenin attenuates LPS-induced neurotoxicity and cognitive impairment in mice via promoting mitochondrial fusion/mitophagy: role of SIRT3/PINK1/Parkin pathway

RationaleAlteration of the NAD+ metabolic pathway is proposed to be implicated in lipopolysaccharide (LPS)-induced neurotoxicity and mitochondrial dysfunction in neurodegenerative diseases. Apigenin, a naturally-occurring flavonoid, has been reported to maintain NAD+ levels and to preserve various metabolic functions.ObjectivesThis study aimed to explore the effect of apigenin on mitochondrial SIRT3 activity as a mediator through which it could modulate mitochondrial quality control and to protect against intracerebrovascular ICV/LPS-induced neurotoxicity.MethodsMice received apigenin (40 mg/kg; p.o) for 7 consecutive days. One hour after the last dose, LPS (12 µg/kg, icv) was administered.ResultsApigenin robustly guarded against neuronal degenerative changes and maintained a normal count of intact neurons in mice hippocampi. Consequently, it inhibited the deleterious effect of LPS on cognitive functions. Apigenin was effective in preserving the NAD+/NADH ratio to boost mitochondrial sirtuin-3 (SIRT3), activity, and ATP production. It conserved normal mitochondrial features via induction of the master regulator of mitochondrial biogenesis, peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α), along with mitochondrial transcription factor A (TFAM) and the fusion proteins, mitofusin 2 (MFN2), and optic atrophy-1 (OPA1). Furthermore, it increased phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) and parkin expression as well as the microtubule-associated protein 1 light chain 3 II/I ratio (LC3II/I) to induce degradation of unhealthy mitochondria via mitophagy.ConclusionsThese observations reveal the marked neuroprotective potential of apigenin against LPS-induced neurotoxicity through inhibition of NAD+ depletion and activation of SIRT3 to maintain adequate mitochondrial homeostasis and function.Graphical abstract

Sexually dimorphic effects of SARM1 deletion on cardiac NAD+ metabolism and function.

Nicotinamide adenine dinucleotide (NAD+) decline is repeatedly observed in heart disease and its risk factors. Although strategies promoting NAD+ synthesis to elevate NAD+ levels improve cardiac function, whether inhibition of NAD+ consumption can be therapeutic is less investigated. In this study, we examined the role of sterile-α and TIR motif containing 1 (SARM1) NAD+ hydrolase in mouse hearts, using global SARM1-knockout mice (KO). Cardiac function was assessed by echocardiography in male and female KO mice and wild-type (WT) controls. Hearts were collected for biochemical, histological, and molecular analyses. We found that the cardiac NAD+ pool was elevated in female KO mice, but only trended to increase in male KO mice. SARM1 deletion induced changes to a greater number of NAD+ metabolism transcripts in male mice than in female mice. Body weights, cardiac systolic and diastolic function, and geometry showed no changes in both male and female KO mice compared with WT counterparts. Male KO mice showed a small, but significant, elevation in cardiac collagen levels compared with WT counterparts, but no difference in collagen levels was detected in female mice. The increased collagen levels were associated with greater number of altered profibrotic and senescence-associated inflammatory genes in male KO mice, but not in female KO mice.NEW & NOTEWORTHY We examined the effects of SARM1 deletion on NAD+ pool, transcripts of NAD+ metabolism, and fibrotic pathway for the first time in mouse hearts. We observed the sexually dimorphic effects of SARM1 deletion. How these sex-dependent effects influence the outcomes of SARM1 deficiency in male and female mice in responses to cardiac stresses warrant further investigation. The elevation of cardiac NAD+ pool by SARM1 deletion provides evidence that targeting SARM1 may reverse disease-related NAD+ decline.

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.

β-lapachone: A Promising Anticancer Agent with a Unique NQO1 Specific Apoptosis in Pancreatic Cancer.

Cancer, one of the major health problems all over the world, requires more competent drugs for clinical use. One recent possible chemotherapeutic drug under research is β-lapachone. β- lapachone (1,2-naphthoquinone) has promising activity against those tumors showing raised levels of Nicotinamide di-phosphate Quinone Oxidoreductases-1 (NQO1). NQO1 is found to be up-regulated in pancreatic tumor cells, and thus β-lapachone could generate cytotoxicity in various cancers like pancreatic tumors. β-lapachone harborage independent growth and clonogenic cell survival in agar. The cell-killing effects of β-lapachone can be stopped by using dicumarol, an inhibitor of NAD(P)H Quinone Oxidoreductases-1. In previously established pancreatic cancer xenografts in mice, β- lapachone inhibited the tumor growth when given orally rather than when combined with cyclodextrin to improve its bioavailability.

NAD+ and its possible role in gut microbiota: Insights on the mechanisms by which gut microbes influence host metabolism.

Nicotinamide adenine dinucleotide (NAD+) is an enzyme cofactor, co-substrate, and redox factor in all living cells and is necessary for maintaining cell metabolism. It has been shown that appropriate supplementation of NAD+ precursors or inhibition of NAD+-depleting enzymes can promote mitochondrial oxidative phosphorylation and improve host energy utilization efficiency. In addition, increasing evidence indicates that the gut microbiota plays a pivotal role in host metabolism. Theoretically, there should be a close correlation among NAD+, gut microbiota, and host metabolism; however, the information is limited. In this review, we summarize the metabolic process of NAD+ and its impact on host metabolism, the link between gut microbiota and host metabolism, as well as the potential effects of NAD+ on microbial metabolism, providing a new perspective on the interaction between gut microbiota and host metabolism.

Abstract 2081: Poly ADP-ribose polymerase inhibition enhances T cell cytotoxicity and anti-tumor function by altering NAD+ levels

Abstract Poly ADP-ribose polymerase (PARP) is an NAD-dependent DNA repair enzyme. Inhibition of PARP in BRCA-mutant cancers has been a clinically approved therapy for several years. While the PARP inhibitor, olaparib, has been approved for the treatment of BRCA-mutant breast and ovarian cancers, it has not been used in other cancers because its mechanism of action relies on synthetic lethality. We have found that olaparib may also have use in other cancers due to its ability to modulate the immune system directly. Specifically, we have found that olaparib is able to increase the proliferation, activation, and cytotoxicity of T cells in vitro and reduce tumor growth in vivo at low doses. Recent papers have proposed that olaparib is able to enhance T cell function through the stimulator of interferon genes (STING) pathway by enhancing antigen presenting cell activity and resulting in more granzyme B expression and enhanced tumor killing. Using purified T cells from both wild type and STING knockout mice, we have shown that olaparib can also cause an increase in granzyme B and perforin expression, an increase in T cell proliferation, and enhanced tumor cell killing in a STING-independent manner. Because PARP is an NAD-consuming enzyme, we examined the role of NAD+ in the activation T cells and the production of granzyme B and perforin. By using an inhibitor of NAD+ recycling, FK866, we show that T cells have reduced NAD+ levels, reduced granzyme B and perforin expression, and even fail to activate in the presence of CD3 and CD28 when NAD+ is limited; however, adding olaparib to these cells rescues the effects of FK866 and allows proliferation and granzyme B production. In vivo we can see that even at olaparib doses that are significantly below those which induce synthetic lethality of tumor cells, olaparib is able to inhibit tumor growth in models of both colorectal and breast cancer. This suggests that olaparib can modulate the activity of T cells directly and has implications for enhancing the anti-tumor activity of these cells both alone and in combination with other forms of immunotherapy. Citation Format: Matthew J. Dean, Liqin Zheng, Phaethon Philbrook, Augusto C. Ochoa, A. Hamid Boulares. Poly ADP-ribose polymerase inhibition enhances T cell cytotoxicity and anti-tumor function by altering NAD+ levels [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 2081.

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.

Long-chain fatty acids regulate SIRT3 expression by affecting intracellular NAD+ levels in large yellow croaker (Larimichthys crocea)

Sirtuin 3 (SIRT3) is a major deacetylase in the mitochondria and sensitive to diverse nutrient signals. However, how dietary fatty acids regulate SIRT3 expression remains poorly understood. Here, sirt3 gene from large yellow croaker was characterized and can be activated by the oxidized nicotinamide adenine dinucleotide (NAD+) precursor, nicotinamide mononucleotide (NMN). Moreover, the expression of SIRT3 is regulated differently by dietary fatty acids. In vivo, soybean oil increased the mRNA expression of sirt3 in head kidney. In vitro, stearic acid (SA) decreased the expression of SIRT3 in macrophages, while DHA and EPA increased the mRNA expression of sirt3. Meanwhile, all long-chain polyunsaturated fatty acids (LC-PUFAs) enhanced the protein levels of SIRT3, which was consistent with the trend of fatty acids affecting NAD+ levels. Furthermore, inhibition of NAD+de novo synthesis blocked DHA-induced increases in SIRT3 expression, and NMN supplement reversed SA-induced decreases in SIRT3 expression. In conclusion, these findings suggest that dietary fatty acids may regulate SIRT3 expression by affecting intracellular NAD+ synthesis. Regarding the important role of SIRT3 in regulating mitochondrial function, appropriate activation of SIRT3 may be an important way to alleviate metabolic disorders in aquaculture.

NAD+ and Vascular Dysfunction: From Mechanisms to Therapeutic Opportunities.

Nicotinamide adenine dinucleotide (NAD+) is an essential and pleiotropic coenzyme involved not only in cellular energy metabolism, but also in cell signaling, epigenetic regulation, and post-translational protein modifications. Vascular disease risk factors are associated with aberrant NAD+ metabolism. Conversely, the therapeutic increase of NAD+ levels through the administration of NAD+ precursors or inhibitors of NAD+-consuming enzymes reduces chronic low-grade inflammation, reactivates autophagy and mitochondrial biogenesis, and enhances oxidative metabolism in vascular cells of humans and rodents with vascular pathologies. As such, NAD+ has emerged as a potential target for combatting age-related cardiovascular and cerebrovascular disorders. This review discusses NAD+-regulated mechanisms critical for vascular health and summarizes new advances in NAD+ research directly related to vascular aging and disease, including hypertension, atherosclerosis, coronary artery disease, and aortic aneurysms. Finally, we enumerate challenges and opportunities for NAD+ repletion therapy while anticipating the future of this exciting research field, which will have a major impact on vascular medicine.

Sirtuin 6 Protects Against Oxidative Stress and Vascular Dysfunction in Mice.

Objective: Sirtuin deacetylases are major regulators of organismal aging, and while depletion of sirtuin 6 (SIRT6) in mice results in a profound progeroid phenotype, the role of SIRT6 in the regulation of vasomotor function is unknown. Thus, our objective was to test the hypothesis that reductions in SIRT6 elicit endothelial dysfunction in young, genetically altered mice.Results and Approach: We used young (3 month old), littermate-matched, SIRT6 wild-type (WT), and SIRT6 heterozygous (HET) mice. SIRT6 expression (qRT-PCR) was reduced by 50% in HET mice. Carotid vessel responses to acetylcholine, sodium nitroprusside, U46619, and serotonin were examined in isolated organ chamber baths. Relaxation in response to acetylcholine (ACH) was impaired in HET mice compared to littermate-matched WT controls (67 ± 3% versus 76 ± 3%, respectively; p < 0.05), while responses to sodium nitroprusside were unchanged. Short-term incubation of carotid rings with the NAD(P)H oxidase inhibitor, apocynin, significantly improved in vessels from HET mice but not their WT littermates. Peak tension generated in response to either U46619 or serotonin was significantly blunted in HET mice compared to their WT littermates.Conclusion: These data suggest that SIRT6 is a key regulator of vasomotor function in conduit vessels. More specifically, we propose that SIRT6 serves as a tonic suppressor of NAD(P)H oxidase expression and activation, as inhibition of NAD(P)H oxidase improved endothelial function in SIRT6 haploinsufficient mice. Collectively, SIRT6 activation and/or histone acetyltransferase inhibition may be useful therapeutic approaches to reduce endothelial dysfunction and combat age-associated cardiovascular disease.