Nicotinamide Mononucleotide Administration Triggers Macrophages Reprogramming and Alleviates Inflammation During Sepsis Induced by Experimental Peritonitis.

Peritonitis and subsequent sepsis lead to high morbidity and mortality in response to uncontrolled systemic inflammation primarily mediated by macrophages. Nicotinamide adenine dinucleotide (NAD+) is an important regulator of oxidative stress and immunoinflammatory responses. However, the effects of NAD+ replenishment during inflammatory activation are still poorly defined. Hence, we investigated whether the administration of ß-nicotinamide mononucleotide (ß-NMN), a natural biosynthetic precursor of NAD+, could modulate the macrophage phenotype and thereby ameliorate the dysregulated inflammatory response during sepsis. For this purpose, C57BL6 mice were subjected to the cecal ligation and puncture (CLP) model to provoke sepsis or were injected with thioglycolate to induce sterile peritonitis with recruitment and differentiation of macrophages into the inflamed peritoneal cavity. ß-NMN was administered for 4 days after CLP and for 3 days post thioglycolate treatment where peritoneal macrophages were subsequently analyzed. In the CLP model, administration of ß-NMN decreased bacterial load in blood and reduced clinical signs of distress and mortality during sepsis. These results were supported by transcriptomic analysis of hearts and lungs 24 h post CLP-induction, which revealed that ß-NMN downregulated genes controlling the immuno-inflammatory response and upregulated genes involved in bioenergetic metabolism, mitochondria, and autophagy. In the thioglycolate model, a significant increase in the proportion of CD206 macrophages, marker of anti-inflammatory M2 phenotype, was detected on peritoneal exudate macrophages from ß-NMN-administered mice. Transcriptomic signature of these macrophages after bacterial stimulation confirmed that ß-NMN administration limited the pro-inflammatory M1 phenotype and induced the expression of specific markers of M2 type macrophages. Furthermore, our data show that ß-NMN treatment significantly impacts NAD + metabolism. This shift in the macrophage phenotype and metabolism was accompanied by a reduction in phagolysosome acidification and secretion of inflammatory mediators in macrophages from ß-NMN-treated mice suggesting a reduced pro-inflammatory activation. In conclusion, administration of ß-NMN prevented clinical deterioration and improved survival during sepsis. These effects relied on shifts in the metabolism of organs that face up an increased energy requirement caused by bacterial infection and in innate immunity response, including reprogramming of macrophages from a highly inflammatory phenotype to an anti-inflammatory/pro-resolving profile.

Nicotinamide Mononucleotide Administration Prevents Doxorubicin-Induced Cardiotoxicity and Loss in Physical Activity in Mice.

Doxorubicin (Doxo) is a widely used antineoplastic drug with limited clinical application due to its deleterious dose-related side effects. We investigated whether nicotinamide mononucleotide (NMN) could protect against Doxo-induced cardiotoxicity and physical dysfunction in vivo. To assess the short- and long-term toxicity, two Doxo regimens were tested, acute and chronic. In the acute study, C57BL6/J (B6) mice were injected intraperitoneally (i.p.) once with Doxo (20 mg/kg) and NMN (180 mg/kg/day, i.p.) was administered daily for five days before and after the Doxo injection. In the chronic study, B6 mice received a cumulative dose of 20 mg/kg Doxo administered in fractionated doses for five days. NMN (500 mg/kg/day) was supplied in the mice's drinking water beginning five days before the first injection of Doxo and continuing for 60 days after. We found that NMN significantly increased tissue levels of NAD+ and its metabolites and improved survival and bodyweight loss in both experimental models. In addition, NMN protected against Doxo-induced cardiotoxicity and loss of physical function in acute and chronic studies, respectively. In the heart, NMN prevented Doxo-induced transcriptomic changes related to mitochondrial function, apoptosis, oxidative stress, inflammation and p53, and promyelocytic leukemia nuclear body pathways. Overall, our results suggest that NMN could prevent Doxo-induced toxicity in heart and skeletal muscle.

Role of CD38 in Adipose Tissue: Tuning Coenzyme Availability?

Nicotinamide adenine dinucleotide (NAD+) is a fundamental molecule in the regulation of energy metabolism, representing both a coenzyme and a substrate for different NAD+ degrading enzymes. Among these enzymes, CD38 can be seen under two perspectives: as the enzyme synthesizing Ca2+-mobilizing second messenger, starting from NAD+, and as the major NAD+-consumer, to be inhibited to increase NAD+ levels. Indeed, the regulation of NAD+ availability is a key event during different processes. In this review, we examine the recent studies related to the modulation of CD38 expression and activity, and the consequent changes in NAD(P)(H), in adipose tissue, during inflammation and cold-induced thermogenesis.

Safety evaluation after acute and sub-chronic oral administration of high purity nicotinamide mononucleotide (NMN-C®) in Sprague-Dawley rats.

ß-nicotinamide mononucleotide (NMN) is a natural molecule intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD+). Preclinical evidences point to the beneficial effect of NMN administration on several age-related conditions. The present work aimed at studying mutagenicity, and genotoxicity, acute oral toxicity and subchronic oral toxicity of a high purity synthetic form of NMN (NMN-C®) following the OECD guidelines. In the experimental conditions tested, NMN-C® was not mutagenic or genotoxic. Acute toxicity assay revealed that at an oral limit dose of 2666 mg/kg, NMN-C® did not lead to any mortality or treatment-related adverse signs. Over a 90-day sub-chronic period of repeated oral administration of NMN-C® at doses of 375, 750 and 1500 mg/kg/d followed by a 28-day treatment-free recovery period, NMN-C® appeared to be safe and did not promote toxic effects as seen from body weight change, food and water consumption, feed conversion efficiency, biochemical and blood parameters as well as organ toxicity and histological examinations of main organs. In conclusion, we provide the first data highlighting the safety of short to intermediate term (sub-chronic) oral administration of NMN and our experimental results allowed to determine a No-Observable Adverse Effect Level (NOAEL) for NMN-C® to be ≥ 1500 mg/kg/d.

NAD+ metabolism and its roles in cellular processes during ageing.

Nicotinamide adenine dinucleotide (NAD+) is a coenzyme for redox reactions, making it central to energy metabolism. NAD+ is also an essential cofactor for non-redox NAD+-dependent enzymes, including sirtuins, CD38 and poly(ADP-ribose) polymerases. NAD+ can directly and indirectly influence many key cellular functions, including metabolic pathways, DNA repair, chromatin remodelling, cellular senescence and immune cell function. These cellular processes and functions are critical for maintaining tissue and metabolic homeostasis and for healthy ageing. Remarkably, ageing is accompanied by a gradual decline in tissue and cellular NAD+ levels in multiple model organisms, including rodents and humans. This decline in NAD+ levels is linked causally to numerous ageing-associated diseases, including cognitive decline, cancer, metabolic disease, sarcopenia and frailty. Many of these ageing-associated diseases can be slowed down and even reversed by restoring NAD+ levels. Therefore, targeting NAD+ metabolism has emerged as a potential therapeutic approach to ameliorate ageing-related disease, and extend the human healthspan and lifespan. However, much remains to be learnt about how NAD+ influences human health and ageing biology. This includes a deeper understanding of the molecular mechanisms that regulate NAD+ levels, how to effectively restore NAD+ levels during ageing, whether doing so is safe and whether NAD+ repletion will have beneficial effects in ageing humans.

Differential modulation of SIRT6 deacetylase and deacylase activities by lysine-based small molecules.

Sirtuin 6 (SIRT6) is an NAD+-dependent deacetylase regulating important functions: modulators of its enzymatic activity have been considered as possible therapeutic agents. Besides the deacetylase activity, SIRT6 also has NAD+-dependent deacylase activity, whereby it regulates the secretion of cytokines and proteins. We identified novel SIRT6 modulators with a lysine-based structure: compound 1 enhances SIRT6 deacylase while inhibiting the deacetylase activity. As expected based on the biological effects of SIRT6 deacetylase activity, compound 1 increased histone 3 lysine 9 acetylation and the activity of glycolytic enzymes. Moreover, the fact that compound 1 enhanced SIRT6 deacylase activity was accompanied by an increased TNF-α release. In conclusion, new SIRT6 modulators with a lysine-like structure were identified, with differential effects on specific SIRT6 activities. The novel SIRT6 modulator concomitantly inhibits deacetylase and enhances deacylase activity.

Slc12a8 is a nicotinamide mononucleotide transporter.

Nicotinamide mononucleotide (NMN) is a biosynthetic precursor of NAD+ known to promote cellular NAD+ production and counteract age-associated pathologies associated with a decline in tissue NAD+ levels. How NMN is taken up into cells has not been entirely clear. Here we show that the Slc12a8 gene encodes a specific NMN transporter. We find that Slc12a8 is highly expressed and regulated by NAD+ in the murine small intestine. Slc12a8 knockdown abrogates the uptake of NMN in vitro and in vivo. We further show that Slc12a8 specifically transports NMN, but not nicotinamide riboside, and that NMN transport depends on the presence of sodium ion. Slc12a8 deficiency significantly decreases NAD+ levels in the jejunum and ileum, which is associated with reduced NMN uptake as traced by doubly labeled isotopic NMN. Finally, we observe that Slc12a8 expression is upregulated in the aged murine ileum, which contributes to the maintenance of ileal NAD+ levels. Our work identifies the first NMN transporter and demonstrates that Slc12a8 has a critical role in regulating intestinal NAD+ metabolism.

Author Correction: Slc12a8 is a nicotinamide mononucleotide transporter.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

SIRT6 deacetylase activity regulates NAMPT activity and NAD(P)(H) pools in cancer cells.

Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the NAD+ salvage pathway from nicotinamide. By controlling the biosynthesis of NAD+, NAMPT regulates the activity of NAD+-converting enzymes, such as CD38, poly-ADP-ribose polymerases, and sirtuins (SIRTs). SIRT6 is involved in the regulation of a wide number of metabolic processes. In this study, we investigated the ability of SIRT6 to regulate intracellular NAMPT activity and NAD(P)(H) levels. BxPC-3 cells and MCF-7 cells were engineered to overexpress a catalytically active or a catalytically inactive SIRT6 form or were engineered to silence endogenous SIRT6 expression. In SIRT6-overexpressing cells, NAD(H) levels were up-regulated, as a consequence of NAMPT activation. By immunopurification and incubation with recombinant SIRT6, NAMPT was found to be a direct substrate of SIRT6 deacetylation, with a mechanism that up-regulates NAMPT enzymatic activity. Extracellular NAMPT release was enhanced in SIRT6-silenced cells. Also glucose-6-phosphate dehydrogenase activity and NADPH levels were increased in SIRT6-overexpressing cells. Accordingly, increased SIRT6 levels reduced cancer cell susceptibility to H2O2-induced oxidative stress and to doxorubicin. Our data demonstrate that SIRT6 affects intracellular NAMPT activity, boosts NAD(P)(H) levels, and protects against oxidative stress. The use of SIRT6 inhibitors, together with agents inducing oxidative stress, may represent a promising treatment strategy in cancer.-Sociali, G., Grozio, A., Caffa, I., Schuster, S., Becherini, P., Damonte, P., Sturla, L., Fresia, C., Passalacqua, M., Mazzola, F., Raffaelli, N., Garten, A., Kiess, W., Cea, M., Nencioni, A., Bruzzone, S. SIRT6 deacetylase activity regulates NAMPT activity and NAD(P)(H) pools in cancer cells.

SIRT6 inhibitors with salicylate-like structure show immunosuppressive and chemosensitizing effects.

The NAD+-dependent deacetylase SIRT6 is an emerging cancer drug target, whose inhibition sensitizes cancer cells to chemo-radiotherapy and has pro-differentiating effects. Here we report on the identification of novel SIRT6 inhibitors with a salicylate-based structure. The new SIRT6 inhibitors show improved potency and specificity compared to the hit inhibitor identified in an in silico compound screen. As predicted based on SIRT6 biological roles, the new leads increase histone 3 lysine 9 acetylation and glucose uptake in cultured cells, while blocking TNF-α production and T lymphocyte proliferation. Notably, the new SIRT6 inhibitors effectively sensitize pancreatic cancer cells to gemcitabine. Finally, studies of compound fingerprinting and pharmacokinetics defined the drug-like properties of one of the new SIRT6 inhibitors, potentially allowing for subsequent in vivo proof-of-concept studies. In conclusion, new SIRT6 inhibitors with a salicylate-like structure were identified, which are active in cells and could potentially find applications in disease conditions, including cancer and immune-mediated disorders.

Pharmacological Sirt6 inhibition improves glucose tolerance in a type 2 diabetes mouse model.

Sirtuin 6 (SIRT6) is a sirtuin family member involved in a wide range of physiologic and disease processes, including cancer and glucose homeostasis. Based on the roles played by SIRT6 in different organs, including its ability to repress the expression of glucose transporters and glycolytic enzymes, inhibiting SIRT6 has been proposed as an approach for treating type 2 diabetes mellitus (T2DM). However, so far, the lack of small-molecule Sirt6 inhibitors has hampered the conduct of in vivo studies to assess the viability of this strategy. We took advantage of a recently identified SIRT6 inhibitor, compound 1, to study the effect of pharmacological Sirt6 inhibition in a mouse model of T2DM (i.e., in high-fat-diet-fed animals). The administration of the Sirt6 inhibitor for 10 d was well tolerated and improved oral glucose tolerance, it increased the expression of the glucose transporters GLUT1 and -4 in the muscle and enhanced the activity of the glycolytic pathway. Sirt6 inhibition also resulted in reduced insulin, triglycerides, and cholesterol levels in plasma. This study represents the first in vivo study of a SIRT6 inhibitor and provides the proof-of-concept that targeting SIRT6 may be a viable strategy for improving glycemic control in T2DM.-Sociali, G., Magnone, M., Ravera, S., Damonte, P., Vigliarolo, T., Von Holtey, M., Vellone, V. G., Millo, E., Caffa, I., Cea, M., Parenti, M. D., Del Rio, A., Murone, M., Mostoslavsky, R., Grozio, A., Nencioni, A., Bruzzone S. Pharmacological Sirt6 inhibition improves glucose tolerance in a type 2 diabetes mouse model.

Long-Term Administration of Nicotinamide Mononucleotide Mitigates Age-Associated Physiological Decline in Mice.

NAD+ availability decreases with age and in certain disease conditions. Nicotinamide mononucleotide (NMN), a key NAD+ intermediate, has been shown to enhance NAD+ biosynthesis and ameliorate various pathologies in mouse disease models. In this study, we conducted a 12-month-long NMN administration to regular chow-fed wild-type C57BL/6N mice during their normal aging. Orally administered NMN was quickly utilized to synthesize NAD+ in tissues. Remarkably, NMN effectively mitigates age-associated physiological decline in mice. Without any obvious toxicity or deleterious effects, NMN suppressed age-associated body weight gain, enhanced energy metabolism, promoted physical activity, improved insulin sensitivity and plasma lipid profile, and ameliorated eye function and other pathophysiologies. Consistent with these phenotypes, NMN prevented age-associated gene expression changes in key metabolic organs and enhanced mitochondrial oxidative metabolism and mitonuclear protein imbalance in skeletal muscle. These effects of NMN highlight the preventive and therapeutic potential of NAD+ intermediates as effective anti-aging interventions in humans.

Quinazolinedione SIRT6 inhibitors sensitize cancer cells to chemotherapeutics.

The NAD(+)-dependent sirtuin SIRT6 is highly expressed in human breast, prostate, and skin cancer where it mediates resistance to cytotoxic agents and prevents differentiation. Thus, SIRT6 is an attractive target for the development of new anticancer agents to be used alone or in combination with chemo- or radiotherapy. Here we report on the identification of novel quinazolinedione compounds with inhibitory activity on SIRT6. As predicted based on SIRT6's biological functions, the identified new SIRT6 inhibitors increase histone H3 lysine 9 acetylation, reduce TNF-α production and increase glucose uptake in cultured cells. In addition, these compounds exacerbate DNA damage and cell death in response to the PARP inhibitor olaparib in BRCA2-deficient Capan-1 cells and cooperate with gemcitabine to the killing of pancreatic cancer cells. In conclusion, new SIRT6 inhibitors with a quinazolinedione-based structure have been identified which are active in cells and could potentially find applications in cancer treatment.

Synthesis, structural characterization and effect on human granulocyte intracellular cAMP levels of abscisic acid analogs.

The phytohormone abscisic acid (ABA), in addition to regulating physiological functions in plants, is also produced and released by several mammalian cell types, including human granulocytes, where it stimulates innate immune functions via an increase of the intracellular cAMP concentration ([cAMP]i). We synthesized several ABA analogs and evaluated the structure-activity relationship, by the systematical modification of selected regions of these analogs. The resulting molecules were tested for their ability to inhibit the ABA-induced increase of [cAMP]i in human granulocytes. The analogs with modified configurations at C-2' and C-3' abrogated the ABA-induced increase of the [cAMP]i and also inhibited several pro-inflammatory effects induced by exogenous ABA on granulocytes and monocytes. Accordingly, these analogs could be suitable as novel putative anti-inflammatory compounds.

Nicotinamide phosphoribosyltransferase promotes epithelial-to-mesenchymal transition as a soluble factor independent of its enzymatic activity.

Boosting NAD(+) biosynthesis with NAD(+) intermediates has been proposed as a strategy for preventing and treating age-associated diseases, including cancer. However, concerns in this area were raised by observations that nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in mammalian NAD(+) biosynthesis, is frequently up-regulated in human malignancies, including breast cancer, suggesting possible protumorigenic effects for this protein. We addressed this issue by studying NAMPT expression and function in human breast cancer in vivo and in vitro. Our data indicate that high NAMPT levels are associated with aggressive pathological and molecular features, such as estrogen receptor negativity as well as HER2-enriched and basal-like PAM50 phenotypes. Consistent with these findings, we found that NAMPT overexpression in mammary epithelial cells induced epithelial-to-mesenchymal transition, a morphological and functional switch that confers cancer cells an increased metastatic potential. However, importantly, NAMPT-induced epithelial-to-mesenchymal transition was found to be independent of NAMPT enzymatic activity and of the NAMPT product nicotinamide mononucleotide. Instead, it was mediated by secreted NAMPT through its ability to activate the TGFß signaling pathway via increased TGFß1 production. These findings have implications for the design of therapeutic strategies exploiting NAD(+) biosynthesis via NAMPT in aging and cancer and also suggest the potential of anticancer agents designed to specifically neutralize extracellular NAMPT. Notably, because high levels of circulating NAMPT are found in obese and diabetic patients, our data could also explain the increased predisposition to cancer of these subjects.

Discovery of novel and selective SIRT6 inhibitors.

SIRT6 is an NAD(+)-dependent deacetylase with a role in the transcriptional control of metabolism and aging but also in genome stability and inflammation. Broad therapeutic applications are foreseen for SIRT6 inhibitors, including uses in diabetes, immune-mediated disorders, and cancer. Here we report on the identification of the first selective SIRT6 inhibitors by in silico screening. The most promising leads show micromolar IC50s, have significant selectivity for SIRT6 versus SIRT1 and SIRT2, and are active in cells, as shown by increased acetylation at SIRT6 target lysines on histone 3, reduced TNF-α secretion, GLUT-1 upregulation, and increased glucose uptake. Taken together, these results show the value of these compounds as starting leads for the development of new SIRT6-targeting therapeutic agents.

Selection and characterization of single stranded DNA aptamers for the hormone abscisic Acid.

The hormone abscisic acid (ABA) is a small molecule involved in pivotal physiological functions in higher plants. Recently, ABA has been also identified as an endogenous hormone in mammals, regulating different cell functions including inflammatory processes, stem cell expansion, insulin release, and glucose uptake. Aptamers are short, single-stranded (ss) oligonucleotidesable to recognize target molecules with high affinity. The small size of the ABA molecule represented a challenge for aptamer development and the aim of this study was to develop specific anti-ABA DNA aptamers. Biotinylated abscisic acid (bio-ABA) was immobilized on streptavidin-coated magnetic beads. DNA aptamers against bio-ABA were selected with 7 iterative rounds of the systematic evolution of ligands by exponential enrichment method (SELEX), each round comprising incubation of the ABA-binding beads with the ssDNA sequences, DNA elution, electrophoresis, and polymerase chain reaction (PCR) amplification. The PCR product was cloned and sequenced. The binding affinity of several clones was determined using bio-ABA immobilized on streptavidin-coated plates. Aptamer 2 and aptamer 9 showed the highest binding affinity, with dissociation constants values of 0.98 ± 0.14 µM and 0.80 ± 0.07 µM, respectively. Aptamers 2 and 9 were also able to bind free, unmodified ABA and to discriminate between different ABA enantiomers and isomers. Our findings indicate that ssDNA aptamers can selectively bind ABA and could be used for the development of ABA quantitation assays.

CD73 protein as a source of extracellular precursors for sustained NAD+ biosynthesis in FK866-treated tumor cells.

NAD(+) is mainly synthesized in human cells via the "salvage" pathways starting from nicotinamide, nicotinic acid, or nicotinamide riboside (NR). The inhibition with FK866 of the enzyme nicotinamide phosphoribosyltransferase (NAMPT), catalyzing the first reaction in the "salvage" pathway from nicotinamide, showed potent antitumor activity in several preclinical models of solid and hematologic cancers. In the clinical studies performed with FK866, however, no tumor remission was observed. Here we demonstrate that low micromolar concentrations of extracellular NAD(+) or NAD(+) precursors, nicotinamide mononucleotide (NMN) and NR, can reverse the FK866-induced cell death, this representing a plausible explanation for the failure of NAMPT inhibition as an anti-cancer therapy. NMN is a substrate of both ectoenzymes CD38 and CD73, with generation of NAM and NR, respectively. In this study, we investigated the roles of CD38 and CD73 in providing ectocellular NAD(+) precursors for NAD(+) biosynthesis and in modulating cell susceptibility to FK866. By specifically silencing or overexpressing CD38 and CD73, we demonstrated that endogenous CD73 enables, whereas CD38 impairs, the conversion of extracellular NMN to NR as a precursor for intracellular NAD(+) biosynthesis in human cells. Moreover, cell viability in FK866-treated cells supplemented with extracellular NMN was strongly reduced in tumor cells, upon pharmacological inhibition or specific down-regulation of CD73. Thus, our study suggests that genetic or pharmacologic interventions interfering with CD73 activity may prove useful to increase cancer cell sensitivity to NAMPT inhibitors.

Rejuvenating sirtuins: the rise of a new family of cancer drug targets.

Sirtuins are a family of NAD+-dependent enzymes that was proposed to control organismal life span about a decade ago. While such role of sirtuins is now debated, mounting evidence involves these enzymes in numerous physiological processes and disease conditions, including metabolism, nutritional behavior, circadian rhythm, but also inflammation and cancer. SIRT1, SIRT2, SIRT3, SIRT6, and SIRT7 have all been linked to carcinogenesis either as tumor suppressor or as cancer promoting proteins. Here, we review the biological rationale for the search of sirtuin inhibitors and activators for treating cancer and the experimental approaches to their identification.