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.

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.

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.

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.

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.

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.

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.

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.

The NAD+-dependent histone deacetylase SIRT6 promotes cytokine production and migration in pancreatic cancer cells by regulating Ca2+ responses.

Cytokine secretion by cancer cells contributes to cancer-induced symptoms and angiogenesis. Studies show that the sirtuin SIRT6 promotes inflammation by enhancing TNF expression. Here, we aimed to determine whether SIRT6 is involved in conferring an inflammatory phenotype to cancer cells and to define the mechanisms linking SIRT6 to inflammation. We show that SIRT6 enhances the expression of pro-inflammatory cyto-/chemokines, such as IL8 and TNF, and promotes cell migration in pancreatic cancer cells by enhancing Ca(2+) responses. Via its enzymatic activity, SIRT6 increases the intracellular levels of ADP-ribose, an activator of the Ca(2+) channel TRPM2. In turn, TRPM2 and Ca(2+) are shown to be involved in SIRT6-induced TNF and IL8 expression. SIRT6 increases the nuclear levels of the Ca(2+)-dependent transcription factor, nuclear factor of activated T cells (NFAT), and cyclosporin A, a calcineurin inhibitor that reduces NFAT activity, reduces TNF and IL8 expression in SIRT6-overexpressing cells. These results implicate a role for SIRT6 in the synthesis of Ca(2+)-mobilizing second messengers, in the regulation of Ca(2+)-dependent transcription factors, and in the expression of pro-inflammatory, pro-angiogenic, and chemotactic cytokines. SIRT6 inhibition may help combat cancer-induced inflammation, angiogenesis, and metastasis.

The plant hormone abscisic acid increases in human plasma after hyperglycemia and stimulates glucose consumption by adipocytes and myoblasts.

The plant hormone abscisic acid (ABA) is released from glucose-challenged human pancreatic ß cells and stimulates insulin secretion. We investigated whether plasma ABA increased during oral and intravenous glucose tolerance tests (OGTTs and IVGTTs) in healthy human subjects. In all subjects undergoing OGTTs (n=8), plasma ABA increased over basal values (in a range from 2- to 9-fold). A positive correlation was found between the ABA area under the curve (AUC) and the glucose AUC. In 4 out of 6 IVGTTs, little or no increase of ABA levels was observed. In the remaining subjects, the ABA increase was similar to that recorded during OGTTs. GLP-1 stimulated ABA release from an insulinoma cell line and from human islets, by ∼10- and 2-fold in low and high glucose, respectively. Human adipose tissue also released ABA in response to high glucose. Nanomolar ABA stimulated glucose uptake, similarly to insulin, in rat L6 myoblasts and in murine 3T3-L1 cells differentiated to adipocytes, by increasing GLUT-4 translocation to the plasma membrane. Demonstration that a glucose load in humans is followed by a physiological rise of plasma ABA, which can enhance glucose uptake by adipose tissues and muscle cells, identifies ABA as a new mammalian hormone involved in glucose metabolism.

Autocrine abscisic acid mediates the UV-B-induced inflammatory response in human granulocytes and keratinocytes.

UV-B is an abiotic environmental stress in both plants and animals. Abscisic acid (ABA) is a phytohormone regulating fundamental physiological functions in plants, including response to abiotic stress. We previously demonstrated that ABA is an endogenous stress hormone also in animal cells. Here, we investigated whether autocrine ABA regulates the response to UV-B of human granulocytes and keratinocytes, the cells involved in UV-triggered skin inflammation. The intracellular ABA concentration increased in UV-B-exposed granulocytes and keratinocytes and ABA was released into the supernatant. The UV-B-induced production of NO and of reactive oxygen species (ROS), phagocytosis, and cell migration were strongly inhibited in granulocytes irradiated in the presence of a monoclonal antibody against ABA. Moreover, presence of the same antibody strongly inhibited release of NO, prostaglandin E2 (PGE(2)), and tumor necrosis factor-α (TNF-α) by UV-B irradiated keratinocytes. Lanthionine synthetase C-like protein 2 (LANCL2) is required for the activation of the ABA signaling pathway in human granulocytes. Silencing of LANCL2 in human keratinocytes by siRNA was accompanied by abrogation of the UV-B-triggered release of PGE(2), TNF-α, and NO and ROS production. These results indicate that UV-B irradiation induces ABA release from human granulocytes and keratinocytes and that autocrine ABA stimulates cell functions involved in skin inflammation.

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.

Binding of abscisic acid to human LANCL2.

The phytohormone abscisic acid (ABA) is the central regulator of abiotic stress in plants and plays important roles during plant growth and development. In animal cells, ABA was shown to be an endogenous hormone, acting as a stress signal and stimulating cell functions involved in inflammatory responses and in insulin release. Recently, we demonstrated that Lanthionine synthetase component C-like protein 2 (LANCL2) is required for ABA binding to the plasmamembrane of granulocytes and for the activation of the signaling pathway triggered by ABA in human granulocytes and in rat insulinoma cells. In order to investigate whether ABA activates LANCL2 via direct interaction, we performed specific binding studies on human LANCL2 recombinant protein using different experimental approaches (saturation binding, scintillation proximity assays, dot blot experiments and affinity chromatography). Altogether, results indicate that human recombinant LANCL2 binds ABA directly and provide the first demonstration of ABA binding to a mammalian ABA receptor.

Functional characterization of a synthetic abscisic acid analog with anti-inflammatory activity on human granulocytes and monocytes.

The phytohormone abscisic acid (ABA), in addition to regulating several important physiological functions in plants, is also produced and released by human granulocytes and monocytes where it stimulates cell activities involved in the innate immune response. Here we describe the properties of an ABA synthetic analog that competes with the hormone for binding to human granulocyte membranes and to purified recombinant LANCL2 (the human ABA receptor) and inhibits several ABA-triggered inflammatory functions of granulocytes and monocytes in vitro: chemotaxis, phagocytosis, reactive oxygen species production and release of prostaglandin E(2) (PGE(2)) by human granulocytes, release of PGE(2) and of monocyte chemoattractant protein-1 by human monocytes. This observation provides a proof of principle that ABA antagonists may represent a new class of anti-inflammatory agents.

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.

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.

LANCL2 is necessary for abscisic acid binding and signaling in human granulocytes and in rat insulinoma cells.

Abscisic acid (ABA) is a plant hormone regulating fundamental physiological functions in plants, such as response to abiotic stress. Recently, ABA was shown to be produced and released by human granulocytes, by insulin-producing rat insulinoma cells, and by human and murine pancreatic beta cells. ABA autocrinally stimulates the functional activities specific for each cell type through a receptor-operated signal transduction pathway, sequentially involving a pertussis toxin-sensitive receptor/G-protein complex, cAMP, CD38-produced cADP-ribose and intracellular calcium. Here we show that the lanthionine synthetase C-like protein LANCL2 is required for ABA binding on the membrane of human granulocytes and that LANCL2 is necessary for transduction of the ABA signal into the cell-specific functional responses in granulocytes and in rat insulinoma cells. Co-expression of LANCL2 and CD38 in the human HeLa cell line reproduces the ABA-signaling pathway. Results obtained with granulocytes and CD38(+)/LANCL2(+) HeLa transfected with a chimeric G-protein (G alpha(q/i)) suggest that the pertussis toxin-sensitive G-protein coupled to LANCL2 is a G(i). Identification of LANCL2 as a critical component of the ABA-sensing protein complex will enable the screening of synthetic ABA antagonists as prospective new anti-inflammatory and anti-diabetic agents.

The cholinergic system and cancer.

The finding that the cholinergic system may be present in non-neuromuscular cells and tissues was, at the beginning, not easily accepted by the scientific community (since 1970-1982). Today the entire set of cholinergic molecules and their functions in cell-to-cell communication, mediated by intracellular ion dynamics, is well-established. Nicotinic and muscarinic (nAChR, mAChR) are functionally present on different human cell types and build a part of an autocrine-proliferative network. In synthesis Ach via nAChR or mAChR appears to be involved in the regulation of vital cell functions: proliferation, differentiation, organization of the cytoskeleton, cell-cell contact, ciliary activity, migration, secretion and absorption of ions, water and mucus. Understanding the role of the non-neuronal cholinergic system in different diseases (i.e. inflammation and or cancer) needs to be clarified in more detail, in order to optimize a future targeted-therapy, as well as precautions in the design of interventional drugs.

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.