NAD+ Anabolism Disturbance Causes Glomerular Mesangial Cell Injury in Diabetic Nephropathy

The homeostasis of NAD⁺ anabolism is indispensable for maintaining the NAD⁺ pool. In mammals, the mainly synthetic pathway of NAD⁺ is the salvage synthesis, a reaction catalyzed by nicotinamide mononucleotide adenylyltransferase (NAMPT) and nicotinamide mononucleotide adenylyltransferase (NMNATs) successively, converting nicotinamide (NAM) to nicotinamide mononucleotide (NMN) and NMN to NAD⁺, respectively. However, the relationship between NAD⁺ anabolism disturbance and diabetic nephropathy (DN) remains elusive. Here our study found that the disruption of NAD⁺ anabolism homeostasis caused an elevation in both oxidative stress and fibronectin expression, along with a decrease in Sirt1 and an increase in both NF-κB P65 expression and acetylation, culminating in extracellular matrix deposition and globular fibrosis in DN. More importantly, through constitutively overexpressing NMNAT1 or NAMPT in human mesangial cells, we revealed NAD⁺ levels altered inversely with NMN levels in the context of DN and, further, their changes affect Sirt1/NF-κB P65, thus playing a crucial role in the pathogenesis of DN. Accordingly, FK866, a NAMPT inhibitor, and quercetin, a Sirt1 agonist, have favorable effects on the maintenance of NAD⁺ homeostasis and renal function in db/db mice. Collectively, our findings suggest that NMN accumulation may provide a causal link between NAD⁺ anabolism disturbance and diabetic nephropathy (DN) as well as a promising therapeutic target for DN treatment.     

Different Effects of RNAi-Mediated Downregulation or Chemical Inhibition of NAMPT in an Isogenic IDH Mutant and Wild-Type Glioma Cell Model

The IDH1^R132H mutation in glioma results in the neoenzymatic function of IDH1, leading to the production of the oncometabolite 2-hydroxyglutarate (2-HG), alterations in energy metabolism and changes in the cellular redox household. Although shifts in the redox ratio NADPH/NADP⁺ were described, the consequences for the NAD⁺ synthesis pathways and potential therapeutic interventions were largely unexplored. Here, we describe the effects of heterozygous IDH1^R132H on the redox system in a CRISPR/Cas edited glioblastoma model and compare them with IDH1 wild-type (IDH1^wt) cells. Besides an increase in 2-HG and decrease in NADPH, we observed an increase in NAD⁺ in IDH1^R132H glioblastoma cells. RT-qPCR analysis revealed the upregulation of the expression of the NAD⁺ synthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT). Knockdown of NAMPT resulted in significantly reduced viability in IDH1^R132H glioblastoma cells. Given this dependence of IDH1^R132H cells on NAMPT expression, we explored the effects of the NAMPT inhibitors FK866, GMX1778 and GNE-617. Surprisingly, these agents were equally cytotoxic to IDH1^R132H and IDH1^wt cells. Altogether, our results indicate that targeting the NAD⁺ synthesis pathway is a promising therapeutic strategy in IDH mutant gliomas; however, the agent should be carefully considered since three small-molecule inhibitors of NAMPT tested in this study were not suitable for this purpose.     

COVID-19: Are We Facing Secondary Pellagra Which Cannot Simply Be Cured by Vitamin B3?

Immune response to SARS-CoV-2 and ensuing inflammation pose a huge challenge to the host’s nicotinamide adenine dinucleotide (NAD⁺) metabolism. Humans depend on vitamin B3 for biosynthesis of NAD⁺, indispensable for many metabolic and NAD⁺-consuming signaling reactions. The balance between its utilization and resynthesis is vitally important. Many extra-pulmonary symptoms of COVID-19 strikingly resemble those of pellagra, vitamin B3 deficiency (e.g., diarrhoea, dermatitis, oral cavity and tongue manifestations, loss of smell and taste, mental confusion). In most developed countries, pellagra is successfully eradicated by vitamin B3 fortification programs. Thus, conceivably, it has not been suspected as a cause of COVID-19 symptoms. Here, the deregulation of the NAD⁺ metabolism in response to the SARS-CoV-2 infection is reviewed, with special emphasis on the differences in the NAD⁺ biosynthetic pathway’s efficiency in conditions predisposing for the development of serious COVID-19. SARS-CoV-2 infection-induced NAD⁺ depletion and the elevated levels of its metabolites contribute to the development of a systemic disease. Acute liberation of nicotinamide (NAM) in antiviral NAD⁺-consuming reactions potentiates “NAM drain”, cooperatively mediated by nicotinamide N-methyltransferase and aldehyde oxidase. “NAM drain” compromises the NAD⁺ salvage pathway’s fail-safe function. The robustness of the host’s NAD⁺ salvage pathway, prior to the SARS-CoV-2 infection, is an important determinant of COVID-19 severity and persistence of certain symptoms upon resolution of infection.     

Nicotinamide Mononucleotide Supplementation Improves Mitochondrial Dysfunction and Rescues Cellular Senescence by NAD+/Sirt3 Pathway in Mesenchymal Stem Cells

In vitro expansion-mediated replicative senescence has severely limited the clinical applications of mesenchymal stem cells (MSCs). Accumulating studies manifested that nicotinamide adenine dinucleotide (NAD⁺) depletion is closely related to stem cell senescence and mitochondrial metabolism disorder. Promoting NAD⁺ level is considered as an effective way to delay aging. Previously, we have confirmed that nicotinamide mononucleotide (NMN), a precursor of NAD⁺, can alleviate NAD⁺ deficiency-induced MSC senescence. However, whether NMN can attenuate MSC senescence and its underlying mechanisms are still incompletely clear. The present study herein showed that late passage (LP) MSCs displayed lower NAD⁺ content, reduced Sirt3 expression and mitochondrial dysfunction. NMN supplementation leads to significant increase in intracellular NAD⁺ level, NAD⁺/ NADH ratio, Sirt3 expression, as well as ameliorated mitochondrial function and rescued senescent MSCs. Additionally, Sirt3 over-expression relieved mitochondrial dysfunction, and retrieved senescence-associated phenotypic features in LP MSCs. Conversely, inhibition of Sirt3 activity via a selective Sirt3 inhibitor 3-TYP in early passage (EP) MSCs resulted in aggravated cellular senescence and abnormal mitochondrial function. Furthermore, NMN administration also improves 3-TYP-induced disordered mitochondrial function and cellular senescence in EP MSCs. Collectively, NMN replenishment alleviates mitochondrial dysfunction and rescues MSC senescence through mediating NAD⁺/Sirt3 pathway, possibly providing a novel mechanism for MSC senescence and a promising strategy for anti-aging pharmaceuticals.     

Role of PGC-1α in the Mitochondrial NAD+ Pool in Metabolic Diseases

Mitochondria play vital roles, including ATP generation, regulation of cellular metabolism, and cell survival. Mitochondria contain the majority of cellular nicotinamide adenine dinucleotide (NAD⁺), which an essential cofactor that regulates metabolic function. A decrease in both mitochondria biogenesis and NAD⁺ is a characteristic of metabolic diseases, and peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) orchestrates mitochondrial biogenesis and is involved in mitochondrial NAD⁺ pool. Here we discuss how PGC-1α is involved in the NAD⁺ synthesis pathway and metabolism, as well as the strategy for increasing the NAD⁺ pool in the metabolic disease state.     

NAD+ Precursors Repair Mitochondrial Function in Diabetes and Prevent Experimental Diabetic Neuropathy

Axon degeneration in diabetic peripheral neuropathy (DPN) is associated with impaired NAD⁺ metabolism. We tested whether the administration of NAD⁺ precursors, nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR), prevents DPN in models of Type 1 and Type 2 diabetes. NMN was administered to streptozotocin (STZ)-induced diabetic rats and STZ-induced diabetic mice by intraperitoneal injection at 50 or 100 mg/kg on alternate days for 2 months. mice The were fed with a high fat diet (HFD) for 2 months with or without added NR at 150 or 300 mg/kg for 2 months. The administration of NMN to STZ-induced diabetic rats or mice or dietary addition of NR to HFD-fed mice improved sensory function, normalized sciatic and tail nerve conduction velocities, and prevented loss of intraepidermal nerve fibers in skin samples from the hind-paw. In adult dorsal root ganglion (DRG) neurons isolated from HFD-fed mice, there was a decrease in NAD⁺ levels and mitochondrial maximum reserve capacity. These impairments were normalized in isolated DRG neurons from NR-treated mice. The results indicate that the correction of NAD⁺ depletion in DRG may be sufficient to prevent DPN but does not significantly affect glucose tolerance, insulin levels, or insulin resistance.     

A Method to Monitor the NAD+ Metabolome—From Mechanistic to Clinical Applications

Nicotinamide adenine dinucleotide (NAD⁺) and its reduced form (NADH) are coenzymes employed in hundreds of metabolic reactions. NAD⁺ also serves as a substrate for enzymes such as sirtuins, poly(ADP-ribose) polymerases (PARPs) and ADP-ribosyl cyclases. Given the pivotal role of NAD(H) in health and disease, studying NAD⁺ metabolism has become essential to monitor genetic- and/or drug-induced perturbations related to metabolic status and diseases (such as ageing, cancer or obesity), and its possible therapies. Here, we present a strategy based on liquid chromatography-tandem mass spectrometry (LC-MS/MS), for the analysis of the NAD⁺ metabolome in biological samples. In this method, hydrophilic interaction chromatography (HILIC) was used to separate a total of 18 metabolites belonging to pathways leading to NAD⁺ biosynthesis, including precursors, intermediates and catabolites. As redox cofactors are known for their instability, a sample preparation procedure was developed to handle a variety of biological matrices: cell models, rodent tissues and biofluids, as well as human biofluids (urine, plasma, serum, whole blood). For clinical applications, quantitative LC-MS/MS for a subset of metabolites was demonstrated for the analysis of the human whole blood of nine volunteers. Using this developed workflow, our methodology allows studying NAD⁺ biology from mechanistic to clinical applications.     

Modulation of Cellular NAD+ Attenuates Cancer-Associated Hypercoagulability and Thrombosis via the Inhibition of Tissue Factor and Formation of Neutrophil Extracellular Traps

Cancer-associated thrombosis is the second-leading cause of mortality in patients with cancer and presents a poor prognosis, with a lack of effective treatment strategies. NAD(P)H quinone oxidoreductase 1 (NQO1) increases the cellular nicotinamide adenine dinucleotide (NAD⁺) levels by accelerating the oxidation of NADH to NAD⁺, thus playing important roles in cellular homeostasis, energy metabolism, and inflammatory responses. Using a murine orthotopic 4T1 breast cancer model, in which multiple thrombi are generated in the lungs at the late stage of cancer development, we investigated the effects of regulating the cellular NAD⁺ levels on cancer-associated thrombosis. In this study, we show that dunnione (a strong substrate of NQO1) attenuates the prothrombotic state and lung thrombosis in tumor-bearing mice by inhibiting the expression of tissue factor and formation of neutrophil extracellular traps (NETs). Dunnione increases the cellular NAD⁺ levels in lung tissues of tumor-bearing mice to restore the declining sirtuin 1 (SIRT1) activity, thus deacetylating nuclear factor-kappa B (NF-κB) and preventing the overexpression of tissue factor in bronchial epithelial and vascular endothelial cells. In addition, we demonstrated that dunnione abolishes the ability of neutrophils to generate NETs by suppressing histone acetylation and NADPH oxidase (NOX) activity. Overall, our results reveal that the regulation of cellular NAD⁺ levels by pharmacological agents may inhibit pulmonary embolism in tumor-bearing mice, which may potentially be used as a viable therapeutic approach for the treatment of cancer-associated thrombosis.     

Substituting N��-thioacetyl-lysine for N��-acetyl-lysine in Peptide Substrates as a General Approach to Inhibiting Human NAD+-dependent Protein Deacetylases

Inhibitors of human NAD⁺-dependent protein deacetylases possess great value for deciphering the biology of these enzymes and as potential therapeutics for metabolic and age-related diseases and cancer. In the current study, we have experimentally demonstrated that, the potent inhibition we obtained previously for one of these enzymes (i.e. sirtuin type 1 (SIRT1)) by simply replacing N^ε-thioacetyl-lysine for N^ε-acetyl-lysine in its peptide substrate, represented a general and efficient strategy to develop potent and selective inhibitors of human NAD⁺-dependent protein deacetylase enzymes. Indeed, by using this simple inhibition strategy, potent (low-micromolar) and selective (≤40-fold) SIRT2 and SIRT3 inhibitors, which were either comparable or superior to currently existing inhibitors, have also been quickly identified in the current study. These inhibitors could be used as chemical biological tools or as lead compounds for further focused structure-activity optimization.     

Poly(neutral red) as a NAD reduction catalyst and a NADH oxidation catalyst: Towards the development of a rechargeable biobattery

In this paper, we have established that poly(neutral red), PNR, functions as an electrocatalyst for the reduction and oxidation of NAD⁺/NADH in a rechargeable biobattery environment. The reversibility of this catalyst was possible only with the addition of Zn²⁺ for complexation to the redox polymer. The zinc ion complexation with the polymer facilitates electron and proton transfer to/from the substrate and the NAD⁺/NADH coenzyme without forming covalent bonds between the nicotinamide and the substrate surface. This research presents use of this reversible catalyst in a rechargeable biobattery. The rechargeable battery includes a Prussian blue cathode and a bioanode including NAD⁺-dependent alcohol dehydrogenase and zinc complexed PNR. This bioanode was coupled to the cathode with Nafion^® 212 acting as the ion exchange membrane separator between the two compartments. The biobattery has an open circuit potential of 0.545(±0.009) V when first assembled and 0.053(±0.005) V when fully discharged. However, when fully charged, the biobattery has an open circuit potential of 1.263(±0.051) V, a maximum power density of 16.3(±4.03) μW cm⁻³ and a maximum current density of 221(±13.2) μA cm⁻³. The efficiency and stability of the biobattery were studied by cycling continuously at a discharging rate of 1 C and the results obtained showed reasonable stability over 50 cycles.     

Synthesis of Stable NAD+ Mimics as Inhibitors for the Legionella pneumophila Phosphoribosyl Ubiquitylating Enzyme SdeC

Stable NAD⁺ analogues carrying single atom substitutions in either the furanose ring or the nicotinamide part have proven their value as inhibitors for NAD⁺-consuming enzymes. To investigate the potential of such compounds to inhibit the adenosine diphosphate ribosyl (ADPr) transferase activity of the Legionella SdeC enzyme, we prepared three NAD⁺ analogues, namely carbanicotinamide adenosine dinucleotide (c-NAD⁺), thionicotinamide adenosine dinucleotide (S-NAD⁺) and benzamide adenosine dinucleotide (BAD). We optimized the chemical synthesis of thionicotinamide riboside and for the first time used an enzymatic approach to convert all three ribosides into the corresponding NAD⁺ mimics. We thus expanded the known scope of substrates for the NRK1/NMNAT1 enzyme combination by turning all three modified ribosides into NAD⁺ analogues in a scalable manner. We then compared the three NAD⁺ mimics side-by-side in a single assay for enzyme inhibition on Legionella effector enzyme SdeC. The class of SidE enzymes to which SdeC belongs was recently identified to be important in bacterial virulence, and we found SdeC to be inhibited by S-NAD⁺ and BAD with IC₅₀ values of 28 and 39 μM, respectively.     

Effect of Divalent Metal Ion on the Structure,Stability and Function of Klebsiella pneumoniae Nicotinate-Nucleotide Adenylyltransferase: Empirical and Computational Studies

The continuous threat of drug-resistant Klebsiella pneumoniae justifies identifying novel targets and developing effective antibacterial agents. A potential target is nicotinate nucleotide adenylyltransferase (NNAT), an indispensable enzyme in the biosynthesis of the cell-dependent metabolite, NAD⁺. NNAT catalyses the adenylation of nicotinamide/nicotinate mononucleotide (NMN/NaMN), using ATP to form nicotinamide/nicotinate adenine dinucleotide (NAD⁺/NaAD). In addition, it employs divalent cations for co-substrate binding and catalysis and has a preference for different divalent cations. Here, the biophysical structure of NNAT from K. pneumoniae (KpNNAT) and the impact of divalent cations on its activity, conformational stability and substrate-binding are described using experimental and computational approaches. The experimental study was executed using an enzyme-coupled assay, far-UV circular dichroism, extrinsic fluorescence spectroscopy, and thermal shift assays, alongside homology modelling, molecular docking, and molecular dynamic simulation. The structure of KpNNAT revealed a predominately α-helical secondary structure content and a binding site that is partially hydrophobic. Its substrates ATP and NMN share the same binding pocket with similar affinity and exhibit an energetically favourable binding. KpNNAT showed maximum activity and minimal conformational changes with Mg²⁺ as a cofactor compared to Zn²⁺, Cu²⁺ and Ni²⁺. Overall, ATP binding affects KpNNAT dynamics, and the dynamics of ATP binding depend on the presence and type of divalent cation. The data obtained from this study would serve as a basis for further evaluation towards designing structure-based inhibitors with therapeutic potential.     

Electrochemical processes in the photoelectrolysis of nicotinamide adenine dinucleotide on the chlorophyll electrodes

The photoelectrolysis, in which redox compounds are electrolysed on a pair of photo-excitable electrodes by supplying photo—energy in place of electric energy, has been performed. The photo—excitable electrodes were prepared by coating a platinum plate with a thin layer of a chlorophyll—quinone composite. These electrodes were called chlorophyll electrodes. The chlorophyll electrode of chlorophyll—naphthoquinone composite worked as a cathode and that of chlorophyll—anthrahydroquinone composite as an anode when they were illuminated. The chlorophyll electrode of chlorophyll—naphthoquinone composite was characterized by an electrochemical behavior of p-type semiconductor electrode. Reduction of nicotinamide adenine dinucleotide (NAD⁺) was carried out on the chlorophyll electrode under illumination at various controlled electrode potentials. NAD was reduced at extremely noble electrode potentials are compared with the reductive potential of NAD⁺ to NADH. Electron transfer accompanied with the photoelectrochemical reactions is discussed.     

The Effects of NAD+ on Apoptotic Neuronal Death and Mitochondrial Biogenesis and Function after Glutamate Excitotoxicity

NAD⁺ is an essential co-enzyme for cellular energy metabolism and is also involved as a substrate for many cellular enzymatic reactions. It has been shown that NAD⁺ has a beneficial effect on neuronal survival and brain injury in in vitro and in vivo ischemic models. However, the effect of NAD⁺ on mitochondrial biogenesis and function in ischemia has not been well investigated. In the present study, we used an in vitro glutamate excitotoxicity model of primary cultured cortical neurons to study the effect of NAD⁺ on apoptotic neuronal death and mitochondrial biogenesis and function. Our results show that supplementation of NAD⁺ could effectively reduce apoptotic neuronal death, and apoptotic inducing factor translocation after neurons were challenged with excitotoxic glutamate stimulation. Using different approaches including confocal imaging, mitochondrial DNA measurement and Western blot analysis of PGC-1 and NRF-1, we also found that NAD⁺ could significantly attenuate glutamate-induced mitochondrial fragmentation and the impairment of mitochondrial biogenesis. Furthermore, NAD⁺ treatment effectively inhibited mitochondrial membrane potential depolarization and NADH redistribution after excitotoxic glutamate stimulation. Taken together, our results demonstrated that NAD⁺ is capable of inhibiting apoptotic neuronal death after glutamate excitotoxicity via preserving mitochondrial biogenesis and integrity. Our findings provide insights into potential neuroprotective strategies in ischemic stroke.     

Selective Cardiomyocyte Oxidative Stress Leads to Bystander Senescence of Cardiac Stromal Cells

Accumulation of senescent cells in tissues during normal or accelerated aging has been shown to be detrimental and to favor the outcomes of age-related diseases such as heart failure (HF). We have previously shown that oxidative stress dependent on monoamine oxidase A (MAOA) activity in cardiomyocytes promotes mitochondrial damage, the formation of telomere-associated foci, senescence markers, and triggers systolic cardiac dysfunction in a model of transgenic mice overexpressing MAOA in cardiomyocytes (Tg MAOA). However, the impact of cardiomyocyte oxidative stress on the cardiac microenvironment in vivo is still unclear. Our results showed that systolic cardiac dysfunction in Tg MAOA mice was strongly correlated with oxidative stress induced premature senescence of cardiac stromal cells favoring the recruitment of CCR2⁺ monocytes and the installation of cardiac inflammation. Understanding the interplay between oxidative stress induced premature senescence and accelerated cardiac dysfunction will help to define new molecular pathways at the crossroad between cardiac dysfunction and accelerated aging, which could contribute to the increased susceptibility of the elderly to HF.     

Exploring the Ion Channel TRPV2 and Testicular Macrophages in Mouse Testis

The cation channel TRPV2 is known to be expressed by murine macrophages and is crucially involved in their functionality. Macrophages are frequent cells of the mouse testis, an immune-privileged and steroid-producing organ. TRPV2 expression by testicular macrophages and possible changes associated with age or inflammation have not been investigated yet. Therefore, we studied testes of young adult and old wild-type (WT) and AROM⁺ mice, i.e., transgenic mice overexpressing aromatase. In these animals, inflammatory changes are described in the testis, involving active macrophages, which increase with age. This is associated with impaired spermatogenesis and therefore AROM⁺ mice are a model for male infertility associated with sterile inflammation. In WT animals, testicular TRPV2 expression was mapped to interstitial CD206⁺ and peritubular MHC II⁺ macrophages, with higher levels in CD206⁺ cells. Expression levels of TRPV2 and most macrophage markers did not increase significantly in old mice, with the exception of CD206. As the number of TRPV2⁺ testicular macrophages was relatively small, their possible involvement in testicular functions and in aging in WT mice remains to be further studied. In AROM⁺ testis, TRPV2 was readily detected and levels increased significantly with age, together with macrophage markers and TNF-α. TRPV2 co-localized with F4/80 in macrophages and further studies showed that TRPV2 is mainly expressed by unusual CD206⁺MHC II⁺ macrophages, arising in the testis of these animals. Rescue experiments (aromatase inhibitor treatment and crossing with ERαKO mice) restored the testicular phenotype and also abolished the elevated expression of TRPV2, macrophage and inflammation markers. This suggests that TRPV2⁺ macrophages of the testis are part of an inflammatory cascade initiated by an altered sex hormone balance in AROM⁺ mice. The changes in testis are distinct from the described alterations in other organs of AROM⁺, such as prostate and spleen. When we monitored TRPV2 levels in another immune-privileged organ, namely the brain, we found that levels of TRPV2 were not elevated in AROM⁺ and remained stable during aging. In the adrenal, which similar to the testis produces steroids, we found slight, albeit not significant increases in TRPV2 in both AROM⁺ and WT mice, which were associated with age. Thus, the changes in the testis are specific for this organ.     

Synthesis of Nicotinamide Mononucleotide from Xylose via Coupling Engineered Escherichia coli and a Biocatalytic Cascade

β-Nicotinamide mononucleotide (NMN) has recently gained attention for a nutritional supplement because it is an intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD⁺). In this study, we developed NMN synthesis by coupling two modules. The first module is to culture E. coli MG1655 ▵tktA ▵tktB ▵ptsG to metabolize xylose to generate D-ribose in the medium. The supernatant containing D-ribose was applied in the second module which is composed of EcRbsK-EcPRPS-CpNAMPT reaction to synthesize NMN, that requires additional enzymes of CHU0107 and EcPPase to remove feedback inhibitors ADP and pyrophosphate. The second module can be rapidly optimized by comparing NMN production determined by the cyanide assay. Finally, 10 mL optimal biocascade reaction generated NMN with a good yield of 84 % from 1 mM D-ribose supplied from the supernatant of E. coli MG1655 ▵tktA ▵tktB ▵ptsG. Our results can further guide researchers to metabolically engineer E. coli for NMN synthesis.