Editorial: NAD+ metabolism as a novel target against infection–Volume II

Fundamental discoveries in the history of science, including research from four Nobel laureates, have identified the pyrimidine nucleotide, nicotinamide adenine dinucleotide (NAD+) as an essential metabolite and the most important cross-kingdom electron carrier. While a wealth of scientific studies in the last century have highlighted the important role of NAD+ in cellular energy generation and redox biology, recent studies have provided evidence for the paramount importance of the non-redox functions of NAD+. Alteration in intracellular NAD+ concentrations is critical in several pathological conditions, including but not limited to neurodegenerative diseases, cancer, diabetes, ischemia injury, dysmetabolic diseases, and inflammatory disorders. Recent advances in our understanding of the biological functions of NAD+-consuming enzymes and the role of NAD+ biosynthetic routes have shown that targeting NAD+ metabolism has very promising potential for therapeutic treatment of infectious diseases. Bacterial resistance to antibiotics is considered one of the greatest threats to human health. Cases of antibiotic resistance are constantly emerging, and the time needed for bacteria to become resistant to newly introduced antibiotics, is getting shorter. Therefore, it is crucial to identify antimicrobials with new mechanism of action to use as therapeutic tools. In this context, modulation of host NAD+ metabolism by the activation or inhibition of key enzymes could influence NAD+ signaling pathways in pathogens and their ability to colonize host cells. Further, the discovery of the enzymes involved in NAD+ turnover in the host during infection could increase our knowledge of bacterial pathogenesis and may be relevant for the development of targeted drug therapies against antibiotic resistant bacteria. However, detailed mechanisms by which NAD+ acts as a regulator and how we can target NAD+ metabolism during microbial infections is still poorly understood

Enzymatic and Chemical Syntheses of Vacor Analogs of Nicotinamide Riboside, NMN and NAD

It has recently been demonstrated that the rat poison vacor interferes with mammalian NAD metabolism, because it acts as a nicotinamide analog and is converted by enzymes of the NAD salvage pathway. Thereby, vacor is transformed into the NAD analog vacor adenine dinucleotide (VAD), a molecule that causes cell toxicity. Therefore, vacor may potentially be exploited to kill cancer cells. In this study, we have developed efficient enzymatic and chemical procedures to produce vacor analogs of NAD and nicotinamide riboside (NR). VAD was readily generated by a base-exchange reaction, replacing the nicotinamide moiety of NAD by vacor, catalyzed by Aplysia californica ADP ribosyl cyclase. Additionally, we present the chemical synthesis of the nucleoside version of vacor, vacor riboside (VR). Similar to the physiological NAD precursor, NR, VR was converted to the corresponding mononucleotide (VMN) by nicotinamide riboside kinases (NRKs). This conversion is quantitative and very efficient. Consequently, phosphorylation of VR by NRKs represents a valuable alternative to produce the vacor analog of NMN, compared to its generation from vacor by nicotinamide phosphoribosyltransferase (NamPT).publishedVersio

Impact of dietary phosphorous in diploid and triploid Atlantic salmon (Salmo salar L.) with reference to early skeletal development in freshwater

In order to assess the effect of dietary phosphorus (P) in reducing vertebral malformations and improving freshwater (FW) performance in triploid Atlantic salmon (Salmo salar), both triploid and diploid Atlantic salmon were fed three different dietary P inclusion levels (low: 4.9, medium: 7.7, and high: 9.7 g available P kg−1) from first feeding until smolt. Somatic and skeletal response was assessed at fry (~0.5 g), parr (~5 g) and smolt (~45 g) stages. Triploid parr initially grew faster on the high P diet, while groups fed low P resulted in a significantly higher weight at smolt. Image analysis of double stained Alcian blue and Alizarin red S fry revealed that low P fed triploid fish presented less well mineralised vertebrae, and significantly more malformed vertebrae in both parr and smolt stages following x-ray radiographic assessment. Triploid parr fed high and medium P had similar numbers of malformed vertebrae relative to their diploid counterparts but greater numbers than at smolt. Low P fed triploids had the highest prevalence of jaw and vertebral malformations as well as the highest number of deformed vertebrae in the central caudal vertebral region, which was more pronounced at parr than at smolt. Shorter vertebrae dorso-ventral lengths were observed throughout the spinal column (R1–R4) in parr fed low P and only in the caudal region (R3) at smolt. In parr, both ploidies showed reduced phosphate homeostasis protein fgf23 gene expression in vertebrae when fed low P diets, while triploids showed greater down-regulation of osteogenic factors (alp, opn and igf1r) between diets relative to diploids, suggesting possible greater active suppression of mineralisation and reduced osteogenic potential in triploids. No effects of diet or ploidy on gene expression were evident at smolt. Comparisons between development stages suggest early P supplementation in triploids is crucial for skeletal development. Ultimately, reducing vertebral deformities observed at smolt with higher P supplementation in triploids could contribute towards improving skeletal performance and welfare of the stocks in the marine phase

Solvent-controlled regioselective protection of allyl-4,6-benzylidene glucopyranosides

Abstract We wish to report a simple synthetic procedure, which permits the regiospecific mono-acylation, alkylation and silylation at the 2-position of allyl 4,6-O-benzylidene α-D-glucopyranoside in high yields and which does not require the use of catalysts

Dietary phosphorous and protein supplementation enhances seawater growth and reduces severity of vertebral malformation in triploid Atlantic salmon (Salmo salar L.)

Diploid (2N) and triploid (3N) sibling post-smolts were divided between six sea pens and fed: a standard commercial nutrient package diet (2×2N SP, 2×3N SP), or an iso-energetic nutrient boosted (higher dietary protein and phosphorous) package (2×3N BP) until market size. 3N groups initially grew significantly faster than 2N, and by harvest, 3N BP weighed significantly more (3210±87g) than 2N SP or 3N SP (3007±64g; 2965±88g), while there was no significant difference in weight between ploidy in SP diet. Higher visible vertebral (9.6±0.4%) and jaw deformities (10.6±1.2%) were observed in 3N compared to 2N (0.9±0.1%; 1.3±0.5%). However, x-ray radiography revealed that 3N BP and 2N SP had comparable levels of severely affected individuals at time of sea transfer, while 3N SP showed a 3 fold increase in the severity of malformed individuals. The tail region (R3) in 3N SP fish had both the lowest vertebral strength and stiffness and the highest number of deformed vertebrae. Fillet quality attributes were comparable between diet and ploidy. These findings show that triploid growth rate can be sustained until harvest throughout the seawater phase, and more importantly the progression of spinal deformity beyond that at sea transfer can be stabilised by increasing dietary P during the marine phase

Formulation of Antimicrobial Tobramycin Loaded PLGA Nanoparticles via Complexation with AOT

Tobramycin is a potent antimicrobial aminoglycoside and its effective delivery by encapsulation within nanoparticle carriers could increase its activity against infections through a combination of sustained release and enhanced uptake. Effective antimicrobial therapy against a clinically relevant model bacteria (Pseudomonas aeruginosa) requires sufficient levels of therapeutic drug to maintain a drug concentration above the microbial inhibitory concentration (MIC) of the bacteria. Previous studies have shown that loading of aminoglycoside drugs in poly(lactic-co-glycolic) acid (PLGA)-based delivery systems is generally poor due to weak interactions between the drug and the polymer. The formation of complexes of tobramycin with dioctylsulfosuccinate (AOT) allows the effective loading of the drug in PLGA-nanoparticles and such nanoparticles can effectively deliver the antimicrobial aminoglycoside with retention of tobramycin antibacterial function

Equilibrative Nucleoside Transporters Mediate the Import of Nicotinamide Riboside and Nicotinic Acid Riboside into Human Cells

Nicotinamide riboside (NR), a new form of vitamin B3, is an effective precursor of nicotinamide adenine dinucleotide (NAD+) in human and animal cells. The introduction of NR into the body effectively increases the level of intracellular NAD+ and thereby restores physiological functions that are weakened or lost in experimental models of aging and various pathologies. Despite the active use of NR in applied biomedicine, the mechanism of its transport into mammalian cells is currently not understood. In this study, we used overexpression of proteins in HEK293 cells, and metabolite detection by NMR, to show that extracellular NR can be imported into cells by members of the equilibrative nucleoside transporter (ENT) family ENT1, ENT2, and ENT4. After being imported into cells, NR is readily metabolized resulting in Nam generation. Moreover, the same ENT-dependent mechanism can be used to import the deamidated form of NR, nicotinic acid riboside (NAR). However, NAR uptake into HEK293 cells required the stimulation of its active utilization in the cytosol such as phosphorylation by NR kinase. On the other hand, we did not detect any NR uptake mediated by the concentrative nucleoside transporters (CNT) CNT1, CNT2, or CNT3, while overexpression of CNT3, but not CNT1 or CNT2, moderately stimulated NAR utilization by HEK293 cells.publishedVersio

Metabolomic profiling to dissect the role of visceral fat in cardiometabolic health

OBJECTIVE: Abdominal obesity is associated with increased risk of type 2 diabetes (T2D) and cardiovascular disease. The aim of this study was to assess whether metabolomic markers of T2D and blood pressure (BP) act on these traits via visceral fat (VF) mass. METHODS: Metabolomic profiling of 280 fasting plasma metabolites was conducted on 2,401 women from TwinsUK. The overlap was assessed between published metabolites associated with T2D, insulin resistance, or BP and those that were identified to be associated with VF (after adjustment for covariates) measured by dual‐energy X‐ray absorptiometry. RESULTS: In addition to glucose, six metabolites were strongly associated with both VF mass and T2D: lactate and branched‐chain amino acids, all of them related to metabolism and the tricarboxylic acid cycle; on average, 38.5% of their association with insulin resistance was mediated by their association with VF mass. Five metabolites were associated with BP and VF mass including the inflammation‐associated peptide HWESASXX, the steroid hormone androstenedione, lactate, and palmitate. On average, 29% of their effect on BP was mediated by their association with VF mass. CONCLUSIONS: Little overlap was found between the metabolites associated with BP and those associated with insulin resistance via VF mass

Metabolomic Profiling of Long-Term Weight Change:Role of Oxidative Stress and Urate Levels in Weight Gain

OBJECTIVE: To investigate the association between long-term weight change and blood metabolites. METHODS: Change in BMI over 8.6 ± 3.79 years was assessed in 3,176 females from the TwinsUK cohort (age range: 18.3-79.6, baseline BMI: 25.11 ± 4.35) measured for 280 metabolites at follow-up. Statistically significant metabolites (adjusting for covariates) were included in a multivariable least absolute shrinkage and selection operator (LASSO) model. Findings were replicated in the Cooperative Health Research in the Region of Augsburg (KORA) study (n = 1,760; age range: 25-70, baseline BMI: 27.72 ± 4.53). The study examined whether the metabolites identified could prospectively predict weight change in KORA and in the Prostate, Lung, Colorectal, and Ovarian Cancer (PLCO) study (n = 471; age range: 55-74, baseline BMI: 27.24 ± 5.37). RESULTS: Thirty metabolites were significantly associated with change in BMI per year in TwinsUK using Bonferroni correction. Four were independently associated with weight change in the multivariable LASSO model and replicated in KORA: namely, urate (meta-analysis β [95% CI] = 0.05 [0.040 to 0.063]; P = 1.37 × 10-19 ), gamma-glutamyl valine (β [95% CI] = 0.06 [0.046 to 0.070]; P = 1.23 × 10-20 ), butyrylcarnitine (β [95% CI] = 0.04 [0.028 to 0.051]; P = 6.72 × 10-12 ), and 3-phenylpropionate (β [95% CI] = -0.03 [-0.041 to -0.019]; P = 9.8 × 10-8 ), all involved in oxidative stress. Higher levels of urate at baseline were associated with weight gain in KORA and PLCO. CONCLUSIONS: Metabolites linked to higher oxidative stress are associated with increased long-term weight gain

Purine nucleoside phosphorylase controls nicotinamide riboside metabolism in mammalian cells

Nicotinamide riboside (NR) is an effective precursor of nicotinamide adenine dinucleotide (NAD) in human and animal cells. NR supplementation can increase the level of NAD in various tissues and thereby improve physiological functions that are weakened or lost in experimental models of aging or various human pathologies. However, there are also reports questioning the efficacy of NR supplementation. Indeed, the mechanisms of its utilization by cells are not fully understood. Herein, we investigated the role of purine nucleoside phosphorylase (PNP) in NR metabolism in mammalian cells. Using both PNP overexpression and genetic knockout, we show that after being imported into cells by members of the equilibrative nucleoside transporter family, NR is predominantly metabolized by PNP, resulting in nicotinamide (Nam) accumulation. Intracellular cleavage of NR to Nam is prevented by the potent PNP inhibitor Immucillin H in various types of mammalian cells. In turn, suppression of PNP activity potentiates NAD synthesis from NR. Combining pharmacological inhibition of PNP with NR supplementation in mice, we demonstrate that the cleavage of the riboside to Nam is strongly diminished, maintaining high levels of NR in blood, kidney, and liver. Moreover, we show that PNP inhibition stimulates Nam mononucleotide and NAD+ synthesis from NR in vivo, in particular, in the kidney. Thus, we establish PNP as a major regulator of NR metabolism in mammals and provide evidence that the health benefits of NR supplementation could be greatly enhanced by concomitant downregulation of PNP activity.publishedVersio