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Charles Brenner - One of the best experts on this subject based on the ideXlab platform.
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Nrt1 and Tna1-Independent Export of NAD+ Precursor Vitamins Promotes NAD+ Homeostasis and Allows Engineering of Vitamin Production
PloS one, 2011Co-Authors: Peter Belenky, Katrina L. Bogan, Charles R. Evans, Rebecca C. Stebbins, Charles BrennerAbstract:NAD(+) is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+) consuming enzymes. NAD(+) biosynthesis is required for two different regimens that extend lifespan in yeast. NAD(+) is synthesized from tryptophan and the three vitamin precursors of NAD(+): nicotinic acid, Nicotinamide and Nicotinamide riboside. Supplementation of yeast cells with NAD(+) precursors increases intracellular NAD(+) levels and extends replicative lifespan. Here we show that both Nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the Nicotinamide riboside transporter, Nrt1, leads to increased export of Nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular Nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular Nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like Nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD(+) metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD(+) metabolism by balancing import and export of NAD(+) precursor vitamins.
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Nrt1 and Tna1-Independent Export of NAD + Precursor Vitamins Promotes NAD + Homeostasis and Allows Engineering of Vitamin Production
2011Co-Authors: Peter Belenky, Katrina L. Bogan, Charles R. Evans, Rebecca Stebbins, Charles BrennerAbstract:NAD + is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD + consuming enzymes. NAD + biosynthesis is required for two different regimens that extend lifespan in yeast. NAD + is synthesized from tryptophan and the three vitamin precursors of NAD +: nicotinic acid, Nicotinamide and Nicotinamide riboside. Supplementation of yeast cells with NAD + precursors increases intracellular NAD + levels and extends replicative lifespan. Here we show that both Nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the Nicotinamide riboside transporter, Nrt1, leads to increased export of Nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular Nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular Nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like Nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD + metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD + metabolism by balancing import and export of NAD
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Nrt1 and Tna1-independent export of NAD+ precursor vitamins promotes NAD+ homeostasis and allows engineering of vitamin production. PLoS ONE6:e19710. doi: 10.1371/journal.pone.0019710
2011Co-Authors: Peter Belenky, Katrina L. Bogan, Charles R. Evans, Rebecca Stebbins, Charles BrennerAbstract:NAD+ is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD+ consuming enzymes. NAD+ biosynthesis is required for two different regimens that extend lifespan in yeast. NAD+ is synthesized from tryptophan and the three vitamin precursors of NAD+: nicotinic acid, Nicotinamide and Nicotinamide riboside. Supplementation of yeast cells with NAD+ precursors increases intracellular NAD+ levels and extends replicative lifespan. Here we show that both Nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the Nicotinamide riboside transporter, Nrt1, leads to increased export of Nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular Nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular Nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like Nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD+ metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD+ metabolism by balancing import and export of NAD
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identification of isn1 and sdt1 as glucose and vitamin regulated Nicotinamide mononucleotide and nicotinic acid mononucleotide 5 nucleotidases responsible for production of Nicotinamide riboside and nicotinic acid riboside
Journal of Biological Chemistry, 2009Co-Authors: Katrina L. Bogan, Peter Belenky, Charles R. Evans, Peng Song, Charles F. Burant, Robert T. Kennedy, Charles BrennerAbstract:Recently, we discovered that Nicotinamide riboside and nicotinic acid riboside are biosynthetic precursors of NAD(+), which are utilized through two pathways consisting of distinct enzymes. In addition, we have shown that exogenously supplied Nicotinamide riboside is imported into yeast cells by a dedicated transporter, and it extends replicative lifespan on high glucose medium. Here, we show that Nicotinamide riboside and nicotinic acid riboside are authentic intracellular metabolites in yeast. Secreted Nicotinamide riboside was detected with a biological assay, and intracellular levels of Nicotinamide riboside, nicotinic acid riboside, and other NAD(+) metabolites were determined by a liquid chromatography-mass spectrometry method. A biochemical genomic screen indicated that three yeast enzymes possess Nicotinamide mononucleotide 5'-nucleotidase activity in vitro. Metabolic profiling of knock-out mutants established that Isn1 and Sdt1 are responsible for production of Nicotinamide riboside and nicotinic acid riboside in cells. Isn1, initially classified as an IMP-specific 5'-nucleotidase, and Sdt1, initially classified as a pyrimidine 5'-nucleotidase, are additionally responsible for dephosphorylation of pyridine mononucleotides. Sdt1 overexpression is growth-inhibitory to cells in a manner that depends on its active site and correlates with reduced cellular NAD(+). Expression of Isn1 protein is positively regulated by the availability of nicotinic acid and glucose. These results reveal unanticipated and highly regulated steps in NAD(+) metabolism.
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Nicotinamide riboside and nicotinic acid riboside salvage in fungi and mammals. Quantitative basis for Urh1 and purine nucleoside phosphorylase function in NAD+ metabolism.
The Journal of biological chemistry, 2008Co-Authors: Peter Belenky, Kathryn C. Christensen, Francesca S. Gazzaniga, Alexandre A. Pletnev, Charles BrennerAbstract:NAD+ is a co-enzyme for hydride transfer enzymes and an essential substrate of ADP-ribose transfer enzymes and sirtuins, the type III protein lysine deacetylases related to yeast Sir2. Supplementation of yeast cells with Nicotinamide riboside extends replicative lifespan and increases Sir2-dependent gene silencing by virtue of increasing net NAD+ synthesis. Nicotinamide riboside elevates NAD+ levels via the Nicotinamide riboside kinase pathway and by a pathway initiated by splitting the nucleoside into a Nicotinamide base followed by Nicotinamide salvage. Genetic evidence has established that uridine hydrolase, purine nucleoside phosphorylase, and methylthioadenosine phosphorylase are required for Nrk-independent utilization of Nicotinamide riboside in yeast. Here we show that mammalian purine nucleoside phosphorylase but not methylthioadenosine phosphorylase is responsible for mammalian Nicotinamide riboside kinase-independent Nicotinamide riboside utilization. We demonstrate that so-called uridine hydrolase is 100-fold more active as a Nicotinamide riboside hydrolase than as a uridine hydrolase and that uridine hydrolase and mammalian purine nucleoside phosphorylase cleave nicotinic acid riboside, whereas the yeast phosphorylase has little activity on nicotinic acid riboside. Finally, we show that yeast nicotinic acid riboside utilization largely depends on uridine hydrolase and Nicotinamide riboside kinase and that nicotinic acid riboside bioavailability is increased by ester modification.
Liqiang Chen - One of the best experts on this subject based on the ideXlab platform.
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5 3 amidobenzyl oxy Nicotinamides as sirtuin 2 inhibitors
Journal of Medicinal Chemistry, 2016Co-Authors: Teng Ai, Swati S More, Daniel J Wilson, Liqiang ChenAbstract:Derived from our previously reported human sirtuin 2 (SIRT2) inhibitors that were based on a 5-aminonaphthalen-1-yloxy Nicotinamide core structure, 5-((3-amidobenzyl)oxy)Nicotinamides offered excellent activity against SIRT2 and high isozyme selectivity over SIRT1 and SIRT3. Selected compounds also exhibited generally favorable in vitro absorption, distribution, metabolism, and excretion properties. Kinetic studies revealed that a representative SIRT2 inhibitor acted competitively against both NAD+ and the peptide substrate, an inhibitory modality that was supported by our computational study. More importantly, two selected compounds exhibited significant protection against α-synuclein aggregation-induced cytotoxicity in SH-SY5Y cells. Therefore, 5-((3-amidobenzyl)oxy)Nicotinamides represent a new class of SIRT2 inhibitors that are attractive candidates for further lead optimization in our continued effort to explore selective inhibition of SIRT2 as a potential therapy for Parkinson’s disease.
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discovery of potent and selective sirtuin 2 sirt2 inhibitors using a fragment based approach
Journal of Medicinal Chemistry, 2014Co-Authors: Zeeshan Kamal, Teng Ai, Swati S More, Yanli Xu, Daniel J Wilson, Liqiang ChenAbstract:Sirtuin 2 (SIRT2) is one of the sirtuins, a family of NAD+-dependent deacetylases that act on a variety of histone and non-histone substrates. Accumulating biological functions and potential therapeutic applications have drawn interest in the discovery and development of SIRT2 inhibitors. Herein we report our discovery of novel SIRT2 inhibitors using a fragment-based approach. Inspired by the purported close binding proximity of suramin and Nicotinamide, we prepared two sets of fragments, namely, the naphthylamide sulfonic acids and the naphthalene–benzamides and −Nicotinamides. Biochemical evaluation of these two series provided structure–activity relationship (SAR) information, which led to the design of (5-benzamidonaphthalen-1/2-yloxy)Nicotinamide derivatives. Among these inhibitors, one compound exhibited high anti-SIRT2 activity (48 nM) and excellent selectivity for SIRT2 over SIRT1 and SIRT3. In vitro, it also increased the acetylation level of α-tubulin, a well-established SIRT2 substrate, in both...
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discovery of potent and selective sirtuin 2 sirt2 inhibitors using a fragment based approach
Journal of Medicinal Chemistry, 2014Co-Authors: Huaqing Cui, Swati S More, Zeeshan Kamal, Daniel J Wilson, Liqiang ChenAbstract:Sirtuin 2 (SIRT2) is one of the sirtuins, a family of NAD(+)-dependent deacetylases that act on a variety of histone and non-histone substrates. Accumulating biological functions and potential therapeutic applications have drawn interest in the discovery and development of SIRT2 inhibitors. Herein we report our discovery of novel SIRT2 inhibitors using a fragment-based approach. Inspired by the purported close binding proximity of suramin and Nicotinamide, we prepared two sets of fragments, namely, the naphthylamide sulfonic acids and the naphthalene-benzamides and -Nicotinamides. Biochemical evaluation of these two series provided structure-activity relationship (SAR) information, which led to the design of (5-benzamidonaphthalen-1/2-yloxy)Nicotinamide derivatives. Among these inhibitors, one compound exhibited high anti-SIRT2 activity (48 nM) and excellent selectivity for SIRT2 over SIRT1 and SIRT3. In vitro, it also increased the acetylation level of α-tubulin, a well-established SIRT2 substrate, in both concentration- and time-dependent manners. Further kinetic studies revealed that this compound behaves as a competitive inhibitor against the peptide substrate and most likely as a noncompetitive inhibitor against NAD(+). Taken together, these results indicate that we have discovered a potent and selective SIRT2 inhibitor whose novel structure merits further exploration.
David J Chaplin - One of the best experts on this subject based on the ideXlab platform.
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further evaluation of Nicotinamide and carbogen as a strategy to reoxygenate hypoxic cells in vivo importance of Nicotinamide dose and pre irradiation breathing time
British Journal of Cancer, 1993Co-Authors: David J Chaplin, Michael R. Horsman, Dietmar W. SiemannAbstract:The combination of Nicotinamide and carbogen breathing is awaiting clinical evaluation as a strategy to overcome tumour hypoxia and thus enhance radiation response. We have continued our evaluation of this approach in the murine SCCVII tumour with the aim of determining the importance of Nicotinamide dose and the pre-irradiation breathing time (PIBT) for carbogen. For carbogen breathing alone maximal enhancement of radiation response was observed with PIBT's of between 5 and 30 min. When Nicotinamide (1,000 mg kg-1 IP) was administered 60 min prior to irradiation little or no variation in radiation response was observed for all the PIBT's examined (5-90 min). Indeed at all PIBT's the cell survival obtained for the carbogen Nicotinamide and radiation combination was indistinguishable from that expected for a fully aerobic response. For PIBT's of 15 and 60 min we examined the influence of Nicotinamide doses between 50 and 1,000 mg kg-1. Significant radiosensitizing effects were observed for all Nicotinamide doses tested above 50 mg kg-1. Moreover for doses of 250 mg kg-1 and above the cell survival data was consistent with that expected for a fully aerobic response. No additional benefit accrued from raising the Nicotinamide dose above 250 mg kg-1. These results indicate that significant radiosensitization may be expected even with clinically achievable Nicotinamide doses when it is combined with carbogen breathing. Furthermore, the use of Nicotinamide may reduce the critical importance of PIBT on the radiosensitization observed with carbogen.
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biochemical and physiological changes induced by Nicotinamide in a c3h mouse mammary carcinoma and cdf1 mice
International Journal of Radiation Oncology Biology Physics, 1992Co-Authors: Michael R. Horsman, David J Chaplin, Paul E G Kristjansen, Masao Mizuno, Kent L Christensen, Bjorn Quistorff, Jens OvergaardAbstract:Abstract We have continued our investigation into the mechanism by which Nicotinamide can enhance radiation damage in tumors, using a C3H mouse mammary carcinoma grown in CDF1 mice. Biochemical analysis of tumor extracts showed that Nicotinamide (1000 mg/kg; i.p.) increased the ATP/Pi and ATP/ADP + AMP ratios. This change in metabolic activity was consistent with Nicotinamide increasing tumor oxygenation. Moreover, the greatest effect occurred 0.5–2.5 hr after drug injection, a time at which radiosensitization by Nicotinamide in this tumor had previously been shown to be maximal. These changes were observed without any apparent modification in tumor blood perfusion, measured using the 86-RbCl uptake procedure, and occurred despite Nicotinamide producing a 50% decrease in mean arterial blood pressure, estimated directly by a carotid cannulation technique.
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Nicotinamide fluosol da and carbogen a strategy to reoxygenate acutely and chronically hypoxic cells in vivo
British Journal of Cancer, 1991Co-Authors: David J Chaplin, Michael R. Horsman, D S AokiAbstract:The effect of Nicotinamide and/or treatment with Fluosol DA and Carbogen breathing on the radiation response of 500-750 mg SCCVII and KHT tumours has been evaluated. Pretreatment with Fluosol DA/Carbogen or Nicotinamide resulted in relatively modest enhancements of radiation damage with enhancement factors of 1.1 and 1.3 being observed using an in vivo/in vitro clonogenic end-point. A combination of Nicotinamide and Fluosol DA/Carbogen resulted in a larger enhancement factor of 1.6 over the radiation dose ranges studied. These modification factors reflect a value close to that expected for a fully aerobic response in this survival range. Growth delay studies in the SCCVII tumour provided similar results. Using a recently developed fluorescence activated cell sorting technique, which utilizes the in vivo pharmacokinetic and DNA binding properties of the bisbenzamide stain Hoechst 33342, the effect of Nicotinamide and/or Fluosol DA/Carbogen schedules on the occurrence of acute hypoxia was assessed. The results clearly show that Nicotinamide significantly reduces the amount of 'acute hypoxia', but has a lesser effect on 'chronic' hypoxic cells. However, combinations of Nicotinamide and Fluosol DA/Carbogen significantly increase the response of both 'acutely' and 'chronically hypoxic' cells. The results provide evidence that a combination of Nicotinamide and Fluosol DA/Carbogen can provide an effective way of reoxygenating both acutely and chronically hypoxic cells.
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Nicotinamide, Fluosol DA and carbogen: a strategy to reoxygenate acutely and chronically hypoxic cells in vivo
1991Co-Authors: David J Chaplin, Michael R. Horsman, D S AokiAbstract:Summary The effect of Nicotinamide and/or treatment with Fluosol DA and Carbogen breathing on the radiation response of 500-750 mg SCCVII and KHT tumours has been evaluated. Pretreatment with Fluosol DA/Carbogen or Nicotinamide resulted in relatively modest enhancements of radiation damage with enhancement factors of 1.1 and 1.3 being observed using an in vivo/in vitro clonogenic end-point. A combination of Nicotinamide and Fluosol DA/Carbogen resulted in a larger enhancement factor of 1.6 over the radiation dose ranges studied. These modification factors reflect a value close to that expected for a fully aerobic response in this survival range. Growth delay studies in the SCCVII tumour provided similar results. Using a recently developed fluorescence activated cell sorting technique, which utilizes the in vivo pharmacokinetic and DNA binding properties of the bisbenzamide stain Hoechst 33342, the effect of Nicotinamide and/or Fluosol DA/Carbogen schedules on the occurrence of acute hypoxia was assessed. The results clearly show that Nicotinamide significantly reduces the amount of 'acute hypoxia', but has a lesser effect on 'chronic ' hypoxic cells. However, combinations of Nicotinamide and Fluosol DA/Carbogen significantly increase the response of both 'acutely ' and 'chronically hypoxic ' cells. The results provide evidence that a combination of Nicotinamide and Fluosol DA/Carbogen can provide an effective way of reoxygenating both acutely and chronically hypoxic cells. Overcoming the problem of radiation resistant hypoxic cells continues to be a major focus of interest in radiation biolog
Keith R Martin - One of the best experts on this subject based on the ideXlab platform.
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improvement in inner retinal function in glaucoma with Nicotinamide vitamin b3 supplementation a crossover randomized clinical trial
Clinical and Experimental Ophthalmology, 2020Co-Authors: Flora Hui, Ia Trounce, Jessica Tang, Peter A Williams, Myra B Mcguinness, Xavier Hadoux, Robert J Casson, Michael Coote, Keith R MartinAbstract:Importance Retinal ganglion cells endure significant metabolic stress in glaucoma but maintain capacity to recover function. Nicotinamide, a precursor of NAD+ , is low in serum of glaucoma patients and its supplementation provides robust protection of retinal ganglion cells in preclinical models. However, the potential of Nicotinamide in human glaucoma is unknown. Background To examine the effects of Nicotinamide on inner retinal function in glaucoma, in participants receiving concurrent glaucoma therapy. Design Crossover, double-masked, randomized clinical trial. Participants recruited from two tertiary care centres. Participants Fifty-seven participants, diagnosed and treated for glaucoma. Methods Participants received oral placebo or Nicotinamide and reviewed six-weekly. Participants commenced 6 weeks of 1.5 g/day then 6 weeks of 3.0 g/day followed by crossover without washout. Visual function measured using electroretinography and perimetry. Main outcome measures Change in inner retinal function, determined by photopic negative response (PhNR) parameters: saturated PhNR amplitude (Vmax), ratio of PhNR/b-wave amplitude (Vmax ratio). Results PhNR Vmax improved beyond 95% coefficient of repeatability in 23% of participants following Nicotinamide vs 9% on placebo. Overall, Vmax improved by 14.8% [95% CI: 2.8%, 26.9%], (P = .02) on Nicotinamide and 5.2% [-4.2%, 14.6%], (P = .27) on placebo. Vmax ratio improved by 12.6% [5.0%, 20.2%], (P = .002) following Nicotinamide, 3.6% [-3.4%, 10.5%], (P = .30) on placebo. A trend for improved visual field mean deviation was observed with 27% improving ≥1 dB on Nicotinamide and fewer deteriorating (4%) compared to placebo (P = .02). Conclusions Nicotinamide supplementation can improve inner retinal function in glaucoma. Further studies underway to elucidate the effects of long-term Nicotinamide supplementation.
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improvement in inner retinal function in glaucoma in response to Nicotinamide vitamin b3 supplementation a crossover randomized clinical trial
medRxiv, 2020Co-Authors: Flora Hui, Ia Trounce, Jessica Tang, Peter A Williams, Myra B Mcguinness, Xavier Hadoux, Robert J Casson, Michael Coote, Keith R MartinAbstract:Importance: Retinal ganglion cells endure significant metabolic stress with ageing and glaucoma-related stressors. Injured cells require increased energy for repair but maintain capacity to recover function despite periods of functional loss. Nicotinamide, a precursor of redox co-factor and metabolite, NAD+, is low in serum of patients with primary open-angle glaucoma and its supplementation provides robust protection of retinal ganglion cells by targeting mitochondrial health in glaucoma models. However, the potential of Nicotinamide to improve retinal ganglion cell function in humans with glaucoma is yet unknown. Objective: To determine whether Nicotinamide supplementation taken in conjunction with conventional IOP-lowering therapy leads to early improvement in retinal ganglion cell function in people with glaucoma. Design: Crossover, double-masked, randomized clinical trial conducted between October 2017 to January 2019. Setting: Study participants recruited from two tertiary care centers in Melbourne, Australia. Participants: Adults diagnosed and treated for primary glaucoma. Ninety-four participants assessed for study eligibility. Intervention: Participants randomized to first receive oral placebo or Nicotinamide and reviewed six-weekly. Accelerated dosing method utilized; participants commenced 6-week course of 1.5 grams/day followed by 6 weeks of 3.0 grams/day. After 12 weeks, participants crossed over to other intervention for 12 weeks without washout. At each visit, visual function measured using full-field flash electroretinography and white-on-white perimetry. Main outcome measures: Primary endpoint was change in inner retinal function determined a-priori as change in photopic negative response (PhNR) parameters: saturated PhNR amplitude (Vmax), ratio of PhNR/b-wave amplitude (Vmax ratio). Results: Fifty-seven participants (65.5±10.0 years, 39% female) enrolled. PhNR Vmax improved beyond 95% coefficient of repeatability (COR) in 23% of participants following 12 weeks of Nicotinamide versus 9% on placebo. Conversely, PhNR Vmax deteriorated in 9% on placebo and 7% on Nicotinamide. Overall, Vmax improved by 14.8% [95% CI: 2.8%, 26.9%], (p=0.02) on Nicotinamide and 5.2% [-4.2%, 14.6%], (p=0.27) on placebo. Vmax ratio improved on average by 12.6% [5.0%, 20.2%], (p=0.002) following Nicotinamide and 3.6% [-3.4%, 10.5%], (p=0.30) on placebo. A concomitant trend for improved visual field mean deviation was observed with 27% improving ≥1dB on Nicotinamide and fewer deteriorating ≥1dB (4%) compared to placebo (p=0.02). Moderate correlation was observed between PhNR and visual field change with treatment. Participants demonstrated excellent treatment adherence rates (>94%) and Nicotinamide was well tolerated with minimal side effects. Conclusions and Relevance: Nicotinamide supplementation can improve inner retinal function in patients receiving concurrent IOP-lowering glaucoma therapy. Further studies are underway to elucidate the effects of long-term Nicotinamide supplementation on glaucoma progression.
Peter Belenky - One of the best experts on this subject based on the ideXlab platform.
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Nrt1 and Tna1-Independent Export of NAD+ Precursor Vitamins Promotes NAD+ Homeostasis and Allows Engineering of Vitamin Production
PloS one, 2011Co-Authors: Peter Belenky, Katrina L. Bogan, Charles R. Evans, Rebecca C. Stebbins, Charles BrennerAbstract:NAD(+) is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+) consuming enzymes. NAD(+) biosynthesis is required for two different regimens that extend lifespan in yeast. NAD(+) is synthesized from tryptophan and the three vitamin precursors of NAD(+): nicotinic acid, Nicotinamide and Nicotinamide riboside. Supplementation of yeast cells with NAD(+) precursors increases intracellular NAD(+) levels and extends replicative lifespan. Here we show that both Nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the Nicotinamide riboside transporter, Nrt1, leads to increased export of Nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular Nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular Nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like Nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD(+) metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD(+) metabolism by balancing import and export of NAD(+) precursor vitamins.
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Nrt1 and Tna1-Independent Export of NAD + Precursor Vitamins Promotes NAD + Homeostasis and Allows Engineering of Vitamin Production
2011Co-Authors: Peter Belenky, Katrina L. Bogan, Charles R. Evans, Rebecca Stebbins, Charles BrennerAbstract:NAD + is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD + consuming enzymes. NAD + biosynthesis is required for two different regimens that extend lifespan in yeast. NAD + is synthesized from tryptophan and the three vitamin precursors of NAD +: nicotinic acid, Nicotinamide and Nicotinamide riboside. Supplementation of yeast cells with NAD + precursors increases intracellular NAD + levels and extends replicative lifespan. Here we show that both Nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the Nicotinamide riboside transporter, Nrt1, leads to increased export of Nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular Nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular Nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like Nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD + metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD + metabolism by balancing import and export of NAD
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Nrt1 and Tna1-independent export of NAD+ precursor vitamins promotes NAD+ homeostasis and allows engineering of vitamin production. PLoS ONE6:e19710. doi: 10.1371/journal.pone.0019710
2011Co-Authors: Peter Belenky, Katrina L. Bogan, Charles R. Evans, Rebecca Stebbins, Charles BrennerAbstract:NAD+ is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD+ consuming enzymes. NAD+ biosynthesis is required for two different regimens that extend lifespan in yeast. NAD+ is synthesized from tryptophan and the three vitamin precursors of NAD+: nicotinic acid, Nicotinamide and Nicotinamide riboside. Supplementation of yeast cells with NAD+ precursors increases intracellular NAD+ levels and extends replicative lifespan. Here we show that both Nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the Nicotinamide riboside transporter, Nrt1, leads to increased export of Nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular Nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular Nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like Nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD+ metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD+ metabolism by balancing import and export of NAD
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identification of isn1 and sdt1 as glucose and vitamin regulated Nicotinamide mononucleotide and nicotinic acid mononucleotide 5 nucleotidases responsible for production of Nicotinamide riboside and nicotinic acid riboside
Journal of Biological Chemistry, 2009Co-Authors: Katrina L. Bogan, Peter Belenky, Charles R. Evans, Peng Song, Charles F. Burant, Robert T. Kennedy, Charles BrennerAbstract:Recently, we discovered that Nicotinamide riboside and nicotinic acid riboside are biosynthetic precursors of NAD(+), which are utilized through two pathways consisting of distinct enzymes. In addition, we have shown that exogenously supplied Nicotinamide riboside is imported into yeast cells by a dedicated transporter, and it extends replicative lifespan on high glucose medium. Here, we show that Nicotinamide riboside and nicotinic acid riboside are authentic intracellular metabolites in yeast. Secreted Nicotinamide riboside was detected with a biological assay, and intracellular levels of Nicotinamide riboside, nicotinic acid riboside, and other NAD(+) metabolites were determined by a liquid chromatography-mass spectrometry method. A biochemical genomic screen indicated that three yeast enzymes possess Nicotinamide mononucleotide 5'-nucleotidase activity in vitro. Metabolic profiling of knock-out mutants established that Isn1 and Sdt1 are responsible for production of Nicotinamide riboside and nicotinic acid riboside in cells. Isn1, initially classified as an IMP-specific 5'-nucleotidase, and Sdt1, initially classified as a pyrimidine 5'-nucleotidase, are additionally responsible for dephosphorylation of pyridine mononucleotides. Sdt1 overexpression is growth-inhibitory to cells in a manner that depends on its active site and correlates with reduced cellular NAD(+). Expression of Isn1 protein is positively regulated by the availability of nicotinic acid and glucose. These results reveal unanticipated and highly regulated steps in NAD(+) metabolism.
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Nicotinamide riboside and nicotinic acid riboside salvage in fungi and mammals. Quantitative basis for Urh1 and purine nucleoside phosphorylase function in NAD+ metabolism.
The Journal of biological chemistry, 2008Co-Authors: Peter Belenky, Kathryn C. Christensen, Francesca S. Gazzaniga, Alexandre A. Pletnev, Charles BrennerAbstract:NAD+ is a co-enzyme for hydride transfer enzymes and an essential substrate of ADP-ribose transfer enzymes and sirtuins, the type III protein lysine deacetylases related to yeast Sir2. Supplementation of yeast cells with Nicotinamide riboside extends replicative lifespan and increases Sir2-dependent gene silencing by virtue of increasing net NAD+ synthesis. Nicotinamide riboside elevates NAD+ levels via the Nicotinamide riboside kinase pathway and by a pathway initiated by splitting the nucleoside into a Nicotinamide base followed by Nicotinamide salvage. Genetic evidence has established that uridine hydrolase, purine nucleoside phosphorylase, and methylthioadenosine phosphorylase are required for Nrk-independent utilization of Nicotinamide riboside in yeast. Here we show that mammalian purine nucleoside phosphorylase but not methylthioadenosine phosphorylase is responsible for mammalian Nicotinamide riboside kinase-independent Nicotinamide riboside utilization. We demonstrate that so-called uridine hydrolase is 100-fold more active as a Nicotinamide riboside hydrolase than as a uridine hydrolase and that uridine hydrolase and mammalian purine nucleoside phosphorylase cleave nicotinic acid riboside, whereas the yeast phosphorylase has little activity on nicotinic acid riboside. Finally, we show that yeast nicotinic acid riboside utilization largely depends on uridine hydrolase and Nicotinamide riboside kinase and that nicotinic acid riboside bioavailability is increased by ester modification.
