Nicotinamide Mononucleotide Research Articles

Effects of Nicotinamide Mononucleotide on Glucose and Lipid Metabolism in Adults: A Systematic Review and Meta-analysis of Randomised Controlled Trials.

Supplementation of nicotinamide mononucleotides (NMN) has been claimed to improve metabolic function. We reviewed human randomised controlled trials (RCTs) of NMN to evaluate its effect on markers of glucose and lipid metabolism. Eight RCTs on NMN (dosage ranged 250-2000mg/d for a duration of 14days to 12weeks) involving a total of 342 middle-age/older adults (49% females, mainly non-diabetic) reporting at least one outcome on glucose control or lipid profile published in 2021-2023 were reviewed. The random-effects meta-analyses indicated no significant benefit of NMN on fasting glucose, fasting insulin, glycated hemoglobin, homeostatic model assessment for insulin resistance and lipid profile. Based on the small number of RCTs involving mainly relatively healthy adults, short-term supplementation of NMN of 250-2000mg/d did not show significantly positive impacts on glucose control and lipid profile.

Nampt/SIRT2/LDHA pathway-mediated lactate production regulates follicular dysplasia in polycystic ovary syndrome

Decreased nicotinamide adenine dinucleotide (NAD+) content has been shown to contribute to metabolic dysfunction during aging, including polycystic ovary syndrome (PCOS). However, the effect of NAD+ on ovulatory dysfunction in PCOS by regulating glycolysis has not been reported. Based on the observations of granulosa cells (GCs) transcriptome data from the Gene Expression Omnibus (GEO) database, the signal pathways including glycolysis and nicotinate-nicotinamide metabolism were significantly enriched, and most genes of the above pathway like LDHA and SIRT2 were down-regulated in PCOS patients. Therefore, the PCOS rat model was established by combining letrozole with a high-fat diet (HFD), we demonstrate that in vivo supplementation of nicotinamide mononucleotide (NMN) significantly improves the ovulatory dysfunction by facilitating the follicular development, promoting luteal formation, as well the fertility in PCOS rats. Furthermore, target energy metabolomics and transcriptome results showed that NMN supplementation ameliorates the lactate production by activating glycolytic process in the ovary. In vitro, when NAD+ synthesis and SIRT2 expression were inhibited, lactate content in KGN cells was decreased and LDHA expression was significantly inhibited. We confirmed that FK866 can enhance the acetylation of LDHA on 293T cells by Co-immunoprecipitation (Co-IP) assay. We also observed that inhibition of NAD+ synthesis can reduce the activity and increase the apoptosis of KGN cells. Overall, these benefits of NMN were elucidated and the Nampt/SIRT2/LDHA pathway mediated lactate production in granulosa cells played an important role in the improvement of follicular development disorders in PCOS. This study will provide experimental evidence for the clinical application of NMN in the treatment of PCOS in the future.

De novo biosynthesis and nicotinamide biotransformation of nicotinamide mononucleotide by engineered yeast cells.

β-Nicotinamide mononucleotide (NMN) is a precursor of NAD+ in mammals. Research on NAD+ has demonstrated its crucial role against aging and disease. Here two technical paths were established for the efficient synthesis of NMN in the yeast Pichia pastoris, enabling the production of NMN from the low-cost nicotinamide (NAM) or basic carbon sources. The yeast host was systematically modified to adapt to the biosynthesis and accumulation of NMN. To improve the semi-biosynthesis of NMN from NAM, nicotinamide phosphoribosyltransferases were expressed intracellular to evaluate their catalytic activities. The accumulation of extracellular NMN was further increased by the co-expression of an NMN transporter. Fine-tuning of gene expression level produced 72.1 mg/L NMN from NAM in flasks. To achieve de novo biosynthesis NMN, a heterologous biosynthetic pathway was reassembled in yeast cells. Fine-tuning of pathway nodes by the modification of gene expression level and enhancement of precursor generation allowed efficient NMN synthesis from glucose (36.9 mg/L) or ethanol (57.8 mg/L) in flask. Lastly, cultivations in a bioreactor in fed-batch mode achieved an NMN titre of 1004.6 mg/L at 165 h from 2 g NAM and 868 g glucose and 980.4 mg/L at 91 h from 160 g glucose and 557 g ethanol respectively. This study provides a foundation for future optimization of NMN biosynthesis by engineered yeast cell factories.

Protein Engineering of Nicotinamide Riboside Kinase Based on a Combinatorial Semirational Design Strategy for Efficient Biocatalytic Synthesis of Nicotinamide Mononucleotides.

Industrial biosynthesis of β-nicotinamide mononucleotide (β-NMN) lacks a highly active nicotinamide riboside kinase for the phosphorylation process. Cumbersome preprocessing steps and excessive ATP addition contribute to its increased cost. To tackle these challenges, a docking combination simulation (DCS) semirational mutagenesis strategy was designed in this study, combining various modification strategies to obtain a mutant NRK-TRA with 2.9-fold higher enzyme activity. Molecular dynamics simulations and structural analysis demonstrate the enhancement of its structural stability. High-density fermentation was achieved through a 5 L fermentation tank, with a titer reaching 208.3 U/mL, the highest in the current report. An ATP-cycling whole-cell catalytic system was employed and optimized by introducing a polyphosphate kinase 2 (PPK2) recombinant strain, and 15.16 g/L β-NMN was obtained through a series of batch transformation experiments. This study provides a new strategy for the efficient screening of highly active enzyme variants and offers a green and promising biotransformation system for NMN production.

Supplementation with NAD+ Precursors for Treating Alzheimer's Disease: A Metabolic Approach.

Alzheimer's disease (AD) is a progressive neurocognitive disorder. There is no cure for AD. Maintenance on intracellular levels of nicotinamide adenine dinucleotide (NAD+) has been reported to be a promising therapeutic strategy for the treatment of AD. NAD+ precursors that represent candidate targets include nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR). This systematic review provides insights into the potential therapeutic value of NAD+ precursors including NMN and NR, for the treatment of AD using preclinical and clinical studies published in the last 5 years. The Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) protocol was followed to systematically search the literature using two databases. We found 3 studies that used NMN to treat AD in preclinical murine models. However, human clinical trials using NMN as a therapeutic intervention in AD was not available in the current literature. We also found 4 studies that investigated the potential benefits of NR for the treatment of AD in preclinical models. We also found 2 human clinical trials that showed marked improvements in plasma and neuroimaging biomarkers, and cognitive measures following supplementation with NR. Results of preclinical and clinical studies confirm the potential benefits of NAD+ precursors for the treatment of AD. However, further clinical studies are required to confirm the increasingly important value of NAD+ precursors as effective pharmacological interventions in the clinic.

Nicotinamide Mononucleotide Improves Endometrial Homeostasis in a Rat Model of Polycystic Ovary Syndrome by Decreasing Insulin Resistance and Regulating the Glylytic Pathway.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder that can lead to insulin resistance (IR) and dysregulation of glucose metabolism, resulting in an imbalance in the endometrial environment, which is unfavorable for embryo implantation of PCOS. This study aims to investigate whether nicotinamide mononucleotide (NMN) improves the stability of the endometrium in a rat model of PCOS and identifies whether it is related to reduce IR and increase glycolysis levels and its potential signaling pathway. Female Sprague-Dawley (SD) rats are fed letrozole and a high-fat diet (HFD) to form the PCOS model, then the model rats are treated with or without NMN. It randomly divided into control, PCOS, and PCOS-NMN groups according to the feeding and treating method. Compared with the PCOS group, the regular estrous cycles are restored, the serum androgen (p<0.01) and fasting insulin levels (p<0.05) are reduced, and endometrial morphology (p<0.05) is improved in NMN-PCOS group. Furthermore, NMN inhibits endometrial cell apoptosis, improves endometrial decidualization transition, reduces IR, restores the expression of glycolysis rate-limiting enzymes, and activates the PI3K/AKT pathway in the uterus. These results suggest that NMN enhances endometrial tissue homeostasis by decreasing uterine IR and regulating the glycolysis through the PI3K/AKT pathway.

Nicotinamide enhances Treg differentiation by promoting Foxp3 acetylation in immune thrombocytopenia.

Imbalanced nicotinamide adenine dinucleotide (NAD+) homeostasis has been reported in multiple autoimmune diseases and supplementation with NAD+ precursors has consistently demonstrated positive therapeutic benefits for these conditions. Immune thrombocytopenia (ITP) is an acquired autoimmune disease, in which the decreased number and impaired function of regulatory T cells (Tregs) contribute to the main pathogenesis. Here we found NAD+ level was decreased in the plasma and CD4+ T cells of ITP patients. Supplementation with NAD+ precursor nicotinamide (NAM), but not nicotinamide mononucleotide (NMN), increased Treg frequency and ameliorated thrombocytopenia in an ITP murine model. Moreover, whilst both NAM and NMN restored cytosolic NAD+ level in the CD4+ T cells from ITP patients, only NAM promoted Treg differentiation. Mechanistically, Sirtuin1 (Sirt1), a major consumer of NAD+, was highly expressed in the CD4+ T cells of ITP patients, potentially contributing to the low level of NAD+. NAM, which could act as Sirt1 inhibitor, promoted Foxp3 acetylation and stability in induced Tregs derived from naïve CD4+ T cells of ITP patients. These findings suggest that NAM holds promise as a novel therapeutic strategy for restoring immune balance in ITP.

Targeting NAD+ Metabolism Vulnerability in FH-Deficient Hereditary Leiomyomatosis and Renal Cell Carcinoma with the novel NAMPT Inhibitor OT-82.

Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) is an inherited cancer syndrome caused by germline pathogenic variants in the fumarate hydratase (FH) gene. Affected individuals are at risk for developing cutaneous and uterine leiomyomas and aggressive FH-deficient renal cell carcinoma (RCC) with a papillary histology. Due to a disrupted TCA cycle, FH-deficient kidney cancers rely on aerobic glycolysis for energy production, potentially creating compensatory metabolic vulnerabilities. This study conducted a high-throughput drug screen in HLRCC cell lines, which identified a critical dependency on nicotinamide adenine dinucleotide (NAD), a redox cofactor produced by the biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT). Human HLRCC tumors and HLRCC-derived cell lines exhibited elevated NAMPT expression compared to controls. FH-deficient HLRCC cells, but not FH-restored HLRCC or normal kidney cells, were sensitive to NAMPT inhibition. HLRCC cell line viability was significantly decreased in both 2D and 3D in vitro cultures in response to the clinically relevant NAMPT inhibitor OT-82. NAMPT inhibition in vitro significantly decreased the total amount of NAD+, NADH, NADP, NADPH, and PAR levels and the effects of NAMPT inhibition could be rescued by the downstream NAD precursor nicotinamide mononucleotide, confirming the on-target activity of OT-82. Moreover, NAMPT inhibition by OT-82 in two HLRCC xenograft models resulted in severely reduced tumor growth. OT-82 treatment of HLRCC xenograft tumors in vivo inhibited glycolytic flux as demonstrated by reduced lactate/pyruvate ratio in hyperpolarized 13C-pyruvate magnetic resonance spectroscopic imaging experiments. Overall, our data define NAMPT inhibition as a potential therapeutic approach for FH-deficient HLRCC-associated renal cell carcinoma.

NAD+-boosting agent nicotinamide mononucleotide potently improves mitochondria stress response in Alzheimer’s disease via ATF4-dependent mitochondrial UPR

Extensive studies indicate that mitochondria dysfunction is pivotal for Alzheimer’s disease (AD) pathogenesis; while cumulative evidence suggests that increased mitochondrial stress response (MSR) may mitigate neurodegeneration in AD, explorations to develop a MSR-targeted therapeutic strategy against AD are scarce. We combined cell biology, molecular biology, and pharmacological approaches to unravel a novel molecular pathway by which NAD+-boosting agent nicotinamide mononucleotide (NMN) regulates MSR in AD models. Here, we report dyshomeostasis plasma UPRmt-mitophagy-mediated MSR profiles in AD patient samples. NMN restores NAD+ metabolic profiles and improves MSR through the ATF4-dependent UPRmt pathway in AD-related cross-species models. At the organismal level, NAD+ repletion with NMN supplementation ameliorates mitochondrial proteotoxicity, decreases hippocampal synaptic disruption, decreases neuronal loss, and brain atrophy in mice model of AD. Remarkably, omics features of the hippocampus with NMN show that NMN leads to transcriptional changes of genes and proteins involved in MSR characteristics, principally within the astrocyte unit rather than microglia and oligodendrocytes. In brief, our work provides evidence that MSR has an active role in the pathogenesis of AD, as reducing mitochondrial homeostasis via atf4 depletion in AD mice aggravates the hallmarks of the disease; conversely, bolstering mitochondrial proteostasis by NMN decreases protein aggregation, restores memory performance, and delays disease progression, ultimately translating to increased healthspan.

Quantitative dynamics of intracellular NMN by genetically encoded biosensor

Nicotinamide mononucleotide (NMN) is the direct precursor and a major booster of NAD+ with increasing applications in NAD+- and aging-related pathologies. However, measuring live cell NMN dynamics was not possible, leaving key questions in NMN uptake and intracellular regulation unanswered. Here we developed genetically encoded bioluminescent and fluorescent sensors to quantify subcellular NMN in live cells by engineering specific NMN-responsive protein scaffolds fused to luciferase and fluorescent proteins. The sensor dissected the multimechanistic uptake of exogenous NMN and nicotinamide riboside (NR) in live cells and further measured the NMN levels across different subcellular compartments, as well as the perturbed NMN/NAD+ ratios by external supplements. Moreover, we measured the NMN regulation by NAD(H) hydrolase Nudts and peroxisomal carrier Pxmp2 and identified Slc25a45 as a potential mitochondrial NMN regulator for its unique fingerprint on the local NMN/NAD+ ratio. Collectively, the genetically encoded sensors provide a useful tool for visualizing NMN metabolism.

Sirt6 Mono-ADP-Ribosylates YY1 to Promote Dystrophin Expression for Neuromuscular Transmission.

The degeneration of the neuromuscular junction (NMJ) and the decline in motor function are common features of aging, but the underlying mechanisms have remained largely unclear. This study reveals that Sirt6 is reduced in aged mouse muscles. Ablation of Sirt6 in skeletal muscle causes a reduction of Dystrophin levels, resulting in premature NMJ degeneration, compromised neuromuscular transmission, and a deterioration in motor performance. Mechanistic studies show that Sirt6 negatively regulates the stability of the Dystrophin repressor YY1 (Yin Yang 1). Specifically, Sirt6 mono-ADP-ribosylates YY1, causing its disassociation from the Dystrophin promoter and allowing YY1 to bind to the SMURF2 E3 ligase, leading to its degradation. Importantly, supplementation with nicotinamide mononucleotide (NMN) enhances the mono-ADP-ribosylation of YY1 and effectively delays NMJ degeneration and the decline in motor function in elderly mice. These findings provide valuable insights into the intricate mechanisms underlying NMJ degeneration during aging. Targeting Sirt6 could be a potential therapeutic approach to mitigate the detrimental effects on NMJ degeneration and improve motor function in the elderly population.

Stoichiometric Regeneration of Biomimetic Nicotinamide Coenzyme Powered by Biomass Sugars via In Vitro Synthetic Enzymatic Biosystems.

Biomimetic nicotinamide coenzymes, including nicotinamide mononucleotide (NMN+), have been demonstrated as promising low-cost alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P)+) in biocatalysis. Herein, to efficiently regenerate NMNH from NMN+ in vitro powered by biomass sugars, a thermophilic NADP+-dependent glucose 6-phosphate dehydrogenase from Thermotoga maritima (TmG6PDH) was engineered to increase the activity toward NMN+. The catalytic efficiency (kcat/Km) of optimal mutant (TmG6PDH-R7) toward NMN+ increased by 71.7-fold than TmG6PDH-WT. As a result, compared to the wild type, the coenzyme specificity ([kcat/Km]NMN+/[kcat/Km]NADP+) of TmG6PDH-R7 increased by ~2.0×105-fold. The structural analysis revealed that the introduced hydrophobic and bulky residues lead to the formation of a smaller binding pocket, which resulting in a higher affinity for NMN+ with small size than NADP+. Then several in vitro synthetic enzymatic biosystems (ivSEBs) comprising this thermophilic TmG6PDH-R7 and a previously engineered thermophilic 6-phosphogluconate dehydrogenase were constructed. These ivSEBs harnessed the complete oxidation of renewable biomass sugars to facilitate the stoichiometric regeneration of 12 molecules of NMNH from 1 molecule of glucose, thereafter producing various products such as levodione, 2,3-butanediol or bioelectricity, over a wide temperature range. This study could pave the way for using stable and low-cost biomimetic coenzymes in ivSEBs for industrial biomanufacturing.

8700 Mono(ADP-Ribosyl)ation of Ribosomal Proteins Promotes Differentiation During Adipogenesis

Abstract Disclosure: X. Tan: None. S. Challa: None. C.V. Camacho: None. T.S. Nandu: None. M.S. Stokes: None. W.L. Kraus: Other; Self; W.L.K. is a founder and stockholder for Ribon Therapeutics, Inc; a founder, member of the SAB, member of the BOD, and a stockholder for and ARase Therapeutics, Inc. He is also coholder of U.S. Patent. ADP-ribosylation, a crucial post-translational modification, involves the covalent attachment of ADP-ribose units to amino acids on proteins through ADP-ribosyl transferase (ART) enzymes, utilizing NAD+ synthesized by nicotinamide mononucleotide adenylyl transferases (NMNATs). We previously showed that NMNAT-2, a cytoplasmic NAD+ synthase, increases during adipogenesis, leading to reduced nuclear NAD+ levels through compartmentalized NMN depletion and enhanced differentiation of preadipocytes. However, the implications of increased cytoplasmic NAD+ synthesis by NMNAT-2 in adipocytes remain unknown. Our current findings demonstrate elevated ribosome MARylation during 3T3-L1 preadipocyte differentiation, driven by NMNAT-2 induction. Inhibiting NAD+ biosynthesis by knockdown of NMNAT-2 or FK866 results in reduced ribosome MARylation, with PARP16 identified as the primary ART mediating MARylation. Knockdown of NMNAT-2 or PARP16 impaired global protein synthesis and preadipocyte differentiation. Treatment with the PARP16 inhibitor DB008 hindered ribosomal MARylation and preadipocyte differentiation. Immunofluorescence and subcellular fractionation analysis demonstrated that MARylated ribosomes are mostly associated with the endoplasmic reticulum. Using TMT mass spectrometry, we identified numerous high-confidence ADP-ribosylation sites on glutamate and aspartate residues in both undifferentiated and differentiated cells. We examined five proteins with specific sites of MARylation that are consistently more modified in the differentiated state. Among these, ADP-ribosylation of RPSA significantly promote preadipocyte differentiation; mutation of the site inhibits this effect. Ongoing investigations involve studying the impact of Parp16 knockout on high fat diet-induced obesity in mice to further determine the biological role of ribosome protein MARylation during adipogenesis. This work is supported by a grant from the NIH/NIDDK (R01 DK069710) and the DOD OCRP (OC200311). Presentation: 6/1/2024

Nicotinamide mononucleotide ameliorates ionizing radiation-induced spermatogenic dysfunction in mice by modulating the glycolytic pathway.

Radiotherapy, a common cancer treatment, leads to infertility in male cancer survivors, particularly young and middle-aged patients. Nicotinamide mononucleotide (NMN), a precursor of nicotinamide adenine dinucleotide (NAD +), plays crucial roles in energy metabolism, DNA repair, and gene expression. The purpose of this study is to investigate the protective effects and underlying mechanisms of NMN against ionizing radiation (IR)-induced testicular injury and spermatogenic dysfunction in an adult male mouse model. To assess the effects of NMN, single whole-body γ-ray irradiation is used to induce testicular injury and spermatogenic dysfunction in adult male mice. NMN is orally administered at 500 mg/kg before and after IR exposure. The structural and cellular damage to the testes caused by 5 Gy γ-ray irradiation, as well as the protective effect of NMN on testicular spermatogenic dysfunction, are evaluated. The serum hormone testosterone, LH, and FSH levels, as well as testicular NAD +, lactate, and pyruvate levels, are detected. Furthermore, the expressions of the apoptosis-related genes Bcl-2, Bax, and Caspase-3 and the rate-limiting enzymes HK2, PKM2, and LDHA, which are potentially associated with the mechanism of injury, are examined. The results demonstrate that 5 Gy γ-ray irradiation exposure causes a decrease in the serum testosterone, LH, and FSH levels in adult male mice, as well as in the testicular NAD +, lactate, and pyruvate levels, and causes damage to the testicular structure and cells. Morphometric analysis reveal a decrease in the testis mass, seminiferous tubule diameter, and height of the germinal epithelium. The sperm quantity, motility, and testicular volume are reduced in the 5 Gy group but are restored by NMN supplementation. NMN intervention downregulates the expressions of proapoptotic genes ( Bax and Caspase-3) and upregulates the expression of an antiapoptotic gene ( Bcl- 2). Sertoli cells marker genes ( WT-1, GATA-4, SOX9, and vimentin) and glycolysis rate-limiting enzyme-encoding genes ( HK2, PKM2, LDHA) are significantly upregulated. In summary, NMN has a positive regulatory effect on testicular spermatogenic dysfunction in male mice induced by ionizing radiation. This positive effect is likely achieved by promoting the proliferation of spermatogenic cells and activating glycolytic pathways. These findings suggest that NMN supplementation may be a potential protective strategy to prevent reproductive damage to male subjects from ionizing radiation.

Chronically Low NMNAT2 Expression Causes Sub-lethal SARM1 Activation and Altered Response to Nicotinamide Riboside in Axons.

Nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) is an endogenous axon survival factor that maintains axon health by blocking activation of the downstream pro-degenerative protein SARM1 (sterile alpha and TIR motif containing protein 1). While complete absence of NMNAT2 in mice results in extensive axon truncation and perinatal lethality, the removal of SARM1 completely rescues these phenotypes. Reduced levels of NMNAT2 can be compatible with life; however, they compromise axon development and survival. Mice born expressing sub-heterozygous levels of NMNAT2 remain overtly normal into old age but develop axonal defects in vivo and in vitro as well as behavioural phenotypes. Therefore, it is important to examine the effects of constitutively low NMNAT2 expression on SARM1 activation and disease susceptibility. Here we demonstrate that chronically low NMNAT2 levels reduce prenatal viability in mice in a SARM1-dependent manner and lead to sub-lethal SARM1 activation in morphologically intact axons of superior cervical ganglion (SCG) primary cultures. This is characterised by a depletion in NAD(P) and compromised neurite outgrowth. We also show that chronically low NMNAT2 expression reverses the NAD-enhancing effect of nicotinamide riboside (NR) in axons in a SARM1-dependent manner. These data indicate that low NMNAT2 levels can trigger sub-lethal SARM1 activation which is detectable at the molecular level and could predispose to human axonal disorders.

NMNAT1 Is Essential for Human iPS Cell Differentiation to the Retinal Lineage.

The gene encoding nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), a nicotinamide adenine dinucleotide synthetase localized in the cell nucleus, is a causative factor in Leber's congenital amaurosis, which is the earliest onset type of inherited retinal degeneration. We sought to investigate the roles of NMNAT1 in early retinal development. We used human induced pluripotent stem cells (hiPSCs) and established NMNAT1-knockout (KO) hiPSCs using CRISPR/cas9 technology to reveal the roles of NMNAT1 in human retinal development. NMNAT1 was not essential for the survival and proliferation of immature hiPSCs; therefore, we subjected NMNAT1-KO hiPSCs to retinal organoid (RO) differentiation culture. The expression levels of immature hiPSC-specific genes decreased in a similar manner after organoid culture initiation up to 2 weeks in the control and NMNAT1-KO. Neuroectoderm-specific genes were induced in the control and NMNAT1-KO organoids within a few days after starting the organoid culture; PAX6 and TUBB3 were higher in NMNAT1-KO organoids up to 7days than in the control organoids. However, the induction of genes involving retinal early development, such as RAX, which was induced at around day 10 in this culture, was considerably reduced in NMNAT1-KO organoids. Morphological examination also showed failure of retinal primordial structure formation, which became visible at around 2 weeks of the control culture, in the NMNAT1-KO organoids. Decreased intracellular NAD levels and poly(ADP-ribosyl)ation were observed in NMNAT1-KO organoids at 7 to 10days of the culture. Mass spectrometry analysis of inhibited proteins in the poly(ADP-ribosyl)ation pathway identified poly(ADP-ribosyl)ation of poly(ADP-ribose) polymerase 1 (PARP1) as a major protein. These results indicate that NMNAT1 was dispensable for neural lineage differentiation but essential for the commitment of retinal fate differentiation in hiPSCs. The NMNAT1-NAD-PARP1 axis may play a critical role in the appropriate development of human retinal lineage differentiation.

Long-Term Administration of Nicotinamide Mononucleotide Mitigates High-Fat-Diet-Induced Physiological Decline in Aging Mice

BackgroundNicotinamide adenine dinucleotide (NAD+) levels decline with age, and boosting it can improve multi-organ functions and lifespan. ObjectivesNicotinamide mononucleotide (NMN) is a natural NAD+ precursor with the ability to enhance NAD+ biosynthesis. Numerous studies have shown that a high-fat diet (HFD) can accelerate the process of aging and many diseases. We hypothesized that long-term administration of NMN could exert protective effects on adipose, muscle, and kidney tissues in mice on an HFD act by affecting the autophagic pathway. MethodsMice at 14 mo of age were fed an HFD, and NMN was added to their drinking water at a dose of 400 mg/kg for 7 mo. The locomotor ability of the mice was assessed by behavioral experiments such as grip test, wire hang test, rotarod, and beam-walking test. At the end of the behavioral experiments, the pathological changes of each peripheral organ and the expression of autophagy-related proteins, as well as the markers of the senescence and inflammaging were analyzed by pathological staining, immunohistochemical staining, and western blotting, respectively. ResultsWe found that NMN supplementation increased NAD+ levels and ultimately attenuated age- and diet-related physiological decline in mice. NMN inhibited HFD-induced obesity, promoted physical activity, improved glucose and lipid metabolism, improved skeletal muscle function and renal damage, as well as mitigated the senescence and inflammaging as demonstrated by p16, interleukin 1β, and tumor necrosis factor α levels. In addition, the present study further emphasizes the potential mechanisms underlying the bidirectional relationship between NAD+ and autophagy. We detected changes in autophagy levels in various tissue organs, and NMN may play a protective role by inhibiting excessive autophagy induced by HFD. ConclusionsOur findings demonstrated that NMN administration attenuated HFD-induced metabolic disorders and physiological decline in aging mice.

Whole-cell synthesis of nicotinamide mononucleotide by recombinant Saccharomyces cerevisiae from glucose and nicotinamide

β-Nicotinamide mononucleotide is recognized as a significant bioactive nucleotide, which is can be used in the fields of health industries. Many studies on the synthesis of NMN have involved Escherichia coli and the current methods are limited by safety problems as well as the expense of the substrate. Herein, GRAS-grade Saccharomyces cerevisiae was chosen as chassis cells to synthesize NMN using the substrates glucose and nicotinamide. First, the gene for the key enzyme nicotinamide phosphoribosyltransferase (Nampt) was screened from different sources, and site-directed mutation was performed to improve the synthesis of NMN. The concentration of intracellular NMN in yeast expressing the D83N-Nampt mutant derived from Chitinophaga pinensis reached 413.4 mg/L, which was 3.7 times higher than that of yeast expressing wild enzymes. The synthesis of NMN was further enhanced by overexpressing Nampt combined with weakening of the further metabolism of NMN. Subsequently, the supply of precursor phosphate ribose pyrophosphate (PRPP) was increased by overexpressing the PRPP synthase mutant, which led to the concentration of intracellular NMN increased to 775.9 mg/L from 537.8 mg/L. Finally, the concentration of intracellular NMN reached 1.2 g/L at 6 h after whole-cell catalytic optimization, which is the highest titer achieved by S. cerevisiae from inexpensive substrate glucose and nicotinamide. This study provides the synthesis of NMN by S. cerevisiae with a new and promising method.

Nicotinamide Mononucleotide (NMN) Ameliorates Free Fatty Acid-Induced Pancreatic β-Cell Dysfunction via the NAD+/AMPK/SIRT1/HIF-1α Pathway.

As the sole producers of insulin under physiological conditions, the normal functioning of pancreatic β cells is crucial for maintaining glucose homeostasis in the body. Due to the high oxygen and energy demands required for insulin secretion, hypoxia has been shown to play a critical role in pancreatic β-cell dysfunction. Lipid metabolism abnormalities, a common metabolic feature in type 2 diabetic patients, are often accompanied by tissue hypoxia caused by metabolic overload and lead to increased free fatty acid (FFA) levels. However, the specific mechanisms underlying FFA-induced β-cell dysfunction remain unclear. Nicotinamide mononucleotide (NMN), a naturally occurring bioactive nucleotide, has garnered significant attention in recent years for its effectiveness in replenishing NAD+ and alleviating various diseases. Nevertheless, studies exploring the mechanisms through which NMN influences β-cell dysfunction remain scarce. In this study, we established an in vitro β-cell dysfunction model by treating INS-1 cells with palmitate (PA), including control, PA-treated, and PA combined with NMN or activator/inhibitor groups. Compared to the control group, cells treated with PA alone showed significantly reduced insulin secretion capacity and decreased expression of proteins related to the NAD+/AMPK/SIRT1/HIF-1α pathway. In contrast, NMN supplementation significantly restored the expression of pathway-related proteins by activating NAD+ and effectively improved insulin secretion. Results obtained using HIF-1α and AMPK inhibitors/activators further supported these findings. In conclusion, our study demonstrates that NMN reversed the PA-induced downregulation of the NAD+/AMPK/SIRT1/HIF-1α pathway, thereby alleviating β-cell dysfunction. Our study investigated the mechanisms underlying PA-induced β-cell dysfunction, examined how NMN mitigates this dysfunction and offered new insights into the therapeutic potential of NMN for treating β-cell dysfunction and T2DM.

Nicotinamide mononucleotide supplementation rescues mitochondrial and energy metabolism functions and ameliorates inflammatory states in the ovaries of aging mice.

Noninvasive pharmacological strategies like nicotinamide mononucleotide (NMN) supplementation can effectively address age-related ovarian infertility by maintaining or enhancing oocyte quality and quantity. This study revealed that ovarian nicotinamide adenine dinucleotide levels decline with age, but NMN administration significantly restores these levels, preventing ovarian atrophy and enhancing the quality and quantity of ovulated oocytes. Improvements in serum hormone secretion and antioxidant factors, along with decreased expression of proinflammatory factors, were observed. Additionally, a significant increase in the number of ovarian follicles in aging individuals was noted. Scanning electron microscopy data indicated that NMN significantly alters the density and morphology of lipid droplets and mitochondria in granulosa cells, suggesting potential targets and mechanisms. Transcriptomic analysis and validation experiments collectively suggested that the beneficial effects of NMN on aging ovaries are mediated through enhanced mitochondrial function, improved energy metabolism, and reduced inflammation levels. Our results suggest that NMN supplementation could improve the health status of aging ovaries and enhance ovarian reserve, offering new insights into addressing fertility challenges in older women through assisted reproductive technology.