Multiomics analysis of the NAD+-PARP1 axis reveals a role for site-specific ADP-ribosylation in splicing in embryonic stem cells.

The differentiation of embryonic stem cells (ESCs) into a lineage-committed state is a dynamic process involving changes in cellular metabolism, epigenetic modifications, post-translational modifications, gene expression, and RNA processing. Here we integrated data from metabolomic, proteomic, and transcriptomic assays to characterize how alterations in NAD+ metabolism during the differentiation of mouse ESCs lead to alteration of the PARP1-mediated ADP-ribosylated (ADPRylated) proteome and mRNA isoform specialization. Our metabolomic analyses indicate that mESCs use distinct NAD+ biosynthetic pathways in different cell states: the de novo pathway in the pluripotent state, and the salvage and Preiss-Handler pathways as differentiation progresses. We observed a dramatic induction of PARP1 catalytic activity driven by enhanced nuclear NAD+ biosynthesis during the early stages of mESC differentiation (e.g., within 12 h of LIF removal). PARP1-modified proteins in mESCs are enriched for biological processes related to stem cell maintenance, transcriptional regulation, and RNA processing. The PARP1 substrates include core spliceosome components, such as U2AF35 and U2AF65, whose splicing functions are modulated by PARP1-mediated site-specific ADP-ribosylation. Finally, we observed that splicing is dysregulated genome-wide in Parp1 knockout mESCs. Together, these results demonstrate a role for the NAD+-PARP1 axis in the maintenance of mESC state, specifically in the splicing program during differentiation.

Dynamics of SLC25A51 reveal preference for oxidized NAD+ and substrate led transport.

SLC25A51 is a member of the mitochondrial carrier family (MCF) but lacks key residues that contribute to the mechanism of other nucleotide MCF transporters. Thus, how SLC25A51 transports NAD+ across the inner mitochondrial membrane remains unclear. To elucidate its mechanism, we use Molecular Dynamics simulations to reconstitute SLC25A51 homology models into lipid bilayers and to generate hypotheses to test. We observe spontaneous binding of cardiolipin phospholipids to three distinct sites on the exterior of SLC25A51's central pore and find that mutation of these sites impairs cardiolipin binding and transporter activity. We also observe that stable formation of the required matrix gate is controlled by a single salt bridge. We identify binding sites in SLC25A51 for NAD+ and show that its selectivity for NAD+ is guided by an electrostatic interaction between the charged nicotinamide ring in the ligand and a negatively charged patch in the pore. In turn, interaction of NAD+ with interior residue E132 guides the ligand to dynamically engage and weaken the salt bridge gate, representing a ligand-induced initiation of transport.

Circadian NAD(P)(H) cycles in cell metabolism.

Intrinsic circadian clocks are present in all forms of photosensitive life, enabling daily anticipation of the light/dark cycle and separation of energy storage and utilization cycles on a 24-h timescale. The core mechanism underlying circadian rhythmicity involves a cell-autonomous transcription/translation feedback loop that in turn drives rhythmic organismal physiology. In mammals, genetic studies have established that the core clock plays an essential role in maintaining metabolic health through actions within both brain pacemaker neurons and peripheral tissues and that disruption of the clock contributes to disease. Peripheral clocks, in turn, can be entrained by metabolic cues. In this review, we focus on the role of the nucleotide NAD(P)(H) and NAD+-dependent sirtuin deacetylases as integrators of circadian and metabolic cycles, as well as the implications for this interrelationship in healthful aging.

The structure of NAD+ consuming protein Acinetobacter baumannii TIR domain shows unique kinetics and conformations.

Toll-like and interleukin-1/18 receptor/resistance (TIR) domain-containing proteins function as important signaling and immune regulatory molecules. TIR domain-containing proteins identified in eukaryotic and prokaryotic species also exhibit NAD+ hydrolase activity in select bacteria, plants, and mammalian cells. We report the crystal structure of the Acinetobacter baumannii TIR domain protein (AbTir-TIR) with confirmed NAD+ hydrolysis and map the conformational effects of its interaction with NAD+ using hydrogen-deuterium exchange-mass spectrometry. NAD+ results in mild decreases in deuterium uptake at the dimeric interface. In addition, AbTir-TIR exhibits EX1 kinetics indicative of large cooperative conformational changes, which are slowed down upon substrate binding. Additionally, we have developed label-free imaging using the minimally invasive spectroscopic method 2-photon excitation with fluorescence lifetime imaging, which shows differences in bacteria expressing native and mutant NAD+ hydrolase-inactivated AbTir-TIRE208A protein. Our observations are consistent with substrate-induced conformational changes reported in other TIR model systems with NAD+ hydrolase activity. These studies provide further insight into bacterial TIR protein mechanisms and their varying roles in biology.

EvdS6 is a bifunctional decarboxylase from the everninomicin gene cluster.

The everninomicins are bacterially produced antibiotic octasaccharides characterized by the presence of two interglycosidic spirocyclic ortho-δ-lactone (orthoester) moieties. The terminating G- and H-ring sugars, L-lyxose and C-4 branched sugar ß-D-eurekanate, are proposed to be biosynthetically derived from nucleotide diphosphate pentose sugar pyranosides; however, the identity of these precursors and their biosynthetic origin remain to be determined. Herein we identify a new glucuronic acid decarboxylase from Micromonospora belonging to the superfamily of short-chain dehydrogenase/reductase enzymes, EvdS6. Biochemical characterization demonstrated that EvdS6 is an NAD+-dependent bifunctional enzyme that produces a mixture of two products, differing in the sugar C-4 oxidation state. This product distribution is atypical for glucuronic acid decarboxylating enzymes, most of which favor production of the reduced sugar and a minority of which favor release of the oxidized product. Spectroscopic and stereochemical analysis of reaction products revealed that the first product released is the oxidatively produced 4-keto-D-xylose and the second product is the reduced D-xylose. X-ray crystallographic analysis of EvdS6 at 1.51 Å resolution with bound co-factor and TDP demonstrated that the overall geometry of the EvdS6 active site is conserved with other SDR enzymes and enabled studies probing structural determinants for the reductive half of the net neutral catalytic cycle. Critical active site threonine and aspartate residues were unambiguously identified as essential in the reductive step of the reaction and resulted in enzyme variants producing almost exclusively the keto sugar. This work defines potential precursors for the G-ring L-lyxose and resolves likely origins of the H-ring ß-D-eurekanate sugar precursor.

Coenzyme biosynthesis in response to precursor availability reveals incorporation of ß-alanine from pantothenate in prototrophic bacteria.

Coenzymes are important for all classes of enzymatic reactions and essential for cellular metabolism. Most coenzymes are synthesized from dedicated precursors, also referred to as vitamins, which prototrophic bacteria can either produce themselves from simpler substrates or take up from the environment. The extent to which prototrophs use supplied vitamins and whether externally available vitamins affect the size of intracellular coenzyme pools and control endogenous vitamin synthesis is currently largely unknown. Here, we studied coenzyme pool sizes and vitamin incorporation into coenzymes during growth on different carbon sources and vitamin supplementation regimes using metabolomics approaches. We found that the model bacterium Escherichia coli incorporated pyridoxal, niacin, and pantothenate into pyridoxal 5'-phosphate, NAD, and coenzyme A (CoA), respectively. In contrast, riboflavin was not taken up and was produced exclusively endogenously. Coenzyme pools were mostly homeostatic and not affected by externally supplied precursors. Remarkably, we found that pantothenate is not incorporated into CoA as such but is first degraded to pantoate and ß-alanine and then rebuilt. This pattern was conserved in various bacterial isolates, suggesting a preference for ß-alanine over pantothenate utilization in CoA synthesis. Finally, we found that the endogenous synthesis of coenzyme precursors remains active when vitamins are supplied, which is consistent with described expression data of genes for enzymes involved in coenzyme biosynthesis under these conditions. Continued production of endogenous coenzymes may ensure rapid synthesis of the mature coenzyme under changing environmental conditions, protect against coenzyme limitation, and explain vitamin availability in naturally oligotrophic environments.

NAD+ overconsumption by poly (ADP-ribose) polymerase (PARP) under oxidative stress induces cytoskeletal disruption in vascular endothelial cell.

Vascular endothelial cytoskeletal disruption leads to increased vascular permeability and is involved in the pathogenesis and progression of various diseases. Oxidative stress can increase vascular permeability by weakening endothelial cell-to-cell junctions and decrease intracellular nicotinamide adenine dinucleotide (NAD+) levels. However, it remains unclear how intracellular NAD+ variations caused by oxidative stress alter the vascular endothelial cytoskeletal organization. In this study, we demonstrated that oxidative stress activates poly (ADP-ribose [ADPr]) polymerase (PARP), which consume large amounts of intracellular NAD+, leading to cytoskeletal disruption in vascular endothelial cells. We found that hydrogen peroxide (H2O2) could transiently disrupt the cytoskeleton and reduce intracellular total NAD levels in human umbilical vein endothelial cells (HUVECs). H2O2 stimulation led to rapid increase in ADPr protein levels in HUVECs. Pharmaceutical PARP inhibition counteracted H2O2-induced total NAD depletion and cytoskeletal disruption, suggesting that NAD+ consumption by PARP induced cytoskeletal disruption. Additionally, supplementation with nicotinamide mononucleotide (NMN), the NAD+ precursor, prevented both intracellular total NAD depletion and cytoskeletal disruption induced by H2O2 in HUVECs. Inhibition of the NAD+ salvage pathway by FK866, a nicotinamide phosphoribosyltransferase inhibitor, maintained H2O2-induced cytoskeletal disruption, suggesting that intracellular NAD+ plays a crucial role in recovery from cytoskeletal disruption. Our findings provide further insights into the potential application of PARP inhibition and NMN supplementation for the treatment and prevention of diseases involving vascular hyperpermeability.

Advancements in NMN biotherapy and research updates in the field of digestive system diseases.

Nicotinamide mononucleotide (NMN), a crucial intermediate in NAD + synthesis, can rapidly transform into NAD + within the body after ingestion. NMN plays a pivotal role in several important biological processes, including energy metabolism, cellular aging, circadian rhythm regulation, DNA repair, chromatin remodeling, immunity, and inflammation. NMN has emerged as a key focus of research in the fields of biomedicine, health care, and food science. Recent years have witnessed extensive preclinical studies on NMN, offering valuable insights into the pathogenesis of age- and aging-related diseases. Given the sustained global research interest in NMN and the substantial market expectations for the future, here, we comprehensively review the milestones in research on NMN biotherapy over the past 10 years. Additionally, we highlight the current research on NMN in the field of digestive system diseases, identifying existing problems and challenges in the field of NMN research. The overarching aim of this review is to provide references and insights for the further exploration of NMN within the spectrum of digestive system diseases.

Probing the Site of Glutathione Reduction by Thioredoxin/Glutathione Reductase from Schistosoma mansoni under Anaerobic Conditions.

Thioredoxin/glutathione reductase from Schistosoma mansoni (SmTGR) is a multifunctional enzyme that catalyzes the reduction of glutathione (GSSG) and thioredoxin, as well as the deglutathionylation of peptide and non-peptide substrates. SmTGR structurally resembles known glutathione reductases (GR) and thioredoxin reductases (TrxR) but with an appended N-terminal domain that has a typical glutaredoxin (Grx) fold. Despite structural homology with known GRs, the site of glutathione reduction has frequently been reported as the Grx domain, based primarily on aerobic, steady-state kinetic measurements and x-ray crystallography. Here, we present an anaerobic characterization of a series of variant SmTGRs to establish the site of GSSG reduction as the cysteine pair most proximal to the FAD, Cys154/Cys159, equivalent to the site of GSSG reduction in GRs. Anaerobic steady-state analysis of U597C, U597S, U597C+C31S, and I592STOP SmTGR demonstrate that the Grx domain is not involved in the catalytic reduction of GSSG, as redox silencing of the C-terminus results in no modulation of the observed turnover number (∼0.025 s-1) and redox silencing of the Grx domain results in an increased observed turnover number (∼0.08 s-1). Transient-state single turnover analysis of these variants corroborates this, as the slowest rate observed titrates hyperbolically with GSSG concentration and approaches a limit that coincides with the respective steady-state turnover number for each variant. Numerical integration fitting of the transient state data can only account for the observed trends when competitive binding of the C-terminus is included, indicating that the partitioning of electrons to either substrate occurs at the Cys154/Cys159 disulfide rather than the previously proposed Cys596/Sec597 sulfide/selenide. Paradoxically, truncating the C-terminus at Ile592 results in a loss of GR activity, indicating a crucial non-redox role for the C-terminus.

Excess glucose alone depress young mesenchymal stromal/stem cell osteogenesis and mitochondria activity within hours/days via NAD+/SIRT1 axis.

BACKGROUND: The impact of global overconsumption of simple sugars on bone health, which peaks in adolescence/early adulthood and correlates with osteoporosis (OP) and fracture risk decades, is unclear. Mesenchymal stromal/stem cells (MSCs) are the progenitors of osteoblasts/bone-forming cells, and known to decrease their osteogenic differentiation capacity with age. Alarmingly, while there is correlative evidence that adolescents consuming greatest amounts of simple sugars have the lowest bone mass, there is no mechanistic understanding on the causality of this correlation. METHODS: Bioinformatics analyses for energetics pathways involved during MSC differentiation using human cell information was performed. In vitro dissection of normal versus high glucose (HG) conditions on osteo-/adipo-lineage commitment and mitochondrial function was assessed using multi-sources of non-senescent human and murine MSCs; for in vivo validation, young mice was fed normal or HG-added water with subsequent analyses of bone marrow CD45- MSCs. RESULTS: Bioinformatics analyses revealed mitochondrial and glucose-related metabolic pathways as integral to MSC osteo-/adipo-lineage commitment. Functionally, in vitro HG alone without differentiation induction decreased both MSC mitochondrial activity and osteogenesis while enhancing adipogenesis by 8 h' time due to depletion of nicotinamide adenine dinucleotide (NAD+), a vital mitochondrial co-enzyme and co-factor to Sirtuin (SIRT) 1, a longevity gene also involved in osteogenesis. In vivo, HG intake in young mice depleted MSC NAD+, with oral NAD+ precursor supplementation rapidly reversing both mitochondrial decline and osteo-/adipo-commitment in a SIRT1-dependent fashion within 1 ~ 5 days. CONCLUSIONS: We found a surprisingly rapid impact of excessive glucose, a single dietary factor, on MSC SIRT1 function and osteogenesis in youthful settings, and the crucial role of NAD+-a single molecule-on both MSC mitochondrial function and lineage commitment. These findings have strong implications on future global OP and disability risks in light of current worldwide overconsumption of simple sugars.

1,25-Dihydroxyvitamin D3 Suppresses UV-Induced Poly(ADP-Ribose) Levels in Primary Human Keratinocytes, as Detected by a Novel Whole-Cell ELISA.

Photoprotective properties of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to reduce UV-induced DNA damage have been established in several studies. UV-induced DNA damage in skin such as single or double strand breaks is known to initiate several cellular mechanisms including activation of poly(ADP-ribose) (pADPr) polymerase-1 (PARP-1). DNA damage from UV also increases extracellular signal-related kinase (ERK) phosphorylation, which further increases PARP activity. PARP-1 functions by using cellular nicotinamide adenine dinucleotide (NAD+) to synthesise pADPr moieties and attach these to target proteins involved in DNA repair. Excessive PARP-1 activation following cellular stress such as UV irradiation may result in excessive levels of cellular pADPr. This can also have deleterious effects on cellular energy levels due to depletion of NAD+ to suboptimal levels. Since our previous work indicated that 1,25(OH)2D3 reduced UV-induced DNA damage in part through increased repair via increased energy availability, the current study investigated the effect of 1,25(OH)2D3 on UV-induced PARP-1 activity using a novel whole-cell enzyme- linked immunosorbent assay (ELISA) which quantified levels of the enzymatic product of PARP-1, pADPr. This whole cell assay used around 5000 cells per replicate measurement, which represents a 200-400-fold decrease in cell requirement compared to current commercial assays that measure in vitro pADPr levels. Using our assay, we observed that UV exposure significantly increased pADPr levels in human keratinocytes, while 1,25(OH)2D3 significantly reduced levels of UV-induced pADPr in primary human keratinocytes to a similar extent as a known PARP-1 inhibitor, 3-aminobenzamide (3AB). Further, both 1,25(OH)2D3 and 3AB as well as a peptide inhibitor of ERK-phosphorylation significantly reduced DNA damage in UV-exposed keratinocytes. The current findings support the proposal that reduction in pADPr levels may be critical for the function of 1,25(OH)2D3 in skin to reduce UV-induced DNA damage.

Structural characterization of Linum usitatissimum hydroxynitrile lyase: A new cyanohydrin decomposition mechanism involving a cyano-zinc complex.

Hydroxynitrile lyase from Linum usitatissimum (LuHNL) is an enzyme involved in the catabolism of cyanogenic glycosides to release hydrogen cyanide upon tissue damage. This enzyme strictly conserves the substrate- and NAD(H)-binding domains of Zn2+-containing alcohol dehydrogenase (ADH); however, there is no evidence suggesting that LuHNL possesses ADH activity. Herein, we determined the ligand-free 3D structure of LuHNL and its complex with acetone cyanohydrin and (R)-2-butanone cyanohydrin using X-ray crystallography. These structures reveal that an A-form NAD+ is tightly but not covalently bound to each subunit of LuHNL. The restricted movement of the NAD+ molecule is due to the "sandwich structure" on the adenine moiety of NAD+. Moreover, the structures and mutagenesis analysis reveal a novel reaction mechanism for cyanohydrin decomposition involving the cyano-zinc complex and hydrogen-bonded interaction of the hydroxyl group of cyanohydrin with Glu323/Thr65 and H2O/Lys162 of LuHNL. The deprotonated Lys162 and protonated Glu323 residues are presumably stabilized by a partially desolvated microenvironment. In summary, the substrate binding geometry of LuHNL provides insights into the differences in activities of LuHNL and ADH, and identifying this novel reaction mechanism is an important contribution to the study of hydroxynitrile lyases.

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.

A Reactive Oxygen Species-Responsive Targeted Nanoscavenger to Promote Mitophagy for the Treatment of Alzheimer's Disease.

Mitophagy modulators are proposed as potential therapeutic intervention that enhance neuronal health and brain homeostasis in Alzheimer's disease (AD). Nevertheless, the lack of specific mitophagy inducers, low efficacies, and the severe side effects of nonselective autophagy during AD treatment have hindered their application. In this study, the P@NB nanoscavenger is designed with a reactive-oxygen-species-responsive (ROS-responsive) poly(l-lactide-co-glycolide) core and a surface modified with the Beclin1 and angiopoietin-2 peptides. Notably, nicotinamide adenine dinucleotide (NAD+ ) and Beclin1, which act as mitophagy promoters, are quickly released from P@NB in the presence of high ROS levels in lesions to restore mitochondrial homeostasis and induce microglia polarization toward the M2-type, thereby enabling it to phagocytose amyloid-peptide (Aß). These studies demonstrate that P@NB accelerates Aß degradation and alleviates excessive inflammatory responses by restoring autophagic flux, which ameliorates cognitive impairment in AD mice. This multitarget strategy induces autophagy/mitophagy through synergy, thereby normalizing mitochondrial dysfunction. Therefore, the developed method provides a promising AD-therapy strategy.

The complexity of nicotinamide adenine dinucleotide (NAD), hypoxic, and aryl hydrocarbon receptor cell signaling in chronic kidney disease.

Early-stage detection of chronic kidney diseases (CKD) is important to treatment that may slow and occasionally halt CKD progression. CKD of diverse etiologies share similar histologic patterns of glomerulosclerosis, tubular atrophy, and interstitial fibrosis. Macro-vascular disease and micro-vascular disease promote tissue ischemia, contributing to injury. Tissue ischemia promotes hypoxia, and this in turn activates the hypoxia-inducible transcription factors (HIFs). HIF-1α and HIF-2α, share a dimer partner, HIF-1ß, with the aryl hydrocarbon receptor (AHR) and are each activated in CKD and associated with kidney cellular nicotinamide adenine dinucleotide (NAD) depletion. The Preiss-Handler, salvage, and de novo pathways regulate NAD biosynthesis and gap-junctions regulate NAD cellular retention. In the Preiss-Handler pathway, niacin forms NAD. Niacin also exhibits crosstalk with HIF and AHR cell signals in the regulation of insulin sensitivity, which is a complication in CKD. Dysregulated enzyme activity in the NAD de novo pathway increases the levels of circulating tryptophan metabolites that activate AHR, resulting in poly-ADP ribose polymerase activation, thrombosis, endothelial dysfunction, and immunosuppression. Therapeutically, metabolites from the NAD salvage pathway increase NAD production and subsequent sirtuin deacetylase activity, resulting in reduced activation of retinoic acid-inducible gene I, p53, NF-κB and SMAD2 but increased activation of FOXO1, PGC-1α, and DNA methyltransferase-1. These post-translational responses may also be initiated through non-coding RNAs (ncRNAs), which are additionally altered in CKD. Nanoparticles traverse biological systems and can penetrate almost all tissues as disease biomarkers and drug delivery carriers. Targeted delivery of non-coding RNAs or NAD metabolites with nanoparticles may enable the development of more effective diagnostics and therapies to treat CKD.

Mechanisms governing PARP expression, localization, and activity in cells.

Poly-(ADP)-ribose polymerases (PARPs) are a family of 17 enzymes in humans that have diverse roles in cell physiology including DNA damage repair, transcription, innate immunity, and regulation of signaling pathways. The modular domain architecture of PARPs gives rise to this functional diversity. PARPs catalyze the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD+) to targets-proteins and poly-nucleic acids. This enigmatic post-translational modification comes in two varieties: the transfer of a single unit of ADP-ribose, known as mono-ADP-ribosylation (MARylation) or the transfer of multiple units of ADP-ribose, known as poly-ADP-ribosylation (PARylation). Emerging data shows that PARPs are regulated at multiple levels to control when and where PARP-mediated M/PARylation occurs in cells. In this review, we will discuss the latest knowledge regarding the regulation of PARPs in cells: from transcription and protein stability to subcellular localization and modulation of catalytic activity.

PPARα agonist WY-14,643 induces the PLA2/COX-2/ACOX1 pathway to enhance peroxisomal lipid metabolism and ameliorate alcoholic fatty liver in mice.

Peroxisome proliferator-activated receptor α (PPARα) regulates fatty acid oxidation (FAO). Usually, very-long chain fatty acids are first activated by acyl-CoA synthetase (ACS) to generate acyl-CoA for oxidation by acyl-CoA oxidase (ACOX) in peroxisomes, and the resultant shorter chain fatty acids will be further oxidized in mitochondria. ACS long-chain family member 4 (ACSL4) preferentially uses arachidonic acid (AA) as substrates to synthesize arachidonoyl-CoA. Arachidonoyl-CoA is usually esterified into phospholipids. When AA is released by phospholipase A2 (PLA2) from phospholipids, it will be used for prostaglandin synthesis by cyclooxygenases (COX). In this study, when PPARα agonist WY-14,643 was mixed in liquid Lieber-DeCarli ethanol or control diets and fed to mice, liver PLA2, COX-2, and ACOX1 were induced but ACSL4 was inhibited, suggesting that AA released by PLA2 from phospholipid will be metabolized to prostaglandin via COX-2 instead of being synthesized into acyl-CoA by ACSL4. However, liver prostaglandin E2 (PGE2), a major component of prostaglandin, was not increased with the induced COX-2 but decreased by WY-14,643. ACOX1 specific inhibitor mixed in the liquid diets restored both the WY-14,643-suppressed liver TG and PGE2, but COX-2 specific inhibitor celecoxib mixed in the liquid diets reversed the WY-14,643-suppressed liver TG but not liver PGE2 contents. These results suggest that induction of PLA2, COX-2 and ACOX1 orchestrates to increase oxidation of AA/PGE2, which constitutes one new mechanism by which PPARα induces peroxisomal FAO and inhibits ethanol-induced liver fat accumulation.

NAD+, Sirtuins and PARPs: enhancing oocyte developmental competence.

Oocyte quality is the limiting factor in female fertility. It is well known that maternal nutrition plays a role in reproductive function, and manipulating nutrition to improve fertility in livestock has been common practice in the past, particularly with respect to negative energy balance in cattle. A deficiency in nicotinamide adenine dinucleotide (NAD+) production has been associated with increased incidences of miscarriage and congenital defects in humans and mice, while elevating NAD+ through dietary supplements in aged subjects improved oocyte quality and embryo development. NAD+ is consumed by Sirtuins and poly-ADP-ribose polymerases (PARPs) within the cell and thus need constant replenishment in order to maintain various cellular functions. Sirtuins and PARPs play important roles in oocyte maturation and embryo development, and their activation may prove beneficial to in vitro embryo production and livestock breeding programs. This review examines the roles of NAD+, Sirtuins and PARPs in aspects of fertility, providing insights into the potential use of NAD+-elevating treatments in livestock breeding and embryo production programs.

Structural Basis of Redox-Sensing Transcriptional Repressor Rex with Cofactor NAD+ and Operator DNA.

The transcriptional repressor Rex plays important roles in regulating the expression of respiratory genes by sensing the reduction-oxidation (redox) state according to the intracellular NAD+/NADH balance. Previously, we reported on crystal structures of apo, NAD+-bound, and NADH-bound forms of Rex from Thermotoga maritima to analyze the structural basis of transcriptional regulation depending on either NAD+ or NADH binding. In this study, the crystal structure of Rex in ternary complex with NAD+ and operator DNA revealed that the N-terminal domain of Rex, including the helix-turn-helix motif, forms extensive contacts with DNA in addition to DNA sequence specificity. Structural comparison of the Rex in apo, NAD+-bound, NADH-bound, and ternary complex forms provides a comprehensive picture of transcriptional regulation in the Rex. These data demonstrate that the conformational change in Rex when binding with the reduced NADH or oxidized NAD+ determines operator DNA binding. The movement of the N-terminal domains toward the operator DNA was blocked upon binding of NADH ligand molecules. The structural results provide insights into the molecular mechanism of Rex binding with operator DNA and cofactor NAD+/NADH, which is conserved among Rex family repressors. Structural analysis of Rex from T. maritima also supports the previous hypothesis about the NAD+/NADH-specific transcriptional regulation mechanism of Rex homologues.

Reduced Nicotinamide Mononucleotide (NMNH) Potently Enhances NAD+ and Suppresses Glycolysis, the TCA Cycle, and Cell Growth.

Decreased cellular NAD+ levels are causally linked to aging and aging-associated diseases. NAD+ precursors in oxidized form such as nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) have gained much attention and been well studied for their ability to restore NAD+ levels in model organisms. Less is known about whether NAD+ precursors in reduced form can also efficiently increase the tissue and cellular NAD+ levels and have different effects on cellular processes than NMN or NR. In the present study, we developed a chemical method to produce dihydronicotinamide mononucleotide (NMNH), which is the reduced form of NMN. We demonstrated that NMNH was a better NAD+ enhancer than NMN both in vitro and in vivo, mediated by nicotinamide mononucleotide adenylyltransferase (NMNAT). Additionally, NMNH increased the reduced NAD (NADH) levels in cells and in mouse livers. Metabolomic analysis revealed that NMNH inhibited glycolysis and the TCA cycle. In vitro experiments demonstrated that NMNH induced cell cycle arrest and suppressed cell growth. Nevertheless, NMNH treatment did not cause an observable difference in mouse weight. Taken together, our work demonstrates that NMNH is a potent NAD+ enhancer and suppresses glycolysis, the TCA cycle, and cell growth.