Synthesis Of Nicotinamide Adenine Dinucleotide Phosphate Research Articles

Detection of NADH and NADPH levels in vivo identifies shift of glucose metabolism in cancer to energy production.

Profound changes in the metabolism of cancer cells have been known for almost 100 years, and many aspects of these changes have continued to be actively studied and discussed. Differences in the results of various studies can be explained by the diversity of tumours, which have differing processes of energy metabolism, and by limitations in the methods used. Here, using fluorescence lifetime needle optical biopsy in a hepatocellular carcinoma (HCC) mouse model and patients with HCC, we measured reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) in control liver, and in HCC tumours and their adjacent regions. We found that NADH level (mostly responsible for energy metabolism) is increased in tumours but also in adjacent regions of the same liver. NADPH level is significantly decreased in the tumours of patients but increased in the HCC mouse model. However, in the ex vivo tumour slices of mouse HCC, reactive oxygen species production and glutathione level (both dependent on NADPH) were significantly suppressed. Thus, glucose-dependent NADH and NADPH production in tumours changed but with a more pronounced shift to energy production (NADH), rather than NADPH synthesis for redox balance.

Glutamine promotes the proliferation of intestinal stem cells via inhibition of TP53-induced glycolysis and apoptosis regulator promoter methylation in burned mice.

Intestinal stem cells (ISCs) play a pivotal role in maintaining intestinal homeostasis and facilitating the restoration of intestinal mucosal barrier integrity. Glutamine (Gln) is a crucial energy substrate in the intestine, promoting the proliferation of ISCs and mitigating damage to the intestinal mucosal barrier after burn injury. However, the underlying mechanism has not yet been fully elucidated. The objective of this study was to explore the mechanism by which Gln facilitates the proliferation of ISCs. A mouse burn model was established to investigate the impact of Gln on intestinal function. Subsequently, crypts were isolated, and changes in TP53-induced glycolysis and apoptosis regulator (TIGAR) expression were assessed using real-time quantitative polymerase chain reaction (RT-qPCR), western blotting, immunohistochemistry, and immunofluorescence. The effects of TIGAR on cell proliferation were validated through CCK-8, EdU, and clonogenicity assays. Furthermore, the effect of TIGAR on Yes-associated protein (YAP) nuclear translocation and ferroptosis was examined by western blotting and immunofluorescence staining. Finally, dot blot analysis and methylation-specific PCR were performed to evaluate the effect of Gln on TIGAR promoter methylation. The mRNA and protein levels of TIGAR decreased after burn injury, and supplementation with Gln increased the expression of TIGAR. TIGAR accelerates the nuclear translocation of YAP, thereby increasing the proliferation of ISCs. Concurrently, TIGAR promotes the synthesis of nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione to suppress ferroptosis in ISCs. Subsequent investigations demonstrated that Gln inhibits TIGAR promoter methylation by increasing the expression of the demethylase ten-eleven translocation. This change increased TIGAR transcription, increased NADPH synthesis, and reduced oxidative stress, thereby facilitating the restoration of intestinal mucosal barrier integrity post-burn injury. Our data confirmed the inhibitory effect of Gln on TIGAR promoter methylation, which facilitates YAP translocation into the nucleus and suppresses ferroptosis, ultimately promoting the proliferation of ISCs.

Mathematical model for glutathione dynamics in the retina

The retina is highly susceptible to the generation of toxic reactive oxygen species (ROS) that disrupt the normal operations of retinal cells. The glutathione (GSH) antioxidant system plays an important role in mitigating ROS. To perform its protective functions, GSH depends on nicotinamide adenine dinucleotide phosphate (NADPH) produced through the pentose phosphate pathway. This work develops the first mathematical model for the GSH antioxidant system in the outer retina, capturing the most essential components for formation of ROS, GSH production, its oxidation in detoxifying ROS, and subsequent reduction by NADPH. We calibrate and validate the model using experimental measurements, at different postnatal days up to PN28, from control mice and from the rd1 mouse model for the disease retinitis pigmentosa (RP). Global sensitivity analysis is then applied to examine the model behavior and identify the pathways with the greatest impact in control compared to RP conditions. The findings underscore the importance of GSH and NADPH production in dealing with oxidative stress during retinal development, especially after peak rod degeneration occurs in RP, leading to increased oxygen tension. This suggests that stimulation of GSH and NADPH synthesis could be a potential intervention strategy in degenerative mouse retinas with RP.

NADPH-dependent ROS accumulation contributes to the impaired osteogenic differentiation of periodontal ligament stem cells under high glucose conditions.

Diabetes mellitus is an established risk factor for periodontal disease that can aggravate the severity of periodontal inflammation and accelerate periodontal destruction. The chronic high glucose condition is a hallmark of diabetes-related pathogenesis, and has been demonstrated to impair the osteogenic differentiation of periodontal ligament stem cells (PDLSCs), leading to delayed recovery of periodontal defects in diabetic patients. Reactive oxygen species (ROS) are small molecules that can influence cell fate determination and the direction of cell differentiation. Although excessive accumulation of ROS has been found to be associated with high glucose-induced cell damage, the underlying mechanisms remain unclear. Nicotinamide adenine dinucleotide phosphate (NADPH) is an important electron donor and functions as a critical ROS scavenger in antioxidant systems. It has been identified as a key mediator of various biological processes, including energy metabolism and cell differentiation. However, whether NADPH is involved in the dysregulation of ROS and further compromise of PDLSC osteogenic differentiation under high glucose conditions is still not known. In the present study, we found that PDLSCs incubated under high glucose conditions showed impaired osteogenic differentiation, excessive ROS accumulation and increased NADPH production. Furthermore, after inhibiting the synthesis of NADPH, the osteogenic differentiation of PDLSCs was significantly enhanced, accompanied by reduced cellular ROS accumulation. Our findings demonstrated the crucial role of NADPH in regulating cellular osteogenic differentiation under high glucose conditions and suggested a new target for rescuing high glucose-induced cell dysfunction and promoting tissue regeneration in the future.

Effectiveness and mechanism study of glutamine on alleviating hypermetabolism in burned rats.

This study aimed to explore the effects of glutamine on hypermetabolic reactions in burned rats and its underlying mechanism. Fifty-five Sprague-Dawley rats were randomly divided into three groups, namely, the control (C), burned (B), and burned+glutamine (B+G) groups. Rats in the glutamine treatment group were supplemented with 1 g glutamine per kg body weight. Changes in body weight and resting energy expenditure in all groups were observed daily. Blood glucose and glucose tolerance level were measured on days 1, 3, 7, 10 and 14 after burn injury. On days 7 and 14 after injury, the rats were sacrificed, and the weight and protein content of the skeletal muscle were measured. Moreover, the level of glutamine, inflammatory mediator, nicotinamide adenine dinucleotide phosphate (NADPH), glutathione, and the activity of glutamine metabolic enzymes were measured. The hypermetabolic reaction after burn injury was significantly inhibited by glutamine administration, and the range of variations in the resting energy expenditure and body weight indicators was narrowed remarkably (P < 0.05 or 0.01), whereas the weight and protein content of the skeletal muscle returned to normal (P < 0.05 or 0.01). Glutamine could increase glutaminase activity in various tissues, promote the utilization of glutamine, and appropriately reduce the degree of organ damage and inflammatory response (P < 0.05 or 0.01). Furthermore, glutamine could promote the synthesis of the reducing substances NADPH and glutathione (P < 0.05 or 0.01). Glutamine administration effectively reduces hypermetabolic reactions by promoting NADPH synthesis, inhibiting oxidative stress, and improving glutamine utilization after burn injury.

Measurement of Chloroplastic NAD Kinase Activity and Whole Tissue NAD Kinase Assay.

Nicotinamide adenine dinucleotide phosphate (NADP) synthesis requires nicotinamide adenine dinucleotide (NAD) kinase activity, substrate NAD and ATP. The NAD kinase responds to various environmental stimuli and its activity is regulated via various regulatory pathways, such as Ca2+-dependent and redox-dependent signals. Conventional in vitro NAD kinase assay has been useful to evaluate enzyme activity; however, recent reports revealed a dynamics of NADP pool (the sum of NADP+ and NADPH) under fluctuating light condition, indicating that the rate of NADP synthesis is not always determined by NAD kinase activity. Here, we developed a novel method for the estimation of chloroplastic NAD kinase activity by quantifying the changes in the NADP amounts in response to illumination. As our approach does not involve protein extraction, it saves time (compared to the in vitro assay), thereby allowing for a sequence of assays, and provides several clues in the investigation of regulatory mechanisms behind NADP synthesis under various environmental conditions.

Betaine supplementation improved l-threonine fermentation of Escherichia coli THRD by upregulating zwf (glucose-6-phosphate dehydrogenase) expression

Abstract Background The supplementation of betaine, an osmoprotective compatible solute, in the cultivation media has been widely used to protect bacterial cells. To explore the effects of betaine addition on industrial fermentation, Escherichia coli THRD, an l -threonine producer, was used to examine the production of l -threonine with betaine supplementation and the underlying mechanism through which betaine functions was investigated. Results Betaine supplementation in the medium of E. coli THRD significantly improved l -threonine fermentation parameters. The transcription of zwf and corresponding enzyme activity of glucose-6-phosphate dehydrogenase were significantly promoted by betaine addition, which contributed to an enhanced expression of zwf that provided more nicotinamide adenine dinucleotide phosphate (NADPH) for l -threonine synthesis. In addition, as a result of the betaine addition, the betaine-stimulated expression of enhanced green fluorescent protein (eGFP) under the zwf promoter within a plasmid-based cassette proved to be a transcription-level response of zwf. Finally, the promoter of the phosphoenolpyruvate carboxylase gene ppc in THRD was replaced with that of zwf, while l -threonine fermentation of the new strain was promoted by betaine addition. Conclusions We reveal a novel mode of betaine that facilitates the microbial production of useful compounds. Betaine supplementation upregulates the expression of zwf and increases the NADPH synthesis, which may be beneficial for the cell growth and thereby promote the production of l -threonine. This finding might be useful for the production of NADPH-dependent amino acids and derivatives in E. coli THRD or other E. coli strains. How to cite: Li Y, Zhang D, Cai N, et al. Betaine supplementation improved l -threonine fermentation of Escherichia coli THRD by upregulating zwf (glucose-6-phosphate dehydrogenase) expression. Electron J Biotechnol 2019;39. https://doi.org/10.1016/j.ejbt.2019.03.004 .

Topology of TROL protein in thylakoid membranes of Arabidopsis thaliana.

Thylakoid rhodanase-like protein (TROL) is a nuclear-encoded protein of thylakoid membranes required for tethering of ferredoxin:nicotinamide adenine dinucleotide phosphate (NADPH) oxydoreductase (FNR). It has been proposed that the dynamic interaction of TROL with flavoenzyme FNR, influenced by environmental light conditions, regulates the fate of photosynthetic electrons, directing them either to NADPH synthesis or to other acceptors, including reactive oxygen species detoxification pathways. Inside the chloroplasts, TROL has a dual localization: an inner membrane precursor form and a thylakoid membrane mature form, which has been confirmed by several large-scale chloroplast proteomics studies, as well as protein import experiments. Unlike the localization, the topology of TROL in the membranes, which is a prerequisite for further studies of its properties and function, has not been experimentally confirmed yet. Thermolysin was proven to be a valuable protease to probe the surface of chloroplasts and membranes in general. By treating the total chloroplast membranes using increasing protease concentration, sequential degradation of TROL was observed, indicating protected polypeptides of TROL and possible domain orientation. To further substantiate the obtained results, TROL-overexpressing Arabidopsis line (OX) and line in which the central rhodanase-like domain (RHO) has been partially deleted (ΔRHO), were used as well. While OX line showed the same degradation pattern of TROL as the wild-type, surprisingly, TROL from ΔRHO membranes was not detectable even at the lowest protease concentration applied, indicating the importance of this domain to the integrity of TROL. In conclusion, TROL is a polytopic protein with a stroma-exposed C-terminal FNR-binding region, and the thylakoid lumen-located RHO domain.

Functional identification and subcellular localization of NAD kinase in the protozoan parasite Giardia intestinalis

Nicotinamide adenine dinucleotide phosphate (NADP) is an essential biomolecule that participates in the redox homeostasis and synthesis of signaling compounds. NAD kinase (NADK) (EC 2.7.1.23 / 2.7.1.86) is the only enzyme capable of synthesizing NADP. This study offers an approach to the NADP metabolism in the parasite Giardia intestinalis, the etiologicalagent of giardiasis, a disease of high prevalence in America, Asia and Africa. Through bioinformatics tools a NADK enzyme candidate was identified, whose tertiary structure modeling demonstrated distinctive and universal motifs of characterized NADKs. The corresponding recombinant protein (His-GINADK) was expressed in Escherichia coliBL21 (DE3) and its partial purification was achieved by nickel affinity chromatography. Functional identification, which showed NADP synthesis, was completed through enzymatic assays evaluated by RP-HPLC. A cytosolic localization of the endogenous GINADK enzyme was observed in trophozoites throughout indirect immunofluorescence analysis, using polyclonal antibodies produced in mice by its immunization with the His-GINADK protein, purified from inclusion bodies. Taken together, our results contribute to the understanding of the NADP metabolism and the physiological role of NADK in the Giardiamodel.

Aspirin inhibits glucose‑6‑phosphate dehydrogenase activity in HCT 116 cells through acetylation: Identification of aspirin-acetylated sites.

Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first reaction in the pentose phosphate pathway, and generates ribose sugars, which are required for nucleic acid synthesis, and nicotinamide adenine dinucleotide phosphate (NADPH), which is important for neutralization of oxidative stress. The expression of G6PD is elevated in several types of tumor, including colon, breast and lung cancer, and has been implicated in cancer cell growth. Our previous study demonstrated that exposure of HCT 116 human colorectal cancer cells to aspirin caused acetylation of G6PD, and this was associated with a decrease in its enzyme activity. In the present study, this observation was expanded to HT-29 colorectal cancer cells, in order to compare aspirin-mediated acetylation of G6PD and its activity between HCT 116 and HT-29 cells. In addition, the present study aimed to determine the acetylation targets of aspirin on recombinant G6PD to provide an insight into the mechanisms of inhibition. The results demonstrated that the extent of G6PD acetylation was significantly higher in HCT 116 cells compared with in HT-29 cells; accordingly, a greater reduction in G6PD enzyme activity was observed in the HCT 116 cells. Mass spectrometry analysis of aspirin-acetylated G6PD (isoform a) revealed that aspirin acetylated a total of 14 lysine residues, which were dispersed throughout the length of the G6PD protein. One of the important amino acid targets of aspirin included lysine 235 (K235, in isoform a) and this corresponds to K205 in isoform b, which has previously been identified as being important for catalysis. Acetylation of G6PD at several sites, including K235 (K205 in isoform b), may mediate inhibition of G6PD activity, which may contribute to the ability of aspirin to exert anticancer effects through decreased synthesis of ribose sugars and NADPH.

Abstract B47: Modulating the NQO1-dependent ‘kiss of death’ mechanism of action of NQO1 bioactivatable drugs

Abstract Metabolic adaptations in tumors are necessary to balance the need for cellular energy supply, macromoleclar biosynthesis and maintenance of redox balance. A critical molecule produced as a result of this altered metabolism is nicotinamide adenine dinucleotide phosphate (NADPH). NADPH is a critical co-factor that provides reducing power to maintain cytoprotective concentrations of reduced glutathione against reactive oxygen species (ROS) that are produced during rapid growth and proliferation. In pancreatic cancers, NADPH can be formed by the NAD+ salvage pathway through the enzyme, Nicotinamide phosphoribosyltransferase (NamPT), as well as through the non-canonical metabolism of glutamine through glutaminase-1 (GLS1). While NamPT and GLS1 inhibitors have shown some anti-tumor activity in vitro and in vivo, these inhibitors lack tumor specificity and can have an extensive toxicity profile due to the requirement of NADPH synthesis in proliferating normal cells/tissue. We recently discovered that NAD(P)H:quinone oxidoreductase 1 (NQO1) levels were elevated 5- to 40-fold in &amp;gt;80% of pancreatic tumors vs associated normal tissue. As important, we noted that catalase levels were inversely expressed comparatively, elevated in normal and suppressed in tumor tissue. Thus, NQO1 represents a unique target to exploit for the therapeutic elimination of pancreatic cancers. While NQO1 is normally a detoxifying (Phase II) enzyme, we have discovered two unique classes of compounds that are ‘bioactivated’ in a futile redox cycle by the enzyme. NQO1 bioactivatable drugs, such as β-lapachone (β-lap) and deoxynyboquinone (DNQ), generate hydrogen peroxide as a mechanism to hyperactivate poly(ADP-ribose) polymerase 1 (PARP1), and to rapidly deplete NAD+ and ATP in a selective manner to kill tumors. Normal cells are protected due to low NQO1 and relatively high Catalase levels. We found that the tumor specificity and efficacy of NamPT or GLS1 inhibition can be greatly increased when small molecule NamPT inhibitors (e.g., FK866) or GLS1 inhibitors (e.g., BPTES) are used in combination with NQO1-bioactivatable drugs. With combination treatment, cells are primed for ROS-mediated damage due to reduced glutathione and NADPH levels; they are unable to recover ATP, NAD+, and NADPH synthesis even after only a 2 h exposure to NQO1 bioactivatable drugs, and they rapidly die through a caspase-independent, parthanatos (programmed necrosis) mechanism. Our findings indicate that NQO1-bioactivatable drugs may improve the sensitivities of clinical grade GLS1 and NamPT inhibitors in pancreatic cancer patients. This work was supported by an AACR/Pancreatic Cancer Action Network - George and June Block Foundation - Innovator Award and an NIH/NCI R01 CA102971 grant to DAB. Citation Format: Zachary Moore, Gaurab Chakrabarti, Costas Lyssiotis, Ralph Deberardinis, Boothman A. David. Modulating the NQO1-dependent ‘kiss of death’ mechanism of action of NQO1 bioactivatable drugs. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B47.

Glutamine: Role in the Fetus and Low-birthweight Infant

After completing this article, readers should be able to: 1. Describe the glutamine-glutamate shuttle. 2. Delineate the effects of glutamine supplementation during total parenteral nutrition, chemotherapy, and radiotherapy in animals. 3. Describe the beneficial effects of glutamine supplementation in low-birthweight infants and in critically ill adults. 4. List the primary actions of glutamine in the intestine. The importance of glutamine for optimal growth of cells in culture has been known since the 1950s, when Eagle published his landmark paper in Science .1 His work demonstrated that of all the amino acids, cell growth in culture relied most on glutamine. Numerous subsequent studies have established the importance of glutamine for a variety of metabolic processes in the entire organism and are beginning to demonstrate that nutritional supplementation of this amino acid may be highly beneficial under certain circumstances. Glutamine and glutamate are structurally similar five-carbon amino acids. Glutamine is a neutral amino acid at pH 7.0 that contains an alpha-amino group and a deltaamide group. In glutamate, the amide nitrogen of glutamine is replaced by a carboxy-group, conferring to it a net negative charge at pH 7.0. This at least partially accounts for the transport of these two amino acids across cell membranes via different transport systems. The presence or absence of the amide group is responsible for different metabolic interactions of these amino acids. The array of metabolic processes in which glutamine is involved is highlighted by its relationship to its metabolites and the citric acid cycle (Fig. 1⇓ ).Glutamine, via glutamate, is readily converted to alpha-ketoglutarate, an integral component of the citric acid cycle. Deamidation of glutamine via glutaminase produces glutamate, a precursor of gamma-amino butyric acid (GABA), a neurotransmission inhibitor. Proline also is produced by the cyclization of glutamate. Proline is an important amino acid component of collagen and …

Control of nicotinamide-adenine dinucleotide phosphate synthesis in the livers of rats treated with ethionine.

INJECTION of tryptophan, nicotinic acid or, particularly, nicotinamide leads to high hepatic levels of NAD but very little increase in the hepatic NADP content1–3. This suggests that at super-normal levels of NAD factors other than NAD may be rate-limiting in NADP synthesis. NADP synthesis is a single-step reaction requiring only three components: two substrates, NAD and ATP, and one supernatant enzyme, NAD+-kinase (E.C. 2.7.1.23). Using the observation of Shull4 that ethionine injection causes a drastic fall in the hepatic ATP concentration in vivo, Greenbaum, Clark and McLean5 studied the effect of a lowered ATP concentration on the synthesis of NAD and NADP 5 h after ethionine was injected. They found that neither the enzyme synthesizing NAD (NMN adenylyl transferase, E.C. 2.7.7.1) nor the enzyme forming NADP (NAD+-kinase) was affected at this particular time. However, while NAD fell only slightly, NADP was reduced to about 60 per cent of the control value. It was suggested that ATP was involved in the control of NADP synthesis.