Fig 3 - uploaded by Manoj N Sonavane
Content may be subject to copyright.
NRH induced PUMA and BAX-mediated apoptosis in HepG3 cells. Apoptosis in HEK293T and HepG3 cells were assessed using immunoblot after 24, 48, and 72 h of NRH exposure. Non-significant increases in the BAX and PUMA protein expression levels were observed in HEK293T cells (A). However, significantly increased expression of the apoptotic markers, BAX and PUMA were observed in HepG3 (D). The graph shows the protein expression levels relative to controls in HEK293T (B and C) and in HepG3 (E and F) cells. Results are expressed as the average of at least two biological replicates ± SEM. Statistical significance: � P < 0.05, �� P < 0.01, ���� P < 0.001. https://doi.org/10.1371/journal.pone.0242174.g003
Source publication
Nicotinamide adenine dinucleotide (NAD+), the essential cofactor derived from vitamin B3, is both a coenzyme in redox enzymatic processes and substrate in non-redox events; processes that are intimately implicated in all essential bioenergetics. A decrease in intracellular NAD+ levels is known to cause multiple metabolic complications and age-relat...
Contexts in source publication
Context 1
... examine the mechanism by which NRH exposure induced cell death, HEK293T and HepG3 cells were exposed to NRH at IC 90 concentration (i.e., 500 μM) for 24 h, then assessed the protein expression levels of the apoptotic markers after 24, 48, and 72 h using immunoblot (Fig 3). HEK293T cells exposed to NRH showed no significant change in the apoptotic markers, BAX or the p53 upregulated modulator of apoptosis (PUMA), at all the time points ( Fig 3A-3C). ...
Context 2
... examine the mechanism by which NRH exposure induced cell death, HEK293T and HepG3 cells were exposed to NRH at IC 90 concentration (i.e., 500 μM) for 24 h, then assessed the protein expression levels of the apoptotic markers after 24, 48, and 72 h using immunoblot (Fig 3). HEK293T cells exposed to NRH showed no significant change in the apoptotic markers, BAX or the p53 upregulated modulator of apoptosis (PUMA), at all the time points ( Fig 3A-3C). As expected, HepG3 cells showed a significant increase in BAX levels at 24 h (40 ± 3.5% increase, relative to control), which further increases at 48 h (50 ± 8.6%) and attained maximum expression at 72 h (110 ± 13.8%) (Fig 3D and 3E). ...
Context 3
... cells exposed to NRH showed no significant change in the apoptotic markers, BAX or the p53 upregulated modulator of apoptosis (PUMA), at all the time points ( Fig 3A-3C). As expected, HepG3 cells showed a significant increase in BAX levels at 24 h (40 ± 3.5% increase, relative to control), which further increases at 48 h (50 ± 8.6%) and attained maximum expression at 72 h (110 ± 13.8%) (Fig 3D and 3E). We further demonstrated that this BAX-induced apoptosis is mediated by PUMA, which showed significantly increased expression levels at 48 h (140 ± 41.2%) and 72 h (150 ± 2.5%) of NRH exposure ( Fig 3D and 3F). ...
Context 4
... expected, HepG3 cells showed a significant increase in BAX levels at 24 h (40 ± 3.5% increase, relative to control), which further increases at 48 h (50 ± 8.6%) and attained maximum expression at 72 h (110 ± 13.8%) (Fig 3D and 3E). We further demonstrated that this BAX-induced apoptosis is mediated by PUMA, which showed significantly increased expression levels at 48 h (140 ± 41.2%) and 72 h (150 ± 2.5%) of NRH exposure ( Fig 3D and 3F). ...
Context 5
... is an immortalized cell line more comparable to primary hepatocytes than HepG2 due to its higher metabolic capacity from cytochrome P-450, high albumin, and alpha fetoprotein production [47]. NRH exposure increased BAX and PUMA protein expression levels, inducing mitochondrial-mediated apoptosis (Fig 3D-3F) [48,49]. These observations suggest that cell-specific responses to NAD increases can cause an imbalance in intra-organelle NAD pools causing cellular dysfunction and eventually inducing cell death. ...
Citations
... Unlike NR, which is phosphorylated to NMN by Nrk, NRH is phosphorylated by adenosine kinase (AK) and converted to NMNH and subsequently to NADH and NAD + [26,27]. NRH can improve mitochondrial function and regulate oxidative stress [28]. NMNH is the reduced form of NMN, which is converted to NAD + by NMNAT and inhibits glycolysis, the TCA cycle, and cell growth [29]. ...
Nicotinamide adenine dinucleotide (NAD⁺) is an essential coenzyme involved in many pathophysiological processes. Supplementation with NAD⁺ and its precursors has been demonstrated as an emerging therapeutic strategy for the diseases. NAD⁺ also plays an important role in the reproductive system. Here, we summarize the function of NAD⁺ in various reproductive diseases and review the application of NAD⁺ and its precursors in the preservation of reproductive capacity and the prevention of embryonic malformations. It is shown that NAD⁺ shows good promise as a therapeutic approach for saving reproductive capacity.
... H9c2 cells were plated at 0.1 × 10 6 density in 60-mm dishes and were left to incubate for two days in a 5% CO 2 incubator at 37 • C. After incubation, cells were dosed with either 1, 2, or 14 mM DHA or 100 μM NRH for 24 h as a positive control [28,34]. Compounds were prepared in a phenol-free medium as the phenol interfered with the autofluorescence detection. ...
... We also checked the highly cytotoxic 14 mM DHA dose, and no changes in NAD(P)H levels were measured (Suppl Fig. 10). We confirmed changes are specific to the NAD(P)H pool by also dosing cells with 100 μM dihydronicotinamide riboside hydride (NRH), which directly boosts the NAD(P)H pool [28,34]. Interestingly, the increase in NAD(P)H produced by NRH is equivalent to that produced by 2 mM DHA (Fig. 13B). ...
... This is also true for melatonin, an endogenous and non-specific inhibitor of QR2, a function that is thought to convey some of the antioxidant properties of melatonin (14). Loss of these QR2-controlling elements is injurious twofold -first, the removal of QR2 from interneurons is critical for novel memory formation and this process is lost (7); and second, chronically elevated levels of QR2 cause oxidative/metabolic stress (6,8,15). Importantly, in a rodent model of scopolamine-induced amnesia this pathway is blocked and QR2 levels remain high (16), but by directly inhibiting QR2 in this model, memory deficits are reversed (6). ...
... There is evidence of QR2 mediated metabolic stress in human cells (6,8,15). However, it is unknown how QR2 activity may affect the proteome in response to the chronic stress it generates. ...
... In each section, the same region of CA1 was acquired, using the same image frame size and resolution, at all possible depths of the section (Z-stack of the whole section), to allow measurement of the fluorescent antibody marker volume, which was normalized to the brain volume from which it was taken. 15 while controls showed far greater variability ( Figure 7A, right histogram, and image panels). ...
Biological aging can be described as accumulative, prolonged metabolic stress, and is the major risk factor for cognitive decline and Alzheimer's disease (AD). Recently, we identified and described a quinone reductase 2 (QR2) pathway in the brain, in which QR2 acts as a removable memory constraint and metabolic buffer within neurons. QR2 becomes over-expressed with age, and is possibly a novel contributing factor to age-related metabolic stress and cognitive deficit. We found that in human cells, genetic removal of QR2 produces a shift in the proteome opposing that found in AD brains, while simultaneously reducing oxidative stress. We therefore created highly specific QR2 inhibitors (QR2i's), enabling evaluation of chronic QR2 inhibition as a novel way to reduce biological-age related metabolic stress and cognitive decline. QR2i's replicated results obtained by genetic removal of QR2 while local QR2i microinjection improved hippocampal and cortical dependent learning in rats and mice. Continuous consumption of QR2i's in drinking-water improved cognition and reduced pathology in the brains of AD-model mice (5xFAD), with a noticeable between-sex effect on treatment duration. These results demonstrate the importance of QR2 activity- and pathway function in the healthy and neurodegenerative brain, and the great therapeutic potential of QR2i's as first-in-class drugs.
... The catabolites that are known and measured see their abundance and distribution change depending on the precursor applied, the animal model, the clinical condition being investigated, and the biospecimens being measured [68,[71][72][73]. Overall, the nature and the effects of catabolites of NAD + and NADH and their sustained endogenous increase in disease or upon supplementation remain poorly understood, although concerns have been raised [73][74][75][76][77]. This warrants the need for more robust identification and characterization of the catabolites of NAD(P)(H). ...
Dietary vitamin B3 components, such as nicotinamide and nicotinic acid, are precursors to the ubiquitous redox cofactor nicotinamide adenine dinucleotide (NAD+). NAD+ levels are thought to decline with age and disease. While the drivers of this decline remain under intense investigation, strategies have emerged seeking to functionally maintain NAD+ levels through supplementation with NAD+ biosynthetic intermediates. These include marketed products, such as nicotinamide riboside (NR) and its phosphorylated form (NMN). More recent developments have shown that NRH (the reduced form of NR) and its phosphorylated form NMNH also increases NAD+ levels upon administration, although they initially generate NADH (the reduced form of NAD+). Other means to increase the combined levels of NAD+ and NADH, NAD(H), include the inhibition of NAD+-consuming enzymes or activation of biosynthetic pathways. Multiple studies have shown that supplementation with an NAD(H) precursor changes the profile of NAD(H) catabolism. Yet, the pharmacological significance of NAD(H) catabolites is rarely considered although the distribution and abundance of these catabolites differ depending on the NAD(H) precursor used, the species in which the study is conducted, and the tissues used for the quantification. Significantly, some of these metabolites have emerged as biomarkers in physiological disorders and might not be innocuous. Herein, we review the known and emerging catabolites of the NAD(H) metabolome and highlight their biochemical and physiological function as well as key chemical and biochemical reactions leading to their formation. Furthermore, we emphasize the need for analytical methods that inform on the full NAD(H) metabolome since the relative abundance of NAD(H) catabolites informs how NAD(H) precursors are used, recycled, and eliminated.
... A careful examination of the tradeoff between benefits and risk factors is necessary. Secondly, determining a safety range for NAD boosting is essential since excessive levels of NAD can be harmful, as shown by reported oxidative cytotoxicity induced by the potent NAD precursor NRH [207]. Lastly, since NAD is in a dynamic equilibrium between synthesis and consumption, it is perhaps more important to keep NAD in a healthy salvaging state rather than elevating the stable state of NAD levels. ...
As the aging population continues to grow rapidly, age-related diseases are becoming an increasing burden on the healthcare system and a major concern for the well-being of elderly individuals. While aging is an inevitable process for all humans, it can be slowed down and age-related diseases can be treated or alleviated. Nicotinamide adenine dinucleotide (NAD) is a critical coenzyme or cofactor that plays a central role in metabolism and is involved in various cellular processes including the maintenance of metabolic homeostasis, post-translational protein modifications, DNA repair, and immune responses. As individuals age, their NAD levels decline, and this decrease has been suggested to be a contributing factor to the development of numerous age-related diseases, such as cancer, diabetes, cardiovascular diseases, and neurodegenerative diseases. In pursuit of healthy aging, researchers have investigated approaches to boost or maintain NAD levels. Here, we provide an overview of NAD metabolism and the role of NAD in age-related diseases and summarize recent progress in the development of strategies that target NAD metabolism for the treatment of age-related diseases, particularly neurodegenerative diseases.
... Gene & Protein in Disease NQO2 and dopamine toxicity versus detoxification the synthetic N-benzyl-dihydronicotinamide (BNAH) or the natural ones: N-methyl-dihydronicotinamide (NMH) and N-ribosyl-dihydronicotinamide (NRH). The concentration of the latter remains mostly elusive in resting tissues [14][15][16] . This very fact has led to a key controversy on whether NQO2 is an enzyme with catalytic activity or a pseudo-enzyme without catalytic activity [17] , although it remains clear that the same enzyme, NQO2, is present in the genome for several millions of years with the same unique co-substrate recognition as it has been cloned from Anas platyrhynchos and Alligator mississippiensis [17] . ...
... In brief, the amount of available UGT co-substrate, UDP-glucuronic acid, is enough to permit the elimination of dopamine as glucuronide, and most importantly, to identify and quantify the amount of NQO2 co-substrate essential to its activity. Among the possible co-substrates are either NRH, sometimes described as resulting from NADH catabolism, or various nicotinamide catabolites [15,16] . What would make the most sense would be that, under lethal stress, NADH breakdown occurs because cells are in search of adenosine source. ...
NQO2 and tyrosine hydroxylase are co-expressed in dopaminergic neurons. These neurons produce dopamine, a diol, which, under aerobic conditions, can spontaneously revert to the more stable form, the o-quinone. O-quinones are preferred substrates of NQO2 over p-quinones. In ad hoc conditions, NQO2 reduces o-quinones into the original diols, leading to a futile cycle, the endpoint of which is a strong local production of reactive oxygen species that is deadly for the cells. This futile cycle can be interrupted by the conjugation of dopamine with UDP-glucuronic acid, leading to a glucuronide that cannot be part of the cycle because the glucuronide is not a substrate of NQO2. In this paper, we confer whether this futile cycle could be one of the causes of the specific death of dopaminergic neuronal population that is the signature of some degenerative diseases.
... To determine mitochondrial superoxide and other reactive oxygen species formed by DHAexposed HepG3 cells, MitoSOX live imaging experiments are performed using the mitochondrial-targeted superoxide-sensitive fluorogenic probe MitoSOX TM Red (Life Technologies, Carlsbad, CA, USA) as previously described [20]. Briefly, cells were plated at 20,000 cells per well in an 8-well chamber dish and left ON to attach at 37˚C in a 5% CO 2 incubator. ...
Dihydroxyacetone (DHA) is the active ingredient in sunless tanning products and a combustion product from e-juices in electronic cigarettes (e-cigarettes). DHA is rapidly absorbed in cells and tissues and incorporated into several metabolic pathways through its conversion to dihydroxyacetone phosphate (DHAP). Previous studies have shown DHA induces cell cycle arrest, reactive oxygen species, and mitochondrial dysfunction, though the extent of these effects is highly cell-type specific. Here, we investigate DHA exposure effects in the metabolically active, HepG3 (C3A) cell line. Metabolic and mitochondrial changes were evaluated by characterizing the effects of DHA in metabolic pathways and nutrient-sensing mechanisms through mTOR-specific signaling. We also examined cytotoxicity and investigated the cell death mechanism induced by DHA exposure in HepG3 cells. Millimolar doses of DHA were cytotoxic and suppressed glycolysis and oxidative phosphorylation pathways. Nutrient sensing through mTOR was altered at both short and long time points. Increased mitochondrial reactive oxygen species (ROS) and mitochondrial-specific injury induced cell cycle arrest and cell death through a non-classical apoptotic mechanism. Despite its carbohydrate nature, millimolar doses of DHA are toxic to liver cells and may pose a significant health risk when higher concentrations are absorbed through e-cigarettes or spray tanning.
... In contrast to other NAD + precursors, however, NRH can manifest cellular toxicity. HepG3 cells treated with NRH showed some signs of cellular toxicity, related to an increase in mitochondrial superoxide production [266]. Interestingly, these effects were cell-specific, as NRH did not prompt toxic effects in HEK293 [266]. ...
... HepG3 cells treated with NRH showed some signs of cellular toxicity, related to an increase in mitochondrial superoxide production [266]. Interestingly, these effects were cell-specific, as NRH did not prompt toxic effects in HEK293 [266]. Nevertheless, this could be in line with the increased PARylation that has been observed in response to NRH treatment in cultured cells as well as in mouse liver and kidney tissues [128,264,266,267]. ...
... Interestingly, these effects were cell-specific, as NRH did not prompt toxic effects in HEK293 [266]. Nevertheless, this could be in line with the increased PARylation that has been observed in response to NRH treatment in cultured cells as well as in mouse liver and kidney tissues [128,264,266,267]. ...
Alterations in cellular nicotinamide adenine dinucleotide (NAD⁺) levels have been observed in multiple lifestyle and age-related medical conditions. This has led to the hypothesis that dietary supplementation with NAD⁺ precursors, or vitamin B3s, could exert health benefits. Among the different molecules that can act as NAD⁺ precursors, Nicotinamide Riboside (NR) has gained most attention due to its success in alleviating and treating disease conditions at the pre-clinical level. However, the clinical outcomes for NR supplementation strategies have not yet met the expectations generated in mouse models. In this review we aim to provide a comprehensive view on NAD⁺ biology, what causes NAD⁺ deficits and the journey of NR from its discovery to its clinical development. We also discuss what are the current limitations in NR-based therapies and potential ways to overcome them. Overall, this review will not only provide tools to understand NAD⁺ biology and assess its changes in disease situations, but also to decide which NAD⁺ precursor could have the best therapeutic potential.
... Recently, two novel and potent NAD reduced precursors, NRH and NMNH, have been described and their roles been explored in vitro and in vivo (14)(15)(16)(17)(18). NRH and NMNH boost NAD + levels faster, and to much higher levels than NR and NMN (14,(16)(17)(18). ...
... Recently, two novel and potent NAD reduced precursors, NRH and NMNH, have been described and their roles been explored in vitro and in vivo (14)(15)(16)(17)(18). NRH and NMNH boost NAD + levels faster, and to much higher levels than NR and NMN (14,(16)(17)(18). Also, the NAD + boosting occurs independent of the enzymes NR kinase (NRK) and NAMPT (14), which are involved in NAD production in the salvage pathway. ...
... In addition, investigating the effects of these reduced NAD precursors can help us understand the physiological consequences of extreme boosting in NAD + levels, and to develop therapies to metabolically reprogram immune cells. In addition, unlike other NAD precursors, NRH is converted to NADH first, before it is oxidized to NAD + (14,16). Therefore, in addition to boosting NAD + , NRH supplementation may overwhelm cells with NADH, which may have significant consequences to cell function. ...
Nicotinamide adenine dinucleotide (NAD) metabolism plays an important role in the regulation of immune function. However, a complete picture of how NAD, its metabolites, precursors, and metabolizing enzymes work together in regulating immune function and inflammatory diseases is still not fully understood. Surprisingly, few studies have compared the effect of different forms of vitamin B3 on cellular functions. Therefore, we investigated the role of NAD boosting in the regulation of macrophage activation and function using different NAD precursors supplementation. We compared nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), and nicotinamide (NAM) supplementation, with the recently described potent NAD precursor NRH. Our results show that only NRH supplementation strongly increased NAD ⁺ levels in both bone marrow-derived and THP-1 macrophages. Importantly, NRH supplementation activated a pro-inflammatory phenotype in resting macrophages, inducing gene expression of several cytokines, chemokines, and enzymes. NRH also potentiated the effect of lipopolysaccharide (LPS) on macrophage activation and cytokine gene expression, suggesting that potent NAD ⁺ precursors can promote inflammation in macrophages. The effect of NRH in NAD ⁺ boosting and gene expression was blocked by inhibitors of adenosine kinase, equilibrative nucleoside transporters (ENT), and IκB kinase (IKK). Interestingly, the IKK inhibitor, BMS-345541, blocked the mRNA expression of several enzymes and transporters involved in the NAD boosting effect of NRH, indicating that IKK is also a regulator of NAD metabolism. In conclusion, NAD precursors such as NRH may be important tools to understand the role of NAD and NADH metabolism in the inflammatory process of other immune cells, and to reprogram immune cells to a pro-inflammatory phenotype, such as the M2 to M1 switch in macrophage reprogramming, in the cancer microenvironment.
... 46 DNR did appear to show metabolic dysregulation that altered mitochondrial respiration. 47 NMNH also demonstrated differences over its oxidized form, including raising NAD levels more rapidly and doubling the levels of NAD available. 48 Although heightened levels of NMNH did appear to deliver many health benefits such as renal tubular epithelial healing in mice. ...
This scoping review aims to perform a brief but comprehensive assessment of existing peer‐reviewed literature and determine whether raising nicotinamide adenine dinucleotide can prevent or promote tumorigenesis. The examination of extensive peer‐reviewed data regarding the synthesis of nicotinamide adenine dinucleotide has been performed with a focus on nuclear dynamics and the deoxyribose nucleic acid repair pathway. Various enzymatic protective functions have been identified from nicotinamide adenine dinucleotide levels, as well as the threat role that is also explored. Nicotinamide adenine dinucleotide precursors and sirtuin‐activating compounds are becoming ubiquitous in the commercial market. Further research into whether elevating levels of nicotinamide adenine dinucleotide or overexpression of sirtuins can increase the potential for neoplasm or other age‐related pathophysiology is warranted due to the high energy requirements of certain diseases such as cancer. Pathways modulating NAD+ content in mammals. Intermediates of the amidated on the left side of NAD+ and deamidated routes on the right side of NAD+, respectively. Orange color indicates NAD+‐consuming enzymes competing with sirtuins for NAD+ availability. MNA and AMS are metabolites not recycled in the NAD+ synthesis pathway.
