Nicotinamide phosphoribosyltransferase delays cellular senescence by upregulating SIRT1 activity and antioxidant gene expression in mouse cells

Senescent cells accumulate in tissues of aged animals and deteriorate tissue functions. The elimination of senescent cells from aged mice not only attenuates progression of already established age‐related disorders, but also extends median lifespan. Nicotinamide phosphoribosyltransferase (NAMPT), the rate‐limiting enzyme in mammalian NAD⁺ salvage pathway, has shown a protective effect on cellular senescence of human primary cells. However, it still remains unclear how NAMPT has a protective impact on aging in vitro and in vivo. In this study, we found that primary mouse embryonic fibroblast (MEF) cells undergo progressive decline of NAMPT and NAD⁺ contents during serial passaging before becoming senescent. Furthermore, we showed that constitutive Nampt over‐expression increases cellular NAD⁺ content and delays cellular senescence of MEF cells in vitro. We further found that constitutive Nampt over‐expression increases SIRT1 activity, increases the expression of antioxidant genes, superoxide dismutase 2 and catalase and promotes resistance against oxidative stress. These findings suggest that Nampt over‐expression in MEF cells delays cellular senescence by the mitigation of oxidative stress via the upregulation of superoxide dismutase 2 and catalase gene expressions by SIRT1 activation.     

USP7 Deubiquitinates and Stabilizes SIRT1

The NAD⁺-dependent protein deacetylase silent information regulator 1 (SIRT1) targets multiple proteins for deacetylation, and it has been implicated in a variety of cellular pathways and human diseases. However, it remains unclear how the abundance of SIRT1 is regulated. Here, by mass spectrometry analysis of SIRT1-containing protein complexes, we revealed that SIRT1 is physically associated with the ubiquitin-specific protease USP7. Importantly, we found that USP7 cleaves K48-linked polyubiquitin chains of SIRT1 and promotes SIRT1 stabilization. Accordingly, we demonstrated that treatment of cells with an enzymatic inhibitor of USP7 led to a decreased level of SIRT1 expression and accumulation of SIRT1 polyubiquitination. Collectively, our findings indicate that USP7 is a critical regulator of SIRT1 and provide a new pathway for the maintenance of SIRT1 abundance in cells. Anat Rec, 303:1337–1345, 2020. © 2019 American Association for Anatomy     

Role of SIRT1 and AMPK in mesenchymal stem cells differentiation

The differentiation capabilities of mesenchymal stem cells (MSCs) compromise with age and with in vitro passages which could impair the efficacy of cell therapy and tissue engineering. However, how to maintain these capabilities is not fully understood. Calorie restriction (CR, decreasing caloric intake by 30–40%) could extend longevity and reduce aging-related diseases. Recent studies revealed that CR could influence the lineage determination of stem cells including MSCs. Two important mediators of CR might be silent mating type information regulation 2 homolog 1 (SIRT1), a NAD⁺-dependent deacetylase, and AMP-activated protein kinase (AMPK), an energy-sensing kinase. Evidences are mounting that both SIRT1 and AMPK play important roles in cell fate determination of MSCs. Herein, we intend to sum up our understanding about the role of SIRT1 and AMPK in osteogenic and adipogenic potential of MSCs. Metabolic process of MSCs differentiation and the putative interplay of SIRT1 and AMPK in this process was also discussed.     

Exercise alters SIRT1, SIRT6, NAD and NAMPT levels in skeletal muscle of aged rats

Silent information regulators are potent NAD⁺-dependent protein deacetylases, which have been shown to regulate gene silencing, muscle differentiation and DNA damage repair. Here, changes in the level and activity of sirtuin 1 (SIRT1) in response to exercise in groups of young and old rats were studied. There was an age-related increase in SIRT1 level, while exercise training significantly increased the relative activity of SIRT1. A strong inverse correlation was found between the nuclear activity of SIRT1 and the level of acetylated proteins. Exercise training induced SIRT1 activity due to the positive effect of exercise on the activity of nicotinamide phosphoribosyltransferase (NAMPT) and thereby the production of sirtuin-fueling NAD⁺. Exercise training normalized the age-associated shift in redox balance, since exercised animals had significantly lower levels of carbonylated proteins, expression of hypoxia-inducible factor-1alpha and vascular endothelial growth factor. The age-associated increase in the level of SIRT6 was attenuated by exercise training. On the other hand, aging did not significantly increase the level of DNA damage, which was in line with the activity of 8-oxoguanine DNA glycosylase, while exercise training increased the level of this enzyme. Regular exercise decelerates the deleterious effects of the aging process via SIRT1-dependent pathways through the stimulation of NAD⁺ biosynthesis by NAMPT.     

SIRT6/NF-κB signaling axis in ginsenoside Rg1-delayed hematopoietic stem/progenitor cell senescence

Objective: To investigate the role of SIRT6/NF-κB signaling axis in ginsenoside Rg1-delayed hematopoietic stem/progenitor cell senescence and to provide theoretical and experimental evidence for delaying HSC/HPC senescence pathway. Methods: After the separation and purification by immunomagnetic sorting, Sca-1⁺HSC/HPC was divided into: normal control group; aging group; positive control group; Rg1 delaying group and Rg1 treatment group. Senescence-associated β-galactosidase (SA-β-Gal) staining, flow cytometry analysis of cell cycle and hematopoietic progenitor cells mixed colony (CFU-Mix) culture were performed to determine the delaying or curing roles of Rg1 in Sca-1⁺HSC/HPC senescence. Quantitative PCR and Western blotting were used to detect the mRNA and protein expression of senescence regulatory molecules, such as SIRT6 and NF-κB. Results: Compared with the aging group, the positive rate of SA-β-gal staining cells and the proportion of cells in G1 phase decreased; the number of CFU-Mix increased; mRNA and protein expression of SIRT6 increased; mRNA and protein expression of NF-κB was down-regulated in Rg1 delaying and treatment groups; the changes of the indicators in Rg1 delaying group were more significant than those in Rg1 treatment group. Conclusion: Rg1 may fight against Sca-1⁺HSC/HPC senescence induced by t-BHP through regulating SIRT6-NF-κB signaling pathway.     

Production of panic-like symptoms by lactate is associated with increased neural firing and oxidation of brain redox in the rat hippocampus

Lactate uses an unknown mechanism to induce panic attacks in people and panic-like symptoms in rodents. We tested whether intraperitoneal (IP) lactate injections act peripherally or centrally to induce panic-like symptoms in rats by examining whether IP lactate directly affects the CNS. In Long-Evans rats, IP lactate (2 mmol/kg) injection increased lactate levels in the plasma and the cerebrospinal fluid. IP lactate also induced tachycardia and behavioral freezing suggesting the production of panic-like behavior. To enter intermediate metabolism, lactate is oxidized by lactate dehydrogenase (LDH) to pyruvate with co-reduction of NAD⁺ to NADH. Therefore, we measured the ratio of NADH/NAD⁺ to test whether IP lactate altered lactate metabolism in the CNS. Lactate metabolism was studied in the hippocampus, a brain region believed to contribute to panic-like symptoms. IP lactate injection lowered the ratio of NADH/NAD⁺ without altering the total amount of NADH and NAD⁺ suggesting oxidation of hippocampal redox state. Lactate oxidized hippocampal redox since intrahippocampal injection of the LDH inhibitor, oxamate (50 mM) prevented the oxidation of NADH/NAD⁺ by IP lactate. In addition to oxidizing hippocampal redox, IP lactate rapidly increased the firing rate of hippocampal neurons. Similar IP pyruvate injections had no effect. Neural discharge also increased following intrahippocampal lactate injection suggesting that increased discharge was a direct action of lactate on the hippocampus. These studies show that oxidation of brain redox and increased hippocampal firing are direct actions of lactate on the CNS that may contribute to the production of lactate-induced panic.     

Expression and mechanism of mammalian target of rapamycin in age-related renal cell senescence and organ aging

The mammalian target of rapamycin (mTOR) is relevant to cell senescence and organismal aging. This study firstly showed that the level of mTOR expression increased with aging in rat kidneys, rat mesangial cells and WI-38 cells (P < 0.05). The levels of phosphorylated-mTOR (p-mTOR), cyclin D1 and p21^{WAF1/CIP1/SDI1} expression were significantly higher in WI-38 cells treated with l-leucine (an activator of mTOR) (P < 0.05). The positive staining ratio of senescence-associated β-galactosidase, number of cells in G1 phase, and cellular volume were all increased in WI-38 cells treated with l-leucine when the cellular population doubling (PD) number was 34, while the above phenotypes did not appear in control group until its PD number reached 40 (P < 0.05). The levels of p-mTOR, cyclin D1, and p21^{WAF1/CIP1/SDI1} as well as the aging-related phenotypes were all reduced in cells treated with rapamycin (an inhibitor of mTOR) than in control cells (P < 0.05). These results demonstrated that the level of mTOR was increased in kidney with aging, and that mTOR may promote cellular senescence by regulating the cell cycle through p21^{WAF1/CIP1/SDI1}, which might provide a new target for preventing renal aging.     

Defective induction of the proteasome associated with T‐cell receptor signaling underlies T‐cell senescence

The proteasome degradation machinery is essential for a variety of cellular processes including senescence and T‐cell immunity. Decreased proteasome activity is associated with the aging process; however, the regulation of the proteasome in CD4⁺ T cells in relation to aging is unclear. Here, we show that defects in the induction of the proteasome in CD4⁺ T cells upon T‐cell receptor (TCR) stimulation underlie T‐cell senescence. Proteasome dysfunction promotes senescence‐associated phenotypes, including defective proliferation, cytokine production and increased levels of PD‐1⁺ CD44^High CD4⁺ T cells. Proteasome induction by TCR signaling via MEK‐, IKK‐ and calcineurin‐dependent pathways is attenuated with age and decreased in PD‐1⁺ CD44^High CD4⁺ T cells, the proportion of which increases with age. Our results indicate that defective induction of the proteasome is a hallmark of CD4⁺ T‐cell senescence.     

Nonlinear Regression Models for Determination of Nicotinamide Adenine Dinucleotide Content in Human Embryonic Stem Cells

Recent evidence suggests that energy metabolism contributes to molecular mechanisms controlling stem cell identity. For example, human embryonic stem cells (hESCs) receive their metabolic energy mostly via glycolysis rather than mitochondrial oxidative phosphorylation. This suggests a connection of metabolic homeostasis to stemness. Nicotinamide adenine dinucleotide (NAD) is an important cellular redox carrier and a cofactor for various metabolic pathways, including glycolysis. Therefore, accurate determination of NAD cellular levels and dynamics is of growing importance for understanding the physiology of stem cells. Conventional analytic methods for the determination of metabolite levels rely on linear calibration curves. However, in actual practice many two-enzyme cycling assays, such as the assay systems used in this work, display prominently nonlinear behavior. Here we present a diaphorase/lactate dehydrogenase NAD cycling assay optimized for hESCs, together with a mechanism-based, nonlinear regression models for the determination of NAD⁺, NADH, and total NAD. We also present experimental data on metabolic homeostasis of hESC under various physiological conditions. We show that NAD⁺/NADH ratio varies considerably with time in culture after routine change of medium, while the total NAD content undergoes relatively minor changes. In addition, we show that the NAD⁺/NADH ratio, as well as the total NAD levels, vary between stem cells and their differentiated counterparts. Importantly, the NAD⁺/NADH ratio was found to be substantially higher in hESC-derived fibroblasts versus hESCs. Overall, our nonlinear mathematical model is applicable to other enzymatic amplification systems.     

Casein kinase 2 regulates the active uptake of the organic osmolyte taurine in NIH3T3 mouse fibroblasts

Inhibition of the constitutively active casein kinase 2 (CK2) with 2-dimethyl-amino-4,5,6,7-tetrabromo-1H-benzimidasole stimulates the Na⁺-dependent taurine influx via the taurine transporter TauT in NIH3T3 cells. CK2 inhibition reduces the TauT mRNA level and increases the localization of TauT to ER but has no detectable effect on TauT protein expression. On the other hand, CK2 inhibition increases the affinity of TauT towards Na⁺ and reduces the Na⁺/taurine stoichiometry for active taurine uptake. It is suggested that CK2 controls the cellular taurine uptake in unperturbated NIH3T3 cells, i.e., inhibition of CK2 increases the affinity of TauT towards Na⁺ and hence Na⁺-dependent taurine uptake.     

Over‐expression of Nicotinamide phosphoribosyltransferase in mouse cells confers protective effect against oxidative and ER stress‐induced premature senescence

A main feature of aged organisms is the accumulation of senescent cells. Accumulated senescent cells, especially stress‐induced premature senescent cells, in aged organisms lead to the decline of the regenerative potential and function of tissues. We recently reported that the over‐expression of NAMPT, which is the rate‐limiting enzyme in mammalian NAD⁺ salvage pathway, delays replicative senescence in vitro. However, whether Nampt‐overexpressing cells are tolerant of stress‐induced premature senescence remains unknown. Here, we show that primary mouse embryonic fibroblasts derived from Nampt‐overexpressing transgenic mice (Nampt Tg‐MEF cells) possess resistance against stress‐induced premature senescence in vitro. We found that higher oxidative or endoplasmic reticulum (ER) stress is required to induce premature senescence in Nampt Tg‐MEF cells compared to wild‐type cells. Moreover, we found that Nampt Tg‐MEF cells show acute expression of unfolded protein response (UPR)‐related genes, which in turn would have helped to restore proteostasis and avoid cellular senescence. Our results demonstrate that NAMPT/NAD⁺ axis functions to protect cells not only from replicative senescence, but also from stress‐induced premature senescence in vitro. We anticipate that in vivo activation of NAMPT activity or increment of NAD⁺ would protect tissues from the accumulation of premature senescent cells, thereby maintaining healthy aging.     

Sirtuin SIRT6 suppresses cell proliferation through inhibition of Twist1 expression in non-small cell lung cancer

SIRT6 is a member of the NAD⁺-dependent class III deacetylase sirtuin family. Current studies have revealed that SIRT6 plays important roles in the epigenetic regulation of genes expression and contribute to the proliferation, differentiation and apoptosis of cancer cells. However, the biological function of SIRT6 in lung cancer has not been elucidated. The present study showed that the mRNA and protein levels of SIRT6 were decreased in human non-small cell lung cancer (NSCLC) tissues and cell lines. MTT assay showed that overexpression of SIRT6 could inhibit the proliferation in NSCLC cells. In contrast, SIRT6 knockdown using small interfering RNA promoted NSCLC cells proliferation. On the molecular level, we found that SIRT6 inhibited the expression of Twist1 both at the mRNA and protein levels in NSCLC cells. Taken together, these results demonstrated for the first time that SIRT6 suppressed NSCLC cells proliferation via down-regulation of Twist1 expression and might provide novel therapeutic targets in the treatment of lung cancer.     

Role of ADP-Ribosyl Cyclase in the Pathogenesis of Neurological Disorders after Coronary Artery Bypass Surgery and Experimental Ischemia

The pathogenesis of neuronal dysfunction was evaluated from the viewpoint of cellular disturbances in NAD⁺ metabolism and changes in activity of NAD⁺-utilizing enzymes (e.g., ADP-ribosyl cyclase/CD38). S-100B concentration and CD38 expression on peripheral blood lymphocytes were altered in patients after surgery for coronary heart disease with extracorporeal circulation. These changes in patients during the early postoperative period correlated with variations in CD38 expression on neuronal cells from postischemic rats with cognitive dysfunction.     

The Protective Effect of Fenofibrate Against TNF-α-Induced CD40 Expression through SIRT1-Mediated Deacetylation of NF-κB in Endothelial Cells

Fenofibrate, as a lipid-lowering drug in clinic, participates in the regulation of inflammatory response. Recently, increasing studies have indicated that sirtuin1 (SIRT1), a NAD⁺-dependent deacetylase, has potential anti-inflammatory effect in endothelial cells. However, whether the regulatory effect of fenofibrate on inflammation response is mediated by SIRT1 remains unclear. The aim of this study was to investigate the effect of fenofibrate on the expressions of SIRT1 and pro-inflammatory cytokine CD40 in endothelial cells and explore the underlying mechanisms. The results showed that fenofibrate upregulated SIRT1 expression and inhibited CD40 expression in TNF-α-stimulated endothelial cells, but these effects were reversed by peroxisome proliferator-activated receptor-α (PPARα) antagonist GW6471. Furthermore, SIRT1 inhibitors sirtinol/nicotinamide (NAM) or SIRT1 knockdown could attenuate the effect of fenofibrate on CD40 expression in endothelial cells. Importantly, NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) augmented the effect of fenofibrate on CD40 expression. Further study found that fenofibrate decreased the expression of acetylated-NF-κB p65 (Ac-NF-κB p65) in TNF-α-stimulated endothelial cells, which was abolished by SIRT1 knockdown. These results indicate that fenofibrate has protective effect against TNF-α-induced CD40 expression through SIRT1-mediated deacetylation of the p65 subunit of NF-κB.     

Alcohol dehydrogenase (ADH) in yeasts II. NAD+-and NADP+-dependent alcohol dehydrogenases in Saccharomycopsis lipolytica

In Sm. lipolytica one NAD⁺-dependent and three NADP⁺-dependent alcohol dehydrogenases are detectable by polyacrylamide gelelectrophoresis. The NAD⁺-dependent ADH (ADH I), with a molecular weight of 240,000 daltons, reacts more intensively with long-chain alcohols (octanol) than with short-chain alcohols (methanol, ethanol). The ADH I is not or only minimally subject to glucose repression. Besides the ADH I band no additional inducible NAD⁺-dependent ADH band is gel-electrophoretically detectable during growth of yeast cells in medium containing ethanol or paraffin. The ADH I band is very probably formed by two ADH enzymes with the same electrophoretic mobility. The NADP⁺-dependent alcohol dehydrogenases (ADH II–IV) react with methanol, ethanol and octanol with different intensity. In polyacrylamide gradients two bands of NADP⁺-dependent ADH are detectable: one with a molecular weight of 70,000 daltons and the other with 120,000 daltons. The occurrence of the three NADP⁺-dependent alcohol dehydrogenases is regulated by the carbon source of the medium. Sm. lipolytica shows a high tolerance against allylalcohol. Resistant mutants can be isolated only at concentrations of 1 M allyalcohol in the medium. All isolates of allylalcohol-resistant mutants show identical growth in medium containing ethanol as the wild type strain.     

The lymphocyte surface antigen CD38 acts as a nicotinamide adenine dinucleotide glycohydrolase in human T lymphocytes

The extracellular domain of the lymphocyte surface antigen CD38 has been recently shown to share a high sequence homology with a nicotinamide adenine dinucleotide (NAD⁺)-specific hydrolyzing enzyme cloned from the ovotestis of the gastropod Aplysia (E. States, D. J., Walseth, T. F., Lee, H. C, Trends Biochem. Sci. 1992. 17: 495). In agreement with this finding, we present here evidence that CD38-overexpressingT cells, such as human thymocytes and cells from the human HPB-ALL T cell line, exhibit a NAD⁺-hydrolyzing enzymatic activity present on the outer surface of the cell membrane. In contrast, T lymphocytes with relatively low levels of CD38 marker, such as the human Jurkat cell line, display a lower activity. This suggests a relationship between ecto-NAD⁺ glycohydrolase activity and CD38 expression, as confirmed here when comparing wild-type Jurkat cells and a Jurkat cell variant overexpressing the CD38 molecule. Moreover, CD38 immunoprecipitates from thymocytes behave as an authentic NAD⁺ glycohydrolase enzyme: it transforms NAD⁺ stoichiometrically into nicotinamide plus adenosine 5′-diphosphoribose. Altogether these results strongly support the assumption that CD38 is actually a lymphocyte-specific NAD⁺-hydrolyzing enzyme, a finding that give new prospects to understand the in vivo function of this cell membrane protein.     

Rapid stimulation of Na+/H+ exchange by 1,25-dihydroxyvitamin D3; interaction with parathyroid-hormone-dependent inhibition

We have examined the rapid effect of 1,25-dihydroxyvitamin-D₃ [1,25(OH)₂D₃] on apical Na⁺/H⁺ exchange activity in opossum kidney (OK) cells and in MCT cells (a culture of simian-virus-40-immortalized mouse cortical tubule cells) grown on filter support. Addition of 1,25(OH)₂D₃ (10 nM) for 1 min increased apical Na⁺/H⁺ exchange activity [recovery from an acid load; measured by 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein] in OK cells (by 56%) and in MCT cells (by 36%). The cellular mechanisms involved in 1,25(OH)₂D₃-dependent stimulation of Na⁺/H⁺ exchange were analysed in OK cells; stimulation of Na⁺/ H⁺ exchange by 1,25(OH)₂D₃ was not prevented by actinomycin D. Applying parathyroid hormone (PTH) reduced Na⁺/H⁺ exchange activity in OK cells (by 34% at 10 nM, 5 min); 1,25(OH)₂D₃ reversed PTH-induced inhibition, either when PTH was added prior to 1,25(OH)₂D₃ or when the two agonists were applied together. 1,25(OH)₂D₃ had no effect on basal OK cell cAMP content or on [Ca²⁺]_i (fura-2). 1,25(OH)₂D₃ attenuated PTH-induced cAMP accumulation and had no effect on the PTH-dependent increase in [Ca²⁺]_i. These data suggest a regulatory control (stimulation) of proximal tubular brush-border Na⁺/H⁺ exchange by 1,25(OH)₂D₃. This effect is non-genomic and might in part be explained by a release from cAMP-dependent control of transport activity.     

The SLC13 gene family of sodium sulphate/carboxylate cotransporters

The SLC13 gene family consist of five sequence-related members that have been identified in a variety of animals, plants, yeast and bacteria. Proteins encoded by these genes are divided into two functionally unrelated groups: the Na⁺-sulphate (NaS) cotransporters and the Na⁺-carboxylate (NaC) cotransporters. Members of this family include the renal Na⁺-dependent inorganic sulphate transporter-1 (NaSi-1, SLC13A1), the Na⁺-dependent dicarboxylate transporters NaDC-1/SDCT1 (SLC13A2), NaDC-3/SDCT2 (SLC13A3), the sulphate transporter-1 (SUT-1, SLC13A4) and the Na⁺-coupled citrate transporter (NaCT, SLC13A5). The general characteristics of the SLC13 proteins are that they encode multi-spanning proteins with 8–13 transmembrane domains, have a wide tissue distribution with most being expressed in the epithelial cells of the kidney and the gastrointestinal tract. They are Na⁺-coupled symporters, DIDS-insensitive, with strong cation preference for Na⁺, with a Na⁺:anion coupling ratio of around 3:1 and have a substrate preference for divalent anions, which include tetraoxyanions (for the NaS cotransporters) or Krebs cycle intermediates, including mono-, di-, and tri-carboxylates (for the NaC cotransporters). The purpose of this review is to provide an update on the most recent advances and to summarize the biochemical, physiological and structural aspects of the vertebrate SLC13 gene family.