NAMPT maintains mitochondria content via NRF2-PPARα/AMPKα pathway to promote cell survival under oxidative stress

Mitochondria plays a key role in regulating cell death process under stress conditions and it has been indicated that NAMPT overexpression promotes cell survival under genotoxic stress by maintaining mitochondrial NAD⁺ level. NAMPT is a rate-limiting enzyme for NAD⁺ production in mammalian cells and it was suggested that NAMPT and NMNAT3 are responsible for mitochondrial NAD⁺ production to maintain mitochondrial NAD⁺ pool. However, subsequent studies suggested mitochondrial may lack the NAMPT-NMANT3 pathway to maintain NAD⁺ level. Therefore, how NAMPT overexpression rescues mitochondrial NAD⁺ content to promote cell survival in response to genotoxic stress remains elusive. Here, we show that NAMPT promotes cell survival under oxidative stress via both SIRT1 dependent p53-CD38 pathway and SIRT1 independent NRF2-PPARα/AMPKα pathway, and the NRF2-PPARα/AMPKα pathway plays a more profound role in facilitating cell survival than the SIRT1-p53-CD38 pathway does. Mitochondrial content and membrane potential were significantly reduced in response to H2O2 treatment, whereas activated NRF2-PPARα/AMPKα pathway by NAMPT overexpression rescued the mitochondrial membrane potential and content, suggesting that maintained mitochondrial content and integrity by NAMPT overexpression might be one of the key mechanisms to maintain mitochondrial NAD⁺ level and subsequently dictate cell survival under oxidative stress. Our results indicated that NRF2 is a novel down-stream target of NAMPT, which mediates anti-apoptosis function of NAMPT via maintaining mitochondrial content and membrane potential.     

Fasting promotes the expression of SIRT1, an NAD+-dependent protein deacetylase, via activation of PPARα in mice

Calorie restriction (CR) extends lifespans in a wide variety of species. CR induces an increase in the NAD⁺/NADH ratio in cells and results in activation of SIRT1, an NAD⁺-dependent protein deacetylase that is thought to be a metabolic master switch linked to the modulation of lifespans. CR also affects the expression of peroxisome proliferator-activated receptors (PPARs). The three subtypes, PPARα, PPARγ, and PPARβ/δ, are expressed in multiple organs. They regulate different physiological functions such as energy metabolism, insulin action and inflammation, and apparently act as important regulators of longevity and aging. SIRT1 has been reported to repress the PPARγ by docking with its co-factors and to promote fat mobilization. However, the correlation between SIRT1 and other PPARs is not fully understood. CR initially induces a fasting-like response. In this study, we investigated how SIRT1 and PPARα correlate in the fasting-induced anti-aging pathways. A 24-h fasting in mice increased mRNA and protein expression of both SIRT1 and PPARα in the livers, where the NAD⁺ levels increased with increasing nicotinamide phosphoribosyltransferase (NAMPT) activity in the NAD⁺ salvage pathway. Treatment of Hepa1-6 cells in a low glucose medium conditions with NAD⁺ or NADH showed that the mRNA expression of both SIRT1 and PPARα can be enhanced by addition of NAD⁺, and decreased by increasing NADH levels. The cell experiments using SIRT1 antagonists and a PPARα agonist suggested that PPARα is a key molecule located upstream from SIRT1, and has a role in regulating SIRT1 gene expression in fasting-induced anti-aging pathways.     

PARP-1 inhibition does not restore oxidant-mediated reduction in SIRT1 activity

Sirtuin1 (SIRT1) deacetylase and poly(ADP-ribose)-polymerase-1 (PARP-1) respond to environmental cues, and both require NAD⁺ cofactor for their enzymatic activities. However, the functional link between environmental/oxidative stress-mediated activation of PARP-1 and SIRT1 through NAD⁺ cofactor availability is not known. We investigated whether NAD⁺ depletion by PARP-1 activation plays a role in environmental stimuli/oxidant-induced reduction in SIRT1 activity. Both H₂O₂ and cigarette smoke (CS) decreased intracellular NAD⁺ levels in vitro in lung epithelial cells and in vivo in lungs of mice exposed to CS. Pharmacological PARP-1 inhibition prevented oxidant-induced NAD⁺ loss and attenuated loss of SIRT1 activity. Oxidants decreased SIRT1 activity in lung epithelial cells; however increasing cellular NAD⁺ cofactor levels by PARP-1 inhibition or NAD⁺ precursors was unable to restore SIRT1 activity. SIRT1 was found to be carbonylated by CS, which was not reversed by PARP-1 inhibition or selective SIRT1 activator. Overall, these data suggest that environmental/oxidant stress-induced SIRT1 down-regulation and PARP-1 activation are independent events despite both enzymes sharing the same cofactor.     

Nicotinamide phosphoribosyltransferase/sirtuin 1 pathway is involved in human immunodeficiency virus type 1 Tat‐mediated long terminal repeat transactivation

Tat is a multifunctional transactivator encoded by human immunodeficiency virus type 1 (HIV‐1). Tat transactivating activity is controlled by nicotinamide adenine nucleotide⁺ (NAD⁺)‐dependent deacetylase sirtuin 1 (SIRT1). Nicotinamide phosphoribosyltransferase (Nampt) is a rate‐limiting enzyme in the conversion of nicotinamide into NAD⁺, which is crucial for SIRT1 activation. Thus, the effect of Nampt on Tat‐regulated SIRT activity was studied in Hela‐CD4‐β‐gal (MAGI) cells. We demonstrated that Tat caused NAD⁺ depletion and inhibited Nampt mRNA and protein expression in MAGI cells. Resveratrol reversed Tat‐induced NAD⁺ depletion and inhibition of Nampt mRNA and protein expression. Further investigation revealed that Tat‐induced inhibition of SIRT1 activity was potentiated in Nampt‐knockdown by Nampt siRNA compared to treatment with Tat alone. Nampt siRNA potentiated Tat‐induced HIV‐1 transactivation in MAGI cells. Altogether, these results indicate that Nampt is critical in the regulation of Tat‐induced inhibition of SIRT1 activity and long terminal repeat (LTR) transactivation. Nampt/SIRT1 pathway could be a novel therapeutic tool for the treatment of HIV‐1 infection. J. Cell. Biochem. 110: 1464–1470, 2010. © 2010 Wiley‐Liss, Inc.     

Tumor growth of neurofibromin-deficient cells is driven by decreased respiration and hampered by NAD+ and SIRT3

Neurofibromin loss drives neoplastic growth and a rewiring of mitochondrial metabolism. Here we report that neurofibromin ablation dampens expression and activity of NADH dehydrogenase, the respiratory chain complex I, in an ERK-dependent fashion, decreasing both respiration and intracellular NAD⁺. Expression of the alternative NADH dehydrogenase NDI1 raises NAD⁺/NADH ratio, enhances the activity of the NAD⁺-dependent deacetylase SIRT3 and interferes with tumorigenicity in neurofibromin-deficient cells. The antineoplastic effect of NDI1 is mimicked by administration of NAD⁺ precursors or by rising expression of the NAD⁺ deacetylase SIRT3 and is synergistic with ablation of the mitochondrial chaperone TRAP1, which augments succinate dehydrogenase activity further contributing to block pro-neoplastic metabolic changes. These findings shed light on bioenergetic adaptations of tumors lacking neurofibromin, linking complex I inhibition to mitochondrial NAD⁺/NADH unbalance and SIRT3 inhibition, as well as to down-regulation of succinate dehydrogenase. This metabolic rewiring could unveil attractive therapeutic targets for neoplasms related to neurofibromin loss.Subject terms: Cancer metabolism, Cell biology     

The phosphorylation status of T522 modulates tissue‐specific functions of SIRT1 in energy metabolism in mice

SIRT1, the most conserved mammalian NAD⁺‐dependent protein deacetylase, is an important metabolic regulator. However, the mechanisms by which SIRT1 is regulated in vivo remain unclear. Here, we report that phosphorylation modification of T522 on SIRT1 is crucial for tissue‐specific regulation of SIRT1 activity in mice. Dephosphorylation of T522 is critical for repression of its activity during adipogenesis. The phospho‐T522 level is reduced during adipogenesis. Knocking‐in a constitutive T522 phosphorylation mimic activates the β‐catenin/GATA3 pathway, repressing PPARγ signaling, impairing differentiation of white adipocytes, and ameliorating high‐fat diet‐induced dyslipidemia in mice. In contrast, phosphorylation of T522 is crucial for activation of hepatic SIRT1 in response to over‐nutrition. Hepatic SIRT1 is hyperphosphorylated at T522 upon high‐fat diet feeding. Knocking‐in a SIRT1 mutant defective in T522 phosphorylation disrupts hepatic fatty acid oxidation, resulting in hepatic steatosis after high‐fat diet feeding. In addition, the T522 dephosphorylation mimic impairs systemic energy metabolism. Our findings unveil an important link between environmental cues, SIRT1 phosphorylation, and energy homeostasis and demonstrate that the phosphorylation of T522 is a critical element in tissue‐specific regulation of SIRT1 activity in vivo.     

SCD1 regulates the AMPK/SIRT1 pathway and histone acetylation through changes in adenine nucleotide metabolism in skeletal muscle

Stearoyl-CoA desaturase (SCD) is a rate-limiting enzyme that catalyzes the synthesis of monounsaturated fatty acids. It plays an important role in regulating skeletal muscle metabolism. Lack of the SCD1 gene increases the rate of fatty acid β-oxidation through activation of the AMP-activated protein kinase (AMPK) pathway and the upregulation of genes that are related to fatty acid oxidation. The mechanism of AMPK activation under conditions of SCD1 deficiency has been unclear. In the present study, we found that the ablation/inhibition of SCD1 led to AMPK activation in skeletal muscle through an increase in AMP levels whereas muscle-specific SCD1 overexpression decreased both AMPK phosphorylation and the adenosine monophosphate/adenosine triphosphate (AMP/ATP) ratio. Changes in AMPK phosphorylation that were caused by SCD1 down- and upregulation affected NAD⁺ levels following changes in NAD⁺-dependent deacetylase sirtuin-1 (SIRT1) activity and histone 3 (H3K9) acetylation and methylation status. Moreover, mice with muscle-targeted overexpression of SCD1 were more susceptible to high-fat diet-induced lipid accumulation and the development of insulin resistance compared with wild-type mice. These data show that SCD1 is involved in nucleotide (ATP and NAD⁺) metabolism and suggest that the SCD1-dependent regulation of muscle steatosis and insulin sensitivity are mediated by cooperation between AMPK- and SIRT1-regulated pathways. Altogether, the present study reveals a novel mechanism that links SCD1 with the maintenance of metabolic homeostasis and insulin sensitivity in skeletal muscle.     

Nicotinamide phosphoribosyltransferase can affect metastatic activity and cell adhesive functions by regulating integrins in breast cancer

NAD⁺ metabolism is an essential regulator of cellular redox reactions, energy pathways, and a substrate provider for NAD⁺ consuming enzymes. We recently demonstrated that enhancement of NAD⁺/NADH levels in breast cancer cells with impaired mitochondrial NADH dehydrogenase activity, through augmentation of complex I or by supplementing tumor cell nutrients with NAD⁺ precursors, inhibits tumorigenicity and metastasis. To more fully understand how aberrantly low NAD⁺ levels promote tumor cell dissemination, we here asked whether inhibition of NAD⁺ salvage pathway activity by reduction in nicotinamide phosphoribosyltransferase (NAMPT) expression can impact metastasis and tumor cell adhesive functions. We show that knockdown of NAMPT, the enzyme catalyzing the rate-limiting step of the NAD⁺ salvage pathway, enhances metastatic aggressiveness in human breast cancer cells and involves modulation of integrin expression and function. Reduction in NAMPT expression is associated with upregulation of select adhesion receptors, particularly αvβ3 and β1 integrins, and results in increased breast cancer cell attachment to extracellular matrix proteins, a key function in tumor cell dissemination. Interestingly, NAMPT downregulation prompts expression of integrin αvβ3 in a high affinity conformation, known to promote tumor cell adhesive interactions during hematogenous metastasis. NAMPT has been selected as a therapeutic target for cancer therapy based on the essential functions of this enzyme in NAD⁺ metabolism, cellular redox, DNA repair and energy pathways. Notably, our results indicate that incomplete inhibition of NAMPT, which impedes NAD⁺ metabolism but does not kill a tumor cell can alter its phenotype to be more aggressive and metastatic. This phenomenon could promote cancer recurrence, even if NAMPT inhibition initially reduces tumor growth.     

NAD+ ameliorates endotoxin‐induced acute kidney injury in a sirtuin1–dependent manner via GSK‐3β/Nrf2 signalling pathway

Acute kidney injury (AKI) is a substantial worldwide public health concern with no specific and effective therapies in clinic. NAD⁺ is a pivotal determinant of cellular energy metabolism involved in the progression of AKI; however, its mechanism in kidney injury remains poorly understood. Sirtuin 1 (SIRT1) is an NAD⁺‐dependent deacetylase associated with renal protection and acute stress resistance. In this study, we have investigated the role of NAD⁺ in AKI and the potential mechanism(s) involved in its renoprotective effect. NAD⁺ was notably decreased and negatively correlated with kidney dysfunction in AKI, restoring NAD⁺ with NMN significantly ameliorates LPS‐induced oxidative stress and apoptosis and attenuates renal damage. We also found that the protection of NAD⁺ is associated with SIRT1 expressions and performs in a SIRT1‐dependent manner. Inhibition of SIRT1 blunted the protective effect of NAD⁺ and up‐regulated the activity of glycogen synthase kinase‐3β (GSK‐3β) that was concomitant with mitigated Nrf2 nuclear accumulation, thereby exacerbates AKI. These findings suggest that NAD⁺/SIRT1/GSK‐3β/Nrf2 axis is an important mechanism that can protect against AKI which might be a potential therapeutic target for the treatment of AKI.     

SIRT2 controls the pentose phosphate switch

The most common enzyme defect in humans is glucose‐6‐phosphate dehydrogenase (G6PD) deficiency, which affects more than 400 million people. G6PD shunts glucose into the pentose phosphate pathway (PPP) to generate nucleotides and reducing potential in the form of NADPH. In this issue, Wang et al ( 2014 ) show that G6PD activity is post‐translationally regulated by SIRT2, a cytoplasmic NAD⁺‐dependent deacetylase, thereby linking NAD⁺ levels to DNA repair and oxidative defences, and identifying potential new approaches to treating this common genetic disease.     

Biochemical effects of SIRT1 activators

SIRT1 is the closest mammalian homologue of enzymes that extend life in lower organisms. Its role in mammals is incompletely understood, but includes modulation of at least 34 distinct targets through its nicotinamide adenine dinucleotide (NAD⁺)-dependent deacetylase activity. Recent experiments using small molecule activators and genetically engineered mice have provided new insight into the role of this enzyme in mammalian biology and helped to highlight some of the potentially relevant targets. The most widely employed activator is resveratrol, a small polyphenol that improves insulin sensitivity and vascular function, boosts endurance, inhibits tumor formation, and ameliorates the early mortality associated with obesity in mice. Many of these effects are consistent with modulation of SIRT1 targets, such as PGC1α and NFκB, however, resveratrol can also activate AMPK, inhibit cyclooxygenases, and influence a variety of other enzymes. A novel activator, SRT1720, as well as various methods to manipulate NAD⁺ metabolism, are emerging as alternative methods to increase SIRT1 activity, and in many cases recapitulate effects of resveratrol. At present, further studies are needed to more directly test the role of SIRT1 in mediating beneficial effects of resveratrol, to evaluate other strategies for SIRT1 activation, and to confirm the specific targets of SIRT1 that are relevant in vivo. These efforts are especially important in light of the fact that SIRT1 activators are entering clinical trials in humans, and “nutraceutical” formulations containing resveratrol are already widely available.     

Upregulation of SIRT1 deacetylase in phenylephrine-treated cardiomyoblasts

The sirtuin SIRT1 is an ubiquitous NAD⁺ dependent deacetylase that plays a role in biological processes such as longevity and stress response. In cardiac models, SIRT1 is associated to protection against many stresses. However, the link between SIRT1 and heart hypertrophy is complex and not fully understood. This study focuses specifically on the response of SIRT1 to the α-adrenergic agonist phenylephrine in H9c2 cardiac myoblasts, a cell model of cardiac hypertrophy. After 24 and 48 h of phenylephrine treatment, SIRT1 expression and deacetylase activity were significantly increased. SIRT1 upregulation by phenylephrine was not associated to changes in NAD⁺ levels, but was blocked by inhibitors of AMP-activated Protein Kinase (AMPK) or by AMPK knockdown by siRNA. When SIRT1 was inhibited with sirtinol or downregulated by siRNA, H9c2 cell viability was significantly decreased following phenylephrine treatment, showing that SIRT1 improves cell survival under hypertrophic stress. We so then propose that the increase in SIRT1 activity and expression in H9c2 cells treated with phenylephrine is an adaptive response to the hypertrophic stress, suggesting that adrenergic stimulation of heart cells activates hypertrophic programming and at the same time also promotes a self-protecting and self-regulating mechanism.     

AMPK phosphorylates NAMPT to regulate NAD+ homeostasis under ionizing radiation

Radiation-induced oral mucositis is the most common complication for patients who receive head/neck radiotherapy. Nicotinamide adenine dinucleotide (NAD⁺) is vital for DNA damage repair under ionizing radiation, through functioning as either the substrate for protein poly(ADP-ribosyl)ation at DNA break sites or the cofactor for multiple DNA repair-related enzymes, which therefore can result in a significant consumption of cellular NAD⁺ during DNA repair. Mammalian cells produce NAD⁺ mainly by recycling nicotinamide via the salvage pathway, in which the rate-limiting step is governed by nicotinamide phosphoribosyltransferase (NAMPT). However, whether NAMPT is co-opted under ionizing radiation to timely fine-tune NAD⁺ homeostasis remains elusive. Here we show that ionizing radiation evokes NAMPT activation within 30 min without apparent changes in its protein expression. AMPK rapidly phosphorylates NAMPT at S314 under ionizing radiation, which reinforces the enzymatic activity of NAMPT by increasing NAMPT binding with its substrate phosphoribosyl pyrophosphate (PRPP). AMPK-mediated NAMPT S314 phosphorylation substantially restores NAD⁺ level in the irradiated cells and facilitates DNA repair and cell viability. Our findings demonstrate a new post-translational modification-based signalling route, by which cells can rapidly orchestrate NAD⁺ metabolism to support DNA repair, thereby highlighting NAMPT as a potential target for the prevention of ionizing radiation-induced injuries.     

Nicotinamide Phosphoribosyltransferase Promotes Epithelial-to-Mesenchymal Transition as a Soluble Factor Independent of Its Enzymatic Activity

Boosting NAD⁺ biosynthesis with NAD⁺ intermediates has been proposed as a strategy for preventing and treating age-associated diseases, including cancer. However, concerns in this area were raised by observations that nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in mammalian NAD⁺ biosynthesis, is frequently up-regulated in human malignancies, including breast cancer, suggesting possible protumorigenic effects for this protein. We addressed this issue by studying NAMPT expression and function in human breast cancer in vivo and in vitro. Our data indicate that high NAMPT levels are associated with aggressive pathological and molecular features, such as estrogen receptor negativity as well as HER2-enriched and basal-like PAM50 phenotypes. Consistent with these findings, we found that NAMPT overexpression in mammary epithelial cells induced epithelial-to-mesenchymal transition, a morphological and functional switch that confers cancer cells an increased metastatic potential. However, importantly, NAMPT-induced epithelial-to-mesenchymal transition was found to be independent of NAMPT enzymatic activity and of the NAMPT product nicotinamide mononucleotide. Instead, it was mediated by secreted NAMPT through its ability to activate the TGFβ signaling pathway via increased TGFβ1 production. These findings have implications for the design of therapeutic strategies exploiting NAD⁺ biosynthesis via NAMPT in aging and cancer and also suggest the potential of anticancer agents designed to specifically neutralize extracellular NAMPT. Notably, because high levels of circulating NAMPT are found in obese and diabetic patients, our data could also explain the increased predisposition to cancer of these subjects.     

Structural and Functional Analysis of Human SIRT1

SIRT1 is a NAD⁺-dependent deacetylase that plays important roles in many cellular processes. SIRT1 activity is uniquely controlled by a C-terminal regulatory segment (CTR). Here we present crystal structures of the catalytic domain of human SIRT1 in complex with the CTR in an open apo form and a closed conformation in complex with a cofactor and a pseudo-substrate peptide. The catalytic domain adopts the canonical sirtuin fold. The CTR forms a β hairpin structure that complements the β sheet of the NAD⁺-binding domain, covering an essentially invariant hydrophobic surface. The apo form adopts a distinct open conformation, in which the smaller subdomain of SIRT1 undergoes a rotation with respect to the larger NAD⁺-binding subdomain. A biochemical analysis identifies key residues in the active site, an inhibitory role for the CTR, and distinct structural features of the CTR that mediate binding and inhibition of the SIRT1 catalytic domain.     

Kallistatin reduces vascular senescence and aging by regulating microRNA‐34a‐SIRT1 pathway

Kallistatin, an endogenous protein, protects against vascular injury by inhibiting oxidative stress and inflammation in hypertensive rats and enhancing the mobility and function of endothelial progenitor cells (EPCs). We aimed to determine the role and mechanism of kallistatin in vascular senescence and aging using cultured EPCs, streptozotocin (STZ)‐induced diabetic mice, and Caenorhabditis elegans (C. elegans). Human kallistatin significantly decreased TNF‐α‐induced cellular senescence in EPCs, as indicated by reduced senescence‐associated β‐galactosidase activity and plasminogen activator inhibitor‐1 expression, and elevated telomerase activity. Kallistatin blocked TNF‐α‐induced superoxide levels, NADPH oxidase activity, and microRNA‐21 (miR‐21) and p16^INK4a synthesis. Kallistatin prevented TNF‐α‐mediated inhibition of SIRT1, eNOS, and catalase, and directly stimulated the expression of these antioxidant enzymes. Moreover, kallistatin inhibited miR‐34a synthesis, whereas miR‐34a overexpression abolished kallistatin‐induced antioxidant gene expression and antisenescence activity. Kallistatin via its active site inhibited miR‐34a, and stimulated SIRT1 and eNOS synthesis in EPCs, which was abolished by genistein, indicating an event mediated by tyrosine kinase. Moreover, kallistatin administration attenuated STZ‐induced aortic senescence, oxidative stress, and miR‐34a and miR‐21 synthesis, and increased SIRT1, eNOS, and catalase levels in diabetic mice. Furthermore, kallistatin treatment reduced superoxide formation and prolonged wild‐type C. elegans lifespan under oxidative or heat stress, although kallistatin's protective effect was abolished in miR‐34 or sir‐2.1 (SIRT1 homolog) mutant C. elegans. Kallistatin inhibited miR‐34, but stimulated sir‐2.1 and sod‐3 synthesis in C. elegans. These in vitro and in vivo studies provide significant insights into the role and mechanism of kallistatin in vascular senescence and aging by regulating miR‐34a‐SIRT1 pathway.     

SIRT2 induces the checkpoint kinase BubR1 to increase lifespan

Mice overexpressing the mitotic checkpoint kinase gene BubR1 live longer, whereas mice hypomorphic for BubR1 (BubR1^H/H) live shorter and show signs of accelerated aging. As wild‐type mice age, BubR1 levels decline in many tissues, a process that is proposed to underlie normal aging and age‐related diseases. Understanding why BubR1 declines with age and how to slow this process is therefore of considerable interest. The sirtuins (SIRT1‐7) are a family of NAD⁺‐dependent deacetylases that can delay age‐related diseases. Here, we show that the loss of BubR1 levels with age is due to a decline in NAD⁺ and the ability of SIRT2 to maintain lysine‐668 of BubR1 in a deacetylated state, which is counteracted by the acetyltransferase CBP. Overexpression of SIRT2 or treatment of mice with the NAD⁺ precursor nicotinamide mononucleotide (NMN) increases BubR1 abundance in vivo. Overexpression of SIRT2 in BubR1^H/H animals increases median lifespan, with a greater effect in male mice. Together, these data indicate that further exploration of the potential of SIRT2 and NAD⁺ to delay diseases of aging in mammals is warranted.