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1.
J Biol Chem ; 297(6): 101388, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34762911

RESUMO

Nicotinamide phosphoribosyltransferase (NAMPT) converts nicotinamide to NAD+. As low hepatic NAD+ levels have been linked to the development of nonalcoholic fatty liver disease, we hypothesized that ablation of hepatic Nampt would affect susceptibility to liver injury in response to diet-induced metabolic stress. Following 3 weeks on a low-methionine and choline-free 60% high-fat diet, hepatocyte-specific Nampt knockout (HNKO) mice accumulated less triglyceride than WT littermates but had increased histological scores for liver inflammation, necrosis, and fibrosis. Surprisingly, liver injury was also observed in HNKO mice on the purified control diet. This HNKO phenotype was associated with decreased abundance of mitochondrial proteins, especially proteins involved in oxidoreductase activity. High-resolution respirometry revealed lower respiratory capacity in purified control diet-fed HNKO liver. In addition, fibrotic area in HNKO liver sections correlated negatively with hepatic NAD+, and liver injury was prevented by supplementation with NAD+ precursors nicotinamide riboside and nicotinic acid. MS-based proteomic analysis revealed that nicotinamide riboside supplementation rescued hepatic levels of oxidoreductase and OXPHOS proteins. Finally, single-nucleus RNA-Seq showed that transcriptional changes in the HNKO liver mainly occurred in hepatocytes, and changes in the hepatocyte transcriptome were associated with liver necrosis. In conclusion, HNKO livers have reduced respiratory capacity, decreased abundance of mitochondrial proteins, and are susceptible to fibrosis because of low NAD+ levels. Our data suggest a critical threshold level of hepatic NAD+ that determines the predisposition to liver injury and supports that NAD+ precursor supplementation can prevent liver injury and nonalcoholic fatty liver disease progression.


Assuntos
Hepatócitos/metabolismo , Mitocôndrias Hepáticas/metabolismo , NAD/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Animais , Citocinas/deficiência , Citocinas/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias Hepáticas/genética , NAD/genética , Nicotinamida Fosforribosiltransferase/deficiência , Nicotinamida Fosforribosiltransferase/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Fosforilação Oxidativa , Fenótipo
2.
J Cereb Blood Flow Metab ; 41(8): 2116-2131, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33563078

RESUMO

Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the NAD+ salvage pathway. Our previous study demonstrated that deletion of NAMPT gene in projection neurons using Thy1-NAMPT-/- conditional knockout (cKO) mice causes neuronal degeneration, muscle atrophy, neuromuscular junction abnormalities, paralysis and eventually death. Here we conducted a combined metabolomic and transcriptional profiling study in vivo in an attempt to further investigate the mechanism of neuronal degeneration at metabolite and mRNA levels after NAMPT deletion. Here using steady-state metabolomics, we demonstrate that deletion of NAMPT causes a significant decrease of NAD+ metabolome and bioenergetics, a buildup of metabolic intermediates upstream of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in glycolysis, and an increase of oxidative stress. RNA-seq shows that NAMPT deletion leads to the increase of mRNA levels of enzymes in NAD metabolism, in particular PARP family of NAD+ consumption enzymes, as well as glycolytic genes Glut1, Hk2 and PFBFK3 before GAPDH. GO, KEGG and GSEA analyses show the activations of apoptosis, inflammation and immune responsive pathways and the inhibition of neuronal/synaptic function in the cKO mice. The current study suggests that increased oxidative stress, apoptosis and neuroinflammation contribute to neurodegeneration and mouse death as a direct consequence of bioenergetic stress after NAMPT deletion.


Assuntos
Morte Celular/genética , Citocinas/genética , Metabolismo Energético/genética , Neurônios/metabolismo , Nicotinamida Fosforribosiltransferase/genética , Estresse Oxidativo/genética , Trifosfato de Adenosina/metabolismo , Animais , Citocinas/deficiência , Regulação para Baixo , Feminino , Glicólise , Masculino , Metabolômica , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , NAD/metabolismo , Nicotinamida Fosforribosiltransferase/deficiência , Análise de Componente Principal , Regulação para Cima
3.
Am J Physiol Heart Circ Physiol ; 317(4): H711-H725, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31347918

RESUMO

The heart requires high-energy production, but metabolic ability declines in the failing heart. Nicotinamide phosphoribosyl-transferase (Nampt) is a rate-limiting enzyme in the salvage pathway of nicotinamide adenine dinucleotide (NAD) synthesis. NAD is directly involved in various metabolic processes and may indirectly regulate metabolic gene expression through sirtuin 1 (Sirt1), an NAD-dependent protein deacetylase. However, how Nampt regulates cardiac function and metabolism in the failing heart is poorly understood. Here we show that pressure-overload (PO)-induced heart failure is exacerbated in both systemic Nampt heterozygous knockout (Nampt+/-) mice and mice with cardiac-specific Nampt overexpression (Tg-Nampt). The NAD level declined in Nampt+/- mice under PO (wild: 377 pmol/mg tissue; Nampt+/-: 119 pmol/mg tissue; P = 0.028). In cultured cardiomyocytes, Nampt knockdown diminished mitochondrial NAD content and ATP production (relative ATP production: wild: 1; Nampt knockdown: 0.56; P = 0.0068), suggesting that downregulation of Nampt induces mitochondrial dysfunction. On the other hand, the NAD level was increased in Tg-Nampt mice at baseline but not during PO, possibly due to increased consumption of NAD by Sirt1. The expression of Sirt1 was increased in Tg-Nampt mice, in association with reduced overall protein acetylation. PO-induced downregulation of metabolic genes was exacerbated in Tg-Nampt mice. In cultured cardiomyocytes, Nampt and Sirt1 cooperatively suppressed mitochondrial proteins and ATP production, thereby promoting mitochondrial dysfunction. In addition, Nampt overexpression upregulated inflammatory cytokines, including TNF-α and monocyte chemoattractant protein-1. Thus endogenous Nampt maintains cardiac function and metabolism in the failing heart, whereas Nampt overexpression is detrimental during PO, possibly due to excessive activation of Sirt1, suppression of mitochondrial function, and upregulation of proinflammatory mechanisms.NEW & NOTEWORTHY Nicotinamide phosphoribosyl-transferase (Nampt) is a rate-limiting enzyme in the salvage pathway of nicotinamide adenine dinucleotide synthesis. We demonstrate that pressure overload-induced heart failure is exacerbated in both systemic Nampt heterozygous knockout mice and mice with cardiac-specific Nampt overexpression. Both loss- and gain-of-function models exhibited reduced protein acetylation, suppression of metabolic genes, and mitochondrial energetic dysfunction. Thus endogenous Nampt maintains cardiac function and metabolism in the failing heart, but cardiac-specific Nampt overexpression is detrimental rather than therapeutic.


Assuntos
Citocinas/metabolismo , Metabolismo Energético , Insuficiência Cardíaca/enzimologia , Mitocôndrias Cardíacas/enzimologia , Miócitos Cardíacos/enzimologia , Nicotinamida Fosforribosiltransferase/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Aorta Torácica/fisiopatologia , Aorta Torácica/cirurgia , Células Cultivadas , Citocinas/deficiência , Citocinas/genética , Modelos Animais de Doenças , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/fisiopatologia , Mediadores da Inflamação/metabolismo , Ligadura , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Cardíacas/patologia , Miócitos Cardíacos/patologia , NAD/metabolismo , Nicotinamida Fosforribosiltransferase/deficiência , Nicotinamida Fosforribosiltransferase/genética , Sirtuína 1/genética , Sirtuína 1/metabolismo
4.
Kidney Int ; 93(5): 1073-1085, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29398136

RESUMO

Circulating inorganic phosphate exhibits a remarkable daily oscillation based on food intake. In humans and rodents, the daily oscillation in response to food intake may be coordinated to control the intestinal absorption, renal excretion, cellular shifts, and extracellular concentration of inorganic phosphate. However, mechanisms regulating the resulting oscillation are unknown. Here we investigated the roles of the sodium phosphate cotransporter SLC34 (Npt2) family and nicotinamide phosphoribosyltransferase (Nampt) in the daily oscillation of plasma inorganic phosphate levels. First, it is roughly linked to urinary inorganic phosphate excretion. Second, expression of renal Npt2a and Npt2c, and intestinal Npt2b proteins also exhibit a dynamic daily oscillation. Analyses of Npt2a, Npt2b, and Npt2c knockout mice revealed the importance of renal inorganic phosphate reabsorption and cellular inorganic phosphate shifts in the daily oscillation. Third, experiments in which nicotinamide and a specific Nampt inhibitor (FK866) were administered in the active and rest phases revealed that the Nampt/NAD+ system is involved in renal inorganic phosphate excretion. Additionally, for cellular shifts, liver-specific Nampt deletion disturbed the daily oscillation of plasma phosphate during the rest but not the active phase. In systemic Nampt+/- mice, NAD levels were significantly reduced in the liver, kidney, and intestine, and the daily oscillation (active and rest phases) of the plasma phosphate concentration was attenuated. Thus, the Nampt/NAD+ system for Npt2 regulation and cellular shifts to tissues such as the liver play an important role in generating daily oscillation of plasma inorganic phosphate levels.


Assuntos
Ritmo Circadiano , Citocinas/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Fosfatos/sangue , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIc/metabolismo , Animais , Biomarcadores/sangue , Biomarcadores/urina , Citocinas/antagonistas & inibidores , Citocinas/deficiência , Citocinas/genética , Inibidores Enzimáticos/farmacologia , Feminino , Intestinos/enzimologia , Rim/enzimologia , Fígado/enzimologia , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , NAD/metabolismo , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Nicotinamida Fosforribosiltransferase/deficiência , Nicotinamida Fosforribosiltransferase/genética , Fosfatos/urina , Eliminação Renal , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/deficiência , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/deficiência , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIc/deficiência , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIc/genética , Fatores de Tempo
5.
J Pharmacol Exp Ther ; 365(1): 96-106, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29420256

RESUMO

Lower plasma nicotinamide phosphoribosyltransferase (NAMPT) levels are associated with improved response to methotrexate (MTX) in patients with juvenile idiopathic arthritis. Cell-based studies confirmed that reduced cellular NAMPT activity potentiates the pharmacologic activity of MTX; however, the mechanism of this interaction has yet to be defined. Therefore, in this study, we investigate the mechanism of enhanced pharmacologic activity of MTX in NAMPT-deficient A549 cells. Small interfering RNA-based silencing of NAMPT expression resulted in a greater than 3-fold increase in sensitivity to MTX (P < 0.005) that was completely reversed by supplementation with folinic acid. Despite a 68% reduction in cellular NAD levels in NAMPT-deficient cells, no change in expression or activity of dihydrofolate reductase was observed and uptake of MTX was not significantly altered. MTX did not potentiate the depletion of cellular NAD levels, but NAMPT-deficient cells had significant elevations in levels of intermediates of de novo purine biosynthesis and were 4-fold more sensitive to depletion of ATP by MTX (P < 0.005). Supplementation with hypoxanthine and thymidine completely reversed the antiproliferative activity of MTX in NAMPT-deficient cells and corresponded to repletion of the cellular ATP pool without any effect on NAD levels. Together, these findings demonstrate that increased MTX activity with decreased NAMPT expression is dependent on the antifolate activity of MTX and is driven by enhanced sensitivity to the ATP-depleting effects of MTX. For the first time, these findings provide mechanistic details to explain the increase in pharmacological activity of MTX under conditions of reduced NAMPT activity.


Assuntos
Trifosfato de Adenosina/metabolismo , Citocinas/deficiência , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Metotrexato/farmacologia , Nicotinamida Fosforribosiltransferase/deficiência , Células A549 , Transporte Biológico , Proliferação de Células/efeitos dos fármacos , Citocinas/genética , Ácido Fólico/metabolismo , Inativação Gênica , Homeostase/efeitos dos fármacos , Humanos , Nicotinamida Fosforribosiltransferase/genética
6.
Circ Res ; 120(12): 1889-1902, 2017 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-28356339

RESUMO

RATIONALE: The thoracic aortic wall can degenerate over time with catastrophic consequences. Vascular smooth muscle cells (SMCs) can resist and repair artery damage, but their capacities decline with age and stress. Recently, cellular production of nicotinamide adenine dinucleotide (NAD+) via nicotinamide phosphoribosyltransferase (Nampt) has emerged as a mediator of cell vitality. However, a role for Nampt in aortic SMCs in vivo is unknown. OBJECTIVES: To determine whether a Nampt-NAD+ control system exists within the aortic media and is required for aortic health. METHODS AND RESULTS: Ascending aortas from patients with dilated aortopathy were immunostained for NAMPT, revealing an inverse relationship between SMC NAMPT content and aortic diameter. To determine whether a Nampt-NAD+ control system in SMCs impacts aortic integrity, mice with Nampt-deficient SMCs were generated. SMC-Nampt knockout mice were viable but with mildly dilated aortas that had a 43% reduction in NAD+ in the media. Infusion of angiotensin II led to aortic medial hemorrhage and dissection. SMCs were not apoptotic but displayed senescence associated-ß-galactosidase activity and upregulated p16, indicating premature senescence. Furthermore, there was evidence for oxidized DNA lesions, double-strand DNA strand breaks, and pronounced susceptibility to single-strand breakage. This was linked to suppressed poly(ADP-ribose) polymerase-1 activity and was reversible on resupplying NAD+ with nicotinamide riboside. Remarkably, we discovered unrepaired DNA strand breaks in SMCs within the human ascending aorta, which were specifically enriched in SMCs with low NAMPT. NAMPT promoter analysis revealed CpG hypermethylation within the dilated human thoracic aorta and in SMCs cultured from these tissues, which inversely correlated with NAMPT expression. CONCLUSIONS: The aortic media depends on an intrinsic NAD+ fueling system to protect against DNA damage and premature SMC senescence, with relevance to human thoracic aortopathy.


Assuntos
Aneurisma da Aorta Torácica/enzimologia , Citocinas/biossíntese , Dano ao DNA/fisiologia , Genoma/fisiologia , Miócitos de Músculo Liso/fisiologia , Nicotinamida Fosforribosiltransferase/biossíntese , Túnica Média/fisiologia , Adulto , Idoso , Animais , Aorta/enzimologia , Aorta/patologia , Aneurisma da Aorta Torácica/genética , Aneurisma da Aorta Torácica/patologia , Células Cultivadas , Citocinas/deficiência , Citocinas/genética , Feminino , Humanos , Microdissecção e Captura a Laser/métodos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Miócitos de Músculo Liso/patologia , Nicotinamida Fosforribosiltransferase/deficiência , Nicotinamida Fosforribosiltransferase/genética , Túnica Média/patologia
7.
Cell Rep ; 17(1): 69-85, 2016 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-27681422

RESUMO

Photoreceptor death is the endpoint of many blinding diseases. Identifying unifying pathogenic mechanisms in these diseases may offer global approaches for facilitating photoreceptor survival. We found that rod or cone photoreceptor-specific deletion of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the major NAD(+) biosynthetic pathway beginning with nicotinamide, caused retinal degeneration. In both cases, we could rescue vision with nicotinamide mononucleotide (NMN). Significantly, retinal NAD(+) deficiency was an early feature of multiple mouse models of retinal dysfunction, including light-induced degeneration, streptozotocin-induced diabetic retinopathy, and age-associated dysfunction. Mechanistically, NAD(+) deficiency caused metabolic dysfunction and consequent photoreceptor death. We further demonstrate that the NAD(+)-dependent mitochondrial deacylases SIRT3 and SIRT5 play important roles in retinal homeostasis and that NAD(+) deficiency causes SIRT3 dysfunction. These findings demonstrate that NAD(+) biosynthesis is essential for vision, provide a foundation for future work to further clarify the mechanisms involved, and identify a unifying therapeutic target for diverse blinding diseases.


Assuntos
Citocinas/genética , Retinopatia Diabética/metabolismo , Mitocôndrias/metabolismo , NAD/metabolismo , Nicotinamida Fosforribosiltransferase/genética , Degeneração Retiniana/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Morte Celular , Citocinas/deficiência , Retinopatia Diabética/induzido quimicamente , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/patologia , Mononucleotídeo de Nicotinamida/metabolismo , Nicotinamida Fosforribosiltransferase/deficiência , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Transdução de Sinais , Sirtuína 3/genética , Sirtuína 3/metabolismo , Sirtuínas/genética , Sirtuínas/metabolismo , Estreptozocina , Visão Ocular/fisiologia
8.
Cell Rep ; 16(7): 1851-60, 2016 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-27498863

RESUMO

Obesity is associated with adipose tissue dysfunction and multi-organ insulin resistance. However, the mechanisms of such obesity-associated systemic metabolic complications are not clear. Here, we characterized mice with adipocyte-specific deletion of nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting NAD(+) biosynthetic enzyme known to decrease in adipose tissue of obese and aged rodents and people. We found that adipocyte-specific Nampt knockout mice had severe insulin resistance in adipose tissue, liver, and skeletal muscle and adipose tissue dysfunction, manifested by increased plasma free fatty acid concentrations and decreased plasma concentrations of a major insulin-sensitizing adipokine, adiponectin. Loss of Nampt increased phosphorylation of CDK5 and PPARγ (serine-273) and decreased gene expression of obesity-linked phosphorylated PPARγ targets in adipose tissue. These deleterious alterations were normalized by administering rosiglitazone or a key NAD(+) intermediate, nicotinamide mononucleotide (NMN). Collectively, our results provide important mechanistic and therapeutic insights into obesity-associated systemic metabolic derangements, particularly multi-organ insulin resistance.


Assuntos
Adipócitos/enzimologia , Tecido Adiposo/enzimologia , Citocinas/genética , Resistência à Insulina/genética , Nicotinamida Fosforribosiltransferase/genética , Obesidade/genética , Adipócitos/efeitos dos fármacos , Adipócitos/patologia , Adiponectina , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/patologia , Animais , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/metabolismo , Citocinas/deficiência , Ácidos Graxos não Esterificados/sangue , Feminino , Regulação da Expressão Gênica , Hipoglicemiantes/farmacologia , Fígado/enzimologia , Fígado/patologia , Masculino , Camundongos , Camundongos Knockout , Músculo Esquelético/enzimologia , Músculo Esquelético/patologia , Mononucleotídeo de Nicotinamida/farmacologia , Nicotinamida Fosforribosiltransferase/deficiência , Obesidade/enzimologia , Obesidade/patologia , PPAR gama/genética , PPAR gama/metabolismo , Fosforilação , Rosiglitazona , Transdução de Sinais , Tiazolidinedionas/farmacologia
9.
Cell Metab ; 24(2): 269-82, 2016 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-27508874

RESUMO

NAD is an obligate co-factor for the catabolism of metabolic fuels in all cell types. However, the availability of NAD in several tissues can become limited during genotoxic stress and the course of natural aging. The point at which NAD restriction imposes functional limitations on tissue physiology remains unknown. We examined this question in murine skeletal muscle by specifically depleting Nampt, an essential enzyme in the NAD salvage pathway. Knockout mice exhibited a dramatic 85% decline in intramuscular NAD content, accompanied by fiber degeneration and progressive loss of both muscle strength and treadmill endurance. Administration of the NAD precursor nicotinamide riboside rapidly ameliorated functional deficits and restored muscle mass despite having only a modest effect on the intramuscular NAD pool. Additionally, lifelong overexpression of Nampt preserved muscle NAD levels and exercise capacity in aged mice, supporting a critical role for tissue-autonomous NAD homeostasis in maintaining muscle mass and function.


Assuntos
Homeostase , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , NAD/metabolismo , Administração Oral , Envelhecimento/fisiologia , Animais , Disponibilidade Biológica , Metabolismo Energético , Glucose/metabolismo , Inflamação/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Força Muscular , Músculo Esquelético/enzimologia , Músculo Esquelético/fisiopatologia , Necrose , Niacinamida/administração & dosagem , Niacinamida/análogos & derivados , Niacinamida/metabolismo , Niacinamida/farmacologia , Nicotinamida Fosforribosiltransferase/deficiência , Nicotinamida Fosforribosiltransferase/metabolismo , Tamanho do Órgão , Condicionamento Físico Animal , Compostos de Piridínio , Transcrição Gênica
10.
Sci Rep ; 6: 26746, 2016 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-27229177

RESUMO

NAMPT has been suggested association with atherosclerosis and insulin resistance. However, the impact of NAMPT on atherosclerosis remained unknown. Therefore, the objective of this study was to use a NAMPT loss-of-function approach to investigate the effect of NAMPT on atherosclerosis in hypercholesterolemic mice. We demonstrated that a specific NAMPT knockdown increased plasma HDL-C levels, reduced the plaque area of the total aorta en face and the cross-sectional aortic sinus, decreased macrophage number and apoptosis, and promoted RCT in HFD-fed ApoE KO mice. These changes were accompanied by increased PPARα, LXRα, ABCA1 and ABCG1 expressions in the liver. NAMPT knockdown also facilitated cholesterol efflux in RAW264.7 cells. We further investigated the effect of NAMPT knockdown on the PPARα-LXRα pathway of cholesterol metabolism with MK886 (a selective inhibitor of PPARα) in RAW264.7 macrophages. MK886 abolished the ability of NAMPT knockdown to decrease intracellular cholesterol levels to enhance the rate of (3)H-cholesterol efflux and to increase ABCA1/G1 and LXRα expressions in RAW264.7 macrophages. Our observations demonstrate that NAMPT knockdown exerted antiatherogenic effects by promoting cholesterol efflux and macrophage RCT through the PPARα- LXRα- ABCA1/G1pathway in vitro and in vivo.


Assuntos
Apolipoproteínas E/deficiência , Aterosclerose , HDL-Colesterol/sangue , Citocinas/deficiência , Gorduras na Dieta/efeitos adversos , Resistência à Insulina , Nicotinamida Fosforribosiltransferase/deficiência , Animais , Aterosclerose/sangue , Aterosclerose/induzido quimicamente , Aterosclerose/genética , Aterosclerose/patologia , Gorduras na Dieta/farmacologia , Técnicas de Silenciamento de Genes , Masculino , Camundongos , Camundongos Knockout , Células RAW 264.7
11.
PLoS One ; 9(6): e98972, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24905194

RESUMO

Nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme for nicotinamide adenine dinucleotide (NAD+) synthesis, and Sirt1, an NAD+-dependent histone deacetylase, protect the heart against ischemia/reperfusion (I/R). It remains unknown whether Nampt mediates the protective effect of ischemic preconditioning (IPC), whether nicotinamide mononucleotide (NMN, 500 mg/kg), a product of Nampt in the NAD+ salvage pathway, mimics the effect of IPC, or whether caloric restriction (CR) upregulates Nampt and protects the heart through a Sirt1-dependent mechanism. IPC upregulated Nampt protein, and the protective effect of IPC against ischemia (30 minutes) and reperfusion (24 hours) was attenuated at both early and late phases in Nampt +/- mice, suggesting that Nampt plays an essential role in mediating the protective effect of IPC. In order to mimic the effect of Nampt, NMN was administered by intraperitoneal injection. NMN significantly increased the level of NAD+ in the heart at baseline and prevented a decrease in NAD+ during ischemia. NMN protected the heart from I/R injury when it was applied once 30 minutes before ischemia or 4 times just before and during reperfusion, suggesting that exogenous NMN protects the heart from I/R injury in both ischemic and reperfusion phases. The protective effect of NMN was accompanied by decreases in acetylation of FoxO1, but it was not obvious in Sirt1 KO mice, suggesting that the effect of NMN is mediated through activation of Sirt1. Compared to control diet (90% calories), CR (60% calories for 6 weeks) in mice led to a significant reduction in I/R injury, accompanied by upregulation of Nampt. The protective effect of CR against I/R injury was not significant in cardiac-specific Sirt1 KO mice, suggesting that the protective effect of CR is in part mediated through the Nampt-Sirt1 pathway. In conclusion, exogenous application of NMN and CR protects the heart by both mimicking IPC and activating Sirt1.


Assuntos
Coração/efeitos dos fármacos , Isquemia Miocárdica/complicações , NAD/biossíntese , Mononucleotídeo de Nicotinamida/metabolismo , Mononucleotídeo de Nicotinamida/farmacologia , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/prevenção & controle , Animais , Restrição Calórica , Cardiotônicos/metabolismo , Cardiotônicos/farmacologia , Coração/fisiopatologia , Precondicionamento Isquêmico , Masculino , Camundongos , Infarto do Miocárdio/prevenção & controle , Nicotinamida Fosforribosiltransferase/deficiência , Nicotinamida Fosforribosiltransferase/metabolismo , Traumatismo por Reperfusão/metabolismo , Sirtuína 1/metabolismo , Regulação para Cima/efeitos dos fármacos
12.
EMBO J ; 33(12): 1321-40, 2014 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-24811750

RESUMO

Neural stem/progenitor cell (NSPC) proliferation and self-renewal, as well as insult-induced differentiation, decrease markedly with age. The molecular mechanisms responsible for these declines remain unclear. Here, we show that levels of NAD(+) and nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in mammalian NAD(+) biosynthesis, decrease with age in the hippocampus. Ablation of Nampt in adult NSPCs reduced their pool and proliferation in vivo. The decrease in the NSPC pool during aging can be rescued by enhancing hippocampal NAD(+) levels. Nampt is the main source of NSPC NAD(+) levels and required for G1/S progression of the NSPC cell cycle. Nampt is also critical in oligodendrocytic lineage fate decisions through a mechanism mediated redundantly by Sirt1 and Sirt2. Ablation of Nampt in the adult NSPCs in vivo reduced NSPC-mediated oligodendrogenesis upon insult. These phenotypes recapitulate defects in NSPCs during aging, giving rise to the possibility that Nampt-mediated NAD(+) biosynthesis is a mediator of age-associated functional declines in NSPCs.


Assuntos
Células-Tronco Adultas/metabolismo , Envelhecimento/fisiologia , Citocinas/metabolismo , Células-Tronco Neurais/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Animais , Bromodesoxiuridina , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Proliferação de Células , Cruzamentos Genéticos , Cuprizona , Citocinas/deficiência , Imunofluorescência , Camundongos , Camundongos Transgênicos , Mononucleotídeo de Nicotinamida , Nicotinamida Fosforribosiltransferase/deficiência
13.
Biochem Biophys Res Commun ; 434(1): 117-23, 2013 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-23537654

RESUMO

Despite emerging data showing that metabolic changes occur with stem cell differentiation, the cross-talk between factors governing energy metabolism and epigenetic modification is not understood. Nicotinamide adenine dinucleotide (NAD) participates in both energy metabolism and protein modification processes. Changes of the intracellular NAD concentration have been shown to correlate with differentiation of adult and embryonic stem cells. In the present study, we investigated the expression pattern of Nampt, the rate-limiting enzyme in NAD salvaging pathway, during osteogenic differentiation of the multipotent mouse fibroblast C3H10T1/2 and the omnipotent preosteoblast MC3T3-E1 cells. We found that Nampt was increasingly expressed during differentiation in both cell models. The increase of Nampt was associated with higher NAD concentration and Sirt1 activity. Knockdown of Nampt or addition of its specific inhibitor FK866 leads to lower intracellular NAD concentration and decline in osteogenesis. These findings indicate that osteogenic differentiation correlates with intracellular NAD metabolism in which Nampt plays a regulatory role.


Assuntos
Diferenciação Celular/fisiologia , Citocinas/biossíntese , Citocinas/genética , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Nicotinamida Fosforribosiltransferase/biossíntese , Nicotinamida Fosforribosiltransferase/genética , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/genética , Células 3T3 , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Citocinas/deficiência , Técnicas de Silenciamento de Genes , Camundongos , Células-Tronco Multipotentes/efeitos dos fármacos , NAD/metabolismo , Nicotinamida Fosforribosiltransferase/deficiência , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Sirtuína 1/metabolismo
14.
Neuro Endocrinol Lett ; 33(5): 559-64, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23090276

RESUMO

OBJECTIVES: Very little is known about the role of adipokines in atopic asthma in children. Studies about the potential role of novel proinflammatory adiopcytokine - visfatin are lacking. DESIGN AND SETTING: In this cross-sectional study serum visfatin (VISF) levels were measured in 89 asthmatic out-patients (61 boys and 28 girls aged 7.0-17.0 years) and in 33 healthy children. MATERIAL AND METHODS: Among examined asthmatics 59 (19 girls and 40 boys) had normal weight and 30 (9 girls and 21 boys) were obese. Serum VISF was evaluated using standard ELISA tests and the results were analysed in relation to body weight, asthma severity, and gender were analysed. RESULTS: The mean serum levels of visfatin were significantly (p<0.001) lower both in obese (2.13±0.07npg/ml) and non-obese asthmatic children (2.05±0.06 npg/ml) than in healthy children (2.57±0.07 pg/ml) regardless of gender. After stratifying by gender, there was a significant decrease (p<0.001) in visfatin levels in obese asthmatic girls (2.30±0.08 ng/ml) and asthmatic girls with normal weight girls (2.21±0,09 ng/ml) compared with girls from control group (2.69 ±0.06 ng/ml). In obese asthmatic boys and non-obese asthmatic boys also significantly lower (p<0.01) mean values of visfatin (2.03±0.06 ng/ml and 1.9±0.06 pg/ml respectively) were observed than in control boys (2.391±0.09 ng/ml). No relationships between examined adipokine levels and asthma severity, spirometric parameters, degree of allergic sensitization, BMI, were observed. CONCLUSION: Visfatin is not potential biomarker in atopic asthma in children and further studies evaluating the possible role of this proinflammatory adipokine in childhood atopic asthma pathogenesis are needed.


Assuntos
Asma/sangue , Asma/imunologia , Nicotinamida Fosforribosiltransferase/sangue , Nicotinamida Fosforribosiltransferase/imunologia , Adolescente , Biomarcadores/sangue , Índice de Massa Corporal , Criança , Feminino , Humanos , Hipersensibilidade/sangue , Hipersensibilidade/imunologia , Masculino , Nicotinamida Fosforribosiltransferase/deficiência , Obesidade/sangue , Obesidade/imunologia , Índice de Gravidade de Doença
15.
J Bone Miner Res ; 26(11): 2656-64, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21812028

RESUMO

Human aging is associated with a progressive decline in bone mass and an accumulation of marrow fat. We found that osteoblast differentiation was reduced and adipocyte formation increased in bone marrow stromal cells derived from aged mice compared with young controls. The increased adipogenesis correlated with a relatively lower Sirt1 activity and a lower intracellular NAD(+) concentration. We suppose that these effects were caused by age-related reduction of nicotinamide phosphoribosyltransferase (Nampt), the enzyme catalyzing NAD resynthesis from nicotinamide (NAM). In support of this hypothesis, treatment with Nampt inhibitor FK866 increased adipocyte formation and reduced mineralization in primary cultured bone marrow stromal cells. In addition, knockdown of Nampt in the mouse mesenchymal cell line C3H10T1/2 cells resulted in decreased Sirt1 activity and enhanced adipogenesis. Interestingly, although Nampt deficiency resulted in both decreased intracellular NAD(+) and increased NAM, the cell differentiation could be controlled only by regulation of NAM. These results indicate that the lineage fate determination of mesenchymal stem cells (MSCs) is influenced by cell energy metabolism and points to a possible mechanism for the development of senile osteoporosis. Furthermore, we suggest that side effects on bone should be considered when evaluating the long-term safety of NAD-interfering pharmaceuticals.


Assuntos
Adipogenia , Envelhecimento/metabolismo , Linhagem da Célula , Citocinas/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/enzimologia , Nicotinamida Fosforribosiltransferase/metabolismo , Osteogênese , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Envelhecimento/efeitos dos fármacos , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Carbazóis/farmacologia , Linhagem Celular , Linhagem da Célula/efeitos dos fármacos , Meios de Cultura/farmacologia , Citocinas/deficiência , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , NAD/metabolismo , Niacinamida/metabolismo , Nicotinamida Fosforribosiltransferase/deficiência , Osteogênese/efeitos dos fármacos , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/metabolismo , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo
16.
Mol Cell Biol ; 29(21): 5872-88, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19703994

RESUMO

GMX1777 is a prodrug of the small molecule GMX1778, currently in phase I clinical trials for the treatment of cancer. We describe findings indicating that GMX1778 is a potent and specific inhibitor of the NAD(+) biosynthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT). Cancer cells have a very high rate of NAD(+) turnover, which makes NAD(+) modulation an attractive target for anticancer therapy. Selective inhibition by GMX1778 of NAMPT blocks the production of NAD(+) and results in tumor cell death. Furthermore, GMX1778 is phosphoribosylated by NAMPT, which increases its cellular retention. The cytotoxicity of GMX1778 can be bypassed with exogenous nicotinic acid (NA), which permits NAD(+) repletion via NA phosphoribosyltransferase 1 (NAPRT1). The cytotoxicity of GMX1778 in cells with NAPRT1 deficiency, however, cannot be rescued by NA. Analyses of NAPRT1 mRNA and protein levels in cell lines and primary tumor tissue indicate that high frequencies of glioblastomas, neuroblastomas, and sarcomas are deficient in NAPRT1 and not susceptible to rescue with NA. As a result, the therapeutic index of GMX1777 can be widended in the treatment animals bearing NAPRT1-deficient tumors by coadministration with NA. This provides the rationale for a novel therapeutic approach for the use of GMX1777 in the treatment of human cancers.


Assuntos
Cianetos/uso terapêutico , Guanidinas/uso terapêutico , NAD/biossíntese , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Nicotinamida Fosforribosiltransferase/deficiência , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cianetos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Guanidinas/farmacologia , Humanos , Camundongos , Modelos Biológicos , Modelos Moleculares , Niacina/administração & dosagem , Niacina/farmacologia , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Nicotinamida Fosforribosiltransferase/genética , Nicotinamida Fosforribosiltransferase/metabolismo , Fosforilação/efeitos dos fármacos , Ribose/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
17.
J Immunol ; 181(7): 4685-95, 2008 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-18802071

RESUMO

Nicotinamide phosphoribosyl transferase (Nampt)/pre-B cell colony-enhancing factor (PBEF)/visfatin is a protein displaying multiple functional properties. Originally described as a cytokine-like protein able to regulate B cell development, apoptosis, and glucose metabolism, this protein also plays an important role in NAD biosynthesis. To gain insight into its physiological role, we have generated a mouse strain expressing a conditional Nampt allele. Lack of Nampt expression strongly affects development of both T and B lymphocytes. Analysis of hemizygous cells and in vitro cell lines expressing distinct levels of Nampt illustrates the critical role of this protein in regulating intracellular NAD levels. Consequently, a clear relationship was found between intracellular Nampt levels and cell death in response to the genotoxic agent MNNG (N-methyl-N'-nitro-N-nitrosoguanidine), confirming that this enzyme represents a key regulator of cell sensitivity to NAD-consuming stress secondary to poly(ADP-ribose) polymerases overactivation. By using mutant forms of this protein and a well-characterized pharmacological inhibitor (FK866), we unequivocally demonstrate that the ability of the Nampt to regulate cell viability during genotoxic stress requires its enzymatic activity. Collectively, these data demonstrate that Nampt participates in cellular resistance to genotoxic/oxidative stress, and it may confer to cells of the immune system the ability to survive during stressful situations such as inflammation.


Assuntos
Diferenciação Celular/imunologia , Citocinas/fisiologia , Imunidade Celular , Linfócitos/enzimologia , Linfócitos/imunologia , Nicotinamida Fosforribosiltransferase/fisiologia , Animais , Morte Celular/genética , Morte Celular/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Citocinas/biossíntese , Citocinas/deficiência , Citocinas/genética , Deleção de Genes , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Celular/genética , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Linfócitos/efeitos dos fármacos , Metilnitronitrosoguanidina/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Células NIH 3T3 , Nicotinamida Fosforribosiltransferase/biossíntese , Nicotinamida Fosforribosiltransferase/deficiência , Nicotinamida Fosforribosiltransferase/genética , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Estresse Oxidativo/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia
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