RESUMO
NAD(P)H: quinone oxidoreductase 1 (NQO1) is a flavin protease highly expressed in various cancer cells. NQO1 catalyzes a futile redox cycle in substrates, leading to substantial reactive oxygen species (ROS) production. This ROS generation results in extensive DNA damage and elevated poly (ADP-ribose) polymerase 1 (PARP1)-mediated consumption of nicotinamide adenine dinucleotide (NAD+), ultimately causing cell death. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD+ salvage synthesis pathway, emerges as a critical target in cancer therapy. The concurrent inhibition of NQO1 and NAMPT triggers hyperactivation of PARP1 and intensive NAD+ depletion. In this study, we designed, synthesized, and assessed a novel series of proqodine A derivatives targeting both NQO1 and NAMPT. Among these, compound T8 demonstrated potent antitumor properties. Specifically, T8 selectively inhibited the proliferation of MCF-7 cells and induced apoptosis through mechanisms dependent on both NQO1 and NAMPT. This discovery offers a promising new molecular entity for advancing anticancer research.
Assuntos
NAD , Nicotinamida Fosforribosiltransferase , Humanos , NAD/metabolismo , Linhagem Celular Tumoral , Espécies Reativas de Oxigênio/metabolismo , Nicotinamida Fosforribosiltransferase/genética , Nicotinamida Fosforribosiltransferase/metabolismo , Citocinas/metabolismo , Quinonas , OxirredutasesRESUMO
Depletion of nicotinamide adenine dinucleotide (NAD+) is associated with aging and disease, spurring the study of dietary supplements to replenish NAD+. The catabolism of NAD+ to nicotinamide (NAM) requires the salvage of NAM to replenish cellular NAD+, which relies on the rate-limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT). Pharmacological activation of NAMPT provides an alternative to dietary supplements. Screening for activators of NAMPT identified small molecule NAMPT positive allosteric modulators (N-PAMs). N-PAMs bind to the rear channel of NAMPT increasing enzyme activity and alleviating feedback inhibition by NAM and NAD+. Synthesis of over 70 N-PAMs provided an excellent correlation between rear channel binding affinity and potency for enzyme activation, confirming the mechanism of allosteric activation via binding to the rear channel. The mechanism accounts for higher binding affinity leading to loss of efficacy. Enzyme activation translated directly to elevation of NAD+ measured in cells. Optimization led to an orally bioavailable N-PAM.
Assuntos
NAD , Nicotinamida Fosforribosiltransferase , Nicotinamida Fosforribosiltransferase/química , Nicotinamida Fosforribosiltransferase/metabolismo , NAD/metabolismo , Niacinamida/farmacologia , Linhagem Celular Tumoral , Citocinas/metabolismo , Relação Estrutura-AtividadeRESUMO
Regular physical activity and the use of nutritional supplements, including antioxidants, are recognized as efficacious approaches for the prevention and mitigation of obesity-related complications. This study investigated the effects of 12 weeks of CrossFit training combined with astaxanthin (ASX) supplementation on some plasma adipokines in males with obesity. Sixty-eight males with obesity (BMI: 33.6 ± 1.4 kg·m-2) were randomly assigned into four groups: the control group (CG; n = 11), ASX supplementation group (SG; n = 11), CrossFit group (TG; n = 11), and training plus supplement group (TSG; n = 11). Participants underwent 12 weeks of supplementation with ASX or placebo (20 mg/day capsule daily), CrossFit training, or a combination of both interventions. Plasma levels of semaphorin 3C (SEMA3C), apelin, chemerin, omentin1, visfatin, resistin, adiponectin, leptin, vaspin, and RBP4 were measured 72 h before the first training session and after the last training session. The plasma levels of all measured adipokines were significantly altered in SG, TG, and TSG groups (p < 0.05). The reduction of resistin was significantly higher in TSG than in SG (p < 0.05). The plasma levels of omentin1 were significantly higher in both training groups of TG and TSG than SG (p < 0.05), although such a meaningful difference was not observed between both training groups (p > 0.05). Significant differences were found in the reductions of plasma levels of vaspin, visfatin, apelin, RBP4, chemerin, and SEMA3C between the SG and TSG groups (p < 0.05). The study found that a 12-week intervention using ASX supplementation and CrossFit exercises resulted in significant improvements in several adipokines among male individuals with obesity. Notably, the combined approach of supplementation and training had the most pronounced results. The findings presented in this study indicate that the supplementation of ASX and participation in CrossFit exercise have the potential to be effective therapies in mitigating complications associated with obesity and enhancing metabolic health.
Assuntos
Adipocinas , Semaforinas , Humanos , Masculino , Resistina/metabolismo , Apelina , Nicotinamida Fosforribosiltransferase/metabolismo , Obesidade , Suplementos Nutricionais , Semaforinas/metabolismo , Proteínas Plasmáticas de Ligação ao RetinolRESUMO
In aging and disease, cellular nicotinamide adenine dinucleotide (NAD+) is depleted by catabolism to nicotinamide (NAM). NAD+ supplementation is being pursued to enhance human healthspan and lifespan. Activation of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting step in NAD+ biosynthesis, has the potential to increase the salvage of NAM. Novel NAMPT-positive allosteric modulators (N-PAMs) were discovered in addition to the demonstration of NAMPT activation by biogenic phenols. The mechanism of activation was revealed through the synthesis of novel chemical probes, new NAMPT co-crystal structures, and enzyme kinetics. Binding to a rear channel in NAMPT regulates NAM binding and turnover, with biochemical observations being replicated by NAD+ measurements in human cells. The mechanism of action of N-PAMs identifies, for the first time, the role of the rear channel in the regulation of NAMPT turnover coupled to productive and nonproductive NAM binding. The tight regulation of cellular NAMPT via feedback inhibition by NAM, NAD+, and adenosine 5'-triphosphate (ATP) is differentially regulated by N-PAMs and other activators, indicating that different classes of pharmacological activators may be engineered to restore or enhance NAD+ levels in affected tissues.
Assuntos
NAD , Nicotinamida Fosforribosiltransferase , Humanos , Citocinas/metabolismo , Longevidade , NAD/metabolismo , Niacinamida/farmacologia , Niacinamida/metabolismo , Nicotinamida Fosforribosiltransferase/química , Nicotinamida Fosforribosiltransferase/metabolismo , Sítio AlostéricoRESUMO
Research studies on NAD+ have proven its crucial role in aging and disease. Nicotinamide mononucleotide (NMN), as the key intermediate of NAD+, plays a significant role in supplying and maintaining NAD+ levels. In the present study, a biocatalytic method for the efficient synthesis of NMN was established. First, Escherichia coli was systematically modified to make it more conducive to the biosynthesis and accumulation of NMN. Next, the performance of nicotinamide phosphoribosyltransferase from Vibrio bacteriophage KVP40 (VpNadV) was determined, which has the best catalytic activity to produce NMN from nicotinamide. The accumulation of extracellular NMN was further increased after the introduction of an NMN transporter. Fine-tuning of gene expression and copy number led to the synthesis of NMN at the yield of 2.6 g/L at the shake flask level. The introduction of a nicotinamide transporter, BcniaP, could not obviously increase the production of NMN at the shake flask level, but it decreased the production of NMN at the bioreactor level. Finally, the titer of NMN reached 16.2 g/L with a conversion ratio of 97.0% from nicotinamide, both of which are highest according to currently available reports. The fed-batch fermentation with direct supplementation of nicotinamide could facilitate the industrial-scale production of NMN compared to that achieved by the whole-cell catalysis process. These results also represent the highest reported yield of NMN synthesized from nicotinamide in E. coli.
Assuntos
Mononucleotídeo de Nicotinamida , Nicotinamida Fosforribosiltransferase , Escherichia coli/genética , Escherichia coli/metabolismo , NAD/metabolismo , Niacinamida/metabolismo , Mononucleotídeo de Nicotinamida/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismoRESUMO
Securidaca inappendiculata (SI) Hassk. is a traditional medicine used to treat rheumatoid arthritis. Recent studies have reported that macrophages are the primary regulators of joint homeostasis and their polarization is closely related to their metabolic mode. Here, we aimed to investigate the relationship between the joint protective effect of SI's xanthone-rich fraction (XRF) on collagen-induced arthritis (CIA) in rats and the nicotinamide phosphoribosyltransferase (NAMPT)-glycolysis-polarization axis of macrophages. CIA model rats were treated with oral XRF and therapeutic efficacy was assessed based on arthritis score, degree of paw swelling, histological examination, and immunohistochemical analysis. Serum levels of cytokines, cellular metabolite concentrations, and protein and mRNA expression were determined by enzyme-linked immunosorbent assay (ELISA), western blotting (WB), and quantitative real-time PCR (RT-qPCR), respectively. The effects of dihydroxy-3,4-dimethoxyxanthone (XAN), a representative SI-derived compound, on RAW264.7 macrophages was analyzed in vitro using confocal laser scanning and flow cytometry. We found that XRF treatment significantly alleviated disease severity in CIA model rats. Levels of pro-inflammatory cytokines in the serum and M1 markers in synovium were reduced after XRF treatment, accompanied by an increase in the levels of anti-inflammatory cytokines and M2 markers. Further, XRF significantly suppressed synovial glycolysis by regulating NAMPT. In vitro studies further showed that XAN induced repolarization of lipopolysaccharide (LPS)-induced RAW264.7 macrophages with M1-M2 phenotype. Moreover, XAN negatively regulated glycolysis in the LPS-induced RAW264.7 macrophages in correlation with changes in NAMPT expression. Overall, the findings of this study suggest that the joint protective effects of XRF are achieved by inhibiting the NAMPT/glycolysis pathway and thereby regulating macrophage polarization.
Assuntos
Artrite Experimental , Securidaca , Xantonas , Animais , Artrite Experimental/patologia , Citocinas/metabolismo , Glicólise , Lipopolissacarídeos/farmacologia , Macrófagos , Nicotinamida Fosforribosiltransferase/metabolismo , Ratos , Securidaca/metabolismo , Xantonas/farmacologia , Xantonas/uso terapêuticoRESUMO
Obesity poses a global health challenge and is a major risk factor for diabetes mellitus, cardiovascular diseases, hypertension, stroke and certain kinds of cancers. Although the effects of nicotinamide (NAM) on liver metabolism and diseases were well documented, its effects on adipose tissue are yet to be characterized. Herein, we found that NAM supplementation significantly reduced fat mass and improved glucose tolerance in obese mice. Proteomic analysis revealed that NAM supplementation upregulates mitochondrial proteins while quantitative polymerase chain reaction showed that PPARα and PGC1α were both upregulated in adipose tissues, proposing that NAM increased mitochondrial biogenesis in adipose tissue. Indeed, NAM treatment increased proteins related to mitochondrial functions including oxidative phosphorylation, fatty acid oxidation, and TCA cycle. Furthermore, isotope-tracing assisted metabolic profiling revealed that NAM activated NAMPT and increased cellular NAD+ level by 30%. Unexpectedly, we found that NAM also increased glucose derived amino acids to enhance glutathione synthesis for maintaining cellular redox homeostasis. Taken together, our results demonstrated that NAM reprogramed cellular metabolism, enhanced adipose mitochondrial functions to ameliorate symptoms associated with obesity.
Assuntos
NAD , Niacinamida , Tecido Adiposo/metabolismo , Animais , Glucose/metabolismo , Camundongos , NAD/metabolismo , Niacinamida/metabolismo , Niacinamida/farmacologia , Nicotinamida Fosforribosiltransferase/metabolismo , Obesidade/metabolismo , Biogênese de Organelas , ProteômicaRESUMO
Non-small cell lung cancer (NSCLC) is one of the prevalent and deadly cancers worldwide. Cisplatin (CDDP) has been used as a standard adjuvant therapy for advanced NSCLC patients, while chemoresistance is one of the most challenging problems to limit its clinical application. Our data showed that the expression of visfatin was significantly increased in CDDP resistant NSCLC cells as compared with that in their parental cells, while knockdown of visfatin or its neutralization antibody can restore the CDDP sensitivity of resistant NSCLC cells. The upregulation of visfatin in CDDP resistant NSCLC cells was due to the increased mRNA stability and promoter activity. Further, we found that signal transducer and activator of transcription 3 (STAT3), which was increased in chemoresistant cells, can increase the transcription of visfatin. While tristetraprolin (TTP), which can decease mRNA stability of visfatin, was decreased in chemoresistant cells. Inhibition of STAT3 or over expression of TTP can restore CDDP sensitivity of resistant NSCLC cells. Collectively, our data showed that STAT3 and TTP-regulated expression of visfatin was involved in CDDP resistance of NSCLC cells. It indicated that targeted inhibition of visfatin should be a potential approach to overcome CDDP resistance of NSCLC treatment.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/fisiopatologia , Citocinas/metabolismo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Neoplasias Pulmonares/fisiopatologia , Nicotinamida Fosforribosiltransferase/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Estabilidade de RNA/fisiologia , Fator de Transcrição STAT3/metabolismo , Tristetraprolina/metabolismo , Regulação para Cima/fisiologiaRESUMO
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 , FenótipoRESUMO
Maternal obesity is associated with multiple adverse reproductive outcomes, whereas the underlying molecular mechanisms are still not fully understood. Here, we found the reduced nicotinamide phosphoribosyl transferase (NAMPT) expression and lowered nicotinamide adenine dinucleotide (NAD+ ) content in oocytes from obese mice. Next, by performing morpholino knockdown assay and pharmacological inhibition, we revealed that NAMPT deficiency not only severely disrupts maturational progression and meiotic apparatus, but also induces the metabolic dysfunction in oocytes. Furthermore, overexpression analysis demonstrated that NAMPT insufficiency induced NAD+ loss contributes to the compromised developmental potential of oocytes and early embryos from obese mice. Importantly, in vitro supplement and in vivo administration of nicotinic acid (NA) was able to ameliorate the obesity-associated meiotic defects and oxidative stress in oocytes. Our results indicate a role of NAMPT in modulating oocyte meiosis and metabolism, and uncover the beneficial effects of NA treatment on oocyte quality from obese mice.
Assuntos
Citocinas/metabolismo , NAD/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Obesidade Materna/metabolismo , Oócitos/metabolismo , Transdução de Sinais/genética , Animais , Citocinas/genética , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Desenvolvimento Embrionário/genética , Feminino , Técnicas de Silenciamento de Genes , Meiose/efeitos dos fármacos , Meiose/genética , Camundongos , Camundongos Endogâmicos ICR , Niacina/administração & dosagem , Nicotinamida Fosforribosiltransferase/genética , Obesidade Materna/tratamento farmacológico , Obesidade Materna/etiologia , Oócitos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Gravidez , Transdução de Sinais/efeitos dos fármacos , Resultado do TratamentoRESUMO
KPT-9274 is a phase 1 first-in-class dual PAK4/NAMPT inhibitor for solid tumor and non-Hodgkin's lymphoma. It demonstrates pre-clinical efficacy toward a broad spectrum of acute myeloid leukemia (AML) subtypes by inhibiting NAMPT-dependent NAD+ production. NAMPT is the rate-limiting enzyme in the salvage metabolic pathway leading to NAD+ generation. Tumor cells which are deficient in de novo pathway enzyme NAPRT1 are addicted to NAMPT. In clinical trials, treatment with NAMPT inhibitors resulted in dose-limiting toxicities. In order to dissect the mechanism of toxicity, mice were treated with KPT-9274 and resulting toxicities were characterized histopathologically and biochemically. KPT-9274 treatment caused gender-dependent stomach and kidney injuries and anemia. Female mice treated with KPT-9274 had EPO deficiency and associated impaired erythropoiesis. KPT-9274 treatment suppressed SIRT3 expression and concomitantly upregulated acetyl-manganese superoxide dismutase (MnSOD) in IMCD3 cells, providing a mechanistic basis for observed kidney toxicity. Importantly, niacin supplementation mitigated KPT-9274-caused kidney injury and EPO deficiency without affecting its efficacy. Altogether, our study delineated the mechanism of KPT-9274-mediated toxicity and sheds light onto developing strategies to improve the tolerability of this important anti-AML inhibitor.
Assuntos
Acrilamidas/efeitos adversos , Aminopiridinas/efeitos adversos , Anemia/induzido quimicamente , Antineoplásicos/efeitos adversos , Nefropatias/induzido quimicamente , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Acetilação/efeitos dos fármacos , Anemia/etiologia , Anemia/metabolismo , Anemia/patologia , Animais , Eritropoese/efeitos dos fármacos , Feminino , Humanos , Nefropatias/etiologia , Nefropatias/metabolismo , Nefropatias/patologia , Masculino , Camundongos , Nicotinamida Fosforribosiltransferase/metabolismo , Fatores Sexuais , Sirtuína 3/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Visfatin appears to be an energy sensor involved in the regulation of female fertility, which creates a hormonal link integrating the control of energy homeostasis and reproduction. This study evaluates the expression levels of visfatin gene and protein in selected areas of the porcine hypothalamus responsible for gonadotropin-releasing hormone synthesis: the mediobasal hypothalamus (MBH) and preoptic area (POA), and visfatin concentrations in the blood plasma. The tissue samples were harvested from gilts on days 2-3, 10-12, 14-16, and 17-19 of the estrous cycle, and on days 10-11, 12-13, 15-16, 27-28 of pregnancy. Visfatin was localized in the cytoplasm and nucleus of cells creating both studied hypothalamic structures. The study demonstrated that visfatin gene and protein expression in MBH and POA depends on hormonal status related to the phase of the estrous cycle or early pregnancy. Blood plasma concentrations of visfatin during the estrous cycle were higher on days 2-3 in relation to other studied phases of the cycle, while during early pregnancy, the highest visfatin contents were observed on days 12-13. This study demonstrated visfatin expression in the porcine hypothalamus and its dependence on the hormonal milieu related to the estrous cycle and early pregnancy.
Assuntos
Estro , Hipotálamo/metabolismo , Nicotinamida Fosforribosiltransferase/sangue , Prenhez/sangue , Animais , Feminino , Nicotinamida Fosforribosiltransferase/genética , Nicotinamida Fosforribosiltransferase/metabolismo , GravidezRESUMO
Dynamic phosphorus MRS (31 P-MRS) is a method used for in vivo studies of skeletal muscle energetics including measurements of phosphocreatine (PCr) resynthesis rate during recovery of submaximal exercise. However, the molecular events associated with the PCr resynthesis rate are still under debate. We assessed vastus lateralis PCr resynthesis rate from 31 P-MRS spectra collected from healthy adults as part of the CALERIE II study (caloric restriction), and assessed associations between PCr resynthesis and muscle mitochondrial signature transcripts and proteins (NAMPT, NQO1, PGC-1α, and SIRT1). Regression analysis indicated that higher concentration of nicotinamide phosphoribosyltransferase (NAMPT) protein, a mitochondrial capacity marker, was associated with faster PCr resynthesis. However, PCr resynthesis was not associated with greater physical fitness (VO2 peak) or messenger ribonucleic acid levels of mitochondrial function markers such as NQO1, PGC-1α, and SIRT1, suggesting that the impact of these molecular signatures on PCr resynthesis may be minimal in the context of an acute exercise bout. Together, these findings suggest that 31 P-MRS based PCr resynthesis may represent a valid non-invasive surrogate marker of mitochondrial NAMPT in human skeletal muscle.
Assuntos
Espectroscopia de Ressonância Magnética , Músculo Esquelético/diagnóstico por imagem , Músculo Esquelético/metabolismo , Fosfocreatina/metabolismo , Fósforo/metabolismo , Adulto , Citocinas/metabolismo , Feminino , Humanos , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Nicotinamida Fosforribosiltransferase/metabolismo , Oxigênio/metabolismo , Fatores de TempoRESUMO
There is evidence in rodents to suggest that theacrine-based supplements modulate tissue sirtuin activity as well as other biological processes associated with aging. Herein, we examined if a theacrine-based supplement (termed NAD3) altered sirtuin activity in vitro while also affecting markers of mitochondrial biogenesis. The murine C2C12 myoblast cell line was used for experimentation. Following 7 days of differentiation, myotubes were treated with 0.45 mg/mL of NAD3 (containing ~2 mM theacrine) for 3 and 24 h (n = 6 treatment wells per time point). Relative to control (CTL)-treated cells, NAD3 treatments increased (p < 0.05) Sirt1 mRNA levels at 3 h, as well as global sirtuin activity at 3 and 24 h. Follow-up experiments comparing 24 h NAD3 or CTL treatments indicated that NAD3 increased nicotinamide phosphoribosyltransferase (NAMPT) and SIRT1 protein levels (p < 0.05). Cellular nicotinamide adenine dinucleotide (NAD+) levels were also elevated nearly two-fold after 24 h of NAD3 versus CTL treatments (p < 0.001). Markers of mitochondrial biogenesis were minimally affected. Although these data are limited to select biomarkers in vitro, these preliminary findings suggest that a theacrine-based supplement can modulate select biomarkers related to NAD+ biogenesis and sirtuin activity. However, these changes did not drive increases in mitochondrial biogenesis. While promising, these data are limited to a rodent cell line and human muscle biopsy studies are needed to validate and elucidate the significance of these findings.
Assuntos
Músculos/metabolismo , NAD/metabolismo , Sirtuínas/metabolismo , Ácido Úrico/análogos & derivados , Ácido Úrico/administração & dosagem , Animais , Biomarcadores/metabolismo , Citocinas/metabolismo , Humanos , Mitocôndrias/metabolismo , Mioblastos/metabolismo , NAD/uso terapêutico , Nicotinamida Fosforribosiltransferase/metabolismo , RNA Mensageiro , Roedores , Sirtuína 1/metabolismoRESUMO
Obesity is a prominent metabolic disease that predisposes individuals to multiple comorbidities, including type 2 diabetes mellitus, cardiovascular diseases, and cancer. Elevated circulating levels of fatty acids contribute to the development of obesity, in part, by targeting the hypothalamus. Palmitate, the most abundant circulating saturated fatty acid, has been demonstrated to dysregulate NAMPT and circadian clock proteins, as well as induce neuroinflammation. These effects ultimately result in hypothalamic dysregulation of feeding behavior and energy homeostasis. NAMPT is the rate-limiting enzyme of the NAD+ salvage pathway and its expression is under the control of the circadian clock. NAD+ produced from NAMPT can modulate the circadian clock, demonstrating bidirectional interactions between circadian and metabolic pathways. Using NPY/AgRP-expressing mHypoE-46 neurons as well as the novel mHypoA-BMAL1-WT/F and mHypoA-BMAL1-KO/F cell lines, we studied whether there were any interactions between NAMPT and the core circadian clock protein BMAL1 in the palmitate-mediated induction of neuroinflammation. We report that palmitate altered Nampt, Bmal1, Per2 and the inflammatory genes Nf-κb, IκBα, Il-6, and Tlr4. Contrary to studies performed with peripheral tissues, the palmitate-mediated induction in Nampt was independent of BMAL1, and basal Nampt levels did not appear to exhibit rhythmic expression. Palmitate-induced downregulation of Bmal1 and Per2 was independent of NAMPT. However, NAMPT and BMAL1 were both involved in the regulation of Nf-κb, IκBα, Il-6, and Tlr4, as NAMPT inhibition resulted in the repression of basal Nf-κb and IκBα and normalized palmitate-mediated increases in Il-6, and Tlr4. On the other hand, BMAL1 deletion repressed basal Nf-κb, but increased basal Il-6. We conclude that NAMPT and BMAL1 do not interact at the transcriptional level in hypothalamic neurons, but are independently involved in the expression of inflammatory genes.
Assuntos
Fatores de Transcrição ARNTL/fisiologia , Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hipotálamo/patologia , Inflamação/patologia , Neurônios/patologia , Nicotinamida Fosforribosiltransferase/metabolismo , Palmitatos/farmacologia , Animais , Citocinas/genética , Feminino , Hipotálamo/efeitos dos fármacos , Hipotálamo/imunologia , Hipotálamo/metabolismo , Inflamação/induzido quimicamente , Inflamação/imunologia , Inflamação/metabolismo , Mediadores da Inflamação , Masculino , Camundongos , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/imunologia , Neurônios/metabolismo , Nicotinamida Fosforribosiltransferase/genéticaRESUMO
Nicotinamide adenine dinucleotide (NAD), a cofactor for hundreds of metabolic reactions in all cell types, plays an essential role in metabolism, DNA repair, and aging. However, how NAD metabolism is impacted by the environment remains unclear. Here, we report an unexpected trans-kingdom cooperation between bacteria and mammalian cells wherein bacteria contribute to host NAD biosynthesis. Bacteria confer resistance to inhibitors of NAMPT, the rate-limiting enzyme in the amidated NAD salvage pathway, in cancer cells and xenograft tumors. Mechanistically, a microbial nicotinamidase (PncA) that converts nicotinamide to nicotinic acid, a precursor in the alternative deamidated NAD salvage pathway, is necessary and sufficient for this protective effect. Using stable isotope tracing and microbiota-depleted mice, we demonstrate that this bacteria-mediated deamidation contributes substantially to the NAD-boosting effect of oral nicotinamide and nicotinamide riboside supplementation in several tissues. Collectively, our findings reveal an important role of bacteria-enabled deamidated pathway in host NAD metabolism.
Assuntos
Amidas/metabolismo , Vias Biossintéticas , Mamíferos/microbiologia , Mycoplasma/fisiologia , NAD/metabolismo , Administração Oral , Animais , Linhagem Celular Tumoral , Citocinas/antagonistas & inibidores , Citocinas/metabolismo , Metabolismo Energético , Feminino , Microbioma Gastrointestinal , Humanos , Masculino , Metaboloma , Camundongos Endogâmicos C57BL , Niacinamida/análogos & derivados , Niacinamida/metabolismo , Nicotinamidase/metabolismo , Mononucleotídeo de Nicotinamida/administração & dosagem , Mononucleotídeo de Nicotinamida/química , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Nicotinamida Fosforribosiltransferase/metabolismo , Compostos de Piridínio/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of motor neurons. Astrocytes from diverse ALS models induce motor neuron death in co-culture. Enhancing NAD+ availability, or increasing the expression of the NAD+-dependent deacylases SIRT3 and SIRT6, abrogates their neurotoxicity in cell culture models. To determine the effect of increasing NAD+ availability in ALS mouse models we used two strategies, ablation of a NAD+-consuming enzyme (CD38) and supplementation with a bioavailable NAD+ precursor (nicotinamide riboside, NR). Deletion of CD38 had no effect in the survival of two hSOD1-linked ALS mouse models. On the other hand, NR-supplementation delayed motor neuron degeneration, decreased markers of neuroinflammation in the spinal cord, appeared to modify muscle metabolism and modestly increased the survival of hSOD1G93A mice. In addition, we found altered expression of enzymes involved in NAD+ synthesis (NAMPT and NMNAT2) and decreased SIRT6 expression in the spinal cord of ALS patients, suggesting deficits of this neuroprotective pathway in the human pathology. Our data denotes the therapeutic potential of increasing NAD+ levels in ALS. Moreover, the results indicate that the approach used to enhance NAD+ levels critically defines the biological outcome in ALS models, suggesting that boosting NAD+ levels with the use of bioavailable precursors would be the preferred therapeutic strategy for ALS.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Vias Biossintéticas/fisiologia , Neurônios Motores/metabolismo , NAD/metabolismo , Medula Espinal/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Neurônios Motores/patologia , Nicotinamida Fosforribosiltransferase/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Sirtuína 3/metabolismo , Sirtuínas/metabolismo , Medula Espinal/patologia , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismoRESUMO
AIM: Neurons in the arcuate nucleus of the hypothalamus are involved in regulation of food intake and energy expenditure, and dysregulation of signalling in these neurons promotes development of obesity. The role of the rate-limiting enzyme in the NAD+ salvage pathway, nicotinamide phosphoribosyltransferase (NAMPT), for regulation energy homeostasis by the hypothalamus has not been extensively studied. METHODS: We determined whether Nampt mRNA or protein levels in the hypothalamus of mice were affected by diet-induced obesity, by fasting and re-feeding, and by leptin and ghrelin treatment. Primary hypothalamic neurons were treated with FK866, a selective inhibitor of NAMPT, or rAAV carrying shRNA directed against Nampt, and levels of reactive oxygen species (ROS) and mitochondrial respiration were assessed. Fasting and ghrelin-induced food intake was measured in mice in metabolic cages after intracerebroventricular (ICV)-mediated FK866 administration. RESULTS: NAMPT levels in the hypothalamus were elevated by administration of ghrelin and leptin. In diet-induced obese mice, both protein and mRNA levels of NAMPT decreased in the hypothalamus. NAMPT inhibition in primary hypothalamic neurons significantly reduced levels of NAD+ , increased levels of ROS, and affected the expression of Agrp, Pomc and genes related to mitochondrial function. Finally, ICV-induced NAMPT inhibition by FK866 did not cause malaise or anhedonia, but completely ablated fasting- and ghrelin-induced increases in food intake. CONCLUSION: Our findings indicate that regulation of NAMPT levels in hypothalamic neurons is important for the control of fasting- and ghrelin-induced food intake.
Assuntos
Jejum/metabolismo , Grelina/metabolismo , Hipotálamo/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Acrilamidas/administração & dosagem , Proteína Relacionada com Agouti/genética , Proteína Relacionada com Agouti/metabolismo , Animais , Linhagem Celular , Ingestão de Alimentos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Piperidinas/administração & dosagem , Pró-Opiomelanocortina/genética , Pró-Opiomelanocortina/metabolismoRESUMO
Visfatin is an adipocytokine with important roles in endothelial angiogenesis. Hyperbaric oxygen (HBO) has been widely used to treat various medical illness with enhanced angiogenesis. The molecular effects of HBO on visfatin under hypoxia are poorly understood. This study aimed to investigate the effect of HBO on visfatin in hypoxic human coronary arterial endothelial cells (HCAECs). HCAECs under chemical hypoxia (antimycin A, 0.01 mmol/L) were exposed to HBO (2.5 atmosphere absolute; ATA) for 2-4 hours. Western blot, real-time polymerase chain reaction, electrophoretic mobility shift assay, luciferase promoter activity, migration and tube formation assay, and in vitro glucose uptake were measured. Visfatin protein expression increased in hypoxic HCAECs with earlier angiotensin II (AngII) secretion and c-Jun N-terminal kinase (JNK) phosphorylation, which could be effectively suppressed by the JNK inhibitor (SP600125), AngII antibody or AngII receptor blocker (losartan). In hypoxic HCAECs, HBO further induced earlier expression of visfatin and AngII. Hypoxia significantly increased DNA-protein binding activity of hypoxia-inducible factor-1α (HIF-1α) and visfatin. Hypoxia, hypoxia with HBO and exogenous addition of AngII also increased promoter transcription to visfatin; SP600125 and losartan blocked this activity. In HCAECs, glucose uptake, migration and tube formation were increased in the presence of hypoxia with HBO, but were inhibited by visfatin small interfering RNA, SP600125 and losartan. In conclusion, HBO activates visfatin expression and angiogenesis in hypoxic HCAECs, an effect mediated by AngII, mainly through the JNK pathway.
Assuntos
Angiotensina II/metabolismo , Vasos Coronários/metabolismo , Citocinas/metabolismo , Células Endoteliais/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Neovascularização Patológica/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Oxigênio/metabolismo , Antracenos/farmacologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Células Cultivadas , Vasos Coronários/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Glucose/metabolismo , Humanos , Oxigenoterapia Hiperbárica/métodos , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Losartan/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
KEY POINTS: This is the first long-term human clinical trial to report on effects of nicotinamide riboside (NR) on skeletal muscle mitochondrial function, content and morphology. NR supplementation decreases nicotinamide phosphoribosyltransferase (NAMPT) protein abundance in skeletal muscle. NR supplementation does not affect NAD metabolite concentrations in skeletal muscle. Respiration, distribution and quantity of muscle mitochondria are unaffected by NR. NAMPT in skeletal muscle correlates positively with oxidative phosphorylation Complex I, sirtuin 3 and succinate dehydrogenase. ABSTRACT: Preclinical evidence suggests that the nicotinamide adenine dinucleotide (NAD+ ) precursor nicotinamide riboside (NR) boosts NAD+ levels and improves diseases associated with mitochondrial dysfunction. We aimed to determine if dietary NR supplementation in middle-aged, obese, insulin-resistant men affects mitochondrial respiration, content and morphology in skeletal muscle. In a randomized, placebo-controlled clinical trial, 40 participants received 1000 mg NR or placebo twice daily for 12 weeks. Skeletal muscle biopsies were collected before and after the intervention. Mitochondrial respiratory capacity was determined by high-resolution respirometry on single muscle fibres. Protein abundance and mRNA expression were measured by Western blot and quantitative PCR analyses, respectively, and in a subset of the participants (placebo n = 8; NR n = 8) we quantified mitochondrial fractional area and mitochondrial morphology by laser scanning confocal microscopy. Protein levels of nicotinamide phosphoribosyltransferase (NAMPT), an essential NAD+ biosynthetic enzyme in skeletal muscle, decreased by 14% with NR. However, steady-state NAD+ levels as well as gene expression and protein abundance of other NAD+ biosynthetic enzymes remained unchanged. Neither respiratory capacity of skeletal muscle mitochondria nor abundance of mitochondrial associated proteins were affected by NR. Moreover, no changes in mitochondrial fractional area or network morphology were observed. Our data do not support the hypothesis that dietary NR supplementation has significant impact on skeletal muscle mitochondria in obese and insulin-resistant men. Future studies on the effects of NR on human skeletal muscle may include both sexes and potentially provide comparisons between young and older people.