SIRT1-Mediated eNAMPT Secretion from Adipose Tissue Regulates Hypothalamic NAD+ and Function in Mice.
ABSTRACT: Nicotinamide phosphoribosyltransferase (NAMPT), the key NAD(+) biosynthetic enzyme, has two different forms, intra- and extracellular (iNAMPT and eNAMPT), in mammals. However, the significance of eNAMPT secretion remains unclear. Here we demonstrate that deacetylation of iNAMPT by the mammalian NAD(+)-dependent deacetylase SIRT1 predisposes the protein to secretion in adipocytes. NAMPT mutants reveal that SIRT1 deacetylates lysine 53 (K53) and enhances eNAMPT activity and secretion. Adipose tissue-specific Nampt knockout and knockin (ANKO and ANKI) mice show reciprocal changes in circulating eNAMPT, affecting hypothalamic NAD(+)/SIRT1 signaling and physical activity accordingly. The defect in physical activity observed in ANKO mice is ameliorated by nicotinamide mononucleotide (NMN). Furthermore, administration of a NAMPT-neutralizing antibody decreases hypothalamic NAD(+) production, and treating ex vivo hypothalamic explants with purified eNAMPT enhances NAD(+), SIRT1 activity, and neural activation. Thus, our findings indicate a critical role of adipose tissue as a modulator for the regulation of NAD(+) biosynthesis at a systemic level.
Project description:Nicotinamide phosphoribosyltransferase (Nampt) catalyzes the rate-limiting step in the salvage pathway for nicotinamide adenine dinucleotide (NAD(+)) biosynthesis, and thereby regulates the deacetylase activity of sirtuins. Here we show accommodative regulation of myocardial NAD(+) by monocyte-derived extracellular Nampt (eNampt), which is essential for hemodynamic compensation to pressure overload. Although intracellular Nampt (iNampt) expression was decreased in pressure-overloaded hearts, myocardial NAD(+) concentration and Sirt1 activity were preserved. In contrast, iNampt was up-regulated in spleen and monocytes, and circulating eNampt protein and nicotinamide mononucleotide (NMN), a key precursor of NAD(+), were significantly increased. Pharmacological inhibition of Nampt by FK866 or depletion of monocytes/macrophages by clodronate liposomes disrupted the homeostatic mechanism of myocardial NAD(+) levels and NAD(+)-dependent Sirt1 activity, leading to susceptibility to cardiomyocyte apoptosis and cardiac decompensation in pressure-overloaded mice. These biochemical and hemodynamic defects were prevented by systemic administration of NMN. Our studies uncover a crucial role of monocyte-derived eNampt in myocardial adaptation to pressure overload, and highlight a potential intervention controlling myocardial NAD(+) against heart failure.
Project description:Intracellular nicotinamide phosphoribosyltransferase (iNampt) is an essential enzyme in the NAD biosynthetic pathway. An extracellular form of this protein (eNampt) has been reported to act as a cytokine named PBEF or an insulin-mimetic hormone named visfatin, but its physiological relevance remains controversial. Here we show that eNampt does not exert insulin-mimetic effects in vitro or in vivo but rather exhibits robust NAD biosynthetic activity. Haplodeficiency and chemical inhibition of Nampt cause defects in NAD biosynthesis and glucose-stimulated insulin secretion in pancreatic islets in vivo and in vitro. These defects are corrected by administration of nicotinamide mononucleotide (NMN), a product of the Nampt reaction. A high concentration of NMN is present in mouse plasma, and plasma eNampt and NMN levels are reduced in Nampt heterozygous females. Our results demonstrate that Nampt-mediated systemic NAD biosynthesis is critical for beta cell function, suggesting a vital framework for the regulation of glucose homeostasis.
Project description:Nicotinamide phosphoribosyltransferase (NAMPT) is a key enzyme for nicotinamide adenine dinucleotide (NAD) biosynthesis, and can be found either intracellularly (iNAMPT) or extracellularly (eNAMPT). Studies have shown that both iNAMPT and eNAMPT are implicated in aging and age-related diseases/disorders in the peripheral system. However, their functional roles in aged brain remain to be established. Here we showed that upon aging, NAMPT level increased in serum but decreased in brain, decreased in cortex and hippocampus but remained unchanged in cerebellum and striatum in brain, and increased in microglia but likely decreased in neuron. Accordingly, total NAD (tNAD) level significantly decreased in hippocampus, cerebellum and striatum in aged brain. Application of recombinant NAMPT, mimicking the elevated serum NAMPT level, enhanced the susceptibility of cerebral endothelial cells to ischemic injury, while inhibition of iNAMPT by FK866, a specific inhibitor, reduced intracellular NAD level and induced neuronal death. Taken together, we have revealed a region- and cell-specific change of NAMPT level in brain and serum upon aging, deduced its potential consequences, which suggests that NAMPT is a regulatory factor in aging and age-related brain diseases.
Project description:Aging is a significant risk factor for impaired tissue functions and chronic diseases. Age-associated decline in systemic NAD+ availability plays a critical role in regulating the aging process across many species. Here, we show that the circulating levels of extracellular nicotinamide phosphoribosyltransferase (eNAMPT) significantly decline with age in mice and humans. Increasing circulating eNAMPT levels in aged mice by adipose-tissue-specific overexpression of NAMPT increases NAD+ levels in multiple tissues, thereby enhancing their functions and extending healthspan in female mice. Interestingly, eNAMPT is carried in extracellular vesicles (EVs) through systemic circulation in mice and humans. EV-contained eNAMPT is internalized into cells and enhances NAD+ biosynthesis. Supplementing eNAMPT-containing EVs isolated from young mice significantly improves wheel-running activity and extends lifespan in aged mice. Our findings have revealed a novel EV-mediated delivery mechanism for eNAMPT, which promotes systemic NAD+ biosynthesis and counteracts aging, suggesting a potential avenue for anti-aging intervention in humans.
Project description:As recombinant tissue plasminogen activator is the only drug approved for the clinical treatment of acute ischemic stroke, there is an urgent unmet need for novel stroke treatments. Endogenous defense mechanisms against stroke may hold the key to new therapies for stroke. A large number of studies suggest that nicotinamide phosphoribosyl-transferase (NAMPT is an attractive candidate to improve post-stroke recovery. NAMPT is a multifunctional protein and plays important roles in immunity, metabolism, aging, inflammation, and stress responses. NAMPT exists in both the intracellular and extracellular space. As a rate-limiting enzyme, the intracellular form (iNAMPT catalyzes the first step in the biosynthesis of nicotinamide adenine dinucleotide (NAD from nicotinamide. iNAMPT closely regulates energy metabolism, enhancing the proliferation of endothelial cells, inhibiting apoptosis, regulating vascular tone, and stimulating autophagy in disease conditions such as stroke. Extracellular NAMPT (eNAMPT is also known as visfatin (visceral fat-derived adipokine and has pleotropic effects. It is widely believed that the diverse biological functions of eNAMPT are attributed to its NAMPT enzymatic activity. However, the effects of eNAMPT on ischemic injury are still controversial. Some authors have argued that eNAMPT exacerbates ischemic neuronal injury non-enzymatically by triggering the release of TNF-? from glial cells. In addition, NAMPT also participates in several pathophysiological processes such as hypertension, atherosclerosis, and ischemic heart disease. Thus, it remains unclear under what conditions NAMPT is beneficial or destructive. Recent work using in vitro and in vivo genetic/ pharmacologic manipulations, including our own studies, has greatly improved our understanding of NAMPT. This review focuses on the multifaceted and complex roles of NAMPT under both normal and ischemic conditions.
Project description:BACKGROUND:There remains a serious need to prevent the progression of invasive prostate cancer (PCa). We previously showed that secreted extracellular nicotinamide phosphoribosyltransferase (eNAMPT) is a multifunctional innate immunity regulator via TLR4 ligation which has been implicated in PCa progression. Here we investigate the role of eNAMPT as a diagnostic biomarker and therapeutic target in the progression of PCa. METHODS:Tumor NAMPT expression and plasma eNAMPT level were evaluated in human subjects with various PCa tumor stages and high risk subjects followed-up clinically for PCa. The genetic regulation of NAMPT expression in PCa cells and the role of eNAMPT in PCa invasion were investigated utilizing in vitro and in vivo models. FINDINGS:Marked NAMPT expression was detected in human extraprostatic-invasive PCa tissues compared to minimal expression of organ-confined PCa. Plasma eNAMPT levels were significantly elevated in PCa subjects compared to male controls, and significantly greater in subjects with extraprostatic-invasive PCa compared to subjects with organ-confined PCa. Plasma eNAMPT levels showed significant predictive value for diagnosing PCa. NAMPT expression and eNAMPT secretion were highly upregulated in human PCa cells in response to hypoxia-inducible factors and EGF. In vitro cell culture and in vivo preclinical mouse model studies confirmed eNAMPT-mediated enhancement of PCa invasiveness into muscle tissues and dramatic attenuation of PCa invasion by weekly treatment with an eNAMPT-neutralizing polyclonal antibody. INTERPRETATION:This study suggests that eNAMPT is a potential biomarker for PCa, especially invasive PCa. Neutralization of eNAMPT may be an effective therapeutic approach to prevent PCa invasion and progression.
Project description:Metastatic melanoma carrying BRAF mutations represent a still unmet medical need as success of BRAF inhibitors is limited by development of resistance. Nicotinamide phosphoribosyltransferase (NAMPT) is a key enzyme in NAD biosynthesis. An extracellular form (eNAMPT) possesses cytokine-like functions and is up-regulated in inflammatory disorders, including cancer. Here we show that eNAMPT is actively released in culture supernatants of melanoma cell lines. Furthermore, cells that become resistant to BRAF inhibitors (BiR) show a significant increase of eNAMPT levels. Plasma from mice xenografted with BiR cell lines contain higher eNAMPT levels compared to tumor-free animals. Consistently, eNAMPT levels are elevated in 113 patients with BRAF-mutated metastatic melanoma compared to 50 with localized disease or to 38 healthy donors, showing a direct correlation with markers of tumor burden, such as LDH, or aggressive disease (such as PD-L1). eNAMPT concentrations decrease in response to therapy with BRAF/MEK inhibitors, but increase again at progression, as inferred from the serial analysis of 50 patients. Lastly, high eNAMPT levels correlate with a significantly shorter overall survival. Our findings suggest that eNAMPT is a novel marker of tumor burden and response to therapy in patients with metastatic melanoma carrying BRAF mutations.
Project description:Intracellular nicotinamide phosphoribosyltransferase (iNAMPT) in neuron has been known as a protective factor against cerebral ischemia through its enzymatic activity, but the role of central extracellular NAMPT (eNAMPT) is not clear. Here we show that eNAMPT protein level was elevated in the ischemic rat brain after middle-cerebral-artery occlusion (MCAO) and reperfusion, which can be traced to at least in part from blood circulation. Administration of recombinant NAMPT protein exacerbated MCAO-induced neuronal injury in rat brain, while exacerbated oxygen-glucose-deprivation (OGD) induced neuronal injury only in neuron-glial mixed culture, but not in neuron culture. In the mixed culture, NAMPT protein promoted TNF-? release in a time- and concentration-dependent fashion, while TNF-? neutralizing antibody protected OGD-induced, NAMPT-enhanced neuronal injury. Importantly, H247A mutant of NAMPT with essentially no enzymatic activity exerted similar effects on ischemic neuronal injury and TNF-? release as the wild type protein. Thus, eNAMPT is an injurious and inflammatory factor in cerebral ischemia and aggravates ischemic neuronal injury by triggering TNF-? release from glia cells, via a mechanism not related to NAMPT enzymatic activity.
Project description:Periodontal disease is one of the most prevalent chronic disorders worldwide. It is accompanied by inflammation of the gingiva and destruction of periodontal tissues, leading to alveolar bone loss. Here, we focused on the role of adipokines, which are locally expressed by periodontal tissues, in the regulation of catabolic gene expression leading to periodontal inflammation. The expression of the nicotinamide phosphoribosyltransferase (NAMPT) adipokine was dramatically increased in inflamed human and mouse gingival tissues. NAMPT expression was also increased in lipopolysaccharide- and proinflammatory cytokine-stimulated primary cultured human gingival fibroblasts (GF). Adenovirus-mediated NAMPT (Ad-Nampt) overexpression upregulated the expression and activity of COX-2, MMP1 and MMP3 in human GF. The upregulation of IL-1?- or Ad-Nampt-induced catabolic factors was significantly abrogated by the intracellular NAMPT (iNAMPT) inhibitor, FK866 or by the sirtuin (SIRT) inhibitor, nicotinamide (NIC). Recombinant NAMPT protein or extracellular NAMPT (eNAMPT) inhibition using a blocking antibody did not alter NAMPT target gene expression levels. Moreover, intragingival Ad-Nampt injection mediated periodontitis-like phenotypes including alveolar bone loss in mice. SIRT2, a part of the SIRT family, was positively associated with NAMPT actions in human GF. Furthermore, in vivo inhibition of the NAMPT-NAD+-SIRT axis by NIC injection in mice ameliorated the periodontal inflammation and alveolar bone erosion caused by intragingival injection of Ad-Nampt. Our findings indicate that NAMPT is highly upregulated in human GF, while its enzymatic activity acts as a crucial mediator of periodontal inflammation and alveolar bone destruction via regulation of COX-2, MMP1, and MMP3 levels.
Project description:Intracellular nicotinamide phosphoribosyltransferase (iNAMPT) is the rate-limiting enzyme of the mammalian NAD+ biosynthesis salvage pathway. Using inducible and conditional knockout (cKO) mice, we show that Nampt gene deletion in adult projection neurons leads to a progressive loss of body weight, hypothermia, motor neuron (MN) degeneration, motor function deficits, paralysis, and death. Nampt deletion causes mitochondrial dysfunction, muscle fiber type conversion, and atrophy, as well as defective synaptic function at neuromuscular junctions (NMJs). When treated with nicotinamide mononucleotide (NMN), Nampt cKO mice exhibit reduced motor function deficits and prolonged lifespan. iNAMPT protein levels are significantly reduced in the spinal cord of amyotrophic lateral sclerosis (ALS) patients, indicating the involvement of NAMPT in ALS pathology. Our findings reveal that neuronal NAMPT plays an essential role in mitochondrial bioenergetics, motor function, and survival. Our study suggests that the NAMPT-mediated NAD+ biosynthesis pathway is a potential therapeutic target for degenerative MN diseases.