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 the 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 β cell function, suggesting a vital framework for the regulation of glucose homeostasis.
NAD+ acts not only as a co-factor for cellular respiration, but also as a substrate for NAD+-dependent enzymes, such as Sirt1. The cellular NAD+ synthesis is regulated by both the de novo and the salvage pathways. Nicotinamide phosphoribosyltransferase (Nampt) is a rate-limiting enzyme in the salvage pathway.
Here we investigated the role of Nampt in mediating NAD+ synthesis in cardiac myocytes and the function of Nampt in the heart in vivo.
Methods and Results
Expression of Nampt in the heart was significantly decreased by ischemia, ischemia/reperfusion and pressure overload. Upregulation of Nampt significantly increased NAD+ and ATP concentrations, while downregulation of Nampt significantly decreased them. Downregulation of Nampt increased caspase 3 cleavage, cytochrome c release, and TUNEL positive cells, which were inhibited in the presence of Bcl-xL, but did not increase hairpin 2 positive cells, suggesting that endogenous Nampt negatively regulates apoptosis but not necrosis. Downregulation of Nampt also impaired autophagic flux, suggesting that endogenous Nampt positively regulates autophagy. Cardiac specific overexpression of Nampt in transgenic mice increased NAD+ content in the heart, prevented downregulation of Nampt and reduced the size of myocardial infarction and apoptosis in response to prolonged ischemia and ischemia/reperfusion.
Nampt critically regulates NAD+ and ATP contents, thereby playing an essential role in mediating cell survival by inhibiting apoptosis and stimulating autophagic flux in cardiac myocytes. Preventing downregulation of Nampt inhibits myocardial injury in response to myocardial ischemia and reperfusion. These results suggest that Nampt is an essential gatekeeper of energy status and survival in cardiac myocytes.
NAD+; Apoptosis; Myocardial ischemia
Type 2 diabetes (T2D) has become an epidemic in our modern lifestyle, likely due to calorie-rich diets overwhelming our adaptive metabolic pathways. One such pathway is mediated by nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in mammalian NAD+ biosynthesis, and the NAD+-dependent protein deacetylase SIRT1. Here we show that NAMPT-mediated NAD+ biosynthesis is severely compromised in metabolic organs by high-fat diet (HFD). Strikingly, nicotinamide mononucleotide (NMN), a product of the NAMPT reaction and a key NAD+ intermediate, ameliorates glucose intolerance by restoring NAD+ levels in HFD-induced T2D mice. NMN also enhances hepatic insulin sensitivity and restores gene expression related to oxidative stress, inflammatory response, and circadian rhythm, partly through SIRT1 activation. Furthermore, NAD+ and NAMPT levels show significant decreases in multiple organs during aging, and NMN improves glucose intolerance and lipid profiles in age-induced T2D mice. These findings provide critical insights into a potential nutriceutical intervention against diet- and age-induced T2D.
Tumor cells have increased metabolic requirements to maintain rapid growth. In particular, a highly lipogenic phenotype is a hallmark of many tumor types, including prostate. Cancer cells also have increased turnover of nicotinamide adenine dinucleotide (NAD+), a coenzyme involved in multiple metabolic pathways. However, a specific role for NAD+ in tumor cell lipogenesis has yet to be described. Our studies demonstrate a novel role for the NAD+-biosynthetic enzyme Nicotinamide phosphoribosyltransferase (Nampt) in maintaining de novo lipogenesis in prostate cancer (PCa) cells. Inhibition of Nampt reduces fatty acid and phospholipid synthesis. In particular, short chain saturated fatty acids and the phosphatidylcholine (PC) lipids into which these fatty acids are incorporated were specifically reduced by Nampt inhibition. Nampt blockade resulted in reduced ATP levels and concomitant activation of AMP-activated protein kinase (AMPK) and phosphorylation of acetyl-CoA carboxylase (ACC). In spite of this, pharmacological inhibition of AMPK was not sufficient to fully restore fatty acid synthesis. Rather, Nampt blockade also induced protein hyperacetylation in PC-3, DU145, and LNCaP cells, which correlated with the observed decreases in lipid synthesis. Moreover, the sirtuin inhibitor Sirtinol, and the simultaneous knockdown of SIRT1 and SIRT3, phenocopied the effects of Nampt inhibition on fatty acid synthesis. Altogether, these data reveal a novel role for Nampt in the regulation of de novo lipogenesis through the modulation of sirtuin activity in PCa cells.
We examined in HepG2 cells whether glucose-induced changes in AMP-activated protein kinase (AMPK) activity could be mediated by SIRT1, an NAD-dependent histone/protein deacetylase that has been linked to the increase in longevity caused by caloric restriction. Incubation with 25 vs. 5 mM glucose for 6 h concurrently diminished the phosphorylation of AMPK (Thr 172) and ACC (Ser 79), increased lactate release, and decreased the abundance and activity of SIRT1. In contrast, incubation with pyruvate (0.1 and 1 mM) for 2 h increased AMPK phosphorylation and SIRT1 abundance and activity. The putative SIRT1 activators resveratrol and quercetin also increased AMPK phosphorylation. None of the tested compounds (low or high glucose, pyruvate, and resveratrol) significantly altered the AMP/ATP ratio. Collectively, these findings raise the possibility that glucose-induced changes in AMPK are linked to alterations in SIRT1 abundance and activity and possibly cellular redox state.
AMPK; redox state; SIRT1; resveratrol
The ability to adapt and respond to nutrients is an ancient cellular function, conserved from unicellular to the most complex multicellular organisms, including mammals. Mammals adapt to changes in nutritional status through the modulation of tissue-specific metabolic pathways so as to maintain energy homeostasis. At least two proteins are activated in response to reduced nutrient availability: AMP-activated protein kinase (AMPK) and NAD+-dependent deacetylase SIRT1. AMPK functions as a sensor of cellular energy status and as a master regulator of metabolism. When ATP levels decrease, AMPK is activated to boost ATP production and to inhibit ATP usage, thus restoring energy balance. Similarly, SIRT1 is activated in response to changes in the energy status to promote transcription of genes that mediate the metabolic response to stress, starvation, or calorie restriction. Several observations support a model where, in response to stress and reduced nutrients, a metabolic pathway is activated within which AMPK and SIRT1 concordantly function to ensure an appropriate cellular response and adaptation to environmental modifications. In this perspective, we compare and contrast the roles of SIRT1 and AMPK in several metabolic tissues and propose a working model of how the AMPK-SIRT1 axis may be regulated to control functions relevant to organismal physiology and pathophysiology.
SIRT1; AMPK; Nampt; PGC1-α; Calorie Restriction; Starvation; Gluconeogenesis; Insulin
Nicotinamide phosphoribosyltransferase (Nampt) catalyzes the rate-limiting step of nicotinamide adenine dinucleotide (NAD+) synthesis and is required for cell growth, survival, DNA replication and repair, and angiogenesis. Nampt expression increases gene expression which promotes cell survival and increases SirT1 activity, promoting angiogenesis, and it is increased in several human malignancies. Recently, others have shown that ovarian serous adenocarcinomas (OSAs) express high levels of activated Stat3. Since Nampt expression is increased by Stat3, we hypothesized that Nampt protein might be highly expressed in OSAs. Using tissue microarray (TMA) and the avidin-biotin complex immunohistochemical technique we examined Nampt expression in 47 samples of benign ovarian tissue and 49 samples of ovarian serous adenoacarcinomas. Our data show that Nampt protein expression is significantly increased in OSAs as compared to benign ovarian tissue (0.49+/-0.12 benign vs. 4.78+/-0.46 malignant; +/-standard error of the mean). This is the first report demonstrating Nampt overexpression in OSA, which may shed light on the pathogenesis of OSA. Further studies of the role of Nampt overexpresion in OSA may shed light on the prognosis and clinical course of OSA. Last, since an effective pharmacologic Nampt inhibitor is currently in clinical use, further studies of Nampt overexpression in OSA may be used in selecting patients for Nampt inhibitor therapy.
Nicotinamide phosphoribosyltransferase; serous adenocarcinoma; ovarian cancer; nicotinamide adenine dinucleotide; Stat3; Interleukin-6
Nicotinamide phosphoribosyltransferase (NAMPT), also known as visfatin, is the rate-limiting enzyme in the salvage pathway of NAD biosynthesis from nicotinamide. Since its expression is upregulated during inflammation, NAMPT represents a novel clinical biomarker in acute lung injury, rheumatoid arthritis, and Crohn's disease. However, its role in disease progression remains unknown. We report here that NAMPT is a key player in inflammatory arthritis. Increased expression of NAMPT was confirmed in mice with collagen-induced arthritis, both in serum and in the arthritic paw. Importantly, a specific competitive inhibitor of NAMPT effectively reduced arthritis severity with comparable activity to etanercept, and decreased pro-inflammatory cytokine secretion in affected joints. Moreover, NAMPT inhibition reduced intracellular NAD concentration in inflammatory cells and circulating TNFα levels during endotoxemia in mice. In vitro pharmacological inhibition of NAMPT reduced the intracellular concentration of NAD and pro-inflammatory cytokine secretion by inflammatory cells. Thus, NAMPT links NAD metabolism to inflammatory cytokine secretion by leukocytes, and its inhibition might therefore have therapeutic efficacy in immune-mediated inflammatory disorders.
Nicotinamide phosphoribosyltransferase (Nampt) converts nicotinamide to nicotinamide mononucleotide (NMN), a key NAD intermediate. Previously identified as a cytokine PBEF and controversially claimed as an insulin-mimetic hormone visfatin, Nampt has recently drawn much attention in different fields, including NAD biology, metabolism, and inflammation. As an NAD biosynthetic enzyme, Nampt regulates the activity of NAD-consuming enzymes such as sirtuins and influences a variety of metabolic and stress responses. Nampt also plays an important role in the regulation of insulin secretion in pancreatic β cells. Nampt appears to have another function as an immunomodulatory cytokine and plays a role in inflammation. This review summarizes these various functional aspects of Nampt and discusses its potential roles in diseases, including type 2 diabetes and cancer.
AMP-activated protein kinase (AMPK) is widely recognized as an important regulator of glucose transport in skeletal muscle. The p38 mitogen-activated protein kinase (MAPK) has been proposed to be a component of AMPK-mediated signaling. Here we used several different models of altered AMPK activity to determine whether p38 MAPK is a downstream intermediate of AMPK-mediated signaling in skeletal muscle. First, L6 myoblasts and myotubes were treated with AICAR, an AMPK stimulator. AMPK phosphorylation was significantly increased, but there was no change in p38 MAPK phosphorylation. Similarly, AICAR incubation of isolated rat extensor digitorum longus (EDL) muscles did not increase p38 phosphorylation. Next, we used transgenic mice expressing an inactive form of the AMPKα2 catalytic subunit in skeletal muscle (AMPKα2i TG mice). AMPKα2i TG mice did not exhibit any defect in basal or contraction-induced p38 MAPK phosphorylation. We also used transgenic mice expressing an activating mutation in the AMPKγ1 subunit (γ1R70Q TG mice). Despite activated AMPK, basal p38 MAPK phosphorylation was not different between wild type and γ1R70Q TG mice. In addition, muscle contraction-induced p38 MAPK phosphorylation was significantly blunted in the γ1R70Q TG mice. In conclusion, increasing AMPK activity by AICAR and AMPKγ1 mutation does not increase p38 MAPK phosphorylation in skeletal muscle. Furthermore, AMPKα2i TG mice lacking contraction-stimulated AMPK activity have normal p38 MAPK phosphorylation. These results suggest that p38 MAPK is not a downstream component of AMPK-mediated signaling in skeletal muscle.
Antigen-dependent stimulation of T cells plays a critical role in adaptive immunity and host defense. Activation of major histocompatibility complex II (MHC II) molecules, dictated by Class II transactivator (CIITA), is considered a pivotal step in this process. The mechanism underlying differential regulation of CIITA activity by the post-translational modification machinery (PTM) and its implications are not clearly appreciated. Here, we report that SIRT1, a type III deacetylase, interacts with and deacetylates CIITA. SIRT1 activation augments MHC II transcription by shielding CIITA from proteasomal degradation and promoting nuclear accumulation and target binding of CIITA. In contrast, depletion of SIRT1 upregulates CIITA acetylation and attenuates its activity. Nicotinamide phosphoribosyltransferase (NAMPT) that synthesizes NAD+ required for SIRT1 activation exerts similar effects on CIITA activity. Two different types of stress stimuli, hypobaric hypoxia and oxidized low-density lipoprotein (oxLDL), induce the acetylation of CIITA and suppress its activity by inhibiting the SIRT1 expression and activity. Thus, our data link SIRT1-mediated deacetylation of CIITA to MHC II transactivation in macrophages and highlight a novel strategy stress cues may employ to manipulate host adaptive immune system.
Both genetic and environmental factors contribute to the pathogenesis of type 2 diabetes, and it is critical to understand the interplay between these factors in the regulation of insulin secretion and insulin sensitivity to develop effective therapeutic interventions for type 2 diabetes. For the past several years, studies on the mammalian NAD-dependent protein deacetylase SIRT1 and systemic NAD biosynthesis mediated by nicotinamide phosphoribosyltransferase (NAMPT) have demonstrated that these two regulatory components together play a critical role in the regulation of glucose homeostasis, particularly in the regulation of glucose-stimulated insulin secretion in pancreatic beta cells. These components also contribute to the age-associated decline in beta cell function, which has been suggested to be one of the major contributing factors to the pathogenesis of type 2 diabetes. In this review article, the roles of SIRT1 and NAMPT-mediated systemic NAD biosynthesis in glucose homeostasis and the pathophysiology of type 2 diabetes will be summarized, and their potential as effective targets for the treatment and prevention of type 2 diabetes will be discussed.
SIRT1; NAMPT; NAD Biosynthesis; Insulin Secretion; Insulin Sensitivity; Type 2 Diabetes; Pancreatic β Cells; Liver; Skeletal Muscle; Adipose Tissue; PBEF; visfatin; Review
The circadian clock is encoded by a transcription-translation feedback loop that synchronizes behavior and metabolism with the light-dark cycle. Here we report that both the rate-limiting enzyme in mammalian NAD+ biosynthesis, nicotinamide phosphoribosyltransferase (NAMPT), and levels of NAD+, display circadian oscillations which are regulated by the core clock machinery in mice. Inhibition of NAMPT promotes oscillation of the clock gene Per2 by releasing CLOCK:BMAL1 from suppression by SIRT1. In turn, the circadian transcription factor CLOCK binds to and up-regulates Nampt, thus completing a feedback loop involving NAMPT/NAD+ and SIRT1/CLOCK:BMAL1.
Nicotinamide phosphoribosyltransferase (Nampt) is a rate-limiting enzyme in the mammalian NAD+ biosynthesis of a salvage pathway and exists in 2 known forms, intracellular Nampt (iNampt) and a secreted form, extracellular Nampt (eNampt). eNampt can generate an intermediate product, nicotinamide mononucleotide (NMN), which has been reported to support insulin secretion in pancreatic islets. Nampt has been reported to be expressed in the pancreas but islet specific expression has not been adequately defined. The aim of this study was to characterize Nampt expression, secretion and regulation by glucose in human islets. Gene and protein expression of Nampt was assessed in human pancreatic tissue and isolated islets by qRT-PCR and immunofluorescence/confocal imaging respectively. Variable amounts of Nampt mRNA were detected in pancreatic tissue and isolated islets. Immunofluorescence staining for Nampt was found in the exocrine and endocrine tissue of fetal pancreas. However, in adulthood, Nampt expression was localized predominantly in beta cells. Isolated human islets secreted increasing amounts of eNampt in response to high glucose (20 mM) in a static glucose-stimulated insulin secretion assay (GSIS). In addition to an increase in eNampt secretion, exposure to 20 mM glucose also increased Nampt mRNA levels but not protein content. The secretion of eNampt was attenuated by the addition of membrane depolarization inhibitors, diazoxide and nifedipine. Islet-secreted eNampt showed enzymatic activity in a reaction with increasing production of NAD+/NADH over time. In summary, we show that Nampt is expressed in both exocrine and endocrine tissue early in life but in adulthood expression is localized to endocrine tissue. Enzymatically active eNampt is secreted by human islets, is regulated by glucose and requires membrane depolarization.
Silent information regulators are potent NAD+-dependent protein deacetylases, which have been shown to regulate gene silencing, muscle differentiation and DNA damage repair. Here, changes in the level and activity of sirtuin 1 (SIRT1) in response to exercise in groups of young and old rats were studied. There was an age-related increase in SIRT1 level, while exercise training significantly increased the relative activity of SIRT1. A strong inverse correlation was found between the nuclear activity of SIRT1 and the level of acetylated proteins. Exercise training induced SIRT1 activity due to the positive effect of exercise on the activity of nicotinamide phosphoribosyltransferase (NAMPT) and thereby the production of sirtuin-fueling NAD+. Exercise training normalized the age-associated shift in redox balance, since exercised animals had significantly lower levels of carbonylated proteins, expression of hypoxia-inducible factor-1alpha and vascular endothelial growth factor. The age-associated increase in the level of SIRT6 was attenuated by exercise training. On the other hand, aging did not significantly increase the level of DNA damage, which was in line with the activity of 8-oxoguanine DNA glycosylase, while exercise training increased the level of this enzyme. Regular exercise decelerates the deleterious effects of the aging process via SIRT1-dependent pathways through the stimulation of NAD+ biosynthesis by NAMPT.
Sirtuins; Exercise; DNA repair; Skeletal muscle; Aging
Nicotinamide phosphoribosyltransferase (Nampt) inhibitors such as FK866 are potent inhibitors of NAD+ synthesis that show promise for the treatment of different forms of cancer. Based on Nampt upregulation in activated T lymphocytes and on preliminary reports of lymphopenia in FK866 treated patients, we have investigated FK866 for its capacity to interfere with T lymphocyte function and survival. Intracellular pyridine nucleotides, ATP, mitochondrial function, viability, proliferation, activation markers and cytokine secretion were assessed in resting and in activated human T lymphocytes. In addition, we used experimental autoimmune encephalomyelitis (EAE) as a model of T-cell mediated autoimmune disease to assess FK866 efficacy in vivo. We show that activated, but not resting, T lymphocytes undergo massive NAD+ depletion upon FK866-mediated Nampt inhibition. As a consequence, impaired proliferation, reduced IFN-γ and TNF-α production, and finally autophagic cell demise result. We demonstrate that upregulation of the NAD+-degrading enzyme poly-(ADP-ribose)-polymerase (PARP) by activated T cells enhances their susceptibility to NAD+ depletion. In addition, we relate defective IFN-γ and TNF-α production in response to FK866 to impaired Sirt6 activity. Finally, we show that FK866 strikingly reduces the neurological damage and the clinical manifestations of EAE. In conclusion, Nampt inhibitors (and possibly Sirt6 inhibitors) could be used to modulate T cell-mediated immune responses and thereby be beneficial in immune-mediated disorders.
Resveratrol induces mitochondrial biogenesis and protects against metabolic decline but whether SIRT1 mediates these benefits is the subject of debate. To circumvent the developmental defects of germ-line SIRT1 knockouts, we have developed the first inducible system that permits whole-body deletion of SIRT1 in adult mice. Mice treated with a moderate dose of resveratrol showed increased mitochondrial biogenesis and function, AMPK activation and increased NAD+ levels in skeletal muscle, whereas SIRT1 knockouts displayed none of these benefits. A mouse overexpressing SIRT1 mimicked these effects. A high dose of resveratrol activated AMPK in a SIRT1-independent manner, demonstrating that resveratrol dosage is a critical factor. Importantly, at both doses of resveratrol no improvements in mitochondrial function were observed in animals lacking SIRT1. Together these data indicate that SIRT1 plays an essential role in the ability of moderate doses of resveratrol to stimulate AMPK and improve mitochondrial function both in vitro and in vivo.
SIRT1, the mammalian homolog of SIR2 in Saccharomyces cerevisiae, is an NAD-dependent deacetylase implicated in regulation of lifespan. By designing effective short hairpin RNAs and a silent shRNA-resistant mutant SIRT1 in a genetically defined system, we show that efficient inhibition of SIRT1 in telomerase-immortalized human cells enhanced cell growth under normal and nutrient limiting conditions. Hematopoietic stem cells obtained from SIRT1-deficient mice also showed increased growth capacity and decreased dependency on growth factors. Consistent with this, SIRT1 inhibition was associated with increased telomerase activity in human cells. We also observed a significant increase in AMPK levels up on SIRT1 inhibition under glucose limiting conditions. Although SIRT1 suppression cooperated with hTERT to promote cell growth, either overexpression or suppression of SIRT1 alone had no effect on life span of human diploid fibroblasts. Our findings challenge certain models and connect nutrient sensing enzymes to the immortalization process. Furthermore, they show that in certain cell lineages, SIRT1 can act as a growth suppressor gene.
For the past several years, it has been demonstrated that the NAD-dependent protein deacetylase Sirt1 and nicotinamide phosphoribosyltransferase (Nampt)-mediated systemic NAD biosynthesis together play a critical role in the regulation of metabolism and possibly aging in mammals. Based on our recent studies on these two critical components, we have developed a hypothesis of a novel systemic regulatory network, named “NAD World”, for mammalian aging. Conceptually, in the NAD World, systemic NAD biosynthesis mediated by intra- and extracellular Nampt functions as a driver that keeps up the pace of metabolism in multiple tissues/organs, and the NAD-dependent deacetylase Sirt1 serves as a universal mediator that executes metabolic effects in a tissue-dependent manner in response to changes in systemic NAD biosynthesis. This new concept of the NAD World provides important insights into a systemic regulatory mechanism that fundamentally connects metabolism and aging and also conveys the ideas of functional hierarchy and frailty for the regulation of metabolic robustness and aging in mammals.
NAD World; metabolism; aging; Sirt1; Nampt; systemic NAD biosynthesis; pancreatic β cells; neurons; robustness; frailty
A number of cancers show increased expression of Nicotinamide phosphoribosyl transferase (Nampt). However, the mechanism through which Nampt is upregulated is unclear. In our study, we found that the Nampt-specific chemical inhibitor FK866 significantly inhibited cell survival and reduced nicotinamide adenine dinucleotide (NAD) levels in LoVo and SW480 cell lines. Bioinformatics analyses suggested that miR-26b targets Nampt mRNA. We identified Nampt as a new target of miR-26b and demonstrated that miR-26b inhibits Nampt expression at the protein and mRNA levels by binding to the Nampt 3′-UTR. Moreover, we found that miR-26b was down regulated in cancer tissues relative to that in adjacent normal tissues in 18 colorectal cancer patients. A statistically significant inverse correlation between miR-26b and Nampt expression was observed in samples from colorectal cancer patients and in 5 colorectal cell lines (HT-29, SW480, SW1116, LoVo, and HCT116). In addition, over expression of miR-26b strongly inhibited LoVo cell survival and invasion, an effect partially abrogated by the addition of NAD. In conclusion, this study demonstrated that the NAD-salvaging biosynthesis pathway involving Nampt might play a role in colorectal cancer cell survival. MiR-26b may serve as a tumor suppressor by targeting Nampt.
Many countries are facing social and economic problems due to increased elderly demographics. With these demands, it is now critical to understand the fundamental regulatory mechanism for aging and longevity in mammals. Our studies on the mammalian NAD-dependent deacetylase SIRT1 and nicotinamide phosphoribosyltransferase (NAMPT)-mediated systemic NAD biosynthesis led us to propose a comprehensive model for the systemic regulatory network connecting metabolism and aging, termed the “NAD World.” In this article, I will discuss the importance of SIRT1 and NAMPT-mediated NAD biosynthesis in the NAD World and the system dynamics of this hierarchical network for the connection between metabolism and aging.
aging; longevity; SIRT1; NAMPT; NAD biosynthesis; NAD World
Ghrelin is a stomach-derived peptide that increases food intake through the activation of hypothalamic AMP-activated protein kinase (AMPK). However, the molecular mechanisms initiated by the activation of the ghrelin receptor, which in turn lead to AMPK activation, remain unclear. Sirtuin 1 (SIRT1) is a deacetylase activated in response to calorie restriction that acts through the tumor suppressor gene p53. We tested the hypothesis that the central SIRT1/p53 pathway might be mediating the orexigenic action of ghrelin.
RESEARCH DESIGN AND METHODS
SIRT1 inhibitors, such as Ex527 and sirtinol, and AMPK activators, such as AICAR, were administered alongside ghrelin in the brain of rats and mice (wild-type versus p53 knockout [KO]). Their hypothalamic effects on lipid metabolism and changes in transcription factors and neuropeptides were assessed by Western blot and in situ hybridization.
The central pretreatment with Ex527, a potent SIRT1 inhibitor, blunted the ghrelin-induced food intake in rats. Mice lacking p53, a target of SIRT1 action, failed to respond to ghrelin in feeding behavior. Ghrelin failed to phosphorylate hypothalamic AMPK when rats were pretreated with Ex527, as it did in p53 KO mice. It is noteworthy that the hypothalamic SIRT1/p53 pathway seems to be specific for mediating the orexigenic action of ghrelin, because central administration of AICAR, a potent AMPK activator, increased food intake in p53 KO mice. Finally, blockade of the central SIRT1 pathway did not modify ghrelin-induced growth hormone secretion.
Ghrelin specifically triggers a central SIRT1/p53 pathway that is essential for its orexigenic action, but not for the release of growth hormone.
Chronic Inflammation is a key link between obesity and insulin resistance. We previously showed that two nutrient sensors AMP-activated protein kinase (AMPK) and SIRT1 interact to regulate macrophage inflammation. AMPK is also a molecular target of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), which has been shown to reduce insulin resistance in various animal models. Here we aim to determine whether the therapeutic effects of AICAR against insulin resistance involve its anti-inflammatory function, which requires macrophage SIRT1. Long-term administration of low-dose AICAR significantly suppressed adipose inflammation in established diet-induced obese mice. This was associated with improved glucose homeostasis and insulin sensitivity without changes of body weight. In contrast, SIRT1 deletion in myeloid SIRT1 knockout (MSKO) mice increased infiltration of classically activated M1 macrophages and decreased alternatively activated M2 macrophages in adipose tissue. As a result, MSKO mice on high fat (HF) diets exhibited impaired insulin signaling in skeletal muscle, fat, and liver, and developed systemic insulin resistance in glucose tolerance tests, insulin tolerance tests, and hyperinsulinemic-euglycemic clamp experiments. Interestingly, the beneficial effects of AICAR on adipose inflammation and insulin sensitivity were absent in MSKO mice fed HF diets, suggesting that the full capacity of AICAR to antagonize obesity-induced inflammation and insulin resistance requires myeloid SIRT1. In summary, AICAR negatively regulates HF diet-induced inflammation, which requires myeloid SIRT1, thereby contributing to the protection against insulin resistance. Myeloid SIRT1 is a therapeutic target of the anti-inflammatory and insulin-sensitizing effects of AICAR.
Nicotinamide (Nam) phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in mammalian NAD synthesis, catalyzing nicotinamide mononucleotide (NMN) formation from Nam and 5-phosphoribosyl 1-pyrophosphate (PRPP). NAMPT has also been described as an adipocytokine visfatin with a variety of actions, although physiological significance of this protein remains unclear. It has been proposed that possible actions of visfatin are mediated through the extracellular formation of NMN. However, we did not detect NMN in mouse blood plasma, even with a highly specific and sensitive liquid chromatography/tandem mass spectrometry. Furthermore, there is no or little ATP, the activator of NAMPT, in extracellular spaces. We thus questioned whether visfatin catalyzes the in situ formation of NMN under such extracellular milieus. To address this question, we here determined Km values for the substrates Nam and PRPP in the NAMPT reaction without or with ATP using a recombinant human enzyme and found that 1 mM ATP dramatically decreases Km values for the substrates, in particular PRPP to its intracellular concentration. Consistent with the kinetic data, only when ATP is present at millimolar levels, NAMPT efficiently catalyzed the NMN formation at the intracellular concentrations of the substrates. Much lower concentrations of Nam and almost the absence of PRPP and ATP in the blood plasma suggest that NAMPT should not efficiently catalyze its reaction under the extracellular milieu. Indeed, NAMPT did not form NMN in the blood plasma. From these kinetic analyses of the enzyme and quantitative determination of its substrates, activator, and product, we conclude that visfatin does not participate in NMN formation under the extracellular milieus. Together with the absence of NMN in the blood plasma, our conclusion does not support the concept of “NAMPT-mediated systemic NAD biosynthesis.” Our study would advance current understanding of visfatin physiology.
Aging science has recently drawn much attention, and discussions on the possibility of anti-aging medicine have multiplied. One potential target for the development of anti-aging drugs is the SIR2 (silent information regulator 2) family of NAD-dependent deacetylases/ADP-ribosyltransferases, called “sirtuins.” Sirtuins regulate many fundamental biological processes in response to a variety of environmental and nutritional stimuli. In mammals, the mammalian SIR2 ortholog SIRT1 has been most studied, and small molecule SIRT1 activators (STACs), including a plant-derived polyphenolic compound resveratrol, have been developed. On the other hand, sirtuin activity is regulated by NAD biosynthetic pathways, and nicotinamide phosphoribosyltransferase (NAMPT) plays a critical role in the regulation of mammalian sirtuin activity. Recent studies have provided a proof of concept for the idea that nicotinamide mononucleotide (NMN), the NAMPT reaction product, can be used a nutriceutical to activate SIRT1 activity. Based on these recent findings, the possibility of sirtuin-targeted nutriceutical development will be discussed.
NAD-dependent deacetylases; sirtuins; SIRT1; NAD biosynthesis; nicotinamide phosphoribosyltransferase; NAMPT; anti-aging medicine; pharmaceuticals; nutriceuticals; aging; metabolism