A one and a two … expanding roles for poly(ADP-ribose) polymerases in metabolism

In two related papers in this issue, Bai et al. (2011a, 2011b) observe that PARP-1- or PARP-2-deficient mice show increased energy expenditure and protection against diet-induced obesity. This metabolic phenotype is achieved, in part, through SIRT1 activation, either by increasing NAD(+) levels or by promoting SIRT1 expression.     

The NAD(+) precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet-induced obesity

As NAD(+) is a rate-limiting cosubstrate for the sirtuin enzymes, its modulation is emerging as a valuable tool to regulate sirtuin function and, consequently, oxidative metabolism. In line with this premise, decreased activity of PARP-1 or CD38-both NAD(+) consumers-increases NAD(+) bioavailability, resulting in SIRT1 activation and protection against metabolic disease. Here we evaluated whether similar effects could be achieved by increasing the supply of nicotinamide riboside (NR), a recently described natural NAD(+) precursor with the ability to increase NAD(+) levels, Sir2-dependent gene silencing, and replicative life span in yeast. We show that NR supplementation in mammalian cells and mouse tissues increases NAD(+) levels and activates SIRT1 and SIRT3, culminating in enhanced oxidative metabolism and protection against high-fat diet-induced metabolic abnormalities. Consequently, our results indicate that the natural vitamin NR could be used as a nutritional supplement to ameliorate metabolic and age-related disorders characterized by defective mitochondrial function.     

Herpes simplex virus 1 infection activates poly(ADP-ribose) polymerase and triggers the degradation of poly(ADP-ribose) glycohydrolase

Herpes simplex virus 1 infection triggers multiple changes in the metabolism of host cells, including a dramatic decrease in the levels of NAD(+). In addition to its role as a cofactor in reduction-oxidation reactions, NAD(+) is required for certain posttranslational modifications. Members of the poly(ADP-ribose) polymerase (PARP) family of enzymes are major consumers of NAD(+), which they utilize to form poly(ADP-ribose) (PAR) chains on protein substrates in response to DNA damage. PAR chains can subsequently be removed by the enzyme poly(ADP-ribose) glycohydrolase (PARG). We report here that the HSV-1 infection-induced drop in NAD(+) levels required viral DNA replication, was associated with an increase in protein poly(ADP-ribosyl)ation (PARylation), and was blocked by pharmacological inhibition of PARP-1/PARP-2 (PARP-1/2). Neither virus yield nor the cellular metabolic reprogramming observed during HSV-1 infection was altered by the rescue or further depletion of NAD(+) levels. Expression of the viral protein ICP0, which possesses E3 ubiquitin ligase activity, was both necessary and sufficient for the degradation of the 111-kDa PARG isoform. This work demonstrates that HSV-1 infection results in changes to NAD(+) metabolism by PARP-1/2 and PARG, and as PAR chain accumulation can induce caspase-independent apoptosis, we speculate that the decrease in PARG levels enhances the auto-PARylation-mediated inhibition of PARP, thereby avoiding premature death of the infected cell.     

The cAMP/PKA pathway rapidly activates SIRT1 to promote fatty acid oxidation independently of changes in NAD(+)

The NAD(+)-dependent deacetylase SIRT1 is an evolutionarily conserved metabolic sensor of the Sirtuin family that mediates homeostatic responses to certain physiological stresses such as nutrient restriction. Previous reports have implicated fluctuations in intracellular NAD(+) concentrations as the principal regulator of SIRT1 activity. However, here we have identified a cAMP-induced phosphorylation of a highly conserved serine (S434) located in the SIRT1 catalytic domain that rapidly enhanced intrinsic deacetylase activity independently of changes in NAD(+) levels. Attenuation of SIRT1 expression or the use of a nonphosphorylatable SIRT1 mutant prevented cAMP-mediated stimulation of fatty acid oxidation and gene expression linked to this pathway. Overexpression of SIRT1 in mice significantly potentiated the increases in fatty acid oxidation and energy expenditure caused by either pharmacological β-adrenergic agonism or cold exposure. These studies support a mechanism of Sirtuin enzymatic control through the cAMP/PKA pathway with important implications for stress responses and maintenance of energy homeostasis.     

Regulation of SIRT 1 mediated NAD dependent deacetylation: a novel role for the multifunctional enzyme CD38

The SIRT 1 enzyme is a NAD dependent deacetylase implicated in ageing, cell protection, and energy metabolism in mammalian cells. How the endogenous activity of SIRT 1 is modulated is not known. The enzyme CD38 is a multifunctional enzyme capable of synthesis of the second messenger, cADPR, NAADP, and ADPR. However, the major enzymatic activity of CD38 is the hydrolysis of NAD. Of particular interest is the fact that CD38 is present on the inner nuclear membrane. Here, we investigate the modulation of the SIRT 1 activity by CD38. We propose that by modulating availability of NAD to the SIRT1 enzyme, CD38 may regulate SIRT1 enzymatic activity. We observed that in CD38 knockout mice, tissue levels of NAD are significantly increased. We also observed that incubation of purified recombinant SIRT1 enzyme with CD38 or nuclear extracts of wild-type mice led to a significant inhibition of its activity. In contrast, incubation of SIRT1 with cellular extract from CD38 knockout mice was without effect. Furthermore, the endogenous activity of SIRT1 was several time higher in nuclear extracts from CD38 knockout mice when compared to wild-type nuclear extracts. Finally, the in vivo deacetylation of the SIRT1 substrate P53 is increased in CD38 knockout mice tissue. Our data support the novel concept that nuclear CD38 is a major regulator of cellular/nuclear NAD level, and SIRT1 activity. These findings have strong implications for understanding the basic mechanisms that modulate intracellular NAD levels, energy homeostasis, as well as ageing and cellular protection modulated by the SIRT enzymes.     

H2O2 accelerates cellular senescence by accumulation of acetylated p53 via decrease in the function of SIRT1 by NAD+ depletion.

It has been reported that p53 acetylation, which promotes cellular senescence, can be regulated by the NAD(+)-dependent deacetylase SIRT1, the human homolog of yeast Sir2, a protein that modulates lifespan. To clarify the role of SIRT1 in cellular senescence induced by oxidative stress, we treated normal human diploid fibroblast TIG-3 cells with H(2)O(2) and examined DNA cleavage, depletion of intracellular NAD(+), expression of p21, SIRT1, and acetylated p53, cell cycle arrest, and senescence-associated beta-galactosidase (SA-beta-gal) activity. DNA cleavage was observed immediately in TIG-3 cells treated with H(2)O(2), though no cell death was observed. NAD(+) levels in TIG-3 cells treated with H(2)O(2) were also decreased significantly. Pre-incubation with the poly (ADP-ribose) polymerase (PARP) inhibitor resulted in preservation of intracellular NAD(+) levels. The amount of acetylated p53 was increased in TIG-3 cells at 4h after H(2)O(2) treatment, while there was little to no decrease in SIRT1 protein expression. The expression level of p21 was increased at 12h and continued to increase for up to 24h. Additionally, exposure of TIG-3 cells to H(2)O(2) induced cell cycle arrest at 24h and increased SA-beta-gal activity at 48h. This pathway likely plays an important role in the acceleration of cellular senescence by oxidative stress.     

SIRT4 inhibits glutamate dehydrogenase and opposes the effects of calorie restriction in pancreatic beta cells

Sir2 is an NAD-dependent deacetylase that connects metabolism with longevity in yeast, flies, and worms. Mammals have seven Sir2 homologs (SIRT1-7). We show that SIRT4 is a mitochondrial enzyme that uses NAD to ADP-ribosylate and downregulate glutamate dehydrogenase (GDH) activity. GDH is known to promote the metabolism of glutamate and glutamine, generating ATP, which promotes insulin secretion. Loss of SIRT4 in insulinoma cells activates GDH, thereby upregulating amino acid-stimulated insulin secretion. A similar effect is observed in pancreatic beta cells from mice deficient in SIRT4 or on the dietary regimen of calorie restriction (CR). Furthermore, GDH from SIRT4-deficient or CR mice is insensitive to phosphodiesterase, an enzyme that cleaves ADP-ribose, suggesting the absence of ADP-ribosylation. These results indicate that SIRT4 functions in beta cell mitochondria to repress the activity of GDH by ADP-ribosylation, thereby downregulating insulin secretion in response to amino acids, effects that are alleviated during CR.     

Nicotinamide mononucleotide, a key NAD(+) intermediate, treats the pathophysiology of diet- and age-induced diabetes in mice

Type 2 diabetes (T2D) has become 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.     

Myocardial postischemic injury is reduced by polyADPripose polymerase-1 gene disruption

BACKGROUND: PolyADPribose polymerase (PARP) is activated by DNA strand breaks to catalyze the addition of ADPribose groups to nuclear proteins, especially PARP-1. Excessive polyADPribosylation leads to cell death through depletion of NAD+ and ATP. MATERIALS AND METHODS: In vivo PARP activation in heart tissue slices was assayed through conversion of [33P]NAD+ into polyADPribose (PAR) following ischemia-reperfusion (I/R) and also monitored by immunohistochemical staining for PAR. Cardiac contractility, nitric oxide (NO), reactive oxygen species (ROS), NAD+ and ATP levels were examined in wild type (WT) and in PARP-1 gene-deleted (PARP-1(-/-)) isolated, perfused mouse hearts. Myocardial infarct size was assessed following coronary artery occlusion in rats treated with PARP inhibitors. RESULTS: Ischemia-reperfusion (I/R) augmented formation of nitric oxide, oxygen free radicals and PARP activity. I/R induced decreases in cardiac contractility and NAD+ levels were attenuated in PARP-1(-/-) mouse hearts. PARP inhibitors reduced myocardial infarct size in rats. Residual polyADPribosylation in PARP-1(-/-) hearts may reflect alternative forms of PARP. CONCLUSIONS: PolyADPribosylation from PARP-1 and other sources of enzymatic PAR synthesis is associated with cardiac damage following myocardial ischemia. PARP inhibitors may have therapeutic utility in myocardial disease.     

Blockade of PARP activity attenuates poly(ADP-ribosyl)ation but offers only partial neuroprotection against NMDA-induced cell death in the rat retina

Recent reports have linked neuronal cell death by necrosis to poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation. It is believed that under stress, the activity of this enzyme is up-regulated, resulting in extensive poly(ADP-ribosyl)ation of nuclear proteins, using NAD(+) as its substrate, which, in turn, leads to the depletion of NAD(+). In efforts to restore the level of NAD(+), depletion of ATP occurs, resulting in the shutdown of ATP-dependent ionic pumps. This results in cell swelling and eventual loss of membrane selectivity, hallmarks of necrosis. Reports from in vitro and in vivo studies in the brain have shown that NMDA receptor activation stimulates PARP activity and that blockade of the enzyme provides substantial neuroprotection. The present study was undertaken to determine whether PARP activity is regulated by NMDA in the rat retina, and whether blockade of PARP activity provides protection against toxic effects of NMDA. Rat retinas exposed to intravitreal injections containing NMDA, with or without the PARP inhibitor N-(6-oxo-5, 6-dihydrophenanthridin-2-yl)-(N,-dimethylamino) acetamide hydrochloride (PJ-34), were assessed for changes in PARP-1 activity as evidenced by poly(ADP-ribosyl)ation (PAR), loss of membrane integrity, morphological indicators of apoptosis and necrosis, and ganglion cell loss. Results showed that: NMDA increased PAR formation in a concentration-dependent manner and caused a decline in retinal ATP levels; PJ-34 blockade attenuated the NMDA-induced formation of PAR and decline in ATP; NMDA induced the loss of membrane selectivity to ethidium bromide (EtBr) in inner retinal neurons, but loss of membrane selectivity was not prevented by blocking PARP activity; cells stained with EtBr, or reacted for TUNEL-labeling, displayed features characteristic of both apoptosis and necrosis. In the presence of PJ-34, greater numbers of cells exhibited apoptotic features; PJ-34 provided partial neuroprotection against NMDA-induced ganglion cell loss. These findings suggest that although blockade of PARP activity fully attenuates NMDA-induced PAR formation and loss of retinal ATP content, and improves the survival of select populations of ganglion cells, this approach does not provide full neuroprotection. In contrast, blockade of PARP activity promotes apoptotic-like cell death in the majority of cells undergoing cell death. Furthermore, these studies show that the loss of membrane selectivity is not dependent upon PAR formation or the resulting decline of ATP, and suggests that an alternative pathway, other than PARP activation, exists to mediate this event.     

Poly(ADP-Ribose) Polymerase 1–Sirtuin 1 Functional Interplay Regulates LPS-Mediated High Mobility Group Box 1 Secretion

Pathophysiological conditions that lead to the release of the prototypic damage-associated molecular pattern molecule high mobility group box 1 (HMGB1) also result in activation of poly(ADP-ribose) polymerase 1 (PARP1; now known as ADP-ribosyl transferase 1 [ARTD1]). Persistent activation of PARP1 promotes energy failure and cell death. The role of poly(ADP-ribosyl)ation in HMGB1 release has been explored previously; however, PARP1 is a versatile enzyme and performs several other functions including cross-talk with another nicotinamide adenine dinucleotide- (NAD⁺) dependent member of the Class III histone deacetylases (HDACs), sirtuin-1 (SIRT1). Previously, it has been shown that the hyperacetylation of HMGB1 is a seminal event prior to its secretion, a process that also is dependent on HDACs. Therefore, in this study, we seek to determine if PARP1 inhibition alters LPS-mediated HMGB1 hyperacetylation and subsequent secretion due to its effect on SIRT1. We demonstrate in an in vitro model that LPS treatment leads to hyperacetylated HMGB1 with concomitant reduction in nuclear HDAC activity. Treatment with PARP1 inhibitors mitigates the LPS-mediated reduction in nuclear HDAC activity and decreases HMGB1 acetylation. By utilizing an NAD⁺-based mechanism, PARP1 inhibition increases the activity of SIRT1. Consequently, there is an increased nuclear retention and decreased extracellular secretion of HMGB1. We also demonstrate that PARP1 physically interacts with SIRT1. Further confirmation of this data was obtained in a murine model of sepsis, that is, administration of PJ-34, a specific PARP1 inhibitor, led to decreased serum HMGB1 concentrations in mice subjected to cecal ligation and puncture (CLP) as compared with untreated mice. In conclusion, our study provides new insights in understanding the molecular mechanisms of HMGB1 secretion in sepsis.     

Nutrient-sensitive mitochondrial NAD+ levels dictate cell survival

A major cause of cell death caused by genotoxic stress is thought to be due to the depletion of NAD(+) from the nucleus and the cytoplasm. Here we show that NAD(+) levels in mitochondria remain at physiological levels following genotoxic stress and can maintain cell viability even when nuclear and cytoplasmic pools of NAD(+) are depleted. Rodents fasted for 48 hr show increased levels of the NAD(+) biosynthetic enzyme Nampt and a concomitant increase in mitochondrial NAD(+). Increased Nampt provides protection against cell death and requires an intact mitochondrial NAD(+) salvage pathway as well as the mitochondrial NAD(+)-dependent deacetylases SIRT3 and SIRT4. We discuss the relevance of these findings to understanding how nutrition modulates physiology and to the evolution of apoptosis.     

Liver steatosis and increased ChREBP expression in mice carrying a liver specific SIRT1 null mutation under a normal feeding condition

SIRT1, a homolog of yeast Sir2, is a type III NAD(+) dependent histone and protein deacetylase. Previous studies of mice carrying liver specific deletion of exon 4 of the Sirt1 gene revealed opposite responses of mutant mice to a high-fat diet in terms of fatty liver formation, which obscures the function of SRIT1 in liver development and lipid metabolism. To investigate this, we deleted exons 5 and 6 of Sirt1 in the liver by using a Cre-loxP approach. Western blot using an antibody to N-terminal SIRT1 does not detect a truncated protein in the liver of the mutant mice (Sirt1(flox5-6/flox5-6);Alb-Cre), suggesting a null mutation for SIRT1 is generated in the liver. Unlike the previously reported phenotypes, the Sirt1(flox5-6/flox5-6);Alb-Cre mice develop fatty liver under a normal feeding condition. The disease starts at two months of age and incidence increases as the animals become older, affecting 78% of them when they are over one year of age. We showed that the steatosis is accompanied by altered expression of a number of genes, including increased expression of ChREBP, which acts as one of the central determinants of lipid synthesis in the liver. This data uncovers an important role of SIRT1 in regulating lipid metabolism in the liver, and the SIRT1 mutant mice may serve as an animal model for studying human fatty liver disease and facilitate the development of effective therapeutic approach for the disease.     

Sirtuins与PARP-1在细胞凋亡过程中的相互作用

哺乳动物Sirtuins家族目前共发现7个成员：SIRT1～SIRT7,它们均为NAD+依 赖性且从细菌到人类都保守的一类酶.人们已经对这7种去乙酰化酶进行了亚细胞定位 .目前,对其研究主要集中在对细胞发育相关的重要转录因子如p53、FOXO家族及相关 蛋白的去乙酰化修饰.Sirtuins对许多生理过程有着重要的调节作用,尤其是当发现 它们对寿命延长的调控作用后,Sirtuins引起了人们极大的关注,且都发表在世界顶 级刊物上.聚ADP核糖聚合酶(poly ADP-ribose polymerase, PARP)是一类存在于大多 数真核细胞中的蛋白质翻译后修饰酶,尤其是聚ADP核糖聚合酶1(PARP-1)在细胞内 DNA损伤修复等过程中起着重要作用,该酶同样以NAD+作为催化反应的底物.有研究发 现,Sirtuins家族成员与PARP-1在细胞内某些重要生理过程中存在着相互作用.本文评 述了Sirtuins家族成员、PARP-1的生物学特点,并就其参与哺乳动物细胞凋亡的调控 机制和相关信号通路进行了详细的论述,以期对Sirtuins家族成员、PARP-1生物学功 能及其相互作用的研究提供理论指导.     

Systemic SIRT1 insufficiency results in disruption of energy homeostasis and steroid hormone metabolism upon high-fat-diet feeding

SIRT1 is a highly-conserved NAD(+)-dependent protein deacetylase that plays essential roles in the regulation of energy metabolism, genomic stability, and stress response. Although the functions of SIRT1 in many organs have been extensively studied in tissue-specific knockout mouse models, the systemic role of SIRT1 is still largely unknown as a result of severe developmental defects that result from whole-body knockout in mice. Here, we investigated the systemic functions of SIRT1 in metabolic homeostasis by utilizing a whole-body SIRT1 heterozygous mouse model. These mice are phenotypically normal under standard feeding conditions. However, when chronically challenged with a 40% fat diet, they become obese and insulin resistant, display increased serum cytokine levels, and develop hepatomegaly. Hepatic metabolomic analyses revealed that SIRT1 heterozygous mice have elevated gluconeogenesis and oxidative stress. Surprisingly, they are depleted of glycerolipid metabolites and free fatty acids, yet accumulate lysolipids. Moreover, high-fat feeding induces elevation of serum testosterone levels and enlargement of seminal vesicles in SIRT1 heterozygous males. Microarray analysis of liver mRNA indicates that they have altered expression of genes involved in steroid metabolism and glycerolipid metabolism. Taken together, our findings indicate that SIRT1 plays a vital role in the regulation of systemic energy and steroid hormone homeostasis.