Nicotinamide Improves Aspects of Healthspan, but Not Lifespan, in Mice.

The role in longevity and healthspan of nicotinamide (NAM), the physiological precursor of NAD+, is elusive. Here, we report that chronic NAM supplementation improves healthspan measures in mice without extending lifespan. Untargeted metabolite profiling of the liver and metabolic flux analysis of liver-derived cells revealed NAM-mediated improvement in glucose homeostasis in mice on a high-fat diet (HFD) that was associated with reduced hepatic steatosis and inflammation concomitant with increased glycogen deposition and flux through the pentose phosphate and glycolytic pathways. Targeted NAD metabolome analysis in liver revealed depressed expression of NAM salvage in NAM-treated mice, an effect counteracted by higher expression of de novo NAD biosynthetic enzymes. Although neither hepatic NAD+ nor NADP+ was boosted by NAM, acetylation of some SIRT1 targets was enhanced by NAM supplementation in a diet- and NAM dose-dependent manner. Collectively, our results show health improvement in NAM-supplemented HFD-fed mice in the absence of survival effects.

Sirt1 inhibition promotes in vivo arterial thrombosis and tissue factor expression in stimulated cells.

AIMS: The mammalian silent information regulator-two 1 (Sirt1) blunts the noxious effects of cardiovascular risk factors such as type 2 diabetes mellitus and obesity. Nevertheless, the role of Sirt1 in regulating the expression of tissue factor (TF), the key trigger of coagulation, and arterial thrombus formation remains unknown. METHODS AND RESULTS: Human as well as mouse cell lines were used for in vitro experiments, and C57Bl/6 mice for in vivo procedures. Sirt1 inhibition by splitomicin or sirtinol enhanced cytokine-induced endothelial TF protein expression as well as surface activity, while TF pathway inhibitor protein expression did not change. Sirt1 inhibition further enhanced TF mRNA expression, TF promoter activity, and nuclear translocation as well as DNA binding of the p65 subunit of nuclear factor-kappa B (NFκB/p65). Sirt1 siRNA enhanced TF protein and mRNA expression, and this effect was reduced in NFκB/p65(-/-) mouse embryonic fibroblasts reconstituted with non-acetylatable Lys(310)-mutant NFκB/p65. Activation of the mitogen-activated protein kinases p38, c-Jun NH(2)-terminal kinase, and p44/42 (ERK) remained unaffected. In vivo, mice treated with the Sirt1 inhibitor splitomicin exhibited enhanced TF activity in the arterial vessel wall and accelerated carotid artery thrombus formation in a photochemical injury model. CONCLUSION: We provide pharmacological and genetic evidence that Sirt1 inhibition enhances TF expression and activity by increasing NFκB/p65 activation in human endothelial cells. Furthermore, Sirt1 inhibition induces arterial thrombus formation in vivo. Hence, modulation of Sirt1 may offer novel therapeutic options for targeting thrombosis.

SRT1720 improves survival and healthspan of obese mice.

Sirt1 is an NAD(+)-dependent deacetylase that extends lifespan in lower organisms and improves metabolism and delays the onset of age-related diseases in mammals. Here we show that SRT1720, a synthetic compound that was identified for its ability to activate Sirt1 in vitro, extends both mean and maximum lifespan of adult mice fed a high-fat diet. This lifespan extension is accompanied by health benefits including reduced liver steatosis, increased insulin sensitivity, enhanced locomotor activity and normalization of gene expression profiles and markers of inflammation and apoptosis, all in the absence of any observable toxicity. Using a conditional SIRT1 knockout mouse and specific gene knockdowns we show SRT1720 affects mitochondrial respiration in a Sirt1- and PGC-1α-dependent manner. These findings indicate that SRT1720 has long-term benefits and demonstrate for the first time the feasibility of designing novel molecules that are safe and effective in promoting longevity and preventing multiple age-related diseases in mammals.

AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity.

AMP-activated protein kinase (AMPK) is a metabolic fuel gauge conserved along the evolutionary scale in eukaryotes that senses changes in the intracellular AMP/ATP ratio. Recent evidence indicated an important role for AMPK in the therapeutic benefits of metformin, thiazolidinediones and exercise, which form the cornerstones of the clinical management of type 2 diabetes and associated metabolic disorders. In general, activation of AMPK acts to maintain cellular energy stores, switching on catabolic pathways that produce ATP, mostly by enhancing oxidative metabolism and mitochondrial biogenesis, while switching off anabolic pathways that consume ATP. This regulation can take place acutely, through the regulation of fast post-translational events, but also by transcriptionally reprogramming the cell to meet energetic needs. Here we demonstrate that AMPK controls the expression of genes involved in energy metabolism in mouse skeletal muscle by acting in coordination with another metabolic sensor, the NAD+-dependent type III deacetylase SIRT1. AMPK enhances SIRT1 activity by increasing cellular NAD+ levels, resulting in the deacetylation and modulation of the activity of downstream SIRT1 targets that include the peroxisome proliferator-activated receptor-gamma coactivator 1alpha and the forkhead box O1 (FOXO1) and O3 (FOXO3a) transcription factors. The AMPK-induced SIRT1-mediated deacetylation of these targets explains many of the convergent biological effects of AMPK and SIRT1 on energy metabolism.

Small molecule activators of SIRT1 replicate signaling pathways triggered by calorie restriction in vivo.

BACKGROUND: Calorie restriction (CR) produces a number of health benefits and ameliorates diseases of aging such as type 2 diabetes. The components of the pathways downstream of CR may provide intervention points for developing therapeutics for treating diseases of aging. The NAD+-dependent protein deacetylase SIRT1 has been implicated as one of the key downstream regulators of CR in yeast, rodents, and humans. Small molecule activators of SIRT1 have been identified that exhibit efficacy in animal models of diseases typically associated with aging including type 2 diabetes. To identify molecular processes induced in the liver of mice treated with two structurally distinct SIRT1 activators, SIRT501 (formulated resveratrol) and SRT1720, for three days, we utilized a systems biology approach and applied Causal Network Modeling (CNM) on gene expression data to elucidate downstream effects of SIRT1 activation. RESULTS: Here we demonstrate that SIRT1 activators recapitulate many of the molecular events downstream of CR in vivo, such as enhancing mitochondrial biogenesis, improving metabolic signaling pathways, and blunting pro-inflammatory pathways in mice fed a high fat, high calorie diet. CONCLUSION: CNM of gene expression data from mice treated with SRT501 or SRT1720 in combination with supporting in vitro and in vivo data demonstrates that SRT501 and SRT1720 produce a signaling profile that mirrors CR, improves glucose and insulin homeostasis, and acts via SIRT1 activation in vivo. Taken together these results are encouraging regarding the use of small molecule activators of SIRT1 for therapeutic intervention into type 2 diabetes, a strategy which is currently being investigated in multiple clinical trials.

Specific SIRT1 activation mimics low energy levels and protects against diet-induced metabolic disorders by enhancing fat oxidation.

The NAD(+)-dependent deacetylase SIRT1 controls metabolic processes in response to low nutrient availability. We report the metabolic phenotype of mice treated with SRT1720, a specific and potent synthetic activator of SIRT1 that is devoid of direct action on AMPK. SRT1720 administration robustly enhances endurance running performance and strongly protects from diet-induced obesity and insulin resistance by enhancing oxidative metabolism in skeletal muscle, liver, and brown adipose tissue. These metabolic effects of SRT1720 are mediated by the induction of a genetic network controlling fatty acid oxidation through a multifaceted mechanism that involves the direct deacetylation of PGC-1alpha, FOXO1, and p53 and the indirect stimulation of AMPK signaling through a global metabolic adaptation mimicking low energy levels. Combined with our previous work on resveratrol, the current study further validates SIRT1 as a target for the treatment of metabolic disorders and characterizes the mechanisms underlying the therapeutic potential of SIRT1 activation.

Sirtuins--novel therapeutic targets to treat age-associated diseases.

Sirtuins post-translationally modulate the function of many cellular proteins that undergo reversible acetylation-deacetylation cycles, affecting physiological responses that have implications for treating diseases of ageing. Potent small-molecule modulators of sirtuins have shown efficacy in preclinical models of metabolic, neurodegenerative and inflammatory diseases, and so hold promise for drug discovery efforts in multiple therapeutic areas. Here, we discuss current knowledge and data that strengthens sirtuins as a druggable set of enzymes for the treatment of age-associated diseases, including activation of SIRT1 in type 2 diabetes.

Resveratrol delays age-related deterioration and mimics transcriptional aspects of dietary restriction without extending life span.

A small molecule that safely mimics the ability of dietary restriction (DR) to delay age-related diseases in laboratory animals is greatly sought after. We and others have shown that resveratrol mimics effects of DR in lower organisms. In mice, we find that resveratrol induces gene expression patterns in multiple tissues that parallel those induced by DR and every-other-day feeding. Moreover, resveratrol-fed elderly mice show a marked reduction in signs of aging, including reduced albuminuria, decreased inflammation, and apoptosis in the vascular endothelium, increased aortic elasticity, greater motor coordination, reduced cataract formation, and preserved bone mineral density. However, mice fed a standard diet did not live longer when treated with resveratrol beginning at 12 months of age. Our findings indicate that resveratrol treatment has a range of beneficial effects in mice but does not increase the longevity of ad libitum-fed animals when started midlife.

Sirtuins: novel targets for metabolic disease.

Sirtuins represent a novel family of enzymes that are collectively well situated to help regulate nutrient sensing and utilization, metabolic rate and ultimately metabolic disease. Activation of one of these enzymes, SIRT1, leads to enhanced activity of multiple proteins, including peroxisome-proliferator activated receptor coactivator-1alpha (PGC-1alpha), which helps to mediate some of the in vitro and in vivo effects of sirtuins. As such, enhanced SIRT1 activity decreases glucose levels, improves insulin sensitivity, increases mitochondrial number and function, decreases adiposity, improves exercise tolerance and potentially lowers body weight. SRT-501 is a proprietary formulation of resveratrol with improved bioavailability. As such, SRT-501 represents the first in a novel class of SIRT1 activators that has proven to be safe and well-tolerated in humans. Clinical trials in type 2 diabetic patients are currently underway.

The influence of corticosteroids on the release of novel biomarkers in human endotoxemia.

OBJECTIVE: Sepsis intervention studies need better patient stratification methods, and one way to realize this is the introduction of stable biomarkers. A set of recently developed novel biomarkers, based upon precursor-fragments of short-lived hormones, was previously shown to be increased during sepsis. However, it is not known whether these biomarkers are influenced by sepsis intervention strategies. Therefore we investigated the markers in a model of human endotoxemia intervened by increasing doses of prednisolone. DESIGN AND SETTING: Prospective, open-label study in a specialized clinical research unit of a university hospital. SUBJECTS: Thirty-two healthy male volunteers. INTERVENTIONS: Subjects received prednisolone orally at doses of 0, 3, 10 or 30 mg (n=8 per group) at 2 h before intravenous injection of Escherichia coli lipopolysaccharide (LPS) (4 ng/kg). Blood samples were drawn during 24 h after LPS injection. MEASUREMENTS AND RESULTS: LPS injection caused an increase in levels of midregional pro-adrenomedullin (MR-proADM), midregional pro-atrial natriuretic peptide (MR-proANP), C-terminal pro-arginine-vasopressin (CT-proAVP) and procalcitonin (PCT). Prednisolone caused a dose dependent inhibition of MR-proADM, MR-proANP and CT-proAVP levels. CONCLUSIONS: These results show that a set of novel, highly stable sepsis biomarkers was increased during human endotoxemia and was dose-dependently inhibited by corticosteroid pre-treatment.

Effects of prednisolone on the systemic release of mediators of cell-mediated cytotoxicity during human endotoxemia.

Corticosteroids are widely used for the suppression of cell-mediated cytoxicity. This process is mediated by natural killer cells and cytotoxic T lymphocytes, and their activation can be monitored by levels of the chemokines CXCL9 and CXCL10, the degranulation product granzymes A and B, and by levels of secretory phospholipase A2. The current study aimed to determine the effects of increasing doses of prednisolone on the release of these mediators in healthy humans exposed to LPS. Therefore, 32 healthy men received prednisolone orally at doses of 0, 3, 10, or 30 mg (n = 8 per group) at 2 h before intravenous injection of Escherichia coil LPS (4 ng/kg). Prednisolone dose-dependently attenuated the LPS-induced rises in the plasma concentrations of the chemokines CXCL9 and CXCL10, as well as of granzymes A and B levels. CXCL10 and granzyme B release were most sensitive to prednisolone, with a significant inhibition already achieved at the lowest prednisolone dose (3 mg). The levels of secretory phospholipase A2 were increased after LPS administration but were not significantly affected by prednisolone. This study demonstrates that prednisolone differentially inhibits the systemic release of mediators involved in cell-mediated cytotoxicity in humans in vivo.

Small molecule activators of SIRT1 as therapeutics for the treatment of type 2 diabetes.

Calorie restriction extends lifespan and produces a metabolic profile desirable for treating diseases of ageing such as type 2 diabetes. SIRT1, an NAD+-dependent deacetylase, is a principal modulator of pathways downstream of calorie restriction that produce beneficial effects on glucose homeostasis and insulin sensitivity. Resveratrol, a polyphenolic SIRT1 activator, mimics the anti-ageing effects of calorie restriction in lower organisms and in mice fed a high-fat diet ameliorates insulin resistance, increases mitochondrial content, and prolongs survival. Here we describe the identification and characterization of small molecule activators of SIRT1 that are structurally unrelated to, and 1,000-fold more potent than, resveratrol. These compounds bind to the SIRT1 enzyme-peptide substrate complex at an allosteric site amino-terminal to the catalytic domain and lower the Michaelis constant for acetylated substrates. In diet-induced obese and genetically obese mice, these compounds improve insulin sensitivity, lower plasma glucose, and increase mitochondrial capacity. In Zucker fa/fa rats, hyperinsulinaemic-euglycaemic clamp studies demonstrate that SIRT1 activators improve whole-body glucose homeostasis and insulin sensitivity in adipose tissue, skeletal muscle and liver. Thus, SIRT1 activation is a promising new therapeutic approach for treating diseases of ageing such as type 2 diabetes.

Prednisolone dose-dependently influences inflammation and coagulation during human endotoxemia.

The effects of steroids on the outcome of sepsis are dose dependent. Low doses appear to be beneficial, but high doses do not improve outcome for reasons that are insufficiently understood. The effects of steroids on systemic inflammation as a function of dose have not previously been studied in humans. To determine the effects of increasing doses of prednisolone on inflammation and coagulation in humans exposed to LPS, 32 healthy males received prednisolone orally at doses of 0, 3, 10, or 30 mg (n = 8 per group) at 2 h before i.v. injection of Escherichia coli LPS (4 ng/kg). Prednisolone dose-dependently inhibited the LPS-induced release of cytokines (TNF-alpha and IL-6) and chemokines (IL-8 and MCP-1), while enhancing the release of the anti-inflammatory cytokine IL-10. Prednisolone attenuated neutrophil activation (plasma elastase levels) and endothelial cell activation (von Willebrand factor). Most remarkably, prednisolone did not inhibit LPS-induced coagulation activation, measured by plasma concentrations of thrombin-antithrombin complexes, prothrombin fragment F1+2, and soluble tissue factor. In addition, activation of the fibrinolytic pathway (tissue-type plasminogen activator and plasmin-alpha(2)-antiplasmin complexes) was dose-dependently enhanced by prednisolone. These data indicate that prednisolone dose-dependently and differentially influences the systemic activation of different host response pathways during human endotoxemia.

The proteasome inhibitor bortezomib (Velcade) sensitizes some human tumor cells to Apo2L/TRAIL-mediated apoptosis.

On testing a panel of different human cancer cell lines, we observed that the proteasome inhibitor bortezomib could dramatically sensitize some lines to the apoptotic effects of Apo2L/TRAIL. Certain renal, colon, or breast tumor cell lines were dramatically sensitized, whereas other tumor lines from the same tissue of origin remained resistant. This sensitization did not correlate with either the p53 status of the individual tumor cell lines or their intrinsic sensitivity to Apo2L/TRAIL. Colon cancer cell lines lacking p53 or Bax were sensitized by bortezomib, suggesting that neither p53 nor Bax levels were crucial for sensitization. Although the molecular basis of bortezomib sensitization of tumor cells to Apo2L/TRAIL remains to be determined, this combination can have an enhanced apoptotic effect over either agent alone for certain human cancer cells.

Systematic discovery of multicomponent therapeutics.

Multicomponent therapies, originating through deliberate mixing of drugs in a clinical setting, through happenstance, and through rational design, have a successful history in a number of areas of medicine, including cancer, infectious diseases, and CNS disorders. We have developed a high-throughput screening method for identifying effective combinations of therapeutic compounds. We report here that systematic screening of combinations of small molecules reveals unexpected interactions between compounds, presumably due to interactions between the pathways on which they act. Through systematic screening of approximately 120,000 different two-component combinations of reference-listed drugs, we identified potential multicomponent therapeutics, including (i) fungistatic and analgesic agents that together generate fungicidal activity in drug-resistant Candida albicans, yet do not significantly affect human cells, (ii) glucocorticoid and antiplatelet agents that together suppress the production of tumor necrosis factor-alpha in human primary peripheral blood mononu-clear cells, and (iii) antipsychotic and antiprotozoal agents that do not exhibit significant antitumor activity alone, yet together prevent the growth of tumors in mice. Systematic combination screening may ultimately be useful for exploring the connectivity of biological pathways and, when performed with reference-listed drugs, may result in the discovery of new combination drug regimens.

Proteasome inhibition: a new anti-inflammatory strategy.

The ubiquitin-proteasome pathway has a central role in the selective degradation of intracellular proteins. Among the key proteins modulated by the proteasome are those involved in the control of inflammatory processes, cell cycle regulation, and gene expression. Consequently proteasome inhibition is a potential treatment option for cancer and inflammatory conditions. Thus far, proof of principle has been obtained from studies in numerous animal models for a variety of human diseases including cancer, reperfusion injury, and inflammatory conditions such as rheumatoid arthritis, asthma, multiple sclerosis, and psoriasis. Two proteasome inhibitors, each representing a unique chemical class, are currently under clinical evaluation. Velcade (PS-341) is currently being evaluated in multiple phase II clinical trials for several solid tumor indications and has just entered a phase III trial for multiple myeloma. PS-519, representing another class of inhibitors, focuses on the inflammatory events following ischemia and reperfusion injury. Since proteasome inhibitors exhibit anti-inflammatory and antiproliferative effects, diseases characterized by both of these processes simultaneously, as is the case in rheumatoid arthritis or psoriasis, might also represent clinical opportunities for such drugs.

Mechanisms of proteasome inhibitor PS-341-induced G(2)-M-phase arrest and apoptosis in human non-small cell lung cancer cell lines.

PURPOSE: PS-341 is a novel dipeptide boronic acid proteasome inhibitor with in vitro and in vivo antitumor activity that induces mechanisms of apoptosis by unknown mechanisms. EXPERIMENTAL DESIGN: Human non-small cell lung cancer cell lines were used to investigate effects PS-341 on cell proliferation, cell cycle progression, and the induction of apoptosis. RESULTS: PS-341 was 38-360-fold more cytotoxic against H460 cells when compared with the proteasome inhibitors MG-132 and PSI. Differential PS-341 cytotoxic effects were found with respect to P53 function: H322 cells (p53 mutant) were 6-fold less sensitive as compared with H460 cells (p53 wild type); and H358 cells (p53 null) were 1.6-fold more sensitive as compared with H460 cells (p53 wild type). A concentration- and time-dependent cell cycle blockade at G(2)-M phase was seen for H460 cells without any direct effects on microtubule polymerization or depolymerization. PS-341 exposure in H460 cells led to stabilization of p53, induction of p21(cip/waf-1) and MDM2 expression, an increase in cyclin B and cyclin A, and the activation of cyclin B and cyclin A kinases. MDM2 induction was found only in H460 cells, whereas in H322 and H358 cells, G(2)-M-phase arrest, p21(cip/waf-1) induction, and an increase in cyclin B1 were found. The commitment of G(2)-M-phase cells to apoptosis was verified by the activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase in drug-free medium. CONCLUSIONS: Our data suggest that the PS-341-induced G(2)-M-phase arrest may be associated with the inhibition of degradation of cell cycle regulators and that the up-regulation of p21(cip/waf-1) expression may be via p53-dependent and/or -independent pathways. The resulting disturbance of cell cycle progression leads either to growth inhibition or to the initiation of apoptotic pathways.

The proteasome inhibitor PS-341 sensitizes neoplastic cells to TRAIL-mediated apoptosis by reducing levels of c-FLIP.

Because of the pivotal role the proteasome plays in apoptosis, inhibitors of this enzyme, such as PS-341, provide a great opportunity for exploring synergy between proteasome inhibition and other apoptosis-inducing agents. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively induce apoptosis in tumor cells. In overnight assays, combinations of PS-341 and TRAIL were much more effective than either agent alone in promoting apoptosis of a murine myeloid leukemia, C1498, and a murine renal cancer, Renca. For C1498 cells, apoptosis sensitization by PS-341 affected neither the activity of nuclear factor kappaB (NF-kappaB) nor the levels of most antiapoptotic proteins. However, reductions in the antiapoptotic protein c-FLIP in response to PS-341 were observed in both C1498 and Renca cells. Treatment of normal bone marrow mixed with C1498 tumor cells for 18 hours with a combination of PS-341 and TRAIL resulted in a specific depletion of the tumor cells. Upon transfer to irradiated syngeneic recipient mice, mixtures treated with the PS-341 plus TRAIL combination resulted in enhanced long-term tumor-free survival of mice. These data therefore support the targeting of apoptotic pathways in tumor cells, using combinations of agents such as PS-341 and TRAIL that interact synergistically to preferentially promote tumor cell apoptosis.

Proteasome inhibition ablates activation of NF-kappa B in myocardial reperfusion and reduces reperfusion injury.

Both acute coronary occlusion and reperfusion of an infarct-related artery lead to significant myocardial cell death. Recent evidence has been presented that activation of the transcription factor nuclear factor-kappaB (NF-kappaB) plays a critical role in reperfusion injury. NF-kappaB is usually bound to its inhibitor, IkappaB, and classic activation of NF-kappaB occurs when the 20S proteasome degrades IkappaB that has been phosphorylated and ubiquitinated. In this study, activation of NF-kappaB was inhibited by systemic administration of a 20S proteasome inhibitor (PS-519) in a porcine model of myocardial reperfusion injury. The experimental protocol induced myocardial ischemia in the distribution of the left anterior descending coronary artery for 1 h with subsequent reperfusion for 3 h. A single systemic treatment with PS-519 reduced 20S proteasome activity; blocked activation of NF-kappaB induced by reperfusion; reduced creatine kinase, creatine kinase-muscle-brain fraction, and troponin I release from the myocardium; preserved regional myocardial function measured by segmental shortening; significantly reduced the size of myocardial infarction; and exhibited no acute toxicity. These data show that myocardial reperfusion injury can be inhibited by using proteasome inhibitors, which likely function through the inhibition of NF-kappaB activation.