Increased SIRT1 Activity Research Articles

Chitosan/hesperidin nanoparticles formulation: a promising approach against ethanol-induced gastric ulcers via Sirt1/FOXO1/PGC-1α/HO-1 pathway.

Hesperidin (Hes) protects different organs from damage by acting as a potent antioxidant and anti-inflammatory. This study aims to evaluate the gastroprotective effects of free hesperidin and its chitosan nanoparticles (HNPs) against ethanol-induced gastric ulcers in rats, hypothesizing that HNPs will enhance bioavailability and therapeutic efficacy due to improved solubility and targeted delivery. HNPs were synthesized via ion gelation and characterized using TEM, SEM, and zeta potential analyses. Key assessments included gastric acidity, histological analysis, and markers of inflammation, oxidative stress, and apoptosis. HNPs significantly decreased gastric acidity, reduced inflammatory and apoptotic markers, and enhanced antioxidant enzyme activities compared to free hesperidin and esomeprazole. Furthermore, Sirt-1, PGC-1α, HO-1, and FOXO1 gene expression were also evaluated. HNPs raised Sirt-1, PGC-1α, HO-1, and downregulated FOXO1, and they suppressed the activities of NF-κB p65, COX-2, IL-1β, CD86, FOXO1 P53, and caspase-3 and increased Sirt-1 activity. HNPs treatment notably restored antioxidant enzyme activity, reduced oxidative stress and inflammatory markers, and improved histological outcomes more effectively than free hesperidin and esomeprazole. These results indicate that chitosan nanoparticles significantly enhance the gastroprotective effects of hesperidin against ethanol-induced gastric ulcers, potentially offering a more effective therapeutic strategy. Further research should explore the clinical applications of HNPs in human subjects.

Preventing loss of sirt1 lowers mitochondrial oxidative stress and preserves C2C12 myotube diameter in an invitro model of cancer cachexia.

Cancer cachexia is a multifactorial syndrome associated with advanced cancer that contributes to mortality. Cachexia is characterized by loss of body weight and muscle atrophy. Increased skeletal muscle mitochondrial reactive oxygen species (ROS) is a contributing factor to loss of muscle mass in cachectic patients. Mice inoculated with Lewis lung carcinoma (LLC) cells lose weight, muscle mass, and have lower muscle sirtuin-1 (sirt1) expression. Nicotinic acid (NA) is a precursor to nicotinamide dinucleotide (NAD+) which is exhausted in cachectic muscle and is a direct activator of sirt1. Mice lost body and muscle weight and exhibited reduced skeletal muscle sirt1 expression after inoculation with LLC cells. C2C12 myotubes treated with LLC-conditioned media (LCM) had lower myotube diameter. We treated C2C12 myotubes with LCM for 24 h with or without NA for 24 h. C2C12 myotubes treated with NA maintained myotube diameter, sirt1 expression, and had lower mitochondrial superoxide. We then used a sirt1-specific small molecule activator SRT1720 to increase sirt1 activity. C2C12 myotubes treated with SRT1720 maintained myotube diameter, prevented loss of sirt1 expression, and attenuated mitochondrial superoxide production. Our data provides evidence that NA may be beneficial in combating cancer cachexia by maintaining sirt1 expression and decreasing mitochondrial superoxide production.

LARP7 overexpression alleviates aortic senescence and atherosclerosis.

Atherosclerosis, characterized by the accumulation of lipid plaques on the inner walls of arteries, is the leading cause of heart attack, stroke and severe ischemic injuries. Senescent cells have been found to accumulate within atherosclerotic lesions and contribute to the progression of atherosclerosis. In our previous study, we discovered that suppressing Larp7 accelerates senescence by inhibiting Sirt1 activity, resulting in increased atherosclerosis in high-fat diet (HFD) fed and ApoE deficient (ApoEKO) mice. However, there has been no direct evidence demonstrating Larp7 per se could attenuate atherosclerosis. To this end, we generated a tetO-controlled and Cre-activated Larp7 gain-of-function mouse. Through RT-PCR and western blotting, we confirmed Larp7 overexpression in the aortas of HFD-fed ApoEKO; Larp7tetO mice. Larp7 overexpression led to increased Sirt1 activity and decreased cellular senescence signals mediated by p53/p65 in the aortas. Additionally, Larp7 overexpression reduced the presence of p16-positive senescent cells in the aortic lesions. Furthermore, Larp7 overexpression resulted in a decrease in pro-inflammatory macrophages and SASP factors. Consequently, Larp7 overexpression led to a reduction in the area of atherosclerotic lesions in HFD-fed ApoEKO; Larp7tetO mice. In summary, our study provides evidence that Larp7 overexpression holds promise as an approach to inhibit cellular senescence and prevent atherosclerosis.

Alcohol-associated skeletal muscle mitochondrial dysfunction: insights into Sirt1 regulation

Alcohol-induced skeletal muscle dysfunction affects approximately 50% of individuals with alcohol use disorder (AUD). Alcohol dysregulates mitochondrial oxidative metabolism and promotes oxidative stress. However, the impact of alcohol on skeletal muscle bioenergetic capacity and cellular redox balance remain to be fully elucidated. The expression and activity of Sirtuin 1 (SIRT1), an NAD+-dependent deacetylase, is dysregulated with ethanol metabolism. This disruption has the potential to play a significant role in the intricate regulatory network responsible for alcohol-induced mitochondrial dysfunction in skeletal muscle. Interventional strategies, such as exercise, have the potential to enhance mitochondrial function and promote overall skeletal muscle health. We hypothesize that alcohol dysregulates myotube mitochondrial bioenergetics by reducing mitochondrial respiration in a Sirt1-dependent manner, and we predict that simulating exercise through electrical pulse stimulation will increase Sirt1 expression. Myoblasts were differentiated into myotubes in the presence of ethanol (0 and 50 mM) for 3 days prior to Mito Stress Test using the Seahorse XF Pro Analyzer. Differentiated myotubes treated with ethanol (0 and 50 mM, for 3 days), were exposed to electrical pulse stimulation simulating exercise (30V, 1Hz, 2ms pulses) for 24 h. A mixed-effects model with Šidák correction and repeated samples t-test was used. Means with p-value of less than 0.05 were considered statistically significant. Preliminary analysis indicates significant decrease in ATP-linked mitochondrial respiration (p<0.05) in the presence of ethanol. Ethanol also decreased myotube Sirt1 activity (p<0.05) and Sirt3 protein expression. There was a main effect of electrical pulse stimulation to increase Sirt1 activity (p<0.001) in both the control and ethanol-treated groups. Ongoing analysis will identify alcohol-mediated changes in proteins involved in mitochondrial dynamics regulated by Sirt1. These results show that ethanol significantly decreases mitochondrial bioenergetics in differentiated human myotubes. The ethanol induced changes in Sirt1 activity and Sirt3 protein expression, suggest a potential role of Sirt1-mediated mitochondrial dysregulation with alcohol. This research was supported by NIH/NIAAA T32AA007577, P60AA009803 (PEM) and R21AA030869 (LS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

SIRT1 mediates breast cancer development and tumorigenesis controlled by estrogen-related receptor β.

Silent mating type information regulation 2 homolog 1 (SIRT1) is a class III histone deacetylase (HDAC) that is NAD + dependent and essential for metabolism, senescence, and cell survival. SIRT1 is overexpressed in several cancers, including breast cancer. SIRT1 is a well-known target gene of the estrogen receptor alpha (ER alpha) and is closely related to ER alpha deacetylation. Transcription factor Estrogen-related receptors (ERRs) share sequence homology with ERs in the DNA-binding domain, therefore, the possibility of sharing target genes between them is high. Our current research aims togain insight into the function of ERRβ in regulating the activity of SIRT1 during the progression of breast cancer. ER-positive (ER + ve) breast cancer cells and tissues had considerably enhanced SIRT1 expression. Six potential ERRE sites were identified by analysis of the 5' upstream region of SIRT1, and both in vitro and in vivo experiments supported their presence. We found SIRT1 to be up-regulated in ERRβ overexpressed ER + ve breast cancer cells. Furthermore, our findings suggested that ectopic production of ERR and PCAF would increase SIRT1 activity. Our findings also indicated that ectopic production of ERRβ and PCAF increased SIRT1 activity. With sufficient evidence demonstrating the substantial involvement of SIRT1 in cell proliferation, migration, and colony formation capability, we were also able to illustrate the tumorigenic role of SIRT1. Overall, our findings highlight SIRT1's tumorigenic influence on breast cancer and suggest that SIRT1 inhibitors might serve as potential therapeutic drugs for the treatment of breast cancer.

ACMSD mediated de novo NAD+ biosynthetic impairment in cardiac endothelial cells as a potential therapeutic target for diabetic cardiomyopathy

Object: The highly conserved α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) is the key enzyme that regulates the de novo NAD+ synthesis from tryptophan. NAD+ metabolism in diabetic cardiomyopathy (DCM) was not elucidated yet. MethodsMice were assigned to non-diabetic (NDM) group, streptozocin (STZ)-induced diabetic (DM) group, and nicotinamide (NAM) treated (DM+NAM) group. ACMSD mediated NAD+ metabolism were studied both in mice and patients with diabetes. ResultsNAD+ level was significantly lower in the heart of DM mice than that of the NDM group. Supplementation with NAM could partially increased myocardial capillary density and ameliorated myocardial fibrosis by increasing NAD+ level through salvage pathway. Compared with NDM mice, the expression of ACMSD in myocardial endothelial cells of DM mice was significantly increased. It was further confirmed that in endothelial cells, high glucose promoted the expression of ACMSD. Inhibition of ACMSD could increase de novo NAD+ synthesis and improve endothelial cell function by increasing Sirt1 activity. Targeted mass spectrometry analysis indicated increased ACMSD enzyme activity in diabetic patients, higher ACMSD activity increased risk of heart diastolic dysfunction. ConclusionIn summary, increased expression of ACMSD lead to impaired de novo NAD+ synthesis in diabetic heart. Inhibition of ACMSD could potentially improve DCM.

Roles of adenosine monophosphate-activated protein kinase (AMPK) in the kidney.

Roles of adenosine monophosphate-activated protein kinase (AMPK) in the kidney.

Thymoquinone and Melatonin Boost Both Sirt1 and Nrf2 Activities in a Complementary Way – A Strategy for Controlling Oxidative Stress and Inflammation

The oil of black cumin seed (Nigella sativa) has been employed therapeutically in Asian traditional medicine, and rodent studies with it or its most prominent and intriguing component, thymoquinone, suggest that it may indeed have the potential to provide benefit in a wide range of pathologies. There is reason to suspect that thymoquinone’s versatile utility may reflect, in large part, its ability to boost the activity of the sirtuin 1 (Sirt1) deacetylase while concurrently activating the Nrf2 transcription factor. Increased Sirt1 activity promotes autophagy, mitochondrial biogenesis, and FOXO-mediated expression of antioxidant enzymes, while suppressing NF-kappaB-mediated inflammatory signaling; Nrf2 promotes expression of a wide range of antioxidant enzymes, as well as the enzyme rate limiting for glutathione synthesis. Thymoquinone is a substrate for NAD(P)H quinone oxidoreductase 1 (NQO1); this reaction gives rise to NAD+ - obligate substrate for Sirt1 activity – while generating thymohydroquinone, a potent scavenging antioxidant. Thymoquinone, which is electrophilic, promotes Nrf2 activation through covalent reaction with its functional inhibitor Keap1. We suggest that supplemental melatonin may be an ideal complement to thymoquinone, as it can promote increased expression of both Sirt1and Nrf2 – likely via activation of the “clock” transcription factor Bmal1 – whereas thymoquinone boosts the biological activities of the pre-formed proteins.

Nicotinamide Mononucleotide Prevents Free Fatty Acid-Induced Reduction in Glucose Tolerance by Decreasing Insulin Clearance.

The NAD-dependent deacetylase SIRT1 improves β cell function. Accordingly, nicotinamide mononucleotide (NMN), the product of the rate-limiting step in NAD synthesis, prevents β cell dysfunction and glucose intolerance in mice fed a high-fat diet. The current study was performed to assess the effects of NMN on β cell dysfunction and glucose intolerance that are caused specifically by increased circulating free fatty acids (FFAs). NMN was intravenously infused, with or without oleate, in C57BL/6J mice over a 48-h-period to elevate intracellular NAD levels and consequently increase SIRT1 activity. Administration of NMN in the context of elevated plasma FFA levels considerably improved glucose tolerance. This was due not only to partial protection from FFA-induced β cell dysfunction but also, unexpectedly, to a significant decrease in insulin clearance. However, in conditions of normal FFA levels, NMN impaired glucose tolerance due to decreased β cell function. The presence of this dual action of NMN suggests caution in its proposed therapeutic use in humans.

Abstract 12268: Overexpression of Endothelial p25 Causes Vascular Remodeling by Inducing Premature Aging

Introduction: Cardiovascular diseases are the leading cause of disability and death worldwide. The cyclin-dependent kinase 5 (CDK5) signaling pathway has been implicated in the regulation of SIRT1, a mammalian longevity regulator involved in the cellular homeostasis and aging. However, the participation of p25, the regulatory subunit of CDK5 in cardiovascular diseases has not been fully understood. Hypothesis: Selective overexpression of p25 in the vascular endothelium leads to vascular remodeling by accelerating vascular aging. Methods: A transgenic mouse model with selective overexpression of p25 in endothelial cells (EC-p25) was established. Aortas were collected from 12- or 30-weeks old EC-p25 and control mice. Vascular tone response, vascular senescence, aortic stiffness and aortic wall hypertrophy were evaluated with wire myography, senescence-associated β-galactosidase (SA-β-gal), alizarin red and heamatoxylin-eosine staining respectively. Protein levels were measured by Western blotting. Primary porcine aortic endothelial cells (PAECs) were transfected with p25, Sirt1 and Sirt1 lacking deacetylase activity, cell death and senescence were evaluated. Sirt1 activity was measured in EC-p25 and Control mice. A p-value p<0.05 was considered as statistically significant. Results: Young EC-p25 mice exhibited increased vascular tone compared to the control mice. Alizarin red staining was more abundant in the aorta of 30 weeks old EC-p25 compared with the control mice, whereas SA-β-gal staining and aortic wall thickness were increased in EC-p25 mice. Overexpression of p25 in PAECs induced endothelial senescence and cell death. EC-p25 mice showed increased Sirt1 activity levels. Western blot analysis demonstrated that SIRT1 protein was fragmented in aorta of EC-p25 mice with increased levels of shorter fragments of Sirt1. PAECs transfected with Sirt1 mutant exhibited increased senescence. Conclusion: Selective overexpression of p25 in the vascular endothelium may regulate vascular remodeling by increasing vascular tone, vascular and endothelial senescence, aortic stiffness and aortic wall hypertrophy possible by modulating Sirt1 activity and integrity in the aorta of EC-p25 mice, accelerating premature vascular aging.

Telomere Length, SIRT1, and Insulin in Male Master Athletes: The Path to Healthy Longevity?

Lower SIRT1 and insulin resistance are associated with accelerated telomere shortening. This study investigated whether the lifestyle of master athletes can attenuate these age-related changes and thereby slow aging. We compared insulin, SIRT1, and telomere length in highly trained male master athletes (n=52; aged 49.9±7.2 yrs) and age-matched non-athletes (n=19; aged 47.3±8.9 yrs). This is a cross-sectional study, in which all data were collected in one visit. Overnight fasted SIRT1 and insulin levels in whole blood were assessed using commercial kits. Relative telomere length was determined in leukocytes through qPCR analyses. Master athletes had higher SIRT1, lower insulin, and longer telomere length than age-matched non-athletes (p<0.05 for all). Insulin was inversely associated with SIRT1 (r=-0.38; p=0.001). Telomere length correlated positively with SIRT1 (r=0.65; p=0.001), whereas telomere length and insulin were not correlated (r=0.03; p=0.87). In conclusion, master athletes have higher SIRT1, lower insulin, and longer telomeres than age-matched non-athletes. Furthermore, SIRT1 was negatively associated with insulin and positively associated with telomere length. These findings suggest that in this sample of middle-aged participants reduced insulin, increased SIRT1 activity, and attenuation of biological aging are connected.

Promotion of Momordica Charantia polysaccharides on neural stem cell proliferation by increasing SIRT1 activity after cerebral ischemia/reperfusion in rats

The deacetylase SIRT1 has been reported to play a critical role in regulating neurogenesis, which may be an adaptive processes contributing to recovery after stroke. Our previous work showed that the antioxidant capacity of Momordica charantia polysaccharides (MCPs) could protect against cerebral ischemia/reperfusion (I/R) after stroke. However, whether the protective effect of MCPs on I/R injury is related to neural stem cell (NSC) proliferation remains unclear. In the present study, we designed invivo and invitro experiments to elucidate the underlying mechanisms by which MCPs promote endogenous NSC proliferation during cerebral I/R. Invivo results showed that MCPs rescued the memory and learning abilities of rats after I/R damage and enhanced NSC proliferation in the rat subventricular zone (SVZ) and subgrannular zone (SGZ) during I/R. Invitro experiments demonstrated that MCPs could stimulate the proliferation of C17.2 cells under oxygen-glucose deprivation (OGD) conditions. Further studies revealed that the proliferation-promoting mechanism of MCPs relied on increasing the activity of SIRT1, decreasing the level of acetylation of β-catenin in the cytoplasm, and then triggering the translocation of β-catenin into the nucleus. These data provide experimental evidence that the up-regulation of SIRT1 activity by MCPs led to an increased cytoplasmic deacetylation of β-catenin, which promoted translocation of β-catenin to the nucleus to participate in the signaling pathway involved in NSC proliferation. The present study reveals that MCPs function as a therapeutic drug to promote stroke recovery by increasing the activity of SIRT1, decreasing the level of acetylated β-catenin, promoting the nuclear translocation of β-catenin and thereby increasing endogenous NSC proliferation.

Resveratrol Suppresses Severe Acute Pancreatitis-Induced Microcirculation Disturbance through Targeting SIRT1-FOXO1 Axis.

Background Resveratrol (RSV), one of the SIRT1 agonists, has the ability of alleviating severe acute pancreatitis (SAP); however, the concrete protective mechanism remains unknown. It is noteworthy that microcirculation disturbance plays a vital role in SAP, and the SIRT1/FOX1 axis can regulate microcirculation. Therefore, this study is aimed at ascertaining what is the underlying mechanism of the protective effect of RSV on SAP, and whether it is associated with alleviating microcirculation disturbance by regulating the SIRT1/FOX1 axis. Method The model of SAP was induced by retrograde injection of sodium taurodeoxycholate into the bile duct of the rats. The pancreatic wet/dry weight, ET/NO, and TXB2/6-keto-PGF1α ratios; microcirculatory function; and SIRT1 activity were examined. ELISA was used to examine the serum level of lipase, amylase, hemorheology, ET, NO, TXB2, and 6-keto-PGF1α and the content of SIRT1, VEGF, Ang I, and Ang II in the pancreas. RT-PCR was used to examine the mRNA level of VEGF, Ang I, and Ang II. Western blotting was used to detect SIRT1, FOXO1, and acetyl-FOXO1. Immunoprecipitation was used to examine the interaction of SIRT1 and FOXO1. Results Resveratrol can significantly decrease the expression of lipase, amylase, acetyl-FOXO1, VEGF, Ang II, ET, NO, TXB2, and 6-keto-PGF1α and the ratio of wet/dry weight, ET/NO, and TXB2/6-keto-PGF1α by improving microcirculatory dysfunction and blood viscosity in SAP. Moreover, resveratrol can also promote the interaction of SIRT1 and FOXO1 and increase SIRT1 activity and the expression of SIRT1 and Ang I. The SIRT1 inhibitor, Sirtinol (EX527), obliviously reversed the effects of RSV on SAP. Conclusion Resveratrol can protect rats against SAP, and its protective mechanism is associated with suppressing microcirculation disturbance through activating SIRT1-FOXO1 axis.

NaSH increases SIRT1 activity and autophagy flux through sulfhydration to protect SH-SY5Y cells induced by MPP~+

ABSTRACT Parkinson’s disease (PD) is one of the most prevailing aging diseases around the world. The present study was to investigate the potential effect of hydrogen sulfide (H2S) and silent mating type information regulation 2 homolog 1 (SIRT1) in MPP~+ induced SH-SY5Y cells and its underlying mechanisms in PD. SH-SY5Y cells were induced by MPP~+ and treated with the H2S donor NaHS to detect the effect of H2S on the molecular behaviors of MPP~+ induced SH-SY5Y cells. NaHS reduced the apoptosis rate and expressions of MDA, 4-HNE and p62, while increased cell viability, autophagy flux and expressions of LC3 II/I and Beclin1 in MPP~+ induced SH-SY5Y cells. Then, levels of autophagy-related proteins and inflammation-related proteins (TNF-α, IL-Iβ) were detected, indicating that Chloroquine and Sirtinol reversed the protective effect of H2S on SH-SY5Y cells induced by MPP~+. We further explored the particular function of H2S, SH-SY5Y cells treated with MPP~+, NaHS chloroquine, and SIRT1 inhibitor (Sirtinol). The results showed that H2S increased SIRT1 expression and sulfhydration. Finally, a PD mouse model verified the above results. In a word, H2S ameliorated SIRT1 activity through acceleration of SIRT1 sulfhydration to increase the autophagy flux and attenuate damage of SH-SY5Y cells induced by MPP~+. H2S and SIRT1 activator might be a target in the treatment of PD patients.

Modulation of Energy Sensing by Leucine Synergy with Natural Sirtuin Activators: Effects on Health Span.

Sirt1 and 5' adenosine monophosphate-activated protein kinase (AMPK) are energy-sensing systems that work cooperatively and regulate mitochondrial biogenesis and fuel metabolism, and mediate, in part, the salutary effects of caloric restriction on lifespan and healthspan. We have shown that leucine activates Sirt1 and enables synergy with sirtuin co-activators. Resveratrol is a widely recognized activator of Sirt1; however, poor bioavailability and rapid metabolism limit effective clinical translation of promising animal data. However, we found that combining low resveratrol doses with leucine increased skeletal muscle and adipocyte Sirt1 activity, mitochondrial biogenesis and fatty acid oxidation; these effects result in increased lifespan and marked reductions in insulin resistance, inflammatory markers, body weight, and visceral adiposity in preclinical models. To translate these data to humans, we assessed the effects of resveratrol (50 mg)/leucine (1.11 g) on glucose dynamics in a 4-week placebo-controlled trial of 36 prediabetic subjects. Leucine-resveratrol reduced insulin resistance (homeostatic model assessment for insulin resistance) 33% with corresponding reductions in glucose and insulin area under the curve in oral glucose tolerance tests. We extended these concepts in preclinical studies using both direct Sirt1 activators and Sirt1 pathway activators. Low-dose (10 nM) NAD+ precursors (nicotinic acid, nicotinamide mononucleotide, and nicotinamide riboside) synergized with leucine to increase Sirt1 activity in adipocytes, hepatocytes, and muscle cells (30-100%, P < .01) and lifespan in Caenorhabditis elegans (25%, P = .025) and to significantly regress atherosclerotic lesion size and macrophage infiltration in a mouse model of atherosclerosis. Thus, synergistic activation of Sirt1 using leucine and a co-activator exerts pleiotropic effects impacting cardiometabolic endpoints.

Sirt1 sustains female fertility by slowing age-related decline in oocyte quality required for post-fertilization embryo development.

The NAD+‐dependent sirtuin deacetylase, Sirt1, regulates key transcription factors strongly implicated in ageing and lifespan. Due to potential confounding effects secondary to loss of Sirt1 function from the soma in existing whole‐animal mutants, the in vivo role of Sirt1 in oocytes (oocyte‐Sirt1) for female fertility remains unknown. We deleted Sirt1 specifically in growing oocytes and study how loss of oocyte‐Sirt1 affects a comprehensive range of female reproductive parameters including ovarian follicular reservoir, oocyte maturation, oocyte mitochondrial abundance, oxidative stress, fertilization, embryo development and fertility during ageing. Surprisingly, eliminating this key sirtuin from growing oocytes has no effect in young females. During a 10‐month‐long breeding trial, however, we find that 50% of females lacking oocyte‐Sirt1 become prematurely sterile between 9 and 11 months of age when 100% of wild‐type females remain fertile. This is not due to an accelerated age‐related decline in oocyte numbers in the absence of oocyte‐Sirt1 but to reduced oocyte developmental competence or quality. Compromised oocyte quality does not impact in vivo oocyte maturation or fertilization but leads to increased oxidative stress in preimplantation embryos that inhibits cleavage divisions. Our data suggest that defects emerge in aged females lacking oocyte‐Sirt1 due to concurrent age‐related changes such as reduced NAD+ and sirtuin expression levels, which compromise compensatory mechanisms that can cover for Sirt1 loss in younger oocytes. In contrast to evidence that increasing Sirt1 activity delays ageing, our data provide some of the only in vivo evidence that loss of Sirt1 induces premature ageing.

2131-P: BET Bromodomain Inhibition Upregulates SIRT1 In Pancreatic ß-Cells

Diabetes develops as a result of both genetic and environmental influences; however, low concordance rates of type 1 diabetes between monozygotic twins and increased penetrance of type 2 diabetes during aging suggests an, as yet, poorly understood epigenetic role in diabetes development and progression. The epigenetic code, of which lysine acetylation (Kac) is a major histone post-translational modification, regulates the structure and dynamics of chromatin leading to epigenetically-controlled changes in gene accessibility toward transcription factors. Histone acetylation is regulated by histone acetyltransferases and deacetylases which write and erase sites of histone acetylation, respectively. SIRT1 is a key protein deacetylase in the control of glucose homeostasis. Decreased SIRT1 can lead to impaired insulin signaling, therefore drugs that enhance either SIRT1 expression or enzymatic activity may treat insulin resistance. Bromodomains are epigenetic readers that interpret the complex pattern of histone acetylation by binding Kac residues on histones and other nuclear proteins. Of particular interest are the bromodomain and extraterminal domain (BET) family of bromodomain-containing proteins, which have recently emerged as a potential target to treat or prevent diabetes. Interestingly, we recently found that treatment of β-cells with BET bromodomain inhibitors leads to increased SIRT1 expression levels, although the underlying mechanism was unclear. Here, we use an insulinoma β-cell line (INS832/13) and primary rat islets to determine the mechanism through which BET proteins suppress SIRT1 expression. To modulate SIRT1 and BET protein levels and activity, we used a combination of small molecules that either inhibit or degrade BET proteins, in conjunction with CRISPR/Cas9 knockout β-cell lines of both SIRT1 and the individual BET proteins. In summary, BET inhibitors may provide an indirect means to increase SIRT1 activity in β-cells and improve β-cell function in the context of diabetes. Disclosure R.A. Jones Lipinski: None. S. Wynia-Smith: None. J. Nord: None. B.C. Smith: None. Funding American Diabetes Association (1-18-IBS-068 to B.C.S.); National Institute of Diabetes and Digestive and Kidney Diseases (R01DK119359)

Sirtuin 1 Inhibiting Thiocyanates (S1th)-A New Class of Isotype Selective Inhibitors of NAD+ Dependent Lysine Deacetylases.

Sirtuin 1 (Sirt1) is a NAD+ dependent lysine deacetylase associated with the pathogenesis of various diseases including cancer. In many cancer types Sirt1 expression is increased and higher levels have been associated with metastasis and poor prognosis. However, it was also shown, that Sirt1 can have tumor suppressing properties and in some instances even a dual role for the same cancer type has been reported. Increased Sirt1 activity has been linked to extension of the life span of cells, respectively, organisms by promoting DNA repair processes and downregulation of tumor suppressor proteins. This may have the downside of enhancing tumor growth and metastasis. In mice embryonic fibroblasts depletion of Sirt1 was shown to decrease levels of the DNA damage sensor histone H2AX. Impairment of DNA repair mechanisms by Sirt1 can promote tumorigenesis but also lower chemoresistance toward DNA targeting therapies. Despite many biological studies, there is currently just one small molecule Sirt1 inhibitor in clinical trials. Selisistat (EX-527) reached phase III clinical trials for treatment of Huntington's Disease. New small molecule Sirt1 modulators are crucial for further investigation of the contradicting roles of Sirt1 in cancer. We tested a small library of commercially available compounds that were proposed by virtual screening and docking studies against Sirt1, 2 and 3. A thienopyrimidone featuring a phenyl thiocyanate moiety was found to selectively inhibit Sirt1 with an IC50 of 13 μM. Structural analogs lacking the thiocyanate function did not show inhibition of Sirt1 revealing this group as key for the selectivity and affinity toward Sirt1. Further analogs with higher solubility were identified through iterative docking studies and in vitro testing. The most active compounds (down to 5 μM IC50) were further studied in cells. The ratio of phosphorylated γH2AX to unmodified H2AX is lower when Sirt1 is depleted or inhibited. Our new Sirtuin 1 inhibiting thiocyanates (S1th) lead to similarly lowered γH2AX/H2AX ratios in mouse embryonic fibroblasts as Sirt1 knockout and treatment with the reference inhibitor EX-527. In addition to that we were able to show antiproliferative activity, inhibition of migration and colony forming as well as hyperacetylation of Sirt1 targets p53 and H3 by the S1th in cervical cancer cells (HeLa). These results reveal thiocyanates as a promising new class of selective Sirt1 inhibitors.

NED416, a novel synthetic Sirt1 activator, promotes cutaneous wound healing via the MAPK/Rho pathway.

Cutaneous wound healing is a highly complex biological process involving major events such as cell migration, angiogenesis, and tissue development. Sirtuin 1 (Sirt1) and its regulators have been suggested to play a role in cell migration and tissue repair. The aim of the present study was to determine the effects of a novel Sirt1 activator, the piper amide derivative (E)-3-(2,4-dichlorophenyl)-N-phenylacrylamide, also known as NED416, on cutaneous wound healing. The effects of NED416 on Sirt1 activity, Sirt1 expression, and angiogenesis were measured in skin and endothelial cells (epidermal keratinocytes, dermal fibroblasts and vascular endothelial cells) using a Sirt1 activity assay kit, western blot analysis and tube formation assays, respectively. The effects of NED416 on the rate of wound closure and collagen deposition were measured via H&E staining and Masson's trichrome staining, respectively. Levels of migration-related [Rac1, cell division cycle 42 (Cdc42) and α-p21-activated kinase] and mitogen-activated protein kinase (MAPK) signaling pathway proteins were measured in hairless mice via western blot analysis. NED416 significantly increased Sirt1 activity in dermal fibroblasts and epidermal keratinocytes to a greater extent than resveratrol, leading to increased cell migration and angiogenesis through Rac1/Cdc42 and ERK/JNK activation. Furthermore, NED416 accelerated wound closure, macrophage infiltration, and epithelium and collagen formation in vivo. The present study demonstrated a role of Sirt1 in cutaneous wound healing, and suggested that NED416 as a Sirt1 activator is more potent than resveratrol in promoting wound healing through Rac1/Cdc42 and MAPK signaling without toxicity, thus serving as a promising candidate for treatment.

Cardiac Rehabilitation Increases SIRT1 Activity and β-Hydroxybutyrate Levels and Decreases Oxidative Stress in Patients with HF with Preserved Ejection Fraction

Purpose Exercise training induces beneficial effects also by increasing levels of Sirtuin 1 (Sirt1) and β-hydroxybutyrate (βOHB). Up to date, no studies investigated the role of exercise training-based cardiac rehabilitation (ET-CR) programs on βOHB levels. Therefore, the present study is aimed at investigating whether a supervised 4-week ET-CR program was able to induce changes in Sirt1 activity and βOHB levels and to evaluate the possible relationship between such parameters, in Heart Failure with preserved Ejection Fraction (HFpEF) patients. Methods A prospective longitudinal observational study was conducted on patients consecutively admitted to the Cardiology and Cardiac Rehabilitation Units of “San Gennaro dei Poveri” Hospital in Naples, Italy. In fifty elderly patients affected by HFpEF, in NYHA II and III class, Sirt1 activity, Trolox Equivalent Antioxidant Capacity (TEAC), βOHB, and Oxidized Low-Density Lipoprotein (Ox-LDL) levels were measured before and at the end of the ET-CR program. A control group of 20 HFpEF patients was also recruited, and the same parameters were evaluated 4 weeks after the beginning of the study. Results ET-CR induced an increase of Sirt1 activity, βOHB levels, and antioxidant capacity. Moreover, it was associated with a rise in NAD+ and NAD+/NADH ratio levels and a reduction in Ox-LDL. No changes affected the controls. Conclusion The characterization of the ET-CR effects from a metabolic viewpoint might represent an important step to improve the HFpEF management.