SIRT2 Activity Research Articles

SIRT6 Maintains Redox Homeostasis to Promote Porcine Oocyte Maturation.

SIRT6, the sixth member of the sirtuin family proteins, has been characterized as a crucial regulator in multiple molecular pathways related to aging, including genome stability, DNA damage repair, telomere maintenance, and inflammation. However, the exact roles of SIRT6 during female germ cell development have not yet been fully determined. Here, we assessed the acquisition of meiotic competency of porcine oocytes by inhibition of SIRT6 activity. We observed that SIRT6 inhibition led to the oocyte meiotic defects by showing the impairment of polar body extrusion and cumulus cell expansion. Meanwhile, the compromised spindle/chromosome structure and actin dynamics were also present in SIRT6-inhibited oocytes. Moreover, SIRT6 inhibition resulted in the defective cytoplasmic maturation by displaying the disturbed distribution dynamics of cortical granules and their content ovastacin. Notably, we identified that transcript levels of genes related to oocyte meiosis, oxidative phosphorylation, and cellular senescence were remarkably altered in SIRT6-inhibited oocytes by transcriptome analysis and validated that the meiotic defects caused by SIRT6 inhibition might result from the excessive reactive oxygen species (ROS)-induced early apoptosis in oocytes. Taken together, our findings demonstrate that SIRT6 promotes the porcine oocyte meiotic maturation through maintaining the redox homeostasis.

Cigarette smoke-inactivated SIRT1 promotes autophagy-dependent senescence of alveolar epithelial type 2 cells to induce pulmonary fibrosis

AimsThe senescence of alveolar epithelial type 2 (AT2) cells is implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Cigarette smoke (CS) is a strong risk factor for IPF and it is also a pro-senescent factor. Here we aimed to investigate whether and how CS induces AT2 cells senescence via a SIRT1/autophagy dependent pathway. Our results showed that CS extract (CSE) reduced autophagy and mitophagy and increased mitochondrial reactive oxygen species (mitoROS) in MLE-12 cells, an AT2 cell line. The autophagy inducer rapamycin (RAPA) and the mitochondria-targeted antioxidant mitoquinone (mitoQ) inhibited CSE-related senescence and decreased mitoROS. Next, we found that CSE promoted DNA damage, downregulated the nicotinamide adenine dinucleotide (NAD+)/nicotinamide adenine dinucleotide (NADH) ratio and suppressed SIRT1 activity. Activating SIRT1 with its activator SRT1720 attenuated senescence through an autophagy-dependent pathway. The NAD+ precursor nicotinamide mononucleotide and the poly ADP-ribose polymerase (PARP1) inhibitor olaparib also exerted anti-senescent effects by activating SIRT1. Moreover, the results showed that mitoQ and RAPA, in turn, elevated SIRT1 activity by inhibiting DNA damage. Consistent with these results, SRT1720 and mitoQ mitigated CS-induced AT2 cells senescence and lung fibrosis in vivo. Moreover, autophagy in AT2 cells was rescued by SRT1720. Taken together, our results suggested that CS-induced senescence of AT2 cells was due to decreased autophagy mediated by SIRT1 inactivation, which was attributed to competitive consumption of NAD+ caused by DNA damage-induced PARP1 activation. The reduction in autophagy, in turn, decreased SIRT1 activity by promoting mitochondrial oxidative stress-related DNA damage, thereby establishing a positive feedback loop between SIRT1 and autophagy in CS-induced AT2 cells senescence. Consequently, CS-inactivated SIRT1 promoted autophagy-dependent senescence of AT2 cells to induce pulmonary fibrosis.

SL010110, a lead compound, inhibits gluconeogenesis via SIRT2-p300-mediated PEPCK1 degradation and improves glucose homeostasis in diabetic mice.

Suppression of excessive hepatic gluconeogenesis is an effective strategy for controlling hyperglycemia in type 2 diabetes (T2D). In the present study, we screened our compounds library to discover the active molecules inhibiting gluconeogenesis in primary mouse hepatocytes. We found that SL010110 (5-((4-allyl-2-methoxyphenoxy) methyl) furan-2-carboxylic acid) potently inhibited gluconeogenesis with 3 μM and 10 μM leading to a reduction of 45.5% and 67.5%, respectively. Moreover, SL010110 caused suppression of gluconeogenesis resulted from downregulating the protein level of phosphoenolpyruvate carboxykinase 1 (PEPCK1), but not from affecting the gene expressions of PEPCK, glucose-6-phosphatase, and fructose-1,6-bisphosphatase. Furthermore, SL010110 increased PEPCK1 acetylation, and promoted PEPCK1 ubiquitination and degradation. SL010110 activated p300 acetyltransferase activity in primary mouse hepatocytes. The enhanced PEPCK1 acetylation and suppressed gluconeogenesis caused by SL010110 were blocked by C646, a histone acetyltransferase p300 inhibitor, suggested that SL010110 inhibited gluconeogenesis by activating p300. SL010110 decreased NAD+/NADH ratio, inhibited SIRT2 activity, and further promoted p300 acetyltransferase activation and PEPCK1 acetylation. These effects were blocked by NMN, an NAD+ precursor, suggested that SL010110 inhibited gluconeogenesis by inhibiting SIRT2, activating p300, and subsequently promoting PEPCK1 acetylation. In type 2 diabetic ob/ob mice, single oral dose of SL010110 (100 mg/kg) suppressed gluconeogenesis accompanied by the suppressed hepatic SIRT2 activity, increased p300 activity, enhanced PEPCK1 acetylation and degradation. Chronic oral administration of SL010110 (15 or 50 mg/kg) significantly reduced the blood glucose levels in ob/ob and db/db mice. This study reveals that SL010110 is a lead compound with a distinct mechanism of suppressing gluconeogenesis via SIRT2-p300-mediated PEPCK1 degradation and potent anti-hyperglycemic activity for the treatment of T2D.

SIRT6 enhances telomerase activity to protect against DNA damage and senescence in hypertrophic ligamentum flavum cells from lumbar spinal stenosis patients.

Lumbar spinal stenosis (LSS) is a condition wherein patients exhibit age-related fibrosis, elastin-to-collagen ratio reductions, and ligamentum flavum hypertrophy. This study was designed to assess the relationship between SIRT6 and telomerase activity in hypertrophic ligamentum flavum (LFH) cells from LSS patients. We observed significant reductions in SIRT6, TPP1, and POT1 protein levels as well as increases in telomerase reverse transcriptase (TERT) levels and telomerase activity in LFH tissues relative to non- hypertrophic ligamentum flavum (LFN) tissues. When SIRT6 was overexpressed in these LFH cells, this was associated with significant increases in telomerase activity and a significant reduction in fibrosis-related protein expression. These effects were reversed, however, when telomerase activity was inactivated by hTERT knockdown in these same cells. SIRT6 overexpression was further found to reduce the frequency of senescence-associated β-galactosidase (SA-β-Gal)-positive LFH cells and to decrease p16, MMP3, and L1 mRNA levels and telomere dysfunction-induced foci (TIFs) in LFH cells. In contrast, hTERT knockdown-induced telomerase inactivation eliminated these SIRT6-dependent effects. Overall, our results indicate that SIRT6 functions as a key protective factor that prevents cellular senescence and telomere dysfunction in ligamentum flavum cells, with this effect being at least partially attributable to SIRT6-dependent telomerase activation.

The role of PARP1 in neurodegenerative diseases and aging.

Neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD), are characterized by progressive memory loss and motor impairment. Aging is a major risk factor for neurodegenerative diseases. Neurodegenerative diseases and aging often develop in an irreversible manner and cause a significant socioeconomic burden. When considering their pathogenesis, many studies usually focus on mitochondrial dysfunction and DNA damage. More recently, neuroinflammation, autophagy dysregulation, and SIRT1 inactivation were shown to be involved in the pathogenesis of neurodegenerative diseases and aging. In addition, studies uncovered the role of poly (ADP-ribose)-polymerase-1 (PARP1) in neurodegenerative diseases and aging. PARP1 links to a cluster of stress signals, including those originated by inflammation and autophagy dysregulation. In this review, we summarized the recent research progresses on PARP1 in neurodegenerative diseases and aging, with an emphasis on the relationship among PARP1, neuroinflammation, mitochondria, and autophagy. We discussed the possibilities of treating neurodegenerative diseases and aging through targeting PARP1.

"Cell Membrane Theory of Senescence" and the Role of Bioactive Lipids in Aging, and Aging Associated Diseases and Their Therapeutic Implications.

Lipids are an essential constituent of the cell membrane of which polyunsaturated fatty acids (PUFAs) are the most important component. Activation of phospholipase A2 (PLA2) induces the release of PUFAs from the cell membrane that form precursors to both pro- and ant-inflammatory bioactive lipids that participate in several cellular processes. PUFAs GLA (gamma-linolenic acid), DGLA (dihomo-GLA), AA (arachidonic acid), EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) are derived from dietary linoleic acid (LA) and alpha-linolenic acid (ALA) by the action of desaturases whose activity declines with age. Consequently, aged cells are deficient in GLA, DGLA, AA, AA, EPA and DHA and their metabolites. LA, ALA, AA, EPA and DHA can also be obtained direct from diet and their deficiency (fatty acids) may indicate malnutrition and deficiency of several minerals, trace elements and vitamins some of which are also much needed co-factors for the normal activity of desaturases. In many instances (patients) the plasma and tissue levels of GLA, DGLA, AA, EPA and DHA are low (as seen in patients with hypertension, type 2 diabetes mellitus) but they do not have deficiency of other nutrients. Hence, it is reasonable to consider that the deficiency of GLA, DGLA, AA, EPA and DHA noted in these conditions are due to the decreased activity of desaturases and elongases. PUFAs stimulate SIRT1 through protein kinase A-dependent activation of SIRT1-PGC1α complex and thus, increase rates of fatty acid oxidation and prevent lipid dysregulation associated with aging. SIRT1 activation prevents aging. Of all the SIRTs, SIRT6 is critical for intermediary metabolism and genomic stability. SIRT6-deficient mice show shortened lifespan, defects in DNA repair and have a high incidence of cancer due to oncogene activation. SIRT6 overexpression lowers LDL and triglyceride level, improves glucose tolerance, and increases lifespan of mice in addition to its anti-inflammatory effects at the transcriptional level. PUFAs and their anti-inflammatory metabolites influence the activity of SIRT6 and other SIRTs and thus, bring about their actions on metabolism, inflammation, and genome maintenance. GLA, DGLA, AA, EPA and DHA and prostaglandin E2 (PGE2), lipoxin A4 (LXA4) (pro- and anti-inflammatory metabolites of AA respectively) activate/suppress various SIRTs (SIRt1 SIRT2, SIRT3, SIRT4, SIRT5, SIRT6), PPAR-γ, PARP, p53, SREBP1, intracellular cAMP content, PKA activity and peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1-α). This implies that changes in the metabolism of bioactive lipids as a result of altered activities of desaturases, COX-2 and 5-, 12-, 15-LOX (cyclo-oxygenase and lipoxygenases respectively) may have a critical role in determining cell age and development of several aging associated diseases and genomic stability and gene and oncogene activation. Thus, methods designed to maintain homeostasis of bioactive lipids (GLA, DGLA, AA, EPA, DHA, PGE2, LXA4) may arrest aging process and associated metabolic abnormalities.

SIRT1 and SIRT2 Activity Control in Neurodegenerative Diseases.

Sirtuins are NAD+ dependent histone deacetylases (HDAC) that play a pivotal role in neuroprotection and cellular senescence. SIRT1-7 are different homologs from sirtuins. They play a prominent role in many aspects of physiology and regulate crucial proteins. Modulation of sirtuins can thus be utilized as a therapeutic target for metabolic disorders. Neurological diseases have distinct clinical manifestations but are mainly age-associated and due to loss of protein homeostasis. Sirtuins mediate several life extension pathways and brain functions that may allow therapeutic intervention for age-related diseases. There is compelling evidence to support the fact that SIRT1 and SIRT2 are shuttled between the nucleus and cytoplasm and perform context-dependent functions in neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD). In this review, we highlight the regulation of SIRT1 and SIRT2 in various neurological diseases. This study explores the various modulators that regulate the activity of SIRT1 and SIRT2, which may further assist in the treatment of neurodegenerative disease. Moreover, we analyze the structure and function of various small molecules that have potential significance in modulating sirtuins, as well as the technologies that advance the targeted therapy of neurodegenerative disease.

EGCG inhibits pressure overload-induced cardiac hypertrophy via the PSMB5/Nmnat2/SIRT6-dependent signalling pathways.

Epigallocatechin-3-gallate (EGCG), the major polyphenol found in green tea, exerts multiple protective effects against cardiovascular diseases, including cardiac hypertrophy. However, the molecular mechanism underlying its anti-hypertrophic effect has not been clarified. This study revealed that EGCG could inhibit pressure overload-induced cardiac hypertrophy by regulating the PSMB5/Nmnat2/SIRT6-dependent signalling pathway. Quantitative real-time polymerase chain reaction and western blotting were used to determine the expression of mRNA and protein respectively. A fluorometric assay kit was used to determine the activity of SIRT6, a histone deacetylase. Luciferase reporter gene assay and electrophoretic mobility shift assay were employed to measure transcriptional activity and DNA binding activity respectively. EGCG could significantly increase Nmnat2 protein expression and enzyme activity in cultured neonatal rat cardiomyocytes stimulated with angiotensin II (Ang II) and heart tissues from rats subjected to abdominal aortic constriction. Nmnat2 knockdown by RNA interference attenuated the inhibitory effect of EGCG on cardiac hypertrophy. EGCG blocked NF-κB DNA binding activity induced by Ang II, which was dependent on Nmnat2 and the subsequent SIRT6 activation. Moreover the activation of PSMB5 (20S proteasome subunit β-5, chymotrypsin-like) was required for EGCG-induced Nmnat2 protein expression. Additionally, we demonstrated that EGCG might interact with PSMB5 and inhibit the activation of the proteasome. These findings serve as the first evidence that the effect of EGCG against cardiac hypertrophy may be, at least partially, attributed to the modulation of the PSMB5/Nmnat2-dependent signalling pathway, suggesting the therapeutic potential of EGCG in the prevention and treatment of cardiac hypertrophy.

CK2 Down-Regulation Increases the Expression of Senescence-Associated Secretory Phenotype Factors through NF-κB Activation.

Senescent cells secrete pro-inflammatory factors, and a hallmark feature of senescence is senescence-associated secretory phenotype (SASP). The aim of this study is to investigate the protein kinase CK2 (CK2) effects on SASP factors expression in cellular senescence and organism aging. Here CK2 down-regulation induced the expression of SASP factors, including interleukin (IL)-1β, IL-6, and matrix metalloproteinase (MMP) 3, through the activation of nuclear factor-κB (NF-κB) signaling in MCF-7 and HCT116 cells. CK2 down-regulation-mediated SIRT1 inactivation promoted the degradation of inhibitors of NF-κB (IκB) by activating the AKT-IκB kinase (IKK) axis and increased the acetylation of lysine 310 on RelA/p65, an important site for the activity of NF-κB. kin-10 (the ortholog of CK2β) knockdown increased zmp-1, -2, and -3 (the orthologs of MMP) expression in nematodes, but AKT inhibitor triciribine and SIRT activator resveratrol significantly abrogated the increased expression of these genes. Finally, antisense inhibitors of miR-186, miR-216b, miR-337-3p, and miR-760 suppressed CK2α down-regulation, activation of the AKT-IKK-NF-κB axis, RelA/p65 acetylation, and expression of SASP genes in cells treated with lipopolysaccharide. Therefore, this study indicated that CK2 down-regulation induces the expression of SASP factors through NF-κB activation, which is mediated by both activation of the SIRT1-AKT-IKK axis and RelA/p65 acetylation, suggesting that the mixture of the four miRNA inhibitors can be used as anti-inflammatory agents.

Resveratrol Mitigates Hippocampal Tau Acetylation and Cognitive Deficit by Activation SIRT1 in Aged Rats following Anesthesia and Surgery.

Postoperative cognitive dysfunction (POCD) is a sever postsurgical neurological complication in the elderly population. As the global acceleration of population ageing, POCD is proved to be a great challenge to the present labor market and healthcare system. In the present study, our findings showed that tau acetylation mediated by SIRT1 deficiency resulted in tau hyperphosphorylation in the hippocampus of the aged POCD model and consequently contributed to cognitive impairment. Interestingly, pretreatment with resveratrol almost restored the expression of SIRT1, reduced the levels of acetylated tau and hyperphosphorylated tau in the hippocampus, and improved the cognitive performance in the behavioral tests. What is more, we observed that microglia-derived neuroinflammation resulting from SIRT1 inhibition in microglia probably aggravated the tau acetylation in cultured neurons in vitro. Our findings supported the notion that activation SIRT1 provided dually beneficial effect in the aged POCD model. Taken together, our findings provided the initial evidence that tau acetylation was associated with cognitive impairment in the aged POCD model and paved a promising avenue to prevent POCD by inhibiting tau acetylation in a SIRT1-dependent manner.

Nitro-fatty acids as activators of hSIRT6 deacetylase activity

Sirtuin 6, SIRT6, is critical for both glucose and lipid homeostasis and is involved in maintaining genomic stability under conditions of oxidative DNA damage such as those observed in age-related diseases. There is an intense search for modulators of SIRT6 activity, however, not many specific activators have been reported. Long acyl-chain fatty acids have been shown to increase the weak in vitro deacetylase activity of SIRT6 but this effect is modest at best. Herein we report that electrophilic nitro-fatty acids (nitro-oleic acid and nitro-conjugated linoleic acid) potently activate SIRT6. Binding of the nitro-fatty acid to the hydrophobic crevice of the SIRT6 active site exerted a moderate activation (2-fold at 20 μm), similar to that previously reported for non-nitrated fatty acids. However, covalent Michael adduct formation with Cys-18, a residue present at the N terminus of SIRT6 but absent from other isoforms, induced a conformational change that resulted in a much stronger activation (40-fold at 20 μm). Molecular modeling of the resulting Michael adduct suggested stabilization of the co-substrate and acyl-binding loops as a possible additional mechanism of SIRT6 activation by the nitro-fatty acid. Importantly, treatment of cells with nitro-oleic acid promoted H3K9 deacetylation, whereas oleic acid had no effect. Altogether, our results show that nitrated fatty acids can be considered a valuable tool for specific SIRT6 activation, and that SIRT6 should be considered as a molecular target for in vivo actions of these anti-inflammatory nitro-lipids.

A novel SIRT6 activator ameliorates neuroinflammation and ischemic brain injury via EZH2/FOXC1 axis

Ischemic stroke is the second leading cause of death worldwide with limited medications and neuroinflammation was recognized as a critical player in the progression of stroke, but how to control the overactive neuroinflammation is still a long-standing challenge. Here, we designed a novel SIRT6 activator MDL-811 which remarkably inhibited inflammatory response in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and primary mouse microglia, which were abolished by silencing SIRT6. RNA-seq screening identified the forkhead box C1 (Foxc1) is a key gene evoked by MDL-811 stimulation and is required for the anti-inflammatory effects of MDL-811. We found MDL-811-activated SIRT6 directly interacted with enhancer of zeste homolog 2 (EZH2) and promoted deacetylation of EZH2 which could bind to the promoter of Foxc1 and upregulate its expression to modulate inflammation. Moreover, our data demonstrated that MDL-811 not only ameliorated sickness behaviors in neuroinflammatory mice induced by LPS, but also markedly reduced the brain injury in ischemic stroke mice in addition to promoting long-term functional recovery. Importantly, MDL-811 also exhibited strong anti-inflammatory effects in human monocytes isolated from ischemic stroke patients, underlying an interesting translational perspective. Taken together, MDL-811 could be an alternative therapeutic candidate for ischemic stroke and other brain disorders associated with neuroinflammation.

Exosomal LncRNA\u2013NEAT1 derived from MIF-treated mesenchymal stem cells protected against doxorubicin-induced cardiac senescence through sponging miR-221-3p

BackgroundThe chemotherapy drug doxorubicin (Dox) is widely used for treating a variety of cancers. However, its high cardiotoxicity hampered its clinical use. Exosomes derived from stem cells showed a therapeutic effect against Dox-induced cardiomyopathy (DIC). Previous studies reported that exosomes derived from mesenchymal stem cells (MSCs) pretreated with macrophage migration inhibitory factor (MIF) (exosomeMIF) showed a cardioprotective effect through modulating long noncoding RNAs/microRNAs (lncRNAs/miRs). This study aimed to investigate the role of exosomeMIF in the treatment of DIC.ResultsExosomes were isolated from control MSCs (exosome) and MIF-pretreated MSCs (exosomeMIF). Regulatory lncRNAs activated by MIF pretreatment were explored using genomics approaches. Fluorescence-labeled exosomes were tracked in vitro by fluorescence imaging. In vivo and in vitro, miR-221-3p mimic transfection enforced miR-221-3p overexpression, and senescence-associated β-galactosidase assay was applied to test cellular senescence. Exosomal delivering LncRNA-NEAT1 induced therapeutic effect in vivo was confirmed by echocardiography. It demonstrated that exosomesMIF recovered the cardiac function and exerted the anti-senescent effect through LncRNA–NEAT1 transfer against Dox. TargetScan and luciferase assay showed that miR-221-3p targeted the Sirt2 3′-untranslated region. Silencing LncRNA–NEAT1 in MSCs, miR-221-3p overexpression or Sirt2 silencing in cardiomyocytes decreased the exosomeMIF-induced anti-senescent effect against Dox.ConclusionsThe results indicated exosomeMIF serving as a promising anti-senescent effector against Dox-induced cardiotoxicity through LncRNA–NEAT1 transfer, thus inhibiting miR-221-3p and leading to Sirt2 activation. The study proposed that exosomeMIF might have the potential to serve as a cardioprotective therapeutic agent during cancer chemotherapy.

RASSF1A Regulates Spindle Organization by Modulating Tubulin Acetylation via SIRT2 and HDAC6 in Mouse Oocytes.

Dynamic changes in microtubules during cell cycle progression are essential for spindle organization to ensure proper segregation of chromosomes. There is growing evidence that post translational modifications of tubulins are the key factors that contribute to microtubule dynamics. However, how dynamic properties of microtubules are regulated in mouse oocytes is unclear. Here, we show that tumor suppressor RASSF1A is required for tubulin acetylation by regulating SIRT2 and HDAC6 during meiotic maturation in mouse oocytes. We found that RASSF1A was localized at the spindle microtubules in mouse oocytes. Knockdown of RASSF1A perturbed meiotic progression by impairing spindle organization and chromosome alignment. Moreover, RASSF1A knockdown disrupted kinetochore-microtubule (kMT) attachment, which activated spindle assembly checkpoint and increased the incidence of aneuploidy. In addition, RASSF1A knockdown decreased tubulin acetylation by increasing SIRT2 and HDAC6 levels. Notably, defects in spindle organization and chromosome alignment after RASSF1A knockdown were rescued not only by inhibiting SIRT2 or HDAC6 activity, but also by overexpressing acetylation mimicking K40Q tubulin. Therefore, our results demonstrated that RASSF1A regulates SIRT2- and HDAC6-mediated tubulin acetylation for proper spindle organization during oocyte meiotic maturation.

Dihydromyricetin improves mitochondrial outcomes in the liver of alcohol-fed mice via the AMPK/Sirt-1/PGC-1α signaling axis.

Alcoholic liver disease (ALD), due to the multifactorial damage associated with alcohol (ethanol) consumption and metabolism, is one of the most prevalent liver diseases in the United States. The liver is the primary site of ethanol metabolism and is subsequently injured due to the production of reactive oxygen species (ROS), acetaldehyde, and metabolic stress. Building evidence suggests that dihydromyricetin (DHM), a bioactive flavonoid isolated from Hovenia dulcis, provides hepatoprotection by enhancing ethanol metabolism in the liver by maintaining hepatocellular bioenergetics, reductions of oxidative stress, and activating lipid oxidation pathways. The present study investigates the utility of DHM on hepatic mitochondrial biogenesis via activation of the AMP-activated protein kinase (AMPK)/Sirtuin (Sirt)-1/PPARG coactivator 1 (PGC)-1α signaling pathway. We utilized a forced drinking ad libitum study that chronically fed 30% ethanol to male C57BL/6J mice over 8 weeks and induced ALD pathology. We found that chronic ethanol feeding resulted in the suppression of AMPK activation and cytoplasmic Sirt-1 and mitochondrial Sirt-3 expression, effects that were reversed with daily DHM administration (5mg/kg; intraperitoneally [i.p.]). Chronic ethanol feeding also resulted in hepatic hyperacetylation of PGC-1α, which was improved with DHM administration and its mediated increase of Sirt-1 activity. Furthermore, ethanol-fed mice were found to have increased expression of mitochondrial transcription factor A (TFAM), reduced mitochondrial content as assessed by mitochondrial DNA to nuclear DNA ratios, and significantly lower levels of hepatic ATP. In contrast, DHM administration significantly increased TFAM expression, hepatic ATP concentrations, and induced mitochondrial expression of respiratory complex III and V. In total, this work demonstrates a novel mechanism of DHM that improves hepatic bioenergetics, metabolic signaling, and mitochondrial viability, thus adding to the evidence supporting the use of DHM for treatment of ALD and other metabolic disorders.

Tanshinone IIA prevents LPS-induced inflammatory responses in mice via inactivation of succinate dehydrogenase in macrophages.

Metabolic reprogramming is associated with NLRP3 inflammasome activation in activated macrophages, contributing to inflammatory responses. Tanshinone IIA (Tan-IIA) is a major constituent from Salvia miltiorrhiza Bunge, which exhibits anti-inflammatory activity. In this study, we investigated the effects of Tan-IIA on inflammation in macrophages in focus on its regulation of metabolism and redox state. In lipopolysaccharides (LPS)-stimulated mouse bone marrow-derived macrophages (BMDMs), Tan-IIA (10 μM) significantly decreased succinate-boosted IL-1β and IL-6 production, accompanied by upregulation of IL-1RA and IL-10 release via inhibiting succinate dehydrogenase (SDH). Tan-IIA concentration dependently inhibited SDH activity with an estimated IC50 of 4.47 μM in LPS-activated BMDMs. Tan-IIA decreased succinate accumulation, suppressed mitochondrial reactive oxygen species production, thus preventing hypoxia-inducible factor-1α (HIF-1α) induction. Consequently, Tan-IIA reduced glycolysis and protected the activity of Sirtuin2 (Sirt2), an NAD+-dependent protein deacetylase, by raising the ratio of NAD+/NADH in activated macrophages. The acetylation of α-tubulin was required for the assembly of NLRP3 inflammasome; Tan-IIA increased the binding of Sirt2 to α-tubulin, and thus reduced the acetylation of α-tubulin, thus impairing this process. Sirt2 knockdown or application of Sirt2 inhibitor AGK-2 (10 μM) neutralized the effects of Tan-IIA, suggesting that Tan-IIA inactivated NLRP3 inflammasome in a manner dependent on Sirt2 regulation. The anti-inflammatory effects of Tan-IIA were observed in mice subjected to LPS challenge: pre-administration of Tan-IIA (20 mg/kg, ip) significantly attenuated LPS-induced acute inflammatory responses, characterized by elevated IL-1β but reduced IL-10 levels in serum. The peritoneal macrophages isolated from the mice displayed similar metabolic regulation. In conclusion, Tan-IIA reduces HIF-1α induction via SDH inactivation, and preserves Sirt2 activity via downregulation of glycolysis, contributing to suppression of NLRP3 inflammasome activation. This study provides a new insight into the anti-inflammatory action of Tan-IIA from the respect of metabolic and redox regulation.

Nicotinamide inhibits melanoma in vitro and in vivo

BackgroundEven though new therapies are available against melanoma, novel approaches are needed to overcome resistance and high-toxicity issues. In the present study the anti-melanoma activity of Nicotinamide (NAM), the amide form of Niacin, was assessed in vitro and in vivo.MethodsHuman (A375, SK-MEL-28) and mouse (B16-F10) melanoma cell lines were used for in vitro investigations. Viability, cell-death, cell-cycle distribution, apoptosis, Nicotinamide Adenine Dinucleotide+ (NAD+), Adenosine Triphosphate (ATP), and Reactive Oxygen Species (ROS) levels were measured after NAM treatment. NAM anti-SIRT2 activity was tested in vitro; SIRT2 expression level was investigated by in silico transcriptomic analyses. Melanoma growth in vivo was measured in thirty-five C57BL/6 mice injected subcutaneously with B16-F10 melanoma cells and treated intraperitoneally with NAM. Interferon (IFN)-γ-secreting murine cells were counted with ELISPOT assay. Cytokine/chemokine plasmatic levels were measured by xMAP technology. Niacin receptors expression in human melanoma samples was also investigated by in silico transcriptomic analyses.ResultsNAM reduced up to 90% melanoma cell number and induced: i) accumulation in G1-phase (40% increase), ii) reduction in S- and G2-phase (about 50% decrease), iii) a 10-fold increase of cell-death and 2.5-fold increase of apoptosis in sub-G1 phase, iv) a significant increase of NAD+, ATP, and ROS levels, v) a strong inhibition of SIRT2 activity in vitro. NAM significantly delayed tumor growth in vivo (p ≤ 0.0005) and improved survival of melanoma-bearing mice (p ≤ 0.0001). About 3-fold increase (p ≤ 0.05) of Interferon-gamma (IFN-γ) producing cells was observed in NAM treated mice. The plasmatic expression levels of 6 cytokines (namely: Interleukin 5 (IL-5), Eotaxin, Interleukin 12 (p40) (IL12(p40)), Interleukin 3 (IL-3), Interleukin 10 (IL-10) and Regulated on Activation Normal T Expressed and Secreted (RANTES) were significantly changed in the blood of NAM treated mice, suggesting a key role of the immune response. The observed inhibitory effect of NAM on SIRT2 enzymatic activity confirmed previous evidence; we show here that SIRT2 expression is significantly increased in melanoma and inversely related to melanoma-patients survival. Finally, we show for the first time that the expression levels of Niacin receptors HCAR2 and HCAR3 is almost abolished in human melanoma samples.ConclusionNAM shows a relevant anti-melanoma activity in vitro and in vivo and is a suitable candidate for further clinical investigations.

Structural Basis for Activation of Human Sirtuin 6 by Fluvastatin.

Sirtuins are NAD+-dependent protein lysine deacylases that are considered attractive drug targets for aging-related diseases. Sirt6 deacetylates, e.g., transcription factors and histone H3, and regulates metabolic processes and stress responses. It has been implicated in lifespan extension and tumor suppression. Sirt6 deacetylase activity can be stimulated with small molecules, and fluvastatin, an FDA-approved synthetic statin, was recently described as a novel Sirt6 activator. We studied the molecular details of this effect on Sirt6 in deacylation assays and by solving a crystal structure of a Sirt6/fluvastatin complex. We find that fluvastatin inhibits Sirt1-3 at higher concentrations but has a unique, activating effect on Sirt6. The complex structure reveals that fluvastatin occupies the Sirt6 substrate acyl channel exit, similar to other, unrelated activator families, providing interaction details that will support the development of potent, druglike Sirt6 activators.

Simultaneous Inhibition of SIRT2 Deacetylase and Defatty-Acylase Activities via a PROTAC Strategy.

As a member of the sirtuin family of enzymes, SIRT2 promotes tumor growth and regulates various biological pathways through lysine deacetylation and defatty-acylation. In the past few years, many SIRT2-selective small molecule inhibitors have been developed, but none have demonstrated simultaneous inhibition of both SIRT2 activities in cells. To further scrutinize the physiological importance and significance of SIRT2 deacetylase and defatty-acylase activities, small molecules that can selectively inhibit both activities of SIRT2 in living cells are needed. Here, we have applied the Proteolysis Targeting Chimera (PROTAC) strategy and synthesized a new SIRT2 inhibitor (TM-P4-Thal) to degrade SIRT2 selectively, which led to simultaneous inhibition of its deacetylase and defatty-acylase activities in living cells. Additionally, this compound exemplifies the advantage of the PROTAC strategy that allows complete eradication of an enzyme and its activity in biological settings.

Development of activity-based probes for the protein deacylase Sirt1

Sirtuins are NAD+-dependent protein deacylases that remove acyl modifications from acyl-lysine residues, resulting in essential cellular signaling. Recognized for their role in lifespan extension, humans encode seven sirtuin isoforms (Sirt1-7), and loss of sirtuin deacylase activity is implicated in many aging-related diseases. Despite being intriguing therapeutic targets, cellular studies of sirtuins are hampered by the lack of chemical probes to measure sirtuin activity independent of sirtuin protein levels. Here, we use a modular, peptide-based approach to develop activity-based probes (ABPs) that directly measure Sirt1 activity in vitro and in cell lysates. ABPs were synthesized containing four elements: (1) thioacetyl-lysine for mechanism-based affinity towards only active sirtuins, (2) either histone H3 lysine-14 (H3K14) or p53 sequences for Sirt1 specificity, (3) a diazirine for covalent labeling upon UV irradiation, and (4) an alkyne for bioorthogonal conjugation to a fluorophore for gel-based detection of active Sirt1. Compared to the H3K14 ABP, the p53 ABP showed increased sensitivity and selective labeling of active Sirt1. Acyl-lysine peptide competition, pharmacological inhibition, and inhibitory post-translational modification of Sirt1 resulted in the loss of p53 ABP labeling both in vitro and in HEK293T cell lysates, consistent with the ABP measuring decreased Sirt1 activity. Furthermore, the p53 ABP measured subcellular Sirt1 activity in MCF7 breast cancer cells. The development of a Sirt1-selective ABP that detects Sirt1 activity with an order of magnitude increased sensitivity compared to previous approaches demonstrates the utility of a modular, peptide-based approach for selective-targeting of the sirtuin protein family and provides a framework for further development of sirtuin-selective chemical probes.