marine drugs Article The Identification of a SIRT6 Activator from Brown Algae Fucus distichus Minna K. Rahnasto-Rilla 1,2, Padraig McLoughlin 3, Tomasz Kulikowicz 1, Maire Doyle 1, Vilhelm A. Bohr 1, Maija Lahtela-Kakkonen 2, Luigi Ferrucci 1, Maria Hayes 3 and Ruin Moaddel 1,* 1 Biomedical Research Center, National Institute on Aging, National Institutes of Health, 251 Bayview Boulevard, Baltimore, MD 21224, USA; minna.rahnasto@uef.fi (M.K.R.-R.); [email protected] (T.K.); [email protected] (M.D.); [email protected] (V.A.B.); [email protected] (L.F.) 2 School of Pharmacy, University of Eastern Finland, Kuopio FI-70210, Finland; maija.lahtela-kakkonen@uef.fi 3 Food Biosciences Department, Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland; [email protected] (P.M.); [email protected] (M.H.) * Correspondence: [email protected]; Tel.: +1-410-558-8294 Received: 22 May 2017; Accepted: 16 June 2017; Published: 21 June 2017 Abstract: Brown seaweeds contain many bioactive compounds, including polyphenols, polysaccharides, fucosterol, and fucoxantin. These compounds have several biological activities, including anti-inflammatory, hepatoprotective, anti-tumor, anti-hypertensive, and anti-diabetic activity, although in most cases their mechanisms of action are not understood. In this study, extracts generated from five brown algae (Fucus dichitus, Fucus vesiculosus (Linnaeus), Cytoseira tamariscofolia, Cytoseira nodacaulis, Alaria esculenta) were tested for their ability to activate SIRT6 resulting in H3K9 deacetylation. Three of the five macroalgal extracts caused a significant increase of H3K9 deacetylation, and the effect was most pronounced for F. dichitus. The compound responsible for this in vitro activity was identified by mass spectrometry as fucoidan. Keywords: sirtuin activators; fucoidan; brown seaweed 1. Introduction SIRT6 is an NAD+-dependent histone deacetylase (HDACs EC 3.5.1.98) that functions as a regulator of many cellular processes, is evolutionarily conserved, and exists in a variety of organisms from eukaryotes to humans [1,2]. SIRT6 controls healthy ageing by regulating genomic stability, oxidative stress, and glucose metabolism, and it is considered a promising target for age-associated diseases such as chronic inflammation, diseases associated with metabolic syndrome, obesity, and insulin resistant type-2 diabetes [3–8]. In a recent study, it was demonstrated that SIRT6 inhibition could improve glycemia in a mouse model of type 2 diabetes [9]. Also, it was shown that SIRT6-deficient mice have a premature aging phenotype with a shortened lifespan, while the overexpression of SIRT6 prolongs the lifespan in male mice and prevents diet-induced obesity [4,10]. SIRT6 activity affects the development of several cancer subtypes, but it is still unclear whether it is a tumor suppressor or promoter, or both [2,11]. Some compounds that enhance SIRT6 activity have been identified, including fatty acids and endogenous fatty acid ethanolamides [12,13]. Phenolic compounds, such as quercetin and luteolin, also enhance SIRT6 deacetylation activity, albeit at very high concentrations [13]. Brown macroalgal species [14–16], specifically Fucaceae and Cystoseira, are rich in molecules that exert a large range of biological activities including phenolic compounds such as phlorotannins, and polysaccharides, such as laminarans and fucoidans. Phlorotannins protect cells against Ultraviolet Mar. Drugs 2017, 15, 190; doi:10.3390/md15060190 www.mdpi.com/journal/marinedrugs Mar. Drugs 2017, 15, 190 2 of 12 (UV)-B-induced DNA modifications by inducing the nucleotide excision repair (NER) pathway of DNA repair [17]. Fucoidans are a complex heterogeneous group of sulfated polysaccharides composed of L-fucose and sulphate ester groups with minor amounts of monosaccharides that have robust anti-inflammatory and anti-proliferative effects [18,19]. Considering the elicited physiological actions of brown seaweeds and their overlap with the reported bioactivity of SIRT6, the aim of this work was to screen brown seaweed species for novel SIRT6 modulators as potential candidates that can be used in the prevention of age-associated diseases and metabolic syndrome associated disorders, including cancer, obesity, and insulin-resistant diabetes. We used anMar. accelerated Drugs 2017, 15, 190 solvent extraction (ASE®) method with acetone:water2 of (70:30 12 v/v)[15,19] as an extractionDNA solvent repair [17]. to generateFucoidans are phlorotannin- a complex heterogeneous and fucoidan-rich group of sulfated extracts polysaccharides from five species of brown macroalgae,composed namely of L‐fucoseFucus and sulphate distichus ester, Fucus groups vesiculouswith minor amounts, Cytoseira of monosaccharides tamariscofolia that, haveCytoseira nodacaulis, robust anti‐inflammatory and anti‐proliferative effects [18,19]. and Alaria esculentaConsidering. The the generated elicited physiological extracts actions were of testedbrown seaweeds for their and their ability overlap to with deacetylate the H3K9, a proxy measurereported of SIRT6 bioactivity activity. of SIRT6, Three the aim of of the this five work macroalgalwas to screen brown extracts seaweed significantly species for novel enhanced SIRT6 SIRT6 modulators as potential candidates that can be used in the prevention of age‐associated activity, and thediseases effect and was metabolic most syndrome pronounced associated for disorders,F. distichus including. Herein,cancer, obesity, we identifyand insulin‐ fucoidan as the compound responsibleresistant diabetes. for SIRT6 activation from F. distichus extract using liquid chromatography and We used an accelerated solvent extraction (ASE®) method with acetone:water (70:30 v/v) [15,19] mass spectrometry.as an extraction solvent to generate phlorotannin‐ and fucoidan‐rich extracts from five species of brown macroalgae, namely Fucus distichus, Fucus vesiculous, Cytoseira tamariscofolia, Cytoseira 2. Results nodacaulis, and Alaria esculenta. The generated extracts were tested for their ability to deacetylate H3K9, a proxy measure of SIRT6 activity. Three of the five macroalgal extracts significantly enhanced 2.1. Screening ofSIRT6 Brown activity, Algae and the effect was most pronounced for F. distichus. Herein, we identify fucoidan as the compound responsible for SIRT6 activation from F. distichus extract using liquid chromatography and mass spectrometry. In this study, we used a previously developed HPLC deacetylation assay that estimates SIRT6 activity by measuring2. Results changes in the level of deacetylated peptide H3K9, over the substrate (H3K9Ac) [13,2.1.20 ],Screening to determine of Brown Algae SIRT6 activity in complex matrices. Five species of brown algae were tested for SIRT6In this modulating study, we used a activity previously at developed two concentrations HPLC deacetylation (Figure assay that1 ).estimates Of these, SIRT6 the A. esculenta ASE® extract hadactivity no by activity, measuring while changesC. in nodacaulisthe level of deacetylateddisplayed peptide SIRT6 H3K9, stimulating over the substrate activity, (H3K9Ac) with a ~five-fold increase at 1 mg/mL[13,20], to compared determine SIRT6 to control.activity in complex The ASE matrices.® extracts Five species from of brownF. distichus algae were, F. tested vesiculosus for (Linnaeus), SIRT6 modulating activity at two concentrations (Figure 1). Of these, the A. esculenta ASE® extract and C. tamariscofoliahad no activity,all displayed while C. nodacaulis an approximate displayed SIRT6 ~35-foldstimulating activity, increase with ina ~five SIRT6‐fold increase activity at when assayed at a concentration1 mg/mL of compared 1 mg/mL. to control. While The the ASE® stimulation extracts from F. ofdistichus SIRT6, F. vesiculosus activity (Linnaeus), was dose-dependent and C. for all tamariscofolia all displayed an approximate ~35‐fold increase in SIRT6 activity when assayed at a species tested, concentrationF. distichus ofdisplayed 1 mg/mL. While the the strongest stimulation of activity SIRT6 activity at 0.5 was mg/mL dose‐dependent with for a all ~10-fold species increase. As a result, F. distichustested,was F. distichus studied displayed further. the strongest activity at 0.5 mg/mL with a ~10‐fold increase. As a result, F. distichus was studied further. Figure 1. SIRT6 deacetylation activity in the presence of five species of brown algae extracts. The Figure 1. SIRT6change deacetylation of SIRT6 deacetylation activity in in the the presence presence of 0.5 mg/mL of five (grey) species and 1.0 mg/mL of brown (light algaegrey) extracts extracts. The change of SIRT6 deacetylationis compared to in controls the presence with 500 μM of NAD 0.5+ and mg/mL 40 μM H3K9Ac (grey) with and 30 min 1.0 of mg/mL incubation time. (light The grey) extracts is compared to controlsdata are presented with 500 as meansµM NAD ± SD, n+ = and3. 40 µM H3K9Ac with 30 min of incubation time. The data are presented as means ± SD, n = 3. 2.2. Separation of F. distichus F. distichus was separated into eight sub-fractions (F1–F8) using an XDB-C18 column (Zorbax Eclipse) guided by the SIRT6 H3K9Ac deacetylation HPLC-based assay (Figure2). Of the Mar.Mar. DrugsDrugs 20172017,, 1515,, 190190 33 ofof 1212 2.2.Mar.2.2. Separation DrugsSeparation2017, 15 ofof, F. 190F. distichusdistichus 3 of 12 F.F. distichusdistichus waswas separatedseparated intointo eighteight subsub‐‐fractionsfractions (F1–F8)(F1–F8) usingusing anan XDBXDB‐‐C18C18 columncolumn (Zorbax(Zorbax Eclipse)eightEclipse) sub-fractions, guidedguided