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    Lemon Verbena (Lippia Citriodora) Polyphenols Alleviate Obesity-Related

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    Lemon Verbena (Lippia Citriodora) Polyphenols Alleviate Obesity-Related

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    Lemon verbena (Lippia citriodora) polyphenols alleviate obesity-related

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    Lemon Verbena (Lippia Citriodora) Polyphenols Alleviate Obesity-Related

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    Phycamediine 222015) 605-614 Ear) Contents lists available at ScienceDirect Phytomedicine ELSEVIER journal homepage: www.elsevier.conviocate/shymed Lemon verbena (Lippia citriodora) polyphenols alleviate obesity-related disturbances in hypertrophic adipocytes through AMPK-dependent mechanisms Qo Maria Herranz-L6pez’, Enrique Barrajén-Catalén*, Antonio Segura-Carretero”, Javier A. Menéndez‘, Jorge Joven“, Vicente Micol**''* insta de Bla Melee y Cer BHC} Uivesided Miguel Herne. ee, cae, Spin "Deparment fae Cherry Faulty af Scenes Unvery of Granda rood Spain “iaetabtem Concer ra. Translate esorehebetry,catln Pete of Oncology an mec! eras ne, Giana. Spa na de Recerca ome, Hepa Unies de Santon, SPV rivers Soe Vr) Santa gn. 43201 Res, pa ER (C829030038, Fonte In Obes a urn, BERD Itt de Sa Carle I, pai ARTICLE INFO ABSTRACT Background: There is growing evidence that natural products, mostly plant-derived polyphenols, are impor- {ant in the relationship between nutrients and health in humans Parpose: We amid wo investigate i verbascoside (VB) ad other lemon verbena polyphenols could ameliorate obesiy-induced metabolic disturbances, a wells their putative mechanism Study design: We used an insulin-resistant hypertrophic 3T3-LI-adipocyte mode cst the effect of VB oF Teyworts lemon verbena extract on triglyceride accumlation, inflammation and oxidative stress and a murine model Verbscosde of detinduced obesity to assess the in vv metabolic response, pte tnedora Results: Polyphenols decreased wiglyceride accumblation, the generation of reactive oxygen species (ROS) Aaipoete and restored mitochondrial membrane potential in adipocytes. The underlying mechanisms seemed 19 o¢cur Aapenecin ‘a ROS-mediated downregulation of nuclear factor kappa-B transcription factor (NF-e3) and peroxisome Diliferator-atvated receptor gama (PPAR-y pendent transcriptional upregulation of adiponectin, We also observed apotent activation of AMP-activated protein kinase (AMPK) the mRNA expression upregulation fof PPAR and the mRNA expression dvencegulation of ty ac synthase. Experiments in mice suggested 2 significant improvement in fat metabolism. Conclusion: Decreased lipogenesis, enhanced fatty acid oxidation and the activation ofthe energy sensor Ft metablim| ‘AMPK. probably throug activating transeriptional factors, are involved in the observed ben lan those observed witht is proposed. The polypharmacologica effect of plant effects were ess potent al eects. VE act sba potential synergistic, mult-argeted action rived polyphenals from lemon verbena may have ‘he potential for clinical apliations in obesity, 1© 2015 Elsevier GmbH Alright reserve, ‘Abbrevatin IC Lipps civadora eta: VB, verbascorid; FS, fetal bovine sero: IX, rb -meeieanbine: DEX dexamethasone Noe sles terkappatignchain ance efactvated cel AMPK aéenosine monophosphate scvseg protein nave: C2, chemin [C-Cmatil gan TN tomar nero factorigas nas ite alpha HoIf neki] el nein *Cotesponding author at insta de Belg Melua y Cllr, Uvessidad Miguel ierdnden Avda dela UnivesiadSN.03202 Eee, lease, Spin Tel: $34 ‘6858430. ax 13496 6558758. mal edéesvicleume ioe cente@gmaior(V Mic, upd or 10 1016jphymed 201503015 Introduction In recent times, the close relationship between lifestyle, diet and the risk of major human diseases is becoming more evident, Un- fortunately, while the prevalence of obesity has increased, current remedies or pharmaceutical drugs to fight obesity are of limited ef- fectiveness and changes in lifestyle are difficult to accomplish (Burke ‘and Wang 2011). The increase in dietary polyphenols supposes a po- {ential alternative but mechanisms require elucidation because they usually hit multipe targets. Dietary energy excess causes lipid accu- ‘mulation in adipocytes and other cell resulting in obesity, metabolic stress and low-grade chronic inflammation, which tends to perpet- uate an imbalance between metabolic and immune cells (Gustalson etal 2009; Kwon and Pessin 2013; Snel etal, 2012) 0s 1M Herne-ipe el Prycomedine 222015) 605-514 Nuclear factor kappa-light-chain-enhancer of activated B cells, (NFHeB) is a critical regulator of several genes that ate involved in immune and inflammation responses. NF-«B activity is increased in high glucose-induced hypertrophic adipocytes, leading to a pro- inflammatory state resembling the effect of nutritional overload and low physical activity (Baker et al, 2011; Han et al. 2007). In this scenario, the CCL2 (chemokine [C-C motif] ligand 2), through its functional receptor (CCR2) induces monocyte recruitment, inlam- ‘mation and metabolic stress (over et al. 2012b; Rull etal. 2010). As a result there is a cellular decrease in the activity of S/-adenosine ‘monophosphate-activated protein kinase (AMPK), an energy sen- sor involved in the survival of affected cells (Joven et al. 20120, “Menendez et al, 2013), Interestingly, AMPK signaling inhibits the in- ammatory responses induced by the NF-«B system in adipocytes (Salminen et al. 2011), an effect that is most likely mediated by adiponectin (Hattori etal, 2008; Joven etal, 2012), n obese state, a release of free fatty acids (FFAs) into the circulation takes place in hypertrophic adipocytes leading to systemic effects, ie. ectopic FFAs accumulation and FA-induced insulin resistance in tissues such as muscle and liver (Rezzee 2013), In this process, the role of pes- ‘oxisome proliferator-activated receptors (PPARs) have been also re- ported (Okada-Iwabu etal. 2013). In this scenario, we have previously suggested that polyphenols, ‘modulate triglyceride accumulation, oxidative stress and inflamma- tion in both humans and murine models (Beltran-Debon et al. 2010, Herranz-Lopez et al, 2012; Joven et al. 2012a; Joven et al, 20126), ‘These effects were observed using complex Hibiscus sabdarifa ex- tracts containing anthocyanins, glycosylated flavonols and organic acids, Nevertheless, bioguided ‘fractionation studies and immuno- histochemical detection evidenced that flavonols (quercetin deriva- tives) appeared to be the best candidates mediating these beneficial effects, although with evident synergistic interactions (Fernancez- Arroyo et al. 2012; Herranz-Loper et al. 2012: Joven et al. 20122), “These effects were observed using Hibiscus sabdarifa (Malvaceae) extractsin which elucidating therapeutic mechanismsis achallenging task Similar anti-in flammatory and radical scavenging activities and beneficial effects have also been observed with extracts from lemon verbena (Lippia ciriodora Palau) Kunth (Verbenaceae) (LC) or Aloysia triphylla) (U'Hér.) Britton (Verbenaceae). Lemon verbena leaves are ‘widely used as a species to add lemony flavor in food and also used to make herbal teas and refreshing sotbets (Funes et al, 2008). In the last decade, the potential of lemon verbena extract supplemen- tation as a nutraceutical to decrease muscular damage, blood oxida- tive stress and proinflammatory cytokines in sport and joint health has been explored (Carrera-Quintanar et al. 2014; Carrera-Quintanar et al. 2012; Caturla et al 2011; Funes et al. 2011; Quirantes-Pine et al, 2013), LC leaves ae rich in phenylpropanoids, glucuronidated flavonoids and ridoid glycosides Supplementary Table 1) (Quirantes- Pine etl. 2013; Funes et al, 2009). Verbascoside (VB) (Supplementary Fig. 1), phenylpropanoid glycoside isthe most abundant compound in this plant (Funes etal. 2008), The antioxidant, antiinflammatory and chemopreventive activity of verbascoside, its biotechnologi- ‘al production, occurrence and uses have been recently reviewed (lipieva etal. 2014), We then reasoned that the effects of the extract could be simplified by the use of vesbascoside. We hypothesized that we could demonstrate in a single polyphenol an inherent poten- {ial to exert polypharmacological effects other than redox mod- tlation. The potential of polyphenols to interact and modulate different proteins would demonstrate the simultaneous modul3- tion of inflammation and energy-related pathways. thereby cor- roborating their multitargeted character. We present here, for the first time, that VB and associated polyphenols hit different ‘molecular targets, which were beneficial in high glucose-induced insulin-resistant hypertrophic adipocytes and in a murine model of hyperlipidemia, Materials and methods Chemicals and reagents Dexamethasone (DEX), 3-isobutyl-I-methylxanthine (1BMX) insulin, crystal violet, paraformaldehyde solution and 2:7 dichlorodihydrofluorescein diacetate (H2DCP-DA) were obtained from Sigma-Aldrich (Madrid, Spain), Dilbecco's modified Eagle's ‘medium was purchased from Gibco (Grand Island, NY, USA), Polyvinyldif_uoride (PVD) filters (0.22 um) were obtained from Mil lipore (Bectord, MA. USA). AdipoRed™ Assay Reagent was obtained vom Lanza (Walkersville, MD, USA). Pure, isolated VB was obtained through preparative HPLC, as previously reported, from a previously characterized lemon verbena aqueous extract (Funes et al. 2010), UC extract (27% VB, wiv, as determined by HPLC, Supplementary ‘Table 1) was kindly provided by Monteloeder, St. (Elehe, Spain). The extract was freshly prepared before use, dissolved in culture media and filtered Cellular experimental model and measurement of intracellular reactive ‘oxygen species (ROS) The 3T3-L1 preadipocytes were purchased from the American Type Culture Collection (Manassas. VA, USA), propagated and dif ferentiated according to previously described procedures (Green and Kehinde 1975), Adipocyte eifferentiation was induced by adding adi pogenic agents (0.5 mM IBMX. 1M DEX, and 1 MINS) to the cul- {ure medium for 2 days. The medium was freshly replaced every 48h The phenotypic change of adipogenesis was observed under a micro- scope. In all experiments, more than 90% of the cells were mature adipocytes after 8-10 days of incubation. To induce cellular hyper trophy, adipocytes were exposed to high glucose (25 mM) for at least 18 days (Yeop Han et al, 2010) Purther details on this cellular model are provided in Supplementary Fig. 2, indicating the accumulation of intracellular lipid droplets ane the effect of glucose supplementation, during the transformation process. Differential effects on mature or hypertrophic adipocytes were assayed by adding LC or VB for 48 h, in pre-designed concentrations tothe media, The absence of cytotoxicity ‘was ascertained using the crystal violet method, ROS generation was assessed in hypertrophic adipocytes using 2,,'-dichlorodihydro-fluorescein diacetate (H;DCF-DA) as described ((feop Han etal 2010). Fluorescent microphatographs were captured. Via fluorescent microscopy (Eclipse TE2000-U, Nikon Microscope. Melville, NY), Western blot analysis High glucose-induced hypertrophic adipocytes were cultured for the indicated times and treated for 48 h with various concentrations ‘of LC and VB, After incubation, cell extracts were analyzed by West- ern blot. Hypertrophic adipocyte were lysed with ice-cold lysis buffer (20 mW Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% CHAPS, 1 mM Pe- fabloc and 1% phosphatase inhibitor cocktail no. 2, Sigma-Aldrich Inc, Steinheim, Germany), Protein concentrations were determined by 4 NanoDrop spectrophotometer (NanoDrop Technologies, Wilm- ington, DE). Electrophoresis was performed in NuPAGE 4-12% Bis Tris gradient or 4-20% Tris-glycine polyacrylamide gels (Invitrogen, Barcelona, Spain), MES was used for AMPK, pAMPK, PPAR-a and actin electrophoresis, while Tris-Cly buffer (Invitrogen) was used for FASN electrophoresis. Proteins were transferred to nitrocelhulose mem- branes using the iBlot transfer system (Invitrogen). Antibodies used ‘were Rabbit anti-AMPK (#2532, Cell Signaling Tech, Danvers, MA, USA), rabbit anti-pAMPK (Thr172) (#2531, Cell Signaling Tech), rab- Dit anti-PPAR-a (H-98, St. Cruz Biotech, Heidelberg, Getmany), rabbit nti-FASN (3180, Cell Signaling Tech, and rabbit ant+-actin(H-300, St 1M eron-Lipe ea Pytomedine 2 (205) 65-614 on (A) % Tateeride eeges >» » » w Ze ‘ow glucose ih acoso LC (400g!) VB (108m) (B) > i Low aucose ih cose LC (400g!) VB (108m) (c) 3 Low geass LC0 int 16250 gi mi foes Fa High oboe VB 135 yom VBErSigm VB 108)9m1 Fig. 1. Leman verbena polyphenols decease the gyerde acum ad the HOS generat i hgh-lacose mute (A) ot byperophic (BC) adipocytes. Representative Dlotomicogtapis of tacellar pds, as assessed by Adee stain, and acl Measurements campated wh hose abused i cos, i peccenlge ate cepted Inuacella RO generation (: xidaton) was assessed by using the HOS sense otescent probe HjDCF-DA The data ae expressed asthe ean LD. em hie ineependent ‘experiments prferined i ezulate Data pons atthe abscissa aus cana equaeat VS ences fo the exatt and the pute conpeusé LG Lipa ceca versed, (©) rashid Ee ee C) wee DOSS SS ACN ee AMP Soe tow gcse 3" al oe it igh glucose 2 e § os mi Fas ao : 2. ol MMB win sella - a Tt ee To ae ao Se ig. 2. 1 and VB decrease FASN and stimulate FPAKr MENA expression, activating ANDK i igh uceseinguceé hyperuope adipocytes. Hypesiopicalpeyts were ‘ured in media contig LC or VB for 48h The crude call extats weve ued for Westen lot analy (15 gane} Bund intents were taesuted ing nage Lab Software Dain the gaph ae pressed asthe mean 25D. (t= 3} os M Herne-ipe el Prycomedine 2 2015) 605-514 Cruz Biotech). The secondary antibodies were goat anti-rabbit-HRP (Dako, Glostrup, Denmark) and anti-goat-HRP (Dako). Chemilumines- cent detection was performed sing the ECL Advance Western Blot- {ing Detection kit (Amersham, GE Healthcare, Barcelona, Spain}. and membranes were analyzed in a ChemuiDoc system (Bio-Rad, Spain}, Immune-reactive protein levels were quantified by band densicom- tty normalized to -actin signal using software Image Lab (Version 430 build 11, Bio-Rad, Madrid, Spain), Immmunoftuorescence study For NF-cB and adiponectin detection, fixed cells were incubated overnight with each antibody, .e.a polyclonal anti-RelA/p85 (Thermo Fisher Scientific Inc) or monoclonal anti-adiponectin (Abcam Inc, USA). Cellswere then washed with P8S and incubated for2 hwith each, corresponding secondary antibody, Anti-Rabbit IgG-TRITC (Sigma) and Anti-Mouse Polyvalent Immunoglobulins (G.AM)-FTTC. Stained cells were photographed with an inverted fluorescence microscope (Nikon Eclipse TE2O00-U; Nikon Instruments, Ine, NY) provided with a digital camera (Nikon DS-10M), and fluorescence was mea- sured by Muorimeter in a multiwel plate reader (POLARstar Omega microplate). Gene expression assays ‘The expression of selected genes was measured by quantitative real-time PCR analysts (QKT-PCR) of DNA samples as previously re- ported (Joven et al. 2012a), Gene and primer information is shown in Supplementary Table 2. Total RNA was putified and converted to cDNA using RNeasy (Qiagen, Valencia, CA, USA) and Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen, Carlsbae, CA, USA) following the manufacturers instructions. The expression levels were measuted by real-time RT-PCR and results are expressed as fold change using B-actin as housekeeping gene. Plasmids and promoter analysis ofthe human adiponectin gene ‘The luciferase reporter construct driven by the S'-flanking region of human adiponectin gene {p(-808)/LUC wt] and a mutated con- struct [p(-S08)/LUC PPRE mut) containing point mutations from GG (0 AA at ~276[-275 in the PPAR response element (FPRE). prepared as previously described (Iwaki etal. 2003), were kindly provided by the Osaka University (Japan). The transfection was performed int ma- ture adipocytes after 5 days of differentiation using the Neon trans- fection system from Invitrogen according to the manufacturer's in- structions, Following electroporation, the mature adipocytes were incubated with either LC or VB for 24 h, after which the luciferase activities were assayed using a Luciferase Assay System (Promega, Madison. WI, USA). Non-transfected cells exhibited similar luciferase signal than controls transfected withthe empty construct pGL3-basic (data not shown) Fluorescence detection of mitochondrial membrane potential To evaluate the mitochondrial membrane potential, high glucose- induced hypertrophic adipocytes were labeled with 100 nM Mito- ‘Tracker Red CMXRos (Mred) and MitoTracker Green FM (Mgreen) (Molecular Probes, Invitrogen, Carlsbad, CA, USA) for 30 min at 437 °C and washed three times with pre-warmed PBS: images being captured as mentioned above ‘Animal experimental model IDL receptor-deficient male mice in a CS7BL/6) background (LoLr!-) were the progeny of animals obtained from the Jackson Laboratory. The animal handling, sample preparation, sampling, sac rifice and the calculation of sample size were performed as described (oven et al, 2007). At 10 weeks of age, the animals (n = 16) with equivalent body weight were assigned to two study groups (n = 8 each) and were fed a high-fat, high-cholesterol diet (HF, 20% fat and (0.25% cholesterol, w/w), One of these treatment groups received tap deionized water asa unique liguid source (control, while the other {group received the LC extract dissolved in tap deionized water(5 gf), which was freshly prepared every day. Although water andjor ex tract were administered ad libitum, LC extract solution intake was measured and average daily dose was estimated for treated animals ‘obtaining a value of 750 mg/kg bw. (202.5 mgikg b.w. of VB). Blood and tissue samples were obtained ana processed as previously de- sctibed (Beltrin-DebGn et al. 2011). The ora fat test and the glucose tolerance test were performed as described elsewhere (Kull et al 2007), Liver steatosis was qualitatively evaluated on a scale of 0-3, ‘here 0 represented an absence of steatosis and 3 indicated a major arade of steatosis (=65%). All procedures in LDL receptor-dleficient ‘mice and experimental protocols were examined and approved by the Ethical Committee for Animal Experimentation of the University ‘Miguel Hernander.(1BM-VMM-003-12), Statistical anatyses ‘Values are represented as the mean + standard deviation (S.D.) of, ‘the mean. The values were subjected to statistical analysis (one-way ANOVA. Student's -test for unpaited samples and Tukey's test for ‘multiple comparisons), The differences were considered statistically significant at p < 0.05 All analyses were pesformed using GraphPad. Prism software (GraphPad, San Diego, CA). p = 0.05, "*p = 0.01 and **p-<0001 onbarsindicate statistically significant ciflerences versus control, unless otherwise stated. Horizontal lines indicat statistically, significant differences between bats, All cellular measurements de- rive from three independent experiments, wherein each performed in octuplicates, unless specified. Results {Cand VB decreas lipid deposition and ROS generation in mature and hypertrophic adipocytes: a possible role for mechanisms involved in Tipogenesis and mitochondrial dysfunction ‘To explore the effects of LC or VB on adipacyte model, we consid ered either mature orhypertrophicadipocytes thaseinthe 8thor 18th day after the start of differentiation respectively. Polyphenols were added at increasing concentrations in these time-points and cells ‘were further incubated for two additional days. In mature adipocytes incubated with high glucose levels, there was a dose-lependent,sim- ilar decrease (20%) in trglyceride content withthe maximum concen- tration of LC extract and VB utilized (400 ugiml LC and 108 ygiml VB, contained equivalent verbascoside concentrations; Fig, 1A) The effect was also similar in high ghicose-induced hypertrophic adipocytes: 31.6% and 29% respectively (Fig. 1B) Similarly, both LC extract and VB decreased intracellular ROS generation (approximately 40%) in hgh glucose-induced hypertrophic adipocytes. Consequently, subse- {quent analyses were limited to hypertrophic adipocytes. To further elucidate the molecular mechanism involved in the suppressive ef= fect of LC and VB on lipid accumulation, we investigated the effects of LC and VB on the mRNA expression and protein levels of FPAR- «2, a major regulator of lipid metabolism fatty acid synthase (FASN}, lipogenic-related gene, and the central metabolic sensor AMPK. After 48 h of incubation of high glucose-induced hypertrophic adipocytes in the presence of LC or VB, we found an increase in PPAR-c, a de- ‘crease in FASN and a significant activation of AMPK (Fig. 2). Significant diflerences were detected between LC and VB at equivalent VB con- centrations, revealing a higher effect in the presence of LC extract. 1M eron-Lipe ea Pytomedine 2 (205) 65-14 co Phase contrast Mgreen Low sluease hic adipocytes MitoTracker CMMs Med} and Green (Maren) were use ‘Also, and to determine the effect of LC or Vi on intracellular KOS gen- ‘ration and their impact on mitochondrial function, we analyzed the accumlation of MitoTracker CMXRos (Mred), which is dependent ‘on the mitochondrial membrane potential, and MitoTracker Green (Mgreen), a general mitochondrial marker (Fig. 3). Huorescence mi ‘rographs show that high glucose-induced hypertrophic adipocytes revealed a dramatic loss of Mred staining without the loss of Mgreen staining, indicating a decrease in mitochondrial membrane potential which was partially restored by both IC and VB treatments, reveal. ing a possible mechanism to explain the decrease in intracelfilar ROS generation (Fig. 1C) 1C and VB upregulate adiponectin gene expression but only LC downregulates NF-«B in hypertrophic adipocytes ‘We then explored the effects of polyphenols on NF-xB, as a reg- Uulator of the oxidative stress-induced inflammatory response and ‘on adiponectin, a perceived antiinflammatory cytokine (Fig, 4). For this purpose, hypertrophic adipocytes were incubated with LC or VB for 48 h. Under these experimental conditions, the LC extract significantly upregulated the adiponectin gene expression as com- pated to the equivalent concentration of purified VB. Although LC and VB treatments showed small but significant decreases in NE-«8 _gene expression compared ro the contol, there were no differences Detween their capacities to downregulate NF-«B gene expression ig 4A) When protein levels were quantitated, both LC and VB showed. dose-dependent significant nereases in adiponectin expression com- pated to the control and also, a cifferentil effect of the LC extract and VB was confirmed on adiponectin expression. However, only LC ‘exhibited a significant effect on NEB expression levels (Fig, 4B) Cellular assays using immunofluorescence resulted in similar values (Fig. 4C and D), These eflects on the anti-inlammatory action were further confirmed when we measured the gene expression of se- lected cytokines in the same experimental cell. We found significant reductions with both. extract and purified compound. with respect to controls of IL-1, IL-6, TNF-a and CCL2/MCP-1 but there were no changes in IL-1a and leptin expression levels (Supplementary Fis. 3). To clarify whether the effect of LC or VB on the transcriptional ‘upregulation of adiponectin was mediated by PPAR-y , a critical tran- scription factor involved in adiponectin expression (Pouskila et al 2008; Iwaki etal. 2003), we transfected the adipocytes with difer- ent constructs containing either the wild type or mutated adiponectin luciferase promoter. Luciferase activity driven by the adiponectin pro- ‘moter in transfected adipocytes incubated inthe presence or absence of either LCor VB was determined, Atthe highest concentrations, cells treated with either LC or VB showed a significant dose-dependent increase in basal luciferase activity ofthe wild-type adiponectin pro- ‘moter [p{-908)/LUC wt] as compared to controls (Fi, 5A), When we used the human adiponectin promoter bearing the mutated PPAR response element [p(-808)/1UC PPRE mut] there were no apprecia- ble changes in luciferase activity indicating a possible major role for PPAR-y in the activation of adiponectin promoter (Fig. 5B) Lemon verbena polyphenols alleviate diet-induced obesity in an ‘animal model To reinforce our interpretation we decided to test the influence of polyphenols in an animal model. When fed a HE diet, LDLr de- ficient mice, consistently develop obesity-associated metabolic dis- turbances and hypertrophic adipocytes (Beltn-Debén et al. 2011 Joven et al. 20123). The effects of LC extract rather than purified VB 7 Sea 2 il h H BY Lc 400 High glucose VB 108 Fig. Theinfuence of LC and VB onthe gene expression and Nevertheless a sight decrease on NFB gene expression ws observed by LC oc V8 1M Herane-tipe el Pycomedcine 22 2015) 605-514 (B) "rom oo |] (0) High glucose LC 400 ‘VB 108 sin eves of algenectin and NF-H lucte induced hyperophi adipocyes were nite with 200 mn The ex stony upeguated the aipeaecn gee expression comnpated 1 VB. When uvesced protein levels however, the exact but net VB, showed sgncat tect in oth protels(),Photemicrograph bain using an nnunetuoescence cella sry centimed there eu for bth adgenechn (ane Nec (0) respective Data the graph ate expressed asthe meas 5D.(0=3) “ ©) 2 pGis-basic am OD pots-basic z fa S080 ws fm peo0eytucmt Eas mt 1s mi : . Ae Bw Fg 5. IC uprerulates PPAR -mecatedaeiponecin the naman aipenetin promoter [9-08 wt ing the save promt were analyzed, due to logistic reasons, in combination with high- fat and high-cholesterol diet during 14 weeks. During this time, the LC extract prevented the expected gain in body weight as compared to placebo-treated controls (Supplementary Fig. 4). Despite food in- {ake was similar in both groups, there were consistent decreases in the weight of most organs and tissues (Supplementary information ‘Table 3), Notably, LC consumption markedly decreased epididymal and inguinal white adipose tissue: the difference being ~38% be- tween groups (p = 0.005) The amount of brown adipose tissue was essentially the same in both groups and we found no differences in insulin resistance as assessed by glucose tolerance tests (Fig. 6A). In contrast, beneficial effects were observed in lipid metabolism, ‘There was a significant reduction ofthe triglyceride values at 60 and 120 min (mgidl} after the ingestion ofan oral olive il bolus associated with LC extract consumption indicating that treatment induced im- proved triglyceride clearance (Fig. 6B). Also, treated animals depict ression Matte ainectes were transfected wih berate constr lctng with the PPAR espose element mated [p-908) PERE at] (8). AEE 24 he or VB andthe fferse ates mere eased. The teat expressed ashe mean = SD. {n— 8). im remote (pGL3-base containing significantly lower serum cholesterol and wiglycerides concentra tions than the controls (Fig. 6C and D). Among obesity-associated. disturbances in this animal model, liver steatosis isa constant find- ‘ng. The administration of the LC extract alleviated the accumulation, ‘of neutral lipids and trigiycerides inthe liver without altering serum. biomarkers of hepatic toxicity (Fig. 6E-C). Discussion ‘The 373-L1 cell line is a well characterized and widely accepted ‘model of n vitro adipogenesis and lipid accumulation that becomes hypertrophic and insulin resistant when induced with high-glucose conditions (Green and Kehinde 1975; Han etal. 2007; Herranz-Lope7 etal, 2012; J etal. 2014: Yoshizaki etal, 2012; Zebisch etal, 2012). In this model, we have assayed the capacity of LC and VB to ameliorate obesity-induced metabolic disturbances ernie a Pytomedine 2 (205) 605 ) co) ° Seton core (c) () e we a Baa shes) i i i al. ° cholesterol ‘Triglycerides Fy () . mac Bilirubin ast ig-6. Lemon verbena plypherls improve trihcerie clearance and prevent the velopment of ative iseaseinhyperipidemic mie LCsupplemesation not modity lose tolerance (A) but icant eeceased the tgheene ves t 60 and 120 ein mg) ae the ingestion ofa ofl Neo us (Band decreased cholesterol nd {seer plasma levels sn Hea mice (Cand D) The ver sete iduceé by the HE-et was ignicanty decrease by Uc poyphena supplement (woot ecg ‘he serum bomarhss of erin The representative stele setons strate the preventive eet "p= 0.0 denotes the signin ferences ecween the Fa HE 1c groups Polyphenols are the most intensively studied natural products as a recognized source of pharmacologic compounds. Several lines ‘of evidence suggest a significant impact of polyphenols in obesity. ‘which is an increasingly prevalent condition. Ie is commonly ac cepted that adipocyte hypertrophy compromises cell function in the progression of obesity associated metabolic disturbances and is as sociated to ROS generation and inflammation (Han et al. 2007), in agreement fo our results. Despite the limitations of a cellular model (Green and Kehinde 1975: ji et al. 2014: Zebisch et al, 2012), we found that LC polyphenolic extract and its major compound, VB, pre= vented most of the expected deleterious effects. These results are also partially confirmed in an animal model of diet-induced obesity. \VBis the most abundant (>25%) polyphenol in LC aqueous extracts. Both have similar effects, decreasing lipid deposition in adipocytes and the consequent intracellular ROS generation. Contrarily the anti- inflammatory action differs, indicating a potential synergistic effect in the extract, ‘A.common diet contains ~500 different polyphenols: extracts re- duce the number but they are also a complex mixture and whether this complexity is relevant to our health remains an elusive point. In this study we have exposed cell lines to an individual polyphenol, VE, and responses are similar to those obtained with the LC extract. We ‘obviously recognize the inherent limitation in using high doses and non-metabolized polyphenols but we consider that VB may be added to the growing list of isolated polyphenols with a potential impact ‘on health. This is important to facilitate the identification of promis ing functional elements in polyphenols. We have previously found the strong free radical scavenging capacity of LC polyphenols and their ability to enliance the activity of antioxidant enaymes (Carrera ‘Quintanar etal, 2012; Funes etal, 2011; Funes et al, 2009), Despite the popular antioxidant hypothesis there is, however, no evidence that the antioxidant properties of polyphenols improve the antioxidant function in the cel (et al. 2014), In this study we identify some previously unrecognized mecha- istic clues. Particularly, these polyphenols maintain mitochondrial ‘membrane potential and mitochondeial viability. These findings in- dicate that further consideration should be paid to other actions of polyphenols that may be extended to the direct modulation of mi tochondrial events affecting the whole cell (eg. energy generation, ‘mitochondrial biogenesis o cell death control; Chung etal 2010), We have considered a variety of molecular targets related to metabolic stress inadipocytes. For example, the activation ofthe redox-sensitive transcription factors such as NF-«B is accepted as a deleterious ef- fect in the pathogenesis of common diseases via the modulation of a large number of genes mediating immune and inflammatory re- sponses (Jiang etal 2011; Li and Karin 1999). Our results reveal that both LC and VB decreased NF-xB and increased adiponectin gene ex- pression, Curiously, only the LC extract decreased the NF-B protein levels but the increase in adiponectin protein levels was observed ‘with both the extract and the individual polyphenols, Moreover. the adiponectin expression in hypertrophic adipocytes was mediated by 8 transcriptional PPAR-y-dependent mechanism. Further, the anti inflammatory action of adiponectin was accompanied by the down- Fegulation of selected inflammatory genes and a significant activation ‘of AMPK in hypertrophic adipocytes. Adipanectin has been proposed as a systemic functional link involved in the activation of AMPK in diferent tissues Hattori et al. 2008; Iwabu et al. 2010; Okada-Iwabu etal, 2013), probably through different actions of adiponectin recep- tor (AdipoR) (Fang etal. 2010). This is important because the ability of polyphenols to act on both, adiponectin and AMPK. may represent important regulators of glucose and lipid metabolism, modulators of inflammation, oxidative stress and insulin resistance (lard et al. 2012; Salminen and Kearnicanta 2012) and consequently a therapeu- ticopportunity inthe management of obesity. Therefore, confirmed, the role of VB as an AMPK activator could have relevant implications {Grahame Hardie 2014), o2 1M Herne-tipe el Prycomedine 222015) 605-514 Aion a a De 7 Th puta chin 8 npn rt igh edd abo dsb eb soe ase fey eg Inflammation, xidave stress and mtochenéial esc So eadgto the (amen) “Taemmation ROS Polyphenols — a Mit Dysfunction —_Totvcorn Ft T EPADAA Te iv (GRAY > saronecin Nucleus reduce nicl 205 reer sensor AMPK Leman verbens polyp nd inflammation and reestablish michondil vblty(j AMP) ane AMPK activation that elt in PPAR“ -neised adpenecn upregulation and PPAR action. Ths equation nay lead toa cecteasein Ipogenesis and choles syne, Whi zs tei ree abésunlates ty aid natn a several sss apes sue, Iverané seta muse) erative os henes may aypocheoclly extracel 5 Ape agonist leading the Ser/Thr knases-medated activation of AMPK fas eet PEAR soni Kappa bina, OF note, the effect of VB and LC extract increasing adiponectin se- cretion, restoring mitochondrial function and decreasing the size of hypertrophic adipocytes may be linked to previous studies indicat that a reduction in mitochondrial mass or function cases the hyper= trophy of adipocytes, and these mitochondrial changes are linked to decreased adiponectin synthesis (Koh etal. 2007). On the other hand, these polyphenols increased PPAR-ar mRNA expression withthe con- sequent upregulation of downstream genes involved in fatty acid ox- ‘dation and mitochondrial machinery. Concomitantly, we observed a decrease in FASN mRNA expression, which most likely contributed to suppress lipid accumulation in che hypertrophic adipocytes (lipoge- esis), Additionally, LC seemed to be more effective than VB in most of these cellular effects. Therefore, the activation of both AMPK and PPAR-a and adecrease in FASN are consistent with a putative action of these polyphenotsin promoting fatty acid oxidation and decreasing lipid accumulation in adipocytes and the liver (Yeop Han et al, 2010}, “This is in agreement with results obtained using the whole extract in an animal model that closely resembles the metabolic syndrome (Rodriguez-Sanabriaet al.2010)in which lemon verbena polyphenols prevented the expected weight gan, liver steatosis and hypertrophic adipocytes. In this model, the improved triglyceride clearance fur- ther suggests thatthe modulation of fat utilization by liver and white adipose tissue may be one of the primary mechanisms of action of these polyphenols. To elucidate whether the intrinsic mechanism is ‘mediated by polyphenol metabolites acting at intracellular level or by {interacting to membrane receptors remains amajor challenge. Recent findings indicate that agonists of AdipoR, which mimic the effect of adiponectin and activate AMPK and PPAR-a, ameliorate insulin resis {ance (Okada-Iwabu et al. 2013). Because polyphenols depict reason- able structural similarities with these agonists, we propose a hypo- thetical sequence of events outlined in Fig 7. In agreement with this assumption, the potential binding ofthe lavone moiety to PPARs has also been predicted by computational modeling to explain ts capacity, ‘to modulate PPARs in adipocyte cell model (Lu etal. 2013) Inconclusion, although an additional value ofthe complete LC ex- tract, via synergistic or complementary effects cannat be discarded, VB deserves further attention as a therapeutic aid in the manage- iment of obesity andjor associated disturbances. The conversion of the cose utilized in out animal study to human equivalent dose re- sulted in several grams per day of lemon verbena extract. Despite of appearing to be a high dose, the use of up to 1.8 g of lemon verbena, extract for at least 1 month revealed to be safe in human studies (Funes et al 2011}, Whether lower doses show potential for clinical applications in obesity needs to be verified in human studies. Our findings indicate interactions with numerous endogenous proteins related to energy-sensing pathways, such as AMPK sensor, at cellular Ievel and the normalization of fat metabolism in animal model, Never= theless, we are fully aware that this fact diminishes the interestin drug IM eron-Lipe a Phomediine 2 (205) 605-614 ea developers despite being the case in many marketed drugs (eg. sal. ieylates), Further studies including the detected metabolites of this polyphenol and ongoing metabolomic studies may help to unravel ‘the potential health effects in humans. Conflict of interest “The authors declare no confit of interest, Acknowledgments ‘This work was supported by AGI2011-29857-C03-03, BFU2014- 52433-C3-I-R and IDI-20120751 grants (Spanish Ministry of Science and Innovation), PROMETEO/20:2/007 and ACOMP/2013/093 grants from Generalitat Valenciana, and CIBER (CB12/03/30038, Fisiopa- tologia de la Obesidad y 1a Nutricién, CIBERobn, Instituto de Salud Carlos Il). MH. isa recipient ofa VALI

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