Printer Friendly

Lemon verbena (Lippia citriodora) polyphenols alleviate obesity-related disturbances in hypertrophic adipocytes through AMPK-dependent mechanisms.

ABSTRACT

Background: There is growing evidence that natural products, mostly plant-derived polyphenols, are important in the relationship between nutrients and health in humans.

Purpose: We aimed to investigate if verbascoside (VB) and other lemon verbena polyphenols could ameliorate obesity-induced metabolic disturbances, as well as their putative mechanism.

Study design: We used an insulin-resistant hypertrophic 3T3-L1-adipocyte model to test the effects of VB or lemon verbena extract on triglyceride accumulation, inflammation and oxidative stress and a murine model of diet-induced obesity to assess the in vivo metabolic response.

Results: Polyphenols decreased triglyceride accumulation, the generation of reactive oxygen species (ROS) and restored mitochondrial membrane potential in adipocytes. The underlying mechanisms seemed to occur via ROS-mediated downregulation of nuclear factor kappa-B transcription factor (NF-[kappa]B) and peroxisome proliferator-activated receptor gamma (PPAR-[gamma])-dependent transcriptional upregulation of adiponectin. We also observed a potent activation of AMP-activated protein kinase (AMPK), the mRNA expression upregulation of PPAR-a and the mRNA expression downregulation of fatty acid synthase. Experiments in mice suggested a significant improvement in fat metabolism.

Conclusion: Decreased lipogenesis, enhanced fatty acid oxidation and the activation of the energy sensor AMPK, probably through activating transcriptional factors, are involved in the observed beneficial effects. VB effects were less potent than those observed with the extract, so a potential synergistic, multi-targeted action is proposed. The polypharmacological effects of plant-derived polyphenols from lemon verbena may have the potential for clinical applications in obesity.

Keywords:

Verbascoside

Lippia citriodora

Adipocyte

Adiponectin

AMPK

Fat metabolism

Introduction

In recent times, the close relationship between lifestyle, diet and the risk of major human diseases is becoming more evident. Unfortunately, while the prevalence of obesity has increased, current remedies or pharmaceutical drugs to fight obesity are of limited effectiveness and changes in lifestyle are difficult to accomplish (Burke and Wang 2011). The increase in dietary polyphenols supposes a potential alternative but mechanisms require elucidation because they usually hit multiple targets. Dietary energy excess causes lipid accumulation in adipocytes and other cells resulting in obesity, metabolic stress and low-grade chronic inflammation, which tends to perpetuate an imbalance between metabolic and immune cells (Gustafson et al. 2009; Kwon and Pessin 2013; Snel et al. 2012).

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-[kappa]B) is a critical regulator of several genes that are involved in immune and inflammation responses. NF-[kappa]B activity is increased in high glucose-induced hypertrophic adipocytes, leading to a proinflammatory 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, inflammation and metabolic stress Qoven et al. 2012b; Rull et al. 2010). As a result there is a cellular decrease in the activity of 5'-adenosine monophosphate-activated protein kinase (AMPK), an energy sensor involved in the survival of affected cells (Joven et al. 2012b; Menendez et al. 2013). Interestingly, AMPK signaling inhibits the inflammatory responses induced by the NF-[kappa]B system in adipocytes (Salminen et al. 2011), an effect that is most likely mediated by adiponectin (Hattori et al. 2008; Joven et al. 2012b). In obese state, a release of free fatty acids (FFAs) into the circulation takes place in hypertrophic adipocytes leading to systemic effects, i.e. ectopic FFAs accumulation and FA-induced insulin resistance in tissues such as muscle and liver (Rezaee 2013). In this process, the role of peroxisome proliferator-activated receptors (PPARs) have been also reported (Okada-Iwabu et al. 2013).

In this scenario, we have previously suggested that polyphenols modulate triglyceride accumulation, oxidative stress and inflammation in both humans and murine models (Beltran-Debon et al. 2010; Herranz-Lopez et al. 2012; Joven et al. 2012a; Joven et al. 2012b). These effects were observed using complex Hibiscus sabdariffa extracts containing anthocyanins, glycosylated flavonols and organic acids. Nevertheless, bioguided fractionation studies and immunohistochemical detection evidenced that flavonols (quercetin derivatives) appeared to be the best candidates mediating these beneficial effects, although with evident synergistic interactions (FernandezArroyo et al. 2012; Herranz-Lopez et al. 2012; Joven et al. 2012a). These effects were observed using Hibiscus sabdariffa L. (Malvaceae) extracts in which elucidating therapeutic mechanisms is a challenging task. Similar anti-inflammatory and radical scavenging activities and beneficial effects have also been observed with extracts from lemon verbena (Lippia citriodora (Palau) Kunth (Verbenaceae) (LC) or Aloysia triphylla) (L'Her.) 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 sorbets (Funes et al., 2009). In the last decade, the potential of lemon verbena extract supplementation as a nutraceutical to decrease muscular damage, blood oxidative 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 are rich in phenylpropanoids, glucuronidated flavonoids and iridoid glycosides (Supplementary Table 1) (QuirantesPine et al. 2013; Funes et al. 2009). Verbascoside (VB) (Supplementary Fig. 1), a phenylpropanoid glycoside, is the most abundant compound in this plant (Funes et al. 2009). The antioxidant, anti-inflammatory and chemopreventive activity of verbascoside, its biotechnological production, occurrence and uses have been recently reviewed (Alipieva et al. 2014).

We then reasoned that the effects of the extract could be simplified by the use of verbascoside. We hypothesized that we could demonstrate in a single polyphenol an inherent potential to exert polypharmacological effects other than redox modulation. The potential of polyphenols to interact and modulate different proteins would demonstrate the simultaneous modulation of inflammation and energy-related pathways, thereby corroborating 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-isobutyi-l-methylxanthine (IBMX), insulin, crystal violet, paraformaldehyde solution and 2',Tdichlorodihydrofluorescein diacetate (H2DCF-DA) were obtained from Sigma-Aldrich (Madrid, Spain). Dulbecco's modified Eagle's medium was purchased from Gibco (Grand Island, NY, USA). Polyvinyldifluoride (PVD) filters (0.22 [micro]m) were obtained from Millipore (Bedford, MA, USA). AdipoRed[TM] Assay Reagent was obtained from Lonza (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). LC extract (27% VB, w/w, as determined by HPLC, Supplementary Table 1) was kindly provided by Monteloeder, S.L. (Elche, 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 differentiated according to previously described procedures (Green and Kehinde 1975). Adipocytic differentiation was induced by adding adipogenic agents (0.5 mM IBMX, 1 p.M DEX, and 1 [micro]M INS) to the culture medium for 2 days. The medium was freshly replaced every 48 h. The phenotypic change of adipogenesis was observed under a microscope. In all experiments, more than 90% of the cells were mature adipocytes after 8-10 days of incubation. To induce cellular hypertrophy, adipocytes were exposed to high glucose (25 mM) for at least 18 days (Yeop Han et al. 2010). Further details on this cellular model are provided in Supplementary Fig. 2, indicating the accumulation of intracellular lipid droplets and 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 to the media. The absence of cytotoxicity was ascertained using the crystal violet method.

ROS generation was assessed in hypertrophic adipocytes using 2',7'-dichlorodihydro-fluorescein diacetate ([H.sub.2]DCF-DA) as described (Yeop Han et al. 2010). Fluorescent microphotographs 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 Western blot. Hypertrophic adipocyte were lysed with ice-cold lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% CHAPS, 1 mM Pefabloc and 1% phosphatase inhibitor cocktail no. 2, Sigma-Aldrich Inc., Steinheim, Germany). Protein concentrations were determined by a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, 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-Gly buffer (Invitrogen) was used for FASN electrophoresis. Proteins were transferred to nitrocellulose membranes using the iBlot transfer system (Invitrogen). Antibodies used were Rabbit anti-AMPK (#2532, Cell Signaling Tech., Danvers, MA, USA), rabbit anti-pAMPK (Thrl 72) (#2531, Cell Signaling Tech.), rabbit anti-PPAR-a (H-98, St. Cruz Biotech., Heidelberg, Germany), rabbit anti-FASN (3180, Cell Signaling Tech.) and rabbit anti-actin (H-300, St. Cruz Biotech.). The secondary antibodies were goat anti-rabbit-HRP (Dako, Glostrup, Denmark) and anti-goat-HRP (Dako). Chemiluminescent detection was performed using the ECL Advance Western Blotting Detection kit (Amersham, GE Healthcare, Barcelona, Spain), and membranes were analyzed in a ChemiDoc system (Bio-Rad, Spain). Immune-reactive protein levels were quantified by band densitometry normalized to [beta]-actin signal using software Image Lab (Version 3.0 build 11, Bio-Rad, Madrid, Spain).

Immunofluorescence study

For NF-[kappa]B and adiponectin detection, fixed cells were incubated overnight with each antibody, i.e. a polyclonal anti-RelA/p65 (Thermo Fisher Scientific Inc.) or monoclonal anti-adiponectin (Abeam Inc., USA). Cells were then washed with PBS and incubated for 2 h with each corresponding secondary antibody, Anti-Rabbit IgG-TRlTC (Sigma) and Anti-Mouse Polyvalent Immunoglobulins (G,A,M)-FITC. Stained cells were photographed with an inverted fluorescence microscope (Nikon Eclipse TE2000-U; Nikon Instruments, Inc., NY) provided with a digital camera (Nikon DS-1QM), and fluorescence was measured by fluorimeter in a multiwell plate reader (POLARstar Omega microplate).

Gene expression assays

The expression of selected genes was measured by quantitative real-time PCR analysis (qRT-PCR) of cDNA samples as previously reported (Joven et al. 2012a). Gene and primer information is shown in Supplementary Table 2. Total RNA was purified and converted to cDNA using RNeasy (Qiagen, Valencia, CA, USA) and Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. The expression levels were measured by real-time RT-PCR and results are expressed as fold change using [beta]-actin as housekeeping gene.

Plasmids and promoter analysis of the human adiponectin gene

The luciferase reporter construct driven by the 5'-flanking region of human adiponectin gene [p(-908)/LUC wt] and a mutated construct [p(-908)/LUC PPRE mut] containing point mutations from GG to AA at -276/-275 in the PPAR response element (PPRE), prepared as previously described (Iwaki et al. 2003), were kindly provided by the Osaka University (Japan). The transfection was performed in mature adipocytes after 5 days of differentiation using the Neon transfection system from Invitrogen according to the manufacturer's instructions. 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 with the 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 MitoTracker Red CMXRos (Mred) and MitoTracker Green FM (Mgreen) (Molecular Probes, Invitrogen, Carlsbad, CA, USA) for 30 min at 37 [degrees]C and washed three times with pre-warmed PBS; images being captured as mentioned above.

Animal experimental model

LDL receptor-deficient male mice in a C57BL/6J background ([LDLr.sup.-/-]) were the progeny of animals obtained from the Jackson Laboratory. The animal handling, sample preparation, sampling, sacrifice and the calculation of sample size were performed as described (Joven 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 as a unique liquid source (control), while the other group received the LC extract dissolved in tap deionized water (5 g/1), which was freshly prepared every day. Although water and/or extract 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 b.w. (202.5 mg/kg b.w. of VB). Blood and tissue samples were obtained and processed as previously described (Beltran-Debon et al. 2011). The oral fat test and the glucose tolerance test were performed as described elsewhere (Rull et al. 2007). Liver steatosis was qualitatively evaluated on a scale of 0-3, where 0 represented an absence of steatosis and 3 indicated a major grade of steatosis (>66%). All procedures in LDL receptor-deficient mice and experimental protocols were examined and approved by the Ethical Committee for Animal Experimentation of the University Miguel Hernandez (IBM-VMM-003-12).

Statistical analyses

Values are represented as the mean [+ or -] standard deviation (S.D.) of the mean. The values were subjected to statistical analysis (one-way ANOVA, Student's t-test for unpaired samples and Tukey's test for multiple comparisons). The differences were considered statistically significant at p < 0.05. All analyses were performed using GraphPad Prism 5 software (GraphPad, San Diego, CA). * p < 0.05, ** p < 0.01 and *** p < 0.001 on bars indicate statistically significant differences versus control, unless otherwise stated. Horizontal lines indicate statistically significant differences between bars. All cellular measurements derive from three independent experiments, wherein each performed in octuplicates, unless specified.

Results

LC and VB decrease lipid deposition and ROS generation in mature and hypertrophic adipocytes: a possible role for mechanisms involved in lipogenesis and mitochondrial dysfunction

To explore the effects of LC or VB on adipocyte model, we considered either mature or hypertrophic adipocytes those in the 8th or 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-dependent, similar decrease (20%) in triglyceride content with the maximum concentration of LC extract and VB utilized (400 [micro]g/ml LC and 108 [micro]g/ml VB, contained equivalent verbascoside concentrations; Fig. 1 A). The effect was also similar in high glucose-induced hypertrophic adipocytes; 31.6% and 29% respectively (Fig. IB). Similarly, both LC extract and VB decreased intracellular ROS generation (approximately 40%) in high glucose-induced hypertrophic adipocytes. Consequently, subsequent analyses were limited to hypertrophic adipocytes. To further elucidate the molecular mechanism involved in the suppressive effect of LC and VB on lipid accumulation, we investigated the effects of LC and VB on the mRNA expression and protein levels of PPAR[alpha], 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-a, a decrease in FASN and a significant activation of AMPK (Fig. 2). Significant differences were detected between LC and VB at equivalent VB concentrations, revealing a higher effect in the presence of LC extract. Also, and to determine the effect of LC or VB on intracellular ROS generation and their impact on mitochondrial function, we analyzed the accumulation of MitoTracker CMXRos (Mred), which is dependent on the mitochondrial membrane potential, and MitoTracker Green (Mgreen), a general mitochondrial marker (Fig. 3). Fluorescence micrographs 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 LC and VB treatments, revealing a possible mechanism to explain the decrease in intracellular ROS generation (Fig. 1C).

LC and VB upregulate adiponectin gene expression but only LC downregulates NF-[kappa]B in hypertrophic adipocytes

We then explored the effects of polyphenols on NF-[kappa]B, as a regulator of the oxidative stress-induced inflammatory response and on adiponectin, a perceived anti-inflammatory 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 compared to the equivalent concentration of purified VB. Although LC and VB treatments showed small but significant decreases in NF-[kappa]B gene expression compared to the control, there were no differences between their capacities to downregulate NF-[kappa]B gene expression (Fig. 4A).

When protein levels were quantitated, both LC and VB showed dose-dependent significant increases in adiponectin expression compared to the control and also, a differential effect of the LC extract and VB was confirmed on adiponectin expression. However, only LC exhibited a significant effect on NF-[kappa]B expression levels (Fig. 4B). Cellular assays using immunofluorescence resulted in similar values (Fig. 4C and D). These effects on the anti-inflammatory action were further confirmed when we measured the gene expression of selected cytokines in the same experimental cells. We found significant reductions with both, extract and purified compound, with respect to controls, of IL-[beta], IL-6, TNF-a and CCL2/MCP-1 but there were no changes in IL-T a and leptin expression levels (Supplementary Fig. 3).

To clarify whether the effect of LC or VB on the transcriptional upregulation of adiponectin was mediated by PPAR-[gamma], a critical transcription factor involved in adiponectin expression (Bouskila et al. 2005; Iwaki et al. 2003), we transfected the adipocytes with different constructs containing either the wild type or mutated adiponectin luciferase promoter. Luciferase activity driven by the adiponectin promoter in transfected adipocytes incubated in the presence or absence of either LC or VB was determined. At the highest concentrations, cells treated with either LC or VB showed a significant dose-dependent increase in basal luciferase activity of the wild-type adiponectin promoter [p(-908)/LUC wt] as compared to controls (Fig. 5A). When we used the human adiponectin promoter bearing the mutated PPAR response element [p(-908)/LUC PPRE mut] there were no appreciable changes in luciferase activity indicating a possible major role for PPAR-[gamma] 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 HF diet, LDLr deficient mice, consistently develop obesity-associated metabolic disturbances and hypertrophic adipocytes (Beltran-Debon et al. 2011; Joven et al. 2012a). The effects of LC extract rather than purified VB 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 intake 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% between 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 of the triglyceride values at 60 and 120 min (mg/dl) after the ingestion of an oral olive oil bolus associated with LC extract consumption, indicating that treatment induced improved triglyceride clearance (Fig. 6B). Also, treated animals depict significantly lower serum cholesterol and triglycerides concentrations than the controls (Fig. 6C and D). Among obesity-associated disturbances in this animal model, liver steatosis is a constant finding. The administration of the LC extract alleviated the accumulation of neutral lipids and triglycerides in the liver without altering serum biomarkers of hepatic toxicity (Fig. 6E-G).

Discussion

The 3T3-L1 ceil line is a well characterized and widely accepted model of in vitro adipogenesis and lipid accumulation that becomes hypertrophic and insulin resistant when induced with high-glucose conditions (Green and Kehinde 1975; Han et al. 2007; Herranz-Lopez et al. 2012; Ji et al. 2014; Yoshizaki et al. 2012; Zebisch et al. 2012). In this model, we have assayed the capacity of LC and VB to ameliorate obesity-induced metabolic disturbances.

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. It is commonly accepted that adipocyte hypertrophy compromises cell function in the progression of obesity associated metabolic disturbances and is associated to ROS generation and inflammation (Han et al. 2007), in agreement to 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, prevented most of the expected deleterious effects. These results are also partially confirmed in an animal model of diet-induced obesity. VB is 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 reduce 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, VB, 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 promising functional elements in polyphenols. We have previously found the strong free radical scavenging capacity of LC polyphenols and their ability to enhance the activity of antioxidant enzymes (CarreraQuintanar et al. 2012; Funes et al. 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 cell (Ji et al. 2014).

In this study we identify some previously unrecognized mechanistic clues. Particularly, these polyphenols maintain mitochondrial membrane potential and mitochondrial viability. These findings indicate that further consideration should be paid to other actions of polyphenols that may be extended to the direct modulation of mitochondrial events affecting the whole cell (e.g. energy generation, mitochondrial biogenesis or cell death control; Chung et al. 2010). We have considered a variety of molecular targets related to metabolic stress in adipocytes. For example, the activation of the redox-sensitive transcription factors such as NF-[kappa]B is accepted as a deleterious effect in the pathogenesis of common diseases via the modulation of a large number of genes mediating immune and inflammatory responses (Jiang et al. 2011; Li and Karin 1999). Our results reveal that both LC and VB decreased NF-[kappa]B and increased adiponectin gene expression. Curiously, only the LC extract decreased the NF-[kappa]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 a transcriptional PPAR-[gamma]-dependent mechanism. Further, the anti-inflammatory action of adiponectin was accompanied by the downregulation of selected inflammatory genes and a significant activation of AMPK in hypertrophic adipocytes. Adiponectin has been proposed as a systemic functional link involved in the activation of AMPK in different tissues (Hattori et al. 2008; Iwabu et al. 2010; Okada-Iwabu et al. 2013), probably through different actions of adiponectin receptor (AdipoR) (Fang et al. 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 (Hardie et al. 2012; Salminen and Kaarniranta 2012) and consequently a therapeutic opportunity in the management of obesity. Therefore, if confirmed, the role of VB as an AMPK activator could have relevant implications (Grahame Hardie 2014).

Of note, the effect of VB and LC extract increasing adiponectin secretion, restoring mitochondrial function and decreasing the size of hypertrophic adipocytes may be linked to previous studies indicating that a reduction in mitochondrial mass or function causes the hypertrophy of adipocytes, and these mitochondrial changes are linked to decreased adiponectin synthesis (Koh et al. 2007). On the other hand, these polyphenols increased PPAR-a mRNA expression with the consequent upregulation of downstream genes involved in fatty acid oxidation and mitochondrial machinery. Concomitantly, we observed a decrease in FASN mRNA expression, which most likely contributed to suppress lipid accumulation in the hypertrophic adipocytes (lipogenesis). 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 a decrease in FASN are consistent with a putative action of these polyphenols in promoting fatty acid [beta]-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-Sanabria et al. 2010) in which lemon verbena polyphenols prevented the expected weight gain, liver steatosis and hypertrophic adipocytes. In this model, the improved triglyceride clearance further suggests that the 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 a major challenge. Recent findings indicate that agonists of AdipoR, which mimic the effect of adiponectin and activate AMPK and PPAR-a, ameliorate insulin resistance (Okada-Iwabu et al. 2013). Because polyphenols depict reasonable structural similarities with these agonists, we propose a hypothetical sequence of events outlined in Fig. 7. In agreement with this assumption, the potential binding of the flavone moiety to PPARs has also been predicted by computational modeling to explain its capacity to modulate PPARs in adipocyte cell model (Lu et al. 2013).

In conclusion, although an additional value of the complete LC extract, via synergistic or complementary effects cannot be discarded, VB deserves further attention as a therapeutic aid in the management of obesity and/or associated disturbances. The conversion of the dose utilized in our animal study to human equivalent dose resulted 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 level and the normalization of fat metabolism in animal model. Nevertheless, we are fully aware that this fact diminishes the interest in drug developers despite being the case in many marketed drugs (e.g. salicylates). Further studies including the detected metabolites of this polyphenol and ongoing metabolomic studies may help to unravel the potential health effects in humans.

ARTICLE INFO

Article history:

Received 20 October 2014

Revised 6 March 2015

Accepted 23 March 2015

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

This work was supported by AGL2011-29857-C03-03, BFU201452433-C3-1-R and IDI-20120751 grants (Spanish Ministry of Science and Innovation), PROMETEO/2012/007 and ACOMP/2013/093 grants from Generalitat Valenciana, and CIBER (CB12/03/30038, Fisiopatologia de la Obesidad y la Nutricion, CIBERobn, Instituto de Salud Carlos III). M.H. is a recipient of a VALi+D fellowship from Generalitat Valenciana (ACIF/2010/162). We thank E. Rodriguez-Gallego and R.M. Medina-Gali for their invaluable help in gene and protein expression experiments and to Monteloeder, SL (Alicante, Spain) for the lemon verbena extract.

Supplementary Materials

Supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.phymed.2015.03.015.

References

Alipieva, K., Korkina, L, Orhan, I.E., Georgiev, M.I., 2014. Verbascoside--a review of its occurrence, (bio)synthesis and pharmacological significance. Biotechnol. Adv. 32, 1065-1076.

Baker, R.G., Hayden, M.S., Ghosh, S., 2011. NF-kappaB, inflammation, and metabolic disease. Cell Metab. 13, 11-22.

Beltran-Debon, R., Alonso-Villaverde, C, Aragones, G.. Rodriguez-Medina, I., Rull, A., Micol, V., Segura-Carretero, A., Fernandez-Gutierrez, A., Camps, J., Joven, J., 2010. The aqueous extract of Hibiscus sabdariffa calices modulates the production of monocyte chemoattractant protein-1 in humans. Phytomedicine 17, 186-191.

Beltran-Debon, R., Rull, A., Rodriguez-Sanabria, F., Iswaldi, I., Herranz-L6pez, M., Aragones, G., Camps, J., Alonso-Villaverde, C., Menendez, JA, Micol, V., SeguraCarretero, A., Joven, J., 2011. Continuous administration of polyphenols from aqueous rooibos (Aspalathus linearis) extract ameliorates dietary-induced metabolic disturbances in hyperlipidemic mice. Phytomedicine 18, 414-424.

Bouskila, M., Pajvani, U.B., Scherer, P.E., 2005. Adiponectin: a relevant player in PPARgamma-agonist-mediated improvements in hepatic insulin sensitivity? int. J. Obes. (Lond.) 29 (Suppl. 1), S17-S23.

Burke, L.E., Wang, J., 2011. Treatment strategies for overweight and obesity. J. Nurs. Sch. 43, 368-375.

Carrera-Quintanar, L., Funes, L., Vicente-Salar, N., Blasco-Lafarga, C., Pons, A., Micol, V., Roche, E., 2014. Effect of polyphenol supplements on redox status of blood cells: a randomized controlled exercise training trial. Eur.J.Nutr. DOI: 10.1007/S0039400014-00785-x.

Carrera-Quintanar, L., Funes, L., Viudes, E., Tur, J., Micol, V., Roche, E., Pons, A., 2012. Antioxidant effect of lemon verbena extracts in lymphocytes of university students performing aerobic training program. Scand. J. Med. Sci. Sports 22, 454-461.

Caturla, N., Funes, L., Perez-Fons, L., Micol, V., 2011. A randomized, double-blinded, placebo-controlled study of the effect of a combination of lemon verbena extract and fish oil omega-3 fatty acid on joint management. J. Altern. Complement. Med. 17, 1051-1063.

Chung, S., Yao, H., Caito, S., Hwang, J.W., Arunachalam, G., Rahman, I., 2010. Regulation of SIRT1 in cellular functions: role of polyphenols. Arch. Biochem. Biophys 501, 79-90.

Fang, X., Palanivel, R., Cresser, J., Schram, K., Ganguly, R., Thong, F.S., Tuinei, J., Xu. A., Abel, E.D., Sweeney, G., 2010. An APPL1-AMPK signaling axis mediates beneficial metabolic effects of adiponectin in the heart. Am. J. Physiol. Endocrinol. Metab. 299, E721-E729.

Fernandez-Arroyo, S., Herranz-Lopez, M., Beltran-Debon, R., Borras-Linares, I., BarrajonCatalan, E., Joven, J., Fernandez-Gutierrez, A., Segura-Carretero, A., Micol, V., 2012. Bioavailability study of a polyphenol-enriched extract from Hibiscus sabdariffa in rats and associated antioxidant status. Mol. Nutr. Food Res. 56, 1590-1595.

Funes, L, Carrera-Quintanar, L., Cerdan-Calero, M., Ferrer, M.D., Drobnic, F., Pons, A., Roche, E., Micol, V.. 2011. Effect of lemon verbena supplementation on muscular damage markers, proinflammatory cytokines release and neutrophils' oxidative stress in chronic exercise. Eur.J. Appl. Physiol 111, 695-705.

Funes, L., FernSndez-Arroyo. S., Laporta, 0., Pons, A., Roche, E., Segura-Carretero, A.. Ferndndez-Gutierrez. A.. Micol, V.. 2009. Correlation between plasma antioxidant capacity and verbascoside levels in rats after oral administration of lemon verbena extract. Food Chem. 117, 589-598.

Funes, L., Laporta, 0., Cerdan-Calero, M., Micol, V., 2010. Effects of verbascoside, a phenylpropanoid glycoside from lemon verbena, on phospholipid model membranes. Chem. Phys. Lipids 163, 190-199.

Grahame Hardie, D., 2014. AMP-activated protein kinase: a key regulator of energy balance with many roles in human disease. J. intern. Med. 276 (6), 543-559.

Green, H., Kehinde, O., 1975. An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion. Cell 5, 19-27.

Gustafson, B., Gogg, S., Hedjazifar, S., jenndahl, L., Hammarstedt, A, Smith, U., 2009. Inflammation and impaired adipogenesis in hypertrophic obesity in man. Am. J. Physiol. Endocrinol. Metab. 297, E999-E1003.

Han, C.Y., Subramanian, S., Chan, C.K., Omer, M., Chiba, T., Wight, T.N., Chait, A., 2007. Adipocyte-derived serum amyloid A3 and hyaluronan play a role in monocyte recruitment and adhesion. Diabetes 56, 2260-2273.

Hardie, D.G., Ross, F.A., Hawley, S.A., 2012. AMP-activated protein kinase: a target for drugs both ancient and modern. Chem. Biol. 19, 1222-1236.

Hattori, Y., Nakano, Y., Hattori, S., Tomizawa, A., Inukai, K., Kasai, K., 2008. High molecular weight adiponectin activates AMPK and suppresses cytokine-induced NF-[kappa]B activation in vascular endothelial cells. FEBS Lett. 582, 1719-1724.

Herranz-Lopez, M., Fernandez-Arroyo, S., Perez-Sanchez, A, Barrajon-Catalan, E., Beltran-Debon, R., Menendez, JA., Alonso-Villaverde, C., Segura-Carretero, A., Joven, J., Micol, V., 2012. Synergism of plant-derived polyphenols in adipogenesis: perspectives and implications. Phytomedicine 19, 253-261.

Iwabu, M., Yamauchi, T., Okada-Iwabu, M., Sato, K., Nakagawa, T., Funata, M., Yamaguchi, M., Namiki, S., Nakayama, R., Tabata, M., Ogata, H., Kubota, N., Takamoto, I., Hayashi, Y.K., Yamauchi, N., Waki, H., Fukayama, M., Nishino, I., Tokuyama, K., Ueki, K., Oike, Y., Ishii, S., Hirose, K., Shimizu, T., Touhara, K., Kadowaki.T., 2010. Adiponectin and AdipoRl regulate PGC-l[agr] and mitochondria by [Ca.sup.2+] and AMPK/SIRT1. Nature 464, 1313-1319.

Iwaki, M., Matsuda, M., Maeda, N., Funahashi, T., Matsuzawa, Y., Makishima, M., Shimomura, I., 2003. Induction of adiponectin, a fat-derived antidiabetic and antiatherogenic factor, by nuclear receptors. Diabetes 52, 1655-1663,

Ji, E., Jung, M.Y., Park, J.H., Kim, S., Seo, C.R., Park, K.W., Lee. E.K., Yeom, C.H., Lee, S., 2014. Inhibition of adipogenesis in 3T3-L1 cells and suppression of abdominal fat accumulation in high-fat diet-feeding C57BL/6J mice after downregulation of hyaluronic acid. Int. J. Obes. (Lond.) 38, 1035-1043.

Jiang, F., Zhang, Y., Dusting, G.J., 2011. NADPH oxidase-mediated redox signaling: roles in cellular stress response, stress tolerance, and tissue repair. Pharmacol. Rev. 63, 218-242.

Joven, J., Espinel, E., Rull, A., Aragones, G., Rodriguez-Gallego, E., Camps, J., Micol, V., Herranz-Lopez, M., Menendez, J.A., Borras, L, Segura-Carretero, A., Alonso-Villaverde, C., Beltran-Debon, R., 2012. Plant-derived polyphenols regulate expression of miRNA paralogs miR-103/107 and miR-122 and prevent diet-induced fatty liver disease in hyperlipidemic mice. Biochim. Biophys. Acta 1820,894-899.

Joven, J., Rull, A., Ferre, N., Escola-Gil, J.C., Marsillach, J., Coll, B., Alonso-Villaverde, C., Aragones, G., Claria, J., Camps, J., 2007. The results in rodent models of atherosclerosis are not interchangeable: the influence of diet and strain. Atherosclerosis 195, e85-e92.

Joven, J., Rull, A., Rodriguez-Gallego, E., Camps, J., Riera-Borrull, M., HerndndezAguilera, A., Martin-Paredero, V., Segura-Carretero, A., Micol, V., AlonsoVillaverde, C., Menendez, J .A., 2012. Multifunctional targets of dietary polyphenols in disease: a case for the chemokine network and energy metabolism. Food Chem. Toxicol. 51, 267-279.

Koh, E.H., Park, J.Y., Park, H.S., Jeon, M.J., Ryu, J.W., Kim, M., Kim, S.Y., Kim, M.S., Kim, S.W., Park, I.S., Youn, J.H., Lee, K.U.. 2007. Essential role of mitochondrial function in adiponectin synthesis in adipocytes. Diabetes 56, 2973-2981.

Kwon, H., Pessin, J.E., 2013. Adipokines mediate inflammation and insulin resistance. Front Endocrinol. 4, 71.

Li, N., Karin, M., 1999. Is NF-[kappa]B the sensor of oxidative stress? FASEB J. 13, 1137-1143.

Lu, K, Han, M, Ting, HL, Liu, Z, Zhang, D, 2013. Scutellarin from Scutellaria baicalensis suppresses adipogenesis by upregulating PPARa in 3T3-L1 cells. J. Nat. Prod. 76, 672-678.

Menendez, J .A., Joven, J., Aragones, G., Barrajon-Catalan, E., Beltran-Debon, R.. BorrasLinares, I., Camps, J., Corominas-Faja, B., Cufi, S., Fernandez-Arroyo, S., GarciaHeredia, A., Hernandez-Aguilera, A., Herranz-Lopez, M., Jimenez-Sanchez, C., Lopez-Bonet, E., Lozano-Sanchez, J., Luciano-Mateo, F., Martin-Castillo, B., MartinParedero, V., Perez-Sanchez, A., Oliveras-Ferraros, C., Riera-Borrull, M., RodriguezGallego, E., Quirantes-Pine, R., Rull, A., Tomas-Menor, L., Vazquez-Martin, A., Alonso-Villaverde, C., Micol, V., Segura-Carretero, A., 2013. Xenohormetic and antiaging activity of secoiridoid polyphenols present in extra virgin olive oil: a new family of gerosuppressant agents. Cell Cycle 12, 555-578.

Okada-Iwabu, M., Yamauchi, T., Iwabu, M., Honma, T., Hamagami, K., Matsuda, K., Yamaguchi, M., Tanabe, H., Kimura-Someya, T., Shirouzu, M., Ogata, H., Tokuyama, K., Ueki, K., Nagano, T., Tanaka, A., Yokoyama, S., Kadowaki, T., 2013. A small-molecule AdipoR agonist for type 2 diabetes and short life in obesity. Nature 503, 493-499.

Quirantes-Pine, R., Herranz-Lopez, M., Funes, L., Borras-Linares, 1., Micol, V., Segura-Carretero, A., Fernandez-Gutierrez. A., 2013. Phenylpropanoids and their metabolites are the major compounds responsible for blood-cell protection against oxidative stress after administration of Lippia citriodora in rats. Phytomedicine 20, 1112-1118.

Rezaee, F., Dashty, M., 2013. Role of adipose tissue in metabolic system disorders adipose tissue is the initiator of metabolic diseases. J. Diabetes Metab. S13, 008. doi: 10.4172/2155-6156.S 13-008.

Rodriguez-Sanabria, F., Rull, A., Aragones, G., Beltran-Debon, R., Alonso-Villaverde, C., Camps, J., Joven, J., 2010. Differential response of two models of genetically modified mice fed with high fat and cholesterol diets: relationship to the study of nonalcoholic steatohepatitis. Mol, Cell. Biochem. 343, 59-66.

Rull, A., Camps, J., Alonso-Villaverde, C., Joven, J., 2010. Insulin resistance, inflammation, and obesity: role of monocyte chemoattractant protein-1 (or CCL2) in the regulation of metabolism. Mediators Inflamm. 2010, 326-580.

Rull, A., Escola-Gil. J.C., Julve, J., Rotllan, N., Calpe-Berdiel, L., Coll, B., Aragones, G., Marsillach, J., Alonso-Villaverde, C., Camps, J., Blanco-Vaca, F., Joven, J., 2007. Deficiency in monocyte chemoattractant protein-1 modifies lipid and glucose metabolism. Exp. Mol. Pathol. 83, 361-366.

Salminen, A., Hyttinen, J.T., Kaamiranta, K., 2011. AMP-activated protein kinase inhibits NF-[kappa]B signaling and inflammation: impact on healthspan and lifespan. J. Mol. Med. 89, 667-676.

Salminen, A., Kaarniranta, K., 2012. AMP-activated protein kinase (AMPK) controls the aging process via an integrated signaling network. Ageing Res. Rev. 11, 230-241.

Snel, M.,Jonker, J.T., Schoones, J., Lamb, H., de Roos, A., Pijl, H., Smit, J.W., Meinders, A.E., Jazet, I.M., 2012. Ectopic fat and insulin resistance: pathophysiology and effect of diet and lifestyle interventions. Int.J. Endocrinol. 2012, 983814.

Yeop Han. C., Kargi, A.Y., Omer, M., Chan, C.K., Wabitsch, M., O'Brien, K.D., Wight, T.N., Chait, A., 2010. Differential effect of saturated and unsaturated free fatty acids on the generation of monocyte adhesion and chemotactic factors by adipocytes: dissociation of adipocyte hypertrophy from inflammation. Diabetes 59, 386-396.

Yoshizaki, T., Kusunoki, C., Rondo, M., Yasuda, M., Kume, S., Morino, K., Sekine. O., Ugi, S., llzu, T., Nishio, Y., Kashiwagi, A., Maegawa, H., 2012. Autophagy regulates inflammation in adipocytes. Biochem. Biophys. Res. Commun. 417, 352-357.

Zebisch, K., Voigt, V., Wabitsch, M., Brandsch, M., 2012. Protocol for effective differentiation of 3T3-Ll cells to adipocytes. Anal. Biochem. 425, 88-90.

Maria Herranz-Lopez (a), Enrique Barrajon-Catalan (a), Antonio Segura-Carretero (b), Javier A. Menendez (c), Jorge Joven (d, 1), Vicente Micol (a, e), (1), *

(a) Institute) de Biologia Molecular y Celular (IBMC), Universidad Miguel Hemindez, Elche, Alicante, Spain

(b) Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Granada, Spain

(c) Metabolism & Cancer Group, Translational Research Laboratory, Catalan Institute of Oncology and Biomedical Research Institute, Girona, Spain

(d) Unitat de Recerca Biomedica, Hospital Universitari deSant Joan, IISPV, Universitat Rovira i Virgili, C/Sant Joan s/n, 4320I Reus, Spain

(e) CIBER (CB12/03/30038, Fisiopatologia de la Obesidad y la Nutrition, CIBERobn, Instituto de Salud Carlos III), Spain

Abbreviations: LC, Lippia citriodora extract; VB, verbascoside; FBS, fetal bovine serum; IBMX, 3-isobutyl-l-methylxanthine; DEX, dexamethasone; NF-[kappa]B, nuclear factor kappa-light-chain-enhancerof activated B cells; AMPK, adenosine monophosphateactivated protein kinase; CCL2, chemokine [C-C motif] ligand 2; TNF-[alpha], tumor necrosis factor-alpha; IL-l[alpha], interleukin-1 alpha; IL-1[beta], interleukin-1 beta; 1L-6, interleukin 6; PPAR-[gamma], peroxisome proliferator-activated receptor gamma; CCL2/MCP-1, monocyte chemotactic protein-1; HF, high-fat, high-cholesterol diet; [LDLr.sup.-/-], low density lipoprotein receptor knock-out mice; PPAR-[alpha], peroxisome proliferator-activated receptor [alpha]; FASN, fatty acid synthase; FFAs, free fatty acids; TG, triglycerides; ROS, reactive oxygen species; AST, aspartate aminotransferase.

* Corresponding author at: Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, Avda. de la Universidad S/N, 03202 Elche, Alicante, Spain. Tel.: +34 96 6658430; fax: +34 96 6658758.

E-mail address; vmicol@umh.es. bioelx.vicente@gmail.com (V. Micol).

(1) These authors share co-senior authorship.

http://dx.doi.org/10.1016/j.phymed.2015.03.015
COPYRIGHT 2015 Urban & Fischer Verlag
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2015 Gale, Cengage Learning. All rights reserved.

 
Article Details
Printer friendly Cite/link Email Feedback
Author:Herranz-Lopez, Maria; Barrajon-Catalan, Enrique; Segura-Carretero, Antonio; Menendez, Javier A.; Jov
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Jun 15, 2015
Words:6614
Previous Article:Red ginseng represses hypoxia-induced cyclooxygenase-2 through sirtuin1 activation.
Next Article:Selective inhibitory effects of machilin A isolated from Machilus thunbergii on human cytochrome P450 1A and 2B6.
Topics:

Terms of use | Privacy policy | Copyright © 2018 Farlex, Inc. | Feedback | For webmasters