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

ARTICLE IN PRESSJID: PHYMED [m5G;April 14, 2015;12:13]

Phytomedicine xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Phytomedicine

journal homepage: www.elsevier.com/locate/phymed

Lemon verbena (Lippia citriodora) polyphenols alleviate obesity-related

disturbances in hypertrophic adipocytes through AMPK-dependent

mechanisms

María Herranz-López a, Enrique Barrajón-Catalán a, Antonio Segura-Carretero b,Q1

Javier A. Menéndez c, Jorge Joven d,1, Vicente Micol a,e,1,∗

a Instituto de Biología Molecular y Celular (IBMC), Universidad Miguel Hernández, Elche, Alicante, Spainb Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Granada, Spainc Metabolism & Cancer Group, Translational Research Laboratory, Catalan Institute of Oncology and Biomedical Research Institute, Girona, Spaind Unitat de Recerca Biomèdica, Hospital Universitari de Sant Joan, IISPV, Universitat Rovira i Virgili, C/ Sant Joan s/n, 43201 Reus, Spaine CIBER (CB12/03/30038, Fisiopatología de la Obesidad y la Nutrición, CIBERobn, Instituto de Salud Carlos III), Spain

a r t i c l e i n f o

Article history:

Received 20 October 2014

Revised 6 March 2015

Accepted 23 March 2015

Available online xxx

Keywords:

Verbascoside

Lippia citriodora

Adipocyte

Adiponectin

AMPK

Fat metabolism

a b s t r a c t

Background: There is growing evidence that natural products, mostly plant-derived polyphenols, are impor-

tant 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-κB) and peroxisome

proliferator-activated receptor gamma (PPAR-γ )-dependent transcriptional upregulation of adiponectin. We

also observed a potent activation of AMP-activated protein kinase (AMPK), the mRNA expression upregulation

of PPAR-α 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.

© 2015 Published by Elsevier GmbH.

Abbreviations: LC, Lippia citriodora extract; VB, verbascoside; FBS, fetal bovine

serum; IBMX, 3-isobutyl-1-methylxanthine; DEX, dexamethasone; NF-κB, nuclear fac-

tor kappa-light-chain-enhancer of activated B cells; AMPK, adenosine monophosphate-

activated protein kinase; CCL2, chemokine [C–C motif] ligand 2; TNF-α, tumor necrosis

factor-alpha; IL-1α, interleukin-1 alpha; IL-1β , interleukin-1 beta; IL-6, interleukin

6; PPAR-γ , peroxisome proliferator-activated receptor gamma; CCL2/MCP-1, mono-

cyte chemotactic protein-1; HF, high-fat, high-cholesterol diet; LDLr−/− , low density

lipoprotein receptor knock-out mice; PPAR-α, peroxisome proliferator-activated re-

ceptor α; FASN, fatty acid synthase; FFAs, free fatty acids; TG, triglycerides; ROS, reac-

tive oxygen species; AST, aspartate aminotransferase.∗ Corresponding author at: Instituto de Biología Molecular y Celular, Universidad

Miguel Hernández, Avda. de la Universidad S/N, 03202 Elche, Alicante, Spain. Tel.: +34

96 6658430; fax: +34 96 6658758.

E-mail address: [email protected], [email protected] (V. Micol).1

These authors share co-senior authorship.

Introduction 1

In recent times, the close relationship between lifestyle, diet and

Q2

2

the risk of major human diseases is becoming more evident. Un- 3

fortunately, while the prevalence of obesity has increased, current 4

remedies or pharmaceutical drugs to fight obesity are of limited ef-Q3

5

fectiveness and changes in lifestyle are difficult to accomplish (Burke 6

and Wang 2011) . The increase in dietary polyphenols supposes a po- 7

tential alternative but mechanisms require elucidation because they 8

usually hit multiple targets. Dietary energy excess causes lipid accu- 9

mulation in adipocytes and other cells resulting in obesity, metabolic 10

stress and low-grade chronic inflammation, which tends to perpet- 11

uate an imbalance between metabolic and immune cells (Gustafson 12

et al. 2009; Kwon and Pessin 2013; Snel et al. 2012). 13

http://dx.doi.org/10.1016/j.phymed.2015.03.015

0944-7113/© 2015 Published by Elsevier GmbH.

Please cite this article as: M. Herranz-López et al., Lemon verbena (Lippia citriodora) polyphenols alleviate obesity-related disturbances in

hypertrophic adipocytes through AMPK-dependent mechanisms, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.03.015


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2 M. Herranz-López et al. / Phytomedicine xxx (2015) xxx–xxx


Nuclear factor kappa-light-chain-enhancer of activated B cells14

(NF-κB) is a critical regulator of several genes that are involved in15

immune and inflammation responses. NF-κB activity is increased16

in high glucose-induced hypertrophic adipocytes, leading to a pro-17

inflammatory state resembling the effect of nutritional overload and18

low physical activity (Baker et al. 2011; Han et al. 2007). In this19

scenario, the CCL2 (chemokine [C–C motif] ligand 2), through its20

functional receptor (CCR2) induces monocyte recruitment, inflam-21

mation and metabolic stress (Joven et al. 2012b; Rull et al. 2010).22

As a result there is a cellular decrease in the activity of 5′ adeno-23

sine monophosphate-activated protein kinase (AMPK), an energy24

sensor involved in the survival of affected cells (Joven et al. 2012b;25

Menendez et al. 2013). Interestingly, AMPK signaling inhibits the in-26

flammatory responses induced by the NF-κB system in adipocytes27

(Salminen et al. 2011), an effect that is most likely mediated by28

adiponectin (Hattori et al. 2008; Joven et al. 2012b). In obese state,29

a release of free fatty acids (FFAs) into the circulation takes place30

in hypertrophic adipocytes leading to systemic effects, i.e. ectopic31

FFAs accumulation and FA-induced insulin resistance in tissues such32

as muscle and liver (Rezaee 2013). In this process, the role of per-33

oxisome proliferator-activated receptors (PPARs) have been also re-34

ported (Okada-Iwabu et al. 2013).35

In this scenario, we have previously suggested that polyphenols36

modulate triglyceride accumulation, oxidative stress and inflamma-37

tion in both humans and murine models (Beltran-Debon et al. 2010;38

Herranz-Lopez et al. 2012; Joven et al. 2012a; Joven et al. 2012b).39

These effects were observed using complex Hibiscus sabdariffa ex-40

tracts containing anthocyanins, glycosylated flavonols and organic41

acids. Nevertheless, bioguided fractionation studies and immunohis-42

tochemical detection evidenced that flavonols (quercetin derivatives)43

appeared to be the best candidates mediating these beneficial effects,44

although with evident synergistic interactions (Fernandez-Arroyo45

et al. 2012; Herranz-Lopez et al. 2012; Joven et al. 2012a). These46

effects were observed using Hibiscus sabdariffa extracts in which elu-47

cidating therapeutic mechanisms is a challenging task. Similar anti-48

inflammatory and radical scavenging activities and beneficial effects49

have also been observed with extracts from lemon verbena (Lippia50

citriodora (LC) or Aloysia triphylla). Lemon verbena leaves are widely51

used as a species to add lemony flavor in food and also used to52

make herbal teas and refreshing sorbets (Funes et al., 2009). In the53

last decade, the potential of lemon verbena extract supplementa-54

tion as a nutraceutical to decrease muscular damage, blood oxida-55

tive stress and proinflammatory cytokines in sport and joint health56

has been explored (Carrera-Quintanar et al. 2014; Carrera-Quintanar57

et al. 2012; Caturla et al. 2011; Funes et al. 2011; Quirantes-Pine58

et al. 2013). LC leaves are rich in phenylpropanoids, glucuronidated59

flavonoids and iridoid glycosides (Supplementary Table 1) (Quirantes-60

Pine et al. 2013; Funes et al. 2009). Verbascoside (VB) (Supplementary61

Fig. 1), a phenylpropanoid glycoside, is the most abundant compound62

in this plant (Funes et al. 2009). The antioxidant, anti-inflammatory63

and chemopreventive activity of verbascoside, its biotechnologi-64

cal production, occurrence and uses have been recently reviewed65

(Alipieva et al. 2014).66

We then reasoned that the effects of the extract could be67

simplified by the use of verbascoside. We hypothesized that we68

could demonstrate in a single polyphenol an inherent poten-69

tial to exert polypharmacological effects other than redox mod-70

ulation. The potential of polyphenols to interact and modulate71

different proteins would demonstrate the simultaneous modula-72

tion of inflammation and energy-related pathways, thereby cor-73

roborating their multitargeted character. We present here, for74

the first time, that VB and associated polyphenols hit different75

molecular targets, which were beneficial in high glucose-induced76

insulin-resistant hypertrophic adipocytes and in a murine model of77

hyperlipidemia.78

Materials and methods 79

Chemicals and reagents 80

Dexamethasone (DEX), 3-isobutyl-1-methylxanthine (IBMX), 81

insulin, crystal violet, paraformaldehyde solution and 2′,7′- 82

dichlorodihydrofluorescein diacetate (H2DCF-DA) were obtained 83

from Sigma-Aldrich (Madrid, Spain). Dulbecco’s modified Eagle’s 84

medium was purchased from Gibco (Grand Island, NY, USA). 85

Polyvinyldifluoride (PVD) filters (0.22 μm) were obtained from Mil- 86

lipore (Bedford, MA, USA). AdipoRedTM Assay Reagent was obtained 87

from Lonza (Walkersville, MD, USA). Pure, isolated VB was obtained 88

through preparative HPLC, as previously reported, from a previously 89

characterized lemon verbena aqueous extract (Funes et al. 2010). 90

LC extract (27% VB, w/w, as determined by HPLC, Supplementary 91

Table 1) was kindly provided by Monteloeder, S.L. (Elche, Spain). The 92

extract was freshly prepared before use, dissolved in culture media 93

and filtered. 94

Cellular experimental model and measurement of intracellular reactive 95

oxygen species (ROS) 96

The 3T3-L1 preadipocytes were purchased from the American 97

Type Culture Collection (Manassas, VA, USA), propagated and dif- 98

ferentiated according to previously described procedures (Green and 99

Kehinde 1975). Adipocytic differentiation was induced by adding adi- 100

pogenic agents (0.5 mM IBMX, 1 μM DEX, and 1 μM INS) to the cul- 101

ture medium for 2 days. The medium was freshly replaced every 48 h. 102

The phenotypic change of adipogenesis was observed under a micro- 103

scope. In all experiments, more than 90% of the cells were mature 104

adipocytes after 8–10 days of incubation. To induce cellular hyper- 105

trophy, adipocytes were exposed to high glucose (25 mM) for at least 106

18 days (Yeop Han et al. 2010). Further details on this cellular model 107

are provided in Supplementary Fig. 2, indicating the accumulation of 108

intracellular lipid droplets and the effect of glucose supplementation 109

during the transformation process. Differential effects on mature or 110

hypertrophic adipocytes were assayed by adding LC or VB for 48 h, in 111

pre-designed concentrations to the media. The absence of cytotoxicity 112

was ascertained using the crystal violet method. 113

ROS generation was assessed in hypertrophic adipocytes using 114

2′,7′-dichlorodihydro-fluorescein diacetate (H2DCF-DA) as described 115

(Yeop Han et al. 2010). Fluorescent microphotographs were captured 116

via fluorescent microscopy (Eclipse TE2000-U, Nikon Microscope, 117

Melville, NY). 118

Western blot analysis 119

High glucose-induced hypertrophic adipocytes were cultured for 120

the indicated times and treated for 48 h with various concentrations 121

of LC and VB. After incubation, cell extracts were analyzed by West- 122

ern blot. Hypertrophic adipocyte were lysed with ice-cold lysis buffer 123

(20 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 1% CHAPS, 1 mM Pe- 124

fabloc and 1% phosphatase inhibitor cocktail no. 2, Sigma–Aldrich 125

Inc., Steinheim, Germany). Protein concentrations were determined 126

by a NanoDrop spectrophotometer (NanoDrop Technologies, Wilm- 127

ington, DE). Electrophoresis was performed in NuPAGE 4–12% Bis– 128

Tris gradient or 4–20% Tris-glycine polyacrylamide gels (Invitrogen, 129

Barcelona, Spain). MES was used for AMPK, pAMPK, PPAR-α and actin 130

electrophoresis, while Tris-Gly buffer (Invitrogen) was used for FASN 131

electrophoresis. Proteins were transferred to nitrocellulose mem- 132

branes using the iBlot transfer system (Invitrogen). Antibodies used 133

were Rabbit anti-AMPK (#2532, Cell Signaling Tech., Danvers, MA, 134

USA), rabbit anti-pAMPK (Thr172) (#2531, Cell Signaling Tech.), rab- 135

bit anti-PPAR-α (H-98, St. Cruz Biotech., Heidelberg, Germany), rabbit 136

anti-FASN (3180, Cell Signaling Tech.) and rabbit anti-actin (H-300, St. 137




M. Herranz-López et al. / Phytomedicine xxx (2015) xxx–xxx 3


(A)

(B)

(C)

Fig. 1. Lemon verbena polyphenols decrease the triglyceride accumulation and the ROS generation in high-glucose mature (A) or hypertrophic (B, C) adipocytes. Representative

photomicrographs of intracellular lipids, as assessed by AdipoRed staining, and actual measurements compared with those obtained in controls, in percentage, are depicted.

Intracellular ROS generation (% oxidation) was assessed by using the ROS sensitive fluorescent probe H2DCF-DA. The data are expressed as the mean ± S.D. from three independent

experiments performed in octuplicate. Data points at the abscissa axis contain equivalent VB concentrations for the extract and the pure compound. LC, Lippia citriodora; VB,

verbascoside.

F

c

s

ig. 2. LC and VB decrease FASN and stimulate PPAR-α mRNA expression, activating AMP

ultured in medium containing LC or VB for 48 h. The crude cell extracts were used for W

oftware. Data in the graph are expressed as the mean ± S.D. (n = 3).

Please cite this article as: M. Herranz-López et al., Lemon verbena (Lippi

hypertrophic adipocytes through AMPK-dependent mechanisms, Phytom

K in high glucose-induced hypertrophic adipocytes. Hypertrophic adipocytes were

estern blot analysis (15 μg/lane). Band intensities were measured using Image Lab

a citriodora) polyphenols alleviate obesity-related disturbances in

edicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.03.015




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Cruz Biotech.). The secondary antibodies were goat anti-rabbit-HRP

(Dako, Glostrup, Denmark) and anti-goat-HRP (Dako). Chemilumines-

cent detection was performed using the ECL Advance Western Blot-

ting 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 densitom-

etry normalized to β-actin signal using software Image Lab (Version

3.0 build 11, Bio-Rad, Madrid, Spain).

Immunofluorescence study

For NF-κB 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 (Abcam Inc.,

USA). Cells were then washed with PBS and incubated for 2 h with each

corresponding secondary antibody, Anti-Rabbit IgG-TRITC (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 mea-

sured 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 re-

ported (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 β-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 con-

struct [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 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 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 Mito-

Tracker Red CMXRos (Mred) and MitoTracker Green FM (Mgreen)

(Molecular Probes, Invitrogen, Carlsbad, CA, USA) for 30 min at

37 °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 ) were the progeny of animals obtained from the Jackson c
Please cite this article as: M. Herranz-López et al., Lemon verbena (Lipp


aboratory. The animal handling, sample preparation, sampling, sac-

ifice and the calculation of sample size were performed as described

Joven et al. 2007). At 10 weeks of age, the animals (n = 16) with

quivalent body weight were assigned to two study groups (n = 8,

ach) and were fed a high-fat, high-cholesterol diet (HF, 20% fat and

.25% cholesterol, w/w). One of these treatment groups received tap

eionized water as a unique liquid source (control), while the other

roup received the LC extract dissolved in tap deionized water (5 g/l),

hich was freshly prepared every day. Although water and/or extract

ere administered ad libitum, LC extract solution intake was mea-

ured and average daily dose was estimated for treated animals ob-

aining a value of 750 mg/kg b.w. (202.5 mg/kg b.w. of verbascoside).

lood and tissue samples were obtained and processed as previously

escribed (Beltrán-Debón et al. 2011). The oral fat test and the glu-

ose tolerance test were performed as described elsewhere (Rull et al.

007). Liver steatosis was qualitatively evaluated on a scale of 0–3,

here 0 represented an absence of steatosis and 3 indicated a major

rade of steatosis (>66%). All procedures in LDL receptor-deficient

ice and experimental protocols were examined and approved by

he Ethical Committee for Animal Experimentation of the University

iguel Hernández (IBM-VMM-003-12).

tatistical analyses

Values are represented as the mean ± standard deviation of the

ean. The values were subjected to statistical analysis (one-way

NOVA, Student’s t-test for unpaired samples and Tukey’s test for

ultiple comparisons). The differences were considered statistically

ignificant at p < 0.05. All analyses were performed using GraphPad

rism 5 software (GraphPad, San Diego, CA). ∗p < 0.05, ∗∗p < 0.01 and∗∗p < 0.001 on bars indicate statistically significant differences versus

ontrol, unless otherwise stated. Horizontal lines indicate statistically

ignificant differences between bars. All cellular measurements de-

ive from three independent experiments, wherein each performed

n octuplicates, unless specified.

esults

C and VB decrease lipid deposition and ROS generation in mature and

ypertrophic adipocytes: a possible role for mechanisms involved in

ipogenesis and mitochondrial dysfunction

To explore the effects of LC or VB on adipocyte model, we consid-

red either mature or hypertrophic adipocytes those in the 8th or 18th

ay after the start of differentiation respectively. Polyphenols were

dded at increasing concentrations in these time-points and cells

ere further incubated for two additional days. In mature adipocytes

ncubated with high glucose levels, there was a dose-dependent, sim-

lar decrease (20%) in triglyceride content with the maximum concen-

ration of LC extract and VB utilized (400 μg/ml LC and 108 μg/ml VB,

ontained equivalent verbascoside concentrations; Fig. 1A). The effect

as also similar in high glucose-induced hypertrophic adipocytes;

1.6% and 29% respectively (Fig. 1B). Similarly, both LC extract and

B decreased intracellular ROS generation (approximately 40%) in

igh glucose-induced hypertrophic adipocytes. Consequently, subse-

uent analyses were limited to hypertrophic adipocytes. To further

lucidate the molecular mechanism involved in the suppressive ef-

ect of LC and VB on lipid accumulation, we investigated the effects

f LC and VB on the mRNA expression and protein levels of PPAR-

, a major regulator of lipid metabolism, fatty acid synthase (FASN),

ipogenic-related gene, and the central metabolic sensor AMPK. After

8 h of incubation of high glucose-induced hypertrophic adipocytes

n the presence of LC or VB, we found an increase in PPAR-α, a de-

rease in FASN and a significant activation of AMPK (Fig. 2). Significant

ifferences were detected between LC and VB at equivalent VB con-

entrations, revealing a higher effect in the presence of LC extract. 253

ia citriodora) polyphenols alleviate obesity-related disturbances in





Fig. 3. LC and VB restore mitochondrial membrane potential in high glucose-induced hypertrophic adipocytes. MitoTracker CMXRos (Mred) and Green (Mgreen) were used to

evaluate the mitochondrial function in hypertrophic adipocytes treated with LC or VB. Representative photomicrographs show the loss of red fluorescence in high glucose-induced

hypertrophic adipocytes compared to low glucose incubated cells. Hypertrophy was decreased in the presence of LC or VB, and red-fluorescence (mitochondrial potential) was also

re-established compared to the control (10× magnification). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this

article.) Q4

Also, and to determine the effect of LC or VB on intracellular ROS gen-254

eration and their impact on mitochondrial function, we analyzed the255

accumulation of MitoTracker CMXRos (Mred), which is dependent256

on the mitochondrial membrane potential, and MitoTracker Green257

(Mgreen), a general mitochondrial marker (Fig. 3). Fluorescence mi-258

crographs show that high glucose-induced hypertrophic adipocytes259

revealed a dramatic loss of Mred staining without the loss of Mgreen260

staining, indicating a decrease in mitochondrial membrane potential,261

which was partially restored by both LC and VB treatments, reveal-262

ing a possible mechanism to explain the decrease in intracellular ROS263

generation (Fig. 1C).264

LC and VB upregulate adiponectin gene expression but only LC265

downregulates NF-κB in hypertrophic adipocytes266

We then explored the effects of polyphenols on NF-κB, as a reg-267

ulator of the oxidative stress-induced inflammatory response and268

on adiponectin, a perceived anti-inflammatory cytokine (Fig. 4). For269

this purpose, hypertrophic adipocytes were incubated with LC or270

VB for 48 h. Under these experimental conditions, the LC extract271

significantly upregulated the adiponectin gene expression as com-272

pared to the equivalent concentration of purified VB. Although LC273

and VB treatments showed small but significant decreases in NF-κB274

gene expression compared to the control, there were no differences275

between their capacities to downregulate NF-κB gene expression276

(Fig. 4A).277

When protein levels were quantitated, both LC and VB showed278

dose-dependent significant increases in adiponectin expression com-279

pared to the control and also, a differential effect of the LC extract280

and VB was confirmed on adiponectin expression. However, only LC281

exhibited a significant effect on NF-κB expression levels (Fig. 4B).282

Cellular assays using immunofluorescence resulted in similar values 283

(Fig. 4C and D). These effects on the anti-inflammatory action were 284

further confirmed when we measured the gene expression of se- 285

lected cytokines in the same experimental cells. We found significant 286

reductions with both, extract and purified compound, with respect 287

to controls, of IL-1β , IL-6, TNF-α and CCL2/MCP-1 but there were no 288

changes in IL-1a and leptin expression levels (Supplementary Fig. 3). 289

To clarify whether the effect of LC or VB on the transcriptional 290

upregulation of adiponectin was mediated by PPAR-γ , a critical tran- 291

scription factor involved in adiponectin expression (Bouskila et al. 292

2005; Iwaki et al. 2003), we transfected the adipocytes with differ- 293

ent constructs containing either the wild type or mutated adiponectin 294

luciferase promoter. Luciferase activity driven by the adiponectin pro- 295

moter in transfected adipocytes incubated in the presence or absence 296

of either LC or VB was determined. At the highest concentrations, cells 297

treated with either LC or VB showed a significant dose-dependent 298

increase in basal luciferase activity of the wild-type adiponectin pro- 299

moter [p(-908)/LUC wt] as compared to controls (Fig. 5A). When we 300

used the human adiponectin promoter bearing the mutated PPAR 301

response element [p(-908)/LUC PPRE mut] there were no apprecia- 302

ble changes in luciferase activity indicating a possible major role for 303

PPAR-γ in the activation of adiponectin promoter (Fig. 5B). 304

Lemon verbena polyphenols alleviate diet-induced obesity in an 305

animal model 306

To reinforce our interpretation we decided to test the influence of 307

polyphenols in an animal model. When fed a high-fat diet, LDLr de- 308

ficient mice, consistently develop obesity-associated metabolic dis- 309

turbances and hypertrophic adipocytes (Beltrán-Debón et al. 2011; 310

Joven et al. 2012a). The effects of LC extract rather than purified VB 311






nectin

μg/ml

(A). W

cellul

were

ter w

the l

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Fig. 4. The influence of LC and VB on the gene expression and protein levels of adipo

and 400 μg/ml of LC and its corresponding concentration of purified VB (54 and 108

Nevertheless, a slight decrease on NF-κB gene expression was observed by LC or VB

effects in both proteins (B). Photomicrographs obtained using an immunofluorescence

Data in the graph are expressed as the mean ± S.D. (n = 3).

Fig. 5. LC upregulates PPAR-γ -mediated adiponectin expression. Mature adipocytes

the human adiponectin promoter [p(-908)/LUC wt] (A) or containing the same promo

transfected cells were incubated for 24 h with different concentrations of LC or VB, and

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-

take 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 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 im-

proved triglyceride clearance (Fig. 6B). Also, treated animals depict



and NF-κB. High glucose-induced hypertrophic adipocytes were incubated with 200

). The extract strongly upregulated the adiponectin gene expression compared to VB.

hen assessed as protein levels, however, the extract, but not VB, showed significant

ar assay confirmed these results for both adiponectin (C) and NF-κB (D) respectively.

transfected with luciferase constructs lacking a promoter (pGL3-basic), containing

ith the PPAR-response element mutated [p(-908)/LUC PPRE mut] (B). After 24 h, the

uciferase activities were measured. The data are expressed as the mean ± S.D. (n = 8).

ignificantly lower serum cholesterol and triglycerides concentra-

ions than the controls (Fig. 6C and D). Among obesity-associated

isturbances in this animal model, liver steatosis is a constant find-

ng. The administration of the LC extract alleviated the accumulation

f neutral lipids and triglycerides in the liver without altering serum

iomarkers of hepatic toxicity (Fig. 6E–G).

iscussion

The 3T3-L1 cell line is a well characterized and widely accepted

odel of in vitro adipogenesis and lipid accumulation that becomes

ypertrophic and insulin resistant when induced with high-glucose

onditions (Green and Kehinde 1975; Han et al. 2007; Herranz-Lopez

t al. 2012; Ji et al. 2014; Yoshizaki et al. 2012; Zebisch et al. 2012). In

his model, we have assayed the capacity of LC and VB to ameliorate

besity-induced metabolic disturbances.






Fig. 6. Lemon verbena polyphenols improve triglyceride clearance and prevent the development of fatty liver disease in hyperlipidemic mice. LC supplementation did not modify

glucose tolerance (A) but significantly decreased the triglyceride values at 60 and 120 min (mg/dl) after the ingestion of an oral olive oil bolus (B), and decreased cholesterol and

triglyceride plasma levels in HF-fed mice (C and D). The liver steatosis induced by the HF-diet was significantly decreased by LC polyphenol supplementation (E) without affecting

the serum biomarkers of liver injury (F, G). The representative histological sections illustrate the preventive effect of LC. ∗p < 0.05 denotes the significant differences between the

HF and HF + LC groups.

Polyphenols are the most intensively studied natural products342

a343

o344

w345

c346

p347

s348

a349

(350

f351

v352

a353

V354

B355

a356

i357

i358

359

d360

t361

t362

a363

o364

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t366

o367

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t370

Q371

p372

t373

f374

In this study we identify some previously unrecognized mecha- 375

n 376

m 377

d 378

p 379

t 380

m 381

h 382

s 383

t 384

f 385

a 386

s 387

b 388

p 389

l 390

w 391

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a 393

i 394

r 395

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d 398

e 399

t 400

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i 403

2 404

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( 407

s a recognized source of pharmacologic compounds. Several lines

f evidence suggest a significant impact of polyphenols in obesity,

hich is an increasingly prevalent condition. It is commonly ac-

epted that adipocyte hypertrophy compromises cell function in the

rogression of obesity associated metabolic disturbances and is as-

ociated to ROS generation and inflammation (Han et al. 2007), in

greement to our results. Despite the limitations of a cellular model

Green and Kehinde 1975; Ji et al. 2014; Zebisch et al. 2012), we

ound that LC polyphenolic extract and its major compound, VB, pre-

ented most of the expected deleterious effects. These results are

lso partially confirmed in an animal model of diet-induced obesity.

B is the most abundant (>25%) polyphenol in LC aqueous extracts.

oth have similar effects, decreasing lipid deposition in adipocytes

nd the consequent intracellular ROS generation. Contrarily, the anti-

nflammatory action differs, indicating a potential synergistic effect

n the extract.

A common diet contains >500 different polyphenols; extracts re-

uce the number but they are also a complex mixture and whether

his complexity is relevant to our health remains an elusive point. In

his study we have exposed cell lines to an individual polyphenol, VB,

nd responses are similar to those obtained with the LC extract. We

bviously recognize the inherent limitation in using high doses and

on-metabolized polyphenols but we consider that VB may be added

o the growing list of isolated polyphenols with a potential impact

n health. This is important to facilitate the identification of promis-

ng functional elements in polyphenols. We have previously found

he strong free radical scavenging capacity of LC polyphenols and

heir ability to enhance the activity of antioxidant enzymes (Carrera-

uintanar et al. 2012; Funes et al. 2011; Funes et al. 2009). Despite the

opular antioxidant hypothesis there is, however, no evidence that

he antioxidant properties of polyphenols improve the antioxidant

unction in the cell (Ji et al. 2014).



istic clues. Particularly, these polyphenols maintain mitochondrial

embrane potential and mitochondrial viability. These findings in-

icate that further consideration should be paid to other actions of

olyphenols that may be extended to the direct modulation of mi-

ochondrial events affecting the whole cell (e.g. energy generation,

itochondrial biogenesis or cell death control; Chung et al. 2010). We

ave considered a variety of molecular targets related to metabolic

tress in adipocytes. For example, the activation of the redox-sensitive

ranscription factors such as NF-κB is accepted as a deleterious ef-

ect in the pathogenesis of common diseases via the modulation of

large number of genes mediating immune and inflammatory re-

ponses (Jiang et al. 2011; Li and Karin 1999). Our results reveal that

oth LC and VB decreased NF-κB and increased adiponectin gene ex-

ression. Curiously, only the LC extract decreased the NF-κB protein

evels but the increase in adiponectin protein levels was observed

ith both the extract and the individual polyphenols. Moreover, the

diponectin expression in hypertrophic adipocytes was mediated by

transcriptional PPAR-γ -dependent mechanism. Further, the anti-

nflammatory action of adiponectin was accompanied by the down-

egulation of selected inflammatory genes and a significant activation

f AMPK in hypertrophic adipocytes. Adiponectin has been proposed

s a systemic functional link involved in the activation of AMPK in

ifferent tissues (Hattori et al. 2008; Iwabu et al. 2010; Okada-Iwabu

t al. 2013), probably through different actions of adiponectin recep-

or (AdipoR) (Fang et al. 2010). This is important because the ability

f polyphenols to act on both, adiponectin and AMPK, may represent

mportant regulators of glucose and lipid metabolism, modulators of

nflammation, oxidative stress and insulin resistance (Hardie et al.

012; Salminen and Kaarniranta 2012) and consequently a therapeu-

ic opportunity in the management of obesity. Therefore, if confirmed,

he role of VB as an AMPK activator could have relevant implications

Grahame Hardie 2014).






Fig. 7. The putative mechanism of VB and LC polyphenols to prevent high glucose-induced metabolic disturbances. Metabolic stress as a result of energy excess triggers chronic

inflammation, oxidative stress and mitochondrial dysfunction, leading to the inhibition of the energy sensor AMPK. Lemon verbena polyphenols reduce intracellular ROS generation

and inflammation and reestablish mitochondrial viability (⇓ AMP) and AMPK activation that results in PPAR-γ -mediated adiponectin upregulation and PPAR-α activation. This

regulation may lead to a decrease in lipogenesis and cholesterol synthesis, which normalizes the lipid profile and stimulates fatty acid oxidation in several tissues (adipose tissue,

liver and skeletal muscle). Alternatively, polyphenols may hypothetically act extracellularly as AdipoR agonists leading to the Ser/Thr kinases-mediated activation of AMPK or also

as direct PPAR agonists (IKK, I kappa B kinase).

Of note, the effect of VB and LC extract increasing adiponectin se-408

cretion, restoring mitochondrial function and decreasing the size of409

hypertrophic adipocytes may be linked to previous studies indicating410

that a reduction in mitochondrial mass or function causes the hyper-411

trophy of adipocytes, and these mitochondrial changes are linked to412

decreased adiponectin synthesis (Koh et al. 2007). On the other hand,413

these polyphenols increased PPAR-α mRNA expression with the con-414

sequent upregulation of downstream genes involved in fatty acid ox-415

416

417

418

419

420

421

422

423

424

425

426

427

428

429

430

431

mediated by polyphenol metabolites acting at intracellular level or by 432

interacting to membrane receptors remains a major challenge. Recent 433

findings indicate that agonists of AdipoR, which mimic the effect of 434

adiponectin and activate AMPK and PPAR-α, ameliorate insulin resis- 435

tance (Okada-Iwabu et al. 2013). Because polyphenols depict reason- 436

able structural similarities with these agonists, we propose a hypo- 437

thetical sequence of events outlined in Fig. 7. In agreement with this 438

assumption, the potential binding of the flavone moiety to PPARs has 439

a 440

t 441

442

t 443

V 444

m 445

t 446

s 447

o 448

e 449

( 450

a 451

fi 452

r 453

l 454

t 455

idation and mitochondrial machinery. Concomitantly, we observed a

decrease in FASN mRNA expression, which most likely contributed to

suppress lipid accumulation in the hypertrophic adipocytes (lipoge-

nesis). Additionally, LC seemed to be more effective than VB in most

of these cellular effects. Therefore, the activation of both AMPK and

PPAR-α and a decrease in FASN are consistent with a putative action of

these polyphenols in promoting fatty acid β-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 fur-

ther 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



lso been predicted by computational modeling to explain its capacity

o modulate PPARs in adipocyte cell model (Lu et al. 2013).

In conclusion, although an additional value of the complete LC ex-

ract, via synergistic or complementary effects cannot be discarded,

B deserves further attention as a therapeutic aid in the manage-

ent of obesity and/or associated disturbances. The conversion of

he dose utilized in our animal study to human equivalent dose re-

ulted in several grams per day of lemon verbena extract. Despite

f appearing to be a high dose, the use of 1.8 g of lemon verbena

xtract for at least 1 month revealed to be safe in human studies

Funes et al. 2011). Whether lower doses show potential for clinical

pplications in obesity needs to be verified in human studies. Our

ndings indicate interactions with numerous endogenous proteins

elated to energy-sensing pathways, such as AMPK sensor, at cellular

evel and the normalization of fat metabolism in animal model. Never-

heless, we are fully aware that this fact diminishes the interest in drug






developers despite being the case in many marketed drugs (e.g. sal-456

icylates). Further studies including the detected metabolites of this457

polyphenol and ongoing metabolomic studies may help to unravel458

the potential health effects in humans.459

Conflict of interest460

The authors declare no conflict of interest.461

Uncited referenceQ5

462

Quirantes-Pine et al. (2009).463

Acknowledgments464

This work was supported by AGL2011-29857-C03-03, BFU2014-465

52433-C3-1-R and IDI-20120751 grants (Spanish Ministry of Science466

and Innovation), PROMETEO/2012/007 and ACOMP/2013/093 grants467

from Generalitat Valenciana, and CIBER (CB12/03/30038, Fisiopa-468

tología de la Obesidad y la Nutrición, CIBERobn, Instituto de Salud469

Carlos III). M.H. is a recipient of a VALi+D fellowship from Generalitat470

Valenciana (ACIF/2010/162). We thank E. Rodríguez-Gallego and R.M.471

Medina-Gali for their invaluable help in gene and protein expression472

experiments and to Monteloeder, SL (Alicante, Spain) for the lemon473

verbena extract.474

Supplementary Materials475

Supplementary material associated with this article can be found,476

in the online version, at doi:10.1016/j.phymed.2015.03.015.477

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