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Lycopene-supplemented diet ameliorates metabolic syndrome induced by

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DOI: 10.1016/j.jff.2020.104098

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 Journal of Functional Foods 73 (2020) 104098

Contents lists available at ScienceDirect

Journal of Functional Foods

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

Lycopene-supplemented diet ameliorates metabolic syndrome induced by T

fructose in rats
⁎
Pedro Ferreira-Santos, Rubén Aparicio, Rosalía Carrón, M. José Montero, M. Ángeles Sevilla
Department of Physiology and Pharmacology, Faculty of Pharmacy, University of Salamanca, 37007 Salamanca, Spain
Biomedical Research Institute of Salamanca (IBSAL), Hospital Virgen de la Vega, 37007 Salamanca, Spain

A R T I C LE I N FO A B S T R A C T

Keywords: This study aimed to evaluate the effect of a lycopenesupplemented diet on a model of metabolic syndrome
Lycopene induced by fructose. Male Wistar rats receiving a normal diet plus tap water with 20% fructose (F) were used;
Fructose metabolic syndrome half were treated simultaneously with 0.01% lycopene (FL). Rats receiving a normal diet were also divided into
Hypertension two groups, a control group untreated (C) and another with lycopene treatment (L). Fructose intake progres-
Dyslipidemia
sively increased the blood pressure, caused cardiac hypertrophy and endothelial dysfunction, as well as meta-
Endotelial-dysfunction
Oxidative-stress
bolic changes. Lycopene treatment significantly attenuated the development of hypertension and endothelial
dysfunction and prevented cardiac hypertrophy, but had no effect in rats fed with standard diet. Others mani-
festations of metabolic syndrome like insulin resistance, dyslipidemia, liver enlargement, accumulation of in-
traperitoneal fat or oxidative stress were also improved by lycopene. In conclusion, our results suggest that
lycopene significantly attenuates pathophysiological condition of metabolic syndrome caused by fructose con-
sumption.

1. Introduction
Metabolic syndrome (MS) is a complex pathophysiologic state
whose prevalence continues to rise worldwide representing a major and
escalating public health problem (Ahima, 2016; Saklayen, 2018). Al-
though there is no international consensus to define MS, the majority of
scientific societies contemplate the presence of three of the following
five criteria: obesity with excess of abdominal fat evidenced by waist
circumference ≥102 cm in men or ≥88 cm in woman, fasting plasma
glucose ≥100 mg/dL; blood pressure ≥130/85 mmHg; triglycerides
(TG) ≥150 mg/dL; high density lipoprotein cholesterol (HDLc) < 40
mg/dL in men or < 50 mg/dL (Grundy, 2016).
A meta-analysis showed that MS doubled the risk of cardiovascular
disease (CVD) and increased all-cause mortality by up to 50%, even
excluding cases of diabetes or subjects with previous CVD (Mottillo
et al., 2010). CVD remain a major cause of premature death and chronic
disability for all regions of the world (Roth et al., 2017). These diseases
have remained as the leading causes of death globally in the last
15 years (Com, 2018).
Redox signaling via reactive oxygen species (ROS) has quite recently
become the focus of much attention in numerous pathological contexts,
including obesity, hypertension, diabetes, and dyslipidemia, all com-
prised in MS (Matsuda & Shimomura, 2013; Togliatto, Lombardo, &
Brizzi, 2017). ROS play a role as second messengers in signal trans-
duction for vascular homeostasis and cell signaling. However when
they are overproduced or when antioxidant capacity is decreased, they
can inflict damage as inflammation, migration and deposition of ex-
tracellular matrix, endothelial dysfunction, hypertrophy and cardiac
dysfunction, fibrosis, etc. (Guzik & Touyz, 2017; Montezano & Touyz,
Montezano & Touyz, 2014, 2012).
Several strategies have been proposed to fight MS, including phar-
macological approaches, promotion of physical activity and dietary
changes. Various studies shown beneficial effects of some antioxidants
in diabetes, obesity and hypertension (Abdali, Samson, & Grover, 2015;

Abbreviations: ACh, acetylcholine; AI, atherogenic index; BMI, body mass index; BW, body weight; CVD, cardiovascular diseases; GOT, aspartate transaminase; GPT,
alanine transaminase; HDLc, high density lipoprotein-cholesterol; HOMA-IR, homeostasis model assessment index; LDLc, low density lipoprotein-cholesterol; LH,
liver hypertrophy; LVH, left ventricular hypertrophy; MRA, mesenteric resistance arteries; MS, metabolic syndrome; NADPH, nicotinamide adenosine dinucleotide
phosphate; O2−, superoxide anion; OGTT, oral glucose tolerance test; PE, phenylephrine; RLU, relative luminescence units; ROS, reactive oxygen species; SBP,
systolic blood pressure; SEM, standard error of the mean; SNP, sodium nitroprusside; TBARS, thiobarbituric acid reactive substances; TC, Total cholesterol; TG,
triglycerides
⁎
Corresponding author.
E-mail address: masevilla@usal.es (M.Á. Sevilla).

https://doi.org/10.1016/j.jff.2020.104098
Received 13 April 2020; Received in revised form 16 June 2020; Accepted 29 June 2020
1756-4646/ © 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
P. Ferreira-Santos, et al.
Ahmad et al., 2017). In this context, has been suggested that a diet rich
in antioxidants can bring health benefits (Varadharaj et al., 2017) and,
therefore, is gaining interest. Recent research and meta-analysis have
reported that adherence to the Mediterranean diet have a protective
effect against CVD risk factors, several types of cancer, and, most re-
cently, Alzheimer and Parkinson’s disease (Tosti & Bertozzi, 2017), and
it is associated with a lower risk of MS (Babio et al., 2014; Godos et al.,
2017; Grosso et al., 2014; Kastorini et al., 2011; Kesse-Guyot et al.,
2013). Mediterranean diet, which includes high consumption of fruits,
vegetables, legumes, nuts, seeds, olive oil, and moderate intake of red
wine, is very rich in antioxidant compounds like polyphenols, car-
otenoids, etc.
Lycopene, compound of the carotenoids family, is a red pigment
found in tomatoes and other red fruits and vegetables included in the
Mediterranean diet, with antioxidant properties and anti-free radical
action and beneficial effects on cardiovascular health (Cheng et al.,
2017; Li & Xu, 2013; Müller, Caris-Veyrat, Lowe, & Böhm, 2016; Thies,
Mills, Moir, & Masson, 2017; Willcox, Catignani, & Lazarus, 2003; Zeng
et al., 2019).
Previous studies from our group also have shown the ability of a diet
supplemented with lycopene to prevent the cardiovascular remodeling
and oxidative stress parameters linked to hypertension induced by an-
giotensin-II in rats (Ferreira-Santos et al., 2018). This work focuses on
studying if lycopene, besides cardiovascular benefit, may also improve
alterations characteristic of MS such as dyslipidemia, resistance to in-
sulin, obesity or accumulation of abdominal fat. We investigate the
ability of lycopene to prevent the onset of this pathological status.
2. Materials and methods
2.1. Experimental protocol
Male Wistar rats, supplied by the Animal Experimentation Service of
the University of Salamanca (PAE-SA001; Salamanca, Spain), were
utilized according to the EU Directive 2010/63/EU for animal experi-
ments and the protocol approved by the Bioethics Committee of the
University of Salamanca (Register 006N°201400039292). The rats eight
weeks old (225 ± 1 g) were randomly assigned to four groups of seven
rats per group: (1) C: the animals in the control group were fed a
standard rodent chow and tap water; (2) L: the rats were fed a standard
rodent diet supplemented with 0.01% of lycopene and tap water; (3) F:
the rats in the fructose group had the same standard rodent chow and
additional 20% fructose in tap water; (4) F-L: in this group the rats
received a standard rodent diet supplemented with 0.01% of lycopene
and tap water with 20% fructose. Rats were housed at 23 ± 2 °C with
12 h light/dark cycles with ab libitum access to water and food. The
study lasted for 12 weeks. The dose of lycopene was selected taking into
account a previous work by our group (Ferreira-Santos et al., 2018).
Body weight changes and caloric ingestion were monitored weekly
during the whole experiment and fluid intake daily.
At the end of study, fasted rats were anaesthetized with pento-
barbital (60 mg/kg bw, i.p.). Blood was collected into tubes, then
plasma was separated by centrifugation and samples were stored at
−80 °C until analysis. Heart, liver, abdominal fat and mesenteric and
aorta arteries were harvested in chilly Krebs solution (composition in
mM: NaCl, 118; KCl, 4.7; CaCl2, 2.5; KH2PO4, 1.2; MgSO4, 1.2;
NaHCO3, 25 and glucose, 11, pH = 7.4) and properly processed for
carrying out morphometric, histological or functional studies. Finally,
the length of the tibia was recorded.
2.2. Measurement of blood pressure
Systolic blood pressure (SBP) was measured by the tail-cuff method
with an automated system (CODA tail-cuff blood pressure, Kent
Scientific, Torrington, USA), as we described elsewhere (Sánchez et al.,
2011), at the beginning and monthly during the study.
2
Journal of Functional Foods 73 (2020) 104098
2.3. Glucose tolerance and insulin resistance
Oral Glucose Tolerance Test (OGTT) was performed just before the
end of study using the Accu-Check glucometer monitor and strips
(Roche Diagnostics, West Sussex, UK). After an overnight fast, baseline
blood glucose (time 0) was determined; then a glucose solution (2 g/kg)
was given to the rats by gavage, followed by the determination of blood
glucose levels at 30, 60, and 120 min. The area under the curve (AUC)
of glucose was calculated.
Fasted plasma insulin level was estimated using rat insulin enzyme
linked immunosorbent assay (ELISA) kits (RayBiotech Inc., Norcross,
United States). Insulin resistance was evaluated by the Homeostasis
Model Assessment index (HOMA-IR) using the formula described by
Matthews (Matthews et al., 1985): [insulin (mIU/L) × glucose (mmol/
L)]/22.5.
2.4. Morphometric determinations
2.4.1. Left ventricular hypertrophy
The atrium was removed from the heart and all the epicardial fat
was scraped off. The right and the left ventricles were separated, re-
garding the interventricular septum as an integral part of the left ven-
tricle, and this portion was weighed. The left ventricular hypertrophy
(LVH) index was calculated using left ventricle weight/tibial length
ratio (mg/mm).
2.4.2. Liver hypertrophy
Isolated liver was washed with chilly Krebs solution and moisture
was eliminated with filter paper. The liver hypertrophy (LH) index was
determined using liver weight/tibial length ratio (g/mm).
2.4.3. Body mass index and adiposity measurement
The body mass index (BMI) was calculated by dividing body weight
(g) to body length squared (cm2). For each rat the abdominal adipose
tissue (retroperitoneal and epididymal fat) were excised and weighed.
The adiposity index was calculated as abdominal fat weight versus ti-
bial length (g/mm).
2.5. Histological studies
Liver and retroperitoneal adipose tissue were fixed in 4% paraf-
ormaldehyde, included in paraffin, cut in 5 μm thick sections, and
stained with hematoxylin and eosin (H&E). Images were examined
under an optical microscope and captured using a high-resolution di-
gital camera (Olympus DP50, Tokyo, Japan).
Size of hepatocytes was measured as an average of 20 hepatocytes
taken from different zones of histological sections using ImageJ soft-
ware (US National Institutes of Health, http://rsb.info.nih.gov/ij/).
Adipocyte-size analysis was performed by measuring cross-sectional
area with ImageJ software and calculating the average based on the
values of 5 adipocytes in 5 random fields per slice.
2.6. Plasma lipid profile and hepatic transaminases
Lipid profile and plasmatic levels of hepatic transaminases (aspar-
tate aminotransferase (GOT), alanine aminotransferase (GPT)) were
determined by enzymatic methods using reagent strips for quantitative
measurement (SpotchemTM II, Arkray, Shiga, Japan) in a fully auto-
matic biochemical analyzer (SpotchemTM EZ sp-4430, Arkray, Shiga,
Japan). TG, total cholesterol (TC) and HDLc were determined and low
density lipoprotein-cholesterol (LDLc) was calculated according to the
formula: LDLc = TC − (HDLc + TG/5). The atherogenic index (AI)
was calculated as follows: AI = (TC − HDLc)/HDLc (Olmez et al.,
2015).
P. Ferreira-Santos, et al.
2.7. Vascular reactivity
The thoracic aorta and the third-order mesenteric resistance arteries
(MRA) were dissected and placed in Krebs solution at 4 °C.
Isolated thoracic aorta rings (3 mm in length) were placed in organ
bath with Krebs solution at 37 °C aerated with carbogen as described
elsewhere (Ferreira-Santos, Carrón, Recio, Sevilla, & Montero, 2017).
After one-hour equilibration period at a resting tension of 2 g, the
vessels were exposed to phenylephrine (PE, 10−6 M) and, at the steady
maximal contraction, cumulative concentration–response curves were
obtained for acetylcholine (ACh 10−8 −10−4 M) or sodium ni-
troprusside (SNP, 10−9−10−4 M).
MRA segments (2 mm in length) were mounted in a small vessel
dual chamber myograph (DMT A/S, Hinnerup, Denmark) for mea-
surement of isometric tension. Two steel wires (40 µm diameter) were
introduced through the lumen of the segments and mounted according
to the method described by Mulvany and Halpern (Mulvany & Halpern,
1977). After a 30 min equilibration period in Krebs solution at 37 °C
bubbled with carbogen, segments were stretched to their optimal lumen
diameter for the active tension development.
After a new 30 min equilibration period, the vessels were exposed to
a 120 mM KCl solution to check their functional integrity. After 15 min
washout, cumulative concentration response curves to ACh (10−8 to
10−4 M) or SNP (10−9 to10−4M) were performed in arteries previously
contracted with PE (3 × 10−7 M).
Responses to ACh and SNP were expressed as percentage of PE
contraction.
2.8. Parameters of oxidative stress
2.8.1. Detection of superoxide anion
Superoxide anion)%O2−) production was assessed by lucigenin-en-
hanced chemiluminescence assay. Briefly, segments of thoracic aorta
were incubated in ROS phosphate buffer (composition in mM: K2H2PO4
50, EGTA 1, sucrose 150 mM and ammonium diethyldithiocarbamate,
10 mM), gassed with carbogen and maintained at 37 °C for 15 min.
Then, samples were transferred into tubes containing ROS buffer, ni-
cotinamide adenosine dinucleotide phosphate (NADPH, 10-4 M) and
lucigenin (5 μM). Lucigenin chemiluminescence was recorded every
10 s for 5 min in a luminometer (LUMAT LB-9507, Berthold
Technologies, Bad Wildbad, Germany). Production of %O2− was ex-
pressed as relative luminescence units (RLU)/min/mg dry tissue.
2.8.2. Lipid peroxidation
Damage to the lipid membrane was evaluated by the formation of
products of lipid peroxidation (malondialdehyde, MDA) in plasma
using the thiobarbituric acid reactive substances (TBARS) method, as
described previously by Kassan et al. (Kassan, Montero, & Sevilla,
2009). The results were expressed as MDA concentration (nmol/mL).
3. Statistical analysis
Data are expressed as the mean ± standard error of the mean
(SEM). The cumulative concentration–response curves by ACh and SNP
were fitted to a logistic equation and the concentration producing half
maximal effect was calculated. Comparison of concentration–response
curves was performed according to the extra sum of squares F-test
principle. The level of statistical significance was determined by one-
way analysis of variance (ANOVA) followed by Bonferroni’s test for
multiple comparisons and two-way ANOVA for blood pressure data.
Significance was accepted at P < 0.05. Fitting and statistical analyses
were performed using GraphPad Prism® software (version 5.0;
GraphPad Software, Inc., San Diego, USA).
3
Journal of Functional Foods 73 (2020) 104098
Fig. 1. Evolution of systolic blood pressure (SBP) during the study in control
rats (C), control rats treated with lycopene (L), untreated rats fed with fructose
enriched-diet (F) and rats fed with fructose and treated with lycopene (F-L).
Values are expressed as mean ± SEM of 7–8 rats. *P < 0.05 vs. C, and
#
P < 0.05 vs. F.
4. Results
4.1. Systolic blood pressure
The periodical control of blood pressure showed that lycopene had
no effect on SBP of normotensive rats. Fructose supplementation pro-
gressively increased this parameter up to hypertension levels (from
105 ± 1 mmHg to 141 ± 1 mmHg at the end of treatment) and the
concurrent treatment with lycopene reduced this increase
(120 ± 1 mmHg), although it did not reach the values of the control
animals (Fig. 1).
4.2. Glucose tolerance and insulin resistance
At the end of the study, fasting glucose concentrations were con-
sistently lower in C group compared with F group (91 ± 2 mg/dL vs.
101 ± 2 mg/dL, P < 0.05). After the oral glucose load, F group
reached a higher glucose peak and cleared the glucose load from the
circulation more slowly than did the others (Fig. 2A). AUC was calcu-
lated to estimate glucose tolerance. As shown in Fig. 2A no statistical
difference was found between L and C groups. As expected, AUC was
increased, about 20%, in fructose fed rats compared with control group,
and significantly decreased in lycopene supplementation group.
Concomitantly, insulin levels were measured and enabled the cal-
culation of HOMA-IR. As shown in the Fig. 2B and C, both parameters
were increased in fructose fed rats with respect to control group, and
significantly decreased in F-L group. Lycopene supplied alone did not
change these parameters when compared to the control group.
4.3. Morphometric determinations
Body weight was slightly higher in F group at the end of the study
but, no significant changes were observed when compared to control
group. On the other hand, lycopene was able to reduce significantly the
body weight as in fructose fed rats as in animals fed unsupplemented
diet (Table 1).
As shown in Table 1, abdominal fat, BMI, LVH and liver index were
similar in C and L groups, but significantly higher in F group when
compared to C animals. These parameters were restored to normal le-
vels when the diet was supplemented with lycopene.
There was no significant difference in caloric intake among the
groups (data not shown).
P. Ferreira-Santos, et al. Journal of Functional Foods 73 (2020) 104098

Fig. 2. Oral glucose tolerance test and calculated area under the curve (AUC) (A), plasmatic insulin values (B) and HOMA-IR (C) in control rats (C), control rats
treated with lycopene (L), untreated rats fed with fructose enriched-diet (F) and rats fed with fructose and treated with lycopene (F-L). Values are expressed as
mean ± SEM of 7 rats. *P < 0.05 vs. C, and #P < 0.05 vs. F.

4.4. Histological studies The observation of images captured from histological sections re-
vealed lipid accumulation in the liver of rats fed with fructose, which
Histological sections stained with H&E were analyzed for observing was markedly reduced by lycopene (Fig. 3B).
the morphology changes in hepatocytes and adipocytes. Both were
significantly greater in F group than in C group, the lycopene treatment
diminished the hepatocytes and adipocytes size (Fig. 3 A and C).

Table 1
Effects of diet on obesity parameters, hepatic and cardiac index in rats at the end of study (12 weeks).
Parameters C L F F-L

Body weight (g) 454 ± 15 416 ± 11 481 ± 15 416 ± 17#

Body mass index (g/cm2) 0.72 ± 0.01 0.68 ± 0.01 0.78 ± 0.02 0.70 ± 0.03#
Abdominal fat (g/mm tibial length) 0.58 ± 0.06 0.51 ± 0.03 0.79 ± 0.05* 0.57 ± 0.05#
Liver Index (g/mm tibial length) 0.23 ± 0.01 0.21 ± 0.01 0.32 ± 0.02* 0.24 ± 0.01#
LVH Index (mg/mm tibial length) 14.4 ± 0.5 14.0 ± 0.4 16.4 ± 0.4* 13.1 ± 0.4#

Left Ventricular Hypertrophy (LVH), control rats (C), control rats treated with lycopene (L), untreated rats fed with fructose enriched-diet (F) and rats fed with
fructose and treated with lycopene (F-L). Values are expressed as mean ± SEM of 7–8 rats.*P < 0.05 vs. C, and #P < 0.05 vs. F.

4
P. Ferreira-Santos, et al. Journal of Functional Foods 73 (2020) 104098

Fig. 3. Area of hepatocytes (A) measured in histological sections of liver (B) and adipocytes area (C) measured in histological sections of retroperitoneal adipose
tissue (D), of control rats (C), control rats treated with lycopene (L), untreated rats fed with fructose enriched-diet (F) and rats fed with fructose and treated with
lycopene (F-L). Values are expressed as mean ± SEM of 5 rats. *P < 0.05 vs. C, and #P < 0.05 vs. F.

4.5. Plasma lipid profile and hepatic transaminases 4.6. Vascular reactivity

As reported in Table 2, fructose fed rats displayed an increase in
plasma cholesterol, TG, AI and GOT levels compared to control rats.
Diet with lycopene reversed these increases and had no effect when
administered to control animals.
As shown in Fig. 4, the fructose supplementation involved en-
dothelial dysfunction manifested in the reduction of relaxation to ACh
compared to the relaxing response from control animals in both aortic
(Emax: 65 ± 4% in control rats vs. 41 ± 5% in fructose group,
P < 0.05) and mesenteric rings (Emax: 90 ± 3% in control rats vs.

Table 2
Effects of diet on plasmatic biochemical parameters and hepatic transaminases at the end of study (12 weeks).
Parameters C L F F-L

Serum lipids
TC (mg/dL) 77.8 ± 9.6 61.2 ± 3.0 106.5 ± 9.8* 71.2 ± 3.2#
TG (mg/dL) 101.2 ± 17.2 70.8 ± 6.6 210.2 ± 31.5* 130.5 ± 7.4#
HDLc (mg/dL) 26.2 ± 3.4 23.5 ± 2.3 20.2 ± 2.9 22.8 ± 1.0
LDLc (mg/dL) 31.4 ± 7.5 22.7 ± 1.1 44.3 ± 9.1 22.2 ± 3.2
AI 2.2 ± 0.5 1.5 ± 0.1 5.0 ± 1.2* 2.2 ± 0.1#

Liver status
GOT (IU/L) 31.2 ± 2.5 31.0 ± 2.5 98.5 ± 19.2* 41.8 ± 6.7#
GTP (IU/L) 15.4 ± 2.2 12.6 ± 1.3 35.7 ± 10.2 19.0 ± 2.2

Control rats (C), control rats treated with lycopene (L), untreated rats fed with fructose enriched-diet (F) and rats fed with fructose and treated with lycopene (F-
L).Triglycerides (TG), total cholesterol (TC), high density lipoprotein-cholesterol (HDLc), low density lipoprotein-cholesterol (LDLc), atherogenic index (AI), as-
partate aminotransferase (GOT) and alanine aminotransferase (GPT). Values are expressed as mean ± SEM of 6 rats. *P < 0.05, vs. C. #P < 0.05 vs. F.

5
P. Ferreira-Santos, et al.
Fig. 4. Cumulative concentration–response curves to acetylcholine and sodium nitroprusside in thoracic aorta (A) mesenteric arteries (B) from control rats (C),
control rats treated with lycopene (L), untreated rats fed with fructose enriched-diet (F) and rats fed with fructose and treated with lycopene (F-L). Values are
expressed as mean ± SEM of 7 rats. *P < 0.05 vs. C, and #P < 0.05 vs. F.
72 ± 6% in F group, P < 0.05). Lycopene had not effect on control
animals, but improved the loss of relaxation caused by fructose (Emax:
51 ± 3%) in aorta and restored ACh induced vasorelaxation to the
levels observed in the control in mesenteric arteries (Emax: 89 ± 2%).
Neither the assays carried out in aorta nor mesenteric bed showed
significant effect of lycopene in the endothelium-independent vasor-
elaxation induced by SNP.
4.7. Parameters of oxidative stress
We evaluated the effects of lycopene on %O2− production in aortic
rings and on damage to the lipid membrane by the formation of pro-
ducts of lipid peroxidation (MDA) in plasma.%O2− production stimu-
lated by NADPH was higher in aortic rings of the F group than of the C
group. Lycopene treatment prevented the increase of %O2− production
as a consequence of the fructose supplementation, but did not modify
the levels in control animals (Fig. 5A).
In Fig. 5B we can see that rats receiving supplementation with
fructose had a significant elevation of MDA content compared to the
untreated control group (8.1 ± 0.8 nmol/mL vs. 4.4 ± 0.4 nmol/mL
P < 0.05). Chronic treatment with lycopene significantly attenuated
this increase (5.4 ± 0.4 nmol/mL) in rats receiving fructose and,
again, did not modify the levels in the animals fed with standard diet.
5. Discussion
In the present study, we evaluated the capacity of a carotenoid
6
Journal of Functional Foods 73 (2020) 104098
present in some food included in Mediterranean diet to prevent the
onset of MS. We demonstrated the beneficial effects of lycopene man-
ifested by decreasing blood pressure, reducing insulin resistance, and
maintaining lipid-homeostasis besides achieve reversion of endothelial
dysfunction in resistance arteries using a rat model of MS. The cluster of
conditions that occur together in MS represents an important health
problem, mainly in developed countries (Ahima, 2016; Saklayen, 2018)
and it is necessary to provide new approaches for preventing or treating
it. The rising prevalence and the elevated consumption of fructose and
sucrose during last years, leads scientists to establish a relationship
between high fructose diets and the development of this syndrome
(Aydin et al., 2014; Pereira et al., 2017; Taskinen, Packard, & Borén,
2019), so our study has been carried out using fructose-enriched diet.
Unlike glucose, fructose does not trigger insulin release by pan-
creatic β cells (Johnson et al., 2009), moreover, fructose is less able to
promote satiety so it stimulates a higher consumption of food and alter
the metabolism of lipids and carbohydrates, which favors the synthesis
and accumulation of fat (Stanhope, 2016). Fructose is commonly used
in experimental animal models to induce MS. However, studies vary
significantly in fructose doses administered as a component of diet,
from supraphysiological doses (60–70%) (Hidayati et al., 2019) to the
concentration of fructose beverages (often between 10% and 30%)
(Malakul, Pengnet, Kumchoom, & Tunsophon, 2018; Moreno-
Fernández et al., 2018). In our study the administration of a 20%
fructose-enriched diet to rats for 12 weeks induced features of MS such
as hypertension, hypertriglyceridemia, and hyperglycemia, besides
endothelial dysfunction or increase of oxidative stress markers. A recent
P. Ferreira-Santos, et al.
Fig. 5. Vascular superoxide anion (·O2−) production in aortic rings (A) and assessment of lipid peroxidation through the quantification of malondialdehyde (MDA) in
plasma (B) of control rats (C), control rats treated with lycopene (L), untreated rats fed with fructose enriched-diet (F) and rats fed with fructose and treated with
lycopene (F-L). Values are expressed as mean ± SEM of 6 rats. *P < 0.05 vs. C, and #P < 0.05 vs. F.
meta-analysis has shown that fructose beverage consumption, in-
dependent of variations in study design and duration, also resulted in
an increase in rodent BW, beside to the above mentioned changes (Toop
& Gentili, 2016). In our study fructose supplementation did not sig-
nificantly increase the BW over 12 weeks, but other indicators of obe-
sity such as BMI or adiposity index were increased. Other researchers
also did not observe increases in the weight of rats that received
10–25% fructose in the diet for 12 or 20 weeks respectively (Lozano
et al., 2016; Malakul et al., 2018; Moreno-Fernández et al., 2018),
confirming that the effect of fructose in this point has contradictory
results. The lack of BW changes in our rats could be justified because
there were not differences in the caloric intake. However, BMI values
and adiposity index were normalized by co-administration of lycopene.
Our findings are consistent with those recently obtained by other re-
searchers (Costa et al., 2019; Han, Soliman, Meza, Islam, & Watanabe-
Galloway, 2016; Ni et al., 2019).
There is abundant literature on diets rich in fructose and metabolic
abnormalities including hyperglycemia, hyperinsulinemia, hyperlipi-
demia and obesity. Although the precise mechanism whereby a fruc-
tose-rich diet induces the MS remains controversial, it has been pos-
tulated that oxidative stress plays a key role. The high MDA
concentration demonstrates the increased ROS in our rats fed with
fructose. This process would simultaneously trigger several dysfunc-
tions characteristic of MS phenotype. In this work, we have observed
that lycopene significantly lowers cholesterol and triglycerides when
such parameters were increased after consuming fructose. Furthermore,
it should be noted that there is also a slight decrease due to lycopene
treatment, even in rats without SM. HDL and LDL cholesterol were not
significantly modified in any group, although the slight variations ob-
served improved de LDL/HDL ratio and shows a favorable effect of
lycopene on the atherogenic index. Similar results are observed in other
studies underlining the benefits of lycopene in initial stages of athero-
sclerosis (Mozos et al., 2018). Our study shows that consuming high
fructose diet had also effect on steatosis and lycopene was able to
prevent the excessive accumulation of fat in liver. Recently Ni et al.
reported that lycopene prevents the progression of lipotoxicity-induced
nonalcoholic steatohepatitis by decreasing oxidative stress in mice (Ni
et al., 2019). The results obtained by Costa and collaborators, using the
same dose of lycopene as we do in obese rats, point in the same di-
rection (Costa et al., 2019). The depositions of abdominal adipose tissue
are important components in the development of dyslipidemia, hy-
perglycemia, and hypertension (Kershaw & Flier, 2004). Adipocytes are
the main cells present in the adipose tissue. In obesity, these cells first
undergo hypertrophy and them hyperplasia when they have reached
7
Journal of Functional Foods 73 (2020) 104098
the maximum size (Björntorp, 1991). Our results shown hypertrophy of
adipocytes without hyperplasia in F group, probably owing to study
length. This finding match those of other researchers who observed
hypertrophy without increase in the number of adipocytes in Wistar
rats fed with fructose 20% or 25% for 8 weeks (Mamikutty et al., 2014).
The beneficial effect of lycopene could be associated with decreased
oxidative in cells. Supporting this hypothesis, a study tested the effect of
an inhibitor of NADPH oxidase in rats fed with 10% fructose revealing
that treatment prevents the development of fructose-induced abdom-
inal adipose tissue dysfunction (Farina et al., 2013). We cannot rule out
either, the fact that some effects of carotenoids are due to the provi-
tamin A properties and mediated by the involvement of RAR (re-
tinoid × receptor). Additionally, lycopene has shown ability to inhibit
proinflammatory cytokine and chemokine expression in adipocytes
which could suppose a favourable impact on adipose tissue (Mounien,
Tourniaire, & Landrier, 2019).
Insulin resistance plays a central etiological role in MS. Free fatty
acids are recognized as a significant risk factor contributing to the de-
velopment of insulin resistance in several organs. In particular, hepatic
insulin resistance correlates with increased liver fat content and nu-
merous studies in animal models clearly reveal this link (Asrih &
Jornayvaz, 2015). In our study, after feeding on high-fructose, there
were significant increases in fasted blood glucose and insulin levels
compared with control group and the HOMA-IR was increased by
115%. All these parameters were reduced after lycopene intervention.
In the oral glucose tolerance test, the blood glucose in the F group was
significantly higher than that in the C group at 30 min, 60 min and
120 min and also the AUC, confirming the insulin resistance. Again,
lycopene treatment was able to improve glucose tolerance. Our findings
are consistent with other studies (Ejike et al., 2018; Hashimoto,
Tominaga, Wakagi, & Ishikawa-Takano, 2019; Yin, Zheng, & Jiang,
2019; Zheng, Yin, Lu, & Jiang, 2019) which report the benefits of ly-
copene in glucose homeostasis.
In the cardiovascular system ROS play a physiological role in con-
trolling endothelial function, vascular tone, and cardiac function, and a
pathophysiological role in inflammation, hypertrophy, proliferation,
fibrosis, etc., all of which are important processes contributing to en-
dothelial dysfunction and cardiovascular remodelling in hypertension
and other CVD (Montezano & Touyz, 2012). In a previous study we
demonstrated the beneficial cardiovascular effects of a diet supple-
mented with lycopene in an experimental model of hypertension with a
high component of oxidative stress, in which the levels of angiotensin-II
are continually elevated (Ferreira-Santos et al., 2018). In the present
work, we also observed that fructose intake caused an important
P. Ferreira-Santos, et al.
increase of blood pressure, associated with cardiovascular remodeling
as evidenced by the high rates of LVH. This point was already reported
by Jalal et al ten years ago using the data collected from a National
Health and Nutrition Examination Survey (NHANES). The study in-
volving 4528 adults whose high fructose intake, in the form of added
sugar, was associated with higher blood pressure levels among US
adults without a history of hypertension (Jalal, Smits, Johnson, &
Chonchol, 2010). Trials investigating the role of lycopene in regulating
BP have provided conflicting results. Several studies demonstrated that
daily oral supplementation with lycopene, tomato extract or tomato
juice significantly decreased BP (Kim et al., 2011; Paran, Novack,
Engelhard, & Hazan-Halevy, 2009; Ried & Fakler, 2011), while others
showed no relation (Hozawa et al., 2009). Our results confirm the an-
tihypertensive potential of lycopene without the risk of causing hypo-
tension, and corroborate its beneficial effects in preventing hypertrophy
cardiac related to high levels of oxidative stress. One published recent
paper reported the involvement of oxidative stress in the pathophy-
siology of cardiac hypertrophy and suggested that lycopene might
mediate these cardioprotective effects by ameliorating ROS production,
inhibiting ROS-dependent signaling pathways, improving mitochondria
function and upregulating antioxidant enzymes (Zeng et al., 2019).
The endothelial dysfunction has been suggested as a common un-
derlying mechanism that links insulin resistance, hyperinsulinemia and
hypertension. Several authors have linked fructose overconsumption
with hyperuricemia and increase of oxidative stress. It is well known
the role of uric acid in the development of endothelial dysfunction (Jia,
Aroor, Whaley-Connell, & Sowers, 2014). This compound has an in-
jurious effect once it enters cells like endothelial cell, vascular smooth
muscle cell or adipocytes. The damage includes inhibition on NO pro-
duction, induction of platelet aggregation and pro-inflammatory ac-
tivity, disturbances observed in developing of hypertension and CVD.
The antioxidant capacity of lycopene was verified in this work, on one
hand, through the decrease of vascular %O2− generation stimulated by
NADPH on aortic rings and on the other hand, by the lowering of lipid
peroxidation. Both results could explain the greater relaxing response to
ACh in the arteries from animals treated which, especially in resistance
arteries, would justify that animals with a diet rich in lycopene main-
tain blood pressure values close to normotension.
Nevertheless, this research also has some limitations that need to be
considered. The lack of differences in body weigh after consumption of
fructose enriched diet lead us to think this rat model does not com-
pletely mirror the development and progression of metabolic syndrome
in humans. Although we observed an increase in liver fat in histological
slices, a specific stain such as oily red would have allowed its quanti-
fication.
6. Conclusions
Our results show that lycopene could be useful in the management
of the metabolic syndrome caused by a high-fructose intake. The role of
this carotenoid involves prevention of blood pressure rising, regulation
of blood glucose levels, possibly by avoiding insulin resistance, and
lipid homeostasis maintain. The increase of oxidative stress markers
and endothelial dysfunction in resistance arteries that we observed after
fructose consumption does not occur if lycopene is administered. The
antioxidant activity of lycopene may explain these beneficial effects
while the underlying mechanism will be explore in future experiments.
The findings of our group for lycopene join those of other researchers,
providing information that supports the use, either lycopene or the
foods that contain it, in the maintenance of health.
Ethical statement
Declare that all the experiments were performed according to the
European Union guidelines for the ethical care and use of laboratory
animals, and the protocol was approved by the Bioethics Committee of
8
Journal of Functional Foods 73 (2020) 104098
the University of Salamanca (Registry No.: 006N°201400039292).
CRediT authorship contribution statement
Pedro Ferreira-Santos: Validation, Investigation, Formal analysis,
Visualization. Rubén Aparicio: Validation, Investigation, Formal ana-
lysis, Visualization. Rosalía Carrón: Conceptualization, Resources,
Writing - original draft, Visualization, Supervision. M. José Montero:
Conceptualization, Resources, Writing - original draft, Visualization,
Supervision, Project administration, Funding acquisition. M. Ángeles
Sevilla: Conceptualization, Resources, Writing - original draft,
Visualization, Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
Pedro Ferreira Santos was the recipient of a fellowship funded by a
project Art.83/LOU of University of Salamanca. Rubén Aparicio was
supported by a grant of the University of Salamanca-Banco Santander.
Lycopene was kindly supplied by DSM NUTRITIONAL PRODUCTS.
Funding
This work was supported by Ministry of Health of the Junta de
Castilla y León (project BIO/SA86/13, SACYL) and University of
Salamanca (Spain)
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