nutrients

Article
High Dietary Fructose Intake on Cardiovascular
Disease Related Parameters in Growing Rats
SooYeon Yoo 1 , Hyejin Ahn 1 and Yoo Kyoung Park 1,2, *
1 Department of Medical Nutrition, Kyung Hee University, 1732 Deogyeong-daero, Giheung-gu,
Yongin 17104, Korea; [email protected] (S.Y.Y.); [email protected] (H.A.)
2 Research Institute of Medical Nutrition, Kyung Hee University, 26, Kyungheedae-ro, Dongdaemun-gu,
Seoul 02447, Korea
* Correspondence: [email protected]; Tel.: +82-31-201-3816; Fax: +82-31-203-3816

Received: 29 October 2016; Accepted: 15 December 2016; Published: 26 December 2016

Abstract: The objective of this study was to determine the effects of a high-fructose diet
on cardiovascular disease (CVD)-related parameters in growing rats. Three-week-old female
Sprague Dawley rats were randomly assigned to four experimental groups; a regular diet group
(RD: fed regular diet based on AIN-93G, n = 8), a high-fructose diet group (30Frc: fed regular diet
with 30% fructose, n = 8), a high-fat diet group (45Fat: fed regular diet with 45 kcal% fat, n = 8) or a
high fructose with high-fat diet group (30Frc + 45Fat, fed diet 30% fructose with 45 kcal% fat, n = 8).
After an eight-week treatment period, the body weight, total-fat weight, serum glucose, insulin, lipid
profiles and pro-inflammatory cytokines, abdominal aortic wall thickness, and expressions of eNOS
and ET-1 mRNA were analyzed. The result showed that total-fat weight was higher in the 30Frc,
45Fat, and 30Frc + 45Fat groups compared to the RD group (p < 0.05). Serum triglyceride (TG) levels
were highest in the 30Frc group than the other groups (p < 0.05). The abdominal aorta of 30Frc, 45Fat,
and 30Frc + 45Fat groups had higher wall thickness than the RD group (p < 0.05). Abdominal aortic
eNOS mRNA level was decreased in 30Frc, 45Fat, and 30Frc + 45Fat groups compared to the RD
group (p < 0.05), and also 45Fat and 30Frc + 45Fat groups had decreased mRNA expression of eNOS
compared to the 30Frc group (p < 0.05). ET-1 mRNA level was higher in 30Frc, 45Fat, and 30Frc + 45Fat
groups than the RD group (p < 0.05). Both high fructose consumption and high fat consumption in
growing rats had similar negative effects on CVD-related parameters.

Keywords: high fructose diet; cardiovascular disease (CVD); growing rat

1. Introduction
Sugar-sweetened beverages and processed foods are the main source of fructose [1]. According to
The Korea National Health and Nutrition Examination Survey (KNHNES) [2], consumption of beverage
products increased 3.7 folds from 1998 (45.5 ± 2.0 g) to 2014 (167.4 ± 5.2 g). Based on data collected
from a 2010 study from The Korea Health Industry Development Institute (KHIDI) [3], consumption
of sugar-sweetened beverages and processed foods in adolescents were higher than in the adult group.
Recently, a considerable volume of research was performed in both animals and humans dedicated
to clarify the link between dietary fructose and health risk markers such as obesity and cardiovascular
disease. Consumption of sugar-sweetened beverages has a positive correlation with body weight
gain [4,5] in human, and in animals [6–8]. Bocarsly et al. [9] reported that rats fed with water
containing high fructose corn syrup for eight weeks had increased body weight and body fat,
and Crescenzo et al. [10] showed significant increases in body fat in rats that consumed fructose
for eight weeks, with no significant difference in body weight. These observations are particularly
important because accumulation of body fats leads to an increase of pro-inflammatory cytokines
(TNF-α, IL-6, PAI-1) [11].

Nutrients 2017, 9, 11; doi:10.3390/nu9010011 www.mdpi.com/journal/nutrients

Nutrients 2017, 9, 11 2 of 12

In addition, a high fructose diet is known to lead hypertension and insulin resistance in
animals [12,13]. Insulin resistance has been proposed as an underlying mechanism that links
endothelial dysfunction factors such as endothelial nitric oxide synthase (eNOS) and Endothelin-1
(ET-1) [14]. Also, chronic exposure of dyslipidemia has a major effect on cardiovascular disease
(CVD) [15]. De Castro et al. [16] reported that rats fed with a high fructose diet had significantly
increased levels of in serum total-cholesterol and triglyceride. These changes are significantly
associated with an increased incidence of cardiovascular disease [17].
Changes of CVD-related parameters in childhood is correlated with development into CVD
which affects CVD risks later in life [18]. Although high-fructose affects CVD-related parameters in
adult human and animals, these effects have not been investigated in adolescent or growing animals.
Therefore, this study investigates the effects of a high-fructose diet and compares the results with a
high-fat diet on CVD-related parameters in growing rats.

2. Materials and Methods

2.1. Experimental Design and Diet

The experimental protocol was approved by the Animal Care Use Review Committee of Kyung
Hee University (IACUC, protocol number: KHP-2014-01-1). Three-week-old female Sprague-Dawley
rats (n = 32) were provided by SLC, Inc. (Shizuoka, Japan). Rats were housed individually in
polycarbonate cages in temperature-controlled rooms (22 ± 2 ◦ C) with a relative humidity of 55% ± 5%,
and a 12-h light/dark cycle. The rats were fed a pellet chow diet, and given water ad libitum for
an adaptation period of 10 days. All rats were weighed weekly, and food intake was measured
daily. After a 10-day adaption period, animals were randomized selected into the four different
groups: regular diet group (RD) (n = 8) rats were fed an AIN-93G (D10012G, Research Diets Inc.,
New Brunswick, NJ, USA) diet, high fructose diet group (30Frc) (n = 8) rats were fed a 30% fructose
(D14010101, Research Diets Inc.) diet, high fat diet group (45Fat) (n = 8) rats were fed a 45 kcal%
fat as soy bean oil and lard (D14010102, Research Diets Inc.) diet, and high fat diet with high
fructose diet group (30Frc + 45Fat) (n = 8) rats were fed a diet with 30% fructose and 45 kcal% fat
(D14010103, Research Diets Inc.). All animals were maintained on these diets ad libitum for eight
weeks. Composition of experimental diets is shown in Table 1.

Table 1. Ingredient composition of experimental diets.

RD 30Frc 45Fat 30Frc + 45Fat

% g kcal g kcal g kcal g kcal
Protein 20 20 20 20 24 20 24 20
Carbohydrate 64 64 64 64 41 35 41 35
Fat 7 16 7 16 24 45 24 45
Total 100 100 100 100
kcal/gm 4.0 4.0 4.8 4.8
Ingredient g kcal g kcal g kcal g kcal
Casein, 80 Mesh 200 800 200 800 200 800 200 800
L -Cystine 3 12 3 12 3 12 3 12
Corn Starch 397.5 1590 229.5 918 137 548 0 0
Maltodextrin 10 132 528 100 400 100 400 37 148
Sucrose 100 400 0 0 100 400 0 0
Fructose 0 0 300 1200 0 0 300 1200
Cellulose 50 0 50 0 50 0 50 0
Soybean Oil 70 630 70 630 26 234 26 234
t-Butylhydroquinone 0.014 0 0.014 0 0.014 0 0.014 0
Lard 0 0 0 0 174 1566 174 1566
Mineral Mix 35 0 35 0 35 0 35 0
Vitamin Mix 10 40 10 40 10 40 10 40
Choline Bitartrate 2.5 0 2.5 0 2.5 0 2.5 0
Total 1000 4000 1000 4000 837.5 4000 837.5 4000
RD: rats received a regular diet based on AIN-93G (4.0 kcal/g diet); 30Frc: rats received a 30% fructose-diet
based on regular diet (4.0 kcal/g diet); 45Fat: rats received a 45 kcal% fat-diet (4.8 kcal/g diet); 30Frc + 45Fat:
rats received a 45 kcal% fat -diet with 30% fructose (4.8 kcal/g diet).
Nutrients 2017, 9, 11 3 of 12

2.2. Body Weight, Food Consumption, and Fat Mass

Body weights and food consumption were measured weekly and daily, respectively.
The food efficiency ratio (FER) was calculated using the following formula: (weight gain
(g)/week)/(food consumed (kcal)/week). At the end of the eight-week experimental period, total fat
was removed and weighed immediately after killing. The total fat was measured by combining the
weight of subcutaneous fat and visceral fat.

2.3. Analysis of Blood Parameters

Blood was collected at the end of experiment, following a 12-h overnight fast. Rats were
anesthetized with a small amount of ethyl ether, and blood samples were taken by heart puncture.
Blood samples were immediately collected into serum-separating tubes (SST) and were centrifuged
at 3000 rpm for 15 min at 4 ◦ C. Serum was stored at −70 ◦ C until used in assays. Serum triglyceride
(TG), Total cholesterol (T-Chol), HDL-cholesterol (HDL-C), and glucose levels were determined using
commercial kits (Asan Co. Ltd., Seoul, Korea). Atherogenic Index (AI) was calculated using the
following formula: AI = (total cholesterol − HDL-cholesterol)/HDL-cholesterol. Serum Insulin
concentrations were determined using an ELISA rat/mouse insulin kit (ALPCO Diagnostics, Salem,
NH, USA). Insulin resistance was calculated by a homeostasis assessment model (HOMA-IR) and
calculated from fasting insulin and glucose concentration according to the formula: (fasting insulin
(ng/mL) × fasting glucose (mg/dL))/22.5. Quantitative insulin sensitivity check index (QUICKI) was
also calculated using the following formula: QUICKI = 1/(log fasting insulin (mg/dL) + log fasting
glucose(mg/dL)). Pro-inflammatory cytokines (TNF-α, IL-6, and PAI-1) were measured in duplicate
using Milipore’s MILLIPLEX rat CVD cytokine panel (Millipore, Billerica, MA, USA).

2.4. Analysis of Abdominal Aorta

Abdominal aorta samples were obtained after rats were sacrificed at the end of
experiment. The abdominal aortas were fixed in a 4% formalin solution, followed by sequential
dehydration (70% ethanol, 100% ethanol, and acetone), xylene clearance, and paraffin embedding.
The paraffin-embedded aortas were cut into 5-micron slices with a microtome (820 II; Reichert-Jung,
Bensheim, Germany). Then the sliced aortas were stained with Harris hematoxylin and eosinY
(H & E). Wall thickness and lumen diameter of abdominal aortas were determined on five isotropic
uniform random sections per animal at a magnification of 500:1 and phase contrast. All sections were
photographed using a microscope (model Axiovert S100, Zeiss, Oberkochen, Germany) connected to a
camera (model AxioCam, Zeiss) and MetaMorph software (Molecular Devices, Sunnyvale, CA, USA).
Wall thickness and lumen diameter were determined as the mean of the minimal and maximal value.

2.5. Quantitative Real-Time Polymerase Chain Reaction (PCR)

Total RNA was extracted from abdominal aorta using an RNeasy mini kit (Qiagen, Gaithersburg,
MD, USA) according to the manufacturer’s instructions. Real-time quantitative PCR was
performed using an Applied Bio-systems (Applied Bio-systems, Foster City, CA, USA); 1 µL
with 100 nM of each primer (forward and reverse) reaction consisted of 10 µL of the SYBR
Green Super-mix (iQ SYBR Green Super-mix, Bio-Rad Laboratories Inc. Hercules, CA, USA),
according to the manufacturer’s instructions. Thermal cycling was initiated by denaturation
at 95 ◦ C for 10 min, followed by 40 cycles of 95 ◦ C for 15 s and 60 ◦ C for 30 s, then an
annealing step was performed at adequate temperature in function of the primers and 72 ◦ C
for 30 s for extension. After the final cycle, melting curves were monitored from 55 to 65 ◦ C
(0.05 ◦ C/s). The primer sequences were: rat eNOS, 50 -CAACAAACCGAGGCAATCTTC-30 (forward),
50 -CCCGGCCAGCGTAGCT-30 (reverse), and rat ET-1, 50 -TGGACATCATCTGGGTCAACA-30
(forward), 50 -GCTTAGACCTAGAAGGGCTTCCTAGT-30 (reverse), and rat glyceraldehyde
Nutrients 2017, 9, 11 4 of 12

30 -phosphate dehydrogenase (GAPDH), 50 -TGGCCTCCAAGGAGTAAGAAAC-30 (forward),

50 -GGCCTCTCTCTTGCTCTCAGTATC-30 (reverse). The detected genes were eNOS and ET-1.

2.6. Statistical Analysis

All measurements were performed in duplicate, and statistical calculations were performed with
Statistical Package for the social Sciences (SPSS, version 20.0, IBM Corp., Armonk, NY, USA) software.
All data were presented as mean ± SD. Differences in measured parameters among the experimental
groups were analyzed by the one-way ANOVA and Duncan’s multiple range tests. The respective
effects of operation and diet were analyzed by two-way ANOVA. The differences were considered to
be significant when the p value was less than 0.05.

3. Results

3.1. Body Weights, Food Intakes, Calorie Intakes, and Food Efficiency Ratios
The effect of the diet on body weight, body weight gain, food intake, calorie intake, and food
efficiency ratio is shown in Table 2.

Table 2. Body weights, food intakes, calorie intakes, and food efficiency ratio.

RD 30Frc 45Fat 30Frc + 45Fat

(n = 8) (n = 8) (n = 8) (n = 8)
Initial body weight (g) 74.6 ± 2.6 74.9 ± 5.0 74.5 ± 4.71 74.4 ± 4.1
Final body weight (g) 251.3 ± 9.6 b 263.3 ± 15.0 a,b 278.1 ± 22.1 a 277.2 ± 16.6 a
Weight gain (g) 177.2 ± 9.8 b 188.0 ± 13.4 a,b 204.3 ± 22.6 a 201.9 ± 17.7 a
Food intake (g/day) 12.5 ± 0.8 a 13.2 ± 0.9 a 11.3 ± 0.9 b 11.5 ± 0.8 b
Calorie intake (kcal/day) 49.8 ± 2.9 52.4 ± 4.4 53.8 ± 4.6 54.7 ± 4.7
Food efficiency ratio 0.05 ± 0.00 0.05 ± 0.00 0.05 ± 0.01 0.05 ± 0.00
All values are presented as means ± standard deviation (SD). RD: rats received a regular diet based on
AIN-93G (4.0 kcal/g diet); 30Frc: rats received a 30% fructose-diet based on control-diet (4.0 kcal/g diet);
45Fat: rats received a 45 kcal% fat-diet (4.8 kcal/g diet); 30Frc + 45Fat: rats received a 45 kcal% fat-diet with
30% fructose (4.8 kcal/g diet); Food efficiency ratio = (weight gain (g)/week)/(food consumed (kcal)/week).
Statistical differences between the experimental groups were based on one-way ANOVA and Duncan’s multiple
range tests at p < 0.05. Means with different alphabetical superscripts are significantly different (p < 0.05).

The initial body weights were not significantly different among the experimental groups.
Feeding of high fat diet groups (45Fat, 30Frc + 45Fat) resulted in a significantly higher body weight
than the RD and 45Fat groups (278.1 ± 22.1 g, 277.2 ± 16.6 g vs. 251.3 ± 9.6 g, 263.3 ± 15.0 g, p < 0.05).
The food intake was significantly higher in the RD (12.5 ± 0.8 g/day) and 30Frc (13.2 ± 1.0 g/day)
groups compared to that of 45Fat (11.3 ± 0.9 g/day) and 30Frc + 45Fat (11.5 ± 0.8 g/day) groups
(p < 0.05). However, total calorie intakes and food efficiency ratios were not significantly different
among the experimental groups. Total calorie was calculated from food intake (g) × calorie
density (kcal/g).

3.2. Total-Fat Weights

The total-fat weights of the experimental groups are shown in Figure 1.
 Nutrients 2017, 9, 011  5 of 12 

3.2. Total‐Fat Weights 
Nutrients 2017, 9, 11 5 of 12
The total‐fat weights of the experimental groups are shown in Figure 1. 

 
Figure  1.  Total‐fat  weight  in  the  experimental  groups.  RD:  rats  received  a  regular  diet  based  on 
Figure 1. Total-fat weight in the experimental groups. RD: rats received a regular diet based on
AIN‐93G (4.0 kcal/g diet); 30Frc: rats received a 30% fructose‐diet based on control‐diet (4.0 kcal/g 
AIN-93G (4.0 kcal/g diet); 30Frc: rats received a 30% fructose-diet based on control-diet (4.0 kcal/g diet);
diet); 45Fat: rats a received 45 kcal% fat‐diet (4.8 kcal/g diet); 30Frc + 45Fat: rats received a 45 kcal% 
45Fat: rats a received 45 kcal% fat-diet (4.8 kcal/g diet); 30Frc + 45Fat: rats received a 45 kcal% fat-diet
fat‐diet with 30% fructose (4.8 kcal/g diet). Statistical differences between the experimental groups 
with 30% fructose (4.8 kcal/g diet). Statistical differences between the experimental groups were based
were based on one‐way ANOVA and Duncan’s multiple range tests at p < 0.05. Means with different 
on one-way ANOVA and Duncan’s multiple range tests at p < 0.05. Means with different alphabetical
alphabetical letters are significantly different (p < 0.05). 
letters are significantly different (p < 0.05).
The average total‐fat weights were 15.2 ± 3.6, 22.2 ± 3.8, 25.1 ± 3.5, and 25.0 ± 2.4 g in the RD, 
30Frc,  45Fat,  and 
The average 30Frc weights
total-fat +  45Fat  groups. 
were 15.2 ± total‐fat 
The  ± 3.8, 25.1
3.6, 22.2weights  ± significantly 
were  ± 2.4 gin in30Frc, 
3.5, and 25.0higher  the RD,
45Fat, and 30Frc + 45Fat groups than the RD group (p < 0.05). 
30Frc, 45Fat, and 30Frc + 45Fat groups. The total-fat weights were   significantly higher in 30Frc, 45Fat,
and 30Frc + 45Fat groups than the RD group (p < 0.05).
3.3. Serum Levels of Glucose and Insulin, HOMA‐IR, and QUICKI 
3.3. Serum Levels of Glucose and Insulin, HOMA-IR, and QUICKI
The blood glucose and insulin levels, HOMA‐IR, and QUICKI are shown in Table 3. The serum 
glucose and insulin levels did not differ among the experimental groups. Also, no differences were 
The blood glucose and insulin levels, HOMA-IR, and QUICKI are shown in Table 3. The serum
observed in HOMA‐IR and QUICKI among groups. 
glucose and insulin levels did not differ among the experimental groups. Also, no differences were
observed in HOMA-IR and QUICKI among groups.
Table 3. Serum levels of glucose and insulin. 

RD3. Serum levels

Table of glucose and45Fat
30Frc insulin. 30Frc + 45Fat   
 
(n = 8)  (n = 8)  (n = 8)  (n = 8) 
Glucose (mg/dL)  RD
143.6 ± 10.3  30Frc
142.0 ± 9.2  45Fat
141.4 ± 14.9  30Frc + 45Fat
136.6 ± 15.8 
Insulin (ng/mL)  (n = 8)
1.2 ± 1.4 (n = 8)
2.2 ± 1.7 (n = 8)
0.9± 0.3 (n = 8)
1.3 ± 0.9 
Glucose
HOMA‐IR  (mg/dL) 143.6 ± 10.3 12.7 ± 11.7 
8.1 ± 9.1  142.0 ± 9.2 141.4 ± 14.9
5.9± 2.4  136.6 ± 15.8
8.3 ± 6.2 
Insulin
QUICKI (ng/mL) 1.2 ± 1.4
0.5 ± 0.07  2.2 ± 1.7
0.4 ± 0.07  0.9 ± 0.3
0.5± 0.05  1.3 ± 0.9
0.5 ± 0.07 
HOMA-IR 8.1 ± 9.1 12.7 ± 11.7 5.9± 2.4 8.3 ± 6.2
All values are presented as means ± standard deviation (SD). RD: rats received a regular diet based 
QUICKI 0.5 ± 0.07 0.4 ± 0.07 0.5± 0.05 0.5 ± 0.07
on AIN‐93G (4.0 kcal/g diet); 30Frc: rats received a 30% fructose‐diet based on control‐diet (4.0 kcal/g 
All values are presented as means ± standard deviation (SD). RD: rats received a regular diet based on
diet); 45Fat: rats received a 45 kcal% fat‐diet (4.8 kcal/g diet); 30Frc + 45Fat: rats received a 45 kcal% 
AIN-93G (4.0 kcal/g diet); 30Frc: rats received a 30% fructose-diet based on control-diet (4.0 kcal/g diet);
fat‐diet 
45Fat: ratswith  30% afructose 
received 45 kcal%(4.8  kcal/g 
fat-diet (4.8diet), 
kcal/g HOMA‐IR: 
diet); 30Frchomoeostasis  model  assessment 
+ 45Fat: rats received of  insulin 
a 45 kcal% fat-diet with
resistance 
30% fructose was  calculated 
(4.8 kcal/g by  (fasting 
diet), HOMA-IR: insulin  (ng/mL); 
homoeostasis QUICKI:  1/(log(fasting 
model assessment insulin 
of insulin resistance wasng/mL)  + 
calculated
bylog(fasting 
(fasting insulin (ng/mL);
glucose  QUICKI:
mg/dL))  1/(log(fasting
×  fasting  insulin ng/mL)There 
glucose  (mg/dL))/22.5.  + log(fasting
were  no glucose mg/dL))
significant  × fasting
differences 
glucose (mg/dL))/22.5. There were no significant differences among groups according to ANOVA and Duncan’s
among groups according to ANOVA and Duncan’s multiple range tests. 
multiple range tests.

 
3.4. Serum Levels of Lipid Profiles
 

The serum lipid profile levels are shown in Table 4. Serum Total-C, HDL-C, and atherogenic
index were not different among groups. However, the serum TG levels in the 30Frc group
(108.6 ± 25.2 mg/dL) were significantly higher than the RD, 45Fat, and 30Frc + 45Fat groups
(71.4 ± 19.8, 88.6 ± 26.9, and 93.9 ± 24.1 mg/dL, respectively) (p < 0.05).
Nutrients 2017, 9, 11 6 of 12

Table 4. Serum levels of lipid profiles.

RD 30Frc 45Fat 30Frc + 45Fat

(n = 8) (n = 8) (n = 8) (n = 8)
TG (mg/dL) 71.4 ± 19.8 b 108.6 ± 25.2 a 88.6 ± 26.9 a,b 93.9 ± 24.1 a,b
Total-C (mg/dL) 107.9 ± 19.7 114.6 ± 17.5 97.8 ± 15.4 96.3 ± 18.4
HDL-C (mg/dL) 98.1 ± 12.4 103.8 ± 10.9 92.1 ± 13.4 91.8 ± 14.7
AI 0.09 ± 0.07 0.10 ± 0.05 0.06 ± 0.03 0.05 ± 0.05
All values are presented as means ± standard deviation (SD). RD: rats received a regular diet based on
AIN-93G (4.0 kcal/g diet); 30Frc: rats received a 30% fructose-diet based on control-diet (4.0 kcal/g diet);
45Fat: rats received a 45 kcal% fat-diet (4.8 kcal/g diet); 30Frc + 45Fat: rats received a 45 kcal% fat-diet
with 30% fructose (4.8 kcal/g diet); TG: Triglyceride; Total-C: Total cholesterol; AI: Atherogenic index
= (total cholesterol − HDL-cholesterol)/HDL-cholesterol. Statistical difference between the experimental
groups were based on one-way ANOVA and Duncan’s multiple range tests at p < 0.05. Means with different
alphabetical superscripts are significantly different (p < 0.05).

3.5. Serum Levels of Pro-Inflammatory Cytokines

The serum pro-inflammatory cytokines levels are shown in Table 5. The mean levels of serum
pro-inflammatory cytokines levels (TNF-α, IL-6, PAI-1) did not differ among the experimental groups.

Table 5. Serum levels of pro-inflammatory cytokines.

RD 30Frc 45Fat 30Frc + 45Fat

(n = 8) (n = 8) (n = 8) (n = 8)
TNF-α (pg/mL) 55.1 ± 4.9 51.5 ± 5.5 47.0 ± 4.8 48.3 ± 10.9
IL-6 (pg/mL) 11.6 ± 4.2 18.2 ± 11.6 12.6 ± 4.0 15.5 ± 8.3
PAI-1 (pg/mL) 100.8 ± 13.3 101.5 ± 9.4 96.0 ± 4.8 99.4 ± 20.5
All values are presented as means ± standard deviation (SD). RD: rats received a regular diet based on AIN-93G
(4.0 kcal/g diet); 30Frc: rats received a 30% fructose-diet based on control-diet (4.0 kcal/g diet); 45Fat: rats
received a 45 kcal% fat-diet (4.8 kcal/g diet); 30Frc + 45Fat: rats received a 45 kcal% fat-diet with 30% fructose
(4.8 kcal/g diet); TNF-α: Tumor Necrosis α; IL-6: Interleukin-6; PAI-1: Plasminogen activator inhibitor-1.
There were no significant differences among groups according to ANOVA and Duncan’s multiple range tests.

3.6. Analysis of Abdominal Aorta Wall Thickness and Lumen Diameter

The abdominal aorta wall thickness and lumen diameter are shown in Table 6 and Figure 2.
The aorta wall thickness was significantly increased in 30Frc, 45Fat, and 30Frc + 45Fat groups
(23.6 ± 0.9 µm, 23.7 ± 2.8 µm, and 22.5 ± 2.2 µm, respectively, p < 0.05) than the RD group
(18.5 ± 0.5 µm). The lumen diameter was not different among the groups. The wall thickness:lumen
ratio was increased in 30Frc, 45Fat and 30Frc + 45Fat groups (0.096 ± 0.001 µm, 0.098 ± 0.014 µm,
and 0.085 ± 0.005 µm, respectively, p < 0.05) than the RD group (0.061 ± 0.009 µm).

Table 6. Abdominal aorta wall thickness and lumen diameter.

RD 30Frc 45Fat 30Frc + 45Fat

(n = 8) (n = 8) (n = 8) (n = 8)
Wall thickness (µm) 18.5 ± 0.5 b 23.6 ± 0.9 a 23.7 ± 2.8 a 22.5 ± 2.2 a
Lumen diameter (µm) 306.8 ± 54.4 248.4 ± 25.1 243.9 ± 29.4 266.9 ± 29.0
Wall thickness/
0.061 ± 0.009 b 0.096 ± 0.001 a 0.098 ± 0.014 a 0.085 ± 0.005 a
Lumen ratio (µm/µm)
All values are presented as means ± standard deviation (SD). RD: rats received a regular diet based on AIN-93G
(4.0 kcal/g diet); 30Frc: rats received a 30% fructose-diet based on control-diet (4.0 kcal/g diet); 45Fat: rats
received a 45 kcal% fat-diet (4.8 kcal/g diet); 30Frc + 45Fat: rats received a 45 kcal% fat-diet with 30% fructose
(4.8 kcal/g diet). Statistical differences between the experimental groups were based on one-way ANOVA
and Duncan’s multiple range tests at p < 0.05. Means with different alphabetical superscripts are significantly
different (p < 0.05).
 Lumen ratio (μm/μm) 
All values are presented as means ± standard deviation (SD). RD: rats received a regular diet based 
on AIN‐93G (4.0 kcal/g diet); 30Frc: rats received a 30% fructose‐diet based on control‐diet (4.0 kcal/g 
diet); 45Fat: rats received a 45 kcal% fat‐diet (4.8 kcal/g diet); 30Frc + 45Fat: rats received a 45 kcal% 
fat‐diet with 30% fructose (4.8 kcal/g diet). Statistical differences between the experimental groups 
were based on one‐way ANOVA and Duncan’s multiple range tests at p < 0.05. Means with different 
Nutrients 2017, 9, 11 7 of 12
alphabetical superscripts are significantly different (p < 0.05). 

 
Figure 2. Abdominal aortic wall thickness and lumen diameter taken at eight weeks, pertaining to
Figure 2. Abdominal aortic wall thickness and lumen diameter taken at eight weeks, pertaining to 
the respective groups (A) RD; (B) 30Frc; (C) 45Fat; (D) 30Frc + 45Fat. Hematoxylin and eosin (H & E) 
the respective groups (A) RD; (B) 30Frc; (C) 45Fat; (D) 30Frc + 45Fat. Hematoxylin and eosin (H & E)
stained stained abdominal aorta × 
abdominal aorta × 500.500. Magnification bars 50 μm. RD: rats received a regular diet based on 
Magnification bars 50 µm. RD: rats received a regular diet based on
AIN‐93G (4.0 kcal/g diet); 30Frc: rats received a 30% fructose‐diet based on control‐diet (4.0 kcal/g 
AIN-93G (4.0 kcal/g diet); 30Frc: rats received a 30% fructose-diet based on control-diet (4.0 kcal/g diet);
diet); 45Fat: rats received a 45 kcal% fat‐diet (4.8 kcal/g diet); 30Frc + 45Fat: rats received a 45 kcal% 
45Fat: rats received a 45 kcal% fat-diet (4.8 kcal/g diet); 30Frc + 45Fat: rats received a 45 kcal% fat-diet
fat‐diet with 30% fructose (4.8 kcal/g diet).   
with 30% fructose (4.8 kcal/g diet).
3.7. Abdominal Aortic eNOS and ET‐1 mRNA Expression Measured by qRTPC 
3.7. Abdominal Aortic eNOS and ET-1 mRNA Expression Measured by qRTPC
The abdominal aortic eNOS and ET‐1 mRNA expression of the experimental groups are shown 
in  Figures  3  and  4.  The  aortic  eNOS  mRNA  expression  was  significantly  decreased  in  45Fat  and 
The abdominal aortic eNOS and ET-1 mRNA expression of the experimental groups are shown
30Frc + 45Fat groups (0.3 ± 0.06 and 0.4 ± 0.07, respectively, p < 0.05) compared to the RD and 30Frc 
in Figures 3 and
groups  (1.0 4. Theand 
±  0.00  aortic eNOS
0.7  ±  mRNA expression
0.05,  respectively).  Also,  for  was significantly
the  30Frc  group  that decreased inhigh 
was  fed  the  45Fat and
45Fat groups (0.3 ± 0.06 and 0.4 ± 0.07, respectively, p < 0.05) compared to the RD and 30Frc
30Frc + fructose diet constantly, the aortic eNOS mRNA expression was significantly decreased compared to 
groups (1.0 ± 0.00
the  RD  group and (p 0.7 ± 0.05,
<  0.05).  The respectively). Also,expression 
aortic  ET‐1  mRNA  for the 30Frc group
of  the  30Frc that wasgroup
+  45Fat  fed the high
  was  the fructose
highest among the four groups (10.3 ± 1.8 vs. 1.0 ± 0.4, 3.3 ± 0.3, and 7.4 ± 0.7, respectively, for the RD, 
diet constantly, the aortic eNOS mRNA expression was significantly decreased compared to the RD
group (p30Frc, and 45Fat groups, p < 0.05). 
< 0.05). The aortic ET-1 mRNA   expression of the 30Frc + 45Fat group was the highest among

the four groups (10.3 ± 1.8 vs. 1.0 ± 0.4, 3.3 ± 0.3, and 7.4 ± 0.7, respectively, for the RD, 30Frc,
 Nutrients
and 45Fat groups, p < 0.05).
2017, 9, 011 
 
8 of 12 

 
Figure 3.Figure 
Aortic3.  Aortic  eNOS  mRNA  expression  measured  by  quantitative  real‐time  polymerase  chain 
eNOS mRNA expression measured by quantitative real-time polymerase chain reaction
reaction  (qRTPCR).  RD: rats received a regular diet based on AIN‐93G (4.0 kcal/g diet); 30Frc: rats 
(qRTPCR). RD: rats received a regular diet based on AIN-93G (4.0 kcal/g diet); 30Frc: rats received
received a 30% fructose‐diet based on control‐diet (4.0 kcal/g diet); 45Fat: rats received a 45 kcal% 
a 30% fructose-diet based on control-diet (4.0 kcal/g diet); 45Fat: rats received a 45 kcal% fat-diet
fat‐diet (4.8 kcal/g diet); 30Frc + 45Fat: rats received a 45 kcal% fat‐diet with 30% fructose (4.8 kcal/g 
(4.8 kcal/g diet); 30Frc + 45Fat: rats received a 45 kcal% fat-diet with 30% fructose (4.8 kcal/g diet).
diet). Statistical differences between the experimental groups were based on one‐way ANOVA and 
Statistical differences between the experimental groups were based on one-way ANOVA and Duncan’s
Duncan’s multiple range tests at p < 0.05. Means with different alphabetical letters are significantly 
multipledifferent (p < 0.05). 
range tests at p < 0.05. Means with different alphabetical letters are significantly different
(p < 0.05).
 reaction  (qRTPCR).  RD: rats received a regular diet based on AIN‐93G (4.0 kcal/g diet); 30Frc: rats 
received a 30% fructose‐diet based on control‐diet (4.0 kcal/g diet); 45Fat: rats received a 45 kcal% 
fat‐diet (4.8 kcal/g diet); 30Frc + 45Fat: rats received a 45 kcal% fat‐diet with 30% fructose (4.8 kcal/g 
diet). Statistical differences between the experimental groups were based on one‐way ANOVA and 
Duncan’s multiple range tests at p < 0.05. Means with different alphabetical letters are significantly 
Nutrients 2017, 9, 11 8 of 12
different (p < 0.05). 

 
Figure Aortic
4. 4. 
Figure  ET-1
Aortic  ET‐1 mRNA
mRNA expression
expression measured
measured  by by qRTPCR. 
qRTPCR.RD:  RD:rats 
ratsreceived 
received a regular
a  regular  diet
diet 
based on AIN‐93G (4.0 kcal/g diet); 30Frc: rats received a 30% fructose‐diet based on control‐diet (4.0 
based on AIN-93G (4.0 kcal/g diet); 30Frc: rats received a 30% fructose-diet based on control-diet
(4.0kcal/g diet); 45Fat: rats received a 45 kcal% fat‐diet (4.8 kcal/g diet); 30Frc + 45Fat: rats received a 45 
kcal/g diet); 45Fat: rats received a 45 kcal% fat-diet (4.8 kcal/g diet); 30Frc + 45Fat: rats received a
kcal%  fat‐diet 
45 kcal% fat-diet with 
with 30% 
30% fructose 
fructose (4.8 
(4.8 kcal/g 
kcal/gdiet). 
diet).Statistical 
Statisticaldifferences 
differencesbetween 
between the  experimental 
the experimental
groups were based on one‐way ANOVA and Duncan’s multiple range tests at p < 0.05. Means with 
groups were based on one-way ANOVA and Duncan’s multiple range tests at p < 0.05. Means with
different alphabetical letters are significantly different (p < 0.05). 
different alphabetical letters are significantly different (p < 0.05).

4. Discussion 
4. Discussion
The purpose of this study was to determine the effects of a high‐fructose diet on cardiovascular 
The purpose of this study was to determine the effects of a high-fructose diet on cardiovascular
disease (CVD)‐related parameters in growing rats.   
disease It 
(CVD)-related parameters
was  known  that  rats  fed in growing
high‐fat  rats.increased  body  weight  and  body  fat  [19,20].  The 
diets 
It was known that rats fed high-fat diets increased body weight and body fat [19,20]. The present
present study showed that the consumption of a high‐fat diet (45Fat, 30Frc + 45Fat) in growing rats 
study showed that the consumption
significantly increased body  weight  and of body 
a high-fat diet (45Fat, 30Frc + and 
fat  compared to regular diet  45Fat) in growing rats
high‐fructose diet 
significantly increased body weight and body fat compared to regular diet and high-fructose
groups. The high‐fructose diet did not affect the body weight, whereas it significantly increased the  diet
groups.
weight of body fat compared to the regular diet fed group. The previous study reported that rats the
The high-fructose diet did not affect the body weight, whereas it significantly increased
weight of body fat compared to the regular diet fed group. The previous study reported that rats fed
fed with the high‐fructose diet (60%) for 10 weeks increased body weight [7]. However, Crescenzo 
et the
with al. high-fructose
[10]  showed  increased 
diet (60%)white 
for 10 adipose  tissue  (WAT) 
weeks increased in  rats [7].
body weight fed However,
fructose  (60%),  despite 
Crescenzo no 
et al. [10]
difference  in  body  weight  gain.  An  increase  in  body  weight  alone  does  not  necessarily 
showed increased white adipose tissue (WAT) in rats fed fructose (60%), despite no difference in body indicate 
obesity, it has to be considered along with other factors, such as changes in body composition [21]. 
weight gain. An increase in body weight alone does not necessarily indicate obesity, it has to be
considered along with other factors, such as changes in body composition [21]. The increase in body
fat reflected the obesogenic property. This suggests that obesity can be induced by high-fructose diet,
as well as high-fat diet.
Dyslipidemia, especially hypertriglyceridemia and insulin resistance are major factors associated
with CVD in rats fed a high-fructose diet (60%) [22,23]. Our data showed that the 30Frc group
had significantly increased serum TG levels compared to the other groups. In the liver, fructose is
divided into glyceraldehyde and dihydroxyacetone phosphate, ultimately becoming triglyceride [24].
Therefore, exposure to high fructose levels to rapidly increased levels of triglyceride synthesis.
We consider that excessive fructose consumption can lead to dyslipidemia and obesity; these changes
are caused CVD.
Insulin resistance is closely linked to dyslipimenia [25]. Many previous studies have reported
that the high-fructose diet can induce hyperglycemia (35%, 66%) [26,27]. However, this study showed
no difference in serum glucose and insulin levels. The previous study suggested that although
fructose does not appear to acutely increase insulin levels, chronic exposure seems to indirectly
cause insulin resistance and obesity through other mechanisms, such as GLUT5 fructose transporters
and inflammation [28]. Iida et al. [29] reported that rats fed 40% fructose for eight weeks displayed
no difference in serum insulin levels. Another study showed that rats fed with 66% fructose for
two weeks increased plasma TG, even though there was no change in plasma glucose, insulin, and body
weight [30]. Increased triglyceride levels in response to high-fructose diet could have resulted in insulin
resistance by reducing the insulin signaling pathway [27]. Our data, together with the previously
Nutrients 2017, 9, 11 9 of 12

established literature showed, suggest that chronic exposure to a high-fructose diet can result in
hypertriglycemia, and that this change could cause insulin resistance.
Enlarged body fat induces the expression of pro-inflammatory cytokines [11]. A previous study
reported that a high fructose diet can increase hepatic mRNA expression of TNF-α, IL-6, and the weight
of epididymal fat pads in rats [31]. However, the present study had shown that rats fed a high-fructose
diet significantly increased the total body fat, but the serum pro-inflammatory cytokines (TNF-α, IL-6,
PAI-1) did not change in between groups. Chronic inflammation is an important pathogenic factor
in the development of CVD [32,33]. Large amounts of markers for inflammatory cytokines can be
released from the adipose tissue [34]. Yudkin et al. [35] reported that adipose tissue can also synthesize
pro-inflammatory cytokines such as TNF-α and IL-6. In this way, increased body fat itself promotes
inflammation. According to this hypothesis, high fructose consumption leads to increased body fat
and obesity, which can cause changes to inflammatory cytokines. In the present study we showed
that rats fed a high-fructose diet significantly increased their total-fat weight, although the serum
pro-inflammatory cytokines did not change.
Changes in the aorta wall thickness is significantly associated with serum lipid profiles and
hypertension, which begins in childhood and may develop into cardiovascular disease [35–38].
According to a previous study, the tunica intima-media layer was increased in the high-fructose
diet group [31]. Autopsy studies have reported that the first atherosclerotic lesions actually begin to
develop in the abdominal aorta [39]. Therefore, we chose the abdominal aorta to conduct the present
experiment. In the present study we showed that the abdominal aorta of high-fructose diet and high
fat diet rats were thicker in comparison to the regular diet group. We consider that high-fructose
consumption as well as high-fat consumption, can have a significant effect on abdominal aorta wall
thickness in the growing rats. These early changes can possibly lead to endothelial dysfunction.
Endothelial dysfunction is a systemic disorder in the pathogenesis of atherosclerosis [40],
which plays an important role in hypertension [41] and CVD [42]. The endothelium maintains the
balance between vasoconstriction and vasodilation, but when this balance is disrupted, endothelial
dysfunction occurs which can be lead to CVD [43]. A major vasodilator NO is synthesized by nitric
oxide synthases [44]. Endothelial dysfunction may occur as a result of decreased eNOS activity
or reduced bioavailability of NO [14]. The endothelium also produces vasoconstrictors, such as
endothelin and angiotension II. Endothelin is the most potent endogenous vasoconstrictor [43].
As such, in this study, we analyzed vasodilator eNOS and vasoconstrictor ET-1. A previous study
showed that NO synthesis inhibited rats had elevated blood pressure [45]. Endemann et al. [14]
reported that the eNOS activity of rats fed high-fructose diet decreased in the aorta. In the present
study, mRNA expression of eNOS in the abdominal aorta in 45Fat and 30Frc + 45Fat groups was
significantly decreased in comparison to those of the other groups. Additionally, the 30Frc group had
significantly decreased mRNA expression of eNOS compared to the RD group. ET-1 is an important
vasoconstrictor produced by endothelial cells that contributes to enhanced blood pressure [43,46].
A previous study reported that rats fed a high-fructose diet for nine weeks had increased ET-1
levels [47]. Similarly, our study showed that the abdominal aortic ET-1 mRNA expression in 30Frc,
45Fat, and 30Frc + 45Fat groups were significantly higher than the RD group. Our data, together
with the previously established literature, suggest that high-fructose diet, as well as the high-fat diet,
negatively affected endothelium-derived relaxing and contracting factors. These changes are important
factors for the development of hypertension and vascular dysfunction.

5. Conclusions
Collectively, the high-fructose diets increased the total-fat weight and serum TG levels in
growing rats. Additionally, it had negative effects on abdominal aortic thickness and eNOS,
ET-1 mRNA expression.
The strength of our study is that we fed 30% fructose-diets to the animals. Numerous animal
studies have used extreme doses of ~60% fructose, which, as White [48] suggested, show results that
Nutrients 2017, 9, 11 10 of 12

are not physiological, and likely cause abnormal metabolism, and therefore cannot be depended on to
assess human risk. In the present study we were able to show that 30% fructose can induce total-fat
weight gain, increase aorta wall thickness, and affect eNOS and ET-1 mRNA expression, which are
related to CVD risk factors. One potential limitation in the present study was that the sample size was
small, which may lessen the significance of the results.
In conclusion, we confirmed high fructose consumption, as well as high fat consumption,
in growing rats had negative effects on CVD-related parameters, such as total-fat weight, serum TG,
aorta wall thickness, and eNOS, ET-1 mRNA expression of abdominal aorta.

Acknowledgments: This work was supported by Korea Institute of Planning and Evaluation for Technology
in Food, Agriculture, Forestry and Fisheries (IPET), funded by Ministry of Agriculture, Food and Rural Affairs
(MAFRA) (316055-3).
Author Contributions: The authors’ responsibilities were as follows: S.Y. and H.A. designed animal model;
S.Y. and H.A. conducted research; S.Y. and Y.K.P. analyzed data; S.Y., H.A., and Y.K.P. drafted and revised the
paper; all authors read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Kim, S.D.; Moon, H.; Park, J.S.; Lee, Y.C.; Shin, G.Y.; Jo, H.B.; Kim, B.S.; Kim, J.H.; Chae, Y.Z.
Macromineral intake in non-alcoholic beverages for children and adolescents: Using the fourth Korea
national health and nutrition examination survey (KNHANES IV, 2007–2009). Korean J. Nutr. 2013, 46, 50–60.
[CrossRef]
2. The Korea National Health and Nutrition Examination Survey (KNHNES). Dietary Intake Survey of Infant,
Children and Adolescents; The Korea National Health and Nutrition Examination Survey: Cheongju,
Korea, 2011.
3. Korea Health Industry Development Institute (KHIDI). In-Depth Analysis on the Dietary Intake Survey of Infant,
Children and Adolescents (II); Korea Health Industry Development Institute: Cheongju, Korea, 2010.
4. Bandini, L.G.; Vu, D.; Must, A.; Cyr, H.; Goldberg, A.; Dietz, W.H. Comparison of high-calorie,
low-nutrient-dense food consumption among obese and non-obese adolescents. Obes. Res. 1999, 7, 438–443.
[CrossRef] [PubMed]
5. Berkey, C.S.; Rockett, H.R.; Field, A.E.; Gillman, M.W.; Colditz, G.A. Sugar-added beverages and adolescent
weight change. Obes. Res. 2004, 12, 778–788. [CrossRef] [PubMed]
6. Nakagawa, T.; Hu, H.; Zharikov, S.; Tuttle, K.R.; Short, R.A.; Glushakova, O.; Ouyang, X.; Feig, D.I.;
Block, E.R.; Herrera-Acosta, J.; et al. A causal role for uric acid in fructose-induced metabolic syndrome.
Am. J. Physiol. Ren. Physiol. 2006, 290, F625–F631. [CrossRef] [PubMed]
7. Mohamed, S.S.; Nallasamy, P.; Muniyandi, P.; Periyasami, V.; Carani, V.A. Genistein improves liver function
and attenuates non-alcoholic fatty liver disease in a rat model of insulin resistance. J. Diabetes 2009, 1, 278–287.
[CrossRef] [PubMed]
8. Shih, C.; Lin, C.; Lin, W.; Wu, J. Momordica charantia extract on insulin resistance and the skeletal muscle
GLUT4 protein in fructose-fed rats. J. Ethnopharmacol. 2009, 123, 82–90. [CrossRef] [PubMed]
9. Bocarsly, M.E.; Powell, E.S.; Avena, N.M.; Hoebel, B.G. High-fructose corn syrup causes characteristics of
obesity in rats: Increased body weight, body fat and triglyceride levels. Pharmacol. Biochem. Behav. 2010, 97,
101–106. [CrossRef] [PubMed]
10. Crescenzo, R.; Bianco, F.; Coppola, P.; Mazzoli, A.; Valiante, S.; Liverini, G.; Iossa, S. Adipose tissue
remodeling in rats exhibiting fructose-induced obesity. Eur. J. Nutr. 2014, 53, 413–419. [CrossRef] [PubMed]
11. Muniyappa, R.; Montagnani, M.; Koh, K.K.; Quon, M.J. Cardiovascular actions of insulin. Endocr. Rev. 2007,
28, 463–491. [CrossRef] [PubMed]
12. Song, D.; Arikawa, E.; Galipeau, D.; Battell, M.; McNeill, J.H. Androgens are necessary for the development
of fructose-induced hypertension. Hypertension 2004, 43, 667–672. [CrossRef] [PubMed]
13. Behr-Roussel, D.; Oudot, A.; Compagnie, S.; Gorny, D.; Le Coz, O.; Bernabe, J.; Wayman, C.; Alexandre, L.;
Giuliano, F. Impact of a long-term sildenafil treatment on pressor response in conscious rats with insulin
resistance and hypertriglyceridemia. Am. J. Hypertens. 2008, 21, 1258–1263. [CrossRef] [PubMed]
Nutrients 2017, 9, 11 11 of 12

14. Endemann, D.H.; Schiffrin, E.L. Endothelial dysfunction. J. Am. Soc. Nephrol. 2004, 15, 1983–1992. [CrossRef]
[PubMed]
15. Austin, M.A.; King, M.C.; Vranizan, K.M.; Krauss, R.M. Atherogenic lipoprotein phenotype: A proposed
genetic marker for coronary heart disease risk. Circulation 1990, 82, 495–506. [CrossRef] [PubMed]
16. De Castro, U.G.; dos Santos, R.A.; Silva, M.E.; de Lima, W.G.; Campagnole-Santos, M.J.; Alzamora, A.C.
Age-dependent effect of high-fructose and high-fat diets on lipid metabolism and lipid accumulation in liver
and kidney of rats. Lipids Health Dis. 2013, 12. [CrossRef] [PubMed]
17. Yamauchi, T.; Kadowaki, T. Physiological and pathophysiological roles of adiponectin and adiponectin
receptors in the integrated regulation of metabolic and cardiovascular diseases. Int. J. Obes. 2008, 32, S13–S18.
[CrossRef] [PubMed]
18. Ross, R. The pathogenesis of atherosclerosis: A perspective for the 1990s. Nature 1993, 362, 801–809.
[CrossRef] [PubMed]
19. Sinitskaya, N.; Gourmelen, S.; Schuster-Klein, C.; Guardiola-Lemaitre, B.; Pevet, P.; Challet, E. Increasing the
fat-to-carbohydrate ratio in a high-fat diet prevents the development of obesity but not a prediabetic state in
rats. Clin. Sci. 2007, 113, 417–425. [CrossRef] [PubMed]
20. Matveyenko, A.V.; Gurlo, T.; Daval, M.; Butler, A.E.; Butler, P.C. Successful versus failed adaptation to
high-fat diet-induced insulin resistance: The role of IAPP-induced beta-cell endoplasmic reticulum stress.
Diabetes 2009, 58, 906–916. [CrossRef] [PubMed]
21. Ouguerram, K.; Nguyen, P. Lipid profile and insulin sensitivity in rats fed with high-fat or high-fructose
diets. Br. J. Nutr. 2011, 106, S206–S210.
22. Tran, L.T.; Yuen, V.G.; McNeill, J.H. The fructose-fed rat: A review on the mechanisms of fructose-induced
insulin resistance and hypertension. Mol. Cell. Biochem. 2009, 332, 145–159. [CrossRef] [PubMed]
23. Miller, A.; Adeli, K. Dietary fructose and the metabolic syndrome. Curr. Opin. Gastroenterol. 2008, 24, 204–209.
[CrossRef] [PubMed]
24. Angelopoulos, T.J.; Lowndes, J.; Zukley, L.; Melanson, K.J.; Nguyen, V.; Huffman, A.; Rippe, J.M. The effect
of high-fructose corn syrup consumption on triglycerides and uric acid. J. Nutr. 2009, 139, 1242S–1245S.
[CrossRef] [PubMed]
25. Shulman, G.I. Cellular mechanisms of insulin resistance. J. Clin. Investig. 2000, 106, 171–176. [CrossRef]
[PubMed]
26. Blakely, S.R.; Hallfrisch, J.; Reiser, S.; Prather, E.S. Long-term effects of moderate fructose feeding on glucose
tolerance parameters in rats. J. Nutr. 1981, 111, 307–314. [PubMed]
27. Tappy, L.; Le, K.A. Metabolic effects of fructose and the worldwide increase in obesity. Physiol. Rev. 2010, 90,
23–46. [CrossRef] [PubMed]
28. Basciano, H.; Federico, L.; Adeli, K. Fructose, insulin resistance, and metabolic dyslipidemia. Nutr. Metab.
2005, 2, 5–20. [CrossRef] [PubMed]
29. Iida, T.; Yamada, T.; Hayashi, N.; Okuma, K.; Izumori, K.; Ishii, R.; Matsuo, T. Reduction of abdominal fat
accumulation in rats by 8-week ingestion of a newly developed sweetener made from high fructose corn
syrup. Food Chem. 2013, 138, 781–785. [CrossRef] [PubMed]
30. Catena, C.; Giacchetti, G.; Novello, M.; Colussi, G.; Cavarape, A.; Sechi, L.A. Cellular mechanisms of insulin
resistance in rats with fructose-induced hypertension. Am. J. Hypertens. 2003, 16, 973–978. [CrossRef]
31. Kho, M.C.; Lee, Y.J.; Ahn, Y.M.; Choi, Y.H.; Kim, A.Y.; Kang, D.G.; Lee, H.S. Effects of ethanol extract of
gastrodia elata blume on high-fructose induced metabolic syndrome. FASEB J. 2013, 27, 1108–1113.
32. Dandona, P.; Aljada, A.; Bandyopadhyay, A. Inflammation: The link between insulin resistance, obesity and
diabetes. Trends Immunol. 2004, 25, 4–7. [CrossRef] [PubMed]
33. Fantuzzi, G. Adipose tissue, adipokines, and inflammation. J. Allergy Clin. Immunol. 2005, 115, 911–999.
[CrossRef] [PubMed]
34. Rudin, E.; Barzilai, N. Inflammatory peptides derived from adipose tissue. Immun. Ageing 2005, 2. [CrossRef]
[PubMed]
35. Yudkin, J.S.; Stehouwer, C.D.; Emeis, J.J.; Coppack, S.W. C-reactive protein in healthy subjects: Associations
with obesity, insulin resistance, and endothelial dysfunction: A potential role for cytokines originating from
adipose tissue? Arterioscler. Thromb. Vasc. Biol. 1999, 19, 972–978. [CrossRef] [PubMed]
Nutrients 2017, 9, 11 12 of 12

36. Davis, P.H.; Dawson, J.D.; Riley, W.A.; Lauer, R.M. Carotid intimal-medial thickness is related to
cardiovascular risk factors measured from childhood through middle age: The Muscatine study. Circulation
2001, 104, 2815–2819. [CrossRef] [PubMed]
37. Berenson, G.S.; Srinivasan, S.R.; Bao, W.; Newman, W.P.; Tracy, R.E.; Wattigney, W.A. Association between
multiple cardiovascular risk factors and atherosclerosis in children and young adults. N. Engl. J. Med. 1998,
338, 1650–1656. [CrossRef] [PubMed]
38. McGill, H.C., Jr.; McMahan, C.A.; Zieske, A.W.; Malcom, G.T.; Tracy, R.E.; Strong, J.P. Effects of nonlipid
risk factors on atherosclerosis in youth with a favorable lipoprotein profile. Circulation 2001, 103, 1546–1550.
[CrossRef] [PubMed]
39. McGill, H.C., Jr.; McMahan, C.A.; Herderick, E.E.; Tracy, R.E.; Malcom, G.T.; Zieske, A.W.; Strong, J.P.
Effects of coronary heart disease risk factors on atherosclerosis of selected regions of the aorta and right
coronary artery. Arterioscler. Thromb. Vasc. Biol. 2000, 20, 836–845. [CrossRef] [PubMed]
40. Bonetti, P.O.; Lerman, L.O.; Lerman, A. Endothelial dysfunction: A marker of atherosclerotic risk.
Arterioscler. Thromb. Vasc. Biol. 2003, 23, 168–175. [CrossRef] [PubMed]
41. Pepine, C.J. Clinical implications of endothelial dysfunction. Clin. Cardiol. 1998, 21, 795–799. [CrossRef]
[PubMed]
42. Brunner, H.; Cockcroft, J.R.; Deanfield, J.; Donald, A.; Ferrannini, E.; Halcox, J.; Kiowski, W.; Lüscher, T.F.;
Mancia, G.; Natali, A. Endothelial function and dysfunction. Part II: Association with cardiovascular risk
factors and diseases. A statement by the working group on endothelins and endothelial factors of the
european society of hypertension. J. Hypertens. 2005, 23, 233–246. [CrossRef] [PubMed]
43. Davignon, J.; Ganz, P. Role of endothelial dysfunction in atherosclerosis. Circulation 2004, 109, 27–32.
[CrossRef] [PubMed]
44. Dawson, V.; Brahmbhatt, H.; Mong, J.; Dawson, T. Expression of inducible nitric oxide synthase causes
delayed neurotoxicity in primary mixed neuronal-glial cortical cultures. Neuropharmacology 1994, 33,
1425–1430. [CrossRef]
45. Rees, D.D.; Palmer, R.M.; Moncada, S. Role of endothelium-derived nitric oxide in the regulation of blood
pressure. Proc. Natl. Acad. Sci. USA 1989, 86, 3375–3378. [CrossRef] [PubMed]
46. Oliver, F.J.; de la Rubia, G.; Feener, E.P.; Lee, M.E.; Loeken, M.R.; Shiba, T.; Quertermous, T.; King, G.L.
Stimulation of endothelin-1 gene expression by insulin in endothelial cells. J. Biol. Chem. 1991, 266,
23251–23256. [PubMed]
47. Verma, S.; Bhanot, S.; McNeill, J.H. Effect of chronic endothelin blockade in hyperinsulinemic hypertensive
rats. Am. J. Physiol. 1995, 269, H2017–H2021. [PubMed]
48. White, J.S. Challenging the fructose hypothesis: New perspectives on fructose consumption and metabolism.
Adv. Nutr. 2013, 4, 246–256. [CrossRef] [PubMed]

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