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R E S E A R C H Open Access
Age-dependent effect of high-fructose and
high-fat diets on lipid metabolism and lipid
accumulation in liver and kidney of rats
Uberdan Guilherme Mendes de Castro
2
, Robson Augusto Souza dos Santos
3
, Marcelo Eustáquio Silva
2
,
Wanderson Geraldo de Lima
1,2
, Maria José Campagnole-Santos
3
and Andréia Carvalho Alzamora
1,2,4*
Abstract
Background: The metabolic syndrome (MS) is characterized by variable coexistence of metabolic and
pathophysiological alterations which are important risk factors for developing of type II diabetes and/or cardiovascular
diseases. Increased of MS patients in worldwide has stimulated the development of experimental models. However, it is
still challenging to find an dietetic model that most closely approximates human MS and, in addition, is not yet fully
established the effect of different diets of MS in lipid metabolism in rats of different ages. The aim of this study was to
evaluate the effect of different diets of MS in lipid metabolism and ectopic fat deposition and define the most
appropriate diet for inducing the characteristic disturbances of the human MS in rats of different ages.
Methods: Young (4 weeks old) and adult rats (12 weeks old) were given a high-fat (FAT) or high-fructose diet (FRU) for
13 weeks and biochemical, physiological, histological and biometric parameters were evaluated.
Results: In young rats, the FAT diet induced increased mean blood pressure (MAP) and heart rate (HR), body weight
after 6 to 10 weeks, and in the 13th week, increased the liver, mesenteric, retroperitoneal and epididymal fat weights,
fasting glucose, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and reduced HDL cholesterol; and
also induced non-alcoholic fatty liver disease (NAFLD) and renal inflammatory infiltrates. In adult rats, the FRU diet
induced transient elevations of MAP and HR in the 6th week, and, at 13 weeks, increased fasting glucose, triglycerides,
total cholesterol, AST and ALT; increased liver, kidneys and retroperitoneal fat weights; and induced macrovesicular and
microvesicular NAFLD, the presence of fat cells in the kidney, glomerular sclerosis, and liver and kidney inflammation.
Additionally, the FAT and FRU diets induced, respectively, increases in liver glycogen in adults and young rats.
Conclusions: Our data show that FRU diet in adult rats causes biggest change on metabolism of serum lipids and lipid
accumulation in liver and kidney, while the FAT diet in young rats induces elevation of MAP and HR and higher
increased visceral lipid stores, constituting the best nutritional interventions to induce MS in rats.
Keywords: High-fat diet, High-fructose diet, Metabolic syndrome, Rats of different ages, NAFLD
Background
Metabolic syndrome (MS) is a pathological condition in
which three or more of the following risk factors are
present: central obesity, high plasma triglyceride levels,
low plasma HDL levels, high cholesterol, hyperglycemia,
atherosclerosis, non-alcoholic fatty liver disease (NAFLD),
endothelial dysfunction, insulin resistance (IR), and/or
hypertension [1,2]. These metabolic abnormalities ob-
served during MS are important risk factors for develop-
ing cardiovascular disease [3,4] and are associated with the
development of type II diabetes [5,6].
The development and establishment of MS are mainly
related to the consumption of high-fat diets and/or high-
carbohydrate diets [7]. Epidemiological studies have
shown that consumption of high-fat diets (≥30% of en-
ergy from fat) is correlated with high rates of overweight,
central obesity and MS [8,9]. Increasing evidence also as-
sociates the consumption of a diet high in carbohydrates,
* Correspondence: andreiaalzamora@iceb.ufop.br
1
Departamento de Ciências Biológicas, Instituto de Ciências Exatas e
Biológicas, Universidade Federal de Ouro Preto, Morro do Cruzeiro, Ouro
Preto, MG 35 400-000, Brazil
2
Núcleo de Pesquisa em Ciências Biológicas, Universidade Federal de Ouro
Preto, Ouro Preto, MG, Brazil
Full list of author information is available at the end of the article
© 2013 de Castro et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
de Castro et al. Lipids in Health and Disease 2013, 12:136
http://www.lipidworld.com/content/12/1/136
such as fructose, with high prevalences of obesity, IR,
hypertension and MS [10]. Therefore, the increasing con-
sumption of fructose-rich beverages and/or foods sweet-
ened with table sugar, corn syrup and other preparations
and/or processed products containing fructose is of great
concern [11].
The increased number of MS patients worldwide has
stimulated the development of experimental models that
mimic the characteristics of human MS [12], in attempts
to understand the biochemical, physiological and patho-
logical alterations involved in the development and
maintenance of excess body fat and MS [7]. Several gen-
etic models mimic many of the features of MS occurring
in humans, such as obese Zucker rats, obese spontan-
eously hypertensive rats (Koletsky rats) and Stroke-
prone SHR-fatty (fa/fa) rats [13]. However, animal
models that develop characteristics of MS without gen-
etic manipulation, but only through consumption of spe-
cific nutritionally unbalanced diets are increasingly
important [14] for use in simulating the most common
cause of human MS.
Despite the large volume of published studies using
experimental models of MS, it is still challenging to find
a model that most closely approximates human MS. Ex-
perimental models of diet-induced MS vary widely in the
induction of MS disturbances [15-18], mainly due to the
wide variation in the proportion and/or the types of nu-
trients that compose the diets and/or the different ages
of the animals used. This wide variability of the proto-
cols used in studies on MS limits the reproducibility of
the dietary treatments used and comparisons of pub-
lished data, making clear the need for a precise defin-
ition of the nutritional intervention to induce in animal
models, the disturbances typical of human MS. In
addition, is not yet fully established the effect of different
diets of MS in lipid metabolism and ectopic fat depos-
ition in rats of different ages.
This study submitted rats of different ages (young and
old) to the two main diets used in the literature to in-
duce MS, the high-fat diet [7,19] and high-fructose diet
[20,21], and evaluated different biometric, physiological,
biochemical and histological parameters in order to de-
fine the most appropriate dietary treatment to induce
the characteristic disturbances of human MS, and evalu-
ate the effect of different diets of MS in lipid metabolism
and ectopic fat deposition in rats of different ages.
Methods
Animals
The study used male Fischer rats, newly weaned at
4 weeks of age (young rats, 40 –60 g) and rats at
12 weeks of age (adult rats, 200 –250 g) from the La-
boratory of Experimental Nutrition (LABNEX) of the
Federal University of Ouro Preto (UFOP, Brazil). The
animals were kept in individual cages under controlled
temperature (25 ± 1°C) and a 12 h –12 h light–dark
cycle in the Animal Science Center (CCA/UFOP).
Throughout the experiment, the animals had free access
to water and diets. All procedures were performed in ac-
cordance with the Guidelines for Ethical Care of Experi-
mental Animals. The protocol was approved by the
Animal Ethics Committee of the Federal University of
Ouro Preto Protocol No. 2011/31.
Experimental protocol
Young and adult rats were randomly divided into three
groups subjected to different diets: AIN-93 control diet
(CT) [22], high-fat diet (FAT) or high-fructose diet
(FRU) for 13 weeks. The composition of the diets is
shown in Table 1. All groups of adult rats were fed the
CT diet from shortly after weaning until the start of the
experiment.
The experimental groups were:
1) Young –CT: young rats (4 weeks old) given the
control diet (AIN-93G, CT) immediately after
weaning during a period of 13 weeks;
2) Young –FAT: young rats (4 weeks old) given a diet
containing 40% fat (FAT) immediately after weaning,
for 13 weeks;
3) Young –FRU: young rats (4 weeks old) given a diet
containing 60% fructose (FRU) after weaning, for
13 weeks;
4) Adult –CT: adult rats (12 weeks old) given the
control diet (AIN-93 M, CT) for 13 weeks;
5) Adult –FAT: adult rats (12 weeks old) given a diet
containing 40% fat (FAT) for 13 weeks;
6) Adult –FRU: adult rats (12 weeks old) given a 60%
fructose diet (FRU) for 13 weeks.
Arterial pressure measurements
Mean arterial pressure (MAP, mmHg) and heart rate
(HR, beats/min) were assessed in awake rats in all
groups, in the second, sixth and tenth weeks of the diet,
by digital tail plethysmography (Panlab, LE5001). For
direct assessment of blood pressure (BP) and HR, rats
were anesthetized with a mixture of ketamine and
xylazine (50 mg/kg and 10 mg/kg respectively, ip) and a
polyethylene catheter was inserted into the abdominal
aorta through the femoral artery 48 h before the arterial
pressure measurement in awake rats. Pulsatile arterial
pressure was monitored by a Gould pressure transducer
(PM-1000, CWE) coupled to a blood pressure signal
amplifier (UIM100A, Powerlab System). The MAP and HR
were determined from the arterial pressure wave. All vari-
ables were continuously recorded with a PowerLab digital
acquisition system (PowerLab 4/25, ADInstruments) with
an 800 Hz sampling rate.
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Evaluation of animal and organ weights
All animals were weighed every two weeks until the end
of the experimental protocol. At the end of the 13 weeks
on the diets, the animals were euthanized, the wet
weights of the liver, heart and kidneys (g/100 g rat) were
measured, and these organs were then stored for histo-
logical analyses. The pancreas and retroperitoneal, epi-
didymal and mesenteric fat depots were dissected and
weighed (g/100 g rat).
Plasma analysis
At the end of the experiment, after euthanasia of animals
subjected to an overnight fasting, blood samples (2 to
3 ml) were collected. Then, these samples were centrifuged
(8000 g, 4°C, 6 min) to separate the plasma for determin-
ation of fasting glucose (blood treated with the anticoagu-
lant Glistab, containing EDTA and potassium fluoride) or
serum for total and HDL cholesterol, triglycerides, total
protein, albumin, creatinine, urea and alanine aminotrans-
ferase (ALT) and aspartate aminotransferase (AST). The
plasma and serum were aliquoted and stored at (−80°C) to
conduct the biochemical analyses. The analyses were
performed using individual commercial kits (Labtest, Lagoa
Santa, MG, Brazil) according to the instructions provided by
the manufacturer.
Histological analyses
For histopathological analysis, fragments of approxi-
mately 1.0 × 1.0 × 0.2 cm of liver, heart and kidneys
were fixed in 10% formalin. After 72 h of fixation, the
fragments were dehydrated, cleared, and embedded in
paraffin. Paraffin blocks were cut into 4 μm thick sec-
tions and stained by Hematoxylin and Eosin (H&E) for
assessment of architectural damage and the inflamma-
tory process (optical microscopy), or by Periodic Acid
Schiff (PAS) for detection of excessive hepatic glycogen.
By optical microscopy, the presence or absence of
microvesicular and macrovesicular NAFLD, necrosis, in-
flammation and fibrosis areas in the sections of stained
liver tissue were observed. The presence of hepatocytes
with glycogen deposits was also evaluated. In the kid-
neys, the presence or absence of glomerular sclerosis,
Table 1 Diet composition and energy contents of diet
Ingredients (g/Kg) AIN-93 FRU FAT
G (Young)* M (Adult)* Young Adult Young Adult
Corn starch 529,50 620,70 ----
Sucrose 100,00 100,00 ----
Fructose - - 600,00 600,00 33,0 34,20
Casein 200,00 140,00 200,00 200,00 180,50 180,50
Condensed milk - - - - 316,00 316,00
Soybean oil 70,00 40,00 40,00 40,00 - -
Lard - - - - 370,00 370,00
Fiber (cellulose) 50,00 50,00 - - 50,00 50,00
Wheat bran - - 109,5 108,3 - -
Mineral mix (AIN-93G-MX)* 35,00 - 35,00 - 35,00 -
Mineral mix (AIN-93 M-MX)* - 35,00 - 35,00 - 35,00
Vitamin mix (AIN-93G-VX)* 10,00 10,00 10,00 10,00 10,00 10,00
DL-Methionine 3,00 1,80 3,00 1,80 3,00 1,80
Choline Chloride 2,50 2,50 2,50 2,50 2,50 2,50
Macronutrients (% by weight)
Carbohydrate 62,95 72,07 60,00 60,00 20,68 20,80
Fat 5,00 4,00 4,44 4,43 39,53 39,53
Protein 20,00 14,00 21,97 21,95 20,26 20,26
Macronutrients (% Kcal)
Carbohydrate 66,82 75,82 65,25 65,28 15,92 16,00
Fat 11,95 9,46 10,86 10,84 68,48 68,42
Protein 21,23 14,72 23,89 23,88 15,60 15,58
Kcal/g 3,77 3,80 3,68 3,68 5,19 5,20
Kj/g 15,78 15,91 15,41 15,41 21,73 21,77
Mmaintenance diet, Ggrowth diet. * See Reeves [22].
de Castro et al. Lipids in Health and Disease 2013, 12:136 Page 3 of 11
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necrosis, inflammation and fibrosis areas and deposition
of lipid cells was evaluated. In the heart, the presence or
absence of areas of necrosis, inflammation and fibrosis
was evaluated. Representative photomicrographs were
obtained with a Leica BM5000 microscope coupled to a
Leica DFC 300 FX camera in RGB mode, using a 40×
magnification objective.
Results
Evaluation of BP and HR
The indirect evaluation, by tail plethysmography, revealed
that both the Young –FAT and the Young –FRU groups
showed higher values of MAP and HR in the 6th week of
the diet, compared to the Young –CT group. In the tenth
week of the diet, the values of MAP and HR of the Young –
FAT group remained higher than in the Young –CT, while
the Young –FRU group showed only increased HR com-
pared to Young –CT (Figure 1A and C). With respect to
adult animals, the Adult –FAT group showed an increase in
MAP only up to the tenth week of the diet compared to
Adult –CT.However,intheAdult–FRU group the MAP
washigherthanintheAdult–CT, only in the sixth week of
the diet (Figure 1B and D).
The direct evaluation performed at the 13th week of
the diet confirmed the results of tail plethysmography
and revealed that the Young –FAT animals showed high
levels of MAP and HR compared to Young –CT, while
the rats of the Young –FRU group showed only an
0 2 4 6 8 10
90
100
110
120
130
140
150
#*
*
*
#
weeks of diet
MAP, (mmHg)
0 2 4 6 8 10
90
100
110
120
130
140
150
#
*
*
#
#
weeks of diet
MAP, (mmHg)
0 2 4 6 8 10
300
350
400
450
500
550
*
*
*
*
##
#
weeks of diet
HR, (beats/min)
0 2 4 6 8 10
300
350
400
450
500
550
#
#
#
#
#
#
weeks of diet
HR, (beats/min)
AB
CD
CT FRU
FAT
0
50
100
150
Young Adult
**
MAP, (mmHg)
150
250
350
450
550
Young Adult
*
*
HR, (beats/min)
CT FAT FRU
EF
Figure 1 Evolution of Mean Blood Pressure (MAP) and Heart Rate (HR) evaluated indirectly for tail pletismography (panels A, B, C
andD)anddirectly(panelsEandF)inanimalsthatstartedtheexperimentatfourweeksofage(Young,panelsAandC)andat12weeks
old (Adult, panels B and D) submitted to the control AIN-93 CT (n = 7–10), high-fat (FAT; n = 7–9) or high-fructose (FRU; n = 8–12 adults) diet
for 13 weeks. * P <0.05 compared to the respective group that consumed the CT diet. # P <0.05 compared to data after 2 weeks on the diet.
de Castro et al. Lipids in Health and Disease 2013, 12:136 Page 4 of 11
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increase in HR. Furthermore, the Adult –FAT rats only
showed increased MAP (Figure 1E and F).
Evaluation of body and organ weights
Adult –CT animals showed increases in the liver weight
and retroperitoneal, epididymal and mesenteric fat de-
pots and total visceral fat (TVF) weights compared to
the Young –CT group. However, the Adult –CT group
showed lower body weight (BW) gain at the end of the
13th week of the diet compared to the Young –CT
group (Table 2).
The Young –FAT group showed a higher mean BW
from the 6th to 10th week of diet (Figure 2), and at the
13th week showed increases in liver weight and mesen-
teric, retroperitoneal and epididymal fat deposits and TVF
weight compared to the Young –CT group (Table 2). The
Young –FRU group showed a lower mean BW beginning
with the 6th week of the diet (Figure 2), and lower weights
of the retroperitoneal, mesenteric, and epididymal fat
depots and a decrease in TVF weight and in final BW
compared to the Young –CT group. At the 13th week
showed increased liver and kidney weights compared to
the Young –CT group.
In adult animals, no differences were observed in BW
between all groups in any of the weeks of diet evaluated
(Figure 2). However, at the 13th week, the Adult –FAT
group showed a greater relative weight of the retroperi-
toneal fat depot and a lower BW gain compared to the
Adult –CT group. The Adult –FRU animals showed in-
creases in the weights of the liver, kidneys and retroperi-
toneal fat depots compared to the Adult –CT group,
but no significant differences in the other parameters. In
addition, no differences were observed in the relative
weights of the heart and pancreas among all groups of
young animals (Table 2).
Plasma analysis
The biochemical analyses performed in the 13th week of
diets showed that Adult –CT rats showed high serum
triglycerides and albumin compared to the Young –CT
group (Table 3).
The Young –FAT animals showed increases in fasting
glucose and the ALT and AST transaminases, and reduced
serum HDL cholesterol compared to the Young –CT
group. The animals of the Young –FRU group also showed
decreases in HDL cholesterol and increases in ALT and
AST serum, and reduced levels of total protein and albu-
min compared to the Young –CT group (Table 3).
In relation to adult animals, the Adult –FAT group
showed only elevated serum levels of ALT compared to
Adult –CT. The Adult –FRU animals showed elevated
levels of fasting glucose, triglycerides, total cholesterol,
ALT and AST compared to Adult –CT (Table 3).
Histological analyses
The hepatic and renal histology showed that the animals
of the Young –FAT group presented microvesicular
NAFLD (25%, n = 8), inflammatory infiltrates in the kid-
neys (50%, n = 8) and hepatic glycogen depots similar to
the Young –CT group. The Young –FRU animals
showed macrovesicular (20%, n = 10) and microvesicular
NAFLD (30%, n = 10), increased hepatic glycogen stores,
and showed deposition of fat cells in the kidney (100%,
n = 10) (Table 4; Figure 3A to F and Figure 4A to C).
Regarding adult rats, the Adult –FAT group presented
microvesicular NAFLD (25%, n = 8) and increased hep-
atic glycogen stores; there were no significant renal
histological changes in this group. The Adult –FRU
animals showed macrovesicular (60%, n = 10) and mic-
rovesicular NAFLD (100%, n = 10), inflammatory infil-
trates in the liver (30%, n = 10), and glycogen depots
Table 2 Relative weights of organs from young and adult rats submitted to different diets
Weights Young Adult
CT FAT FRU CT FAT FRU
Liver 2,46 ± 0,08 2,81 ± 0,07* 2,81 ± 0,11* 3,13 ± 0,09†3,28 ± 0,72 4,08 ± 0,09*
Kidney 0,27 ± 0,02 0,26 ± 0,01 0,29 ± 0,02* 0,29 ± 0,01 0,32 ± 0,01 0,36 ± 0,01*
Heart 0,30 ± 0,01 0,31 ± 0,01 0,30 ± 0,03 0,27 ± 0,01 0,29 ± 0,01 0,28 ± 0,01
Pancreas 0,78 ± 0,07 0,72 ± 0,01 0,81 ± 0,03 0,84 ± 0,06 0,70 ± 0,06 0,79 ± 0,04
Mesenteric fat 1,05 ± 0,06 1,23 ± 0,05* 0,75 ± 0,10* 1,78 ± 0,18†1,80 ± 0,14 1,67 ± 0,11
Retroperitoneal fat 1,50 ± 0,06 3,37 ± 0,09* 0,95 ± 0,05* 2,85 ± 0,17†3,59 ± 0,15* 3,36 ± 0,12*
Epididymal fat 1,63 ± 0,09 2,64 ± 0,06* 1,14 ± 0,07* 2,47 ± 0,17†2,74 ± 0,16 2,73 ± 0,12
TVF 4,18 ± 0,19 7,24 ± 0,17* 2,85 ± 0,18* 7,09 ± 0,47†8,13 ± 0,41 7,77 ± 0,24
Weight gain 228,60 ± 16,25 241,90 ± 8,59 74,42 ± 4,83* 163,90 ± 14,09†132,20 ± 3,80* 162,70 ± 6,06
BW 284,2 ± 15,66 302,1 ± 5,6 131,8 ± 5,48* 402,5 ± 5,99 369,9 ± 6,34 397,6 ± 5,99
N121812101110
Nnumber of animals, BW Body Weight, TVF Total visceral fat. * P <0.05 compared to the respective group that consumed the CT diet. †P <0.05 compared to the
Young - CT group.
de Castro et al. Lipids in Health and Disease 2013, 12:136 Page 5 of 11
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similar to the Adult –CT group. The Adult –FRU group
also showed several renal disorders, including the depos-
ition of fat cells in the kidney (100%, n = 10), inflammatory
infiltrates (50%, n = 10) and glomerulosclerosis (20%,
n = 10), compared to the Adult –CT group (Table 4;
Figures 3G, 3L and 4D to 4F). In addition, both the young
and adult groups that consumed FAT or FRU diets for
13 weeks showed no significant changes in cardiac hist-
ology compared to the control groups (data not shown).
Discussion
The literature data diverge widely regarding the charac-
terization of MS disorders in rats. These differences are
attributable mainly to differences among experimental
protocols, such as (1) the variability in the composition
of diets; (2) different forms of fructose administration,
such as in the diet [23] or water [24]; (3) duration of
dietary treatments, from weeks [24] to months [15];
(4) use of different rodent models [15,23]; and differ-
ent age of animals, i.e., young [17,23] or adults [24].
To minimize these biases, in the present study we
compared several biochemical, physiological, histo-
logical and biometric parameters of newly weaned young
rats and adult rats submitted to the FRU and FAT di-
ets, i.e., those that are most often used to induce distur-
bances related to MS. Taken together, our data revealed
that FRU diet for adult rats and FAT diet for young rats are
the best nutritional interventions to induce MS in rats and
differentlychangethelipidmetabolism and the ectopic fat
deposition in the liver and kidney in rats of different ages.
Studies in animals and humans have shown that over-
weight and central obesity, characteristic of MS, are asso-
ciated with important changes in autonomic regulation,
such as an increase in the sympathetic nervous system ac-
tivity [25] and a decrease in the parasympathetic nervous
system activity [26]. In addition, consumption of a high-fat
0 2 4 6 8 10 12 14
0
100
200
300
400
500
*
*
*
*
*
**
*
CT FAT FRU
weeks of diet
Weight (g)
0 2 4 6 8 10 12 14
0
100
200
300
400
500
weeks of diet
Weight (g)
A
B
Figure 2 Evolution of body weight of rats that started the
experiment at four weeks of age (Young; panel A) and at
12 weeks of age (Adult, panel B) submitted to the AIN-93 (CT,
n = 12), high-fat (FAT, n = 11–18) or high-fructose (FRU, n = 12)
diet for 13 weeks. * P <0.05 compared to the respective group
that consumed the CT diet.
Table 3 Biochemical parameters of young and adult rats submitted to different diets
Biochemical parameters Young Adult
CT FAT FRU CT FAT FRU
Fasting glucose (mmol/L) 5,87 ± 0,14 7,18 ± 0,13* 5,77 ± 0,11 5,84 ± 0,17 5,42 ± 0,63 7,37 ± 0,19*
Triglycerides (mmol/L) 0,39 ± 0,07 0,27 ± 0,03 0,34 ± 0,06 0,64 ± 0,06†0,68 ± 0,06 1,02 ± 0,11*
Total cholesterol (mmol/L) 2,29 ± 0,24 2,11 ± 0,12 2,26 ± 0,09 1,81 ± 0,16 2,27 ± 0,11 2,97 ± 0,23*
HDL cholesterol (mmol/L) 1,52 ± 0,08 0,37 ± 0,01* 0,38 ± 0,03* 1,63 ± 0,06 1,44 ± 0,24 1,80 ± 0,07
ALT (U/I) 17,31 ± 1,32 24,96 ± 0,92* 30,09 ± 2,78* 18,15 ± 1,80 26,67 ± 3,02* 32,87 ± 1,25*
AST (U/I) 38,56 ± 4,33 72,80 ± 2,27* 87,15 ± 5,35* 30,01 ± 1,01 28,51 ± 2,06 63,43 ± 3,02*
Creatinine (μmol/L) 53,81 ± 5,33 53,44 ± 8,93 57,98 ± 8,13 72,24 ± 14,63 51,06 ± 5,36 52,85 ± 11,85
Urea (mmol/L) 4,33 ± 0,66 4,28 ± 0,56 4,45 ± 0,22 5,05 ± 0,32 5,85 ± 0,50 4,96 ± 0,28
Total protein (g/L) 69,53 ± 1,23 68,17 ± 2,95 45,98 ± 3,03* 67,59 ± 1,04 66,47 ± 1,23 68,19 ± 1,54
Albumin (μmol/L) 352,90 ± 8,27 342,10 ± 20,69 260,0 ± 13,68* 389,00 ± 4,57†378,60 ± 7,97 373,50 ± 13,63
N121888109
* P <0.05 compared to the respective group that consumed the CT diet. †P <0.05 compared to the Young - CT group.
de Castro et al. Lipids in Health and Disease 2013, 12:136 Page 6 of 11
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diet increases the MAP and HR in both adult obesity-
prone rats (32% fat diet) [27] and in humans [28]. Simi-
larly, our results showed that the Young –FAT group
increased MAP and HR from the sixth week of the diet.
Published work has shown that a high-fructose diet in-
duced an increase in systolic BP [29,30] and MAP in rats
[31,32]; however, D’Angelo et al. [33] showed that adult
Sprague–Dawley rats given a diet containing 66% fructose
for 8 weeks did not show increases in BP and HR. In the
present study, the FRU diet induced a transient increase in
MAP only in the sixth week of the diet, in both young and
adult animals. This suggests that counter-regulatory
mechanisms to increased BP are efficiently activated, so
that from the tenth week on, the groups that consumed
Table 4 Percentage of occurence (%) of histological changes in the liver and kidney
Liver Kidney
Tissue
inflammation
n (%)
Tissue
fibrosis
n(%)
Micro-
esteatosis
n (%)
Macro-
esteatosis
n (%)
Excessive
glycogen
deposition n (%)
Adipocytes
cell presence
n (%)
Tissue
inflammation
n (%)
Tissue
fibrosis
n (%)
Glomerular
sclerosis
n (%)
Tissue
amyloidosis
n (%)
Young –CT: n=8 0 (00.0) 0 (00.0) 0 (00.0) 0 (00.0) 2 (40.0) 0 (00.0) 0 (00.0) 0 (00.0) 0 (00.0) 0 (00.0)
Young –FAT: n=8 0 (00.0) 0 (00.0) 2 (25.0) 0 (00.0) 0 (00.0) 0 (00.0) 4 (50.0) 0 (00.0) 1 (12.5) 0 (00.0)
Young –FRU: n=10 0 (00.0) 0 (00.0) 3 (30.0) 2 (20.0) 5 (100.0) 10 (100.0) 0 (00.0) 0 (00.0) 0 (00.0) 0 (00.0)
Adult –CT: n=8 0 (00.0) 0 (00.0) 0 (00.0) 0 (00.0) 0 (00.0) 0 (00.0) 1 (12.5) 0 (00.0) 0 (00.0) 0 (00.0)
Adult –FAT: n=8 0 (00.0) 0 (00.0) 2 (25.0) 1 (12.5) 5 (100.0) 0 (00.0) 1 (12.5) 0 (00.0) 0 (00.0) 0 (00.0)
Adult –FRU: n=10 3 (30.0) 0 (00.0) 10 (100.0) 6 (60.0) 0 (00.0) 10 (100.0) 5 (50.0) 0 (00.0) 2 (20.0) 0 (00.0)
Figure 3 Histological examination of the liver with hematoxylin and eosin (H & E) staining and determining the excessive deposition
of glycogen by periodic acid-Schiff (PAS) in rats that started the experiment at four weeks of age (Young; panels A to F) and at
12 weeks (Adult, panels G to L) submitted to AIN-93 (CT), high-fructose (FRU) or high-fat (FAT) diet for 13 weeks. The arrowhead ( )
represents the non-alcoholic microvesicular fatty liver disease (NAFLD), and the long arrow ( ) represents the occurrence of macrovesicular
NAFLD. Panels Eand Fshow higher glycogen stores in the Young –FRU and Adult –FAT groups. 40× magnification.
de Castro et al. Lipids in Health and Disease 2013, 12:136 Page 7 of 11
http://www.lipidworld.com/content/12/1/136
the FRU diet exhibited BP values similar to their respect-
ive control groups.
Interestingly, in this study in the second week was ob-
served in all control animals, especially in the adult
group, increased in HR. The probably explanation must
be the tail plethysmography methodology used for evalu-
ating cardiovascular parameters. This methodology
could be a source of greater stress for adult animals in
relation to young animals. Furthermore, the second
measurement (6 weeks) onwards, the level of HR in con-
trol animals was decreased, probably because the ani-
mals have adapted to the procedure, for this reason was
carried also direct evaluation of MAP and HR in the
13th week of the diets.
In addition, previous studies have shown that young
C57B1/6J mice fed a diet rich in saturated fats, for
7 weeks, developed central obesity especially [34]. Simi-
larly, our data showed that the Young –FAT animals
showed increases in BW only in the 6th, 8th and 10th
week of the diet, and increased fat depots (mesenteric,
retroperitoneal and epididymal) in the 13th week. Our
data, together with the literature reports, suggest that
the FAT diet induces considerable central obesity, which
seems to be related to the deleterious factors associated
with MS, rather than to the increase in BW per se [14].
Previous studies showed that adult rats consuming a
high-fructose diet (60 g/100 g) for 8 to 10 weeks showed
increases in BW [35,36]. However, Moura et al. [21]
found that adult rats fed a high-fructose diet (60%) for
8 weeks increased the amount of retroperitoneal fat, but
not BW. Similarly, our study showed that Adult –FRU
rats did not have increased BW, but did show an in-
creased retroperitoneal fat depot.
The high-fat diet reduces the expression of insulin re-
ceptors, inhibits the oxidation of fatty acids (FA) in skel-
etal muscles, diminishes the mRNA expression and
intracellular protein content of GLUT4, and reduces the
translocation of GLUT4 to the cell membrane [37,38],
factors that may be responsible for the observed hyper-
glycemia and hyperinsulinemia in animals [39]. In our
study, the Young –FAT animals showed elevated plasma
levels of fasting glucose, suggesting, together with the
evidence from the literature, that the FAT diet alters glu-
cose metabolism, mainly in young animals. Moreover,
the FAT diet was not effective in increasing serum trigly-
ceride (TG) in both young and adult rats. Similar find-
ings [40,41] showed that a high-fat diet can also reduce
the synthesis of VLDL in hepatocytes, since excess TG
consumed can be directed to an oxidative route to form
ketones and carbon dioxide. Another plausible explan-
ation is that the high-fat diet has provided low amount of
carbohydrates for synthesis of triglycerides and/or the high
concentration of saturated fat in the FAT diet could some-
how reduce the hepatic secretion of VLDLs [41].
A high-fructose diet greatly increases the amount of
this monosaccharide in the liver, independently of insu-
lin action. This event accelerates the production of pyru-
vate and glycerol 3-P, which in turn stimulates de novo
lipogenesis, leading to up-regulating of lipogenic en-
zymes that increase the synthesis, esterification, secre-
tion and accumulation of cholesterol and FA in
hepatocytes [42]. Our data agreed with these findings,
and showed that the Adult –FRU group had increased
serum levels of TG and cholesterol.
The Young –FRU rats had low levels of total protein
and albumin, and showed no change in fasting glucose,
Figure 4 Histological examination of the kidney with hematoxylin and eosin (H & E) staining in rats that started the experiment at
four weeks of age (Young; panels A to C) and 12 weeks of age (Adult, panels D to F) given the AIN-93 (CT), high-fructose (FRU) or
high-fat (FAT) diet for 13 weeks. The arrow ( ) indicates the occurrence of inflammatory infiltration in the Young –FAT and Adult –FRU
groups. The asterisk (*) indicates the presence of fat cells in the kidneys of animals in the Young –FRU and Adult –FRU groups. 40× magnification.
de Castro et al. Lipids in Health and Disease 2013, 12:136 Page 8 of 11
http://www.lipidworld.com/content/12/1/136
total cholesterol and TG. These data, combined with the
reduction in visceral fat depots and BW in these animals,
suggest that the FRU diet is not the most appropriate for
induction of MS in young rats. This concords with the
findings of Moura et al. [21], who noted that a high-
fructose diet induces MS more effectively in adult than in
young rats.
NAFLD induces increased hepatic transaminases,
nonalcoholic steatohepatitis (NASH), hepatic inflamma-
tion, and fibrosis, in both humans and animals [43]. In
addition, Lieber et al. [44] demonstrated the presence of
NAFLD and NASH in rats that consumed a high-fat diet
(71% of energy from fat) for three weeks. Similarly, our
data showed that the Young –FAT group had increased
liver weight and showed microvesicular NAFLD along
with high serum levels of ALT and AST. These data
showed that young rats subjected to a FAT diet, in
addition to presenting important changes characteristic
of MS, such as central obesity and hyperglycemia, also
shows damage and ectopic fat deposition in the liver.
Previous studies showed that the FAT diet decreases in-
sulin sensitivity, while the high ratio of saturated fatty acid
induces hepatic steatosis [45,46] and hepatic IR [47]. Stud-
ies in dogs have found that a high-fat, high-fructose diet
impairs glucose uptake, lactate production, the flow of the
glycolytic pathway, and the synthesis of hepatic glycogen
[47], through interfering with the action of insulin in the
liver [45]. Similarly, our findings show that although the
rats in the Young –FAT group showed elevated fasting
glucose, and consumed high levels of simple carbohydrates
in the FAT diet, this group showed no increased glycogen
stores, unlike the Adult –FAT group, which showed nor-
mal fasting glucose and high glycogen depots. This sug-
gests that the young animals showed greater alterations in
the hepatic metabolism in comparison to the adult rats.
The Adult –FRU animals showed more types of liver
abnormalities, including increased liver weight, higher
percentages of occurrence of and microvesicular and
macrovesicular NAFLD, and the presence of inflamma-
tory infiltrates, along with increases in ALT and AST
serum levels. Similarly, previous studies in rats consum-
ing diets with high fructose concentrations, both in the
feed (60%) [48] and in drinking water (10%) [49], showed
microvesicular NAFLD [43,50], intralobular inflamma-
tion, and increased expression of IL-6 and TNF-αin the
liver [43]. These results together confirm that the con-
sumption of high-fructose diets induces NAFLD and
NASH; and in addition, our data showed that these dis-
turbances were most evident in adult rats.
In the early stages of MS induced by fructose, the syn-
thesis of P-trioses increases, which exceeds the oxidative
capacity of the liver, leading to stimulation of glucose
(50%), lactate (25%) and TG production and glycogen
synthesis (15%) [50,51]. Accordingly, Francini et al. [52]
showed that in Wistar rats given drinking water
containing 10% fructose over three weeks, glycogen
stores in the liver increased. However, in more advanced
stages, the large input of fructose in the liver produces an
allosteric negative regulation of the enzyme phosphofruc-
tokinase, reducing the uptake of glucose by the liver [53],
increasing hepatic gluconeogenesis, and inducing a high
intrahepatic accumulation of lipids, which, in turn, occupy
a large volume within hepatocytes and disorganize the
hepatic structure and functions such as glycogen storage,
as well as reducing hepatic IGF-1 synthesis and inducing
hepatic IR [50,54]. Our data showing an increase in glyco-
gen stores of the Young –FRU group, unlike the results
for the Adult –FRU group, suggest that these groups are
at different stages of MS and hepatic IR induced by fruc-
tose. Adult animals appear to be more susceptible to the
development of alterations in the hepatic metabolism of
glucose and lipids.
Several studies [55-57] have shown that an increase in BP
and development of central obesity are followed by renal
disorders, such as vasodilation, glomerular hyperfiltration
and inflammation. Similarly, our data showed that although
serum levels of creatinine and urea did not change, the
Young –FAT group showed renal inflammatory infiltrates,
along with central obesity and increases in BP and HR,
suggesting that there is an association between these factors.
Evidence shows that adult rats consuming a high-
fructose diet (60 g/100 g) [24] and diabetic rats at different
ages [58] also have various renal impairments, such as
hypertrophy, arteriolopathy, cortical vasoconstriction, and
hypertension and glomerular hyperfiltration [59] due to
increased blood glucose. Our findings that Adult –FRU
rats had renal alterations such as accumulation of fat cells,
increases in kidney weight, glomerular sclerosis and in-
flammatory infiltrates, along with elevated blood-glucose
levels, reinforce the idea that glycosylation of proteins, the
increased release of proinflammatory cytokines, oxidative
stress, and the accumulation of lipid peroxidation prod-
ucts may be the cause of kidney damage [58,60]. Regard-
ing the accumulation of fat cells in the kidneys on the
groups that consumed the FRU diet, still is not fully
understood the exact mechanism by which the FRU diet
induced it, but we believe that some renal glucose trans-
porters, such as GLUT2, GLUT5, NaGLT1, SGLT4
[61,62] and the recently reported SGLT5, which is exclu-
sively expressed in the kidney and transports fructose and
mannose [63], may be involved [64].
In the present study, we observed no significant
changes in cardiac histology in any group submitted to
the FAT or FRU diets. Similarly, studies by Carroll et al.
[65] and Mellor et al. [66], which submitted, respectively,
adult rats to a high-fat diet (32% kcal fat) and to a diet
with 60% fructose, also found no hypertrophy [66] or
changes in heart function [65].
de Castro et al. Lipids in Health and Disease 2013, 12:136 Page 9 of 11
http://www.lipidworld.com/content/12/1/136
Conclusions
Taken together, our results showed that FRU diet in
adult rats causes biggest change in metabolism of serum
lipids and induces ectopic fat accumulation in liver and
kidney, while the FAT diet in young rats induces eleva-
tion of MAP and HR and increased visceral lipid stores,
constituting the most effective nutritional interventions
in inducing, to a large extent, the biochemical, physio-
logical and histological features of human MS.
Statistical analysis was performed using Prism 5 for
Windows (GraphPad Software, Inc., San Diego, CA,
USA). Data are expressed as means with their standard
errors of the mean for animals given each diet. All pa-
rameters were analyzed by one-way ANOVA. Post-test
comparisons were made using a Bonferroni multiple-
comparison test. A Pvalue < 0.05 was considered statis-
tically significant.
Competing interests
The authors declare that they have no conflict of interest.
Authors’contributions
UGMC participated in developing protocol, data collection, data analysis and
interpretation of data and writing manuscript. ACA contributed in
conception, co-ordination, supervision and design of the study, acquisition
of funding and critically revising the manuscript. WGL carried out for
histological analysis, MES carried out for biochemistry analysis, RASS and
MJCS contributed in acquisition of funding and critically revising the
manuscript. All authors read and approved the final manuscript.
Acknowledgements
This study was supported by Universidade Federal de Ouro Preto (UFOP),
FAPEMIG-RedeToxifar (Fundação de Amparo à Pesquisa do Estado de Minas
Gerais), CNPq (Conselho Nacional de Desenvolvimento Científico e
Tecnológico), INCT-FAPEMIG-CNPq, Pronex Project Grant (FAPEMIG/ CNPq)
and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior).
Uberdan Guilherme Mendes de Castro received a CAPES fellowship (Master’s
Degree) in the “Programa de Pós-graduação Ciências Biológicas”, NUPEB,
UFOP.
Author details
1
Departamento de Ciências Biológicas, Instituto de Ciências Exatas e
Biológicas, Universidade Federal de Ouro Preto, Morro do Cruzeiro, Ouro
Preto, MG 35 400-000, Brazil.
2
Núcleo de Pesquisa em Ciências Biológicas,
Universidade Federal de Ouro Preto, Ouro Preto, MG, Brazil.
3
Departamento
de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade Federal
de Minas Gerais, Belo Horizonte, MG, Brazil.
4
Departamento de Ciências
Biológicas, Núcleo de Pesquisa em Ciências Biológicas, Universidade Federal
de Ouro Preto, Ouro Preto, MG, Brazil.
Received: 28 August 2013 Accepted: 6 September 2013
Published: 18 September 2013
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doi:10.1186/1476-511X-12-136
Cite this article as: de Castro et al.:Age-dependent effect of high-
fructose and high-fat diets on lipid metabolism and lipid accumulation
in liver and kidney of rats. Lipids in Health and Disease 2013 12:136.
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