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Atherosclerosis 154 (2001) 71 – 77
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Gelatin intake increases the atheroma formation in apoE knock
out mice
Dirce R. Oliveira a, Luciane R. Portugal a, Denise C. Cara b, Enio C. Vieira a,
Jacqueline I. Alvarez-Leite a,*
a
Departamento de Bioquı́mica e Imunologia, Laboratório de Gnotobiologia e Nutrição, Instituto de Ciências Biológicas (ICB),
Uni6ersidade Federal de Minas Gerais (UFMG), Caixa Postal 486, 30.161 -970 Belo Horizonte, MG, Brazil
b
Departamento de Patologia Geral, Instituto de Ciências Biológicas (ICB), Uni6ersidade Federal de Minas Gerais (UFMG), Caixa Postal 486,
30.161 -970 Belo Horizonte, MG, Brazil
Received 9 March 1999; received in revised form 2 February 2000; accepted 2 March 2000
Abstract
The effect of gelatin ingestion on cholesterol metabolism and on atheroma formation was evaluated in both wild type (n =14)
and apoprotein E (apoE) knock out (apoE − / − ) (n=20) C57BL/6 7-week-old mice. Animals were fed a cholesterol-free isoproteic
semi-purified diet containing 20% of casein (control diet) or 10% of casein plus 10% of gelatin (gel diet) for 8 weeks. In wild type
mice, dietary gelatin caused a reduction in the serum triacylglycerols levels associated with an increase in the fecal excretion. No
difference in blood cholesterol was seen at the sixth week of experiment. At the eighth week of experiment, there was a modest
but significant reduction of serum total and high density lipoprotein (HDL) cholesterol in apoE − / − mice fed on gel diet
compared to the control. Total cholesterol/HDL cholesterol ratio was 2-fold higher in the gel group than that seen in the control
group (14.39 and 7.84, respectively). Histological analyzes showed a 2.2-fold increase in the dimension of the atherosclerotic
plaques in the proximal aorta in apoE − / − mice fed on a gel diet compared to those fed on a control diet. The gel diet also
promoted a reduction in the fecal excretion of bile acids. Hepatic cholesterol was similar in both groups. In conclusion, although
gelatin reduced total serum cholesterol, this reduction was associated to a decrease of HDL cholesterol and consequent increase
of total cholesterol/HDL cholesterol ratio, resulting in an acceleration of atherogenesis. © 2001 Elsevier Science Ireland Ltd. All
rights reserved.
Keywords: Gelatin; Dietary protein; Cholesterol metabolism; Atherosclerosis; ApoE − / − mice
1. Introduction
Animal and vegetal proteins influence cholesterol
metabolism in humans and in experimental models [1].
It has been reported that animal proteins such as casein
induce hypercholesterolemia, whereas vegetal proteins
such as soybean protein have a hypocholesterolemic
effect [2]. The mechanisms of these effects are still
unclear and include: (i) alterations in digestion and
intestinal absorption of steroids due to the amino acid
composition of proteins or to physico-chemical activities of either proteins or their constituent amino acids
* Corresponding author. Tel.: +55-31-4992652; fax: + 55-314415963.
E-mail address: [email protected] (J.I. Alvarez-Leite).
[3]; and (ii) metabolic factors related to differences in
serum concentration of amino acids [4] or in lipoprotein
metabolism [5].
Gelatin is a mixture of small and large peptides with
a typical amino acid composition: 30% glycine, 30%
proline and hydroxyproline and absence of tryptophan
[6]. Notwithstanding the consumption of gelatin all
over the world, very few studies deal with the effect of
its ingestion on lipid metabolism [3,7–10] and there is
no consensus about its role in hypercholesterolemia and
atherosclerosis. Aust et al. [7] described serum triacylglycerols lowering the effect of a mixture of casein and
gelatin (1:1) in rats. With a similar protein mixture,
Gibney [8] found a hypocholesterolemic effect in rabbits. In contrast, Popescu et al. [9] reported a decrease
of hepatic triglycerides, total- and free-cholesterol in
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PII: S0021-9150(00)00460-3
D.R. Oli6eira et al. / Atherosclerosis 154 (2001) 71–77
72
rats fed on a diet containing gelatin (12%) and casein
(8%) as the protein source. Ratnayake et al. [10] evaluating the effect of different protein sources on the rat
serum lipids and liver polyunsaturated fatty acids,
found that gelatin had a hypocholesterolemic effect and
also suppressed the incorporation of arachidonic and
docosahexaenoic acids in the liver phospholipids. In
that study, the levels of serum triacylglycerols and high
density lipoproteins (HDL) were 5 and 30% lower in
rats fed on the gelatin diets compared with those fed on
the casein diets.
Apoprotein E (apoE) is a glycoprotein that mediates
a high affinity binding of remnant chylomicrons and
very low density lipoprotein (VLDL) and a HDL subclass to two different receptors: LDL receptor (B, E
receptor) and apoE receptor [11]. ApoE deficient mice
(apoE − / − ) fed on a low fat, low cholesterol diet show
serum cholesterol levels six to eight times higher than
the wild type mice due to a massive accumulation of
remnant lipoproteins [12]. Consequently, these animals
spontaneously develop atherosclerotic plaques, which
were similar to human lesions [13].
To obtain further information on the effects of
gelatin on the levels of total cholesterol and lipoproteins, wild type and apoE − / − mice were fed a
gelatin rich diet for 8 weeks. The influence of gelatin
ingestion on atherogenesis was studied by analyzing
lesional development in the proximal aorta of apoE − / −
mice. The results showed that gelatin reduced the serum
levels of total and HDL cholesterol and accelerated
atheroma formation in apoE − / − mice when compared
to mice in the control diet (20% of casein).
2. Materials and methods
2.1. Diets
The composition of the diets is shown in Table 1.
Diets were prepared every 2 weeks and stored at −
4°C.
Table 1
Composition (g/kg) of control and gel diet given to wild type or
apoE−/− mice for 8 weeks
Component
Control
Maize starch
Casein
Gelatina
Sucrose
Cellulose
Soybean oil
Vitamin mixtureb
Mineral mixtureb
Choline
533
200
0
100
50
70
20
25
2
a
b
Sigma, St. Louis, MO.
AOAC [14].
Gel
533
100
100
100
50
70
20
25
2
2.2. Animals and experimental design
The homozygous apoE deficient (apoE − / − ) mice (in
C57BL/6 background) [12,15] were purchased from
Jackson Laboratory (USA). Wild type C57BL/6 mice
were obtained from the ‘Instituto de Ciências Biológicas’, UFMG, Brazil. Fourteen female wild type and 20
apoE − / − 7-week-old female mice were used in the
experiment. Wild type mice were fed the control diet
(n=7) or gelatin (gel) diet (n= 7) for 8 weeks. ApoE − /
− mice were fed a control diet (n= 10) or gel diet
(n=10) and were distributed in two identical experiments using five animals in each group. The animals
were distributed based on their initial weight (16.59 1 g
for the wild type groups and 189 1 g for apoE − / −
groups) and initial serum cholesterol (969 15 mg/dl for
the wild type groups and 4709 60 mg/dl for apoE − / −
groups). The mice were housed in plastic cages and
kept in a room with a controlled light/dark cycle (12:12
h). Free access to food and water was provided. Individual weight and food intake were recorded weekly.
Blood samples from the ocular plexus were collected for
cholesterol analysis at the beginning and sixth week of
the diet feeding experiment. At the end of the experiment, the animals were fasted for eight hours and killed
under ether anesthesia. Blood was collected from the
axillary plexus for lipoprotein fractions and amino acid
determinations. The liver was removed, weighted and
frozen at − 20°C. The aorta was removed from the
root at the heart to the iliac bifurcation and fixed for
histopathological analysis. The relative liver weight
(RLW) ratio (liver weight/body weight × 100) was determined and used as an indirect parameter of nutritional status. The animal feeding and treatment
protocol was reviewed and approved by the Animal
Care Committee of the Instituto de Ciências Biológicas,
UFMG, Brazil.
2.3. Analytical methods
Blood samples were centrifuged at 12 000× g (3500
rpm) for 10 min and the sera was used for determination of lipids, protein and free amino acids.
Total cholesterol [16] and triacylglycerols [17] in
serum, liver and feces were determined enzymatically
using commercial kits (kits Analisa-Belo Horizonte,
Brazil). Sera of apoE − / − mice were diluted 1:2 in 0.85%
NaCl before cholesterol determination to keep the absorbance in the proper range of the test. Total protein
and albumin were measured using commercial kits from
Analisa (Belo Horizonte, Brazil).
Three samples from serum from each group (pooled
serum of animals 1 and 2, pooled serum of animals 3
and 4 and serum of animal 5) were used for free amino
acids and lipoprotein determination.
D.R. Oli6eira et al. / Atherosclerosis 154 (2001) 71–77
Table 2
Weight gain (g), relative liver weight (RLW) (liver weight/body
weight×100) and blood parameters of wild type and apoE−/− mice
fed diets containing 20% casein (control) or 10% casein+10% gelatin
(gel) as the protein source for 8 weeks
Parameter
Weight gain (g)
RLW
Total proteins
(g/dl)
Albumin (g/dl)
Total cholesterol
(mg/dl)
Triacylglycerols
(mg/dl)
Wild typea
ApoE−/−b
Control
Gel
Control
Gel
4.8 90.7c
3.5 90.6
5.4 90.2
3.1 9 0.7
3.69 0.4
5.79 0.2
2.09 1.0
4.09 0.3
6.4 9 0.4
2.8 9 0.7
4.0 9 0.2
6.7 9 0.6
3.7 90.1
125 98
3.89 0.2
1219 7
4.09 0.1
7059 60
4.3 9 0.2
558 9 69*
99 920
629 12*
859 22
89 9 29
a
n= 7 in each group.
n= 5 in each group. A second experiment using the same number
of animals yielded similar results.
c
Average 9 S.D.
* Significant difference (PB0.05) from the corresponding control.
73
root) separated by 200 mm intervals were obtained and
stained with hematoxylin-eosin. Histological sections
were examined under a light microscope by one individual who had no access to the codes. The lesion size was
evaluated morphometrically using an image analyzer
(KS 300 program) attached to a microcamera and Zeiss
microscope.
2.4. Statistical analysis
Evaluation of differences between diets was analyzed
within the genotype (wild type or apoE − / − mice).
Statistical analysis were performed using the unpaired
2-tailed Student’s t-test. A value of P B0.05 was used
to define statistical significance. The results were considered significant at PB 0.05 using unpaired Student’s
t-test.
b
For free amino acid analysis, 10 ml of serum and 50
mg of liver (perfused with phosphate buffered saline
solution and dried on filter paper) were precipitated in
methanol (1:4) and, after ultracentrifugation, the supernatant was diluted 1:1 in milliQ water, filtered, vacuum
dried and frozen for HPLC analysis [18]. To obtain
better resolution, liver samples were diluted 10 times in
run buffer (sodium acetate 0.14 M, pH 5.8, containing
0.05% of triethylamine) just before HPLC.
Serum lipoproteins were separated by FPLC analysis
as described by Fazio et al. [19]. Cholesterol concentration was determined in each fraction using commercial
kits (Analisa) adapted to a microplate assay [19].
Feces collected during the last 7 days of the experimental period, were pooled, dried, weighed and powdered. Fecal bile acids were extracted by the method of
van der Meer et al. [20] and the supernatants were
enzymatically assayed as previously described [21].
Hepatic and fecal total lipids were extracted by the
method of Folch et al. [22], gravimetrically quantified
and assayed for triacylglycerols and cholesterol. Hepatic free cholesterol content was also determined by the
same procedure except that cholesterol esterase was not
added to the reagent preparation [23]. Cholesterol ester
was indirectly determined from total and free cholesterol levels.
Aorta and liver samples were obtained from apoE − /
− and wild type mice and immediately fixed in 10%
formalin in PBS solution. The heart with the aorta was
removed and 10 ml of formalin solution was injected
into the heart. The heart and aorta were gently
stretched in a dish with formalin until the vessel wall
become rigid. Sample were embedded in paraffin and
processed. Fourteen 4 mm aortic sections (from aortic
3. Results
There were no differences in food intake (data no
shown) or RLW ( =liver weight/body weight×100)
between groups (Table 2). Animals had a good nutritional development, as documented by weight gain and
serum protein levels (Table 2). No differences were seen
in serum total protein and albumin levels (Table 2). No
differences were seen in total serum cholesterol at the
beginning and sixth week of experiment when apoE − /
− gel and wild type gel groups were compared to their
respective controls (data not shown). At the end of the
experiment, apoE − / − mice fed on the control and gel
diets had serum cholesterol levels 5.6 and 4.6 times
higher than the wild type group that received the same
diet. By 8 weeks, gelatin ingestion reduced triglyceridemia in wild type mice and total cholesterol in
apoE − / − mice (Table 2). Lipoprotein profiles were
similar in both wild type groups (Fig. 1A). However,
when the lipoprotein profile was determined in apoE − /
− mice, the reduction in total cholesterol was found to
be mainly due to a reduction in the HDL fraction (Fig.
1B, fractions 20–26). The total cholesterol concentration in atherogenic fractions (VLDL+ intermediate
density lipoproteins (IDL) and low density lipoproteins
(LDL)) were similar in both apoE − / − groups (Fig. 1B,
fractions 6–19). Consequently, total cholesterol/HDL
cholesterol ratio was approximately the twofold higher
than that seen in control group (14.39 and 7.84, respectively). The reduction in HDL-cholesterol and increase
of total cholesterol/HDL ratio paralleled to a 5-fold
increase in atherosclerotic lesions observed in apoE − / −
mice fed the gel diet compared to their respective
control (26 2009 3889 vs. 11 8009 2967) (Fig. 2). Animals from the gel group showed fatty streaks that
contained more fat deposition and macrophage infiltration (foam cells) when compared to the control group
74
D.R. Oli6eira et al. / Atherosclerosis 154 (2001) 71–77
Fig. 1. Cholesterol distribution in serum lipoprotein of wild type (A) and apoE − / − mice (B) fed diets containing 20% casein (control) or 10%
casein+ 10% gelatin (gel). A second experiment using the same number of apoE − / − mice yielded similar results. Total high density lipoproteins
(HDL0 cholesterol (sum of fractions 20–25) and very low density lipoproteins (VLDL)/intermediate density lipoproteins (IDL)/low density
lipoproteins (LDL) cholesterol (sum of fractions 6–19) are: 38 and 8 mg in wild type control group; 38 and 13 mg in wild type gel group; 25 and
200 mg in apoE − / − control group and 11 and 185 mg in apoE − / − gel group, respectively. For apoE − / − and wild type groups n =3.
(Fig. 3A and B), suggesting an enhanced lesional development in the mice that consumed gelatin. The histological analysis of livers of both groups showed fat
accumulation, but no significant differences were found
(data not shown).
Lesions were found in the seven first sections from
the aortic root in both apoE − / − groups. Morphological and morphometrical analysis was performed using
the largest lesion area from each animal. Control and
gel groups showed that largest lesions were located 400
and 200 mm from the aortic root, respectively. The gel
group had a 5-fold increase in atherosclerotic lesion
size, which were closer to the aortic root when compared with the lesions of the control group (P = 0.003).
Amino acid levels were determined in blood and liver
from mice in an attempt to clarify whether the effect of
gelatin was related to differences in amino acid profiles.
Compared to the control group, apoE − / − mice fed the
gel diet had a 38% increase on serum and a 40%
reduction of liver concentrations of total free amino
acids (23 872 vs. 32 933 pmol/ml serum and 329.61 vs.
235.50 nmol/g of liver, respectively). In wild-type mice,
those differences were not seen. The concentrations of
amino acids described as having an effect on serum
cholesterol (i.e. methionine, arginine, and lysine) were
similar in both apoE − / − groups.
Differences in the excretion of fecal steroids could
contribute for the effects of dietary intervention. There
was a lower excretion of bile salts in both wild type and
Fig. 2. Area of the atherosclerotic lesion in the proximal aorta at the
base of the heart to thoracic aorta) in apoE − / − mice fed diets
containing 20% casein (control group, n =5) or 10% casein +10%
gelatin (gel group, n =5) as the protein source for 8 weeks. Circles
represent individual measurement. Bars represent the average of each
group.
D.R. Oli6eira et al. / Atherosclerosis 154 (2001) 71–77
75
pattern; i.e. the serum levels of triacylglycerols were not
affected whereas hepatic concentrations were significantly reduced. Since there was a lower fecal excretion
in apoE − / − mice of gel group, one may suggest that
the uptake of triacylglycerols by extra hepatic tissues
was increased. Since hepatic synthesis of triacylglycerols
is not affected by the addition of gelatin to the diet [7],
the lower levels of total free amino acids in liver of
apoE − / − mice could have led to a lower protein synthesis, including apolipoproteins or membrane receptors involved on the lipoprotein uptake.
Wild type mice are highly resistant to the development of atherosclerosis, due to the low levels of total
serum cholesterol, a high HDL fraction (70%) and very
low levels of LDL fraction [12,26–30]. ApoE − / − mice
develop atherosclerotic lesions similar to those found in
humans, mainly in the proximal aorta [12,28,30]. Therefore, the use of apoE − / − mice, as a model for studies
of gelatin on atherosclerosis development, is justified.
To the authors’ knowledge, this is the first study that
shows the effect of gelatin on the atherogenesis, using
apoE − / − animals. Some authors reported the hypocholesterolemic effect of gelatin in rats and rabbits [8,10]
and assumed that gelatin can protect against atherosclerosis. However, those studies did not include analysis of
Fig. 3. Histology of proximal aorta from apoE − / − mice fed diets
containing (A) 20% casein (control) or (B) 10% casein +10% gelatin
(gel) as the protein source for 8 weeks. Note the accumulation of
foam cells in the subendothelium. Hematoxilin-eosin. Magnification
bars: 50 mm. The sections correspond to the biggest lesion seen in
each group: In (A) 400 mm from aortic root (1st section) and (B) 200
mm from aortic root (2nd section).
knock out mice fed on gelatin, suggestive of a higher
absorption or a lower bile acid production. There were
no differences in liver and fecal concentrations of total
cholesterol. However, in the liver, the gel diet promoted
a non-significant reduction in free cholesterol in
apoE − / − mice and of cholesteryl ester in wild type
mice (Table 3).
4. Discussion
Although gelatin can reduce the weight gain in rats
and rabbits [7–9,24], in these experiments, the addition
of 10% of gelatin to the diet did not affect the nutritional status of both apoE − / − and wild-type mice as
measured by weight gain, liver weight/body weight
ratio and serum total protein and albumin levels.
Regarding triglycerides, gelatin ingestion did not affect the hepatic concentrations in wild-type mice but
caused a reduction in serum triglycerides levels that can
be explained by the rise in the fecal excretion. In
apoE − / − mice, gelatin ingestion showed a different
Table 3
Biochemical parameters determined in feces and liver from wild type
and apoE−/− mice fed diets containing 20% casein (control) or 10%
casein+10% gelatin (gel) as the protein source for 8 weeksa
Wild typeb
Control
Feces d
Bile acids
(mmol/g)
Total
cholesterol
(mg/g)
Triacylglycer
ols
(mg/g)
Li6er (mg/g)
Total
cholesterol
Cholesterol
ester
Free
cholesterol
Gel
Control
Gel
8.5 90.2
6.8 90.2*
7.0 9 0.5
3.0 90.1
2.9 90.1
2.05 9 0.3
2.15 9 0.2
1.2 90.4
2.4 90.3*
2.5 9 0.4
1.9 90.3
5.7 9 1.2
4.9 9 1.0
8.6 91.8
7.7 9 0.9
1.6 90.5
1.3 9 0.2
2.2 90.8
3.2 9 0.9
4.1 91.0
3.9 9 0.8
6.7 91.8
4.5 9 0.8*
33.1 97.8
44.4 96.8
31.4 9 5.7*
Triacylglycer 33.7 9 11.3
ols
a
ApoE−/−c
3.7 9 0.6*
The results are the average9S.D.
For the hepatic parameters: n =7 in each group.
c
For the hepatic parameters: n =5 in each group.
d
Samples were pooled and three different extractions were performed.
* Significant difference (PB0.05) from the corresponding control.
b
76
D.R. Oli6eira et al. / Atherosclerosis 154 (2001) 71–77
atherosclerotic lesions or amino acid profile in animal
tissues. In rats, like in these experiments, this effect of
gelatin was associated with a decrease in serum triglycerides and in serum total cholesterol (due to HDL
cholesterol) [10]. The authors assumed that differences
of amino acid composition in gelatin compared to
casein are responsible for these actions in lipid
metabolism. Appreciable differences were not found in
free amino acids composition in the liver and serum of
mice fed the gel diet, especially regarding amino acids
that influence cholesterolemia (methionine, lysine and
arginine). It must be pointed out that the amino acid
determinations were done in fasting mice. The levels
reached in post absorptive state were not analyzed in
this work. These results are in agreement with those
reported by Kurowska and Carrol [25] who found no
correlation between serum levels of cholesterol and
specific amino acids either in starved or fed rabbits.
The lipoprotein pattern in the wild-type mice in this
study is in agreement with those reported in the literature [12,26,30]: HDL is the main cholesterol carrier in
the blood and VLDL, IDL and LDL fractions were
found only in trace amounts. In these animals, dietary
gelatin did not affect the distribution of cholesterol in
lipoprotein fractions. However, gelatin ingestion promoted a lower fecal excretion of bile acids with no
alterations in serum cholesterol or plaque formation in
wild type mice. It is possible that these animals have an
efficient compensation mechanism that involved enzymes of hepatic metabolism of cholesterol, such as
cholesterol 7-a-hydroxylase and b-hydroxy-b-methylglutaryl CoA reductase. Although the activities of these
enzymes were not measured in the present study, it is
well known that bile acid synthesis is regulated by bile
acids returning to the liver, in order to maintain the bile
acid homeostasis [31].
ApoE-/ − mice fed the control diet showed a plasma
lipoprotein pattern characterized by high levels of
atherogenic fractions and lower levels of HDL compared to wild-type mice (Fig. 1). In apoE − / − mice
ingesting gelatin, the decrease of HDL cholesterol accelerated plaque formation, which can be explained by
the reduced efflux of cholesterol from subendothelium,
which contribute to a faster foam cells formation.
The reduction of HDL cholesterol (and the consequent increase of total cholesterol/HDL ratio) was
probably the main factor involved on the increase of
lesional area in gelatin fed mice. The mechanism of
action of gelatin in reducing cholesterol in HDL fraction and on increasing the size of atherosclerotic lesions
has not been well explained. Saeki and Kiriama [32]
report that dietary protein-induced alterations in the
levels of HDL were directly related to the LCAT activity involved in free cholesterol esterification by HDL.
These authors concluded that the effect of dietary
proteins on serum cholesterol is not explained by the
differences in intestinal absorption and fecal excretion
of steroids or by the modification in lipoprotein (mainly
HDL) metabolism. Some amino acids can also affect
cholesterol metabolism in rabbits, partly through alteration of liver phospholipids [33], an important component of HDL. Differences may not have been detected
under the present conditions, because the animals were
fasting when they were sacrificed. The results also revealed that gelatin ingestion causes a reduction in the
fecal excretion of bile acids, which may reflect a higher
intestinal absorption, or a lower conversion of cholesterol into bile acids and its secretion in the bile, both
contributing for the faster development of aorta lesions.
The hepatic levels of total cholesterol were not increased in these animals, probably due to some compensating mechanism such a higher hepatic excretion of
VLDL or a lower HDL uptake. The LDL/HDL ratio is
also in agreement with the higher risk of atherosclerosis
development. It was concluded that gelatin ingestion
has a negative effect on cholesterol metabolism, reducing bile acid excretion and HDL cholesterol, resulting
in a faster lesional development.
Acknowledgements
The authors are grateful to Jamil Silvano de Oliveira
and Tiago Rennó dos Mares-Guia for the skillful technical help and to Dr Marcelo Matos Santoro and Dr
Leonides Resende Junior for their assistance. This work
was supported by Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq), Coordenação
de Pessoal de Ensino Superior (CAPES) and Fundação
de Amparo à Pesquisa do Estado de Minas Gerais
(FAPEMIG).
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