https://doi.org/10.52973/rcfcv-e34320
Received: 11/09/2023 Accepted: 28/11/2023 Published: 26/02/2024
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Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34320
ABSTRACT
Metabolic syndrome is a worldwide common disorder that coexists
with abdominal obesity, high blood pressure, dyslipidemia, and
high blood sugar. The purpose of this investigation was to examine
the effects of metabolic syndrome on enteroendocrine cell count
and histological structure in duodenum. The rats were separated
into control and metabolic syndrome groups. Duodenum tissue
sections were stained with hematoxylin–eosin for histomorphological
examinations. Additionally, immunohistochemical analysis of
duodenum showed the presence of somatostatin and gastrin
immunoreactive cells. In duodenum, it was found that villus height,
increased in metabolic syndrome group compared to control group.
Similarly, it was observed that the numbers of somatostatin and
in metabolic syndrome group compared to control group. As a result,
it was revealed that metabolic syndrome caused structural disorders
in duodenum and changed enteroendocrine cell population. Thus, it
can be assumed that intestinal functions may be negatively affected
due to metabolic syndrome and digestive physiology may be disrupted.
Key words: Duodenum; gastrin; metabolic syndrome; rat;
somatostatin
RESUMEN
El síndrome metabólico es un trastorno común a nivel mundial en el
que coexisten obesidad abdominal, presión arterial alta, dislipidemia
y niveles altos de azúcar en sangre. En el estudio presentado, el
objetivo fue evaluar los efectos del síndrome metabólico en la
estructura histológica y las células enteroendocrinas del duodeno.
Las ratas se dividieron aleatoriamente en dos grupos: control y
síndrome metabólico. Mientras que las ratas del grupo de control
recibieron agua del grifo durante 16 semanas, las ratas del grupo
con síndrome metabólico recibieron agua del grifo que contenía un
20% de D–fructosa. Se tomaron muestras de duodeno del intestino
con hematoxilina y eosina a secciones de tejido para exámenes
histomorfológicos. Además, se detectaron inmunohistoquímicamente
células inmunorreactivas de somatostatina y gastrina en el duodeno.
Se determinó que la altura de las vellosidades, el ancho de las
vellosidades, el área de las vellosidades y el grosor de la túnica
muscular en el duodeno aumentaron significativamente en el
grupo con síndrome metabólico en comparación con el grupo de
control. De manera similar, se observó que el número de células
inmunorreactivas de somatostatina y gastrina en el duodeno
en comparación con el grupo de control. Como resultado, se reveló
que el síndrome metabólico causa trastornos estructurales en el
duodeno y cambia la población de células enteroendocrinas. Por
tanto, se puede pensar que las funciones intestinales pueden verse
debido al síndrome metabólico.
Palabras clave: Síndrome metabólico; duodeno; somatostatina;
gastrina; rata
The effect of fructose–induced metabolic syndrome on the histological
structure and enteroendocrine cells in duodenum of rats
Efecto del síndrome metabólico inducido por fructosa sobre la estructura
histológica y las células enteroendocrinas en el duodeno de ratas
Mustafa Yildiz
1
* , Sadiye Kum
2
1
Canakkale Onsekiz Mart University, Can Faculty of Applied Sciences, Department of Occupational Health and Safety. Can, Canakkale, Türkiye.
2
Aydin Adnan Menderes University, Faculty of Veterinary Medicine, Department of Histology and Embryology. Isikli, Aydin, Türkiye.
*Corresponding author: mustafayildiz17@yahoo.com
The effect of fructose-induced metabolic syndrome on duodenum / Yildiz and Kum _______________________________________________
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INTRODUCTION
Metabolic syndrome (MS) is a disease characterized by insulin
resistance, hypertension, dyslipidemia and an increase in body mass
index [1]. An essential factor in development of this disease is the rise
decline in physical activity [2]. Additionally, people with metabolic
syndrome have a higher chance of developing cardiovascular disease,
which is the main cause of mortality. Nowadays, frequency of metabolic
serious health issue on a global scale [3]. In addition, loss in economy
due to care of metabolic syndrome patients has reached high costs [2].
Fructose is a six–carbon monosaccharide. It is consumed as a
syrup with high fructose [4]. Fructose mediates weight gain by stimulating
lipogenesis. Besides, excessive fructose consumption has become an
important factor in development of metabolic syndrome, as it can cause
an increase in blood pressure and triglycerides as well as insulin resistance
[5]. In addition, it has been reported that an experimental metabolic
syndrome model can be created in rodents with high fructose intake [1].
The metabolic syndrome induced by feeding with a high fructose
or fatty diet causes various degrees of damage such as irregularity
in microvilli [5], epithelial swelling, epithelial degeneration, villus
[6]. Also, it increases intestinal permeability, decreases intestinal
barrier integrity and stimulates the formation of apoptosis [7].
Furthermore, metabolic syndrome has been linked to an increase
in the number of goblet cells [8], villus length [9], crypt depth and
mucosal thickness in small intestine [5]. In addition, metabolic
syndrome increases small intestine weight, mitotic activity, and
absorption of short–chain fatty acids. On the other hand, metabolic
syndrome induces oxidative stress in intestinal tissue by increasing
lipid peroxidation and increases the levels of cytokines interleukin
β1 (IL–β1) and interleukin 6 (IL–6) [10].
Enteroendocrine cells are found in digestive tract and produce
peptide hormones. It is reported that they consist of at least 15 cell
types. Somatostatin and gastrin hormones are secreted by D and
G cells, respectively [11]. The somatostatin hormone inhibits the
secretions of insulin and glucagon [12
contracting smooth muscle in intestine [13]. The gastrin hormone
stimulates gastric acid secretion [14] and angiogenesis [15]. Besides,
it suppresses apoptosis [16] while increasing mucosal growth [14].
The purpose of this study was to look into the effects of fructose–
induced metabolic syndrome on histological structure and
enteroendocrine cells in duodenum that secrete somatostatin and
gastrin hormones.
MATERIALS AND METHODS
Ethical approval
Local ethics committee for animal experiments at Aydin Adnan
Menderes University granted permission for study (Decision number:
64583101/2020/042).
Animals
Sprague Dawley rats (Rattus norvegicus
t 22 ± 1°C with a 12–hour light/dark cycle.
Experimental design
The rats were randomly divided into two groups as control (n=10)
and metabolic syndrome (n=10). Tap water and food ad libitum were
given to the rats in control group for 16 weeks. The rats of metabolic
syndrome group were fed with tap water containing 20% D–fructose
(Merck D(–)–fructose for biochemistry 104007.0250) [17] and food ad
libitum for 16 weeks [18, 19].
Histomorphological analysis
At the end of experiment, rats were killed by cervical dislocation
under ether anesthesia. Then, duodenum samples were taken from
for 24 hours. After routine histological procedures, tissue samples
µm thickness were
taken. Tissue sections were stained with hematoxylin–eosin staining
method and two sections from each animal were examined. The
area, crypt depth and thickness of tunica muscularis were made in
two sections from each animal. The measurements were carried out
with a light microscope (SOIF BK5000–TR/L, Denmark) equipped with
an image analysis system (MShot Digital Imaging System, China). In
addition, photographs were taken with a camera (MShot MD 50, China)
from the necessary parts of the sections.
Immunohistochemical analysis
sections were boiled three times for 5 min each time in a microwave
2
O
2
that was prepared with distilled water for 10 min to
remove endogenous peroxidase activity. After that, sections were
kept in blocking solution for 5 min. After this process, sections
were incubated (NUVE, FN 055, NUVE Factory, Turkey) in primary
antibody (anti–gastrin, bs–1189R; anti–somatostatin, bs–1132R)
diluted 1/100 for 2 hours at 37°C. Then, sections were incubated
for 10 min each, respectively. Subsequently, sections were kept
in 3,3'–diaminobenzidine (DAB) for 3–5 min. Finally, sections were
The numbers of enteroendocrine cells were determined by manually
counting on the lamina epithelialis and crypts in randomly selected
of each animal. Then, the microscopic area was calculated and the
results were normalized to a unit area of 1 mm
2
.
Statistical analysis
The data were statistically analyzed with package program SPSS
20.00. t–test and Mann–Whitney U test were used to determine whether
or not there were differences between groups. The data were presented
using mean ± standard deviation format. The values with differences
of P<0.05 (*) and P<0.001 (***) were deemed statistically si
FIGURE 1. Microscopic views of the duodenum in the control (A, B) and metabolic
syndrome groups (C, D). It is seen that villus height (black lines), villus width
(blue lines), villus area (inner regions of green line) and thickness of the tunica
muscularis (brown lines) increased in the metabolic syndrome group compared
to the control group. Hematoxylin–eosin staining method. Bars: 100 μm
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RESULTS AND DISCUSSIONS
Studies have stated that metabolic syndrome increased the
mucosal thickness [5], villus height [9] and mitotic activity in small
intestine [10]. In the present study, it was determined that the villus
this study shows parallelism with the literature. In addition, it has
been detected that the increase in villi height is associated with an
increase in intestinal surface area and absorption of nutrients [20]. It
is also known that metabolic syndrome causes obesity [1]. Therefore,
in the present study; villi height increasing in duodenum as a result
of metabolic syndrome may increase the absorptive capacity of
small intestine and may play a role in pathogenesis of weight gain
in metabolic syndrome by leading to excessive nutrient absorption.
TABLE I
Histomorphological values in duodenums of
control and metabolic syndrome groups
Parameters
Control
(n=10)
Metabolic syndrome
(n=10)
P–value
Number of villus 5.81 ± 1.12 5.80 ± 1.04 NS
Villus height
(µm)
570.73 ± 122.98
b
685.10 ± 81.37
a
***
Villus width
(µm)
120.11 ± 26.24
b
137.63 ± 28.38
a
***
Villus area
(µm
2
)
65703.69 ± 54084.47
b
81226.94 ± 19579.99
a
***
Crypt depth
(µm)
252.07 ± 55.32 252.33 ± 54.86 NS
Thickness of tunica
muscularis (µm)
92.56 ± 23.03
b
99.28 ± 24.52
a
*
a,b
: Dierent superscripts in the same row indicate the signicant dierence. NS:
Non–signicant, *
P<0.05, ***P<0.001
It has been reported that feeding a high sucrose and fatty diet
increased blood tumor necrosis factor–alpha (TNFα) and IL–6
cytokines, lipopolysaccharide (LPS) endotoxin levels, as well as
caused swelling of villus epithelium in small intestine [6]. In addition,
it has been determined that high–fat diet together with sedentary
behavior triggered plasmacytoid and lymphocyte infiltration in
intestine, causing an increase in villus width [21]. Similarly, it has been
22].
In the present study, it was noticed that villus width and villus area
compared to control group (TABLE I; FIG. 1). In light of this information,
it can be thought that feeding with a high fructose diet may have
villi in the present study.
In the studies, it has been stated that the increase in thickness
of intestinal muscle layer mostly resulted from hypertrophy. The
as hypertrophic stimuli for smooth muscle cells [23]. Also, it has
been found that increased muscle thickness in intestine may be
[24]. In the present study, it was determined that the thickness of
group compared to control group (TABLE I; FIG. 1). Based on this
data, it is considered that a high fructose diet may have induced
muscle cells or connective tissue accumulation between muscle
cells. On the other hand, it has been emphasized that there may be
an increase in the thickness of tunica muscularis in order to increase
the digestibility of nutrients due to an increase in food intake during
pregnancy [25]. Also, it has been detected that the increase in the
thickness of tunica muscularis increased the contact between the
intestinal contents and mucosa [26
can be deduced that the muscle layer thickness may have increased
in order to increase digestive and absorption capacity of duodenum
as a result of feeding with a high fructose diet in our study.
Somatostatin regulates gastric acid secretion. It decreases smooth
13]. It has been
found that somatostatin immunoreactive cells were localized in
villus epithelium and crypts of small intestine [11]. In addition, it has
been determined that the number of somatostatin–producing D cells
increased in cardia and antrum parts of stomach but decreased
in corpus part of stomach in streptozotocin and fructose diet–
administered rats compared to control group [27]. Also, it has been
detected that 3–aminoisobutyric acid (BAIBA) metabolite was higher
in plasmas of subjects with high L and D cell density in duodenal
biopsies of individuals with metabolic syndrome. Thus, it has been
emphasized that there may be a link between increased L and D
cell density, plasma metabolites and clinical features of metabolic
syndrome [28]. In the current study, somatostatin immunoreactive
cells were found in epithelial layer and crypts of duodenum. It was
noticed that they were more intensely localized in crypts. In addition,
FIGURE 2. Microscopic views of somatostatin (A, C) and gastrin immunoreactive
cells (B, D) in the duodenum (arrows). It is observed that the number of
enteroendocrine cells increased in the metabolic syndrome group (C, D)
compared to the control group (A, B). Polymer–based immunohistochemical
method. Bars: 50 μm
The effect of fructose-induced metabolic syndrome on duodenum / Yildiz and Kum _______________________________________________
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it was detected that the number of somatostatin immunoreactive cells
are in parallel with the literature. On the basis of this information, it is
possible to conclude that the increase in the number of somatostatin
immunoreactive cells with effect of metabolic syndrome may cause
problems in functions such as regulation of gastric acid secretion in
stomach, blood circulation and motility in duodenum.
Gastrin immunoreactive cells are observed in villus epithelium
and crypts in all segments of small intestine [11]. Gastrin is a
regulator of gastric acid secretion. It stimulates cell proliferation
and angiogenesis [15]. It has been reported that it reduced apoptosis
in crypt cells in intestinal damage induced by ischemia reperfusion
[16]. It has been determined that the number of gastrin–positive
endocrine cells decreased in corpus and antrum parts of stomach
in streptozotocin and fructose diet administered rats compared
to control group [27]. In this study, gastrin immunoreactive cells
were localized in epithelial layer and crypts. Also, they were found
to be more common in crypts. On the other hand, the number of
in metabolic syndrome group compared to control group (TABLE II)
et al
[27]. The reason for obtaining different results in the studies may be
due to the differences in the tissue, experimental model and duration.
In addition, it can be considered that the increase in the number of
gastrin immunoreactive cells in duodenum with effect of metabolic
syndrome may induce disturbances in regulation of gastric acid
secretion as well as intestinal apoptosis and proliferation processes.
CONCLUSIONS
In conclusion, it was revealed that metabolic syndrome caused
changes in enteroendocrine cell population and histomorphological
structure in duodenum. Furthermore, it was drawn attention that
these alterations may lead to digestive physiology issues by adversely
present study will be a reference for future studies that affect
digestive system.
Conict of interests
with the publication of this manuscript.
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2
)
Enteroendocrine cells
Control
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Metabolic syndrome
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