Department of Basic Sciences, Histology Section, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran
Stem Cell and Transgenic Technology Research Center of Shahid Chamran University of Ahvaz, Ahvaz, Iran
Department of Biochemistry and Molecular Biology Section, Faculty of Veterinary Medicine, Shahid Chamran University
of Ahvaz, Ahvaz, Iran
P.O. BOX: 61357-831351
Department of Basic Sciences
Faculty of Veterinary Medicine
University of Ahvaz
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Erfani Majd Naeem,
Tabandeh Mohammad Reza,
Okra (Abelmoscus esculentus) Improved Islets Structure, and
Down-Regulated PPARs Gene Expression in Pancreas of
High-Fat Diet and Streptozotocin-Induced Diabetic Rats.
Okra (Abelmoschus esculentus) is a tropical vegetable that is rich in carbohydrates, fibers, proteins and
natural antioxidants. The aim of the present study was to evaluate the effects of Okra powder on pancreatic islets
and its action on the expression of PPAR-γ and PPAR-α genes in pancreas of high-fat diet (HFD) and streptozotocin-
induced diabetic rats.
Materials and Methods
In this experimental study, diabetes was induced by feeding HFD (60% fat) for 30 days
followed by an injection of streptozotocin (STZ, 35 mg/kg). Okra powder (200 mg/kg) was given orally for 30 days after
diabetes induction. At the end of the experiment, pancreas tissues were removed and stained by haematoxylin and
Eozine and aldehyde fuchsin for determination of the number of β-cells in pancreatic islets. Fasting blood sugar (FBS),
Triglycerides (TG), cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), and insulin levels were
measured in serum. Moreover, PPAR-γ and PPAR-α mRNAs expression were measured in pancreas using real time
polymerase chain reaction (PCR) analysis.
Okra supplementation significantly decreased the elevated levels of FBS, total cholesterol, and TG and attenuated
homeostasis model assessment of basal insulin resistance (HOMA-IR) index in diabetic rats. The expression levels of PPAR-γ
and PPAR-α genes that were elevated in diabetic rats, attenuated in okra-treated rats (P<0.05). Furthermore, okra improved
the histological damages of pancreas including vacuolization and decreased β-cells mass, in diabetic rats.
Our findings confirmed the potential anti-hyperglycemic and hypolipidemic effects of Okra. These changes
were associated with reduced pancreatic tissue damage. Down-regulation of PPARs genes in the pancreas of diabetic
rats after treatment with okra, demonstrates that okra may improve glucose homeostasis and β-cells impairment in
diabetes through a PPAR-dependent mechanism.
Type 2 diabetes (Fig T2D,) and obesity are the most frequent
endocrine-metabolic diseases that are characterized
by hyperglycemia and impaired insulin action and
secretion (i.e. insulin resistance) (1). Increasing evidence
from epidemiological studies indicates that genetic
predisposition and environmental factors including
obesity and sedentary life style are major risk factors
for the development of diabetes. Consumption of high
amounts of prepared foods rich in sugar and fat increases
the risk of dyslipidaemia, obesity, insulin resistance and
diabetes (2). Most of the individuals diagnosed with T2D
are found to be obese. Similarly, consumption of a high-
fat diet (HFD) increases the risk of acute insulin resistance
in rodents (3).
Obesity is accompanied by various metabolic
complications including dyslipidemia, hyperglycemia
and increased levels of circulating cytokines. Both
dyslipidemia and hyperglysemia contribute to loss of
ß-cells function and impairment of insulin secretion in
obesity and diabetes. Fatty acids (FAs) have been shown
to be pro-apoptotic for ß-cells. Proliferative capacity
of ß-cells is inhibited after a prolonged exposure to
increased glucose concentrations. Considering the
role of ß-cells pathophysiology in progression of T2D,
genetic background seems to be crucially important
and recent studies have shown that ß-cells dysfunction
in hyperglycemic state is associated with down/up
regulation of key islets genes (4).
Peroxisome proliferator-activated receptors (PPARα,
PPARγ and PPARβ/δ) are the members of the nuclear
receptor superfamily which play crucial roles in regulating
lipid and glucose homeostasis and controlling cellular
proliferation in pancreatic endocrine tissue. The function
of PPAR-γ on insulin sensitivity is due to its ability to
channel FAs into adipose tissue; therefore, diminishing
plasma FAs concentration and reducing lipotoxicity in the
pancreas (5). Also PPAR-γ regulates insulin release from
the pancreatic ß-cells by activating glucokinase and glucose
transporter (GLUT2). Also, PPAR-α is expressed in rat
pancreatic islets and severe activation of PPAR-α induces
mitochondrial ß oxidation of FA and potentiates glucose-
stimulated insulin secretion (GSIS) in rat islets (6). Therefore,
PPAR-α agonists improve pancreatic ß-cells function in
insulin-resistant rodents (7) and manipulation of PPAR
signaling pathway is one of the most attractive approaches in
drug discovery for the treatment of diabetes.
In recent years, the use of different medicinal plants for
the treatment of diabetes mellitus has increased. Medicinal
herbs could be extensively used for the treatment of diabetic
patients due to beneficial actions of their bioactive compounds
on ß-cells function, as well as insulin action, production and
resistance. Although, herbal medicines are considered for the
treatment of diabetes mellitus, the efficacy of these medicinal
plants and their derivatives in regulation of metabolic
disorders is not completely investigated and many of them
Abelmoschus esculentus belonging to Malvaceae
family, is a plant native to Africa and India and has
been a part of the diet in various parts of the world (8).
Phytochemical studies exhibited that polysaccharides,
polyphenols, flavonoids, tannins, sterols and triterpenes
are the major components of A. esculentus with various
biological activities (9). It has been reported that the okra
powder plays antidiabetic and antihyperlipidemic roles
in diabetic rats. Dietary fibers and polyphenols which are
abundantly found in A. esculentus, may contribute to the
hypoglycemic and hypolipidemic effects of A. esculentus
as suggested previously (10).
In spite of beneficial effects of A. esculentus for
treating metabolic complications of diabetic patients,
its impact on pancreatic histological and molecular
changes associated with HFD-induced diabetes has not
been clarified. This study was conducted to evaluate the
effects of A. esculentus (okra) on histological change and
PPAR-γ and PPAR-α gene expression in pancreas of HFD/
streptozotocin (STZ)-induced diabetic animal model.
Materials and Methods
A. esculentus (a native cultivar of Khuzestan, Iran) was
collected from local farms in Ahvaz, South-West of Iran. The
plants were kindly identified by Plant Taxonomy laboratory,
Faculty of Agriculture Science, Shahid Chamran University
of Ahvaz, Ahvaz, Iran. The fruit (seed and peel) were washed
with water and shade-dried at room temperature. The dried
materials were grounded into fine powder using a mixer
grinder. Then, the powder was weighed and kept away
from moisture in plastic vials in desiccator for further use.
Moisture of dried fruits was calculated based on the following
formula: % moisture (w/w)=[(WsplWdry)/Wspl]×100, where
Wspl was the weight of sample before drying and Wdry was
the weight of dried material. Total protein and sugar contents
were determined using Bradford and PhenolSulfuric acid
methods, respectively and reported as g/100 g dry material.
Measurement of flavonoids content
The flavonoids content (FC) was determined using
the method suggested by Huang et al. (11) with minor
modifications. For this purpose, 5 mL of 2% aluminium
trichloride (AlCl3) in methanol was mixed with the same
volume of A. esculentus powder (0.4 mg/ mL). Absorption
of the resulting solution was read at 367 nm using UV-
visible spectrophotometer (BioTek, CA, USA) against a
blank sample containing 5 mL extract mixture dissolved
in 5 mL methanol without AlCl3. FC was determined using
a standard curve plotted using quercetin (0.2-1 mg/ml) as
the standard and expressed as mg of quercetin equivalents
per 100 g dry extract. All experiments were performed in
Measurement of phenolic content
Phenolic content (PC) of the samples were measured
using Folin-Ciocalteu colorimetric method (12) with
slight modifications. Here, 100 µL of A. esculentus
powder was mixed with 0.5 mL Folin-Ciocalteu reagent
(10 times diluted with distilled water). Next, 7 mL of
distilled water was added to the mixture and it was
left at room temperature for 5 minutes. Then, 1.5 mL
sodium bicarbonate solution (60 mg/ ml) was added to
the mixture and left at room temperature in the dark for
2 hours. Absorbance was read at 725 nm against blank
using UV-visible spectrophotometer (BioTek, CA, USA).
A calibration curve was constructed using a standard
solution of gallic acid (0.2-1 mg/ml). Results were
expressed as mg gallic acid per 100 g dry extract. All
experiments were performed in triplicate.
Animals and diets
In this experimental study, healthy adult female Wistar rats
(200-220 g) were obtained from the experimental animal
holding of Joundishapour University of Medical Sciences,
Ahvaz, Iran. The animals were housed in standard cages and
maintained at controlled room temperature (23 ± 1°C), with a
relative humidity of 60 ± 5%, and 12 hour/12 hour light/darkcycles. They had ad libitum access to rat chow (Pars, Iran)
and water. All animals were used according to the guidelines
for the care and use of laboratory animals provided by thenational academy of sciences (National Institutes of Healthpublication No. 86-23). Approval for animal studies was
obtained from the animal Ethics Committee of Faculty ofVeterinary Medicine, Shahid Chamran University of Ahvaz,
Ahvaz, Iran. Initially, all rats were acclimatized to new
environmental conditions for 1 week before the beginning of
the experiment. Animals were randomly divided into 5 equal
groups (n=5 in each group) as follow: group I: rats were fed
with standard diet, group II: HFD-STZ-induced diabetic
rats, group III: HFD-STZ-induced diabetic rats received A.
esculentus (200 mg/kg) (13). The A. esculentus powder was
mixed with normal diet and administrated orally. Group IV:
HFD-STZ-induced diabetic rats received metformin (200 mg/
kg) (14), group V: rats received normal diet and A. esculentus
(200 mg/kg) (13). Groups II, III and IV were fed with HFD
for 4 weeks, whereas groups I and V consumed normal diet
during the same period.
The fat content of HFD was adjusted to 60% by addition
of beef tallow into normal diets (15). Normal diet contained
pellet chow of standard composition containing all the
recommended macro and micro elements (56% carbohydrate,
18.5% protein, 8% fat, 12% fiber and adequate levels of
minerals and vitamins). After 4 weeks of feeding the animals
with HFD, rats were treated with a single dose of STZ (35 mg/
kg, i.v) (Sigma, Germany) that was prepared in citrate buffer
0.1 M (pH=4) (16). Five days after STZ treatment, glucose
was measured by a hand-held glucometer (Medisign, China)
and diabetes induction was confirmed if blood sugar was >250
mg/dl. The day after diabetes confirmation was considered as
day 0 of treatment. After confirmation of diabetes induction,
animals of groups III and V were orally treated with A.
esculentus powder at the dose of 200 mg/kg body weight for
30 days and group IV was treated with oral metformin (200
mg/kg) for the same period. Animals in groups II, III and IV
received a high-calorie diet throughout the experiment, while
rats in groups I and V had access to standard diet during the
experiment. The body weight and fasting blood sugar (FBS)
were determined every week during the experimental period.
Serum biochemical assays
After overnight (12 hour) fasting, the rats were anesthetized
using ketamine 100 mg/kg and xylazine 10 mg/kg on day 30
after initiation of the treatment. Heart blood samples were
collected, and sera were separated and stored at -20°C for
future use. Serum glucose was measured using a commercial
kit (Pishtazteb, Iran) according to the manufacturer protocol.
Insulin concentration was measured using a species specific
ELISA kit (Koma Biotech Inc., South Korea) in a multiplate
ELISA reader (BioTek, CA, USA) based on the protocol
recommended by the manufacturer. Lipid profile including
triglycerides (TG), total cholesterol (TC) and high density
lipoprotein-cholesterol (HDL-c), was evaluated by enzymatic
assay kits (Pars Azmoon, Iran). The serum low-density
lipoprotein-cholesterol (LDL-c) and very-low-density
lipoprotein-cholesterol (VLDL-c) concentrations were
calculated using the Friedewald formula: LDL-c=TC-(HDLc+
VLDL-c) and VLDL-c=TG/5 (17).
Homeostasis Model Assessment of Basal Insulin
For homeostasis model assessment of basal insulin
resistance (HOMA-IR) the following equation was
used: HOMA-IR=Fasting insulin level (µU/ml)×fasting
blood glucose (mmol/l)/22.5. Lower HOMA-IR values
demonstrated greater insulin sensitivity, and higher
HOMA-IR values demonstrated lower insulin sensitivity
(insulin resistance) (18).
RNA isolation and cDNA synthesis
At the end of the experiment, animals were scarified and
pancreas tissues were immediately collected and frozen
at -70°C. Total RNA was isolated using RNX TM reagent
according to the manufacturer’s procedure (CinnaGen,
Iran). Concentration of extracted RNA was calculated at a
wavelength of 260 nm using nano drop spectrophotometry
(Eppendorf, Germany). To detect the purity of RNA, its
optical density (OD) ratio at 260/280 nm was determined and
samples with a ratio of >1.8 were used for cDNA synthesis.
Reverse transcription was carried out using the Rocket Script
RT PreMix kit using 1 µg of RNA and random hexamer,
based on manufacturer’s protocol (Bioneer Corporation,
South Korea). Reverse transcription was carried out at 42°C
for 90 minutes followed by incubation at 80°C for 3 minutes.
cDNAs were stored at -20°C until used in the real-time
polymerase chain reaction (PCR).
To evaluate the expression levels of PPAR-γ and
in the pancreas, real-time PCR analysis was
performed using qPCRTM Green Master Kit for SYBR
Green I® (Jena Biosciense, Germany) on a Lightcycler®
Detection System (Roche, USA). Relative expression
level of PPAR-γ and PPAR-α transcripts were compared
to rat GAPDH as the housekeeping gene. Reactions
were performed using 12.5 µl mixtures containing 6.25
µl qPCRTM Green Master Kit for SYBR Green I® (Jena
Biosciense, Germany), 0.25 µl of each primer (200 nM),
3 µl cDNA (100 ng), and 2.25 µl nuclease-free water. The
PCR protocol consisted of a 5-minute denaturation at 94°C
followed by 45 cycles at 94oC for 15 seconds, and at 60°C
for 30 seconds. Reactions were performed in triplicate.
Two separate reactions without cDNA or with RNA were
performed in parallel as controls. Relative quantification
was performed according to the comparative 2-ΔΔCt method
using Lightcycler 96® software. Validation of assay, in
order to check that primer for target genes and GAPDH
had similar amplification efficiencies, was performed as
described previously. All qPCR analyses were performed
according to The Minimum Information for Publication
of Quantitative Real-Time PCR Experiments (MIQE)
Rat pancreas samples were taken from all groups for
histological studies. The samples were taken from gastric
and splenic regions of pancreas. They were fixed in 10%
buffered formalin immediately upon removal. Next,
samples were dehydrated by passing through a graded
series of ethanol and embedded in paraffin blocks. Then,
5-6-µm sections were prepared using routine paraffin
embedding methods and the sections were stained by
H&E. To clarify the effect of diabetes induction on ß-cells
mass, the aldehyde fuchsin staining was performed on
paraffin embedded sections of pancreas samples (20).
Histological parameters including the number of ß cells,
size of islets, cytoplasmic vacuolization and tonality of
insulin granules within the cytoplasm of ß-cells were
evaluated in histological analysis.
For an exact estimation of the number of ß-cells, in each
slide, islets were divided into two categories of large islets
(A) and small islets (B) according to their approximate
diameter. The larger islets (>200 µm) (21) had open spaces
and higher numbers of ß-cells per islet and the small islets
(<200 µm) had little space and fewer ß-cells per islet.
In order to count ß-cells, 30 slides from the pancreas
of each group were randomly selected for histometrical
analysis. Then, 10 microscopic fields of equal size were
screened. The number of ß-cells in large and small
islets, separately, was assessed by counting all nuclei
of purple-violet stained cells inside one islet in the field
(22). Approximately 10 islets were examined on each
section. For each animal, 5 sections were counted and a
total number of 250 large and small islets of each group
were counted. All measurements were performedunder light microscope using Dino Lite lens (with Dino
capture software, FDP2, Taiwan) at ×40 magnification.
Data analyses were done using SPSS 16.0 software package
(SPSS Inc., Chicago, IL, USA). The data are reported
as mean ± SD. One way analysis of variance (ANOVA)
followed by Tukey’s post-test for multiple comparisons were
used to assess the variations in means among the groups. The
level of significance for all tests was set at P<0.05.
Proximate composition, and flavonoid and phenolic
contents of A. esculentus
The results of the present study showed that
concentrations of the total phenolic and flavonoid
compounds in the A. esculentus extract were 141 mg
gallic acid/g of dry extract and 147 mg quercetin/g of dry
extract, respectively. The moisture of dried A. esculentus
was 13.7%. Carbohydrates, proteins and ash contents of
dried plant were 1.6, 8.4 and 0.63 g/100 g, respectively.
Effects of A. esculentus on serum diabetes markers
Serum glucose level was significantly increased in
HFD-treated diabetic rats compared to the control group.
Treatment of HFD-treated diabetic rats with A. esculentus
for 30 days, significantly reduced blood glucose level
compared to the untreated diabetic rats (P<0.05). The
reduction of blood glucose level was not significantly
different between HFD-treated diabetic rats treated with
A. esculentus and those treated with metformin. Also,
A.esculentus had no significant effects on blood glucose
level in control group (Fig .1A,).
Fasting serum insulin level and HOMA-IR in HFD-
treated diabetic rats following treatment with A. esculentus
are shown in Table 1. HOMA-IR that had higher level
in HFD-treated diabetic rats compared to control rats
(P<0.05), was significantly decreased after administration
of A. esculentus and metformin (P<0.05). HOMA-IR was
decreased in control rats which received A. esculentus.
Conversely, serum insulin level was decreased in HFD-
treated diabetic rats compared to normal ones, while it was
increased following treatment with A. esculentus for 30
days. Metformin had no obvious effects on serum insulin
level in HFD-treated diabetic rats (P>0.05). A. esculentus
had no significant effect on insulin level of healthy rats
which received A. esculentus (Table 1,).
Effects of A. esculentus on diabetic rats’ body weight
At the end of 5-week HFD feeding, the mean weight
gain of the HFD-treated diabetic rats was not significantly
changed, while body weight was significantly decreased in
HFD-treated group after STZ administration as compared
to control group. HFD-treated diabetic rats which received
A.esculentus and metformin for 30 days showed a significant
increase in body weight as compared to HFD-treated diabetic
rats (P<0.05). The effect of metformin on improvement of
weight loss of HFD-treated diabetic rats was similar to thatof A. esculentus. No obvious body weight changes were
observed in control rats treated with A. esculentus (Fig .1B,).
Serum lipid profiles, insulin levels and HOMA-IR in in different groups
46.99 ± 2.32a
117.90 ± 9.05a
79.43 ± 9.54a
9.39 ± 0.46a
28.94 ± 1.41a
87.08 ± 9.2a
10.56 ± 3.32a
93.22 ± 25.12b
140.13 ± 19.5b
91.15 ± 18.17b
16.91 ± 5.02b
22.03 ± 3.04b
47.9 ± 3.2b
21.77 ± 1.45b
Diabetic received A.esculentus
52.83 ± 1.92a
98.89 ± 5.02a
63.79 ± 4.87a
12.16 ± 1.63ab
22.93 ± 1.01b
62.06 ± 3.96c
17.8 ± 4.47c
Diabetic received metformin
57.03 ± 3.96a
97.60 ± 2.32a
65.34 ± 8.48a
11.40 ± 0.79ab
22.90 ± 1.15b
51.05 ± 6.09b
15.86 ± 4.82c
Control received A. esculentus
53.85 ± 4.54a
100.37 ± 2.46a
72.20 ± 6.70a
10.77 ± 0.90a
25.06 ± 0.34b
65.83 ± 9.48c
8.62 ± 0.54d
Values are mean ± SD, n=5 animals per group. Different letters in each column denote significant differences (P<0.05).
TG; Triglyceride, TC; Total cholesterol, LDL-c; Low density lipoprotein-cholesterol, HDL-c; High density lipoprotein-cholesterol, VLDL-c; Very-low-density
lipoprotein-cholesterol, and HOMA-IR; Homeostasis model assessment of basal insulin resistance.
- The serum glucose levels and body weight changes in different
groups. Different numbers of * show significant difference (P<0.05). A and
B. Are serum glucose level and body weight changes, respectively.
Effect of A. esculentus on serum lipid profile of diabetic rats
Lipid profile including TG, cholesterol, HDL-C, LDL-C,
and VLDL-C, of treated and untreated diabetic rats are
presented in Table 1. Diabetic rats showed significantlyhigher levels of cholesterol, TG, and VLDL-C compared tothe non-diabetic group. Serum LDL-C levels in diabetic ratswere similar to those of control animal (P>0.05). In diabeticrats treated with A. esculentus and metformin for 30 days,
serum TG and cholesterol levels were significantly reduced,
while LDL-C and VLDL-C showed no significant changes(P>0.05). Serum HDL-C level was significantly decreased(P<0.05) in diabetic rats, while it remained unchanged inanimals treated with A. esculentus and metformin. Treatment
of healthy rats with A. esculentus had no significant effect on
serum lipids profile (P>0.05) (Table 1,).
Effect of A. esculentus on pancreatic expression of
PPAR-γ and PPAR-α
The expression levels of PPAR-γ and PPAR-α were
significantly increased in the pancreas of HFD/STZtreated
diabetic rats compared to the control rats (P<0.05).
Treatment of diabetic animals with A. esculentus or
metformin resulted in down-regulation of PPAR-γ and
PPAR-α in the pancreas (P<0.05, Fig .2A, B,).
PPAR-α mRNA expression level in rats treated with A.
esculentus and metformin was the same as that of control
rats (Fig .2B,). There were no significant changes in mRNA
expression of pancreatic PPAR-γ and PPAR-α genes in healthy
rats treated with A. esculentus (P>0.05, Fig .2A, B,).
- The PPAR-γ and PPAR-α
mRNA gene expression in the pancreas
in different groups. Data were presented as mean ± SD. Different letters
denote significant differences (P<0.05). A and B. Are PPAR-γ and PPAR-α
mRNA gene expression in the pancreas, respectively.
Effect of Okra powder on histological changes of
Histological examination of the pancreatic islet tissues
of experimental rats are presented in Figures 3 and 4. As
seen in Figure 3A, the pancreatic islets of normal animals
showed normal architecture. In contrast, islets of HFD
treated diabetic rats showed severe pancreatic disruption, and
vacuolization, as well as reduced islets’ size and relatively
decreased number of ß-cells (Fig .3C, D,). The severity of the
above-mentioned changes was reduced in rats treated with A.
esculentus and metformin compared to the untreated diabetic
rats (Fig .3E, F,).
The number of ß-cells was assessed by counting all nuclei of
the purple-violet stained cells inside the large and small islets
of pancreas. The numbers of ß-cells in both large and small
islets were decreased significantly in the pancreas of HFD-
treated diabetic rats compared to the control rats (P<0.05).
There was a significant increase in the number of ß-cells in
large pancreatic islets of A. esculentus and metformin-treated
groups compared to the HFD-treated diabetic rats (P<0.05).
The number of ß-cells in small pancreatic islets in HFD-
treated diabetic rats was increased significantly (P<0.05)
following treatment with metformin, while A. esculentus
had no significant effect on ß-cell mass of small pancreatic
islets in HFD-treated diabetic rats (P>0.05). Treatment
of healthy rats with A. esculentus caused no significant
changes in the number of ß-cells in both large and small
islets (P>0.05, Table 2,).
The results of the aldehyde fuchsin staining are shown in
Figure 4. The normal cells in the islets of Langerhans showed
distinct granules that were strongly stained in purple (Fig .4A,
B,). Diabetic rats (Fig .4C, D,) demonstrated significant
reductions in ß-cells and few surviving ß-cells were observed
in the islets of Langerhans (P<0.05). Analyses of the pancreas
of rats treated with A. esculentus and metformin (Fig .4E, F,)
showed remarkable increases in ß-cell, as reflected by purple
- Histological changes of pancreatic islets in different experimental groups (H&E staining, ×40). A. Normal control rats, B. Normal control rats
that received okra had normal pancreatic islets and ß-cells pancreatic composition, C, D. Pancreas of high-fat diet (HFD)-treated diabetic rats showed
vacuolization (arrows), as well as reduction of islets size and ß-cells numbers, E. Pancreas of okra, and F. Metformin-treated diabetic HFD rats showed
increased pancreatic islets size and ß -cells number (arrows), and decreased vacuolization.
- Aldehyde fuchsin staining of pancreatic islets in different experimental groups. A. Normal control rats (×40), B. (×100) showing normal ß-cells in
the islets of Langerhans as well as distinct insulin granules filling the entire islets of Langerhans that are strongly stained in deep purple violet. High-fat
diet (HFD)-treated diabetic rats, C. (×40), D. (×100) showing few surviving ß-cells in the islets of Langerhans and deficiency of their cytoplasmic tonality
compared to control rats. The decreases in the reaction and the number of ß-cells are considerable (arrows, ×40), E. diabetic HFD rats treated with
metformin, and F. diabetic HFD rats treated with okra showing remarkable increases in ß-cells mass, with heavily stained insulin granules.
The number of β-cells in large and small islets in the pancreas in different groups
Large islets (A)
Small islets (B)
264.91 ± 16.92a
44.5 ± 2.13a
119.43 ± 15.15b
22.08 ± 1.89b
Diabetic received A. esculentus
172.6111 ± 14.91c
25.27 ± 0.7bc
Diabetic received metformin
192.29 ± 16.55c
30.41 ± 3.01c
Control received A. esculentus
253.91 ± 13.078a
47.16 ± 2.11a
Values are presented as mean ± SD, n=5 animals per group. Different letters in each column denote significant differences (P<0.05).
T2D is associated with decreased pancreatic ß-cells
mass and function. Recently, focus on plant research has
increased all over the world and a large body of evidence
has shown the beneficial effects of medicinal plants on
pancreatic dysfunction in diabetic patients (23). Recent
findings have shown that A. esculentus can attenuate
metabolic disturbances and insulin resistance related
to diabetes in experimental animals (18), but its impact
on pancreas histology and PPARs-dependent regulation
in diabetes has not been clarified. In the present study,
the effect of administration of A. esculentus on insulin
resistance markers, serum lipid profile, pancreas structure
and pancreatic expression of PPARs genes was determined
in HFD/STZ-induced diabetic rats.
In the present study, HFD/STZ-induced diabetic rats
displayed elevated fasting blood glucose and HOMA-IR,
accompanied by decreased serum insulin levels confirming
the induction of diabetes in these animals. A. esculentus
administration decreased blood glucose levels and HOMAIR
and improved insulin resistance in HFD/STZ-treated
rats. In accordance with our findings, Ramachandran et al.
(24) have reported anti-diabetic activity of A. esculentus
in alloxan-induced diabetic rats. Moreover, Sabitha et al.
(25) have reported antidiabetic and antihyperlipidemic
potential of okra peel and seed powder in STZ-induced
diabetic rats. It has been found that administration of peel
and seed powder of okra to diabetic rats reduces blood
glucose level and increases body weight as compared
to diabetic control (13). Various mechanisms have been
proposed for antidiabetic action of A. esculentus. High
concentrations of fiber and polysaccharides in fruits of A.
esculentus can stabilize blood sugar by curbing the rate at
which sugar is absorbed from intestinal tract (26). Khatun
et al. (27) have also shown that water-soluble fraction of
A. esculentus reduced the absorption of glucose from the
Previous studies have shown that the extract of
A. esculentus contains quercetin and its analogues.
Recently, it has been found that quercetin decreases
blood glucose levels in HFD/STZ-induced diabetic rats
(18). Furthermore, quercetin and its analogues ameliorate
insulin resistance in diabetic mice (28). Therefore, the
phytoconstituents of okra might be responsible for
antidiabetic property of this plant in diabetic rats.
One major finding of our study was contrasting effects
of A. esculentus on serum insulin level in diabetic and
control animals. In accordance with previous works,
our results showed that diabetes was associated with
decreased pancreatic ß-cells mass and reduced serum
insulin level. Therefore, we propose the hypothesis that
A. esculentus ability to increase ß-cells mass was the key
factor in restoration of insulin production and secretion
in diabetic animals. ß-cells mass of control rats that
received A. esculentus, had no significant difference with
that of control, untreated rats, while serum insulin was
obviously reduced in healthy A. esculentus-treated rats.
Although the mechanism of insulin-lowering effect of
A. esculentus in healthy animals is unknown, it may be
indirectly related to increased ß-cells responsiveness to
glucose as manifested by reduced HOMA-IR and insulin
secretion. Thus, as insulin sensitivity increases in healthy
A. esculentus treated rats, first-phase of insulin release
may decrease proportionately to maintain normal glucose
levels. However, further studies are needed to confirm
In the present study, diabetes was accompanied by
an increase in TC, LDL-C, and TG and a reduction in
HDL-C in HFD/STZ-induced diabetic rats. Treatment
of HFD/STZ-induced diabetic rats with okra powder
could profoundly improve lipid disturbances. Treatment
of diabetic rats with A. esculentus notably reduced
serum TG and TC levels while HDL-C levels remained
unchanged. These results are in agreement with previous
studies (29) that have shown that treatment with A.
esculentus can reduce TC, total lipids and TG levels in
rats fed with a HFD. Different mechanisms may underlie
the improvement of lipid disturbances in diabetic rats
after treatment with A. esculentus. Previous studies have
shown that addition of A. esculentus to diet decreases the
gene expression of SREBP1c and FAS (two key modulator
of FA and cholesterol biosynthesis), which may finally
reduce serum levels of TG and TC. Abundant dietary
fibers present in A. esculentus are also capable of binding
to bile acids consequently lowering TC through interfering
with bile acids reabsorption (29). Roy et al. (30) reported
that A. esculentus polysaccharides could decrease blood
glucose levels in normal mice.
PPAR-α and PPAR-γ are nuclear hormone receptors that
maintain homeostasis of glucose in the pancreas. PPARs
exhibit beneficial effects on metabolic abnormalities
associated with T2D and also control the expression
of various genes that are important for lipid and
glucose metabolism (31). Studies have indicated strong
correlations between PPARs activation and anti-diabetic
effects of many herbal plants (23). More than 200 natural
compounds, especially flavonoids, have been identified
as agonist or antagonist of PPAR-γ receptors and may
play roles in the prevention and treatment of metabolic
disorders. In this regard, recent data has shown that A.
esculentus can activate PPAR-γ in the liver of HFD-
induced obese C57BL/6 mice. Because PPARs have
insulin-sensitizing effects in peripheral tissues as well as
the ability to sense blood glucose in pancreatic ß-cells,
we attempted to evaluate whether okra may affect PPARs
gene expression in the pancreas of diabetic rats.
Quantitative real time PCR data showed that mRNA
levels of PPAR-γ and PPAR-α were increased in HFD/
STZ-induced diabetic rats compared to control group rats.
In accordance with our results, Zhou et al. (32) reported
that, PPAR-γ expression is increased >5-fold in islets from
Zucker diabetic fatty (ZDF) rats. In T2D, blood glucose
and free FA levels are elevated, resulting in intracellular
accumulation of TG within the pancreatic islets (33) and
ß-cells secretory failure. Intracellular TG accumulation
and overabundance of islet lipid, induce apoptosis in ß-cells
by increasing free radicals formation. Over expression or
activation of PPAR-γ and PPAR-α, up-regulates of key
enzymes of mitochondrial and peroxisomal ß-oxidation
and enhances FAs oxidation. It is reported that forced
activation of PPAR-γ in the islets leads to stimulation of
multiple metabolic pathways that help the disposal of FAs
(34). Kakuma et al. (35) reported that long-term activation
of PPAR-γ can reduce the lipid content of ZDF rat islets.
Also, PPAR-α regulates the expression of genes involved
in FAs and lipid metabolism (36). Studies performed in
rodent models of insulin resistance indicated that PPAR-α
activation by natural (FAs) or synthetic (fibrates) ligands,
enhances insulin sensitivity by decreasing the lipid content
of adipose and nonadipose tissues (7) or decreasing the
endogenous glucose production (33). Based on these
findings, we concluded that over-expression of PPARs
in the pancreas of diabetic rats may be a compensatory
mechanism for improvement of glucose sensitivity and
ß-cells function. To confirm this hypothesis, previous
studies have demonstrated that PPARγ over-expression
can protect ß-cells function, morphology, and mass in
rodent models of diabetes (34).
Interestingly, PPAR-γ antagonists may ameliorate
metabolic disorders such as obesity, insulin resistance and
dyslipidemia, by inhibition of adipocyte differentiation.
PPAR-γ antagonists, tanshinone IIAand ß-cryptoxanthine,
have been reported to reduce body weight, blood glucose
and serum TG in HFD-induced obese mice (37). Rieusset
reported that dimethyl a-(dimethoxyphosphinyl)pchlorobenzyl
phosphate (SR-202) as a selective synthetic
inhibitor of PPAR-γ inhibits adipocyte differentiation and
improves insulin sensitivity in diabetic ob/ob mice (38).
Thus, PPARγ antagonists may be useful for the treatment
of obesity-related insulin resistance. Our data showed that
PPAR-γ and PPAR-α mRNA levels declined in diabetic
animals treated with A. esculentus powder, confirming the
PPAR antagonistic effect of A. esculentus. These results
were in accordance with the findings of Fan et al. (18)
that showed that okra consumption inhibits PPAR-γ and
PPAR-α transcription in the liver of HFD-induced obese
Together, we suggest that okra may improve metabolic
disorders related to diabetes through suppression of PPARs
signaling. In addition to the above-described mechanisms, it
seems that reduction of PPAR-γ and PPAR-α expression might
be a consequence of improvement of hyperinsulinemia. In
other words, increased PPARs expression during insulin
resistance state, can improve insulin resistance, attenuate
hyperlipidemia and increase overall glucose utilization,
while improvement of insulin resistance after A. esculentus
treatment results in down-regulation of PPARs in the pancreas.
Based on these observations, it has been suggested that an
increase in PPARs expression may induce a compensatory
mechanism against progression of insulin resistance in obese
patients (7, 18, 34).
Additionally, our histological data showed that the size
of islets and population of insulin-producing ß-cells were
reduced in the pancreas of HFD/STZ-induced diabetic
rats. Aldehyde fuchsin staining also demonstrated that
A. esculentus powder could restore pancreatic ß-cells
mass and reverse the ß-cells damage caused by HFD/STZ
treatment. These results were in accordance with previous
studies that demonstrated that hyperglycemia leads to a
progressive decline in ß-cells function, the insufficiency
of insulin secretion by the pancreatic ß -cells (39) and
increased apoptosis in pancreatic islets (40).
Based on our data, okra could improve metabolic
complications in an animal model of diabetes. Our results
revealed that A. esculentus had beneficial effect on the
pancreas of diabetic rats by restoration of ß-cell mass and
modulation of PPAR-dependent pathways. The results of
the present study provide new scientific evidence about
therapeutic benefits of A. esculentus in diabetes.
Thanks for the financial support of Shahid Chamran
University of Ahvaz. The authors declare that there is no
conflicts of interest.
N.E.M.; Was supervisors and collaborated in
histological studies and wrote the manuscript. M.R.T.;
Was advisor and collaborated in molecular study. A.Sh.;
Was supervisors and collaborated in biochemistry study.
Z.S.; Contributed for her thesis. All authors read and
approved the final study.
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