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Cytotechnology
Incorporating Methods in Cell Science
International Journal of Cell Culture and
Biotechnology
ISSN 0920-9069
Volume 72
Number 3
Cytotechnology (2020) 72:357-366
DOI 10.1007/s10616-020-00382-y
Effect of β-sitosterol on glucose homeostasis
by sensitization of insulin resistance via
enhanced protein expression of PPRγ and
glucose transporter 4 in high fat diet and
streptozotocin-induced diabetic rats
Sundaram Ramalingam, Meenatchi
Packirisamy, Muthu Karuppiah, Ganesh
Vasu, et al.
1 23
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ORIGINAL ARTICLE
Effect of b-sitosterol on glucose homeostasis by sensitization
of insulin resistance via enhanced protein expression
of PPRcand glucose transporter 4 in high fat diet
and streptozotocin-induced diabetic rats
Sundaram Ramalingam .Meenatchi Packirisamy .Muthu Karuppiah .
Ganesh Vasu .Rahul Gopalakrishnan .Kirubananthan Gothandam .
Muthusamy Thiruppathi
Received: 30 May 2019 / Accepted: 22 February 2020 / Published online: 2 March 2020
ÓSpringer Nature B.V. 2020
Abstract
Ethnopharmacological relevance b-Sitosterol is a
plant derived compound similar to cholesterol struc-
ture and used in the treatment of hypercholes-
terolemia, prostate cancer, breast cancer and
coronary artery disease. But no studies have been
reported the effect of b-sitosterol on glucose home-
ostasis by sensitization of insulin resistance via
enhanced protein expression of peroxisome prolifer-
ator-activated receptor c(PPARc) and glucose trans-
porter 4 (GLUT4) in insulin dependent tissues of high
fat diet and streptozotocin-induced diabetic rats.
Materials and methods Type 2 diabetes was induced
in male albino Wistar rats by feeding them with high
fat diet comprising of 84.3% standard laboratory
chow, 5% lard, 10% yolk powder, 0.2% cholesterol
and 0.5% bile salt for 2 weeks. After 2 weeks, the
animals were kept in an overnight fast and injected
with low dose of streptozotocin (35 mg/kg, dissolved
in 0.1 M sodium citrate buffer, pH 4.5). Analysis of
blood glucose, insulin, hemoglobin and glycated
hemoglobin were done by commercially available
diagnostic kits. The PPARcand GLUT4 were ana-
lyzed by western blotting using respective primary and
secondary antibodies.
Results Upon administration of b-sitosterol at a dose
of 15 mg/kg body weight per day to high fat diet and
streptozotocin induced diabetic rats for 30 days sig-
nificantly decreased the levels of plasma glucose,
homeostatic model assessment of insulin resistance
and glycosylated hemoglobin and increased the levels
S. Ramalingam G. Vasu R. Gopalakrishnan
Central Research Laboratory, Meenakshi Academy of
Higher Education and Research, West K.K. Nagar,
Chennai, Tamil Nadu, India
S. Ramalingam (&)
Department of Biochemistry, Saveetha Dental College &
Hospital, Saveetha Institute of Medical & Technical
Sciences, Vellapanchavadi, Chennai, Tamil Nadu 600077,
India
e-mail: sundaram1477@gmail.com
M. Packirisamy
PG & Research Department of Biochemistry, Mohamed
Sathak College of Arts and Science, Chennai,
Tamil Nadu, India
M. Karuppiah
Department of Chemistry, Manomanium Sundaranar
University, Tirunelveli, Tamil Nadu, India
K. Gothandam
Department of Biotechnology, University of Madras,
Guindy Campus, Chennai, Tamil Nadu, India
M. Thiruppathi
Department of Kinesiology and Nutrition, University of
Illinois at Chicago, Chicago, IL, USA
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Cytotechnology (2020) 72:357–366
https://doi.org/10.1007/s10616-020-00382-y(0123456789().,-volV)(0123456789().,-volV)
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of insulin, hemoglobin and protein expression of
PPARcand GLUT4 in insulin dependent tissues.
Furthermore, b-sitosterol administration prevented the
body weight loss and excessive intake of food and
water.
Conclusion These finding suggest that b-sitosterol
can replace the commercial drugs which could lead to
reduction in toxicity and side effect caused by the later
as well as reduce the secondary complications.
Keywords Coccinia grandi b-Sitosterol
Diabetes Metabolic enzymes Insulin resistant
GLUT4
Introduction
Type 2 diabetes mellitus, the frequent form of
diabetes, represents more than 90% of all diabetic
patients (Tripathi and Srivastava 2006). Insulin resis-
tance and impaired insulin secretion are two main
characteristics of type 2 diabetes (Sharma et al. 2011).
Insulin resistance in type 2 diabetes mellitus could be
provoked by glucotoxicity, oxidative stress, lipotox-
icity and inflammation (Weir and Bonner Weir 2004).
Recently, the International Diabetes Federation (IDF)
reported that the number of diabetic patients was 415
million in 2015 and is expected to rise to 642 million
by 2040 (International Diabetes Federation 2015). If
ineffectively controlled in diabetic patients, chronic
hyperglycemia can cause serious complications in
different organs (International Diabetes Federation
2015). Diet, exercise and several pharmacological
agents are treatment approaches for type 2 diabetes
mellitus. However, the use of currently used pharma-
cological agents is associated with adverse side effects
(Geirch 2003). Therefore, research is focused on
medicinal plants which are used in the practices and
development of newer drug leads from phytocon-
stituents with more potential and effective agents with
lesser side effects than the existing hypoglycemic
agents (Chandramohan et al. 2008). Many medicinal
plants are currently used in India for the treatment of
diabetes and scientifically its efficacy has been proved
earlier (Are et al. 2011).
Coccinia grandis belongs to the family Cucur-
bitaceae and grows abundantly in India. It is a
climbing perennial herb and its fruits are widely used
for culinary purposes as a vegetable and traditional
treatment of diabetes (Venkateswaran and Pari 2002).
Many scientific investigations have been proved that
the efficacy of leaf and root extracts of Coccinia
grandis reduced the diabetic complications and pro-
gression of the disease (Kumar et al. 1993; Venka-
teswaran and Pari 2003; Akhtar et al. 2007). The
present was aimed to isolate and characterize the b-
sitosterol from the unripe fruits of Coccinia grandis
and investigate its potential on glucose homeostasis by
sensitization of insulin resistance via enhanced protein
expression of PPARcand glucose transporter 4 in high
fat diet and streptozotocin—induced diabetic rats as
there are no scientific reports available on these
aspects so far.
Materials and methods
Collection of plant material
The unripe fruits of Coccinia grandis were procured
from local place, identified and authentified by Dr.
KN. Sunil kumar, Pharmacognosy, Siddha Research
Institute (Central council for Research in Siddha,
Chennai, Ministry of AYUSH, Government of India)
(Voucher no: C17112901G).
Extraction and isolation
Two thousand gram of unripe fruits of Coccinia
grandis was purchased from local vegetables market,
Chennai. Coccinia grandis was sliced into many
pieces and allowed to shade dry for 15 days. After
shade drying, the G. grandis was pulverized and
soaked in 2.5 L of methanol and kept in refrigerator
for 3 days. Then the filtrate was filtered through
Whatmann filter paper No 1 and this was repeated
three to four times until the filtrate gave no coloration
and concentrated using vacuum rotary evaporator at
40 °C. The methanolic concentrate was checked on
thin layer chromatography with hexane and ethyl
acetate in the ratio of 8:2 which showed four spots
(compounds). The methanolic concentrate was chro-
matographed on silica gel column and eluted with
hexane and ethyl acetate (80:20 ratio). Fractions were
collected at an interval of 10 mL each and monitored
by thin layer chromatography (precoated silica gel
merk—60 F
254
0.25 mm thick plate). Fractions
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358 Cytotechnology (2020) 72:357–366
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formed as a pale yellow and showed single spot on
TLC and pooled together in a clean vial and evapo-
rated to dryness. This process was repeated until
getting satisfactory yield of the compound (500 mg/
2 kg of crude extract). The physical nature of the
compound was creamish pale yellow. The structure of
the compound was confirmed by b-sitosterol on the
basis of their
1
H&
13
C NMR spectral data. The
molecular weight of b-sitosterol was m/z: 414.39 and
molecular formula was C
29
H
50
O. The rest of the three
spots (three compounds) are under the isolation
process. The physical and spectroscopic data of
isolated b-sitosterol are comparable with those
reported in literature (Hwang et al. 2008; Gupta
et al. 2011). The isolated compound displayed [90%
purity as estimated by the examination of NMR and
Mass spectra.
Experimental animals
Adult male albino Wistar rats weighing about
200–220 g were obtained from Sri Muthukumaran
Medical College Hospital & Research Institute, Man-
gadu, Chennai, Tamil Nadu, India. Rats were housed
in clean, sterile and polypropylene cages under
standard vivarium conditions 12 h light/12 h dark
cycle and constant temperature (25 ±2°C) with free
access to standard commercial rat chow (Pranav Agro
Industries Ltd., Pune, Maharashtra, India) and water.
The experimental protocol was approved by the
Ministry of Social Justices and Empowerment,
Government of India and Institutional Animal Ethics
Committee Guidelines (IAEC No: No. 32/02/2014).
Induction of type 2 diabetes in rats
The animals were divided into six groups of six
animals each. The rats were fed with high fat diet
consisting of 84.3% standard laboratory chow, 5%
lard, 10% yolk powder, 0.2% cholesterol and 0.5%
bile salt for 2 weeks (Xie et al. 2005). After 2 weeks,
the animals were kept in an overnight fast and injected
with low dose of streptozotocin (35 mg/kg, dissolved
in 0.1 M sodium citrate buffer, pH 4.5), (Wu et al.
2012). Fasting blood glucose was measured 3 days
after the injection. The rats with fasting blood glucose
levels above 250 mg/dL were considered diabetic.
The diabetic rats were fed on the high-fat diet for
another 4 weeks.
Experimental design
A total of 36 rats (30 diabetic rats and 6 normal rats)
were used and experimental animals were divided into
six groups, each group consists of a minimum of six
rats (n = 6) detailed as given below. b-sitosterol was
dissolved in 0.5 mL of olive oil and administered
orally at different doses using an intragastric tube for a
period of 30 days. Metformin was dissolved in 1 mL
of distilled water used as standard drug.
Group I: Normal control rats.
Group II: Diabetic control rats.
Group III: Diabetic ?b-sitosterol (5 mg/kg b.wt).
Group IV: Diabetic ?b-sitosterol (10 mg/kg b.wt).
Group V: Diabetic ?b-sitosterol (15 mg/kg b.wt).
Group VI: Diabetic ?Metformin (500 mg/kg
b.wt).
Body weight of all the animals was recorded prior
to the treatment and sacrifice. Food and water intake of
all groups of animals were monitored on a daily basis
for 30 days at a fixed time. Fixed amount of rat chow
and fluid was given to each rat and replenished the next
day. At the end of the treatment period (30 days), the
rats were fasted overnight, anaesthetized and killed by
cervical decapitation. Blood samples were collected in
tubes containing potassium oxalate and sodium fluo-
ride (3:1) mixture for the estimation of plasma glucose
and insulin. Hb and HbA1c levels were estimated in
whole blood samples. The liver tissues was dissected
out, washed in ice-cold saline and weighed. Tissue
was minced and homogenized (10%, w/v) with 0.1 M
Tris -HCl buffer (pH 7.4) and centrifuged (30009gfor
10 min). The resulting supernatant was used for
enzyme assays.
Biochemical analysis
Plasma glucose was estimated by the method of
Trinder (1969) using a reagent kit. Haemoglobin (Hb)
and glycosyated haemoglobin (HbA1c) were esti-
mated by the method of Drabkin and Austin (1932)
and Sudhakar and Pattabiraman (1981), respectively.
The plasma insulin was measured by the method of
Burgi et al. (1988).
HOMA - IR ¼fasting insulin
fasting blood sugar=405:
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Western blot analysis of PPAR cand GLUT4
Briefly, 100 mg of adipose tissue and skeletal muscle
were taken individually and homogenized in buffer
containing 20 mM Tris–HCl (pH 7.8), 300 mM NaCl,
2 mM ethylenediaminetetraacetic acid (EDTA),
2 mM dithiothreitol (DTT), 2% NP-40, 0.2% SDS,
0.2% sodium deoxycholate, 0.5 mM phenylmethyl-
sulfonyl fluoride (PMSF), 50 mM sodium fluoride
(NaF), 25 mM sodium pyrophosphate, 40 mM b-
glycerophosphate, 2 mM sodium orthovanadate (Na
3-
VO
4
) and protease inhibitor cocktail (Sigma) using a
polytron equipped homogenizer at a precise low
setting on ice. The homogenate was centrifuged at
10,0009gfor 10 min 4 °C and then the supernatant
was collected. The resultant supernatant was sampled
as the protein for PPARcand GLUT4. Protein
concentration was estimated by Lowry et al. (1951)
using bovine serum albumin (BSA) as a standard.
Briefly, each sample (50 lg) was subjected to heat
denaturation at 100 °C for 5 min with Laemmli buffer.
Proteins were resolved by SDS-PAGE on 10% poly-
acrylamide gels and then transferred to PVDF mem-
brane (Amersham Biosciences, Little Chalfont
Buckinghamshire, UK). The membrane was blocked
with 5% blocking buffer (Amersham Biosciences,
Little Chalfont Buckinghamshire, UK) in TBS-T
(Tris-buffered saline and Tween 20) for 1 h at room
temperature followed by incubation with mouse
monoclonal primary antibodies to PPARc(SC-7273,
Santa Cruz Biotechnology) and GLUT 4 (SC-53566,
Santa Cruz Biotechnology, California, USA) at a
dilution of 1:1000. The membrane was subjected for
repeated wash for 3 times with TBS-T and then
incubated for 1 h in horse radish peroxidase (HRP)-
conjugated rabbit mouse secondary antibody by
1:7500 dilutions in TBS-T. The membrane was again
subjected for repeated wash for 3 times with TBS and
TBS-T. Protein bands were visualized in Chemidoc
using Enhanced Chemiluminescence Reagents (ECL;
Amersham Biosciences, Little Chalfont Bucking-
hamshire, UK). The detected bands were quantified
by Quantity one software (BioRad). Later, the mem-
branes were incubated in stripping buffer (50 mL,
containing 62.5 mM Tris HCl (pH 6.7), 1 g SDS and
0.34 mL b-mercaptoethanol) at 55 °C for 40 min.
Following this, the membrane was re-probed using b-
actin antibody (1:5000). In the present study, rat b-
actin was used as loading control.
Histopathological studies
The pancreas tissues of the experimental rats was fixed
in 10% formaldehyde, dehydrated in a graded series of
ethanol and embedded in paraffin. Pancreatic sec-
tions (5 lm thick) were obtained using a microtome,
then dewaxed and rehydrated. Sections were then
stained with hematoxylin–eosin and viewed under the
light microscope (1009).
Statistical analysis
All the grouped data were statistically evaluated with
SPSS 17.0 software. Hypothesis testing methods
included one way analysis of variance (ANOVA)
followed by least significant difference (LSD) test;
Pvalue less than 0.05 were considered to indicate
statistical significance. All the results were expressed
as the mean ±SD for six animals in each group.
Results
Dose dependent effect of b-sitosterol on blood
glucose, plasma insulin levels and HOMA-IR
index
There was a significant increase in the level of plasma
glucose and decrease in the level of plasma insulin in
diabetic rats compared to normal control rats. b-
Sitosterol was administered orally to diabetic rats at
different doses (5, 10 and 15 mg/kg b.wt.) for 30 days
significantly decreased the level of plasma glucose and
homeostatic model assessment of insulin resistance
whereas the levels of plasma insulin were significantly
increased in a dose dependent manner. The effect was
more pronounced at a dose of 15 mg/kg b.wt. than 5
and 10 mg/kg b.wt. and it was comparable to that of
metformin. Therefore, 15 mg of b-sitosterol was fixed
as an effective dose and used for further analysis
(Table 1).
Effect of b-sitosterol on body weight gain
Table 2depicts the values of the initial and final body
weights of the normal and diabetic rats. Body weight
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significantly decreased in diabetic rats compared to
normal control rats. Oral administration of b-sitosterol
and metformin to diabetic rats prevented the body
weight loss compared to untreated diabetic rats.
Effect of b-sitosterol on the levels of hemoglobin
and glycated hemoglobin
Table 3represents the levels of Hb and HbA1c in
control and experimental rats. The levels of Hb were
significantly decreased whereas the levels of HbA1c
were significantly increased in high fat diet and
streptozotocin induced diabetic rats and when treated
with b-sitosterol and metformin, these values were
brought toward near normal level.
Effect of b-sitosterol on the protein expression
of PPARcin adipose tissue
Figure 1depicts the protein expression levels of
PPARcin adipose tissue of control and experimental
animals. The expression of PPARcprotein was found
to be significantly decreased in the adipose tissue of
diabetic rats when compared to control rats. However,
PPARcprotein expression was significantly increased
in the adipose tissue of diabetic rats upon treatment
with b-sitosterol and metformin.
Effect of b-sitosterol on the protein expression
of GLUT4 in skeletal muscle
The protein expression levels of GLUT4 in skeletal
muscle were significantly decreased in diabetic rats
when compared to control rats. However, those
proteins levels were elevated in diabetic rats after
treatment with b-sitosterol and metformin (Fig. 2).
Effect of b-sitosterol on histopathological changes
in pancreas
Based on H and E stained pancreatic tissue sections
(Fig. 3), normal control rat showing b-cells with
displayed granulated cytoplasm and uniform nuclei
and there were no notable changes observed in the
pancreas of normal rats (A). In contrast, microvesic-
ular, macro vesicular changes and decreased number
of the bislets were observed in the pancreas of high fat
diet and streptozotocin induced diabetic rats (B).
Table 1 Dose dependent of b-sitosterol on the levels of blood glucose, Insulin and Homeostatic model assessment of insulin
resistant of control and experimental animals
Group Plasma glucose (mg/dL) Plasma ins ulin (IU/mL) HOMA-IR
Before
treatment
After treatment Before
treatment
After
treatment
Before
treatment
After
treatment
Control 84.10 ±8.02 92.02 ±5.09 20.01 ±2.00 18.51 ±1.81 4.15 ±0.55 4.22 ±0.57
Diabetes induced 260.16 ±12.91 300.55 ±11.7a 9.23 ±0.86 7.53 ±0.73
a
5.93 ±0.68 5.59 ±0.49
a
Diabetes ?b-sitosterol (5 mg/
kg b.wt)
254.99 ±24.35 191.29 ±9.16b 10.66 ±0.79 11.59 ±0.87
b
6.71 ±0.71 5.48 ±0.54
b
Diabetes ?b-sitosterol
(10 mg/kg b.wt)
246.09 ±18.06 172.85 ±8.53c 11.36 ±0.91 12.58 ±1.00
c
6.90 ±0.73 5.36 ±0.43
c
Diabetes ?b-sitosterol
(15 mg/kg b.wt)
235.71 ±15.59 127.03 ±9.48d 11.82 ±0.72 14.98 ±0.83
d
6.90 ±0.79 4.70 ±0.42
d
Diabetes ?Metformin
(500 mg/kg b.wt)
240.18 ±13.65 122.45 ±9.05 11.71 ±0.41 16.23 ±0.74 6.95 ±0.44 4.91 ±0.27
Values are given as mean ±S.D for six animals in each group
Values are considered significantly different at P\0.05 with post-hoc LSD test *P\0.05
a
Control vs diabetic rats
b
Diabetic rats vs. diabetic rats treated with bsitosterol (5 mg/kg b.wt)
c
Diabetic rats vs diabetic rats treated with bsitosterol (10 mg/kg b.wt)
d
Diabetic rats vs. diabetic rats treated with bsitosterol (15 mg/kg b.wt)
e
Diabetic rats treated with bsitosterol (15 mg/kg b.wt) vs Diabetic rats treated with Metformin (500 mg/kg b.wt)
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Treatment with b-sitosterol and metformin showed
increase in the cellularity of b-cell islets of pancreas
when compared to diabetic rats (C and D).
Discussion
High fat diet and low dose streptozotocin are known to
induce rapid destruction of pancreatic b-cells leading
to impaired glucose stimulated insulin release and
insulin resistance, both of which are marked features
of type 2 diabetes (Farswan et al. 2009). The elevated
blood glucose is a result of reduced glucose uptake in
muscle and adipose tissue and increased gluconeoge-
nesis, hepatic glucose production and glycogen
breakdown (Sundaram et al. 2013). In the present
study, a significant decrease in the levels of blood
glucose, HOMA-IR and significant increase in the
levels of insulin were observed in diabetic rats treated
with b-sitosterol and metformin. b-sitosterol might
bring about glucose lowering action by stimulate the
surviving b-cells of islets of Langerhans to release
more insulin through its antioxidant potential (Gupta
et al. 2011).
The decreased body weight observed in diabetic
rats compared to control rats indicates the excessive
breakdown or loss of structural tissue proteins due to
altered carbohydrate metabolic enzymes (Chen and
Ianuzzo 1982). Previous study reported that protein
synthesis is decreased in all tissues due to decreased
Table 2 Effect of bsitosterol on body weight of control and experimental rats
Group Body weight(g)
Initial Final
Control 186.16 ±4.87 208.7 ±7.67
Diabetes induced 188.83 ±4.09
a
147.70 ±10.64
a
Diabetes ?b-sitosterol (5 mg/kg b.wt) 185.70 ±6.24
b
187.42 ±6.18
b
Diabetes ?b-sitosterol (10 mg/kg b.wt) 181.59 ±5.59
c
191.60 ±5.16
c
Diabetes ?b-sitosterol (15 mg/kg b.wt) 185.02 ±4.22
d
197.96 ±5.59
d
Diabetes ?Metformin (500 mg/kg b.wt) 182.44 ±6.010 200.59 ±8.89
Values are given as mean ±S.D for six animals in each group
Values are considered significantly different at P \0.05 with post-hoc LSD test *P \0.05
a
Control vs diabetic rats
b
Diabetic rats vs. diabetic rats treated with bsitosterol (5 mg/kg b.wt)
c
Diabetic rats vs diabetic rats treated with bsitosterol (10 mg/kg b.wt)
d
Diabetic rats.vs diabetic rats treated with bsitosterol (15 mg/kg b.wt)
e
Diabetic rats treated with bsitosterol (15 mg/kg b.wt) vs diabetic rats treated with Metformin 500 mg/kg b.wt)
Table 3 Effect of b-sitosterol on the levels of hemoglobin and glycated hemoglobin in control and experimental rats
Parameters Control Diabetes
Induced
Diabetes ?b-sitosterol (15 mg/kg
b.wt)
Diabetes ?Metformin (500 mg/kg
b.wt)
Haemoglobin (g/
dL)
13.12 ±1.19 8.85 ±0.83
a
11.57 ±0.65
b
12.27 ±0.72
HbA1c (%) 5.03 ±0.48 10.90 ±0.92
a
7.10 ±0.67
b
6.72 ±0.54
Values are given as mean ±S.D for six animals in each group.Values are considered significantly different at P \0.05 with post-hoc
LSD test *P \0.05
a
Control vs diabetic rats
b
Diabetic rats vs Diabetic rats treated with bSitoterol
c
Diabetic rats treated with bsitosterol (15 mg/kg) vs Diabetic rats treated with Metformin (500 mg/kg)
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a
b
0
20
40
60
80
100
120
140
160
180
Normal
Control
Diabetes
Induced
Diabetes +
β˗Sitosterol
(15 mg/kg b.wt.)
Diabetes +
Metformin
(500 mg/kg b.wt.)
OD units of PPRγ relative to β˗actin
PPRγ
β˗actin
Fig. 1 Effect of bsitosterol on PPRcexpression in adipose
tissue of control and experimental rats. Values are given as
mean ±SD for six animals in each group. Values are
considered significantly different at P\0.05 with post-hoc
LSD test *P\0.05.aControl vs Diabetic rats. bDiabetic rats
vs. Diabetic rats treated with b-sitosterol (15 mg/kg b.wt.). cb-
sitosterol treated diabetic rats (15 mg/kg b.wt.) vs. Metformin
(500 mg/kg b.wt.)
a
b
0
20
40
60
80
100
120
140
160
Normal
Control
Diabetes
Induced
Diabetes +
β˗Sitosterol
(15 mg/kg b.wt.)
Diabetes +
Metformin
(500 mg/kg b.wt.)
OD units of GLUT4 relative to β˗actin
GLUT4
β˗actin
Fig. 2 Effect of bsitosterol on GLUT 4 expression in skeletal
muscle of control and experimental rats. Values are given as
mean ±SD for six animals in each group. Values are
considered significantly different at P\0.05 with post-hoc
LSD test *P\0.05.aControl vs Diabetic rats. bDiabetic rats
vs. Diabetic rats treated with b-sitosterol (15 mg/kg b.wt.). cb-
Sitosterol treated diabetic rats (15 mg/kg b.wt.) vs. Metformin
(500 mg/kg b.wt.)
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Cytotechnology (2020) 72:357–366 363
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production of ATP and absolute or relative deficiency
of insulin (Bender 2009). Oral administration of b-
sitosterol and metformin improved body weight of
diabetic rats which might result from an improvement
in glycemic control.
During diabetes the excess of glucose present in
blood reacts with hemoglobin to form increased
glycosylated hemoglobin (Sheela and Augusti 1992;
Sundaram et al. 2013). Estimation of HbA1c has been
found to be useful in monitoring the effectiveness of
therapy in diabetes and also a vital biochemical
marker for the diagnosis and management of ambient
glycemia during a period of 3 month (Goldstein et al.
1995). Prolonged administration of b-sitosterol and
metformin to diabetic rats significantly reversed the
Hb andHbA1c to near normal levels.
PPAR-cis a member of the nuclear hormone
receptor super family of ligand-dependent transcrip-
tion factors and regulates the expression of genes
involved in insulin signaling, carbohydrate and lipid
metabolism (Herzig et al. 2003). PPARcis highly
expressed in white adipose tissue and its elevation
enhances insulin sensitivity in rodent type 2 diabetes
mellitus models (Hevener et al. 2003; Escher et al.
2001, Sharma et al. 2011). Tissue specific PPARc
down regulation in rats causes insulin resistance
a
b
0
10
20
30
40
50
60
70
80
90
Normal
Control
Diabetes
Induced
Diabetes +
β˗Sitosterol
(15 mg/kg b.wt.)
Diabetes +
Metformin
(500 mg/kg b.wt.)
β cells (%)
Fig. 3 Histological observations (H&E staining 9100) of
pancreatic tissues from control and experimental rats. Control
(a), diabetes induced (b), Diabetic ?b-sitosterol (c), Dia-
betic ?Metformin (d), Quantification of bcells (e). Values are
given as mean ±SD for six animals in each group. Values are
considered significantly different at P\0.05 with post-hoc
LSD test *P\0.05.aControl vs Diabetic rats. bDiabetic rats
vs. Diabetic rats treated with b-sitosterol (15 mg/kg b.wt.). cb-
sitosterol treated diabetic rats (15 mg/kg b.wt.) vs. Metformin
(500 mg/kg b.wt.)
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(Barroso et al. 1999; Rangwala et al. 2003). In our
study, the administration of b-sitosterol and met-
formin decreased insulin resistance by increasing
PPAR-cexpression in diabetic rats.
The ability of insulin to reduce blood glucose levels
results from the suppression of hepatic glucose
production and increased glucose uptake in muscle
and adipose tissue via GLUT4 (Singla et al. 2010). Our
findings in the present study indicated decreased
expression of muscle GLUT4 in high fat diet and
streptozotocin induced diabetic rats which could be
due the reduced hexokinase activity, decreased glyco-
gen content and elevated plasma glucose levels.
However, diabetic rats treated with b-sitosterol and
metformin showed significant improvement in muscle
GLUT4 expression. The improved expression of
muscle GLUT4 and adipose tissue PPARcare sug-
gested that b-sitosterol is an insulin sensitizing agent
and it can be used as an alternative drug for the
treatment of type 2 diabetes.
Streptozotocin causes abnormalities on bcells
function and it is also documented further that NO
and free nitrous radicals (peroxynitrite) may be an
aggravating factors in the toxicity of streptozotocin
(Elsner et al. 2000). In addition, reactive oxygen
species which are generated during ATP degradation
by xanthine oxidase from hypoxanthine is also
responsible for toxic effect of streptozotocin (Naik
et al. 2013). The alkylating potency of streptozotocin
known to cause ATP depletion that results in hypoxia
and ischemia, a crucial factor for b-cell toxicity,
reduced and shrunken of islets. However, diabetic rats
showed normal architecture of pancreas with
increased number of islets after treatment with b-
sitosterol. This result indicates that antidiabetic poten-
tial of b-sitosterol which could be mediated through
the amelioration of oxidative stress which finally
resulted in the prevention of degeneration of pancre-
atic b-islets and able to preserve its histoarchitecture.
Conclusion
The administration of b-sitosterol significantly
reduced blood glucose and increased the protein
expression PPARcand GLUT4 on insulin target
tissues which indicate that b-sitosterol improved
insulin sensitivity. Decreased blood glucose levels
and reduced insulin resistance are key characteristics
of treatments for diabetes mellitus; hence, b-sitosterol
could be an effective antidiabetic agent and may
possibly be used as an alternative to supportive
treatment for type 2 diabetes mellitus in old-aged
individuals.
Compliance with ethical standards
Conflicts of interest All authors declare that there are no
conflicts of interest.
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