Stem Cells / Research Article
Cells Tissues Organs Accepted after revision: December 28, 2018
DOI: 10.1159/000496506
Naringin (4′,5,7-Trihydroxyflavanone 7-Rhamnoglucoside)
Attenuates β-Cell Dysfunction in Diabetic Rats through
Upregulation of PDX-1
Manickam Subramanian Balaji Thotakura Swathi Priyadarshini Chandra Sekaran
Ashok kumar Jyothi Indumathi Sundaramurthi
Department of Anatomy, Chettinad Hospital and Research Institute, Chettinad Academy of Research and Education,
Chennai, India
Keywords
Naringin · PDX-1 · Diabetes · Insulin
Abstract
Background: Pancreatic duodenal homeobox-1 (PDX-1) is a
key transcription factor which regulates Insulin gene expres-
sion and insulin secretion in adult β-cells and helps to main-
tain β-cells mass. Naringin, a flavanone, owing to its anti­
oxidant property, is reported to have antidiabetic effects.
Objectives: The present study tries to evaluate the role of
naringin on the β-cell-specific transcription factor PDX-1 in
diabetic rats. Methods: Diabetes was induced in male rats
using streptozotocin and treated with naringin (100 mg/kg)
orally for 4 and 8 weeks. Serum insulin level, Pdx-1 and Insu-
lin gene expression, and PDX-1 protein expression were as-
sessed in the rat pancreas. Histopathological and ultrastruc-
tural changes in the islet and β-cells were observed. Results:
Naringin prevented leukocytic infiltration in the pancreas of
diabetic rats and recouped the β-cells with adequate secre-
tory granules. Naringin-treated diabetic rats showed signifi-
cantly increased mRNA expression of Pdx-1 and Insulin
genes, increased expression of transcription factor PDX-1,
and higher serum insulin levels than the diabetic control an-
© 2019 S. Karger AG, Basel
E-Mail karger@karger.com
www.karger.com/cto
Published online: March 18, 2019
imals. These changes were more pronounced in the 8-week
naringin-treated diabetic animals. Conclusions: Naringin
was found to be an effective antidiabetic agent which in-
creased Insulin gene expression and insulin secretion by up-
regulating the PDX-1 gene and protein expression.
© 2019 S. Karger AG, Basel
Introduction
Diabetes mellitus is a metabolic disease affecting 425
million people globally and around 73 million people in
India in particular, placing it second among the countries
affected with diabetes. Its prevalence in India is extrapo-
lated to double by 2045 to reach 134 million [Ogurtsova,
2017]. It is characterized by hyperglycemia, and the dis-
ease process includes dysfunction of pancreatic β-cells,
reduction in β-cell mass, and impairment in insulin se-
cretion irrespective of the type of diabetes [Bell and Po-
lonsky, 2001; Soleimanpour et al., 2015]. Maintenance of
β-cell mass and insulin secretion are regulated by multi-
ple factors. Pancreatic duodenal homeobox-1 (PDX-1) is
one of the cardinal transcription factors which binds to
the promoter region of the insulin gene and regulates its
129.127.145.240 - 3/20/2019 2:12:09 PM
Manickam Subramanian
Department of Anatomy, Chettinad Hospital and Research Institute
Chettinad Academy of Research and Education, Rajiv Gandhi Salai
Kelambakkam, Tamil Nadu 603 103 (India)
Univ.of Adelaide
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E-Mail gandhiayu @ hotmail.com
expression [Ahlgren et al., 1998]. PDX-1 expression is
necessary for postnatal rodent β-cells to mature and be-
come glucose-responsive cells [Guo et al., 2013] and for
maintaining the adult β-cell mass [Holland et al., 2005].
PDX-1 regulates the expression of Insulin, Somatostatin,
Glucokinase, Glucose transporter 2 (GLUT2), and Islet
amyloid polypeptide (IAPP) genes specific to β-cells [Bell
and Polonsky, 2001]. Autosomal dominant mutation of
the Pdx-1 gene leads to diabetes in adults at a younger age
called maturity onset diabetes of the young 4 (MODY4)
[Kushner et al., 2002].
There is an ever-growing need for newer drugs, which
could combat the disease and probably reduce its compli-
cations, in addition to the currently available therapies
like insulin and oral antihyperglycemic agents. In this
context, pharmacological intervention with natural prod-
ucts could be a better alternative and might be effective
and economical as well.
Naringin (4′,5,7-trihydroxyflavanone 7-rhamnoglu-
coside) is a flavanone found in citrus fruits, grapefruits,
beans, cherries, cocoa, oregano, and tomatoes [Bharti et
al., 2014]. Being a common constituent of dietary prod-
ucts, humans are exposed to naringin in some form or the
other. Previous in vitro and in vivo studies show naringin
has antidiabetic activity, and almost all studies attribute
that effect to its antioxidant property and its ability to
counter oxidative stress [Meier et al., 2008; Pari and
Suman, 2010; Liu et al., 2016; Nzuza et al., 2016; Ahmed
et al., 2017; Lim et al., 2018]. Most of the studies show that
naringin has a beneficial effect on diabetes through extra-
pancreatic mechanisms like reduced glucose absorption
[Reshmi et al., 2017; Taslimi et al., 2017], regulation of
hepatic enzyme dysfunction [Punithavathi et al., 2008;
Sharma et al., 2011; Pu et al., 2012; Ahmed et al., 2017;
Pari and Chandramohan, 2017] and improvement in pe-
ripheral resistance [Priscilla et al., 2015; Ahmed et al.,
2017]. Available literature shows that naringin could in-
crease insulin levels [Pari and Suman, 2010; Sharma et al.,
2011; Ahmed et al., 2012; Pu et al., 2012; Priscilla et al.,
2015; Lim et al., 2018] in diabetic animals, but the mech-
anism proposed for such an activity was rather a general
antioxidative, anti-inflammatory, and antiapoptotic
function. There are few data on the central effect of na­
ringin on the endocrine pancreas and mechanism by
which it modulates insulin secretion.
The present study tries to explain the plausible role
of naringin on the β-cell-specific transcription factor
PDX-1 in upregulating insulin gene expression and in
turn modulating insulin production in experimentally in-
duced diabetes in rats.
2 Cells Tissues Organs
DOI: 10.1159/000496506
Materials and Methods
Animals
Male adult Wistar albino rats (250–300 g) were procured from
the Tamil Nadu University of Veterinary and Animal Sciences
(TANUVAS), Madhavaram (Chennai, India), and maintained at a
room temperature of 25 ± 1 ° C on a 12-h-light/12-h-dark cycle.
The animals were fed with rat/mouse pellet obtained from VRK
Nutritional Solutions, Maharashtra. The food and water were pro-
vided ad libitum. The study was approved by the Institutional An-
imal Ethics Committee of the Chettinad Hospital and Research
Institute (Reg. No. 944/AC/06/CPCSEA, approval No. IAEC1/
Desp. No. 60 and IAEC1/Desp. No. 1 dated 09.06.2014 and
25.01.2016, respectively).
Chemicals
Streptozotocin (STZ) and naringin (95% purity) were pur-
chased from Himedia Laboratories Pvt. Ltd., Mumbai, India (No.
RM6099). Primary rabbit polyclonal anti-PDX-1 antibody (No.
ab47267; Abcam, USA) was used in the study. A REAL EnVision
detection system, peroxidase/diaminobenzidine (DAB), rabbit/
mouse kit (Dako, Denmark) was used for primary antibody detec-
tion and labeling. All other chemicals used in this study were of
analytical grade and obtained from Himedia.
Induction of Diabetes
Animals were subjected to overnight fasting prior to the induc-
tion of diabetes mellitus, and their fasting blood glucose levels were
recorded. Animals were given a single dose of STZ (50 mg/kg body
weight [BW] i.p., dissolved in 0.1 M cold citrate buffer, pH 4.5).
The control and nondiabetic group animals were given intraperi-
toneal injection of citrate buffer. Twenty percent glucose solution
was given to STZ-injected animals for 1 day to prevent mortality
due to hypoglycemia. Three days after STZ injection, animals were
once again tested for their fasting blood glucose level. Animals
with a fasting blood glucose level > 250 mg/dL were considered
diabetic and were chosen for the study.
Experimental Design
A total of 84 rats were used for the study and were divided into
the following 7 groups (n = 12 animals/group):
• I: normal control animals treated with vehicle alone
• II: normal rats + naringin for 4 weeks
• III: normal rats + naringin for 8 weeks
• IV: diabetic control treated with vehicle alone for 4 weeks
• V: diabetic control treated with vehicle alone for 8 weeks
• VI: diabetic rats treated with naringin for 4 weeks
• VII: diabetic rats treated with naringin for 8 weeks
Drug Administration
Naringin (100 mg/kg BW) was mixed in distilled water as ve-
hicle (1 mL/kg BW) and was administered once daily orally be-
tween 10 and 11 a.m. using an intragastric tube. Control and un-
treated animals were given equal volumes of vehicle alone.
Sample Collection
At the end of the study, all the animals were sacrificed by an
overdose of thiopentone sodium (75 mg/kg BW). The ventral ab-
dominal wall of the animals was cut open by midline incision, and
the pancreas was dissected out along with the spleen. Fat sur-
rounding the pancreas was carefully removed by dissection. Pan-
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Chandra Sekaran/Jyothi/Sundaramurthi
Univ.of Adelaide
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 Table 1. PCR primer sequences
Genes Forward primer
Gapdh (NM_017008.4) 5′-AGTTCAACGGCACAGTCAAG-3′
Pdx-1 (NM_022852.3) 5′-GGTATAGCCAGCGAGATGCT-3′
Insulin I 5′-ACCTTTGTGGTCCTCACCTG-3′
creatic tissues were stored in neutral buffered formalin for histo-
pathological and immunohistochemical studies and in glutaralde-
hyde for electron-microscopic studies. For Western blot analysis
and mRNA extraction, pancreatic tissues were stored at –80 ° C.
    
For insulin assays, 2-mL venous blood samples were collected
by retro-orbital venous puncture after giving ketamine (22 mg/kg
BW i.p.). Blood was centrifuged at 4,000 rpm for 10 min, and se-
rum separated and stored at –20 ° C.
    
Serum Insulin
A rat insulin ELISA kit (RayBiotech, USA) was used to measure
fasting serum insulin levels; 100-µL serum samples were used ac-
cording to the manufacturer’s instruction and read using a micro-
plate reader at 450 nm.
Histopathology
Pancreatic tissue fixed in 10% neutral buffered formalin was
processed and embedded in paraffin, and 4-µm-thick serial sec-
tions were cut. The sections were stained with hematoxylin and
eosin (H&E). The sections were examined under an Axio Lab A1
microscope (Carl Zeiss, Germany), and photomicrographs were
taken using an Axiocam ERc5s camera attached to the microscope.
Immunohistochemistry
The 4-µm-thick pancreatic sections were placed on slides coat-
ed with poly-L-lysine. Heat-induced antigen retrieval was done by
boiling the sections in a pressure cooker using 10 mM citrate buffer
(pH 6) for 20 min. The endogenous enzymes were blocked by in-
cubation with 3% hydrogen peroxide. The sections were incubated
with primary antibody against PDX-1 (Abcam) diluted with Ven-
tana antibody diluents (Roche Diagnostics, USA) at a ratio of 1:10
for 45 min. The sections were incubated with secondary antibody
labeled with horseradish peroxidase (HRP) (Dako Envision, USA)
and with DAB for 30 and 10 min, respectively. Finally, the sections
were counterstained with Harris hematoxylin.
Transmission Electron Microscopy
Pancreatic tissues were fixed in 2.5% glutaraldehyde and post-
fixed in 1% aqueous osmium tetraoxide and embedded in Araldite
6005 resin. Ultrathin sections of 60-nm thickness were cut using an
ultramicrotome (Leica Ultracut UCT-GA-D/E-1/100) and stained
with saturated aqueous uranyl acetate and counterstained with lead
citrate. The sections were viewed under a transmission electron mi-
croscope (Hitachi H-700, Japan), and photographs were taken at
RUSKA Labs, College of Veterinary Science, P.V. Narsimha Rao
Telangana Veterinary University, Hyderabad, India.
PDX-1Western Blot Analysis
The pancreatic tissues stored at –80 ° C were thawed and washed
with cold physiological saline. The tissue (100 µg) was completely
Naringin Upregulates PDX-1 in
Diabetic Rats
Reverse primer
5′-TACTCAGCACCAGCATCACC-3′
5′-TCAGGTGGGAGCCTGATTCT-3′
5′-AGCTCCAGTTGTGGCACTTG-3′
homogenized using Tomy Microsmash tissue homogenizer
(Tomy, Japan) in RIPA buffer. The homogenate was centrifuged
at 12,000 rpm for 15 min at 4 ° C, and the supernatant was collect-
ed and used for protein estimation and Western blot analysis. Pro-
tein concentrations in the pancreatic tissue lysates of the different
groups were determined using the previously published method of
Lowry et al. [1951] using bovine serum albumin as standard. The
cell lysates were subjected to centrifugation at 14,000 rpm for 10
min at 4 ° C, and supernatant was separated. Protein extract (50 µg)
was separated on 12% SDS-polyacrylamide gels and transferred
onto polyvinylidene difluoride membranes. Then, the membranes
were incubated with primary antibody to PDX-1 (Abcam) at 1:
1,000 dilutions in Tris-buffered saline overnight at 4 ° C followed
by detection using an enzymatically labeled HRP conjugated sec-
ondary antibody (Merck, India) at 1: 5,000 dilutions. Protein ex-
pression was visualized by a chemiluminescent method using Che-
mi Doc XRS Imaging System (BioRad, USA). Band intensity was
quantified by ImageJ software.
Gene Expression Studies
Total RNA was extracted from the rat pancreas using TRIzol
reagent (TakaraBio, USA) according to the manufacturer’s in-
structions. The RNA extracted was quantified using a UV spectro-
photometer at 260–280 nm. Then, complementary deoxyribonu-
cleic acid (cDNA) was obtained with 5 µL total RNA using Prime
script cDNA master mix (TakaraBio, USA) and using 1 µL of
cDNA, amplifications were done in a PCR system (Syngene, UK)
with different primers (Table 1). PCR products were separated us-
ing agarose gel electrophoresis and visualized using an ultraviolet
gel documentation system. Band intensity was quantified using
ImageJ software. Glyceraldehyde 3-phosphate dehydrogenase
(Gapdh) was used as an endogenous control, and the transcription
of Pdx-1 and Insulin was compared in relation to Gapdh in the dif-
ferent groups. Each value represented the mean value of at least 3
independent experiments.
Morphometric Analysis
Ten random H&E- and PDX-1-immunostained sections were
selected per animal in each group. From the selected sections, 5
random fields were studied. Totally, 50 fields per animal were ob-
served. The following parameters were measured:
(A) The islet diameter was calculated from H&E-stained sec-
tions at a magnification of ×100 using the following equation:
L+B
Axial ratio (mm) =
2
where L is maximum islet length and B is maximum islet breadth,
which is perpendicular to L.
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Cells Tissues Organs 3
DOI: 10.1159/000496506
Univ.of Adelaide
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 (B) The numerical density of the islets (H&E-stained section
at ×100 magnification) per unit volume of reticule with 121 inter-
sections was calculated using the following equation:
NA
NV (number of cells/mm 3 ) =
A (D + T)
where NV is the numeric density or number of islets per cubic mil-
limeter; NA the average number of islets counted within the reti-
cule; A the area of the reticule in square millimeters; T the thick-
ness of the section in millimeters; and D the mean diameter of islets
in millimeters.
(C) The fractional volume of PDX-1-positive β-cells in PDX-
1-immunostained sections (×400 magnification), using a reticule
with 121 intersections, was calculated using the following equation
(modified Delesse’s method):
P weeks (group IV).
Vf = int
Ptot
where Vf is the volume fraction of PDX-1-positive cells; Pint the
number of intersections hitting the PDX-1-positive cells; and Ptot
the number of points hitting the whole section.
Statistical Analysis
All the data are expressed as the mean ± standard error of mean
(SEM) and subjected to statistical analysis using one-way analysis
of variance (ANOVA). The t test was performed to determine sig-
nificant differences between the individual groups using comput-
er-based software (SPSS, version 21; IBM, USA). p < 0.05 was con-
sidered statistically significant.
Results
Fasting Serum Insulin
Groups I, II, and III had similar fasting insulin levels,
while the diabetic untreated groups (groups IV and V)
presented significantly lower (p < 0.05) insulin levels than
the control group. In group VI and VII animals, insulin
levels were significantly higher (13.30 and 15.21 µU/
mL, respectively) compared to group IV and V animals
(Table 2).
Histopathology
H&E-stained pancreatic sections of groups I, II, and III
showed normal parenchyma and islets (Fig. 1a, b, 2a, b).
The diabetic groups IV and V exhibited shrunken and ir-
regularly shaped islets. A lobular pattern of the exocrine
part was observed due to extensive lymphocytic infiltra-
tion (Fig. 1c, d, 2c, d).
Group VI animals had less pronounced pathological
changes than the diabetic groups (Fig. 1e, 2e). The dia-
betic rats treated with naringin for 8 weeks (group VII)
showed almost normal parenchyma and islets, and ab-
sence of lymphocytic infiltration. Despite the smaller size
4 Cells Tissues Organs
DOI: 10.1159/000496506
Table 2. Serum insulin levels of the different groups
Group Insulin, µU/mL
I Control 18.26±0.33
II Naringin 4 weeks 17.71±0.41
III Naringin 8 weeks 18.58±0.37
IV DM 4 weeks 9.81±0.39A
V DM 8 weeks 8.56±0.41A
VI DM + naringin 4 weeks 13.30±0.42A, C
VII DM + naringin 8 weeks 15.21±0.35A, B
Mean values were significantly different by t test following
ANOVA: A p < 0.05 vs. normal controls (group I); B p < 0.05 vs.
diabetes mellitus (DM) 8 weeks (group V); C p < 0.05 vs. DM 4
of the islets, the regular contour and absence of other
pathological changes showed that the rats were recover-
ing from the insult caused by STZ (Fig. 1f, 2f).
Morphometric analysis showed significant decreases
in islet diameter and density in group IV and V diabetic
animals compared to group I (Fig. 3a, b). This effect was
reversed in the naringin-treated groups VI and VII. This
proves the proliferative effect of naringin on islet cells.
These changes were more conspicuous in group VII when
compared to the corresponding untreated group V ani-
mals. In normal rats administered naringin, these param-
eters were similar to the control group.
Transmission Electron Microscopy
β-Cells of control animals (group I) and naringin con-
trol animals (groups II and III) had large secretory gran-
ules in the cytoplasm, which were electron lucent with the
characteristic halo around them. Numerous small vesi-
cles were seen without secretory content (Fig. 4a).
Group IV showed damaged islets with absence of se-
cretory granules thereby making the β-cell inconspicu-
ous. The nuclei of islets were pyknotic and distorted with
signs of degeneration (Fig. 4b). Group V animals exhib-
ited similar pathology with swollen nuclei and indistinct
organelles (Fig. 4c).
β-Cell nuclei of group VI animals had less heterochro-
matin and dilated nuclear pores. Cytoplasm exhibits
agranular vesicles and distorted endoplasmic reticulum
(Fig. 4d). In the 8-week naringin-treated animals (group
VII), β-cells demonstrated increased number of charac-
teristic small secretory granules suggesting β-cell regen-
eration (Fig. 4e).
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 Is Is Ac

Is
Ac Ac

Is
Is
Is
a b Is c

Is
Is

Ac
Is
Ac Ac
Is
d e f

Fig. 1. a–f H&E staining of rat pancreatic tissue. ×100. Scale bar, of lymphocytes damaging the islet. e Group VI exhibits shrunken
1 mm. Group I (a) and group III (b) show normal acinar and islet islets and lymphocytic infiltration but less pronounced than group
architecture. c Group IV shows shrunken islet and lymphocytic V. f Group VII demonstrates near normal islets. Arrows indicate
infiltration. d Group V shows shrunken and extensive infiltration lymphocytic infiltration. Is, Islets; Ac, Acini.

a b c

d e f

Fig. 2. a–f H &E staining of rat pancreatic tissue. ×400. Scale bar,
100 µm. Group I (a) and group III (b) reveal normal islets. c Group
IV shows islets with extensive lymphocytic infiltration. d Group V
demonstrates islets with fewer cells and lymphocytic infiltration.
e Group VI shows small islets with few lymphocytes. f Group VII
demonstrates near normal islets with very few lymphocytes. Ar-
rowhead indicates lymphocytic infiltration and arrow indicates
the islets.
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Naringin Upregulates PDX-1 in Cells Tissues Organs 5

Diabetic Rats DOI: 10.1159/000496506
Univ.of Adelaide
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 14
120 30 $

Fractional volume of PDX1-

Islet density, n of cells/mm3
12 $
$ #
100 25
10
Islet diameter, µm

positive cells, %
* #
80 * 20
8
* *
60 15 6
40 10 4
* *
20 5 2
0 0 0
I II III IV V VI VII I II III IV V VI VII I II III IV V VI VII
a Groups b Groups c Groups

Fig. 3. Morphometric analysis of the rat pancreas. Graphs show the islet diameter (a), islet density (b), and frac-
tional volume of PDX-1-positive β-cells in the islets (c) of the different groups. Mean values were significantly
different by one-way ANOVA. * p < 0.05 vs. control; # p < 0.05 vs. group IV; $ p < 0.05 vs. group V (t test).

Immunohistochemistry PDX-1 Immunoblot

Groups I, II, and III showed PDX-1-immunopositive Western blot analysis of PDX-1 protein showed a
nuclei in the central portion of the islets suggesting the statistically significant difference between the groups
expression and localization of PDX-1 in the nuclei of (Fig. 7). Protein expression was significantly downregu-
β-cells (Fig. 5a, b). In contrast, group IV and V islets ex- lated in group IV and group V in comparison to group I.
hibited very mild reactivity to PDX-1 immunostaining When compared to group IV, group V animals (left un-
showing positivity in very few nuclei (Fig. 5c, d). Group treated) had very low expression of PDX-1 protein. PDX-1
VI islets expressed weak reactivity for PDX-1 in few nu- expression was significantly enhanced in the diabetic
clei (Fig.  5e). Group VII islets were bigger when com- group treated with naringin (group VI) when compared
pared to groups IV and V and displayed more PDX-1-im- to its corresponding untreated diabetic group IV (Fig. 7b).
munopositive cells (Fig. 5f). Moreover, group VII had an almost twofold increase in
The fractional volume of PDX-1-positive β-cells dras- PDX-1 expression when compared to group V, showing
tically reduced in the diabetic untreated groups IV and V. significant increase in the transcription factor which
The diabetic animals treated with naringin (group VI and could influence the upregulatory target genes like Insulin
VII) showed significantly higher PDX-1-positive cells (Fig.  6d). PDX-1 expression was also higher in normal
when compared with the corresponding untreated groups rats treated with naringin for 8 weeks (group III) than the
(groups IV and V) (Fig. 3c). controls.

Gene Expression
Figure 6a, b shows the effect of naringin administra- Discussion
tion on Pdx-1 gene expression. The animals exhibited low
Pdx-1 expression in diabetic groups (groups IV and V), STZ-induced diabetes is a well-accepted animal model
and the same pattern was found in Insulin mRNA expres- for experimental induction of diabetes causing selective
sion (Fig.  6c, d). However, mRNA expression of Pdx-1 destruction of β-cells. It also generates reactive oxygen
and Insulin was increased in the diabetic groups treated species, which accelerates β-cell destruction [Lenzen,
with naringin (groups VI and VII), and they were found 2008]. In the present study, we established the model of
to be significantly higher in group VII than the diabetic experimental diabetes in rats using STZ to investigate
untreated counterparts (group V). Naringin administra- molecular mechanisms through which naringin exerts a
tion to normal rats (groups II and III) did not alter the central effect on the endocrine pancreas.
expression of Pdx-1 and Insulin genes much when com- Untreated diabetic rats show leukocyte infiltration in
pared to control animals. pancreatic parenchyma, perivascular fibrosis, and de-
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6 Cells Tissues Organs Subramanian/Thotakura/

DOI: 10.1159/000496506 Chandra Sekaran/Jyothi/Sundaramurthi
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 ER N

N
N N N

2.5 µm b 25 µm 25 µm
a c

2.5 µm 2.5 µm
d e

Fig. 4. Electron micrographs of pancreatic β-cells of the different
groups (×7,720). a Normal β-cell with characteristic secretory
granules. b Group IV islet with indistinguishable cell types and
abnormally shaped nuclei. c Group V islet with indistinguishable
cell types and noncharacteristic granules. d Group VI β-cell with
empty granules and distorted endoplasmic reticulum. e Group VII
β-cell with numerous small characteristic granules. N, nucleus; ER,
endoplasmic reticulum; *, artifact. Arrows indicate secretory gran-
ules with characteristic halo around them. Arrowheads indicate
agranular vesicles.

a b c

d e f

Fig. 5. Photomicrographs of pancreatic islets of various groups im-
munostained with PDX-1 antibody (×400, scale bar, 100 µm).
a, b Group I and III islets with nuclear localization of PDX-1 in
β-cells present in the center of the islet. c, d Group IV and V islets
with few PDX-1-positive cells. e, f Group VI and VII islets of
naringin-treated diabetic rats with immunopositivity for PDX-1.
Arrowheads show the immunopositive nuclei of the islets.
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Naringin Upregulates PDX-1 in Cells Tissues Organs 7

Diabetic Rats DOI: 10.1159/000496506
Univ.of Adelaide
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 Pdx-1
50 $

Relative mRNA expression,

40 *
* *
Groups *

% of GAPDH
30
I IV V VI VII II III

Pdx-1 20

10
Gapdh
0
I IV V VI VII II III
a b Groups

Insulin
70

Relative mRNA expression,

$
60
Groups

% of GAPDH
I IV V VI VII II III
50 *#
Insulin 40
*
30 *
Gapdh
20
I IV V VI VII II III
c d Groups

Fig. 6. Effect of naringin on mRNA expression of Pdx-1 and Insulin genes in rat pancreas. Representative gene
expression (a) and quantification of Pdx-1 gene (b) in the different groups. Representative gene expression (c)
and quantification of Insulin gene (d) in the different groups (n = 6). Mean values were significantly different by
one-way ANOVA: * p < 0.05 vs. control; # p < 0.05 vs. group IV; $ p < 0.05 vs. group V (t test).

struction of islets. Naringin treatment reversed these
changes showing markedly reduced leukocyte infiltration
and reappearance of normal islet architecture (Fig. 1, 2),
and increased islet diameter and density (Fig.  3a, b).
These changes confirm the regeneration of the diabetic
pancreas and islets in particular from the necrosis caused
by STZ. This strongly reaffirms the results of Lim et al.
[2018] that naringin protects the pancreas against leuko-
cyte infiltration and proinflammatory cytokine produc-
tion.
Ultrastructural changes in the β-cells of naringin-
treated diabetic rats include the presence of characteristic
secretory granules and increased endoplasmic reticular
network. This indicates the β-cells have recovered from
the STZ-created insult, and they are functionally active.
This is in accordance with the results of Sharma et al.
[2011], where administration of naringin (100 mg/kg) to
type II diabetic rats reduced the endoplasmic reticular
distension and maintained adequate secretory granules.
In contrast, the diabetic animals had necrosis of β-cells,
which made them indistinguishable from other islet cell
types and devoid of secretory granules.
It is established that naringin reduces the β-cell oxida-
tive stress and increases the insulin secretion in a dose-
dependent manner in protease inhibitor-treated RIN5F
cell lines [Nzuza et al., 2016]. Similarly, in the present
study, administration of naringin increased fasting serum
insulin levels in diabetic animals. Moreover, the secretory
granules of the β-cells in diabetic rats increased notice-
ably followed by increasing serum insulin levels with ad-
ministration of naringin. These changes confirm that
there was a significant improvement in insulin synthesis
and secretion in diabetic animals treated with naringin.
The increased fractional volume of PDX-1-positive
cells in naringin-treated animals (Fig. 3) correlates well
with the increase in their Pdx-1 transcript level and pro-
tein level indicating that β-cells are regenerating follow-
ing naringin treatment. This accounts for increased ex-
pression of Insulin gene and insulin secretion. Thus, in
diabetic rats, the administration of naringin increases the
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8 Cells Tissues Organs Subramanian/Thotakura/

DOI: 10.1159/000496506 Chandra Sekaran/Jyothi/Sundaramurthi
Univ.of Adelaide
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 4 weeks
#
100
* *

Relative protein expression

compared to GAPDH
Groups 80
I IV VI II 60
PDX-1
40

20
GAPDH
0
I IV VI II
a b Groups

8 weeks
140
*$ *

Relative protein expression

120

compared to GAPDH
Fig. 7. Effect of naringin on PDX-1 protein
expression in rat pancreas. Representative Groups 100
Western blot (a) and quantification of I V VII III 80
PDX-1 (b) in pancreas of groups I, II, IV, *
PDX-1 60
and VI. Representative Western blot (c)
40
and quantification of PDX-1 (d) in pancre-
as of groups I, III, V, and VII (n = 6). Mean GAPDH 20
values were significantly different by one- 0
way ANOVA. * p < 0.05 vs. control; # p < I V VII III
0.05 vs. group IV; $ p < 0.05 vs. group V (t c d Groups
test).

expression of PDX-1, which sequentially influences the
expression of the Insulin gene and in turn modulates in-
sulin hormone secretion.
Moreover, the results show that the protective effect of
naringin against STZ-induced diabetes was more pro-
nounced in 8-week naringin-treated animals. This sug-
gests the need for prolonged usage of this phytochemical
to abate the adverse changes involved in the diabetic
pathophysiology.
In β-cells, glucose enters the cell through GLUT2
transporters and a signaling pathway involving phos-
phoinositide 3-kinase (PI3K) which phosphorylates
PDX-1. The phosphorylated PDX-1 is translocated from
the cytoplasm to the nucleus and binds the A element lo-
cated in the 5′ flanking sequence of the Insulin gene pro-
moter region and activates Insulin gene transcription
[Vaulont et al., 2000]. Chronic glucotoxicity shuttles the
PDX-1 from the nucleus to the cytoplasm and downregu-
lates Insulin gene transcription and insulin biosynthesis
[Kitamura et al., 2002]. Figure 5 shows the absence of
PDX-1 immunoreactivity in the nucleus of diabetic islets
and the increased expression of PDX-1 and its nuclear
localization in the islets of control, naringin-treated nor-
mal animals, and naringin-treated diabetic animals. Lo-
calization of PDX-1 in β-cell nuclei might facilitate its ac-
tion on Insulin gene expression in diabetic rats.
A previous study suggests that PDX-1 is a signaling
target of insulin, and insulin causes nuclear localization
of PDX-1 and decreases the apoptosis of islets through
mediation of PDX-1 expression both in vitro and in vivo
[Johnson et al., 2006]. This elucidates an additional anti-
apoptotic role exhibited by PDX-1.
Peroxisome proliferator-activated receptor γ (PPARɣ)
plays a significant role in cellular proliferation and differ-
entiation, glucose homeostasis, and lipid metabolism. It
regulates the expression of PDX-1 in β-cells and thereby
improves β-cell function by regulating the downstream
targets of PDX-1 [Jung et al., 2014]. Naringin upregulates
the PPARɣ receptors in the liver and kidney of rats and in
adipose tissue of db/db mice, and decreases insulin resis-
tance and proinflammatory cytokines [Jung et al., 2006;
Sharma et al., 2011]. Similar regulation of PPARɣ expres-
sion in β-cells by naringin has to be ascertained to under-
stand the mechanism resulting in upregulation of PDX-1.
Chronic hyperglycemia causes oxidative stress and de-
terioration of β-cells via c-Jun N-terminal kinase (JNK)
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Naringin Upregulates PDX-1 in Cells Tissues Organs 9

Diabetic Rats DOI: 10.1159/000496506
Univ.of Adelaide
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pathway activation. Translocation of PDX-1 to the cyto-
plasm and its reduced binding to the insulin gene are
linked to the activation of JNK. Further, oxidative stress
downregulates Akt expression which phosphorylates an-
other important negative regulator of PDX-1, namely
Forkhead box protein O1 (FoxO1), leading to nucleocy-
toplasmic translocation of PDX-1 [Tabatabaie and Yaz-
danparast, 2017]. Naringin, a potent antioxidant flava-
none, may counteract the hyperglycemia-induced β-cell
oxidative stress. We hypothesize that naringin by virtue
of its antioxidant property might probably maintain the
normal localization and increased expression of PDX-1
through JNK kinase pathway suppression and downregu-
lation of FoxO1.
The results of the present study confirm that increased
expression of PDX-1 in diabetic rats treated with narin-
gin upregulates the insulin gene expression and insulin
secretion. This in turn can eventually result in a reduction
in hyperglycemia. Research on the effect of naringin on
the Pdx-1 gene and protein, the key controllers of insulin
secretion, has not been reported and hence the need for
the present study.
In summary, the present study demonstrates naringin
as an effective antidiabetic agent and reveals the mecha-
nism by which naringin increases insulin secretion in an
experimental model of diabetes. Increased expression of
PDX-1 by naringin positively modulates the Insulin gene
and its expression resulting in increased insulin biosyn-
thesis and secretion. Further research is required to inves-
tigate the up- and downregulatory mechanisms by which
naringin regulates the transcription factor PDX-1 to es-
tablish its clinical value.
Acknowledgments
The authors thank the following individuals for their contribu-
tions to this article: S. Govindaraju for statistical support and R.
Vijayashree for pathological interpretation.
Statement of Ethics
Animal experiments were conducted according to the guide-
lines of the Committee for the Purpose of Control and Supervision
of Experiments on Animals (CPCSEA). The experimental proto-
col was approved by the Institutional Animal Ethics Committee of
the Chettinad Hospital and Research Institute (Reg. No. 944/
AC/06/CPCSEA, approval No. IAEC1/Desp. No. 60 and IAEC1/
Desp. No. 1 dated 09.06.2014 and 25.01.2016, respectively).
Disclosure Statement
The authors have no conflict of interest to declare.
Funding Sources
The study was supported by the Chettinad Academy of Re-
search and Education.
Author Contributions
Study conception and design: Manickam Subramanian, Balaji
Thotakura, and Indumathi Sundaramurthi; acquisition, analysis,
and interpretation of data: Manickam Subramanian, Swathi Priya-
darshini Chandra Sekaran, and Ashok Kumar Jyothi; drafting of
the manuscript: Manickam Subramanian; critical revision: Balaji
Thotakura and Indumathi Sundaramurthi, and final approval of
the version to be published: Manickam Subramanian, Balaji
Thotakura, Indumathi Sundaramurthi, Swathi Priyadarshini
Chandra Sekaran, and Ashok Kumar Jyothi.

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