Chemico-Biological Interactions 186 (2010) 72–81

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Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint

Insulinotropic effect of cinnamaldehyde on transcriptional regulation of pyruvate

kinase, phosphoenolpyruvate carboxykinase, and GLUT4 translocation in
experimental diabetic rats
Prachi Anand, Murali K.Y. 1 , Vibha Tandon, P.S. Murthy ∗ , Ramesh Chandra ∗
Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110007, India

a r t i c l e i n f o a b s t r a c t

Article history: Diabetes mellitus is a chronic metabolic disorder affecting about 6% of population worldwide with its com-
Received 1 January 2010 plications and is rapidly reaching epidemic scale. Cinnamomum zeylanicum is widely used in alternative
Received in revised form 22 March 2010 system of medicine for treatment of diabetes. In the present study, we have performed bioassay guided
Accepted 25 March 2010
fractionation of chloroform extract of C. zeylaniucm and identified cinnamaldehyde (CND) as an active
Available online 2 April 2010
principle against diabetes. In continuation to it, a detailed study was undertaken to elucidate its mode
of antidiabetic action in STZ induced diabetic rats. Oral administration of CND (20 mg/kg bw) to diabetic
Keywords:
rats for 2 months showed significant improvement (p < 0.001) in muscle and hepatic glycogen content. In
Cinnamaldehyde
Glycogen
vitro incubation of pancreatic islets with CND enhanced the insulin release compared to glibenclamide.
GLUT4 The insulinotropic effect of CND was found to increase the glucose uptake through glucose transporter
Insulin (GLUT4) translocation in peripheral tissues. The treatment also showed a significant improvement in
Pancreatic ␤ cells altered enzyme activities of pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK) and
their mRNA expression levels.
Furthermore, the median lethal dose (LD50 ) of CND could not be obtained even at 20 times (0.4 g/kg bw)
of its effective dose. With the high margin of safety of CND, it can be developed as a potential therapeutic
candidate for the treatment of diabetes.
© 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction From the reports on the potential effectiveness of herbal reme-

Diabetes mellitus is one of the most common metabolic dis-
orders prevalent in society [1,2]. Hyperglycemia is one of the
manifestations of diabetes and is believed to be due to the
decreased utilization and increased production of glucose [3–5].
Insulin-stimulated glucose utilization is the major site for regula-
tion of plasma glucose concentrations [6]. Glucose transport is the
rate-limiting step in carbohydrate metabolism [7,8] which is facili-
tated by glucose transporters (GLUT) across the cell membrane. The
subtype 4 form (GLUT4) is the insulin sensitive glucose transporter;
reduction in insulin-mediated glucose uptake is reported to be
caused by decreasing GLUT4 protein [9,10]. Therefore, compounds
facilitating GLUT4 translocation can be potentially beneficial for the
treatment of diabetes.
Abbreviations: bw, Body weight; CND, Cinnamaldehyde; STZ, streptozotocin.
∗ Corresponding authors. Tel.: +91 11 27666272; fax: +91 11 27666248.
E-mail addresses: panand@acbr.du.ac.in (P. Anand), psmurthy 2000@yahoo.com
(P.S. Murthy), acbrdu@hotmail.com (R. Chandra).
1 gluconeogenic enzyme in the liver and kidney of STZ induced dia-
Present address: Dept. of Immunodermatology and Allergy Research, Hannover
Medical School, Hannover, Germany.
dies against diabetes, it is believed that the natural products play
a major role in the management of diabetes [11]. Cinnamaldehyde
(CND) is an active compound isolated from the stem bark of C. zey-
lanicum, a traditional oriental medicinal herb. In a recent study
cinnamaldehyde has been shown to possess antihyperglycemic
activity in diabetic rats [12,13]. It has also been shown to possess
inhibition of tumor cell proliferation [14,15], antioxidative [16] and
anti-inflammatory activities in suppressing nitric oxide production
by LPS-stimulated macrophages [17]. Dimeric cinnamaldehydes
have been reported to inhibit tumor cell growth through PARP
degradation via caspase-3 pathway or cell cycle arrest in G2/M
phase [18]. However the mechanism of antidiabetic action of this
compound remained unknown. The objective of present investiga-
tion was to ascertain the scientific basis for its use in the treatment
of diabetes and its margin of safety.
In the present study, we have evaluated cinnamaldehyde for its
antidiabetic effect. CND possesses a direct insulin-like effect caus-
ing an increase in glucose uptake through GLUT4 translocation in
peripheral tissues. In addition to it, we have demonstrated that
CND affects the activities and mRNA levels of key glycolytic and
betic rats. The LD50 of CND is high (no death even with 20 times of

0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.cbi.2010.03.044
 P. Anand et al. / Chemico-Biological Interactions 186 (2010) 72–81
effective dose) indicating high margin of safety. Hence, CND can be
developed as a potential drug candidate for diabetes.
2. Materials and methods
2.1. Chemicals
Streptozotocin, all coenzymes, purified enzymes, substrates,
standards and buffers were purchased from Sigma–Aldrich Co.,
USA. All the other chemicals used were of analytical grade and
purchased from S.D. Fine Chemicals, India. ELISA kit for insulin esti-
mation was purchased from DRG Diagnostics, Germany, while the
other kits for serum estimations (total protein, total bilirubin, SGOT,
SGPT, creatinine) were purchased from Bayer diagnostics India. Kit
for the estimation of blood glucose was purchased from Ranbaxy,
New Delhi, India. Kit for the estimation of glycosylated hemoglobin
(HbA1c ) was purchased from (Erba Diagnostics Mannheim GmbH,
Germany.
2.2. Animals
Male Wistar rats (6–7 weeks old) obtained from the animal
research facility (350/CPCSEA) of our center (Dr. B. R. Ambedkar
Center for Biomedical Research, University of Delhi, Delhi, India)
were used. The animals were kept in the air-conditioned animal
house at 25–30 ◦ C and 45–55% relative humidity, acclimatized with
free access to food (Golden feed, Delhi, India) and water ad libitum
under 12 h light, 12 h dark cycle through out the study. All ani-
mals were carefully monitored and experimental protocols were in
accordance with the ethical recommendations of the Institutional
Animal Ethical Committee.
2.3. Study design
Twenty-four rats were divided into four equal groups as follows:
(i) Healthy control group: received orally 0.5% DMSO in water
(0.5 ml/100 g bw/day).
(ii) Diabetic control group: received intraperitoneal injection
of STZ (50 mg/kg bw in citrate buffer) [19] to make type
1 diabetic model and after 7 days 0.5% DMSO in water
(0.5 ml/100 g bw/day) for next 60 days.
(iii) Diabetic + CND supplement group: diabetic rats were
orally given CND 20 mg/kg bw in 0.5% DMSO in water
(0.5 ml/100 g bw/day) after 7 days of STZ injection for next 60
days with canula.
(iv) Diabetic + glibenclamide supplement group: diabetic rats were
orally given glibenclamide 200 mg/kg bw in 0.5% DMSO in
water (0.5 ml/100 g bw/day) after 7 days of STZ injection for
next 60 days.
The treatment was continued until the day of the sacrifice. At the
end of the antidiabetic treatment (2 months) general body param-
eters (i.e. body weight, fluid intake, tissues weight, fasting blood
glucose, HbA1c and serum insulin) were estimated and then the
rats were killed by cervical dislocation. Liver, kidneys and hind limb
muscle were removed, weighed, rapidly frozen in liquid nitrogen,
and stored at −80 ◦ C until further analysis. Three more groups of
animals of 6 rats each (3 males and 3 females) were used for acute
toxicity studies.
2.4. Isolation of pancreatic islets from experimental animals
Pancreatic islets were prepared by collagenase digestion and
density gradient purification method [20] with slight modifications
73
as described earlier. [21]. Pancreas was removed from the anaes-
thetized healthy animals into HBBS (Hanks bicarbonate buffered
solution containing 3% glucose, 1% BSA, pH 7.4), cut into small
pieces and washed with HBBS to remove pre-existing insulin and
centrifuged at 5000 rpm for 15 min at 4 ◦ C and the supernatant
was discarded. The residue was transferred into conical flasks and
incubated at 37 ◦ C with rapid magnetic stirring in HBBS contain-
ing collagenase (3 mg/ml) having protease inhibitor aprotinin (2%,
v/v) specifically formulated for pancreatic islet isolation. At optimal
period of digestion, the residue was subjected to density gradient
centrifugation for 10 min at 800 g with different gradients (25%,
23%, 20% and 11%) of Ficoll [22]. Islets from the 20–11% interface
were separated and used to measure the insulin release.
2.5. In vitro release of insulin from pancreatic islets
Islets were transferred to small Petridishes containing bovine
serum albumin (BSA) and 10 mM glucose, pH 7.4 and preincu-
bated under the atmosphere of 95% O2 , 5% CO2 for 1 h. These islets
show good release of insulin and served as the initial standard for
the respective groups. The islets were then incubated with 200 ␮l
(2 mg/ml) of either CND or the standard drug glibenclamide for 2 h.
0.5% DMSO in distilled water was added to islets for the control
group. Incubation was continued for 2 h then the mixture was cen-
trifuged at 16,000 g for 10 min at 37 ◦ C. Supernatant obtained was
used for ELISA to estimate the release of insulin from pancreatic ␤
cells.
2.6. Estimation of glycogen content in liver and skeletal muscle
tissue
Liver and skeletal muscles were removed, cut into pieces and the
glycogen was purified [23]. It was then washed with 80% ethanol
and dissolved in a suitable volume of water. Samples containing
100–200 ␮g glycogen were mixed with 5 ml of anthrone reagent
(100 mg anthrone and 200 mg thiourea in 20 ml of 70% H2 SO4 ) and
kept in boiling water bath for 15 min. After cooling, the optical den-
sity (OD) of the samples was measured at 620 nm. A known amount
of glucose was used as standard and run simultaneously and the OD
obtained was multiplied by 0.9 for conversion to glycogen.
2.7. Determination of enzyme activities
The tissue homogenates were prepared as described previously
[24]. Protein concentration of the tissue homogenates was deter-
mined by Bradford method [25]. The supernatants were used for
the determination of enzyme activities of pyruvate kinase (PK)
and phosphoenol pyruvate carboxykinase (PEPCK). PK (EC 2.7.1.40)
activity was measured by the method of Blair et al. [26]. Two con-
centrations of PEP were used to distinguish between the active
(0.15 mmol/l) and the total forms (5 mmol/l) of the PK. PEPCK (EC
4.1.1.32) activity was measured by the decarboxylation assay as
described in literature [27].
2.8. RNA extraction and reverse transcriptase-polymerase chain
reaction analysis
cDNA (1 ␮g) was reverse transcribed from 5 ␮g of total cellu-
lar RNA from liver and kidney tissues prepared using TRI reagent
(Sigma Chemicals Co., USA), oligo (dT) primers and M-MuLV reverse
transcriptase [Fermentas Life Sciences, USA (Revert Aid First strand
cDNA synthesis kit)]. The specific primers used were as follows: PK,
5 -ATT GCT GTG ACT GGA TCT GC-3 (sense), 5 -CCC GCA TGA TGT
TGG TAT AG-3 (antisense), 229 bp, PEPCK 5 -TTT ACT GGG AAG GCA
TCG AT-3 (sense), 5 -TCG TAG ACA AGG GGG ACA C-3 (antisense),
74 P. Anand et al. / Chemico-Biological Interactions 186 (2010) 72–81

Table 1
Effect of 60 days treatment with the CND and glibenclamide on body weight, fluid intake, liver and kidney weight in rats.

Parameters Control Untreated diabetic CND treated Glibenclamide treated

Body weight (g) 224 ± 16 139 ± 12a

209 ± 10b
195 ± 16b
Fluid intake (ml/day) 40 ± 3.2 175 ± 16c 74 ± 8.0d 91 ± 8.9d
Liver weight (g) 7.2 ± 0.61 3.9 ± 0.41c 6.1 ± 0.58 5.4 ± 0.52
Liver weight (g)/100 g body weight 3.21 ± 0.03 2.8 ± 0.02c 2.92 ± 0.026 2.76 ± 0.022
Kidney weight (g) 0.51 ± 0.03 1.42 ± 0.13c 0.63 ± 0.02d 0.69 ± 0.02d
Kidney weight g/100 g body weight 0.23 ± 0.02 1.02 ± 0.05c 0.301 ± 0.02d 0.35 ± 0.03d

Number of animals per group is 6; values represent the mean ± S.D.

a
p < 0.01 when compared with normal control group.
b
p < 0.01 when compared with untreated diabetic group.
c
p < 0.001 when compared with normal control group.
d
p < 0.001 when compared with untreated diabetic group.

236 bp, ␤-actin 5 -CCA TCT ACG AGG GCT ATT CT-3 (sense), 5 -TTT
GAT GTC ACG CAC GAT TT-3 (antisense), 242 bp.
After RT-PCR, the content of each independent reaction tube was
subjected to electrophoresis on 1.5% agarose gels containing ethid-
ium bromide. Images from electrophoresed gels were captured by
a camera in a computer assisted gel documentation system (alpha
imager). Relative band intensities of each sample were calculated
after being normalized with the band intensity of ␤-actin.
2.9. Sub-cellular fractionation for measuring glucose transporter
protein (GLUT4) levels
To obtain membrane fractions from skeletal muscle of the
treated animals 1 g of muscle tissue was minced well and
homogenized (1/10, w/v) by polytron 2 × 5 s, (setting 4–5), in
homogenization buffer (20 mmol/l NaHCO3 pH 7.0, 0.25 mol/l
sucrose, 5 mmol/l Na3 N, 1 mmol/l leupeptin, 1 mmol/l pepstatin,
1 mmol/l aprotinin) at 4 ◦ C. The homogenate was centrifuged at
1200 g for 10 min and the pellet was resuspended in above buffer,
homogenized and recentrifuged to remove debris. The combined
supernatants were centrifuged at 9000 g for 10 min at 4 ◦ C for sed-
imenting nuclei and mitochondria. The resulting supernatant was
then centrifuged at 1,90,000 g for 60 min at 4 ◦ C to obtain total
membrane protein fraction [28]. The membrane pellets were resus-
pended in homogenization buffer and total membrane protein
content was measured by Nanodrop (ND1000) spectrophotometer.
To get the whole homogenate, minced muscle tissue was
homogenized at full speed in the homogenization buffer at 4 ◦ C
and subjected to centrifugation at 1000 g to pellet down the cell
debris. The supernatant was collected and the protein content was
measured by Nanodrop (ND1000) spectrophotometer.
2.10. GLUT4 protein analysis
2.11. Determination of median lethal dose (LD50 )
Animals were divided into three groups (groups 1–3) contain-
ing 6 rats (3 of each sex). Group 1 animals received 5 times of
ED (20 mg/kg bw) i.e. 0.1 g/kg bw. General behavior of rats was
observed for food consumption and behavioral signs such as
excitement, nervousness, dullness, alertness, ataxia or death con-
tinuously for 1 h. Observation was continued intermittently for 4 h
and thereafter over a period of 24 h. Blood parameters for liver and
kidney function tests (total protein, alkaline phosphatase, SGPT,
SGOT, total bilirubin, creatinine and lipid profile) were also esti-
mated pre- and post-treatment. Careful observation of the animals
was continued up to 4 days. Since, there were no deaths or any
adverse effects in the first group with 5 times of ED, the second
step of the experiment was carried out in group 2 animals with 10
times of ED i.e. 0.2 g/kg bw and the animals were observed after
the administration of CND as mentioned above. Since there were
no deaths even in group 2 rats, the third step of the experiment was
carried out in group 3 animals with 20 times of ED (0.4 g/kg bw) as
above.
2.12. Calculation of enzyme activity and definition of units
The activity of all enzymes is expressed as units/g tissue/min.
One unit of enzyme is defined as the enzyme required to oxidize
one micromole of NADH at 25 ◦ C. The following formula was used
to calculate the enzyme activity:
enzyme activity
 OD × total assay volume × dilution of extract
=
amount of the extract × time of incubation × 6.22
where 6.22 is the molar extinction coefficient of NADH at 340 nm
in cm2 /␮M at 25 ◦ C and  OD is the change in the optical density
in the assay mixture.

The amount of GLUT4 in the membrane fraction and total
homogenate was measured by Western blotting (BIO RAD – Trans
Blot, Semidry transfer cell). A 10% SDS-PAGE was run for skeletal
muscle membrane fractions and whole homogenate (25 ␮g each
lane) and the resolved proteins were transferred to polyvinyli-
dene fluoride (PVDF) membrane. After transfer the membrane
was blocked in 2.5% BSA and incubated with a 1:500 dilution
of polyclonal GLUT4 antibody. The antigen antibody complexes
were detected with horse radish peroxidase-conjugate secondary
antibodies using diamino benzidine tetrahydrochloride (DAB) as
a substrate for the colour development reaction. In each case the
Western blot revealed a single band of 45 kDa, compatible with the
molecular weight of the transporter. Quantification was performed
by densitometry after scanning the blots with gel documentation
system (alpha imager). Membrane is further incubated with a pos-
itive control gene i.e. ␤-actin after stripping the GLUT4 antibody.
2.13. Statistical analysis
The results were expressed as mean ± SD. The statistical analysis
involving two groups was performed by means of two-tailed paired
t-test, whereas analysis of variance (ANOVA) followed by Dunnett’s
multiple comparison test were also used. All the data were pro-
cessed with Graph Pad Prism version 4.01 software. p < 0.05 was
considered significant and p < 0.001 was considered more signifi-
cant.
3. Results
3.1. Effect of treatment on general parameters
Changes in various physical parameters like the weight of the
total body, internal organs like liver, kidney and fluid intake were
 P. Anand et al. / Chemico-Biological Interactions 186 (2010) 72–81 75

Table 2
Effect of the purified fraction CND (20 mg/kg bw) and glibenclamide (200 mg/kg bw) given for 60 days to STZ induced diabetic rats on FBG and HbA1c levels.

Group of animals (n = 6) Fasting blood glucose (mg/dl) Glycosylated hemoglobin (% Hb A1c )

Day 0 Day 30 Day 60 Day 0 Day 30 Day 60

Healthy control 73 ± 7.5 84 ± 7.4 75 ± 6.8 4.3 ± 0.23 4.5 ± 0.27 4.4 ± 0.30
Untreated diabetic 282 ± 10.5 323 ± 10.5 396 ± 10.3a 5.1 ± 0.49 8.9 ± 0.53 12.7 ± 0.46b
CND treated 289 ± 9.2 248 ± 6.8 152 ± 7.8c , a 4.8 ± 0.47 7.6 ± 0.36 6.6 ± 0.43c
Glibenclamide treated 294 ± 7.6 253 ± 10.3 164 ± 8.2c , a 4.9 ± 0.5 7.1 ± 0.33 6.4 ± 0.37c

n = number of animals per group; values are presented as mean ± S.D.

a
p < 0.001 when compared with the initial value of the same group.
b
p < 0.001 as compared with the initial value of same group.
c
p < 0.001 when compared with the corresponding value of untreated diabetic control.

studied in normal (healthy controls), STZ induced untreated and end of the experiment. Results in Table 2 show that 2 months
treated (with CND and the standard drug glibenclamide) diabetic administration of CND (20 mg/kg bw) to diabetic animals signif-
rats (Table 1). icantly (p < 0.001) brought down the FBG from the initial value
of 289 ± 9.2 mg/dl by 47.4% to 152 ± 7.8 mg/dl which is slightly
3.1.1. Body weight higher than the fall of 44.2% (p < 0.001) (from 294 ± 7.6 mg/dl to
In STZ induced diabetes there was a significant decrease 164 ± 8.2 mg/dl produced by 200 mg/kg bw of the standard drug
(p < 0.01) of about 37.9% in the body weight (139 ± 12 g) after 60 glibenclamide (group 4). On the other hand in the untreated dia-
days of diabetes induction compared to healthy control group betic animals (group 2), there was an increase of 40.4% (p < 0.001)
(224 ± 16 g). The treatment of diabetic rats with CND and gliben- from 282 ± 10.5 mg/dl to 396 ± 10.3 mg/dl. While glycosylated
clamide restored the body weight to near normal values of hemoglobin (measured as % of HbA1c ) remained more or less
209 ± 10 g and 195 ± 16 g respectively. the same in healthy controls (4.3 ± 0.23–4.4 ± 0.30%), it was sig-
nificantly increased (p < 0.001) in untreated diabetic rats from
3.1.2. Fluid intake 5.1 ± 0.49% to 12.7 ± 0.46%. The elevation of HbA1c was lesser in
The fluid intake of the untreated diabetic rats was signifi- glibenclamide (from 4.9 ± 0.5% to 6.4 ± 0.37%) and CND treated
cantly increased (p < 0.001) by about 4.37 times (175 ± 16 ml/day) (from 4.8 ± 0.47% to 6.6 ± 0.43%) animals than in the untreated
as compared to the control rats (40 ± 3.2 ml/day). The fluid intake group (Table 2). The treated values were brought closer to normal
(polydipsea) was decreased to around 2 times the normal lev- values by the drug treatment.
els after treatment of the diabetic animals for 60 days with CND As is known the initial 0 day serum insulin values of dia-
(74 ± 8 ml/day). betic animals were less (9.6 ± 0.47 ␮U/ml) than the healthy
controls (13.2 ± 0.74 ␮U/ml). In the untreated diabetic rats the
serum insulin values decreased from the initial (0 day) value of
3.1.3. Liver weight
9.6 ± 0.47–5.5 ± 0.62 ␮U/ml (Table 3) on 60th day. In the CND and
The weight of the liver of the untreated diabetic rats
glibenclamide treated groups the serum insulin levels which were
(3.9 ± 0.41 g) was significantly decreased (p < 0.001) by 45.8% after
less on 0 day (9.1 ± 0.48 ␮U/ml and 9.0 ± 0.46 ␮U/ml) increased to
60 days in comparison to the control rats (7.2 ± 0.61 g). Treatment
12.5 ± 0.59 and 11.7 ± 0.65 ␮U/ml respectively on day 60. In the
with antidiabetic compounds (CND and standard drug gliben-
healthy control animals, they remained more or less the same
clamide) for 60 days increased the liver weights to near normal
(13.2–13 ␮U/ml). It is thus evident from the above results in
values of 6.1 ± 0.58 g and 5.4 ± 0.52 g respectively. However, when
Tables 2 and 3 that administration of CND for 60 days significantly
expressed as liver weight/100 g body weight for functional com-
reversed the elevated FBG, HbA1c and decreased serum insulin val-
parison, the value was only slightly less in untreated diabetic
ues of the diabetic animals.
group 2.8 ± 0.02 g when compared to 3.21 ± 0.03 in the normal
animals (p < 0.001). There was an increase of 2.92 ± 0.026 g in the
CND group. But in the glibenclamide treated group it is still less 3.3. Effect of treatment on in vitro release of insulin from
2.76 ± 0.022 g. This indicates that its favorable effect on liver is pancreatic ˇ cells
slight.
The incubation of islets from normal healthy rats with CND
3.1.4. Kidney weight in the presence of 10 mM glucose for 2 h resulted in signifi-
After 60 days the kidney weight of the untreated diabetic rats cant (p < 0.001) stimulation of the release of insulin, which is
increased significantly (p < 0.001) to 1.42 ± 0.13 g (178%) as com- (41.2 ± 1.3 pM/IEQ islets) more than 2-fold when compared with
pared to the healthy control animals (0.51 ± 0.03 g). The increase in
kidney weight was found to be more prominent when expressed as Table 3
kidney weight/100 g bw 1.02 ± 0.06 g in untreated diabetic (343%) Effect of CND (20 mg/kg bw) and glibenclamide (200 mg/kg bw) on serum insulin
when compared to 0.23 ± 0.02 g in controls (p < 0.001). In the CND levels given for 60 days to STZ induced diabetic rats.
and glibenclamide treated groups the kidney weight increased only Group of animals (n = 6) Serum insulin (␮U/ml)
slightly to 0.301 ± 0.02 g (23.5%) and 0.35 ± 0.03 g (52.2%) respec-
Day 0 Day 30 Day 60
tively compared to normal healthy group. Thus treatment with CND
prevented the increase of kidney weight to a significant (p < 0.001) Healthy control 13.2 ± 0.74 12.8 ± 0.83 13.0 ± 0.61
extent. Untreated diabetic 9.6 ± 0.47 7.2 ± 0.54 5.5 ± 0.62a
CND treated 9.1 ± 0.48 11.1 ± 0.83 12.5 ± 0.59b , c
Glibenclamide treated 9.2 ± 0.46 10.4 ± 0.9 11.7 ± 0.65b , a
3.2. Effect of treatment on FBG, HbA1c and serum insulin levels
n = number of animals per group; values are presented as mean ± S.D.
a
p < 0.001 when compared with initial value of same group.
The levels of FBG, HbA1c and serum insulin were monitored b
p < 0.001 as compared with untreated diabetic group of final day.
c
before starting the experiment, at 2 weeks intervals and at the p < 0.01 when compared with the initial value of same group.
76 P. Anand et al. / Chemico-Biological Interactions 186 (2010) 72–81

Table 4
Effect of CND and glibenclamide on in vitro release of insulin from pancreatic ␤ cells
in STZ induced diabetic rat.

Group Concentration of glucose in Insulin release (pM

the incubation medium IEQ−1 2h−1 )
(mmoles/l)

Control 10 18.7 ± 0.8

CND treated 10 41.2 ± 1.3a
Glibenclamide treated 10 39.5 ± 1.1a

Values are presented as mean ± S.D. IEQ – absolute number of islets.

a
p < 0.01 compared to corresponding value of control.

respect to control (18.7 ± 0.8 pM/IEQ islets) and even slightly

greater than glibenclamide treated islets (39.5 ± 1.1 pM/IEQ islets).
These results indicate that CND is acting directly on pancreas and
stimulating the release of insulin from it in the presence of glu-
cose.
It is interesting that the effect of CND was more than gliben-
clamide (Table 4).

3.4. Effect of treatment on glycogen level in tissues

Glycogen contents of the liver and muscle tissues in healthy

control, diabetic control and diabetic treated with CND and gliben-
clamide groups are shown in Table 5. Hepatic and skeletal muscle
glycogen contents were significantly decreased in diabetic rats with
respect to control (p < 0.001). However, treatment with CND led to
an insignificant decrease in liver (17.6%) and muscle glycogen con-
tents (18.07%) over healthy control animals. While a lesser decrease
(25.16% in hepatic glycogen and 26.6% in muscle glycogen) was
observed in the animals treated with glibenclamide. This indicates
that the defective glycogen storage of the diabetic state was par-
tially corrected by the treatment of CND.

3.5. Effect of treatment on pyruvate kinase activity

The activity of total and active pyruvate kinase was significantly

lowered by 18.6 ± 2.0 U/g tissue/min (p < 0.01) and 10.5 ± 0.95 U/g
tissue/min (p < 0.001) respectively in the liver of diabetic rats in
comparison to the normal control values 36.4 ± 4.2 U/g tissue/min
and 23.7 ± 2.5 U/g tissue/min (Fig. 1(a)). Diabetic rats treated with
CND and glibenclamide exhibited 34.5 ± 4.5 U/g tissue/min and
31.7 ± 4.2 U/g tissue/min respectively of total pyruvate kinase
activity to, which is near to normal control values.
A significantly higher activity (p < 0.05) of 29.7% (from
20.5 ± 1.5 U/g tissue/min of normal control to 26.6 ± 2.0 U/g tis-
sue/min of diabetic control) was observed in the active form of
pyruvate kinase in the kidney of diabetic rats (Fig. 2(a)). While CND
treated diabetic animals exhibited almost similar activity as the
control activities. But there was no significant difference observed
in glibenclamide treated animals showing that CND is more effec-
tive than glibenclamide.
Fig. 1. (a) Activity of pyruvate kinase in the liver of control, diabetic and diabetic rats
treated with antidiabetic agents for 60 days. Values are presented as mean ± SD of
four separate experiments; a p < 0.01, d p < 0.001 compared with corresponding val-
ues of healthy control; b p < 0.01, c p < 0.05, e p < 0.001 compared with corresponding
values of untreated diabetic control. (b) Pyruvate kinase and ␤-actin mRNA levels
in the liver of control, untreated diabetic and diabetic rats treated with CND and
glibenclamide for 60 days. M – marker, *C – control, D – diabetic, D + CND diabetic
treated with CND, D + glib – diabetic treated with glibenclamide, *taken as 100%.
3.6. Effect of treatment on phosphoenol pyruvate carboxykinase
(PEPCK) activity
In the liver of diabetic untreated animals a significantly (p < 0.01)
increased activity of 60% was observed (from 1.85 ± 0.03 U/g tis-
sue/min in the healthy controls to 2.97 ± 0.04 U/g tissue/min in
diabetic rats). Treatment of CND and glibenclamide to diabetic

Table 5
Effect of CND and glibenclamide treatment for 60 days on the tissue glycogen levels in STZ induced diabetic rats.

Group of animals Liver tissue (mg/g) % Fall Muscle tissue (mg/g) % Fall

Healthy control 46.5 ± 4.1 – 8.3 ± 0.75 –

Untreated diabetic 17.8 ± 1.87a 61.6 3.06 ± 0.31a 63.1
CND treated 38.3 ± 3.74b 17.6 6.8 ± 0.42b 18.07
Glibenclamide treated 34.8 ± 3.45b 25.16 6.09 ± 0.36b 26.6

Values are presented as mean ± S.D.

a
p < 0.001 compared to the healthy controls.
b
p < 0.01 compared to the diabetic controls.
 P. Anand et al. / Chemico-Biological Interactions 186 (2010) 72–81
Fig. 2. (a) Activity of pyruvate kinase in kidney of control, diabetic and diabetic rats
treated with antidiabetic agents for 60 days. Values are mean ± SD of four separate
experiments; a p < 0.05 compared with the corresponding values of healthy con-
trol animals. (b) Pyruvate kinase and ␤-actin mRNA levels in the kidney of control,
untreated diabetic and diabetic rats treated with the fraction CND and glibenclamide
for 60 days. *C – control, D – diabetic, D + CND – diabetic treated with CND, D + glib
– diabetic treated with glibenclamide, M – marker, *taken as 100%.
rats resulted in good reversal of PEPCK activity to 1.96 ± 0.04
and 2.03 ± 0.03 U/g tissue/min (Fig. 3(a)). But the values were still
slightly above normal.
There was a significant (p < 0.01) increase of 85% in kidney PEPCK
activity of diabetic rats 0.85 ± 0.06 U/g tissue/min in comparison
to the 0.46 ± 0.04 U/g tissue/min of normal controls. Treatment of
diabetic animals with CND and glibenclamide for 60 days prevented
the increase in PEPCK levels. The values of CND treated animals
were 0.50 ± 0.04 U/g tissue/min, which is very close to the normal
value. But the enzyme activity in glibenclamide treated animals
(0.54 ± 0.04 U/g tissue/min) is still slightly higher than the normal
control values (Fig. 4(a)).
3.7. Effect of treatment on mRNA expression of key regulatory
enzyme of glucose metabolism
3.7.1. Effect on pyruvate kinase in liver and kidney tissues
The expression of liver pyruvate kinase (L-PK) mRNA as esti-
mated by semi quantitative RT-PCR, it was significantly (p < 0.01)
decreased by 64% as indicated by band intensity in diabetic rats in
77
Fig. 3. (a) Activity of phosphoenol pyruvate carboxykinase in the liver of control,
untreated diabetic and diabetic rats treated with antidiabetic agents for 60 days.
Values are mean ± SD of four separate experiments; a p < 0.01 compared with the
corresponding values of healthy control animals; b p < 0.05, c p < 0.001 compared with
the corresponding values of diabetic control animals. (b) PEPCK and ␤-actin mRNA
levels in the liver of control, diabetic and diabetic rats treated with CND and gliben-
clamide for 60 days. *C – control, D – diabetic, D + CND – diabetic treated with CND,
D + glib – diabetic treated with glibenclamide, M – marker, *taken as 100%.
comparison to healthy controls, which is taken as 100% (Fig. 1(b)).
Treatment of diabetic rats with CND and glibenclamide resulted in
reversal of L-PK mRNA levels to 95.5% and 89% respectively which
are close to normal values. The changes in L-PK mRNA levels are in
agreement with the changes in pyruvate kinase activity observed
in the liver of diabetic treated and untreated animals.
Treatment of diabetic rats with CND and glibenclamide resulted
in reversal of increased PK mRNA levels to 87% and 90% in kidney
tissues. The changes in kidney PK mRNA levels are in agreement
with the changes in the pyruvate kinase activity observed in the
kidneys of diabetic untreated and treated animals (Fig. 2(b)).
3.7.2. Effect on phosphoenol pyruvate carboxykinase in liver and
kidney tissues
There was a 73% increase in PEPCK mRNA level in untreated dia-
betic liver, which is significantly (p < 0.05) different from the normal
control (Fig. 3(b)). Treatment with CND and glibenclamide for 60
days normalized the PEPCK mRNA levels of treated diabetic ani-
78 P. Anand et al. / Chemico-Biological Interactions 186 (2010) 72–81
Fig. 4. (a) Activity of phosphoenol pyruvate carboxykinase in the kidney of control,
diabetic and diabetic rats treated with antidiabetic agents for 60 days. Values are
mean ± SD of four separate experiments; a p < 0.01 compared with the correspond-
ing values of healthy control animals; b p < 0.001 compared with the corresponding
values of diabetic control animals. (b) PEPCK and ␤-actin mRNA levels in the kidney
of the control, untreated diabetic and diabetic rats treated with CND and gliben-
clamide for 60 days. C – control, D – diabetic, D + CND – diabetic treated with CND,
D + glib – diabetic treated with glibenclamide, M – marker, *taken as 100%.
mals. This is in conformity with changes observed in the activity of
PEPCK in control, untreated diabetic and treated diabetic rats.
Fig. 4(b) showed the level of mRNA encoding PEPCK of kidney
tissue. It was elevated nearly 2-fold in untreated diabetic rats com-
pared with normal rats. This increase of mRNA level in the kidney
of diabetic rats was reduced to near control values after 60 days
treatment with CND and glibenclamide.
3.8. Effect of treatment on translocation of GLUT4 protein
Since in CND treated diabetic rats there was an increase in serum
insulin levels as well as in vitro release from isolated rat pancreatic
islets and because of the physiological importance of insulin-
dependent GLUT4 translocation to the cell membrane, attempts
Fig. 5. GLUT4 levels in the skeletal muscle total protein fraction and (b) mem-
brane fraction of healthy control – C, diabetic – D and diabetic rats treated with
cinnamaldehyde (CND). Each value is the mean ± SD of four separate experiments.
c
p < 0.01 compared to the healthy control; d p < 0.05 compared to the diabetic control.
a
Total protein; b membrane protein.
have been made to see the effect of CND treatment on muscle tis-
sue membrane GLUT4 levels. In the immunoblot of total protein
fractions the density of all the bands was found similar which is
expected. In the membrane fractions of diabetic rats, the translo-
cation of GLUT4 was only about 42.8% when compared with that
of band density of healthy controls. This is quite rational since
the deficiency of insulin in the diabetic state would decrease the
translocation of GLUT4 from the vesicles to cell membranes. Treat-
ment with CND resulted in the reversal of membrane GLUT4 levels,
i.e. after 60 days of treatment GLUT4 levels were restored to 73.1%
in comparison to the healthy controls which was taken as 100%. The
modulation of GLUT4 protein could thus be one of the mechanisms
of antidiabetic properties of CND. GLUT4 levels of normal control,
diabetic and CND treated diabetic rats after 60 days treatment, are
shown in Fig. 5.
3.9. Determination of median lethal dose (LD50 )
In healthy rats, administered 5, 10 and 20 times of effective dose
(ED) of the CND, no behavioral signs such as excitement, nervous-
ness, dullness, alertness, ataxia or death were observed. Values of
serum glucose, total protein, SGOT, SGPT, creatinine, total biliru-
bin, alkaline phosphatase and lipid profile remained in the normal
range (Tables 6 and 7) throughout the study (data not shown with
5 times of ED). In 20 times of ED administered animals after 72 h
(3 days), a slight but insignificant increase was observed in SGOT,
SGPT and ALKP values, which however returned to their initial val-
ues within 120 h i.e. 5 days of CND administration. Thus CND seems
to have no toxic effects even with 20 times of ED and its margin of
safety is much higher as is already reported by others [29]. Food
consumption was also normal in all animals treated with all the
three doses (data not shown).
 P. Anand et al. / Chemico-Biological Interactions 186 (2010) 72–81 79

Table 6
Effect of 10 times effective dose of CND in normal healthy rats.

Blood parameters Before fraction CND administration After CND administration

24 h 72 h

Glucose (mg/dl) 79 ± 8.6 75.7 ± 6.2 74 ± 8.7

Total protein (g/l) 6.5 ± 0.3 7.0 ± 0.4 6.7 ± 0.7
SGPT (U/L) 16.6 ± 4.3 17.4 ± 5.1 15.8 ± 4.1
SGOT (U/L) 27.6. ± 4.2 34.3 ± 4.7 30.7 ± 3.4
ALKP (U/L) 67.5 ± 5.9 74.2 ± 6.6 72.5 ± 6.0
Creatinine (U/L) 0.38 ± 0.03 0.49 ± 0.02 0.52 ± 0.04
Bilurubin (mg/dl) 0.20 ± 0.02 0.17 ± 0.03 0.24 ± 0.03
TC (mg/dl) 85.9 ± 5.4 84.2 ± 6.4 87.7 ± 6.1
TG (mg/dl) 81.1 ± 5.7 77.3 ± 3.4 78.6 ± 6.5
HDLC (mg/dl) 23.7 ± 4.0 25.2 ± 3.4 24..4 ± 4.2

Values are presented as mean ± S.D; 6 animals were used in both the groups. All the values are with in the normal range.

4. Discussion Glycogen is the primary intracellular storable form of glucose

Diabetes is a chronic metabolic disorder affecting a major pro-
portion of the population worldwide. A sustained reduction in
hyperglycemia will decrease the risk of developing microvascu-
lar diseases and reduce their complications [30]. The conventional
therapies for diabetes have many shortcomings like side effects and
high rate of secondary failure. On the other hand herbal drugs are
expected to have similar efficacy without side effects as that of
conventional drugs. Present study is focused to explore the mode
of antidiabetic activity and margin of safety of one such compound
cinnamaldehyde in STZ induced diabetic rats.
The bioassay guided fractionation of chloroform extract of CZ
yielded an active compound, CND, which reduced the glucose levels
by 47.1% in GTT (glucose tolerance test) in STZ induced experimen-
tal animals. CND not only reduced blood glucose levels by 47.4%
in long term treated animals it has also reduced the increase in
HbA1c . It also showed increase in serum insulin levels by 37.36%
compared to its initial value while in untreated group insulin levels
were decreased by 42.7%. These experiments were repeated with
commercially available cinnamaldehyde.
Earlier studies by Subash Babu et al. [12] and our group demon-
strated that 45 days treatment of CND (purified and isolated from
CZ extract) increases serum insulin levels in STZ induced diabetic
rats. Further, to understand how CND ameliorates hyperglycemia,
in vitro insulin secretion studies were carried out using isolated rat
primary islet culture. This study suggests that CND acts by enhanc-
ing release of insulin through ␤-cells stimulation [23,31,32] in the
similar manner to sulphonylureas [33] (Table 1) i.e.; it exerts a
direct insulin releasing effect. The enhancement of insulin secretion
by the CND correlates with the decreased blood glucose whereas
increased circulating insulin level can be attributed to the stimula-
tion of ␤-cells by CND which in turn exerts an antihyperglycemic
action.
and its level in various tissues especially in liver and skeletal mus-
cles indicate direct reflection of insulin activity since it regulates
glycogen deposition by stimulating glycogen synthase and inhibit-
ing glycogen phosphorylase [34–36]. Since STZ causes selective
destruction of ␤-cells of islets of Langerhans resulting in marked
decrease in insulin levels, it could be predicted that glycogen levels
in tissues (muscle and liver) decrease as the influx of glucose in the
liver is inhibited in the absence of insulin and recovers on insulin
treatment [37–39]. Our results showed that upon supplementation
of diabetic rats with CND there is significant elevation in both mus-
cle and hepatic glycogen content. Restoration of glycogen content
in CND treated group was higher than the glibenclamide treated
group.
Further, in diabetic rats CND was also found to improve PK
and PEPCK enzyme levels, two major enzymes involved in glucose
homeostasis. On treatment with CND, the levels of pyruvate kinase
and phosphoenolpyruvate carboxykinase in liver tissues increased
and decreased respectively, almost to the level of control animals.
This result is similar to that of earlier work, where several potential
herbal plant extract and purified compounds have been shown to
improve the diabetic condition [40,41]. The PK activity decreases
as the result of diabetes and increases by the administration of
insulin to diabetic rats in the liver tissues [42]. The altered activ-
ity during diabetic conditions could be expected to diminish the
metabolism of glucose and ATP production. Hence, the observed
decline in the activity of PK in the liver tissue of streptozotocin
induced diabetic rats is promptly responsible for the reduced gly-
colysis and amplified gluconeogenesis signifying that these two
pathways are distorted in diabetes [43]. But in the case of insulin
independent tissues like kidney and lens [44]) increase in pyru-
vate kinase activity as well as mRNA levels can be attributed to
the increased flux of glucose. In these tissues glucose uptake is
concentration dependent and not mediated by transporters and

Table 7
Effect of 20 times effective dose of CND in normal healthy rats.

Serum parameters Before CND administration After CND administration

24 h 72 h

Glucose (mg/dl) 75.8 ± 4.5 70.7 ± 7.0 78.1 ± 6.9

Total protein (g/l) 6.9 ± 0.4 7.3 ± 0.6 7.1 ± 0.3
SGPT (U/L) 27.3 ± 2.4 23.2 ± 3.0 25.3 ± 3.6
SGOT (U/L) 28.8 ± 3.4 34.3 ± 4.1 37.7. ± 3.8
ALKP (U/L) 81.3 ± 7.1 85.3 ± 6.5 84.5 ± 7.3
Creatinine (U/L) 0.46 ± 0.03 0.57 ± 0.05 0.60 ± 0.03
Bilurubin (mg/dl) 0.20 ± 0.02 0.28 ± 0.04 0.31 ± 0.03
TC (mg/dl) 78.9 ± 6.2 75.3 ± 4.3 81.8 ± 6.1
TG (mg/dl) 65.6 ± 5.2 68.2 ± 5.5 70.2 ± 6.8
HDLC (mg/dl) 26.6 ± 3.2 30.3 ± 2.7 33.4 ± 3.6

Values are presented as mean ± SD; 6 animals were used in both the groups. All the values are with in the normal range.
80 P. Anand et al. / Chemico-Biological Interactions 186 (2010) 72–81
insulin as in case of insulin-dependent tissues. Therefore glucose
overutilization occurs in these tissues [45]). The increased activity
of glycolytic enzyme, here pyruvate kinase highlights the over uti-
lization of glucose through glycolytic pathway which was observed
in present study. The treatment of CND to diabetic rats showed a
notable increase in plasma insulin that induces a decrease in ATP,
a known allosteric inhibitor of PK, thereby increasing the PK activ-
ity to near normalcy. Treatment of diabetic rats with CND resulted
in reversal of altered PK activity and its expression in 2 months
treatment (Figs. 1(b) and 2(b)). PEPCK, which catalyzes a regulatory
step in gluconeogenesis, is one of the key enzymes of hepatic car-
bohydrate metabolism and insulin deficiency is clearly associated
with changes in hepatic metabolism including increased expres-
sion of PEPCK [27]. In our study, CND treatment reversed increase
of hepatic PEPCK mRNA expression in STZ diabetic rats and this
attenuation of the hepatic PEPCK mRNA expression was associated
with plasma glucose-lowering activity of CND. Our findings sug-
gest that CND exerts its glucose-lowering effect mainly through an
enhancement of glucose utilization of skeletal muscle and a reduc-
tion of hepatic gluconeogenesis. CND treatment normalized PEPCK
activity as well as the mRNA expression both in kidney and liver
tissues and the effect produced was equal to that of standard drug
(Figs. 3(a and b) and 4(a and b)).
A key role of insulin is to facilitate the uptake of glucose from
blood into muscle and adipose tissue [46,47]. In muscle tissue,
two glucose transporter isoforms GLUT1 and GLUT4 are expressed.
The GLUT4 isoform is quantitatively more abundant in adult rat
muscle and is distributed among intracellular compartments in
the basal state, from where it rapidly translocates to the plasma
membrane in response to insulin or excercise [47–49]]. GLUT1 is
considered to be responsible for basal glucose transport. GLUT4
expression is downregulated when there is relative insulin defi-
ciency, such as in STZ induced diabetes [50]. GLUT4 protein levels
were measured by immunoblot analysis in the whole homogenate
and membrane fraction of skeletal muscle. The GLUT4 protein in the
plasma membranes of skeletal muscle tissue was decreased in dia-
betic control group (Fig. 5). After treatment with CND, the contents
of GLUT4 protein were restored to near normal values in skeletal
muscle membrane fractions and prevented the insulin resistance
and hyperglycemia like pharmacological therapeutic approach of
PPAR ␥ [51,52]. The decrease in GLUT4 levels is essentially as one
of the main reasons of hyperglycemia in the diabetic state, which is
due to decreased uptake of glucose by the skeletal muscle. Restora-
tion of GLUT4 levels would therefore, enhance the uptake of glucose
in the skeletal muscle and thus help to combat hyperglycemic con-
ditions.
The median lethal dose (LD50 ) gives a measure of the immediate
or acute toxicity of a chemical in a particular animal species being
tested and also gives an idea of margin of safety. The basic idea of
this test is to take healthy animals and force feed them enough drug
to kill approximately 50% of them. The LD50 test is expected to give
an idea at what dose CND produces side effects if any and at what
dose 50% of the animals will be killed. LD50 studies of CND seem
to have no toxic effects even with the administration of 20 times
of effective dose suggesting its margin of safety is much higher
(Tables 6 and 7). Food consumption (results are not shown) was
also normal in all animals treated with all the doses (5, 10 and 20
times of ED).
4.1. Conclusion
Our study clearly suggests that CND is a potent antidiabetic
agent. In vitro insulin release assay revealed that it increases the
insulin release at increased glucose concentrations, which is sim-
ilar to the glucose levels in diabetic state. It is an evidence for the
compound acting directly on pancreatic islets. Long term effects
of CND on key regulatory enzymes of carbohydrate metabolism in
diabetic state showed the restoration in altered enzyme activities
and their mRNA expressions. CND had produced similar antihyper-
glycemic effects at 20 mg/kg bw which glibenclamide produced at
200 mg/kg bw. It had also been shown to increase the translocation
of glucose transporter protein GLUT4, which stimulates glucose
transport across the membranes in skeletal muscle tissues in com-
parison to the untreated diabetic animals. Liver and kidney function
tests (SGPT, SGOT, ALKP, creatinine, bilirubin) estimated of treated
animals serum revealed no adverse effect even at higher doses (5,
10 and 20 times of ED) which confirmed its higher margin of safety.
Hence, CND can be developed as a new drug entity for diabetes how-
ever extensive investigations are necessary to establish the use of
CND in human subjects.
Conflict of interest
The authors declare that there are no conflicts of interest asso-
ciated with this manuscript.
Acknowledgements
The authors acknowledge the financial support from Univer-
sity Grant Commission. P. Anand and M.K. Yanda are recipients of
senior research fellowship from UGC and ICMR, respectively. We
are also grateful to S. Cushman, National Institute of Health, USA
for providing antiGLUT4 sera.
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