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Research Paper
May-June 2018 Indian Journal of Pharmaceutical Sciences 453
Metabolic syndrome (MS) is a conglomerate of
interconnected disorders[1]. Almost 25 % of the adults
worldwide suffer from MS. It increases the risk of
type 2 diabetes, heart attack and stroke in developing
nations[2]. More than 17 % of the European population
in the age group of 44-60 y suffer from MS and 80 %
of them has elevated blood pressure[3]. MS is one of
the commonest lifestyle problems in different parts of
Asia that include China and India[4]. Among Indians,
MS are predominantly common in women due to
high carbohydrate intake especially in Asian Indian
phenotype[5,6]. Late complications of MS coexist with
various pathological complications like neuropathy,
colorectal cancer, menstrual disturbances and arthritis
thus broadening the co-morbidities[7]. Antioxidant-
rich diet especially food containing cinnamon and
hydroxycinnamic acids has been shown to prevent
MS-associated cardiovascular complications[8]. MS
has also been associated with CNS disorders like
anxiety and depression[9]. Anxiety disorders have been
independently associated in MS population[10]. On the
other hand, higher evening cortisol levels are common
in patients suffering from a major depressive disorder.
Bardet-Biedl syndrome (BBS), a condition similar to
MS, is associated with obesity, hyperlipidaemia, liver
dysfunction as well as elevated insulin and leptin[11].
BBS individuals have been shown to be more prone
to anxiety disorders. Obesity, induced by carbohydrate
diet followed by induction of stress may promote the
development of anxiety-like behaviour compared to
Effect of Metabolic Syndrome on Anxiety in Mice
A. MUKHERJEE AND S. BANERJEE1*
Department of Pharmacology, Gupta College of Technological Sciences, Asansol-713 301, 1Department of Pharmaceutical
Sciences and Technology, Birla Institute of Technology, Mesra, Ranchi-835 215, India
Mukherjee and Banerjee: Metabolic Syndrome Associated Anxiety
Metabolic syndrome is a combination of obesity, dyslipidemia, insulin resistance and hypertension. Clinical
evidence indicated the coexistence of metabolic syndrome and depression. However, relatively few studies
have been attempted to determine the pathogenesis of metabolic syndrome-associated anxiety. In the present
study, the role of metabolic syndrome in the development of anxiety and the role of neurotransmitters in
metabolic syndrome-associated anxiety were evaluated in Swiss albino mice. A high-fat and high-carbohydrate
diet was used to develop the metabolic syndrome in mice while monitoring elevated fasting blood glucose,
hyperlipidaemia and hypertension. Anxiety levels were measured using elevated plus maze and marble
burying test with corresponding determination of serum corticosterone levels. The role of γ-aminobutyric
acid and serotonin in metabolic syndrome-associated anxiety were also evaluated. The high-fat and high-
carbohydrate fed animals developed metabolic syndrome, characterized by signicant high fasting blood
glucose and insulin resistance compared to controls. These animals also had signicant increase in body
weight and waist circumference, low-density lipoprotein and triglyceride levels, reduced high-density
lipoprotein content and high blood pressure. Both metabolic syndrome and water avoidance-anxiety groups
spent signicantly lower time and showed fewer entries into the open arm of elevated plus maze. They also
buried more marbles, thus showing clear signs of anxiety when compared to controls. The corticosterone
level in metabolic syndrome and anxiety-induced animals were higher than controls. GABA agonists showed
a dose-dependent reduction in metabolic syndrome-associated anxiety as revealed by more time spent on the
open arm of plus maze with a corresponding decrease in plasma corticosterone levels. Metabolic syndrome
animals spontaneously developed anxiety-like behaviour. GABA agonists partially reversed the metabolic
syndrome-associated anxiety, suggesting a role for GABAergic pathway.
Key words: Anxiety, GABA, metabolic syndrome, obesity, type 2 diabetes
*Address for correspondence
E-mail: sbanerjee@bitmesra.ac.in
This is an open access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-ShareAlike 3.0 License, which
allows others to remix, tweak, and build upon the work non-commercially,
as long as the author is credited and the new creations are licensed under
the identical terms
Accepted 19 March 2018
Revised 05 August 2017
Received 20 February 2017
Indian J Pharm Sci 2018;80(3):453-459
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May-June 2018
Indian Journal of Pharmaceutical Sciences
454
animals on normal diet[12]. A transgenic animal model
of obesity and diabetes, the db/db mice made by a point
mutation of leptin receptor gene also showed anxiety-
like behaviour[13].
Though there is signicant clinical evidence of
the coexistence of depression, MS and established
common therapeutic objective to treat anxiety and
depression associated with MS, there are relatively
few animal studies trying to determine the pathways
linking MS and anxiety. Obesity or type 2 diabetes has
been shown to have a bidirectional link with depressive
disorder[14]. However, whether metabolic disorders
may produce anxiety or depression and the effect of
antidepressant therapy on the later conditions is less
well-characterized.
Many transgenic and drug or diet-induced MS
models have been established and are in practice,
but a correlation model of anxiety and the metabolic
association is less established. In this study, we evaluated
the relationship between different behavioural and
biochemical aspects of anxiety in association with
diet-induced MS on Swiss albino mice. We also
determined the primary neurotransmitters responsible
for MS-associated anxiety in these animals
MATERIAL AND METHODS
Normal pellets, maize starch, lard, casein, vitamins and
mineral mixture and sodium chloride were purchased
from VRK nutritional solutions, Pune, India. Casein
(Charotar Casein Company, Vadodara, India). Fructose
(Loba Chemie Pvt. Ltd, Mumbai India), diazepam and
gabapentin (Sun Pharma Industries Ltd, Mumbai India)
phenobarbitone (Piramal Health Care, Mumbai, India)
hydroxyzine (Uni-med India, Mumbai, India) and
uoxetine (Cipla Ltd, Mumbai, India) were procured.
Triglyceride (TG), total cholesterol (TC) and high
density lipid (HDL) were measured using respective
kits (Coral Clinical system, Goa, India).
Experimental animals:
Male Swiss albino outbred mice (18-22 g) were used for
the study. Initially, the animals were housed in colony
cages and maintained under standard environmental
conditions at 25°, 12:12 h light:dark cycle, and
50-55 % relative humidity, with free access to food
and water ad libitum. The Institutional Animal Ethical
Committee approved the protocol (GCTS/IAEC/2013-
SEPT/08) for the study. Groups (6 animals/group):
group-1, control group were fed with standard diet kept
in normal condition; group-2, anxiety group induced
by water avoidance test; group-3, MS group on high-
fat diet and supplemented with 20 % fructose solution
for 4 w (Table 1).
Ingredients for the high-fat diets were mixed, formed
into a dough with water, rolled into pellets, wrapped
with plastic wrap, and stored at –20° until use to
minimize oxidation. These small pellets were given
to mice every day. Drinking water was supplemented
with 20 % fructose solution as the source of high-
carbohydrate in the diet.
Modied water avoidance test:
Apparatus consisted of a plexiglass tank (45×25×
25 cm) with a block (6×4.5×5 cm) at the centre of the
oor. The tank was lled with water at 25° covering up
to 1 cm of the central block. Mice were placed on the
block for 1 h daily in the daytime for three consecutive
days keeping at least 18 h interval[15]. All mice were
assessed for measuring anxiety using elevated plus
maze (EPM). Food intake/mouse/day, the weight of
the mice and waist circumference were calculated and
lipid prole, oral glucose tolerance, fasting blood sugar,
brain corticosterone, blood pressure were measured.
The high-fat and high-carbohydrate (HFHC)-fed mice
showing ≥30 g body weight, fasting blood glucose of
140 mg/dl and systolic blood pressure of 130 mm Hg
were considered as MS.
In the next set of experiment, mice were fed with high-
fat diet and 20 % fructose water. After 4 w MS mice
were divided into 11 groups of six animals each. One
group was kept drug-untreated. Rest of the 10 groups
were treated with the following compounds: diazepam
(0.5 and 1 mg/kg)[16,17]; phenobarbitone (20 and 40 mg/
kg)[18]; gabapentin (10 and 30 mg/kg)[19]; hydroxyzine
(5 and 10 mg/kg)[20]; uoxetine (5 and 10 mg/kg)[21]
successively just before the behavioural and brain
corticosterone measurement. Changes in food intake,
weight of the animal, waist circumference, systolic
blood pressure, fasting blood sugar, oral glucose
tolerance, and lipid prole were assessed for animals.
Fasting blood glucose and glucose tolerance test:
Mice were fasted for 6 h before oral glucose tolerance
test (OGTT). At 0 h fasting blood glucose was measured
by tail prick method using one touch glucose strips.
Oral gavage of glucose solution 2 g/kg (40 % aqueous
solution) was given. After glucose administration,
glucose levels were measured at 30, 60, 90 and 120
min[22].
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Indian Journal of Pharmaceutical Sciences 455
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Lipid prole:
After eight weeks, the mice were fasted overnight and
anesthetized using ketamine and cardiac puncture-
collected blood samples were used to measure TC,
HDL and TG in plasma. Low-density lipid (LDL) was
calculated using the Eqn., LDL = TC–HDL–TG/5.0
(mg/dl)[23].
Measurement of blood pressure:
Non-invasive blood pressure system (IITC Life
Sciences, USA) was used to measure systolic and
diastolic blood pressure. Mice were placed in a clear
plastic restrainer tube with black nose cone and tail hole
pieces at either end. Mice were introduced in the tubes
and placed on a platform heated to 35° and allowed to
warm for 5-15 min before the experiment. Mice were
habituated to the procedure with 10-30 preliminary
cycles; after that data measurements were recorded
for each mouse. Measurements were performed at the
same time each day between 9 am to 2 pm in a closed
room to avoid noise.
Plasma corticosterone assessment:
Blood was collected retro-orbitally from all the groups
of mice at 5.30 pm in the same room under similar
condition. Corticosterone was estimated in plasma
samples using UV/Vis spectrophotometer (UV/Vis-
NIR spectrophotometer Agilent) using the procedure
of Bartos and Pesez[24].
EPM test:
EPM is made up of two closed and two open arms
connected to a central platform. Mice were placed
individually in the centre of the EPM, head facing open
arm. The time spent in both the open and closed arms
were recorded for 5 min. The numbers of entries into
the open and closed arms were also counted during
the test. An entry was dened as having all four paws
within the arms. Data obtained from the experiment
were expressed as mean±SEM[25].
Marble burying test:
Mice were placed in separate cages containing 20
glass marbles (10 mm diameter) evenly spaced on saw
dust. After 30 min exposure to the marbles, mice were
removed. The number of marbles buried (>50 % under
saw dust) was determined.
RESULTS AND DISCUSSION
HFHC diet signicantly increased the body weight and
waist circumference (p<0.001) of the animals compared
to controls indicating the development of central
obesity. Initially, the food intake was normal for all the
animals, which increased signicantly after four weeks
in case of HFHC diet animals. Biochemical parameters
associated with MS including fasting blood glucose
levels were found to be signicantly high (p<0.001)
after two weeks in HFHC diet (Table 2). OGTT for mice
was also assessed to conrm the insulin resistance. The
area under the curve for elevated blood glucose was
much higher for HFHC animals when compared to
healthy controls (g. 1) suggesting glucose tolerance
an indication of insulin resistance[26]. Elevated systolic
blood pressures, a diagnostic characteristic for MS[27]
were measured after 4 w of HFHC diet. Systolic blood
pressure in HFHC group mice was signicantly higher
(p<0.001) compare to control animals. Lipid prole of
HFHC mice was measured, which also been reported
in MS[28]. HFHC diet led to signicant increase in LDL
and TG and reduction in HDL levels compared to
mice receiving normal pelleted diet (Table 2). Hence,
it could be concluded that Swiss albino mice on high
fat and 20 % fructose water developed signs of MS
including high fasting blood glucose, glucose tolerance,
hyperlipidaemia including hypertriglyceridemia and
hypertension all suggestive of MS development.
To access anxiety levels in HFHC diet-fed mice,
EPM test was used. Water avoidance was used to
induce anxiety, thus serving as anxiety controls.
Mice undergoing the water avoidance stress spent the
lowest time in the open arm, which was comparable
with HFHC group mice. Both these groups spent
signicantly lower (p<0.001) time and showed fewer
number of entries (p<0.001) in the open arm as
compared to control group (g. 2). Mice undergoing
water avoidance stress and on HFHC diet buried
signicantly more marbles (p<0.001) when compared
to mice on normal chow (g. 2). Both the above
observations suggest the development of anxiety in
HFHC group mice, which developed MS. Plasma
corticosterone substantiated the behavioural studies.
Plasma corticosterone levels were measured for all
three groups of mice. While, water avoidance stress
Component g/kg of diet
Maize starch 367
Lard 316
Cassien 255
Vitamin and mineral mixture 61
Sodium chloride 1
TABLE 1: INGREDIENTS FOR THE HIGH-FAT DIET
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Indian Journal of Pharmaceutical Sciences
456
mice showed higher plasma corticosterone levels,
the corticosterone level in HFHC group mice was
comparable with both the groups showing signicantly
higher plasma (p<0.001) corticosterone levels when
compared to control animals (g. 2).
Primary neurotransmitters involved in MS-
associated anxiety were determined using
various pharmacological agents by EPM test and
corresponding plasma corticosterone levels. Drugs
acting as gamma-aminobutyric acid (GABAA) agonists
including diazepam, phenobarbitone and gabapentin
all showed (p<0.001) dose-dependent recovery from
MS-associated anxiety as evidenced by more time
spent on the open arm of the EPM and a corresponding
decrease (p<0.001) in plasma corticosterone levels
(g. 3A and B). However, serotonin modulators like
uoxetine and hydroxyzine failed to reverse the MS-
associated anxiety and plasma corticosterone levels in
HFHC-fed animals. The above results suggested that
MS may result in anxiety disorder in mice, which may
be GABAA receptor mediated.
350
300
250
200
150
100
50
0
0 30 60 90 120
min
Blood glucose (mg/dI)
Fig 1: Development of glucose tolerance by HFHC diet
▬▲▬ Concentration of glucose; ▬○▬ HFFC, all values are
expressed as mean±SEM for n= 6
40
30
20
10
0
80
60
40
20
0
20
15
10
5
0
C Ai MS D
a b
cd
Time (sec)
Number
Plasma Corticosterone
(µg/100ml)
6
4
2
0
C Ai MS D
C Ai MS C Ai MS
number of entries
(1 mg/kg)
Fig 2: Development of anxiety in MS animals
(a) Time spent in the open arm of elevated plus maze by control
(C), metabolic syndrome (MS) and anxiety-induced (Ai) groups
and diazepam (D). (b) Number of entries into the open arm of
elevated plus maze by control, MS and anxiety-induced groups.
(c) Number of marbles buried by control, MS and anxiety-
induced groups. (d) Plasma corticosterone levels in control,
MS and anxiety-induced groups. All values are expressed as
mean±SEM for n= 6, ***p<0.001
75
60
45
30
15
0
a
80
60
40
20
0
C Ai MS D (0.5) D (1) Pb (20) Pb (40) Gp (10) Gp (30) Hx (5) Fx (5)
C Ai MS D (0.5) D (1) Pb (20) Pb (40) Gp (10) Gp (30) Hx (5) Fx (5)
Time (sec)
Plasma Coticosterone
µg/100ml
b
Fig. 3: Effect of antianxiety drugs in MS associated anxiety
(a) Effect of control (C) diazepam (0.5 mg/kg: D0.5 and 1 mg/
kg: D1), phenobarbitone (20 mg/kg: Pb20 and 40 mg/kg: Pb40),
gabapentin (10 mg/kg: Gb10 and 30 mg/kg: Gb30), hydroxyzine
(5 mg/kg: Hx5) and uoxetine (5 mg/kg: Fx5) on time spent in
the open arm of elevated plus maze in MS animals. (b) Effect
of diazepam, phenobarbitone, gabapentin, hydroxyzine and
uoxetine on plasma corticosterone levels of MS animals. All
values are expressed as mean±SEM for n= 6, ***p<0.001
Control High-fat and high-
carbohydrate
Fasting blood
glucose 115.5±2.33 mg/dl 264.8±2.72 mg/dl***
LDL 31.00±0.58 mg/dl 85.33±4.41 mg/dl***
HDL 53.67±1.86 mg/dl 42.67±2.33 mg/dl
Triglyceride 107.0±2.65 mg/dl 142.3±3.53 mg/dl
Body weight 22.0±1.15 g 32.3±0.88 g
Waist
circumference 7.47±0.06 cm 8.87±0.19 cm***
Systolic blood
pressure 113.3±2.19 mm Hg 132.7±1.2 mm Hg
TABLE 2: EFFECT OF HFHC DIET ON
DEVELOPMENT OF MS IN MICE
All values are expressed as mean±SEM; ***p<0.001
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Indian Journal of Pharmaceutical Sciences 457
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mutants showed hyper excitable brain primarily at the
amygdala and hippocampal regions. These animals
also showed increased levels of anxiety, while GAD65
decient animals were found to be less prone to external
stress[36]. Restraint stress has also shown reduced
GABAA currents in the amygdala resulting in excessive
amygdalar excitability[37]. This stress model might
also decrease levels of the cortical and hippocampal
GABAA receptor, thus modulating the functions of HPA
axis[38]. Studies on human post traumatic stress disorder
(combat post traumatic stress disorder patients) using
magnetic resonance have shown abnormalities
in cortical or parito-occipital-temporal cortical
GABA[39-41]. In separation anxiety models (maternal
separation; MS) role of GABA has also been
documented. MS might enhance tonic GABA currents
promoting subsequent neurogenesis and differentiation
of GABA neurons during adulthood[42]. MS during
breastfeeding affects the expression and function of one
of the GABAA subunits in a gender specic manner[43].
This study demonstrated that diazepam, phenobarbitone
and gabapentin, which were all involved in activation
of GABAA receptors could dose-dependently reverse
anxiety-like behaviour in MS induced mice. The above
observations suggested the role of GABA pathway in
MS-associated anxiety in these animals. However, the
brain GABA levels remained unaltered in MS animals
when compared to control animals. Hydroxyzine and
uoxetine, which act on the serotonergic system were
unable to reverse the MS-associated anxiety disorders
suggesting less involvement of serotonergic pathways
in the above process.
In the current study, a cost-effective MS model was
successfully developed using high-fat diet and 20 %
fructose water in Swiss albino mice. These mice also
exhibited anxiety-like behaviour with elevated plasma
corticosterone levels. GABAA agonists partially
reversed the MS-induced anxiety thus suggesting the
predominant role of the GABAergic pathways in the
above process.
Acknowledgement:
Authors express their gratitude to Prof Late Debesh
Chandra Majumdar, Chairman, Trinity trust, Dr.
Kalyan K. Sen, principal and all the faculty members
of Gupta College of Technological Sciences Asansol,
for their constant support and encouragement. Authors
also thank the Department of Pharmaceutical Sciences
and Technology, BIT, Mesra for its support.
In the present study, MS was successfully induced
using HFHC diet in Swiss albino mice, which was
characterized by elevated fasting blood glucose levels,
insulin resistance, hyperlipidaemia, and hypertension.
Diet-induced model of MS appear to have various
advantages over genetic models. The MS in human is
a multifactorial disease, which in most cases does not
involve mutations in the leptin gene a standard feature
in most transgenic animals. Thus the human form of MS
is different from the genetic mice models of obesity and
MS[29]. The diet-induced models of MS including high-
fat or high-carbohydrate diet or their combinations
were shown to correlate better with the human form of
the disease. However, diet-induced murine models of
MS mostly involve various strains of rat or C57bl/6j
mice[29]. In this investigation high-fat diet was used
along with 20 % fructose water for four weeks to
induce MS for the rst time in Swiss albino mice; the
most commonly used outbred laboratory mice. Mice
developed distinguishing characteristics of human MS
including high fasting blood glucose levels and glucose
tolerance, high cholesterol and TG levels, increased
central obesity, body weight, and hypertension. Animals
on high-carbohydrate diet have been shown to be more
prone to anxiety[12], while db/db obese mice have
shown greater anxiety-like behaviour compared to their
db+ counterparts. Anxiety-like behaviour attributes to
neuroinammatory changes with increased levels of
cytokines including interleukin-1β, tumor necrosis
factor-α and interleukin-6 and a parallel reduction in
hippocampal brain-derived neurotrophic factor[30,31].
Other studies on Bbs4-null mice, with a mutation that
also leads to obesity with elevated insulin and leptin
levels similar to MS animals, showed anxiety-related
responses and reduced social dominance[32]. While
a recent study also demonstrated that high-fat diet-
induced diabetic mice showed depressive behaviour
with impaired 5-HT function[33]. In the present study,
diet induced-MS-associated increase in anxiety could
be demonstrated as measured by less time spent in the
open arm of plus maze and number of marbles buried.
These behavioural parameters could be correlated with
increased plasma corticosterone levels; an established
biomarker increased during stress in rodents. Induction
of anxiety in neonatal rats might result in upregulation
of genes related to serotonin (5-HT) and GABA[34] in
amygdala region. Inescapable foot shock has also been
reported to cause hippocampal cell damage along with
glutamate/GABA imbalance[35]. The GAD65 enzyme
is critically involved in regulation of GABA. GAD65
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Financial support and sponsorship:
Nil.
Conict of interest:
The authors declare that this paper content has no
conict of interests.
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