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YHBEH-03993; No. of pages: 11; 4C: Hormones and Behavior xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Hormones and Behavior

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Sita Sharan Patel a,c, Vineet Mehta a, Harish Changotra b, Udayabanu Malairaman a,⁎

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Article history: Received 11 June 2015 Revised 5 September 2015 Accepted 25 November 2015 Available online xxxx

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Department of Pharmacy, Jaypee University of Information Technology, Waknaghat, Himachal Pradesh, India Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Himachal Pradesh, India Department of Pharmacology, Lakshmi Narain College of Pharmacy, Bhopal, Madhya Pradesh, India

a b s t r a c t

Comorbidity of depression and diabetes is a serious risk factor worsening the complications such as cognitive function and locomotion. Treatment under this condition becomes extremely complicated. Insulin signaling and autophagy pathways are involved in modulation of learning and memory. Rosiglitazone (ROSI) ameliorate cognitive deficit associated with depression and insulin resistance. In the present study, we investigated the effect of ROSI against chronic unpredictable stress (CUS) induced depression as a risk factor for diabetes and behavioral dysfunctions. Adult male Swiss albino mice were exposed to CUS alongside ROSI (5 mg/kg/day) treatment for 21 days. Thereafter, animals were subjected to different behavioral studies to assess depressive like behavior, cognition and locomotion. The effect of ROSI on insulin signaling, autophagy and apoptosis were evaluated in the hippocampus. CUS resulted in depressive like behavior, cognitive impairment and hypolocomotion associated with oxidative stress, impaired glucose tolerance and hypercorticosteronemia. CUS significantly impaired hippocampal insulin signaling, membrane translocation of glucose transporter type 4 (GLUT4) as well as decreased the expression of autophagy5, autophagy7, B-cell lymphoma 2 and apoptosis inhibitory protein 2. ROSI significantly reduced depressive like behavior, postprandial blood glucose, hypercorticosteronemia, oxidative and inflammatory stress, and apoptosis in stressed mice. Moreover, ROSI treatment effectively improved hippocampal insulin signaling, GLUT4 membrane translocation and cognitive performance in depressed mice. ROSI administration might prove to be effective for neurological disorders associated with depressive like behavior and impaired glucose tolerance. © 2015 Published by Elsevier Inc.

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Depression mediates impaired glucose tolerance and cognitive dysfunction: A neuromodulatory role of rosiglitazone

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journal homepage: www.elsevier.com/locate/yhbeh

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Keywords: Chronic stress Cognition Depression Glucose tolerance Rosiglitazone

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Introduction

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The chronic unpredictable stress (CUS) model is widely used for the induction of depressive like behavior in rodents, which consists of repeated exposure to an array of unpredictable stressors over a sustained period of time (Katz, 1982; Kessler et al., 1985). Repeated stressors are associated with hyperactivation of hypothalamic–pituitary–adrenal axis (HPA axis) which is known to induce neurodegeneration,

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Abbreviations: AIP2, apoptosis inhibitory protein 2; BCL2, B-cell lymphoma 2; CUS, chronic unpredictable stress; FST, forced swim test; GLP1, glucagon like peptide 1; GLUT4, glucose transporter type 4; HPA axis, hypothalamic–pituitary–adrenal axis; INSG1, insulin induced gene 1; ILGF 1r, insulin like growth factor 1 receptor; IR, insulin receptor; IRS1, insulin receptor substrate 1; IRS2, insulin receptor substrate 2; MAPK1, mitogen activated protein kinase 1; MWM, Morris water maze; NO, nitric oxide; OGTT, oral glucose tolerance test; PA task, passive avoidance step-through task; PPARγ, peroxisome proliferator activated receptor gamma; PI3K, phosphoinositide 3-kinase; PKB, protein kinase B; ROSI, rosiglitazone; STL, step through latency; TST, tail suspension test; TBARS, thiobarbituric acid reactive substances. ⁎ Corresponding author at: Jaypee University of Information Technology, Waknaghat, Himachal Pradesh 173234, India. E-mail address: [email protected] (U. Malairaman).

depression and cognitive dysfunction (Rossetti et al., 2014; Sousa et al., 2008). Chronic activation of HPA axis mediates hypercorticosteronemia, inhibits insulin secretion from pancreatic βcells, reduces glucose uptake and utilization, stimulates glucagon secretion and induces type 2 diabetes like state (Ghaisas et al., 2009; Jatwa et al., 2007) as well as impairs neuronal plasticity in hippocampus (Grillo et al., 2009; Piroli et al., 2007). Depressed diabetics show impaired cognitive performance in attention and information processing (Watari et al., 2006). Hypercorticosteronemia induces neuronal oxidative stress and inflammation resulting in cognitive impairment (Pariante and Miller, 2001; Peng et al., 2012; Suwanjang et al., 2013). Reactive nitrogen species such as nitric oxide (NO) has been implicated in stress mediated inflammation and cognitive deficit (Peng et al., 2012). In addition, NO impairs autophagy in rat cortical neurons, which is known to modulate neuronal health (Sarkar et al., 2011). Dysfunctioning of neuronal peroxisome proliferator-activated receptor-γ (PPARγ), insulin receptor (IR), brain derived neurotrophic factor and mitogen activated protein kinase (MAPK) have been observed in brain regions during depressive like behavior and cognitive impairment (Gottschalk et al., 1999;

http://dx.doi.org/10.1016/j.yhbeh.2015.11.010 0018-506X/© 2015 Published by Elsevier Inc.

Please cite this article as: Patel, S.S., et al., Depression mediates impaired glucose tolerance and cognitive dysfunction: A neuromodulatory role of rosiglitazone, Horm. Behav. (2015), http://dx.doi.org/10.1016/j.yhbeh.2015.11.010

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Behavioral assessment

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Forced swim test (FST) The animals were individually forced to swim in a cylinder with radius 24 cm and height 25 cm filled with water (26 ± 2 °C) up to a height of 18 cm. An animal was considered immobile whenever it remained floating passively in the water in a slightly hunched but upright position and its nose just above the water surface. The total immobility period of each animal during the 6 min test was recorded (Kulkarni and Mehta, 1985).

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Materials and methods

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Animals

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Experimental design

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Animals were divided into four groups: group I received 0.3% carboxymethyl-cellulose (0.3% CMC, p.o.) and served as control; group II was exposed to CUS and received vehicle (0.3% CMC, p.o.); group III was subjected to CUS and received ROSI (5 mg/kg, p.o.) and group IV received ROSI (5 mg/kg, p.o.). Dose of ROSI was selected from previous study (Lee et al., 2006) and administered once daily for 21 days. The animals were subjected to CUS paradigm as described previously (Bhutani et al., 2009; Katz, 1981) with modifications. Animals underwent stress paradigm once a day over a period of 21 days (Fig. 1). After 3 weeks of CUS and drug treatment, animals were

Morris water maze task (MWM) Spatial memory was assessed using MWM, which consisted of a white circular pool of 1 m diameter, filled with water at room temperature and had a submerged transparent escape platform kept 1 cm below the water surface. The pool was made opaque with addition of nontoxic water-soluble white paint, which makes the submerged platform invisible to the mice. The pool was divided into four hypothetical quadrants. Each mouse was individually allowed to swim freely (habituation trial) in the maze for 5 min (without platform) on day 21. During training trial (days 22–25) the platform was positioned in the centre of a quadrant and each mouse was released facing toward the wall of the pool in the randomly selected quadrant. The mice were allowed to search the platform spontaneously within 60 s. Mice that failed to find the submerged platform within 60 s were placed onto the platform by the experimenter and allowed to remain on the platform for 5 s (learning trial). Each

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Male Swiss albino mice weighing 25–30 g were housed under a 12 h light/dark cycle at 26 ± 2 °C. The animals had access to food and water ad libitum. All animal experiments were carried out in accordance with CPCSEA guidelines and Institutional Animal Ethical Committee. All efforts were made to minimise pain or discomfort.

Tail suspension test (TST) The animals were individually suspended on the edge of a shelf by adhesive tape placed approximately 1 cm from the tip of the tail. Animals were considered immobile when they hang passively and motionless. The duration of immobility was recorded for the periods of 6 min during the test (Steru et al., 1985).

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subjected to different behavioral studies to assess depressive like behavior, cognition and locomotion. On day 25, immediately after behavioral studies (3–5 pm) blood was collected via retro-orbital puncture. Serum and plasma were separated for biochemical estimation. Animals were sacrificed by cervical dislocation and the hippocampus was dissected for further studies.

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Greene-Schloesser et al., 2014; Grillo et al., 2011; Sadaghiani et al., 2011; Zhang et al., 2012). Rosiglitazone (ROSI) is a selective agonist for nuclear PPARγ receptor, known to increase glucose influx in adipose tissue and muscle by enhancing synthesis and translocation of glucose transporters (Hardman and Limberd, 2001; Tripathi, 2013). ROSI improves cognitive performance, β-cell functions, attenuates insulin resistance and inflammation in diabetics (Abbatecola et al., 2010; Awara et al., 2005; Hanley et al., 2010; Hsu et al., 2005). ROSI attenuates depression associated insulin resistance (Rasgon et al., 2010) and impaired hippocampal neurogenesis (Cheng et al., 2015). The present study was designed to investigate the effect of CUS induced depression as a risk factor for diabetes and associated behavioral dysfunction. We investigated the involvement of insulin signaling and autophagy in the hippocampus region of brain during chronic stress. Further, we studied the neuromodulatory role of ROSI, an antidiabetic drug, against CUS induced depression.

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S.S. Patel et al. / Hormones and Behavior xxx (2015) xxx–xxx

Fig. 1. CUS procedure and experimental design: C — cold swim (8 °C, 3 min); T — tail pinch (1 min); F — food and water deprivation (24 h); I1 — immobilization (3 h); O — overnight illumination; FS — foot shock (20 trials, 0.5 mA, 5.0 s maximum duration, 1 min intervals); T1 — tail pinch (2 min); C1 — cold swim (10 °C, 5 min); FS1 — foot shock (20 trials, 0.5 mA, 5.0 s maximum duration, 30 s intervals); I2 — immobilization (4 h); T2 — tail pinch (3 min); O1 — overnight illumination with wet cage; C2 — cold swim (6 °C, 3 min); I3 — immobilization (5 h); OT — overnight illumination with tilted cage.


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Locomotor activity

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The locomotor activity of mice was assessed by using digital actophotometer (Inco Ambala, India). Animals were individually placed at the centre of the square arena (35 cm × 35 cm) of apparatus. After an initial familiarization period (3 min), the digital locomotor score was recorded for the next 10 min (Mahesh et al., 2012).

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Real-time quantitative reverse transcription PCR

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After behavioral studies, the animals were sacrificed, hippocampus was dissected and total RNA was isolated using TRIzol reagent (Invitrogen). The integrity of RNA was checked on 2% agarose gel and quantified using nano-drop (ND-2000C, Thermo Scientific). The reverse transcription of 3 μg of total RNA was performed using First strand cDNA synthesis kit (Fermentas life-sciences). qPCR amplifications were performed in a CFX96™ Real-Time PCR Detection System (Bio-Rad) using the iQ™ SYBR green supermix (Bio-Rad). Reactions were carried out in total volumes of 12.5 μl, included 2.5 pM of each primer (Table 1) and 1 μl of cDNA template containing 100 ng cDNA. The thermal cycler conditions for cDNA amplification were as follows: Step 1, 95 °C for 3:00 min; Step 2, 95 °C for 10 s, 52–58 °C for 30 s and 72 °C for 2:20 s (35 cycles). GAPDH was used as an internal control and thermal cycler

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Table 1 Sequence of oligonucleotides used for qRT-PCR.

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1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

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PA task was used to evaluate the associative memory, which consisted of a 25 cm long box partitioned into light (10 cm × 14 cm × 16 cm) and dark (10 cm × 10 cm × 16 cm) compartments. The light compartment was illuminated with 60 W bulb, kept 60 cm above the apparatus. Initially, all mice were given one habituation trial to explore both compartments for 120 s. On day 1 of acquisition trial the animals were allowed to explore the light chamber for 5 s. The guillotine door was opened and time taken by mouse to enter the dark chamber was recorded as step through latency (STL). Each animal received an inescapable electric shock for 2 s on a grid floor in the dark chamber. On day 2 of acquisition, memory retention was tested for each mouse in the same manner, but the shock was not delivered (Udayabanu et al., 2012).

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Hippocampus was homogenized in 400 μl ice-cold RIPA buffer containing protease inhibitor cocktail (MP Biomedicals) and then centrifuged at 4 °C 1000 × g for 10 min. The supernatant was collected, centrifuged at 16,000 ×g for 15 min to remove cytosolic fraction. The pellet was resuspended in 500 μl cell lysis RIPA buffer containing 1% Triton X-100, centrifuged at 4 °C 16,000 ×g for 15 min and the supernatant was collected as a crude membrane fraction (Potapenko et al., 2012) for GLUT4 expression. Immunoprecipitation of GLUT4 from crude membrane fraction was then performed with modifications (Mayat et al., 1995). Briefly, 50 μl of Protein A/G PLUS-Agarose (Santa Cruz Biotechnology) was added to 500 μl of membrane lysate and left to incubate for 1 h in rotor. The mixture was then centrifuged (3000 × g) for 3 min at 4 °C. Supernatant was then transferred into fresh tubes containing 10 μg of GLUT4 antibody (Santa Cruz Biotechnology) and subjected to overnight incubation on a rotor. Thereafter, 50 μl of Protein A/G PLUSAgarose slurry was added and incubated in rotor for 1 h. The complex was pelleted by centrifugation 3000 ×g for 10 min at 4 °C and washed five times with 500 μl of ice cold PBS. The washed-immune complex was then resuspended in 50 μl of 1 × Laemmli buffer and boiled for 3 min. After centrifugation, the supernatant was subjected to 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The electrophoretically separated GLUT4 protein was analyzed by Coomassie staining and band density analysis was performed using densitometer (GS-800 Calibrated densitometer, BioRad).

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Passive avoidance step-through (PA) task

Glucose transporter type 4 (GLUT4) translocation

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condition for GAPDH was as follows: Step 1, 95 °C for 3:00 min; Step 181 2, 95 °C for 10 s, 57.6 °C for 30 s and 72 °C for 2:20 s (35 cycles). 182

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mouse received four learning session per day with 10 min interval between each trial. In each trial, the time taken by the mouse to find the hidden platform was recorded as escape latency. Probe trial test was conducted on day 25 to evaluate the index of memory in which the number of crossings across the platform area was recorded (Udayabanu et al., 2012).

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Biochemical estimation

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Blood glucose levels were measured in blood samples collected from the tail vein using Accu-check (Roach Diagnostics) blood glucose monitoring system. Oral glucose tolerance test (OGTT) was performed on 16 h fasted mice using 2 g glucose/kg body weight. Blood was collected from the animals by tail snipping at 0, 0.5, 1.0, 1.5 and 2.0 h after glucose load. The level of serum insulin was determined by chemiluminescent immunoassay using a commercially available kit (AccuLite CLIA Microwells, Monobind Inc., USA). An HPLC-UV system was used for quantification of plasma corticosterone using dexamethasone as an internal standard. Briefly, 500 μl of plasma containing known quantity of dexamethasone was extracted with 5 ml of dichloromethane (DCM). The DCM extract was evaporated to dryness and dissolved in 100 μl of mobile phase. 20 μl of extract was injected into HPLC system for quantification. Mobile phase was composed of methanol:water (70:30) and flow was adjusted at 1.2

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Gene

Forward primer 5′ → 3′

Reverse primer 5′ → 3′

PPARγ IR ILGF 1r GLP1 IRS1 IRS2 PI3K PKB GLUT4 INSG1 MAPK1 BCL2 AIP2 Autophagy5 Autophagy7 GAPDH

AGG GCG ATC TTG ACA GGA AA TTT GTC ATG GAT GGA GGC TA GTG GGG GCT GCT CGT GTT TCT C TCA GAG ACG GTG CAG AAA TGG CGA TGG CTT CTC AGA CGT G CTG CGT CCT CTC CCA AAG TG CGA GAG TGT CGT CAC AGT GTC TGC CCA CAC GCT TAC TGA GA GAT GGG CTT TCT CCG TCC CAC GAC CAC GTC TGG AAC TAT CCC TTA GAC ACT GTG ACG GT GGC TGA GCA CTA CCT TCA GTA AGC TTG GTG TCT GTT CTC TGT CTC GCT AGA TGG AAC CAC TGG CTG CTA CTT CTG CAA TGA TGT TTC ACC ACC ATG GAG AAG GC

CGA AAC TGG CAC CCT TGA AA CCT CAT CTT GGG GTT GAA CT GAT CAC CGT GCA GTT TTC CA ATC AAA GGT CCG GTT GCA GAA CAG CCC GCT TGT TGA TGT TG GGG GTC ATG GGC ATG TAG C TGT TCG CTT CCA CAA ACA CAG CAA AGC AGA GGC GGT CGT GTG TGG CAA GAG TTC AGT GG TGA GAA GAG CAC TAG GCT CCG CAC AGT CCC AAA GCC ACA AA TGG CGG TAT CTA TGG ATT CCA C TGG AGG GAA GAT AGG TCC CAC AGT GGT CCT GTG TGT CTC CAG GAC AGA GAC CAT CAG CTC CAC GGC ATG GAC TGT GGT CAT GA


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All data were analyzed using GraphPad prism 5 software. Two-way ANOVA with Bonferroni post hoc test was used to analyze MWM and OGTT data. The statistical significance of other data was assessed by one-way ANOVA followed by Tukey's post hoc test for differences among the groups. Further, two-way ANOVA for main effect and interaction was performed for all data. A confidence level of p b 0.05 was considered as significant. Eta squared effect sizes (η2) for ANOVA results and Cohen's d effect size estimates for pair-wise comparisons were performed.

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Results

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Behavioral assessment

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FST

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A two-way ANOVA of depressive behavior in FST did not demonstrate a significant CUS/ROSI interaction [F (1,20) = 2.59, p N 0.05, η2 = 0.05], a main effect of CUS [F (1,20) = 21.18, p b 0.001, η2 = 0.41 and d = 2.12] and ROSI [F (1,20) = 7.87, p b 0.05, η2 = 0.15 and d = 3.03]. Post hoc comparisons demonstrated that CUS significantly increased the duration of immobility whereas chronic ROSI administration significantly improved the mobility period in stressed mice (Fig. 2A).

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PA task A one-way ANOVA revealed that chronically stressed mice showed no significant alteration in STL [F (7,40) = 41.32, p N 0.05, R2 = 0.87] on day 23 (memory retention trial) when compared with their respective day 22 (acquisition trial) STL. A two-way ANOVA of associative memory performance during memory retention trial revealed a significant CUS/ROSI interaction [F (1,20) = 24.8, p b 0.001, η2 = 0.26] and a main effect of CUS [F (1,20) = 27.49, p b 0.001, η2 = 0.29 and d = 4.38] and ROSI [F (1,20) = 20.17, p b 0.001, η2 = 0.21 and d = 4.24]. Post hoc comparisons during memory retention trial demonstrated that CUS significantly decreased STL, and the administration of ROSI prevented the decrease of STL (Fig. 3C).

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Locomotor activity A two-way ANOVA of locomotor activity in the actophotometer did not reveal a significant CUS/ROSI interaction [F (1,20) = 0.87, p N 0.05, η2 = 0.01], a main effect of CUS [F (1,20) = 52.23, p b 0.001, η2 = 0.71 and d = 3.18] and ROSI [F (1,20) = 0.32, p N 0.05, η2 = 0.004 and d = 0.21]. Post hoc comparisons revealed that CUS exposure significantly reduced the locomotor activity, and the administration of ROSI did not reverse hypolocomotion (Fig. 4).

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Determination of blood glucose level

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Fasting blood glucose A two-way ANOVA of fasting blood glucose levels in mice did not reveal a significant CUS/ROSI interaction [F (1,20) = 2.12, p N 0.05, η2 = 0.06], a main effect of CUS [F (1,20) = 4.30, p N 0.05, η2 = 0.13 and d = 1.37] and ROSI [F (1,20) = 6.01, p b 0.05, η2 = 0.18 and d =

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TST

A two-way ANOVA of depressive behavior in TST revealed a significant CUS/ROSI interaction [F (1,20) = 5.40, p b 0.05, η2 = 0.14] and a main effect of CUS [F (1,20) = 6.30, p b 0.05, η2 = 0.16 and d = 1.84] and ROSI [F (1,20) = 6.64, p b 0.05, η2 = 0.17 and d = 2.68]. Post hoc

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MWM task A two-way ANOVA of spatial memory in MWM task did not reveal a significant CUS/ROSI interaction [F (1,20) = 3.10, p N 0.05, η2 = 0.13], a main effect of CUS [F (1,20) = 0.04, p N 0.05, η2 = 0.001 and d = 0.62] and ROSI [F (1,20) = 0.45, p N 0.05, η2 = 0.01 and d = 0.45] on day 1 of training. On day 2, there was a significant CUS/ROSI interaction [F (1,20) = 14.49, p b 0.01, η2 = 0.24] and a main effect of CUS [F (1,20) = 23.57, p b 0.001, η2 = 0.40 and d = 4.0] and ROSI [F (1,20) = 0.27, p N 0.05, η2 = 0.004 and d = 1.20]. On day 3, there was a significant CUS/ROSI interaction [F (1,20) = 27.3, p b 0.001, η2 = 0.36] and a main effect of CUS [F (1,20) = 26.57, p b 0.001, η2 = 0.35 and d = 3.41] and ROSI [F (1,20) = 0.34, p N 0.05, η2 = 0.004 and d = 1.96]. On day 4, there was a significant CUS/ROSI interaction [F (1,20) = 25.77, p b 0.001, η2 = 0.22] and a main effect of CUS [F (1,20) = 36.73, p b 0.001, η2 = 0.31 and d = 3.67] and ROSI [F (1,20) = 32.94, p b 0.001, η2 = 0.28 and d = 3.26]. Post hoc comparisons revealed that there was no significant alteration in the learning pattern between the groups on day 1 of training (day 22). During training trial, CUS significantly increased the escape latency on day 2, day 3 and day 4. Chronic ROSI treatment significantly decreased the escape latency on day 3 and day 4 in stressed animals. Control animals treated with ROSI showed a significant increase in escape latency on day 2 and day 3 but not on day 4 (Fig. 3A). A two-way ANOVA of probe trial test demonstrated a significant CUS/ROSI interaction [F (1,20) = 8.88, p b 0.01, η2 = 0.17] and a main effect CUS [F (1,20) = 8.88, p b 0.01, η2 = 0.17 and d = 2.76] and ROSI [F (1,20) = 13.89, p b 0.01, η2 = 0.26 and d = 3.22]. Post hoc comparisons revealed that the number of crossings across the platform area was significantly decreased in stressed mice. Chronic ROSI treatment significantly increased the number of crossings across the platform area in stressed mice (Fig. 3B).

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comparisons revealed that CUS significantly increased the duration of 262 immobility, and the administration of ROSI prevented the increase of 263 immobility period caused by CUS (Fig. 2B). 264

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ml/min. Corticosterone was detected at 250 nm using photodiode array detector (Sheikh et al., 2007). Lipid peroxidation level in plasma was measured by the quantification of thiobarbituric acid-reactive substances (TBARS) according to the method of Ohkawa et al. (1979). Plasma NO level was estimated as reported earlier (Patel and Udayabanu, 2014). Briefly, the nitrates were reduced to nitrite by using 2% ammonium molybdate and 4% ferrous ammonium sulphate and quantified by using Griess reagent at 540 nm. Catalase level was measured by the method of Kuhad and Chopra (2007). Briefly, the rate of decomposition of H2O2 was measured spectrophotometrically at 240 nm. The level of total thiol in plasma was determined as per More et al. (2012) using 10 mM DTNB and measured at 412 nm.

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Fig. 2. Effect of ROSI on CUS-induced behavioral alterations in FST (A) and TST (B). Data were mean ± SEM values (n = 6). Significant differences: #CTRL vs. CUS; *CUS vs. CUS + ROSI. *p b 0.05, ***p b 0.001. CTRL = control; CUS = chronic unpredictable stress; ROSI = rosiglitazone; FST = forced swim test; TST = tail suspension test.


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Fig. 4. Effect of ROSI on CUS-induced alteration in locomotor activity. Data were mean ± SEM values (n = 6). Significant differences: #CTRL vs. CUS. ###p b 0.001. CTRL = control; CUS = chronic unpredictable stress; ROSI = rosiglitazone.

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1.47]. Post hoc comparisons revealed that the fasting blood glucose level 322 did not change significantly in all groups (Fig. 5A). 323 324 325

Determination of corticosterone level A two-way ANOVA of corticosterone levels in the plasma of mice revealed a significant CUS/ROSI interaction [F (1,12) = 47.4, p b 0.001, η2 = 0.13] and a main effect CUS [F (1,12) = 278.3, p b 0.001, η2 = 0.77 and d = 12.50] and ROSI [F (1,12) = 21.64, p b 0.001, η2 = 0.06 and d = 5.51]. Post hoc comparisons revealed that ROSI significantly reversed the hypercorticosteronemia in mice caused by CUS exposure (Fig. 5C).

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Determination of serum insulin level A two-way ANOVA of insulin levels in the serum of mice did not demonstrate a significant CUS/ROSI interaction [F (1,12) = 0.40, p N 0.05, η2 = 0.03], a main effect of CUS [F (1,12) = 0.93, p N 0.05, η2 = 0.07 and d = 1.41] and ROSI [F (1,12) = 0.03, p N 0.05, η2 = 0.002 and d = 0.17]. Post hoc comparisons revealed that the serum insulin level did not change significantly in all groups (Fig. 5D).

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OGTT A two-way ANOVA of blood glucose levels in the OGTT did not reveal a significant CUS/ROSI interaction [F (1,16) = 1.96, p N 0.05, η2 = 0.09], a main effect of CUS [F (1,16) = 2.15, p N 0.05, η2 = 0.09 and d = 1.14] and ROSI [F (1,16) = 1.45, p N 0.05, η2 = 0.06 and d = 1.03] at 0 h. There was a significant CUS/ROSI interaction at 0.5 h [F (1,16) = 12.65, p b 0.01, η2 = 0.20], 1 h [F (1,16) = 62.04, p b 0.001, η2 = 0.25], 1.5 h [F (1,16) = 10.97, p b 0.01, η2 = 0.19] and 2 h [F (1,16) = 15, p b 0.01, η2 = 0.34]. The main effect of CUS at 0.5 h, 1 h, 1.5 h and 2 h was observed as [F (1,16) = 23.33, p b 0.001, η2 = 0.37 and d = 3.30], [F (1,16) = 73.23, p b 0.001, η2 = 0.29 and d = 5.89], [F (1,16) = 20.45, p b 0.001, η2 = 0.36 and d = 3.19] and [F (1,16) = 8.86, p b 0.01, η2 = 0.20 and d = 2.33], respectively. The main effect of ROSI at 0.5 h, 1 h, 1.5 h and 2 h was observed as [F (1,16) = 9.70, p b 0.01, η2 = 0.15 and d = 3.34], [F (1,16) = 95.49, p b 0.001, η2 = 0.38 and d = 9.45], [F (1,16) = 9.16, p b 0.01, η2 = 0.16 and d = 2.65] and [F (1,16) = 3.14, p N 0.05, η2 = 0.07 and d = 2.03], respectively. Post hoc comparisons revealed that CUS significantly increased the level of blood glucose from 0.5 to 2 h, and chronic ROSI administration significantly reversed the glucose load caused by CUS exposure (Fig. 5B).

Fig. 3. Effect of ROSI on CUS-induced behavioral alterations in Morris water maze task (A), probe trial (number of crossings) (B) and passive avoidance step through task (C). Data were mean ± SEM values (n = 6). Significant differences: #CTRL vs. CUS; *CUS vs. CUS + ROSI; + CTRL vs. CTRL + ROSI. *p b 0.05, **p b 0.01, ***p b 0.001. CTRL = control; CUS = chronic unpredictable stress; ROSI = rosiglitazone.


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Insulin signaling A two-way ANOVA of PPARγ mRNA expression in the hippocampus of mice demonstrated a significant CUS/ROSI interaction [F (1,20) = 5.44, p b 0.05, η2 = 0.14] and a main effect of CUS [F (1,20) = 6.05, p b 0.05, η2 = 0.15 and d = 1.56] and ROSI [F (1,20) = 6.62, p b 0.05, η2 = 0.17 and d = 2.03]. Post hoc comparisons revealed that CUS exposure significantly downregulated the PPARγ expression and the administration of ROSI prevented the downregulation of PPARγ expression in the hippocampus of mice (Fig. 6A). A two-way ANOVA of IR expression in the hippocampus of mice revealed a significant CUS/ROSI interaction [F (1,12) = 9.93, p b 0.01, η2 = 0.17] and a main effect of CUS [F (1,12) = 17.92, p b 0.01, η2 = 0.31 and d = 2.96] and ROSI [F (1,12) = 17.76, p b 0.01, η2 = 0.30 and d = 3.15]. Post hoc comparisons demonstrated that CUS exposure significantly downregulated the IR expression and ROSI administration prevented the downregulation in the expression of IR (Fig. 6B). A two-way ANOVA of insulin like growth factor 1 receptor (ILGF 1r) expression in the hippocampus of mice did not reveal a significant CUS/ ROSI interaction [F (1,12) = 0.87, p N 0.05, η2 = 0.06], a main effect of CUS [F (1,12) = 1.15, p N 0.05, η2 = 0.08 and d = 1.04] and ROSI [F (1,12) = 0.16, p N 0.05, η2 = 0.01 and d = 0.22]. Post hoc comparisons revealed that the hippocampal ILGF 1r expression did not change significantly in all groups (Fig. 6C). A two-way ANOVA of glucagon like peptide 1 (GLP1) expression in the hippocampus of mice did not reveal a significant CUS/ROSI interaction [F (1,12) = 2.04, p N 0.05, η2 = 0.10], a main effect of CUS [F (1,12) = 5.98, p b 0.05, η2 = 0.29 and d = 0.52] and ROSI [F (1,12) = 0.06, p N 0.05, η2 = 0.003 and d = 0.58]. Post hoc comparisons demonstrated that the hippocampal GLP1 expression did not change significantly in all groups (Fig. 6D). A two-way ANOVA of insulin receptor substrate 1 (IRS1) expression in the hippocampus of mice demonstrated a significant CUS/ROSI interaction [F (1,16) = 19.95, p b 0.001, η2 = 0.30] and a main effect of CUS

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Fig. 5. Effect of ROSI on CUS-induced alterations in the level of fasting blood glucose (A), OGTT (B), plasma corticosterone (C) and serum insulin (D). Data were mean ± SEM values (n = 4– 6). Significant differences: #CTRL vs. CUS; *CUS vs. CUS + ROSI. **p b 0.01, ***p b 0.001. CTRL = control; CUS = chronic unpredictable stress; OGTT = oral glucose tolerance test; ROSI = rosiglitazone.

[F (1,16) = 13.15, p b 0.01, η2 = 0.19 and d = 4.51] and ROSI [F (1,16) = 17.28, p b 0.001, η2 = 0.26 and d = 4.66]. Post hoc comparisons demonstrated that CUS exposure significantly downregulated the IRS1 expression and ROSI administration prevented the downregulation in the expression of IRS1 (Fig. 6E). A two-way ANOVA of insulin receptor substrate 2 (IRS2) expression in the hippocampus of mice revealed a significant CUS/ROSI interaction [F (1,12) = 9.80, p b 0.01, η2 = 0.17] and a main effect of CUS [F (1,12) = 17.11, p b 0.01, η2 = 0.29 and d = 3.58] and ROSI [F (1,12) = 18.46, p b 0.01, η2 = 0.32 and d = 4.16]. Post hoc comparisons demonstrated that CUS exposure significantly downregulated the IRS2 expression and chronic ROSI administration significantly reversed the downregulation of hippocampal IRS2 expression in stressed mice (Fig. 6F). A two-way ANOVA of phosphoinositide 3-kinase (PI3K) expression in the hippocampus of mice demonstrated a significant CUS/ROSI interaction [F (1,12) = 18.06, p b 0.01, η2 = 0.11] and a main effect of CUS [F (1,12) = 77.6, p b 0.001, η2 = 0.49 and d = 9.38] and ROSI [F (1,12) = 49.14, p b 0.001, η2 = 0.31 and d = 4.68]. Post hoc comparisons revealed that ROSI significantly reversed the downregulation of PI3K expression on the hippocampus of mice caused by CUS exposure (Fig. 6G). A two-way ANOVA of protein kinase B (PKB) expression in the hippocampus of mice revealed a significant CUS/ROSI interaction [F (1,12) = 6.42, p b 0.05, η2 = 0.20] and a main effect of CUS [F (1,12) = 6.42, p b 0.05, η2 = 0.20 and d = 2.13] and ROSI [F (1,12) = 6.79, p b 0.05, η2 = 0.21 and d = 2.23]. Post hoc comparisons demonstrated that CUS exposure significantly downregulated the PKB expression and ROSI administration prevented the downregulation in the expression of PKB (Fig. 6H). A two-way ANOVA of GLUT4 expression in the hippocampus of mice demonstrated a significant CUS/ROSI interaction [F (1,12) = 6.12, p b 0.05, η2 = 0.17] and a main effect of CUS [F (1,12) = 7.71, p b 0.05, η2 = 0.21 and d = 4.56] and ROSI [F (1,12) = 9.86, p b 0.01,


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A two-way ANOVA of insulin induced gene 1 (INSG1) expression in the hippocampus of mice revealed a significant CUS/ROSI interaction [F (1,12) = 19.79, p b 0.001, η2 = 0.46] and a main effect of CUS [F (1,12) = 6.34, p b 0.05, η2 = 0.14 and d = 4.25] and ROSI [F (1,12) = 4.61, p N 0.05, η2 = 0.10 and d = 3.07]. Post hoc comparisons demonstrated that CUS exposure significantly downregulated the INSG1 expression and chronic ROSI administration significantly reversed the downregulation of hippocampal INSG1 expression in stressed mice (Fig. 6J). A two-way ANOVA of MAPK1 expression in the hippocampus of mice revealed a significant CUS/ROSI interaction [F (1,12) = 7.26, p b 0.05, η2 = 0.20] and a main effect of CUS [F (1,12) = 10.16, p b 0.01, η2 = 0.28 and d = 5.68] and ROSI [F (1,12) = 6.06, p b 0.05, η2 = 0.17 and d = 2.40]. Post hoc comparisons demonstrated that CUS exposure significantly downregulated the MAPK1 expression and chronic ROSI administration significantly reversed the downregulation of hippocampal MAPK1 expression in stressed mice (Fig. 6K).

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GLUT4 translocation A two-way ANOVA of GLUT4 protein expression in the hippocampus of mice revealed a significant CUS/ROSI interaction [F (1,12) = 220.6, p b 0.001, η2 = 0.35] and a main effect of CUS [F (1,12) = 103.4, p b 0.001, η2 = 0.16 and d = 30.12] and ROSI [F (1,12) = 294.2, p b 0.001, η2 = 0.46 and d = 14.23]. Post hoc comparisons demonstrated that GLUT4 membrane protein was significantly downregulated in the hippocampus of chronically stressed mice and chronic ROSI treatment significantly reversed the downregulation of GLUT4 membrane protein in stressed mice (Fig. 7).

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η2 = 0.27 and d = 4.92]. Post hoc comparisons revealed that ROSI significantly reversed the downregulation of GLUT4 mRNA expression on the hippocampus of mice caused by CUS exposure (Fig. 6I).

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Apoptosis and neuronal survival A two-way ANOVA of B-cell lymphoma 2 (BCL2) expression in the hippocampus of mice revealed a significant CUS/ROSI interaction [F (1,12) = 7.62, p b 0.05, η2 = 0.19] and a main effect of CUS [F (1,12) = 13.77, p b 0.01, η2 = 0.36 and d = 3.70] and ROSI [F (1,12) = 4.83, p b 0.05, η2 = 0.12 and d = 3.16]. Post hoc comparisons demonstrated that CUS exposure significantly downregulated the BCL2 expression and ROSI administration prevented the downregulation in the expression of BCL2 (Fig. 8A). A two-way ANOVA of apoptosis inhibitory protein 2 (AIP2) expression in the hippocampus of mice did not demonstrate a significant CUS/ROSI interaction [F (1,12) = 0.99, p N 0.05, η2 = 0.01], a main effect of CUS [F (1,12) = 50.43, p b 0.001, η2 = 0.74 and d = 2.73] and ROSI [F (1,12) = 3.85, p N 0.05, η2 = 0.05 and d = 1.32]. Post hoc comparisons demonstrated that CUS exposure significantly downregulated the AIP2 expression and ROSI administration did not modulate the expression of AIP2 in stressed mice (Fig. 8B). A two-way ANOVA of autophagy5 expression in the hippocampus of mice did not reveal a significant CUS/ROSI interaction [F (1,12) = 1.98, p N 0.05, η2 = 0.06], a main effect of CUS [F (1,12) = 17.98, p b 0.01, η2 = 0.55 and d = 4.35] and ROSI [F (1,12) = 0.61, p N 0.05, η2 = 0.01 and d = 1.50]. Post hoc comparisons revealed that ROSI administration did not reverse the downregulation of autophagy5 expression on the hippocampus of mice caused by CUS exposure (Fig. 8C). A two-way ANOVA of autophagy7 expression in the hippocampus of mice did not demonstrate a significant CUS/ROSI interaction [F

Fig. 6. Effect of ROSI on CUS-induced alterations in the mRNA expression of hippocampal PPARγ (A), IR (B), ILGF 1r (C), GLP1 (D), IRS1 (E), IRS2 (F), PI3K (G), PKB (H), GLUT4 (I), INSG1 (J) and MAPK1 (K). Data were mean ± SEM values (n = 4). Significant differences: # CTRL vs. CUS; *CUS vs. CUS + ROSI. *p b 0.05, **p b 0.01, ***p b 0.001. CTRL = control; CUS = chronic unpredictable stress; ROSI = rosiglitazone; PPARγ = peroxisome proliferator activated receptor gamma; IR = insulin receptor; ILGF 1r = insulin like growth factor 1 receptor; GLP1 = glucagon like peptide 1; IRS1 = insulin receptor substrate 1; IRS2 = insulin receptor substrate 2; PI3K = phosphoinositide 3-kinase; PKB = protein kinase B; GLUT4 = glucose transporter type 4; INSG1 = insulin induced gene 1; MAPK1 = mitogen activated protein kinase 1.


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a main effect of CUS [F (1,20) = 11.89, p b 0.01, η2 = 0.25 and d = 3.41] and ROSI [F (1,20) = 8.56, p b 0.01, η2 = 0.18 and d = 1.83]. Post hoc comparisons revealed that ROSI significantly reversed the increase of TBARS level on the plasma of mice caused by CUS exposure (Fig. 9A).

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A two-way ANOVA of NO levels in the plasma of mice revealed a significant CUS/ROSI interaction [F (1,20) = 12.92, p b 0.01, η2 = 0.13] and a main effect of CUS [F (1,20) = 55.04, p b 0.001, η2 = 0.58 and d = 4.37] and ROSI [F (1,20) = 5.50, p b 0.05, η2 = 0.05 and d = 2.77]. Post hoc comparisons revealed that ROSI significantly reversed the increase of NO level on the plasma of mice caused by CUS exposure (Fig. 9B).

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TBARS A two-way ANOVA of TBARS levels in the plasma of mice revealed a significant CUS/ROSI interaction [F (1,20) = 5.6, p b 0.05, η2 = 0.12] and

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Fig. 8. Effect of ROSI on CUS-induced alterations in the mRNA expression of hippocampal BCL2 (A), AIP2 (B), autophagy5 (C) and autophagy7 (D). Data were mean ± SEM values (n = 4). Significant differences: #CTRL vs. CUS; *CUS vs. CUS + ROSI. *p b 0.05, **p b 0.01, ***p b 0.001. CTRL = control; CUS = chronic unpredictable stress; ROSI = rosiglitazone; BCL2 = B-cell lymphoma 2; AIP2 = apoptosis inhibitory protein 2.

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Chronic stress often precipitates depression and affects the normal physiological state of body, interfering with the emotional, cognitive and physical aspects of health. Clinically, depression has been reported to induce neurocognitive dysfunctions (Shimizu et al., 2013). Chronic stress has been reported to induce disruption of spatial and associative memory (Bisaz et al., 2011; Gumuslu et al., 2013) as well as motor function (Strekalova et al., 2004). In the present study, exposure of mice to CUS resulted in increased duration of immobility in FST and TST, depicting depressive like behavior. We observed that CUS increased the escape latency in MWM task, a reliable index of spatial memory dysfunction. In addition, CUS lead to associative memory dysfunction in mice as evident from decreased STL in PA task. The depressive like behavior in stressed mice was attenuated by ROSI administration. ROSI treatment significantly reversed spatial and associative memory dysfunction associated with depressive like behavior. ROSI is known to induce neurogenesis and attenuate depressive like behavior (Cheng et al., 2015) as well as improve learning and memory (O'Reilly and Lynch, 2012; Pipatpiboon et al., 2011). Further, CUS induced hypolocomotion was not altered by chronic ROSI administration. The major neuro-endocrine responses to chronic stress involve the release of glucocorticoids leading to depressogenic-like effect, hyperglycemia and cognitive impairment (Detka et al., 2013; Haynes et al., 2001; Patel and Udayabanu, 2014; Wróbel et al., 2014). CUS exhibited insulin resistance, hypoinsulinemia and hyperglycemia in animal models (Lin et al., 2005; Pan et al., 2013). In the present study, CUS significantly increased the level of plasma corticosterone, impaired glucose tolerance but did not alter the level of fasting blood glucose and serum insulin. It has been reported that fasting glucose level is often normal but with glucose load hyperglycemia occurs depicting insulin resistance during type 2 diabetes (O'Rahilly et al., 1994). Chronic ROSI administration

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(1,12) = 0.17, p N 0.05, η2 = 0.002], a main effect of CUS [F (1,12) = 50.65, p b 0.001, η2 = 0.75 and d = 3.80] and ROSI [F (1,12) = 4.07, p N 0.05, η2 = 0.06 and d = 1.18]. Post hoc comparisons revealed that ROSI administration did not reverse the downregulation of autophagy7 expression on the hippocampus of mice caused by CUS exposure (Fig. 8D).

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Total thiol A two-way ANOVA of total thiol levels in the plasma of mice revealed a significant CUS/ROSI interaction [F (1,20) = 7.97, p b 0.05, η2 = 0.18] and a main effect of CUS [F (1,20) = 10.13, p b 0.01, η2 = 0.23 and d = 2.13] and ROSI [F (1,20) = 4.44, p b 0.05, η2 = 0.10 and d = 1.69]. Post hoc comparisons demonstrated that CUS exposure significantly decreased the total thiol level and chronic ROSI administration significantly reversed the decrease of plasma total thiol in stressed mice (Fig. 9D).

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Fig. 7. Effect of ROSI on CUS-induced alteration in the level of hippocampal GLUT4 membrane protein. Data were mean ± SEM values (n = 4). Significant differences: #CTRL vs. CUS; *CUS vs. CUS + ROSI. ***p b 0.001. CTRL = control; CUS = chronic unpredictable stress; ROSI = rosiglitazone; GLUT4 = glucose transporter type 4.

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Catalase A two-way ANOVA of catalase levels in the plasma of mice did not demonstrate a significant CUS/ROSI interaction [F (1,20) = 4.33, p N 0.05, η2 = 0.08], a main effect of CUS [F (1,20) = 15.52, p b 0.001, η2 = 0.29 and d = 2.85] and ROSI [F (1,20) = 12.64, p b 0.01, η2 = 0.24 and d = 2.06]. Post hoc comparisons demonstrated that CUS exposure significantly decreased the catalase level and chronic ROSI administration significantly reversed the decrease of plasma catalase in stressed mice (Fig. 9C).

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significantly reversed hypercorticosteronemia and impaired glucose tolerance in stressed mice. PPARγ agonist is known to induce insulin sensitizing action (Kavak et al., 2008), improve glucose uptake and reduce hypercorticosteronemia (Yang et al., 2007). Insulin signaling in the hippocampus is involved in synaptic plasticity, energy metabolism, neuroprotection and cognitive function (Datusalia and Sharma, 2014; Mielke and Wang, 2011). Alterations in insulin signaling mechanism have been observed in the hippocampus of depressed mice (Detka et al., 2013; Hölscher, 2011; Patel and Udayabanu, 2014). We observed that, CUS induced depressed mice have impaired glucose tolerance which might be due to hypercorticosteronemia or downregulation of IR. Hypercorticosteronemia or downregulation of IR leads to dysregulation of insulin signaling components like PPARγ, IRS1, IRS2, PI3K, PKB, MAPK1, INSG1 and GLUT4, resulting in impaired memory performance and depressive like behavior (Agrawal et al., 2011; Hölscher, 2011; Patel and Udayabanu, 2014; Qi et al., 2012; Vannucci et al., 1998). CUS induced depression significantly impaired insulin signaling and GLUT4 membrane translocation in the hippocampus, which was revered by ROSI. We did not observe any significant alteration in the levels of ILGF 1r and GLP1 in hippocampus. ROSI treatment increases IR expression via PPARγ gene transcription resulting in improved insulin sensitivity and GLUT4 membrane translocation (Hernandez et al., 2003; Tripathi, 2013). During neuronal activity, IR → Src → MAPK pathway activates gene transcription essential for glucose homeostasis, synapse growth and cell repair (Hölscher, 2011). Induction of INSG1 via MAPK pathway plays a role in cellular glucose homeostasis (Krapivner et al., 2007). Activation of IR induces GLUT4 membrane translocation and suppresses apoptosis through stimulation of IRS1/2 binding to PI3K, and activation of PI3K and Akt/PKB (Hölscher, 2011; Schubert et al.,

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Fig. 9. Effect of ROSI on CUS-induced alterations in TBARS level (A), NO level (B), catalase level (C) and total thiol level (D) in plasma. Data were mean ± SEM values (n = 6). Significant differences: #CTRL vs. CUS; *CUS vs. CUS + ROSI. *p b 0.05, **p b 0.01, ***p b 0.001. CTRL = control; CUS = chronic unpredictable stress; ROSI = rosiglitazone; TBARS = thiobarbituric acid reactive substances; NO = nitric oxide.

2003). Further, GLUT4 membrane translocation in hippocampal neurons is associated with neurocognitive improvement (Piroli et al., 2007). It has been well accepted that, exposure to stress increases the number of apoptotic cells in the hippocampus resulting in cognitive impairment (Lucassen et al., 2001; Rinwa and Kumar, 2014; Zhang et al., 2007), whereas antidepressants upregulate BCl2 expression and improve neural plasticity (Manji et al., 2001). The ability of AIP2 to inhibit caspase-2 indicates that it occupies a unique position in programmed cell death (Cheung et al., 2006). In the present study, CUS significantly downregulated the level of hippocampal BCl2 and AIP2. Chronic ROSI treatment significantly upregulated hippocampal BCL2 but did not modulate AIP2 in stressed mice. Earlier study demonstrated that, ROSI protect the neurons against advanced glycation end products-induced injury via its antiapoptotic property, mediated by PPARγ activation (Wang et al., 2011). In contrast to apoptosis, autophagy has a protective role against the pathogenesis of a number of neurodegenerative diseases. In the present study, ROSI treatment did not modulate the CUS mediated downregulation of autophagy5 and autophagy7 in hippocampus. Dysfunction in autophagy results in neuronal damage accompanied by ubiquitinated protein aggregates, suggesting that basal levels of autophagy are essential for neuronal health (Cherra and Chu, 2008). CUS plays an important role in induction of various clinical disorders by inducing oxidative stress and inflammation (Ahmad et al., 2010; Rinwa and Kumar, 2014). In the present study, CUS resulted in elevation in the level of oxidative and nitrative stress markers as well as depletion of antioxidants such as catalase and total thiol. Chronic treatment with ROSI reversed CUS induced alteration in oxidative and nitrative stress, which is in line with a previous report (García-Bueno et al., 2005). In conclusion, CUS induces depression and diabetic like state associated with cognitive impairment, dysregulation of neuronal insulin


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Authors would like to thank Council of Scientific & Industrial Research for financial assistance as senior research fellowship (09/957 (0002)/2012-EMR-I) and Defence Research and Development Organisation (DLS/81/48222/LSRB-175/FSB/2008) (Ministry of Defence, Govt. of India) for funding.

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