Amelioration of Diabetes-induced Cognitive Deficitsby GSK-3β Inhibition is Attributed to Modulationof Neurotransmitters and Neuroinflammation

Ashok Kumar Datusalia & Shyam Sunder Sharma

Received: 25 October 2013 /Accepted: 2 January 2014# Springer Science+Business Media New York 2014

Abstract Chronic type 2 diabetes (T2D) causes cognitivedeficits which are debilitating to the young as well as the olderpopulation. Glycogen synthase kinase-3β (GSK-3β) signal-ing has been reported to be impaired in insulin-resistant andT2D animal models. In this study, we have investigated theinvolvement of GSK-3β in cognitive deficits associated withT2D using SB216763, a GSK-3 β inhibitor. In high-fat diet-streptozotocin (HFD-STZ) model of T2D in rats, cognitivedeficits appeared on the 15th week after induction of diabetes.Treatment with GSK-3β inhibitor SB216763 (i.p. daily for3 weeks) reversed impaired cognitive performance in theMorris water maze, Y-maze, and passive avoidance tests.Administration of SB216763 also significantly improved ace-tylcholine esterase activity, GABA, and glutamate levels inthe hippocampus and cortex of diabetic rats. Importantly,GSK-3β inhibition showed an increase in pGSK-3β andpCREB expression and reduction in pNF-κB-p65 expressionin both hippocampus and cortex. Neuroinflammation wasreduced by SB216763 in diabetic rats as evident from reduc-tion in IL-6, TNF-α, COX-2, and inducible nitric oxide syn-thase levels. This study suggests that cognitive deficits asso-ciated with diabetes involved intricate compartmental interac-tion between transcription factors and neurotransmitterhomeostasis/energy metabolism, and GSK-β might play acentral role in diabetes-induced cognitive impairment.

Keywords Glycogen synthase kinase-3β . Type 2 diabetes .

Cognitive deficits . Neurotransmitter . Neuroinflammation

Introduction

Diabetes mellitus (DM) is one of the most prevalent devastat-ing chronic diseases. It is currently affecting 371 millionpeople worldwide, and the number of people with type 2diabetes (T2D) is increasing at an alarming rate in most ofthe countries [1, 2]. DM is a systemic disease that can damageseveral organs of the body, and the complications associatedwith diabetes include pathologic changes involving both smalland large vessels, cranial and peripheral nerves, kidneys, andeyes. Recently, efforts have beenmade to investigate the effectof diabetes on the brain, and diabetes has been implicated inthe development of neurological co-morbidities [3-8]. A lessaddressed and not well-recognized complication of DM iscognitive dysfunction. Like diabetes, cognitive dysfunctionrepresents another serious problem and is rising in prevalenceworldwide, especially among the elderly. Further, DM hasbeen implicated as a risk factor not only for dementia ofvascular type but also for Alzheimer’s disease (AD) [9].Patients with type 1 diabetes (T1D) and T2D have been foundto present cognitive deficits, associated with reduced perfor-mance on multiple domain of cognitive function [10]. Cogni-tive domain which is being affected during diabetes differswith respect to the type, severity, and onset of diabetes [10].Many studies suggest that the risk of cognitive decline andneurodegeneration is increased not only in diabetic patients,but also in patients with prediabetic and metabolic syndrome[11, 12]. Insulin resistance at peripheral tissues has beenshown to influence central insulin resistance with reducedbrain insulin level and affect cognition [13]. The pathophys-iology for cognitive decline in prediabetic and metabolicsyndrome patients might differ from elderly diabetic patients.

A. K. Datusalia : S. S. SharmaMolecular Neuropharmacology Laboratory, Department ofPharmacology and Toxicology, National Institute of PharmaceuticalEducation and Research (NIPER), Sector 67, SAS Nagar(Mohali) 160062, Punjab, India

S. S. Sharma (*)Department of Pharmacology and Toxicology, National Institute ofPharmaceutical Education and Research (NIPER), Sector 67, SASNagar 160062, Punjab, Indiae-mail: sssharma@niper.ac.inURL: www.niper.gov.in

Mol NeurobiolDOI 10.1007/s12035-014-8632-x

Reduced cognitive ability has been observed in obese and type2 diabetic patients. Hyperglycemia, vascular disease, and al-tered insulin signaling play a significant role in the pathophys-iology of cognitive impairment, but the mechanism of cogni-tive impairment in diabetes looks complex and yet notcompletely understood [10].

Glycogen synthase kinase-3 (GSK-3), a ubiquitous serine/threonine kinase, has attracted wide attention due to its mul-tifunctional role in physiology to disease pathogenesis. Itmodulates many fundamental cell processes and is evenemerging among the most promising therapeutic target forAD [14-18]. GKS-3β is a key kinase required for AD-typeabnormal hyperphosphorylation of tau protein and overex-pression of GSK-3β results in tau hyperphosphorylation anddisruption of microtubules in transgenic mice [19, 20]. In vitrostudies have shown that GSK-3α isoform regulates amyloidprecursor protein (APP) processing and amyloid-β (Aβ) pro-duction, and its overexpression has been shown to promoteapoptotic neuronal cell death [19-21]. Transgenic mice over-expressing GSK-3β in CA1 and dentate gyrus showed im-paired spatial memory and long-term potentiation (LTP) [22].Although many studies have shown the role of GSK-3β inmemory and cognition using in vitro and in vivo models ofAlzheimer’s disease, the role of GSK-3β in diabetes-inducedcognitive deficits is yet not investigated. Therefore, the pur-pose of the current study was to elucidate the role of GSK-3βin cognitive deficit induced by type 2 diabetes usingSB216763, a GSK-3β inhibitor.

Materials and Methods

Animals

Male Sprague–Dawley rats (130±10 g of body weight; 5–6 weeks of age) were procured from the Central AnimalFacility of the Institute (NIPER, SAS Nagar, India). Theexperimental study protocol was duly approved by the Insti-tutional Animal Ethics Committee (IAEC), NIPER and strict-ly carried out in accordance with the guidelines of the Com-mittee for the Purpose of Control and Supervision of

Experiments on Animals (CPCSEA), Government of India.The rats were provided with a regular rodent pellet diet(Ashirwad Industries, Chandigarh) and high-fat diet adlibitum [23].

Induction of Type 2 Diabetes and Experimental Design

Type 2 diabetes was induced in rats by a combination of high-fat diet (HFD) feeding and low dose of streptozotocin (STZ)treatment as described elsewhere [23]. Briefly, the rats werefed with HFD for 2 weeks and then injected with single lowdose of STZ (35 mg/kg, i.p.) to induce diabetes. Diabetes wasconfirmed by blood glucose levels. Only those rats withplasma glucose level of ≥250 mg/dl at the end of 2 weekswere considered diabetic and selected for the study. Normalcontrol (normal pellet diet, NPD) (n=7) and type 2 diabeticanimals (HFD + STZ) (n=14) were screened for cognitiveimpairment during the 15th week after induction of diabetes.Another pool of animals was randomly assigned to the fol-lowing experimental groups, namely, (1) normal control(NPD) (n=9), (2) diabetic control + vehicle treatment (HFD+ STZ) (n=14), (3) diabetic + SB216763 (0.3 mg/kg) treat-ment (n=9), (4) diabetic + SB216763 (0.6 mg/kg) treatment(n=12), and (5) diabetic + SB216763 (1.2 mg/kg) treatment(n=9). Treatment was given for 3 weeks of daily singleintraperitoneal injection starting from the 16th to the 18thweek after diabetes induction (Fig. 1). Dose of SB216763and treatment duration were selected based on literature[24]. DMSO was used as a vehicle. No exogenous insulintreatment was given.

Behavioral Tasks

Place Preference Task Using Y-maze

Place preference task consists of two trials—trial 1 and trial 2.Trial 1 was acquisition, in which animals were placed in thestart arm, facing the wall, and were allowed to explore for20 min, by closing one arm. Trial 2 was retention after 2 h ofthe first trial, in which animals were placed in the start arm,facing the wall, and allowed to explore for 10 min, by opening

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Fig. 1 Schematic representationof the study design for diabetes-induced cognitive deficits andtreatment schedule

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the closed arm. Chocolate flavor was mixed in bedding mate-rial to increase the exploratory behavior of animals. Variouscues of different shapes were placed in the two arms, in orderto make a distinction between them for the rats while explor-ing. All experiments were recorded and analyzed usingANYMAZE™ software (Stoelting Co., USA). Duration oftime spent in each arm during the initial 2 min of the secondtrial was calculated and analyzed [25].

Step-Through Passive Avoidance Task

Rats were trained on a one-trial step-through passiveavoidance task. A two-compartment step-through passiveavoidance apparatus duly software operated (PACS-30)was used in the study (Columbus Instruments, USA).Two compartments were separated by a guillotine door.One compartment was illuminated and the second com-partment consisted with a series of metal rod, throughwhich an electric shock could be delivered. Rats wereinitially placed in the lighted compartment, facing awayfrom the dark compartment and allowed to explore for15 s. After 15 s, the door was raised and animals wereallowed to explore freely. When the rat entered the darkcompartment with all four paws, the guillotine door wasclosed and returned to the home cage. After 1 h ofhabituation, acquisition trial was performed. Each animalwas placed in the lighted compartment and allowed for a15-s habituation period before the door was opened. Oncethe rat entered the dark compartment with all four paws,the door was closed behind the rat and a foot shock(0.6 mA, 3 s duration) was delivered. Approximately,10 s later, the rat was returned to its home cage. Thelatency for the rats to enter the dark compartment, i.e.,acquisition latency was recorded. On test day (24 h afteracquisition trial), the inhibitory avoidance response wasrecorded. This was done by placing each rat in an illumi-nated compartment, as described above. The latency toenter into the dark compartment was recorded as a mea-sure of retention task. Rats which did not enter the darkcompartment within 5 min were given a ceiling score of300 s [26].

Morris Water Maze Test

Morris water maze test is commonly used to assess learn-ing and memory. Briefly, it consisted of a pool dividedinto four equal quadrants, partially filled with opaquewater and a hidden platform in one of the quadrants.The animal was kept in one quadrant facing toward thewall and was given a 120-s trial to find the platform. Thiswas repeated for five consecutive days with four trialseach day to each animal (intra-trial intervals is 10 min).All experiments were recorded and analyzed using

ANYMAZE™ software. Escape latency was measuredto assess the learning and acquisition tasks. Alternatively,probe trial was also performed for spatial memory assess-ment, and the time spent in the respective quadrants wasrecorded. Path tracking analysis was used to assess thig-motaxis behavior. The cumulative distance to platformparameter is the sum of the distance between the platformin the water maze and each sample of the animal’s posi-tion collected by image analysis system. To address thespecificity for the learned location, the same calculationswere done for arbitrary platform position in the otherthree quadrants (i.e., the adjacent quadrants left and rightand opposite to platform position). Percentage of cumu-lative distance to the platform was calculated as the cu-mulative distance to platform divided by the sum of thecumulative distance to left, right, opposite, and the plat-form location, multiplied by 100. According to traditionalcriteria, individual swim patterns were labeled nonspatial/random and spatial/persistent. A nonspatial/random pat-tern was scored as such when no preference for theplatform location relative to the other possible locationscould be observed. A spatial/persistent swim pattern ischaracterized by a clear preference for the platform posi-tion (i.e., ≥30 % time spent in the platform quadrant and ashorter latency to the platform location) [27].

Biochemical Estimations

Plasma Glucose, Insulin, and Glycosylated Hemoglobin(HBA1c) Levels

Briefly, blood was collected from the tail vein inmicrocentrifuge tubes containing heparin. Glucose level wasestimated from glucose oxidase–peroxidase kit (AccurexMumbai, India). Plasma insulin was determined using anELISA kit (Millipore, USA). Glycosylated hemoglobin wasestimated by ion-exchange resin method using a kit (CrestBiosystems, Goa, India).

Acetylcholinesterase (AChE Activity)

Hippocampus and cortex were homogenized in 0.1 M phos-phate buffer (pH 8); 0.4 ml aliquot of the homogenate wasadded to a cuvette containing 2.6 ml phosphate buffer (0.1 M,pH 8) and 100 μl of 5,5′-dithiobis-(2-nitrobenzoic acid)(DTNB). The contents of the cuvette were mixed thoroughlyby bubbling. Twenty microliters of substrate (0.075 Macetylthiocholine) was added and change in absorbance wasrecorded for a period of 10 min at intervals of 2 min. Theenzyme activity was calculated using the formula: R=5.74×10−4×A/CO, where R=rate in moles of substrate hydrolyzedper minute per gram tissue, A=change in absorbance per

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minute, and CO = original concentration of the tissue (milli-grams per milliliter) [28].

Brain TNF-α and IL-6 Levels

Brain tissues (hippocampus and frontal cortex) were homog-enized in PBS buffer containing phenylmethanesulfonyl fluo-ride (PMSF) and protease inhibitor cocktail. The homogenatewas kept in ice-cold water for 30 min, sonicated, and centri-fuged at 10,000 rpm for 10 min in a centrifuge at 4 °C. Thesupernatant fraction was separated and used for estimations.Commercially available kits from eBiosciences (USA) wereused for assaying TNF-α and IL-6. The principle of the assaywas sandwiched ELISA. The plates were read using anELISA plate reader. The protein level of the supernatant wasestimated and the TNF-α and IL-6 levels were expressed aspicomolars per milligram of protein.

Neurotransmitter Estimation

Rats were sacrificed by decapitation immediately after theprobe trial, and various brain regions were quickly dissectedout. GABA and glutamate levels were measured by theHPLC-EC method. Briefly, brains were isolated on ice-coldPetri dish. Immediately, the hippocampi were removed andhomogenized in ice-cold perchloric acid (0.05 M) containingL-tyrosine (0.1 mg/ml or 5.5 μmol/ml). Homogenized sampleswere centrifuged and the supernatant was derivatized with o-phthalaldehyde (OPA). Derivatizing agent was prepared bydissolving 100 mg of o-phthaldehyde in 2 ml of HPLC grademethanol and added into 200 ml of NaHCO3 (0.5 M, pH 9.5).Finally, 20 μl of β-mercaptoethanol was added into it and thesolution was kept in the dark and cold (2–8 °C). Samples werederivatized with 30:50 ratios of sample and derivatizing re-agent with excessive shaking for 2 min. Mobile phase wasprepared by dissolving 10.92 g of Na2HPO4 and 148.8 mg ofEDTA in 500 ml of ultra pure Millipore water. Then, 1.5 mltetrahydrofuran and 450 ml of HPLC grade methanol wereadded. After adjusting the pH to 5.25 with o-phosphoric acid,the volume was made up to 1,000 ml. Fifty microliters ofderivatized samples were injected using an autosampler (Wa-ters, 717) into a column (Neucleosil®: RP18, 5 μm, 4.6×250,30 °C). The sample was run for 30 min at a flow of 1 ml/min(Pump: Waters 515) and components present in the samplewere detected by an electrochemical detector (Waters 2465:mode: DC, Ec +0.80 V, 30 °C). Glutamate and GABA wereidentified using corresponding standards and quantified usingan internal standard method [29].

Western Blotting

Brain hippocampus and frontal cortex were homogenized inlysis buffer (1 % Triton X-100, 150 mM NaCl, 1 mM EDTA,

2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate,1 mM Na3VO4, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and20 mM Tris, pH 7.5). Equal amount of proteins was separatedby SDS-PAGE and transferred to a nitrocellulose membrane(Pall Life Sciences, FL, USA). The blots were blocked bynonfat dried milk (Sigma Aldrich, USA) for 1 h and wereprobed with primary antibody GSK-3β (1:1,000), pGSK-3β(1:1,000), NF-κB (1:500), pNF-κB (1:1,000), cyclic AMPresponse element-binding protein (CREB) (1:1,000), pCREB(1:500), and COX-2 (1:250) obtained from Santa Cruz Bio-technologies, USA; inducible nitric oxide synthase (iNOS)(1:1,000) was obtained from Cell Signaling Technology,USA and β-actin (1:1,000) from Sigma Aldrich, USA, inTBS overnight at 4 °C and then detected with HRP-conjugated secondary antibody [30].

Statistical Analysis

The data are presented as mean ± standard error of mean(SEM). In comparing the difference between the twogroups, Student’s t test was used. Comparisons betweenmultiple groups were done using one-way analysis ofvariance (ANOVA). Morris water acquisition performancedata were analyzed using two-way repetitive measureANOVA followed by post hoc Greenhouse-Geisser/Bonferroni correction to examine the differences betweenindividual treatments (GraphPad Prism 5). For the westernimmunoblotting data, the results were statistically evalu-ated by one-way ANOVA, followed by a Tukey’s post hoctest (GraphPad Prism 5). p<0.05 was considered statisti-cally significant.

Results

Body Weight and Plasma Glucose Levels

Fifteen weeks after STZ injection, diabetic rats exhibitedsignificantly (p<0.001) elevated (338±31.13 mg/dl)plasma glucose levels compared with the control group(105.0±7.0 mg/dl) (Table 1). HBA1c (percent of totalhaemoglobin; percent of THb) was also increased sig-nificantly (p<0.001) 15 weeks after induction of diabe-tes. Moreover, the plasma insulin level was decreasedsignificantly (p<0.001) compared to age-matched con-trols as displayed in Table 1. Chronic (3 weeks) treat-ment with SB216763 (GSK-3β inhibitor) at the doses of0.3, 0.6, and 1.2 mg/kg did not show any significanteffect on plasma glucose, body weight, plasma insulin,and HbA1c level. No mortality and blindness symptom(visual cliff experiment) [31] were observed due todiabetes and drug treatment.

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Type 2 Diabetes Adversely Affected Cognitive Skills After15 Weeks of Diabetic Induction

Diabetic rats and their age-matched control rats were assessedfor cognitive skills on different time points (data not shown).Y-maze is a suitable method to investigate spatial and nonspa-tial one-trial, nonaversive, and short-term memory. Moreover,this task is an ethological paradigm that captures latent learn-ing in the absence of explicit rewards. Diabetes significantlyaffected place preference after 15 weeks. Similar altered be-havior was observed on the 18th week. During training,diabetic and control subjects spent equivalent time investigat-ing the objects, and no differences between groups wereobserved in ambulation, grooming, or risk-assessing behav-iors. In a standard short-term memory paradigm (2 h afterhabituation/training; see Fig. 2a), control rats demonstrated astrong preference for the novel place and novel objects, butdiabetic animals failed to do so. Rats with diabetes alsoexhibited behavioral deficits in terms of shorter avoidanceresponse than the age-matched control group after 15 weeksof induction of diabetes in the passive avoidance test (Fig. 2b).

Escape latency, average distance from the platform, swim-ming path length, and mean swimming velocity to the hiddenplatform during acquisition trials are shown in Fig. 2c–h.Overall performance was decreased after the 15th week ofdiabetes induction compared to age-matched nondiabetic rats.We alsomeasured the mean swim speed throughout the test, inorder to exclude differences in navigation speed, which couldaccount for the differences observed in the previous parame-ters. Importantly, we did not observe a significant decrease inthe mean swim speed throughout the test (F(4, 277)=1.3604,p>0.05). This indicates that spatial navigation impairmentwas not due to a deficit in swimming speed. To assess theaccuracy of the rat’s goal-oriented behavior during learning,we calculated the average rat-to-platform distance during atrial (Fig. 2d). Diabetic rats showed a longer mean distance

relative to the platform than control animals over the entiretraining period (group (F(1, 11)=8.0000; p<0.05), day (F(4,239)=23.2486; p<0.001), and group × day (F(4, 239)=3.1447;p<0.05). These results indicate that the trajectories of diabeticrats toward the platform were less direct than those used bycontrol rats. The results obtained with the Morris water mazesuggested that diabetic rats could learn to locate the platformbut they executed non-optimal goal-directed trajectories after15 weeks of diabetic induction. The special navigation im-pairment was also evidently observed during probe trial. Di-abetic rats were less focused in their search for the location ofthe platform compared to the control group. Diabetic animalsdid not show significant difference between times spent intarget quadrant vs nontarget quadrants compared to controlanimals (Fig. 2g). Spatial/persistent and goal-directed swimstrategies were observed by the control animals, whereasnonspatial/random strategy was followed by diabetic animalsto solve the Morris water maze task.

GSK-3β Inhibition Ameliorated Memory Deficits in Type 2Diabetic Rats

SB216763 treatment at doses 0.6 and 1.2 mg/kg showed asignificant increase (p<0.001) in preference of the previouslyvisited arm during acquisition trial as compared to the diabeticcontrol group (Fig. 3a). This tendency to retain more in theexplored arm was observed as opposite behavior compared tothe normal control group. This behavior might be due toincreased anxiety behavior after 18 weeks of diabetes, and insuch cases, the animal identified the previously visited arm butfailed to explore a novel place. This effect was not observed inthe lower dose (0.3 mg/kg) of SB216763.

The antiamnesic effect of GSK-3β inhibitor (SB216763)treatment followed a dose-dependent trend, although the 0.3-mg/kg dose failed to show a significant increase in avoidanceresponse compared to diabetic rats. However, rats treated with

Table 1 Effect of SB216763 on body weight and biochemical parameters

Body weight (g) Plasma glucose (mg/dl) Plasma insulin (pmol/l) HbA1c (% of THb)

15 weeks

NPD 350.0±10 105.0±7.0 304.4±7.5 5.4±0.4

HFD+STZ 311.8±31.5 338.0±31.1*** 130.0±6.6*** 16.2±2.6***

18 weeks

NPD 443.0±8.0 106.0±6.0 301.2±7.5 8.4±0.5

HFD+STZ 323.0±23.6* 328.5±6.1*** 139.7±8.1*** 18.3±0.14***

SB 0.3 mg/kg 314.3±18.4** 335.8±18.1*** 166.5±13.6*** 19.8±2.1***

SB 0.6 mg/kg 339.5±11.2* 343.8±23.4*** 124.6±1.2*** 22.9±0.9***

SB 1.2 mg/kg 327.6±20.7* 312.2±12.1*** 146.1±5.2*** 18.2±2.08***

Data are expressed as mean±SEM. There were 7–13 animals in each experimental group and at each time point

HbA1c glycated hemoglobin, THb total hemoglobin* p<0.05, ** p<0.01, *** p<0.001 vs age-matched NPD group

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SB216763 at the doses of 0.6 and 1.2 mg/kg did not differsignificantly in avoidance response compared to each other.Latency to enter the dark compartment during acquisition andretention trials is illustrated in Fig. 3b. Rats receivingSB216763 at a dose of 1.2 mg/kg showed a maximum reten-tion of inhibitory avoidance response.

To verify the effect of SB216763 treatment on spatiallearning and memory, we examined rats using the Morriswater maze (MWM) test (Fig. 3c–f). A repeated measuresANOVA with a Greenhouse-Geisser correction determinedthat learning differed significantly between time points(F(3.95, 176.566)=103.374, p<0.001). Post hoc tests using theBonferroni correction revealed that SB216763 at a dose of0.3 mg/kg had a significant impairment in learning comparedto control (p<0.01) and did not show any effect on learningcompared to diabetic control (p=1.00). Further, impairment inlearning was completely reversed in diabetic animals on0.6 mg/kg treatment (p=1.00). Further, there is no significantimprovement at 1.2 mg/kg compared to the 0.6-mg/kg dose(p=1.00). Therefore, it can be concluded that SB216763 at adose of 0.6 mg/kg elicits a statistically significant improve-ment in diabetes-induced learning deficits. Animals showedmore tendencies to swim in the platform quadrant (SW)compared to other quadrants (Fig. 3d). Despite evidence foracquisition of the platform position in control, diabetic,SB216763-treated rats, the probe trial given 24 h after the lasttesting day revealed that SB-treated (0.6 and 1.2 mg/kg) ratsfocused their search on the former location of the platform(Fig. 3f). They spent significantly (p<0.05) more time in theappropriate quadrant when compared to the oppositequadrant.

GSK-3β Inhibition Reversed Diabetes-Induced BrainNeurotransmitter Changes

Cholinergic dysfunction was assessed by AChE activity in thehippocampus and frontal cortex. A variable effect of diabeteson brain AChE activity was observed in different brain

regions on the 18th week after induction of diabetes (Fig. 4).Twofold increases in the AChE activity was observed in thehippocampus of diabetic rats compared to normal control rats.AChE activity of the cortical regions was increased in diabeticrats but failed to show a significant difference (p=0.432)compared to control. Treatment with SB216763 restored thechanges in AChE activity in rat hippocampus but did not showa significant effect in frontal cortex.

Glutamate and GABA levels were measured in NPD-,HFD + STZ-, and SB216763-treated groups in brain regionsand depicted in Fig. 5a, b. Diabetes after 18 weeks of induc-tion significantly affected brain glutamate level. Glutamatelevels were significantly (p<0.05) decreased in the hippocam-pus of diabetic rats compared to age-matched control rats.Similarly, an effect of diabetes was also observed in the frontalcortex (p<0.01) region. Increase in brain glutamate level wasobserved in the hippocampus and cortex region on treatmentwith SB216763 which further supported the improvement inmemory in the behavioral paradigm test. Further, GABAwassignificantly increased (threefold) in the hippocampus of dia-betic rats (p<0.001) (Fig. 5b). Similarly, in the frontal cortex,GABA levels were observed significantly (p<0.05) higher in18-week diabetic animals compared to the age-matched con-trol group. Treatment with SB216763 showed a variable effectin restoration of GABA level to normal. Lower doses(0.3 mg/kg) showed a higher effect in the hippocampus com-pared to higher doses. Moreover, a decrease in GABA levelwas observed in the frontal cortex which was similar at allthree doses used in the study.

GSK-3β Inhibition Increased CREB Phosphorylationin the Rat’s Hippocampus and Frontal Cortex

Diabetes significantly decreased phosphorylation of GSK-3βin both hippocampus (p<0.01) and cortex (p<0.05). However,total GSK-3β levels were not found to be significantly altered.Treatment with SB216763 significantly increased the inhibi-tory phosphorylation of GSK-3β (Ser9) in both hippocampusand cortex in a dose-dependent manner (Fig. 6a, b). To furtherinvestigate whether diabetes significantly affects CREB activ-ity in the hippocampus and cortex, total CREB and phosphor-ylation of CREB were measured. CREB is the downstreamsignalingmolecule of GSK-3β and well known to be involvedin memory and learning processes. The basal CREB level wasfound to be unchanged in the cortex and hippocampus of type2 diabetic rats compared to NPD-nourished and type 2 dia-betic rats after 18 weeks (Fig. 7a, b). Activation of CREB wasevidently observed in parallel to increased Morris water mazeand other behavioral performance on GSK-3β inhibition.Elevated pCREB (Ser133) levels was observed onSB216763 treatment in both hippocampus and cortex com-pared to age-matched diabetic rats at all three dose levels.Moreover, GSK-3β inhibition also significantly increased

�Fig. 2 Effect of 15-week type 2 diabetes on apercentage of time spent inthe respective zones during the Y-maze performance test, **p<0.01 novelarm vs explored arm (Student’s t test); b transfer latency during thepassive avoidance task, ***p<0.001 acquisition vs retention trial (Stu-dent’s t test), ###p<0.001 NPD vs STZ-HFD retention trial (two-wayANOVA); c latency to find platform during the Morris water maze(MWM) learning trial, *p<0.05, **p<0.01, and ***p<0.001 NPD vsSTZ-HFD; d average distance from platform during MWM learningtrials, ***p<0.001 NPD vs STZ-HFD; e average swimming speed inMWM learning trials; f integrated path length to platform during learningtrials, **p<0.01 and ***p<0.001 NPD vs STZ-HFD; g percentage of timespent in respective quadrant during theMWMprobe trial, ***p<0.001NEvs SW quadrant (Student’s t test); h searching strategy during the MWMprobe trial. Numerical values are presented as mean ± SEM. SW south–west, NW north–west, SE south–east, NE north–east, NPD normal pelletdiet, HFD high-fat diet, D day

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SB (0.3

mg/kg

)

SB (0.6

mg/kg

)

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mg/kg

)0

10

20

30

40

NENW SE SW

***

**

(d)

Tim

e (s

)

(e) (f)

Fig. 3 Effect of GSK-3β inhibitor SB216763 on a percentage of timespent in the respective zones during the Y-maze performance test,***p<0.001 novel vs explored arm (Student’s t test); b transfer latencyduring the passive avoidance task, *p<0.05 and **p<0.01 vs NPD reten-tion trial; ##p<0.01 and ###p<0.001 vs HFD + STZ retention trial (one-way ANOVA post hoc Tukey’s test); c latency to find platform during the

Morris water maze (MWM) learning trial; d time spent in the respectivequadrant during the MWM probe trial, *p<0.05 and ***p<0.001 NE vsSW quadrant (Student’s t test); e searching strategy during the MWMprobe trial; f representative track plot of the MWM probe trial. Numericalvalues are presented as mean ± SEM (n=7–14). SW south–west, NWnorth–west, SE south–east, NE north–east, NPD normal pellet diet, HFD

high-fat diet, SB SB216763, D day

Mol Neurobiol

CREB activation in the cortex on 0.3 mg/kg (p<0.05) and1.2 mg/kg (p<0.01) compared to age-matched nondiabeticrats (Fig. 7b).

GSK-3β Inhibition Decreased Diabetes-InducedNeuroinflammation

NF-κB, iNOS, and COX-2 Expression

Total NF-κB was found to be significantly higher in thehippocampus and cortex of diabetic rats (Fig. 8a, b).SB216763 (GSK-3β inhibitor) treatment showed a dose-dependent effect in the reduction of the total NF-κBlevel in diabetic rats. NF-κB-mediated transcription ofproinflammatory mediators is activated by phosphoryla-tion of p65(Ser536) unit of NF-κB. pNFκB-p65/β-actinlevel was significantly increased in the hippocampusand cortex of diabetic rats. However, pNFκB-p65/NFκB-p65 ratio did not significantly change in diabeticrats which showed a parallel increase in synthesis andactivation of NF-κB. Moreover, GSK-3β inhibitionshowed a significant decrease in total pNFκB-p65 levelin a dose-dependent manner. However, pNFκB-p65/NFκB-p65 ratio was found significantly lower comparedto NPD control rats at a higher dose (1.2 mg/kg) of

SB216763 in both hippocampus and cortex. Reductionin pNF-κB level was in line with the reduction of totalNF-κB.

COX-2 and iNOS have been shown to be the keyenzymes involved in neuroinflammation and diabetes indownstream to NF-κB. Thus, to assess the effect ofdiabetes-associated neuro-inflammatory stress, we exam-ined two marker enzymes (COX-2 and iNOS) of inflam-mation in brain hippocampus. A significant increase inexpression of both iNOS (p<0.05) and COX-2 (p<0.001)was observed in the hippocampus of diabetic rats(Fig. 9a, b). GSK-3β inhibition by SB216763 showed adose-dependent effect on elevated expression of iNOS inthe hippocampus. Moreover, SB216763 treatment at adose of 1.2 mg/kg showed significantly lowered(p<0.01) expression of iNOS in the hippocampus of dia-betic rats compared to age-matched control rats. However,a significant (p<0.01) decrease in COX-2 expression inthe hippocampus was observed only at the highest dose(1.2 mg/kg).

IL-6 and TNF-α Levels

IL-6, a naturally occurring cytokine, plays a central role inthe inflammatory response in immediate, acute, and

Hippocampus F. Cortex0

10

20

30

40

50NPD

HFD+STZ

SB (0.3 mg/Kg)

SB (0.6 mg/Kg)

SB (1.2 mg/Kg)

***

###

Ac

hE

ac

tiv

ity

(m

ole

sh

yd

roly

ze

d/m

in/g

tis

su

e)

Fig. 4 Effect of SB216763 on brain AChE activity in diabetic rats. Results are expressed as mean ± SEM (n=3–7). Data were analyzed by ANOVAfollowed by post hoc Tukey’s test. ***p<0.001 vs NPD and ###p<0.001 vs HFD-STZ. NPD normal pellet diet, HFD high-fat diet, SB SB216763

Hippocampus F. Cortex0.000

0.005

0.010

0.015

0.020

0.025 NPD

HFD+STZ

SB (0.3 mg/Kg)

SB (0.6 mg/Kg)

SB (1.2 mg/Kg)

**

##

**

###

(a)

Glu

tam

ate

leve

ls(m

M/g

of

tiss

ue)

Hippocampus F. Cortex0.000

0.005

0.010

0.015

0.020 NPD

HFD+STZ

SB (0.3 mg/Kg)

SB (0.6 mg/Kg)

SB (1.2 mg/Kg)#

##

*(b)

*

#

GA

BA

leve

ls(m

M/g

of

tiss

ue)

Fig. 5 Effects of SB216763 on brain neurotransmitters: a glutamate andbGABA level in diabetic rats. Data are represented as mean ± SEM (n=3–6) and analyzed by ANOVA followed by post hoc Tukey’s test.

*p<0.05 and **p<0.01 vs NPD; #p<0.05, ##p<0.01, and ###p<0.001 vsHFD + STZ. NPD normal pellet diet, HFD high-fat diet, SB SB216763

Mol Neurobiol

chronic inflammation. Brain hippocampus of diabetic an-imals contained approximately two times of the IL-6compared to normal animals (Fig. 10a). However, frontalcortex IL-6 levels in diabetic rats were not significantlydifferent compared to age-matched control animals.SB216763 treatment to diabetic animals showed a dose-dependent effect on elevated hippocampus IL-6 levels.SB216763 treatment with a higher dose (1.2 mg/kg) sig-nificantly (p<0.05) lowered the hippocampal IL-6 levelscompared to age-matched nondiabetic rats. Similarly, thenegative impact of diabetes on TNF-α level was observedon the 18th week of diabetic rat brain. A significantlyhigher level of TNF-α was present in the hippocampus ofdiabetic rats (p<0.05) and cortex (p<0.01) compared tonondiabetic rats (Fig. 10b). SB216763 showed a signifi-cant effect on TNF-α level in diabetic animal’s hippocam-pus as well as in the cortex. GSK-3β inhibition in thehippocampus of diabetic rats showed a dose-dependenteffect on TNF-α level.

Discussion

This study demonstrated the protective effects of SB216763, aGSK-3β inhibitor in diabetes-induced cognitive deficits. Anumber of reports have implicated that both T1D and T2D

have a negative correlation with cognitive function. Diabetesand long-term hyperglycemia can induce multifaceted chang-es ranging from neurovascular to synapses [10, 32]. Cognitivedeficits associated with T2D are still not extensively studied.In the present study, we used a high-fat diet combined with asingle low-dose injection of STZ to induce type 2 diabetes,and cognitive functions were assessed using Y-maze, passiveavoidance, and Morris water maze test. Fifteen weeks of T2Dduration resulted in a significant decrease in cognitive func-tions. Significant impairment in learning and memory in dia-betic animals was observed in the Morris water maze test. Thestrategy to find the platform, the indicator of executive func-tion, was also impaired in diabetic rats. Short-term memoryfunctions were assessed using Y-maze paradigm, and diabeticanimals showed a significant alteration in memory comparedto control animals. Moreover, the 15-week diabetes durationalso showed a negative impact on fear memory in the passiveavoidance task. Similarly, cognitive deficits have been report-ed in animal models of type 2 diabetes and obesity; however,the time course of development of cognitive deficits differs inthese models [11, 33, 34]. The 3-week treatment withSB216763 (GSK-3β inhibitor) reversed significantly thediabetes-induced cognitive deficits. Although per se the effectof SB216763 on memory parameters in control (NPD) ani-mals was not investigated in present study, Guo et al. reportedthat SB216763 treatment in control mice had no effect on

GSK-3 / -Actin pGSK-3 / -Actin pGSK-3 /GSK-30

50

100

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200

NPD

HFD+STZSB (0.3 mg/kg)SB (0.6mg/kg)SB (1.2 mg/kg)

####

**

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**

####

**

####

**

######

**

Rel

ativ

e de

nsit

y (%

to N

PD

)GSK-3 / -Actin pGSK-3 / -Actin pGSK-3b/GSK-3b

0

50

100

150

# ##

*

###

**

Rel

ativ

e de

nsit

y (%

to N

PD

)

(a)

(b)

Fig. 6 Immunoblotting analysis of GSK-β expression in the a hippo-campus and b frontal cortex. Data are represented as mean ± SEM (n=3)and analyzed byANOVA followed by post hoc Tukey’s test. *p<0.05 and

**p<0.01 vs NPD; #p<0.05, ##p<0.01, and ###p<0.001 vs HFD + STZ.NPD normal pellet diet, HFD high-fat diet, SB SB216763

Mol Neurobiol

cognition [24]. SB216763 inhibited the GSK-3β kinase ac-tivity and increased levels of pGSK-3β (Ser9) in the hippo-campus and cortex of diabetic rats. Moreover, SB216763treatment increased the expression of transcription factor pro-tein such as pCREB. NF-κB activation was also associatedwith diabetes in the brain that was inhibited by SB216763treatment. Improved cognition by GSK-3β inhibition wasfurther supported by reversal of change in glutamate andGABA levels. Similarly, AChE activity was also improvedin the study on SB216763 treatment in diabetic rats. Takentogether, cognitive deficits induced by diabetes were associ-ated with decreased CREB-mediated transcription, disruptionof neurotransmitter homeostasis, and increased neuroinflam-mation which can be ameliorated by inhibition of GSK-3β.

Diabetes-induced cognitive impairment is the conse-quence of prolonged uncontrolled hyperglycemia [10,32, 35]. Antihyperglycemics, insulin sensitizers (PPAR-γactivators), and antioxidants have been found to be ben-eficial in diabetes-induced cognitive dysfunctions [33, 34,36, 37]. Moreover, insulin resistance and type 2 diabetesis responsible for memory deficits [38]. Insulin is also

reported to improve memory dose-dependently that indi-cates insulin receptors and its signaling are present in thebrain and have a role in memory [39]. GSK-3β is aconstitutively active kinase, which regulates multiple bi-ological processes. Numerous stimuli lead to inactivationof GSK-3β through the phosphorylation at Ser9. GSK-3βalso plays a significant role ranging from neuronal differ-entiation and survival to synaptic plasticity [16]. More-over, GSK-3β is found to be involved in the pathology ofneurodegenerative and memory disorders [40, 41]. Phar-macological inhibition of GSK-3β has been found to bebeneficial and provide an opportunistic target for learningand memory disorders [16]. In peripheral tissues, insulinand its signaling are reported to be a major contributor forinhibitory phosphorylation of GSK-3β at Ser9 anddiabetes/insulin resistance was found to reduce inhibitoryphosphorylation of GSK-3β at Ser9 [42]. There are a fewand contradictory reports available regarding the impactof diabetes or insulin resistance on GSK-3β activity in thebrain. Clodfelder-Miller et al. reported increased phos-phorylation of GSK-3β in db/db insulin-resistant mice

CREB/ -Actin CREB/ -Actin pCREB/CREB0

50

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NPDHFD+STZSB (0.3 mg/kg)SB (0.6mg/kg)SB (1.2 mg/kg)

**

##

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**

Rel

ativ

e de

nsit

y (%

to N

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CREB/ -Actin pCREB/ -Actin pCREB/CREB0

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****

****

##

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**

**

###

##

Rel

ativ

e de

nsi

ty (

% t

o N

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)

(a)

(b)

Fig. 7 Immunoblotting analysis of CREB expression in the ahippocam-pus and b frontal cortex. Data are represented as mean ± SEM (n=3) andanalyzed by ANOVA followed by post hoc Tukey’s test. *p<0.05,

**p<0.01, and ***p<0.001 vs NPD; #p<0.05, ##p<0.01, and ###p<0.001vs HFD + STZ.NPDnormal pellet diet,HFDhigh-fat diet, SBSB216763

Mol Neurobiol

after 4 weeks [43]. Contradictory to that, decreased phos-phorylation of GSK-3β in the hippocampus of STZ andHFD + STZ diabetic rats were also reported [44-46]. Inthe present study, we observed unchanged total GSK-3βlevel but decrease in phosphorylation in both hippocam-pus and cortex of diabetic rats after 18 weeks of diabetesinduction in HFD- and STZ-induced T2D model. De-creased phosphorylation of GSK-β at Ser9 is evidentlyassociated with decreased performance in memory para-digm (MWM and passive avoidance). Further, in thepresent study, increased pGSK-3β level with parallel im-provement in cognitive performance was observed on

SB216763 treatment in both hippocampus and cortex ofdiabetic rats.

GSK-3β phosphorylates and modulates the activity ofvarious substrates including transcription factors such asCREB, Nrf-2, and NF-κB. CREB is a key transcription reg-ulator involved in several critical functions of the brain in-cluding learning, memory, neuronal plasticity, and survival[47, 48]. In the present study, we observed a significantdecrease in Ser133 phosphorylation of CREB in diabetic rats.Decrease in pCREB (Ser133) level was reported by manyinvestigators, and our results are also in line with the previousreports [48, 49]. GSK-3β is found to be a negative regulator

NF B-p65/ -Actin pNFkB-p65/ -Actin pNFkB-p65/NFkB-p650

50

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NPD

HFD+STZ

SB (0.3 mg/kg)

SB (0.6mg/kg)

SB (1.2 mg/kg)#

##

***

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##

***

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Rel

ativ

e de

nsit

y (%

of N

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)

NFkB-p65/b-Actin pNFkB-p65/b-Actin pNFkB-p65/NFkB-p650

50

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***

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##

***###

**

##

******###

####

Rel

ativ

e de

nsit

y (%

of N

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)

**(b)

(a)

β β

Fig. 8 Immunoblotting analysis of NF-κB expression in the a hippo-campus and b frontal cortex. Data are represented as mean ± SEM (n=3)and analyzed by ANOVA followed by post hoc Tukey’s test. **p<0.01

and ***p<0.001 vs NPD; #p<0.05, ##p<0.01, and ###p<0.001 vs HFD +STZ. NPD normal pellet diet, HFD high-fat diet, SB SB216763

iNOS COX-20

100

200

300 NPDHFD+STZSB (0.3 mg/kg)SB (0.6mg/kg)SB (1.2 mg/kg)

##**

**

***

###

Rel

ativ

e de

nsit

y(%

to

NP

D)

Fig. 9 Immunoblotting analysis of iNOS and COX-2 expression in thehippocampus. Data are represented as mean±SEM (n=3) and analyzedby ANOVA followed by post hoc Tukey’s test. *p<0.05 and **p<0.01 vs

NPD; #p<0.05 and ##p<0.01 vs HFD + STZ. NPD normal pellet diet,HFD high-fat diet, SB SB216763

Mol Neurobiol

for CREB function. The increased kinase activity of GSK-3βis reported to inhibit CREB-CBP complex formation which isessential for DNA binding of CREB which can be facilitatedby lithium [50]. Moreover, GSK-3β also represses severalgenes downstream to the CREB playing a transcriptional role[51].We observed that pharmacological inhibition of GSK-3βreversed the pCREB (Ser133) level which supports improve-ment in cognition by SB2167633 treatment. Similarly, NF-κBis another transcriptional factor affected in diabetes and fun-damental to the pathophysiology of diabetes. Activation ofNF-κB-mediated transcription is very well reported and isknown to involve in peripheral and CNS complications ofdiabetes [30, 52]. Similar findings were observed in the pres-ent study with increased phosphorylation of p65 componentof NF-κB in diabetic rats compared to age-matched control.NF-κB plays a central role in the regulation of proinflamma-tory and inflammatory mediators such as TNF-α, IL-6, COX-2, and iNOS, which leads to alteration in neuronal microen-vironment and seems to be a key factor in the developmentand progression of memory-related disorders [53, 54].TNF-α, IL-6, and COX-2 are found to have a detrimentaleffect on long-term potentiation and memory formation ininflammatory conditions [54]. Our results also support thenegative correlation of the elevated inflammatory markerswith reduced cognitive function in diabetic rats. Moreover,NF-κB function is strikingly affected by GSK-3β. NF-κB andCREB share similar transcriptional coactivator p300/CBP.Activated GSK-3β increases the phosphorylation of p65 pro-tein of NF-κBwhich recruited complex CBP and binding withthe promoter region of DNA [55]. Inhibition of GSK-3β bySB216763 showed a significant decrease in phosphorylationof p65 component and reduced the level of inflammatorymediators in the hippocampus and cortex in the present study.Our results are supported by earlier findings related to thetranscriptional association between CREB and NF-κB.

Cholinergic transmission dysfunction is evidently ob-served in neurological disorders and dementia. Acetylcho-line, a neurotransmitter associated with learning and memo-ry, is degraded by the enzyme acetylcholinesterase,

terminating the physiological action of the neurotransmitter.The changes in AChE activity might reflect impairment inbiosynthesis, degradation, or insertion into the plasma mem-brane. Diabetes is known to cause membrane alterations thatcan affect the kinetic properties of the membrane-boundcholinesterases [56, 57]. The activity of AChE was evidentlyaffected in diabetes and varies in the brain region. Increase inAChE activity was observed in the cerebral cortex of STZ-induced diabetes rats [58]. Furthermore, AChE activity wassignificantly increased in the hippocampus of 18-week dia-betic animals in this study. Metabolism and neurotransmis-sion are closely linked, especially through the major excit-atory neurotransmitter glutamate. Glutamate is primarily syn-thesized and stored in glutamatergic neurons, released uponexcitation and removed mostly by uptake into astrocytesthrough glutamate-aspartate transporter (GLAST) and gluta-mate transporter (GLT) and forms a glutamate-glutaminecycle [59]. Glutamate plays a key role in synaptic plasticityand cognition [60]. Glutamate was found to decrease signif-icantly in the hippocampus and cortex of diabetic rats ascompared to age-matched control animals in line with thestudy of Zucker fatty diabetic rats by Sickmann et al. [61]. Adecrease in glutamate pool was observed with altered synap-tic plasticity and cognitive deficits, and similar results wereobserved in the present study on diabetic animals [60-62].Reduction of the glutamate level in the present study may bea contributory effect of impaired glutamate-glutamine turn-over and gap junction function between neuron and astro-cytes [63]. However, further studies are warranted to explorethe impact of diabetes in various enzymes associated withglutamate-glutamine cycling. In the present study, GABAlevel was found to increase with reduction in glutamate levelin hippocampal and cortical regions of diabetic brain. Simi-larly, decreased glutamate/GABA was observed in Zukardiabetic fatty rats by Sickmann et al. [61, 64]. Many studiessupport the concept of inhibition of memory formation bythe GABAergic system. Drugs which modulate theGABAergic system at different stages like GABA synthesis,GABA binding site, or chloride channels significantly affect

Hippocampus F. Cortex0

5

10

15NPDHFD+STZSB (0.3 mg/Kg)SB (0.6 mg/Kg)SB (1.2 mg/Kg)

*

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**

IL-6

(p

g/m

g o

f p

rote

in)

Hippocampus F. Cortex0.0

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NPDHFD+STZSB (0.3 mg/Kg)SB (0.6 mg/Kg)SB (1.2 mg/Kg)

*

###

###**

(b)

**

*

**

###

TNF

- (

pg

/mg

of

pro

tein

)

Fig. 10 Analysis of a IL-6 and b TNF-α levels in the hippocampus andfrontal cortex. Data are represented as mean±SEM (n=3) and analyzedby ANOVA followed by post hoc Tukey’s test. *p<0.05 and **p<0.01 vs

NPD; #p<0.05, ##p<0.01, and ###p<0.001 vs HFD + STZ. NPD normalpellet diet, HFD high-fat diet, SB SB216763

Mol Neurobiol

memory and cognitive ability [65]. Similarly, GABAantagonist and some GABA inhibiting steroids likepregenolone-sulfate show significant improvement inlearning and memory [66, 67]. We observed an increasein GABA level in the hippocampus of diabetic rats and itmay be one of the possible reasons for the impairedavoidance response in diabetic animals. In the T2D mod-el, GABA levels were increased suggesting that brainglycogen serves a role in maintaining a proper ratiobetween excitatory and inhibitory neurotransmitters inT2D. In the present study, inhibition of GSK-3 restoredthe neurotransmitter homeostasis which may be due toretained glycogen-glutamate-GABA homeostasis in thebrain. Further, neuroinflammation induced by diabetesalso impaired the neuron-astrocytes gap junction commu-nication [68]. Inhibition of NF-κB mediates transcription,and proinflammatory cytokines by SB216763 may be anadditive effect to maintain neurotransmitter homeostasis.Thus, diabetes causes metabolic disturbances in neuronaland astrocyte pathways related to energy metabolism,glucose storage, and the excitatory and inhibitoryneurotransmission.

In conclusion, the data presented here strongly supportsthat overactivation of GSK-3β activity may cause cognitivedeficits in type 2 diabetes. Cognitive deficits induced bydiabetes involved intricate compartmental interaction betweentranscription factor and neurotransmitter homeostasis/energymetabolism, and GSK-3β might play a central role. Pharma-cological inhibition of GSK-3β ameliorated diabetes-inducedcognitive deficits.

Acknowledgments We would like to thank the Department of Phar-maceuticals, Ministry of Chemicals and Fertilizers, Government of Indiafor financial support. Mr Ashok Kumar Datusalia is a recipient of aresearch fellowship from University Grant Commission, New Delhi.

Conflict of Interest The authors declare that they have no conflicts ofinterest.

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