Debapriya Garabadu and Sairam Krishnamurthy. 1. Introduction

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1 Hindwi Publishing Corportion BioMed Reserch Interntionl Volume 214, Article ID 69374, 15 pges Reserch Article Dizepm Potentites the Antidibetic, Antistress nd Anxiolytic Activities of Metformin in Type-2 Dibetes Mellitus with Cooccurring Stress in Experimentl Animls Debpriy Grbdu nd Sirm Krishnmurthy Neurotherpeutics Lb, Deprtment of Phrmceutics, Indin Institute of Technology (Bnrs Hindu University), Vrnsi 2215, Indi Correspondence should be ddressed to Sirm Krishnmurthy; Received 22 Februry 214; Revised 7 My 214; Accepted 9 My 214; Published 4 June 214 Acdemic Editor: Andre Mencrelli Copyright 214 D. Grbdu nd S. Krishnmurthy. This is n open ccess rticle distributed under the Cretive Commons Attribution License, which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. Psychologicl stress is considered s one of the limiting fctors in the mngement of type-2 dibetes mellitus (T2DM). Therefore, the bsic objective of the present study ws to evlute the ntidibetic effect of metformin, dizepm, nd their combintion in cooccurring T2DM nd stress condition (). T2DM ws induced in the mle rts by dministering streptozotocin (45 mg/kg, i.p.) nd nicotinmide (11 mg/kg, i.p.) with time lg of 15 min. Rts were subjected to two sessions of cold restrint stress prdigm for one hour on the sixth nd seventh dy fter streptozotocin injection. Administrtion of metformin (25 mg/kg, p.o.) nd dizepm (1 mg/kg, p.o.) in combintion from the seventh to thirteenth dy fter streptozotocin injection showed better improvement in glucose tolernce nd insulin sensitivity compred to monotherpy of either drug. In ddition, the combintion significntly ttenuted -induced hyperglycemi, hypertriglyceridemi, hypercorticosteronemi, nxiety-like behvior, nd insulin resistnce through modulting insulin signling pthwy in the liver compred to monotherpy. Further, improvement of mitochondril function, integrity, nd oxidtive stress in hippocmpus, hypothlmus, prefrontl cortex, stritum, mygdl, nd nucleus ccumbens ws observed with the combintion. Therefore, metformin in combintion with dizepm my be better therpeutic option in the mngement of T2DM with cooccurring stress condition. 1. Introduction Dibetes mellitus is the most common chronic endocrine metbolic disorder. The prevlence of dibetes is rising rpidly worldwide with projected estimtion of bout 439 million dibetic people globlly by 23 [1]. The mjority of the dibetic ptients belong to the type-2 clss of dibetes mellitus (T2DM). Considerble reserch hs confirmed biologicl nd behviorl vribles s risk fctors for the development of T2DM [2]. Psychologicl distress hs long been considered s one of the predisposing fctors in the development of T2DM. Stress is n versive stimulus which disturbs physiologicl homeostsis nd is reported to ply n importnt role in the genesis nd pthophysiology of different psychologicl disorders [3].Stresscnbemesured s the ctul exposure to events ssumed to be stressful, or s n individul s interprettion nd perception of stressors nd cn be ssessed cross ll contexts of life. However, the role of psychosocil risk fctors, including psychologicl chrcteristics, socil reltionships, nd stressors, in the development of T2DM hs received much less ttention. Only smll number of studies hve explored the role of psychosocil stress on the development of dibetes. Clinicl studies suggest tht there is strong reltionship between psychologicl stress nd dibetes. One study reports tht there is one mesure of work stress, effort-rewrd imblnce ( model bsedontheimportnceofthereciproclblncebetween theeffortspenttworkndtheintrinsicndextrinsicrewrds received), nd dibetes incidence in men but not in women [4]. In contrst, nother mesure of work stress, iso-strin (the combintion of high demnds, low control, nd socil isoltion) s predictor of dibetes incidence in women but not in men [5]. A met-nlysis of the work stress-dibetes literture filed to show sttisticlly significnt reltionships

2 2 BioMed Reserch Interntionl between ny individul spect of work-relted stress nd risk of dibetes [6]. Recently, preclinicl study revels tht psychologiclstressftert2dminductionggrvtesthe progression of dibetes [7]. Thus, psychologicl stress could be n importnt fctor in the mngement of T2DM. Insulin resistnce is considered s n importnt pthophysiologicl mechnism for the progression to both T2DM nd neurodegenertion conditions [8 1]. In most cses, insulin resistnce is ssocited with complex network of signling pthwys, including reduced insulin-stimulted tyrosine phosphoryltion of insulin receptor (IR) nd insulin receptorsubstrte(irs)swellsaktserinephosphoryltion in the min trget tissues of insulin, including the liver, skeletl muscle, nd dipose tissue [11, 12]. Severl studies hve recommended tht the phosphoryltion levels of IRS- 1 on serine residue 37 (IRS-1 ser37 ) nd of Akt on serine residue 473 (Akt ser473 ) in rodents could be used s insulin resistnce mrkers [1, 13, 14]. Thus, informtion regrding insulin resistnce would be crucil in the development of novel drugs in the phrmcotherpy of cooccurring T2DM nd stress condition. Mitochondrireproducedinthecellbody,trnsported to specific neuronl loctions of incresed energy demnds such s synpses [15], nd ply n importnt role in the neuronl ctivity pertinent to specific neurotrnsmitters [16, 17]. Brin mitochondril function hs been reported to declineinbothdibetesndstressconditions[18], indicting tht mitochondril dysfunction is common to both disorders. Recently, it hs been reported tht mitochondril electron trnsport chin enzyme ctivity incresed while mitochondril integrity decresed with the cooccurring T2DM nd stress condition in experimentl nimls [7]. Moreover, both hyperglycemi [19] nd stress [2] re reported to elicit n increse in rective oxygen species production in brin. Incresed mitochondril biogenesis is prt of the cellulr response to oxidtive stress [21]. Therefore, s mitochondri re the common substrte for both T2DM nd stress, drugs trgeted to mitochondri would be better therpeutic option in the mngement of T2DM with cooccurring stress condition. Metformin (N, N-dimethylimidodicrbonimidic dimide) is one of the most widely used ntihyperglycemic gents s the first-line drug therpy for mngement of T2DM [22, 23]. Metformin inhibits gluconeogenesis through mechnisms linked to perturbtion of mitochondril function [24].ComplexIofmitochondrilrespirtionchinis considered s one of the possible trgets of metformin ction [25]. Metformin lso inhibits mitochondril trnsition pore nd mitochondri-linked cell deth [26]. Benzodizepines re commonly prescribed nxiolytics to T2DM ptients with history of stress [27, 28]. Dizepm is widely prescribed for the tretment of nxiety, insomni, or stress disorders. Dizepm ugmented the blood glucose level in hyperglycemic rts; however, it did not lter the sme in presence of metformin in the nimls [29]. Thus, the ntihyperglycemic ctivity of metformin is not impired in the presence of dizepm. Despite its promising neuroprotective properties, the exct mechnism of dizepm in neuroprotection is not fully understood. It hs lso been reported tht dizepm cts on trnsloctor proteins prt from other ctions such s gmm-mino butyric cid-a receptor stimultion nd hypothermi [3]. Trnsloctor proteins locted t the contct site between outer nd inner mitochondril membrne ndthusregultethemitochondri-linkedpoptosis[31, 32]. Therefore,bothmetforminnddizepmhvemitochondril effects prt from other reported mechnisms. As stress cn influence dibetogensis, the mngement of T2DM by the combintion therpy could be better choice over monotherpy. Therefore, the study evlutes the ntihyperglycemic nd nti-hypertriglyceridemic ctivity of metformin nd dizepm in T2DM rts with repeted CRS exposure. The level of corticosterone in the blood nd ulcers in the stomch region ws estimted s mesure of stress in the T2DM rts with cooccurring stress condition. Further, the potentil nxiolytic-like effect of metformin nd dizepm ws evluted in the elevted plus mze (EPM) in the bove condition. In ddition, the ntidibetic, ntistress, nd nxiolytic-like ctivity of metformin ws evluted in presence of dizepm in the bove condition. At the moleculr level, the extent of phosphoryltion of IRS-1 nd Akt ws evluted to elborte on the insulin resistnce in the bove condition. At the subcellulr level, mitochondril function nd integrity were investigted in six brin regions such s hippocmpus (HIP), hypothlmus (HYP), prefrontl cortex (PFC), stritum (STR), mygdl (AMY), nd nucleus ccumbens (NAC) to elborte on the mitochondril bsis of the combintion therpy. Oxidtive stress mrkers such s extent of lipid peroxidtion (LPO) nd ntioxidnt enzyme ctivities like superoxide dismutse (SOD) nd ctlse (CAT) were estimtedinthebovebrinregionstostudythemitochondrildependent ntioxidnt mechnism. 2. Mterils nd Methods 2.1. Animls. Mle Chrles Foster strin lbino rts (2 25 g) were purchsed from the Centrl Animl House, Institute of Medicl Sciences, Bnrs Hindu University (BHU), nd were housed in polypropylene cges under controlled environmentl conditions (25 ± 1 C, 45 55% reltive humidity nd 12 : 12 h light/drk cycle). All experiments were conducted in complince with the principles of lbortory niml cre (NIH publiction number 85 23, revised 1985) guidelines. Experiments on nimls were pproved by the Institutionl Animl Ethics Committee of BHU, Vrnsi, Indi (Protocol number Den/11-12/CAEC/328). The nimls hd free ccess to commercil rt feed (Doodh dhr Pshu Ahr, Indi) nd wter d libitum unless stted otherwise during the experiment. Animls were cclimtized for t lest one week before using them for experiments nd exposed only once to every experiment Chemicls. Streptozotocin, thiobrbituric cid (TBA), tetr methyl rhodmine methyl ester (TMRM),nd dexmethsone were procured from Sigm (St. Louis, MO, USA). Antibodies such s phospho-irs-1 ser37, totl IRS,

3 BioMed Reserch Interntionl 3 phosphor-akt ser473,totlakt,ndbet-ctinwerepurchsed from Abcm Plc., Cmbridge, USA. All other chemicls nd regents were vilble commercilly from locl suppliers nd were of nlyticl grde Induction of Cooccurring T2DM nd Repeted CRS (). The T2DM ws induced in overnight fsted rts by single injection of streptozotocin (45 mg/kg, i.p.), 15 min fter nicotinmide (11 mg/kg, i.p.) dministrtion. Streptozotocin ws dissolved in.1 M citrte buffer (ph 4.5) nd nicotinmide ws dissolved in physiologicl sline [33]. Further, two stress sessions 24 hr prt were performed during 8: hr to 12: hr on the 6th nd 7th dy of streptozotocin injection nd were consisting of 1 hr restrint period (rt restriners were trnsprent plstic tubes 15 cm long 6.5 cm width) in 4 Croom[7, 34] The Experimentl Design. The experimentl design consists of three sets of experiments. The nimls were cclimtized for seven dys nd were rndomly divided into five groups, nmely, control, dibetes with repeted CRS (),,, nd + DZ in ech of the experiment. The experimentl protocol ws followed for 13 dys for ll experiments. The dy nimls received the streptozotocin injection ws considered s dy-1(d-1). The rts were exposed to repeted CRS procedures to ll the group nimls except control group rts on D-6 nd D-7. On D-7, fter 1 hr to CRS prdigm, metformin (25 mg/kg, p.o.; [35]) wsdministeredto+metnd+met+dz group nimls while dizepm (1 mg/kg, p.o.; [36, 37]) ws dministered to nd + DZ group rts fter 3 min to metformin tretment. This tretment schedule ws continued for seven consecutive dys, tht is, D-13 of the experimentl design. The experiments 1 nd 2 were performed for the orl glucose tolernce test (OGTT) nd insulin tolernce test (ITT), respectively. In experiment 3,llthenimlswerekilledfter1hrtothelstdose on D-13 of the experimentl schedule by decpittion. The blood nd liver were stored immeditely t 8 Ctillfurther study. The brins were removed nd microdissected [38] into hippocmpus (HIP), hypothlmus (HYP), prefrontl cortex (PFC), stritum (STR), mygdl (AMY), nd nucleus ccumbens (NAC) nd stored immeditely t 8 C till further study Orl Glucose Tolernce Test (OGTT). Orl glucose tolernce test (OGTT) is considered s clssicl nd modelbsedestimteofbet-cellfunction[39]. The OGTT ws performed on overnight fsted rts on the 13th dy of the experimentl schedule. Metformin, dizepm, their combintion, nd vehicle were dministered 6 min prior to glucose dministrtion (2 g/kg, i.g.). The blood smples were collected through retroorbitl puncture just before glucose lod ( min) nd t 3, 6, nd 12 min fter glucose dministrtion. Plsm glucose concentrtions were determined with glucose GOD PAP kit (Primn Instrument Pvt. Ltd., Indi) bsedonglucoseoxidsemethod[4] Insulin Tolernce Test (ITT). Insulin tolernce test (ITT) is simple nd relible method of estimting insulin sensitivity [41, 42]. The ITT ws performed on overnight fsted rts on the 13th dy of the experimentl schedule. Metformin, dizepm, their combintion, nd vehicle were dministered 6 min prior to insulin dministrtion (.4 IU/kg, s.c.). The blood smples were collected through retroorbitl puncture just before glucose lod ( min) nd t 3, 6, nd 9 min fter glucose dministrtion. Plsm glucose concentrtions were determined with glucose GOD PAP kit (Primn Instrument Pvt. Ltd., Indi) bsed on glucose oxidse method [4] Estimtion of Plsm Glucose nd Triglyceride Level. On the D-1, 3, 7, nd 13 of the experimentl protocol, 1 ml ofbloodwscollectedthroughretroorbitlpuncturend centrifuged t 3 gfor5mint4 C[43]toobtinplsm for mesuring the glucose, triglyceride, nd corticosterone levels. The plsm glucose nd triglyceride ws determined spectrophotometriclly (Beckmn Coulter DU 74 UV-VIS Spectrophotometer, Fullerton, CA) in triplicte using the glucose GOD PAP kit (Primn Instrument Pvt. Ltd., Indi) nd triglyceride GPO-PAP kit (Spn Dignostics Ltd., Indi), respectively Estimtion of Plsm Corticosterone Level. The plsm corticosterone ws quntified in High Performnce Liquid Chromtogrphy (HPLC) with Ultrviolet (UV) detector system (Wters, USA), ccording to Woodwrd nd Emery [44] with minor modifictions using dexmethsone s n internl stndrd [45]. Briefly, 5 μl of plsm contining known quntity of dexmethsone ws extrcted with 5 ml of dichloromethne. The dichloromethne extrct ws evported to dryness nd dissolved in 1 μl ofmobilephse. Twenty microliters of extrct ws injected into HPLC system forquntifiction.mobilephseconsistedofmethnol:wter (7 : 3) t flow rte of 1.2 ml/min nd CORT ws detected t 25 nm using UV detector (Model 2849, Wters, USA). The chromtogrm ws recorded nd nlyzed with Empower softwre (Version 2.) Estimtion of Ulcer Index. The stomch ws cut through gretercurvturendblindobserverclcultedtheulcer index by following stndrd protocol [46] Anxiety-Like Behvior in EPM Test. The plus mze consisted of two opposite open rms, 5 1 cm, crossed with two opposite open rms of the sme dimensions with wlls of 4cmhigh.Thermswereconnectedwithcentrlsqure (1 1 cm) to give the pprtus plus-sign ppernce. The mzewskeptelevted5cmbovethefloorindimlylit room.thertswereplcedindividullyonthecentrlsqure of the plus mze fcing n enclosed rm. The percentge time spent nd the numbers of entries mde by the rt, during the next 5 min, on the open rms were recorded s n index of nxiety. Further, the totl rm entries were recorded s n index of locomotor ctivity. An rm entry ws defined when ll four limbs of the rt were on the rm [47].

4 4 BioMed Reserch Interntionl Western Blot Anlysis. For Western blot nlysis, the liver tissues were lysed in buffer contining complete protese inhibitor cocktil. Protein concentrtions were determined ccording to Brdford (1976; [48]). A stndrd plot ws generted using bovine serum lbumin. An liquot of ech smple ws electrophoresed in 1% SDS-PAGE gels for phospho- IRS-1 ser37, totl IRS, phosphor-akt ser473, nd totl Akt proteins, trnsferred to polyvinylidene fluoride membrnes nd probed with specific ntibodies. The membrne ws incubted overnight with polyclonl rbbit nti-phospho- IRS-1 ser37 (1 : 1,, Abcm Plc., Cmbridge, USA), monoclonl nti-totl IRS (1 : 1, Abcm Plc., Cmbridge, USA), monoclonl nti-phospho-akt ser473 (1 : 1,, Abcm Plc., Cmbridge, USA), nd polyclonl nti-totl Akt (1 : 1, Abcm Plc., Cmbridge, USA) primry ntibodies. After detection with the desired ntibodies ginst the proteins of interest, the membrne ws stripped with stripping buffer (25 mm Glycine ph 2., 2% SDS) for 3 min t room temperture nd reprobed overnight with rbbit nti β-ctin polyclonl primry ntibody (Abcm Plc., Cmbridge, USA) t dilution of 1 : 5 to confirm equl loding of protein. Further, membrne ws probed with corresponding secondry ntibodies. Immunorective bnd of proteins were detected by chemiluminescence using enhnced chemiluminescence (ECL) regents (Amershm Bioscience, USA). Quntifiction of the results ws performed by densitometric scn of films. The immunorective re ws determined by densitometric nlysis using Biovis gel documenttion softwre Assessment of Mitochondril Function, Integrity, nd Oxidtive Stress Mitochondri Isoltion Procedure. Mitochondri were isolted by following stndrd procedure of Pedersen et l. [49]. Briefly, the brin regions were homogenized in (1 : 1, w/v) ice cold isoltion buffer (25 mm sucrose, 1 mm EGTA, nd 1 mm HEPES-KOH, ph 7.2) followed by centrifugtion t 6 g/5 min. The resultnt superntnt ws centrifuged t 1, g/15 min. The resultnt pellets were suspended in 1 ml medium (25 mm sucrose,.3 mm EGTA, nd 1 mm HEPES-KOH, ph 7.2) nd gin centrifuged t 14, g/1 min. All centrifugtion procedures were performed t 4 C. The finl mitochondril pellet ws resuspended in medium (25 mm sucrose nd 1 mm HEPES- KOH,pH7.2)ndusedwithin3h.Themitochondrilprotein content ws estimted using the method of Lowry et l. [5] Estimtion of Mitochondril Succinte Dehydrogense (SDH) Activity. The mitochondril SDH ws determined by following the method of Old nd Johnson [51] bsed on the progressive reduction of NBT to diformzn (dfz) mesured t 57 nm. The men SDH ctivity of ech region ws expressed s micromole formzn produced per min per milligrm of protein Estimtion of Mitochondril Membrne Potentil (MMP). TheRhodminedyetkenupbymitochondriws mesured with spectrofluorometer (Hitchi, F-25, Jpn; [52]). Briefly, the mitochondril suspension ws mixed with TMRM solution nd incubted for 5 min t 25 Cfollowed by frequent wshings (four times) to remove ny unbound TMRM. The florescence emission ws red t n excittion λ of 535 ± 1 nm nd emission λ of 58 ± 1 nm using slit no. 1. The pek fluorescence intensity recorded ws round 57 ± 5 nm. The intensity of fluorescence ws recorded which ws considered to be proportionl to MMP Estimtion of Mitochondril Lipid Peroxidtion (LPO). Mitochondril MDA content ws mesured s mrker for LPO described by Uchiym nd Mihr [53] nd modified by Sundermn et l. [54]. Briefly, the chromophore formed in the rection ws mesured t 532 nm. The MDA concentrtions re expressed s micromoles of MDA/mg of protein Estimtion of Mitochondril Superoxide Dismutse (SOD) Activity. The ctivity of SOD ws ssyed by the method of Kkkr et l. [55] bsed on the formtion of NADH-phenzine methosulphte-nitro blue tetrzolium formzn mesured t 56 nm ginst butnol s blnk. A single unit of the enzyme ws expressed s 5% inhibition of NBT reduction/min/mg of protein under the ssy conditions Estimtion of Mitochondril Ctlse (CAT) Activity. DecompositionofhydrogenperoxideinpresenceofCATws followed t 24 nm [56]. The results were expressed s units (U) of CAT ctivity/min/mg of protein Dt Anlysis. The results were expressed s men ± S.E.M. The sttisticl significnce for time-course effects on plsm glucose, TG, nd CORT levels nd prmeters in the EPM test prdigm ws nlyzed by two-wy nlysis of vrince (ANOVA) followed by post hoc Bonferroni test. All other dtsets were nlyzed by one-wy ANOVA followed by post hoc Student Newmn-Keuls test. P <.5 ws considered to be sttisticlly significnt throughout the experimentl dt nlysis. 3. Results 3.1. Metformin nd Dizepm Combintion Enhnced Glucose Tolernce during OGTT in Rts More Thn Metformin or Dizepm Monotherpy. Tble 1 illustrtes the effect of metformin or dizepm or their combintion on plsm glucose levels during OGTT in exposed rts. Repeted mesures of two-wy ANOVA reveled tht there were significnt differences in plsm glucose levels mong group (F (4, 1) = 1432; P <.5), time (F (3, 1) = 418.1; P <.5), nd n interction (F (12, 1) = 52.5; P <.5) between group nd time. Post hoc nlysis showed tht metformin but not dizepm monotherpy decresed the glucose level t 3 min fter glucose loding compred to rts. Moreover, the combintion of metformin nd dizepm showed remrkble improvement in glucose response t 3 min fter glucose loding compred to metformin monotherpy. This effect persisted up to 12 min fter glucose loding during OGTT.

5 BioMed Reserch Interntionl 5 Tble 1: Effect of metformin, dizepm, or their combintion on plsm glucose level during OGTT in cooccurring T2DM nd RS exposed rts. Groups Plsm glucose level (mg/dl) min 3 min 6 min 12 min 74.3 ± ± ± ± ± ± ± ± ± 6.6,b 69.6 ± 25.5,b 69. ± 23.5,b ± 24.2,b 499. ± 9.7,c ± 23.,c ± 16.2,c ± 29.,c + DZ 26.9 ± 4.3,b,c,d ± 24.,b,c,d ± 26.,b,c,d ± 9.,b,c,d Allvluesremen± SEM (n =6). P <.5 compred to control, b P <.5 compred to, c P <.5 compred to, nd d P <.5 compred to (repeted mesures of two-wy ANOVA followed by Bonferroni post hoc test). Tble 2: Effect of metformin, dizepm, or their combintion on plsm glucose level during ITT in cooccurring T2DM nd RS exposed rts. Groups Plsm glucose level (mg/dl) min 3 min 6 min 9 min 73.2 ± ± ± ± ± ± ± ± ± 5.6,b ± 4.5,b ± 3.7,b 39. ± 4.1,b ± 9.2,c ± 9.5,c ± 1.6,c 427. ± 9.4,c + DZ 23.1 ± 5.,b,c,d ± 3.9,b,c,d ± 3.8,b,c,d 14. ± 3.3,b,c,d Allvluesremen± SEM (n =6). P <.5 compred to control, b P <.5 compred to, c P <.5 compred to, nd d P <.5 compred to (repeted mesures of two-wy ANOVA followed by Bonferroni post hoc test) Metformin nd Dizepm Combintion Incresed Insulin Sensitivity during ITT in Rts More Thn Metformin or Dizepm Monotherpy. Tble 2 illustrtes the effect of metformin or dizepm or their combintion on plsm glucose levels during ITT in exposed rts. Repeted mesures of two-wy ANOVA reveled tht there were significnt differences in plsm glucose levels mong group (F (4, 1) = 1392; P <.5), time (F (3, 1) = 28.5; P <.5), nd n interction (F (12, 1) = 1.5; P <.5) between group nd time. Post hoc nlysis showed tht metformin but not dizepm monotherpy decresed significntly the plsm glucose level t 3 min fter insulin injection compred to rts. Moreover, the combintion of metformin nd dizepm showed remrkble improvement in insulin sensitivity t 3 min fter insulin injection compred to metformin monotherpy. This effect persisted up to 9 min fter insulin injection during ITT Metformin nd Dizepm Combintion Reduced - Induced Increse in the Plsm Glucose, Triglyceride, nd Corticosterone Levels in Rts More Thn Metformin or Dizepm Monotherpy. Tble 3 illustrtes the effect of metformin or dizepm or their combintion on -induced ltertions in plsm glucose, triglyceride, nd corticosterone levels. Repeted mesures of two-wy ANOVA reveled tht there were significnt differences in plsm glucose, triglyceride, nd corticosterone levels mong group ((F (4, 1) = 622; P <.5), (F (4, 1) = 746.2; P <.5) nd (F (4, 1) = 35.7; P <.5), resp.), time ((F (3, 1) = 1557; P <.5), (F (3, 1) = 227; P <.5) nd (F (3, 1)=1525;P <.5), resp.) nd n interction ((F (12, 1) = 1568; P <.5), (F (12, 1) = 231.3; P <.5), nd (F (12, 1) = 126.9; P <.5), resp.) between group nd time. Post hoc nlysis showed tht there were no significnt differences mong groups in plsm glucose or triglyceride or corticosterone levels on D-1. Streptozotocin injection cused significnt increse in the plsm glucose, triglyceride, nd corticosterone levels on D-3 of the experimentl schedule compred to vehicle treted rts. Further, exposure to repeted CRS ugmented the levels of plsm glucose, triglyceride, nd corticosterone on D-7 compred to control nimls. Metformin tretment significntly decresed the -induced increse in ll the biochemicl prmeters in the plsm on D-13 of the experimentl schedule. However, dizepm significntly reduced the -induced increse in the level of corticosterone only in the plsm of the rts. Furthermore, metformin nd dizepm combintion significntly reduced the -induced increse in ll the biochemicl prmeters in the plsm on D-13 compred to metformin nd dizepm monotherpy Dizepm in Combintion with Metformin Reduced Gstric Ulcer in Exposed Rts More Thn Either Metformin or Dizepm Monotherpy. The effect of metformin or dizepm or their combintion on -induced gstric ulcer in rts is depicted in Figure 1. Sttisticl nlysis by onewy ANOVA reveled tht there were significnt differences in gstric ulcer index (F (4, 25) = 49.29; P <.5) mong groups on D-13. Post hoc test showed tht induced significnt increse in the gstric ulcers in rts compred to vehicle treted nimls. Administrtion of either metformin or dizepm significntly reduced the -induced increse in the gstric ulcers in rts. Moreover, dizepm treted rts showed significnt decrese in the -induced

6 6 BioMed Reserch Interntionl Tble 3: Effect of metformin, dizepm, or their combintion on plsm glucose, triglyceride, nd corticosterone levels in cooccurring T2DM nd RS exposed rts. Groups D-1 D-3 D-7 D-13 Plsm glucose (mg/dl) 76.3 ± ± ± ± ± ± ± ± ± ± ± ± 4.7,b 75.3 ± ± ± ± 1.8,c + DZ 74.9 ± ± ± ± 5.2,b,c,d Plsm triglyceride (mg/dl) 85.9 ± ± ± ± ± ± ± ± ± ± ± ± 9.,b 91.3 ± ± ± ± 9.6,c + DZ 91.7 ± ± ± ± 7.1,b,c,d Plsm corticosterone (μg/dl) 15.8 ± ± ± ± ± ± ± ± ± ± ± ± 3.9,b 15.2 ± ± ± ± 2.6,b,c + DZ 15.4 ± ± ± ± 5.5,b,c,d Allvluesremen± SEM (n =6). P <.5 compred to control, b P <.5 compred to, c P <.5 compred to, nd d P <.5 compred to (repeted mesures of two-wy ANOVA followed by Bonferroni post hoc test). Gstric ulcer index , c, c, d Figure 1: The effect of metformin (MET), dizepm (DZ), nd their combintion on gstric ulcer in T2DM nd repeted CRS prdigm () exposed rts. All vlues re men ± SEM (n = 6). P <.5 compred to control, b P <.5 compred to, c P <.5 compred to, nd d P <.5 compred to (one-wy ANOVA followed by Student Newmnn-Keuls test). gstric ulcers compred to metformin dministered nimls. Furthermore, dministrtion of both dizepm nd metformin significntly decresed the -induced increse in the gstric ulcers compred to metformin nd dizepm monotherpy. + DZ 3.5. Metformin nd Dizepm Combintion Exhibited Better Anxiolytic Activity in Exposed Rts More Thn Their Monotherpy. Figure 2 illustrtes the effect of metformin or dizepm or their combintion on percentge of open rm entries to totl rm entries Figure 2() ndpercentgeof open rm time spent to totl rm time spent Figure 2(b),nd totl rm entries Figure 2(c) in EPM test prdigm of exposed rts. Sttisticl nlysis by repeted mesures of twowy ANOVA reveled tht there were significnt differences in percentge open rm entries nd time spent mong groups ((F (4, 5) = 4.97; P <.5) nd (F (4, 5) = 34.65; P <.5), resp.), time ((F (1, 5) = 52.99; P <.5) nd (F (1, 5) = 22.8; P <.5), resp.) nd n interction between group nd time ((F (4, 5) = 7.66; P <.5) nd (F (4, 5) = 4.79; P <.5), resp.). However, there were no significnt differences in totl rm entries in EPM prdigm mong group (F (4, 5) =.72; P >.5), time (F (1, 5) = 9.92; P >.5), nd there ws no significnt interction between group nd time (F (4, 5) =.9; P >.5). Post hoc nlysis showed tht prdigm cused significnt increse in the percentge open rm entries nd time spent on D-7 compred to control rts nd ws sustined up to D-13. Metformin nd dizepm significntly meliorted the -induced increse in the percentge open rm entries nd time spent in the EPM test. Moreover, tretment of both dizepm nd metformin significntly reduced the -induced increse in the percentge open rm entries nd time spent compred to metformin nd dizepm dministrtion Metformin nd Dizepm Combintion Enhnced the Serine Phosphoryltion of IRS-1 ser37 nd Akt ser473 in the Liver

7 BioMed Reserch Interntionl 7 25 b, c, d 1 Open rm entries (%) Open rm time spent (%) b, c, d D-7 D-13 D-7 D-13 + DZ + DZ () (b) 1 Totl rm entries (in number) D-7 D-13 (c) + DZ Figure 2: The effect of MET, DZ, nd their combintion on -induced ltertions in percentge of open rm entries to totl rm entries (), percentge of open rm time spent to totl rm time spent (b), nd totl rm entries (c) in EPM. All vlues re men ± SEM (n =6). P <.5 compred to control, b P <.5 compred to, c P <.5 compred to, nd d P <.5 compred to (repeted mesure two-wy ANOVA followed by Bonferroni test). of Exposed Rts More Thn Their Monotherpy. Figures 3 nd 4 depict the effect of metformin nd dizepm or their combintion on the -induced ltertions in the level of phosphoryltion of IRS-1 ser37 nd Akt ser473 in the liver tissues, respectively. Sttisticl nlysis by one-wy ANOVA reveled tht there were significnt differences in the level of expression of p-irs-1 ser37 (F (4, 1) = 62.14; P <.5) nd p-akt ser473 (F (4, 1) = 75.4; P <.5), nd rtio of p-irs-1 ser37 /totl IRS (F (4, 1) = 34.18; P <.5) ndp- Akt ser473 /totlakt(f(4,1)=56.26;p <.5) intheliver tissues mong groups. However, there were no significnt differences in the level of expression of totl IRS-1 (F (4, 1) =.47; P >.5) nd Akt (F (4, 1) =.21; P >.5) in the liver tissues mong groups. Post hoc nlysis showed tht exposure significntly reduced the extent of phosphoryltion of IRS-1 ser37 nd Akt ser473 in the rt liver tissues. Metformin but not dizepm significntly mitigted the -induced decrese in the extent of phosphoryltion of IRS-1 ser37 nd Akt ser473 in the liver. Moreover, metformin with dizepm combintion further meliorted the -induced decrese in the extent of phosphoryltion of IRS-1 ser37 nd Akt ser473 in the liver compred to metformin monotherpy Effect of Metformin or Dizepm or Their Combintion on -Induced Altertions in Mitochondril SDH nd MMP in Discrete Brin Regions. Figure 5 depicts the effect of metformin, dizepm, or their combintion on -induced chnges in mitochondril SDH nd MMP in different brin regions. Sttisticl nlysis by one-wy ANOVA reveled tht there were significnt differences in percentge in mitochondril SDH nd MMP in HIP ((F (4, 25) = 72.12; P <.5) nd(f(4,25)=75.19;p <.5), resp.), HYP ((F (4, 25) = 5.29; P <.5) nd (F (4, 25) = 7.1; P <.5), resp.), PFC ((F (4, 25) = 32.54; P <.5) nd (F (4, 25) = 43.8; P <.5), resp.), STR ((F (4, 25) = 6.91; P <.5) nd(f (4,25)=56.4;P <.5), resp.), AMY ((F (4, 25) = 54.94;

8 8 BioMed Reserch Interntionl p-irs-1 ser37 Totl IRS β-actin + DZ Rtio of intensity of p-irs /β-ctin ser , c, c, d + DZ () (b) Rtio of intensity of totl IRS/β-ctin Rtio of intensity of p-irs-1 ser37 /totl IRS , c, c, d + DZ + DZ (c) (d) Figure 3: The effect of MET, DZ, nd their combintion on -induced chnges in the level of expression of phospho-irs ser37 (p-irs ser37 ) nd totl IRS in the liver tissues. The blots re representtive of p-irs ser37 nd totl IRS () in the liver tissues. The results in the histogrm re expressed s rtio of reltive intensity of levels of protein expression of either p-irs ser37 or totl IRS to β-ctin nd rtio of reltive intensity of level of expression of p-irs ser37 to totl IRS. All vlues re men ± SEM of three seprte sets of independent experiments. P <.5 compred to control, b P <.5 compred to, c P <.5 compred to, nd d P <.5 compred to (one-wy ANOVA followed by Student Newmnn-Keuls test). P <.5) nd (F (4, 25) = 5.94; P <.5), resp.) nd NAC ((F (4, 25) = 3.4; P <.5) nd (F (4, 25) = 35.41; P <.5), resp.) mong groups. Post hoc test showed tht metformin nd dizepm monotherpy significntly reversed the-inducedincresenddecreseinthemitochondril SDH nd MMP, respectively, in ll the brin regions. Furthermore, dministrtion of combintion of metformin nd dizepm cused significnt reduction in the induced increse nd decrese in the mitochondril SDH nd MMP, respectively, in ll the brin regions compred to their monotherpy Effect of Metformin or Dizepm or Their Combintion on -Induced Altertions in Mitochondril LPO nd Activities of SOD nd CAT in Discrete Brin Regions. The effect of metformin or dizepm or their combintion on -induced chnges in mitochondril LPO nd ctivities of SOD nd CAT in different brin regions is illustrted in Figure 6.Onewy ANOVA reveled tht there were significnt differences in percentge in mitochondril LPO nd ctivities of SOD nd CAT in HIP ((F (4, 25) = 38.73; P <.5), (F (4, 25) = 22.26; P <.5) nd (F (4, 25) = 35.26; P <.5), resp.), HYP ((F (4, 25) = 57.62; P <.5), (F (4, 25) = 25.43; P <.5) nd (F (4, 25) = 43.28; P <.5), resp.), PFC ((F (4, 25) = 54.3; P <.5), (F (4, 25) = 41.21; P <.5) nd (F (4, 25) = 32.11; P <.5), resp.), STR ((F (4, 25) = 31.44; P <.5), (F (4, 25) = 21.71; P <.5) nd (F (4, 25) = 52.8; P <.5), resp.), AMY ((F (4, 25) = 24.; P <.5), (F (4, 25) = 63.25;

9 BioMed Reserch Interntionl 9 p-akt ser473 Totl Akt β-actin + DZ Rtio of intensity of p-akt ser473 /β-ctin , c, c, d + DZ () (b) Rtio of intensity of totl Akt /β-ctin Rtio of intensity of p-akt ser473 /totl Akt , c, c, d + DZ + DZ (c) (d) Figure 4: The effect of MET, DZ, nd their combintion on -induced chnges in the level of expression of phospho-akt ser473 (p-akt ser473 ) ndtotlaktinthelivertissues.theblotsrerepresenttiveofp-akt ser473 nd totl Akt () in the liver tissues. The results in the histogrm re expressed s rtio of reltive intensity of levels of protein expression of either p-akt ser473 or totl Akt to β-ctin nd rtio of reltive intensity oflevelofexpressionofp-akt ser473 to totl Akt. All vlues re men ± SEM of three seprte sets of independent experiments. P <.5 compred to control, b P <.5 compred to, c P <.5 compred to, nd d P <.5 compred to (one-wy ANOVA followed by Student Newmnn-Keuls test). P <.5) nd (F (4, 25) = 42.3; P <.5), resp.) nd NAC ((F (4, 25) = 29.49; P <.5), (F (4, 25) = 45.42; P <.5) nd (F (4, 25) = 46.26; P <.5), resp.) mong groups. Post hoc test reveled tht metformin nd dizepm monotherpy significntly decresed the -induced increse in the mitochondril LPO in ll the brin regions. Administrtion of both metformin nd dizepm further decresed the inducedincreseinthemitochondrillpoinllthebrin regions compred to metformin nd dizepm monotherpy. Moreover, metformin nd dizepm monotherpy cused significnt increse in the -induced decrese in the ctivities of mitochondril SOD nd CAT in ll the brin regions. Administrtion of metformin nd dizepm combintion further incresed the -induced decrese in the mitochondril SOD nd CAT in ll the brin regions compred to their monotherpy. 4. Discussion The primry objective of the present study ws to evlute the ntidibetic efficcy of metformin nd dizepm in cooccurring T2DM nd repeted CRS exposed rts. Additionlly, the study lso exmined the effect of codministrtion of metformin nd dizepm for ntidibetic, ntistress, nd nxiolytic-like ctivities in the bove model. Metformin exhibited ntidibetic, ntistress, nd nxiolytic-like ctivities in the cooccurring condition of T2DM nd stress. However,

10 1 BioMed Reserch Interntionl Mitochondril SDH ctivity (%) Mitochondril membrne potentil (%), c, d, c, d, c, d 35, c, d 3, c, d, c b, c, d HIP HYP PFC STR AMY NAC , c, d (), c, d, c, d, c, d, c, d, c, d HIP HYP PFC STR AMY NAC (b) + DZ Figure 5: The effect of MET, DZ, nd their combintion on mitochondril succinte dehydrogense (SDH) ctivity nd membrne potentil in discrete brin regions in exposed rts. All vlues re men ± SEM (n = 6). P <.5 compred to control, b P <.5 compred to, c P <.5 compred to, nd d P <.5 compred to (one-wy ANOVA followed by Student Newmnn-Keuls test). dizepm showed ntistress nd nxiolytic-like ctivities but not ntidibetic ctivity in the bove condition. Metformin nd dizepm combintion enhnced ntidibetic, ntistress, nd nxiolytic-like ctivities compred to metformin tretment in cooccurring T2DM nd repeted CRS exposed rts. We for the first time report tht the combintion therpy improved the glucose tolernce nd insulin sensitivity in the cooccurring T2DM nd stress condition. Further, the combintion improved the insulin resistnce in the liver of cooccurring T2DM nd repeted CRS exposed rts. The combintion therpy showed mrked improvement in mitochondril function, integrity, nd oxidtive stress in ll the brin regions in the bove condition more thn their monotherpy. Thus, metformin in combintion with dizepm would be better therpeutic option in the mngement of cooccurring T2DM nd stress condition. To ssess the effect of metformin, dizepm, nd their combintion on cooccurring T2DM nd stress conditioninduced disturbnce in the glucose homeostsis, the OGTT nd ITT were performed. OGTT is n importnt index for the evlution of bet cell function while ITT indictes the insulin sensitivity [57]. The impirment in glucose tolernce nd insulin sensitivity during T2DM is reported to be improved by metformin tretment [4, 58]. In the present study, the co-occurring condition impired glucose tolernce nd insulin sensitivity. Metformin, but not dizepm monotherpy, improved glucose tolernce in the bove cooccurring condition. Interestingly, the improvement ofglucosetolernceinthecooccurringconditionwsbetter with combintion therpy thn metformin monotherpy. Moreover, the combintion improved insulin sensitivity in the cooccurring condition of T2DM nd stress compred to metformin monotherpy. These results suggest the fct tht the combintion my hve potentil therpeutic effect on the insulin signling pthwy in the bove condition. Experimentl s well s clinicl studies report hyperglycemi nd berrnt lipid profile in T2DM with stress condition [7, 59 61]. Similr to erlier findings, the levels of glucose nd triglyceride were elevted with the cooccurring T2DM nd stress exposure. Metformin exhibited ntidibetic ctivity in terms of reducing glucose s well s triglyceride in the rts exposed to both T2DM nd stress prdigm. Metformin decreses T2DM-induced increse in the level of glucose nd triglyceride in the plsm of both nimls nd ptients [62, 63]. However, this is the first report of effect of metformin on the rts exposed to both T2DM nd stress prdigm. Further, metformin in presence of dizepm exhibited pronounced ntihyperglycemic nd ntihypertriglyceridemic effect thn metformin monotherpy in cooccurring T2DM nd stress exposed rts. In n erlier report, it hs been documented tht dizepm tretment ugmented the hyperglycemic condition in hyperglycemic rts nd this effect ws meliorted with metformin dministrtion [29]. However, in the present study dizepm did not lter the hyperglycemic condition in the cooccurring condition of T2DM nd stress. This discrepncy could be due to both the mode of induction of dibetes nd the presence of stress. These effects indicte tht this combintion shows prominent ntidibetic effect in terms of reducing blood glucose nd triglyceride levels in cooccurring T2DM nd stress condition. It is demonstrted tht hypercorticosteronemi is observed in both T2DM nd stress conditions nd lso in their cooccurring sitution [7, 64 66], result of overctivtion of hypothlmic-pituitry-drenl cortex- (HPA-) xis function [67]. Similr to erlier observtions, there ws significnt increse in the level of corticosterone in theplsmincooccurringt2dmndstressexposednimls in the current study. In ddition, this cooccurring condition exhibited significnt ulcers in the stomch of the nimls providing further evidence s peripherl mrker of HPAxis dysfunction. Dizepm potentited the ntistress ctivity of metformin in the T2DM rts exposed to repeted stress prdigm in terms of reducing in the plsm corticosterone nd gstric ulcertion more thn metformin nd dizepm monotherpy. Similr to our findings, metformin regulted plsm corticosterone in T2DM subjects [68, 69]. However, the mechnism of reduction of corticosterone by metformin is still not cler. It hs lso been postulted tht metformin exhibits the ntidibetic effect by downregulting the glucocorticoid receptors in the brin tissues of rts with hypercorticosteronemi [68]. Dizepm modultes HPAxis function during stress conditions nd thus reduces the level of plsm corticosterone in both nimls nd ptients [27, 3]. It hs been suggested tht ctivtion

11 BioMed Reserch Interntionl 11 Mitochondril LPO (%) , c, d, c, d, c, d, c, d, c, d b, c, d HIP HYP PFC STR AMY NAC () Mitochondril CAT ctivity (%) , c, d Mitochondril SOD ctivity (%) b, c, d, c, d, c, d, c, d, c, d b, c, d HIP HYP PFC STR AMY NAC, c, d b, c, d b, c, d, c, d HIP HYP PFC STR AMY NAC (b), c, d (c) + DZ Figure 6: The effect of MET, DZ, nd their combintion on mitochondril lipid peroxidtion (LPO) nd superoxide dismutse (SOD) nd ctlse (CAT) ctivities in discrete brin regions in exposed rts. All vlues re men ± SEM (n = 6). P <.5 compred to control, b P <.5 compred to, c P <.5 compred to, nd d P <.5 compred to (one-wy ANOVA followed by Student Newmnn-Keuls test). of corticosteroid receptors plys significnt role in the pthogenesis of nxiety-like behviors in dibetic rts [7]. Further, corticosteroid receptor ntgonists synergize the nxiolytic-like ctivity of dizepm in the dibetic rts [7]. Thus, it cn be ssumed tht the metformin nd dizepm in combintion exhibited ntistress effect in cooccurring dibetic-stress condition probbly cting through common corticosteroid-dependent mechnism. This contention, however,hstobefurtherinvestigted. T2DM ptients more vulnerble to stress-relted disorders such s nxiety hve been ssocited with poor glycemic control [71, 72].Dibetic ptients with nxiety disorders hve shown much pronounced hyperglycemi [73]. Thus, it is ssumed tht successful tretment of nxiety my improve glycemic control in T2DM subjects. In the present study, cooccurring T2DM nd CRS condition exhibited nxietylike symptoms on D-7 of the streptozotocin injection in the EPM test prdigm nd this effect ws sustined up to D- 13 of the experimentl schedule. The nxiety-like behvior is well documented in dibetes [74]. However, this is the first study where T2DM rts exposed to repeted stress prdigm showed nxiety-like behviour. Metformin nd dizepm tretment for seven consecutive dys ttenuted the nxiety-like symptoms in experimentl rts exposed to both T2DM nd repeted CRS prdigm. Metformin in presence of dizepm cused remrkble reduction in the nxiety-like symptoms in experimentl nimls exposed to both T2DM nd repeted CRS prdigm. The results indicte better nxiolytic-like effect of the combintion over either metformin or dizepm monotherpy in these conditions in ddition to ntidibetic ctivity. The combined results of OGTT nd ITT demonstrte tht thereisimpirmentintheinsulinsignlingpthwyinthe cooccurring T2DM nd stress exposed nimls. Both metformin nd its combintion with dizepm therpy showed improvement in the insulin signling pthwy in the bove condition. To elborte on the insulin resistnce, we evluted the extent of phosphoryltion of IRS-1 ser37 nd Akt ser473 in the liver. It hs lredy been documented tht there is reduction in the phosphoryltion of IRS-1 ser37 nd Akt ser473 in the liver of either T2DM or neurodegenertion [11, 12, 75 77]. In the present study, we for the first time report tht the phosphoryltion of IRS-1 ser37 nd Akt ser473 ws reduced in the cooccurring condition of T2DM nd stress. Further, the combintion of metformin nd dizepm improved the insulin resistnce in prt by incresing the phosphoryltion of IRS-1 ser37 nd Akt ser473 in the liver of the cooccurring T2DM nd stress exposed nimls. Mitochondri function nd integrity re impired in both T2DMndstressndlsointhecooccurringconditionof T2DMndstressinbrintissues[7, 18]. Similr to erlier observtions, the present study reveled tht the cooccurring T2DM nd stress condition cuse hyperctivity of the mitochondril SDH enzyme nd loss in the mitochondril integrity in ll the brin regions [7, 18]. Further, metformin nd dizepm reversed the mitochondril SDH hyperctivity nd loss in integrity in ll the brin regions in the T2DM rts lso exposed to repeted stress prdigm. It hs been reported

12 12 BioMed Reserch Interntionl tht metformin restores the brin mitochondril function in dibetic condition [78, 79]. However, this is the first report on effect of metformin in repeted stress exposed dibetic rts. In ddition, metformin nd dizepm combintion further ttenuted the mitochondril SDH hyperctivity nd loss in integrity in ll the brin regions in cooccurring T2DM nd stress condition. Dizepm hs been shown to modulte mitochondril ctivity in the brin tissues [3]. Thus, the combintion therpy of metformin nd dizepm my hve potentil mitochondril-dependent ctivity in ddition to glucocorticoid-medited mechnism in this condition. The reltionship between lipid oxidtive dmge nd mitochondril function nd integrity is well estblished in T2DM, stress, nd their cooccurring condition [7, 21, 8]. Similr to erlier findings, the present study revels tht the extent of LPO ws higher in the cooccurring condition of T2DMndstressinllthebrinregions[7, 21, 8]. Metformin nd dizepm mitigted the extent of LPO in ll the brin regions in T2DM rts lso exposed to repeted stress. It hs been reported tht metformin decreses dibetesinduced increse in LPO in the brin regions [81]. However, this is the first time we report the effect of metformin nd dizepm on the extent of LPO in different brin regions in cooccurring T2DM nd stress prdigm. The combintion of metformin nd dizepm ttenuted the increse in the extent of LPO in ll the brin regions in T2DM rts subjected to repeted CRS prdigm. Moreover, reports lso suggest tht there is ttenution in the ntioxidnt defense system in the brin tissues in T2DM, stress, nd their cooccurring condition [7, 82]. In the present study, the combintion of metformin nd dizepm mitigted the decrese in the ntioxidnt enzyme ctivities such s SOD nd CAT in ll the brin regions in T2DM rts subjected to repeted stress thn either monotherpy. Hence, it cn be ssumed tht by improving the ntioxidnt defense system the combintion of metformin nd dizepm restores mitochondril function nd integrity nd ttenutes mitochondril oxidtive dmge in ll the brin regions. In conclusion, metformin exhibited ntidibetic effect in ddition to ntistress nd nxiolytic-like ctivity in the cooccurring condition of T2DM nd stress. Further, metformin long with dizepm showed pronounced ntidibetic, ntistress, nd nxiolytic-like effects compred to metformin monotherpy in the bove condition. This indictes synergistic effect of the combintion. Further, the combintion improved glucose tolernce, insulin sensitivity, nd resistnce in the bove condition. The combined regimen improved the mitochondril function, integrity, nd oxidtive stress in ll the brin regions in the cooccurring condition of T2DM nd stress. Thus, metformin in combintion with dizepm my be better therpeutic cndidte in the phrmcotherpy of cooccurring condition of T2DM nd stress. Conflict of Interests The uthors declre tht there is no conflict of interests regrding the publiction of this pper. Acknowledgment Debpriy Grbdu is thnkful to the Council of Scientific nd Industril Reserch (CSIR), New Delhi, for the Senior Reserch Fellowship (9/13 (366)/211-EMR-I). References [1] J.E.Shw,R.A.Sicree,ndP.Z.Zimmet, Globlestimtesof theprevlenceofdibetesfor21nd23, Dibetes Reserch nd Clinicl Prctice,vol.87,no.1,pp.4 14,21. [2]D.J.Mglino,E.L.M.Brr,P.Z.Zimmetetl., Glucose indices, helth behviors, nd incidence of dibetes in Austrli: the Austrlin dibetes, obesity nd lifestyle study, Dibetes Cre,vol.31,no.2,pp ,28. [3]S.M.Southwick,M.Vythilingm,ndD.S.Chrney, The psychobiology of depression nd resilience to stress: implictions for prevention nd tretment, Annul Review of Clinicl Psychology, vol. 1, pp , 25. [4] M. Kumri, J. Hed, nd M. 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